Liver targeting of hepatitis-B antiviral lamivudine using the HepDirect prodrug technology

Article abstract of DOI:10.1081/NCN-200059781

A new class of phosphate and phosphonate prodrugs, called HepDirect prodrugs, has been developed to deliver drugs to the liver while simultaneously diminishing drug exposure to extra-hepatic tissues. The technology combines liver-selective cleavage and ki

Full text of DOI:10.1081/NCN-200059781

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LIVER TARGETING OF HEPATITIS-B ANTIVIRAL
LAMIVUDINE USING THE HEPDIRECT™ PRODRUG
TECHNOLOGY
K. Raja Reddy a , Timothy J. Colby a , James M. Fujitaki a , Paul D. van Poelje a & Mark D.
Erion a
a Departments of Chemistry and Biochemistry , Metabasis Therapeutics, Inc. , San Diego,
California, USA
Published online: 15 Nov 2011.
To cite this article: K. Raja Reddy , Timothy J. Colby , James M. Fujitaki , Paul D. van Poelje & Mark D. Erion (2005) LIVER
TARGETING OF HEPATITIS-B ANTIVIRAL LAMIVUDINE USING THE HEPDIRECT™ PRODRUG TECHNOLOGY, Nucleosides, Nucleotides
and Nucleic Acids, 24:5-7, 375-381, DOI: 10.1081/NCN-200059781
To link to this article: http://dx.doi.org/10.1081/NCN-200059781
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):375–381, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059781
LIVER TARGETING OF HEPATITIS-B ANTIVIRAL LAMIVUDINE USING
THE HEPDIRECTTM PRODRUG TECHNOLOGY
K. Raja Reddy, Timothy J. Colby, James M. Fujitaki, Paul D. van Poelje, and
5
Mark D. Erion
Departments of Chemistry and Biochemistry, Metabasis Therapeutics, Inc.,
San Diego, California, USA
A new class of phosphate and phosphonate prodrugs, called HepDirectTM prodrugs, has been
5
developed to deliver drugs to the liver while simultaneously diminishing drug exposure to extra-hepatic
tissues. The technology combines liver-selective cleavage and kinase by pass with high plasma and tissue
stability to achieve increased drug levels in the liver. Lamivudine (LMV), a nucleoside analogue, is a
currently approved treatment for hepatitis B infection, but shows modest efficacy and significant drug
resistance due to inefficient phosphorylation. LMV is inadequately phosphorylated to the corresponding
nucleoside triphosphate in rat and human hepatocytes. A HepDirect prodrug of LMV monophosphate
generated 34-fold higher levels of the triphosphate in rat hepatocytes and 320-fold higher triphosphate
levels in the liver of treated rats relative to LMV.
INTRODUCTION
Hepatitis B virus (HBV) infection is a serious global health problem with
2 billion people infected worldwide, and 350 million suffering from chronic HBV
infection. HBV infections result in 500,000 to 1.2 million deaths per year.[1] The
three currently approved treatments for HBV, interferon-a, lamivudine (LMV), and
adefovir dipivoxil, are inadequate. Interferon-a has a treatment benefit in only
approximately 20% of patients,[2] LMV therapy is associated with modest efficacy
(2.5–3 log HBV DNA reduction)[3] and significant drug resistance,[4] while the
therapeutic potential of adefovir dipivoxil[5] is limited by renal toxicity.[6]
The modest efficacy of LMV and associated drug resistance may be partially
explained by the poor conversion of LMV to the corresponding triphosphate.
Phosphorylation of LMV to the triphosphate (LMV-TP) is a requirement for the
inhibition of the target enzyme, HBV DNA polymerase.[7] L-Nucleoside analogs like
LMV are known to be poor substrates for deoxycytidine kinase, the enzyme
believed to be responsible for the conversion of LMV to its monophosphate.
Address correspondence to K. Raja Reddy, Departments of Chemistry and Biochemistry, Metabasis
Therapeutics, Inc., 9390 Towne Centre Drive, Building 300, San Diego, CA 92121, USA
375
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376
K. Raja Reddy et al.
FIGURE 1 Activation of HepDirect prodrugs.
HepDirectTM prodrugs represent a new class of phosphate and phosphonate
prodrugs that selectively deliver drugs to the liver.[8] HepDirect prodrugs are
resistant to esterase cleavage and therefore are stable in blood and most tissues. In
the liver, HepDirect prodrugs undergo oxidative cleavage by the cytochrome P450
isoenzyme CYP3A to produce the corresponding nucleoside monophosphate
(NMP), which is rapidly converted to the active nucleoside triphosphate (NTP) by
nucleotide kinases.[9] Activation of the HepDirect prodrugs thus bypasses the need
for the initial, oftentimes rate-limiting, phosphorylation of nucleosides to NMPs.
The objective of the work described herein was to apply the HepDirect prodrug
technology to LMV and thereby improve its conversion to LMV-TP in liver and
provide a more efficacious therapy for HBV.
PRODRUG DESIGN AND MECHANISM OF ACTIVATION
HepDirect prodrugs are cyclic-1,3-propanyl esters substituted with groups that
promote an oxidative cleavage reaction. Oxidation of the C4-methine via a CYP3A-
catalyzed reaction results in ring-opening to produce an intermediate that
undergoes a beta-elimination to the NMP. The NMP subsequently undergoes
further phosphorylation by nucleotide kinases to form the active drug metabolites
(Figure 1). The resulting aryl vinyl ketone byproduct undergoes conjugation with
liver-abundant glutathione (GSH).
SYNTHESIS
Synthesis of the LMV HepDirect prodrug containing a 4-aryl substituent started
from the highly reactive P(III) reagent 3. 1-Aryl-propane-1,3-diol 2 was synthesized
via an aldol condensation involving 3-chlorobenzaldehyde and the enolate of ethyl
acetate generated by LDA. The resulting b-hydroxy ester[12] was reduced with
LAH to yield 3(3-chlorophenyl)-propane-1,3-diol (2). Commercially available di-
isopropylphosphoramidous dichloride was reacted with diol 2 in the presence of

K. Raja Reddy et al.
377
SCHEME 1
triethylamine to give a stable 6-membered ring phosphoramidite 3. The
phosphoramidite was purified by a quick filtration through a silica gel column
and was distilled under vacuum to give pure 3 as a low melting solid.
LMV[13] (4) was reacted with phosphoramidite 3 in the presence of 5-
(methylthio)-1H-tetrazole in DMF to result in the corresponding phosphite.[14] The
intermediate phosphite was then oxidized in situ using a 5-6M tBuOOH solution in
heptane at low temperature (À40°C) to avoid decomposition from the resulting
exothermic oxidation reaction to give a diastereomeric mixture of oxidized
products (5 and 6).[15] Diastereomers at the phosphorus center were isolated by
column chromatography to give 70% overall yield of 5 and 6 from 4. The
stereochemistry of 5 and 6 was assigned based on the known 6-membered
phosphorinane ring systems[16,17] and NOE studies on similar prodrugs. The
diastereomer with the 2- and 4-substituents on different sides of the ring plane is
called the trans isomer (5) and the diastereomer with the 2- and 4-substituents on
the same side is called the cisisomer (6).
METHODS
The kinetics of activation of 5 and 6 by CYP3A were evaluated in pooled
human and rat liver microsomes as measured by the GSH byproduct capture
assay previously described.[8] For hepatocyte activation studies, 4, 5 and 6 were
incubated with freshly isolated rat hepatocytes (n = 2) and intracellular levels of
LMV-TP were determined.[8,9] To evaluate the liver targeting potential of 6, male
Simonsen albino (S.A.) rats were administered an IV bolus of either 230 mg/kg
SCHEME 2

378
K. Raja Reddy et al.
FIGURE 2 LMV-TP generation in rat hepatocytes.
LMV (4) or HepDirect-LMV (6) (30 mg/kg LMV molar equivalents). At 0.33, 1,
3, 6, 12, and 24 hr following dosing, blood and liver were collected under
anesthesia (n = 4 rats/time point/test compound), processed, and subjected to
HPLC analysis. Blood samples were analyzed for LMV (4) and liver samples
analyzed for LMV-TP.[8,9]
RESULTS
Activation of 5 and 6 in Rat and Human Liver Microsomes
1
The mean ( /2 range) Km and Vmax values for the activation of 6 in pooled
mixed human liver microsomes were 73.3 13 mM and 0.19 0.01 nmol/min/mg,
respectively. The intrinsic clearance value was 2.6 0.6 mL/min/mg. The
corresponding mean ( s.d.) Km, Vmax, and CLint values in pooled male rat liver
microsomes were calculated to be 63.9 3 mM, 0.51 0.03 nmol/min/mg, and
8.0 0.4 mL/min/mg, respectively. Activation of 5 was undetectable, indicating a
microsomal enzyme preference for the cis-diastereomer 6.
FIGURE 3 Liver targeting of LMV vs. Prodrug 6 in the rat.

K. Raja Reddy et al.
379
TABLE 1 AUCs of LMV and LMV-TP in Rat Liver and Plasma
*
*
Test compound
Liver NTP (nmol g/h)
Plasma LMV, (mM h)
LMV (4), 230 mg/kg
Prodrug (6), 30 mg/kg
2.6
29.4
617
<12.8
Activation of 6 in Isolated Rat Hepatocytes
The concentration-time profiles of LMV-TP levels following incubation of
freshly isolated rat hepatocytes with 100 mM of 6 and LMV (4) are shown in
*
Figure 2. The AUC0–4h (mean half range) of LMV-TP was 648 88 nmol h/g for
*
6 compared to 18.9 0.3 nmol h/g for LMV (4). In accordance with the
microsomal activation data, LMV-TP formation was not seen with diastereomer 5.
Liver Targeting of 6 versus LMV (4) in the Rat
The temporal profile of LMV 4 (230 mg/kg) and 6 (30 mg/kg, LMV molar
equivalents) in plasma and liver following IV administration to rats is shown in
Figure 3. Despite high levels of nucleoside in plasma following administration of
LMV (4), LMV-TP was evident in liver at low concentrations at a single time point
only (0.9 nmoles/g at 20 min). Rats administered 6, in contrast, showed no
detectable LMV (4) in plasma and significant LMV-TP generation in liver at all
time points examined. The mean ( s.d.) Cmax and half-life values of LMV-TP in
liver were 6.9 0 and ꢀ5 h, respectively, following administration of 6. The AUCs
of LMV (4) in plasma and LMV-TP in liver for both test compounds are shown in
Table 1. Based on the AUC values, the liver-targeting index (liver LMV-TP–to-
plasma LMV) of 6 and LMV (4) was calculated to be > 2.3 and 0.007, respectively.
This represents a >320-fold enhancement in liver targeting of 6 relative to LMV (4).
DISCUSSION
HepDirect prodrugs achieve liver selectivity by virtue of being activated via
the action of a microsomal cytochrome P450 enzyme, CYP3A,[8] which is pre-
dominantly expressed in the liver. In humans, the CYP3A isoenzyme responsible
for HepDirect prodrug activation, CYP3A4, is largely expressed in the liver, with
lower levels in the intestine, stomach, and colon and undetectable levels in other
tissues.[18] Accordingly, HepDirect prodrugs are capable of the delivery of NMPs
selectively to the liver. Delivery of the NMP is an important characteristic since
phosphorylation of nucleosides to their corresponding monophosphates is generally
the rate-limiting step in their conversion to NTPs. This is clearly the case for LMV
(4), which is a poor substrate for nucleoside kinases and consequently results in
modest levels of LMV-TP in rat hepatocytes as well as following intravenous
administration of LMV at a high dose (230 mg/kg) to rats. In contrast, the HepDirect
prodrug of LMV, 6, generated ꢀ35-fold higher LMV-TP levels in rat hepatocytes

380
K. Raja Reddy et al.
versus an equivalent test concentration of LMV (4), and >10-fold higher liver LMV-
TP levels in rats versus a 7.6-fold higher molar equivalent dose of LMV (4). The
enhanced NTP generation by 6 is likely a reflection of its efficient microsomal
activation in hepatocytes to generate high levels of intracellular LMV-MP. The latter
bypasses the slow nucleoside kinase step that limits the conversion of LMV (4) to
LMV-MP and ultimately to the active antiviral metabolite, LMV-TP.
Another important finding from this study was that following administration of
6 to rats and liver-specific mechanism of activation, there was no detectable LMV
in plasma. While toxicity of systemically circulating LMV is not a major concern,
nucleoside exposure to the periphery is a potential issue for other therapeutic
agents. The therapeutic potential of adefovir, for instance, is limited by extrahepatic
exposure that leads to renal toxicity.[6] Whether a liver-targeted HepDirect prodrug
will significantly improve the therapeutic window for this antiviral nucleoside is
currently being tested clinically.[19]
In summary, the favorable cellular and in vivo profile of 6 relative to LMV (4),
supports the potential clinical utility of a HepDirect prodrug of LMV. Enhanced
hepatic LMV-NTP formation by means of the HepDirect prodrug strategy is likely
to confer more rapid and complete suppression of HBV replication and decrease
the emergence of drug-resistant HBV strains, a major problem with LMV and other
nucleoside-based therapies.
REFERENCES
1. Lavanchy, D.J. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging
prevention and control measures. Viral Hepat. 2004, 11(2), 97–107.
2. Wong, D.K.H.; Cheung, A.M.; O’Rourke, K.; Naylor, C.D.; Detsky, A.S.; Heathcote, J. Effect of alpha-
interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B. A meta-analysis. Ann.
Intern. Med. 1993, 119, 312–323.
3. Lai, C.L.; Chien, R.N.; Leung, N.W.Y.; Chiang, T.T.; Guan, R.; Tai, D.I.; Ng, K.Y.; Wu, P.C.; Dent, J.C.;
Barber, J.; Stephenson, S.L.; Gray, D.F. A one-year trial of lamivudine for chronic hepatitis B. Asia hepatitis
lamivudine study group. N. Eng. J. Med. 1998, 339, 61–68.
4. Lok, A.S.; Hussain, M.; Cursano, C.; Margotti, M.; Gramenzi, A.; Grazi, G.L.; Jovine, E.; Benardi, M.;
Andreone, P. Evolution of hepatitis B virus polymerase gene mutations in hepatitis B e antigen-negative
patients receiving lamivudine therapy. Hepatology 2000, 32(5), 1145–1153.
5. Marcellin, P.; Chang, T.T.; Lim, S.G.; Tong, M.J.; Sievert, W.; Shiffman, M.L.; Jeffers, L.; Goodman, Z.;
Wulfsohn, M.S.; Xiong, S.; Fry, J.; Brosgart, C.L. Adefovir dipivoxil for the treatment of hepatitis B e antigen-
positive chronic hepatitis B. N. Engl. J. Med. 2003, 348(9), 808–816.
6. Tanji, N.; Tanji, K.; Kambham, N.; Markowitz, G.S.; Bell, A.; D’agati, V.D. Adefovir nephrotoxicity: possible
role of mitochondrial DNA depletion. Human Pathol. 2001, 32(7), 734–740.
7. Kewn, S.; Hoggard, P.G.; Sales, S.D.; Johnson, M.A.; Back, D.J. The intracellular activation of lamivudine
(3TC) and determination of 2’-deoxycytidine-5’-triphosphate (dCTP) pools in the presence and absence of
various drugs in HepG2 cells. Br. J. Clin. Pharmacol. 2000, 50, 597–604.
8. Erion, M.D.; Reddy, K.R.; Boyer, S.H.; Matelich, M.C.; Gomez-Galeno, J.; Lemus, R.H.; Ugarkar, B.G.;
Colby, T.J.; Schanzer, J.; van Poelje, P.D. Design, synthesis, and characterization of a series of cytochrome
P(450) 3A-activated prodrugs (HepDirect prodrugs) useful for targeting phosph(on)ate-based drugs to the
liver. J. Am. Chem. Soc. 2004, 126, 5154–5163.
9. Erion, M.D.; van Poelje, P.D.; MacKenna, D.A.; Colby, T.J.; Montag, A.; Fujitaki, J.M.; Linemeyer, D.L.;
Bullogh, D.A. Liver-targeted drug delivery using HepDirectTM prodrugs. J. Pharm. Exp. Ther. 2005, 308,
in press.

K. Raja Reddy et al.
381
10. Schultz, C. Prodrugs of biologically active phosphate esters. Bioorg. Med. Chem. 2003, 11(6), 885–898.
11. Meunier, B.; de Visser, S.P.; Ahaik, S. Mechanism of oxidation reactions catalyzed by cytochrome P450
enzymes. Chem. Rev. 2004, 104, 3947–3980.
12. Turner, J.A. A general approach to the synthesis of 1,6-, 1,7-, and 1,8-naphthyridines. J. Org. Chem. 1990,
55, 4744–4750.
13. Milton, J.; Brand, S.; Jones, M.F.; Rayner, C.M. Enantioselective enzymatic synthesis of the anti-viral agent
lamivudine (3TCTM). Tetrahedron Lett. 1995, 36(38), 6961–6964.
14. Beaucage, S.L.; Iyer, R.P. The synthesis of modified oligonucleotides by the phosphoramidite approach and
their applications. Tetrahedron 1993, 49, 6123–6194.
15. Bentrude, W.G.; Sopchik, A.E.; Tadeusj, G. Stereo-and regiochemistries of the oxidations of 2-methoxy-5-tert-
butyl-1,3,2-dioxaphosphorinane and the cyclic methyl 3’,5’-phosphite of thymidine by H2O/I2 and O2/AIBN to
P-chiral phosphates. J. Am. Chem. Soc. 1989, 111, 3981–3987.
16. Mosbo, J.; Verkade, J.G.A. Dipole moment, nuclear magnetic resonance, and infrared studies of phosphorus
configurations and equilibriums in 2-R-2-oxo-1,3,2-dioxaphosphorinanes. J. Org. Chem. 1977, 42, 1549–
1555.
17. Cooper, D.B.; Harrison, J.M.; Inch, T.D.; Lewis, G.J. Use of carbohydrate derivatives for studies of
phosphorus stereochemistry. Part II. Synthesis and configurational assignments of 1,-3,2-oxathiaphosphorinan-
2-ones and 1,3,2-dioxaphosphorinan-2-thiones. J. Chem. Soc., Perkin Trans. 1 1974, 1049–1052.
18. de Waziers, I.; Cugnenc, P.H.; Yang, C.S.; Leroux, J.P.; Beaune, P.H. Cytochrome P 450 isoenzymes, epoxide
hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues. J. Pharmacol. Exp.
Ther. 1990, 253(1), 387–394.
19. Lin, C.-C.; Chao, Y.-C.; Lai, M.-Y.; Chang, T.-T.; Chuang, W.-L.; Yang, S.-S.; Braeckman, R.; Chen, D-S.
Safety, tolerance, pharmacokinetics and pharmacodynamics of Remofovir, a liver-targeting prodrug of PMEA
in HBV patients following daily dosing for 28 days. Hepatology 2004, 40(Suppl. 1), A1141.
20. Phosphoramidite (3): Rf = 0.50 in 5% EtOAc in hexanes. mp 44–46°C. 1HNMR (200Mz, DMSO-d6)
7.5–7.2 (m, 5H), 5.2–5.0 (m, 1H), 4.2–4.0(m, 2H), 3.9–3.6 (m, 2H), 1.8–1.5 (m, 2H), 1.3–1.0 (12H, series of
1
d). trans-isomer (5): Rf = 0.45 (10% MeOH in CH2Cl2). H NMR (200Hz, DMSO-d6): 7.80–7.60 (m, 2 H),
7.52–7.16 (m, 4 H), 6.32–6.16 (m, 1 H), 5.80–5.36 (m, 4 H), 4.56–4.20 (m, 4 H), 3.46–3.22 (m, 1 H), 3.18–
2.98 (m, 1 H), 2.26–2.00 (m, 2 H). Anal. calcd for C17H19ClN3O6PS. 0.5 H2O. 0.1 CH2Cl2: C, 42.82 H, 4.25
N, 8.74. Found: C, 42.71 H, 3.82 N, 8.54. cis-isomer (6): mp 110–113°C. Rf = 0.40 (10% MeOH in CH2Cl2).
1H NMR (200Hz, DMSO-d6): 7.66 (s, 1 H), 7.62 (s, 1 H), 7.50–7.21 (m, 4 H), 6.22 (t, J = 6 Hz, 1 H), 5.78–5.62
(m, 3 H), 5.42–5.30 (m, 1 H), 4.62–4.20 (m, 4 H), 3.45–3.24 (m, 1 H), 3.18–2.97 (m, 1 H), 2.40–2.05 (m, 2
H). Anal. calcd for C17H19ClN3O6PS. 0.75H2O: C, 43.14 H, 4.37 N, 8.88. Found: C, 43.00 H, 3.95 N, 8.70.