A Simplified Tissue Extraction Method and Plasma Bioanalysis of a Liposome-Encapsulated Immunostimulatory

Oligonucleotide, mODN 6303: Qualification and Pharmacokinetic Profiles in Sprague Dawley Rats
G. A. Tremblay1, G. K. Toor1, L. M. Chagnon1, S. Carriero1, P. R. Oldfield1, A. J. Bartlett1, and S. C. Semple2
1
Charles River Laboratories Preclinical Services Montreal Inc., 22022 Transcanadienne, Senneville, Quebec, Canada H9X 3R3
2
Tekmira Pharmaceuticals Corporation, 8900 Glenlyon Parkway, Burnaby, BC, Canada V5J 5J8

Abstract Material and Methods Results

Purpose. mODN 6303 is an immunostimulatory, liposome-encapsulated
phosphodiester oligonucleotide drug currently in preclinical development for the
treatment of cancer. Bioanalysis of oligonucleotide compounds in tissues are
typically performed using a liquid-liquid phenol-chloroform extraction procedure.
In our hands, the liquid-liquid extraction yielded limited quantities of mODN 6303
from rat tissues. An alternative procedure for the tissue extraction of mODN 6303 is
A. Ligation-based hybridization ELISA in tissue and plasma
Quantitation of mODN is performed with a ligation-based hybridization ELISA. The
ligation step of the hybridization ELISA is shown in Figure 1.
The ligation-based hybridization ELISA provided greater sensitivity, with typically a In vitro degradation profiles of the liposomal mODN 6303 versus the
Figure 4: In vivo PK profile in plasma of the liposome-formulated mODN 6303 in rats at a dose of 30 mg/kg.
lower limit of quantitation (LLOQ) of 0.2 ng/mL in plasma, and 2 ng/g in tissues unformulated mODN in rat serum
using the SPK extraction method.
To answer the aforementioned question, we have compared the unformulated
The ligation-based hybridization ELISA is highly specific for the full-length mODN, mODN with the liposome-encapsulated mODN 6303 in Figure 6, by spiking the test
as we have detected less than 1.5% of signal using an n-1 truncated 0.6
Sample 1 article in fresh rat serum and incubating the samples at 37ºC.

mODN 6303 in plasma (mg/mL)

described. Pharmacokinetic (PK) profiles in the rat are presented.
Methods. The simplified tissue extraction method, which is a slight modification of
the plasma method, is based on: i) The use of proteinase K for degradation of
proteins and disruption of potential protein:nucleic acid interactions, ii) Fine
homogenization of tissues using a sonicating bath and, iii) Heat-denaturation at
90ºC for the hybridization of mODN 6303 to a biotinylated sequence-specific
capture probe, along with concomitant disruption of potential interactions of mODN
6303 with tissue components. A sensitive ligation-based hybridization ELISA was
produced.
Figure 1: Schematic representation of the ligation step in the hybridization ELISA. The mODN test article (red) was oligonucleotide variant of mODN (results not shown).
first hybridized to the template probe (blue) and immobilized to a microtiter plate (left). A universal 5'-phosphorylated Sample 2
0.5 We demonstrate that the free, unformulated mODN is quickly degraded and almost
digoxigenin-labeled ligation probe (green) complementary to the template probe is ligated using T4 DNA ligase disappears after 2 hours, whereas the formulated, liposome-encapsulated mODN
onto the 3'-end of the mODN test article (right). The amount of test article is proportional to the amount of ligated In tissues, using a classical LLE (phenol-chloroform based extractor), which is
probe; quantitation of the labeled ligated probe ensues via fluorogenic development. typically used in the industry, a lesser sensitive LLOQ of 25 ng/g was achieved with 0.4 6303 is still present after 24 hours in serum. Therefore, the persistence of mODN
mODN 6303. In fact, our simplified extraction method with sonication and 6303 in blood is likely due to the increased stability due to the liposomal vehicle.
proteinase K (SPK) is typically about 20-fold more sensitive than the LLE method. 0.3
LABEL
Also, the run success rate for the assays with SPK as opposed to LLE increased This is also indicative that the gradual decrease of mODN 6303 at 6 and 24 hours
significantly. (Figure 3) may be due to clearance, and not degradation.
Ligation Ligation Probe 0.2
Site
3’
OH Figure 2 shows a representative standard curve obtained in tissues such as liver, 0.1

LIGATION
kidney or spleen using the simplified SPK extraction method. The upper limit of
Results. Precision and accuracy parameters for the tissue method were satisfactory: mODN
quantitation (ULOQ) is 100 ng/g with an LLOQ of 2 ng/g, giving a 50-fold curve 0
CV was ≤ 5% and recovery within 12% of the theoretical concentrations. The tissue Test Article
5’
PO4 range. 15 minutes 2 hours 6 hours 24 hours Figure 6: In vitro degradation profile of the free, unformulated mODN versus the liposome-formulated mODN
procedure is faster and less tedious than liquid-liquid extractions, and overall Time post-dose 6303 in fresh rat serum. Error bar: range of results at two different dilutions.
provides for better results regarding CV, recovery and standard curve parameters. Template Figure 2: Typical standard curve obtained with the sonication and proteinase K (SPK) extraction method. Here the
The PK profiles for mODN 6303 in rat liver tissue and plasma were consistent with Probe standards were spiked in a monkey kidney homogenate. High sensitivity and specificity in tissues is achieved
expectations. using a ligation-based hybridization ELISA.
In liver, the amount of mODN peaked at 2 hours, whereas the plasma concentration 0.6
STD Curve Liposomal ( mODN 6303 )
peaked at 15 minutes post-dose. mODN was still high in the plasma at 6 hours, at
BIOTIN BIOTIN

mODN 6303 concentration ( mg/mL )

Conclusion. Qualification data demonstrate that the procedure is suitable for
bioanalysis of mODN 6303 in rat tissue and plasma. The method is readily adapted Streptatividin-coated plate Streptatividin-coated plate
a concentration similar to that at 2 hours, and was still detectable at 24 hours at 0.5
Unformulated ( mODN )
to other oligonucleotides, tissues and species.
100000 the high dose of 30 mg/kg.

Relative Fluorescence Units (RFU)

80000
The % difference between the two samples for the time-points at 15 minutes, and 0.4
Concentration in Rat Liver Intra-Assay Precision 1. Standards, quality control (QC) samples and study samples were prepared in 2 and 6 hours in both tissues (Figure 3) and plasma (Figure 4) were within 25%. For
Tissue Homogenate and Accuracy the 24 hour time-point, the difference was higher than 25%, which is not surprising
rat tissue homogenate or in rat plasma.
( ng / g ) (n=6) 60000 for samples at low concentrations. 0.3
Theoretical
Theoretical Measured
Determined CV
CV (( %
%) Recovery
Recovery (( %
%)) 2. For the plasma method, no extractions were performed.
LLOQ 1.70 1.89 2.7 111.4 40000
QC1 4.00 4.32 2.4 108.0 3. For tissues, liposomes were extracted by: 0.2
In vivo unformulated mODN pilot study in plasma
AAPS, Atlanta, GA

QC2 10.00 10.33 1.8 103.3 a) Sonication; 20000

QC3 40.00 40.97 2.0 102.4 b) Proteinase K digestion; We also wanted to document the residence time of the unformulated mODN in 0.1
ULOQ 100.00 96.65 4.9 96.6 c) Heat denaturation of potential interactions of mODN with tissues; plasma, as well as the reproducibility of the plasma bioanalysis method.
0
d) Use of the non-ionic detergent Triton X-100. 0.1 1 10 100 1000
As expected, free mODN is quickly degraded (or cleared) in vivo, since after 0
15 minutes of circulation in the bloodstream, based on results obtained in
Introduction
4. Plasma and tissue samples were denatured at 90ºC, followed by hybridization Concentration ( ng / g ) 0 min. 5 min. 15 min. 30 min. 2 hr 6 hr 24 hr
at 37ºC of the mODN analyte to a complementary biotinylated template probe. triplicate, only about half of the mODN is still detectable (Figure 5). This seems
to indicate that the delivery vehicle increases the half-life of mODN in vivo in Incubation time at 37ºC
Oligonucleotide-based pharmaceuticals are currently investigated in preclinical and 5. The mODN:template probe duplex was bound to a streptavidin-coated 96-well The standard curve in Figure 2 was obtained with a different test article than mODN comparison to the unformulated variant. The rapid decline of the unformulated
clinical settings for various disease conditions. plate. 6303 and with a different matrix, i.e. monkey kidney, which serves to illustrate that mODN is in sharp contrast with the persistence observed in plasma with the
the method is readily adapted to different oligonucleotides; standard curves and formulated mODN 6303 (Figure 4).
Un-modified, naked oligonucleotides are swiftly degraded by nucleases found in 6. A 5'-phosphorylated, digoxigenin-labeled ligation probe was ligated onto the parameters obtained with mODN 6303 were comparable.
plasma, and therefore favorable delivery vehicles are sought for increased plasma immobilized duplex, at the 3'-end of the mODN test article (Figure 1). The results obtained in Figure 5 are reproducible within 25% difference between
stability, as well as specific cellular targeting.
7. The 96-well plate was incubated with an anti-digoxigenin POD conjugate and
was measured using a sensitive fluorogenic substrate of POD. In vivo PK assessment of mODN 6303
different plasma aliquots of the same animal (5 minutes and 15 minutes), and
within 25% of difference between varying dilutions of the same sample (error bars). Conclusion
mODN 6303 is a liposome formulation of a methylated CpG immunomodulatory
DNA oligonucleotide (mODN) that targets Toll-like receptor 9. The liposome delivery A method for the quantitation of a liposome-formulated oligonucleotide pharmaceutical
The formulated mODN 6303 was injected intravenously with 30 mg/kg mODN 6303 Figure 5: In vivo degradation profile of the unformulated mODN at 5 minutes versus 15 minutes post-injection for compound has been developed in both plasma and tissue of Sprague Dawley rats.
vehicle is composed of a cationic lipid (DODAP), a neutral lipid (DSPC), cholesterol and both plasma and liver tissues were obtained from 8 female Sprague Dawley three plasma samples obtained in two animals. Error bar: range of results at two different dilutions.
and PEG-DMG. B. Dosage in Sprague Dawley rats rats. The analysis was carried-out in duplicate for each animal (Figures 3 and 4) The SPK tissue extraction method is innovative in its simplicity. It makes use of
using the SPK method. proteinase K and sonication to achieve reproducible results that favorably compare
Preclinical studies have demonstrated the capability of this liposome-encapsulated • For the in vivo PK assessment of mODN 6303, the liposome-formulated to the more traditional LLE method, which can be considered tedious.
mODN to enhance natural killer cell (NK) activity and to improve the anti-tumor mODN 6303 was administered by iv injection in rats (30 mg/kg). Whole blood 2
effectiveness of monoclonal antibodies when tested in lymphoma and breast cancer Figure 3: mODN in rat liver after injection of the liposome-formulated mODN 6303 at a dose of 30 mg/kg.
was collected in K 2 EDTA tubes, placed on ice, centrifuged and stored at Animal 1 Plasma and tissue quantitations rely upon a robust, sensitive and specific ligation-
models. ~-80ºC. Whole tissues were rinsed in saline solution, and the resultant plasma based hybridization ELISA to determine PK profiles in rats.

mODN in plasma ( µg/mL )

Animal 2
stored at ~-80ºC. 40 1.5
11/2008 Following administration to animals in preclinical toxicology studies, mODN 6303 Residence time for the liposome-encapsulated mODN 6303 and the unformulated
Sample 1
is determined using a bioanalytical method. Although no extraction is performed • For the in vivo rat plasma pilot study, the naked mODN were injected iv at
mODN 6303 in liver ( µg/g )

35
Sample 2 mODN test article were determined in vivo and in vitro, in tissue and plasma. The
in the plasma method, oligonucleotides in tissues are typically extracted using 30 mg/kg in two rats. Whole blood was collected in K 2 EDTA tubes, placed formulated test article is stable in plasma and accumulates in liver tissue, whereas
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30 1
a liquid:liquid extract (LLE) phenol-chloroform based method. The procedure is on ice, centrifuged, and the resultant plasma stored at ~-80ºC. Study samples the unformulated test article is degraded rapidly in the blood.
tedious, it requires the use of toxic solvents which need to be disposed of were analyzed in triplicate. 25
appropriately, and it takes two days to perform. The SPK extraction method followed by the ligation-based hybridization ELISA are
• For in vitro degradation profiles of mODNs, whole blood from rat was placed in 20 0.5 readily adapted to different formulated oligonucleotide test articles.
We have developed a tissue immunoassay in which the extraction is performed with serum separator tubes, allowed to clot at room temperature for 15 minutes, and
15
sonication and proteinase K (SPK). The SPK method is performed in one day, it is the fresh serum separated after centrifugation. The serum from 2 rats was
simpler and the results favorably compare to those obtained with an LLE in terms of pooled and mixed. The mODNs were incubated at a concentration of 0.6 mg/mL 10 0
standard curve, QC and validation parameters; overall the success rate of at 37ºC. Study samples were frozen at given time points. 5 minutes 15 minutes
hybridization assays using SPK increased significantly. 5

0
A sensitive, highly specific and robust ligation-based hybridization ELISA was suitable
to determine PK profiles of formulated and unformulated mODN in tissue, serum or
15 minutes 2 hours 6 hours 24 hours Is the favorable persistence of mODN 6303 solely due to increased stability, or is it
plasma of Sprague Dawley rats. For tissues, the SPK method of extraction is used. rather due to slower clearance, accumulation in, or affinity towards, given tissues?
Time post-dose