Determining the Effects of

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

THC 11-OH-THC 11-COOH-THC 11-COOH-THC-gluc

Fatty Acid Binding Proteins (FABPs)
• Fatty acid binding proteins (FABPs)
• Member of the superfamily of intracellular lipid binding proteins (iLBPs)
• Regulates the homeostasis of endogenous compounds
• FABPs are found everywhere in the body
Gene Common Name Localization
FABP1 Liver FABP Liver, intestine, pancreas, kidney, lung, stomach
FABP2 Intestinal FABP Intestine, liver
FABP3 Heart FABP Cardiac and skeletal muscle, brain, kidney, lung, stomach, testis, adrenal gland,
mammary gland, placenta, ovary, brown adipose tissue
FABP4 Adipocyte FABP Adipocyte, macrophages, dendritic cells, skeletal muscle fibers
FABP5 Epidermal FABP Skin, tongue, adipocyte, macrophage, dendritic cells, mammary gland, brain,
stomach, intestine, kidney, liver, lung, heart, skeletal muscle, testis, retina,
lens, spleen, placenta
 FABP and Albumin Changed the
Metabolic Rate of THC in a CYP Specific Manner

• Formation of 11-OH-THC and

3A4M1 decreased in
Supersomes and HLMs in the
presence of HSA and FABP5
• Except 11-OH-THC in HLMs
with 0.1% HSA
• Decrease in metabolite
formation was greater for
CYP3A4 than CYP2C9
What is the role of FABP1
on THC Metabolism?
• THC and subsequent
metabolites bind with high
affinity to FABP1
• Knockout of FABP1 in mouse
hepatocytes was shown to
decrease the formation and
clearance of 11-OH-THC
• Metabolism of 11-COOH-THC
stayed relatively the same

Elmes et al., 2019

 What is the effect of binding proteins
on THC-COOH metabolism by UGTs?
⇌ ⇌
apo-binding protein holo-binding protein THC-COOH
Cytosol

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

• Reaction conditions: 5.00E-01

4.50E-01

• 0.1mg/mL HLM 4.00E-01

3.50E-01

• 5uM 11-COOH-THC

Average PAR
3.00E-01

2.50E-01
• 10µg/mL alamethicin 2.00E-01

• 10mM MgCl2 1.50E-01

1.00E-01

• 2mM UDPGA 5.00E-02

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

• 5uM 11-COOH-THC 7.00E-02

• 2.5µg/mL alamethicin 6.00E-02

• 10mM MgCl2 5.00E-02

• 2mM UDPGA 4.00E-02

PAR
• Crash 1:1 w/ ACN + IS + FA 3.00E-02

• 5min incubation 2.00E-02

• Non-linear formation of 3.1 11- 1.00E-02

COOH-THC-gluc metabolite with 0.00E+00

0 0.1 0.2 0.3 0.4 0.5 0.6

respect to protein concentration HLM (mg/mL)

➢Test lower HLM concentrations

Formation of the both glucuronide metabolites
are linear with respect to HLM concentration
• Tested 0.025, 0.05, 0.1, 0.2mg/mL
• Reaction conditions: Protein Linearity 2
• 5uM 11-COOH-THC 9.00E-02
• 10µg/mL alamethicin 8.00E-02

• 10mM MgCl2 7.00E-02

• 2mM UDPGA 6.00E-02

• Crash 1:3 w/ ACN + IS + FA 5.00E-02

PAR
4.00E-02
• 5min incubation 3.00E-02

• Re-running samples showed no 2.00E-02

change in metabolite PAR 1.00E-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

• 0.05mg/mL HLM 1.00E+01

• 5uM 11-COOH-THC 8.00E+00

• 2.5µg/mL alamethicin

PAR
6.00E+00

• 10mM MgCl2 4.00E+00

• 2mM UDPGA 2.00E+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)

metabolites with respect to time

Incubations in Donor HLMs
150

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

• Crash 1:3 w/ ACN + IS 4

• 10min incubation

2.8 min Peak (PAR)

3
• Formation of each metabolite
had low variability between 2

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?