Intrathecal carbachol produces consistent analgesia in animals without appreciable adverse effects. Little is known about the ability of this drug to provide analgesia as stimulus intensity is increased. Likewise, there are few data regarding interactions between carbachol and other intrathecal analgesics.
Using two different noxious radiant heat intensities, one applied to each hind limb, analgesic effects of 1, 3, 10, and 30 micrograms intrathecal carbachol on paw withdrawal latencies were measured. Similar testing was done for intrathecal morphine and clonidine. ED50 fractions (1/2, 1/4, 1/8, 1/16) of drug combinations of carbachol-morphine and carbachol-clonidine were administered, responses to the low intensity stimulus were recorded, and the ED50 of each combination was established and isobolographic analysis of the drug interactions was carried out.
The 30-micrograms dose of carbachol was associated with transient agitation, salivation, and hind limb weakness. No other adverse effects were noted. The ED50 (95% confidence interval) of intrathecal carbachol was 2.34 micrograms (1.34-4.04) for low intensity stimulation and 12.64 micrograms (4.18-38.25) for high intensity. There was no significant difference between high- and low-intensity ED50 values for intrathecal morphine and clonidine. The analgesic effect of the carbachol-morphine and carbachol-clonidine combinations were significantly greater than the calculated additive effects. The ED50 for the carbachol-morphine combination was 12% of the expected additive value and the ED50 for the carbachol-clonidine combination was 30% of the expected additive value.
Intrathecal carbachol provides analgesia to noxious thermal stimulation of the hind paw in rats. It is relatively less effective at providing analgesia than intrathecal morphine or clonidine when stimulus intensity is raised. Intrathecal carbachol is synergistic when combined with intrathecal morphine or clonidine.
Methods: Using two different noxious radiant heat intensities, one applied to each hind limb, analgesic effects of 1, 3, 10, and 30 micro gram intrathecal carbachol on paw withdrawal latencies were measured. Similar testing was done for intrathecal morphine and clonidine. ED50fractions (1/2, 1/4, 1/8, 1/16) of drug combinations of carbachol-morphine and carbachol-clonidine were administered, responses to the low intensity stimulus were recorded, and the ED50of each combination was established and isobolographic analysis of the drug interactions was carried out.
Results: The 30-micro gram dose of carbachol was associated with transient agitation, salivation, and hind limb weakness. No other adverse effects were noted. The ED50(95% confidence interval) of intrathecal carbachol was 2.34 micro gram (1.34-4.04) for low intensity stimulation and 12.64 micro gram (4.18-38.25) for high intensity. There was no significant difference between high- and low-intensity ED50values for intrathecal morphine and clonidine. The analgesic effect of the carbachol-morphine and carbachol-clonidine combinations were significantly greater than the calculated additive effects. The ED50for the carbachol-morphine combination was 12% of the expected additive value and the ED50for the carbachol-clonidine combination was 30% of the expected additive value.
Conclusions: Intrathecal carbachol provides analgesia to noxious thermal stimulation of the hind paw in rats. It is relatively less effective at providing analgesia than intrathecal morphine or clonidine when stimulus intensity is raised. Intrathecal carbachol is synergistic when combined with intrathecal morphine or clonidine.
Key words: Anesthetic techniques: spinal. Cholinergic analgesics, intrathecal: carbachol; clonidine; morphine.
INTRATHECAL cholinergic agonists and acetylcholinesterase inhibitors produce analgesia in animals [1-4]and acetylcholinesterase inhibitors produce analgesia in humans. The analgesic effects appear to be related to muscarinic rather than nicotinic receptor activation. [2,4]Intrathecal nicotinic agonists, conversely, appear to be associated with irritability and possibly hyperalgesia, as demonstrated by the appearance of gnawing, vocalization, and hyperactivity after intrathecal nicotinic agonist administration. In addition, Naguib and Yaksh observed irritability, vocalization, and truncal rigidity in animals given intrathecal neostigmine after atropine pretreatment.
Intrathecally administered cholinesterase inhibitors are synergistic with alpha2-adrenergic agonists, and, provide at least additive analgesia in conjunction with spinal opioids. At doses that are analgesic in human volunteers, they are associated with nausea, vomiting, and lower extremity weakness. In animals, and at large doses in humans, spinally administered neostigmine produces hypertension. The combination of very small doses of intrathecal cholinesterase inhibitors with opioids or alpha2-adrenergic agents may, however, result in improved analgesia without these adverse side effects.
Several animal studies have documented the analgesic effect of carbachol (carbamyl choline). [1-3,8-11]While intrathecally administered cholinesterase inhibitors were observed to produce abnormal posturing (exaggerated thoracic kyphosis and generalized irritability), our preliminary studies of the analgesic effect of carbachol did not demonstrate these abnormalities. Therefore, we sought to determine whether carbachol, a potentially better-tolerated cholinergic agonist than neostigmine, would produce synergistic interactions with morphine and clonidine, further reducing the possibility of adverse effects.
Analgesic efficacy is often expressed as intrinsic activity, which refers to the percent receptor occupation required to provide a given analgesic effect. Drugs with high intrinsic activity, such as sufentanil, are able to produce analgesia by activation of a relatively small percent of the available opiate receptors, whereas morphine requires a higher percent of receptor occupation. A measure of the intrinsic activity of an analgesic drug is its ability to produce effective analgesia as the intensity of the noxious stimulus is increased. Saeki and Yaksh found that there is a relatively small increase in the ED50of intrathecal sufentanil (a drug with high intrinsic activity) to tail immersion testing as the bath temperature is increased from 52 to 60 degrees Celsius, while there is a sharp increase in the ED sub 50 of intrathecal morphine (a drug with a relatively low intrinsic activity) as the temperature is increased.
Little is known about the intrinsic activity of cholinergic agonists. Abram and Winne showed that intrathecal neostigmine failed to produce effective analgesia on the tail immersion test at doses that produced complete analgesia to hotplate testing. The mean control response latency to tail immersion using those paradigms was 2 s, while the mean control response latency in the hotplate test was 10 s. This suggests that the tail immersion test produced more rapid skin heating and that perhaps intrathecal neostigmine has a relatively low intrinsic activity. We sought to characterize the intrinsic activity of intrathecal muscarinic agonists by assessing the dose-response characteristics of intrathecal carbachol using two different intensities of noxious thermal stimulation.
Materials and Methods
The following investigations were carried out under a protocol approved by the Animal Care Committee of the Zablocki VA Medical Center, Milwaukee, Wisconsin.
In male Sprague-Dawley rats weighing 250-350 g lumbar intrathecal catheters were implanted via an incision in the atlantooccipital membrane under halothane anesthesia as previously described by Yaksh and Rudy. Catheters were advanced 11 cm caudally and externalized through the anterior portion of the scalp. Animals showing neurologic deficits on emergence from anesthesia were killed by barbiturate overdose. To ensure correct placement of the catheters, 20 micro liter 2% lidocaine was injected, followed by 10 micro liter 0.9% saline to flush the catheter on the day of the operation. Only animals that developed transient bilateral motor and sensory blockade in the hind legs were included in the study. Intrathecal injection studies were carried out at least 5 days postoperatively.
Drugs and Injection
All intrathecal drug doses were dissolved in normal saline and administered via micrometer driven injection device in a volume of 10 micro liter, followed by 10 micro liter saline to flush the catheter. The following drugs were used in the study: morphine (morphine sulfate; molecular weight = 334; Mallinckrodt, St. Louis, MO), clonidine (clonidine hydrochloride; molecular weight = 230.1; Sigma, St. Louis, MO), carbachol (carbamyl choline chloride; molecular weight 187.2; Sigma).
The general behavior of all of the rats was carefully observed and tested. The following tests of motor function and coordination were carried out: observation of gait; righting reflex; and placing/stepping reflex (the dorsum of either hind paw was drawn across the edge of a table, which normally results in the animal lifting the paw and placing it on the table surface). The presence of allodynia was examined by looking for agitation (escape, aggression, or vocalization) evoked by lightly stroking the fur.
Response latency to noxious thermal stimulation of the hind paw was assessed using a device similar to that previously reported by Hargreaves et al. Rats were confined in individual clear plastic cages placed on an elevated 2-mm thick glass surface. A movable radiant heat source (50 W, 8 V halogen projector lamp, Ushio, Tokyo, Japan) with a 4-mm aperture was situated below the glass surface. The chamber below the glass was thermostatically heated to maintain the glass temperature at 30 degrees Celsius. The radiant heat source was positioned to focus on that portion of the plantar surface of the hind paw in contact with the glass. Activation of the radiant heat source initiated a timer. Intensities were calibrated to produce either a 5-s latency to brisk paw withdrawal (high intensity) or a 10-s latency (low intensity) in control animals. The high-intensity stimulus was used on the right hind paw of each animal, while the low-intensity one was used on the left. For the low-intensity stimulus, if an animal failed to respond within a cutoff time of 20 s, the stimulus was terminated and a latency of 20 s was recorded. Similarly a cutoff time of 10 s was used for the high-intensity stimulus. Baseline measurements were recorded twice at each intensity and the mean was calculated.
After baseline testing, animals were give intrathecal injections of study drug using the following doses: carbachol 1, 3, 10, and 30 micro gram; morphine 0.3, 1, 3, and 10 micro gram; clonidine 1, 3, 10, and 30 micro gram. Testing was repeated at 10, 20, and 30 min after injection. These times were chosen on the basis of preliminary studies showing near maximal analgesia at these times for all three drugs tested. Five to eight animals were tested at each drug dose.
To characterize the interactions among the drugs tested, an equal dose ratio isobolographic analysis was carried out. After calculation of the ED50for the low intensity noxious radiant heat stimulus for each drug, ED50fractions (1/2, 1/4, 1/8, 1/16) of drug combinations of carbachol-morphine and carbachol-clonidine were administered, responses to the low-intensity stimulus were recorded at the 10-, 20-, and 30-min time intervals, and the ED50of each combination was established. These data were then compared statistically to the values that would be expected if the interactions were strictly additive.
The %MPE (% maximum possible effect) for each test of response latency was calculated as: Equation 1.
Calculation of the area under the time-response curve from 10 through 30 min was calculated by a trapezoidal rule, and dose-response curves were constructed from the area under the %MPE curves.
The ED50values and 95% confidence intervals for individual drugs and drug combinations were calculated using the pharmacologic software programs of Tallarida and Murray. The ratios of the ED50for high-intensity versus low-intensity stimulation were compared for each drug to provide an estimate of each drug's intrinsic activity. In addition, the %MPE for the high- and low-intensity stimulation was compared for each dose of each drug using the paired Student's t test.
Statistical analysis of drug interactions was conducted according to procedures of Tallarida et al. The variances for the individual drugs in each mixture were calculated from the variance of the total dose. The difference between the theoretical additive point and the experimentally derived ED50was compared using the Student's t test. For experimental values that were lower than theoretical additive values, a P value < 0.05 for the differences in both the X and Y directions was interpreted as a significant synergistic interaction.
No behavioral abnormalities were noted for any of the morphine-treated animals or for any animals receiving carbachol-morphine or carbachol-clonidine combinations. Animals receiving 30 micro gram intrathecal carbachol exhibited mild transient agitation for 10-12 min after injection (6 of 8 animals), mild, transient (10-15 min) hind limb paresis (3 of 8), and salivation (4 of 8). Transient conjunctival bleeding was noted just after intrathecal injection in one animal at that dose. No animals receiving smaller doses of carbachol exhibited any adverse effects. Animals that received 10 or 30 micro gram intrathecal clonidine exhibited brisk diuresis after injection.
The ED50values for both high- and low-intensity stimulation of the three drugs tested are shown in Table 1. The ratio of the high- to low-intensity ED50values for carbachol (5.40) is greater than that of either morphine (2.15) or clonidine (2.12). There was no overlapping of the 95% confidence limits for the high- and low-intensity ED50values of carbachol and there were significant differences between the high- and low-intensity analgesic effects for the 3, 10, and 30 micro gram carbachol doses (P < 0.05; Figure 1(A)). There was considerable overlapping of the 95% confidence intervals for the high- and low-intensity ED50values for both morphine and clonidine and there were no significant differences in analgesic effect between the high- and low-intensity stimuli for any dose of those drugs (Figure 1(B and C)).
For both the carbachol-morphine and carbachol-clonidine combinations, the combined effect of the drugs was significantly greater than the calculated additive effect (Figure 2(A and B)). Experimental values that lie inside the line of additivity, which connects the ED50values of the two drugs, are considered to have greater-than-additive, or synergistic effects. For the carbachol-morphine combination, the experimental ED50values (plus/minus SEM) were: morphine 0.06 plus/minus 0.01 micro gram; carbachol 0.13 plus/minus 0.03 micro gram. The expected additive values for this combination are: morphine 0.51 plus/minus 0.01 micro gram; carbachol 1.17 plus/minus 0.06 micro gram. For the carbachol-clonidine combination, the experimental ED50values were: clonidine 0.23 plus/minus 0.01 micro gram; carbachol 0.33 plus/minus 0.03 micro gram. The expected additive values for this combination are: clonidine 0.79 plus/minus 0.03 micro gram; carbachol 1.17 plus/minus 0.05 micro gram. The differences between additive and experimental values were significant for both drug combinations (P < 0.05).
It is generally assumed that the intrathecal analgesic effect of cholinergic agonists is the result of muscarinic rather than nicotinic receptor stimulation. Support for this assumption is fairly convincing. The highly specific muscarinic agonist (+)-cis-methyldioxolane is capable of producing analgesia on both the hotplate and tail-flick tests. Most studies evaluating the analgesic effect of intrathecal nicotine fail to demonstrate an appreciable analgesia, [1,6,9]although one study showed a fleeting (< 5 min) analgesic effect. In addition, intrathecal atropine, [1,2,4,6,8-10]but not mecamylamine, reliably reverses or prevents the analgesic effects of intrathecal cholinergic agonists.
Intrathecal carbachol produces profound analgesia to moderate levels of noxious thermal stimulation with minimal adverse effects. This is in contrast with neostigmine, which produces prolonged irritability and abnormal posturing at doses that are less profoundly analgesic. The reason for the lack of side effects of carbachol compared to neostigmine may be related to carbachol's relative lack of nicotinic effect. While carbachol has been characterized as a muscarinic agonist, it is thought to have at least some nicotinic effect. It is not clear whether the side-effect profile of carbachol in humans would be different from that of neostigmine, whose principle side effect is nausea and vomiting. Neither drug is associated with vomiting in rat studies.
Carbachol is relatively ineffective at producing analgesia when stimulus intensity is increased to produce rapid skin heating. At the largest dose tested (30 micro gram) the analgesic effect on the high-intensity stimulus was only about 60% (larger doses were not tested because of the occurrence of profound motor blockade, marked agitation, and salivation at doses higher than 30 micro gram in preliminary studies). This suggests that intrathecal cholinergic agonists may be relatively ineffective in treating severe cancer pain, or intense incident pain, such as rib fracture. Another characteristic of drugs with low intrinsic activity is a profound loss of effect with continuous or repetitive administration, i.e., rapid development of tolerance. Svensson et al. demonstrated that tolerance to analgesia for both thermal and mechanical nociception developed after 5 days of daily intrathecal carbachol injections (10 micro gram). These observations suggest that cholinergic agonists may not be ideal analgesic agents for long-term use.
While the relative inability of carbachol to provide analgesia at the higher stimulus intensity suggests that it may not be an ideal analgesic, the dramatic reduction in doses associated with combining the drug with either morphine or clonidine is quite encouraging. This effect suggests that postoperative intrathecal morphine doses might be dramatically reduced by the addition of carbachol, reducing the risk of respiratory depression and possibly reducing the incidence of side effects of either drug. Likewise, combinations of carbachol and clonidine may be useful in patients who experience severe opioid side effects or who have become tolerant of opioids.
The ability of intrathecal morphine to produce analgesia at the higher stimulus intensity in this study was surprising in light of previous studies demonstrating that drug's very poor analgesic effects with high-intensity stimuli. [13,21]We suggest a possible explanation for this discrepancy: The high-intensity stimulus we used may not have been intense enough to test the upper limits of morphine's efficacy (although it was obviously adequate to do so for carbachol). In the studies by Saeki and Yaksh and Yaksh, the cutoff times for the high-intensity stimuli were the same as for low intensity, while our cutoff times were adjusted to twice the baseline for each stimulus. Therefore, the high-intensity stimuli in their studies probably represented a much more marked increase in stimulus intensity, perhaps to the point of tissue injury.
The potential of intrathecal carbachol to produce neurologic damage in humans remains an issue. The drug has been used in many rat studies without reports of adverse effects except for transient hind limb weakness at large doses. A neurotoxicologic study carried out in rats receiving intrathecal carbachol for 14 days failed to demonstrate any histologic differences between carbachol- and saline-treated animals. However, more extensive testing in other species would be required before the drug can be tested in humans. That there are no commercially available preservative-free preparations of carbachol makes it unlikely that it will be made available for human use. However, two other muscarinic agonists, bethanechol and methacholine, are in clinical use, and may prove to have analgesic applications if safety and efficacy can be established.
The authors thank Dr. James Fujimoto, Blythe Holmes, and Jody Rady for their assistance and advice.