WO1998041224A1

WO1998041224A1 - Use of protegrins for periodontal indications

Info

Publication number: WO1998041224A1
Application number: PCT/US1998/005362
Authority: WO
Inventors: Robert I. Lehrer; Kenneth T. Miyasaki
Original assignee: The Regents Of The University Of California
Priority date: 1997-03-20
Filing date: 1998-03-19
Publication date: 1998-09-24
Also published as: AU6567098A

Abstract

The invention is directed to a method to treat periodontal disease which method comprises administering to a subject afflicted with such disease an amount of a protegrin effective to treat said disease; wherein said protegrin contains the amino acid sequence (1): A1-A2-A3-A4-A5-C6-A7-C8-A9-A10-A11-A12-C13-A14-C15-A16-A17-A18, wherein said protegrin contains 10-30 amino acid residues, wherein the amino acid sequence of formula (1) may be extended at the N and/or C-terminus by additional noninterferring amino acids; and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, said protegrin either in the optionally -SH stabilized linear or in a disulfide-bridged form wherein each of C6, C8, C13 and C15 is independently a cysteine, homocysteine, or penicillamine, or wherein one or more of C6, C8, C13 and C15 is independently replaced by a basic, hydrophobic, large/polar or small amino acid or wherein C8 and/or C13 is not present; each of A1-A5 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid; each of A7 and A14 is independently a hydrophobic or a small amino acid; A9-A12 are capable of effecting a β-turn when contained in the compound of formula (1) and at least one of A9-A12 must be a basic amino acid and wherein A9 and/or A12 may be present or not present; each of A16-A18 is independently present or not present, and if each present each is independently a basic, hydrophobic, polar/large or small amino acid; wherein in said protegrin at least about 15 % to about 50 % of the amino acids are basic amino acids, and wherein the protegrin compound has a net positive charge of at least +1 at physiological pH.

Description

USE OF PROTEGRINS FOR PERIODONTAL INDICATIONS

This invention was made with funding from U.S.-PHS Grant No. DE08161 from NIH-NIDR and from a U.S.-PHS Grant No. A122839 from NIH-NIAID. The U.S. Government has certain rights in this invention.

Technical Field

The invention relates to the field of treating periodontal pathogens. In particular, the invention concerns the use of protegrins in the context of such conditions .

Background Art One of the defense mechanisms against infection by both animals and plants is the production of 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 of the literature on this subject. Such peptides include tachyplesins, which are 17-18 amino acid peptides containing four invariant cysteines, the defensins, β-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.

The antimicrobial peptides used in the present invention are members of a class designated "protegrins". Representative members of protegrins have been isolated from porcine leukocytes; however, the isolated peptides are simply exemplary members of a class of peptides that are effective against periodontal disease.

The isolation of certain protegrin peptides and related peptides or of DNA encoding them has been reported by Kokryakov, V.N. et al . FEBS Lett (1993) 337:231-236; Zhao, C. et al . , FEBS Lett (1994) 346:285- 288; Mirgorodskaya, O.A. et al . FEBS Lett (1993) 330:339- 342; Storici, P. et al . Biochem Biophys Res Comm (1993) 196:1363-1367; Harwig, S.S.L., et al . J Peptide Sci

(1995) 3:207; Zhao, C, et al . FEBS Lett (1995) 376:130- 134; Zhao, C. et al . FEBS Lett (1995) 368:197-202; Miyakawa, Y. et al . Infect Immun (1996) 64:926-932; Yasin, B. et al . Infect Immun (1996) 64:709-713; Qu, X-D et al . Infect Immun (1996) 64:1240-1245; Aumelas, A. et al . Eur J. Biochem (1996) 237:575-583; angoni, M.E. et al . FEBS Lett (1996) 383:93-98; and a paper from the Eighth International Symposium on Staph Infections, Aix les Bains, France, June 23-26, 1996, Steinberg, D.A. et al . , entitled "Protegrins: Fast Acting Bactericidal

Peptides." The general protegrin group of peptides is also disclosed in U.S. Serial No. 08/499,523 and in U.S. Serial No. 08/690,921, the contents of which are incorporated herein by reference in their entirety. The peptide class designated "protegrins" has a wide range of antimicrobial activity and varies in activity spectrum by member of class.

The protegrins have 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. 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).

The present invention is directed to the use of the protegrins to treat periodontal conditions. Specifically, the protegrins have been found effective in bactericidal or bacteriastatic activity against Gram negative, facultative periodontal pathogens.

Disclosure of the Invention

The present invention is directed to treatment periodontal disease. Specifically, the protegrins alone or in combination are effective in ameliorating the symptomology caused by infection with Actinobacillus act inomycetemcomi tans and Capnocytophaga Spp.

Accordingly, in one aspect, the invention is directed to a method to treat periodontal conditions which method comprises administering to a subject afflicted with periodontal disease an amount of a compound that includes the amino acid sequence of formula 1, alone or in combination with other medicaments, in an amount effective to ameliorate or otherwise treat said periodontal condition. The protegrins of the invention contain an amino acid sequence of the formula

A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈- ₉-Aιo- ₁₁- ₁₂-Cι₃- i₄-C₁₅-A₁₆- ₁₇-Aι₈ (1)

and contains 10-30 amino acid residues. The sequence shown as (1) can be extended at the N and/or C-terminus with non-interferring amino acids or peptide sequence. Also included are the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, and the protegrins may be either in the optionally -SH stabilized linear or in a disulfide-bridged form.

Each of Cβ, Cs, C₁₃ and C₁₅ shown in amino acid sequence (1) is independently a cysteine, homocysteine, or penicillamine; or alternatively, one or more of C₆, C₈, C₁₃ and C15 is independently replaced by a basic, hydrophobic, large/polar or small amino acid; or C₈ and/or Cχ₃ is not present; each of A1-A₅ is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid; each of A₇ and A₁₄ is independently a hydrophobic or a small amino acid;

A₉-Aχ₂ are capable of effecting a β-turn when contained in the protegrin and at least one of A₉-Aι₂ must be a basic amino acid, optionally A₉ and/or Aj_₂ may not be present; each of A1₆-A1₈ is independently present or not present, and if each present each is independently a basic, hydrophobic, polar/large or small amino acid. In all the compounds containing the sequence of formula (1) at least about 15% and no more than about 50% of the amino acids must be basic amino acids, and the compounds must have a net charge of at least +1 at physiological pH. The invention is also related to periodonital compostions containings the protegrins.

Brief Description of the Drawings

Figure 1 shows the structures of PG-1, PG -2, PG-3, PG-4 and PG-5.

Figure 2A, 2B and 2C are graphic representations of the activity of various concentrations of PG-1 versus Gram-negative bacteria important in periodontal disease. Figure 3A-3F are graphic representations of the activity of various protegrins against Gram-negative bacteria causative of periodontal disease.

Figures 4A-4C are graphical representations of the effect of serum on the bactericidal activity of PG-1 against periodontal microorganisms.

Figure 5 is the graphic representation of bactericidal activity of PG-1 as affected by salt concentration .

Modes of Carrying Out the Invention The protegrins useful in the invention include the amino acid sequence:

A₁-A2-A₃-A4-A₅-C₆-A₇-C₈-A₉-Aιo-A₁₁-A₁₂-Cι₃-A₁4-Ci5-Aι₆-A₁₇-Ai8 (1)

and its defined modified forms. The designation A_n in each case represents an amino acid at the specified position in the peptide. As defined, Aι-A₅, C₈, A₉, Aι₂, Cι₃, i6, ι₇ and/or ι₈ may or may not be present.

However, the peptides of the invention contain 10-30 amino acids. Thus, the amino acid sequence shown as formula (1) may contain extensions at the N and/or C-terminus of additional amino acids or peptide sequence. The positions of the cysteine, homocysteine or penicillamine residues, shown as C in the formula, are invariant in one embodiment of the peptides of the invention; however, in the modified forms, also included within the scope of the invention, one or more of these cysteine, homo-cysteine or penicillamines may be replaced by a small, basic or hydrophobic amino acid. All of the protegrins useful in the invention, however, have a net positive charge; approximately 15% but no more than about 50% of the amino acids must be basic amino acids, and the compounds must have a net charge of at least +1 at physiological pH. For embodiments having as few as 10 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 protegrin contains as many as 15 amino acid residues, two basic residues are required. It is preferred that at least 20% of the amino acids in the sequence be basic, more preferably 30%.

The active protegrins are also preferably contain a β turn bracketed by two strands that form a β sheet.

While not intending to be bound by any theory, applicants believe that antimicrobial activity of the compounds of formula (1) is associated with such a β-turn bracketed by two strands that form a β sheet structure. The amino acids A₉-Aχ₂ must be capable of effecting a β-turn, which can be encouraged by hydrogen bonding between A₉ and Aι₂. The presence of proline at Aio and/or n does not interfere with the β-turn stabilized by the presence of a hydrophobic amino acid at positions A₉ or A_i2. As used herein, "β-turn" refers to a recognized subclass of reverse-turns. Typically, a "β-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 β-sheet secondary structure. Generally, the two internal amino acid residues of the β-turn are not involved in the hydrogen-bonding of the β-sheet; the two amino acid residues on either side of the internal residues are included in the hydrogen-bonding of the β-sheet. The term "β-turn" expressly includes all types of peptide β- turns commonly known in the art including, but not limited to, type-I, type-II, type-Ill, type-I', type-II' , and type-Ill' β-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 of the four invariant cysteine, homo- cysteine or penicillamine residues is helpful in effecting the β-sheet structure; however, by properly choosing the substitutions, one or more of the cysteine, homocysteine or penicillamine residues can be replaced without substantially disturbing the three-dimensional shape of the molecule.

The β sheets are believed to be effected by C₆-A₇-C₈ and Cι₃- ι₄-Ci5. Thus, in the unmodified forms of the compound, A₇ and Aχ₄ are preferably hydrophobic amino acids. The cysteine, homocysteine or penicillamine residues may also, then, be replaced by other residues which do not affect the maintenance of the β sheet formation; these substitutions would include basic, hydrophobic or small amino acids.

The amino terminus of the protegrin 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-lOC, more preferably 1-8C. The hydrocarbyl group is saturated or unsaturated, straight chain or cyclic, and is typically, for example, methyl, ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, octyl, decyl, eicanosyl and the like.

The C-terminus of the protegrin 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. In some embodiments, it is difficult to make salts since the remainder of the 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 of the formula ROH, or may be amidated by an amine of the formula NH₃, or RNH₂, or R₂NH, 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 CONH₂ are preferred.

Addition of lipophilic groups at the C- and/or N-terminus facilitates the transition of the peptide into the membrane of the target microbe. Choice of optimum substitution is determined by evaluation with respect to the lipid content of the target microbe.

As the protegrins contain substantial numbers of basic amino acids, the peptides of the invention may be supplied in the form of the 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 protegrins that contain at least two cysteine, homocysteine or penicillamines may be in straight-chain or cyclic form, due to disulfide bond formation. The cyclic forms are the result of the formation of disulfide linkages among all or some of the four invariant cysteine, homocysteine or penicillamine residues. 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 linear compounds can be stabilized by addition of a suitable alkylating agent such as iodoacetamide.

The native forms of the protegrins contain two disulfide bonds are between the cysteine, homocysteine or penicillamine at position 6 and the cysteine, homocysteine or penicillamine at position 15 and the other between the cysteine, homocysteine or penicillamine at position 8 and the cysteine, homocysteine or penicillamine at position 13. Accordingly, in those embodiments having two disulfide linkages, the C₆-Cι₅, C₈- C₁₃ form is preferred. However, 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 C₆-Ci5 alone and by C₈-Cι₃ alone.

Forms containing a C₆-Ci₅ disulfide as the only disulfide linkage are generally designated "bullet" forms of the protegrins; those wherein the sole disulfide is C₈- C₁3 are designated the "kite" forms. The bullet and kite forms can most conveniently be made by replacing the cysteine, homocysteine or penicillamines at the positions not to be involved in a disulfide linkage preferably with a small amino acid such as glycine, serine, alanine or threonine. Alternatively, C₈, C_i3 or both may be absent. As the linearalized or "snake" forms of the 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 protegrins also include linearalized forms which are stabilized with suitable reagents. As defined herein, "SH-stabilized" forms of the peptides of the invention contain sulfhydryl groups reacted with standard reagents to prevent reformation into disulfide linkages. Alternatively the cysteine, homocysteine or penicillamine residues are replaced by small or basic amino acids as set forth above. It is preferred that all 4 cysteine, homocysteine or penicillamine residues be replaced in order to minimize the likelihood of the formation of intermolecular disulfide links.

The amino acids denoted by A_n (and the cysteine, homocysteine or penicillamine residues) may be those encoded by the gene or analogs thereof, and may also be the D-isomers thereof. One preferred embodiment is that form wherein all of the residues are in the

D-configuration thus conferring resistance to protease activity while retaining antimicrobial or antiviral properties. The resulting protegrins are themselves enantiomers of the native L-amino acid-containing forms. In one class of protegrins, either the hydrophobic amino acids found in the native protegrins at A₅ and/or Ai₆ are replaced with a basic amino acid and/or at least one of A₁-A₄ is hydrophobic and/or at least one, and preferably all four of amino acids Ai and A₄ found in the native forms are deleted; and/or one or more of A₅, C₈,

A₉, Aχ₂, C₁₃ and A_i6 is absent. By suitable manipulation of these and other features, the range of conditions under which the class of protegrins of the present invention are effective can be varied. Furthermore, the spectrum of microbes against which they are effective can also be modified. This is further described hereinbelow.

The amino acid notations used herein are conventional and are as follows:

The amino acids not encoded genetically are abbreviated as indicated in the discussion below. In the specific peptides shown in the present application, the L-form of any amino acid residue having an optical isomer is intended unless the D-form is expressly indicated by a dagger superscript (^f) .

The compounds 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. Basic: 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 necessarily seek an inner position in the conformation of the peptide in which it is contained when the peptide is in aqueous medium. Depending on the conditions, and on the remaining amino acids in the sequence, the residue may reside either in the inner space or at the surface of the protein.

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. It is understood, of course, that in a statistical collection of individual residue molecules some molecules will be charged, and some not, and there will be an attraction for or repulsion from an aqueous medium to a greater or lesser extent. To fit the definition of "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, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassification according to the foregoing scheme is as follows .

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, homocysteine or penicillamine residues are also not included in these classifications since their capacity to form disulfide bonds to provide secondary structure is critical in the compounds of the present invention.

Certain commonly encountered amino acids, which are not encoded by the genetic code, include, for example, β-Alanine (β-Ala) , or other omega-amino acids, such as 3-aminopropionic, 2, 3-diaminopropionic (2,3-diaP), 4-aminobutyric and so forth, α-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) ; β-2- thienylalanine (Thi) ; methionine sulfoxide (MSO) ; and homoarginine (Har) . These also fall conveniently into particular categories.

Based on the above definitions,

Sar, β-Ala, and Aib are small; t-BuA, t-BuG, N-Melle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic are hydrophobic;

Orn, 2,3-diaP and Har are basic;

Cit, Acetyl Lys, and MSO are polar/large.

The various omega-amino acids are classified according to size as small (β-Ala and 3-aminopropionic) or as large and hydrophobic (all others) .

Other amino acid substitutions of those encoded in the gene can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme according to their structure.

In all of the peptides of the invention, one or more amide linkages (-CO-NH-) may optionally be replaced with another linkage which is an isostere such as -CH₂NH-, -CH₂S-, -CH₂CH₂, -CH=CH- (cis and trans), -C0CH₂-, -CH(OH)CH₂- and -CH₂SO-. 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, B. Weinstein, eds . , Marcel Dekker, New York, p. 267 (1983) (general review); Morley, J.S., Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. , et al . , Int J Pept Prot Res (1979) 14:177-185 (-CH₂NH-, -CH₂CH₂-); Spatola, A.F., et al . , Life Sci (1986) 38:1243- 1249 (-CH₂-S); Hann, M.M., J Chem Soc Perkin Trans I

(1982) 307-314 (-CH-CH-, cis and trans); Almquist, R.G., et al . , J Med Chem (1980) 23:1392-1398 (-COCH₂-) ;

Jennings-White, C, et al . , Tetrahedron Lett (1982) 23:2533 (-COCH₂-) ; Szelke, M., et al . , European Application EP 45665 (1982) CA:97:39405 (1982) (-CH(OH)CH₂-) ; Holladay, M.W., et al . , Tetrahedron Lett (1983) 24:4401-4404 (-C (OH) CH₂-) ; and Hruby, V.J., Life Sci (1982) UB .- 189-199 (-CH2-S-) .

In addition to analogs which contain isosteres in place of peptide linkages, the peptides or proteins of the 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. The compounds of formula (1) are generally defined as set forth in the Disclosure of the Invention set forth above .

In preferred embodiments, all of the cysteine, homocysteine or penicillamines at positions 6, 8, 13 and 15 are present as are A₉ and A₁₂ .

In addition, or alternatively, each of A₇ and A_i4 is a hydrophobic amino acid, preferably lie, Val, Leu, Trp, Phe, or Tyr. In another set of preferred embodiments, all of Aι-A₄ are not present or at least one, and preferably two of Aι-A₄ is a hydrophobic amino acid, preferably lie, Val, Leu, Trp, Phe or Tyr.

In another set of preferred embodiments, g-A.2 contain at least one hydrophobic amino acid residue, preferably Phe, Tyr or Trp.

Other preferred embodiments include those wherein each of Ai and Ag is independently selected from the group consisting of R, K and Har; more preferably, both Aj_. and A₉ are R; however, each of A_x and Ag may be absent. In another class of preferred embodiments, each of A₂ and A₃ is independently selected from the group consisting of G, A, S and T, or I, V, L, F, Y, or W; more preferably, A₂ and A₃ are G, W, F, Y, L, or V; however, A₂ and/or A₃ may be absent. In another set of preferred embodiments, A₄ is selected from the group consisting of R, K, H, Orn, Har, G, A, S, T, F, Y and W; more preferably, A₄ is R, G or W; however, A₄ may be absent.

In another set of preferred embodiments, each of A₅ and A]_₆ is independently selected from the group consisting of I, V, L, Nle, W, Y, and F, preferably I, V, L, W, F and Y. However, A₅ and/or ι₆ may be absent.

In another set of preferred embodiments, each of A₇ and Aχ is independently selected from the group consisting of I, V, L, W, Y and F, preferably A₇ is I, F, Y or W and A_i4 is I, V, L, W, Y, or F.

In another set of preferred embodiments, one of Ag and A_i2 is R, K, H, Orn or Har, preferably R and the other is I, V, L, NLe, W, Y or F, preferably R, F or W. In another set of preferred embodiments, A_i0 is R, G, W or P.

In another set of preferred embodiments, n is R, G, W or P.

Aχ₇ is preferably absent, but when present, is preferably G, A, S or T; Axe is preferably absent, but when present, is preferably R, K, H, Orn or Har, most preferably R.

Also preferable when all four amino acids Aι-A₄ are present, preferably Aj. and A₄ are basic and A₂ and A₃ are small amino acids, and at least one of Aι-A₄ is a small or hydrophobic amino acid. Preferred embodiments of A₁-A₄ include R-G-G-R, R-G-W-R, R-L-L-R and the like.

As described above, the compounds of formula (1) are either in disulfide or noncyclic (linearalized) form or may be modified wherein one or more cysteine, homocysteine or penicillamines is replaced by a small amino acid residue, a basic amino acid residue or a hydrophobic amino acid residue. If the linearalized forms of the compound of formula (1) are prepared, or if linearalized forms of those modified peptides which contain at least two cysteine, homocysteine or penicillamines are prepared, it is preferred that the sulfhydryl groups be stabilized by addition of a suitable reagent. Preferred embodiments for the basic amino acid to replace cysteine, homocysteine or penicillamine residues are R, K, H and Har, preferably R or K. Preferred small amino acids to replace the cysteine, homocysteine or penicillamine residues include G, A, S and T, most preferably A and T. The compounds of the invention may thus contain either two disulfide bonds, one disulfide bond, or no disulfide bonds. Where two disulfide bonds are present, as described above, those corresponding to the disulfide bonds in naturally occurring protegrins are preferred — i.e., C₆-C₁₅ and C₈-Cι₃. Where only one disulfide bond is present, it is preferred that the protegrin be in the bullet or kite form. This can be assured by replacing at least one noninvolved cysteine, homocysteine or penicillamine with an alternative amino acid as described above. The parent applications herein describe members of the protegrin family which are isolated from porcine leukocytes. Five such protegrins have been found, PG-1 through PG-5 with the following amino acid sequences:

Unmodified forms

PG-1 R-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-V-G-R PG-2 R-G-G-R-L-C-Y-C-R-R-R-F-C-I-C-V PG-3 R-G-G-G-L-C-Y-C-R-R-R-F-C-V-C-V-G-R PG-4 R-G-G-R-L-C-Y-C-R-G-W-I-C-F-C-V-G-R PG-5 R-G-G-R-L-C-Y-C-R-P-R-F-C-V-C-V-G-R

In the form present in the porcine leukocytes, it is believed that the C-terminal amino acids are amidated and that there are two disulfide linkages as described above. However, linearalized forms of these native protegrins are also biologically active. Particularly preferred compounds of the invention are those which are similar to the native forms but are in the enantiomeric form—i.e. all of the amino acids are in this D-configuration. Thus, particularly preferred forms of the unmodified protegrins include the following and their D-enantiomers :

Protegrin form-21

R-G-G-R-R-C-Y-C-R I I \

R 21a R

I I / V-C-V-C-F

R-G-G-R-L-C-Y-C-R

R 21b R

/

R-C-V-C-F R-G-G-R-R-C-Y-C-R I I \

R 21d R

I I / R-C-V-C-F

R-C-Y-C-R

R 21e R

I I / V-C-V-C-F

L-C-Y-C-R I I \ I I R 21f

I I R I I /

R-C-V-C-F

R-C-Y-C-R I I \

I I R 21g I I R 1 1 /

R-C-V-C-F

L-C-Y-C-R I I \

I I R 21h I I R I I / V-C-V-C-F

Protegrin form-22

R-G-G-R-R-C-Y-C-R I I \

I I R 22a

I I R I I / V-C-I-C-F R-G-G-R-L-C-Y-C-R I I \

I I R 22b I I R I I / R-C-I-C-F

R-G-G-R-R-C-Y-C-R

R 22c

R

I I / R-C-I-C-F

R-C-Y-C-R I I \

R 22d R

I I / V-C-I-C-F

L-C-Y-C-R

I I \

R 22e R

I I / R-C-I-C-F

R-C-Y-C-R I I \ I I R 22f

I I R I I / R-C-I-C-F

L-C-Y-C-R I I \ I I R 22g

I I R 1 1 /

V-C-I-C-F Protegrin form-23

R-G-G-R-R-C-Y-C-R

I I \ I I R 23a

I I R I I / V-C-V-C-F

R-G-G-R-L-C-Y-C-R

I I \

I I R 23b

I I R 1 1 /

R-C-V-C-F

R-G-G-R-R-C-Y-C-R

R 23c R

/

R-C-V-C-F

R-C-Y-C-R

\

R 23d

R

/

V-C-V-C-F

L-C-Y-C-R

I I \

R 23e R

I I / R-C-V-C-F

R-C-Y-C-R I I \ I I R 23f

I I R I I / R-C-V-C-F L-C-Y-C-R

I I \

I I R 23g

I I R

I I / V-C-V-C-F

Protegrin form-24

R-G-G-R-R-C-Y-C-R

I I \

I I R 24a

I I R 1 1 /

V-C-F-C-I

R-G-G-R-L-C-Y-C-R

R 24b

R

I I /

R-C-F-C-I

R-G-G-R-R-C-Y-C-R

\

R 24c

R

/

R-C-F-C-I

R-C-Y-C-R

I I \

I I R 24d

I I R

I I / V-C-F-C-I

L-C-Y-C-R

I I \ I I R 24e

I I R I I / R-C-F-C-I R-C-Y-C-R

I I \

I I R 24 f

I I R I I / R-C-F-C-I

L-C-Y-C-R I I \

R 24g R

I I /

V-C-F-C-I

Protegrin form-25

R-G-G-R-R-C-Y-C-R

\

R 25a R

/

V-C-V-C-F

R-G-G-R-L-C-Y-C-R

I I \

R 25b R

I I / R-C-V-C-F

R-G-G-R-R-C-Y-C-R

I I \ I I R 25c

I I R I I / R-C-V-C-F

R-C-Y-C-R I I \ I I R 25d

I I R I I /

V-C-V-C-F L-C-Y-C-R

I I \

I I R 25e

I I R I I / R-C-V-C-F

R-C-Y-C-R

R 25f R

I I / R-C-V-C-F

L-C-Y-C-R I I \

I I R 25g I I R

I I / V-C-V-C-F and the foregoing protegrins wherein A₇ is W and/or

A_i2 is W and/or wherein A_i is W and/or wherein Aι₆ is W and/or wherein Aι₇ is G and Aι₈ is R; and/or wherein at least one of A₅, Ag, A_i2 and ι₆ is not present; including the linearalized forms thereof and the N-terminal acylated and C-terminal amidated forms thereof. In the terminology set forth above, protegrin form 21 consists of compounds which are characteristic of the present class but which are otherwise similar to PG-1; forms labeled 22 contain the characteristics of the present class but are modeled after PG-2; classes 23-25 are similarly related to PG-3, PG-4 and PG-5. Similarly, those compounds of the invention which contain one disulfide bond are preferably selected from the group consisting of the following, including their enantiomeric forms: Bullet-21

R-G-G-R-L-C-Y-X-R I \ I R 21a

I R I / V-C-V-X-F

R-G-G-R-R-C-Y-X-R

I \

I R 21b

I R I /

V-C-V-X-F

R-G-G-R-L-C-Y-X-R

R 21c R

R-C-V-X-F

R-G-G-R-R-C-Y-X-R I \

R 21d R

I / R-C-V-X-F

R-C-Y-X-R

I \

I R 21e

I R I / V-C-V-X-F

L-C-Y-X-R I \ I R 21f

I R I /

R-C-V-X-F R-C-Y-X-R

I \

I R 21g

I R

I / R-C-V-X-F

L-C-Y-X-R

R 21h R

I / V-C-V-X-F

Bullet 22

R-G-G-R-L-C-Y-X-R I \

R 22a R

V-C-I-X-F

R-G-G-R-R-C-Y-X-R

\

R 22b R

V-C-I-X-F

R-G-G-R-L-C-Y-X-R

I \

I R 22c I R I / R-C-I-X-F

R-G-G-R-R-C-Y-X-R I \ I R 22d

I R I / R-C-I-X-F R-C-Y-X-R

I \

I R 22e

I R I /

V-C-I-X-F

L-C-Y-X-R

R 22f R

/

R-C-I-X-F

R-C-Y-X-R

R 22g R

R-C-I-X-F

L-C-Y-X-R

I \

R 22h R

I / V-C-I-X-F

Bullet 23 R-G-G-R-L-C-Y-X-R

I \

I R 23a

I R I / V-C-V-X-F

R-G-G-R-R-C-Y-X-R I \ I R 23b

I R I / V-C-V-X-F R-G-G-R-L-C-Y-X-R I \

I R 23c I R I / R-C-V-X-F

R-G-G-R R-C-Y-X-R

R 23d R

/

R-C-V-X-F

R-C-Y-X-R

R 23e R

/

V-C-V-X-F

L-C-Y-X-R

I \

R 23f R

I / R-C-V-X-F

R-C-Y-X-R I \ I R 23g

I R I /

R-C-V-X-F

L-C-Y-X-R

I \

I R 23h

I R I /

V-C-V-X-F Bullet 24

R-G-G-R-L-C-Y-X-R I \ I W 24a

I G I / V-C-F-X-I

R-G-G-R-R-C-Y-X-R

I \

I W 24b I G I /

V-C-F-X-I

R-G-G-R-L-C-Y-X-R

W 24c G

R-C-F-X-I

R-G-G-R-R-C-Y-X-R I \

W 24d G

I / R-C-F-X-I

R-C-Y-X-R

I \

I W 24e I G I / V-C-F-X-I

L-C-Y-X-R

I \ I W 24f

I G I / R-C-F-X-I R-C-Y-X-R I \

I W 24g I G I /

R-C-F-X-I

L-C-Y-X-R I \

I W 24h I G I / V-C-F-X-I

Bullet 25

R-G-G-R-L-C-Y-X-R I \

I P 25a I R I / V-C-V-X-F

R-G-G-R-R-C-Y-X-R I \

I P 25b I R

I / V-C-V-X-F

R-G-G-R-L-C-Y-X-R

P 25c R

/

R-C-V-X-F

R-G-G-R-R-C-Y-X-R I \ I P 25d

I R I / R-C-V-X-F R-C-Y-X-R

I \

I P 25e

I R I /

V-C-V-X-F

L-C-Y-X-R

P 25f R

/

R-C-V-X-F

R-C-Y-X-R I \

I P 25g I R

I / R-C-V-X-F

L-C-Y-X-R

I \

I P 25h

I R

I / V-C-V-X-F and the foregoing bullet forms wherein A₇ is W and/or _i2 is W and/or wherein _i4 is W and/or wherein Aiβ is W and/or wherein Aι₇ is G and Aι₈ is R; and/or wherein at least one of A₅, Ag, A_i2 and A_i6 is not present, and the amidated forms thereof, wherein each X is independently a hydrophobic, a small, or a large polar amino acid residue; and the following kite forms, including their enantiomers:

Kite form-21

R-G-G-R-L-X-Y-C-R I \

I R 21a I R

I / V-X-V-C-F R-G-G-R-R-X-Y-C-R

R 21b R

I / V-X-V-C-F

R-G-G-R-L-X-Y-C-R

R 21c R

/

R-X-V-C-F

R-G-G-R-R-X-Y-C-R I \

I R 21d I R I / R-X-V-C-F

R-X-Y-C-R I \

I R 21e I R

I / V-X-V-C-F

L-X-Y-C-R

I \

R 21f R

I / R-X-V-C-F

R-X-Y-C-R I \ I R 21g

I R I /

R-X-V-C-F L-X-Y-C-R

I \

I R 21h

I R I /

V-X-V-C-F

Kite form-22

R-G-G-R-L-X-Y-C-R

R 22a R

/

V-X-I-C-F

R-G-G-R-R-X-Y-C-R I \

I R 22b I R I / V-X-I-C-F

R-G-G-R-L-X-Y-C-R I \

I R 22c I R

I / R-X-I-C-F

R-G-G-R-R-X-Y-C-R

I \ I R 22d

I R I / R-X-I-C-F

R-X-Y-C-R I \ I R 22e

I R I / V-X-I-C-F L-X-Y-C-R

I \

I R 22f

I R I /

R-X-I-C-F

R-X-Y-C-R

R 22g R

/

R-X-I-C-F

L-X-Y-C-R I \

R 22h R

I / V-X-I-C-F

Kite form-23

R-G-G-R-L-X-Y-C-R I \

I R 23a I R

I / V-X-V-C-F

R-G-G-R-R-X-Y-C-R

I \

I R 23b I R I / V-X-V-C-F

R-G-G-R-L-X-Y-C-R I \ I R 23c

I R I / R-X-V-C-F R-G-G-R-R-X-Y-C-R I \ I R 23d

I R I /

R-X-V-C-F

R-X-Y-C-R

R 23e R

I / V-X-V-C-F

L-X-Y-C-R I \

R 23f R

I / R-X-V-C-F

R-X-Y-C-R

\

R 23g R

I / R-X-V-C-F

L-X-Y-C-R I \ I R 23h

I R I /

V-X-V-C-F

Kite form-24

R-G-G-R-L-X-Y-C-R I \ I W 24a

I G I / V-X-F-C-I R-G-G-R-R-X-Y-C-R

I \

I W 24b

I G I /

V-X-F-C-I

R-G-G-R-L-X-Y-C-R

W 24c G

I / R-X-F-C-I

R-G-G-R-R-X-Y-C-R

\

W 24d G

I / R-X-F-C-I

R-X-Y-C-R

I \

W 24e G

I / V-X-F-C-I

L-X-Y-C-R I \ I W 24f

I G I /

R-X-F-C-I

R-X-Y-C-R

I \

I W 24g I G I /

R-X-F-C-I L-X-Y-C-R

I \

I W 24h I G I /

V-X-F-C-I

Kite form-25

R-G-G-R-L-X-Y-C-R

R 25a R

/

V-X-V-C-F

R-G-G-R-R-X-Y-C-R I \

I R 25b I R I / V-X-V-C-F

R-G-G-R-L-X-Y-C-R

I \

I R 25c I R

I / R-X-V-C-F

R-G-G-R-R-X-Y-C-R

I \

I R 25d

I R I / R-X-V-C-F

R-X-Y-C-R

I \ I R 25e

I R I / V-X-V-C-F L-X-Y-C-R

I \

I R 25f

I R

I / R-X-V-C-F

R-X-Y-C-R

R 25g R

I / R-X-V-C-F

L-X-Y-C-R I \

I R 25h I R

I / V-X-V-C-F and the foregoing kite forms wherein A₇ is W and/or

Aj_.2 is W and/or wherein A_i4 is W and/or wherein Aι₆ is W and/or wherein Aι₇ is G and Aι₈ is R; and/or wherein at least one of ₅, Ag, A_i2 and Rie is not present, and the amidated forms thereof, wherein each X is independently a hydrophobic, small, or large polar amino acid. These preferred forms also include the linearalized forms, as well as the N-acylated and C-amidated forms.

The designation "X" refers to the replacement amino acid as described herein, preferably X is A, S, T or G, most preferably A or T. Preferred forms which are completely modified by replacement of all cysteine, homocysteine or penicillamine residues are selected from the group consisting of Snake form-21 R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-V

R-G-G-R-R-X-Y-X-R-R-R-F-X-V-X-V

R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-R

R-G-G-R-R-X-Y-X-R-R-R-F-X-V-X-R R-X-Y-X-R-R-R-F-X-V-X-V

L-X-Y-X-R-R-R-F-X-V-X-R R-X-Y-X-R-R-R-F-X-V-X-R L-X-Y-X-R-R-R-F-X-V-X-V

Snake form-22 R-G-G-R-L-X-Y-X-R-R-R-F-X- I-X-V

R-G-G-R-R-X-Y-X-R-R-R-F-X-I-X-V

R-G-G-R-L-X-Y-X-R-R-R-F-X-I-X-R

R-G-G-R-R-X-Y-X-R-R-R-F-X-I-X-R

R-X-Y-X-R-R-R-F-X-I-X-V L-X-Y-X-R-R-R-F-X-I-X-R

R-X-Y-X-R-R-R-F-X-I-X-R L-X-Y-X-R-R-R-F-X-I -X-V

Snake form-23 R-G-G-G-L-X-Y-X-R-R-R-F-X-V-X-V R-G-G-G-R-X-Y-X-R-R-R-F-X-V-X-V

R-G-G-G-L-X-Y-X-R-R-R-F-X-V-X-R

R-G-G-G-R-X-Y-X-R-R-R-F-X-V-X-R

R-X-Y-X-R-R-R-F-X-V-X-V

L-X-Y-X-R-R-R-F-X-V-X-R R-X-Y-X-R-R-R-F-X-V-X-R

L-X-Y-X-R-R-R-F-X-V-X-V

Snake form-24 R-G-G-R-L-X-Y-X-R-G-W-I-X-F-X-V

R-G-G-R-R-X-Y-X-R-G-W-I-X-F-X-V R-G-G-R-L-X-Y-X-R-G-W-I-X-F-X-R

R-G-G-R-R-X-Y-X-R-G-W-I-X-F-X-R

R-X-Y-X-R-G-W-I-X-F-X-V

L-X-Y-X-R-G-W-I-X-F-X-R

R-X-Y-X-R-G-W- I-X-F-X-R L-X-Y-X-R-G-W-I-X-F-X-V Snake form-25 R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-V

R-G-G-R-R-X-Y-X-R-R-R-F-X-V-X-V

R-G-G-R-L-X-Y-X-R-R-R-F-X-V-X-R R-G-G-R-R-X-Y-X-R-R-R-F-X-V-X-R

R-X-Y-X-R-R-R-F-X-V-X-V L-X-Y-X-R-R-R-F-X-V-X-R R-X-Y-X-R-R-R-F-X-V-X-R L-X-Y-X-R-R-R-F-X-V-X-V and the foregoing snake forms wherein A₇ is W and/or i₂ is W and/or wherein A_i4 is W and/or wherein Aι₆ is W and/or wherein Aι₇ is G and Aι₈ is R; and/or wherein at least one of A₅, Ag, Aχ₂ and Aι₆ is not present, and the amidated forms thereof, wherein each X is independently a hydrophobic, small, or basic amino acid.

All of these embodiments also include the N-acylated and C-amidated forms. X is preferably S, A, T or G, most preferably A or T. In all of the foregoing cases, the enantiomeric forms, wherein all of the amino acids are in the D- configuration are also preferred for use in the methods of the invention.

A multiplicity of protegrins have been prepared, and these include:

PC11: LCYCRRRFCVCVGR

PC12: RCYCRRRFCVCV

PC15: RGGRLCYCRRRFCVCR

PC16: RCYCRRRFCVCR P PCC1177:: LCYCRRRFCVCV

PC18: LCYARRRFAVCV

PC19: RCYARRRFAVCR

PC20: LAYCRRRFCVAV

PC21: RAYCRRRFCVAR P PCC2222:: RGGRLCY RR VCV PC31: GGRLCYCRRRFCVCV PC32: RGRLCYCRRRFCVCV PC33: GRLCYCRRRFCVCV PC34: RRLCYCRRRFCVCV PC35: RLCYCRRRFCVCV PC36: RRCYCRRRFCVCV PC37: CYCRRRFCVCV PC44: RGGRLCYCRRRFCVC PC47: RGGRLCY RRRF VCV PC48 : RGWRLCYCRRRFCVCV PC37a: CYCRRRFCVCVGR PC45 : RGGRLCYCRRRFCV

PC72 LCYCRRRFCVC

PC64 LCYTRRRFTVCV

PC64a: LTYCRRRFCVTV

PC31a: GGRLCYCRRRFCVCVGR

PC32a: RGRLCYCRRRFCVCVGR

PC33a: GRLCYCRRRFCVCVGR

PC34a: RRLCYCRRRFCVCVGR

PC35a: RLCYCRRRFCVCVGR

PC36a: RRCYCRRRFCVCVGR

PC44a: RGGRLCYCRRRFCVCR

PC47a: RGGRLCY RRRF VCVGR

PC48a: RGWRLCYCRRRFCVCVGR PC54: RGWRLAYCRRRFCVAVGR PC61: RCYCRRRFCVCV PC62: LCYCRRRFCVCR PC63: VCYCFRRFCYCV PC65: LCYTRPRFTVCV PC66: LCYTRGRFTVCV PC67: LCYFRRRFIVCV PC68: LCYFRPRFIVCV PC69: LCYTFRPRFVCV PC70: LCYTFRGRFVCV PC74: CYCFRRFCVC PC77: LCYCRRRRCVCV

PC78: LCYCFRRRCVCV

PC79: LCYCRFRRCVCV

PC80: LCYCRRFRCVCV

PC81: LCYCRRFFCVCV

PC82: LCYCRFFRCVCV

PC83: LCYCFFRRCVCV

PC84: LCYCFRRFCVCV

PC85: LCYCFRFRCVCV

PC86: LCYCRFRFCVCV

PC87: LCYCFRFFCVCV

PC88: LCYCFFRFCVCV

PC89: LCYCFFFRCVCV

PC90: LCYCRFFFCVCV RGGRLCY RR VCVGR

PC91 YCYCRRRFCVCVGR

PC95 ICYCRRRFCVCVGR

PC96 FCYCRRRFCVCVGR

PC97 WCYCRRRFCVCVGR

PC99 RCYCRRRFCVCVGR

PC109: RLCYTRGRFTVCV

PC110: LCYTRGRFTVCVR

PC111: RLCYTRGRFTVCVR

PC112: LCYCHHHFCVCV

PC113: LCYTHHHFTVCV RGGLCYCRRRFCVCVGR RGGRLCYCRRRFCVCVGR RGGGLCYCRRRFCVCVGR RGGGLCYCRRGFCVCFGR RGGGLCYCRRPFCVCVGR RGGGLCYCRPRFCVCVGR RGGRLCYCRXRFCVCVGR (X=NMeG)

RGGLCYCRGRFCVCVGR RGGRLCYCXGRFCVCVGR (X=Cit) XGGRLCYCRGRFCVCVGR (X=Cit) RGGRVCYCRGRFCVCVGR RGGGLCYCFPKFCVCVGR RGWGLCYCRPRFCVCVGR RGWRLCYCRXRFCVCVGR (X=NMeG) 5 RGWRLCYCRGRFCVCVGR

RGWRLCYCXPRFCVCVGR (X=Cit)

RWRLCYCRPRFCVCVGR

RGWRLCYCRPRFCVCVGR

RGWRACYCRPRFCACVGR

10 GWRLCYCRPRFCVCVGR

RWRLCYCKGKFCVCVGR RGWRLCYCRXRFCVCVGR (X=NMeG) GGWRLCYCRGRFCVCVGR RGGWLCYCRGRFCVCVGR 15 RLLRLCYCRXRFCVCVGR (X=NMeG)

RLLRACYCRXRFCVCVGR (X=NMeG) RLLRLCYCRRRFCVCVGR RGLRXCYCRGRFCVCVGR (X=Cha) RGGRLCYCRXRZCVCWGR (X=NMeG) (Z=Cha) 20 RGGRWCVCRXRZCYCVGR (X=NMeG) (Z=Cha)

RGLRXCYCRGRFCVCVGR (X=Cha) RGGRWCVCRGRXCYCVGR (X=Cha) RGGRLCYCRRRFCXCVGR (X=NMeV) LCYCRRRFCVCV 25 LCYCRRCFCVCV

LCYCRRRFCVCF LCACRRRACVCV LCYCRXRFCVCV (X=D-Arg) LCWCRRRFCVCV 30 WCYCRRRFCVCV

LCYCRRRXCVCV (X=homoPhe) LCYCRRRXCVCV (X=P-ClPhe) XCYCRRRFCVCV (X=Cha ) LCYCRRRFCXCV (X=DHis) 35 LCYCRRRXCVCV (X=NMeGly) LCYCRRRXCVCV (X=NMePhe) LCYCRRRFCXCV (X=NMeVal) LCXCRRRXCVCV (X=Cha) LCGCRRRGCVCV 5 LCACRGRACVCV

RACYCRPRFCACV RLCYCRPRFCVCF RLCYCRPRFCVCV KLCYCKPKFCVCV 10 RLCACRGRACVCV

RLCYCRXRFCVCV (X=NMeGly) RXCFCRPRFCVCV (X=Cha) RWCFCRPRFCVCV WLCYCRRRFCVCV 15 WLCFCRRRFCVCV

FLCFCRRRFCVCV WLCFCRRRXCVCV (X=NMeF) WYCYCRRRFCVCV WXCYCRRRFCVCV (X=Cha) 20 RXCFCRGRZCVCV (X=Cha) (Z=NMeF)

XLCFCRRRZCVCV (X=Cha) (Z=NMeF) RLCYCRPRFCVCVGR WLCYCRRRFCVCVGR WXCYCRRRFCVCVGR (X=Cha) 25 RLCYCRGPFCVCR

RRWCFVCYAGFCYRCR

RRCYCRGRFCGCVGR

RWRCYCGRRFCGCVGR

RARCYCGRRFCGCVGR

30 GWRCYCRGRFCGC

RGWACYCRGRFCVC RRCYGRRRFGVCVGR RGWRLCYGRGRFKVC RGWRLCYCRGRFCVC 35 CYCRRRFCVCF RGWRLCYCRXRFCVC (X=NMeG) RGWRGCYCRXRFCGC (X=NMeG) LCYCRPRFCVCVGR LCYCKPKFCVCVGK 5 LCYCRGRFCVCVGR

LCYCRPRFCVCVGRGR RRWCYCRPRFCVCVR WRLCYCRPRFCVCVGR GWLCYCRGRFCVCVGR 10 RWLCYCRGRFCVCVGR

RLLCYCRGRFCVCVGR RWRLCYCRPRFCVCV

RXRLCYCRZRFCVCV (X=Cha) (Z=NMeG) RGWRLCYCRGRXCVCV (X=Cha) 15 RGGRVCYCRGRFCVCV

LCYCRXRFCVCV (X=D-Ala) LCYCKPKFCVCV VCYCRPRFCVCV LCYCRPRFCVCW

20 LCYRRPRFRVCV

RGWRLCYCRGRXCVCV (X=Cha) RXRLCYCRZRFCVCV (X=Cha) (Z=NMeG) RXRLCYCRGRFCVCV (X=Cha)

RGGGLCYARGWIAFCVGR 25 RGGGLCYARGFIAVCFGR

RGGGLCYARPRFAVCVGR

RGGGLCYTRPRFTVCVGR

RGGGLCYARKGFAVCVGR

RGGRLCYARRRFAVCVGR

30 RGGGLCYKRGFIKVCFGR

RGGGLCYKRGWIKFCVGR RGGGLCYRLPKFRVCVGR RGGGLCYRLPGFRVCVGR RGWRGCYKRGRFKGCVGR 35 LCYKRGRFKVCV ICYRPRFVCVGR

Preferred such compounds include the free acid and amidated forms thereof either in linear or disulfide- bridged form, and in the L- or D-enantiomeric forms.

Preparation of the Invention Compounds

The protegrins are essentially peptide backbones which may be modified at the N- or C-terminus and also may contain one or two 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 disulfide bond formation. As applied to the protegrins herein, "cyclic forms" refers to those forms which contain cyclic portions by virtue of the formation of disulfide linkages between cysteine, homocysteine or penicillamine residues in the peptide. If the 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, homocysteine or penicillamine residues.

Standard methods of synthesis of peptides the size of protegrins are known. 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 and has considerable benefits for large scale production. 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 of the invention is to employ these standard chemical synthesis techniques.

In addition to providing the peptide backbone, the N- and/or 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.

At the carboxy terminus, 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 NH₄ ⁺, 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. However, when the compound of formula (1) contains a multiplicity of basic residues, salt formation may be difficult or impossible. The carboxy terminus may also be esterified using alcohols of the formula ROH wherein R is hydrocarbyl (1-6C) as defined above. Similarly, the carboxy terminus may be amidated so as to have the formula -CONH₂, -CONHR, or -CONR₂, 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.

If the peptides of the invention are prepared under physiological conditions, the side-chain amino groups of the basic amino acids will be in the form of the relevant acid addition salts.

For synthesis of linear peptide with a C-terminal amide, the peptide sequence is conveniently synthesized on a Fmoc Rink amide solid support resin (Bachem) using F oc chemistry on an automated ABI 433 peptide synthesizer (ABD, Perkin Elmer, Foster City, CA) according to the manufacturer's standard protocols. Cleavage is typically carried out in 10 ml of thioanisole/EDT/TFA (1/1/9) for 2 hours at room temperature. Crude cleavage product is precipitated with t-butyl methyl ether, filtered and dried.

Formation of 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 of the air to effect these linkages. Various methods are known in the art. Processes useful for disulfide bond formation have been described by Tarn, 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. Giralt et al . , ESCOM Leiden, The Netherlands (1991) 164-166. An additional alternative is described by Kamber, B. et al . , Helv Chim Acta (1980) 63:899-915. A method conducted on solid supports is described by Albericio Int J Pept Protein Res (1985) 26:92-97.

A particularly preferred method is solution oxidation using molecular oxygen. This method has been used by the inventors herein to refold synthetic PG-1, PG-3 in its amide or acid forms, enantio PG-1 and the two unidisulfide PG-1 compounds (C₆-C__.5 and C₈-Cι₃) . Recoveries are as high as 65-90%.

In this preferred method to form disulfide linkages, the crude peptide is dissolved in DMSO and added to 20 mM ammonium acetate buffer, pH 7. The final concentration of the peptide in the solution is between 1-8 μg/mL, the pH ranges from 7.0-7.2, and the DMSO concentration ranges from 5-20%. The peptide solution is stirred overnight at room temperature. The pH of the solution is adjusted to pH5 with concentrated acetic acid and the sample purified on Prep LC. After loading, the column is washed with 10% acetonitrile/H₂0 (0.1% TFA) until the UV absorbance decreases to the baseline. The gradient is then started.

Column: Vydac Cat#218TP101522, 2.2 x 25 cm, C₁₈ peptides & proteins; UVλ: 235 nm; Flow Rate: 10 ml/min.

Solvent A is 100% 0.1% TFA/H₂0; Solvent B is 100% 0.08% TFA/ACN. The gradient is as follows.

Fractions are analyzed by analytical HPLC and those that contain the desired peptide are combined. The acetonitrile is stripped and the resulting aqueous solution lyophilized. The resulting amide, containing sulfide bonds, is confirmed by mass spectrum.

If 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 hereinabove. Depending on the host organism used for recombinant production and the animal source from which the protein is isolated, some or all of these conversions may already have been effected.

For recombinant production, 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. Thus, 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. Indeed, 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 protegrins can be produced in a form that will result in their secretion from the host cell by fusing to the DNA encoding the protegrin, a DNA encoding a suitable signal peptide, or may be produced intracellularly. 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.

Thus, the protegrins can be produced in a variety of modalities including chemical synthesis and recombinant production or some combination of these techniques.

Compositions Containing the Protegrins and Methods of Use The protegrins of the invention are effective in inactivating Gram-negative bacteria that are the cause of periodontal disease.

For use as a treatment of periodontal conditions, the protegrins of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired — e.g., prevention, prophylaxis, therapy; the protegrins 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 protegrins can be used in animal subjects both as therapeutic and prophylactic treatments; by "treating" an infection is meant either preventing it from occuring, ameliorating the symptoms, inhibiting the growth of the microbe in the subject, and any other negative effect on the microbe which is beneficial to the subject. Thus, "treating" or "treatment" have both prophylactic and therapeutic aspects.

The protegrins are particularly attractive as an active ingredient in pharmaceutical compositions useful in treatment of periodontal diseases. 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.

In general, for use in therapy or prophylaxis of periodontal disease, the protegrins of the invention may be used alone or in combination with other antibiotics such as erythromycin, tetracycline, macrolides, for example azithromycin and the cephalosporins . Depending on the mode of administration, the protegrins will be formulated into suitable compositions to permit facile delivery to the affected areas. The protegrins may be used in forms containing one or two disulfide bridges or may be in linear form. In addition, use of the enantiomeric forms containing all D-amino acids may confer advantages such as resistance to those proteases, such as trypsin and chymotrypsin, to which the protegrins containing L-amino acids are less resistant. Of course, mixtures of protegrins can be used.

The protegrins can be administered singly or as mixtures of several protegrins 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, intraperitoneal 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 protegrins can be administered also in liposomal compositions or as microemulsions .

If systemic administration is to be oral, the protegrins of the invention should be protected from degradation in the digestive tract 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. The protegrins are relatively acid stable, however, some degree of enteric coating may still be required.

For use in treating periodontal disease, of course, it is preferred to use topical compositions that can be applied directly in the mouth. It is significant that the protegrins are effective antimicrobials in the presence of saliva. The compositions can be applied directly to the affected areas using standard techniques known in the art.

The following examples illustrate but do not limit the invention: Preparation A Preparation Of Test Bacteria Bacteria, including A. actinomycetemcomi tans ATCC 29523, FDC-Y4, NCTC 9709, Capnocytophaga sputigena ATCC 33123, Capnocytophaga gingivalis ATCC 33124, and Capnocytophaga ochracea ATCC 27872 were grown on Laked blood agar overnight, and suspended in trypticase soy broth (BBL Microbiology; Cockeysville, Md. ) containing 0.1% sodium bicarbonate, 0.05% equine hemin III (Sigma Chemical Co.; St. Louis, Mo.), 0.0001% menadione (Sigma) and 0.1% yeast extract (Difco Laboratories, Detroit, Mich.). The bacteria were incubated an additional 4 h to early log growth phase.

Preparation B

Preparation of Protegrins Protegrin l(PG-l), an enantiomer of protegrin 1 comprised of all D-amino acides (D-PG-1) , protegrin 2 (PG-2), protegrin 3 (PG-3) , and protegrin 5 (PG-5) were synthesized using Fmoc chemistry (SynPep; Dublin, CA) . The crude, synthetic peptides were reduced with dithiothreitol and purified by reversed phase HPLC on a Vydac Ciesilica column (1 x 25 cm; The Separations Group; Hesperia, CA) using an acetonitrile gradient in the presence of 0.1% aqueous trifluoracetic acid, and concentrated by vacuum centrifugation (Speed-Vac; Savant Instruments, Farmington, NY). The reduced peptides (0.1- 0.2 μg/mL) were subjected to air oxidation, 24-48 h, in 0.1 mol/L tris, pH 7.7, to allow the formation of intramolecular cystine disulfide bonds. The peptides were purified by reversed phase HPLC using the Vydac Cι₈ silica column. The peptide preparations used in this study were homogenous as assessed by reversed phase HPLC, acid-urea polyacrylamide gel electrophoresis, and fast atom bombardment-mass spectrometry. Stock solutions were prepared in glass distilled water. Concentrations of PG-1 were verified upon an extinction coefficient of 1.280 (mrnol/L)^"1 cm^"1 at 280 nm.

Example 1 Sensitivity of Periodontal Bacteria to the Protegrins In all of the assays conducted, bacterial concentrations were adjusted turbidometrically such that the final concentration in the bactericidal assay was approximately 10⁷ cells/mL in Hank's balanced salt solution (HBSS; Sigma Chemical Co., St. Louis MO), pH 7.0. The bacterial suspension, protegrin, and any other additive (such as serum) were admixed in a final volume of 40 μL The mixture was incubated at 37°C in a temperature block for the time periods specified in the results. The reaction was terminated by dilution, 1:100 in HBSS, and plating using a Spiral plater, Model D (Spiral Biotech, Inc.; Bethesda, MD) . Colony-forming units (CFU) were enumerated after 48-72 h incubation.

Bactericidal activity was expressed as the logio reduction in CFU (δLogio) . The 99% effective dose (ED₉₉) is the theoretical concentration of protegrin peptide at which the δLogio is 2. The effect of PG-1 on various strains of

Actinobacillus actinomycetemcomi tans and Capnocytophaga Spp . are shown in Table 1.

Table 1

Organism Strain ED₉₉,μg/mL^a n

A. actinomycetemcomitans ATCC 29523 1.3 + 1.0 5

A. actinomycetemcomi tans FDC-Y4 0.9 + 0.7 5

A. actinomycetemcomi tans NCTC 9709 0.7 + 0.2 5

C. sputigena ATCC 33123 14.6 + 11.1 5

C. gingivalis ATCC 3124 6.2 + 4.2 5

C. ochracea ATCC 27872 21.2 + 10.7 5 ^a Incubated 1 h, 37 °C. Means +_ standard deviations from values interpolated from dose-response curves The results are shown as EDgg, 1 hour—i.e., 99% effective doses. In general, strains of A. actinomycetemcomi tans were an order of magnitude more sensitive than those of Capnocytophaga Spp. Kinetic analysis showed exponential killing although there was occasionally a brief lag phase. An alternative presentation of these results is shown in Figure 2.

In addition to PG-1, its D-enantiomer and various synthetic protegrins including PG-2, PG-3 and PG-5 were tested, with the results set forth in Figure 3. Figure 3A represents killing of A. actinomycetemcomi tans ATCC 29523; panel 3B shows killing of another strain of this organism, FDC-Y4; and panel 3C shows killing of the NCTC-9709 strain. Panels 3D, 3E and 3F show killing of C. sputigena ATCC 33123; C. gingivalis ATCC 33124, and C. ochracea ATCC 27872 respectively.

Bacterial activity is shown as δLogio (please explain) and the bars represent the mean and standard deviation of three trials. The clear bar represents no additions; and, subsequently reading left to right the bars represent 100 μg/mL, 10 μg/mL, 1 μg/mL and 0.1 μg/mL of the protegrin respectively.

It is seen that all of these protegrins were affective against A. actinomycetemcomi tans between 1- 10 μg/mL and against strains of Capnocytophaga Spp between 10-100 μg/mL. While all strains of A. actinomycetemcomi tans were comparably affected by all congeners, for the strains of Capnocytophaga , the profile of sensitivities was D-PG-l=PG-5 > PG-2 = PG-1 > equal to PG-3.

Example 2 Effect of Serum and Tonicity The method of Example 1 was repeated in the presence of 20% normal human serum. The results are shown in Figure 4A, 4B and 4C. Human serum antagonizes the bactericidal effects of PG-1 against both serum sensitive A . actinomycetemcomitans and serum sensitive

Capnocytophaga . This was not clearly demonstrable against Capnocytophaga Spp. unless the serum was first inactivated by heat. Serum, 20% (v/v) , usually did not block the bactericidal effects of PG-1 at concentrations of PG-1 equal to or greater than 100 μg/mL. When the concentration of PG-1 was reduced to 10 μg/mL, no killing was observed above 10% (v/v) serum (Fig. 4C) .

Figure 4C shows that serum at 10% (v/v) or less does not inhibit bactericidal activity of 10 μg/mL PG-1. The protegrins were bactericidal in HBSS, thus the protegrins are relatively insensitive to tonicity. The concentrations of NaCl, KCl, and NaBr in 10 mmol/L sodium phosphate buffer, pH 7.2 were varied; the protegrins lost their bactericidal activity only under hypertonic conditions, above 0.5 mol/L NaCl, KCl, and NaBr (Fig. 5).

Claims

1. A method to treat periodontal disease which method comprises administering to a subject afflicted with such disease an amount of a protegrin effective to treat said disease; wherein said protegrin contains the amino acid sequence:

A₁-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁o-A╬╣╬╣-A₁₂-C₁₃-A₁₄-C₁₅-A₁₆-A₁₇-A₁₈ (1)

wherein said protegrin contains 10-30 amino acid residues, wherein the amino acid sequence of formula (1) may be extended at the N and/or C-terminus by additional noninterferring amino acids; and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, said protegrin either in the optionally -SH stabilized linear or in a disulfide-bridged form wherein each of C₆, C₈, C╬╣₃ and C╬╣₅ is independently a cysteine, homocysteine, or penicillamine, or wherein one or more of C₆, C₈, C╬╣₃ and C₁₅ is independently replaced by a basic, hydrophobic, large/polar or small amino acid or wherein C₈ and/or C₁₃ is not present; each of A₁-A₅ is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid; each of A₇ and A_i4 is independently a hydrophobic or a small amino acid; g- ╬╣₂ are capable of effecting a ╬▓-turn when contained in the compound of formula (l)and at least one of Ag-A_i2 must be a basic amino acid and wherein Ag and/or A╬╣₂ may be present or not present; each of ╬╣₆- ╬╣₈ is independently present or not present, and if each present each is independently a basic, hydrophobic, polar/large or small amino acid; wherein in said protegrin at least about 15% to about 50% of the amino acids are basic amino acids, and wherein the protegrin compound has a net positive charge of at least +1 at physiological pH.

2. The method of claim 1 wherein said protegrin contains two disulfide bridges.

3. The method of claim 1 wherein said protegrin contains one disulfide bridge.

4. The method of claim 1 wherein said protegrin is in the linear form.

5. The method of any of claims 1-4 wherein A₇ and 14 are hydrophobic, and/or wherein A₅ and A╬╣₆ are hydrophobic or small; and/or wherein at least one of Ag and A╬╣₂ is hydrophobic; and/or wherein A╬╣₀ and n is each independently proline, or small or a basic or hydrophobic amino acid; and/or wherein C₈ and C╬╣₃ are independently cysteine, homocysteine or penicillamine and the amino acids at Ag and ╬╣₂ are present; and/or wherein at least one of A╬╣~A₅ is not present; and/or wherein at least one of A╬╣-A₄ is hydrophobic.

6. The method of claim 5 wherein each of A₅ and A╬╣₆ is independently selected from the group consisting of I, V, L, NLe, W, Y and F; and/or wherein each of A and A╬╣₄ is independently selected from the group consisting of I, V, L, W, Y and F; and/or wherein said protegrin is in the D-enantiomeric form.

7. The method of claim 6 wherein said protegrin is PG-1, PG-2, PG-3, PG-4 or PG-5 or the enantiomeric form thereof.

8. A pharmaceutical composition for treatment of periodontal disease use which comprises the protegrin of any of claims 1-7 in admixture with at least one pharmaceutically acceptable excipient.

9. Use of a protegrin to prepare a medicament to treat periodontal disease wherein said protegrin contains the amino acid sequence:

A╬╣-A₂-A₃-A₄-A₅-C₆-A₇-C₈-A₉-A₁₀-A₁₁-A₁₂-C╬╣₃-Ai₄-Ci5-A╬╣₆-A₁₇-A₁₈ (1)

wherein said protegrin contains 10-30 amino acid residues, wherein the amino acid sequence of formula (1) may be extended at the N and/or C-terminus by additional noninterferring amino acids; and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, said protegrin either in the optionally -SH stabilized linear or in a disulfide-bridged form wherein each of C╬▓, C₈, C₁3 and C15 is independently a cysteine, homocysteine, or penicillamine, or wherein one or more of C╬▓, C₈, C╬╣₃ and C15 is independently replaced by a basic, hydrophobic, large/polar or small amino acid or wherein C₈ and/or C╬╣₃ is not present; each of A₁-A5 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid; each of A₇ and A_i4 is independently a hydrophobic or a small amino acid;

Ag-A_i2 are capable of effecting a ╬▓-turn when contained in the compound of formula (l)and at least one of Ag-A_i2 must be a basic amino acid and wherein Ag and/or A_i2 may be present or not present; each of A╬╣₆-A╬╣₈ is independently present or not present, and if each present each is independently a basic, hydrophobic, polar/large or small amino acid; wherein in said protegrin at least about 15% to about 50% of the amino acids are basic amino acids, and wherein the protegrin compound has a net positive charge of at least +1 at physiological pH.

10. The use of claim 9 wherein said protegrin contains two disulfide bridges.

11. The use of claim 9 wherein said protegrin contains one disulfide bridge.

12. The use of claim 9 wherein said protegrin is in the linear form.

13. The use of claim 9 wherein A₇ and A_i4 are hydrophobic, and/or wherein A₅ and ╬╣₆ are hydrophobic or small; and/or wherein at least one of A₉ and A_i2 is hydrophobic; and/or wherein A╧ço and An is each independently proline, or small or a basic or hydrophobic amino acid; and/or wherein C₈ and C╧ç₃ are independently cysteine, homocysteine or penicillamine and the amino acids at Ag and A╧ç₂ are present; and/or wherein at least one of A╬╣-A₅ is not present; and/or wherein at least one of A╬╣-A is hydrophobic.

14. The use of claim 9 wherein each of A₅ and A╬╣₆ is independently selected from the group consisting of I, V, L, NLe, W, Y and F; and/or wherein each of A₇ and A_i4 is independently selected from the group consisting of I, V, L, W, Y and F; and/or wherein said protegrin is in the D-enantiomeric form.

15. The use of claim 9 wherein said protegrin is PG-1, PG-2, PG-3, PG-4 or PG-5 or the enantiomeric form thereof.