Amide naphthotube as a novel supramolecular sequestration agent for tetracaine and decamethonium

Rationale: Anesthetics are widely used for optimizing surgical conditions, postoperative pain management, and treating various chronic pain conditions. Tetracaine and decamethonium are representative drugs of local anesthetics and neuromuscular blocking agents, respectively. However, overdose and toxicity of the drugs always lead to serious adverse events. Thus, there is a strong demand for effective antidotes. Methods: The binding interactions of amide naphthotubes with tetracaine and decamethonium were systematically studied using 1H NMR, ITC, and DFT calculations. The antidotal effects of amide naphthotube to tetracaine toxicity were assessed in vitro and in vivo, and the mechanism of detoxification was explored at a cellular level. Additionally, mouse models were established to evaluate the reversal activities of amide naphthotube on decamethonium-induced mortality and muscle relaxation, and the reversal mechanism was investigated through pharmacokinetic experiments. Results: We have demonstrated that the anti-isomer of amide naphthotube exhibits significant binding affinities towards tetracaine (Ka = 1.89×107 M-1) and decamethonium (Ka = 1.01×107 M-1) in water. The host displayed good biocompatibility both in vitro and in vivo. The administration of amide naphthotube following tetracaine overdose in mouse models notably increased the overall survival rate, indicating its effective antidotal properties. The host could reverse the tetracaine-induced Na+ channels blockage at the cellular level. Moreover, the injection of amide naphthotube also reversed the mortality and paralysis induced by decamethonium in mouse models following a pharmacokinetic mechanism. Conclusion: An emerging artificial receptor, amide naphthotube, has strong binding affinities towards tetracaine and decamethonium. It functions as a supramolecular antidote for tetracaine poisoning and a reversal agent for decamethonium by selectively sequestering these compounds in vivo.

(b) their equimolar mixture in PB buffer (pH =7.4).In the spectrum of their mixture, the aromatic protons of the host underwent obvious shifts, while the aromatic protons of the guest became broadened or disappeared into the baseline, supporting the binding between 1a and tetracaine.tetracaine@1a-conformer 1 tetracaine@1a-conformer 2 -42.58 kJ/mol 0 kJ/mol Figure S16.Energy-minimized structures of two representative conformers of TC@1a using the wB97XD/def2-SVP method (ma-def2-SVP basis set was used for anionic parts) in water (PCM).The results show that TC@1a-conformer 1 is more energetically stable than TC@1a-conformer 2.

Chemicals and Reagents
Lidocaine hydrochloride used as an internal standard (IS) was obtained from Aladdin.
The purity of C10, IS and 1b are all above 98.0%.LC-MS grade acetonitrile and methanol were supplied by Merck KGaA (Germany).Formic acid was procured from Thermo Fisher Scientific Ltd (USA).All other chemicals and reagents required for the experiment were of analytical grade.

LC/MS analysis
The LC/MS analysis was carried out by Liquid chromatography-mass spectrometry (Waters ACQUITY UPLC/Xevo TQ-S).Separation was performed on a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.7 μm), using 0.1 % (v/v) formic acid in water (A) and acetonitrile (B) as mobile phase.The auto-sampler was kept at 10 ℃ and the injection volume was 2 μL.An optimized gradient program was established as follows: 5% B at 0-0.5 min, 5-95 % B at 0.5-4.0min, 95-5 % B at 4.0-4.1 min, and 5 % B at 4.1-7 min.The flow rate was 0.3 mL/min, and the column temperature was set at 40 ℃.The parameters of the ESI source were set as follows: Capillary voltages, 1.0 kV; desolvation temp, 500 ℃; desolvation gas flow, 1000 L/Hr; Cone gas flow, 50 L/Hr; The analyte confirmation was performed by using retention time and multi-reaction monitoring (MRM) in positive ionization modes according to the optimized condition of each analyte (Table 1).

Administration and collection of plasma samples
Male Kunming mice (30±2g) were randomly divided into 5 groups, namely 1b group (dose 80mg/kg, concentration 20mg/ml), C10 group (dose 0.45mg/kg, concentration 0.125mg/ml, C10 injection for 30s followed by the same volume of normal saline), 1b combined with C10 group (C10 dose 0.45 mg/kg, C10 concentration 0.125mg/ml, C10 injection for 30s followed by 1b 6 mg/kg, 1b concentration 1.5 mg/ml).Drugs were injected through the tail vein of mice.In group 1b, eyeball blood and liver and kidney tissues were collected at 0.25, 0.5, 1,1.5, 2, 4, 6, 8, 12, 24, 36 and 48 hours after administration, with 6 mice in each time point.In the other four groups, blood and liver and kidney tissues were collected from eyeballs at 3min, 5min, 10min, 15min, 30min, 45min and 1h after administration.Six mice were selected at each time point.Blood samples were placed in heparin sodium tubes and centrifuged at 6000rpm for 20 minutes at 4℃ to obtain plasma samples.They were stored in a refrigerator at -80 ° C and frozen until analysis.Plasma, liver and kidney tissues were stored in a freezer at -80 ℃ and then frozen until analysis was performed.

Mouse plasma sample preparation
The plasma to be tested was thawed at room temperature, and 100 μL of mouse plasma samples were collected in 1.5 mL EP tubes.10 μL of IS solution (10 ng/mL) and 10 μL of methanol were mixed followed by the addition of 400 μL ethyl methanol-acetonitrile (1:1 v / v), and then the tubes were vortex mixed for 3.0 min.
After centrifugation at 13,000 rpm for 20 min at 4 °C, the supernatant was transferred to a nitrogen blower, and the solvent was dried by maintaining a steady flow of nitrogen.After drying the solvent, reconstituted with 100 μL 20% aqueous methanol solution, vortexed for 1 min, and centrifuged at 13000 rpm for 20 min, 2 μL of the supernatant was used for analysis in LC/MS.

Mouse liver and kidney sample preparation
Accurately weigh 200 mg of mouse liver or kidney samples, add 800 μL saline to the tissue grinding machine, centrifuge (4000 rpm / min, 4℃, 20min) with 200 μl supernatant, add 600 μl of methanol-acetonitrile (1:1 v / v) as protein precipitant, precision add 10 μL methanol solution (or QC solution) and 10 μL IS solution.The later experimental procedures refer to mouse plasma sample preparation.

Method Validation
This study was carried out in accordance with the 2020 Chinese Pharmacopoeia Part IV: Guidelines for Verification of Quantitative analytical methods for biological samples and the 2018 FDA Guidelines for Validation of biological sample analytical methods, etc. Low, medium and high quality control samples were selected for methodological investigation.The survey included the specificity of the analysis method, linearity, precision and accuracy, extraction recovery, and matrix effects.

Specificity
Specificity was determined by comparison with blank plasma samples and chromatograms of blank plasma samples supplemented with C10, 1b, and IS and plasma samples obtained after intravenous injection.

Standard curve and the linear range
The concentration of each compound in plasma was taken as the abscissa (X), the ratio of the peak area of the compound to the peak area of IS was taken as the ordinate (Y), and the weighted (W=1 / X) least squares linear regression was used to obtain the regression equation of each compound.

Precision and Accuracy
Precision and accuracy were obtained by analyzing QC samples six times in duplicate on the same day (intra-day) and on three consecutive days (inter-day) at three concentration levels (low, medium, and high concentration).Relative standard deviation (RSD) was used to evaluate precision, and relative error (RE) was used to evaluate accuracy.

Extraction Recovery and Matrix Effect
A total of 100μL of blank mouse plasma was previously removed, and QC sample solutions of low, medium and high concentrations were added, respectively, with 6 aliquots of each concentration in parallel.According to the above plasma sample processing procedures and LC/MS analysis methods, the peak areas of each tested

Data handling
The Masslynx 4.1 software was used to obtain the peak area of the tested compounds and internal standards in each plasma sample and calculate the standard curve formula.
Their plasma concentration data were processed using DAS 3.2.8software and pharmacokinetic parameters were calculated.

Specificity
By performing specialized analysis on blank mouse plasma, spiked mouse plasma, and actual mouse plasma samples, as depicted in Figure below, it was found that the blank mouse plasma did not contain target compounds.The retention times for 1b, C10, and lidocaine were 4.20 min, 2.76 min, and 3.26 min, respectively.Each compound exhibited good peak shapes, and there were no interfering peaks observed in the blood samples.
Figure S1. 1 H NMR spectra (500 MHz, 0.5 mM, 298 K) of (a) tetracaine, (c) 1a, and Figure S2. 1 H NMR spectra (500 MHz, 0.5 mM, 298 K) of (a) decamethonium, (c) 1a, and (b) their equimolar mixture in PB buffer (pH =7.4).In the spectrum of their mixture, the signals of the guest become de-symmetrized obviously and the upfield shifts of the proton signals of the methylenes indicate the guest is within the cavity and experiences the shielding effect of aromatic rings.

Figure S4 .
Figure S3. 1 H NMR spectra (500 MHz, 0.5 mM, 298 K) of (a) decamethonium, (c) 1a, and (b) their equimolar mixture in PB buffer (pH =7.4).In the spectrum of their mixture, the signals of the guest become de-symmetrized obviously and the upfield shifts of the proton signals of the methylenes indicate the guest is within the cavity and experiences the shielding effect of aromatic rings.

Figure S19 .Figure S21 .Figure S22 .Figure S23 .
Figure S19.Hemolysis assay of 1b.Data are presented as mean ± SD (n = 3) Standards and Quality Control (QC) Samples 1b, C10, and IS were dissolved separately in methanol to get a stock solution concentration of 1.0 mg/mL.The stock solution of the drug to be tested was then diluted with methanol to prepare the working solution.Calibration standards and QC samples were prepared by diluting the working solution with methanol.The final concentrations of C10 were 25, 250 and 1000 ng/mL, 1b final concentration of 150, 1500 and 6000 ng/mL.The final concentration of IS is 10 ng/mL.
compound and internal standard were determined and recorded as A; (2) The blank rat plasma (100 μL) was treated by the plasma sample processing method mentioned above, and then QC sample solution of low, medium and high concentration was added to redissolve in 6 aliquots in parallel.The peak areas of each compound were determined by injection analysis and recorded as B. (3) The peak areas of each compound were directly determined in 6 parallel samples of QC samples with low, medium and high concentrations and recorded as C. The extraction recovery rate R=A/B×100%; Matrix effect E=B/C×100%.

Figure S24
Figure S24 Specialized mass spectra of target compounds and internal standards: (A) Blank plasma; (B) Blank plasma spiked with QC and internal standards; (C) Post-dose mouse plasma; where a corresponds to C10, b corresponds to lidocaine, c corresponds to 1b.

Table S1
Summary of target analytes and corresponding MRM parameters.

Table S2 .
Standard curves and linear ranges for 1b and C10 in mouse plasma

Table S3 .
Precision and accuracy data of 1b and C10 in mouse plasma (n = 6).

Table S4 .
Recovery and matrix effect (%) data for the analytes in mouse plasma(n = 6)

Table S5 .
Standard curves and linear ranges for 1b in mouse liver and kidney