Anesth Analg 2004;98:672-676
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000100742.87447.C1
ANESTHETIC PHARMACOLOGY
Local Anesthetic Properties of a Novel Derivative, N-Methyl Doxepin, Versus Doxepin and Bupivacaine
Yukari Sudoh, MD*,
Elaine Elliott Cahoon, BS*,
Umberto De Girolami, MD
, and
Ging Kuo Wang, PhD*
*Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital; and
Department of Pathology, Brigham and Women’s Hospital and Children’s Hospital and Harvard Medical School, Boston, Massachusetts
Address correspondence and reprint requests to Ging Kuo Wang, PhD, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Address e-mail to wang{at}zeus.bwh.harvard.edu
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Abstract
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Among various tricyclic antidepressants, doxepin and amitriptyline are also long-acting local anesthetics. We synthesized a new compound, N-methyl doxepin, and investigated whether this derivative possesses local anesthetic properties. N-methyl doxepin and doxepin were tested in a rat sciatic nerve model at 2.5, 5.0, and 10 mM. Proprioceptive, motor, and nociceptive blockade were evaluated and compared with those induced by 0.5% bupivacaine. Block of Na+ channels by N-methyl doxepin and doxepin was assessed in cultured pituitary tumor cells under voltage clamp conditions. N-methyl doxepin elicited complete nociceptive blockade that generally lasted longer than that caused by doxepin (e.g., approximately 7.4 h versus 5.3 h at 10 mM). Significant differences were observed for full recovery of function at all concentrations and for the duration of complete blockade except at 2.5 mM. Bupivacaine at 0.5% (15.4 mM) was less effective in producing complete blockade (approximately 1.5 h) than N-methyl doxepin and doxepin. Both doxepin and N-methyl doxepin were potent Na+ channel blockers, although N-methyl doxepin displayed a slower wash-in rate. No morphological alterations were detected in cross-sectioned sciatic nerve specimens with these three drugs. We conclude that N-methyl doxepin is a potent Na+ channel blocker and a long-acting local anesthetic for rat sciatic nerve blockade.
IMPLICATIONS: N-methyl doxepin and doxepin are both potent Na+ channel blockers; they elicit rat sciatic nerve block lasting longer than that induced by bupivacaine and seem to be nontoxic to peripheral nerves at concentrations up to 10 mM.
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Introduction
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Doxepin is a dibenzoxepin tricyclic compound that has been used as an antidepressant for >40 yr. Doxepin blocks 
2-adrenergic, N-methyl-D-aspartate, and histaminergic H2 receptors, inhibits the re-uptake of 5-HT serotonin and norepinephrine, and functions as an antidepressant predominantly in the central nervous system (1–4). Doxepin taken orally alleviates chronic pain (5). McCleane (4) reported that topical application of doxepin cream also has an analgesic effect on neuropathic pain. Furthermore, preemptive administration of doxepin reduces postoperative pain and decreases the need for opioids (6). As we reported previously, doxepin is a potent Na+ channel blocker that elicits sciatic nerve blockade of much longer duration than bupivacaine in rats (7). The fact that doxepin acts as a potent local anesthetic may partially explain the efficacy of doxepin in neuropathic pain.
In this study, we synthesized a new quaternary ammonium (QA) derivative of doxepin (N-methyl doxepin; Fig. 1). We postulated that this QA compound could penetrate the axonal membranes and would be trapped within the axonal cytoplasm because of its amphipathic property. We compared the potencies of N-methyl doxepin, its parent drug doxepin, and bupivacaine, the most potent local anesthetic. We used rat pituitary tumor GH3 cells to measure Na+ currents and their sensitivity toward these drugs in vitro and used the rat sciatic nerve model to examine proprioceptive, motor, and nociceptive functions in vivo. In addition, to assess the neurotoxicity of this derivative, we undertook detailed neurobehavioral follow-up and histopathological evaluation of cross-sectioned sciatic nerve specimens.
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Figure 1. Chemical structures of doxepin and N-methyl doxepin. Addition of a methyl group to the tertiary amine of doxepin yields N-methyl doxepin, which is permanently charged.
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Methods
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Bupivacaine-HCl and doxepin-HCl were purchased from Sigma (St. Louis, MO). N-methyl doxepin bromide was custom synthesized by Sigma-RBI (Natick, MA) in white powder form (molecular weight, 374.3). The purity of this product was 99% by high-performance liquid chromatography. All drugs were freshly prepared and dissolved in 0.9% sodium chloride, with pH ranging from 6.4 to 6.6.
The protocol for neurobehavioral studies was reviewed and approved by the Harvard Medical Area Standing Committee on Animals. Male Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, MA) and kept in animal housing facilities with controlled humidity (20%–30% relative humidity), room temperature (24°C), and a 12-h (6:00 AM–6:00 PM) light-dark cycle. Before the experiments, rats were handled daily for 7–10 days to familiarize them with the experimental environment and procedures and thus reduce animal stress during experiments.
All rats (200–250 g) were anesthetized before injection by inhalation of a small concentration of sevoflurane. A volume of 200 µL of N-methyl doxepin or doxepin at various concentrations (2.5, 5, and 10 mM) or bupivacaine at 15.4 mM (0.5%) was injected with a 27-gauge needle at the sciatic notch of the left hind limb. Rats recovered from sevoflurane anesthesia in 1.0–1.5 min. The right hind limb served as a control in functional assays. The drugs injected were blinded from the examiner. Functional changes were evaluated at appropriate intervals (before, 4, 6, 8, 10, 20, and 30 min after injection and continued 1 or 2 h until fully recovered) and determined as percentage of maximal possible effect (% MPE). Details of neurobehavioral assays can be found elsewhere (8). In brief, the nocifensive function was evaluated by the withdrawal reflex and vocalization to pinch at the fifth toe of the hind limbs. The nocifensive reaction was graded as 4 (normal or 0% MPE), 3 (25% MPE), 2 (50% MPE), 1 (75% MPE), or 0 (absent or 100% MPE). Motor function of hind limbs was evaluated by measuring extensor postural thrust on a digital scale. The force of the hind limb to push against the platform of the scale was measured. The control was considered a 0% MPE. Any reduction in force, resulting from extensor muscle tone, was considered motor deficit. A force <20 g was considered absence of extensor postural thrust or 100% motor block. Proprioception evaluation was based on the resting posture and postural reactions ("tactile placing" and "hopping"). The functional deficit was graded as 3 (normal or 0% MPE), 2 (slightly impaired), 1 (severely impaired), or 0 (complete or 100% MPE). Hopping response was evoked by lifting the front half of the animal off the ground and then lifting one hind limb at a time off the ground so that the animal moved laterally.
Two weeks after drug injection, all rats were killed by overdoses of sevoflurane. Sciatic nerves with the injection site near the center were removed, trimmed, and rinsed with saline solution. Each nerve (approximately 3 cm) was placed in a vial containing 4% glutaraldehyde in 0.1 M cacodylate buffer. Cross-sectioning and staining of sciatic nerves were performed as described (9).
Rat clonal pituitary GH3 cells (which generated growth hormone) were purchased from the American Type Culture Collection (Rockville, MD), and maintained in Dulbecco’s modified Eagle’s medium (Hyclone Labs, Logan, UT) supplemented with L-glutamine (1%), penicillin/streptomycin (1%), hydroxyethylpiperazineethane sulfonic acid (HEPES, 20 mM), and heat-inactivated fetal bovine serum (10%), as described before (10). The whole-cell configuration of the patch-clamp technique was used to record Na+ currents in rat clonal GH3 cells at room temperature (22° ± 2°C) (11). The pipette electrodes had a tip resistance ranging from 0.5 to 1.0 M
. Command voltages were controlled by pCLAMP software (Axons Instruments, Inc., Foster City, CA) and delivered by a List-EPC7 voltage clamp device (List-Electronic, Darmstadt/Eberstadt, Germany). Under whole-cell configuration, cells were dialyzed for approximately 20 min before data were acquired. Data were filtered at 3 kHz, collected, and stored with pCLAMP software. Leak and capacitance were subtracted. Pipette electrodes were filled with an internal solution containing 100 mM NaF, 30 mM NaCl, 10 mM ethyleneglycolbis (ß-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA) and 10 mM HEPES titrated with CsOH to pH 7.2. The external solution consisted of 65 mM NaCl, 85 mM choline Cl, 10 mM HEPES, and 2 mM CaCl2 and was titrated with TMA-OH to pH 7.4. The identity of Na+ currents was confirmed by 300 nM tetrodotoxin, which blocked these currents completely.
Statistical analysis was performed by means of two-way analysis of variance, followed by multiple comparisons testing with the Tukey-Kramer post hoc test to compare the functional effects in each group. Results are presented as means ± SE. Statistical significance was defined as P < 0.05 using the software StatView 5.0 (SAS Institute Inc., Cary, NC).
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Results
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After injection of doxepin or N-methyl doxepin (5 mM or 10 mM) into the sciatic notch, the rat hind leg showed complete nociceptive blockade within 10 min (Fig. 2, A and B, open circles and triangles). At 2.5 mM of either drug, it required approximately 20 min to reach complete nociceptive blockade (Fig. 2, A and B, open squares). All rats recovered completely from nociceptive deficits (Fig. 2, A and B); no rats displayed sedative reaction or other behavioral abnormality.
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Figure 2. Time courses of nociceptive functional impairment in percentage of maximal possible effect after injection via the sciatic notch. A, Neurological evaluations were performed before, 4, 6, 8, 10, 20, and 30 min after injection with 0.2 mL of doxepin at concentrations of 2.5 mM (n = 10), 5 mM (n = 12), or 10 mM (n = 7). Evaluations continued every 1 or 2 h until fully recovered. B, Neurological evaluations were performed after the administration of 0.2 mL of N-methyl doxepin at concentrations of 2.5 mM (n = 6), 5 mM (n = 7), or 10 mM (n = 10). The effects of 10 mM N-methyl doxepin lasted almost 1 day. The time course after injection of 0.2 mL of bupivacaine at 15.4 mM (0.5%) was included for comparison.
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In the 15.4 mM (0.5%) bupivacaine group, sciatic nerve functions were completely blocked for 1.5 ± 0.2 h (Fig. 3A; n = 6). In the 2.5 mM doxepin group, complete blockade lasted for 2.4 ± 0.2 h for proprioception and motor, and 2.3 ± 0.2 h for nociception (n = 7) whereas in the 5 mM group, 3.3 ± 0.2 h for proprioception and nociception and 3.2 ± 0.1 h for motor (n = 12). In the 10 mM doxepin group, duration of complete blockade was further prolonged; 5.5 ± 0.6 h for both proprioception and motor, and 5.3 ± 0.4 h for nociception (n = 10) (Figs. 2A and 3A). In comparison, the duration of blockade produced by 2.5 mM N-methyl doxepin was 2.4 ± 0.2, 2.3 ± 0.2, and 2.9 ± 0.3 h for proprioceptive, motor, and nociception, respectively (n = 6). At 5 mM, N-methyl doxepin produced blockade longer than 0.5% bupivacaine by more than threefold (P < 0.001); duration was 5.0 ± 0.5 h for proprioception and motor, and 5.3 ± 0.3 h for nociception (n = 7). N-methyl doxepin at 10 mM elicited complete blockade of proprioception, motor, and nociception: 6.8 ± 0.4, 6.3 ± 0.5, and 7.4 ± 0.7 h, respectively (n = 10) (Figs. 2B and 3A). Evidently, the larger the concentration of doxepin or N-methyl doxepin, the longer the complete nerve block.
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Figure 3. Duration of the complete blockade and full recovery of sciatic nerve functions by bupivacaine, doxepin, or N-methyl doxepin. A, Duration of complete sciatic nerve blockade by bupivacaine at 15.4 mM or at 2.5, 5, and 10 mM doxepin or N-methyl doxepin. The blockade duration of all functions by 5 mM N-methyl doxepin and of nociception by 10 mM N-methyl doxepin was significantly longer than that of doxepin (P < 0.001). B, The time until full recovery of sciatic nerve functions after the administration of bupivacaine at 15.4 mM or doxepin or N-methyl doxepin at 3 different concentrations. The time to reach the full recovery of all functions after N-methyl doxepin was significantly longer than that after doxepin at each given concentration (P < 0.001).
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The duration of full recovery was also significantly different among doxepin, N-methyl doxepin, and bupivacaine. At 15.4 mM bupivacaine, proprioceptive, motor, and nociceptive functions recovered fully after 2.6 ± 0.2, 2.7 ± 0.1, and 2.6 ± 0.2 h, respectively (Fig. 3B, n = 6). In comparison, full recovery time for 2.5 mM doxepin was 5.4 ± 0.1, 5.7 ± 0.1, and 5.5 ± 0.1 h for proprioception, motor, and nociception, respectively. At 5 mM versus 10 mM doxepin, full recovery duration of proprioception was 5.8 ± 0.1 versus 12.6 ± 1.4 h, motor was 7.2 ± 0.1 versus 13.4 ± 1.3 h, and proprioception was 6.3 ± 0.1 versus 12.4 ± 1.2 h (Figs. 2A and 3B). At 2.5 mM N-methyl doxepin, full recovery of proprioception, motor, and nociception was 6.7 ± 0.2, 7.2 ± 0.2, and 6 ± 0 h, respectively. At 5 mM N-methyl doxepin, full recovery was 3 times longer than that of 0.5% bupivacaine: 9.5 ± 0.3, 10.3 ± 0.2, and 9.9 ± 0.2 for proprioception, motor, and nociceptive function, respectively (P < 0.001). At 10 mM N-methyl doxepin, full recovery was even longer duration than that of doxepin: 17.1 ± 1.9, 20.6 ± 1.1, and 19.2 ± 1.4 h for proprioception, motor, and nociception, respectively (Figs. 2B and 3B, P < 0.001). Again, the larger the concentration of doxepin and N-methyl doxepin, the longer the duration for the full recovery of functions. However, no rats showed functional blockade for >24 h.
Possible morphological alterations were assessed by light microscopic examination in the cross-sectioned sciatic nerves, which were administrated 0.5% (15.4 mM) bupivacaine, 10 mM doxepin, or 10 mM N-methyl doxepin. A normal density of axons and an absence of axonal degeneration were observed in all transverse sections at a magnification of 400. Thus, there was no evidence of neurotoxicity by these three drugs at the concentrations specified.
Finally, we measured block of Na+ currents by N-methyl doxepin and doxepin under voltage clamp conditions. Superimposed current traces are shown in Figure 4, A and B before and after 250 µM doxepin and N-methyl doxepin applications, respectively. The time course of the block by N-methyl doxepin reached approximately 80% blockade in 40 min (Fig. 4C, closed circle; n = 10). In contrast, doxepin reached its steady-state block rapidly; most of Na+ currents were inhibited within 10 min (Fig. 4C, closed rectangular; n = 5). Evidently, N-methyl doxepin blocked Na+ channels with a slower wash-in rate than its parent drug doxepin as expected, but this derivative remained a potent Na+ channel blocker.
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Figure 4. Tonic inhibition of Na+ currents by doxepin and N-methyl doxepin in neuronal GH3 cells. The tonic effects of doxepin (A) and N-methyl-doxepin (B) on Na+ currents at 250 µM were measured. Current traces were superimposed and labeled with the time after drug wash-in. The holding potential was -140 mV. A test pulse of +30 mV for 3.8 ms was applied at 30-s intervals (A, bottom). C, Peak Na+ currents were measured, normalized, and plotted against time. Doxepin blocked approximately 95% of Na+ current within 2 min. However, N-methyl doxepin was slow to wash in; it took 40 min to reach approximately 80% blockade.
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Discussion
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We demonstrated that N-methyl doxepin and doxepin elicit longer duration of sciatic nerve block than the long-acting local anesthetic, bupivacaine. We also showed that both drugs are nontoxic for peripheral nerve block at concentrations up to 10 mM. Thus, N-methyl doxepin and doxepin may be useful as long-acting local anesthetics.
Doxepin has a relatively high octanol/buffer partition coefficient (P0 and P+ for neutral and protonated drug, respectively). The calculated logP0 value is 3.99 for neutral doxepin and logP+ is 1.66 for protonated doxepin (www.logp.com). The observed logP0 value is 3.41 for neutral bupivacaine and logP+ is 0.18 for protonated bupivacaine (12). In comparison, N-methyl doxepin has a calculated logP+ value of 2.02. The relative high logP+ value of N-methyl doxepin suggests that this compound is able to penetrate nerve sheath in vivo and to block Na+ currents in vitro as shown in Figure 4B. N-methyl doxepin generally elicits a longer duration of sciatic nerve blockade than doxepin. This may be attributed to the trapping of charged N-methyl doxepin within the cytoplasm. Other QA derivatives of tricyclic antidepressants (TCAs) with comparable amphipathic characteristics may also possess similar local anesthetic properties. It may be fruitful to design and test a series of such TCA derivatives as long-acting local anesthetics in the future.
As we have reported, injection of amitriptyline at 10 mM blocked nociceptive function of sciatic nerve for 10.9 hours, whereas subcutaneous injection of 0.25% (7.95 mM) amitriptyline blocked cutaneous nociceptive function for 18.9 hours (13,14). Because local anesthetics diffuse slowly within subcutaneous tissues (15) and because the optimum percutaneous logP value for absorption is 2.6 (16), both doxepin and N-methyl doxepin might also be suitable for subcutaneous anesthesia.
Although oral administration of doxepin causes several adverse effects, including drowsiness, dry mouth, blurred vision, and hypotension, doxepin nonetheless is less cardiotoxic than other TCAs (17–20). For topical administration, transient somnolence is the only known side effect. This is because the serum level for topical application with doxepin cream is 25 times less than that required for central nervous system modulation (21). During our animal studies, rats behaved normally after N-methyl doxepin or doxepin administration, suggesting that side effects by local TCA injections will be minimal when used for peripheral nerve block. Finally, all local anesthetics become neurotoxic at concentrations more than their clinical range. The neurotoxicity of local anesthetics seems proportional to the local anesthetic potency (22). In this study, both doxepin and N-methyl doxepin at 10 mM (or 0.63 mg per 200 µL of doxepin) blocked the sciatic nerve for an exceptionally long duration without showing any neurotoxicity. The dosage is only a fraction of a 150-mg oral dose given once to patients with depression.
In summary, N-methyl doxepin elicits a prolonged nerve blockade without being neurotoxic. Both doxepin and N-methyl doxepin display dose-dependent peripheral nerve blockade in rats and may be potentially applicable as long-acting local anesthetics.
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Acknowledgments
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This study was supported by National Institutes of Health Grant GM48090.
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Accepted for publication September 29, 2003.
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Abstract
Introduction
