(T1330-06-39) Pharmacokinetic and Pharmacodynamic Correlations of Riluzole and Etoricoxib in a Long-Evan Rat Model with Traumatic Brain Injury

Purpose: Traumatic brain injury (TBI) refers to brain dysfunction caused by a forceful impact, such as a sport-related injury or car accident involving the head. In 2019, 223,135 TBI-related hospitalizations were reported¹. TBI impacts individuals of every age group. However, currently, no FDA-approved therapeutics are available to treat TBI². In this study, we evaluated riluzole and etoricoxib as a single or combination regimen for TBI. Riluzole has been shown safe and promisingly efficacious in treating cervical spinal cord injury³, while etoricoxib inhibits the inducible cyclo-oxygenase-2 (COX-2) pathway to reduce inflammation caused by TBI⁴. Our aim was to establish the pharmacokinetic/pharmacodynamic (PK/PD) correlations of riluzole and etoricoxib in a rat model of TBI by using the TBI-specific biomarker, glial fibrillary acidic protein (GFAP).

Methods: The TBI model of Long-Evan (L.E.) rats was generated using the Marmarou weight drop injury approach. L.E. rats were treated with either co-administration of riluzole (5 mg/kg) and etoricoxib (5 mg/kg) or with etoricoxib alone therapy 30 min after injury through the IV route. Drug concentrations in plasma, brain, and spinal cord were quantified by a validated liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay with LLOQ of 0.25 ng/mL. Using Phoenix WinNonlin^® Version 8.3, a PK 2-compartment model analysis was conducted. For the PD assessment, GFAP concentrations in the samples were quantified through enzyme-linked immunosorbent assay (ELISA, Abbexa Co., London, UK). Then, PK/PD correlations were established by analyzing the relationship between GFAP concentrations versus drug exposures (AUC_0-24h) as well as the C_max of both drugs. Using GraphPad Version 9, statistical analysis was conducted by Student’s t-test for TBI and healthy groups and f-test for correlations, with a significance level set at p< 0.05.

Results: In the combination group, PK analysis showed that the peripheral volume of distribution (V₂) of riluzole was smaller in the TBI group than that in the healthy group (0.83 vs 1.85 L/kg) (Table 1). No significance was shown in riluzole PK profiles between TBI and healthy combo group (Figure 1 A). However, the PK of etoricoxib in combo group was affected by TBI, as evidenced by the significant decrease of plasma concentration 15 min after dosing in rats with TBI (4.87±0.59 µg/mL), compared to the healthy rats (7.10±1.13 µg/mL) (p < 0.05, Figure 1 B). Additionally, the V₂ of etoricoxib was also decreased to 0.67 L/kg after TBI in combo therapy group verse in the healthy rats (1.07 L/kg) (Table 1). In the etoricoxib alone group, the plasma concentration of the drug was significantly lower at 24 hours post injury (17.15±12.87 ng/mL) compared to the healthy group (60.40±25.86 ng/mL) (p < 0.05) (Figure 1 B). The clearance of etoricoxib was faster with TBI (0.48 L/kg*hr) than that in healthy rats (0.35 L/kg*hr) (Table 1). Compared to the healthy rats, where the GFAP concentrations measured 0.69±0.23 µg/mL in the brain and 1.66±0.58 µg/mL in the spinal cord, while rats with severe TBI exhibited elevated GFAP levels. At 3 hours and 24 hours after injury, GFAP concentrations in the brain increased to 0.74±0.09 µg/mL and 0.96±0.30 µg/mL, respectively. Likewise, GFAP concentrations in the spinal cord increased to 1.84±0.90 µg/mL at 3-hour and 2.48±1.65 µg/mL at 24-hour post injury. In contrast, both combination and etoricoxib alone therapy resulted in significant reduction of GFAP levels in both brain and spinal cord at 24 hours post-dose (p < 0.05, Figure 2). Specifically, in the brain, the GFAP concentration was 0.47±0.29 µg/mL in the combination therapy group and 0.63±0.06 µg/mL in the etoricoxib alone group. Similarly, in the spinal cord, the combination therapy led to a GFAP concentration of 1.04±0.40 µg/mL, while the etoricoxib resulted in a concentration of 0.63±0.43 µg/mL. Although both treated groups exhibited a significant decline in GFAP concentration as compared to the sham group (p < 0.05, Figure 2), no statistically significant difference was observed between the two treatment groups. Lastly, an inverse correlation was established between GFAP concentrations and C_max of etoricoxib in etoricoxib alone treatment (R²=0.990). In the combination group, the GFAP concentrations was inversely correlated with the plasma AUC_(0-24h) of etoricoxib (R²=0.893) and riluzole (R²=0.911).

Conclusion: The PK profiles of riluzole and etoricoxib were altered by TBI, resulting in a decreased V₂ and lower plasma concentrations at 15-minute of both drugs in TBI rats than in healthy rats in combination group, and the etoricoxib concentration in alone group was also significantly reduced in plasma with TBI at the 24-hour time point compared to the healthy group. GFAP was a well-established diagnostic biomarker of TBI, which was upregulated post-injury. Both the combination therapy and etoricoxib alone group significantly reduced GFAP levels in the brain and spinal cord at the 24-hour post the single dose treatment. In addition, the decrease in GFAP concentrations in the brain and spinal cord was inversely correlated with the drug exposure (AUC_0-24h) of etoricoxib and riluzole in the combination treatment and C_max of etoricoxib in alone treatment group.

References: 1. Centers for Disease Control and Prevention. National Center for Health Statistics: Mortality Data on CDC WONDER. Accessed April 2023 https://wonder.cdc.gov/mcd.html.
2. Abbasloo, E., Amiresmaili, S., Shirazpour, S., Khaksari, M., Kobeissy, F., & Thomas, T. C. (2023). Satureja khuzistanica Jamzad essential oil and pure carvacrol attenuate TBI-induced inflammation and apoptosis via NF-κ B and caspase-3 regulation in the male rat brain. Scientific Reports, 13(1), 4780. https://doi.org/10.1038/s41598-023-31891-3
3. Siahposht-Khachaki, A., & Farzin, D. (2022). The effect of Riluzole on neurological outcomes, blood-brain barrier, brain water and neuroinflammation in traumatic brain injury. Brain Disorders, 8, 100052. https://doi.org/10.1016/j.dscb.2022.100052
4. Keshavarzi, Z., Khaksari, M., Razmi, Z., Soltani Hekmat, A., Naderi, V., & Rostami, S. (2012). The effects of cyclooxygenase inhibitors on the brain inflammatory response following traumatic brain injury in rats. Iranian journal of basic medical sciences, 15(5), 1102–1105.

Acknowledgements: This work was a proud collaboration with the late Dr. Robert G Grossman (Methodist) and the late Dr. Raymond Grill (UMMC). The funding source was from the Institute for Drug Education and Research (IDER), College of Pharmacy, University of Houston.

Figure 1 PK profiles (Mean ± SD) of riluzole and etoricoxib in combination and alone groups in healthy and TBI rats, respectively
Note: Multiple unpaired t-test was used for the statistical analysis. (A) Riluzole PK profiles in TBI combo group and healthy groups; (B) Etoricoxib PK profiles in TBI and healthy groups; “*” showed a significantly higher plasma concentration of etoricoxib at 15-minute in the healthy combination group compared to TBI combination group (p < 0.05). “#” showed a significant difference at 24 hours between the healthy alone group and TBI alone group (p < 0.05).

Figure 2 GFAP concentrations (Mean ± SD) in brain and spinal cord
Note: (A): GFAP concentrations in the brain; (B): GFAP concentrations in the spinal cord; Sham: injury without treatment; Combo: injury with combination treatment of riluzole and etoricoxib; Etc: injury with treatment of etoricoxib. Multiple unpaired t-test was used for statistical significance denoted by an “*” for p< 0.05, “**” p<0.01, “***” p<0.001.

Table 1 PK parameters of intravenously administered combo and etoricoxib single groups in
Long-Evan (L.E.) rats with 2-compartment models
Note: TBI: traumatic brain injury; Combo: administrated with riluzole and etoricoxib; Single: riluzole or etoricoxib treatment; V: volume of distribution; V2: peripheral volume of distribution; Cl: clearance; Cl2: inter-compartmental clearance; “§” for riluzole and “¢” for etoricoxib P<0.05 between TBI and healthy combo groups by Student’s t-test. Values in the table were Mean (CV%).