COMPOSITIONS ET TRAITEMENT DE PEPTIDES BIOLOGIQUEMENT ACTIFS CONTENANT DES RESIDUS D'ACIDES D-AMINES

COMPOSITIONS OF AND TREATMENT WITH BIOLOGICALLY ACTIVE PEPTIDES HAVING D-AMINO ACID RESIDUES

Abrégé anglais

ABSTRACT OF THE DISCLOSURE
A biologically active peptide wherein each amino acid
residue of the peptide is a D-amino acid residue or a glycine
residue. Examples of such peptides wherein each amino acid
residue is a D-amino acid residue or a glycine residue include
deletion and substitution analogues of magainin peptides, CPF
peptides, PGLa peptide, XPF peptide, and derivatives of
cholecystokinin. Such peptides have increased resistance to
proteolytic enzymes while retaining biological activity.

Revendications

WHAT IS CLAIMED IS:
1. A compound comprising
an analogue of Magainin I peptide or Magainin II peptide,
said Magainin I peptide or Magainin II peptide being in an
amide-or carboxy-terminated form, and wherein Magainin I is
represented by the following structural formula using the single
letter amino acid code and the numbers below each amino acid
residue refer to the position of the residue in the peptide:
G I G K F L H S A G K F G K A F V G E I M K S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23, and
wherein each amino acid residue is a D-amino acid residue or a
glycine residue, and wherein Magainin II is represented by the
following structural formula using the single letter amino acid
code and the numbers below each amino acid residue refer to the
position of the residue in the peptide:
G I G K F L H S A G K F G K A F V G E I M N S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23,
and wherein each amino acid residue is a D-amino acid residue or
a glycine residue, and wherein said Magainin I or Magainin II
peptide is substituted in at least one of positions 1-23, wherein
the substituents which may be employed in each of positions 1-23
are shown in the following table:

Residue No. Substituent
1 D-Lys,D-Ala
2 D-Lys,D Ala,D-Ile,D-Arg,D-Leu,D-Val,D-His,D-Met
3 D-Lys,D-Ala,D-Trp,D-Arg,D-His
4 D-Lys,D-Ala,D-Arg,D-His
D-Phe,D-Ala,D-Lys,D-Trp,D-Leu,D-Ile,D-Val,D-Met
6 D-Leu,D-Lys,D-Ala,D-Ile,D-Val,D-Met
7 D-Lys,D-His,D-Ala,D-Arg,D-Ile,D-Val,D-Met
8 D-Ala,D-Lys,D-Ser,D-Trp,D-Met,D-Ile,D-Arg,D-His,
D-Thr,D-Leu,D-Val
9 D-Lys,D-Ala,D-Trp,D-Arg,D-His,D-Leu,D-Ile,D-Val
D-Lys
D-Ala,D-Trp,D-Arg,D-His,D-Leu,D-Ile,D-Val
11 D-Lys,D-Arg,D-His
12 D-Phe,D-Lys,D-Trp,D-Arg,D-His
13 D-Ala,D-Lys,D-Trp,D-Met,D-Arg,D-His,D-Phe,D-Leu,
D-Ile,D-Val
14 D-Ala,D-Lys,D-Arg,D-His
D-Lys,D-Trp,D-D-Ala,D-Arg,D-His,D-Phe
16 D-Ala,D-Lys,D-Phe,D-Ile,D-Val,D-Met,D-Leu
17 D-Ala,D-Lys,D-Val,D-Trp,D-Arg,D-His,D-Met,D-Leu
18 D-Ala,D-Lys,D-Trp,D-Arg,D-His,D-Leu,D-Met
19 D-Ala,D-Lys,D-Glu,Gly,D-Arg,D-His,D-Leu
D-Ile,D-Phe,D-Asn
D-Ile,D-Ala,D-Lys,D-Trp,D-Leu,D-Phe,D-Val,D-Met
21 D-Lys,D-Pro,D-Ala,D-His,D-Leu,D-Arg,D-Ile,D-Phe
22 D-Lys,D-Ala,D-Asn,D-Gln,D-Arg,D-His
23 D-Ser,D-Lys,D-Ala,D-Thr,Gly,D-Leu,D-Ile,
D-Gln,D-Asn
2. The compound of Claim 1 wherein said Magainin I or
Magainin II peptide is substituted in at least one of positions
1-23, wherein each of the substituents in each of positions 1-23
is shown in the following table:

-36-
Residue No. Substituent
1 D-Lys,D-Ala
2 D Lys,D-Ala,D-Ile
3 D-Lys,D-Ala,D-Trp
4 D-Lys,D-Ala
D-Phe,D-Ala,D-Lys,D-Trp
6 D-Leu,D-Lys,D-Ala
7 D-Lys,D-His,D-Ala
8 D-Ala,D-Lys,D-Ser,D-Trp
9 D-Lys,D-Ala,D-Trp
D-Lys,D-Ala,D-Trp
11 D-Lys
12 D-PhegD-Lys,D-Trp
13 D-Ala,D-Lys,D-Trp
14 D-Ala,D-Lys
D-Lys,D-Trp,D-Ala
16 D-Ala,D-Lys,D-Phe
17 D-Ala,D-Lys,D-Val,D-Trp
18 D-Ala,D-Lys,D-Trp
19 D-Ala,D-Lys,D-Glu
D-Ile,D-Ala,D-Lys,D-Trp
21 D-Lys,D-Pro,D-Ala
22 D-Lys,D-Ala,D-Asn
23 D-Ser,D-Lys,D-Ala
3. A compound comprising:
an analogue of <againin I peptide or Magainin II peptide 9
said Magainin I peptide or Magainin II peptide being in an
amide-or carboxy-terminated form, and wherein Maginin I is
represented by the following structural formula using the single
letter amino acid code and the numbers below each amino acid
residue refer to the position of the residue in the peptide:

-37-
G I G K F L H S A G K F G K A F V G E I M K S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23, and
wherein each amino acid residue is a D-amino acid residue or a
glycine residue, and wherein Magainin II is represented by the
following structural formula using the single letter amino acid
code and the numbers below each amino acid residue refer to the
position of the residue in the peptide:
G I G K F L H S A K K F G K A F V G E I M N S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23,
and wherein each amino acid residue is a D-amino acid residue or
a glycine residue, and wherein at least one of amino acid
residues 15-23 is omitted, and wherein said Magainin I or
Magainin II peptide is substituted in at least one of the
remaining positions 1-23, wherein the substituents which may be
employed in each of positions 1-23 are shown in the following
table:

-38-
<IMG>
4. The compound of Claim 3 wherein said Magainin I or
Magainin II peptide is substituted in at least one of the

-39-
remaining positions 1-23, wherein each of the substituents in
each of positions 1-23 is shown in the following table:
Residue No. Substituent
l D-Lys,D-Ala
2 D-Lys,D-Ala,D-Ile
3 D-Lys,D-Ala,D-Trp
4 D-Lys,D-Ala
S D-Phe,D-Ala,D-Lys,D-Trp
6 D-Leu,D-Lys,D-Ala
7 D-Lys,D-His,D-Ala
8 D-Ala,D-Lys,D-Ser,D-Trp
9 D-Lys,D-Ala,D-Trp
D-Lys,D-Ala,D-Trp
11 D-Lys
12 D-Phe,D-Lys,D-Trp
13 D-Ala,D-Lys,D-Trp
14 D-Ala,D-Lys
D-Lys,D-Trp,D-Als
16 D-Ala,D-Lys,D-Phe
17 D-Ala,D-Lys,D-Val,D-Trp
18 D-Ala,D-Lys,D-Trp
19 D-Ala,D-Lys,D-Glu
D-Ile,D-Ala,D-Lys,D-Trp
21 D-Lys,D-Pro,D-Ala
22 D-Lys D-Ala,D-Asn
23 D-Ser,D-Lys,D-Ala
5. A compound, comprising:
a deletion analogue of an amide or carboxy terminated
Magainin I wherein Magainin I is represented by the following
structural formula using the single letter amino acid code and
the numbers below each amino acid residue refer to the position
of the residue in the peptide:

-40-
G I G R F L H S A G, K F G K A F V G E I M K S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23,
and wherein each amino acid residue is a D-amino acid residue or
a glycine residue, and at least one of amino acids 15 through 23
is omitted.
6. The compound of Claim 5 wherein at least one of amino
acids 15, 16, 18, 19, 21, 22 and 23 is omitted.
7. A compound comprising:
a deletion analogue of an amide or carboxy terminated
Magainin II wherein Magainin II is represented by the following
structural formula using the single letter amino acid code and
the numbers below each amino acid residue refer to the position
of the residue in the peptide:
G I G K F L H S A K K F G K A F V G E I M N S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23,
and wherein each amino acid residue is a D-amino acid residue or
a glysine residue, and at least one of amino acids 15 through 22
is omitted.
8. The compound of Claim 7 wherein at least one of amino
acids 15, 18, 19, 20, 21, and 22 is omitted.
9. A peptide comprising:
said peptide containing at least eight amino acids, wherein
each amino acid is a D-amino acid or glycine, said peptide
containing at least two groups of amino acids A-B wherein each
A-B group is separated from another A-B group by at least one
D-amino acid other than a hydrophobic amino acid and at least two
groups of D-amino acids C-D wherein each C-D group is separated
from another C-D group by at least one D-amino acid or glycine
other than a hydrophilic D-amino acid, wherein each of A and B is
a hydrophobic D-amino acid or glycine wherein each of A and B may
be the game or different amino acids and one of C and D is a
basic hydrophilic D-amino acid ant the other of C and D is a
basic or neutral hydrophilic D-amino acid.

-41-
10. The peptide of Claim 9 wherein the peptide has from 8
to 15 amino acids.
11. The peptide of Claim 10 wherein the peptide includes
one of the following sequences:
(i) (Wl)a(A-B-C-D)n(Z1)b
(ii) (W2)a(B-C-D-A)n(Z2)b
(iii) (W3)8(C-D-A-B)n(Z3)b
(IV) (W4)a(D-A-B-C)n(Z4)b
wherein W1 is D-, C-D-, B-C-D-
W2 is A-, D-A-, C-D-A-
W3 is B-, A-B-, D-A-B-
W4 is C-, B-C-, A-B-C-
Z1 is -A, -A-B, -A-B-C
Z2 is -B, -B-C, -B-C-D
Z3 is -C, -C-D, -C-D-A
Z4 is -D, -D-A, -D-A-B
n is 2 or 3, a is 0 or 1 and b is 0 or 1.
12. The peptide of Claim 9 wherein the peptide contains at
least 16 amino acids and there are at least four groups of amino
acids A-B ant at least 3 groups of D-amino acids C-D.
13. The peptide of Claim 12 wherein there are at least four
groups of D-amino acids C-D.
14. The peptide of Claim 13 wherein the peptide includes at
least four groups of the peptide sequence -A-B-C-D, -B-C-D-A-,
-C-D-A-B, -D-A-B-C.
15. The peptide of Claim 14 wherein the peptide includes
one of the following sequences:
(X1)a(A-B-C-D)n(Y1)
(X2)a(B-C-D-A)n(Y2)b
(X3)a(C-D-A-B)n(Y3)b
(X4)a(D-A-B-C)n(Y4)b

-42-
wherein X1 is D; C-D- or B-C-D-, Y1 is -A or -A-B or -A-B-C
X2 is A-, D-A- or C-D-A-
Y2 is -B, -B-C or B-C-D
X3is B-, A-B-, D-A-B-
Y3 is -C, -C-D, -C-D-A
X4is C-, B-C-, A-B-C-
Y4 is -D, -D-A, -D-A-B
a is 0 or 1; b is 0 or 1;
and n is at least 4.
16. The peptide of Claim 13 wherein said hydrophobic amino
acids are selected from the class keynoting of D-Ala, D-Phe,
Gly, D-Ile, D-Leu, D-Met, D-Val, D-Trp, and D-Tyr and D-Cys, said
neutral hydrophilic D-amino acids are selected from the class
consisting of D-Asn, D-Ser, D-Gln, and D-Thr, and said basic
hydrophilic D-amino acids are selected from the class consisting
of D-Lys, D-Arg, D-His, and D-ornithine.
17. The polypeptide of Claim 16 wherein at least one of
said at least four groups of said four amino acids is of the
sequence A-B-C-D, wherein A and B are hydrophobic amino acids,
one of C and D is a neutral or basic hydrophilic amino acid, the
other of C or D is a basic hydrophilic amino acid.
18. The polypeptide of Claim 13 wherein said polypeptide
includes the following sequence:
-(D-Ala-D-Phe-D-Ser-D-Lys)4-
19. The polypeptide of Claim 14 wherein said polypeptide
comprises a chain of at least 20 amino acids and no greater than
50 amino acids.
20. The polypeptide of Claim 14 wherein said polypeptide
includes five groups of said sequence.
21. The polypeptide of Claim 14 wherein said polypeptide
includes six groups of said sequence.

-43-
22. The polypeptide of Claim 9 wherein said polypeptide
includes repeating groups of said four amino acids.
23. A compound, comprising:
a peptide, said peptide including the following basic
structure X:
-R1-R1-R2-R2-R1-R1-R3-R1-
-R1-R1-R3-R1-R1-R4-R5-R1
wherein R1 is a hydrophobic amino acid; R2 is a basic
hydrophilic amino acid or a hydrophobic amino acid, R3 is a basic
hydrophilic amino acid, R4 is a hydrophobic or neutral
hydrophilic amino acid, and R5 is a basic or neutral hydrophilic
amino acid, and each amino acid residue of said peptide is a
D-amino acid or glycine.
24. The composition of Claim 23 wherein the peptide
includes the following structure:
Y - X -
wherein X is the basic peptide structure of Claim 2
and Y is R5-; R2-R5-; R1-R2-R5- or R2-R1-R2-R5.
25. The composition of Claim 23 wherein the peptide
includes the following basic structure
- X - Z-
wherein X is the basic peptide structure of Claim 2 and Z
R1; R1-R1; R1-R1-R4; R1-R1-R4-R4; R1-R1-R4-R4-R6;
R1-R1-R4-R4-R6-D-Gln; or
R1-R1-R4-R4-R6-D-Gln-D-Gln, wherein
R6 is D-proline or a hydrophobic amino acid.
26. The composition of Claim 23 wherein the peptide
includes the following basic structure
(Y)a - X - (Z)b
wherein Y and Z are as previously defined in Claims 24 and
25,
a is 0 or 1, and b is 0 or 1.

-44-
27. The composition of Claim 23 wherein the peptide has the
following structure:
G12S3LG4ALKA5LKIG678LGG9(10)QQ
Where:
1 = F, L
2 = G, A
3 = F, L
4 = K, L
= A, G, T
6 = A, T
7 = H, N
8 = A, M, F, L
9 = A, S, T
= P, L
28. A composition comprising at least one biologically
active peptide, said at least one biologically active peptide
including a chain of at least 20 amino acids of the structure
X10, wherein each of said at least 20 amino acids is a D-amino
acid or glycine, and wherein X10 is of the formula:
-R11-R12-R11-R11-R13-R13-R11-R11-R12-R11-R11-R15-R14-R11-R12-R13-
R11-Rl6-R15-R17-, wherein:
R11 is a hydrophobic amino acid;
R12 is a basic hydrophilic amino acid;
R13 is a hydrophobic or basic hydrophilic amino acid;
R14 is a natural hydrophilic amino acid, or a basic
hydrophilic amino acid;
R15 is neutral hydrophilic amino acid,
R16 is D-proline or a hydrophobic amino acid; and
R17 is neutral hydrophilic, basic hydrophilic or
hydrophobic amino acid,
said at least one biologically active peptide having no
greater than 35 amino acids.
29. The composition of Claim 28 wherein said at least one
biologically active peptide has no greater than 30 amino acids.

-45-
30. The composition or Claim 28 wherein said at least one
biologically active peptide is of the structure:
-R11-R11-X-.
31. The composition of Claim 30 wherein said at least one
biologically active peptide is of the structure:
D-Leucine-D-Leucine-X-.
32. The composition of Claim 23 wherein said at least one
biologically active peptide is of the structure:
-X-R12-R11-R15-R11-R11-.
33. A biologically active amphiphilic peptide, said peptide
including the following basic structure X12:
-[R21-R22-R22-R23-R21-R22-R22]-n, wherein
R21 is a basic hydrophilic amino acid,
R22 is a hydrophobic amino acid, R23 is a neutral
hydrophilic or hydrophobic amino acid, and n is from 2 to 5, and
wherein each amino acid residue of said peptide is a D-amino acid
residue or a glycine residue.
34. The peptide of Claim 1 wherein the peptide includes the
following structure:
Y12-X12, wherein X12 is the basic peptide structure of Claim
1, and Y12 is:
(i) R22;
(ii) R22-R22;
(iii) R21-R22-R22;
(iv) R23-R21-R22-R22;
(v) R22-R23-R21-R22-R22;or
(vi) R22-R22-R23-R21-R22-R22.
35. The peptide of Claim 33 wherein the peptide includes
the following basic structure:
X12-Z12-, wherein X12 is the basic peptide structure of
Claim 1 and Z12 is:
(i) R21;
(ii) R21-R22;
(iii) R21-R22-R22;

(iv) R21-R22-R22-R23;
(v) R21-R22-R22-R23-R21;
(vi) R21-R22-R22-R23-R21-R22.
36. The peptide of Claim 33 wherein the peptide includes
the following basic structure:
(Y12)a-X12-(Z12)b, wherein Y12 and Z12 are as previously
defined in Claims 34 and 35, a is 0 or l, and b is 0 or 1.
37. The peptide of Claim 33 wher3in n is 3.
38. The peptide of Claim 37 wherein said peptide is of the
following structural formula as indicated by the single letter
amino acid code.
[KIAGRIA]3.
39. The peptide of Claim 33 wherein n is 2.
40. The peptide of Claim 39 wherein said peptide is of the
following structural formula as indicated by the single letter
amino acid code:
KIA(KIAGKIA)2KIAG.
41. A biologically active amphiphilic peptide, said peptide
including the following basic structure: X14:
R21-R22-R22-R23-R21-R22-R22-R21-R22-R22-R22-R21-R22-R22,
wherein R21 is a basic hydrophilic amino acid, R22 is a
hytrophobic amino acid, and R23 is a neutral hydrophilic or a
hydrophobic amino acid, and wherein each amino acid residue of
said peptide is a D-amino acid residue or a glycine residue.
42. The peptide of Claim 41 wherein the peptide includes
the following structure:
Y14-X14, wherein X14 is the basic peptide structure of Claim
41, and Y14 is:
(i) R22;
(ii) R22-R22;
(iii) R21-R22-R22;
(iv) R23-R21-R22-R22;
(v) R22-R23-R21-R22-R22;
(vi) R22-R22-R23-R21-R22-R22; or

-47-
(vii) R21-R22-R22-R23-R21-R22-R22.
43. The peptide of Claim 41 wherein the peptide includes
the following structure:
X14-Z14, wherein X14 is the basic peptide structure of Claim
41 and Z14 is:
(i) R21;
(ii) R21-R22;
(iii) R21-R22-R22;
(iv) R21-R22-R22-R23;
(v) R21-R22-R22-R23-R21;
(vi) R21-R22-R22-R23-R21-R22; or
(vii) R21-R22-R22-R23-R21-R22-R22.
44. The peptide of Claim 41 wherein the peptide includes
the following basic structure:
(Y14)a-(X14)-(Z14)b, wherein Y14 and Z14 are as previously
described in Claims 42 and 43, a is 0 or 1 and b is 0 or 1.
45. The peptide of Claim 44 wherein said peptide is of the
following structural formula as indicated by the single letter
amino acid code:
KLASKAGKIAGKIAKVALKAL.
46. The peptide of Claim 44 wherein said peptide is of the
following structural formula as indicated by the single letter
amino acid code:
KIAGKIAKIAGOIAKIAGKIA.
47. A biologically active àmphiphilic peptide, said peptide
including the following basic structure X16:
--R31-R33-R32-R31-R34-R34-R31-R31-R34-
R32-R31-R31-R32-R31-R31-R31-R32-,
wherein R31 is a hydrophobic amino acid, R32 is a basic
hydrophilic amino acid, R33 is a neutral hydrophilic, basic
hydrophilic, or hydrophobic amino acid, and R34 is a hydrophobic

-48-
or basic hydrophilic amino acid, and wherein each amino acid
residue of said peptide is a D-amino acid residue or a glycine
residue.
48. The peptide of Claim 47 wherein the peptide includes
the following structure Y16-X16, wherein X16 is the basic peptide
structure of Claim 47, and Y16 is:
(i) R31; or
(ii) R34-R31.
49. The peptide of Claim 47 wherein the peptide includes
the following structure:
X16 -Z16: wherein X16 is the basic peptide structure of
Claim 47 and Z16 is:
(i) R31; or
(ii) R31-R31.
50. The peptide of Claim 47 wherein the peptide includes
the following basic structure:
(Y16)a - (X16) -(Z16)b, wherein Y16 and Z16 are as
previously described in Claims 48 and 49, a is 0 or 1, and b is 0
or 1.
51. A biologically active amphiphilic peptide, said
peptide including the following basic structure X18:
--R31-R33-R32-R31-R34-R35-R33-R31-34-
R32-R31-R31-R32-R31-R31-R31-R32-(R36)n-R31-,
wherein R31 is a hydrophobic amino acid, R32 is a basic
hydrophilic amino acid, R33 is a neutral hydrophilic, basic
hydrophilic, or hydrophobic amino acid, R34 is a hydrophobic or
basic hydrophilic amino acid, R35 is glutamine or asparagine, or
a basic hydrophilic, or hydrophobic amino acid, ant R36 is
glutamic acid, aspartic acid, a hydrophobic amino acid, or a
basic hydrophilic amino acid, ant n is 0 or 1, and wherein each
amino acid residue is a D-amino acid residue or glycine.
52. The peptide of Claim 51 wherein said peptide includes
the following structure:
Y18 - X18, wherein X18 is the basic peptide structure of
Claim 51, and Y18 is:

-49-
(i) R31; or
(ii) R34-R31.
53. The peptide of Claim 51 wherein said peptide includes
the following structure:
X18-Z18, where X18 is the basic peptide structure of Claim
51, and Z18 is:
(i) R31;
(ii) R31-R35; or
(iii) R31-R35-D-Proline; or
(iv) R31-R35-D-Proline-R32.
54. The peptide of Claim 51 wherein the peptide includes
the following basic structure:
(Y18)a - (X18) - (Z18)b,
wherein Y18 and Z18 are as previously described in Claims 52 and
53, a is 0 or 1, and b is 0 or 1.
55. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the compound
of Claim 1.
56. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the compound
of Claim 3.
57. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the compound
of Claim 5.
58. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the compound
of Claim 7.
59. A process for inhibiting growth of a target cell or
virus, comprising:

-50-
administering to a host an effective amount of the peptide
of Claim 9.
60. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the peptide
of Claim 23.
61. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the
composition of Claim 28.
62. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the peptide
of Claim 33.
63. A process for inhibiting growth of target cell or
virus, comprising:
administering to 8 host an effective amount of the peptide
of Claim 41.
64. A process for inhibiting growth of 6 target cell or
virus, comprising:
administering to 8 host an effective amount of the
peptide of Claim 47.
65. A process for inhibiting growth of a target cell or
virus, comprising:
administering to a host an effective amount of the
peptide of Claim 51.

Description

C (~ . .,r l D ~ 8
2 ~, G~&
~//~/.,~0
PATAP390
COMPOSITIONS O~ ~N3 TR~AT~E~T WITH
~IOLOGIC~LLY ACTIYE 2~PTID~5 H~IN~
: D-A~I~O ~CID ~SIDU~S
.
Th~s invention relate~ to biolo~ically ac~ive peptides, and
more particularly to blolo~ically actiYe p~ptides wherein each
amino acid residue of ~uch peptid~s i~ a D-a~ino acit residue or
Ycine~
In accordance with an a~pect oP the present invention, there
is provldod a compound &omprisln~ a~ analo~ue of Ma~ainin I
Peptide or Ma8ainin II peptide. ~ach ~mino acid resitue o~ the
Magainin I peptid~ and the M~gainin II peptite is a D-amino acit
residue or ~lyclne. Ths Ma~a~ni~ I o~ Ma8ainin II peptide i~ in
an amid~ or carbo~y t~rminat~d form. Magainln I is repre3en~ed
by thc follow~ng structural formuls u~in~ tha ~in~le letter amino
acid code and the numbers b~low ~sch amino acid re~idu~ refer to
th~ position o~ ~he ~esitua in th~ pep~id~
G I G R F L H S ~ a ~ F G K ~ F V ~ E I M ~ S
1 2: 3.4 5-~ 7~8 9 10 lI 12 13 14 15 16 17 18 l9 20 2~ 22 23
MaBainln II 1A r~preqented ~y ~he follsw~ng ~tructural ~o~mula
u3ing tho si~gl~ Iettcr amino acit cod~ a~d th~ numbers below
each a~ino acid residu~ refer ~o ~ho po~ on of the re~idu~ in
th~ p~ptid~
G I G K F ~ H ~ A ~ ~ F G R ~ P V a ~: I M N S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 IB 19 20 21 22 23
'
, - ' ~ ' ` ` ` ' -

` '` . C ~ 2~ D ~ `.3, ~ 3 ~
The Magainin I orC~againin II peptide ls substig~uted ~ L~ ~2~
one of poYition3 1-23. The ~ubstituents which may be employed in
esch of positions 1-23 are shown in the following table:.
Re~idue No. Substituent
l D-Lys,D-Ala
2 D-Lys,D-Ala, D~ ,D-Ar~,D-Leu,D-Val,D-His,D-Met
3 D-Ly3,D-Al~,D-Trp,D-Ar~,D-His
4 D-Ly~,D-Al~,D-Ar6,D-Hl~
S D-P~Q,D-Ala,D-I.y8,D-Trp,D-Leu,D-Ile,D-Val,D-Met
6 D-Leu,D-Lys,~-Ala,D~ ,D-Vsl,D-Met
7 D^Ly~,D-Hls,D-Ala,D-Arg,D-Il~,D-Val,D-Met
8 D-Ala,D-Lys,D-Ser,D-Trp,D-M2t,D-Ile,D-Ar~,D-Hi~,
D-Thr,D-Leu,D-Yal
9 D-Ly3,D-Al`a,D-Trp,D Arg,D-Hls,D-L~u,D-Ile,D-Val
D-Lys
D-Ala,D-Trp,D-Arg,D-Hi~,D-Leu,D-Il@,D-Val
11 D-Ly~,D-Arg,D-His
12 D-Phe,D-Ly3,D-Trp,D-Arg,D-His
13 D-Ala,D-Lys,D-Trp,D-M~t,D-Arg,D-His,D-Phe,D-Leu,
D-Il2,D-~al
14 D-~la,D-Lys,D-Ar~,D-~i~
lS D-Ly~,D-Trp,D-Ala,D-~rg,D-His,D-Phe
16 D-Ala,D-Lys,D~Ph~,D-Ilo,D-V~l,D-Met,D-Leu
17 D-Ala,D-Ly~,D-Val,D-Trp,D-~rggD-~i~,D-Met,D-Leu
18 D-Ala,D-Lys,D-Trp,D-~rg,D-His,D-Leu,D-Met
19 D-~13,D-Lys,D-Glu,Gly,D-~r~,D-His,D-Leu
D~ ,D-Phe,D-A3n
D-Il~,D-Ala,D-~y~,D-Trp,D-L~u,D-Ph~,D-Val,D-Met
21 D-Ly~,D-Pro,D-Als,D-His,D~L~u,D-Arg,D-IlQ,D-Ph~
22 D-Ly3,D-Ala,D A~n,D-Gln,D-Arg,D-Hls
23 D-S~r,D-Lys,D-~la,D-Thr,Gly,D-Leu,D-Ile
D-Gln,D-A~n
'

r ~ F N T ~
Preferably, ~h~ ~bstituents are ~s f~ll-o.w~
Residue No. Substituent
1- D-Lys,D-Ala
2 D-Ly~,D-Ala,D-Ile
3 D-Lys,D-Ala,D-Trp
4 D-Ly3,D-Ala
D-Pho,D-~la,D-Lys!D-Trp
6 D-L~u,D-Lys,D-Ala
7 D-Lys,D-Hi3,D-Ala
8 D-Ala,D-Lys,D-Ser,D-Trp
9 ~-Lys,D-Ala,D-Trp
D-Lys
D-~la,D-Trp
11 D-Lys
12 D-Ph~,D-Ly~,D-Trp
13 D-Ala,D-Lys,D-Trp
14 D-Ala,D-Lys
D-Lys,D-Trp,D-Ala
16 D-Ala,D-Ly~,D-Phe
17 D-Ala,D-Ly~,D-Val,D-Trp
18 D-Ala,D-Lys,D-Trp
19 D-Ala,D-Lys,D-Glu
D-Ile,D-Ala,D-Lys9D-Trp
21 D-Lys,D-Pro,D-~la
22 D-Lya,D-Al~,D-Asn
23 D-S~r,D-Ly~,D-Ala
It i~ to b~ undorstood tha~ for purpo~e~ of th~ present
invention, that th~ D-~ryptophan rasidu~ m~y bR protected wi~h a
formyl gro~p or be unpro~ct~d. Tho ~-phenylalanlno re~idues,
wheth~r ~uch r~sidu~ is pre~nt in lt~ normal poaition, or
employ~d as a ~ub~tltution re~idu~, may bQ a normal
D-phenylalan~n~ rQ~idu~ or an iodinat~d D-ph~nylal~nine re~idue.
-
.: - ~
.
' ' . ,

C (~ r ~ T I ~ 2 ~ !~ 2 ~ ~ 8
In accordàn;~ë'wieh one embadiment.,-the peptld~ i'3 a Magainin
I peptide~ and at lea~t one of smino acid residues 8, 10, 13, 16,
18 and 19 is sub~tituted with a D-~lanine r~sidue. In another
embodiment, the Magainin I peptite has D-alanine residue
~ubstitution~ at each of sminQ scid re~idue3 3, 8, 16, 19 and 23.
In accordanoe with another embodiment, the Ma8sinin I
peptide has D-alanine r~idue substltutions at four of amino acid
residues 3, 8, 16, 19 and 23, and the remalning one of these
re9idue~ ls sub9tltuSed with a D-ly~n~ r~idu~.
In accortance with yet a further embodiment, amino acid 21
of a Magainin I peptide i9 substituted with a D-proline residue.
In accordance with another embodiment, at lea~t one of amino
acid residu~s 3, 7, 8, 10, 18-21, and 23 of Magainin I i~
substituted wlth a D-ly~in~ residu~.
In accordance with anoth~r embodi~ent, amino acid re~idue~
3, 8, 9, 19 and 23 of Ma8ainin I a~a ~ach substltuted with a
D-lysine residu~ and amino acld residu~ 15 i~ ~ubstituted with a
D-alanine re~itu~.
In sccordance with a further embodiment, at least one of
amino acid residue~ 3, 5, 8, 10, 12, 13, 15, 18, and 20 of
MaKainin I is sub~tituted with 8 protect2d D-tryptophan residue,
wherein the protecting group is prefera~ly a ~ormyl group. In
accordance with anothor embodim~nt, at laast one of amino acid
residues 9, 13, 15 and 17 of Magainin I is substituted with an
unprotected D-tryptophan re~ldu~.
In accordancQ with anoth~r embodiment, the peptide ls a
Magainin II poptld~ , and at least ono of a~ino acid re~idues 1- 8,
10, 13, 14, 16 as3d la-23 is a D-alanine re~idut!.
In accordan~ wlth ano~h~r embodlment, at le89t OII~ of amino
a,cid residuQ~ 1-3, 5-9, 12, 13 snd 15-23 o Magainin II ls a
D- lyslne re~idu~ .
Xn accordanca wlth anoth~r asp~c~ o~E th~ present invention,
there i~ provided an an~logu~ of Ma~3~inin I or Magainln II
peptide, ~ald Ma~ainin I or Magainin II pep~id~ being in an
.~

f~F~ ,T~ f2~7 ~
amide- or carbo~,y-t naeed form sn~ having the stru~tural ~
formulas hereinabove described, where.ln each amino acid residue
of ~aid Magslnin I or Msgsinin II peptide i3 a D-smino acid
residue or glycine, and wherein st lesst one o amino acid
residue~ 15-23 is omitted and st lea~t one of the remaining amino
acid re~idues is substituted. Th~ substituent~ which may be
employed in st lesst one of positlons 1-23 sre shown in the
following table:
' ' .
' '
.

C (? ~ i T IRe~idue No. ~ . Sub~tituent
1 D-Lys,D-~ls ~.~.: ; ;. . ..
2 D-Ly~D-Als,
D-Ile,D-Arg,D-Leu,D-Val,D-His,D-Met
3 D-Lys,D-Ala,D-Trp,D-Arg,D-His
4 D-Lys,D-Ala,D-Arg,D-His
D-Phc,D-Ala,D-Ly3,D-T~p,D-Leu,D-Ile,D-Val,D-Met
6 D-Leu,D-L~s,D-Al~,D-Ile,D-Val,D-Met
7 D-Lys,D-His,D-Ala,D-Arg,D-Il~,D-Val,D-Met
8 D-Als,D-Ly9,D-Ser,D-Trp,D-Met,D-Ile,D-Arg,
D-Hi~,D-Thr,D-L~u,D-Val
9 D-Ly~,D-Ala,D-Trp;D-Arg,D-His9D-Leu,D-Il~,D-Val
D-Lys,
D-~la,D-Trp,D-Ar~,D-Hi~,D-L~u,D-Il~,D-Val
ll D-Ly~,D-Arg,D-Hi~
12 D-Phe,D-Ly~,D-Trp,D-Ar~,D-Hi~
13 D-Ala,D-Ly~,D-Trp,D-M~t,D-Ar~,D-His,D-Phe,
D-Leu,D-Il~,D-Vsl
14 D-Ala,D-Lys,D-Arg,D-Hi~
D-Ly~,D-Trp,D-Ala,D-Ar~,D-His5D-Phe
16 D-Ala,D-Ly~,D-Phe,D-Ile,D-Val,D-Met,D-Leu
17 D-Ala,D-Ly~,D-Val,D-Trp,D-Arg,D-His,D-Met~D-Leu
18 D-~la,D-Ly~,D-Trp,D-Arg,D-Hi~,D-Leu,D-Met,D-Phe
l9 D ~la,D-Ly~,D-Glu,Gly,D-~rg,D-Hi~,D-Leu,
D-Ile,D-Phe,D-~s~
D-Ile,D-Ala,D-Lys,D-Txp,D-Leu,D~Phe,D-Ysl,D-Met
21 D-Ly~,D-Pro,D-Ala,D-His,D~Leu9D-Arg,~ ,D-Ph
22 D-Lys,D-~l~,D-~n,D-Gln,D-Ar~,D-His
23 D-52r,D-Lys,D-~la,~-Thr,Gly,D-Leu,D-Ile,
D-Gln,D-Asn
,
,

r~ A~ 2~8
Preferably, the s~ituents are 83 follow~
Residue No. ~ Sub~tituent . .
1 D-Lys,D-Ala
2 D-Lys,D-Ala,D-Ile
3 D-Lys,D-Ala,D-Trp
4 D-Lys,D-Ala
D-Phe,D-Ala,D-Ly~,D-Trp
6 D-Leu,D-Lys,D-Ala
7 D-Lys,D-His,D-Ala
8 D-Ala,D-Ly~,D-Ser,D~Trp
9 D-Lys,D-Ala,D-Trp
D-Ly~,D-Ala,D-Trp
11 D-Lys
12 D-Phe,D-Lys,D-Trp
13 D-~la,D-Ly~,D-Trp
14 D-Ala,D-Lys
D-Ly~,D-Trp,D-Ala
16 D-Als,D-Lys,D-Ph~
17 D-Ala,D-Ly~,D-Val,D-Trp
18 D-Ala,D-Ly~,D-Trp
19 D-Ala,D-Lys,D-Glu
.D-Il~,D-Ala,D-Lys,D-Trp
21 D-Lys,D-Pro,D-Ala
22 D-Ly~,D-Ala,D-~n
23 D S~rtD-LyY,D-~la
In accord~n~ with a particular embodiment, smino acid
re3idu~ 16-23 o~ Magainin I ar~ d~ et, a~d amino acid residues
3, 8 and 10 ar~ ~ub~tituted with a D-a`lan~ne re3itue.
In accordanc~ wlth yQt anothsr embodi~nt, amino acid
residue 19 of Ma~ainin I i~ deleted,~and pr~orably a~ino acid
residue3 5~ 8, 9 a~d 16 are each sub~tltuted w~h a D-1ysine
r~situe, amino acid re~id~ 21 i~ ~ub~t~tuted with a D-leucine
.
~, , ''
'

6~ ,fr~ ~ ~ r ~ f~
re~idue, ant acid residues 18 a~d-2.3:ar~ sub~tituted with a
D-alanine r~sidue.
In accordance with another embodiment, in carboxy- or
amide-terminat~d (preferably amide-termlnated) Msgainin I or
Magainin II peptide, amino acid re~idues 17-23 or 16-23 or 15-23
are deleted, and 8mino acid residues 3, 7, and 8 are each
substituted with a D-lysinc residu~, and optionally amino acid
residue 13 and/or amino acld residuQ 10 is sub3tituted with a
D-alanine re~idue. Preferred peptides are as follows:
GIKKFLKKAGKFGK-NH2
GIKKFLKKAGKFGKAF-NH~
GIKKFLKKAXKFGKA-NH2
GI~KFLKXAKKFARA-NH2
GIKKFLKKAAKFAKA NH2
Preliminary ~tudies indicate thdt th~ abo~e-mentioned preferred
peptides pos~ess low hemolyt~c act~vity. In accord~nce with a
further embodimQnt, amino acid residu~ 21 of Magainin I may be
del~ted, and preferably amino acid resldue~ 5,10, 18, and 19 are
each substituted with a D-lysine resldu~, smino acid residue 7 i9
sub~tituted with a D-phenylalanin~ re3idue, and amino acid
residue 22 i8 substituted wlth ~ D-alanlno re~$due.
In accordanc~ with ~noth~r embodlment, ln carboxy or smide
terminated ~preferably amide ~erminated) Magainin I or Magainin
II tPrefersblY Ma8ainin II), amino acid residue 19 i~ omitted,
and st least ono o~ amino acid r~idu2s 3, 7, B, 10, 13, 15, 16,
18 21, 22 or 23 ia substituted with anothe~ amino acid ~g
~ollow~:
!i~ll!~ Substituen~
3 D-L~u
7 D-Ly~
8 D-Lys, D-~la
D-Ala, D-Lys
13 D-Trp, D-Leu, D-Ph~, D~Aia
D-Ph~
.,
.

16
lB D-Lys, D-Ala, DrPh;~, ~; . .
21 D-Lys, D-Ile - r!~
22 D-Ly~
23 D-Ly3, D-Ser
In a preferred embodiment, the peptide is a Magainin II
peptide, a~d the substitution analogue wherein amino acid 19 is
deleted is sel~cted from the claa~ con~istin~ o~ the following
~ubstitution analo~ues:
GIGKFLHSAXXFGRAFVGIMKS;
GIGXFLHSAKKFGRAF~AIMXS;
GXGKFLHSA~FFR~FVFIMNS;
GIGRFLKSARRFGKAFVFIMNS;
GIGXFLHKAKKFAKAFVFIMNS;
GIG~FLKSAKKFAKAF~FIMNS;
GIGKFLHRAKRFARAFVFI~
GIGKFLX~AKKFGKAFYFIMXK; and
GIGKFLHSAXXF9RAFVK**IMNS; wherein
K** i9~- F ~oc-ly~in~ w~ 0/~
Prellminary studiea indicato that the~o pr~erred analogues
possess low hemolytic actl~ity.
In accordance with another 2~bodiment, ther~ are provided
derivatives of Magainin I or Mag&lnln II (carbo~y- or :
a~ide-ter~inated), pr~erably Ma6ainin II, whQrein a portion of
th~ basic peptit~ i8 telated and at lea~t one of the remaining
amino acit re~iduo~ i~ subst~tutQt a~ h~reiQabo~e de~cribed; in
partisular, ~m~no acid re~idu~ 19 i8 omltted and in addition
: eithar a~ino 8cid ra9idues 1-4 or 3,5, and 6 sre omitted.
Pref~rred p~ptid~ ar~ as follow~:
FLHSAKR~GK~FYFIMNS
GIXHSAKKFAKAF~IMNS
FLRSAKKFGR~FVGIM~S
FLKSAgKF~XAFVGI~NS
,i
- :
",

C O ~ 13~ T ~` 1 2~
In accd~j,ance,~ith another aspec~.~of.the presant i~vention,
there i~ provided a compound comprislng ~ pepti'de deletion
analogue of an amide or carboxy terminated Magsinin I, wherein
Magainin I i9 represented by the following structural formula
using the sin~le letter amino acld code and the numbers below
each amino acid residue refer to the pO9~ tion of the residue in
the peptide:
G I G K F L H S A G K F G K A F V G E I M K S
1 2 3 4 5 6 7 8 9 10 11 1~ 13 14 15 16 17 L8 19 20 21 22 23
and wherein each amino acid re~idue 19 a D-smino acld residue or
a gly~ine resldue; and at lea~t one of sm~no acid residues 15
through 23 is omitted. I~ a pre~rrQd embodiment, at least one
of amino acid re~idues 1~, 16, 18, 19, 21, 22 and 23 i9 omitted.
In one embodim~n~, at lea~t a~ino acid residu~ 18 i~ omitted
whereas in another embodiment, at lea~t amino acit residue 19 is
omittad, and in yet anoth~r ambodimant ~t l~t ~ino acid
residue 2$ i~ o~itted. In preferr~d embodlments, only one of
amino acid residue3 18, 19, and 21 t re9p~ctively, i~ omitted. In
other preferred embodiments, amino ac~d re~it~es 21, 22, and 23
are omittad, amino acid residue~ 19 through 23 are omitted, amino
acid residues 18 through 23 are omittet, and amino acid residue~
17 throu~h 23 are omitted. The compound can be a deletion
analo6ue of amid~ - or carbo~y-t~rminated Magainin I.
In accordance wlth a~other asp~ct of the pre~ent invention,
there is provided a compound comprl~ln~ a peptide that i8 a
deletion analo~u~ o an smid~ - or carboxy-terminated Magainin
II, wher~i~ M~88in~n II is repr~nted by the followin~
structural fornula UBing the slngl~ letter amino acit code and
the nu~bsr~ b~low ~ach amino acid r~ldu~ s~ar to ~h~ posltion
o~ th~ rQsitu~ in th~ peptldQ:
G I G K F L H S A K K F G K A F V G ~ I M N S
1 2 3 4 5 6 7 ~ 9 10 11 12 13 14 lS 16 17 1~ 19 20 21 22 23,
wherein each amino scid residu~ is a D-s~ino acid residua or
glycine, ant at l~a~t on~ of amino ac~d~ 15 through 22 ls
l .

- ; C ~ ~ 3 ~ L ~ ~ ~ g
`_ . L ,, , ~,
omitted. In a pr'eerred,emb,odiment, at lea~t,,one of ami'no a~ids
15, 18, 19, 20, 21, and 22 i~ omitted. In one embodim~nt, at
lea~t amino acid 18 i9 omitted. In another embodiment, at least
amino acid 19 i9 omitted. Another embodiment omit~ at least
amino acid 21, and yet othe~ embodiment omits at least ami~o acid
22. In preferred ~bodimenta, only ono of amino acids 18, 19,
21, and 22, re~pectively, is omitted. The compound can be a
deletion analoguc o amide-terminated Magainin II.
In accordance with yet another aspect of the present
inven~ion, there i9 provided a baslc tpositi~ely charg~d)
polypeptide having at lea~t ~ixteen amino aclds wherein the
polypeptide includes at least eight hydrophobic amino acids and
at least eight hydrophilic amino acid~. Each of the amino acid
re~itues of the polypeptid~ i8 a D-amino acid resldue or a
glycin~ re~iduQ. Still more partlcularly, the hydroph~bi~ amino
acids are in groups of two ad~acent ~mino acids wherein the amino
acid~ are D-amino ac1ds or glycine, and each group of two
hydrophobic amino acids i~ spac~d from anoth~r group of two
hydrophobic amino aclds by at l~a~t on~ D-amlno acid other than a
hydrophobic amino acld (pr~ferably at least two D-amino acids)
and generally by no greater than four D-amino acids, and the
D-amino acids betwe~n pairs of hydrophobic amino acids may or may
not be hydrophilic.
Th~ hydrophilic ami~o acids ar~ generally al~o in group~ of
two ad~acent D-amino acids in which at lea~ one of the two
D-amino acid~ i~ a ba~ic hydrophiliG a~ino scid, with such groups
of ~wo ~yd~ophilic D-amino scld~ bein8 ~pac~d from each other by
at least on~ ~ino acid, other than a hydrophillc D-amino acid,
wherein 2~ch o~ ~ald at lea~t on~ amino acld(s) is a D-amino acid
or glycin~ ~pr~f~rably at lea~t two smino acid~3 and g n rally no
greater than four amino acid~, and tho ~mino acid~ b~tween pairs
of hytrophili~ D-a~ino acids ~ay or may not ba hydrophobic.
~ .
,. ~ .
~: .
.

i ~ D
In accordanc~ with a particutarl~ pref~rred"embodiment,~the
polypeptide compri~e~ a chain of st les~t ~our group~ of'amino
acidq wher~in each amino acid i9 a D-amino acid or glycin~, with
each group consisting of four amino acids wherein each ~f the at
least four amino acids i9 a D-amino acld or glycine. Two of the
four amino acids in each group are hydrophobic amino acids, and
two of the four amino acids in esch gsoup are hydrophilic, with
at lea~t one of the hydrophilic amino acids in each group being a
basic hydrophilic amino acid and the other being a basic or
neutral hydrophilic amino acld.
The hydrophobic amino acid~ may be seleoted from the class
consisting of D-Ala, D7Cys, D-Phe, D-Ile, D-Leu, D-Met, D~Ysl,
D-Trp, D-Tyr, snd Gly. The neutrQl hydrophilic D-smlno acid3 may
be selected from th~ clas~ consisting of D-Ser, D-Asn, D-Gln, and
D-Thr. Th~ basic hydrophilic D-amino aoids may be ~elected from
the class consisting of D-Ly3, D-Ar~, and D-Hi~, snd D-ornithine.
Eaoh of the group~ of our amino scids may be of the
sequenoe ABCD, BCDA, CDAB, or DA~C~ wher~in ~ snd B ara each
hydrophobic amino acids and may be the sam~ or different, one of
C or D is a ba~io hydrophilic amino acid, ant the oth~r of C or D
i~ a bssic or neutral hydrophilic amino acid ant may be the ssme
or different. In a preferred embodlm~nt, the polypeptide chain
may comprise 5 or 6 groups of this sQquenoe. In each group, each
o~ A, ~, C and D ~ay be tha sa~ in ~om~ or all of the groups or
may be di~fsrent in 30m~ or all of th~ groups.
Tho polypeptid~ chain preferably ha~ a~ lca~t 20 amino
acids, and no ~roatQr than ~0 amino ~cids wh~rein each amino acid
is a j-a~o acid or glycine. Xt is to be unders~ood, however,
that thQ polyp~p~id~ does not hav~ to con~l~t entirely of the
groups describet above. The polyp~p~lde may have amino acids
extending from either or bo~h ends o~ thæ noted groups forming
the polypeptido chain and/or thQr~ may b~ amino ~cits b2tw~en one
or more of ~h~ at lea~ four group~ and ~tlll remain w~thin the
, ~

~cope of th~ invention, provided that.each amino.s,c~d;-residue of
the polypeptide chain-is a D'am~o acld residue or glycine.
The groups of amino acid~ may be repeatln~ groups of amino
acids, or the amino acids in the various groups may vary pro~ided
that in each gro~p of the at least four groups of amino acids
there are two hydrophobic amino acids wherein each amino acid i9
a D-amino acid or glycine, and two hydrophilic D-amino acid~ as
hereinabove noted.
Th~s, in 8 preferrad embodiment, th~ biolo~ically active
polypeptide comprises a chai~ includlng at lea~t four groups of
smino acids, each containing four amlno acids wherein each amino
acid is a D-amino acid or glycina. Two of tha fous amino acids
in each group are hydrophobic, wh~reln ~ach hydrophobic amino
acid i~ a D-smino acid or glyclne, at lea~t ono ami~o acid i9 a
basic hydrophilic D-amino acld, and tha remslning one is a basic
or neutral hydrophilic D-smlno acid, wlth the polyp~p~lde chain
preferably having at least 20 s~ino aclds but no greater.than ~0
amino acids.
In one embodlment, each of the ~t least four group~ of
amino acit~ which ar~ in the p~ptido chain i~ of the sequence
A-B-C-D, B-C-D-A, C-D-~-B or D-A-~-C wher~in A and ~ are
hydrophobic, on~ o~ C or D i~ basic hydrophilic, and the other of
C or D is ba~ic or neu~ral hydrophilic. The re~ulting
polypeptite chain, th~refore, may hav~ on~ of th2 following
seque~ce~:
(A-B-C-D)n(Y~)b ~ /30/~
/q~ (~21~~C~D~A)n(Y2)b
(1~3)a(C~D~A~~)n(Y3)b
(a~4)a(D-A-~-C)r,(Y~)b
wherein ~1 18 D; C-D- or 8-C-D-, Y~ or -A-~ or -A-B-C
X is A-, D-A- or C-D-A-
Y2 ~8 -B, -B-C or B-C-D

~3i~ B~, A-B- D~A-B~
Y3 is -C, -C-D, -C-D-A
~4i9 ~-, B-C-, A-B-C_
Y4 i3 -D, -D-A, -D-A-B
a iq o or l; b is o or 1
and n is at lea~t 4.
It i9 to be under~tood thAt th2 p~ptlde chaln may include
D-amino acids or glycine between the hereinabove noted groups of
four amino acids pro~ided thst the spacing between such ~roups
and the ch~rge on the D-amino acidx or glycine does not change
the characteristlc~ of the peptide chaln which provide
amphiphilicity and a positive charge and do not adversely affect
the foldin~ characteri3tics of the chain to that which i~
significantly different ~rom onc in whlch th~ hereinabove noted
~roup~ o~ four amino acid9 are ~ot ~paced ~rom çach other.
As reprosentative ~amplc~, th~re may bo mo~tloned.
I D-Ala-D-Phe-D-Sar-~-Ly~-D-Ala-D-Phe-D-Ser-D-Lys-D-Ala-D-Phe-D-Ser-
D-Lys-D-Ala-D-Phe-D-Ser-D-Ly~-D-Ala-D-Ph~-D-Ser-D-I.ys
II D-Ala-D-Phe-D-Ser-D-Ly~-D-Ala-D-Phe-D-Ser-D-Ly~-D-Ala-D-Phe-D-Ser-
D-Lys-D-Ala-D-Ph~-D-Ser-D-Ly~-D-Ala-D-Phss-D-S~r-D-Lys-D-Ala-D-Phe-
D-Ser-D-Ly~.
III D-Phe-D-Ser-D-Ly~-D-Ala-D-Ph~-D-Ser-
D-Lys-D-Ala-D-PhQ-D-Ser-D-Ly8-D-Ala-
D-Phe-D-Ser-D-Ly~-D-~la-
IV D-S~s-D-Ly~-D-Ala-D-Phe-D-S~r-D-Lys-D-Ala-
D-Pho-D-~r-D-Ly~-D-Ala-D-Ph~-D-Sar-D-Ly~-D-Ala-
D-Ph~-D-Ser D-Lys-D-Ala D-Phe-
V D-L~--Ala-D-Phe-D-Ser-D-Ly~-D-Al~-D-Phe-D-Ser-D-Ly~
D-~18~D-Ph~-D-S~r-D-Ly~-D-Al~-D-PhQ-D-Sor
The peptid~ may hav~ amino acit~ e~tandin~ from either end
of the chain~ For example, the chaln~ ~ay h~ a D-Ser-D-Ly~
sequenc~ ba~ore the "D-Ala" end, ant/or a D-~la-D-Phe ~equence
after th2 "D-Ly~" snt. Other amino acid ~equences may al~o be
attachod to the "D-Ala" and/or ~h~ "D-L~" end.
-
-
.

CO~ 3 ~ ~2t~ j
Similar~y, in.any poly~eptid~..chaln of.the.pr~se~t ~~
invention having atnlea~foù~rrgroups-o~ amino acid~;;of.ithe
sequence as described above, the chain may hsve, for exsmple ~ a
C-D sequence before the first A-B-C-D group. Also other acid
sequences including D-amino acid~ or glycine may be attached to
the "A" and/or the "D" end of one of these polypeptide chains.
Also there may be D-amino acids or glycine residues in thP chain
which sp~ce one or more groups of the hereinabove noted four
amino acids from esch other.
Such polypeptides ~re generally water-soluble to a
concentration of at lea~t 20 mg/ml at n~utral pH in water. Such
polypeptides are n~n-hemolytic, l.e., it will not rupture red
blood cell~ at effective antimlcrobial conc~ntrations. The
~tructure of quch polypeptide~ provides for flexibility of the
polypeptide molecule. When tha polypeptide is placed in water,
it does not as6um~ sn amphiphilic structur~. When th~
polypeptide enco~nt~r~ an oily surfacQ or membrane, the
polypeptid~ chain fold~ upon it~elf lnto a rod llke structure.
In accordanc~ wlth a furth~r 8~pact of tha present
invention, there ls provited a peptide (polypeptidc) having from
eight to fifteen amino aclds comprised of at least four
hydrophobic amlno acids ant four hyd~ophilic amino scids. Each
amino acid residu~ is a D-am~no ac~t re~idue or glycine. The
hydrophobic amino acids are in groupY of two ad~acent hydrophobic
D-amino acids or glycine wherein each group o~ two hydrophobic
D-amino acids or ~17cinQ is spaced from each other by at least
one D-amino acid other than a hydrophobio D-amino acid or glycine
(preferably ~t l~a~t tw~ D-amino acids) and generally no greater
than four D-amino acids, ant th~ D-amino acid(s) b~tween pairs of
hydrophobic a~ino scgts wh~r~in e~ch am~no acid ia a D-amino acid
or glycine9 may or may not bQ hydrophilic. The hytrophilic
D-amino acid~ ara gen~rally alao ~ groupa of two ad3acent
D-ami~o acid~ in which at lea3t one of th~ two n-amino acids i~ a
basic hydrophilic D-amino acid snd th~ oth~r of th~ two i~ ba~ic
.

or neutral. Tfi~group3 of two .D-hydrophilic ~min~,açid~ sre
~paced fro~:~ach othjer by.-at l.ë~ o~e'~ mino!.acid'.ot.~er than a
hydrophilic D-amino acid, and wh~rein the at least one amino acid
i9 a D-ami~o acid or glycin~ (preferably at le~st two amino
acids) and ~enerally no greater than 4 ~mino acids, and the amino
acids between pair~ o~ hydrophilic amino acid~ may or may not be
` hydrophoblc.
The peptide havin~ ~rom 8 to 15 amino acids is amphiphilic
snd i~ po~ltively charged (basic).
The 8 to 15 amino scid peptldo hereinabove described may or
may not be bioactive, and in the ca~e where such peptide is not
bioactive, it hss utility a~ an lnterm~diate in provid$n~ the
hereinsbove noted peptide~ which h~va at lea~t 16 amino acids and
which are bioactive. For exampl~, two pQptid~s, one having eight
D-amino acid3 and the other ha~ing twel~e D-amino acid~ may b~
coupled to each other to form a peptido havin~ 20 D-amino acids
of the type hereinabove described and whtch i8 bioactive. The
peptides may be coupled by standard peptite chemi~try technique~.
Thu3, for example, such peptides may b~ conden~et in solution by
the technique disclosed by Johnson, ~t al. PePt~te~, pages 239-42
(Walter de Gruzter & Co., 19~6).
A3 repre~entative e~amples of such peptides, there may be
mentionet peptid~Y rapresent~t by th~ following structure wherein
A, ~, C and D are as defined prQ~iously;
(i) (Wl)a(A-E~~C~D)n(Zl)b
(ii) (w2)a(B-c-D-~)n(~2)b
(~li) (w3)~a(c-~-A^B)n(z3)b
(IV) (w4)a(D-A-~-c3n(z4)b
wherein Wl i~ D-, C-D-~ B-C-D-
W ~ A-, D-~-, C-D-~-
W is B- A-B-, D-A-B-
W4 is G-, B-C-, A-B-C-
Z i8 -A, -~-B, -~-B-C
:, ~
,~ ' , .

2~7~
Z2 i~.-Bj :B-~C, -9-C-D-. "~
Z3 lS~ D~ D-A~
Z4 is -D, -D-A, -D-A-B
n ig 2 or 3, a i9 o or 1 snd b i3 o or 1.
In accordance with yet another sspect of the present
invention, there is pro~ided a peptide which include~ the
following ba~ic peptide 9tructure ~:
-Rl-Rl-R2-R2-Rl-Rl-R3 ~1
-Rl-Rl-R3-Rl-Rl-R4 R~ ~1
whereln Rl is a hydrophobic amlno acid;
R2 i~ a hydrophobic amino acid or a b~sic hydrophilic amino
acid;
R3 is ~ basic hydrophilic ~mlno acid;
R4 i8 a hydrophobic or n~utral hydrophilic amino acid;
R5 is a ba~ic or neutral hytrophillc amino acld, and each
amino acid residu~ of said peptito i~ a D-amino acid ra~idue or
glycine.
The her~inabov~ basic st~cturo i~ har~nafter ~ymboIically
lndic~ted a8 ~. Paptide~ formed in 6ccortanc~ with this ba3ic
~tructure ar~ com~only re~erred to a~ CPF peptide3.
The CPF peptides of the preaant invention may includ~ only
the hereinabov~ noted D-amino acids or glycine or may include
additional D-amino ac~ds or ~lycino r~ldu~a at th~ amino and/or
carbo~yl end or both th~ amino and carbo~yl end. ~n general, the
peptide does not include more than 40 amino acid~, wherein each
amino acid 1~ a D-a~ino acid or 61ycine.
Th~ CP~ poptid~s incl~ding th~ abov~ ba8ic 9tructur~
preferabl~ h~ rom 1 to 4 atditional amino acid~ at the smino
end, wh~Ql~ ~ch amino acid is a D-ami~o acit or glyclne.
Accordingly, ~uch pr~f~rrQd paptida~ may b~ r~pse~ented by
the structural ~os~ula:
y
whereln ~ i8 thc hereinabov~ d~crlbet ba~lc peptide
3tructure ant Y 18
.
.

O ~ d~ 2 ,~
(ii) '~. ( R2-R5~.;,or-~
(iii) Ri-R2-R5; or
(iv) R2-Rl-R2-R5i pre~erably
Glycine - Rl-~2-R5.
wherein Rl, R2 and R5 are a~ previously dafined.
The carbo~yl end of the baslc peptida struoture may also
have additional amino acits which m~y range from 1 to 13
additional amino acids.
In a preferr2d embodiment, tho basic structure msy have
from 1 to 7 additionsl smino scid~ at the carboxyl end, which may
be represented as follow~:
-X - Z whereln
is the h~reinabove dQfined ba~lc peptide structure and-Z
i3
(i) Rl-, or
(ii) ~1 Rl ; or
(iii) ~l-Rl-R4; or
(iv) Rl Rl R4 R4; r
(v) R~ -R4 R4-R~; or
(vi) R -Rl-R4-R~-R6-D-Gl~; or
(vii) Rl-Rl-R4-R4-R6-D-~ln-D-Gln, wherein Rl and R4
are a~ previou~ly tefined, and R6 1~ D-prolin~ or a hydrophobic
amino acid.
Pre~errad peptida3 may b~ repre~ont~d by the following
structural ~o~ula
(Y)~ - X ~ (~)b
whæse~n ~, Y ant Z are a~ prQ~io~sly d~fined snd a is O or
1 and b 18 0 or 1.
A3 repre~ntativa e~ample~ of CPF peptld~ used ln the
present inve~tion, th2re may b~ ~ntioned peptide~ represented by
the followlng (~in~le l~tter anino ~clt code):
Gl2S3LG4ALKASLKIG678LCG9(10~QQ
.
, . " , ~ ., ~
. - ' ~
I

- C (~) N F ~ iT ~ A~ f5 ~ ~
bhere ~ s~ - ,
2 - G, A
3 ~ F, L
4 ~ K, L
5 = A, G, T
6 = A, T
7 ~ H, N
8 = A, M, F, L
9 = A, S, T
10 = P, L
Preferred CPF peptide~ are of th~ following ~equences:
(1) GFGSFLGLALKAALKIG~NALG~APQQ
(2)~ GLASFLGKALRAGLKIGAHLLGGAPQQ
(3) GLASLLGX~L~AGLKIGTHFLGGAPQQ
(4) GLAsLLGKALKATL~lGTHFLGGApQQ
: (5) GFAS~LGXAlX~ALXIGANMLGBTPQQ
: (6) ~FGSFLGK~LKAALKIGANALÇGAPQQ
: (7~ ~FGSFLGRALKA~LKIGANALGGSPQQ
:: (8) GFASFLG ~ LKIGANLLGGTPQQ
In accordanc~ with an aspect of th~ pre~nt invention,
there is provided a compo3ition compri~ing at les~t on~
biologically activc poptido whlch incIudes at l~a~t a chain`of
th~ followin~ 20 a~ino acid r~3idu~ h~vin~ the following peptide
structure ~10 wh~r2 ~lO i9
11 12 11 ll R13 R13 Rll Rll R12^~ R15 ~l4^Rll^R -R
: Rll R16 ~15 R17, wh~rein
~ : ~R~ a ~ydrophobic amino acid;
; Rlæ i~ :a ba~ic hydrophilic~amino ~cid; ~
:R13 l~ a~hydrophobic:~or:ba~lc~:hydrophilic amino acid; ~ :
Rl4 i8 a n~tural hydrophillc~a~ino~;-c~d,~or a ba~ic :
hyd~ophilic amlno acid;
R15 ls a neutral hydrophll~c a~ino acit; Rl~ is ~^proline
or a hydrophobic a~o acit; and:R17 1~ a n~tural hydrophilic,
.: : :
:: : ~ '
;:
: ~ :
.
- ' : ' ' : : . . ~
.
,
.

~ C O ~ ,. '30 , . ~ J~8
ba3ic hydrophilic or hydrophobic am~no acid. Each;of~th~ ~mino
acid re~idu~a of the peptide i9 a D-amlno acid residue or
glycine. Th~ at l~a~t one biologically active peptide which
include~ peptide residus X10 has at la~st the hereinabove
described twenty amino acid peptide and generally no greater than
35 amino acids, preferably no grc~ter than 30 ~mino acid~. Such
peptides are derived from fra8ments of the human hormone
cholecystokinin, or may be derivsti~e~ o such fragments.
In one embodiment, the at lea~t one blologically active
peptide i9 of the fo~mula Rll-Rll-X-,
wherein Rll i9 a hydrophobic amino Rcid as de~cribed abo~e. Mo~t
preferably, the biolo~ically activ~ peptide of thls emhodiment
include~ a chsin of at least th~ following amino acids:
D-Leucine-D-Leucine-X-.
In another embodiment, th~ at l~a9t on2 biologicslly active
petpide includes a chain of at leaat the fcllowing amino acids:
12 11 15 Rll Rll , wher~ln Rll ' R12 ~ ~nd R15 are
amino acids of the type3 hereinabove descrlb2d.
The peptidQ may be amide-t~rminated or carbo~y-terminated.
In ~ccordance with still anoth~r aspQct of the pre~ant
invention, there ~ 8 provid~d a biolo~lc~lly ~ctiv~ ~mphiphilic
peptide which include3 the following ba~lc structure X12:
~ R21 R22 R22 R~3~R21~R22~R22~n- whoreln R21 i~ a basic
hydrophilic amino acid, R22 ia a hydrophobic amino acid, R23 is a
neutral hydrophilic or hytrophobic amlno acid, n i3 from 2 to 5,
and esch am~no acid re~iduQ ~f the pep~it~ i~ a D-amino acid
residu~ o~ ~lycin~.
In ~ccostanco with one e~bodl~nt 3 the peptide may include
th~ followln~ st~ucture:
Y12-~12, wh~r~in X~2 i9 a3 h~in~boY~ de~cribed, and Y i9:
(i) R22;
(ii) ~22-R22;
(ii~) R21-R22 R22;
(iv) R23-R2l-R22 ~22;
,
.

CO~ 3 ~A~ 2~
tv) ~22-R23-R21-R22 R22;
t ) R22 R22 R23 R21 R22~R22~ whPrein R21, R22, and R23
are a~ h~reinabove de~cribed
In accordance with another ~mbodiment, the peptide may
include the following ~tructure:
X12-Zl2, wharein X12 is a~ herelnabove de~cribed, and Z12
i9 :
(i) ~21i
(ii~ R21-R22;
(iii) R21-~22 R22;
(iv) Rzl-R22 R22 R23;
(~r) R21-R22-R22-R23 X21;
(vi) RZl-R22-R22-R23 R21 22
I~ accordance wlth y~t anothor ombodi~en~, the peptid~ may
include thc followi~g ~tructure:
(Yl2)a ~12 (Z12)b, wherein Y and Z ~r~ a~ previously
defined, a is 0 or l, and b i8 0 or 1.
lr~ a preferred embodiment, n i~ 3, and mo~t preferably the
peptide i~ of th2 following structur~ as indicated by the single
letter amino acid code:
~KIAGKIA]3~
In another embodiment, n i~ 2, and th~ peptide preferably
i9 of the following ~tructur~ a8 indlca~et by th~ single letter
amino acid cota:
KIA(K~AG~IA)2~IAG.
In accordanc~ *ith another a~pcct o~ the pre~ent i~v~ntion,
ther~ 18 pro~id~d a blologic811y ~ctlv~ amphlphlllc p~p~ide which
includ~s th~ ~ollowi~g basic structur~ ~14:
R21 R22 R22 R23 ~21 R22 ~22 R21 R22-~22-R22-R21-R22-X22'
wh~rein R21, R22 and R23 are a~ h~rslnabov~ de~crlbed, and
esch ~mino acid reoiduo o~ th~ p~ptid~ io a D-amino aclt r~3idue
or gl~cin2.
In accordance with one ~mbott~nt 7 thQ peptld~ msy include
the ~ollowin~ 9tructure:

C ~ L~
Y~ w~reln ~l4 i~ as-herelnabove describêd, and Y14
i9 :
(i) R22;
(ii) R22-R22;
(iii) R21 R22 R22;
(~v) R23-~21-R22-R22;
(v) R22 R23 R21 ~22 R22;
(~i) R22 R22-R23-R2l-R22-R22l or
(vii) R21 R22 R22-R23-R2l-R22-R22~wherein R21, R22
and R23 are as hereinabov~ described.
In accordance with anoth~r embodiment, th~ peptide may
include tha following structure:
X~ 4, wborein ~14 i~ a~ h~r~ns~ove d~scribed And Z14
is:
(i) R21;
(ii) R21-R22;
(iii) R21 R22 ~22;
(lv~ R2l-R22-R22-R23;
~v) R2.1-R22-R22-R23 a21;
~vi) R21 R22-R22-R23-R2l-R22; or
(vii) R21 R22 ~22-R23-R~}-R22-R22, wh~r~in R21, R22
and R23 are as her~inabove de~crib~d.
In acco~tanca with yet anoth~r ~mbodiment th~ pept~de may
include th~ following structuro:
(Y14)a-~14-(Z14)b, wh~rein ~ and Y ar~ a~ pre~iou~ly
definet, 8 i~ O or 1, and b i8 0 or 1. In a preferred
emboti~nt, th~ p~ptld~ is o~ th~ followin~ structural ~ormula a~
indicat~d by th~ Sin~lQ letter amino aoid cod~:
KLAS~AG~I~GKI~XV ~ .
In anoth~r prQf~rr~d ~mbodi~nt, tho p~ptid~ i5 of the
following ~tructural formula 8S indlcat~d ~y th~ ~lngle l~ttes
amino acid cod~:
KIAGKIA~IAGOI~RIA~KIA.
, , . , ., . :, .. .. .
,. ::
.

C (~ r ~ NT ~ V { ~ i2 ~ ~
In accordance with a further emb.odiment,.the peptide may
include the followlng basic peptide structure X16:
-R31-R33-R32-R31-R34-R34-R31-
R3l R34 R32 R31 R31 R32 R31
R3l R31 R32
wherein R31 i9 a hydrophobic amino acid, R32 is a basic
hydrophilic smino acid, R33 i~ a neutral hydrophilic, b~sic
hydrophilic, or hydrophobic amino acld, and R34 is a hydrophobic
or basic hydrophilic ami~o scid, and ~ach amino acid re~itue i~ a
D-amino acid resitu~ or glycine. Pr~ferably9 R33 i9 a neutral
hydrophilic amino acid.
Such peptid~s ~re commonly reforred to as PGLa poptite~.
The PGLa peptides generally inclu~e at least seventeen
amino acids and may include a~ ~any ~8 forty amino acids.
Accortingly, the hQreinabov~ describ2d ba~ic peptide structure
for a PGLa peptide msy includ~ additionsl amino acids at the
amino end or at th~ carboxyl ~nd or ~t both thQ amino and
carboxyl end.
Thus, for example, 8 PGLa paptido may havQ thQ following
structure:
- Y16 - X16
wh~ra X16 is a pre~ioualy d~fined; and Y16 i8:
(i) R31; or
(ii) R34-R31
wher~ R31 and R34 are as previously defined.
For Q~ampl~j a IGLa peptidQ may ~lso hav~ the following
structuro:
~16 Z16
wh~r~ ~16 ia a pr~viou~ly d~ined; ant Z16 is:
Y16 i9
(i) R31; or
(ii) Et'31-R31
where R31 is as previou31y de~ined.
A PGLa peptid~ may also have the following struotre:
,

C O ~ 2 ~ 8
(Y16)a 1-~ ~ 16)b
wh~r~ ~16~ Y16, and Z16 ar~ as previously defined, a i~ O
or 1 and b is O or 1.
In accordance with another embodiment, the peptide may
include the following basic peptide struct~re X18:
R31 R33 R32 R31 R34 R35 ~33
R31-R34-R32-R31 R31 R32
R31 R31 R31 R32-(R36)~-R31--' wherein R31, R32, R33, and
R34 are as previou~ly deflned, and R35 i~ glutamlne or a~paragine
or a basic hydrophilic, or hydrophobic amino acid, R36 i3
glutamic acid, aspartic acid, a hydrophobic amino acid or a basic
hydrophilic amind acid, and n is 0 or 1. Each amino acid residue
is a D-amino acid re~idue or glycin~
Such peptides are commonly referred to a~ XPF peptides.
The XPF peptide~ generally includ~ at l~a~t nlneteen amino
acids and may include up to forty amino a~id~. Accordingly, the
hereinabove described basic peptite 3tructure oP XPF may include
additional amino acids at the aminn and carboxyl end~.
Thus, for example, an XPF peptlte may includa the following
~tructure:
-Y18 -X18 -
where ~18 is a~ prevlouQly dePinet ~nd YlB i3
(i) R31 or
(ii) R34-~31
where R31 ant R34 are ~g prevlou~ly defined.
An gP~ pop~ido may include the followln~ structure:
-X18-Z18-
whQr~ ~18 i8 a~ pr~io~sly d~1n~d nd Z18 i9
(i) R31; or
(~i) R31-R35; or
(iii) R31-R35-D-Prolin~; or
(iv) R31-R3,~-D-Pr~ e R32
An ~PF peptide ~ay al#o hsve the following 3tructure;
~Yl8~8 ~18(Z18)b
.j

wh~r~ , Y18land Z18 ar~ a~ pre~;iously defin~dj a is 0 or
1, and b is 0 or 1.
Preferred are ~PF or PGLa peptides, which sre characterized
by the following primary amino acid sequence (single letter amino
acid code):
PGLa: GMASKAaAIAG~IARVALKAL (NH2)
XPF: ~SKIGQT~KIARVGLKELIQP~
A review of XPF and PGLa can be ~ound in Hoffmann et al.,
EMBO J. 2:711-714, 1983; Andreu et al., J.~iochem, 149:531-535,
1985; Gibson et al. J. ~iol Chem. 261:5341-5349, 1986; and
Giovannini et al, ~iochem J. 243:113-1~0, 1987.
Applicant~ unexpectedly have found that the above-mentioned
peptid~, all of which consist entirely of D-a~ino acids or
glycine resldue~, have biological activity.
The abo~e-mention~d peptide~ alao hsve increa~ed resi~tance
to proteolytic enzymes while retsininB their biological activity.
In general, the peptide~ hereinabo~e described, and/or
analogues or derivatives thereo~ are generally water soluble to a
concentration of at least 20 m3/ml ~t neutral pH in water. In
addition, such pcptides are non-hemolytlc; l.e., they will not
ruptur~ blond cell~ at effactlvo c~ncontrations. The peptide~
cau~e less than 5X hemolysis of human erythrocytes at a
concentration of 500 ~glml. In ~ddition, the structure of ~uch
peptlde provides for fle~ibilitr of eh~ p~ptida molecule. When
the peptide i3 placet in water, it does not s~sume an amphiphilic
structure. When tho p~ptid~ encount~r~ an oily ~urface or
membran~, thQ peptid~ chain ~old~ upon its~lf lnto a rod-like
s'sructur~ .
Th~ peptldes m~y b~ C- tern~ln~l aclds or a~id~s .
The pept~d~s may be admini~tered to a ho~t; ~or example a
human or non-hu~an animal, in an a~ount ffectiv~ to inhibit
growth o~ a tar~t c~ll or ~irus. Thus, for e~mple, the
peptides and/or analo~ues or derivativ~ theroo~ may b~ used as
antimicrobial agen~s, anti-viral agent~; antibiotic~, anti-tumor
:
:~
. ' :

,C~ ..2~F,N"T,l,~ ,J~t~
agPnts, sntlpar~iti;c agent,s, spermicide~.,.as-well a~ ~xhibiting
other bioacti~e functions.
The term "antimicrobial" 88 used herein means that the
peptides of the pre~ent invention lnhlblt, prevent, or destroy
the growth or proliferation of microbes such a3 bacteria, fungi,
or ~he like.
The term "~ntlbiutic" as used h~rein means that the
peptides employed in the present inv~ntion produce effects
adverse to the normal blological functlons of ~he cell, tissue,
or organism includlng teath or d~structlon snd prevention of the
growth or proliferation of the biological ~ystem when contacted
with the peptide3.
The term "spermicidsl" 8~ u~ed herein means that the
peptides employed ln the present in~ntlon, lnhibit, prevent, or
d~stroy tha mot~ y of ~perm.
The term "antiYiral" as u~ed herein means that the peptides
employed in the present inv~ntion inhibit, prevent, or destroy
the growth or prollferatlon of virusa~.
The term anti-tumor a~ u~d hor~in means that the peptide
inhibit~ the growth of or de~troy~ tumor~.
The term "antlparasit~c" a~ uset herein means that ~he
peptides of the pr~s~nt ln~ntlon may be usQd to inhibit the
growth of or destroy para~ita~.
Th~ pap~itos of ~ho pr~sQn~ inv~ntion hava a broad ran8e of
potent antibiotic ac~ivity a6ainst 8 plurality of microorganisms
includin~ Gr~m-posltivo and Gram-ne~atlve baoteria, fungi,
protozoa, ~ h~ lik~, as well a~ parsslt~3. The pep~id2s of
the pras~nt lnvontlon allow a m~thod for treatin8 or controlling
microblal in~ction c~usQd by or~ani~m~ whlch aro ~n~itiv~ to
the peptid~s. Such t~atm~n~ ~ay comprlse sdmlnistering to a
host organism or ~lasu~ suaceptlbl~ to or af~lliat2d with a
microbial lnfection an antimicrobial amount of at l~a~t one of
th~ peptites.
!~ . . .

CO~2ir~ )E~`' ~ ' `'~ I
3 L~ 2 ~ ~ ~
B~cau~ of the intiblotlc propertles of th~ peptid~s, they
may also be u~d as preservative~ or sterilant~ of materials
9US ceptible to microbial contamination.
The peptide may be administered in combination with a
non-to~ic pharmaceutical carrier or vehlcle ~uch as a filler,
non-to~ic buffer, or physiologicsl saline solution. Such
pharmaceutical composition~ may be used topically or systemically
and may be in any suitable form such a8 8 liquid, solid,
semi-solid, in~ectable solu~ion, tablet, ointment, lotion, paste,
capsule, or the like. The peptide compositions msy also be u~ed
in combination with ad~uvants, protea~e i~hibitor~, or compatible
drugs where such a combination is aeen to be desirable or
advsnta~ous in co~trollin~ inaction cau~d by harmful
microor~anisms including protozoa vlru~e~, and ~he like, ~ well
as by parasite~.
The peptides of th~ pre~ent invention may be administered
to a host; in particular an animal, in an effec~ive antibiotic
and/or anti-tumor and/or anti-viral ant/or anti-microbisl and/or
anti-para81tic and/or an anti~per~icidal amoun~.
Depentin~ on th~ u8e, A compo~iSlon ln accord~nce with the
invention will contaln sn Qf~ectiva anti-microbial amount andlor
an effective antisp~rmicidal amount and/or an effec~lve
anti-viral amount and/or an effective anti-tumor amount and/or an
effective antlbiotlc amount snd/or anti-parasitic a~ount of one
or mora o~ the herelnabove described paptide~ which hsve.such
activity.
Th~ pQptidQ of the present invention may al90 be employed
in promotin3 or ~timulating healing of a wound in a host.
The ter~ "wound h~aling" a~ us~d herein lncludes various
aspect~ of th~ would healin~ process.
These a~p2ct9 include 9 but are not llmited toS increa~ed
contraction of thQ wound, increa~ed d~poaitlon of connactlve
ti~3ue, a~ evitenced by, for e~s~ple, lncreas~d depo~ition of
collagen in the wound, 8nd incr~ d t~n~lle s~rength of th~

C~ 8~NT'i,','
wound, i.Q. ,~:the peptides increase wou~drbrea~ing strength. The
peptides of the present in~ention may alao be employed ~o a~ to
rever~e th~ inhibition of wound healin~ cau~ad by steroids such
a~ cortisone.
The peptides of the presant invention may bo used in the
treatment of externsl burn~ and to treat snd/or prevent skin and
burn infections. In particular, tho peptlde~ may be used to
treat skin and burn infection9 cau~ed by or~anisms such as, but
not limited to, P. aeruRinosa and S. aureus.
The p~ptides are also u~eful in the prevention or treatment
of eye infections. Such inf~ction~ may be caused by bacteria
such as, but not limited to, P. aeru~lnosa, S. aureu~, and N.
onorrhoeae 9 by fungi such a~ but not limited to C. albicans and
A. fumi~atus, by parasit~ ch aa but not liml~d to A
ca~t~llani, or by ~iru~e9.
The peptidQs may al~o b~ eff~ctlve in killing cy9t8,
~pores, or trophozoites o infection - c~using or~anism~. Such
organi~ms include, but are not limitcd to Acanth~moeba which
forms trophozoit~ or cyst~, C. albicsn~, which form~ spore~, and
A. fumiRatus, which forms spore~ Q~ w~
In general, the peptid~ ia employed to provide peptide
dos~es of from 0.1 mg. to 5G0 m~. per kilogram of host weight.
When admini3tered topically, tha pQptide i~ u~ed in a
concentration of ~ro~ .05% to 10%.
The peptide~ may be protuced by known techniques and
obtainod in subatantlally pure form. For e~smple, the peptldes
may bo syntho~iz~d on an auto~atic synthe~iz~r. Journal of the
American ~h~lcal SocietY, Vol. 85, pg~. 2149-54 (1963). It i~
al~o pos~lbl~ to produce such pQptid~ by g-netic en8~neering
techniques.
The peptidcs of the pr~s~nt in~ntion ~ay bo at~inistercd
alona, or ln combination with oth~r ~uch paptide8. The peptites
may al~o b~ ~d~iniatered in comblnstion wi~h biologically active
peptidea or prot~ns con~aining L-amino acid re~iduo~, huch as
,.
:
~, :

C C~ ~ t ' , ~ 2 ~ ~ g
ion channel-fo ~ ng protein~, or the p2ptides of:the presenc
invention may be admini~tered in combinat~on with other active
components, such as to~ic ions or antiblotics.
BiologicAlly active peptid~ containlng L-amino acid
re~due~ which may be administer~d ln combination with the
peptide~ o~ the preaent invention lnclude magainln peptides, PGLa
peptides, XPF peptide~, cecropin~, and sarcotoxins. Magainin
peptide~ ar~ described in Proc. N~tl. Ac~d. Sci., Vol. 84, pg9
544~-53 (AU8~ 1987). A review o~ ~PF ~nd PGLa peptides may be
found in Hoffman, et al., EM~OJ., 2:711-714 (1983). Andreu, et
al., J. Biochem, 149:531-S35 ~198S); Gib~on, et al., Biochem J.,
243:113-120 (1987).
The cecropins and analogue~ and derivatives thereoP sre
described in Ann. R~. Microbiol., Vol. 41, p~, 103-26 (1987),
and Christensen, et al., PNAS, Vol. 85, pga. 5072-5076. ~1988).
Sarcoto~ins and anAlogue~ and tarivati~es thereof are tescribed
in Molecular Entomotogy, pg5 369-7B, Alan R. Li~s, Inc. (1987).
Ion chann~l-fonmin~ protein~ or pQptidQs which may be
employed in combination with ~he p~ptld~s of the present
inYention includ~ dQfe~sin~, al80 known A~ human n~utrophil
antimicrobial peptites (HNP), ma~or ba~ic protein (MBP) ~f
eosinophil~, bactaricidal pormaability-increa~$n~ protein (BPI),
ant a pore-forming cytoto~in cAllod varlously parforin,
cytolysin, or pore-formin~ protoin. Do~n~ins are de~c~ibed in
Selstet, et al., J._Clin. Inv~st., Vol. 76, pgs. 1436-1439
(198S). M9P protQins are d~cribed ln Was~oQn, et al., J. Biol.
Che~., Vol. 263, p~. 12559-12563 (1988). BPI protQin~ are
describ~d in Ooi, ~t al., J. Biol. Chem. Vol. 262, pg8.
14891-14894 (198~). Perforin is d~cribsd in Henkart, et al., J.
Exp. Med., 160:75 ~1984), and ln Podack3 ~t al., J. EXP. Med.,
160:695 (1984). Th~ abo~e articl~ ara h~reby ~ncosporated by
reference.
To~lc ion~ which msy be e~ploy~d ln co~binstion with the
peptid~3 o~ the prQsent inYen~ion includ~ anion~ 3uch as

~ ,~ CO ~ rl ~- r,~ l T ~ r,~
fluorida, p~ x~di~ and bicarbqnate a ~ ~, and cations ~uch as
silver~ zinc~ mèrcury~ ~rsenic~ copper,. ~ atin~m, antimon~, gold,
thallium, nickel, selenium, bi~muth, and ca~mium cation~.
A to~ic ion is one which when introduced into a target cell
inhibits and/or pre~ents and/or destroys the growth of the target
cell.
Such a toxic ion is one which in the ab~ence o~ an ion
channel forminR peptide is unable to cro~s a natural or ~ynthetic
lipid membrane; in particular a cell membrane, in sufficient
amounts to affect a cell adversely.
Tha pQptide and the to~ic ion, whether administered or
prepared in a singla composition or in s~parate compositions, are
employed in amounts effective to inhibit and/or prevent and/or
destroy tho growth of the target cell. In affcct, the ion
potantiates the action of the peptite, i . Q ., the amount of toxic
ion i9 effectiv~ to rsduc~ ~he ma~lmum cffectiv~ concentration of
thc peptid~ or protein for inhlbitlng ~rowth of a target cell.
Antibiotic~ which may be employ~d in combination with the
peptides of the pre~nt invention includ~ b~citr~cins,
aminoglyco~ide8, hydrophobic ~ntibiotics, lnclutln~ macrolide
antibiotics, penicillins, monobactam~, or deri~atives or
analogues thereof.
Other antibiotic~ which may be u~ed (wh~ther or not
hydrophobic) in combinstion with the p~ptlde~ of the present
in~ention are ant$biotic~ which are 50-S ribosome inhlbitors such
as linco~yci~; cllnda~ycin; and chlora~phenicol; etc.; and
antibio~ic- whlch have a lsrge lipld li~e lactone ring, such as
mystat~n; pi~aricin, etc.
Th~ p~ptid~ and antib$otic may be ad~in~tored by direct
administration to a tar~t cell or by ~y~temlc or top~cal
administration to a host which lnclud~ the targat cell, in order
to pre~ent, de~troy or inhibit th~ ~rowth of a targ~ c~ll.
Tar8et cells whos~ growth may bo pr~onted, inhibited, or
de~troy~t by thc admlnistrstion of th~ p~ptld~ ant sntlbiotic
:- ,
,

~ ,Y~ T ~ 2 ~ ~ ~
include Gr~-posItiv~ and Gram- 2 bacteria as w~ll aQ
fungal cells.
The in~entlon will now be furthar described with respect to
the following examples; however, tha acope of the present
invention i9 not to be llmited th~r~by.
EXAMPLE
The procedure ~or the following antibacterial a3say is
based upon the guidelines of the Nstional Committee for Clinical
Laboratory Standards, Document M7-T2, Volume 8, No. 8, 1988. ~ l~b
A stock solution of 8 p~ptlde of th~ followi ~8 struc~ al
formula:
KIAGKIARIAGKIAKIAGKIA-smide, ~ Sl ~
wherein each amino acid re~ldue i8 a D-amino acit re~idue
or glycine, was prepsred ~ a concentratiQn of 512 ~g/ml in
sterile deionizet tistill2t water ~nd atored at -70C.
The stock peptid~ 301ution ia dllut~d in a~rial dilutions
(1:2) down the wells of a microtiter platQ ~o that the final
concentrations o~ peptide~ in th~ w~ ar¢ 0.25, 0.50, 1, 2, 4,
8, 16, 32, 64, 128, ~nd 256 I~g/ml. 1.5 ~1 105 CFlJ~/ml of elther
S. aureus ATCC 25923, E. coli ATCC 25922, or P. aeru~inosa ATCC
27853 were added to the wells in full strength Mueller Hinton
broth (B~L 11443 ~ from a Islid- loE~ cultur~ . Th~ inoculum i8
standarized spectrophotom~trically at 600nm and i8 ~ertified by
colony counts. Th~ platQa ar~ incubated or 16-20 hour~ a~ 37C,
and thQ ~ini~al inhibitory concentration (MIC~ for each peptide
is tat~r~in~d. Minimal inhibitory conc~rltration is d~fin~d as
the low~at conc~ntration of peptid~ which p~oduce~ a clea~ well
in the mlcrotitar plat~.
Th~ ~lni~l inhi~itory concontrat~on o th~ p~ptid~ was 8
~/ml a8sin~t S. aureus, 32 ~g/ml a~aln~t P. aeru ino~a, ant 4
l against ~. coli. For compari~on, a atoc~ ~olution o~ the
above-mentioned peptid~ wa~ prepar~d as h~rainabov~ d~cribet
wherein each amino scid r~idue wa8 not ~ D-amlno acid resldue~
,
.

~CeQ ~ 2 ~ 8
This peptid~ wa s~ed~as ~ bove de~cribed f4r M~C
against S. a~eùs,- P. aeruRinosa, and E. coli. The MIC o~ the
peptide was 8 ~g/mi a8ainst S. aureus, 16 ~g/ml agai~st P.
aeruRinosa, and 4 ~g/ml against E. coli.
The above result~ indicate that wh2n ~11 residues of
biologically sctive peptide are D-amino scid residues or glycine
resldues, the peptide retains it~ biological actlvity.
The peptid~9 of the pre~nt invantion, whether sdministered
alone or in combination with agents s~ch as to~ic ions,
antibiotics, or other biolo~lcally activ~ peptides or proteins as
hereinabove d~scrlbed, m~y be employed in a wita variety of
pharmaceutlcal compositions in comblnatlon with a non-tox~c
pharmaceutica~ carrier or vehiclQ such a~ a filler, non-to~ic
buffer, or physiological ~aline ~olution. Such phsrmaceutical
composition~ mQy b~ used topic~lly or ay~temic~lly and m~y be in
~ny suitabl~ form ~uch a~ a liquid9 ~olid, ~emi-solid, inJectable
301ution~ tablet, ointment, lotion, pa~te, cap~le or the like.
The peptide and/or a8ent a~ hereinAbove de~cribad may also be
u~ed in combination with ad~uvant~, protea8e inhibitor~, or
compatible drug~ where such 8 combinatlon i~ ~e~n to be desirable
or advanta~eous in controlling inf~ction caused by harmful
microorganism~ including protozoa, viru~es ~ and the lik~.
The peptide may be admini~t~rd to a host, in particular an
animal, in an eff~ctive antibiotic ant/or anti-tumor and/or
an~iviral snd/or a~ti~icrobial and/or anti~permicidal and/or
antipara~itic a~ount, or in ~n amount ef~ctive to stimulate
wound hQaling ln a host. The pRptid~ may b~ admini~terd ~ither
alone or 1~ co~blns~io~ with a to~ic lon, antibiotic, or ion
channel for~in~ p~ptide or pro~ein ~a horeinabove described.
When the peptlto i~ atmlni~tered in co~blnation wlth a toxic ion,
ths actlvity of tha peptid~ is potentiat~t.
When tha p~ptidQ 18 adminiatQr~d in combination with an
agent as h~reinabove de~cribed, 1~ i~ pos~ible to administer the
peptide and agent in aepar~te ~orm~. For e~mpl~, the agent msy

C Q
be admini~t~rëd 8y8;temically and tho pe~id~:may bs admini~tered
topically.
When the peptide i~ adminitared topioally, it may be
admini~tered in combination with a wat2r-301uble vehicle, said
water-~oluble vehicle bein~ in the form of an ointment, cream,
lotion, paste or the like. E~amples of water-~oluble vehicles
which may be employed include, but arc not limited to, glycol~,
9UC~ as polyethylen~ glycol, hydroxyc~llulos~, and KY J~lly. The
water-~olubl~ vehicle is preferably free of an olly sub~tance.
The peptld~ may slso be employet alone, or in co~bination
with other p~ptide~ of the p~e~ent invention, or in combinatlon
with peptides containing L-amino acid residues, or in combination
with othar active component~ (eg., to~ic ions, ~ntibiotic~) as
herainabove described in the form o~ ~n oral composition for oral
hygiene. Such a composition mBy be lncorporated into a wide
variety of compo~ition~ and matQrials used for oral hy~iene
purpose~, which include, but ar~ not llmit~d ~o, toothpastes,
mouthwashe~, tooth gels, snd tooth powd~r~. Such compo~ltion may
thus be u~et to treat or pr~vent p~r~odontal di~ea~, to prevent
or reduce plaque, and/or to prevent or tr~at or r~duc~ dental
csries. The peptide may be u~ed to inhibit, preYent, or destroy
the growth of 5trePtococcu~ mutans, whlch i9 a~sociated with
dental carle~ ant per$odontsl di~as~.
Numerou~ modification~ ant vari~t~on~ of the present
invention are po~siblQ ln light of thQ above teaching~ and,
therefore, within tho 8COp~ of the a~companying claims, the
invention ~ay b~ practiced other than ~ psrticularly described.