Anesthesiology 2001; 95:1198–1204 © 2001 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Local Anesthetic Properties of Prenylamine Mustafa G. Mujtaba, Ph.D.,* Peter Gerner, M.D.,† Ging Kuo Wang, Ph.D.‡ Background: Local anesthetics that produce analgesia of long of the L-type calcium channels.3 Both ␣-subunits form a duration with minimal impairment of autonomic functions are fourfold pseudosymmetry with four repeated domains, highly desirable for pain management in the clinic. Pre- each with six transmembrane segments.3 There are nylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels drugs that can block both calcium channels and 4,5 have not been studied thus far. NaChs. It has been reported that there is a correlation Methods: The authors characterized the tonic and use-depen- between the potencies of calcium channel blockers in ؉ dent prenylamine block of native Na channels in cultured rat preventing neurotoxicity induced by veratridine in brain neuronal GH cells during whole cell voltage clamp conditions Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/95/5/1198/334086/0000542-200111000-00025.pdf by guest on 02 October 2021 3 neuronal cultures and their binding affinity for and the local anesthetic effect of prenylamine by neurologic 3 6 evaluation of sensory and motor functions of sciatic nerve dur- [H ]batrachotoxinin-B binding sites. This raises a possi- ing neural block in rats. bility that some calcium channel blockers may block .Results: Prenylamine elicits both use-dependent block of Na؉ Naϩ channels with a high affinity channels during repetitive pulses (3 ␮M prenylamine produced Few LAs for pain management produce analgesia of 50% block at 5 Hz) and tonic block for both resting and inacti- ؉ long duration. The development and availability of such vated Na channels. The 50% inhibitory concentration for pre- -nylamine was 27.6 ؎ 1.3 ␮M for resting channels and 0.75 ؎ LAs that can cause reversible blockade of neural func 0.02 ␮M for inactivated channels. Furthermore, in vivo data tions is desirable and advantageous for certain pain man- show that 10 mM prenylamine produced a complete sciatic agement in the clinic. Prenylamine, a coronary vasodila- nerve block of motor function, proprioceptive responses, and tor, is a calcium channel blocker formerly used in the nociceptive responses that lasted approximately 27, 34, and treatment of angina pectoris but now superseded by 24 h, respectively. Rats injected with 15.4 mM bupivacaine, a 7–10 known local anesthetic currently used for pain management, safer antianginal drugs. The general pharmacologic had a significantly shorter duration of blockade (< 2 h) com- activities, including surface anesthesia, of prenylamine pared with rats injected with prenylamine. and some of its cycloalipahtic derivatives have been Conclusions: The data presented here demonstrate that pre- previously studied.11 The LA properties of prenylamine nylamine possesses local anesthetic properties in vitro and on voltage-gated NaChs have not been characterized elicits prolonged local anesthesia in vivo. thus far. We selected prenylamine because it contains large hydrophobic moieties (fig. 1). Previous results in- TRADITIONAL local anesthetics (LAs) block voltage- dicate that LAs with two large hydrophobic moieties are gated sodium channels (NaChs) and inhibit the propaga- more potent in vivo.12,13 This study examines the LA tion of action potentials in excitable membranes. Volt- properties of prenylamine in vitro by electrophysiologic age-gated NaChs are the main target of LAs. LAs are studies of the NaCh on neuronal GH cells and assesses known to block nerve, skeletal, muscle, and cardiac 3 its usefulness as a long-acting LA in vivo by neurobehav- muscle NaChs, which are primarily responsible for ex- ioral examination of sciatic nerve block in the rat in citability of their respective tissue. LA potency, as Naϩ comparison with the known LA anesthetic bupivacaine. channel blockers, is governed by channel state, with open and inactivated states being favored over resting states. Voltage-dependent conformational changes of the Materials and Methods LA binding site can explain the changes of LA affinity between resting, open, and inactivated states.1 Prenylamine and Bupivacaine There are structural similarities between voltage-gated Prenylamine was purchased from Sigma Chemical Co. sodium and calcium channel proteins, even though (St. Louis, MO). Bupivacaine was a gift from AstraZeneca, these two channels differ strongly in their ion selectivi- (Westborogh, MA). For the electrophysiologic experi- ty.2 The homologous domains of the ␣-subunit of NaChs ments, prenylamine and bupivacaine were dissolved in can be aligned with 32–37% sequence identity with that dimethyl sulfoxide at 100 mM and were diluted shortly before the experiments. For sciatic nerve blockade, pre- nylamine was dissolved in 5% dextrose solution and * Research Fellow, † Instructor in Anesthesia, ‡ Associate Professor of bupivacaine in 0.9% sodium chloride solution. Pre- Anesthesia. nylamine is insoluble in saline solutions; thus, 5% dex- Received from the Department of Anesthesia Research Laboratories, Brigham trose was used as the vehicle of transfer for injection. and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Submitted for publication February 21, 2001. Accepted for publication May 30, Neither 0.9% sodium chloride nor 5% dextrose solution 2001. Supported by grant No. GM48090 from the National Institutes of Health, had any analgesic effect. Bethesda, Maryland (to Dr. Wang). Address reprint requests to Dr. Mujtaba: Department of Anesthesia Research Laboratories, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massa- Neurologic Evaluation of Sciatic Nerve Block in the Rat chusetts 02115. Address electronic mail to: [email protected]. In- dividual article reprints may be purchased through the Journal Web site, The following protocol, which we and other investi- www.anesthesiology.org. gators have used previously for the evaluation of sciatic Anesthesiology, V 95, No 5, Nov 2001 1198 PRENYLAMINE AS A POTENT LOCAL ANESTHETIC 1199 reflex or vocalization to pinch of a skin fold over the lateral metatarsus (coetaneous pain) and of the distal phalanx of the fifth toe (deep pain). Nocifensive reaction is graded 4 (normal: vocalization and withdrawal of hind limb, or 0% MPE), 3 (more slowly than control, or 25% MPE), 2 (not complete response, or 50% MPE), 1 (weak attempts to withdraw, or 75% MPE), and 0 (absent re- sponse, or 100% MPE). Whole Cell Voltage Clamp Experiments Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/95/5/1198/334086/0000542-200111000-00025.pdf by guest on 02 October 2021 Fig. 1. Chemical structures of prenylamine and bupivacaine. Rat clonal pituitary GH3 cells were purchased from the American Type Culture Collection (Rockville, MD). Cell nerve block in the rat,13,14 was approved by the Harvard cultures were split twice a week and maintained in Medical Area Standing Committee on Animals (Boston, Dulbecco modified Eagle medium supplemented with MA). Male Sprague Dawley rats were purchased from 1% penicillin–streptomycin and heat-inactivated 10% fe- Charles Rivers Laboratories (Cambridge, MA) and used tal bovine serum (Hyclone Labs, Logan, UT) in a 5% for in vivo experiments. Neurobehavioral examination carbon dioxide incubation chamber at 37°C. For record- consisted of evaluation of motor function, propriocep- ϩ ing of Na current, GH3 cells were replated in a 35-mm tion, and nocifensive reaction immediately before injec- culture dish, which was then used as a recording cham- tion of 0.2 ml of the drug solution and at various timed ber. The whole cell configuration of the patch clamp intervals after injection. Changes of function were esti- technique was used to record macroscopic Naϩ currents mated as percentage of maximal possible effect (MPE). at room temperatures ranging from 21 to 23°C. Pipette Motor function was evaluated by measuring the “exten- electrodes were fabricated with a tip resistance ranging sor postural thrust” of the hind limbs by holding the rat from 0.8 to 1.2 M⍀. Command voltages were controlled upright with the hind limb extended so that the distal by pCLAMP software (Axons Instruments, Inc., Foster metatarsus and toes support the animal’s weight and City, CA) and delivered by a List-EPC7 patch clamp measuring the extensor thrust as the gram force applied amplifier (List-Electronics, Darmstadt/Eberstadt, Germa- to a digital platform balance, the force that resists con- ny). After the establishment of whole cell configuration, tact of the platform by the heel. The preinjection control value is considered 0% of the MPE. The reduction in this cells were dialyzed for 20–30 min to equilibrate with the force, representing reduced extensor muscle contrac- pipette solution before data were acquired. Data were tion caused by motor blockade, was calculated as a filtered at 5 kHz, sampled at 50 kHz, collected, and percentage of the control force. A force less than 20 g stored with pCLAMP software. Voltage error was gener- ϩ (also referred to as weight of the “flaccid limb”) is con- ally less than 3 mV at 30 mV after series resistance sidered 100% MPE. Proprioception evaluation was based compensation. Leak and capacitance currents were sub- on the resting posture and postural reaction (“tactile tracted by P/-4 protocol, which was not applied in the ϩ placing” and “hopping”). The functional deficit was use-dependent block of Na currents. Pipette electrodes graded as 3 (normal) or 0% MPE, 2 (slightly impaired), 1 were filled with an internal solution containing 100 mM (severely impaired), and 0 (complete) or 100% MPE. NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES Keeping the rat in the normal resting posture with titrated with CsOH to pH 7.2. The external solution the toes flexed and the dorsi of the feet placed on the consisted of 85 mM choline Cl, 65 mM NaCl, 2 mM CaCl2, supporting surface, we evaluated tactile placing as the and 10 mM HEPES titrated with tetramethylammonium- ability of the animal to reposition its toes.