U.S. patent application number 17/002053 was filed with the patent office on 2021-01-21 for mineral salt-sulfonic acid compositions and methods of use.
This patent application is currently assigned to BMG PHARMA S.P.A.. The applicant listed for this patent is BMG PHARMA S.P.A.. Invention is credited to William A. Goolsbee, Jeffrey L. Lillard.
Application Number | 20210015855 17/002053 |
Document ID | / |
Family ID | 1000005165051 |
Filed Date | 2021-01-21 |
United States Patent
Application |
20210015855 |
Kind Code |
A1 |
Goolsbee; William A. ; et
al. |
January 21, 2021 |
MINERAL SALT-SULFONIC ACID COMPOSITIONS AND METHODS OF USE
Abstract
The present disclosure generally relates to the medical use of
compositions comprising a mineral salt and a sulfonic acid for
prevention and/or treatment of one or more mucosal diseases,
disorders, or conditions or one or more dermal diseases, disorders,
or conditions.
Inventors: |
Goolsbee; William A.;
(Gardnerville, NV) ; Lillard; Jeffrey L.; (Gig
Harbor, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BMG PHARMA S.P.A. |
Milano |
|
IT |
|
|
Assignee: |
BMG PHARMA S.P.A.
Milano
IT
|
Family ID: |
1000005165051 |
Appl. No.: |
17/002053 |
Filed: |
August 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16449632 |
Jun 24, 2019 |
10786531 |
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17002053 |
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14582839 |
Dec 24, 2014 |
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16449632 |
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13797360 |
Mar 12, 2013 |
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14582839 |
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13264690 |
May 31, 2012 |
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PCT/US2010/031319 |
Apr 15, 2010 |
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13797360 |
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61169540 |
Apr 15, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0014 20130101;
A61K 31/79 20130101; A61K 33/30 20130101; A61K 31/191 20130101;
A61K 9/0053 20130101 |
International
Class: |
A61K 33/30 20060101
A61K033/30; A61K 31/191 20060101 A61K031/191; A61K 31/79 20060101
A61K031/79 |
Claims
1. Method of treating mucosal disorders with a composition
comprising zinc gluconate, taurine and polyvinylpyrrolidone (PVP),
said method comprising: applying to each dermal area in need
thereof a pharmaceutically effective amount of said
composition.
2. The method according to claim 1, wherein said composition
comprises between 0.25% w/w to 5.5% w/w zinc gluconate, between
0.25% w/w to 30% w/w taurine and from 0.04% w/w to 15% w/w PVP.
3. The method according to claim 2, wherein said composition
comprises from between 0.20% w/w to 5.5% w/w zinc gluconate.
4. The method according to claim 2, wherein said composition
comprises from between 0.5% w/w to 8.0% w/w taurine.
5. The method according to claim 2, wherein said composition
comprises from between 0.5% w/w/ to 4.0% w/w taurine.
6. The method according to claim 1, wherein said composition
comprises 0.5% w/w zinc gluconate, 1.0% w/w taurine and 4.0% w/w
PVP.
7. The method according to claim 1, wherein said composition
comprises comprises 0.5% w/w zinc gluconate, 1.0% w/w taurine and
8.0% w/w PVP.
8. The method according to claim 1, wherein said composition
comprises comprises 2.0% w/w zinc gluconate, 4.0% w/w taurine and
4.0% w/w PVP.
9. The method according to claim 1, wherein said composition is in
the form of a liquid, a solid, a gel, a paste, an emulsion, an
ointment, a foam or a spray.
10. The method according to claim 1, wherein said composition is
delivered by a vehicle selected from the group consisting of a
sponge, gel cap, suppository and a lozenge.
11. The method according to claim 1, wherein said composition has a
pH between 3.5 and 4.5.
12. The method according to claim 1, wherein said composition has a
pH between 5.5 and 7.5.
13. The method according to claim 1, wherein said mucosal disorder
comprises mucositis.
14. The method according to claim 1, wherein said mucosal disorder
is selected from oral stomatitis, oral mucositis, an oral
ulceration.
15. The method according to claim 1, wherein said composition is
administered one or more times per day, once every day, once every
other day, once weekly or once a month.
16. The method according to claim 1, wherein said composition is
administered topically, orally or topically and orally.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/449,632 filed Jun. 24, 2019, which is a
continuation of of U.S. patent application Ser. No. 14/582,839
filed Dec. 24, 2014, which is a continuation of U.S. patent
application Ser. No. 13/797,360 filed Mar. 12, 2013, now abandoned,
which is a continuation of U.S. patent application Ser. No.
13/264,690, filed on May 31, 2012, now abandoned, which is U.S.
national stage application filed under 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2010/031319, filed on
Apr. 15, 2010, which claims priority to and the benefit of U.S.
Provisional Application No. 61/169,540 filed on Apr. 15, 2009, the
contents of all of which are incorporated herein by reference in
their entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
170169_401C2_SEQUENCE_LISTING.txt. The text file is 84 KB, was
created on Dec. 23, 2014 and is being submitted electronically via
EFS-Web.
BACKGROUND
Field
[0003] The present disclosure generally relates to the medical use
of compositions comprising a mineral salt and a sulfonic acid for
prevention and/or treatment of one or more mucosal or dermal
diseases, disorders, or conditions.
Description of the Related Art
[0004] Prolonged and recurrent inflammation associated with dermal
or mucosal disorders can result in extensive damage to the mucosal
epithelium, leading to severe ulceration, pain, and infection and
may ultimately require surgery. In addition, damaged mucosal
epithelium may be associated with severe malabsorption of
nutrients, diarrhea, weight loss, and the frequent need for oral
and parenteral nutrient supplementation. Currently, few therapies
are available for patients that act to enhance nutrient absorption
and restore the functional integrity of a mucosal or dermal
epithelium.
[0005] Atrophic vaginitis (AV) is known to affect many women. Women
who are in mid-life or beyond and who have declining estrogen
levels often present symptoms of AV. An estimated 10-40% of
postmenopausal women have symptoms of AV; however, despite the
prevalence of symptoms, only 20-25% of symptomatic women seek
medical attention (Cardozo et al., Obstet. Gynecol. 92:722-27,
1998; Pandit et al., Am. J. Med. Sci. 314:228-31, 1997). Through
identification of and intervention in this often overlooked and
under-diagnosed condition, the urogenital health and quality of
life of a large patient population could be improved.
[0006] A number of over-the-counter (OTC) vaginal moisturizer and
lubricant products are considered first-line nonhormonal treatments
for vaginal dryness. This option is appropriate for women concerned
about hormone use, who have minimal physiologic changes or
symptoms, or those who are not candidates for estrogen treatment.
For example, REPLENS.RTM., a polycarbophil-based vaginal
moisturizing gel has been shown to restore vaginal pH and improve
cytological morphology (Dupont et al., Maturitas 13:297-311, 1991;
Leiblum et al., JAMA 249:2159-98, 1983). However, definitive
efficacy data are lacking for almost all OTC preparations used for
treating atrophic vaginitis.
[0007] Moreover, some women may experience sensitivity or allergy
to components of moisturizers or lubricants. OTC products may
contain warming additives, dyes, perfume, bactericides, or
spermicides that can further irritate already sensitive, dry
vaginal mucosa. Other common vaginal and vulvar irritants include
benzocaine, chlorhexidine, preservatives (parabens and propylene
glycol), and condoms made of latex or containing lanolin. Thus, an
unfulfilled need remains for a first-line product that can treat or
relieve symptoms of atrophic vaginitis.
[0008] Another unmet medical need includes treatment of persons
with oral mucositis. Oral mucositis is a significant side effect of
cancer therapy and bone marrow transplantation, but it is not
adequately managed by current approaches (Sonis, "Oral
Complications," In Cancer Medicine, pp. 2381-2388, 1993a; Holland
et al., Eds., Lea and Febiger, Philadelphia; Sonis, "Oral
Complications in Cancer Therapy," In Principles and Practice of
Oncology, pp. 2385-2394, 1993b; DeVitta et al., Eds., J. B.
Lippincott, Philadelphia). Oral mucositis occurs in almost 100% of
patients receiving chemotherapy and radiotherapy for head and neck
tumors and in about 90% of children with leukemia. About 40% of
patients treated with chemotherapy for other tumors develop oral
problems during each exposure to the chemotherapeutic agent (Sonis,
1993b, supra). Additionally, approximately 75% of patients
undergoing bone marrow transplantation, both autologous and
allogeneic, develop mucositis (Woo et al., Cancer 72:1612-1617,
1993). Current estimates indicate that about 400,000 patients
suffer from oral mucositis annually in the United States alone
(Graham et al., Cancer Nursing 16:117-122, 1993). Given that
patients often receive multiple cycles of chemo- and/or
radiotherapy, an estimated 1,000,000 incidences of oral mucositis
occur per year in the United States.
[0009] A variety of approaches for treating oral mucositis,
including mitigation of the potential for subsequent oral
infections, have been tested with limited success. For example, the
use of an allopurinol mouthwash, an oral sucralfate slurry, and
pentoxifylline were reported in preliminary studies to result in a
decrease in mucositis. However, subsequent randomized and
controlled studies have failed to demonstrate any benefit (Loprinzi
et al., Sem. Oncol. 22 (53):95-97, 1995; Epstein et al., Int. J.
Radiat. Oncol. Biol. Phys. 28:693-698, 1994; Verdi et al., Oral
Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 80:36-42,
1995).
[0010] Other treatments have been directed at decreasing oral flora
and minimizing extent of infection as means to manage oral
ulcerations. For example, systemic treatment with
granulocyte-macrophage colony-stimulating factor (GM-CSF) has been
shown to result in a decreased incidence of oral mucositis,
presumably by allowing for more rapid neutrophil recovery and thus
an improved ability to combat infection (Chi et al., J. Clin.
Oncol. 13:2620-2628, 1995). However, in at least one study GM-CSF
was reported to exacerbate mucositis (Cartee et al., Cytokine
7:471-477, 1994).
[0011] Benzydamine hydrochloride, a nonsteroidal drug with
analgesic and antimicrobial properties, has been studied both in
patients undergoing radiation therapy and in patients receiving
intra-arterial chemotherapy (Epstein et al., Oral Surg. Oral Med.
Oral Pathol. 62:145-148, 1986; Epstein et al., Int. J. Radiat.
Oncol. Biol. Phys. 16:1571-1575, 1989). Chlorhexidine, an
antimicrobial mouth rinse, has also been used extensively in the
treatment and prevention of oral mucositis (Ferretti et al., Bone
Marrow Transplan. 3:483-493, 1990; Weisdorf et al., Bone Marrow
Transplan. 4:89-95, 1989). However, the efficacy of chlorhexidine
has been observed to be significantly decreased in saliva, and this
compound is relatively ineffective against the Gram negative
bacteria that tend to colonize the oral cavity in patients
undergoing radiation therapy (Spijkervet et al., Oral Surg. Oral
Med. Oral Pathol. 69:444-449, 1990). In addition, at least one
study has shown that the use of chlorhexidine may be detrimental
and result in a higher incidence of mucositis (Foote et al., J.
Clin. Oncol. 12:2630-2633, 1994). Several studies have shown that
the use of a vancomycin paste and antibiotic lozenges containing
polymixin B, tobramycin, and amphotericin B in patients undergoing
myelosuppressive chemotherapy or radiation therapy can result in a
decrease in oral mucositis and in the incidence of sepsis due to
alpha hemolytic streptococci (Barker et al., 1995, J. Ped. Hem.
Oncol. 17:151-155; Spijkervet et al., 1991, In Irradiation
Mucositis, Munksgaard Press, pp. 43-50).
[0012] However, despite the clear need for therapeutic compositions
to simply and reliably treat oral mucositis, no drugs are currently
approved for this indication. As a result no standard treatment is
available for this mucosal disorder, and an unmet need remains.
BRIEF SUMMARY
[0013] Provided herein are methods for treating mucosal and dermal
disorders. In one embodiment, a method is provided for treating a
mucosal disorder or a dermal disorder in a subject, which method
comprises administering to the subject a physiologically acceptable
composition that comprises a mineral salt and a sulfonic acid. In
one embodiment, the mucosal disorder comprises mucositis. In
specific embodiments, mucositis comprises inflammation of mucosa of
the gastrointestinal tract, bladder, esophagus, vagina, rectum,
lung, a nasal cavity, an ear, or ocular mucosa. In another
embodiment, the mucosal disorder comprises oral stomatitis, oral
mucositis, an oral ulceration, inflammatory bowel disease
(including Crohn's disease and ulcerative colitis), periodontitis,
interstitial cystitis, or a wound. In yet other embodiments, the
mucosal disorder comprises vaginal dryness, vaginal burning,
vaginal ulceration, dyspareunia, leukorrhea, vulvar pruritus,
vulvar burning, or atrophic vaginitis. In certain embodiments, the
mucosal disorder is consequent to any one or more of hormone
insufficiency, bone marrow transplant, chemotherapy, radiation
therapy, viral infection, fungal infection, and bacterial
infection. In a more specific embodiment, the mucosal disorder is
consequent to one or both of chemotherapy and radiation therapy
administered to the subject for treatment of a head and neck tumor,
a leukemia, breast cancer, prostate cancer, pancreatic cancer,
ovarian cancer, melanoma, liver cancer, lung cancer, urinary
cancer, colon cancer, or HIV/AIDS. In still another specific
embodiment, the viral infection is caused by a Herpes Simplex Virus
or Varicella zoster virus. In other embodiments, the dermal
disorder comprises diaper rash, skin dryness, dermatitis, eczema,
psoriasis, erythema, acne, xerosis, and radical oxygen
species-induced skin damage.
[0014] With respect to the methods described above and herein, in
certain embodiments, the mineral salt comprised within the
composition comprises (a) a mineral moiety selected from zinc,
calcium, iron, copper, magnesium, manganese, cobalt, chromium,
selenium, and vanadium and (b) a salt moiety selected from
gluconate, acetate, ascorbate, and sulfate. In a more specific
embodiment, the mineral salt is zinc gluconate, and the composition
comprises from 0.25% (w/w) to 5.5% (w/w) zinc gluconate. In other
specific embodiments, the mineral salt is zinc gluconate, and the
composition comprises from between 0.20% (w/w) to 5.5% (w/w) zinc
gluconate. Further with respect to the methods described above and
herein, in certain embodiments, the sulfonic acid comprised within
the composition is taurine and the composition comprises from
between 0.25% (w/w) to 30% (w/w) taurine. In certain embodiments,
the composition comprises from between 0.25% (w/w) to 5.5% (w/w)
zinc gluconate and comprises from between 0.25% (w/w) to 30% (w/w)
taurine. In more specific embodiments, the sulfonic acid is
taurine, and the composition comprises from between 0.5% (w/w) and
4.0% (w/w) taurine. In another more specific embodiment, the
sulfonic acid is taurine and the composition comprises from between
0.5% (w/w) and 8.0% (w/w) taurine. In still other specific
embodiments, the composition comprises from between 0.25% (w/w) to
5.5% (w/w) zinc gluconate and from between 0.5% (w/w) and 8.0%
(w/w) taurine. In other embodiments, the composition further
comprises one or more of a flavoring agent, a mucoadhesive agent, a
pH adjusting agent, a solubilizing agent, a viscosity modulating
agent, and a stabilizing agent. In a more particular embodiment,
the compositions described above and herein further comprise one or
more of (a) from between 0.05% to 3.0% (w/w) glycyrrhetinic acid;
(b) from between 0.04% to 15% (w/w) polyvinylpyrrolidone (PVP); (c)
from between 0.01% to 5.0% (w/w) hyaluronic acid; and (d) from
between 0.05% to 3.0% (w/w) glycerin. In other specific
embodiments, the compositions comprising a mineral salt and a
sulfonic acid (as described above and herein) further comprise one
or more of (a) from between 0.05% to 3.0% (w/w) glycyrrhetinic
acid; (b) from between 0.04% to 15% (w/w) polyvinylpyrrolidone
(PVP); (c) from between 0.01% to 5.0% (w/w) hyaluronic acid; and
(d) from between 0.05% to 5.0% (w/w) glycerin. In another specific
embodiment, the compositions comprise (a) 0.5% (w/w) zinc
gluconate, 1.0% (w/w) taurine, and 4.0% PVP (w/w); (b) 0.5% (w/w)
zinc gluconate, 1.0% (w/w) taurine, and 8.0% PVP (w/w); or (c) 2.0%
(w/w) zinc gluconate, 4.0% (w/w) taurine, and 4.0% PVP (w/w). In
other specific embodiments, the composition comprises 0.5% (w/w)
zinc gluconate and 1.0% (w/w) taurine. In still another specific
embodiment, the composition comprises 2.5% (w/w) zinc gluconate and
5.0% (w/w) taurine. In still other particular embodiments, these
compositions that comprise a mineral salt and a sulfonic acid, as
described above and herein, further comprise a lactoferrin.
[0015] In particular embodiments, any one of the compositions
described above and herein that is administered according to the
methods described above and herein is a liquid, a solid, a gel, a
paste, an emulsion, an ointment, a foam, or a spray. In other
particular embodiments, the composition is delivered by a vehicle
selected from a sponge, gel cap, suppository, and a lozenge. In
certain embodiments, the composition has a pH between 3.0 and 8.5.
In certain particular embodiments, the pH is between 3.5 and 4.5;
and in such particular embodiments, the mucosal disorder is
atrophic vaginitis. In other particular embodiments, the pH is
between 5.5 and 7.5, and in a specific embodiment, the mucosal
disorder is oral mucositis.
[0016] With respect to the methods described above and herein, the
composition is administered one or more times per day, once every
day, once every other day, once weekly, once biweekly, or once a
month. In a particular embodiment, the compositions described
herein and above are administered topically, or orally, or
topically and orally. In certain specific embodiments, wherein any
of the compositions described above and herein comprises a
lactoferrin, the composition is administered orally. In another
specific embodiment, wherein any of the compositions described
above and herein comprises a lactoferrin, the composition is
administered topically, or orally, or topically and orally. In
particular embodiments, the methods described above and herein
further comprise orally administering a second physiologically
acceptable composition, wherein the second composition comprises a
lactoferrin.
[0017] In another embodiment, a method is provided for treating a
mucosal disorder in a subject who is receiving or who will receive
chemotherapy or radiation therapy for treatment of a malignancy
(i.e., cancer), said method comprising administering to the subject
a therapeutically effective amount of a physiologically acceptable
composition that comprises a mineral salt and a sulfonic acid. In
certain specific embodiments, the mineral salt is zinc gluconate
and the sulfonic acid is taurine. In other embodiments, these
methods comprise administering in certain embodiments, the mineral
salt comprised within the composition comprises (a) a mineral
moiety selected from zinc, calcium, iron, copper, magnesium,
manganese, cobalt, chromium, selenium, and vanadium and (b) a salt
moiety selected from gluconate, acetate, ascorbate, and sulfate. In
a more specific embodiment, the mineral salt is zinc gluconate, and
the composition comprises from 0.2% (w/w) to 5.5% (w/w) zinc
gluconate. In other specific embodiments, the mineral salt is zinc
gluconate, and the composition comprises from between 0.25% (w/w)
to 5.5% (w/w) zinc gluconate. Further with respect to the methods
described above and herein, in certain embodiments, the sulfonic
acid comprised within the composition is taurine and the
composition comprises from between 0.25% (w/w) to 30% (w/w)
taurine. In certain embodiments, the composition comprises from
between 0.25% (w/w) to 5.5% (w/w) zinc gluconate and comprises from
between 0.25% (w/w) to 30% (w/w) taurine. In more specific
embodiments, the sulfonic acid is taurine and the composition
comprises from between 0.5% (w/w) and 4.0% (w/w) taurine. In
another more specific embodiment, wherein the sulfonic acid is
taurine, the composition comprises from between 0.5% (w/w) and 8.0%
(w/w) taurine. In still other specific embodiments, the composition
comprises from between 0.25% (w/w) to 5.5% (w/w) zinc gluconate and
from between 0.5% (w/w) and 8.0% (w/w) taurine. In other
embodiments, the composition further comprises one or more of a
flavoring agent, a mucoadhesive agent, a pH adjusting agent, a
solubilizing agent, a viscosity modulating agent, and a stabilizing
agent. In a more particular embodiment the compositions described
above and herein further comprise one or more of from between 0.05%
to 3.0% (w/w) glycyrrhetinic acid; from between 0.04% to 15% (w/w)
polyvinylpyrrolidone (PVP); from between 0.01% to 5.0% (w/w)
hyaluronic acid; and from between 0.05% to 5.0% (w/w) glycerin. In
another embodiment, the composition comprises from between
0.25-5.5% (w/w) zinc gluconate; from between 0.5%-8% (w/w) taurine;
and from between 0.04%-15% (w/w) PVP. In another specific
embodiment, the compositions comprise (a) 0.5% (w/w) zinc
gluconate, 1.0% (w/w) taurine, and 4.0% PVP (w/w); (b) 0.5% (w/w)
zinc gluconate, 1.0% (w/w) taurine, and 8.0% PVP (w/w); or (c) 2.0%
(w/w) zinc gluconate, 4.0% (w/w) taurine, and 4.0% PVP (w/w). In
other specific embodiments, the composition comprises 0.5% (w/w)
zinc gluconate and 1.0% (w/w) taurine. In still another specific
embodiment, the composition comprises 2.5% (w/w) zinc gluconate and
5.0% (w/w) taurine. In other specific embodiments, the pH of the
composition is adjusted to between 3.5 and 4.5 or is adjusted to
between 5.5 and 7.5. In still other particular embodiments, these
compositions further comprise a lactoferrin.
[0018] Also provided herein is a composition comprising 0.5%-2%
(w/w) zinc gluconate; 0.5%-4% (w/w) taurine; and at least one of
(a) 0.5%-2.5% (w/w) glycyrrhetinic acid; (b) 0.25%-10% (w/w)
polyvinylpyrrolidone (PVP); (c) 0.05%-0.25% (w/w) hyaluronic acid;
and (d) 0.05%-0.25% (w/w) glycerin. In certain embodiments, the
composition comprises 0.5%-2% (w/w) zinc gluconate; 0.5%-4% (w/w)
taurine; and 4-8% (w/w) PVP. In other specific embodiments, the
composition comprises 0.5% (w/w) zinc gluconate, 1.0% (w/w)
taurine, 1.0% (w/w) glycyrrhetinic acid, 8.0% (w/w) PVP, 0.1% (w/w)
hyaluronic acid, and 0.1% (w/w) glycerin. In still another
embodiment, the composition comprises 0.5% (w/w) zinc gluconate,
1.0% (w/w) taurine, 1.0% (w/w) glycyrrhetinic acid, 8.0% (w/w) PVP,
0.1% (w/w) hyaluronic acid, and 0.1% (w/w) glycerin. In other
specific embodiments, the pH of the composition is adjusted to
between 3.5 and 4.5 or is adjusted to between 5.5 and 7.5. In yet
another embodiment, the composition further comprises a
lactoferrin.
[0019] Also provided herein, is a method for supplementing a
mineral deficiency in a subject, said method comprising
administering a composition comprising a mineral salt, a sulfonic
acid, and one or more of 0.05% to 3.0% (w/w) glycyrrhetinic acid;
0.04% to 15% (w/w) polyvinylpyrrolidone (PVP); 0.01% to 5.0% (w/w)
hyaluronic acid; and 0.05% to 5.0% (w/w) glycerin. In particular
embodiments of this method, the mineral salt comprises (a) a
mineral moiety selected from zinc, calcium, iron, copper,
magnesium, manganese, cobalt, chromium, selenium, and vanadium and
(b) a salt moiety selected from gluconate, acetate, ascorbate, and
sulfate. In a more specific embodiment, the mineral moiety is zinc
and the salt moiety is gluconate. In still other specific
embodiments, the sulfonic acid is taurine. In yet another
embodiment, the method for supplementing a mineral deficiency in a
subject comprises administering a composition comprising from
between 0.25% (w/w) to 5.5% (w/w) zinc gluconate and from between
0.25% (w/w) to 30% (w/w) taurine. In still another embodiment, the
method for supplementing a mineral deficiency in a subject
comprises administering a composition comprising from between 0.25%
(w/w) to 5.5% (w/w) zinc gluconate and from between 0.5% (w/w) to
8% (w/w) taurine.
[0020] In another embodiment, a method is provided for inhibiting
disruption of intercellular junctions between adjacent cells,
comprising contacting the cells with a composition comprising a
physiologically acceptable composition that comprises a mineral
salt and a sulfonic acid. In certain embodiments, the cells are
epithelial cells, and in other certain embodiments, the cells are
endothelial cells. In still another embodiment, the cells are
present in a subject who has or who is at risk for developing a
mucosal disorder or a dermal disorder. In a more specific
embodiment, the mucosal disorder comprises mucositis. In particular
embodiments, the mucosal disorder is selected from oral stomatitis,
oral mucositis, an oral ulceration, Crohn's disease, periodontitis,
interstitial cystitis, and a wound; vaginal dryness, vaginal
burning, vaginal ulceration, dyspareunia, leukorrhea, vulvar
pruritus, vulvar burning, and atrophic vaginitis; a mucosal
disorder that is consequent to any one or more of hormone
insufficiency, bone marrow transplant, chemotherapy, radiation
therapy, viral infection, fungal infection, and bacterial
infection; and a mucosal disorder that is consequent to one or both
of chemotherapy and radiation therapy administered to the subject
for treatment of a head and neck tumor, a leukemia, breast cancer,
prostate cancer, pancreatic cancer, ovarian cancer, melanoma, liver
cancer, lung cancer, urinary cancer, colon cancer, or HIV/AIDS. In
yet another specific embodiment, the dermal disorder comprises
diaper rash, skin dryness, dermatitis, eczema, psoriasis, erythema,
acne, xerosis, or radical oxygen species-induced skin damage. In
particular embodiments, the mineral salt comprises (a) a mineral
moiety selected from zinc, calcium, iron, copper, magnesium,
manganese, cobalt, chromium, selenium, and vanadium and (b) a salt
moiety selected from gluconate, acetate, ascorbate, and sulfate. In
more specific embodiments, the mineral salt is zinc gluconate, and
the composition comprises from between 0.25% (w/w) to 5.5% (w/w)
zinc gluconate. In another specific embodiment, the sulfonic acid
is taurine and the composition comprises from between 0.25% (w/w)
to 30% (w/w) taurine. In yet other embodiments, the mineral salt is
zinc gluconate, and the composition comprises from between 0.25%
(w/w) to 5.5% (w/w) zinc gluconate, and the sulfonic acid is
taurine and the composition comprises from between 0.25% (w/w) to
30% (w/w) taurine. In other particular embodiments, the sulfonic
acid comprised in the composition is from between 0.5% (w/w) and
8.0% (w/w) taurine. In yet other embodiments, the mineral salt is
zinc gluconate, and the composition comprises from between 0.2%
(w/w) to 5.5% (w/w) zinc gluconate. In still another embodiment,
the composition comprises from between 0.25% (w/w) to 5.5% (w/w)
zinc gluconate and between 0.5% (w/w) and 8.0% (w/w) taurine. In
yet other certain embodiments, the composition further comprises a
lactoferrin. In still other particular embodiments, the pH of the
composition is between 3.0 and 8.5; in more specific embodiments,
the pH is between 3.5 and 4.5; and yet in still more specific
embodiments, the pH is between 5.5 and 7.5.
[0021] Also provided herein is a use for a composition comprising a
mineral salt and a sulfonic acid for the manufacture of a
medicament for treating and/or preventing a mucosal disorder,
disease, or condition or a dermal disorder, disease, or condition.
In other embodiments, a physiologically acceptable composition
comprising a mineral salt and a sulfonic acid for use in treating
and/or preventing a mucosal disorder, disease, or condition or a
dermal disorder, disease, or condition is provided. The
compositions, mucosal and dermal diseases and disorders and
conditions and other embodiments are described in detail above and
herein.
[0022] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a compound" or "a composition" includes a plurality of such
compounds or compositions, respectively. Similarly, reference to "a
cell" or "the cell" includes reference to one or more cells and
equivalent terms (e.g., plurality of cells) known to those skilled
in the art, and so forth. Use of the conjunction "or" is meant to
illustrate choice or possibilities and unless stated otherwise, the
use of "or" does not mean that the terms or phrases joined by the
conjunction are alternatives that are exclusive of each other. When
referring to a number or a numerical range means that the number or
numerical range referred to is an approximation within experimental
variability (or within statistical experimental error), and thus
the number or numerical range may vary between 1% and 20% of the
stated number or numerical range. The term "comprising" (and
related terms such as "comprise" or "comprises" or "having" or
"including") is not intended to exclude that in other certain
embodiments, for example, an embodiment of any composition of
matter, composition, method, or process, or the like, described
herein, may "consist of" or "consist essentially of" the described
features.
[0023] As used herein, any concentration range, percentage range,
ratio range, or integer range is understood to include the value of
any integer within the recited range and, when appropriate,
fractions thereof (such as one tenth and one hundredth of an
integer), unless otherwise indicated. Also, any number range
recited herein relating to any physical feature, such as polymer
subunits, size, thickness, height, weight, mass, volume, molarity,
or pH are to be understood to include any integer or fraction
thereof within the recited range, unless otherwise indicated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates the effect of zinc (Z) and taurine (T) to
reduce production of the proinflammatory cytokine IL-6 in
lipopolysaccharide (LPS)-stimulated CaCo2 cells. The concentrations
for each of zinc gluconate and taurine (Z/T) are indicated in mM on
the x-axis.
[0025] FIG. 2 illustrates the effect of zinc (Z) and taurine (T) to
reduce production of the proinflammatory cytokine IL-8 in
lipopolysaccharide-stimulated CaCo2 cells. The concentrations for
each of zinc gluconate and taurine (Z/T) are indicated in mM on the
x-axis.
[0026] FIG. 3 illustrates the effect of zinc (Z) and taurine (T) to
reduce production of the proinflammatory cytokine IL-8 in
doxorubicin-stimulated CaCo2 cells. The concentrations for each of
zinc gluconate and taurine (Z/T) are indicated in mM on the
x-axis.
[0027] FIG. 4 shows the effect of zinc (Z) alone, taurine ((T);
also indicated as "Taurin") alone, and the combination of zinc and
taurine on production of IL-8 in lipopolysaccharide-stimulated
CaCo2 cells. The concentration (pg/ml) of IL-8 detected is shown on
the y-axis. Controls include CaCo-2 cells that were not exposed to
LPS (UNTR), and CaCo-2 cells that were exposed to LPS (LPS) but not
to either zinc gluconate or taurine or both zinc gluconate and
taurine together.
DETAILED DESCRIPTION
[0028] The methods described herein relate generally to treatment
of mucosal and dermal diseases, disorders, and conditions using
compositions comprising a mineral salt (e.g., a zinc salt, for
example, zinc gluconate) and a sulfonic acid (e.g., taurine). These
methods provide benefit, at least in part, by inhibiting disruption
of intercellular junctions between adjacent and neighboring cells,
which also inhibits disruption of membrane barrier integrity and
function of the cells, thus reducing undesired membrane barrier
permeability.
[0029] A composition (GelX.RTM. Oral Gel, BMG Pharma, Gardnerville,
Nev.) has been approved by the U.S. Food and Drug Administration
for use as a mechanical device for management of pain and for
relief of pain by its adherence to the mucosal surface of the
mouth. Polyvinyl pyrrolidone (PVP) is included in GelX.RTM. Oral
Gel as the main active ingredient of the device because it forms a
protective film over the mucosal surface. Two other ingredients
included in GelX.RTM. Oral Gel are a zinc salt and taurine, which
were added as preservatives and which were less toxic than other
commonly used preservatives.
[0030] A small clinical study was initiated in which patients with
cancer (i.e., a malignancy) who had radiation-induced mucositis as
a consequence of radiotherapy were treated with compositions
comprising PVP, a zinc salt (zinc gluconate), and taurine. The
expected results after such treatment were that a palliative
benefit would be observed in which the stinging, burning, and
general pain associated with mucositis would be temporarily
reduced, and that over the course of typically several weeks of
therapy, chemical irritation would be reduced and a slightly
improved rate of healing might occur, even as ulceration from the
underlying cause of radiation therapy continued to develop. In
addition to the immediate palliative benefits anticipated, the
following were observed: an abrupt reduction in the severity of
ulceration; a reduction of the primary symptoms of ulceration; and
improvement in secondary markers of mucositis including, a
reduction of xerostomia (dryness due to lack of saliva), and a
return of the ability to taste.
[0031] Treatment of dermal conditions using compositions comprising
a zinc salt (zinc gluconate), and taurine also provided therapeutic
benefit beyond palliative relief. By way of example, as described
herein, application of an exemplary composition comprising zinc
gluconate and taurine halted development of edema, redness, and
reduced pain related to a second degree burn.
[0032] Consistent with these human in vivo observations was that
the combination of a mineral salt and a sulfonic acid (for example,
zinc gluconate and taurine, respectively) inhibited production of
pro-inflammatory cytokines (e.g., IL-6 and IL-8) in a cell culture
model used for assessing epithelial cell tight junction structure
and function (see Example 3). Additional analysis with respect to
inhibition of production of the pro-inflammatory cytokine, IL-8,
indicated that the effect of zinc and taurine in combination was
synergistic compared with each of zinc and taurine alone. As
presently understood in the art, IL-8 production is a
prognosticator of intercellular junction damage. The cell culture
model studies in conjunction with the in vivo observations
indicates that therapeutic benefit relates, at least in part, to
the capability of the combined mineral salt and sulfonic acid to
prevent, inhibit, and/or reduce, disruption of intercellular
junctions that affects the structural and functional integrity of
the cells.
[0033] While zinc deficiency has been associated in vivo with
inflammatory bowel disease and Helicobacter pylori-induced gastric
mucosa inflammation (see, e.g., Sturniolo et al., Inflamm. Bowel
Dis. 7:94-98 (2001); Sempertegui et al., Heliobacter 12:43-48
(2007); Finamore et al., J. Nutr. 138:1664-70 (2008)), and in vitro
induces membrane barrier damage in a Caco-2 cell model (see, e.g.,
Finamore et al., supra), the suggested treatment of patients with
these disorders has been to increase zinc intake by dietary
improvement and zinc supplementation (e.g., with zinc sulfate) to
provide improvement in chronic conditions over the long-term (see,
e.g., Finamore et al., supra). By contrast as described herein, by
combining a mineral salt, such as a zinc mineral salt, with a
sulfonic acid such as taurine, the mineral salt is effectively
trafficked (i.e., delivered) to the affected cells and tissue. In
addition, because taurine is capable of penetrating skin (see,
e.g., da Silva et al., Pharmaceut. Res. 25:1846-1850 (2008)), a
mineral salt, such as zinc, may be delivered to damaged dermal
tissue when combined with taurine, thus providing zinc to cells and
tissue that zinc not in combination with taurine would not
otherwise have contact. Without wishing to be bound by theory, the
sulfonic acid taurine, which can act as an anti-oxidant and which
also inhibits cytokine production, also contributes to the
effectiveness of the compositions described herein in healing and
restoration of cellular integrity of affected tissue.
[0034] As described herein, an exemplary composition comprising a
zinc salt and taurine in combination have anti-inflammatory
activity and are capable of inhibiting production of
anti-inflammatory cytokines (such as, by way of nonlimiting
example, IL-6 and IL-8) in cells. Accordingly, as described in
greater detail herein, methods are provided for treating and/or
preventing inflammation associated with mucosal diseases,
disorders, and conditions and dermal diseases, disorders, and
conditions described in greater detail herein.
[0035] Methods are provided herein for treating and/or preventing
dermal diseases, disorders, and conditions, and for treating and/or
preventing mucosal diseases, disorders, and conditions, including
inflammatory dermal and mucosal diseases, disorders, and
conditions. These methods comprise administering compositions
comprising at least one mineral salt and at least one sulfonic
acid.
[0036] Mucosal diseases, condition, and disorders that are
treatable by the methods and compositions described herein include,
but are not limited to, mucositis (e.g., oral mucositis), which is
an inflammation of a mucous membrane and may also include
ulceration of the mucous membrane. In other embodiments, methods
and compositions are provided herein for treating atrophic
vaginitis (AV) and for treating conditions (e.g., vaginal dryness)
that may precede or are associated with atrophic vaginitis.
Additional dermal diseases, disorders, and conditions and mucosal
diseases, disorders, and conditions that may be treatable with the
compositions described herein include, but are not limited to,
vaginal ulcerations (including micro-lesions); dermal conditions
and mucosal conditions that are side effects (i.e., adverse
effects) of radiation therapy and/or chemotherapy (e.g., oral
mucositis) (i.e., radiation therapy or chemotherapy induced
mucositis); viral infections such as shingles and herpes simplex,
HIV/AIDS; and chronic skin disorders such as eczema, psoriasis, and
dermatitis. Dermal and mucosal diseases, conditions, and disorders
treatable using the compositions and methods described herein are
discussed in greater detail below.
[0037] In one embodiment, compositions (pharmaceutically and
physiologically acceptable) are provided herein, and methods of
using the compositions, for treating dermal disorders or mucosal
disorders, which occur as side effects of radiation therapy and/or
chemotherapy. Dermal or mucosal disorders occur in subjects who are
receiving radiation therapy and/or chemotherapy, including those
who receive treatment of head and neck tumors, and also occur in
about 90% of children with leukemia. These side effects include
oral mucositis (including micro-lesions) and oral stomatitis. Side
effects (also called adverse effects) may also result in a mucosal
disorder of any one or more mucosa including oral mucosa,
intestinal mucosa, rectal mucosa, and the like consequent to
chemotherapy or radiotherapy treatment of any one or more of a wide
variety of solid or non-solid cancers or lymphomas (for example,
breast, prostate, pancreatic, ovarian, liver, lung, urinary, and
colon cancer, Kaposi's sarcoma, and melanoma).
[0038] The compositions and methods described herein may also be
used for preventing or treating a mucosal disorder, including but
not limited to, atrophic vaginitis, vaginal micro-lesions,
inflammatory bowel disease (including Crohn's disease and
ulcerative colitis), periodontitis, interstitial cystitis, wound
healing, an inflammatory condition, dyspareunia, burning,
leucorrhea, xerosis (i.e., dry skin, atopic dermatitis), vaginal
dryness, vulvar pruritus, vaginal pruritus, vulvar burning, vaginal
burning, vulvar dystrophy, vaginal malodor, candidiasis,
trichomoniasis, or bacterial vaginosis; and urinary disorders such
as dysuria, hematuria, frequency, stress incontinence, and tract
infection; complications resulting from antiestrogen medications
including menopausal sexual dysfunction, among other symptoms;
viral infections including shingles, herpes simplex, HIV/AIDS; and
chronic skin disorders such as eczema, psoriasis and dermatitis;
irritation due to oral surgery, aging, traumatic ulcers caused by
braces or ill fitting dentures, diffuse aphthous ulcers, or
medication, or disease; and other dermal disorders, diseases, and
conditions described herein.
[0039] In a certain embodiment, the methods described herein, which
comprise administering a composition comprising a mineral salt and
a sulfonic acid, are used for treating or preventing (i.e.,
reducing or decreasing the likelihood of occurrence in a
statistically, biologically, or clinically significant manner)
inflammation or an inflammatory response that is associated with a
mucosal or dermal disease, disorder or condition. In one
embodiment, the methods described herein may reduce inflammation of
a mucosa or dermis, thereby treating conditions such as oral
mucositis and AV. In other embodiments, the methods provided herein
may reduce the likelihood of occurrence of inflammation of the
dermis or of a mucous membrane by administering the mineral salt
and sulfonic acid to a subject at risk of developing an
inflammation of a mucous membrane or dermis (by way of nonlimiting
example, a subject who has vaginal dryness or who is beginning
radiotherapy and/or chemotherapy for treatment of a cancer or
malignancy).
[0040] Also provided herein are physiologically acceptable (i.e.,
physiologically suitable and pharmaceutically suitable and
acceptable) compositions that may be administered to a subject for
treating or preventing inflammation associated with a dermal or
mucosal disease, disorder, or condition. These compositions
comprise at least one mineral salt and at least one sulfonic acid.
In particular embodiments, the compositions comprise
therapeutically effective concentrations of a mineral salt of zinc
and a sulfonic acid. In more particular embodiments, the mineral
salt is zinc gluconate and the sulfonic acid is taurine. In certain
embodiments, the compositions further comprise a lactoferrin.
[0041] Compositions described herein may be administered in forms
and in a manner, described in greater detail herein and understood
in the art, to deliver the composition and the active ingredients
thereof in an amount sufficient to produce a therapeutic benefit.
In certain embodiments, one or more of these compositions may be
administered topically; in other embodiments one or more of the
compositions is delivered orally; in yet other embodiments, one or
more of the compositions is administered topically and orally. The
compositions are therefore formulated to be physiologically
acceptable (i.e., pharmaceutically suitable or acceptable) for
administration to a subject, including a human subject. These
compositions comprise a mineral salt (e.g., a zinc salt, for
example, zinc gluconate) formulated with a sulfonic acid (e.g.,
taurine) and may be administered to prevent and treat dermal or
mucosal disorders, diseases, and conditions, including but not
limited to mucositis (including oral mucositis and oral stomatitis)
or atrophic vaginitis. As used herein, the term atrophic vaginitis
is interchangeable with the terms, urogenital atrophy and vaginal
atrophy. Mucositis is an inflammation of a mucous membrane, which
is a painful disorder involving a mucous membrane located at one or
more of an oral cavity, gastrointestinal tract, bladder, esophagus,
vagina, rectum, lung, a mucosal surface of a nasal cavity, ear, or
ocular mucosa.
[0042] In certain embodiments, a composition comprising a mineral
salt (e.g., a zinc salt, for example, zinc gluconate) and a
sulfonic acid (e.g., taurine) may further comprise a lactoferrin
(or variant or fragment thereof). Such a composition may be
administered locally (e.g., topically to a mucosal surface, for
example, oral or vaginal mucosa) and/or orally. In other
embodiments, methods are provided herein that comprise
administering a physiologically acceptable composition (a first
composition) comprising a mineral salt and a sulfonic acid, which
composition may, but not necessarily, also comprise a lactoferrin,
which first composition is administered sequentially (either prior
to or after) or concurrently with administration of a separate (or
second) physiologically acceptable composition comprising a
lactoferrin and one or more physiologically acceptable carriers
(excipients) but which lacks a mineral salt and a sulfonic acid. In
particular embodiments, the composition comprising the lactoferrin
and lacking a mineral salt and a sulfonic acid is administered
orally.
[0043] As described in greater detail herein, methods for treating
mucositis, (including oral mucositis and oral stomatitis), atrophic
vaginitis, vaginal dryness, and other mucosal conditions and
disorders described herein may decrease inflammation; promote
restoration and healing of the mucous membrane including
minimizing, preventing, and inhibiting ulceration; and/or slow,
inhibit, or prevent further loss of mucous membrane integrity. By
minimizing, preventing, or reducing ulceration, the compositions
provide the added benefit of reducing the susceptibility of the
mucous membrane to invasion and colonization by microorganisms,
thus decreasing the likelihood of microbial infection and/or
decreasing the recurrence and frequency of microbial infections.
Moreover, compositions comprising a sulfonic acid and a mineral
salt, such as taurine and zinc gluconate, respectively, have
antimicrobial activity.
[0044] In other embodiments of the methods described above and
herein for treating or preventing a dermal disease, disorder, or
condition or a mucosal, disease, disorder or condition, the methods
further comprise identifying a subject who is need of receiving a
composition comprising a mineral salt (e.g., a zinc salt, for
example, zinc gluconate) and a sulfonic acid (e.g., taurine). As
described in greater detail herein, the subject may have mucositis
(e.g., oral mucositis or oral stomatitis) or may be at risk of
developing mucositis (for example, a patient who is receiving or
who is about to receive chemotherapy and/or radiation therapy). By
way of additional example, a subject in need may be a female
subject who has AV or who is at risk of developing AV (e.g., a
woman who presents symptoms that may precede clinical manifestation
of AV, such as vaginal dryness).
[0045] In another embodiment, methods are provided for inhibiting
(i.e., reducing, abrogating, or decreasing, or reducing the
likelihood of occurrence of) disruption of intercellular junctions
between adjacent (i.e., neighboring) cells, which methods comprise
contacting the cells with a physiologically acceptable composition
comprising a mineral salt (e.g., a zinc salt, for example, zinc
gluconate) and a sulfonic acid (e.g., taurine). The step of
contacting, in some manner, permits or enables interaction between
the cells and the composition. Such methods maintain or restore the
structure and function of the membrane barrier, which in turn,
maintains or restores membrane barrier permeability, which in the
absence of cellular contact with the composition would result in
loss of intercellular junction integrity and function, increasing
permeability of the cells. The methods thus inhibit, reduce,
prevent, and/or maintain membrane barrier integrity of a cell.
[0046] Intercellular junctions refer to intercellular junctional
complexes that are formed between adjacent cells. Intercellular
junctions include tight junctions (TJ) and adherens junctions (AJ)
that form circumferential zones of contact between adjacent cells.
Disruption refers to loss, destruction, or other undesired or
deleterious effect to structural and/or functional integrity of the
intercellular junctions. The methods provided herein may inhibit,
reduce, prevent loss of structural and/or functional integrity of
the intercellular junctions in a statistically significant,
clinically significant, or biologically significant manner.
Disruption may adversely affect the structure and/or function of
one or more membrane or cytoskeletal proteins and disrupt their
respective interactions that help maintain the structure integrity
and function of the intercellular junction. These methods thereby
inhibit, prevent, or reduce loss of integrity (functional and/or
structural) of the TJ and/or AJ. In certain embodiments, the cells
are endothelial cells, and in other particular embodiments, the
cells are epithelial cells. Endothelial and epithelial cells
include, by way of non-limiting example, gastrointestinal,
oropharyngeal, bladder, esophageal, vaginal, rectal, pulmonary,
nasal, ear, or ocular endothelial or epithelial cells,
respectively.
[0047] In certain embodiments, the cells (i.e., epithelial or
endothelial cells) comprise tissue in a subject. In particular
embodiments, the subject has or is at risk of developing a dermal
disease, disorder, or condition or has or is at risk of developing
a mucosal disease, disorder, or condition. As described in detail
herein, mucosal and dermal disorders include mucositis (e.g., oral
mucositis, oral stomatitis, AV). Mucosal and dermal disorders also
include, for example, the dermal or mucosal disorders that occur as
side effects of radiation therapy and/or chemotherapy associated
with the radioactive and/or chemotherapeutic treatment of cancers
(i.e., malignancies), including, head and neck tumors and leukemia.
Such side effects include oral mucositis (including micro-lesions)
and oral stomatitis. Dermal or mucosal disorders may also occur as
side effects of radiation therapy and/or chemotherapy of other
cancers, including, any one or more of a wide variety of solid or
non-solid cancers or lymphomas (for example breast, prostate,
pancreatic, ovarian, melanoma, liver, lung, urinary, salivary
gland, and colon cancers; Kaposi's sarcoma), which may affect any
one or more mucosa including oral mucosa, intestinal mucosa, rectal
mucosa, and the like. Mucosal disorders also include atrophic
vaginitis, vaginal micro-lesions, inflammatory bowel disease
(including Crohn's disease and ulcerative colitis), eczema,
psoriasis, periodontitis, interstitial cystitis, wound healing, or
an inflammatory condition, dyspareunia, burning, leucorrhea,
xerosis, vaginal dryness, vulvar pruritus, vaginal pruritus, vulvar
burning, vaginal burning, vulvar dystrophy, vaginal malodor,
candidiasis, trichomoniasis or bacterial vaginosis, and urinary
disorders such as dysuria, hematuria, frequency, stress
incontinence and tract infection, among other symptoms, including
menopausal sexual dysfunction, complications resulting from
antiestrogen medications, viral infections including shingles,
herpes simplex, HIV/AIDS; and chronic skin disorders such as
eczema, psoriasis and dermatitis, irritation due to oral surgery,
aging and traumatic ulcers caused by braces or ill fitting
dentures, diffuse aphthous ulcers, medication, or disease.
[0048] In another embodiment, the methods for inhibiting (i.e.,
reducing, abrogating, or decreasing, or reducing the likelihood of
occurrence of) disruption of intercellular junctions between
adjacent (i.e., neighboring) cells may comprise contacting cells
with a mineral salt and taurine (mixing, combining, or in some
manner permitting interaction) in vitro. Such methods may thus be
useful as in vitro assays for monitoring pharmacokinetics of a
mineral salt and sulfonic acid during pre-clinical, clinical, and
post-marketing studies; evaluating (including quality control and
quality assurance) biological activity of compositions comprising a
mineral salt and sulfonic acid; evaluating, measuring, and/or
monitoring the biological effect of other agents that may be
included in a composition comprising a mineral salt and sulfonic
acid; among others.
[0049] As noted above, the cells may be endothelial cells or the
cells may be epithelial cells. For in vitro assays, the cells may
be present in a biological sample. Such a biological sample may be
a biopsy specimen, a body fluid (e.g., lung lavage, ascites,
mucosal washings, synovial fluid) that contains the endothelial
and/or epithelial cells, bone marrow, lymph nodes, tissue explant,
organ culture, or any other tissue or cell preparation from a
subject or a biological source. A sample may further refer to a
tissue or cell preparation in which the morphological integrity or
physical state has been disrupted, for example, by dissection,
dissociation, solubilization, fractionation, homogenization,
biochemical or chemical extraction, pulverization, lyophilization,
sonication, or any other means for processing a sample derived from
a subject or biological source. In certain embodiments, the subject
or biological source may be a human or non-human animal, a primary
cell culture (e.g., immune cells, virus infected cells), or culture
adapted cell line, including but not limited to, genetically
engineered cell lines that may contain chromosomally integrated or
episomal recombinant nucleic acid sequences, immortalized or
immortalizable cell lines, somatic cell hybrid cell lines,
differentiated or differentiatable cell lines, transformed cell
lines, and the like. Cell lines that may be used in the in vitro
methods include cultured monolayers of polarized epithelial cell
lines, such as MDCK, T84, and Caco-2, which provide model systems
for the study of tight junction structure and function (see, e.g.,
Clayburgh et al., BioRad Protocol Guide, BioRad 2008; Clayburgh et
al., J. Biol. Chem. 279:55506-13 (2004)).
[0050] The in vitro methods described herein may be performed by
using techniques such as propagation of cells (i.e., cell culture)
and detection methods all of which are routinely practiced in the
art and with which the skilled person will be familiar. Conditions
for a particular assay include temperature, buffers (including
salts, cations, media), and other components that maintain the
integrity of the mineral salt, the sulfonic acid, and cells, with
which a person skilled in the art will be familiar and/or which can
be readily determined. Persons skilled in the art are also familiar
with assay design such that appropriate controls will be performed
to enable determination of the capability of the composition (and
its components) to inhibit disruption of intercellular junctions.
The mineral salt and/or sulfonic acid may be contacted (mixed,
combined with, or in some manner permitted to interaction) with the
cells, under conditions and for a time sufficient to permit
interaction between the component or components of the
composition.
[0051] Appropriate conditions for permitting interaction of the
reaction components according to this method and other methods
described herein include, for example, appropriate concentrations
of reagents and components (including a sulfonic acid and mineral
salt), temperature, and buffers with which a skilled person will be
familiar. Concentrations of reaction components, buffers,
temperature, and time period sufficient to permit interaction of
the reaction components can be determined and/or adjusted according
to methods described herein and with which persons skilled in the
art are familiar. To practice the methods described herein, a
person skilled in the art will also readily appreciate and
understand which controls are appropriately included when
practicing these methods.
[0052] In addition to the methods and techniques described above
and herein, which include determination of cytokine
production/inhibition in epithelial and endothelial cells,
additional assays may be performed to determine the effect and
capability of the compositions described herein to inhibit
disruption and/or destruction of intercellular junctions (i.e.,
inhibit, reduce, prevent loss of structural and/or functional
integrity of the intercellular junctions in a statistically
significant, clinically significant, or biologically significant
manner). Such assays include membrane permeability assays (see,
e.g., Ferruzza et al., Toxicol. In Vitro 16:399-404 (2002));
neutrophil transmigration assays (see, e.g., Finamore, supra;
Roselli et al., Br. J. Nutr. 95:1177-84 (2006)); immunolocalization
of junction proteins (see, e.g., Ara et al., Cell Commun. Adhes.
11:13-23 (2004)).
[0053] In other embodiments, methods are provided for administering
a bioactive mineral to a subject who has a mineral insufficiency or
who would otherwise benefit from the therapeutic effect(s) of being
treated with a composition comprising the mineral. The compositions
described herein may also be formulated for use in cosmetics.
Mineral Salts
[0054] The compositions described herein for use in the methods
described herein comprise at least one mineral salt. The mineral
moiety of a mineral salt may be any mineral that is an inorganic
element that is essential in some amount to normal biological
function of a human (e.g., zinc, calcium, magnesium, manganese,
cobalt, chromium, selenium, vanadium, copper, iron, nickel,
silicon, boron, arsenic, molybdenum, sodium, potassium, phosphorus,
sulfur, chlorine, fluorine, iodine, and lithium). In certain
embodiments, the mineral moiety of a mineral salt may be zinc,
calcium, magnesium, manganese, cobalt, chromium, selenium,
vanadium, copper, iron. In particular embodiments, the mineral
moiety of the mineral salt is zinc. The salt moiety of the mineral
salt may be any suitable inorganic or organic acid, including but
not limited to, gluconate, acetate, ascorbate, and sulfate. In
particular embodiments, the salt moiety is gluconate. In a specific
embodiment, the mineral salt is zinc gluconate wherein the mineral
moiety is zinc and the salt moiety is gluconate.
[0055] In certain embodiments, the compositions comprise a
sufficient concentration of a mineral salt of zinc (e.g., zinc
gluconate) to treat effectively a dermal or mucosal disorder and
yet are intended to comprise zinc at a concentration that is less
irritating than other zinc-containing compositions. The
compositions described herein may be formulated to increase the
bioavailability of a mineral salt, such as zinc, thereby reducing
the amount of the mineral salt required to treat a mucosal or
dermal disorder and/or to reduce or prevent inflammation associated
with the mucosal or dermal disorder. Reduction of the concentration
and amount of one or more active ingredients in a composition used
for treating a disease or disorder may minimize toxic effects and
thus increase patient compliance, reduce unwanted complications
associated with higher amounts of one or more active ingredients,
and/or reduce the cost of manufacturing.
[0056] In certain embodiments, the composition may contain a
sulfonic acid (e.g., taurine) that is in the form of a mineral salt
of zinc, calcium, magnesium or manganese. By way of example, the
sulfonic acid taurine may be prepared or be in the form of a
taurate salt, having the general formula
H.sub.2N--CH.sub.2--CH.sub.2--SO.sub.3.sup.-).sub.2X.sup.2+,
wherein X may be zinc, magnesium, calcium, or manganese. In certain
embodiments, the physiologically acceptable composition may
comprise a zinc taurate salt, a calcium taurate salt, or a
magnesium taurate salt, which may be used in the methods described
herein for treating a dermal or mucosal disease, disorder, or
condition.
[0057] Because metals are highly charged molecules, many minerals
are not absorbed well by tissues and are not readily transported,
actively or passively, into cells, even if available in the serum.
Some minerals are also unpleasant for a subject to consume or
apply. For example, zinc supplements taken orally can produce
nausea, vomiting, and diarrhea; zinc compounds applied topically
are astringent and can cause irritation and burns. Even though zinc
oxide is neutral and can be applied topically, it is not readily
absorbed into the tissue. Water soluble zinc salts such as zinc
acetate, zinc chloride, and zinc sulfate are highly acidic and
cannot be neutralized with sodium bicarbonate, sodium hydroxide or
the like to provide a physiologically suitable composition that can
be administered to a subject. For example, to neutralize these
water soluble zinc salts with sodium bicarbonate requires as much
as a molar ratio of 5 to 1 to obtain a solution at pH 7, yielding a
composition that has undesirable elevated sodium content. When
sodium hydroxide is used to neutralize these water soluble zinc
salts, the neutralized composition readily precipitates on
standing.
[0058] As described herein, methods for treating dermal and mucosal
disorders comprise administering a composition comprising a mineral
salt, which in certain embodiments is a zinc salt, such as zinc
gluconate. Zinc is essential for the function of at least 70
enzymes and is involved in a variety of metabolic processes,
including tissue growth and repair. Even though zinc salts have
been administered to subjects for treatment of various diseases and
disorders, many of the zinc salts used to date have undesired
effects.
[0059] Zinc salts have been used to inhibit bacterial and viral
growth in subjects who are infected or who are at risk of becoming
infected. Ophthalmic preparations of zinc sulfate to treat herpetic
keratitis have been recommended since 1943. Zinc oxide, zinc
sulfate, and zinc chloride have each been used in treating chronic
and acute wounds. Oral preparations of zinc citrate have been used
for treating gingivitis and periodontitis to reduce plaque
formation and to inhibit bacterial growth. Oral preparations of
several different zinc salts have been marketed for reducing the
symptoms and duration of the common cold caused by rhinovirus;
however, the preparations are unpalatable and cause mouth
irritation and nausea. A more palatable and less irritating
formulation has been developed that contains a zinc salt and an
amino acid (see, e.g., U.S. Pat. No. 4,229,430).
[0060] In addition, topical application of certain available zinc
solutions can cause painful or irritating side effects unless zinc
is present in very low concentrations, which may be insufficient to
effectively treat the disorder or condition intended to be treated.
Zinc sulfate solutions of 0.2-1% can cause severe irritation,
unpleasant dryness and stimulate the emetic reflex when applied
circumorally. Reports of dermal irritancy in animal dermal abrasion
models that are used for studying wound healing show the following:
1% aqueous zinc chloride is a severe irritant; 20% aqueous zinc
acetate is a slightly less irritant; 20% suspension zinc oxide, 1%
aqueous zinc sulfate, and 20% suspension zinc pyrithione, are not
overtly irritant. The less irritant zinc salts, such as zinc oxide,
which is only slightly soluble in water, were only marginally
effective in stimulating epidermal healing in comparison to the
more irritating and more water-soluble zinc salts (see, e.g., U.S.
Pat. No. 6,558,710).
[0061] Previously described compositions comprising zinc salts,
such as zinc gluconate and zinc ascorbate, comprise at least one
amino acid that is formulated with the zinc salt to improve
solubility at neutral pH (see, e.g., U.S. Pat. Nos. 4,711,780 and
4,937,234). The amino acids used in the compositions were typically
either a sulfur containing amino acid (e.g., cysteine) or a basic
amino acid (e.g., lysine, arginine, or histidine). However, in
certain circumstances when lysine is, for example, used for
treating a subject vaginally, the amino acid lysine may be
decarboxylated by vaginal bacteria to produce the diamine
cadaverine, a toxic foul smelling molecule similar to putrescine,
both of which are produced by the breakdown of amino acids in
living and dead organisms and both can be toxic. Cadaverine and
putrescine are largely responsible for the foul odor of putrefying
flesh and also contribute to the odor of processes related to bad
breath (i.e., halitosis) and bacterial vaginosis. Use of other
amino acids may also produce toxic metabolites or malodors
(including tryptophan to tryptamine, phenylalanine to
phenylethylamine, tyrosine to tyramine, histidine to histamine,
serine to ethanolamine, and the like). Accordingly, in certain
embodiments, provided herein are compositions comprising minerals
in bioavailable form that are readily adsorbed but that do not
require the addition of an amino acid that may produce toxic
metabolites or malodors. In certain embodiments, methods for
treating a vaginal infection, such as bacterial vaginosis, comprise
administering a composition comprising a mineral salt (e.g., zinc
gluconate) and a sulfonic acid (e.g., taurine) that is amino acid
free (i.e., a composition that lacks an amino acid, either a
standard amino acid or non-standard amino acid).
[0062] As discussed herein, a zinc salt alone may not be delivered
in an adequate and effective amount to tissue and to the cells of
the tissue to provide therapeutic benefit. Without wishing to be
bound by any particular theory, combining the mineral salt, such as
a zinc salt (including zinc gluconate), with a sulfonic acid (e.g.,
taurine) may improve delivery to a site of the mucosal or dermal
injury, damage, and/or inflammation. Thus, the mineral salt can
effect, among other benefits, inhibition of endothelial or
epithelial cell junction damage, including inhibiting disruption or
dissociation of intercellular junction complexes.
Sulfonic Acids
[0063] The compositions described herein that are useful for
treating dermal diseases, conditions, and disorders and mucosal
diseases, conditions, and disorders, (including those that are
inflammatory disorders or that are associated with inflammation),
comprise both a mineral salt and a sulfonic acid. As defined
herein, a sulfonic acid is an organic acid that is represented by
the formula R--S(.dbd.O).sub.2--OH (also depicted as R--SO.sub.3H)
wherein R is an alkyl that may be optionally substituted. "Alkyl"
means a straight chain or branched, noncyclic or cyclic,
unsaturated or saturated aliphatic hydrocarbon containing from 1 to
8 carbon atoms, while the term "C.sub.1-8 alkyl" has the same
meaning. Representative saturated straight chain alkyls include
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like,
while saturated branched alkyls include isopropyl, sec-butyl,
isobutyl, tert-butyl, heptyl, n-octyl, isopentyl, 2-ethylhexyl and
the like. Representative saturated cyclic alkyls include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
--CH.sub.2cyclopropyl, --CH.sub.2cyclobutyl, --CH.sub.2cyclopentyl,
--CH.sub.2cyclohexyl, and the like; unsaturated cyclic alkyls
include cyclopentenyl and cyclohexenyl, and the like. Cyclic
alkyls, also referred to as "homocyclic rings," include di- and
poly-homocyclic rings such as decalin and adamantyl. Unsaturated
alkyls contain at least one double or triple bond between adjacent
carbon atoms (referred to as an "alkenyl" or "alkynyl,"
respectively).
[0064] As used herein, the term "substituted" in the context of
alkyl means that at least one hydrogen atom of the alkyl is
replaced with a substituent. In the instance of an oxo substituent
(".dbd.O"), two hydrogen atoms are replaced. A "substituent" as
used within the context of this disclosure includes oxo, halogen,
hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl,
alkoxy, thioalkyl, haloalkyl, substituted alkyl, heteroalkyl,
substituted alkyl, heteroalkyl, aryl, substituted aryl, arylalkyl,
substituted arylalkyl, heteroaryl, substituted heteroaryl,
heteroarylalkyl, substituted heteroarylalkyl, heterocycle,
substituted heterocycle, heterocycloalkyl, substituted
heterocycloalkyl, --NR.sub.aR.sub.b, --NR.sub.aC(.dbd.O)R.sub.b,
--NR.sub.aC(.dbd.O)NR.sub.aR.sub.b,
--NR.sub.aC(.dbd.O)OR.sub.b--NR.sub.aS(.dbd.O).sub.2R.sub.b,
--OR.sub.a, --C(.dbd.O)R.sub.a--C(.dbd.O)OR.sub.a,
--C(.dbd.O)NR.sub.aR.sub.b, --OCH.sub.2C(.dbd.O)NR.sub.aR.sub.b,
--OC(.dbd.O)NR.sub.aR.sub.b, --SH, --SR.sub.a, --SOR.sub.a,
--S(.dbd.O).sub.2NR.sub.aR.sub.b, --S(.dbd.O).sub.2R.sub.a,
--SR.sub.aC(.dbd.O)NR.sub.aR.sub.b, --OS(.dbd.O).sub.2R.sub.a and
--S(.dbd.O).sub.2OR.sub.a (also written as --SO.sub.3R.sub.a),
wherein R.sub.a and R.sub.b are the same or different and
independently hydrogen, alkyl, haloalkyl, substituted alkyl,
alkoxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl,
arylalkoxy, heteroaryl, substituted heteroaryl, heteroarylalkyl,
substituted heteroarylalkyl, heterocycle, substituted heterocycle,
heterocycloalkyl or substituted heterocycloalkyl.
[0065] In specific embodiments, R is an alkyl substituted with an
amino group ("amino" refers to the --NH.sub.2 radical). In yet
another specific embodiment, R is a straight chain alkyl,
substituted with amino (e.g., R is --(CH.sub.2).sub.nNH.sub.2
wherein n is 1-6). In a more specific embodiment, the compositions
used in the methods described herein comprise the sulfonic acid
taurine (2-aminoethanesulfonic acid), having the formula
NH.sub.2CH.sub.2CH.sub.2SO.sub.3H. Without wishing to be bound by
theory, the amino group of taurine would be expected to bind to
metal ions and although the affinity of a sulfonate group for metal
ions is weak, the presence of the amino group may further serve as
an `anchor` allowing the formation of stable six-membered chelate
rings. Thus, taurine may coordinate to metal ions in a monodentate
or bidentate manner (see, e.g., "Interaction of Taurine with Metal
Ions", O'Brien, et al., Advances in Experimental Medicine and
Biology, Springer Netherlands, 2002).
[0066] As described herein, taurine is a sulfonic acid and is not a
standard amino acid and, consequently, is not incorporated into a
polypeptide by the process of protein synthesis, either naturally
occurring or synthetic. Taurine does not contain a carboxyl group
that is necessary for peptide bond formation, and taurine is not a
substrate for tRNA synthetase or charged to a transfer RNA (tRNA).
Sulfonic acids may also be derived from other amino acids such as
methionine and homocysteine, and related molecules such as
S-adenosylmethionine, by decarboxylation (e.g., a decarboxylated
methionine). Taurine, and other sulfonic acids, may be synthesized
by methods described and routinely practiced in the art (see, e.g.,
Kosswigg, "Sulfonic Acids, Aliphatic," In Ullmann's Encyclopedia of
Industrial Chemistry (John Wiley & Sons, 2000), and the
reactants are available commercially. Taurine for pharmaceutical
use is also commercially manufactured and available.
[0067] Taurine is a naturally occurring sulfonic acid, and in
mammals is synthesized in the liver via the cysteine sulfinic
pathway wherein cysteine is an initial reactant. Studies have
described that taurine is involved in numerous physiological
processes, including neurological, metabolic, cardiovascular, and
skeletal muscular functions.
[0068] Physiologically Acceptable Compositions and Methods of
Administration and Dosing
[0069] Provided herein are physiologically acceptable (i. e.,
physiologically suitable and pharmaceutically suitable and
acceptable) compositions that may be administered to a subject for
treating a dermal or mucosal disease, disorder, or condition. As
described herein these compositions comprise at least one mineral
salt and at least one sulfonic acid. In more particular
embodiments, the mineral salt is zinc gluconate and the sulfonic
acid is taurine. In certain embodiments, the compositions further
comprise a lactoferrin. The physiologically acceptable compositions
described herein may be a sterile (or in some instances
non-sterile) aqueous or non-aqueous solution, suspension or
emulsion, or solid (all of which are described in greater detail
herein), which typically additionally comprise a physiologically
acceptable excipient (pharmaceutically acceptable or suitable
excipient, diluent, or carrier) (i.e., a non-toxic material that
does not interfere with the activity of the active ingredient).
[0070] Many existing topical formulations are inadequate because
they produce local irritation and are not well tolerated.
Therefore, provided herein are compositions (e.g., a topical
formulation) comprising a mineral salt, for example, a zinc salt
(e.g., zinc gluconate) or a mineral gluconate salt, and a sulfonic
acid (e.g., taurine) or a sulfonic acid of a decarboxylated
sulfur-containing amino acid that addresses deficiencies in
presently available treatments.
[0071] In certain embodiments, the methods described herein
comprise administering a composition that includes the mineral
salt, such as zinc gluconate, and the sulfonic acid, such as
taurine, as the active ingredients and that lacks any amino acids
(i.e., the compositions are amino acid free). A composition that is
amino-acid free lacks the presence of a naturally occurring or
synthetically produced amino acid. Unlike certain previously
described compositions (see, e.g., U.S. Pat. Nos. 4,937,234;
4,711,780), an amino acid is not required in the compositions
described herein that comprise a zinc salt. As understood in the
art, an amino acid is an organic molecule comprising both a
carboxyl group (COOH) and an amino group (NH.sub.2), which form
peptide bonds with other amino acids to form peptides and
polypeptides. Thus, compositions that are amino acid free lack the
twenty standard amino acids encoded by codons of the genetic code.
These compositions also lack non-standard amino acids described in
the art including those rarely encoded by the genetic code, such as
selenocysteine and pyrrolysine, and those not encoded by the
genetic code, such as but not limited to lanthionine,
2-aminoisobutyric acid, and dehydroalanine. The term, amino
acid-free, is intended to encompass an amino acid molecule and is
not intended to encompass a peptide and polypeptide that are formed
by peptide bonding of amino acids.
[0072] The compositions comprising a mineral salt and a sulfonic
acid (for example, zinc gluconate and taurine, respectively) also
have antimicrobial activity. The antimicrobial activity includes
bactericidal activity against Gram negative and Gram positive
bacteria; and anti-fungal activity (e.g., anti-Aspergillus
activity), including anti-fungal activity against yeast (e.g.,
Candida albicans). In certain embodiments, the compositions used in
the methods described herein lack an additional agent that is known
to have anti-viral, anti-bacterial, or anti-fungal activity.
Accordingly, in certain embodiments, a composition useful for
treating mucosal and dermal disorders is a composition that
comprises a mineral salt (e.g., zinc gluconate) and a sulfonic acid
(e.g., taurine) and that lacks the presence of a third active
ingredient with antimicrobial activity (such as, for example but
not limited to, methylparaben, polymixin B, tobramycin, and
amphotericin B, or vancomycin that is present in sufficient amounts
as would be understood by a person skilled in the infectious
disease art to inhibit, prevent, treat, or abrogate a microbial
infection in the subject). In other embodiments, the compositions
described herein that comprise a sulfonic acid (e.g., taurine) and
a mineral salt (e.g., a zinc salt, for example, zinc gluconate) may
further comprise at least one third active ingredient that is an
antimicrobial agent. In other particular embodiments, the
compositions described herein do not include (i.e., lack) ascorbic
acid or a salt thereof.
[0073] As described herein, the mineral moiety of a mineral salt
may be, but is not limited to, zinc, calcium, magnesium, manganese,
cobalt, chromium, selenium, vanadium, copper, and iron. In
particular embodiments, the mineral moiety of the mineral salt is
zinc. The salt moiety of the mineral salt may be any suitable
inorganic or organic acid, including but not limited to, gluconate,
acetate, ascorbate, and sulfate. In particular embodiments, the
salt moiety is gluconate. In a specific embodiment, the mineral
salt is zinc gluconate wherein the mineral moiety is zinc and the
salt moiety is gluconate. In more specific embodiments,
compositions disclosed herein may contain a mineral salt (e.g.,
zinc gluconate) from 0.01% (w/w) to 30% (w/w), from 0.1% (w/w) to
30%, from 0.25% (w/w) to 5.5% (w/w), from 0.5% (w/w) to 20% (w/w),
from 1% (w/w) to 15% (w/w), from 1% (w/w) to 5% (w/w), from 2%
(w/w) to 5% (w/w), about 0.5% (w/w), about 1% (w/w), about 2%
(w/w), about 2.5% (w/w), about 3% (w/w), about 4% (w/w), about 4.5%
(w/w), about 5% (w/w), or about 5.5% (w/w). In yet more specific
embodiments, compositions described herein comprise the mineral
salt (e.g., a zinc salt, for example, zinc gluconate) at 0.5%,
1.0%, 1.5%, 2.0%, or 2.5% (w/w).
[0074] In certain embodiments these compositions contain a sulfonic
acid (for example, taurine) at a percent weight in the composition
at a percent within the range from 0.01% (w/w) to 35% (w/w), from
0.1% (w/w) to 35%, from 0.25% (w/w) to 30% (w/w), from 0.5% (w/w)
to 8% (w/w), from 0.5% (w/w) to 20% (w/w), from 1% (w/w) to 15%
(w/w), from 1% (w/w) to 5% (w/w), from 2% (w/w) to 5% (w/w), from
0.5% to 4% (w/w), or at about 0.5% (w/w), 1% (w/w), 2% (w/w), 3%
(w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), or 8% (w/w). In
specific embodiments, the sulfonic acid is taurine. The ingredients
(also called herein, components) of the compositions described
herein are presented in percent weight of each ingredient to the
composition. In certain embodiments, the solute (or diluent) used
to formulate the composition is water and in other certain
embodiments, the solute is saline (both of which are understood to
be from pharmaceutically suitable sources when the compositions are
to be used for administration to a subject). When the solute is
water, the percentage by weight of each ingredient will be similar
to weight per volume of the composition.
[0075] In any composition disclosed herein, the molar ratio of a
mineral salt (e.g., a zinc salt, for example, zinc gluconate)
relative to a sulfonic acid (e.g., taurine) may be from 0.5 to 1.0,
at 1 to 1, from 1 to 1.5, from 1 to 2, from 1 to 2.5, from 1 to 3,
from 1 to 5, from 1 to 10, or from 1 to 20, 1 to 50, or 1 to 100.
In particular embodiments, the compositions comprise a mineral salt
and sulfonic acid at a ratio of 1 to 2 (1:2). In a specific
embodiment, a composition is provided comprising zinc gluconate and
taurine at a molar ratio of 1:2. In certain specific embodiments,
the compositions for use in the methods described herein (e.g., for
treating a mucosal or dermal disorder and/or for inhibiting
disruption of intercellular junctions between adjacent cells)
comprise a mineral salt (e.g., a zinc salt, for example, zinc
gluconate) at a percent weight in the composition of between about
0.25% (w/w)-5.5% (w/w) or from about 0.5% (w/w)-2.5% (w/w). These
compositions also comprise a sulfonic acid (e.g., taurine) at a
percent weight in the composition of about 0.25% (w/w)-30% (w/w),
0.5% (w/w) to 8% (w/w), or about 1.0% (w/w)-5.0% (w/w). In more
specific embodiments, the composition comprises a mineral salt
(e.g., a zinc salt, for example, zinc gluconate) at a percent
weight in the composition of about 0.5% (w/w) and a sulfonic acid
(e.g., taurine) at a percent weight in the composition of about
1.0% (w/w); in other more specific embodiments, the composition
comprises a mineral salt (e.g., a zinc salt, for example, zinc
gluconate) at a percent weight in the composition of about 2.5%
(w/w) and a sulfonic acid (e.g., taurine) at a percent weight in
the composition of about 5.0% (w/w).
[0076] The compositions described herein may be formulated at a pH
appropriate and effective for the condition to be treated. The
physiologically acceptable compositions described herein,
therefore, may include at least one buffering agent (also referred
to herein as a pH adjusting agent) or any combination or mixture of
more than one buffering agent. Exemplary buffering agents include
sodium hydroxide, hydrochloric acid, and sodium bicarbonate. A
buffering agent may have a pKa ranging from about 3.5 to about 9,
or from about 4 to about 5, or from about 4.5 to about 5.5, or from
about 6 to about 8, etc., whichever is suitable for maintaining the
desired pH of the composition.
[0077] The pH of a composition described herein may be adjusted
depending upon the intended site of administration. The composition
may be formulated to have a pH range from between pH 3 and pH 8,
from between pH 4.5 and pH 5.5, from between pH 5.5 and pH 6.5,
from between pH 6.5 and pH 7.5, from between pH 7.5 and pH 8.5,
from between pH 6 and pH 7, from between pH 7 and pH 8, or from
between pH 3.5 and pH 5.5. Such compositions may be used in the
methods described herein that comprise orally and/or topically
administering the composition for treating any of the mucosal or
dermal conditions, diseases, or disorders described herein. For
example, the composition may be administered topically to the oral
mucosa, and administered orally to treat a mucosal disorder of the
gastrointestinal or oropharyngeal tract, such as oral mucositis. In
a particular embodiment, the composition used in methods for
administration orally and for administration orally and/or
topically to the oral mucosa, such as for treating oral mucositis
and other oral mucosal disorders, a composition having a neutral pH
is formulated and has a pH from between pH 5.5 and pH 7.5.
[0078] In other embodiments, the compositions for use in the
methods described herein that comprise a mineral salt and a
sulfonic acid, such as taurine, may be formulated at an acid pH
such as between pH 3.5-4.5, which is a pH range normally found at
the vaginal mucosa or intestinal tract. Accordingly, for treating
urogenital diseases, disorders, or conditions, (including but not
limited to atrophic vaginitis, vaginal dryness, vaginal burning,
vaginal ulceration, dyspareunia, leukorrhea, vulvar pruritus, and
vulvar burning), a pH range between pH 3 and pH 5, from between pH
3.5 and 4.5, from between pH 4 and pH 5, from between pH 4.5 and pH
5.5, from between pH 5.5 and pH 6.5, or between pH 5 and pH 6 may
be used. In particular embodiments, the composition used in methods
for urogenital administration, for example to treat atrophic
vaginitis, has a pH from between 3.5 and 4.5. Because the sulfonic
group of taurine has a low pKa (1.5), taurine is expected to remain
negatively charged within the pH range normally found in the
vaginal mucosa.
[0079] The compositions described herein may additionally comprise
a physiologically acceptable excipient (pharmaceutically acceptable
or suitable excipient or carrier) (i.e., a non-toxic material that
does not interfere with the activity of the active ingredient).
Physiologically acceptable compositions may also contain other
components, which may be biologically active or inactive.
Compositions comprising a mineral salt and a sulfonic acid may
further comprise one or more agents and compounds, such as a
viscosity modulating agent, a flavoring agent, a mucoadhesive
agent, a solubilizing agent, a mucosal absorption-promoting agent,
a penetration-promoting agent, and a stabilizing agent. As
discussed herein, the compositions may further comprise acidifying
agents, alkalizing agents, and/or buffering agents. The
compositions may also include one or more pharmaceutically
acceptable additives such as antimicrobial preservatives,
antioxidants, chelating agents, complexing agents, solubilizing
agents, emulsifying agents, humectants, solvents, suspending and/or
tonicity agents, wetting agents and other biocompatible materials.
The inclusion of such agents will depend upon the disease or
disorder to be treated, the administration route, and the part of
the body or tissue to be treated and to which the composition is
administered.
[0080] In certain embodiments, the compositions described herein
further comprise a viscosity modulating agent, such as a thickening
agent (also called a thickener), which includes but is not limited
to a viscosity enhancer (also called a viscosity enhancing agent or
a viscosity increasing agent). A thickening agent increases the
viscosity of the composition. For example, viscosity-enhancing
agents include polyvinylpyrrolidone (PVP) and hyaluronic acid. Each
of PVP and hyaluronic are available in mixtures of polymers of
varying molecular weights (e.g., K60, K85, K95K100, which are
available from commercial vendors). The compositions described
herein, in certain embodiments, may be formulated with from about
0.04 to about 15% by weight of a K60 to K100 PVP. A
viscosity-enhancing agent such as PVP (including K60 to K90 PVP)
may be formulated at low percent weight of the composition (e.g.,
from 0.5% (w/w) to 5.0% (w/w)) to achieve a composition of low
viscosity, or at higher percent weight of the composition (e.g.,
from about 5.1% (w/w) to about 10% (w/w), 12.5% (w/w), 15% (w/w) or
higher) to achieve a composition of high viscosity. In other
embodiments, PVP is from about K85 and K95 and is from about 3 and
10% by weight of the composition. In still another embodiment, PVP
is from about 7%-10% (w/w). (See U.S. Pat. No. 6,828,308, which is
incorporated by reference in its entirety.)
[0081] The compositions described herein comprising a mineral salt
and a sulfonic acid may further comprise hyaluronic acid. In
certain embodiments, the compositions comprise a mineral salt, a
sulfonic acid, hyaluronic acid, and PVP. The compositions described
herein may comprise from about 0.01 to about 5% by weight of
hyaluronic acid (i.e., between from about 0.01-5.0% (w/w)), or a
pharmaceutically acceptable salt thereof, having a molecular weight
from between about 1.6 and 2.2 million Daltons. In other
embodiments, hyaluronic acid, or the pharmaceutically acceptable
salt thereof, is from about 1.8 to about 2.0 million Daltons and
from about 0.01 to about 2% by weight. In still another embodiment,
hyaluronic acid, or the pharmaceutically acceptable salt thereof,
is from about 1.8 to about 2.0 million Daltons and from about 0.01
to about 2% by weight of the composition.
[0082] Solubilizing agents useful for including in the compositions
described herein include, but are not limited to, at least one or
more of cyclodextrin, hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, and
methyl-.beta.-cyclodextrin. A sulfonic acid (e.g., taurine) as
described herein may also act as a solubilizing agent, solubilizing
a mineral salt (e.g., a zinc salt, for example, zinc gluconate),
which may increase bioavailability of the mineral salt. The
addition of a sulfonic acid (for example, taurine) allows for
subsequent pH adjustment with an appropriate acid or base without
consequent precipitation of the mineral salt. The pH of the
compositions may then be adjusted with the appropriate pH adjusting
agent (i.e., an acid or base), neutralizing the mineral salt when
the composition is applied for treating conditions for which a
neutral pH is desired (e.g., oral mucositis) or maintaining or
adjusting to an acid pH when the composition is applied for
treating conditions for which an acid pH is desired (e.g., atrophic
vaginitis) as well as solubilizing a mineral salt (e.g., zinc
gluconate).
[0083] The methods described herein for treatment of mucosal
diseases, disorders, and conditions may be more effective when the
composition comprises one or more agents that increase (i.e.,
enhance) the residence time of the mineral salt and the sulfonic
acid at the mucosal delivery site (e.g., oral mucosa or vaginal
mucosa) (i.e., maintain the presence of the mineral salt and
sulfonic acid for a longer period of time at the mucosal site to
which the composition is delivered or administered than would occur
in the absence of such an agent). Accordingly, polymeric delivery
vehicles and other agents that contribute to increasing (i.e.,
improving) residence time, for example, sustained release-enhancing
formulations, such as polyethylene glycol (PEG) (e.g., PEG-40), and
methods for delivery same are described herein.
[0084] In certain embodiments, the compositions may be formulated
for mucosal delivery. The physiologically acceptable compositions
comprising at least one mineral salt, such as a zinc salt, and one
or more sulfonic acids (e.g., taurine) may be combined or
coordinately administered with a suitable carrier or vehicle for
delivery to a mucosal or epithelial surface. By coordinate delivery
is meant that at least two compositions are sequentially
administered to a subject in need of treatment, which can be
prophylactic or therapeutic in its use. By way of example, a
composition that increases the bioavailability, such as by
increasing or maintaining the residence time of the mineral salt
and/or the sulfonic acid, may be administered before or after the
composition comprising the mineral salt and sulfonic acid.
[0085] The compositions described herein that comprise a mineral
salt and a sulfonic acid may further comprise an absorption
promoting agent. The amount of each active ingredient that is
formulated in a composition with one or more additional agents to
produce a single dosage form will vary depending upon the
particular mode of administration. While the mechanism of
absorption promotion may vary with different mucosal
delivery-enhancing agents, useful reagents in this context will not
adversely affect the mucosal tissue in a biologically or
statistically significant manner and will be selected according to
the physicochemical characteristics of the particular mineral salt
(e.g., a zinc salt, for example, zinc gluconate) and sulfonic acid
(e.g., taurine), and other agents included in the composition.
Delivery-enhancing agents that increase penetration or permeability
of mucosal tissues may cause some alteration of the protective
permeability barrier of the mucosa. A delivery-enhancing agent
useful for the methods described herein are those that if
administration of such an agent causes significant changes in
permeability of the mucosa that these changes may be reversible
within a time frame appropriate to the desired duration of drug
delivery. Furthermore, a suitable delivery-enhancing agent has no
substantial, cumulative toxicity, and does not induce permanent
deleterious changes in the barrier properties of the mucosa,
particularly when the compositions described herein are intended
for long-term use.
[0086] The compositions described herein comprising a mineral salt
(e.g., a zinc salt, for example, zinc gluconate) and a sulfonic
acid (e.g., taurine) may also include absorption-promoting agents.
An absorption-promoting agent may be selected from small
hydrophilic molecules, including but not limited to, dimethyl
sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and
the 2-pyrrolidones. Alternatively, long-chain amphipathic
molecules, for example, deacylmethyl sulfoxide, azone, sodium
laurylsulfate, oleic acid, and the bile salts, may be employed to
enhance mucosal delivery or penetration. In further embodiments,
surfactants (e.g., polysorbates such as polysorbate 20 and
polysorbate 80) are employed as adjunct compounds, processing
agents, or formulation additives to enhance mucosal delivery.
[0087] Other mucosal absorption-promoting agents are selected from
a variety of compounds, compositions, and molecules that enhance
mucosal delivery, stability, activity or trans-epithelial
penetration. These include, inter alia, cyclodextrins (e.g.,
cyclodextrin) and .beta.-cyclodextrin derivatives (e.g.,
hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, methyl-.beta.-cyclodextrin and
heptakis(2,6-di-O-methyl-.beta.-cyclodextrin)). These compounds,
optionally conjugated with one or more of the active ingredients
and further optionally formulated in an oleaginous base, enhance
bioavailability of the one or more active ingredients (such as the
mineral salt, sulfonic acid, and/or a lactoferrin). Yet additional
absorption-enhancing agents adapted for mucosal delivery include
medium-chain fatty acids, including mono- and diglycerides (e.g.,
sodium caprate--extracts of coconut oil, CAPMUL.RTM.), and
triglycerides (e.g., amylodextrin, Estaram 299, MIGLYOL.RTM.
810).
[0088] Compositions described herein may also be supplemented with
any suitable penetration-promoting agent that facilitates
absorption, diffusion, or penetration of a mineral salt (e.g., zinc
gluconate) or sulfonic acid (e.g., taurine) across mucosal
barriers. The penetration-promoting agent may be any such agent
that is pharmaceutically acceptable. Thus, in certain embodiments,
compositions are provided that incorporate one or more of the
penetration-promoting agents selected from sodium salicylate and
salicylic acid derivatives (acetyl salicylate, choline salicylate,
salicylamide, etc.). Also provided as penetration-promoting agents
are substances that are generally used as emulsifiers (e.g., sodium
oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate,
sodium myristyl sulfate, polyoxyethylene alkyl ethers,
polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid,
malic acid and citric acid and alkali metal salts thereof,
pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid
esters, N-alkylpyrrolidones, proline acyl esters, and the like.
[0089] A physiologically acceptable carrier or excipient includes a
pharmaceutically acceptable solid or liquid filler, diluent, or
encapsulating material. A water-containing liquid carrier can
contain pharmaceutically acceptable additives such as any one or
any combination of acidifying agents, alkalizing agents,
antimicrobial preservatives, antioxidants, buffering agents,
chelating agents, complexing agents, solubilizing agents,
emulsifying agents, humectants, solvents, suspending and/or
viscosity-increasing agents (e.g., a thickener), tonicity agents,
wetting agents or other biocompatible materials.
[0090] Exemplary materials that may be included in the compositions
described herein as pharmaceutically acceptable carriers or
excipients are sugars, such as lactose, glucose, sodium saccharin
and sucrose; starches such as corn starch and potato starch;
cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, castor oil (e.g.,
hydrogenated castor oil), corn oil and soybean oil; glycols, such
as propylene glycol; polyols such as glycerin, sorbitol, mannitol
and polyethylene glycol; esters such as ethyl oleate and ethyl
laurate; agar; buffering agents such as magnesium hydroxide, sodium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water,
purified water; isotonic saline, acetate, lactate, formate, and
glycolate, Ringer's solution, ethyl alcohol and phosphate buffer
solutions, as well as other non toxic compatible substances
typically used or generally regarded as safe (GRAS) to use in
pharmaceutical formulations. Such agents may be used individually
or in any combination, or at any concentration. In certain
embodiments, the compositions described herein comprise glycerin as
an excipient, which is formulated at a percent weight from about
0.01 to about 3% by weight of the composition.
[0091] The compositions may also include humectants including, but
are not limited to, propylene glycol, glycerin, glyceryl
triacetate, a polyol, a polymeric polyol, lactic acid, and urea.
The physiologically acceptable compositions described herein may
comprise one humectant or any combination or mixture of more than
one (i.e., at least two) humectants.
[0092] The compositions described herein may also comprise one or
more wetting agents, emulsifiers, and lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions disclosed herein. Exemplary flavoring agents include
essential oils, synthetic flavors, fruit essences, anise, flavor
oils, citrus oil, peppermint oil, spearmint oil, mint oil, clove
oil, oil of wintergreen, menthol, eucalyptol, thymol, and the like,
and combinations thereof. The compositions may also include an
agent that is described in the art as a flavor altering agent, such
as glycyrrhetinic acid, that masks the flavor of an otherwise less
palatable or less pleasant tasting composition. A flavoring agent
and/or flavor altering agent may be used in a composition disclosed
herein at a concentration of from 0.05% to 3.0% (w/w), from 0.1%
(w/w) to 20% (w/w), from 0.5% (w/w) to 1.5% (w/w), from 1.5% (w/w)
to 2% (w/w), from 2% (w/w) to 3% (w/w), from 3% (w/w) to 4%, or
from 5% to 10%, 15%, or 17% (w/w). In a particular embodiment,
glycyrrhetinic acid, or a pharmaceutically acceptable salt thereof,
may be formulated with the compositions described herein at a
percent weight from about 0.01 to about 3% by weight of the
composition.
[0093] As used herein, examples of emulsifying agents include
lecithin, Cm to C12 fatty acids, mono and diacyl glycerides, ox
bile extract, polyglycerol esters, polyethylene sorbitan esters,
propylene glycol, sorbitan monopalmitate, sorbitan monosterate,
sorbitan tristerate, enzyme modified lecithin, hydroxylated
lecithins, and combinations thereof. Examples of pharmaceutically
acceptable antioxidants include water soluble antioxidants such as
ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite and the like; oil-soluble
antioxidants such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol and the like; and metal-chelating agents such as
citric acid, ethylenediamine tetraacetic acid (EDTA), ethylene
glycol tetraacetic acid (EGTA), sorbitol, tartaric acid, phosphoric
acid and the like.
[0094] Other pharmaceutically acceptable agents having any one or
more of the properties described herein can be found in the U.S.
Pharmacopeia National Formulary, 1990, 1857-1859. Any suitable
excipient or carrier known to those of ordinary skill in the art
for use in pharmaceutical compositions may be employed in the
compositions described herein. Excipients for therapeutic use are
well known, and are described, for example, in Remington: The
Science and Practice of Pharmacy (Gennaro, 21.sup.st Ed. Mack Pub.
Co., Easton, Pa. (2005)). In general, as discussed herein, the type
of excipient is selected on the basis of the mode of
administration. Pharmaceutical compositions may be formulated for
any appropriate manner of administration, including, for example,
topical, oral, nasal, intrathecal, buccal, rectal, vaginal,
intraocular, subconjunctival, sublingual or parenteral
administration, including subcutaneous, intravenous, intramuscular,
intrasternal, intracavernous, intrameatal or intraurethral
injection or infusion.
[0095] The compositions may further comprise ingredients that act
as delivery vehicles, including but not limited to aluminum salts,
water-in-oil emulsions, biodegradable oil vehicles, oil-in-water
emulsions, biodegradable microcapsules, and liposomes. While any
suitable excipient or carrier known and available to a person
having skill in the art may be employed in the compositions
described herein, the type of carrier will vary depending on the
mode of administration and whether a sustained release is desired.
In the methods described herein, a pharmaceutical composition may
be administered through use of insert(s), bead(s), timed-release
formulation(s), patch(es) or fast-release formulation(s).
[0096] For parenteral administration, such as subcutaneous
injection, the carrier preferably comprises water, saline, alcohol,
a fat, a wax or a buffer, and the composition is sterile. For oral
administration, any of the above carriers or a solid carrier, such
as mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, and magnesium carbonate, may
be employed. Biodegradable microspheres (e.g., polylactic
galactide) may also be used as carriers for the compositions
described herein. Suitable biodegradable microspheres described,
for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In
particular embodiments in which the composition is combined with a
microsphere, the microsphere is larger than approximately 25
microns. A composition described herein may be lyophilized or
otherwise formulated as a lyophilized product using one or more
appropriate excipient solutions (e.g., sucrose) as diluents upon
administration.
[0097] A pharmaceutical composition (or a physiologically
acceptable formulation) disclosed herein may be intended for
topical administration, in which case the carrier may suitably
comprise a solution, emulsion, ointment or gel base. The base, for
example, may comprise one or more of the following: petrolatum,
lanolin, polyethylene glycols, beeswax, mineral oil, diluents such
as water and alcohol, and emulsifiers and stabilizers. As discussed
in greater detail herein, thickening agents may be present in a
pharmaceutical composition for topical administration (e.g., oral
or vaginal). The compositions described herein may be administered
by using any one of several delivery vehicles described herein and
used in the art, including but not limited to a sponge, gel cap,
suppository, gauze (or other suitable fabric for application to the
tissue to be treated), and a lozenge. With respect to certain
delivery vehicles, such as a sponge, fabric, or gauze, the
composition is attached to, absorbed by, adsorbed to, or in some
manner applied to the vehicle that permits release of the
composition upon contact with the tissue to be treated.
[0098] A composition disclosed herein may be intended for rectal
(for treatment of a rectal mucosa), oral, or vaginal
administration, in the form, e.g., of a suppository or lozenge,
which will melt in the rectum, oral, or vaginal space, and release
the drug or components of the composition. A composition described
herein that is administered orally may also be in the form of a
liquid. The composition for rectal administration may contain an
oleaginous base as a suitable nonirritating excipient. Such bases
include, without limitation, lanolin, cocoa butter and polyethylene
glycol.
[0099] The compositions described herein may be endotoxin free,
particularly when delivered parenterally. An endotoxin free
composition that comprises a mineral salt and a sulfonic acid and
that further comprises one or more agents described herein is
substantially free of endotoxins and/or related pyrogenic
substances (i.e., an endotoxin is not detectable by methods
accepted by regulatory agencies to demonstrate with sufficient
sensitivity whether an endotoxin is present). Endotoxins include
toxins that are present in viable microorganisms and include toxins
that are released only when the microorganisms lack cell integrity
or die. Pyrogenic substances include fever-inducing, thermostable
substances (lipopolysaccharides and glycoproteins) located in the
outer membrane of bacteria and other microorganisms. These
substances can cause fever, hypotension, and shock when
administered to humans. Manufacturing compositions that are
endotoxin-free can require special equipment, expert artisans, and
can be significantly more expensive than making formulations that
are not endotoxin-free.
[0100] In specific embodiments, the physiologically acceptable
compositions that comprise a mineral salt (for example, from
between 0.25%-5.5% (w/v) zinc gluconate) and a sulfonic acid (for
example, from between 0.5%-8% (w/w) taurine) further comprise one
or more of 0.05% to 3.0% (w/w) glycyrrhetinic acid; 0.04% to 15%
(w/w) polyvinylpyrrolidone (PVP); 0.01% to 5.0% (w/w) hyaluronic
acid; and 0.05% to 3.0% (w/w) glycerin (or from between 0.05% to 5%
glycerin). In other specific embodiments, the composition comprises
0.5% (w/w) zinc gluconate, 1.0% (w/w) taurine, and 4.0% PVP (w/w);
or alternatively, 0.5% (w/w) zinc gluconate, 1.0% (w/w) taurine,
and 8.0% PVP (w/w); or in certain other embodiments, 2.0% (w/w)
zinc gluconate, 4.0% (w/w) taurine, and 4.0% PVP (w/w). Without
wishing to be bound by theory, inclusion of PVP and hyaluronic acid
promote adherence of the composition to mucosa, which provides a
protective coating of any exposed nerve endings, thereby reducing
pain, promoting cicatrisation and healing of any ulceration,
lesion, or microlesion of the mucosa. In other specific
embodiments, PVP is absent from the compositions described herein
that comprise a mineral salt (e.g., a zinc salt, for example, zinc
gluconate) and a sulfonic acid (e.g., taurine).
[0101] In other certain embodiments, the methods described herein
may include administering a composition that is formulated to
contain purified water, PVP, taurine, zinc gluconate, PEG-40,
hydrogenated castor oil, sodium saccharin, sodium hydroxide and a
flavoring agent. In other specific embodiments, the composition
comprises purified water, PVP, taurine, zinc gluconate, PEG-40,
hydrogenated castor oil, sodium saccharin, sodium hydroxide, and a
flavoring agent. In a specific embodiment, a composition is
provided that comprises 0.5% (w/w) zinc gluconate, 1.0% (w/w)
taurine, 1.0% (w/w) glycyrrhetinic acid (a flavoring agent, also
referred to herein as a flavor modifying or altering agent), 1.0%
(w/w) polyvinylpyrrolidone (PVP, which acts as a viscosity
enhancing or thickening agent), 0.1% (w/w) hyaluronic acid, and
0.1% (w/w) glycerin. In a specific embodiment, the pH of the
composition is adjusted to be a neutral pH, for example, between pH
5.5 and 7.5. In other certain embodiments, the pH of the
composition is adjusted to between 3.5 and 4.5. Because the above
composition has a PVP percent by weight of 1.0%, this formulation
would be considered low viscosity.
[0102] In another embodiment, the composition may be formulated to
comprise 0.5% (w/w) zinc gluconate, 1.0% (w/w) taurine, 1.0% (w/w)
glycyrrhetinic acid, 8.0% (w/w) polyvinylpyrrolidone (PVP), 0.1%
(w/w) hyaluronic acid, 0.1% (w/w) glycerin. In certain embodiments,
the pH of the composition is adjusted to between pH 3.5 and 4.5.
Such a composition may be used to treat urogenital mucosal diseases
and disorders described herein, including but not limited to
vaginal dryness and atrophic vaginitis.
[0103] In other specific embodiments, the methods described herein
comprise administering a composition comprising a mineral salt and
a sulfonic acid with the proviso that a composition consisting of
deionized water, zinc gluconate, ascorbic acid, methylcellulose,
taurine, methylparaben, propylparaben, and F.D. & C. Blue No:1
and a composition consisting of deionized water, zinc gluconate,
carboxymethylcellulose, taurine, methylparaben, propylparaben, and
F.D. & C. Blue No:1 are each excluded.
[0104] In certain embodiments, the methods for treating a mucosal
or dermal disease, disorder, or condition comprise administering
the compositions comprising a mineral salt and a sulfonic acid
(which are described in detail herein) enterally (i.e., orally) or
topically. Topical administration refers to administration of the
composition to the surface of the tissue to be treated and to which
the composition will have a beneficial effect, such as to a mucous
membrane, including but not limited to, an oropharyngeal mucous
membrane, vaginal mucous membrane, or anal mucous membrane. Enteral
administration includes oral administration (i.e., administered
orally) to the oropharyngeal cavity. When a composition is
administered orally, the components of the composition are likely
absorbed systemically and have a systemic effect and may also be
absorbed topically (or have a topical effect). In certain specific
embodiments, the compositions described herein that comprise a
mineral salt (e.g., zinc gluconate) and a sulfonic acid (e.g.,
taurine) may be administered topically in the form of a gel.
[0105] In other embodiments, the compositions described herein
further comprise a protein or polypeptide that exhibits properties
and characteristics that are useful for treating one or more of the
mucosal and dermal disorders and associated inflammation described
herein. In certain embodiments, the polypeptide is a transferrin,
and in a specific embodiment, the transferrin is lactoferrin.
[0106] The preparations and compositions described herein comprise
a lactoferrin, which is a globular, cationic, non-heme iron-binding
protein. Lactoferrin is a prominent glycoprotein in milk, other
secretory fluids, and white blood cells, and is synthesized by
exocrine glands and by neutrophils at infection and inflammation
sites. has been shown to have antibacterial, antiviral, and
anti-fungal, and anti-inflammatory properties (see, e.g., Conneely,
J. Amer. Coll. Nutr. 20(5):389S-395S, 2001; van der Strate et al.,
Antiviral Res. 52: 225-39 (2002); Antonini. Cell. Mol. Life Sci.
62: 2576-87 (2006); Ward et al., Cell. Mol. Life Sci. 62: 2540-48
(2006); Bellamy et al., Bochim. Biophys. Acta 1121:130-36 (1992)).
Without wishing to be bound by theory, lactoferrin may reduce
inflammation by reducing and/or maintaining the production of
proinflammatory factors such as IL-1.beta., IL-6, IL-8,
TNF-.alpha., and NF-.kappa.B, for example, to a level that reduces,
abrogates, prevents, minimizes destructive inflammatory effects
(see, e.g., International Application Publication No. WO
2007/065482, which is incorporated herein by reference in its
entirety).
[0107] Lactoferrin typically contains two bound Fe.sup.+3 (also
referred to as iron III or FeIII) ions. Full-length lactoferrin has
a molecular weight of approximately 80 kDa, and belongs to the
transferrin family of proteins. The molecular weight of lactoferrin
has also been reported to be 78 kDa. The difference in reported
molecular size may represent the presence or absence of one
N-linked oligosaccharide modification.
[0108] Lactoferrin belongs to the family of transferrin proteins,
which also includes serum transferrin (see, e.g., Baker et al.,
Biochem. Cell Biol. 80:27-34 (2002) and references cited therein).
Lactoferrins between species share approximately 70% sequence
identity (see, e.g., Baker, Adv. Inorg. Chem. 41:389-463 (1994)).
The amino acid sequence of lactoferrin contains a two-fold internal
repeat, and the N-terminal half has approximately 40% sequence
identity with the C-terminal half, which results in the protein
folding into two homologous halves. Compared with serum
transferrin, lactoferrin has a more potent iron-withholding
activity: lactoferrin retains iron at a ph as low as pH 3.5,
whereas, serum transferrin begins to lose iron at pH 6 (see, e.g.,
Mazurier et al., Biochim. Biophys. Acta 629:399-408 (1986);
Peterson et al., Biochemistry 39:6625-33 (2000)).
[0109] Lactoferrin may be human lactoferrin, bovine lactoferrin,
murine lactoferrin, or buffalo lactoferrin. In certain embodiments,
the compositions described herein comprise bovine lactoferrin; in
other embodiments, the compositions comprise human lactoferrin.
Bovine lactoferrin can be produced in large quantities by isolating
the polypeptide from cow's milk. Lactoferrin may also be obtained
from commercial sources. Lactoferrin may also be produced
recombinantly according to methods routinely practiced in the
molecular biology and protein expression arts.
[0110] The majority of full-length human lactoferrin polypeptide
species that have been sequenced are 711 amino acids in length,
which includes a 19-amino acid signal peptide. Accordingly, an
exemplary mature (without the signal peptide) lactoferrin
polypeptide has 692 amino acids. Exemplary amino acid sequences for
human lactoferrin are located in the GenBank database (National
Center for Biotechnology Information (NCBI)) and include but are
not in any way limited to Accession Nos. AAA59511.1 (SEQ ID NO:1),
ACF19793.1 (SEQ ID NO:2), and AAW71443.1 (SEQ ID NO:3) (see also
AAR12276.1). The amino acid sequences of mature human lactoferrin
(i.e., without the 19-amino acid signal peptide) are provided in
SEQ ID NOS:8, 9, and 10, respectively.
[0111] The full-length bovine lactoferrin polypeptide species that
have been sequenced are 708 amino acids, and the bovine lactoferrin
polypeptides also include a 19-amino acid signal peptide.
Accordingly, an exemplary mature (without the signal peptide)
lactoferrin polypeptide has 689 amino acids. Exemplary amino acid
sequences available in the art for bovine lactoferrin include, but
are not limited to, GenBank Accession Nos. AAA30610.1 (SEQ ID
NO:4), AAA30617.1 (SEQ ID NO:5), AAA30609.1 (SEQ ID NO:6), and
AAA21722.1 (SEQ ID NO:7). The amino acid sequences of mature bovine
lactoferrin (i.e., without the 19-amino acid signal peptide) are
provided in SEQ ID NOS:11-14, respectively. The encoding
polynucleotide sequences for lactoferrins can be readily obtained
in a similar manner from publicly available and privately (i.e.,
for a fee or supporting membership) available databases or by
deducing an encoding polynucleotide sequence from the amino acid
sequence.
[0112] In certain embodiments, the methods described herein
comprise administering lactoferrin, wherein the lactoferrin is a
lactoferrin polypeptide fragment (see, e.g., U.S. Pat. No.
7,420,033). As described herein, certain lactoferrin polypeptide
fragments retain antimicrobial activity such as a cationic domain
at the amino terminal end of lactoferrin, which retains
antimicrobial activity (see, e.g., Bellamy, et al., supra;
Conneely, supra; Nakamura et al., Protein Exp. Purif. 21; 424-31
(2001); Tanaka et al., Biochem. Cell Biol. 81: 349-354 (2003)). The
antimicrobial activity of lactoferrin is structurally distinct and
separate from its iron binding activity. Other fragments described
in the art include fragments called lobe N and lobe C, and smaller
fragments within each of lobe N and lobe C (see, e.g.,
International Application Publication No. WO 2007/065482). The
amino terminal half of lactoferrin is referred to as the N-lobe (or
Lobe N) and the carboxy terminal half is referred to as the C-lobe
(or Lobe C). Both lobes have the same fold, which is consistent
with the high percent sequence identity between the lobes. Each
lobe is subdivided into two domains that are separated by an
interdomain cleft that includes an iron binding site (see, e.g.,
Baker et al., Biochem. Cell Biol., supra). Exemplary human
lactoferrin fragments of the amino terminal region of lactoferrin
(i.e., Lobe N) include but are not limited to a lactoferrin
fragment from amino acid at position 1 to about position 280 of the
mature human polypeptide. Lactoferrin fragments of lobe C include
but are not limited to a lactoferrin fragment from about amino acid
at position 285 to amino acid 692 of the mature human lactoferrin
polypeptide. Certain exemplary N lobe fragments of bovine
lactoferrin include amino acids at positions 1-333 (see SEQ ID
NOS:11-14), which may in certain embodiments, also include the
inter-lobe region (typically residues at positions 334-344) (see,
e.g., Bai et al., Biometals 2010 Feb. 10, epub ahead of print). The
N lobe and/or the C lobe may be obtained by recombinant expression
of the lobe polypeptide using molecular biology and protein
expression methods known and routinely practiced in the art or the
lobe of interest may be obtained by proteolytic digest of the
lactoferrin.
[0113] Lactoferrin belongs to the family of transferrin proteins,
which also includes serum transferrin (see, e.g., Baker et al.,
Biochem. Cell Biol. 80:27-34 (2002) and references cited therein).
Lactoferrins between species share approximately 70% sequence
identity (see, e.g., Baker, Adv. Inorg. Chem. 41:389-463 (1994)).
The amino acid sequence of lactoferrin contains a two-fold internal
repeat, and the N-terminal half has approximately 40% sequence
identity with the C-terminal half, which results in the protein
folding into two homologous halves. Compared with serum
transferrin, lactoferrin has a more potent iron-withholding
activity: lactoferrin retains iron at a ph as low as pH 3.5,
whereas, serum transferrin begins to lose iron at pH 6 (see, e.g.,
Mazurier et al., Biochim. Biophys. Acta 629:399-408 (1986);
Peterson et al., Biochemistry 39:6625-33 (2000)).
[0114] The protein surface of the lactoferrin molecule has regions
with high concentrations of positive charge that result in a high
isoelectric point (.about.pI 9) for the polypeptide. For example,
in human lactoferrin, one region of positive charge includes the
N-terminus portion of the mature lactoferrin that has an amino acid
sequence of GRRRRS (SEQ ID NO:15) (see, for example, amino acid
residues 1-6 of SEQ ID NOS:8, 9, and 10), which projects from the
protein surface-terminus of the polypeptide chain, together with
the adjacent carboxy terminal portion of helix 1, which includes a
positively charged region, for example, the amino acid sequence,
RKVR (SEQ ID NO:16) or RRVR (SEQ ID NO:17). This region provides a
site for binding heparin (see, e.g., Van Berkel et al., Biochem. J.
328:145-151 (1997)) and glycosaminoglycans (see, e.g., Mann et al.,
J. Biol. Chem. 269: 2366-23667 (1994)) and may be the site that
binds to DNA. The N-terminal portion is contiguous with helix 1 of
the N-lobe, which forms the main part of the bactericidal domain
(see, e.g., Bellamy et al., Biochim. Biophys. Acta 1121:130-136
(1992)), which is characterized by the presence of surface arginine
residues. Despite their virtually identical fold, other
transferrins do not share this bactericidal activity, presumably
because they lack the necessary surface features (see, e.g, Baker,
Biochem. Cell Biol., supra).
[0115] While bovine lactoferrin (bLf) does not share the same
N-terminal repeat of arginine residues, bLf also has a highly
positively charged region at its N terminus. In bovine lactoferrin,
the domain responsible for bactericidal activity, which is also the
heparin binding domain, includes residues 17-42 of the mature bLf
polypeptide
(Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile--
Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala (SEQ ID NO:18)) (see, for example,
SEQ ID NO:11-14) (see, e.g., Bellamy Biochim. Biophys. Acta, supra;
Shimazaki et al., J. Dairy Sci. 81:2841-49 (1998)). Motifs, KCRR
(SEQ ID NO:19) at positions 18-21 and RMKK (SEQ ID NO:20) at
positions 25-28 (see SEQ ID NOS:11-14), are believed to be
particularly important for heparin binding (Shimazaki et al.,
supra).
[0116] In certain embodiments, the methods described herein
comprise administering a composition comprising a mineral salt and
a sulfonic acid and further comprising lactoferrin, wherein the
lactoferrin is a lactoferrin polypeptide fragment (see, e.g., U.S.
Pat. No. 7,420,033). As described herein, certain lactoferrin
polypeptide fragments retain antimicrobial activity such as a
cationic domain at the amino terminal end of lactoferrin, which
retains antimicrobial activity (see, e.g., Bellamy, et al., supra;
Conneely, supra; Nakamura et al., Protein Exp. Purif. 21; 424-31
(2001); Tanaka et al., Biochem. Cell Biol. 81: 349-354 (2003)). The
antimicrobial activity of lactoferrin is structurally distinct and
separate from its iron binding activity. Other fragments described
in the art include fragments called lobe N and lobe C, and smaller
fragments within each of lobe N and lobe C (see, e.g.,
International Application Publication No. WO 2007/065482). The
amino terminal half of lactoferrin is referred to as the N-lobe (or
Lobe N) and the carboxy terminal half is referred to as the C-lobe
(or Lobe C). Both lobes have the same fold, which is consistent
with the high percent sequence identity between the lobes. Each
lobe is subdivided into two domains that are separated by an
interdomain cleft that includes an iron binding site (see, e.g.,
Baker et al., Biochem. Cell Biol., supra). Exemplary human
lactoferrin fragments of the amino terminal region of lactoferrin
(i.e., Lobe N) include but are not limited to a lactoferrin
fragment from amino acid at position 1 to about position 280 of the
mature human polypeptide. Lactoferrin fragments of lobe C include
but are not limited to a lactoferrin fragment from about amino acid
at position 285 to amino acid 692 of the mature human lactoferrin
polypeptide. Certain exemplary N lobe fragments of bovine
lactoferrin include amino acids at positions 1-333 (see SEQ ID
NOS:11-14), which may in certain embodiments, also include the
inter-lobe region (typically residues at positions 334-344) (see,
e.g., Bai et al., Biometals 2010 Feb. 10, epub ahead of print). The
N lobe and/or the C lobe may be obtained by recombinant expression
of the lobe polypeptide using molecular biology and protein
expression methods known and routinely practiced in the art or the
lobe of interest may be obtained by proteolytic digest of the
lactoferrin.
[0117] Lactoferrin has the capability to tightly but reversibly
bind two Fe.sup.+3 (also referred to as ferric ions, iron III, or
FeIII) ions together with two carbonate ions (C03.sup.2-). The
presence of lactoferrin in tissues and secretions and transferrin
in blood assures that iron is tightly complexed. The high affinity
for iron (approximately 10.sup.22 M) ensures that the concentration
of free iron does not exceed 10.sup.-18 M, at which point ferric
hydroxides would precipitate (see, e.g., Aisen et al., "Physical
biochemistry of the transferrins" in Iron carriers and iron
proteins, vol. 5, Loehr, ed. VCH Publishers, New York, pp. 241-351
(1989); Baker et al., Cell. Mol. Life Sci. 62:2531-2539 (2005)),
and which also contributes to lack of microbial growth and
formation of reactive oxygen species (see, e.g., Weinberg, Biochim.
Biophys. Acta 1790:600-605 (2009)). Lactoferrin is an important
regulator of systemic iron homeostasis and is capable of restoring
hematological parameters in hypoferremia and IDA (see, e.g.,
Paesano et al., Biochimie, supra; Paesano et al., Biochem. Cell
Biol. 84:377-380 (2006)). As discussed herein, several functions,
dependent and independent of iron binding ability, have been
attributed to lactoferrin (see, e.g., Valenti et al. Cell Mol. Life
Sci. 62: 2576-87 (2005)).
[0118] The biological effects of a lactoferrin may be achieved when
the lactoferrin has any degree of saturation of the binding sites
for iron (III), wherein "any degree" is understood not to exceed
100%. Even when no iron is bound by the lactoferrin (referred to as
the "apo" form wherein the degree of saturation of iron sites is
equal to 0%), biological effects may be observed. The biological
effects of a lactoferrin may also be achieved when the lactoferrin
is partially saturated, or when the lactoferrin is completely
saturated (referred to as the "holo" form of the lactoferrin when
the degree of saturation of iron sites is equal to 100%). A
lactoferrin used in the methods described herein may have any
degree of saturation of the iron binding sites ranging from 0%
saturation to and including 100% saturation, including but not
limited to saturation from 0%-20%, 0-40%, 10-30%, 10-35%, 10-40%,
10-50%, 10-60%, 15-30%, 15-40%, 15-50%, 15-60%, 10-70%, or 10-80%.
In a specific embodiment, a lactoferrin has a degree of saturation
of the iron binding sites ranging from 15-30%. In another specific
embodiment, a lactoferrin has a degree of saturation of the iron
binding sites ranging from 10-30% or from 10%-35%. The level of
saturation may be achieved by using a mixture of lactoferrin
molecules that have differing percent saturation to achieve a
desired level of saturation. The binding sites for iron can be
occupied at any degree of saturation by Fe (III) and/or optionally,
with different kinetics and affinities, by one or more other
transition metals that have similar chemical and physical
properties. These metals can be, for example, one or more zinc
(Zn), copper (Cu), aluminum (Al), gallium (Ga), chromium (Cr) and
manganese (Mn). Accordingly, in one embodiment, lactoferrin used in
the methods and compositions described herein has any degree of
saturation by Fe (III) and one or more of Zn, Cu, and Mn. In
another certain embodiment, lactoferrin has any degree of
saturation with one or more of Zn, Cu, and Mn.
[0119] Lactoferrin can be prepared by isolating the protein from a
source of milk or colostrum. Isolated lactoferrin means that the
protein is removed (i.e., partially purified, or totally purified
such that other components present in the source of lactoferrin are
not detectable) from its original environment (e.g., the natural
environment if it is naturally occurring). For example, when the
polypeptide present in a living animal, it is not considered to be
isolated; however, the same polypeptide, separated from some or all
or most of the co-existing materials in the natural system, is
considered isolated. Alternatively, lactoferrin may be produced
recombinantly according to methods routinely practiced by a person
skilled in the molecular biology art, particularly given that the
polypeptide sequence of lactoferrin and encoding nucleotide
sequence have been long known in the art.
[0120] The compositions described herein may comprise full length
lactoferrin, or truncated lactoferrin, or a fragment of
lactoferrin. Truncated molecules are polypeptides that comprise
less than the full-length amino acid sequence of the polypeptide.
As used herein "deletion" has its common meaning as understood by
those familiar with the art, and may refer to molecules that lack
one or more of a portion of a sequence from either terminus or from
a non-terminal region, relative to a corresponding full length
molecule, for example, as in the case of truncated molecules
provided herein. Truncated molecules that are linear biological
polymers such as polypeptides may have one or more of a deletion
from either terminus of the molecule or a deletion from a
non-terminal region of the molecule, where such deletions may be
deletions of any number of amino acids including a deletion that is
one amino acid up to about 675 amino acids. Also provided herein
are compositions that comprise a polypeptide that comprises a
fragment of lactoferrin as described herein. A lactoferrin fragment
may comprise any number of contiguous amino acids between at least
10 and 700 amino acids (including but not limited to at least 10,
20, 40, 60, 80, 100, 120, 150, 200, 300, 400, and 500 amino acids
and any whole number of amino acids between 10 and 690).
[0121] In certain embodiments, a lactoferrin polypeptide also
includes lactoferrin species that have one or more amino acid
substitutions, insertions, or deletions (also called herein a
lactoferrin variant). Conservative substitutions of amino acids are
well known and may occur naturally in the polypeptide or may be
introduced when the polypeptide is recombinantly produced. Amino
acid substitutions, deletions, and additions may be introduced into
a polypeptide using well-known and routinely practiced mutagenesis
methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory
Manual, 3d ed., Cold Spring Harbor Laboratory Press, N Y 2001)).
Oligonucleotide-directed site-specific (or segment specific)
mutagenesis procedures may be employed to provide an altered
polynucleotide that has particular codons altered according to the
substitution, deletion, or insertion desired. Deletion or
truncation variants of proteins may also be constructed by using
convenient restriction endonuclease sites adjacent to the desired
deletion. Subsequent to restriction, overhangs may be filled in and
the DNA religated. Alternatively, random mutagenesis techniques,
such as alanine scanning mutagenesis, error prone polymerase chain
reaction mutagenesis, and oligonucleotide-directed mutagenesis may
be used to prepare lactoferrin polypeptide variants and fragment
variants (see, e.g., Sambrook et al., supra). A bovine lactoferrin
variant includes a polypeptide that has at least 85%, 90%, 95%, or
99% amino acid sequence identity to any of the exemplary bovine
lactoferrin amino acid sequences known in the art and/or provided
in the sequence listing. Similarly, a human lactoferrin variant
includes a polypeptide that has at least 85%, 90%, 95%, or 99%
amino acid sequence identity to any of the exemplary human
lactoferrin amino acid sequences known in the art and/or provided
in the sequence listing.
[0122] Given the description in the art regarding regions of
lactoferrin that exhibit particular activities (e.g., the
N-terminal region comprising anti-microbial activity) and the amino
acids that are the ligands for binding to FeIII (see, e.g., Baker
et al., Biochem. Cell Biol., 2002, supra; see also, e.g., Chapple,
Antimicrob. Agents Chemother. 48:2190-98 (2004); Shimazaki et al.,
J. Dairy Sci. 81:2841-49 (1998); Tanaka et al., Biochem. Cell Biol.
81:349-54 (2003); Nakamura et al., Protein Expression and
Purification 21:424-31 (2001); Chapple et al., Adv. Exp. Med. Biol.
443:215-20 (1998)), persons skilled in the art can readily
determine which regions of lactoferrin may be more amenable to
alteration (i.e., substitution, deletion, or addition of one or
more amino acids) and which regions may not be amenable to change.
Also given the description herein and given the many molecular
biology, protein expression, and protein isolation techniques and
methods routinely practiced in the art for introducing mutations in
a polypeptide, preparing polypeptide fragments, isolating the
fragments and variants, and analyzing same, lactoferrin variants
and fragments having the desired biological activities can be made
readily and without undue experimentation.
[0123] Assays for assessing whether the lactoferrin variant folds
into a conformation comparable to the non-variant polypeptide or
fragment include, for example, the ability of the protein to react
with mono- or polyclonal antibodies that are specific for native or
unfolded epitopes, the retention of ligand-binding functions, and
the sensitivity or resistance of the mutant protein to digestion
with proteases (see Sambrook et al., supra). Lactoferrin variants
as described herein can be identified, characterized, and/or made
according to these methods described herein or other methods known
in the art, which are routinely practiced by persons skilled in the
art.
[0124] Lactoferrin polypeptides, variants and fragments thereof,
can be prepared without altering the biological activity of the
resulting protein molecule (i.e., without altering one or more
functional activities in a statistically significant or
biologically significant manner). For example, such substitutions
are generally made by interchanging within the groups of polar
residues, charged residues, hydrophobic residues, small residues,
and the like. The effect of any amino acid substitution may be
determined empirically merely by testing the resulting modified
protein for the ability to function in a biological assay, or to
bind to a cognate ligand or target molecule. By way of example, the
capability of the lactoferrin variant or fragment to bind iron,
exhibit antimicrobial activity, and/or exhibit anti-inflammatory
activity can be determined according to methods practiced by a
person skilled in the art.
[0125] Lactoferrin, a variant or fragment thereof, may be included
in a composition comprising a mineral salt (e.g., zinc gluconate)
and a sulfonic acid (e.g., taurine) by combining all three
components and, optionally, other agents as described herein.
Alternatively, lactoferrin, a variant or fragment thereof, may be
administered in a separate composition and administered prior to,
concurrent with, or subsequent to administration of a composition
comprising a mineral salt and a sulfonic acid (also referred to as
coordinate administration). A composition comprising lactoferrin
may be administered topically or systemically, or administered both
topically (e.g., to a mucosal membrane, for example, orally or
vaginally) and systemically at the same time or at varying times.
Accordingly, in certain embodiments, a composition comprising a
mineral salt, sulfonic acid, and lactoferrin (or a variant or
fragment thereof) may be administered locally (e.g., to a mucosal
surface, for example, oral or vaginal mucosa). In other
embodiments, methods comprise administering a separate
physiologically acceptable composition comprising lactoferrin (or a
variant or fragment thereof) and one or more physiologically
acceptable carriers (or excipients) and that lacks a mineral salt
and a sulfonic acid, which is administered sequentially (either
prior to or after) or concurrently with administration of a
physiologically acceptable composition comprising a mineral salt
and a sulfonic acid, which composition may, but not necessarily,
also comprise lactoferrin. When two separate compositions are
administered, the compositions may be in the same form (i.e.,
solid, liquid, spray, gel, past, emulsion, ointment, or foam or
other form) or a different form. The two compositions may be
delivered in the same or different manner, such as by lozenge, gel
cap, suppository, sponge, or by another delivery vehicle described
herein or with which a person skilled in the art is familiar.
[0126] In one embodiment, methods provided herein for treating a
mucosal disease or disorder comprise administering a first
composition that is a physiologically acceptable composition
comprising a mineral salt (e.g., zinc gluconate) and a sulfonic
acid (e.g., taurine) and administering a second composition
comprising lactoferrin in the absence of (i.e., that lacks) each of
a mineral salt and a sulfonic acid. The second composition may be
administered prior to, concurrent with, or subsequent to
administration of the first composition. In one particular
embodiment, the first composition (i.e., comprising a mineral salt
and a sulfonic acid) is administered topically to the tissue to be
treated (e.g., administered topically to the oral mucosa, vaginal
mucosa, or anal mucosa) and the second composition (i.e.,
comprising lactoferrin) is administered orally, which may have a
topical effect as well as a systemic effect. In another particular
embodiment, the first composition comprises a mineral salt (e.g.,
zinc gluconate) and a sulfonic acid (e.g., taurine) and
lactoferrin. In certain embodiments, the first composition
comprising the mineral salt and sulfonic acid is in the form of a
gel and the second composition comprising lactoferrin is in a form
appropriate for oral administration (which are described herein and
known in the art).
[0127] Lactoferrin may be formulated according to well known
methodologies in a composition described herein or in a separate
composition for administration. Any physiological or
pharmaceutically suitable excipient or carrier known to those of
ordinary skill in the art for use in pharmaceutical compositions
may be employed in the compositions described herein that comprise
lactoferrin. Excipients for therapeutic use are well known, and are
described, for example, in Remington: The Science and Practice of
Pharmacy (Gennaro, 21' Ed. Mack Pub. Co., Easton, Pa. (2005)). For
example, saline and phosphate-buffered saline at physiological pH
may be used. Preservatives, stabilizers, dyes and even flavoring
agents may be provided in the composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. In addition, antioxidants and suspending
agents may be used. An injectable pharmaceutical composition is
preferably sterile.
[0128] An optimal dose of lactoferrin may generally be determined
using experimental models and/or clinical trials. The optimal dose
may depend upon the body mass, weight, or blood volume of the
patient. Results of animal studies with lactoferrin have indicated
that lactoferrin is well tolerated with minimum toxic effects at
doses of 2000 mg/kg/day (see, e.g., Yamauchi et al., Food Chem.
Toxicol. 38:503-12 (2000)). In certain embodiments of the methods
provided herein, lactoferrin may be administered from about 5 to 50
mg lactoferrin per day, from about 50 to 400 mg lactoferrin per
day, from about 400 to 1000 mg per day, from about 1 gram to 5
grams per day, or from about 5 grams to 10 grams per day, or from
about 10 to about 15 grams per day, which may be administered in
one or in multiple doses. The use of the minimum dosage that is
sufficient to provide effective therapy is usually preferred.
Patients may generally be monitored for therapeutic or prophylactic
effectiveness using any one or more of observations, physical
examination, clinical history, hematological profile, immunological
assays and/or any other methods and techniques suitable for
assessing a patient who has the condition being treated or
prevented, which methods and techniques will be familiar to those
having ordinary skill in the art.
[0129] The compositions described herein that comprise one or more
mineral salts (e.g., zinc gluconate) and a sulfonic acid (e.g.,
taurine) may be in any form that allows for the composition to be
administered to a subject. For example, the composition may be in
the form of a solid, liquid, emulsion, ointment, suspension, gel,
or gas (aerosol). A gel may also be referred to herein and in the
art as a medical device or device. For example, a composition may
be in the form of a device that is a gel and that comprises a
mineral salt (e.g., zinc gluconate), a sulfonic acid (e.g.,
taurine), and one or more agents described in detail herein, such
as hyaluronic acid and polyvinylpyrrolidone (PVP). As used herein,
a device that is a gel may be used for treating a vaginal mucosal
disorder, such as vaginal dryness and atrophic vaginitis, and may
be applied intravaginally. The composition may also be embedded or
impregnated in a separate device, such as a sponge. Typical routes
of administration include, without limitation, oral, topical,
parenteral, sublingually or buccally, rectal, vaginal, or
intranasal. The term parenteral as used herein includes
subcutaneous injection, intravenous, intramuscular, intrasternal,
intracavernous, intrathecal, intrameatal, intraurethral injection,
or infusion techniques. A composition is formulated in a manner
that allows the active ingredients contained therein to be
bioavailable upon administration of the composition to a subject.
Compositions that will be administered to a subject may be prepared
in a form that comprises one or more dosage units. For example, a
tablet may be a single dosage unit, and a container (e.g., a bottle
or ampoule) comprising a composition described herein may contain a
plurality of dosage units.
[0130] As described herein, for oral administration, an excipient
and/or binder may be present. Exemplary excipients include sucrose,
kaolin, glycerin, starch dextrins, sodium alginate,
carboxymethylcellulose and ethyl cellulose. Coloring and/or
flavoring agents may also be present. A coating shell may be
employed.
[0131] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection (or infusion),
as two examples. When intended for oral administration, and as
described in detail herein, compositions may further comprise, in
addition to one or more mineral salt (e.g., zinc gluconate) and a
sulfonic acid (e.g., taurine), at least one of sweetening agent, a
preservative, a dye/colorant, a flavor enhancer, a surfactant, a
preservative, a wetting agent, a dispersing agent, a suspending
agent, a buffer, a viscosity enhancing or a thickening agent, a
stabilizer, and an isotonic agent.
[0132] A liquid composition as used herein, whether in the form of
a solution, suspension or other form, may include one or more of
the following excipients: sterile diluents such as water for
injection, saline solution, preferably physiological saline,
Ringer's solution, isotonic sodium chloride, fixed oils such as
synthetic mono or digylcerides that may serve as the solvent or
suspending medium, polyethylene glycols, glycerin, propylene glycol
or other solvents; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
(EDTA) acid; buffers such as acetates, citrates or phosphates and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The composition can be enclosed in ampoules, disposable
syringes, bottles or multiple dose vials made of glass or plastic.
In certain embodiments, the composition may comprise antimicrobial
agents such as benzyl alcohol or methylparaben that act as
preservatives (i.e., prevent growth in the composition of any
microorganism that may contaminate or be unintentionally introduced
into the composition).
[0133] The compositions described herein that comprise a mineral
salt (e.g., zinc gluconate) and a sulfonic acid (e.g., taurine)
(which may be formulated with one or more mucosal
delivery-enhancing agents as described herein), may be delivered at
a dose and for a time sufficient to treat a dermal or mucosal
disease or disorder. A dose release may be substantially normalized
and/or sustained for an effective delivery period ranging from
0.001 to 1.0 hours, from 0.001 to 0.01 hours, from 0.025 to 0.05
hours, from 0.01 to 0.05 hours, from 0.1 to 2.0 hours; from 0.4 to
1.5 hours; from 0.7 to 0.5 hours; or from 0.8 to 1.0 hours. In some
methods of administration, the delivery period may be from many
hours or days as necessary to provide benefit to the subject. A
composition disclosed herein can be administered once a day, or
multiple times a day (e.g., twice, three, four, or five times a
day), once every other day, once weekly, once biweekly, or once a
month as necessary to treat or prevent, or modulate the appearance
of a symptom of, or otherwise improve the outcome of a mucosal or
dermal disorder. Administration of a composition disclosed herein
can begin prior to the initiation or detection of symptoms of a
dermal or mucosal disease, disorder, or condition, or prior to
initiation or detection of associated or related inflammation. By
way of example, prior to a first administration of chemotherapy or
radiotherapy that is expected to have a side effect such as oral
mucositis, the compositions described herein may be administered to
the subject. Alternatively, the compositions described herein may
be administered after chemotherapy or radiotherapy treatment but
before initiation of a symptom of oral mucositis, or administration
can begin after a symptom of oral mucositis (or oral stomatitis) is
detected.
[0134] A composition described herein for the treatment of vaginal
mucosal disorders, diseases, and conditions, including but not
limited to vaginal dryness and atrophic vaginitis, may be
administered daily, weekly or bi-weekly, or otherwise over a
suitable period of time, for example, for ten, twenty, thirty,
thirty-five or more days (e.g., 12 weeks). Such treatment may
continue until one or more of the following occur: vaginal
epithelial thickness increases (relative to epithelial thickness
prior to initiating treatment); the maturation index increases
(relative to maturation index prior to initiating treatment); in
vaginal pH decreases (relative to pH prior to initiating
treatment); the severity of a bothersome symptom (e.g., vaginal
dryness, vaginal irritation/itching, vaginal soreness, difficulty
passing urine, frequent urination, pain during intercourse, or
bleeding after intercourse) decreases relative to the same prior to
initiating treatment. As described in greater detail herein, Pap
smears may be performed and the number of parabasal, intermediate
and superficial cells may be counted, and the percentage of each
cell type calculated in order to determine a Maturation Index. A
maturation index represents the degree of proliferation and
maturation of, for example, vaginal cells. A maturation index may
be reported as a percentage of parabasal, intermediate, and
superficial cells as determined by calculations known in the art.
For example, the percentages may then be used in the following
equation to determine a Maturation Index=(% Parabasal cells X 0)+(%
Intermediate cells X 0.5)+(% Superficial cells X 1.0).
[0135] Dermal and Mucosal Diseases, Disorders, and Conditions and
Treatment Thereof
[0136] Methods are provided herein for treating dermal and mucosal
diseases, disorders, and conditions, and include inflammatory
mucosal diseases (e.g., mucositis and atrophic vaginitis) and
disorders and conditions. Mucositis refers to inflammation and
ulceration of a mucous membrane and may occur at one or more
mucosal sites located at a mucosal surface of the oral cavity (or
oropharyngeal cavity), gastrointestinal tract, vagina, bladder,
rectum, anus, lung, esophagus, mucosal surface of the nasal cavity,
ear, and ocular mucosa. In one specific embodiment, methods are
provided herein for treating and/or preventing mucositis that
comprises oral mucositis or oral stomatitis by administering the
compositions described herein. In another specific embodiment, the
mucosal disorder prevented or treated by the methods described
herein includes atrophic vaginitis, vaginal dryness, vaginal
burning, vaginal ulceration, vulvar burning), dyspareunia,
leukorrhea, and vulvar pruritus. In other certain embodiments, the
mucosal disorder comprises an oral ulceration, inflammatory bowel
disease (including Crohn's disease and ulcerative colitis),
periodontitis, interstitial cystitis, and a wound. In still other
embodiments, the methods provided herein are useful for treating
and/or preventing a dermal disease, disorder, or condition,
including but not limited to eczema, psoriasis, xerosis, erythema,
radical oxygen species-induced skin damage, and other dermal
diseases, conditions, and disorders, including other inflammatory
dermal disorders. In certain embodiments, the mucosal disorder
treatable by the methods and compositions described herein is
consequent to any one or more of hormone insufficiency, bone marrow
transplant, chemotherapy, radiation therapy, viral infection,
fungal infection, and bacterial infection. In a particular
embodiment, the mucosal disorder results from or is associated with
either one or both of chemotherapy and radiation therapy for
treatment of a head and neck tumor, Kaposi's sarcoma, a leukemia,
breast cancer, prostate cancer, pancreatic cancer, ovarian cancer,
melanoma, liver cancer, lung cancer, urinary cancer, and/or colon
cancer. In other specific embodiments, the methods described herein
may be used for treating hormone insufficiency, which in particular
embodiments is estrogen insufficiency that is consequent to,
associated with, or results from perimenopause or menopause.
[0137] Oral mucositis may be initiated by the cytotoxic effects of
chemotherapy and/or radiotherapy on the rapidly dividing epithelial
cells of the oropharyngeal mucosa, which is exacerbated by
infection with both endogenous oral flora and opportunistic
bacterial and fungal pathogens. Complications related to oral
mucositis vary in the different patient populations affected, but
include pain, poor oral intake with consequent dehydration and
weight loss, and systemic infection with organisms originating in
the oral cavity (Sonis, 1993b, supra). The pain associated with
oral mucositis may be severe requiring narcotic analgesics, and
difficulty in eating can result in patients receiving total
parenteral nutrition. The damaged oral epithelium of the oral
mucosa and defective immune response often found in these patients
offers a ready route for entry of organisms from the mouth into the
systemic circulation, and the potential for sepsis. Due to these
complications, oral mucositis can be a direct dose-limiting
toxicity of radiation or chemotherapy treatment, thereby resulting
in an inadequate cancer therapy.
[0138] Therapies used for treating tumors as well as the very
aggressive chemotherapy protocols used in bone marrow transplant
are associated with a high incidence of oral mucositis. Younger
patients seem to have an even higher incidence, which may be due to
their more rapid epithelial cell turnover and hence susceptibility
to cytotoxic drugs (Sonis 1993a, supra). Incidence of oral
mucositis is also related to the choice of chemotherapeutic agent,
with commonly used chemotherapeutic agents such as carmustine
(BCNU), chlorambucil (LEUKERAN), cisplatin (PLATINOL), cytarabine,
doxorubicin (ADRIAMYCIN), fluorouracil (5-FU), methoxetrate
(MEXATE) and plicamycin (MITHRACIN) being known for their direct
stomatotoxic potential (Sonis, 1993b, supra). The likelihood that a
patient receiving chemotherapy or radiotherapy will develop
mucositis is increased in patients who smoke, have poor oral or
dental health, use chewing tobacco, drink alcohol, are dehydrated,
and are suffering from underlying diseases such as kidney disease,
diabetes, or HIV/AIDS.
[0139] Localized topical application of agents that may be used to
treat oral disorders of mucosal epithelia such as oral mucositis
presents unique problems. For example, due to salivation and/or
food or fluid intake, oftentimes sufficient mucoadhesion and
residence time in the mouth may be difficult to attain for the
agent to be effective. Other difficulties associated with topical
oral application of drugs include tooth discoloration and patient
compliance. The oral formulations of the compositions described
herein provide good mucoadhesion and residence time in the mouth,
while at the same time providing high levels of patient
compliance.
[0140] Provided herein are methods for treating a mucosal disorder
such as oral mucositis comprising administering compositions that
comprise a mineral salt such as a zinc salt (e.g., zinc gluconate)
and a sulfonic acid (e.g., taurine) that may result in broad
spectrum reduction in mucositis (including reducing production of
inflammatory cytokines and reducing inflammation), antimicrobial
activity, reduction of pain, good stability, mucoadhesion and
residence time in the mouth, and high levels of patient compliance.
As a consequence of administering the compositions described herein
for treating oral mucositis, which may decrease inflammation and
promote restoration and healing of the mucous membrane including
minimizing or inhibiting occurrence of microlesions and ulceration,
the susceptibility of the mucous membrane to invasion and
colonization by microorganisms (such as Candida albicans (causative
agent of thrush)) may be decreased, thus preventing or decreasing
the likelihood of microbial infection, and/or decreasing the
recurrence and frequency of microbial infections.
[0141] Oral or gastrointestinal mucositis is a painful inflammation
and ulceration of a mucous membrane lining the digestive tract. The
pathophysiology of mucositis can be divided into 5 stages,
including an initiation phase, a message generation phase, a
signaling and amplification phase, an ulceration phase, and a
healing phase. The initiation phase is often associated with the
production of free radicals (caused by chemotherapy or
radiotherapy, for example), which damage the DNA of cells and
upregulate inflammatory cytokines. At the time when inflammatory
cytokines are upregulated marks the beginning of the ulceration
phase. The healing phase involves the movement of epithelial cells
to the site of the ulcer and the initiation of reepithelialization
of the ulcer. The methods described herein may be used for treating
(prophylactically or therapeutically) any one or a combination of
the pathophysiological stages of mucositis, including oral
mucositis, in a subject having or likely to have mucositis or
stomatitis (including intestinal cystitis).
[0142] Oral stomatitis is an inflammation of the mucous lining of
the structures of the mouth, including the cheeks, gums, tongue,
lips, throat, and roof or floor of the mouth. Stomatitis may be
caused by poor oral hygiene, poorly fitted dentures, or from mouth
burns from hot food or drinks, medications, allergic reactions,
radiation therapy, or infections. Iron deficiency anemia can also
lead to stomatitis, including irritation and fissuring in the
corners of the lips (e.g., angular stomatitis or angular
cheilitis).
[0143] Subjects with cancer (i.e., a malignancy) usually display
symptoms of mucositis (or stomatitis) four or five days after
beginning treatment (e.g., chemotherapy), reaching a peak at about
day 10 and slowly improving over the course of a few weeks.
Mucositis associated with administration of radiotherapy usually
appears at the end of the second week of treatment and may last for
six to eight weeks. Furthermore, recovery from mucositis may be
slowed and complicated by infection with one or more opportunistic
viruses, bacteria, or fungi that infect the sores or ulcerations
that are symptomatic of mucositis. A loss of taste perception (as a
result of mucositis) often makes eating more difficult for a
patient, which, in turn, leads to weight loss. Thus, in certain
embodiments, methods are provided for preventing, delaying the
onset of, reducing the duration of, or otherwise treating oral
mucositis by administering the compositions described herein.
[0144] Treatment of oral mucositis or oral stomatis, and any
associated or related inflammation, may be monitored throughout the
treatment regimen and may be evaluated at the end of treatment by
determining quantity of food and liquid intake by the subject,
weight loss or gain, evaluating pain, which may be determined
subjectively as well as objectively, for example, by monitoring use
of need for analgesics, including over the counter medications and
narcotics. Healing of tissue may be evaluated by a clinical
provider during the course of treatment according to methods and
standards in the medical art.
[0145] In other embodiments, methods are provided for treating or
preventing atrophic vaginitis that includes an inflammation of the
vagina, sometimes extending to the outer urinary tract, which may
be associated with a thinning or shrinking of mucosal tissues or
associated epithelial layers. A decrease in vaginal lubrication
(i.e., vaginal dryness) is often associated with atrophic
vaginitis.
[0146] Any one or more of a variety of causes (e.g., postmenopausal
estrogen reduction) may lead to the progressive development of
atrophic vaginitis. Throughout a woman's life cycle, the vaginal
epithelium undergoes changes in response to the level of
circulating estrogen. Stimulated by maternal estrogen, the vaginal
epithelium is rugated and rich in glycogen in the newborn. During
childhood, the epithelium remains thin until puberty, when it again
thickens as a result of estrogen stimulation. After menopause,
circulating estrogen levels (mainly estradiol) are dramatically
reduced from greater than 120 pg per ml to about 18 pg per ml.
Postmenopausal women may present numerous cytologic transformations
follow estrogen reduction, including proliferation of connective
tissue, fragmentation of elastin, and hyalinization of collagen.
These changes may result in granulation, fissures, ecchymoses,
telangiectases and ulcerations of the vaginal mucosa (Rigg L. A.,
Int. J. Fertil. 31:29-34, 1986). Postmenopausal changes in tissue
composition are not limited to the genital tract but also include
the urinary tract because of the shared common embryologic origin.
Therefore, both vaginal and urethral epithelia of associated mucosa
are estrogen dependent and adversely changed in an
estrogen-deprived environment.
[0147] Menopause is a leading cause of decreased levels of
circulating estrogen and is commonly associated with atrophic
vaginitis. A long-term decrease in estrogen stimulation is often
required before symptoms of atrophic vaginitis arise, an early sign
of which is a decrease in vaginal lubrication. However, even in
nonmenopausal women, production of ovarian estrogen can be
interrupted by radiation therapy, chemotherapy, immunologic
disorders, and oophorectomy. Side effects of antiestrogen
medications, such as medroxyprogesterone (PROVERA), tamoxifen
(NOLVADEX), danazol (DANOCRINE), leuprolide (LUPRON), and nafarelin
(SYNAREL), are also implicated as causes of atrophic vaginitis
(Beard, Postgrad. Med. 91:257-60, 1992). An increase in the
severity of symptoms occurs in women who are naturally
premenopausally estrogen deficient, smoke cigarettes, have not
given birth vaginally, receive radiotherapy or chemotherapy or
other medications described herein, have an immune disorder, have
had the ovaries removed, after pregnancy, lactating, or who exhibit
nonfluctuating levels of estrogen (Pandit et al., Am. J. Med. Sci.
314:228-31, 1997; Kalogeraki et al., In Vivo 10:597-600, 1996;
Dupont et al., Maturitas 13:297-311, 1991). Milder atrophy occurs
in postmenopausal women who participate in coitus, have higher
androgen levels, and have not undergone vaginal surgery (Pandit et
al., supra; Beard, supra; Kalogeraki, supra; Dupont et al., supra;
Leiblum et al., JAMA 249:2159-98, 1983). In addition, many younger
women and those in perimenopause (i.e., the menopausal transitional
period) may also experience periodic vaginal dryness and associated
problems.
[0148] Postmenopausal genital symptoms include dryness, burning,
dyspareunia, loss of vaginal secretions, leukorrhea, vulvar
pruritus, feeling of pressure, itching and yellow malodorous
discharge (Pandit et al., supra; Beard, supra; Mettler et al.,
Maturitas 14:23-31, 1991). Urinary symptoms of urethral discomfort,
frequency, hematuria, urinary tract infection, dysuria, and stress
incontinence may be presenting symptoms of atrophic vaginitis
(Pandit et al., supra; Beard, supra; Bachmann, Maturitas
22(Suppl):S1-5, 1995; Mettler et al., supra). Furthermore, many if
not all symptoms of atrophic vaginitis can be exacerbated by a
simultaneous infection of candidiasis, trichomoniasis or bacterial
vaginosis.
[0149] As the life expectancy of women increases, the impact of
vaginal dryness (which may precede as well as be a symptom of or
condition associated with atrophic vaginosis) on quality of life,
sexual functioning, and urogynecologic health is becoming more
evident in the day-to-day practice of medicine. Because women's
life expectancy is increasing, insult to the vaginal mucosa also
increases (e.g., vaginitis/vaginosis and exposure to medications
treating these conditions). In addition, the number of routine
physical examinations performed throughout a woman's life increase
as well, which can result in traumatization of vaginal mucosa
during digital or pelvic examination because of vaginal dryness.
Petechiae or small hemorrhages on the vaginal lining and evidence
of vaginal micro-lesions may also be observed. In addition, the
vaginal introitus may be narrowed; the epithelial surface (of the
vaginal mucosa) is typically very friable and may be ulcerated
(Rigg, Int. J. Fertil. 31:29-34, 1986).
[0150] A decrease in estrogen (estrogen insufficiency) associated
with post-menopausal women is a significant cause of atrophic
vaginitis. However, premenopausal women may also experience
significant discomfort associated with atrophic vaginitis, which
can be initiated as a side effect of radiotherapy or chemotherapy,
immune disorder, removal of ovaries, after pregnancy, during
lactation, or because of consumption of various medications such as
TOMOXAFEN, DANAZOL, MEDROXYPROGESTERONE, LEUPROLIDE, and NAFARELIN.
A decline in estrogen can cause a reduction of tissue mass, thus
increasing or contributing to declining health of the vaginal
tissue, which increases the likelihood that microlesions will
develop, which in turn, increase the susceptibility to microbial
infection. Presently available treatments received by women with
declining estrogen may exacerbate the declining integrity of
vaginal tissue and contribute to a cycle of declining vaginal
health. The conditions of atrophic vaginitis can be further
exacerbated by opportunistic infection by Gram-positive aerobic
microorganisms, Gram-negative aerobic micro-organisms,
Gram-positive anaerobic microorganisms, and Gram-negative anaerobic
microorganisms in the vaginal tract of pregnant and non-pregnant
women. Atrophic vaginitis is particularly exacerbated in
Gram-positive aerobic vaginitis caused by group B streptococci
(Streptococcus agalactiae).
[0151] The symptoms of atrophic vaginitis include vaginal soreness,
itching, painful intercourse and bleeding after intercourse, and
varying degrees of ulceration. Atrophic vaginitis is often
associated with a reduction in epithelial thickness of the vaginal
mucosa. Urinary symptoms of atrophic vaginitis include painful
urination, increased frequency, blood in urine, incontinence, and
an increase likelihood of microbial infection, which may result
from a change in vaginal pH.
[0152] Thus, in certain embodiments, methods are provided for
preventing, delaying the onset of, reducing the duration of, or
otherwise treating atrophic vaginitis by administering the
compositions described herein. As a consequence of administering
the compositions described herein for treating atrophic vaginitis,
which may decrease inflammation and promote restoration and healing
of the urogenital mucous membrane including minimizing or
inhibiting ulceration, the susceptibility of the mucous membrane to
invasion and colonization by microorganisms may be decreased, thus
preventing or decreasing the likelihood of microbial infection,
and/or decreasing the recurrence and frequency of vaginal
infections, including yeast infections, bacterial vaginosis,
aerobic vaginitis, and trichomonas.
[0153] The methods provided herein may be useful for treating any
one or more of the conditions that may, but not necessarily,
precede, indicate a predisposition to developing atrophic
vaginitis, are associated with or a symptom of atrophic vaginitis,
or result from or are consequent to atrophic vaginitis. In certain
embodiments, methods are provided for preventing, delaying the
onset of, reducing the duration of, reducing recurrence of, or
otherwise treating any one or more of genital dryness, itching, and
burning (e.g., vaginal dryness, vaginal burning, vaginal
ulceration, vulvar burning), dyspareunia, leukorrhea, and vulvar
pruritus by administering the compositions described herein
comprising a mineral salt and a sulfonic acid. The compositions
described herein may also be administered to maintain the pH of the
vagina, which is normally an acid pH (e.g., between pH 3.5 and 5)
or to reduce the pH of the vagina if the vaginal pH is greater than
the pH considered normal vaginal pH. The methods described herein
are also useful for treating these conditions when the etiology is
unrelated to atrophic vaginitis. Administration of the compositions
described herein over a period of time may thus, in general,
improve and maintain the health of the vaginal mucosa.
[0154] Without wishing to be bound by theory, methods described
herein comprising administering a composition comprising a mineral
salt of zinc and a sulfonic acid for the prevention or treatment of
mucosal disorders, such as oral mucositis or atrophic vaginitis,
may relate to a capability of the composition to down regulate
proinflammatory cytokines associated with these disorders or
conditions. For example, Mainnemare et al. (J. Dent. Res. 83(11):
823-831, 2004) found that intracellular taurine-N-monochloramine
(TauCl) in combination with hypochlorous acid (HOCl) appears to
play a crucial role in the periodontal inflammatory process by
neutralizing IL-6 and several metalloproteinases. Gurujeyalakshmi
et al. (J. Pharmacol. Exp. Ther. 293:82-9, 2000) reported that
taurine and niacin blocked lung injury and fibrosis by
down-regulating bleomycin-induced activation of NF-kB in mice.
Taurine and niacin attenuated the induced proinflammatory cytokines
IL-1.alpha., TNF.alpha., IL-6 and TGF.beta.. However, neither of
Mainnemare et al. or Gurujeyalakshmi et al. describe topically
applying a composition comprising a mineral salt (e.g., zinc
gluconate) and a sulfonic acid (e.g., taurine) in the treatment of
a mucosal disorder such as oral mucositis or atrophic
vaginitis.
[0155] In a large study of 631 women in prenatal care, Donder et
al. (BJOG 109: 34-43, 2002) defined a new class of abnormal vaginal
flora that is distinct from bacterial vaginosis: aerobic vaginitis.
Unlike bacterial vaginosis, aerobic vaginitis, characterized by
group B streptococci and E. coli aerobic microorganisms results in
a host immune response that leads to the production of IL-6,
IL-1.beta. and leukemia inhibitory factor in vaginal fluid. Donder
et al. suggest that aerobic vaginitis may be a better candidate
than bacterial vaginosis as the cause of pregnancy complications
and preterm delivery.
[0156] Doderlein's lactobacilli depend on glycogen from sloughed
vaginal cells (Pandit et al., Am. J. Med. Sci. 314:228-31, 1997),
which in turn produce lactic acid and thereby lower vaginal pH
levels to between 3.5 and 4.5. A vaginal pH from about 3.5 to 4.5
is essential for the body's natural defense against vaginal and
urinary tract infections (Semmens et al., JAMA 248:445-48, 1982).
Increased vaginal pH levels predispose the vagina to bacterial
infection by streptococci, staphylococci, coliforms and diphtheroid
(Pandit et al., supra). In fact, vaginal pH is typically greater
than 5.0 in patients with atrophic vaginitis due to opportunistic
bacterial infections. Thus, the methods provided herein for
preventing, delaying the onset of, reducing the duration of, or
otherwise treating vaginitis, vaginosis, and atrophic vaginitis by
administering the compositions described herein that are formulated
at an acid pH, such as between 3.5 and 4.5, may provide benefit to
affected subjects in need of such treatment.
[0157] In other embodiments, compositions described herein may be
used in methods for treating skin irritations (e.g., itching and
dry skin), treating skin (i.e., dermal) wounds, dermatitis, eczema,
and psoriasis. In still other embodiments, a composition comprising
a mineral salt (e.g., zinc gluconate) and a sulfonic acid (e.g.,
taurine) may be administered topically in a method for treating a
subject with a dermal disorder (e.g., a surface epithelium). Such a
disorder of a surface epithelium includes damage caused by burning
(e.g., chemical, electrical, radioactive or thermal burning).
Thermal burning may result from exposure to a flame, hot object, or
to steam or a hot liquid, for example. In still other embodiments,
the methods described herein may be used for topical application to
skin to reduce or decrease discomfort and/or to reduce decrease
visual size and/or color of varicose veins.
[0158] In still other embodiments, compositions described herein
may be used in methods for treating a mucosal disorder resulting
from the treatment (e.g., chemotherapy or radiation therapy) of a
malignancy. In still another embodiment, compositions described
herein may be used in methods for treating a dermal disorder (also
referred to as a skin disorder), which includes a chronic skin
disorder. Exemplary dermal disorders treatable with the
compositions described herein include, but are not limited to,
diaper rash, skin dryness, dermatitis, eczema, psoriasis, erythema,
acne, and xerosis, and other dermal diseases, conditions, and
disorders, including other inflammatory dermal disorders. The
methods and compositions described herein are thus useful for
promoting normal epithelial cell growth and healing of dermal
tissue, and which reduce colonization and infection by
microorganisms, such as bacteria, viruses, fungus, and yeast.
[0159] The methods and compositions described herein may also be
useful for treating tissue and cell damage caused by reactive
oxygen species in mammals, including humans. Free radicals such as
superoxide ions, hydroxy radicals, and oxides are known as a major
factor of degeneration and thus the ageing of the skin (see, e.g.,
U.S. Pat. No. 6,462,067). These free radicals effect destruction of
the proteins and lipids of the cellular membrane, affect the DNA,
and also cause decomposition of hyaluronic acid, a key substance of
the skin. Under normal biological conditions an equilibrium ratio
exists between generation of free radicals and their removal from a
cell by endogenous chemical or enzymatic systems. Additional
outside environmental stress factors such as pollution, tobacco
smoke, and ultraviolet radiation, for example, may overload these
inherent immune systems and shift the equilibrium in favor of the
free radicals Inflammation or ageing phenomena of the skin may
occur. Among principal enzymes that have an effect on aging
process, catalase, glutathione peroxidase, ascorbate peroxidase,
and superoxide dismutase are most important. The enhancement of
superoxide dismutase (SOD) activity as a method to control various
human ailments including aging has been studied extensively, for
example, by Dugas et al. (U.S. Pat. No. 6,426,068), Anggard et al.
(U.S. Pat. No. 6,455,542), Hellstrand et al. (U.S. Pat. Nos.
6,462,067; 6,407,133), Golz-Berner et al. (U.S. Pat. No.
6,426,080), and others. Approaches for treating skin conditions
related to oxygen radical tissue damage include design of
low-molecular weight SOD mimics (synzymes) that would mimic the
natural SOD enzyme in removing superoxide radical anion, [O2-.],
and the perhydroxyl radical, [HO2.], as well as preventing
formation of peroxynitrite anion, [ONO2-]. The methods described
herein provide an alternative method for treating oxygen radical
species-induced skin and tissue damage, by administering a
composition comprising a mineral salt and a sulfonic acid, which
are considered safe to administer to a human subject.
[0160] In other embodiments, the compositions described herein for
treating or preventing a dermal disorder may also be considered to
have cosmetic use. The compositions described herein may be used as
effective inhibitors of UV rays in topical sunscreens, and in
treating dandruff and other scalp conditions. In still other
embodiments, the compositions described herein may be used in
preventing or treating diaper rash and skin dryness. As exemplified
herein, cosmetic uses of a composition comprising a mineral salt
(e.g., zinc gluconate) and a sulfonic acid (e.g., taurine) may be
suitable in particular shampoos, scalp creams, deodorizing
body-cleansing compositions, care compositions and emulsions. In
addition, a composition described herein may be used in hair
rinses, hair treatments, hair regeneration products, hair tonics,
blow-drying lotions, hairsprays, styling creams, styling gels, hair
oils, hair pomades, products modulating hair brilliance, shower
preparations, soaps, liquid soaps, deodorant sticks, deodorant
spray, emulsions. Products comprising a composition described
herein may applied transiently (i.e., for a short period of time)
or applied for a longer period of time (e.g., as a leave-in or
leave-on hair care product). Additional cosmetic uses include foot
sprays or creams, antibacterial face washes, and body lotions.
[0161] In certain other embodiments, methods are provided for
treating a viral infection. In one embodiment, the virus causing
the infection is a herpes simplex virus. In another embodiment,
methods are provided for treating a viral infection such as caused
by herpes zoster that is characterized by painful rash and
blisters. Zinc salts irreversibly inhibit herpes virus replication
in vitro and are effective in treating herpes infections in vivo.
Herpes of the lips occurs in 50% of the population, and genital
herpes is one of the most common sexually transmitted diseases.
Zinc ions irreversibly inhibit herpes simplex virus (HSV)
glycoprotein functions by accumulating in the sulfhydryl groups of
glycoprotein B in the viral membrane, blocking synthesis of DNA. In
the closely related rhinovirus, research scientists have theorized
that free zinc ions may sequester in the membrane, inhibiting viral
binding with ICAM receptor sites in mucous membranes.
[0162] The genome and encoded polypeptides of Herpes simplex virus
(HSV) share significant homology with Varicella-Zoster virus (VSV)
(a pox virus). Varicella-Zoster virus is the cause of chickenpox
and shingles. Eruptions of herpes zoster associated with shingles
are thought to be more frequent in the elderly not because of
immune dysfunction, but because of slowed mobilization of the
immune system. Prompt treatment with the compositions described
herein may be beneficial by decreasing the viral load and reducing
painful lesions independent of immune system activation. Moreover,
the compositions described herein provide an alternative therapy
that is less expensive than current standard of care
(administration of an anti-viral such as acyclovir, valacylovir,
and famciclovir with or without a steroid), and that has more than
a palliative effect such as use of calamine lotion. In certain
embodiments, the methods provided herein for treating or preventing
a viral infection, such as an infection caused by Herpes Simplex
Virus or Varicella zoster virus, comprise administering a
composition comprising a mineral salt (e.g., zinc gluconate) and a
sulfonic acid (e.g., taurine) and an amino acid; in certain
particular embodiments, the amino acid is lysine.
[0163] In certain other embodiments, methods are provided herein
for supplementing a mineral deficiency in a subject, that is,
treating a subject who has a mineral insufficiency or deficiency or
who is in need of mineral supplementation. By way of example,
administration of a composition comprising a mineral required for
enzyme function, for example, zinc, may promote healing by
providing the mineral to the cells of the tissue such that enzymes,
cofactors, and other cellular substituents that require the mineral
may properly function. In one embodiment, a method is provided
wherein the method comprises administering a composition described
herein that comprises a mineral salt, a sulfonic acid, and one or
more of 0.05% to 3.0% (w/w) glycyrrhetinic acid; 0.04% to 15% (w/w)
polyvinylpyrrolidone (PVP); 0.01% to 5.0% (w/w) hyaluronic acid;
and 0.05% to 3.0% (w/w) glycerin (or 0.05% to 5.0% (w/w) glycerin).
In particular embodiments, the mineral insufficiency or deficiency
is related to lower than normal levels (i.e., a normal level refers
to the level or amount of a mineral that is available for normal
cellular and biological function in the absence of disease or
dysfunction) of any one or more of zinc, calcium, magnesium,
manganese, cobalt, chromium, selenium, vanadium, copper, iron,
nickel, silicon, boron, arsenic, molybdenum, sodium, potassium,
phosphorus, sulfur, chlorine, fluorine, iodine, and lithium. In
certain other embodiments, the mineral moiety of the mineral salt
included in the compositions described herein is any one of zinc,
calcium, iron, copper, magnesium, manganese, cobalt, chromium,
selenium, and vanadium. The mineral may be in the form of a
gluconate, acetate, ascorbate, or sulfate salt. In still another
specific embodiment, the sulfonic acid is taurine. In a certain
embodiment, methods comprise administering a composition comprising
0.25% (w/w) to 5.5% (w/w) zinc gluconate and 0.25% (w/w) to 30%
(w/w) taurine. In other particular embodiments, the compositions
that are useful for supplementing a mineral deficiency or treating
a subject who has a mineral deficiency comprise a mineral salt
(e.g., zinc gluconate), a sulfonic acid (e.g., taurine), and
further comprise one or more of a flavoring agent, a mucoadhesive
agent, a pH adjusting agent, a solubilizing agent, a viscosity
modulating agent, and a stabilizing agent. These compositions may
comprise at least one of 0.05% to 3.0% (w/w) glycyrrhetinic acid;
0.04% to 15% (w/w) polyvinylpyrrolidone (PVP); 0.01% to 5.0% (w/w)
hyaluronic acid; and 0.05% to 3.0% (w/w) glycerin (or 0.05% to 5%
(w/w) glycerin), and lactoferrin.
[0164] The physiologically acceptable compositions described herein
may be administered in a manner appropriate to the disease to be
treated (or prevented) as determined by persons skilled in the
medical arts. Treatment of a subject or patient refers to the
medical management of the disease, disorder, or condition (see,
e.g., Stedman's Medical Dictionary). An appropriate dose and a
suitable duration and frequency of administration will be
determined by such factors as the condition of the patient, the
type and severity of the patient's disease, the particular form of
the active ingredient(s), and the method of administration. In
general, an appropriate dose and treatment regimen provides the
composition(s) in an amount sufficient to provide therapeutic
and/or prophylactic benefit (e.g., an improved clinical outcome,
such as more frequent complete or partial remissions, longer
disease-free status (i.e., decreasing the likelihood or the
propensity that a subject will present symptoms on the basis of
which a diagnosis of a disease is made); and/or overall survival;
decrease in the number of symptoms presented, or a lessening of
symptom severity; decrease or abrogation of pain; improved quality
of life). By way of example, treatment of mucositis may be
effected, in part, by decreasing the number and/or size of lesions,
reducing or decreasing the time to which one or more lesions heal,
and/or reducing or eliminating associated pain, and/or by
decreasing or reducing production of inflammatory cytokines and
other inflammatory factors. With respect to oral mucositis,
treatment may be indicated, in part, by the ability of the patient
to retain or regain all or partial ability to taste food and drink.
For prophylactic use, a dose should be sufficient to prevent, delay
the onset of, or diminish the severity of a dermal or mucosal
disease or disorder or a symptom, condition, or sequelae thereof
(which includes, for example, inflammation). A "therapeutically
effective amount" or "effective amount" means an amount of an
active pharmaceutical ingredient, composition or formulation, or
agent that is sufficient, in the subject (e.g., a mammal) in need
thereof and to which it is administered, to treat (i.e.,
effectively manage) or prevent (i.e., decrease or reduce the
likelihood of occurrence of) the stated disease, disorder, or
condition.
[0165] Optimal doses may generally be determined using experimental
models and/or clinical trials. The optimal dose may depend upon the
body mass, weight, or blood volume of the patient. The use of the
minimum dosage that is sufficient to provide effective therapy is
usually preferred. Patients may generally be monitored for
therapeutic or prophylactic effectiveness using assays and methods
suitable for the condition being treated or prevented, which
assays, methods, and techniques are discussed herein will be
familiar to those having ordinary skill in the art. For subjects
who receive the compositions described herein according to the
methods described herein, monitoring likely includes clinical
observations, personal history (e.g., to determine comfort or pain,
quality of life, ability to eat and drink without discomfort or
pain), and physical examination.
[0166] For example, for methods comprising administration of the
compositions described herein for treating any one or more of the
mucosal diseases, disorders, or conditions affecting the urogenital
tract of women, particularly the vaginal mucosa, a multicenter,
open, non-controlled study may be designed to evaluate safety in a
composition described herein is applied vaginally to about ten
women for ten, twenty, thirty, thirty-five or more days.
Alternatively or in addition, a study evaluating about 100
post-menopausal women may be initiated, with vaginal application of
the composition daily, weekly, or bi-weekly. An endpoint of such
studies may include the level of vaginal dryness, measured
according to standard methods such as a visual analogue scale.
Other endpoints may include evaluation and assessment of itching,
burning, dysareunia, vaginal inflammation and edema and rash may
also be assessed by a four-point scale, which may include
determination of vaginal abrasions and disepithelialisation.
[0167] Papanicolaon ("Pap") smears may be obtained from the upper
one-third lateral vaginal wall in the routine manner for maturation
index, and evaluated to determine the ratio of superficial,
intermediate, and parabasal cells. As necessary, biopsy specimens
may be obtained from the upper one-third of the vagina using a
Kevorkian forceps after application of topical 4% lidocaine.
Specimens for histology may be evaluated for epithelial thickness
and the presence or absence of glycogen, which is a measure of
vaginal epithelial maturation. Changes that may indicate
improvement in atrophic vaginitis (e.g., vaginal epithelial
thickness, maturation index, vaginal pH, or severity of bothersome
self-assessed symptom(s)) can be measured or otherwise quantified
over a suitable period of time (e.g., 6-12 weeks or longer). In
parallel, the mean change in vaginal pH between baseline and end of
treatment may be calculated by measuring the vaginal pH, for
example, by inserting a standardized pH paper into the vagina and
comparing the results to the manufacturer's color chart.
[0168] A maturation index of a vaginal mucosa can be measured at
the beginning (baseline) and at end of treatment (patient's last
visit), which is determined by counting the number of parabasal,
intermediate, and superficial cells and calculating the percentage
of each cell type. The percentages are then used in the following
equation to determine the maturation index: Maturation Index=(%
Parabasal cells X 0)+(% Intermediate cells X 0.5)+(% Superficial
cells X 1.0). The most bothersome symptoms of a patient can be
identified by the patient from a list of (for example) the seven
different symptoms of atrophic vaginitis measured at the baseline
visit. Such symptoms include one or more of (1) vaginal dryness;
(2) vaginal irritation/itching; (3) vaginal soreness; (4)
difficulty passing urine; (5) frequent urination; (6) pain during
intercourse; and (7) bleeding after intercourse.
[0169] Overall, a method of treating a mucosal disorder as
described herein, such as atrophic vaginitis and other vaginal
mucosal diseases, disorders, and conditions, is intended to be
safe, tolerated, and effective. With regard to atrophic vaginitis,
effectiveness includes a statistically significant increase or
biologically significant increase in the maturation index or a
statistically significant, clinically significant, or biologically
significant decrease in vaginal pH, or reduction in the severity of
a most bothersome symptom. The reduction and severity of symptoms
may be determined subjectively and may be determined objectively by
metrics for determining quality of life that are familiar to
persons skilled in the art.
[0170] Other assays and techniques that may be used for evaluating
the effect of treatment using a composition described herein
include in vitro cell culture assay systems routinely practiced by
persons skilled in the relevant art. For example, normal human
vaginal epithelial cells, such as a cell line commercially
available from Clonetics (NHVE 5164), may be subcultured in basal
PrEBM (Clonetics CC 3165) using 96 well plates at 37.degree. C., 5%
CO.sub.2. Cells may be exposed to medium containing various
concentrations of the composition used for treatment. In parallel,
appropriate controls are included, such as media of control cells
that is devoid of such composition. Cell proliferation and/or
viability may then be determined at various times according to
methods routinely practiced in the art.
[0171] Inflammation and the inflammatory response, including
cytokine induction and production can be determined by methods
routinely practiced in the art. The increased or decreased level of
inflammatory factors and cytokines in a biological sample obtained
from the subject before, during, and or after treatment may be
readily determined by methods and assays described herein and
practiced routinely in the art to monitor the effect of treatment.
An immune response in a host or subject may be determined by any
number of well-known immunological techniques and methods with
which those having ordinary skill in the art will be readily
familiar. Such assays include, but need not be limited to, in vivo
or in vitro determination of soluble antibodies, soluble mediators
such as cytokines (e.g., IL-6, IL-1.beta., leukemia inhibitory
factor, TNF-.alpha., IFN-.gamma., IL-2, IL-4, IL-10, IL-12, and
TGF-.beta.), lymphokines, chemokines, hormones, growth factors, and
the like, as well as other soluble small peptide, carbohydrate,
nucleotide and/or lipid mediators; cellular activation state
changes as determined by altered functional or structural
properties of cells of the immune system, for example cell
proliferation, altered motility, induction of specialized
activities such as specific gene expression or cytolytic behavior;
cellular differentiation by cells of the immune system, including
altered surface antigen expression profiles or the onset of
apoptosis (programmed cell death). Procedures for performing these
and similar assays are may be found, for example, in Lefkovits
(Immunology Methods Manual: The Comprehensive Sourcebook of
Techniques, 1998). See also Current Protocols in Immunology; Weir,
Handbook of Experimental Immunology, Blackwell Scientific, Boston,
Mass. (1986); Mishell and Shigii (eds.) Selected Methods in
Cellular Immunology, Freeman Publishing, San Francisco, Calif.
(1979); Green and Reed, Science 281:1309 (1998) and references
cited therein).
[0172] The compositions described herein may exhibit broad spectrum
reduction in mucositis; and may exhibit anti-inflammatory activity,
antimicrobial activity, reduction of pain, good stability,
mucoadhesion, and when used in oral applications may provide
adequate residence time in the mouth and high levels of patient
compliance. As described in detail herein, compositions
(pharmaceutically or physiologically acceptable) and methods of
using the compositions are provided herein for treating dermal or
mucosal disorders including side effects of radiation therapy
and/or chemotherapy associated with the treatment of head and neck
tumors and that also occurs in about 90% of children with leukemia.
Such side effects include oral mucositis (including micro-lesions),
and oral stomatitis. Such side effects (also called adverse
effects) of chemotherapy or radiotherapy treatment of any one or
more of a wide variety of solid or non-solid cancers (i.e.,
malignancies) or lymphomas (for example breast, prostate,
pancreatic, ovarian, melanoma, liver, lung, urinary, and colon
cancers; Kaposi's sarcoma) may also result in a mucosal disorder of
any one or more mucosa including oral mucosa, intestinal mucosa,
rectal mucosa, and the like. The compositions and methods described
herein may also be used for preventing or treating a mucosal
disorder such as atrophic vaginitis, vaginal micro-lesions,
inflammatory bowel disease (including Crohn's disease and
ulcerative colitis), eczema, psoriasis, periodontitis, interstitial
cystitis, wound healing, or an inflammatory condition, dyspareunia,
burning, leucorrhea, xerosis, vaginal dryness, vulvar pruritus,
vaginal pruritus, vulvar burning, vaginal burning, vulvar
dystrophy, vaginal malodor, candidiasis, trichomoniasis or
bacterial vaginosis, and urinary disorders such as dysuria,
hematuria, frequency, stress incontinence and tract infection,
among other symptoms, including menopausal sexual dysfunction,
complications resulting from antiestrogen medications, viral
infections including shingles, herpes simplex, HIV/AIDS; and
chronic skin disorders such as eczema, psoriasis and dermatitis,
irritation due to oral surgery, aging and traumatic ulcers caused
by braces or ill fitting dentures, diffuse aphthous ulcers,
medication, or disease.
[0173] A subject (host or patient) in need of treatment as
described herein may be a human or may be a non-human primate or
other animal (i.e., veterinary use) who is afflicted with a dermal
or mucosal disorder and has developed symptoms of a dermal or
mucosal disease, disorder, or condition, or who may be free of
detectable disease but is at risk for developing a dermal or
mucosal disease, disorder, or condition. Accordingly, the
compositions described herein may be administered to a subject who
has an existing disease, or the treatment may be prophylactic,
administered to a subject who is at risk for developing the disease
or condition. Examples of non-human primates and other animals
include but are not limited to farm animals, pets, and zoo animals
(e.g., horses, cows, buffalo, llamas, goats, rabbits, cats, dogs,
chimpanzees, orangutans, gorillas, monkeys, elephants, bears, large
cats, etc.). In certain embodiments, the subject is a human. In
other certain embodiments, the compositions and methods described
herein are excluded from veterinary use (i.e., use in any non-human
animal).
[0174] A "biological sample" as used herein may be a blood sample
(from which serum or plasma may be prepared), biopsy specimen, body
fluids (e.g., lung lavage, ascites, mucosal washings, synovial
fluid), bone marrow, lymph nodes, tissue explant, organ culture, or
any other tissue or cell preparation from a subject or a biological
source. A sample may further refer to a tissue or cell preparation
in which the morphological integrity or physical state has been
disrupted, for example, by dissection, dissociation,
solubilization, fractionation, homogenization, biochemical or
chemical extraction, pulverization, lyophilization, sonication, or
any other means for processing a sample derived from a subject or
biological source. In certain embodiments, the subject or
biological source may be a human or non-human animal, a primary
cell culture (e.g., immune cells, virus infected cells), or culture
adapted cell line, including but not limited to, genetically
engineered cell lines that may contain chromosomally integrated or
episomal recombinant nucleic acid sequences, immortalized or
immortalizable cell lines, somatic cell hybrid cell lines,
differentiated or differentiatable cell lines, transformed cell
lines, and the like.
[0175] A derivative of a chemical compound (e.g., of a sulfonic
acid or mineral salt) is structurally similar to a parent compound
and (actually or theoretically) derivable from that parent
compound. As used herein, derivatives include pharmaceutically
acceptable derivatives of a compound used in the compositions
described herein, which derivatives include salts, esters, enol
ethers, enol esters, acetals, ketals, orthoesters, hemiacetals,
hemiketals, acids, bases, solvates, hydrates or prodrugs thereof.
Such derivatives may be readily prepared by those of skill in this
art using known methods for such derivatization. The compounds
produced may be administered to animals or humans without
substantial toxic effects and are either pharmaceutically active or
are prodrugs.
[0176] Also provided herein are methods of manufacturing the
compositions described herein. Such methods comprise formulating
the compositions described herein comprising a mineral salt and a
sulfonic acid and which may further comprise lactoferrin with other
agents, excipients, and diluents as described herein. The
manufacturing methods may further comprise adjusting the pH of the
composition, and placing the composition in a suitable container
for delivery to a health care provider or pharmacist.
EXAMPLES
Example 1
Bactericidal Activity of Compositions Comprising Zinc Gluconate and
Taurine
[0177] This example illustrates the microbicidal activity of a
composition comprising zinc gluconate and taurine.
[0178] GelX.TM. ORAL GEL was formulated as a viscous gel comprised
of purified water, polyvinyl pyrrolidone (PVP), taurine, zinc
gluconate, PEG-40 hydrogenated castor oil, sodium saccharin, sodium
hydroxide, and flavoring. GelX.TM. ORAL GEL has a mechanical action
which provides pain relief by adhering to the mucosal surface of
the mouth and throat soothing lesions.
[0179] Antimicrobial activity of a composition comprising zinc
gluconate and taurine was determined by a method typically used to
demonstrate whether one or more agents may be included as a
preservative in a composition formulated under conditions such that
the composition will be considered a non-sterile composition
according to regulatory authorities.
[0180] The minimum inhibitory concentration of solutions containing
differing amounts of zinc gluconate and taurine (adjusted to a
neutral pH with sodium hydroxide) were determined according to a
method referred to as the Challenge Test that meets the
requirements according to the Italian X Pharmacopoeia, which
requirements are similar to U.S. requirements.
[0181] Testing was performed with solutions comprising 2% (w/w)
zinc gluconate and 2% (w/w) taurine; 1% (w/w) zinc gluconate and 1%
(w/w) taurine; 0.5% (w/w) zinc gluconate and 1% (w/w) taurine;
0.25% (w/w) zinc gluconate and 0.5% (w/w) taurine; and 0.1% (w/w)
zinc gluconate and 0.1% (w/w) taurine. The inoculum of the
microorganism used for the study is based on the presence of CFU of
the microorganism per gram GelX.TM. ORAL GEL.
[0182] The acceptance criteria are defined in terms of decay of the
number of microorganisms. [0183] Microbiological Total Viable Count
Before Challenge Test: [0184] Total Aerobic Mesophilic Bacteria
Count: <10 CFU/1 g [0185] Mold and Yeast Total Count: <10
CFU/1 g
[0186] Five different ATCC (Manassas, Va.) microbial strains were
evaluated for microbial growth decay at four different intervals
(48 hours, 7 days, 14 days and 28 days). The inoculum of the five
microbial strains for each of the solutions tested is provided in
the following Table. One set of experiments was performed to
determine the antimicrobial activity of zinc gluconate/taurine at
0.5%/1.0% (w/w), and a second set of experiments were performed to
determine the antimicrobial activity of zinc gluconate/taurine at
four different ratios as indicated in Table 1 below.
TABLE-US-00001 TABLE 1 Antimicrobial Activity of Zinc Gluconate and
Taurine Compositions 0.1%/0.1% 0.25%/0.5% Zn 1.0%/1.0%
gluconate/taurine 0.5%/1.0% 2.0%/2.0% (w/w) (%) INOCULUM INOCULUM
Microorganism (CFU/g product) (CFU/g product) Staphylococcus aureus
8.80 .times. 10.sup.6 1.67 .times. 10.sup.6 ATCC 6538 Pseudomonas
aeruginosa 2.10 .times. 10.sup.6 2.02 .times. 10.sup.7 ATCC 9027
Escherichia coli 1.00 .times. 10.sup.7 5.35 .times. 10.sup.6 ATCC
8739 Candida albicans 1.20 .times. 10.sup.7 9.80 .times. 10.sup.6
ATCC 10231 Aspergillus niger 4.30 .times. 10.sup.6 1.20 .times.
10.sup.6 ATCC 16404
[0187] Five plates of the product tested were prepared, and each
test sample was inoculated with a different microbial stump. The
inoculated samples were maintained at 20.degree. C.-25.degree. C.
and protected from light between intervals of inoculation. At the
prescribed time intervals (48 hours, 7 days, 14 days, and 28 days),
samples of 1 g/ml were taken from each inoculated sample.
[0188] Aliquots from the inoculated samples were applied to Petri
dishes, which were incubated at 32.degree. C. (bacteria) or at
20.degree. C. (molds and yeast) for a time sufficient to allow
microbial growth of (3 days for bacteria, 5-7 days for molds and
yeast). The number of countable colonies was corrected for the
proscribed dilution factor giving the number of CFU (Colony Forming
Units) per gram of product. Samples were prepared at 48 hours, 7
days, 14 days and 28 days, to evaluate the trend of microbial
growth. The cultures were routinely observed through the course of
the weeks for which each sample was under study, and the
differences in colony population for each microorganism were noted
and recorded. Antimicrobial activity was also described in terms of
log reduction of the number of viable microorganisms compared to
the inoculated number of microorganisms.
[0189] Solutions at each of 2% (w/w) zinc gluconate and 2% (w/w)
taurine; 1% (w/w) zinc gluconate and 1% (w/w) taurine; and 0.5%
(w/w) zinc gluconate and 1% (w/w) taurine exhibited a significant
reduction of microorganisms (CFU<10, or undetectable) present in
each solution sample taken at each time point for each
microorganism. Accordingly, the log reduction for each sample
containing 0.5% zinc gluconate and 1.0% taurine was as follows:
6.94 for S. aureus inoculated samples, 6.32 for P. aeruginosa
inoculated samples, 7.00 for E. coli inoculated samples, 7.08 for
C. albicans inoculated samples, and 6.63 for A. niger inoculated
samples. The log reduction for each sample containing 1.0% zinc
gluconate/1.0% taurine and for each sample containing 2% zinc
gluconate/2.0% taurine was as follows: 7.22 for S. aureus
inoculated samples, 7.30 for P. aeruginosa inoculated samples, 6.73
for E. coli inoculated samples, 6.99 for C. albicans inoculated
samples, and 6.08 for A. niger inoculated samples.
[0190] Solutions at each of 0.25% (w/w) zinc gluconate and 0.5%
(w/w) taurine and 0.1% (w/w) zinc gluconate and 0.1% (w/w) taurine
exhibited a significant reduction of microorganisms (CFU<10, or
undetectable) present in each solution sample taken at each time
point for each of S. aureus, P. aeruginosa, C. albicans, and A.
niger. With respect to CFUs determined for E. coli at each time
point in samples containing 0.1% (w/w) zinc gluconate and 0.1%
(w/w) taurine, 10 CFUs E. coli were detected for each of 48 hours,
7, 14, and 28 days. For samples containing 0.25% (w/w) zinc
gluconate and 0.5% (w/w) taurine, 10 E. coli CFUs were detected in
samples at 48 hours and at 7 days but the number of bacteria in
samples evaluated at 14 days and 28 days was <10 or
undetectable.
Example 2
Treatment of Oral Aphthous Stomatitis
[0191] This example describes the effectiveness of using a
composition comprising zinc gluconate and taurine and polyvinyl
pyrrolidone (PVP) for treatment of oral aphthous stomatitis.
[0192] A painful ulcer inside the oral cavity caused by a rupture
of the membrane is defined by the term aphthous. An aphthous ulcer
is also called a canker sore. When multiple aphthous ulcers occur
and/or when the condition is chronic (or recurrent), the condition
is called aphthous stomatitis (see, e.g., Natah et al., Int. J.
Oral. Maxillofac. Surg. 33:221-34 (2004)).
[0193] Over a period of eleven months, in a dentist's surgery in
Italy, 150 patients who were affected by minor aphthous were
recruited to participate in a clinical study. The patients (87
female and 63 male) were between the ages of 18 and 71 and provided
informed consent to participate. The patients were randomly divided
into five groups, thirty people each. [0194] Group A: Patients were
treated with 100% pure Vitamin E (Vea Oil, Hulka srl, Rovigo,
Italy. [0195] Group B: Patients were treated with a gel containing
an Aloe Vera extract (ALOVEX gel, Recordati spA, Milan, Italy).
[0196] Group C: Patients were not prescribed any treatment and
formed a non-treatment control group. [0197] Group D: Patients were
treated with a BMG0902-03 gel, (BMG Pharma, Gardnerville, Nev.; lot
101108). The BMG0902-03 gel is viscous gel containing 12% w/w
polyvinyl pyrrolidone (PVP), 1% (w/w) taurine, 0.5% (w/w) zinc
gluconate, and also containing PEG-40 hydrogenated castor oil,
sodium saccharin, sodium hydroxide, flavoring, and purified water.
[0198] Group E: Patients were treated with a product containing
only Vitamin A associated with other polymers.
[0199] The patients were advised to apply the assigned product 4
times per day (after main meals and in the evening before going to
bed) and were advised to avoid eating or drinking for at least an
hour after application. The patients in groups A, B, D and E were
asked to cover the entire sore with the prescribed gel. All
patients were scheduled for a follow-up visit seven days after
beginning treatment. After the required seven days, patients who
did not report to the dentists' office were excluded from the
study. Excluded were five patients from group A; nine from group B;
five from group C; seven from group D; and five from group E.
Therefore, patients who completed the study included 25 from group
A; 21 from group B; 25 from group C; 23 from group D; and 25 from
group E.
[0200] Clinical progress was evaluated based on the presence of
oral ulcers and the number and distribution of the sores. A
photographic record was made at the first visit and at the check-up
seven days later. Healing was defined for the purpose of this study
as (1) absence of painful and burning symptoms; and (2) absence of
ulcerous lesions. The results were evaluated according to the
following scoring system: complete healing (+++); lesion present,
but asymptomatic (++); reduction by half of symptomatology (+);
persistence of the symptoms and signs of aphthous ulcer (-); side
effects ({circumflex over ( )}). The data are presented in Table
2.
TABLE-US-00002 TABLE 2 Treatment of Minor Aphthous Ulcers Group A
Group B Group C Group D Group E Complete Healing 85% 80% 40% 90%
20% (+++) lesion present, but 13% 15% 20% 5% 30% asymptomatic (++)
reduction by half of 2% 5% 10% 5% 20% symptomatology (+)
persistence of 0 0 30% 0 30% symptoms and signs (-) side effects
({circumflex over ( )}) 0 0 0 0 0
Example 3
Inhibition of IL-6 and IL-8 Production in Cells by Zinc and
Taurine
[0201] This example describes the capability of zinc (e.g., zinc
gluconate) in combination with taurine to inhibit production of
cytokines, IL-6 and IL-8, in cells stimulated by either
lipopolysaccharide (LPS) or doxorubicin, a chemotherapeutic
agent.
[0202] CaCo2 cells (human intestinal (colonic) epithelial cells)
were grown in culture in 24-well tissue culture dishes according to
methods routinely practiced in the cell culture art. Zinc gluconate
and taurine were combined at varying concentrations and added to
the cells in the absence and presence of proinflammatory agents
(lipopolysaccharide, doxorubicin, dextran-sulfate sodium (DSS)). In
one series, zinc gluconate and taurine were combined with
lipopolysaccharide (LPS) (1 .mu.g/ml) for six hours. In a second
series, zinc gluconate and taurine were combined with doxorubicin
(3 .mu.M) for 24 hours. In a third series, zinc gluconate and
taurine were combined with DSS (5% w/w) for 24 hours. For detecting
inhibition of IL-6 or IL-8 production in the cells stimulated with
each of the pro-inflammatory agents, the zinc/taurine (Z/T)
concentrations (mM) were as follows: Z/T: 0.1/0.8; Z/T: 0.3/2.4;
Z/T: 1/8; Z/T: 3/24; Z/T: 10/80; and Z/T: 30/240. After
stimulation, the cell supernatants were harvested. The presence of
IL-6 and IL-8 in cell supernatants from cells stimulated with the
agents was determined using commercially available reagents for
detection of each according to the manufacturer's instructions
(ORGENIUM ELISA kits, Anibiotech Oy, Orgenium Laboratories
Division, Vantaa, Finland). Inhibition of production of IL-6 and
IL-8 by zinc gluconate and taurine in cells stimulated with LPS is
presented in FIG. 1 and FIG. 2, respectively. The data are
presented in FIGS. 1 and 2. Inhibition of production of IL-8 by
zinc gluconate and taurine in cells stimulated with doxorubicin is
presented in FIG. 3. In CaCo-2 cells to which DSS was added, IL-8
production was inhibited to background levels at each of the
zinc/taurine concentrations tested.
[0203] In a second set of experiments, CaCo-2 cells were stimulated
with LPS in the presence of zinc gluconate alone (0.15 mM, 0.2 mM,
and 0.3 mM), taurine alone (1.2 mM, 1.6 mM, and 2.4 mM), and both
zinc gluconate (Z) and taurine (T) (Z/T: 0.15 mM/1.2 mM; Z/T: 0.2
mM/1.6 mM; and Z/T: 0.3 mM/2.4 mM). The level of IL-8 (pg/ml) in
the cell supernatants was then determined as described above. The
results are presented in FIG. 4.
Example 4
Treatment of Mucositis with Zinc and Taurine
[0204] This example describes treatment of five patients who had
radiation therapy-induced-mucositis with a muco-adhesive gel
containing 0.5% (w/w) zinc gluconate and 1.0% (w/w) taurine.
[0205] Five patients with head or neck cancer, each of whom had
moderate to severe mucositis, were treated with a muco-adhesive gel
containing 0.5% (w/w) zinc gluconate and 1.0% taurine (w/w) on an
open label basis. Each patient was being treated for recurrent
disease, and each had previously been diagnosed with radiation
therapy-induced mucositis during a previous treatment.
[0206] The patients were provided with the muco-adhesive gel in
each of two different presentations: (1) 450 ml bottles with 15 ml
dispensing cups, and (2) 150 ml bottle with a long neck spray tip
to be able to reach specific lesions. Patients were instructed to
use the product as needed at least 3 but not more than 5 times each
day, including preferably one hour prior to meals.
[0207] Observations of the effects of the treatment were made by
the treating physician at approximately seven days after initiating
treatment; observations included incorporation of patient
statements. Following are the results of this open label study.
[0208] (1) Two patients deemed to have severe oral mucositis were
observed to have clinically significant reductions in the extent of
their lesions, including a notable reduction in common indications
of inflammation, such as edema. No new lesions occurred in either
patient even as radiation therapy continued. Each of these two
patients reported significant and increasingly sustained reductions
of pain associated with their lesions, and each reported an
increased ability to eat and drink, which had been problematic for
each during the peak of their mucositis symptoms.
[0209] (2) One additional patient was observed to have the same or
similar reduction in the general symptoms of mucositis.
Additionally, this patient reported the return of his ability to
taste food after only three days of treatment, a faculty previously
lost during treatment. The return of this ability was accompanied
by an increase in saliva production, which had been dramatically
reduced as a consequence of radiation therapy.
[0210] (3) Two patients deemed to have moderate mucositis with
lesions toward the back of their mouths and throats used localized
therapy with the spray tip package. Each reported near immediate
reduction of pain and enhanced ability to swallow. Clinical
observations were consistent with the three other patients (see (1)
and (2) above) with regard to a reduction of severity of the
lesions and observable characteristics of inflammation.
Example 5
Treatment of Dermal Conditions with Zinc and Taurine
[0211] This example describes treatment of dermal conditions with
compositions comprising zinc gluconate and taurine.
[0212] (1) Thermal Injury. An adult woman experienced a severe burn
with boiling water that resulted in immediate skin blistering,
erythema, and intense pain. The burn affected approximately 70% of
her left breast, the entire nipple, and portions of her left
abdomen. Within 5 minutes of the injury, a zinc gluconate (0.5%
w/w) and taurine (1.0% w/w) aqueous gel composition was applied to
the injured area. An additional component of the gel included 1%
(w/w) PVP. Fifteen minutes later an additional application of the
gel was applied. When the skin was examined 30 minutes after the
injury, nearly all erythema had dissipated, resulting in a skin
tone and color nearly identical to adjacent non-injured skin, and
the pain associated with the injury had resolved.
[0213] (2) Diaper Dermatitis. A two year old child experienced
diaper rash, redness, and irritation. The child was treated with a
gel containing zinc gluconate (2.5% w/w) and taurine (5% w/w) for
the condition. Twelve hours after the first application, most of
the redness and irritation had disappeared. The child was treated
again the following day, once in the morning and again in the
evening. The following day the child's condition resolved.
[0214] (3) Intertrigo (inflammation/rash of a body fold). A woman
developed a skin fold infection that presented as a painful,
inflamed, reddish-brown rash. The rash was treated with a gel
containing zinc gluconate (2.5% w/w) and taurine (5% w/w), twice a
day over a three day period. After the treatment, the rash
resolved.
Example 6
Treatment of Oral Mucositis Associated with Head and Neck
Cancer
[0215] This example provides a study of oral mucositis associated
with radiotherapy, chemotherapy, or a combination of radiotherapy
and chemotherapy for the treatment of head and neck cancer. This
study evaluates any one or more of, for example, the onset of oral
mucositis, pain, severity of oral mucositis, or decrease in the
time to resolution of oral mucositis. This study includes a
treatment period lasting through the duration of radiotherapy
and/or chemotherapy, continuing until resolution of oral mucositis.
This study involves 4 groups of subjects receiving one of the
following formulations: (1) Placebo (sterile water), (2) a GelX.TM.
Oral Gel (medium viscosity) (0.5% w/w zinc gluconate, 1.0% w/w
taurine and 4.0% w/w PVP), (3) GelX Oral Gel (high viscosity) (0.5%
w/w zinc gluconate, 1.0% w/w taurine and 8.0% w/w PVP), or (4) a
GelX Oral Gel 4.times. (medium viscosity) (2.0% w/w zinc gluconate,
4.0% w/w taurine and 4.0% w/w PVP).
[0216] Formulations to be evaluated are administered locally as a
mouth rinse for a period of time such as 30 seconds, one minute, or
1.5 minutes. Administration is once, twice or three times a day or
as needed, with drinking and eating withheld for at least thirty
minutes thereafter.
[0217] Results are evaluated based upon any one or more of: (1)
time of onset of oral mucositis, (2) time to resolution of oral
mucositis, (3) mouth soreness, (4) percentage of subjects
developing oral mucositis, (5) safety, (6) average number of doses
administered, and (7) dry mouth. Results of the study are evaluated
using the WHO Oral Mucositis Assessment Scale, according to Table 3
below.
TABLE-US-00003 TABLE 3 WHO Oral Mucositis Assessment Scale Grade 0
1 2 3 4 None Soreness Erythema, Ulcers with Mucositis to and/or
ulcers, and extensive extent that erythema patient can erythema and
alimentation is swallow patient cannot not possible solid food
swallow solid food
[0218] The above criteria are evaluated at least on the basis of
pain (1) in general, (2) of the mouth, and (3) of the throat; and
for saliva based upon (1) swallowing, (2) amount, (3)
consistency.
[0219] All U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications
and non-patent publications referred to in this specification
and/or listed in the Application Data Sheet, are incorporated
herein by reference, each in their entirety.
[0220] From the foregoing a person skilled in the art will
appreciate that, although specific embodiments have been described
herein for purposes of illustration, various modifications may be
made. Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, many
equivalents to the specific embodiments described herein. Such
equivalents are intended to be encompassed by the following claims.
In general, in the following claims, the terms used should not be
construed to limit the claims to the specific embodiments disclosed
in the specification and the claims, but should be construed to
include all possible embodiments along with the full scope of
equivalents to which such claims are entitled. Accordingly, the
claims are not limited by the disclosure.
Sequence CWU 1
1
201711PRTHomo sapiens 1Met Lys Leu Val Phe Leu Val Leu Leu Phe Leu
Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Gly Arg Arg Arg Arg Ser Val
Gln Trp Cys Ala Val Ser 20 25 30Gln Pro Glu Ala Thr Lys Cys Phe Gln
Trp Gln Arg Asn Met Arg Lys 35 40 45Val Arg Gly Pro Pro Val Ser Cys
Ile Lys Arg Asp Ser Pro Ile Gln 50 55 60Cys Ile Gln Ala Ile Ala Glu
Asn Arg Ala Asp Ala Val Thr Leu Asp65 70 75 80Gly Gly Phe Ile Tyr
Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro 85 90 95Val Ala Ala Glu
Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr 100 105 110Tyr Ala
Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu 115 120
125Leu Gln Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly
130 135 140Trp Asn Val Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp
Thr Gly145 150 155 160Pro Pro Glu Pro Ile Glu Ala Ala Val Ala Arg
Phe Phe Ser Ala Ser 165 170 175Cys Val Pro Gly Ala Asp Lys Gly Gln
Phe Pro Asn Leu Cys Arg Leu 180 185 190Cys Ala Gly Thr Gly Glu Asn
Lys Cys Ala Phe Ser Ser Gln Glu Pro 195 200 205Tyr Phe Ser Tyr Ser
Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly 210 215 220Asp Val Ala
Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp225 230 235
240Glu Ala Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg
245 250 255Lys Pro Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val
Pro Ser 260 265 270His Ala Val Val Ala Arg Ser Val Asn Gly Lys Glu
Asp Ala Ile Trp 275 280 285Asn Leu Leu Arg Gln Ala Gln Glu Lys Phe
Gly Lys Asp Lys Ser Pro 290 295 300Lys Phe Gln Leu Phe Gly Ser Pro
Ser Gly Gln Lys Asp Leu Leu Phe305 310 315 320Lys Asp Ser Ala Ile
Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser 325 330 335Gly Leu Tyr
Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg 340 345 350Lys
Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys 355 360
365Ala Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu
370 375 380Ser Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu
Asp Cys385 390 395 400Ile Ala Leu Val Leu Lys Gly Glu Ala Asp Ala
Met Ser Leu Asp Glu 405 410 415Gly Tyr Val Tyr Thr Ala Gly Lys Cys
Gly Leu Val Pro Val Leu Ala 420 425 430Glu Asn Tyr Lys Ser Gln Gln
Ser Ser Asp Pro Asp Pro Asn Cys Val 435 440 445Asp Arg Pro Val Glu
Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser 450 455 460Asp Thr Ser
Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His465 470 475
480Thr Ala Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu
485 490 495Phe Asn Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser
Gln Ser 500 505 510Cys Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys
Ala Leu Cys Ile 515 520 525Gly Asp Glu Gln Gly Glu Asn Lys Cys Val
Pro Asn Ser Asn Glu Arg 530 535 540Tyr Tyr Gly Tyr Thr Gly Ala Phe
Arg Cys Leu Ala Glu Asn Ala Gly545 550 555 560Asp Val Ala Phe Val
Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly 565 570 575Asn Asn Asn
Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala 580 585 590Leu
Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser 595 600
605Cys His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp
610 615 620Lys Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala
Lys Phe625 630 635 640Gly Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe
Cys Leu Phe Gln Ser 645 650 655Glu Thr Lys Asn Leu Leu Phe Asn Asp
Asn Thr Glu Cys Leu Ala Arg 660 665 670Leu His Gly Lys Thr Thr Tyr
Glu Lys Tyr Leu Gly Pro Gln Tyr Val 675 680 685Ala Gly Ile Thr Asn
Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu 690 695 700Ala Cys Glu
Phe Leu Arg Lys705 7102711PRTHomo sapiens 2Met Lys Leu Val Phe Leu
Val Leu Leu Phe Leu Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Gly Arg
Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser 20 25 30Gln Pro Glu Ala
Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Arg 35 40 45Val Arg Gly
Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln 50 55 60Cys Ile
Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp65 70 75
80Gly Gly Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro
85 90 95Val Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His
Tyr 100 105 110Tyr Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln
Leu Asn Glu 115 120 125Leu Gln Gly Leu Lys Ser Cys His Thr Gly Leu
Arg Arg Thr Ala Gly 130 135 140Trp Asn Val Pro Ile Gly Thr Leu Arg
Pro Phe Leu Asn Trp Thr Gly145 150 155 160Pro Pro Glu Ser Ile Glu
Ala Ala Val Ala Arg Phe Phe Ser Ala Ser 165 170 175Cys Val Pro Gly
Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu 180 185 190Cys Ala
Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro 195 200
205Tyr Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly
210 215 220Asp Val Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu
Ser Asp225 230 235 240Glu Ala Glu Arg Asp Glu Tyr Glu Leu Leu Cys
Pro Asp Asn Thr Arg 245 250 255Lys Pro Val Asp Lys Phe Lys Asp Cys
His Leu Ala Arg Val Pro Ser 260 265 270His Ala Val Val Ala Arg Ser
Val Asn Gly Lys Glu Asp Ala Ile Trp 275 280 285Asn Leu Leu Arg Gln
Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro 290 295 300Lys Phe Gln
Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe305 310 315
320Lys Asp Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser
325 330 335Gly Leu Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn
Leu Arg 340 345 350Lys Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg
Val Val Trp Cys 355 360 365Ala Val Gly Glu Gln Glu Leu Arg Lys Cys
Asn Gln Trp Ser Gly Leu 370 375 380Ser Glu Gly Ser Val Thr Cys Ser
Ser Ala Ser Thr Thr Glu Asp Cys385 390 395 400Ile Ala Leu Val Leu
Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly 405 410 415Gly Tyr Val
Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala 420 425 430Glu
Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val 435 440
445Asp Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser
450 455 460Asp Thr Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser
Cys His465 470 475 480Thr Ala Val Asp Arg Thr Ala Gly Trp Asn Ile
Pro Met Gly Leu Leu 485 490 495Phe Asn Gln Thr Gly Ser Cys Lys Phe
Asp Glu Tyr Phe Ser Gln Ser 500 505 510Cys Ala Pro Gly Ser Asp Pro
Arg Ser Asn Leu Cys Ala Leu Cys Ile 515 520 525Gly Asp Glu Gln Gly
Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg 530 535 540Tyr Tyr Gly
Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asp Ala Gly545 550 555
560Asp Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly
565 570 575Asn Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp
Phe Ala 580 585 590Leu Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr
Glu Ala Arg Ser 595 600 605Cys His Leu Ala Met Ala Pro Asn His Ala
Val Val Ser Arg Met Asp 610 615 620Lys Val Glu Arg Leu Lys Gln Val
Leu Leu His Gln Gln Ala Lys Phe625 630 635 640Gly Arg Asn Gly Ser
Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser 645 650 655Glu Thr Lys
Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg 660 665 670Leu
His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val 675 680
685Ala Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu
690 695 700Ala Cys Glu Phe Leu Arg Lys705 7103711PRTHomo sapiens
3Met Lys Leu Val Phe Leu Val Leu Leu Phe Leu Gly Ala Leu Gly Leu1 5
10 15Cys Leu Ala Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val
Ser 20 25 30Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met
Arg Arg 35 40 45Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser
Pro Ile Gln 50 55 60Cys Ile Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala
Val Thr Leu Asp65 70 75 80Gly Gly Phe Ile Tyr Glu Ala Gly Leu Ala
Pro Tyr Lys Leu Arg Pro 85 90 95Val Ala Ala Glu Val Tyr Gly Thr Glu
Arg Gln Pro Arg Thr His Tyr 100 105 110Tyr Ala Val Ala Val Val Lys
Lys Gly Gly Ser Phe Gln Leu Asn Glu 115 120 125Leu Gln Gly Leu Lys
Ser Cys His Thr Gly Leu Arg Arg Asn Ala Gly 130 135 140Trp Asn Val
Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly145 150 155
160Pro Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser
165 170 175Cys Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys
Arg Leu 180 185 190Cys Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser
Ser Gln Glu Pro 195 200 205Tyr Phe Ser Tyr Ser Gly Ala Phe Lys Cys
Leu Arg Asp Gly Ala Gly 210 215 220Asp Val Ala Phe Ile Arg Glu Ser
Thr Val Phe Glu Asp Leu Ser Asp225 230 235 240Glu Ala Glu Arg Asp
Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg 245 250 255Lys Pro Val
Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser 260 265 270His
Ala Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp 275 280
285Asn Leu Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro
290 295 300Lys Phe Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu
Leu Phe305 310 315 320Lys Asp Ser Ala Ile Gly Phe Ser Arg Val Pro
Pro Arg Ile Asp Ser 325 330 335Gly Leu Tyr Leu Gly Ser Gly Tyr Phe
Thr Ala Ile Gln Asn Leu Arg 340 345 350Lys Ser Glu Glu Glu Val Ala
Ala Arg Arg Ala Arg Val Val Trp Cys 355 360 365Ala Val Gly Glu Gln
Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu 370 375 380Ser Glu Gly
Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys385 390 395
400Ile Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly
405 410 415Gly Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val
Leu Ala 420 425 430Glu Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp
Pro Asn Cys Val 435 440 445Asp Arg Pro Val Glu Gly Tyr Leu Ala Val
Ala Val Val Arg Arg Ser 450 455 460Asp Thr Ser Leu Thr Trp Asn Ser
Val Lys Gly Lys Lys Ser Cys His465 470 475 480Thr Ala Val Asp Arg
Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu 485 490 495Phe Asn Gln
Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser 500 505 510Cys
Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile 515 520
525Gly Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg
530 535 540Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asp
Ala Gly545 550 555 560Asp Val Ala Phe Val Lys Gly Val Thr Val Leu
Gln Asn Thr Asp Gly 565 570 575Asn Asn Asn Glu Ala Trp Ala Lys Asp
Leu Lys Leu Ala Asp Phe Ala 580 585 590Leu Leu Cys Leu Asp Gly Lys
Arg Lys Pro Val Thr Glu Ala Arg Ser 595 600 605Cys His Leu Ala Met
Ala Pro Asn His Ala Val Val Ser Arg Met Asp 610 615 620Lys Val Glu
Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe625 630 635
640Gly Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser
645 650 655Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu
Ala Arg 660 665 670Leu His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly
Pro Gln Tyr Val 675 680 685Ala Gly Ile Thr Asn Leu Lys Lys Cys Ser
Thr Ser Pro Leu Leu Glu 690 695 700Ala Cys Glu Phe Leu Arg Lys705
7104708PRTBos taurus 4Met Lys Leu Phe Val Pro Ala Leu Leu Ser Leu
Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Ala Pro Arg Lys Asn Val Arg
Trp Cys Thr Ile Ser Gln 20 25 30Pro Glu Trp Phe Lys Cys Arg Arg Trp
Gln Trp Arg Met Lys Lys Leu 35 40 45Gly Ala Pro Ser Ile Thr Cys Val
Arg Arg Ala Phe Ala Leu Glu Cys 50 55 60Ile Arg Ala Ile Ala Glu Lys
Lys Ala Asp Ala Val Thr Leu Asp Gly65 70 75 80Gly Met Val Phe Glu
Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val 85 90 95Ala Ala Glu Ile
Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr 100 105 110Ala Val
Ala Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu 115 120
125Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
130 135 140Val Ile Pro Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr
Glu Ser145 150 155 160Leu Glu Pro Leu Gln Gly Ala Val Ala Lys Phe
Phe Ser Ala Ser Cys 165 170 175Val Pro Cys Ile Asp Arg Gln Ala Tyr
Pro Asn Leu Cys Gln Leu Cys 180 185 190Lys Gly Glu Gly Glu Asn Gln
Cys Ala Cys Ser Ser Arg Glu Pro Tyr 195 200 205Phe Gly Tyr Ser Gly
Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp 210 215 220Val Ala Phe
Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys225 230 235
240Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala
245 250 255Pro Val Asp Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro
Ser His 260 265 270Ala Val Val Ala Arg Ser Val Asp Gly Lys Glu Asp
Leu Ile Trp Lys 275 280 285Leu Leu Ser Lys Ala Gln Glu Lys Phe Gly
Lys Asn Lys Ser Arg Ser 290 295 300Phe Gln Leu Phe Gly Ser Pro Pro
Gly Gln Arg Asp Leu Leu Phe Lys305 310 315 320Asp Ser Ala Leu Gly
Phe Leu Arg Ile Pro Ser Lys Val Asp Ser Ala 325 330 335Leu Tyr Leu
Gly
Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu 340 345 350Thr Ala
Glu Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala 355 360
365Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser
370 375 380Gly Gln Asn Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp
Cys Ile385 390 395 400Val Leu Val Leu Lys Gly Glu Ala Asp Ala Leu
Asn Leu Asp Gly Gly 405 410 415Tyr Ile Tyr Thr Ala Gly Lys Cys Gly
Leu Val Pro Val Leu Ala Glu 420 425 430Asn Arg Lys Thr Ser Lys Tyr
Ser Ser Leu Asp Cys Val Leu Arg Pro 435 440 445Thr Glu Gly Tyr Leu
Ala Val Ala Val Val Lys Lys Ala Asn Glu Gly 450 455 460Leu Thr Trp
Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val465 470 475
480Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln
485 490 495Thr Gly Ser Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys
Ala Pro 500 505 510Gly Arg Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys
Ala Gly Asp Asp 515 520 525Gln Gly Leu Asp Lys Cys Val Pro Asn Ser
Lys Glu Lys Tyr Tyr Gly 530 535 540Tyr Thr Gly Ala Phe Arg Cys Leu
Ala Glu Asp Val Gly Asp Val Ala545 550 555 560Phe Val Lys Asn Asp
Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr 565 570 575Ala Asp Trp
Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys 580 585 590Leu
Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu 595 600
605Ala Val Ala Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala
610 615 620His Val Lys Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly
Lys Asn625 630 635 640Gly Lys Asn Cys Pro Asp Lys Phe Cys Leu Phe
Lys Ser Glu Thr Lys 645 650 655Asn Leu Leu Phe Asn Asp Asn Thr Glu
Cys Leu Ala Lys Leu Gly Gly 660 665 670Arg Pro Thr Tyr Glu Glu Tyr
Leu Gly Thr Glu Tyr Val Thr Ala Ile 675 680 685Ala Asn Leu Lys Lys
Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala 690 695 700Phe Leu Thr
Arg7055708PRTBos taurus 5Met Lys Leu Phe Val Pro Ala Leu Leu Ser
Leu Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Ala Pro Arg Lys Asn Val
Arg Trp Cys Thr Ile Ser Gln 20 25 30Pro Glu Trp Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu 35 40 45Gly Ala Pro Ser Ile Thr Cys
Val Arg Arg Ala Phe Ala Leu Glu Cys 50 55 60Ile Pro Gly Ile Ala Glu
Lys Lys Ala Asp Ala Val Thr Leu Asp Gly65 70 75 80Gly Met Val Phe
Glu Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val 85 90 95Ala Ala Glu
Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr 100 105 110Ala
Val Ala Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu 115 120
125Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
130 135 140Ile Ile Pro Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr
Glu Ser145 150 155 160Leu Glu Pro Leu Gln Gly Ala Val Ala Lys Phe
Phe Ser Ala Ser Cys 165 170 175Val Pro Cys Ile Asp Arg Gln Ala Tyr
Pro Asn Leu Cys Gln Leu Cys 180 185 190Lys Gly Glu Gly Glu Asn Gln
Cys Ala Cys Ser Ser Arg Glu Pro Tyr 195 200 205Phe Gly Tyr Ser Gly
Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp 210 215 220Val Ala Phe
Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys225 230 235
240Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala
245 250 255Pro Val Asp Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro
Ser His 260 265 270Ala Val Val Ala Arg Ser Val Asp Gly Lys Glu Asp
Leu Ile Trp Lys 275 280 285Leu Leu Ser Lys Ala Gln Glu Lys Ser Gly
Lys Asn Lys Ser Arg Ser 290 295 300Phe Gln Leu Phe Gly Ser Pro Pro
Gly Gln Arg Asp Leu Leu Phe Lys305 310 315 320Asp Ser Ala Leu Gly
Phe Leu Arg Ile Pro Ser Lys Val Asp Ser Ala 325 330 335Leu Tyr Leu
Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu 340 345 350Thr
Ala Glu Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala 355 360
365Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser
370 375 380Gly Gln Asn Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp
Cys Ile385 390 395 400Val Leu Val Leu Lys Gly Glu Ala Asp Ala Leu
Asn Leu Asp Gly Gly 405 410 415Tyr Ile Tyr Thr Ala Gly Lys Cys Gly
Leu Val Pro Val Leu Ala Glu 420 425 430Asn Arg Lys Ser Ser Lys His
Ser Ser Leu Asp Cys Val Leu Arg Pro 435 440 445Thr Glu Gly Tyr Leu
Ala Val Ala Val Val Lys Lys Ala Asn Glu Gly 450 455 460Leu Thr Trp
Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val465 470 475
480Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln
485 490 495Thr Gly Ser Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys
Ala Pro 500 505 510Gly Ala Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys
Ala Gly Asp Asp 515 520 525Gln Gly Leu Asp Lys Cys Val Pro Asn Ser
Lys Glu Lys Tyr Tyr Gly 530 535 540Tyr Thr Gly Ala Phe Arg Cys Leu
Ala Glu Asp Val Gly Asp Val Ala545 550 555 560Phe Val Lys Asn Asp
Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr 565 570 575Ala Asp Trp
Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys 580 585 590Leu
Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu 595 600
605Ala Val Ala Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala
610 615 620His Val Lys Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly
Lys Asn625 630 635 640Gly Lys Asn Cys Pro Asp Lys Phe Cys Leu Phe
Lys Ser Glu Thr Lys 645 650 655Asn Leu Leu Phe Asn Asp Asn Thr Glu
Cys Leu Ala Lys Leu Gly Gly 660 665 670Arg Pro Thr Tyr Glu Glu Tyr
Leu Gly Thr Glu Tyr Val Thr Ala Ile 675 680 685Ala Asn Leu Lys Lys
Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala 690 695 700Phe Leu Thr
Arg7056708PRTBos taurus 6Met Lys Leu Phe Val Pro Ala Leu Leu Ser
Leu Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Ala Pro Arg Lys Asn Val
Arg Trp Cys Thr Ile Ser Gln 20 25 30Pro Glu Trp Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu 35 40 45Gly Ala Pro Ser Ile Thr Cys
Val Arg Arg Ala Phe Ala Leu Glu Cys 50 55 60Ile Arg Ala Ile Ala Glu
Lys Lys Ala Asp Ala Val Thr Leu Asp Gly65 70 75 80Gly Met Val Phe
Glu Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val 85 90 95Ala Ala Glu
Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr 100 105 110Ala
Val Ala Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu 115 120
125Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
130 135 140Ile Ile Pro Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr
Glu Ser145 150 155 160Leu Glu Pro Leu Gln Gly Ala Val Ala Lys Phe
Phe Ser Ala Ser Cys 165 170 175Val Pro Cys Ile Asp Arg Gln Ala Tyr
Pro Asn Leu Cys Gln Leu Cys 180 185 190Lys Gly Glu Gly Glu Asn Gln
Cys Ala Cys Ser Ser Arg Glu Pro Tyr 195 200 205Phe Gly Tyr Ser Gly
Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp 210 215 220Val Ala Phe
Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys225 230 235
240Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala
245 250 255Pro Val Asp Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro
Ser His 260 265 270Ala Val Val Ala Arg Ser Val Asp Gly Lys Glu Asp
Leu Ile Trp Lys 275 280 285Leu Leu Ser Lys Ala Gln Glu Lys Phe Gly
Lys Asn Lys Ser Arg Ser 290 295 300Phe Gln Leu Phe Gly Ser Pro Pro
Gly Gln Arg Asp Leu Leu Phe Lys305 310 315 320Asp Ser Ala Leu Gly
Phe Leu Arg Ile Pro Ser Lys Val Asp Ser Ala 325 330 335Leu Tyr Leu
Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu 340 345 350Thr
Ala Glu Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala 355 360
365Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser
370 375 380Gly Gln Asn Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp
Cys Ile385 390 395 400Val Leu Val Leu Lys Gly Glu Ala Asp Ala Leu
Asn Leu Asp Gly Gly 405 410 415Tyr Ile Tyr Thr Ala Gly Lys Cys Gly
Leu Val Pro Val Leu Ala Glu 420 425 430Asn Arg Lys Ser Ser Lys His
Ser Ser Leu Asp Cys Val Leu Arg Pro 435 440 445Thr Glu Gly Tyr Leu
Ala Val Ala Val Val Lys Lys Ala Asn Glu Gly 450 455 460Leu Thr Trp
Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val465 470 475
480Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln
485 490 495Thr Gly Ser Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys
Ala Pro 500 505 510Gly Ala Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys
Ala Gly Asp Asp 515 520 525Gln Gly Leu Asp Lys Cys Val Pro Asn Ser
Lys Glu Lys Tyr Tyr Gly 530 535 540Tyr Thr Gly Ala Phe Arg Cys Leu
Ala Glu Asp Val Gly Asp Val Ala545 550 555 560Phe Val Lys Asn Asp
Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr 565 570 575Ala Asp Trp
Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys 580 585 590Leu
Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu 595 600
605Ala Val Ala Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala
610 615 620His Val Lys Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly
Lys Asn625 630 635 640Gly Lys Asn Cys Pro Asp Lys Phe Cys Leu Phe
Lys Ser Glu Thr Lys 645 650 655Asn Leu Leu Phe Asn Asp Asn Thr Glu
Cys Leu Ala Lys Leu Gly Gly 660 665 670Arg Pro Thr Tyr Glu Glu Tyr
Leu Gly Thr Glu Tyr Val Thr Ala Ile 675 680 685Ala Asn Leu Lys Lys
Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala 690 695 700Phe Leu Thr
Arg7057708PRTBos taurus 7Met Lys Leu Phe Val Pro Ala Leu Leu Ser
Leu Gly Ala Leu Gly Leu1 5 10 15Cys Leu Ala Ala Pro Arg Lys Asn Val
Arg Trp Cys Thr Ile Ser Gln 20 25 30Pro Glu Trp Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu 35 40 45Gly Ala Pro Ser Ile Thr Cys
Val Arg Arg Ala Phe Ala Leu Ala Cys 50 55 60Ile Arg Ala Ile Ala Glu
Lys Lys Ala Asp Ala Val Thr Leu Asp Gly65 70 75 80Gly Met Val Phe
Glu Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val 85 90 95Ala Ala Glu
Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr 100 105 110Ala
Val Ala Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu 115 120
125Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
130 135 140Val Ile Pro Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr
Glu Ser145 150 155 160Leu Glu Pro Leu Gln Gly Ala Val Ala Lys Phe
Phe Ser Ala Ser Cys 165 170 175Val Pro Cys Ile Asp Arg Gln Ala Tyr
Pro Asn Leu Cys Gln Leu Cys 180 185 190Lys Gly Glu Gly Glu Asn Gln
Cys Ala Cys Ser Ser Arg Glu Pro Tyr 195 200 205Phe Gly Tyr Ser Gly
Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp 210 215 220Val Ala Phe
Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys225 230 235
240Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala
245 250 255Pro Val Asp Ala Phe Lys Glu Tyr His Leu Ala Gln Val Pro
Ser His 260 265 270Pro Val Val Ala Arg Ser Val Asp Ala Lys Glu Asp
Leu Ile Trp Lys 275 280 285Leu Leu Arg Lys Ala Gln Glu Lys Phe Gly
Lys Asn Lys Ser Arg Ser 290 295 300Phe Gln Leu Phe Gly Ser Pro Pro
Gly Gln Arg Asp Leu Leu Phe Lys305 310 315 320Asp Ser Ala Leu Gly
Phe Leu Arg Ile Pro Ser Lys Val Asp Ser Ala 325 330 335Leu Tyr Leu
Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu 340 345 350Thr
Ala Glu Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala 355 360
365Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser
370 375 380Gly Gln Asn Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp
Cys Ile385 390 395 400Val Leu Val Leu Lys Gly Glu Ala Asp Ala Leu
Asn Leu Asp Gly Gly 405 410 415Tyr Val Tyr Thr Ala Gly Lys Cys Gly
Leu Val Pro Val Leu Ala Glu 420 425 430Asn Arg Lys Ser Ser Lys His
Ser Ser Leu Asp Cys Val Leu Arg Pro 435 440 445Thr Glu Gly Tyr Leu
Ala Val Ala Val Val Arg Lys Ala Asn Glu Gly 450 455 460Leu Thr Trp
Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val465 470 475
480Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln
485 490 495Thr Gly Ser Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys
Ala Pro 500 505 510Gly Ala Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys
Ala Gly Asp Asp 515 520 525Gln Gly Leu Asp Lys Cys Val Pro Asn Ser
Lys Glu Lys Tyr Tyr Gly 530 535 540Tyr Thr Gly Ala Phe Arg Cys Leu
Ala Glu Asp Val Gly Asp Val Ala545 550 555 560Phe Val Lys Asn Asp
Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr 565 570 575Ala Asp Trp
Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys 580 585 590Leu
Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu 595 600
605Ala Val Ala Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala
610 615 620His Val Lys Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly
Lys Asn625 630 635 640Gly Lys Asn Cys Pro Asp Lys Phe Cys Leu Phe
Lys Ser Glu Thr Lys 645 650 655Asn Leu Leu Phe Asn Asp Asn Thr Glu
Cys Leu Ala Lys Leu Gly Gly 660 665 670Arg Pro Thr Tyr Glu Glu Tyr
Leu Gly Thr Glu Tyr Val Thr Ala Ile 675 680 685Ala
Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala 690 695
700Phe Leu Thr Arg7058692PRTHomo sapiens 8Gly Arg Arg Arg Arg Ser
Val Gln Trp Cys Ala Val Ser Gln Pro Glu1 5 10 15Ala Thr Lys Cys Phe
Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly 20 25 30Pro Pro Val Ser
Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile Gln 35 40 45Ala Ile Ala
Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly Phe 50 55 60Ile Tyr
Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala Ala65 70 75
80Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr Ala Val
85 90 95Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu Gln
Gly 100 105 110Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly
Trp Asn Val 115 120 125Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp
Thr Gly Pro Pro Glu 130 135 140Pro Ile Glu Ala Ala Val Ala Arg Phe
Phe Ser Ala Ser Cys Val Pro145 150 155 160Gly Ala Asp Lys Gly Gln
Phe Pro Asn Leu Cys Arg Leu Cys Ala Gly 165 170 175Thr Gly Glu Asn
Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr Phe Ser 180 185 190Tyr Ser
Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val Ala 195 200
205Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala Glu
210 215 220Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys
Pro Val225 230 235 240Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val
Pro Ser His Ala Val 245 250 255Val Ala Arg Ser Val Asn Gly Lys Glu
Asp Ala Ile Trp Asn Leu Leu 260 265 270Arg Gln Ala Gln Glu Lys Phe
Gly Lys Asp Lys Ser Pro Lys Phe Gln 275 280 285Leu Phe Gly Ser Pro
Ser Gly Gln Lys Asp Leu Leu Phe Lys Asp Ser 290 295 300Ala Ile Gly
Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly Leu Tyr305 310 315
320Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys Ser Glu
325 330 335Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala
Val Gly 340 345 350Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly
Leu Ser Glu Gly 355 360 365Ser Val Thr Cys Ser Ser Ala Ser Thr Thr
Glu Asp Cys Ile Ala Leu 370 375 380Val Leu Lys Gly Glu Ala Asp Ala
Met Ser Leu Asp Glu Gly Tyr Val385 390 395 400Tyr Thr Ala Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr 405 410 415Lys Ser Gln
Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg Pro 420 425 430Val
Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr Ser 435 440
445Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala Val
450 455 460Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe
Asn Gln465 470 475 480Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser
Gln Ser Cys Ala Pro 485 490 495Gly Ser Asp Pro Arg Ser Asn Leu Cys
Ala Leu Cys Ile Gly Asp Glu 500 505 510Gln Gly Glu Asn Lys Cys Val
Pro Asn Ser Asn Glu Arg Tyr Tyr Gly 515 520 525Tyr Thr Gly Ala Phe
Arg Cys Leu Ala Glu Asn Ala Gly Asp Val Ala 530 535 540Phe Val Lys
Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn Asn Asn545 550 555
560Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu Cys
565 570 575Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys
His Leu 580 585 590Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met
Asp Lys Val Glu 595 600 605Arg Leu Lys Gln Val Leu Leu His Gln Gln
Ala Lys Phe Gly Arg Asn 610 615 620Gly Ser Asp Cys Pro Asp Lys Phe
Cys Leu Phe Gln Ser Glu Thr Lys625 630 635 640Asn Leu Leu Phe Asn
Asp Asn Thr Glu Cys Leu Ala Arg Leu His Gly 645 650 655Lys Thr Thr
Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala Gly Ile 660 665 670Thr
Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Glu 675 680
685Phe Leu Arg Lys 6909692PRTHomo sapiens 9Gly Arg Arg Arg Arg Ser
Val Gln Trp Cys Ala Val Ser Gln Pro Glu1 5 10 15Ala Thr Lys Cys Phe
Gln Trp Gln Arg Asn Met Arg Arg Val Arg Gly 20 25 30Pro Pro Val Ser
Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile Gln 35 40 45Ala Ile Ala
Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly Phe 50 55 60Ile Tyr
Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala Ala65 70 75
80Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr Ala Val
85 90 95Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu Gln
Gly 100 105 110Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly
Trp Asn Val 115 120 125Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp
Thr Gly Pro Pro Glu 130 135 140Ser Ile Glu Ala Ala Val Ala Arg Phe
Phe Ser Ala Ser Cys Val Pro145 150 155 160Gly Ala Asp Lys Gly Gln
Phe Pro Asn Leu Cys Arg Leu Cys Ala Gly 165 170 175Thr Gly Glu Asn
Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr Phe Ser 180 185 190Tyr Ser
Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val Ala 195 200
205Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala Glu
210 215 220Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys
Pro Val225 230 235 240Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val
Pro Ser His Ala Val 245 250 255Val Ala Arg Ser Val Asn Gly Lys Glu
Asp Ala Ile Trp Asn Leu Leu 260 265 270Arg Gln Ala Gln Glu Lys Phe
Gly Lys Asp Lys Ser Pro Lys Phe Gln 275 280 285Leu Phe Gly Ser Pro
Ser Gly Gln Lys Asp Leu Leu Phe Lys Asp Ser 290 295 300Ala Ile Gly
Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly Leu Tyr305 310 315
320Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys Ser Glu
325 330 335Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala
Val Gly 340 345 350Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly
Leu Ser Glu Gly 355 360 365Ser Val Thr Cys Ser Ser Ala Ser Thr Thr
Glu Asp Cys Ile Ala Leu 370 375 380Val Leu Lys Gly Glu Ala Asp Ala
Met Ser Leu Asp Gly Gly Tyr Val385 390 395 400Tyr Thr Ala Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr 405 410 415Lys Ser Gln
Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg Pro 420 425 430Val
Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr Ser 435 440
445Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala Val
450 455 460Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe
Asn Gln465 470 475 480Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser
Gln Ser Cys Ala Pro 485 490 495Gly Ser Asp Pro Arg Ser Asn Leu Cys
Ala Leu Cys Ile Gly Asp Glu 500 505 510Gln Gly Glu Asn Lys Cys Val
Pro Asn Ser Asn Glu Arg Tyr Tyr Gly 515 520 525Tyr Thr Gly Ala Phe
Arg Cys Leu Ala Glu Asp Ala Gly Asp Val Ala 530 535 540Phe Val Lys
Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn Asn Asn545 550 555
560Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu Cys
565 570 575Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys
His Leu 580 585 590Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met
Asp Lys Val Glu 595 600 605Arg Leu Lys Gln Val Leu Leu His Gln Gln
Ala Lys Phe Gly Arg Asn 610 615 620Gly Ser Asp Cys Pro Asp Lys Phe
Cys Leu Phe Gln Ser Glu Thr Lys625 630 635 640Asn Leu Leu Phe Asn
Asp Asn Thr Glu Cys Leu Ala Arg Leu His Gly 645 650 655Lys Thr Thr
Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala Gly Ile 660 665 670Thr
Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Glu 675 680
685Phe Leu Arg Lys 69010692PRTHomo sapiens 10Gly Arg Arg Arg Arg
Ser Val Gln Trp Cys Ala Val Ser Gln Pro Glu1 5 10 15Ala Thr Lys Cys
Phe Gln Trp Gln Arg Asn Met Arg Arg Val Arg Gly 20 25 30Pro Pro Val
Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile Gln 35 40 45Ala Ile
Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly Phe 50 55 60Ile
Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala Ala65 70 75
80Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr Ala Val
85 90 95Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu Gln
Gly 100 105 110Leu Lys Ser Cys His Thr Gly Leu Arg Arg Asn Ala Gly
Trp Asn Val 115 120 125Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp
Thr Gly Pro Pro Glu 130 135 140Pro Ile Glu Ala Ala Val Ala Arg Phe
Phe Ser Ala Ser Cys Val Pro145 150 155 160Gly Ala Asp Lys Gly Gln
Phe Pro Asn Leu Cys Arg Leu Cys Ala Gly 165 170 175Thr Gly Glu Asn
Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr Phe Ser 180 185 190Tyr Ser
Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val Ala 195 200
205Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala Glu
210 215 220Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys
Pro Val225 230 235 240Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val
Pro Ser His Ala Val 245 250 255Val Ala Arg Ser Val Asn Gly Lys Glu
Asp Ala Ile Trp Asn Leu Leu 260 265 270Arg Gln Ala Gln Glu Lys Phe
Gly Lys Asp Lys Ser Pro Lys Phe Gln 275 280 285Leu Phe Gly Ser Pro
Ser Gly Gln Lys Asp Leu Leu Phe Lys Asp Ser 290 295 300Ala Ile Gly
Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly Leu Tyr305 310 315
320Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys Ser Glu
325 330 335Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala
Val Gly 340 345 350Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly
Leu Ser Glu Gly 355 360 365Ser Val Thr Cys Ser Ser Ala Ser Thr Thr
Glu Asp Cys Ile Ala Leu 370 375 380Val Leu Lys Gly Glu Ala Asp Ala
Met Ser Leu Asp Gly Gly Tyr Val385 390 395 400Tyr Thr Ala Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr 405 410 415Lys Ser Gln
Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg Pro 420 425 430Val
Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr Ser 435 440
445Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala Val
450 455 460Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe
Asn Gln465 470 475 480Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser
Gln Ser Cys Ala Pro 485 490 495Gly Ser Asp Pro Arg Ser Asn Leu Cys
Ala Leu Cys Ile Gly Asp Glu 500 505 510Gln Gly Glu Asn Lys Cys Val
Pro Asn Ser Asn Glu Arg Tyr Tyr Gly 515 520 525Tyr Thr Gly Ala Phe
Arg Cys Leu Ala Glu Asp Ala Gly Asp Val Ala 530 535 540Phe Val Lys
Gly Val Thr Val Leu Gln Asn Thr Asp Gly Asn Asn Asn545 550 555
560Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu Cys
565 570 575Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys
His Leu 580 585 590Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met
Asp Lys Val Glu 595 600 605Arg Leu Lys Gln Val Leu Leu His Gln Gln
Ala Lys Phe Gly Arg Asn 610 615 620Gly Ser Asp Cys Pro Asp Lys Phe
Cys Leu Phe Gln Ser Glu Thr Lys625 630 635 640Asn Leu Leu Phe Asn
Asp Asn Thr Glu Cys Leu Ala Arg Leu His Gly 645 650 655Lys Thr Thr
Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala Gly Ile 660 665 670Thr
Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Glu 675 680
685Phe Leu Arg Lys 69011689PRTBos taurus 11Ala Pro Arg Lys Asn Val
Arg Trp Cys Thr Ile Ser Gln Pro Glu Trp1 5 10 15Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 20 25 30Ser Ile Thr Cys
Val Arg Arg Ala Phe Ala Leu Glu Cys Ile Arg Ala 35 40 45Ile Ala Glu
Lys Lys Ala Asp Ala Val Thr Leu Asp Gly Gly Met Val 50 55 60Phe Glu
Ala Gly Arg Asp Pro Tyr Lys Leu Arg Pro Val Ala Ala Glu65 70 75
80Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr Ala Val Ala
85 90 95Val Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu Gln Gly
Arg 100 105 110Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
Val Ile Pro 115 120 125Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr
Glu Ser Leu Glu Pro 130 135 140Leu Gln Gly Ala Val Ala Lys Phe Phe
Ser Ala Ser Cys Val Pro Cys145 150 155 160Ile Asp Arg Gln Ala Tyr
Pro Asn Leu Cys Gln Leu Cys Lys Gly Glu 165 170 175Gly Glu Asn Gln
Cys Ala Cys Ser Ser Arg Glu Pro Tyr Phe Gly Tyr 180 185 190Ser Gly
Ala Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp Val Ala Phe 195 200
205Val Lys Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys Ala Asp Arg
210 215 220Asp Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala Pro
Val Asp225 230 235 240Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro
Ser His Ala Val Val 245 250 255Ala Arg Ser Val Asp Gly Lys Glu Asp
Leu Ile Trp Lys Leu Leu Ser 260 265 270Lys Ala Gln Glu Lys Phe Gly
Lys Asn Lys Ser Arg Ser Phe Gln Leu 275 280 285Phe Gly Ser Pro Pro
Gly Gln Arg Asp Leu Leu Phe Lys Asp Ser Ala 290 295 300Leu Gly Phe
Leu Arg Ile Pro Ser Lys Val Asp Ser Ala Leu Tyr Leu305 310 315
320Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn Leu Arg Glu Thr Ala Glu
325 330 335Glu Val Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala Val
Gly Pro 340 345 350Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln
Ser Gly Gln Asn 355 360 365Val Thr Cys Ala Thr Ala Ser Thr Thr
Asp Asp Cys Ile Val Leu Val 370 375 380Leu Lys Gly Glu Ala Asp Ala
Leu Asn Leu Asp Gly Gly Tyr Ile Tyr385 390 395 400Thr Ala Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu Asn Arg Lys 405 410 415Thr Ser
Lys Tyr Ser Ser Leu Asp Cys Val Leu Arg Pro Thr Glu Gly 420 425
430Tyr Leu Ala Val Ala Val Val Lys Lys Ala Asn Glu Gly Leu Thr Trp
435 440 445Asn Ser Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val Asp
Arg Thr 450 455 460Ala Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn
Gln Thr Gly Ser465 470 475 480Cys Ala Phe Asp Glu Phe Phe Ser Gln
Ser Cys Ala Pro Gly Arg Asp 485 490 495Pro Lys Ser Arg Leu Cys Ala
Leu Cys Ala Gly Asp Asp Gln Gly Leu 500 505 510Asp Lys Cys Val Pro
Asn Ser Lys Glu Lys Tyr Tyr Gly Tyr Thr Gly 515 520 525Ala Phe Arg
Cys Leu Ala Glu Asp Val Gly Asp Val Ala Phe Val Lys 530 535 540Asn
Asp Thr Val Trp Glu Asn Thr Asn Gly Glu Ser Thr Ala Asp Trp545 550
555 560Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg Leu Leu Cys Leu Asp
Gly 565 570 575Thr Arg Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu
Ala Val Ala 580 585 590Pro Asn His Ala Val Val Ser Arg Ser Asp Arg
Ala Ala His Val Lys 595 600 605Gln Val Leu Leu His Gln Gln Ala Leu
Phe Gly Lys Asn Gly Lys Asn 610 615 620Cys Pro Asp Lys Phe Cys Leu
Phe Lys Ser Glu Thr Lys Asn Leu Leu625 630 635 640Phe Asn Asp Asn
Thr Glu Cys Leu Ala Lys Leu Gly Gly Arg Pro Thr 645 650 655Tyr Glu
Glu Tyr Leu Gly Thr Glu Tyr Val Thr Ala Ile Ala Asn Leu 660 665
670Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala Phe Leu Thr
675 680 685Arg12689PRTBos taurus 12Ala Pro Arg Lys Asn Val Arg Trp
Cys Thr Ile Ser Gln Pro Glu Trp1 5 10 15Phe Lys Cys Arg Arg Trp Gln
Trp Arg Met Lys Lys Leu Gly Ala Pro 20 25 30Ser Ile Thr Cys Val Arg
Arg Ala Phe Ala Leu Glu Cys Ile Pro Gly 35 40 45Ile Ala Glu Lys Lys
Ala Asp Ala Val Thr Leu Asp Gly Gly Met Val 50 55 60Phe Glu Ala Gly
Arg Asp Pro Tyr Lys Leu Arg Pro Val Ala Ala Glu65 70 75 80Ile Tyr
Gly Thr Lys Glu Ser Pro Gln Thr His Tyr Tyr Ala Val Ala 85 90 95Val
Val Lys Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu Gln Gly Arg 100 105
110Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Ile Ile Pro
115 120 125Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr Glu Ser Leu
Glu Pro 130 135 140Leu Gln Gly Ala Val Ala Lys Phe Phe Ser Ala Ser
Cys Val Pro Cys145 150 155 160Ile Asp Arg Gln Ala Tyr Pro Asn Leu
Cys Gln Leu Cys Lys Gly Glu 165 170 175Gly Glu Asn Gln Cys Ala Cys
Ser Ser Arg Glu Pro Tyr Phe Gly Tyr 180 185 190Ser Gly Ala Phe Lys
Cys Leu Gln Asp Gly Ala Gly Asp Val Ala Phe 195 200 205Val Lys Glu
Thr Thr Val Phe Glu Asn Leu Pro Glu Lys Ala Asp Arg 210 215 220Asp
Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala Pro Val Asp225 230
235 240Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro Ser His Ala Val
Val 245 250 255Ala Arg Ser Val Asp Gly Lys Glu Asp Leu Ile Trp Lys
Leu Leu Ser 260 265 270Lys Ala Gln Glu Lys Ser Gly Lys Asn Lys Ser
Arg Ser Phe Gln Leu 275 280 285Phe Gly Ser Pro Pro Gly Gln Arg Asp
Leu Leu Phe Lys Asp Ser Ala 290 295 300Leu Gly Phe Leu Arg Ile Pro
Ser Lys Val Asp Ser Ala Leu Tyr Leu305 310 315 320Gly Ser Arg Tyr
Leu Thr Thr Leu Lys Asn Leu Arg Glu Thr Ala Glu 325 330 335Glu Val
Lys Ala Arg Tyr Thr Arg Val Val Trp Cys Ala Val Gly Pro 340 345
350Glu Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser Gly Gln Asn
355 360 365Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp Cys Ile Val
Leu Val 370 375 380Leu Lys Gly Glu Ala Asp Ala Leu Asn Leu Asp Gly
Gly Tyr Ile Tyr385 390 395 400Thr Ala Gly Lys Cys Gly Leu Val Pro
Val Leu Ala Glu Asn Arg Lys 405 410 415Ser Ser Lys His Ser Ser Leu
Asp Cys Val Leu Arg Pro Thr Glu Gly 420 425 430Tyr Leu Ala Val Ala
Val Val Lys Lys Ala Asn Glu Gly Leu Thr Trp 435 440 445Asn Ser Leu
Lys Asp Lys Lys Ser Cys His Thr Ala Val Asp Arg Thr 450 455 460Ala
Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln Thr Gly Ser465 470
475 480Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys Ala Pro Gly Ala
Asp 485 490 495Pro Lys Ser Arg Leu Cys Ala Leu Cys Ala Gly Asp Asp
Gln Gly Leu 500 505 510Asp Lys Cys Val Pro Asn Ser Lys Glu Lys Tyr
Tyr Gly Tyr Thr Gly 515 520 525Ala Phe Arg Cys Leu Ala Glu Asp Val
Gly Asp Val Ala Phe Val Lys 530 535 540Asn Asp Thr Val Trp Glu Asn
Thr Asn Gly Glu Ser Thr Ala Asp Trp545 550 555 560Ala Lys Asn Leu
Asn Arg Glu Asp Phe Arg Leu Leu Cys Leu Asp Gly 565 570 575Thr Arg
Lys Pro Val Thr Glu Ala Gln Ser Cys His Leu Ala Val Ala 580 585
590Pro Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala His Val Lys
595 600 605Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly Lys Asn Gly
Lys Asn 610 615 620Cys Pro Asp Lys Phe Cys Leu Phe Lys Ser Glu Thr
Lys Asn Leu Leu625 630 635 640Phe Asn Asp Asn Thr Glu Cys Leu Ala
Lys Leu Gly Gly Arg Pro Thr 645 650 655Tyr Glu Glu Tyr Leu Gly Thr
Glu Tyr Val Thr Ala Ile Ala Asn Leu 660 665 670Lys Lys Cys Ser Thr
Ser Pro Leu Leu Glu Ala Cys Ala Phe Leu Thr 675 680
685Arg13689PRTBos taurus 13Ala Pro Arg Lys Asn Val Arg Trp Cys Thr
Ile Ser Gln Pro Glu Trp1 5 10 15Phe Lys Cys Arg Arg Trp Gln Trp Arg
Met Lys Lys Leu Gly Ala Pro 20 25 30Ser Ile Thr Cys Val Arg Arg Ala
Phe Ala Leu Glu Cys Ile Arg Ala 35 40 45Ile Ala Glu Lys Lys Ala Asp
Ala Val Thr Leu Asp Gly Gly Met Val 50 55 60Phe Glu Ala Gly Arg Asp
Pro Tyr Lys Leu Arg Pro Val Ala Ala Glu65 70 75 80Ile Tyr Gly Thr
Lys Glu Ser Pro Gln Thr His Tyr Tyr Ala Val Ala 85 90 95Val Val Lys
Lys Gly Ser Asn Phe Gln Leu Asp Gln Leu Gln Gly Arg 100 105 110Lys
Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Ile Ile Pro 115 120
125Met Gly Ile Leu Arg Pro Tyr Leu Ser Trp Thr Glu Ser Leu Glu Pro
130 135 140Leu Gln Gly Ala Val Ala Lys Phe Phe Ser Ala Ser Cys Val
Pro Cys145 150 155 160Ile Asp Arg Gln Ala Tyr Pro Asn Leu Cys Gln
Leu Cys Lys Gly Glu 165 170 175Gly Glu Asn Gln Cys Ala Cys Ser Ser
Arg Glu Pro Tyr Phe Gly Tyr 180 185 190Ser Gly Ala Phe Lys Cys Leu
Gln Asp Gly Ala Gly Asp Val Ala Phe 195 200 205Val Lys Glu Thr Thr
Val Phe Glu Asn Leu Pro Glu Lys Ala Asp Arg 210 215 220Asp Gln Tyr
Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala Pro Val Asp225 230 235
240Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro Ser His Ala Val Val
245 250 255Ala Arg Ser Val Asp Gly Lys Glu Asp Leu Ile Trp Lys Leu
Leu Ser 260 265 270Lys Ala Gln Glu Lys Phe Gly Lys Asn Lys Ser Arg
Ser Phe Gln Leu 275 280 285Phe Gly Ser Pro Pro Gly Gln Arg Asp Leu
Leu Phe Lys Asp Ser Ala 290 295 300Leu Gly Phe Leu Arg Ile Pro Ser
Lys Val Asp Ser Ala Leu Tyr Leu305 310 315 320Gly Ser Arg Tyr Leu
Thr Thr Leu Lys Asn Leu Arg Glu Thr Ala Glu 325 330 335Glu Val Lys
Ala Arg Tyr Thr Arg Val Val Trp Cys Ala Val Gly Pro 340 345 350Glu
Glu Gln Lys Lys Cys Gln Gln Trp Ser Gln Gln Ser Gly Gln Asn 355 360
365Val Thr Cys Ala Thr Ala Ser Thr Thr Asp Asp Cys Ile Val Leu Val
370 375 380Leu Lys Gly Glu Ala Asp Ala Leu Asn Leu Asp Gly Gly Tyr
Ile Tyr385 390 395 400Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu
Ala Glu Asn Arg Lys 405 410 415Ser Ser Lys His Ser Ser Leu Asp Cys
Val Leu Arg Pro Thr Glu Gly 420 425 430Tyr Leu Ala Val Ala Val Val
Lys Lys Ala Asn Glu Gly Leu Thr Trp 435 440 445Asn Ser Leu Lys Asp
Lys Lys Ser Cys His Thr Ala Val Asp Arg Thr 450 455 460Ala Gly Trp
Asn Ile Pro Met Gly Leu Ile Val Asn Gln Thr Gly Ser465 470 475
480Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys Ala Pro Gly Ala Asp
485 490 495Pro Lys Ser Arg Leu Cys Ala Leu Cys Ala Gly Asp Asp Gln
Gly Leu 500 505 510Asp Lys Cys Val Pro Asn Ser Lys Glu Lys Tyr Tyr
Gly Tyr Thr Gly 515 520 525Ala Phe Arg Cys Leu Ala Glu Asp Val Gly
Asp Val Ala Phe Val Lys 530 535 540Asn Asp Thr Val Trp Glu Asn Thr
Asn Gly Glu Ser Thr Ala Asp Trp545 550 555 560Ala Lys Asn Leu Asn
Arg Glu Asp Phe Arg Leu Leu Cys Leu Asp Gly 565 570 575Thr Arg Lys
Pro Val Thr Glu Ala Gln Ser Cys His Leu Ala Val Ala 580 585 590Pro
Asn His Ala Val Val Ser Arg Ser Asp Arg Ala Ala His Val Lys 595 600
605Gln Val Leu Leu His Gln Gln Ala Leu Phe Gly Lys Asn Gly Lys Asn
610 615 620Cys Pro Asp Lys Phe Cys Leu Phe Lys Ser Glu Thr Lys Asn
Leu Leu625 630 635 640Phe Asn Asp Asn Thr Glu Cys Leu Ala Lys Leu
Gly Gly Arg Pro Thr 645 650 655Tyr Glu Glu Tyr Leu Gly Thr Glu Tyr
Val Thr Ala Ile Ala Asn Leu 660 665 670Lys Lys Cys Ser Thr Ser Pro
Leu Leu Glu Ala Cys Ala Phe Leu Thr 675 680 685Arg14689PRTBos
taurus 14Ala Pro Arg Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro
Glu Trp1 5 10 15Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu
Gly Ala Pro 20 25 30Ser Ile Thr Cys Val Arg Arg Ala Phe Ala Leu Ala
Cys Ile Arg Ala 35 40 45Ile Ala Glu Lys Lys Ala Asp Ala Val Thr Leu
Asp Gly Gly Met Val 50 55 60Phe Glu Ala Gly Arg Asp Pro Tyr Lys Leu
Arg Pro Val Ala Ala Glu65 70 75 80Ile Tyr Gly Thr Lys Glu Ser Pro
Gln Thr His Tyr Tyr Ala Val Ala 85 90 95Val Val Lys Lys Gly Ser Asn
Phe Gln Leu Asp Gln Leu Gln Gly Arg 100 105 110Lys Ser Cys His Thr
Gly Leu Gly Arg Ser Ala Gly Trp Val Ile Pro 115 120 125Met Gly Ile
Leu Arg Pro Tyr Leu Ser Trp Thr Glu Ser Leu Glu Pro 130 135 140Leu
Gln Gly Ala Val Ala Lys Phe Phe Ser Ala Ser Cys Val Pro Cys145 150
155 160Ile Asp Arg Gln Ala Tyr Pro Asn Leu Cys Gln Leu Cys Lys Gly
Glu 165 170 175Gly Glu Asn Gln Cys Ala Cys Ser Ser Arg Glu Pro Tyr
Phe Gly Tyr 180 185 190Ser Gly Ala Phe Lys Cys Leu Gln Asp Gly Ala
Gly Asp Val Ala Phe 195 200 205Val Lys Glu Thr Thr Val Phe Glu Asn
Leu Pro Glu Lys Ala Asp Arg 210 215 220Asp Gln Tyr Glu Leu Leu Cys
Leu Asn Asn Ser Arg Ala Pro Val Asp225 230 235 240Ala Phe Lys Glu
Tyr His Leu Ala Gln Val Pro Ser His Pro Val Val 245 250 255Ala Arg
Ser Val Asp Ala Lys Glu Asp Leu Ile Trp Lys Leu Leu Arg 260 265
270Lys Ala Gln Glu Lys Phe Gly Lys Asn Lys Ser Arg Ser Phe Gln Leu
275 280 285Phe Gly Ser Pro Pro Gly Gln Arg Asp Leu Leu Phe Lys Asp
Ser Ala 290 295 300Leu Gly Phe Leu Arg Ile Pro Ser Lys Val Asp Ser
Ala Leu Tyr Leu305 310 315 320Gly Ser Arg Tyr Leu Thr Thr Leu Lys
Asn Leu Arg Glu Thr Ala Glu 325 330 335Glu Val Lys Ala Arg Tyr Thr
Arg Val Val Trp Cys Ala Val Gly Pro 340 345 350Glu Glu Gln Lys Lys
Cys Gln Gln Trp Ser Gln Gln Ser Gly Gln Asn 355 360 365Val Thr Cys
Ala Thr Ala Ser Thr Thr Asp Asp Cys Ile Val Leu Val 370 375 380Leu
Lys Gly Glu Ala Asp Ala Leu Asn Leu Asp Gly Gly Tyr Val Tyr385 390
395 400Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Arg
Lys 405 410 415Ser Ser Lys His Ser Ser Leu Asp Cys Val Leu Arg Pro
Thr Glu Gly 420 425 430Tyr Leu Ala Val Ala Val Val Arg Lys Ala Asn
Glu Gly Leu Thr Trp 435 440 445Asn Ser Leu Lys Asp Lys Lys Ser Cys
His Thr Ala Val Asp Arg Thr 450 455 460Ala Gly Trp Asn Ile Pro Met
Gly Leu Ile Val Asn Gln Thr Gly Ser465 470 475 480Cys Ala Phe Asp
Glu Phe Phe Ser Gln Ser Cys Ala Pro Gly Ala Asp 485 490 495Pro Lys
Ser Arg Leu Cys Ala Leu Cys Ala Gly Asp Asp Gln Gly Leu 500 505
510Asp Lys Cys Val Pro Asn Ser Lys Glu Lys Tyr Tyr Gly Tyr Thr Gly
515 520 525Ala Phe Arg Cys Leu Ala Glu Asp Val Gly Asp Val Ala Phe
Val Lys 530 535 540Asn Asp Thr Val Trp Glu Asn Thr Asn Gly Glu Ser
Thr Ala Asp Trp545 550 555 560Ala Lys Asn Leu Asn Arg Glu Asp Phe
Arg Leu Leu Cys Leu Asp Gly 565 570 575Thr Arg Lys Pro Val Thr Glu
Ala Gln Ser Cys His Leu Ala Val Ala 580 585 590Pro Asn His Ala Val
Val Ser Arg Ser Asp Arg Ala Ala His Val Lys 595 600 605Gln Val Leu
Leu His Gln Gln Ala Leu Phe Gly Lys Asn Gly Lys Asn 610 615 620Cys
Pro Asp Lys Phe Cys Leu Phe Lys Ser Glu Thr Lys Asn Leu Leu625 630
635 640Phe Asn Asp Asn Thr Glu Cys Leu Ala Lys Leu Gly Gly Arg Pro
Thr 645 650 655Tyr Glu Glu Tyr Leu Gly Thr Glu Tyr Val Thr Ala Ile
Ala Asn Leu 660 665 670Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala
Cys Ala Phe Leu Thr 675 680 685Arg156PRTHomo sapiens 15Gly Arg Arg
Arg Arg Ser1 5164PRTHomo sapiens 16Arg Lys Val Arg1174PRTHomo
sapiens 17Arg Arg Val Arg11826PRTBos taurus 18Phe Lys Cys Arg Arg
Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro1 5 10 15Ser Ile Thr Cys
Val Arg Arg Ala Phe Ala 20 25194PRTBos taurus 19Lys Cys Arg
Arg1204PRTBos taurus 20Arg Met Lys Lys1
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