Combinations of 9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl]guanine or a pharmaceutically acceptable salt thereof, and interferon show synergy in their activity against herpes virus infections.

FIELD OF THE INVENTION 
This invention relates to pharmaceutical compositions, and more 
particularly to pharmaceutical compositions useful for treatment of viral 
infections such as herpes infections in mammals. 
BACKGROUND OF THE INVENTION 
Herpes simplex virus (HSV) infections are widespread in human populations, 
and pose a particularly difficult health problem. Genital herpes poses a 
serious health threat to women, in particular. Pregnant women with active 
genital herpes infections at the time of delivery have a 50--50 chance of 
passing it on to their babies. The American Academy of Pediatrics states 
that 60% of those babies born with HSV infections will die, and half of 
the survivors will suffer severe damage to the brain, nervous system and 
eyes ("Pediatrics" 66, 147-9, 1980). It has also been proposed that HSV2 
may have a role in the onset of cervical cancer. There has been observed 
an association between sexual intercourse and cervical cancer, which may 
be explained by transmission of HSV-2. 
Unlike other sexually transmitted diseases such as gonorrhea, syphilis and 
nongonococcal urethritis, there is currently no cure for herpes 
infections. Many of the drugs currently in clinical use may not be 
effective in reducing the severity or the duration of the symptoms. Even 
after the symptoms disappear, herpes virus tends to remain dormant in 
nerve tissue, only to be reactivated at a later date to an active phase of 
infection, causing lesions ("cold sores") and other symptoms to recur. A 
drug can be considered effective if it diminishes the severity of the 
lesions, allows for more rapid healing, extends the period between 
recurrences of herpes infections or stops recurrences altogether. 
Herpes simplex virus is one member of the family "Herpetoviridae"; other 
members of this family which infect humans are varicella-zoster, 
cytomegalovirus and Epstein-Barr virus. The family also includes various 
members which attack animals. For example, there are three types of equine 
herpesvirus, a swine herpesvirus, a canine herpesvirus and a feline 
herpesvirus, among others. 
As with all viruses, herpes virus invades healthy host cells on which it 
relies to provide its needs for replication. Herpes viruses code for some 
of the enzymes they need for replication, instead of relying completely on 
the host cell for all their needs. Hence, herpes viruses are subject to 
selective inhibition by certain drugs that interfere specifically with 
viral enzymes. 
BRIEF REFERENCE TO THE PRIOR ART 
A variety of drugs have been proposed and tested for treatment of HSV 
infections. For example, U.S. Pat. No. 4,199,574 Schaeffer, issued Apr. 
22, 1980 discloses a wide variety of compounds said to be useful in such 
treatments, extensive testing of one of which (acycloguanosine or 
acyclovir, 9-[2-hydroxyethoxymethyl]guanine) has been reported in the 
literature, with sometimes promising results. Another drug which has been 
explored is 5-iododeoxyuridine (IDU), but this has been reported to be 
effective only against herpes infections of the eyes. It also has 
undesirable side effects, associated with toxicity to normal cells. 
Adenine arabinoside (ara-A), phosphonoformic acid (PFA), phosphonoacetic 
acid (PAA), 2-deoxy-D-glucose (2DG), and 
5-(2-halogenovinyl)-2'-deoxyuridines as exemplified by 
bormovinyl-deoxyuridine (BVDU) and its iodo-analog are other drugs which 
have some demonstrated activity against human herpesviruses. 
U.S. Pat. No. 3,767,795 Schleicher et al, assigned to Abbott Laboratories, 
describes a method of preventing or treating herpesvirus infections in 
animals by administering phosphonoacetic acid or its salts. 
U.S. Pat. No. 4,215,113 Eriksson et al, assigned to Astra Lakemedel AB, 
describes a method of treating virus infections, including herpesvirus, by 
administering phosphonoformic acid or its salts to infected animals. 
U.S. patent application Ser. No. 302,790 Ogilvie, filed Sept. 16, 1981, 
describes 9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl]guanine and its 
activity against herpes virus. 
Patent Co-operation Treaty Application US82/00/182 K. O. Smith and ens Bio 
Logicals inc., describes synergistic mixtures of 
9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl]guanine and PFA or PAA or 
salts thereof, for use in treatment of herpes virus infected cells. 
SUMMARY OF THE INVENTION 
It has now been found that therapeutic compositions having good activity 
against herpes virus can be prepared from combinations of interferon and 
9-[[(2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl] guanine, or a 
pharmaceutically acceptable salt thereof. These combinations are much more 
effective than one could have predicted from a consideration of the 
activities of the compounds individually, indicating that some form of 
synergistic effect is taking place. 
Thus, according to the present invention, there is provided a therapeutic 
composition having activity against herpes virus infections and comprising 
in combination at least one interferon compound in admixture with 
9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl] guanine, or a 
pharmaceutically acceptable salt thereof. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Interferon is a low molecular weight polypeptide produced as an excretion 
from various types of viable mammalian cells. Its properties, chemical 
nature and methods of preparation and recovery have been extensively 
studied and documented in recent years, because of its potentially 
valuable pharmacological properties. It is known to exist in several 
types. Whilst all types of interferon are useful in composition of the 
present invention, it is preferred to use interferon-.alpha.. Interferon 
concentrations are commonly expressed as standard "units" which are 
internationally accepted and documented, and relate to the potency of a 
given quantity of interferon to inhibit a virus replication under standard 
conditions. 9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl] guanine, 
(hereinafter sometimes referred to a G*) and its processes for preparation 
are described in aforementioned U.S. application Ser. No. 302,790 Ogilvie. 
The compositions of the invention may of course include more than two 
active ingredients. G* may be present as the free base, or alternatively 
as a pharmaceutically acceptable salt such as hydrochloride. 
The preferred compositions of the present invention appear to be active 
against a wide representative variety of strains of HSV, both types I and 
II. The compositions may also be active against equine herpesvirus of 
various types, and swine herpesvirus (pseudorabies virus). 
Nearly every HSV strain produces virus particles which are partially 
resistant to each of the drugs mentioned above. For example, if one 
examines a typical titration curve which shows the effect of varying drug 
concentrations upon HSV plaque formations (the viral plaque titration 
method of Roizman and Roane referred to in more detail below), the curve 
is sigmoid, i.e. there are a few viral plaques which emerge in the 
presence of drug concentration which readily suppresses other plaques. It 
is these partially drug-resistant virus plaques which can sometimes be 
suppressed by a second drug possible having a mode of action different 
from the first drug. The result may be synergistic action between the 
first and second drugs, as set out below. 
The relative amounts of the drugs in the compositions according to the 
invention can be varied over wide limits. The optimum amount of each drug 
varies according to the nature of the formulation in which the drugs are 
to be applied, the type and strain of HSV to be treated, and the severity 
and location of the infection, among other factors. The amount of 
interferon, as noted above, is commonly expressed in standard units. 
Generally, compositions containing from about 50-500,000 units interferon 
per microgram of G* are effective. Preferred compositions are those 
containing from about 1,000-200,000 units of interferon per microgram of 
G*. 
For administration to patients, the compositions of the invention may be 
applied topically as ointment, cream or powder, parenterally, 
interthecally, as nose drops, eye drops or as an aerosol for inhalation, 
again depending upon the nature and location of the infection to be 
treated. Effective unit doses for administration of the compositions 
interthecally or parenterally are suitably in the range from about 0.1-100 
mg of total drugs in the chosen combinations, per kg mammal body weight, 
most suitably in the 0.5-20 mg per kg and most preferably about 5 mg per 
kg, on the basis of a dosage administered from 2-4 times daily. It is 
preferred to treat the infection with relatively large doses of the 
combination of drugs at the outset, so as to limit the chances of 
development of resistant viral strains in the infection. 
For topical administration, ointments or creams in conventional inert bases 
(e.g. petrolatum, etc.) can be formulated, in the known way. An amount 
from about 0.1-10 weight percent of total drugs, preferably from about 
0.5-5 weight percent of total drugs, provides a suitable concentration in 
an ointment or cream, for topical administration 1-4 times per day. Such 
topically applied formulations are effectively holding a reservoir of the 
active drugs against the infected site, so that the concentrations of 
drugs in the formulations are not critical, provided of course that a 
dosage level harmful to surrounding skin areas is not used.

DETAILED DESCRIPTION OF SPECIFIC PREFERRED EMBODIMENTS 
The invention is further illustrated in the following specific experimental 
results and examples. 
EXAMPLE 1 
Human fetal fibroblasts (HFF) derived from fetal tissues were used in these 
experiments. Cells were grown and maintained in Basal Medium Eagle (BME) 
supplemented with 0.112% sodium bicarbonate, 2 mML-glutamine, 2 mg% 
Neomycin and 20% (vol/vol) calf serum. 
HSV-1-Patton strains, an old established, well-known strain of herpes 
simplex type 1 virus was used in the tests, as set out in the Tables 
presented below. 
A viral plaque titration method (Roizman and Roane, 1961) was used to 
determine the titer of the HSV strain. Tissue culture dishes (35 by 10 mm, 
Corning) were seeded with cells and used for assays when they were 
approximately 75% monolayer. Volumes (0.2 ml) of logarithmic dilutions of 
the virus strain were inoculated onto each of two tissue culture dishes 
and adsorbed for 1 hr with intermittent shaking, the inoculum was removed 
and 2 ml of 20% BME containing 0.5% human immune serum globulin was added. 
After a 48 hr. incubation period at 36.degree. C. in a 5% CO.sub.2 
atmosphere, the overlay medium was removed, and the cell sheets were 
stained with a 0.05% aqueous crystal violet solution. The plaque numbers 
were counted with the aid of a Nikon profile projector which magnified the 
dishes 10X. Duplicate results were averaged, and the number of 
plaque-forming units (PFU) was calculated. The virus titre is thus 
expressed as a number of plaque forming units to be seen after growth 
under these conditions. 
As antiviral drugs in these experiments, there were used combinations of G* 
and interferon-.gamma. and .alpha.. 
In order to make comparisons between the activities of the various drugs 
and combinations, experiments were conducted by plaque titration to 
observe the antiviral potency, by observing and counting the number of 
PFUs per dish after growth of the infected cells in culture medium. For 
this purpose, tissue culture dishes (35 by 10 mm) with HFF cell monolayers 
at 75% confluence were inoculated with approximately 50 plaque-forming 
units of virus per 0.2 ml, and the virus was allowed to adsorb for 1 hr 
with intermittent shaking. After removal of the inoculum, 2 ml of 20% BME 
with 0.5% immune globulin and threefold dilutions of the appropriate drug 
were added to duplicate dishes. One set of dishes received no drug. After 
a 48 hr incubation period at 36.degree. C. in a 5% CO.sub.2 atmosphere, 
the overlay medium was removed, the cells were stained as described above, 
and plaques were counted. The counts of duplicate plates were averaged. 
Combinations of the drugs were tested for activity by viral plaque 
titration, at various concentrations, and the drugs were individually 
plaque titrated for comparison purposes. 
The results are given in the following Table. The virus titer results are 
expressed in plaque-forming units (PFU) per dish and, of course, the lower 
the figure the greater the inhibitory, anti-viral effect of the tested 
drug. 
TABLE 1 
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Plaque Inhibition Tests against 
HSV-1-Patton Infected HFF cells 
Interferon-.gamma. concentration (units per ml) 
10 100 1000 0 
PFU PFU PFU PFU 
per dish 
per dish per dish per dish 
______________________________________ 
G* concentra- 
0.02 27 23 24 31 
tion (micro- 
0.064 24 26 19 29 
grams per ml) 
0.2 18 20 7 18 
0.64 1 0 0 1 
2.0 0 0 0 0 
0 30 24 24 36 
______________________________________ 
These figures show that, whilst interferon-.gamma. alone has only slight 
effect on the virally infected cells and G* has effect at relatively high 
concentration, the combination in suitable proportions is much more 
effective, and permits the amount of compound G* to be reduced very 
significantly. 
EXAMPLE 2 
The procedures of Example 1 were essentially repeated, but using mixtures 
of equal proportion of interferon-.gamma. and interferon-.alpha., with 
various amounts of G*, and again using HFF cells infected with 
HSV-1-Patton. In each experiment, the amount of interferon was 1000 units 
per ml of interferon-.gamma. and 1000 units per ml of interferon-.alpha., 
for a total amount of 2000 units per ml of interferon. The virus titer 
results, in this case, are expressed as plaque forming units per ml, 
.times.10.sup.5. The figure for "Fold Titer Reduction" is the ratio of the 
virus titer for the control, i.e. no drug, to that of the drug-present 
experiment. The results are presented in Table 2 below. 
TABLE 2 
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With interferon 
No interferon 
G* Concentration, 
PFU/ml Fold titre 
PFU/ml Fold titre 
micrograms/ml 
.times. 10.sup.5 
reduction 
.times. 10.sup.5 
reduction 
______________________________________ 
0.02 30 4.0 115 1.0 
0.064 5 24.0 90 1.3 
0 100 1.2 120 1.0 
______________________________________ 
The results show that synergistic action occurs in these combinations of 
drugs, against the tested HSV strains. At least the same degree of synergy 
is to be expected in vivo. 
EXAMPLE 3 
In a further experiment, similarly virally infected HFF cells were 
incubated as described, and plaque titrated with various combinations of 
G* and an interferon-.gamma./interferon-.alpha. combination. In one set of 
dishes, no G* was used, and in the other set of dishes 0.05 micrograms per 
ml of G* was used. The interferon was in each case a mixture of equal 
amounts of interferon-.gamma. and interferon-.alpha., the numbers shown in 
the following results being the individual amounts of each component in 
the mixture. 
The results are shown in Table 3. The figures are presented as "fold titre 
reduction", with the dishes containing no drugs being assigned the value 
of unity. 
TABLE 3 
______________________________________ 
Interferon Concentration 
(units/ml) 
0 10 100 1000 
______________________________________ 
Fold titre reduction 
1.0 1.1 1.1 1.6 
with 0 micrograms/ml G* 
Fold titre reduction 
1.0 1.3 3.0 &gt;27 
with 0.05 micrograms/ml G* 
______________________________________ 
The relative amounts and concentrations of drugs used in these examples are 
appropriate for in vitro testing and to demonstrate the synergy of the 
combinations, but are illustrative only. Relative amounts in combinations 
for in vivo use and in practical administration for HSV treatments are as 
discussed previously, and may not bear close relationship to the 
proportions shown in the specific examples herein.