Mechanism of Activation and Liver Targeting of Hepavir B, a Prodrug of Adefovir

[Guest guest]

54th Annual Meeting of the American Association

for the Study of Liver Diseases

October 24 - 28, 2003, Boston, MA

Mechanism of Activation and Liver Targeting of Hepavir B, a Prodrug of

Adefovir

Adefovir dipivoxil (Hepsera), a prodrug of the nucleotide adefovir, has

demonstrated efficacy in naïve and lamivudine-resistant patients without

the development of drug resistance, but its maximal efficacy is limited by

renal toxicity associated with high systemic adefovir exposure.

Hepsera is FDA-approved for treatment of chronic HBV infection.

MB6866 (Hepavir is a HepDirectTM prodrug of adefovir designed to enhance

the therapeutic index of adefovir by targeting it specifically to the

liver.

Ninety-one HepDirectTM prodrugs of adefovir were synthesized and

characterized by means of in vitro microsomal activation assays and in vivo

liver targeting studies (p.o.). The lead prodrug identified, MB6866, was

further evaluated in cellular metabolism, tissue distribution,

pharmacokinetic studies in the rat and/or dog.

Prodrug activation was evaluated either by reverse phase HPLC quantitation

of the adefovir liberated or by reverse phase HPLC quantitation of a

glutathione conjugate of a byproduct of prodrug activation. Adefovir

diphosphate quantitation in perchloric acid extracted and neutralized

cellular or tissue samples was performed by ion exchange HPLC.

Study Results

MB6866 was activated to adefovir in the subcellular microsome fraction of

rat (Vmax = 1.2 nmol/mg/min, Km = 25 µM) and human liver (Vmax = 1.3

nmol/mg/min, Km = 119 µM). Ketoconazole, a cytochrome P450 3A4 selective

inhibitor, blocked its activation (IC50 = 0.5 µM).

Testing across a panel of human, recombinant P450 enzymes confirmed that

MB6866 was activated efficiently and primarily by CYP3A4. In isolated

primary rat hepatocytes, MB6866 was activated to adefovir by the rat enzyme

homolog, CYP3A1/2, and subsequently phosphorylated to its active

metabolite, adefovir diphosphate.

Consistent with the in vitro activation studies, liver activation of MB6866

in the rat was associated with the generation of a vinylketone byproduct,

which was eliminated following its rapid conjugation with glutathione.

Oral bioavailability of MB6866 was 20% in the rat and 82% in the dog, based

on urinary excretion of prodrug and metabolites. Radiolabeled tissue

distribution studies in the rat versus adefovir dipivoxil (at 30 mg/kg

adefovir equivalents, p.o.) showed that MB6866 enhanced the delivery of

adefovir and its metabolites to the liver by 3.1-fold (AUC0-24h) while

reducing kidney exposure of adefovir and metabolites by 3.8-fold

(AUC0-24h).

Mass balance studies in the rat (3H-MB6866) indicated that both biliary

clearance and renal clearance contributed equally to the clearance of

MB6866. Four-day repeat dose studies with MB6866 (31, 103.7, 311 mg/kg)

demonstrated dose-dependent formation of adefovir diphosphate in liver and

indicated that the liver/kidney targeting index of approximately 12-fold

was maintained throughout the treatment period.

Conclusions

(1) MB6866 is activated by an abundant hepatic P450 enzyme, CYP3A4,

which forms the basis of its liver selectivity profile;

(2) MB6866 had a >10-fold improved liver/kidney targeting index

relative to adefovir dipivoxil;

(3) The vinylketone byproduct of MB6866 activation was eliminated

following glutathione conjugation.

In HBV patients, MB6866 is expected to increase the therapeutic index of

adefovir and thereby enable improved antiviral activity relative to

adefovir dipivoxil therapy.

11/05/03

Reference

PD van Poelje and others. MB6866 (HEPAVIR , A HEPDIRECTTM PRODRUG OF

ADEFOVIR: MECHANISM OF ACTIVATION AND LIVER TARGETING. Abstract 1143

(poster). 54th Annual Meeting of the American Association for the Study of

Liver Diseases. October 24-28, 2003. Boston, MA.

[Guest guest]

54th Annual Meeting of the American Association

for the Study of Liver Diseases

October 24 - 28, 2003, Boston, MA

Mechanism of Activation and Liver Targeting of Hepavir B, a Prodrug of

Adefovir

Adefovir dipivoxil (Hepsera), a prodrug of the nucleotide adefovir, has

demonstrated efficacy in naïve and lamivudine-resistant patients without

the development of drug resistance, but its maximal efficacy is limited by

renal toxicity associated with high systemic adefovir exposure.

Hepsera is FDA-approved for treatment of chronic HBV infection.

MB6866 (Hepavir is a HepDirectTM prodrug of adefovir designed to enhance

the therapeutic index of adefovir by targeting it specifically to the

liver.

Ninety-one HepDirectTM prodrugs of adefovir were synthesized and

characterized by means of in vitro microsomal activation assays and in vivo

liver targeting studies (p.o.). The lead prodrug identified, MB6866, was

further evaluated in cellular metabolism, tissue distribution,

pharmacokinetic studies in the rat and/or dog.

Prodrug activation was evaluated either by reverse phase HPLC quantitation

of the adefovir liberated or by reverse phase HPLC quantitation of a

glutathione conjugate of a byproduct of prodrug activation. Adefovir

diphosphate quantitation in perchloric acid extracted and neutralized

cellular or tissue samples was performed by ion exchange HPLC.

Study Results

MB6866 was activated to adefovir in the subcellular microsome fraction of

rat (Vmax = 1.2 nmol/mg/min, Km = 25 µM) and human liver (Vmax = 1.3

nmol/mg/min, Km = 119 µM). Ketoconazole, a cytochrome P450 3A4 selective

inhibitor, blocked its activation (IC50 = 0.5 µM).

Testing across a panel of human, recombinant P450 enzymes confirmed that

MB6866 was activated efficiently and primarily by CYP3A4. In isolated

primary rat hepatocytes, MB6866 was activated to adefovir by the rat enzyme

homolog, CYP3A1/2, and subsequently phosphorylated to its active

metabolite, adefovir diphosphate.

Consistent with the in vitro activation studies, liver activation of MB6866

in the rat was associated with the generation of a vinylketone byproduct,

which was eliminated following its rapid conjugation with glutathione.

Oral bioavailability of MB6866 was 20% in the rat and 82% in the dog, based

on urinary excretion of prodrug and metabolites. Radiolabeled tissue

distribution studies in the rat versus adefovir dipivoxil (at 30 mg/kg

adefovir equivalents, p.o.) showed that MB6866 enhanced the delivery of

adefovir and its metabolites to the liver by 3.1-fold (AUC0-24h) while

reducing kidney exposure of adefovir and metabolites by 3.8-fold

(AUC0-24h).

Mass balance studies in the rat (3H-MB6866) indicated that both biliary

clearance and renal clearance contributed equally to the clearance of

MB6866. Four-day repeat dose studies with MB6866 (31, 103.7, 311 mg/kg)

demonstrated dose-dependent formation of adefovir diphosphate in liver and

indicated that the liver/kidney targeting index of approximately 12-fold

was maintained throughout the treatment period.

Conclusions

(1) MB6866 is activated by an abundant hepatic P450 enzyme, CYP3A4,

which forms the basis of its liver selectivity profile;

(2) MB6866 had a >10-fold improved liver/kidney targeting index

relative to adefovir dipivoxil;

(3) The vinylketone byproduct of MB6866 activation was eliminated

following glutathione conjugation.

In HBV patients, MB6866 is expected to increase the therapeutic index of

adefovir and thereby enable improved antiviral activity relative to

adefovir dipivoxil therapy.

11/05/03

Reference

PD van Poelje and others. MB6866 (HEPAVIR , A HEPDIRECTTM PRODRUG OF

ADEFOVIR: MECHANISM OF ACTIVATION AND LIVER TARGETING. Abstract 1143

(poster). 54th Annual Meeting of the American Association for the Study of

Liver Diseases. October 24-28, 2003. Boston, MA.

[Guest guest]

54th Annual Meeting of the American Association

for the Study of Liver Diseases

October 24 - 28, 2003, Boston, MA

Mechanism of Activation and Liver Targeting of Hepavir B, a Prodrug of

Adefovir

Adefovir dipivoxil (Hepsera), a prodrug of the nucleotide adefovir, has

demonstrated efficacy in naïve and lamivudine-resistant patients without

the development of drug resistance, but its maximal efficacy is limited by

renal toxicity associated with high systemic adefovir exposure.

Hepsera is FDA-approved for treatment of chronic HBV infection.

MB6866 (Hepavir is a HepDirectTM prodrug of adefovir designed to enhance

the therapeutic index of adefovir by targeting it specifically to the

liver.

Ninety-one HepDirectTM prodrugs of adefovir were synthesized and

characterized by means of in vitro microsomal activation assays and in vivo

liver targeting studies (p.o.). The lead prodrug identified, MB6866, was

further evaluated in cellular metabolism, tissue distribution,

pharmacokinetic studies in the rat and/or dog.

Prodrug activation was evaluated either by reverse phase HPLC quantitation

of the adefovir liberated or by reverse phase HPLC quantitation of a

glutathione conjugate of a byproduct of prodrug activation. Adefovir

diphosphate quantitation in perchloric acid extracted and neutralized

cellular or tissue samples was performed by ion exchange HPLC.

Study Results

MB6866 was activated to adefovir in the subcellular microsome fraction of

rat (Vmax = 1.2 nmol/mg/min, Km = 25 µM) and human liver (Vmax = 1.3

nmol/mg/min, Km = 119 µM). Ketoconazole, a cytochrome P450 3A4 selective

inhibitor, blocked its activation (IC50 = 0.5 µM).

Testing across a panel of human, recombinant P450 enzymes confirmed that

MB6866 was activated efficiently and primarily by CYP3A4. In isolated

primary rat hepatocytes, MB6866 was activated to adefovir by the rat enzyme

homolog, CYP3A1/2, and subsequently phosphorylated to its active

metabolite, adefovir diphosphate.

Consistent with the in vitro activation studies, liver activation of MB6866

in the rat was associated with the generation of a vinylketone byproduct,

which was eliminated following its rapid conjugation with glutathione.

Oral bioavailability of MB6866 was 20% in the rat and 82% in the dog, based

on urinary excretion of prodrug and metabolites. Radiolabeled tissue

distribution studies in the rat versus adefovir dipivoxil (at 30 mg/kg

adefovir equivalents, p.o.) showed that MB6866 enhanced the delivery of

adefovir and its metabolites to the liver by 3.1-fold (AUC0-24h) while

reducing kidney exposure of adefovir and metabolites by 3.8-fold

(AUC0-24h).

Mass balance studies in the rat (3H-MB6866) indicated that both biliary

clearance and renal clearance contributed equally to the clearance of

MB6866. Four-day repeat dose studies with MB6866 (31, 103.7, 311 mg/kg)

demonstrated dose-dependent formation of adefovir diphosphate in liver and

indicated that the liver/kidney targeting index of approximately 12-fold

was maintained throughout the treatment period.

Conclusions

(1) MB6866 is activated by an abundant hepatic P450 enzyme, CYP3A4,

which forms the basis of its liver selectivity profile;

(2) MB6866 had a >10-fold improved liver/kidney targeting index

relative to adefovir dipivoxil;

(3) The vinylketone byproduct of MB6866 activation was eliminated

following glutathione conjugation.

In HBV patients, MB6866 is expected to increase the therapeutic index of

adefovir and thereby enable improved antiviral activity relative to

adefovir dipivoxil therapy.

11/05/03

Reference

PD van Poelje and others. MB6866 (HEPAVIR , A HEPDIRECTTM PRODRUG OF

ADEFOVIR: MECHANISM OF ACTIVATION AND LIVER TARGETING. Abstract 1143

(poster). 54th Annual Meeting of the American Association for the Study of

Liver Diseases. October 24-28, 2003. Boston, MA.

[Guest guest]

54th Annual Meeting of the American Association

for the Study of Liver Diseases

October 24 - 28, 2003, Boston, MA

Mechanism of Activation and Liver Targeting of Hepavir B, a Prodrug of

Adefovir

Adefovir dipivoxil (Hepsera), a prodrug of the nucleotide adefovir, has

demonstrated efficacy in naïve and lamivudine-resistant patients without

the development of drug resistance, but its maximal efficacy is limited by

renal toxicity associated with high systemic adefovir exposure.

Hepsera is FDA-approved for treatment of chronic HBV infection.

MB6866 (Hepavir is a HepDirectTM prodrug of adefovir designed to enhance

the therapeutic index of adefovir by targeting it specifically to the

liver.

Ninety-one HepDirectTM prodrugs of adefovir were synthesized and

characterized by means of in vitro microsomal activation assays and in vivo

liver targeting studies (p.o.). The lead prodrug identified, MB6866, was

further evaluated in cellular metabolism, tissue distribution,

pharmacokinetic studies in the rat and/or dog.

Prodrug activation was evaluated either by reverse phase HPLC quantitation

of the adefovir liberated or by reverse phase HPLC quantitation of a

glutathione conjugate of a byproduct of prodrug activation. Adefovir

diphosphate quantitation in perchloric acid extracted and neutralized

cellular or tissue samples was performed by ion exchange HPLC.

Study Results

MB6866 was activated to adefovir in the subcellular microsome fraction of

rat (Vmax = 1.2 nmol/mg/min, Km = 25 µM) and human liver (Vmax = 1.3

nmol/mg/min, Km = 119 µM). Ketoconazole, a cytochrome P450 3A4 selective

inhibitor, blocked its activation (IC50 = 0.5 µM).

Testing across a panel of human, recombinant P450 enzymes confirmed that

MB6866 was activated efficiently and primarily by CYP3A4. In isolated

primary rat hepatocytes, MB6866 was activated to adefovir by the rat enzyme

homolog, CYP3A1/2, and subsequently phosphorylated to its active

metabolite, adefovir diphosphate.

Consistent with the in vitro activation studies, liver activation of MB6866

in the rat was associated with the generation of a vinylketone byproduct,

which was eliminated following its rapid conjugation with glutathione.

Oral bioavailability of MB6866 was 20% in the rat and 82% in the dog, based

on urinary excretion of prodrug and metabolites. Radiolabeled tissue

distribution studies in the rat versus adefovir dipivoxil (at 30 mg/kg

adefovir equivalents, p.o.) showed that MB6866 enhanced the delivery of

adefovir and its metabolites to the liver by 3.1-fold (AUC0-24h) while

reducing kidney exposure of adefovir and metabolites by 3.8-fold

(AUC0-24h).

Mass balance studies in the rat (3H-MB6866) indicated that both biliary

clearance and renal clearance contributed equally to the clearance of

MB6866. Four-day repeat dose studies with MB6866 (31, 103.7, 311 mg/kg)

demonstrated dose-dependent formation of adefovir diphosphate in liver and

indicated that the liver/kidney targeting index of approximately 12-fold

was maintained throughout the treatment period.

Conclusions

(1) MB6866 is activated by an abundant hepatic P450 enzyme, CYP3A4,

which forms the basis of its liver selectivity profile;

(2) MB6866 had a >10-fold improved liver/kidney targeting index

relative to adefovir dipivoxil;

(3) The vinylketone byproduct of MB6866 activation was eliminated

following glutathione conjugation.

In HBV patients, MB6866 is expected to increase the therapeutic index of

adefovir and thereby enable improved antiviral activity relative to

adefovir dipivoxil therapy.

11/05/03

Reference

PD van Poelje and others. MB6866 (HEPAVIR , A HEPDIRECTTM PRODRUG OF

ADEFOVIR: MECHANISM OF ACTIVATION AND LIVER TARGETING. Abstract 1143

(poster). 54th Annual Meeting of the American Association for the Study of

Liver Diseases. October 24-28, 2003. Boston, MA.