# Patent

## Title
Methods for use of short bioactive peptides

## Abstract

Short bioactive peptides containing phenylalanine, leucine, alanine, and lysine residues are disclosed. The peptides can be used in antibacterial, antifungal, anticancer, and other biological applications.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part application of co-pending application Ser. No. 09/820,053 filed Mar. 28, 2001, now issued as U.S. Pat No. 6,875,744, and claims priority to US provisional application 60/279,505 filed Mar. 28, 2001.


FIELD OF THE INVENTION
The invention relates to short length peptides containing phenylalanine, leucine, alanine, and lysine amino acid residues (F, L, A, and K; “FLAK peptides”) in their primary sequence. In particular, FLAK peptides having desirable antimicrobial, antifungal, anticancer, and other biological activities are disclosed.
BACKGROUND OF THE INVENTION
Various bioactive peptides have been reported in both the scientific literature and in issued patents. Peptides historically have been isolated from natural sources, and have recently been the subject of structure-function relationship studies. Additionally, natural peptides have served as starting points for the design of synthetic peptide analogs.
A review of peptide antibiotics was published by R. E. W. Hancock in 1997 (Lancet 349: 418-422). The structure, function, and clinical applications of various classes of peptides were discussed. An additional review of cationic peptide antibiotics was published in 1998 (Hancock, R. E. W. and Lehrer, R. Trends Biotechnol. 16: 82-88). The peptides are typically cationic amphipathic molecules of 12 to 45 amino acids in length. The peptides permeabilize cell membranes leading to the control of microbial agents. The clinical potential of host defense cationic peptides was discussed by R. E. W. Hancock in 1999 (Drugs 57(4): 469-473; Antimicrobial Agents and Chemotherapy 43(6): 1317-1323). The antibacterial, antifungal, antiviral, anticancer, and wound healing properties of the class of peptides are discussed.
Reviews of the structural features of helical antimicrobial peptides, and their presumed mechanisms of action have been published (see, for example, Dathe, M. and Wieprecht, T. Biochimica et Biophysica Acta 1462: 71-87 (1999); Epand, R. M. and Vogel H. J. Biochimica et Biophysica Acta 1462: 11-28 (1999)). Structural parameters believed to be capable of modulating activity and selectivity include helicity, hydrophobic moment, hydrophobicity, angle subtended by the hydrophilic/hydrophobic helix surfaces, and charge.
A wide array of naturally occurring alpha helical peptides have been reported. The following are representative of the many references in the field.
Cecropins are a family of α-helical peptides isolated from insects. Cecropins are known for their antibacterial properties, as described in U.S. Pat. Nos. 4,355,104 and 4,520,016. The cecropins were generally found to have activity against gram-negative bacteria, but not against all gram-negative bacteria. Cecropins were found not to have activity against eucaryotic cells (Andreu, et al., Biochemistry 24: 163-188 (1985); Boman, et al., Developmental and Comparative Immunol. 9: 551-558 (1985); Steiner et al., Nature 292: 246-248 (1981)). Cecropins from Drosophila and Hyalphora were presented as having activity against various strains of fungi (Ekengren, S. and Hultmark, D., Insect Biochem. and Molec. Biol. 29: 965-972 (1999)). Cecropin A from mosquito Aedes aegypti is reportedly different from most insect cecropins in that it lacks tryptophan and C-terminal amidation (Lowenberger, C. et al., J. Biol. Chem. 274(29): 20092-20097 (1999)).
Frogs from the genus Rana produce a wide array of antimicrobial peptides in their skin (Goraya, J. et al., Eur. J. Biochem. 267: 894-900 (2000)). Peptides as short as 13 amino acids were reported, and were grouped into structural families. The sequences showed little or no sequence identity to peptides isolated from frogs of other genera, such as the magainin and dermaseptin peptides.
U.S. Pat. No. 5,962,410 disclosed the inhibition of eucaryotic pathogens, and the stimulation of lymphocytes and fibroblasts with lytic peptides such as cecropins and sarcotoxins. Various peptides presented include Cecropin B, Cecropin SB-37, Cecropin A, Cecropin D, Shiva-1, Lepidopteran, Sarcotoxin 1A, Sarcotoxin 1B, and Sarcotoxin 1C.
Transgenic mice producing the Shiva-1 cecropin class lytic peptide were reported by Reed, W. A. et al., Transgenic Res. 6: 337-347 (1997). Infection of the transgenic mice with a Brucella abortus challenge resulted in a reduction of the number of bacteria relative to infection of non-transgenic mice.
Magainin is an α-helical 23 amino acid peptide isolated from the skin of the African frog Xenopus laevis (Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 84: 5449-5453 (1987).
Cathelin associated α-helical peptides of 23 to 38 amino acids are found in the blood cells of sheep, humans, cattle, pigs, mice, and rabbits (Zanetti, M. et al., FEBS Lett. 374: 1-5 (1995)).
The antimicrobial activities of buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin were reported by Giacomette, A. et al. (Peptides 20: 1265-1273 (1999)). The peptides showed variable activities against bacteria and yeast.
Various synthetic peptides have been prepared and assayed both in vitro and in vivo.
U.S. Pat. No. 5,861,478 disclosed synthetic lytic peptides of about 20 to 40 amino acids which adopt an α-helical conformation. The peptides are effective in the treatment of microbial infections, wounds, and cancer. The peptides disclosed include cecropin B, SB-37*, LSB-37, SB-37, Shiva 1 and 10-12, β-fibrin signal peptide, Manitou 1-2, Hecate 1-3, Anubis 1-5 and 8, and Vishnu 1-3 and 8.
Hecate was described as a synthetic peptide analog of melittin by Baghian, A. et al. (Peptides 18(2): 177-183 (1997)). The peptides differ in their charge distribution, but not in their amphipathic alpha helical conformation. Hecate inhibited herpes simplex virus (HSV-1) while not adversely affecting cell growth and protein synthesis.
Synthetic peptides D2A21, D4E1, D2A22, D5C, D5C1, D4E, and D4B were described in Schwab, U. et al., Antimicrob. Agents and Chemotherapy 43(6): 1435-1440 (1999). Activities against various bacterial strains were presented.
Hybrid peptides made of cecropin and melittin peptides were reportedly prepared and assayed by Juvvadi, P. et al. (J. Peptide Res. 53: 244-251 (1999)). Hybrids were synthesized to investigate the effects of sequence, amide bond direction (helix dipole), charge, amphipathicity, and hydrophobicity on channel forming ability and on antibacterial activity. Sequence and amide bond direction were suggested to be important structural requirements for the activity of the hybrids.
A 26 amino acid insect cecropin—bee melittin hybrid, and analogs thereof, were described in a study of salt resistance (Friedrich, C. et al., Antimicrobial Agents and Chemotherapy 43(7): 1542-1548 (1999)). A tryptophan residue in the second position was found to be critical for activity. Modest changes in sequence were found to lead to substantial changes in the properties of the peptides.
The effects of proline residues on the antibacterial properties of α-helical peptides has been published (Zhang, L. et al., Biochem. 38: 8102-8111 (1999)). The addition of prolines was reported to change the membrane insertion properties, and the replacement of a single proline may change an antimicrobial peptide into a toxin.
A series of peptides having between 18 and 30 amino acids were prepared in order to test the effects of changes in sequence and charge on antibacterial properties (Scott, M. G., et al., Infect. Immun. 67(4): 2005-2009 (1999)). No significant correlation was found between length, charge, or hydrophobicity and the antimicrobial activity of the peptides. A general trend was found that shorter peptides were less active than longer peptides, although the authors expressed that this effect would probably be sequence dependent.
“Modellins”, a group of synthetic peptides were prepared and assayed to compare sequence and structure relationships (Bessalle, R. et al. J. Med. Chem. 36: 1203-1209 (1993)). Peptides of 16 and 17 amino acids having hydrophobic and hydrophilic opposite faces were highly hemolytic and antibacterial. Smaller peptides tended to have lower biological activities.
A cecropin-melittin hybrid peptide and an amidated flounder peptide were found to protect salmon from Vibrio anguillarum infections in vivo (Jia, X. et al., Appl. Environ. Microbiol. 66(5): 1928-1932 (2000)). Osmotic pumps were used to deliver a continuous dose of either peptide to the fish.
Amphipathic peptides have been reported as being capable of enhancing wound healing and stimulating fibroblast and keratinocyte growth in vivo (U.S. Pat. Nos. 6,001,805 and 5,561,107). Transgenic plants have been reportedly prepared expressing lytic peptides as a fusion protein with ubiquitin (U.S. Pat. No. 6,084,156). Methylated lysine rich lytic peptides were reportedly prepared, displaying improved proteolytic resistance (U.S. Pat. No. 5,717,064).
While a number of natural and synthetic peptides exist, there exists a need for improved bioactive peptides and methods for their use.
SUMMARY OF THE INVENTION
Short (i.e. no more than 23 amino acids in length) peptides containing phenylalanine, leucine, alanine, and lysine amino acid residues in their primary sequence are disclosed. The peptides display desirable antibacterial, antifungal, anticancer biological activities, and also cause stimulation and proliferation of human fibroblasts and lymphocytes.
DESCRIPTION OF THE SEQUENCE LISTINGS
The following sequence listings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these sequences in combination with the detailed description of specific embodiments presented herein.










TABLE 1





SEQ ID

P-



NO:
Name
No.
Primary sequence


















1
Hecate AC #1010
1
FALALKALKKALKKLKKALKKAL-COOH


2
Hecate AM
2
FALALKALKKALKKLKKALKKAL-NH2


3
SB-37 AC #1018
5
MPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-





COOH


4
Shiva 10 AM
11
FAKKLAKKLKKLAKKLAKLALAL-NH2


5
SB-37 AM
12
MPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-





NH2


6
Shiva 10 AC #1015
13
FAKKLAKKLKKLAKKLAKLALAL-COOH


7
Magainin 2
16
GIGKFLHSAKKFGKAFVGGIMNS-NH2


8
FLAK01 AM
23
FALAAKALKKLAKKLKKLAKKAL-NH2


9
FLAK03 AM
24
FALALKALKKLLKKLKKLAKKAL-NH2


10
FLAK04 AM
25
FALALKALKKLAKKLKKLAKKAL-NH2


11
FLAK05 AM
26
FALAKLAKKAKAKLKKALKAL-NH2


12
FLAK06 AM
27
FALALKALKKLKKALKKAL-NH2


13
FLAK06 AC
28
FALALKALKKLKKALKKAL-COOH


14
FLAK06 R-AC
29
FAKKLAKKLKKLAKLALAL-COOH


15
KAL V
30
VALALKALKKALKKLKKALKKAL-NH2


16
FLAK 17 AM
34
FALALKKALKALKKAL-NH2


17
FLAK 26 AM
35
FAKKLAKLAKKLAKLAL-NH2


18
FLAK 25 AM
36
FAKKLAKLAKKLAKLALAL-NH2


19
Hecate 2DAc
37
FALALKALKKAL-(D)-K-(D)-KLKKALKKAL-COOH


20
FLAK43 AM
38
FAKKLAKLAKKLLAL-NH2


21
FLAK44 AM
39
FAKKLAKLAKKALAL-NH2


22
FLAK62 AM
40
FALAKKALKKAKKAL-NH2


23
FLAK 06R-AM
41
FAKKLAKKLKKLAKLALAK-NH2


24
MSI-78 AM
42
GIGKFLKKAKKFGKAFVKILKK-NH2


25
FLAK50
43
FAKLLAKLAKKLL-NH2


26
FLAK51
44
FAKKLAKLALKLAKL-NH2


27
FLAK57
45
FAKKLAKKLAKLAL-NH2


28
FLAK71
46
FAKKLKKLAKLAKKL-NH2


29
FLAK77
47
FAKKALKALKKL-NH2


30
FLAK50V
48
VAKLLAKLAKKLL-NH2


31
FLAK50F
49
FAKLLAKLAKKL-NH2


32
FLAK26V AM
50
VAKKLAKLAKKLAKLAL-NH2


33
CAME-15
53
KWKLFKKIGAVLKVL-NH2


34
FLAK50C
54
FAKLLAKLAKKAL-NH2


35
FLAK50D
55
FAKLLAKALKKLL-NH2


36
FLAK50E
56
FAKLLKLAAKKLL-NH2


37
FLAK80
57
FAKLLAKKLL-NH2


38
FLAK81
58
FAKKLAKALL-NH2


39
FLAK82
59
FAKKLAKKLL-NH2


40
FLAK83M
60
FAKLAKKLL-NH2


41
FLAK 26 Ac
61
FAKKLAKLAKKLAKLAL-COOH


42
Indolicidin
63
ILPWKWPWWPWRR-NH2


43
FLAK 17C
64
FAKALKALLKALKAL-NH2


44
FLAK 50H
65
FAKLLAKLAKAKL-NH2


45
FLAK 50G
66
FAKLLAKLAKLKL-NH2


46
Shiva Deriv
70
FAKKLAKKLKKLAKKLAKKWKL-NH2



P69 + KWKL


47
Shiva 10 (1-18 AC)
71
FAKKLAKKLKKLAKKLAK-COOH


48
Shiva 10 peptide
72
FAKKLAKKLKKLAKKLAKKWKL-COOH



71 + KWKL


49
CA(1-7)Shiva10(1-
73
KWKLFKKKTKLFKKFAKKLAKKL-NH2



16)


50
FLAK 54
74
FAKKLAKKLAKAL-NH2


51
FLAK 56
75
FAKKLAKKLAKLL-NH2


52
FLAK 58
76
FAKKLAKKLAKAAL-NH2


53
FLAK 72
77
FAKKLAKKAKLAKKL-NH2


54
FLAK 75
79
FAKKLKKLAKKL-NH2


55
Shiva 10 (1-16) Ac
80
KTKLFKKFAKKLAKKLKKLAKKL-COOH


56
CA(1-7)Shiva10
81
KWKLFKKKTKLFKKFAKKLAKKL-COOH



(1-16)-COOH


57
Indolocidin-ac
91
ILPWKWPWWPWRR-COOH


58
FLAK50B
92
FAKALAKLAKKLL-NH2


59
FLAK50J
93
FAKLLAKLAKKAA-NH2


60
FLAK50I
94
FAKLLALALKLKL-NH2


61
FLAK50K
95
FAKLLAKLAKAKA-NH2


62
FLAK50L
96
FAKLLAKLAKAKG-NH2


63
Shiva-11
98
FAKKLAKKLKKLAKKLAKLALALKALALKAL-NH2


64
Shiva 11
99
FAKKLAKKLKKLAKKLIGAVLKV-COOH



[(1-16)ME(2-9]-



COOH


65
FLAK 50N
101
FAKLLAKALKLKL-NH2


66
FLAK 50O
102
FAKLLAKALKKAL-NH2


67
FLAK 50P
103
FAKLLAKALKKL-NH2


68
CA(1-
104
KWKLFKKALKKLKKALKKAL-NH2



& Hecate(11/23)


69
PYL-ME
105
KIAKVALAKLGIGAVLKVLTTGL-NH2


70
FLAG26-D1
106
FAKKLAKLAKKL-NH2


71
Vishnu3
107
MPKEKVFLKIEKMGRNIRN-NH2


72
Melittin
108
GIGAVLKVLTTGLPALISWIKRKRQQ-NH2


73
FLAK26-D2
109
FAKKLAKLAKKLAKAL-NH2


74
FLAG26-D3
110
FAKKLLAKALKL-NH2


75
FLAK50 Q1
111
FAKFLAKFLKKAL-NH2


76
FLAK50 Q2
112
FAKLLFKALKKAL-NH2


77
FLAK50 Q3
113
FAKLLAKFLKKAL-NH2


78
FLAK50 Q4
114
FAKLLAKAFKKAL-NH2


79
FLAK50 Q5
117
FAKLFAKAFKKAL-NH2


80
FLAK50 Q6
118
FAKLLAKALKKFL-NH2


81
FLAK50 Q7
119
FAKLLAKALKKFAL-NH2


82
FLAK50 Q8
120
FAKLLAKLAKKFAL-NH2


83
FLAK50 Q9
121
FAKLFAKLAKKFAL-NH2


84
FLAK50 Q10
122
FKLAFKLAKKAFL-NH2


85
FLAK50 T1
123
FAKLLAKLAK-NH2


86
FLAK50 T2
124
FAKLLAKLAKKVL-NH2


87
FLAK50 T3
125
FAKLLAKLAKKIL-NH2


88
FLAK50 T4
126
FAKLLAKLAKKEL-NH2


89
FLAK50 T5
127
FAKLLAKLAKKSL-NH2


90
FLAK90
128
FAKLA-NH2


91
FLAK91
129
FAKLF-NH2


92
FLAK92
130
KAKLF-NH2


93
FLAK93
131
KWKLF-NH2


94
FLAK50 Z1
132
FGKGIGKVGKKLL-NH2


95
FLAK50 Z2
133
FAFGKGLGKVGKKLL-NH2


96
FLAK50 Z3
134
FAKAIAKIAFGKGIGKVGKKLL-NH2


97
FLAK50 Z4
135
FAKLWAKLAFGKGIGKVGKKLL-NH2


98
FLAK50 Z5
136
FAKLWAKLAKKL-NH2


99
FLAK50 Z6
137
FAKGVGKVGKKAL-NH2


100
FLAK50 Z7
138
FAFGKGIGKIGKKGL-NH2


101
FLAK50 Z8
139
FAKIIAKIAKIAKKIL-NH2


102
FLAK50 Z9
140
FAFAKIIAKIAKKII-NH2


103
FLAK94
141
FALALKA-NH2


104
FLAK93B
142
KWKLAKKALALL-NH2


105
FLAK50 Z10
143
FAKIIAKIAKKI-NH2


106
FLAK96
144
FALALKALKKAL-NH2


107
FLAK97
145
FALKALKK-NH2


108
FLAK98
146
KYKKALKKLAKLL-NH2


109
FKRLA
147
FKRLAKIKVLRLAKIKR-NH2


110
FLAK91B
148
FAKLAKKALAKLL-NH2


111
FLAK92B
149
KAKLAKKALAKLL-NH2


112
FLAK99
150
KLALKLALKALKAAKLA-NH2


113
FLAK50T6
151
FAKLLAKLAKK-NH2


114
FLAK50T7
152
FAKLLAKLAKKGL-NH2


115
FLAK95
153
FALKALKKLKKALKKAL-NH2


116
FLAK50T8
154
VAKLLAKLAKKVL-NH2


117
FLAK50T9
155
YAKLLAKLAKKAL-NH2


118
FLAK100-CO2H
156
KLLKLLLKLYKKLLKLL-COOH


119
FAGVL
157
FAVGLRAIKRALKKLRRGVRKVAKDL-NH2


120
Modelin-5
159
KLAKKLAKLAKLAKAL-NH2


121
Modelin-5-CO2H
160
KLAKKLAKLAKLAKAL-COOH


122
Modelin-8
161
KWKKLAKKW-NH2


123
Modelin-8-CO2H
162
KWKKLAKKW-COOH


124
Modelin-1
163
KLWKKWAKKWLKLWKAW-NH2


125
Modelin-1-CO2H
164
KLWKKWAKKWLKLWKA-COOH


126
FLAK120
165
FALALKALKKL-NH2


127
FLAK121
166
FALAKALKKAL-NH2


128
FLAK96B
167
FALALKLAKKAL-NH2


129
FLAK96G
168
FALLKL-NH2


130
FLAK96F
169
FALALKALKK-NH2


131
FLAK96C
170
FALKALKKAL-NH2


132
FLAK96D
171
FALLKALKKAL-NH2


133
Modelin-8B
172
KWKK-NH2


134
Modelin-8C
173
KWKKL-NH2


135
Modelin-8D
174
KFKKLAKKF-NH2


136
Modelin-8E
175
KFKKLAKKW-NH2


137
Flak 96
176
FALALKALKKA-NH2


138
Flak 96I
177
FALLKALLKKAL-NH2


139
Flak 96J
178
FALALKLAKKL-NH2


140
Flak 96L
179
LKKLAKLALAF-NH2


141
FLAK-120G
180
VALALKALKKL-NH2


142
FLAK-120D
181
FALALKLKKL-NH2


143
FLAK-120C
182
FALALKAKKL-NH2


144
FLAK-120B
183
FALA-NH2


145
FLAK-120F
184
WALAL-NH2


146
Magainin2wisc
300
GIGKFLHAAKKFAKAFVAEIMNS-NH2


147
D2A21
301
FAKKFAKKFKKFAKKFAKFAFAF-NH2


148
KSL-1
302
KKVVFKVKFK-NH2


149
KSL-7
303
FKVKFKVKVK-NH2


150
LSB-37
306
LPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-





NH2


151
Anubis-2
307
FAKKLAKKLKKLAKKLAKLAKKL-NH2


152
FLAK17CV
501
VAKALKALLKALKAL-NH2


153
FLAK50Q1V
502
VAKFLAKFLKKAL-NH2


154
D2A21V
503
VAKKFAKKFKKFAKKFAKFAFAF-NH2


155
FLAK25AMV
504
VAKKLAKLAKKLAKLALAL-NH2


156
FLAK43AMV
505
VAKKLAKLAKKLLAL-NH2


157
FLAK50DV
506
VAKLLAKALKKLL-NH2


158
HECATE AMV
507
VALALKALKKALKKLKKALKKAL-NH2


159
HECATE ACV
508
VALALKALKKALKKLKKALKKAL-COOH


160
FLAK04AMV
509
VALALKALKKLAKKLKKLAKKAL-NH2


161
FLAK03AMV
510
VALALKALKKLLKKLKKLAKKAL-NH2


162
D-Shiva 10 AC
67
(D)-FAKKLAKKLKKLAKKLAKLALAL-COOH


163
Shiva 11 AC
100
FAKKLAKKLKKLAKKLAKLALALKALALKA-COOH


164
Shiva 10 (1-18)AM
69
FAKKLAKKLKKLAKKLAK-NH2


165
FLAK 50M
97
FAKLLALALKKAL-NH2









DETAILED DESCRIPTION OF THE INVENTION
The invention is generally directed towards peptides having desirable biological properties, and their use. It is surprising that the peptides are efficacious due to their short length as compared to other peptides described in the art.
Peptides
One embodiment of the invention is directed towards an isolated peptide comprising phenylalanine, leucine, alanine, and lysine residues, wherein the peptide is about 5 to about 23 amino acids in length. The peptide can have a minimum length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or about 18 amino acids. The peptide can have a maximum length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or about 23 amino acids. The peptide can be about 5 to about 20 amino acids in length. The peptide can consist essentially of, or consist of phenylalanine, leucine, alanine, and lysine residues. The peptide can have a percent amino acid composition of phenylalanine, leucine, alanine, and lysine residues of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. The peptide can generally be any of the listed SEQ ID NOS which fall within these various guidelines, and more preferably is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:152, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, and SEQ ID NO:165. The peptide is preferably not hecate-1, anubis-1, anubis-2, anubis-5, anubis-8, vishnu-1, vishnu-2, vishnu-3, vishnu-8, or shiva-10.
The peptide can be similar to any of the above described peptides, and preferably is similar to SEQ ID NO:2 (or SEQ ID NO:16 or SEQ ID NO:126), SEQ ID NO:4 (or SEQ ID NO:14 or SEQ ID NO:17), SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:75, SEQ ID NO:84, SEQ ID NO:115, SEQ ID NO:126, or SEQ ID NO:132 as determined by percent identity. The percent identity between the peptides is preferably at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Percent identity is determined using a sequence alignment by the commercial product CLUSTALW. The number of aligned amino acids are divided by the length of the shorter peptide, and the result is multiplied by 100% to determine percent identity. If the length of the shorter peptide is less than 10 amino acids, the number of aligned amino acids are divided by 10, and the result is multiplied by 100% to determine percent identity.
The peptides can comprise D- or L-amino acids. The peptides can comprise all D-amino acids. The peptides can have an acid C-terminus (—CO2H) or an amide C-terminus (—CONH2, —CONHR, or —CONR2).
Methods of Use
An additional embodiment of the invention is directed towards methods of using the above described peptides. The methods of use preferably do not cause injury or kill normal uninfected mammalian cells. The methods of use at therapeutic dose levels preferably do not cause injury to or kill normal uninfected or non-neoplastic mammalian cells. The methods of use may involve the use of a single peptide, or may involve the use of multiple peptides.
An embodiment of the invention is the use of the above described peptides to inhibit or kill microbial cells (microorganisms). The microorganisms may be bacterial cells, fungal cells, protozoa, viruses, or eucaryotic cells infected with pathogenic microorganisms. The method generally is directed towards the contacting of microorganisms with the peptide. The contacting step can be performed in vivo, in vitro, topically, orally, transdermally, systemically, or by any other method known to those of skill in the art. The contacting step is preferably performed at a concentration sufficient to inhibit or kill the microorganisms. The concentration of the peptide can be at least about 0.1 μM, at least about 0.5 μM, at least about 1 μM, at least about 10 μM, at least about 20 μM, at least about 50 μM, or at least about 100 μM. The methods of use can be directed towards the inhibition or killing of microorganisms such as bacteria, gram positive bacteria, gram negative bacteria, mycobacteria, yeast, fungus, algae, protozoa, viruses, and intracellular organisms. Specific examples include, but are not limited to, Staphylococcus, Staphylococcus aureus, Pseudomonas, Pseudomonas aeruginosa, Escherichia coli, Chlamydia, Candida albicans, Saccharomyces, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trypanosoma cruzi, or Plasmodium falciparum. The contacting step can be performed by systemic injection, oral, subcutaneous, IP, IM, IV injection, or by topical application. For injection, the dosage can be between any of the following concentrations: about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, and about 100 mg/kg. The contacting step can be performed on a mammal, a cat, a dog, a cow, a horse, a pig, a bird, a chicken, a plant, a fish, or a human.
Presently preferred peptides for antibacterial applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:93, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, and SEQ ID NO:165.
Presently preferred peptides for antifungal applications include SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:163, and SEQ ID NO:165.
An additional embodiment of the invention is the use of any of the above described peptides to inhibit or kill cancer cells. The method generally is directed towards the contacting of cancer cells with the peptide. The contacting step can be performed in vivo, in vitro, topically, orally, transdermally, systemically, or by any other method known to those of skill in the art. The contacting step is preferably performed at a concentration sufficient to inhibit or kill the cancer cells. The concentration of the peptide can be at least about at least about 0.1 μM, at least about 0.5 μM, at least about 1 μM, at least about 10 μM, at least about 20 μM, at least about 50 μM, or at least about 100 μM. The cancer cells can generally be any type of cancer cells. The cancer cells can be sarcomas, lymphomas, carcinomas, leukemias, breast cancer cells, colon cancer cells, skin cancer cells, ovarian cancer cells, cervical cancer cells, testicular cancer cells, lung cancer cells, prostate cancer cells, and skin cancer cells. The contacting step can be performed by subcutaneous, IP injection, IM injection, IV injection, direct tumor injection, or topical application. For injection, the dosage can be between any of the following concentrations: about 0.1 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, and about 100 mg/kg. The contacting step can be performed on a mammal, a cat, a dog, a cow, a horse, a pig, a bird, a chicken, a plant, a fish, a goat, a sheep, or a human. The inhibition of cancer cells can generally be any inhibition of growth of the cancer cells as compared to the cancer cells without peptide treatment. The inhibition is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and ideally 100% inhibition of growth. The inhibition may be achieved by lysis of the cancer cells or by other means. The cancer inhibiting peptide can be used synergistically with other cancer chemotherapeutic agents.
Presently preferred peptides for anticancer applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:152, and SEQ ID NO:162.
An additional embodiment of the invention is directed towards a method for promoting the stimulation and/or proliferation of cells. The method can comprise contacting the cells and a composition, wherein the composition comprises a peptide. The peptide can be any of the above described peptides. The concentration of the peptide in the composition can be about 0.01 μM to about 500 μM, about 0.1 μM to about 100 μM, about 1 μM to about 50 μM, or about 1 μM to about 10 μM. The cells can generally be any type of cells, and preferably are mammalian cells, specifically including, but not limited to fibroblast and leukocyte cells, including lymphocyte and phagocytic cells. The metabolic stimulation and/or proliferation of the cells is preferably increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the same cells not contacted with the composition. The composition can further comprise a growth factor. The stimulatory and proliferative properties of some of the FLAK peptides hold promise for their application in skin care, wound healing, and in immunomodulation of compromised mammalian immune systems.
Presently preferred peptides for stimulation and proliferation applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:164, and SEQ ID NO:165.
An additional embodiment of the invention is directed towards a method for promoting wound healing of skin or ocular and internal body tissues damaged by normal aging, disease, injury, or by surgery or other medical procedures. The method can comprise administering to the wound of an animal a composition, wherein the composition comprises any of the above described peptides. The concentration of the peptide in the composition can be about 0.01 μM to about 500 μM, about 0.1 μM to about 100 μM, about 1 μM to about 50 μM, or about 1 μM to about 10 μM. The composition can be administered to the wound topically or by systemic delivery. The animal can generally be any kind of animal, preferably is a mammal, and more preferably is a human, cow, horse, cat, dog, pig, goat, or sheep. The promotion of wound healing is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the same wound not contacted with the composition.
Presently preferred peptides for wound healing applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, and SEQ ID NO:164.
A further embodiment of the invention is directed towards methods for the additive or synergistic enhancement of the activity of a therapeutic agent. The method can comprise preparing a composition, wherein the composition comprises a peptide and a therapeutic agent. Alternatively, the method may comprise co-therapy treatment with a peptide (or peptides) used in conjunction with other therapeutic agents. The peptide can be any of the above described peptides. The therapeutic agent can generally be any therapeutic agent, and preferably is an antibiotic, an antimicrobial agent, a growth factor, a chemotherapy agent, an antimicrobial agent, lysozyme, a chelating agent, or EDTA. Preferably, the activity of the composition is higher than the activity of the same composition containing the therapeutic agent but lacking the peptide. The composition or co-therapy can be used in in vitro, in vivo, topical, oral, IV, IM, IP, and transdermal applications. The enhancement of the activity of the composition containing the therapeutic agent and the peptide is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the activity of the therapeutic agent alone.
Generally, any peptide which is active on a stand-alone basis against a target is preferred for use to increase either additively or synergistically the activity of another therapeutic agent against that target. If several peptides are candidates for a given synergy application, then the less toxic peptides would be more favorably considered.
A further additional embodiment of the invention is directed towards methods for the treatment of patients diagnosed with Cystic Fibrosis (CF). CF causes, among other effects, inflammation and infection in the lungs. The above described peptides of the instant invention can be used in treating such lung infections, which are often caused by P. aeruginosa. The inventive peptides may possess anti-inflammatory properties, making them further useful for the treatment of lung infections in CF patients. The peptide can be administered to the CF patient by any acceptable method including inhalation or systemic delivery. The peptide can be administered in a single dose, in multiple doses, or as a continuous delivery.
An additional embodiment of the invention is directed towards methods of treating sexually transmitted diseases (STDs). Many of the fungal species responsible for STDs are inhibited or killed by the inventive peptides described above. Examples of such species include C. albicans, C. glabrata, and C. tropicalis. The inventive peptides may additionally be used against other agents responsible for STDs including viruses and bacteria. The peptides can be administered to an STD patient by any acceptable method, such as topical, oral, or systemic delivery. The peptide can be administered in a single dose, in multiple doses, or as a continuous delivery. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid.
A further additional embodiment of the invention is directed towards methods for the treatment of acne. The inventive peptides have activity against the bacteria isolated from acne sores, Propionibacterium acnes, and may further possess anti-inflamatory properties. The peptide can be present in a clinical therapeutic composition or in a cosmeceutical composition. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid. The peptide can be administered in any acceptable manner, such as topical administration. The peptide can be used in a treatment method, or in a preventative manner to reduce or eliminate future outbreaks of acne.
Yet a further embodiment is directed towards cosmetic compositions. The inventive peptides have been shown to stimulate collagen and fibroblasts, and to promote wound healing. The inclusion of the inventive peptides in cosmetic formulations may be useful in the anti-aging and rejuvination markets.
An additional embodiment of the invention is directed towards the use of peptides in promoting wound healing. The inventive peptides have high potency against the bacteria most associated with wound infections: S. aureus, S. pyogenes, and P. aeruginosa. The peptides also promote wound healing and reducing of inflammation. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid. The peptide can be administered in any acceptable manner, such as topical administration or systemic administration.
The following Examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.


EXAMPLES
Example 1
Microbial Strains
The following table lists the various microorganisms used throughout the Examples.








TABLE 2





Microorganism
Reference or source







Escherichia coli
ATCC25922


Staphylococcus aureus
ATCC6538 and ATCC25923


Pseudomonas aeruginosa
ATCC9027 and ATCC27853


Staphylococcus intermedius
ATCC19930 and ATCC20034


Candida albicans
ATCC10231


Escherichia coli UB1005
D. Clark, FEMS Microb. Lett. 21: 189-195, 1984


Salmonella typhimurium 14028S
Fields et al, Science 243: 1059-1062, 1989


Staphylococcus aureus SAP0017
Methicillin resistant clinical isolate from Prof. T.



Chow, Vancouver General hospital


Staphylococcus epidermidis C621
clinical isolate from David. Speer


Streptococcus pyogenes
ATCC19615


Streptococcus pyogenes M76
From Prof. R. Gallo (UCSD)


Streptococcus pneumoniae
ATCC6305-C718


Streptococcus pneumoniae
ATCC49619-C719


Pseudomonas aeruginosa H187
Angus, et al., AAC 21: 299-309, 1982


Pseudomonas aeruginosa H374
Masuda, N., et al., AAC, 36: 1847-1851, 1992


(nfxB efflux mutant)


Pseudomonas aeruginosa H744
Poole, K., et al. J. Bacteriol. 175-7363-7372, 1993


nalB multiple resistant efflux


mutant


Pseudomonas aeruginosa 100609
Tobramycin resistant strain from Prof. D. Woods



(U. Calgary)


Pseudomonas aeruginosa 105663
Tobramycin resistant strain from Prof. D. Woods



(U. Calgary)


Candida albicans 105
From Prof Barbara Dill (UBC)


Candida guilliermondii
ATCC8492


Candida tropicalis
ATCC13803


Candida glabrata
ATCC15126


Propionibacterium acnes
ATCC6919


Propionibacterium acnes
ATCC11827


Acinetobacter baumannii
ATCC19606









Example 2
Antimicrobial Assays I
The data for the following antimicrobial assay of the peptides have been obtained by making OD measurements in in vitro cell culture experiments with and without added peptide. The protocol used is as follows.
Cell lines included Staphylococcus aureus ATCC 6538 or 25923, Pseudomonas aeruginosa ATCC 9027 or 27853. Medium used were Antibiotic Medium 3 (Difco), Antibiotic Medium 2 (Difco), and 0.85% saline. Controls used were physiological saline, and gentamycin at 50, 25, 10, 5, 1, and 0.1 ppm.
The preparation of all media, stock solutions, and dilutions took place in a laminar flow hood to prevent contamination. Bacterial cells were freshly grown on antibiotic medium 2 agar slants (pH 7.0 at 25° C.). Bacteria were suspended and diluted in antibiotic medium 3 to about 104 cfu/ml and used as the inoculum. Sample solutions (100 μl/well) were added to plates according to the plate layout. Inoculum (100 μl/well) was added to achieve a final concentration of 5×103 cfu/ml. Negative controls received 100 μl saline and 100 μl growth medium. Positive controls received 100 μl saline and 100 μl inoculum. Bacterial plates were incubated at 37° C. for 24 hours.
Absorbance was read at 620 nm after shaking to resuspend cells. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptide that completely inhibits the growth of the test organism.
The yeast assay was performed in RPMI 1640 media (pH 7.0 at 25° C.).
The data presented in Table 3 were obtained using the above protocol. However, the data for Table 4 were obtained with a modified protocol wherein the medium was tryptic soy broth, inocolum strength was approximately 104 CFU per ml, and values determined were minimum bactericidal concentrations (MBC) or minimum fungicidal concentrations (MFC).
The following Table 3 describes the antimicrobial properties of the peptides measured as MIC or MFC values in μg/mL. Staph6538 is Staphylococcus aureus ATCC accession number 6538; paerug9027 is Pseudomonas aeruginosa ATCC accession number 9027, yeast is Saccharomyces cerevisiae.












TABLE 3






SEQ ID
P





Name
NO:
Number
staph6538
paerug9027
yeast




















Hecate AC #1010
1
1
5
10
>


Hecate AM
2
2
25
100
25


SB-37 AC #1018
3
5
100
50
>


SB-37 AM
5
12
>
100
>


Shiva 10 AC
6
13
10
>
>


#1015


FLAK01 AM
8
23
5
50
100


FLAK04 AM
10
25
10
5
25


FLAK05 AM
11
26
10
15
>


FLAK06 AM
12
27
10
10
25


KAL V
15
30
>
>
ND


FLAK 17 AM
16
34
5
50
25


FLAK 26 AM
17
35
5
200
25


Hecate 2DAc
19
37
5
100
50


FLAK43 AM
20
38
5
50
50


FLAK44 AM
21
39
100
25
100


FLAK62 AM
22
40
100
25
100


FLAK 06R-AM
23
41
10
10
ND


MSI-78 AM
24
42
10
>
200


FLAK50
25
43
5
100
25


FLAK51
26
44
5
5
50


FLAK57
27
45
5
100
100


FLAK71
28
46
10
5
50


FLAK77
29
47
200
100
50


FLAK50V
30
48
5
5
25


FLAK50F
31
49
10
200
50


FLAK26V AM
32
50
5
15
50


CAME-15
33
53
5
15
50


FLAK50C
34
54
5
50
50


FLAK50D
35
55
5
5
25


FLAK 50E
36
56
200
5
50


FLAK80
37
57
100
200
200


FLAK81
38
58
100
100
200


FLAK82
39
59
>
>
>


FLAK83M
40
60
200
100
200


FLAK 17 C
43
64
5
>
200


FLAK 50H
44
65
15
50
200


FLAK 50G
45
66
5
50
100


Shiva deriv
46
70
10
>
100


P69 + KWKL


Shiva 10 (1-18—
47
71
15
15
200


AC


CA(1-7)Shiva
49
73
50
15
100


10(1-16)


FLAK 54
50
74
15
5
100


FLAK 56
51
75
5
5
50


FLAK 58
52
76
10
100
200


FLAK 72
53
77
200
100
200


FLAK 75
54
79
100
200
100


Shiva 10 (1-16) Ac
55
80
10
100
100


CA(1-7)Shiva10 (1-
56
81
10
>
>


16)-COOH


Indolocidin-ac
57
91
10
>
>


FLAK50B
58
92
5
5
50


FLAK50I
60
94
10
>
>


FLAK50K
61
95
100
200
>


FLAK50L
62
96
>
>
>


Shiva-11
63
98
>
>
>


Shiva 11[(1-
64
99
100
>
>


16)ME(2-9)]-


COOH


FLAK 50N
65
101
10
25
100


FLAK 50O
66
102
5
10
50


FLAK 50P
67
103
10
25
100


CA(1-
68
104
10
10
200


&Hecate(11/23)


PYL-ME
69
105
200
200
>


FLAG26-D1
70
106
100
25
100


Vishnu3
71
107
>
>
>


Melittin
72
108
5
>
25


FLAK26-D2
73
109
>
200
200


FLAG26-D3
74
110
>
200
200


FLAK50 Q1
75
111
5
100
200


FLAK50 Q2
76
112
50
200
100


FLAK50 Q3
77
113
10
200
200


FLAK50 Q4
78
114
50
15
100


FLAK50 Q5
79
117
100
200
200


FLAK50 Q6
80
118
10
100
100


FLAK50 Q7
81
119
50
25
50


FLAK50 Q8
82
120
50
200
200


FLAK50 Q9
83
121
50
>
100


FLAK50 T1
85
123
50
200
100


FLAK50 T2
86
124
5
100
100


FLAK50 T3
87
125
10
100
50


FLAK50 T4
88
126
>
>
>


FLAK50 T5
89
127
100
25
100


FLAK90
90
128
>
100
200


FLAK91
91
129
100
25
100


FLAK92
92
130
200
200
200


FLAK93
93
131
25
10
100


FLAK50 Z1
94
132
>
100
>


FLAK50 Z2
95
133
>
>
>


FLAK50 Z3
96
134
100
>
200


FLAK50 Z4
97
135
15
10
50


FLAK50 Z5
98
136
100
50
100


FLAK50 Z6
99
137
>
>
>


FLAK50 Z7
100
138
>
>
>


FLAK50 Z8
101
139
50
25
200


FLAK50 Z9
102
140
>
>
>


FLAK94
103
141
15
50
200


FLAK93B
104
142
100
50
100


FLAK50 Z10
105
143
100
50
200


FLAK 96
106
144
5
50
50


FLAK 97
107
145
200
100
200


FLAK 98
108
146
10
10
50


FKRLA
109
147
5
5
200


FLAK91B
110
148
>
200
200


FLAK92B
111
149
50
100
200


FLAK99
112
150
100
10
>


FLAK50T6
113
151
>
>
200


FLAK50T7
114
152
100
50
100


FLAK95
115
153
5
25
100


FLAK50T8
116
154
100
100
50


FLAK50T9
117
155
>
>
>


FLAK100-CO2H
118
156
15
>
>


FAGVL
119
157
200
>
>


FLAK120
126
165
10
25
25


FLAK121
127
166
>
>
>


FLAK96B
128
167
10
25
100


FLAK96G
129
168
50
100
>


FLAK96F
130
169
100
100
100


FLAK96C
131
170
200
100
100


FLAK96D
132
171
25
50
100


FLAK96
137
176
>
>
>


FLAK96J
139
178
200
100
>


FLAK96L
140
179
50
50
100


FLAK-120G
141
180
200
>
>


FLAK-120D
142
181
100
200
100


FLAK-120C
143
182
>
>
>


FLAK-120B
144
183
200
100
200


FLAK-120F
145
184
25
100
100


FLAK 50M
165
97
5
50
50





> indicates greater than 200 μg/mL;


ND = not determined.






The following Table 4 describes describes the antimicrobial properties of the peptides measured as minimum bactericidal or minimum fungicidal (Candida) concentrations. MBC or MFC values are in μg/mL. E. coli is Escherichia coli ATCC accession number 25922; P. aerug is Pseudomonas aeruginosa ATCC accession number 27853, S. aur. is Stapholococcus aureus ATCC accession number 25923; Candida is Candida albicans ATCC accession number 10231.












TABLE 4







E. coli
P. aerug
S. aur
Candida


SEQ ID NO:
P #
A.25922
A.27853
A.25923
A.10231




















1
1
25
30
25
>50


2
2
25
10
25
>50


3
5
50
>60
40
ND


4
11
40
25
25
>50


5
12
50
>60
75
ND


6
13
8
15
30
>50


8
23
15
25
30
>50


9
24
>80
30
>40
>50


10
25
40
30
40
>50


11
26
>80
>40
>40
>50


12
27
10
8
8
>50


13
27B
40
10
>40
>40


14
27C
10
4
>40
>40


15
30
10
15
40
>50


16
34
15
15
40
>40


17
35
8
8
10
>40


18
36
30
15
10
>40


19
37
8
8
40
>50


20
38
15
30
15
ND


21
39
>40
>40
>40
ND


22
40
30
40
>40
ND


23
41
40
40
40
ND


24
42
10
30
10
ND


25
43
8
15
4
15


26
44
10
55
30
>50


27
45
30
40
80
>50


29
47
>50
>50
>50
>50


30
48
8
25
4
10


31
49
40
30
50
30


32
50
50
25
25
>50


33
53
15
15
10
30


34
54
15
40
15
30


35
55
4
10
4
25


36
56
50
10
55
30


37
57
>50
>50
>50
>50


38
58
>50
>50
>50
>50


39
59
>50
>50
>50
>50


40
60
>50
>50
>50
>50


41
61
4
50
>80
>40


42
63
10
50
15
60


43
64
10
30
4
>50


44
65
>55
>50
>55
>50


45
66
40
50
30
40


46
70
40
30
40
>50


47
71
50
40
>50
>50


48
72
>50
40
>50
>50


50
74
>55
50
>55
>55


51
75
40
30
>55
30


52
76
40
>55
>55
>50


53
77
>50
>50
>50
>50


54
79
>50
>50
>50
>50


55
80
30
15
>50
>50


58
92
40
25
15
25


59
93
>50
>50
>50
>50


60
94
>50
>50
>50
>50


61
95
>50
>50
>50
>50


62
96
>50
>50
>50
>50


65
101
300
>50
>50
40


66
102
25
30
25
15


67
103
30
30
>50
25


69
105
25
>50
ND
>50


70
106
50
>50
ND
>50


71
107
ND
>50
>50
>50


72
108
>50
>50
25
>50


73
109
ND
ND
80
>50


74
110
8
>50
>50
>50


75
111
30
ND
40
INACT


76
112
30
INACT
INACT
INACT


77
113
INACT
INACT
INACT
40


79
117
INACT
INACT
INACT
INACT


80
118
8
25
10
25


81
119
15
30
4
25


82
120
INACT
INACT
INACT
INACT


83
121
INACT
INACT
INACT
50


84
122
30
30
25
15


85
123
40
INACT
INACT
25


86
124
10
40
8
15


87
125
40
40
INACT
40


88
126
INACT
INACT
INACT
INACT


89
127
INACT
INACT
INACT
INACT


90
128
INACT
INACT
INACT
INACT


91
129
INACT
INACT
INACT
INACT


92
130
INACT
INACT
INACT
INACT


93
131
INACT
INACT
INACT
INACT


94
132
INACT
INACT
INACT
INACT


95
133
INACT
INACT
INACT
INACT


96
134
INACT
INACT
INACT
INACT


97
135
INACT
40
INACT
25


98
136
INACT
INACT
INACT
INACT


99
137
INACT
INACT
INACT
INACT


100
138
INACT
INACT
INACT
INACT


101
139
INACT
INACT
INACT
INACT


102
140
INACT
INACT
INACT
INACT


103
141
INACT
INACT
INACT
INACT


104
142
INACT
INACT
INACT
INACT


105
143
INACT
INACT
INACT
INACT


106
144
10
25
25
25


107
145
INACT
INACT
INACT
100


108
146
10
>250
75
10


109
147
25
75
>250
>250


110
148
150
>250
>250
100


111
149
150
>250
>250
100


112
150
75
>250
>250
50


113
151
>250
>250
>250
100


114
152
150
150
>250
50


115
153
10
25
5
25


116
154
50
100
>250
25


117
155
>250
>250
>250
>250


118
156
100
>250
>250
>250


119
157
75
>250
>250
>250


120
159
10
10
>250
50


121
160
>250
>250
>250
>250


122
161
150
>250
>250
25


123
162
50
>250
>250
100


124
163
25
50
25
25


125
164
25
25
25
25


126
165
10
25
25
10


127
166
>250
>250
>250
>250


128
167
25
>250
10
25


129
168
75
100
>250
150


130
169
200
>250
>250
75


131
170
25
>250
150
25


132
171
75
100
>250
50


133
172
>250
>250
>250
>250


134
173
>250
>250
>250
150


162
67
25
30
30
>50


165
97
25
>50
25
25





INACT refers to no detectable activity.


ND indicates no data available.






Example 3
Antimicrobial Assays II
Anti-microbial activity against a broader range of pathogens (including clinical strains) than were tested in Example 2. It should be noted that somewhat different protocols were employed for the assays in Example 2 and Example 3.
MICs were determined for this Example using a slightly modified version of the NCCLS (National Committee for Clinical Laboratory Standards) broth microdilution method as described previously (Steinberg et al., AAC 41: 1738, 1997). Briefly, antimicrobial agents were prepared as 10X concentrates in the most appropriate solvent. For the peptide, 0.01% acetic acid containing 0.2% bovine serum albumin as a carrier protein was used. Inocula were prepared by resuspending colonies from a BAP in medium and adjusting the suspension to match that of a 0.5 McFarland standard. The suspension was diluted into fresh medium (as recommended by NCCLS for the organism) to give 2×105 to 7×105 CFU/ml for bacteria or 2×103 to 7×103 CFU/ml for Candida. After dispensing 100 μl aliquots of the microbial suspension into each well of a 96-well polypropylene microtiter plate, 11 μl of test compound was added. The MIC was defined as the lowest concentration of drug which prevented visible turbidity after 16 to 20 hours (bacteria) or 46 to 50 hours (Candida) at 35° C. For facultative anaerobes incubation was performed in 7% carbon dioxide and for strict anaerobes in an oxygen free environment maintained using a standard anaerobic “jar”. All MICs were performed three times and the mean value determined.







TABLE 5







Activity against gram positive bacteria











S. aureus
S. epidermidis
S. pyogenes


Peptide (SEQ ID NO:)
(MRS A)
C621
M76













P23 (8) 
32
16
16


P25 (10)
16
4
8


P26 (11)
32
4
4


P27 (12)
16
4
4


P34 (16)
16
8
4


P35 (17)
8
4
4


P37 (19)
8
4
8


P41 (23)
64
4
8


P42 (24)
16
2
4


P43 (25)
4
2
2


P44 (26)
8
4
4


P46 (28)
64
8
8


P49 (31)
64
8
8


P50 (32)
4
4
8


P54 (34)
16
8
8


P55 (35)
4
2
4


P59 (39)
8
8
2


P60 (40)
32
4
8


P61 (41)
32
8
16


P63* (42) 
32
16
8


P64* (43) 
8
4
4


P72 (48)
16
4
16


P73 (49)
16
4
16


P75 (51)
32
8
8


P94* (60) 
16
8
8


 P97 (165)
8
4
4


P105* (69) 
32
8
16


P111 (75) 
8
4
4


P119 (81) 
8
4
8


P124 (86) 
8
4
16


P146 (108)
16
8
8


P153 (115)
16
4
2


P157 (119)
32
4
8


P177 (138)
8
4
8


P301 (147)
8
4
8


P504 (155)
4
4
8


P510 (161)
8
4
8


P2 (2)
32
8
4


P27 (12)
8
4
4





Bold indicates broad spectrum activity; *indicates gram-positive selective













TABLE 6







Activity against gram positive bacteria











Peptide






(SEQ ID NO:)
S. pyogenes
S. pneumoniae
S. pneumoniae
P. acne














P23 (8) 
8
16
16
4


P25 (10)
8
64
8
2


P26 (11)
4
>128
16
4


P27 (12)
4
32
8
4


P34 (16)
4
8
8
8


P35 (17)
16
4

4


P37 (19)
8
64
16
4


P41 (23)
8
64
32
4


P42 (24)
4
32
8
2


P43 (25)
2
8
4
2


P44 (26)
4
8
16
4


P46 (28)
16
64
128


P49 (31)
8
64
32


P50 (32)
4
32
16
4


P54 (34)
8
64
64


P55 (35)
2
8
4
4


P59 (39)
2
16
4
2


P60 (40)
8
128
>128
4


P61 (41)
16
128
32
2


P63* (42) 
8
128
16


P64* (43) 
4
8
2
2


P72 (48)
16
>128
16
2


P73 (49)
16
>128
64
4


P75 (51)
4
>128
64
16


P94* (60) 
8
64
128


 P97 (165)
4
32
16
8


P105* (69) 
16
64
32
16


P111 (75) 
2
16
4
4


P119 (81) 
8
128
32
8


P124 (86) 
16
>128
64
8


P146 (108)
8
>128
128
16


P153 (115)
2
32
8
4


P157 (119)
8
128
16
4


P177 (138)
4
32
16
8


P301 (147)
8
>128
8
2


P504 (155)
16
64
8
4


P510 (161)
8
64
16
2


 P2A* (2)  
8
128
32


 P97 (165)
8
32
32
16


P27 (12)
4
16
4
4





Bold indicates broad spectrum activity; *indicates gram-positive selective; S. pyogenes ATCC19615; S. pneumoniae C718; S. pneumoniae C719; P.acne ATCC 6919













TABLE 7







Activity against gram-negative bacteria











E.coli
S. typhimurium
P. aeruginosa


Peptide (SEQ ID NO:)
UB1005
14028S
H374













P12 (5) 
1
4
8


P39 (21)
4
16
16


P41 (23)
2
4
4


P46 (28)
4
8
4


P61 (41)
2
4
4


P71 (47)
2
8
4


P100 (163)
0.5
4
8


P109 (73) 
16
32
8


P110 (74) 
16
32
8


P157 (119)
8
8
8


P306 (150)
4
4
8


P46 (28)
8
16
4


P29 (14)
8
8
16
















TABLE 8







Activity against gram-negative bacteria










P. aeruginosa
C. glabrata


Peptide
H187
ATCC15126












P12 (5) 
16
128


P39 (21)
32
16


P41 (23)
8
32


P46 (28)
16
32


P61 (41)
8
32


P71 (47)
8
32


P100 (163)
32
>128


P109 (73) 
64
128


P110 (74) 
64
128


P157 (119)
8
64


P306 (150)
16
>128


P46 (28)
8
32


P29 (14)
32
128
















TABLE 9







Activity against Pseudomonas bacterial strains













P.
P.



Peptide
P.
aerugi-
aerugi-
P.


(SEQ ID
aeruginosa
nosa
nosa
aeruginosa


NO:)
H374
H187
Tb 105663
Tb 100609














P12 (5) 
8
16
8
8


P25 (10)
8
8
8
8


P27 (12)
8
8
16
16


P35 (17)
8
8
4
4


P37 (19)
8
8
16
16


P39 (21)
16
32
32
32


P41 (23)
4
8
8
8


P42 (24)
4
8
8
8


P43 (25)
8
8
8
8


P44 (26)
8
8
16
8


P45 (27)
8
16
32
32


P46 (28)
4
16
32
16


P50 (32)
4
4
8
4


P55 (35)
8
8
16
8


P59 (39)
8
8
8
8


P61 (41)
4
8
8
16


P71 (47)
4
8
16
16


P72 (48)
4
8
8
8


P73 (49)
8
16
16
16


 P97 (165)
8
16
16
16


P111 (75) 
8
8
32
16


P119 (81) 
8
16
16
16


P124 (86) 
16
32
64
64


P146 (108)
2
4
8
8


P153 (115)
4
8
8
8


P157 (119)
8
8
16
16


P177 (138)
16
16
32
32


P301 (247)
4
8
8
8


P306 (150)
8
16
32
16


P504 (155)
8
8
16
8


P510 (161)
8
8
16
16


P2 (2)
16
16
16
32


P13 (6) 
16
16
16
16


P27 (12)
8
8
8
8


P11 (4) 
16
16
16
16





Bold indicates broad spectrum activity.






The following tables compare the anti-fungal and anti-bacterial properties of a representative sample of peptides.







TABLE 10







Comparison of anti-fungal and anti-bacterial


activities of selected peptides










Peptide

C. tropicalis
C. glabrata


(SEQ ID NO:)
C. albicans 105
ATCC13803
ATCC15126













 P40 (22)
32
1
32


 P47 (29)
32
1
64


 P49 (31)
16
2
16


 P74 (50)
16
1
16


 P77 (53)
16
1
64


 P79 (54)
32
2
128


P101 (65)
32
4
32


P103 (67)
16
2
16


P106 (70)
32
2
64


P113 (77)
32
4
32


P122 (84)
32
4
64


 P154 (116)
64
8
128


 P167 (128)
64
8
128


 P169 (130)
64
8
128
















TABLE 11







Comparison of anti-fungal and anti-bacterial


activities of selected peptides













S.




Peptide
E. coli
typhimurium
P. aeruginosa
S. aureus


(SEQ ID NO:)
UB1005
14028S
H187
SAP0017














P40 (22)
64
>128
>128
>128


P47 (29)
64
>128
64-128
>128


P49 (31)
32
64
16-64 
64


P74 (50)
16
64
32-128
>128


P77 (53)
64
>128
64-128
>128


P79 (54)
32
>128
>128
>128


P101 (65) 
32
128
32-128
128


P103 (67) 
32
128
64
64


P106 (70) 
64
>128
>128
>128


P113 (77) 
32
44
32-128
32


P122 (84) 
64
128
32-128
128


P154 (116)
64
>128
>128
>128


P167 (128)
32
64
128
128


P169 (130)
32
64
128
>128









Many of the disclosed FLAK peptides have activity against a wide array of microorganisms. The following tables illustrate these properties for a representative sample of peptides.







TABLE 12







Broad spectrum activities













S.




Peptide
E. coli
typhimurium
P. aeruginosa
P. aeruginosa


(SEQ ID NO:)
UB1005
1402S
H374
H187














P25 (10)
8
8
8
8


P27 (12)
8
16
8
8


P35 (17)
2
4
8
8


P37 (19)
4
8
8
8


P42 (24)
4
8
4
8


P43 (25)
8
8
8
8


P44 (26)
1
4
8
8


P45 (27)
4
32
8
16


P50 (32)
2
4
4
4


P55 (35)
4
4
8
8


P59 (39)
8
8
8
8


P72 (48)
2
8
4
8


P73 (49)
8
16
8
16


 P97 (165)
8
16
8
16


P111 (75) 
16
16
8
8


P119 (81) 
4
8
8
16


P124 (86) 
16
16
16
32


P146 (108)
2
4
2
4


P153 (115)
8
8
4
8


P177 (138)
8
16
16
16


P301 (147)
8
8
4
8


P504 (155)
4
4
8
8


P510 (161)
8
16
8
8
















TABLE 13







Broad spectrum activities











Peptide
S. aureus
S. epidermis
C. albicans
C. glabrata


(SEQ ID NO:)
SAP0017
C621
105
ATCC15126














P25 (10)
16
4
32
32


P27 (12)
16
4
32
32


P35 (17)
8
4
32
16


P37 (19)
8
4
32
32


P42 (24)
16
2
32
64


P43 (25)
4
2
8
16


P44 (26)
8
4
8
16


P45 (27)
32
16
16
16


P50 (32)
4
4
16
16


P55 (35)
4
2
16
8


P59 (39)
8
8
32
16


P72 (48)
16
4
32
64


P73 (49)
16
4
32
128


 P97 (165)
8
4
16
16


P111 (75) 
8
4
32
32


P119 (81) 
8
4
16
16


P124 (86) 
8
4
16
16


P146 (108)
16
8
8
16


P153 (115)
16
4
16
16


P177 (138)
8
4
16
16


P301 (147)
8
4
32
32


P504 (155)
4
4
64
64


P510 (161)
8
4
32
64


P27 (12)
8
4
16
16









While FLAK peptides are generally active against an array of microbial targets, not all peptides are equally effective against all microorganisms. The following tables present some combinations of peptides and microorganisms in which the peptide was observed to have poor activity.







TABLE 14







Low observed anti-microbial activities











E. coli
S. typhimurium
P. aeruginosa


Peptide (SEQ ID NO:)
UB1005
14028S
H374













 P57 (37)
>128
>128
>128


 P58 (38)
>128
>128
>128


 P65 (44)
128
>128
64


 P76 (52)
16
128
64


 P93 (59)
128
>128
128


 P95 (61)
>128
>128
>128


 P96 (62)
>128
>128
>128


P107 (71)
>128
>128
>128


P112 (76)
>128
>128
>128


P114 (78)
32
128
>128


P120 (82)
>128
>128
128


P121 (83)
>128
>128
>128


P123 (85)
64
>128
>128


P126 (88)
>128
>128
>128


P127 (89)
128
>128
>128


P128 (90)
128
>128
>128


P129 (91)
64
>128
>128


P130 (92)
>128
>128
>128


P131 (93)
>128
>128
>128


P132 (94)
128
>128
>128


P133 (95)
>128
>128
>128


P134 (96)
128
>128
128


P136 (98)
128
>128
>128


P137 (99)
>128
>128
>128


 P138 (100)
>128
>128
>128


 P139 (101)
64
>128
>128


 P140 (102)
>128
>128
>128


 P141 (103)
>128
>128
>128


 P142 (104)
64
128
>128


 P143 (105)
>128
>128
>128


 P145 (107)
>128
>128
>128


 P147 (109)
64
128
128


 P148 (110)
128
>128
>128


 P149 (111)
32
>128
128


 P151 (113)
>128
>128
128


 P152 (114)
32
>128
>128


 P155 (117)
>128
>128
>128


 P166 (127)
>128
>128
>128


 P168 (129)
128
>128
128


 P169 (130)
64
64
128


 P170 (131)
64
>128
>128


 P171 (132)
32
>128
>128


 P174 (135)
>128
>128
>128


 P175 (136)
>128
>128
>128


 P180 (141)
>128
>128
>128
















TABLE 15







Low observed anti-microbial activities












P.

S.



Peptide
aeruginosa
S. aureus
epidermidis
C. albicans


(SEQ ID NO:)
H187
SAP0017
C621
105














 P57 (37)
>128
>128
>128
128


 P58 (38)
>128
>128
>128
64


 P65 (44)
>128
>128
>128
64


 P76 (52)
>128
>128
>128
64


 P93 (59)
>128
>128
>128
64


 P95 (61)
>128
>128
>128
>128


 P96 (62)
>128
>128
>128
>128


P107 (71)
>128
>128
>128
>128


P112 (76)
>128
>128
64
128


P114 (78)
>128
>128
64
64


P120 (82)
>128
>128
>128
64


P121 (83)
>128
>128
>128
64


P123 (85)
>128
>128
16
64


P126 (88)
>128
>128
>128
>128


P127 (89)
>128
>128
64
32


P128 (90)
>128
>128
128
128


P129 (91)
>128
>128
32
128


P130 (92)
>128
>128
>128
>128


P131 (93)
>128
>128
>128
>128


P132 (94)
>128
>128
>128
128


P133 (95)
>128
>128
>128
>128


P134 (96)
>128
>128
128
64


P136 (98)
>128
>128
128
64


P137 (99)
>128
>128
>128
>128


 P138 (100)
>128
>128
>128
>128


 P139 (101)
128
>128
64
128


 P140 (102)
>128
>128
>128
>128


 P141 (103)
>128
>128
>128
>128


 P142 (104)
>128
>128
128
64


 P143 (105)
>128
>128
>128
>128


 P145 (107)
>128
>128
>128
64


 P147 (109)
>128
>128
64
64


 P148 (110)
>128
>128
128
128


 P149 (111)
>128
>128
>128
128


 P151 (113)
>128
>128
>128
128


 P152 (114)
>128
>128
32
128


 P155 (117)
>128
>128
>128
>128


 P166 (127)
>128
>128
>128
>128


 P168 (129)
128
>128
128
128


 P169 (130)
>128
>128
32
64


 P170 (131)
>128
0.128
>128
128


 P171 (132)
>128
>128
128
>128


 P174 (135)
>128
>128
>128
>128


 P175 (136)
>128
>128
>128
>128


 P180 (141)
>128
>128
>128
>128









Example 4
Anti-Cancer Assays
Cancer cell assays were performed in a manner similar to the anti-microbial assays described above, except that the assay procedure used the MTT dye protocol. Viability of cells is determined by the dye response. In the following procedure, approximately 1.5×104 cells per well were added and viability was determined with the cells in a semi-confluent state. The assay was performed in a 96-well microtiter plate. After addition of peptide, the plate was set for 24 hours. MTT (5 mg/ml in phenol red-free RPMI-1640, 20 μl) was added to each well including positive control wells untreated with peptide. The plate was incubated at 37° C. for 4 hours. The liquid contents of each well was removed, and isopropanol with 0.1 M HCl (100 μl) was added to each well. The plate was sealed with parafilm to prevent evaporation of the isopropanol. The plate is allowed to rest for 5-10 minutes in order to solubilize the precipitate. Purified water (100 μl) was added to each well. Absorbance was determined with an ELISA Reader instrument. Color intensity at 540 nm is proportional to viability of cells. Results for each concentration of peptide are plotted relative to untreated controls, and LD50 values are determined from the graphs.
WI38 (ATCC No. CCL75) is a normal fibroblast line of lung diploid cells, MCF7 (ATCC No. HTB22) is a breast adenocarcinoma tumor cell line, SW480 (ATCC No. CCL228) is a colon adenocarcinoma tumor cell line, BMKC is a cloned melanoma line derived from Bowes melanoma line HMCB (ATCC No. CRL9607), H1299 (ATCC No. CRL5803) is a lung large cell carcinoma tumor line, HeLaS3 (ATCC No. CCL2.2) is a cervical epitheleal carcinoma tumor cell line, and PC3 (ATCC No. CRL1435) is a prostate adenocarcinoma tumor cell line. Numbers are LD50 values (μg/mL). Data on the six targets are presented in the following Tables 16 and 17.













TABLE 16






SEQ ID







Name
NO:
P No.
WI38
MCF7
SW480
BMKC





















HECATE AC
1
1
27
54
6
72


HECATE AM
2
2
66
23
46
128


SB37COOH
3
5
130
175
82
120


SB-37 AM
5
12
950
540
>
>


SHIVA 10 AC
6
13
57
>
ND
ND


FLAK01 AM
8
23
34
62
5
27


FLAK03 AM
9
24
55
26
38
85


FLAK04 AM
10
25
24
10
12
36


FLAK05 AM
11
26
96
74
8
94


FLAK06 AM
12
27
37
14
26
44


FLAK06 AC
13
27B
101
65
59
93


FLAK06 R-AC
14
27C
520
140
210
300


KAL V
15
30
93
72
62
140


FLAK 17AM
16
34
40
21
35
53


FLAK 26 AM
17
35
8
9
14
7


FLAK 25 AM
18
36
19
9
30
56


HECATE 2DAc
19
37
80
14
57
150


FLAK43 AM
20
38
12
17
13
21


FLAK44 AM
21
39
300
130
435
510


FLAK62 AM
22
40
>
760
>
>


FLAK 06R-AM
23
41
175
98
120
290


MSI-78 AM
24
42
67
31
34
140


FLAK50
25
43
5
9
9
7


FLAK51
26
44
36
140
32
47


FLAK57
27
45
200
260
180
160


FLAK71
28
46
200
300
160
150


FLAK77
29
47
>
575
>
700


FLAK50V
30
48
41
23
47
43


FLAK50F
31
49
135
40
100
115


FLAK26V AM
32
50
43
32
46
40


CAME-15
33
53
32
45

40


FLAK50C
34
54
97
60

90


FLAK50D
35
55
32
16
14
16


FLAK 50E
36
56
250
500
215
205


FLAK80
37
57
900
>
740
740


FLAK81
38
58
>
>
>
>


FLAK82
39
59
77
31
42
155


FLAK83M
40
60
>
>
>
>


FLAK 26 Ac
41
61
93
105
100
140


INDOLICIDIN
42
63
ND
64
345
200


FLAK 17 C
43
64
37
80

35


FLAK 50H
44
65
320
475
345
250


FLAK 50G
45
66
240
90
145
200


SHIVA DERIV
46
70
34
44
11
94


P69 + KWKL


SHIVA 10 (1-18—
47
71
355
190
250
445


AC


SHIVA 10
48
72
125
93
82
290


PEPTIDE


71 + KWKL


CA(1-7)Shiva10(1-
49
73
160
150
70
360


16)


FLAK 54
50
74
335
465
340
460


FLAK 56
51
75
80
42
17
24


FLAK 58
52
76
445
970
400
750


FLAK 72
53
77
>
>
>
125


FLAK 75
54
79
>
540
>
830


SHIVA 10 (1-16)
55
80
28
29
35
76


Ac


CA(1-7)Shiva10(1-
56
81
8
63
13
12


16)-COOH


INDOLOCIDIN-ac
57
91
9
12
30
180


FLAK50B
58
92
43
23
51
46


FLAK50I
60
94
6
65
ND
11


FLAK50K
61
95
250
>
>
820


FLAK50L
62
96
>
>
>
>


Shiva-11
63
98
47
96
125
94


SHIVA 11 [(1-
64
99
34
95
120
94


16)ME(2-9]-


COOH


FLAK 50N
65
101
300
250
170
160


FLAK 50O
66
102
73
60
57
60


FLAK 50P
67
103
26
46
90
75


CA(1-
68
104
24
11
54
100


&HECATE(11/23)


PYL-ME
69
105
430
635
>
ND


FLAG26-D1
70
106
>
620
570
690


VISHNU3
71
107
>
>
>
>


MELITTIIN
72
108
16
9
23
18


FLAK26-D2
73
109
>
>
>
>


FLAG26-D3
74
110
45
180
325
400


FLAK50 Q1
75
111
24
35
27
26


FLAK50 Q2
76
112
420
500
800
445


FLAK50 Q3
77
113
170
150
180
115


FLAK50 Q4
78
114
>
730
>
>


FLAK50 Q5
79
117
>
>
>
>


FLAK50 Q6
80
118
170
70
115
135


FLAK50 Q7
81
119
45
54
46
36


FLAK50 Q8
82
120
600
730
630
660


FLAK50 Q9
83
121
625
400
800
670


FLAK50 Q10
84
122
720
360
570
700


FLAK50 T1
85
123
600
615
>
635


FLAK50 T2
86
124
21
18
9
10


FLAK50 T3
87
125
90
90
125
220


FLAK50 T4
88
126
>
>
>
>


FLAK50 T5
89
127
760
440
400
535


FLAK90
90
128
500
500
530
330


FLAK91
91
129
>
>
550
>


FLAK92
92
130
>
>
>
>


FLAK93
93
131
>
600
555
>


FLAK50 Z1
94
132
>
>
>
>


FLAK50 Z2
95
133
>
>
>
>


FLAK50 Z3
96
134
>
>
740
>


FLAK50 Z4
97
135
110
54
80
155


FLAK50 Z5
98
136
>
500
600
530


FLAK50 Z6
99
137
>
>
>
>


FLAK50 Z7
100
138
>
>
>
>


FLAK50 Z8
101
139
550
625
>
525


FLAK50 Z9
102
140
>
>
>
>


FLAK94
103
141
420
430
560
465


FLAK93B
104
142
73
44
38
38


FLAK50 Z10
105
143
>
>
>
>


FLAK96
106
144
750
150
285
250


FLAK97
107
145
>
>
>
>


FLAK98
108
146
270
110
380
185


FKRLA
109
147
83
106
185
110


FLAK91B
110
148
380
315
>
330


FLAK92B
111
149
>
>
>
>


FLAK99
112
150
125
160
235
190


FLAK50T6
113
151
>
>
>
>


FLAK50T7
114
152
620
430
740
>


FLAK95
115
153
130
64
61
165


FLAK50T8
116
154
600
315
750
330


FLAK50T9
117
155
>
>
>
>


FLAK100-CO2H
118
156
230
135
345
520


FAGVL
119
157
500
240
530
600


Modelin-5
120
159
82
61
140
140


Modelin-5-CO2H
121
160
700
320
370
220


FLAK120
126
165
470
360
240
240


FLAK121
127
166
>
>
>
>


FLAK96B
128
167
260
230
360
240


FLAK96G
129
168
>
630
>
590


FLAK96F
130
169
>
510
>
530


FLAK96C
131
170
>
940
>
>


FLAK96D
132
171
615
305
770
600


Modelin-8D
135
174
>
>
>
>


Modelin-8E
136
175
>
>
70
>


Flak 96H
137
176
>
>
>
>


Flak 96I
138
177
270
190
310
310


Flak 96J
139
178
405
770
>
640


Flak 96L
140
179
540
555
>
920


FLAK-120G
141
180
940
950
600
770


FLAK-120D
142
181
500
550
870
830


FLAK-120C
143
182
>
>
>
>


FLAK-120B
144
183
>
>
>
>


FLAK-120F
145
184
800
260
440
600


Magainin2wisc
146
300
52
22
60
130


D2A21
147
301
66
64
76
140


KSL-1
148
302
800
340
>
700


KSL-7
149
303
355
315
530
330


LSB-37
150
306
320
50
240
170


Anubis-2
151
307
75
38
73
83


FLAK 17 CV
152
501
26
23
ND
ND


FLAK50 Q1V
153
502
64
92
ND
ND


D2A21V
154
503
150
210
ND
ND


FLAK 25 AM V
155
504
110
130
ND
ND


FLAK43 AM V
156
505
85
86
ND
ND


FLAK50D V
157
506
75
45
ND
ND


HECATE AM V
158
507
285
340
ND
ND


HECATE AC V
159
508
190
160
ND
ND


FLAK04 AM V
160
509
95
84
ND
ND


03 AM V
161
510
77
62
ND
ND


D-Shiva 10 AC
162
67
4
7
ND
ND


Shiva 11 AC
163
100
95
175
82
120


Shiva 10 (1-18)AM
164
69
101
45
63
66





Note:


> indicates greater than 1000;


ND indicates not determined;


numbers are in μg/mL.



















TABLE 17






SEQ ID







Name
NO:
P No.
WI38
H1299
HeLaS3
PC3





















HECATE AC
1
1
27
44
95
61


HECATE AM
2
2
66
140
50
44


SB37COOH
3
5
130
220
150
ND


SB-37 AM
5
12
950
720
>
630


SHIVA 10 AC
6
13
57
>
>
83


FLAK01 AM
8
23
34
64
82
41


FLAK03 AM
9
24
55
72
145
38


FLAK04 AM
10
25
24
37
20
12


FLAK05 AM
11
26
96
84
150
125


FLAK06 AM
12
27
37
16
25
8


FLAK06 AC
13
27B
101
54
80
16


FLAK06 AM
14
27C
520
170
260
280


KAL V
15
30
93
125
190
65


FLAK 17 AM
16
34
40
24
62
9


FLAK 26 AM
17
35
8
16
27
5


FLAK 25 AM
18
36
19
57
ND
19


HECATE 2DAc
19
37
80
150
ND
64


FLAK43 AM
20
38
12
33
35
10


FLAK44 AM
21
39
300
420
620
310


FLAK62 AM
22
40
>
>
>
435


FLAK 06R-AM
23
41
175
245
185
140


MSI-78 AM
24
42
67
150
ND
66


FLAK50
25
43
5
6
15
12


FLAK51
26
44
36
72
22
45


FLAK57
27
45
200
330
160
170


FLAK71
28
46
200
290
280
280


FLAK77
29
47
>
>
>
>


FLAK50V
30
48
41
17
44
32


FLAK50F
31
49
135
140
ND
77


FLAK26V AM
32
50
43
7
33
54


CAME-15
33
53
32
65
30
40


FLAK50C
34
54
97
80
190
90


FLAK50D
35
55
32
7
15
47


FLAK 50E
36
56
250
370
300
435


FLAK80
37
57
900
>
830
>


FLAK81
38
58
>
>
>
>


FLAK82
39
59
77
180
ND
81


FLAK83M
40
60
>
>
>
>


FLAK 26 Ac
41
61
93
127
170
66


INDOLICIDIN
42
63
ND
270
345
290


FLAK 17 C
43
64
37
30
30
46


FLAK 50H
44
65
320
450
210
470


FLAK 50G
45
66
240
130
140
170


SHIVA DERIV
46
70
34
63
28
82


P69 + KWKL


SHIVA 10(1-18—
47
71
355
320
570
270


AC


SHIVA 10
48
72
125
160
240
63


PEPTIDE


71 + KWKL


CA(1-7)Shiva10(1-16)
49
73
160
115
270
97


FLAK 54
50
74
335
670
260
660


FLAK 56
51
75
80
80
74
54


FLAK 58
52
76
445
860
380
675


FLAK 72
53
77
>
>
>
>


FLAK 75
54
79
>
>
>
>


SHIVA 10(1-16)
55
80
28
64
97
28


Ac


CA(1-7)Shiva10(1-
56
81
8
22
19
170


16)-COOH


Indolocidin-ac
57
91
9
64
20
31


FLAK50B
58
92
43
25
670
83


FLAK50J
59
93
530
320
>
690


FLAK50I
60
94
6
ND
>
ND


FLAK50K
61
95
250
>
>
>


FLAK50L
62
96
>
>
>
>


Shiva-11
63
98
47
53
175
52


SHIVA 11[(1-
64
99
34
54
180
28


16)ME(2-9]—


COOH


FLAK 50N
65
101
300
340
170
730


FLAK 50O
66
102
73
27
43
66


FLAK 50P
67
103
26
150
70
330


CA(1-
68
104
24
52
130
18


&HECATE(11/23)


PYL-ME
69
105
430
>
>
ND


FLAG26-D1
70
106
>
920
700
>


VISHNU3
71
107
>
>
>
>


MELITTIIN
72
108
16
25
35
13


FLAK26-D2
73
109
>
>
>
>


FLAG26-D3
74
110
45
95
540
>


FLAK50 Q1
75
111
24
8
7
11


FLAK50 Q2
76
112
420
470
660
640


FLAK50 Q3
77
113
170
50
190
240


FLAK50 Q4
78
114
>
>
>
>


FLAK50 Q5
79
117
>
>
>
>


FLAK50 Q6
80
118
170
74
87
330


FLAK50 Q7
81
119
45
33
30
140


FLAK50 Q8
82
120
600
620
810
>


FLAK50 Q9
83
121
625
460
830
>


FLAK50 Q10
84
122
720
830
780
800


FLAK50 T1
85
123
600
>
940
>


FLAK50 T2
86
124
21
30
14
10


FLAK50 T3
87
125
90
76
220
145


FLAK50 T4
88
126
>
>
>
>


FLAK50 T5
89
127
760
770
610
>


FLAK90
90
128
500
>
700
>


FLAK91
91
129
>
790
550
>


FLAK92
92
130
>
>
>
>


FLAK93
93
131
>
>
>
>


FLAK50 Z1
94
132
>
>
>
>


FLAK50 Z2
95
133
>
>
>
>


FLAK50 Z3
96
134
>
>
>
>


FLAK50 Z4
97
135
110
115
215
310


FLAK50 Z5
98
136
>
450
400
900


FLAK50 Z6
99
137
>
>
>
>


FLAK50 Z7
100
138
>
>
>
>


FLAK50 Z8
101
139
550
850
>
>


FLAK50 Z9
102
140
>
>
285
>


FLAK94
103
141
420
>
>
ND


FLAK93B
104
142
73
115
55
60


FLAK50 Z10
105
143
>
>
>
>


FLAK96
106
144
750
225
275
350


FLAK97
107
145
>
>
240
>


FLAK98
108
146
270
93
640
440


FKRLA
109
147
83
93
>
340


FLAK91B
110
148
380
660
>
>


FLAK92B
111
149
>
>
>
>


FLAK99
112
150
125
185
320
74


FLAK50T6
113
151
>
>
>
>


FLAK50T7
114
152
620
410
>
>


FLAK95
115
153
130
50
140
97


FLAK50T8
116
154
600
400
>
640


FLAK50T9
117
155
>
>
>
ND


FLAK100-CO2H
118
156
230
ND
>
260


FAGVL
119
157
500
315
>
375


Modelin-5
120
159
82
74
275
145


Modelin-5-CO2H
121
160
700
470
550
450


FLAK120
126
165
470
56
400
340


FLAK121
127
166
>
>
>
>


FLAK96B
128
167
260
300
325
320


FLAK96G
129
168
>
>
>
>


FLAK96F
130
169
>
640
>
>


FLAK96C
131
170
>
>
>
>


FLAK96D
132
171
615
540
820
600


Modelin-8D
135
174
>
>
>
>


Modelin-8E
136
175
>
>
510
>


Flak 96H
137
176
>
>
>
>


Flak 96I
138
177
270
240
380
120


Flak 96J
139
178
405
>
>
>


Flak 96L
140
179
540
>
>
>


FLAK-120G
141
180
940
>
760
>


FLAK-120D
142
181
500
>
>
>


FLAK-120C
143
182
>
>
>
>


FLAK-120B
144
183
>
>
>
>


FLAK-120F
145
184
800
370
302
570


Magainin2wisc
146
300
52
60
125
45


D2A21
147
301
66
77
170
45


KSL-1
148
302
800
720
>
>


KSL-7
149
303
355
345
>
530


LSB-37
150
306
320
120
250
370


Anubis-2
151
307
75
160
100
66


D-Shiva 10 AC
163
100
95
220
150
ND


Shiva 10 (1-18)AM
164
69
101
71
190
81





Note:


> indicates greater than 1000;


ND indicates not determined; numbers are in μg/mL.






It can be seen from Tables 16 and 17 that all targets challenged were inhibited by one or more of the peptides to an appreciable extent (i.e. LD50 less than 50 μg/ml). Table 18 below shows that 44 (29%) of the 150 peptides tested were active with some LD50 values at or below 50; 26 of the peptides were active on some targets at or below the LD50 value of 25; and 16 peptides were very active on one or more target strains with LD50 values at or below 10.
Table 19 below shows a broad spectrum of activity against six cancer cell types for various active peptides. It is noted that each target has one or more lead candidate peptides inhibitory to cell growth at an LD50 level of 10 or less.







TABLE 18







FLAK peptides showing substantial


activity against cancer cell lines









LD50
Number of
Percent of 150


values
“active” peptides
peptides tested





< or = 50 μg/ml
44
29%


< or = 25 μg/ml
26
17%


< or = 10 μg/ml
16
11%
















TABLE 19







Activity and specificity of FLAK peptides


against six cancer cell targets









Number of active peptides per target



















PC3



MCF7
SW480
BMKC
H1299
HeLaS3
(pros-


LD50
(breast)
(colon)
(melanoma)
(lung)
(cervix)
tate)
















< or = 50
31
25
19
19
17
20


μg/ml


< or = 25
17
13
8
10
8
11


μg/ml


< or = 10
6
5
3
4
1
5


μg/ml









Example 5
Stimulation and Proliferation of Leukocytes
In vitro viability of human leukocyte cells in the presence of different peptides at different concentrations was determined by an Alamar Blue protocol. Alamar Blue (Promega, Madison, Wis.) is an indicator dye, formulated to measure quantitatively the proliferation and cytotoxicity of the cells. The dye consists of an oxidation-reduction (redox) indicator that yields a colorimetric change and a fluorescent signal in response to cellular metabolic activity.
Assay protocol: Blood from a 50 year old male human was drawn and centrifuged at 1500 rpm for 15 minutes at room temperature. The buffy coat cells at the plasma-red blood cell interface were aspirated. Buffy coat cells (mainly lymphocyte cells) were then transferred into 15 ml centrifuge tubes containing 5 ml of RPMI-1640 medium+10% Fetal Bovine Serum (Gibco, Grand Island, N.Y.). Additional medium was added to the tubes to bring the volume up to 10 ml. The buffy coat suspension was then carefully layered on 5 ml of Histopaque (Sigma Chemical Co., St. Louis, Mo.) and centrifuged at 1500 rpm for 30 minutes at room temperature. The interface which is mostly PBMCs (peripheral mononuclear cells) was aspirated and transferred to a 15 ml conical centrifuge tube and, resuspended in 2 ml cold RPMI-1640 and brought up to 15 ml with cold RPMI-1640 medium. Cells were centrifuged at 1500 rpm for 10 minutes. The supernatant was then aspirated and discarded. The cell pellet was re-suspended in 1 ml of cold RPMI 1640 and brought up to 15 ml with RPMI medium. This step was repeated twice, except that in the last step, the cells were resuspended with 1 ml of cold RPMI-1640 medium and cell counts were performed with a hemocytometer according to the Sigma cell culture catalogue.
Pokewood mitogen was used as a control along with positive and negative controls. Negative control cells were killed with 70% methanol. Positive (+) control cells were incubated in RPMI medium (untreated). 20 ml of AlamarBlue was added to the cells, and readings were taken after 24 hours, 48 hours, 72 hours, and 96 hours using a fluorimeter (excitation 544/transmission 590 nm).
Calculations were performed using the following formula. The peptide treated sample and positive control were adjusted for negative control.




  
    %
    ⁢
    
      
    
    ⁢
    
      
        treated  cell  stimulation
      
      /
      
        proliferation
      
    
  
  =
  
    
      
        Peptide  treated  sample
      
      
        Positive  control
      
    
    ×
    100
    ⁢
    %
  




Using the protocol described immediately above, about 100-150 peptides were screened for their stimulatory and/or inhibitory actions upon the growth of human leukocyte (“WBC”) cells as compared to the growth of untreated positive control cells. The data in Table 20 below show that various selected FLAK peptides are stimulatory at low concentrations (0.1 to 1.0 μg/ml), whereas certain of the peptides become inhibitory (causing cell death) at higher concentrations. Several of the peptides (i.e. SEQ ID NOS:5, 143, and 160) are stimulatory (and/or proliferative) at all concentrations through 500 μg/ml.
The Alamar Blue stain used in the protocol permeates both cell and nuclear membranes, and is metabolized in the mitochondria to cause the change in color. The resulting fluorometric response is therefore a result of total mitochondrial activity caused by cell stimulation and/or mitosis (cell proliferation). The increase in values (for treated cells, as a percent of values for untreated cells) with increased incubation time (120 hours vs. 48 hours) may be attributed to increased cell proliferation in addition to stimulation of cell metabolic activity caused by the peptide.
Table 20 presents peptide treated cell stimulation/proliferation, as percent of untreated positive control, for human leukocytes (white blood cells, “WBC”) in the presence of selected FLAK peptides. The table also shows for each of these peptides its toxicity (LD50 values) to human red blood cells (RBC) and to human fibroblast cells (WI38). Those certain peptides which are stimulatory to WBCs at low peptide concentrations (i.e. 10 μg/ml or less) and are inhibitory or toxic to WBCs at higher concentrations are also relatively more toxic to RBCs and to fibroblasts than those peptides which are stimulatory and not inhibitory to WBC growth even at concentrations as high as 500 μg/ml.
In limited experiments with other than the Alamar Blue protocol described above, it has been qualitatively determined that those peptides which cause stimulation and proliferation of leukocytes are active upon both the phagocytic and lymphocyte cell components of the mammalian lymphatic system. As such, certain of the stimulatory FLAK peptides which are relatively non-toxic to mammalian cells at therapeutic dose levels may be used as immunomodulators to treat humans or other mammals with compromised immune systems. Such treatment may be administered systemically in vivo or by extra-corporeal treatment of whole blood or blood components to be reinfused to the donor. Such therapy would serve to counteract immune deficiency in neutropenic patients caused by age, disease, or chemotherapy and would stimulate natural immune responses to prevent or combat pathogenic infections and growth of certain cancer cell lines or to enhance wound healing processes involving the lymphoid system. Table 21 is a more detailed example (with one peptide, SEQ ID NO:10) of the phenomenon showing the relationships of concentration and time as they effect stimulation, proliferation, and inhibition of the leukocytes.







TABLE 20







Human lymphocyte (WBC) stimulation/proliferation


by selected FLAK peptides









Selected peptides
Peptide treated cell activity
Peptide toxicity











SEQ ID

Percent stimulation relative to control
RBC
WI-38















NO.
P NO.
0.1 μg/ml
1 μg/ml
10 μg/ml
100 μg/ml
500 μg/ml
LD/50
LD/50


















 2
2
117
118
119
121
119
30
66


  5*
12
111
115
118
116
101
>1000
950


 10
25
117
104
99
27
27
60
24


 12
27
108
110
99
30
23
125
37


 17
35
82
76
61
18
16
200
8


 20
38
79
82
78
37
36
350
12


 25
43
78
82
71
14
12
20
5


 30
48
74
68
62
13
13
130
60


 58
92
112
112
98
35
26
300
25


 61
95
110
115
116
124
114
>1000
>1000


165
97
107
109
106
27
22
350
850


 66
102
100
102
97
37
17
500
210


 71
107
101
100
108
109
110
>1000
>1000


115
153
93
92
37
72
29
780
130


 119*
157
88
108
54
117
89
850
500


 147*
301
100
94
83
22
20
10
66


 150*
306
97
101
94
109
112
>1000
320





*not a FLAK peptide;


incubation times were 48 hours for all samples













TABLE 21







Human leukocyte (WBC) stimulation/proliferation and inhibition


by FLAK peptide SEQ ID NO: 10 (P25)












Time of







incubation
0.1 μg/ml
1 μg/ml
10 μg/ml
100 μg/ml
500 μg/ml















 24 hours
111
98
88
10
10


 48 hours
117
104
99
27
27


 72 hours
119
105
102
31
32


 96 hours
128
112
110
38
40


120 hours
135
118
119
43
45





Note:


Number values are percent peptide treated cell stimulation/proliferation relative to control cells (100%)






Example 6
Stimulation and Proliferation of Fibroblasts
The cyQUANT cell proliferation assay provides a convenient, rapid and sensitive procedure for determining the density of cells in culture. The assay has a linear detection range extending from 50 or fewer to at least 50,000 cells in 200 μl volumes using a single dye concentration. The assay is ideal for cell proliferation studies as well as for routine cell counts and can be used to monitor the adherence of cells to surfaces.
Procedure: Different cell lines were maintained with different medium according to the ATCC. Cells were trypsinized with 8 ml of Trypsin (0.25%, Fisher, Pittsburgh, Pa.). The cell suspension was centrifuged for 10 minutes at 100 rpm. The supernatant was removed and discarded without disturbing the cell pellet. A concentrated cell suspension was prepared in 1.0 ml of medium to obtain a density of about 105 to 106 cells/ml. The actual cell density was determined by counting the cells using a hemocytometer with the Trypan Blue method. Cell numbers were adjusted to obtain equal number of cells per 200 μl volume. Cells were plated with 0% FBS, 2% FBS, 3% FBS and 5% FBS. The plates were incubated at 37° C. for a time sufficient to allow the cells to attach. For long-term proliferation studies, 100 μl of medium was removed from each well each day and replaced with fresh medium.
At the desired time, the medium was removed from the adherent cells in a 96 well plate. These cells were already treated with test agents. The cells were frozen in the plate at −70° C. for 30 minutes. The cells were thawed at room temperature. CyQuant GR dry/Cell Lysis Buffer (200 μl) was added to each sample cell. The cells were incubated at room temperature for 15 minutes while protected from the light. Fluorescence was measured using fmax at 485-538 nm.
The above CyQuant protocol was used to examine possible peptide stimulation and/or proliferation of fibroblasts. In the following Table 22, data are shown for selected peptides demonstrating their effect on human fibroblast cells (WI38). In the table, the substantial stimulatory and/or proliferative property of selected peptides, as a function of concentration is evident. Table 23 shows that the fibroblast stimulation and/or proliferation effect is enhanced for certain peptides in the presence of other growth factors. This is shown by the addition of Fetal Bovine Serum (FBS) to the medium. Number values shown in Tables 2,2 and 23 are cell stimulation/proliferation activity expressed as a percent of control (untreated cells). Control cells and peptide treated cells are with medium and FBS as indicated. Values below 100% (for control) indicate inhibitory action of the peptide, especially at concentrations above 10 μg/ml.







TABLE 22







Human fibroblast (WI-38) cell stimulation by selected FLAK peptides









Peptide treated cell activity











Inc. Time
% FBS in
Stimulation relative to control














SEQ ID NO:
P No.
(hrs)**
serum
0.1 μg/ml
1 μg/ml
10 μg/ml
100 μg/ml

















 2
2
48
2.0
125
156
122
35


 4
11
48
2.0
149
145
166
113


  5*
12
48
3.0
111
116
109
120


 10
25
48
2.0
137
143
120
73


 12
27
48
2.0
134
115
104
116


 25
43
48
3.0
93
99
83
14


 30
48
48
3.0
117
117
109
110




72
3.0
119
123
139
144


 32
50
72
3.0
108
123
127
56


 35
55
48
3.0
101
109
116
25




72
3.0
91
98
101
6


 61
95
72
3.0
101
90
94
93


 66
102
72
3.0
123
121
126
122


 71*
107
72
3.0
114
104
98
86


 80
118
72
3.0
163
193
192
184


108
146
72
3.0
109
101
84
74


115
153
72
3.0
125
125
132
106


 119*
157
72
3.0
126
118
104
119


126
165
72
3.0
133
119
79
129


 147*
301
48
3.0
87
98
95
58


 150*
306
48
3.0
102
103
101
94





*not a FLAK peptide;


**incubation time in hours.













TABLE 23







Effect of growth factors on human fibroblast


(WI38) cell stimulation









Peptide concentration













SEQ ID
P
% FBS in



100


NO:
Number
serum
0.1 μg/ml
1 μg/ml
10 μg/ml
μg/ml
















2
2
0
−27
−3
27
−82




2.5
26
57
23
−66


4
11
0
19
34
50
−40




2.5
50
52
62
14


8
23
0
21
78
10
−48




2.5
16
23
58
75


80
118
0
12
−4
−7
−1




3
61
70
68
72





Note:


Number values are percent cell viability above or below control.






Example 7
Toxicity Assay—Red Blood Cell (RBC) Hemolysis and Leukocyte (WBC) and Fibroblast (WI38) Inhibition
Table 24 below summarizes the RBC, WBC, and WI38 toxicity data for typical FLAK peptides. The three RBC, WBC, and WI38 values (LD50) are generally consistent directional indicators of peptide toxicity. In choosing a peptide for possible treatment of a given indication it is important to match the therapeutic activity and specificity of the peptide with its possible toxic properties. The, SEQ ID NO:5 peptide is not a FLAK peptide, but rather it is SB-37, a close homolog of Cecropin B. It has previously been shown not to be as active as the FLAK peptides as an antibacterial agent, but to possess wound healing properties as demonstrated in vivo in a rat model. This probably results from its stimulatory and proliferative effects on both mammalian leukocytes and fibroblasts.
The protocols for WBC and WI38 stimulation have been discussed above. The RBC protocol follows Table 24.







TABLE 24







In vitro toxicity of selected FLAK peptides on red blood cells


(RBC), human leukocytes (WBC), and human fibroblasts (WI38)













RBC LD50
WBC LD50
WI38 LD50


SEQ ID NO:
P Number
μg/ml
μg/ml
μg/ml














5
12
>1000
>500
60


10
25
60
79
60


11
26
900
185
100


12
27
125
78
60


16
34
200
77
200


17
35
200
64
25


20
38
350
160
100


25
43
20
70
25


30
48
130
78
70


35
55
30
80
28


58
92
300
51
400


66
102
300
115
45









The RBC protocol is as follows. Well positions of each dilution and untreated controls are recorded on the lid of a 96-well plate. When the cells were confluent, the media is removed, and replaced with freshly prepared sample dilutions to a final volume of 200 μl. Test agent was added into designed wells of the 96-well plate. The 200 μl fresh medium was added to positive control wells; and 200 μl of 70% ethanol was added to negative control wells. The plate was incubated overnight at 37° C., 5% CO2, and at leasy 90% humidity. Room temperature AlamarBlue solution (20 μl) was added to all wells. The plates were read spectrofluorometrically (excitation 544 nm, emission 590 nm). The plates were incubated for 3 hours at 37° C., 5% CO2, and at least 90% humidity. The plates were read again at 3 and 24 hours incubation. The LD50 endpoint was determined from the graph by reading from where the 50 percent point intercepts the Dose Response Curve to the concentration along the x-axis. That concentration is the LD50 value. The LD50 value for test agents within a single test agent class can be used to rank-order their relative toxicities or to correlate with in vivo data.
This hemolytic assay is based upon that presented in Journal of Peptide Research 53: 82-90 (1999). Preparation of all media, stock solutions and dilutions were performed in a laminar flow hood to minimize or prevent contamination. All procedures were performed according to safety protocols pertaining to the handling and disposal of human body fluids.
Red blood cells (RBCs) were washed three times with PBS (35 mM phosphate buffer 0.15 M NaCl, pH 7.0). RBCs suspended in PBS (0.4% (v/v); about 10 ml per 15 peptides) were prepared. Suspensions (100 μl) were aliquoted to each sample and control tube. Serially diluted peptide solutions (100 μl) were pipetted into the sample tubes. Negative control tubes contained 100 μl PBS; positive control tubes contained 100 μl 1% Triton-X100 detergent. All tubes were incubated for 1 hour at 37° C. The tubes were removed from the incubator and centrifuged at 1000 g for 5 minutes. Supernatant (100 μl) was pipetted to a 96-well polyvinyl chloride plate. The absorbance at 414 nm (A414) was measured, and used to calculate the percent hemolysis according to the following formula.




  
    
      (
      
        
          
            A
            414
          
          ⁢
          
            
          
          ⁢
          in
          ⁢
          
            
          
          ⁢
          peptide
          ⁢
          
            
          
          ⁢
          solution
        
        -
        
          
            A
            414
          
          ⁢
          
            
          
          ⁢
          in
          ⁢
          
            
          
          ⁢
          PBS
        
      
      )
    
    
      (
      
        
          
            A
            414
          
          ⁢
          
            
          
          ⁢
          in
          ⁢
          
            
          
          ⁢
          Triton
        
        -
        
          X
          ⁢
          
            
          
          ⁢
          100
        
        -
        
          
            A
            414
          
          ⁢
          
            
          
          ⁢
          in
          ⁢
          
            
          
          ⁢
          PBS
        
      
      )
    
  
  ×
  100
  ⁢
  %




Percent hemolysis is plotted against peptide concentration, and the concentration at which 50% hemolysis is determined (LD50). The following Table 25 details the results of the hemolytic assay using the peptides discussed herein.










TABLE 25





Peptide name
SEQ ID NO:
P Number
LD50 μg/mL


















Hecate AC #1010
1
 1
100


Hecate AM
2
 2
10


SB-37 AC #1018
3
 5
>


Shiva 10 AM
4
 11
76


SB-37 AM
5
 12
>


Shiva 10 AC #1015
6
 13
50


Magainin 2
7
 16
550


FLAK01 AM
8
 23
300


FLAK03 AM
9
 24
10


FLAK04 AM
10
 25
16


FLAK05 AM
11
 26
90


FLAK06 AM
12
 27
125


FLAK06 AC
13
  27B
700


FLAK06 R-AC
14
  27C
250


KALV
15
 30
150


FLAK 17 AM
16
 34
200


FLAK 26 AM
17
 35
200


FLAK 25 AM
18
 36
85


Hecate 2DAc
19
 37
30


FLAK43 AM
20
 38
350


FLAK44 AM
21
 39
>


FLAK62 AM
22
 40
>


FLAK 06R-AM
23
 41
40


MSI-78 AM
24
 42
300


FLAK50
25
 43
20


FLAK51
26
 44
90


FLAK57
27
 45
700


FLAK71
28
 46
900


FLAK77
29
 47
>


FLAK50V
30
 48
200


FLAK50F
31
 49
225


FLAK26V AM
32
 50
420


CAME-15
33
 53
20


FLAK50C
34
 54
250


FLAK50D
35
 55
20


FLAK 50E
36
 56
600


FLAK80
37
 57
>


FLAK81
38
 58
>


FLAK82
39
 59
1000


FLAK83M
40
 60
>


FLAK 26 Ac
41
 61
390


Indolicidin
42
 63
375


FLAK 17 C
43
 64
6


FLAK 50H
44
 65
950


FLAK 50G
45
 66
600


Shiva deriv P69 + KWKL
46
 70
80


Shiva 10 (1-18_AC
47
 71
>


Shiva 10 peptide 71 + KWKL
48
 72
110


CA(1-7)Shiva10(1-16)
49
 73
90


FLAK 54
50
 74
>


FLAK 56
51
 75
750


FLAK 58
52
 76
>


FLAK 72
53
 77
>


FLAK 75
54
 79
>


Shiva 10 (1-16) Ac
55
 80
900


CA(1-7)Shiva10(1-16)-COOH
56
 81
8


Indolocidin-ac
57
 91
40


FLAK50B
58
 92
300


FLAK50J
59
 93
>


FLAK50I
60
 94
350


FLAK50K
61
 95
>


FLAK50L
62
 96
>


Shiva-11
63
 98
60


Shiva 11 [(1-16)ME(2-9)]-
64
 99
25


COOH


FLAK 50N
65
101
550


FLAK 50O
66
102
500


FLAK 50P
67
103
650


CA(1-&Hecate(11/23)
68
104
70


PYL-ME
69
105
ND


FLAG26-D1
70
106
>


Vishnu3
71
107
>


Melittin
72
108
<1


FLAK26-D2
73
109
>


FLAG26-D3
74
110
>


FLAK50 Q1
75
111
60


FLAK50 Q2
76
112
>


FLAK50 Q3
77
113
1000


FLAK50 Q4
78
114
>


FLAK50 Q5
79
117
>


FLAK50 Q6
80
118
700


FLAK50 Q7
81
119
400


FLAK50 Q8
82
120
>


FLAK50 Q9
83
121
>


FLAK50 Q10
84
122
>


FLAK50 T1
85
123
1000


FLAK50 T2
86
124
55


FLAK50 T3
87
125
>


FLAK50 T4
88
126
>


FLAK50 T5
89
127
>


FLAK90
90
128
>


FLAK91
91
129
>


FLAK92
92
130
>


FLAK93
93
131
>


FLAK50 Z1
94
132
>


FLAK50 Z2
95
133
>


FLAK50 Z3
96
134
>


FLAK50 Z4
97
135
900


FLAK50 Z5
98
136
>


FLAK50 Z6
99
137
>


FLAK50 Z7
100
138
20


FLAK50 Z8
101
139
>


FLAK50 Z9
102
140
>


FLAK94
103
141
900


FLAK93B
104
142
900


FLAK50 Z10
105
143
>


FLAK96
106
144
600


FLAK97
107
145
>


FLAK98
108
146
180


FKRLA
109
147
300


FLAK91B
110
148
>


FLAK92B
111
149
>


FLAK99
112
150
650


FLAK50T6
113
151
>


FLAK50T7
114
152
880


FLAK95
115
153
800


FLAK50T8
116
154
450


FLAK50T9
117
155
>


FLAK100-CO2H
118
156
10


FAGVL
119
157
850


Modelin-5
120
159
ND


Modelin-5-CO2H
121
160
>


FLAK120
126
165
350


FLAK121
127
166
>


FLAK96B
128
167
200


FLAK96G
129
168
600


FLAK96F
130
169
>


FLAK96C
131
170
>


FLAK96D
132
171
550


Modelin-8D
135
174
>


Modelin-8E
136
175
>


Flak 96
137
176
>


Flak 96I
138
177
400


Flak 96J
139
178
>


Flak 96L
140
179
850


FLAK-120G
141
180
>


FLAK-120D
142
181
>


FLAK-120C
143
182
>


FLAK-120B
144
183
>


FLAK-120F
145
184
850


Magainin2wisc
146
300
250


D2A21
147
301
10


KSL-1
148
302
>


KSL-7
149
303
500


LSB-37
150
306
>


Anubis-2
151
307
>


FLAK17CV
152
501
15


FLAK50Q1V
153
502
100


D2A21V
154
503
20


FLAK25AMV
155
504
70


FLAK43AMV
156
505
620


FLAK50DV
157
506
120


HECATE AMV
158
507
20


HECATE ACV
159
508
70


FLAK04AMV
160
509
40


FLAK03AMV
161
510
10


D-Shiva 10 AC
162
 67
40


Shiva 11 AC
163
100
>


Shiva 10 (1-18) AM
164
 69
900





Note:


> indicates greater than 1000; ND = not determined.






Example 8
Effects of Valine Substitution
Changing a peptide sequence where the first amino acid is valine, and particularly when the first amino acid is changed from phenylalanine to valine, can lead to desirable properties. The red blood cell and fibroblast cell (WI38) toxicity can be decreased, while not significantly decreasing other desirable properties. Table 26 below shows numerous examples (14) of reducing the indicated toxicity of a peptide as seen from increase in viability of both red blood cells and fibroblast cells when treated with peptide. LD50 values are in μg/ml.











TABLE 26








Hemo-



SEQ.


lysis


ID
P

RBC
WI-38


NO:
No.
Sequence
LD50
LD50



















2
2
FALALKALKKALKKLKKALKKAL-NH2
12
66


15
30
VALALKALKKALKKLKKALKKAL-NH2
150
93


17
35
FAKKLAKLAKKLAKLAL-NH2
150
25


32
50
VAKKLAKLAKKLAKLAL-NH2
420
45


 


25
43
FAKLLAKLAKKLL-NH2
20
25


30
48
VAKLLAKLAKKLL-NH2
130
160


 


86
124
FAKLLAKLAKKVL-NH2
55
21


116
154
VAKLLAKLAKKVL-NH2
870
110


 


126
165
FALALKALKKL-NH2
350
850


141
180
VALALKALKKL-NH2
850
1000


 


43
64
FAKALKALLKALKAL-NH2
6
37


152
501
VAKALKALLKALKAL-NH2
15
26


 


75
111
FAKFLAKFLKKAL-NH2
5
25


153
502
VAKFLAKFLKKAL-NH2
100
64


 


147
301
FAKKFAKKFKKFAKKFAKFAFAF-NH2
10
66


154
503
VAKKFAKKFKKFAKKFAKFAFAF-NH2
20
150


 


18
36
FAKKLAKLAKKLAKLALAL-NH2
12
19


155
504
VAKKLAKLAKKLAKLALAL-NH2
70
110


 


20
38
FAKKLAKLAKKLLAL-NH2
350
100


156
505
VAKKLAKLAKKLLAL-NH2
620
85


 


35
55
FAKLLAKALKKLL-NH2
20
32


157
506
VAKLLAKALKKLL-NH2
120
75


 


1
1
FALALKALKKALKKLKKALKKAL-COOH
20
27


159
508
VALALKALKKALKKLKKALKKAL-COOH
70
190


 


10
25
FALALKALKKLAKKLKKLAKKAL-NH2
16
24


160
509
VALALKALKKLAKKLKKLAKKAL-NH2
40
95


 


9
24
FALALKALKKLLKKLKKLAKKAL-NH2
10
55


161
510
VALALKALKKLLKKLKKLAKKAL-NH2
10
77









Although the effects of reduction of toxicity to mammalian cells by valine substitution is accompanied by modest reductions of therapeutic activity against microbial pathogens and cancer cells, there are some cases in which the valine substitution results in a desirable increase in therapeutic activity. This can be seen in the following Table 27 where it is shown that the valine substitution in some cases has increased the peptide's activity against the gram negative bacterium Pseudomonas.
Hemolysis and WI38 values represent LD50 values. P. aerug values represent MIC values in μg/mL against Pseudomonas aeruginosa ATCC accession number 9027.












TABLE 27





SEQ ID
P



P.


NO:
No.
Sequence
Hemolysis
WI38
aerug




















17
35
FAKKLAKLAKKLAKLAL
100
25
200


32
50
VAKKLAKLAKKLAKLAL
420
45
15


 


25
43
FAKLLAKLAKKLL
20
25
100


30
48
VAKLLAKLAKKLL
200
160
5


 


86
124
FAKLLAKLAKKVL
300
21
100


116
154
VAKLLAKLAKKVL
450
110
100









Changing a peptide sequence where the second amino acid is tyrosine can lead to desirable properties. FLAK98 (P-146, SEQ ID NO:108) is an a typical FLAK peptide due to the presence of a tyrosine (Y) at the second position. The significance of this modification and the peptide's overall sequence is that the structure produced is likely to fold readily into an alpha-helix at neutral pH (Montserret et al., Biochemistry 39: 8362-8373, 2000). The ability to assume an alpha-helical structure at neutral pH may account for the potency and broad spectrum of activity seen with this peptide. Montserret et al. demonstrated that sequences such as these are driven into folding not only by hydrophobic but also by electrostatic forces. The substitution of tyrosine for an amino acid in FLAK peptides may generally lead to improved properties.
Example 10
Presently Preferred Peptides
Preferred peptides can be selected from the above described experimental data. Preferred antimicrobial peptides for gram positive or gram negative bacteria can be selected as having MIC values of less than or equal to about 10 μg/ml, or as having MBC values of less than or equal to about 25 μg/ml. Preferred antifungal peptides can be selected as having MIC or MBC values of less than or equal to about 25 μg/ml. Preferred anticancer peptides can be selected as having LD50 values of less than or equal to about 25 μg/ml.
The following Table 28 lists representative presently preferred peptides, where an ‘X’ indicates that the peptide is a preferred peptide for that column's property. The peptide's “length” is the number of amino acid residues in the sequence.












TABLE 28





SEQ ID

Length
Anti-




NO:
P-number
(AA)
bacterial
Anti-fungal
Anti-cancer




















1
1
23
X

X


2
2
23
X
X
X


4
11
23
X


6
13
23
X


8
23
23
X

X


10
25
23
X
X


11
26
21
X
X
X


12
27
19
X
X


13
27B
19
X
X
X


14
27C
19
X


15
30
23
X


16
34
16
X
X
X


17
35
17
X
X
X


18
36
19
X

X


19
37
23
X

X


20
38
15
X

X


23
41
19
X


25
43
13
X
X
X


26
44
15
X

X


27
45
14
X


28
46
15
X


29
47
12


X


30
48
13
X
X
X


31
49
12
X


32
50
17
X

X


34
54
13
X


35
55
13
X
X
X


36
56
13
X


39
59
10
X


41
61
15
X


43
64
15
X


45
66
13
X


46
70
23
X

X


47
71
18
X


48
72
22
X


50
74
13

X


51
75
13
X

X


52
76
14
X


55
80
23
X


56
81
23
X

X


57
91
15
X

X


58
92
13
X
X
X


60
94
13
X

X


65
101
13
X


66
102
13
X
X


67
103
12
X
X


68
104
20
X

X


74
110
12
X


75
111
13
X

X


77
113
13
X


80
118
13
X
X


81
119
14
X
X


84
122
13
X
X


85
123
10

X


86
124
13
X
X
X


87
125
13
X


93
131
5
X


106
144
12
X
X


108
146
13
X
X


112
150
17
X


115
153
17
X
X


116
154
13

X


126
165
11
X
X


128
167
12
X
X


131
170
10

X


143
182
10

X


152
501
15
X

X


155
504
13
X


157
506
23
X

X


161
510
23
X
X


162
67
23
X

X


163
100
13
X
X


164
69
23
X


165
97
13
X
X









Preferred peptides for stimulation and proliferation can also be selected. The following Table 29 lists representative preferred peptides, where an ‘X’ indicates that the peptide is a preferred peptide for that column's property. Peptides which are stimulatory for leukocytes at 0.1 μg/ml to 1.0 μg/ml concentration are preferred, as at this concentration the peptides are not toxic to red blood cells, WI-38 fibroblasts, or to human leukocytes. Peptides which are stimulatory for fibroblasts at 0.1 μg/ml to 1.0 μg/ml are preferred, as at this concentration the peptides are not toxic.

    
    
        In Table 29 please add peptides P146 (SEQ 108) (Length=13) and P97 (SEQ 165) (Length=13). Both of these peptides should have X in the Leukocyte and in the Fibroblast columns.
    
    









TABLE 29







Preferred peptides for leukocyte and


fibroblast stimulation/proliferation











SEQ ID NO:
P-number
Length
Leukocyte
Fibroblast














1
29
23
X
X


2
 2
23
X
X


5
12
38
X
X


6
13
23
X
X


8
23
23
X
X


10
25
23
X
X


11
26
21
X
X


12
27
19
X
X


13
  27B
19
X
X


14
  27C
19
X
X


15
30
23
X
X


16
34
16
X
X


17
35
17
X
X


20
38
15

X


27
45
14

X


28
46
15

X


30
48
13

X


32
50
17

X


34
54
13
X


45
66
13
X
X


46
70
23
X
X


50
74
13
X
X


51
75
13
X
X


55
80
23

X


56
81
23

X


57
91
15
X
X


58
92
13
X
X


59
93
13

X


60
94
13

X


61
95
13
X
X


65
101 
13

X


66
102 
13

X


71
107 
19
X
X


74
110 
12

X


75
111 
13

X


77
113 
13

X


80
118 
13

X


81
119 
14

X


87
125 
13
X
X


90
128 
5
X
X


91
129 
5

X


92
130 
5

X


108
146 
13
X
X


115
153 
17

X


116
154 
13
X


126
165 
11

X


127
166 
11

X


129
168 
6
X
X


132
171 
11

X


137
176 
11
X


138
177 
12
X


139
178 
11
X
X


140
179 
11
X
X


141
180 
11
X
X


142
181 
10
X
X


143
182 
10
X
X


144
183 
5
X
X


145
184 
5
X
X


159
508 
23
X
X


162
67
23
X
X


164
69
18

X


165
97
13
X
X









Example 11
Synergistic Effects with Lysozyme
Synergy between lytic peptides and lysozyme was assayed. Sterilized milk was inoculated with bacteria to 5×105 per ml. Peptide Shiva-10 (SEQ ID NO:4) was added to 10 μg/ml, and chicken lysozyme was added to 1 mg/ml. The percent killing of bacteria was determined.










TABLE 30







Staph. aureus
Pseud. aeruginosa


















Peptide and lysozyme
0%
100% 


Peptide
0%
0%


Lysozyme
0%
0%









Synergy between cecropin SB-37 (SEQ ID NO:5) and lysozyme was determined against Pseudomonas syringae pv. tabaci (PSPT), Pseudomonas solanacearum (PS), Erwinia caratovora subsp. carotova (EC), and Xanthomonas campestris pv. campestris (XC). LD50 (μM) values were determined.











TABLE 31







SB-37
Lysozyme
SB-37 and Lysozyme





















PSPT
5.20
>
0.19



PS
64.0
>
16.0



EC
1.48
>
0.44



XC
0.57
>
0.027







> indicates greater than 1000.






Synergy between Shiva-1 and lysozyme was determined. The percent viability of Pseudomonas aeruginosa was determined relative to blank controls. Lysozyme was used at the same molar concentration as the peptide.











TABLE 32





Peptide



Shiva-1 and


concentration


Lysozyme
Lysozyme


(μM)
SB-37
Shiva-1
(1×)
(1×)



















0
100
100
100
100


0.01
100
100
100
56.6


0.1
79.4
69.6
82.2
25.8


1
48.8
37.9
52.1
4.4


5
38.5
1.5
7.9
0.2


7.5
0.7
0.1
0.6
0


25
0
0
0.4
0









Synergy between Shiva-1 and lysozyme was determined. The percent viability of gram positive S. intermedius 19930, S. intermedius 20034, and S. aureus was determined relative to blank controls. Lysozyme was used at ten times the molar concentration as the







TABLE 33







S. intermedius 19930











Peptide



Shiva-1 and


concentration


Lysozyme
Lysozyme


(μM)
SB-37
Shiva-1
(10×)
(10×)














0
100
100
100
100


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


1
42.5
42.1
53
43


5
36.1
35.2
49.5
17.2


10
5.6
1.2
34.4
1.1


50
0
0
22
0
















TABLE 34







S. intermedius 20034











Peptide






concentration


Lysozyme
Shiva-1 and


(μM)
SB-37
Shiva-1
(10×)
Lysozyme (10×)














0
100
100
100
100


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


0.75
62.2
60.1
42.3
41.0


5
35.1
4.1
38.3
4.3


50
0
0
10.0
0
















TABLE 35







S. aureus











Peptide






concentration


Lysozyme
Shiva-1 and


(μM)
SB-37
Shiva-1
(10×)
Lysozyme (10×)














0
100
100
100
100


0.01
100
100
100
100


0.1
100
100
100
100


0.5
81.0
50.1
100
100


1
47.5
24.4
51.0
31.2


5
31.8
15.9
18.4
8.2


10
5.6
4.5
13.3
4.5


50
1.9
1.6
9.5
1.4









Synergy experiments can also be performed using peptides in the presence of EDTA, which potentiates the peptides additively or synergistically.
Example 12
Synergistic Effects with Antibiotics
Synergy between peptide Shiva-10 (SEQ ID NO:4) and various antimicrobial agents was investigated against Escherichia coli 25922. The following table illustrates the beneficial effects of combining the peptide with the agents, where the numbers are the minimum bactericidal concentration (MBC; μg/mL).











TABLE 36







Agent
Without peptide
With peptide




















Shiva-10
50
n/a



Ticarcillin
100
50 (15 μg/mL peptide)



Cefoperazone
150
2.5 (15 μg/mL peptide) 



Doxycycline
5
 1 (15 μg/mL peptide)



Neomycin
100
5 (5 μg/mL peptide)



Amikacin
150
50 (5 μg/mL peptide) 



Tetracycline
10
2.5 (5 μg/mL peptide)  










Synergy between peptide Shiva-10 (SEQ ID NO:4) and various antimicrobial agents was investigated against Staph. aureus 29213. The following table illustrates the beneficial effects of combining the peptide with the agents, where the numbers are the minimum bactericidal concentration (MBC; μg/mL).











TABLE 37







Agent
Without peptide
With 5 μg/mL peptide




















Shiva-10
200
n/a



Ampicillin
5
2.5



Ticarcillin
25
15



Cefoperazone
10
2.5



Tobramycin
25
10



Tetracycline
10
1










Synergy between peptide FLAK 26AM (P35; SEQ ID NO:17) and various antimicrobial agents was investigated against Staph. aureus 29213 MBC. The following table illustrates the beneficial effects of combining the peptide with the agents, where the numbers are the minimum bactericidal concentration (MBC; μg/mL). This experiment determined the peptide MBC in the absence of the antimicrobial agent, or in the presence of the indicated concentration of antimicrobial agent










TABLE 38







Agent
MBC of peptide









FLAK 26AM alone
50



Vancomycin (1 ppm)
32



Cefoperazone (0.25 ppm)
20










Synergy between doxacycline and various peptides was investigated against P. aeruginosa 27853. The following table illustrates the beneficial effects of combining doxacycline and the peptides, where the numbers are the minimum bactericidal concentration (MBC; μg/mL). When combined with the peptides, the doxacycline was held at 10 ppm concentration.









TABLE 39






Without
With


Agent
doxacycline
doxacycline

















Doxacycline
n/a
100


SB-37 (P5; SEQ ID NO: 3)
200
30


FLAK 26AM (P35; SEQ ID NO: 17)
50
32









Synergy between tetracycline and various peptides was investigated against Escherichia coli 25922 MBC. The following table illustrates the beneficial effects of combining tetracycline and the peptides, where the numbers are the minimum bactericidal concentration (MBC; μg/mL). When combined with the peptides, the concentration of tetracycline was held at 1.5 ppm.









TABLE 40






Without
With


Agent
tetracycline
tetracycline







Tetracycline
n/a
10


FLAK 06AM (P27; SEQ ID NO: 12)
75
25


FLAK 26AM (P35; SEQ ID NO: 17)
50
20









Example 13
Synergistic Effects with Chemotherapy Agents
Other investigators have reported that lytic peptides which are inhibitory to cancer cells will act synergistically with conventional cancer chemotherapy drugs. The FLAK peptides are no exception. Table 41 below demonstrates for example that selected FLAK peptides are synergistic with Tamoxifen in the inhibition of the MCF7 line of breast cancer cells. Table 42 lists other more active anti-cancer peptide candidates for synergistic application with Tamoxifen or other cancer therapy drugs.
Tables 41 and 42 also show toxicity of the selected peptides against RBCs, WBCs, and WI38 cells. When used at very low non-toxic levels selected anti-cancer peptides can synergistically potentiate other chemotherapy agents to permit their effective use at substantially lower dose levels with consequently fewer side effects.







TABLE 41





Synergy of FLAK peptides with tamoxifen on MCF7 cells


















Active agent
LD50 on MCF7 cells














MCF7
Peptide
Tamox.
Total


SEQ ID NO:

LD50 
conc.
conc.
conc.


(P No.)
Agent
μg/ml
μg/ml
μg/ml
μg/ml






Tamoxifen
20
0
20
20


164 (69) 
Alone
79
2.5
4.6
7.1



With Tamox.


145 (184)
Alone
240
10
4
14



With Tamox.


121 (160)
Alone
240
11
3.7
14.7



With Tamox.


106 (144)
Alone
310
35
7.7
42.7



With Tamox.














SEQ ID NO:
MCF7 LD50
RBC LD50
WI38 LD50
WBC LD50


(P No.)
μg/ml
μg/ml
μg/ml
μg/ml





164 (69) 
79
900
60
140


145 (184)
240
850
1000
410


121 (160)
240
>1000
700
900


106 (144)
310
600
740
320


17 (35)
9
200
25
25


32 (50)
32
420
40
420


20 (38)
17
350
100
54
















TABLE 42







Other highly active peptide candidates


for synergistic anti-cancer applications











SEQ ID NO:
MCF7 LD50
RBC LD50
WI38 LD50
WBC LD50


(P No.)
μg/ml
μg/ml
μg/ml
μg/ml














17 (35)
9
200
25
25


32 (50)
32
420
40
420


20 (38)
17
350
100
54









Example 14
Synergistic Effects with Growth Factors
It has been shown above in Example 17 and Table 23 that certain of the FLAK peptides are synergistic with other mitogens or growth factors in the stimulatory and/or proliferative properties of the peptides.
Example 15
Synergistic Effects with Nalidixic Acid and Chloramphenicol
The synergistic effects of the inventive peptides with either chloramphenicol or nalidixic acid against efflux mutants of Pseudomonas aeruginosa were investigated. The MIC values were determined for either nalidixic acid or chloramphenicol alone as baselines. Peptides were added at their 1⁄4 MIC concentration, and the concentration of either nalidixic acid or chloramphenicol to arrive at the MIC was determined. Table 43 shows the peptides' synergistic effects with nalidixic acid against P. aeruginosa H374, Table 44 shows the peptides' synergistic effects with nalidixic acid against P. aeruginosa H774, and Table 45 shows the peptides' synergistic effects with chloramphenicol against P. aeruginosa H374. The fractional inhibitory concentration (FIC) index was used to determine synergy between peptides and antibiotics. Two-fold serial dilutions of antibiotic were tested in the presence of a constant amount of peptide, equal to one quarter of peptide MIC. The FIC index was determined as follows: FIC=0.25+MICantibiotic in combination/MICantibiotic alone. An FIC index of 0.5 or less is considered as synergy.











TABLE 43









Peptide in
P. aeruginosa H374












Combination
MICNal-comb




(1⁄4 MIC)
(μg/ml)
FIC*Index















Nal alone
5000
—



P12
2500
0.75



P23
2500
0.75



P24
5000
1.25



P25
2500
0.75



P26
2500
0.75



P27
2500
0.25



P30
5000
1.25



P31
2500
0.75



P34
2500
0.75



P35
10,000
2.25



P37
2500
0.75



P39
1250
0.5



P41
5000
1.25



P42
5000
1.25



P43
5000
1.25



P44
5000
1.25



P45
2500
0.75



P46
2500
0.75



P49
2500
0.75



P50
5000
1.25



P54
5000
1.25



P55
5000
1.25



P56
2500
0.75



P59
2500
0.75



P60
1250
0.5



P61
5000
1.25



P64
5000
1.25



P66
5000
1.25



P69
2500
0.75



P71
2500
0.75



P72
2500
0.75



P73
2500
0.75



P75
2500
0.75



P80
2500
0.75



P81
5000
1.25



P97
5000
1.25



P100
2500
0.75



P101
5000
1.25



P102
5000
1.25



P103
625
0.375



P109
2500
0.75



P110
2500
0.75



P111
2500
0.75



P118
2500
0.75



P119
2500
0.75



P124
2500
0.75



P146
625
0.375



P150
1250
0.5



P153
5000
1.25



P157
2500
0.75



P177
5000
1.25



P300
312
0.312



P301
625
0.375



P306
5000
1.25



P307
625
0.375



P504
5000
1.25



P508
5000
1.25



P510
625
0.375




















TABLE 44









P. aeruginosa H744












Peptide in
MICNal-comb.




combination
(μg/ml)
FIC*Index















Nal alone
624
—



P12
312
0.75



P23
624
1.25



P24
624
1.25



P25
156
0.5



P26
624
1.25



P27
624
1.25



P30
624
1.25



P31
624
1.25



P34
624
1.25



P35
624
1.25



P37
624
1.25



P39
624
1.25



P41
624
1.25



P42
624
1.25



P43
624
1.25



P44
624
1.25



P45
624
1.25



P46
624
1.25



P49
624
1.25



P50
624
1.25



P54
624
1.25



P55
624
1.25



P56
624
1.25



P59
624
1.25



P60
624
1.25



P61
624
1.25



P64
624
1.25



P66
624
1.25



P69
312
0.75



P71
624
1.25



P72
312
0.75



P73
624
1.25



P75
624
1.25



P80
624
1.25



P81
624
1.25



P97
78
0.375



P100
624
1.25



P101
624
1.25



P102
624
1.25



P103
624
1.25



P109
624
1.25



P110
624
1.25



P111
624
1.25



P118
624
1.25



P119
624
1.25



P124
624
1.25



P146
624
1.25



P150
312
0.75



P153
624
1.25



P157
624
1.25



P177
312
0.75



P300
156
0.5



P301
624
1.25



P306
312
0.75



P307
156
0.5



P504
1248
2.25



P510
624
1.25





















TABLE 45









Peptide in
P. aeruginosa H374












Combination
MICCm-comb




(1⁄4 MIC)
(μg/ml)
FIC*Index















Cm alone
16
—



P12
16
1.25



P23
8
0.75



P24
16
1.25



P25
4
0.5



P26
8
0.75



P27
8
0.75



P30
16
1.25



P31
16
1.25



P34
16
1.25



P35
16
1.25



P37
4
0.5



P39
8
0.75



P41
16
1.25



P42
16
1.25



P43
16
1.25



P44
16
1.25



P45
16
1.25



P46
8
0.75



P49
8
0.75



P50
16
1.25



P54
16
1.25



P55
16
1.25



P56
16
1.25



P59
8
0.75



P60
4
0.5



P61
16
1.25



P64
16
1.25



P66
16
1.25



P69
8
0.75



P71
8
0.75



P72
8
0.75



P73
8
0.75



P75
8
0.75



P80
4
0.5



P81
16
1.25



P97
16
1.25



P100
16
1.25



P101
16
1.25



P102
16
1.25



P103
8
0.75



P109
16
1.25



P110
16
1.25



P111
16
1.25



P113
16
1.25



P118
16
1.25



P119
16
1.25



P124
16
1.25



P146
4
0.5



P150
8
0.75



P153
8
0.75



P157
8
0.75



P177
8
0.75



P300
16
1.25



P301
16
1.25



P306
8
0.75



P307
2
0.375



P504
16
1.25



P508
8
0.75



P510
4
0.5










Example 16
Activity Against Drug Resistant Strains
Peptides were assayed for their activity against tobramycin sensitive and resistant strains. As shown in the following Table 46, peptides P56 (SEQ ID NO:36), P74 (SEQ ID NO:50), and P125 (SEQ ID NO:87) showed greater activity against tobramycin resistant (tr) Pseudomonas ATCC 13096 than against tobramycin sensitive (ts) Pseudomonas ATCC 27853. The same three peptides showed greater activity against clinical tobramycin resistant strain 960890198-3c (Table 46).









TABLE 46





Peptide
tr Pseudomonas 13096
ts Pseudomonas 27853

















SEQ ID NO: 36 (P56)
16
125


SEQ ID NO: 50 (P74)
16
125


SEQ ID NO: 87 (P125)
4
31




















TABLE 47








tr Pseudomonas
ts Pseudomonas



Peptide
960890198-3c
27853




















SEQ ID NO: 36 (P56)
>50
125



SEQ ID NO: 50 (P74)
25
125



SEQ ID NO: 87 (P92)
50
63










Example 17
Wound Healing
The inventive peptides can be used in compositions for topical or systemic delivery in wound healing applications. The compositions can be a liquid, cream, paste, or other pharmaceutically acceptable formulation. The compositions may contain other biologically active agents. The compositions may contain pharmaceutically acceptable carriers.
FLAK peptides have demonstrated high potency against the bacteria most associated with wound infections, S. aureus, S. pyogenes and P. aeruginosa (e.g. Tables 5, 6, and 7). The peptides have also demonstrated the ability to aid in the healing of wounds and perhaps reduce inflammation. These properties are all essential attributes of wound and wound infection treatment products.
Those peptides presently preferred for wound healing, shown in Table 48 below, are peptides that were preferred for either, or both, leukocyte or fibroblast stimulation and for anti-bacterial properties.







TABLE 48







Presently preferred peptides for wound healing










SEQ ID NO:
P No.














1
1



2
2



5
12



6
13



8
23



10
25



11
26



12
27



13
27B



14
27C



15
30



16
34



17
35



20
38



27
45



28
46



30
48



32
50



34
54



45
66



46
70



50
74



51
75



55
80



56
81



57
91



58
92



59
93



60
94



61
95



65
101



66
102



71
107



74
110



75
111



77
113



80
118



81
119



87
125



90
128



91
129



92
130



93
131



108
146



115
153



116
154



126
165



127
166



129
168



132
171



137
176



138
177



139
178



140
179



141
180



142
181



143
182



144
183



145
184



159
508



162
67



164
69



165
97










Example 18
Wound Healing with FLAK Peptides Demonstrated in-vivo
U.S. Pat. No. 5,861,478 disclosed in vivo wound healing in a rat model in which the healing agent was the peptide LSB-37. LSB-37 is identified herein as SEQ. NO. 150 (peptide P306), and is evaluated herein by way of comparision with the smaller FLAK peptides which are the subject of the present invention. As set forth in Example 17 the FLAK peptides, based on extensive in vitro assays, offer promise as wound healing agents. This has been demonstrated in in vivo testing of selected FLAK (and other) peptides in a small animal topical wound healing model developed for this purpose.
The objective of the study was to evaluate the effects of certain selected peptides on (i) the rate of wound closure, (ii) inflammatory response, and (iii) epidermal thickening on a chemically induced skin burn wound. The hairless rat was chosen as a suitable test model. Female hairless rats of 100 to 150 grams weight and 8 to 12 weeks age were used in the study.
Phenol based skin peels reported in the literature and in private communications were found to be systemically toxic for use in this study, where six separate test patches (peels) with a total surface area of >2 square inches were induced on a single animal. As an alternative, 70% trichloroacetic (TCA) dissolved in 70% ethanol was employed to induce the dermal erosion patches. With 30 minute peel occlusions resulted in third degree burns with complete erosion of the epidermis and dermis. As the chemical burn agent, the TCA treatment inflicted on the rats far less trauma and mortality than occurred with the Phenol model.
The experimental Protocol procedure steps were as follows:

    1. The animal was anesthetized (40 mg/kg Phenobarbital).
    2. Color photographs of the animal's back (with six separate peels) were taken before each treatment and daily thereafter.
    3. Rat skin surface was prepared by wiping with 70% ethanol. Filter paper discs (1.1 cm diameter) were soaked in 70% TCA/ethanol.
    4. The discs were placed on the back of the hairless rat for 30 minutes [6 disks providing for 2 control (no peptide treatment) disks and 4 disks for peels to receive peptide treatment.]
    5. After a 30 minute burn the discs were removed. Twenty four hours later, different peptide solutions (1500 ppm in saline) were applied to four peels, and saline was applied to the two control peels.
    6. Peptide solutions (and saline for the controls) were applied to the six wounds with a soft brush each day thereafter.
    7. It took approximately one month for the wounds to heal (complete skin closure with stabilized epidermis), after which the animal was sacrificed.
    8. The treated skin was harvested, section stained with trichrome, and mounted on slides.


The percentage of wound closure for each peel (six sites) was measured each day until the animal was sacrificed. The percentage closure was determined by measuring on the animal photographs the area of the remaining scab relative to the area of the initial scar after the burn. These measurements were made by digitizing and analyzing the peels using the Sigma Plot ProScan 4 program.
After full wound closure, a portion of each peel still had a red, inflamed area which was quantitated by the Sigma Plot analysis of the animal photgraph, as a percentage of the total healed scar. This provided a measure of the post-TCA burn treatment of the inflammatory response in each peel site.
The extent of epidermal thickening (hyperkeratosis) at each site was also determined by measurement with the Sigma Plot program applied to the stained section slides of the various wound areas and the normal untreated skin (control) surrounding the peels. At magnifications of 100× to 320×, the microphotographs of the color slides provided a powerful tool for such quantification of the extent of hyperkeratosis evident in each peel.
Treatment of the section slides with selective stains produced identifiable evidence of the presence of both leukocyte and fibroblast cells in the wound areas. This was also quantified by the Sigma Plot program. It proved to be a useful tool in determining, in vivo, the mechanisms by which different peptides affected the wound healing process, including leukocyte stimulation/proliferation and fibroblast stimulation/proliferation and chemotactic effects of the peptides in wound healing in-vivo.
The above described animal model and protocols were employed in the testing of approximately 20 of the peptides listed in Table 48 (and other peptides for comparison) as preferred FLAK peptides for wound healing. By way of example, the following results on an experiment with four peptides evaluated in a single animal are shown in Table 49. These peptides are SEQ ID NO:66 (P102), SEQ ID NO:71 (P107), SEQ ID NO:115 (P153), and SEQ ID NO:119 (P157). Peptide SEQ ID NO:71 (P107) is not a FLAK peptide, but is a derivative of LSB-37 (SEQ ID NO:150; P06). In earlier experiments these two peptides have been shown to have very similar wound healing properties in vivo. SEQ ID NO:119 (P157) is a non-FLAK peptide, reported in the literature, which is a comparison peptide.
Table 49 supports the conclusion that several peptides evaluated for post wound treatments demonstrated the ability to limit post-TCA burn inflammatory responses. SEQ ID NO:71 and SEQ ID NO:115 were superior in this respect and also showed the lowest evidence of hyperkeratosis (epidermal thickening). Since the experiment was carried to full wound closure at 26 days, these same two peptides displayed a small advantage in rate of wound closure over the other peptides and no peptide in post wound treatment. These two peptides also showed substantially no hyperkeratosis as compared to the TCA burn untreated control.
Overall the best wound healing activity was displayed by the two above cited peptides. However, the experiment was conducted under sterile conditions that do not usually occur in real life animal wound situations. Because such topical wounds are subject to infection, it must be considered that the superior anti-bacterial properties of both SEQ ID NO:66 (P102) and SEQ ID NO:115 (P153) make them logical candidates for wound healing applications.







TABLE 49







Selected in-vivo FLAK peptide wound healing example (Rat model)
















Leukocyte
Fibroblast



Wound
Inflammatory
Epidermal
cells in test
cells in test



closure
response area
thickening
area
area



% of initial
% of healed
% of control
% of normal
% of normal



wound
scar
(TCA only)
skin
skin










SKIN SAMPLE












Normal skin
N/A
N/A
N/A
100
100


TCA burn untreated
98.4
15
30
200
275


(control)







Burns treated by peptide:












SEQ ID NO: 66 (P102)
96.7
27
50
370
220


SEQ ID NO: 71 (P107)
100
0
33
400
420


SEQ ID NO: 115 (P153)
99.1
7
25
235
350


SEQ ID NO: 119 (P157)
95.2
25
80
265
450









Example 19
Treatment of Cystic Fibrosis (CF)
CF is the most common autosomal recessive genetic disorder in North America, causing inflammation and infection in the lungs of 30,000 children a year in the USA. Over 90% of CF lung infections are caused by P. aeruginosa and over 95% of these patients die from lung damage. Certain FLAK peptides are active against multi-drug resistant strains Pseudomonas aeruginosa and against clinical isolates from CF patients (Tables 9, 43 and 44). These include strains resistant to TOBI, the current drug of choice for this condition. In addition, peptides such as these (alpha-helical peptides) have previously been shown to have anti-inflammatory properties (Scott et al., J. Immunol. 165: 3358-3365, 2000) and it would therefore not be surprising if FLAK peptides also exhibited this property. The combination of an anti-inflammatory and an anti-infective role makes these peptides extremely good candidates as novel therapeutics for the CF lung.
Example 20
Treatment of Sexually Transmitted Diseases (STDs)
Sexually transmitted diseases (STD) are a significant problem in North America costing the US alone $10 billion a year in treatment costs. One of the key problems is the increasing incidence of anti-fungal, primarily fluconazole, resistant strains of Candida including species such as C. albicans, C. glabrata and C. tropicalis. Certain FLAK peptides have demonstrated significant activity against all three of these species (Tables 13 and 10) and present a very viable opportunity for the development of a topical anti-fungal agent to prevent the spread of fungal disease. There is evidence in the literature suggesting that FLAK peptides may also have activity against other STD agents including viruses and bacteria which suggests that a broad spectrum application may also be possible. Certain FLAK peptides demonstrate a broad spectrum of activity (Tables 12 and 13).
Example 21
Treatment of Acne
Acne is caused by a combination of infection and inflammation that leads to tissue damage and scarring. FLAK peptides have demonstrated activity against the primary bacteria isolated from acne sores, Propionibacterium acne and also will likely exhibit anti-inflammatory activities (Scott et al., J. Immunol. 165: 3358-3365, 2000). In addition, the FLAK peptides have also shown a propensity to increase the speed and efficiency of wound healing, increase the proliferation of fibroblasts and increase collagen and laminin production. All of these attributes provide compelling evidence for the application of FLAK peptides to the treatment of acne either as a clinical therapeutic or as a cosmeceutical.
Example 22
Cosmetics Applications
The attributes of FLAK peptides such as collagen stimulation, fibroblast stimulation and wound healing make the potential for the use of such peptides in cosmetics such as anti-aging and rejuvination products very appealing.
Example 23
Use of FLAK Peptides in the Food Industry
The primary causes of diseases related to the food industry are Gram-negative bacteria such as Salmonella typhimurium and Escherichia coli. A number of FLAK peptides demonstrated specific activity against these organisms (Tables 7 and 12). The application of such peptides to the treatment and also prevention of food borne disease is therefore an appealing application. For example the use of such peptides for the decontamination of food preparation surfaces is a specific and potentially novel application.
Example 24
Systemic Application of Peptides in Serum
A series of peptides were introduced into sheep serum at 1280 ug/ml and incubated at 37° C. for either 30 minutes or 2 hours (Table 50). Subsequently, the serum MICs against Pseudomonas aeruginosa were conducted to determine extent of serum inactivation of the peptides. Of the peptides tested, two (P153 and P508) were soluble at 1280 μg/ml in 70% serum and their activities were only modestly decreased by exposure to serum. This suggests that P153 and P508 are able to function in serum and are good candidates for a systemic application.







TABLE 50







Serum inactivation of peptides










MIC 30 min treatment
MIC 2 hr treatment










Peptide
Solubility
(μg/ml)
(μg/ml)















P24
Precipitated
40
20
20
20


P31
Precipitated
20
20
20
20


P69
Precipitated
20
20
20
20


P81
Precipitated
20
20
20
20


P153
Soluble
10
5
20
5


P508
Soluble
40
20
40
20


KB142
Precipitated
20
20
20
20


KB146
Precipitated
20
20
20
20









Example 25
Collagen and Laminin Stimulation by FLAK Peptides
Fibroblast cell lines were cultured under standard conditions and assayed for collagen and laminin using an ELISA system manufactured by Panvera (Madison, Wis.). Antibodies for collagen and laminin manufactured by Takara Shuzo Co., Ltd Japan. Table 51 below shows that one of the four peptides displayed significant stimulation of collagen and laminin production. The other three peptides tested neither stimulated nor inhibited production (i.e. no effect was observed).







TABLE 51







Collagen and laminin stimulation









Peptide
Collagen stimulation
Laminin stimulation












TGFβ (control)
60%
—


P153 (SEQ ID NO: 115)
120%
32%


P165 (SEQ ID NO: 126)
0%
0%


P94 (SEQ ID NO: 60)
0%
0%


P12 (SEQ ID NO: 5)
0%
0%









All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

## Claims

1. A method for promoting the stimulation and/or proliferation of cells wherein the activity of a therapeutic agent is enhanced, the method comprising preparing a composition, wherein:
the composition comprises a peptide and a therapeutic agent;
the peptide comprises phenylalanine, leucine, alanine, and lysine residues;
the peptide is about 5 to about 23 amino acids in length; and
the peptide comprises at least about 50% phenylalanine, leucine, alanine, and lysine residues, and the peptide has no more than 20% phenylalanine and tryptophan residues; and whereby the activity of the composition is higher than the activity of the same composition containing the therapeutic agent but lacking the peptide.

2. The method of claim 1, wherein the therapeutic agent is an antibiotic, a growth factor, or an antimicrobial agent.

3. The method of claim 1, wherein the peptide is SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:164.

4. The method of claim 1, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:129, SEQ ID NO:142, SEQ ID NO:143, or SEQ ID NO:145.

5. The method of claim 1, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or SEQ ID NO:145.

6. The method of claim 1, wherein the peptide is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:162, or SEQ ID NO:164.

7. The method of claim 1, wherein the peptide is SEQ ID NO:15, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:116, SEQ ID NO:141, or SEQ ID NO:159.

8. The method of claim 1, wherein the concentration of the peptide in the composition is 0.01 μM to 500 μM.

9. The method of claim 1, wherein the cells are mammalian cells.

10. The method of claim 1, wherein the cells are lymphocyte cells.

11. The method of claim 1, wherein the composition further comprises a growth factor.

12. A method for promoting wound healing, comprising administering to the wound of an animal a composition, wherein:
the composition comprises an isolated peptide;
the peptide is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, or SEQ ID NO:164; and
wherein the composition is administered to the wound in an effective amount and for an effective amount of time sufficient to promote healing of the wound.

13. The method of claim 12, wherein the peptide is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:164.

14. The method of claim 12, wherein the peptide is SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:164.

15. The method of claim 12, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:129, SEQ ID NO:142, SEQ ID NO:143, or SEQ ID NO:145.

16. The method of claim 12, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or SEQ ID NO:145.

17. The method of claim 12, wherein the peptide is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:162, or SEQ ID NO:164.

18. The method of claim 12, wherein the peptide is SEQ ID NO:15, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:116, SEQ ID NO:141, or SEQ ID NO:159.

19. The method of claim 12, wherein the concentration of the peptide in the composition is 0.01 μM to 500 μM.

20. The method of claim 12, wherein the composition is administered to the wound topically, systemically, or transdermally.

21. A method for treating acne comprising administering to the skin of a mammal a composition, wherein:
the composition comprises an isolated peptide;
the peptide is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, or SEQ ID NO:164; and
wherein the composition is administered to the skin of a mammal in an effective amount for an effective amount of time sufficient to treat the acne.

22. The method of claim 21, wherein the mammal is a human.

23. The method of claim 21, wherein the peptide is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:164.

24. The method of claim 21, wherein the peptide is SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:164.

25. The method of claim 21, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:129, SEQ ID NO:142, SEQ ID NO:143, or SEQ ID NO:145.

26. The method of claim 21, wherein the peptide is SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or SEQ ID NO:145.

27. The method of claim 21, wherein the peptide is is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:162, or SEQ ID NO:164.

28. The method of claim 21, wherein the peptide is SEQ ID NO:15, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:116, SEQ ID NO:141, or SEQ ID NO:159.

29. The method of claim 21, wherein the concentration of the peptide in the composition is 0.01 μM to 500 μM.