CA2226121A1 - Parevins and tachytegrins - Google Patents

Abstract

Compounds based on the antimicrobial protegrins and tachyplesins are disclosed having a net positive charge of at least +1 at physiological pH, comprising from 15 % to 50 % basic amino acids, and having an amino acid sequence of the formula: A1-A2-A3-C4*-C5*-C6*-A7-C8-A9-A10-A11-A12-C13-A14-C15*-C16*-C17*-A18, within a size range of 11-24 amino acids when the above formula is extended at either the N- or C-terminus by additional, non-interfering, amino acids and wherein the N-terminus may be acylated and the C-terminus may be amidated or esterified and disulfide bridges may be present or absent in a -SH stabilized linear form. In one embodiment, C8 and C13 denote cysteine, homocysteine or penicillamine; C4*-C6* and C15*-C16* denote the preceding or any naturally-occurring amino acid save glutamic acid, aspartic acid and proline, except when either or both of C4* or C17* are optionally absent wherein only proline is excluded at C5* and C15*; A1-A3 and A18 may be present or absent and denote any naturally-occurring amino acid except glutamic acid, aspartic acid, proline and cysteine; A7 and A14 denote a hydrophobic or a small amino acid;
A9-A12 must be capable of forming a .beta.-turn in the compound; and, at least one of A9-A12 must be a basic amino acid.

Description

CA 02226121 1998-01-0~

PAREVINS AND TACHYTEGRINS

This invention was made with fi~nding from NIH Grant No. A122839. The U.S.
Government has certain rights in this invention.

Techni~l Field The invention relates to the field of a,~Lil,i~,lic peptides. In particular, the invention concerns short peptides with unique patterns of cysteine type residues and cor~llllaLions that have a wide range of ~ntim:crobial activities.

Bacl~:loulld Art One of the defense m~c.~l~ni~m~ against infection by both animals and plants is the production of peptides that have ~ntimirrobial and antiviral activity. Various classes of these peptides have been isolated from tissues of both plants and ~nim~l~ PCT application WO 95/03325 published 2 February 1995 contains a review ofthe li~e.~lul~ on this subject.
Such peptides inc!ude tachyplesins, which are 17-18 amino acid peptides co.,~ g four invariant cysteines, the d~re,-si--s, ~-d~:re--sins, and insect der~ sins, which are somewhat longer peptides characterized by six invariant ~,y~,L~illes, and antifi~ng~l and ~ntibacterial peptides and proteins which have been found in plants.
The applications in the series of which WO 95/03325 is a part provide a new class of antimicrobial and antiviral peptides, deei~n~ted "protegrins", representative members of ~,vhich have been isolated from porcine leukocytes. These peptides are useful as antibacterial antiviral and ~ntifi-ng~l agents in both plants and ~nim~lc The isolation of some of the protegrin peptides was reported in a paper by Kokryakov, V.N. et al. FEBS (1993) 337:231-236 (July issue). A later publication described the presence of a new protegrin, whose sequence and that of its ple~iul~or were ded~lced from its isolated cDNA clone. Zhao, C et al, FEBSLetters (1994) 346:285-288. An additional paper disclosing cationic peptides from porcine neutrophils was published by Mirgorodskaya, O.A.
et al. FEBS (1993) 330:339-342. Storici, P. et al. Biochem Biophys Res Comm (1993) 196: 1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte antirnicrobial peptide with a cathelin-like prosequence. The peptide is reported to be one of the protegrins. ~ 1ition~1 publications related to pLotegrills are Harwig, S. S.L., et al. J
Peptide Sci (1995) in press; Zhao, C., et al. FEBS Lett (1995) 376: 130-134; Zhao, C. et al.
FEBS Lett (1995) 368: 197-202.

SUBSTITUTE SHEET (RULE 26) CA 02226l2l l998-Ol-0~

The protegrins have also been found to bind to endotoxins -- i.e., the lipopolysaccharide (LPS) compositions derived from gram-negative bacteria which are believed .t;~on~;l,le for gram-negative sepsis. The protegrins are also effective in hlhibiLi.lg the growth of or~ni~mx that are associated with sexually ll~ led ~ P~es such as Chlamydia t,.lc~lv".atis and Neisseria g~,~.J"hoeae.
Protegrins are also effective against the microolg~ a~sori~ted with oral mucositis, a signific~nt side effect of cancer therapy and bone marrow transplantation that is not adequately m~n~gecl 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 Oncologv. J.B. Lip~,hlcoLL, Phil~-lçlrhi~
(1993b) pp. 2385-2394). Oral mucositis is ;..;~ ecl by the cytotoxic effects of these therapies on the rapidly dividing epitheli~l cells ofthe olophhlyngeal mucosa, and is exacerbated by infection with both endogenous oral flora and opportunistic bacterial and fungal pathogens.
Oral mucositis is associated with extreme discon~l L and pain, especially when eating. In the case of head and neck cancers, oral mucositis is frequently a dose limiting toxicity causing delay in the completion of the therapeutic regimen. Such a delay in trç~tment can have a negative impact on the final therapeutic outcome. Tnfectecl oral lesions are also increasingly recognized as a ~i~nific~nt portal of entry of microolg~l-ix...~ to the immnnosupplessed patient rçslllting in sepsis (Sonis, 1993 a,b, supra).
The invention described below relates to peptide type compounds that are related to the protegrins described above, but reflect displ~ce. . .~ of the protegrin cysteines at positions 6 and 15. The availability ofthese compounds, the ple~"ed forms of which are clecign~te~
parevins and tachytegrins, ç~r~n~ls the lept;,Lc,ile of antimicrobial peptides and permits more exquisite m~tching of inrlic~tion~ to antimicrobial formulations. Although at least one of C4 ', C5, C 16 or C 17 in the formula set forth below must be cysteine, the common name terminlogy of these components reflects particularly perferred situations wherein both of C4 and Cl7 are cysteine type residues (the tacllyleglins) or where both C 5 and C 16 are cysteine type residues (the parevins).

SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

Disclosure of the Invention The invention provides compounds which retain generally the antimicrobial activity of the ploLe~ ,s ~ c~lqse~ above, but differ in conrolllldlion due to the dislocation ofthe cysteine residues at positions 6 and/or 15 of these protegrins. Surprisingly, these modified compounds exhibit activity spectra which are analogous to those ofthe plol~,ins, but offer the oppo~tunity to fine-tune the biological activity of antibiotics and antivirals. All of these peptides can be produced synthetic~lly and those that contain only gene-encoded amino acids can also be produced reco~ A~ y. These compounds are usefi~l as preservatives or in pharm~celltic~l compositions in treating or preventing infection in anim~l~ Alternatively, the peptides can be form~ ted into compositions which can be applied to plants to protect them against viral or rnicrobial infection. In still another approach, the DNA encoding the peptides can be expressed in situ, in animals or plt;Çt;,~bly in plants, to combat infections. The peptides are also useful as standards in antimicrobial assays and in binding endotoxins.
Accoldingly, in one aspect, the invention is directed to a purified and isolated or ;collll)inhlllly or synthetically produced compound which contains the amino acid sequence Al-A2-A3-C-C~C~A7-C8-Ag-AIo-Al ,-A,2-C,3-A,4-C~5-C~6-C~;-A,8 ( 1 ) said compound co~ g 11-24 amino acid residues. The sequence shown as (I) can be e~ctçndecl at the N and/or C terminus with non-intelrelling amino acids or sequence.
The compounds also include the N-terminal acylated and/or C-terminal ami~l~ted or esterified forms and may be either in the, optionally -SH stabilized, linear or in a tli~ fi~e-bridged forrn.
In the amino acid seq~l~nt~e shown, each of Al-A3 is independently present or not present, and if present each is independently a basic, hydlophobic, polar/large, or small amino acid;
each of C4*, C5 *, C6*, C,5*, C,6* and Cl7* is independently cysteine, homocysteine or p~nicill~min~ or a basic, hydrophobic, polar/large, or small amino acid, and C4* and/or Cl~*
may be present or not pleselll, C6* and/or Cl5* may also be acidic;
each of C8 and Cl3 is independently cysteine, homocysteine or pPniç~ mine;

SUBSTITUTE SHEET (RULE 26) each of A~ and Al4 is in~epen-l~.ntly a hydrophobic or a small amino acid;
Ag-Al2 must be capable of ~jl ;..g a 13-turn when contained in the compound and at Ieast one of Ag-Al2 must be a basic amino acid;
Al8 is present or not present, and if present, is a basic, hydrophobic, polar/large or small amino acid.
The compounds of the invention may, in the alternative, contain a lnodified form of formula (1) wherein one or both of Cg and Cl3 is indep~n-lently replaced by a basic, hydrophobic, polar/large, acidic, or small amino acid.
In all of the compounds of the invention 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 +1 at physiological pH;
with the proviso that at least one of C~ 6 arld C~; must be cysteine, homocysteine or p~nicill~mine; and only one of C4, C5, and C6, and only one of Cl5, Cl6 and Cl, can be cysteine, homocysteine or penicill~mine.
A particular advantage of some of the peptides of the invention, especially those which contain fewer amino acids, lies in their reduced size. As a result of this, they are less costly to produce, generally are expected to provide better distribution in tissue, and are less imml~nogenic. As they provide alternative structures, they are likely to have di~e pharmacokinetic and toxicological profiles.
In still other aspects, the invention is directed to reco-,-bi.la..l materials useful for the production of the peptides of the invention as well as plants or animals modified to contain ~.es~ion systems for the production ofthese peptides. The invention is also directed to pharm~celltic~l compositions and compositions for application to plants co..~ ;..g the peptides of the invention as active ingredients or compositions which contain c~l~-ession systems for production of the peptides or for in si~u ~ es~ion of the nucleotide sequence encoding these peptides. The invention is also directed to methods to prepare the invention peptides synthetic~lly~ to antibodies specific for these peptides, and to the use ofthe peptides as preservatives.

SUBSTITUTE SHEET (RULE ~6) CA 02226l2l l998-Ol-0~
WO 97/022x7 PCT/US96/11323 Specific infections against which some of the peptides, especially the parevins and tachytegrins are particularly effective are those associated with oral mucositis, infections, such as stomach ulcers associated with H. pylori, and infections caused by Pseudomonas or MRSA.

In other aspects, the invention is directed to the use of the compounds of the invention as ~Lan~alds in antimicrobial assays. The compounds many also be used as antirnicrobials in solutions useful in eye care, such as contact lens solutions, and in topical or other pharns~sce~lfic~sl compositions for tre~stmpnt of sexually L~ ed t~i~ç~eps (STDs). The invention is also directed to use of the invention compounds as preservatives for foods or other perishables. As the invention peptides can inactivate endotoxin, the invention is also directed to a method to inactivate endotoxins using the compounds of the invention and to treat gram-negative sepsis by taking advantage of this pl opc:l ~y.

Brief Description of the Drawin~s Figure 1 shows antibacterial activity of two of the parevins against E. Coli;
Figure 2 shows ~sntibactPrial activity of two of the parevins against Listeria monocytogenes;
Figure 3 shows antifi-ng~l activity of two of the parevins against Candida albicans;
Figure 4 shows ~ntihacterial activity of a tachytegrin against E. coli.
Figure 5 shows ~nfil~actPrial activity of a tachytegrin against ~. subtilis.
Figure 6 shows ,snti~acterial activity of a tachytegrin against S. typhimurium.

Modes of Carrying Out the Invention The peptides of the invention are characterized by the amino acid sequence:

Al-A2-A3-C4 -C5 -C6 -A7-c8-A9-Alo-All-Al2-cl3-Al4-cl5 -Cl6 -Cl7 -Al8- (1) and its defined modified forms. Any of these peptides which may coin-;ident~lly occur in nature must be in purified and isolated form or plep~ed recombinantly or synthetically.
The dç~i~n~tiQn An in each case ,eplese-lls an amino acid at the specified position in the peptide. As defined Al-A3~ C4, Cl7 and/or Al8 may or may not be present. However, the peptides of the invention contain 1 1-24 amino acids. Thus, the sequence sho wn as ( 1 ) can SUBSTtTUTE SHEET (RULE 26) be extended at the N and/or C terrninus with non~ Lc-rcl~ing amino acids or sequence. The positions ofthe cysteine, homocysteine or pçnirill~minl? r~idtlee, shown as C in formula (1), are invariant in one embodiment of the peptides of the invention; however, in the modified forms ofthe peptides co~ g the sequence offormula (1), also inclnded within the scope of the invention, one or more of these cysteines may be replaced by a small, basic acidic or hydlophobic amino acid. However, at least one of C, C~ C~ and (~; must be cysteine, homocysteine peniçill~min~
All of the peptides of the invention, however, have a net positive charge of at least + 1 at physiological pH; appl~J~ill.alely 15%-50% ofthe amino acid residues in~l~lded in the sequence should be basic. For embo~imente having as few as 11 amino acids, there may be only one basic amino acid residue; however, at least two basic resid~çs, even in this short-chain residue, are pl crel I ed. If the peptide contains as many as 15 amino acid residues, two basic residues are required. It is plcrcl I cd that at least 20% of the amino acids in the sequence be basic, more preferably 30%, but not more than 50%.
The active peptides also preferably contain a ~ turn bracketed by two strands that form a ,~ sheet. While not intP.n~ling to be bound by any theory, applicants believe that antimicrobial activity ofthe compounds co..~;";..g the sequence offormula (1) is associated with such a ~-turn bracketed by two strands that form a ,~ sheet structure. The amino acids A9-AI2 must be capable of e~ g a 13 turn, which can be encouraged by the cystine bond between C8 and Cl3 as well as by hydrogen bonding between Ag and Al2. The presence of proline at Alo and/or All does not interfere with the ~-tum stabilized by the presence of a hydrophobic amino acid at positions Ag or Al2.
As used herein, "~-turn" refers to a recognized sub-class of reverse-turns. Typically, a ",~-turn" is a four amino acid residue peptide segm~nt that reverses the direction of a polypeptide chain so as to allow a single polypeptide chain to adopt an anti-parallel ,13-sheet secondary structure. Generally, the two internal amino acid residues of the ,B-turn are not ~
involved in the hydrogen-bonding of the ~-sheet; the two amino acid residues on either side of the internal residues are inclllded in the hydrogen-bonding ofthe 13-sheet. The terrn "~-turn"
C~.~JI cs~ly incllldes all types of peptide ,13-turns commonly known in the art inci~ ing~ but not limited to, type-I, type-II, type-III, type-I', type-II', and type-III' ,~-turns (see, Rose ef al., SUBSTITUTE SH EET (RULE 26) CA 02226121 1998-01-0~

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; Tr~mont~no et al., 1989, o~eins; Struct. Funct. Genet. 6:382-394).
The presence ofthe four invariant cysteines ofthe proteg~ s or ofthe C8 and Cl3 cysteines, homocysteine or peniç~ mine of the compounds of the present invention is helpful in e~e~ g the 13-turn co,~""a~ion; however, by p,o~e,ly choosing the s~bstit~ltions~ one or both of the cysteine, homocysteine or penirill~mine residues at C8 or Cl3 can be replaced ~,vithout subst~nti~lly disturbing the three-dimensional shape of the molecule.
The ,B sheets are believed to be ~ffected by the sequences surrounding C8 and Cl3, and are inclusive of these residues. Thus, in the unmodified forms of the compound, A, and Al4 are preferably hydrophobic amino acids. The cysteine residues may also, then, be replaced by other residues which do not affect the m~inten~nce of the ,~ sheet formation; these substitutions would include acidic, basic, hydrophobic polar or small amino acids.
The amino terminus of the peptide may be in the free amino form or may be acylated by a group ofthe formula RCO-, wherein R lep,ese,lls a hydrocarbyl group of 1-6C. The hydrocarbyl group is saturated or unsaturated and is typically, for example, methyl, ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, and the like.
The C-terminus of the peptides of the invention may be in the form of the underivatized carboxyl group, either as the free acid or an acceptable salt, such as the pot~eeil~m sodium, calcium, m~n~eillm, or other salt of an inorganic ion or of an organic ion such as caffeine. In some embo~1imçnts7 it is difficult to make salts since the rçm~inrler 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 ~mid~ted by an amine of the formula NH3, or RNH2, or R2NH, wherein each R is independently hydrocarbyl of 1-6C as defined above. Amidated forms of the peptides wherein the C-terminus has the formula CONH2 are pl~re"t;d.
As the peptides of the invention 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 i"o,~ a,fic ions such as chloride, bromide, iodide, fluoride or the SU85TITUTE SHEET (RULE 26) , CA 02226121 1998-01-0~

like, sulfate, nitrate, or phosphate, or may be salts of organic anions such as acetate, formate, bel~zoale and the like. The acceptability of each of such salts is dependent on the intçnded use, as is co~ lolllyunderstood.
The peptides of the invention that contain at least two cysteines, homocysteine or pPnir.ill~minç may be in straight-chain or cyclic form. The straight-chain forms are convertible to the cyclic forms, and vice versa. Methods for rulll~il~ ~ielllfide bonds to create the cyclic peptides are well known in the art, as are methods to reduce di.elllfides to form the linear compounds. The linear compounds can be stabilized by addition of a suitable alkylating agent such as iodo~cet~mide.
The cyclic forms are the result of the formation of die~llfide link~ges among all or some of the four cysteine, homocysteine or p~n:~ill~mine residues that may be present. Cyclic forms of the invention include all possible permutations of ~liel~lfi(le bond formation; if the -SH
co."~il.;..g amino acids are numbered in order oftheir occurrence starting at the N-terminus as C6 C8, Cl3, C~6, (~; or ~8, these permutations include, when two ~lielllfid~os are present:
a) C4-CI7 and C8-CI3;
b) C4-C~6 and C8-CI3;
c) C4-CIs and C8-CI3;
d) C5-C~7 and C8-CI3, e) C5-CI6 and C8-CI3;
f) C5-CI5 and C8-CI3;
g) C6-CI7 and C8-CI3;
h) C6-CI6 and C8-CI3;
i) C4-C8 and Cl3-C17;
j) C4-C8 and C,3-CI6;
k) C5-C8 and Cl3-CI7; and 1) C5-C8 and Cl3-CI6;

When one fli.elllfide is present, these pelllluL~Lions include:
C4-CI7;
C4-CI6;

SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

C4-CI5;
C5-C I7;
C5-C16;
C5-CI5;
C6-C 17;
C6-C16;
C8-CI3;
C4-C8;
C5-C8;
Cl3-CI7; and Cl3-CI6.
In the modified forms of the peptides, where 1 or 2 ~;y~leilles, homoey~Lei"e orp~nicill~mine are replaced, similar permutations are available as in when 2-3 cysteines, homocysteine or p~n~ mine are present.
The linearalized 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 iodo~cet~mide. The compounds of the invention also include linearalized forms which are stabilized with suitable reagents. As defined herein, "SH-stabilized" forms ofthe peptides ofthe invention contain sulfhydryl groups reacted with standard reagents to prevent lerc~ ion into di~--lfide linkages.
An alternative approach to providing linear forms of the invention compounds comprises use of the ~odifie~ form of the peptides where residues at Cg and/or Cl3 are replaced by amino acia~s which do not form cystine linkages, in cc,ml,inaLion with st~bili7.~tion of any cysteine, homocysteine or peniçill~mine residues at C~ or C~and/or (~, C~, C~;.
Forms of the invention compounds which have only one ~ fide bond are conveniently obtained by replacing the cysteine, homocysteine or p~nicill~mine residues at C8 and/or Cl3, preferably both, with amino acids which do not form di~llfide link~ges The amino acids denoted by An may be those encoded by the gene or analogs thereof, and may also be the D-isomers thereof. One pler~"ed embodiment of the peptides of the invention is that form whel eill all of the residues are in the D-configuration thus co~" hlg SUBSTITUTE 5H EET (RULE 26) resi~t~nee to protease activity while ~ ntimicrobial or antiviral propt;,Lies. The resulting peptides are enantiomers ofthe native L-amino acid-co..~ g forrns.
In one class of peptides described herein, either one or both of the residues found at C5 and/or C,6 is a basic amino acid and/or at least one of Al-A3 and C4 iS hydrophobic and/or at least one, and pler~l~ly all four ofthese amino acids are deleted. By suitable m~nipul~ti~n of these and other features, the range of con~litions under which the class of peptides ofthe present invention are effective can be varied. F~ llnole, the s~e.illul~, 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:

SUBSTITUTE SHFET (RULE 26) CA 02226121 l998-0l-05 One-Letter Three Letter Amino AcidSymbol Symbol Alanine A Ala Argilune R Arg ~ J~ ;"r. N Asn Aspartic acid D Asp Cysteine C Cys .... ;.. e Q Gln t'rl~t~mic acid E Glu Glycine G Gly IIi~li~LR H His i~olc~lrin~ I lle Leucine L Leu Lysine K Lys ~cthinnine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tly~Jtc~h~ W Trp Tyrosine Y Tyr Valine V Val 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 intçn-le~i unless the D-form is expressly indicated by a dagger sul)e~,lipl (t) The compounds of the invention are peptides which are partially defined in terrns of amino acid residues of de~ign~ted classes. Amino acid residues can be generally subclassified into major subclasses as follows:

SUBSTITUTE SHEE-r (RULE 26) CA 02226121 lsss-ol-os 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 co"ru""alion of a peptide in which it is co~ d when the peptide is in aqueous mç~ m 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 co"ru"llalion of a peptide in which it is contained when the peptide is in aqueous merlil-m 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 co,~l",ation of a peptide in which it is col~ ed when the peptide is in aqueous me~ m Polar/large: The residues are not charged at physiolûgical pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the col~ll"alion of a peptide in which it is conlai"ed when the peptide is in aqueous mP~illm This description also characterizes certain neutral amino acids as "small" since their side chains are not sufficiently large, even if polar groups are l~rL in~ 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 st~ti~t~ collection of individual residuemolecules some molecules will be charged, and some not, and there will be an attraction for or repulsion from an aqueous merlillm to a greater or lesser extent. To fit the definition of "charged," a ~ignific~nt percentage (at least a~)pro~;...~tçly 25%) of the individual molecules are charged at physiological pH. The degree of attraction or repulsion required for rl~.cific~ti~ n as polar or nonpolar is ~bill~y and, therefore, amino acids specifically colllelllplated by the invention have been classified as one or the other. Most amino acids not specifically named can be çl~sified on the basis of known behavior.
Amino acid residues can be further s~b~ ified as cyclic or noncyclic, and aromatic or nonalû",aLic, self-explanatory çl~c.cific~fions with respect to the side-chain substituent groups of the rç~ ç~, and as small or large. The residue is con~idçred small if it contains a total of SUBSTITUTE SH EET (RULE 26) CA 02226l2l lsss-0l-05 four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar ~ substituent is plesellL, three or less if not. Small residues are, of course, always non~u~aLic.
For the naturally occurring protein amino acids, sub~l~.c.~ifi~tiQn acco,-lh~g to the rO~goil~g scheme is as follows.

Acidic Aspartic acid and Glutamic acid Basic l'~l~,y~,lic. A~ginine, Lysine Cyclic: T-Tictit1in.o Small Glycine, Serine, Alanine, Threonine Po~ arge ~ TI-~
Hy~ G Tyrosine, Valine, T! ~ Leucine, Pl~ c~ T.,~

The gene-encoded secondary amino acid proline is a special case due to its knowneffects on the seCon~ry co"ru...,ation of peptide chains, and is not, the~ro,e, in~ ded in a group. Cysteine and other -SH co.~l~;..;ng amino acid residues are also not in~ ded in these çl~ceific~tions since their capacity to form ~liclllfi~e bonds to provide secondary structure is critical in the compounds ûf the present invention.
Certain cornrnonly encountered arnino acids, which are not encoded by the genetic code, include, for example, 13-Alanine (~-Ala), or other omega-amino acids, such as 3-aminopropionic, 2,3-~ minopropionic (2,3-diaP), 4-aminobutyric and so forth, a-aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); J-2-thienylalanine (Thi); methionine sulfoxide (MSO); and homoa-~inine (Har). These also fall conveniently into particular categories.
Based on the above clçfinitions, Sar, ~-AIa, and Aib are small, t-BuA, t-BuG, N-MeIle, Me, 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.

SUBSTITUTE 5H EET (RULE 26) CA 02226121 1998-01-0~

The various omega-amino acids are classified accoldil~, to size as small (,B-Ala and 3-all.i.,o~ )ionic) or as large and L~dlc"ohobic (all others).
Other amino acid substitutions ofthose encoded in the gene can also be inrllldrd in peptide compounds within the scope of the invention and can be çl~c~ifie(l within this general scheme accol~lh~g to their structure.
In all of the peptides of the invention, one or more amide link~es (-CO-NH-) mayoptionally be replaced with another linkage which is an isostere such as -CH2NH-, -CH2S-, -CH2CH2, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2- and -CH2SO-. This repl~cçm~nt can be made by methods known in the art. The following references describe ple;~al~LiOn of peptide analogs which include these alternative-linking m~ie~ies Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Spatola, A.F., in ChellllsLly and Bioçh~mistry of Arnino Acids Peptides and Proteins.
B. Wein~t~in~ 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 JPept Prot Res (1979) 14:177-185 (-CH2NH-, -CH2CH2-); Spatola, A.F., etal., 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., etal., JMed 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. etal. JMed 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.
One class of plere-,~d embo~limentc ofthe compounds invention includes the "unrnodified" forms where positions 8 and 13 are independently cysteine, homocysteine or penirill~mine residues, especially in the r~ lfide bonded form.

SUBSTITUTE SHEET (RULE ~6) CA 02226l2l lsss-ol-o~

In addition, or alternatively, each of A7 and Al4is a hydl ophobic acid, p,ere.~bly Ile, Val, Leu, NLe, Trp, Tyr or Phe, or is a small amino acid, Ala, Gly, Ser or Thr.
In another set of plGrelled embot~im~nts, all of Al-A3 are not present or at least one, and preferably two of Al-A3 is a hydlophol~ic amino acid, preferably Ile, Val, Leu, NLe, Trp, Tyr or Phe.
In another set of plerélled embo~im~nt~C4 and/or C1, is not present or, if present, is a cysteine, homocysteine or p~nicill~mine or a llydlophobic amino acid, plërel~bly Ile, Val, Leu,.NLe, Trp, Tyr or Phe, or a small amino acid, preferably S, A, G or T.
In another set of plerélled embo-limPnt~, C5$ and/or Cl6* is a cysteine, homocysteine or p~nic.ill~mine or a hydrophobic amino acid, preferably Ile, Val, Leu,.NLe, Trp, Tyr or Phe, or a small amino acid, plere.~bly S, A, G or T.
In another set of plerelled embo~1im~nte7 Ag-Al2 contain at least one hydlOphobic amino acid residue, plerel~bly Phe, Tyr or Trp.
Other pleÇelled embodiments include those wherein each of Al and A9isindepPn(lently selected from the group consisting of R, K and Har; more preferably, both Al and A9 are R;
however, each of Al may be absent.
In another class of p~ert:--ed embo-iim~nte, each of A2 and A3is independently selected from the group consisting of G, A,S and T; more preferably, A2 and A3 are G; however, A2 and/or A3 may be absent.
In another set of plerellèd embodim~nte, one of A9 and Al2 is R, K, Har, Orn or H
plerel~bly R and the other is I, V, L, NLe, W, Y or F, preferably R, F or W, or is S, G, A or T.
In another set of plefelled embo~1im~nt~ each of Alo and All is independently proline or a small, basic or hydrophobic amino acid, preferably R, G, W or P.
Al8 is preferably absent, but when present, is preferably E~, K or Har, most preferably R.
Also preferably when all four amino acids Al-A3 and C4 are present, Alis basic, C4 iS
C or basic, and A2 and A3 are small amino acids, or at least one of Al-A3 and C4 iS
Lydlophobic. ~lèrelled embodiments of Al-A3 include R-G-G, K-G-S, K-S-G, and the like.
As described above, the compounds of Formula (1 ) are either in cyclic or noncyclic SUBSTITUTE SH EET (RULE 26) CA 02226l2l l998-Ol-0~

(linearalized) form or may be modified wherein 1 or 2 of the cysteines, homocysteine or p~nir.ill~mine at C8 and Cl3 are replaced by a small, hydrophobic, or a basic amino acid residue. Such modification is plert;~ d when compounds con~ ;..g only one ~iisulfide bond are prepared. If the linearalized forms ofthe compound of Forrnula (1) are prep~ed, or if linearalized forms of those modified peptides which contain at least two cysteines are pl ~ ~d, it is pl c;rt;llt;d that the sulfhydryl groups be stabilized by addition of a suitable reagent. Plerellt;d embodim~nt~ for the hydrophobic amino acid to replace cysteine, homocysteine or p~nirill~min~ residues at C8 and/or C13 are I, V, L and Me, plerel~bly I, V or L. Preferred small amino acids to replace the cysteine, homocysteine or p~ni~.ill~mine residues include G, A, S and T, more preferably G. Pl~rt;ll~;d basic amino acids are R and K.
Where the compounds of the invention have two ~ lfide bridges, particularly pler~lled are the pairs of bridges:
a) C5-C16 and C8-Cl3;
b) C5-C17 and C8-CI3;
C) C5-C8 and Cl3-CI6;
d) C4-CI6 and C8-CI3; and e) C4-Cl7 and C8-CI3.
F.speçi~lly pl~rt;lled are the bridges:
C5-CI6 and C8-CI3; and C4-CI7 and C8-CI3.
Where the compound has only one di~l~lfide bridge, particularly plefelled are:
C4-CI7and C5-CI6.
Particularly plt;rt;lled compounds ofthe invention, incl~in~ the N-terminal acylated and C-terminal ~mi~l~ted forms thereof are the parevins, wherein C~and C~6 are both cysteine, homocysteine or penic~ mine and the tachytegrins wherein both C;and (:~; are cysteine, homocysteine or peni~ min~o Also prerelled are the di~ llfide forms ofthese compounds, cis-parevins wherein the two diel~!fide bridges are C5-C8 and Cl3-CI6; the trans-parevins wherein the ~lielllfi(le bridges are C5-CI6 and C8-CI3; and the trans-tachytegrins wherein the ll1fide bridges are C4-CI7 and C8-Cl3. Particularly prt;~:llc;d are the following parevins and tachytegrins:

SUBSTITUTE SH EET (RULE 26) CA 02226121 lgss-ol-o~
wo 97/02287 PCT/USg6/11323 Parevin-ltrans_ and cis R-G-G-R-i~ L-Y l-R
R
R
R-G-C-V-V-C-F
L...... l Parevin-2trans_ and cis R-G-G-R-l-L-Y l-R
R
R
R-G-C-V-I-C-F
L...... l Parevin-3trans_ and cis R-G-G-G-l L-Y-l-R
R
R
R-G-C-V-V-C-F

SUBSTITUTE SHEET (RULE 26) WO 97/02287 PCTtUS96tl 1323 Parevin-4 trans_ and cis R-G-G-R-~ L-Y-~-R
\
R
R-G-C-V-V-C-F
1...... 1 Parevin-S trans_ and cis R-G-G-R-l-L-Y-~-R
\
R
R-G-C-V-V-C-F
1...... 1 Tachyte~rin- 1 trans_ R-G-G-C-R-L-Y-C-R
R

R
R-C-G-V-V-C-F

SUBSTITUTE SHEET (RULE 26) . CAo22261211998-01-05 W0971022x7 PCT~S96/11323 Tachyte~rin-2 trans_ - R-G-G-C-R-L-Y-C-R
R
R

R-C-G-V-I-C-F

Tachyte~rin-3 trans_ R-G-G-C-G-L-Y-C-R
R

R
R-C-G-V-V-C-F

Tachyte~rin-4 trans_ R-G-G-C-R-L-Y-C-R
G
W
R-C-G-V-F-C-I

Tachyte~rin-S trans_ R-G-G-C-R-L-Y-C-R
p R
R-C-G-V-V-C-F

Particularly prt;r~,.ed are cis- and trans-parevin-l and trans-tachytegrin-l.
Typical compounds of the invention include:

Unmodified forms R-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-R-R-F-C-I-V-C-G;

SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

R-G-G-C-R-L-Y-C-R-R-R-F-C-I-V-G-C;
R-G-G-G-C-L-Y-C-R-R-R-F-C-V-V-C-G-R;
R-G-G-C-G-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-G-W-I-C-F-V-C-G-R;
R-G-G-C-R-L-Y-C-R-G-W-I-C-F-V-G-C-R;
R-G-G-R-C-L-Y-C-R-P-R-F-C-V-V-C-G-R;
R-a G-C-R-L-Y-C-R-P-R-F-C-V-V-G-C-R;
R-G-G-R-C-V-Y-C-R-R-R-F-C-V-V-C-G;
R-G-G-C-R-V-Y-C-R-R-R-F-C-V-I-G-C;
K-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G;
K-G-G-C-R-I-Y-C-R-R-R-F-C-V-I-G-C;
R-G-G-Har-C-L-Y-C-R-R-R-F-C-V-V-C;
R-G-G-C-Har-L-Y-C-R-R-R-F-C-V-I-C;
R-G-G-Har-C-L-Y-C -Har-R-R-F-C-V-V-C-G-R;
R-G-G-C-Har-L-Y-C-Har-R-R-F-C-V-I-G-C-R;
R-G-G-R-C-V-Y-C-R-Har-R-F-C-V-V-C-G-R;
R-G-G-C-R-V-Y-C-R-Har-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-K-K-W-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-K-K-W-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-Har-R-Y-C-V-V-C-G-R;
R-G-G-C -R-L-Y-C-R-Har-R-Y-C-V-V-A-C-R;
R-G-S-G-C-L-Y-C-R-R-K-W-C-V-V-C-G-R;
R-G-S-C-G-L-Y-C-R-R-K-W-C-V-V-G-C-R;
R-A-T-R-C-I-F-C-R-R-R-F-C-V-V-C-G-R;
R-A-T-C-R-I-F-C-R-R-R-F-C-V-I-G-C-R;
R-G-G-K-C-V-Y-C-R-Har-R-F-C-V-V-C-G-R;
R-G-G-C-K-V-Y-C-R-Har-R-F-C -V-I-G-C-R;
R-A-T-R-C-I-F-C-Rt-R-R-F-C-V-V-C-G-Rt;
R-A-T-C-R-I-F-C-Rt-R-R-F-C-V-V-G-C-Rt R-G-G-K-C-V-Y-C-R-Hart-R-F-C-V-V-C-G-R;

SU8STITUTE SH EET (RULE 26) CA 02226l2l lsss-ol-o~

R-G-G-C-K-V-Y-C-R-Hart-R-F-C-V-V-G-C-R;
- R-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G-R (all t);
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-R-R-F-C-I-V-C-G (all t);
R-G-G-C-R-L-Y-C-R-R-R-F-C-I-V-G-C (all t);
R-G-G-G-C-L-Y-C-R-R-R-F-C-V-V-C-G-R (all t);
R-GG-C-G-L-Y-C-R-R-R-F-C-V-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-G-W-I-C-F-V-C-G-R (all t);
R-G-G-C-R-L-Y-C-R-G-W-I-C-F-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-P-R-F-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-P-R-F-C -V-V-G-C-R;
R-G-G-C-L-R-Y-C-R-P-R-F-C-V-R-V-C-R
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-R;
R-G-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-F-R;
R-W-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-L-R;
R-G-W-R-V-C-L-K-Y-C-R-G-R-F-C-V-K-L-C-L-R;
R-G-G-R-V-C-L-R-Y-C-R-G-K-F-C -V-R-L-C-L-R;

both the linear and mono- and bicyclic forms thereof, and incl-l-lin~ the N-terminal acylated and C-terminal ami(~ted forms;
Particularly pl~re-led are the cyclic forms and C-terminal ~mid~ted forms of R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C -R
R-G-G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-C-R-P-R-F-C-V-R-V-C-R
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C -R;
R-G-V-C-L-R-Y-C-R-G-R-F-C -V-R-L-C-R;
R-G-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C -F-R;
R-W-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-L-R;

SUBSTIl UTE SHEET (RULE 26) CA 02226121 lsgs-ol-o~

R-G-W-R-V-C-L-K-Y-C-R-G-R-F-C-V-Kl-C-L-R;
R-G-G-R-V-C-L-R-Y-C-R-G-K-F-C-V-R-L-C-L-R;

Modified forms R-G-G-R-C-L-Y-A-R-R-R-F-A-V-V-C-G-R;
R-G-aR-C-L-Y-A-R-R-R-F-S-I-V-C;
R-G-G-G-C-L-Y-S-R-R-R-F-A-V-V-C-G-R;
R-G-G-R-C-L-Y-A-R-R-R-F-G-V-V-C;
K-G-G-R-C-L-Y-V-R-R-R-F-I-V-V-C;
R-G-G-Har-C-L-Y-A-R-R-R-F-V-G-C-V;
R-G-G-Har-C-L-Y-A-Har-R-R-F-S-V-V-C-G-R;
R-G-G-C-Har-L-Y-A-Har-R-R-F-S-V-V-G-C-R;
R-G-G-R-C-V-Y-V-R-Har-R-F-L-V-C-V-G-R;
R-G-G-R-C-L-Y-S-R-K-K-W-A-V-S-C-G-R;
R-G-G-R-C-L-Y-S-R-Har-R-Y-S-V-I-C-G-R;
R-G-S-G-C-I-Y-C-R-R-K-W-G-V-V-aC-R;
R-A-T-R-C-I-F-S-R-R-R-F-S-V-V-C-G-R;
R-G-G-K-C-V-Y-G-R-Har-R-F-S-V-V-C-G-R;
R-A-T-R-C-I-F-G-Rt-R-R-F-G-V-V-C-G-Rt;
R-G-G-K-C-V-Y-L-R-Hart-R-F-L-V-V-C-G-R;
R-G-G-R-C-V-F-L-R-P-R-I-G-V-V-C-G-R;
R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-F -C -L-R-Y-T-V-P-R-F-T-V-R-F-C -V-R;
R-G-F-C-L-R-Y-K-V-GR-F-K-V-R-F-C-V-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG);
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C -R;

SUBSTITUTE SHEET (RULE 26) CA 02226l2l l998-Ol-0~

R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X---NMeG) both the linear and cyclic (where possible) forms thereof, and inclllding the N-terrninal acylated and C-terminal ~mi~l~ted forms.
Particularly plert;l-ed are the cyclic forms and C-terrninal ~mit~te~ forms of R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C-R
R-a G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (~=NMeG);
R-G-F-C-L-R-Y-T-V-P-R-F-T-V-R-F-C-V-R;
R-G-F-C-L-R-Y-K-V-G-R-F-K-V-R-F-C-V-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG);
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG) Pl ~al ~Lion of the Invention Compounds The invention compounds are ess~nti~lly peptide backbones which may be modified at the N- or C-terminus and also may contain one or two cystine diclllfide lin~s The peptides may first be synthP~i~ed in noncyclized forrn. These peptides may then be converted to the cyclic peptides if desired by standard methods of cystine bond forrnation. As applied to the compounds herein, "Gyclic forms" refers to those forms which contain cyclic portions by virtue of the formation of dielllfide linkages between cysteine residues in the peptide. If the straight-chain forrns are prerel I ed, it is pl ert;l ~ble to stabilize the sulfhydryl groups for any peptides of the invention which contain two or more cysteine residues.
- Standard methods for synthesis of peptides can be used. Most commonly used currently are solid phase synthesis techniques; indeed, automated equipment for syst~m~tic~lly constructing peptide chains can be purchased. Solution phase synthesis can also be used but is considerably less convenient. When synth~ei~ed using these standard techniques, amino acids not encoded by the gene and D-lo.n~ntiom~rs can be employed in the synthesis. Thus, one very SUBSTITUTE SHEET (RULE 26) , CA 02226121 1998-01-0~
WO 97/02287 PCT/US96tll323 practical way to obtain the compounds of the invention is to employ these standard chemi~
sy,l~lle~is techniques.
In addition to providing the peptide backbone, the N- and/or C-terminus can be de~iva~i~ed, again using conv~ontion~l rh~m;-~l techniques. The compounds of the invention may optionally contain an acyl group, ~ ;rel~bly an acetyl group at the amino terrninus.
Methods for acetylating or, more generally, acylating, the free amino group at the N-terrninus are generally known in the art; in addition, the N-terminal amino acid may be supplied in the syll~hesis in acylated form.
At the carboxy terrninus, the carboxyl group may, of course, be present in the form of a salt; in the case of pharm~ce~lti~ l compositions this will be a pharm~ce~-tic~lly acceptable salt. Suitable salts include those formed with inol ~,anic 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 m~ltipli~.ity of basic resi~ e~, 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 ~rni(l~ted so as to have the formula -CONH2, -CONHR, or -CONR2, wherein each R is independently hydrocarbyl (1 -6C) as herein df~fine~ 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.
Formation of ~ -lfi(le link~es, if desired, is conrl~lcted in the presence of rnild oxitli7:in~ agents. Chemical oxidizing agents may be used, or the compounds may simply be exposed to the oxygen of the air to effect these link~es. Various methods are known in the art. Processes useful for ~ lfide bond formation have been described by Tam, J.P. et al., Synthesis (1979) 955-957; Stewart, J.M. et al., "Solid Phase Peptide Synthesis" 2d Ed. Pierce Chemic~l Company Rockford, IL (1984); Ahmed A.K. et al., J Biol Chem (1975) 250:8477-8482 and Pennin~on M.W. et al., Peptides 1990, E. Giralt et al., ESCOM Leiden, The Netherlands (1991) 164-166. An additional alternative is described by Karnber, B. et al., Helv SUBSmUTE SH EET (RULE 26) CA 02226121 1998-01-0~

2~7 PCT/US96/11323 Chim Acta (1980) 63 :899-915. A method con~ cted on solid supports is described by Albericio Int J Pept Protein Res (1985) 26:92-97.
A particularly prer~ d method is solution oxidation using molecular oxygen. Thismethod has been used by the inventors herein to refold the compounds of the invention.
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 synthPci7~d using recollll)inalllDNA techniques. The DNA en~o-ling the peptides of the invention may itself be ,y"l1~ee;,.ed using collllllelc;ally available equipl-,ell~, codon choice can be illlegl,lLed into the synthesis depending on the nature of the host.
Synfh~ci7ed and lecol.,b;l.al.Lly produced forms ofthe compounds may require subsequent derivatization to modify the N- and/or C-terminus and, depending on the isolation procedure, to effect the formation of cystine bonds as described hereinabove. Depending on the host organism used for recombi.~ production, some or all of these conversions may already have been effected.
For I eco..ll~;nal.L production, the DNA encoding the peptides of the invention is incl~ldecl in an ~ ,ression system which places these coding sequences under control of a suitable promoter and other control sequences colllpalible with an intçn~led host cell. Types of host cells available span almost the entire range of the plant and animal kingdoms. Thus, the compounds 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 resi~ strains) as well as in animal cells, insect cells and plant cells. Indeed, modified plant cells can be used to regenel~e plants co.,~ g the relevant e,.,u~es~ion systems so that the resulting transgenic plant is capable of self protection vis-à-vis these infective agents.
The compounds of the invention can be produced in a form that will result in their secretion from the host cell by fusing to the DNA encoding the peptide, a DNA encoding a suitable signal peptide, or may be produced intrac~ rly. They may also be produced as fusion proteins with additional amino acid seq~l~nce which may or may not need to be subsequently removed prior to the use of these compounds as ~ntimiçrobials or antivirals.
Thus, the compounds of the invention can be produced in a variety of modalities in~.hlrling ~h~mic~l synthesis, recolllbin~l~ production, isolation from natural sources, or some SUBSTITUTE S}IEET (RULE 26) CA 02226l2l l998-Ol-0~

co...l)i..~tion ofthese techniques.
Any In~ Cl:i ofthe invention class which coinsidpnt~lly occur naturally must be supplied in purified and i~ol~ted form. By "purified and isolated" is meant free from the en~,i,ol,..,~;"l in which the peptide normally occurs (in the case of such naturally occurring peptides) and in a form where it can be used practically. Thus, "purified and isolated" form means that the peptide is s~ lly pure, i.e., more than 90% pure, pr~fe.~bly more than 95% pure and more preferably more than 99% pure or is in a completely di~ltillL context such as that of a pharm~ce~ltic~l preparation.

Antibodies Antibodies to the peptides of the invention may also be produced using standard immllnological techniques for production of polyclonal antisera and, if desired, immortalizing the antibody-producing cells ofthe i"....~.;,e(l host for sources of monoclonal antibody production. Teçhniqllec for producing antibodies to any substance of interest are well known.
It may be necessa,y to e l~h~nse the immnnogenicity of the substance, particularly as here, where the material is only a short peptide, by coupling the hapten to a carrier. Suitable carriers for this purpose include substances which do not Ihe,,,selves produce an immnne l~spollse in the .. A~.. ~I to be a-lmini~t~red the hapten-carrier conjugate. Common carriers used include keyhole limpet hemocyanin (KLH), diphtheria toxoid, serum ~lknmin~ and the viral coat protein of rotavirus, VP6. Coupling of the hapten to the carrier is ~ffected by standard techniques such as cont~ting the carrier with the peptide in the presence of a dehydrating agent such as dicyclohexylcarbodiimide or through the use of linkers such as those available through Pierce Chemical Company, Chicago, IL.
The peptides ofthe invention in immllnogenic form are then injected into a suitable ",A.. ~ n host and antibody titers in the serum are monitored. It should be noted, however, that some forrns of the peptides require modification before they are able to raise antibodies, due to their reei~t~nse to antigen processing. For example, peptides co..~ g two cystine bridges may be nG~ n-)genic when ~ el ed without coupling to a larger carrier and may be poor imml-nogens even in the presence of potent adjuvants and when coupled in certain formats such as using glutaraldehyde or to KLH. Any lack of immnnogenicity may SUBSTITUTE SHEET (RULE 26) CA 02226l2l lsss-ol-o~
WO 97/02287 PCT/US96tll323 ll~elerc"e result from rÇ~iet~nre to procç~ing to a linear form that can fit in the antigen-plese~ g pocket ofthe ~lese~l;..g cell. Tmmllnogenicity ofthese forrns ofthe peptides can be enh~ncecl by cleaving the ~ fi~le bonds.
Polyclonal antisera may be harvested when titers are s -ffiei~ntly high. Alternatively, antibody-producing cells ofthe host such as spleen cells or peli~hel~l blood lymphocytes may be harvested and hlllllol Lalized. The immortalized cells are then cloned as individual colonies and s~ elled for the production of the desired monoclonal antibodies.
Reco".l,i.lallL techniques are also available for the production of antibodies, and thus, the antibodies of the invention include those that can be made by genetic ç~ ee~ ;,-g techniques. For eY~m~le, single-chain forms, such as Fv forms, chimeric antibodies, and antibodies modified to mimic those of a particular species, such as hllm~n~ can be produced using sLalldaud metho~5 Thus, the antibodies ofthe invention can be prepared by i~ol~ting or modifying the genes encoding the desired antibodies and producing these through e,~les~ion in recombill~,L host cells, such as CHO cells.
The antibodies ofthe invention are, of course, useful in immllnoassays for dele.l.~ g the amount or presence of the peptides. Such assays are ~c~.nti~l in quality controlled production of compositions co..l~ g the peptides ofthe invention. In addition, the antibodies can be used to assess the efficacy of l~co--.bi"~,l production of the peptides, as well as sclee,fillg eAp,~ssion libraries for the presence of peptide encoding genes.

Compositions Co..~ the Invention Peptides and Methods of Use The peptides of the invention are effective in inactivating a wide range of microbial and viral targets, in~ tling gram-positive and gram-negative bacteria, yeast, protozoa and certain strains of virus. Acco~L"gly, they can be used in di~i"re-;La,lL compositions and as preservatives for materials such as foodstuffs, co~metiÇs7 medic,....~ i, or other materials ~ cor.l~;.. i~-g nutrients for o~ . For use in such contexts, the peptides are supplied either as a single peptide, in admixture with several other peptides of the invention, or in admixture with additional antimicrobial agents or both. In general, as these are preservatives in this context, they are usually present in relatively low amounts, of less than 5%, by weight of the total composition, more pr~elably less than 1%, still more preferably less than 0.1%.

SU8STITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

The peptides ofthe invention are also useful as ~L~Idalds in ~ntimir.robial assays and in assays for delellllillaLion of capability of test compounds to bind to endotoxins such as lipopolysaccharides.
For use as ~ntimirrobials or antivirals for lle.,~ of ani~nal subjects, the peptides of the invention can be form~ ted as pharm~ce-ltic~l or veterinary compositions. Depending on the subject to be treated, the mode of ~ lion, and the type of 11 e ~ desired -- e.g., prevention, prophylaxis, therapy; the invention peptides are forrmll~ted in ways consollarl with these p~ clers. A summary of such teçhni~ es is found in Remin~on's Pharrn~- e~ltic~
Sçiencee, latest edition, Mack Publishillg Co., Easton, PA.
The peptides of the invention can also be used as active ingredients in pharrn~ceuti compositions useful in l, ~ of sexually L~ ed ~liceA~es~ inr l~lding those caused by Chlamydiatrachomatis, Treponemap~ ~ Neisseria~ J"hoeae, Trichomonas vaginalis, Herpes simplex type 2 and HIV. Topical formulations are ~crc~ed 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 L,~ or prophylaxis of STDs, the peptides of the invention may be used alone or in col,lbhlalion with other antibiotics such as erythlolllycill, tetracycline, macrolides, for example azill~lul"yci" and the cephalosporins. Depending on the mode of arl-";--;~ lion, the peptides will be formlll~ted into suitable compositions to permit facile delivery to the affected areas. The tachytegrins may be used in forms c~ ;..;..g one or two disulfide bridges or may be in linear form. In addition, use of the enantiomeric forms co..~ g all D-amino acids may confer advantages such as r~eict~nce to those proteases, such as trypsin and chymol,y~ , to which the peptides co~ L-amino acids are less reci~t~nt The peptides of the invention can be ~ , ed singly or as mixtures of severalpeptides or in co",bh~dLion with other pharm~t~e~lti~ lly active components. The formulations may be prep~ed in a manner suitable for systemic ~mini~tration or topical or local lion Systemic formulations include those designed for injection (e.g., intr~mll~clll~r, intravenous or subc~lt~n~oll~ injection) or may be p,eya,ed for transdermal, tr~n~mllcosal, or oral a~ lion. The formulation will generally include a diluent as well SU8STITUTE SHEET (RULE 26) CA 02226l2l l998-Ol-0~

as, in sorne cases, adjuvants, buffers, preservatives and the like. The tachytegrins can be mini~tçred also in liposomal compositions or as microemulsions.
If ~imini~tration is to be oral, the peptidles of the invention must be plote~;Led from degradation in the stomach using a suitable enteric coatin~ This may be avoided to some extent by lltili7ing amino acids in the D-configuration, thus providing ~ nre to protease.
However, the peptide is still susceptible to hydrolysis due to the acidic con~iition~ of the stom~ch; thus, some degree of enteric coating may still be required.
The peptides of the invention also retain their activity against microbes in the context of borate solutions that are commonly used in eye care products. Also, it is i..,po~ that the peptides retain their activity under physiological conditions incl~ltling relat*ely high saline and in the presence of serum. In addition, the peptides are dr~m~tir.~lly less ~;ylolo~ic with respect to the cells of higher or~ni~m~ as colllpal ~d with their toxicity to microbes. These pl.~pel lies, make them particularly suitable for in vivo and the-~l~eu~ic use.
By app. up.iately choosing the member or ~ )tl :i of the peptide class of the invention, it is possible to adapt the ~ntimir.robial activity to .~ .;...;7e its effectiveness ~,-vith respect to a particular target microbe. As used herein, "microbe" will be used to include not only yeast, bacteria, and other unicellular org~nicm~, but also viruses. The particular peptide used can also be chosen to be advantageous in a particular context, such as low salt or physiological salt, the presence or human serum, or conditions that mimic the conditions found in blood and tissue fluids.
The peptides ofthe invention may also be applied to plants or to their en-vho--l--e--~ to prevent virus- and microbe-inrl-lced ~ e~eçs in these plants. Suitable compositions for this use will typically contain a diluent as well as a spreading agent or other ancillary a~ l eç~ s b~nPfici~l to the plant or to the ellvilulllllc;llL.
Thus, the peptides of the invention may be used in any context wherein an antimicrobial and/or antiviral action is required. This use may be an entirely in vitro use, or the peptides may be ~(lmini~tered to ol~
In addition, the ~ntimi~robial or antiviral activity may be generated in situ bymini~t~ring an ~ u- es~ion system suitable for the production of the peptides of the invention.
Such e~,-es~ion systems can be supplied to plant and animal subjects using known SUBSTITUTE SHEET (RULE ~6) CA 02226121 1998-01-0~

te~hniq~les. For example, in ~nim~le, pox-based c,.l~lession vectors can be used to generate the peptides in situ. Similarly, plant cells can be ~ r~,.,.ed with ~ s:,ion vectors and then ene~led into whole plants which are capable oftheir own production ofthe peptides.
The peptides of the invention are also capable of inactivating endotoxins derived from gram-negative bacteria -- i.e., lipopolysaccharides (LPS) and may be used under any circ~ ee where inactivation of LPS is desired. One such eit~l~tiQn is in the llc~ .r~ll or amelioration of gram-negalive sepsis.

Conditions Relevant to AntimicrobiaVAntiviral Activity It has been stated above that as used herein "antimicrobial" activity refers to inhibition with respect both to traditional m-icroorg~ llle and to viruses, although occasionally, "5mtimjcrobial" and "antiviral" are both specifically in-1ic~ted.
Media for testing antimicrobial activity are de~ei~ned to m-imic certain specific conditions. The standard buffer metiillm, me~ m A, uses an underlay agar with the following composition: 0.3 mg/ml oftrypticase soy broth powder, 1% w/v agarose and 10 mM sodium phosphate buffer (final pH 7.4). This will be c~e.ei~n~ted either "merlillm A" or "standard in vitro conditions" herein.
All ofthe ~c~ ;..g media contain these same components. However, in addition:
A second metlillm co.-L~i--s 100 mM NaCI in order to mimic the salt levels in blood and tissue fluids. This will be dçei~n~ted "medillm B" or "salt mP~ lm" herein.
A third merlillm is supplemented with 2.5% normal human serum; however, it is of low ionic strength and thus does not mimic body fluids. This me-iillm will be cleei n~ted "me~illm C" or "serum-co..~ me~illm~ herein.
A fourth me~ m co.-L~ns 80% RPMI-1640, a standard tissue culture m~dium which co..lai..s the principal ions and amino acids found in blood and tissue fluids. In addition, it cont~ine 2.5% norrnal human serum. This will be desi~n~ted "medi~lm D" or "physiological m~ m" herein.
Particularly prere,.ed is the ~mi~ted form ofthis peptide.

SUBSTITUTE SHEET ~RULE 26) CA 02226l2l lsss-ol-o~

Summary The peptides ofthe invention thelerole lep,ese ll a peculiarly useful class of compounds because of the following prope. lies:
1) They have an ~ntimicrobial effect with respect to a broad SpC~,~IUlll oftarget microbial systems, inr.lllrling viruses, in~ din~ retroviruses, bacteria, fungi, yeast and pro~ozoa.
2) Their ~ c~ ol)ial activity is effective under physiological con-iitiQns - i.e., physiological saline and in the presence of serum.

3) They are much less toxic to the cells of higher or~nicmcth~n to microbes.

4) They can be prepared in no~immllnogenic form thus ~,Yt~ntlin~ the number of species to which they can be ~i., .;.,i~lp., ed.
S) They can be plepaled in forms which are resistant to certain proteases s~ gesting they are ~ntimicrobial even in Iysosomes.
6) They can be prepared in forrms that resist degradation when autoclaved, thus simplifying their prep~lion as components of pharm~ce~ltic~lc.
7) They can be modified in amino acid sequence so as to oplillll e the specificity with respect to target.
8) They can be modified structurally so as to accommodate the conditions under which ~ntimicrobial activity is to be exhibited.

The following examples are int~n~1ecl to illustrate but not to limit the invention.

Example I
Synthesis of the Invention Compounds The peptides of the invention are synthçci~ed using conventional Fmoc ~.h~.mictry on solid-phase supports. The crude synthetic peptides are refolded, purified and characterized as follows.
The crude synthetic peptide is reduced by adding an amount of dithiothreitol (DTT) equal in weight to that of the synthetic peptide, which has been dissolved at 10 mg/ml in a solution cc"~li.;..;..g 6M ~l~ni~ine HCI, 0.5M Tris buffer and 2 mmol EDTA, pH 8.05 and SUBSTITUTE SHEET (RULE 26) CAo22261211998-01-05 W097/02287 PCT~S96/11323 incl~h2ted for 2 hours at 52~C under nitrogen. The rnixture is passed through a 0.45 um, filter, ~ci~ified with 1/20 v/v glacial acetic acid and subjected to conventional RP HPLC purification with a C 18 column.
The HPLC-purified, reduced peptides are partially concentrated by vacuum centrifugation in a Speed Vac and allowed to fold for 24 hours at room telll?~ re and air.
The folding is accomplished in 0. lM Tris, pH 7.7 at 0.1 mg peptide/rnl to .~ e formation of i,l~elcl~ cystine di~ ~lfides. The folded compounds are concentrated and acidified with 5%
acetic acid. The purity of the final products is verified by AU-PAGE, analytical HPLC and FAB-mass spec.
Using this procedure, the compounds trans-parevin-l (or the "hairpin" isoform), cis-parevin-l ~or "cloverleaf" isoforrn) and trans-tachytegrin-l were prepared. These compounds are of the formulas Trans-parevin- 1:
R-G-G-R-C-L-Y-C-R
R

R
R-G-C-V-V-C-F

Cis-parevin- 1:

R-G-G-R-C-L-Y-~-R
R
R
R-G-C-V-V-C-F
L...... l SUBSTITUTE SHEET (RULE 26) CA022261211998-01-0~

W097/02287 PCT~S96/11323 Trans-tachyte~rin- 1:
R-G-G-C-R-L-Y-C-R
R

R

R-C-G-V-V-C-F

Example 2 Antimicrobial Activity The radial diffusion assay in agarose gels is con~ ted using radiodiffusion and gel overlay techniques as described by Lehrer, R.I. et al. J Tmm~lnol Meth (1991) 137:167-173.
Briefly, the underlay agars used for all o~ had a final pH of 7.4 and co..~ çd lO mM
sodium phosphate buffer, 1% w/v agarose and 0.30 ug/ml tryptocase soy broth powder (BBL
Cockeysville, MD). In some cases, the underlay was suppllo.m~.nted with 100 mM NaCI. The units of activity in the radial diffusion assay were measured as described; 10 units COIIespond to a 1 mm ~ meter clear zone around the sample well. Figures 1-6 show the results against five test or~ni~m~ in units des~,libed as above. A synthetic protegrin (PG-l) cont~inin~ two cystines (sPG-l) or PG-l in linear form were used as controls.
Figure 1 shows the results for trans-parevin and tachytegrin with respect to E coli both with and without the addition of l OO mM NaCI. Both of these peptides were slightly more effective than sPG-1 although slightly less effective than linear PG-1 in the absence of salt. However, in the presence of l OO mM NaCl, all four peptides were colllpal ~bly effective.
Figure 2 shows the results of the same detel.n,llalion with respect to L. monocytogenes. With respect to this organism, all four peptides were roughly similarly effective in the absence of salt; the presence of 100 mM NaCI, however, greatly reduced the effectiveness of linear PG-1. The Ir.."~il.;..g three peptides rem~ined effective under these conditions.
Figure 3 shows the results of the same experiment using C. albicans as the target olg~islll. All four peptides were conlpal~bly effective in the absence of salt; again, the effectiveness of linear PG- l was greatly reduced in the pl esence of 100 mM NaCl, while the ,~."~ g three peptides ..-~ ed their effectiveness under these conditions.

SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

Figures 4-6 show the results of similar expelinlcllls using, as test peptides, the two isomers of parevin, trans-parevin (hairpin) and cis-parevin (cloverleaf). sPG- 1 was used as a control. As shown in Figure 4, the two parevins were colllpal ~bly effective in the absence of salt and both were more effective than sPG- 1. In the presellce of 100 mM NaCI, all three peptides ...~ d their effectiveness and were col~ ble.
Figure S shows results of the same e;A~lhllent con~ cte(~ with B. subtilis as target olg~islll. Again, both forms of parevin were colllp~bly effective and both were slightly more effective than sPG-l; in the presence of 100 mM NaCI, all three peptides r~.m~ined effective ~ntimicrobials and had about the same activity.
The results obtained with respect to S. typ*imurium are also similar, as shown in Figure 6. Again, the two parevins were more effective than sPG1 in the absence of salt and all three peptides had collll al~ble effectiveness when 100 mM NaCI was added.

Example 3 Ability to Bind Endotoxin The compounds of the invention are tested for their ability to bind the lipid polysaccharide (LPS) of the gram-negative bacterium E. coli strain 0.55B5, using the Limulus amebocyte Iysate (LAL) test for endotoxins con~ucte~l in the presence and absence of the test compounds. The test is cond~lcted using the procedure desclibed in Sigma Technical Bulletin No. 210 as revised in December 1992 and published by Sigma Chemic~l Company, St. Louis, MO.
The LAL test is based on the ability of LPS to effect gelation in the commercialreagent E-ToxateO which is plepaled from the Iysate of circ~ ting amebocytes of the Horseshoe Crab ~int~ pol.~ ",us. As described in the teçhnicAI bulletin, when exposed to minute quantities of LPS, the Iysate h~ eases in opacity as well as viscosity and may gel depending on the concentration of endotoxin. The technical bulletin goes on to speculate that the mec.l1~..i.c.-- appears analogous to the clotting of ".A~",-AliAn blood and involves the steps of activation of a trypsin-like preclotting enzyrnes by the LPS in the presence of calcium ion, followed by enzymic modifications of a "coagulogen" by proteolysis to produce a clottable protein. These steps are believed tied to the biologically active or "pyrogenic" portion of the SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~
wO 97/02287 PCT/US96/11323 molecllle. It has been shown previously that detc xified LPS (or endotoxin) gives a negative LAL test.
The test compounds are used at various conce~ LLions from 0.25 ug-10 ug in a final volume of 0.2 ml and the test llli~lu.es conlahled LPS at a final conc~ Lion of 0.05 endotoxin unit/ml and E-Toxate~M at the sarne cQncf;~ lion. The test co~ oullds are in-.~tb~tecl together with the LPS for 15 mimltes before the E-ToxateTM is added to a final volume af'~er E-ToxateTM addition of 0.2 ml. The tubes are then inc~lb~ted for 30 rninutes at 37~C and ex~mined for the formation of a gel.
In a ffillow-up cA~e,illlent, the concentration of LPS is varied from 0.05-0.25 endotoxin units (E.U.).

Example 4 Antimicrobial Activit,v Under Conditions Suitable for Tl e~ of the Eye Contact lens solutions are typically form~ ted with borate buffered physiological saline and may or may not contain EDTA in addition. The compounds of the invention are tested generally in the assay described in E~ ,le 2 ~Leleill all underlay gels contain 25 mM
borate buffer, pH 7.4, 1% (v/v) trypticase soy broth (0.3 ug/ml TSB powder) and 1% agarose.
Additions include either 100 mM NaCI, 1 mM EDTA or a co",l)illalion thereof. Other test compounds used as controls are the defensin NP-l and lysozyme, and dose response curves are dete",lined.

Example 5 Plep~lion of Enanfio Trans-parevin Using standard solid phase techniques, a peptide having the amino acid sequence of ~ trans-parevin, but wherein every amino acid is in the D form is prepared. This form is tested against E. coli, L. monocytogenes, C. albicans and other microbes in the absence and presence of protease and otherwise as described for the radiodiffusion assay in agarose gels set forth in Example 2.

SUBSTITUTE SHEET (RULE 26) CA 02226121 1998-01-0~

Example 6 Activity A~ainst STD Patho~ens The colllpounds of the invention are tested for ~ntimic robial activity against various STD pathogens. These include E~V-1, Chlamydia l,dc~lv,,.atis, Treponema palli~um, Neisseriag~J"u"h~eae, Trichomonas vaginalis, Herpes simplex type 2, Herpes simplex type 1, Hemophilus ducreyi, and Human papilloma virus. The results are provided in a form whelc;ill "active" means that the peptide is effective at less than 10 ug/ml; moderately active in~iç~tes that it is active at 10-25 ug/ml; and slightly active means activity at 25-50 ug/ml. If no effect is obl~ined at 50-200 ug/ml the compound is corl~id~red inactive.
The compounds of the invention are tested for their ~ntimirrobial activity against Chlamydia using the "gold standard" chlamydial culture system for clinical ~,l,e~
described by Clarke, L.M. in Clinical Microbiology Procedures Handbook II (1992), Isenberg, H.T. Ed. Am. Soc. Microbiol. W~hin~on, D.C.; pp. 8Ø1 to 8.24.3.9.
In the assays, C. trachomatis serovar L2 (L2/434Bu) described by Kuo, C.C. et al. in Nongl,~nococcal Urethritis and Related Infections (1977), Taylor-Robinson, D. et al. Ed. Am.
Soc. Microbiol. Washington, D.C., pp. 322-326 is used. The seed is pl~paled from a sonic~ted culture in L929 mouse fibroblast cells, and partially purified by centrifugation. Since host protein is still present in the seed aliquots, each seed batch is titered at the time of pl~ion with serial ten-fold dilutions to 2 x 10~. The seed co..~ ng 9.2 x 106 IFlJ/ml is thawed quickly at 37~C and diluted to 10-2 with sucrose/phosphate salts/glycine to produce IFU of about 200 after room temperature pr.?inc~b~tion and to dilute background eukaryotic protein.
In the initial assays, the peptides to be tested are prepared as stock solutions in 0.01%
glacial acetic acid. 100 ul ofthe diluted chlall,y-lial seed are aliquoted into 1.~ ml eppendorf tubes and 200 ul of the antibiotic peptide was added per tube. Aliquots of the peptide stock (and controls) are inc~lb~ted with the seed at room te.,.,)e-~ re for one hour, two hours and four hours. About 10 mimltes before the end of each incub~tion period, ,,.~ el~ ce media are aspi~led from the McCoy vials in p-e~a ~lion for standard inoculation and culture.
Culture is then pelrc,lllled in the presence and absence of the peptides; in some cases, the peptides are added to final concentration in the culture media in addition to the preculture SUBSTITUTE Sl IEET (RULE 26) CA 02226l2l l998-Ol-0~

in~ b~tion. The test is evaluated microscopically.
- In another series of cAI~G~ lcllL~, various collc~ntrations of tachyleg,in (1 ug, 12.5 ug, 25 ug and 50 ug) are used in the two-hour pr~inc~ ation.
The effect ofthe presence of serum is also tested. The Chlamydia seed is pr~inrl~b~ted for two hours with and without 10% FBS and also with or without test compound at 25 ug.
The expe~ lellLs are re~eaLed but adding 25 ug of compound after the start of the chlamydial culture, i.e., after centrifugation and final medil~m mix and one hour into the beginning of the 48-hour culture period. Finally, the colllpo~nd (at 25 ug) is added to the chlamydial seed and the mix then immçdi~t~ly cultured.
The effect of serum is particularly important since for a topical agent to be effective in c~ Chl~mydia infection~ it must act in the pl~sGnce of serum.
In addition, there are several mouse-based models for Chlamydia infection which can be used to assess the efficacy of the tachytegrins. These include those described by Patton, D.L. et al. in Chlamydial Infections (1990) Bowie, W.R. et al. Eds. Cambridge Universproviding a source of complement. Ten ul of a suspension of T. pallidum co.~ -g about 5 x 107/ul or~nicm.C is added to each tube and the mixtures with the apploplia~e peptides are inrllb~ted at 34~C under 95% N2 and 5% CO2. At time zero, just prior to in-,ub~tion, 4 hours and 16 hours, 25 randomly s~lected orf~;~ni.cmc are ~ ..;..ed for the presence or absence of motility. The 50% immobilizing end point (IE50) iS r~ ted to inrlic~te the conct;n~ ion needed to immobilize 50% ofthe spirochetes. Tacl.y~Jle~i-- IE50s are 5.231 ug and 2.539 ug for 0 and 4 hours, in contrast to HNP and NP plt;p~alions which show little immobilizing ability.
For Herpes Simplex Virus, using viral stocks p. t;pal ed in VERO cells, grown inminim~l es~enti~l m~ m (~M) with 2% fetal calf serum, the effect of various peptides on HSV 1 MacIntyre strain, a pool of ten clinical HSV 1 isolates, HSV-2G, and a pool of ten ~ clinical HSV 2 isolates, all sensitive to 3 uM acyclovir are tested. Two fibroblast cell lines, human W138 and equine CCL57, are used as targets and tests are done by direct viral neutr~li7~tic-n and delayed peptide addition.
In the direct neutralization format, the virus is pr~inc~lbated with the peptides for 90 min before it is added to the tissue culture monolayers. In the delayed peptide addition SUBSTITUTE SH EET (RULE 26) CA 02226121 1998-01-0~

format, the virus is added and allowed 50 min to adsorb to the target cells, then the monolayers are washed and peptides are added for 90 min. Finally, the monolayer is washed to remove the peptide and the cells are fed with peptide-free MEM and cultured until the untreated h~iled monolayers exhibit 4+ ~iylopalllic effect (CPE) (about 60 hours).
For Trichomonas vaginallis, strain Cl (ATCC 30001) is grown as described by Gorrell, T.E. etal., C~l~be.~, Res Comm (1984) 49:259-268. In ~,~elilllc;llL~ pelrulllled in RPMI + 1% heat-activated fetal calf serum, within a few mim~t~s after exposure to S0 ug/ml PG-l, T. vaginallis (heretofore vigolously motile) becollles ~Liona,y. Soon thereafter, the Olg~ become permeable to trypan blue, and, over the ensuing 15-30 min~lte~ Iyse. As c,~l.e.;Led, such org~ .C fail to grow when introduced into their customary growth m~ lm (Diamond's medium). o~ ",.c exposed to 25 ug/ml of PG-3 retain their motility.

Example 6 ~ ;Llo~iral Activity The invention compounds are tested for antiviral activity against strains of HIV using the method described in Miles, S.A. et al., Blood (1991) 78:3200-3208. Briefly, the mononuclear cell fraction is recovered from normal donor leukopacs from the Arnerican Red Cross using a Ficoll-hypaque density gradient. The mononuclear cells are resuspended at 1 x 106 ce11s per ml in RPMI 1640 me~ m with 20% fetal bovine serum, 1% penn/strep with fullgiGol1e and 0.5% PHA and inrllb~ted 24 hours at 37~C in 5% COz. The cells are centrifuged, washed and then exp~n-led for 24 hours in growth me~ lm Non-laboratory adapted, cloned HIV~R~SF and ~V.1R-FL are ele~llopol~led into thehuman peripheral blood mononllr.le~r cells plepared as described above. Titers are delel,l,illed and in general, multiplicities of infection (MOI) of about 4,000 infectuous units per cell are used (which cc,llt;s~ollds to 25-40 picograms per ml HIV p24 antigen in the S~ ;lllaLa"l).
In the assay, the HIV stocks prepared as above are diluted to the correct MOI and the PBM are added to 24 well plates at a concentration of 2 x 106 per ml. One ul total volume is added to each well. The peptide to be tested is added in growth merli-lm to achieve the final desired conce~ lion. Then the a~ rc,plial~ number of MOI are added. To assay viral growth, 200 ul of supelllalalll is removed on days 3 and 7 and the concentration of p24 SUBSTITUTE SHEET (RULE 26) CA 02226121 lsss-ol-o~

wo 97/02287 PCT/US96/11323 antigen is d~ ed using a commercial assay (Coulter Tmml-nology, Hialeah, Florida).
Controls include ~llrlic~te wells co~ cells alone, cells plus peptide at S ug/ml cells with virus but not peptide and cells with virus in the pre~nce of AZT at 10-5 M - 10-8 M.
The time of addition of peptide can be varied. Cells preLl~,a~ed for 2 hours prior to addition of virus, at the time of ad(litiQn of virus, or 2 hours after infection show antiviral activity for the peptide.
Example 7 P~ Lion and Activity of Tachytegrins Several illustrative tachytegrins were synth~ ed as described in Example 1 and tested for activity against Staphylococcus aureus (~SA), Ps~ nnas (Psa), VREF, Candic~a and E.coli as described in Example 2. The results shown in Table 1 as minim~l inhibitory concentration (MIC) in ug/ml were obtained with the C-terminal ~mitl~te~l forms except for the last two which were tested as the free acids, as in~ ted by *.

SEQUENCE MRSA Psa ¦VREF¦Candida¦ E. Coli RGGCLRYAVPRFAVRVCR >128 0.05 RGCCLRYTKPKFTVRVCR
RCCCLRYAVGRFAVRVCR
RC(CLRYARXRFAVRVCR (X=NMeG) >32 5.7 RC-:'CLRYTVPRFTVRFCVR 1.88 0.57 0.99 RCFCLRY~XVGRFKVRFCVR >64 2.7 R(FCLRYXVt--RFXVRFCVR (X=NMeG) RC-GCLRYCRPRFCVRVC:. 9.8 0.18 9.68 0.2 RC-GCRLYCR:.RFCW GC~ 53.3 3.3 4 4 RGVCLRYCRGRFCVRLC............. 8 2 RGRVCLRYCRCRFCV.LCF:. 6.7 RWRVCLRYCRtRFCV.LCL:. 4 4 R~WRVCLKYC.~R:'CJKLC_R
RC-GRVCL:.YCRt-K'CVRLC_R 8 0.75 :.CGCLRY~RXR:'AVRVC. (X=NMeG) .. GGCLRYAVGR:'AVRVC. >32 5.3 :.GFCLRYXVGR:'XVRFCVR(X=NMeG) >32 12 SUBSTITUTE SHEET (RULE 26) -

Claims (40)

Claims

1. A purified and isolated, or synthetically or recombinantly produced, compoundcontaining the amino acid sequence:

A1-A2-A3-C*4-C*5-C*6-A7-C8-A9-A1o-A11-A12-C13-A14--C*15-C*16-C*17-A18 (1) said compound containing 11-24 amino acid residues, wherein the amino acid sequence of formula (1) may be extended at the N and/or C terminus by additionalnoninterfering amino acids;
and the N-terminal acylated and/or C-terminal amidated or esterified forms thereof, which is either in the optionally -SH stabilized linear or in a disulfide-bridged form wherein each of A1-A3 is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid;
wherein each of C4*, C5 *, C6*, C15*, C16* and C17* is independently a cysteine,homocysteine or penicillamine or a basic, hydrophobic, polar/large, or small amino acid, and C4* and/or C17* may be present or not present and C6 and/or C15 may also be acidic;
C8 and C13 denote cysteine, homocysteine or penicillamine;
each of A7 and A14 is independently a hydrophobic or a small amino acid;
A9-A12 must be capable of effecting a .beta.-turn when contained in the compoundand at least one of A9-A12 must be a basic amino acid;
A18 is present or not present, and if present, is a basic, hydrophobic, polar/large, or small amino acid; or said compound containing a modified form of the sequence of formula (I) wherein one or both of C8 and C13 is independently replaced by an acidic, basic, hydrophobic, polar/large, or small amino acid;
wherein at least about 15% to about 50% ofthe amino acids are basic amino acids, and the compound has a net positive charge of at least +1 at physiological pH;
with the proviso that at only one of C4*, C5*, and C6*, and only one of C15*, C16*
and C17* can be cysteine, homocysteine or penicillamine and at least one of C4*, C5* C16* and C17*

must be cysteine, homocysteine or penicillamine.

2. The compound of claim 1 which contains two disulfide bridges.

3. The compound of claim 1 which contains one disulfide bridge.

4. The compound of claim 1 which is in the linear form.

5. The compound of claim 1 wherein A, and A14 are hydrophobic.

6. The compound of claim 1 wherein C4* and C17* are independently cysteine, homocysteine or penicillamine.

7. The compound of claim 6 wherein C4* and C17* are linked by a disulfide bridge.

8. The compound of claim 1 wherein C5* and C16* are independently cysteine, homocysteine or penicillamine.

9. The compound of claim 8 wherein C5* and C16* are linked by a disulfide bridge.

10. The compound of claim 1 wherein at least one of A9 and A12 is hydrophobic orsmall.

11. The compound of claim 1 wherein A10 and A11 is each independently proline, abasic, a hydrophobic, or a small amino acid.

12. The compound of claim 1 in the unmodified form wherein each C shown at positions 8 and 13 is present independently as a cysteine, homocysteine or penicillamine.

13. The compound of claim 12 wherein C8 and C13 form a disulfide bridge.

14. The compound of claim 1 wherein at least one of A1-A3 is not present.

15. The compound of claim 14 wherein all of A1-A3 are absent.

16. The compound of claim 1 wherein at least one of A1-A3 is hydrophobic.

17. The compound of claim 1 wherein each of C5* and C16* is independently selected from the group consisting of C, homocysteine, penicillamine, I, V, L, NLe, W, Y, F, A, S, G and T.

18. The compound of claim 1 wherein each of C4* and C17* is independently selected from the group consisting of C, homocysteine, penicillamine, I, V, L, NLe, W, Y, F, A, S, G and T.

19. The compound of claim 1 wherein each of A7 and A14 is independently selectedfrom the group consisting of I, V, L, NLe, W, Y, F, A, S, G and T.

20. The compound of claim 1 where one of A9 and A12 is R, K, Har, Orn or H and the other is I, V, L, NLe, W, Y, F, A, S, G or T.

21. The compound of claim 1 which is selected from the group consisting of RGGRCLYCRRRFCVVCGR
RGGCRLYCRRRFCVVGCR
RGGRCLYCRRRFCVVGCR
RGGCRLYCRRRFCVVCGR
RGGRCLYCRRRFCVCVGR
RGGCRLYCRRRFCVCVGR
RGGRLCYCRRRFCVVCGR
RGGRLCYCRRRFCVVGCR

RGGCLRYAVPRFAVRVCR
RGGCLRYTKPKFTVRVCR
RGGCLRYAVGRFAVRVCR
RGGCLRYARXRFAVRVCR (X=NMeG);
RGFCLRYTVPRFTVRFCVR;
RGFCLRYKVGRFKVRFCVR;
RGFCLRYXVGRFXVRFCVR (X=NMeG);
RGGCLRYCRPRFCVRVCR
RGGCRLYCRRRFCVVGCR;
RGVCLRYCRGRFCVRLCR;
RGRVCLRYCRGRFCVRLCFR;
RWRVCLRYCRGRFCVRLCLR;
RGWRVCLKYCRGRFCVKLCLR;
RGGRVCLRYCRGKFCVRLCLR;
RGGCLRYARXRFAVRVCR (X=NMeG);
RGGCLRYAVGRFAVRVCR; and RGFCLRYXVGRFXVRFCVR (X=NMeG) and the amidated forms thereof either in linear or disulfide-bridged form.

22. The compound of claim 21 which is selected from the group consisting of RGGRCLYCRRRFCVVCGR;
RGGCRLYCRRRFCVVGCR;
RGGCRLYCRRRFCVVGC;
RGGGCLYCRRRFCVVCGR;
RGGCGLYCRRRFCVVGCR;
RGGRCLYCRRRFCVVCGR;
RGGCRLYCRRRFCVVGCR;
RGGRCLYCRPRFCVVCGR; and RGGCRLYCRRRFCVVGCR
and the amidated forms thereof either in linear or disufide-bridged form.

23. The compound of claim 22 which is selected from the group consisting of RGGRCLYCRRRFCVVCGR; and RGGCRLYCRRRFCVVGCR
and the amidated forms thereof either in linear or disulfide-bridged form.

24. The compound of claim 21 which is selected from the group consisting of RGGCLRYAVPRFAVRVCR
RGGCLRYTKPKFTVRVCR
RGGCLRYAVGRFAVRVCR
RGGCLRYARXRFAVRVCR (X=NMeG);
RGFCLRYTVPRFTVRFCVR;
RGFCLRYKVGRFKVRFCVR;
RGFCLRYXVGRFXVRFCVR (X=NMeG);
RGGCLRYCRPRFCVRVCR
RGGCRLYCRRRFCVVGCR;
RGVCLRYCRGRFCVRLCR;
RGRVCLRYCRGRFCVRLCFR;
RWRVCLRYCRGRFCVRLCLR;
RGWRVCLKYCRGRFCVKLCLR;
RGGRVCLRYCRGKFCVRLCLR;
RGGCLRYARXRFAVRVCR (X=NMeG);
RGGCLRYAVGRFAVRVCR; and RGFCLRYXVGRFXVRFCVR (X=NMeG) and the amidated forms thereof either in linear or disulfide-bridged form.

25. The compound of claim 24 which is RGRVCLRYCRGRFCVRLCFR; or the amidated form thereof, either in linear or disulfide-bridged form.

26. The compound of claim 1 wherein all amino acids are in the D-configuration.

27. A recombinant expression system for production of an antimicrobial peptide containing the amino acid sequence of the compound of claim 1 which expression system comprises a nucleotide sequence encoding said peptide operably linked to control sequences for effecting expression.

28. A recombinant host cell modified to contain the expression system of claim 27.

29. A method to produce an antimicrobial or antiviral peptide or intermediate peptide therefor which method comprises culturing the modified host cells of claim 28 under conditions wherein said peptide is produced; and recovering the peptide from the culture.

30. The method of claim 29 which further comprises effecting disulfide linkages of said peptide and/or modifying the N-terminus and/or C-terminus of said peptide.

31. A pharmaceutical composition for antimicrobial or antiviral use which comprises the compound of claim 1 in admixture with at least one pharmaceutically acceptable excipient.

32. A composition for application to plants or plant environments for conferring resistance to microbial or viral infection in plants which comprises the compound of claim 1 in admixture with at least one environmentally acceptable diluent.

33. A method to prevent the growth of a virus or microbe which method comprises contacting a composition which supports the growth of said virus or microbe with an amount of the compound of claim 1 effective to prevent said growth.

34. A method to inactivate the endotoxin of gram-negative bacteria, which method comprises contacting said endotoxin with an amount of the compound of claim 1 effective to inactivate said endotoxin.

35. Antibodies specifically reactive with the compound of claim 1.

36. A method to treat a microbial or viral infection in a subject which method comprises administering to a subject in need of such treatment an amount of the compound of claim 1 effective in the composition in which it is contained to ameliorate said infection in the subject.

37. The method of claim 36 wherein the microbial infection is oral mucositis.

38. The method of claim 36 wherein the microbial infection is of Staphylococcus aureus.

39. The method of claim 36 wherein the microbial infection is a Pseudomonas infection.

40. The method of claim 36 wherein the microbial infection is an infection ofH. pylori.