Method for combating virus infections

A method for the selective treatment of virus infections in animals and man, comprising administering to a host so infected a therapeutically effective amount of phosphonoformic acid or a physiologically acceptable salt thereof.

FIELD OF THE INVENTION 
The present invention relates to novel pharmaceutical compositions and to a 
novel method for selectively combating virus infections such as influenza 
virus infections, herpes virus infections, etc. in animals including man. 
GENERAL OUTLINE OF THE INVENTION 
There are mainly three approaches for the prophylactic and/or therapeutic 
treatment of virus infections, namely by use of vaccines, by use of 
chemotherapeutic agents, and by use of interferon. Of these, vaccines and 
chemotherapeutic agents have gained the largest medicinal use. 
However, the existing vaccines, in particular the influenza vaccines, are 
not deemed to be sufficiently effective. With regard to influenza virus, 
this is partly because it is difficult to prepare a vaccine in time 
against a prevailing modified influenza virus. No antiviral 
chemotherapeutic agent exhibiting good antiviral effect and acceptable 
side effects has yet been found. One especially undesired effect with 
available chemotherapeutic antiviral agents is that they may interact not 
only with the virus but also with components in the host cell. 
An effective selective antiviral agent with acceptable side effects should 
have a selective inhibiting effect on a specific viral function of the 
virus to be combated. It is, therefore, one object of the present 
invention to provide a novel method for combating virus infections using 
an antiviral agent which exerts a selective inhibiting effect on viral 
functions but which exerts only a negligible inhibiting effect on 
functions of the host cells. 
The invention also relates to novel pharmaceutical compositions containing 
the antiviral agent. 
Although the present invention relates broadly to a novel method for 
selectively combating virus infections in animals and man, and 
pharmaceutical preparations to be used at such treatment, it will be 
particularly useful in the treatment of herpes virus infections and 
influenza virus infections. 
Influenza is one of the most frequent diseases of man, but it is so far 
very difficult to prevent or treat. 
There are two major varities of influenza, commonly designated influenza A 
and influenza B. Another variety of influenza, designated influenza C also 
exists, but is not as frequently occuring as influenza A and B. These 
types of influenza are caused by virus commonly denoted influenza virus 
type A, B and C, respectively. 
An especially important area of use for the compositions of the present 
invention is in the treatment of herpes virus infections. Among the herpes 
viruses may be mentioned Herpes simplex type 1 and 2, varicella (Herpes 
zoster), virus causing infections mononucleosis (i.e. Epstein-Barr virus) 
and cytomegalovirus. Other herpes infections to which the present 
invention is applicable are Herpes dermatitis, Herpes genitalis, Herpes 
keratitis, and Herpes encephalitis. Other areas of use for the 
compositions of the present invention are in the treatment of infections 
caused by viruses such as papaloma virus (i.e. warts), adenoviruses, 
poxviruses, hepatitis virus A and hepatitis virus B. Other possible areas 
of use for the compositions of the present invention are in the treatment 
of infections caused by picornaviruses, arboviruses, leucoviruses, 
arenaviruses, coronaviruses, rhabdoviruses and paramyxoviruses and for 
inhibiting the growth of virus transformed cells in animals and man.

DETAILED DESCRIPTION OF THE INVENTION 
It has been found according to the present invention that phosphonoformic 
acid of the structural formula 
##STR1## 
and physilogically acceptable salts thereof, selectively inhibits certain 
viral functions which are essential for the replication of the virus. 
It has been found that phosphonoformic acid selectively inhibits a specific 
function of influenza virus, namely the influenza virion-associated RNA 
polymerase, while it does not affect either of the corresponding host cell 
polymerases, that is calf thymus DNA dependent RNA polymerase A and B. The 
said polymerases are enzymes which catalyze the synthesis of RNA in the 
host cell. It has also been found that phosphonoformic acid inhibits a 
specific function of herpes virus, namely the induced herpes simplex type 
1 DNA polymerase. It is not active on E. coli DNA dependent RNA polymerase 
and Micrococcus lysodeicticus DNA dependent DNA polymerase. Inhibition of 
the viral polymerases means that the virus cannot replicate and thus the 
viral infection is prevented. 
The phosphonoformic acid may be formulated for use in human and veterinary 
medicine for therapeutic and prophylactic use. The compounds may be used 
in the form of a physiologically acceptable salt. Suitable salts are e.g. 
amine salts, e.g. dimethylamine and triethylamine salt, ammonium salt, 
tetrabutylammonium salt, cyclohexylamine salt, dicyclohexylamine salt; and 
metal salts, e.g. mono-, di- and trisodium salt, mono-, di- and 
tripotassium salt, magnesium salt, calcium salt and zinc salt. 
In clinical practice the phosphonoformic acid will normally be administered 
topically, orally, intranasally, by injection or by inhalation in the form 
of a pharmaceutical preparation comprising the active ingredient in the 
form of the original compound or optionally in the form of a 
pharmaceutically acceptable salt thereof, in association wjth a 
pharmaceutically acceptable carrier which may be a solid, semi-solid or 
liquid diluent or an ingestible capsule, and such preparations comprise a 
further aspect of the invention. The compound may also be used without 
carrier material. As examples of pharmaceutical preparations may be 
mentioned tablets, drops such as nasal drops, preparations for topical 
application such as ointments, jellies, creams and suspensions, aerosols 
for inhalation, nasal spray, liposomes, etc. Usually the active substance 
will comprise between 0.05 and 99, or between 0.1 and 99% by weight of the 
preparation, for example between 0.5 and 20% for preparations intended for 
injection and between 0.1 and 50% for preparations intended for oral 
administration. In addition, the present invention may be practiced by the 
treatment of virus infections in animals and may by the method in which 
the phosphonoformic acid or a pharmaceutically acceptable salt thereof is 
formed in the body. 
To produce pharamaceutical preparations in the form of dosage units for 
oral application containing a compound of the invention the active 
ingredient may be mixed with a solid, pulverulent carrier, for example 
lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, 
corn starch, amylopectin, laminaria powder or citrus pulp powder, a 
cellulose derivative or gelatine and also may include lubricants such as 
magnesium or calcium stearate or a Carbowax.RTM. or other polyethylene 
glycol waxes and compressed to form tablets or cores for dragees. If 
dragees are required, the cores may be coated for example with 
concentrated sugar solutions which may contain gum arabic, talc and/or 
titanium dioxide, or alternatively with a film forming agent dissolved in 
easily volatile organic solvents or mixtures of organic solvents. 
Dyestuffs can be added to these coatings, for example, to distinguish 
between different contents of active substance. For the preparation of 
soft gelatine capsules consisting of gelatine and, for example, glycerol 
as a plasticizer, or similar closed capsules, the active substance may be 
admixed with a Carbowax.RTM. or a suitable oil as e.g. sesame oil, olive 
oil, or arachis oil. Hard gelatine capsules may contain granulates of the 
active substance with solid, pulverulent carriers such as lactose, 
saccharose, sorbitol, mannitol, starches (for example potato starch, corn 
starch or amylopectin), cellulose derivatives or gelatine, and may also 
include magnesium stearate or stearic acid as lubricants. 
By using several layers of the active drug, separated by slowly dissolving 
coatings sustained release tablets are obtained. Another way of preparing 
sustained release tablets is to divide the dose of the active drug into 
granules with coatings of different thicknesses and compress the granules 
into tablets together with the carrier substance. The active substance can 
also be incorporated in slowly dissolving tablets made for instance of fat 
and wax substances or evenly distributed in a tablet of an insoluble 
substance such as a physiologically inert plastic substance. 
In order to obtain dosage units of oral preparations--tablets, capsules 
etc.--which are designed so as to prevent release of and possible 
decomposition of the active substance in the gastric juice, the tablets, 
dragees etc. may be enteric-coated, that is provided with a layer of a 
gastric juice-resistant enteric film or coating having such properties 
that it is not dissolved at the acidic pH in the gastric juice. Thus, the 
active substance will not be released until the preparation reaches the 
intestines. As example of such known enteric coatings may be mentioned 
cellulose acetate phtalate, hydroxypropylmethylcellulose phtalates such as 
those sold under the trade names HP 55 and HP 50, and Edragit.RTM.L and 
Eudragit.RTM.S. 
Effervescent powders are prepared by mixing the active ingredient with 
non-toxic carbonates or hydrogen carbonates of e.g. sodium, potassium or 
calcium, such as calcium carbonate, potassium carbonate and potassium 
hydrogen carbonate, solid, non-toxic acids such as tartaric acid, ascorbid 
acid, and citric acid, and for example aroma. 
Liquid preparations for oral application may be in the form of elixirs, 
syrups or suspensions, for example solutions containing from about 0.1% to 
20% by weight of active substance, sugar and a mixture or ethanol, water, 
glycerol, propylene glycol and optionally aroma, saccharine and/or 
carboxymethylcellulose as a dispersing agent. 
For parenteral application by injection preparations may comprise an 
aqueous solution of a water soluble pharmaceutically acceptable salt of 
the active acids according to the invention, desirably in a concentration 
of 0.5-10%, and optionally also a stabilizing agent and/or buffer 
substances in aqueous solution. Dosage units of the solution may 
advantageously be enclosed in ampoules. 
For topical application, especially for the treatment of herpes virus 
infections on skin, the preparations are suitably in the form of an 
ointment, gel, suspension, cream or the like. The amount of active 
substance may vary, for example between 0.05-20% by weight of the active 
substance. Such preparations for topical application may be prepared in 
known manner by mixing the active substance with known carrier materials 
such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene 
glycol, etc. The pharmaceutically acceptable carrier may also include a 
known chemical absorption promoter. Examples of absorption promoters are 
e.g. dimethylacetamide (U.S. Pat. No. 3,472,931), trichloroethanol or 
trifluoroethanol (U.S. Pat. No. 3,891,757), certain alcohols and mixtures 
thereof (British Pat. No. 1,001,949). A carrier material for topical 
application to unbroken skin is also described in the British patent 
specification No. 1,464,975, which discloses a carrier material consisting 
of a solvent comprising 40-70% (v/v) isopropanol and 0-60% (v/v) glycerol, 
the balance, if any, being an inert constituent of a diluent not exceeding 
40% of the total volume of solvent. 
The dosage at which the active ingredients are administered may vary within 
a wide range and will depend on various factors such as for example the 
severity of the infection, the age of the patient, etc., and may have to 
be individually adjusted. As a possible range for the amount of 
phosphonoformic acid which may be administered per day may be mentioned 
from about 0.1 mg to about 2000 mg or from about 1 mg to about 2000 mg. 
The pharmaceutical compositions containing the active ingredients may 
suitably be formulated so that they provide doses within these ranges 
either as single dosage units or as multiple dosage units. 
Thus, it has been found according to the invention that phosphonoformic 
acid, and the physiologically acceptable salts thereof can be used to 
selectively inhibit viral functions of herpes virus and influenza virus. 
Since the functions in question are essential for the replication of the 
virus, phosphonoformic acid and physiologically acceptable salts thereof 
are useful in therapeutic and/or prophylactic treatment of virus 
infections. 
The preferred aspect of the invention is the use of phosphonoformic acid or 
a physiologically acceptable salt thereof, in the treatment of herpes 
virus infections. 
Phosphonoformic acid is a known compound. Its synthesis and its trisodium 
salt are described e.g. by Nylen, Chem. Berichte 57B: 1023-1038 (1924). 
Since phosphonoformic acid is unstable in its free acid form, it is 
preferably used in the form of its salts. 
BIOLOGICAL TESTS 
The Inhibiting effect of phosphonoformic acid on influenza and herpes virus 
was tested using the methods described below. The test method for testing 
of the effect of phosphonoformic acid on host cell functions are also 
described below. In the experiments A-K phosphonoformic acid was used in 
the form of its trisodium salt. 
I. Inhibition of viral and cellular polymerases 
Polymerase, nucleic acid template, nucleoside triphosphates, of which 
guanosine triphosphate is tritium-lbelled, salt and buffer are mixed at 
0.degree. C. in a total volume of 125 .mu.l (for herpes DNA polymerase, 
see F. below). The concentrations of the nucleoside triphosphates UTP 
(uridine triphosphate), CTP (cytidinetriphosphate), GTP 
(guanosinetriphosphate) and ATP (adenosinetriphosphate) were generally 
400, 400, 400, and 2000 .mu.M, respectively. The test compound is also 
added in various concentrations to the mixture obtained. A standard 
mixture without test compound is also prepared using the same amounts of 
the ingredients. The enzyme reaction, i.e. synthesis of nucleic acid, is 
started by incubating the mixture at 37.degree. C. For influenza RNA 
polymerases the temperature is 33.degree. C. The reaction is allowed to 
proceed for 60 minutes in the assay of influenza and Micrococcus 
polymerases. For E. coli DNA dependent RNA polymerase the time is 20 min. 
and for herpes DNA polymerase 30 minutes. The incubation time for calf 
thymus polymerases is 10 minutes. The incorporation of labelled monomer 
into trichloroacetic acid insoluble nucleic acid product was measured in 
the following way. Before and after the incubation period 50 .mu.l are 
withdrawn from the mixture and applied to filter discs. These are put in 
5% trichloroacetic acid solution and washed several times. After drying 
the discs are measured for radioactivity in a liquid scintillation 
counter. the difference in radioactivity between samples with and without 
added compound is used to calculate the inhibition of the polymerase 
activity. The inhibition is expressed as percentage inhibition using the 
radioactivity of the standard sample as basis. 
A. Inhibition by phosphonoformic acid of type A influenza virion associated 
RNA polymerase 
Influenza A.sub.2 Aichi virus was purified according to the method of Pons 
and Hirst, Virology 34 385 (1968). The assay mixture is described by 
Bishop, Obijeski and Simpson, J. Virol. 8 66 (1971). The inhibitory effect 
of phosphonoformic acid is shown in Table 1 below. 
Table 1 
______________________________________ 
Inhibition by phosphonoformic acid of type A 
influenza virion-associated RNA polymerase 
Conc. of phosphonoformic acid 
Inhibition.sup.a 
(.mu.M) (%) 
______________________________________ 
0.1 21 
0.50 67 
1.00 79 
10 91 
100 95 
500 93 
______________________________________ 
.sup.a mean of two experiments 
B. Inhibition by phosphonoformic acid of type B influenza virionassociated 
RNA polymerase 
Polymerase from influenza B Hongkong 8/73 was assayed in the same way as 
for influenza A.sub.2 Aichi in experiment A. The inhibitory effect of 
phosphonoformic acid is shown in Table 2 below. 
Table 2 
______________________________________ 
Inhibition by phosphonoformic acid of Type B 
influenza virion associated RNA polymerase.sup.a 
Conc. of phosphonoformic acid 
Inhibition 
(.mu.M) (%) 
______________________________________ 
1.0 77 
500 100 
______________________________________ 
.sup.a In this experiment the concentration of GTP was 135 .mu.M. 
C. Inhibition by phosphonoformic acid of calf thymus DNA dependent RNA 
polymerase 
Purification of the enzymes was carried out according to the method of 
Kedinger et al, Eur. J. Biochem. 28 269 (1972). The enzyme fractions DCB 
and DCA were used for all experiments. The assay mixture of Kedinger, 
loc.cit., was used. The test results are given in Table 3 below. 
Table 3 
______________________________________ 
Inhibition by phosphonoformic acid of calf thymus 
DNA dependent RNA polymerase fractions B and A 
Conc. of phosphonoformic acid 
Inhibition % 
(.mu.M) DCB DCA 
______________________________________ 
500 0 -3 
______________________________________ 
D. Inhibition by phosphonoformic acid of E. coli DNA dependent RNA 
polymerase 
The enzyme was bought from Sigma. The template used was DNA extracted from 
E. coli according to Marmur, J. Mol. Biol. 3 208 (1961) and the assay 
mixture essentially that described by Burgess, J. Biol. Chem. 244 6160 
(1969). The test results are given in Table 4. 
Table 4 
______________________________________ 
Inhibition by phosphonoformic acid of E. coli 
dependent RNA polymerase 
Conc. of phosphonoformic acid 
Inhibition 
(.mu.M) (%) 
______________________________________ 
500 3 
______________________________________ 
E. Inhibition by phosphonoformic acid of Micrococcus luteus DNA dependent 
DNA polymerase 
The polymerase was bought from Sigma and assayed essentially according to 
Harwood et al., J. Biol, Chem. 245 5614 (1970). 
The test results are given in Table 5 below. 
Table 5 
______________________________________ 
Inhibition by phosphonoformic acid of Micrococcus 
luteus DNA dependent DNA polymerase 
Conc. of phosphonoformic acid 
Inhibition 
(.mu.M) (%) 
______________________________________ 
500 6 
______________________________________ 
F. Inhibition by phosphonoformic acid of herpes simplex virus type 1 
induced DNA polymerase 
Purification of the enzyme was carried out according to the method of 
Weissbach et al., J. Biol. Chem. 248 6270 (1973). 
The assay mixture (200 .mu.l) contained 200 .mu.g/ml activated calf thymus 
DNA and 0.05 mM [3H] dTTP (specific activity 130 cpm per pmole). 
All other ingredients were according to Weissbach (see above). 
The results are given in table 6. 
Table 6 
______________________________________ 
Inhibition by phosphonoformic acid of herpes simplex 
virus type 1 induced DNA polymerase 
Conc. of phosphonoformic acid 
Inhibition 
(.mu.M) (%) 
______________________________________ 
5 15 
20 43 
100 76 
500 90 
______________________________________ 
II. Inhibition of virus multiplication in cell cultures 
The inhibition of influenza virus and herpes virus by phosphonoformic acid 
has been measured as plaque reduction according to the following 
procedures. 
G. Inhibition by phosphonoformic acid of influenza (WSN Wilson Smith 
Neurotropic type A.) plaque 
The method for plaque assay of influenza has been described by Bentley et 
al., Archiv fur die Gesamte Virusforschung 33 (1971) 234. Monolayers of 
MDCK (Mardin Darby Canine Kidney) cells on 5 cm plastic petri dishes were 
inoculated with 100 plaque-forming units of influenza virus (WSN). After 
virus adsorption, 5 ml of agarose overlay containing different 
concentrations of phosphonoformic acid was added and the plates were 
incubated at 34.degree. C. for 4 days. The plaques formed at this time 
were counted. The results are shown in Table 7. 
Table 7 
______________________________________ 
Inhibition by phosphonoformic acid of influenza virus 
(WSN) plaque on MDCK monolayers. 
Conc. of phosphonoformic acid 
Inhibition.sup.a 
(.mu.M) (%) 
______________________________________ 
100 10 
250 50 
500 95 
______________________________________ 
.sup.a a mean of three different experiments 
H. Inhibition by phosphonoformic acid of herpes simplex type 1 plaque 
The plaque reduction assay for herpes simplex type 1 was performed on GMK 
(Green Monkey Kidney) cells as described by Ejereito et el., J. Gen. 
Virol. 2 (1968) 357. Monolayers on 5 cm petri dishes were used and after 
virus adsorption phosphonoformic acid was added in the medium. The results 
are shown in table 8. 
Table 8 
______________________________________ 
Inhibition by phosphonoformic acid of herpes simplex 
type 1 plaque on GMK monolayers. 
Conc. of phosphonoformic acid 
Inhibition.sup.a 
(.mu.M) (%) 
______________________________________ 
1 0 
15 50 
100 90 
______________________________________ 
a means of three different experiments 
I. Inhibition by phosphonoformic acid of herpes simplex type 2 plaque 
The plaque reduction assay for herpes simplex type 2 was performed in the 
same way as in experiment H. The results are shown in table 9. 
Table 9 
______________________________________ 
Inhibition by phosphonoformic acid of herpes simplex 
type 2 patient isolates plaque on SIRC (Staatens Serum- 
institut Rabbit Cornea) monolayers. 
Conc. of phosphonoformic acid 
Inhibition 
(.mu.M) (%) 
______________________________________ 
500 &gt;99.9 
______________________________________ 
J. Acute toxicity 
Phosphonoformic acid was tested for acute toxicity in mice. The compound 
(as its sodium salt) was given as solution i.p. in doses of 250, 500, 
1000, 2000, and 4000 .mu.mol/kg. Groups of 4 male mice of NMRI strain 
weighing 18.5-20.5 g were used for each dose. LD50 was found to be between 
4000 and 2000 .mu.mole/kg bodyweight. 
K. Other animal experiments 
Preliminary experiments on cutaneous herpes type 1 infected guinea pigs 
have shown that phosphonoformic acid as its trisodium salt in topical 
preparations according to examples 15, 16 and 17 below has a therapeutic 
effect. 
Discussion of the test results 
The purpose of tests A. B and G above is to ascertain the effect of the 
phosphonoformic acid against influenza viruses. The purpose of tests F, H 
and I is to ascertain the effect of phosphonoformic acid against herpes 
viruses. The purpose of test C, D and E is to ascertain the absence of 
effect of phosphonoformic acid on cellular polymerases. As seen in tables 
1 and 2, respectively, phosphonoformic acid inhibits the influenza A virus 
polymerase activity to more than 50% at a concentration of 0.5 .mu.M and 
influenza B virus polymerase to more than 50% at a concentration of 1.0 
.mu.M. As seen in table 7, phosphonoformic acid inhibits the corresponding 
plaque formation to 50% at a concentration of 250 .mu.M. As seen in tables 
6, 8 and 9, respectively, phosphonoformic acid inhibits the herpes simplex 
virus type 1 induced DNA polymerase activity to more than 50% at a 
concentration of 100 .mu.M, the plaque formation of herpes simplex virus 
type 1 to 50% at a concentration of 15 .mu.M and plaque formation of the 
herpes virus type 2 to more than 99.9% at 500 .mu.M. In tables 3, 4 and 5 
it is seen that phosphonoformic acid has no significant effect against 
calf thymus DNA dependent RNA polymerases, or 0% and -3% inhibition 
respectively, at a concentration of 500 .mu.M; that it is practically 
inactive against E. coli DNA dependent RNA polymerase, or 3% inhibition at 
a concentration of 500 .mu.M, and that it is practically inactive against 
Micrococcus luteus DNA dependent DNA polymerase, or 6% inhibition at a 
concentration of 500 .mu.M. The acute toxicity of phosphonoformic acid is 
low, i.e. LD50 between 2000 and 4000 .mu.mole/kg i.p. in mice. Thus, 
phosphonoformic acid exerts a selective effect on influenza and herpes 
viruses. The selective effect of phosphonoformic acid on the viral 
polymerases gives a molecular basis for a selective antiviral effect in 
animals including man. 
Salts of phosphonoformic acid 
Physiologically acceptable salts of phosphonoformic acid are prepared by 
methods known in the art as illustrated in the following. Metal salts can 
be prepared by reacting a metal hydroxide with an alkylester of 
phosphonoformic acid. Examples of metal salts of phosphonoformic acid 
which can be prepared in this way are salts containing Li, Na, K, Ca, Mg, 
Zn, Mn and Ba. A less soluble metal salt can be precipitated from a 
solution of a more soluble salt by addition of a suitable metal compound. 
Thus for examples, Zn, Mg and Mn salts of phosphonoformic acid can be 
prepared from phosphonoformic sodium salts. The metal ion of a metal salt 
of phosphonoformic acid can be exchanged by hydrogen ions, other metal 
ions, ammonium ion and ammonium ions substituted by one or more organic 
radicals by using a cation exchanger as shown in the following examples. 
EXAMPLE 1. Disodium salt of phosphonoformic acid 
Phosphonoformic acid trisodium salt (1.30 g) was dissolved in 50 ml of 
water. To this solution a cation exchanger Dowex 50 W.times.2 in acid form 
was added with stirring until a pH of 5.35 was obtained. The ion exchanger 
was filtered off and the filtrate evaporated at a reduced pressure. The 
residue was triturated with ethanol with cooling. The yield of disodium 
salt was 0.65 g. It contained 8.3% water. Titration as base gave 
equivalent weight 168.2 (corrected for 8.3% water) and Na 27.3% (corrected 
for water). Calculated for CHO.sub.5 P.2Na, formula weight 170.0, Na 
27.1%. 
EXAMPLE 2. Monocyclohexylammonium salt of phosphonoformic acid 
A cation exchanger Dowex 50 W.times.2 (75 g) on acid form was saturated 
with cyclohexylamine (10 g), poured into a column (diameter 2 cm) and 
washed free from excess of cyclohexylamine. A water solution of 
phosphonoformic acid trisodium salt (1.3 g in 40 ml of water) was slowly 
passed through the column followed by about 100 ml of water and the 
combined eluate evaporated at a reduced pressure. The salt obtained was 
recrystallized from ethanol-water. The final product was crystalline and 
hand a melting-point of 215.degree.-218.degree. C. (decomposition) and 
contained 27.0% of water. Titration as acid gave equivalent weight 220.5 
(corrected for 27.0% water). Calculated for CH.sub.3 O.sub.5 P.C.sub.6 
H.sub.13 N formula weight 225.2. The NMR spectrum indicated that the salt 
contained only one cyclohexylamine residue (a minor impurity in form of 
ethanol was detected). 
Examples of other useful salts which can be prepared in this way are the 
salts of the formula 
##STR2## 
in which formula n is 1, 2 or 3 and X is a salt-forming component such as 
NH.sub.3, CH.sub.3 NH.sub.2, C.sub.2 H.sub.5 NH.sub.2 C.sub.3 H.sub.7 
NH.sub.2, C.sub.4 H.sub.9 NH.sub.2, C.sub.5 H.sub.11 NH.sub.2, C.sub.6 
H.sub.13 NH.sub.2, (CH.sub.3).sub.2 NH, (C.sub.2 H.sub.5).sub.2 NH, 
(C.sub.3 H.sub.7).sub.2 NH, (C.sub.4 H.sub.9).sub.2 NH, (C.sub.5 
H.sub.11).sub.2 NH, (C.sub.6 H.sub.13).sub.2 NH, (CH.sub.3).sub.3 N, 
(C.sub.2 H.sub.5).sub.3 N, (C.sub.3 H.sub.7).sub.3 N, (C.sub.4 
H.sub.9).sub.3 N, (C.sub.5 H.sub.11).sub.3 N, (C.sub.6 H.sub.13).sub.3 N, 
C.sub.6 H.sub.5 CH.sub.2 NH.sub.2, HOCH.sub.2 CH.sub.2 NH.sub.2, 
(HOCH.sub.2 CH.sub.2).sub.2 NH, (HOCH.sub.2 CH.sub.2).sub.3 N, C.sub.2 
H.sub.5 NH(CH.sub.2 CH.sub.2 OH), C.sub.2 H.sub.5 N (CH.sub.2 CH.sub.2 
OH).sub.2, (HOH.sub.2 C).sub. 3 CNH.sub.2 and 
##STR3## 
Further examples of other useful salts which can be prepared by the ion 
exchange technique are quaternary ammonium salts of phosphonoformic acid, 
i.e. salts in which 1-3 of the hydrogens in phosphonoformic acid 
(structural formula I) have nbeen substituted with quaternary ammonium 
ions such as (CH.sub.3).sub.4 N, (C.sub.2 H.sub.5).sub.4 N, (C.sub.3 
H.sub.7).sub.4 N, (C.sub.4 H.sub.9).sub.4 N, (C.sub.5 H.sub.11).sub.4 N, 
(C.sub.6 H.sub.13).sub.4 N and C.sub.2 H.sub.5 N(CH.sub.2 CH.sub.2 
OH).sub.3. Lipophilic salts of this type can also be prepared by mixing a 
salt of phosphonoformic acid with a quaternary ammonium salt in water and 
extracting out the resulting quaternary ammonium salt of phosphonoformic 
acid with an organic solvent such as dichloromethane, chloroform, ethyl 
acetate and methyl isobutyl ketone. 
Pharmaceutical compositions 
The following examples illustrate the preparation of pharmaceutical 
compositions of the invention. The phosphonoformic acid is preferably used 
in the form of its sodium salt. 
EXAMPLE 3. Aerosol for inhalation 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
1.00 g 
Miglyol.RTM. 0.20 g 
Frigen.RTM. 11/12/113/114 
ad 100.0 g 
______________________________________ 
EXAMPLE 4. Tablets 
______________________________________ 
Each tablet contains: 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
20.0 mg 
Maize starch 25.0 mg 
Lactose 190.0 mg 
Gelatin 1.5 mg 
Talc 12.0 mg 
Magnesium stearate 1.5 mg 
250.0 mg 
______________________________________ 
EXAMPLE 5. Suppositories 
______________________________________ 
Each suppository contains: 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
20.0 mg 
Ascorbyl palmitate 1.0 mg 
Suppository base (Imhausen H or Witepsol.RTM. H) 
ad 2000.0 mg 
______________________________________ 
EXAMPLE 6. Syrup 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
0.200 g 
Liquid glucose 30.0 g 
Sucrose 50.0 g 
Ascorbic acid 0.1 g 
Sodium pyrosulfite 0.01 g 
Disodium edetate 0.01 g 
Orange essence 0.025 g 
Certified colour 0.015 g 
Purified water ad 100.0 g 
______________________________________ 
EXAMPLE 7. Injection solution 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
0.500 mg 
Sodium pyrosulfite 0.500 mg 
Disodium edetate 0.100 mg 
Sodium chloride 8.500 mg 
Sterile water for injection 
ad 1.00 ml 
______________________________________ 
EXAMPLE 8. Inhalation solution 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
5.00 g 
Sodium pyrosulfite 0.10 g 
Disodium edetate 0.10 g 
Sodium chloride 0.85 g 
Purified water ad 100.0 ml 
______________________________________ 
EXAMPLE 9. Sublingual tablets 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
5.0 mg 
Lactose 85.0 mg 
Talc 5.0 mg 
Agar 5.0 mg 
100.0 mg 
______________________________________ 
EXAMPLE 10. Drops 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
2.00 g 
Ascorbic acid 1.00 g 
Sodium pyrosulfite 0.10 g 
Disodium edetate 0.10 g 
Liquid glucose 50.00 g 
Absolute alcohol 10.00 g 
Purified water ad 100.0 ml 
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EXAMPLE 11. Syrup 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
0.200 g 
Liquid glucose 30.0 g 
Sucrose 50.0 g 
Ascorbic acid 0.1 g 
Disodium edetate 0.01 g 
Orange essence with solubilizer 
0.25 g 
Hydrochloric acid to pH 6.0-6.5 
Purified water ad 100.0 g 
______________________________________ 
EXAMPLE 12. Solution for injection 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
0.500 mg 
Disodium edetate 0.100 mg 
Sodium chloride 8.500 mg 
Hydrochloric acid to pH 6.5-7.0 
Sterile water for injection 
ad 1.00 ml 
______________________________________ 
EXAMPLE 13. Solution for inhalation 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
5.00 g 
Disodium edetate 0.10 g 
Sodium chloride 0.85 g 
Hydrochloric acid to pH 6.5-6.9 
Purified water ad 100.0 ml 
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EXAMPLE 14. Drops 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
2.00 g 
Citric acid 1.00 g 
Disodium edetate 0.10 g 
Liquid glucose 50.00 g 
Ethanol 95% 10.00 g 
Sodium hydroxide and hydrochloric acid to 
pH 6.2-6.8 
Purified water ad 100.0 ml 
______________________________________ 
EXAMPLE 15. Solution for topical use 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
2.00 g 
Isopropanol 38.0 g 
Glycerol 13.6 g 
Hydrochloric acid to pH 5.0-7.0 
Purified water ad 100.0 g 
______________________________________ 
Preparations containing 0.2, 0.5 and 1.0 g of phosphonoformic acid 
trisodium salt have also been prepared. 
EXAMPLE 16. Jelly 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
4.0 g 
Methocel.RTM. 4.0 g 
Methyl paraoxybenzoate 0.12 g 
Propyl paraoxybenzoate 0.05 g 
Sodium hydroxide and hydrochloric acid to pH 6.7 
Destilled water ad 100.0 ml 
______________________________________ 
EXAMPLE 17. Ointment I 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
2.5 g 
Cetyltrimethylammonium bromide 
0.6 g 
Stearyl alcohol 2.25 g 
Cetanol 6.75 g 
Liquid paraffine 17.0 g 
Glycerol 12.0 g 
Hydrochloric acid to pH 6.5 
Destilled water ad 100.0 g 
______________________________________ 
Preparations containing 0.2, 0.5, 1.0 and 2.0 g of phosphonoformic acid 
trisodium salt have also been prepared. 
EXAMPLE 18. Ointment II 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
2.5 g 
Polyethylene grycol 1500 50 g 
Polyethylene glycol 4000 15 g -Propylene glycol ad 100 
______________________________________ 
g 
EXAMPLE 19. Ointment III 
______________________________________ 
Phosphonoformic acid (as its trisodium salt) 
3.0 g 
Sorbitan monoleate 5.0 g 
Petrolatum ad 100 g 
______________________________________ 
EXAMPLE 20. Gastric juice-resistant tablets 
Tablets according to Example 4 are coated with an enteric coating solution 
with the following composition: 
______________________________________ 
Cellulose acetate phtalate 
120.0 g 
Propylene glycol 30.0 g 
Sorbitan monoleate 10.0 g 
Ethanol 95% 450.0 ml 
Acetone q.s. ad 1000.0 ml 
______________________________________ 
The coating is carried out by a pouring procedure in a conventional coating 
pan or by spraying the tablets in a pan spray tablet coater.