Note: Descriptions are shown in the official language in which they were submitted.
WO95/11038 21 S 2 3 7 ~ PCT/CA94/00590
TREATMENT OF CYTOMEGA~uvlKuS lN~lON
This invention relates to anti-viral compounds, and
particularly to the use of peptide-based anti-viral agents
for the treatment of cytomegalovirus infection.
Background to the Invention
Cytomegalovirus (CMV) is a mem.ber of the herpesvirus
family, other well-known mem.bers of which include herpes
simplex virus, types I and II, Epstein-Barr virus and
Varicella Zoster virus. Although these viruses are related
taxonomically as double-stranded DNA viruses, each
manifests in a clinically distinct m~nnPr. In the case of
CMV, medical conditions arising from congenital infection
include jaundice, respiratory distress and convulsive
seizures which may result in mental retardation, neurologic
disability or death. Infection in adults is frequently
asymptomatic, but may manifest as mononucleosis, hepatitis,
pneumonitis or retinitis, particularly in ;mm~nocompromised
patients such as AIDS sufferers, chemotherapy patients and
organ transplant patients undergoing tissue rejection
therapy.
A variety of drugs have been developed to treat
herpesvirus infection, including natural occurring proteins
and synthetic nucleoside analogs. For example, the natural
antiviral protein, interferon, has been used in the
treatment of herpesvirus infections, as have the nucleoside
analogs, cytosine-arabinoside, ~pn;ne arabinoside,
iodoxyuridine and acyclovir, which is presently the
treatment of choice for herpes simplex type I infection.
Unfortunately, drugs such as acyclovir that have
proven effective to treat infection by certain
herpesviruses are not sufficiently effective to treat CMV.
And, drugs currently used to treat CMV infection, such as
WO9S/ll038 pcTlcA94loos9o
-
~5?-3~3 2 -
9-[(1,3-dihydroxy-2-propoxy)methyl]gllAn;n~ (ganciclovir,
DHPG) and phosphonoformic acid (foscarnet), lack the
acceptable side effect and safety profiles of the drugs
approved for other treatment of other herpesviruses.
Moreover, such drugs are ineffective to treat certain
strains of CMV that have acquired drug resistance. Thus,
despite recent advances in the development of anti-
herpesvirus drugs, there rem~;n~ a need for therapeutic
agents effective to treat CMV infection.
In co-pending patent application WO92/07871 (published
14 May 1992), there are disclosed oligopeptides that are
effective to control replication of the human
immunodeficiency virus (HIV). In co-pending patent
application WO93/21941 (published 11 November 1993), there
is disclosed the discovery that these oligopeptides are
also effective to prevent replication of certain viruses
within the herpesvirus family, particularly the herpes
simplex viruses. Further investigation of the anti-viral
properties of these oligopeptides has now surprisingly
revealed their ability to control replication of
cytomegalovirus, including human CMV. It has now also been
discovered that these oligopeptides act synergistically
with current anti-CMV drugs, particularly ganciclovir, to
control replication of CMV.
Accordingly, it is an object of the present invention
to provide a method useful to control CMV replication, and
further to provide a method for controlling cytomegaloviral
infection in a m~mm~l.
It is another object of the present invention to
provide pharmaceutical compositions and combinations useful
to treat cytomegaloviral infection.
~ WO95/ll038 21 S 2 3 7 3 PCTlCA94/nOS9O
SUMMARY OF THE INVENTION
Accordingly, there is provided in one aspect of the
~ present invention, a method for controlling cytomegalovirus
infection in a m~mm~l, which comprises the step of
~m; n;stering to the m~mm~ 1 a composition containing a
therapeutically effective amount of a compound of Formula
(I):
R1 - (A)y - [X] - (B)z - R2
wherein
R1 is H or an N-terminal protecting group;
R2 is OH or a C-terminal protecting group;
X represents an oligopeptide consisting of 'n' amino acids,
wherein n is an integer from 6 to 12, said
oligopeptide comprising at least one D-amino acid and
having a net positive charge selected from n, n-1 and
n-2;
y is 0 or 1;
z is 0 or 1; and
A and B collectively represent from 1 to 20 amino acid
residues.
In embodiments of the invention, the ~m; n;stered
compound is one in which both y and z are 0, and X is an
oligopeptide consisting of 7 to 10 basic amino acids in the
D-isomer form.
For use in controlling cytomegalovirus infection in a
patient, the present invention further provides, as an
article of manufacture, a pharmaceutically acceptable
package, such as an ampoule or vial, cont~;n;ng an
effective amount of an anti-CMV compound of the invention,
the package further comprising an information label or
sheet instructing use of the contents for the control of
CMV infection.
In another of its aspects, the invention provides a
method for controlling CMV infection, in which there is
WO95/11038 PCT/CA94/ooSgo
3~
~m; n;stered to a mAmm~l a therapeutically useful
com.bination of a compound of Formula I and an anti-CMV
compound such as phosphonoformic acid or ganciclovir. In
a preferred em.bodiment, such com.bination therapy entails
the ~m;n;stration of synergistically effective amounts of
ganciclovir and a compound of Formula I.
Embodiments of the present invention are now described
in greater detail with reference to the accompanying
drawings in which:
Brief Reference to the Drawings
Figure l shows the tissue distribution of a compound
according to the present invention when administered by IV
injection;
Figure 2 shows the tissue distribution of the Figure
l compound when ~m;n;stered by subcutaneous injection;
Figure 3 shows the death pattern of murine CMV-
infected, cyclophosphamide-;mml]nosuppressed mice treated
with a compound of the invention, and with ganciclovir; and
Figure 4 shows the synergy/antagonism plot tA) and
contour plot (B) for a combination of compound of the
invention and ganciclovir in treatment of human CMV-
infected MRC cells.
Description of the Invention and Preferred Em.bodiments
The present invention provides an anti-cytomegaloviral
composition that is effective to control CMV infection.
Compositions that are effective to "control CMV infection"
have the property of slowing, interrupting, arresting or
stopping replication of at least one CMV strain, as
determined by a cell culture assay used conventionally in
' W O 95/11038 21 S 2 3 7 3 PCT/CA94/00590
the art, such as the well established plaque reduction
assay. In the context of the plaque reduction assay for
example, the anti-cytomegaloviral nature of a given
compound, i.e., its ability to control CMV infection, is
indicated by a statistically significant reduction in
plaque number and/or size, relative to an untreated
control.
The term "cytomegalovirus" is meant to encompass
laboratory and other cytomegalovirus strains that infect
hnmAn~, as well as cytomegalovirus strains that infect
other mAmmAls including mice, rats, cats, dogs and horses
as well as livestock such as sheep and cattle.
In one aspect of the invention, the anti-CMV
compositions of the invention comprise, as active anti-CMV
ingredient, a peptidic compound represented by the general
Formula (I) provided hereinabove. In a preferred
embodiment, the active ingredient is a compound of Formula
I in which both y and z are zero, i.e., a compound of
Formula (Ia):
Rl - ~X] - R2 (Ia)
in which R1, R2 and X are as defined above. Preferred
compounds of Formula (Ia) are those in which Rl and R2 are
protecting groups, i.e. chemical substituents used commonly
in the art of peptide chemistry to stabilize and protect
the N- and C-terminal peptide ends from undesired attack,
particularly by endogenous exopeptidases. The R1 and R2
groups include chemical substituents attached to the
nitrogen atom at the N-terminus of the compound, or the
oxygen or carbon atoms of the C-terminal carboxyl group of
the compound. By "undesired attack" is meant any type of
enzymatic, chemical or biochemical breakdown of the
compound at its termini which is likely to affect the
function of the compound as an anti-cytomegaloviral agent,
WO 95/11038 PCT/CA94/00590
~5~,3'13 6
i.e. sequential degradation of the compound initiated at a
terminal end thereof.
The anti-CMV compounds of the present invention
incorporate a core oligopeptide designated 'X' in each of
the above formulae I and Ia. Oligopeptide X consists of
from 6 to 12 amino acids, coupled for instance by amide
linkage. Preferably, X consists of from 7 to 11 amino
acids, and in particular, from 8 to 10 amino acids. The
amino acid constituents of oligopeptide X are selected to
confer on the oligopeptide a net positive charge of 'n',
'n-1' and 'n-2', where ~n' represents the number of amino
acids incorporated within oligopeptide X. In other words,
X is an oligopeptide consisting either entirely of
positively charged amino acids (in the case where the net
positive charge is 'n') or of substantially all positively
charged amino acids (in the case where the net positive
charge is 'n-1' and 'n-2'). The term ~net positive charge"
refers to the charge on the oligopeptide X as a whole, and
is calculated simply by adding the number of positively
charged amino acids resident in oligopeptide X and
subtracting from that total the number of non-positively
charged amino acids resident in oligopeptide X. For
instance, an oligopeptide X in which all but one amino acid
is positively charged will have a "net" positive charge of
'n-1' in the case where the one amino acid has a neutral
charge. The net charge on oligopeptide X will be
'n-2' in the case where the single non-positively charged
amino acid is negatively charged rather than neutral. A
charge of 'n-2' is also realized when X includes two amino
acids carrying a neutral charge and all other amino acids
incorporated in X are positively charged. For the purposes
of calculating net positive charge, the term "positively
charged" refers to an amino acid having a side chain,
suitably a B-carbon side chain but desirably an ~-carbon
side chain, that is cationic in aqueous solution at neutral
pH. The term "negatively charged" refers to an amino acid
WO 95/11038 2 1 S 2 3 7 3 PCTICA94/00590
-- 7
having a side chain that is anionic in aqueous solution at
neutral pH. Amino acids having a neutral charge carry a
side chain that exhibits either no charge (e.g. alanine) or
exhibits both positive and negative charges (e.g.
glut~m; n~ ) in aqueous solution at neutral pH. Preferred
compounds for incorporation into the present anti-
cytomegaloviral composition are those in which the net
positive charge on oligopeptide X is 'n' or 'n-1'.
The terms "amino acid" and "~-amino acid residue" are
used interchangeably herein with reference to naturally
occurring and synthetic amino acids in either D- or L-
form. Unless otherwise stated, the amino acid is the
naturally occurring L-amino acid. Included, unless
otherwise stated, are: (1) the amino acids having a neutral
charge such as glycine; those amino acids having an
aliphatic ~-carbon side chain such as alanine, valine,
norvaline, leucine, norleucine, isoleucine and proline;
those having aromatic ~-carbon side-ch~i n~ such as
phenylalanine, tyrosine and tryptophan; (2) the negatively
charged amino acids, including those having acidic ~-carbon
side ch~; n~ such as aspartic acid and glutamic acid; those
having side ch~;nS which incorporate a hydroxyl group such
as serine, homoserine, hydroxynorvaline, hydroxyproline and
threonine; those having sulfur-cont~;n;ng ~-carbon side
ch~;ns such as cysteine and methionine; and those having
side cha; n~ incorporating an amide group such as glutamine
and asparaginei and (3) the positively charged amino acids,
including those having basic ~-carbon side ch~; n~ such as
lysine, arginine, histidine, and ornithine (also herein
referred to as "basic amino acids").
According to the present invention, the oligopeptide
X comprises at least one amino acid in the D-isomer form.
Preferably, oligopeptide X comprises more than one amino
acid in D-isomer form to result in a compound comprising,
for example, a random combination of L- and D-amino acids,
WO 95/11038 PCT/CA94/00590
~513~3 8-
alternating L- and D-amino acids, or alternating blocks of
L- and D-amino acids. In a most preferred embodiment, the
oligopeptide consists of D-amino acids.
In specific embodiments of the present invention,
oligopeptide X in the above Formulae I and Ia has a
sequence selected from among the group consisting of;
i) an oligopeptide consisting of from 6 to 11 basic
amino acids and one amino acid other than a basic amino
acid, wherein each basic amino acid is independently
selected from among the group consisting of arginine,
lysine, histidine and ornithine, and the single non-basic
amino acid is selected from among the group consisting of
glutamine, serine, histidine, asparagine and homoglutamine.
Especially suitable oligopeptides are those in which each
basic amino acid is independently selected from arginine
and lysine, and the non-basic amino acid is glut~m;ne; and
ii) an oligopeptide consisting essentially of from 7
to 12 basic amino acids, wherein each basic amino acid
residue is independently selected from among the group
consisting of lysine and arginine.
According to specific embodiments of the present
invention, X represents an oligopeptide selected from among
the group consisting of:
i) an oligopeptide comprising amino acids arranged in the
sequence Arg-Y2-Y3-Arg-Arg-Y4-Arg-Arg-Arg wherein each of
Y2, Y3 and Y4 is a basic amino acid, and at least one of
Y2, Y3 and Y4 is arginine;
ii) an oligopeptide comprising 6 to 11 arginines and one
glutamine;
iii) an oligopeptide homopolymer consisting of 7 to 12
arginlnes .
According to preferred em.bodiments of the present
invention, X in the above Formulae I and Ia represents an
oligopeptide, preferably consisting essentially of D-amino
WO95/11038 2 1 5 2 3 7 3 PCT/CA94/00590
acids, having an amino acid sequence selected from:
Arg-Lys-Lys-Arg-Arg-Lys-Arg-Arg-Arg;
Arg-Lys-Lys-Arg-Arg-Ser-Arg-Arg-Arg;
Arg-Lys-Lys-Arg-Arg-His-Arg-Arg-Arg;
Arg-Lys-Lys-Arg-Arg-Asn-Arg-Arg-Arg;
Arg-Lys-Lys-Arg-Arg-homoGln-Arg-Arg-Arg;
Arg-Lys-Lys-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Lys-Arg-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Lys-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Lys-Arg-Arg-Lys-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Lys-Arg-Arg-Arg;
Arg-Gln-Arg-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Gln-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gln-Arg;
Arg-Arg-Arg-Gln-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg;
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg; and
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg.
Especially preferred are anti-CMV peptides are those
in which X in Formulae I and Ia represents an oligopeptide
consisting essentially of D-amino acids, and which have a
sequence selected from:
D-[Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg];
D-[Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg]; and
D-[Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg].
In another preferred embodiment, compounds of the
present invention are those compounds in which Rl and R2
woss/11038 PCT/CA94/00590
- 10 -
31~J
are N- and C-terminal protecting groups conventionally
employed in the art of peptide chemistry. Suitable N-
terminal protecting groups include, for example, lower
alkanoyl groups of the formula R-C(O)- wherein R is a
linear or branched C1s alkyl chain. A preferred group for
protecting the N-terminal end of the present compounds is
the acetyl group, CH3C(O)-. Also suitable as N-terminal
protecting groups are amino acid analogues lacking the
amino function. Suitable C-terminal protecting groups
include groups which form ketones or amides at the carbon
atom of the C-terminal carboxyl, or groups which form
esters at the oxygen atom of the carboxyl. Ketone and
ester-forming groups include alkyl groups, particularly
branched or unbranched C15alkyl groups, e.g. methyl, ethyl
and propyl groups, while amide-forming groups include amino
functions such as primary amines (-NH2), or alkylamino
functions, e.g. mono-C15alkylamino and di-C15alkylamino
groups such as methylamino, ethylamino, dimethylamino,
diethylamino, methylethylamino and the like. Amino acid
analogues are also suitable for protecting the C-terminal
end of the present compounds, for example, decarboxylated
amino acid analogues such as agmatine. Of course, N- and
C-terminal protecting groups of even greater structural
complexity may alternatively be incorporated to protect the
N- and C-terminal ends of the compound from attack provided
that the anti-cytomegaloviral activity of the compound is
not adversely affected by the incorporation thereof, as
determined using the plaque reduction assay.
The most preferred anti-CMV peptide is acetyl-[(D-
Arg)g]-NH2-
It will be appreciated that the oligopeptide portionof the present compounds may be conjugated, either through
its C-terminus or its N-terminus, to other amino acids
without necessarily sacrificing the anti-cytomegaloviral
activity exhibited by the oligopeptide, as determined by
- ~ ~ S2373
11
the assays herein described. The present invention thus further
embraces anti-cytomegaloviral polypeptide compounds which
incorporate the oligopeptides described herein and which conform
to the general formula (I), i.e.
R1 - (A)y - [X] - (B)z - R2 (I)
wherein at least one of y and z is 1, A and B collectively
represent from 1 to 20 amide-linked, amino acids, and preferably
from 1 to 10 amino acids, and R1, R2 and X are as specified
above.
Specifically contemplated compounds of formula I are
anti-cytomegaloviral compounds in which the oligopeptide X is
flanked at the C-terminus and/or at the N-terminus by another
unit of oligopeptide X, or by partial units of oligopeptide X.
The additional units, i.e. A and B, may be linked directly by
amide bonding. Alternatively, A and B may comprise peptide
linkers of from 1 to about 10 amino acids in length, which bind
the additional oligopeptide units to the core oligopeptide X.
Preparation of the anti-CMV peptides is described in
the co-pending published patent applications referenced
hereinabove, i.e., WO93/21941 and WO92/07871. It will
nonetheless be readily apparent that, as peptides, the anti-CMV
compounds can be prepared by standard, well-established solid-
phase peptide synthesis methods (SPPS), general descriptions of
which appear, for example, in J.M. Stewart and J.D. Young, Solid
Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical
Company, Rockford, Illinois; and in M. Bodanszky and A.
Bodanszky, The Practice of Peptide Synthesis, 1984, Springer-
Verlag, New York; Applied Biosystems 430A Users Manual, 1987, ABI
Inc., Foster City, California.
, .,
WO95111038 PCT/CA94/00590
~ 3~3 12 -
To incorporate N- and/or C- protecting groups,
protocols conventional to solid phase peptide synthesis
methods can also be applied. To incorporate C-terminal
protecting groups, for example, synthesis of the desired
peptide is typically performed using, as solid phase, a
supporting resin that has been chemically modified so that
cleavage from the resin results in a peptide having the
desired C-terminal protecting group. To provide peptides
in which the C-terminus bears a primary amine as protecting
group, for instance, synthesis is performed using a p-
methylbenzhydrylamine (MBHA) resin so that, when peptide
synthesis is completed, treatment with hydrofluoric acid
releases the desired C-terminally amidated peptide.
Similarly, incorporation of an N-methylamine protecting
group at the C-terminus is achieved using N-
methylaminoethyl-derivatized DVB resin, which upon HF
treatment releases peptide bearing an N-methylamidated C-
terminus. Protection of the C-terminus by esterification
can also be achieved using conventional procedures. This
entails use of resin/blocking group combination that
permits release of side-chain protected peptide from the
resin, to allow for subsequent reaction with the desired
alcohol, to form the ester function. FMOC protecting
groups, in combination with DVB resin derivatized with
methoxyalkoxybenzyl alcohol or equivalent linker, can be
used for this purpose, with cleavage from the support being
effected by TFA in dichloromethane. Esterification of the
suitably activated carboxyl function e.g. with DCC, can
then proceed by addition of the desired alcohol, followed
by deprotection and isolation of the esterified peptide
product.
Incorporation of N-terminal protecting groups can be
achieved while the synthesized peptide is still attached to
the resin, for instance by treatment with suitable
anhydride and nitrile. To incorporate an acetyl protecting
group at the N-terminus, for instance, the resin-coupled
2152373
WO 95/11038 ~ PCT/CA~ ,0~30
- 13 -
peptide can be treated with 20~ acetic anhydride in
acetonitrile. The N-protected peptide product can then be
cleaved from the resin, deprotected and subsequently
isolated.
Once the desired peptide sequence has been
synthesized, cleaved from the resin and fully deprotected,
the peptide is then purified to ensure the recovery of a
single oligopeptide having the selected amino acid
sequence. Purification can be achieved using any of the
standard approaches, which include reversed-phase high-
pressure liquid chromatography (RP-HPLC) on alkylated
silica columns, e.g. C4-, C8-, or Cl8- silica. Such column
fractionation is generally accomplished by running linear
gradients, e.g. 0-50~, of increasing ~ organic solvent,
e.g. acetonitrile, in aqueous buffer, usually containing a
small amount of TFA, e.g. 0.1%. Alternatively, ion-
exchange HPLC can be employed to separate peptide species
on the basis of their charge characteristics. Column
fractions are collected, and those cont~; n; ng peptide of
the desired/required purity are pooled together. The
peptide is typically then treated to exchange the cleavage
acid (e.g. TFA) with a pharmaceutically acceptable acid,
such as acetic, hydrochloric, phosphoric, maleic, tartaric,
succinic and the like, to provide a water soluble salt of
the peptide.
For use as an anti-CMV agent, the oligopeptide
compounds of the invention are desirably of "pharmaceutical
grade" purity, a term used herein with reference to an
oligopeptide preparation that migrates as a single peak
using HPLC, exhibits uniform and authentic amino acid
composition and sequence upon analysis thereof, and
otherwise meets standards set by the various national
bodies which regulate ~uality of pharmaceutical products.
It will be appreciated that strict st~nA~rds of purity may
not be required for use of the present compounds and
~p95/1l038 PCT/CA94/00590
~5a~3 - 14 -
compositions in laboratory research and in the veterinary
field.
For use in controlling CMV infection, the chosen anti-
CMV peptide is formulated in an effective amount with an
appropriately selected carrier. The expression "an
effective amount" is meant to encompass amounts of the
anti-CMV peptide sufficient to prevent or cause a reduction
in cytomegaloviral replication. For in vitro use, for
instance to control CMV infection in cultured cells, the
carrier may simply be culturing medium appropriate for
maintaining the cells under culture. In this instance, the
effective amount of anti-CMV peptide is that which yields
a concentration in the medium sufficient to inhibit CMV
replication in the cultured host.
For therapeutic use, the chosen anti-CMV peptide is
formulated with a carrier that is pharmaceutically
acceptable and is appropriate for delivering the peptide by
the chosen route of ~Am; n; stration. Such pharmaceutical
compositions contain the chosen peptide in a
therapeutically effective amount, i.e., an amount
sufficient to reduce CMV burden in the patient being
treated. Such reduction is most properly revealed by
assaying virus titer in samples of biological fluid, such
as blood and urine, obtained from the patient before and
after treatment.
Suitable pharmaceutically acceptable carriers are
those used conventionally with peptide-based drugs, such as
diluents, excipients and the like. Reference may be made
to "Remington's Pharmaceutical Sciences", 17th Ed., Mack
Publishing Company, Easton, Penn., 1985, for guidance on
drug formulations generally. In one emboA;mPnt of the
invention, the compounds are formulated for ~m; n; stration
by infusion, or by injection either sub-cutaneously or
intravenously, and are accordingly utilized as aqueous
WO95/11038 21 5 2 3 7 3 PCT/CA94/00590
_.
solutions in sterile and pyrogen-free form and optionally
buffered or made isotonic. Thus, the compounds may be
~m;n; stered in distilled water or, more desirably, in
saline, phosphate-buffered saline or 5~ dextrose solution.
The compounds herein designated as preferred compounds are
substantially water-soluble. Water solubility of these and
other compounds of the invention may be enhanced, if
desired, by incorporating a solubility enhancer, such as
cetyltrimethyl~mmo~;um bromide or chloride.
For use in controlling CMV infection in a m~mm~l
including a human, the present invention provided in one of
its aspects a package, in the form of a sterile-filled vial
or ampoule, that contains a therapeutically effective
amount of the anti-CMV peptide, in either unit dose or
multi-dose amounts, wherein the package incorporates a
label instructing use of its contents for the control of
CMV infection. In one embofl;mPnt of the invention, the
package contains the peptide and the desired carrier, as an
~m; n; stration-ready formulation. Alternatively, and
according to another em.bodiment of the invention, the
package provides the anti-CMV peptide in a form, such as a
lyophilized form, suitable for reconstitution in a suitable
carrier, such as phosphate-buffered saline.
In a preferred em.boflimPnt, the package is a sterile-
filled vial or ampoule containing an injectable solution
which comprises an effectve amount of an anti-CMV peptide
of the formula Rl-[D-Arg]g-R2 wherein Rl and R2 are as
defined by Formula I, dissolved in neutral phosphate buffer
(pH 6.5-7.5) to a peptide concentration of O.l - lO mg/mL
or greater, e.g. l-2 mg/mL.
As an alternative to injectable formulations, the
stability particularly of the all D-form peptides permits
their formulation for ~m; n; stration by other routes.
Compositions for topical application, such as eye drops,
WO95/11038 PCT/CA94/00590
~S23~ ~ 16 -
creams, lotions, or ointments may be useful, as may aerosol
inhalable formulations. Oral dosage forms, such as
tablets, capsules and the like, formulated in accordance
with standard pharmaceutical practise, may also be
employed. Cream, lotion and ointment formulations will be
useful particularly for application to virally-induced skin
lesions. Appropriate triglyceride bases and gels can be
used to prepare creams and ointments, which may include
conventional surfactants and antimicrobial agents.
The anti-CMV peptides may be ~m;n;stered in
conjunction with other therapeutics, for example, other
therapeutic agents useful for the treatment of
cytomegaloviral infection including, but not limited to,
ganciclovir, foscarnet and HMPA. Such a combination
therapy may involve ~m;n;stration of discrete compositions
cont~;n;ng a single therapeutic, i.e. a composition
containing an anti-CMV peptide of the present invention and
a second active anti-CMV compound, or may involve
~m;n;stration of a composition containing both the anti-
CMV peptide and the second anti-CMV compound. As noted
above, such compositions will be prepared with a
pharmaceutically acceptable carrier selected for its
suitability in delivering the therapeutic agent to the site
of infection.
In a preferred em.bodiment, such combination therapy
entails the ~m~n;stration of synergistically effective
amounts of ganciclovir and an anti-CMV peptide of the
formula Rl-[D-Arg]g-R2, wherein Rl and R2 are as defined by
Formula I.
The compositions of the invention are ~m; n;stered
particularly to treat patients diagnosed with CMV
infection. Candidates for treatment are those patients in
an ;mmllnocompromised condition, including AIDS patients,
patients undergoing cancer chemotherapy and organ and
W O 95/11038 21 ~ 2 3 7 3 PC~r/CA94/00590
- 17 -
tissue transplant patients undergoing tissue rejection
therapy. Clinically effective doses of the anti-CMV
peptides are determined using clinical trial protocols
established for other anti-CMV drugs, such as ganciclovir.
It is expected that the dosing schP~ e will vary during
the course of treatment, moving from an initial loading
dose at the high end of the effective range to control
current infection followed by maintenance at a lower dose
and/or frequency to control recurrence. It is anticipated,
on the basis of the results reported herein, that an
effective treatment regimen for patients infected with
cytomegalovirus will require ~m;n; stration, either daily
or every other day, of doses in the range of from 0.01 mg
to about 5 mg per kg, e.g., between about 0.1 mg/kg to
about 4 mg/kg.
Specific embodiments of the present invention are
described in more detail in the following examples which
are not to be construed as limiting.
~xample 1 - Synthesis of the acetyl-[D-Arg]g-NH2
oli~opeptide
The title compound, designated compound AV-9, was
synthesized using p-methylbenzhydrylamine (MBHA) resin as
solid support to provide the C-terminal blocking amine on
the resultant peptide. Synthesis proceeded using D-
arginine residues in which the amino function was blocked
with the t-BOC group, and the guanidino function was
blocked with the Tos group. Couplings were carried out
using excess hydroxybenzotriazole (HOBt)-activated ester of
BOC-L-Arg(Tos). Removal of the BOC protecting group after
each cycle was effected with TFA. When coupling cycles
were completed, the resin-bound peptide was treated with
20~ acetic anhydride in acetonitrile, to incorporate an
acetyl protecting group at the N-terminus thereof.
Liberation of peptide from the resin, and lel.,ovdl of Tos
~ ~ 5~3~
18
groups, were achieved by treatment with hydrofluoric acid,
yielding the C-terminally amidated, title compound. After
removal of hydrofluoric acid, the resin/peptide mixture was
washed with diethyl ether and extracted with aqueous acetic acid.
The crude peptide was lyophilized, then reconstituted and
fractionated by RP-HPLC on a Cl8 silica column using a gradient
of 2-40~ acetonitrile in a 0.1~ TFA. Fractions were collected
and checked by analytical RP-HPLC. Those containing 295~ of the
major product were combined and lyophilized. High resolution
mass spectrometry showed the product to be the desired compound,
characterized as a white to off-white very hygroscopic powder
(op. Rot. +52~, water sol. ~lg/mL).
The resulting peptide was formulated as follows: Flint
glass vials (Type I, 5mL, 13mm) were first prepared by boiling
in HPLC-grade water for 10 minutes and allowed to dry in a 65~C
oven. Stoppers (V32, 13mm) were washed with isopropanol, then
boiled in HPLC-grade water for 10 minutes and then dried in a
65~C oven. The peptide (99.8mg) was weighed, transferred to a
lOOmL Class A volumetric flask, and taken to volume with
phosphate buffered saline (PBS). The PBS (pH7.02) was prepared
by combining 400mL of 8g/L -sodium phosphate monobasic
(dihydrate), 600mL of 9.47g/L sodium phosphate dibasic
(anhydrous), and 4.61g NaCl in a lL volumetric flask. This
solution was taken to volume with HPLC grade water. The peptide
solution was then filtered through a 0.22~m Millex-GV (trade-
mark) Millipore filter, and allocated into lmL fractions into
clean glass vials, each containing lmg of drug solution. The
vials were stoppered, capped, crimped, labeled and stored at 4~C.
Example 2 - Inhibition of CMV replication
The compound of example 1, acetyl-[(D-Arg)g]-NH2 as the
acetate salt (hereinafter referred to as AV-9), was
2152373
WO 95/11038 PCT/CA94/00590
formulated as a 10 mM stock in water for i vitro and cell
culture procedures. The stock was then diluted into
buffers used for specific assays, or into cell culture
media.
The following procedures were then used to determine
inhibitory effects of AV-9 on the replication of CMV.
First, confluent monolayers of the human diploid cell line,
MRC-5 (ATCC# CCL 171) in 24 well cell culture plates were
pretreated with specified concentrations (20~M, lO~M, 5~M,
2.5~M, l.O~M, 0.5~M and O.O~M) of AV-9 for 24 hours. This
was accomplished by diluting the stock peptide solution in
growth medium (10~ fetal bovine serum and 10 ~g/ml
gentamicin in Dulbecco's MEM (DMEM)) used to overlay the
monolayers.
After pretreatment with AV-9, the monolayers were
overlaid with 0.1 ml log10 dilutions of virus (CMV strain
AD-169) ranging from 10~1 to 10-6. Virus was then allowed to
adsorb for 1 hour at 37~C. The virus inoculum was removed
and the monolayers were overlayed with DMEM containing 2~
FBS, 10 ug/ml gentamicin and a specified concentration of
AV-9. Virus was next allowed to replicate for 7 to 10 days
until the plaques were judged to be well developed, and
then the monolayers were fixed and stained with a solution
of 1~ crystal violet in 1~ formaldehyde, 70~ ethanol.
Finally, plaques (each representing a single viable virion)
were counted and checked microscopically.
In a like m~nn~r, plaque reduction assays were also
performed against other CMV strains and another CMV host,
with comparison against ganciclovir (GCV) and foscarnet
(FCV). Results were as follows:
WO 95/11038 PCT/CA94/00590
2~S~313 - 20 -
TABLE 1
ICs~(~M)
CMV Strain Cell Line AV-9 GCVFCV
AD-169 MRC-51 2.6 1.996.0
MRC-5 4.8 c3.6 -
MRC-5 2.7 14.4 -
HFF2 5 8~t 5.8~
HFF 12.0~ 2.0~ -
Davis MRC-5 2.3 - -
Towne HFF 8.7~ 6.5~ -
CPE assay, all others are plaque reduction assays.
Without 24h drug pre-treatment, all other assays were
with 24h drug pre-treatment
1 human embryonic lung
2 human foreskin fibroblast
Example 3 - Inhibition of Dru~-Resistant CMV strains
The anti-CMV activity of peptide AV-9 was evaluated
against two ganciclovir-resistant and two foscarnet-
resistant laboratory strains of human CMV in the
continuous, hllm~n embryonic lung cell line MRC-5.
To perform the studies, peptide AV-9 was dissolved as
a solid in sterile, deionized water to a final stock
concentration of lOmM. This stock solution was
subsequently diluted in cell culture medium to obtain the
test concentrations used in the study. Ganciclovir (GCV)
was dissolved in sterile, deionized water, and the drug
concentration measured spectrophotometrically (7.8mM).
Foscarnet (phosphonoformic acid, PFA) was dissolved in
sterile, deionized water to a final concentration of lOOmM.
Aliquots of the stock solutions were stored at -70~C.
Drug-resistant human CMV strains 759rD100, GDGrK17,
PFArD100 and PFArB300 were obtained from Dr. Donald M. Coen,
Harvard Medical School, Boston, Massachusetts. These
strains were generated in the laboratory from the standard
WO95/11038 21 5 2 3 7 3 PCT/CA94/00590
- 21 -
CMV strain AD-169 via multiple passages in the presence of
either ganciclovir or foscarnet.
Anti-CMV activity of peptide AV-9, ganciclovir (GCV)
and foscarnet (PFA) was assessed by standard plaque
reduction assay using 24-well microtiter plates. The drug
stock solutions were diluted with fresh culture medium to
obtain the desired test concentrations in the range 0.5-
2500~M.
MRC-5 cells (4-5x105 per well) were grown for 24 hours
at 37~C in 5~ CO2 atmosphere in D-MEM/F-12FBS medium
containing varying concentrations of drug. At the end of
the pre-treatment period, the cells were infected with a
suspension containing 50 plaque forming units of CMV
(MOI=0.0001). The virus was allowed to absorb for 60
minutes at 37~C in the absence of drug, at which time the
virus innoculum was washed away, and the cells fed with
fresh growth medium containing drug. The cells were
incubated for 7 to 10 days, until viral plaques were
visible. The D-MEM/F-12/FBS medium + drug was replaced
with fresh lmL aliquots on a routine basis during the
incubation period. Cell cultures were inspected visually
for evidence of cytopathic effects and/or cytotoxicity.
Viral plaques were counted under a microscope after
removing the culture medium from the wells, staining the
cells with 1.1~ Crystal Violet solution, and w~h;ng twice
with tap water. Positive control cultures were treated
either with GCV or PFA. Negative control cultures were
treated as above with D-MEM/F-12/FBS medium only.
Formation of viral plaques was used as an indication
of CMV infection. The absence or a reduction in the number
of plaques relative to untreated, control cultures
indicated an inhibition of virus replication. ICso and ICgo
values, the concentrations of drug required to inhibit CMV
replication by 50~ and 90~ respectively, were calculated.
WO95/11038 PCT/CA94/00590
2~S~3~ 3 - 22 -
In summary, the results showed that peptide AV-9
exhibited appreciable inhibitory activity versus both the
ganciclovir-resistant CMV strains 759~100 and GDGrKl7, and
the foscarnet-resistant CMV strains PFA~l00 and PFArB300 in
a standard plaque reduction assay when host cells were
treated with drug, both pre- and post-infection. IC50
values for peptide AV-9 ranged from l.3 to 2.l ~M. By
comparison, the IC50's for ganciclovir versus strains
759~100 and GDGrK17 were determined to be 30. 5 and 6.l,
respectively, while the IC50's for foscarnet versus strains
PFA~l00 and PFA~300 in MRC-5 cells were 233.8 and 384.9
~M, respectively.
Example 4 - Assessment of peptide distribution, in vivo
The distribution and localization of a 14C-acetyl form
of peptide AV9 was determined following ~m; n; stration by
intravenous and sub-cutaneous injection. Ten mice were
injected intravenously in the tail vein with 0.25 mL of a
solution of 26~g of l4C-labelled compound in l0mL PBS, and
ten mice were given the same dose by subcutaneous injection
in the abdomen. One mouse from each group was sacrificed
at the time points noted in Figures l and 2, and the noted
organs were weighed and digested to homogeneity for
scintillation counting. Counts were measured and used to
calculate the amount and concentration of drug in each
organ. It will be noted that both i.v. and s.c. injection
bring about rapid distribution of drug to tissues. The
highest and most prolonged levels are attained in the
liver, followed by the kidneys and spleen.
Blood levels in mice resulting from single
subcutaneous injection of a 53~g dose of the labelled
compound were also evaluated. It was revealed that near-
peak blood levels around l ~g/ml were reached 5 minutes
following injection. Peak levels were maintained for about
20 minutes. At 40 minutes and 2 hours, blood levels had
3~ ~
declined to about 80~ of peak values. A rapid decline in blood
levels followed, so that the 4 hour level was about 10~ of the
peak, and blood levels further declined to the threshold of
detection at 8 hours and 24 hours. Evidently, the compound AV-9
reaches the bloodstream rapidly. Near peak levels are maintained
for 20-120 min, and the drug is then cleared rapidly from blood.
Example 5 - Assessment of anti-CMV activity, in vivo
The anti-CMV activity of compound AV-9 was evaluated
in mice which had been immunosuppressed by cyclophosphamide.
This immunosuppression model mimics the human patient situation,
wherein the HCMV-induced disease in immunosuppressed individuals
can often be fatal. The particular model used for evaluating
potential antiviral activity has been described by Smee et al in
J. Infect. Dis., 1991, 164:958.
The Smith strain of murine CMV, obtained originally
from the American Type Culture Collection (Rockville, MD) was
used. A mouse salivary gland preparation was used for the virus
pool. An inoculation of 2 x 104 plaque forming units of the
virus was used in the antiviral study. Female BALB/c mice
weighing 10-12g were quarantined 24 hr prior to use, and
maintained on Wayne Lab Blox (trade-mark) and tap water ad
libi tum the duration of the study.
Peptide AV-9 was provided in phosphate buffered saline
at a concentration of 1.7 mg/ml. Ganciclovir (DHPG) was
purchased from a commercial source, and cyclophosphamide was
purchased from Sigma Chemical Co. (St. Louis, MO).
To determine tissue virus titre, homogenates of
infected mouse organs were titrated for virus using C127/I cells
in 96-well microplates. Each tissue sample was homogenized and
titrated separately with 3 micro wells used
woss/11038 PCT/CA94/00590
~3~3 24 -
per dilution; plates were read by microscopic ~x~m; n~Ation
for viral-induced cytopathic effect, with 50~ end points
determined by the Reed-Muench method. Mean viral titers
were expressed at 50~ cell culture infectious doses
(CCIDsO)/gram of tissue.
The mice were immunosuppressed by intraperitoneal
(i.p.) injection with lO0 mg/kg/day of cyclophosphamide
administered on days -l, +3, +6, +lO, +14 and +18 relative
to virus inoculation. For treatment, peptide AV-9 was
administered i.p. in doses of 2.0, 0.7, 0.2 and 0.07
mg/kg/day on days -2, 0, 2, 4, 6, 8 and lO. In a second
portion of the study, the compound was administered i.p. in
a dose of O.l mg/kg/day once daily from day -2 through +lO.
Ganciclovir in a dose of 50 mg/kg/day was injected i.p.
once daily for lO days beginning 24 hr post-virus exposure
(days +l +lO). A total of 15 infected mice/dose were
used. Five were designated to be killed on day +9 for
determination of virus titers in spleen, lung and salivary
gland. The remA;ning lO were to be held for 33 days with
deaths recorded daily. Five toxicity control mice were
used at each dose; these mice were treated in parallel with
the infected mice, and were weighed on the initial day of
treatment and again 24 after the final treatment. They,
too, were observed for a 33-day period. Normal controls
were weighed and held in parallel with the toxicity
controls. A total of 30 placebo-treated, infected mice
were run in parallel as virus controls. Of these, lO mice
were killed for virus titer determinations in their
tissues.
For statistical evaluation, increases in mean day to
death and reductions in viral titers were assessed using
the two-tailed Mann-Whitney U test. Total survivor
increases were evaluated by chi square analysis with
Yates'. The results of this study are summarized in Tables
2 and 3 and in Figure 3.
WO 95/11038 21 5 2 3 7 3 PCT/CA91/~5~0
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WO 95/11038 PCT/CA94/00590
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WO95/11038 2 15 2 3 7 3 pcTlcAs~Joo53o
As seen in Table 2, treatment with peptide AV-9 using a dose of
0.2 mg/kg/day ~m;n;stered every other day resulted in a
significant increase in mean day to death. The death pattern
seen in this study is summarized in Figure 3. The animals began
dying after treatment had terminated; this was seen in both the
peptide AV-9- and the DHPG-treated ~n;m~l and is an anticipated
occurrence resulting from virus rem~;n;ng in the tissues after
chemotherapy ceased which, in the ;mml]nocompromised ~n;m~l, will
eventually kill the mouse. The peptide AV-9 treatment at this
dose also reduced splenic virus titers; of the five animals
sampled, three had no detectable virus in their spleens, and one
had a titer of lo25, while the last ~n;m~l had a spleen titer of
IOs ~. Assuming the three spleens having undetectable virus
titers at the m;n;mllm dilution used (10-2'5) had titers of "0",
the mean titer was markedly less than that seen in the placebo
(Pc0.0l). Among the lungs of the AV-9 treated group, one had no
detectable virus, with the result that the mean titer was reduced
by 0.6 log10 in the lungs compared to the placebo-treated
controls. No effect was seen on salivary gland virus titers.
Treatment with the lowest dose (0.07 mg/kg/day) of peptide
AV-9 ~m;n;stered on an every other day schedule also resulted
in a moderately significant increase (P~0.05) in mean survival
time (Table 2, Figure 3).
Although l0 animals were used in each drug-treated group,
with 20 assigned as virus controls, it can be noted in Table 2
that less than these numbers are shown in the "Surv/Total"
column. On the day ~n;m~ls were to be killed for virus titer
P~m;n~tions, a number in each group except those receiving DHPG
had already died for reasons unrelated to the experimentation.
The known active drug, DHPG, exerted the positive effect
expected, with no animals dying until 21 days after initiation
of the infection. Although DHPG treatment reduced virus titers
significantly, only in the lung was no virus detected. It would
be expected that the DHPG ~n;m~ls would have survived longer than
WO 95/110.3~, PCTICA94/00590
?.3rl~ -
- 28 -
the AV-9-treated mice since less virus was present in the DHPG-
treated group. It would take additional days for the titers to
increase to the point the ~n;m~l S would die. Also, DHPG was
~Am;n;stered for one additional day, and was ~m;n;stered at an
optimal dose.
Peptide AV-9 appeared reasonably non-toxic in this study
(Table 3), with all mice gaining weight during the treatment
period. No other signs of toxicity (hunching, prostration,
ruffled fur, tremors) were seen.
These results show that peptide AV-9, when ~m;n; stered i.p.
on an every other day treatment schedule for a total of 7
treatments beginning 48 hr pre-virus inoculation was
significantly inhibitory to MCMV infections in BALB/c mice
;mmllnosuppressed by cyclophosphamide. The antiviral effect was
seen as increased mean survival time and decreased spleen and
lung virus titer. This effect was seen at a dose of 0.2
mg/kg/day. Treatment with the peptide at a dose of 0.07
mg/kg/day ~m; n; stered every other day appeared to also have a
moderate antiviral effect based on a significant increase in mean
day to death.
Example 6 - Druq combination study
For use in this study, HCMV strain AD-169 was obtained from
the American Type Culture Collection, Rockville, MD and
ganciclovir-resistant clinical isolate of HCMV, strain D16, was
obtained from Kenneth D. Thompson, Loyola University Medical
Center, Maywood, IL. Host MRC-5 cells were cultured in a medium
of Basal Medium Eagle (BME) (GIBCO), fetal bovine serum (FBS)
(Hyclone Laboratories), 0.035~ NaHCO3 and without antibiotics.
Test medium for dilution of virus and for preparation and
dilution of compounds was Dulbecco's modified Eagle medium
(DMEM), 2~ FBS, 0.1~ NaHCO3, 50~g gentamicin/ml.
For drug combination experiments, each of AV-9, DHPG,
WO9S/11038 215 2 3 7 3 PCT/CA94J~590
- 29 -
foscarnet, and ALT was prepared in test medium at double the
highest concentration used. Each of these compound preparations
was then diluted by serial 2-fold dilutions in test medium
(except for AZT, which was diluted by serial half-log dilutions).
A uniform volume of each concentration of peptide AV-9 was placed
in each of 8 sterile tubes (56 total tubes). An equal volume of
test medium (without compound) was place in one tube of each of
the 8-tube sets (to give the 7 final concentrations of peptide
without the combination compound). An equal volume of each
concentration of the combination compound (DHPG, foscarnet, or
AZT) was placed in one tube of each of the 8-tube sets (to give
proper final concentrations of the mixtures of peptide and the
combination compound). Empty sterile tubes (7) were used to mix
equal volumes of test medium, without drug, with each of the 2X
concentrations of the combination compound (to give the 7 final
concentrations of combination compound without peptide AV-9).
Growth medium was decanted from established monolayers of
MRC-5 cells in 24-well tissue culture plates. Compound dilutions
were placed in designated wells of the plates at 0.8 ml/well,
with test medium only placed in virus control or cell control
wells, and the plates were placed at 37~C. Four wells were used
for each different compound dilution. After 24 hr of pre-
treatment incubation, the medium was aspirated from each well of
the plates. One ml of virus, diluted in test medium was placed
in each well except those to be used for cell controls or those
to be used for toxicity controls. One ml of sterile test medium
was placed in each of these cell or toxicity control wells. The
plates were centrifuged at 2200 rpm for 30 minutes at room
temperature to allow the virus to adsorb. Medium was aspirated
from each well of the plates. The proper individual compound
concentrations or combinations were placed in test wells (0.8
ml/well, 4 wells/dilution) or in toxicity control wells. Test
medium without compound was added (0.8 ml/well) to each cell
control and virus control well. All plates were incubated at
37~C in a moist atmosphere of 5~ CO2, 95~ air. When virus
plaques had formed in virus control wells, the cells were
WO 95/11038 PCT/CA94/00590
,~ $~,3~ 3 30 -
observed microscopically for morphological changes due to
compound cytotoxicity, the medium was aspirated from all wells,
and the cells were stained by adding 0.3ml of 0.2~ crystal violet
in 10~ buffered formalin to each well. After 15 minutes, the
stain was Le,lloved, cells were rinsed in tap water until the water
was clear, and the plates were inverted and dried at room
temperature. Plaques were counted by use of a dissecting
microscope. The plaque counts were entered in the MacSynergy~
II program of Prichard and Shipman and 3-D plots were made.
In experiments with DHPG-resistant HCMV, growth medium was
decanted from established monolayers of MRC-5 cells in 24-well
tissue culture plates. The selected concentrations of compounds
were added in duplicate to wells of the plates at 0.8 ml/well.
Cell controls (2 wells/plate) each received 0.8 ml of test
medium. Plates were placed in a incubator at 37~C in a moist
atmosphere of 5~ CO2, 95~ air for 24 hr. All medium was
aspirated from each plate and 1.0 ml of virus, diluted in test
medium was placed in each well except those to be used for cell
controls. One ml of sterile test medium was placed in each of
these cell control wells. Virus was allowed to adsorb to the
cells during centrifugation at 2200 rpm for 30 minutes at room
temperature. Medium was aspirated from each well of the plates.
Eight tenths (0.8) ml of the proper compound dilution was placed
in each of the test wells. Test medium without compound was
added (0.8 ml/well) to each cell control and virus control well.
Plates were returned to the 37~C incubator until plaques could be
distinguished in the virus control wells. Cells were observed
microscopically for morphological changes due to compound
cytotoxicity before the medium was aspirated from all wells and
the cells stained by adding 0.3 ml of 0.2~ crystal violet in 10~
buffered formalin to each well. After 15 minutes, the stain was
e,lloved and the plates were inverted and dried at room
temperature. Plaques were counted by use of a dissecting
microscope. Effective dose, 50~ endpoint (ED50) was calculated
by regression analysis of the viral plaque data.
WO95/11038 _ 31 _ PCTIC~94/00590
Results of these experiments are shown in Figure 4. The
intense synergy seen when peptide AV-9 was used in combination
with DHPG indicates that these compounds work synergistically.
When phosphonoformic acid (foscarnet) was used in combination
with peptide AV-9, a moderate amount of synergy was seen as well
as an insignificant amount of antagonism (not shown). No synergy
was seen in the combination of AZT and AV-9.
