Derivative compounds derived from or based on...

Details Derivative compounds derived from or based on... Derivative compounds derived from or based on...

1999-06-25

2002-03-12

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S015800, C514S016700, C514S017400, C514S018700, C530S327000, C530S328000, C530S329000, C530S330000, C530S345000

Reexamination Certificate

active

06355616

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to derivatized compounds that are peptide-based constructs derived from or based on Domain III (amino acids 142-169) of bactericidal/permeability-increasing protein (BPI) and therapeutic uses of such compounds.
BACKGROUND OF THE INVENTION
Bactericidal/permeability-increasing protein (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, 1979,
J. Biol. Chem.
254: 11000) or
E. coli
affinity chromatography (Weiss, 1987, et al.,
Blood
69: 652). BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have bactericidal activity against 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, 1989, et al.,
J. Biol. Chem.
264: 9505. The Gray et al. DNA and amino acid sequences are set out in SEQ ID NOS: 53 and 54 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., 1987,
J. Bio. Chem.
262: 14891-14894). 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 and some endotoxin neutralizing activity. (Ooi et al., 1991,
J. Exp. Med.
174: 649). 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, including anti-endotoxin activity against gram-negative organisms (Gazzano-Santoro et al., 1992,
Infect. Immun.
60: 4754-4761). In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI
23
). The vector was constructed to encode the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ ID NOS: 53 and 54 taken from 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, also referred to as rBPI, has also been produced having the sequence set out in SEQ ID NOS: 53 and 54 taken from Gray et al., supra, with the exceptions noted for rBPI
23
. An N-terminal fragment analog designated rBPI
21
or rBPI
21
&Dgr;cys has been described in co-owned, co-pending U.S. Pat. No. 5,420,019. This analog comprises the first 193 amino acids of BPI holoprotein as set out in SEQ ID NOS: 53 and 54 but wherein the cysteine at residue number 132 is substituted with alanine, and with the exceptions noted for rBPI
23
.
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, 1992, Inflammation:
Basic Principles and Clinical Correlates,
eds. Gallin et al., Chapter 30, Raven Press, Ltd. BPI is commonly thought to be non-toxic for other microorganisms, including yeast, and for higher eukaryotic cells. Elsbach and Weiss, 1992, supra, reported that BPI exhibits anti-bacterial activity towards a broad range of gram-negative bacteria at concentrations as low as 10
−8
to 10
−9
M, but that 100- to 1,000-fold higher concentrations of BPI were non-toxic to all of the gram-positive bacterial species, yeasts, and higher eukaryotic cells tested at that time. It was also reported that BPI at a concentration of 10
6
M or 160 &mgr;g/ml had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the gram-positive organisms
Staphylococcus aureus
(four strains),
Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis, Micrococcus lysodeikticus,
and
Listeria monocytogenes.
BPI at 10
−6
M reportedly had no toxic effect on the fungi
Candida albicans
and
Candida parapsilosis
at pH 7.0 or 5.5, and was non-toxic to higher eukaryotic cells such as human, rabbit and sheep red blood cells and several human tumor cell lines. See also, Elsbach and Weiss, 1981,
Advances in Inflammation Research,
ed. G. Weissmann, Vol. 2, pages 95-113, Raven Press. 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.
The precise mechanism by which BPI kills gram-negative bacteria is not yet known, 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 proposed 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. It has been reported that bacteria at this stage can be rescued by growth in serum albumin supplemented media (Mannion et al., 1990,
J. Clin. Invest.
85: 853-860). The second stage, defined by growth inhibition that was not reversed by serum albumin, occurring after prolonged exposure of the bacteria to BPI and 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 in the KDO region 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., 1980,
J. Clin. Invest.
65: 619-628). This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring the presence of high divalent cations and synthesis of new LPS (Weiss et al., 1984,
J. Immunol.
132: 3109-3115). 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., 1990,
J. Clin. Invest.
86: 631-641). Specific investigation of this possibility has shown tha

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