This document summarizes research investigating the effect of intravenous t-TUCB administration on cerebral blood flow (CBF) following asphyxial cardiac arrest (ACA) in immature rats. Key findings include: 1) An assay was validated to measure t-TUCB concentrations in plasma and brain tissue. 2) Pharmacokinetic studies found t-TUCB present in the brain at twice the inhibitory concentration for 6 hours after dosing. 3) However, post-ACA CBF was not significantly different between rats treated with t-TUCB and vehicle controls. Future studies are needed to determine if t-TUCB increases protective epoxyeicosatrienoic acid levels and improves neurological outcomes.

1. The Effect of Intravenous
t-TUCB on Post Asphyxial
Cardiac Arrest CBF
Vince Netto
School of Pharmacy
Center for Clinical Pharmaceutical Sciences

2. Asphyxial Cardiac Arrest
 Cardiac arrest (CA) brought on by obstructed breathing
(or asphyxiation)
 70% of those that survive suffer permanent
neurological damage
 No approved pharmacological treatment exists to
prevent neurological damage

3. ACA and Cerebral Blood Flow
SHAM CA-NS
CA-
Albumin
CA-
PNABase
line
5 min
30 min
60 min
90 min
150 min
Sham CA NS CA albumin CA PNA
ProbeTrial
Time(sec)
0
10
20
30
40
50
60
Morris Water Maze

4. sEH Inhibitors: t-TUCB
Role of EETs in CBF
COOH
Arachidonic Acid
PLA2
Epoxyeicosatrienoic Acids (EETs)
(Vasodilatory and neuroprotective)
Soluble Epoxide Hydrolase
(sEH)
Dihydroxyeicosatrienoic Acids (DiHETEs)
(Inactive)
CYP450

5. Hypothesis
 Intravenous administration of t-TUCB in PND 17 rats
resuscitated from ACA will raise EET levels in the brain
and consequently impart neurological protection by
increasing cerebral blood flow.

6. Objectives
 Develop and validate an assay to test plasma and brain
concentrations of t-TUCB
 Develop and test an IV formulation of t-TUCB
 Discover the optimal dose of t-TUCB via
Pharmacokinetic Study
 Determine the effect of t-TUCB on cortical CBF
following ACA

7. Methods
 T-TUCB and ARA metabolite quantification with Waters
Acuity liquid chromatographer and TSQ triple
quadrapole mass spectrometer
Isoflurane Fentanyl 50 mcg/kg/h
Vecuronium 5 mg/kg/h
PREPARATION
Intubation
Catheters
ANOXIA
12 min
Sham
BASELINE
CBF
RESUSCITATION
Chest compressions
Epinephrine
Sodium bicarbonate
Normal Saline
POST RESUSCITATION
Serial CBF
Laser Speckle Contrast
Analysis (Pericam PSI)
5, 10, 15, 30, 45 min
1, 1.5, 2, hr

8. Regional CBF After Asphyxial CA in
Immature 17 Day Old Rats*
*Manole et al, JCBFM 2009
5 min 120 min sham
Cortical8,9-EET
0
10
20
30
40
50
60
70
5 min 120 min sham
Cortical8,9-EET
0
10
20
30
40
50
60
70
9 min
12 min
EET After ACA
EET After ACA

9. Assay Validation
 Diluted 20 times in Serum/DI water matrix
 T-AUCB (sEH inhibitor) as internal standard
 Duplicate standard curve run each of 3 days
 6 QCs on days 1 and 2
 12 QCs on day 3

10. Pharmacokinetics
6 hours Post Dose Half Life
Mean Brain [C] t-TUCB 30.42 nM
13 hours
Times Above IC50 (16 nM) 1.9
• 1 mg/Kg
• 10% Hydroxypropyl Beta
Cyclodextrin
• Lyophilization
• 0.1667 mg/mL t-TUCB
y = -14.594x + 335.41
R² = 0.6224
10
100
1000
0 2 4 6 8
t-TUCBconcentration(ng/mL)
Hours After IV Injection
Mean Plasma t-TUCB [C]
(ng/mL) vs Time (hours)

11. Mean Blood:Brain t-TUCB at
2 Hours
 T-TUCB is about 15 to 20 times higher in Plasma
 Brain concentrations at 2 hours were well above IC 50
0
5
10
15
20
25
30
1
Blood:Brainratiot-TUCB
Experimental Groups
ACA Drug
Sham Drug
ACA Vehicle

12. Test of Treatment [C]
 We tested the IV formulations given to each group of
rats for t-TUCB concentration
 Concentration was about 75 % of target
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
1
Concentrationt-TUCB
(mg/mL)
Experimental Groups
ACA Drug
Sham Drug
ACA Vehicle

13. CBF Post Resuscitation
No significant Difference between rats treated with t-
TUCB and rats treated with Vehicle
0
0.2
0.4
0.6
0.8
1
1.2
1.4
CBF(%ofBaseline)
Time Point
Cortical CBF (% baseline) vs Time
ACA Drug
ACA Vehicle
Sham Drug
Vehicle T-TUCBSham
5
min
60
min
120
min

14. Conclusions
 A validated assay has been developed to measure t-TUCB
in rat plasma
 An IV formulation of t-TUCB has been developed, though
batch to batch variation in [C] exists
 T ½ is estimated to be 13 hours in PND 17 rats
 T-TUCB was present in the brain at ~ 2 times IC 50 at both 2
and 6 hours
 Post ACA CBF was no different between t-TUCB and vehicle
groups

15. Future Directions
 Test whether EET levels are raised at a dose of 1
mg/Kg t-TUCB
 Demonstrate whether the increase in EET
concentrations have activated known signal
transduction pathways for neurological protection
(PI3K/AKT or ERK 1/2)
 Test the effect of 1 mg/Kg t-TUCB on neurological
outcomes (Morris Water Maze and histological
assessment of neuron survival)

16. Acknowledgements
 Li Lingjue
 Dr. Poloyac and Dr. Manole
 S.K. Bhasha
 Safar Center For Resuscitation Research
 Funding: Pittsburgh Research and Investigational
Summer Experience Program (AHA)