U.S. patent application number 11/398989 was filed with the patent office on 2006-08-03 for method of treating chronic cardiac disease.
This patent application is currently assigned to XOMA Technology Ltd.. Invention is credited to Brett P. Giroir, Patrick J. Scannon.
Application Number | 20060171937 11/398989 |
Document ID | / |
Family ID | 22368911 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060171937 |
Kind Code |
A1 |
Giroir; Brett P. ; et
al. |
August 3, 2006 |
Method of treating chronic cardiac disease
Abstract
New therapeutic uses for BPI protein products that involve
treatment of chronic cardiac disease.
Inventors: |
Giroir; Brett P.; (Dallas,
TX) ; Scannon; Patrick J.; (San Francisco,
CA) |
Correspondence
Address: |
ANNE DOLLARD;XOMA (US) LLC
2910 SEVENTH STREET
BERKELEY
CA
94710
US
|
Assignee: |
XOMA Technology Ltd.
Berkeley
CA
THE UNIVERSITY OF TEXAS
San Antonio
TX
|
Family ID: |
22368911 |
Appl. No.: |
11/398989 |
Filed: |
April 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10342169 |
Jan 14, 2003 |
7045501 |
|
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11398989 |
Apr 5, 2006 |
|
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09488979 |
Jan 21, 2000 |
6509317 |
|
|
10342169 |
Jan 14, 2003 |
|
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60116736 |
Jan 22, 1999 |
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Current U.S.
Class: |
424/94.1 ;
514/12.4; 514/16.4; 514/2.1; 514/20.6 |
Current CPC
Class: |
A61K 38/1751 20130101;
A61P 9/00 20180101; A61P 31/04 20180101; A61P 9/04 20180101 |
Class at
Publication: |
424/094.1 ;
514/012 |
International
Class: |
A61K 38/43 20060101
A61K038/43 |
Claims
1. A method of treating a human with chronic cardiac disease
comprising the step of administering a therapeutically effective
amount of a bactericidal/permeability-increasing (BPI) protein
product to said human.
2. The method of claim 1 wherein the BPI protein product is
rBPI.sub.21.
3. The method of claim 1 wherein the chronic cardiac disease is
chronic congestive heart failure.
4. The method of claim 1 wherein the chronic cardiac disease is
cardiomyopathy.
5. The method of claim 1 wherein the chronic cardiac disease is a
congenital heart defect.
6. The method of claim 1 wherein the human exhibits elevated levels
of circulating LPS.
7. The method of claim 1 wherein the human exhibits elevated levels
of circulating LBP.
8. The method of claim 1 wherein the human exhibits elevated levels
of circulating LPS and circulating LBP.
9. The method of claim 1 further comprising concurrently
administering a second therapeutic agent for treating the chronic
cardiac disease state.
10. The method of claim 9 wherein said chronic cardiac disease
state is chronic congestive heart failure and the second
therapeutic agent is selected from the group consisting of
diuretics, positive inotropic agents, vasodilators and
beta-blockers.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/342,169, filed Jan. 14, 2003, which is a
continuation of U.S. patent application Ser. No. 09/488,979, filed
Jan. 21, 2000, now U.S. Pat. No. 6,509,317, and which claims
priority of U.S. provisional application Ser. No. 60/11.6,736 filed
Jan. 22, 1999, the disclosures of which are all incorporated herein
by reference.
[0002] The present invention relates generally to novel therapeutic
uses of BPI protein products that involve treatment of chronic
cardiac disease including, but not limited to, chronic states such
as congestive heart failure and cardiomyopathy.
BACKGROUND OF THE INVENTION
[0003] Chronic cardiac disease is a leading cause of mortality and
morbidity in the developed world. One type of chronic cardiac
disease is cardiomyopathy, which is actually a diverse group of
diseases characterized by myocardial dysfunction that is not
related to the usual causes of heart disease such as coronary
atherosclerosis, valvular dysfunction and hypertension.
Cardiomyopathies are categorized hemodynamically into dilated,
hypertrophic, restrictive and obliterative cardiomyopathy, and can
be of known or idiopathic etiology. Among the etiologies of dilated
cardiomyopathy are pregnancy, drugs and toxins, such as alcohol,
cocaine and chemotherapeutic agents (including doxorubicin and
daunorubicin, dactinomycin, dacarbazine, cyclophosphamide,
mitomycin, and anthracycline), and infectious and autoimmune
processes. Hypertrophic cardiomyopathy is hereditary in more than
50% of cases and has a distinctive pattern of myocardial
hypertrophy (thickening of muscle) usually with a pattern of
asymmetrical thickening of the interventricular septum (also called
asymmetrical septal hypertrophy). Restrictive cardiomyopathies are
usually the product of an infiltrative disease of the myocardium,
such as amyloidosis, hemochromatosis or a glycogen storage disease,
and may also be seen in certain diabetic patients. Obliterative
cardiomyopathy can be caused by endomyocardial fibrosis and
hypereosinophilic syndrome. A common complication of all of the
cardiomyopathies is progressive congestive heart failure.
[0004] Congestive heart failure is often defined as the inability
of the heart to deliver a supply of oxygenated blood sufficient to
meet the metabolic needs of peripheral tissues at normal filling
pressures. Chronic congestive heart failure can result as a
consequence of coronary artery disease, cardiomyopathy,
myocarditis, aortic stenosis, hypertension, idiopathic asymmetrical
septal hypertrophy, coarctation of the aorta, aortic regurgitation,
mitral regurgitation, left-to-right shunts, hypertrophied muscle,
pericardial tamponade, constrictive pericarditis, mitral stenosis,
left atrial mzxoma, left atrial thrombus, cor triatriatum and
numerous other conditions. Congestive heart failure is generally
distinguished from other causes of inadequate oxygen delivery, such
as circulatory collapse from hemorrhage or other causes of severe
volume loss, congestion caused by fluid overload and high-output
failure caused by increased peripheral demands which occurs in
conditions such as thyrotoxicosis, arteriovenous fistula, Paget's
disease and anemia. Therapy for congestive heart failure typically
focuses on the treating the underlying etiology and the symptoms of
fluid overload and heart failure. Chronic congestive heart failure
that persists after correction of reversible causes is treated with
diuretics (including thiazides such as chlorothiazide and
hydrochlorothiazide, loop diuretics such as ethacrynic acid,
furosemide, torsemide and bumetanide, potassium sparing agents such
as spironolactone, triamterene and amiloride, and others such as
metolazone and other quinazoline-sulfonamides), vasodilators
(including nitroglycerin, isosorbide dinitrate, hydralazine, sodium
nitroprusside, prostacyclin, captopril, enalapril, lisinopril,
quinapril and losartan), positive inotropic agents (such as
digitalis or digoxin), occasionally beta blockers, or combinations
of these measures.
[0005] Recent studies indicate that an increase in pro-inflammatory
cytokines is seen in diverse cardiac diseases, including congestive
heart failure, cardiomyopathy, and myocarditis. Hegewisch S, et al.
Lancet 1990;2:294-295; Levine B, et al., N. Engl. J. Med. 1990;323
(4):236-241; Mann D L, et al., Chest 1994;105:897-904; and Givertz
M M, et al., Lancet 1998;352:34-38 For example, the cytokine tumor
necrosis factor-* (TNF) is synthesized by human cardiac myocytes,
and the level of TNF expression correlates with the degree of
cardiac dysfunction in patients. Torre-Amione G, et al., J. Am.
Coll. Cardiol. 1996;27:1201-1206; Torre-Amione G, R D, et al.
Circulation 1995;92:1487-1493; and Torre-Amione G, et al.,
Circulation 1996;93:704-711 In animals, synthesis of TNF by the
heart is itself sufficient to cause cardiomyopathy and lethal
cardiac failure. Bryant D, et al., Circ. 1998;97:175-183 and Kubota
T, et al. J. Am. Coll. Cardiol. 1997;346A(Abstract) Furthermore,
early human trials have demonstrated that antagonism of TNF
improves cardiac failure in humans with NYHA Class III heart
failure or idiopathic dilated cardiomyopathy. Deswal et al.,
Circulation 96: 1-323 (1997); and Sliwa et al., Lancet 351:
1091-1093 (1998) However, the primary stimulus for cytokine
secretion remains unknown.
[0006] Bacterial endotoxin, or lipopolysaccharide (LPS), is a
primary inducer of TNF production during sepsis. With respect to
cardiac diseases, the role of endotoxin has been examined primarily
in the context of cardiopulmonary bypass, driven by the hypothesis
that endotoxin may be present in the extracorporeal circuit, or may
be translocated across the intestine secondary to non-pulsatile,
low flow perfusion. Riddington D W, et al. JAMA 1996;275:1007-1012
and Wan S, et al., Chest 1997;1 12:676-692 These studies have
demonstrated only transient low-level endotoxemia during
cardiopulmonary bypass, with rapid resolution following completion
of cardiopulmonary bypass in the majority of patients. Nilsson L, J
Thorac Cardiovasc Surg 1990;100:777-780; Casey W F, Crit. Care Med.
1992;20 (8):1090-1096; Khabar K S, et al., Clin Immunol
Immunopathol 1997;85:97-103; Jansen N J, Ann Thorac Surg
1992;54:744-747. Bennett-Guerrero E et al., JAMA 1997;277:646-650
reported that lower levels of anti-endotoxin antibodies
pre-operatively were associated with an increased risk of
post-operative complications and hypothesized that this difference
was due to a poor immunity to endotoxin.
[0007] Investigators have thus far failed to demonstrate, or failed
to attempt to demonstrate, persistent endotoxemia in a majority of
patients with cardiac disease Nilsson L, J Thorac Cardiovasc Surg
1990; 100:777-780; Casey W F, Crit. Care Med. 1992;20
(8):1090-1096; Khabar K S, et al., Clin Immunol Immunopathol
1997;85:97-103; Jansen N J, Ann Thorac Surg 1992;54:744-747. See
also Niebauer J, Eur. Heart J. 1998;19:174, which reported elevated
levels of plasma endotoxin in adults with edemetous chronic
congestive heart failure that was not associated with elevated
levels of LBP or anti-endotoxin antibodies (indicators of long-term
endotoxin exposure).
[0008] BPI is a protein isolated from the granules of mammalian
polymorphonuclear leukocytes (PMNs or neutrophils), which are blood
cells essential in the defense against invading microorganisms.
Human BPI protein has been isolated from PMNs by acid extraction
combined with either ion exchange chromatography [Elsbach, J Biol.
Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss,
et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is
referred to herein as natural BPI and has been shown to have potent
bactericidal activity against a broad spectrum of gram-negative
bacteria. The molecular weight of human BPI is approximately 55,000
daltons (55 kD). The amino acid sequence of the entire human BPI
protein and the nucleic acid sequence of DNA encoding the protein
have been reported in FIG. 1 of Gray et al., J Biol. Chem.,
264:9505 (1989), incorporated herein by reference. The Gray et al.
amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat.
No. 5,198,541 discloses recombinant genes encoding and methods for
expression of BPI proteins, including BPI holoprotein and fragments
of BPI.
[0009] BPI is a strongly cationic protein. The N-terminal half of
BPI accounts for the high net positive charge; the C-terminal half
of the molecule has a net charge of -3. [Elsbach and Weiss (1981),
supra.] A proteolytic N-terminal fragment of BPI having a molecular
weight of about 25 kD possesses essentially all the anti-bacterial
efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi
et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the
N-terminal portion, the C-terminal region of the isolated human BPI
protein displays only slightly detectable anti-bacterial activity
against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649
(1991).] An N-terminal BPI fragment of approximately 23 kD,
referred to as "rBPI.sub.23," has been produced by recombinant
means and also retains anti-bacterial activity against
gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun.
60:4754-4761 (1992).] An N-terminal analog of BPI, rBPI.sub.21, has
been produced as described in Horwitz et al., Protein Expression
Purification, 8:28-40 (1996).
[0010] The bactericidal effect of BPI was originally reported to be
highly specific to gram-negative species, e.g., in Elsbach and
Weiss, Inflammation: Basic Principles and Clinical Correlates, eds.
Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise
mechanism by which BPI kills gram-negative bacteria is not yet
completely elucidated, but it is believed that BPI must first bind
to the surface of the bacteria through electrostatic and
hydrophobic interactions between the cationic BPI protein and
negatively charged sites on LPS. In susceptible gram-negative
bacteria, BPI binding is thought to disrupt LPS structure, leading
to activation of bacterial enzymes that degrade phospholipids and
peptidoglycans, altering the permeability of the cell's outer
membrane, and initiating events that ultimately lead to cell death.
[Elsbach and Weiss (1992), supra]. LPS has been referred to as
"endotoxin" because of the potent inflammatory response that it
stimulates, i.e., the release of mediators by host inflammatory
cells which may ultimately result in irreversible endotoxic shock.
BPI binds to lipid A, reported to be the most toxic and most
biologically active component of LPS.
[0011] BPI protein products have a wide variety of beneficial
activities. BPI protein products are bactericidal for gram-negative
bacteria, as described in U.S. Pat. Nos. 5,198,541 and 5,523,288,
both of which are incorporated herein by reference. International
Publication No. WO 94/20130 (incorporated herein by reference)
proposes methods for treating subjects suffering from an infection
(e.g. gastrointestinal) with a species from the gram-negative
bacterial genus Helicobacter with BPI protein products. BPI protein
products also enhance the effectiveness of antibiotic therapy in
gram-negative bacterial infections, as described in U.S. Pat. No.
5,523,288 and International Publication No. WO 95/08344
(PCT/US94/11255), which are incorporated herein by reference. BPI
protein products are also bactericidal for gram-positive bacteria
and mycoplasma, and enhance the effectiveness of antibiotics in
gram-positive bacterial infections, as described in U.S. Pat. Nos.
5,578,572 and 5,783,561 and International Publication No. WO
95/19180 (PCT/US95/00656), which are incorporated herein by
reference. BPI protein products exhibit anti-fungal activity, and
enhance the activity of other anti-fungal agents, as described in
U.S. Pat. No. 5,627,153 and International Publication No. WO
95/19179 (PCT/US95/00498), and further as described for anti-fungal
peptides in U.S. Pat. No. 5,858,974, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/504,841 filed
Jul. 20, 1994 and corresponding International Publication Nos. WO
96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), all of
which are incorporated herein by reference. BPI protein products
exhibit anti-protozoan activity, as described in U.S. Pat. No.
5,646,114 and International Publication No. WO 96/01647
(PCT/US95/08624), which are incorporated herein by reference. BPI
protein products exhibit anti-chlamydial activity, as described in
co-owned, co-pending U.S. application Ser. No. 08/694,843 filed
Aug. 9, 1996 and WO 98/06415 (PCT/US97/13810), which are
incorporated herein by reference. Finally, BPI protein products
exhibit anti-mycobacterial activity, as described in co-owned,
co-pending U.S. application Ser. No. 08/626,646 filed Apr. 1, 1996,
which is in turn a continuation of U.S. application Ser. No.
08/285,803 filed Aug. 14, 1994, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/031,145 filed
Mar. 12, 1993 and corresponding International Publication No.
WO94/20129 (PCT/US94/02463), all of which are incorporated herein
by reference.
[0012] The effects of BPI protein products in humans with endotoxin
in circulation, including effects on TNF, IL-6 and endotoxin are
described in U.S. Pat. No. 5,643,875, which is incorporated herein
by reference.
[0013] BPI protein products are also useful for treatment of
specific disease conditions, such as meningococcemia in humans (as
described in co-owned, co-pending U.S. application Ser. No.
08/644,287 filed May 10, 1996 and continuation Ser. No. 08/927,437
filed Sep. 10, 1997 and International Publication No. WO97/42966
(PCT/US97/08016), all of which are incorporated herein by
reference), hemorrhagic trauma in humans, (as described in U.S.
Pat. No.5,756,464, U.S. application Ser. No. 08/862,785 filed May
23, 1997 and corresponding International Publication No. WO
97/44056 (PCT/US97/08941), all of which are incorporated herein by
reference), burn injury (as described in U.S. Pat. No. 5,494,896,
which is incorporated herein by reference), ischemia/reperfusion
injury (as described in U.S. Pat. No. 5,578,568, incorporated
herein by reference), and liver resection (as described in
co-owned, co-pending U.S. application Ser. No. 08/582,230 filed
Jan. 3, 1996, which is in turn a continuation of U.S. application
Ser. No. 08/318,357 filed Oct. 5, 1994, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/132,510 filed
Oct. 5, 1993, and corresponding International Publication No. WO
95/10297 (PCT/US94/11404), all of which are incorporated herein by
reference).
[0014] BPI protein products also neutralize the anti-coagulant
activity of exogenous heparin, as described in U.S. Pat. No.
5,348,942, incorporated herein by reference, and are useful for
treating chronic inflammatory diseases such as rheumatoid and
reactive arthritis and for inhibiting angiogenesis and for treating
angiogenesis-associated disorders including malignant tumors,
ocular retinopathy and endometriosis, as described in U.S. Pat.
Nos. 5,639,727, 5,807,818 and 5,837,678 and International
Publication No. WO 94/20128 (PCT/US94/02401), all of which are
incorporated herein by reference.
[0015] BPI protein products are also useful in antithrombotic
methods, as described in U.S. Pat. No. 5,741,779 and U.S.
application Ser. No. 09/063,465 filed Apr. 20, 1998 and
corresponding WO 97/42967 (PCT/US7/08017), all of which are
incorporated herein by reference.
SUMMARY OF THE INVENTION
[0016] The present invention provides novel therapeutic uses for
BPI protein products that involve treatment of subjects with
chronic cardiac disease. Uses of BPI protein products according to
the invention are specifically contemplated for prophylactic or
therapeutic treatment of chronic cardiac disease states or
conditions in humans, particularly humans with chronic cardiac
disease who exhibit elevated levels of circulating LPS and
circulating LBP (in plasma or serum). Chronic cardiac disease
states or conditions include but are not limited to
cardiomyopathies, chronic congestive heart failure, and congenital
heart defects.
[0017] Chronic congestive heart failure as used herein includes
long-term congestive heart failure (i.e., congestive heart failure
persisting more than two weeks, or more than three weeks, or more
than one month, or more than two months, or more than three
months), congestive heart failure that persists after correction of
reversible causes, and congestive heart failure not immediately
associated with an acute myocardial infarction or an acute
infectious process.
[0018] Congenital heart defects, which may result in congestive
heart failure or cyanotic heart disease, include pulmonary atresia,
total anomalous pulmonary venous return, ventricular septal defect,
hypoplastic left heart syndrome, double outlet right ventricle,
right pulmonary artery stenosis, interrupted aortic arch,
Ebsteins's anomaly, tetralogy of Fallot, atrioventricular canal,
transposition of the great arteries and truncus arteriosus.
[0019] It is contemplated that the administration of a BPI protein
product may be accompanied by the concurrent administration of
other known therapeutic agents for treating the chronic cardiac
disease state. For example, agents that are known in the art for
treating congestive heart failure include diuretics (including
thiazides such as chlorothiazide, hydrochlorothiazide and
metolazone, loop diuretics such as ethacrynic acid, furosemide,
torsemide and bumetanide and their congeners, potassium sparing
agents such as spironolactone, canrenone, triamterene and
amiloride, and others such as metolazone and other
quinazoline-sulfonamides), vasodilators (including
nitrovasodilators such as nitroglycerin, isosorbide dinitrate, and
sodium nitroprusside, hydralazine, prostacyclin, ACE inhibitors
such as captopril, enalapril, lisinopril, quinapril and ramipril,
and angiotensin II antagonists such as losartan), positive
inotropic agents (such as cardiac glycosides, including digitalis
or digoxin), phosphodiesterase inhibitors (such as amrinone and
milrinone, primarily useful for short term support), occasionally
beta-adrenergic receptor antagonists (beta blockers such as
propanolol, metoprolol, atenolol, pindolol, acebutolol, labetalol,
carvedilol and celiprolol), or combinations of these measures. See,
e.g., Ch. 34, Goodman and Gilman, The Pharmacological Basis of
Therapeutics, McGraw Hill, New York (1996), incorporated herein by
reference.
[0020] The invention also contemplates use of a BPI protein product
in the preparation of a medicament for the prophylactic or
therapeutic treatment of a chronic cardiac disease state.
[0021] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon consideration
of the following detailed description of the invention which
describes presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A displays plasma LPS levels for all patients
completing the study protocol (n=29). FIG. 1B displays data from
patients with endotoxemia pre-operatively (n=11), while FIG. 1C
displays data from patients without endotoxemia pre-operatively
(n=18).
[0023] FIG. 2 displays plasma LBP levels from all patients
completing the study protocol (n=29).
[0024] FIG. 3 displays plasma IL-6 levels from all patients
completing the study protocol (n=29).
[0025] FIGS. 4A and 4B displays pre-operative plasma LPS (FIG. 4B)
and LBP (FIG. 4A) levels in patients with a severe (n=15), versus
less severe (n=15), post-operative clinical course.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides novel therapeutic uses for
BPI protein products, particularly BPI-derived peptides, that
involve treatment of chronic cardiac disease. "Treatment" as used
herein encompasses both prophylactic and therapeutic treatment. The
invention contemplates methods for treatment of subjects suffering
from chronic heart disease which comprise the administration of
therapeutically effective amounts of
bactericidal/permeability-increasing protein (BPI) protein products
to those subjects so as to alleviate the negative physiological
effects of endotoxemia.
[0027] The invention is based on the discovery that a substantial
proportion of subjects suffering from chronic heart disease exhibit
evidence of endotoxemia associated with the chronic heart disease
prior to surgery, and that this endotoxemia correlates to a poorer
prognosis for these subjects. Thus, one basis for the invention is
the expectation that endotoxemia is not simply a side effect of
chronic cardiac disease but is a significant contributing factor to
the pathology of chronic cardiac disease.
[0028] Therapeutic uses of BPI protein products are specifically
contemplated for treatment of mammals, including humans, suffering
from chronic cardiac disease as distinguished from acute cardiac
disease states such as myocardial infarction, circulatory collapse
from hemorrhage and the like.
[0029] Another aspect of the present invention is the treatment of
patients undergoing cardiopulmonary bypass, on the basis that the
severity of endotoxemia during and after cardiopulmonary bypass is
correlated with poorer post-surgical outcome. Thus, treatment with
BPI protein product is expected to improve post-surgical
outcome.
[0030] As used herein, "BPI protein product" includes naturally and
recombinantly produced BPI protein; natural, synthetic, and
recombinant biologically active polypeptide fragments of BPI
protein; biologically active polypeptide variants of BPI protein or
fragments thereof, including hybrid fusion proteins and dimers;
biologically active polypeptide analogs of BPI protein or fragments
or variants thereof, including cysteine-substituted analogs; and
BPI-derived peptides. The BPI protein products administered
according to this invention may be generated and/or isolated by any
means known in the art. U.S. Pat. No. 5,198,541, the disclosure of
which is incorporated herein by reference, discloses recombinant
genes encoding, and methods for expression of, BPI proteins
including recombinant BPI holoprotein, referred to as rBPI and
recombinant fragments of BPI. U.S. Pat. No. 5,439,807 and
corresponding International Publication No. WO 93/23540
(PCT/US93/04752), which are all incorporated herein by reference,
disclose novel methods for the purification of recombinant BPI
protein products expressed in and secreted from genetically
transformed mammalian host cells in culture and discloses how one
may produce large quantities of recombinant BPI products suitable
for incorporation into stable, homogeneous pharmaceutical
preparations.
[0031] Biologically active fragments of BPI (BPI fragments) include
biologically active molecules that have the same or similar amino
acid sequence as a natural human BPI holoprotein, except that the
fragment molecule lacks amino-terminal amino acids, internal amino
acids, and/or carboxy-terminal amino acids of the holoprotein.
Nonlimiting examples of such fragments include an N-terminal
fragment of natural human BPI of approximately 25 kD, described in
Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant
expression product of DNA encoding N-terminal amino acids from 1 to
about 193 to 199 of natural human BPI, described in Gazzano-Santoro
et al., Infect. Immun. 60:4754-4761 (1992), and referred to as
rBPI.sub.23. In that publication, an expression vector was used as
a source of DNA encoding a recombinant expression product
(rBPI.sub.23) having the 31-residue signal sequence and the first
199 amino acids of the N-terminus of the mature human BPI, as set
out in FIG. 1 of Gray et al., supra, except that valine at position
151 is specified by GTG rather than GTC and residue 185 is glutamic
acid (specified by GAG) rather than lysine (specified by AAG).
Recombinant holoprotein (rBPI) has also been produced having the
sequence (SEQ ID NOS: 1 and 2) set out in FIG. 1 of Gray et al.,
supra, with the exceptions noted for rBPI.sub.23 and with the
exception that residue 417 is alanine (specified by GCT) rather
than valine (specified by GTT). A fragment consisting of residues
10-193 of BPI has been described in co-owned, co-pending U.S.
application Ser. No. 09/099,725 filed Jun. 19, 1998, incorporated
herein by reference. Other examples include dimeric forms of BPI
fragments, as described in U.S. Pat. No. 5,447,913 and
corresponding International Publication No. WO 95/24209
(PCT/US95/03125), all of which are incorporated herein by
reference.
[0032] Biologically active variants of BPI (BPI variants) include
but are not limited to recombinant hybrid fusion proteins,
comprising BPI holoprotein or biologically active fragment thereof
and at least a portion of at least one other polypeptide, and
dimeric forms of BPI variants. Examples of such hybrid fusion
proteins and dimeric forms are described in U.S. Pat. No. 5,643,570
and corresponding International Publication No. WO 93/23434
(PCT/US93/04754), which are all incorporated herein by reference
and include hybrid fusion proteins comprising, at the
amino-terminal end, a BPI protein or a biologically active fragment
thereof and, at the carboxy-terminal end, at least one constant
domain of an immunoglobulin heavy chain or allelic variant
thereof.
[0033] Biologically active analogs of BPI (BPI analogs) include but
are not limited to BPI protein products wherein one or more amino
acid residues have been replaced by a different amino acid. For
example, U.S. Pat. No. 5,420,019 and corresponding International
Publication No. WO 94/18323 (PCT/US94/01235), all of which are
incorporated herein by reference, discloses polypeptide analogs of
BPI and BPI fragments wherein a cysteine residue is replaced by a
different amino acid. A stable BPI protein product described by
this application is the expression product of DNA encoding from
amino acid 1 to approximately 193 or 199 of the N-terminal amino
acids of BPI holoprotein, but wherein the cysteine at residue
number 132 is substituted with alanine and is designated
rBPI.sub.21*cYs or rBPI.sub.21. Production of this N-terminal
analog of BPI, rBPI.sub.21, has been described in Horwitz et al.,
Protein Expression Purification, 8:28-40 (1996). Similarly, a
fragment consisting of residues 10-193 of BPI in which the cysteine
at position 132 is replaced with an alanine (designated
"rBPI(10-193)C132A" or "rBPI(10-193)ala.sup.132") has been
described in co-owned, co-pending U.S. application Ser. No.
09/099,725 filed Jun. 19, 1998. Other examples include dimeric
forms of BPI analogs; e.g. U.S. Pat. No. 5,447,913 and
corresponding International Publication No. WO 95/24209
(PCT/US95/03125), all of which are incorporated herein by
reference.
[0034] Other BPI protein products useful according to the methods
of the invention are peptides derived from or based on BPI produced
by synthetic or recombinant means (BPI-derived peptides), such as
those described in International Publication No. WO 97/04008
(PCT/US96/03845), which corresponds to U.S. application Ser. No.
08/621,259 filed Mar. 21, 1996, and International Publication No.
WO 96/08509 (PCT/US95/09262), which corresponds to U.S. Pat. No.
5,858,974, and International Publication No. WO 95/19372
(PCT/US94/10427), which corresponds to U.S. Pat. No. 5,652,332, and
International Publication No. WO94/20532 (PCT/US94/02465), which
corresponds to U.S. Pat. No. 5,763,567 which is a continuation of
U.S. Pat. No. 5,733,872, which is a continuation-in-part of U.S.
application Ser. No. 08/183,222, filed Jan. 14, 1994, which is a
continuation-in-part of U.S. application Ser. No. 08/093,202 filed
Jul. 15, 1993 (corresponding to International Publication No. WO
94/20128 (PCT/US94/02401)), which is a continuation-in-part of U.S.
Pat. No. 5,348,942, as well as International Application No.
PCT/US97/05287, which corresponds to U.S. Pat. No. 5,851,802, the
disclosures of all of which are incorporated herein by
reference.
[0035] Presently preferred BPI protein products include
recombinantly-produced N-terminal analogs and fragments of BPI,
especially those having a molecular weight of approximately between
20 to 25 kD such as rBPI.sub.21 or rBPI.sub.23, rBPI(10-193)C132A
(rBPI(10-193)ala.sup.132), dimeric forms of these N-terminal
proteins (e.g., rBPI.sub.42 dimer), and BPI-derived peptides.
[0036] The administration of BPI protein products is preferably
accomplished with a pharmaceutical composition comprising a BPI
protein product and a pharmaceutically acceptable diluent,
adjuvant, or carrier. The BPI protein product may be administered
without or in conjunction with known surfactants or other
therapeutic agents. A stable pharmaceutical composition containing
BPI protein products (e.g., rBPI.sub.23) comprises the BPI protein
product at a concentration of 1 mg/ml in citrate buffered saline (5
or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1 % by weight
of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.)
and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas
Inc., Wilmington, Del.). Another stable pharmaceutical composition
containing BPI protein products (e.g., rBPI.sub.21) comprises the
BPI protein product at a concentration of 2 mg/ml in 5 mM citrate,
150 mM NaCl, 0.2% poloxamer 188 and 0.002% polysorbate 80. Such
preferred combinations are described in U.S. Pat. Nos. 5,488,034
and 5,696,090 and corresponding International Publication No. WO
94/17819 (PCT/US94/01239), the disclosures of all of which are
incorporated herein by reference. As described in U.S. application
Ser. No. 08/586,133 filed Jan. 12, 1996, which is in turn a
continuation-in-part of U.S. application Ser. No. 08/530,599 filed
Sep. 19, 1995, which is in turn a continuation-in-part of U.S.
application Ser. No. 08/372,104 filed Jan. 13, 1995, and
corresponding International Publication No. WO96/21436
(PCT/US96/01095), all of which are incorporated herein by
reference, other poloxamer formulations of BPI protein products
with enhanced activity may be utilized.
[0037] Therapeutic compositions comprising BPI protein product may
be administered systemically or topically. Systemic routes of
administration include oral, intravenous, intramuscular or
subcutaneous injection (including into a depot for long-term
release), intraocular and retrobulbar, intrathecal, intraperitoneal
(e.g. by intraperitoneal lavage), intrapulmonary (using powdered
drug, or an aerosolized or nebulized drug solution), or
transdermal.
[0038] When given parenterally, BPI protein product compositions
are generally injected in doses ranging from 1 *g/kg to 100 mg/kg
per day, preferably at doses ranging from 0.1 mg/kg to 20 mg/kg per
day, more preferably at doses ranging from 1 to 20 mg/kg/day and
most preferably at doses ranging from 2 to 10 mg/kg/day. The
treatment may continue by continuous infusion or intermittent
injection or infusion, at the same, reduced or increased dose per
day for, e.g., 1 to 3 days, and additionally as determined by the
treating physician. When administered intravenously, BPI protein
products are preferably administered by an initial brief infusion
followed by a continuous infusion. The preferred intravenous
regimen is a 1 to 20 mg/kg brief intravenous infusion of BPI
protein product followed by a continuous intravenous infusion at a
dose of 1 to 20 mg/kg/day, continuing for up to one week. A
particularly preferred intravenous dosing regimen is a 1 to 4 mg/kg
initial brief intravenous infusion followed by a continuous
intravenous infusion at a dose of 1 to 4 mg/kg/day, continuing for
up to 72 hours.
[0039] Those skilled in the art can readily optimize effective
dosages and administration regimens for therapeutic compositions
comprising BPI protein product, as determined by good medical
practice and the clinical condition of the individual patient.
[0040] "Concurrent administration," or "co-administration," as used
herein includes administration of the agents, in conjunction or
combination, together, or before or after each other. The BPI
protein product and second agent(s) may be administered by
different routes. For example, the BPI protein product may be
administered intravenously while the second agent(s) is(are)
administered intravenously, intramuscularly, subcutaneously, orally
or intraperitoneally. The BPI protein product and second agent(s)
may be given sequentially in the same intravenous line or may be
given in different intravenous lines. Alternatively, the BPI
protein product may be administered in a special form for gastric
delivery, while the second agent(s) is(are) administered, e.g.,
orally. The formulated BPI protein product and second agent(s) may
be administered simultaneously or sequentially, as long as they are
given in a manner sufficient to allow all agents to achieve
effective concentrations at the site of action.
[0041] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples. Example 1 addresses a study in which thirty children with
complex chronic heart disease were tested for markers of
endotoxemia prior to and at 1, 8, 24, 48 and 72 hours following
cardiopulmonary bypass surgery.
EXAMPLE 1
[0042] The experimental protocol, approved by the Institutional
Review Board at the University of Texas Southwestern Medical
Center, was an unblinded, prospective study in which 30 children
with severe congenital heart disease were sequentially enrolled
while awaiting surgical repair and/or palliation. One patient with
hypoplastic left heart syndrome died intra-operatively, and
therefore data on this child are included only in the pre-operative
analysis. Patients with clinical evidence of preoperative infection
were excluded from the study.
[0043] The 30 enrolled children ranged in age from 4 days to 402
days (median age 59 days), and in weight from 2.0 to 9.5 kg (median
weight 4.0 kg) The genders, ages, cardiac diagnoses, and surgical
repairs are listed in Table 1 below. TABLE-US-00001 TABLE 1 PATIENT
CHARACTERISTICS Age- Wt Sex days (kg) Diagnosis* Procedure M 349
8.90 Pulmonary Atresia RVOT Reconstruction M 6 5.70 TAPVR TAPVR
Repair F 265 6.60 TAPVR TAPVR Repair M 5 3.30 Interrupted Aortic
Arch Aortic Arch Repair M 44 3.50 Ebstein's anomaly, VSD VSD Repair
M 245 8.09 VSD VSD Repair F 7 2.50 HLHS Norwood Procedure F 10 3.4
HLHS Norwood Procedure F 210 6.70 VSD VSD Repair M 163 5.00 DORV,
RPA Stenosis DKS F 395 8.20 TOF Tetralogy Repair M 10 2.50 TGA,
DORV Arterial Switch M 75 3.60 AV Canal AV Canal Repair M 4 4.40
TGA Arterial Switch M 4 3.60 TGA Arterial Switch M 4 3.35 LV
Rhabdomyosarcoma Tumor Resection M 26 3.70 TAPVR TAPVR Repair F 110
3.20 TAPVR TAPVR Repair F 126 2.00 Truncus Arteriosus RVOT
Reconstruction M 402 9.50 TOF Tetralogy Repair M 25 2.60 Pulmonary
Atresia RVOT Reconstruction F 105 4.40 AV Canal AV Canal Repair M
178 5.80 TOF Tetralogy Repair M 5 4.40 TGA Arterial Switch M 102
4.10 TAPVR TAPVR Repair F 5 3.70 TAPVR TAPVR Repair M 6 3.10 HLHS
Norwood Procedure F 120 5.40 AV Canal AV Canal Repair M 4 3.80 TGA
Arterial Switch M 335 7.50 TOF Tetralogy Repair
[0044] Anesthesia was induced with sevoflurane, nitrous oxide and
oxygen; intubation was facilitated with intravenous rocuronium and
fentanyl. Anesthesia was maintained with fentanyl (30-50 mcg/kg),
isoflurane, and pancuronium. Nine patients received tranexamic acid
(50-100 mg/kg) and 3 received aprotinin (dosed to achieve 350
units/mL total blood volume).
[0045] Cardiopulmonary bypass was performed as follows. The
extracorporeal circuit consisted of a roller pump, membrane
oxygenator, and cardiotomy filters Prior to the institution of
cardiopulmonary bypass, the patients' blood was anticoagulated with
heparin (300 units/kg). 13 patients underwent deep hypothermic
circulatory arrest (core temp 16-18.degree. C.) and the remainder
were cooled to a core temperature of 25-30.degree. C. for the
completion of surgery. Hemofiltration was performed prior to
completion of cardiopulmonary bypass on all patients in an attempt
to remove excess free water and attain a hemoglobin >12
gm/dL.
[0046] Blood samples for the determination of LPS, LBP, and IL-6
were obtained prior to surgery and at 1, 8, 24, 48 and 72 hours
after completion of cardiopulmonary bypass. The pre-operative
sample was obtained from a newly placed central venous catheter,
immediately after the induction of anesthesia and endotracheal
intubation. For determination of endotoxin levels, blood samples
were collected into heparinized Vacutainer.TM. tubes
(Becton-Dickson, Rutherford N.J.) selected for low endotoxin
content (BioWhitaker, Walkersville, Md.), immediately placed on
ice, and walked to the laboratory by an investigator. Platelet-rich
plasma was obtained by centrifugation (180.times.g, 10 min, 2-8
.degree. C.). Samples were stored at -70.degree. C. until
assay.
[0047] LPS, LBP and IL-6 assays were conducted in a blinded
fashion. The level of LPS in the platelet-rich plasma was
determined by using a kinetic chromogenic Limulus amebocyte lysate
assay (Endochrome-K.TM., Endosafe, Charleston, S.C.) according to
the manufacturer's instructions. LPS concentrations are expressed
in terms of endotoxin units (EU) per ml relative to an E. coli
055:B5 control standard endotoxin. LBP levels were determined by
ELISA as described in Meszaros S, et al., Infect. Immun.
1995;63:363-365. IL-6 was measured using a sandwich ELISA (R&D
Systems, Minneapolis).
[0048] A severe (versus less severe) post operative course was
prospectively defined. Severity of post-operative myocardial
dysfunction was estimated according to an adaptation of the scale
utilized by Wemovsky et al., Circulation 1995;92:2226-2235.
Specifically, an inotropic support score was calculated as follows:
each 1.0 mcg/kg/min of dopamine or dobutamine, and each 0.01
mcg/kg/min of epinephrine yielded a score of 1. Children with a net
positive fluid balance of >40 cc/kg in the first 24 hours and an
inotropic support score of >12, or perioperative death, were
considered to have a severe clinical course. Post-operative
severity of illness was scored prior to knowledge of LPS, LBP, or
IL-6 values.
[0049] All statistical analyses were performed on the Statistical
Package for the Social Sciences (SPSS). Wilcoxon signed rank tests
for non-parametric data were performed to determine if a
significant rise in LPS, LBP or IL-6 had occurred post-operatively.
A Mann-Whitney test for non-parametric data was performed to
determine if there was a significant difference in LPS or LBP
concentrations between the patients who had a more severe clinical
course compared to those with a less severe clinical course.
[0050] Twenty-nine of the thirty patients (96%) had evidence of
endotoxemia during the study period, either by detection of
elevated LPS directly or by detection of an elevated LBP plasma
level >2SD above the mean for healthy adults. The LPS, LBP and
IL-6 levels for all patients are displayed in FIGS. 1A, 2 and 3.
FIG. 1A displays data from all children completing the study
protocol and demonstrate elevated LPS at all time points.
Differences between pre-operative and post-operative LPS levels are
statistically non-significant. To better elucidate endotoxin
kinetics, we divided patients into two groups: those who were
endotoxemic prior to cardiopulmonary bypass, and those who were not
endotoxemic prior to cardiopulmonary bypass (CPB). Levels for these
two groups are shown in FIGS. 1B and 1C, respectively.
[0051] Prior to CPB, 12 patients had significant elevation of
plasma endotoxin. In these patients, endotoxin tended to decline
following completion of cardiopulmonary bypass, likely due to
hemodilution/partial exchange transfusion (FIG. 1B); but endotoxin
levels remained abnormally elevated throughout the study period. In
those children without pre-operative endotoxemia, the level of
plasma endotoxin rose significantly following bypass, achieving a
peak value at 1 hour post bypass, and remaining significantly
elevated thereafter (p<0.0001) (FIG. 1C).
[0052] There was a transient, but significant decrease in plasma
LBP immediately after completion of cardiopulmonary bypass and
hemofiltration. The decrease in plasma LBP at 1 hour following
cardiopulmonary bypass was statistically significant (p<0.0001),
and the increase in LBP at all points thereafter was also highly
consistent and statistically significant (p<0.0001) compared to
preoperative levels. This rise in LBP was similar for patients who
were and were not endotoxemic preoperatively. Similarly, there was
a significant rise in IL-6 at all time points following CPB
(p<0.05) compared to preoperative levels (FIG. 3).
[0053] Finally, it was determined whether children who experienced
a more severe clinical course, defined prospectively, might differ
from less severe patients when pre-operative LPS and LBP levels
were compared. In this comparison, the more severely ill children
had significantly higher pre-operative plasma LBP (p<0.02) (FIG.
4A), and tended toward higher pre-operative LPS (p<0.05)
compared to patients who experienced a less severe post-operative
course (FIG. 4B). Additionally, of the 12 patients who were
endotoxemic prior to surgery, there were 3 deaths (25%), compared
to 0 deaths in the 18 patients who were not endotoxemic prior to
surgery (p=0.054).
[0054] The underlying biology of peri-operative endotoxemia is
clarified by dividing those patients who were or were not
endotoxemic-pre operatively. In patients who were endotoxemic
pre-operatively, endotoxin levels initially fell following
cardiopulmonary bypass, but remained abnormally elevated throughout
the study period. This initial decrease may have been secondary to
a dilution effect of cardiopulmonary bypass, given the infants'
small blood volumes, or perhaps due to clearance of endotoxin by
hemofiltration prior to completion of cardiopulmonary bypass.
Millar A B, Ann Thorac Surg 1993;56:1499-1502 It is also possible
that these patients, who were endotoxemic pre-operatively, may have
induced and enhanced mechanisms for endotoxin clearance, compared
to patients who were not endotoxemic pre-operatively. Dentener M A,
et al., Journal of Infectious Diseases 1997;175:108-117; Dentener M
A, Journal of infectious Diseases 1995;171:739-743; and
Gazzano-Santoro et al., Infect. Immunol. 1994;62,
No4:1185-1191.
[0055] In contrast, patients who were not endotoxemic
pre-operatively demonstrated a significant elevation of plasma
endotoxin at one and eight hours after cardiopulmonary bypass. A
number of factors have been invoked to explain endotoxemia during
cardiopulmonary bypass. First, there are many sources of endotoxin
including the extracorporeal circuit, infusion solutions, drugs,
and surgical materials. More importantly, increased intestinal
permeability during cardiopulmonary bypass has been documented in
adult patients, allowing for bacterial translocation and release of
endotoxin into the circulation. Measures such as pulsatile
perfusion or higher flow during bypass to improve gut perfusion and
aggressive antibiotic regimens to decrease intestinal bacterial
load prior to bypass have resulted in lower plasma LPS level.
Watarida S, et al., J Thorac Cardiovasc Surg 1994;108:620-625; and
Quigley R L, et al. Perfusion 1995;10:27-31.
[0056] The finding of elevated LPS and LBP levels pre-operatively
in a substantial proportion of children with chronic cardiac
disease indicates that endotoxemia is associated with the chronic
cardiac disease itself. The finding that severity of outcome
correlated with endotoxemia as measured by elevated LPS and LBP
levels indicates that measurement of endotoxemia can be used to
predict prognosis. Consequently, the treatment of endotoxemia with
BPI protein product is expected to ameliorate the signs and
symptoms of chronic cardiac disease and to improve the prognosis of
these patients.
[0057] Numerous modifications and variations of the above-described
invention are expected to occur to those of skill in the art.
Accordingly, only such limitations as appear in the appended claims
should be placed thereon.
Sequence CWU 1
1
2 1 1813 DNA Homo sapiens CDS (31)..(1491) mat_peptide
(124)..(1491) 1 caggccttga ggttttggca gctctggagg atg aga gag aac
atg gcc agg ggc 54 Met Arg Glu Asn Met Ala Arg Gly -30 -25 cct tgc
aac gcg ccg aga tgg gtg tcc ctg atg gtg ctc gtc gcc ata 102 Pro Cys
Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile -20 -15 -10
ggc acc gcc gtg aca gcg gcc gtc aac cct ggc gtc gtg gtc agg atc 150
Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile -5
-1 1 5 tcc cag aag ggc ctg gac tac gcc agc cag cag ggg acg gcc gct
ctg 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala
Leu 10 15 20 25 cag aag gag ctg aag agg atc aag att cct gac tac tca
gac agc ttt 246 Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser
Asp Ser Phe 30 35 40 aag atc aag cat ctt ggg aag ggg cat tat agc
ttc tac agc atg gac 294 Lys Ile Lys His Leu Gly Lys Gly His Tyr Ser
Phe Tyr Ser Met Asp 45 50 55 atc cgt gaa ttc cag ctt ccc agt tcc
cag ata agc atg gtg ccc aat 342 Ile Arg Glu Phe Gln Leu Pro Ser Ser
Gln Ile Ser Met Val Pro Asn 60 65 70 gtg ggc ctt aag ttc tcc atc
agc aac gcc aat atc aag atc agc ggg 390 Val Gly Leu Lys Phe Ser Ile
Ser Asn Ala Asn Ile Lys Ile Ser Gly 75 80 85 aaa tgg aag gca caa
aag aga ttc tta aaa atg agc ggc aat ttt gac 438 Lys Trp Lys Ala Gln
Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 90 95 100 105 ctg agc
ata gaa ggc atg tcc att tcg gct gat ctg aag ctg ggc agt 486 Leu Ser
Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser 110 115 120
aac ccc acg tca ggc aag ccc acc atc acc tgc tcc agc tgc agc agc 534
Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser 125
130 135 cac atc aac agt gtc cac gtg cac atc tca aag agc aaa gtc ggg
tgg 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser Lys Val Gly
Trp 140 145 150 ctg atc caa ctc ttc cac aaa aaa att gag tct gcg ctt
cga aac aag 630 Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala Leu
Arg Asn Lys 155 160 165 atg aac agc cag gtc tgc gag aaa gtg acc aat
tct gta tcc tcc aag 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn
Ser Val Ser Ser Lys 170 175 180 185 ctg caa cct tat ttc cag act ctg
cca gta atg acc aaa ata gat tct 726 Leu Gln Pro Tyr Phe Gln Thr Leu
Pro Val Met Thr Lys Ile Asp Ser 190 195 200 gtg gct gga atc aac tat
ggt ctg gtg gca cct cca gca acc acg gct 774 Val Ala Gly Ile Asn Tyr
Gly Leu Val Ala Pro Pro Ala Thr Thr Ala 205 210 215 gag acc ctg gat
gta cag atg aag ggg gag ttt tac agt gag aac cac 822 Glu Thr Leu Asp
Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 cac aat
cca cct ccc ttt gct cca cca gtg atg gag ttt ccc gct gcc 870 His Asn
Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala 235 240 245
cat gac cgc atg gta tac ctg ggc ctc tca gac tac ttc ttc aac aca 918
His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 250
255 260 265 gcc ggg ctt gta tac caa gag gct ggg gtc ttg aag atg acc
ctt aga 966 Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met Thr
Leu Arg 270 275 280 gat gac atg att cca aag gag tcc aaa ttt cga ctg
aca acc aag ttc 1014 Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Arg
Leu Thr Thr Lys Phe 285 290 295 ttt gga acc ttc cta cct gag gtg gcc
aag aag ttt ccc aac atg aag 1062 Phe Gly Thr Phe Leu Pro Glu Val
Ala Lys Lys Phe Pro Asn Met Lys 300 305 310 ata cag atc cat gtc tca
gcc tcc acc ccg cca cac ctg tct gtg cag 1110 Ile Gln Ile His Val
Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln 315 320 325 ccc acc ggc
ctt acc ttc tac cct gcc gtg gat gtc cag gcc ttt gcc 1158 Pro Thr
Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala 330 335 340
345 gtc ctc ccc aac tcc tcc ctg gct tcc ctc ttc ctg att ggc atg cac
1206 Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly Met
His 350 355 360 aca act ggt tcc atg gag gtc agc gcc gag tcc aac agg
ctt gtt gga 1254 Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn
Arg Leu Val Gly 365 370 375 gag ctc aag ctg gat agg ctg ctc ctg gaa
ctg aag cac tca aat att 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu
Glu Leu Lys His Ser Asn Ile 380 385 390 ggc ccc ttc ccg gtt gaa ttg
ctg cag gat atc atg aac tac att gta 1350 Gly Pro Phe Pro Val Glu
Leu Leu Gln Asp Ile Met Asn Tyr Ile Val 395 400 405 ccc att ctt gtg
ctg ccc agg gtt aac gag aaa cta cag aaa ggc ttc 1398 Pro Ile Leu
Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 410 415 420 425
cct ctc ccg acg ccg gcc aga gtc cag ctc tac aac gta gtg ctt cag
1446 Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu
Gln 430 435 440 cct cac cag aac ttc ctg ctg ttc ggt gca gac gtt gtc
tat aaa 1491 Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val
Tyr Lys 445 450 455 tgaaggcacc aggggtgccg ggggctgtca gccgcacctg
ttcctgatgg gctgtggggc 1551 accggctgcc tttccccagg gaatcctctc
cagatcttaa ccaagagccc cttgcaaact 1611 tcttcgactc agattcagaa
atgatctaaa cacgaggaaa cattattcat tggaaaagtg 1671 catggtgtgt
attttaggga ttatgagctt ctttcaaggg ctaaggctgc agagatattt 1731
cctccaggaa tcgtgtttca attgtaacca agaaatttcc atttgtgctt catgaaaaaa
1791 aacttctggt ttttttcatg tg 1813 2 487 PRT Homo sapiens 2 Met Arg
Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val -30 -25 -20
Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val -15
-10 -5 -1 1 Asn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp
Tyr Ala 5 10 15 Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys
Arg Ile Lys 20 25 30 Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys
His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met Asp Ile
Arg Glu Phe Gln Leu Pro Ser 50 55 60 65 Ser Gln Ile Ser Met Val Pro
Asn Val Gly Leu Lys Phe Ser Ile Ser 70 75 80 Asn Ala Asn Ile Lys
Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe 85 90 95 Leu Lys Met
Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile 100 105 110 Ser
Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120
125 Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His
130 135 140 145 Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe
His Lys Lys 150 155 160 Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Ser
Gln Val Cys Glu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu
Gln Pro Tyr Phe Gln Thr Leu 180 185 190 Pro Val Met Thr Lys Ile Asp
Ser Val Ala Gly Ile Asn Tyr Gly Leu 195 200 205 Val Ala Pro Pro Ala
Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys 210 215 220 225 Gly Glu
Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro 230 235 240
Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly 245
250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu
Ala 260 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile Pro
Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr
Phe Leu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe Pro Asn Met Lys
Ile Gln Ile His Val Ser Ala Ser 310 315 320 Thr Pro Pro His Leu Ser
Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val Asp Val
Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser Leu
Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360 365
Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370
375 380 385 Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu
Leu Leu 390 395 400 Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val
Leu Pro Arg Val 405 410 415 Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu
Pro Thr Pro Ala Arg Val 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln
Pro His Gln Asn Phe Leu Leu Phe 435 440 445 Gly Ala Asp Val Val Tyr
Lys 450 455
* * * * *