Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL TREATMENT
This invention relates to the use of a nucleoside analogue active against
hepatitis B
virus (HBV), or another class of antiviral active against HBV, such as y
interferon or a
nucleotide analogue and an HBV vaccine in the treatment of hepatitis B virus
infections.
Chronic hepatitis B virus (HBV) infection, for which there is currently no
effective
cure, constitutes a global public health problem of enormous dimensions.
Chronic
carriers of HBV, estimated to number more than 300 million world-wide, are at
risk
for development of chronic active hepatitis, cirrhosis and primary
heptocellular
carcinoma.
EP-A-388049 (Beecham Group p.l.c.), discloses the use of
penciclovir/famciclovir in
the treatment of hepatitis B virus infection. All references herein to
penciclovir/famciclovir include pharmaceutically acceptable salts, such as the
hydrochloride, and solvates, such as hydrates.
EP-A-494119 (IAF Biochem. International Inc.) discloses the use of 1,3-
oxathiolane
nucleoside analogues, including lamivudine, in treatment of Hepatitis B.
The present invention provides a pharmaceutical pack comprising as active
ingredients ( 1 ) an antiviral agent active against hepatitis B virus and (2)
a vaccine for
the prophylaxis and/or treatment of hepatitis B infection, the active
ingredients being
for simultaneous or sequential use.
By pharmaceutical pack is meant a pack or dispenser device which may contain
one
or more unit dosage forms containing the active ingredients. The pack may for
example comprise metal or plastic foil, such as a blister pack. The pack or
dispenser
device may be accompanied by instructions for administration. Where the
antiviral
agent and the HBV vaccine are intended for administration as two separate
compositions these may be presented in the form of, for example, a twin pack.
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The invention may be used for either the treatment or prophylaxis of hepatitis
B
infections. The invention is most particularly of value for treatment, for
example, of
chronic hepatitis B infections.
In one aspect, the antiviral agent as used in the pharmaceutical pack is a
nucleoside
agent. In a further aspect the antiviral agent is a nucleotide agent. Suitable
agents for
use in the invention include penciclovir, famciclovir, lamivudine,
ganciclovir,
lobucavir, adefovir, ribavirin, BMS200,475, vidarabin or ARA-AMP. Preferred
nucleoside analogues include penciclovir, famciclovir and lamivudine.
A further potential antiviral agent is an interferon. Alpha - interferon is
especially
preferred.
Information with respect to structure and activity of nucleoside analogues may
be
obtained from well known pharmaceutical industry references, such as
"Pharmaprojects", PJB publications Limited, Richmond, Surrey, U.K. or from 'R
& D
Focus', isssued by IMS World publications, 364 Euston Road, London NW 1 3BL.
References to an anti-hepatitis B virus nucleoside analogue, including the
specific
compounds mentioned hereinbefore and salts thereof, include solvates such as
hydrates.
Examples of pharmaceutically acceptable salts are as described in the
aforementioned
Patent reference in the name of Beecham Group p.l.c. and references quoted
therein,
the subject matter of which are incorporated herein by reference.
It will be appreciated that the anti-hepatitis B virus nucleoside or
nucleotide analogue
and HBV vaccine of this invention may be administered in combination with
other
pharmacologically active agents, in particular, other antivirals.
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In this invention the vaccine for the prophylaxis and/or treatment of
hepatitis B
infection includes all vaccines containing HBV antigens (such as surface
antigen, core
and polymerise) and therapeutic vaccines.
In one aspect of the invention the hepatitis B virus antigen is the hepatitis
B surface
antigen (HbsAg). The preparation of Hepatitis B surface antigen is well
documented.
See for example, Harford et. al. in Develop. Biol. Standard 54, page 125
(1983),
Gregg et. al. in Biotechnology, S, page 479 (1987), EP-A- 0 226 846, EP-A-0
299 108
and references therein.
As used herein the expression 'Hepatitis B surface antigen' or'HBsAg' includes
any
HBsAg antigen or immunogenic derivative thereof, particularly fragments
thereof,
displaying the antigenicity of HBV surface antigen. It will be understood that
in
addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais
et. al.
Nature, 317, 489 (1985) and references therein) HBsAg as herein described may,
if
desired, contain all or part of a pre-S sequence as described in the above
references
and in EP-A- 0 278 940. HBsAg as herein described can also refer to variants,
for
example the 'escape mutant' described in WO 91/14703. In a further aspect the
HBsAg may comprise a protein described as L* in European Patent Application
Number 0 414 374, that is to say a protein, the amino acid sequence of which
consists
of parts of the amino acid sequence of the hepatitis B virus large (L) protein
(ad or ay
subtype), characterised in that the amino acid sequence of the protein
consists of
either:
(a) residues 12 - 52, followed by residues 133 - 145, followed by residues
175 - 400 of the said L protein; or
(b) residue 12, followed by residues 14 - 52, followed by residues 133 -
145, followed by residues 175 - 400 of the said L protein.
HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0 304
578.
Normally the HBsAg will be in particle form. It may comprise S protein alone
or may be as composite particles, for example (S, L*) wherein L* is as defined
above
and S denotes the S-protein of hepatitis B surface antigen.
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A preferred hepatitis B antigen is this composite particle, defined as S,L*.
A further preferred hepatitis B antigen is the 226 amino acid sequence of the
HBV
surface antigen, in particle form.
Such a vaccine may advantageously include a pharmaceutically acceptable
excipient
such as a suitable adjuvant. Suitable adjuvants include an aluminium salt such
as
aluminium hydroxide gel (alum) or aluminium phosphate (as described in
W093/24148), but may also be a salt of calcium, iron or zinc, or may be an
insoluble
suspension of acylated tyrosine, or acylated sugars, cationically or
anionically
derivatised polysaccharides, or polyphosphazenes.
Advantageously, the hepatitis B virus may be formulated with strong adjuvant
systems. Thus in the formulation of the invention, it is preferred that the
adjuvant
composition induces an immune response comprising THl aspects. Suitable
adjuvant
systems include, for example a combination of monophosphoryl lipid A,
preferably 3-
de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium
salts.
A vaccine comprising hepatitis B surface antigen in conjunction with 3D-MPL
was
described in European Patent Application 0 633 784.
An enhanced system involves the combination of monophosphoryl lipid A and a
saponin derivative particularly the combination of QS21 and 3D-MPL as
disclosed in
WO 94/00153, or a less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96133739.
Other known adjuvants which may be included are CpG containing
oligonucleotides
(see University of Iowa; W09602555).
In a preferred embodiment of the present invention there is provided a vaccine
comprising an HBV antigen, adjuvanted with a monophosphoryl lipid A or
derivative
thereof.
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Preferably the vaccine additional comprises a saponin, more preferably QS21.
Preferably the formulation additional comprises an oil in water emulsion and
tocopherol.
A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol
in
an oil in water emulsion is described in WO 95/17210.
The present invention also provides a method of treatment and/or prophylaxis
of
hepatitis B virus infections, which comprises administering to a human or
animal
subject, suffering from or susceptible to Hepatitis B virus infection, either
either
simultaneously or sequentially in any order, a safe and effective amount of 1)
an
antiviral agent active against hepatitis B virus and 2) a vaccine for the
prophylaxis
and/or treatment of hepatitis B infection.
The antiviral such as penciclovir/famciclovir and the HBV vaccine or a
pharmaceutically acceptable salt or ester thereof, may be co-administered in
the form
of two separate pharmaceutical compositions for simultaneous or sequential
use.
Normally the active ingredients will be administered separately according to
the
normal dosage and administration regimen for the ingredients given alone.
Commencement of administration may be either with the vaccine or the
antiviral.
The present invention also provides for the use of an antiviral compound in
the
manufacture of a medicament for the treatment of patients already primed with
a
hepatitis B vaccine and suffering from a hepatitis B virus infection. The
invention
further provides for the use of a hepatitis B vaccine in the manufacture of a
medicament for the treatment of patients already primed with an antiviral
compound
and suffering from a hepatitis B virus infection. The preferred antiviral is a
nucleoside analogue, most preferably penciclovir/famciclovir or lamivudine.
Preferred hepatitis B vaccines are identified hereinabove.
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The unit doses of the nucleoside or nucleotide analogue may be administered,
for
example, 1 to 4 times per day. The exact dose will depend on the route of
administration and the severity of the condition being treated, and it will be
appreciated that it may be necessary to make routine variations to the dosage
depending on the age and weight of the patient and immunocompromised patients
may require an increased dosage.
Vaccines are administered in multiple doses at various intervals. This is
usually 6 -
12 doses at biweekly or monthly intervals.
The preferred ingredients in the pharmaceutical pack when administered
simlutaneously are given as separate preparations, for example, as
vaccinations in
each arm. It is however possible to consider simultaneous administration by
mixing
the ingredients before administration. The ingredients may be given enterally,
such as
orally or parenterally (e.g. intramuscularly or, more particularly,
intravenously).
The anitviral agents of the invention may be formulated as a tablet prepared
by
conventional means. Compositions for oral use such as tablets and capsules may
be
prepared by conventional means with pharmaceutically acceptable excipients
such as
binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g. lactose, micro -crystalline
cellulose or
calcium hydrogen phosphate); lubricant (e.g. magnesium stearate, talc or
silica);
disintegrants (e.g. potato starch or sodium starch glycollate); or wetting
agent (e.g.
sodium lauryl sulphate). Tablets may be coated by methods well known in the
art.
Liquid preparations for oral administration may take the form of, for example,
solutions, syrups or suspensions, or they may be presented as a dry product
for
constitution with water or other suitable vehicle before use. Such liquid
preparations
may be prepared by conventional means with pharmaceutically acceptable
additives
such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or
hydrogenated
edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous
vehicles (e.g.
almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and
preservatives
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(e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations
may also
contain buffer salts, flavouring, colouring and sweetening agents as
appropriate.
Preparations for oral administration may be suitably formulated to give
controlled
release of one or both active ingredients.
For parenteral administration the compositions may be presented in a form
suitable for
bolus injection or continuous infusion. Formulations for injection may be
presented
in unit dosage form e.g. in syringes, ampoules or in mufti-dose containers,
with an
added preservative.
The active antiviral agent 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 active ingredients
may be in
powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-
free water,
before use.
For rectal administration the active antiviral agents may be formulated as
suppositories or retention enemas, e.g. containing conventional suppository
bases
such as cocoa butter or other glycerides.
The active antiviral agents of the invention may be prepared according to
conventional techniques well known in the pharmaceutical industry. Thus, for
example, the lamivudinelpenciclovir/famciclovir may be admixed, if desired,
with
suitable excipients. Tablets may be prepared, for example, by direct
compression of
such a mixture. Capsules may be prepared by filling the blend along with
suitable
excipients into gelatin capsules, using a suitable filling machine. Controlled
release
forms for oral or rectal administration may be formulated in a conventional
manner
associated with controlled release forms.
Anti-hepatitis B virus nucleoside analogues may be identified by standard
methods,
such as tests involving studies in in vitro primary duck hepatocytye cultures
infected
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with the duck hepatitis B virus (DHBV). Changes in the levels of preS 1 and/or
viral
DNA in cultures treated with such anologs would indicate activity.
Alternatively,
analogues may be identified by the ability to interfere with normal acylation
of
synthetic peptides representing the N-terminal amino acids of DHBV or
hepatitis B
viruses of man, woodchucks, ground squirrels or other animals.
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EXAMPLES
Hepatitis B surface antigen yaccine/Lamivudine pharmacolcinetics interaction
study in dogs
METHODS
The following vaccine composition was employed. The HBV surface antigen was
equivalent to the antigen employed in the commercially available Engerix-B
vaccine
TM (Smithkline Beecham Biologicals), except that it was lyophilised.
Lyophilized Ag:
HBsAg 100pg
Sucrose 12.6 mg
NaCl 20.3mM
NaH2P04 l Na2HP04 1.35 mM
Adjuvant system:
oil in water emulsion: 250 ~l
- Squalene 10.7
mg
- DL a-tocopherol 11.9
mg
polyoxyethylenesorbitan
monooleate (Tween 80) 4.8 mg
Monophosphoryl lipid A 100 pg
QS21 100 pg
Water for injection q.s. ad 0.5
ml
Na~HP04 575 pg
KHZPO4 100 pg
KCl 100 pg
NaCI 4mg
pH 6.8+/-0.2
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Lamivudine (Zeffix T"', GlaxoWellcome) was administered daily by oral capsule
to
three male and three female dogs at a dose level of 100 mg/dog/day for 6
weeks. On
Days 14, 28 and 42 the HBs/adjuvant vaccine as described above was
administered by
5 intramuscular injection immediately before administration of Lamivudine.
Blood
samples were taken at pre-dose, 0.5, 0.75, l, 2, 4, 6, 8, 12 and 24 hours
after dosing of
Lamivudine on Days 7, 14, 28 and 42. The separated plasma was frozen at -
20°C prior
to despatch to Pharma Bio-Research for analysis of plasma concentrations of
Lamivudine.
Sera were collected on days 0, 29 and 43 for anti-HBs antibody evaluation.
RESULTS
Lamivudine pharmacokinetics
Blood samples were taken on Days 7, 14, 28 and 42 of a 6-week toxicity study
in order
to assess the systemic exposure of male and female dogs to Lamivudine
following
daily oral administration of Lamivudine at a dose level of 100 mg/dog/day and
intramuscular administration of HBs vaccine on Days 14, 28 and 42 immediately
before administration of Lamivudine. Plasma concentrations of Lamivudine in
samples
taken up to 24 hours post-dose were measured by Pharma Bio-Research.
The maximum mean plasma concentrations of Lamivudine occurred at 2 hours post-
dose on all the sampling days except for females on Day 7 where the maximum
mean
plasma Lamivudine concentration occurred at 1 hour post-dose. On Day 28, the
maximum mean plasma concentrations of Lamivudine were lower than those values
on Day 7, 14 and 42. After the maximum, the mean plasma concentrations of
Lamivudine declined in an apparently biexponential manner.
Mean maximum plasma concentrations (Cmax) of Lamivudine and the areas
under the plasma Lamivudine concentration-time curves estimated up to 24
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hours post-dose (AUC24) on Days 7, 14, 28 and 42 are summarised below with
standard deviations in parentheses:
Cmax (nglm I)
Day 7 Day 14 Day28 Day 42
Males Females Males Females Males Females Males Females
3045 4290 3176 3555 2053 2542 3277 3287
(1516) (3335) (871) (1901) (515) (1255) (567) (1256)
A UC24 (ng. hlml)
Day 7 Day 14 Day 28 Day 42
Males Females Males Females Males Females Males Females
12541 11514 12858 13567 11629 8883 12585 11049
(2211) (4324) (3231) (5957) (2694) (2534) (1182) (4334)
The times at which the maximum plasma concentrations occurred (Tmax) in
individual dogs were generally 2 hours, and in the range 0.75 to 4 hours and
appeared
to be independent of administration of the HBs vaccine.
Plasma concentrations of Lamivudine were quantifiable in male animal numbers
71
and 73 and in female animal number 70 at all time points on Days 7, 14, 28 and
42,
therefore, these animals were continuously exposed to quantifiable
concentrations of
Lamivudine during a dosing interval.
The rate (Cmax) of systemic exposure of female dogs to Lamivudine was slightly
higher
than that in male dogs. The extent (AUCz4) of systemic exposure of female dogs
to
Lamivudine was generally slightly lower than that in male dogs. However, there
was no
statistically significant evidence for any sex-related differences in systemic
exposure (p
_> 0.57).
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On Days 14, 28 and 42 the rate (Cmax) and extent (AUC24) of systemic exposure
of
dogs to Lamivudine were generally similar to those values on Day 7, however,
the
Cmax values in female dogs on Days 14, 28 and 42 appeared to be lower than
those
values on Day 7. Overall, there was no statistically significant evidence for
any time
(day of sampling) related differences in the rate and extent of systemic
exposure (p
0.08). The mean values of accumulation ratios, based on AUC24 values are
summarised
below
Accumulation ratio
Males Females
Day 14/Day 7 1.0 1.2
Day 28/Day 7 0.9 0.8
Day 42/Day 7 1.0 1.0
The mean accumulation ratios were generally close to or less than one
indicating that little or no accumulation of Lamivudine occurred following
administration of HBs vaccine.
The terminal rate constants, and corresponding terminal half lives, of
Lamivudine on
Days 7, 14, 28 and 42 are presented in Tables 5 - 8. The terminal rate
constant, where
it could be calculated ranged from 0.3239 to 0.1364 hours-' corresponding to a
terminal half life of Lamivudine of 2.1 to 5.1 hours.
Ser- ology
Methodology
Quantitation of anti-HBs antibody was performed by ELISA using HBs (Hep 286)
as
coating antigen. Antigen and antibody solutions were used at 100 ~l per well.
Antigen
was diluted at a final concentration of 1 ~.g/ml in PBS and was adsorbed
overnight at
4°C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate,
Nunc,
Denmark). The plates were then incubated for lhr 30 min at 37°C
with PBS
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containing 5% non fat powder milk and 0.1% Tween 20. Two-fold dilutions of
sera
(starting at 1/50 or 1/200 dilution) in PBS containing 0.5% Gloria milk and
0.1%
Tween 20 were added to the HBs-coated plates and incubated for 1 hr at
37°C. The
plates were washed four times with PBS 0.1% Tween 20. HRPO-conjugated anti-dog
IgG (Rockland, USA) diluted 1/40000 in 0.5% non fat powder milk and 0.1% Tween
20 buffer was added to each well and incubated for 1 hr at RT. After a washing
step,
plates were incubated for 10 min at RT with a solution of Tetramethyl
benzidine
(TMB) (Biorad, USA) 2-fold diluted in Citrate buffer (O.1M pH=5.8). The
reaction
was stopped with HzS04 0.5N and plates were read at 450/630 nm. ELISA titers
were expressed as midpoint titers.
Results
The anti-HBs serologic response was measured by ELISA at day 0, 29 and 43.
Midpoint titers are presented in the following table
Midpoint of anti-HBs antibody titers
Dog # Day 0 Day 29 Day 43
69 25 679 7258
71 25 389 3780
73 25 705 6496
70 25 63 1027
72 25 176 3821
74 25 582 11482
Average 25 383 5321
The mid-point average titers at the different timepoint are the respectively
25 on Day
0 (arbitrary 1/2 of first dilution), 383 on day 29 and 5321 on day 43. This
clearly
indicate the induction of an immune response.
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CONCLUSION
In conclusion, the rate and extent of systemic exposure of dogs to Lamivudine
following repeated oral administration of Lamivudine at a dose level of 100
mg/dog/day appeared to be independent of the administration of HBs vaccine on
Days
14, 28 and 42 o the 6-week pharmacokinetic interaction study. There was no
evidence
of a difference in the rate and extent of systemic exposure to Lamivudine
between
male and female dogs.
Administration of the pharmaccine appeared to be immunogenic and induced high
circulating levels of anti-HBs antibodies, validating the use of the Beagle
dog as an
animal species for this PK interaction study.
