An anchor-tether 'hindered' HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model

Abstract

Background: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier.

Methods: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties.

Results: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing.

Conclusions: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Keywords: HCN1; antihyperalgesia; ion channel; nerve injury; neuropathic pain; rat.

Fig 1

Molecular dynamic modelling demonstrates functionally tolerated para position anchors control within-membrane distribution of the 2,6-di-tert-butylphenol (26DTB-P) pharmacophore. (a) Left: structures of zero-length linker anchors used to examine the effect of charge vs volume on hyperpolarisation-activated cyclic nucleotide-regulated (HCN) 1 inverse agonist activity. Right: shift in the V_1/2 (ΔV_1/2) of HCN1 gating determined in two-electrode voltage clamp (TEVC) for 2,6-di-iso-propylphenol (26DIP-P also known as propofol) and 4-substituted derivatives thereof (4-TBP, 4-tert-butyl-propofol; 4-AP, 4-amino-propofol; 4-TMAP, 4-trimethylamino-propofol). 26DIP-P was at 100 μM (the concentration that is saturating with respect to HCN1 inhibition); 4-TBP, 4-AP, and 4-TMAP were each at 200 μM. The effects of 26DTB-P, 4-TBP, and 4-AP are different to the absence of effect of 4-TMAP but not different to each other (see Supplementary Table S1). (b) ChemDraw representations of an unanchored (BP4C-10:0:1) and short and long tether diol-anchored variants of 26DTB-P (BP4L-10:0:1 and BP4L-18:1:1, respectively). (c) Schematic representation of the conceived coupling between a tether-anchored alkylphenol inverse agonist and an HCN1 channel. The cyan hexagon represents the alkylphenol pharmacophore. The thin black line represents the hydrophobic tether that, in the molecules presented in (b), are saturated or partially unsaturated acyl chains. The red rectangle is a hydrophilic coupling element that links the tether to the dark blue anchor. In the molecules studied here, the coupling element and the anchor are collectively the diol unit. The channel structure is adapted from Lee and MacKinnon. (d) In each of the four pairs of panels, the left-hand graphic is a still image from a high-resolution molecular dynamics simulation, and the right is a normalised density distribution depicting the Z-plane occupancy within the simulation cube of the water molecules, phosphate head groups of the phospholipids, the 26DTB-P group, and the anchor–tether complex (when present) plotted with respect to the membrane midline. The behaviour of the anchor–tether complex was determined by following the distal terminal carbon (i.e., the carbon atom furthest from the pharmacophore). In each density plot, the midline of the membrane (blue dashed line) was established with respect to the distance between the phosphate peaks. Distances (in nm) are shown relative to the midline. Increasingly positive values represent the outer leaflet and extracellular medium. The location of the blue dotted line representing the polar interface was located at the maximal occupancy of the diol anchor in BP4L-18:1:1. The membrane lipid acyl chains are omitted from view for clarity.

Fig 2

BP4L-18:1:1 is a potent and efficacious hyperpolarisation-activated cyclic nucleotide-regulated (HCN) 1 inverse agonist. (a) Exemplar families of HCN1 two-electrode voltage clamp (TEVC) current traces (left, full length; right, the tail currents on expanded scales). Current traces in red were those obtained upon activation at –65 mV. The green lines are superimposed fits of a single exponential function that simultaneously optimises the time constant and the pre-exponential lag. Human HCN1 was similarly inhibited by BP4L-18:1:1 (data not shown). (b) Representative normalised tail current activation curves, each fits with the Boltzmann function. Each curve is from a different cell, but all are from the same donor frog recorded on the same day. (c, d) Activation time constants (c) and their inverse (d), plotted as a function of the step potential with respect to the V_1/2 with data aggregated into 10 mV bins. Errors around the dependent variables and around the binned V–V_1/2 are standard deviation (sd). Where no error is seen, it is smaller than the symbol. Data are from nine, six, and six separate recordings (control, 10 μM, and 30 μM, respectively) paired by day and donor frog. (e) Shift in the V_1/2 of HCN1 gating, as determined in TEVC in the presence of BP4L-18:1:1 (filled blue circle, blue line is the superimposed fit of the Hill equation). Data are mean [sd]; the number of cells are indicated below each symbol. The potency and efficacy of HCN1 inhibition by BP4L-18:1:1 (IC₅₀=6.4 μM; ΔV_{1/2 MAX}=–38.9 mV; Hill coefficient=1.0) is comparable with that by free 2,6-di-tert-butylphenol (26DTB-P) (IC₅₀=4.2 μM; ΔV_{1/2 MAX}=–41.3 mV; Hill coefficient=1.1; the red line is the Hill fit to the shift in the V_1/2 of HCN1 gating in response to 26DTB-P (see Supplementary Fig. S9). ΔV_1/2 in 3, 10, and 30 μM BP4L-18:1:1 (but not 1, 0.3, or 0.1) are significantly different from vehicle controls (Supplementary Table S2) but are not different from the respective ΔV_1/2 in 3, 10, and 30 μM 26DTB-P (Supplementary Table S3). (f) Representative current–voltage relationships for HCN1 I_h currents in the absence and presence of 100 μM BP4L-10:0:1 (short diol-anchored molecule) or BP4C-10:0:1 (the equally short but unanchored chlorine variant) indicate BP4L-10:0:1 is less effective than BP4C-10:0:1 (mean ΔV_1/2 values [sd] were –3.6 [5.5], n=5 and –24 [6.3], n=5; –21.3 [2.4], n=5, respectively) and either the longer diol-anchored molecule, BP4L-18:1:1 (a–e) or the unanchored synthesis intermediate, BP4K-10:0:1 (mean ΔV_1/2 [sd], –21.3 [2.4], n=5 at 30 μM; not shown). The ΔV_1/2 values in BP4C-10:0:1 and BP4K-10:0:1 are significantly different from BP4L-10:0:1 but not from each other (Supplementary Table S4).

Fig 3

BP4L-18:1:1 is orally bioavailable following peanut oil gavage. (a) Whole blood concentrations of BP4L-18:1:1 following a single gavage of BP4L-18:1:1 with tail vein blood draws at the indicated times. Smooth lines are simultaneous fits of equation (3) to the low- and high-dose male data and the high-dose female data. Based on the approximate equilibration between major organs and blood (see Fig. 2), we assumed the volume of distribution (V) was equal to the whole-body volume (250 ml for a 250 g rat). As there is no a priori reason to attribute the observed difference in males and female peak blood concentration following the same (high) dose to any of the free parameters (F, k_a, and k_e), we ran fits, wherein F, k_a, and k_e were each allowed to be gender divergent with the other two parameters constrained to be equal between males and females. The thick dark blue and cyan lines are from the fit, where k_e was variant between males and females (lowest entry in (b)). The red and green lines are fits, wherein F and k_a were gender divergent, respectively. (b) Parameter estimates for fits of equation (3), as shown in (a). (c) Whole blood concentrations of BP4L-18:1:1 following a once-daily gavage with blood draws 6 h after the first gavage and 6 h after the seventh daily gavage. The trend lines are anchored at either end by the mean of low-dose and high-dose populations. Whilst the general trends suggest there is a degree of repeat dose summation (as is anticipated from the single-dose time courses in (a)), the scatter in each of the sex/dose populations suggests there is marked gavage-to-gavage variation that precludes this aspect of the current data.

Fig 4

BP4L-18:1:1 is excluded from the CNS and shows variable access to peripheral tissues. (a, b) Following once-daily dosing by gavage for 7 days, blood and tissue samples were taken from 12 randomly selected animals (three animals from each of the four plus-BP4L-18:1:1 behavioural cohorts reported in Fig. 5). The box plots in (a) report the BP4L-18:1:1 accumulation in each tissue relative to the blood concentration in the same animal. Note that tissue loads of pmol mg⁻¹ wet weight are equivalent to μM, assuming the tissue density is 1^, and the drug can distribute freely across the entirety of the tissue. Based on the overlap in the ratios of the high and low dose and male and female populations (as per (a)) and a lack of difference between the tissue loads of male vs female (P=0.464) and high dose vs low dose (P=0.430) (as per Student's t-test comparisons), (b) reports the mean tissue loads [standard deviation] across the combined samples (males plus females at both high and low doses). (c) A Friedman repeated measures analysis of variance on the tissue loads indicates brain, dorsal root ganglia (DRG), and kidney are protected organs relative to blood, whereas lung, heart, and liver are in near equilibrium with blood. For full statistical comparisons, see Supplementary Table S5.

Fig 5

BP4L-18:1:1 relieves sciatic spared nerve injury-induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner in male and female rats. The top four panels examine mechanical allodynia by plotting the withdrawal threshold of the paw ipsilateral to SNI injury in response to mechanical stimuli applied using (a) electronic and (b) manual von Frey procedures. Hypersensitivity to (c) cold and (d) hot thermal stimuli were determined by observing the length of time an animal lifted/licked the injured/tested paw (cold) and the latency before the animal withdrew its foot from the heat source (hot). In all panels, the timing of baseline and post-surgical measurements is in days (‘d’) with respect to the day of surgery (as detailed below (d)). Treatment is 1 or 7 days of once-daily dosing with vehicle or the indicated dose of BP4L-18:1:1 with timing relative to the SNI+28d determination. Thirty-six animals were used in total, with 12 animals each randomly assigned to vehicle, low-dose, and high-dose groups. Low dose, 0.58 mmol kg⁻¹; high dose, 1.74 mmol kg⁻¹. Data are presented as mean [standard deviation].

Fig 6

BP4L-18:1:1 is devoid of adverse effects on cardiovascular function and motor activity and lacks abuse potential. (a) Cardiovascular measurements were taken 1 h after dosing. Twenty-four animals total (12 males; 12 females). Data for each animal are from 20 individual HR and BP (MAP) measurements. Doses 1 and 7 indicate the total number of days dosed. Statistics: repeated measures analysis of variance. (b) Motor measurements were taken 1 h after dosing. Twenty-four animals total (12 males; 12 females). Data for each animal are from 10 replicates using the Ugo Basile Rota-Rod ramping from 5 to 80 rpm. Motor activity was measured using the Stoelting ANY-box automated tracking system. Total activity was measured over 20 min. (c) Schematic representation of the conditioned place paradigm training and dosing schedule (D, drug; V, vehicle). (d) An example of a preference trail map for a single rat. (e) BP4L-18:1:1 (5.8 mmol kg⁻¹) did not lead to place preference in either male (left panel) or female (right panel) rats. In contrast, morphine (5 mg kg⁻¹, s.c.) produced a robust place preference (P<0.0001 vs vehicle and BP4L-18:1:1, and P<0.0001 for morphine pre- and post-conditioning).