Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/03—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
• • 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
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

• the present invention relates to the use of
• the present invention relates to the use of protegrin peptides and congeners thereof to treat oral mucositis in animals including humans.
• chemotherapeutic agent with agents such as carmustine
• Cytarabine Cytarabine, doxorubicin (Adriamycin), fluorouracil (5-FU), methoxetrate (Mexate) and plicamycin (Mithracin) being known for their direct stomatotoxic potential (Sonis, 1993b, supra) and hence incidence of oral mucositis.
• doxorubicin Adriamycin
• fluorouracil 5-FU
• methoxetrate Methoxetrate
• Mithracin plicamycin
• Oral mucositis is initiated by the cytotoxic effects of chemotherapy and/or radiotherapy on the rapidly dividing epithelial cells of the oropharyngeal mucosa, and is
• Benzydamine hydrochloride a nonsteroidal drug with analgesic and antimicrobial properties, has been studied both in patients undergoing radiation therapy and in patients receiving intraarterial chemotherapy (Epstein et al., 1986, Oral Surg. Oral Med. Oral Pathol. 62:145-148; Epstein et al., 1989, Int. J. Radiation Oncology Biol. Phys. 16:1571-1575).
• Chlorhexidine an antimicrobial mouth rinse, has also been used extensively in the treatment and prevention of oral mucositis (Ferretti et al ., 1990, Bone Marrow Transplan.
• myelosuppresive chemotherapy or radiation therapy can result in a decrease in oral mucositis and in the incidence of sepsis due to alpha hemolytic streptococci (Barker et al., 1995, J. Ped. Hem. Oncol. 17:151-155; Spijkervet et al . ,
• Topical application of agents useful to treat oral diseases such as oral mucositis presents unique problems.
• compositions and methods for treating oral mucositis which exhibit broad spectrum
• the present invention in one aspect relates to methods of treating and/or preventing oral mucositis with antimicrobial peptides.
• antimicrobial peptides useful in the methods of the invention are protegrin peptides and/or congeners thereof.
• other broad spectrum antimicrobial peptides such as magainins,
• dermaseptins PGLa or XPF peptides, adrenoregulins, BPI protein and peptides, caeruleins, perforins, insect defensins or sapecins, rabbit or human cationic antimicrobial peptides (CAP-18), porcine myeloid antibacterial peptides (PMAP) , aibellins, acerins, brevenins, esculentins, lactoferrins, cecropin-raellitin hybrids (CEMA peptides), bombenins, tachyplesins, polyphemusins and defensins may be used as well.
• CAP-18 human cationic antimicrobial peptides
• PMAP porcine myeloid antibacterial peptides
• aibellins acerins
• brevenins esculentins
• lactoferrins lactoferrins
• protegrin peptides useful for the treatment or prophylaxis of oral mucositis are peptides having the formula: ( I ) X 1 - X 2 - X 3 - X 4 - X 5 - C 6 - X 7 - C 8 - X 9 - X 10 - X 11 - X 12 - C 1 3 - X 14 -C 15 - X 16 - X 17 - X 18 or pharmaceutical salts thereof, wherein: each of C 8 and C 13 is independently present or not present, and if present each is independently a cysteine-like, basic, small,
• each of C 6 and C 15 is independently a cysteine-like, basic, small, polar/large or hydrophobic amino acid
• each of X 1 -X 5 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid;
• each of X 7 and X 14 is independently a hydrophobic or a small amino acid
• each of X 9 and X 12 is independently present or not present;
• X 9 -X 12 taken together are capable of effecting a reverse-turn when contained in the amino acid sequence of formula (I) and at least one of X 9 -X 12 must be a basic amino acid;
• each of X 16 -X 18 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large or small amino acid;
• amino acids comprising said antimicrobial peptide are basic amino acids such that said antimicrobial peptide has a net charge of at least +1 at physiological pH.
• the present invention is directed to a pharmaceutical formulation suited to topical application of antimicrobial agents to the oral cavity of animals, including humans.
• the pharmaceutical formulation of the invention generally comprises an antimicrobial compound in admixture with a gel-like vehicle.
• the gel-like vehicle generally comprises a mixture of a water-soluble gelling agent and a humectant, and may optionally contain other ingredients such as sweetening agents, preservatives, etc.
• the gel-like formulation provides superior mucoadhesion properties and residence time in the mouth, and has favorable moistening and flavor properties associated with high patient compliance.
• the gel-like formulation is particularly suited for use with the methods described herein.
• FIG. 1 is a graphical representation of the effect of peptide OM-3 (SEQ ID NO:3) on colony forming units (“CFUs”) of microflora in pooled normal human saliva;
• FIG. 2 is a graphical representation of the effect of peptide OM-3 (SEQ ID NO:3) on reduction of oral flora in hamsters;
• FIG. 3 is a graphical representation of the effect of peptide PG-1 (SEQ ID NO:1) treatment on oral mucositis in hamsters;
• FIG. 4 is a graphical representation of the effect of peptide PG-1 (SEQ ID NO:1) treatment on the body weight of hamsters with experimentally-induced oral mucositis;
• FIG. 5 is a graphical representation of the effect of peptide OM-3 (SEQ ID NO:3) treatment on oral mucositis in hamsters;
• FIG. 6 is a graphical representation of the effect of peptide OM-3 (SEQ ID NO:3) treatment on the body weight of hamsters with experimentally-induced oral mucositis;
• FIG. 7 is an illustration of a peptide ⁇ -sheet secondary structure.
• the present invention relates to compositions and methods for the treatment or prevention of oral mucositis in animals, including humans.
• oral mucositis develops in a significant number of cancer and bone marrow transplantation patients receiving chemotherapy and/or radiotherapy.
• Complications related to oral mucositis vary in the different patient populations affected, but include, pain, poor oral intake with consequent dehydration and weight loss, and systemic infection with organisms originating in the oral cavity (Sonis, 1993b).
• the pain associated with oral mucositis may be severe requiring narcotic analgesics, and the difficulty in eating can result in patients receiving total parenteral nutrition.
• the oral cavity is colonized by a variety of organisms including alpha and non-hemolytic streptococci, group D streptococci, Corynebacterium species, staphylococci, lactobacilli, Neisseria species, Branhamella catarrhalis , Hemophilus paramfluenzae , Hemophilus mfluenzae ,
• agents useful to treat oral mucositis must exhibit broad spectrum antimicrobial activity, particularly against Gram-negative microorganisms (Sonis, 1993, "Oral
• properties of agents useful to treat oral mucositis include fast kill kinetics and low frequency of microbial resistance.
• Protegrin peptides are a recognized class of naturally occurring antimicrobial peptides that exhibit broad spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, yeast, fungi and certain viruses (for a review of the properties of protegrin peptides see, U.S.
• Protegrins PG-1 through PG-5 are amidated at the C-terminus and have two disulfide linkages; one between C 6 and C 15 and another between C 8 and C 13 .
• antimicrobial peptides such as the protegrin peptides are capable of exhibiting broad spectrum antimicrobial activity against the normal oral flora of animals, as well as against opportunistic pathogens associated with oral mucositis.
• this antimicrobial activity is effected within the natural environment of the oral cavity, particularly in saliva. Additionally, it was unexpectedly discovered that treatment with protegrins prevents the onset of mucositis. Based on these surprising discoveries, it was surmised that such peptides would be effective to treat or prevent oral infections such as oral mucositis.
• protegrins exhibit antimicrobial activity against pathogens and opportunistic infections associated with mucositis, particularly against the Gram-negative bacteria observed in cancer patients.
• protegrins kill oral pathogens in minutes rather than hours, making them ideally suited for topical application in the mouth where it is difficult to achieve the long residence times necessary for other treatments to be effective. And unlike traditional antibiotics such as vancomycin, protegrins exhibit a low frequency of resistance, making them ideally suited for treating infections such as oral mucositis.
• compositions of the invention provide myriad advantages as well.
• the compositions provide superior mucoadhesion, permitting the active ingredient to remain in contact with the mucosa for long durations. Additionally, through the use of a humectant, the formulations act to moisten the oral mucosa, leading to higher patient
• the humectant also acts as a water barrier, preventing the treatment from being washed away by saliva or other fluids.
• the vehicle also has a pleasant flavor, an important factor in achieving high patient compliance.
• Antimicrobial peptides useful for treating or preventing oral mucositis according to the invention include virtually any broad spectrum antimicrobial peptides that exhibit efficacy against pathogens associated with oral mucositis in the oral environment of the subject being treated.
• Such antimicrobial peptides include, but are not limited to, cationic amphipathic peptides such as
• lactoferrin or analogues or derivatives thereof (U.S. Patent No. 5,317,084; U.S. Patent No. 5,304,633; European Patent Application No. 519,726 A2; European Patent Application No. 503,939 A1; PCT Application WO 93/22348, published November 11, 1993; PCT Application WO 90/13642; and Tomita et al . , In : Lactoferrin Structure and Function, Hutchens, T.W., et al .
• tachyplesins and analogues of tachyplesins such as polyphemusins (Nakamura et al . , 1988, J. Biol. Chem. 263:16709-16713; Miyata et al . , 1989, J. Biochem. 106:663-668), defensins (Lehrer et al . , 1991, Cell 64:229-230; Lehrer et al., 1993, Ann. Rev. Immunol. 11:105-128; U.S. Patent No. 4,705,777; U.S. Patent No. 4,659,692; U.S. Patent No.
• protegrin peptides such as those described, for example in U.S. Patent No.
• antimicrobial peptides useful in the methods of the invention will have minimum inhibitory concentrations (MICs) against Gram-positive and Gram-negative bacteria of less than about 128 ⁇ g/mL, preferably less than about 64 ⁇ g/mL and most preferably less than about 32 ⁇ g/mL as measured using the assays provided in the Examples.
• MICs minimum inhibitory concentrations
• useful peptides will generally exhibit at least a two log reduction in oral colony forming units (CFUs) in saliva in 15 minutes at a peptide concentration of about 0.001% (w/w) to 5% (w/w).
• CFUs oral colony forming units
• protegrin peptides useful for the treatment or prophylaxis of oral mucositis are peptides having the formula:
• the designation X n in each case represents an amino acid at the specified position in the peptide.
• the designation C n represents an amino acid at the specified position and further represents those positions in the peptides of formula (I) which may optionally contain amino acid residues capable of forming disulfide interlinkages.
• amino acid residues denoted by X n or C n may be the genetically encoded L-amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of all of the above.
• amino acid notations used herein for the twenty genetically encoded L-amino acids and common non-encoded amino acids are conventional and are as follows:
• Illustrative compounds useful in the methods of the 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 is not sufficiently repelled by aqueous solutions so that it would necessarily seek an inner position in the conformation of the peptide in which it is contained when the peptide is in aqueous medium.
• the residue may reside either in the inner space or at the surface of the protein.
• Cysteine-Like Residues having a side chain capable of participating in a disulfide linkage.
• cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group, such as cysteine, homocysteine, penicillamine, etc.
• Small Certain neutral amino acids having side chains that 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.
• the gene-encoded secondary amino acid proline (as well as proline-like imino acids such as 3-hydroxyproline and 4-hydroxyproline) is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in a group.
• charged a significant percentage (at least approximately 25%) of the individual molecules are charged at physiological pH.
• the degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.
• 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 of the residues.
• Certain commonly encountered amino acids which are not genetically encoded of which the peptides of the invention may be composed include, but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); ⁇ -aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit);
• t-butylalanine t-BuA
• t-BuG t-butylglycine
• MeIle N-methylisoleucine
• Phg phenylglycine
• cyclohexylalanine Cha); norleucine (Nle); 1-naphthylalanine (1-Nal); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); ⁇ -2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (Har); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,4-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(
• Table 1 The classifications of the above-described genetically encoded and non-encoded amino acids are summarized in Table 1, below. It is to be understood that Table 1 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that may comprise the illustrative peptides described herein.
• amide linkages may optionally be replaced with a linkage other than amide.
• the peptides of the invention are characterized by a structure containing two main elements or motifs: a reverse-turn region bracketed by two strands that form an anti-parallel ⁇ -sheet. While not intending to be bound by theory, it is believed that the antimicrobial activity of the compounds of formula (I) is in part associated with such a core structure.
• the ⁇ -sheet region of the peptides comprises an N-strand (residues X 1 -C 8 ) and a C-strand (residues C 13 -X 18 ).
• the N-strand and C-strand are arranged anti-parallel to one another and are non-covalently linked together via backbone-backbone hydrogen bonds (for a detailed description of the structure of ⁇ -sheets the reader is referred to Creighton, 1993,
• the ⁇ -sheet region of the peptides is amphiphilic, i.e., one surface of the ⁇ -sheet has a net hydrophobic character and the other surface has a net hydrophilic character.
• the side chains of L-amino acid residues adjacent to one another intrastrand-wise (residues n, n+1, n+2, etc.) point in opposite directions so as to be positioned on opposite surfaces of the ⁇ -sheet.
• the side chains of L-amino acid residues adjacent to one another interstrand-wise point in the same direction so as to be positioned on the same surface of the ⁇ -sheet.
• an amphiphilic antiparallel ⁇ -sheet is obtained by selecting amino acids at each residue position so as to yield a ⁇ -sheet having hydrophobic side chains positioned on one surface of the sheet and hydrophilic side chains positioned on the other.
• each side chain comprising a particular surface need not be hydrophobic or hydrophilic.
• the surfaces may contain side chains that do not significantly alter the net character of the surface.
• both the hydrophobic and hydrophilic surfaces may contain small amino acid side chains, as these side chains do not significantly contribute to the net character of the surface.
• the ⁇ -sheet region of the peptides of formula I may contain from one to four cysteine-like amino acids,
• C 8 , C 13 and C 15 which may participate in interstrand disulfide bonds.
• Peptides that contain at least two cysteine-like amino acid residues may be in straight-chain or cyclic form, depending on the extent of disulfide bond formation.
• the cyclic forms are the result of the formation of disulfide linkages among all or some of the four invariant cysteine-like amino acids. Cyclic forms of the invention include all possible permutations of disulfide bond formation.
• 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 native forms of the protegrins contain two disulfide bonds; one between cysteine C 6 -C 15 and another between cysteine C 8 -C 13 (Harwig et al . , 1995, J. Peptide Sci. 3:207). Accordingly, in those embodiments having two disulfide linkages, the C 6 -C 15 , C 8 -C 13 form is preferred. Such peptides are designated "native" forms. However, it has been found that forms of the protegrins containing only one disulfide linkage are active and easily prepared. Preferred among embodiments having only one disulfide linkage are those represented by C 6 -C 15 alone and by C 8 -C 13 alone.
• Forms containing a C 6 -C 15 disulfide as the only disulfide linkage are generally designated “bullet” forms of the protegrins; those wherein the sole disulfide is C 8 -C 13 are designated the “kite” forms.
• the bullet and kite forms can most conveniently be made by replacing each of the cysteine-like amino acid residues at the positions that are not involved in a disulfide linkage with amino acids that do not participate in disulfide bonds, preferably with small amino acids such as glycine, serine, alanine or threonine.
• C 8 and/or C 13 may be absent.
• the peptides of the invention include linearized forms wherein the sulfhydryl (SH) groups are chemically stabilized with suitable reagents.
• SH sulfhydryl
• cysteine-like amino acid residues are replaced by other amino acids as set forth above. It is preferred that all four cysteine-like amino acid residues be SH-stabilized or replaced in order to minimize the likelihood of the formation of intermolecular disulfide linkages.
• the sulfur atoms involved in an interstrand disulfide bridge in a ⁇ -sheet are not positioned within the plane defined by the interstrand backbone-backbone hydrogen-bonds; the sulfur atoms are at an angle with respect to the
• C 6 , C 9 , C 13 and C 15 are each independently a cysteine-like amino acid
• X 7 and X 14 are each independently a hydrophobic or small amino acid and
• C 6 , C 8 , C 13 and C 15 are each cysteine and X 7 and X 14 are each independently
• the ⁇ -sheet secondary structure illustrated in FIG. 7 is composed entirely of L-amino acids.
• L-amino acids substituting an L-amino acid with its corresponding D-enantiomer at a specific residue position may disrupt the structural stability or amphiphilicity of amphiphilic anti-parallel ⁇ -sheet region.
• the degree to which any particular enantiomeric substitution disrupts the structural stability or amphiphilicity depends, in part, on the size of the amino acid side chain and position of the residue within the ⁇ -sheet.
• the ⁇ -sheet region of the peptides of formula I will contain mixtures of L- and D-amino acids that do not significantly affect the stability or amphiphilicity of the ⁇ -sheet region as compared to peptides containing the corresponding all D- or all
• hydrophobic, basic, polar/large and cysteine-like amino acids comprising the ⁇ -sheet region are either all L-enantiomers or all
• Small amino acids comprising the ⁇ -sheet region may be either L-enantiomers or D-enantiomers.
• the reverse-turn region of the peptides of formula I links the strands of the anti-parallel ⁇ -sheet.
• the reverse-turn region comprises a structure that reverses the direction of the polypeptide chain so as to allow a region of the peptide to adopt an anti-parallel ⁇ -sheet secondary structure.
• the reverse-turn region of the molecule generally comprises two, three or four amino acid residues (residue X 9 and/or X 12 may be absent).
• An important feature of the illustrative protegrin peptides described herein is the presence of a positive charge in the turn region of the molecule.
• one of X 9 -X 12 preferably two of X 9 -X 12 , must be a basic amino acid.
• Such two, three and four amino acid segments capable of effecting a turn in a peptide are well known and will be apparent to those of skill in the art.
• the reverse-turn is a three amino acid residue ⁇ -turn.
• Virtually any ⁇ -turn sequence known in the art may be used in the peptides described herein, including those described, for example, in 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. 203:221-232; Sibanda et al . , 1989, J. Mol. Biol 206:759-777; and Tramontano et al . , 1989, Proteins: Struct. Funct. Genet. 6:382-394.
• the reverse-turn is a four amino acid residue ⁇ -turn.
• the two internal amino acid residues of the turn are usually not involved in the hydrogen-bonding of the anti-parallel ⁇ -sheet; the two amino acid residues on either side of the internal residues are usually included in the hydrogen-bonding of the ⁇ -sheet. While not intending to be bound by theory, it is believed that such hydrogen bonding helps stabilize the ⁇ -sheet region of the molecule.
• conformations and sequences of many peptide ⁇ -turns have been well-described in the art and include, by way of example and not limitation, type-I, type-I', type-II, type-II', type-III, type-III', type-IV, type-V, type-V, type-VIa, type-VIb, type-VII and type-VIII (see, Richardson, 1981, Adv. Protein Chem. 34: 167-339; Rose et al., 1985, Adv. Protein Chem. 37:1-109; Wilmot et al . , 1988, J. Mol. Biol.
• peptide ⁇ -turn structures and sequences are specifically contemplated by the invention.
• the specific conformations of short peptide turns such as ⁇ -turns depend primarily on the positions of certain amino acid residues in the turn (usually Gly, Asn or Pro).
• the type-I ⁇ -turn is compatible with any amino acid residue at positions X 9 through X 12 , except that Pro cannot occur at position X 11 .
• Asp, Asn, Ser and Cys residues frequently occur at position X 9 , where their side chains often hydrogen-bond to the NH of residue X 11 .
• type-II turns Gly and Asn occur most frequently at position X 11 , as they adopt the required backbone angles most easily.
• type-I' turns have Gly at positions X 10 and X 11
• type-II' turns have Gly at position X 10 .
• Type-III turns generally can have most amino acid residues, but type-III' turns usually require Gly at positions X 10 and X 11 .
• Type-VIa and VIb turns generally have a cis peptide bond and Pro as an internal residue.
• Preferred ⁇ -turn sequences include those wherein X 9 is a basic amino acid (preferably R, K, Orn or Dab) or a
• hydrophobic amino acid preferably W, F, Y or Cha
• X 10 is a basic amino acid (preferably R), a small amino acid
• X 11 is a basic amino acid
• X 12 is a hydrophobic amino acid (preferably W, F, Y, I or Cha).
• protegrin peptides useful is the methods of the invention are generally basic, i.e., they have a net positive charge at physiological pH. While not intending to be bound by theory, it is believed that the presence of positively charged amino acid residues, particularly in the turn region of the molecule, is important for antimicrobial activity.
• the amino acids must be basic amino acids, and the compounds must have a net charge of at least +1 at physiological pH.
• the illustrative peptides will have a net charge of at least +3 at physiological pH.
• the amino terminus of the illustrative peptides may be in the free amino form or may be acylated by a group of the formula RCO-, wherein R represents a hydrocarbyl group of 1-25C, preferably 1-10C, more preferably 1-8C.
• hydrocarbyl group can be saturated or unsaturated, straight chain, branched or cyclic, and is typically, for example, methyl, ethyl, isopropyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, octyl, decyl, eicanosyl and the like.
• amino terminus may be substituted with aromatic groups such as naphthyl, etc.
• aromatic groups such as naphthyl, etc.
• Such peptides can be conveniently prepared by incorporating appropriate amino acids, such as 1-naphthylalanine and 2-naphthylalanine at the N-terminus of the peptide.
• amino terminus of the peptides may also be N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• the peptides can be conveniently modified at the N-terminus with catechol using catechol-NHS activated ester.
• the C-terminus of peptides useful in the methods described herein may be in the form of the 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.
• the carboxyl terminus may also be derivatized by formation of an ester with an alcohol of the formula ROH, or may be amidated by an amine of the formula NH 3 , or RNH 2 , or R 2 NH, wherein each R is independently hydrocarbyl of 1-25C as defined and with preferred embodiments as above.
• Amidated forms of the peptides wherein the C-terminus has the formula CONH 2 are preferred.
• each of C 8 and C 13 is independently present or not present, and if present each is independently a cysteine-like, basic, small, polar/large or hydrophobic;
• each of C 6 and C 15 is independently a cysteine-like, basic, small, polar/large or hydrophobic amino acid
• each of X 1 -X 5 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid;
• each of X 7 and X 14 is independently a hydrophobic or a small amino acid
• each of X 9 and X 12 is independently present or not present;
• X 9 -X 12 taken together are capable of effecting a reverse turn when contained in the amino acid sequence of formula (I) and at least one of X 9 -X 12 must be a basic amino acid;
• each of X 16 -X 18 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large or small amino acid; and wherein at least about 15% up to about 50% of the amino acids comprising said antimicrobial peptide are basic amino acids such that said antimicrobial peptide has a net charge of at least +1 at physiological pH.
• the illustrative peptides have the formula:
• X 1 is either present or absent, and if present is a basic amino acid
• X 2 is either present or absent, and if present is a small, basic or hydrophobic amino acid
• X 3 is either present or absent, and if present is a small or hydrophobic amino acid
• X 4 is either present or absent, and if present is a small, basic or hydrophobic amino acid
• X 5 is a small, basic or hydrophobic amino acid
• C 6 is a cysteine-like amino acid
• X 7 is a small or hydrophobic amino acid
• C 8 is a cysteine-like, small, basic or hydrophobic amino acid
• X 9 is a basic or hydrophobic amino acid
• X 10 is a small or basic amino acid or proline
• X 11 is a basic or hydrophobic amino acid
• X 12 is a hydrophobic amino acid
• C 13 is a cysteine-like, small, basic or hydrophobic amino acid
• X 14 is a small or hydrophobic amino acid
• C 15 is a cysteine-like amino acid
• X 16 is either present or absent, and if present is a hydrophobic amino acid
• X 17 is either present or absent, and if present is a small amino acid
• X 18 is either present or absent, and if present is a basic amino acid.
• Particularly preferred peptides are those wherein X 1 is R; X 2 is absent or R, G or L; X 3 is absent or G, L, W or Cha; X 4 is absent or R, G or W; X 5 is R, G, A, L, V, W or Cha; C 6 is C; X 7 is A, Y, F or Cha; C 8 is C, K, A or T; X 9 is R, F, W, Y or L; X 10 is R, G, MeGly or P; X 11 is R, W, F or Cha; X 12 is F, I, Y, W or Cha; C 13 is C, K, A or T; X 14 is G, A, V or F; X 15 is C; X 16 is absent or V, F; X 17 is absent or G; and X 18 is absent or R.
• the peptide of formula (I) is selected from
• Certain peptides useful in the methods of the invention can be isolated from procine leukocytes as described in U.S. Patent No.
• C-terminus can be derivatized, again using conventional chemical techniques.
• the compounds of the 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, as previously described.
• the carboxyl terminus may also be esterified using alcohols of the formula ROH wherein R is hydrocarbyl (1-6C) as defined above.
• carboxyl 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.
• the sulfhydryl groups of cysteine-like amino acids can be stabilized by reacting with alkylating agents using well-known methods.
• 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 of the invention may itself be synthesized using commercially available equipment; codon choice can be integrated into the synthesis depending on the nature of the host.
• Recombinantly produced forms of the protegrins may require subsequent derivatization to modify the N- and/or C-terminus and, depending on the isolation procedure, to effect the formation of disulfide bonds as described
• the DNA encoding the protegrins of the 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.
• Types of host cells available span almost the entire range of the plant and animal kingdoms.
• the protegrins of the 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-à-vis these infective agents.
• the methods of the invention generally involve topically applying to the oral cavity of the subject being treated an amount of antimicrobial protegrin peptide effective to treat or prevent oral mucositis.
• therapeutically effective dose refers to that amount of protegrin peptide sufficient to result in amelioration of symptoms associated with oral mucositis and/or in a reduction in the mouth of the subject of the number colony forming units ("CFUs") of flora associated with oral mucositis as compared to the number of CFUs observed prior to treatment.
• CFUs colony forming units
• a reduction of CFUs on the order of 3-4 log is considered to be therapeutically effective, however, even reductions on the order of 1-2 log may provide significant amelioration of symptoms, and hence therapeutic benefit.
• protegrins are particularly effective when used prophylactically.
• a therapeutically effective dose also refers to an amount of protegrin peptide sufficient to prevent the onset of oral mucositis. Accordingly, the prophylactic use of protegrin peptides in patients at risk for developing oral mucositis, such as those receiving chemotherapy or radiotherapy, is an important aspect of the invention.
• a therapeutically effective dose can be estimated initially from in vitro tests such as, for example, MICs and saliva kill kinetics. Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art.
• protegrin peptides will be most important.
• treatment will begin when a patient is considered to be at high risk for
• the protegrin peptides may be applied when the patient begins to feel oral inflammation, or even after lesions have appeared.
• protegrins will typically be administered in the form of a topical oral formulation. Such formulations will generally comprise about 0.001% (w/w) to 2.5% (w/w) active ingredient; however, concentration ranges such as 0.005% (w/w) to 0.75% (w/w) or even 0.03% (w/w) to 0.3% (w/w) are expected to be effective.
• the protegrins may be applied topically several times per day, depending in part on the concentration of the applied dose and the frequency of food and fluid intake by the patient. Thus, depending on the particular topic
• protegrins may be applied 2, 3, 4 or even as many as 6 times per day.
• application may enhance the effectiveness of treatment.
• the protegrin treatment will be administered for about 1-4 weeks, but treatment regimens as short as 3-4 days may also provide prophylactic or therapeutic benefit. In some instances, it may be desirable to treat the patient for the entire period during which the patient receives
• the actual amount of antimicrobial peptide administered as well as the dosing schedule of peptide administered will, of course, depend on factors such as the age of the patient, the severity of the affliction, the aggressiveness of chemotherapy or radiotherapy being pursued, and, of course, on the judgement of the prescribing physician.
• protegrins can be administered singly or in
• protegrins or antimicrobial peptides or other agents including, for example, painkillers (lidocaine, etc.) or anti-inflammatories.
• the peptides are applied to the oral cavity in the form of a topical pharmaceutical formulation.
• Formulations suitable for topical oral application include oral emulsions, magmas, gels, swishes, lozenges, pastes, creams, oral solutions, gums, etc., as are well known in the art. Any of these topical oral vehicles can be used in conjunction with the methods of the invention. Exact formulations, as well as methods of their preparation, will be apparent to those of skill in the art (see, e.g., Ansel et al . , 1995, Pharmaceutical Dosage Forms and Drug Delivery, Williams & Wilkins, Malvern, PA; Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA).
• the peptides are administered in a topical gel-like formulation comprising about 0.001% (w/w) to 2.5% (w/w), preferably about 0.005% (w/w) to 0.75% (w/w), more preferably about 0.03% (w/w) to 0.3% (w/w) and most preferably about 0.025% (w/w) to 0.15% (w/w) active peptides (s) in admixture with a gel-like vehicle.
• the gel-like vehicle generally comprises a water-soluble gelling agent, a humectant and water, and has a viscosity of about 500 to 100,000 cps, preferably about
• the gelling agent provides the formulation with good mucoadhesion properties; the humectant with good moisturizing and
• Gelling agents suitable for use with the vehicle of the invention include, e.g., agar, bentonite, carbomer (e.g., carbopol), water soluble cellulosic polymers (e.g.,
• carboxyalkyl cellulose hydroxyalkyl cellulose, alkyl cellulose, hydroxyalkyl alkylcellulose), povidone, kaolin, tragacanth and veegum, with hydroxylalkyl alkyl celluloses such as hydroxypropyl methylcellulose being preferred.
• Humectants suitable for use with the gel-like vehicle of the invention include, e.g., glycerin, propylene glycol and sorbitol, with sorbitol being preferred.
• the vehicle comprises about 0.1% (w/w) to 10% (w/w) water-soluble gelling agent, with about 0.25% (w/w) to 5% (w/w) being preferred and about 0.5% (w/w) to 3% (w/w) being most preferred and about 0.1% (w/w) to 20% (w/w) humectant.
• concentration ranges are for guidance only.
• concentration of gelling agent will depend, in part, on the polymer selected, the supplier and the specific lot number.
• the actual concentrations of other ingredients will likeise affect the viscosity of the gel-like formulation. Choosing appropriate concentrations to yield a gel-like formulation with the desirable viscosity and other properties described herein is within the capabilities of ordinarily skilled artisans.
• the gel-like vehicle of the invention may include antimicrobial preservatives.
• Antimicrobial preservatives Antimicrobial
• preservatives useful with the compositions of the invention include, but are not limited to, antifungal preservatives such as benzoic acid, alkylparabens, sodium benzoate and sodium propionate; and antimicrobial preservatives such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal, with antifungal preservatives such as benzoic acid, alkylparabens, sodium benzoate and sodium propionate; and antimicrobial preservatives such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal, with
• alkylparabens such as methylparaben, propylparaben and mixtures thereof being preferred.
• antimicrobial preservative (s) effective for use with the formulations of the invention will be apparent to those of skill in the art and will depend, in part, on the antimicrobial agent (s) used. Typical concentrations range from about 0.01% (w/w) to about 2% (w/w).
• composition of the invention may also contain from about 1% (w/w) to 10% (w/w) of a sweetening agent such as aspartame, dextrose, glycerin, malitol, mannitol, saccharin sodium, sorbitol, sucrose and xylitol.
• a sweetening agent such as aspartame, dextrose, glycerin, malitol, mannitol, saccharin sodium, sorbitol, sucrose and xylitol.
• sweetening agents are believed to aid patient compliance.
• the pH of the composition will depend on the active ingredient (s) contained in the composition.
• compositions of the invention include, but are not limited to, acidifying agents such as acetic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid and nitric acid; alkalinizing agents such as ammonia solution, ammonium carbonate, diethanolamine,
• This Example describes preferred methods of synthesizing peptides of the invention having a C-terminal amide.
• concentration of peptide was about 1-8 mg/mL, the pH ranged from 7.0-7.2 and the DMSO concentration ranged from about 5- 20%.
• the solution was stirred overnight at room temperature, and the pH of the solution was adjusted to pH 5 with
• the oxidized peptide was loaded onto a preparative reverse-phase HPLC column (Vydac C18, 2.2cm ⁇ 25cm, Cat. No. 218TP101522), the column was washed with buffer (10% v/v acetonitrile, 0.1% v/v TFA in water) until absorbance of the effluent (measured at 235 cm) reached baseline and the pure product was eluted at 10 mL/min. using the following buffers and gradient:
• This Example provides preferred methods for preparing the preferred topical formulations of the invention.
• Methylparaben and propylparaben were dissolved in hot aqueous lactate buffer solution (pH 4.2). Hydroxypropyl methylcellulose (HPMC) was dispersed in the hot solution followed by addition of sorbitol, xylitol and aqueous OM-3. The mixture was cooled to room temperature, becoming viscous with cooling.
• HPMC Hydroxypropyl methylcellulose
• Staphylococcus aureus (ATCC 33591) were obtained from the American Type Culture Collection, Rockville, MD. P.
• mirabilis was an isolated strain obtained from the cheek pouch of a hamster.
• Microorganisms from other sources such as, for example, clinical isolates, can be used interchangeably with the above-described microorganisms in the assays described herein.
• TSA Trypticase Sov Agar
• Cockeysville, MD, BBL #4311768) dissolve 30 g in 1 Liter deionized water, autoclave 121°C, 20 minutes, and store at room temperature.
• 2X Trypticase Soy Broth (2X TSB) : dissolve 60 g in 1 Liter deionized water, autoclave 121°C, 20 minutes, and store at room temperature.
• Glycerol (20% v/v): mix 20 mL glycerol with 80 mL deionized water, Filter sterilize with 0.20 ⁇ filter and store at room temperature.
• Monobasic phosphate buffer 100 mM: dissolve 13.7 g sodium phosphate monobasic (Fisher #S368-500) in 1 Liter deionized water. Filter sterilize with 0.20 ⁇ filter and store at room temperature.
• Dibasic phosphate buffer 100 mM: dissolve 14.2 g sodium phosphate dibasic (Fisher #S374-500) in 1 Liter deionized water. Filter sterilize with 0.45 ⁇ filter and store at room temperature.
• PBS Phosphate-buffered saline
• Phosphate buffer (100 mM, pH 6.5): mix 40 mL dibasic phosphate buffer (100 mM) with 160 mL monobasic phosphate buffer (100 mM). Adjust pH if necessary, filter sterilize with 0.45 ⁇ filter and store at room temperature.
• Liguid Testing Medium aseptically combine the following sterile ingredients: 10 mL Phosphate buffer (100 mM, pH 6.5), 1.0 mL TSB, 2 mL NaCl (5 M) and 87 mL deionized water. Store at room temperature.
• Acetic acid (0.01% v/v): mix 10 ⁇ L acetic acid with 100 mL sterile deionized water.
• Agarose mix 1 g agarose (Sigma #S6013) in 80 mL deionized water, autoclave 121°C, 20 minutes.
• Agarose Underlay Medium combine 10 mL Phosphate buffer (100 mM, pH 6.5), 1.0 mL TSB, 2 mL NaCl (5 M) and 7 mL deionized water with 80 mL tempered (50°C) agarose.
• 2X TSB Agarose Overlay Medium dissolve 60 g TSB and 10 g agarose in 1 Liter deionized water, aliquot 100 mL per bottle, autoclave 121°C, 20 minutes, and store at room temperature.
• Preparation of Microorganism Slants Each strain was cultured on TSA. Isolated colonies were transferred into TSB (10 mL in a sterile 50 mL Erlenmeyer flask) using a sterile, disposable loop and the flask incubated at 37°C (bacteria) or 30°C (yeast) with shaking (200 RPM) for 16-18 hours.
• the MCZ assay uses minimal amounts of test materials to determine the sensitivity of microorganisms to various antimicrobial compounds. Cells are grown to
• microbroth dilution method accommodates large numbers of samples and is more amenable to automation than the MCZ assay and the data analysis is direct and simple.
• a key step in this assay is combining microorganisms and peptide in a defined minimal nutrient buffer system that minimizes interference with the peptide's biological
• HSA human serum albumin
• HSA Human Serum Albumin
• MRSA turbidity (cell growth). Often, MRSA will settle out and form a pellet at the bottom of the well. MRSA can be evaluated by placing the microtiter plate on a stand and examining the bottom of the well using a tilted mirror.
• the minimum concentration for inhibition of growth in broth medium is defined as the lowest concentration of peptide that inhibits all visible growth. If the MCB values for each of the triplicate samples differ, the MCB is obtained by averaging the results of the three samples.
• the minimum concentration of peptide showing 100% biocidal activity is determined by incubating a 10 ⁇ L aliquot from each well on a TSA plate for 24 hours at 37°C (bacteria) or 30°C (yeast) (for plating, 1.5 mL TSA in each well of a 24-well plate minimizes cross contamination).
• NCLS Mueller-Hinton Broth
• protegrin peptides precipitate in MHB at concentrations greater than 128 ⁇ g/mL.
• the following modified NCCLS assay is the preferred method for determining MICs of protegrin peptides.
• precipitation is avoided by preparing concentrated (10X) stock solutions of test peptide in a buffer that is suitable for the peptide and which does not exhibit deleterious effects on the
• microorganisms 0.01% v/v acetic acid, 0.1% w/v HSA
• HSA Human Serum Albumin
• BSA bovine serum albumin
• MRSA turbidity (cell growth). Often, MRSA will settle out and form a pellet at the bottom of the well. MRSA can be evaluated by placing the microtiter plate on a stand and examining the bottom of the well using a tilted mirror.
• the minimum inhibitory concentration (MIC) is defined as the lowest peptide concentration that inhibits all visible growth. If the MIC values for each of the triplicate samples differ, the MIC is obtained by averaging the results of the three samples.
• the minimum concentration of peptide showing 100% biocidal activity is determined by incubating a 10 ⁇ L aliquot from each well on a TSA plate for 24 hours at 37°C (bacteria) or 30°C (yeast) (for plating, 1.5 mL TSA in each well of a 24-well plate minimizes cross contamination).
• the following assay is used to determine the rate at which a protegrin peptide kills a target microorganism, as well as to determine if a particular peptide is bactericidal or bacteriostatic.
• the peptide In order to assess an effect using this assay, the peptide must reduce the CFUs by at least one log (i.e., at least 800 CFUs per plate). Although such numbers are higher than recommended for accuracy (30-300 CFUs/plate), log-order changes in recoverable CFUs indicate significant bacteriocidal efficacy.
• PG-l exhibits broad spectrum anti-microbial activity, being extremely effective against L . monocytogenes, strain EGD, C. alibicans, S . aureus , K .
• tuberculosis tuberculosis . Antimicrobial activity is observed in the presence of 90% fetal calf serum.
• This Example demonstrates the antimicrobial activity of the native form of preferred peptide OM-3 against pathogen associated with oral mucositis. Although more than 200 species of microorganisms have been isolated from the oropharynx, individual surfaces of the oral cavity are dominated by specific subgroups (Liljemark et al . , 1994, In : Oral Microbiology and Immunology, pp. 120-128, Nisengard and Newman, Eds., Saunders Co., Philadelphia). In the saliva and buccal mucosa, alpha and non-hemolytic streptococci are most prevalent (Loeshe, 1994, In : Oral Microbiology and
• S. salivarius a representative species of this group, was tested in the radial diffusion assay described above (except that fetal calf serum was added to the overlay at 10%) to determine susceptibility to peptide OM-3.
• Gram-positive bacteria are the most common flora in the oral cavity, lower levels of Gram-negative bacteria and fungi are also present.
• MICs methicillin sensitive and resistant Staphylococcus aureus (MSSA, MRSA, respectively), vancomycin resistant E . faecium (VREF), P. aeruginosa,
• MSSA methicillin sensitive and resistant Staphylococcus aureus
• VREF vancomycin resistant E . faecium
• P. aeruginosa P. aeruginosa
• the MICs obtained for OM-3 are provided in Tables 4 and 5, below.
• the MIC values represent the average of three determinations of the concentration in the first well with no growth.
• the data represents the average of three determinations for each individual strain tested.
• the MIC values obtained for each organism were equal to the minimum bactericidal concentration (MBC) observed for the same organism (data not shown).
• This Example demonstrates the ability of preferred peptide OM-3 to decrease CFUs of bacteria found in pooled normal human saliva.
• Peptide OM-3 (20 mM sodium acetate, pH 5) or placebo vehicle was mixed 1:1 with saliva. At 1, 2, 4, 8 and 16 minutes after mixing, aliquots were plated onto Trypticase Soy Agar containing 10% fetal bovine serum, and the plates were incubated overnight at 37°C. 6.2 Results
• This Example demonstrates the effect of saliva on the antimicrobial activity of a variety of protegrin peptides, as measures by radial diffusion and reduction in CFUs.
• the media in the underlay agar contained phosphate buffer at 10 mM, pH6.5, 100 mM NaCl, 1% TSB, 1% agarose.
• the media in the overlay contain 10 mM phosphate buffer, pH 6.5, 100 mM NaCl, 2XTSB, 1% agarose.
• the peptides were diluted from 10X stock made up in 0.01% acetic acid either with 10 mM acetate buffer (pH 5) or with saliva.
• the initial inoculum contained approximately 4 ⁇ 10 7 CFUs/mL saliva.
• Peptides (320 ⁇ g/mL) were dissolved in 0.01% acetic acid and added as 1/10 volume to saliva. 7.2 Results
• the results are given as the minimal concentration required to produce a detectable zone of clearance, or MCZ - - i.e., an extrapolated value to the x-axis when the concentration of peptide is plotted against the diameter of the zone.
• the results of the radial diffuion assay are shown in Table 6; the reduction in CFUs in Table 7.
• a large number of the peptides tested showed comparable or even improved activity in the presence of saliva. Many of the peptides tested exhibited greater than a two-log reduction in oral CFUs, even at a low
• This Example demonstrates the efficacy of preferred peptide OM-3 in reducing the CFUs of natural oral microflora in the cheek pouches of hamsters.
• Peptide OM-3 (1 mg/ml or 5 mg/ml in an aqueous formulation containing hydroxypropylmethylcellulose) was delivered to one cheek pouch of hamsters in a volume of 0.25 ml three times per day for 4 days.
• the cheek pouches were swabbed approximately 4 or 15 hours after treatment.
• the swabs were placed in culture tubes containing lmL of 0.87% (w/v) NaCl and 0.1% (w/v) Tween-80 and refrigerated. All culture tubes were mixed virorously within 24 hours of sample collection. After mixing, a 0.1 mL aliquot of undiluted sample and 0.1 mL aliquots of two 100-fold serial dilutions were spread onto standard blood agar plates. Following incubation at 37°C for at least 24 hours, the number of CFUs per plate was determined.
• open squares represent treatment with 1 mg/mL peptide; filled squares treatment with 5 mg/mL.
• the arrows on the X-axis labelled "Rx" indicate the times at which the peptide was applied.
• the number of CFUs in untreated hamsters is generally between 10 6 and 10 7 per swab.
• a 1,000 to 10,000-fold (3-4 log) reduction in oral CFUs was consistently present 4 hours after OM-3 was applied.
• An expected regrowth of oral flora occurred by 15 hours after treatment. Comparable results were obtained with PG-1.
• hamsters were subsequently randomized into two groups, and beginning on day 6, were treated by direct application of 0.5 ml placebo vehicle (2% methocel K4M, 10% propylene glycol, 10% glycerol, 10mM acetate buffer) or PG-1
• FIGS. 3 and 4 oral mucositis scores are presented in FIG. 3; percent change in body weight in FIG. 4.
• open squares designate treatment with placebo vehicle; closed squares treatment with PG-1.
• This Example demonstrates the ability of OM-3 to reduce the severity of oral mucositis in the hamster pre-clinical model.
• test formulation placebo HPMC gel (group 1; vehicle
• FIG. 5 is a graphical representation of the progression of oral mucositis in each of the four treatment groups
• FIG. 6 is a graphical representation of the change in body weight of each of the four treatment groups.
• triangles represent treatment with placebo vehicle; squares with chlorhexidine, diamonds with
• mucositis scores were significantly lower (p ⁇ 0.05) in hamsters treated with OM-3 when compared to the vehicle-treated controls. There was no apparent difference in averaged scores between animals given 0.25 or 1.0 mg of OM-3 per application (0.5 and 2.0 mg/ml, respectively). Among hamsters administered chlorhexidine, mucositis scores tended to be lower than in the vehicle controls on days 11, 12 and 13; however, none of the
• This Example demonstrates the dose-response of OM-3 (SEQ ID NO:3) treatment in the hamster pre-clinical oral mucositis model.

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