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Medicilon Preclinical Research (Shanghai) LLC
Test Article: ALC-0159
Study No.: 01049-20021
1
In Vitro Metabolic Stability of ALC -0159 in CD -1/ICR Mouse, Sprague
Dawley Rat, Cynomolgus Monkey, and Human Liver S9 Fractions
Sponsor Acuitas Therapeutics Inc.
6190 Agronomy Road, Suite 402
Vancouver BC V6T 1Z3
Canada
Testing Facility Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Rd, Pudong
Shanghai 201299
China
Study Monitor
Acuitas Therapeutics Inc.
Study Director
Medicilon Preclinical Research (Shanghai) LLC
Alternate Contact
Medicilon Preclinical Research (Shanghai) LLC
Study Identification 01049-20021
Experimental Start Date 2020-06-19
Experimental Completion Date 2020-06-24
Number of Pages in Report 31
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Test Article: ALC-0159
Study No.: 01049-20021
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SUMMARY
This study evaluated the in vitro metabolic stability of ALC-0159 in liver S9 fractions of CD-1/ICR
mouse, Sprague Dawley r at, cynomolgus monkey, and h uman. ALC-0159 was stable after an
approximately 2 -hour incubation with liver S9 from all these species.
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Test Article: ALC-0159
Study No.: 01049-20021
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1. OBJECTIVE
To evaluate the in vitro metabolic stability of ALC-0159 in liver S9 fractions from different
species.
2. MATERIALS
2.1 Test Article
Name: ALC-0159
Molecular Formula: C30H60NO (C2H4O) n (n = 45~50)
MW (g/mol): ~2400-2600
2.2 Positive Controls
Compound Name Vendor CAS No. Cat. No. Lot No. Molecular Weight
Testosterone Aladdin 58-22-0 T102169 K1505051 288.42
7-hydroxycoumarin J&K Scientific 93-35-6 153384 L630I04 162.14
2.3 Internal Standard
Compound Name Vendor CAS No. Cat. No. Lot No. Molecular Weight
Verapamil hydrochloride TCI 152-11-4 V0118 RFMWJ-RL 491.06
Tolbutamide Sigma-Aldrich 64-7-7 46968 BCBV8457 270.35
2.4 Liver S9 Fractions
The following pooled liver S9 fractions of CD-1/ICR mouse, Sprague Dawley rat, cynomolgus
monkey, and human were stored in a -70oC ultra low temperature freezer prior to use.
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Species Manufacturer Cat. No. Lot No. Protein Concentration
（mg/mL）
CD-1/ICR mouse (male) XenoTech M1000.S9 1310210 20
Sprague Dawley rat (male) XenoTech R1000.S9 1310212 20
Cynomolgus monkey (male) XenoTech P2000.S9 0910273 20
Human (mixed gender) BioreclamationIVT X008023 BKY 20
2.5 Coenzymes and Pore-forming Agent
NADPH (reduced β-nicotinamide adenine dinucleotide 2′-phosphate ) tetrasodium salt was stored
at 2~8oC in a refrigerator prior to use. UDPGA (uridine-diphosphate-glucuronic acid trisodium
salt) and alamethicin were stored in a -20oC freezer prior to use.
Compound Name Manufacturer Cat. No. Molecular Weight Purity
NADPH Roche Diagnostic 10621706001 833.35 97%
UDPGA Sigma U6751 646.23 ≥98
Alamethicin Aladdin A132913 1964.3078 99%
3. EXPERIMENTAL PROCEDURES
3.1 Stock solutions preparation :
1.90 mg of ALC-0159 was weighed and dissolved in 7 6 μL of DMSO to obtain a 10 mM stock
solution. 2.547 mg of testosterone was weighed and dissolved in 551.93 μL of DMSO to obtain
a 16 mM solution. A 10 mM testosterone stock solution was then prepared by adding 60 μL
DMSO to 100 μL of the 16 mM testosterone solution. 3.97 mg of 7-hydroxycoumarin was
weighed and dissolved in 1224.25 μL of DMSO to obtain a 20 mM solution. A 10 mM
7-hydroxycoumarin stock solution was then prepared by adding 100 μL DMSO to 100 μL of
the 20 mM 7-hydroxycoumarin solution. 5 mg of alamethicin was weighed and dissolved in
495 μL of 100 mM potassium phosphate buffer to obtain a 10 mg/mL alamethicin solution.
3.2 0.5 mM spiking solutions preparation:
Spiking Solution of Test Article or Positive Control
Conc. of Stock Solution
(mM) Volume of Stock Solution
(μL) Volume of MeOH
(μL) Final Concentration
(mM)
10 10 190 0.5
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3.3 Preparation of 1.5× liver S9 suspensions (with alamethicin) containing test article or positive
control:
1.5× Liver S9 Suspension (with Alamethicin ) Containing Test Article or Positive Control
Livers S9 0.5 mM
Spiking
Solution
(μL) 10 mg/ml
Alamethicin
Solution 100 mM potassium
phosphate buffer
containing 5 mM of
MgCl2 (pH 7.4) (μL) Final Concentration
Conc. of
Stock
Solution
(mg/mL) Volume of
Stock
Solution (μL) Liver S9
Protein
(mg/mL) Compound
(μM)
20 37.5 1.5 1.9 459.1 1.5 1.5
3.4 1.5× liver S9 suspensions (with alamethicin) containing test article or positive control were
kept on ice for 15 minutes.
3.5 28.51 mg of NADPH was weighed and dissolved in 2.851 mL of 100 mM potassium phosphate
buffer to obtain a 12 mM NADPH working solution. 25 mg of UDPGA was weighed and
dissolved in 6.448 mL of 100 mM potassium phosphate buffer to obtain a 6 mM UDPGA
working solution. 2.8 mL of 12 mM NADPH working solution was added into 2.8 mL of 6
mM UDPGA working solution to obtain a 6 mM NADPH and 3 mM UDPGA mixed working
solution. This mixed working solution was then pre-warmed at 37oC.
3.6 30µL of liver S9 suspension (with alamethicin) containing 1.5 µM test article or positive
control was added to 96-well plates in duplicate for each time point (0, 15, 30, 60, 90, and 120
min).
3.7 96-well incubation plates were pre-warmed at 37oC for 5 min.
3.8 For 0 min samples: 450 µL of ethanol containing internal standard (IS solution) was added
before 15 µL of pre-warmed mixed working solution (6 mM NADPH and 3 mM UDPGA) was
added.
3.9 For other samples (15, 30, 60, 90, and 120 min): 15 µL of pre-warmed mixed working solution
(6 mM NADPH and 3 mM UDPGA) was added to initiate the reaction.
Volume (μL) Final Concentration in Incubation Mixture
1.5× Liver S9 Suspension
Containing Test Article or
Positive Control 3×NADPH
Working
Solution Total Liver S9 Protein
(mg/mL) Test Article or
Positive
Control (μM) NADPH
(mM)
30 15 45 0.5 1 2
The samples were incubated at 37oC and 450 µL of IS solution was added to stop the reaction at
the corresponding time points (15, 30, 60, 90, and 120 min).
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Test Article: ALC-0159
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3.10 After quenching, the plates were shaken at 600 rpm for 10 min and then centrifuge at 6, 000
rpm for 15 min.
3.11 Then 200 μL of the supernatant from each well was transferred into a 96-well sample plate
for LC-MS/MS analysis.
4. BIOANALYSIS
4.1 Instruments
SHIMADZU ：UPLC system
Sciex Triple Quad 6500+ with ESI ion source
4.2 LC/MS/MS Conditions
Column: Agilent Zorbax SB-CN 3.5 m (100 mm*2.1 mm)
Gradient for ALC-0159:
Time (min) Solvent A (%) Solvent B (%)
0.00 80 20
0.40 30 70
1.60 10 90
2.70 10 90
2.71 80 20
3.00 80 20
Solvent A: 0.1% formic acid in water
Solvent B: 0.1% formic acid in acetonitrile
Flow rate ：600 μL/min
Column temperature ：40oC
Autosampler temperature: 4oC
MS Conditions: MRM detection
Compound Q1(m/z) Q3(m/z) DP CE Retention Time (min)
ALC-0159 1164.00 494.70 45 71 ~1.33
Tolbutamide (IS) 271.10 172.00 70 18 ~1.03
4.3 Detection of ALC-0159
Representative chromatograms of ALC-0159 in each matrix are shown in Appendix 1 .
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5. DATA ANALYSIS
The % remaining parent compound (ALC-0159 or positive control, testosterone and
7-hydroxycoumarin) was calculated by dividing the peak area ratio (test article peak area/
internal standard peak area) by the time zero peak area ratio. The natural logarithm
of % remaining parent compound was plotted against time, and the slope of the regression line
was determined. The elimination constant and half-life were calculated, when possible, as
indicated below.
Elimination rate constant (k) = - slope
Half-life (t 1/2) = 0.693/k
6. RESULTS
A summary of the % remaining parent compound and half-life of ALC-0159 obtained from a
2-hour incubation with liver S9 from CD-1/ICR mouse, Sprague Dawley rat, cynomolgus
monkey, and human is presented in Table 1. The stability of ALC-0159 over time in each matrix
is shown in Figure 1. Raw data is presented in Appendix 2 .
The liver S9 used in this study were tested for activity using metabolism control substrates under
incubation conditions identical to those used for ALC-0159 . The enzymes were found to exhibit
satisfactory activity as determined by significant consumption of the positive control compounds
(testosterone and 7-hydroxycoumarin) during the 2-hour incubation period, hence the test
systems were considered to have yielded valid results. A summary of the % remaining parent
compound and half-life of testosterone and 7-hydroxycoumarin is provided in Table 1. The
stability of testosterone and 7-hydroxycoumarin over time in each matrix is shown in Figure 2
and Figure 3, respectively. Raw data for controls is presented in Appendix 3 (testosterone) and
Appendix 4 (7-hydroxycoumarin).
7. CONCLUSIONS
This study evaluated the in vitro metabolic stability of ALC-0159 in liver S9 fractions of
CD-1/ICR mouse, Sprague Dawley rat, cynomolgus monkey, and h uman. ALC-0159 was stable
after an approximately 2-hour incubation with li
ver S9 from all these species.
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Table 1. Summary of Liver S9 Stability of ALC-0159 , Testosterone and 7-Hydroxycoumarin
Compounds Species Percent Remaining (%) T1/2
(minute) 0 min 15 min 30 min 60 min 90 min 120 min
ALC-0159 CD-1/ICR
Mouse Mean 100.00 98.93 91.10 102.85 90.75 106.76 >120 RSD of Area Ratio 0.02 0.03 0.03 0.00 0.04 0.03
Sprague
Dawley Rat Mean 100.00 84.38 90.87 97.97 93.51 92.70 >120 RSD of Area Ratio 0.12 0.02 0.08 0.06 0.03 0.03
Cynomolgus
Monkey Mean 100.00 91.30 97.96 105.56 108.33 105.74 >120 RSD of Area Ratio 0.02 0.08 0.01 0.11 0.05 0.13
Human Mean 100.00 106.73 107.60 104.97 109.36 119.59 >120 RSD of Area Ratio 0.05 0.00 0.01 0.00 0.01 0.03
Testosterone CD-1/ICR
Mouse Mean 100.00 59.38 35.71 6.89 BQL BQL 15.5 RSD of Area Ratio 0.05 0.11 0.01 0.16 N/A N/A
Sprague
Dawley Rat Mean 100.00 BQL BQL BQL BQL BQL N/A RSD of Area Ratio 0.05 N/A N/A N/A N/A N/A
Cynomolgus
Monkey Mean 100.00 81.12 68.58 45.76 27.19 16.74 46.6 RSD of Area Ratio 0.11 0.11 0.02 0.06 0.02 0.08
Human Mean 100.00 63.69 17.40 BQL BQL BQL 11.9 RSD of Area Ratio 0.00 0.02 0.09 N/A N/A N/A
7-Hydroxycoumarin CD-1/ICR
Mouse Mean 100.00 40.06 16.68 3.57 1.89* 1.54* 12.5 RSD of Area Ratio 0.00 0.11 0.02 0.17 0.18 0.20
Sprague
Dawley Rat Mean 100.00 71.60 48.00 26.17* 13.71* 11.92* 28.3 RSD of Area Ratio 0.08 0.04 0.07 0.06 0.00 0.04
Cynomolgus
Monkey Mean 100.00 80.70 64.55 38.33 23.76 16.34 44.8 RSD of Area Ratio 0.04 0.04 0.05 0.00 0.01 0.04
Human Mean 100.00 69.01 43.17 19.16 8.29 3.68 25.0 RSD of Area Ratio 0.02 0.00 0.04 0.03 0.03 0.12
* The compound showed biphasic metabolic kinetics, i.e., an initial fast disappearance phase was followed by a slow disappearance phase. The data points marked with * were in the
slow disa
ppearance phase and were excluded from half-life calculation. BQL = Below quantification limit; N/A = not applicable
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Figure 1. Stability of ALC-0159 in Mouse, Rat, Monkey and Human Liver S9
CD-1/ICR Mouse Sprague Dawley Rat
Cynomolgus Monkey Human
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Figure 2 . Stability of Testosterone in Mouse, Rat, Monkey and Human Liver S9
CD-1/ICR Mouse Sprague Dawley Rat
Cynomolgus Monkey Human
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Figure 3 . Stability of 7 -Hydroxycoumarin in Mouse, Rat, Monkey and Human Liver S9
CD-1/ICR Mouse Sprague Dawley Rat
Cynomolgus Monkey Human
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8.APPENDICES
Appendix 1 – Representative Chromatograms of ALC-0 159 in Mouse, Rat, Monkey and Human Liver S9
Appendix 2 – Stability of ALC-0 159 in Mouse, Rat,
Monkey and Human Liver S9 – Raw Data
Appendix 3 – Stability of Testosterone
in Mouse, Rat, Monkey and Human Liver S9 – Raw Data
Appendix 4 – Stability of 7-Hydroxycoumarin in Mouse, Rat, Monkey and Human Liver S9 – Raw Data
Appendix 5 – 01049-2002 1
-S9 stability_protocol
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APPENDIX 1
Representative Chromatograms of ALC-0159 in Mouse, Rat, Monkey and Human Liver S9
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CD 1/ICR mouse
Sprague Dawley rat
Cynomolgus monkey
Human
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APPENDIX 2
Stability of ALC-0159 in Mouse, Rat, Monkey and Human Liver S9 – Raw Data
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Compounds Species Time(min) Raw Data
Analyte
Peak Area
(counts) Analyte
Peak Area
(counts) IS Peak
Area
(counts) IS Peak
Area
(counts) Area Ratio Area Ratio
ALC-0159 CD-1/ICR
Mouse 0 5.08E+04 5.05E+04 3.57E+07 3.62E+07 0.001 0.001
15 5.09E+04 4.82E+04 3.58E+07 3.55E+07 0.001 0.001
30 4.48E+04 4.74E+04 3.58E+07 3.61E+07 0.001 0.001
60 5.11E+04 5.11E+04 3.52E+07 3.56E+07 0.001 0.001
90 4.47E+04 4.65E+04 3.59E+07 3.56E+07 0.001 0.001
120 5.21E+04 5.42E+04 3.55E+07 3.55E+07 0.001 0.002
ALC-0159 Sprague
Dawley Rat 0 5.46E+04 6.41E+04 2.42E+07 2.40E+07 0.002 0.003
15 5.31E+04 5.16E+04 2.51E+07 2.51E+07 0.002 0.002
30 5.82E+04 5.24E+04 2.46E+07 2.47E+07 0.002 0.002
60 6.09E+04 5.58E+04 2.43E+07 2.40E+07 0.003 0.002
90 5.90E+04 5.65E+04 2.49E+07 2.51E+07 0.002 0.002
120 5.66E+04 5.43E+04 2.42E+07 2.44E+07 0.002 0.002
ALC-0159 Cynomolgus
Monkey 0 6.60E+04 6.44E+04 2.41E+07 2.42E+07 0.003 0.003
15 5.61E+04 6.26E+04 2.42E+07 2.40E+07 0.002 0.003
30 6.35E+04 6.29E+04 2.39E+07 2.39E+07 0.003 0.003
60 6.38E+04 7.33E+04 2.44E+07 2.38E+07 0.003 0.003
90 7.46E+04 6.96E+04 2.47E+07 2.46E+07 0.003 0.003
120 6.16E+04 7.28E+04 2.37E+07 2.34E+07 0.003 0.003
ALC-0159 Human 0 5.93E+04 5.62E+04 3.36E+07 3.40E+07 0.002 0.002
15 6.18E+04 6.07E+04 3.37E+07 3.34E+07 0.002 0.002
30 6.23E+04 6.19E+04 3.37E+07 3.38E+07 0.002 0.002
60 6.08E+04 6.08E+04 3.40E+07 3.37E+07 0.002 0.002
90 6.34E+04 6.28E+04 3.37E+07 3.38E+07 0.002 0.002
120 7.07E+04 6.69E+04 3.38E+07 3.35E+07 0.002 0.002
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APPENDIX 3
Stability of Testosterone in Mouse, Rat, Monkey and Human Liver S9 – Raw Data
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Compound s Species Time(min) Raw Data
Analyte
Peak Area
(counts) Analyte
Peak Area
(counts) IS Peak
Area
(counts) IS Peak
Area
(counts) Area Ratio Area Ratio
Testosterone CD-1/ICR
Mouse 0 1.74E+04 1.71E+04 5.35E+05 5.68E+05 0.032 0.030
15 1.06E+04 1.16E+04 6.22E+05 5.77E+05 0.017 0.020
30 6.44E+03 6.29E+03 5.72E+05 5.69E+05 0.011 0.011
60 1.10E+03 1.37E+03 5.79E+05 5.69E+05 0.002 0.002
90 LOD LOD 5.84E+05 5.74E+05 LOD LOD
120 LOD LOD 5.70E+05 5.75E+05 LOD LOD
Testosterone Sprague
Dawley Rat 0 1.52E+04 1.62E+04 6.17E+05 6.14E+05 0.025 0.026
15 LOD LOD 6.05E+05 5.74E+05 LOD LOD
30 LOD LOD 6.53E+05 5.96E+05 LOD LOD
60 LOD LOD 5.75E+05 5.65E+05 LOD LOD
90 LOD LOD 6.06E+05 5.80E+05 LOD LOD
120 LOD LOD 5.98E+05 6.00E+05 LOD LOD
Testosterone Cynomolgus
Monkey 0 1.68E+04 1.43E+04 5.88E+05 5.86E+05 0.029 0.024
15 1.22E+04 1.32E+04 6.16E+05 5.72E+05 0.020 0.023
30 1.08E+04 1.08E+04 6.04E+05 5.85E+05 0.018 0.018
60 6.82E+03 7.12E+03 5.88E+05 5.64E+05 0.012 0.013
90 4.29E+03 4.11E+03 5.87E+05 5.79E+05 0.007 0.007
120 2.78E+03 2.38E+03 5.94E+05 5.69E+05 0.005 0.004
Testosterone Human 0 1.66E+04 1.57E+04 5.95E+05 5.63E+05 0.028 0.028
15 1.02E+04 1.05E+04 5.81E+05 5.81E+05 0.018 0.018
30 3.05E+03 2.85E+03 5.88E+05 6.27E+05 0.005 0.005
60 LOD LOD 5.92E+05 5.75E+05 LOD LOD
90 LOD LOD 6.07E+05 6.32E+05 LOD LOD
120 LOD LOD 5.50E+05 5.76E+05 LOD LOD
LOD = Limit of detection
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APPENDIX 4
Stability of 7-Hydroxycoumarin in Mouse, Rat, Monkey and Human Liver S9 – Raw Data
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Compounds Species Time
(min) Raw Data
Analyte
Peak Area
(counts) Analyte
Peak Area
(counts) IS Peak
Area
(counts) IS Peak
Area
(counts) Area
Ratio Area
Ratio
7-
Hydroxycoumarin CD-1/ICR
Mouse 0 2.69E+04 2.63E+04 5.42E+05 5.29E+05 0.050 0.050
15 1.20E+04 1.01E+04 5.59E+05 5.52E+05 0.021 0.018
30 4.62E+03 4.53E+03 5.49E+05 5.54E+05 0.008 0.008
60 1.11E+03 8.20E+02 5.54E+05 5.27E+05 0.002 0.002
90 5.99E+02 4.34E+02 5.62E+05 5.30E+05 0.001 0.001
120 3.69E+02 4.87E+02 5.59E+05 5.58E+05 0.001 0.001
7-
Hydroxycoumarin Sprague
Dawley Rat 0 2.60E+04 2.51E+04 5.20E+05 5.61E+05 0.050 0.045
15 1.88E+04 1.82E+04 5.37E+05 5.52E+05 0.035 0.033
30 1.29E+04 1.20E+04 5.39E+05 5.52E+05 0.024 0.022
60 6.94E+03 6.43E+03 5.38E+05 5.40E+05 0.013 0.012
90 3.57E+03 3.60E+03 5.49E+05 5.56E+05 0.007 0.006
120 2.92E+03 3.11E+03 5.32E+05 5.34E+05 0.005 0.006
7-
Hydroxycoumarin Cynomolgus
Monkey 0 2.46E+04 2.60E+04 5.32E+05 5.32E+05 0.046 0.049
15 2.18E+04 2.06E+04 5.53E+05 5.50E+05 0.039 0.037
30 1.65E+04 1.74E+04 5.57E+05 5.45E+05 0.030 0.032
60 9.83E+03 9.77E+03 5.40E+05 5.35E+05 0.018 0.018
90 6.29E+03 6.06E+03 5.52E+05 5.40E+05 0.011 0.011
120 4.19E+03 4.03E+03 5.23E+05 5.34E+05 0.008 0.008
7-
Hydroxycoumarin Human 0 2.65E+04 2.65E+04 5.32E+05 5.16E+05 0.050 0.051
15 1.88E+04 1.83E+04 5.37E+05 5.24E+05 0.035 0.035
30 1.23E+04 1.15E+04 5.49E+05 5.42E+05 0.022 0.021
60 5.42E+03 5.06E+03 5.48E+05 5.33E+05 0.010 0.009
90 2.28E+03 2.37E+03 5.55E+05 5.52E+05 0.004 0.004
120 9.57E+02 1.11E+03 5.60E+05 5.53E+05 0.002 0.002
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Test Article: ALC-0159
Study No.: 01049-20021
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APPENDIX 5
01049-20021-S9 stability_protocol
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In Vitro Metabolic Stability of ALC- 0159 in CD-1/ICR Mouse,
Sprague Dawley Rat, Cynomolgus Monkey, and Human Liver S9
Fractions
Testing Facility
Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Road, Pudong
Shanghai 201299, China
Study Number
01049-20021
Study Director
Sponsor
Acuitas Therapeutics Inc.
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2 CONTENTS
1.INTRODUCTION .........................................................................................................3
1.1. Study Number .........................................................................................................3
1.2. Study Title ..............................................................................................................3
1.3. Sponsor Representative ..........................................................................................3
1.4. Objective .................................................................................................................3
1.5. Compliance .............................................................................................................3
1.6. Testing Facility .......................................................................................................3
1.7. Personnel ................................................................................................................3
1.8. Study Schedule .......................................................................................................4
2.MATERIALS ................................................................................................................4
2.1. Test Article .............................................................................................................4
2.2. Positive Control and Internal Standard ...................................................................4
2.3. Liver Microsomes and Cofactor .............................................................................4
3.EXPERIMENTAL PROCEDURES .............................................................................5
4.BIOANALYSIS ............................................................................................................6
4.1. Instruments .............................................................................................................6
4.2. LC/MS/MS Conditions ...........................................................................................6
5.DATA ANALYSIS .......................................................................................................7
6.FINAL REPORT ...........................................................................................................7
7.SIGNATURES ..............................................................................................................8
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1. INTRODUCTION
1.1. Study Number
01049-20021
1.2. Study Title
In Vitro Metabolic Stability of ALC-0159 in CD-1/ICR Mouse, Sprague Dawley Rat,
Cynomolgus Monkey, and Human Liver S9 Fractions
1.3. Sponsor Representative
Acuitas Therapeutics Inc.
6190 Agronomy Road, Suite 402
Vancouver BC V6T 1Z3
Canada
1.4. Objective
To evaluate the in vitro metabolic stability of ALC-0159 in liver S9 from different
species.
1.5. Compliance
This is a non-GLP study and will be conducted according to the Standard Operating
Procedures (SOPs) of Medicilon Preclinical Research (Shanghai) LLC.
1.6. Testing Facility
Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Road, Pudong, Shanghai 210299, China
1.7. Personnel
1.7.1. Study Director
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1.7.2. Alternate Contact
1.8. Study Schedule
Study Initiation Date: Signature date by Study Director
Experiment Start Date: To be included in the final report
Experime nt Termination Date: To be included in the final report
Draft Report Issue Date: To be included in the final report
2. MATERIALS
2.1. Test Article
Name: ALC-0159
Molecular Formula: C 30H60NO (C2H4O) n (n = 45~50)
MW (g/mol): ~2400-2600
2.2. Positive Control and Internal Standard
Testosterone and 7 -hydroxycoumarin will be used as positive controls . Tolbutamide
will be used as internal standard. The sources will be documented in experiment al
records and presented in the report.
2.3. Liver Microsomes and Cofactor
Liver S9 fractions o f CD-1/ICR mouse, Sprague Dawley rat, cynomolgus monkey and
human were purchased from qualified supplier and stored in a -7 0oC ultra low
temperature freezer.
NADPH (reduced β-Nicotinamide adenine dinucleotide 2′-phosphate tetrasodium salt )
were purchased from qualified supplier and stored at 2 -8oC in a refrigerator.
UDPGA (uridine-diphosphate -glucuronic acid trisodium salt) and alamethicin were
purchased from qualified suppliers and stored in a -20oC freezer.
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The source and lot numbers will be documented in the experiment al records and
presented in the final report .
3. EXPERIMENTAL PROCEDURES
(1) Preparation of stock solutions : Appropriate amount of test article or positive
control is weighed and dissolved in DMSO to obtain a 10 mM stock solution.
(2) Preparation o f 0.5 mM spiking solutions:
Spiking Solution of Test Article or Positive Control
Conc. of stock solution Volume of stock solution (μL) Volume of MeOH (μL) Final Concentration
10 mM 10 190 0.5 mM
(3) Preparation of 1.5× liver S9 suspensions with alamethicin containing test article
or positive control:
1.5× Liver S9 Suspension with Alamethicin c ontaining Test article or Positive control
Livers S9
0.5 mM
spiking
solution
(μL) 10 mg/ml
Alamethicin 100 mM potassium
phosphate buffer
containing
5 mM of MgCl 2 (pH
7.4)
(μL) Final Concentration
Conc. of
stock
solution
(mg/mL) Volume of stock
solution (μL) Liver S9
protein
(mg/mL) Compound
(μM)
20 37.5 1.5 1.9 459.1 1.5 1.5
(4) 1.5× liver S9 suspensions with alamethicin containing test article or positive
control are kept on ice for 15 minutes.
(5) 3× master mix of cofactors (6 mM NADPH and 3 mM UDPGA in 100 mM
potassium phosphate buffer containing 5 mM of MgCl 2, pH7.4) is prepared and
then pre-warmed to 37oC.
(6) 30 µL of liver S9 suspension with alamethicin containing 1. 5 µM test article or
positive control is added to 96-well plates in duplicate for each time point (0, 15,
30, 60, 90, and 120 min).
(7) 96-well incubation plates (from Step 6) are pre-warmed at 37 oC for 5 min.
(8) For 0-min samples: 450 µL ethanol containing internal standard (IS solution) is
added, followed by 15 µL pre-warmed 3× master mix of cofactors.
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(9) For the 15, 30, 60, 90, and 120 min samples, 15 µL pre-warmed 3× master mix of
cofactors is added to initiate reaction.
Volume of final incubation system (μL) Final Concentration
1.5× Liver S9
Suspension
with
Alamethicin
containing Test
article or
Positive control 3× Master
Mix of
Cofactors Total
Volume Liver S9
protein
(mg/mL) Compound
(μM) UDPGA
(mM) NADPH
(mM)
30 15 45 1 1 1 2
The samples are incubated at 37 oC and 450 µL IS solution (10ng/mL v erapamil
in ethanol ) is added to stop the reaction at the corresponding time points (15, 30,
60, 90, and 120 min).
(10) After quenching, shake the plates at 600 rpm for 10 min and then centrifuge them
at 6,000 rpm for 15 min.
(11) The plates are sealed and stored at -20 oC in a freezer until bioanalysis.
(12) Thaw the plates at room temperature, centrifuge them at 6, 000 rpm for 15 min ,
then transfer 200 μL of the supernatant from each well into a 96-well sample
plate for LC- MS/MS analysis.
4. BIOANALYSIS
4.1. Instruments
SHIMADZU ：UPLC system
Sciex Triple Quad 6500+ with ESI ion source
4.2. LC/MS/MS Conditions
Column：Agilent Zorbax SB- CN 3.5um (100mm*2.1mm)
Gradient Chromatography Parameters for ALC-0159:
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Time (min) Solvent A (%) Solvent B (%)
0.00 80 20
0.40 30 70
1.60 10 90
2.70 10 90
2.71 80 20
3.00 80 20
Solvent A: 10mM ammonium formate, 0.1% Formic acid in water
Solvent B: 10mM ammonium format e, 0.1% Formic acid in acetonitrile
Flow rate ：500 μL/min
Column temperature ：40 oC
Autosampler temperature: 4oC
MS Conditions: MRM detection
Compound Q1(m/z) Q3(m/z) DP CE Retention
ALC-0159 1164.00 494.70 45 71 ~1.30
Tolbutamide (IS)
271.10 172.00 70 18 ~1.02
5. DATA ANALYSIS
The % remaining will be calculated by dividing the peak area ratio (test article
peak area/ internal standard peak area) by the time zero peak area ratio. The
natural logarithm of % remaining will be plotted against time, and the slope of the
regression line will be determined. Then elimination constant and half-life will be
calculated as below.
Elimination rate constant (k) = - slope
Half-life (t 1/2) = 0.693/k
6. FINAL REPORT
After completion of the study, a draft report including the results, analysis and
discussion will be sent to the Sponsor in Microsoft Word format.
One month after issuance of the draft report, if no requested revisions or instructions
to finalize have been communicated by the Sponsor, the draft report will be issued as
a final report, signed by the Study Director, and submitted to the Sponsor in Adobe
Acrobat PDF format, containing hyperlinks, as applicable. Any modifications or
changes to the draft report requested one month after issuance of the draft will be
performed at additional cost to the Sponsor.
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