Note: Descriptions are shown in the official language in which they were submitted.
2~7
WO 94/02154 PCI/EP93/01846
ANTIVIRAL COMBINATIONS
The present invention relates to combinations of antiviral agents. More
specifically it is concerned with combinations of 1,3-oxathi~olane nucleoslde
5 analogues with other antiviral agents, in particular agents effective against HIV.
Human immunodeficiency virus (HIV) c~uses a variety of clinical conditions
including the acquired immune deficiency syndrome (AIDS) and chronic
neurological disorders. Nucleosides such as AZT, ddC and ddl inhibit HIV
replication in vitro. and appear to exert their antiviral activity on the
10 virus-encoded reverse transcriptase enzyme after metabolism by the cell to their
5'-triphosphate derivatives.
AZT reduces morbidity and mortality in patients with AIDS. However, HIV
infection of cells results in integration of the virus genome into the host
chromosome, and so it has been necessary to continue AZT treatment for long
15 periods of time. The consequences of long-term AZT therapy are associated
bone-marrow toxicity and the appearance of AZT-resistant variants of HIV-1.
Similarly, some AIDS patients treated with ddC develop peripheral neuropathy
and ddl has been shown to induce pancreatitis and peripheral neuropathy.
The use of combinations of compounds may give rise to an equivalent antiviral
20 effect with reduced toxicity, or an increase in drug efficacy if synergy between
compounds occurs. Lower overall drug doses will possibly also reduce the
frequency of occurrence of drug-resistant variants of HIV. Many different
methods have been used to examine the effects of combinations of compounds
acting together in different assay systems. All of these methods have limitations
25 and for example, some methods have been applied to systems other than those
for which they were derived. AZT demonstrates synergistic antiviral activity in
vitro in combination with agents that act at HIV-1 replicative steps other than
reverse transcription, including recombinant soluble CD4 castanospermine and
recombinant interferon alpha. However, it must be noted that combinations of
30 compounds can give rise to increased cytotoxicity. AZT and recombinant
WO 94/02154 PCI'/EP93/01846-
2 i 40237 2
interferon alpha have an increased cytotoxic effect on normal human bone
marrow progenitor cells.
The compound (2R, cis)-4-amino-1-(2-hydroxymethyl-1 ,3-oxathiolan-5-yl) -(1 H)-
pyridmidin-2-one, also known as 3TC, is currently undergoing clinical trials forthe treatment of conditions associated with infection by HIV.
We have now found that 3TC exhibits unexpected advantages when used in
combination with certain non-nucleoside inhibtors of HIV replication.
There is thus provided in a first aspect of the invention a combination
comprising 3TC or a pharmaceutically acceplable derivitive thereof and a non-
nucleoside inhibitor of HIV selected from a {[(benzoxazol-2-yl)methyl]amino}-5-
alkyl-6-alkyl-2-(1 H)-pyridinone or a derivative thereof bearing an unsubstituted
or substituted pyridyl or phenyl substituent.
In a preferred aspect the invention provides a combination of 3TC and a
compound selected from
3-~l(4,7-dichloroberl70~Yz,7QI-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-
pyndlnone,
3-~[(4,7-dimethylhenzoaxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-
pyridinone,
3-~[(7-chlorobenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-~[(7-methylbenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-~[(4-fluorobenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-~[(7-fluorobenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-{[(benzoaxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-{[(4-chlorobenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-{[(4-fluoro-7-chlorobenzoxazol-2-yl)methyl]amino)-5-ethyl-6-methyl-2-(1 H)-
pyridinone,
3-[2-benzoaxazol-2-yl)ethyl]-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-1N-(5-ethyl-2-methoxy-6-methyl-3-pyridylmethyl)-amino]-5-ethyl-6-methyl-2-
(1 H)-pyridinone
3-[N-(5,6-dimethyl-2-methoxy-3-pyridylmethyl)amino]-5-ethyl-6-methyl-2-(1 H)-
pyridinone,
wo 94/02154 214 0 2 3 7 PCr/EP93/01846
3-[N-(5-ethyl-2-methoxybenzyl)amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone,
3-[N-(2-methoxy-4,5-dimethylbenzyl)amino)-5-ethyl-6-methyl-2-(1 H)-pyridinone
and
3-[N-(2,6-dimethoxybenzyl)amino]-5-ethyl-6-methyl-2(1 H)-pyridinone.
5 3TC will normally be provided substantially free of the corresponding
(+)-enantiomer, that is to say no more than about 5% w/w of the (+)-enantiomer,
preferably no more than about 2%, in particular less than about 1% w/w will be
present.
By "a pharmaceutically acceptable derivative" is meant any pharmaceutically
10 acceptable salt, ester, or salt of such ester, of a parent compound or any other
compound which, upon administration to the recipient, is capable of providing
(directly or indirectly) the parent compound or an antivirally active metabolite or
residue thereof.
It will be appreciated by those skilled in the art that 3TC may be modified to
15 provide pharmaceutically acceptable derivatives thereof at functional groups in
both the base moiety and at the hydroxymethyl group of the oxathiolane ring.
Modification at all such functional groups are included within the scope of the
invention. However of particular interest are pharmaceutically acceptal,le
derivatives obtained by modification of the 2-hydroxymethyl group of the
20 oxathiolane ring.
Preferred esters of 3TC include the compounds in which the hydrogen of the 2-
hydroxymethyl group is replaced by an acyl function R-CO- in which the non-
carbonyl moiety R of the ester is selected from hydrogen, straight or branched
chain alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g.
25 methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl
(e.g. phenyl optionally substituted by halogen, C1 4 alkyl or C1 4 alkoxy);
sulphonate esters such as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl);
amino acid esters (e.g. L-valyl or L-isoleucyl) and mono-, di- or tri-phosphate
esters.
WO 94/02154 PCI/EP93/01846 -
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With regard to the above described''é'sters, unless otherwise specified, any alkyl
moiety present advantageo~ ~sly contains 1 to 16 carbon atoms, particularly 1 to4 carbon atoms. Any aryl moiety present in such esters advantageously
comprises a phenyl group.
5 In particular the esters may be a C1 -1 6alkyl ester, an uns~bstitllted benzyl ester
or a benzyl ester substituted by at least one halogen (bromine, chlorine, fluorine
or iodine), C1- 6alkyl, C1 6alkoxy, nitro or trifluoromethyl groups.
Pharmaceutically acceptable salts of 3TC include those derived from
pharmaceutically acceplable inorganic and organic acids and bases. Examples
10 of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric,
fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene-p-
sulphonic, tartaric, acetic, citric, methanesulphonic, formic, benzoic, malonic,naphthalene-2-sulphonic and benzenesulphonic acids. Other acids such as
oxalic, while not in themselves pharmaceutically acceptable, may be useful as
15 intermediates in obtaining the compounds of the invention and their
pharmaceutically acce~J~able acid addition salts.
Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline
earth metal (e.g. magnesium), ammonium and NR4+ (where R is C1 4alkyl)
salts.
20 3TC is either synergistic with the second component of the combination and/or reduces the cytotoxic effects of the second component.
The advantageous effects of 3TC and the non-nucleoside inhibitors of HIV are
realised over a wide ratio for example 1:250 to 250:1 preferably 1:50 to 50:1,
particularly about 1 :10 to 10:1 by weight. Conveniently each compound will be
25 employed in the combination in an amount at which it exhibits antiviral activity
when used alone.
It is expected that the present combinations will be generally useful against viral
infections or virus-associated tumours in humans, and the method of their use to
~1~0237
WO 94/02154 PCI/EP93/01846
inhibit viral infectivity or tumour growth in vitro or in vivo is also within the scope
of the present invention.
Thus there is provided in a second aspect a method for the treatment of a viral
infection in a mammal, including man, comprising co-ad",inisl,alion of 3TC or a
pharmaceutically acceptable derivative thereof and a non-nucleoside inhibitor ofHIV replication as defined herein. Therapeutic methods comprising
administration of a combination of a 3TC and more than one of the non-
nucleoside inhibitors of HIV, either together or in a plurality of paired
combinations, is also within the scope of the invention.
It will be appreciated that 3TC and the second antiviral agent may be
administered either simultaneously (either separately or in combination) or
sequentially. If admini;,l,alion is sequential, the delay in administering the
second of the active ingredients should not be such as to lose the benefit of any
synergistic effect of the combination. Preferably administration will be
1 5 simultaneous.
It will be appreciated by those skilled in the art that reference herein to
treatment extends to prophylaxis as well as the treatment of established
infections or symptoms.
It will be further appreciated that the amount of a combination of the inventionrequired for use in treatment will vary not only with the particular compounds
selected but also with the route of administration, the nature of the condition
being treated and the age and condition of the patient and will be ultimately atthe discretion of the aller,dant physician or veterinarian. In general however asuitable dose will be in the range of from about 1 to about 750mg/kg e.g. from
about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 120mg
per kilogram body weight of the recipient per day, preferably in the range of 6 to
90mg/kg/day, most preferably in the range of 15 to 6ûmg/kg/day of each of the
active ingredients of the combination.
WO 94/02154 ' PCI'/EP93/01846
2~4~23~
~ `
The desired dose may conveniently be presented in a single dose or as divided
doses administered at appropriate intervals, for example as two, three, four or
more sub-doses per day.
The combination is conveniently administered in unit ~iosage form; for example
containing 10 to 1 500mg, conveniently 20 to 1 OOOmg, most conveniently 50 to
700mg of each active ingredient per unit dos~ge form.
Ideally the combinations should be administered to achieve peak plasma
concentrations of each of the active compound of about 1 to about 75,uM,
preferably about 2 to 50,uM, most preferably about 3 to about 30,uM. This may
be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of
the active ingredients, optionally in saline, or orally administered as a bolus
containing about 1 to about 1 OOmg of each active ingredient. Desirable blood
levels may be r"ainlained by a continuous infusion to provide about 0.01 to
about 5.0mg/kg/hour or by intermittent infusions cGnlaining about 0.4 to about
15mg/kg of each active ingredient.
While it is possible that, for use in therapy, the active ingredients of the
combination may be administered as the raw chemical it is preferable to present
combinations as a pharmaceutical formulation.
The invention thus further provides a pharmaceutical formulation comprising
3TC or a pharmaceutically acceptable derivative thereof and a non-nucleoside
inhibitor of HIV replication as defined herein together with one or more
pharmaceutically acceptable carriers therefor and, optionally, other therapeuticand/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the
sense of being compatible with the other ingredients of the formulation and not
deleterious to the recipient thereof.
Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical
(including buccal and sub-lingual), vaginal or parenteral (including
intramuscular, sub-cutaneous and intravenous) administration or in a form
suitable for administration by inhalation or insufflation. The formulations may,where appropriate, be conveniently presented in discrete dosage units and may
WO94/02154 21 ~ D ? 3 7 PCI'/EP93/01846
be prepared by any of the methods well known in the art of pharmacy. All
methods include the step of bringing into association the active compound with
liquid carriers or finely divided solid carriers or both and then, if necessary,shaping the product into the desired formulation.
5 Pharmaceutical formulations suitable for oral administration may conveniently
be presented as ~lisc,~le units such as c~pslJles, cachets or tablets each
containing a predetermined amount of the active ingredient; as a powder or
granules; as a solution, a suspension or as an emulsion. The active ingredient
may also be presented as a bolus, electuary or paste. Tablets and capsules for
10 oral admini~lralion may contain conventional excipients such as binding agents,
fillers, lubricants, disintegrates, or wetting agents. The tablets may be coatedaccording to methods well known in the art. Oral liquid preparations may be in
the form of, for exa""~l~, aqueous or oily suspensions, solutions, emulsions,
syrups or elixirs, or may be presented as a dry product for constitution with
15 water or other suitable vehicle before use. Such liquid preparations may
contain conventional additives such as suspending agents, emulsifying agents,
non-aqueous vehicles (which may include edible oils), or preservatives.
The compounds according to the invention may also be formulated for
parenteral admini~;tlalion (e.g. by injection, for example bolus injection or
20 continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an addedpreservative. The con"~osilions may take such forms as suspensions, solutions,
or emulsions in oily or aqueous vehicles, and may contain formulatory agents
such as suspending, stabilising and/or dispersing agents. Alternatively, the
25 active ingredient may be in powder form, obtained by aseptic isolation of sterile
solid or by Iyophilisation from solution, for constitution with a suitable vehicle,
e.g. sterile, pyrogen-free water, before use.
For topical administration to the epidermis the compounds according to the
invention may be formulated as ointments, creams or lotions, or as a
30 transdermal patch. Ointments and creams may, for example, be formulated with
an aqueous or oily base with the addition of suitable thickening and/or gelling
WO 94/02154 PCI/EP93/01846
o231
agents. Lotions may be formulated with a~,àqueous or oily base and will in
general also contain one or more emu!sif,ylng agents, stabilising agents,
dispersing agents, suspending agents, thickening agents, or colouring agents.
Formulations suitable for topical administration in the mouth include lozenges
5 comprising active ingredient in a flavoured base, usually sucrose and acacia or
tragacanth; pastilles comprising the active ingredient in an inert base such as
gelatin and glycerin or sucrose and ~r~ci~; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
Pharmaceutical formulations suitable for rectal admini~ lion wherein the
10 carrier is a solid are most preferably presented as unit dose suppositories.
Suitable carriers include cocoa butter and other materials commonly used in the
art, and the suppositories may be conveniently formed by admixture of the
active compound with the softened or melted carrier(s) followed by chilling and
shaping in moulds.
15 Formulations suitable for vaginal ad",inislralion may be presented as pessaries,
tampons, creams, gels, pastes, foams or sprays containing in addition to the
active ingredient such carriers as are known in the art to be appropriate.
For intra-nasal administration the compounds of the invention may be used as a
liquid spray or dispersible powder or in the form of drops.
20 Drops may be formulated with an aqueous or non-aqueous base also
comprising one more dispersing agents, solubilising agents or suspending
agents. Liquid sprays are conveniently delivered from pressurised packs.
For administration by inhalation the compounds according to the invention are
conveniently delivered from an insufflator, nebuliser or a pressurised pack or
25 other convenient means of delivering an aerosol spray. Pressurised packs may
comprise a suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorote(ra~luoroethane, carbon dioxide or other
suitable gas. In the case of a pressurised aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
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_ WO 94/02154 - PCr/EP93/01846
Alternatively, for administration by inhalation or insufflation, the compounds
according to the invention may take the form of a dry powder composition, for
example a powder mix of the compound and a suitable powder base such as
l~ctose or starch. The powder composition may be presented in unit dosage
5 form in, for example, capsules or cartridges or e.g. gelatin or blister packs from
which the powder may be administered with the aid of an inhalator or insufflator.
When desired the above described formulations adapted to give sustained
release of the active ingredient may be employed.
The pharmaceutical compositions according to the invention may also contain
10 other active ingredients such as an~i",ic,~,bial agents, or preservatives.
3TC may be obtained as described in International Patent Application No.
WO91/1 71 59.
The non-nucleoside inhibitors of HIV replication may be obtained as clescril,ed
in EP0484071.
15 The following examples illustrate the invention but are not intended as a
limitation thereof.
FXAMPI F 1
Antiviral Activities Alone or in Combin~tion
Compounds are first serially-diluted in 2-fold decrements in 96-well microtitre
20 plates. Chequerboard titrations are prepared by mixing 251ul aliquots from each
compound dilution both alone or in combination (to a final volume of 501J1 in new
96-well microtitre plates). Aliquots of MT-4 cells (1 o6 cells/ml) in RPMI 1640
growth medium are infected with HIV-1 strain RF at a moi of 2 x 10-3 infectious
doses/cell. Virus is adsorbed at room temperature for 90 minutes, after which
25 the cells are washed in RPMI 1640 growth medium to remove unadsorbed virus
and resuspended at 106cells/ml in RPMI 1640 growth medium. 50~ul of infected
cell suspension are inoculated into wells containing compound or growth
medium only. 50,ul of mock-infected cell suspension are inoculated into wells
WO 94/02154 PCI/EP93/01846 -
,2~40~3~
not containing compound. The plates are then incubated for 7 days at 37C in
5% CO2/air.
After incubation, 10~1 of 3-[4,5-dimethyl thiazol-2-yll-2,5- diphenylle~r~olium
bromide (MTT) at 7.5mg/ml are added to all wells and the plates incuh~terl for afurther 90 minutes at 37C. 150~1 of 10% (v/v) Triton X-100 in isopropanol are
then added and the cells resuspended. After 15 minutes at room temperature
the plates are analysed in a Multiskan MC (Flow Laboratories, Irvine, UK)
reader at 405nm. Conversion of yellow MTT to its for",~an derivative is
maximum in the uninfected untreated cells, and absent in unlre~ted infected
1 0 cells.
Dose-response curves are plotted for each compound alone (IC50% values)
and for reciprocal titrations of each compound at a fixed concentration of the
second compound. Isobolograms of all compound combindlions giving IC50%
values are plotted.
If the IC50% values of compound combination lies on a line joining the IC50%
values of each compound on its own, then the two compounds act additively. If
the combination IC50% lie to the left of the line, the compounds are acting
synergistically.
FXAMPl F ~
Cytotoxicities of Compounds Alone and in Combination.
Cytotoxicity is determined by examination of the cells employed in Example 1
following drug treatment and/or by comparing the cytotoxlcities of 3TC and the
non-nucleoside HIV inhibitors alone and in combination (at l~g/ml ratios of 1:1,1:5 and 5:1 ) in uninfected peripheral blood Iymphocytes and an established T-
lymphocyte cell line; cytotoxicity is measured using a [3H]-thymidine uptake
assay.
