Metabolism, pharmacokinetics, tissue distribution, and stability studies of the prodrug analog of an anti-hepatitis B virus dinucleoside phosphorothioate

Article abstract of DOI:10.1124/dmd.111.044446

The alkoxycarbonyloxy dinucleotide prodrug R_p, S_p-2 is an orally bioavailable anti-hepatitis B virus agent. The compound is efficiently metabolized to the active dinucleoside phosphorothioate R _p, S_p-1 by human liver microsomes and S9 fraction without cytochrome P450-mediated oxidation or conjugation. The conversion of R _p, S_p-2 to R_p, S_p-1 appears to be mediated by liver esterases, occurs in a stereospecific manner, and is consistent with our earlier reported studies of serum-mediated hydrolytic conversion of R_p, S_p-2 to R_p, S_p-1. However, further metabolism of R_p, S_p-1 does not occur. The presence of a minor metabolite, the desulfurized product 10 was noted. The prodrug R_p, S_p-2 was quite stable in simulated gastric fluid, whereas the active R_p, S_p-1 had a half-life of <15 min. In simulated intestinal fluid, the prodrug 2 was fully converted to 1 in approximately 3 h, whereas 1 remained stable. To ascertain the tissue distribution of the prodrug 2 in rats, the synthesis of ³⁵S-labeled R_p, S_p-2 was undertaken. Tissue distribution studies of orally and intravenously administered radiolabeled [³⁵S]2 demonstrated that the radioactivity concentrates in the liver, with the highest liver/plasma ratio in the intravenous group at 1 h being 3.89 (females) and in the oral group at 1 h being 2.86 (males). The preferential distribution of the dinucleotide 1 and its prodrug 2 into liver may be attributed to the presence of nucleoside phosphorothioate backbone because phosphorothioate oligonucleotides also reveal a similar tissue distribution profile upon intravenous administration. Copyright

Full text of DOI:10.1124/dmd.111.044446

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2012/02/10/dmd.111.044446.DC1.html
1521-009X/12/4005-970–981$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:970–981, 2012
Vol. 40, No. 5
44446/3764310
Metabolism, Pharmacokinetics, Tissue Distribution, and Stability
Studies of the Prodrug Analog of an Anti-Hepatitis B Virus
□
S
Dinucleoside Phosphorothioate
John E. Coughlin, Rajendra K. Pandey, Seetharamaiyer Padmanabhan,
Kathleen G. O’Loughlin, Judith Marquis, Carol E. Green, Jon C. Mirsalis, and
Radhakrishnan P. Iyer
Spring Bank Pharmaceuticals, Inc., Milford, Massachusetts (J.E.C., R.K.P., S.P., R.P.I.); SRI International, Toxicology &
Pharmacokinetics, Menlo Park, California (K.G.O., C.E.G., J.C.M.); and Genzyme Corporation, Marlborough, Massachusetts (J.M.)
Received December 28, 2011; accepted February 10, 2012
ABSTRACT:
The alkoxycarbonyloxy dinucleotide prodrug Rp, Sp-2 is an orally converted to 1 in approximately 3 h, whereas 1 remained stable. To
bioavailable anti-hepatitis B virus agent. The compound is effi- ascertain the tissue distribution of the prodrug 2 in rats, the syn-
ciently metabolized to the active dinucleoside phosphorothioate thesis of 35S-labeled Rp, Sp-2 was undertaken. Tissue distribution
Rp, Sp-1 by human liver microsomes and S9 fraction without cyto- studies of orally and intravenously administered radiolabeled
chrome P450-mediated oxidation or conjugation. The conversion
of Rp, Sp-2 to Rp, Sp-1 appears to be mediated by liver esterases, with the highest liver/plasma ratio in the intravenous group at 1 h
occurs in a stereospecific manner, and is consistent with our being 3.89 (females) and in the oral group at 1 h being 2.86 (males).
[
35S]2 demonstrated that the radioactivity concentrates in the liver,
earlier reported studies of serum-mediated hydrolytic conversion The preferential distribution of the dinucleotide 1 and its prodrug 2
of Rp, Sp-2 to Rp, Sp-1. However, further metabolism of Rp, Sp-1 into liver may be attributed to the presence of nucleoside phos-
does not occur. The presence of a minor metabolite, the desulfu- phorothioate backbone because phosphorothioate oligonucleo-
rized product 10 was noted. The prodrug Rp, Sp-2 was quite stable tides also reveal a similar tissue distribution profile upon intrave-
in simulated gastric fluid, whereas the active Rp, Sp-1 had a half-life
of <15 min. In simulated intestinal fluid, the prodrug 2 was fully
nous administration.
Introduction
agonists and antagonists of specific nucleotide-protein and nucleic acid-
protein interactions should have broad therapeutic applications against
multiple diseases (Ellington and Conrad, 1995).
Small molecule nucleic acid hybrids (SMNH), which are two to six
nucleotides long, represent a new class of small molecule chemical
entities that can be rationally designed to target key nucleotide-protein
and nucleic acid-protein interactions involved in a disease process.
Indeed, reports suggest that mono-, di-, tri-, and short-chain oligonu-
cleotides (ODNs) possess significant and diverse biological activities
that can be exploited for therapeutic applications (Wagner et al., 1996;
Nucleotides are among the most important ligands for a number of
intracellular and extracellular proteins involved in biochemical reactions
and cell signaling (Saito et al., 2006). In addition, nucleic acid-protein
interactions constitute essential events in a variety of cellular processes
(Sanger, 1983; Ollis and White, 1987). Therefore, rationally designed
This study was supported by the National Institutes of Health National Institute
of Allergy and Infectious Diseases Extramural Activities [Grants R01-AI094469,
U01-AI058270] (under a Research Project Cooperative Agreement Grant Award;
to R.P.I.). The radiolabeled synthesis and tissue distribution studies were con- Hannoush et al., 2004; Iyer et al., 2004, 2005b).
ducted by SRI International (Menlo Park, CA) with support from the National
In previous studies, we have shown that in general, the SMNH class
Institutes of Health National Institute of Allergy and Infectious Diseases Extramu-
ral Activities [Contract Number N01-AI60011] (to J.M.).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
of molecules has unique metabolic and pharmacokinetic properties
that are distinct from traditional small molecule drugs (Iyer et al.,
2004a, 2006). Thus, in contrast to the hydrophobic small molecule
compounds, which are subject to cytochrome P450 (P450)-mediated
Phase I oxidative processes and Phase II conjugation reactions,
SMNH analogs are not metabolized by P450 enzyme systems. Fur-
http://dx.doi.org/10.1124/dmd.111.044446.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: SMNH, small molecule nucleic acid hybrids; P450, cytochrome P450; ODN, oligonucleotide; PS-ODN, phosphorothioate
oligonucleotide; ADME, absorption, distribution, metabolism, and excretion; siRNA, small interfering RNA; HBV, hepatitis B virus; SGF, simulated
gastric fluid; SIF, simulated intestinal fluid; DMT-NBz-dA, 5Ј-O-4,4-dimethoxytriphenylmethyl-N6-benzoyl-2Ј-deoxyadenosine; ETT, 5-ethylthio-
tetrazole; DCA, dichloroacetic acid; DCM, dichloromethane; CPG, controlled pore glass; DMT-T, 5Ј-O-4,4-dimethoxytriphenylmethyl-thymidine;
dA, 2Ј-deoxyadenosine; HPLC, high-performance liquid chromatography; MS, mass spectrometry; LC/MS, liquid chromatography/mass spec-
trometry; AUC, area under the plasma concentration-time profile; Cmax, maximal plasma concentration.
970

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
971
dimethylformamide, obtained from Sigma-Aldrich (St. Louis, MO) were freshly
distilled from CaH2 before use. Other reagents such as 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide, N,N-dimethylaminopyridine, 5-ethylthio-
tetrazole (ETT), dichloroacetic acid (DCA), and triethylamine were obtained
from Sigma-Aldrich and were used as received. Controlled pore glass
(CPG) support was obtained from Prime Synthesis, Inc. (Aston, PA).
Capping reagent A and capping reagent B were obtained from American
International Chemicals (Natick, MA).
thermore, in in vivo studies, we have reported that a representative
dinucleoside phosphorothioate 1 does not seem to be metabolized by
Phase I or Phase II processes and is mostly eliminated as intact
compound (Iyer et al., 2006). Lack of P450 metabolism of SMNH
molecules is a unique pharmaceutical attribute that confers distinct
advantages in that such molecules are not subject to the “first-pass
effect,” and they can be combined with different classes of drugs
without the potential for drug-drug interactions (Gibaldi et al., 1971).
In the context of antiviral therapeutics, a cocktail of drugs each with
Synthesis of the Dinucleotide 1. The dinucleotide 1 was synthesized on a
multimillimole scale using 2Ј-deoxyadenosine (dA)-loaded CPG support in
unique mechanisms of action that can act synergistically with minimal conjunction with solid-phase phosphoramidite chemistry (Beaucage and Iyer,
1992) (Fig. 1). For the synthesis, we used a specially fabricated LOTUS reactor
as described previously (Iyer et al., 2005a). Nucleoside-loaded support (112 g,
89 mmol) prepared as described previously (Padmanabhan et al., 2005) was
detritylated using DCA in dichloromethane (DCM) (2.5%, DCA/DCM, 3 ϫ
400 ml) with DCM washes (3 ϫ 400 ml) between each DCA/DCM treatment,
and the support was subsequently washed thoroughly with DCM (5 ϫ 400 ml)
and later with acetonitrile (low water, Ͻ30 ppm). Detritylated nucleoside was
coupled with 2Ј-O-methyluridine phosphoramidite (5 Eq) in the presence of
ETT (0.4 M, 10 Eq) in anhydrous acetonitrile under argon. After acetonitrile
washings, sulfurization was performed using 3H-benzodithiole-3-one-1,1-di-
toxicity is particularly important for the treatment of chronic infec-
tions (Perrillo, 2004). Consequently, the development of SMNH an-
alogs as antiviral agents is of great interest.
SMNH compounds are small molecule counterparts of the more
widely studied class of long-chain phosphorothioate oligonucleotides
(PS-ODN). Extensive absorption, distribution, metabolism, and ex-
cretion (ADME) studies of PS-ODN reveal a pattern in which after
intravenous administration, they are 1) rapidly cleared from plasma
after absorption, 2) preferentially distributed to liver, kidney, spleen,
and bone marrow, 3) metabolized by exo- and endo-nucleases into oxide reagent (5 Eq) (Iyer et al., 1990a,b) to give the fully protected CPG-
bound dinucleotide. The 5Ј-DMT group was removed using 2.5% DCA/DCM
(3 ϫ 400 ml), and the CPG was washed with DCM (5 ϫ 400 ml), acetonitrile
(3 ϫ 400 ml), and finally water (3 ϫ 500 ml). The dinucleotide 1 was released
from the support by deprotection and cleavage using NH4OH (200 ml, 28–
30%) at ϳ30°C under orbital shaking overnight. The ammoniacal solution was
acidified with glacial acetic acid to pH 6 under cooling followed by filtration
to give crude 1, which was purified by three-stage preparative high-perfor-
mance liquid chromatography (HPLC) as described below.
shorter ODN fragments, and 4) excreted primarily through kidneys
(Crooke et al., 2000; Peng et al., 2001; Geary, 2009). Studies of
duplexed synthetic ODNs as small interfering RNA (siRNA) (Tuschl,
2001; Hannon, 2002) show a similar ADME trend, although the
overall pharmacokinetic and tissue distribution profile is dependent on
the extent of chemical modification and the conjugating moieties (van
de Water et al., 2006; Li and Liang, 2010). It was therefore of interest
to evaluate the ADME properties of SMNH compounds.
Desalting of the dinucleotide 1 solution was performed in a 500-ml
stainless steel C-18 column. Initially, the column was equilibrated with
Chemically modified antisense ODNs (Szymkowski, 1996; Dias
and Stein, 2002) as “hybrid” or “gapmers” have been reported to be NH4OAc (0.1 M, pH 7.0, at a flow rate of 20 ml/min) followed by loading
of dinucleotide 1 solution. The dinucleotide 1 was eluted from the column
using a gradient of acetonitrile and water. The desalted dinucleotide 1
solution was concentrated to 20% of volume in vacuo, diluted with water,
and was subjected to ion-exchange chromatography using Toyopearl Super
Q 650M resin (Tosoh Bioscience, King of Prussia, PA). After equilibration
of the column with NaOAc (0.1 M), 1 was loaded onto the column, which
was washed with 3 column volumes of NaOAc (0.1 M, pH 7.0). The
dinucleotide 1 was eluted with NaOAc (0.1 M) and NaCl (1 M) buffer and
was further purified by reversed-phase HPLC on a C-18 column. The
sodium salt of 1 was eluted with acetonitrile/water (20:80). The eluent was
orally bioavailable in vivo (Agrawal et al., 1995; Tillman et al., 2008),
although the extent of pharmaceutical bioavailability and mechanisms
of absorption are not known. In contrast, in vitro studies using Caco-2
cell lines and in vivo studies in rats revealed that none of the SMNH
analogs has potential for oral bioavailability, presumably because of
the negative charge(s) on their backbone that impedes their passive
diffusion across the cellular lipid bilayer (Iyer et al., 2004a, 2006;
Coughlin et al., 2010).
As part of further development of the SMNH compounds, we have
undertaken preclinical studies to develop the anti-hepatitis B virus lyophilized to obtain the dinucleotide 1 as a white fluffy solid.
Synthesis of 2. The prodrug 2 was prepared by the S-alkylation of the
dinucleotide 1 with iodomethyl isopropyl carbonate (3). The iodo-compound 3
was prepared as described previously (Padmanabhan et al., 2006). The product
3 was characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, and
(HBV) SMNH analog 1 as an orally bioavailable compound. The
analog 1 was not stable in simulated gastric fluid (SGF), because of its
susceptibility to acid-catalyzed depurination, but was stable in simu-
lated intestinal fluid (SIF). An ideal SMNH analog is required to be
stable in the gastrointestinal tract; therefore, the development of a
prodrug strategy for oral delivery appeared logical.
1
mass spectrometry (MS). H NMR (CDCl3): ␦ 1.2 ppm (6H, s), 4.8 (1H, m),
5.8 (2 H, s); 13C NMR (CDCl3): ␦ 154, 76, 36, and 22 ppm; electrospray
ionization (positive mode)-MS, 244.
We have reported the synthesis and evaluation of various orally
bioavailable prodrug derivatives of 1 (Padmanabhan et al., 2006).
These analogs are designed to mask the negative charge on the
backbone of the dinucleotide and to improve their hydrophobicity to
facilitate passive diffusion through cell membrane. As a part of the
continuing development of a prodrug analog 2, we evaluated its
ADME properties, its stability in SGF and SIF, and its oral bioavail-
ability. The results are presented here.
The S-alkylation protocol was essentially as described previously (Coughlin
et al., 2010). After HPLC purification, 2 was obtained as a lyophilized powder
as an Rp, Sp diastereomeric mixture (ϳ55:45, 52%).
Spectral Data of Rp, Sp-2. 1H NMR (CD3OD), ␦ 1.16 (s, 3H), 1.24 (s, 3H),
2.49 (m, 1H), 2.89 (m, 1H), 3.45 (s, 3H), 3.75 (s, 2H), 4.21 (m, 1H), 4.3 (d,
1H), 4.45 (m, 2H), 4.77 (m, 1H), 4.85 (m, 1H), 5.15 (m, 1H), 5.42 (d, 1H), 5.44
(d, 1H), 5.71 (d, 1H), 6.01 (d, 1H), 6.46 (t, 1H), 7.98 (d, 1H), 8.21 (s, 1H), 8.29
(d, 1H) ppm; 13C NMR (CD3OD), ␦ 22.62, 40.68, 59.34, 59.42, 61.59, 61.84,
67.81, 67.92, 69.03, 72.35, 74.79, 76.78, 83.09, 83.14, 83.44, 85.22, 85.99,
86.11, 86.61, 103.39, 103.47, 120.52, 120.72, 141.09, 141.22, 142.28, 150.61,
152.39, 152.47, 154.00, 154.68, 154.93, 157.39, 165.06 ppm; 31P NMR
Materials and Methods
Materials for Chemical Synthesis. 5Ј-O-4,4Ј-Dimethoxytriphenylmethyl- (CD3OD), ␦ 27.7 and 28.6 ppm; MS, m/z 703.6.
N6-benzoyl-2Ј-deoxyadenosine (DMT-NBz-dA), 5Ј-O-4,4Ј-dimethoxytriphenylm-
ethyl-thymidine (DMT-T), and 4,4Ј-dimethoxytriphenylmethyl-2Ј-O-methyl-
Synthesis of Radiolabeled Compounds. Synthesis of [35S]2. The dinucleo-
side phosphorothioate ester [35S]2 [34.2 mg, total activity 4.44 mCi (91.81
uridine (DMT-U2Ј-OMe) were obtained from Reliable Biopharmaceuticals (St. ␮Ci/␮mol)] was prepared by radiosynthesis (Fig. 1). The 35S label was
Louis, MO) and were used as such. T-phosphoramidite monomer was obtained installed on the phosphorothioate backbone using 35S-3H-benzodithiole-3-one-
from Rasayan Chemicals (Encinitas, CA). Anhydrous pyridine, triethylamine, and 1,1-dioxide (Iyer et al., 1994). [35S]2 had a radiochemical purity of Ͼ98% as

972
COUGHLIN ET AL.
FIG. 1. A, synthetic scheme depicting the solid-phase synthesis of 1. B, synthesis of the prodrug 2 from 1.
assessed by radio-HPLC (see Supplemental Fig. 1). Detailed methods of
A (0.1 M ammonium acetate) to 100% B [0.1 M ammonium acetate/
radiosynthesis and analysis are reported elsewhere (S.-W. Rhee, R. P. Iyer, acetonitrile (20:80)] with a flow rate of 0.5 ml/min over 41 min was used.
J. E. Coughlin, S. Padmanabhan, J. P. Malerich, and M. J. Tanga, unpublished
observations). All materials were stored at Ϫ20°C until ready to use.
Liquid Chromatography/mass spectrometry (LC/MS) analysis of the sam-
ples was performed on an Agilent 1100 Series HPLC with UV detection and
In Vitro Metabolism and Stability Studies of 2. Metabolism studies using MSD system (Agilent Technologies, Santa Clara, CA). For the liquid chro-
liver microsomes and S9 fractions were performed as described previously matography analysis of the samples, a Phenomenex Luna C18 column (5 ␮M,
(Dalvie et al., 2009).
4.6 ϫ 250 mm) operating at 25°C was used (Phenomenex, Torrance, CA).
Materials. Pooled human liver microsomes, S9 from liver, NADPH, al- Other chromatographic conditions were as follows: injection volume, 50 ␮l;
amethicin, UDP-GlcUA, adenosine 3Ј-phosphate 5Ј-phosphosulfate lithium elution gradient, 5 to 50% acetonitrile in 0.1 M NH4OAc over 45 min; and flow
salt hydrate, S-(5Ј-adenosyl)-L-methionine iodide, CoASAc sodium salt, pep-
rate, 0.5 ml/min. In the mass spectrum, the molecular mass signals represented
sin and pancreatin were purchased from Sigma-Aldrich.
[M ϩ H] and/or [M ϩ Na] ions. The MS instrument was an electrospray
The metabolite samples were analyzed by reversed-phase HPLC using ionization, ion-trap mass spectrometer. The mass spectra were obtained in full
Waters instrument equipped with 600E Controller, PDA 996 detector, Waters ion-scan mode with a range between 100 and 1200 m/z. Other parameters were
717 Auto sampler with Millennium software (Waters, Milford, MA). A Waters
as follows: resolution, 13,000 m/z per s; dry temperature, 350°C; capillary
Nova-Pak C18 4-␮m (3.9 ϫ 300 mm) column operating on a gradient of 100%
ramp range, 1500 to 4500 V; nebulizer pressure, 60 psi; dry gas flow, 10 l/min;

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
973
trap target 30,000; and maximal accumulation time, 100 ms. Instrument
calibration was verified using seven compounds of known molecular mass
between 118 and 2722 Da. The predicted chemical formulae and calculated
accurate mass were obtained from ChemDraw Standard 8.0 (CambridgeSoft
Corporation, Cambridge, MA) on the basis of the proposed metabolic path-
ways and putative structures.
g) was dissolved in water (250 ml), and NaOH (0.2 N, 77 ml) and H2O (500
ml) were added. Pancreatin (10 g) was added and mixed, and the solution was
adjusted to pH 6.8 with 0.2 N HCl and finally diluted with water to 1 liter.
A stock solution of 2 was prepared (2 mg in 100 ␮l of dimethyl sulfoxide). To
10 ␮l of stock solution, 200 ␮l of either SGF or SIF was added and incubated at
37°C. At different time points (15 min, 30 min, 1 h, and 3 h), aliquots were drawn
In Vitro Metabolism of 2 Using Liver Microsomes. A stock solution of and quenched with acetonitrile. The pH of the SGF sample was adjusted to 7.0
prodrug 2 was prepared in deionized water at 1 mM concentration and diluted
further to make a 10-␮M solution. The incubation mixture contained liver micro-
using ammonium acetate buffer. The precipitate was removed by centrifugation.
The supernatant was lyophilized, and the residue was reconstituted in 200 ␮l of
somes (1 mg/ml), NADPH (1.3 mM), UDP-GlcUA (5 mM), alamethicin (10 water for HPLC analysis as described before.
␮g/ml) in 1 ml of 1ϫ phosphate-buffered saline buffer, at 10 ␮M final concen-
Tissue Distribution and Metabolism of [35S]2. Tissue distribution and
tration of 2. The liver microsomes were treated with alamethicin in an ice bath for metabolism studies were performed using Sprague-Dawley rats obtained from
15 min before use. NADPH and UDP-GlcUA were added, and the incubate was
maintained at 37°C in a shaking water bath. Samples were taken out at 1, 4, 6, and
8 h and were quenched with acetonitrile (2 ml). The resulting precipitate was
removed by centrifugation for 10 min, and supernatant was lyophilized and
reconstituted in 200 ␮l of water for HPLC analysis. The metabolite samples were
also independently evaluated by LC/MS analysis.
Harlan (Livermore, CA) and Charles River Laboratories (Wilmington, MA). A
total of 18 animals (9 males and 9 females), 8 to 13 weeks old, weighing 245
to 303 g were used. Animals were housed in a facility accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care.
General procedures for animal care and housing were performed in accordance
with the National Research Council Guide for the Care and Use of Laboratory
Metabolism of 2 in S9 Fractions from Human Liver. The prodrug 2 (10 Animals (Institute of Laboratory Animal Resources, 1996) and the Animal
␮M) was incubated with a mix containing S9 fraction (pooled from human) (1 mg/ml), Welfare Standards incorporated in 9 Code of Federal Regulations Part 3, 1991.
NADPH (1.3 mM), UDP-GlcUA (5 mM), and alamethicin (10 ␮g/ml) in 1 ml of 1ϫ Animals destined for the 24-h sacrifice were housed in glass metabolism cages
PBS buffer. S-(5Ј-adenosyl)-L-methionine iodide (0.1 mM), adenosine 3Ј-phosphate to allow for urine and feces collection. The light cycle was 12-h light/dark; the
5Ј-phosphosulfate lithium salt hydrate (0.1 mM), and CoASAc (1 mM).
temperature range was 65–72°F; and the number of ventilations was 10 room
volumes per hour, with no recirculation of air. Purina Certified Rodent Chow
#5002 ad libitum was used (Purina, St. Louis, MO). Water (purified, reverse
osmosis) was provided ad libitum.
The S9 fraction was treated with alamethicin on ice for 15 min before use.
Incubations were performed at 37°C in a shaking water bath. Samples were
removed at 1, 4, and 6 h and were quenched with 2 ml of acetonitrile. After 60 min
at room temperature, the resulting precipitate was removed by centrifugation. The
The prodrug [35S]2 was administered by bolus intravenously via a tail vein or
supernatant was lyophilized and reconstituted in 200 ␮l of water for HPLC oral gavage at a single dose. The dosing volume was 5 ml/kg, and the experimental
analysis. The lyophilized samples were also independently evaluated by LC/MS duration was 24 h. Evaluation parameters included radioactivity levels in blood,
analysis.
tissues, carcass, and excreta. For intravenous administration, the vehicle used was
Stability Studies of 2 in SGF and SIF. SGF and SIF were prepared as per a mixture of 25% polyethylene glycol 400 and 75% sterile saline for injection
The United States Pharmacopeia (1993). In brief, SGF was prepared by
dissolving NaCl (2.0 g), purified pepsin (3.2 g, from porcine stomach mucosa),
and HCl (36%, 7 ml) and diluting with water to 1 liter. This test solution had
a pH of 1.2. For the preparation of SIF, monobasic potassium phosphate (6.8
(0.9% sodium chloride for injection; The United States Pharmacopeia, 1993), and
that for oral administration was 0.05 M citric acid buffer, pH 2.5.
Formulations. Intravenous and oral dose formulation(s) were prepared by
adding the appropriate amount of 2 in the vehicle to achieve the target
FIG. 2. Representative HPLC profile at different time points of aliquots from the incubation of Rp, Sp-2 with liver microsomes.

974
COUGHLIN ET AL.
concentration and specific activity. [35S]2 was added to the unlabeled drug
formulation (under reduced light conditions) to give the correct final drug and
radioactivity concentration. Dose formulations were prepared fresh at room
temperature on the day of administration and were used within 2 h.
approximately Ϫ20°C. The tissues were analyzed for radioactivity by liquid
scintillation counting after homogenization and treatment with a tissue solu-
bilizer. Total radioactivity (disintegrations per minute) was determined and
calculated as a concentration (microgram-equivalent per milliliter or gram) and
Experimental Procedures (In-Life Evaluations). Mortality/morbidity as a percentage of the administered dose.
evaluations were performed at least once daily, and animals were examined for
clinical signs related to the pharmacology and toxicology of the test article,
gross motor and behavioral activity, and observable changes in appearance.
Urine and feces were collected ϳ1, 4, and 24 h postdose. Blood (ϳ200–300
The total radioactivity in plasma, presented as microgram-equivalent per
milliliter, was also evaluated using WinNonlin Professional (version 5.2;
Pharsight, Mountain View, CA), noncompartmental approaches with sparse
sampling feature to determine the elimination t1/2, time to maximal plasma
␮l) was collected from the retro-orbital sinus under 60% CO2/40% O2 anes- concentration, observed maximal plasma concentration (Cmax), and area under
thesia, just before necropsy, into tubes containing EDTA in wet ice. To
generate plasma, samples were centrifuged within 15 min of collection at
1750g for 6 to 8 min. Plasma and whole blood were transferred to cryovials
and stored at Ϫ20°C for storage until analysis for radioactivity.
All animals were euthanized at their scheduled sacrifice with an overdose of
sodium pentobarbital administered intraperitoneally at 1, 4, or 24 h. Liver,
the plasma concentration-time profile (AUC). Cmax and AUC were presented
as the mean Ϯ S.E. of the data.
Results
Synthesis of Compounds. The prodrug 2 was prepared by chemo-
lung, kidney, heart, brain, spleen, and tail were collected and stored frozen at selective S-alkylation of the dinucleotide 1 (Fig. 1). For our studies,
FIG. 3. Mass spectrum of the prodrug 2. Peak at 704 represents the [M ϩ H] ion.

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
975
we used the ϳ55:45 Rp/Sp mixture of 1, which was synthesized in carbonate was prepared by halogen exchange reaction with the cor-
large scale using nucleoside-loaded CPG support in conjunction with responding chloro-compound as described previously (Padmanabhan
solid-phase phosphoramidite chemistry, using a specially fabricated et al., 2006). After HPLC purification and lyophilization, 2 was
LOTUS reactor. In brief, the dA-linked CPG support was prepared obtained in overall isolated yields of 50 to 52% from 1. 31P NMR of
using our recently developed ultrafast functionalization and loading 2 showed two peaks representing a ϳ55:45 ratio of the Rp/Sp isomers,
process for solid supports. Coupling of the 2Ј-OMe uridine phos- and the mass spectrum was consistent with the expected molecular ion
phoramidite to the dA-loaded support was achieved using a solution of 703 for 2.
of ETT as a coupling reagent. The sulfurization of the internucleotidic
In Vitro Metabolic Studies of 2 Using Liver Microsomes. The
dinucleoside phosphite-coupled product 5 was performed using 3H- metabolism protocols followed were essentially as described previ-
1,2-benzodithiole-3-one-1,1,-dioxide. After processing, chromato- ously (Dalvie et al., 2009), with slight modifications. Thus, exposure
graphic purification, and lyophilization, the sodium salt of Rp,Sp-1 of the prodrug Rp, Sp-2 to liver microsomes up to 8 h resulted in its
(ϳ60:40 mixture) was obtained, which was characterized by 31P and stereospecific conversion to the dinucleotide Rp, Sp-1 (Fig. 2). Indeed,
1H NMR and was Ͼ96% pure.
the LC/MS evaluation of the microsomal incubate revealed that the
The chemoselective S-alkylation of Rp,Sp-1 with iodomethyl iso- major product(s) of metabolism was the Rp, Sp-dinucleotide 1 (Figs. 3
propyl carbonate gave the prodrug Rp,Sp-2. In turn, the iodomethyl and 4). A minor amount of the desulfurized product 10 (Ͻ5%) was
FIG. 4. Mass spectrum of the dinucleoside phosphorothioate 1 resulting from the metabolism of 2 by liver microsomes. The peak at 588 Da represents the [M ϩ H] ion.

976
COUGHLIN ET AL.
also detected as determined by MS analysis (m/e, 571) (Fig. 5). A few ylated derivatives of 1 or other phase II conjugation products. In all
minor metabolites (Ͻ10%) were also seen (Fig. 2). However, the cases, in the microsomal metabolism, both Rp and Sp isomers of
identity of these metabolites could not be firmly established on the prodrug 2 underwent stereospecific conversion to the active 1 with
basis of molecular ion. There was no evidence for any quantifiable only minor amounts of desulfurized product (10) (corresponding to 1)
formation of the deaminated analog 9 (the inosine analog correspond- being observed (Fig. 5). It was also gratifying to find that there
ing to 1 that could be formed by the action of adenosine deaminase). appears to be no significant rate differences in the conversion of the
The absence of 9 was additionally confirmed by independent synthe- individual Rp and Sp isomers of 2 to Rp and Sp isomers of 1, respec-
sis and cochromatography along with the metabolite incubate. None tively. These results are consistent with serum-mediated biorevers-
of the other predicted metabolites (Fig. 6) such as O-dealkylated ibility studies previously reported by us (Coughlin et al., 2010).
ribonucleoside analog of 2 (i.e., 10) or that of 1 and the 8-oxo-
Similar to the serum-mediated conversion of 2 to 1 in the case of
deoxyadenosine analog of 1 (i.e., 11) and 2 (i.e., 12) was apparent on microsomes, liver esterases appear to be the major metabolizing
the basis of molecular ion analysis in LC/MS. Our curiosity in the enzymes involved in the hydrolytic conversion of 2 to 1. Mechanis-
formation of 11 and 12 was triggered by the reported susceptibility of tically, it appears that the formation of 1 from 2 occurs by nucleophilic
position 8 of purine nucleosides to oxidation (Ahmad and Mond, attack of the serine hydroxyl group of the esterase on the carbonyl
1985). In addition, there was no apparent formation of 5Ј-phosphor- carbon of 2 to give 11, followed by the intramolecular attack of the
FIG. 5. Mass spectrum of the desulfurized product 10 resulting from the metabolism of 2 by liver microsomes. The peak at 572.1 Da represents the [M ϩ H] ion.

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
977
FIG. 6. Predicted pathways for phase I biotransformation of Rp-,Sp-2 by liver microsomes.
incipient oxyanion on the phosphoryl group to give the cyclic inter- equal ease. In our earlier bioreversibility studies of Rp-, Sp-2 using serum,
mediate 12 (Fig. 7). The transient species 12 could reorganize to give the we have found that the individual isomers are stereospecifically con-
trigonal bipyramidal intermediates 13 and 14 that could interconvert by verted to Rp-, Sp-1 at almost equal rates (Coughlin et al., 2010). However,
pseudorotation (Westheimer, 1968). Although in the intermediate 14, the the bioreversion of the corresponding S-alkyl derivatives of the dinucle-
S-acyloxyalkyl group is favorably poised to depart from an apical direc- otides 3Ј,5Ј-linked thymidine-thymidine phosphorothioate and 3Ј,5Ј-
tion (that could cause the formation of desulfurized product 10), its linked deoxyadenosine-thymidine phosphorothioate-5Ј (both of which
formation is presumably disallowed because of the energetics involved in lack the 2Ј-OMe substituent) occurs with significant rate differences
the reorganization of the initially formed enzyme-substrate complex 12. between Rp and Sp isomers (Padmanabhan et al., 2006; Coughlin et al.,
Consequently, the hydrolytic pathway is directed to occur via 13 to yield 2010). These results suggest that subtle conformational effects are in play
the desired 1 with minimal formation of 10. It appears that the 2Ј-OMe in the enzyme-mediated hydrolysis of 2. The facile bioconversion of 2 to
substituent in the dinucleotide structure facilitates hydrolysis of the ester 1 in liver and plasma is also consistent with broad substrate specificity of
group in each of the isomers with almost equal ease. We hypothesize that the ubiquitous esterase enzymes.
the 2Ј-OMe substitution in the furanose ring of Rp-, Sp-2 favors a
In Vitro Metabolism of 2 by S9 Fractions. Similar to liver micro-
C3Ј-endo versus a C2Ј-endo conformation that might optimally orient the somal studies, exposure of the prodrug 2 to S9 fractions also resulted in
ester group in both isomers for enzyme-mediated nucleophilic attack with stereospecific conversion of 2 to the dinucleotide 1 (Supplemental Fig. 2).

978
COUGHLIN ET AL.
FIG. 7. Proposed mechanism of bioconver-
sion of Rp-, Sp-2 to Rp-, Sp-1 by liver es-
terases. The proposed mechanism is based
on an earlier publication (Coughlin et al.,
2010) and references cited therein.
Evaluation of the incubate by LC/MS revealed that besides the major
product 1, minor amounts of the desulfurized product 10 (Ͻ5%) were
also formed. A few minor metabolites were also observed. The pattern of
metabolites was similar to that observed with liver microsomes. There
was no apparent evidence of any phase II conjugative reactions of 2 or
that of the initially formed active metabolite 1.
Stability Studies of 2 in SGF and SIF. An important requirement
for oral bioavailability of the prodrug 2 is its stability in gastric fluid.
Given the known susceptibility of the dinucleotide 1 to acid-catalyzed
decomposition, it was not known whether 2 would have adequate
stability gastric fluid. Therefore, we evaluated the stability of 2 and
the dinucleotide 1 in SGF and SIF by HPLC analysis of aliquots at
different time points. Whereas the dinucleotide 1 decomposed rapidly
in SGF (t1/2 Ͻ 15 min), we were gratified to find that the prodrug 2
displayed high stability in SGF with t1/2 Ͼ 3 h (Fig. 8). In SIF, 2 was
almost completely converted to 1 in ϳ3 h, whereas 1 was stable in SIF
(Supplemental Fig. 3). Our stability data on prodrug 2 is consistent
with that reported for S-acyl-2-thioethyl pronucleotides (Shafiee et al.,
2001). However, the mechanistic rationale for the greater stability of
2 in SGF compared with 1 is not established as yet.
A larger fraction of the total dose of radioactivity was found in the
carcass of females in the oral group (and the intestines were observed
to contain more fecal material), suggesting that excretion by this route
is probably similar in male and female rats, but that females had not
yet eliminated feces during the study period. Comparing the same
time points, the highest fraction of the dose was present in liver Ͼ
blood Ϸ plasma Ͼ kidney Ͼ lung, after both intravenous and oral
administration. Brain, spleen, and heart had 0.5% or less of the dose
at all time points and for both intravenous and oral routes of admin-
istration. Supplemental Figs. 4 and 5 are graphical representations of
the time course of the presence of radioactivity in the principal tissues
in male and female rats, respectively. After intravenous administra-
tion, the concentration of radioactivity in liver and kidney was higher
than that of the plasma levels at the three time points 1, 4, and 24 h,
whereas lung had a similar concentration of radioactivity compared
with that of plasma. After oral administration of [35S]2, the highest
concentration of radioactivity was observed at 4 h postdose. On the
basis of the concentration ratios of tissue/plasma, radioactivity from
[35S]2 tended to concentrate in the liver and kidney after both intra-
venous or oral administration (Fig. 9). The time of peak concentration
after an oral dose was 4 h for plasma, blood, and tissues. Tissue
distribution evaluation demonstrated that the radioactivity concen-
trates in the liver, with the highest liver/plasma ratio in the intravenous
group at 1 h, 3.89 (females), and in the oral group at 1 h, 2.86 (males).
Table 2 summarizes the pharmacokinetic parameters for total ra-
dioactivity in plasma, calculated as microgram-equivalent per milli-
liter. The elimination t1/2 for radioactivity after an intravenous dose
was 8 to 9 h. The observed Cmax of radioactivity was approximately
2.5 ␮g-Eq/ml in the oral dose group at 4 h and approximately twice
that in the intravenous group at 0.25 h. The ratio of the AUC in the
oral dose group to AUC in the intravenous dose group was 0.50 and
0.78 for males and females, respectively.
Tissue Distribution and Excretion of [35S]2. Each rat was admin-
istered [35S]2, 10 mg/kg, by either the intravenous or the oral route.
The intravenous dose of radioactivity was 15.5 Ϯ 0.64 (males) and
14.2 Ϯ 0.15 ␮Ci (females). The oral dose of radioactivity was 50.1 Ϯ
0.91 (males) and 48.0 Ϯ 2.02 ␮Ci (females). The total recovery of
radioactivity determined at 24 h after intravenous administration of
[35S]2 was 83.7 Ϯ 0.32 and 84.9 Ϯ 0.36% for male and female rats
and after oral administration was 83.0 Ϯ 3.59 and 80.4 Ϯ 6.79% for
male and female rats, respectively.
Table 1 summarizes the excretion of radioactivity after intravenous
and oral dosing of [35S]2. After intravenous administration of [35S]2,
approximately 20% of the dose was excreted in the urine in the first
hour. In both males and females, the major route of excretion after
intravenous dosing was in the urine (55–60% of the radioactive dose),
whereas approximately 10 to 20% of the dose was excreted in the
feces in 24 h. Oral administration of [35S]2 resulted in 35 to 40% of
the dose eliminated in the urine in 24 h. In male rats, a similar fraction
of the dose was excreted in the feces, whereas female rats excreted a
much smaller fraction in the feces, ϳ3% in 24 h.
Although individual fractions from tissues and plasma at different
time points were not analyzed by radio-HPLC, on the basis of the
metabolism and stability studies of 1 and 2, it is reasonable to assume
that the radioactivity in tissues and plasma corresponds mostly to that
of [35S]1 or [35S]2 and minimally to 35S-containing metabolites or
elemental 35S.

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
979
FIG. 8. Representative time course HPLC profiles of incubates of Rp-, Sp-2 in SGF that demonstrates the stability of 2 in the acidic environment of stomach.
Discussion
1. In acidic pH, 2 is likely to be protonated in the ring nitrogen of
adenine, and consequently 2 may not be absorbed from stomach by
passive diffusion. It is hence likely that oral absorption of the prodrug
2 occurs predominantly in the upper duodenal region where a signif-
icant portion of the molecule may be in the un-ionized form. It is not
known whether nucleotide transporters in the gastrointestinal tract
have any role in the active transport-mediated absorption of 2 and 1
(Ritzel et al., 2001; Zheng et al., 2004).
These observations further support the hypothesis that the observed
anti-HBV activity of 1 and 2 (Iyer et al., 2004a,b; Coughlin et al.,
2010) is associated with the intact dinucleotide structure. The tissue
distribution studies show that after the oral administration of 2 in rats,
the compound is rapidly absorbed and is readily distributed signifi-
cantly to the liver, the target organ for HBV. The compound is
converted rapidly to the active 1 via plasma and/or liver esterases and
is concentrated in the liver. Once inside the cell, it is possible that the
negatively charged 1 remains trapped in the intracellular compartment
and is made available for sustained antiviral effect.
The studies described in this article were undertaken as part of the pre-
clinical development of a prodrug 2 of the dinucleoside phosphorothioate 1,
a representative SMNH compound, having anti-HBV activity. Metabolic
studies of Rp-, Sp-2 using liver microsomes and S9 fractions show that it is
stereospecifically converted to Rp-,Sp-1 via hydrolysis mediated by esterases.
Neither 2 nor 1 was susceptible to oxidative metabolism by P450- or exo- and
endo-nuclease-mediated fragmentation. Furthermore, there was no evidence
of phase II conjugation reactions. The prodrug 2 was quite stable in SGF
compared with the dinucleotide 1.
Tissue distribution studies of orally and intravenously administered
[35S]2 demonstrated that the radioactivity concentrates in the liver.
According to the metabolic studies, it is likely that most of the
radioactivity in the liver is associated with either [35S]2 or [35S]1. The
preferential distribution of the dinucleotide 1 and its prodrug 2 into
liver may be attributed to the presence of nucleoside phosphorothioate
backbone because PS-ODN also reveal similar tissue distribution
profile upon intravenous administration (Geary, 2009).
The metabolism and tissue distribution studies of the prodrug 2
The dinucleoside phosphorothioate prodrug 2 is quite stable in SGF
and appears to be less stable in SIF where it is converted to the active have revealed a number of interesting findings that may have impli-

980
COUGHLIN ET AL.
TABLE 1
Excretion data after oral and intravenous administration of [35S]2
Data are presented as mean and S.D. values derived at each time point from nine male and nine female rats.
Route
Sex
Time
Urine
Feces
h
% of dose mean
S.D.
% of dose mean
S.D.
Intravenous
M
0–1
1–4
4–24
0–24
0–1
22.83
11.33
29.60
56.15
15.81
6.34
36.79
58.94
NS
NC
6.72
11.46
5.13
5.16
2.86
1.11
1.87
NC
0.33
5.20
5.14
NC
NS
0.01
16.90
16.90
NS
NC
NC
4.57
4.57
NC
Intravenous
F
M
F
1–4
NS
NC
4–24
0–24
0–1
9.44
9.44
0.01
0.01
35.71
35.71
0.02
0.08
2.68
2.72
0.18
0.18
NC
Oral
Oral
1–4
0.86
NC
4–24
0–24
0–1
1–4
4–24
0–24
33.73
34.59
0.16
2.04
34.78
36.88
7.40
7.40
NC
NC
2.31
2.34
1.20
5.31
6.26
M, male; F, female; NC, not calculated; NS, not significant.
cations in the ADME of the longer chain PS-ODN counterparts. As into much shorter fragments including dinucleotides, which are then
mentioned before, ADME studies of PS-ODN and oligos with differ-
ent chemical modifications reveal a “class pattern” in which after
intravenous administration, they are rapidly cleared from plasma after
absorption and are preferentially distributed to liver, kidney, spleen,
and bone marrow. In contrast, our evaluation of ADME properties of
SMNH compounds reveals that they are preferentially distributed in
liver and kidney with less distribution to other tissues. Our studies
suggest that distribution to tissue compartments other than liver and
kidney may depend on the length and charge of the PS-ODN.
In toxicology studies, intravenous administration of PS-ODN
shows a number of dose-dependent effects such as complement acti-
vation, splenomegaly, elevation of liver transaminases, lymphoid hy-
perplasia, immune stimulation manifested as multiorgan mixed mono-
nuclear cell infiltrate with fibroblast proliferation, increased IgM,
cytokine production, etc. that are charge- and sequence-dependent as
well as species-dependent (Henry et al., 1997, 1999). siRNA is also
known to induce immune-stimulatory effects via activation of Toll-
like receptors 7 and 8 (Marques and Williams, 2005) that constitute
one of their important off-target effects. On the other hand, 7-
and 14-day dose-ranging toxicology and toxicokinetic studies of 2
do not show these toxicological characteristics (C. E. Green, K. G.
O’Loughlin, J. Marquis, R. P. Iyer, and J. C. Mirsalis, unpublished
results) that are hallmarks of PS-ODN and siRNA.
eliminated. In this context, it is pertinent to mention that PS-ODNs,
including those that carry additional sugar modifications, appear to be
retained in the tissues over extended periods and slowly metabolized
predominantly in liver and kidney over time. Although several reports of
metabolic studies of ODN with different sequences and chemical modi-
fications have been reported (Geary, 2009), the biological effects and fate
of the shorter fragments that result from metabolism of the parent ODN
remain largely unknown. Our studies show that SMNH compounds,
particularly dinucleotides, may represent the terminal fragments of nu-
clease-mediated metabolism of longer chain ODNs.
Furthermore, as described before, SMNH analogs have a variety of
biological activities. We have found (Iyer et al., 2010) that certain SMNH
analogs can activate cellular cytosolic pathogen recognition receptors
such as retinoic acid-inducible gene (RIG-I) (Katze et al., 2002; Akira et
al., 2006; Saito et al., 2007; Myong et al., 2009) and nucleotide oligomer-
ization domain protein 2 (Sabbah et al., 2009; Ting et al., 2010) that cause
stimulation of innate immune pathways, interferon production and induc-
tion of antiviral state (Saito et al., 2007).
In conclusion, ADME studies of SMNH compounds suggest that they
1) are not subject to P450 metabolism and 2) can be delivered orally via
a prodrug strategy. In addition, the prodrug derivatization facilitates
distribution into the liver, the target organ for hepatitis viruses. A greater
understanding of biological activities of SMNH analogs may help to
design ODNs with minimal off-target effects for application as antisense,
PS-ODN and siRNA are primarily metabolized by exo- and endo-
nucleases into shorter ODN fragments and are excreted primarily through
kidneys. It is possible that most of the ODNs are metabolized eventually RNA interference, aptamers, and immunomodulatory compounds. Fur-
FIG. 9. Ratio of liver to plasma radioactivity concentration after
intravenous and oral administration of [35S]2 in male and female
rats. Data is presented as mean values derived at each time point
from nine male and nine female rats.

ADME STUDIES OF AN ANTI-HBV DINUCLEOSIDE PHOSPHOROTHIOATE
981
as an improved sulfurizing reagent in the solid-phase synthesis of oligodeoxyribonucleoside
phosphoramidites. J Am Chem Soc 112:1253–1254.
TABLE 2
Pharmacokinetic parameters for total radioactivity in plasma after oral and
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Iyer RP, Tan W, Yu D, and Agrawal S (1994) Synthesis of [35S]-3H-1, 2-benzodithiol-3-one-
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Intravenous Dose
Oral Dose
Iyer RP, Jin Y, Roland A, Morrey JD, Mounir S, and Korba B (2004a) Phosphorothioate di- and
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Male
Female
Male
Female
Iyer RP, Roland A, Jin Y, Mounir S, Korba B, Julander JG, and Morrey JD (2004b) Anti-
hepatitis B virus activity of ORI-9020, a novel phosphorothioate dinucleotide, in a transgenic
mouse model. Antimicrob Agents Chemother 48:2318–2320.
Iyer RP, Coughlin JE, and Padmanabhan S (2005a) Rapid functionalization and loading of solid
supports. Org Prep Proc Int 37:205–212.
Iyer RP, Padmanabhan S, Zhang G, Morrey JD, and Korba BE (2005b) Nucleotide analogs as
novel anti-hepatitis B virus agents. Curr Opin Pharmacol 5:520–528.
Iyer RP, Coughlin J, and Padmanabhan S (2006) Metabolism and pharmacokinetic studies of SB
9000–a novel anti-HBV agent. Abstracts of the 19th International Conference on Antiviral
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t1/2, h
Tmax, h
Cmax, ␮g-Eq/ml
Cmaxp.o./Cmaxi.v.
7.8
NA
6.51 Ϯ 0.60
NA
9.0
NA
4.49 Ϯ 0.16
NA
NC
4
2.53 Ϯ 0.30
NC
4
2.50 Ϯ 0.22
0.56
0.39
AUC, h-␮g-Eq/ml 53.58 Ϯ 1.37 35.21 Ϯ 3.23 26.86 Ϯ 3.52 27.37 Ϯ 2.52
AUCp.o./AUCi.v. NA NA 0.50 0.78
NC, not calculated; NA, not applicable; AUCp.o., area under the plasma concentration-time
profile with oral dose administration; AUCi.v., area under the plasma concentration-time profile
with intravenous dose administration.
Iyer RP, Coughlin, JE, Padmanabhan S, Korba, BE, and Myong S (2010) Activation of retinoic
acid inducible gene (RIG-I) by nucleotide analogs–a potential novel mechanism for antiviral
discovery. Abstracts of the 23rd International Conference on Antiviral Research; 2010 Apr
25–27; San Francisco, CA. International Society for Antiviral Research, Washington, DC.
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Immunol 2:675–687.
ther studies on the preclinical development of SMNH compounds are
planned and results will be reported in due course.
Li J and Liang Z (2010) The consideration of synthetic short interfering RNA for therapeutic use.
Basic Clin Pharmacol Toxicol 106:22–29.
Acknowledgments
We thank C. Griesberger of PPG Fine Chemicals for the generous gift of
chloromethyl isopropyl carbonate. We also thank Ceclia Paculba, Sandra Phillips,
and Janice Tugade Corpuz for providing technical support in the ADME study.
Marques JT and Williams BR (2005) Activation of the mammalian immune system by siRNAs.
Nat Biotechnol 23:1399–1405.
Myong S, Cui S, Cornish PV, Kirchhofer A, Gack MU, Jung JU, Hopfner KP, and Ha T (2009)
Cytosolic viral sensor RIG-I is a 5Ј-triphosphate-dependent translocase on double-stranded
RNA. Science 323:1070–1074.
Ollis DL and White SW (1987) Structural basis of protein-nucleic acid interactions. Chem Rev
87:981–995.
Authorship Contributions
Participated in research design: Pandey, Padmanabhan, Marquis, Green,
Mirsalis, and Iyer.
Padmanabhan S, Coughlin JE, Zhang G, Kirk CJ, and Iyer RP (2006) Anti-HBV nucleotide
prodrug analogs: synthesis, bioreversibility, and cytotoxicity studies. Bioorg Med Chem Lett
16:1491–1494.
Padmanabhan S, Coughlin JE, and Iyer RP (2005) Microwave-assisted functionalization of solid
supports. Application in the rapid loading of nucleosides on controlled-pore-glass (CPG).
Tetrahedron Lett 46:343–347.
Peng B, Andrews J, Nestorov I, Brennan B, Nicklin P, and Rowland M (2001) Tissue distribution
and physiologically based pharmacokinetics of antisense phosphorothioate oligonucleotides
ISIS 1082 in rat. Antisense Nucleic Acid drug Dev 11:15–27.
Conducted experiments: Coughlin, Pandey, Padmanabhan, O’Loughlin, and Iyer.
Contributed new reagents or analytic tools: Coughlin and Pandey.
Performed data analysis: Coughlin, Pandey, Padmanabhan, Green, and Iyer.
Wrote or contributed to the writing of the manuscript: Iyer, Pandey, Pad-
manabhan, Marquis, and Mirsalis.
Perrillo RP (2004) Overview of treatment of hepatitis B: key approaches and clinical challenges.
Semin Liver Dis 24 (Suppl 1):23–29.
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Address correspondence to: Radhakrishnan P. Iyer, Spring Bank Pharmaceuticals,
Inc., S-7, 113 Cedar St., Milford, MA 01757. E-mail: kiyer@springbankpharm.com
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