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Approach to analyzing nucleic acid pharmaceuticals

Approach to analyzing nucleic acid pharmaceuticals

In recent years, next generation pharmaceuticals have become more actively developed all over the world. Among them, nucleic acid pharmaceuticals have been on the market every year since 2016 and are typical novel modalities which are expected to be further development in the future. In drug development, evaluation of ‘drug metabolism and disposition’ is important, and quantitative analysis of drugs in biological samples is essential.

Mediford Corporation conducts method development, method validation, and determination of nucleic acid pharmaceuticals in biological samples (plasma, tissue etc.) under GLP organization. Metabolite screening studies of nucleic acid pharmaceuticals are conducted under reliability standards.

Service Details

Drug concentration determination in biological samples

Analysis of nucleic acid pharmaceuticals by LC-MS/MS

We offer contract services of method development, method validation, and determination. As LC-MS/MS has higher selectivity than Hybridization-ECLIA, it can be applied to metabolite determination of nucleic acid pharmaceuticals. LC-MS/MS method is attracting attention by its benefits of high versatility of analytical method and ability of detecting metabolites directly. It is suitable for rapid method development and determination required in screening stages of pharmaceuticals since probe synthesis is not necessary unlike Hybridization assays.

Case of analyzing antisense nucleic acid pharmaceuticals (sample: 50 microliters of plasma)
LC-MS/MS (Triple Quad 5500)
Calibration curve
Analyte: Nusinersen
Range: 0.2-100 ng/mL in plasma
Nexera X2 (Shimadzu) / Triple Quad 5500 (Sciex)

Analysis of nucleic acid pharmaceuticals by Hybridization-ECLIA

Contract services such as designing and arrangement of probes, labeling detection reagents (Ruthenium, etc.), method development, method validation, and determination are available. Hybridization-Electro Chemi Luminescence Immuno Assay (Hybridization-ECLIA) is effective for analyzing many samples because of its simple sample preparation and high throughput analysis. It can quantitate with smaller samples since it has higher sensitivity than LC-MS/MS.

Case of analyzing antisense nucleic acid pharmaceuticals (sample: 1.25 microliters of plasma)
Hybridization-ECL (MESO QuickPlex SQ120)
Calibration curve
Analyte: Nusinersen
Range: 0.7-713 ng/mL in plasma
MESO QuickPlex SQ120
(Meso Scale Diagnostics, LLC)

Determination results by analysis method

LC-MS/MS method and Hybridization-ECLIA method presented good correlation in determination results of rats with a single intravenous dosing of nusinersen (1mg/kg).

Comparison of concentrations of Nusinersen between two quantification methods,
LC-MS/MS (Triple Quad 5500) and Hybridization-ECLIA (MESO QuickPlex SQ120)
Administration: Crl:CD(SD), intravenous, 1 mg/kg, single dosing

Metabolite screening of nucleic acid pharmaceuticals

In general, drugs taken into the body are metabolized into structures easy to excrete, although sometimes active metabolites are produced. Thus, it is very important in safety assessment of pharmaceuticals to quantitate concentrations of drugs and their metabolites in biological samples. Domestic guideline*1 mentions that non-clinical safety assessment is necessary for metabolites containing chemical modifications derived from nucleic acid pharmaceuticals. Mediford Corporation conducts metabolite screening services for nucleic acid pharmaceuticals (cold/hot) by LC-MS (Orbitrap) under reliability standards.

*1: ICH-M3(R2)

Instrument  
LC Nexera X2 System (Shimadzu)
MS/MS (Orbitrap) Q Exactive Focus (Thermo Fisher Scientific)
Maximum Resolution: 70,000@m/z=200
Mass error < ±3 ppm (external standard). < ±1 ppm (internal standard)
Software Xcalibur 4.1. Compound Discoverer 2.1 (Thermo Fisher Scientific)

Example data of metabolite screening

Metabolites of nucleic acid pharmaceuticals in rat serum after 72 hours of incubation at 37℃
LNA-A: 5-CTAgttcacgaaTGC-3
Upper case: LNA, lower case: DNA, C: 5-C, all phosphorothioate backbone
Metabolite estimation in rat serum after 72 hours incubation at 37℃

Simultaneous determination of unchanged form and metabolites of nucleic acid pharmaceuticals

Unchanged form and metabolites of nucleic acid pharmaceuticals can be determined simultaneously by LC-MS/MS.

Simultaneous determination of Nusinersen and metabolites (3’N-1, 3’N-2)
by LC-MS/MS（QTRAP 6500+）
Administration: Crl:CD(SD), intravenous, 1 mg/kg, single dosing