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Medicilon Preclinical Research (Shanghai) LLC
Test Article: ALC-0159
Study No.: 01049-20020
In Vitro Metabolic Stability of ALC -0159 in CD-1/ICR Mouse,
Sprague Dawley Rat, Wistar Han Rat, Cynomolgus Monkey , and
Human Liver Microsomes
Sponsor Acuitas Therapeutics Inc.
6190 Agronomy Road, Suite 402
Vancouver BC V6T 1Z3
Canada
Testing Fa cility Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Rd, Pudong
Shanghai 201299
China
Study Monitor
Acuitas Therapeutics Inc.
Stud y Director
Medicilon Preclinical Research (Shanghai) LLC
Alternate Contact
Medicilon Preclini cal Research (Shanghai) LLC
Study Identification 01049-20020
Experimental Start Date 2020-06-04
Experimental Completion Date 2020-06-08
Number of Pages in Report 28
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FDA-CBER-2021-5683-0709311
FDA-CBER-2021-5683-0709312
Medicilon Preclinical Research (Shanghai) LLC
Test Article: ALC-0159
Study No.: 01049-20020
SUMMARY
This study evaluated the in vitro metabolic stability of ALC-0159 in liver microsom es of
CD-1/ICR mouse, Sprague Dawley r at, Wistar Han r at, cynomolgus monkey, and human. ALC-
0159 was stable after an approximately 2-hour incubation with liver microsomes from all these
species.
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Test Article: ALC-0159
Study No.: 01049-20020
1. OBJECTIVE
To evaluate the in vitro metabolic stability of ALC-0159 in liver microsomes from different
species.
2. MATERIALS
2.1 Test Article
Name: ALC-0159
Molecular Formula: C 30H60NO (C 2H4O) nOCH3 n = 45-50
MW (g/mol): ~2400-2600
2.2 Positive Control
Compound
Name Vendor CAS No. Cat. No. Lot No. Molecular Weight
Ketanserin TCI 74050-98-9 K0051 NPGAF-CO 395.43
2.3 Internal Standard
Compound
Name Vendor CAS No. Cat. No. Lot No. Molecular Weight
Tolbutamide Sigma-
Aldrich 64-7-7 46968 BCBV8457 270.35
2.4 Liver Microsomes and Cofactor
The following pooled liver
microsomes of CD-1/ICR mouse, Sprague Dawley rat, Wistar Han
rat, cy
nomolgus monkey, and human were stored in a -70oC ultra low temperature freezer prior
to use.
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Test Article: ALC-0159
Study No.: 01049-20020
Species Manufacturer Cat. No. Lot No. Protein
Concentration
（mg/mL）
CD-1/ICR mouse (male) XenoTech M1000 1910002 20
Sprague Dawley rat (male) XenoTech R1000 1910100 20
Wistar Han rat BioIVT BCF 201801BCF 20
Cynomolgus monkey (male) RILD Shanghai LM-SXH-02M NXNN 20
Human (mixed gender) XenoTech H0610 1810003 20
2.5 Coenzyme
NADPH (reduced β-nicotinamide adenine dinucleotide 2′-phosphate ) tetrasodium salt was stored
at 2-8oC in a refrigerator prior to use.
Compound Name Manufacturer Cat. No. Molecular Weight Purity
NADPH Roche Diagnostic 10621706001 833.35 97%
3. EXPERIMENTAL PROCEDURES
3.1 Stock solution : 1.90 mg of ALC-0159 was weighed and dissolved in 76 μL of DMSO to
obtain a 10 mM
stock solution. 3.237 mg of ketanserin was weighed and dissolved in 818.60
μL of DMSO to obtain a
10 mM stock solution.
3.2 0.5 mM spiking solution:
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
3.3 1.5× liver microsomes suspension containing test article or positive control:
1.5× Liver Microsome s Suspension Containing Test Article or Positive Control
Liver Microsomes 0.5 mM
spiking
solution
(μL) 100 mM potassium
phosphate buffer (pH 7.4)
(μL) Final Concentration
Conc. of stock
suspension
(mg/mL) Volume of stock
suspension
(μL) Liver microsomal
protein
(mg/mL) Compound
(μM)
20 18.75 1.5 479.75 0.75 1.5
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Study No.: 01049-20020
3.4 22.98 mg of NADPH was weighed and dissolved in 4.596 m L of 100 mM potassium
phosphate buffer to obtain a 6 mM NADPH working solution. This working solution was
then pre-warmed
at 37oC.
3.5 30 µL of 1.5× liver microsomes suspension containing 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).
3.6 96-well incubation plates were pre-warmed at 37 oC for 5 min.
3.7 For 0-min samples: 450 µL of ethanol containing internal standard (IS solution) was added
before 15 µL of p
re-warmed NADPH working solution (6 mM) was added.
3.8 For other samples (15, 30, 60, 90, and 120 min): 15 µL of pre-warmed NADPH working
solution (6 mM ) was added to initiate the reaction.
Volume (μL) Final Concentration in Incubation Mixture
1.5× Liver Microsome s
Suspension Containing Test
Article or Positive Control 3×NADPH
working
solution Total Liver microsomal
protein
(mg/mL) Test Article or
Positive Control
(μM) NADPH (mM)
30 15 45 0.5 1 2
The samples were incubated at 37 oC and 450 µL of IS solution was added to stop the reaction at
the corresponding ti
me points (15, 30, 60, 90, and 120 min).
3.9 After quenching, the plates were shaken at 600 rpm for 10 min and then centrifuged at 6,000
rpm for 15 min.
3.10 200 μL of supernatant was transferred from each well into a 96-well sample plate for LC-
MS/MS analysis.
4. BIOANALYSIS
4.1 Instruments
SHIMADZU：UPLC system
Sciex Triple Qua
d 6500+ with ESI ion source
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Test Article: ALC-0159
Study No.: 01049-20020
4.2 LC/MS/MS Conditions
Column：Agilent Zorbax SB-CN 3.5um (100mm*2.1mm)
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 ：40 oC
Autosampler temperature: 4oC
MS Conditions: MRM detection
Compound Q1(m/z) Q3(m/z) DP CE Retention Time
ALC-0159 1164.00 494.70 45 71 ~1.31
Tolbutamide(IS) 271.10 172.00 70 18 ~1.01
4.3 Detection of ALC-0159
Representative chromatog
rams of ALC-0159 in each matrix are shown in Appendix 1 .
5. DATA ANALYSIS
The % remaining parent compound (ALC-0159 or positive control, ketanserin) 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 li
ne was determined. The elimination constant and half-life was
calculated, when possible,
as indicated below.
Elimination rate constant (k) = - slope
Half-life (t 1/2) =
0.693/k
The in vitro intrinsic clearance, CL′ int, was calculated from the t 1/2 as follows:
CL′int = (0.693/t 1/2) × (1/ (microsomal protein concentration (0.5 mg/mL))) × Scaling Factor
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Study No.: 01049-20020
The scaling factors are listed in Table 1.
Table 1. Scaling Factors for Intrinsic Clearanc
e Prediction
in Mouse, Rat, Monkey, and Human Liver Microsomes
Species Microsomal Protein (mg)
per Gram of Liver Liver Weight (g) per
kg Body Weight Scaling Factor
(mg/kg)a Hepatic Blood Flow
(mL/min/kg)
Mouse 45 87.5 3937.5 90
Rat 44.8 40 1792 55.2
Monkey 45 32.5 1462.5 44
Human 48.8 25.7 1254.2 20.7
aMicrosomal protein (mg/g liver) × liver weight (g)/kg body weight
6. RESULTS
A summary of the % remaining parent compound, CL’ int and half-life of ALC-0159 obtained
from a 2-hour incubation of ALC-
0159 with liver microsomes from CD-1/ICR mouse, Sprague
Dawley rat, W
istar Han rat, cynomolgus monkey, and human is presented in Table 2. The
stability of ALC-0159 over time in each matrix is shown in Figure 1. Raw data is presented in
Appendix 2 .
The liver microsomes used in this study were tested for activity using a metabolism control
substrate under incubation conditions identical to those used for ALC-0159. The enzymes were
found to exhibit satisfactory activity as de
termined by significant consumption of the positive
control compound (ketanserin ) during the 2 -hour incubation period, hence the test systems were
considered to have yielded valid results. A summary
of the % remaining parent compound, CL’ int
and half-life of ketanserin is provide
d in Table 2. The stability of ketanserin over time in each
matrix is shown in Figure 2. Raw data is presented in Appendix 3 .
7. CONCLUSIONS
This study evaluated the in vitro metabolic stability of ALC-0159 in liver microsomes of
CD-1/ICR mouse, Sprague Dawley rat, Wist
ar Han rat, cynomolgus monkey, and human. ALC-
0159 was stable after an approximately 2-hour incubation with liver microsomes from all these
species.
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Test Article: ALC-0159
Study No.: 01049-20020
Table 2. Summary of Liver Microsomal Stability of ALC-0159 and Ketanserin
Test
Article Species Percent Remaining (%) t1/2
(minute) CL’int
(mL/min/kg) 0 min 15 min 30 min 60 min 90 min 120 min
ALC-0159 CD-1/ICR mouse Mean 100.00 82.27 86.40 85.54 85.41 95.87 >120 <45.5 RSD of Area Ratio 0.07 0.09 0.11 0.01 0.05 0.18
Sprague Dawley rat Mean 100.00 101.24 93.78 98.34 95.44 97.10 >120 <20.7 RSD of Area Ratio 0.09 0.03 0.08 0.03 0.05 0.11
Wistar Han rat Mean 100.00 112.11 102.69 105.38 100.90 108.97 >120 <20.7 RSD of Area Ratio 0.01 0.06 0.06 0.01 0.04 0.13
Cynomolgus monkey Mean 100.00 100.83 85.12 86.36 94.63 93.39 >120 <16.9 RSD of Area Ratio 0.06 0.07 0.03 0.03 0.04 0.05
Human Mean 100.00 99.59 92.28 95.53 97.97 93.09 >120 <14.5 RSD of Area Ratio 0.01 0.11 0.03 0.05 0.02 0.02
Ketanserin CD-1/ICR mouse Mean 100.00 61.73 37.16 17.24* 10.16* 6.43* 21.0 260 RSD of Area Ratio 0.04 0.01 0.02 0.05 0.01 0.05
Sprague Dawley rat Mean 100.00 74.03 51.43 26.11 16.08* 10.01* 30.7 80.9 RSD of Area Ratio 0.04 0.02 0.03 0.05 0.03 0.03
Wistar Han rat Mean 100.00 54.03 25.10 6.76 2.35 1.18* 16.4 151 RSD of Area Ratio 0.02 0.02 0.01 0.07 0.04 0.06
Cynomolgus monkey Mean 100.00 71.44 47.42 24.00 13.05* 8.35* 28.9 70.1 RSD of Area Ratio 0.03 0.02 0.01 0.02 0.04 0.02
Human Mean 100.00 77.74 57.56 38.26 26.22* 24.46* 43.1 40.3 RSD of Area Ratio 0.09 0.01 0.01 0.04 0.12 0.05
* Compound showed biphasic metabolic kinetics, i.e., an initial fast disappearance phase was followed by a slow disappearance phase. The data points marked in * were in the slow
disappearance phase and were excluded from half-life calculation.
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Test Article: ALC-0159
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Figure 1. Stability of ALC-0159 in Mouse, Rat, Monkey and Human Liver Microsomes
CD-1/ICR Mouse Sprague Dawley Ra t Wistar Han Rat
Cynomolgus Monkey Human
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Study No.: 01049-20020
Figure 2. Stability of Ketanserin in Mouse, Rat, Monkey and Human Liver Microsomes
CD-1/ICR Mouse Sprague Dawley Rat Wistar Han 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 Microsomes
Appendix 2 – Stability of ALC-0 159 in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
Appendix 3 – Stability
of Ketanserin
in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
Appendix 4 –01049-20020-microsomal stability
protocol
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APPENDIX 1
Representative Chromatograms of ALC-0159 in Mouse, Rat, Monkey and Human Liver
Microsomes
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Study No.: 01049-20020
CD-1/ICR mouse
Sprague Dawley rat
Wistar Han rat
Cynomolgus monkey
Human
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APPENDIX 2
Stability of ALC-0159 in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
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Test Article: ALC-0159
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Stability of ALC-0159 in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
Compound 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 2.04E+04 2.24E+04 1.77E+07 1.77E+07 0.001 0.001
15 1.43E+04 1.88E+04 1.54E+07 1.78E+07 0.001 0.001
30 2.04E+04 1.50E+04 1.80E+07 1.56E+07 0.001 0.001
60 1.88E+04 1.85E+04 1.83E+07 1.79E+07 0.001 0.001
90 1.80E+04 1.90E+04 1.81E+07 1.78E+07 0.001 0.001
120 1.88E+04 2.31E+04 1.86E+07 1.77E+07 0.001 0.001
ALC-0159 Sprague
Dawley rat 0 2.03E+04 2.23E+04 1.79E+07 1.74E+07 0.001 0.001
15 2.12E+04 2.24E+04 1.79E+07 1.78E+07 0.001 0.001
30 1.93E+04 2.16E+04 1.81E+07 1.81E+07 0.001 0.001
60 2.01E+04 2.11E+04 1.74E+07 1.75E+07 0.001 0.001
90 1.97E+04 2.08E+04 1.78E+07 1.75E+07 0.001 0.001
120 1.95E+04 2.17E+04 1.81E+07 1.72E+07 0.001 0.001
ALC-0159 Wistar Han
rat 0 1.97E+04 1.98E+04 1.78E+07 1.76E+07 0.001 0.001
15 2.27E+04 2.13E+04 1.75E+07 1.77E+07 0.001 0.001
30 2.00E+04 2.15E+04 1.82E+07 1.81E+07 0.001 0.001
60 2.06E+04 2.09E+04 1.77E+07 1.77E+07 0.001 0.001
90 1.96E+04 1.94E+04 1.70E+07 1.78E+07 0.001 0.001
120 2.27E+04 1.89E+04 1.71E+07 1.72E+07 0.001 0.001
ALC-0159 Cynomolgus
monkey 0 2.31E+04 2.12E+04 1.83E+07 1.83E+07 0.001 0.001
15 2.14E+04 2.36E+04 1.84E+07 1.85E+07 0.001 0.001
30 2.00E+04 1.91E+04 1.91E+07 1.90E+07 0.001 0.001
60 1.90E+04 2.03E+04 1.86E+07 1.89E+07 0.001 0.001
90 2.08E+04 2.14E+04 1.88E+07 1.82E+07 0.001 0.001
120 2.04E+04 2.18E+04 1.87E+07 1.86E+07 0.001 0.001
ALC-0159 Human 0 2.23E+04 2.15E+04 1.80E+07 1.76E+07 0.001 0.001
15 2.30E+04 2.02E+04 1.74E+07 1.79E+07 0.001 0.001
30 2.08E+04 2.02E+04 1.80E+07 1.82E+07 0.001 0.001
60 2.03E+04 2.13E+04 1.80E+07 1.75E+07 0.001 0.001
90 2.14E+04 2.10E+04 1.75E+07 1.76E+07 0.001 0.001
120 2.01E+04 2.01E+04 1.77E+07 1.74E+07 0.001 0.001
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APPENDIX 3
Stability of Ketanserin in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
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Stability of Ketanserin in Mouse, Rat, Monkey and Human Liver Microsomes – Raw Data
Compound 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
Ketanserin CD-1/ICR
mouse 0 1.93E+06 1.99E+06 8.68E+05 8.45E+05 2.22 2.36
15 1.18E+06 1.17E+06 8.32E+05 8.31E+05 1.42 1.41
30 7.30E+05 7.08E+05 8.43E+05 8.45E+05 0.87 0.84
60 3.42E+05 3.24E+05 8.37E+05 8.49E+05 0.41 0.38
90 1.94E+05 1.94E+05 8.29E+05 8.36E+05 0.23 0.23
120 1.20E+05 1.28E+05 8.43E+05 8.39E+05 0.14 0.15
Ketanserin Sprague
Dawley rat 0 2.00E+06 1.93E+06 8.58E+05 8.74E+05 2.33 2.21
15 1.42E+06 1.46E+06 8.57E+05 8.57E+05 1.66 1.70
30 9.99E+05 1.00E+06 8.34E+05 8.78E+05 1.20 1.14
60 5.01E+05 5.15E+05 8.76E+05 8.37E+05 0.57 0.61
90 3.16E+05 3.07E+05 8.43E+05 8.62E+05 0.37 0.36
120 1.91E+05 1.89E+05 8.55E+05 8.14E+05 0.22 0.23
Ketanserin Wistar Han
rat 0 2.02E+06 2.08E+06 8.55E+05 8.52E+05 2.36 2.44
15 1.08E+06 1.09E+06 8.41E+05 8.28E+05 1.28 1.31
30 5.31E+05 5.23E+05 8.76E+05 8.71E+05 0.61 0.60
60 1.29E+05 1.41E+05 8.41E+05 8.24E+05 0.15 0.17
90 4.80E+04 4.97E+04 8.74E+05 8.55E+05 0.05 0.06
120 2.31E+04 2.42E+04 8.56E+05 8.22E+05 0.03 0.03
Ketanserin Cynomolgus
monkey 0 2.07E+06 2.07E+06 8.64E+05 8.34E+05 2.40 2.49
15 1.43E+06 1.46E+06 8.30E+05 8.23E+05 1.72 1.77
30 9.68E+05 9.82E+05 8.42E+05 8.42E+05 1.15 1.17
60 4.84E+05 4.88E+05 8.40E+05 8.18E+05 0.58 0.60
90 2.68E+05 2.75E+05 8.65E+05 8.40E+05 0.31 0.33
120 1.69E+05 1.65E+05 8.19E+05 8.19E+05 0.21 0.20
Ketanserin Human 0 2.11E+06 1.97E+06 8.12E+05 8.57E+05 2.60 2.30
15 1.57E+06 1.56E+06 8.30E+05 8.13E+05 1.89 1.92
30 1.09E+06 1.19E+06 7.77E+05 8.37E+05 1.40 1.42
60 7.23E+05 6.78E+05 7.52E+05 7.42E+05 0.96 0.91
90 6.14E+05 5.18E+05 8.82E+05 8.80E+05 0.70 0.59
120 4.40E+05 4.88E+05 7.60E+05 7.88E+05 0.58 0.62
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APPENDIX 4
01049-20020-microsomal stability protocol
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In Vitro Metabolic Stability of ALC- 0159 in CD-1/ICR Mouse,
Sprague Dawley Rat, Wistar Han R at, Cynomolgus Monkey, and
Human Liver Microsomes
Testing Facility
Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Road
Pudong, Shanghai 201299
China
Study Number
01049-20020
Study Director
Sponsor
Acuitas Therapeutics Inc.
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1. INTRODUCTION
1.1. Study Number
01049-20020
1.2. Study Title
In Vitro Metabolic Stability of ALC-0159 in CD-1/ICR Mouse, Sprague Dawley Rat,
Wistar Han R at, Cynomolgus Monkey, and Human Liver Microsomes
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 microsomes from
different species and to determine intrinsic clearance in each 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
1.7.2. Alternate Contact
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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
Ketanserin and verapamil will be used as positive control and internal standard,
respectively. The sources will be documented in experiment al records and presented
in the report.
2.3. Liver Microsomes and Cofactor
Liver microsomes of CD-1/ICR mouse, Sprague Dawley rat, Wistar Han rat,
cynomolgus monkey, and human were purchased from qualified suppliers and stored
in a -70oC ultra low temperature freezer. NADPH (reduced β-n icotinamide adenine
dinucleotide 2′-phosphate) tetrasodium salt were purchased from a qualified supplier
and stored at 2-8oC in a refrigerator . The source and lot numbers will be documented
in the experiment al records and presented in the final report.
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3. EXPERIMENTAL PROCEDURES
(1) Preparation of stock solution : Appropriate amount of test article or positive
control is weighed and dissolved in DMSO to obtain a 10 mM stock solution.
(2) Preparation of 0.5 mM spiking solution:
Spiking Solution of Test Article or Po sitive Control
Conc. of stock solution
(mM) Volume of stock solution
(μL) Volume of MeOH
(μL) Final Concentration
(mM)
10 10 190 0.5
(3) Preparation of 1.5× liver microsomes suspension containing test article or
positive control:
1.5× Liver Microsomes Suspension Containing Test Article or Positive Control
Liver Microsomes 0.5 mM
spiking
solution
(μL) 100 mM potassium
phosphate buffer (pH 7.4)
(μL) Final Concentration
Conc. of stock
suspension
(mg/mL) Volume of stock
suspension
(μL) Liver microsomal
protein
(mg/mL) Compound
(μM)
20 18.75 1.5 479.75 0.75 1.5
(4) 3×NADPH working solution (6 mM; 5 mg/mL) will be prepared by dissolving
NADPH in 100 mM pH 7.4 potassium phosphate buff er. The working solution is
then pre-warmed at 37oC.
(5) 30 µL of 1.5× liver microsomes suspension containing test article or positive
control is added to 96-well plates in duplicate for each time point (0, 1 5, 30, 60,
90, and 120 min).
(6) 96-well incubation plates are pre-warmed at 37 oC for 5 min.
(7) For 0-min samples: 450 µL ethanol containing internal standard (IS solution) is
added before 15 µL pre-warmed NADPH working solution (6mM) is added.
(8) For other samples (15, 30, 60, 90, and 120 min): 15 µL pre-warmed NADPH
working solution
(6 mM) is added to initiate reaction.
Volume (μL) Final Concentration in incubation mixture
1.5× Liver Microsome s
Suspensio n Containing Test
Article or Positive Control 3×NADPH
working
solution Total Liver microsomal
protein
(mg/mL) Test Article or
Positive Control
(μM) NADPH
(mM)
30 15 45 0.5 1 2
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The samples are incubated at 37 oC and 450 µL IS solution is added to stop the
reaction at the corresponding time points (15, 30, 60, 90, and 120 min).
(9) Af
ter quenching, shake the plates at 600 rpm for 10 min and then centrifuge them
at 6,000 rpm for 15 min.
(10) The plates are sealed and stored at -20 oC in a freezer until bioanalys is.
(11) 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 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
A: 0.1%Formic acid in water
B: 0.1%Formic acid in acetonitrile
Flow rate ：600 μL/min
Column temperature ：40 oC
Autosampler temperature: 4oC
Compound Q1(m/z) Q3(m/z) Retention Time (min)
ALC-0159 1164.00 494.70 ~1.30
Tolbutamide (IS) 271.10 172.00 ~1.02
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5. TA 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 n atural 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
The in vitro intrinsic clearance, CL′ int, will be calculated from the t 1/2 as follows:
CL′int = (0.693/T 1/2) × (1/(microsomal protein concentration (0.5 mg/mL))) × Scaling Factor
The scaling factors are listed in Table 1.
Table 1. Scaling Factors for Intrinsic Clearance Prediction
in Mouse, Rat, Monkey, and Human Liver Microsomes
Species Microsomal Protein (mg)
per Gram of Liver Liver Weight (g) per
kg Body Weight Scaling Factor
(mg/kg)a Hepatic Blood
Flow (mL/m in/kg)
Mouse 45 87.5 3937.5 90
Rat 44.8 40 1792 55.2
Monkey 45 32.5 1462.5 44
Human 48.8 25.7 1254.2 20.7
aMicrosomal protein (mg/g liver) × liver weight (g)/kg body weight
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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