WO1999015175A1 - Procede d'inhibition du developpement du cancer du foie et de l'augmentation de la survivance de l'infection a virus adn de l'hepatite chronique - Google Patents

Procede d'inhibition du developpement du cancer du foie et de l'augmentation de la survivance de l'infection a virus adn de l'hepatite chronique Download PDF

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WO1999015175A1
WO1999015175A1 PCT/US1998/019983 US9819983W WO9915175A1 WO 1999015175 A1 WO1999015175 A1 WO 1999015175A1 US 9819983 W US9819983 W US 9819983W WO 9915175 A1 WO9915175 A1 WO 9915175A1
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hbv
group
week
serum
development
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PCT/US1998/019983
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English (en)
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Bud C. Tennant
Simon F. Peek
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Cornell Research Foundation, Inc.
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Priority to EP98949470A priority Critical patent/EP1023071A4/fr
Priority to CA002309357A priority patent/CA2309357A1/fr
Priority to AU95786/98A priority patent/AU738472B2/en
Publication of WO1999015175A1 publication Critical patent/WO1999015175A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • This invention relates to a novel use of a -L(-)- nucleoside analog. More particularly, the present invention relates to the method of using a ⁇ -l ⁇ ( - ) - nucleoside analog to inhibit the development and growth of hepatocellular carcinomas during Hepatitis B infection.
  • Hepatitis B virus is a partly double-stranded DNA virus belonging to the family Hepadnaviridae . In the majority of individuals infected with HBV, a self limiting acute infection occurs. However, in some individuals, the viral infection persists and can result in chronic persistent hepatitis or chronic active hepatitis. Chronic viral hepatitis can lead to progressive liver disease (e.g., liver cirrhosis); and is associated with the development of hepatocellular carcinoma (HCC) . HCC is one of the most common cancers in humans. The mechanism(s) by which HBV indirectly or directly causes HCC is not completely understood.
  • HBV chronically infected with HBV.
  • Preventative therapy includes vaccination of those individuals at a particular high risk of HBV infection.
  • Treatment of chronic HBV infection includes the administration of any one of a number of drugs.
  • Interferon ⁇ -2b is one of the most widely used drugs for the treatment of chronic HBV infection.
  • HBV is a DNA virus, its replication involves the use of the enzyme reverse transcriptase .
  • nucleosides and nucleoside analogs have been evaluated for antiviral activity against HBV.
  • 5,567,688 discloses that 2'- fluoro-5-methyl-j ⁇ -L-arabino-furanosyluracil, known as "L(-)FMAU", and derivatives thereof, have antiviral activity against HBV in cultured human hepatoma cells in vi tro .
  • U.S. Patent No. 5,631,239 discloses that l-(2,3- Dideoxy-beta-L-ribofuranosyl) -5-fluorocytosine (known as /3-L-FddC) and derivatives thereof, have antiviral activity against HBV in cultured human hepatoma cells in vi tro .
  • 5,627,160 discloses that l-(2,3- didehydro-dideoxy-beta-L-ribofuranosyl) -5- fluorocytosine, and derivatives thereof, have antiviral activity against HBV in cultured human hepatoma cells in vi tro .
  • U.S. Patent Nos . 5,486,520 and 5,532,246 disclose certain 1,3 oxathiolane nucleoside analogs as having antiviral activity against HBV in cultured duckling primary hepatocytes infected with duck hepatitis B virus in vi tro .
  • 5,559,100 discloses treating HBV infection with a therapeutic nucleoside comprising 2-aminopurine-9-beta-D-2 ' , 3'- dideoxy-ribofuranoside.
  • U.S. Patent No. 5,444,063 describes certain -D-dioxolanyl nucleoside analogs as having antiviral activity against HBV in cultured cells in vi tro .
  • 3TC One jS-L (-) -nucleoside analog which has been described, and is widely used, for its antiviral activity against the human immunodeficiency virus in combination with other antiviral agents (see, e.g., U.S. Patent No. 5,627,186) is commonly known as 3TC.
  • Other names for 3TC include lamivudine, L(-)-SddC, and 2',3'- dideoxy-3 ' -thia-3-L-cytidine. It has been demonstrated that 3TC has antiviral activity against HBV in vi tro (Chang et al . , 1992, J. Biol . Chem . 267:13938-13942).
  • HBV develops resistance to 3TC.
  • one or more amino acid substitutions in the active site of the HBV DNA polymerase contributes to 3TC resistance in HBV isolated from patients following liver transplantation (Tipples et al . , 1996, Hepatology 24:714-717; Ling et al . , 1996, Hepatology 24 : 711-713 ) .
  • HCC HBV-associated hepatocellular carcinoma
  • L(-)-OddC showed effective in vivo activity against HepG2 tumors in nude mice (Bridges and Cheng, Chapter 9 in Progress in Liver Diseases, 1995, eds . J.L. Boyer and R.K. Ockner, publs. W.B. Saunders Co.) .
  • L(-)-0ddC is extremely cytotoxic to cells (CEM cells) in vi tro (Bridges and Cheng, 1995, supra) .
  • a method for inhibiting the development and growth of hepatocellular carcinoma in an individual infected with HBV, and particularly a human chronically infected with HBV comprises prolonged administration to the HBV-infected individual of a prophylactically effective amount of 3TC, thereby impairing or inhibiting the development of hepatocellular carcinoma.
  • 3TC alone is administered to the HBV-infected individual.
  • 3TC is administered in combination with one or more antiviral agents having antiviral activity against HBV.
  • FIG. 1 is a graph illustrating the mean body weight values observed for the placebo group (•) and treatment group (O) .
  • FIG. 2A is a graph illustrating the mean serum ALT (alanine aminotransferase) levels observed for the placebo group (•) and treatment group (O) .
  • FIG. 2B is a graph illustrating the mean serum AST (aspartate aminotransferase) levels observed for the placebo group (•) and treatment group (O) .
  • FIG. 3 is a graph illustrating the mean serum GGT
  • FIG. 4 is a graph illustrating the mean serum amylase levels observed for the placebo group (•) and treatment group (O) .
  • FIG. 5A is a graph illustrating the mean serum viral DNA levels observed for the placebo group (•) and treatment group (O) .
  • FIG. 5B is a graph illustrating the mean genomic equivalents observed for the placebo group (•) and treatment group (O) .
  • FIG. 6 is a graph showing percent free from hepatocellular carcinoma in the treatment group (O) and placebo group (•) during the 136 weeks of the study period.
  • FIG. 7 is a graph showing the survival curve for the treatment group (O) and placebo group (•) during the 136 weeks of the study period.
  • 3TC is used herein, for purposes of the specification and claims, to mean L(-)-SddC, 2 ',3'- dideoxy-3 ' -thia-j ⁇ -L-cytidine, or a physiologically acceptable derivative such as 3TC prodrug (see, e.g.,
  • 3TC can be prepared using methods known to those skilled in the art.
  • the term "prolonged” as related to usage or administration of 3TC, is used herein for purposes of the specification and claims to mean more than 52 weeks of 3TC treatment .
  • antiviral agents is used herein, for purposes of the specification and claims, to mean drugs having antiviral activity against HBV. These drugs include, but are not limited to, interferon; ddC; D4T (2' ,3' -didehydro-2' , 3 ' -dideoxythymidine) ; L(-)-FTC; L(- )-FddC; L(-)-FMAU; 1- (2 , 3-didehydro-dideoxy-beta-L- ribofuranosyl) -5-fluorocytosine ; 1,3 oxathiolane nucleoside analogs; 2-aminopurine-9-beta-D-2 ' , 3 ' - dideoxy-ribofuranoside; and ⁇ -D-dioxolanyl nucleoside analogs .
  • a prophylactically effective amount of 3TC is used herein, for purposes of the specification and claims, to mean an amount of 3TC effective to impair or inhibit the development of hepatocellular carcinoma in an HBV-infected individual, without causing serious toxic effects in the treated individual .
  • concentration of 3TC will depend on factors such as the drug's rates of solubility, absorption, excretion, and inactivation (stability) , and drug formulation, and mode of administration; as well as individual factors such as size, age, and weight; and other factors as known to those skilled in the art.
  • 3TC may be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid form, or in solid form.
  • concentrations of 3TC which may be used in the method according to the present invention include 25 mg to 1000 mg daily.
  • Oral daily dosages in humans which have shown antiviral activity against HBV include 25 mg, 100 mg, 300 mg (Lai et al . , 1997, supra;
  • HBV hepatocellular carcinoma
  • the members of the hepadnavirus family are similar in DNA sequence, antigenic structures, morphological appearance, and replicative strategies.
  • WHV woodchuck hepatitis virus
  • HBV hepatitis B virus
  • nucleic acid homology and immunological cross-reactivity between HBV and the duck hepatitis virus
  • HBV hepatitis B virus
  • DNA polymerases of WHV and HBV showing striking similarities in conditions for optimal activity, and susceptibility to inhibition by certain nucleotide triphosphates in vitro.
  • WHV has been accepted by those skilled in the art as an experimental model for HBV.
  • Woodchucks Marmota monax have been used by those skilled in the art as an acceptable animal model for HBV infection in humans and for studying the efficacy of antiviral agents against HBV in vivo (Fourel et al . , 1990, Antimicrob . Agents Chemother. 34:473-75; Ikeda et al., 1994, J. Antimicrob. Chemother. 33:83-89).
  • WHV-infected woodchucks have a high incidence of hepatocellular carcinoma (Rajagopalan et al . , 1996, supra) .
  • the pharmacokinetics of 3TC in woodchucks has been determined; thus, providing a physiological basis for scaling up the therapeutic agents from the woodchuck model to humans (see, e.g., Rajagopalan et al . , 1996, supra) .
  • the following embodiments used to illustrate the invention represent a lifetime study of the effect of 3TC during prolonged treatment of woodchucks chronically infected with WHV, as an in vivo model of chronic HBV infection in humans, including the effects, if any, of prolonged 3TC treatment on: antiviral activity and potential for viral resistance thereto; toxicity associated with drug administration; chronic liver disease; and the prevention or delay in the development of hepatocellular carcinoma.
  • This example illustrates the development of chronically infected woodchucks .
  • the woodchucks were the offspring of laboratory-raised females and males seronegative for markers of WHV infection at breeding.
  • the individual woodchucks were inoculated subcutaneously with 100 ⁇ l of a 10 "1 dilution of the WHV7-P-1 pool of woodchuck Hepatitis virus.
  • WHs surface antigen of WHV
  • persistence of WHs antigenemia was confirmed.
  • each woodchuck included in the lifetime study tested positive for serum WHV DNA and anti-WHV core antibody at six months of age.
  • liver enzyme gamma- glutamyl transpeptidase (GGT) were determined to be less than 5 IU/L. According to these criteria, two groups of twenty 7 to 8 month old chronic WHV carrier woodchucks were selected and assigned to a treatment group or a control group. Each group was stratified on the basis of gender and body weight.
  • GTT liver enzyme gamma- glutamyl transpeptidase
  • This example illustrates the protocols for 3TC administration used to treat chronically infected woodchucks .
  • 3TC was synthesized by Dr. Raymond F. Schinazi. Subsequently, a commercially available form of 3TC (available from Glaxo Wellcome) was used. 3TC was administered orally in an aqueous solution to each woodchuck in the treatment group. The woodchucks in the control group received water as a placebo. The water or 3TC was added to 2 to 3 ml of a semipurified liquid dietary food to ensure consumption; and was administered via an oral dose syringe. The amount of 3TC or placebo administered was adjusted to reflect changes in body weight each time the woodchucks were weighed during the study. The treatment group received 3TC at a dosage of 5 mg/kg body weight/day orally from the start of the study to week 42 of treatment. After week 42, the treatment group received a dosage of 15 mg/kg/day.
  • EXAMPLE 3 This example illustrates analyses of the effects of 3TC treatment or placebo treatment of chronically infected woodchucks on their body weight and on various parameters measured in sequential blood samples. As shown in FIG. 1, during the first 136 weeks of the study, there were no differences in mean body weight observed between the placebo group (•) and treatment group (O) . Each group demonstrated a gradual increase in body weight until week 40, followed by a slight decrease coincident with normal circannual rhythm.
  • the mean serum ALT (FIG. 2A) and AST (FIG. 2B) activities increased slightly in both the treatment group (O) and the placebo group (•) during the first 64 weeks of the study, but rose more dramatically between week 64 and week 88.
  • Mean AST and ALT activities then decreased at week 96 and week 104 in the control group.
  • the mean serum activities of both ALT and AST fell in the 3TC group at week 104.
  • Mean serum AST and ALT activities subsequently increased in both groups through week 136. Elevations in the levels of these enzymes are usually associated with hepatocellular damage. Thus, the increase seen in both groups are likely due to moderate portal and parenchymal hepatitis.
  • the serum GGT activity of all but 4 animals of the treatment group remained constant; thus, the steady rise observed for the mean serum GGT activity treatment group during the first 72 weeks of the study (O; FIG. 3) , represents the development of HCC (identified by ultrasound) in 4 individuals.
  • the rise in mean serum GGT activity of the placebo group (•; FIG. 3) during the first 50 weeks represents several individuals developing HCC.
  • the reduction after week 50 reflects the loss of a total of 6 individuals with advanced HCC in the placebo group whose elevated GGT activities no longer contribute to the calculation of the mean value.
  • the mean serum GGT activity in the control group subsequently increased between week 72 and week 112 in association with the development of HCC in a further 13 woodchucks.
  • the mean GGT activity in the control group rose again at weeks 128 and 136 in association with enlarging HCC in all 7 surviving control animals.
  • the mean serum GGT activity in the 3TC group rose gradually during the first 72 weeks of the study, before increasing more rapidly between week 72 and 96 in association with the development of HCC in 10 individuals.
  • the reduction in mean serum GGT in the 3TC group between week 96 and week 104 was due to the loss of four 3TC treated woodchucks with advanced HCC whose elevated GGT activities no longer contributed to the mean value .
  • a steady increase in mean serum GGT activity in the 3TC group was observed between week 104 and week 136 in association with the development of HCC in 5 woodchucks and the continued growth of tumors whose detection predated the week 104 time point in 3 other 3TC treated woodchucks .
  • Mean serum sorbitol dehydrogenase (SDH) and alkaline phosphatase (AP) activity increased in both groups at 80 and 88 weeks.
  • Subsequent reduction in the mean activity in both groups at weeks 96 and 104 were observed in association with either the death of individual animals with HCC or with the return of serum enzyme activity levels to a lower point.
  • individual serum bilirubin values of greater than 0.8 mg/dl were recorded in association with advanced HCC in two placebo and two 3TC recipients.
  • mean hematocrit mean segmented neutrophil counts or mean platelet count.
  • FIG. 5A This example illustrates analyses of the antiviral activity of 3TC during prolonged treatment of chronically infected woodchucks.
  • 3TC resulted in a reduction in mean serum WHV DNA (Figure 5A) first noticed at 2 weeks after initiation of treatment.
  • Figure 5B depicts the same data converted to genomic equivalents/ml of serum based upon a calculated weight for one WHV genome of 3.09 x 10 18 g.
  • a continued decrease in serum WHV DNA was observed in the 3TC treatment group until week 12 by which time the mean serum WHV DNA level had fallen from a pretreatment level of 142 ng/ml to 7.22 ng/ml, representing an approximately 1.5 log reduction compared to pretreatment and control levels .
  • WHV DNA was isolated from serum and analyzed by nucleic acid amplification for the presence of specific mutations known to occur in the Hepatitis viral DNA polymerase gene (e.g. in the portion encoding the polymerase YMDD motif) . These techniques detected only wild-type sequence with no evidence of mutations known to confer resistance to 3TC.
  • WHV DNA was isolated from serum and analyzed by nucleic acid amplification for the presence of specific mutations known to occur in the hepatitis viral DNA polymerase gene. These techniques failed to identify at 46-54 weeks, any mutations known to confer in vivo resistance to 3TC. However, at 80 weeks, four samples from the 3TC treated group contained a mutation at the amino acid position 565 of the WHV DNA polymerase. There were no mutations at this position in any of the placebo group.
  • This example illustrates an evaluation as to whether prolonged treatment of chronically infected woodchucks with 3TC affects the development of hepatocellular carcinoma in the treated individuals.
  • Transabdominal ultrasound was performed at bimonthly intervals, beginning at week 24, in order to identify and monitor the development of hepatocellular carcinoma in the placebo group and treatment group during the study period.
  • 3TC had a significant effect on the course of hepatic disease and the development of hepatocellular carcinoma.
  • a total of 11 animals in the placebo group had developed ultrasonographic lesions consistent with a diagnosis of hepatocellular carcinoma by week 72.
  • 3TC lacks the potent cytotoxicity that is characteristic of potential anticancer agents such as L(-)-OddC.
  • the mean values for the assessed hematological parameters appeared normal, e.g., evidence of the lack of toxicity of 3TC.
  • viral resurgence occurs during prolonged treatment with 3TC to levels that reflect chronic persistence of the virus despite continued 3TC treatment .
  • Chronic persistent hepatitis is associated with the development of HCC. Yet despite this chronic persistence of the virus during 3TC treatment, there is a statistically significant reduction in the development and growth of HCC in individuals receiving prolonged treatment with 3TC. It is unexpected that 3TC would inhibit the development of HCC in these individuals in view of the persisting viral titers.

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Abstract

L'invention concerne un procédé d'inhibition du développement et de la croissance du carcinome hépatocellulaire chez un sujet infecté de manière chronique par le virus de l'hépatite B. Ledit procédé consiste à administrer de manière prolongée une dose efficace sur le plan prophylactique de 3TC au sujet infecté de manière chronique. De plus, il consiste à administrer du 3TC de manière prolongée conjointement avec un autre agent antiviral ayant une activité dirigée contre le virus de l'hépatite B.
PCT/US1998/019983 1997-09-25 1998-09-24 Procede d'inhibition du developpement du cancer du foie et de l'augmentation de la survivance de l'infection a virus adn de l'hepatite chronique WO1999015175A1 (fr)

Priority Applications (3)

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EP98949470A EP1023071A4 (fr) 1997-09-25 1998-09-24 Procede d'inhibition du developpement du cancer du foie et de l'augmentation de la survivance de l'infection a virus adn de l'hepatite chronique
CA002309357A CA2309357A1 (fr) 1997-09-25 1998-09-24 Procede d'inhibition du developpement du cancer du foie et de l'augmentation de la survivance de l'infection a virus adn de l'hepatite chronique
AU95786/98A AU738472B2 (en) 1997-09-25 1998-09-24 Method for inhibiting development of liver cancer and increasing survival in chronic hepadnavirus infection

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US93770497A 1997-09-25 1997-09-25
US08/937,704 1997-09-25

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030329A2 (fr) * 1999-10-29 2001-05-03 Glaxo Group Limited Combinaisons antivirales

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts Service (C A S); 1 January 1900 (1900-01-01), XP002915270, Database accession no. 124-164383 *
GANEM D.: "HEPADNAVIRIDAE AND THEIR REPLICATION.", FIELDS VIROLOGY, XX, XX, vol. 02., 1 January 1996 (1996-01-01), XX, pages 2703 - 2737., XP002915271 *
See also references of EP1023071A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030329A2 (fr) * 1999-10-29 2001-05-03 Glaxo Group Limited Combinaisons antivirales
WO2001030329A3 (fr) * 1999-10-29 2001-11-01 Glaxo Group Ltd Combinaisons antivirales
JP2003512421A (ja) * 1999-10-29 2003-04-02 グラクソ グループ リミテッド 抗ウィルス複合薬

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EP1023071A1 (fr) 2000-08-02
AU738472B2 (en) 2001-09-20
CN1279610A (zh) 2001-01-10
EP1023071A4 (fr) 2002-05-29
AU9578698A (en) 1999-04-12
CA2309357A1 (fr) 1999-04-01

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