In Vitro and In Vivo Sequestration of Phencyclidine by Me4 Cucurbit[8]uril*

Abstract

We report investigations of the use of cucurbit[8]uril (CB[8]) macrocycles as an antidote to counteract the in vivo biological effects of phencyclidine. We investigate the binding of CB[8] and its derivative Me₄ CB[8] toward ten drugs of abuse (3-9, 12-14) by a combination of ¹ H NMR spectroscopy and isothermal titration calorimetry in phosphate buffered water. We find that the cavity of CB[8] and Me₄ CB[8] are able to encapsulate the 1-amino-1-aryl-cyclohexane ring system of phencyclidine (PCP) and ketamine as well as the morphinan skeleton of morphine and hydromorphone with K_d values ≤50 nm. In vitro cytotoxicity (MTS metabolic and adenylate kinase cell death assays in HEK293 and HEPG2 cells) and in vivo maximum tolerated dose studies (Swiss Webster mice) which were performed for Me₄ CB[8] indicated good tolerability. The tightest host⋅guest pair (Me₄ CB[8]⋅PCP; K_d =2 nm) was advanced to in vivo efficacy studies. The results of open field tests demonstrate that pretreatment of mice with Me₄ CB[8] prevents subsequent hyperlocomotion induction by PCP and also that treatment of animals previously dosed with PCP with Me₄ CB[8] significantly reduces the locomotion levels.

Keywords: cucurbituril; hyperlocomotion; molecular recognition; phencyclidine; sequestration agents.

Figure 1.

Structure of CB[n] and acyclic CB[n]-type receptors M1 and M2.

Figure 2.

Structure of the water soluble CB[8] derivative (Me₄CB[8]) that was used in this study.

Figure 3.

Chemical structures of competitive guests and drugs of abuse used in this study.

Figure 4.

¹H NMR spectra recorded (600 MHz, RT, D₂O) for a) PCP 8 (0.4 mM), b) an equimolar mixture of Me₄CB[8] and 8 (0.2 mM), c) a mixture of 8 (0.4 mM) and Me₄CB[8] (0.2 mM), d) fentanyl 4 (0.4 mM), e) a equimolar mixture of 4 (0.2 mM) and Me₄CB[8] (0.2 mM), f) a mixture of 4 (0.4 mM) and Me₄CB[8] (0.2 mM), g) a mixture of 4 (0.8 mM) and Me₄CB[8] (0.2 mM).

Figure 5.

Cross-eyed stereoview of an MMFF minimized geometry of the Me₄CB[8]•8 complex. Color code: C, grey; H, white; N, blue; O, red.

Figure 6.

(a) Plot of DP vs time from the titration of Me₄CB[8] (104 μM) and 11 (200 μM) in the cell with 8 (1.0 mM) in the syringe in 20 mM NaH₂PO₄ buffer (pH = 7.4); (b) plot of ΔH as a function of molar ratio of Me₄CB[8] to 8. The solid line represents the best non-linear fit of the data to the competition binding model (K_a = (5.35 ± 0.19) × 10⁸ M⁻¹ and ΔH = (–8.39 ± 0.01) kcal•mol⁻¹

Figure 7.

In vitro cytotoxicity experiments performed for Me₄CB[8]: a) HEPG2 cell viability assay after incubating the cells with Me₄CB[8] container for 24 h (UT = Untreated). This figure is the average SEM values representative of two replicate experiments. Statistical analysis is one-way ANOVA with Dunnett’s multiple comparisons test. **P = 0.001 – 0.01; ****P< 0.0001. b) HEK293 cell viability assay performed after incubation with Me₄CB[8] container for 24 h (UT = Untreated). This figure is the average SEM values representative of two replicate experiments. Statistical analysis is one-way ANOVA with Dunnett’s multiple comparisons test. *P = 0.01 – 0.05; ****P< 0.0001. c) HEPG2 cell death after incubation with Me₄CB[8]. AK assay was performed using the supernatant from cells seeded for MTS assay (UT = untreated). This figure is the average and SEM values representative of two replicate experiments. Statistical analysis is one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.001, d). HEK293 cell death after incubation with Me₄CB[8]. AK assay was performed using the supernatant from cells seeded for MTS assay (UT = untreated). This figure is the average and SEM values representative of two replicate experiments. Statistical analysis is one-way ANOVA with Dunnett’s multiple comparisons test. ****P<0.0001.

Figure 8.

MTD study performed for Me₄CB[8]. Female Swiss Webster mice (n = 5 per group) were dosed via tail vein injection (0.150 mL) on days 0 and 2 (denoted by *) with different concentrations of Me₄CB[8] or phosphate buffered saline (PBS). The normalized average weight change per study group is indicated. Error bars represent SEM.

Figure 9.

In vivo reversal of PCP-induced hyperlocomotion by Me₄CB[8]. a) Average locomotion counts for male Swiss Webster mice (N = 8) (black bars) treated with nothing (REF), saline (SAL), Me₄CB[8] alone (CON), PCP (PCP, 8) or a premixed solution of PCP and Me₄CB[8] (PCPC). Error bars represent SEM. Star signifies significant increase in locomotion counts (p’s < 0.05) for PCP compared to REF, SAL, CON and PCPC. b) Average locomotion counts for male Swiss Webster mice (n = 9) (grey bars) treated with nothing (REF), saline (SAL), sequential administration of Me₄CB[8] followed 30 s later by PCP (CON + PCP), sequential administration of PCP followed 30 s later by Me₄CB[8] (PCP + CON), or PCP alone (PCP). Error bars represent SEM. Star signifies significant increase in locomotion counts (p’s < 0.05) for PCP compared to REF, SAL, CPCP and PCPC.