U.S. patent application number 09/993959 was filed with the patent office on 2003-09-04 for method of treating rhinitis or sinusitis.
This patent application is currently assigned to B.M.R.A. Corporation B.V.. Invention is credited to Grouzmann, Eric, Lacroix, Jean-Silvain, Monod, Michel.
Application Number | 20030165489 09/993959 |
Document ID | / |
Family ID | 25162086 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165489 |
Kind Code |
A1 |
Grouzmann, Eric ; et
al. |
September 4, 2003 |
Method of treating rhinitis or sinusitis
Abstract
The present invention is directed to methods of treating mucosal
inflammation associated with rhinitis or sinusitis by administering
peptidases that recognize and cleave polypeptides at Xaa-Pro
sequences. In addition, the invention encompasses therapeutic
packages in which pharmaceutical compositions containing the
peptidases are preloaded in a device suitable for intranasally
delivering drug.
Inventors: |
Grouzmann, Eric; (La
Conversion, CH) ; Lacroix, Jean-Silvain; (Geneva,
CH) ; Monod, Michel; (Lausanne, CH) |
Correspondence
Address: |
MICHAEL A SANZO
FITCH EVEN TABIN & FLANNERY
1801 K STREET N W
SUITE 401L
WASHINGTON
DC
20006-1201
US
|
Assignee: |
B.M.R.A. Corporation B.V.
|
Family ID: |
25162086 |
Appl. No.: |
09/993959 |
Filed: |
November 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09993959 |
Nov 27, 2001 |
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09794236 |
Feb 28, 2001 |
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6337069 |
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Current U.S.
Class: |
424/94.63 ;
435/810 |
Current CPC
Class: |
A61P 11/02 20180101;
C12Y 304/14005 20130101; A61K 38/4813 20130101; A61P 29/00
20180101; C12Y 304/13009 20130101; A61K 38/178 20130101 |
Class at
Publication: |
424/94.63 ;
435/810 |
International
Class: |
A61K 038/48; C12N
001/00 |
Claims
What is claimed is:
1. A method of treating a patient for mucosal inflammation
associated with rhinitis, sinusitis or both, comprising:
intranasally administering to said patient a therapeutically
effective amount of a peptidase that cleaves at Xaa-Pro
sequences.
2. The method of claim 1, wherein said peptidase is an exopeptidase
selected from the group consisting of: dipeptidyl peptidase IV;
quiescent cell proline dipeptidase; dipeptidyl peptidase 8; and
attractin.
3. The method of claim 2, wherein said exopeptidase is dipeptidyl
peptidase IV.
4. The method of claim 2, wherein said exopeptidase is dipeptidyl
peptidase 8.
5. The method of claim 2, wherein said exopeptidase is quiescent
cell proline dipeptidase.
6. The method of claim 2, wherein said exopeptidase is
attractin.
7. The method of claim 2, wherein said exopeptidase is administered
at a dose of between 1 .mu.g and 1 mg.
8. The method of any one of claims 1-7, wherein said rhinitis,
sinusitis or both, is the result of allergies or asthma.
9. A therapeutic package comprising a device for intranasally
delivering drug to a patient, preloaded with a solution or powder
comprising a peptidase that cleaves at Xaa-Pro sequences.
10. The therapeutic package of claim 9, wherein said peptidase is
an exopeptidase selected from the group consisting of: dipeptidyl
peptidase IV; quiescent cell proline dipeptidase; dipeptidyl
peptidase 8; and attractin.
11. The therapeutic package of claim 9, wherein said exopeptidase
is dipeptidyl peptidase IV.
12. The therapeutic package of claim 9, wherein said exopeptidase
is quiescent cell proline dipeptidase.
13. The therapeutic package of claim 10, wherein said exopeptidase
is dipeptidyl peptidase 8.
14. The therapeutic package of claim 9, wherein said exopeptidase
is attractin.
15. The therapeutic package of claim 10, wherein said exopeptidase
is present in a solution at a concentration of between 1 .mu.g/ml
and 10 mg/ml.
16. A method of treating a patient for mucosal inflammation
associated with rhinitis, sinusitis or both, comprising:
intranasally administering to said patient a therapeutically
effective amount of an agent that inhibits the binding of SP to the
NK1 receptor.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to methods of treating
inflammation associated with rhinitis or sinusitis by intranasally
delivering peptidases to a patient. The invention also encompasses
therapeutic packages in which a peptidase is preloaded in a device
designed for intranasally delivering drug.
BACKGROUND OF THE INVENTION
[0002] Rhinitis, an inflammation of the nasal mucosal membrane, is
characterized by sneezing, rhinorrhea, nasal congestion, and
increased nasal secretion. It is often accompanied by sinusitis, an
inflammation of the sinuses. When these conditions persist for a
period of more than three weeks, they are termed "chronic." More
than 37 million Americans, particularly those with allergies or
asthma, suffer from these conditions, making them the most common
chronic medical problems in the United States.
[0003] Chronic "rhinosinusitis" or sinusitis is difficult to treat
successfully. In general, treatment consists of a combination of
antibiotics and decongestants or antihistamines. In addition,
steroid nasal sprays are commonly used to reduce inflammation. For
patients with severe chronic sinusitis, oral steroids, such as
prednisone, may also be prescribed. However, the long-term safety
of steroid administration, especially in children, is not fully
understood and oral steroids often have significant side effects.
When drug therapy fails, surgery is usually the only
alternative.
[0004] The mucosal tissue lining the nasal and sinus passages is
densely packed with sensory neurons (Alving, et al., Cell Tissue
Res. 264:529-538 (1991); Saria, et al., Am. Rev. Respir. Dis.
147:1330-1335 (1988)). When activated, these neurons release a
variety of bioactive peptides that contribute to inflammation by
causing vasodilation, stimulating mucosal gland secretion, and
promoting infiltration by inflammatory mast cells, lymphocytes and
eosinophils (Stead, et al., Immunol. Rev. 10:333-359 (1987);
Mygind, et al., Eur. J. Respir. Dis. 64(S128):1-379 (1983)).
Included among the released bioactive peptides are substance P,
calcitonin-gene related peptide, neuropeptide Y and vasoactive
intestinal peptide (Lundblad, et al., Acta. Physiol. Scand.
529:1-42 (1984)). Means for controlling the activity of these
peptides should provide an effective treatment for the inflammation
associated with both rhinitis and sinusitis.
SUMMARY OF THE INVENTION
[0005] The present invention stems from the discovery that
dipeptidyl peptidase IV, an exopeptidase that cleaves Xaa-Pro
dipeptides from the N-terminus of polypeptides, is present in human
nasal mucosa at levels that are inversely related to inflammation.
Thus, low levels of dipeptidyl peptidase IV are associated with a
high density of inflammatory cells, and high levels of dipeptidyl
peptidase IV are associated with a low density of inflammatory
cells. This is important because dipeptidyl peptidase IV degrades
peptides that contribute to the pathophysiology of rhinitis,
sinusitis and asthma (Mentlein, et al., Regul. Peptides 49:133-144
(1993); Heymann, et al., FEBS Lett. 91:360-364 (1978); Beauvais, et
al., Fum. Infect. Immun. 65:3042-3047 (1997)). Among the
inflammation-related peptides cleaved are NPY, SP, and desArg1
bradykinin. Based upon these observation and further experiments,
the concept has been developed that the intranasal administration
of dipeptidyl peptidase IV can reduce inflammation in the mucosal
tissue that lines both the nasal cavity and sinuses. Other
proteases that possess the same proteolytic activity should also
produce a positive therapeutic effect. These proteases include
quiescent cell proline dipeptidase (Underwood, et al., J. Biol.
Chem. 274:34053-34058 (1999)), dipeptidyl peptidase 8 (Abbott, et
al., Eur. J. Biochem. 20 267:6140-6150 (2000)), and attractin
(Duke-Cohan, et al., Proc. Nat'l. Acad. Sci. U.S.A. 95:11336-11341
(1998)).
[0006] In its first aspect, the invention is directed to a method
of treating a patient for inflammation of the nasal or sinus
mucosa. The method involves intranasally administering a
therapeutically effective amount of a peptidase (preferably an
exopeptidase) that cleaves at Xaa-Pro residues, where Xaa
represents any of the 20 amino acids commonly found in animals. A
"therapeutically effective" dose is defined as an amount sufficient
to produce a significant reduction in inflammation as evidence by
either a reduced number of inflammatory cells in mucosal tissue or
by a significant improvement in one or more symptoms associated
with the inflammation. For example, in the case of rhinitis or
sinusitis, a therapeutically effective dose would be a sufficient
amount to produce a significant reduction in sneezing, coughing,
sinus-related headaches, nasal obstruction, mucosal secretion, or
other discomfort associated with these conditions. Inflammatory
cells include mast cells, lymphocytes and eosinophils. In general,
it is expected that a therapeutically effective dose for any of the
proteases used will be between I microgram and 1 milligram and,
typically, between 5 micrograms and 500 micrograms.
[0007] The preferred peptidase for use in the method is dipeptidyl
peptidase IV. Other peptidases that can be used include quiescent
cell proline dipeptidase; dipeptidyl peptidase 8, and attractin. In
each case, it is the human form of the peptidase that is preferred.
However, peptidases from other species (e.g., that secreted by
Aspergilus Fumigatus, see Examples section) may also be used
provided that they have the ability to cleave at the Xaa-Pro
sequence. Although the method will work for rhinitis and sinusitis
caused by any disease or condition, it is expected that the most
common causes will be allergies or asthma.
[0008] In another aspect, the invention is directed to a
therapeutic package in which a device for intranasally delivering
drug to a patient is preloaded with a solution or powder containing
one or more of the peptidases described above. The invention is
compatible with any intranasal delivery device (including
encapsulated dosage forms) and with any of the numerous
compositions that have been described for delivering drugs by means
of the nasal cavity. When liquid compositions are used in the
device, it is expected that peptidase will be present at a
concentration of between 1 .mu.g/ml and 10 mg/ml, and more
typically, at a concentration of between 10 .mu.g/ml. and 1
mg/ml.
[0009] The invention also encompasses the concept that SP is
particularly important in causing inflammation in lung and nasal
mucosa. Any method that reduces the local activity of this peptide
should be useful in the treatment of rhinitis or sinusitis. A
reduction in activity may be accomplished either using a peptidase
that degrades SP (e.g., one of the peptidases described above) or
by administering an agent that inhibits the binding of SP to the
NK1 receptor (see Examples section).
DETAILED DESCRIPTION OF THE INVENTION
[0010] A. Preparation of Peptidases
[0011] The present invention is directed to treatment methods which
utilize peptidases that have the common characteristic of cleaving
at Xaa-Pro sites. These may be purchased commercially or obtained
using any of the procedures described in the relevant literature.
For example, the gene corresponding to the peptidase can be
isolated and used for recombinant protein production. Especially
preferred peptidases, along with references relevant to their
isolation and recombinant production, are: human dipeptidyl
peptidase IV, shown herein as SEQ ID NO: 1 (Misumi, et al.,
Biochim. Biophys. Acta 15:1131 (1992); Darmoul, et al., J Biol.
Chem. 267:4824-4833 (1992); Abbott, et al., Immunogenetics
40:331-338 (1994)); human quiescent cell proline dipeptidase, shown
herein as SEQ ID NO: 2 (Underwood, et al., J. Biol. Chem.
274:34053-34058 (1999)); human attractin, shown herein as SEQ ID
NO: 3 (Duke-Cohan, et al., Proc. Nat'l. Acad. Sci. USA
95:11336-11341 (1998); Nagase, et al., DNA Res. 5:31-39 (1998));
and human dipeptidyl peptidase 8, shown herein as SEQ ID NO: 4
(Abbott, et al., Eur. J Biochem. 267:6140-6150 (2000)). In addition
to being made recombinantly, these proteins can be synthesized
using methods that are well-known in the art.
[0012] B. Making of Pharmaceutical Compositions
[0013] Compositions for intranasally delivering peptidases can be
made in accordance with methods that are standard in the art (see,
e.g., Remington's Pharmaceutical Sciences, 16.sup.th ed. A. Oslo
Editor, Easton Pa. (1980)). Enzymes will typically be prepared in
admixture with conventional excipients. Suitable carriers may
include, but are not limited to: water; salt solutions; alcohols;
vegetable oils; polyethylene glycols; gellatin; carbohydrates such
as lactose, amylose or starch; talc; hydroxymethylcellulose etc.
The pharmaceutical preparations can be sterilized and, if desired,
mixed with auxiliary agents such as preservatives or stabilizers.
The invention is compatible with any of the numerous compositions
that have been disclosed in the art for nasal delivery including
those in: U.S. Pat. Nos. 6,054,462; 4,946,870; 5,897,858; and
4,476,116. The concentration of peptidase present can vary over a
wide range. Typically, in liquid formulations, peptide should be
present in an amount of between 1 .mu.g/ml and 10 mg/ml and, more
commonly, at a concentration of between 10 .mu.g/ml and 1
mg/ml.
[0014] Treatment Methods
[0015] The present invention is directed to methods for treating
inflammation present in the mucosal lining of the nasal or sinus
passages. It depends upon the direct administration of sufficient
peptidase to proteolytically cleave peptides known to contribute to
inflammation. The total dosage of peptidase to be administered to a
patient should be at least the amount required to achieve this
objective as reflected by a reduction or elimination of symptoms
associated with inflammation. For example, a patient being treated
for rhinosinusitis should receive sufficient compound to reduce or
eliminate the frequency or intensity of sinus headache, reduce
coughing, congestion, sneezing, respiratory obstruction or other
discomforts associated with the condition.
[0016] In general, a patient may begin by self-administering a
relatively small dose of compound and then repeat administration as
necessary. For example, a patient may begin by administering 0.1 mg
per day and then increase the dosage upward using changes in
inflammation-related symptoms as a guide. Typically, it is expected
that patients will receive a daily dose of between 1 .mu.g and I mg
per day, and, more typically, between 5 .mu.g and 500 .mu.g. Daily
dosages may be provided in either a single or multiple regimen with
the latter being generally preferred. These are simply guidelines,
since the actual dose will be determined by the patient and their
physician based upon a variety of clinical factors.
[0017] Therapeutic Packages
[0018] In addition to the pharmaceutical compositions described
above, the invention includes therapeutic packages for the
intranasal delivery of the compositions. A therapeutic package is
comprised of a device designed for the intranasal inhalation of
medication which has been preloaded with a pharmaceutical
composition containing one or more of the peptidases described
above. In general, spray devices are preferred, such as those
disclosed in U.S. Pat. No. 6,145,703; WO 95/00195; U.S. Pat. No.
5,307,953; EP 0388651; U.S. Pat. Nos. 4,017,007; 5,301,846;
3,176,883; or 4,286,735. Devices for administering powders or nasal
drops may also be used. When the pharmaceutical composition is in
the form of a solution, it is expected that peptidase should
generally be present at a concentration of between 1 .mu.g/ml and
10 mg/ml, and, more typically, at a concentration of between 10
.mu.g/ml and 1 mg/ml.
EXAMPLES
[0019] I. Materials and Methods
[0020] Recombinant Dipeptidyl Peptidase IV (DPPIV)
[0021] A soluble form of DPPIV secreted from Aspergilus Fumigatus
has been previously characterized and was used in the present
experiments (Beauvais et al., Infection Immun. 65 3042-3047
(1997)). The enzyme has an apparent molecular weight of 95 kDa. It
was expressed in the yeast Pichia Pastoris and purified to more
than 99% purity as assessed by electrophoresis and gel-filtration
to reach a specific activity of 40 units/mg of protein. SP (1.8
.mu.g) co-incubated with 0.016 .mu.g of DPPIV for ten minutes at
37.degree. C. is degraded to SP5-11 as identified by
mass-spectrometry. If an excess of SP is added (3.5 .mu.g) a
partial of digestion of the peptide is observed.
[0022] Patients
[0023] Forty-five patients, 23 males and 22 females, suffering from
nasal obstruction, rhinorrhea and headaches for more than eighteen
months were included in the study. Pre-operative rhinoscopy
revealed septal deviation associated with concha bullosa of the
middle turbinate. All patients underwent septoplasty and partial
middle turbinectomy under endoscopic control with general
anesthesia. The age range was 14 to 64 years with the average
patient being 39 years of age. Patients with allergy, nasal polyps
or tumors were excluded.
[0024] Tissue Processing
[0025] Samples of middle turbinate mucosa from patients undergoing
partial turbinectomy were fixed immediately in ice cold acetone
with 2 mM phenyl methyl sulphonyl fluoride and 20 mM iodoacetamide
and incubated overnight at -20.degree. C. Biopsies were embedded in
glycol methacrylate resin and allowed to polymerize overnight at
4.degree. C.
[0026] Antibodies
[0027] The following monoclonal antibodies were used: CD26 (clone
BA5, DAKO) directed against DPPIV protease, diluted 1:20; CD1A
(Biogenex) for dendritic cells, diluted 1:20; CD31 (DAKO)
recognizing the adhesion molecule PECAM on endothelial cells,
diluted 1:20; and the polyclonal antibody CD3 (DAKO) directed
against T cells and used at a dilution of 1:20.
[0028] Immunohistochemical Staining
[0029] Serial sections, 2 mm thick, were cut using a Reichert-ung
microtome equipped with a glass knife. Immunohistochemical staining
was performed using the streptavidin biotin-peroxidase method with
aminoethyl-carbazole (AEC) as substrate.
[0030] Quantification of Inflammation in Nasal Biopsies and
Intensity of Symptoms
[0031] Mucosal samples of the middle turbinate from both sides
(N=90) were fixed in formaldehyde and dehydrated, embedded in
paraffin, and colored by haematoxylin-eosin. They were then
examined under a Zeiss microscope at 40.times. magnification.
Histological analysis included defining the integrity of the
pseudo-stratified columnar epithelium, noting the presence or
absence of edema and quantifying the number of inflammatory cells
within the submucosa. This was accomplished using a scale graded
from 0 to +++, where 0 meant no inflammatory cells and +++
represented abundant inflammatory cells. Using the Rank Spearman
correlation test, the correlation between DPPIV and the degree of
inflammation of the nasal mucosa was examined. The intensity of
nasal obstruction, rhinorrhea and headache was recorded by means of
a visual analog scale graded from 0 to 5, where 0 corresponds to
the absence of symptoms and 5 corresponds to severely intense
symptoms. Nasal airway resistance was recorded by means of anterior
rhinomanometry (rhinotest).
[0032] The Determination of DPPIV Activity in Human Mucosa
Biopsies
[0033] DPPIV activity was determined according to Scharpe, et al.
(Clin. Chim. Acta 195: 125-132 (1990)) with the following
modifications: nasal biopsies were sonicated in the presence of 0.5
ml of 100 mM Tris-HCl, pH 8, for 2 minutes on ice using a Branson
sonifier (output 4) and centrifuged for 10 minutes at 15,000 rpm in
a microfuge at 4.degree. C. The supernatant was recovered and the
pellet was treated with 0.5 ml of 100 mM Tris-HCl, pH8, containing
2% Triton X100. The suspension was vortexed for one minute and
centrifuged for 10 minutes at 15,000 rpm in a microfuge at
4.degree. C. The supernatant was recovered, pooled with the one
obtained previously, and stored at -20.degree. C. DPPIV activity
was determined on 1, 2.5 and 5 .mu.l of supernatant
fluorometrically using Gly-Pro-AMC (Novabiochem) at 5 mM final
concentration for 60 minutes at 37.degree. C. under agitation in an
Eppendorf thermomixer in 25 .mu.l of 100 mM Tris-HCl, pH 8. The
reaction was stopped by the addition of 2.5 .mu.l of pure acetic
acid. The incubation mixture was recovered in 3 ml of water. A
blank value was obtained by incubating the substrate in the absence
of enzyme and a standard curve was determined using AMC
fluorescence measurement on a fluorometer. The DPPIV activities
were standardized based on wet tissue weight and specific
activities expressed as pmoles of substrate converted per mg of
tissue per minute.
[0034] Experiments in the Pig In Vivo
[0035] Experiments were performed on pigs of both sexes (body
weight 18-25 kg). All animals were premedicated with atropine (0.05
mg/kg) and ketamine (20 mg/kg i.m.). They were anesthesized with
thiopentone (5 mg/kg i.v.). After tracheostomy, animals were
intubated and artificially ventilated by a volume regulated
ventilator. During surgery, animals were given a continuous i.v.
infusion of Ringer's solution, pancuronium bromide (0.25 mg/kg) and
heparin. Each experiment lasted approximately 8 hours. Catheters
were placed in the femoral artery for systemic blood pressure and
heart rate monitoring and in the femoral vein for thiopentone,
heparin and fluid administration (300 ml/hr). The contralateral
femoral vein was canulated for blood sampling. Surgical preparation
of the internal maxillary artery was done in accordance with
Lacroix (Acta Physiol. Scand. 136:1-63 (1989)). Selective recording
of the nasal arterial blood flow was performed with a Transonic
flow probe (probe 2.4RB 143) of 2.4 mm diameter placed around the
sphenopalatine artery. The flow probe was connected to a T202S
ultrasonic blood flowmeter. All the arterial branches situated
downstream of the flow probe were ligated and cut except for the
superficial temporal artery which was cannulated with a PE 90
catheter for infusions or injections.
[0036] Variations in the area under the curve (AUC) of the
sphenopalatine artery vascular resistance (Vr), derived from both
mean arterial blood pressure and systemic blood flow curves, were
analyzed over time. Durations of the responses of the vascular
resistance were compared and all results were expressed in percent
of baseline. Vascular parameters were recorded simultaneously using
a 6 pen trace recorder.
[0037] All animals were intranasally administered DPIV (50 .mu.g,
26.5 pmoles/kg), subjected to sympathetic nerve stimulation (SNS,
15V, 5 ms, 10 Hz for 2 minutes) and then infused with histamine
(0.1-25 .mu.g), capsacin (0.01-25 .mu.g), bradykinin (0.001-10
.mu.g), SP or its C-terminal fragment SP 5-11, DPPIV, or NK1
antagonist L733060 in the superficial temporal artery under
controlled conditions. In each case, the vascular response of the
animal was measured. This entire procedure was then repeated after
local i.a. pretreatment with the alpha-adrenergic receptor
antagonist phenoxybenzanime (1 mg/kg).
[0038] Vascular Responses to DPPIV in Domestic Pigs In Vivo
[0039] The basal blood flow in the internal maxillary artery of the
pig under control conditions was 4.92.+-.0.7 ml min.sup.-1
kg.sup.-1. After section of the sympathetic nerves on the left
side, the homolateral nasal arterial flow was 6.1.+-.0.26 ml
min.sup.-1 kg.sup.-1 (representing a 19.34%.+-.4.3% increase). The
mean arterial blood pressure (MAP) was not modified by the section
of the sympathetic nerves. Electrical stimulation of the
sympathetic nerve induced a frequency-depended increase of the SVR
whereas the MAP was not significantly modified. Sympathetic nerve
stimulation at 10 Hz for 5 minutes reduced the blood flow in the
maxillary artery from 4.92.+-.0.7 ml min.sup.-1 kg.sup.-1 to
1.40.+-.0.47 ml min.sup.-1 kg.sup.-1, representing an increase in
SVR of 71.5% and lasting more than 6 minutes. The i.a. infusion of
phenoxybenzamine induced a MAP reduction of 10.+-.2%. The basal
blood flow in the sphenopalatine artery, the heart rate and the MAP
were not significantly affected by the administration of exogenous
dipeptidyl peptidase IV.
[0040] Statistical Analysis
[0041] All values were expressed in terms of mean .+-.SEM.
Statistical analysis was done by analysis of variance, ANOVA. A
value of p<0.05 was taken as statistically significant.
[0042] II. Results
[0043] DPPIV is Expressed in Endothelial Cells and Submucosal
Seromucus Glands from Patients
[0044] Using immunohistochemistry, DPPIV-like immunoreactivity
(-LI) was detected in submucosal seromucus glands and leukocytes.
In submucosal seromucus glands, DPPIV-LI appeared to be located in
the apical position. Endothelial cells in blood vessels expressed
weak DPPIV-LI. Some epithelial cells located in the human nasal
mucosa were also DPPIV positive. However, these cells did not show
any positive immunoreactivity for CD1A and Protein 100, suggesting
they were not Langerhans cells.
[0045] Correlation Between DPPIV and Inflammation in the Human
Nasal Mucosa
[0046] Nasal mucosa biopsies were sampled from both nostrils in 45
patients suffering from rhinosinusitis to determine if DPPIV
activity was affected by the mucosal inflammation. DPPIV activity
was found to vary from undetectable to 707 pmol/min/mg.
Histological analysis revealed marked differences in the density of
inflammatory cells within the submucosa of the nasal biopsies
studied. A low activity of DPPIV was associated with a high density
of inflammatory cells in the nasal mucosa of patients with chronic
rhinosinusitis. When the density of inflammatory cells observed was
plotted against DPPIV activity, the regressive correlation was
found to be statistically significant (p<0.001). Nasal mucosa
samples obtained from smokers exhibited a significantly lower DPPIV
activity than samples obtained from non-smokers having the same low
density of inflammatory cells (p<0.01).
[0047] When the density of inflammatory cells in each biopsy was
plotted against the subjective evaluation of nasal obstruction, the
data fitted a statistically significant correlation (p<0.01),
suggesting that nasal inflammation increased in parallel with
subjective nasal obstruction severity. In addition, nasal mucosa
biopsies were obtained from 10 patients with significant
improvement of their symptoms (p<0.001), 6 months after
endonasal surgery. The DPPIV activity was significantly increased
in all of the samples studied when compared to the preoperative
state (p<0.001) indicating that DPPIV activity can be restored
when chronic rhinosinusitis is cured. Since there was a clear
negative correlation between DPPIV activity and nasal mucosal
inflammation, the hemodynamic effect of SP in the presence of
recombinant DPPIV was studied in pig nasal mucosa.
[0048] DPPIV Modulates Inflammatory Response Mediated by Histamine
in Pigs
[0049] Histamine is responsible for the early-phase allergic
reaction and exerts both direct and indirect effects on sensory
nerves, glands and blood vessels of the nasal mucosa (Alving, Acta.
Physiol. Scand. 597:1-64 (1991)). There is direct evidence that
histamine can release peptides from capsaicin sensitive sensory
nerves in the lung by activation of H.sub.I-receptors (Alving, et
al., Acta. Physiol. Scand. 138:49-60 (1990)). In addition,
endogenous or exogenous histamine stimulates sensory fibers,
possibly by acting on specific receptors to increase the release of
CGRP and SP (Tani, et al., Neurosci. Lett. 115:171-176 (1990).
[0050] Histamine i.a. local injection following sympathetic nerve
stimulation (SNS) at 10 Hz for 5 minutes showed a significant
reduction in duration at 5.4.times.10.sup.-09 moles (33%) and at
1.4.times.10.sup.-07 moles (32%) when compared to control
conditions. After i.a. administration of 50 .mu.g of DPPIV and SNS,
histamine challenge showed no significant variation in the area
under the curve (AUC) or vascular resistance (Vr) when compared to
control. However, the duration of histamine effect was reduced by
33.7% at a dose of 5.4.times.10.sup.-08 moles (p<0.05) and by
50.7% at a dose of 1.4.times.10.sup.-07 moles (p<0.01). After
phenoxybenzamine (PBZ) pretreatment, DPPIV and SNS, histamine
vasodilatory effects were markedly reduced. When compared to
control, the AUC showed a reduction of 81.9% at a dose of
5.4.times.10.sup.-08 moles (p<0.001) and a reduction of 81.8% at
a dose of 1.4.times.10.sup.-07 moles (p<0.001). Vr showed a
reduction of 67.9% at 5.4.times.10.sup.-08 moles (p<0.01) and a
reduction of 61.1% at 1.4.times.10.sup.-07 moles (p<0.05).
Duration of effect was also reduced at doses of
5.4.times.10.sup.-09 moles (42.6%, p<0.01); 5.4.times.10.sup.-08
moles (55.6%, p<0.001); and 1.4.times.10.sup.-07 moles (62.5%,
p<0.001).
[0051] DPPIV Modulates the Vasodilation Evoked by Capsaicin in
Pigs
[0052] Capsaicin (8-methyl-N-vanillyl-6-nonenamide), by activating
unmylinated sensory C-fibers, acts in many respects like histamine.
Capsaicin has been shown to release tachykinins (Hua, et al.,
Neurosci. 19:313-319 (1986)) as well as CGRP (Stjarne, et al.,
Regul. Pept. 33:251-262 (1991)) both in vitro and in vivo.
Capsaicin injection following SNS showed a dose-dependent decrease
of Vr similar that to observed under control conditions. The
vasodilatory effect of capsaicin after SNS and PBZ pretreatment
showed no significant variation at any doses. However, the duration
of vasodilation evoked at 8.2.times.10.sup.-08 moles was
significantly reduced (39.9%, p<0.01) and AUC was reduced by
26.6% at 8.2.times.10.sup.-08 moles (p<0.05). Capsaicin effects
were also modified after DPPIV and SNS. The AUC observed at a dose
of 8.2.times.10.sup.-08 moles showed a reduction by 56.6%
(p<0.01). The Vr response was reduced by 34.8% (p<0.05) and
the duration of effect at doses of 3.3.times.10.sup.-09 moles and
8.2.times.10.sup.-08 moles was reduced by 55.4% (p<0.05) and
50.5% (p<0.001) respectively. After PBZ, DPPIV and SNS, there
was marked reduction in Vr, duration of effect, and AUC. Vr at
8.2.times.10.sup.-08 moles was reduced by 39.9% (p<0.01). At
3.3.times.10.sup.-09 moles and 8.2.times.10.sup.-08 moles, the
duration of the vascular response was reduced by 64.3% (p<0.05)
and 56.8% (p<0.001). At 8.2.times.10.sup.-08 moles, AUC was
reduced by 66.7% (p<0.01).
[0053] DPPIV Modulates the Vasodilatory Response Mediated by
Bradykinin in Pigs
[0054] Bradykinin (BK) is a polypeptide involved in nociception and
humoral regulation of vascular tonicity and permeability. BK
produces marked vasodilation, increases capillary permeability and
is involved in most inflammatory reactions, including rhinitis
(Svensson, et al., J. Allergy Clin. Immunol. 85:828-833 (1990)).
Like histamine and capsaicin, BK stimulates nociceptive sensory
nerves to produce CGRP and SP (Hua, et al., Can. J. Physiol.,
Pharmacol. 73:999-1006 (1995); Vasco, et al., J. Neurosci.
14:4987-4997 (1994)).
[0055] BK injection following SNS showed a dose-dependent decrease
of Vr similar to that observed under control conditions. After
DPPIV and SNS, BK showed only variation in the duration of effect
at doses of 9.4.times.10.sup.-11 moles (34%, p<0.01) and
9.4.times.10.sup.-09 moles (38.3%, p<0.001). After PBV, DPPIV
and SNS, vascular parameters were reduced to a smaller extent than
that seen with histamine and capsaicin. For example, AUC after i.a.
administration of BK was reduced by 29.3% at 9.4.times.10.sup.-09
moles (p<0.01) and by 40% at 9.4.times.10.sup.-11 moles
(p<0.01). At 9.4.times.10.sup.-10 moles duration of effect was
reduced by 32.2% (p<0.05) and at 9.4.times.10.sup.-09 moles,
duration was reduced by 39.4% (p<0.001).
[0056] DDPIV Modulates the Vasodilatory Response Mediated by SP in
Pigs
[0057] Repeated injection of high doses of SP caused a reproducible
decrease in sphenopalatine vascular resistance, indicating that the
desensitization of neurokinin (NK) receptors did not occur.
Therefore, the same animal was used to perform dose-response curves
with SP both before and after pretreatment with DPPIV. A
dose-response curve for duration of vasodilation and area under the
curve was constructed using doses of SP in the range of 1 pg to 0.1
.mu.g. A reproducible dose-response curve was obtained over a 45
minute period using 6 doses of SP. Vasodilation ranged from 15.1%
.+-.3.3% to 37.3% .+-.3.3% after local intraarterial administration
of 1 pg and 100 ng of SP respectively. To determine whether
vasodilation evoked by SP was affected by pretreatment with DPPIV,
two doses of SP (100 pg and 1 ng) were administered and a reduction
in SVR of 13.1% and 14.9% was observed. The same animals received
50 .mu.g (530 pmoles) of DPPIV and similar doses of SP were
injected. Recombinant DPPIV had no vascular effect per se.
Subsequent administration of the SP resulted in a dramatic
diminution of SP-evoked vasodilatory response (by 66 and 71%
respectively) when compared to controls. Since DPPIV cleaves SP
into SP 5-11, experiments were conducted to determine whether SP
5-11 is also capable of decreasing SVR. Similar to what was
observed with respect to SP, a dose response curve was obtained
with 10 .mu.g to 1 .mu.g of SP 5-11 (5 to 35% decrease in SVR
respectively). However, the vasodilatory effect of SP 5-11 was
considerably lower when compared to SP. Based on molarity, SP is
200 fold more potent than SP 5-11 as a vasodilator. Therefore,
DPPIV administration to pigs might result in an almost immediate
conversion of SP 5-11.
[0058] To further characterize the nature of neurokinin receptor
subtype involved in the reduction of SVR, pigs were pretreated with
a NK1 antagonist, L-733060, at 114 nmoles/kg to evaluate the
blockade of SP-induced decreased in SVR. It was found that the
antagonist has a vasoconstrictive effect. A clear inhibition of the
SP effect was observed at all doses of SP used except 100 ng. Thus,
the SP effect on SVR is likely to mediated by the NK1 receptor.
[0059] III. Discussion
[0060] The localization of the enzyme DPPIV in the apical position
of nasal exocrine cells in seromucus glands suggests a role of this
enzyme in the protective function of nasal mucus. The presence of
DPPIV in vascular endothelial cells and T-cells is in line with
earlier histochemical studies of DPPIV in mammalian tissue (Sannes,
J. Histochem. Cytochem. 31:684-690 (1983)) as well as with more
recent reports on the distribution of human DPPIV (Van. Der. Elden,
et al., Clin. Exp. Allergy 28:110-120 (1998)). DPPIV
immunoreactivity was also observed in some intraepithelium cells of
nasal mucosa. These cells were probably not Langerhans cells since
they did not express CD1A or Protein 100 immunoreactivity on their
surface.
[0061] Correlation between DPPIV activity and inflammation of nasal
mucosa showed a marked decrease of enzyme activity in the presence
of severe inflammation. In agreement with Van der Velden, et al.
(Id.), who showed that DPPIV activity is reduced in the serum of
healthy smokers compared to non-smokers, DPPIV activity was reduced
in nasal samples with severe inflammation. In contrast, high DPPIV
enzymatic activity was correlated with a small number of
inflammatory cells in the nasal mucosa. The fact that enzyme
activity increased after treatment of chronic rhinosinusitis is
also consistent with the involvement of the enzyme in this
pathology. Pretreatment with 50 ug of DPPIV significantly decreased
duration of vasodilation and spenopalatine artery vascular
resistance when DPPIV was used alone or with the adrenoreceptor
blocker phenoxybenzamine.
[0062] DPPIV significantly decreased both peak and duration of the
vasodilation evoked by SP. It was found that the SP 5-11 was at
least 100 fold less potent than SP at causing vasodilation. These
results suggest that a lost of DPPIV expression occurs during
chronic rhinosinusitis and that the resulting reduction of SP
degradation contributes to the maintenance of nasal mucosa
inflammation. In this regard, Nieber, et al. have shown that
allergic patients have higher baseline levels of SP-ir in nasal
lavage fluids than non-allergic controls and that intranasal
allergen increases SP levels in nasal lavage fluids only in
subjects with grass pollen allergy (J. Allergy Clin. Immunol. 4
pt1:646-652 (1992)). The antibody used for the SP assay in the
Nieber study required both an intact N- and C-terminus peptide
sequence. The data is in line with that presented herein and could
be explained by the fact that allergic patients expressing less
DDPIV than normal volunteers produce intact SP which could be
measured by their antibody. In contrast, normal volunteers cleave
their SP into SP 5-11 fragments which are no longer detected in the
SP assay. SP effects on the vascular bed of the nasal mucosa are
maintained only when allergenic stimulation persists in subjects
with nasal allergies (i.e., subjects without DPPIV).
[0063] In parallel to SP cleavage by DPPIV into SP 5-11, a
N-terminal fragment, SP 1-4, is generated and has been reported to
exert an antagonist affect on SP action (Sakurada, et al., J.
Pharm. Sci. 88: 1127-1132 (1999)). Thus, DPPIV might modulate SP
action, not only by producing a less active NK1 agonist, but also
by interfering with SP effects. Since SP 5-11 (but not SP) is
subject to cellular uptake, DPPIV may also act to terminate the
action of SP via conversion to a form which is removed by cellular
processes (Nakata, et al., J. Neurochem. 37:1529-1534 (1981)). Once
SP is cleaved by DPPIV into SP 5-11, this fragment is further
processed with a high efficiency by aminopeptidase M into inactive
fragments. Thus, DPPIV first converts SP to a much less active
form, SP 5-11, which is then completely inactivated by the action
of aminopeptidase M. Finally, DPPIV could potentially act upon
other peptides which are known to be involved in promoting
inflammation and which have structures that lend themselves to
degradation by this enzyme.
[0064] All references cited are fully incorporated by reference.
Having now fully described the invention, it will be understood by
those of skill in the art that the invention may be performed
within a wide and equivalent range of conditions, parameters and
the like, without affecting the spirit or scope of the invention or
any embodiment thereof.
Sequence CWU 1
1
4 1 766 PRT Homo sapiens 1 Met Lys Thr Pro Trp Lys Val Leu Leu Gly
Leu Leu Gly Ala Ala Ala 1 5 10 15 Leu Val Thr Ile Ile Thr Val Pro
Val Val Leu Leu Asn Lys Gly Thr 20 25 30 Asp Asp Ala Thr Ala Asp
Ser Arg Lys Thr Tyr Thr Leu Thr Asp Tyr 35 40 45 Leu Lys Asn Thr
Tyr Arg Leu Lys Leu Tyr Ser Leu Arg Trp Ile Ser 50 55 60 Asp His
Glu Tyr Leu Tyr Lys Gln Glu Asn Asn Ile Leu Val Phe Asn 65 70 75 80
Ala Glu Tyr Gly Asn Ser Ser Val Phe Leu Glu Asn Ser Thr Phe Asp 85
90 95 Glu Phe Gly His Ser Ile Asn Asp Tyr Ser Ile Ser Pro Asp Gly
Gln 100 105 110 Phe Ile Leu Leu Glu Tyr Asn Tyr Val Lys Gln Trp Arg
His Ser Tyr 115 120 125 Thr Ala Ser Tyr Asp Ile Tyr Asp Leu Asn Lys
Arg Gln Leu Ile Thr 130 135 140 Glu Glu Arg Ile Pro Asn Asn Thr Gln
Trp Val Thr Trp Ser Pro Val 145 150 155 160 Gly His Lys Leu Ala Tyr
Val Trp Asn Asn Asp Ile Tyr Val Lys Ile 165 170 175 Glu Pro Asn Leu
Pro Ser Tyr Arg Ile Thr Trp Thr Gly Lys Glu Asp 180 185 190 Ile Ile
Tyr Asn Gly Ile Thr Asp Trp Val Tyr Glu Glu Glu Val Phe 195 200 205
Ser Ala Tyr Ser Ala Leu Trp Trp Ser Pro Asn Gly Thr Phe Leu Ala 210
215 220 Tyr Ala Gln Phe Asn Asp Thr Glu Val Pro Leu Ile Glu Tyr Ser
Phe 225 230 235 240 Tyr Ser Asp Glu Ser Leu Gln Tyr Pro Lys Thr Val
Arg Val Pro Tyr 245 250 255 Pro Lys Ala Gly Ala Val Asn Pro Thr Val
Lys Phe Phe Val Val Asn 260 265 270 Thr Asp Ser Leu Ser Ser Val Thr
Asn Ala Thr Ser Ile Gln Ile Thr 275 280 285 Ala Pro Ala Ser Met Leu
Ile Gly Asp His Tyr Leu Cys Asp Val Thr 290 295 300 Trp Ala Thr Gln
Glu Arg Ile Ser Leu Gln Trp Leu Arg Arg Ile Gln 305 310 315 320 Asn
Tyr Ser Val Met Asp Ile Cys Asp Tyr Asp Glu Ser Ser Gly Arg 325 330
335 Trp Asn Cys Leu Val Ala Arg Gln His Ile Glu Met Ser Thr Thr Gly
340 345 350 Trp Val Gly Arg Phe Arg Pro Ser Glu Pro His Phe Thr Leu
Asp Gly 355 360 365 Asn Ser Phe Tyr Lys Ile Ile Ser Asn Glu Glu Gly
Tyr Arg His Ile 370 375 380 Cys Tyr Phe Gln Ile Asp Lys Lys Asp Cys
Thr Phe Ile Thr Lys Gly 385 390 395 400 Thr Trp Glu Val Ile Gly Ile
Glu Ala Leu Thr Ser Asp Tyr Leu Tyr 405 410 415 Tyr Ile Ser Asn Glu
Tyr Lys Gly Met Pro Gly Gly Arg Asn Leu Tyr 420 425 430 Lys Ile Gln
Leu Ser Asp Tyr Thr Lys Val Thr Cys Leu Ser Cys Glu 435 440 445 Leu
Asn Pro Glu Arg Cys Gln Tyr Tyr Ser Val Ser Phe Ser Lys Glu 450 455
460 Ala Lys Tyr Tyr Gln Leu Arg Cys Ser Gly Pro Gly Leu Pro Leu Tyr
465 470 475 480 Thr Leu His Ser Ser Val Asn Asp Lys Gly Leu Arg Val
Leu Glu Asp 485 490 495 Asn Ser Ala Leu Asp Lys Met Leu Gln Asn Val
Gln Met Pro Ser Lys 500 505 510 Lys Leu Asp Phe Ile Ile Leu Asn Glu
Thr Lys Phe Trp Tyr Gln Met 515 520 525 Ile Leu Pro Pro His Phe Asp
Lys Ser Lys Lys Tyr Pro Leu Leu Leu 530 535 540 Asp Val Tyr Ala Gly
Pro Cys Ser Gln Lys Ala Asp Thr Val Phe Arg 545 550 555 560 Leu Asn
Trp Ala Thr Tyr Leu Ala Ser Thr Glu Asn Ile Ile Val Ala 565 570 575
Ser Phe Asp Gly Arg Gly Ser Gly Tyr Gln Gly Asp Lys Ile Met His 580
585 590 Ala Ile Asn Arg Arg Leu Gly Thr Phe Glu Val Glu Asp Gln Ile
Glu 595 600 605 Ala Ala Arg Gln Phe Ser Lys Met Gly Phe Val Asp Asn
Lys Arg Ile 610 615 620 Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val
Thr Ser Met Val Leu 625 630 635 640 Gly Ser Gly Ser Gly Val Phe Lys
Cys Gly Ile Ala Val Ala Pro Val 645 650 655 Ser Arg Trp Glu Tyr Tyr
Asp Ser Val Tyr Thr Glu Arg Tyr Met Gly 660 665 670 Leu Pro Thr Pro
Glu Asp Asn Leu Asp His Tyr Arg Asn Ser Thr Val 675 680 685 Met Ser
Arg Ala Glu Asn Phe Lys Gln Val Glu Tyr Leu Leu Ile His 690 695 700
Gly Thr Ala Asp Asp Asn Val His Phe Gln Gln Ser Ala Gln Ile Ser 705
710 715 720 Lys Ala Leu Val Asp Val Gly Val Asp Phe Gln Ala Met Trp
Tyr Thr 725 730 735 Asp Glu Asp His Gly Ile Ala Ser Ser Thr Ala His
Gln His Ile Tyr 740 745 750 Thr His Met Ser His Phe Ile Lys Gln Cys
Phe Ser Leu Pro 755 760 765 2 492 PRT Homo sapiens 2 Met Gly Ser
Ala Pro Trp Ala Pro Val Leu Leu Leu Ala Leu Gly Leu 1 5 10 15 Arg
Gly Leu Gln Ala Gly Ala Arg Arg Ala Pro Asp Pro Gly Phe Gln 20 25
30 Glu Arg Phe Phe Gln Gln Arg Leu Asp His Phe Asn Phe Glu Arg Phe
35 40 45 Gly Asn Lys Thr Phe Pro Gln Arg Phe Leu Val Ser Asp Arg
Phe Trp 50 55 60 Val Arg Gly Glu Gly Pro Thr Phe Phe Tyr Thr Gly
Asn Glu Gly Asp 65 70 75 80 Val Trp Ala Phe Ala Asn Asn Ser Gly Phe
Val Ala Glu Leu Ala Ala 85 90 95 Glu Arg Gly Ala Leu Leu Val Phe
Ala Glu His Arg Tyr Tyr Gly Lys 100 105 110 Ser Leu Pro Phe Gly Ala
Gln Ser Thr Gln Arg Gly His Thr Glu Leu 115 120 125 Leu Thr Val Glu
Gln Ala Leu Ala Asp Phe Ala Glu Leu Leu Arg Ala 130 135 140 Leu Arg
Arg Asp Leu Gly Ala Gln Asp Ala Pro Ala Ile Ala Phe Gly 145 150 155
160 Gly Ser Tyr Gly Gly Met Leu Ser Ala Tyr Leu Arg Met Lys Tyr Pro
165 170 175 His Leu Val Ala Gly Ala Leu Ala Ala Ser Ala Pro Val Leu
Ala Val 180 185 190 Ala Gly Leu Gly Asp Ser Asn Gln Phe Phe Arg Asp
Val Thr Ala Asp 195 200 205 Phe Glu Gly Gln Ser Pro Lys Cys Thr Gln
Gly Val Arg Glu Ala Phe 210 215 220 Arg Gln Ile Lys Asp Leu Phe Leu
Gln Gly Ala Tyr Asp Thr Val Arg 225 230 235 240 Trp Glu Phe Gly Thr
Cys Gln Pro Leu Ser Asp Glu Lys Asp Leu Thr 245 250 255 Gln Leu Phe
Met Phe Ala Arg Asn Ala Phe Thr Val Leu Ala Met Met 260 265 270 Asp
Tyr Pro Tyr Pro Thr Asp Phe Leu Gly Pro Leu Pro Ala Asn Pro 275 280
285 Val Lys Val Gly Cys Asp Arg Leu Leu Ser Glu Ala Gln Arg Ile Thr
290 295 300 Gly Leu Arg Ala Leu Ala Gly Leu Val Tyr Asn Ala Ser Gly
Ser Glu 305 310 315 320 His Cys Tyr Asp Ile Tyr Arg Leu Tyr His Ser
Cys Ala Asp Pro Thr 325 330 335 Gly Cys Gly Thr Gly Pro Asp Ala Arg
Ala Trp Asp Tyr Gln Ala Cys 340 345 350 Thr Glu Ile Asn Leu Thr Phe
Ala Ser Asn Asn Val Thr Asp Met Phe 355 360 365 Pro Asp Leu Pro Phe
Thr Asp Glu Leu Arg Gln Arg Tyr Cys Leu Asp 370 375 380 Thr Trp Gly
Val Trp Pro Arg Pro Asp Trp Leu Leu Thr Ser Phe Trp 385 390 395 400
Gly Gly Asp Leu Arg Ala Ala Ser Asn Ile Ile Phe Ser Asn Gly Asn 405
410 415 Leu Asp Pro Trp Ala Gly Gly Gly Ile Arg Arg Asn Leu Ser Ala
Ser 420 425 430 Val Ile Ala Val Thr Ile Gln Gly Gly Ala His His Leu
Asp Leu Arg 435 440 445 Ala Ser His Pro Glu Asp Pro Ala Ser Val Val
Glu Ala Arg Lys Leu 450 455 460 Glu Ala Thr Ile Ile Gly Glu Trp Val
Lys Ala Ala Arg Arg Glu Gln 465 470 475 480 Gln Pro Ala Leu Arg Gly
Gly Pro Arg Leu Ser Leu 485 490 3 1198 PRT Homo sapiens 3 Met Val
Ala Ala Ala Ala Ala Thr Glu Ala Arg Leu Arg Arg Arg Thr 1 5 10 15
Ala Ala Thr Ala Ala Leu Ala Gly Arg Ser Gly Gly Pro His Cys Val 20
25 30 Asn Gly Gly Arg Cys Asn Pro Gly Thr Gly Gln Cys Val Cys Pro
Ala 35 40 45 Gly Trp Val Gly Glu Gln Cys Gln His Cys Gly Gly Arg
Phe Arg Leu 50 55 60 Thr Gly Ser Ser Gly Phe Val Thr Asp Gly Pro
Gly Asn Tyr Lys Tyr 65 70 75 80 Lys Thr Lys Cys Thr Trp Leu Ile Glu
Gly Gln Pro Asn Arg Ile Met 85 90 95 Arg Leu Arg Phe Asn His Phe
Ala Thr Glu Cys Ser Trp Asp His Leu 100 105 110 Tyr Val Tyr Asp Gly
Asp Ser Ile Tyr Ala Pro Leu Val Ala Ala Phe 115 120 125 Ser Gly Leu
Ile Val Pro Glu Arg Asp Gly Asn Glu Thr Val Pro Glu 130 135 140 Val
Val Ala Thr Ser Gly Tyr Ala Leu Leu His Phe Phe Ser Asp Ala 145 150
155 160 Ala Tyr Asn Leu Thr Gly Phe Asn Ile Thr Tyr Ser Phe Asp Met
Cys 165 170 175 Pro Asn Asn Cys Ser Gly Arg Gly Glu Cys Lys Ile Ser
Asn Ser Ser 180 185 190 Asp Thr Val Glu Cys Glu Cys Ser Glu Asn Trp
Lys Gly Glu Ala Cys 195 200 205 Asp Ile Pro His Cys Thr Asp Asn Cys
Gly Phe Pro His Arg Gly Ile 210 215 220 Cys Asn Ser Ser Asp Val Arg
Gly Cys Ser Cys Phe Ser Asp Trp Gln 225 230 235 240 Gly Pro Gly Cys
Ser Val Pro Val Pro Ala Asn Gln Ser Phe Trp Thr 245 250 255 Arg Glu
Glu Tyr Ser Asn Leu Lys Leu Pro Arg Ala Ser His Lys Ala 260 265 270
Val Val Asn Gly Asn Ile Met Trp Val Val Gly Gly Tyr Met Phe Asn 275
280 285 His Ser Asp Tyr Asn Met Val Leu Ala Tyr Asp Leu Ala Ser Arg
Glu 290 295 300 Trp Leu Pro Leu Asn Arg Ser Val Asn Asn Val Val Val
Arg Tyr Gly 305 310 315 320 His Ser Leu Ala Leu Tyr Lys Asp Lys Ile
Tyr Met Tyr Gly Gly Lys 325 330 335 Ile Asp Ser Thr Gly Asn Val Thr
Asn Glu Leu Arg Val Phe His Ile 340 345 350 His Asn Glu Ser Trp Val
Leu Leu Thr Pro Lys Ala Lys Glu Gln Tyr 355 360 365 Ala Val Val Gly
His Ser Ala His Ile Val Thr Leu Lys Asn Gly Arg 370 375 380 Val Val
Met Leu Val Ile Phe Gly His Cys Pro Leu Tyr Gly Tyr Ile 385 390 395
400 Ser Asn Val Gln Glu Tyr Asp Leu Asp Lys Asn Thr Trp Ser Ile Leu
405 410 415 His Thr Gln Gly Ala Leu Val Gln Gly Gly Tyr Gly His Ser
Ser Val 420 425 430 Tyr Asp His Arg Thr Arg Ala Leu Tyr Val His Gly
Gly Tyr Lys Ala 435 440 445 Phe Ser Ala Asn Lys Tyr Arg Leu Ala Asp
Asp Leu Tyr Arg Tyr Asp 450 455 460 Val Asp Thr Gln Met Trp Thr Ile
Leu Lys Asp Ser Arg Phe Phe Arg 465 470 475 480 Tyr Leu His Thr Ala
Val Ile Val Ser Gly Thr Met Leu Val Phe Gly 485 490 495 Gly Asn Thr
His Asn Asp Thr Ser Met Ser His Gly Ala Lys Cys Phe 500 505 510 Ser
Ser Asp Phe Met Ala Tyr Asp Ile Ala Cys Asp Arg Trp Ser Val 515 520
525 Leu Pro Arg Pro Asp Ser Thr Met Met Ser Thr Asp Leu Ala Ile Pro
530 535 540 Ala Val Leu His Asn Ser Thr Met Tyr Val Phe Gly Gly Phe
Asn Ser 545 550 555 560 Leu Leu Leu Ser Asp Ile Leu Val Phe Thr Ser
Glu Gln Cys Asp Ala 565 570 575 His Arg Ser Glu Ala Ala Cys Leu Ala
Ala Gly Pro Gly Ile Arg Cys 580 585 590 Val Trp Asn Thr Gly Ser Ser
Gln Cys Ile Ser Trp Ala Leu Ala Thr 595 600 605 Asp Glu Gln Glu Glu
Lys Leu Lys Ser Glu Cys Phe Ser Lys Arg Thr 610 615 620 Leu Asp His
Asp Arg Cys Asp Gln His Thr Asp Cys Tyr Ser Cys Thr 625 630 635 640
Ala Asn Thr Asn Asp Cys His Trp Cys Asn Asp His Cys Val Pro Arg 645
650 655 Asn His Ser Cys Ser Glu Gly Gln Ile Ser Ile Phe Arg Tyr Glu
Asn 660 665 670 Cys Pro Lys Asp Asn Pro Met Tyr Tyr Cys Asn Lys Lys
Thr Ser Cys 675 680 685 Arg Ser Cys Ala Leu Asp Gln Asn Cys Gln Trp
Glu Pro Arg Asn Gln 690 695 700 Glu Cys Ile Ala Leu Pro Glu Asn Ile
Cys Gly Ile Gly Trp His Leu 705 710 715 720 Val Gly Asn Ser Cys Leu
Lys Ile Thr Thr Ala Lys Glu Asn Tyr Asp 725 730 735 Asn Ala Lys Leu
Phe Cys Arg Asn His Asn Ala Leu Leu Ala Ser Leu 740 745 750 Thr Thr
Gln Lys Lys Val Glu Phe Val Leu Lys Gln Leu Arg Ile Met 755 760 765
Gln Ser Ser Gln Ser Met Ser Lys Leu Thr Leu Thr Pro Trp Val Gly 770
775 780 Leu Arg Lys Ile Asn Val Ser Tyr Trp Cys Trp Glu Asp Met Ser
Pro 785 790 795 800 Phe Thr Asn Ser Leu Leu Gln Trp Met Pro Ser Glu
Pro Ser Asp Ala 805 810 815 Gly Phe Cys Gly Ile Leu Ser Glu Pro Ser
Thr Arg Gly Leu Lys Ala 820 825 830 Ala Thr Cys Ile Asn Pro Leu Asn
Gly Ser Val Cys Glu Arg Pro Ala 835 840 845 Asn His Ser Ala Lys Gln
Cys Arg Thr Pro Cys Ala Leu Arg Thr Ala 850 855 860 Cys Gly Asp Cys
Thr Ser Gly Ser Ser Glu Cys Met Trp Cys Ser Asn 865 870 875 880 Met
Lys Gln Cys Val Asp Ser Asn Ala Tyr Val Ala Ser Phe Pro Phe 885 890
895 Gly Gln Cys Met Glu Trp Tyr Thr Met Ser Thr Cys Pro Pro Glu Asn
900 905 910 Cys Ser Gly Tyr Cys Thr Cys Ser His Cys Leu Glu Gln Pro
Gly Cys 915 920 925 Gly Trp Cys Thr Asp Pro Ser Asn Thr Gly Lys Gly
Lys Cys Ile Glu 930 935 940 Gly Ser Tyr Lys Gly Pro Val Lys Met Pro
Ser Gln Ala Pro Thr Gly 945 950 955 960 Asn Phe Tyr Pro Gln Pro Leu
Leu Asn Ser Ser Met Cys Leu Glu Asp 965 970 975 Ser Arg Tyr Asn Trp
Ser Phe Ile His Cys Pro Ala Cys Gln Cys Asn 980 985 990 Gly His Ser
Lys Cys Ile Asn Gln Ser Ile Cys Glu Lys Cys Glu Asn 995 1000 1005
Leu Thr Thr Gly Lys His Cys Glu Thr Cys Ile Ser Gly Phe Tyr 1010
1015 1020 Gly Asp Pro Thr Asn Gly Gly Lys Cys Gln Pro Cys Lys Cys
Asn 1025 1030 1035 Gly His Ala Ser Leu Cys Asn Thr Asn Thr Gly Lys
Cys Phe Cys 1040 1045 1050 Thr Thr Lys Gly Val Lys Gly Asp Glu Cys
Gln Leu Cys Glu Val 1055 1060 1065 Glu Asn Arg Tyr Gln Gly Asn Pro
Leu Arg Gly Thr Cys Tyr Tyr 1070 1075 1080 Thr Leu Leu Ile Asp Tyr
Gln Phe Thr Phe Ser Leu Ser Gln Glu 1085 1090 1095 Asp Asp Arg Tyr
Tyr Thr Ala Ile Asn Phe Val Ala Thr Pro Asp 1100 1105 1110 Glu Gln
Asn Arg Asp Leu Asp Met Phe Ile Asn Ala Ser Lys Asn 1115 1120 1125
Phe Asn Leu Asn Ile Thr Trp Ala Ala Ser Phe Ser Ala Gly Thr 1130
1135 1140 Gln Ala Gly Glu Glu Met Pro Val Val Ser Lys Thr Asn Ile
Lys 1145 1150 1155 Glu Tyr Lys Asp Ser Phe Ser Asn Glu Lys Phe Asp
Phe Arg Asn 1160 1165 1170 His Pro Asn Ile Thr Phe Phe Val Tyr
Val Ser Asn Phe Thr Trp 1175 1180 1185 Pro Ile Lys Ile Gln Val Gln
Thr Glu Gln 1190 1195 4 310 PRT Homo sapiens 4 Phe Glu Gly Thr Lys
Asp Ser Pro Leu Glu His His Leu Tyr Val Val 1 5 10 15 Ser Tyr Val
Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg Gly Tyr 20 25 30 Ser
His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser Lys 35 40
45 Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser
50 55 60 Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe Trp
Ala Thr 65 70 75 80 Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr
Pro Pro Glu Ile 85 90 95 Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr
Leu Tyr Gly Met Leu Tyr 100 105 110 Lys Pro His Asp Leu Gln Pro Gly
Lys Lys Tyr Pro Thr Val Leu Phe 115 120 125 Ile Tyr Gly Gly Pro Gln
Gly Gln Ile Glu Ile Asp Asp Gln Val Glu 130 135 140 Gly Leu Gln Tyr
Leu Ala Ser Arg Tyr Asp Phe Ile Asp Leu Asp Arg 145 150 155 160 Val
Gly Ile His Gly Trp Ser Tyr Gly Gly Tyr Leu Ser Leu Met Ala 165 170
175 Leu Met Gln Arg Ser Asp Ile Phe Arg Val Ala Ile Ala Gly Ala Pro
180 185 190 Val Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg
Tyr Met 195 200 205 Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu
Gly Ser Val Ala 210 215 220 Met Gln Ala Glu Lys Phe Pro Ser Glu Pro
Asn Arg Leu Leu Leu Leu 225 230 235 240 His Gly Phe Leu Asp Glu Asn
Val His Phe Ala His Thr Ser Ile Leu 245 250 255 Leu Ser Phe Leu Val
Arg Ala Gly Lys Pro Tyr Asp Leu Gln Ile Tyr 260 265 270 Pro Gln Glu
Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr 275 280 285 Glu
Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile 290 295
300 Ala Ala Leu Lys Val Ile 305 310
* * * * *