Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-08-20
2001-01-30
Celsa, Bennett (Department: 1627)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C514S014800, C530S327000, C530S328000, C930S021000
Reexamination Certificate
active
06180604
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to treatment of microorganism-caused infections, and more specifically, to compositions comprising indolicidin analogues, polymer-modified analogues, and their uses in treating infections.
BACKGROUND OF THE INVENTION
For most healthy individuals, infections are irritating, but not generally life-threatening. Many infections are successfully combated by the immune system of the individual. Treatment is an adjunct and is generally readily available in developed countries. However, infectious diseases are a serious concern in developing countries and in immunocompromised individuals.
In developing countries, the lack of adequate sanitation and consequent poor hygiene provide an environment that fosters bacterial, parasitic, fungal and viral infections. Poor hygiene and nutritional deficiencies may diminish the effectiveness of natural barriers, such as skin and mucous membranes, to invasion by infectious agents or the ability of the immune system to clear the agents. As well, a constant onslaught of pathogens may stress the immune system defenses of antibody production and phagocytic cells (e.g., polymorphic neutrophils) to subnormal levels. A breakdown of host defenses can also occur due to conditions such as circulatory disturbances, mechanical obstruction, fatigue, smoking, excessive drinking, genetic defects, AIDS, bone marrow transplant, cancer, and diabetes. An increasingly prevalent problem in the world is opportunistic infections in individuals who are HIV positive.
Although vaccines may be available to protect against some of these organisms, vaccinations are not always feasible, due to factors such as inadequate delivery mechanisms and economic poverty, or effective, due to factors such as delivery too late in the infection, inability of the patient to mount an immune response to the vaccine, or evolution of the pathogen. For other pathogenic agents, no vaccines are available. When protection against infection is not possible, treatment of infection is generally pursued. The major weapon in the arsenal of treatments is antibiotics. While antibiotics have proved effective against many bacteria and thus saved countless lives, they are not a panacea. The overuse of antibiotics in certain situations has promoted the spread of resistant bacterial strains. And of great importance, antibacterials are useless against viral infections.
A variety of organisms make cationic (positively charged) peptides, molecules used as part of a non-specific defense mechanism against microorganisms. When isolated, these peptides are toxic to a wide variety of microorganisms, including bacteria, fungi, and certain enveloped viruses. One cationic peptide found in neutrophils is indolicidin. While indolicidin acts against many pathogens, notable exceptions and varying degrees of toxicity exist.
Although cationic peptides show efficacy in vitro against a variety of pathogenic cells including gram-positive bacteria, gram-negative bacteria, and fungi, these peptides are generally toxic to mammals when injected, and therapeutic indices are usually quite small. Approaches to reducing toxicity have included development of a derivative or delivery system that masks structural elements involved in the toxic response or that improves the efficacy at lower doses. Other approaches under evaluation include liposomes and micellular systems to improve the clinical effects of peptides, proteins, and hydrophobic drugs, and cyclodextrins to sequester hydrophobic surfaces during administration in aqueous media. For example, attachment of polyethylene glycol (PEG) polymers, most often by modification of amino groups, improves the medicinal value of some proteins such as asparaginase and adenosine deaminase, and increases circulatory half-lives of peptides such as interleukins.
None of these approaches are shown to improve administration of cationic peptides. For example, methods for the stepwise synthesis of polysorbate derivatives that can modify peptides by acylation reactions have been developed, but acylation alters the charge of a modified cationic peptide and frequently reduces or eliminates the antimicrobial activity of the compound. Thus, for delivery of cationic peptides, as well as other peptides and proteins, there is a need for a system combining the properties of increased circulatory half-lives with the ability to form a micellular structure.
The present invention discloses analogues of indolicidin, designed to broaden its range and effectiveness, and further provide other related advantages. The present invention also provides methods and compositions for modifying peptides, proteins, antibiotics and the like to reduce toxicity, as well as providing other advantages.
SUMMARY OF THE INVENTION
The present invention generally provides indolicidin analogues. In related aspects, an indolicidin analogue is provided, comprising up to 25 amino acids and containing the formula: RXZXXZXB (SEQ ID NO:1); BXZXXZXB (SEQ ID NO:2) wherein at least one Z is valine; BBBXZXXZXB (SEQ ID NO:6); BXZXXZXBBB
n
(AA)
n
MILBBAGS (SEQ ID NO:4-7); BXZXXZXBB(AA)
n
M (SEQ ID NOS:8 and 9); LBB
n
XZ
n
XXZ
n
XRK (SEQ ID NOS:10-17 ); LK
n
XZXXZXRRK (SEQ ID NOS:18 and 19); BBXZXXZXBBB (SEQ ID NO:20), wherein at least two X residues are phenylalanine; BBXZXXZXBBB (SEQ ID NO:21), wherein at least two X residues are tyrosine; and wherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; AA is any amino acid, and n is 0 or 1. In preferred embodiments, Z is proline, X is tryptophan and B is arginine or lysine. In other aspects, indolicidin analogues having specific sequences are provided. In certain embodiments, the indolicidin analogues are coupled to form a branched peptide. In other embodiments, the analogue has one or more amino acids altered to a corresponding D-amino acid, and in certain preferred embodiments, the N-terminal and/or the C-terminal amino acid is a D-amino acid. Other preferred modifications include analogues that are acetylated at the N-terminal amino acid, amidated at the C-terminal amino acid, esterified at the C-terminal amino acid, modified by incorporation of homoserine/homoserine lactone at the C-terminal amino acid, and conjugated with polyethylene glycol or derivatives thereof.
In other aspects, the invention provides an isolated nucleic acid molecule whose sequence comprises one or more coding sequences of the indolicidin analogues, expression vectors, and host cells transfected or transformed with the expression vector.
Other aspects provide a pharmaceutical composition comprising at least one indolicidin analogue and a physiologically acceptable buffer, optionally comprising an antibiotic agent. Preferred combinations include ILKKFPFFPFRRK (SEQ ID NO:22) and Ciprofloxacin; ILKKFPFFPFRRK (SEQ ID NO:22) and Mupirocin; ILKKYPYYPYRRK (SEQ ID NO:23) and Mupirocin; ILKKWPWWPWRK (SEQ ID NO:24) and Mupirocin; ILRRWPWWPWRRR (SEQ ID NO:25) and Piperacillin; WRIWKPKWRLPKW (SEQ ID NO:26) and Ciprofloxacin; WRIWKPKWRLPK (SEQ ID NO:26) W(SEQ ID NO:26) and Mupirocin; WRIWKPKWRLPKW (SEQ ID NO:26) and Piperacillin; ILRWVWWVWR (SEQ ID NO:26) RK (SEQ ID NO:27) and Piperacillin; and ILKKWPWWPWK (SEQ ID NO:28) and Mupirocin. In other embodiments, the pharmaceutical composition further comprises an antiviral agent, (e.g., acyclovir; amantadine hydrochloride; didanosine; edoxudine; famciclovir; foscarnet; ganciclovir; idoxuridine; interferon; lamivudine; nevirapine; penciclovir; podophyllotoxin; ribavirin; rimantadine; sorivudine; stavudine; trifluridine; vidarabine; zalcitabine and zidovudine); an antiparasitic agent (e.g., 8-hydroxyquinoline derivatives; cinchona alkaloids; nitroimidazole derivatives: piperazine derivatives; pyrimidine derivatives and quinoline derivatives, albendazole; atovaquone; chloroquine phosphate; diethylcarbamazine citrate; eflomithine; halofantrine; iodoquinol; ivermectin; mebendazole; mefloquine hydrochloride; melarsoprol B; metronidazole; niclosamide; nifurtimox; paromomycin; pentamidine isethionate; piperazine; praziqua
Erfle Douglas
Fraser Janet R.
Krieger Timothy J.
Taylor Robert
West Michael H. P.
Celsa Bennett
Micrologix Biotech Inc.
Seed Intellectual Property Law Group PLLC
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