Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model

Abstract

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ET_B) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ET_B receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K⁺ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ET_B receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ET_B receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ET_B receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ET_B receptor upregulation, making it suitable as a drug delivery system.

Keywords: Basilar artery; MEK1/2; U0126; cremophor EL; endothelin-1; optimal dose.

Figure 1.

Concentration-dependence of U0126 (and DMSO) on S6c contractility after 48 h organ culture. Basilar arteries were incubated with different concentrations of (a) U0126 and corresponding concentrations of (b) DMSO. (a,b) Concentration-response curves to S6c show varying EC₅₀ and E_max values. (c,d) Correlation between the concentrations of either U0126 or corresponding concentrations of DMSO (on the double x-axis) of E_max (c) or log EC₅₀ (d) from the concentration-response curves with S6c (a,b). Values are listed in the Table S1, one-way ANOVA with Holm-Sidak post-test (##p < .01 and ###p < .001) or multiple t-tests with Holm-Sidak correction for multiple testing (***p < .001), $=non-overlapping 95% confidence intervals. Data are expressed as mean ± SEM, n = 4–15.

Figure 2.

Concentration-dependence of cremophor on S6c-induced contractility after 48 h organ culture. Basilar arteries were incubated with different concentrations of cremophor. (a) Concentration-response curves with S6c show varying log EC₅₀ and E_max values. Correlation between varying concentrations of cremophor with E_max (b) and log EC₅₀ (c) from the concentration-response curves with S6c. Values are listed in the Table S2, one-way ANOVA with Holm-Sidak post-test (###p < .001). $=non-overlapping 95% confidence intervals. Data are expressed as mean ± SEM, n = 6–7.

Figure 3.

Concentration-dependence of U0126, DMSO, and cremophor on potassium-induced contractility after 48 h organ culture. Basilar arteries were incubated with different concentrations of U0126, corresponding concentrations of DMSO and cremophor. Correlation between the maximal 60 mM potassium contraction (K⁺ E_max) and the concentrations of U0126/DMSO (a) and cremophor (b). Values are listed in the Table S3, one-way ANOVA with Holm-Sidak post-test (##p < .01 and ###p < .001) or multiple t-tests with Holm-Sidak correction for multiple testing (***p < .001). Data are expressed as mean ± SEM, n = 4–15.

Figure 4.

Additive effect of cremophor and U0126 on S6c contractility after 48 h organ culture. (a) Basilar arteries were incubated without DMSO and with 0.0042 M DMSO which is the corresponding concentration for the 3 × 10⁻⁶M U0126. (b) Concentration-response curves with S6c of basilar arteries incubated with cremophor and different concentrations of U0126 in DMSO or cremophor. (c) Comparison of the log EC₅₀ values of the different concentrations of U0126 in DMSO or cremophor based on the concentration-response curves with S6c. When 0.1% is stated it refers to 0.1% cremophor. $ = non-overlapping 95% confidence intervals. Data are expressed as mean ± SEM, n = 6–7.

Figure 5.

Comparison of metabolism between fresh arteries and after 48 h organ culture. Metabolism was measured using cell counting kit 8. There was no change in the control arteries with DMSO (0.14 M) only. U0126 significantly lowered the metabolism. 0.5% (0.039 M) cremophor also lowered the metabolism but to a less extent than 10⁻⁴M U0126. n = 4, paired t-test (***p < .001).