Note: Descriptions are shown in the official language in which they were submitted.
1~790i
Field of the Invention
This invention relates to a pharmaceutical composition of a
combination of a protease inhibitor and a peptide, and to a
method for treating herpes viral infections in a mammal by
administering the combination to the mammal.
Background of the Invention
Herpes viral infections are common and their occurrences
represent serious economic and social problems. Among the most
common forms of these infections are herpes labialis caused by
herpes simplex virus type 1 (HSV-1) and herpes genitalis caused
by herpes simplex virus type 2 (HSV-2). Most of the existing
medications for herpes viral infections are only marginally
effective and can produce side effects. Hence, there is a need
for a safe and effective means to treat herpes viral infec-
tions.
Recently, E.A. Cohen, P. Gaudreau, P. Brazeau and Y.
Langelier, Nature, 321, 441 (1986) reported that the nonapeptide
H-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH, corresponding to the
carboxy-terminal nine amino acids of the smaller of two subunits
of the herpes simplex virus ribonucleotide reductase, inhibits
the action of the reductase, which is an enzyme required for
viral replication. Simultaneously, the same finding was report-
ed by B.M. Dutia et al., Nature, 321, 439(1986).
1 .
` lZ~79~i '
Prior to the actual publications, this finding had led to the
discovery of a new class of antiviral peptides free of delete-
rious effects. The particular antiviral peptides, described in
more detail hereinafter, are disclosed by E. Cohen et al. in
Canadian patent application, serial no. 509527, filed May 20,
1986.
Now it has been found that the activity of the antiviral
peptides can be enchanced significantly by combining the same
with a protease inhibitor, for example, the antibiotic bacitra-
cin. In this context, it is interesting to note that bacitracin,when used alone, has been reported as being inactive against
HSV-1 by A. Alarcon et al., Antiviral Research, 4, 231 (1984);
and that bacitracin in combination with neomycin and
glycyrrhizin has been proposed for treating oral infections, R.
Segal et al., U.K. Patent Application 2167296, published May 29,
1986. Hence, the enhancement of the antiviral activity realized
with the straight forward combination of this invention
represents an unexpected turn of events.
Summary of the Invention
Accordingly, an anti-herpes viral pharmaceutical composi-
tion is provided which comprises an anti-herpes virally effec-
tive amount of a combination of a protease inhibitor, a peptide
of formula 1
. A-R8-R9-R10-Rll-R12-R13-R14-R15-B
"--``` lZ779~1
wherein A i3 L-(aa)0_6-R7- wherein L i~ hydrogen or
lower acyl; aa is an amino acid residue derived from an amino
acid selected from the group of Y ~ CH2CH(NH2)COOH
(wherein Y is halo), norleucine, norvaline, the natural amino
acids excluding cystine, and any of the enantiomorphic forms
thereof (each cf the aa in the radical aa2_6 being the
~ame or a different amino acid residue in relation to aal),
and R7 is an amino acid residue derived from an amino acid
selected from the group of Z ~ CH2CH(NH2)COOH(wherein
Z i~ hydrogen, halo or hydroxy), His and Trp, and any of the
enantiomorphic forms thereof; or A is X ~ CH2CH2CO
wherein X iR hydrogen, halo or hydroxy; and R8, R9, R10,
R13, R14 and R15 are independently an amino acid
re~idue (aa) as defined hereinabove; Rll and R12 are
independently Val, D-Val, Nva, or D-Nva; and B is hydroxy, amino
or lower alkyl amino; or a therapeutically acceptable salt
thereof; and a pharmaceutically acceptable carrier.
A preferred pharmaceutical composition of this invention
comprises an anti-herpes virally effective amount of a combina-
tion of a protease inhibitor; a peptide of formula la
L Rl R2~R3 R4 R5-R6-R7-R8-R9-Rlo-Rll-Rl2-Rl3-Rl -R -B
la
wherein L is hydrogen or lower acyl; Rl to R6, inclusive,
R9, R10 and R13 to R15, inclusive, are indepen-
dently an amino acid residue as defined hereinabove; R7
~;~'779(11
is -NHCH(CH2 ~ Z)CO- wherein Z is hydrogen, halo or
hydroxy, or any of the enantiomorphic forms thereof; R is
Ala, D-Ala, Thr, D-Thr, Leu, D-Leu, Ile or D-Ile; R11 and
Rl2 are independently Val, D-Val, Nva or D-Nva; and B is
hydroxy or aminoi provided that any or all of the residues
to R6, inclusive, may be deleted; or a therapeutically accept-
able salt thereof; and a pharmaceutically acceptable carrier.
Another preferred pharmaceutical composition of this inven-
tion comprises an anti-herpes virally effective amount of a
combination of a protease inhibitor; a peptide of formula lb
X ~ CH2CH2co-R8-R9-Rlo-Rll-Rl2-Rl3-Rl4 Rl5 B
1b
wherein X is hydrogen, halo or hydroxy, and R8, R9, RlO,
R11 R1 2 Rl3~ Rl4 an d Rl5 ar e as de f in ed
hereinabove for formula 1i and B is hydroxy or amino; or a ther-
apeutically acceptable salt thereof; and a pharmaceutically
acceptable carrier.
A method of treating herpes viral infections in a mammal
also is provided. The method comprises administering to the
mammal an anti-herpes virally effective amount of a combination
of a protease inhibitor and a peptide of formula l, formula la
or formula lb, or a therapeutically acceptable salt thereof, as
defined hereinabove.
4.
~ 1Z779Q~
Details of the Invention
In general, the abbreviations used herein ~or designating
the amino acids and the protective groups are based on recommen-
dations of the IUPAC-IUB Commission on Biochemical Nomenclature,
see Biochemistry, 11, 1726-1732 (1972). For in~tance, Ser, Tyr,
D-Trp, Sar, D-Nle, Nva, Leu, Arg and Gly represent the
'residues' of L-serine, L-tyrosine, D-trytophan, L-sarcosine,
D-norleucine, L-norvaline, L-leucine, L-arginine and glycine,
respectively.
The term 'amino acid residue' refers to a radical derived
from the corresponding a-amino acid by eliminating the hydroxyl
of the carboxyl group and one hydrogen of the -amino group.
The term 'natural amino acid' means an amino acid which
occurs in nature or which is incorporated as an amino acid resi-
due in a naturally occurring peptide, exclusive of the amino
acid cystine. Such amino acids are described, for example, in
general textbooks of peptide chemistry; for example, K.D.
Kopple, "Peptides and Amino Acids", W.A. Benjamin, Inc, New
York, 1966, and E. Schroder and K.L. Lubke, "The Peptides",
Vol.l, Academic Press, New York, 1965, and include alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid,
glutamine, glycine, histidine, hydroxylysine, hydroxyproline,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
pyroglutamic acid, sarcosine, serine, threonine, tryptophane,
tyrosine and valine.
5.
~" lZ7790~
The term 'halo' as u~ed hereln mean~ the halo radical ~elected
from bromo, chloro, fluoro or iodo.
The term 'lower alkyl' as used herein means straight chain
alkyl radicals containing one to four carbon atoms and branched
chain alkyl radicals containing three or four carbon atoms and
includes methyl, ethyl, propyl, butyl, l-methylethyl, 2-methyl-
propyl and l,l-dimethylethyl.
The term 'pharmaceutically acceptable carrier' as used
herein means a non-toxic, generally inert vehicle for the active
ingredient which does not adversely affect the ingredient.
The term 'phy~iologically acceptable carrier' as used here-
in mean~ an acceptable cosmetic vehicle of one or more non-toxic
excipients, which do not react with the active ingredients con-
tained therein or reduce their effectiveness.
The peptides of formula 1, disclosed by E.A. Cohen et al in
Canadian patent application, serial no. 509,527, filed May 20,
1986, are prepared by processes described therein; namely, by
exclusive solid phase techniques, by partial solid phase techni-
que~ and/or fragment condensation, or by classical solution
coupling.
The term 'protease inhibitor' means an agent capable of
inhibiting the hydrolysis or proteolysis of peptides by protea-
ses. Suitable protease inhibitors are the commercially
6.
~277901
available amastatin, antipain, epiamastatin, aprotinin, chymo-
statin, leupeptin, and preferably bacitracin or its therapeutic-
ally acceptable salts. Bacitracin and its therapeutically ac-
ceptable salts, for instance, zinc bacitracin, manganese baci-
tracin, sodium bacitracin and bacitracin methylenedisalicylic
acid, are described by G.A. Brewer in 'Analytical Profiles of
Drug Substances', volume 9, Academic Press, New York, N.Y. USA,
1980, pp 1-69.
The term 'effective amount' means a predetermined antiviral
amount of the antiviral agent, i.e. an amount of the combina-
tion sufficient to be effective against the viral organisms in
vivo.
The antiviral activity of the combination of the proteo-
lytic inhibitor and the peptide of formula 1 can be demonstrated
by biochemical, microbiological and biological procedures show-
ing the inhibitory effect of the combination on the replication
of HSV-1 and HSV-2, and other herpes viruses, for example, vari-
cella zoster virus (VZV), Epstein-Barr virus (EBV), equine her-
pes virus (EHV) and pseudorabies virus (PRV).
. . --
~27790~
Noteworthy is the fact that all of the aforementioned vi-
ruses are dependent on ribonucleotide reductase to synthesize
deoxyribonucleotides for their replication. Although this fact
may not be an essential requirement for the antiviral activity
of the combination of this invention, the combination has been
shown so far to have antiviral properties against all viruses
dependent on ribonucleotide reductase to synthesis DNA for their
replication.
A biochemical assay for demonstrating the inhibitory effect
of the combination with respect to the ~pecific inhibition of
herpes ribonucleotide reductase is the assay described by
E.A. Cohen et al., Nature, 321, 441 (1986~.
Noteworthy, in the connection with the specific inhibition
of herpes nucleotide reductase, is the absence of such an effect
by the combination on cellular ribonucleotide reductase activity
required for normal cell replication.
Another method for demonstrating the inhibitory effect of
the combination on viral replication is the cell culture techni-
que; see, for example, T. Spector et al., Proc. Natl. Acad.
Sci. USA, 82, 4254 (1985). This method in a modified form is
exemplified hereinafter.
.. .. ...
,
~2~7901
A method for demonstrating the therapeutic effect of the
combination is the guinea pig model for cutaneous herpes simplex
viral infections; see, for example, S. Alenius and B. Oberg,
Archives of Virology, 58, 277 (1978). A modification of this
method is exemplified hereinafter.
When utilizing the protease inhibitor in combination with
the peptide, or its therapeutically acceptable salt, for treat-
ing viral inPections, the combination is administered topically
to warm blooded animals, e.g. humans, pigs or horses, in a vehi^
cle comprising one or more pharmaceutically acceptable carriers,
the proportion of which is determined by the solubility and
chemical nature of the protease inhibitor and peptide, chosen
route of administration and standard biological practice. For
example, the two active agents (i.e. the protease inhibitor and
the peptide of formula 1 or a therapeutically acceptable salt
thereof) can be formulated in the form of solutions, emulsions,
creams, or lotions in pharmaceutically acceptable vehicles. Such
formulation can contain 0.01-0.2 percent, preferably 0.05 to 0.2
percent, by weight of the peptide and about 0.5 to 3, preferably
0.8 to 1.5, molar equivalents of the protease inhibitor with
respect to the peptide.
lZ779~
One preferred embodlment of thls lnventlon lnvolves an
antiviral pharmaceutical composition for treating herpes viral
lnfections of the eye, mouth or skin. This composition compri-
ses a combination of 0.01 to 0.2 percent by weight of bacitracin
or zinc bacitracin (specific activity ranging from 40 to 65
units per mg) and 0.01 to 0.2 percent of the peptide of formula
la or lb, as defined hereinbefore, together with a pharmaceutic-
ally acceptable carrler. Preferred carriers in this instance
are water soluble ointment bases or water-oil type emulsions.
Examples of suitable excipients or carrlers for the above
mentioned formulations are found in standard pharmaceutical
texts, e.g. in "Remington's Pharmaceutlcal Sciences", 16th ed,
Mack Publishing Company, Easton, Penn., 1980.
The dosage of the combination of the peptide and the pro-
tease inhibitor wlll vary with the form of administration and
the particular active agents for the combination chosen. Fur-
thermore, it will vary with the particular host under treatment.
Cenerally, treatment ls lnltlated wlth small dosages substan-
tially less than the optlmum dose of the combination. There-
after, the dosage is increased by small increments untll theoptimum effect under the circumstances is reached. In general,
the combination is most desirably administered at a concentra-
tion level that will generally afford antiviral effective
results agalnst herpes virus without causing any harmful or
deleterious side effects.
10.
-- 1.2779~1,
The combination is adminlstered topically to the infected area
of the body, e.g. the skin, eye or part of the oral or genital
cavity, in an amount sufficient to cover the infected area. The
treatment should be repeated, for example, every four to 9iX
hours until lesion~ heal, usually within 3 to 4 days. No con-
traindications have been observed.
Although the method of treating herpes viral infections can
be most advantageously practiced by administering the combina-
tion of the protease inhibitor and the peptide o~ formula 1
simultaneously in a formulation, the separate or sequential
administration on a daily basis of the two active agents is also
encompassed within the scope of this invention.
Another embodiment of this invention comprises a cosmetic
composition comprising a herpes viral prophylactic amount of the
combination of the protease inhibitor and the peptide of formula
1, or a therapeutically acceptable salt thereof, together with a
physiologically acceptable cosmetic carrier. Additional compo-
nents, for example, skin softeners, may be included in the for-
mulations. The cosmetic formulation of this invention is used
prophylactically to prevent the outbreak of herpetic lesions.
They can be applied nightly and generally contain less of the
two active agents of the combination than pharmaceutical prepa-
rations. A preferred range for the amount of each of the agents
in the cosmetic composition is 0.01 to 0.1 percent by weight.
~ 1277g~1
Finally, although the formulations di~clo~ed herein are
effective and relatively safe medications for treating herpe~
viral infections, the po~sible concurrent administration of
these formulations with other antiviral medications or a8ents to
obtain beneficial re~ult~ is not excluded. Such other antiviral
medication~ or agents include acyclovir (see Example 1), and
antiviral surface active agent~ or antiviral interferons ~uch a~
tho~e di~clo~ed by S.S. A~culai and F. Rapp in U.S. Patent
4,507,281, March 26, 1985.
The following example~ illustrate further this invention.
In the examples, the term "HSV 7-15 peptide" is used to de~ig-
nate the nonapeptide, H-Tyr-Ala-Gly-Ala-Val-Val-Asn-A~p-Leu-OH,
described by E.A. Cohen et al., Nature, ~upra. Thi~ nonapeptide
is one of the preferred peptide of formula 1 for use according
to the teaching of this disclosure. Other more preferred pepti-
des of formula 1 for which activity can be demonstrated accord-
ing to the exemplified procedures include H-Glu-Cys-Arg-Ser-
Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH, H-Ser-Thr-Ser-
Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH, H-Tyr-Thr-Met-Leu-Val-
Val-Agn-Agp-Leu-OH, H-Val-Ser-Arg-Ser-Thr-Ser-Tyr-Ala-Gly-Ala-
Val-Val-Asn-Asp-Leu-OH and HO ~ CH2CH2CO-Ala-Gly-Ala-
Val-Val-Asn-Asp-Leu-OH.
EXAMPLE 1
Therapeutic Effect of HSV 7-15 Peptlde and Combinations thereof
on Cutaneous Herpes Viru~ Infection in Guinea Pi~s.
Viruses: HSV-1 strain MP-12 having a titer of 4.8 X 107
PFU/ml, and HSV-2 strain HG-52 having a titer of 6 X 107
PFU/ml were utllized. These strains have been described by A.L.
Epstein and B. Jacquemont, J.Gen. Virol.,64,1499(1983) and by Y.
Langelier and G. Buttin, J.Gen. Virol.,57,21tl981), respective-
ly .
Animals: Dunkin-Hartley guinea pigs (550-630g) obtained from
Canadian BreedinB Farm, ~t-Constant, Québec, Canada were used.
Food and drinking water were given ad libitum.
Inoculation Procedure: Seven guinea pigs were anesthetized with
sodium pentobarbltal (50 mg/kg, ip). Their backs were shaved
with an electric razor, depilated with a commercial hair depila-
tor (Neet ~ , washed and dried. Their toenails were cut and
rounded off. The hairless area of their backs was divided into
six distinct zones with a marking pencil. Four animals were
inoculated with the HSV-1 and three were inoculated with the
HSV-2 as follows: an application of 20 ~l of the virus wa~
applied to the center of each zone; thereafter, the area of
application of each zone was punctured 60 times to a depth of l
mm with a 26-5/8 gauge syringe needle. The animals awoke from
their postanesthetic sleep about two hours after being anesthe-
tized and resumed normal activity.
13.
12~9~1
Treatment: The following formulation~ were used. Note: Prior
.
to u~e, the emul~ion~ were blended at high ~peed in an emul~i-
fier (POLYTRON ~ , cooled in an ice bath for 30 minutes and
then qtored at 4C until u~e.
l. Control emulsion: a mixture of 11.25 ml of ~aline (0.85%
NaCl in H20), 13.75 ml of ~esame oil (Sigma Chemical Co., St.
Louis, MO, USA) and 25 mg of bovine serum albumin (Sigma Chemi-
oal Co.).
2. Peptide emulsion: the ~ame as the control emul~ion except
that the BSA is replaced wlth an equal weight o~ the HSV 7-15
peptide.
3. Acyclovir ointment 5~ (ZORIVAX ~, Burroughs Welcome,
Inc.).
4. Bacitracin emulsion: the ~ame as the control emul~ion
except that the BSA i~ replaced with an equal weight of bacitra-
cin (Sigma Chemical Co.).
5. Peptidé-bacitracin emulsion: a mixture of 11.25 ml of
saline, 13.75 ml of sesame oil, 25 mg of HSV 7-15 peptide and 25
mg of bacitracin.
14.
~ lZ77901 ,
Treatment began on the day following the inoculation (noted
as Day l in Tables II and III hereinafter). The treatment com-
pri~ed applying lOO ~l of a ~ormulation to five of the six above
mentioned zones (a different formulation for each zone). The
~ixth zone wa~ given a treatment comprising 50 ~l each of the
peptide - bacitracin emulsion and the acyclovir ointment 5%.
Treatments were performed twice daily, once in the morning and
once in the evening.
Evaluation of Results: The inoculated areas were scored daily,
prior to the morning treatment for the duration of the experi-
ment. The scorlne wa~ done blind according to scorin~ system of
S. Alenius and B. Oberg, Archives of Virology, 58, 277 (1978).
The ~coring sy~tem i8 reproduced in TABLE I. The results are
shown in TABLES II and III. All scoring was confirmed single
blind by an independent observer. The score for any given day
was obtained by the addition of all score numbers read for each
animal in the HSV-1 infected group or the HSV-2 infected group,
divided by the number of animals in that group.
-~ ~z77æ~
TABLL I
ScorinE system for H ~ on guinea pig skin
Appearance of inoculated skin Score
Erythematous and slightly edematous 0.5
Erythema and one or two small vesicles
Erythema and numerou~ small vesicles 2
Numerou~ large vesicles ~ if in close 3
Juxtaposition, coalesced
Vesicles dried, large crusts 3
Crusts fallen off to ca. 50 per cent 2
Circa lO per cent of the crusts remaining
Uninfected or healed area, no crusts or vesicles.
Trauma from the inoculation or traces from
the infection can be present O
16.
~Z779(~
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l.Z~79~1
With respect to shortening the healing time and diminishing the
symptoms of HSV-l infection, TABLE II shows that HSV 7-15 pepti-
de and its combinations with bacitracin and with bacitracin plus
acyclovir are effective, and that the combination of the HSV
7-15 peptide and bacitracin is superior in these two parameters
to either of the latter two compounds alone. The shortening of
the healing time with the combination of the peptide and baci-
tracin i~ noteworthy. Likewise, TABLE III shows that the HSV
7-15 peptide and such combinations are effective against HSV-2
with re~pect to shortening healing time and alleviating the
symptom~ of the virus, and that the peptide bacitracin combina-
tion significantly reduces the symptoms of the herpes simplex as
compared to the peptide and bacitracin alone.
19.
... ~ ~,Z77901,
Example 2
Comparison of HSV 7-i5 Peptide? Bacitracin and the Comblnation
f th T A ents in Inhibitin HSV-2 Re lication U~in Cell
o e wo g g p g
Culture Techniques
a) Preparation of serum-~tarved cells: A medium composed of
alpha medium (Gibco Canada Inc, Burlington, Ontario, Canada) and
fetal calf serum (Gibco Canada Inc.), in a 9:1 volume ratio, was
placed in tissue culture dishes (35 mm, A/S Nunc, Kamstrup,
Denmark). The medium in each dish wa~ seeded with BHK 21/C13
cell~ (1.5 X 10 6 for each dish). (BHK 21/C13 cells have been
de~cribed by Langelier and Buttin, supra.) After 6 hour~, the
medium wa~ replaced with a new medium composed of alpha medium
and fetal calf ~erum (99.5:0.5,v/v). The resultant preparation
was incubated at 37C for 4 days.
b) Cell infection in BBMT medium*without serum: The incubation
medium i~ removed from the cell~. The cell~ are washed twice
with alpha medium (without the ~erum) and once with the BBMT
medium. The cells are then incubated at 37C in BBMT medium for
2 hour~. The ~tock of HSV-2, described in Example 1, i~ dilut-
ad with BBMT medium, with or without the agents (conc. -1 mM)
to be te~ted, to obtain new ~tock preparation~ having a multi-
plicity of infection of 0.02 PFU/cell. Thus four stock
* BBMT medium is described by P. Brazeau et al.,
Proc. Natl. Acad. Sci. USA, 79, 7909(1982). 20.
_ - 1 277901
solutions of the virus were prepared for each virus: one
without any of the agents (control), one with HSV 7-15 peptide
(lmM), one with bacitracin (lmM) and one containing HSV 7-15
peptide (lmM) and bacitracin (lmM).
Each stock solution (250 ~l) is added to separate dishes of the
previously prepared cells in BBMT medium. After one hour for
absorption of the virus, the medium is aspirated from the dishes
and the cells washed twice with BBMT medium. BBMT medium (400
~l), with or without the respective 0.1 mM concentrations of the
agents being assayed, is then added to the dishes containing the
cells.
c) Harvesting: At appropriate times, cells are detached with a
rubber policeman and frozen at -80C until titration.
d) Titration: Virus titration is performed according to the
method of B.B. Wentworth and L. French, Proc. Soc. Exp. Biol.
Med., 131, 588 (1969).
The results are shown in TABLE IV expressed in PFU/cell; B
refers to values obtained before absorption and A refers to
values obtained after absorption.
~ 2779~1
TABLE IV Cell Culture Assay
.
Time Control Peptide Bacitracin Peptide(c=1mM)~
(Hour~, po~t (C~lmM) (C~1mM) Bacitracin (c51~M)
1~f~ctLon) ~ B _ B A B A
4 o.ooo8 o.ooo6 O.OOl 0.0007 0.0005 0.0004 0.0004
12 0.04 o.oo6 o,ol 0.005 0.007 0.0015 0.0014
24 16.0 0.4 0.5 o.o6 0.06 0.010.015
36 32.0 0.8 1.1 0.03 0.06 0.003
The results expressed in TABLE IV show that the combination of bacitracin
and the HSV 7-15 peptide is able to decrease the actual viral production titer
to a much greater degree (i.e. one twenty seven thousandth after 36 hours) than
either of the agents alone.
