A Simplified Tissue Extraction Method and Plasma Bioanalysis of a Liposome-Encapsulated ImmunostimulatoryOligonucleotide, mODN 6303: Qualification and Pharmacokinetic Profiles in Sprague Dawley RatsG. A. Tremblay1, G. K. Toor1, L. M. Chagnon1, S. Carriero1, P. R. Oldfield1, A. J. Bartlett1, and S. C. Semple2

1Charles River Laboratories Preclinical Services Montreal Inc., 22022 Transcanadienne, Senneville, Quebec, Canada H9X 3R32Tekmira Pharmaceuticals Corporation, 8900 Glenlyon Parkway, Burnaby, BC, Canada V5J 5J8

AbstractPurpose. mODN 6303 is an immunostimulatory, liposome-encapsulatedphosphodiester oligonucleotide drug currently in preclinical development for thetreatment of cancer. Bioanalysis of oligonucleotide compounds in tissues aretypically performed using a liquid-liquid phenol-chloroform extraction procedure. In our hands, the liquid-liquid extraction yielded limited quantities of mODN 6303from rat tissues. An alternative procedure for the tissue extraction of mODN 6303 isdescribed. Pharmacokinetic (PK) profiles in the rat are presented.

Methods. The simplified tissue extraction method, which is a slight modification ofthe plasma method, is based on: i) The use of proteinase K for degradation ofproteins and disruption of potential protein:nucleic acid interactions, ii) Finehomogenization of tissues using a sonicating bath and, iii) Heat-denaturation at90ºC for the hybridization of mODN 6303 to a biotinylated sequence-specificcapture probe, along with concomitant disruption of potential interactions of mODN6303 with tissue components. A sensitive ligation-based hybridization ELISA wasproduced.

Results. Precision and accuracy parameters for the tissue method were satisfactory:CV was ≤ 5% and recovery within 12% of the theoretical concentrations. The tissueprocedure is faster and less tedious than liquid-liquid extractions, and overallprovides for better results regarding CV, recovery and standard curve parameters.The PK profiles for mODN 6303 in rat liver tissue and plasma were consistent withexpectations.

Conclusion. Qualification data demonstrate that the procedure is suitable forbioanalysis of mODN 6303 in rat tissue and plasma. The method is readily adaptedto other oligonucleotides, tissues and species.

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Material and Methods A. Ligation-based hybridization ELISA in tissue and plasma

Quantitation of mODN is performed with a ligation-based hybridization ELISA. Theligation step of the hybridization ELISA is shown in Figure 1.

IntroductionOligonucleotide-based pharmaceuticals are currently investigated in preclinical andclinical settings for various disease conditions.

Un-modified, naked oligonucleotides are swiftly degraded by nucleases found inplasma, and therefore favorable delivery vehicles are sought for increased plasmastability, as well as specific cellular targeting.

mODN 6303 is a liposome formulation of a methylated CpG immunomodulatoryDNA oligonucleotide (mODN) that targets Toll-like receptor 9. The liposome deliveryvehicle is composed of a cationic lipid (DODAP), a neutral lipid (DSPC), cholesteroland PEG-DMG.

Preclinical studies have demonstrated the capability of this liposome-encapsulatedmODN to enhance natural killer cell (NK) activity and to improve the anti-tumoreffectiveness of monoclonal antibodies when tested in lymphoma and breast cancermodels.

Following administration to animals in preclinical toxicology studies, mODN 6303 is determined using a bioanalytical method. Although no extraction is performedin the plasma method, oligonucleotides in tissues are typically extracted using a liquid:liquid extract (LLE) phenol-chloroform based method. The procedure istedious, it requires the use of toxic solvents which need to be disposed ofappropriately, and it takes two days to perform.

We have developed a tissue immunoassay in which the extraction is performed withsonication and proteinase K (SPK). The SPK method is performed in one day, it issimpler and the results favorably compare to those obtained with an LLE in terms ofstandard curve, QC and validation parameters; overall the success rate ofhybridization assays using SPK increased significantly.

A sensitive, highly specific and robust ligation-based hybridization ELISA was suitableto determine PK profiles of formulated and unformulated mODN in tissue, serum orplasma of Sprague Dawley rats. For tissues, the SPK method of extraction is used.

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Figure 1: Schematic representation of the ligation step in the hybridization ELISA. The mODN test article (red) wasfirst hybridized to the template probe (blue) and immobilized to a microtiter plate (left). A universal 5'-phosphorylateddigoxigenin-labeled ligation probe (green) complementary to the template probe is ligated using T4 DNA ligaseonto the 3'-end of the mODN test article (right). The amount of test article is proportional to the amount of ligatedprobe; quantitation of the labeled ligated probe ensues via fluorogenic development.

1. Standards, quality control (QC) samples and study samples were prepared in rat tissue homogenate or in rat plasma.

2. For the plasma method, no extractions were performed.

3. For tissues, liposomes were extracted by:

a) Sonication;b) Proteinase K digestion;c) Heat denaturation of potential interactions of mODN with tissues;d) Use of the non-ionic detergent Triton X-100.

4. Plasma and tissue samples were denatured at 90ºC, followed by hybridization at 37ºC of the mODN analyte to a complementary biotinylated template probe.

5. The mODN:template probe duplex was bound to a streptavidin-coated 96-well plate.

6. A 5'-phosphorylated, digoxigenin-labeled ligation probe was ligated onto the immobilized duplex, at the 3'-end of the mODN test article (Figure 1).

7. The 96-well plate was incubated with an anti-digoxigenin POD conjugate and was measured using a sensitive fluorogenic substrate of POD.

B. Dosage in Sprague Dawley rats

• For the in vivo PK assessment of mODN 6303, the liposome-formulated mODN 6303 was administered by iv injection in rats (30 mg/kg). Whole blood was collected in K2EDTA tubes, placed on ice, centrifuged and stored at ~-80ºC. Whole tissues were rinsed in saline solution, and the resultant plasma stored at ~-80ºC.

• For the in vivo rat plasma pilot study, the naked mODN were injected iv at 30 mg/kg in two rats. Whole blood was collected in K2EDTA tubes, placed on ice, centrifuged, and the resultant plasma stored at ~-80ºC. Study samples were analyzed in triplicate.

• For in vitro degradation profiles of mODNs, whole blood from rat was placed in serum separator tubes, allowed to clot at room temperature for 15 minutes, and the fresh serum separated after centrifugation. The serum from 2 rats was pooled and mixed. The mODNs were incubated at a concentration of 0.6 mg/mLat 37ºC. Study samples were frozen at given time points.

Results The ligation-based hybridization ELISA provided greater sensitivity, with typically alower limit of quantitation (LLOQ) of 0.2 ng/mL in plasma, and 2 ng/g in tissuesusing the SPK extraction method.

The ligation-based hybridization ELISA is highly specific for the full-length mODN,as we have detected less than 1.5% of signal using an n-1 truncatedoligonucleotide variant of mODN (results not shown).

In tissues, using a classical LLE (phenol-chloroform based extractor), which istypically used in the industry, a lesser sensitive LLOQ of 25 ng/g was achieved withmODN 6303. In fact, our simplified extraction method with sonication andproteinase K (SPK) is typically about 20-fold more sensitive than the LLE method.Also, the run success rate for the assays with SPK as opposed to LLE increasedsignificantly.

Figure 2 shows a representative standard curve obtained in tissues such as liver,kidney or spleen using the simplified SPK extraction method. The upper limit ofquantitation (ULOQ) is 100 ng/g with an LLOQ of 2 ng/g, giving a 50-fold curverange.

Concentration in Rat Liver Tissue Homogenate

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Theoretical Determined CV ( % ) Recovery ( % ) LLOQ 1.70 1.89 2.7 111.4 QC1 4.00 4.32 2.4 108.0 QC2 10.00 10.33 1.8 103.3 QC3 40.00 40.97 2.0 102.4 ULOQ 100.00 96.65 4.9 96.6

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Figure 2: Typical standard curve obtained with the sonication and proteinase K (SPK) extraction method. Here thestandards were spiked in a monkey kidney homogenate. High sensitivity and specificity in tissues is achievedusing a ligation-based hybridization ELISA.

The standard curve in Figure 2 was obtained with a different test article than mODN6303 and with a different matrix, i.e. monkey kidney, which serves to illustrate thatthe method is readily adapted to different oligonucleotides; standard curves andparameters obtained with mODN 6303 were comparable.

In vivo PK assessment of mODN 6303

The formulated mODN 6303 was injected intravenously with 30 mg/kg mODN 6303and both plasma and liver tissues were obtained from 8 female Sprague Dawleyrats. The analysis was carried-out in duplicate for each animal (Figures 3 and 4)using the SPK method.

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In liver, the amount of mODN peaked at 2 hours, whereas the plasma concentrationpeaked at 15 minutes post-dose. mODN was still high in the plasma at 6 hours, ata concentration similar to that at 2 hours, and was still detectable at 24 hours atthe high dose of 30 mg/kg.

The % difference between the two samples for the time-points at 15 minutes, and2 and 6 hours in both tissues (Figure 3) and plasma (Figure 4) were within 25%. Forthe 24 hour time-point, the difference was higher than 25%, which is not surprisingfor samples at low concentrations.

In vivo unformulated mODN pilot study in plasma

We also wanted to document the residence time of the unformulated mODN inplasma, as well as the reproducibility of the plasma bioanalysis method.

As expected, free mODN is quickly degraded (or cleared) in vivo, since after 15 minutes of circulation in the bloodstream, based on results obtained in triplicate, only about half of the mODN is still detectable (Figure 5). This seemsto indicate that the delivery vehicle increases the half-life of mODN in vivo incomparison to the unformulated variant. The rapid decline of the unformulatedmODN is in sharp contrast with the persistence observed in plasma with theformulated mODN 6303 (Figure 4).

The results obtained in Figure 5 are reproducible within 25% difference betweendifferent plasma aliquots of the same animal (5 minutes and 15 minutes), andwithin 25% of difference between varying dilutions of the same sample (error bars).

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Conclusion A method for the quantitation of a liposome-formulated oligonucleotide pharmaceuticalcompound has been developed in both plasma and tissue of Sprague Dawley rats.

The SPK tissue extraction method is innovative in its simplicity. It makes use of proteinase K and sonication to achieve reproducible results that favorably compareto the more traditional LLE method, which can be considered tedious.

Plasma and tissue quantitations rely upon a robust, sensitive and specific ligation-based hybridization ELISA to determine PK profiles in rats.

Residence time for the liposome-encapsulated mODN 6303 and the unformulatedmODN test article were determined in vivo and in vitro, in tissue and plasma. Theformulated test article is stable in plasma and accumulates in liver tissue, whereasthe unformulated test article is degraded rapidly in the blood.

The SPK extraction method followed by the ligation-based hybridization ELISA arereadily adapted to different formulated oligonucleotide test articles.

Figure 5: In vivo degradation profile of the unformulated mODN at 5 minutes versus 15 minutes post-injection forthree plasma samples obtained in two animals. Error bar: range of results at two different dilutions.

Figure 6: In vitro degradation profile of the free, unformulated mODN versus the liposome-formulated mODN6303 in fresh rat serum. Error bar: range of results at two different dilutions.

Figure 3: mODN in rat liver after injection of the liposome-formulated mODN 6303 at a dose of 30 mg/kg.

Figure 4: In vivo PK profile in plasma of the liposome-formulated mODN 6303 in rats at a dose of 30 mg/kg.In vitro degradation profiles of the liposomal mODN 6303 versus the unformulated mODN in rat serum

To answer the aforementioned question, we have compared the unformulatedmODN with the liposome-encapsulated mODN 6303 in Figure 6, by spiking the testarticle in fresh rat serum and incubating the samples at 37ºC.

We demonstrate that the free, unformulated mODN is quickly degraded and almostdisappears after 2 hours, whereas the formulated, liposome-encapsulated mODN6303 is still present after 24 hours in serum. Therefore, the persistence of mODN6303 in blood is likely due to the increased stability due to the liposomal vehicle.

This is also indicative that the gradual decrease of mODN 6303 at 6 and 24 hours(Figure 3) may be due to clearance, and not degradation.

Is the favorable persistence of mODN 6303 solely due to increased stability, or is itrather due to slower clearance, accumulation in, or affinity towards, given tissues?

Measured CV ( % ) Recovery ( % )Theoretical