Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
PATAP287
COMPOSITION ~ND TREATMENT WITH
PEPTlDe CGM~INATION5
Thi~ invention relate3 to biologically active
peptides, and more particularly to compositions and
uses involving combinations of biologically active
peptides.
In accordance with an a~pect of the present
invention, there iq provided a composition which
includes (a) a magainin peptide OE analogue or
derivative thereof; and (b) at least one member
selected from the group consisting of (i) a PGLa
peptide or analogue or derivative thereof and (ii) an
XPF peptide or analogue or derivative thereof. The
magainin peptide or analogue or derivative thereof,
as well as the PGLa peptide or analogue or derivative
thereof and the ~PF peptide or analogue or derivative
thereof, may be amide terminated or
carboxy-terminated.
In accordance with another aspect of the present
invention, there is provided a process which
compri~Ps admini~terin~ to a ho~t both (a) a magainin
peptide or analogue or derivative thereof and (b) at
lea3t one member selected from the group con~i~ting
of (i) ~n XPF peptide or analogue or derivative
th~reof and (ii) a PGLa peptide or analogue or
derivative thereof. In one embodiment, components
(a) and (b) may be administered in separate
compositions. In a~o~her embodiment, components (a)
and (b) may be administered in a single composition.
In addition, compone~lts (a) and (b) may b~
administered in amoun~s effective to inhibit gro~th
of a target.
Although the present invention is not to be
limited by any theoretical reasoning, it is believed
that the combination of the magainin peptide with the
PCLa peptide or XPF peptide provide~ a syner~istic
effect in the inhibition of growth of a target. The
target, for example, may be bacteria, fungi~
protozoa, virally infected cells, malignant cells, or
sperm cells as compared to normal host cells. The
synergi~m may be due to association of the peptides
to a novel multimeric complex, such as a dimer, which
possesses markedly increased membrane affinity for
the target cells. The complex formed between the two
peptides is believed to possess potent membrane
disruptive properties. It is believed that the
peptides have the capacity to organize within tar~et
cell membranes and to disturb cellular functions.
In ~eneral, the magainin peptide or analogue or
derivative thereof is employed in a dosage of from
about lmg to about lOOmg per kilogram of host weight,
when administered systemioally. When administered
topically, the magainin peptide is used in a
concentration of from about .05% to about . 50%.
The PGLa peptide or analogue or derivative
thereof, when administered 5y~temically, is
adminis~ered in a dosage o~ from about lmg to about
lOOmg per kilogram of host weight. When administered
~ 7~L3
topically, the PGLa peptide is administered in a
concentration of from about 0.5% to about .50~.
The ~PF pep~ide or analogue or derivative
thereof, when administered systemically, is
administered in a dosage of from about lmg to ~bout
lOOmg per kilogram of ho~t weight. When administered
topically, the XPF peptide or analogu~ or derivative
thereof is admi~ister~d in a concentration of from
about .05% to about .50%.
The use of this combination of peptides in
accordance with the present invention is effective as
an antibiotic, and may be employed to inhibit,
prevent or destroy the growth or proliferation of
microbesl such as bacteria, fungi, viruses or the
like. Similarly, such compositions may be employed
as an an~i-viral composition to inhibit, prevent or
destroy the growth or proliferation of viruses.
Such compositions may also be employed as a
spermicide to inhibit, prevent or destroy the
motility of sperm.
Such compositions may also be employed as
anti-tumor agents to in~ibit the growth of or destroy
tumors.
The compo~itions have a broad range of potent
antibiotic activity against a plurality of
microorganisms, including gram-positive and
gram-negative bacteria, fungi, protozoa and the like.
Such compositions may be employed for treating or
controlling microbial infection caused by organisms
which are sensitive to ~uch composition. The
treatment may comprise admini~terin~ to a host
organism or tissue~ acceptable to or affiliated with
a microbial infection an anti-microbial amount of
magainin peptide or analog or derivative thereof and
of PGLa peptide or XPF peptide.
~7~
4-
Th~ compositions may also be used as
preservatives or sterilants for materials susceptible
to microbial contamination.
The magainin peptide may be, for example, a
magainin such as magainin I, II or III or an analogue
or derivative thereof. The magainin peptides
preferably include the following basic peptide
structure X12
11 11 12 R13 Rll R14 ~R12 ~Rll ~
14 12 11 11 11 ~14a (R15)n ~~14a -R14 ~~
wherein R11 is a hydrophobic amino acid, R12 is
a basic hydrophilic amino acid; R13 is a hydrophobic,
neutral hydrophilic or basic hydrophilic amino acid;
R14 and R14a are hydrophobic or basic hydrophilic
amino acids; R15 is glutamic or aspartic acid, or a
hydrophobic or basic hydrophilic amino acid, and n is
O or 1. In a preferred embodi~ent, R13 is a
hydrophobic or neutral hydrophilic amino acid, R14a
is a hydrophobic amino acid, and R15 is glutamic acid
or aspartic acid.
The hydrophobic amino acids may be selected from
the class consisting of Ala, Cys, Phe, Gly, Ile, Leu,
Met, Val, Trp, and Tyr. The neutral hydrophilic
amino acids may be selected from the class consisting
of Asn, Gln, Ser, and Thr. The basic hydrophilic
amino acids may be selected from the class consisting
of Lys, Arg, and His, and ornithine (O).
The.magainin peptides generally include at least
seventeen amino acids and may also include up to
forty amino acids. Accordingly, the hereinabove
described basic peptide structure of a magainin
peptide may include additional amino acids at the
amino end or at the carboxyl end, or at both ends.
Thus, for example, a magainin peptide may
include the following structure:
~7~L3
-Y12 -Xl -
where X12 is the hereinabove described basic
pertide structure and Y12 is
(i) Rl~
( i i ) R14 - R12
t iii ) 11 R14a R12
(iv) R14 ~Rll -R14 -R
11' R12~ R14 and R14a are as previously
defined.
A magai~in pep~ide may also have the following
structure
-X12 Z12-
wherin X12 is as preYiously defined and Z12 is:
(i) R16 where R16 i3 a basic hydrophilicamino acid or asparagine or glutamine.
(ii) R16 -R17 where R17 is a neutral
hydrophilic amino acid, a hydrophobic amino acid, or
a basic hydrophilic amino acid. Preferably R17 is a
neutral hydrophilic amino acid.
A magainin peptide may also have the following
structure:
(Y12) a-X12 (Z12)b
re X12, Y12 and Z12 are as previously defined
and a is O or 1 and b is O or 1.
The magainin peptides may also include the
following basic peptide stmcture X13:
14 11 14a R12 Rll Rll-R12-R13-
Rll R14 R12-Rll-Rll-R12- ~ wherein Rll ,R12 ,R13 . ~1
and R14a are amino acids as hereinabove described.
The magainin peptide may also include the
follo~7ing structure X13-Z13; wherein X13 i~ the
hereinabove described ba~ic peptide struc~ure and Z13
is
(Rll)n (Rll)n (Rll)n (~14a)n (R15)n (R14a)n (R14)n (R16)n
.
-6-
(R17)n wherein Rll, Rl~, R14a~ R15, R16' and R17 are
as hereinabove described, and n is O or 1, and each n
may be the same or dlfferent.
Th~ magainin peptides generally include at least
fourteen amino acids and ~ay include up to forty
amino acids. A magainin peptide preferably has 22 or
23 amino acids. Accordingly, the hereinabove
described basic peptide ~tructure~ of a magainin
peptide may include additional amino acids a~ the
amino end or a~ the carboxyl end, or at both ends.
As representative examples of such magainin
peptides, there may be mentioned peptides having the
following primary sequence (expressed as a single
letter code) as well as appropriate analogues and
deriatives thereof:
(a) (NH2) GIGKFLHSAGKFGKAFVGEIMKS (OH) or (NH2)
(Magainin I)
(b) (NH2) GIGKFLHSAKKFGXAFVG~I~NS (OH) or (NH2)
(Magainin II)
(c) (NH2) GIGKFLHSAKKFGKAFVGEIMN (OH) or (NH2)
(Magainin III)
The following are examples of peptide
derivative~ or analogs of the basic s~ructure:
(d)(NH2) IGKFLHSAKKFGKAFVGEIMNS (OH) or (NH2)
(e)~NH2) GKFLHSAKKFGKAFVGEIMNS (OH) or (NH2)
(f)(NH2) KFLHSAKKFGKAFVGEIMNS (OH) or (NH2)
Magainin peptides are described in Proc. Natl.
Acad Sci. Yol 84 pp. 5449-53 ¢Aug. 87). The term
"magainin peptides" as used herein refers to the
basic magainin structure as well as derivatives and
analogs thereof, including but not limited to the
representative derivative~ or analogs.
The peptide employed in conJunction with the
magainin peptide is a PGLa peptide or an XPF peptide.
;~ 3
-7-
A PGLa peptide i5 ~ither PCLa or an analogue or
deriva~ive thereof. The PGLa peptides preferably
include the following basic structure X14:
11 17 12 Rll ~14 R14 Rll ~
11 14 12 Rll Rll R12 -Rll -
11 Rll R12
Rll' Rl2' R14~ and R17 are as previou~ly
defined.
The PGLa peptides generally include at least
seventeen amino acids and may include as many as
forty amino acids. Accordingly, the hereinabove
described basic peptide sturcture for a PGLa peptide
may include additional amino acids a~ the amino end
or at the carboxyl end or at both the amino and
carboxyl end.
Thus, for example, a PGLa peptide may have the
following sturcture:
-Y14 -X 4-
where Xl4 is as previously defined and
Y14 iS
(i) Rll;
(ii) R14 R
where Rll and R14 are as previously defined.
For example, a PGLa peptide may also have the
following structure:
-Xl4 -Z14-
where X14 is as previou~ly defined; and Z14 is:
(i) Rll; or
(ii) Rll-Rll, where Rll i~ a~ previously
defined.
A PGLa peptide may also have the following
structure: (yl4)a-xl4-(z14)b
re X14; Y14 and Z14 are a~ previously
d~fined, a is O or 1 and b is 0 or 1.
An XPF peptide is eith~r XPF or a~ analogue or
d~rivative thereof. The XPF like peptides preferably
include the following basis peptide structure Xl6:
11 17 12 Rll R14 Rl8 R17-
Rll-Rl4-Rl2-Rll-Rll-Rl2-
11 11 11 R12 (R15)n R~
Rll ~ R12 t Rl4~ Rls and R17 are as
previously defined and Rl8 is glutamine or
asparagi~e, or a basic hydrophilic, or hydrophobic
amino acid, and n is O or 1.
The XPF peptid2s generally include at least
nineteen amino acids and may include up to forty
amino acids. Accordingly, the hereinabove described
basic peptide structure of XPF may include additional
amino acids at the amino end, or at the carboxyl end
or at both the amino and carboxyl ends.
Thus, for example, an XPF peptide may include
the following struct~re:
-Y16 -X16-
where X16 is as previously defined and Y16 is
(i) Rll or
~ ii ) R14 -Rll
where R~l and R14 are as previously de~ined.
An XPF peptide may include the follwing
structure:
-X16 ~ Z16 ~
where X16 is a~ previously defined and Z16 is
(i) Rll; or
(ii) Rll -Rl~ i or
(iii) Rll -Rlg -Proline; or
( iv) Rll -R18 -Proline R12
An XPF peptide may also have the following
structure:
(Y16 )a X16 ( Z16 )b
2~
~ here X16, Y16 and Z16 are as prevlously defined
: a is O or 1 and b is 0 or 1.
Preferred are XPF or PGLa peptides, which are
characteriæed by the following primary amino acid
sequence (single letter amino aoid code);
PGLa: GMASKAGAIAGKIAKVALKAL (NH2)
XPF: GWASKIGQTLGKIAKVGLKELIQPK
A re~iew of XPF and PGLa can be found in Hof~man
et al, EMBO J. 2:711-714, 1983; Andreu et al, J.
Biochem. 149:531-535, 1985; Gibson e~ al J. Biol.
Chem. 261:5341-5349, 1986; and Giovanni et al,
Biochem J. 241:113-120, 19&7.
The present invention will be further described
with re~pect to the following examples, however, the
scope of the present invention is not to be limited
thereby.
EXAMPLE 1
About 105 bacteria/ml were added to a small
volume of Luria broth. Magainin 2 (MGN2), PGLa
peptidej or a mixture of MGN2 and PGLa in a 1:1 molar
ratio was added to the broth in increasing
concentration. The minimal inhibitory concentration
(MIC), which inhibits microbial growth completely at
24 hours i~ noted below in Table 1.
~J7~3
-10-
TA~LE 1
ANTIBACTERXAL ACTIVITY OF ~OMBINATION OF PGLa AND
~AGAININ 2
ORGANISM MINIMAL INHIBITORY CONCENTRATION (~
~GN2 PGLa PGLa/MGN2
(1:1 molar ratio)
S. aureus >500 >500 10
P. ~eruginosa 250 250 10
C. albicans 250 240 10
Micrococcus 125 125 10
Diphtheroids125 125 10
E. coli 50 50 10
The above result~ indicate an increase in
antibacterial activity when a combination of Magainin
2 and PGLa peptides is added to a culture bro~h of
bacteria in a 1:1 molar ratio of Magainin 2 to PGLa
over either peptide added alone.
EXAMPLE 2
In this example, the concentration of S. aureus
killed by 100 ~g/ml of preparations of Magainin 2,
PGLa, and a preparation of Magainin 2 and PGLa in a
1:1 molar ratio is noted. It was found that a 100
~g/ml preparation of Magainin 2 does not completely
~ill S. aureus at concentrationq of bacteria at less
than 10 bacterial/ml. Similar re~ults were also
obtained for PGLa. The 100 yg/ml preparation of the
equimolar miture of Magainin 2 and PGLa, however,
achieved complete killi~g o~ a concentration of 105
bacteria/ml. Thus, the equimolar mixture of Magainin
~ ;37~3
2 and PGLa achiev~d an inc~ea~e in bac~ericidal
potency over either peptide by greater than 105.
EXAMPLE 3
Approximately 103 S. aureus bacteria were added
per/ml of Luria broth. Peptid~ preparations
con~aining varying molar ratio amounts of PGLa to
Magainin 2 (mole PGLa/mole MGN2) were added to the
broths to increasing concentration of peptide
(~g/ml). The microbial inhibitory concentration
(MIC), at which point no growth was evid~nt after 24
hours was measured for each peptide preparation. The
results are given in Table 2 below.
TABLE 2
Mole PGLa/
Mole MGN2~IC v~. S. aureus (~g/ml)
lO/0 >500
10/ 1 ~$
7.5/2.5 15
5/5 a
2.5/7.5 15
1/10 25
0/lO >500
The above results indicate that maximum
bactericital activity is achieved when an equimolar
mixture of PGLa and Magainin 2 is added to the S.
aureus culture. It is also shown, however, that
preparations containing both peptides in any molar
ratio achieved greater bactericidal activity than
either peptide alone.
The peptide combinatio~s, in acoordance wlth the
present invention, may be employed for 'creating a
wide variety of hosts. In accordance ~ith a
preferred embodiment, a host may be an animal, and
such animal may be a human or non-human animal. The
magainin peptide and the PGLa and/or XPF peptide may
b¢ employed together in a single composition, or in
~eparate compositions.
The magainin peptide and PGLa and/or XPF peptide
may be employed in a wide variety of pharmaceutical
compositions in combination wi~h a non-toxic
pharmaceutical carrier or vehicle such as a filler,
non-toxic buffer, or physiological saline solution.
Such pharmaceutical compositions may be used
topically or systemically and msy be in any suitable
form such as a liquld, solid, semi-solid, injectable
solution, tablet, ointmen~, lotion, paste, capsule,
or the like. It i~ also contemplated that the
magainin peptide and the PGLa and/or XPF peptide may
be delivered or administered in different form~. The
magainin peptide and PGLa and/or XPF peptide may also
be used in combination with ad~uvants, protease
inhibitors, or compatible drugs where such a
combination is seen to be desirable or advantageous
in controlling infection cau~ed by harmful
microorganisms including protozoa viruses, and the
like.
The peptidets) of the present invention may be
administered to a host; in particular an animal, in
an e~fective antibiotic and/or anti-tumor and/or
anti-viral and/or anti-microbial and/or a spermicidal
amount.
Numerous modification~ and variations of the
present invention are pos~ible in light of the above
teachin~s, and, therefore, within the ~cope of the
accompanying claim~, the invention may be practiced
other than as particularly described.
