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Medicilon Preclinical Research (Shanghai) LLC
Test Article: ALC-0315
Study No.: 01049-20010
1
In Vitro Metabolic Stability of ALC -0315 in CD-1/ICR Mouse,
Sprague Dawley Rat, Wistar Han Rat, Cynomolgus Monkey , and
Human Hepatocytes
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 01049-20010
Experimental Start Date 2020-07-20
Experimental Completion Date 2020-07-22
Number of Pages in Report 32
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Test Article: ALC-0315
Study No.: 01049-20010
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SUMMARY
This study evaluated the in vitro metabolic stability of ALC-0315 in hepatocytes of CD-1/ICR
mouse, Sprague Dawley rat, Wistar Han rat, cynomolgus monkey, and human. ALC-0315 was
stable after an approximately 4-hour incubation with hepatocytes from all these species.
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Test Article: ALC-0315
Study No.: 01049-20010
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1. OBJECTIVE
To evaluate the in vitro metabolic stability of ALC-0315 in hepatocytes from different species.
2. MATERIALS
2.1 Test Article
Name: ALC-0315
Molecular Formula: C 48H95NO5
MW (g/mol): 766.27
Exact Mass: 765.72
2.2 Positive Control
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 Hepatocytes
The following cryopreserved hepatocytes of CD-1/ICR mouse, Sprague Dawley rat, Wistar Han
rat, cynomolgus monkey, and human were purchased from qualified suppliers and stored in
liquid nitrogen until use.
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Test Article: ALC-0315
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Species Manufacturer Cat. No. Lot No. Assured Minimum Yield
（cells per vial ）
CD-1/ICR mouse (male) XenoTech MPCH1000 1810242 2.0×106
Sprague Dawley rat (male) XenoTech RPCH1000 1810189 5.0×106
Wistar Han rat BioIVT M00065 YMV 5.0×106
Cynomolgus monkey (male) RILD Shanghai HP-SXH-02M CJJC 5.0×106
Human (mixed gender) XenoTech HPCH10 1810156 5.0×106
3. EXPERIMENTAL PROCEDURES
3.1 Stock solution :
2.06 mg of ALC-0315 was weighed and dissolved in 268.83 μL of DMSO to obtain a 10 mM
stock solution. 3. 31 mg of testosterone was weighed and dissolved in 1147.60 μL of DMSO to
obtain a 10 mM stock solution. 2.81 mg of 7-hydroxycoumarin was weighed and dissolved in
882.70 μL of DMSO to obtain a 10 mM stock solution.
3.2 4 mM spiking solution:
Spiking Solution of Test Ar ticle or Positive Control
Compound Conc. of Stock
Solution (mM) Volume of Stock
Solution (μL) Volume of DMSO
(μL) Final Concentration
(mM)
ALC-0315 10 20 30 4
Testosterone &
7-Hydroxycoumarin 10 20 10 4
3.3 2 µM dosing solution (2×):
Dosing Solution (2×) of Test Article or Positive Control
Conc. of
Spiking Solution
(mM) Volume of
Spiking Solution
(μL) Volume of
William's E Medium
(μL) Final Concentration
(μM)
4 2 3998 2
3.4 Preparation of hepatocyte suspension:
Cryopreserved hepatocytes were thawed in a 37°C water bath, transferred to hepatocyte
thawing medium (William's E Medium with 30% percoll and 5% FBS), and then centrifuged
at 100×g for 10 min at room temperature. The cell pellet was resuspended with William's E
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Medium, cell viability was determined by trypan blue exclusion analysis, and the density of
viable cells was calculate d. The hepatocytes were diluted with incubation medium to an
appropriate density (2×106 viable cells/mL) and then pre-warm ed at 37 °C for 10 min.
3.5 40 µL of each hepatocyte suspension was added to 96-well plates in duplicate for each time
point (0, 30, 60, 90, 120, 180, and 240 min).
3.6 For 0 min samples: 4 80 µL of internal standard solution (IS solution, 10 ng/mL verapamil in
ethanol) was added, followed by 40 µL of pre-warmed 2× dosing solution. The final
concentration of test article or positive control in the incubation mixture was 1 M.
3.7 For the 30, 60, 90, 120, 180, and 240 min samples, 40 µL of pre-warmed 2× dosing solution
was added to initiate the reaction. The final concentration of test article or positive control in
the incubation mixture was 1 M.
3.8 Samples were incubated at 37 °C. At 30, 60, 90, 120, 180, and 240 min time points, the
reaction was stopped by adding 480 µL ethanol containing internal standard to all of the
duplicate wells.
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 The plates were sealed and stored at -20 °C until bioanalysis.
3.11 Plates were thawed at room temperature, centrifuged at 6,000 rpm for 15 min, and 200 μL
of the supernatants were transferred from each well into a 96-well sample plate for LC-MS/MS.
4. BIOANALYSIS
4.1 Instruments
Waters Acuity UPLC system
Sciex Triple Quad 6500+ with ESI ion source
4.2 LC/MS/MS Conditions
Column: ACQUITY UPLC BEH HILIC 1.7 μm (2.1*100 mm)
Gradient for ALC-0315:
Time (min) Solvent A (%) Solvent B (%)
0.01 5 95
1.50 40 60
2.00 40 60
2.01 5 95
2.50 5 95
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Solvent A: 10mM ammonium formate, 0.1% formic acid in water
Solvent B: 10mM ammonium formate, 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 Time
ALC-0315 766.90 510.60 100 66 ~1.21
Verapamil (IS) 455.30 165.20 49 28 ~1.32
4.3 Detection of ALC-0315
Representative chromatograms of ALC-0315 in each matrix are shown in Appendix 1 .
5. DATA ANALYSIS
The % remaining parent compound (ALC-0315 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 was calculated, when possible, as indicated below.
Elimination rate constant (k) = - slope
Half-life (T1/2) (minutes) = 0.693/k
Intrinsic clearance, predicted from the in vitro hepatocyte stability study, was calculated as
shown below:
CL’int (mL/min/kg) = k × V (1 mL incubation/106 cells) × Scaling Factor (106 cells/kg),
Scaling Factor (106 cells/kg) = Hepatocellularity (106 cells/g liver) × Normalized Liver Weight
(g liver/kg body weight)
The scaling factors are listed in Table 1.
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Table 1. Scaling Factors for Intrinsic Clearance Prediction in Mouse, Rat, Monkey, and
Human Hepatocytes
Species Hepatocellularity
(106 cells/g liver) Liver Weight
(g/kg BW) Scaling Factor
(106 cells/kg)
Mouse 135 87.5 11812.5
Rat 117 40 4680
Monkey 120 32 3840
Human 99 25.7 2544.3
6. RESULTS
A summary of the % remaining parent compound, CL’ int and half-life of ALC-0315 obtained
from a 4-hour incubation with hepatocytes from CD-1/ICR mouse, Sprague Dawley rat, Wistar
Han rat, cynomolgus monkey, and human is presented in Table 2. The stability of ALC-0315
over time in each matrix is shown in Figure 1. Raw data is presented in Appendix 2 .
The hepatocytes used in this study were tested for activity using metabolism control substrates
under incubation conditions identical to those used for ALC-0315. The enzymes were found to
exhibit satisfactory activity as determined by significant consumption of the positive control
compounds (testosterone and 7-hydroxycoumarin) during the 4-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 testosterone and 7-hydroxycoumarin is provided in
Table 2. 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-0315 in hepatocytes of CD-1/ICR
mouse, Sprague Dawley rat, Wistar Han rat, cynomolgus monkey, and human. ALC-0315 was
stable after an approximately 4-hour incubation with hepatocytes from all these species.
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Test Article: ALC-0315
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Table 2. Summary of Hepatocyte Stability of ALC-0315 , Testosterone and 7-Hydroxycoumarin
Test Article Species Percent Remaining (%) T1/2
(minute) CL’int
(mL/min/kg) 0 min 30 min 60 min 90 min 120 min 180 min 240 min
ALC-0315 CD-1/ICR
mouse Mean 100.00 101.15 100.77 101.92 98.85 101.15 99.62 >240 <34.1 RSD of Area Ratio 1.63 1.07 0.54 0.00 2.19 0.00 1.09
Sprague
Dawley rat Mean 100.00 97.75 98.50 99.25 97.38 98.88 101.12 >240 <13.5 RSD of Area Ratio 1.59 3.79 3.76 1.60 2.18 4.29 2.10
Wistar Han
rat Mean 100.00 102.70 102.32 103.09 99.61 103.47 100.00 >240 <13.5 RSD of Area Ratio 0.55 1.06 1.60 0.53 1.10 3.17 7.10
Cynomolgus
monkey Mean 100.00 96.36 97.82 100.00 96.36 95.64 93.82 >240 <11.3 RSD of Area Ratio 1.54 1.60 2.63 3.60 3.74 1.61 4.39
Human Mean 100.00 100.72 101.44 100.36 100.72 98.92 99.64 >240 <7.35 RSD of Area Ratio 2.03 1.01 3.01 2.53 0.00 0.51 0.51
Testosterone CD-1/ICR
mouse Mean 100.00 16.60 BQL BQL BQL BQL BQL 11.6 707 RSD of Area Ratio 5.81 11.78 N/A N/A N/A N/A N/A
Sprague
Dawley rat Mean 100.00 7.23 BQL BQL BQL BQL BQL 7.92 410 RSD of Area Ratio 3.17 N/A N/A N/A N/A N/A N/A
Wistar Han
rat Mean 100.00 BQL BQL BQL BQL BQL BQL N/A N/A RSD of Area Ratio 8.03 N/A N/A N/A N/A N/A N/A
Cynomolgus
monkey Mean 100.00 10.07 BQL BQL BQL BQL BQL 9.06 298 RSD of Area Ratio 2.81 41.26 N/A N/A N/A N/A N/A
Human Mean 100.00 15.92 BQL BQL BQL BQL BQL 11.3 156 RSD of Area Ratio 4.34 7.16 N/A N/A N/A N/A N/A
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7-
Hydroxycou
marin CD-1/ICR
mouse Mean 100 35.05 3.2 BQL BQL BQL BQL 12.1 677 RSD of Area Ratio 1.22 15.06 8.46 N/A N/A N/A N/A
Sprague
Dawley rat Mean 100 20.97 BQL BQL BQL BQL BQL 13.3 244 RSD of Area Ratio 2.99 10.49 N/A N/A N/A N/A N/A
Wistar Han
rat Mean 100 19.11 BQL BQL BQL BQL BQL 12.6 258 RSD of Area Ratio 1.97 16.89 N/A N/A N/A N/A N/A
Cynomolgus
monkey Mean 100 17.03 BQL BQL BQL BQL BQL 11.7 230 RSD of Area Ratio 0.85 2.27 N/A N/A N/A N/A N/A
Human Mean 100 40.7 18.53 3.36 BQL BQL BQL 24.7 71.5 RSD of Area Ratio 1.52 1.67 8.47 0.73 N/A N/A N/A
* 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.
BQL = Below quantification limit; N/A = not applicable
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Figure 1. Stability of ALC-0315 in Mouse, Rat, Monkey and Human Hepatocytes
CD-1/ICR Mouse Sprague Dawley Rat Wistar Han Rat
Cynomolgus Monkey Human
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Figure 2 . Stability of Testosterone in Mouse, Rat, Monkey and Human Hepatocytes
CD-1/ICR Mouse Sprague Dawley Rat Wistar Han Rat
Cynomolgus Monkey Human
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Figure 3 . Stability of 7 -Hydroxycoumarin in Mouse, Rat, Monkey and Human Hepatocytes
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-0315 in Mouse, Rat, Monkey and Human Hepatocytes
Appendix 2 – Stability of ALC-0315 in Mouse, Rat, Monkey and Human Hepatocytes – Raw Data
Appendix 3 – Stability of Testosterone in Mouse, Rat, Monkey and Human Hepatocytes – Raw Data
Appendix 4 – Stability of 7-Hydroxycoumarin in Mouse, Rat, Monkey and Human Hepatocytes – Raw Data
Appendix 5 – 01049-20010-ALC-0 315
-Hepatocytes Stability_Protocol
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APPENDIX 1
Representative Chromatograms of ALC-0315 in Mouse, Rat, Monkey and Human Hepatocytes
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CD 1/ICR mouse
Sprague Dawley rat
Wistar Han rat
Cynomolgus monkey
Human
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APPENDIX 2
Stability of ALC-0315 in Mouse, Rat, Monkey and Human Hepatocyte – Raw Data
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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-0315 CD-1/ICR
mouse 240 6.00E+05 5.90E+05 4.65E+06 4.50E+06 0.129 0.131
180 5.99E+05 5.96E+05 4.54E+06 4.53E+06 0.132 0.132
120 5.86E+05 5.84E+05 4.60E+06 4.45E+06 0.127 0.131
90 5.88E+05 5.83E+05 4.43E+06 4.38E+06 0.133 0.133
60 5.83E+05 5.92E+05 4.47E+06 4.47E+06 0.131 0.132
30 5.94E+05 5.89E+05 4.53E+06 4.43E+06 0.131 0.133
0 5.92E+05 5.65E+05 4.48E+06 4.39E+06 0.132 0.129
ALC-0315 Sprague
Dawley rat 240 6.40E+05 5.94E+05 4.69E+06 4.47E+06 0.137 0.133
180 6.18E+05 5.83E+05 4.53E+06 4.56E+06 0.136 0.128
120 5.97E+05 5.77E+05 4.53E+06 4.51E+06 0.132 0.128
90 6.08E+05 5.81E+05 4.53E+06 4.45E+06 0.134 0.131
60 6.08E+05 5.75E+05 4.51E+06 4.48E+06 0.135 0.128
30 6.06E+05 5.67E+05 4.53E+06 4.47E+06 0.134 0.127
0 6.09E+05 5.82E+05 4.50E+06 4.41E+06 0.135 0.132
ALC-0315 Wistar Han
rat 240 5.55E+05 6.12E+05 4.50E+06 4.51E+06 0.123 0.136
180 5.91E+05 6.06E+05 4.52E+06 4.44E+06 0.131 0.137
120 5.68E+05 5.83E+05 4.45E+06 4.49E+06 0.128 0.13
90 5.91E+05 5.94E+05 4.40E+06 4.48E+06 0.134 0.133
60 5.82E+05 5.99E+05 4.46E+06 4.48E+06 0.131 0.134
30 6.04E+05 5.94E+05 4.51E+06 4.49E+06 0.134 0.132
0 5.87E+05 5.88E+05 4.55E+06 4.51E+06 0.129 0.13
ALC-0315 Cynomolgus
monkey 240 6.17E+05 5.78E+05 4.65E+06 4.64E+06 0.133 0.125
180 6.09E+05 5.91E+05 4.59E+06 4.54E+06 0.133 0.13
120 6.28E+05 5.85E+05 4.61E+06 4.55E+06 0.136 0.129
90 6.42E+05 6.07E+05 4.55E+06 4.55E+06 0.141 0.134
60 6.38E+05 5.95E+05 4.66E+06 4.50E+06 0.137 0.132
30 6.03E+05 6.02E+05 4.61E+06 4.49E+06 0.131 0.134
0 6.32E+05 6.17E+05 4.54E+06 4.55E+06 0.139 0.136
ALC-0315 Human 240 6.30E+05 6.38E+05 4.52E+06 4.64E+06 0.139 0.138
180 6.44E+05 6.19E+05 4.66E+06 4.52E+06 0.138 0.137
120 6.49E+05 9.24E+05 4.65E+06 4.54E+06 0.14 0.204
90 6.51E+05 6.20E+05 4.60E+06 4.52E+06 0.142 0.137
60 6.53E+05 6.27E+05 4.54E+06 4.54E+06 0.144 0.138
30 6.42E+05 6.20E+05 4.54E+06 4.46E+06 0.141 0.139
0 6.42E+05 6.22E+05 4.56E+06 4.55E+06 0.141 0.137
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APPENDIX 3
Stability of Testosterone in Mouse, Rat, Monkey and Human Hepatocyte – Raw Data
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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
Testosterone CD-1/ICR
mouse 240 LOD LOD 7.53E+05 7.48E+05 LOD LOD
180 LOD LOD 7.77E+05 7.83E+05 LOD LOD
120 LOD LOD 7.44E+05 7.99E+05 LOD LOD
90 LOD LOD 7.60E+05 7.89E+05 LOD LOD
60 LOD LOD 7.39E+05 7.46E+05 LOD LOD
30 5.29E+03 6.16E+03 7.70E+05 7.58E+05 0.007 0.008
0 3.64E+04 3.41E+04 7.73E+05 7.88E+05 0.047 0.043
Testosterone Sprague
Dawley rat 240 LOD LOD 8.19E+05 8.01E+05 LOD LOD
180 LOD LOD 7.97E+05 7.54E+05 LOD LOD
120 LOD LOD 7.48E+05 8.25E+05 LOD LOD
90 LOD LOD 8.12E+05 7.45E+05 LOD LOD
60 LOD LOD 7.59E+05 7.44E+05 LOD LOD
30 LOD 2.38E+03 8.25E+05 8.19E+05 LOD 0.003
0 3.38E+04 3.38E+04 8.23E+05 8.59E+05 0.041 0.039
Testosterone Wistar Han
rat 240 LOD LOD 7.72E+05 8.57E+05 LOD LOD
180 LOD LOD 7.61E+05 7.44E+05 LOD LOD
120 LOD LOD 7.87E+05 7.53E+05 LOD LOD
90 LOD LOD 7.87E+05 7.71E+05 LOD LOD
60 LOD LOD 7.29E+05 7.93E+05 LOD LOD
30 LOD LOD 7.78E+05 7.87E+05 LOD LOD
0 3.34E+04 3.39E+04 8.20E+05 7.44E+05 0.041 0.046
Testosterone Cynomolgus
monkey 240 LOD LOD 8.17E+05 8.22E+05 LOD LOD
180 LOD LOD 8.26E+05 8.16E+05 LOD LOD
120 LOD LOD 8.22E+05 8.12E+05 LOD LOD
90 LOD LOD 8.44E+05 7.91E+05 LOD LOD
60 LOD LOD 8.47E+05 7.85E+05 LOD LOD
30 4.32E+03 2.37E+03 8.24E+05 8.22E+05 0.005 0.003
0 3.45E+04 3.26E+04 8.72E+05 7.93E+05 0.04 0.041
Testosterone Human 240 LOD LOD 8.02E+05 8.22E+05 LOD LOD
180 LOD LOD 8.65E+05 8.75E+05 LOD LOD
120 LOD LOD 8.29E+05 8.22E+05 LOD LOD
90 LOD LOD 8.60E+05 8.16E+05 LOD LOD
60 LOD LOD 8.21E+05 8.47E+05 LOD LOD
30 6.13E+03 5.10E+03 8.78E+05 8.09E+05 0.007 0.006
0 3.25E+04 3.56E+04 8.02E+05 8.26E+05 0.04 0.043
LOD = limit of detection
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APPENDIX 4
Stability of 7-Hydroxycoumarin in Mouse, Rat, Monkey and Human Hepatocyte – Raw Data
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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
7-
Hydroxycoumarin CD-1/ICR
mouse 240 LOD LOD 6.12E+05 6.29E+05 LOD LOD
180 LOD LOD 6.12E+05 6.09E+05 LOD LOD
120 LOD LOD 6.11E+05 5.99E+05 LOD LOD
90 LOD LOD 6.29E+05 6.06E+05 LOD LOD
60 1.33E+03 1.21E+03 6.10E+05 6.25E+05 0.002 0.002
30 1.25E+04 1.57E+04 6.23E+05 6.31E+05 0.02 0.025
0 3.97E+04 4.12E+04 6.25E+05 6.37E+05 0.064 0.065
7-
Hydroxycoumarin Sprague
Dawley rat 240 LOD LOD 6.30E+05 6.18E+05 LOD LOD
180 LOD LOD 6.29E+05 6.25E+05 LOD LOD
120 LOD LOD 6.36E+05 6.49E+05 LOD LOD
90 LOD LOD 6.11E+05 6.30E+05 LOD LOD
60 LOD LOD 6.19E+05 6.07E+05 LOD LOD
30 8.21E+03 9.55E+03 6.30E+05 6.32E+05 0.013 0.015
0 3.98E+04 4.10E+04 6.06E+05 5.99E+05 0.066 0.068
7-
Hydroxycoumarin Wistar Han
rat 240 LOD LOD 6.23E+05 6.17E+05 LOD LOD
180 LOD LOD 6.51E+05 6.11E+05 LOD LOD
120 LOD LOD 6.05E+05 6.24E+05 LOD LOD
90 LOD LOD 6.10E+05 6.15E+05 LOD LOD
60 LOD LOD 6.36E+05 6.05E+05 LOD LOD
30 6.78E+03 8.59E+03 6.20E+05 6.18E+05 0.011 0.014
0 4.01E+04 3.94E+04 6.09E+05 6.14E+05 0.066 0.064
7-
Hydroxycoumarin Cynomolgus
monkey 240 LOD LOD 5.82E+05 6.25E+05 LOD LOD
180 LOD LOD 6.01E+05 6.18E+05 LOD LOD
120 LOD LOD 6.38E+05 6.14E+05 LOD LOD
90 LOD LOD 6.38E+05 6.07E+05 LOD LOD
60 LOD LOD 6.28E+05 6.20E+05 LOD LOD
30 7.22E+03 6.96E+03 6.42E+05 6.39E+05 0.011 0.011
0 4.21E+04 4.15E+04 6.44E+05 6.43E+05 0.065 0.065
7-
Hydroxycoumarin Human 240 LOD LOD 6.04E+05 6.05E+05 LOD LOD
180 LOD LOD 6.45E+05 6.24E+05 LOD LOD
120 LOD LOD 6.28E+05 6.50E+05 LOD LOD
90 1.43E+03 1.40E+03 6.42E+05 6.21E+05 0.002 0.002
60 7.22E+03 8.24E+03 6.20E+05 6.28E+05 0.012 0.013
30 1.69E+04 1.68E+04 6.27E+05 6.10E+05 0.027 0.028
0 4.06E+04 3.99E+04 6.01E+05 6.03E+05 0.068 0.066
LOD = limit of detection
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Test Article: ALC-0315
Study No.: 01049-20010
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APPENDIX 5
01049-20010 -ALC-0315-Hepatocytes Stability_Protocol
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In Vitro Metabolic Stability of ALC- 0315 in CD- 1/ICR Mouse,
Sprague Dawley Rat, Wistar Han Rat , Cynomolgus Monkey, and
Human Hepatocytes
Testing Facility
Medicilon Preclinical Research (Shanghai) LLC
585 Chuanda Road
Pudong, Shanghai 201299
China
Study Number
01049-200 10
Study Director
Sponsor
Acuitas Therapeutics Inc.
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Medicilon Study Number: 01049-20010
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. Hepatocytes ............................................................................................................4
3.EXPERIMENTAL PROCEDURES .............................................................................4
4.BIOANALYSIS ............................................................................................................6
4.1. Instruments .............................................................................................................6
4.2. LC/MS/MS Conditions ...........................................................................................6
5.DATA ANALYSIS .......................................................................................................6
6.FINAL REPORT ...........................................................................................................7
7.SIGNATURES ..............................................................................................................8
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1. INTRODUCTION
1.1. Study Number
01049-20010
1.2. Study Title
In Vitro Metabolic Stability of ALC-0315 in CD-1/ICR Mouse, Sprague Dawley Rat,
Wistar Han R at, Cynomolgus Monkey, and Human Hepatocytes
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-0315 in Hepatocytes from
different spe
cies 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-0315
Molecular Formula: C 48H95NO5
MW (g/mol): 766.27
Exact Mass: 765.72
2.2. Positive Control and Internal Standard
Testosterone and 7-hydroxycoumarin will be used as positive controls. Verapamil will
be used as internal standard. The sources will be documented in the experiment al
records and presented in the report.
2.3. Hepatocytes
Cryopreserved hepatocytes of CD-1/ICR mouse, Sprague Dawley rat, Wistar Han rat,
cynomolgus monkey, and human were purchased from qualified suppliers and stored
in liquid nitrogen until use. The source(s) and lot numbers will be documented in the
experiment al records and presented in the final report.
3. EXPERIMENTAL PROCEDURES
(1) Preparation of stock solution : Appropriate amount of test article or positive
control is weighed and dissolved in d imethyl sulfoxide (DMSO) to obtain a
10 mM stock solution.
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(2) Preparation of 4 mM spiking solution:
Spiking Solution of Test Article or Positive Control
Conc. of Stock Solution
(mM) Volume of Stock Solution
(μL) Volume of DMSO
(μL) Final Concentration
(mM)
10 20 30 4
(3) Preparation of 2 µM dosing solution(2×) of test article or positive control:
Dosing Solution (2×) of Test Article or Positive Control
Conc. of
Spiking Solution
(mM) Volume of
Spiking Solution
(μL) Volume of
William's E Medium
(μL) Final Concentration
(μM)
4 2 3998 2
(4) Preparation of hepatocyte suspension : Thaw cryopreserved hepatocytes in a 37°C
water bath. Transfer the hepatocytes to hepatocyte thawing medium (William's E
Medium with 30% percoll and 5% FBS) and centrifuge at 100×g for 10 min at
room temperature. Resuspend the cell pellet with William's E Medium and
determine
cell viability by trypan blue exclusion analysis and calculate the viable
cell density. Dilute the hepatocytes with incubation medium to an appropriate
density (2×106 viable cells/mL) and pre-warm at 37 °C for 10 min.
(5) 40 µL of each hepatocyte suspension is added to 96-well plates in duplicate for
each time point (0, 30, 60, 90, 120, 180, and 240 min).
(6) For 0 min samples: 4 80 µL of internal standard solution (IS solution, 10 ng/mL
verapamil in ethanol ) is added, followed by 40 µL of pre-warmed 2× dosing
solution. The final concentration of test article or positive control in the
incubation mixture is 1 M.
(7) For the 30, 60, 90, 120 , 180, and 240 min samples, 40 µL of pre-warmed 2×
dosing solution is added to initiate reaction. The final concentration of test article
or positive control in the incubation mixture is 1 M.
(8) The samples are incubated at 37 °C. At 30, 60, 90, 120, 180, and 240 min time
points, stop the reaction by adding 480 µL ethanol containing internal standard to
all of the duplicate wells.
(9) After que
nching, 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 °C freezer until bioanalysis.
(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.
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4. BIOANALYSIS
4.1. Instruments
Waters Acquity UPLC system
Sciex Triple Quad 6500+ with ESI ion source
4.2. LC/MS/MS Conditions
Column：ACQUITY UPLC BEH HILIC 1.7 μm (2.1*100mm)
Gradient for ALC-0315 :
Time (min) Solvent A (%) Solvent B (%)
0.01 5 95
1.50 40 60
2.00 40 60
2.01 5 95
2.50 5 95
A: 10mM ammonium formate, 0.1% Formic acid in water
B: 10mM ammonium formate, 0.1% Formic acid in acetonitrile
Flow rate ：500 μL/min
Column temperature ：40 oC
Autosampler temperature: 4oC
Compound Q1(m/z) Q3(m/z) Retention Time (min)
ALC-0315 766.90 510.60 ~1.07
Verapamil (IS) 455.30 165.20 ~1.19
5. DATA ANALYSIS
The % remaining (parent compound) will be calculated by dividing the peak area ratio
(compound peak area/ internal standard peak area) by the 0 min peak area ratio. The
natural logarithm of % remaining is plotted against time and the slope of the fitted line
will be determined as follows:
Elimination rate constant (k) = - slope
Half-life (T 1/2) (minutes) = 0.693/k
Intrinsic clearance predicted from the in vitro hepatocyte stability study will be calculated
as shown below:
CL’int (mL/min/kg) = k * V (1 mL incubation/106 cells) * Scaling Factor (106 cells/kg),
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Scaling Factor (106 cells/kg) = Hepatocellularity (106 cells/g liver) * Normalized Liver
Weight (g liver/kg body weight)
The scaling factors are listed in Table 1.
Table 1. Scaling Factors for Intrinsic Clearance Prediction
in Mouse,
Rat, Monkey, and Human Hepatocytes
Species Hepatocellularity Liver Weight Scaling Factor
(106 cells/g liver) (g/kg BW) (106 cells/kg)
Mouse 135 87.5 11812.5
Rat 117 40 4680
Monkey 120 32 3840
Human 99 25.7 2544.3
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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