In general, anesthetics are administered IV in clinical practice because of the need for rapid onset of action. However, when IV access is difficult to establish, as in small children and infants, muscle relaxants often have to be administered IM. Succinylcholine is the neuromuscular blocking drug for which the effects of IM administration have been the most studied (1), but it is associated with frequent adverse effects (2,3). Although some nondepolarizing neuromuscular blocking drugs have been used IM, the results have been less than optimal because the onset of action is slow (4–6). Some drugs, such as epinephrine, have been administered intratracheally (IT), and it has been shown that the IT route of administration may provide similar pharmacologic effects to IV administration of a drug, provided a larger dose is used (7). Prompted by these results that suggest that the IT route may also serve as a suitable alternative route of administration for muscle relaxants, we conducted a study in rats to ascertain the efficacy of vecuronium administered via the IT route, by comparing pharmacodynamic parameters after IT, IV, and IM administration of vecuronium. We also examined the pharmacokinetics of the drug to clarify the mechanism of its actions.
After obtaining approval from our institutional committee on animal research, we procured male Sprague-Dawley rats (7–8 wk old) for conducting the experiments. The animal rearing and experiments were conducted in accordance with the animal study guidelines established by Jikei University School of Medicine.
The animals were anesthetized by intraperitoneal injection of a mixture of pentobarbital sodium (40 mg/kg), urethane (500 mg/kg), and atropine sulfate (0.03 mg/kg), and the anesthetized rats were fixed in the supine position. The trachea was intubated without the administration of any neuromuscular blocking drug. An 18-gauge IV catheter was used as the tracheal tube. To avoid leakage from the glottis, a rubber stopper had been fixed at a point 15 mm from the tip of the IV catheter. Controlled mechanical ventilation was instituted using a Harvard 683 rodent ventilator (Harvard Apparatus, MA) with 100% O2, tidal volume 10 mL/kg and respiratory rate 84 breaths/min.
The right sciatic nerve was exposed to attach a small slide electrode (special order; Unique Medical, Tokyo, Japan). The tendon of the anterior tibial muscle was severed to connect a force transducer (Type-45196A; NEC San-ei, Tokyo, Japan). The sciatic nerve was stimulated using an electric stimulator (8EN-7203; NihonKohden, Tokyo, Japan) and an isolator (SS302-J; NihonKohden), at supramaximal stimulation (stimulation frequency, 0.1 Hz; stimulation width, 300 μs). Single twitch responses were amplified using a tension amplifier (AS1202; NEC, Tokyo, Japan) and a biological amplifier (AB-621G; NihonKohden) and recorded using a mini-polygraph (RM-6100; NihonKohden). After stabilization of the single twitch responses for 10 min, the rats were randomly assigned to one of 3 groups: the IT, IV, and IM administration groups. Vecuronium bromide was then administered in the following manner:
In the IT group, a tube used to administer vecuronium was tightly fitted in the tracheal tube. It was passed through the tracheal tube and fixed at 1 mm beyond the tip of the tracheal tube, and the drug was administered via this tube (Fig. 1). In the IV group, the drug was given via a 24-gauge IV catheter inserted into a jugular vein. In the IM group, a 26-gauge injection needle was used to inject the drug into the center of the left gastrocnemius muscle.
Measurements were commenced before the start of drug administration, and the data were continuously recorded until complete recovery of block was achieved.
A preliminary experiment was carried out to determine the dose of vecuronium for this experiment. The dose for each route was escalated in steps. Based on the results, the doses required to achieve 0%–100% block were estimated and set as follows.
In the IT group, 7 rats in each dose group were administered 1.0, 1.125, 1.250, 1.375, or 1.5 mg/kg vecuronium in a diluent solution volume of 0.175 mL/kg. In the IV group, 7 rats each were administered 0.150, 0.200, 0.250, or 0.300 mg/kg of vecuronium. In the IM group, 7 rats each were administered 1.750, 1.875, 2.000, 2.125, or 2.250 mg/kg of vecuronium in a diluent solution volume of 0.5 mL/kg.
After drug administration, the maximum depression of a single twitch was determined, and the suppression rate, percentage of the maximum depression of a single twitch for control, was calculated. Fifty percent and 95% effective doses were determined from a four-variable logistic sigmoidal dose-response model fitted to the dose-response curves, using the GraphPad Prism analysis software (GraphPad Software, San Diego, CA). Results were expressed as mean and 95% confidence interval.
Onset and Duration of Action and Recovery Index
The doses for the IT, IV, and IM groups were set at 1.50, 0.300, and 2.25 mg/kg, respectively, and administered to 7 rats each from these groups. These doses were selected (based on the results of the preceding experiment.) to obtain 90%–100% twitch depression, on average, for each route. The onset and duration of action and recovery index were determined as follows: onset of action was defined as the time from drug administration to 50% twitch depression. Duration of action was defined as the duration from drug administration to 25% recovery of twitch height. Recovery index was defined as the time from 25%–75% recovery of twitch height. Results were expressed as mean and 95% confidence interval.
Pharmacokinetics After Administration of Vecuronium by Different Routes
A carotid artery was exposed, and a 24-gauge arterial catheter was inserted to secure a route for collection of blood. The rats were randomly assigned to the one of the three drug administration routes (IT, IV, and IM administration group); the drug administration procedures were the same as in the pharmacodynamic study.
In all of the groups, about double the 50% effective dose of vecuronium was administered: 2.50, 0.400, and 3.75 mg/kg, respectively, based on the dose-response curves obtained in the pharmacodynamic study. A blood samples (0.9 mL) was collected from 3–6 rats at 0.5, 1, 2, 4, 8, 16, 32, or 64 min after drug administration using a syringe containing 0.1 mL of 3.8 w/v% trisodium citrate. The sample was centrifuged at 3000 rpm at 4°C for 10 min, 400 μL plasma was mixed with 60 μL of 1 M NaH2PO4, and the samples were stored at −20°C. 17β-deacetyl-OrgNC45 was used as the internal standard. The plasma concentrations of vecuronium were then determined using high-performance liquid chromatography with Kaseisorb LC60-5 (4.6 × 250 mm; Tokyo Kasei, Tokyo, Japan), in accordance with the method described by Paanakker and Van de Laar (8).
The analysis of the pharmacokinetics was performed using the GraphPad Prism analysis software. Analyses were performed by nonlinear regression analysis using three-compartment models. The following pharmacokinetic parameters were calculated: maximum plasma concentration of vecuronium (Cmax), time to reach the Cmax (Tmax), absorption rate constant (Ka), area under the plasma concentration– time curve (AUC), total body clearance (Cl), and bioavailability (F). Cmax and Tmax were obtained from the fitted formulae. Cl was calculated based on the AUC and the dose for the IV administration group. It was hypothesized that the Cl, distribution volume, and each rate constant after movement into the central compartment would be the same in the IT and IM administration groups as in the IV administration group. The AUC and F after IT and IM administration were calculated after adjusting for the absorption lag time. The absorption lag time after a dose through the IT or IM route was defined as the time at which the plasma concentrations calculated using the fitted formulae was 0. These values were calculated using standard equations used in pharmacokinetics.
Blood Gas Analysis Before and After IT Administration of Vecuronium
The IT drug administration method was the same as in the pharmacodynamic study, and the dose was set at 1.50 mg/kg, which yielded between 90% and 100% twitch suppression, on average. The rats were divided into 5 groups (n = 4 each) and blood samples were collected for blood gas analysis before, and 2, 7, 15, or 30 min after IT administration of vecuronium.
Values that were markedly different in each group were rejected based on the Smirnov-Grubbs test. The onset of action among the IT, IV, and IM groups was compared by analysis of variance, followed by multiple comparison testing using the Tukey-Kramer method, with the level of significance set at P < 0.05. The duration of action and recovery index were also compared among the three groups. The Pao2 among the before, and 2, 7, 15, and 30 min after IT administration groups was compared by analysis of variance, followed by multiple comparison testing using the Tukey-Kramer method, with the level of significance set at P < 0.05. Paco2 and pH were also compared among the five groups.
Pharmacodynamics After Administration of Vecuronium by Different Routes
Figure 2 shows the dose-response curves for the IT, IV, and IM groups. Table 1 shows the 50% and 95% effective doses in the IT, IV, and IM groups. Table 2 shows the onset and duration of action and time to recovery in the IT, IV, and IM groups. In the analysis of the onset of action, a seemingly rather high value was obtained in the IM group. The Smirnov-Grubbs test allowed this value to be rejected at a significance level of 1%. Therefore, the data on the onset of action, the duration of action, and the recovery index obtained from this subject were excluded from the analysis.
The onset of action in the IT group was significantly faster than that in the IM group, and significantly later than that in the IV group (P < 0.05). Although the duration of action in the IT group was significantly longer than that in the IV group (P < 0.05), no significant difference in the duration of action was noted between the IT and IM groups. The recovery index in the IT group was significantly shorter than that in the IM group but significantly longer than that in the IV group (P < 0.05). The onset and duration of action and the recovery index were all significantly longer in the IM group than in the IV group (P < 0.05).
Pharmacokinetics After Administration of Vecuronium by Different Routes
The plasma concentration-time curves in the three groups are shown in Figure 3.
Using the formulae obtained from the nonlinear regression analysis, the absorption lag time after IT and IM administration was calculated as 0.32 and 0.46 min, respectively. The respective AUC values were calculated using integral from the absorption lag time to infinity using the fitted formulae. The values of Cmax, Tmax, Ka, AUC, Cl, and F in the three groups are shown in Table 3.
Blood Gas Analysis Before and After IT Administration of Vecuronium
No significant differences in the Pao2, Paco2, or pH were observed between baseline, and 2, 7, 15, and 30 min after the administration of vecuronium (Table 4).
The key finding in this study was that, in rats, the onset of action of vecuronium after IT administration was slower than that after IV administration, but faster than that after IM administration. Furthermore, the 50% effective dose was about ninefold larger for IM vecuronium and about sixfold larger for IT vecuronium than for IV vecuronium. Regarding pharmacokinetic parameters, it was noted that Ka after IT administration was more than that after IM administration. This is why Tmax after IT vecuronium was shorter than that after IM administration, even though the dose and F of IT vecuronium were smaller than those of IM vecuronium. In the present study, the onset of action was defined as the time from drug administration to 50% block. When the block was approximately 50%, the twitch height had a steep gradient, suggesting that this might be more suitable for comparing the onset of action.
Vecuronium is a quaternary ammonium compound and thus does not readily cross biological membranes. The results of this study, however, demonstrate that vecuronium can be absorbed from the mucosa of the respiratory tract, although the underlying mechanism is unclear. The rapid rate of absorption of vecuronium after IT administration may be attributable to the large surface area of the highly permeable epithelium. The effectiveness of IT administration of drugs, in general, is supposedly influenced by various mechanisms, including the pulmonary blood flow, depth of drug administration in the trachea, the diluent solution used (physiological saline or distilled water), and the volume of the diluent solution used. Various studies have been conducted on IT administration of epinephrine, and the effectiveness of this approach was confirmed in the treatment of cardiopulmonary arrest (7). Because IT epinephrine has been shown to be effective even during resuscitation after cardiopulmonary arrest, when the blood flow is poor, the IT route of administration may be expected to be effective when the blood flow is normal, as was the case with this study.
Various techniques have been used for intratracheal administration, and although several studies have shown differences in efficacy among these various techniques (9–11), others have reported no such differences (12–14). We used our present administration method to prevent the drug solution from remaining inside the tube and allowing it to spread over a broad area. The drug solution was instilled into the area anterior to the tracheal bifurcation without allowing any solution to remain within the tube. The artificial ventilation caused drug aerosols of various particle sizes to be formed, and these were carried to the lung peripheries of both sides by the air flow, allowing absorption from a broad area. Conversely, in the case of IM administration, the absorption area is limited to the site of administration, resulting in a slower absorption rate. In addition, with regard to the diluent solution used for IT administration, several reports have suggested that absorption is better when distilled water rather than physiological saline is used (15,16). Lower osmotic pressures are thought to result in faster absorption. Our use of distilled water as a diluent may have contributed to our favorable results. The volume of the diluent solution volume was set at 0.175 mL/kg in our study. Greater dilution has been reported to result in better absorption, but poorer oxygenation (17). The level of dilution used in the present study was based on what is generally recommended for IT administration in humans (18), and the volume of the diluent solution used was considered to be appropriate for favorable absorption without negatively affecting oxygenation. These may be the reasons why the onset of action of vecuronium after IT administration was faster than that after IM administration.
The duration of action of vecuronium after IT administration was significantly longer than that after IV administration, whereas no significant difference in the duration of drug action was observed between IT and IM administration. The recovery index in the IT group was significantly longer than that in the IV group but significantly shorter than that in the IM group. Concerning pharmacokinetic parameters, it was found that the bioavailability in IT vecuronium was lower. Vecuronium was instilled as an aerosol of various particle sizes by artificial ventilation. Although the site of deposition or the size of the drug particles was not investigated in the present study, it was deduced that the absorption of some droplets via the mucosa may have resulted in the rapid absorption and quick efficacy of the drug. However, most of the droplets may not have adhered to the mucosa and may have been either exhaled or degraded in the pulmonary tissue. Furthermore, some droplets could have been adsorbed over time. These factors would be associated with those pharmacologic effects of IT administration of vecuronium. Incidentally, there was no difference in the duration of drug action between IT and IM administration, despite the observation of a discrepancy in the time courses of changes in the plasma concentrations between the two methods. This might be attributable to the difference in the dose set between the pharmacodynamic study and the pharmacokinetic study.
As for nonpharmacological problems associated with IT administration, the adverse effects of administration on pulmonary tissues must be considered, including the potential reduction of oxygenation. In the present study, no marked changes were seen in blood gas variables, including Pao2, Paco2, and pH after IT drug administration. Thus, we observed no adverse effects of IT drug administration on the respiratory function, at least in the short-term. Distilled water and physiological saline appear to have different effects on oxygenation, and although some studies have found that oxygenation is better preserved with physiological saline (19), others have documented better results with distilled water (16). Thus, further investigations are needed to clearly resolve this issue. In addition, bicarbonate and calcium cannot be administered intratracheally, as they can cause pulmonary injury (18). Although the solution used by us did not appear to cause tissue damage, as the pH was approximately 5, more thorough histopathological investigation is required. Because this study does not examine any long-term damage to the lungs, clinical application should not be attempted before the true safety of IT administration is established.
In the present study conducted using rats, IT administration of vecuronium appears to be more advantageous as compared with IM administration, given the more rapid absorption and faster onset of the drug action. Although more studies must be conducted before clinical use, the present results suggest the possibility of IT administration of vecuronium.
Fifty percent and 95% effective doses were determined from a four-variable logistic sigmoidal dose-response model fitted to the dose-response curves. The equations were expressed as follows:
(X is the logarithm of concentration. Y is the response.)
Plasma concentration of vecuronium for the IV group, Civ(t), was expressed as follows:
Plasma concentration of vecuronium for the IT and IM groups, Cit(t) and Cim(t), was expressed as follows:
AUC for the IV group, AUCiv, was calculated using integration from zero to infinity using formula (1):
The respective AUC values for the IT and IM groups, AUCit and AUCIM, were calculated using integral from the absorption lag time (tlag) to infinity using the formulae (2) and (3):
Cl was calculated based on the AUC and the dose for the IV administration group, AUCIV and XIV, using the following formula:
Fit and Fim were defined and calculated as follows based on AUC (AUCit and AUCim, respectively), the dose (Xit and Xim, respectively) and Cl obtained from Formula (7):
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