AU760574B2 - Novel treatment - Google Patents

Description

WO 00/41463 PCTIEP99/1 0295 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 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 an antiviral agent active against hepatitis B virus and 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.

WO 00/41463 PCTIEP99/1 0295 2 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 NW1 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.

WO 00/41463 PCT/EP99/1 0295 3 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 polymerase) 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, 5, 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 protein (ad or ay subtype), characterised in that the amino acid sequence of the protein consists of either: residues 12 52, followed by residues 133 145, followed by residues 175 400 of the said L protein; or 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 wherein L* is as defined above and S denotes the S-protein of hepatitis B surface antigen.

WO 00/41463 PCT/EP99/10295 4 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 TH1 aspects. Suitable adjuvant systems include, for example a combination of monophosphoryl lipid A, preferably 3de-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 ofQS21 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 96/33739.

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.

WO 00/41463 PCT/EP99/10295 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.

WO 00/41463 PCT/EP99/1 0295 6 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 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 pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers lactose, micro -crystalline cellulose or calcium hydrogen phosphate); lubricant magnesium stearate, talc or silica); disintegrants 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 sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents lecithin or acacia); non-aqueous vehicles (e.g.

almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives WO 00/41463 PCT/EP99/10295 7 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 multi-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 lamivudine/penciclovir/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 WO 00/41463 PCT/EP99/10295 8 with the duck hepatitis B virus (DHBV). Changes in the levels of preS 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.

WO 00/41463 PCT/EP99/10295 9

EXAMPLES

Hepatitis B surface antigen vaccine/Lamivudine pharmacokinetics 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 (Smithikline Beecham Biologicals), except that it was lyophilised.

Lyophilized Ag: HBsAg I 001ig Sucrose 12.6 mg NaCI 20.3mM NaH 2

PO

4 NaJIPO, 1.35 mM Adjuvant system: oil in water emulsion: 250 ptl Squalene 10.7 mg DL ac-tocopherol 11.9 mg polyoxyethylenesorbitan monooleate (Tween 80) 4.8 mg Monophosphoryl lipid A 100 g QS21 10 Pg Water for injection q.s. ad 0.5 ml 4 575 p~g

KH

2

PO

4 100 P KCI 100 g NaCI 4mg 6.8 WO 00/41463 PCT/EP99/1 0295 Lamivudine (Zeffix M, 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 intramuscular injection immediately before administration of Lamivudine. Blood samples were taken at pre-dose, 0.5, 0.75, 1, 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 0 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 postdose 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 WO 00/41463 PCTIEP99/10295 11 hours post-dose (AUC 24 on Days 7, 14, 28 and 42 are summarised below with standard deviations in parentheses: Cmax (ng/ml) 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 UC2 4 (ng.h/ml) 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 (AUC 24 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 2 0.57).

WO 00/41463 PCT/EP99/10295 12 On Days 14, 28 and 42 the rate (Cmax) and extent (AUC 2 4 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 2 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 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.

Serology 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 pl per well. Antigen was diluted at a final concentration of 1 pg/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 0 C with PBS WO 00/41463 PCTIEP99/1 0295 13 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 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 (0.1M pH=5.8). The reaction was stopped with HSO, 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 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.

WO 00/41463 PCT/EP99/10295 14

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.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

Claims (12)

1. A method of treating a patient suffering from or susceptible to Hepatitis B virus infection, comprising administering to a patient in need thereof, either simultaneously or sequentially in any order, a safe and effective amount of (1) an antiviral agent active against hepatitis B virus and a vaccine for the prophylaxis and/or treatment of hepatitis B infection.

2. A method according to claim 1 which comprises use of a pharmaceutical pack comprising as active ingredients an antiviral agent active against hepatitis B virus and a vaccine for the prophylaxis and/or treatment of hepatitis B infection, the active ingredients being for simultaneous or sequential use.

3. A method according to claim 1 or 2 wherein the antiviral agent is a nucleoside analogue.

4. A method according to any one of the preceding claims wherein the antiviral agent is selected from the group comprising: penciclovir, famciclovir or lamivudine. A method according to claim 1 or 2 wherein the antiviral agent is a nucleotide analogue. 25 6. A method according to claim 3 or 5 wherein the antiviral agent is selected from the group comprising: ganciclovir, lobucavir, adefovir, ribavirin, BMS200,475, vidarabin or ARA-AMP.

7. A method according to claim 1 or 2 wherein the antiviral agent is alpha- 30 interferon. ooo GSK specdoc 16

8. A method according to any one of the preceding claims wherein the vaccine active against hepatitis B comprises hepatitis B surface antigen.

9. A method according to any one of the preceding claims wherein the vaccine active against hepatitis B comprises the antigen SL*. A method according to any one of claims 1 to 8 wherein the vaccine active against hepatitis B comprises the 226 amino acid S antigen.

11. A method according to any one of the preceding claims wherein the vaccine comprises an adjuvant.

12. A method according to claim 11 wherein the adjuvant is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, a CpG oligonucleotide, aluminium hydroxide, aluminium phosphate, tocopherol, and an oil in water emulsion or a combination of two or more of the said adjuvants.

13. A method according to claim 12 wherein the adjuvant comprises 3D-MPL and an oil in water emulsion. 000* 14. A method according to claim 13 wherein the oil in water emulsion comprises squalene, tocopherol and polyoxyethylenesorbitan monooleate S• 25 (Tween o0 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. o*o *:0 Ai, 17

16. 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 infection.

17. A method according to claim 1 substantially as hereinbefore described, with reference to any of the examples. DATED: 5 March, 2003 PHILLIPS ORMONDE FITZPATRICK Attorneys for: SMITHKLINE BEECHAM BIOLOGICALS S.A. e*