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Enantioselective relief of neuropathic pain by systemic mexiletine in the rat
Enantioselective Relief of Neuropathic Pain by Systemic Mexiletine in the Rat Catherine J. Sinnott, Joseph M. Garfield, Andrew Zeitlin, Steve Teo, Mingdan Wu, JianHong Chen, Steven L. Shafer, and Gary R. Strichartz Abstract:
The many pharmacologically diverse molecules that are able to reduce neuropathic pain might do so through a common action on neuronal Na+ channels. Here we compare the potency for relieving allodynia from rat nerve root (L5-L6) ligation by mexiletine enantiomers known to stereoselectively inhibit Na+ channels. After programmed intravenous infusions of R(-)- and 5(+)-mexiletine to plasma concentrations of 0.5 to 2.0 pg/mL for 25 minutes 5 to 10 days after unilateral nerve root ligation of male Sprague-Dawley rats (Taconic Farms, Germantown, NY), allodynia was tested by the von Frey filament force necessary for hindpaw withdrawal. Allodynia in operated rats (paw withdrawal threshold [PWT] = 1 to 2 g versus 11 to 12 g in unoperated controls) was unrelieved by S(+)-mexiletine from 0.5 to 1.4 pg/mL; convulsive-like behavior appeared at 2.0 pg/mL S(+)- and R(-)mexiletine. R(-)-mexiletine at 0.5 pg/mL was ineffective, but 1.0, 1.2, and 1.4 pg/mL all raised PWT values to 7 to 8 g, corresponding to 60% recovery. Racemic mexiletine was ineffective at 1.0 pg/mL but equieffective to R(-)-mexiletine at 1.2 and 1.4 ug/mL. The findings of enantioselective pain relief are consistent with the published reports of R(-)-mexiletine’s greater potency for inhibition of Na+ channels. Keywords: Na+ channels, allodynia, local anesthetic, conduction block, abnormal impulses.
s
ystemically administered local anesthetics, antiarrhythmics and anticonvulsants have been shown to relieve various neuropathic pain syndromes in humans.‘-* In vitro studies show that all of these agents can block voltagegated sodium (Na+) channels in tonic and usedependent modes3-4 and, in certain in vivo situations, some also can selectively depress high-frequency trains of impulses in peripheral nerve.5 These observations have led to the hypothesis that the antineuropathic analgesia afforded by these several drug classes arise in common from their actions on Na+ channels6 However, these same agents also affect many other molecular targets,7 and often in the same rank order that they inhibit Na+ channels, so there is no direct evidence for this single mechanism. Intravenous Iidocaine provides relief of allodynia in a rat model involving the tight ligation of
Received December 16, 1999; Revised January 27, 2000; Accepted January 28, 2000 From the Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine Research Laboratories, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA; the Celgene Corporation, Warren, NJ; and the Anesthesiology Service, Veteran’s Administration Medical Center, Stanford University School of Medicine, Palo Alto, CA. Address reprint requests to Gary R. Strichartz, PhD, Pain Research Center, Department of Anesthesia, Brigham and Women’s Hospital, Boston, MA 02115. E-mail: [email protected] 0 2000 by the American Pain Society 1526-5900/00/0102-0009$8.00/O doi:10.1054/xb.2000.6173
128
The Journal
the L5 and L6 nerve roots.8-g Reversal of allodynia is produced both acutely during infusion and persistently for weeks after infusion to a sufficiently high plasma concentration. Mexiletine, an orally effective antiarrhythmic with Na+ channel blocking actions, also appears to be effective in the treatment of neuropathic pain in ratslo-” and humans,1,12-15 although often accompanied by poorly tolerated side effects. Importantly, mexiletine exhibits stereoisomerism and the R(-)enantiomer has a greater inhibitory potency on Na+ channels (and potentially greater cardiotoxicity16) than the S(+)-enantiomer.“-I* Because stereoisomerism is often observed for a single drug target, we designed the present experiments to test the hypothesis that the stereopotency reported for mexiletine on Na+ channels would manifest in the relief of experimental neuropathic pain.
Methods Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), initially weighing either T50165 g or 250-350 g, were used in this study. Rats weighing 150-165 g received L5-L6 nerve ligation and jugular catheters and were used to determine the behavioral effects of mexiletine on mechanical allodynia. Jugular and arterial catheters were implanted in rats weighing 250350 g to measure plasma concentrations of mex-
of Pain, Vol 1, No. 2 (Summer),
2000;
pp
128-137
ORIGINAL REPORT/Sinnott
et al
129
iletine during infusion. All rats were housed individually in plastic cages with soft bedding under a noninverted 12-hour light-dark cycle and fed rat chow without restriction. All experimental procedures, including surgical preparation and behavioral testing, were approved by the Harvard Medical Area Standing Committee on Animals.
Surgical Preparation Anesthesia
and Perioperative
Management
Animals receiving either L5-L6 nerve ligation and jugular catheters or jugular and arterial catheters were anesthetized with pentobarbital sodium using an intraperitoneal dose of 50 mg/kg. Following the onset of surgical anesthesia, animals were placed prone with care to prevent airway obstruction. Upon completion of surgery, animals were placed back in their individual cages and allowed to recover.
Surgical
Procedures
Surgical preparation entailed either L5-L6 nerve ligation and external jugular vein cannulation or external jugular vein cannulation and carotid artery cannulation. For the L5-L6 nerve ligation procedure we followed the protocol of Kim and Chung,17 with the exception that a 6-O silk suture was used for nerve ligation, as opposed to the 3-O silk suture originally described. A 2-cm paraspinal incision was made to the left of the vertebral column at the level L4-S2 to a depth of 3 cm (or at the level of the underlying transverse processes). Enough paraspinous muscle was removed to completely expose the L6 transverse process, which was then removed with microrongeurs, exposing the L5 and L6 nerve segments. Both nerve segments were isolated and tightly ligated using 1 silk suture.
External
Jugular
Vein Cannulation
Under general anesthesia with the animals supine, the left anterior neck area was shaved and a vertical paratracheal incision made over the clavicle down to fascia. The external jugular vein was identified and 2 loose 3-O silk suture ties were placed loosely around the vein. The more cephalad ligature was tied tightly and traction applied to the vein. A small incision was thenmade in the vein wall and a polyethylene catheter (PE-50), 12 cm in length, previously filled with heparinized saline (100 Units/mL; Heparin Sodium Injection, Elkins-Sinn, Inc., Cherry Hill, NJ) was inserted and advanced 2 cm toward the heart. With the catheter in place, as
verified by venous blood upon aspiration, the second tie was tightened securely and a hemostat applied to the catheter 2-3 cm beyond the incision, leaving 6-8 cm of free tubing. We then inserted the free end of the cannula through a 17-gauge needle with the hub removed. The cannula was a tight fit, wedging into the proximal end of the needle. Grasping the needle, the catheter tubing was tunneled subcutaneously to the midline in the posterior cervical area at about the level of the ears, exiting the skin at this point. The needle was then removed and the catheter cut so that approximately 4 cm protruded. The free end was then heated with a match until it melted, allowed to cool slightly and compressed with the jaws of a hemostat to seal it. The anterior neck incision was closed with wound clips and the animal allowed to recover.
Carotid
Artery
Cannulation
Under general anesthesia, the anterior midline area of the neck was shaved and a 3-cm vertical incision was made directly overlying the trachea. The right carotid sheath, lying posterolateral to the trachea was identified and the internal carotid artery dissected free. The method of cannulating and tunneling was identical to that described previously for the external jugular vein, except that the arterial cannula exited 3-4 mm caudad to the jugular cannula. Both jugular and carotid artery cannulae were flushed daily with heparinized saline.
Assessment
of Allodynia
Allodynia was correlated to a reduced paw withdrawal threshold (PWT) in response to the application of von Frey filaments. To measure rat PWTs, animals were placed on a wire mesh platform (0.5-cm mesh) and covered by a transparent box to limit straying. Von Frey filaments (Stoelting, Wood Dale, IL) were applied orthogonally to the midplantar region of the hindpaw with enough force to cause buckling of the filament. The up-down method of Dixon,18 as modified by Chaplan et al,lg was used to determine the filament value in g at which foot withdrawal would occur 50% of the time, and applied to quantitate drug-induced analgesia.8-g
Mexiletine
Administration
Confirmation
Arterial and venous catheters were implanted in a group of 12 rats (group 1; see Table 1) to correlate actual plasma concentrations of mexiletine with the values predicted by the Stanpump infusion program.8 Pump parameters for infusing R(-) and S(+)-mexiletine were set
Acrlons
130 --•-0-
1.0
R-(-)-mexiletine S-(+)-mexiletine
of Mextletce
Enanriome?
(k4) (n=4)
0.9 1 3
0.8
6 2
0.7
c z iii A 3 z i!i f a
1 0.6 0.5 -
.
0.4 0.3 0.2 0.1 -1
v.v-,
~~ 0
lb
!i
1; Time
Figure 1. Mean enantiomers.
Table
plasma
mexiletine
concentration
+ S.E.M.
DRUG RECEIVED AND SCHEDULE DAY
1 (n = 12)
Mex
2
(n = 8)
3
(n = 8)
4 (n = 8) 5
(n = 8)
6 (n = 8) 7(n= 11)
Administration Verification (unoperated rats). R (n = 4) and S (n = 4) Q 0.25 and 1 .O (d7) pg/mL
(d5),
0.50
(d6),
S Q 1 .O (d5), 1.2 (d6), 1.4 (d7), and R Q 1 .O (d8) @mL R Q 1 .O (d5), 1.2 (d6), 1.4 (d7). and 2.0 (d9) @mL Racemate Q 1 .O (d6), 1.2 (d7), and 1.4 (dl0) pgImL i.p. injections of R and S @ 30 mg/kg i.p. injections of R and 5 Q 30 mg/kg
Abbreviations: Mex, mexiletine; i.p., intraperitoneal. *Schedule day is postoperative
R, R(-)-mexiletine; (surgery
(d7) (d6)
S, S(+)-mexiletine;
on do), listed in parentheses.
according to published pharmacokinetic values for mexiletine enantiomers20; for R(-)-mexiletine the value was VI = 5.95; KIO = 0.027; K12 = 0.0054; and K21 = 0.0106. For S(+)-mexiletine the value was VI = 5.57; KIO = 0.022; K12 = 0.0037; and K21 = 0.0114. The racemic mixture was infused using pump settings that were the average of values for the S(+) and R(-) enantiomers. To verify the infusion procedure, arterial blood samples were drawn from these rats at 5, IO, 20, and 30 minutes during an infusion to a target level of 0.5 pg mexiletine per mL plasma (eg, see Fig 1). Four rats received the R(-)-enantiomer, 4 received the S(+)-enantiomer, and the remaining 4 rats received the racemate. At each time point, 30 FL of whole blood was drawn
2;
;0
3k
(Minutes)
in rats during
1. Schedulefor Mexiletine Dosing
GROUP
2b
a 30-minute
intravenous
infusion
of the
S(+) and
the
R(-)
from each rat and added to 10 pL of heparinized saline to prevent clotting. Samples were centrifuged for 20 minutes at 1065 g and the plasma aliquoted and frozen at -20°C. Plasma samples were extracted with ethyl acetate and assayed for mexiletine by liquid chromatography (LC)-tandem mass spectrometry (see later in this article). Two additional rats were used in this portion of the study to determine the accuracy of this assay. Arterial catheters were implanted in these 2 rats, and 3 30-pL samples of whole blood were drawn from each rat; to each sample was added 10 FL of heparinized saline. Plasma was removed from each sample according to the same protocol described previously, and each plasma sample was then externally doped with either R(-)- or S(+)-mexiletine to achieve a total content of 0.5 pg/mL.
Mexiletine Plasma Determination R(-), S(+) and racemic mexiletine plasma samples were analyzed separately. All chemicals used were reagent grade. Methanol and ethyl acetate were purchased from Fisher Scientific &Voburn, MA) and the formic acid, ammonium hydroxide, and phenacetin were from Sigma-Aldrich (St. Louis, MO). Frozen plasma samples were altowed to come to room temperature. One hundred mL plasma and 10 mL phenacetin internal standard were mixed and vortexed in centrifuge tubes. An assay blank was prepared using 100 mL blank plasma and 10 mL methanol. Calibration curve samples consisted of 100 mL blank plasma and 10 mL spiking solutions consisting of phenacetin at
ORIGINAL REPORThinnott
et al
131
1.0 ug/mL and R(-), S(+) or racemic mexiletine at 0.5, 2.0, 5.0, 15, and 25 ug/mL. This gave calibration curve concentrations 0.05, 0.2, 0.5, 1.5, and 2.5 ug/mL. Low, medium, and high concentration quality control samples were prepared by mixing 100 mL blank plasma and 10 mL spiking solutions consisting of 1 .O g/mL phenacetin and 1.0, 5.0, and 20.0 g/mL of R(-), S(+) or racemic mexiletine. All of the prepared samples were mixed with 1 mL of 2% aqueous ammonia hydroxide solution to increase pH for better extraction efficiency. Three mL of ethyl acetate was added and the mixture vortexed and then shaken for 5 minutes. The tubes were centrifuged at 3000 rpm for 5 minutes and 2 mL of the supernatant was aliquoted and blown dry with a gentle stream of nitrogen. The residue was reconstituted with 400 mL high-performance liquid chromatography (HPLC) mobile phase (see later in this article), vortexed, and shaken. Tubes were again spun and aliquots of the supernatant injected into the LC-MS/MS. The analytical system consisted of a Waters 2690 HPLC (Waters Corporation, Milford, MA) connected to a Micromass Quattro LC tandem mass spectrometer (Micromass, Beverly, MA). The LC system was equipped with a C8 Phenomenex column (50 x 2 mm; Phenomenex, Torrance, CA) kept at a temperature of 30°C. The mobile phase consisted of a 50:50 mixture of methanol and 0.05% formic acid at an isocratic flow rate of 0.2 mUmin. Mass detection was performed in the positive ion electrospray mode using multiple reaction monitoring channels of 179.96 > 57.96 and 179.62 > 109.83 for mexiletine and phenacetin, respectively. For both compounds, the dwell time was 0.20 seconds, whereas the collision energy and cone voltage was 20 V.
Concentration-Dependent Mexiletine Enantiomers
Effects of and Racemate
Groups 2, 3, 4, and 5, each with 8 rats, were used in this component of the study (see Table 1). Preoperative PWTs for all 4 groups of rats were measured 1 day before surgery. The operative procedure consisted of left L5-L6 spinal nerve root ligation along with external jugular vein cannulation (as previously mentioned in this article). The animals in group 2 received 3 30-minutes infusions of R(-) or S(+) mexiletine on days 5, 6, and 7 after surgery, targeted at 0.25, 0.5, and 1 .O ug/mL, respectively (4 of the 8 animals in group 2 received R(-)-mexiletine and the other 4 received S(+)-mexiletine). Our purpose was to explore the threshold dose range for the 2 drugs and so better resolve the concentration-dependence in the active range. Therefore, for these low-concentration studies, the scoring observer was not blinded to the drug being infused.
Experiments with groups 3 and 4 were designed to examine enantioselective actions at the same plasma levels administered at corresponding times after surgery. The investigator measuring PWTs (for these and all subsequent studies, except for the racemate) was blinded as to which enantiomer was being infused. The animals in group 3 received infusions of S(+)-mexiletine on days 5, 6, and 7 after surgery, targeted at 1.0, 1.2, and 1.4 ug/mL, respectively. On day 8 after surgery, these animals received a 30-minute infusion of R(-)-mexiletine targeted at 1 .O pg/mL. The animals in group 4 received infusions of R(-)mexiletine on days 5, 6, and 7 after surgery, targeted at 1 .O, 1.2, and 1.4 pg/mL, respectively. On day 9 after surgery, animals in group 4 received an infusion of R(-)-mexiletine targeted at 2.0 ug/mL. Group 5 animals were used to evaluate the effect of infusing racemic mexiletine on PWTs. These animals received infusions of racemic mexiletine on days 6, 7, and 10 after surgery, targeted at 1 .O, 1.2, and 1.4 ug/mL, respectively.
lntraperitoneal Enantiomers
Delivery
of Mexiletine
Groups 6 and 7 were used for this portion of the study. Experiments with these 2 groups were performed in a blinded, crossover manner. On day 7, the first 4 animals in group f received an intraperitoneal (i.p.) injection of S(+)-mexiletine (30 mg/kg). On day 8, the remaining 4 animals in group 6 received an i.p. injection of R(-)-mexiletine (30 mg/kg). On day 9, the first 4 rats received a second i.p. injection, this time of R(-)-mexiletine (30 mg/kg), and on day 10 the other 4 animals in this group received an i.p injection of S(+)-mexiletine. Animals in group 7 received the same dosing regimen, except that the first dose was given on day 6 after surgery rather than day 7. After each injection, PWTs were measured at 5, 10, 20, 30, 40, 50, 60, 80, 100, and 120 minutes.
Data Analysis/Statistics Data are expressed as the mean percent maximum possible effect (%MPE) * the standard error (S.E.) of the drug on PWTs. This presentation normalizes the span between preoperative (maximum) and postsurgical, predrug (minimum) PWTs; thus, it provides a relative scale, transposable among groups, for assessing druginduced analgesia. Absolute values of PWT (in g, mean * S.E.) also are given for all conditions. Percent MPE caused by mexiletine infusion was determined by the following equation: PW
%Mpr =-----
(g) during
Preoperative
infusion
- Postoperative
___--.---~..~~-
PWT (g) - Postoperative
PW
(g)
-- -_..-~ * 100 PWl
(g)
Actions of Mextletrne
132 --- 0-8-
loo-
Endntmwi
R-(-)-mexlletine S-(+)-mexiletine Racemic mexiletine
goc
=-
80
-10
1 0.9
I 1.0
I 1.1
Target Figure during
2. Mean intravenous
percent maximum infusions targeted
Plasma
I
I
1.2
1.3
Concentration
possible effect 2 S.E.M. of R-(-), S-(+) at 1000, 1200, and 1400 ng/mL.
The Friedman test was used to determine overall significance. Once overall significance was determined for a response set, the Wilcoxon test was used after the fact to determine which postinfusion PWT values differed significantly from the preinfusion values. All analyses were performed using SPSS for Windows, (Version 6.1.3, SPSS Inc., Chicago, IL). Results were considered significant at P < 0.05.
Results Mexifetine Administration
Verification
Mexiletine extraction efficiencies were greater than 90%, and calibration curves were linear (R2 > 0.98). The low-, medium-, and high-concentration quality control samples for R(-)-mexiletine and S(+)-mexiletine were 97, 103, and 131 and 107, 101, and 106% of expected concentrations, respectively. Measured plasma concentrations of mexiletine enantiomers sampled during programmed infusions were within 20% of the target level of 0.5 vg/mL, and, equally important, there was no significant difference between the achieved levels of R(-) and S(+) enantiomers. Fig 1 shows the measured plasma concentrations of R(-)-mexiletine and S(+)-mexiletine at 4 time points during an infusion targeted to 0.5 pg/mL. The levels at 5 minutes were slightly but insignificantty greater than those at later times, and the levels at 10 to 30 minutes were within 1 S.E. of the target concentration. The mean concentration (-c S.E.) of the plasma samples that were
I 1.5
I 1.4
(pg/mL) and
racemic
mexiletine
on paw
withdrawal
thresholds
externally doped to a final content of 500 ng/mL was 405.7 ng * 11 .I ng S(+)-mexiletine /mL and 400.3 ng * 9.0 ng R(-)-mexiletine/mL (each n = 3), showing that 80% of each enantiomer was extractable from plasma by the method used here, and without any stereoisomeric bias. Thus, the infusion pump parameters used here adequately account for the plasma protein binding of both mexiletine enantiomers. In a previous study, using the Stanpump infusion program for lidocaine, a nearly linear correlation from 0.4 to IO g/mL was shown between target and actual plasma lidocaine levels.g The agreement at the single mexiletine level targeted here for S-(+) and R-(-) enantiomers confirms the reliability of this method for these agents.
Concentration-Dependent Mexifetine Enantlomers
Effects of
At plasma levels up to 0.5 pg/mL neither enantiomer relieved mechanical allodynia. An enantioselective action became evident at higher concentrations and with a single crossover procedure in the group receiving S(+)-mexiletine (group 3; n = 8, the mean preoperative PWT of which was 10.4 g f 1.0 g and mean postoperative PWT was 1.5 g * 1.8 g). The %MPE for the elevation of PWT by S(+)-mexiletine was zero at plasma concentrations of 1.0 and 1.4 pg/mL, and, oddly but insignificantly different, about 25% (P = .32), at 1.2 pg/mL (Fig 2). These same rats were then infused with R(-)-mexiletine targeted at 1.0 vg/mL on day 8 after surgery, during which PWTs
ORIGINAL REPORT/Sinnott
Y ;
et al
133
1101
8
loo-
a‘E
90 -
-r a a g
8070-
6 t Eal 3$
60-
i oi: z
30 -
g
lo-
50
*-
40 -
20 -
5
o-10
1 0.9
I 1 .o
I 1.1 Target
Figure on paw
3. Percent withdrawal
maximum thresholds
possible effect of individual
I 1.2 Plasma
I 1.3
Concentration
of intravenous infusions animals in group 4.
I 1.4
I 1.5
(pg/mL)
of R-(-)-mexiletine
-O-0-8-
100
targeted
at 1000,
1200,
and
1400
ng/mL
R-(-)-mex. (1.4 I*g/mL) S-(+)-mex. (1.4 pg/mL) Racemic mex. (1.4 PglmL)
90 ‘1 8070 -
5040 -
1 r
/d
60 -
/
_____c-
30 20-
f
//
10-
J/ e-o-o
O-10
f
f
1 -5
.f
I 0
0
I 10
I 5 Time
Figure 4. Mean percent maximum possible effect sions of R-(-), S-(+) and racemic mexiletine targeted
* S.E.M. at 1400
During on paw ng/mL.
were elevated to a mean value of 6.7 g * 0.8 g (%MPE = 50.0% + 5.8%) suggesting an enantioselective action of the R(-) enantiomer. Infusion of S(+)-mexiletine targeted at 2.0 pg/mL produced convulsions in the first 2 rats in this group within 20 seconds after commencing the infusion, obviating any antiallodynic data for that dose. Concentration-dependent relief of allodynia by R(-) mexiletine was assessed in a separate group of animals (group 4; n = 8) with the doses corresponding to S(+) mexiletine’s dosing regimen given on the corresponding postoperative days
0
I 15
I
I
I
20
25
30
Infusion
(Minutes)
withdrawal
thresholds
during
1 35
a 30-minute
intravenous
infu-
(Fig 2). (The mean preoperative PWT value for this group was 12.0 g + 0.0 g and the mean postoperative value was 1.6 g + 0.3 g). Infusions of the R(-)-enantiomer at 1.0 g/mL resulted in an elevation of PWT to 62.4 f 13.9 %MPE (8.1 g -c 1.4 g), whereas infusions targeted at 1.2 pg/mL and 1.4 ug/mL both elevated PWTs to 53.5 + 13.6 %MPE (7.2 g f 1.4 g), insignificantly different from that at 1 .O pg/mL (P = .16). Each of the individual animals in this group that responded to treatment with R(-) mexiletine (7 of 8) achieved approximately the same degree
Actions
134 -o-o-
loo-
ot Mexlietlne
R-(-)-mex. S-(+)-mex.
(30 mg/kg) (30 mg/kg)
I 100
I 120
Errantlomrs
go80 70 60-
-10
1 -10
I 0
I 10
I 20
I 30
Time
Fiaure 5. an>
S-(+)
maximum Mean percent mexiletine at 30 mg/kg.
possible
effect
I 50
I 40
f S.E.M.
After
I 60 Injection
on paw
of alleviation at all 3 concentrations (Fig 3). Three to 4 were completely returned to preoperative levels, and 3 to 4 had intermediate degrees of relief; 1 rat in this group did not achieve relief at any concentration. Infusions of R(-)-mexiletine targeted at 2.0 ug/mL produced convulsions in 2 of these animals within 1 minute; no other rats were infused to this level. When infused with racemic mexiletine at target concentrations identical to those of the single enantiomers, the rats in group 5 showed a significant concentration-dependent relief of allodynia at 1.2 and 1.4 ug/mL (P = .02; Fig 2). (Preoperative PWTs were 11 .O g + 0.5 g; postoperative values were 1.3 g f 0.1 g). After 30 minutes of infusion targeted to 1.0 pg/mL, racemic mexiletine gave no significant relief (%MPE = 4.0 it: 1.9; P = .08). The elevation afforded by racemic mexiletine at 1.2 and 1.4 pg/mL was midway between those of the 2 enantiomers, as one would expect if simple receptor occupancy accounted for the therapeutic action without any interenantiomer interactions. When the antiallodynic activity of mexiletine was examined over the 30-minute course of drug infusion targeted to 1.4 ug/mL (see Fig 4), the effects of the single enantiomers are constant; R(-)-mexiletine produces exactly the same PWT elevation at each time (P = .82) and S(+)-mexiletine produced no elevation at any time. However, the effect of racemic mexiletine developed over the early course of the infusion; relief at 5 minutes was less than that at the later time points (P = .03).
I 70
I 80
I 90
I 110
I 130
(Minutes)
withdrawal
thresholds
after
intraperitoneal
injections
of R-(-)
Any antiallodynic effect of intravenous (i.v.) mexiletine was temporary. Allodynia had returned to the postoperative level when the animals were examined 24 hours after the infusion, in the period just preceding the next infusion.
lntraperitoneal Enantiomers
Dosing With Mexiletine
Enantioselective actions of mexiletine were not evident after intraperitoneal injections. Fig 5 shows the results of experiments with group 6 (n = 8) and 7 (n = 11) animals. lntraperitoneal injections (30 mg/kg) of both the R(-)- and the S-(+)enantiomer produced a significant 20-minute elevation of PWT values compared with preinjection values (P = .Ol at 5 and 10 minutes; P = .03 [R(-)-mexiletine] and P = .04 [S(+)-mexiletine] at 20 minutes); there was no significant difference between the degree of relief achieved with each enantiomer at any time point after injection (eg, P = .30 [5 minutes]). The order of administration of the enantiomers had no effect; relief by R(-) or S(+) enantiomers was not different in those rats where one was injected first compared with those where it was the second drug injected. I.p. injections of either enantiomer at 50 mg/kg in these animals resulted in convulsions within 3 minutes.
Discussion These experiments show a clear stereosetective action of intravenous mexiletine for temporary relief of experimental mechanical allodynia.
ORIGINAL REPORT/Sinnott
et al
When intravenous concentrations were controlled at predetermined levels by programmed infusions, the pathologically low mechanical threshold to elicit brisk hindpaw withdrawal (1 to 2 g von Frey filament) was raised back toward the normal, preoperative value (10 to 12 g) by the R(-)-mexiletine enantiomer, but not by the S(+) enantiomer. Relief of allodynia by R(-) mexiletine had several characteristics of the previously reported relief by lidocaine, including a minimal threshold concentration for detecting any relief and a ceiling concentration, above which no further relief occurred.8-g Indeed, for R(-) mexiletine, these threshold changes occurred at about the same plasma concentration as for lidocaine, lpg/mL. At 1.4 ug/mL R(-) mexiletine, withdrawal thresholds were no higher than at 1 .O ug/mL, the “ceiling” effect. As with lidocaine, the effective plasma concentration (1 to 1.5 ug/mL), equivalent to 5.6 to 8.4 umol/L R(-) mexiletine, was well below the IC,, values reported for the inhibition of resting or phasically stimulated Na+ channels, 54 or 23 umol/L, respectively (see later in this article). Withdrawal thresholds were not elevated by infusions lower than at 1.0 ug/mL. Doubling the threshold concentration, to 2.0 ug/mL, produced convulsive-like behavior early in the infusion, indicating the toxic limit of systemic mexiletine. Interestingly, signs of central nervous system (CNS) toxicity occurred at about the same early time during infusion with both mexiletine enantiomers at 2 ug/mL, despite the absence of therapeutic activity from S(+)mexiletine at lower concentrations, suggesting that such toxicity might be caused by different mechanisms than those that relieve allodynia. One distinct difference between lidocaine’s and R(-) mexiletine’s antiallodynic actions was the persistence of effect. For days to weeks following 30-minute lidocaine infusions to 1 to 2 ug/mL, allodynic threshold remained elevated.8-g In contrast, relief by R(-)mexiletine never persisted, even though it reached the same mean level as that by lidocaine during infusion (about 60 %MPE). When tested 24 and 48 hours after infusions, rats treated with R(-)-mexiletine had the same very low postoperative withdrawal thresholds. We interpret these results as showing that separate mechanisms account for the transient relief during infusion and that which endures after the drug is long gone. Both lidoCaine and R(-)-mexiletine can effect the former, but only lidocaine yields the latter response. The absolute stereoselectivity of this allodynic action could not be determined in these experiments because of the limit on dosing caused by CNS toxicity. We can only conclude that the R(-)
135
enantiomer is at least 1.4 times more potent than the S(+) enantiomer, where potency refers to a threshold concentration for an all-or-none action and not to a drug level that produces half of a concentration-graded effect. This stereoselectivity order is consistent with IC,, values reported for inhibition of Na+currents (INa) in frog skeletal muscle, 21-22 Na+ currents carried through the m, type of Na+ channel, the local anesthetic pharmacology23 of which is very similar to that of peripheral nerve,24-26 and of central nervous system channels.27-28 DeLuca et all2 found that tonic inhibition of I,, by mexiletine in skeletal muscle was about 2-fold stereoselective for R- over S- (IC,, values: R(-) = 54 umol/L; S(+) = 114 umol/L; R/S (-I+) = 83 umol/L) mexiletine, but that for use-dependent inhibition, measured at a higher frequency of brief test depolarizations (10 Hz), stereoselectivity effectively disappeared (IC,, values: R(-) = 23 pmol/L; S(+) = 27 umol/L; R/S (-/+) = 29 umol/L.21 Inhibition of rat brain Na+ channels by R-mexiletine is also use-dependent, with lower tonic inhibitory potency (IC,, = 305 umol/L) and usedependent potency (IC,, = 148 pmol/L, at 5 Hz) than reported for skeletal muscle; no data on the S-enantiomer were reported.2g Because use-dependent inhibition is caused by additional binding of the drug to channels that are in the open and inactivated states30e3* and their corresponding slower drug dissociation, the disappearance of stereoselectivity at high-frequency depolarization implies that the open or inactivated conformations of the Na+ channels have higher affinities for S(+)- than for R(-)-mexiletine. The additional S(+)-enantiomer-selective binding that occurs during 10 Hz, then sums with the R(-)-enantioselective tonic binding to give equal inhibition from the 2 isomers during repetitive stimulation. Therefore, for the enantioselective relief of allodynia that we observe in vivo to be caused by Na+ channel blockade, it should arise from the R(-)-selective inhibition of infrequently stimulated neurons with Na+ channels predominantly in the resting state, rather than the enantioneutral or S(+)-selective inhibition of open and inactivated channels. Although this conclusion relies strongly on voltage-clamp data from frog skeletal muscle that must be confirmed in mammalian nerve, it directly contradicts the widespread argument that therapeutic relief of neuropathic pain by intravenous local anesthetics and antiarrhythmics (and, in addition, a plethora of amphipathic bases belonging to other drug classes) results from the selective suppression of high-frequency, injury-induced impulses in hyperexcitable nerve fibers.5,33 The apparent equieffectiveness of intraperitoneally injected boluses of S(+)- and R(-) enan-
AC~IOPS of Mexiietine
136
tna~tmr~r~~
tiomers in the transient relief of allodynia in this model is surprising. This was a consistent result in 2 separate groups of animals in which each individual rat received both enantiomers in sequence. The discrepancy between these equivalent effects and the pronounced stereoselectivity during infusion, emphasized by the crossover results in the infusion group treated first with S(+) mexiletine (ineffective at 1.4 pg/mL) then R(-) mexiletine (giving relief at 1.0 ug/mL), might be explained by several possibilities. First, bolus intraperitoneal injections might result in higher concentrations of systemic S(+)- than R(-)-mexiletine, because of their different pharmacokinetic parameters, a difference that is accounted for during the programmed infusion. Clearly, the
anatomic distribution of S(+)-mexiletine differs when delivered by the 2 routes, because toxicity precedes (and obviates) therapy during i.v. infusion but is not apparent during bolus injection (at 30 mg/kg). Second, S(+) mexiletine might undergo an enzyme-catalyzed chiral inversion that changes it to the R(-) conformation, although there is no direct evidence that this occurs. Alternatively, both enantiomers might be transformed to a common metabolite leading to apparent equipotency. However, such a reaction also would occur when S(+) mexiletine is infused intravenously, yet no antiallodynic activity is detected in such a case. At this time, the weak transient equipotency of i.p. administered mexiletine enantiomers remains an anomaly.
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20. lgwemezie L, Beatch GN, Walker MJ, McErlane Tissue distribution of mexiletine enantiomers in Xenobiotica 21:1153-1158,1991 21. De Luca A, Natuzzi F, Lentini V, Conte Camerino D:Stereoselective enantiomers on sodium currents teristics of adult skeletal Schmiedebergs Arch Pharmacol
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The many pharmacologically diverse molecules that are able to reduce neuropathic pain might do so through a common action on neuronal Na+ channels. Here we compare the potency for relieving allodynia from rat nerve root (L5-L6) ligation by mexiletine enantiomers known to stereoselectively inhibit Na+ channels. After programmed intravenous infusions of R(-)- and 5(+)-mexiletine to plasma concentrations of 0.5 to 2.0 pg/mL for 25 minutes 5 to 10 days after unilateral nerve root ligation of male Sprague-Dawley rats (Taconic Farms, Germantown, NY), allodynia was tested by the von Frey filament force necessary for hindpaw withdrawal. Allodynia in operated rats (paw withdrawal threshold [PWT] = 1 to 2 g versus 11 to 12 g in unoperated controls) was unrelieved by S(+)-mexiletine from 0.5 to 1.4 pg/mL; convulsive-like behavior appeared at 2.0 pg/mL S(+)- and R(-)mexiletine. R(-)-mexiletine at 0.5 pg/mL was ineffective, but 1.0, 1.2, and 1.4 pg/mL all raised PWT values to 7 to 8 g, corresponding to 60% recovery. Racemic mexiletine was ineffective at 1.0 pg/mL but equieffective to R(-)-mexiletine at 1.2 and 1.4 ug/mL. The findings of enantioselective pain relief are consistent with the published reports of R(-)-mexiletine’s greater potency for inhibition of Na+ channels. Keywords: Na+ channels, allodynia, local anesthetic, conduction block, abnormal impulses.
s
ystemically administered local anesthetics, antiarrhythmics and anticonvulsants have been shown to relieve various neuropathic pain syndromes in humans.‘-* In vitro studies show that all of these agents can block voltagegated sodium (Na+) channels in tonic and usedependent modes3-4 and, in certain in vivo situations, some also can selectively depress high-frequency trains of impulses in peripheral nerve.5 These observations have led to the hypothesis that the antineuropathic analgesia afforded by these several drug classes arise in common from their actions on Na+ channels6 However, these same agents also affect many other molecular targets,7 and often in the same rank order that they inhibit Na+ channels, so there is no direct evidence for this single mechanism. Intravenous Iidocaine provides relief of allodynia in a rat model involving the tight ligation of
Received December 16, 1999; Revised January 27, 2000; Accepted January 28, 2000 From the Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine Research Laboratories, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA; the Celgene Corporation, Warren, NJ; and the Anesthesiology Service, Veteran’s Administration Medical Center, Stanford University School of Medicine, Palo Alto, CA. Address reprint requests to Gary R. Strichartz, PhD, Pain Research Center, Department of Anesthesia, Brigham and Women’s Hospital, Boston, MA 02115. E-mail: [email protected] 0 2000 by the American Pain Society 1526-5900/00/0102-0009$8.00/O doi:10.1054/xb.2000.6173
128
The Journal
the L5 and L6 nerve roots.8-g Reversal of allodynia is produced both acutely during infusion and persistently for weeks after infusion to a sufficiently high plasma concentration. Mexiletine, an orally effective antiarrhythmic with Na+ channel blocking actions, also appears to be effective in the treatment of neuropathic pain in ratslo-” and humans,1,12-15 although often accompanied by poorly tolerated side effects. Importantly, mexiletine exhibits stereoisomerism and the R(-)enantiomer has a greater inhibitory potency on Na+ channels (and potentially greater cardiotoxicity16) than the S(+)-enantiomer.“-I* Because stereoisomerism is often observed for a single drug target, we designed the present experiments to test the hypothesis that the stereopotency reported for mexiletine on Na+ channels would manifest in the relief of experimental neuropathic pain.
Methods Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), initially weighing either T50165 g or 250-350 g, were used in this study. Rats weighing 150-165 g received L5-L6 nerve ligation and jugular catheters and were used to determine the behavioral effects of mexiletine on mechanical allodynia. Jugular and arterial catheters were implanted in rats weighing 250350 g to measure plasma concentrations of mex-
of Pain, Vol 1, No. 2 (Summer),
2000;
pp
128-137
ORIGINAL REPORT/Sinnott
et al
129
iletine during infusion. All rats were housed individually in plastic cages with soft bedding under a noninverted 12-hour light-dark cycle and fed rat chow without restriction. All experimental procedures, including surgical preparation and behavioral testing, were approved by the Harvard Medical Area Standing Committee on Animals.
Surgical Preparation Anesthesia
and Perioperative
Management
Animals receiving either L5-L6 nerve ligation and jugular catheters or jugular and arterial catheters were anesthetized with pentobarbital sodium using an intraperitoneal dose of 50 mg/kg. Following the onset of surgical anesthesia, animals were placed prone with care to prevent airway obstruction. Upon completion of surgery, animals were placed back in their individual cages and allowed to recover.
Surgical
Procedures
Surgical preparation entailed either L5-L6 nerve ligation and external jugular vein cannulation or external jugular vein cannulation and carotid artery cannulation. For the L5-L6 nerve ligation procedure we followed the protocol of Kim and Chung,17 with the exception that a 6-O silk suture was used for nerve ligation, as opposed to the 3-O silk suture originally described. A 2-cm paraspinal incision was made to the left of the vertebral column at the level L4-S2 to a depth of 3 cm (or at the level of the underlying transverse processes). Enough paraspinous muscle was removed to completely expose the L6 transverse process, which was then removed with microrongeurs, exposing the L5 and L6 nerve segments. Both nerve segments were isolated and tightly ligated using 1 silk suture.
External
Jugular
Vein Cannulation
Under general anesthesia with the animals supine, the left anterior neck area was shaved and a vertical paratracheal incision made over the clavicle down to fascia. The external jugular vein was identified and 2 loose 3-O silk suture ties were placed loosely around the vein. The more cephalad ligature was tied tightly and traction applied to the vein. A small incision was thenmade in the vein wall and a polyethylene catheter (PE-50), 12 cm in length, previously filled with heparinized saline (100 Units/mL; Heparin Sodium Injection, Elkins-Sinn, Inc., Cherry Hill, NJ) was inserted and advanced 2 cm toward the heart. With the catheter in place, as
verified by venous blood upon aspiration, the second tie was tightened securely and a hemostat applied to the catheter 2-3 cm beyond the incision, leaving 6-8 cm of free tubing. We then inserted the free end of the cannula through a 17-gauge needle with the hub removed. The cannula was a tight fit, wedging into the proximal end of the needle. Grasping the needle, the catheter tubing was tunneled subcutaneously to the midline in the posterior cervical area at about the level of the ears, exiting the skin at this point. The needle was then removed and the catheter cut so that approximately 4 cm protruded. The free end was then heated with a match until it melted, allowed to cool slightly and compressed with the jaws of a hemostat to seal it. The anterior neck incision was closed with wound clips and the animal allowed to recover.
Carotid
Artery
Cannulation
Under general anesthesia, the anterior midline area of the neck was shaved and a 3-cm vertical incision was made directly overlying the trachea. The right carotid sheath, lying posterolateral to the trachea was identified and the internal carotid artery dissected free. The method of cannulating and tunneling was identical to that described previously for the external jugular vein, except that the arterial cannula exited 3-4 mm caudad to the jugular cannula. Both jugular and carotid artery cannulae were flushed daily with heparinized saline.
Assessment
of Allodynia
Allodynia was correlated to a reduced paw withdrawal threshold (PWT) in response to the application of von Frey filaments. To measure rat PWTs, animals were placed on a wire mesh platform (0.5-cm mesh) and covered by a transparent box to limit straying. Von Frey filaments (Stoelting, Wood Dale, IL) were applied orthogonally to the midplantar region of the hindpaw with enough force to cause buckling of the filament. The up-down method of Dixon,18 as modified by Chaplan et al,lg was used to determine the filament value in g at which foot withdrawal would occur 50% of the time, and applied to quantitate drug-induced analgesia.8-g
Mexiletine
Administration
Confirmation
Arterial and venous catheters were implanted in a group of 12 rats (group 1; see Table 1) to correlate actual plasma concentrations of mexiletine with the values predicted by the Stanpump infusion program.8 Pump parameters for infusing R(-) and S(+)-mexiletine were set
Acrlons
130 --•-0-
1.0
R-(-)-mexiletine S-(+)-mexiletine
of Mextletce
Enanriome?
(k4) (n=4)
0.9 1 3
0.8
6 2
0.7
c z iii A 3 z i!i f a
1 0.6 0.5 -
.
0.4 0.3 0.2 0.1 -1
v.v-,
~~ 0
lb
!i
1; Time
Figure 1. Mean enantiomers.
Table
plasma
mexiletine
concentration
+ S.E.M.
DRUG RECEIVED AND SCHEDULE DAY
1 (n = 12)
Mex
2
(n = 8)
3
(n = 8)
4 (n = 8) 5
(n = 8)
6 (n = 8) 7(n= 11)
Administration Verification (unoperated rats). R (n = 4) and S (n = 4) Q 0.25 and 1 .O (d7) pg/mL
(d5),
0.50
(d6),
S Q 1 .O (d5), 1.2 (d6), 1.4 (d7), and R Q 1 .O (d8) @mL R Q 1 .O (d5), 1.2 (d6), 1.4 (d7). and 2.0 (d9) @mL Racemate Q 1 .O (d6), 1.2 (d7), and 1.4 (dl0) pgImL i.p. injections of R and S @ 30 mg/kg i.p. injections of R and 5 Q 30 mg/kg
Abbreviations: Mex, mexiletine; i.p., intraperitoneal. *Schedule day is postoperative
R, R(-)-mexiletine; (surgery
(d7) (d6)
S, S(+)-mexiletine;
on do), listed in parentheses.
according to published pharmacokinetic values for mexiletine enantiomers20; for R(-)-mexiletine the value was VI = 5.95; KIO = 0.027; K12 = 0.0054; and K21 = 0.0106. For S(+)-mexiletine the value was VI = 5.57; KIO = 0.022; K12 = 0.0037; and K21 = 0.0114. The racemic mixture was infused using pump settings that were the average of values for the S(+) and R(-) enantiomers. To verify the infusion procedure, arterial blood samples were drawn from these rats at 5, IO, 20, and 30 minutes during an infusion to a target level of 0.5 pg mexiletine per mL plasma (eg, see Fig 1). Four rats received the R(-)-enantiomer, 4 received the S(+)-enantiomer, and the remaining 4 rats received the racemate. At each time point, 30 FL of whole blood was drawn
2;
;0
3k
(Minutes)
in rats during
1. Schedulefor Mexiletine Dosing
GROUP
2b
a 30-minute
intravenous
infusion
of the
S(+) and
the
R(-)
from each rat and added to 10 pL of heparinized saline to prevent clotting. Samples were centrifuged for 20 minutes at 1065 g and the plasma aliquoted and frozen at -20°C. Plasma samples were extracted with ethyl acetate and assayed for mexiletine by liquid chromatography (LC)-tandem mass spectrometry (see later in this article). Two additional rats were used in this portion of the study to determine the accuracy of this assay. Arterial catheters were implanted in these 2 rats, and 3 30-pL samples of whole blood were drawn from each rat; to each sample was added 10 FL of heparinized saline. Plasma was removed from each sample according to the same protocol described previously, and each plasma sample was then externally doped with either R(-)- or S(+)-mexiletine to achieve a total content of 0.5 pg/mL.
Mexiletine Plasma Determination R(-), S(+) and racemic mexiletine plasma samples were analyzed separately. All chemicals used were reagent grade. Methanol and ethyl acetate were purchased from Fisher Scientific &Voburn, MA) and the formic acid, ammonium hydroxide, and phenacetin were from Sigma-Aldrich (St. Louis, MO). Frozen plasma samples were altowed to come to room temperature. One hundred mL plasma and 10 mL phenacetin internal standard were mixed and vortexed in centrifuge tubes. An assay blank was prepared using 100 mL blank plasma and 10 mL methanol. Calibration curve samples consisted of 100 mL blank plasma and 10 mL spiking solutions consisting of phenacetin at
ORIGINAL REPORThinnott
et al
131
1.0 ug/mL and R(-), S(+) or racemic mexiletine at 0.5, 2.0, 5.0, 15, and 25 ug/mL. This gave calibration curve concentrations 0.05, 0.2, 0.5, 1.5, and 2.5 ug/mL. Low, medium, and high concentration quality control samples were prepared by mixing 100 mL blank plasma and 10 mL spiking solutions consisting of 1 .O g/mL phenacetin and 1.0, 5.0, and 20.0 g/mL of R(-), S(+) or racemic mexiletine. All of the prepared samples were mixed with 1 mL of 2% aqueous ammonia hydroxide solution to increase pH for better extraction efficiency. Three mL of ethyl acetate was added and the mixture vortexed and then shaken for 5 minutes. The tubes were centrifuged at 3000 rpm for 5 minutes and 2 mL of the supernatant was aliquoted and blown dry with a gentle stream of nitrogen. The residue was reconstituted with 400 mL high-performance liquid chromatography (HPLC) mobile phase (see later in this article), vortexed, and shaken. Tubes were again spun and aliquots of the supernatant injected into the LC-MS/MS. The analytical system consisted of a Waters 2690 HPLC (Waters Corporation, Milford, MA) connected to a Micromass Quattro LC tandem mass spectrometer (Micromass, Beverly, MA). The LC system was equipped with a C8 Phenomenex column (50 x 2 mm; Phenomenex, Torrance, CA) kept at a temperature of 30°C. The mobile phase consisted of a 50:50 mixture of methanol and 0.05% formic acid at an isocratic flow rate of 0.2 mUmin. Mass detection was performed in the positive ion electrospray mode using multiple reaction monitoring channels of 179.96 > 57.96 and 179.62 > 109.83 for mexiletine and phenacetin, respectively. For both compounds, the dwell time was 0.20 seconds, whereas the collision energy and cone voltage was 20 V.
Concentration-Dependent Mexiletine Enantiomers
Effects of and Racemate
Groups 2, 3, 4, and 5, each with 8 rats, were used in this component of the study (see Table 1). Preoperative PWTs for all 4 groups of rats were measured 1 day before surgery. The operative procedure consisted of left L5-L6 spinal nerve root ligation along with external jugular vein cannulation (as previously mentioned in this article). The animals in group 2 received 3 30-minutes infusions of R(-) or S(+) mexiletine on days 5, 6, and 7 after surgery, targeted at 0.25, 0.5, and 1 .O ug/mL, respectively (4 of the 8 animals in group 2 received R(-)-mexiletine and the other 4 received S(+)-mexiletine). Our purpose was to explore the threshold dose range for the 2 drugs and so better resolve the concentration-dependence in the active range. Therefore, for these low-concentration studies, the scoring observer was not blinded to the drug being infused.
Experiments with groups 3 and 4 were designed to examine enantioselective actions at the same plasma levels administered at corresponding times after surgery. The investigator measuring PWTs (for these and all subsequent studies, except for the racemate) was blinded as to which enantiomer was being infused. The animals in group 3 received infusions of S(+)-mexiletine on days 5, 6, and 7 after surgery, targeted at 1.0, 1.2, and 1.4 ug/mL, respectively. On day 8 after surgery, these animals received a 30-minute infusion of R(-)-mexiletine targeted at 1 .O pg/mL. The animals in group 4 received infusions of R(-)mexiletine on days 5, 6, and 7 after surgery, targeted at 1 .O, 1.2, and 1.4 pg/mL, respectively. On day 9 after surgery, animals in group 4 received an infusion of R(-)-mexiletine targeted at 2.0 ug/mL. Group 5 animals were used to evaluate the effect of infusing racemic mexiletine on PWTs. These animals received infusions of racemic mexiletine on days 6, 7, and 10 after surgery, targeted at 1 .O, 1.2, and 1.4 ug/mL, respectively.
lntraperitoneal Enantiomers
Delivery
of Mexiletine
Groups 6 and 7 were used for this portion of the study. Experiments with these 2 groups were performed in a blinded, crossover manner. On day 7, the first 4 animals in group f received an intraperitoneal (i.p.) injection of S(+)-mexiletine (30 mg/kg). On day 8, the remaining 4 animals in group 6 received an i.p. injection of R(-)-mexiletine (30 mg/kg). On day 9, the first 4 rats received a second i.p. injection, this time of R(-)-mexiletine (30 mg/kg), and on day 10 the other 4 animals in this group received an i.p injection of S(+)-mexiletine. Animals in group 7 received the same dosing regimen, except that the first dose was given on day 6 after surgery rather than day 7. After each injection, PWTs were measured at 5, 10, 20, 30, 40, 50, 60, 80, 100, and 120 minutes.
Data Analysis/Statistics Data are expressed as the mean percent maximum possible effect (%MPE) * the standard error (S.E.) of the drug on PWTs. This presentation normalizes the span between preoperative (maximum) and postsurgical, predrug (minimum) PWTs; thus, it provides a relative scale, transposable among groups, for assessing druginduced analgesia. Absolute values of PWT (in g, mean * S.E.) also are given for all conditions. Percent MPE caused by mexiletine infusion was determined by the following equation: PW
%Mpr =-----
(g) during
Preoperative
infusion
- Postoperative
___--.---~..~~-
PWT (g) - Postoperative
PW
(g)
-- -_..-~ * 100 PWl
(g)
Actions of Mextletrne
132 --- 0-8-
loo-
Endntmwi
R-(-)-mexlletine S-(+)-mexiletine Racemic mexiletine
goc
=-
80
-10
1 0.9
I 1.0
I 1.1
Target Figure during
2. Mean intravenous
percent maximum infusions targeted
Plasma
I
I
1.2
1.3
Concentration
possible effect 2 S.E.M. of R-(-), S-(+) at 1000, 1200, and 1400 ng/mL.
The Friedman test was used to determine overall significance. Once overall significance was determined for a response set, the Wilcoxon test was used after the fact to determine which postinfusion PWT values differed significantly from the preinfusion values. All analyses were performed using SPSS for Windows, (Version 6.1.3, SPSS Inc., Chicago, IL). Results were considered significant at P < 0.05.
Results Mexifetine Administration
Verification
Mexiletine extraction efficiencies were greater than 90%, and calibration curves were linear (R2 > 0.98). The low-, medium-, and high-concentration quality control samples for R(-)-mexiletine and S(+)-mexiletine were 97, 103, and 131 and 107, 101, and 106% of expected concentrations, respectively. Measured plasma concentrations of mexiletine enantiomers sampled during programmed infusions were within 20% of the target level of 0.5 vg/mL, and, equally important, there was no significant difference between the achieved levels of R(-) and S(+) enantiomers. Fig 1 shows the measured plasma concentrations of R(-)-mexiletine and S(+)-mexiletine at 4 time points during an infusion targeted to 0.5 pg/mL. The levels at 5 minutes were slightly but insignificantty greater than those at later times, and the levels at 10 to 30 minutes were within 1 S.E. of the target concentration. The mean concentration (-c S.E.) of the plasma samples that were
I 1.5
I 1.4
(pg/mL) and
racemic
mexiletine
on paw
withdrawal
thresholds
externally doped to a final content of 500 ng/mL was 405.7 ng * 11 .I ng S(+)-mexiletine /mL and 400.3 ng * 9.0 ng R(-)-mexiletine/mL (each n = 3), showing that 80% of each enantiomer was extractable from plasma by the method used here, and without any stereoisomeric bias. Thus, the infusion pump parameters used here adequately account for the plasma protein binding of both mexiletine enantiomers. In a previous study, using the Stanpump infusion program for lidocaine, a nearly linear correlation from 0.4 to IO g/mL was shown between target and actual plasma lidocaine levels.g The agreement at the single mexiletine level targeted here for S-(+) and R-(-) enantiomers confirms the reliability of this method for these agents.
Concentration-Dependent Mexifetine Enantlomers
Effects of
At plasma levels up to 0.5 pg/mL neither enantiomer relieved mechanical allodynia. An enantioselective action became evident at higher concentrations and with a single crossover procedure in the group receiving S(+)-mexiletine (group 3; n = 8, the mean preoperative PWT of which was 10.4 g f 1.0 g and mean postoperative PWT was 1.5 g * 1.8 g). The %MPE for the elevation of PWT by S(+)-mexiletine was zero at plasma concentrations of 1.0 and 1.4 pg/mL, and, oddly but insignificantly different, about 25% (P = .32), at 1.2 pg/mL (Fig 2). These same rats were then infused with R(-)-mexiletine targeted at 1.0 vg/mL on day 8 after surgery, during which PWTs
ORIGINAL REPORT/Sinnott
Y ;
et al
133
1101
8
loo-
a‘E
90 -
-r a a g
8070-
6 t Eal 3$
60-
i oi: z
30 -
g
lo-
50
*-
40 -
20 -
5
o-10
1 0.9
I 1 .o
I 1.1 Target
Figure on paw
3. Percent withdrawal
maximum thresholds
possible effect of individual
I 1.2 Plasma
I 1.3
Concentration
of intravenous infusions animals in group 4.
I 1.4
I 1.5
(pg/mL)
of R-(-)-mexiletine
-O-0-8-
100
targeted
at 1000,
1200,
and
1400
ng/mL
R-(-)-mex. (1.4 I*g/mL) S-(+)-mex. (1.4 pg/mL) Racemic mex. (1.4 PglmL)
90 ‘1 8070 -
5040 -
1 r
/d
60 -
/
_____c-
30 20-
f
//
10-
J/ e-o-o
O-10
f
f
1 -5
.f
I 0
0
I 10
I 5 Time
Figure 4. Mean percent maximum possible effect sions of R-(-), S-(+) and racemic mexiletine targeted
* S.E.M. at 1400
During on paw ng/mL.
were elevated to a mean value of 6.7 g * 0.8 g (%MPE = 50.0% + 5.8%) suggesting an enantioselective action of the R(-) enantiomer. Infusion of S(+)-mexiletine targeted at 2.0 pg/mL produced convulsions in the first 2 rats in this group within 20 seconds after commencing the infusion, obviating any antiallodynic data for that dose. Concentration-dependent relief of allodynia by R(-) mexiletine was assessed in a separate group of animals (group 4; n = 8) with the doses corresponding to S(+) mexiletine’s dosing regimen given on the corresponding postoperative days
0
I 15
I
I
I
20
25
30
Infusion
(Minutes)
withdrawal
thresholds
during
1 35
a 30-minute
intravenous
infu-
(Fig 2). (The mean preoperative PWT value for this group was 12.0 g + 0.0 g and the mean postoperative value was 1.6 g + 0.3 g). Infusions of the R(-)-enantiomer at 1.0 g/mL resulted in an elevation of PWT to 62.4 f 13.9 %MPE (8.1 g -c 1.4 g), whereas infusions targeted at 1.2 pg/mL and 1.4 ug/mL both elevated PWTs to 53.5 + 13.6 %MPE (7.2 g f 1.4 g), insignificantly different from that at 1 .O pg/mL (P = .16). Each of the individual animals in this group that responded to treatment with R(-) mexiletine (7 of 8) achieved approximately the same degree
Actions
134 -o-o-
loo-
ot Mexlietlne
R-(-)-mex. S-(+)-mex.
(30 mg/kg) (30 mg/kg)
I 100
I 120
Errantlomrs
go80 70 60-
-10
1 -10
I 0
I 10
I 20
I 30
Time
Fiaure 5. an>
S-(+)
maximum Mean percent mexiletine at 30 mg/kg.
possible
effect
I 50
I 40
f S.E.M.
After
I 60 Injection
on paw
of alleviation at all 3 concentrations (Fig 3). Three to 4 were completely returned to preoperative levels, and 3 to 4 had intermediate degrees of relief; 1 rat in this group did not achieve relief at any concentration. Infusions of R(-)-mexiletine targeted at 2.0 ug/mL produced convulsions in 2 of these animals within 1 minute; no other rats were infused to this level. When infused with racemic mexiletine at target concentrations identical to those of the single enantiomers, the rats in group 5 showed a significant concentration-dependent relief of allodynia at 1.2 and 1.4 ug/mL (P = .02; Fig 2). (Preoperative PWTs were 11 .O g + 0.5 g; postoperative values were 1.3 g f 0.1 g). After 30 minutes of infusion targeted to 1.0 pg/mL, racemic mexiletine gave no significant relief (%MPE = 4.0 it: 1.9; P = .08). The elevation afforded by racemic mexiletine at 1.2 and 1.4 pg/mL was midway between those of the 2 enantiomers, as one would expect if simple receptor occupancy accounted for the therapeutic action without any interenantiomer interactions. When the antiallodynic activity of mexiletine was examined over the 30-minute course of drug infusion targeted to 1.4 ug/mL (see Fig 4), the effects of the single enantiomers are constant; R(-)-mexiletine produces exactly the same PWT elevation at each time (P = .82) and S(+)-mexiletine produced no elevation at any time. However, the effect of racemic mexiletine developed over the early course of the infusion; relief at 5 minutes was less than that at the later time points (P = .03).
I 70
I 80
I 90
I 110
I 130
(Minutes)
withdrawal
thresholds
after
intraperitoneal
injections
of R-(-)
Any antiallodynic effect of intravenous (i.v.) mexiletine was temporary. Allodynia had returned to the postoperative level when the animals were examined 24 hours after the infusion, in the period just preceding the next infusion.
lntraperitoneal Enantiomers
Dosing With Mexiletine
Enantioselective actions of mexiletine were not evident after intraperitoneal injections. Fig 5 shows the results of experiments with group 6 (n = 8) and 7 (n = 11) animals. lntraperitoneal injections (30 mg/kg) of both the R(-)- and the S-(+)enantiomer produced a significant 20-minute elevation of PWT values compared with preinjection values (P = .Ol at 5 and 10 minutes; P = .03 [R(-)-mexiletine] and P = .04 [S(+)-mexiletine] at 20 minutes); there was no significant difference between the degree of relief achieved with each enantiomer at any time point after injection (eg, P = .30 [5 minutes]). The order of administration of the enantiomers had no effect; relief by R(-) or S(+) enantiomers was not different in those rats where one was injected first compared with those where it was the second drug injected. I.p. injections of either enantiomer at 50 mg/kg in these animals resulted in convulsions within 3 minutes.
Discussion These experiments show a clear stereosetective action of intravenous mexiletine for temporary relief of experimental mechanical allodynia.
ORIGINAL REPORT/Sinnott
et al
When intravenous concentrations were controlled at predetermined levels by programmed infusions, the pathologically low mechanical threshold to elicit brisk hindpaw withdrawal (1 to 2 g von Frey filament) was raised back toward the normal, preoperative value (10 to 12 g) by the R(-)-mexiletine enantiomer, but not by the S(+) enantiomer. Relief of allodynia by R(-) mexiletine had several characteristics of the previously reported relief by lidocaine, including a minimal threshold concentration for detecting any relief and a ceiling concentration, above which no further relief occurred.8-g Indeed, for R(-) mexiletine, these threshold changes occurred at about the same plasma concentration as for lidocaine, lpg/mL. At 1.4 ug/mL R(-) mexiletine, withdrawal thresholds were no higher than at 1 .O ug/mL, the “ceiling” effect. As with lidocaine, the effective plasma concentration (1 to 1.5 ug/mL), equivalent to 5.6 to 8.4 umol/L R(-) mexiletine, was well below the IC,, values reported for the inhibition of resting or phasically stimulated Na+ channels, 54 or 23 umol/L, respectively (see later in this article). Withdrawal thresholds were not elevated by infusions lower than at 1.0 ug/mL. Doubling the threshold concentration, to 2.0 ug/mL, produced convulsive-like behavior early in the infusion, indicating the toxic limit of systemic mexiletine. Interestingly, signs of central nervous system (CNS) toxicity occurred at about the same early time during infusion with both mexiletine enantiomers at 2 ug/mL, despite the absence of therapeutic activity from S(+)mexiletine at lower concentrations, suggesting that such toxicity might be caused by different mechanisms than those that relieve allodynia. One distinct difference between lidocaine’s and R(-) mexiletine’s antiallodynic actions was the persistence of effect. For days to weeks following 30-minute lidocaine infusions to 1 to 2 ug/mL, allodynic threshold remained elevated.8-g In contrast, relief by R(-)mexiletine never persisted, even though it reached the same mean level as that by lidocaine during infusion (about 60 %MPE). When tested 24 and 48 hours after infusions, rats treated with R(-)-mexiletine had the same very low postoperative withdrawal thresholds. We interpret these results as showing that separate mechanisms account for the transient relief during infusion and that which endures after the drug is long gone. Both lidoCaine and R(-)-mexiletine can effect the former, but only lidocaine yields the latter response. The absolute stereoselectivity of this allodynic action could not be determined in these experiments because of the limit on dosing caused by CNS toxicity. We can only conclude that the R(-)
135
enantiomer is at least 1.4 times more potent than the S(+) enantiomer, where potency refers to a threshold concentration for an all-or-none action and not to a drug level that produces half of a concentration-graded effect. This stereoselectivity order is consistent with IC,, values reported for inhibition of Na+currents (INa) in frog skeletal muscle, 21-22 Na+ currents carried through the m, type of Na+ channel, the local anesthetic pharmacology23 of which is very similar to that of peripheral nerve,24-26 and of central nervous system channels.27-28 DeLuca et all2 found that tonic inhibition of I,, by mexiletine in skeletal muscle was about 2-fold stereoselective for R- over S- (IC,, values: R(-) = 54 umol/L; S(+) = 114 umol/L; R/S (-I+) = 83 umol/L) mexiletine, but that for use-dependent inhibition, measured at a higher frequency of brief test depolarizations (10 Hz), stereoselectivity effectively disappeared (IC,, values: R(-) = 23 pmol/L; S(+) = 27 umol/L; R/S (-/+) = 29 umol/L.21 Inhibition of rat brain Na+ channels by R-mexiletine is also use-dependent, with lower tonic inhibitory potency (IC,, = 305 umol/L) and usedependent potency (IC,, = 148 pmol/L, at 5 Hz) than reported for skeletal muscle; no data on the S-enantiomer were reported.2g Because use-dependent inhibition is caused by additional binding of the drug to channels that are in the open and inactivated states30e3* and their corresponding slower drug dissociation, the disappearance of stereoselectivity at high-frequency depolarization implies that the open or inactivated conformations of the Na+ channels have higher affinities for S(+)- than for R(-)-mexiletine. The additional S(+)-enantiomer-selective binding that occurs during 10 Hz, then sums with the R(-)-enantioselective tonic binding to give equal inhibition from the 2 isomers during repetitive stimulation. Therefore, for the enantioselective relief of allodynia that we observe in vivo to be caused by Na+ channel blockade, it should arise from the R(-)-selective inhibition of infrequently stimulated neurons with Na+ channels predominantly in the resting state, rather than the enantioneutral or S(+)-selective inhibition of open and inactivated channels. Although this conclusion relies strongly on voltage-clamp data from frog skeletal muscle that must be confirmed in mammalian nerve, it directly contradicts the widespread argument that therapeutic relief of neuropathic pain by intravenous local anesthetics and antiarrhythmics (and, in addition, a plethora of amphipathic bases belonging to other drug classes) results from the selective suppression of high-frequency, injury-induced impulses in hyperexcitable nerve fibers.5,33 The apparent equieffectiveness of intraperitoneally injected boluses of S(+)- and R(-) enan-
AC~IOPS of Mexiietine
136
tna~tmr~r~~
tiomers in the transient relief of allodynia in this model is surprising. This was a consistent result in 2 separate groups of animals in which each individual rat received both enantiomers in sequence. The discrepancy between these equivalent effects and the pronounced stereoselectivity during infusion, emphasized by the crossover results in the infusion group treated first with S(+) mexiletine (ineffective at 1.4 pg/mL) then R(-) mexiletine (giving relief at 1.0 ug/mL), might be explained by several possibilities. First, bolus intraperitoneal injections might result in higher concentrations of systemic S(+)- than R(-)-mexiletine, because of their different pharmacokinetic parameters, a difference that is accounted for during the programmed infusion. Clearly, the
anatomic distribution of S(+)-mexiletine differs when delivered by the 2 routes, because toxicity precedes (and obviates) therapy during i.v. infusion but is not apparent during bolus injection (at 30 mg/kg). Second, S(+) mexiletine might undergo an enzyme-catalyzed chiral inversion that changes it to the R(-) conformation, although there is no direct evidence that this occurs. Alternatively, both enantiomers might be transformed to a common metabolite leading to apparent equipotency. However, such a reaction also would occur when S(+) mexiletine is infused intravenously, yet no antiallodynic activity is detected in such a case. At this time, the weak transient equipotency of i.p. administered mexiletine enantiomers remains an anomaly.
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