CA1340716C - Inhibition of eucaryotic pathogens and neoplasms and stimulation of fibroblasts and lymphocytes with lytic peptides - Google Patents

Abstract

Inhibition of eucaryotic pathogens and neoplasms and stimulation of lymphocytes and fibroblasts with lytic peptides such as cecropins and sarcotoxins. Eucaryotic cells are contacted with cecropin or sarcotoxin, or a synergistic combination of cecropins or sarcotoxin with lysozyme, in an amount effective to lyse or inhibit the cells. Target cells include eucaryotic microorganisms such as protozoa, e.g. T. cruzi and P. falciparum, mammalian lymphomas and leukemias, and cells infected with intracellular pathogens such as viruses, bacteria and protozoa. Also disclosed is a method for stimulating proliferation of lymphocytes and fibroblasts by contacting such cells with an effective amount of cecropin or sarcotoxin. The methods may be in vitro or in vivo.

Description

INHIBITION OF EUCARYOTIC PATHOGENS
AND NEOPLASMS AND STIMULATION OF FIBROBLASTS
AND LYMPHOCYTES WITH LYTIC PEPTIDES
Field of the Invention The present invention relates to lytic peptides, their use in methods for inhibiting eucaryotic pathogens, cancer cells and intracellularl~r infected cells, and their use in methods for 5 stimulating the proliferation of fibroblasts and lymphocytes.
More particularly, this invention relates to the inhibition of such pathogens, cancers and infected cells, and the stimulation of fibroblasts and lymphocytes in mammals and other higher animals.
Bi

-2-Background of the Invention Many diseases of procaroytic origin, i.e. caused by pathogenic bacteria, are known. Such diseases are in general more easily treated than those of eucaryotic 5 origin because of the marked differences between the invading proc:aryotes and the eucaryotic cells of the host.
Thus, because of the differences between bacterial cells and those of the host, many antibiotics are known to specifi~~ally inhibit the invading bacteria without 10 significant adverse effects on the host. In contrast, it has generally been more difficult to treat diseases of nonbacterial origin, such as malaria, sleeping sickness and Chagas disease.
Thcs property of certain peptides to induce lysis of 15 procaryotic microorganisms such as bacteria are known.
For example, LJ.S. patents 4,355,104 and 4,520,016 to Hultmarlc et al describe the bacteriolytic properties of some cecropins against Gram-negative bacteria. Quite interesi:ingly, the cecropins described in the Hultmark et 20 al patents were not universally effective against all Gram-negative bacteria. For example, the cecropins described therein lysed Serratia marcescens D61108, but not Serratia marcescens D611. Moreover, cecropins have heretofore been reported to have no lytic activity towards 25 eucaryol:ic cells such as insect cells, liver cells and sheep eythrocytes, as reported in the Hultmark patents;
Internai:ional. Patent Publication WO/8604356; Andreu et al, Biochemistry, vol. 24, pp. 1683-88 (1985); Boman et al, Developmental. and Comparative Immunology, vol. 9, pp.
30 551-558 (1985); and Steiner et al, Nature, vol. 292, pp.
246-248 (1981).
Other lytic peptides heretofore known include, for example, the sarcotoxins and lepidopterans. Such peptides general7.y occur naturally in the immune system of 35 Sarcophy ~eregrina and the silkworm, lepidopteran, respectively, as reported in Nakajima et al, The Journal 1340'16

-3-_of Biol.ogical Chemistry, vol. 262) pp. 1665-1669 (1987) and Nakai et a:l, Chem. Abst. 106:214351w (1987).
The mechanism of action of the lytic peptides in the immune systems in which they occur is not entirely clear.
5 There must, of course, be some aspect of the mechanism which regulates the specificity of the lytic peptides for invading pathogens among the cells of the host organism which must generally be preserved from lysis. For example, human complement fixation involves antibodies 10 which are generally specific for certain antigens expressed by the invading pathogen. The activated components of complement attack the membrane of the invading cell to which they are bound by the antigen.-antibody reaction to produce circular lesions 15 which are probably formed as a result of insertion of the C9 protein into the membrane. The more primitive mechanisms involved in insect immunology are less specific, but the peptides involved apparently do not significantly lyse the host cells.
20 There are many differences between the membranes of different types of cells which can affect their susceptibility to lysis by the various lytic peptides. As suggested above, for example, some proteins are capable of lysing only cells expressing an appropriate antigen for 25 the antibody associated with such protein. Thus, it is riot surprisi.nc~ that the less specific lytic peptides such as cecropins are more capable of lysing procaryotes than the euc:aryotic: cells of the insect.
Gram-positive procaryotes generally have a thicker 30 cell wall than Gram-negative ones. Also, Gram-positive cell mc~mbrar~e:c have a cytoplasmic membrane and a cell wall containing mostly peptidoglycans and teichoic acids, whereas Gram-negative cell membranes have an inner cell wall consisting entirely of peptidoglycan and associated 35 proteins surrounded by an outer cell wall comprised of Lipid) lipopolysaccharide and protein. In contrast, eucaryotic cells generally have a plasma membrane 1340'16

-4-comprising a lipid bilayer with proteins and carbohydrates interspersed therein, and also have organelles with their own membrane systems, but generally do not have an outer cell wall. xt is readily appreciated that the

5 considerable variation of membrane structures among bacteria (proc~aryotes) accounts for considerable variation in their susceptibility to lysis by the various insect immune :proteins.
The variation of membrane structures among eucaryotes 10 is also considerable, but these membranes generally comprise phospolipid molecules in a bilayer arrangement with a thickness of about 50A. The hydrophilic portion of the pho;spholip:id is generally oriented to the exterior and interior surfaces of the membrane, while the hydrophobic 15 portion's are generally found in the interior region of the membrane between the hydrophilic surfaces. As reported in Nakajima et al, the presence of cholesterol and the assymet:ric distribution of phospholipids in the cytopla:amic membrane of eucaryotic cells may explain the 20 selective toxicity of sarcotoxin to bacteria. Since cholesterol causes condensation of the phospholipid bilayers, it can hinder the penetration of lytic peptides into tree cytoplasmic membrane of eucaryotic cells.
Similar:Ly, the predominance of neutral phospholipids in 25 the outer monolayer of eucaryotic membranes would result in less affinity to positively charged lytic peptides such as cecropin and sarcotoxin than acidic phospholipids general:Ly located on the cytoplasmic side of the membrane.
A :number of the antibacterial polypeptides have been 30 found to be useful when the genes encoding therefor are incorporated into various plant species. Particularly, when introduced into the plant genome by means of an Agrobaci~erium plasmid vector, the antibacterial palypepl_ide-encoding genes produce plant species much more 35 resistant to certain bacterially induced disease conditions and plant pathogens. Such antibacterial polypepi=ides and the transformation of plants with genes encoding therefor are described in PCT Published Patent Application WO88/00976 (published February 11, 1988).
Polynucleotide molecules expressible in a given host and having the sequence araB promoter operably linked to a 5 gene which. is het:erologous to such host. are also known.
The heterologous gene codes for a biologically active polypeptide. A genetic construct of a first genetic sequence coding for cecropin operably linked to a second genetic sequence coding for a polypeptide which is capable 10 of suppressing the biological effect of the resulting fusion protein towards an otherwise cecropin-sensitive bacterium is also described in International Publication W086/04356, July :31, 1986.
The Eiultmark et al patents mentioned above also 15 mention tY.at there are no known antibodies to cecropin, indicating a wide acceptability for human and veterinary applications, including one apparently useful application for surface infections because of the high activity against pseudomonas. Similarly, EPO publication 182,278 20 ( 1986 ) men.tions that sarcotoxins may be expected to be effective in pharmaceutical preparations and as foodstuff additives, and that antibacterial activity of sarcotoxin can be recognized in the presence of serum. Shiba, Chem.
Abstr. 104: 230430K (1985) also mentions preparation of an 25 injection containing 500 mg lepidopteran, 250 mg glucose and injection water to 5 ml.
Several analogs of naturally-occurring cecropins, sarcotoxin> and lepidopterans have been reported. For example, it is reported in Andreu et al, Proc. Natl. Acad.
30 Sci. USA, vol. 80, pp. 6475-6479 (1983) that changes in either end of the amino acid sequence of cecropin generally result in losses in bactericidal activity in varying degrees against different bacteria. It is reported in Andreu et al (1985) mentioned above that Trp2 35 is <:learl~r important for bactericidal activity of cecropin, .and that other changes in the 4, 6 or 8 position have different effects on different bacteria. Frpm the i

-6-data given in Table II at page 1687 of Andreu et al (1985), it appE:ars that almost any change from natural cecropin generally adversely affects its bactericidal activity.
Cecropin is defined in International Publication 5 W086/04356 t:o include bactericidally active polypeptides from any insect species and analogs, homologs, mutants, isomers and derivatives thereof having bactericidal activity from 1% of the naturally-occurring polypeptides up to 100 i:imes or higher activity of the 10 naturally-occurring cecropin. Other references generally discuss the effects of the a-helix conformation and the amphiph.ilic nature of cecropin and other lytic peptides.
It. is known that lysozyme and attacins also occur in insect homolymph. For example, it is reported in Okada et 15 al, Biochem. J., vol. 229, pp. 453-458 (1985) that lysozym.e participates with sarcotoxin against bacteria, but that the bactericidal actions are diverse. Steiner et al mentioned above suggests that lysozyme plays no role in the antibacterial activity of insect hemolymph other than 20 to remove debris following lysis of bacteria by cecropin.
Merrifield et al, Biochemistry, vol. 21, pp. 5020-5031 (1982) and Andreu et al (1983) mentioned above state that cecropin purified from insect hemolymph may be contaminated with lysozyme, but demonstrate that the 25 synthetically prepared cecropin is as bactericidally active as purified cecropin from insect hemolymph.
Summary' of the Invention It. has now been found that lytic peptides are effective against certain eucaryotic cells which are a 30 source of disease in higher animals. Lytic peptides are capable of lysing protozoa, fungi, cancer cells and eucaryotic cells infected with an intracellular pathogen;
and, yet, by appropriate selection of the lytic peptide, will not generally lyse the normal cells of the host 35 animal. Thus, lytic peptides can be used in vitro to lyse t:he membranes of certain eucaryotic cells. More i.mporta.ntly, t:he lytic peptides can be used in vivo to X3407.16 treat or prevent cancer and pathogenic diseases of eucaryotic origin in higher animals. This discovery is quite surprising and unexpected in view of the apparently unanimous conclusions of prior researchers that lytic peptides do not lyse eucaryotic cells.
Also quite surprisingly, it has been found that certain lytic peptides such as, for example, the cecropins, a,re effective to stimulate the proliferation of mammalian fibroblasts and lymphocytes. Thus, the 10 cecropins are useful in enhancing the production of products obi~ainec! from the in vitro culturing of such cells. More importantly, the cecropins can be used in vivo in the treatment of mammals to accelerate the regenerative processes associated with disease and injury 15 by stimulating lymphocytes and fibroblasts in the injured mammal.
Accordingly, the invention provides a method for lysing euca~-yoti<: cells which includes contacting the cells with a lytic peptide in an amount effective to lyse 20 the cells. The cells are eucaryotic microorganisms, lymphomas, l.eukemias or carcinomas, or eucaryotic cells infected with an intracellular pathogenic microorganism.
The lytic peptide has from about 30 to about 40 amino acids, at least a portion of which are arranged in an 25 amphiphilic a-helical conformation. The peptide has a substantially hydrophilic head with a positive charge density and a substantially hydrophobic tail. The conformation has a predominantly hydrophobic face along the length of the conformation and a predominantly 30 hydrophilic face opposed therefrom.
The invention also provides a method for selectively lysing eucaryotic cells in the presence of cells which are not lysed. The method includes contacting target eucaryotic cells in the presence of non-target cells with 35 a selectively lytic, free peptide in an amount effective to lyse the target cells. The target cells are eucaryotic microorganisms, lymphomas, leukemias or carcinomas, or 1340'16 eucaryotic cells infected with an intracellular pathogenic microorganism. The lytic peptide contains from about 30 to about 40 amino acids, at least a portion of which are arrang~sd in an amphiphilic a-helical conformation.
5 I:n another aspect, the invention provides a method for lysing eucaryotic microorganisms which includes contacting the eucaryotes with an amount of a lytic peptid~s effeci:ive to lyse the microorganisms. The peptide includes from about 30 to about 40 amino acids, at least a 10 portion of which are arranged in an amphiphilic a-helical conformation.
In still another aspect, the invention provides a method for lysing cancer cells. The method includes contacl:ing lymphoma, leukemia or carcinoma cells with an 15 effective amount of a lytic peptide to lyse the cells.
The peptide has from about 30 to about 40 amino acids at least a portion of which are arranged m an amphiphilic a-helical conformation.
Further, the invention provides a method for 20 selectively lysing infected eucaryotic cells. The infected eucaryotic cells are infected with an i.ntrace~llular pathogenic microorganism, such as, for example:, virus, bacteria, fungi or protozoa. The method includes contacting the infected and uninfected cells with 25 a selecaively lytic, free peptide in an amount effective to selectively lyse the infected cells and leave the uninfected cells substantially free of lysis.
Still further, the invention provides a method for inhibiting euc:aryotic cells in a higher animal. The 30 method includes introducing a selectively lytic, free peptide into the higher animal in an amount effective to inhibit therein eucaryotic cells such as eucaryotic microorganisms, mammalian lymphomas) leukemias or carcinomas, or cells infected with an intracellular 35 pathogenic microorganism. , Still further, the invention provides a method for stimulating the proliferation of normal mammalian 1340'716 _g_ fibrobla:>ts and lymphocytes which includes contacting the fibroblasts or :Lymphocytes with a stimulating peptide in an amount effective to stimulate the proliferation thereof. There is also provided a method for stimulating 5 the proliferation of normal fibroblasts and lymphocytes in a mammal which .includes introducing a stimulating peptide into a mammal in an amount effective to stimulate the proliferation of fibroblasts or lymphocytes therein.
In other aspects, the invention provides a 10 synergistic bactericidal composition containing lytic peptide and lysozyme, and novel lytic peptides.
Brief De~;cription of the Drawincls Fig. 1 is an Edmundson helical wheel construct for cecropin B.
15 Fig. 2 is an Edmundson helical wheel construct for cecropin SB-37.
Fig. 3 is an Edmundson helical wheel construct for cecropin A.
Fig. 4 is an Edmundson helical wheel construct for 20 cecropin D.
Fig. 5 is an Edmundson helical wheel construct for Shiva 1.
Fig. 6 is an Edmundson helical wheel construct for lepidopte:ran.
25 Fig. 7 is an Edmundson helical wheel construct for sarcotoxin lA.
Fig. 8 is an Edmundson helical wheel construct for sarcotoxin 1B.
Fig. 9 is <in Edmundson helical wheel construct for 30 sarcotoxin 1C.
Description of the Invention It has been found that lytic peptides having about 30-40 amino acids are capable of lysing or otherwise inhibiting euca:ryotic cells which are used for the 35 production of biological products and/or are involved in the disease cr illness of higher animals. Such eucaryot.ic cells include, for example, protozoa, fungi, algae, cancer cells such as lymphomas, leukemias and carcinomas, and cells infected with intracellular fungi, bacteria, protozoa or viruses. On the other hand, certain lyt:ic peptides have also been found to stimulate the proliferation of lymphocytes and fibroblasts.
As used herein, the term "lytic peptide" includes any polypeptide which lyses the membrane of a cell in an in vivo or in vitro system in which such activity can be measured. Suitable lytic peptides used in the present invention have lytic activity toward one or more eucaryotic cells such as protozoa, fungi, helminths, leukemias, lymphomas or carcinomas, or cells infected by intracellular pathogens. Preferred lytic peptides have from about 30 to about 40 amino acids, but can inclu~9e peptides of fewer amino acids, at least a portion of which are arranged in an amphiphilic a-helical confo:rm;ation having a substantially hydrophilic head with a positive charge'density, a substantially hydrophobic t;~i:l, and a pair of opposed faces along the length of the he=_lical conformation, one such face being predominantly hydrophilic and the other being predominantly hydrophobic. The head of this conformation rnay be taken as either the amine terminus end or t:ze carboxy terminus end, but is preferably the amine terminus end.
A ";>elect:ively lytic peptide" is a lytic peptide which preferentially lyses target cells in a system comprising bot=h target and non-target cells, wherein the=_ target cells are selected from protozoa, fungi, mammalian leul:ernias, lymphomas and carcinomas and eucaryotic cells infected by intracellular pathogens.
The selective7_y lytic peptides used in the present methods are preferably "free peptides", i.e. undirected in action by an antibody and otherwise unbound or unfused t=o another molecular fragment which adversely affects its lyti.c activity.
Suitable lytic peptides generally include cec ropins such as cecropin A, cecropin B, cecropin D, and lepidopteran; sarcotoxins such as sarcotoxin IA, sarcotox:in IB, and sarcotoxin IC; and other polypeptides obtainable from the hemolymph of any ira ect species which have lytic activity against bacteria similar to that of the cecropins and sarcotox:ins. It is also contemplated that lytic peptides may be obtained as the lytically active portion of larger peptides such as attacins; lysozymes;.
certain phage proteins such as S protein of ~ phage, E
protein of PHIX 1.74 phage and P13 protein of P22 phage;
and C9 protein of human complement. As used herein, classes~of lytically active peptides such as, for example, "cercopins", "sarcotoxins" and "phage proteins", and specific peptides within such classes, are meant to include the lytically active analogues, homologues, mutants or isomers thereof unless otherwise indicated by context. Of these exemplary lytic peptides, those having fewer than about 30 amino acids such as the melittins are generally unsuitable in the present invention because of their lack of specificity as indicated by their hemolytic potential, whereas those with more than about 40 amino acids such as attacins and lysozymes are generally not sufficiently lytic to be of use in the present invention. On the other hand, those having between about 30 and about 40 amino acids, such as cercopins and sarcotoxins are generally more :preferred because of their specificity for lysing target cells over non-target cells, but they are lytic peptides of fewer than 30 amino acids that are suitable for the present invention.
Hydrophilic amino acids generally include and generally hav~= the respective relative degree of hydrophobicityy (at pH 7.0; kcal/mol) as follows:
aspartic acid (:D), -7.4; glutamic acid (E) -9.9;
asparagine (N), -0.2; glutamine (Q), -0.3; lysine (K), -4.2; arginin~= (R), -11.2; serine (S), -0.3; and cysteine (C), -2.8. Hydrophobic amino acids generally include and generally have the respective relative -degree of hydro:phobicity as follows: histidine (H), 0.5; threonine (T), 0.4; tyrosine (Y), 2.3; tryptophan (W), 3.4; phenylalanine (F), 2.5; leucine (L), 1.8;
isoleucine (I), 2.5; methionine (M), 1.3; va.line (V), 1.5; and alanine (A), 0.5. Glycine has a relative degree of hydrophobicity of 0 and may be considered to be hydrophilic or hydrophobic.
The amino acid homology of peptides can be readily 5 determined by contrasting the amino acid sequences thereof as is known i.n the art. Similarly, the amphiphilic homology of peptides can be determined by contrasting the hydrophilicity and hydrophobicity of the amino acid sequences. The amino acid sequences of various preferred 10 lytic peptides are compared in Table 1, with their degree of homology to cecropin B indicated by underscoring homologous amino acids. The hydrophobic and hydrophilic conformational faces of lytic peptides are readily observed by constructing an Edmunson helical wheel which 15 is a superimposed-type end view of the peptide with the side chains of the amino acids of the peptide arranged in their relative axial positions which would be assumed in an a-he:Lical conformation. Helical wheels constructed for the peptides listed in Table I are seen in Figs. 1-9.
20 These helical wheel constructs illustrate the hydrophilic face h and the hydrophobic face H of each peptide, the numbering of each sequential amino acid appearing in a shaded circlf: to represent a hydrophobic amino acid or in an unsh<ided circle to represent a hydrophilic amino acid.

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15 . 1340716 Cecropin B is a potent bacteriolytic peptide which occurs naturally and can be obtained from insects as described in the Hultmark et al patents mentioned above, by direct peptide synthesis, or from genetically 5 transformer! host. cells as described in the aforementioned Publication WO/08fi/04356. The amino acid sequence of cecropin I3 is given in Table 1 and the hydrophilicity/hydrophobicity of each amino acid in its sequence i~, indicated immediately therebelow. The helical 10 wheel. construct oi: cecropin B seen in Fig. 1 illustrates that fourteen of the sixteen amino acids on the hydrophobic face are hydrophobic, while eleven of the twenty amino acids on the hydrophilic face are hydrophilic', for a total of eleven "imperfections". It is 15 contemplated that the removal or replacement of G1y23 and Pro24 would result: in a more lytic.peptide with only six imperfections in the amphiphilic helical conformation. In addition, proline is known to disrupt the helical conformation, and its removal may permit a more helical 20 conformation and, hence, more lytic activity. Note that the helica7_ wheel constructs for cecropin SB-37, cecropin D and Shiv~~ 1 have been constructed assuming that the end region prol_ines would disrupt the a-helical conformation there and this is indicated by placing proline and the 25 preceding amino acids outside the "wheel."
Cecropin S13-:37 is an analogue which was prepared using a peptide synthesizer and is about as lytically active as c~ecropin B. It has 94% homology therewith in its amino ;acid :sequence and its amphiphilicity. As seen 30 in the helical wheel construction of Fig. 2, two of the sixteen amino acids are hydrophilic on the hydrophobic face, while nine of twenty are hydrophobic on the hydrophilic: face. It is similarly contemplated that if G1y23 and 1?ro24 were removed or replaced, there would be 35 only five :imperfections in the amphiphilic conformation, and thus be: more lytically active.

Similarly, the other naturally occurring lytic peptides cecropin A, cecropin D, lepidopteran, sarcotoxin lA, sarcotoxin 1B and sarcotoxin 1C fit the amphiphilic helical conformation of cecropin B as indicated in Table 1 5 and Figs. 3, 4 and 6-9. With the exception of cecropin A
which is about as lytically active as cecropin B and SB-37, these peptides are generally less lytically active against eucaryotes than cecropin B. However, it is likewise contemplated that the lytic activity thereof may 10 be improved by removing amino acids from the sequence thereof, for ~°_xample, Val2~ and Ile2° from cecropin D or G1y23 and Pro24 from lepidopteran or cecropin A.
Anol:her peptide designated herein as "Shiva 1" was prepared usin<I a peptide synthesizer and has the amino 15 acid sequence indicated in Table 1 and the corresponding helical wheel construction seen in Fig. 5. While this peptide teas only a 46% amino acid homology with cecropin B, its amphiphilic homology therewith is 100%. Quite surprisingly, however, Shiva 1 is generally much more 20 lyt.ically active than cecropin B, and it is contemplated that its lytic activity may be further enhanced by removal or replacement of Glyz3 and Pro24) A cecropi.n SB-37 homologue designated herein as "*cecropin SB-37" identical thereto except for the 25 substitution of glutamic acid in the fourth position (for threonine; come>sponding to the second position of cecropin B) and lysine in the eighth position (for leucine; corresponding to the sixth position in cecropin B). Substitutions in these positions may reduce the lytic 30 activity of the cecropin SB-37 against procaryotes by as much as 90% as reported in Andreu et al (1985) mentioned above. Quite surprisingly, however, it has been found that the lytic activity of *cecropin SB-37 against eucaryotes such as eucaryotic microorganisms is generally 35 comparable. to c:ec:ropin SB-37.
The lytic peptides may be used alone, or in combination with other lytic peptides. It has been found _1~_ 13 4 0 ? 16 that the lytic activity of the peptides used in the present method may be used to synergistically enhance the lytic activity of a less lytic peptide which may not otherwise be sufficiently lytic to be used in the present methods. For example, the lytic activity of cecropin or sarcotoxin is synergistically enhanced when used in combination with lysozyme. In general, the lytic activity of a mixture of 10 moles lysozyme and 0.1-100 moles cecropin, preferably 1-10 moles cecropin, is more lytically active than a molar' equivalent of either cecropin or lysozyme alone. Such synergistic blends may be used not only to lyse or inhibit eucaryotes, but also bacteria. 1't is contemplated that. such a mixture can be advantageously used in pharmaceutical preparations containing a pharmaceutical carrief for administration to man or other higher animals, in foodstuffs and other products as an antibacterial preservative, and in agricultural applications, for example, in a spray applied in an effective amount to crops to prevent infection by, or to inhibit plant. pathogens.
The present method is effective to lyse various types of eucaryot:ic cells such as eucaryotic microorganisms, mammalian neoplastic cells and cells infected with intracellular pathogenic microorganisms. Eucaryotic microorgani~;ms include, for example, fungi such as yeasts, and protozoans such as sarcodina, mastigophora, ciliata and sporozoa. The lytic peptides are particularly effective against Trypanosoma cruzi and Plasmodium falciparum 'which a.re the causitive agents of Chagas disease and malaria, respectively, and are also contemplated as being particularly effective against Trypanosoma gamb:iense which is the causative agent of African sleeping sickness. The method of the present invention is useful to lyse or inhibit cancer cells such as lymphomas, leuke:mias and carcinomas, and particularly mamma7.ian cancer cells of these types.
-~. _v ~i -18- 134071fi Suitable infected eucaryotes subject to lysis or inhibition according to the present method include cells infected with intracellular pathogenic microorganisms such as viruses, bacteria, fungi, or protozoans. Specific representative examples of such pathogens, the host cells of which are: contemplated as suitable for lysis or inhibition according to the present method, include protozoa such as P. falciparum, T_. cruzi, bacteria such as Listeria monocytogenes, Brucella abortus and viruses such as parainfluenza, measles and herpes simplex II. The method is particularly effective for the treatment of DNA
virus-infected cells, such as herpes simplex II. Such pathogens grow or replicate within the infected cell and are generally protected from inhibition or lysis by the membranE: of the host cell. However, the method of the present invention results in lysis of the host cell so that the intracellular pathogen is subsequently destroyed or subject to lysis or inhibition with the lytic peptides of the present invention or another inhibiting agent since it is no longer protected by the host cell membrane.
In accordance with the present method, the lytic peptide is used to lyse or otherwise inhibit eucaryotic cells by contacting the cells with the peptide. The amount of the l.ytic peptide necessary to induce cell lysis will usually be sufficient to provide a peptide concentration in the medium containing the cells of at least 1 NM, but it is contemplated that less than this amount may be sufficient in some circumstances. On the other hand, lytic peptide concentrations above about 200 NM mill usually not significantly improve cell lysis, a7.though concentrations higher than 200 NM can be used.
Preferably, the lytic peptide concentration is in the range of.' 20-200 ~~M, and especially 50-100~NM.
In a system in which the cells contacted with the lytic peptide are cultured or grown for obtaining biological or biochemical products therefrom, a cytopla~~mic product may be desirably recovered from the lysed cells. For example, the recovery of a product such as interferon m<zy be facilitated by treating cells producing the product with a lytic peptide in combination with cytoske:letal formation inhibitor such as cytochalasin B, avoiding the use of detergents which complicates purification of the desired product.
In a preferred embodiment, the target cells to be lysed are l;~rsed or otherwise inhibited in the presence of non-target cells. For example, the target cells may be an in vitro culture, mixture, or suspension, or may be target cells in a higher animal host, particularly chordate animals and chordate or nonchordate aquacultural animals, and especially mammals such as man. In such a situation, some degree of care is exercised in the selection othe lytic peptide and its concentration to avoid substantial. lysis or inhibition of the non-target cells. For use in vivo, an amount of lytic peptide in the range of from about one mg/kg to about 100 mg/kg is usually sufi:icient to effect the desired inhibition and avoid substantial. :inhibition of the non-target or host cells, although this dosage may be increased or decreased, or repeated in a series of applications. The peptide may be introduc<:d directly into the higher animal in any conventional manner, e.g. by injection of the peptide in a pharmaceutically acceptable carrier intramuscularly, subcutaneously, intravenously, or intraperitoneally, and preferably at or near the site of infection or cancer.
Where there may be a relatively high incidence of target cells in the host, particularly in advanced stages of infection or cancer, additional caution should be exercised since rapid lysis of such a large number of target cells may induce a host reaction to the products of the lytic or inhibitory activity.
Although the present invention is not bound or limited by theory, it is believed that the membranes of the lower eucaryc>tes are generally subject to lysis by lytic peptides because of the differences in their 1340'16 membranes and cytoskeletal components. However, the membranes of normal cells of the higher eucaryotes somehow prevent l.ysis, possibly for example, by the ability of their well-developed cytoskeletons to quickly repair any membrane damage caused by the lytic peptide. On the other hand, higher eucaryotic cells with an aberrant cytoskeleton, such as neoplastic or infected cells, are generally unable to prevent lysis by the :Lytic peptide in the present method.
Quite unexpectedly, it has also been found that the lytic peptide can also stimulate the proliferation of fibroblasts and mitogen activated lymphocytes by contact:Lng the lymphocytes or fibroblasts with an effective amount of a stimulating peptide. As used herein, the term "stimulating peptide" includes not only the preferred lytic peptides described hereinabove having from about 30 to about 40 amino acids, but also includes such peptides having fewer than 30 or more than 40 amino acids containing an active form of the portion of such lytic peptides inducing such stimulation, whether or not they are lytically active.
Specifically contemplated stimulating peptides include peptides having the first approximately 15-20 amino acids from th~? amine terminus of lytic peptides such cecropins and sarcotoxins. In this sense, "stimulation" means an enhancement of proliferation in any system in which it can be observed or measured, and the stimulating property of such peptides may or may not be related to their lytic property. Preferred stimulating peptides are cecropin A, cecropin SB-37, *cecropin SB-37, cecropin B, cecropin D and Shiva 1, and particularly cecropin SB-37 for stimulating fibroblasts and Shiva 1 for stimulating lymphocytes.
For convenien~~e, reference is made hereinbelow to cecropin by way of example with the understanding that other stimula~ing peptides including analogues, homologues, mutants and isomers thereof may be used.
Generally, lymphocytes must be activated by a mitogen, or an antigen reactive with antibodies expressed by the lympho~~ytes, before any stimulation thereof by a -i.
:: <.l t~i40~1~

stimulating peptide such as cecropin. Mitogens include, for example, ph:ytohemagglutinin, pokeweed mitogen, concanavilin A, substances for which lymphocytes have developed "memory" such as tetanus toxoid, and the like.
Thus, in one embodiment, activated lymphocytes (e,g,~
lymphocytes in the presence of a mitogen) or fibroblasts are contacaed with a stimulating peptide in vitro to increase proliferation thereof. In this manner, the production of biological products by lymphocytes, for example, such as interferon and interleukin-2, may be enhanced by culturing the lymphocytes in the presence of, for example, pokeweed mitogen and cecropin.
In accordance with this embodiment, the lymphocytes or fibroblasts to be stimulated are contacted with an effective amount of cecropin, generally an amount sufficient to provide a concentration in the medium containing the Lymphocytes or fibroblasts of from about 0.1 to about 50 NM. 7:n a preferred embodiment in which the stimulating peptides are administered _in vivo, contemplated dosages of cecropin range from about one to about ten :ng/kg. 'The cecropin may be introduced into the animal to be t:re:ated by injection in a suitable pharmaceutical carrier such as saline solution, for example, intramuscularly, subcutaneously, intravenously or intraperitc>neally, preferably at or near the site where such stimuation is desired, e.g. a wound, graft, injury or infection. The treatment may be repeated as necessary to sustain the proliferative stimulation of the lymphocytes; or fibroblasts.
AlthoL.gh the present invention is not bound or limited by any theory, it is believed that the stimulation of fibrobla.sts and lymphocytes results from the binding of the amphiphilic, a-helical amine terminus portion or end of the preferred lytic peptide to lymphocyte and fibroblast cell membranes. No binding activity is believed to be associated with the generally hydrophobic tail or carboxy terminus end or portion of cecropin or s -22- 13 4 0 'l 16 sarcotoxin. Thus, where lytic activity is not beneficial or desired, lymphocytes and/or fibroblasts can be stimulated by a shorter peptide from which the hydrophobic tail has been removed, i.e. the remaining head including 5 the first 15-25, and preferably the first 18-20 amino acids in the cec:ropin or sarcotoxin sequence.
In another embodiment of the invention, a lytic and/or stimulating peptide is introduced into a higher animal by placing cells into the animal which have an 10 expressible: gene coding for the peptide. Such cells may be prepared by genetically transforming cells such as bone marrow cells, embryos, hematapoietic stem cells, and the like. Techniques for such transformation are well known in the art and include, for example, transfection with 15 retroviral vectors, electroporation, microinjection and the like.
The invention is illustrated by way of the following examples.
Example 1 20 In Vitro Effect of Cecropin SB-37 on T. cruzi Trypomastigotes Tr anosoma cruzi trypomastigotes were suspended at 105 cells/ml in minimum essential medium ("MEM") and 10%
heat-inactivated fatal bovine serum ("MEM-FBS") containing 25 100 NM cecropin SB-37. Control trypomastigotes were suspended in the same medium without the cecropin SB-37.
After one hour in suspension, the treated trypomastigotes exhibited a 90% r<sduction in viability, as assessed by microscopically counting viable cells, compared to 30 untreated trypomastigotes under otherwise identical conditions. The reduction in viability was verified by adding the treate d and untreated trypomastigotes suspensions to Vero cells in MEM-FBS at 105 cells/ml in a 1:1 ratio o:E trypomastigotes (as counted prior to cecropin 35 treatment) to Vero cells and culturing 24 hours in microscope glide chambers at 37°C in a 5% C02 atmosphere.
Based on counts of infected Vero cells, the treated -23_ 1~~o7~s trypomastigotes showed a significantly decreased level of parasitemia compared to the Vero cells with the untreated trypomastigotes.
Example 2 In Vitro Effect of Cecropin SB-37 on T. c:ruzi Intracellular Amastiqotes in Vero Cells Vero cells at 105 cells/ml in MEM-FBS were mixed with Trypanosoma cruzi in the trypomastigote stage at 105 cel.ls/ml in the same medium at a 1:1 ratio of Vero cells 10 to trypomastigot.es. The mixture of Vero cells and trypomast:igotes was cultured at 37°C in a 5% COz atmospheres in microscope slide chambers. At 24 hours, cecropin SB-37 was added to the chambers, except for a set of controls, at a final concentration of 100 NM. The 15 number of intracellular amastigotes per hundred Vero cells was determined every 24 hours by fixation with formalin, staining ~:rith geimsa and counting several hundred Vero cells taken along a line down the microscope slide. The results are presented in Table 2.
20 Table 2 In Vitro Effect of Cecropin SB-37 On T. cruzi Intracellular Amastigotes in Vero Cells Hours Post Amastigotes/ Cells Infected 25 T. cruzi Addition 100 Vero Cells Control Treated Control Treated 48 20 15 ~ 15 14 72 50 5 16 2.5 The :__°oregoing technique was used to determine the LD50 of Shiva ~ and cecropin SB-37 (the concentration of each pept:de required to obtain a SO% reduction in the number of Vero cells infected with trypomastigotes over a -24- 134o7~s one hour period relative to the number of infected Vero cells incubated in the absence of the peptide for the same period). The LD50 for cecropin SB-37 was about 90 NM, while that for Shiva 1 was about 9 NM, indicating that Shiva 1 is about ten times as effective as cecropin SB-37 against T. cruzi infection.
Example 3 In Vitro Effect of Cecropin B and Cecropin SB-37 on _P. Fa_lciparum In Human Red Blood Cells Plasmodium f~alciparum was added at 0.0625, 0.125, 0.25 and 0.5 percent parasitized red blood cells (PRBC) to flasks containing 50 ml human red blood cells in RPMI and 10 NM hypoxanthine containing 50 NCi 3H-hypoxanthine at 150 ml final volume. The mixture was incubated one week at 37°C in a 5% COZ atmosphere. Cecropin B and cecropin SB-37 were then added to different flasks at concentrations of 0 (control), 1, 20 and 200 NM. After 24 hours of additional incubation, the red blood cells were harvested by filtration and hypoxanthine uptake was measured by liquid scintillation as an indication of _P.
falciparum viability. The results are seen in Table 3 Table 3 In Vitro Effect of Cecropin B and Cecropin SB-37 on P. Falci~arum In Human Red Blood Cells 3H-Hypoxanthine Uptake (cpm) Cecro in Concentration NM P. falciparum (PRBC) 0.0625 0.125 0.25 0.5 w 1340'~I6 '.Che foregoing procedure was repeated with _P.
falciparum at: 0.5 PPRC with the addition of cecropin SB-37 at 0, 25, 50, 75 and 100 NM. The number of infected red blood cells was determined by microscopically counting 5 infected red blood cells after incubation for 24 hours by fixation staining and counting as described in Example 2.
The reaults are presented in Table 4.
Table 4 In Vitro Effect of Cecropin SB-37 on 10 P. Falciparum In Human Red Blood Cells Concentration (NM) Infected RBC
0 g 15 50 ~.S

The foregoing procedure and techniques were used to 20 determine the: LD50 of Shiva 1 and cecropin SB-37 (the concentratiorn of the peptide required to obtain a 50%
reduction i.n 3H-hypoxanthine uptake over a 24-hour period relative to untreated infected red blood cells). The LD50 for ce:cropin SB-37 was about 22.5 NM, while that for Shiva 25 1 was about :LONM, indicating that Shiva 1 is about twice as effective as cecropin SB-37 against _P. falciparum infection.
Example 4 '' In Vitro Effect of Cecropin B, Cecropin SB-37 30 and Melittin on S. cerevisiae The yeast Saccharomyces cerevisiae was grown to late log phase 1>y placing a small inoculum in 100 ml nutrient broth containing 10 grams tryptose, 5 grams of yeast extracts and 2 grams glucose per liter. The yeast was then -26- 134o7is pellete:d by centrifugation and resuspended at 105 cells/ml in O.O:L M sodium phosphate buffer solution at pH 6.8 (PBS). Cecropin B, cecropin SB-37) melittin and an unrelated control peptide having 15 amino acids were added 5 in varying <~oncentrations to different wells containing 105 cells/well and the wells incubated at 37°C for 1 hour.
Each well was diluted 1000-fold with PBS and plated on glucose agar. The next day the number of surviving cells was determined by taking plate counts. The results are 10 presented in Table 5.
Table 5 I:ff~ect of Lytic Peptides on S. cerevisiae Plate Count (Thousands) 15 Pe tide Concentration N

Control 500 500 500 500 Melittin 500 260 0 -Cecropin B 500 500 75 0 20 Cecropin SB-37 500 400 75 0 Example 5 In Vitro Effect of Cecropin SB-37 and Shiva 1 on Tumor Cells 25 EL-4 lymphoma cells were suspended in RPMI 1640 at 5,000,000/ml with 500,000 cells per well. Various concentrations of cecropin SB-37 and Shiva 1 were added and the wells incubated at 37°C in a 5% C02 atmosphere for one hour. Viability was determined by microscopic 30 observation for trypan blue exclusion. The results are seen in Table 6.

4340~I6 Table 6 In Vitro Effect of Cecropin SB-37 and Shiva 1 on EL-4 Cells 5 f'e tide Concentration (NM) Viability (°
Cecropin SB-37 0 95 Shiva 1 0 95 10 g5 The foregoing results illustrate that both peptides are lytically active against EL-4 cells, and that Shiva 1 is about twice as effective as cecropin SB-37. These 20 results were confirmed by chromium release data obtained by incubating 106 EL-4 cells in 0.5 ml of MEM-FBS
containing 10% fetal calf serum and 1 NCi of 5lCr for 60 minutes at 37°C, centrifuging and washing the incubated EL-4 cells with PBS, and resuspending the EL-4 cells in 25 MEM-FBS. The EL-4 cells were then incubated for 30 minutes in the presence of various peptides at different concentrations. Cytotoxicity was taken as a percentage of chromium released (measured in the culture supernatant) relative' to the. chromium released by EL-4 cells lysed by 30 detergent. The results are presented in Table 7.

Table 7 Effect of Peptides on EL-4 Cells Measured by Chromium Release ~340~10 PercentC totoxicityl Peptide Cecropin'Cecropin Concentration (ihl)SB-37 SB-37 Shiva Melittin 100 36.42 42.47 82.35 102.43 50 6.63 12.19 42.73 106 25 4.49 2.45 16.7 .
106.65 10 0.0 0.0 0.37 109.65 Note for Table 7:
1. Calculated as a percentage of the cpm of culture supernatara divided by the cpm of the supernatant from detergent-lysed cells:
c:ytoxicity = _c~m (treated cell su ernatant) - c m (s ontaneous release) cpm (detergent lysis - cpm spontaneous release .
Similar chromium release data were obtained using the same procedure with a 100 NM concentration of peptide, but with EL-4 cell concentrations of 2x106, 106, and 5x105 cells in 0.5 ml medium. The results are presented in Tak>le 8.
Table 8 Effect of EL-4 Cell Concentration on Cytotoxicity of Peptides Percent C totoxicityl Cells per 0.5 ml Cecropin SB-37 Shiva 1 ~'Cecropin SB-37 Melittin 2x106 93.75 84.88 89.07 105.44 106 89.25 89.22 80.51 --5x105 99.64 98.74 82.48 --Note for Table 8:
1. See Table 7, note 1.

The chromium release data was also obtained by using the supernatant from unlabeled EL-4 cells cultured for 30 minutes in the presence of 100 NM peptide as medium for 57Cr-~.abeled EL-4 cultured as described above. The 5 percent cytotoxicity calculated as described above was 20.16 for cecropin SB-37, 19.73 for *cecropin SB-37, 41.96 for Shiva 1) and 106.67 for melittin.
Example 6 In Vitro Effect of Cecropin SB-37 on 10 Normal and Neoplastic Mammalian Cells The procedure of Example 5 was repeated with 50 and 200 NM cecropin SB-37 using SP2 mouse lymphoma cells, KG-1 human leukemia cells, Daudi human Burkitt's lymphoma cells, non-adherent normal human lymphocytes and adherent 15 3T3 human fi.broblasts. Viability was determined by microscopic observation for trypan blue exclusion. The results are presented in Table 9.
Table 9 Effect of Cecropin SB-37 on 20 Normal and Neoplastic Mammalian Cells Cell Line Survival (percent) 50 uM SB-37 100 NM SB-37 Daudi 85 0 Non-adherent Normal Human Lymphocytes 100 30 This procedure was repeated with 100 NM cecropin SB-37 using non-adherent normal human lymphocytes, Daudi , human :3urkitt's lymphoma cells, KG-1 human leukemia cells, U937 human monocytic leukemia cells, and SP2 murine 35 myeloma cells, and determining viability by the trypan blue exclusion method at 15, 30 and 60 minutes following addition of the peptide. The results are presented in 'table :LO.

r~

Table 10 Effect of 100 NM Cecropin SB-37 on Normal and Neoplastic Mammalian Cells 5 Cell Line _ Viability 15 mm 30 min. 60 min.
.

Non-adherent normal human lymphocytes 75 80 85 Daudi 70 70 65 10 Kgl 50 75 50 Example 7 15 In Vitro Effect of Lytic Peptides on Virus-Infected Cells Simian kidney cells infected with measles virus, parainfluenza virus and herpes simplex II virus were incubcited at 105 cells/ml in 1.0 ml total volume of DMEM
20 and 10% FCS in the presence of 100 NM cecropin SB-37, *cecropin SB-37 and Shiva 1 for 1-4 days. Following incubation, the cells were lysed by addition of equal volume: of sterile water, and 0.1 ml of supernatant was added to 105 simian kidney cells in 1.0 ml total volume of 25 DMEM/7.0% FCS and the mixture was cultured for 24 hours.
The number o.f virus-infected cells in the fresh culture was determined by microscopic slide counting. The results are presented in Table 11.

1340~1b Table 11 Titration of Viruses in the Presence of Lytic Peptides 5 Incubation Number of Cells Infected _ Period (Days) Pe tide MeaslesParam es Simplex f luenza Herp II

1 Cecropin SB-37 107 104 <102 ~-Cecropin SB-37 >108 >108 <102 Shiva 1 >108 108 103 10 Control >108 >108 104 2 Cecropin SB-37 108 107'5 <102 ~Cecropin SB-37 >109 >109 103 Shiva 1 >109 >109 105 Control >109 >109 >107 15 3 Cecropin SB-37 105 10~'S <102 ~Cecropin SB-37 >109 >109 <102 Shiva 1 >109 109 105 Control >109 >109 107 4 Cecropin SB-37 107'5 107 <102 20 -'~Cecropin SB-37 >109 >109 >107 Shiva 1 >109 >109 106 Control >109 >109 >107 These results indicate that lytic peptides are 25 effective in inhibiting virus-infected eucaryotic cells, and particularly effective against DNA viruses such a herpes simplex II. Cecropin SB-37 appears to be more inhibitory than *cecropin SB-37 and Shiva 1.

1~4Q'~~6 Example 8 In Vitro Proliferation of Lymphocytes With Cecropin SB-37 and Shiva 1 Lymphocytes recovered by density gradient centrifugation were t:aken from a human subject 5 months after receiving a tetanus vaccination. The lymphocytes were cultured in IMDM containing 10% Hyclone serum at 3x 106 cells/ml in the absence (control) or presence of cecropin SB-37 o:r Shiva 1 at 50 NM, and 5 Ng tetanus toxoid. After incubation at 37°C in a 5% C02 atmosphere for 96 hours, tr.e relative proliferative response was determined by 3H-~thymidine uptake. A second control was established with lymphacytes, obtained from a human subject who had not had a recent tetanus vaccination, which were cultured in the absence of peptide and tetanus toxoid. Relative to t:he second control, the thymidine uptake was 70% and 8U% greater for the cells cultured in the presence of c:ecropin SB-37 and Shiva 1, respectively, but only 60% greater for the first set of controls (cells recently exposed to tetanus toxoid). This demonstrates the ability of the lytic peptides to stimulate lymphocyte proliferation.
The foregoing procedure was repeated with lymphocytes obtained from four different human subjects. The lymphocytes were cultured as described above, but for a period of 72 hours in the presence of cecropin SB-37 at various concentrations and with other known lymphocyte proliferation inducers. The results are presented in Table 12.

Tahl a l 7 Stimulatory Effect of Cecropin SB-37 on Hum~.a_ n LymF~hocyte Cell Proliferation _ Rang e of Values(cpm/cpm ~ control) Treatment 1CJ.~M 2525 NM 5050 NM 100 NM

SB-37 0.5:3-1.32 0.44-1.83 0.40-1.850.07-1.43 SB-37 +PHA1 1:800 0.10-3.67 0.24-4.15 0.07-2.160.01-0.09 SB-37 +PHA 1:1600 0.15-6.82 0.11-3.52 0.08-2.190.01-0.09 SB-37 +PWM2 1:50 0.28-1.95 0.25-1.98 0.17-1.640.00-0.71 SB-37 +PWM 1:100 0.2,3-2.37 0.29-2.39 0.21-2.200.01-0.81 SB-37 +CONA350 Ng 0.01-3.23 0.01-3.10 0.01-2.100.00-0.80 SB-37 +CONA 25 Ng 0.01-5.75 0.01-6.29 0.01-4.950.00-0.45 1 S Notes for Table 12:
1. PHA - phytohemaggluti.n:in.
2. PWM - pokeweed mitoge~n.
3. CONA - concanavalin A,.
Example 9 In Vitro Stimulation of Fibroblast Proliferation With Cecropin SB-37 3T3 Mouse en~bryon:ic: fibroblasts were cultured in IMDME without serum at 1.04 cells per well for 48 hours with cecropin SB-~37 an<i Shiva 1 at various concentrations with or without insulin at 10 ~g/ml. Proliferation was assessed by 3H-thymidine uptake using a scintillation counter. The re~;ults are presented in Table 13, -34- 1340'16 m~i,, o , ~z Stimulation of Fibroblast Proliferation With Cecropin SB-37 -- 3H-Thymidine Uptake (103 cpm) SB-37 Shiva 1 Peptide and and Concentration (NM) SB-37 Shiva 1 Insulin Insulin 100 2'i 2 30 2 Example 10 In Vivo Screening of Cecropin SB-37 on Mice Nine BALB/C mice, 4 to 5 weeks of age, maintained on commercial rodent: ration fed ad libidum and in general good health were each inoculated with 1.76 mg/day of cecropin SB-37 i:n PBS intramuscularly for 4 consecutive days. No other changE: was made in diet or conditions.
The mice were observed. twice daily and no adverse reactions were noted. A.t the end of the fourth day, three were sacrificed and examined. After 3o-- days, three were given an additional 1.7p mg of cecropin SB-37 each. No adverse reactions were noted.
The remaining mice were all sacrificed 7 days after the last inoculation. i~xamination of organs and tissues indicated no gross pathological changes were present in the organs or at the injection sites. None of these mice produced detectable levels of antibody to the cecropin SB-37.
Following th.e abo~re procedure, three BALB/C mice were given 110 mg/kg of body weight per day of cecropin SB-37 injected intramuscular:Ly in balanced salt solution for 6 days. white blood <:ell counts were determined periodically and are reported in Table 14. Ten days after -35- 1340'~1fi the last inoculation, the mice were again injected intramuscularly with 110 mg/kg of body weight with cecropin :.B-37. Observation revealed no adverse effects during the procedure. All three mice were sacrificed 7 5 days after the final inoculation and tissues were examined. All mice had enlarged spleens but were otherwise unremarkable. No cecropin SB-37 antibodies were detected.
Table 14 10 Effect of Cecropin SB-37 on WBC Count WBC Count (103/ml Mouse 1 Mouse 2 Mouse i 13 13 9 15 3 14 9 i0 20 Example 11 In Vivo Effect of Shiva 1 on Murine Mammary Carcinoma One B~~LB/c mouse was given daily injections of 1.76 mg (88 mg/l~:g) Shiva 1 in PBS for three consecutive days as 25 described .in the foregoing examples. The mouse had a large mammary carcinoma into which the Shiva 1 was directly injected. Following the third injection, this mouse died almost immediately. Autopsy revealed shock as the immediate cau~;e of death, and substantial cell death 30 in the carcinoma. It is believed that lower dosages would have avoided shoclt, but would still have significantly inhibited the carcinoma.

-36- i34o~~s L
Example 12 In Vivo Effect of Cecropin SB-37 on B. Abortus in Mice A total of 18 BALB/C mice, maintained on commercial 5 rodent ratio fed acl libidum, 4 to 5 weeks of age and in good health were inoculated intraperitoneally with 3 to 5x108 Brucella abortus (an intracellular pathogen) in physiological saline. On the twelfth day post infection, six of the mice were each inoculated intramuscularly with 10 0.176 mg/d~ay of cec:ropin SB-37 in PBS, six were treated with 0.176 mg/day of tetracycline and six were given sterile PBS, all l:or a period of 4 days. Half of the mice in each group were sacrificed on the sixteenth day post infection and spleen tissue examined morphologically, 15 histologic<~lly and by culturing according to standard procedures for Brucella abortus. The concentrations found are given _Ln the following Table 15.
Table 15 Concentration of Brucella abortus in BALB/c 20 Mouse Spleen Tissue After a Days Treatment (Number per gram of spleen) Control Tetracycline Cecropin SB-37

8.15x103 4.4x103 1.59x103 The remaining mice were sacrificed on the twenty-third day post infection and examined in the same manner 'and no dii~f~srence in result was observed. This result indicates 'that the lytic peptide cecropin SB-37 is 30 capable of significantly decreasing the level of infection and growth of Bruc:e:Lla abortus in mice.

~'1340~16 Example 13 In Vivo Effect of Cecropin SB-37 on L,. Mono~togenes in Mice The procedure: of Example 12 was repeated except that 5 another intracellular pathogen, Listeria monocytogenes, was used insteaf, of B. Abortus, and the control group was untreated (sterile: saline injections only). The untreated mice sacrificed five days post infection had an average of 46 million L. monocytogenes bacteria in their spleen, in 10 contrast to less than five million in the mice given daily injection of cec:ropin SB-37.
Example 14 Lysozyme~/Cecropin Synergism Against E. Coli To a suitable container was added 50 N1 of the 15 bacteria under investigation, in this instance, _E. coli E/2/64, obtained from Cornell UniversWy, and 50 N1 of phosphate buffered saline solution (PBS), prepared from NaCl 80.Og, KC1 3g, NaZHP04 - 0.73g, f:H2P04 - 0.2g, with dilution of 10 rnl of mixture wth 90 ml of water and 20 sterilization by autoclaving. The _E. coli were collected in late log phase growth at which the majority of cells were in growth phase at 18-24 hours. In addition, the same mixture was prepared with 5 NM of cecropin B or cecropin SB-37. The mixtures were incubated at room 25 tem~eratu:re for 90 minutes. The mixtures were diluted to 10 or 10 with serial dilutions at 7 drops per dilution (10 N1 per drop), and plated on tryptose agar growth media. The plates were incubated for 3 days at room temperature. Plate counts indicated that there was no 30 growth of E. coli..
Comb:Lnation_; of both cecropin B and cecropin SB-37 with lyso:~yme (25:25 N1 mixture) also prevented growth at 0.1 NM concentr~~tions, but lysozyme itself at concentrations o:f 10 Ng) 1 mg, and 10 mg per milliliter -i 35 all showed positive growth of E. coli at both 10 and 10 dilutions. I?urther, lower concentrations of one nanomolar and one micromolar of both cecropin species showed positive E. coli growth.

Example 15 ~~oz a C:ecropin ~ngergism Against B. Abortus The procedure of Example 14 was followed, except the microorganism saas Brucella abortus, isolated by 5 conventional procedure, the cecropin/bacteria mixture was incubated for one, hour at room temperature and then 30 minutes at 37°C, and the plates were incubated at 37°C.
The results are shown in Table 16 below.
10 Effect of Cecropins on Brucella abortus Cecropin Concentration (NM) Results Cecropin E 50 No growth Cecropin fIB-37 50 No growth Com;~inations of cecropin 5B-37 and cecropin B with lysozyme at 1, 10 and 100 Ng/ml were effective to prevent growth only at the highest concentration of lysozyme with cecropin SB-37; otherwise, some growth of the Brucella 20 abortus was noted.
Example 16 Lysozyme L tip-Peptide Synergism Against P. Aeruginosa The procedure of Example 15 was followed with cecropin SB-37 and Shiva 1, alone and in combination with 25 lysozyme at the: same molar cencentration of the lytic peptide, against Pseudomonas aeruginosa. Tt~e results are presented in Table 17.

-39- 13 4 0'~ 1~6 Table 17 P. aeru~inosa Viability in Lytic Peptide/Lysozyme Viabilit Peptide Cecropin Shiva 1 and 5 Concentration (NM) SB-37 Shiva 1 Lysozyme(lx) Lysozyme(lx) 0.01 100 100 100 56.6 0.1 79.4 69.6 82.2 25.8 1.0 48.8 37.9 52.1 4.4 10 5.0 38.5 1.5 7.9 0.2 7.5 0.7 0.1 0.6 0 25. 0 0 0.4 0 Example 17 15 Lysozy;ne Peptide Synergismainst Staphylococci L tic Ag The procedure repeated of Example using 16 was S. intermedius19930, S. intermedius20034 and S. aureus lysozyme at ten the molar with times concentration The results of the lytic are peptide.

20 presented espectively.
in Tables 18, 19 and 20, r Table 18 S. Intermedius yme 19930 Viability in Lytic Peptide/Lysoz Viab ility (%) Peptide Cecropin Shiva 1 and 25 ConcentrationSB-37 Shiva 1 zyme(lOx) yme(lOx) (NM) Lyso Lysoz 0.01 100 100 100 100 0.1 94.7 81.8 100 79.2 0.5 69.4 65.0 81.3 65.1 30 1.0 42.5 42.1 53 43 5.0 36.1 35.2 49.5 17.2 10. 5.6 1.2 34.4 1.1 50. 0 0 22 0 1340'716 Table 19 S. Intermedius Viability in Lytic Peptide/Lysozyme Viability (°/) Peptide Cecropin Shiva 1 and 5 Concentration SB-37 Shiva Lysozyme(lx)Lysozyme(1 (NM) 1 x) 0.01 100 100 100 100 0.25 85.4 87.1 100 85.1 0.5 68.0 80.0 59.0 53.4 10 0.75 62.2 60.1 42.3 41.0 5.0 35.1 4.1 38.3 4.3 50. 0 0 10.0 0 Table 20 15 S. aureus Viability in Lytic Peptide/Lysozyme Viability Peptide Cecropin Shiva 1 and Concentration ( M) SB-37 Shiva 1 Lysozyme~.lx) Lysozyme(lOx) 20 0.01 100 100 100 100 0.1 100 100 100 100 0.5 81.0 50.1 -- --1.0 47.5 24.4 51.0 31.2 5.0 31.8 15.9 18.4 8.2 25 10. 5.6 4.5 13.3 4.5 50. 1.9 1.6 9.5 1.4 Example 18 Lysozyme Lytic Peptide Synergism Against Plant Pathogens 30 The procedu.rca of the foregoing examples with the exception of an incubation temperature of 30°C instead of 37°C, were used to demonstrate the synergistic bactericidal properties of a lytic peptide in combination with lysozyme against common plant pathogens. The results 35 are presented in Table 21.

Table 21 Plant Pathogen LD50 in Cecropin SB-37/Lysozyme Bacteria LD50(NM) Cecropin SB-37 Cecropin SB-37 Lysozyme (+Lysozyme (10x)) Pseudomonas syrin~-,ae 5.20 >1000 0.19 pv, tabaci 10 Pseudomonas 64.0 >1000 16.0 Erwinia _caratovora~_ 1.48 >1000 0.44 subsp. carotova Xanthomonas campes,tris 0.57 >1000 0.027 15 pv. cam estris Hac~ing described the invention above, various modifications of the techniques, procedures, material and equipment will be apparent to those in the art. It is 20 intended that all such variations within the scope and spirit of the appended claims be embraced thereby.

Claims (36)

Claims

1. Use of a selectively lytic peptide for the manufacture of a medicament for the lysis of eucaryotic microorganisms, mammalian neoplastic cells, and eucaryotic cells infected with an intracellular pathogenic microorganism in the presence of non target eucaryotic cells wherein the mammalian neoplastic cells are selected from the group consisting of lymphomas, leukemias, carcinomas and sarcomas.

2. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 1 in which the selectively lytic peptide is a free peptide.

3. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claims 1 or 2 in which the selectively lytic peptide is cecropin, lepidopteran or sarcotoxin and lytically active analogues, homologues, mutants or isomers thereof.

4. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 1, 2 or 3 in which said selectively lytic peptide has a portion which is arranged in an amphiphilic .alpha.-helical conformation.

5. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 4 in which said selectively lytic peptide has a substantially hydrophilic head with a positive charge density, a substantially hydrophobic tail, a predominantly hydrophilic face along the length of said .alpha.-helical conformation and a predominantly hydrophobic face opposed therefrom.

6. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in any one of claims 1, 2, 3, 4 or 5 in which the selectively lytic peptide comprises between 30 and 40 amino acids inclusive.

7. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in any one of claims 1 to 6 in which said intracellular pathogenic microorganism is a virus, bacteria, fungi or protozoa.

8. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 7 in which the virus is a DNA virus, parainfluenza, measles or herpes simplex II.

9. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 7 in which said bacteria is a species of Listeria or Brucella.

10. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 7 in which said intracellular pathogen is a species of Trypanosoma or Plasmodium.

11. A lytic peptide having the amino acid sequence of MP
RWR LFR RID RVG KQI KQG ILR AGP AIA LVG DAR AVG.

12. A lytic peptide having the amino acid sequence of MP
KWK VFK KIE KPHG RNI RNG IVK AGP AIA VLG EAK ALG.

13. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in any one of claims 1 to 12 in which the selectively lytic peptide is in admixture with lypozyme in a synergistic proportion.

14. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 13 in which the selectively lytic peptide is in admixture with lysozyme in a synergistic proportion of 10 moles lysozyme to 0.1-100 moles selectively lytic peptide.

15. Use of a selectively lytic peptide for the manufacture of medicament as claimed in claim 14 in which the selectively lytic peptide is in admixture with lysozyme in a synergistic proportion of 10 moles lysozyme to 1-10 moles selectively lytic peptide.

16. A method of lysing target eucaryotic cells selected from the group consisting essentially of eucaryotic microorganisms, mammalian neoplastic cells, and eucaryotic cells infected with an intracellular pathogenic microorganism in the presence of non target eucaryotic cells characterized in that said target eucaryotic cells are contacted in vitro with a selectively lytic peptide and the mammalian neoplastic cells are selected from the group consisting of lymphomas, leukemias, carcinomas, and sarcomas.

17. Use of a selectively lytic peptide for the manufacture of a medicament for stimulating the proliferation of normal mammalian fibroblasts and lymphocytes.

18. Use of selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 in which the selectively lytic peptide is a free peptide.

19. Use of selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 or 18 in which the selectively lytic peptide is a cecropin, lepidopteran or sarcotoxin and lytically active analogues, homologues, mutants or isomers thereof.

20. Use of selectively lytic peptide for the manufacture of a medicament as claimed in claim 17, 18 or 19 in which the selectively lytic peptide has a portion which is arranged in an amphiphilic .alpha.-helical conformation.

21. Use of selectively lytic peptide for the manufacture of a medicament as claimed in claim 20 in which the selectively lytic peptide has a substantially hydrophilic head with a positive charge density, a substantially hydrophobic tail, a predominantly hydrophilic face along the length of said .alpha.-helical conformation and a predominantly hydrophobic face opposed therefrom.

22. Use of selectively lytic peptide for the manufacture of a medicament as claimed in claim 17, 18, 19, 20 or 21 in which said peptide comprises between 30 and 40 amino acids inclusive.

23. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 17, 18, 19, 20 or 21 in which the lytic peptide comprises from about 15-20 amino acids.

24. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 in which said lytic peptide has the amino acid sequence of MP RWR LFR RID RVG KQI KQG ILR AGP AIA LVG DAR AVG.

25. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 in which said lytic peptide has the amino acid sequence of MP KWK VFK KIE KMG RNI RNG IVK AGP AIA VLG EAK ALG.

26. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 in which said peptide is provided in a serum concentration from about 0.1 to about 200 micromolar.

27. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in claim 17 in which said peptide is available for use in a concentration of about 0.1 to about 200 micromolar.

28. Use of a selectively lytic peptide for the manufacture of a medicament as claimed in any one of claims 17 to 25 in which the selectively lytic peptide is in admixture with other growth factors or mitogens in synergistic proportion.

29. Use of a selectively lytic peptide to inhibit infection from eucaryotic microorganisms.

30. Use of a selectively lytic peptide as claimed in claim 29 in which they selectively lytic peptide is a free peptide.

31. Use of a selectively lytic peptide as claimed in claim 29 or 30 in which the selectively lytic peptide is cecropin, lepidopteran or sarcotoxin and lytically active analogues, homologues, mutants or isomers thereof.

32. Use of a selectively lytic peptide as claimed in claim 29, 30 or 31 in which said selectively lytic peptide has a portion which is arranged in an amphiphilic .alpha.-helical conformation.

33. Use of a selectively lytic peptide as claimed in claim 32 in which said selectively lytic peptide has a substantially hydrophilic head with a positive charge density, a substantially hydrophobic tail, a predominantly hydrophilic face along the length of said .alpha.-helical conformation and a predominantly hydrophobic face opposed therefrom.

34. Use of a selectively lytic peptide as claimed in claim 29, 30, 31, 32 or 33 in which the selectively lytic peptide comprises between 30 and 40 amino acids inclusive.

35. Use of a selectively lytic peptide as claimed in claim 29 in which the eucaryotic microorganisms are selected from the group consisting essentially of fungi and protozoans.

36. Use of a selectively lytic peptide as claimed in claim 29 in which the eucaryotic microorganisms are selected from the group consisting essentially of sarcodina, mastigophora, ciliata and sporozoa.