Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
i 3 4 $~
The invention relates to a new ester of 9-(2-hydroxyethoxymethyl)guanine
having valuable antiviral properties.
This application is a division of Canadian Patent Application 574,613,
filed August 12, 1988.
9-(2-Hydroxyethoxymethyl)guanine, otherwise known as acyclovir, possesses a
potent antiviral activity, particularly against herpes viruses (H. J.
Schaeffer et al, "Nature", 272 583-585 (1978), UK Patent Specification
1523865 and U.S. Patent Specification No. 4199574). Acyclovir is however
only poorly soluble in water, thereby limiting the formulation of the drug
in aqueous pharmaceutical preparations where solubility is required.
.,
Also acyclovir is only poorly absorbed from the gastrointestinal tract
after oral administration ( 15% recovery in the urine when tested in rats
and 20% in humans). Such low bioavailability requires the administration
of large doses of drug in order to achieve and maintain effective
anti-viral levels in the plasma.
' !
European Patent YSpecification 99493 describes amino acid esters of
acyclovir, specifically the glycine and alanine esters which show improved
water-solubility compared with acyclovir.
It has now been discovered that the L-isoleucine ester of acyclovir,
characterised by side-chain branching adjacent to the ~-carbon atom, and
which was not disclosed in European Patent Specification 99493, surprisingly
has improved bioavailability after oral administration compared with the
alanine and glycine esters mentioned therein.
According to one feature of the present invention we provide the compounds
of formula (I)
c
~ f~
I (I)
CH20CH2CH20COCH(Rl)NH2
2 - ~ ~ x O ~ ~ ~
wherein Rl represents a group of fonmula -Cl-l[CH3]CH2CH3 and pharn~ceutically
acceptable salts thereof. The compound of fonmula (I) can also be named as
2-(2-amino-1,6-dihydro-6-oxo-9H(purin-9-yl)methoxy)ethyl L-isoleucinate.
The isoleucine moi~ty is in tile L confiqura-~ion.
In tests in rats, measuring the urinary recovery as acyclovir (% dose
administered) after oral administration, the compounds of the invention
show a large increase in absorption from the gut compared with the other
esters and compared with acyclovir. This enables less drug to be
adminstered while still providin~ equivalent dru~ levels in the plasma
after oral absorption.
In addition to the relatively high bioavailability, the compounds according
to the invention possess substantially the same antiviral effect as
acyclovir in vitro. The advantageous increase in bioavailability of the
compound is thus not gained at the expense of antiviral potency. Indeed,
it has been found that in certain clinical applications, e.g. the treatment
of stromal keratitis, certain amino acid esters have been found to provide
a superior therapeutic effect to acyclovir (EP 99493)
The pharmaceutically acceptable salts of the compounds of formula (I) are
preferably acid addition salts derived from an appropriate acid, e.g.
hydrochloric, sulphuric, phosphoric, maleic, fumaric, citric, tartaric,
lactic, acetic or p-toluenesulphonic acid. Particularly preferred salts
are the hydrochloride salts of compounds of formula (I).
In experiments in animals, it was discovered that the oral administration
of the compounds of formula (I) above produced measurable levels of
acyclovir in the plasma. Thus according to another aspect of the invention
we provide a means of generating acyclovir in vivo by administration of a
compound of formula (I) above or a pharmaceutically acceptable salt thereof
to a mammal.
- 3 - ~ ~400~A
The compounds according to the invention may be prepared in conventional
manner, e.g. by a process as described below.
Thus, according to a curther feature of the present invention there is provide~
a process for the pr~paration of the compound of formula (I) ~bove and pharma-
ceutically acceptable salts thereof which comprises:
a) reacting a compound of formula (II)
~ N
l! /
CH20CH2CH20H
wherein X is an optionally protected hydroxy group, and Y is an optionally
protected amino group with an optionally protected L-isoleucine or
a functional equiYalent thereof;
, Y
b) converting a compound of formula (III)
~ ~ (III)
I
CH20CH2CH20COCH(Rl)NH2
(wherein Rl is as defined above; and M represents a hydroxy group and G
represents an atom or group that can be replaced by or converted to an
amino group; or G represents an amino group and M represents an atom or
group that can be replaced by or converted to a hydroxy group) into a
compound of formula (I) or a pharmaceutically acceptable salt thereof; or
! ~
- 4 - ~ 3 4 ~ ~ ~
c) reacting a compound of formula (IV)
~ \ ~ (IV)
(wherein X and Y are as defined above and Q represents a leaving atom or
group) with a compound of formula (V)
ACH2~CH2CH2~C~CH(Rl)R2
(V)
(wherein Rl is as defined above, A represents a leaving group or atom and
R is an optionally protected aDino group); and optionally effecting one or
more of the following conversions, in any desired sequence:-
i) removal of any protecting groups;
ii) where the resulting product is a compound of formula (I),conversion of the said compound into a pharmaceutically
acceptable salt thereof; and
iii) where the resulting product is a pharmaceutically acceptable salt
of a compound of formula (I), conversion of the said salt into
the parent compound.
With regard to process a), the esterification reaction may be carried out
in conventional manner, for example in a solvent such as pyridine or
dimethylformamide in the presence of a coupling agent such as
N,N'-dicyclohexylcarbodiimide, optionally in the presence of a catalytic
.~.., .~
1 3 ~ 4
base such as 4-dimethylaminopyridine. The water formed during the reaction
may, if desired, be removed in conventional manner, for example by
distillation or by the addition of a water-binding substance.
Subsequently, the ester obtained as reaction product may be isolated in
conventional manner.
As an alternative to the use of L-isoleucine per se, a functional
equivalent of the acid may be employed, e.g. an acid halide such as the
acid chloride, or an acid anhydride. In such a case in order to avoid
undesirable side-reactions, it is advantageous to use an amino-protected
derivative. Examples of preferred amino-protecting groups including acyl,
e.g. Cl 4alkanoyl such as acetyl and aryloxycarbonyl, e.g. benzyloxy
carbonyl. A suitable amino-protected derivative, for example, is one
wherein the amino group of the amino acid is replaced by an azido group.
Conversion of a compound of formula (III) into a compound of formula (I),
by method b), can be achieved by various means. For example G may
represent an azid,el group which can be reduced to an amino group by
catal~tic hydro~enation~ usin~ a suitable catalYst such as ~alladium on
carbon. Alternatively, G may represent a halogen atom or an alkylthio
or alkylsulphonyl group which can be converted to an azide group which in
turn can be converted to an amino group by catalytic hydrogenation using,
for example, hydrogen in the presence of palladium on carbon. For the
preparation of the compound of formula (I), a compound of formula (III)
wherein M is an amino group may be converted to a hydroxy group for example
by treatment with a deaminating enzyme such as adenosine deaminase.
These processes together with other conventional processes are described in
Fused Pyrimidines, Part II, Purines, Ed. , by D.J.Brown (1971), Wiley-
Interscience.
In process (c), the group Q in formula (IV) may, for example, represent a
hydrogen atom; an acyl group, e.g. a Cl 4alkanoyl group such as an acetyl
group or an aroyl group such an a benzoyl group; or a tri-Cl 4alkylsilyl
group such as a trimethylsilyl group. The group A in formula (V) may, for
example, represent a halogen atom (e.g. chlorine) or an acyloxy group
W
1 3 4 ~
- 6
wherein the acyl moiety may be, for example, a C~ ,,alkanoyl group such as
acetyl or an aroyl group such as benzoyl- The group R2 may represent an
amino-protecting group such as for example, Cl 4 alkanoyl (e.g. acetyl) or
aryloxycarbanoyl (e.g. benzyloxycarbonyl) and it may also represent an azido
group. The reaction may be conveniently effected in a strong polar solvent
such as dimethylformamide or hexamethylphosphoramide, advantageously in the
presence of a base such as triethylamine or potassium carbonate.
Alternatively, a thermal condensation may be effected by heating the
compounds of formulae (IV) and (V) in the presence of a catalytic amount of
a strong acid, e.g. sulphuric acid.
Compounds of formulae (II) to (V), employed as intermediates in the
synthesis of the compound of formula (I), can be prepared in conventional
manner, e.g. by procedures described in U.K. Patent Specification No.
1523865. These methods rely on intermediates prepared from simply
substituted purines, which may be available commercially, or prepared
according to techniques which are well known ~ se and which are disclosed
in the literature~such as the aforementioned text-book. Thus, for example,
compounds of formu~a (III) may be generally prepared by using an analogous
procedure to that of process (c), i.e. reacting an appropriate purine with
a compound of formula (V)
The optional conversions i), ii) and iii) may be effected in conventional
manner. Thus, for example, removal of protecting groups in conversion i)
may be effected by hydrolysis, solvolysis or hydrogenolysis as appropriate.
With regard to removal of protecting groups on the amino acid acyl
radicals, hydrogenolysis, e.g. of aryloxycarbonyl protecting groups, and
conversion of azido group, e.g. by catalytic hydrogenation, e.g. using a
palladium catalyst, are preferred. With regard to protection of the groups
in the 2- and/or 6-positions of the purine nucleus, these may ~e selected for
example from arylmethyl groups e.g. benzyl; or tri-Cl ~ alkylsilyl e.g.
trimethylsilyl. Arylmethyl blocking groups may be removed for example by
hydrogenolysis, e.g. by hydrogenation in the presence of Raney nickel or a
palladium catalyst. Trialkylsilyl blocking groups may be removed for
example by solvolysis e.g by alcoholysis.
~'.~
- 7 ~ q~
The conversion of a compound of formula (I) into a pharmaceutically
acceptable salt may be effected in conventional manner, for example, by
treatment of the compound with an appropriate acid to form an acid addition
salt, for example, by lyophilisation of a methanolic solution of the parent
ester with an acid solut~on.
Similarly, conversion of a sàlt into the parent compound of formula (I) may
be effected in conventional manner.
The present invention also provides the compound of formula (I) and
pharmaceutically acceptable salts thereof (hereinafter identified as "the
active compounds") for use in medical therapy e.g. in the treatment or
prophylaxis of a viral disease in an animal, e.g. a mammal such as man.
The compounds are especially useful for the treatment or prophylaxis of
diseases caused by various DNA viruses, such as herpes infections, for
example herpes simplex, variceIla zoster, cytomegalovirus as well aq
diseases caused by hepatitis B or Epstein-Barr viruses or human herpes
virus -6 (HHV-6).~ The active compounds can also be used for the treatment
or prophylaxis of retrovirus infections such as HIV infections and
papilloma or wart virus infections. In addition to their use in human
medical therapy, the compounds of formula (I) can be administered to other
animals for treatment or prophylaxis of viral diseases, e.g. in other
mammals. For example, the active compounds are especially useful for the
treatment of equine rhinopneumonitis.
The present invention also provides a method for the treatment or
prophylaxis of a viral disease in an animal, e.g. a mammal such as man,
which comprises administering to the animal an effective antiviral amount
of a compound of formula (I) or a pharmaceutic,ally acceptable salt thereof.
The present invention also provides the use of a compound of formula (I) in
the manufacture of a medicament for the treatment or prophylaxis of a viral
infection.
The active compounds may be administered by any route appropriate to the
condition to be treated, suitable routes including oral, rectal, nasal,
- 8 - ~ 8 -~
topical (including buccal and sublingual) vaginal and parenteral (including
subcutaneous, intramuscular, intravenous, intradermal, intrathecal and
epidural). It will be appreciated that the preferred route may vary with
for example the condition of the recipient.
For each of the above-indicated utilities and indications the amount
required of an active ingredient (as above defined) will depend upon a
number of factors including the severity of the condition to be treated and
the identity of the recipient and will ultimately be at the discretion of
the attendant physician or veterinarian. In general however, for each of
these utilities and indications, a suitable effective dose will be in the
range 0.1 to 250 mg per kilogram bodyweight of recipient per day,
preferably in the range 1 to 100 mg per kilogram bodyweight per day and
most preferably in the range 5 to 20 mg per kilogram bodyweight per day; an
optimum dose is about 10 mg per kilogram bodyweight per day. (Unless
otherwise indicated, all weights of active ingredient are calculated as the
parent compound of formula (I): for salts thereof the figures would be
increased proportionately.) The desired dose is preferably presented as
two, three, four or more sub-doses administered at appropriate intervals
throughout the day. These sub-doses may be~ administered in unit dosage
forms, for example, containing 10 to 1000 mg~ preferably 20 to 500 mg and
most preferably 100 to 400 mg of active ingredient per unit dosage form.
The compounds of the present invention may be administered alone or in
combination with other therapeutic agents, for example, with
9-(2-hydroxyethoxymethyl)guanine (acyclovir) used to treat herpes virus
infections in particular HSV (I), and with zidovudine used to treat
retroviral infections in particular HIV infections.
While it is possible for the active ingredients to be administered alone,
it is preferable to present them as pharmaceutical formulations. The
formulations, both for veterinary and for human use, of the present
invention comprise at least one active ingredient, as above defined,
together with one or more acceptable carriers therefor and optionally other
therapeutic ingredients. The carrier(s) must be "acceptable" in the sense
9 1 ~ 4
of being compatible with the other ingredients of the formulation and not
deleterious to the recipient thereof.
The formulations include those suitable for oral, rectal, nasal, topical
(including buccal and sublingual), vaginal or parenteral (including
subcutanous, intramuscular, intravenous, intradermal, intrathecal and
epidural) administration. The formulations may conveniently be presented
in unit dosage form and may be prepared by any of the methods well known in
the art of pharmacy. Such methods include the step of bringing into
association the active ingredient with the carrier which constitutes one or
more accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association the active ingredient
with liquid carriers of finely divided solid carriers or both, and then, if
necessary, shaping the product.
Formulations of the present invention suitable for oral administration may
be presented as discrete units such as capsules, cachets or tablets each
containing a predetermined amount of the active ingredient; as a powder or
granules; as a solution or a suspension in an aqueous liquid or a
non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil
liquid emulsion. The active ingredient may also be presented as a bolus,
electuary or paste.
A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient in a free-flowing
form such as a powder or granules, optionally mixed with a binder,
lubricant, inert diluent, preservative, surface active or dispersing agent.
Moulded tablets may be made by moulding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent. The tablets
may optionally be coated or scored and may be formulated so as to provide
slow or controlled release of the active ingredient therein.
For infections of the eye or other external tissues e.g. mouth and skin,
the formulations are preferably applied as a topical ointment or cream
containing the active ingredient in an amount of, for example, 0.075 to 20~
lo .t 3 ~ , 4
w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10~ w/w. When
formulated in an ointment, the active ingredients may be employed with
either paraffinic or a water-miscible ointment base. Alternatively, the
active ingredients may be for~ulated in a cream with an oil-in-water cream
base. In addition topical applications may be made transdermally by means
of an iontophoretic device.
If desired, the aqueous phase of the cream base may include, for example,
at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or
more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol,
sorbitol, glycerol and polyethylene glycol and mixtures thereof. The
topical formulations may desirably include a compound which enhances
absorption or penetration of the active ingredient through the skin or
other affected areas. Examples of such dermal penetration enhancers include
dimethylsulphoxide and related analogues.
Formulations suitable for topical administration to the eye also include
eye drops wherein the active ingredient is dissolved or suspended in a
suitable carrier, especially an aqueous solvent for the active ingredient.
The active ingredient is preferably present in such formulations in a
concentration of 0.5 to 20~, advantageously 0.5 to 10% particularly about
1.5% w/w.
Formulations suitable for topical administration in the mouth include
lozenges comprising the active ingredient in a flavoured basis, usually
sucrose and acacia or tragacanth; pastilles comprising the active
ingredient in an inert basis such as gelatin and glycerine, or sucrose and
acacia; and mouthwashes comprising the active ingredient in a suitable
liquid carrier.
Formulations for rectal administration may be presented as a suppository
with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size for example in the
range 20 to 500 microns which is administered in the manner in which snuff
is taken, i.e. by rapid inhalation through the nasal passage from a
container of powder held close up to the nose. Suitable formulations
wherein the carrier is a liquid, for administration as for example a nasal
spray or as nasal drops, include aqueous or oily solutions of the active
ingredient.
Formulations suitable for vaginal administration may be presented as
pessaries, tampons, creams, gels, pastes, foams or spray formulations
containing in addition to the active ingredient such carriers as are known
in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous andnon- aqueous sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be stored in a
freeze-dried (lyophilized) condition requiring only the addition of the
sterile liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets of the kind previously
described. Formulations for intramuscular administration are particularly
preferred.
Preferred unit dosage formulations are those containing a daily dose orunit daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly
mentioned above the formulations of this invention may include other agents
conventional in the art having regard to the type of formulation in
question, for example those suitable for oral administration may include
flavouring agents.
The present invention further provides veterinary compositions comprising
at least one active ingredient as above defined together with a veterinary
carrier therefor.
Veterinary carriers are materials useful for the purpose of administering
the composition and may be solid, liquid or gaseous materials which are
otherwise inert or acceptable in the veterinary art and are compatible with
the active ingredient. These veterinary compositions may be ~t' ~ni.stered
orally, parenterally or by any other desired route.
For oral A~lni~tration the compositions can be in the form of a tablet,
granule drench, paste, cachet, capsule or feed supplement. Granules may be
made by the well known techniques of wet granulation, precompression or
slugging. They can be administered to animals in an inert liquid vehicle so
as to form a drench, or in a suspension with water or oil base. Preferably
further accessory ingredients such as a dispensing agent are included.
These formulations preferably contain from 15 to 85~ of the active
ingredient. ~'
, ~ /
The following Examples illustrate the present invention
Exam~le 1 2-(2-Amino-1.6-dihydro-6-oxo-9H(purin-9-yl)methoxy)ethyl
L-isoleucinate hydrochloride
a) 2-~(2-Amino-1.6-dihydro-6-oxo-9H-purin-9-yl)methoxylethyl-
N-~(benzyloxy)carbonyll L-isoleucinate
A mixture of acyclovir (1.0 g, 4.4. mmol; Burroughs ~ellcome Co.),
4-dimethylaminopyridine (74 mg, 0.6 mmol; Aldrich Chemical Co. and
Chem.Ber. 89 2921-33 [1956]), 1,3-dicyclohexylcarbodiimidide (1.6 g,
8.0 mmol; Aldrich Chemical Co. and US 2656383),
.., ~
. ~
- 13 - I 3~ ii84
N-carbobenzoxy-L-isoleucine (1.8 g, 6.6 mmol; Sigma Chemical Co. and
Bull.Chem.Soc. Jap (1966) 39 947 or Tetrahedron 40 (24) 5207-11
[1984]), and molecular sieves (0.3 g, Davison type 3A; Fisher
Scientific, Co.) in dry N,N-dimethylformamide (80 ml) was stirred at
room temperature under nitrogen. After 4 days, additonal
1,3-dicyclohexylcarbodiimide (1.6 g, 8.0 mmol) and
N-carbobenzoxy-L-isoleucine (1.8 g, 6.6 mmol) were added. Stirring at
room temperature was continued for 7 days. The resulting mixture was
filtered and the clear filtrate was concentrated in vacuo to a
semi-solid residue. Elution of the residue from silica gel 60 (EM,
230-400 mesh; 8.5x14 cm) with 2.5-5% methanol/methylene chloride gave
2-l2-Amino-1,6-dihydro-6-oxo-9H-
purin-9-yl)methoxy]ethyl-~-[(benzyloxy)carbonyl]-_-isoleucinate 0.8 g,
45%) as a white solid; mp 155-157 C;
W (MeOH); ~ max 255 nm (~17700) ~ sh 279 (9800), ~ min 230 (6100)
NMR (200MHz, ME2SO-d6): 0.73-0.80 ppm (m, 6H), 1.13-1.33 (m, 2H),
1.66-1.70 (m, lH), 3.64 (t,2H), 3.92 (t,lH), 4.16 (m, 2H), 5.00 (s,
2H), 5.32 (s,2H) 6.47 (br s, 2H), 7.33 (s, 5H), 7.65 (d, j-8 Hz, lH),
7.78 (s, lH), 10.59 (br s, lH);
MS (Cl/CH4; 70 C)) m/z 473 (1.0%, M+l), 347 (23.2, M-125), 225 (100.0,
M-247); [~] 20 C~ 3.07~ (c 0.488, 6N HCl).
TLC: one spot on silica gel with 10% MeOH/CH2C12. Rf= 0.38
HPLC: one peak on Supelco LC8 with 50~ MeOH/H2O; 100% K'-7.02
22 28 6 6- 2
Found: C, 53.94; H, 6.20 N, 17.04
b) 2-~(2-Amino-1.6-dihydro-6-oxo-9H-purin-9-yl~ethoxylethyl
L-isoleucinate hydrochloride
A solution of 2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-methoxy]-
l?~ a~
- 14 -
ethyl-N-[(benzyloxy)carbonyl]-L-isoleucinate (1.11 g, 2.2 mmol) in
methanol-tetrahydro furan (1:1, 50 ml) was treated with 0.5 N
hydrochloric acid (5 ml) and 5% palladium on charcoal (0.30g; MCB
Reagents). The mixture was hydrogenated on a Parr hydrogenator at 50
psi for 11 hours, filtered through a Celite pad and the filtrate was
concentarted in vacuo to give 2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-
9-yl)methoxy]ethyl-L-isoleucinate hydrochloride (0.86 g, 92%) as an
off-white solid; mp 180-182 C (effervescent softening ca. 150~C):
W (MeOH): ~ max 254 nm (~13400), ~ sh 272 (9000), ~ min 224 (3600);
NMR (200 MHz, Me2SO-d6): 0.77-0.84 ppm (m, 6H), 1.13-1.37 (m, 2H),
1.82 (m,lH), 3.73 (t,2H), 3.87 (br t, lH), 4.14-4.40 (m, 2H), 5.36 (s,
2H), 6.70 (br s, 2H), 7.97 (s, lH), 8.46 (br s, 3H), 10.87 (s, lH);
MS (Cl/CH4; 150 C): m/z 367 (6.9%, M+29), 339 (100, M+l), 225 (92.9,
M-113); [~]D20 c + 11.15~ (c 2.0, HOAc)-
' !
, ~
TLC: one spot on silica gel with 10% MeOH/CH2C12 Rf-0.12; HPLC: one
peak on Versapack C18 with 10% MeOH/H2O/0.1% F3CCOOH;100% K'-3.74.
Anal Calcd for C14H22N6O4. 1.25 HCl. 1.10 MeOH. 0.25 H2O:
C, 42.81; H, 6.70; N, lg.84;; Cl, 10.46
Found C, 42.82; H, 6.50; N, 19.64; Cl, 10.46
Example 2 : Tablet Formulations
The following formulations A, B and C are p,repared by wet granulation of
the ingredients with a solution of povidone, followed by addition of
magnesium stearate and compression.
Formulation A
mg/tablet mg/tablet
Active ingredient 250 250
Lactose B.P. 210 26
Povidone B.P. 15 9
Sodium Starch Glycollate 20 12
Magnesium Stearate 5 3
500 300
Formulation B
j mg/tablet m~/tablet
Active ingredient 250 250
Lactose 150
Avicel PH 101 (Trade Mark) 60 26
Povidone B.P. 15 9
Sodium Starch Glycollate 20 12
Magnesium Stearate 5
500 300
~ormulation C
mg/tablet
Active ingredient 100
Lactose 200
Starch 50
Povidone B.P. 5
Magnesium stearate . 4
359
The following formulations, D and E, are prepared by direct compression of
the admixed ingredients. The lactose in formulation E is of the
compression type.
t
- 16 -
Formulation D
mg/tablet
Active Ingredient 250
Avicel (Trade Mark) 150
Magnesium Stearate 4
404
Formulation E
mg/tablet
Active Ingredient 250
. ,,
Lactose 150
Avicel (Trade Mark) 100
Magnesium Stearate 5
505
Formulation F (Controlled Release For~ulation)
. I
, i ~
The formulation is prepared by wet granulation of the ingredients (below)
with a solution of povidone followed by the addition of magnesium stearate
and compression.
mg/tablet
Active Ingredient 500
Hydroxypropylmethylcellulose112
(Methocel K4M Premium Trade Mark)
Lactose B.P. 53
Povidone B.P. 28
Magnesium Stearate 7
700
- 17 - ~ ~ ~ g~ ~ L~
Example 3 : Capsule Formulations
Formulation A
A capsule formulation is prepared by admixing the ingredients of
Formulation D in Example 2 above and filling into a two-part hard gelatin
capsule. Formulation B tinfra) is prepared in a similar manner.
Formulation B
mg/capsule
Active ingredient 250
Lactose B.P. 143
Sodium Starch Glycollate 25
Magnesium Stearate 2
420
Formulation C
mg/capsule
. ~
Active ingredient 250
Macrogol(Trademark) 4000 B.P.350
600
Capsules are prepared by melting the Macrogol 4000 BP, dispersing the
active ingredient in the melt and filling the melt into a two-part hard
gelatin capsule.
~,
- 18 - ~ 3~
~ormulation D
mg/capsule
Active ingredient 250
Lecithin 100
Arachis Oil 100
450
Capsules are prepared by dispersing the active ingredient in the lecithin
and arachis oil and filling the dispersion into soft, elastic gelatin
capsules.
Formulation E (Controlled Release Capsule~
The following controlled release capsule formulation is prepared by
extruding ingredients a, b and c using an extruder, followed by
spheronisation of the extrudate and drying. The dried pellets are then
coated with releas~e-controlling membrane (d) and filled into a two-piece,
hard gelatin capsule.
mg/capsule
Active Ingredient 250
Microcrystalline Cellulose 125
Lactose B.P. 125
Ethyl Cellulose 13
513
Example 4 : Ophthalmic Solution
Active ingredient 0.5
Propylene Glycol 0.2 g
Thiomersal 0.001 g
Purified water to 100 ml
pH adjusted to 7.5
k ~
-- 19 --
Example 5: Injectable Formulation
Active Ingredient 0.200 g
Sterile, pyrogen free citrate buffer (pH 7.0) to 10 ml
The active ingredient is dissolved in most of the citrate buffer
(35 -40 C), then made up to volume and filtered through a sterile micropore
filter into a sterile lOml amber glass vial (type l) and sealed with
sterile closures and overseals.
ExamDle 6: Intramuscular injection
Active Ingredient 0.20 g
Benzyl Alcohol 0.10 g
Glycofurol 75 1.45 g
Water for Injection q.s. to 3.00 ml
The active ingredient is dissolved in the glycofurol. The benzyl alcohol
is then added and~dissolved, and water added to 3 ml. The mixture is then
filtered through a sterile micropore filter and sealed in sterile 3 ml
amber glass vials (type 1).
Example 7 : SyruD Suspension
Active Ingredient 0.25 g
Sorbitol Solution 1.50 g
Glycerol 2.00 g
Sodium Benzoate 0.005 g
Flavour, 0.0125 ml
Purified Water q.s. to 5.00 ml
The sodium benzoate is dissolved in a portion of the purified water and the
sorbitol solution added. The active ingredient is added and dissolved.
The glycerol and flavours are added and mixed in. Water is added to a
final volume of 5ml.
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Example 8 : SuPpository
mg/suppository
Active Ingredient (63~m)* 250
Hard Fat, BP (Witepsol H15 (Trade Mark) - 1700
Dynamit Nobel )
1950
*The active ingredient is used as a powder wherein at least 90~ of the
particles are of 63~m diameter or less.
One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45 C
maximum. The active ingredient is sifted through a 200~m sieve and added
to the molten base with mixing, using a silverson fitted with a cutting
head, until a smooth dispersion is achieved. Maint~ining the mixture at
45 C, the remaining Witepsol Hl5 is added to the suspension and stirred to
ensure a homogenous mix. The entire suspension is passed through a 250~m
stainless steel sc~reen and, with continuous stirring, is allowed to cool to
40 C. At a tempe~rYature of 38 C to 40 C, 2.02g of the mixture is filled
into suitable plastic moulds. The suppositories are allowed to cool to
room temperature.
Example 9: Pessaries
mg~pessary
Active ingredient 63~m250
Anhydrous Dextrose 543
Starch 200
Magnesium Stearate 7
1000
The above ingredients are mixed directly and pessaries prepared by direct
compression of the resulting mixture.
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Example IO
a) Antiviral Activity
Herpes Simplex Virus (HSV l) was asssayed in monolayers of Vero cells in
multiwell trays. Activity of compounds was determined in the plaque
reduction assay, in which a cell monolayer was infected with a suspension
of HSV l, and then overlaid with nutrient agarose in the form of a gel to
ensure that there was no spread of virus throughout the culture. A range
of concentrations of compound of known molarity was incorporated in the
nutrient agarose overlay. Plaque numbers at each concentration were
expressed as percentages of the control and a dose-response curve was
drawn. From this curve the 50% inhibitory concentration (IC50) was
estimated.
Compound IC50 ~M
Example l 3.8
Acyclovir ~ 0.08 - 0.1
b) Determination of Oral Bioavailability
Long Evans Rats were adminstered the compound to be tested by gavage at a
dose equivalent to 25 mg/kg acyclovir. The urine was collected for 24 and
48 hours post-dose, ultrafiltered, and analysed by reverse-phase
high-pressure liquid chromatography. The oral bioavailability of the
compound was expressed as the percent of the dose excreted in the urine as
acyclovir.
ComDound Urinary Recovery (~ of dose) as acyclovir
Example l 50
Acyclovir (ACV) 15
Glycyl ester of ACV 30
L -alanyl ester of ACV 34
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d) Toxicity Data
Determination of Growth Inhibition of Uninfected Mammalian Cells
The capability of candidate compounds to inhibit the growth of D98 cells
(human) and L cells (murine) was measured by determination of cell number
following three days exposure of a standard number of cells to various
dilutions of compound (Rideout, J. L., Krenitsky, T.A., Koszalka, G.W.,
Cohn, N.K., Chao, E.Y. Elion, G.B., Latter, V.S., and Williams, R.B. (1982)
J. Med Chem. 25: 1040-1044). The cell number was then compared to the
number obtained in the absence of compound. Cell enumeration was performed
by either direct particle counts following trypsinization of the monolayer,
or by spectrophotometric determination of the amount of vital stain taken
up by the cells. Comparable results were obtained with both methods.
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Data Analysis
The concentration of compound resulting in 50% of control values (IC50) was
calculated either by direct interpolation from graphs of the log of the
compound concentration versus the percent of control value, or from a
computer program which analyses the data according to the same algorithm.
Data in the range of 20% to 80% of control were used in these calculations.
Example Cell Toxicity
(% of control at lOO~m)
D-98 Cells L-Cells
ACV (acyclovir) 99 72
1 .~ 80 83
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