Prodrugs of Nucleoside and Nucleotide Analogs for the Treatment of HCV:

An Overview of Concepts and Chemistry

Michael J. Sofia, Ph.D. Gilead Sciences

7th International Workshop on Hepatitis C – Resistance and New Compounds

Cambridge, MA June 28-29, 2012

What is a Prodrug?

• A compound that undergoes biotransformation prior to eliciting its pharmacological effect – The administered therapeutic agent is inactive but is

transformed into one or more active metabolites – Usually implemented to address a deficiency

associated with the active agent • Chemical or biotransformation of a structural molecular

functionality that produces the actual active agent • Attachment of a promoiety to a pharmacologically active

agent such that the prodrug can overcome the barrier that hinders the optimal use of the active agent

Albert, A., Nature, 1958, 182, 421 Bundgaard, H., In A Textbook of Drug Design and Development , Harwood, Reading, UK, 19921, 113

The Prodrug Concept

Biologial B

arrier

Drug

Drug

Promoiety

Drug

Promoiety

Drug Promoiety +

Biotransformation

Non-toxic & rapidly cleared

Key Issues Addressed by Prodrugs

• Bioavailability – Aqueous solubility – Poor passive intestinal absorption - lipophilicity – Leveraging transporter-mediated intestinal absorption

• Peptide transporters • Nucleoside transporters • Ion transporters • Bile acid transporters • Vitamin transporters

– Fast metabolism • Tissue Selectivity (“magic bullet”)

– Passive enrichment – Transporter targeting – Targeting tissue- or cell-specific enzymes – Targeting surface antigens

Marketed Prodrugs

HO

OO

HO O

Lovastatin

O

OO

NH2

HN

O

Oseltamivir

N

OHO

POO

OOO

Fosinopril

N

N N

N

O

P

NH2

O

O OOO

O O

Adefovir Dipivoxil

NH

NN

N

O

O

O

NH2

O

NH2H

Valaciclovir

Guidances for Prodrug Development

• A comprehensive understanding of the ADME/PK of the prodrug and the parent drug is critical.

• Depending on the rate of conversion and the site of metabolism, prodrugs may alter the tissue distribution, efficacy, and toxicity of the parent drug.

• The promoiety should ideally be safe and rapidly excreted from the body.

• The choice of promoiety should be considered with respect to the disease state, dose, and duration of therapy

Unique Characteristics of Nucleos(t)ides

O

HO

Kinase 1 Kinase 2

Kinase 3

BASE

O

HO

HOBASE

O

HO

BASE O

HO

BASE

= Phosphate

X

Y

X X

Y Y

X

Y

The triphosphate metabolite of a nucleoside is the pharmacologically active agent A nucleoside must be a substrate for each of the kinases needed to produce the active triphosphate metabolite The first phosphorylation event is the most substrate selective. The triphosphate must be a substrate for the viral polymerase and it must get incorporated into the growing RNA or DNA chain – eliciting a chain termination event

Polymerase

O O OO O O

OOOO O

Ade CytUraGua

3'

5'

AdeUraGuaAde UraUraAde

O

HO

BASE

O

Ade

ChainTermination

Y

XHO HO HO HO HO

OHOHOHOHOHOHOH

RNA

O

HO

OBASE

XY

PO

O-OP

O

O-OP

O-

-OO

Poor Drug-like Characteristics of Nucleos(t)ides

• Nucleosides: – Highly polar – Require multiple enzyme mediated steps for activation – Can be enzymatically degraded – nucleosidases &

nucleobase metabolizing enzymes • Nucleotides

– Highly polar – Chemically unstable – Require enzyme mediated activation – Can be enzymatically degraded – nucleosidases,

nucleobase metabolizing enzymes and phosphatases

Ideal Characteristics of a Nucleos(t)ide Prodrug for the Treatment of Hepatitis

• Must have sufficient chemical stability and physical properties to be formulated for oral administration

• Must be stable to conditions of the gastrointestinal tract such that the prodrug reaches the site of absorption intact

• Prodrug must have good absorption properties • Prodrug must not undergo appreciable enzymatic degradation

during the absorption phase • Nucleotides: Once absorbed the prodrug must have sufficient

stability in the blood in order to reach the target organ • Nucleotides: The prodrug must be transported into hepatocytes

and release the free 5’-phosphorylated nucleoside • Achieving a high liver-plasma ratio (liver targeting) is desirable. • The intact prodrug must be biologically inert • The promoiety byproducts should be non-toxic and rapidly

cleared.

Issues Addressed by Nucleoside Prodrugs

• Poor oral bioavailability • Formation of inactive metabolites

O

YHO

BaseHOZ X

Nucleoside Prodrugs: NM283 (Valopicitabine)

O

OHHO

NHON

O

NH2

NM107(EC50 = 1.23 µM)

O

OHO

NHON

O

NH2

O

NH2

NM 283 Valopicitabine

Issues • Poor oral bioavailability in rat • Formation of inactive uridine metabolite

Results • Leveraged peptide transporter • %F = 33% (rat) • No detection of uridine metabolite • Human studies

• 800 mg BID, 14 days gave -1.2 log10 IU/mL reduction in viral load • 800 mg BID + SOC, 28 days ->4 log10 IU/mL reduction in viral load • terminated for GI tox Pierra, C, et al, JMed Chem, 2006, 49, 6614

Nucleoside Prodrugs: RG7128 (Mericitabine)

O

FHO

NHON

O

NH2

PSI-6130 (EC90 = 4.5 µM)

O

FO

NON

O

NH2O

O

RG7128 Mericitabine

Issues • Modest oral bioavailability in humans (~25%) • Formation of inactive uridine metabolite in significant amounts • Liver to plasma ratio ~1

Results • Human oral bioavailablity increased to ~75% • Reduction in formation of uridine metabolite • No improvement in liver to plasma ratio • Human studies

• 1500 mg BID, 14 days, -2.7 log10 IU/mL reduction in viral load • 1000 mg BID + SOC, 28 days, -5 log10 IU/mL reduction in viral load

Sofia,M., et al., J. Med. Chem., 2012, 55, 2481

Nucleoside Prodrugs: R1626 (Balapiravir)

O

OHHO

NHON

O

NH2

N3

O

OO

NON

O

NH2

N3

O

O

O

R1479 (EC50 = 1.28 µM)

R1626 Balapiravir

Issues • Poor animal oral bioavailability • Poor exposure in humans

Results • %F >90% dog • >5-fold increase in oral bioavailability in rat and monkey • Human studies

•4500 mg BID, 14 days, -3.7 log10 reduction in viral load • 1500 mg BID + SOC, 28 days, -5.2 log10 reduction in viral load 81% RVR • Terminated do to hematological tox

Toniutto, P., et al., IDRUGS, 2008, 11, 738

Nucleoside Prodrugs: TMC647078

O

R2O

NR1ON

O

NH2

TMC647078 (EC50 = 7.3 µM)

O

HO

NHON

O

NH2

Issues • Exposure in animals poor • Formation of inactive uridine metabolite

OR1 and/or R2 =

Results • 3’-Monoester 10-fold increase in animal exposure • Diester 24-fold increase in animal exposure • Liver to plasma ratio ~1

Jonckers, T.H., et al., J. Med. Chem., 2010, 53, 8150

Issues Addressed by Nucleotide Prodrugs

O

YHO

BaseOZ X

PO

-OO-

• Highly polar – do not penetrate biological membranes • Chemically unstable • Enzymatically degraded

Kinase Bypass

OBaseHO

X

YOH

OBaseO

X

YOH

PO

HOOH

OBaseO

X

YOH

PO

OOH

PO

OHHO

OBaseO

X

YOH

PO

OOH

PO

OHOP

O

OHHO

OBaseO

X

YOH

PO

X Bypass the non-productive phosphorylation step

Phosphoramidates

PO

O

NH

OH

O R2

Ar

PO

O

HNR2

O

PO

O

NH

OH

O R2

H

PO

O

HO

H

OPO

O

NH

OR1

O R2

Ar

Nucleoside NucleosideO

NucleosideO

NucleosideO NucleosideO

Carboxyesterase

orCathepsin A

HINT-1

H2Ospontaneous

HepDirect

NucleosideOOP

OO

Ar cytochrome P450NucleosideO

OP

OO

Ar OH

spontaneous NucleosideOOP

HOO

Ar

O

spontaneous

NucleosideOOHPHOO

Ar

O

Designed for liver targeting

SATE

NucleosideOPO

OO

S

S

R

R

O

O

NucleosideOPO

OO

S

HS

R

O

esterase

SNucleosideOP

OO

HO

SR

O

S

esterase

NucleosideOPO

HOHO

Cyclic Phosphates

O

X

YO

BASEO

PO

O

O

X

YO

BASEO

PO

NH

EtO O

O

X

YO

BASEO

PO

OSO

O

n-Pr

O

X

YO

BASEO

PO

O

O

X

YO

BASEO

PO

HO

CYP450

CYP3A4

PDE

O

X

YHO

BASEOPO

OHHO

Double Prodrugs

O

YHOX

NO

N

NN

OR

NH2

PO

O

YHOX

NO

N

NN

OR

NH2

PO

HOOH

O

YHOX

NO

N

NNH

NH2

PO

HOOH

ORemoval of phosphate

promoiety ADAL1

Improves overall lipophilicity thus improving cellular uptake

Murakami, E., et al., J. Med. Chem., 2011, 54, 5902

Nucleotide Phosphoramidates: GS-7977

O

FHO

NONH

O

O

PO

ONHO

O

GS-7977 (PSI-7977) EC90 = 0.42 µM)

O

FHO

NHONH

O

O

PSI-6206

Issues • Nucleoside inactive in replicon • TP potent inhibitor of NS5B • Long TP half-life in hepatocytes • Not a substrate for DCK • MP readily converted to TP

Results • Potent inhibitor of HCV replication • High TP levels in hepatocytes in vitro • High TP levels in liver on oral admin. • High liver to plasma ratio – leverages first pass metabolism for liver targeting • Human studies

• Safe and well tolerated to 24 wks • 400 mg QD, 14 days,~5.0 log10 IU/mL reduction in viral load • + RBV, GT2,3 100% SVR12 • +Daclatasvir, GT1, 100% SVR12 Sofia, M.J., et al., J. Med. Chem., 2010, 53, 7202.

Lam, A., et al., ACC, 2010, 54, 3187.

Nucleotide Phosphoramidates: PSI-353661

O

FHO

NHO

N

NNH

O

NH2

O

FHO

NO

N

NN

OMe

NH2

PO

ONHO

O

PSI-353661 (EC90 = 0.008 µM)

(EC90 = 69.2 µM)

Issues • Weak replicon potency • TP is potent inhibitor of NS5B, IC50 = 5.9 µM • Polar molecule • Weak kinase substrate

Results • Potent inhibitor of HCV replication • Conversion to the G-MP derivative occurs readily in hepatocytes • Produced high levels of G-TP in human hepatocytes • Produced a favorable ~5:1 liver to plasma ratio in vivo - leverages first pass metabolism for liver targeting

Chang, W., et al., ACS Med Chem Lett., 2011,2, 130 Furman, P., et al., Antiviral Res., 2011, 91, 120

Nucleotide Phosphoramidates: INX-8189

O

OHHO

NO

N

NN

OCH3

NH2

PO

OHN

O

O

CH3

INX-8189 (EC50 = 0.01 µM)

O

OHHO

NHO

N

NNH

O

NH2

(EC50 = 3.5 µM)

Issues • Low levels of TP detected in cells • G-TP potent inhibitor: IC50 = 0.13 µM • Natural guanosine derivative poor cellular uptake

Results • > 100-fold improvement in replicon potency • Conversion to the G-MP derivative occurs readily in hepatocytes • Substantial levels of G-TP produced in cells. • Human studies

• Monotherapy: 25 mg QD, -1.3 log10 IU/mL decline in viral load

Vernachio, J.H., et al., ACC, 2011, 55, 1843

Nucleotide Phosphoramidates: IDX184

O

OHHO

NO

N

NNH

O

NH2

PO

OHN

SO

OHIDX184

(EC50 = 0.4 µM)

O

OHHO

NHO

N

NNH

O

NH2

(EC50 = 3.5 µM)

Issues • Low levels of TP detected in cells • G-TP potent inhibitor: IC50 = 0.13 µM • Natural guanosine derivative poor cellular uptake

Results • P450 dependent and independent cleavage • High liver TP levels observed in cynomolgus monkeys on PO dosing • 10 mg/kg PO dose to HCV infected chimpanzees resulted in -2.3 log10 IU/mL decline in viral load after 3 days • Human studies

• 25-100mg QD , 3days, max -0.74 log10 IU/mL decline in viral load • 50 – 200 mg QD + SOC, 14 days, -2.7 to -4.1 log10 decline in viral load

Zhou, X.J., et al., AAC, 2011, 55, 76

Cyclic Phosphates: PSI-352938

O

FO

NO

N

NN

OEt

NH2PO

O

PSI-352938 (EC90 = 1.37 µM)

O

FHO

NHO

N

NNH

O

NH2

(EC90 = 69.2 µM)

Issues • Weak replicon potency • TP is potent inhibitor of NS5B, IC50 = 5.9 µM • Polar molecule • Weak kinase substrate

Results • High live G-TP levels in rat and dog after PO dosing • Observe circulating levels of intact prodrug • CYP3A4 mediated cleavage mech. • Human studies

• 100 – 300 mg QD, 7 days, -4.0 -4.6 log10 IU/mL decline in viral load • 300 mg, QD, +SOC, 14 days gave -5.5 log10 IU/mL viral load decline • Clinical hold – liver enzyme elevation Reddy, G.P., et al., BMCL, 2010, 20, 7376

Lam, A., et al., J. Virol., 2011, 85, 12334

HepDirect:

O

OHHO

NHON

O

NH2

O

OHHO

NOPO

O

O

F

F N

NH2

O

(EC50 = 1.23 µM)

Issues • Poor oral bioavailability in rat • Low levels of TP after i.p. dosing • Formation of inactive uridine metabolite

Results • 10-fold increase in TP levels on i.p. dosing • HCV-infected chimpanzees

• 10 mg/kg QD, -1.3 to -1.5 log10 IU/mL decline in viral load vs -1.0 log10 IU/mL decline with 16 mg/kg NM283

Carroll, S.S., et al., AAC, 2011, 55, 3854

Other Nucleotide Prodrugs

O

OHHO

NON

O

NH2

N3

PO

ONH

CH3

O

OPhO

OHHO

NONH

O

O

N3

PO

ONHO

OPh

Sofia, M.J., et al., J. Med. Chem., 2012, 55, 2481

O

OHHO

NON

O

NH2

PO

ONHO

O O

XY

NOPO

O

O

S

S

O

O

N

NH2

O

O

OHO

NON

O

NH2

PO

NH

EtO O

O

OHO

NO

N

NN

N

PO

O

HN SO2CH3

SO

O

n-Pr

O

OO

NO

N

NN

NH2

PO

O

O

Cl

O

N

N N

N

NH2

O P

HO

O

OHO (CH2)2O(CH2)17CH3

Developing a Prodrug: In Vitro and In Vivo Characterization

• Physico-chemical characterization – solubility, chemical stability, and lipophilicity

• Hepatic/microsomal/tissue homogenate/ intestinal fluid/ plasma stability

• Caco-2 and PAMPA permeability • Absorption mechanism(s) • Are the active drug or prodrug substrates for efflux

pumps? • Understand the disposition of both the active parent

and prodrug compounds

Summary

The application of a prodrug strategy is a well established approach to improve the physicochemical, biopharmaceutical or pharmacokinetic properties of a pharmacologically potent compound increasing its developability. In no therapeutic field has the application of prodrug technology seen as wide and impactful an application as has been seen in the development of nucleoside and nucleotide HCV therapies

References

• Prodrugs: Challenges and Rewards, Parts I and II, Biotechnology: Pharmaceutical Aspects, Vol 5, Editors: Stella, V., et al., Publisher: Springer

• Prodrugs: Design and Clinical Applications, Rautio, J., et al., Nature Reviews Drug Discovery, 2008, 7, 255.

• Prodrugs: Some Thoughts and Current Issues, Stella, V., J. Pharma. Sci., 2010, 99, 4755.

• Lessons Learned from Marketed and Investigational Prodrugs, Ettmayer, P., et al., J. Med. Chem., 2004, 47, 2393.

• Prodrugs of Phosphates and Phosphonates, Hecker, S., et al., J. Med. Chem., 2008, 51, 2328.

• Nucleotide Prodrugs for HCV Therapy, Sofia, M.J., Antiviral Chem and Chemother., 2011, 22, 23.

• Nucleoside, Nucleotide, and Non-Nucleoside Inhibitors of Hepatitis C Virus NS5B RNA-Dependent RNA-Polymerase, Sofia, M.J., et al., J. Med. Chem., 2012, 55, 2481.