This study developed an assay to quantify the soluble epoxide hydrolase inhibitor t-TUCB in plasma and brain samples from immature rats. A pharmacokinetic study determined an optimal dose of 1 mg/kg intravenous t-TUCB, which achieved brain concentrations over 2 times the IC50 at 2 and 6 hours. While t-TUCB treatment did not significantly alter cerebral blood flow or epoxyeicosatrienoic acid levels after asphyxial cardiac arrest compared to vehicle, higher doses of t-TUCB may be needed to effectively inhibit sEH and improve outcomes following pediatric cardiac arrest.

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CBF(%ofBaseline)
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Cortical CBF (% baseline) vs Time
ACA Drug ACA Vehicle Sham Drug
THE EFFECT OF A SOLUBLE EPOXIDE HYDROLASE INHIBITOR, T-TUCB, ON
EET FORMATION IN THE IMMATURE RAT BRAIN
Vince Netto1,3, Lingjue Li1,3, Patrick M. Kochanek2,3, Robert S.B. Clark2,3, Henry Alexander2,3, Mioara Manole2,3, Samuel Poloyac1,3
1Center for Clinical Pharmaceutical Science: School of Pharmacy, 2Safar Center for Resuscitation Research, 3University of Pittsburgh: Pittsburgh, PA USA
• An assay has been developed for the quantification of t-TUCB in plasma samples from
pediatric rats.
• t-TUCB was detected in the brain at concentrations 2 times higher than IC 50 at 2 and 6 hrs.
• There were no statistically significant differences in CBF between vehicle and
t-TUCB groups 2 hours after resuscitation from ACA.
• Cerebral EET concentration data did not demonstrate a significant difference between
vehicle and t-TUCB treated rats.
Future Directions:
• Reformulate t-TUCB at a higher dose and test the effect on cerebral EET concentrations.
• Test whether CBF is altered at higher doses of t-TUCB.
• Test neurological outcomes of groups given sufficient doses of t-TUCB to increase
EET levels.
Support: American Heart Association Grand River Affiliates: Pittsburgh Research and Investigational Summer
Experience (VAN); NIH R01 HD075760-01A1 (MDM); S10RR023461 (SMP)
• Develop an assay for quantification of t-TUCB in plasma samples from immature rat
• Determine an optimal dose of t-TUCB in immature rats
• Determine the effect of t-TUCB on cerebral EET levels using LC/MS/MS
• Pediatric asphyxial cardiac arrest (ACA) is a significant cause of morbidity and
mortality. There are currently no pharmacological therapies approved for
prevention of neurological damage following ACA.
• Cerebral blood flow (CBF) after resuscitation from severe ACA is characterized
by cortical hypoperfusion, which may lead to secondary neuronal injury.
Normalization of CBF after ACA could improve neurological outcome after ACA.
• Epoxyeicosatrienoic acids (EET) are potent vasodilatory metabolites of arachidonic
acid. After resuscitation from ACA, cerebral concentration of EET is reduced.
• EETs are degraded into inactive Dihydroxyeicosatrienoic acids (DiHETEs) by
soluble epoxide hydrolases (sEH).
• We hypothesize that pharmacological inhibition of sEH with the agent t-TUCB will
increase cerebral EET levels, normalize cortical CBF, and improve neurological
outcome after ACA.
Develop an assay for quantification of t-TUCB in plasma samples from pediatric rats.
Conduct a pharmacokinetic study (n=6) to test IV formulation of t-TUCB and determine optimal dose.
Measure cortical perfusion at baseline and serially to 120 min post resuscitation. Three groups of rats
(n=6/group): sham treated with t-TUCB, ACA treated with t-TUCB, and ACA treated with vehicle.
Quantification of brain and plasma concentrations of EETs and t-TUCB at 120 min post-resuscitation
(n=6/group): sham treated with t-TUCB, ACA treated with t-TUCB, and ACA treated with vehicle.
Figure 5. Laser Speckle images at
5, 60, and 120 minutes post ROSC
of experimental groups.
Figure 4. Brain concentration data
demonstrated levels of t-TUCB above the
IC50 (16 nM in rats).
Figure 3. A pharmacokinetic study with
(n=6) was conducted with a 1 mg/kg
dosage of t-TUCB. An exponential decay
curve was fitted to the data and it
predicted a half life of 13 hours, which
matches values reported in other models.
Figure 2. An assay for determining plasma concentrations of t-TUCB was validated over three
days, with both intra and inter day coefficients of variation below 10 %.
Materials: Male Sprague Dawley postnatal day (PND) 17 rats were used. Chemicals
for LC/MS analysis were purchased from Cayman Chemical. t-TUCB and t-AUCB
were synthesized and obtained from Dr. Bruce Hammock, UC Davis. EET levels were
quantified using a Fisher Scientific triple quadrapole MS and Waters Acuity LC.
Plasma Sample Preparation: Plasma samples (50 uL) were collected in heparinized
capillary tubes and vortexed with 5 uL of 1% Disodium EDTA for 30 s. After
centrifugation (5 min) an aliquot (10 uL) of the supernatant was then diluted 20 fold
with de-ionized water. T-AUCB (10 uL of 0.2 ug/mL) was added as internal standard,
and a double extraction was performed with ethyl acetate (1 mL). The supernatant was
dried down, reconstituted in 50:50 acetonitrile:water, and run on the LC/MS using
50:50 acetonitrile:water isocratic gradient on positive electrospray.
Brain Sample Preparation: Brains were dissected on ice. The cerebral hemispheres
were collected rapidly into liquid nitrogen and were homogenized in microsomal
incubation buffer with BHT. The homogenates were centrifuged at 10,000 RPM for 30
minutes and 250 uL of supernatant was used for analysis. Analysis was done as stated
above, with t-AUCB as internal standard.
Asphyxial Cardiac Arrest Model:
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150
200
250
300
350
400
450
0 1 2 3 4 5 6 7
t-TUCBconcentration
(ng/mL)
Hours After IV Injection
Mean Plasma t-TUCB [C]
(ng/mL) vs Time (hours)
Isoflurane Fentanyl 50 mcg/kg/h
Vecuronium 5 mg/kg/h
PREPARATION
Intubation
Catheters
ASPHYXIA
12 min
Sham
BASELINE
CBF
Laser Speckle
Flowmetry
RESUSCITATION
Chest compressions
Epinephrine
Sodium bicarbonate
T-TUCB or Vehicle
POST RESUSCITATION
Serial CBF
5, 10, 15, 30, 45 min
1, 1.5, 2, hr
Figure 6. Cortical CBF at baseline and serially after
resuscitation from 12 min ACA or sham surgery and
treatment with t-TUCB.
Sham Vehicle t-TUCB
5 min
60 min
120 min
Figure 7. EET to DiHETE ratios in the brain tissue of sham, t-TUCB, and vehicle groups.
Figure 1. A calibration curve of
t-TUCB extracted from double stripped
serum generated during assay validation.
The X-axis represents the concentration
of t-TUCB standard solution in ng/mL.
The Y-axis represents the relative area
ratio of t-TUCB to t-AUCB—a close
chemical analog used as an internal
standard.
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8,9-EET:DiHETE Ratio 11,12-EET:DiHETE Ratio 14,15-EET:DiHETE Ratio
EET:DiHETERatio
Ratio of EETs to Degradative Products Among Experimental
Groups
Sham t-TUCB Vehicle
Hours After Dose 6
Mean Brain [C] t-TUCB (nM) 30.42 +/- 8.8
Times Above IC 50 1.9
Quality Control (QC) Inter-day Coefficient of Variation of QC Intra-day Coefficient of Variation of QC
Low (15 ng/mL) 2.767 % 2.574 %
Medium (75 ng/mL) 7.988 % 4.573 %
High (350 ng/mL) 6.093 % 6.284 %