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    公开号:AU2005202199A1
    申请号:U2005202199
    申请日:2005-05-20
    公开日:2005-06-23
    发明作者:Jan J Grote；Maartje J Nell
    申请人:Xoma US LLC；
    IPC主号:A61K38-00
    专利说明:
    -1-
    AUSTRALIA
    PATENTS ACT 1990 PATENTS ACT 1990 COMPLETE
    SPECIFICATION
    FOR A STANDARD PATENT
    ORIGINAL
    Name of Applicant: Actual Inventors: Xoma (US) LLC Jan J Grote and Maartje J Nell Address for Service is: SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.
    (02) 9777 1111 (02) 9241 4666 Invention Title: THERAPEUTIC USES OF BPI PROTEIN PRODUCTS IN HUMANS WITH OTITIS MEDIA WITH EFFUSION Details of Original Application No. 52923/00 dated 24 May 2000 The following statement is a full description of this invention, including the best method of performing it known to us:- File: 33824AUP01 500600769 1.DOC/5844
    I
    -la- THERAPEUTIC USES OF BPI PROTEIN PRODUCTS IN HUMANS
    WITH
    OTITIS MEDIA WITH EFFUSION The present application is a divisional application of Australian Application No.
    52923/00, which is incorporated in its entirety herein by reference.
    The present invention relates generally to novel therapeutic uses of BPI protein products for treatment of otitis media with effusion in humans.
    BACKGROUND OF THE INVENTION Otitis media is the most frequently diagnosed disease in children in the United States, and most children will have at least one episode. It is most common in children under 6 years of age. The term "otitis media" refers to a spectrum of diseases in which fluid (effusion) is present in the middle ear space. The effusion may be serous, mucoid, purulent or some combination of these. In acute otitis media, signs and symptoms of acute infection and inflammation accompany middle ear effusion.
    In otitis media with effusion (OME), there is a middle ear effusion behind an intact tympanic membrane without signs or symptoms of acute infection; synonymous terms include "fluid in the ear," "glue ear," and "serous otitis media." OME is defined as chronic when middle ear effusion has been present for at least three months. OME frequently can cause hearing loss at a critical time in a child's development and can interfere with speech, language, behavioural and cognitive development. In most cases, the effusion may resolve spontaneously, but there remain a significant number of children in whom effusion persists even after the acute infection has resolved.
    Approximately 15% of children have chronic problems with longer term conductive hearing loss and sometimes lasting changes of the eardrum and middle ear ossicles. The complications and sequelae often persist into the adult years.
    Persistent effusion, infection or epithelial damage promotes the release of a number of potent inflammatory mediators which, together with numerous bacterial toxins and enzymes present in the effusion, may be stimuli for metaplasia of the middle ear epithelium into a more secretory (mucusproducing) type of epithelium, similar to that found in the lower respiratory tract. The resulting effusion formed in the tympanic cavity will be removed to the pharynx, provided that the mucociliary clearance system (MCS) is functioning normally. The MCS is composed of ciliated cells, secretory cells and a mucus blanket, and propels mucus towards the eustachian tube by means of beating cilia. Mucociliary function can be impaired not only due to lowered ciliary activity but also due to mucus abnormalities. Increased mucus production and altered mucus consistency may seriously affect ciliary beat and coordination.
    The two most important factors preceding the development of OME are a gram-negative bacterial infection and a dysfunction of the eustachian tube. Obstruction of the eustachian tube in germ-carrying rats was reported to result in secretory transformation of the epithelium, a phenomenon not observed in germ-free rats. Kuijpers et al., HistochemicalJ. 16:807-18 (1984).
    Endotoxin is the lipopolysaccharide (LPS) outer membrane constituent of gram-negative bacteria and is a strong inducer of inflammation.
    The lipid A portion of LPS, which accounts for the toxic properties of endotoxin, is structurally similar and serologically cross-reacts among many species of gram-negative bacteria. Endotoxin has been found in human middle ear effusions. DeMaria et al., J Clin. Microbiol. 20(1):15-17 (1984); Ovesen et al., Clin. Otolaryngology 17:531-4 (1992); and Dingman et al., J. Clin.
    Microbiol., 36:3417-19 (1998). Inoculation of endotoxin into the middle ear has been shown to induce morphological changes in the mucociliary transport system, although long term effusion was not observed. [Ohashi et al., Ann.
    Otol. Rhinol. Laryngol. 98:479-84 (1989) and Ohashi et al, Acta Otolaryngol (Stockh), 486:149-61 (1991).] Moreover, injection of viable or non-viable H.
    influenzae or its endotoxin has been shown to induce some inflammatory changes. [DeMaria et al., Ann. Otol. Rhinol. Laryngol 94 (S120):14-6 (1985) and 93:52 (1984).]
    I
    Obstruction of the eustachian tube disables the normal clearance system of the middle ear. The normal middle ear mucosa has a mucociliary clearance system which consists of ciliated cells interspersed with secretory cells. Both the ciliary function and the secretory activity in the tympanic orifice of the middle ear are important for the clearance of the middle ear. During OME this clearance system is often impaired. When an obstruction of the eustachian tube is accompanied by inflammatory changes in the middle ear which lead to dysfunction of the mucociliary clearance system (MCS), an accumulation of fluid in the tympanic cavity occurs.
    A model for OME has been developed in which a combination of eustachian tube obstruction (ETO) and endotoxin injection is used to produce structural changes to and dysfunction of the mucociliary clearance system which persists for 12 weeks. [Nell et al., Eur. Arch. Otorhinolaryngol., 256:167-172 (1999).] Although acute otitis media is typically treated with antibiotic therapy, medical treatment of OME is problematic and has been unsatisfactory.
    The efficacy of treatment with an antihistamine-decongestant combination has been debated; Daley [Pediatrics in Review, 20:85 (1999)] has reported that this therapy is not recommended because these agents are not effective either separately or together. The use of systemic corticosteroids is common, and benefits of this therapy have been demonstrated in several trials, although the risks and side effects of this type of treatment are significant. Sequentially increasing doses of antimicrobial agents and prolonged courses of antibiotic therapy are common treatments but their efficacy has also been debated.
    Myringotomy with tube placement (through the tympanic membrane) has been shown to reduce the frequency of recurrence of acute otitis media and to improve hearing while the tubes remain in place, but is a surgical procedure requiring general anesthesia. Adenoidectomy is generally not recommended but may be beneficial in children older than 4 years of age with chronic OME.
    [Shapiro et al., Postgraduate Medicine, 97:73 (1995); Klein, Clin. Infect. Dis., 19:823 (1994); Daley, supra.]
    I
    in -4- A number of other potential therapies, including antiinflammatory agents, have been tested in various models of otitis media. S- Scarboxymethylcysteine has been reported to reduce damage to ciliated cells and goblet cell hyperplasia in chinchillas with immune-mediated OME, although it did not inhibit infiltration or prevent release of chemical mediators such as histamine and prostaglandin E2. [Hori et al, Ann. Otol. Rhinol. Laryngol 103:567-75 (1994). Treatment of children with secretory otitis media with hydroxyzine, an anti-histamine, has been reported to reduce the rate of relapse and the amount of histamine present in middle ear effusions. [Theoharides et 10 al., Int. Arch. Allergy Immunol. 103:95-101 (1994).] Endotoxin-induced hypertrophic and metaplastic changes of goblet cells in rat nasal respiratory epithelium was reported to be inhibited by intraperitoneal injection of antiinflammatory drugs. [Takahashi et al., Ann. Otol. Rhinol. Larvngol 106:683-7 (1997).] Indomethacin has been reported to inhibit the accumulation of middle ear effusion [Goldie et al., Ann. Otol. Rhinol. LaryngolI 102(12):954-60 (1993)]. In vitro addition of human monoclonal antibody against endotoxin, HA-lA (Centoxin), to medium supplemented with endotoxin has been reported to partially suppress some of the proliferative and morphological effects of endotoxin on cultured rat middle ear epithelium, although the morphology of epithelium cultured in the presence of HA-1A and endotoxin was still altered.
    [Grote et al., Ann. otol. Rhinol. Laryngol 104:226-230 (1995).] Children with OME are currently treated with recurrent placement of ventilation tubes and/or with antibiotics. However, the insertion of tubes will only temporarily remove the middle ear effusion, and antibiotics can be effective in eradicating the infection yet do not reduce the accumulation of fluid.
    Because of the current lack of satisfactory treatment, including the disadvantages of ventilation tubes and the growing resistance of many bacterial species to antibiotics, there remains a need for new therapies which can prevent the occurrence of OME, or more quickly resolve OME once it occurs.
    I I I 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 gramnegative 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 Figure 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. Patent No. 5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.
    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 Nterminal 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 gramnegative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI2," 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,,, has been produced as described in Horwitz et al., Protein Expression Purification, 8:28-40 (1996).
    The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, 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, 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.
    BPI protein products have a wide variety of beneficial activities.
    BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Patent 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 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. Patent 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 grampositive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Patent 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 antifungal agents, as described in U.S. Patent 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. Patent No. 5,858,974, which is in turn a continuation-in-part of U.S. Application Serial No. 08/504,841 filed July 1994 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/092 6 2) 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. Patent 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 Serial No. 08/694,843 filed August 9, 1996 and WO 98/06415 (PCT/US97/13810), which are incorporated herein by reference. Finally, BPI protein products exhibit antimycobacterial activity, as described in co-owned, co-pending U.S. Application Serial No. 08/626,646 filed April 1, 1996, which is in turn a continuation of U.S. Application Serial No. 08/285,803 filed August 14, 1994, which is in turn a continuation-in-part of U.S. Application Serial No. 08/031,145 filed March 12, 1993 and corresponding International Publication No. W094/20129 (PCT/US94/02463), all of which are incorporated herein by reference.
    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.
    Patent No. 5,643,875, which is incorporated herein by reference.
    BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in coowned, co-pending U.S. Application Serial No. 08/644,287 filed May 10, 1996 and continuation No. 08/927,437 filed September 10, 1997 and International Publication No. W097/42966 (PCT/US97/08016), all of which are incorporated herein by reference), hemorrhagic trauma in humans, (as described in U.S. Patent No.5,756,464, U.S. Application Serial 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. Patent No. 5,494,896, which is incorporated herein by reference), ischemia/reperfusion injury (as described in U.S. Patent No. 5,578,568, incorporated herein by reference), and liver resection (as described in co-owned, co-pending U.S. Application Serial No.
    08/582,230 filed January 3, 1996, which is in turn a continuation of U.S.
    Application Serial No. 08/318,357 filed October 5, 1994, which is in turn a continuation-in-part of U.S. Application Serial No. 08/132,510 filed October 1993, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404), all of which are incorporated herein by reference).
    BPI protein products also neutralize the anti-coagulant activity of exogenous heparin, as described in U.S. Patent 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. Patent 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.
    BPI protein products are also useful in antithrombotic methods, as described in U.S. Patent No. 5,741,779 and U.S. Application Serial No.
    09/063,465 filed April 20, 1998 and corresponding WO 97/42967 (PCT/US97/08017), all of which are incorporated herein by reference.
    SUMMARY OF THE INVENTION The present invention provides novel therapeutic uses for BPI protein products for treatment (both prophylactic and therapeutic) of otitis media with effusion (OME) in humans, which results in amelioration of the clinical signs and symptoms, quicker resolution of the signs and symptoms, or a reduction in the occurrence, recurrence or severity of complications associated with the disease. Treatment of subjects with and without tympanostomy tubes is also contemplated.
    It is contemplated that the administration of a BPI protein I product may be accompanied by the concurrent administration of other known therapeutic agents appropriate for treating otitis media or OME, including histamine, corticosteroids, and antibiotics.
    Use of a BPI protein product in the manufacture of a medicament for the treatment of OME is also contemplated.
    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.
    DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel therapeutic uses for BPI protein products for treatment of humans suffering from otitis media with effusion (OME), including recurrent or chronic OME. "Treatment" as used herein encompasses both prophylactic and therapeutic treatment. Treatment is expected to be effective even when the patient shows no signs of acute infection.
    The invention thus contemplates methods for treating a human suffering from OME which comprise administering a therapeutically effective amount of a BPI protein product. Such an amount may be effective to ameliorate clinical signs and symptoms associated with OME, including impaired mucociliary clearance function, histological signs of inflammation or mucociliary system dysfunction, clinical signs of inflammation (including swelling that impinges on drainage through the eustachian tube), the accumulation or presence of effusion, reduced mobility of the tympanic membrane as observed by pneumatic otoscope or as measured by tympanometry, which measures tympanic membrane stiffness [a tympanometric width of >250 dekaPascals (daPa) is considered diagnostic of OME]), hearing loss as measured by an audiogram), especially hearing loss at high frequencies >4000 Hz), local pain and discomfort, and other signs and symptoms. Such an amount may also be effective to more quickly resolve i L reduce the duration of) these clinical signs and symptoms or to reduce the occurrence, recurrence or severity of complications associated with OME, including the development of chronic'OME; recurrence of otitis media or OME; the need for myringotomy or placement oftympanostomy tubes; intratemporal complications such as drainage, chronic suppurative otitis media.
    tympanosclerosis, atrophy, tympanic membrane perforation or retraction, atelectasis, adhesive otitis media, cholesteatoma, facial nerve paralysis and destruction of ossicles; mastoid complications such as mastoiditis, mastoid abscess and petrositis; intracranial complications such as menigitis, lateral sinus thrombosis and extradural abscess; the potential speech, language, behavioral, cognitive or other developmental delays, potential learning disabilities, deficient expressive language skills, or deficient attention skills. Treatment with BPI protein product may assist in the re-establishment of the mucociliary clearance system, may render unnecessary a myringotomy procedure and/or the placement of tympanostomy tubes or may shorten the duration of tube placement. These advantages taken together result in reduced treatment costs and an improved quality of life.
    The treatment of neonates, infants, toddlers and older children, as well as adults, whether healthy or suffering from accompanying illness or infection, is contemplated. Treatment is contemplated not only of subjects suffering from OME (in which an effusion in the middle ear is not accompanied by signs of acute infection), with or without the presence of tympanostomy or ventilation tubes; but also subjects suffering from recurrent OME or chronic OME (present at least three months). Further contemplated is treatment of subjects at risk for development of OME, including subjects with a small eustachian tube, subjects with otherwise impaired middle ear drainage by impaired function of the mucociliary clearance system), subjects with chronic or recurrent episodes of OME, and subjects with other risk factors for a longer duration of OME group child care, exposure to other children, smoke exposure, feeding in supine position, early onset of otitis media, several prior episodes of otitis media, or having a sibling suffering from otitis media).
    I -11- The invention is based on the discovery that a BPI protein product, rBPI,I, was efficacious in a rat model of OME. An OME syndrome O can be produced by a combination of eustachian tube obstruction and endotoxin injection. In this model of otitis media with effusion (OME), histological signs of mucociliary clearance system dysfunction include hyperproliferation of the epithelium, increases in secretory cells of the epithelium and degeneration of cilia, which result in a disturbance of the mucociliary clearance system of the middle ear. Endotoxin injection alone also Sdisturbs the mucociliary clearance system but induces less morphological 1 10 changes in the epithelial layer. As long as these morphological changes are present in the middle ear, the OME will continue. Therefore, re-establishment of the clearance system is expected to be an important step in treating ongoing OME or preventing recurring OME. In this model, injection of rBPI2 1 directly into the middle ear two days or even as late as two weeks after OME induction protected the middle ear mucosa from morphological changes which may disturb the normal mucociliary clearance system of the middle ear and also inhibited the influx ofPMNs and macrophages.
    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. Patent 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. Patent No. 5,439,807 and corresponding International Publication No. WO 93/23540 (PCT/US93/04752), which are all incorporated herein by reference, disclose II I I 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.
    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,. In that publication, an expression vector was used as a source ofDNA encoding a recombinant expression product (rBPI,) 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 Figure 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 Figure 1 of Gray et al., supra, with the exceptions noted for rBPI, 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 Serial No. 09/099,725 filed June 19, 1998, incorporated herein by reference. Other examples include dimeric forms of BPI fragments, as described in U.S. Patent No. 5,447,913 and corresponding International Publication No. WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.
    Biologically active variants of BPI (BPI variants) include but are i -13not 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. Patent 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.
    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. Patent 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, 2 Acys or rBPI,2. Production of this N-terminal analog of BPI, rBPI 2 1 has been described in Horwitz et al., Protein Expression Purification, 8:28-40 (1996). Similarly, a fragment consisting of residues 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 3 2 has been described in co-owned, co-pending U.S. Application Serial No. 09/099,725 filed June 19, 1998. Other examples include dimeric forms of BPI analogs; e.g.
    U.S. Patent No. 5,447,913 and corresponding International Publication No.
    WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.
    Other BPI protein products useful according to the methods of
    I
    S-14-
    O
    the invention are peptides derived from or based on BPI produced by synthetic or recombinant means (BPI-derived peptides), such as those described in SInternational Publication No. WO 97/04008 (PCT/US96/03845), which Scorresponds to U.S. Application Serial No. 08/621,259 filed March 21, 1996, and International Publication No. WO 96/08509 (PCT/US95/09262), which corresponds to U.S. Patent No. 5,858,974, and International Publication No.
    WO 95/19372 (PCT/US94/10427), which corresponds to U.S. Patent No.
    0 5,652,332, and International Publication No. W094/20532 (PCT/US94/02465), which corresponds to U.S. Patent No. 5,763,567 which is 10 a continuation of U.S. Patent No. 5,733,872, which is a continuation-in-part of U.S. Application Serial No. 08/183,222, filed January 14, 1994, which is a continuation-in-part ofU.S. Application Serial No. 08/093,202 filed July 1993 (corresponding to International Publication No. WO 94/20128 (PCT/US94/02401)), which is a continuation-in-part of U.S. Patent No.
    5,348,942, as well as International Application No. PCT/US97/05287, which corresponds to U.S. Patent No. 5,851,802, the disclosures of all of which are incorporated herein by reference.
    Presently preferred BPI protein products include recombinantlyproduced N-terminal analogs and fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI, or rBP12, rBPI(10-193)C132A (rBPI(10-193)ala' 32 dimeric forms of these Nterminal proteins rBP 4 dimer), and BPI-derived peptides.
    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 rBPI2) comprises the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or mM citrate, 150 mM NaCI, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, DE). Another stable pharmaceutical composition containing BPI protein products rBPI,,) comprises the BPI protein product at a concentration of 2 mg/ml in mM citrate, 150 mM NaCI, pH 5.0, 0.2% poloxamer 188 and 0.002% polysorbate 80. Such preferred combinations are described in U.S. Patent 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 Serial No. 08/586,133 filed January 12, 1996, which is in turn a continuation-in-part of U.S.
    Application Serial No. 08/530,599 filed September 19, 1995, which is in turn a continuation-in-part of U.S. Application Serial No. 08/372,104 filed January 13, 1995, and corresponding International Publication No. W096/21436 (PCT/US96/01095), all of which are incorporated herein by reference, other poloxamer formulations of BPI protein products with enhanced activity, with or without polysorbate, may be utilized.
    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 by intraperitoneal lavage), intrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal. Topical routes include administration in the form of salves, ophthalmic drops, ear drops for administration into the ear canal), irrigation fluids (for, e.g., irrigation of wounds) or medicated shampoos. For example, for topical administration in drop form, about 10 to 200 ~pL of a BPI protein product composition may be applied one or more times per day as determined by the treating physician.
    When given parenterally, BPI protein product compositions are generally injected in doses ranging from 1 lg/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 I 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, 1 to 3 days, and additionally as determined by the treating physician.
    Administration of BPI protein product for OME is preferably via instillation of a BPI protein product composition into the middle ear through the tympanic membrane, via a needle inserted for that purpose or via tympanostomy or ventilation tubes already in place, optionally preceded by withdrawal of existing effusion from the middle ear. The amount to be administered can be as much BPI protein product composition as may be accommodated in the middle ear, up to about 2 ml of fluid. The BPI protein product composition may contain BPI protein product at a concentration ranging from, 1 pg/mL to 10 mg/mL, or 0.2 to 2 mg/mL.
    The BPI protein product composition may be administered one time only as a single dose, or additional doses may be administered periodically, once weekly, once every two weeks, once every three weeks, or monthly, continuing until the desired effect is achieved. BPI protein product administration may also be accompanied by any surgical procedure deemed appropriate, e.g.
    myringotomy or tympanostomy tube placement.
    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 subject.
    "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 I t -17- C1 intravenous lines. Alternatively, the BPI protein product may be administered t in a special form for gastric delivery, while the second agent(s) is(are) administered, orally. The formulated BPI protein product and second N 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.
    SOther aspects and advantages of the present invention will be Sunderstood upon consideration of the following illustrative examples. Example S1A addresses the effect of BPI protein product on the signs and symptoms of 1 10 OME when administered concurrently with endotoxin or two days after endotoxin. Example 1B addresses the effect of BPI protein product on the signs and symptoms of OME when administered two weeks after endotoxin.
    Example 2 addresses the effect of BPI protein product in humans suffering from OME.
    EXAMPLE 1 Effect of BPI Protein Product on Otitis Media with Effusion A. Treatment after two days The effect of a BPI protein product, rBPI, 2 was evaluated as follows in a rat model of chronic OME caused by eustachian tube obstruction in combination with endotoxin injection. Sixty-four female Wistar rats (body weight about 200 g, 10 weeks old) were divided into four control groups: control group (no injection), injection control group treated with sterile pyrogen free phosphate buffered saline (PBS), BPI control group treated with 2 mg/ml rBPI 21 and endotoxin (E)/BPI group injected with 2 ig/ml endotoxin [from Salmonella typhimurium, Sigma, L6511] premixed (1:1) with 2 mg/ml rBPI 21
    (E/BPI);
    and four experimental groups: E group, injected with 2 Ag/ml endotoxin, I -18t1 E+BPI group, treated with 2 mg/ml rBPI, after two days.
    ct ETO+E group, which underwent eustachian tube Sobstruction (ETO) in combination with 2 /g/ml endotoxin C'I injection (ETO+E), and ETO+E+BPI group, which underwent ETO in combination with 2 ug/ml endotoxin injection followed by 2 mg/ml rBPI 2 1 C injection after two days.
    CN The rats were anesthetized with nitrous oxide, the Eustachian tube was O accessed by a ventral approach, medially to the posterior belly of the digastric C 10 muscle, and was obstructed by plugging a small piece of Gelfoam® (Upjohn Co.) into the tube. Additionally, tissue glue (Historesin®) was used to keep the Gelfoam® in the tube. This procedure was directly followed by injection of the endotoxin and/or treatment solutions through the tympanic membrane until the solution overflowed. Injection of 50 pl of a solution of 2 Jug/ml endotoxin results in a final concentration of approximately 100 ng endotoxin per ear.
    After 1, 2, 4 or 12 weeks the animals were sacrificed with CO, gas and subsequently decapitated. The middle ear was dissected from the skull, denuded of adhering tissues and further processed for light microscopy and scanning electron microscopy. For light microscopy, the specimens were fixed with a solution of 1.5% glutaraldehyde in cacodylate buffer (0.14 M, pH 7.4), decalcified with a solution of 10% EDTA in 1.5% glutaraldehyde in cacodylate buffer (0.14M, pH 7.4) and subsequently dehydrated in a graded series of ethanols and embedded in glycol methacrylate (JB4, Brunschwig Chemie).
    Sections were stained with toluidine blue for histological studies and with Alcian Blue-PAS for glycoprotein histochemistry. In addition, two middle ears from each time period, prepared and fixed as described for light microscopy, were processed for scanning electron microscopy. The specimens were dehydrated in a graded series of ethanols and critical point dried using liquid
    CO
    2 The distribution of the epithelial cells was studied with a Philips SEM 525M scanning electron microscope at 15 kV after mounting and coating with gold in a Balzers MED010 sputtercoater.
    -19- The numbers of ciliated and secretory goblet cells were counted in duplicate in each ear in standardized areas of the tympanic orifice of the Eustachian tube. The number of macrophages and PMNs, were counted in duplicate in each ear in the submucosal layer of the epitympanum and the hypotympanum of the middle ear bulla. To compare means of the different variables, one-way ANOVA Tukey's HSD test with significance level P 0.05, was performed using the Statistical Package for the Social Science (SPSS). This test uses the studentized range statistic to make all of the pairwise comparisons between groups and sets the experimentwise error rate at the error rate for the collection for all pairwise comparisons. Results are reported as mean cell numbers standard error (SE).
    The results of light- and scanning electron microscopy showed that control middle ears appeared normal during the entire treatment period.
    The hypotympanum of the middle ear consisted of thin, one-layered squamous epithelium, containing very few ciliated cells. In the tympanic orifice of the Eustachian tube a more pseudostratified, cuboidal or cylindrical epithelium (representing the mucociliary clearance system) was observed which contained an abundant number of ciliated cells and a few secretory cells. Inoculation of BPI induced a slight increase in goblet cells after one and twelve weeks but caused no morphological changes to the epithelial layer. In addition, some infiltration of PMNs in the middle ear cavity was observed, but these cells disappeared after two weeks. In comparison to the non-injected controls, the premixed solution of endotoxin and BPI did induce morphological changes in the middle ear. Injection ofrBPIz 2 into the middle ear cavity two days after the induction of OME prevented the induction of morphological changes to the mucociliary clearance system. BPI protein product administration at day two prevented the increases in secretory cells, the hyperproliferation of the epithelial layer and the infiltration of inflammatory cells into the subepithelial layer, and inhibited the formation of cobblestone-like cells. Furthermore, no deformation or degeneration of the cilia was observed in the BPI-treated group.
    ir -Lu-
    O
    SB. Treatment after two weeks Twenty-four female Wistar rats (body weight about 200 g, weeks old) were used in this study. They were divided into three control O groups: untreated control group (no injections), BPI control group injected with 2 mg/ml rBPI 2 and BPI-buffer control group injected with BPI formulation O buffer [5 mM citrate, 150 mM NaCI, pH 5.0, 0.2% poloxamer S. 188 and 0.002% polysorbate and two experimental groups: ETO+E group that underwent eustachian tube obstruction (ETO) in combination with injection of 2 gg/ml endotoxin, ETO+E+BPI group that underwent ETO followed by injection of 2 mg/ml rBPI 21 after two weeks (ETO+E+BPI).
    The BPI protein product was injected directly into the middle ear cavity two weeks after the induction of OME to a final concentration of approximately 100 g per ear, which is approximately 1000 times the endotoxin concentration administered.
    Animals were sacrificed after 4 or 12 weeks and their middle ears were prepared and analyzed as described above in Example 1A. Results are displayed in Table 1 below.
    TABLE 1 Cilia Goblet cells Macrophages 4 wk 12 wk 4wk 12 wk 4wk 12 wk Untreated 26+4 24+3 15+3 14+2 10+4 13+4 rBPI 2 1 23+4 21+3 19+2 16+3 17+5 21+4 BPI-buffer 23+3 22+3 19+3 15+2 11+6 20+9 ETO E 8+3* 6+4* 27+4* 31+4* 25+2* 27+3* ETO+E+rBPI 21 28+4* 26+6* 18+3** 18+4** 11+5** 18+3 I -21represent values significantly different from untreated ears represent values significantly different from ETO E of the same week In the untreated control group, at 12 weeks 14+2 secretory cells and 24+ 3 ciliated cells were counted. In the subepithelial layer of the hypotympanum and the epitympanum, at 12 weeks 13+4 macrophages were present.
    In the BPI control groups, injection of either rBPI2, or BPI formulation buffer into the middle ear cavity did not induce histological changes to the middle ear mucosa. In addition, no significant changes in the numbers of secretory cells nor in the number of ciliated cells were measured.
    Although the numbers ofmacrophages increased to 21+ 4 at 12 weeks after rBPI,, injection, this was not significantly different from the untreated ears.
    In the group with experimentally induced OME (ETO+E), obstruction of the eustachian tube in combination with endotoxin injection induced thickening of the middle ear mucosa. An important parameter of inflammation is the influx ofpolymorphonuclear cells (PMNs) and macrophages. After experimentally induced OME, a direct influx of PMNs occurs in the middle ear which gradually decreases followed by a gradual increase in macrophages. In the subepithelial layer a significant number of macrophages (27+3, at 12 weeks) were counted. The number of macrophages in the submucosal layer of the middle ear cavity was significantly higher than in the untreated ears at both 4 and 12 weeks. Furthermore, in the tympanic orifice a significant increase in the number of secretory cells (31±4) and a significant decrease in the number of ciliated cells were measured at 12 weeks. With scanning electron microscopy it was observed that the epithelial cells in the hypotympanic part of the middle ear cavity contained an abundant number of microvilli and the surface was irregular and swollen. In the tympanic orifice, abnormalities in the MCS were observed, including large areas of secretory cells with sporadic mucus deposits and separation of some epithelial cells.
    In the BPI treatment group (ETO+E+BPI), injection of rBPI 21 -22two weeks after induction of OME resulted in a normal thin squamous epithelium in the hypotympanum, which was not thickened and contained few microvilli. The number ofmacrophages was significantly decreased at 4 weeks (1l1+5 compared to 25+2 for ETO+E). At 12 weeks, the number of macrophages was decreased but not significantly different from ETO+E. In addition, in the tympanic orifice the numbers of secretory cells were significantly decreased at 4 and 12 weeks (18+4) compared to ETO+E. The numbers ofciliated cells were significantly increased at 4 weeks (28±4) and 12 weeks (26+6) compared to ETO+E. Both the number of ciliated and secretory cells and the number ofmacrophages were not significantly different from that of the untreated control group. Finally, with scanning electron microscopy the cilia in the tympanic orifice were observed to have a normal appearance and no mucus deposits or separation of epithelial cells were observed.
    These results show that BPI protein product treatment two weeks after induction of OME inhibited the histological signs of MCS dysfunction and inhibited the influx ofmacrophages associated with this disease state. This result indicates that BPI protein product can re-establish the MCS function of the middle ear and is expected to be effective for treating OME.
    EXAMPLE 2 Clinical studies are performed to test the effect ofrBPI,, in humans suffering from OME. A study is carried out generally as follows.
    Children suffering from OME, preferably chronic OME with recurrent otitis media, are divided into treatment and control groups. In the treatment group, BPI protein product is instilled into the middle ear, e.g. through existing ventilation tubes or via a needle injection. The children are then monitored for up to one month following treatment for, resolution of tympanic membrane stiffness as measured on a tympanogram, improvement in hearing, especially high frequency hearing, by audiogram, and time to resolution of these endpoints. Biopsies of middle ear epithelium to evaluate histological signs of MCS dysfunction may also be performed. Alternatively, rather than dividing the subjects into treatment and control groups, for each subject (with OME in both ears), one ear is the treatment ear and the other ear is the control ear. In another clinical study, treatment modalities are compared among several groups, BPI protein product treatment with tube placement, BPI protein product treatment without tube placement, tube placement alone without BPI protein product treatment, and no treatment and no tube placement. In further or ongoing clinical studies, repeat injections of BPI protein product are performed, every 1, 2, 3 or 4 weeks and the children continue to be monitored.
    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.
    权利要求:
    Claims (6)
    [1] 1. A method of treating a human suffering from otitis media with effusion comprising administering to said human a therapeutically effective amount of a bactericida/permeability-increasing protein (BPI) protein product.
    [2] 2. The method of claim 1 wherein the BPI protein product is rBPI 21
    [3] 3. The method of claim 1 wherein the BPI protein product is an N-terminal fragment of BPI having a molecular weight approximately between about 20 to 25 kd.
    [4] 4. is rBPI(10-193)ala 2 The method of claim I wherein the BPI protein product The method of claim 1 wherein the BPI protein product is rBPIo.
    [5] 6. The method of claim 1 wherein the human has tympanostomy tubes placed in the middle ear. DATED this 20 h day of May 2005 Shelston IP Attorneys for: Xoma (US) LLC -1I- SEQUENCE LISTING ,110> XOMA (US) LLC ,120> THERAPEUTIC USES OF BPI PROTEIN PRODUCTS IN HUMANS WITH OTITIS MEDIA WITH EFFUSION <130> 29715/35634 <140> <141> <150> 60/136,148 <151>
    [6] 1999-05-24 <160> 2 ,170> Patentln Ver. <210> 1 <211> 1813 <212> DNA <213> HOMO sapiens <220> <221> CDS <222> (31) (1491) <220> <221> mat..yepiide <222> (124)--.(1491) <400> 1 caggccttga ggttttggca gciciggagg atg aga gag aac atg gcc agg ggC Met Arg Giu Asn Met Ala Arg Gly cct tgc aac Pro CyS Asn ggc acc gcc Gly Thr Ala ccg aga tg.g gtg icc ctg aig gtg cic Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile gtg aca. gcg gcc gic aac cct ggc gtc gig gic agg atc Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile -1 1 icc Ser cag aag ggc ctg Gin Lys GJly Leu tac gcc agc cag Tyr Ala Ser Gin Gin gg acg gcc gct Giy Thr Aia Ala 150 198 246 294 cag aag gag ctg Gin Lys Giu Leu aag agg Lys Arg atc aag att cci gac tac tca gac Ile Lys Ile Pro ASP Tyr Ser Asp agc ttt Ser Phe aag atc aag cat cit ggg aag ggg cat tat agc tic tac agc atg gac LYS Ile Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met ASP 5o -2- atc cgt gaa tic cag Ile Arg Giu Phe Gin ctt ccc agt Leu Pro Ser tcc cag ata Ser Gin Ile agc aig gtg ccc aat Ser Met Vai Pro Asn gtg ggc Vai Gly ctt aag ttc tcc Leu Lys Phe Ser atc agc Ile Ser 80 aac gcc aat aic aag atc agc Asn Ala Asn Ile Lys Ile Ser Gly aaa Lys tgg aag gca caa Trp Lys Ala Gin aag Lys 95 aga ttc tta aaa Arg Phe Leu Lys atg agc ggc aat~ Met Ser Giy Asn 1o0 gat ctg aag ctg Asp Leu Lys Leu ttt gac Phe Asp 105 ggc agi Giy Ser 120 ctg agc ata gaa Leu Ser Ilie Glu ggc Giy 110 atg tcc att tcg Met Ser Ile Ser gct Ala 115 438 486 534 582 aac ccc acg Asn Pro Thr cac atc aac His Ilie Asn 140 tca Ser 125 ggc aag ccc acc Gly Lys Pro Thr atc acc tgc tcc agc Ile Thr Cys Ser Ser 130 tgc agc agc Cys Ser Ser 135 gtc ggg tgg Vai Gly Trp agt gtc cac gtg Ser Val His Val atc tca aag agc Ile Ser Lys Ser Lys 150 ctg atc Leu Ile 155 caa ctc ttc cac Gin Leu Phe His aaa Lys 160 aaa att gag tct Lys Ile Giu Ser gcg ctt cga aac aag Ala Leu Arg Asn Lys 165 atg Met 170 aac agc cag gtc Asn Ser Gin Val tgc Cys 175 gag aaa gtg acc Giu Lys Vai Thr aat Asn 180 tct gta tcc tcc Ser Val Ser Ser Lys 185 630 678 726 cig caa cct tat Leu Gin Pro Tyr ttc Phe 190 cag act ctg cca Gin Thr Leu Pro gta Val 195 atg acc aaa ata Met Thr Lys Ile gat tct .Asp Ser 200 gtg gct gga Val Ala Gly gag acc ctg Giu Thr Leu 220 atc Ile 205 aac tat ggt ctg Asn Tlyr Gly Leu gtg Val 210 gca cct cca gca Ala Pro Pro Ala acc acg gct Thr Thr Ala 215 gag aac cac Giu Asn His 774 822 gat gia cag atg Asp Val Gin Met aag Lys 225 ggg gag ttt tac Gly Giu Phe Tyr agt Ser 230 cac aat His Asfl 235 cca cct ccc ttt Pro Pro Pro Phe gct Ala 240 cca cca gtg atg Pro Pro Val Met ttt ccc gct gcc Phe Pro Ala Ala cat His 250 gac cgc atg gta Asp Arg Met Val tac TIyr 255 ctg ggc ctc tca Leu Gly Leu Ser gap Asp 260 tac ttc ttc aac Tyr Phe Phe Asn aca Thr 265 870 918 966 gcc ggg ctt gta tac caa gag gct ggg Ala Gly Leu Val Tyr Gin Glu Ala Gly gtc ttg aag atg acc Val Leu Lys Met Thr 275 ctt aga. Leu Arg 280 CA -3- gat gac atg Asp Asp Met itt gga acc Phe Gly Thr 300 at Ile 285 cca aag gag icc Pro Lys Giu Ser aaa Lys 290 ttt cga ctg aca Phe Arg Leu Thr acc aag tic Thr Lys Phe 295 aac atg aag Asn Met Lys 1014 1062 tic cta cct gag Phe Leu Pro Giu gig Val 305 gcc aag aag iii. Ala Lys Lys Phe ccc Pro 310 ata cag Ile Gin 315 aic cat gic ica Ile His Val Ser tcc acc ccg cca Ser Thr Pro Pro cac His 325 ctg tct gig cAg Leu Ser Val Gin ccc Pro 330 acc ggc cii acc Thr Gly Leu Thr iac cci gcc gig Tyr Pro Ala Val gai Asp 340 gic cag gcc ttt Val. Gin Ala Phe gcc Al a 345 1110 1158 1206 gtc ctc ccc aac Val Leu Pro Asn icc ctg gci icc Ser Leu Ala Ser cic Leu 355 ttc cig ait ggc Phe Leu Ile Gly aig cac Met His 360 aca act ggt Thr Thr Gly gag cic aag Glu Leu Lys 380 ggc ccc tic Gly Pro Phe 395 icc Ser 365 aig gag gtc agc Met Glu Val Ser gcc Ala 370 gag icc aac agg Glu Ser Asn Arg cit gtt gga Leu Val Gly 375 ica aai at Ser Asn Ile 1254 1302 1350 ctg gat agg cig Leu Asp Arg Leu cic Leu 385 cig gaa cig aag Leu Giu Leu Lys cac, His 390 ccg git gaa Pro Val Glu tg Leu 400 cig cag gat atc Leu Gin Asp Ile aig Met 405 aac iac ait gia Asn Tyr Ile Val ccc Pro 410 ati cii gig ctg Ile Leu Val Leu ccc Pro 415 agg git aac gag Arg Val Asn Glu aaa Lys 420 cta cag aaa ggc Leu Gln Lys Gly tic Phe 42S 1398 1446 cci etc ccg acg Pro Leu Pro Thr ccg Pro 430 gcc aga gic cag Ala Arg Val Gin ctc Leu 435 tac aac gia gig Tyr Asn Val'Val cti cag Leu Gin 440 cci cac cag Pro His Gin aac Asfl 445 tic ctg cig tic Phe Leu Leu Phe ggi Gly 450 gca gac gii Ala Asp Val gic tat aaa Val Tyr Lys 455 1491 igaaggcacc aggggtgccg ggggcigtca gccgcacctg iiccigaigg gcigtggggc 1551 accggcigcc titccccagg gaatccictc cagatciiaa ccaagagccc ciigcaaaci 1611 iciicgactc agaitcagaa aigaiciaaa cacgaggaaa caitaticat iggaaaagtg i671 catggtgigi aitiaggga itaigagcii ciitcaaggg ciaaggcigc agagatatti 1731 cciccaggaa icgtgiiica atigiaacca agaaaiiicc atiigigcii caigaaaaaa i791 aacticiggi ttticaig ig 1813 -4- <210> 2. <211> 487 <212> PRT <213> Homo sapiens <400> 2 Met Ser Asn Ser Ile His Ser Asa Leu Ser Ile 130 Ile Ile Val Pro Val 210 Gly Arg Leu Pro Gin Pro Tyr Gin Ala Lys Ala Thr Ser Giu Thr Val 1.95 Ala Glu Giu Met Gly Gin Asp Ser Ile Asal Met 100 Asp Cys LYS Ser Asal 180 Met Pro Phe Asn Val Val Gly Tyr Phe Ser Ile Ser Leu Ser Ser Ala 165 Ser Thr Pro Met Aia Arg -25 Leu Vai Ala Val Vai Arg Thr Ala Aia Ser Asp Ser 40 Tyr Ser Met 55 Met Val Pro Lys Ile Ser Gly Asn Phe Lys Leu Giy 120 Ser Cys Ser 135 Lys Vai Gly 150 Leu Arg Asn Vai Ser Ser Lys Ile Asp .200 Aia Thr Thr 215 Ser Giu Asn 230 Giy Pro Cys Asn Ala Pro Arg Trp, Val Ile Ile Leu 25 Phe Asp Asn Gly Asp 105 Ser Ser Trp Lys Lys 185 Ser Al a His Gly Ser 10 Gin Lys Ile Vai Lys 90 Leu Asal His Leu Met 170 Leu Val Giu His Thr Gin LYS Ile Arg Gly 75 Trp Ser Pro Ile Ile 155 Asa Gin Ala Thr Asn 235 Ala I LysC Giu LysI Glu 60 Leu Lys Ile Th r Asa 140 Gin Ser Pro Giy Leu 220 Pro ;iy I jeu ~iis Phe Lys Ala Giu Ser 125 Ser Leu Gin Tyr Ile 205 Asp Pro ~hr jeu 4'S Aeu Glm Phe Gin Gly 110 Gly Vai Phe Val Phe 190 Asn Val Prc Ala Asp Arg Gly Leu Ser Lys Met Lys His His Cys 175 Gin LTyr Gin Phe Jaa Ile Lys Pro Ile Arg Ser Pro Val LYE 160C GliL Th Gil Mel Al 24 Val Al a Lys Giy Ser Ser Phe Sle Thr *His 145 Lys Lys *Leu (Leu Lys 225 a Pro Pro Leu Val Met Ser Asp Glu Phe 245 Tyr Phe 260 Gly V 2 Lys P 290 Ala I Thr Ala Ser Ala 370 Leu Gin Asn Gin Gly 450 ral :75 ,he 'yS Pro Tal Leu 355 Glu Glu Leu Arg Lys Pro Asp 340 Phe Ser Leu Uys I L e u Phe His 325 Val Leu Asf Lys Met Thr Pro 310 Leu Gin Ile Arg His Pro I Phe Thr Thr 295 Asl Ser Ala Gly Leu 375 Ser Leu 280 Lys Met Val Phe Met 360 Val Asn Arg Phe Lys Gin Ala 345 His Gly Ile ksp Phe Ile Pro 330 Val Thr Glu Gly Lsp ;ly 3.l 315 Thr Leu Thr Let Prc Met I Thr I 300 Ile I Gly Pro Gly 1 Lys 380 2 Phe :le he 4is Leu Asn Ser 365 Let Prc Pro I Leu I Val Thr Ser 350 Met Asp D Val la ksn Ala His Asp Arg Met Val Tyr Leu Gly 250 255 Thr Aia Gly Leu Val Tyr Gin Glu Ala' 265 270 ,ys C ro Ser Phe 335 Ser Glu Arg Glu lu 3iu Ala 320 Tyr Leu Val Leu Let Ser Val 305 Ser Pro Ala Ser Leu 385 1 Leu Val g Val u Phe 395 390 Asp Ile Met 405 Glu Lys Leu 420 Leu Tyr Asn 435 Ala Asp Val Asn Gin Val Val Tyr Ile Val Lys Gly Phe 425 Val Leu Gin 440 Tyr Lys 455 Pro 410 Pro Pro Ile Leu His Leu Pro Gin Val Leu Thr Pro 430 Asn Phe 445 Pro Arc 415 Ala Ar! Leu Le
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    同族专利:
    公开号 | 公开日
    AU2005202199B2|2007-08-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5198541A|1987-08-11|1993-03-30|New York University|Dna encoding bactericidal/permeability-increasing proteins|
    法律状态:
    2005-06-23| TH| Corrigenda|Free format text: IN VOL 19, NO 21, PAGE(S) 1355 UNDER THE HEADING COMPLETE APPLICATIONS FILED - NAME INDEX UNDER THENAME XOMA (US) LLC, APPLICATION NO. 2005202199, UNDER INID (54) CORRECT THE TITLE TO READ THERAPEUTIC USES OF BPI PROTEIN PRODUCTS IN HUMANS WITH OTITIS MEDIA WITH EFFUSION. |
    2007-09-13| TC| Change of applicant's name (sec. 104)|Owner name: XOMA TECHNOLOGY LTD Free format text: FORMER NAME: XOMA (US) LLC |
    2007-12-06| FGA| Letters patent sealed or granted (standard patent)|
    2009-12-17| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|
    优先权:
    申请号 | 申请日 | 专利标题
    US60136148||1999-05-24||
    AU52923/00A|AU5292300A|1999-05-24|2000-05-24|Therapeutic uses of bpi protein products in humans with otitis media with effusion|
    AU2005202199A|AU2005202199B2|1999-05-24|2005-05-20|Therapeutic uses of BPI protein products in humans with otitis media with effusion|AU2005202199A| AU2005202199B2|1999-05-24|2005-05-20|Therapeutic uses of BPI protein products in humans with otitis media with effusion|
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