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Metaflumizone is a novel sodium channel blocker insecticide
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Veterinary Parasitology 150 (2007) 182–189 www.elsevier.com/locate/vetpar
Metaflumizone is a novel sodium channel blocker insecticide V.L. Salgado a,*, J.H. Hayashi b a
BASF Corporation, 26 Davis Drive, Research Triangle Park, NC 27709, USA b P.O. Box 786, Princeton Junction, NJ 08550, USA
Abstract Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips–Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide. # 2007 Elsevier B.V. All rights reserved. Keywords: Sodium channel blocker insecticide; Metaflumizone; Indoxacarb; Local anesthetic
1. Introduction Metaflumizone (IUPAC (EZ)-20 -[2-(4-cyanophenyl)1-(a,a,a-trifluoro-m-tolyl)ethylidene]-4-(trifluoromethoxy)carbanilohydrazide, CAS 2-[2-(4-cyanophenyl)-1-[3-(trifluoromethyl)phenyl]ethylidine]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarboxamide) is a new insecticidal compound discovered by Nihon Nohyaku Co. Ltd. (Takagi et al., 2007). This compound is now being co-developed globally by BASF Aktiengesellschaft, Fort Dodge Animal Health, a Division of Wyeth Corporation and Nihon Nohyaku Co. Ltd. Metaflumizone provides good to excellent control of most economically important lepidopterous pests and
* Corresponding author. Tel.: +1 919 547 2244; fax: +1 919 547 2450. E-mail address: [email protected] (V.L. Salgado). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.08.032
certain pests in the orders Coleoptera, Hemiptera, Hymenoptera, Diptera, Isoptera and Siphonaptera (BASF, 2007). Metaflumizone provides long-lasting control of fleas on animals with a single spot-on application and is being developed for this use under the trade names ProMeris1 and ProMeris Duo1 by Fort Dodge Animal Health, Overland Park, KS, USA (Rugg and Hair, 2007). The semicarbazone metaflumizone is an open-ring analog of the 3,4-diphenyl-1-phenylcarbamoyl-2-pyrazolines (Grosscurt et al., 1979), which in turn evolved from the 3-phenyl-1-phenylcarbamoyl-2-pyrazolines discovered at Philips–Duphar in the early 1970s (Mulder et al., 1975). The relationship between these three families is shown in Fig. 1. The pyrazolines paralyze insects and block nerve activity by blocking voltage-dependent sodium channels. The sodium channels are most sensitive at depolarized potentials, so that tonic sensory receptors
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Fig. 1. Chemical evolution of the semicarbazones exemplified by metaflumizone, from the first pyrazoline SCBIs.
and pacemaker neurons are the most sensitive, giving a type of paralysis in which the nervous system shows little or no spontaneous activity and the insects appear paralyzed, but can be stimulated to convulse by activation of phasic sensory receptors. These convulsions cease after several seconds, as more sodium channels become blocked, and the insect lapses back into a pseudo-paralyzed state (Salgado, 1990). There is a refractory period after these convulsions, during which the sodium channels in phasic neurons and axons recover from block. The pyrazolines appear to block sodium channels by binding at the local anesthetic site (Salgado, 1992; Payne et al., 1998). However, in contrast to local anesthetics and other drugs, which can access the binding site through the intracellular mouth of the open pore, entry and exit of the pyrazolines is very slow, on the order of minutes, which precludes the direct block of open channels often seen with local anesthetics. The pyrazolines interact very slowly with the channels, and appear to access the receptor primarily through lateral diffusion from the lipid bilayer, blocking equally well from either face of the membrane (Salgado, 1992). Because of their slow action, the pyrazolines appear to interact specifically with channels in the slow-inactivated state, but when slow and fast-inactivation were progressively removed by treatment of the intracellular-exposed surfaces of sodium channels with trypsin and N-bromoacetamide (NBA), respectively, block of fast-inactivated and open channels by the pyrazoline RH-1211 proceeded at least as rapidly as block of slow-inactivated channels (Salgado, 1992). Indoxacarb, a member of the oxadiazine subclass of SCBIs, was the first commercialized sodium channelblocking insecticide (SCBI) (Wing et al., 2005); metaflumizone is the first SCBI in the animal health market. In the present paper, we confirm, using in vivo and in vitro experiments, that state-dependent block of
voltage-dependent sodium channels is the mechanism of action of metaflumizone. 2. Materials and methods 2.1. Mosquito larvicide test Ten early third instar Aedes aegypti larvae were placed into 2 mL tap water in a well of a 12-well microtitre plate. Compound was dissolved in DMSO and added to the well at a DMSO concentration not exceeding 0.1%, which itself had no observable effect on the larvae. The treated insects were held at room temperature and observed hourly for the first 8 h, and daily thereafter. 2.2. Spodoptera eridania gross electrophysiology Fifth instar larvae of the southern armyworm, S. eridania, weighing 400–600 mg, were injected with compound dissolved in 1 mL of DMSO using a Hamilton syringe fitted with a 30-gauge needle. The tip of the needle was inserted dorsolaterally in the abdomen, just beneath the cuticle. Paralyzed insects were dissected and investigated electrophysiologically with bipolar hook electrode recordings from CNS connectives and segmental nerves, in comparison with control insects. Recordings were made in the insects’ own hemolymph. 2.3. Spodoptera frugiperda stretch receptor For measurements on the abdominal stretch receptor organ, a fifth instar fall armyworm, S. frugiperda, larva was dissected and pinned out in a wax dish. Because the organs lie dorsolaterally, the cut was made dorsolaterally on one side, so that the organs on the other side would not be damaged. The gut and fat body were removed and three or four segments containing nerve cord and body wall were removed and pinned in a
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perfusion chamber. A stretch receptor organ was exposed and perfused directly with Lepidoptera saline, containing (in mM): 128 NaCl, 16 KCl, 29 CaCl2, 18 MgCl2, 175 sucrose, 3 NaH2PO4, at pH 6.5. A suction electrode was used to record the stretch receptor activity. Successful preparations fired continuously at a rate between 20 and 50 s1. Compounds were introduced into the perfusion solution. 2.4. Manduca sexta neurons Techniques and media used for isolation and wholecell recording from primary-cultured insect neurons were as described by Hayashi and Hildebrand (1990). Briefly, nerve cords from fifth instar larvae of the tobacco hornworm, M. sexta, were removed into sterile culture saline. The thoracic and abdominal ganglia were dissected free from the nerve sheath and dissociated by treatment with 0.125 mg ml1 collagenase and 0.5 mg ml1 dispase in Ca2+, Mg2+-free Hank’s solution at 37 8C for 2.5 min, followed by trituration with a fire-polished Pasteur pipette. The enzyme activity was stopped by centrifuging the cells through culture saline (in mM, 149.9 NaCl, 3 KCl, 3 CaCl2, 0.5 MgCl2, 10 TES, 11 Dglucose, plus 2 g L1 lactalbumin hydrolysate, 2 g L1 yeastolate) at 207 g for 8 min at 10 8C, re-suspending the pellet in I-L15 (a supplemented version of Liebovitz’s L-15 medium) and re-centrifuging as before through IL15. The pellet was re-suspended in culture medium (150 mL per ganglion) and plated onto 35 mm Petri dishes with mini-wells that had glass cover slip bottoms coated with a mixture of concanavalin A and laminin. Cells were allowed to adhere for 1.5 h, and the dish was then flooded with 1.5 mL of I-L15 growth medium. Sodium currents could be recorded for up to 2 days in culture. Action potentials and Na+ currents were recorded with standard whole-cell patch clamp methods, (Hamill et al., 1981; Fenwick et al., 1982) using patch pipettes with a resistance between 3 and 5 MV. The pipettes were filled with a Kasp solution, containing (in mM): 150 Kaspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 2 ATP, 5 HEPES, and pH 7.0, with the addition of mannitol to adjust the osmolality to 330 mOsm. The external solution was AIS, containing (in mM): 150 NaCl, 4 KCl, 6 CaCl2, 10 HEPES, 5 glucose, and pH 7.0, with the addition of mannitol to adjust the osmolality to 360 mOsm.
the African clawed frog (Xenopus) were defolliculated with collagenase and injected with Drosophila melanogaster Para sodium channel cRNA (0.2–2 ng), together with D. melanogaster TipE cRNA (0.2– 2 ng), which is known to enhance the expression of insect sodium channels in oocytes (Feng et al., 1995). After incubation for 2–7 days, an oocyte was placed in the recording chamber and superfused with ND96 solution, containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and pH 7.4. Two microelectrodes containing 3 M KCl, with tip resistances of 2–5 MV, were inserted, and the oocyte was voltage clamped with a TEC-03X amplifier (NPI electronic). Test compound was added from a DMSO stock solution to the ND96 superfusion solution at a final DMSO concentration not exceeding 0.1%, a DMSO concentration which had no effect on the sodium currents. 3. Results and discussion 3.1. Mosquito larvicide test Ninety-five percent of third instar A. aegypti larvae exposed to 40 mM metaflumizone were slow or paralyzed at 1 h and 100% were paralyzed within 3 h. The compound was quite active at lower concentrations, giving 100% kill within 24 h at 100 nM, but 0% kill within 3 days at 30 nM.
3.2. S. eridania gross electrophysiology Metaflumizone injected into fifth instar larvae of S. eridania, at a dose of 15 mg g1, produced 100% prostration within 4 h, and paralysis within 24 h. Gross electrophysiological analysis of the paralyzed insects at 24 h after treatment showed that all nerve activity was blocked, as is also seen in insects poisoned by pyrazolines and indoxacarb (Salgado, 1990; Wing et al., 2005). Table 1 Action of increasing concentrations of metaflumizone on the abdominal stretch receptor organ of Spodoptera frugiperda: percent of block within 1 h Concentration (M)
2.5. Xenopus oocyte expression and voltage clamp The procedures for oocyte preparation, cRNA injection and voltage clamp recording are identical to those described by Stu¨hmer (1992). Briefly, oocytes of
7
10 3 107 106 3 106 105
Block (%) 0 0 50 100 100
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Fig. 2. Block of action potentials by 1 mM metaflumizone in a neuron isolated from the CNS of a Manduca sexta larva. The cell had been in culture for 1-day and was current-clamped with a patch pipette at a potential of 60 mV. The external solution was AIS and the internal solution was Kasp. The six panels show successive action potentials overlaid on the control. The stimulus artifact can be seen, which generates an action potential with a positive phase lasting about 1.5 ms, with an amplitude of 55 mV. After addition of metaflumizone, the action potential was gradually blocked. Block was complete at 20 min, when only an electrotonic response remained.
3.3. S. frugiperda stretch receptor Recordings made from abdominal stretch receptor organs of S. frugiperda confirmed the direct blocking effect on action potential generation. These tonic mechanoreceptors normally generate action potentials constantly at a low rate and are very sensitive to block by SCBIs (Salgado, 1990; Wing et al., 2005). Metaflumizone blocked S. frugiperda stretch receptor activity with a threshold concentration of 106 M, but had no effect at 3 107 M within 1 h (Table 1). Sodium channels in sensory organs such as the stretch receptor organ are very sensitive to sodium channel blockers (Salgado, 1990), which are generally selective for activated or inactivated channel states. Because these sensory neurons are constantly active, their Na channels cycle between all states and are therefore available to state-dependent blockers (Salgado, 1992; Wing et al., 2005). 3.4. M. sexta neurons Block of the action potential was investigated directly on neurons isolated from the central nervous system (CNS) of M. sexta larvae and studied in wholecell current clamp mode. Fig. 2 shows the recordings from a M. sexta neuron at a resting potential of 60 mV, demonstrating complete block of the action potential by 106 M metaflumizone over a time course of 20 min. This result confirms that metaflumizone directly affects action potential generation of native Na+ channels.
Fig. 3. Block of the sodium current by 5 mM metaflumizone in a neuron isolated from the CNS of a Manduca sexta larva. The cell had been in culture for 1-day and voltage was clamped with a patch pipette, at a potential of 70 mV. The external solution was AIS containing 30 mM TEA, 20 mM 4-AP and 500 mM Cd2+, and the KAsp internal solution was used. (A) Control family of sodium currents for steps from 70 to +40 mV. (B) Ten minutes in 5 mM metaflumizone. (C) Twenty minutes in 5 mM metaflumizone.
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To assess the effect of metaflumizone on sodium current directly, Na+ channels were studied in primarycultured neurons isolated from the CNS of fifth instar M. sexta larvae, with the whole-cell voltage clamp method. In the presence of external 30 mM tetraethylammonium (TEA) and 20 mM 4-aminopyridine (4AP) to block potassium channels, and 500 mM Cd2+ to block voltage-dependent calcium channels, depolarizing voltage steps activated voltage-gated, tetrodotoxin sensitive, sodium channels that give rise to an inward sodium current. Under maintained step depolarization, about 50% of the channels inactivate, but a current is maintained throughout the duration of the 150 ms depolarizing pulse (Fig. 3A). After application of 5 mM metaflumizone, the peak and maintained portions of the sodium current gradually decreased together and completely disappeared by 20 min (Fig. 3B and C). This confirms that metaflumizone directly blocks the native insect Na+ channels.
3.5. Xenopus oocyte expression and voltage clamp The insect Na+ channel consists of a single channelforming subunit that has been named Para because it is coded for by a gene in which mutations can cause paralysis in Drosophila (Loughney et al., 1989). A small accessory subunit known as TipE, which is unrelated to the beta subunit found in vertebrate sodium channels, is required for functional expression. When Para and TipE are co-expressed in the Xenopus oocyte expression system, voltage-dependent sodium currents are obtained that exhibit all of the excitable and pharmacological properties of normal insect sodium channels (Feng et al., 1995). Fig. 4A shows the Para/ TipE sodium currents evoked by step depolarizations between 60 and +50 mV. The current–voltage relation is plotted in Fig. 4B. After treatment with 10 mM metaflumizone, the currents were completely blocked (Fig. 4C and D).
Fig. 4. Block of Para/TipE sodium channels expressed in Xenopus oocytes, by metaflumizone. Inward currents were observed for 3 days after injection with Para and TipE RNA. External solution consisted of ND96. The oocyte was clamped using the two-electrode method, and the holding potential was set at 70 mV. The current responses to steps to various potentials are superimposed in the lower portion of panel A, with the voltage steps illustrated in the upper portion. These data are plotted in a current/voltage relationship in B. The activation point is approximately 40 mV. Panel C shows that the inward current was blocked by 10 mM metaflumizone. Small outward currents, presumably due to endogenous potassium current, remain, for which the current-voltage relationship is shown in D. Current/Voltage relationship for the data in panel C.
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Fig. 5. Slow inactivation of the Para/TipE sodium channel expressed in Xenopus oocytes. See text for description. The oocyte holding potential was set at 80 mV using the two-electrode voltage clamp method, and currents elicited by voltage steps from 60 to +40 mV were recorded. The oocyte was impaled with the electrodes and observed for 2 min before records were taken, to ensure that the holding current was stable and that the oocyte was in good physiological condition. Panel A shows the inward voltage-sensitive current flowing through the sodium channels. Slow inactivation was elicited by holding the membrane at +20 mV for 2 min. Panel B shows that the sodium current had not fully recovered within 5 s of returning the holding potential to 80 mV. (C) The amplitude of the current returned to its original value within 2 min. (D) 21 min after the return of this oocyte to a holding potential of 70 mV, the amplitude of the currents increased beyond that of our original record, an indication that additional channels had made the transition from slow inactivation to the resting state.
The Na+ channel-blocking action of SCBIs is enhanced under depolarizing conditions, because the compounds selectively block the channels in the slowinactivated state. The Para/TipE Na+ channel expressed in the Xenopus oocyte offers a good opportunity for examining the state dependence of action of metaflumizone. Fig. 5 demonstrates the slow inactivation process and the slow recovery of the channels from slow inactivation. In the control, step depolarizations from the holding potential of 80 mV evoked sodium currents with a peak amplitude of approximately 1 mA. After depolarization to +20 mV for 2 min, sodium current was completely inactivated. In panel B, the currents were still depressed after a 5 s recovery period, but after 2 min they were fully recovered (Fig. 5C). The percentage of channels in the slow-inactivated state varies as a function of the holding potential. If the holding potential is increased, for example to 80 mV, the sodium current amplitude increases gradually over the course of 10 min, as previously inactive channels are
recruited into the pool of resting channels. Since resting channels are not sensitive to SCBIs, the sensitivity to block by metaflumizone should decrease at 80 mV. As can be seen in Fig. 6, metaflumizone, applied for 10 min at 80 mV, had very little effect on the sodium current, up to a concentration of 10 mM. On the other hand, even 0.1 mM metaflumizone significantly depressed the sodium current under depolarizing conditions (Fig. 7), confirming the voltage dependence of its action. In this experiment, the oocyte was clamped at a holding potential of 80 mV, where metaflumizone loses its potency. Panel A shows the currents that were unchanged from control levels by 0.1 mM metaflumizone added to the superfusing saline. Panel B shows that when slow inactivation was induced by a 2 min shift of the holding potential to +20 mV, the continuous presence of metaflumizone abolished the ability of the channels to fully recover. The more positive the holding potential relative to 80 mV, the greater the channel’s sensitivity to metaflumizone, confirming the expected voltage dependence of action.
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Fig. 6. Metaflumizone is inactive when the Para/TipE Na+ channel expressed in Xenopus oocytes channel is prevented from entering slow inactivation by holding at 80 mV. In an attempt to drive the Para/TipE sodium channels out of the slow-inactivated state and into the resting state, this oocyte was held at 80 mV. Current families were elicited in a single oocyte during exposure to increasing concentrations of metaflumizone. In each panel a series of voltage steps from 60 to +40 mV was used to elicit a current family. (A) The family of sodium currents elicited in the pretrial run. (B) A 5 min exposure to 0.1 mM metaflumizone had no effect on the Na+ current. (C) Five minutes after increasing the metaflumizone concentration to 1 mM, there was still no effect. (D) A slight decrease in the maximal current amplitude was observed for 5 min after increasing the metaflumizone concentration to 10 mM.
Fig. 7. Metaflumizone is active at depolarized potentials on Para/TipE Na+ channels expressed in Xenopus oocytes. The holding potential was set at 80 mV and current families were elicited by delivering voltage steps from 60 to +40 mV. (A) As in Fig. 8, treatment with metaflumizone at a holding potential of 80 mV had no effect. (B) Depolarization to +20 mV for 2 min during metaflumizone exposure enabled the compound to block the channels, and recovery was still incomplete for 20 min after return to 80 mV.
4. Conclusion The results shown here confirm that the novel, semicarbazone insecticide metaflumizone is a SCBI,
with the same mode of action on insects, a statedependent block of Na+ channels, as the pyrazolines from which it was chemically derived. Metaflumizone is the first product with this mode of action in the animal
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health market. Indoxacarb, also derived from the pyrazolines, but chemically very different from metaflumizone, is the only other registered insecticide with this mode of action. Indoxacarb is used primarily against lepidopteran pests, but it also controls many species of plant bugs, leafhoppers, fleahoppers, weevils, beetles, flies, cockroaches and ants (Wing et al., 2005). The voltage-dependent Na+ channel-blocking action of SCBIs is similar to that of local anesthetics, Class I anticonvulsants, and Class I antiarrhythmics (Salgado, 1992), a structurally broad range of drugs (Clare et al., 2000; Anger et al., 2001) all known to act at a common blocker site within the Na+ channel pore. Access to the blocker site is facilitated by opening of the activation gate, giving rise to use dependence. Additionally, channel gating also modulates the equilibrium binding affinity of the channel for the molecules: there is little affinity of the blocker molecule for the resting state, but strong binding to the open and inactivated states. Several excellent reviews on the interaction of blocking drugs with Na+ channels have recently been published (Clare et al., 2000; Anger et al., 2001; Wang and Wang, 2003). SCBIs behave like local anesthetics with very slow kinetics, and do not display use dependence (Zhao et al., 2003), suggesting that the insecticides are not reaching their binding site through the internal mouth of the pore. Although metaflumizone shares the same target site as indoxacarb, there is no known target site resistance at that site. Metabolic resistance to indoxacarb has been documented (Shono et al., 2004), but this would not be expected to confer cross-resistance to metaflumizone, because of the structural differences. Consistent with this, a pyrethroid-resistant strain of Spodoptera litura that was cross-resistant to indoxacarb was highly susceptible to metaflumizone (Behm et al., 2004). Furthermore, insect strains resistant to imidacloprid, carbamates, organophosphates and avermectins have not shown cross-resistance to metaflumizone (Cutler et al., 2006; Behm et al., 2004). References Anger, T., Madge, D.J., Mulla, M., Riddall, D., 2001. Medicinal chemistry of neuronal voltage-gated Na+ channel blockers. J. Med. Chem. 44, 115–137. BASF Agricultural products, 2007. Metaflumizone Worldwide Technical Brochure. Behm, J., Farlow, R.A., Oloumi-Sadeghi, H., Mallipudi, N.M., Wolff, M.A., Mahl, T.A., 2004. BAS 320 I: A new insecticide from BASF. Poster presented at annual meeting. Entomological Society of America. Clare, J.J., Tate, S.N., Nobbs, M., Romanos, M.A., 2000. Voltagegated Na+ channels as therapeutic targets. Drug Discov. Today 5, 506–520.
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Cutler, C., Scott-Dupree, C.D., Dugas, M.L., Wang, D.-Y., 2006. Susceptibility of Canadian Colorado potato beetle populations to imidacloprid and metaflumizone. Resistant Pest Manage. Newslett. 16, 33–36. Feng, G., Deak, P., Chopra, M., Hall, L.M., 1995. Cloning and functional analysis of tipE, a novel membrane protein that enhances Drosophila para sodium channel function. Cell 82, 1001–1011. Fenwick, E.M., Marty, A., Neher, E., 1982. A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. 331, 577–597. Grosscurt, A.C., van Hes, R., Wellinga, K., 1979. 1-Phenylcarbonyl-2pyrazolines, a new class of insecticides. 3. Synthesis and insecticidal properties of 3,4-diphenyl-1-phenylcarbamoyl-2-pyrazolines. J. Agric. Food Chem. 27, 406–409. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100. Hayashi, J.H., Hildebrand, J.G., 1990. Insect olfactory neurons in vitro: morphological and physiological characterization of cells from the developing antennal lobes of Manduca Sexta. J. Neurosci. 10, 848–859. Loughney, K., Kreber, R., Ganetzky, B., 1989. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58, 1143–1154. Mulder, R., Wellinga, K., vn Daalen, J.J., 1975. A new class of insecticides. Naturwissenschaften 62, 531–532. Payne, G.T., Deecher, D.C., Soderlund, D.M., 1998. Structure–activity relationships for the action of dihydropyrazole insecticides on mouse brain sodium channels. Pest. Biochem. Physiol. 60, 177– 185. Rugg, D., Hair, J.A., 2007. Dose determination of a novel formulation of metaflumizone plus amitraz for the treatment and control of fleas (Ctenocephalides felis) and ticks (Rhipicephalus sanguineus) on dogs. Vet. Parasitol. 150, 203–208. Salgado, V.L., 1990. Mode of action of insecticidal dihydropyrazoles: selective block of impulse generation in sensory nerves. Pest. Sci. 28, 389–411. Salgado, V.L., 1992. Slow voltage-dependent block of Na+ channels in crayfish nerve by dihydropyrazole insecticides. Mol. Pharmacol. 41, 120–126. Shono, T.L., Zhang, L., Scott, J.G., 2004. Indoxacarb resistance in the house fly, Musca domestica. Pest. Biochem. Physiol. 80, 106–112. Stu¨hmer, W., 1992. Electrophysiological recording from Xenopus oocytes. Methods in Enzymology. Ion Channels, vol. 207. Academic Press, New York, pp. 319–339. Takagi, K., Hamaguchi, H., Nishimatsu, T., Konno, T., 2007. Discovery of metaflumizone, a novel semicarbazone insecticide. Vet. Parasitol. 150, 177–181. Wang, S.Y., Wang, G.K., 2003. Voltage-gated Na+ channels as primary targets of diverse lipid-soluble neurotoxins. Cell. Signal. 15, 151–159. Wing, K.D., Andaloro, J.T., McCann, S.F., Salgado, V.L., 2005. Indoxacarb and the Na+ channel blockers: chemistry, physiology and biology in insects. In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, Insect Control, vol. 6. Elsevier B.V., Oxford, UK, pp. 30–53. Zhao, X., Ikeda, T., Yeh, J.Z., Narahashi, T., 2003. Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW. Neurotoxicology 24, 83–96.
Veterinary Parasitology 150 (2007) 182–189 www.elsevier.com/locate/vetpar
Metaflumizone is a novel sodium channel blocker insecticide V.L. Salgado a,*, J.H. Hayashi b a
BASF Corporation, 26 Davis Drive, Research Triangle Park, NC 27709, USA b P.O. Box 786, Princeton Junction, NJ 08550, USA
Abstract Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips–Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide. # 2007 Elsevier B.V. All rights reserved. Keywords: Sodium channel blocker insecticide; Metaflumizone; Indoxacarb; Local anesthetic
1. Introduction Metaflumizone (IUPAC (EZ)-20 -[2-(4-cyanophenyl)1-(a,a,a-trifluoro-m-tolyl)ethylidene]-4-(trifluoromethoxy)carbanilohydrazide, CAS 2-[2-(4-cyanophenyl)-1-[3-(trifluoromethyl)phenyl]ethylidine]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarboxamide) is a new insecticidal compound discovered by Nihon Nohyaku Co. Ltd. (Takagi et al., 2007). This compound is now being co-developed globally by BASF Aktiengesellschaft, Fort Dodge Animal Health, a Division of Wyeth Corporation and Nihon Nohyaku Co. Ltd. Metaflumizone provides good to excellent control of most economically important lepidopterous pests and
* Corresponding author. Tel.: +1 919 547 2244; fax: +1 919 547 2450. E-mail address: [email protected] (V.L. Salgado). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.08.032
certain pests in the orders Coleoptera, Hemiptera, Hymenoptera, Diptera, Isoptera and Siphonaptera (BASF, 2007). Metaflumizone provides long-lasting control of fleas on animals with a single spot-on application and is being developed for this use under the trade names ProMeris1 and ProMeris Duo1 by Fort Dodge Animal Health, Overland Park, KS, USA (Rugg and Hair, 2007). The semicarbazone metaflumizone is an open-ring analog of the 3,4-diphenyl-1-phenylcarbamoyl-2-pyrazolines (Grosscurt et al., 1979), which in turn evolved from the 3-phenyl-1-phenylcarbamoyl-2-pyrazolines discovered at Philips–Duphar in the early 1970s (Mulder et al., 1975). The relationship between these three families is shown in Fig. 1. The pyrazolines paralyze insects and block nerve activity by blocking voltage-dependent sodium channels. The sodium channels are most sensitive at depolarized potentials, so that tonic sensory receptors
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Fig. 1. Chemical evolution of the semicarbazones exemplified by metaflumizone, from the first pyrazoline SCBIs.
and pacemaker neurons are the most sensitive, giving a type of paralysis in which the nervous system shows little or no spontaneous activity and the insects appear paralyzed, but can be stimulated to convulse by activation of phasic sensory receptors. These convulsions cease after several seconds, as more sodium channels become blocked, and the insect lapses back into a pseudo-paralyzed state (Salgado, 1990). There is a refractory period after these convulsions, during which the sodium channels in phasic neurons and axons recover from block. The pyrazolines appear to block sodium channels by binding at the local anesthetic site (Salgado, 1992; Payne et al., 1998). However, in contrast to local anesthetics and other drugs, which can access the binding site through the intracellular mouth of the open pore, entry and exit of the pyrazolines is very slow, on the order of minutes, which precludes the direct block of open channels often seen with local anesthetics. The pyrazolines interact very slowly with the channels, and appear to access the receptor primarily through lateral diffusion from the lipid bilayer, blocking equally well from either face of the membrane (Salgado, 1992). Because of their slow action, the pyrazolines appear to interact specifically with channels in the slow-inactivated state, but when slow and fast-inactivation were progressively removed by treatment of the intracellular-exposed surfaces of sodium channels with trypsin and N-bromoacetamide (NBA), respectively, block of fast-inactivated and open channels by the pyrazoline RH-1211 proceeded at least as rapidly as block of slow-inactivated channels (Salgado, 1992). Indoxacarb, a member of the oxadiazine subclass of SCBIs, was the first commercialized sodium channelblocking insecticide (SCBI) (Wing et al., 2005); metaflumizone is the first SCBI in the animal health market. In the present paper, we confirm, using in vivo and in vitro experiments, that state-dependent block of
voltage-dependent sodium channels is the mechanism of action of metaflumizone. 2. Materials and methods 2.1. Mosquito larvicide test Ten early third instar Aedes aegypti larvae were placed into 2 mL tap water in a well of a 12-well microtitre plate. Compound was dissolved in DMSO and added to the well at a DMSO concentration not exceeding 0.1%, which itself had no observable effect on the larvae. The treated insects were held at room temperature and observed hourly for the first 8 h, and daily thereafter. 2.2. Spodoptera eridania gross electrophysiology Fifth instar larvae of the southern armyworm, S. eridania, weighing 400–600 mg, were injected with compound dissolved in 1 mL of DMSO using a Hamilton syringe fitted with a 30-gauge needle. The tip of the needle was inserted dorsolaterally in the abdomen, just beneath the cuticle. Paralyzed insects were dissected and investigated electrophysiologically with bipolar hook electrode recordings from CNS connectives and segmental nerves, in comparison with control insects. Recordings were made in the insects’ own hemolymph. 2.3. Spodoptera frugiperda stretch receptor For measurements on the abdominal stretch receptor organ, a fifth instar fall armyworm, S. frugiperda, larva was dissected and pinned out in a wax dish. Because the organs lie dorsolaterally, the cut was made dorsolaterally on one side, so that the organs on the other side would not be damaged. The gut and fat body were removed and three or four segments containing nerve cord and body wall were removed and pinned in a
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perfusion chamber. A stretch receptor organ was exposed and perfused directly with Lepidoptera saline, containing (in mM): 128 NaCl, 16 KCl, 29 CaCl2, 18 MgCl2, 175 sucrose, 3 NaH2PO4, at pH 6.5. A suction electrode was used to record the stretch receptor activity. Successful preparations fired continuously at a rate between 20 and 50 s1. Compounds were introduced into the perfusion solution. 2.4. Manduca sexta neurons Techniques and media used for isolation and wholecell recording from primary-cultured insect neurons were as described by Hayashi and Hildebrand (1990). Briefly, nerve cords from fifth instar larvae of the tobacco hornworm, M. sexta, were removed into sterile culture saline. The thoracic and abdominal ganglia were dissected free from the nerve sheath and dissociated by treatment with 0.125 mg ml1 collagenase and 0.5 mg ml1 dispase in Ca2+, Mg2+-free Hank’s solution at 37 8C for 2.5 min, followed by trituration with a fire-polished Pasteur pipette. The enzyme activity was stopped by centrifuging the cells through culture saline (in mM, 149.9 NaCl, 3 KCl, 3 CaCl2, 0.5 MgCl2, 10 TES, 11 Dglucose, plus 2 g L1 lactalbumin hydrolysate, 2 g L1 yeastolate) at 207 g for 8 min at 10 8C, re-suspending the pellet in I-L15 (a supplemented version of Liebovitz’s L-15 medium) and re-centrifuging as before through IL15. The pellet was re-suspended in culture medium (150 mL per ganglion) and plated onto 35 mm Petri dishes with mini-wells that had glass cover slip bottoms coated with a mixture of concanavalin A and laminin. Cells were allowed to adhere for 1.5 h, and the dish was then flooded with 1.5 mL of I-L15 growth medium. Sodium currents could be recorded for up to 2 days in culture. Action potentials and Na+ currents were recorded with standard whole-cell patch clamp methods, (Hamill et al., 1981; Fenwick et al., 1982) using patch pipettes with a resistance between 3 and 5 MV. The pipettes were filled with a Kasp solution, containing (in mM): 150 Kaspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 2 ATP, 5 HEPES, and pH 7.0, with the addition of mannitol to adjust the osmolality to 330 mOsm. The external solution was AIS, containing (in mM): 150 NaCl, 4 KCl, 6 CaCl2, 10 HEPES, 5 glucose, and pH 7.0, with the addition of mannitol to adjust the osmolality to 360 mOsm.
the African clawed frog (Xenopus) were defolliculated with collagenase and injected with Drosophila melanogaster Para sodium channel cRNA (0.2–2 ng), together with D. melanogaster TipE cRNA (0.2– 2 ng), which is known to enhance the expression of insect sodium channels in oocytes (Feng et al., 1995). After incubation for 2–7 days, an oocyte was placed in the recording chamber and superfused with ND96 solution, containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and pH 7.4. Two microelectrodes containing 3 M KCl, with tip resistances of 2–5 MV, were inserted, and the oocyte was voltage clamped with a TEC-03X amplifier (NPI electronic). Test compound was added from a DMSO stock solution to the ND96 superfusion solution at a final DMSO concentration not exceeding 0.1%, a DMSO concentration which had no effect on the sodium currents. 3. Results and discussion 3.1. Mosquito larvicide test Ninety-five percent of third instar A. aegypti larvae exposed to 40 mM metaflumizone were slow or paralyzed at 1 h and 100% were paralyzed within 3 h. The compound was quite active at lower concentrations, giving 100% kill within 24 h at 100 nM, but 0% kill within 3 days at 30 nM.
3.2. S. eridania gross electrophysiology Metaflumizone injected into fifth instar larvae of S. eridania, at a dose of 15 mg g1, produced 100% prostration within 4 h, and paralysis within 24 h. Gross electrophysiological analysis of the paralyzed insects at 24 h after treatment showed that all nerve activity was blocked, as is also seen in insects poisoned by pyrazolines and indoxacarb (Salgado, 1990; Wing et al., 2005). Table 1 Action of increasing concentrations of metaflumizone on the abdominal stretch receptor organ of Spodoptera frugiperda: percent of block within 1 h Concentration (M)
2.5. Xenopus oocyte expression and voltage clamp The procedures for oocyte preparation, cRNA injection and voltage clamp recording are identical to those described by Stu¨hmer (1992). Briefly, oocytes of
7
10 3 107 106 3 106 105
Block (%) 0 0 50 100 100
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Fig. 2. Block of action potentials by 1 mM metaflumizone in a neuron isolated from the CNS of a Manduca sexta larva. The cell had been in culture for 1-day and was current-clamped with a patch pipette at a potential of 60 mV. The external solution was AIS and the internal solution was Kasp. The six panels show successive action potentials overlaid on the control. The stimulus artifact can be seen, which generates an action potential with a positive phase lasting about 1.5 ms, with an amplitude of 55 mV. After addition of metaflumizone, the action potential was gradually blocked. Block was complete at 20 min, when only an electrotonic response remained.
3.3. S. frugiperda stretch receptor Recordings made from abdominal stretch receptor organs of S. frugiperda confirmed the direct blocking effect on action potential generation. These tonic mechanoreceptors normally generate action potentials constantly at a low rate and are very sensitive to block by SCBIs (Salgado, 1990; Wing et al., 2005). Metaflumizone blocked S. frugiperda stretch receptor activity with a threshold concentration of 106 M, but had no effect at 3 107 M within 1 h (Table 1). Sodium channels in sensory organs such as the stretch receptor organ are very sensitive to sodium channel blockers (Salgado, 1990), which are generally selective for activated or inactivated channel states. Because these sensory neurons are constantly active, their Na channels cycle between all states and are therefore available to state-dependent blockers (Salgado, 1992; Wing et al., 2005). 3.4. M. sexta neurons Block of the action potential was investigated directly on neurons isolated from the central nervous system (CNS) of M. sexta larvae and studied in wholecell current clamp mode. Fig. 2 shows the recordings from a M. sexta neuron at a resting potential of 60 mV, demonstrating complete block of the action potential by 106 M metaflumizone over a time course of 20 min. This result confirms that metaflumizone directly affects action potential generation of native Na+ channels.
Fig. 3. Block of the sodium current by 5 mM metaflumizone in a neuron isolated from the CNS of a Manduca sexta larva. The cell had been in culture for 1-day and voltage was clamped with a patch pipette, at a potential of 70 mV. The external solution was AIS containing 30 mM TEA, 20 mM 4-AP and 500 mM Cd2+, and the KAsp internal solution was used. (A) Control family of sodium currents for steps from 70 to +40 mV. (B) Ten minutes in 5 mM metaflumizone. (C) Twenty minutes in 5 mM metaflumizone.
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To assess the effect of metaflumizone on sodium current directly, Na+ channels were studied in primarycultured neurons isolated from the CNS of fifth instar M. sexta larvae, with the whole-cell voltage clamp method. In the presence of external 30 mM tetraethylammonium (TEA) and 20 mM 4-aminopyridine (4AP) to block potassium channels, and 500 mM Cd2+ to block voltage-dependent calcium channels, depolarizing voltage steps activated voltage-gated, tetrodotoxin sensitive, sodium channels that give rise to an inward sodium current. Under maintained step depolarization, about 50% of the channels inactivate, but a current is maintained throughout the duration of the 150 ms depolarizing pulse (Fig. 3A). After application of 5 mM metaflumizone, the peak and maintained portions of the sodium current gradually decreased together and completely disappeared by 20 min (Fig. 3B and C). This confirms that metaflumizone directly blocks the native insect Na+ channels.
3.5. Xenopus oocyte expression and voltage clamp The insect Na+ channel consists of a single channelforming subunit that has been named Para because it is coded for by a gene in which mutations can cause paralysis in Drosophila (Loughney et al., 1989). A small accessory subunit known as TipE, which is unrelated to the beta subunit found in vertebrate sodium channels, is required for functional expression. When Para and TipE are co-expressed in the Xenopus oocyte expression system, voltage-dependent sodium currents are obtained that exhibit all of the excitable and pharmacological properties of normal insect sodium channels (Feng et al., 1995). Fig. 4A shows the Para/ TipE sodium currents evoked by step depolarizations between 60 and +50 mV. The current–voltage relation is plotted in Fig. 4B. After treatment with 10 mM metaflumizone, the currents were completely blocked (Fig. 4C and D).
Fig. 4. Block of Para/TipE sodium channels expressed in Xenopus oocytes, by metaflumizone. Inward currents were observed for 3 days after injection with Para and TipE RNA. External solution consisted of ND96. The oocyte was clamped using the two-electrode method, and the holding potential was set at 70 mV. The current responses to steps to various potentials are superimposed in the lower portion of panel A, with the voltage steps illustrated in the upper portion. These data are plotted in a current/voltage relationship in B. The activation point is approximately 40 mV. Panel C shows that the inward current was blocked by 10 mM metaflumizone. Small outward currents, presumably due to endogenous potassium current, remain, for which the current-voltage relationship is shown in D. Current/Voltage relationship for the data in panel C.
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Fig. 5. Slow inactivation of the Para/TipE sodium channel expressed in Xenopus oocytes. See text for description. The oocyte holding potential was set at 80 mV using the two-electrode voltage clamp method, and currents elicited by voltage steps from 60 to +40 mV were recorded. The oocyte was impaled with the electrodes and observed for 2 min before records were taken, to ensure that the holding current was stable and that the oocyte was in good physiological condition. Panel A shows the inward voltage-sensitive current flowing through the sodium channels. Slow inactivation was elicited by holding the membrane at +20 mV for 2 min. Panel B shows that the sodium current had not fully recovered within 5 s of returning the holding potential to 80 mV. (C) The amplitude of the current returned to its original value within 2 min. (D) 21 min after the return of this oocyte to a holding potential of 70 mV, the amplitude of the currents increased beyond that of our original record, an indication that additional channels had made the transition from slow inactivation to the resting state.
The Na+ channel-blocking action of SCBIs is enhanced under depolarizing conditions, because the compounds selectively block the channels in the slowinactivated state. The Para/TipE Na+ channel expressed in the Xenopus oocyte offers a good opportunity for examining the state dependence of action of metaflumizone. Fig. 5 demonstrates the slow inactivation process and the slow recovery of the channels from slow inactivation. In the control, step depolarizations from the holding potential of 80 mV evoked sodium currents with a peak amplitude of approximately 1 mA. After depolarization to +20 mV for 2 min, sodium current was completely inactivated. In panel B, the currents were still depressed after a 5 s recovery period, but after 2 min they were fully recovered (Fig. 5C). The percentage of channels in the slow-inactivated state varies as a function of the holding potential. If the holding potential is increased, for example to 80 mV, the sodium current amplitude increases gradually over the course of 10 min, as previously inactive channels are
recruited into the pool of resting channels. Since resting channels are not sensitive to SCBIs, the sensitivity to block by metaflumizone should decrease at 80 mV. As can be seen in Fig. 6, metaflumizone, applied for 10 min at 80 mV, had very little effect on the sodium current, up to a concentration of 10 mM. On the other hand, even 0.1 mM metaflumizone significantly depressed the sodium current under depolarizing conditions (Fig. 7), confirming the voltage dependence of its action. In this experiment, the oocyte was clamped at a holding potential of 80 mV, where metaflumizone loses its potency. Panel A shows the currents that were unchanged from control levels by 0.1 mM metaflumizone added to the superfusing saline. Panel B shows that when slow inactivation was induced by a 2 min shift of the holding potential to +20 mV, the continuous presence of metaflumizone abolished the ability of the channels to fully recover. The more positive the holding potential relative to 80 mV, the greater the channel’s sensitivity to metaflumizone, confirming the expected voltage dependence of action.
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Fig. 6. Metaflumizone is inactive when the Para/TipE Na+ channel expressed in Xenopus oocytes channel is prevented from entering slow inactivation by holding at 80 mV. In an attempt to drive the Para/TipE sodium channels out of the slow-inactivated state and into the resting state, this oocyte was held at 80 mV. Current families were elicited in a single oocyte during exposure to increasing concentrations of metaflumizone. In each panel a series of voltage steps from 60 to +40 mV was used to elicit a current family. (A) The family of sodium currents elicited in the pretrial run. (B) A 5 min exposure to 0.1 mM metaflumizone had no effect on the Na+ current. (C) Five minutes after increasing the metaflumizone concentration to 1 mM, there was still no effect. (D) A slight decrease in the maximal current amplitude was observed for 5 min after increasing the metaflumizone concentration to 10 mM.
Fig. 7. Metaflumizone is active at depolarized potentials on Para/TipE Na+ channels expressed in Xenopus oocytes. The holding potential was set at 80 mV and current families were elicited by delivering voltage steps from 60 to +40 mV. (A) As in Fig. 8, treatment with metaflumizone at a holding potential of 80 mV had no effect. (B) Depolarization to +20 mV for 2 min during metaflumizone exposure enabled the compound to block the channels, and recovery was still incomplete for 20 min after return to 80 mV.
4. Conclusion The results shown here confirm that the novel, semicarbazone insecticide metaflumizone is a SCBI,
with the same mode of action on insects, a statedependent block of Na+ channels, as the pyrazolines from which it was chemically derived. Metaflumizone is the first product with this mode of action in the animal
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health market. Indoxacarb, also derived from the pyrazolines, but chemically very different from metaflumizone, is the only other registered insecticide with this mode of action. Indoxacarb is used primarily against lepidopteran pests, but it also controls many species of plant bugs, leafhoppers, fleahoppers, weevils, beetles, flies, cockroaches and ants (Wing et al., 2005). The voltage-dependent Na+ channel-blocking action of SCBIs is similar to that of local anesthetics, Class I anticonvulsants, and Class I antiarrhythmics (Salgado, 1992), a structurally broad range of drugs (Clare et al., 2000; Anger et al., 2001) all known to act at a common blocker site within the Na+ channel pore. Access to the blocker site is facilitated by opening of the activation gate, giving rise to use dependence. Additionally, channel gating also modulates the equilibrium binding affinity of the channel for the molecules: there is little affinity of the blocker molecule for the resting state, but strong binding to the open and inactivated states. Several excellent reviews on the interaction of blocking drugs with Na+ channels have recently been published (Clare et al., 2000; Anger et al., 2001; Wang and Wang, 2003). SCBIs behave like local anesthetics with very slow kinetics, and do not display use dependence (Zhao et al., 2003), suggesting that the insecticides are not reaching their binding site through the internal mouth of the pore. Although metaflumizone shares the same target site as indoxacarb, there is no known target site resistance at that site. Metabolic resistance to indoxacarb has been documented (Shono et al., 2004), but this would not be expected to confer cross-resistance to metaflumizone, because of the structural differences. Consistent with this, a pyrethroid-resistant strain of Spodoptera litura that was cross-resistant to indoxacarb was highly susceptible to metaflumizone (Behm et al., 2004). Furthermore, insect strains resistant to imidacloprid, carbamates, organophosphates and avermectins have not shown cross-resistance to metaflumizone (Cutler et al., 2006; Behm et al., 2004). References Anger, T., Madge, D.J., Mulla, M., Riddall, D., 2001. Medicinal chemistry of neuronal voltage-gated Na+ channel blockers. J. Med. Chem. 44, 115–137. BASF Agricultural products, 2007. Metaflumizone Worldwide Technical Brochure. Behm, J., Farlow, R.A., Oloumi-Sadeghi, H., Mallipudi, N.M., Wolff, M.A., Mahl, T.A., 2004. BAS 320 I: A new insecticide from BASF. Poster presented at annual meeting. Entomological Society of America. Clare, J.J., Tate, S.N., Nobbs, M., Romanos, M.A., 2000. Voltagegated Na+ channels as therapeutic targets. Drug Discov. Today 5, 506–520.
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Cutler, C., Scott-Dupree, C.D., Dugas, M.L., Wang, D.-Y., 2006. Susceptibility of Canadian Colorado potato beetle populations to imidacloprid and metaflumizone. Resistant Pest Manage. Newslett. 16, 33–36. Feng, G., Deak, P., Chopra, M., Hall, L.M., 1995. Cloning and functional analysis of tipE, a novel membrane protein that enhances Drosophila para sodium channel function. Cell 82, 1001–1011. Fenwick, E.M., Marty, A., Neher, E., 1982. A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. 331, 577–597. Grosscurt, A.C., van Hes, R., Wellinga, K., 1979. 1-Phenylcarbonyl-2pyrazolines, a new class of insecticides. 3. Synthesis and insecticidal properties of 3,4-diphenyl-1-phenylcarbamoyl-2-pyrazolines. J. Agric. Food Chem. 27, 406–409. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100. Hayashi, J.H., Hildebrand, J.G., 1990. Insect olfactory neurons in vitro: morphological and physiological characterization of cells from the developing antennal lobes of Manduca Sexta. J. Neurosci. 10, 848–859. Loughney, K., Kreber, R., Ganetzky, B., 1989. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58, 1143–1154. Mulder, R., Wellinga, K., vn Daalen, J.J., 1975. A new class of insecticides. Naturwissenschaften 62, 531–532. Payne, G.T., Deecher, D.C., Soderlund, D.M., 1998. Structure–activity relationships for the action of dihydropyrazole insecticides on mouse brain sodium channels. Pest. Biochem. Physiol. 60, 177– 185. Rugg, D., Hair, J.A., 2007. Dose determination of a novel formulation of metaflumizone plus amitraz for the treatment and control of fleas (Ctenocephalides felis) and ticks (Rhipicephalus sanguineus) on dogs. Vet. Parasitol. 150, 203–208. Salgado, V.L., 1990. Mode of action of insecticidal dihydropyrazoles: selective block of impulse generation in sensory nerves. Pest. Sci. 28, 389–411. Salgado, V.L., 1992. Slow voltage-dependent block of Na+ channels in crayfish nerve by dihydropyrazole insecticides. Mol. Pharmacol. 41, 120–126. Shono, T.L., Zhang, L., Scott, J.G., 2004. Indoxacarb resistance in the house fly, Musca domestica. Pest. Biochem. Physiol. 80, 106–112. Stu¨hmer, W., 1992. Electrophysiological recording from Xenopus oocytes. Methods in Enzymology. Ion Channels, vol. 207. Academic Press, New York, pp. 319–339. Takagi, K., Hamaguchi, H., Nishimatsu, T., Konno, T., 2007. Discovery of metaflumizone, a novel semicarbazone insecticide. Vet. Parasitol. 150, 177–181. Wang, S.Y., Wang, G.K., 2003. Voltage-gated Na+ channels as primary targets of diverse lipid-soluble neurotoxins. Cell. Signal. 15, 151–159. Wing, K.D., Andaloro, J.T., McCann, S.F., Salgado, V.L., 2005. Indoxacarb and the Na+ channel blockers: chemistry, physiology and biology in insects. In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, Insect Control, vol. 6. Elsevier B.V., Oxford, UK, pp. 30–53. Zhao, X., Ikeda, T., Yeh, J.Z., Narahashi, T., 2003. Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW. Neurotoxicology 24, 83–96.