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DMD#50666
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Title Page
Metabolism Studies of Unformulated Internally
[<sup>3</sup>H]-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.
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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:
karine.litherland@novartis.com
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 [<sup>3</sup>H]-
radiolabeling procedure, where the full-length oligonucleotides were radiolabeled by
Br/<sup>3</sup>H-exchange. Tissue distribution, excretion, and mass balance of
radioactivity were investigated in male CD-1 mice, following a single intravenous
administration of the [<sup>3</sup>H]-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
[<sup>3</sup>H]-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 [<sup>3</sup>H]-MRP4 and [<sup>3</sup>H]-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 [<sup>3</sup>H]-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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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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