Continuous Intrathecal Administration of Short-lasting micro Opioids Remifentanil and Alfentanil in the Rat

Animal surgery and testing was approved by the institutional animal care committee of the University of California, San Diego. All procedures were performed according to this protocol. Rats (male Sprague Dawley, Harlan Industries, Indianapolis IN) weighing 300-325 g were implanted and thereafter kept in separate cages on a 12 h light/dark cycle with water and food supply provided ad libitum. Each rat was implanted with an intrathecal catheter as described later. Animals were randomly assigned to the several treatment groups.

The surgical insertion of the intrathecal catheters was performed using a modified version of the techniques as previously described. [12]For insertion, the animals were anesthetized with halothane (2% or 3% in 50% Oxygen²/air). After shaving and preliminary cleaning of the surgical area on the back of the head and neck with alcohol and betadine, the rat was placed in a stereotaxic head holder for insertion of the intrathecal catheter. Intrathecal catheters were made out of polyethylene tubing (PE-10, ID 0.28 mm, OD 0.61 mm) with a small knot for fixation under the skin. Implantation of the intrathecal catheters was performed after incision of the atlantooccipital membrane. The catheter was inserted into the intrathecal space and passed to the rostral edge of the lumbar enlargement, 8.5 cm from the cisterna magna. The catheter external arm was then subcutaneously tunneled and fixed under a skin suture.

After the surgical procedures, a recovery period of at least 5 days was allowed before initiating the injection series. Neurologic status of the animal was assessed after surgery and before testing after flushing the intrathecal catheter with 10 micro liter normal saline. Animals with impaired motor function or an elevated sensory threshold were killed.

Animals were connected to a microinjection syringe pump (Harvard pump 22, Harvard Apparatus, South Natick, MA) via a length of calibrated PE-50 tubing. Drugs were injected at either a rate of 1.0 micro liter/min (remifentanil in glycine [R^g], alfentanil) or in a rate of 0.1 micro liter/min (R^g, alfentanil) or as a control remifentanil without vehicle (R^s) all over 90 min. Before testing for the 0.1 micro liter/min flow rate, the intrathecal catheter was preloaded with 6.5 micro liter of the different compounds (calibration verified a volume of 6.5 micro liter from one end of the PE-10 tubing to the other).

Drugs given intrathecally in this study were remifentanil hydrochloride with its vehicle glycine (R^g; MW = 413) remifentanil hydrochloride (R^s; MW = 369), and glycine (MW = 75). These drugs were provided by Glaxo. (Research Triangle Park, NC). Alfentanil hydrochloride (MW = 453) by Janssen (Janssen Pharmaceutica, Titusville, NY). Naloxone hydrochloride (Dupont Pharmaceutical, Wilmington, DE) was given intraperitoneally. All drugs were dissolved in sterile normal saline (0.9% USP). The R^gstudies were carried out with solutions prepared from ampules containing 5 mg remifentanil and 15 mg glycine/ampule, the glycine was prepared from ampules containing glycine, 17 mg/ampule (both drugs provided by Glaxo). The remifentanil alone was prepared from the powder. Infusion rates were expressed in micrograms per minute. Doses were prepared in two different concentrations such that the dose per minute was delivered in an infusion rate of 1.0 micro liter/min and 0.1 micro liter/min. Control experiments were carried out with the vehicle (normal saline or glycine) delivered intrathecally.

Typically, rats were used in three to five separate experiments, separated by an interval of 4 or 5 days. Animals were randomized to receive R^g, R^s, alfentanil, glycine, or normal saline (5-8 animals per dose/ group). Agonist dose-response curves were obtained for intrathecal infusions of the different drugs. Antagonist response studies were carried out during the plateau with infusion doses that yielded a just maximal effective analgesic dose of the agonist. Naloxone was injected intraperitoneally in a dose of 1 mg/kg body weight (0.2 ml/kg body weight).

For each drug, the following test measures were obtained periodically before and after injection.

Antinociception. The antinociceptive effect was quantified by measuring the latency of withdrawal evoked by exposing the hind paw to a thermal stimulus. To accomplish these studies, unanesthetized rats were placed in methyl methacrylate polymer cages (9 x 22 x 25 cm) on top of a glass plate. The thermal stimulus was maneuvered under the glass to focus the projection bulb on the plantar surface [13,14](UARDG, Department of Anesthesiology 0818, University of California, San Diego, La Jolla, CA). Initiation of the current to the bulb started a timer. Bulb current and time were automatically terminated when paw elevation was sensed by photodiodes or when an interval of 20 s (cutoff time) had passed. To avoid variations in paw starting temperature, the underglass surface was maintained at 30 degrees C by a feedback-controlled heater fan. The aiming of the focused stimulus was reliably accomplished by a mirror attached to the stimulus, which permitted visualization of the undersurface of the paw. Light beam intensity was monitored by a measurement of bulb current and the stimulus intensity was calibrated daily by assessing the temperature change after 10 s sensed by an underglass thermocouple (T¹/2 = 0.2 s). After placing the rat in the plastic cages, a 20-min adaptation period was allowed. The first measurement was done on both hind paws, the response latencies averaged and counted as baseline score (time 0). Tests were then made at 5, 10, 15, 20, 30, 40, 60, and 90 min during drug delivery and at 3, 5, 10, 15, and 30 min after the end of the infusion.

Supraspinal Effects. To score the behavioral changes before and during treatment of the animal, a supraspinal effects index was employed consisting of four different parameters that have been shown to be dose-dependently blocked by opioids. [15]These measured responses included: pinna reflex, cornea reflex (both evoked by light touch of the surface of pinna or cornea with a small piece of PE-10 tubing), evoked movement (startle reflex evoked by tapping on the cage wall), and signs of spontaneous movement (e.g., grooming, chewing, ambulation). Each parameter was scored as: 0 = normal (brisk pinna/cornea reflex response or startle reflex, spontaneous movement within 30 s of the assessed time point; 1 = attenuated (touch with tubing for pinna or cornea reflex or knocking on cage wall does result in a slow reflex behavior, touch or knocking has to be repeated at least twice); or, 2 = response completely absent (no reflex was shown after 3 times of touching cornea or pinna both sides, no startle behavior was displayed after 3 times knocking against cage wall and no spontaneous movement was observed for > than 1 min). To permit a sensitive assessment of the supraspinal effect, a supraspinal effects index was employed, which consisted of summing the individual scores for the four measurements at each time point, permitting a total score of 8. To determine the reliability of the method, 50 rats were observed by two investigators; one blinded as to treatment and the other not blinded. The overall agreement between the two observers was reflected by the significant correlation coefficient (0.91) obtained with the two sets of observations. Tests for the supraspinal side effects were concomitant with antinociceptive testing performed (at 5, 10, 15, 20, 30, 40, 60, and 90 min during drug delivery and at 3, 5, 10, 15, and 30 min after the end of the infusion).

Motor Impairment. Before, during, and after the administration of the different agents, the hind paw of the animal was displaced with a flexible probe and the rat's reaction was scored as 0 = normal (immediately repositioned); 1 = attenuated (more than 1 s for repositioning); or 2 = completely absent (no repositioning of the hind paw). This test was assessed for both hind paws and the scores were compiled (maximal score = 4/animal/time point).

Data are expressed as mean of 5-8 animals and+/-SEM. For the time course studies, latencies are indicated in seconds, each time point representing 5-8 animals. For further analysis, the thermal latencies and the sedation index scores were converted to %MPE according to the formula: Equation 1.

Dose-response curves are presented as the %MPE. For all drugs, the dose-response analysis as described by Tallarida and Murray [16]was accomplished. The effective dose for 50% of subjects (ED⁵⁰) and the 95% confidence intervals were calculated using a least-squares linear regression model, where the log dose values were used. Changes in the thermal latency and supraspinal index with and without antagonist treatment were tested for significance using an unpaired Student's t test. Critical values of P < 0.05 were considered as statistically significant. Comparison in the time until onset of analgesia or supraspinal side effects and time until recovery until at least 50% MPE were conducted using two-way analysis of variance.

After the initiation of the intrathecal infusion of R^g, R^s, or alfentanil, there was a rapid increase in the thermal escape latency that reached an apparent steady state, as defined by response latencies within 15 min for both remifentanil and alfentanil. These latencies remained essentially stable for the 90-min interval of infusion (Figure 1).

The magnitude of the increase in hind paw response latencies were dependent on the infusion concentration (Figure 2and Figure 3). The ED⁵⁰values for the several agents are presented in Table 1. As indicated, there was no difference between R^s, R^g, and alfentanil. There was no difference between the peak plateau effects produced with the infusion rates of 0.1 or 1.0 micro liter/min, e.g., the effects were proportional to the dose in micrograms of opioid delivered per minute.

Glycine alone, at the highest concentration examined, was without an acute effect (e.g., < 60 min) on the thermal withdrawal latencies at infusion doses that corresponded to those used in the presence of remifentanil, e.g., 3-9 micro gram/min did not produce an increase in the thermal escape latency of the animals at intervals up to 60 min. However, at longer intervals there was a dose-dependent increase, up to approximately a 50% MPE of the thermal withdrawal latency (Figure 4). This increased latency was, however, accompanied by an increase in motor impairment (see later).

After the initiation of infusion of remifentanil or alfentanil, there was a rapid onset of depression of behavioral reactivity, as indicated by an increase in the supraspinal index and this depression remained stable during the 90-min infusion interval (Figure 1). The behavioral depression was characterized by reduced spontaneous activity, as well as reduced corneal and pinnae reflexes. The degree of depression was dose-dependent (Figure 2and Figure 3). The ED⁵⁰values for the supraspinal index are presented in Table 1.

Regarding glycine alone, there were no significant changes in the supraspinal index, during the 90-min infusion interval (Figure 4).

To determine the effect of different flow rates on the time course for analgesia and supraspinal side effects, the time for each rat to reach and return to the individual 50% MPE for both assessments was determined and the group means were calculated (Table 2). Peak plateau effect for analgesia and side effect index were independent of infusion rate. Comparison between R^gand alfentanil revealed an increase of about 30-50%, respectively, in the time to onset in analgesia, when matched by dose for peak analgesic effects within the two different flow rates. However, there appeared to be only a modest increase in the onset for analgesia with remifentanil (flow rate of 0.1 micro liter/min). Regarding the supraspinal action, the time to onset of side effects was decreased at the faster rate (Table 2).

Time until recovery after the standard 90-min infusion revealed a more rapid recovery to 50% effect for analgesia and supraspinal index at the higher flow rate for both R^gand alfentanil (significant for analgesia only for alfentanil; Table 2). Supraspinal side effects were decreased to 50% MPE of the index more rapidly in both flow rates for R sub g than for alfentanil, whereas analgesia only showed a faster decrease for R^g(R^srevealed tendency to a faster recovery, but not significant against R^gas shown in Figure 1) in comparison to alfentanil with the flow rate of 0.1 micro liter/min (Table 2).

Increasing infusion doses of alfentanil or remifentanil delivered in a rate of 0.1 micro liter/min or 1.0 micro liter/min (in both formulations, R^sand R^g) resulted in a dose-dependent increase in the supraspinal index and in the paw withdrawal latency. Plotting the supraspinal index as %MPE versus analgesic effect as %MPE revealed a positive correlation between the two variables for both remifentanil and alfentanil, with the slopes of both being positively inflicted (Figure 5). Comparisons of the slope of the regression line demonstrated that the ordering of the slopes for drug delivery at 1.0 micro liter/min showed a difference (y = 11.4 + 0.6x; r = 0.9 for remifentanil and y = 7.6 + 0.9x r = 1 for alfentanil; P < 0.05 Spearman's rank test). However, there was no difference in the higher concentrations with the slower delivery rate of 0.1 micro liter/min. Comparison of the ED⁵⁰ratio (ED⁵⁰supraspinal index/ED⁵⁰analgesia in micro gram) revealed the following ratio at 1.0 micro liter/min: 1.3 (+/-0.1) for remifentanil (R^g) versus 0.9 (+/-0.3) for alfentanil, whereas at a flow rate of 0.1 micro l/min, a tendency for moderately higher ratios for remifentanil also was seen: 1.6 (+/-0.7) for R^g, 1.2 (+/-0.2) for R^sand 1 (+/-0.1) for alfentanil.

Over the 90-min infusion period and the subsequent recovery period, neither R^snor alfentanil had any effect on motor function. In contrast, R^gresulted in a dose-dependent increase in the motor impairment score. Time until onset of the impairment started around 60 min infusion time and peaked just after termination of the 90-min infusion (e.g., 100 min; Figure 4). All animals recovered by 8-12 h after termination of infusion (data not shown). The ED⁵⁰for the motor dysfunction assessed at 100 min revealed the ED⁵⁰to be 6.4 +/-0.7 micro gram/min (calculated for the total dose over 90 min). There was no difference in the onset or the dose dependency of motor dysfunction between the delivery of glycine alone or R^g(ED⁵⁰in micro gram: 6.6+/-0.9; Figure 6).

Naloxone (1 mg/kg) administered intraperitoneally resulted in a significant reduction in the spinal and supraspinal effects of the highest administered intrathecal doses of the analgesic and supraspinal index response (P < 0.05) of both micro opioids, R^g, R^s, and alfentanil but did not affect saline or glycine animals (Figure 7). Motor impairment in R^gand glycine was not reversed or decreased after intraperitoneal naloxone administration.

Intrathecal continuous infusion of normal saline in a volume of either 1.0 micro liter/min or 0.1 micro liter/min did not produce any significant increase in the thermal withdrawal latency or the supraspinal index (Figure 7).

Remifentanil is an intermediate, lipid-soluble micro opioid agonist that is rapidly metabolized to an inactive isomer by plasma and tissue esterases. [2,3]In humans and animals, systemic delivery [4,6]results in a very short-lasting opioid action, consistent with its rapid clearance from the circulation. Continuous delivery has been shown to result in the rapid achievement of steady state whereas termination of delivery results in a rapid reversal of the opioid effect, consistent with a lack of accumulation. Recently, we have shown that similar to alfentanil, another intermediately lipid-soluble agent, bolus delivery of intrathecal remifentanil would yield a potent short-lasting antinociception that was naloxone reversible. [11].

Because of their rapid kinetics, agents such as remifentanil might serve to establish steady analgesic levels through continuous delivery. Lipid-soluble agents that are slowly metabolized such as alfentanil and sufentanil have been employed using continuous epidural delivery but the rapid clearance of these agents from the spinal space into the vasculature has led to the appearance of a significant plasma level that results in the loss of the therapeutic advantage contemplated for spinally delivered agents, i.e., significant supraspinal side effects are noted. Because of its rapid metabolism through plasma and tissue esterases, continuous spinal infusion of remifentanil might yield an analgesic action with fewer side effects because of rapid plasma inactivation.

In the current study, when the analgesic versus supraspinal index was plotted, a significant linear regression was noted over the range of infusion doses. This differs from our previous observation with bolus delivery, where at low doses of remifentanil, there was little supraspinal action, whereas at the highest dose, an increase in supraspinal activity was noted. [11]While the practical significance of the supraspinal index associated with the action of remifentanil is not clear, it can be used to compare the profile of spinal remifentanil actions with those of other spinally delivered agents in the same model. Thus, after bolus delivery, alfentanil, unlike remifentanil, displayed a close covariance between the increase in the supraspinal index and the analgesic effect.

After bolus delivery, it thus appears that alfentanil at steady state results in a more prominent supraspinal action than remifentanil, presumably reflecting the rapid plasma metabolism of remifentanil as compared to alfentanil. In the current study, the close correlation between analgesia and the supraspinal effects for the two agents was unexpected. The rapid metabolism of the agent was hypothesized to diminish the likelihood of a supraspinal action. We suspect that the supraspinal effects produced by the ongoing infusion likely reflect on the finding that the plasma half-life, although short, is not sufficiently so to preclude a supraspinal redistribution in the rat. The three-compartment model for the distribution of remifentanil also includes the possibility for slow redistribution through tissue stores.

In previous work, we noted that the spinal bolus administration of remifentanil and alfentanil resulted in ED⁵⁰(micro gram) for analgesia of 0.7 for remifentanil and 16 for alfentanil. [11]In the recent study, the continuous administration revealed almost similar relative potencies for analgesia with both micro opioids. We suggest that this similarity is attributable to the rapid clearance and metabolism of remifentanil during continuous administration. Thus, remifentanil appears to be less potent, whereas alfentanil appears to be more active because of its moderate accumulation.

Regarding the time course, the time of onset of spinal opioid action in the rodent animal has been shown to covary with lipid solubility. Remifentanil appeared to produce a faster onset of analgesic response in equianalgesic doses than alfentanil after intrathecal administration only with the slower infusion rate. The more rapid rate of recovery of remifentanil supports the efficiency of the metabolic pathway of remifentanil in the blood after continuous delivery of remifentanil and blood stores are presumably low in contrast to alfentanil. Interestingly, the continuous spinal administration of remifentanil also does not appear to increase the time to recovery in comparison to the bolus administration. [11].

Neither alfentanil nor remifentanil alone had any effect on motor function at the doses employed. In contrast, the observed motor impairment after continuous intrathecal administration of remifentanil occurred in its commercially available formulation with glycine. Separate studies with the glycine excipient alone revealed that the motor effect was associated with a dose-dependent action of the glycine vehicle. Surprisingly, the effect displayed a clear time course with the effect appearing during an interval of infusion. Bolus delivery of the agent was without a similar effect. [11]Consideration of the possible mechanism of the observed motor impairment may suggest several possible components.

First, glycine alone may be able to induce a local effect on receptors in the spinal cord or promote a neurotoxic effect. Glycine is a potent inhibitory transmitter that is found in the central nervous system within the brain [17]and in interneurons in the dorsal and ventral horns that is known to hyperpolarize neurons by increasing Chlorine-conductance. Treatment of spasticity might be achieved with administration of glycine intrathecally. [18]Simpson et al. [19]found in a model of spinal shock that animals showing flaccidity had glycine levels that were 2 or 3 times higher than in spinal cord of animals in which the spinal ischemia resulted in spasticity. These observations supports the suggestion that exogenous glycine (as provided in the placebo control and in the formulation of remifentanil) can function as an inhibitory transmitter in suppression of muscle tone.

Second, glycine is employed in the preparation of the injectable material because (1) it facilitates the preparation of remifentanil as it provides a visible product after lyophilization, and (2) it provides an acidic buffer. While the current studies did not systematically consider the role of pH, it is possible that extended exposure to this acidic pH might prove to have deleterious effects on spinal function. In recent studies examining repeated intrathecal injections in the dog, reversible motor weakness and apparent dysesthesias have been observed with the acidic glycine vehicle (Yaksh, Rathbun, Dragani, and Malkmus, unpublished observations).

In conclusion, in rats, it appears that the spinal action of remifentanil is consistent with its pharmacology as a potent micro-opioid agonist. Its rapid onset after the initiation of a continuous infusion and the rapid development of a dose-dependent elevation in the nociceptive threshold is consistent with this lack of any accumulation of active drug stores, even after continuous spinal infusion. Unexpectedly, in this model, the covariance of supraspinal effect with the analgesic action suggests that despite its rapid inactivation, with constant infusion there remains the likelihood that plasma levels at analgesic doses are sufficient to produce a supraspinal redistribution that leads to measurable side effects. Aside from the time course profile of this agent, the current studies provide an initial report that the glycine vehicle may be associated with a dose-dependent reversible motor weakness after spinal administration. Further studies are required to define the mechanisms of this action.

**Schuster SV, Bilotta JM, Lutz MK: Analgesic activity of the ultrashort acting remifentanil (abstract). FASEB 1991; 5:A860.

**Amin HM, Sopchak AM, Esposito BF, Graham CL, Batenhorst RL, Camporesi, EM: Naloxone reversal of depressed ventilatory response to hypoxia during continuous infusion of remifentanil (abstract). ANESTHESIOLOGY 1993; 79:A1203.

***Glass PSA, Kapila A, Muir KT, Hermann DJ, Shiraishi M: A model to determine the relative potency of mu opioids: Alfentanil versus remifentanil (abstract). ANESTHESIOLOGY 1993; 79:A378.