Anti-chlamydial methods and materials

Details Anti-chlamydial methods and materials Anti-chlamydial methods and materials

2000-10-30

2003-06-24

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S021800, C530S324000, C530S350000, C128S899000, C604S021000, C604S540000, C623S001100

Reexamination Certificate

active

06583116

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to methods of treating chlamydial infections by administration of bactericidal/permeability-increasing (BPI) protein products.
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.
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 has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-terminal fragment of human BPI possesses 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
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).
The bactericidal effect of BPI has been 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). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms. Although BPI was commonly thought to be non-toxic for other microorganisms, including yeast, and for higher eukaryotic cells, it has recently been discovered that BPI protein products, as defined infra, exhibit activity against gram-positive bacteria, mycoplasma, mycobacteria, fungi, and protozoa. [See allowed, co-owned, co-pending U.S. patent application Ser. No. 08/372,783 filed Jan. 13, 1995, the disclosures of which are incorporated herein by reference; co-owned, copending U.S. patent application Ser. No. 08/626,646, the disclosures of which are incorporated herein by reference; co-owned, co-pending U.S. patent application Ser. No. 08/372,105, the disclosures of which are incorporated herein by reference; and co-owned, co-pending U.S. patent application Ser. No. 08/273,470, the disclosures of which are incorporated herein by reference.] It has also been discovered that BPI protein products have the ability to enhance the activity of antibiotics against bacteria. [See U.S. Pat. No. 5,523,288, the disclosures of which are incorporated herein by reference, and allowed, co-owned, co-pending U.S. patent application Ser. No. 08/372,783.]
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. 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.
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]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media [Mannion et al.,
J. Clin. Invest.,
85:853-860 (1990)]. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably result from binding to the anionic groups of LPS, which normally stabilize the outer membrane through binding of Mg
++
and Ca
++
. Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabilization of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long O-chain bearing, “smooth” organisms being more resistant to BPI bactericidal effects than short O-chain bearing, “rough” organisms [Weiss et al.,
J. Clin. Invest.
65: 619-628 (1980)]. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring high concentrations of divalent cations and synthesis of new LPS [Weiss et al.,
J. Immunol.
132: 3109-3115 (1984)]. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al.,
J. Clin. Invest.
86: 631-641 (1990)). Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al.,
Infection and Immunity
56: 1203-1208 (1988)) but the exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.
Chlamydia are nonmotile, gram-negative, obligate intracellular bacteria that have unusual biological properties which phylogenetically distinguish them from other families of bacteria. Chlamydiae are presently placed in their own order, the Chlamydiales, family Chlamydiaceae, with one genus, Chlamydia. [Schachter and Stamm, Chlamydia, in
Manual of Clinical Microbiology
, pages 669-677, American Society for Microbiology, Washington, D.C. (1995).] There are four species,
Chlamydia trachomatis, C. pneumoniae, C. psittaci
and
C. pecorum
, which cause a wide spectrum of human diseases. In developing countries,
C. trachomatis
causes trachoma, the world's leading cause of preventable blindness. Over 150 million children have active trachoma, and over 6 million people are currently blind from this disease. In industrialized countries,
C. trachomatis
is the most prevalen

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