EP0836617A1 - Parevines et tachytegrines - Google Patents

Parevines et tachytegrines

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Publication number
EP0836617A1
EP0836617A1 EP96923662A EP96923662A EP0836617A1 EP 0836617 A1 EP0836617 A1 EP 0836617A1 EP 96923662 A EP96923662 A EP 96923662A EP 96923662 A EP96923662 A EP 96923662A EP 0836617 A1 EP0836617 A1 EP 0836617A1
Authority
EP
European Patent Office
Prior art keywords
compound
ofthe
amino acid
peptides
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96923662A
Other languages
German (de)
English (en)
Other versions
EP0836617A4 (fr
Inventor
Robert I. Lehrer
Sylvia S. L. Harwig
Conway Cheng Chang
Chee Liang Gu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ardea Biociences Inc
Original Assignee
IntraBiotics Pharmaceuticals Inc
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Filing date
Publication date
Application filed by IntraBiotics Pharmaceuticals Inc filed Critical IntraBiotics Pharmaceuticals Inc
Publication of EP0836617A1 publication Critical patent/EP0836617A1/fr
Publication of EP0836617A4 publication Critical patent/EP0836617A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L12/00—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor
    • A61L12/08—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor using chemical substances
    • A61L12/14—Organic compounds not covered by groups A61L12/10 or A61L12/12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of antibiotic peptides.
  • the invention concerns short peptides with unique patterns of cysteine type residues and conformations that have a wide range of antimicrobial activities.
  • peptides that have antimicrobial and antiviral activity.
  • Various classes of these peptides have been isolated from tissues of both plants and animals.
  • PCT application WO 95/03325 published 2 February 1995 contains a review ofthe literature on this subject.
  • Such peptides include tachyplesins, which are 17-18 amino acid peptides containing four invariant cysteines, the defensins, ⁇ -defensins, and insect defensins, which are somewhat longer peptides characterized by six invariant cysteines, and antifungal and antibacterial peptides and proteins which have been found in plants.
  • WO 95/03325 provides a new class of antimicrobial and antiviral peptides, designated "protegrins", representative members of which have been isolated from porcine leukocytes. These peptides are useful as antibacterial antiviral and antifungal agents in both plants and animals.
  • Biochem Biophys Res Comm (1993) 196:1363-1367 report the recovery of a DNA sequence which encodes a pig leukocyte antimicrobial peptide with a cathelin-like prosequence.
  • the peptide is reported to be one of the protegrins. Additional publications related to protegrins are Harwig, S.S.L., et al. J Peptide Sci (1995) in press; Zhao, C, et al. FEBS Lett (1995) 376:130-134; Zhao, C. et al. FEBS Lett (1995) 368:197-202.
  • the protegrins have also been found to bind to endotoxins — i.e., the lipopolysaccharide (LPS) compositions derived from gram-negative bacteria which are believed responsible for gram-negative sepsis.
  • LPS lipopolysaccharide
  • the protegrins are also effective in inhibiting the growth of organisms that are associated with sexually transmitted diseases such as Chlamydia trachomatis and Neisseria gonorrhoeae.
  • Protegrins are also effective against the microorganisms associated with oral mucositis, a significant side effect of cancer therapy and bone marrow transplantation that is not adequately managed by current approaches (Sonis, S.T. In: J.L. Holland et al. Cancer Medicine. 3rd ed. Lea and Febiger, Philadelphia (1993a) pp. 2381-2388; Sonis, S T. In: V. DeVitta et al. (ed ), Principles and Practice of Oncology. J.B. Lippincott, Philadelphia (1993b) pp. 2385-2394).
  • Oral mucositis is initiated by the cytotoxic effects of these therapies on the rapidly dividing epithelial cells ofthe oropharyngeal mucosa, and is exacerbated by infection with both endogenous oral flora and opportunistic bacterial and fungal pathogens.
  • Oral mucositis is associated with extreme discomfort and pain, especially when eating.
  • oral mucositis is frequently a dose limiting toxicity causing delay in the completion ofthe therapeutic regimen. Such a delay in treatment can have a negative impact on the final therapeutic outcome.
  • Infected oral lesions are also increasingly recognized as a significant portal of entry of microorganisms to the immunosuppressed patient resulting in sepsis (Sonis, 1993 a,b, supra).
  • the invention described below relates to peptide type compounds that are related to the protegrins described above, but reflect displacements ofthe protegrin cysteines at positions 6 and 15.
  • C 4 * ', Cs * , C * i6 or C * ⁇ in the formula set forth below must be cysteine
  • the common name terminlogy of these components reflects particularly perferred situations wherein both of C 4 * and C ⁇ * are cysteine type residues (the tachytegrins) or where both C * 5 and C * ⁇ are cysteine type residues (the parevins). Disclosure ofthe Invention
  • the invention provides compounds which retain generally the antimicrobial activity of the protegrins discussed above, but differ in conformation due to the dislocation ofthe cysteine residues at positions 6 and/or 15 of these protegrins. Su ⁇ risingly, these modified compounds exhibit activity spectra which are analogous to those ofthe protegrins, but offer the opportunity to fine-tune the biological activity of antibiotics and antivirals. All of these peptides can be produced synthetically and those that contain only gene-encoded amino acids can also be produced recombinantly. These compounds are useful as preservatives or in pharmaceutical compositions in treating or preventing infection in animals. Alternatively, the peptides can be formulated into compositions which can be applied to plants to protect them against viral or microbial infection. In still another approach, the DNA encoding the peptides can be expressed in situ, in animals or preferably in plants, to combat infections. The peptides are also useful as standards in antimicrobial assays and in binding endotoxins.
  • the invention is directed to a purified and isolated or recombinantly or synthetically produced compound which contains the amino acid sequence
  • the sequence shown as (1) can be extended at the N and/or C terminus with non-interferring amino acids or sequence.
  • the compounds also include the N-terminal acylated and/or C-terminal amidated or esterified forms and may be either in the, optionally -SH stabilized, linear or in a disulfide- bridged form.
  • each of A ⁇ -A 3 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid; each of C *, C 5 *, C ⁇ *, Cu*, Ci ⁇ * and C ⁇ * is independently cysteine, homocysteine or penicillamine or a basic, hydrophobic, polar/large, or small amino acid, and C 4 * and/or C ⁇ * may be present or not present; C 6 * and/or C15* may also be acidic; each of C 8 and C ⁇ 3 is independently cysteine, homocysteine or penicillamine; each of A 7 and A ⁇ 4 is independently a hydrophobic or a small amino acid;
  • Ag-A ⁇ 2 must be capable of effecting a ⁇ -turn when contained in the compound and at least one of A 9 -A ⁇ 2 must be a basic amino acid;
  • Aie is present or not present, and if present, is a basic, hydrophobic, polar/large or small amino acid.
  • the compounds ofthe invention may, in the alternative, contain a modified form of formula (1) wherein one or both of C 8 and C ⁇ is independently replaced by a basic, hydrophobic, polar/large, acidic, or small amino acid.
  • At least about 15% and no more than about 50% ofthe amino acids must be basic amino acids, and the compounds must have a net charge of +1 at physiological pH; with the proviso that at least one of G * G * Ge and G * 7 must be cysteine, homocysteine or penicillamine; and only one of C ⁇ C5 * , and C ⁇ * , and only one of C15 * , Ci ⁇ * and C ⁇ * can be cysteine, homocysteine or penicillamine.
  • a particular advantage of some ofthe peptides ofthe invention lies in their reduced size. As a result of this, they are less costly to produce, generally are expected to provide better distribution in tissue, and are less immunogenic. As they provide alternative structures, they are likely to have different pharmacokinetic and toxicological profiles.
  • the invention is directed to recombinant materials useful for the production ofthe peptides ofthe invention as well as plants or animals modified to contain expression systems for the production of these peptides.
  • the invention is also directed to pharmaceutical compositions and compositions for application to plants containing the peptides ofthe invention as active ingredients or compositions which contain expression systems for production ofthe peptides or for in situ expression ofthe nucleotide sequence encoding these peptides.
  • the invention is also directed to methods to prepare the invention peptides synthetically, to antibodies specific for these peptides, and to the use ofthe peptides as preservatives.
  • Specific infections against which some ofthe peptides, especially the parevins and tachytegrins are particularly effective are those associated with oral mucositis, infections, such as stomach ulcers associated with H. pylori, and infections caused by Pseudomonas or MRSA.
  • the invention is directed to the use ofthe compounds ofthe invention as standards in antimicrobial assays.
  • the compounds many also be used as antimicrobials in solutions useful in eye care, such as contact lens solutions, and in topical or other pharmaceutical compositions for treatment of sexually transmitted diseases (STDs).
  • STDs sexually transmitted diseases
  • the invention is also directed to use ofthe invention compounds as preservatives for foods or other perishables.
  • the invention peptides can inactivate endotoxin
  • the invention is also directed to a method to inactivate endotoxins using the compounds ofthe invention and to treat gram-negative sepsis by taking advantage of this property.
  • Figure 1 shows antibacterial activity of two ofthe parevins against E. Coli
  • Figure 2 shows antibacterial activity of two ofthe parevins against Listeria monocytogenes
  • Figure 3 shows antifungal activity of two ofthe parevins against Candida albicans
  • Figure 4 shows antibacterial activity of a tachytegrin against E. coli
  • Figure 5 shows antibacterial activity of a tachytegrin against B. subtilis
  • Figure 6 shows antibacterial activity of a tachytegrin against S. typhimurium.
  • A represents an amino acid at the specified position in the peptide.
  • a ⁇ -A 3 , C 4 * , C ⁇ * and/or A ⁇ 8 may or may not be present.
  • the peptides ofthe invention contain 11-24 amino acids.
  • sequence shown as (1) can be extended at the N and/or C terminus with non-interferring amino acids or sequence.
  • the positions ofthe cysteine, homocysteine or penicillamine residues, shown as C in formula (1), are invariant in one embodiment ofthe peptides ofthe invention; however, in the modified forms ofthe peptides containing the sequence of formula (1), also included within the scope of the invention, one or more of these cysteines may be replaced by a small, basic acidic or hydrophobic amino acid. However, at least one of G * G * G ⁇ and G * 7 must be cysteine , homocysteine penicillamine.
  • All ofthe peptides ofthe invention have a net positive charge of at least +1 at physiological pH; approximately 15%-50% ofthe amino acid residues included in the sequence should be basic. For embodiments having as few as 11 amino acids, there may be only one basic amino acid residue; however, at least two basic residues, even in this short- chain residue, are preferred. If the peptide contains as many as 15 amino acid residues, two basic residues are required. It is preferred that at least 20% ofthe amino acids in the sequence be basic, more preferably 30%, but not more than 50%.
  • the active peptides also preferably contain a ⁇ turn bracketed by two strands that form a ⁇ sheet. While not intending to be bound by any theory, applicants believe that antimicrobial activity ofthe compounds containing the sequence of formula (1) is associated with such a ⁇ - turn bracketed by two strands that form a ⁇ sheet structure.
  • the amino acids A 9 -A ⁇ must be capable of effecting a ⁇ turn, which can be encouraged by the cystine bond between C 8 and C ⁇ as well as by hydrogen bonding between A 9 and A ⁇ 2 .
  • the presence of proline at Aio and/or An does not interfere with the ⁇ -turn stabilized by the presence of a hydrophobic amino acid at positions A 9 or A ⁇ 2 .
  • ⁇ -turn refers to a recognized sub-class of reverse-turns.
  • a " ⁇ -turn” is a four amino acid residue peptide segment that reverses the direction of a polypeptide chain so as to allow a single polypeptide chain to adopt an anti-parallel ⁇ -sheet secondary structure.
  • the two internal amino acid residues ofthe ⁇ -turn are not involved in the hydrogen-bonding ofthe ⁇ -sheet; the two amino acid residues on either side of the internal residues are included in the hydrogen-bonding ofthe ⁇ -sheet.
  • ⁇ -turn expressly includes all types of peptide ⁇ -turns commonly known in the art including, but not limited to, type-I, type-II, type-Ill, type-F, type-IF, and type-Ill' ⁇ -turns (see, Rose et al, 1985, Adv. Protein Chem. 37:1-109; Wilmer- White et al, 1987, Trends Biochem. Sci. 12:189-192; Wilmot et al, 1988. J. Mol. Biol. 206:759-777: Tramontano et al, 1989, Proteins: Struct. Funct. Genet. 6:382-394).
  • the presence ofthe four invariant cysteines ofthe protegrins or ofthe C 8 and C ⁇ cysteines, homocysteine or penicillamine ofthe compounds ofthe present invention is helpful in effecting the ⁇ -turn conformation; however, by properly choosing the substitutions, one or both ofthe cysteine, homocysteine or penicillamine residues at C 8 or C ⁇ can be replaced without substantially disturbing the three-dimensional shape ofthe molecule.
  • a 7 and A ⁇ 4 are preferably hydrophobic amino acids.
  • the cysteine residues may also, then, be replaced by other residues which do not affect the maintenance ofthe ⁇ sheet formation; these substitutions would include acidic, basic, hydrophobic polar or small amino acids.
  • the amino terminus ofthe peptide may be in the free amino form or may be acylated by a group ofthe formula RCO-, wherein R represents a hydrocarbyl group of 1-6C.
  • R represents a hydrocarbyl group of 1-6C.
  • the hydrocarbyl group is saturated or unsaturated and is typically, for example, methyl, ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, and the like.
  • the C-terminus ofthe peptides ofthe invention may be in the form ofthe underivatized carboxyl group, either as the free acid or an acceptable salt, such as the potassium, sodium, calcium, magnesium, or other salt of an inorganic ion or of an organic ion such as caffeine. In some embodiments, it is difficult to make salts since the remainder ofthe molecule bears a positive charge which may repel the relevant cation.
  • the carboxyl terminus may also be derivatized by formation of an ester with an alcohol ofthe formula ROH, or may be amidated by an amine ofthe formula NH 3 , or RNH 2 , or R 2 NH, wherein each R is independently hydrocarbyl of 1-6C as defined above. Amidated forms ofthe peptides wherein the C-terminus has the formula CONH 2 are preferred.
  • the peptides ofthe invention may be supplied in the form ofthe acid addition salts.
  • Typical acid addition salts include those of inorganic ions such as chloride, bromide, iodide, fluoride or the like, sulfate, nitrate, or phosphate, or may be salts of organic anions such as acetate, formate, benzoate and the like. The acceptability of each of such salts is dependent on the intended use, as is commonly understood.
  • the peptides ofthe invention that contain at least two cysteines, homocysteine or penicillamine may be in straight-chain or cyclic form.
  • the straight-chain forms are convertible to the cyclic forms, and vice versa.
  • Methods for forming disulfide bonds to create the cyclic peptides are well known in the art, as are methods to reduce disulfides to form the linear compounds.
  • the linear compounds can be stabilized by addition of a suitable alkylating agent such as iodoacetamide.
  • Cyclic forms are the result ofthe formation of disulfide linkages among all or some ofthe four cysteine, homocysteine or penicillamine residues that may be present.
  • Cyclic forms ofthe invention include all possible permutations of disulfide bond formation; ifthe -SH containing amino acids are numbered in order of their occurrence starting at the N-terminus as G * G * G * C 8 , C ⁇ , G * 6, G * 7 or Gs, these permutations include, when two disulfides are present:
  • these permutations include:
  • linearalized forms ofthe native cyclic peptides have valuable activities, even when chemically stabilized to preserve the sulfhydryl form of cysteine, homocysteine or penicillamine for example, by reaction with iodoacetamide.
  • the compounds ofthe invention also include linearalized forms which are stabilized with suitable reagents. As defined herein,
  • SH-stabilized forms ofthe peptides ofthe invention contain sulfhydryl groups reacted with standard reagents to prevent reformation into disulfide linkages.
  • Forms ofthe invention compounds which have only one disulfide bond are conveniently obtained by replacing the cysteine, homocysteine or penicillamine residues at C and/or Cn, preferably both, with amino acids which do not form disulfide linkages.
  • amino acids denoted by A- may be those encoded by the gene or analogs thereof, and may also be the D-isomers thereof.
  • One preferred embodiment ofthe peptides ofthe invention is that form wherein all ofthe residues are in the D-configuration thus conferring resistance to protease activity while retaining antimicrobial or antiviral properties.
  • the resulting peptides are enantiomers ofthe native L-amino acid-containing forms.
  • either one or both ofthe residues found at C5 * and/or Ci ⁇ * is a basic amino acid and/or at least one of A ⁇ -A 3 and C 4 * is hydrophobic and/or at least one, and preferably all four of these amino acids are deleted.
  • amino acid notations used herein are conventional and are as follows:
  • amino acids not encoded genetically are abbreviated as indicated in the discussion below.
  • the compounds ofthe invention are peptides which are partially defined in terms of amino acid residues of designated classes.
  • Amino acid residues can be generally subclassified into major subclasses as follows: Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • the residue has a positive charge due to association with H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Polar/large The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • This description also characterizes certain neutral amino acids as "small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity.
  • "Small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not.
  • Amino acid residues can be further subclassified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups ofthe residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive ofthe carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic.
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in a group. Cysteine and other -SH containing amino acid residues are also not included in these classifications since their capacity to form disulfide bonds to provide secondary structure is critical in the compounds ofthe present invention.
  • Certain commonly encountered amino acids include, for example, ⁇ -Alanine ( ⁇ -Ala), or other omega-amino acids, such as 3-aminopropionic, 2,3-diaminopropionic (2,3-diaP), 4-aminobutyric and so forth, ⁇ -aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-Melle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine (2-Nal); 1,2,3,4- tetrahydroisoquinoline-3 -carboxylic acid (Tic); J-2-thienylalanine (Thib), or other omega-amino acids
  • Cit, Acetyl Lys, and MSO are polar/large.
  • the various omega-amino acids are classified according to size as small ( ⁇ -Ala and 3-aminopropionic) or as large and hydrophobic (all others).
  • amino acid substitutions of those encoded in the gene can also be included in peptide compounds within the scope ofthe invention and can be classified within this general scheme according to their structure.
  • This replacement can be made by methods known in the art.
  • the following references describe preparation of peptide analogs which include these alternative-linking moieties: Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Spatola, A.F., in Chemistry and Biochemistry of Amino Acids Peptides and Proteins.
  • the peptides or proteins ofthe invention include peptide mimetics in general, such as those described by Olson, G.L. et al. J Med Chem (1993) 36:3039-3049 and retro-inverso type peptides as described by Chorev, M. et al. Science (1979) 204: 1210-1212; and Pallai, P. V. et al, Int J Pept Protein Res (1983) 21:84-92.
  • positions 8 and 13 are independently cysteine, homocysteine or penicillamine residues, especially in the disulfide bonded form.
  • each of A 7 and A ⁇ 4 is a hydrophobic acid, preferably Ile, Val, Leu, NLe, T ⁇ , Tyr or Phe, or is a small amino acid, Ala, Gly, Ser or Thr.
  • all of A ⁇ -A 3 are not present or at least one, and preferably two of A ⁇ -A 3 is a hydrophobic amino acid, preferably Ile, Val, Leu, NLe, T ⁇ , Tyr or Phe.
  • C * and/or C ⁇ * is not present or, if present, is a cysteine, homocysteine or penicillamine or a hydrophobic amino acid, preferably Ile, Val, Leu, .NLe, T ⁇ , Tyr or Phe, or a small amino acid, preferably S, A, G or T.
  • C 5 * and/or Ci ⁇ * is a cysteine, homocysteine or penicillamine or a hydrophobic amino acid, preferably Ile, Val, Leu, .NLe, T ⁇ , Tyr or Phe, or a small amino acid, preferably S, A, G or T.
  • a 9 -A ⁇ 2 contain at least one hydrophobic amino acid residue, preferably Phe, Tyr or T ⁇ .
  • each of Ai and A 9 is independently selected from the group consisting of R, K and Har; more preferably, both Ai and A are R; however, each of Ai may be absent.
  • each of A 2 and A 3 is independently selected from the group consisting of G, A, S and T; more preferably, A 2 and A 3 are G; however, A 2 and/or A 3 may be absent.
  • one of A 9 and A ]2 is R, K, Har, Orn or H preferably R and the other is I, V, L, NLe, W, Y or F, preferably R, F or W, or is S, G, A or T.
  • each of Aio and An is independently proline or a small, basic or hydrophobic amino acid, preferably R, G, W or P.
  • Ai 8 is preferably absent, but when present, is preferably R, K or Har, most preferably R.
  • Ai is basic
  • C * is C or basic
  • a 2 and A 3 are small amino acids, or at least one of A A 3 and C is hydrophobic.
  • Preferred embodiments of A ⁇ -A 3 include R-G-G, K-G-S, K-S-G, and the like.
  • the compounds of Formula (1) are either in cyclic or noncyclic (linearalized) form or may be modified wherein 1 or 2 ofthe cysteines, homocysteine or penicillamine at C g and C ⁇ are replaced by a small, hydrophobic, or a basic amino acid residue. Such modification is preferred when compounds containing only one disulfide bond are prepared. Ifthe linearalized forms ofthe compound of Formula (1) are prepared, or if linearalized forms of those modified peptides which contain at least two cysteines are prepared, it is preferred that the sulfhydryl groups be stabilized by addition of a suitable reagent.
  • Preferred embodiments for the hydrophobic amino acid to replace cysteine, homocysteine or penicillamine residues at Cg and/or C ⁇ are I, V, L and Nle, preferably I, V or L.
  • Preferred small amino acids to replace the cysteine, homocysteine or penicillamine residues include G, A, S and T, more preferably G.
  • Preferred basic amino acids are R and K.
  • Particularly preferred compounds ofthe invention including the N-terminal acylated and C-terminal amidated forms thereof are the parevins, wherein G * and G * ⁇ are both cysteine, homocysteine or penicillamine and the tachytegrins wherein both G * and G * 7 are cysteine, homocysteine or penicillamine.
  • disulfide forms of these compounds cis-parevins wherein the two disulfide bridges are C 5 -Cg and C ⁇ -C ⁇ 6 ; the trans-parevins wherein the disulfide bridges are C5-C16 and Cg-C ⁇ ; and the trans-tachytegrins wherein the disulfide bridges are C -C ⁇ and Cg-C ⁇ .
  • Particularly preferred are the following parevins and tachytegrins: Parevin-1 trans and cis.
  • Typical compounds ofthe invention include:
  • R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R; R_G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X NMeG)
  • the invention compounds are essentially peptide backbones which may be modified at the N- or C-terminus and also may contain one or two cystine disulfide linkages.
  • the peptides may first be synthesized in noncyclized form. These peptides may then be converted to the cyclic peptides if desired by standard methods of cystine bond formation.
  • cyclic forms refers to those forms which contain cyclic portions by virtue ofthe formation of disulfide linkages between cysteine residues in the peptide. Ifthe straight- chain forms are preferred, it is preferable to stabilize the sulfhydryl groups for any peptides of the invention which contain two or more cysteine residues.
  • Standard methods for synthesis of peptides can be used. Most commonly used currently are solid phase synthesis techniques; indeed, automated equipment for systematically constructing peptide chains can be purchased. Solution phase synthesis can also be used but is considerably less convenient. When synthesized using these standard techniques, amino acids not encoded by the gene and D-enantiomers can be employed in the synthesis. Thus, one very practical way to obtain the compounds ofthe invention is to employ these standard chemical synthesis techniques.
  • the N- and/or C-terminus can be derivatized, again using conventional chemical techniques.
  • the compounds ofthe invention may optionally contain an acyl group, preferably an acetyl group at the amino terminus. Methods for acetylating or, more generally, acylating, the free amino group at the N-terminus are generally known in the art; in addition, the N-terminal amino acid may be supplied in the synthesis in acylated form.
  • the carboxyl group may, of course, be present in the form of a salt; in the case of pharmaceutical compositions this will be a pharmaceutically acceptable salt.
  • Suitable salts include those formed with inorganic ions such as NH , Na + , K + , Mg ++ , Ca ++ , and the like as well as salts formed with organic cations such as those of caffeine and other highly substituted amines.
  • inorganic ions such as NH , Na + , K + , Mg ++ , Ca ++ , and the like
  • salts formed with organic cations such as those of caffeine and other highly substituted amines.
  • the carboxy terminus may also be esterified using alcohols ofthe formula ROH wherein R is hydrocarbyl (1-6C) as defined above.
  • carboxy terminus may be amidated so as to have the formula -CONH 2 , -CONHR, or -CONR 2 , wherein each R is independently hydrocarbyl (1-6C) as herein defined.
  • Techniques for esterification and amidation as well as neutralizing in the presence of base to form salts are all standard organic chemical techniques.
  • the side- chain amino groups ofthe basic amino acids will be in the form ofthe relevant acid addition salts.
  • disulfide linkages if desired, is conducted in the presence of mild oxidizing agents. Chemical oxidizing agents may be used, or the compounds may simply be exposed to the oxygen ofthe air to effect these linkages.
  • oxidizing agents may be used, or the compounds may simply be exposed to the oxygen ofthe air to effect these linkages.
  • Various methods are known in the art. Processes useful for disulfide bond formation have been described by Tam, J.P. et al, Synthesis (1979) 955-957; Stewart, J.M. et al, "Solid Phase Peptide Synthesis” 2d Ed. Pierce Chemical Company Rockford, IL (1984); Ahmed A.K. et al, J Biol Chem (1975) 250:8477- 8482 and Pennington M.W. et al, Peptides 1990. E.
  • a particularly preferred method is solution oxidation using molecular oxygen. This method has been used by the inventors herein to refold the compounds ofthe invention.
  • the peptide backbone is comprised entirely of gene-encoded amino acids, or if some portion of it is so composed, the peptide or the relevant portion may also be synthesized using recombinant DNA techniques.
  • the DNA encoding the peptides ofthe invention may itself be synthesized using commercially available equipment; codon choice can be integrated into the synthesis depending on the nature ofthe host.
  • Synthesized and recombinantly produced forms ofthe compounds may require subsequent derivatization to modify the N- and/or C-terminus and, depending on the isolation procedure, to effect the formation of cystine bonds as described hereinabove. Depending on the host organism used for recombinant production, some or all of these conversions may already have been effected.
  • the DNA encoding the peptides ofthe invention is included in an expression system which places these coding sequences under control of a suitable promoter and other control sequences compatible with an intended host cell.
  • suitable promoter and other control sequences compatible with an intended host cell.
  • Types of host cells available span almost the entire range ofthe plant and animal kingdoms.
  • the compounds ofthe invention could be produced in bacteria or yeast (to the extent that they can be produced in a nontoxic or refractile form or utilize resistant strains) as well as in animal cells, insect cells and plant cells.
  • modified plant cells can be used to regenerate plants containing the relevant expression systems so that the resulting transgenic plant is capable of self protection vis-a-vis these infective agents.
  • the compounds ofthe invention can be produced in a form that will result in their secretion from the host cell by fusing to the DNA encoding the peptide, a DNA encoding a suitable signal peptide, or may be produced intracellulariy. They may also be produced as fusion proteins with additional amino acid sequence which may or may not need to be subsequently removed prior to the use of these compounds as antimicrobials or antivirals.
  • the compounds ofthe invention can be produced in a variety of modalities including chemical synthesis, recombinant production, isolation from natural sources, or some combination of these techniques.
  • purified and isolated is meant free from the environment in which the peptide normally occurs (in the case of such naturally occurring peptides) and in a form where it can be used practically.
  • purified and isolated form means that the peptide is substantially pure, i.e., more than 90% pure, preferably more than 95% pure and more preferably more than 99% pure or is in a completely different context such as that of a pharmaceutical preparation.
  • Antibodies to the peptides ofthe invention may also be produced using standard immunological techniques for production of polyclonal antisera and, if desired, immortalizing the antibody-producing cells ofthe immunized host for sources of monoclonal antibody production. Techniques for producing antibodies to any substance of interest are well known. It may be necessary to enhance the immunogenicity ofthe substance, particularly as here, where the material is only a short peptide, by coupling the hapten to a carrier. Suitable carriers for this pu ⁇ ose include substances which do not themselves produce an immune response in the mammal to be administered the hapten-carrier conjugate.
  • Common carriers used include keyhole limpet hemocyanin (KLH), diphtheria toxoid, serum albumin, and the viral coat protein of rotavirus, VP6. Coupling ofthe hapten to the carrier is effected by standard techniques such as contacting the carrier with the peptide in the presence of a dehydrating agent such as dicyclohexylcarbodiimide or through the use of linkers such as those available through Pierce Chemical Company, Chicago, IL.
  • KLH keyhole limpet hemocyanin
  • VP6 the viral coat protein of rotavirus
  • peptides ofthe invention in immunogenic form are then injected into a suitable mammalian host and antibody titers in the serum are monitored.
  • some forms ofthe peptides require modification before they are able to raise antibodies, due to their resistance to antigen processing.
  • peptides containing two cystine bridges may be nonimmunogenic when administered without coupling to a larger carrier and may be poor immunogens even in the presence of potent adjuvants and when coupled in certain formats such as using glutaraldehyde or to KLH. Any lack of immunogenicity may therefore result from resistance to processing to a linear form that can fit in the antigen- presenting pocket ofthe presenting cell. Immunogenicity of these forms ofthe peptides can be enhanced by cleaving the disulfide bonds.
  • Polyclonal antisera may be harvested when titers are sufficiently high.
  • antibody-producing cells ofthe host such as spleen cells or peripheral blood lymphocytes may be harvested and immortalized.
  • the immortalized cells are then cloned as individual colonies and screened for the production ofthe desired monoclonal antibodies.
  • the antibodies ofthe invention include those that can be made by genetic engineering techniques. For example, single-chain forms, such as F v forms, chimeric antibodies, and antibodies modified to mimic those of a particular species, such as humans, can be produced using standard methods.
  • the antibodies ofthe invention can be prepared by isolating or modifying the genes encoding the desired antibodies and producing these through expression in recombinant host cells, such as CHO cells.
  • the antibodies ofthe invention are, of course, useful in immunoassays for determining the amount or presence ofthe peptides. Such assays are essential in quality controlled production of compositions containing the peptides ofthe invention.
  • the antibodies can be used to assess the efficacy of recombinant production ofthe peptides, as well as screening expression libraries for the presence of peptide encoding genes.
  • compositions Containing the Invention Peptides and Methods of Use are effective in inactivating a wide range of microbial and viral targets, including gram-positive and gram-negative bacteria, yeast, protozoa and certain strains of virus. Accordingly, they can be used in disinfectant compositions and as preservatives for materials such as foodstuffs, cosmetics, medicaments, or other materials containing nutrients for organisms.
  • the peptides are supplied either as a single peptide, in admixture with several other peptides ofthe invention, or in admixture with additional antimicrobial agents or both.
  • peptides ofthe invention are also useful as standards in antimicrobial assays and in assays for determination of capability of test compounds to bind to endotoxins such as lipopolysaccharides .
  • the peptides of the invention can be formulated as pharmaceutical or veterinary compositions.
  • the mode of administration, and the type of treatment desired e.g., prevention, prophylaxis, therapy; the invention peptides are formulated in ways consonant with these parameters.
  • a summary of such techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.
  • the peptides ofthe invention can also be used as active ingredients in pharmaceutical compositions useful in treatment of sexually transmitted diseases, including those caused by Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, He ⁇ es simplex type 2 and HIV.
  • Topical formulations are preferred and include creams, salves, oils, powders, gels and the like. Suitable topical excipient are well known in the art and can be adapted for particular uses by those of ordinary skill.
  • the peptides ofthe invention may be used alone or in combination with other antibiotics such as erythromycin, tetracycline, macrolides; for example azithromycin and the cephalosporins.
  • antibiotics such as erythromycin, tetracycline, macrolides; for example azithromycin and the cephalosporins.
  • the peptides will be formulated into suitable compositions to permit facile delivery to the affected areas.
  • the tachytegrins may be used in forms containing one or two disulfide bridges or may be in linear form.
  • enantiomeric forms containing all D-amino acids may confer advantages such as resistance to those proteases, such as trypsin and chymotrypsin, to which the peptides containing L-amino acids are less resistant.
  • the peptides ofthe invention can be administered singly or as mixtures of several peptides or in combination with other pharmaceutically active components.
  • the formulations may be prepared in a manner suitable for systemic administration or topical or local administration.
  • Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration.
  • the formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like.
  • the tachytegrins can be administered also in liposomal compositions or as microemulsions.
  • the peptides ofthe invention must be protected from degradation in the stomach using a suitable enteric coating. This may be avoided to some extent by utilizing amino acids in the D-configuration, thus providing resistance to protease. However, the peptide is still susceptible to hydrolysis due to the acidic conditions ofthe stomach; thus, some degree of enteric coating may still be required.
  • the peptides ofthe invention also retain their activity against microbes in the context of borate solutions that are commonly used in eye care products. Also, it is important that the peptides retain their activity under physiological conditions including relatively high saline and in the presence of serum. In addition, the peptides are dramatically less cytotoxic with respect to the cells of higher organisms as compared with their toxicity to microbes. These properties, make them particularly suitable for in vivo and therapeutic use.
  • microbe will be used to include not only yeast, bacteria, and other unicellular organisms, but also viruses.
  • the particular peptide used can also be chosen to be advantageous in a particular context, such as low salt or physiological salt, the presence or human serum, or conditions that mimic the conditions found in blood and tissue fluids.
  • the peptides ofthe invention may also be applied to plants or to their environment to prevent virus- and microbe-induced diseases in these plants.
  • Suitable compositions for this use will typically contain a diluent as well as a spreading agent or other ancillary agreements beneficial to the plant or to the environment.
  • the peptides ofthe invention may be used in any context wherein an antimicrobial and/or antiviral action is required. This use may be an entirely in vitro use, or the peptides may be administered to organisms.
  • the antimicrobial or antiviral activity may be generated in situ by administering an expression system suitable for the production ofthe peptides ofthe invention.
  • an expression system suitable for the production ofthe peptides ofthe invention can be supplied to plant and animal subjects using known techniques. For example, in animals, pox-based expression vectors can be used to generate the peptides in situ. Similarly, plant cells can be transformed with expression vectors and then regenerated into whole plants which are capable of their own production ofthe peptides.
  • the peptides ofthe invention are also capable of inactivating endotoxins derived from gram-negative bacteria — i.e., lipopolysaccharides (LPS) and may be used under any circumstances where inactivation of LPS is desired.
  • LPS lipopolysaccharides
  • One such situation is in the treatment or amelioration of gram-negative sepsis.
  • antimicrobial activity refers to inhibition with respect both to traditional microorganisms and to viruses, although occasionally, “antimicrobial” and “antiviral” are both specifically indicated.
  • the standard buffer medium, medium A uses an underlay agar with the following composition: 0.3 mg/ml of trypticase soy broth powder, 1% w/v agarose and 10 mM sodium phosphate buffer (final pH 7.4). This will be designated either “medium A” or “standard in vitro conditions” herein.
  • a second medium contains 100 mM NaCl in order to mimic the salt levels in blood and tissue fluids. This will be designated “medium B” or “salt medium” herein.
  • a third medium is supplemented with 2.5% normal human serum; however, it is of low ionic strength and thus does not mimic body fluids.
  • This medium will be designated “medium C” or “serum-containing medium” herein.
  • a fourth medium contains 80% RPMI- 1640, a standard tissue culture medium which contains the principal ions and amino acids found in blood and tissue fluids. In addition, it contains 2.5% normal human serum. This will be designated “medium D” or “physiological medium” herein.
  • the peptides ofthe invention therefore represent a peculiarly useful class of compounds because ofthe following properties:
  • Example 1 Synthesis ofthe Invention Compounds
  • the peptides ofthe invention are synthesized using conventional Fmoc chemistry on solid-phase supports.
  • the crude synthetic peptides are refolded, purified and characterized as follows.
  • the crude synthetic peptide is reduced by adding an amount of dithiothreitol (DTT) equal in weight to that ofthe synthetic peptide, which has been dissolved at 10 mg/ml in a solution containing 6M guanidine HCl, 0.5M Tris buffer and 2 mmol EDTA, pH 8.05 and incubated for 2 hours at 52°C under nitrogen.
  • DTT dithiothreitol
  • the mixture is passed through a 0.45 um, filter, acidified with 1/20 v/v glacial acetic acid and subjected to conventional RP HPLC purification with a Cl 8 column.
  • HPLC-purified, reduced peptides are partially concentrated by vacuum centrifugation in a Speed Vac and allowed to fold for 24 hours at room temperature and air.
  • the folding is accomplished in 0. IM Tris, pH 7.7 at 0.1 mg peptide/ml to minimize formation of interchain cystine disulfides.
  • the folded compounds are concentrated and acidified with 5% acetic acid. The purity ofthe final products is verified by AU-PAGE, analytical HPLC and FAB-mass spec.
  • trans-parevin-1 or the "hai ⁇ in” isoform
  • cis- parevin-1 or “cloverleaf ' isoform
  • trans-tachytegrin-1 were prepared. These compounds are ofthe formulas
  • the radial diffusion assay in agarose gels is conducted using radiodiffiision and gel overlay techniques as described by Lehrer, R.I. et al. J Immunol Meth (1991) 137:167-173. Briefly, the underlay agars used for all organisms had a final pH of 7.4 and contained 10 mM sodium phosphate buffer, 1% w/v agarose and 0.30 ug/ml tryptocase soy broth powder (BBL Cockeysville, MD). In some cases, the underlay was supplemented with 100 mM NaCl. The units of activity in the radial difiusion assay were measured as described; 10 units conespond to a 1 mm diameter clear zone around the sample well. Figures 1-6 show the results against five test organisms in units described as above. A synthetic protegrin (PG-l) containing two cystines (sPG-1) or PG-l in linear form were used as controls.
  • PG-l protegrin
  • Figure 1 shows the results for trans-parevin and tachytegrin with respect to E. coli both with and without the addition of 100 mM NaCl. Both of these peptides were slightly more effective than sPG-1 although slightly less effective than linear PG-l in the absence of salt. However, in the presence of 100 mM NaCl, all four peptides were comparably effective.
  • Figure 2 shows the results ofthe same determination with respect to L. monocytogenes. With respect to this organism, all four peptides were roughly similarly effective in the absence of salt; the presence of 100 mM NaCl, however, greatly reduced the effectiveness of linear PG-l . The remaining three peptides remained effective under these conditions.
  • Figure 3 shows the results ofthe same experiment using C. albicans as the target organism. All four peptides were comparably effective in the absence of salt; again, the effectiveness of linear PG-l was greatly reduced in the presence of 100 mM NaCl, while the remaining three peptides maintained their effectiveness under these conditions.
  • Figures 4-6 show the results of similar experiments using, as test peptides, the two isomers of parevin, trans-parevin (hai ⁇ in) and cis-parevin (cloverleaf). sPG-1 was used as a control. As shown in Figure 4, the two parevins were comparably effective in the absence of salt and both were more effective than sPG-1. In the presence of 100 mM NaCl, all three peptides maintained their effectiveness and were comparable.
  • Figure 5 shows results ofthe same experiment conducted with B. subtilis as target organism. Again, both forms of parevin were comparably effective and both were slightly more effective than sPG-1; in the presence of 100 mM NaCl, all three peptides remained effective antimicrobials and had about the same activity.
  • the compounds ofthe invention are tested for their ability to bind the lipid polysaccharide (LPS) ofthe gram-negative bacterium E. coli strain 0.55B5, using the Limulus amebocyte lysate (LAL) test for endotoxins conducted in the presence and absence ofthe test compounds.
  • LPS lipid polysaccharide
  • LAL Limulus amebocyte lysate
  • the LAL test is based on the ability of LPS to effect gelation in the commercial reagent E-ToxateO which is prepared from the lysate of circulating amebocytes ofthe Horseshoe Crab Limulus polyphemus. As described in the technical bulletin, when exposed to minute quantities of LPS, the lysate increases in opacity as well as viscosity and may gel depending on the concentration of endotoxin.
  • test compounds are used at various concentrations from 0.25 ug-10 ug in a final volume of 0.2 ml and the test mixtures contained LPS at a final concentration of 0.05 endotoxin unit/ml and E-ToxateTM at the same concentration.
  • the test compounds are incubated together with the LPS for 15 minutes before the E-ToxateTM is added to a final volume after E-ToxateTM addition of 0.2 ml.
  • the tubes are then incubated for 30 minutes at 37°C and examined for the formation of a gel.
  • the concentration of LPS is varied from 0.05-0.25 endotoxin units (E.U.).
  • Example 4 Antimicrobial Activity Under Conditions Suitable for Treatment ofthe Eye Contact lens solutions are typically formulated with borate buffered physiological saline and may or may not contain EDTA in addition.
  • the compounds ofthe invention are tested generally in the assay described in Example 2 wherein all underlay gels contain 25 mM borate buffer, pH 7.4, 1% (v/v) trypticase soy broth (0.3 ug/ml TSB powder) and 1% agarose. Additions include either 100 mM NaCl, 1 mM EDTA or a combination thereof.
  • Other test compounds used as controls are the defensin NP-1 and lysozyme, and dose response curves are determined.
  • Example 5 Preparation o ⁇ Enantio Trans-parevin Using standard solid phase techniques, a peptide having the amino acid sequence of trans-parevin, but wherein every amino acid is in the D form is prepared. This form is tested against E. coli, L. monocytogenes, C. albicans and other microbes in the absence and presence of protease and otherwise as described for the radiodif ⁇ usion assay in agarose gels set forth in Example 2.
  • Example 6 Activity against STD Pathogens
  • the compounds ofthe invention are tested for antimicrobial activity against various STD pathogens. These include HTV-l, Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, He ⁇ es simplex type 2, He ⁇ es simplex type 1, Hemophilus ducreyi, and Human papilloma virus.
  • STD pathogens include HTV-l, Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, He ⁇ es simplex type 2, He ⁇ es simplex type 1, Hemophilus ducreyi, and Human papilloma virus.
  • active means that the peptide is effective at less than 10 ug/ml; moderately active indicates that it is active at 10-25 ug/ml; and slightly active means activity at 25-50 ug/ml
  • the compounds ofthe invention are tested for their antimicrobial activity against Chlamydia using the "gold standard" chlamydial culture system for clinical specimens described by Clarke, L.M. in Clinical Microbiology Procedures Handbook II (1992), Isenberg, H T. Ed. Am. Soc. Microbiol. Washington, D.C; pp. 8.0.1 to 8.24.3.9.
  • C. trachomatis serovar L2 (L2/434Bu) described by Kuo, CC. et al. in Nongynococcal Urethritis and Related Infections (1977), Taylor-Robinson, D. et al. Ed. Am. Soc. Microbiol. Washington, D.C, pp. 322-326 is used.
  • the seed is prepared from a sonicated culture in L929 mouse fibroblast cells, and partially purified by centrifugation. Since host protein is still present in the seed aliquots, each seed batch is titered at the time of preparation with serial ten-fold dilutions to 2 x IO "9 .
  • the seed containing 9.2 x IO 6 IFU/ml is thawed quickly at 37°C and diluted to IO "2 with sucrose/phosphate salts/glycine to produce IFU of about 200 after room temperature preincubation and to dilute background eukaryotic protein.
  • the peptides to be tested are prepared as stock solutions in 0.01% glacial acetic acid. 100 ul ofthe diluted chlamydial seed are aliquoted into 1.5 ml eppendorf tubes and 200 ul ofthe antibiotic peptide was added per tube. Aliquots ofthe peptide stock (and controls) are incubated with the seed at room temperature for one hour, two hours and four hours. About 10 minutes before the end of each incubation period, maintenance media are aspirated from the McCoy vials in preparation for standard inoculation and culture. Culture is then performed in the presence and absence ofthe peptides; in some cases, the peptides are added to final concentration in the culture media in addition to the preculture incubation. The test is evaluated microscopically.
  • Chlamydia seed is preincubated for two hours with and without 10% FBS and also with or without test compound at 25 ug.
  • the effect of serum is particularly important since for a topical agent to be effective in combatting Chlamydia infection, it must act in the presence of serum.
  • the 50% immobilizing end point (IE50) is calculated to indicate the concentration needed to immobilize 50% ofthe spirochetes.
  • Tachyplesin IE 50 S are 5.231 ug and 2.539 ug for 0 and 4 hours, in contrast to HNP and NP preparations which show little immobilizing ability.
  • HSV 1 Maclntyre strain For He ⁇ es Simplex Virus, using viral stocks prepared in VERO cells, grown in minimal essential medium (MEM) with 2% fetal calf serum, the effect of various peptides on HSV 1 Maclntyre strain, a pool often clinical HSV 1 isolates, HSV-2G, and a pool often clinical HSV 2 isolates, all sensitive to 3 uM acyclovir are tested.
  • Two fibroblast cell lines, human W138 and equine CCL57 are used as targets and tests are done by direct viral neutralization and delayed peptide addition.
  • the virus In the direct neutralization format, the virus is preincubated with the peptides for 90 min before it is added to the tissue culture monolayers.
  • the delayed peptide addition format the virus is added and allowed 50 min to adsorb to the target cells, then the monolayers are washed and peptides are added for 90 min. Finally, the monolayer is washed to remove the peptide and the cells are fed with peptide-free MEM and cultured until the untreated infected monolayers exhibit 4+ cytopathic effect (CPE) (about 60 hours).
  • CPE cytopathic effect
  • T. vaginallis For Trichomonas vaginallis, strain Cl (ATCC 30001) is grown as described by Gorrell, T.E. etal, Carlsberg Res Comm (1984) 49:259-268. In experiments performed in RPMI + 1% heat-activated fetal calf serum, within a few minutes after exposure to 50 ug/ml PG-l, T. vaginallis (heretofore vigorously motile) becomes stationary. Soon thereafter, the organisms become permeable to trypan blue, and, over the ensuing 15-30 minutes, lyse. As expected, such organisms fail to grow when introduced into their customary growth medium (Diamond's medium). Organisms exposed to 25 ug/ml of PG-3 retain their motility.
  • the invention compounds are tested for antiviral activity against strains of HIV using the method described in Miles, S.A. et al, Blood (1991) 78:3200-3208. Briefly, the mononuclear cell fraction is recovered from normal donor leukopacs from the American Red Cross using a Ficoll-hypaque density gradient. The mononuclear cells are resuspended at 1 x IO 6 cells per ml in RPMI 1640 medium with 20% fetal bovine serum, 1% penn/strep with fungizone and 0.5% PHA and incubated 24 hours at 37°C in 5% CO 2 . The cells are centrifuged, washed and then expanded for 24 hours in growth medium.
  • Non-laboratory adapted, cloned HIVJ R -CSF and HIVJ R - F are electroporated into the human peripheral blood mononuclear cells prepared as described above. Titers are determined and in general, multiplicities of infection (MOI) of about 4,000 infectuous units per cell are used (which corresponds to 25-40 picograms per ml HIV p24 antigen in the supernatant).
  • MOI multiplicities of infection
  • the HIV stocks prepared as above are diluted to the correct MOI and the PBM are added to 24 well plates at a concentration of 2 x IO 6 per ml. One ul total volume is added to each well. The peptide to be tested is added in growth medium to achieve the final desired concentration. Then the appropriate number of MOI are added.
  • 200 ul of supernatant is removed on days 3 and 7 and the concentration of p24 antigen is determined using a commercial assay (Coulter Immunology, Hialeah, Florida). Controls include duplicate wells containing cells alone, cells plus peptide at 5 ug/ml cells with virus but not peptide and cells with virus in the presence of AZT at 10 "5 M - IO *8 M.
  • the time of addition of peptide can be varied. Cells pretreated for 2 hours prior to addition of virus, at the time of addition of virus, or 2 hours after infection show antiviral activity for the peptide.
  • Example 2 Several illustrative tachytegrins were synthesized as described in Example 1 and tested for activity against Staphylococcus aureus (MRSA), Pseudomonas (Psa), VREF, Candida and E.coli as described in Example 2. The results shown in Table 1 as minimal inhibitory concentration (MIC) in ug/ml were obtained with the C-terminal amidated forms except for the last two which were tested as the free acids, as indicated by *.

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Abstract

On décrit des composés à base d'agents antimicrobiens, des protégrines et des tachyplésines, qui présentent une charge positive nette d'au moins +1 au pH physiologique et comprennent 15 à 50 % d'acides aminés basiques et une séquence d'acides aminés de formule A1-A2-A3-C4*-C5*-C6*-A7-C8-A9-A10-A11-A12-C13-A14-C15*-C16*-C17*-A18, dans une fourchette de taille de 11 à 24 acides aminés quand la formule ci-dessus est prolongée aux terminaisons N- ou C- par des acides aminés complémentaires non intervenants, où la terminaison N- peut être acylatée et la terminaison C- amidatée ou estérifiée, et où des ponts disulfure peuvent être présents ou absents sous une forme linéaire stabilisée -SH. Dans une variante, C8 et C13 représentent cystéine, homocystéine ou pénicillamine; C4*-C6* et C15*-C16* représentent aussi ces éléments ou tout acide aminé naturel sauf les acides glutamique et aspartique et la proline, excepté quand C4*, C17* ou les deux sont éventuellement absents, la proline étant alors seule exclue en C5* et C15*; A1-A3 et A18 peuvent être présent ou absents et représentent tout acide aminé naturel sauf les acides glutamique et aspartique, la proline et la cystéine; A7 et A14 représentent un acide aminé hydrophobe ou court; A9-A12 doivent pouvoir former une spine β dans le composé, et au moins un acide aminé situé entre A9 et A12 doit être basique.
EP96923662A 1995-07-06 1996-07-03 Parevines et tachytegrines Withdrawn EP0836617A4 (fr)

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US6653442B1 (en) 1993-07-20 2003-11-25 Intrabiotics Pharmaceuticals, Inc. Protegrins
US6307016B1 (en) 1995-07-06 2001-10-23 Intrabiotics Pharmaceuticals, Inc. Parevins and tachytegrins
USRE38828E1 (en) * 1995-07-06 2005-10-11 Intrabiotics Pharmaceuticals, Inc. Parevins and tachytegrins
PL326924A1 (en) * 1995-11-22 1998-11-09 Intrabiotics Pharmaceuticals Compositions for and methods of preventing and treating mouth mucositis
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WO1998041224A1 (fr) * 1997-03-20 1998-09-24 The Regents Of The University Of California Utilisation de protegrines pour des indications parodontales
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WO1997002287A1 (fr) 1997-01-23
AU6452996A (en) 1997-02-05
CA2226121A1 (fr) 1997-01-23
JPH11509842A (ja) 1999-08-31
AU714294B2 (en) 1999-12-23

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