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EN3427: A Novel Cationic Aminoindane with Long-Acting Local Anesthetic Properties

Banerjee, Manish PhD*; Baranwal, Atul MVSc*; Saha, Soumya PhD*; Saha, Ashis PhD; Priestley, Tony PhD

doi: 10.1213/ANE.0000000000000629
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Currently approved local anesthetic drugs provide relatively brief local anesthesia that is appropriate and even desirable in some settings, but an extended duration of action beyond their capabilities would be a distinct benefit in other clinical situations. We implemented a drug discovery program that sought to identify novel local anesthetic molecules that specifically demonstrated a long-acting, preferential action on nociceptor sensory afferents that expressed transient receptor potential (TRP) channels. The hypothesis we tested was whether relatively membrane-impermeant local anesthetic molecules could confer long-lasting anesthesia if neuronal access was facilitated by TRP channel activation. The current work describes in vivo studies on a lead molecule that emerged from the discovery program, EN3427, in several rodent pain models.

METHODS: Studies were performed on male Sprague-Dawley rats using 2 models of acute mechanical paw-pinch−evoked and pinprick-evoked nociceptive pain. Behavioral responses to noxious stimuli were assessed at baseline, that is, before any pharmacologic intervention, and at various timepoints after a single perisciatic or subcutaneous administration of either EN3427 alone or in combination with lidocaine. Paw withdrawal thresholds or cutaneous trunci reflexes were quantified, and pre-post drug values were compared statistically with analysis of variance followed by post hoc Dunnett multiple range test.

RESULTS: A single perisciatic injection of lidocaine (2%) produced relief of paw-pinch−evoked pain that was significantly different from baseline through to the 1-hour timepoint (Dunnett multiplicity-adjusted P = 0.0081), as assessed using paw withdrawal or vocalization end points. EN3427 (0.2%), in the same model, produced a long-lasting block, with pain thresholds being significantly above baseline through to the 18-hour timepoint (Dunnett multiplicity-adjusted P = 0.0002); the combination of EN3427 (0.2%) plus lidocaine (2%) produced even longer lasting analgesia, with pain thresholds being significantly above baseline through to the 24-hour timepoint (Dunnett multiplicity-adjusted P = 0.0073). Similar results were obtained with use of the pinprick approach. A single subcutaneous injection of lidocaine (2%) produced complete loss of sensation to cutaneous pinprick through 0.5 hours, but sensitivity thresholds were no different to baseline by the 1-hour timepoint, a similar injection of EN3427 alone (0.2%) produced a loss of sensation that was significantly different from baseline through the 8-hour timepoint (Dunnett multiplicity-adjusted P = 0.0045), and the combination of lidocaine (2%) plus EN3427 (0.2%) appeared to further enhance duration of analgesia, although this was significantly different from baseline only through the 10-hour timepoint (Dunnett multiplicity-adjusted P = 0.0048). Analgesic efficacy was dose related; using the combined injection approach, we found that increases in the dose of EN3427 with a fixed 2% lidocaine led to substantially extended analgesia and increasing doses of lidocaine combined with a fixed dose of EN3427 (0.2%) led to only modestly increased duration of action.

CONCLUSIONS: The present studies demonstrate that a new molecular entity, EN3427, produces effective and long-lasting analgesia in 2 rodent pain models. The analgesic effects of EN3427 are significantly longer-lasting than lidocaine and are further extended when EN3427 is combined with lidocaine. The results are discussed with respect to a possible lidocaine-mediated TRP channel activation and facilitated neuronal access of EN3427, with subsequent entrapment conferring extended-duration efficacy.

From the *TCG LifeSciences, Kolkata, India; TCG LifeSciences, New York, New York; and Discovery and Early Development, Endo Pharmaceuticals, Malvern, Pennsylvania.

Atul Baranwal, MVSc, is currently affiliated with Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.

Accepted for publication October 31, 2014.

Funding: All studies were funded by TCG Lifesciences and Endo Pharmaceuticals.

Conflict of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address for correspondence Tony Priestley, PhD, Discovery and Early Development, Endo Pharmaceuticals, 1400 Atwater Dr., Malvern, PA 19355. Address e-mail to priestley.tony@endo.com.

Millions of patients each year, in the United States alone, elect to have, or require, operations and procedures that necessitate the use of local anesthesia, and many of these procedures are invasive enough to warrant postsurgical pain management. Typically, acute/subchronic postsurgical pain is managed with the use of nonsteroidal anti-inflammatory drugs, opioids, local anesthetics, or combinations of the aforementioned. Localized wound infiltration of, typically, large volumes of local anesthetics, such as lidocaine, bupivacaine, and ropivacaine, usually as part of a multimodal regimen comprising nonsteroidal anti-inflammatory drugs and opioids, has gained in popularity over recent years as a means for providing postsurgical analgesia after total hip or knee arthroplasties.1–3 For pain control after intra-abdominal or thoracic procedures, the same local anesthetics often are administered by continuous delivery via an indwelling catheter.4–6 Despite their growing popularity, each of these approaches has issues, including disputed or inconsistent efficacy,7,8 compounded by the fact that they are usually one component of a cocktail approach to pain management and a general lack of consensus on dose, volume, concentration, and even optimal cannula positioning.1,9 Moreover, numbness and motor paralysis are drawbacks of a local anesthetic approach to protracted analgesia, particularly when used for nerve block in surgeries involving weight-bearing articulating joints1,10 because the resulting muscle weakness can delay the initiation of ambulatory rehabilitation. These are unavoidable consequences, however, of the relatively nonselective effect of currently approved local anesthetic drugs on sensory and motor nerves.

Ideally, an optimal therapeutic in this class would be one that selectively targeted the predominantly nociceptive C and Aδ fibers while leaving tactile Aβ-mediated sensory transmission and motor Aα efferent function intact. Several studies suggested that a membrane-impermeable, positively charged lidocaine analog, QX-314, offered some progress in this regard because studies in rodents revealed the potential for motor-sparing, relatively long-lasting regional pain relief after a single bolus injection.11–14 However, although QX-314 is capable of providing effective, dense anesthesia/analgesia for longer periods than typically seen with other local anesthetics, the duration of efficacy, from a purely clinical perspective, remains suboptimal because it lacks what might reasonably be considered an optimal 36- to 48-hour pain-free window that would more likely meet patient, physician, and payor expectations.

Despite the shortcomings of QX-314, the concept of nociceptor-selective, long-acting analgesia by facilitated entry of a permanently charged cationic molecule through activated transient receptor potential (TRP) channels and into C and Aδ sensory nerve axons/terminals (reviewed in Roberson et al.14) is attractive from a drug development perspective. Accordingly, we sought to exploit the basic principles of TRP channel-mediated access of otherwise relatively impermeable molecules, by implementing a medicinal chemistry campaign aimed at identifying permanently charged QX-314 mimetics with improved affinity as blockers of voltage-gated sodium channels. Candidate compounds were assessed for their analgesic efficacy and duration in acute pain models when delivered as a coinjection with a known TRP channel activator, lidocaine.15 Lidocaine was chosen above the prototypic TRP channel activator, capsaicin,14 in the anticipation that a lidocaine combination formulation might be more clinically acceptable than one comprising capsaicin. We describe the in vivo profile of one such molecule, EN3427, a novel permanently charged cationic sodium channel blocker that is superior to QX-314 and is capable of providing >30 hours of analgesia when administered as a combination injection with lidocaine.

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METHODS

All experiments described in the following studies were performed on male Sprague-Dawley rats and were approved by TCG Lifesciences (Kolkata, India) IRB for animal research. This work was funded entirely by Endo Pharmaceuticals.

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Pinch-Pain Model

Male Sprague-Dawley rats (180–220 g body weight) were used for all experiments; they were assigned randomly to cohorts of 6 per treatment arm and subjected to 3 days of behavioral acclimatization before the designated test day. Pretest acclimatization comprises 3 brief daily sessions (each of approximately 2-minute duration) designed to progressively habituate the animals to the experimenter, the test room, and a mock test procedure. By the end of the acclimatization period, animals tolerated light restraint without overt signs of distress such that, on the actual test day, reliable and consistent paw withdrawal and/or vocalization force (PWF) thresholds were easily obtainable from several cohorts of animals in rapid succession.

A commercially available rodent paw-pinch device (Bioseb, Vitrolles, France) comprising a pair of large, blunt forceps was used to apply an acute noxious stimulus unilaterally to one hindpaw of the animal.16 The forceps were connected to strain gauges that provided a readout of the applied force to an electronic dynamometer. Analgesia testing was conducted by applying the tip of the forceps device at the base of the last phalange, approximately equidistant between the 5th and 4th metatarsus, and manually applying a gradually increasing force until a withdrawal response or vocalization was elicited, at which point the test was immediately terminated. The dynamometer automatically recorded the maximally applied force; an arbitrary “cutoff” force of 500 g was assigned to the instrument to avoid tissue damage. Baseline PWF values were determined for each rat before the administration of test substances.

Test substances, or vehicle (saline), were examined after unilateral injection into the immediate vicinity of a sciatic nerve trunk. Injections were performed exactly as described by Thalhammer et al.,17 except that animals were lightly anesthetized with isoflurane during the procedure. Animals were anesthetized and drug injected on a sequential basis, that is, no group anesthesia was used. After the procedure, animals were monitored visually during the postinjection period and then returned to their home cage once they had fully recovered from anesthesia (typically within 2 minutes). Postinjection PWF values were recorded at intervals (short intervals initially and gradually extended intervals as the experiments progressed) and were compared with preinjection baseline values. Statistical significance was determined using a 1-way analysis of variance (ANOVA) followed by Dunnett multiple range post-test. All studies were performed “blind,” and study scientists were unaware of the treatment assigned to any given animal in the study.

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Cutaneous Trunci Pinprick Model

The cutaneous anesthetic effects of lidocaine and EN3427 were assessed in a series of experiments using pinprick-evoked trunci reflex activity essentially as described previously.18 In summary, male Sprague-Dawley rats (250–300 g body weight) were assigned randomly to study groups in cohorts of 6. Before treatment days, animals underwent a 3-day period of acclimatization to both experimenter and environment that entailed 2 sessions of 30 to 45 minutes’ duration each day.

On the testing day, baseline pinprick responsiveness (PPR) for each rat was determined using 6 presentations of an 18-gauge needle mounted on a 26-g von Frey filament as a nociceptive stimulus. After recording baseline responsiveness, we injected test substances subcutaneously (volume 100 μL), resulting in a clear elevated wheal at the injection site. The extent of the wheal was marked with a nontoxic marker pen to delineate the area for subsequent PPR evaluations. Data are expressed as percentage maximal possible effect (%MPE) according to the equation below:

CV

CV

Postinjection %MPE assessments were made at 0.5-, 2-, 4-, 6-, 8-, 10-, 12-, 26-, 30-, 34-, 48-, 72- and 145-hour timepoints. Injections were performed blind and with the use of gentle restraint; injections for a cohort of 6 rats usually were completed within a 5-minute period. Data were analyzed by comparing postinjection %MPE values with preinjection baseline values. Statistical significance was assessed using a 1-way ANOVA followed by Dunnett multiple range comparison post-test.

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Motor Function Assessments

Motor function was assessed via a “sciatic function index score” that was based on gait analysis observations and a subjectively rated footprint score (FPS).19 Male Sprague-Dawley rats (180–220 g body weight) were assigned randomly to cohorts of 6, and the plantar surface of the hindpaw ipsilateral to the intended hindlimb injection site was gently tapped onto a permanent black ink pad. The animal was then released onto a raised platform onto which was secured a plain white paper cover. The initial footprint was disregarded, and subsequent footprints were analyzed by counting the number of ink marks corresponding to placements of each calli and digit as the animal ambulated along the platform. The number of calli/digit marks was rated, such that an FPS of 11 constituted normal gait and a score of 0 represented a fully splayed hindpaw with no weight bearing. All observations were performed by a skilled observer who was blind to the treatment condition. After obtaining a baseline FPS for each animal, we performed perisciatic injections precisely as described previously for the pinch-pain assessment studies. Injections comprise 200 μg of EN3427 and 2 mg of lidocaine, and FPS assessments commenced after animals had fully recovered from anesthesia and were repeated at intervals of 0.5, 2, 4, 6-, 8, 10, and 25 to 26 hours postinjection.

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Statistical Analyses

All experiments used a repeated-measures design. We did not perform a priori sample size determinations to power each study to detect any predetermined effect size. Pain thresholds were assessed for significance with a repeated-measures 1-way ANOVA followed by Dunnett multiple comparison post hoc (α = 0.01), with each repeated measure being compared with respective predrug baseline control values. When P values are quoted, they are multiplicity adjusted; all comparisons are to baseline pain thresholds and not to the time dimension, and significant differences in durations are therefore reported using the last time point at which pain thresholds were significantly above baseline.

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Materials

Lidocaine HCl was purchased from Sigma-Aldrich (Bangalore, India). EN3427 (bromide salt, Fig. 1) was synthesized by TCG Lifesciences, Kolkata, India. Drugs were dissolved in physiological saline for either perineural or subcutaneous injection. For all experiments involving combination injections, EN3427 was prepared at the appropriate concentration (weight/volume) in a lidocaine solution of the stated concentration (typically 2%).

Figure 1

Figure 1

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RESULTS

Pinch-Pain Experiments

The model was validated using perisciatic injections of the prototypic local anesthetic, lidocaine (2, 3, or 4 mg in 200 μL, equivalent to 1%, 1.5%, and 2%, respectively), that also served as a benchmark reference for analgesic efficacy and duration. Baseline PWF values were between 170 and 200 g force, and in all rats, these were briefly increased to cutoff (500 g) when measurements were taken at 30 minutes after lidocaine injection (all dose levels), consistent with the anticipated dense local anesthetic effects of lidocaine. In agreement with Binshtok et al.,12 we found only a weak dose-duration relationship for analgesia over this narrow lidocaine dose range, and in all cases, PWF thresholds in each animal were not statistically different from predrug baseline beyond the 1-hour timepoint (Fig. 2), regardless of the dose of lidocaine.

Figure 2

Figure 2

Compared with 2% lidocaine, EN3427 (400 μg or 0.2%) produced a substantially longer lasting analgesic response, and pain thresholds remained statistically different from baseline through to the 14-hour timepoint (P = 0.0002; Fig. 3). Combined administration of 4 mg of lidocaine with 400 μg of EN3427 (i.e., 2%/0.2%, respectively) produced a supra-additive analgesic state that manifested as complete insensitivity to the maximal 500-g force stimulus for at least 12 hours in 5 of 6 animals and that remained statistically significantly different from predrug baseline through to the 24-hour timepoint (P = 0.0073; Fig. 3).

Figure 3

Figure 3

In a separate experiment, we compared the analgesic efficacy duration profile of 2% lidocaine plus 0.5% QX-314 with that of 2% lidocaine alone and 2% lidocaine plus 0.2% EN3427 (Fig. 4). The combination of lidocaine and EN3427 produced a significantly longer lasting analgesia to pinch pain than was seen during similar studies using a lidocaine/QX-314 combination injection, as evidenced by the timepoints at which thresholds remained significantly elevated with respect to baseline (10 hours for the QX-314 combination [P = 0.0057] compared with 24 hours for the EN3427 combination [P = 0.0073]).

Figure 4

Figure 4

The duration of analgesia observed after lidocaine combination injections was further extended by increases in the dose/concentration of EN3427. We demonstrated this extension by comparing 200, 600, and 800 μg of EN3427 (equivalent to 0.2, 0.3, and 0.4%, respectively) as combined perisciatic injections with a fixed dose (4 mg; 2%) of lidocaine (Fig. 3). Although the duration of analgesia with lidocaine combinations comprising 600 and 800 μg EN3427 might appear impressive, the lack of recovery with the 800-μg EN3427 combination and the modest recovery with the 600-μg combination were cause for concern because they implied a potentially irreversible drug-induced neurologic deficit or toxicity. Importantly, however, all 6 rats treated with the 2%/4 mg lidocaine/0.2%/400 μg EN3427 combination showed complete recovery of sensory responsiveness by 57 hours after injection, and this treatment produced, on average, approximately 24 hours of analgesia as judged by PWF values being statistically significant (P = 0.0073) from baseline up to this timepoint.

Having established the analgesic qualities of EN3427/lidocaine after perineural delivery, we explored the effects of the combination in a more superficial cutaneous pain context by using the well-established cutaneous trunci reflex model. Subcutaneous injections of lidocaine (2%) alone produced the anticipated rapid onset, dense, but short-lived, analgesia to pinprick-evoked reflexes (Fig. 5). Similar injections of EN3427 (0.2%) alone produced an equally robust dense analgesia that took longer to manifest (approximately 2 hours after injection to reach maximal possible effect compared with within 30 minutes with lidocaine) but that also lasted far longer than was the case with lidocaine, and pain thresholds remained significantly greater than baseline at the 8-hour timepoint (P = 0.0045 as opposed to 0.5 hours, respectively). As expected from the perisciatic experiments, the combination of EN3427 (0.2%) with lidocaine (2%) produced a rapid-onset, dense analgesia with pain thresholds remaining significantly greater than baseline, on average, through to the 10-hour timepoint (P = 0.0048). Return to baseline from the 10-hour timepoint was heterogeneous; some animals remained anesthetic, whereas others fully recovered, as reflected by the variance in the data (Fig. 5).

Figure 5

Figure 5

In a series of additional studies, we examined the effect of varying the concentrations of EN3427 and lidocaine in the combination formulation. Figure 6 shows the effect of maintaining a fixed amount of lidocaine (2 mg, 2%) but varying the amount of EN3427 in the combination. This experiment showed that the duration of pinprick analgesia was dependent on the amount of EN3427 injected; doses of EN3427 as low as 50 μg (0.05%) produced an analgesic response that was clearly longer lasting than seen with 2% lidocaine alone (pain thresholds significantly greater than baseline for 5 hours [P = 0.0007] with EN3427 [0.05%] compared with 0.5 hour with lidocaine [2%]; Figs. 5 and 6). Increasing the concentration of EN3427 to 100 μg (0.1%) produced no apparent improvement, on average, to pain scores, whereas increasing the dose of EN3427 to 200 μg (0.2%) maintained loss of pain sensitivity to approximately 10 hours (P = 0.0052). Importantly, the overall response profile seen in the latter experiment replicated that seen in the earlier study with a 200-μg EN3427/2 mg lidocaine combination, indicating that the effects of the EN3427/lidocaine combination were reproducible over time and across animals. We also explored the converse situation, maintaining a fixed dose of EN3427 (200 μg, 0.2%) and varying the concentration of lidocaine in the combination. Somewhat surprisingly, these experiments revealed no evidence of dose dependency for lidocaine; indeed, all combination injections comprising lidocaine were equivalent in terms of efficacy and duration (Fig. 7).

Figure 6

Figure 6

Figure 7

Figure 7

Pain-related reflex behaviors, such as those described previously, are critically dependent on uncompromised motor function, and it could be argued that the observed “analgesic” efficacy might, in fact, have been attributable to motor paralysis induced by local anesthesia. Typical local anesthetics, such as lidocaine, bupivacaine, and ropivacaine, all produce dense local anesthesia, and although they may vary in their relative sensory versus motor nerve block,20–22 it is clear that as a class they show, at best, marginal bias toward the desired sensory nerve block versus undesirable motor nerve block, and in the case of lidocaine, there is actually a good case for the reverse being true.23,24

Therefore, to determine whether the long-lasting analgesia observed with the EN3427/lidocaine combination was an artifact of complete sensorimotor anesthesia, we performed detailed motor function assessments that measured ambulatory gait behavior after a unilateral perisciatic injection of the drug combination. As expected, a single unilateral injection of the EN3427/lidocaine injection combination (0.2%/400 μg EN3427 + 2%/4 mg lidocaine) into the vicinity of a sciatic nerve produced complete, but temporary, paralysis of the treated limb corresponding to a compromised gait score of 0 to 3 (Fig. 8). Motor function remained significantly compromised (P = 0.0029) in these treated animals for approximately 4 hours but was not significantly different to predrug baseline for all remaining timepoints (Fig. 8). These experiments showed that, of the approximately 24 hours of pinch-pain analgesia produced by perisciatic nerve injections of EN3427/lidocaine (Fig. 3), only 4 hours might be attributed to the inability to produce an appropriate avoidance response due to compromised motor function. The remaining 20 hours presumably reflect insensitivity to pain, constituting analgesia, rather than being the result of a long-lasting disruptive anesthetic state that had compromised motor function of the entire hindlimb.

Figure 8

Figure 8

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DISCUSSION

In this series of studies, we describe the in vivo profile of EN3427, a novel, long-acting, local analgesic that emerged from a medicinal chemistry campaign that was aimed at identifying permanently charged cationic molecules with superior properties to the prototype compound, QX-314. Using a fluorescence-based assay, EN3427 was found to be approximately 30-fold more potent than QX-314 in inhibiting sodium ion flux through constitutively expressed voltage-gated sodium channels in the N1E115 clonal cell line (data not shown). We also studied the in vivo local anesthetic/analgesic properties of EN3427 in 2 different contexts, one involving perineural injection around the sciatic nerve and the second involving interscapular subcutaneous injection. In each experimental setting, we found EN3427 to be more potent than QX-314 and to be capable of producing long-duration analgesia, especially when administered as a combined injection with the prototypic local anesthetic, lidocaine.

Lidocaine is a well-established local anesthetic that is relatively membrane permeable; when applied externally to dissociated neurons or desheathed nerve trunks, lidocaine rapidly partitions into the cytosol, from which it accesses a pharmacophore on voltage-gated sodium channels and effectively blocks channel function.25,26 When used to establish local nerve block in a typical clinical setting, lidocaine is not particularly efficient and high concentrations, well in excess of those required for efficient conduction block in vitro, are needed to secure conduction block, largely because of complex local distribution and elimination kinetics.27 Several in vitro studies, in which investigators used either isolated nerve or dissociated cells, have shown that N-ethyl-lidocaine, or QX-314, is significantly less membrane permeable than lidocaine. This is attributed to a permanent positive charge on a quaternary nitrogen, which effectively decreases lipophilicity and, consequently, externally applied QX-314 is only weakly effective, at best, as a sodium channel blocker.28–30 This property does not, however, hinder the ability of QX-314 to readily permeate through open TRPV1 and TRPA1 channels that act as portals for QX-314 entry into the cell.14,30 In fact, the cationic nature of QX-314 enables the molecule to act as a charge carrier,30 and once inside the cytosol, it is available to block sodium channel function in the same manner as lidocaine.30 The TRP channel–facilitated entry concept is also borne out by in vivo data. Binshtok et al.11 first demonstrated, using essentially the same perisciatic model used in the present experiments, that the analgesic potential of QX-314 was only revealed when the compound was coadministered with the canonical TRPV1 agonist, capsaicin. This first observation has been subsequently replicated13 and extended12 to include QX-314 formulations that used lidocaine,15 rather than capsaicin, as a TRP channel activator.

On the basis of these published studies, we expected EN3427 to behave in a similar manner to QX-314, and indeed, an EN3427-lidocaine combined injection, whether administered perineurally or subcutaneously, produced analgesia that persisted much longer than that observed with either agent alone. These results were consistent with the notion of lidocaine acting to enhance intraneuronal access of EN3427. Unlike the earlier published studies with QX-314,11 however, we also noted a rather prominent analgesic effect of EN3427 alone—findings that would appear to be at odds with the impermeable quaternary ammonium rationale discussed earlier. There is, in fact, ample precedent for QX-314 and other quaternary ammonium local anesthetics being effective in a variety of pain models and by a variety of routes of administration31–35 that is sufficiently expansive as to suggest that the observation by Binshtok et al.11 may be more the exception than the rule.

Several explanations have been proposed to explain the in vitro/in vivo efficacy discrepancy, but perhaps the most parsimonious answer may lie in the simple fact that appreciably greater local concentrations typically are required for in vivo efficacy, and in the face of millimolar concentration gradients and at body temperature that is several degrees higher than typically used for in vitro studies, sufficient permeability of the charged species ensues to enable cytosolic accumulation over time that is eventually sufficient to affect conduction block. Regardless of the fact that QX-314 and EN3427 are effective local anesthetics in their own right, it is also true that their efficiency as such, in vivo, is enhanced by concurrent TRP channel activation, which is consistent with the general concept of facilitated access via open TRP channels.

As expected, lidocaine administered via either the perineural or the subcutaneous routes produced a dense analgesia in both the paw-pinch (all animals reaching either the 500-g cutoff stimulus level) and the pinprick (100% of MPE) pain models, respectively, but the effect was very short-lived (typically 1 hour or less). As noted by Binshtok et al.,12 the actual duration of pinch-pain analgesia varied only marginally over the 2-, 3 and 4-mg lidocaine (equivalent to 1%, 1.5%, and 2%, respectively) dose range. The likely explanation for the brief duration of lidocaine analgesia is that the anesthetic is rapidly removed from the site of injection and into the systemic circulation.36 We attribute the much long-lasting effects of EN3427 to most likely be attributable to the compound being effectively trapped intracellularly. We assume that there is little likelihood of an outward conductance of cytosolic EN3427, even during the period that we might expect lidocaine-activated open TRP channels, because the concentration gradient would be unlikely to ever be conducive to outward flux. Not surprisingly, intracellular accumulation of QX-314 has been suggested as a basis for the prolonged sodium channel block seen with that compound, and whole-cell voltage clamp experiments have revealed that internal QX-314 is incapable of outward conductance through TRPV1 channels but, in fact, actually acts as a potent inhibitor of outward current flux through open TRPV1 channels.30 We suspect that this may also hold true for EN3427, but additional studies would be required to establish whether this is the case. An alternative possibility for long-lasting efficacy with EN3427 would be that the drug persisted for some extended period of time in the tissues at the site of the injection, implying a pharmacokinetic rather than pharmacodynamic basis for the duration of action. We do not believe this to be the case; systemic blood levels of EN3427, sampled at various intervals after a bolus injection of 2% lidocaine with 0.2% EN3427 (200 μL volume), revealed a rapid appearance and elimination of EN3427 from the circulation with essentially 99.9% of the injected mass of EN3427 being eliminated by 2 hours after injection (data not shown).

There have been surprisingly few studies on the intrinsic factors (i.e., as opposed to pharmacokinetic influences) governing duration of action of local anesthetics but, at least for lidocaine and mepivacaine, duration of action appears to be a linear function of the logarithm of the administered concentration.37,38 EN3427 differs in this regard; logarithmic transformation of the concentration-response data revealed nonlinearity, and the reason for the difference is not presently understood but may reflect the novel mechanism discussed earlier or simply the added complexities of interpreting dose-response data in the context of a drug combination.

Assuming that the “trapped EN3427” hypothesis is correct and that accumulated EN3427 is responsible for the long-lasting effects of EN3427, implying a reservoir of EN3427 being available to repeatedly block sodium conductances, it raises an interesting question about the recovery process. What mechanisms or processes are responsible for the eventual elimination of EN3427 leading to the reinstatement of voltage-gated sodium channel activity and action potential generation/propagation? Although we did not address, experimentally, the basis for recovery, 2 possible explanations were considered and these were not necessarily, or even likely to be, mutually exclusive. First, EN3427 may slowly diffuse out of the axon or terminal because we know that it is capable of entering, albeit inefficiently, when applied without exogenous TRP channel activation. Second, we assume that EN3427 will likely diffuse away from the primary site of accumulation over time and will eventually be diluted in the axonal cytoplasm to a point at which it is unable to effectively block sufficient sodium channels.

Our dose-response perineural studies with EN3427 suggested the potential for irreversible loss of sensation, or exceedingly slow recovery, at concentrations approaching 0.5% in a combined solution with 2% lidocaine (Fig. 3). We did not explore the basis for such long-lasting effects in rodents, although pilot studies using subcutaneous injections in a canine pinch-pain model revealed analgesic efficacy lasting, in some cases, up to 11 days with eventual full recovery of sensation (data not shown). Preliminary histopathology assessments have identified clear signs of localized myopathy and neuropathy after single perisciatic injections of 1 mg of EN3427, and this was not unexpected, given that this is a toxicity that is characteristic of many local anesthetics. Toxicity severity was markedly reduced at the lowest dose level (i.e., 0.4 mg), was less obvious after subcutaneous administration, and showed signs of time-dependent reversal, but additional work would be required to characterize this more definitively.

The perisciatic injection studies suggested that 200 μg of EN3427, when combined with 2 mg of lidocaine, provided a reasonable balance between the desire for long-duration analgesia and for full functional recovery. Accordingly, subsequent dose-ranging studies that were performed using the subcutaneous administration route and pinprick model explored doses of EN3427 that were 200 μg or less and also examined the influence of EN3427:lidocaine dose ratio and of dose volume. When combined with a fixed dose of lidocaine (2 mg), EN3427 (50, 100, and 200 μg) produced a clear dose-related effect and although the lower 2 doses were superior, in terms of the duration of effect, to lidocaine alone (see Fig. 4) it was also apparent that the 200-μg dose level was likely to translate into a clinically more meaningful duration of action. With this in mind, we then explored the effect of varying the dose of lidocaine while maintaining a fixed, 200-μg dose of EN3427. Surprisingly, lidocaine doses over the 0.5- to 2-mg range had little, if any, influence on the duration of action of the combination (Fig. 6), suggesting that even 0.5 mg of lidocaine may be sufficiently effective at gating TRP channels to the extent required for efficient access of EN3427.

In summary, we have described the local anesthetic properties of EN3427, a novel quaternary ammonium compound that is capable of providing long-lasting regional analgesia in 2 commonly used rodent models of acute noxious pain. EN3427 was clearly superior to lidocaine and the quaternary ammonium analog of lidocaine (QX-314), in terms of the duration of analgesia provided after a single bolus injection. The duration of analgesia, however, was extended significantly when EN3427 was injected as a combined solution formulation with lidocaine; the combination formulation provided analgesia that persisted up to 24 hours. Assuming the rodent data described herein were to translate to equivalent efficacy/durations in humans, EN3427 may be capable of providing robust analgesia with only brief motor impairment for a substantial part of the critical postsurgical period that follows many common surgical procedures. Should this prove to be the case, EN3427 would present a new and alternative approach to postsurgical care that might be beneficial to patients and physicians and with potential for significant pharmacoeconomic impact.

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ACKNOWLEDGMENTS

The authors thank Dr. Sandeep Gupta for his enthusiastic support of this program and Drs. Dana Shuey, Scott Thompson, Clifford Woolf, and Bruce Bean for thoughtful comments and guidance.

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DISCLOSURES

Name: Manish Banerjee, PhD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Manish Banerjee has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Atul Baranwal, MVSc.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Atul Baranwal has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Soumya Saha, PhD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Soumya Saha has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Ashis Saha, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Ashis Saha has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Tony Priestley, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Tony Priestley has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Conflicts of Interest: Tony Priestley is an employee of the sponsoring organization that funded the research program.

This manuscript was handled by: Jianren Mao, MD, PhD.

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