Locating the site of general anesthetic actions on proteins is difficult (1). One approach applies custom-designed photoactivable general anesthetics. Stable under normal conditions, these drugs covalently insert into their binding site when activated by light. Few of these drugs have been used as clinical anesthetics, but a photoactivable derivative of etomidate [2-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate] has been produced (2,3). The usefulness of this drug, azietomidate [2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate], which introduces a —C(N2)—CH2— group into the 2-ethyl group of etomidate, depends on how faithfully it mimics etomidate. Azietomidate has effects similar to those of etomidate, including a capacity to both abolish the righting reflex in tadpoles and enhance the response of gamma-aminobutyric acid (GABA)A receptors to GABA in vitro (2), but its action on mammals has not been examined.
Etomidate (4,5) purportedly acts primarily by enhancing the response of GABAA receptors to GABA. Confirmation that these anesthetics act in this manner comes from the in vivo observation that mice bearing a single point mutation, N265M, within the β3 subunit of the GABAA receptor resist various anesthetic actions of etomidate. For example, they recover their righting reflexes much more rapidly than wild type (WT) mice (6). It is not known whether the N265 residue forms part of the binding site for etomidate or, rather, whether it changes the conformation of the receptor resulting in insensitivity to these drugs (7).
In the present study, we first asked whether the finding that azietomidate abolishes the righting reflex in tadpoles applies to a higher-order organism, mice. Finding this to be so, we then assessed the relative importance of the GABA-ergic contribution to general anesthesia of etomidate and azietomidate by comparing their abilities to abolish the righting reflexes of WT mice versus mice bearing the above-noted N265M mutation in the β3 subunit of the GABAA receptor.
The Committee on Animal Research of the University of California, San Francisco approved our study of mice of both sexes consisting of WT mice and mice having an altered β3 subunit point mutation (N265M) (129/SvJ × 129/Sv). The embryonic stem cell used for gene targeting (R1) was a 129/SvJ × 129/Sv hybrid. The chimeras were crossed twice with 129/SvJ mice, so that the theoretical contribution of the 129/SvJ substrain was 87.5% and that of the 129/Sv substrain was 12.5%. Breeding pairs that were the result of two generations of homozygous mutant and WT breeding, respectively, were transferred from Zurich to San Francisco, and the first generation offspring of these pairs provided the subjects for the present studies (8). The offspring were 8–14 wk of age when studied. Animals were housed in our animal care facility under 12 h light and dark cycles and had continuous access to standard mouse chow and tap water before the study. Experiments were performed between 0900 and 1500 h.
Etomidate (Bedford Laboratories, Bedford, OH) and azietomidate (synthesized as previously described) (2), both as the R (+) isomer, were dissolved as 2% solutions in 35% propylene glycol. To secure venodilation, test mice were exposed to a warming lamp until they increased activity. They were then confined in restraint devices (Kent Scientific, Torrington, CT) from which their tails protruded. A tail vein was identified and cannulated with a 28-gauge needle connected to a syringe containing the predetermined dose (in mg/kg) of etomidate or azietomidate. After injection of the test drug, the mouse was freed from restraint, laid on its back, and a stopwatch started. The time from injection to spontaneous turning of the mouse to a prone position (defined as a position in which all four paws touched the tabletop) was recorded. The same mouse might be used two or three times to assess the effect of etomidate versus azietomidate or different doses of either drug. Both etomidate and azietomidate appeared to produce anesthesia, but we did not stimulate the mice during the assessment of the time to a return of righting reflexes. The largest doses of both etomidate and azietomidate killed occasional mice (for etomidate, one mouse at 10 mg/kg and two at 15 mg/kg; for azietomidate, one each at 10 and 20 mg/kg).
The statistical power of the comparison between drugs and types of mice may be increased if each complete group of animals, regardless of dose, is combined. To do this, a relationship between the dose and the time to righting must be defined. After an IV bolus of etomidate the plasma concentration decreases from its peak level until it is no longer sufficient to maintain anesthesia and the mouse rights itself at a time tr, the time to righting. The plasma concentration at which this occurs for a given drug will be the same for each animal within normal biological variation. Assuming the plasma concentration decreases following first order kinetics, tr will increase linearly with the logarithm of the dose. That is, the data may be fit to the expression tr = m × Log(dose) − c, where m is the slope and c is the intercept when time to righting is plotted as a function of the logarithm of the dose. The value of m depends on the half-life for elimination of the drug (9). We assumed that this half-life depends only on the drug and that the point mutation in the GABA receptor does not alter the pharmacodynamics of elimination. The latter assumption is supported by the finding that IV steroid anesthetics, whose potency is unaffected by the mutation, have identical sleep times in WT and mutant mice (6). The data for each drug for both WT and mutant mice were then fitted simultaneously by a global least squares procedure with the slope being a common parameter.
Etomidate (Fig. 1) anesthetized WT mice at doses ≥1.25 mg/kg. In general, groups of four to six mice were used, although a trial dose was occasionally abandoned after one or two animals (Figs. 1 and 2). The mean time ± sd to righting increased with dose; it was 3.5 ± 1.9 min at 1.25 mg/kg and 14 ± 8.0 min at 10 mg/kg. Azietomidate (Fig. 2) anesthetized WT mice at doses ≥2.5 mg/kg. The mean time to righting increased from 2.5 ± 2.3 min to 12 ± 3.7 min as the dose was increased from 2.5 to 10 mg/kg. These sleep times were somewhat shorter than those induced by equivalent doses of etomidate. However, because the number of animals in each group was small and the scatter of the data increased with increasing doses, the difference was only significant at the smallest dose. Thus, at 2.5 mg/kg etomidate’s mean time to righting was 3.6 ± 1.93 min longer than azietomidate’s (P = 0.02; two-tailed). Overall, we conclude that azietomidate causes general anesthesia in WT mice with a potency comparable to etomidate.
Etomidate (Fig. 1) anesthetized mutant mice at doses ≥2.5 mg/kg. In general, groups of four to six mice were used. The mean time to righting ± sd increased with dose; it was 0.2 ± 0.11 min at 2.5 mg/kg and 5.8 ± 2.3 min at 15 mg/kg. Azietomidate (Fig. 2) also anesthetized mutant mice at doses ≥2.5 mg/kg. The mean time to righting increased from 0.7 ± 0.34 min to 13 ± 7.5 min as the dose was increased from 5 to 20 mg/kg. A single animal was anesthetized at 2.5 mg/kg, righting itself after 0.6 min. These sleep times tended to be somewhat shorter than those induced by equivalent doses of etomidate. However, for the reasons stated above, the difference was only significant at the smallest dose. Thus, at 5 mg/kg etomidate’s mean time to righting was 2.5 ± 0.77 min longer than azietomidate’s (P < 0.01; two-tailed). Overall, we conclude that azietomidate causes general anesthesia in the mutant mice with a potency comparable to etomidate.
To better compare the shift in potency caused by the mutation, the data of each individual drug were combined by global fitting of the WT and mutant sets to the equation. The results are tabulated in Table 1 and displayed in Figures 1 and 2. The fitted parameters and their errors were then used to calculate the times to righting, tr, at a dose of 7.5 mg/kg, which was chosen to avoid extrapolation beyond the data. These times and their standard deviations are given in Table 2, where it can be seen that the β3 subunit N265M mutation significantly (P < 0.001) decreased tr by 7.6 ± 1.5 min and 7.2 ± 1.8 min (mean ± sd) for etomidate and azietomidate, respectively. These approximately threefold decreases in time to righting induced by the mutation dwarfed the slight tendency for azietomidate sleep times to be shorter than those of etomidate. However, any comparison between etomidate and azietomidate in a given mouse must be treated with reservation because the analysis in Table 1 suggests that azietomidate is eliminated one and a half times more rapidly than etomidate.
Although times to righting allow the comparison of WT to mutant animals in each case, estimating the true shift in potency in terms of dose is inherently complicated by pharmacodynamic factors. However, estimates of potency can be made using the parameters in Table 1. One way to minimize the pharmacodynamic issues is to calculate the threshold dose that causes loss of righting for an infinitely short time because this dose achieves the threshold anesthetic concentration in the brain on the first pass. These calculated values are presented in Table 3. Introduction of the mutation causes a marked increase in the dose to reach threshold for both etomidate and azietomidate by 2.4 and 3.3 mg/kg respectively, confirming that these two drugs are comparably affected by the mutation. Once again, etomidate was found to be somewhat more potent than azietomidate by a margin of 1.1 and 2 mg/kg in WT and mutant mice, respectively.
The actions of the recently developed photoactivable etomidate derivative, azietomidate, have been characterized in tadpoles and in GABAA and nicotinic receptors (2). In the present work, we further characterized the resemblance of its pharmacology to that of etomidate by studying its action in mammals. Azietomidate lacked obvious toxic effects, suggesting that the diazirine group is stable under biological conditions, allowing us to examine its effects in vivo. Husain et al. (2) determined the concentration-response curves for loss of righting reflexes in tadpoles immersed in aqueous solutions of these two drugs, finding them equally potent. Limited supplies of azietomidate precluded such a pseudo-equilibrium approach in the present study; instead we measured the duration of loss of righting reflexes to a single IV bolus of anesthetic. With this approach, our experiments demonstrated that equal doses of IV azietomidate and etomidate produced approximately equal durations of loss of righting reflexes in WT mice. Our conclusion is consistent with a study in rats that concluded that anesthetic potency in etomidate analogs is not particularly sensitive to the substituent on the ester group (10).
The key new finding in the present study resulted from our comparison of the action of azietomidate to etomidate on righting reflexes in mice bearing the N265M mutation in the β3 subunit of the GABAA receptor. This subtle change drastically attenuates the general anesthetic potency of etomidate (6). Our data show that this mutation caused a threefold decrease in the duration of loss of righting reflexes induced by both azietomidate and etomidate, suggesting that these two drugs cause loss of righting reflexes by similar mechanisms. Thus, accumulating evidence suggests that azietomidate’s pharmacology is equivalent to that of etomidate and that its capacity to photolabel will therefore provide useful insights into how etomidate and similar drugs act.
Although N265 in the β3 subunit of the GABAA receptor is a major determinant of the general anesthetic action of etomidate and azietomidate, it is premature to conclude that it is also the location of the etomidate binding site. Mutation at this site might also cause either a subtle allosteric conformation shift that modifies the geometry of a distant etomidate site or a change in the relative thermodynamic stability of conformations of the GABAA receptor that interact preferentially with etomidate. Azietomidate’s ability to photoincorporate into amino acid residues with which it is in contact offers a direct method of addressing such questions. Indeed, it has recently been used to characterize binding sites in the relatively abundant nicotinic acetylcholine receptor from Torpedo (3). This muscle subtype receptor is a close relative of neuronal GABAA receptors, suggesting that photolabeling this much less abundant receptor will provide definitive information on the location of those binding site or sites of etomidate that are involved in anesthesia.
The purpose of our exercise was to determine the usefulness of azietomidate in mechanistic studies of drugs such as etomidate. To be useful, azietomidate would have to act in a manner similar to that of etomidate. Our finding that the time to righting of etomidate and azietomidate decrease by comparable amounts when mutant mice are compared with WT mice (Table 2, Figs. 1 and 2) is consistent with a common mode of action.
1. Miller KW, Addona GH, Kloczewiak MA. Approaches to proving there are general anesthetic sites on ligand gated ion channels. Toxicol Lett 1998;101:139–47.
2. Husain S, Ziebell MR, Ruesch D, et al. 2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: A derivative of the stereoselective general anesthetic etomidate for photolabeling ligand-gated ion channels. J Med Chem 2003;46:1257–65.
3. Ziebell MR, Nirthanan S, Husain SS, et al. Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic. J Biol Chem 2004;279:17640–9.
4. Belelli D, Muntoni AL, Merrywest SD, et al. The in vitro
and in vivo
enantioselectivity of etomidate implicates the GABAA receptor in general anaesthesia. Neuropharmacology 2003;45:57–71.
5. Flood P, Krasowski MD. Intravenous anesthetics differentially modulate ligand-gated ion channels. Anesthesiology 2000;92:1418–25.
6. Jurd R, Arras M, Lambert S, et al. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 2003;17:250–2.
7. Belelli D, Lambert JJ, Peters JA, et al. The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci U S A 1997;94:11031–6.
8. Phillips TJ, Hen R, Crabbe JC. Complications associated with genetic background effects in research using knockout mice. Psychopharmacology (Berl) 1999;147:5–7.
9. Shafer SL. Principles of Pharmacokinetics and Pharmacodynamics. In: Longnecker DE, Tinker JH, Morgan GE, eds. Principles and Practice of Anesthesiology St. Louis: Mosby, 1998:1159–210.
10. Godefroi EF, Janssen PA, Vandereycken CA, et al. Dl-1-(1-arylalkyl)imidazole-5-carboxylate esters: A novel type of hypnotic agents. J Med Chem 1965;56:220–3.