Note: Descriptions are shown in the official language in which they were submitted.
:
This invention relates to a novel therapeutic agent for virus
- diseases, and to its preparation and use.
It is known that several nucleoside compour~s, i.e. ccmpounds
having a sugar moiety bound to a heterocyclic nucleus, have antiviral
activities. Among them may be r~entioned ara-A or 9-(beta-D-
arabinofuranosyl)-adenine (compare French Patent No. 3585 M) and
ribavirin or (l-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
~compare Science, 177, 705, 1972, and Chemotherapy, 21, 505, 1975).
Ara-A has a marked antiviral activity but only for same DNA viruses, and
ribavirin has a broad-spectrum antiviral activity, i.e. an activity
against several RNA and DNA viruses but its utilisation is hampered by
a rather narrow safety rr~rgin.
In accordance with the invention, it has now been found that
(S)-9-(2,3-dihydroxypropyl) adenine of the following formula:
NH2
N ' ~ N~
N N
H2C - CH - CH2
OH O~
exhibits a marked broad-spectrum antiviral activity (against RNA as well
as DNA viruses) and that it has a very low acute toxicity. This is
surprising since it could not be e~pected that the substitution of a
dihydroxypropyl group for a sugar rr~oiety (when ccmpared with ara-A)
would result in any maintenance of antiviral activity. Moreover, it would
: 25 not be expected that the compound of the invention, hereafter termed
(S)-DHPA, would have a broad-spectrum antiviral activity since the antiviral
activity of ara-A is limited only to DNh viruses. Finally, its low
toxicity is surprising in view of the high toxicity of ribavirin.
It should be noted that only the D~glycero form of S-
er~ntiomeric form of 9-(2,3-dihydroxypropyl) adenine is active and not the
I-form or R-enantiomeric form. Further, the RS-form or racemic mixture
,, _
: 1331 P/2 CA - 1 -
, . .
~ `` "
is almost as effective as the S-form.
Further, it should be noted that several related ccmpounds
; having another heterocyclic nucleus or another aliphatic side chain
do not exhibit any antiviral activities, at least not in acceptable doses,
as may appear from the experimental part of this specification. Thus,
the antivirial activity of (S)-D~PA is still more surprising.
Furthermore, it should be noted that (S)-DHPA potentiates
the anitviral activity of other antiviral age~ts, such as ara-A, and can,
therefore, be used in combination with these antivial agents.
.,
On the basis of these findings, the invention provides a novel
therapeutic composition for use in the treatment of virus diseases which
camprises an effective amount of (S)-9-(2,3-dihydroxypropyl)adenine or
(RS) 9-(2,3-dihydroxypropyl)adenine as an active ingredient. (S) or
.
(RS~-9-(2,3-dihydroxypropyl)adenine can be used as the sole active
.: _
ingredient or in ccmbination with other active ingredients, such as ara-A.
Furthermore, the invention provides a method of preparing such a
therapeutic composition by combining (S)-9-(2,3-dihydroxypropyl)adenine or
(RS)-9-(2,3-dihydroxypropyl)adenine with a pharmaceutically acceptable
excipient, and a method for treatment of virus diseases, which camprises
administering the aforementioned therapeutic composition to a patient
suffering from a vi~us disease.
The process for the preparation of both the (S)-enantiomer and
the racemic (RS)-form of 9-(2,3-dihydroxypropyl)adenine is known. The
synthesis of these compounds is therefore no part of the present invention.
Thus, according to procedure published by Holy (Collect. Czech. Chem.
~-- CbmmNn., 40, 187, 1975), both compounds can be prepared by heating
1-0-p-tolylsulfonyl-2,3-0-isopropylidene-D-(or DL-) glycerol of the
fornula A
p-CH3C6H4S020CH2iCH ICH 2
- 30 0 0
X (A)
CH3 CH3
1331 P/2 CA - 2
~ -~'';.
':
L
with the sodium salt of adenine in dimethylformamide solution at 100C
and treatment of the intermediate compound with refluxing 80% acetic acid.
E.G., 11.4 g (40 mmol)of compound A and 7.8 g (50 mmol) sodium salt of
adenine in dimetl~ylformamide (100 ml) is heated for 8 h at 100 C,
evaporated in vacuo and the residue crystallized from methanol. m e
crystalline -product is refluxed with 80% acetic acid for 1 h, evaporated
in vacuo, codistilled with e'~hanol (3 x 50 ml) and crystallized fro~ -
methanol. Yield 50-60% of (S)- or (RS)-9-(2,3-dihydroxypropyl)adenine.
(S)-form : M.p. 202-203& , (a)D -35.4 (c=l, water). (RS)-form :
.. o ~.
M.p. 207-208 C. Uv-Spectra (pH 7) : ln~X 260nm, max 14000, lmin 228 nm-
~ According to the Czechoslovak Author's certificate PV 1787-77
,,!.~"...... (RS)-9-(2,3-dihydroxypropyl)adenine can be obtained by heating of adenine
with glycerol-1,2-cyclic carbonate of the formula B
CH - CH - CH OH
2 1 2
15 0 0
C (B)
O
, and sodium or potassium hydroxide or carbonate in dimethylformamide or
1,4-dioxane solution at 80-140C. E.g., a mixture of adenine (1.35 g),
. ..~. .~
~ 20 glycerol-1,2-cyclic carbonate (2.0 g), sodium carbonate (0.3 g) and
.
dimethylformamide (25 ml) is refluxed for 1 h, evaporated in vacuo and
the residue dissolved in 50 ml boiling water is decolorized by charcoal.
After evaporation in vacuo, the residueaffords on crystallization frcm
,i. . . _
;.
methanol racemic (RS)-9-(2,3-dihydroxypropyl)adenine in 60-70~ yield.
M.p. 205-206C, W-spectra (water) : ~max 260 ~m~ a 14000-
; The antiviral activities of (S)-DHPA will now be described in
detail. Reference is made thereby to the drawings, wherein: Fig. 1 is
a gr~phical representation showing the effect of (S)-DHPA on human skin
:. .
~ fibroblast cultures inoculated with vesicular stcmatitis virus; Fig. 2
;~ 30 is a graphical representation showing the effect of (S)-DHPA in vivo in
mice inoculated intranasally with vesicular stcmatitis virus; Fig. 3 is a
: . .
graphical representation showing the inhibitory effect of (S)-DHPA on
deamination of ara-A by adenos~le-deaminase of calf intestinal mucosa;
1331 P/2 CA - 3 -
. . .~.~'~,
:
Fig. 4 is a graphical representation of the same data as in Fig. 3 but with
reciprocal plotting on the ordinate.
The antiviral activity of (S)-~HPA was explored in a variety
of cell cultures and with a variety of DNA and RNA viruses such as listed
in Table 1. The cells of each cell culture were inoculated with a ce-tain
virus in a dose of about 100 CCID50, that is about 100 times the dose
needed to infect 50% of the cells. One hour after inoculation, (S)-DHPA
was added in varying doses fro~ zero to 40 ~g/ml and sometLmes to more
than 200 ug/ml. For each virus-cell system, the ID50 of (S)-DHPA, that is
the dose of (S)-DHPA needed to suppress the cytopathic effect of the
virus by 50%, was deternL~led. This cytopathic effect (CPE) was measured
in the untreated virus-infected cell cultures ((S)-DHPA does 0) and
recorded as soon as it reached completion (according to the method
described by L.J. Rosenthal and I.L. Shechmaister, in "Tissue Culture",
15 page 510, Academic Press, New York, 1973). The results of these experiments
are shcwn in Table 1.
It appears from Table 1, that several DNA and RNA viruses including
vaccinia, herpes simplex (types 1 and 2), measles and vesicular stomatitis
virus, were inhibited by (S~-DHPA. Other vi~uses such as polio, Coxsackie
and Sindbis virus were not affected.
That the inhibitory effects of (S)-DHPA on virus-induced
cytopathogenicity actually reflected an inhibition of virus mL~tipiication,
was ascertained by measuring virus growth in human skin fibroblast (HSF)
; cultures which had been inoculated with vesicular stomatitis virus (VSV)
and subsequently exposed to (S)-DHPA. In these experim~lts, confluent
` ~,onolayers of HSF cells in plastic petri dishes were inoculated with'
vesicular stomatitis virus (4.5 log 10 CCID50/0.5 ml/petri dish) for 1 h
at 37& and, immediately thereafter, exposed to (S)-DHPA (100 ug/ml).
The cell cultures were then incubated for varying times at 37 C. At the
end of the incubation period, the cells were frozen at -70C, and
the cell hcmogenates were assayed for virus content by plaque foLmation in
- m~use Lr929 fibroblast cultures. mis method is well-known. Each plaque
is produced by a virus having infected a cell, multiplied within the cell
1331 P/2 CA - 4 -
~ .,
and burst the cell. As the cell monolayers are overlaid by a gel-like
medium (e.g. agar) the viruses released upon the destruction of the cell
can only invade neighbour cells, resulting in the formation of a zone
of cell destruction, called plaque. The number of plaques is equivalent to
the virus content. The results are presented graphically in Fig. 1, where
PFU means Plaque Formation Vnits.
It appears frcm Fig. 1 that 100 ~g/ml of (S)-DHPA caused a
dramatic decrease of virus titer, as compared with the control culture where
."
no (S)-DHPA was added. This decrease amounted to approximately 4 log10 for
the virus yields measured at 24 and 48 h after infection.
TABLE 1. Antiviral activity of (S)-DHPA in cell cultures
.
Virus Cell culture*ID50(~g/ml)
.
15 DNA viruses
..... .
Vaccinia PRK 10 - 20
Vaccinia HSF 10 - 20
Herpes simplex-l strain KO6 PRK 10
-~ ~erpes simplex-l strain KOS HSF 20
20 Herpes simplex-2 strain 333 P~ 4 - 10
Herpes simplex-2 strain 333 HSF 7 - 20
RNA viruses
,
.:
-;~ Vesicular stcmatitis PRK 7 - 10
. .
Vesicular stomatitis HSF 2 - 7
- 25 Vesicular stcnatitis ~eLa ;-'200
Polio-l HSF ~200
. .
Polio-l HeLa >200
- Coxsackie B-4 HeLa >200
Coxsackie B-4 Vero >200
30 Measles Vero 4 - 40
Sindbis _ BHK _ ~ 200
.
* Abbreviations : PRK, primary rabbit kidney; HSF, human skin Fibroblast;
Vero, a continuous cell line of green monkey kidney cells; BHK, a
ccntinuous cell line of baby hamster kidney cells.
~ . .
~ 1331 P/2 CA - 5 -
.,
,'` X
3~f''~
The ~otential in vivo activity of (S)-DHPA was assessed in mice
infected with vesicular stomatitis virus (VSV) according to the method
described in J. Gen. Virol., 5, 359 (1969) and J. Clin. Invest., 49,
1565 (1970). r~wenty days old female NMRI mice (average weight 15 g) were
inoculated intranasally with vesicular stcmatitis virus (2.5 log10
CCID50/0.01 ml/mouse). m ereupon, they were repeatedly injected intra-
peritoneally with (S)-DHPA at a dose of 2 mg/mouse (about 133 mg/kg) at
- 1 h and 1, 2, 3 and 4 days after the virus infection~ Deaths were recorded
,.
daily and the cu~ulative mortality was deteremuned. me results for 14
consecutive days are graphically presented in Fig. 2. No deaths were noted
beyond the 14th day after infection.
It appears from Fig. 2 that the repeated doses of (S)-DHPA at 2
mg/mouse brought about a significant increase in the final nurnber of
surviving mice : 67% for the (S)-DHPA-treated mice as compared to 37.5%
for the control group. If the numbers of survivors were compared at 9 days
after infection, the difference between the test group and the control
group was also significant.
The experiments of Fig. 2 were repeated with different (S)-~HPA
levels. Repeated doses of 0.08 mg/mouse of (S)-DHPA (about 5.4 mg/kg~ did
not confer protection, whereas repeated doses of 0.4 mg/m~use (about
: ~7 mg/kg) caused a slight protection (final number of surviving mice 55
as compared to 37.5% for the control group).
Experiments for assessing the acute toxicity of (S)-DHPA have also
been carried out in mice but a LD50 value (dose lethal for 50% of the
mice) could not yet been determined, due to the fact that all mice survived
(without any sign of illness) after they had received 5_)-DXPA doses
up to 1000 mg/kg/day for 3 to 5 days.
From the foregoing experiments, it is evident that (S)-DHPA is a
~` useful therapeutic agent for virus diseases.
In addition to (S)-D~A, various related substances have been
examined in the virus-cell systems of Table 1 in which (S)-D~PA exhibited a
- marked antiviral activity : VSV/PRK, vaccinia/PRK and herpes simplex-l
1331 P/2 CA - 6 -
~,
,.
'
; (KOS)/PRK. Only the ts)-enantiomer of DHPA proved active. The (R)-enantio-
mer, (R)-D~A, was not. The racemic mixture, (RS)-DHPA, was almost as
effective as the (S)-enanticmerD Whereas (S)-DHPA and (RS)-DHPA inhibited
the cytopathic effects of VSV, vaccinia and herpes simple~ OS) at a
. 5 level of about 10 ~g/ml, the following congeners of (S)-DHPA did not
demonstrate an antiviral activity at 100 ~g/ml (the highest level tested):
S-9-(2,3-dihydroxypropyl)hypoxanthine, RS-9-(2-hydroxypropyl)adenine,
9-(2-hydroxyethyl)adenine, 9-(2-a~lnoethyl)adenine, 9-(S-DLralanyl)-
adenine, S-9-(3,4-dihydroxybutyl)adenine, RS-9-(3,4-dihydroxybutyl)-
adenine, RS-threo-9-(2,3,4-trihydroxybutyl)adenine, RS-9-(3,5-dihydroxy-
pentyl)adeine, S-1-(2,3-dihydroxypropyl)thymine, R~1-(2,3-dihydroxypropyl)-
thym me, S-3-(2,3-dihydroxypropyl)thymine, S-1-(3,4-dihydroxybutyl)uracil,
RS-1-(3,5-dihydroxypentyl)uracil, S-1-(2,3-dihydroxypropyl)uracil, 9-(3-
hydroxypropyl)adenine and 2-(9-adeninyl)propane-1,3-diol.
. 15 In the therapy of virus diseases, (S)-DHPA may be used as such and
also in the form of its racemic mixture (the RS-form). Further, it may be
used in ccmbination with other antiviral substances such as ara-A. There
is, in fact, circumstantial evidence for a synergism in the antiviral
; . .
activities of (S)-DHPA and ara-A.
(S)-DHPA was found to be a strong inhibitor of adenosine deaminase.
- Adenosine deaminase is an enzyme that is ubiquitous in cells, tissues and
` ~ biological fluids. It deaminates the aforementioned ara-A to its metabo-
lite ara-Hx (9-(beta-D-arabinofuranosyl)hypoxanthine) which is less
active as an antiviral agent than ara-A (ccmpare Ann. N.Y. Acad. Sci.,
284, 60, 1977). ~llike ara-A, (S)-DHPA did not serve as a substrate for
the adenosine deaminase of extracts of bacterial cells (Escherichia coli,
Salmonella typhimurium) or extracts of mammalian cells (primary rabbit
:"
~ kidney cells). However, (S)-DHPA strongly inhibited the deamination of
- ara-A by adenosine deamunase, extracted from calf intestinal mucosa. The
. .
latter enzyme preparation was a Calbiochem product.
~` Figure 3 is a graphical representation showing the inhibitory
effect of (S)-DHPA on deamination of ara-A by adenosine deaminase of calf
....
~ 1331 P/2 CA - 7 -
'
. . , , r
.
'' ' ' .
r ~ C~
- intestinal mucosa. Deamination of ara-A was measured according to the
` method of Kalckar (compare J. Biol. Chem., 167, 461, 1972), modified by Trams
ard Lauter (compare Biochem. J., 152, 681, 1975). The extent of deamina-
tion is equivalent to the decrease of absorbance at ~(wavelength) =
5 265 nm. m e decrease in absorkance at 265 nm was 0.11 if 50 nmol of ara-A
were used, and was 0.16 if 100 nmD1 of ara-A were used (Fig. 3A). In the
presence of (S)-DHPA, the deamination of ara-A (as reflected by the
absorbance decrease) was inhibited proportionally to the concentration of
(S)-DHPA. Fig. 4 represents a reciprocal plot of the data presented in
Fig. 3.
Adenosine deaminase inhibitors are known to potentiate the
antiviral activity of ara-A. A typical example of such adenosine deaminase
inhibitor is (R)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-
tetrahydroimidazo- (4,5-d)-(1,3)-diazepin-8-ol, referred to as to CO-V or
CO-vidarabine (com~are Ann. N.Y. Acad. Sci., 284, 9 and 284, 60, 1977).
(S)-DHPA markedly potQntiated the antiviral activity of ara-A.
The synergism in the antiviral activities of (S)-DHPA and ara-A was
, :..
determined in PRX (primary rabbit kidney) cultures which had been inoculated
with vaccinia virus. In these experiments, confluent monolayers of PRK
cells in plastic petri dishes-were inoculated with vaccinia virus (4.5 log10
CCID5 ~0.5 ml/petri dish) for 1 h at 37C and, immediately thereafter,
exposed to either (S)-DHPA alone or ara-A alone or ccmbinations of (S)-
` DHPA and ara-A. Both cam~ounds were used at various concentrations (1,3,
10, 30 or 100~ g/ml)O me cell cultures were then incubated for varying
times at 37&. At the end of the incubation period, the cells wQrQ
frozen at -70~C, and the cell hamogenates were assayed for virus content by
, .
plaque formation in PRK cell cultures, according to the plaque formation
procedure described above for vesicular stamatitis virus in mouse Lr929
; fibroblast cultures. rrhe results of a representative exFeriment are
shown in Table 2.
It appears fram Table 2 that a combination of (S)-DHPA (30 ~g/ml)
and ara-A (3 ~g/ml) effected a much greater reduction in virus titer than
1331 P/2 CA - 8 -
;
:,
. .,
'~
did either (S)-DHPA or ara~A when used individually. Hence, (S) DHPA
may be considered to enhance the antiviral activity of ara~A. This
enhancement is most probably due to the inhibitory effect of (S)~DHPA on
the deamination of ara-A by adenosine deaminase.
Ph2rmaceutical compositions comprising (S)-DHPA, or its
racemic mixture (RS)-DHPA, as an active ingredient may take the form of
powders, suspensions, solutions, emulsions as well as ointments and pastes
and may be used for parenteral (intravenous, intradermal, intramuscular,
~-~ intrathecal, ... ) injections, oral, rectal, intravaginal and intranasal
~ 10 administration or topical application (e.g. to lesions of skin, mucosa
and eye). These compositions may be prepared by combining the active
ingredient(s) with pharmaceutically acceptable excipients which are
normally used for this purpose. These excipients may comprise aqueous
~` or non-aqueous solvents,
TABLE 2. Synergism in the antiviral activities of (S)-DHPA and
~ ara-A in primary rabbit kidney (PRK) cell cultures infected with
. .:; , , ,
vacclnla vlrus
Treatment Virus yield
(log10 PFU~)
Days after infection
1 2 3
.
.,
; (1) Control (infected, untreated) 3.6 5.0 5.3
;:
25 (2) (S)-DHPA at 30 ~g/ml 3.64.9 5.4
(3) Ara-A at 3 ~g/ml 2.14.0 5.0
(4) Combined treatment (2) and (3) 2.0 1.8 2.1
. . _
, stabilisers, suspenders, dispersers, wetting agents and the like and will
.. .
~ 30 be kncwn to the pharmaceutical skilled in the art. Further, the
:, .
''
1331 P/2 CA - g -
:
",
'''' ~;~
'''
:
composition may comprise any suitable additives like polyethyleneglycols,
and, if necessary, dyestuffs, perfumes and the like.
The pharmaceutical compositions will contain at least 0.1~
by weight of the active ingredient. The actual concentration will depend
on the disease and on the chosen route of administration. In general,
this concentration will be comprised between 0.1% and 100%.
A particular advantage of (R5)-DHPA and (S)-DHPA is their
~` remarkable stability which renders pDssible the thermic sterilization
of the neutral, and weakly acid or weakly alkaline solutions. In pure
form their stability is practically unlimited. The costs of production
of (RS)-DHPA and (S)-DHPA are significantly lower than the costs of
production of other antiviral drugs such as ara-A or ribavirin.
.:
~ 15
. .
',.,. :
: . .
:,
.', .
,
,.,
.'':,
: 30
''''
1331 P/2 CA - 10 -
.'
