metabolism studies of unformulated...

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Title Page

Metabolism Studies of Unformulated Internally

[3H]-labeled siRNAs in Mice

Jesper Christensen, Karine Litherland, Thomas Faller, Esther van de Kerkhof,

François Natt, Jürg Hunziker, Joel Krauser, and Piet Swart

Novartis Pharma AG, Novartis Institutes for Biomedical Research, Drug Metabolism

and Pharmacokinetics (J.C., K.L., T.F., E.v.d.K., J.K., P.S.), and Biologics Center

(F.N., J.H.), Basel, Switzerland

DMD Fast Forward. Published on March 22, 2013 as doi:10.1124/dmd.112.050666

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 22, 2013 as DOI: 10.1124/dmd.112.050666

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Running Title Page

Running title: ADME-properties of unformulated [3H]-siRNAs (43, max. 60

characters)

Address correspondence to: Dr. Karine Litherland, Novartis Pharma AG, Novartis

Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics,

Fabrikstrasse 14, 1.02, CH-4002 Basel, Switzerland. E-mail:

[email protected]

Number of:

text pages 35

tables 1

figures 4

references 32

Number of words in abstract 256

Number of words in introduction 612

Number of words in discussion 1494

Abbreviations: siRNA, short interfering RNA; RNAi, RNA interference; ADME,

absorption, distribution, metabolism, and excretion; QWBA, quantitative whole-body

autoradiography.


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Abstract

Absorption, distribution, metabolism, and excretion properties of two unformulated

model siRNAs were determined using a single internal [3H]-

radiolabeling procedure, where the full-length oligonucleotides were radiolabeled by

Br/3H-exchange. Tissue distribution, excretion, and mass balance of

radioactivity were investigated in male CD-1 mice, following a single intravenous

administration of the [3H]-siRNAs, at a target dose level of 5 mg/kg.

Quantitative whole-body autoradiography (QWBA) and liquid scintillation counting

techniques were used to determine tissue distribution. Radiochromatogram profiles

were determined in plasma, tissue extracts and urine. Metabolites were separated by

liquid chromatography and identified by radiodetection and high-resolution accurate

mass spectrometry. In general, there was little difference in the distribution of total

radiolabeled components after administration of the two unformulated

[3H]-siRNAs. The radioactivity was rapidly and widely distributed

throughout the body, and remained detectable in all tissues investigated at later time

points (24 and 48 hours for [3H]-MRP4 and [3H]-SSB

siRNA, respectively). After an initial rapid decline, concentrations of total radiolabeled

components in dried blood declined at a much slower rate. A nearly complete mass

balance was obtained for the [3H]-SSB siRNA and renal excretion was

the main route of elimination (38%). The metabolism of the two model siRNAs was

rapid and extensive. Five minutes after administration, no parent compound could be

detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease

hydrolysis were observed. In the metabolism profiles obtained from various tissues

only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly


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metabolized and that the distribution pattern of total radiolabeled components can be

ascribed to small molecular weight metabolites.


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Introduction

Over the last decade, the pharmaceutical industry has shown a significant interest in

RNA interference (RNAi) therapeutics, particularly for the potential of small interfering

RNAs (siRNA) as triggers of this endogenous mechanism, which theoretically can

silence or slow down the production of any protein. This new therapeutic potential

would offer an innovative alternative to treat human diseases which may not be

drugable with traditional approaches. Understanding siRNA biodistribution and

biostability is essential for their development as therapeutics. Although a variety of

non-isotopic labeling techniques exists (e.g. bioluminescence, fluorescence)

(Bogdanov, 2008; Moore and Medarova, 2009), the use of 3H-radiolabeled

oligonucleotides to perform (pre)-clinical in vitro / in vivo studies offers a distinct

advantage of avoiding chemical and structural modifications.

Several pre-clinical studies have been reported using radiolabeled siRNAs.

Nevertheless, the labeling methods applied in those studies had potential drawbacks.

Typically, siRNAs were 3H-labeled at chemically unstable positions (Mook et al.,

2007; Sonoke et al., 2008; Mook et al., 2010) or at problematic sites at the end of the

oligonucleotide, such as by addition of a [32P]-phosphate group to the 5´-end (Beale

et al., 2003; Khan et al., 2004; Ocker et al., 2005; Yanagihara et al., 2006; Malek et

al., 2008; Gao et al., 2009; Malek et al., 2009). End-labeling of siRNAs is not optimal

due to the readily cleavable end from the oligonucleotide in vivo and the loss of label

to follow the distribution.

Alternatively, introduction of radioactive isotopes such as fluorine-18 (Viel et al.,

2008; Hatanaka et al., 2010), copper-64 (Bartlett et al., 2007; Mudd et al., 2010),


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technetium-99m (Kang et al., 2010), indium-111 (van de Water et al., 2006; Merkel et

al., 2009a; Merkel et al., 2009b), or iodine-125 (Braasch et al., 2004) were also

tested. The incorporation of these isotopes often required conjugation with linkers

and/or complexation methods. The possibility for noninvasive imaging techniques is a

clear advantage of utilizing these isotopes (18F, 64Cu, 99mTc, 111In and 125I), which is

not feasible for negative beta emitters (3H, 14C, 32P and 35S), where only invasive

techniques such as scintillation counting or autoradiography have to be used.

Overall, drug candidates with attached pendant groups containing complexed

isotopes are often assumed to not alter the reactivity or metabolism of the molecules.

However, this labeling approach has previously been shown to influence the

pharmacodynamic and pharmacokinetic behavior of large molecules, such as for

proteins by Swart et al. (1996).

Furthermore, biostability of the siRNAs was not considered for the majority of those

studies, and the “intactness” of the oligonucleotides is not understood. In fact, it is

difficult to draw meaningful conclusions on whether the observed distribution of

radioactivity represents the parent compound, metabolites or simply the free

radiolabel.

For our work, a method for labeling siRNAs with tritium at a single internal nucleotide

position (uridine or 2´-O-methyluridine) was applied (Christensen et al., 2012). The

method is thought to eliminate the previously described issues and therefore

provides a suitable research tool for ADME-studies of siRNAs.

With other siRNA ADME studies using randomly labeled oligonucleotides, we here

present results obtained using single internal 3H-radiolabeled siRNAs. The tritium


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was placed in a chemically stable and predetermined internal position by our novel

radiolabeling procedure. The objective of the present studies were to assess the

ADME-properties of two [3H]-siRNAs after intravenous administration to CD-1 mice.

There was little difference in the distribution of the two [3H]-siRNAs tested and renal

excretion was the major route of elimination. Generally, unformulated siRNAs with

modest chemical modifications are considered to be metabolized rapidly in vivo

(Morrissey et al., 2005). This correlates well with our observations. Complete

degradation of the guide strand to radiolabeled nucleosides was observed within 5

min post dosing in plasma.


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Material and Methods

Test compounds and radiolabeling of siRNAs. The ADME properties of two

unformulated siRNAs were studied in mice. For this purpose the siRNA’s were

internally labeled with tritium following procedures previously described (Christensen

et al., 2012). The MRP4 siRNA targets the transporter protein, multidrug resistance

protein isoform 4 (MRP4), which is expressed in the brush-border membrane of the

rat kidney. The second siRNA, the SSB siRNA, targets the ubiquitous gene, Sjögren

Syndrome antigen B (SSB). The 21mer double stranded sequences for the MRP4

and SSB siRNAs were: 5´-ACAGCUCCU9GACACCUCUCdTdT-3´ and 5´-

GAGAGGUGUCAGGAGCUGUdTdT-3´ for the MRP4 guide and passenger strand,

respectively, and 5´-UuAcAUu7AAAGUCUGUUGUuu-3´ and 5´-

AcAAcAGAcuuuAAuGuAAuu-3´ for the SSB guide and passenger strand,

respectively. The MRP4 siRNA was slightly modified by dTdT overhangs (dT =

2´deoxy thymidine), whereas the SSB siRNA was stabilized via selected

incorporation of 2´-O-methyl pyrimidines (u = 2´OMe uridine and c = 2´OMe cytidine)

throughout the sequences. Both siRNAs were uniquely 3H-radiolabeled in the guide

strands. The tritium atom was incorporated into the chemically stable C5-position of a

predetermined uridine or 2´-O-methyluridine (bold typed). The specific radioactivities

of [3H]-MRP4 and [3H]-SSB siRNAs were 5.25 and 6.99 MBq/mg, respectively. Both

siRNAs had a radiochemical purity of 74% by ion pair RP-HPLC.

In vivo experiments. The animal studies were approved by Animal Care and Use

Committees of the Kanton Basel, Switzerland. Male albino CD-1 mice (Charles River

France, 26-32 g, 8-10 weeks) received an intravenous bolus injection in the vena

saphena of either 5 mg/kg [3H]-MRP4 siRNA or 5 mg/kg [3H]-SSB siRNA in 0.9%


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sodium chloride under a light inhalation anesthesia by isoflurane. The radioactive

dose ranged from 26.3 and 35.0 MBq/kg for MRP4 and SSB siRNAs, respectively.

After dosing, blood was collected from either vena saphena (opposite leg than used

for dosing) or vena cava (for end-points) in EDTA containing tubes at 0.083, 0.5, 1, 2,

4, 8, 24 and 48 h post-dose (no 8 and 48 h time points for MRP4 siRNA). From each

end-point, tissues were collected and an aliquot of the blood was processed

immediately to plasma by centrifugation at 3000 g for 10 min at 4ºC. A small aliquot

as taken for total radioactivity detection and the remainder material was snap frozen

and stored at -80ºC until further analysis.

Selected animals were housed in metabolic cages for urine and feces collection

allowing determination of the route of excretion and the mass balance. Quantitative

whole-body autoradiography (QWBA) was performed at 10, 30, 60 min and 2 and 24

h for MRP4 siRNA and at 10, 60 min and 2, 24 and 48 h for SSB siRNA. The use of

animals was minimalized during these studies (11-13 mice for each siRNA), i.e.

every mouse contributed to at least two types of experiments, allowing a good

characterization of its PK, mass balance, routes of excretion, metabolism profiles and

tissue distribution.

In vitro experiments. To support the in vivo metabolism studies, in vitro incubations

with both [3H]-siRNAs in CD-1 mouse serum (mixed gender, SeraLab, West Sussex,

UK) were performed at a concentration of 25 µM (~45 KBq/mL). The mixtures were

incubated at 37°C in a thermo mixer (Thermomixer comfort 5355, Eppendorf,

Hamburg, Germany) for 5, 30 and 60 min and for 2, 4, 8, and 24 h under gentle

mixing. At the respective time points, 200 µL aliquots were mixed with an equal

aliquot of Phenomenex lysis-loading buffer (containing 10 mM TCEP, Phenomenex,


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Torrance, CA, USA) and briefly vortexed. Samples were stored at -80ºC until further

analysis. Additional in vitro metabolic studies were performed for SSB siRNA at 1

and 5 µM under the same experimental conditions. The radiochemical purities of

[3H]-MRP4 and [3H]-SSB siRNAs used for in vitro experiments were 72% and 88%,

respectively.

Analytical Methods

Radiometric analysis. Radioactive signals in the different matrices were quantified

by LSC, using Liquid Scintillation Systems 2500 TR from Packard Instr. Co.

(Meriden, CT, USA). For quench correction, an external standard ratio method was

used. Quench correction curves were established by means of sealed standards

(Packard). Background values were prepared for each batch of samples according to

the respective matrix. The limit of detection was defined as 1.8 times the background

value. All determinations of radioactivity were conducted in weighed samples. In

order to determine possible formation of tritiated water, samples were analyzed

twice: direct measurements of radioactivity and after drying of the sample (at 37ºC for

at least 12 h). Dried or non-dried plasma and urine were mixed with scintillation

cocktail directly, whereas whole blood and feces samples were solubilized before

radioactivity analysis. Measured radioactivity for blood, plasma and tissues was

converted into concentrations (mol equivalents per volume or gram) considering the

specific radioactivity and assuming a matrix density of 1 g/mL.

Tritiated water. The determination of tritiated water was based on blood and plasma

data. The following assumptions were considered: I) Radioactivity was measured in

non-dried blood/plasma and in dried blood/plasma. The difference between the non-


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dried and dried sample was ascribed to tritiated water. II) The derived concentration

of tritiated water in the respective matrix equals the concentration in the body water.

Therefore, the tritiated water concentrations in blood were corrected by the blood cell

content (92.5% of blood was expected to be water). III) The elimination half-life (T1/2)

of water in the mouse was 27.1 h (Richmond et al., 1962) and the body water 72.5%

(Davies and Morris, 1993) of body weight. For the calculation, the concentration-time

course of tritiated water in blood and plasma was analyzed by the deconvolution

method given in Phoenix™ WinNonlin® software (Pharsight Corp., Mountain View,

CA, USA, version 6.1.0.173). As input parameter for the deconvolution, the

elimination rate constant of water and the reciprocal value for body water, derived

from the actual body weight for the mice, were used.

Quantitative whole-body autoradiography (QWBA). Mice were sacrificed by deep

isoflurane inhalation. After deep anesthesia, blood for radioactivity determination by

LSC was collected by vena saphena (the other leg than the one used for dosing).

The animals were then submerged in n-hexane/dry ice at -70°C for approximately 30

minutes. The carcasses were stored at -18°C and all subsequent procedures were

performed at temperatures below -20°C to minimize diffusion of radiolabeled

materials into thawed tissues. Sections were prepared as follows: in brief, 40 µm

thick lengthwise dehydrated whole-body sections were prepared and exposed to Fuji

BAS III imaging plates (Fuji Photo Film Co., Ltd., J-Tokyo) in a lead-shielded box at

room temperature for two weeks, and then scanned in a Fuji BAS 5000 phosphor

imager (Fuji Photo Film) at a 50 µm scanning step. The concentrations of total

radiolabeled components in the tissues were determined by comparative

densitometry and digital analysis of the autoradiogram; blood samples of known


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concentrations of total radiolabeled components processed under the same

conditions were used as calibrators.

Nomenclature and assignment of the metabolites. Metabolites which were clearly

identified, for example by chromatographic retention time and/or mass spectral

information, are named according to the origin of the metabolite; ASx for guide

strand, Sx for passenger strand or Mx for monomer related metabolites (x being a

positive integer). Furthermore, if not stated otherwise, the metabolite is a cleavage

product with no phosphate. A phosphate on the cleavage product is designated by a

P and a cyclic phosphate is designated cycP (e.g. AS4cycP). In some cases, a peak

contains more than one metabolite. The percentage of the minor metabolite, as

based on mass spectrometry responses making up that peak is given. Pronounced

but uncharacterized peaks in radiochromatograms, e.g. because of their abundance,

are named as Px (x representing the approximate retention time).

Structural elucidation of oligonucleotide metabolites. The structural

characterizations of the parent and metabolites were done using LC-MS. A HPLC

method under denaturing conditions was used for profiling the parent guide (and

passenger) strand and oligonucleotide metabolites (LC-method 1). For smaller and

more polar metabolites, which showed no retention on the HPLC method, an UHPLC

method was used (LC-method 2). Mass spectrometric data were obtained using a

LTQ-Orbitrap (ThermoFisher Scientific) in electrospray negative or positive mode.

The radiochromatogram profiles were analyzed and identified with off-line

radioactivity analysis. Metabolites were identified in each profile by MS and/or

corresponding peaks in the radiochromatogram versus a control sample. Full mass

spectra were acquired for the parent compound and each metabolite. An MS


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spectrum of each peak gave a range of charged species up to [M-13H]13-, each with

a typical multiply charged ion envelope.

The spectra were deconvoluted to give the accurate mass of the metabolites which

were compared with the corresponding theoretical masses. The exact mass

calculation was performed using the first isotopic ion of the major multiply charged

ion using the following formula: Exact mass = (Mfirst isotopic ion × n) + n × H, (n = charge

state of the selected ion). ProMass deconvolution software (Novatia, version 2.5) for

Thermo instruments was applied to help with metabolite identification (Zou et al.,

2008).

Sample preparation of matrices for metabolite profiling. For metabolite profiling

of plasma and serum, the oligonucleotides were extracted and isolated by the use of

Clarity OTX, weak anion exchange solid phase extraction cartridges from

Phenomenex (WAX-SPE), according to the manufacturer’s instructions. As low and

high molecular weight material were expected the wash fraction of the SPE was

analyzed by LC-method 2 (low molecular weight material), whereas the higher

molecular weight metabolites were collected within the elution fraction and analyzed

using LC-method 1. For metabolic profiling of urine, a precipitation method was

carried out before analysis using two volumes of acetonitrile. The mixture was

vortexed and centrifuged at 17530 g for 15 min at 4ºC (Beckman GS-15R). The

supernatant was recovered and concentrated to near dryness using an Eppendorf

concentrator 5301 at 30ºC and reconstituted to about 50 µL with water.

Tissues were homogenized using a CryoPrep™ system from Covaris. Equal volumes

of water (by weight) were added to the pulverized tissues, and the homogenates


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were extracted with 3 volumes of acetonitrile overnight at 4ºC in a thermo mixer

(Thermomixer comfort 5355) under agitation (750 RPM). The samples were then

centrifuged at 4ºC, 20000 g for 20 min (Sigma 3K30, Osterode am Harz, Germany).

The supernatants recovered and concentrated under a nitrogen flow to approximately

the initial volume of water, and up to 40 µL of sample were injected on the LC-

systems. Extraction recoveries of radioactivity of all the sample extraction procedures

described were all above 85%.

HPLC-MS-RA metabolite identification of oligonucleotide metabolites (LC-

method 1). Selected plasma, urine, and tissue samples were analyzed for metabolite

identification purposes by ion pairing reverse phase (IP-RP) HPLC-MS-RA.

Separation was accomplished using an HPLC system from Agilent Technologies

(Santa Clara, CA, USA), 1100 Series equipped with a binary capillary pump,

including a degasser, an autosampler, a column oven and a diode array detector.

The operating software monitoring the HPLC system was LC/MSD Chemstation

Version B.04.02 (Agilent Technologies). 10 µL samples were injected on a reverse

phase C18 column (Waters XBridge Shield; 150 × 2.1 mm; 3.5 µm particles)

equipped with a guard column (Waters XBridge; 10 × 2.1 mm; 3.5 µm particles,

Water, Milford, MA, USA). The column was maintained at 70°C (denaturing

conditions), and the flow rate on the column was 0.25 mL/min. Eluent A was made up

of 1.6 mM triethylamine, 100 mM hexafluoroisopropanol (pH ~7.5), and eluent B was

made up of 1.6 mM triethylamine, 100 mM hexafluoroisopropanol in 40% methanol.

The following gradient was applied: 0% B for 10’ → 2.5% B/min → 50% B after 20’ →

450% B/min → 95% B after 0.1’ → 95% B for 4.9’ → -950% B/min → 0% B after

0.1’→ 0% B for 4.9’. After elution from the column, the effluent was split.


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Approximately 20 µL was directed to the MS, while the remaining flow was monitored

by a DAD detector at λ = 260 nm and collected into 96-well Luma plates using a

fraction collector (Gilson FC204, 4.2 seconds fraction size, collect from 0-27 min,

Gilson, Middleton, WI, USA). In order to help with the fraction collection, a make-up

flow composed of mobile phase B was used (Jasco pump, PU-980, 0.25 mL/min,

Essex, UK). The plates were dried using a speed-vac (AES2010 from Savant) and

counted (up to 60 min per well, depending on the level of radioactivity) on a Packard

TopCount instrument (PerkinElmer, Waltham, MA, USA). Mass measurements were

made online using a LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific)

operating under Xcalibur™, version 2.0 for instrument control, data acquisition and

data processing. From 0 to 40 min, the mass spectrometer was set to scan in ESI

negative mode with resolution set at 30000 and an m/z window from 400 to 2000.

Mass spectra were obtained using a spray voltage of 3.6 kV, a sheath gas flow of 16

(arbitrarily flow rate), a source temperature at 275ºC, and a capillary and tube lens

voltage of -53 and -94.3 V, respectively.

UHPLC-MS-RA metabolite identification of monomer metabolites (LC-method

2). Selected plasma, urine, and tissue samples were analyzed for metabolite

identification by UHPLC-MS-RA. Separation was accomplished using a UHPLC

system from Agilent Technologies (Santa Clara, CA, USA), 1290 Infinity equipped

with a binary capillary pump including a degasser, an autosampler and a column

oven. The operating software monitoring the UHPLC system was Chemstation

Version B.04.02 (Agilent Technologies). Typically 10 µL sample was injected on a

reverse phase C18 column (Waters Acquity UPLC HSS T3; 150 × 2.1 mm; 1.7 µm

particles) with a guard Waters Acquity UPLC HSS column attached (5 × 2.1 mm; 1.7


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µm particles). The column was maintained at 30°C, and the flow rate was 0.35

mL/min. Eluent A was made up of 10 mM ammonium formate (pH adjusted to 3), and

eluent B was made up of 0.1% formic acid in methanol. The gradient used was: 0% B

for 2’ → 2.5% B/min → 20% B after 8’ → 37.5% B/min → 95% B after 2’ → 95% B for

3’ → -950% B/min → 0% B after 0.1’→ 0% B for 2.9’. After elution from the column,

the effluent was mixed with 0.20-0.35 mL/min of methanol or ethanol (Jasco pump,

PU-980) to increase the droplets per well during fraction collection. The effluent was

then split for MS and fraction collection (Gilson GX-271 operation software was

Trilution LC) into 384-well Luma plates (3.0-3.2 seconds fraction size). The plates

were dried using a speed-vac (AES2010 from Savant) and counted up to 20 min per

well, depending on the level of radioactivity, using a Packard TopCount instrument

(PerkinElmer). Mass measurements were made online using a LTQ-Orbitrap XL

mass spectrometer (ThermoFisher Scientific) operating under Xcalibur™, version 2.0

for instrument control, data acquisition and data processing. From 0 to 18 min, the

mass spectrometer was set to scan in ESI positive or negative mode with resolution

set at 15000 and a m/z window from 50 to 1000. Mass spectra were obtained using a

spray voltage of ESI+/ESI- 3.6/3.0 kV, a sheath gas flow of 16 (arbitrarily flow rate), a

source temperature at ESI+/ESI- 225/275ºC, a capillary voltage of ESI+/ESI- 10/-53 V

and tube lens voltage of ESI+/ESI- 70/-94.3 V.

Pharmacokinetic analysis. Pharmacokinetic parameters and QWBA data were

calculated using Phoenix™ WinNonlin® software (Pharsight Corp., Mountain View,

CA, USA, version 6.1.0.173), unless stated otherwise. The pharmacokinetic

estimations were based on a non-compartmental analysis model. The tissue to blood

Cmax and AUClast ratios were calculated where possible. The AUClast were calculated


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using the linear trapezoidal rule. For metabolite profiles, the peaks in the

radiochromatograms were integrated, and from the relative peak areas, the

concentrations of individual radiolabeled components in plasma/serum and the

amounts of individual radiolabeled components in excreta were calculated as follows:

Ci,plasma = CRA,plasma % % and Ai,excreta = CRA,excreta % %

With: Ci, plasma Molar concentration of radiolabeled component i in plasma/serum; CRA,

plasma Molar concentration of total radiolabeled components (radioactivity) in plasma;

RSP Recovery of radioactivity after sample preparation (%); RPAi Relative peak area

of radiolabeled component i in radiochromatogram (% of total area under the

radiochromatogram); Ai, excreta Amount of radiolabeled component i in excreta (urine or

feces; % of dose); ARA, excreta Amount of total radiolabeled components (radioactivity)

in excreta (urine or feces; % of dose).


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Results

Blood/plasma pharmacokinetics. After intravenous administration of [3H]-MRP4 or

[3H]-SSB siRNA, the concentrations of radiolabeled components were measurable

throughout the observation periods in blood and plasma (for [3H]-MRP4 siRNA: 24

and 4 h post dose in blood and plasma, respectively (figure 1a), and for [3H]-SSB

siRNA up to 48 h post dose in both blood and plasma (figure 1b)). The concentration

of total radiolabeled components declined in a multi-exponential manner in both

matrices. Following the apparent Cmax at 5 min, the radioactivity declined rapidly

with elimination half-lives below 10 min. Samples dried prior to radioactivity analysis

showed in all cases lower levels of total radioactivity suggesting the formation of

tritiated water. Measurements of dried blood were comparable with results derived

from QWBA data, as shown in figure 1a and 1b for [3H]-MRP4 and [3H]-SSB siRNA,

respectively. Detection and quantitation of the guide strands was only feasible in the

5 min samples. This suggests an extremely fast rate of metabolism and the early

presence of degradation products in the central circulation.

Tissue distribution and mass balance. Following single intravenous administration

of the two [3H]-siRNA’s, the radioactivity was rapidly and widely distributed

throughout the body. At the last time points after administration (24 and 48 h for [3H]-

MRP4 and [3H]-SSB siRNA, respectively), all tissues, including blood, still contained

measurable radioactivity. Tmax was observed in most matrices at the first sampling

point (10 min after administration). In general, the highest concentrations of total

radiolabeled components (Cmax values) were observed in the kidney (10 min p.d.),

which were 7 to 8-fold higher than in blood (141 and 200 nM for [3H]-MRP4 and [3H]-

SSB siRNA, respectively). Additionally, high concentrations of radioactivity were


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determined in the salivary gland being 2 to 6-fold higher than in blood. Lowest

concentrations (but still above LOQ) were generally those in the central nervous

system. Overall, the highest exposures of total radiolabeled components were

observed in kidney, salivary gland, spleen, and liver. In general, there was no notable

difference in the pattern of distribution following [3H]-MRP4 and [3H]-SSB siRNA

administration. The figures 1a and 1b display concentration-time profiles of total

radiolabeled components in dried blood, plasma, and selected tissues after [3H]-

MRP4 and [3H]-SSB siRNA administration, respectively, and Figures 2a and 2b

display selected whole-body autoradioluminograms. Mass balance data obtained for

[3H]-MRP4 and [3H]-SSB siRNA at 24 and 48 hours p.d., respectively, are presented

in table 1. For the [3H]-SSB siRNA tritiated water was calculated to 26% (this could

be caused by the endogenous metabolism of uridine nucleosides. However, on-going

research suggests this is not the case). The mass balances for [3H]-MRP4 and [3H]-

SSB siRNA were in-complete (50%) and complete (87%), respectively.

Metabolic stability of 3H-label. The fraction of the administered radioactive dose

following intravenous administration of [3H]-SSB siRNA converted to tritiated water,

was estimated. Following administration, the percentage of administered radioactivity

transformed to tritiated water increased with time in mice. The average formation of

tritiated water was estimated at 9% of the radioactive dose when considering the

blood and plasma at 2 h after dosing, however, the mean value increased to 26% of

the dose at 48 h after dosing. This increase indicated that the formation of tritiated

water was an ongoing process and an average value, considering all blood and/or

plasma sample time points, could not be given. Therefore, the values derived from 48


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h were used for the mass balance calculation, since this value reflected the total

formation of tritiated water most accurately.

In vivo metabolite profiles in plasma, urine, and tissues.

Plasma. No intact guide strand of either [3H]-siRNAs were observed after their i.v.

administration in any of the analyzed plasma samples, not even 5 min post dosing.

As a results, the half-lives of the parent compounds in plasma could not be

determined, but were clearly lower than 5 minutes for both [3H]-siRNAs. The

metabolites detected in plasma 5 min post dosing included 18- to 13mers (figure 3,

for description of the metabolites of [3H]-MRP4 and [3H]-SSB siRNAs are referred to

supplemental data table S1 and S2 and supplemental data table S3 and S4,

respectively). Furthermore, also complete degradation to the radiolabeled

nucleosides were observed; i.e. 2´-O-methyluridine (2´OMeU) and uridine (M1). The

observed retention times in radiochromatograms of these two nucleoside metabolites

corresponded with their reference substances, and the correct m/z was extracted

with a mass accuracy below 5 ppm (4.67 and 4.94 ppm for 2´OMeU and M1,

respectively). Additionally, [3H]-SSB siRNA O-demethylation of the 2´-O-

methyluridine stabilized guide strand to uridine was observed within 5 min post

dosing in plasma (figure 4). These observations were considered as strong

indications that the guide strand of both [3H]-siRNAs were completely metabolized to

the nucleoside level in the mouse.

Urine. The mean total recovery of radiolabeled components up to 48 h after dosing

of [3H]-SSB siRNA recovered in urine and feces were 47% and 5%, respectively.

Metabolite identification in feces was not conducted due to its low representation of


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the dose. Intact guide strand of [3H]-SSB siRNA was not observed in the urine up to

48 h post dosing, which is consistent with the plasma results. Large molecular weight

oligonucleotide metabolites were identified in urine up to 8 h after dosing, and

represented 31% of the dose for the mouse sacrificed at 24 h. The most prominent

component was a 15mer (AS7, 10% of the dose). In addition, the radiolabeled

nucleoside 2´-O-methyluridine and its corresponding metabolites represented 11% of

the dose in urine up to 8 h. Both nucleosides were also detected in urine at later time

points (up to 48 h after dosing), but only as minor components.

Tissues. No intact material of the guide strand, or even any oligonucleotide

metabolites were detected in kidney, liver or salivary gland 10 min or 1 h post dosing

of [3H]-MRP4 or [3H]-SSB siRNA, respectively. Only uridine (M1) was detected in

selected tissues (heart, kidney, liver, small intestine, and spleen) 1 h post dosing of

[3H]-SSB siRNA. The uridine nucleoside metabolite was also detected in kidney, liver

and salivary gland after 10 min post dosing of [3H]-MRP4 siRNA. For both siRNAs,

the abundance of radiolabeled uridine was the highest in kidneys, amongst the

investigated tissues. These findings supported the results obtained from plasma and

urine samples.

In vitro metabolite profiles in serum. In order to support metabolism identification,

the degradation patterns of both [3H]-siRNAs were investigated via incubations at 25

µM in CD-1 mouse serum for up to 24 h. Concentrations of the guide strands (AS)

were measurable for [3H]-MRP4 siRNA up to 4 h and for [3H]-SSB siRNA up to 8 h.

The half-lives of both guide strands were approximately 1.7 h. For both [3H]-siRNAs,

the smallest degradation products observed at 25 µM incubations were 13mers of

both the guide and passenger strands.


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For both, all observed metabolites of the guide strand and passenger strand

originated from intramolecular cleavages from nuclease activity at the

phosphodiester linkages. Refer to supplemental data for kinetic profiles and mass

spectrometric data for the proposed metabolites and parent compounds of the guide

and passenger strands of the [3H]-siRNAs. The degradation appeared to occur in a

stepwise manner, leading to the consecutive formation of smaller fragments of the

strands (n-1 etc.). For the [3H]-SSB siRNA the degradation occurred primarily from

the 3´-end towards the 5´-end. Most of the metabolites observed were terminal

cleavage products without a phosphate, but phosphate and cyclic phosphate

conjugates were also observed as terminal cleavage products (supplemental data

Figure S1 and Figure S2).

By performing incubations at lower concentrations (5 and 1 µM for 24 h), the

metabolism of the [3H]-SSB siRNA was shown to be concentration dependent. The

metabolism profiles obtained at higher concentrations were different to those

obtained at lower concentrations. Incubations performed at lower concentrations led

to higher turnover of the [3H]-SSB siRNA. Furthermore, complete degradation of the

guide strand to the radiolabeled nucleoside 2´OMeU was also observed in vitro after

24 h incubation at 1 µM. This observation supports the in vivo observations, that the

guide strand of the [3H]-SSB siRNA was completely metabolized to the nucleoside

level.


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Discussion

Understanding siRNA biodistribution and biostability behavior is essential for their

development as therapeutics. A novel 3H-radiolabeling method of oligonucleotides

(Christensen et al., 2012) was used to perform pre-clinical ADME-studies of

unformulated siRNAs. Tritiated oligonucleotides offer several advantages for ADME-

studies including: I) No chemical/structural modification of the oligonucleotide (3H

replacing a 1H), i.e. the reactivity and metabolism of the molecule should be remain

unchanged. II) These [3H]-siRNAs have been proven to be both radio and chemically

stable for at least 6 months at -80ºC (Christensen et al., 2012), which allows a

practical time window to conduct pharmaceutical formulation and pharmacokinetic

studies. III) The 3H-label is not released by metabolic processes from the guide

strand of the siRNA until it has been extensively degraded, and is likely then to be

RNA interference inactive. IV) Simplification of the analysis of metabolites due to the

presence of a single radiolabeled site in the oligonucleotide. V) The method is solely

dependent upon the existence of a uridine or a 2´-O-methyluridine residue in the

sequence. This method should be applicable to chemically modified as well as

unmodified phosphodiester oligonucleotides. However, the observed increase in

formation of tritiated water over time (26% of the dose at 48 h p.d.) remains a

limitation.

To address this issue, positioning of the tritium in the C6-position of a pyrimidine,

instead of the current C5-position, could improve the metabolic stability, thereby

reducing the formation of tritiated water. The chemical stability of the carbon-tritium

bond in the C6-position of pyrimidines is known in the literature to be more stable

than the C5-position (Evans et al., 1970; Noll and Mittag, 1983). The 3H-radiolabeling


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method as used in this study remains a powerful tool for pre-clinical ADME-studies.

The methods presented in this paper could be applicable to a wide range of siRNA

formulations as well as providing useful perspectives for the further development of in

vivo applications of siRNA as therapeutics.

This study presents a comparison of the pharmacokinetics and metabolism of two

unformulated [3H]-siRNAs. The two siRNAs, MRP4 and SSB, differ in sequences and

targets. Previously reported biodistribution studies of unformulated siRNAs generally

have not focused on the biostability of the siRNA, and therefore the intactness of the

oligonucleotides is less clear. In these cases, a conclusion is difficult to draw on what

extent the observed distribution of radioactivity represents the parent compound,

metabolites or simply the free radiolabel. This study reveals the in vivo fate of this

class of compounds in mice.

Metabolite identification of plasma, urine, and tissue samples were conducted to

elucidate the in vivo fate of the [3H]-siRNAs. Metabolites were separated by liquid

chromatography and subsequently identified by radiodetection and high-resolution

accurate mass spectrometry. In addition, in vitro incubations of both [3H]-siRNAs in

CD-1 mouse serum were done to support metabolite profiling and metabolite

identification. The degradation of the [3H]-SSB siRNA was shown to be concentration

dependent. Overall, the rate of in vivo degradation for both siRNAs clearly exceeded

their rate of degradation in vitro. This difference in degradation rates has also been

reported by Viel et al. (2008).

In general, there was little difference in the distribution of these two unformulated

[3H]-siRNAs. After intravenous administration of the [3H]-siRNAs, the radioactivity


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was rapidly and widely distributed throughout the body, which was determined by

QWBA in all tissues investigated up to 24 and 48 h for the MRP4 and SSB siRNA,

respectively. At early time points, the highest concentrations of radioactivity were

located in the kidney. Lowest concentrations (but still above LOQ) were generally

those in the central nervous system. At later time points, the highest concentrations

of total radiolabeled components were observed in kidney, salivary gland, spleen and

liver. Our observations where the kidney, followed by the liver, are the primary

organs for distribution of radioactive components after systemic administration of

unformulated [3H]-siRNAs, are in agreement with other unformulated siRNA

biodistribution studies, reported in mice (Braasch et al., 2004; Ocker et al., 2005;

Bartlett et al., 2007; de Wolf et al., 2007; Mook et al., 2007; Viel et al., 2008; Gao et

al., 2009; Malek et al., 2009; Merkel et al., 2009a; Merkel et al., 2009b; Hatanaka et

al., 2010; Kang et al., 2010; Mook et al., 2010; Mudd et al., 2010).

Among the investigated tissues, the parent guide strand of either siRNAs or any

oligonucleotide metabolites were not observed - even 10 min post dosing for the

MRP4 siRNA. Nevertheless, radiolabeled nucleoside metabolites were observed for

both siRNAs, including the monomer uridine (M1).

Generally, unformulated siRNAs with modest chemical modifications are considered

to be metabolized rapidly in vivo (Morrissey et al., 2005), which correlates well with

the observations presented in this work. Complete degradation of the guide strand of

either [3H]-siRNAs to their radiolabeled nucleosides was observed only 5 min post

dosing in plasma. In addition, within 5 min p.d. in plasma, an O-demethylation of the

radiolabeled 2´OMeU nucleoside to uridine was also observed (figure 4). As parent

guide strand of either siRNAs were not observed in plasma, intact parent compound


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was assumed not to be distributed in the tissues. This observation was supported by

metabolite investigations in selected tissues and in urine. The radiolabeled

nucleosides were also observed in vitro in serum. The extremely rapid degradation to

monomers has also been observed in vivo for the comparable antisense technology

with oligodeoxynucleotides, as reported by Sands et al. (1994 and 1995).

A nearly complete mass balance was obtained for the [3H]-SSB siRNA at 48 h (87%).

Radiolabeled components were excreted predominantly via the urine (38%) and only

to a minor extent via feces (7%). No parent compound was observed in the urine,

and the recovered radioactivity was associated with both oligonucleotide and

nucleoside metabolites. These findings supported the results obtained from plasma

and tissue samples.

The MRP4 siRNA has previously been radiolabeled with 111In and administered

intravenously for a biodistribution study in rats by van de Water et al. (2006). The

labeling was placed using a cDTPA-chelator/[111In]-complex conjugated to an amino

C7 linker at the 3´-end of the guide strand of the MRP4 siRNA. This technique has

some potential drawbacks, i.e. short half-life of 111In (2.8 days), 3´-endlabeling, and

the effect of the chemical modification by the bulky chelator/[111In]-complex on the

distribution and pharmacokinetics is unknown. Studies using proteins have shown

that labeling by 125I affected the pharmacokinetics compared to the molecule labeled

with carbon-14 (Swart et al., 1996).

The siRNA labeled with either 111In or 3H were both found to distribute primarily to the

kidneys and predominantly excreted via the urine. As a standard of reference, the

radioactivity per matrix was determined by calculating the percentage of the injected


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dose per gram matrix (% ID/g). The amounts of radiolabeled components in the

kidneys one hour after injection were 1.7% ID/g and 9.3% ID/g. This on average was

40 and 5 times higher than in comparison to other matrices investigated (blood,

brain, intestine, liver, lung, muscle and spleen) for the 111In- and 3H-labeled MRP4

siRNA, respectively. All observed matrix concentrations, one hour post dosing, for

the [3H]-siRNA were higher than for the [111In]-siRNA. Furthermore, liver distribution

was not observed by van de Water et al. This discrepancy does not appear to be

explained by interspecies differences (rat vs. mouse), since another study in rat (and

mouse) described by Viel et al. (2008) reported kidney and liver distributions in both

rodents. Additionally, for the [3H]-siRNA, significant distribution of radioactivity was

observed in the salivary gland and spleen (8.9% and 2.4% ID/g, respectively). The

salivary gland in contrary to the spleen (Mook et al., 2007; Mook et al., 2010) has not

before been reported as an organ of distribution of total radiolabeled components

after siRNA administration.

The distribution studies indicated most likely not intact siRNA, but more likely the

degraded nucleosides. This was definitely the case for our model [3H]-siRNA’s, as

discussed above. Only a few of the above mentioned studies addressed this: Gao et

al. (2009) by northern blotting, and Malek et al. (2009) by autoradiography on gel. By

these methods, both groups discovered that almost none of the radioactivity taken up

by the kidney corresponded to intact siRNA. Viel et al. (2008) studied the biostability

of the parent compound in plasma by HPLC, and found that the unmodified siRNA

was rapidly degraded with a half-life of approximately 3 min.

The observed differences in distribution and pharmacokinetics between the [111In]-

and [3H]-MRP4 siRNA may be caused by the location of the label (3´-end vs.


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internal), the radiolabeling method used (111In vs. 3H), and/or interspecies differences

(rat vs. mouse). Overall, the 111In-labeled MRP4 siRNA showed different distribution

and pharmacokinetics compared to the molecule labeled with 3H.

In conclusion, the two unformulated [3H]-siRNAs were rapidly metabolized in vivo in

the mouse. The parent guide strands of either siRNAs were not observed in any

matrices (plasma, urine and tissues). Instead, complete degradation of the siRNA

occurred. The distribution of the radioactivity is therefore more associated to the

distribution of radiolabeled nucleoside than that of the siRNA.


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Acknowledgements

We sincerely thank nonclinical PK/PD for their excellent support with the animal and

QWBA studies (Ms Martina Suetterlin, Ms Sandra Wehrle, Ms Denise Schaerer, Ms

Heidrun Reilmann, and Mike Becquet). We would also like to gratefully acknowledge

Alexandre Catoire, Cyril Castel, and Maxime Garnier for their excellent technical

assistance with LC-MS-RA analysis, and finally, Dr. Michael Beverly for his expertise,

guidance and support.


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Authorship Contributions

Participated in research design: Christensen, Litherland, Faller, van de Kerkhof, Natt,

Hunziker, Krauser, and Swart

Conducted experiments: Christensen

Contributed with new reagents or analytical tools: Faller, van de Kerkhof, Swart

Performed data analysis: Christensen, Litherland, Faller, van de Kerkhof, Swart

Wrote or contributed to the writing of the manuscript: Christensen, Litherland, Faller,

Krauser, Swart


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References

Bartlett DW, Su H, Hildebrandt IJ, Weber WA and Davis ME (2007) Impact of Tumor-

Specific Targeting on the Biodistribution and Efficacy of siRNA Nanoparticles

Measured by Multimodality In Vivo Imaging. Proceedings of the National Academy of

Sciences of the United States of America 104:15549-15554.

Beale G, Hollins AJ, Benboubetra M, Sohail M, Fox SP, Benter I and Akhtar S (2003)

Gene Silencing Nucleic Acids Designed by Scanning Arrays: Anti-EGFR Activity of

siRNA, Ribozyme and DNA Enzymes Targeting a Single Hybridization-Accessible

Region Using the Same Delivery System. Journal of Drug Targeting 11:449-456.

Bogdanov AA (2008) Merging Molecular Imaging and RNA Interference: Early

Experience in Live Animals. Journal of Cellular Biochemistry 104:1113-1123.

Braasch DA, Paroo Z, Constantinescu A, Ren G, Oz OK, Mason RP and Corey DR

(2004) Biodistribution of Phosphodiester and Phosphorothioate siRNA. Bioorganic &

Medicinal Chemistry Letters 14:1139-1143.

Christensen J, Natt F, Hunziker J, Krauser J and Swart P (2012) Tritium Labeling of

Full-Lengh Small Interfering RNAs. J Label Compd Radiopharm 55:189-196.

Davies B and Morris T (1993) Physiological-Parameters in Laboratory-Animals and

Humans. Pharmaceutical Research 10:1093-1095.

de Wolf HK, Snel CJ, Verbaan FJ, Schiffelers RM, Hennink WE and Storm G (2007)

Effect of Cationic Carriers on the Pharmacokinetics and Tumor Localization of


at ASPE

T Journals on A

pril 21, 2018dm


ownloaded from


DMD#50666

32

Nucleic Acids After Intravenous Administration. International Journal of

Pharmaceutics 331:167-175.

Evans EA, Sheppard HC and Turner JC (1970) Validity of Tritium Tracers - Stability

of Tritium Atoms in Purines, Pyrimidines, Nucleosides and Nucleotides. Journal of

Labelled Compounds 6:76-87.

Gao S, Dagnaes-Hansen F, Nielsen EJB, Wengel J, Besenbacher F, Howard KA and

Kjems J (2009) The Effect of Chemical Modification and Nanoparticle Formulation on

Stability and Biodistribution of siRNA in Mice. Molecular Therapy 17:1225-1233.

Hatanaka K, Asai T, Koide H, Kenjo E, Tsuzuku T, Harada N, Tsukada H and Oku N

(2010) Development of Double-Stranded siRNA Labeling Method Using Positron

Emitter and Its In Vivo Trafficking Analyzed by Positron Emission Tomography.

Bioconjugate Chem 21:756-763.

Kang L, Wang RF, Yan P, Liu M, Zhang CL, Yu MM, Cui YG and Xu XJ (2010)

Noninvasive Visualization of RNA Delivery With (99m)Tc-Radiolabeled Small-

Interference RNA in Tumor Xenografts. Journal of Nuclear Medicine 51:978-986.

Khan A, Benboubetra M, Sayyed PZ, Ng KW, Fox S, Beck G, Benter IF and Akhtar S

(2004) Sustained Polymeric Delivery of Gene Silencing Antisense ODNs, siRNA,

DNAzymes and Ribozymes: In Vitro and In Vivo Studies. Journal of Drug Targeting

12:393-404.

Malek A, Czubayk F and Aigner A (2008) PEG Grafting of Polyethylenimine (PEI)

Exerts Different Effects on DNA Transfection and siRNA-Induced Gene Targeting

Efficacy. Journal of Drug Targeting 16:124-139.


at ASPE

T Journals on A

pril 21, 2018dm


ownloaded from


DMD#50666

33

Malek A, Merkel O, Fink L, Czubayko F, Kissel T and Aigner A (2009) In Vivo

Pharmacokinetics, Tissue Distribution and Underlying Mechanisms of Various PEI(-

PEG)/siRNA Complexes. Toxicology and Applied Pharmacology 236:97-108.

Merkel OM, Librizzi D, Pfestroff A, Schurrat T, Behe M and Kissel T (2009a) In Vivo

SPECT and Real-Time Gamma Camera Imaging of Biodistribution and

Pharmacokinetics of siRNA Delivery Using an Optimized Radiolabeling and

Purification Procedure. Bioconjugate Chem 20:174-182.

Merkel OM, Librizzi D, Pfestroff A, Schurrat T, Buyens K, Sanders NN, De Smedt SC,

Behe M and Kissel T (2009b) Stability of siRNA Polyplexes From Poly(Ethylenimine)

and Poly(Ethylenimine)-g-Poly(Ethylene Glycol) Under In Vivo Conditions: Effects on

Pharmacokinetics and Biodistribution Measured by Fluorescence Fluctuation

Spectroscopy and Single Photon Emission Computed Tomography (SPECT)

Imaging. Journal of Controlled Release 138:148-159.

Mook OR, Baas F, de Wissel MB and Fluiter K (2007) Evaluation of Locked Nucleic

Acid-Modified Small Interfering RNA In Vitro and In Vivo. Molecular Cancer

Therapeutics 6:833-843.

Mook ORF, Vreijling J, Wengel S, Wengel J, Zhou C, Chattopadhyaya J, Baas F and

Fluiter K (2010) In Vivo Efficacy and Off-Target Effects of Locked Nucleic Acid (LNA)

and Unlocked Nucleic Acid (UNA) Modified siRNA and Small Internally Segmented

Interfering RNA (sisiRNA) in Mice Bearing Human Tumor Xenografts. Artificial DNA:

PNA & XNA 1:36-44.


at ASPE

T Journals on A

pril 21, 2018dm


ownloaded from


DMD#50666

34

Moore A and Medarova Z (2009) Imaging of siRNA Delivery and Silencing, in

Methods in Molecular Biology, siRNA and miRNA Gene Silencing (Sioud M ed) pp

93-110, Humana Press.

Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough

K, Machemer L, Radka S, Jadhav V, Vaish N, Zinnen S, Vargeese C, Bowman K,

Shaffer CS, Jeffs LB, Judge A, MacLachlan I and Polisky B (2005) Potent and

Persistent In Vivo Anti-HBV Activity of Chemically Modified siRNAs. Nature

Biotechnology 23:1002-1007.

Mudd SR, Trubetskoy VS, Blokhin AV, Weichert JP and Wolff JA (2010) Hybrid

PET/CT for Noninvasive Pharmacokinetic Evaluation of Dynamic Poly Conjugates, a

Synthetic siRNA Delivery System. Bioconjugate Chem 21:1183-1189.

Noll S and Mittag E (1983) Investigation of Hydrogen-Exchange in Uracil and Its

Derivatives Labeled With Tritium. Reaction Kinetics and Catalysis Letters 22:35-37.

Ocker M, Neureiter D, Lueders M, Zopf S, Ganslmayer M, Hahn EG, Herold C and

Schuppan D (2005) Variants of Bcl-2 Specific siRNA for Silencing Antiapoptotic Bcl-2

in Pancreatic Cancer. Gut 54:1298-1308.

Richmond CR, Langham WH and Trujillo TT (1962) Comparative Metabolism of

Tritiated Water by Mammals. Journal of Cellular and Comparative Physiology 59:45-

53.

Sands H, Gorey-Feret LJ, Cocuzza AJ, Hobbs FW, Chidester D and Trainor GL

(1994) Biodistribution and Metabolism of Internally 3H-Labeled Oligonucleotides. I.


at ASPE

T Journals on A

pril 21, 2018dm


ownloaded from


DMD#50666

35

Comparison of a Phosphodiester and a Phosphorothioate. Mol Pharmacol 45:932-

943.

Sands H, Goreyferet LJ, Ho SP, Bao YJ, Cocuzza AJ, Chidester D and Hobbs FW

(1995) Biodistribution and Metabolism of Internally H-3 Labeled Oligonucleotides .2.

3',5'-Blocked Oligonucleotides. Mol Pharmacol 47:636-646.

Sonoke S, Ueda T, Fujiwara K, Sato Y, Takagaki K, Hirabayashi K, Ohgi T and Yano

J (2008) Tumor Regression in Mice by Delivery of Bcl-2 Small Interfering RNA With

Pegylated Cationic Liposomes. Cancer Research 68:8843-8851.

Swart PJ, Beljaars E, Smit C, Pasma A, Schuitemaker H and Meijer DKF (1996)

Comparative Pharmacokinetic, Immunologic and Hematologic Studies on the Anti-

HIV-1/2 Compounds Aconitylated and Succinylated HSA. Journal of Drug Targeting

4:109-116.

van de Water FM, Boerman OC, Wouterse AC, Peters JGP, Russel FGM and

Masereeuw R (2006) Intravenously Administered Short Interfering RNA Accumulates

in the Kidney and Selectively Suppresses Gene Function in Renal Proximal Tubules.

Drug Metabolism and Disposition 34:1393-1397.

Viel T, Boisgard R, Kuhnast B, Jego B, Siquier-Pernet K, Hinnen F, Dolle F and

Tavitian B (2008) Molecular Imaging Study on In Vivo Distribution and

Pharmacokinetics of Modified Small Interfering (siRNAs). Oligonucleotides 18:201-

212.

Yanagihara K, Tashiro M, Fukuda Y, Ohno H, Higashiyama Y, Miyazaki Y, Hirakata

Y, Tomono K, Mizuta Y, Tsukamoto K and Kohno S (2006) Effects of Short


at ASPE

T Journals on A

pril 21, 2018dm


ownloaded from


DMD#50666

36

Interfering RNA Against Methicillin-Resistant Staphylococcus Aureus Coagulase In

Vitro and In Vivo. Journal of Antimicrobial Chemotherapy 57:122-126.

Zou Y, Tiller P, Chen IW, Beverly M and Hochman J (2008) Metabolite Identification

of Small Interfering RNA Duplex by High-Resolution Accurate Mass Spectrometry.

Rapid Commun Mass Spectrom 22:1871-1881.


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Figure Legends

Figure 1a: Concentrations of total radiolabeled components in dried blood, plasma

and selected tissues after a single intravenous administration of [3H]-MRP4 siRNA.

Figure 1b: Concentrations of total radiolabeled components in dried blood, plasma

and selected tissues after a single intravenous administration of [3H]-SSB siRNA.

Figure 2a: Selected whole-body autoradioluminographs at 10 min and at 1 and 24 h

after a single intravenous administration of [3H]-MRP4 siRNA (nominal dose:

5 mg/kg) to male CD-1 mice. The whitest area corresponds to the highest

concentration of total radiolabeled components.

Figure 2b: Selected whole-body autoradioluminographs at 10 min and at 1 and 48 h

after a single intravenous administration of [3H]-SSB siRNA (nominal dose: 5 mg/kg)

to male CD-1 mice. The whitest area corresponds to the highest concentration of total

radiolabeled components.

Figure 3: Metabolite profiles of [3H]-siRNAs in plasma following a single i.v.

administration (nominal dose 5 mg/kg, analysis of SPE elution fractions by LC-

method 1).

Figure 4: Metabolite profile of [3H]-SSB siRNA in plasma 5 min post dosing, indicated

complete degradation to single nucleosides (analysis of SPE wash fraction by LC-

method 2).


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Tables

Table 1

[3H]-siRNA Urine

(% of dose)

Feces

(% of dose)

Carcass

(% of dose)

Tritiated water

(% of dose)

Total

(% of dose)

MRP4, 24 h 15 1 27 7 50

SSB, 48 h 38 7 16 26 87

Mass balance; Excretion and recovery of total radiolabeled components of male CD-

1 mice following a single intravenous administration of either [3H]-MRP4 or [3H]-SBB

siRNA (nominal dose: 5 mg/kg).


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