Contractile Response of Bovine Lateral Saphenous Vein to Ergotamine Tartrate Exposed to Molecularly Imprinted Polymers –Physiological Significance of In Vitro Studies

Previously, we have provided evidence from in vitro adsorption assays that specifically developed polymer adsorbents (molecularly imprinted: MIP; non-imprinted: NIP) can bind alkaloids. To aid understanding the physiological significance, here we report on research using ex-vivo models. The objective was to investigate the physiological significance of MIP and NIP application using ex vivo myography and to compare the impact of MIP and NIP on the ergot alkaloid bioavailability. Lateral saphenous veins were collected from 17 steers (2 veins/steer) for 4 independent studies: 1) dose response of ergotamine (ETA), 2) validate adsorbent dose, 3) validate incubation conditions and 4) evaluate MIP and NIP (n=10, 6, 6 and 12, respectively). Products were also evaluated using in vitro isothermal adsorption (ETA [7.813E-07M] vs increasing polymer). For Exp. 1, 2, and 4, one-way ANOVA (GraphPad Prism) was used to analyze data within a CRD. Means were compared using Tukey's test. Percent contractile response normalized to norepinephrine, fitted with a 3-parameter nonlinear regression model (R²=0.95) for ETA concentrations (9.8E-08 to 5.0E-05M) exhibited a sigmoidal response with a maximum of 86.8±7.3%, and Log EC₅₀(-log[ETA]) of 6.59±0.26M. Increasing polymer inclusion using a log₂ (0.625 to 10mg), as compared to a log₁₀ (0.0001 to 1mg), scale improved curve fits. Ergotamine added immediately upon thawing increased (P<0.05) contractile response compared to addition after 1h incubation at 39^oC or 20^oC. Isothermal adsorption showed a maximum adsorption of ≈96% (7.52E-06moles·10mg^-1) for both products, with an affinity coefficient (K_d­, mg·10ml^-1) of 0.51±0.10 (R²=0.92) and 0.57±0.19 (R²=0.83) for MIP and NIP, respectively. At the maximum isothermal adsorption, a maximal reduction of 11.24±2.25 and 9.30±2.74% in contractile response were obtained for MIP and NIP, respectively. Imprinted polymer had better inhibitory response (IC₅₀=0.28±0.19mg) than NIP (IC₅₀ = 0.83±0.26mg). Comparing in vitro and ex vivo, the trend between the two products remained the same, and MIP had a better adsorption affinity (low K_d) to ETA and caused greater inhibition in contractile response (low IC₅₀). Percent contraction was predicted from the adsorption data using the equation y=0.98±0.06(x)+0.15±2.07, R²=0.82, P<0.01 and y=0.92±0.05(x)-1.35±1.74, R²=0.87, P<0.01, for MIP and NIP, respectively. These studies indicated that synthetic polymers are potentially effective adsorbents toward ergot alkaloids that could mitigate their impact provided that they could be used in animals. Although earlier studies pointed towards an increased specificity of MIP towards alkaloids adsorption, no differences were observed between products. The ex-vivo efficacy of both polymers was accurately predicted from in vitro adsorption data.