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日本核酸医薬学会第6回年会

Developmentof Simultaneous Quantification Methodfor Nusinersen and its Metabolites
in Rat Plasma and Tissues using LC-MS/MS〇Tomoko Saito, Rika Dosho, Masaki Hoshino, Ryuta Hosogai,
Shin-ichiro Nitta, Kodo Miura, Tetsuya Ueda

LSI Medience Corporation
1Ⓒ 2021 LSI Medience Corporation All rights reserved

Introduction

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Because drugs are often metabolized to active/toxic metabolites,
it is important to quantify the drug and its metabolites in biologicalmatrices for the evaluation of drug safety.
The LC-MS/MS method is useful for quantification of oligonucleotide
drugs and its metabolites, because the method has high selectivity
and can be quantified simultaneously.
Previously, we administered nusinersen, one of the antisense
oligonucleotide drugs, to rats and identified 3’N-1 and 3’N-2 as its
metabolites.
Therefore, we developed simultaneous quantification method fornusinersen and identified metabolites using LC-MS/MS.
We determined concentrations of nusinersen and its metabolites in rat
samples using this method.
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Precise Metabolite Identification

Equipment and software:
Nusinersen (18mer): 5’-TCACTTTCATAATGCTGG -3’
Q Exactive™ Focus & Xcalibur™ ver. 4.1
3’N-1 (17mer):5’-TCACTTTCATAATGCTG -3’
3’N-2 (16mer):5’-TCACTTTCATAATGCT-3’(Thermo Fisher Scientific, Waltham, MA)
Result:
The metabolites were identified more precisely using UV detection, HILIC condition for achieving baseline
separation, PSO2- detection with an AIF1) method, positive MS and MS/MS spectra.

1) Anal. Chem. 2017, 89, 12, 6821–6826

Rat kidney, 360 min after dosing
Chromatograms

10003′N-2 3′N-10AIF (-)
m/z=1000-1800>94.9

Nusinersen

0100

345Time (min)6[M+3H]3+1000Base Peak (+)
Chromatogram
2[M+3H]3+

100

UV@260 nm

1000MS Spectra

100[M+4H]4+01500

7

RT=3.8 min
3′N-2100

RT=4.6 min
3′N-1100

1113

788
710

0814
710

100

839
710

02500

b3+b4+b5+16261233 839

5′−TC−−−A−−−C−−−T−−−TTCATAATG−−−C−−−T −−−GG−3′
3710

1113

1506

1900

Nusinersen x4+x3+x2+b2+b3+b4+

RT=4.6 min
3′N-11506
1626 1900

RT=5.2 min
Nusinersen

1113

1233 1506
1626 1900

1000

Observed fragments of nusinersen and 3′N-1 metabolite>b2+

RT=3.8 min
3′N-21506

1113 1207

0RT=5.2 min
Nusinersen[M+3H]3+
2000
m/z

MS/MS Spectra from [M+3H]3+m/z

2000

1626

b5+

3000

1207 8145′−TC−−−A−−−C−−−T−−−TTCATAAT−−−G−−−C−−−TG−3′710

11131506

1900

3′N-1x4+x3+x2+

Pre-treatment
50 μL of rat plasma (EDTA-2K) or rat tissue lysate↓ Add lysis buffer (Clarity®OTX* kit reagent)
Mixture↓ Load to Clarity®OTX 25 mg plate↓ Wash with 50mM NaH2PO4 (pH5.5)↓ Wash with 500mM NaH2PO4 (pH5.5)/water/ACN (10:40:50, v/v/v)↓ Elute with 400mM NH4HCO3/ACN (50:50, v/v)
Eluent↓ Dry under N2 stream
Residue↓

Reconstitute with 50 μLof TE buffer/methanol (70:30, v/v)
Filter↓
Filtrate↓

4Inject (7 μL) to LC-MS/MS*: Phenomenex, Torrance, CA

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LC-MS/MS condition
Nexera X2 system(Shimadzu, Kyoto, Japan)

LCColumn
Columntemp.

Mobilephase A*1
Mobilephase B*1
Run time

X Bridge™ BEH C18 2.5μm, 2.1×50mm(Waters, Milford, MA)
60°C

Water/methanol/TEA/HFIP/acetylacetone(900:100:2:40:0.05, v/v/v/v/v)
Methanol/water/TEA/HFIP/acetylacetone(900:100:2:40:0.05, v/v/v/v/v)
27 min*1: Under shading and nitrogen bubbling(Patent pending in Japan,patent application No. 2020-122679)

MS

QTRAP®6500+ and Analyst®(ver. 1.7)
(SCIEX, Framingham, MA)

Ionization
mode

Turbo ion spray

Polarity

Negative

Scan type

MRM

Ion spray
voltage-4500 VTEM

Monitoring
ions

500°C

AnalyteNusinersen
3’N-1
3’N-2I.S.*2

Q1 (m/z)1017.0
957.1
697.7
881.7

Q3 (m/z)

94.9
94.9
94.9
94.9

*2: Nusinersen which its chemical modification of 2’-MOE(2’-O-Methoxyethyl) is changed to 2’-OMe (2’-O-Methyl)
is used as I.S. for this analytical method.
5

Typical chromatograms of validation samples
Nusinersen and its metabolites
in rat plasma and tissues (datanot shown) was analyzed withthe same analytical conditionsof LC-MS/MS.
S/N ratio of LLOQ peak wasenough and carry over peak
was not observed.

Overlay3’N-2I.S.

3’N-1
Nusinersen

Typical chromatograms (plasma)
DoubleblankSingleblank
LLOQ
1 ng/mL
ULOQ
400 ng/mL
Carry overNusinersen

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3’N-1

3’N-2I.S.

Results of validation study (plasma)

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Good linearity, precision and accuracy were observed over the concentration range of
1 to 400 ng/mL each in plasma. Nusinersen and its metabolites in plasma were stable
under various conditions.
Selectivity

Items

Male and female, n=3 each, total n=6Results

No interfering peak

Carry overn=1

No interfering peakWithin-run accuracy andprecision

4 concentrations, n=5 each

Accuracy: 89.0-107.0%
C.V.: 2.8-6.7%

Calibration curve

Post-preparative stability

Stability at room temperaturein plasmaFreeze and thaw stability
in plasma
7Contents

1-400 ng/mL, n=1 each

4°C, 72 hours, 1 concentration, n=324 hours, 1 concentration, n=3
5 cycles, 1 concentration, n=3

Accuracy: 90.3-113.0%, r≥0.9971

Accuracy: 98.0-108.7%

Accuracy: 104.0-109.0%
Accuracy: 88.0-94.3%

Results of validation study (tissue)2P-51

Good linearity, precision and accuracy were observed over the concentration range of
25 to 10000 ng/g in tissues (liver and kidney). Nusinersen and its metabolites in liver
lysate were stable under various conditions.
Carry overItems

Calibration curve

Within-run accuracy andprecision

Post-preparative stability
Stability at room temperaturein tissue lysate

Freeze and thaw stability
in tissue lysate
8n=1

Contents

25-10000ng/g tissue,
n=1 each

4 concentrations,
n=5 each

4°C, 24 hours,
1 concentration, n=3
24 hours,
1 concentration, n=3

5 cycles,1 concentration, n=3Results

No interfering peak (liver and kidney)

Accuracy: 95.6-106.0%, r≥0.9992 (liver)
Accuracy: 93.6-108.0%, r≥0.9986 (kidney)

Accuracy: 94.2-101.6%, C.V.: 1.2-5.1% (liver)
Accuracy: 89.0-107.0%, C.V.: 1.2-6.7% (kidney)

Accuracy: 94.8-103.1% (liver)

Accuracy: 101.5-103.9% (liver)
Accuracy: 99.1-100.9% (liver)Typical chromatograms of biological samples

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Nusinersen
Administration• Rat (Crl:CD(SD))• Intravenous• 1 mg/kg ofnusinersen• Single dosing
The chromatograms of
the samples collected
360 minutes after
dosing are shown.

93’N-1

3’N-2I.S.
Plasma(5-fold diluted)

Liver(Undiluted)

Kidney(5-fold diluted)

2P-51Results of quantitative analysis

Concentrations of nusinersen and its metabolites in plasma tend to decrease
until 360 minutes after dosing. Whereas nusinersen in liver and kidney did not decrease orrather increased after dosing, and its metabolites in liver and kidney tend to increase
until 360 minutes after dosing.
Plasma1000001000010010Concentration (ng/g)

Concentration (ng/g)

Concentration (ng/mL)

1000

1000

10060120180240

Sampling time (min)

300

360

1060120180240

Sampling time (min)

300

360

n=1 each

Mean, n=3 each
10

1000

100

10

100

Kidney100000

10000

10000

1Liver

100000Nusinersen

3’N-1

1060

120

180240Sampling time (min)

300

360

n=1 each

3’N-2

Conclusion

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 The metabolites were identified more precisely using
HILIC condition for achieving baseline separation andseveral mass spectrometric technique.
 We have developed an LC-MS/MS method for
simultaneous quantification of nusinersen and its
metabolites in plasma, liver and kidney.

 Our results showed that nusinersen and its metabolites
distribute in liver and kidney as commonly described foroligonucleotides.
We have no financial relationships to disclose for this presentation.

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