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Determining The Effects of Binding Proteins On Cannabinoid Metabolism in Human Liver Microsomes
Determining The Effects of Binding Proteins On Cannabinoid Metabolism in Human Liver Microsomes
Binding Proteins on
Cannabinoid Metabolism
in Human Liver Microsomes
Wendy Ni, King Yabut, Nina Isoherrenan
Isoherrenan Lab Meeting
03.20.2020
Outline
• Background
• THC and FABP
• Albumin & FABP’s effect on THC metabolism
• Knockout of FABP1 decreased metabolism of 11-OH-THC
• Experimental Results
• Time Linearity
• Protein Linearity
• Unidentified Mass Spec Product Hypothesis
• Literature Search
• Next Steps
Δ-9-tetrahydrocannabinol (THC)
• Δ-9-tetrahydrocannabinol (THC) is the primary psychoactive
compound from the highly abused Cannabis
• Highly lipophilic (logP = 6.97)
• THC is metabolized by both CYP450s and UGTs
gluc
H
CYP2C9 CYP2C9 UGT1A1
CYP2C19 CYP2C19 UGT1A3
ER Membrane
UGT1A1
UGT1A3
UGTs
⇌
THC-COOH THC-COOH-gluc
Formation of the 3.1min glucuronide
metabolite is linear with respect to time
• Tested 1, 5, 15, and 30min Time Linearity 1 y = 0.0135x + 0.0255
R² = 0.9892
4.50E-01
3.50E-01
• 5uM 11-COOH-THC
Average PAR
3.00E-01
2.50E-01
• 10µg/mL alamethicin 2.00E-01
1.00E-01
0.00E+00
• Crash 1:1 w/ ACN + IS + FA 0 5 10 15 20
Incubation Time (min)
25 30 35
Formation of the 3.1min glucuronide metabolite
is nonlinear with respect to HLM concentration
• Tested 0.01, 0.1, 0.5mg/mL
• Reaction conditions: Protein Linearity 1
y = 0.1036x + 0.0134
R² = 0.8405
PAR
• Crash 1:1 w/ ACN + IS + FA 3.00E-02
PAR
4.00E-02
• 5min incubation 3.00E-02
0.00E+00
• Both product are stable 0 0.05 0.1 0.15 0.2 0.25
HLM (mg/mL)
• No cofactor control showed negligible
formation of either product 3.1 PAR 2.8 PAR Linear (3.1 PAR) Linear (2.8 PAR)
LC-MS/MS Profiles
• Both chromatograms show
0.1mg/mL HLM with 5min
incubation
• new and significant 2.8min peak
• 3.1min peak splits
• Internal standard earlier than
3.1min at around 3.08min
• Overlaps with 3.1min peak
Both glucuronide metabolites are better
stabilized with the 1:3 crashing condition
• Repeat of Protein Linearity 2
• Linear formation for both
metabolites
• Additional condition: crashing
1:3 ACN + IS only (no FA)
• When crashing w/o FA:
• Increased formation of both
products
• Increased ratio of the 2.8 product
vs. 3.1 product
➢Continue protocol testing using
crashing conditions w/o FA
Time Linearity 2
• Tested 1, 5, 15, 30 min
• Reaction conditions: 1.20E+01
Time Linearity 2
• 2.5µg/mL alamethicin
PAR
6.00E+00
•
0.00E+00
Crash 1:3 w/ ACN + IS 0 5 10 15 20 25 30 35
Time (min)
• Linear formation of both 3.1 PAR 2.8 PAR Linear (3.1 PAR) Linear (2.8 PAR)
11-COOH-THC-gluc (nM)
• Reaction conditions:
• 0.05mg/mL HLM 100
• 5uM 11-COOH-THC
•
50
2.5µg/mL alamethicin
• 10mM MgCl2
0
• 2mM UDPGA HLM 143 HLM 152 HLM 157
HLMs 1
0
HLM 143 HLM 152 HLM 157
Incubations in Donor HLMs
• Product ratio of 2.8min to 3.1min metabolite was ~1
• Product ratio did not vary greatly between the different donor HLMs
1.5
(2.8 min/3.1 min)
Product Ratios
1.0
0.5
0.0
HLM 143 HLM 152 HLM 157
Unidentified Metabolite – Hypothesis 1
• Glucuronide conjugated to the
hydroxide of THC
• Ester THC-COOH
• Ester THC-COOH is more stable
but a less favorable product than
Acyl THC-COOH
➢Add more transitions to the
H
mass spec
➢Ester glucuronide would not have: gluc
➢ 521 > 299 transition
➢ 327 > 299 transition
Weinmann, 2000
Unidentified Metabolite – Hypothesis 2
• Acyl shift of glucuronic acid
changing its LC retention
time
• Hanisch, 2017
• Observed new peaks with
THC-COOH incubation time
course in aqueous buffer
• Used pure mixture of β-
isomer with minimal α-
isomer impurities
Hanisch, 2018
Unidentified Metabolite – Hypothesis 3
• Glucuronide product of 11-COOH-
THC stereoisomer
➢Quality control check current 11-COOH-
THC stock on LC-UV to confirm racemic
mixture
➢Test new stock of (-)11-COOH-THC in
incubations again to see if 2.8min peak
is present
• Both chromatograms show 0.1mg/mL
HLM with 5min incubation
• Internal standard earlier than 3.1min
at around 3.08min
• Overlaps with 3.1min peak
Next Steps
• Michaelis-Menten 3-point estimate
• Incubations with FABP1 and HSA to examine their effects on 11-
COOH-THC metabolism
Thank you!
Questions?