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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
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
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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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
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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