Title: Therapeutic peptide-based constructs
Abstract: The present invention relates generally to small peptide-based constructs and their therapeutic uses. The sequences of these peptide-based constructs are based on a reverse subsequence derived from Domain II of bactericidal/permeability-increasing protein (BPI).
Patent Number: 6,906,037 Issued on 06/14/2005 to Little, II, et al.
| Inventors:
|
Little, II; Roger G. (Benicia, CA);
Lin; Jong-Jye (Hercules, CA)
|
| Assignee:
|
Xoma Technology Ltd. (BM)
|
| Appl. No.:
|
359013 |
| Filed:
|
February 4, 2003 |
| Current U.S. Class: |
514/14; 514/15; 514/16 |
| Intern'l Class: |
A61K 038/10 |
| Field of Search: |
514/9,12,14,15,16
530/326,327,328
|
References Cited [Referenced By]
U.S. Patent Documents
| 5639727 | Jun., 1997 | Little, II.
| |
| 6423825 | Jul., 2002 | Little et al.
| |
| 6515104 | Feb., 2003 | Little et al.
| |
| Foreign Patent Documents |
| WO 97/0400/8 | Feb., 1997 | WO.
| |
Other References
Lehninger et al. Biochemistry, 2d ed. (1993), pp. 112-116 154-155.
|
Primary Examiner: Russel; Jeffrey Edwin
Attorney, Agent or Firm: McAndrews, Held & Malloy, Ltd.
Parent Case Text
This a continuation of U.S. application Ser. No. 09/344,219, filed Jun. 25,
1999, now U.S. Pat. No. 6,515,104, incorporated by reference in its entirety.
Claims
1. A pharmaceutical composition comprising a peptide-based construct of 8-14
amino acid moieties in length having heparin binding, heparin neutralizing, endothelial
cell proliferation inhibiting, antiangiogenic LPS binding, LPS neutralization,
or antimicrobial properties comprising:
a sequence having the formula:
wherein,
α is a hydrophilic basic amino acid moiety selected from the group consisting
of lysine, arginine, histidine, ornithine, diamiriobutyric acid, citrulline, and
para-amino phenylalanine;
β is a hydrophilic neutral amino acid moiety selected from the group consisting
of asparagine, glutamine, serine, threonine, tyrosine, hydroxyproline, and 7-hydroxy-tetrahydroisoquinoline
carboxylic acid;
χ is a hydrophobic amino acid moiety selected from the group consisting
of alanihe, naphthylalanine, biphenylalanine, valine, leucine, isoleucine, proline,
hydroxyproline, phenylalanine, tryptophan, methionirie, glycine, cyclohexylalanine,
amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline
carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,
dehydroleucine, 2, 2-diethylglycine, 1-amino-1-cyclopentane carboxylic acid, 1-amino-1-cyclohexane
carboxylic acid, amino-benzoic acid, amino-naphthyl carbbxylic acid, γ-amino
butyric acid, beta-alanine, difluorophenylalanine, fluorophenylalanine, nipecotic
acid, aminobutyric acid, thienyl-alanine, and t-butyl-glycine; and
R is an amino acid moiety selected from the group consisting of -χ, -χ-α,
-χ-α-χ, χ-α-χ-β, -χ-α-χ-β-χ,
-χ-α-χ-β-χ-α, -χ-β-χ-χ-β-χ,
-NH
2, -χ-NH
2, -χ-α-NH
2, -χ-α-χ-NH
2,
-χ-α-χ-β-NH
2, -χ-α-χ-β-χ-NH
2,
-χ-α-χ-β-χ-α-NH
2, and -χ-β-χ-χ-β-χ-NH
2,
and
a pharmaceutically acceptable diluent, adjuvant, or carrier.
2. A pharmaceutical composition comprising a composition of 8-14 amino acid moieties
consecutively linked by peptide ttonds, said composition having heparin binding
properties and comprising a sequence of the formula:
wherein R
1 is seleted from the group consisting of K (SEK ID NO: 8),
K(naph-A) (SEQ ID NO: 7), K(naph-A)K (SEQ ID NO: 6), K(naph-A)KG (SEQ ID NO: 5),
K(naph-A)KGS (SEQ ID NO: 4), K(naph-A)KGSI (SEQ ID NO: 3), and K(naph-A)KGSIK (SEQ
ID NO: 2); and
wherein the carboxyl terminal group is amidated or nonamidated,
and conservative substitution variants thereof having hepanin binding properties,
and
a pharmaceutically acceptable diluent, adjuvant, or carrier.
3. The pharmaceutical composition of claim 2 wherein the composition comprises
at least one conservative substitution.
4. The pharmaceutical compositioni of 2 wherein the peptide-based construct or
composition has heparin neutralizing properties.
5. The pharmaceutical composition, of 2 wherein the peptide-based construct or
composition has endothelial cell proliferation inhibiting properties.
6. The pharmaceutical composition of 2 wherein the peptide-based construct or
composition has anti-angiogenic properties.
7. The pharmaceutical composition of 2 comprising a peptide-based construct or
composition wherein the first two amino-terminal amino acid moieties are D-amino
acid moieties and the last two carboxy-terminal amino acid moieties are D-amino
acid moieties.
8. A method of neutralizing heparin in a mammal that has been administered an
exogenous heparin compound comprising the step of administering to said mammal
an amount of the pharmaceutical composition of 2 effective to neutralize the anticoagulant
effect of the exogenous heparin compound.
9. The method of claim 8 wherein the clotting time of said mammal is returned
to normal.
10. A method of inhibiting endothelial cell proliferation in a mammal in need
thereof comprising the step of administering to said mammal an amount of the pharmaceutical
composition of 2 effective, to inhibit endothelial cell proliferation.
11. A method of inhibiting angiogenesis in a mammal in need thereof comprising
the step of administering to said mammal an amount of the pharmaceutical composition
of 2 effective to inhibit angiogenesis.
12. The method of claim 11 wherein said angiogenesis is in the eye.
13. A method of treating a mammal suffering from a disorder involving angiogenesis
comprising the step of administering to said mammal an amount of the pharmaceutical
composition of 2 effective to inhibit angiogenesis.
14. The method of claim 13 wherein said disorder involving angiogenesis is a
chronic inflammatory disease.
15. The method of claim 14 wherein said chronic inflammatory disease is rheumatoid
or reactive arthritis.
16. The method of claim 13 wherein said disorder involving angiogenesis is proliferation
or metastasis of tumor cells.
Description
FIELD OF THE INVENTION
The present invention relates generally to small peptide-based constructs that
have 8 to 15 amino acid moieties. The sequences of these peptide-based constructs
are designed and prepared based on a reverse subsequence (99-85) derived from amino
acids identified and selected from Domain II of bactericidal permeability-increasing
protein (BPI). The invention further relates to therapeutic uses of such peptide-based
constructs due to their properties of heparin binding and neutralization, inhibition
of endothelial cell proliferation and/or inhibition of angiogenesis, e.g., inhibition
of in vivo neovascularization, including in models of chronic inflammatory disease
states and metastatic tumors.
BACKGROUND OF THE INVENTION
Bactericidal/permeability-increasing protein
(BPI) is a protein isolated from the granules of mammalian polymorphonuclear neutrophils
(PMNs), which are blood cells essential in defending a mammal against invading
microorganisms. Human BPI 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 et al., 1987
, Blood
69: 652), and has 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 BPI, have been reported by Gray et al., 1989
, J. Biol. Chem. 264:
9505 (see FIG. 1 in Gray et al.). The Gray et al. DNA and amino acid sequences
are set out in SEQ ID NOS: 12 and 13 hereto.
The bactericidal effect of BPI was originally reported to be highly specific
to sensitive gram-negative species. The precise mechanism by which BPI kills gram-negative
bacteria is not yet known, but it is known that BPI must first attach to the surface
of susceptible gram-negative bacteria. This initial binding of BPI to the bacteria
involves electrostatic interactions between BPI, which is a basic (i.e., positively
charged) protein, and negatively charged sites on lipopolysaccharides (LPS). LPS
is also known as "endotoxin" because of the potent inflammatory response that it
stimulates. LPS induces the release of mediators by host inflammatory cells which
may ultimately result in irreversible endotoxic shock. BPI binds to Lipid A, the
most toxic and most biologically active component of LPS.
BPI is also capable of neutralizing the endotoxic properties of LPS to which
it binds. Because of its gram-negative bactericidal properties and its ability
to bind to and neutralize LPS, BPI can be utilized for the treatment of mammals
suffering from diseases and conditions initiated by infection with gram-negative
bacteria whether the bacteria infect from outside the host or the bacteria infect
from within the host (i.e., gut-derived), including conditions of bacteremia, endotoxenia,
and sepsis. These properties of BPI make BPI particularly useful and advantageous
for such therapeutic administration.
A proteolytic fragment corresponding to the amino-terminal portion of human BPI
possesses the LPS binding and neutralizing activities and antibacterial activity
of BPI holoprotein. In contrast to the amino-terminal portion, the carboxyl-terminal
region of isolated human BPI displays only slightly detectable antibacterial activity
and some endotoxin neutralizing activity (Ooi et al., 1991
, J. Exp. Med. 174:
649). One BPI amino-terminal fragment, referred to as "rBPI
23" (see
Gazzano-Santoro et al., 1992
, Infect. Immun. 60: 4754-4761) has been produced
by recombinant means as a 23 kD protein and comprises an expression product of
DNA encoding the first 199 amino acid residues of the human BPI holoprotein 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: 12 and 13 taken from
Gray et al., supra, with the exceptions noted for rBPI
23. An N-terminal
fragment analog designated rBPI
21 or rBPI
21Δcys or
rBPI (1-193) ala
132 has been described in co-owned U.S. Pat. No. 5,420,019
and corresponding International Publication No. WO 94/18323 (PCT/US94/01235). This
analog comprises the first 193 amino acids of BPI holoprotein as set out in SEQ
ID NOS: 12 and 13 but wherein the cysteine at residue number 132 is substituted
with alanine, and with the exceptions noted for rBPI
23. rBPI
23,
as well as the cysteine substitution analog designated rBPI
21, have
been introduced into human clinical trials. Proinflammatory responses to endotoxin
were significantly ameliorated when rBPI
23 was administered in humans
challenged with endotoxin. (See, e.g., co-owned U.S. Pat. Nos. 5,643,875 and 5,753,620
and corresponding International Publication No. WO 95/19784 (PCT/US95/01151). In
addition, rBPI
21 was administered in humans with meningococcemia and
hemorrhage due to trauma. (See, e.g., U.S. Pat. No. 5,888,977 and corresponding
International Publication No. WO 97/42966 (PCT/US97/08016) and U.S. Pat. No. 5,756,464
and corresponding International Publication No. WO 97/44056 (PCT/US97/08941).
Other endotoxin binding and neutralizing proteins and peptides are known in
the art. One example is
Limulus antilipopolysaccharide factor (LALF) from
horseshoe crab amebocytes (Warren et al., 1992
, Infect. Immunol. 60: 2506-2513).
Another example is a cyclic, cationic lipopeptide from
Bacillus polymyxa,
termed Polymyxin B
1. Polymyxin B
1 is composed of six α,γ-diaminobutyric
acid residues, one D-phenylalanine, one leucine, one threonine and a 6-methyloctanoyl
moiety (Morrison and Jacobs, 1976
, Immunochem. 13: 813-818) and is also
bactericidal. Polymyxin analogues lacking the fatty acid moiety are also known,
which analogues retain LPS binding capacity but are without appreciable bactericidal
activity (Danner et al., 1989
, Antimicrob. Agents Chemother. 33: 1428-1434).
Similar properties have also been found with synthetic cyclized polymyxin analogues
(Rustici et al., 1993
, Science 259: 361-365).
Known antibacterial peptides include cecropins and magainins. The cecropins
are a family of antibacterial peptides found in the hemolymph of lepidopteran insects
(Wade et al, 1990
, Proc. Natl. Acad. Sci. USA 87: 4761-4765), and the magainins
are a family of antibacterial peptides found in
Xenopus skin and gastric
mucosa (Zasloffet al., 1988
, Proc. Natl. Acad. Sci. USA 85: 910-913). These
peptides are linear and range from about 20 to about 40 amino acids in length.
A less active mammalian cecropin has been reported from porcine intestinal mucosa,
cecropin P1 (Boman et al., 1993
, Infect. Immun. 61: 2978-2984). The cecropins
are generally reported to be more potent than the magainins in bactericidal activity
and appear to have less mammalian cell cytotoxicity. The cecropins and magainins
are characterized by a continuous, amphipathic α-helical region which is
necessary for bactericidal activity. The most potent of the cecropins identified
to date is cecropin with the BPI amino acid sequence 90-99 but does not share the
motif of charged and uncharged amino acids specified by the BPI amino acid sequence
90-99. In addition, the other 27 amino acids of cecropin A are necessary for maximal
bactericidal activity and there is no homology with BPI for those 27 amino acids.
The magainins have minimal homology with the BPI amino acid sequence 90-99.
Of interest to the present application are the disclosures in PCT International
Application PCT/US91/05758 [WO 92/03535] relating to compositions comprising BPI
and an anionic compound, which compositions are said to exhibit (1) no bactericidal
activity and (2) endotoxin neutralizing activity. Anionic compounds are preferably
a protein such as serum albumin but can also be a polysaccharide such as heparin.
In addition, Weiss et al., 1975
, J. Clin. Invest. 55: 33-42, disclose that
heparin sulfate and LPS block expression of the permeability-increasing activity
of BPI. However, neither reference discloses that BPI actually binds to and/or
neutralizes the biologic activities of heparin. Heparin binding does not necessarily
imply heparin neutralization. For example, a family of heparin binding growth factors
(HBGF) requires heparin as a cofactor to elicit a biological response. Examples
of HBGF's include: fibroblast growth factors (FGF-1, FGF-2) and endothelial cell
growth factors (ECGF-1, ECGF-2). Antithrombin III inhibition of clotting cascade
proteases is another example of a heparin binding protein that requires heparin
for activity and clearly does not neutralize heparin. Heparin binding proteins
that do neutralize heparin (e.g., platelet factor IV, protanune, and thrombospondin)
are generally inhibitory of the activities induced by heparin binding proteins
that use heparin as a cofactor.
Of particular interest to the present application are the heparin-related activities
of BPI protein products. Specifically, BPI protein products have been shown to
have heparin binding and heparin neutralization activities in co-assigned U.S.
Pat. Nos. 5,348,942; 5,639,727; 5,807,818; 5,837,678; 5,854,214 and corresponding
International Publication No. WO 94/02401 (PCT/US94/02401). For example, rBPI
23
was shown to have high affinity for heparin (see also, Little et al., 1994
,
J. Biol. Chem. 269: 1865-1872, and has been administered in humans to neutralize
heparin (see, e.g. U.S. Pat. No. 5,348,942). These heparin binding and neutralization
activities of BPI protein products are significant due to the importance of current
clinical uses of heparin. Heparin is commonly administered in doses of up to 400
U/kg during surgical procedures such as cardiopulmonary bypass, cardiac catheterization
and hemodialysis procedures in order to prevent blood coagulation during such procedures.
When heparin is administered for anticoagulant effects during surgery, it is an
important aspect of post-surgical therapy that the effects of heparin are promptly
neutralized so that normal coagulation function can be restored. Currently, protamine
is used to neutralize heparin. Protamines are a class of simple, arginine-rich,
strongly basic, low molecular weight proteins. Administered alone, protamines (usually
in the form of protamine sulfate) have anti-coagulant effects. When administered
in the presence of heparin, a stable complex is formed and the anticoagulant activity
of both drugs is lost. However, significant hypotensive and anaphylactoid effects
of protamine have limited its clinical utility. Thus, due to its heparin binding
and neutralization activities, BPI protein products have potential utility as a
substitute for protamine in heparin neutralization in a clinical context without
the deleterious side-effects which have limited the usefulness of the protamines.
The additional antibacterial and anti-endotoxin effects of such BPI protein products
would also be useful and advantageous in post-surgical heparin neutralization compared
with protamine.
Additionally of particular interest, is the activity of BPI protein
products to inhibit angiogenesis due in part to their heparin binding and neutralization
activities (see, e.g., U.S. Pat. Nos. 5,807,818 and 5,837,678 and corresponding
International Publication No. WO 94/02401 (PCT/US94/02401). Angiogenesis, the growth
of new blood vessels (neovascularization) is a complex phenomenon that involves
growth factors, most of which have heparin as a co-factor. In adults, angiogenic
growth factors are released as a result of vascular trauma (wound healing), immune
stimuli (autoimmune disease), inflammatory mediators (prostaglandins) or from tumor
cells. These factors induce proliferation of endothelial cells (which is necessary
for angiogenesis) via a heparin-dependent receptor binding mechanism (see Yayon
et al., 1991
, Cell 64: 841-848). Angiogenesis is also associated with a
number of other pathological conditions, including the growth, proliferation, and
metastasis of various tumors; diabetic retinopathy, macular degeneration, retrolental
fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune
inflammation including rheumatoid arthritis, capillary proliferation within atherosclerotic
plaques, hemangiomas, endometriosis and Kaposi's sarcoma. Thus, it would be desirable
to inhibit angiogenesis in these and other instances, and the heparin binding and
neutralization activities of BPI protein products, including peptides derived from
or based on BPI, are useful to that end.
Heparin binding proteins fall into at least two classes. The first class
consists of those proteins that utilize heparin as a co-factor in eliciting a specific
response. These proteins include heparin-dependent growth factors (e.g., basic
fibroblast growth factor, acidic fibroblast growth factor and vascular endothelial
cell growth factor) which play a major role in angiogenesis. The second class includes
proteins that neutralize the heparin-dependent response. BPI protein products,
including peptides derived from BPI, have been identified as heparin neutralizing
and anti-angiogenic agents. Several other heparin neutralizing proteins are also
known to inhibit angiogenesis. For example, protamine is known to inhibit tumor-associated
angiogenesis and subsequent tumor growth [see Folkman et al., 1992
, Inflammation:
Basic Principles and Clinical Correlates, 2d ed., (Galin et al., eds., Review
Press, N.Y.), Ch. 40, pp. 821-839]. A second heparin neutralizing protein, platelet
factor IV, also inhibits angiogenesis (i.e., is angiostatic). Another known angiogenesis
inhibitor, thrombospondin, binds to heparin with a repeating serine/tryptophan
motif instead of a basic amino acid motif (see Guo et al., 1992
, J. Biol. Chem.
267: 19349-19355). Murine endostatin is also reported to bind heparin and inhibit
angiogenesis (see, e.g., Hohenester et al., 1998
, Embo J. 17: 1656-1664;
O'Reilly et al, 1997
, Cell 88: 277-285).
Another utility of BPI protein products involves pathological conditions
associated with chronic inflammation, which is usually accompanied by angiogenesis
(see, e.g., U.S. Pat. No. 5,639,727). One example of a human disease related to
chronic inflamation is arthritis, which involves inflammation of peripheral joints.
In rheumatoid arthritis, the inflammation is autoimmune, while in reactive arthritis,
inflammation is hypothesized to be associated with initial infection of the synovial
tissue with pyogenic bacteria or other infectious agents followed by aseptic chronic
inflammation in susceptible individuals. Folkman et al., 1992, supra, have also
noted that many types of arthritis progress from a stage dominated by an inflammatory
infiltrate in the joint to a later stage in which a neovascular pannus invades
the joint and begins to destroy cartilage. While it is unclear whether angiogenesis
in arthritis is a causative component of the disease or an epiphenomenon, there
is evidence that angiogenesis is necessary for the maintenance of synovitis in
rheumatoid arthritis. One known angiogenesis inhibitor, AGM1470, has been shown
to prevent the onset of arthritis and to inhibit established arthritis in collagen-induced
arthritis models (Peacock et al., 1992
, J. Exp. Med. 175: 1135-1138). While
nonsteroidal anti-inflammatory drugs, corticosteroids and other therapies have
provided treatment improvements for relief of arthritis, there remains a need in
the art for more effective therapies for arthritis and other inflammatory diseases.
Many additional utilities of BPI protein products, including rBPI
23
and rBPI
21, have been described due to the wide variety of biological
activities of these products. For example, BPI protein products are bactericidal
for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541 and 5,523,288.
International Publication No. WO 94/20130 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). 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). 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 95/191 79 (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, abandoned, and corresponding International Publication Nos.
WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845). BPI protein products
exhibit anti-protozoan activity, as described in U.S. Pat. No. 5,646,114 and International
Publication No. WO 96/01 647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial
activity, as described in co-owned U.S. Pat. No. 5,888,973 and WO 98/06415 (PCT/US97/13810).
Finally, BPI protein products exhibit anti-mycobacterial activity, as described
in co-owned, co-pending U.S. application Ser. No. 08/626,646, issued as U.S. Pat.
No. 6,214,789, which is in turn a continuation of U.S. application Ser. No. 08/285,803,
abandoned, which is in turn a continuation-in-part of U.S. application Ser. No.
08/031,145, abandoned, and corresponding International Publication No. WO 94/20129 (PCT/U594/02463).
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
and corresponding International Publication No. WO 95/19784 (PCT/US95/01151).
BPI protein products are also useful for treatment of specific disease conditions,
such as meningococcemia in humans (as described in co-owned U.S. application Ser.
No. 08/644,287 and U.S. Pat. No. 5,888,977 and International Publication No. WO97/42966
(PCT/US97/08016), hemorrhagic trauma in humans, (as described in U.S. Pat. No.
5,756,464, U.S. application Ser. No. 08/862,785 and corresponding International
Publication No. WO 97/44056 (PCT/US97/08941), burn injury (as described in U.S.
Pat. No. 5,494,896) ischemia/reperfusion injury (as described in U.S. Pat. No.
5,578,568), and liver resection (as described in co-owned, co-pending U.S. application
Ser. No. 08/582,230 which is in turn a continuation of U.S. application Ser. No.
08/318,357, which is in turn a continuation-in-part of U.S. application Ser. No.
08/132,510, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404).
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 and corresponding
International Publication No. WO 97/42967 (PCT/US7/08017).
There continues to exist a need in the art for new products that have one or
more of the biological activities of BPI protein products, particularly products
for use as heparin binding and neutralizing agents and for the inhibition of endothelial
cell proliferation as well as inhibition of angiogenesis (normal or pathological).
Advantageous therapeutic products that are peptide-based would ideally comprise
small active sequences that are serum stable.
SUMMARY OF THE INVENTION
This invention provides compounds and compositions of small peptide-based constructs
having an 8-15 amino acid moiety sequence that is derived from or based on reverse
subsequences from functional domain II (amino acids 65-99) of BPI and having at
least one of the heparin-related biological activities of BPI, such as heparin
binding, heparin neutralization, inhibition of endothelial cell proliferation and/or
inhibition of angiogenesis. A reverse (or retro) sequence is inverted from the
original (e.g., if an original sequence is A-B-C, the inverted sequence is C-B-A).
Such peptide-based constructs according to the invention have reverse subsequences
that consist of a minimum core sequence based on an amino acid motif derived from
amino acids 99-92 of BPI. In a preferred embodiment the reverse subsequence is
a substituted subsequence (for example, amino acids 99-92, 99-91, 99-90, 99-89,
99-88, 99-87, 99-86, or 99-85 wherein the substitutions are at 95 and 91).
Constructs (or compositions) according to the invention include those
that are 8-15 moieties in length having heparin binding, heparin neutralizing,
endothelial cell proliferation inhibiting, or antiangiogenic properties and comprise:
a sequence having the formula: α-χ-χ-α-χ-β-χ-α-R.
In the sequence, α is a hydrophilic basic amino acid moiety selected from
the group consisting of lysine, arginine, histidine, ornithine, diaminobutyric
acid, citrulline, or para-amino phenylalanine; β is a hydrophilic neutral
amino acid moiety selected from the group consisting of asparagine, glutamine,
serine, threonine, tyrosine, hydroxyproline, or 7-hydroxy-tetrahydroisoquinoline
carboxylic acid; χ is a hydrophobic amino acid moiety selected from the group
consisting of alanine, naphthylalanine, biphenylalanine, valine, leucine, isoleucine,
proline, hydroxyproline, phenylalanine, tryptophan, methionine, glycine, cyclohexylalanine,
amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline
carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,
dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentane carboxylic acid, 1-amino-1-cyclohexane
carboxylic acid, amino-benzoic acid, amino-naphthyl carboxylic acid, γ-amino
butyric acid, beta-alanine, difluorophenylalanine, fluorophenylalanine, nipecotic
acid, aminobutyric acid, thienyl-alanine, t-butyl-glycine; and R is a moiety selected
from the group consisting of -χ, -χ-α, -χ-α-χ,
χ-α-χ-β, -χ-α-χ-β-χ, -χ-α-χ-β-χ-α,
-χ-α-χ-β-χ-α-χ, -χ-β-χ-χ-β-χ,
—NH
2, -χ-NH
2, -χ-α-NH
2,
-χ-α-χ-NH
2, -χ-α-χ-β-NH
2,
-χ-α-χ-β-χ-NH
2, -χ-α-χ-β-χ-α-NH
2,
-χ-α-NH
2,-χ-α-χ-β-χ-α-χ-NH
2, -χ-β-χ-χ-β-χ-NH
2.
The invention also provides a composition of 8-15 amino acid moieties consecutively
linked by peptide bonds, said composition having heparin binding properties and
comprising a sequence of the formula:
wherein R
1 is selected from the group consisting of K, K(naph-A),
K(naph-A)K, K(naph-A)KG, K(naph-A)KGS, K(naph-A)KGSI, K(naph-A)KGSIK and K(naph-A)KGSIKI; and
wherein the carboxyl terminal group is amidated or nonamidated,
and conservative substitution variants thereof having heparin binding properties.
Preferably, the variants comprise at least one conservative substitution.
The compositions of the invention preferably comprise compositions wherein the
first two amino-terminal amino acid moieties are D-amino acid moieties and the
last two carboxy-terminal amino acid moieties are D-amino acid moieties.
Also provided are methods of neutralizing heparin in a mammal that has been
administered an exogenous heparin compound (including heparin or heparinoid substances,
such as low molecular weight heparins) comprising the step of administering to
said mammal an amount of the composition of the invention effective to neutralize
the anticoagulant effect of the exogenous heparin compound, preferably in an amount
effective to return the clotting time of said mammal to normal; methods of inhibiting
endothelial cell proliferation in a mammal in need thereof by administering to
said mammal an amount of the compositions of the invention effective to inhibit
endothelial cell proliferation; methods of inhibiting angiogenesis in a mammal
in need thereof by administering to said mammal an amount of such compositions
effective to inhibit angiogenesis, including angiogenesis in the eye; methods of
treating a mammal suffering from a disorder involving angiogenesis, including a
chronic inflammatory disease, such as rheumatoid or reactive arthritis, and including
the growth, proliferation or metastasis of tumor cells.
Additional properties or activities of such constructs may include LPS
binding, LPS neutralization, and/or antimicrobial activity and/or any other previously
known activity or property of BPI protein products. Although three functional domains
of BPI were previously reported and include: domain I, encompassing the amino acid
sequence of BPI from about amino acid 17 to about amino acid 45; domain-II, encompassing
the amino acid sequence of BPI from about amino acid 65 to about amino acid 99;
and domain III, encompassing the amino acid sequence of BPI from about amino acid
142 to about amino acid 169, biologically active reverse (or retro) sequences have
not been previously reported based on subsequences of domain II. Thus, such peptide-based
reverse (retro) sequence constructs according to the invention, which preferably
comprise selected D-amino acid moieties, are particularly useful as therapeutic agents.
DETAILED DESCRIPTION
The present invention provides biologically active novel constructs (or compositions)
having a sequence of 8 to 15 amino acid moieties that is derived from a reverse
subsequence of functional domain II of BPI (i.e., peptide-based constructs). Preferred
are constructs with sequences that contain D-amino acid moieties. Particularly
preferred are constructs with sequences where the D-amino acid moieties are positioned
as the first two and last two moieties of the sequence. Such constructs are particularly
useful for the treatment of heparin-related or heparin-mediated disorders, diseases
or conditions. "Treatment" as used herein encompasses both prophylactic and therapeutic
treatment. Treatment of mammals, including humans, is contemplated.
As used herein, "amino acid moiety" includes typical and a typical amino acid
compounds (including derivatized amino acids and amino acid analogs). "Conservative"
substitutions of one amino acid for another are substitutions of amino acids having
similar structural and/or chemical properties, and are generally based on simlarities
in polarity, charge, hydrophobicity, hydrophilicity and/or the amphipathic nature
of the residues involved. Hydrophobic, polar neutral and polar basic amino acids
include those described above for α, β and χ. Polar acidic amino
acids include aspartic acid and glutamic acid. As a general rule, as the similarity
between the amino acids being substituted decreases, the likelihood that the substitution
will affect activity increases.
For the purposes of this invention, the term "functional domain" is intended
to designate a region of the amino acid sequence of BPI that exhibits one or more
of the biological activities of BPI. These functional domains of BPI were defined
by the activities of proteolytic cleavage fragments, overlapping 15-mer peptides
and other synthetic peptides. Domain I has been defined as the amino acid sequence
of BPI comprising from about amino acid 17 to about amino acid 45. Initial peptides
based on this domain were moderately active in both the inhibition of LPS-induced
LAL activity and in heparin binding assays, and did not exhibit significant antibacterial
activity. Domain II has been defined as the amino acid sequence of BPI comprising
from about amino acid 65 to about amino acid 99. Initial peptides based on this
domain exhibited high LPS and heparin binding capacity and exhibited significant
antibacterial activity. Domain III has been defined as the amino acid sequence
of BPI comprising from about amino acid 142 to about amino acid 169. Initial peptides
based on this domain exhibited high LPS and heparin binding activity, and exhibited
surprising antimicrobial activity, including antifungal and antibacterial (including,
e.g., anti-gram-positive and anti-gram-negative) activity.
For purposes of this invention, the term "biological activity of BPI" is intended
to include, but is not limited to one or more of the biological activities or properties
of a human bactericidal/permeability-increasing (BPI) protein product, including,
for example, a recombinant BPI holoprotein such rBPI (SEQ ID NO: 13), an amino-terminal
fragment of BPI such as rBPI
23, and analogs that are mutated amino-terminal
fragments of BPI such as rBPI
21Δcys and including any of the known
activities of the BPI protein products discussed above. Specifically included is
a biological activity of any peptide-based construct of this invention that is
between 0.1 and 10 times the activity of BPI or of a corresponding peptide encompassing
a corresponding functional domain of BPI. The term "biological activity of BPI"
is intended to include, but is not limited to an activity of heparin binding, heparin
neutralization, inhibition of endothelial cell proliferation or inhibition of angiogenesis
(e.g., inhibition of in vivo neovascularization such as that associated with metastatic
tumors and chronic inflammatory disease states). Also included in this definition
of "biological activity of BPI" is an activity of LPS binding, LPS neutralization,
or antimicrobial activity. Also expressly included in this definition of the "biological
activity of BPI" is a biological activity, for example antimicrobial activity,
that is qualitatively different than the activity of BPI or the corresponding peptide
encompassing the entire corresponding domain of BPI. For example, such qualitative
differences include differences in the spectrum of bacteria or other microorganisms
against which the peptide is effective, relative to the amino acid sequence of
the corresponding functional domain of BPI. This definition thus encompasses antimicrobial
activities, such as antibacterial activity (e.g., against gram-positive bacteria,
mycobacteria and chlamydia) and antifungal activity (e.g., against species of
Candida,
Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces, Basidiobolus,
Conidiobolus, Rhizopus, Rhizomucor, Mucor, Absidia, Mortierella, Cunninghamella,
Saksenaea, Fusarium, Trichophyton, Trichosporon, Microsporum, Epidermophyton, Scytalidium,
Malassezia, Actinomyceies, Sporothrix and
Penicillium).
The invention provides exemplary peptide-based constructs each of which has a
sequence that is derived from or based on reverse substituted subsequences from
functional domain II of human BPI (e.g., amino acids 99-92, 99-91, 99-90, 99-89,
99-88, 99-87, 99-86, or 99-85 wherein the substitutions are at 75 and 91)). Embodiments
of such constructs include the following exemplary domain II basic constructs [single-letter
abbreviations for amino acids can be found in G. Zubay,
Biochemistry (2d.
ed.), 1988 (MacMillen Publishing: N.Y.), p.33]:
| XMP.394 (1) |
(SEQ ID NO:1) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g-s-i-k-i |
| XMP.624 (2) |
(SEQ ID NO:2) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g-s-i-k |
| XMP.625 (3) |
(SEQ ID NO:3) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g-s-i |
| XMP.626 (4) |
(SEQ ID NO:4) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g-s |
| XMP.627 (5) |
(SEQ ID NO:5) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g |
| XMP.628 (6) |
(SEQ ID NO:6) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k |
| XMP.629 (7) |
(SEQ ID NO:7) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a) |
| XMP.630 (8) |
(SEQ ID NO:8) |
| XMP.656 (9) |
(SEQ ID NO:9) |
| k-l-f-r-(naph-a)-q-a-k-(naph-a)-k-g-i-k-i |
| XMP.679 (10) |
(SEQ ID NO:10) |
| k-l-f-k-(naph-a)-q-a-k-(naph-a)-k-g |
| XMP.684 (11) |
(SEQ ID NO:11) |
| (biphenyl-a)-k-l-f-r-(naph-a)-q-a-k |
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. BPI protein products may
be generated and/or isolated by any means known in the art. U.S. Pat. No. 5,198,541,
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), 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.
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., 1991
, J. Exp. Med.,
174:649, 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., 1992
, Infect. Immun. 60:4754-4761, and referred to as rBPI
23.
In that publication, an expression vector was used as a source of DNA encoding
a recombinant expression product (rBPI
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: 12 and 13) set out in FIG.
1 of Gray et al., supra, with the exceptions noted for rBPI
23 and with
the exception that residue 417 is alanine (specified by GCT) rather than valine
(specified by GTT). An analog of an N-terminal fragment consisting of residues
10-193 of BPI has been described in co-owned, co-pending U.S. application Ser.
No. 09/099,725. 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).
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), 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. Pat. No. 5,420,019 and corresponding
International Publication No. WO 94/18323 (PCT/US94/01235), 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
21Δcys or rBPI
21.
Production of this N-terminal analog of BPI, rBPI
21, has been described
in Horwitz et al., 1996
, Protein Expression Purification, 8:28-40. 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
132")
has been described in co-owned, co-pending U.S. application Ser. No. 09/099,725.
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).
Other BPI protein products 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. Pat. No. 5,858,974 and International Publication No. WO 96/08509 (PCT/US95/09262),
which corresponds to U.S. patent application Ser. No. 09/119,858, and International
Publication No. WO 95/19372 (PCT/US94/10427), which corresponds to U.S. Pat. Nos.
5,652,332 and 5,856,438, 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,
which is a continuation-in-part of U.S. application Ser. No. 08/093,202 (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 Publication No. WO 97/35009
(PCT/US97/05287), which corresponds to U.S. Pat. No. 5,851,802.
The present invention defines novel peptide-based constructs that may be defined
as BPI protein products.
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
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
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). As described in U.S. Pat. No. 5,912,228, which
is in turn a continuation-in-part of U.S. application Ser. No. 08/530,599, which
is in turn a continuation-in-part of U.S. application Ser. No. 08/372,104, and
corresponding International Publication No. WO 96/21436 (PCT/US96/01095), other
poloxamer formulations of BPI protein products with enhanced activity may be utilized.
Peptide-based constructs may be formulated like other BPI protein products or may
be formulated in saline or a physiological buffer.
Therapeutic compositions comprising BPI protein product (including the
peptide-based constructs or compositions of the invention) 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. Topical routes include administration in the form of
salves, ophthalmic drops, ear drops, or irrigation fluids (for, e.g., irrigation
of wounds).
When given parenterally, BPI protein product compositions are generally injected
in doses ranging from 1 μg/kg to 100 mg/kg per day, preferably at doses ranging
from 0.1 mg/kg to 20 mg/kg per 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.
Those skilled in the art can readily optimize effective dosages and administration
regimens for therapeutic compositions comprising BPI protein product (including
the constructs or compositions of the present invention), as determined by good
medical practice and the clinical condition of the individual subject.
The constructs or compositions of the invention may be used in any of the therapeutic
uses for which BPI products are known to be effective, including those described
above. The constructs are particularly useful in methods for binding and neutralizing
exogenous heparin, methods for inhibiting endothelial cell proliferation, treating
disorders associated with endothelial cell proliferation, methods for inhibiting
angiogenesis, and treating disorders associated with or involving angiogenesis.
Exogenous heparin compounds are commonly administered during surgical procedures
requiring anticoagulation, such as cardiopulmonary bypass, cardiac catheterization
or angioplasty, and hemodialysis. Exogenous heparin compounds are also administered
to patients at risk of or suffering from thrombosis, e.g. patients suffering from
deep venous thrombosis, acute myocardial infarction, stroke, or pulmonary embolism.
Angiogenesis-associated disorders are disorders in which
angiogenesis plays a role in the initiation or progression of disease. Angiogenesis
is involved in a number of conditions, illustrated below, and inhibition of angiogenesis
is expected to be effective for treating any of these conditions (including inhibiting
progression of the disease and ameliorating signs and symptoms of the disease).
Use of the constructs of the invention in preparation of a medicament for any
of these therapeutic uses is also contemplated.
Angiogenesis is of considerable importance in cancer conditions because
new vessel production is required to support the rapid growth of cancer cells.
Inhibition of angiogenesis thus may promote tumor regression in adult and pediatric
oncology, including reducing growth of solid tumors/malignancies, locally advanced
tumors, metastatic cancer, human soft tissue sarcomas, cancer metastases, including
lymphatic metastases, blood cell malignancies, effusion lymphomas (body cavity
based lymphomas), lung cancer, including small cell carcinoma, non-small cell cancers,
breast cancer, including small cell carcinoma and ductal carcinoma, gastrointestinal
cancers, including stomach cancer, colon cancer, colorectal cancer, polyps associated
with colorectal neoplasia, pancreatic cancer, liver cancer, urological cancers,
including bladder cancer, prostate cancer, malignancies of the female genital tract,
including ovarian carcinoma, uterine endometrial cancers, and solid tumors in the
ovarian follicle, kidney cancer, including renal cell carcinoma, brain cancer,
including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas,
metastatic tumor cell invasion in the central nervous system, bone cancers, including
osteomas, skin cancers, including malignant melanoma, tumor progression of human
skin keratinocytes, and squamous cell cancer, hemangiopericytoma, and Kaposi's sarcoma.
Angiogenesis also plays a role in chronic inflammation, including chronic
pancreatitis, dermatosis associated with chronic inflammation, including psoriasis,
cirrhosis, asthma, multiple sclerosis, arthritis, including rheumatoid arthritis,
reactive arthritis and chronic inflammatory arthritis, autoimmune disorders, including
vasculitis, glomerulonephritis, experimental allergic encephalomyelitis (EAE),
lupus, myasthenia gravis, ulcerative colitis, Crohn's disease, inflammatory bowel
disease, chronic inflammation associated with hemodialysis, granulocyte transfusion
associated syndrome; rejection reactions after allograft and xenograft transplantation,
including graft versus host disease; and other chronic inflammatory disorders.
Angiogenesis in the eye is involved in ocular neovascularization, proliferative
retinopathy, retrolental fibraplasia, macular degeneration, neovascular glaucoma
and diabetic ocular disease, in particular, diabetic iris neovascularization and retinopathy.
Coronary atheroma are highly vascularized by a fragile capillary network,
and rupture of these newly formed capillaries when they are exposed to high intravascular
pressures may lead to hemorrhage into atherosclerotic plaques and coronary occlusion.
Inhibition of angiogenesis thus may reduce the growth of atherosclerotic plaques
and may be useful in the treatment of atherosclerosis, ischemic heart disease,
myocardial infarction, coronary heart disease, restenosis, particularly following
balloon angiography, neointimal hyperplasia, disruption of intercellular junctions
in vascular endothelium, hypertension, vessel injury, arterial ischemia, arterial
stenosis, peripheral-vascular disease, stroke
Angiogenesis also occurs during the female reproductive cycle and is
involved in endometriosis, uterine fibroids, other conditions associated with dysfunctional
vascular proliferation (including endometrial microvascular growth) during the
female reproductive cycle.
Angio
