Note: Descriptions are shown in the official language in which they were submitted.
W O 9~/lg246 2 l ~ 212 ~ PCT/GB92/00803
- 1 -
U5E OF A 1,3--O~A~O~ND~RI~ATIUE FOR T9E. TREATMENT
OF HEPATTTIS B INFECTIoN
The present inventio~ relates to the use of a 1-(2-(hydroxymethyl)-
1,3-oxathiolan-5-yl)-cytosine derivative and physiologically
functional derivatives thereof for the trea~ment of hepatitis B viral
infections.
Hepatitis B virus (HBV) is a viral pathogen of major worldwide
imp~rtance. HBV is most common i~ Asian Countries, and prevalent in
sub-Saharan Africa. The ~irus is etiologically associated with
primary hepatocellular carcinoma and is thou~ht to cause 804 of the
world's livcr cancer. In the United S~ates more than ten thousand
people are hospitalized for HBV illness each year, an average of
250 die with fulminant disease. The United States currently contains
an estimated pool of 500,000 - 1 million infectious carrisrs. Chronic
active hepatitis will develop in over 25~ of carriers and of~en
progresses to cirrhosis. It is est-;mated that 5000 people die from
HBV-related cirrhosis each year in the U.S.A. and ~hat perhaps
1000 die from HBV-related liver cancer. Even when a universal
HBV vaccine is in place, the need ~.Eor effective anti-HBV compounds
will continue. The lar~e reservoir of perslstenely infected carriers,
estimated at 220 million worldwide, will receive no benefit from
vaccination and will continue at high risk for HBV-induced liver
disease. This carrier population serves as the source of infection ~f
susceptible individuals perpetuating the instance of disease
partieularly in endemic areas or high risk groups such as i.v. drug
abusers and homosexuals. Thus, there is a great need for effective
antiviral a~ents, both to control the chronic infection a~d reduce
progression to hepatoc~llular carcinoma.
'~
Clinical effects of infection with ffBV range from headache, fever,
malaise, nausea, vomiting, anorexia and abdominal pains. Replication
.
of the virus is usually controlled by the immune response, with a
course of recovery lasting weeks or months in humans, but infection
~ay be more severe leading to persistent chronic liver disease as
W O ~2/19246 PCT/~B92/008~
2:102120
outlined above. In "Viral Infections of Humans" ~second edition, Ed.
Evans, A.S. (1982) Plenum Publishing Corporation, New York),
Chapter 12 describes the etiology of viral hepatitis in~ections.
European Patent Specification 0 382 526 discloses certain 1,3-oxathio-
lane nucleoside analogues which are effective in inhibiting the
replication of human immunodeficiency virus (HIV~.
Wa have now surprlsingly found that a 1-(2-(hydroxymeehyl)-1,3-oxathi-
olan-5-yl)-cytosine derivative of formula I
.. 2
~ R .
N ~.
(I~
s ` .
(wherein R is a hydrogen a~om or a Cl 3 alkyl group~
and physiologically functional deri~ati~es thereof have potent
acti~ity against HBV.
:
A preferred compound of formula (I) is that in which R is a hydrogen
atom, namely: 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine.
It should be noted that the compound of formula (I) contains two
chiral centers and therefore exists in the for~ of two palrs of
optical isomers (i.e. enantiomers) and mixtures thereof includîng
racemic mixtures. Thus the compound of formula (I) may be either a
cis- or a ~rans- isomer or mixtures thereof. Each cis- and trans-
isomer can exist as one of two enantiomers or mixtures thereof
including racemic mixtures. All such isomers and mixtures thereof
including racemic mixtures are within the scope of the in~ention. The
SUBSTiTUTE SHEET
W O 92/19246 2 1~ ~ ~ 2 ~ PCT/GB92/00803
- 3
cls- isomers of the compound of formula (I) are preferred. The ::
invention includes the optical and geometric isomers and all -:
tautomeric forms of the compound of f~rmula (I); of the cis isomers,
the (-) isomer, ie. (-)-cis-1-(2-(2-hydroxymethyl)-1,3-oxathiolan-5-
yl)-5-fluorocytosine, is especially preferred.
According to one feature of the present i~v4ntîon we provide a
compound of formula (I) or a physiologically unctional derivative -~
thereof for use in the treatment or prophylaxis of a hepatitis B virus :~
infection. Accordin~ to a further feature of the present in~ention we
provide the use of compound of formula (I) or a physiologically
funct~onal derivative thereof, in the manufacture of a medicament for
the treatment or prophylaxis of a hepa~itis B virus infection. ~:
In a further aspect of the presen~ in~ention there is included a
method for the treatment or prophylaxis of a hepatitis B virus
infection in a host, for example, a mammal such as a human which
comprises treating the host with a therapeutically effective amount of
a compound of formula ~I~ or a physiologically functional derivative
thereof.
~,.
By "physiologically functional derivative" i5 meant a pharmaceutically
acceptable salt, ester or salt of an ester of a compound of formula
~I) or any other compo~nd which upon administration t~ the recipient,
is capable of providing (directly or indirectly) the said compound o~
formula (I) or an active metabolite Qr residue thereo.
Preferred esters in accordance with the invention include carboxyIic :~
acid esters in which the non-car~onyl moiety of the carboxylic acid
portion of the~ester grouping is selected from straight or bxanehed
chain alkyl e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl
(e.g. methoxymethyl), arylalkyl (e.g.. benzyl), aryloxyalkyl ~e.g.
phenoxymethyl), and aryl (e.g. phenyl); sulfonate esters such as
alkyl- or arylalkylsulfonyl (e.g. methanesulfonyl); amino acid es~ers
(e.g. L-valyl or L-isoleucyl); dicarboxylic acid esters (e.g-
hemisuccinate); and 5'- mono- di- or tri-phosphate esters. The
W O 92/19246 PCT/GB92tO08~
~11)2120
phosphate esters may be further esterified by, for example, a C1 20
alcohol or reacti~e der;vative thereof, or by a 2,3-di(C6 24)acyl
glycerol.
Any alkyl moiety present in such esters advanta~eously contains 1 to
18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety
present in such esters advantageously comprises a phenyl group
optionally s~bstituted e.g. by halo~en, Cl 4 alkyl, C1 4 alkoxy or
nitro.
In the abo~e-described esters, the cytosine amino ~roup may be present
in the form of an amide, e.g. -NHCOR whexe R is Cl 6 alkyl or aryl
(e.~. phenyl optionally substituted by halogen, Cl 4 alkyl, Cl 4
alkoxy, nitro or hydroxyl~.
Examples of pharmaceutically acceptable salts according to the
invention includ~ base salts, e.g. derived from an approp7iate base,
such as alkali metal (e.g. sodium), alkaline earth metal
(e.g. magnesiu~) salts, ammonium and NX4 (wherein X is Cl 4 alkyl).
Pharmac2utically acceptabIe acld addition salts inc}ude salts of
organic carboxylic acids such as acetic, lactic, ~artaric, malic,
isethionic, lactobionic and succinic aeids; organic sulfonic acids
such as methanesulfonic, ethanesulfonic, benzenesulfonic and
p-toluenesulfonic aoids and inorganio acids such as hydrochlGric,
sulfuric, phosphoric and sulfamic acids.
'~
The amount of the compound of formula (I) (hereinafter also referred
to as the "actlve ingredient") or physiologically functional
deri~ative thereof which is requîred in a medication to aehieve the
desired effect will depend on a number of factors, in particular the
specific application, the nature of the particular compound used, the
mode of administration and the condition of the patient. In general a
suitable dose will be in the range of 3.0 to 120 mg per kilogram body
weight of the recipient per day, preferably in the range of 6 to 90 mg
per kilogram body weight per day and most preferably in the range 15
~1~2~20
W O 92/19246 PCT/GB92/00803
to 60 mg per kilogram body weight per day. The desired dose is
preferably presented as two, three, four, five, six or more sub-doses
adm}nistered at appropriate intervals throughout the day. These
sub-doses may be administered in unit dosage forms, for example,
c~ntaining lO to 1500 mg, preferably 20 to lO00 mg, and most
preferably 50 to 700 mg of active ingredient per unit dosage form.
Ideally, the active ingredient shGuld be ad~inistered to achieve peak
plasma concentrations of the active ingredient of from about l to
about 75~M, preferably about 2 to SO~M, most preferably about 3 to
about 30~M. This may be achieved, for example 7 by the intra~enous
injection of a O.l to 5% solution of the active ingredient, optionally
in saline, or orally administered as a bolus containing about l to
about lO0 m~/kg of the active ingredient. Desirable blood levels may
be maintained by a continuous infusion to provide about O.Ol to about
5.0 mg/kg/h~ur or by intermittent infusions containing about 0.4 to
about 15 mg/kg of the actîve ingredient.
In the manufactl~re of a medicament accoxding to the i~vention,
hereinafter referred to as a "formulation", the compound of formula
(I) or a physiologically functional derivative thereof herein as
"active ingredient', is typically admixed with, la~ , one or
more pharmaceutically acceptable carriers or excipient~ and optionally
other therapeutic agents.
The formulations include those suitable for oral, rectal, nasal~
topical (includin~ transdermal, buccal and sublingual), vaginal or
parenteral (including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods well
known in the art of pharmacy. Such methods include the s~ep of
bringing into association the active ingredient with the carrier which
c~nstitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
W 0 92~19246 ` - 6 - P ~ /GB92/OOX~:~
Zlf~2120
divided solid carriers or both, and then if necessary shaping the
product.
Formulations of the present invention suitable for oral administratîon
may be presented as discrete units such as capsules; cachets or
tablets each containing a predetermined amount of the aceive
ingredient; as a powder or granules; as a solution or suspension in an
aqueous or no~-aqueous liquid; or as an oil-in-water liquid emulsion
or a water-ln-oil liquid emulsion. The active ingredient may also be
presented as a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or
more 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 ~e.g. povidone, gelatin, hydroxypropylmethyl cellulose),
lubricant, inert diluent, preservative, disintegrant (e.g. sodium
starch glycollate, cross-lin~ed povidone, cross-linked sodium
carboxymethyl cellulose) surface active or dispersing agent. Molded~ ~
tablets may be made by molding in a suitahle machine a mixture of ~he ~-
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
using, for example hydroxypropyl~ethyl cellulose in varying
proportions to pr~ovide the desired release profile. Tablets may
optionally be provided with an enteric coating, to provide release in
parts of the gu~ other than the stomach.
Formulations suitable for oral use as described above may also include
buffering agents designed to neutralize stomach acidity. Such buffers
may be chosen from a ~ariety of organic or inorganic agents such as
weak acids or bases admixed with their conjugated salts.
Formulations suitable for topical ad~inistration in the mouth include
lozenges comprising the active ingredient in a flavored basis, usually
W O 92/tg246 21 a ~ 1 ~ 0 P ~ /GB92/00803
- 7 -
sucrose and a~acia or tragacanth; pastilles comprisi~g the active
ingredien~ in an inert basis such as gelatin and glycerin, 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 comprisin~ for-example cocoa butter
or a salicyl~e.
Formulations suitable for vaginal administration may be presented as
pessari~s, 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 sui~able for parenteral administration include aqueous
and non-aqueous isotonic sterile injections solu~ions which may
contain nti-oxidants, buffers, bacteriostats and solutes which render
the formulation isotonic with the bl.ood of the intended recipient; and
aqu ous and non-~queous sterile suspensions which may include
suspending agents ; and thickening agents, as liposomes or other
micropar~iculate systems which are clesigned to targe~ the comp~unds to
blood components or one or more organs. The formulations may be
presented in unit-dose or multi-dose sealed containers, for example, -
ampules and vials, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of the steri1e 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.
,.
Preferred unit dosa~e formulations are those containing a daily dose
or unit, daily sub-dose, as herein above recited, or an appropriate
fraction thereof, of an active ingredient.
W O 92/19246 PCT/GB92tOO~
- 8 -
21(32~2~
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 ~uestion, for example, those suitable for oral
administration may include such further agents as sweeteners,
thickeners and flavoring agents.
The compound of formula (I) may be prepared for ex~mple in accordance
with the processes described in European Patent Specification
O 382 526 or by processes analogous thereto.
Thus, ~he compound of formula I may be prepared for example by:
a~ reacting an optionally protacted 5-R-cytosine compound (wherein R
is as defined above) with a l,3-oxathiolane of $ormula IIA
~o
L
\ J ~IIA~
s
wherein Rl is hydrogen or a hydroxy protecting group and L is a
leaving group; or
b) reacting a compound of formula IIB
~ R
N
1 (IIB)
SUBSTITUTE SHEET
W O 92/19246 2 ~ 0 2 ~ 2 ~ PCT/GB92/00803
(wherein R and Rl are as d~fined above) with an agent serving to
con~ert the oxo group in the 4-position o~ the uracil ring to an
amino group; any remaining protecting groups being removed for
example by acid or base hydrolysis to produce the desired
product.
'~ith regard to process a), the hydxoxy protecting group includes
protecting groups such as acyl (e.g. acetyl), arylacyl (e.g. benzoyl
or substituted benzoyl), trityl or monomethoxytrityl, benzy1 or
substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or
diphenylmethylsilyl. The 5-R-cytosine compound may be optionally
protected with silyl, e.g. trimethyl silyl groups. Such groups may be
removed in con~entional manner. The leaving group L is a leavin~
group typical of those known~ iD the art o~ nucleoside:chemistry e.g.
halogen such as chlorine or bromine, alkoxy such ~as: me:thoxy or ethoxy
or acyl such as acetyl or benzoyl.
The reac~ion~in~proc~ess~a) may be:effected in an crganic solvent (e.g.
l,2-dichloroethane or~acetoni~trile~ in the presence~ f a Lewis:: acid
such as stannic chloride:or trimethylsilyl triflate.
Compouhds of:~formu1a~IIA~ may~be:~obtained ~rom :a:~suitab1y protected
2-hydroxyaceta1dehyde of formu1a 1~
:
~ RlOCh~c~o
:,
wherein Rl is defined above, as described In Can. J~ ~esearch~, 3,;~l2g ~ -
(1933)~ and European Patent Specification 0 382 526. Reaction of
compounds of formula III with a mercaptoacetal HSCH2CH(OR52,~ wherein R ~.
is Cl 4 alkoxy such a5 ~SCH2CH(0C2H5)~, known in the art (Chem. Ber.
85:924-932, 1952), yields compounds: of formula::IIA wherein L is: OR
(a1koxy) e.g. methoxy or e~hoxy. Al:cernati~e1y, compounds of
formula IIA, whereir. L is aIk~xy, may be converted :to compounds of
formula IIA wherein L is halogen or acyl by methods known in the~ arC
of carbohydrate chemistry. : i-
;~
W O 92~l9246 PCT/GB92/00~r~
- 10 -
21~2~2'3
Compounds of formula III may be prepared from 1,2-0-isopropylidene
glycerol by introduction of Rl (e.g. trisubstieu~ed silyl, benzyl or
trityl) and removal of the isopropylidene group with mild acid (e.g.
aqueous formic or acetic acid) or zinc bromide in acetonitrile,
followed by oxidation of the alcohol group with aqueous periodate.
With regard to process b), the compound of ormula IIB is
advantageously treated with 1,2,4-triazole, advantageously together
with 4-chlorophenyl dichlorophosphate, t~ form the corresponding
4-(1,2,4-triazoylyl) compound which is then converted to the desired
4-amino (cytldine) compound by reaction with for example methanol.
The starting materials of for~ula IIB be prepared for example by
reaction of an appropriate (optionally protec~ed) base with a compound
of formula IIA in an analogous~manner to that described in process a).
SeparatiQn of the (+)-cis and (~)-trans isomers f~r example ln a
protected form, ~y ~e accomplished by chromatograp~y on silica gel
with mixtures of organic solYents such as ethyl acetate/m~thanol,
ethylacetate/hexane or dichloromethane/methanol. Any protecting group
may then be rem~ved usin~ the appropriate reagent ~or each group.
Resolution of the ~+)-enantiomers may be accomplished enzymatically
with an esterase such as pig liver esterase (Sigma Chemical o.,
St. Louis, MO 63178~ wherein one enantiomer of a 5'-acyl derivative of
the compound of formula I is de-esterified. After separation, the
remaining esterified compound of formula I may be reacted under basic
conditions (e.g. NH3 in methanol or NaOMe in methannl) to give the
individual (~) and (-)-enantiomers.
Alternatively, the (+)-cis or (+)-tran~i iso~ers of the compound of
formula I may be reacted with a phosphorylating agent (e.g.
phosphorous oxychloride) in an inert solvent such as triethylphos-
phate, acetonitrile or 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimi-
dinone to yield the 5'-monophosphate derivative or a salt thereof.
~vo 92~g246 ~ 1 2 0 PCTJC~92/00~03
Treatment of the 5'-monophosphate derivative with an enzyme such as
snake venom 5'-nucleotidase preferentially hydrolyzes one of the
enantiomers. Separation and s~bsequent dephosphorylation of the other
5'-monophosphat~ enantiomer yields the individual (+)- and
(-)-enantiomers of the co~pound of formula I.
The compound of formula (I) may be converted into a pharmaceutically
acceptable ester by reaction with an appropriate esterlfying agent,
for example, an acid halide or anhydride. The compound of formula (I)
may be converted into a pharmaceutically acceptable salt thereof in a
conventional manner, for example, by treatment with an appropriate
base. An ester or salt of a compound of formula (I) may be converted
into the parent compound, for example, by hydrolysis.
For a better understandin~ of the invention, the following Exa~ples
are given by way of illustration.
Example 1: 1-(2~ ~ydroxYmeth~ l 3-~xa~hi~l~n~ 5-ylh=chD~L~A
: .
Method A: (+)-Cis and (+~-trans-2-ben~oyloxymethyl-5-(N4-acetyl-cyto-
sin-l-yl)-1,3-oxathiolane are prepared and separated~ to the (+)-cis -
~nd (+)trans isomers as described in European Patent (EP) ~-
Specification 0 382 526. The N4-acetyl and 2-benzoyl groups are
removed wieh dimethylamine in ethanol, and the~product, (~)-cis-1-(2-
(hydroxymethyl)-1,3-oxathiolan-S-yl)cytoslne, Ls isolated.
..
Method B: (+)-Cis and (~)-trans-2-benzoyloxymethyl-5-(uracii-1-yl~-
1,3-oxathiolane are prepared as desoribed in EP 0 382 ~26. After
deprotection of the 2-hydroxyl group with saturated methanolic ~:
ammonia, the isomers are separated on silica gel using EtOAc/MeOH as
eluant (EP 0 382 526j. The (+)-cis isomer is reacted with acetic
anhydride in pyridine at room temperature to give the 2-acetate. ~-
Solvent is removed in Yacuo at ~30C. The 2-acetate is then dissol~rd
in CHC13 and washed with aqueous sodium bicarbonate. The separated ~;
organic layer is dried, and CHC13 is evaporated in vacuo, Coslversion ~
W O 92/l9246 PCT/GB92/008~ ~
2102120 - 12 -
of the uracil base to the cytosine base is carried out by preparation
of the 4-(1,2,4-triazol-1-yl) derivati~e according t~ the methods of
C.B. Reese, J~Chem.Soc., Perkins 1, 1171, 1984 and W.L. Sung, Nucleic
Acids Res. 9:6139, 1981, using 1,~,4-triazole and 2 equivalents o~
4-chloro- phenyldichlorophosphate in dry pyridine at ambient
temperature. This con~ersion is followed by reaction with methanol
previously saturated with ammonia at 0C, a~d the 2-acetate is
hydrolyzed to giYe
(+)-cis-1-(2-(hydroxyme~hyl)-1,3-oxathiolan-5-yl)cytosine.
Example 2- 1-(2~ d~roxvmethyl-1,3-oxathi~l~n-~-Yl~-5-meth~ylc~tosine
.. ~
(+)-Cis and (~)-trans 2-benzoyloxymethyl-5-(thymin-N-yl~-1,3-oxathio-
lane are prepared and separated to the (+)-cis and (+)-trans isomers
as described in European Pa~ent Specification 0 382 526. The (+j-cis
isomer is reacted with acetic anhydride in pyridine at room
temperature to give the 2-acetate. Solvent is removed in vacuo at
<300C. The 2-acetate is then dissolved in CHCl3 washed with aqueous
sodium bicarbonate. The separated organic layer is dried, and CHCl3
is evaporated tn vacuo. Conver~ion of the thymine base to the
5-methylcytosine base is carried out by preparation of the
4-(1,2,4-triazol-1-yl) deri~ative according to the methods of C.B.
Reese, J.Chem.Soc., Perkins 1, 1171, 1984 and W.L. Sung, N~cleic Acids
Res. 9:6139, 1981, using 1,2,4-triazole and 2 equivalents of
4-chlorophenyldichlorophosphate in dry pyridine at ambient
temperature. This conversion is followed by reaction with methanol
previously saturated with ammonia at 0 C, and the 2-acetate is
hydrolyzed to give (+)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-
5-methylcytosine.
,;,
Pharmaceutical Formulations
In the following formulation Examples, the "Active Ingredient" is
1-(2-(hydroxy~ethyl~-1,3-oxathiolan-5-yl)cytosine.
~VO 92/l9246 21~ O P ~ /GB92/00803
Exa~le 3
Tablet Formulations
The following formulation~ A, B and C are prepared by wet granulativn
o the ingredients with a solution o povidone, followed by addition
of magnPsium stearate and compression.
Formulation A
mg~table~ ~g~tablet
~a) Active ingredient 250 250
(b) Lactose B.P. 210 26 :~.
(c) Povidone B.P. 15 9
(d) Sodium Starch Glycollate 20 12
(e) Magnesium Stearate 5 3
500 300
Formulation B
mg~tablet mgitablet
(a) Active ingredient 250 250
(b) Lactose 150
(c) Avicel PH 101 60 :26
: (d) Povidone B.P. 15 9
(e) Sodium Starch Glycollate 20~ 12
(f) Ma~nesium Stearate __~ 3
~.
.
~ : ,
W ~ 9~/1g246 PCT/GB92/008~.
2102120 14
Formulation C
., . .. _
m~tablet
Active ingredient 100
Lactose 200
Starch 50
Povidone 5
Magnesium Stearate 4
359
The following formulations, D and E, are prepared ~y dlreet
compression of the admixed ingredients. The lactose in formulation E
is of the direct compression type (Dairy Crest - "Zeparox"j.
Formul~ation D
mg~tablet
:
Active ingredient 250 ;~:
Pregela inized Starch NF15 150~
~: ~ ' ,
~; ~
: Formulation_E : :~
~ee/tablet~ :
: Acti~e ingredient 250
. .
Lactose 150 : :
Avicel ~ 100
500
Formulation F [Cont~r.olled ~elease Formula~ion) :
The formulation is prepared by ~et granulation of the ingredients
(below) with a solution of povidone followed by the addition of
magnesium stearate and compression. :;
r6~ ",,~ r:",.,,"~",.~,~",,.~,,6","h".,~,,"j~~;~ " ~ ,;
W O 92~19246 2 1 ~ 2 ~ 2 ~ PCT~GB92~00803
m~tablet
(a) Active ingredient 500
~b? Hydr~xypropylmethylcellulose 112
(Methocel K4M Premiu~)
(c~ Lactose B.P. 53
(d) Povidone B.P. 28
(e) Magnesium Stearate
700
Drug release takes place over a period of~ about 6-8 hours and is
complete after 12 hours.
Example 4
CaRsule F~rmulations ~;
Formulation_-A
:''
A cap~ule formulation is prepared by admixing the ~ingredients of
~Formula~ion D in Example 3:above and filling into~a two-part hard
gelatin capsule. Formulatlon B (infra~ ~is~prepared :in ::a~ ~slDil~ar
manner.
: ~
Formulation B
mg/ca ule
(a) Active ingredient250
(b) Lactose B.P. 143
(c) Sodiu~ Starch Glycoll~ate 25
~d) Magnesium Stearate ~ 2
: 420
W O 92/19246 PCT/~B9V008~
2102 ~ 16 -
Formulation C
mg~capsule
(a) Active ingredient 250
(b) Macrogol 4000 B.P 350
600 ~
Formulation D -
mg/capsule
Active ingredient 250
Lecithin 100
Arachis Oil 100
450 ;~
Capsules of formulation D are prepared by dispersing the active
ingredient in the Iecithin and arachis oil and filling the dispersion
into soft, elastic gelat1n capsules.
Formulati~n E fC~L~L ~ 5~:l 5 L~
,...
The following controlled release capsule formulation ~is prepared by
extruding in~redients a, b and c using an extruder, followed by
spheronization of the extrudate and drying. The dried pellets are
then coated with controlled-release membrane (d) and filled into a
two-piece, hard gelatin capsule.
mg/capsule :~;
(a) Actîve ingxedient ~ 250 ~ -
(b) MicrocrystalIine Ceilulose 125
(c) Lactose B.P. 125 : :,
~d) Ethyl Cellulose 13
513 :~
: '
'~
VVO 9~/19246 l7PCT/GB92/00803
Example 5 ,~
Injectable Formulation
Fo~rmulation A
Active ingredient 0.200 g
Hydrochloric acid solution, 0.1 M, or
Sodium hydroxide solution, 0.1 M q.s. to pH 4.0 to 7.0
Sterile water q.s. to 10 mL
The active ingredient is dissolved in most of the water (35~C-40C)
., and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid
or the sodium hydroxide as appropriate. The batch is then made up ~o
volume with the water and filtered through a sterile micropore filter
into a sterile 10 mL~amber glass ~ial (type 1) and sealed ~ h sterile
closures and overseals.
,,
Formulation B ~
'. .
Active ingredient 0.125 -~
Sterile, pyrogen-free, pH 7 phosphate
Buffer, : q.s. to 25 mL
Exa~mpl~
Intramus"c~lar iniection ;,
Acti~e ingredient 0.20 g ::~ -
Benzyl Alcohol . 0.10 g
Glycofurol 75 1.45 g ,
Benzy~ Alcohol q.s, t~ 3.00 mL '~
~'~
The active ingredient is dissolved in the glycofurol. The benzyl
alcohol ic then added and d~ssolved, 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~.
W O ~/19246 PCT/GB9~/008
- 18 -
21~212U
Example 7
Syrup
Active in~redient 0.25 g
Sorbi~ol Solution 1.50 g
Glycerol 2.00 g
Sodi~m Benzoate O.005 ~
Flavor, Peach 17.420316 9 0.0125 mL
Purified Water ~.s. to 5.00 mL
The acti~e ingredient is dissolved in a mixture of the ~lycerol and
most of the purified water. An aqueous solution of the sodium
benzoate is then added to the solution, ~ollowed by addition of the
sorbitol solution and finally the flavor. The volume is made up with
purified water and mixed well.
Example 8
Suppos tgrX ~,
m~/suppvsitory
~.
Active ingredient 250
Hard Fat, B.P. (Witepsol H15 - Dynamit Nobel) 1770
2020
One-fifth of the ~itepsol H1 is melted in a steam-jacketed pan at
45C maximum. The active ingredient is sifted through a 200 ~M sieve
and added to the molten base with mixing, using a Silverson fiY~ted
with a cutting head, until smooth dispersion is achieved. Maintaining
the mixture at 45C, the remaining Witepsol H15 is added to the
suspension and stirred to ensure a homo~enous mix. The en~ire
suspension is passed through a 250 M stainless steel screen and, with
continuo~s stirring, is allowed to cool to 40C. At a temperature of
38C ~o 40C, 2.02 g of the mixture is filled into suitable, 2 mL
plasY~ic molds. The suppositories are allowed tc cool to room
tempera~ure.
. W O 92/19246 2 1 a ,~ ~ 2 0 PCT/GB92/00803
- 19 -
Example 9
e~
Active ingredient 250 ;
Anhydrate Dextrose 380
Potato Starch 363
Ma~nesium Stearate
1000 ~
The above ingredients are mixed directly ànd pessaries prepared by
direct compression of thr~ resulting mixture. ~
Antiviral Activitv ~ ainst ~Hepatitis B Virus ~H.BV~ ~:
The compounds, 1-~2 hydroxymethyl3-1,3-oxathiolan-5-yl)cytosine and
1-(2-(2-hydroxy~ethyl~-1,3-oxat~iolan-5-yl)-5-methylcytosine, were
tested as described below.
The human HBV producer oell line of HepG2, 2.2.15, described and
characterized by Sells et al., PNAS 84:1005, 1987 and J. Virol.
62:2836, 1988, has been shown to share many characteris~ics of~the HBV ~:
chronically infected hepatocyte. It is infectious as demonstrated by
the ability to cause disease in chimpanzees. This c211 line was
utilized in vi~ro to identify compounds with anti^HBV ac~ivity.
T~ test compounds for antiviral activity, monolayer cultures were
trea~ed with compound, 50-200~M for ten days. Supexnatant media
containing extracellular virion DNA (Dane particles) were harvested on :~
dsys ~hree, six and ten, treated with proteinase K (1 mg~mL) and
sodium dodecyl sulfate (1%), and incubated at 50C for one hour. DNA
was extracted with equal volumes of phenol followed by chloroform and
then precipita~ed by ammonium acetate and propanol. The DNA
precipitate was dissolved and collected on nitrocellulose using the
W O 92~19246 PCT/GB92~00
- 20 -
21021 20
procedure of Schleicher and Schuell (S ~ S, 10 Optical Ave., Keene,
NH 03431, Publication No. 700, l9B7), and treated as desoribed by
Southern, J. Mol. Biol. 98:503, 1975. Cells were harvested, and the
intracellular DNA was obtained after cell lysis with guanidine
isothiocyanate. The intracellular DNA was handled in the same manner
as the extracellular DNA. After precipitation by ~mmonium acetate and
propanol, the intracellular DNA precipitate was dissolved, cut by
restriction endonuclease, Hind III, applied to agarose gel and then
treated as described by Southern to determine the quantity of
replicative intermediate forms. The antiviral effec~ of the compound
was determined by measuring at least a 100-fold reduction of the
amoun~ of Dane particles extruded into ~he culture medium and a
similar decrease in the intracellular replicative intermediates.
The results are given below:
W O 92/19246 PCT/GB9~/OOY~
210'~120 - 21 -
Effect of l (~ IEy__oxYmethyl~ 3-oxathio
(Compound Al and l-(2-(Hvdroxymethyl)-l.3-oxathiolan-5-y~
5-meth~lcytosine ~Compound B)_on HBV P=roductiGn in 2.2,15
Cell Cultures
_ . .
Intracellular HBY PNA*
(p~/~g cell DNA)
:
Replica- HBV DNA in Culture Medium
Treatment tive (pg/mL)+
Compound Integra- Mono- inter- Day Day Day Day
(~M) ted mer- mediate 0 3 6 lO
- ~,
A. untreated l.l 2.2 88 88 29 46 llO
cells l.l 2.0 67 67 ~32 52 51
.'
25(A) l.l l.O l 50 lO l O
l.O 0.4 0.3 65 28 3 O
25~B) l.l l.O 3 76 12 O O
1.3 0.6 ~ 6~ ~, O O
';~
: 2. untreated l.O 2.l 92 :71 33 120 150
cells l.O 1.8 63 64 130 :43 120 .;
~ -
25(A) l.2 0.9 l lSO 36 lO ~ O `~;
- l.l ~ ~.7 l :120 64 6 0
25(B) l.O 2.l l 46 41 13 0
l.O l.l 2 58 42 24 l
~ . - .. .
* Analysis of intracellular HBV DNA (Dane particles) was 24 hours
following the 10th day of treatment. ;
. .:
+ A "zero" indicates an undectectable level of HBV DNA, sensitivity -
cutoff was 0,1 pg/mL.
