Note: Descriptions are shown in the official language in which they were submitted.
1 68975-92
COMPOSITION AMD TRBATM~MT WITH BIOLOGICALLY
ACTIVE PEPTIDES AND TOXIC CATIONS
This invention relates to biologlcally active peptldes
and protelns, and more partlcularly to composltions and uses
lnvolving blologically actlve peptides or protelns and toxlc
cations, ln partlcular sllver catlons. In par~icular, such
composltlons may be employed ln preventlng and/or treatlng ocular
infectivns.
,Silver-containing cornpositions, such as silver nitrate,
have been used for preventing and/or treating external ocular
infections, such as, for example, the preventlon and treatment o~
ophthalmlc gonorrhea. Darrell, et al., Tr. Am. OPhth. Soc., Vol.
82 (1984), 75-91, discloses the use of silver norfloxacin, or :
sllver sulfadlazlne, or sllver sulfacetamlde to treat corneal
lnfectlons caused by P.aeruqlnosa. These compounds also exhiblt
slgniflcant blolgolcal actlvlty agalnst S.aureus, as well as the
fungl C.alblcan~, and A.fumlqatus.
SUMMARY OF TH~ IMVE~TION
The present lnventlon provldes a use of a composltionjto
inhlbit growth of a target cell or vlrus ln a host, said
composition comprlsing,
~a) at lea~t one biologlcally active amphiphllic pept:lde and/or
blolo~lcally active prote:ln~ ~aid peptlde or ~roteln be:lng an ion
ch~nnel-~orming peptide or protein
(b) a toxic catl.on1 and
(c) an accept~ble pharmaceutical carrier, w~lerein components (a)
and (b) are pre~ent in a combine~ amount e~ect:lve to inhlblt
growth Oe a target ce:ll or vlrus in a ~o~t.
, ~ , . .~
. . . . .
la 68975-92
The present lnventlon also provides a composltion,
comprlslng:
~a) at least one biologically active arnphlphlllc peptide and/or
blologlcally actlve proteln, sald peptlde or proteln belng an lon
channel-forming peptlde or proteln;
(b) a toxlc cation; and
(c) an acceptable pharmaceutlcal carrler, whereln sald cornponents
(a) and (b) are present ln a comblned amount effectlve to lnhlblt
growth of a target cell or vlrus ln a host.
The present lnventlon yet also provldes a use of a
compcsltlon to treat or prevent lnfectlons of the eye ln a host,
said composltlon comprlslng:
(a) at least one biologically active amphlphlllc peptlde and/or
blologlcally active proteln, said peptlde or proteln belng an lon
channel-formlng peptlde or proteln;
(b) a toxlc catlon; and
(c) an acceptable pharrnaceutlcal carrier, wherein cornponents (a)
and (b) are present ln a combined amount effectlve to lnhlblt
growth of a target cell or vlrus in a host;
whereln components (a) and (b) are present in a comblned amount
effectlve to inhiblt growth of a target cell or vlrus ln a host.
The present invention further provides a use o~ a
composltlon to treat or prevent ~kin ln~ectlons ln a host, sald
composltlon comprl~ln~
(~) at l~ast one biologlcally actlve arnphlphlllc peptlde an~/or
blologlcally actlve prote:ln, sald peptlcle or protein beln~ an lon
channel-~orrning peptlde or proteln~
(b) a toxlc catlon; and
. . .
lb
6~975-92
(c) an acceptable pharmaceutlcal carrler, whereln components (a)
and (b) are present ln a combined amount effective to inhibit
growth of a target cell or vlrus in a host.
The present inventlon also provides a use of a
composltlon to treat or prevent burn lnfections ln a host, said
composltion comprising:
(a) at least one blologlcally actlve amphiphlllc peptlde and/or
biologically actlve proteln, sald peptlde or proteln belny an lon
channel-formlng peptlde or proteln~
(b) a toxic catlon; and
(c) an acceptable pharmaceutlcal carrier, wherein components (a~
and ~b) are present ln a comblned amount effectlve to inhibit
growth of a target cell or virus in a host.
The present lnvention yet further provldes a use to
inhlbit the growth of a target cell or a virus in a host, of at
least one biologically active amphlphllic peptide selected from
the group consisting of:
(1) a magalnln peptlde7
(11) a cecropln;
(lii) a sarcotoxin;
(iv) an XPF peptide~
~v) a PG~a peptlde~
~vl) a CPF peptlde~
~vll) a peptide includin~ the ~ollowing bas:Lc ~,tructure X40,
[ ~ 2 ~42 R43 ~ 42 R~2]11
whereln R41 ls a basic hy~rophilic amlno acid, R42 ls a
hyclrophob:lc amino acid, and R~3 ls a ne~t ral hydrophlllc amlno
acid, and n i~ from 2 to 5~
.
, ;
.
lc 6~975-92
(vlil) a peptlde whlch lncludes the followlng basLc structure X50:
41 42 42 43 41- 42 42 R41 R42 R42 R42 R41 R42 R42'
whereln R41 is a baslc hydrophlllc amino acid, R42 ls a
hydrophoblc amlno acld, and R43 ls a neutral hydrophilic or
hydrophoblc amino acld;
(lx) a basic polypeptide having at least sixteen amlno acids,
whereln said baslc polypeptlde lncludes at least eight hydrophobic
amino aclds and at least eight hydrophilic arQlno aclds; and
(b) a toxlc cation, ~aid component~ (a) and (b) belng
adminlstered in a cornbined amount effective to inhibit growth of a
target cell in a host.
: The present lnvention also provldes a composition :~
; comprising:
(a) at least one biologically actlve amphlphilic peptide selected
from the class consisting of:
(i) a magainln peptide;
. (ii) a cecropln;
(lil) a arcotoxin;
(iv) an XPF peptide;
(v) a PGLa peptide;
(vi) a CPF peptide;
(vii) a peptide including the following basic structure X40:
-R42-R~2-R43 R~l R~2 ~42Jn
wherein R41 1~ a ba~ic hydrophilic amlno acld, ~42 L5 a
hydrophobic amlno acid, R43 is a neu-tral hydroph:llic amino acid or
hydrophoblc amlno acld, and n i~ ~rom 2 to 5; and
~vlil) a pep~lde inclu~lng the folLowing baslc ~tructure X50,
R41 R42 ~42 R43 R41-~42 ~42 R41 ~42 ~42 R42 R41 R42 R42'
c~
.: . . . .
. . . .
,,
.
-. , - . .
ld 68975-92
whereln R41 is a baslc hydrophllic amlno acid, R42 ls a
hydrophobic amino acid, and R43 ls a neutral hydrophilic or
hydrophoblc amino acid;
(lx) a baslc polypeptide havlng at least slxteen amlno acids,
wherein sald baslc polypeptide includes at least elght hydrophoblc
amino acids and at least eight hydrophil:Lc arnino acids;
(b) a toxic catlon; and
(c) an acceptable pharmaceutlcal carrler, wherein said cornponents
(a) and (b) are pre.sent in a comblned e~ectlve amount to lnhlblt
growth of a target cell or virus ln a host.
.~
,......................................................................... .
.,
:,~
~ ~.
.' ~
:
. . . : -. - :
- . , . ~
-2-
68975-92
An lon channel-forming peptlde or protein or lonophore iY a
peptide or prot~1n whlch lncrea~ th~ porme~blllty for lons
acro~ a natural or 0ynthetic lipi~ membrane. B. Christen~en et
~1. PMA8 Vol. 85 P. 5072-76 (July, 19~8) d~crlb~ m~t.~lo~o10ay
which indicateu wh~ther or not a peptlde or proteln ha~ ion
channel-formlng propertie~ and t 8 therefore an ionophore. Ae
u~ed herein an lon channel-formlng peptlde or lon channel forming
protaln is Q peptide or proteln whlch ha~ ion channel-forming
propertie~ aH determined by the method of Chri~ten~en et al.
An amphiphllic peptld~ i~ a peptlde whlch include~ both
hydrophoblc and hydrophllic p~ptld~ regionc.
In accordance with an a~pect o tho present lnvent~on
whereln th~ biolo~lc~lly activo poptldo or protoin ~nd toxlc
c~tlon are admini~tered to a ho0t, ~uch biologically active
- p~ptid~ or prot~ln ~nd toxic c~tlon m~y b~ Admlt1l~t~r~d n~ n
slngls compo~tion or in ~2parate compo~itlon~ and the single or
~eparate compo~itlon~ mQy in~lude additlonal material~, actives
and/or inactive~, in addltion to the pept~de ~nd/or protein and
toxt G ~ation.
The compo~ltlon~ of th~ present lnventlon thu~ m~y be u~ed
in the treat~nt of external burnc and to treat Rnd/or prevent
ckin and b~rn infectlonc. In p~rtlcular, the compo~ltlon~ may be
u~ed to treat ~kin Qnd burn inectlon~ caused by organism~ ~uch
R~, but not limited to, P. aoru~ino~a and S. aureu~.
! Thn ~on channel-orming peptide~ employed in the pre~ent
lnvetltion are gon~r~lly w~tor ~olubl~ ~o a conc~ntr~tlol~ o~ nl:
lea~t 20 mg/ml at neutral pH in water. In additlon, ~uch
poptlde~ ~ro non~hemolytlc) i.~., th~y will no~ ruptur~ blood
cell~ at ef~ctiv~ concentr~tion~. In addition, the 0tructure of
~uch peptlde provide~ ~or ~lexibility of the peptide mo:Lecule.
Whon th~ paptide iN plac~d ln water, i~ doe~ not a~uma an
~mphiphllic ot~Uct~r~. Wh~n tha p~ptid~ ~ncQunter~ ~n oily
r
; ~A
.,;
,,
-3-
~urface or membrane, the peptide chain folds upon it~elf into a
rod-like struckure.
In general, such peptides have at least 16 aminc acids, and
preferably at least 20 amino acid~. In most ca~es, such peptides
do not have in exce 9 of 40 amino acid~.
In genera, the toxic cation 1 8 employed as part of a
suitable compound. Toxic cations which may be employed include,
but are not limited to, silver cation~, zinc cation~, mercury
cations, ar~enic cations, copper cation~, platinum cations,
antimony cations, gold cations, thalluim cations, nickel cations,
~elenium cation~, bismuth cations, and cadmium cation3.
In a partlcularly preferred embodiment, the toxic cation i~
a ~ilver cation, which may be employed a~ part of a suitable
compound. Suitable 3ilver-contalning compound~ which may be
employed include 3ilver nitrat2, ~ilver acetate, ~ilver benzoate,
silver carbonate, silver iodate, 311ver iodide, ~ilver lactate,
silver laurate, silver oxlde, ~ilver palmitate, mild silver
protein~, and ~ilver sulfadiazine. It i~ al~o preferred that the
~ilver-containing compo~ition be water-soluble. In accordance
with one embodlment, the toxic cation may be ~ilver nitrate.
Silver nitrate as a 0.5% solution i8 u~ed in the treatment
of external burn~, and ~ilver sufladiaz~ne i~ al80 used to treat
external burns, but bactarial resi~ance to the ~ulfonamides
(sulfadiazlne) can result. S. Harvay, in Goodman and Gilman,
Pharmacological Ba~is of Therapeutic~, 7th ed., pg~. 966-967;
MacM~ llan ( 1985 ) .
In employing both an ion channel-orming biologically active
amphiphillc peptide or an ion channel-forming proteln, and a
to~ic cation, wheth~r admini~tered or prepared in a single
co~po~ition, or in separate compo~i~ion~, khe peptide or protcln
and the toxic cation, are amployed in amount~ o~actlva to
i~hibit and/or preVent and/or da~roy tha growth o~ the target
cell. In e~ect, the toxlc cation potentlate~ the action of the
pept.tde or pro~ein, and the peptide or protein potentiates the
,~ f ~ a
~4~
action of the toxic cation. The term "potentiate," aq employed
hereln, mean~ that th~ amount of toxic cation i~ effective to
reduce the minimum effective concentration of the peptide or
protein for inhibitlng growth of a target cell and the amount of
peptide or protein i~ effective to reduce the minimum effective
concentration of the toxic cation for inhibiting ~rowth of a
target cell.
In general, the peptide or proteln i~ administered topically
at a concentration of from .05% to 5%.
The toxic cation, in general, i~ used topically at a
concentratlon o~ from 0.05% to 2.0%.
The u~e of a combination o peptide or protein and toxic
cation in accordanco with the pre~nt invention i8 ef~ective as
an antibiotic, and may be employed to inhlbit, prevent or destroy
the growth or prollferation of microbe~, such a8 bact~ria and
fungi. Similarly, such compo~itions may be employed as an
anti-viral compo~ition to lnhibit, prevent or destroy the growth
or proliferation of viru~e~.
Such composition3 may al80 be u~ed a3 antiungal agent~ to
inhibit the growth of or de~troy fungi.
Such compo~ition~ may also be u~ad aq anti-paraaitic agents
to inhibit the growth of or destroy parasites.
The composition~ have a broad ranqe of potenk antlb:lotic
activity against a plurality o microorgani~m~, incLuding
gram-po~itive and gram~negative bacteria, fungi, protozoa
para~ite~, and the like. Such compo~itions may be employed for
treating or controlling microblal infection cau~ed by organisms
which are ~en~itive to 3uch compo~ition~. The treatment may
compri~e adminiætering to a ho~t organi~m or tl~ueq acceptable
~o or afiliated wlth a microbial infectlon an anti-microbial
amount o~ peptide or protein and toxlc cation.
The compo~itlon~ may al00 be u~ed as pre~&rvative3 or
~terilantæ for material~ ~u~ceptible to mlcrobial contamlnation.
. .
~v i~
Such compo~ition~ are especially u6eful in the prevention or
treatment of eye infections. Such infection~ may be caused by
: bacteria such as, but not limited to, P.aeruaino~a, S.aureus, and
N.qonorrhoeae, by fungi ~uch as but not limited to C.albicans and
A.fumiaatus, by parasites ~uch as but not limited to
A.castellani, or by viruse~. Applicant~ have found that
significant ~ynergistic effect~ ayain~t such organi~m~ or viru~e3
may be obtained when the ion-channel-forming peptide~ or proteins
are employed in conjunction with a ~llver-containing compo~ition.
Such compositions may al~o be ef~ective in killing cy~ts,
~pore~, or trophozoite~ of infection-causirlg organi~ms. Such
organisms lnclude, but are not limited to Acanthamoeba which
form~ trophozoite~ or cy3ts, C. albicans, which form~ spores, and
: A._umiqatu~, which form~ spore~ a~ well. ~:
In accordance with a preferred ~mbodiment, the peptide u~ed
in conjunct~on with a toxic cation i~ a basic (positively
charged) polypeptide having at least sixteen amino acid~ wherein
the polypeptide include~ at lea~t elght hydrophobic amino acids
and at least eight hydrophilic amino acid~. Still more
particularly, the hydrophobic amino acids ar~ in groups of two
ad~acent amino acid3, and each group of two hydrophobic amino
acid3 is spaced from another group of two hydrophobic amino acids
by at lea~t one amino acid othsr than a hydrophobic amino acid
(prefarably at loa~t two amino acld~) and g~nerally by no gr~ater
than four amino acid~, and the amino acids b~twesn pairs of
hydrophobic amino acid~ may or may not be hydrophilic.
The hydrophillc amino acids are generally al30 in group~ of
two ad~acent amino acids in which at lea~t one of the two amino
acid~ i~ a ba~ic hydrophilic amino acld, with such group~ of two
hydrophlllc amino acida being ~pacod from each other by at least
one amino acid o~her than a hydrophillc amino acid (pre~erably at
lea~ tWo amino acid~ ) and generally no greater than ~our amino
aald~, and the amino acid~ b0tween palr~ of hydrophilic amino
acid~ may or ma~ not be hydrophobic.
..
- 6 - ~ ? ~
In accordance with a particularly preferred embodiment, the
polypeptide comprl~ a chain of at 12a~t four group~ of amino
acids, with each group consisting of four amino acids. Two of
the four amino acid~ in each group are hydrophobic amino acid~,
and two of the four amino acids in each group are hydrophilic,
with at least one of the hydrophilic amino acids in each group
being a basic hydrophilic amino acid and th~ other being a basic
or neutral hydrophilic amino acid.
The hydrophobic amino acids may be 3el~cted from the clas~
con~i~tlng of Ala, Cy~, Phe, Gly, Ile, Leu, Met, Val, Trp, and
Tyr. The neutral hydrophilic amino acid~ may be ~elected from
the cla~ con~i~ting o A~n, Gln, Ser, and Thr. The ba~ic
hydrophilic amino acid~ may be selected from the cla~ con~isting
of Ly~, Arg, ~i~ and ornithine ~0).
Each of the groups of four amino acids may be of the
sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each
hydrophobic amlno acids and may be the ~ame or differ~nt, one o
C or D is a ba~ic hydrophilic amino acid, and the other of C or D
i~ a basic or nautral hydrophllic amino acid and may be the same
or differant. In a preferred embodlment, the polypeptide chain
may compri~ 5 or 6 group~ of this ~equence. In each group, each
of A, B, C and D may be the ~ame in some or all of the groups or
may be differont in some or all of the groups.
The polypeptide chain preferably ha~ at }eas~ 20 amino
acids, and no greater than 50 amino acids. It is to be
under~tood, however, that the polypeptide does not have to
consist entirely of the group~ described above. The polypeptide
may have amino acids extending rom either or both end~ of the
noted group~ ~orming the polypeptide chain and/or there may be
~mino acid~ batween one or more o~ tha at lea~t four group~ and
3till remain within the ~cope o~ ths invention.
! ~he ~roupu o~ amino acid~ may be repeating group~ o~ amino
acld~, or the amino acid~ in the variou~ ~roup~ may vary provided
that ln each ~roup o~ the at le~t ~our group~ oP amino acld~
;
: - .
there ar~ two hydrophobic and two hydroph1lic amlno acid~ a~
hereinabove noted.
Thu~, in a preferred embodiment, the biologically active
polypeptide comprise~ a chaln including at least four groups of
amino acid~, each containing four amino acid~. Two of the four
amino acid~ in each group are hydrophobic, at lea~t one amino
acid i~ basic hydrophilic, and the remaining one i~ basic or
neutral hydrophilic, with the polypeptide chain preferably having
at lea~t 20 amino acids but no greatsr than 50 amino acid~.
In one embodiment, each of the at lea~t four group~ of amino
aclds which ara in the peptide chaln i~ of th~ sequence A-B-C-D,
B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic
amino acld~, one of C or D i~ baslc hydrophillc amino acid, and
the other of C or D is ba~ic or neutral hydrophilic amino acid.
The re~ulting polypeptide chain, therefore, may have one of the
following ~equance~:
(Xl)a(A-B-C-D)n(Yl)b
(X2)(B-~-D-A)~Y2)b
~X3)a(C-D-A-B)n(Y3)b
(X4)a(D-A-B-C)n(Y4)b
wherein Xl i3 D; C-D or B-C-D-, Yl i8 -A or -A-B or -A-B-C
X2 i~ A-, D-A- or C-D-A-
2 ia -B, -B-C or B-C-D
~3i~ B-, A-B-, D-A-B-
Y3 is -C, -C-D, -C-D-A
X4is C-, B-C , A~B-C-
Y4 i9 -D, -D-A, -D-A-B
a i8 o or l; b is o or l
and n i 8 at leaat 4
It i~ to be under~tood that the peptide chain may include
amlno acids between the hereinabove notod ~roup~ o~ four amino
a~id~ provldecl that the ~paclncJ between ~uch group~ and ~he
charye on the amino acid~ doe~ not change the characterl~tics o~
the peptide ch~in wh.tch provldo amphiphilicity and a poBi tive
;l ~
:
2 l~ `~Y ~ c,i
charge and do not adver~ely affect the folding characteristics o~
the chain to that which i9 significantly different from one in
which the hereinabove noted group of four amino acid~ are not
~paced from each other.
As repreeentative example~ of peptide~ in accordance with
the pre~ent invention, there may be mentioned.
I Ala-Phe-Ser-Lys-Ala-Phe-Ser Lys-Ala Phe-5er-
Lys-Ala-Phe-Ser-Ly~-Ala-Phe-Ser-Ly~
II Ala-Phe-Ser-~y~-Ala-Phe-Ser-Lys-Ala-Phe-Ser-
Ly~-Ala-Phe-Ser-Ly~-Ala-Phe-Ser-Lys-Ala-Phe~
Ser-Ly~.
III Pha Ser-Ly3-Ala-Phe-Ser-
Ly~-Ala-Phe-Ser-Lys-Ala-
Phe-Ser-Lys-Ala-
IV Ser-Ly~-Ala-Phe-Ser-Lys-Ala-
Phe-Ser-Ly~-Ala-Phe-Ser-Lys-Alao
Phe~S~r-Ly~-Ala~Phe-
V Ly~-Ala-Phe-Ser-Ly~-Ala-Phe-Ser-Lys~Ala-Pho-Ser~
Ly~-Ala-Ph~-Ser
The peptide, may have amino acid~ extending from either end
of the chain. For example, the chain~ may have a Ser-Ly~
~equence befor~ the "Ala" end, and/or an Ala-Phe ~eguence after
the "Lys" end. Other amino acid sequences may al~o be attached
to the "Ala" and/or the "ly~" end.
Similarly, in any polypeptide chain having at lea~t four
group~ of amino acid~ of the ~equence as de~cribed above, the
chain may have, for example, a C-D ~quence before the fir~t
A-B-C-D group. AL~o other amino acid sequence~ may be at;tached
to the "A~ and/or the l'D" end o one of these polypeptide c~ain~.
Al~o there may be amino acids in the chaln which ~pace one or
mor~ group~ of th2 her3inabovo noted four amino acid~ from each
other.
The peptlde~ may be produced by known technique3 and
obtalned in ~ub~tantlally pure ~orm For example, the p~p~ides
'
,
~. .. -
: .
. . :
. .
may be ~ynthe~ized on an automatic ~ynthe~izer. Journal of
: American Chemical Society, Vol 85 Page~ 2149-54~1963~. It is
also po~ible to produce ~uch peptide~ by genetic engineering
technique~.
In accordance with another preferred e~bodiment, the peptide
employed in con~unction with a to~ic cation may be a magainin
peptide.
A magainin peptide i~ either a magainin ~uch a~ magainin I,
II or III or an analogue or derivative thereof. The magainin
peptide~ preferably include th& following basic peptide ~tructure
XlZ
-- Rll-Rll-R12-R13-Rll-R14-R12~
R14 R12 Rll Rll Rll R14a (~15)n R14a R14
wherein Rll i~ a hydrophobic amlno acid, R12 i~ a baslc
hydrophilic amlno acid; Rl3 is a hydrophobic, neutral
hydrophilic, or basic hydrophilic amino acid; R14 and R14a are
hydrophobic or bA~ic hydrophilic amino acld~; R15 i8 glutamic
.` acid or aspartic acid, or a hydrophobic or a ba~ic hydroph~lic
amino acid, and n i8 0 or l. In a preferred embodiment, R13 is a
hydrophobic or neutral hydrophilic am1no acid, R14a i a
hydrophobic amino acid, and R15 i~ glutamic acid or ~spartic
acid.
Thu~, for example, a magainin peptide may include the
following ~tructurc:
-Y12 -X12 -
where X12 i~ th~ hereinabeva de~cribed basic peptide
structure and Yl2 i~
(i) ~12
( i i ~ R14a R12
~iil) Rll ~14a ~12
( 1~ ) R ~ ~R~ a ~
wher~ Rll, Rl~, R~4 and R14~ ara aa previoualy de~inad.
A ma~aitlln peptlde may al~o hava tha followlng ~tructure:
-X Z
:, . , ,- - . . .:
'
-~0~ 7 .,~JJ
wherein X12 i~ as previou~ly defined and Z12 i~:
(i) R~6 where R16 i9 a basic hydrophilic amino acid or
a~paragine or glutamine.
(ii) R16-R17 where R17 i3 a neutral hydrophilic amino acid,
a hydrophobic amino acid, or a basic hydrophilic amino acid.
Preferably, Rl7 i~ a neutral hydrophilic amino acid~
A magainin peptide may al~o have the following structure:
( 12)a- 12 ( 12)b
where X12, Y12 and Z12 are as previou~31y d~fined and a is O
or 1 and b is O or 1.
The maigainin peptides may al~o include the following ba~ic
peptide structure X13:
~ R14 Rll Rl4a R12 Rll Rll R12 ~13
R -R14-R12~ Rll-R~ wherein Rll,R12,R13, 14 14a
are amino acids a~ h~reinabove described.
The magainin peptide may al~o include the following
structure X13-Z13; wherein X13 1~ the h~reinabove de~crlbed ba~ic
peptide ~tructure and Z13 i 8
(Rll)n (Rll)n (~ll)n ~iR14a)n (iRl5)n (R14a)n tR14~n (R16)n
(R ) wherein Rll, Rl4, R14a~ Rls~ 16' 17
her~inabove de~cribed, and n is O or 1, and each n may be the
same or different.
The magainin peptldes generally include at leaat fourteen
amino acids and may include up to orty amino aclds. A magainin
peptide preferably hac 22 or 23 amino acid~. Accordingly, the
hereinabove deacrlbed ba~ic peptlde ~tructure~ of a magainin
peptide may includo additional amino acids at the amino ~end or at
the carboxyl end, or at both end~.
A~ repre~entative example~ of ~uch magainin peptide3, -there
may be mentloned peptid~ having the ~ollowing prlmary ~e~uence
(e~pre~uod a~ a ~lngle letter code) a~ well a~ appropriate
analoglle~ and d~r~vat:lve~ thereo~:
~A) (NH~) GXGK~L~S~G~GKAFVGEIMK~OH) or (N~)
.,
:;~
,.,
.i'~ ~ .
';,
,;' "
:
,
,
-11- 2 ~
(MAgainin I)
(b) (NH2) GIGKFLHSAKKFGKAFVGEIMNS(OH) o~ (NH2)
(Magainin II)
(c) (NH2~ GIGKFLHSAKKFGKAFVGEIMN(OH) or (NH2)
(Magainin III)
The following are example of p~ptide derivative~ or analogs
of the basic structure:
(d) (NH2) IGKFLHSAKKFGKAFVGEIMNS(OH) or (NH~)
(e) (NH~) GKFLHSAKKFGKAFVGEIMNS(OH) or (NH2)
(f) (NH2) KFLHSAKKEGKAEVGE I MNS (OH) or (NH~)
Magainin peptides are described in Proc._Natl. Acad Sci.
Vol. 84 pp. 5449-53 (Aug. 87). The term "magainin p~ptides" as
used hereln refera to the ba~ic magainin ~tructura as well a~
derivatives and analog~ thereof, including but not limited to the
representative derivative~ or analog~.
In accordance with a further embodiment, the peptide
employed in con~unction with a toxic cation may b~ a PGLa peptide
or an XPF peptide.
A PGLa peptide i8 elther PGLa or an analogue or derivative
th~roof. Th~ PGLa peptlde~ pr~ferably include the followlng
ba~ic peptide ~tructure X14:
Rll R17 R12 E?ll R14 R14-R
Rll R14 R12 Rll Rll R12 R
Rl 1 Rl 1 R12
e R11, R1~, R14, and R17 are as previously defined.
The PGLa peptide~ generally include at lea~t ~evante,en amino
acids and may include as many a~ forty amino acids. Accordingly,
the hereinabove described ba~lc peptlde structure for a PGLa
peptida may lnclude additional am~no acid~ at the amino ~nd or at
the carboxyl ~nd or at both the amlno and carboxyl ~nd.
Thu~, Eor oxamplq, a PGL~ p~ptid~ may havo the followin~
~trUcture:
Y14 ~14
where X~ a~ proviou~ly dein~d and
.
'''
i'
.
,
-12- 2 ~
Ylg i~
(i) Rll;
( ii ) R14-R
whera Rll and R14 are as previoualy defined.
For example, a PGLa peptlde may al~o have the following
~tructure:
-X14-Z14-
where X14 i8 a~ previou~ly defined; and Z14 i~:
(i) Rll; or
( ii ) Rll-Rll
where R~ a~ previou~ly defined.
A PGLa pepticle may al~o have the following structure:
~4 ) a 1~ ( Zl4 )b
, X14; Y14 and Z14 are a~ praviou~ly defin0d, a i O or
: 1 and b i8 0 or 1.
: An XPF peptide i~ either XPF or an analogue or derivative
thereof. The XPF peptide~ preferably include the follow:ing basic
peptlde structur~ Xl6:
. -~Rll~R17~~12 Rll R14 R18 R~7
R ~R -R -R -R -R
: 11 14 12 11 11 12
11 Rll Rll R12~(R15)n~R~ , wherein
Rll, Rl~, Rl~, R15 and R17 are a~ previou~ly d~fined ~nd
R18 i~ glutamine or a~paragine or a basic hydrophilic, or
hydrophobic amino acid and, n i~ O or l.
The XPE peptides generally include at lea~t nineteerl amino
acida and may include up to forty amino acid~. Accordingly, the
hereinabove do~cribed baalc peptide ~tructure of XPF may include
additional amino acid~ at the amino end, or at the carboxyl end
or at both the amino and carboxyl end~.
; Thu~, ~or example, an XPF pep~.ide ~ay include the following.: ~truc~ur~
; -~16 X
wh~re X~ a~ pr~viou~ly d~1ned ~nd Y16 i~
,,
:
'
r
:
~ ~ .
. .
-13
(i) Rll or
( ii ) R14-R1 1
where Rll and R1~ are as previou~ly defined.
An XPF peptide may include the following ~tructure:
-X1 6~
where X16 i~ as previously deflned and Z16 i~
11; r
(ii) R11 R18; or
(iii) 11 R1g~Prline; or
( iv) ~ll-R18-Prline R12
An XPF pep~ide may alBo have the followinc~ structure:
(Y16)a X16(Z16)b
where X16, Yl~ and Z16 are a~ previously deflned: a i~ 0 or
1 and b i 8 0 or 1.
Preforred ar3 XPF or PGLa peptide~, which are characterized ~,
: by the following primary amino acid ~equenc~single letter amino
acid code): ;
PGLa : GMASKAGAIAGKIAKVALKAL ~NH2
. XPF : CWASKIG~TLGKIAKVGLKELIQPK
: A review of XPF and PGLa can be found in Hoffman et al, EMBO
J. 2:711-714, 1983; Andreu et al, J. Biochem. 149:531-535, 19~5;
Glb~on et al J. Biol. Chem. 261:5341-5349, 1986; and GiovAnnini
et al, Biochem J. 243:113-120, 1987.
In accordance with yet another ~mbodiment, the pepti.de
'. employed in coniunction with a toxic cation may be a CPF peptide
or appropriato analogue or derviative thereof.
CPF peptide~ a~ well a~ analo~ue~ and derivative~ thereof
are h~rein ~emetime~ r~ferred to collectively a~ CPF pept:ide~.
Th~ CPF pe~pti~ preferably one which include~ ~he
followin~ paptlde ~tructura X30:
R21 R21 ~22 ~22 ~Zl ~ 3 ~21
-R2~ 3~R21-R~ R~5 21
wherein ~21 i~ a hydrophoblc amino acid;
' .
.
' . .
,~ , . ,
.
,
1 ~ 2 ~
R22 i~ a hydrophobic amino acid or a ba~ic hydrophilic amino
acid;
R23 i~ a basic hydrophilic amino acid; and
R24 is a hydrophobic or neutral hydrophilic amino acid; and
R25 i8 a basic or neutral hydrophilic amino acid.
The hereinabove basic ~tructur~ i~ hereinafter symbolically
indicated as X30.
The hydrophobic amino acids are ~la, Cys, Phe, Gly, Ile,
Leu, Met, Val, Trp, and Tyr.
The neutral hydrophilic amino acid~ are A~ln, Gln, Ser, and
Thr.
The ba~ic hydrophllic amino acld~ are Ly~, Arg, Hi~ and
ornithine.
: The CPF peptide may include only the hereinabove noted amino
acids or may include additional amino acids at the amino end or
carboxyl and or both the amino and carboxyl end. In general, the
pept~de doe~ not include more than 40 amino acids.
. The CPF peptide~ including the absve ba~ic peptide ~tructure
,~ may have from 1 to 4 additional amino acid~ at the amino end.
Accordingly, such preferred peptide~ may be repre~ented by the
~tructural formula:
~30 ~30
wherein X30 i~ the hereinabove de~crib~d ba~ic peptide
.' ~tructure and Y30 i~
.~ (i) R25_, or
(ii) R22 R25; or
iii ) R21 R22 R2s;
(iv) R22 R21 E~22~R25; pre~erably
~l yG 1 n~ -R~l-R2~ R~5
R21, R22, and R25 ar~ a~ prettiou~ly d~fined
Th~ cQrboxyl ond oP the ba~ic p~ptlde ~tructure may al~o
hav~ additlonal amino acid~ which may ran~e ~rom 1 to 13
addltional amlno acld~.
;
'.,~
, ~
.
1 5
: In a prsferred embodiment, the ba~ic structure may have from
1 to 7 addltional amino acid~ at the carboxyl end, whlch may be
represented a~ followa:
-X30-z30 wherein
X30 i~ the hereinabove defined ba~is peptide structure and
Z30 i~
(ii~ R21 R21 ;
R2 1 R2 1 R2 4;
(iv) ~21 R21 R24 R24;
(v) R21-R21-R24-R24 R26;
(vi) R21 R21-R24 ~24~R~6-Gln; or
(vii) R21 R21 R24~R24~R26-Gln-Gln~
wherein R21 and R24 are as previously defined, and R~6 i~ :;
proline or a hydrophobic amino acid.
P-eferred peptides may be repre~ented by the following
structural formula:
~Y30)a X30 (Z30)~
.:
wherein X30, Y30 and Z30 are a~ previou~ly defi~ed and a i :-
. O or 1 and b 1~ 0 or 1.
;.~ Repra~entative example~ of CPF peptide~ which are u~eful in
the pre3ent invention ~ome of which have been de~cribed in the
.; literatur~ and compr~a the followin~ seguencea (~lngle l~tter
amino acid code~:
~1) GFGSFLGLALXAALKIGANALGGAPQQ
: (2) GLASFLGKALKAGLKIGAHLLGGAPQQ
(3) GLASLLGKALKAGLKIGTHFLGGAPQQ
.; (4) GLASLLGKALKAThKIGTHFLGGAPQQ
(5) GFAS~LGKALKAALKIG~NMLGGTPQQ
'~ (li) C;F~FLGK~LE~ L~CIaANALGGAPQQ
(7) GFCSFLCKALKA~LKI~NALGGSPQQ
~) GFAS~LGKALKAALKIGANLLGGTPQQ
., ,
,.,
;'
.
'~
-lB-
A review of the CPF peptide~ can be found in Richter, K.,
Egger, R., and Kreil (1986) J. Biol. Chem. 261, 3676-3680;
Wakabaya~hi, T. Kato, H., and Tachibaba, S. (1985) Nuclelc Acid~
Research 13, 1817-1828; Gib~on, B.W., Poulter, L., William~,
D.H., and Maygio, J.E. (1986) J. Biol. Chem. 261, 5341-5349.
CPF peptides which may be employed in the present invention
are represented by the following (single letter amino acid code):
G12S3LG4ALKA5LKIG678LGG9(10)QQ
Where:
1 = F, L
2 = G, A
-~ 3 ~ F, L
= K, L
= A, G, T
6 = A, T
7 = H, N
B = A, M, F, L
. 9 = A, S, T
j 10 = P, L
The numbered amino acids may be employed a~ described in any
combination to provide either a basic CPF peptide ~ructure or an
analogue or derivative. The term CPF peptide include~ the basic
peptide ~tructure as well a~ analog~ or derivatives thereof.
In accordance with ~tlll anothor embodiment, the
biologically activo peptide may include the following ba~iic
. ~trucutre X40:
[R41 R42 R42 R43 R41-R4~-R42]n, wh~rein R41 is a ba~ic
hydrophillc amino acid, R42 i~ a hydrophobic amino acid, R43 i~ a
neutral hydrophllic or hydrophobic amino acid, and n is from 2 to
:: 5.
In one embodiment, all o~ the amino acid residues are
,~ D-amlno acid re~idues. Thu~, in a preferred embodimenk, each o~
tho amino ncid re~idue~ aro D amino acid re~idue~ or L-amino acid
ra~idue~,
"
~ ' ' ' ', ' '
In one embodiment, ~uch peptide may include the following
~tructure:
Y40-X40, wherein X40 i3 a~ hereinabove de~cribed, and Y40
i~: ;
(i) R42; `
(ii) R42 R42;
(iii) R4l-R42 R42;
(iv) R43-R4l-R42 R42;
(v) R42-R43-R41 R42 R42;
( ) R42 R42 ~43 R4l R42-R42~ wharein R4l, R42, and R~3 are
a~ herelnabove described
In accordance with another embodiment, ~uch peptide may
include the following ~tructure:
X4~Z40, wherein X40 is a~ hereinabove de~cribed, and 240
i3:
' (i) R41; :
(il) R4l R42;
) R4l~R4~ R42;
(iv~ R4l R42 R42 43
(v) R4l-R42-R~2 R43 R4l;
(vi) ~4l-R42~R42~R43 R4l 42
In accordance another embodiment, ~uch peptide ~ay include
the following structure:
. (Y40)a X40 (Z40)b, wherein Y and Z are a~ previously
defined, a i8 0 or l, and b i~ O or l.
In one embodiment, n i8 3, and mo~t preferably the peptide
i~ oP the followinq ~tructure aa indicated by the ~ingle.letter
amino acid code:
l KIAGRIA 1 3,
In ano~her embodiment, n i~ 2, and tho peptide preferably i~
o~ the following ~tr~ctur~ a~ in~icated by the single lett:er
a~ino acld co~a~
~ IA(KIP~C:KI~)2KIAG.
: '
: :
In accordance with yet another embodiment, the biologically
active amphiphilic peptide may be a biologically active
amphiphilic pepttde including the following ba~ic ~tructure X50:
R -R -R4Z-R43-R41-R42-R42-R41-R42 R42 42 41 42 42
wherein R41, R42 and R~3 are as hereinabove de~cribed.
In one embodiment, all of the amino acid re~idue~ are
D-amino acid residue~. Thus, in a pr~ferred embodiment, each of
the amino acid ro~idues in the peptide are D-amino acid re~idues
or L-amino acid re~idue3.
In accordance with one embodiment, such peptide may inclu~e
the followin~ structure:
; Y50--X50, wherein X50 i~ a~ hereinabove d~cribed, and Y50
;,.:.` i~:
: (i) R42;
(ii) R~2-R42;
p (lii) R41-R42 R42;
(iv) R43-R~l-R42-R42;
(v) R~2-R43-R41 R42 4~
., (vi) 42 42 R43 R41 42 42'
( ) R41 R42 R42 R43~R41~R42~R42,Wharein R41, R42 and R43
are as hereinabo~e de~cribed.
In accordance with another embodiment, such peptide may
include the ~ollvwing structure:
X50-Z50, wherein X50 i~ a~ herainabove described and Z50 i~:
( i ) R~
(ii) R41 ~42;
(iii~ R41-R42 R42;
(iv) R~l~R42 ~42 43
.. (v) ~4~-R42~R42~R~3 R41;
4~ R~z ~ 3~R~1-R~2; o~
) 4~ 4~ R42 R43 R4~-R~ 2~ wherein R~1, R~2 and R
are a~ hereinabove de~crlbed.
In accordanc~ with yet another embodiment the peptide may
include tha ~olLowing ~tructura:
-19-
~ Y50)a-X50-(Z50)b~ wherein X and Y are a3 previously
defined, a i~ O or 1, and b iR O or 1. }n one embodiment, the
peptide i~ of the followlng ~tructural formula a~ indicated by
tho Bingle letter amino acid code:
KLASKAGKIAGKIAKVALKAL.
In another embodiment, khe peptide i9 of the following
structural formula as indicated by the ~ing:Le l~tter amino acid
code:
KIAGKIAKIAGOIAKIAGKIA.
In still another embodiment, the peptide employed in
conjunction with a toxic cation i~ a cecropin. The cecropins and
analogs and derivative~ thereof are de~cribed in Ann. Rev.
Microbiol 1987 Vol 41 page~ 103-26, in particular p. 108 and
- Christensen at al PNAS Vol. 85 p. 5072-76, which are hereby
incorporated by reference.
.~. The term cacropin~ include~ the basic ~tructure as well as
analogue~ and dsrivativea.
In yet another embodiment, the peptide employed in
conjunction with the toxic cat1on i9 a ~arcotoxin. The
~arcotoxins and analog~ and deriv~tives thereof are de~cribed in
Molecular Entomology page~ 369-78 in particular p. 375 Alan R.
LiR~ Inc. (1987), which i~ hereby incorporated by reerence.
The term ~arcotoxin include~ the basic materials aa well aq
analogue~ and derivative~.
In another embodiment, an ion channel-forming protein may be
u~ed in con~unction with a toxic cation. Ion channel-forming
protein~ which may be employed include defensins, al~o known as
human neutrophil antimicrobial peptlde~ ~HNP), major ba~ic
protein ~MBP) of eoainophils, bactericidal
p~rm~abiliky-incroa~lng protein (BPI), and a pore-forming
cytotoxin called variou~ly perforin, cytoly0in, or pore-~orming
protein. De~en~inu are described in Selaked, et al., J._Clin.
Inve~t., Vol. 76, pg8. 1436-1439 ~19~5~. MBP protein~ are
de~aribed in Wa~moen, et al., J. ~iol. Ch~m., Vol. 263, p~
:.
'
.. ~
: ' .:
2 C
1~559-12563. (1988). 8PI proteins are de~cribed in Ooi, et al,
J. Biol. Chem., Vol. 262, pg8. 14891-14894 (19B7~. Perforin i9
described in Henkart, et al., J. Ex~. Med., 160: 75 (1984), and
in Podack, et al., J. Ex~. Med., 160:695 (1984). The above
articles are hereby incoroporated by reference.
The term ion channel-forming protein~ include-~ the basic
tructure~ of the ion-forming proteins a3 well a~ analogue~ and
derivative~.
The present invention will be further de~cribed with respect
to the following example~, howeYer, the ~cope of the invantion i~
not to be limited thereby.
"
E~alOV1Q 1
Approximately 1-5 x 105 colony forming unita (CFU3) of P.
aeruainosa strain 27853, dicper~ed in 100 ~1 of Tryp~ica~e ~oy
broth ~TSB), were added to each test well. B13-33 peptide was
added in increa~ing concentratlon~ from 0.25 to 256 ~g/ml in the
absence of or pro~snce of ~lver nltrate. Silver nltrate wa~
al80 added in increasing concentrations from 0.25 to 256 ~g/ml in
the ab~ence or pxesence o~ B13-33 peptide. B13-33 peptide is of
the following ~tructure
GIGKFLKKAKKFGKAFVKIMKK
The following peptide combinations were te~ted or Minimal
Inhibitory Concentr~tion (MIC) of the B13-33 peptlde:
MIC (uq/ml)
l. B13-33 peptide alone 4-16
2. B13~33 peptlde plu8 <0. 03
5 ~g/ml ~ilver nitrate
Th~ Pollowing comblnatlon~ were te~ted for tha MIC o~ ~ilver
ni trato:
MI I u~ml
.
" .:
.
~ . " .
-
., ~
: , ,
'J'`' ~ t
1. Silver nitrate alone 4-8
2. Silver nitrate plus 2-8
1.6 ~g/ml B13-33 peptide
3. Silver nitrate plus <0.25
5 ~g/ml B13-33 p~ptide
As can be 3een, in the ab~ence of ~ilv~sr nitrate, the MIC of
B13-33 peptide against P. aeruqino~a ~train 27853 wa3 from 4-16
~g/ml. In the presence of 5 ~g/ml of ~ilve:r nitrate, the MIC
against the ~ame organi~m fell to lee~ than 0.03 ~g/ml.
In the ab~ence of B13-33 pep-tide, the MIC o silver nitrate
again~t P. aeruqinosa wa~ 2-8 ~g/ml. When 5 ~g/ml of B13-33
peptide waa ad~ed, the MIC fell to le~ than 0.25 ~g/ml.
li~amp19 2
Approximately 1-5 x 105 CFU~ of P. aeruainosa ~train 27853
disper3ed in 100 ~1 of TSB were added to each te~t well. Ei-ther
Peptide 1 or Peptide 2 waa added in increa~ing concentrations
from 0.25 to 256 ~g/ml in the ab~ence o or in the pre~ence of
silver nitrate.
Peptide 1 i~ of the following structure:
[KIAGKIA13
Peptide 2 i8 of the following structure:
KIAGKIA~IAGOIAKIAG~IA.
The following combinations of Peptide 1 or Peptide 2, with
or wlthout ~ilve nitrate, were tested for Minlmal Inhibitory
Concentratlon (M~C) of Peptide 1 or Peptide 2:
M I C L~m~
1. Peptide 1 alone 1~-32
2. Sllv~r n:l~rate alone
3 . P~ptid~ 1 plu~ S ,ug/mL co . 03
~i lver nitra~e
4. Peptlde 2 ~lon~ 16
'~ :
.
:
-22~
5. Peptide 2 plu~ 5 ~g/ml <0.03
sllver nitrate
....
Example 3
. Approximately 1-5 x 105 CFU~ of a clinical i~olate of a
. methicillin resistant S. aureus ~MSRA), di~per~Pd in 100 ~1 of
; TSB were added to each te~t well. Either E~13-33, Peptide 1, or
Peptide 2 was added in increasing concentration~ from 0.25 to 256
~g/ml in the ab~ence of or the pressnce of ~ilver nitrate. The
MIC of each peptide alone, and of ~ilver nitrate alone was
datermined. The MIC of each peptide when 5 ~lg/ml a~d~or 10 ~g/ml
of ~ilver nitrate was added was detarmlned a~ well. Tha MIC of
the variuo~ peptides and combinations of peptides and silver
nitrate are listed below.
.~, .
. MIC (uqiml~
1. B13-33 alone
:.
2. Silver nitrate alone 16
3. Bl3-33 plus 5 ~g/ml
silver nitrate
. 4. B13-33 plu~ 10 ~g/ml <0.03
silver nitrato
5. Peptide 1 alone 8
6. Peptide 1 plus lO
~g/ml ~ilver nltrate
7. Peptide 2 elone 8
8. Peptida 2 plu8 10
~/ml ~ilver nitrate
x~a~
Approximately l-S ~ 105 C~U~ of ~ J~ o~ ~train 27a53
dl~per~d ln lO0 ~l o~ TSB Were added ~o each te~t woll.
I Cecropin A~~ wa~ added in incre~i~g amount~ from 0.25 ~g/ml to
: 256 ~g/ml in the ab~ence o~ or in the pre~enc~ o~ silver nitrate.
. , .
;.
-23~
Cecropin A-D is of the following structure:
KWKLFKKIEKVGQRVRDAVISAGPAVATVAQATALAK.
The following combinations of Cecropin A-D with or without
~ilver nitrate, were tested for Minimal Inhibitory Concentration
-` (MIC) of Cecropin A-D;
.~ .
MIC ~g/ml)
` l. CeGrOpin A-D alone 16
: 2. Silver nitrate alone 8
i 3. Cecrepln A-D plu~ ~0.25
: . 3 ~g/ml silver nitrate
' E~ample 5
Approximately 1-5 x 105 CFU~ of P. aeruqino~a ~train 27853
;: di3per~ed in 100 ~l of TSB were added to each te~t well.
: . .
Magainin II (amide-texminated) wa~ added in increaaing amounts
from 0.25 to 256 ~g/ml in the absence of or in the prs~ence of
ailver nitrate. The following comblnations of Magainin II, with
or without silver nitrate, were te~ted for Minimal Inhlbitory
Concentration (MIC) of Magalnin II:
MIC (~q/ml)
1. Magainin II alone 256
2. Silver nitrate alone 8
3. Magainin II plu~ 1 ~g/ml 32
silver nitrate
4. Magainin II plu~ 2 ~g/ml 16
8i lvar nitrate
5. Magainin II plu~ 3 ~g/ml 8
~llvar nitrate
6. Magainln XI plua ~ ~g/ml ~0.03
~ilver ni~rate
p~ 6
-2~-
Approximately 1-5 x 105 CFU~ of P. aeruaino~a atrain 107 which
.,IJI wa~ gentamicin re~i~tant, di~per~ed irl 100 ~l of TSB were added
~ to each test w~ll. Magainin II (amide-termianted) wa~ added in
. ~ .
increa~ing amount~ from 0.25 to 256 ~g/ml ln the ab~ence of or in
the pre~ence of 3ilver nitrate. The following combination~ of
Magainin II, with or without silver nitrate, were te~ted for
- Minlmum Inhibitory Concentration (MIC) of Magainin II:
. .
MIC (uq/ml)
~ 1. Magainin II alone 256
2. Sllver nitrate alone 8
3. Magalnin II plu~ 1 ~g/ml 32
~ilver nitrate
4. Mag~inin iI plu~ 2 ~g/ml 16
Yer nitrate
5. Magainin II plu~ 3 ~g/ml 16
3ilver nltrate
6. Magainin II plu~ 4 ~g/ml 4
silver nitrate
7. Magainin II plu~ 5 ~g/ml <0.03
8i lver nitrate
Example 7
Th~ procedure for this a~ay, which i~ a checkerboa:rd ~ay,
i~ performed a~ de~cribed ~n Antibiotic~ and LaboratorY Medicine,
2nd ed., Victor Lorian, M.D. Editor, p~. 540-546 (1986).
The checkerboard a~ay wa~ carried out in a 96-well
microtlter plate. The plate ha~ twelve rows (Row~ 1 through 12),
and ei~ht column~ ~Column~ A through H) of well~. 100 ~l of
plain broth i~ added to each well o~ the 9~-wall pl~te. 100~1 o~
4X concentratlon o~ Papt~de 3 i~ addad to Row 1 and 100
~erlall~ dilu~ed 1:2 ~rom Row 1 to Row 11. Concentra~;ton~ o~
Peptide 3 range ~rom 1 to 1,024 ~g/ml. S.tlver nitr~te (~gN03) i~
~arially diluted 1:~ at 4X the inal da~tred concentr~tlon in a
-25- ~ t :`~
~et of tubes. Each 4X ~olution of silver nit~ate i~ then added
to the appropriate column of the microtiter plate. ~Column~ A
through G.~ To Column H, 50~1 of plain broth is ~dded. (No
silver nitrate is added to Column H.) Concentrations of ~ilver
nitrate range from 0.25 to 16 ~g/ml. Peptide 3 has the following
structural formula: GIKKFLKKAGKFGKAF. 50~1 of S. aureu~ strain
25923 is then added to each well. Each well has a concentration
1-5 x 10 organi~ms per well. The plate i~ then incubated at
35-37C for 18 to 24 hours, and the well~ are then examin~d for
the presence of vi~ible growth; i.e., turb:ldity. The
checkerboard assay procedure was carried out in duplicate.
In the checkerboard a~aay in which Peptide 3 and silver
nltrate were tested for activity again~t S. aureus, the MIC of
Peptide 3 was 512~g/ml, and the MIC of silver nitrate was 8ug/ml.
Comblnatlon~ of varylng amounts of Peptide 3 and 3i lver
nitrate which re~ultecl in inhibition of the growth of S. aureus,
and the FIC value~ of each combination, were then determined.
- FIC, or Eractional Inhibitory Index, i~ determined as
follow~:
FIC = MIC of peptide in combination MIC of AgN03 ln comblnation
. ~ MIC of AgN0 alone
MIC of peptlde a}one 3
An FIC value of 0.5 or les~ i8 indicative of ~ynergy, a value of
greater than 0.5 but le~ than 2 i8 indicative of indifference,
ancl a value greater than 2 is indicative of antagoni~m. The
following combinatlonn of Peptide 3 and ~ilver nitrate wer~ ~ound
to be inhibitory, and tho FIC value~ of each combination are
givan herewith.
Combination FIC
2$~g/ml Peptida 3 plu8 0 . 25~g/ml AgN03 0.53
2S6~y/ml Peptide 3 plu~ O.S~g/ml AgN03 0.56
12~g/ml Paptide 3 plu~ l~g/ml AgN03 0.3
16~g/ml Peptide 3 plu~ 2~yfml AgN03 0.~8
2~/ml Peptide 3 plu~ ~g/ml AgN03 0.50
.
. ' :
2 ~ f''~
Th~ above re~ult~ ~ugge~t that ther~ i~ a ~ynergi~tio
tnteraction between Peptlde 3 and allver nitrate wh~n employ~d in
combination to inhibit growth of S.aureus.
The peptide or protein and toxic cati.on, as hereinabove
de~cribed, may be employed for treating a wide variety of ho~t3.
In accordance with a preferred embodiment, a ho~t i9 an anima~,
and such animal may be a human or non-human animal. The peptide
or protein and the toxic cation may be employed together in a
single composition, or in separate composition~. Moreover, the
toxic cation and the peptide or protein may be delivered or
administered in diffarent forms, for example, the toxic cation
may be administered topicaIly, while the peptide or protein may
be administered by IV or IP.
The pept1de or protein and/or toxic cation may be employed
in a wide variety of pharmaceutical compo~ition~ in combination ;~
with a non-toxic pharmaceutical carrier or vehicle f~uch as a
filler, non-toxic buffer, or physiological saline ~olution. Such
pharmaceutical composltions may be u~ed topically or ~ystemically
and may be in any suitable form such a~ a liquld, solid,
semi-~olid, injectable ~olution, tablet, ointment, lo ion, paste,
capsule, or the like. The peptlde or protain and/or toxic cation
may also be u~ed in combination with ad~uvant~, protease
inhibitors, or compatible drug~ where ~uch a combination i~ seen
to be desirable or advantageous in controlling inf ction cau~ed
by harmful microorgani~m~ including protozoa, viruses, parasites
and the like.
The peptide( 8 ) or protein of the present invention may be
admini~tered to a ho~t in particular an animal, in an ef~ective
~ntibiotic and/or anti-viral and/or anti microbial and/or
anti~para~itic amount in con~unction with a toxlc cation for
potentiating the activity of the peptide or protein.
A3 rapro~entatlve examples of administering the peptide or
protein and toxic catlon for topical or local administratlon, the
peptlde could be a~mini~tared in an amount of up to about 1%
''
. ,
-27-
weight to weight and the toxic cation delivared in an amount ofabout 50 mM (about 0.1%). Alternatively, the toxic cation could
be administered topically in conjunction with systemic
administration of the peptide and/or protein. For example, the
peptide or protein may be administered IV or IP to achieve a
~erum dose of 100 microgram~ per milllliter (10 milligram~ per
kilogram) in conjunction with a topical doae of toxic cation of
from about 4 ~g/m7 to about 100 ~g/ml.
Numerous modifications and variationa of the present
invention are possible in light of the above teachinga and,
therefore, within the scope of the appended ciaim~, the invention
may be practiced otherwi~e than a~ particularly de~cribed.
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