A Simplified Tissue Extraction Method And Plasma Bioanalysis Of A Liposome-encapsulated Immunostimulatory Oligonucleotide, Modn 6303: Qualification And Pharmacokinetic Profiles In Sprague Dawley Rats

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 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

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Results

A. Ligation-based hybridization ELISA in tissue and plasma

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

LABEL

Ligation Site OH LIGATION

AAPS, Atlanta, GA www.criver.com

11/2008

LLOQ QC1 QC2 QC3 ULOQ

1.70 4.00 10.00 40.00 100.00

1.89 4.32 10.33 40.97 96.65

2.7 2.4 1.8 2.0 4.9

111.4 108.0 103.3 102.4 96.6

5’

PO4

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

Template Probe

BIOTIN

Streptatividin-coated plate

Streptatividin-coated plate

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) b) c) d)

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

Introduction

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

Oligonucleotide-based pharmaceuticals are currently investigated in preclinical and clinical settings for various disease conditions.

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

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

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).

mODN 6303 is a liposome formulation of a methylated CpG immunomodulatory DNA oligonucleotide (mODN) that targets Toll-like receptor 9. The liposome delivery vehicle is composed of a cationic lipid (DODAP), a neutral lipid (DSPC), cholesterol and PEG-DMG. Preclinical studies have demonstrated the capability of this liposome-encapsulated mODN to enhance natural killer cell (NK) activity and to improve the anti-tumor effectiveness of monoclonal antibodies when tested in lymphoma and breast cancer models. Following administration to animals in preclinical toxicology studies, mODN 6303 is determined using a bioanalytical method. Although no extraction is performed in the plasma method, oligonucleotides in tissues are typically extracted using a liquid:liquid extract (LLE) phenol-chloroform based method. The procedure is tedious, it requires the use of toxic solvents which need to be disposed of appropriately, and it takes two days to perform. We have developed a tissue immunoassay in which the extraction is performed with sonication and proteinase K (SPK). The SPK method is performed in one day, it is simpler and the results favorably compare to those obtained with an LLE in terms of standard curve, QC and validation parameters; overall the success rate of hybridization assays using SPK increased significantly. 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 plasma of Sprague Dawley rats. For tissues, the SPK method of extraction is used.

STD Curve

BIOTIN

100000 Relative Fluorescence Units (RFU)

mODN Test Article

Conclusion. Qualification data demonstrate that the procedure is suitable for bioanalysis of mODN 6303 in rat tissue and plasma. The method is readily adapted to other oligonucleotides, tissues and species. Intra-Assay Precision and Accuracy (n=6) CV Recovery %)) CV (( % %) Recovery (( %

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 of quantitation (ULOQ) is 100 ng/g with an LLOQ of 2 ng/g, giving a 50-fold curve range.

3’

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

Concentration in Rat Liver Tissue Homogenate ( ng / g ) Theoretical Measured Theoretical Determined

Ligation Probe

In tissues, using a classical LLE (phenol-chloroform based extractor), which is typically used in the industry, a lesser sensitive LLOQ of 25 ng/g was achieved with mODN 6303. In fact, our simplified extraction method with sonication and proteinase 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 increased significantly.

60000

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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/mL at 37ºC. Study samples were frozen at given time points.

0.4 0.3

0.1

1

10

100

1000

Concentration ( ng / g )

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

Sample 1

35

Sample 2

30 25 20 15

15 minutes

2 hours

6 hours

24 hours

Figure 6: In vitro degradation profile of the free, unformulated mODN versus the liposome-formulated mODN 6303 in fresh rat serum. Error bar: range of results at two different dilutions.

Time post-dose

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

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 seems to indicate that the delivery vehicle increases the half-life of mODN in vivo in comparison to the unformulated variant. The rapid decline of the unformulated mODN is in sharp contrast with the persistence observed in plasma with the formulated mODN 6303 (Figure 4). The results obtained in Figure 5 are reproducible within 25% difference between 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). Figure 5: In vivo degradation profile of the unformulated mODN at 5 minutes versus 15 minutes post-injection for three plasma samples obtained in two animals. Error bar: range of results at two different dilutions.

Animal 1 Animal 2 1.5

2 hours

6 hours

Time post-dose

24 hours

Liposomal ( mODN 6303 ) Unformulated ( mODN ) 0.5

0.4

0.3

0.2

0.1

0

0 min.

5 min.

15 min.

30 min.

2 hr

6 hr

24 hr

Incubation time at 37ºC

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

Plasma and tissue quantitations rely upon a robust, sensitive and specific ligationbased hybridization ELISA to determine PK profiles in rats. Residence time for the liposome-encapsulated mODN 6303 and the unformulated mODN test article were determined in vivo and in vitro, in tissue and plasma. The formulated test article is stable in plasma and accumulates in liver tissue, whereas the unformulated test article is degraded rapidly in the blood.

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

0.5

15 minutes

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15 minutes

0.6

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

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5 minutes

5

We demonstrate that the free, unformulated mODN is quickly degraded and almost disappears after 2 hours, whereas the formulated, liposome-encapsulated mODN 6303 is still present after 24 hours in serum. Therefore, the persistence of mODN 6303 in blood is likely due to the increased stability due to the liposomal vehicle.

0.1

0

10

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

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Figure 3: mODN in rat liver after injection of the liposome-formulated mODN 6303 at a dose of 30 mg/kg. 40

In vitro degradation profiles of the liposomal mODN 6303 versus the unformulated mODN in rat serum

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.

0.2

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

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

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 K 2 EDTA tubes, placed on ice, centrifuged, and the resultant plasma stored at ~-80ºC. Study samples were analyzed in triplicate.

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In vivo unformulated mODN pilot study in plasma 20000

B. Dosage in Sprague Dawley rats

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Sample 1 Sample 2

40000

In vivo PK assessment of mODN 6303

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 K 2 EDTA tubes, placed on ice, centrifuged and stored at ~-80ºC. Whole tissues were rinsed in saline solution, and the resultant plasma stored at ~-80ºC.

0.6

The % difference between the two samples for the time-points at 15 minutes, and 2 and 6 hours in both tissues (Figure 3) and plasma (Figure 4) were within 25%. For the 24 hour time-point, the difference was higher than 25%, which is not surprising for samples at low concentrations.

80000

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

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Figure 4: In vivo PK profile in plasma of the liposome-formulated mODN 6303 in rats at a dose of 30 mg/kg.

mODN in plasma ( µg/mL )

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

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 truncated oligonucleotide variant of mODN (results not shown).

mODN 6303 in liver ( µg/g )

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.

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

mODN 6303 concentration ( mg/mL )

Material and Methods

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 described. Pharmacokinetic (PK) profiles in the rat are presented.

mODN 6303 in plasma (mg/mL)

Abstract

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