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From the Departments of *Anesthesiology Research, University of California, San Diego, La Jolla, California Cognetix, Inc., Salt Lake City, Utah
Address correspondence and reprint requests to Tony L. Yaksh, PhD, Anesthesiology Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0818. Address e-mail to tyaksh{at}ucsd.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Male Holtzman rats (300–375 g) were obtained from Harlan Industries (Indianapolis, IN) and prepared with intrathecal catheters as previously described (8,9). In brief, rats were anesthetized with isoflurane and a lumbar catheter of stretched polyethylene tubing (PE-10; Clay Adams, Parsippany, NJ) approximately 8.5 cm in length was inserted through the atlanto-occipital membrane to terminate in the L1-3 region. Rats were allowed to recover for approximately 7 days before testing.
Epidural rat catheters were placed in anesthetized rats by making a 1–2 cm midline skin incision over the dorsal thoracolumbar region, and the muscles were bluntly dissected to expose the intervertebral space. The intraspinous ligament was carefully dissected, allowing access to the epidural space. A PE-10 catheter was advanced approximately 2 cm caudal in the epidural space and attached to a second length of tubing, externalized at the dorsal neck. Epidural placement was confirmed by lack of cerebrospinal fluid aspiration and the incision was closed in layers with 3.0 Vicryl. Rats were allowed 2 days recovery before testing.
Purpose-bred beagle dogs (9–12 kg) were obtained from Covance Research Products (Denver, PA). Animals were acclimatized for at least 3 days. Prophylactic antibiotics (sulfamethoxazole-trimethoprim, 200–40 mg per os bid) were started 2 days before surgery and continued for 5 days. Surgical implantation of lumbar intrathecal and epidural catheters was performed in an Association for Assessment of Laboratory Animal Care-approved surgical suite using sterile techniques. After an overnight fast, dogs were premedicated (atropine 0.04 mg/kg, IM) and sedated with xylazine (1.5 mg/kg, IM). Dogs were anesthetized with isoflurane, tracheally intubated, and maintained on 1%–2% isoflurane in 50%:50% N2O:O2. Intraoperative monitoring included heart rate, respiratory rate, oxygen saturation, inspired and end-tidal O2, CO2, N2O, and isoflurane concentrations.
For intrathecal catheterization, the dorsal neck was shaved and scrubbed with chlorhexidine. After sterile draping, the cisterna magna was exposed. A 1–2 mm incision was then made in the cisternal membrane. A customized PE-10 catheter was implanted approximately 38 cm, terminating in the lumbar intrathecal space (L2-3). The catheter was externalized out the dorsal neck and the incision closed in layers with 3.0 Vicryl. Animals were allowed to recover from anesthesia and then given 0.04 mg/kg butorphanol for postoperative analgesia (10). Testing was begun 3 to 5 days after surgery.
For epidural implantation, dogs were prepared as described above except the lower back was shaved and scrubbed. A 2-cm incision was made and an 18-gauge Tuohy needle was inserted into the L7-S1 epidural interspace. A polyethylene catheter (PE-50) was inserted 10 cm to terminate at the L1-3 region. Epidural placement was verified intraoperatively via the loss-of-resistance technique and the catheter was then connected to an injection port that was implanted subcutaneously through a small incision on the lateral aspect of the lower back. The incisions were closed with 3.0 Vicryl and animals were allowed to recover as described above.
After either intrathecal or epidural placements, animals were ambulatory within 30 min and manifested normal neurological function.
For rats, contulakin-G was administered as a 10-µL bolus followed by a 10-µL saline flush for intrathecal and most epidural studies. For the largest dose epidural study, 89 nmol Contulakin-G, a 20-µL bolus was used followed by a 10-µL saline flush. Control animals received a 20-µL or 30-µL saline bolus. Intrathecal Contulakin-G doses were 0.03, 0.1, 0.3, and 3 nmol. Group size for all rat studies was 8 rats, with the exception of intrathecal 3 nmol, which consisted of 4 rats, and the 10-µL epidural saline control group consisted of 6 rats and 89 nmol epidural Contulakin-G was only tested in 2 rats. Nociceptive activity of intrathecal Contulakin-G was assessed in the formalin test. Here, 50 µL of 5% formalin was injected into the paw. A small metal band applied to the injected paw was used in conjunction with an automated nociception analyzer as described previously (11,12). During these studies, systematic behavioral assessments after injections were made that assessed the presence of symmetrical ambulation, hindpaw placing and stepping reflex evoked by drawing the dorsum of the paw across the edge of a surface, and the pinnae and blink reflexes evoked by light touch.
For dogs, contulakin-G or MSO4 was dissolved in 0.9% saline for injection. Injection volume for intrathecal and epidural dosing was 0.3 mL and 0.5 mL, respectively. Dosing was followed by an equal volume saline flush. For intrathecal dosing, dogs pseudo-randomly received 50, 150, or 500 nmol Contulakin-G, 150 nmol or 450 nmol MSO4 or saline. For intrathecal dosing, group size was 4 dogs for all doses except for the saline control and 450 nmol MSO4 groups, which had 3 dogs each. For epidural dosing, dogs (n = 2) received 1000 nmol Contulakin-G, 750 nmol (0.5 mg), and 7500 (5 mg) MSO4.
Physiological variables (heart rate, respiratory rate, mean arterial blood pressure, body temperature) were measured at regular intervals after dosing (Pre dose, 15, 30, 60, 120, 240, 480 min, 24, 48, 72 h). Heart rate and arterial blood pressure were measured using a DynaMap system (Critikon, Tampa, FL) with a pediatric cuff placed at the base of the tail. In addition, a spinally mediated thermally evoked skin twitch response was evaluated as described previously (13). Motor function, muscle tone, and arousal were also assessed using a standardized grading system. Systematic behavioral assessments were made of arousal (–3 = semicomatose to + 3 = uncontrollable fits and continuous agitated activity), motor tone (-3 = paralysis/flaccidity to + 3 = uncontrolled muscle contractions), and coordination (0 = normal to + 3 = complete loss of motor coordination) during the interval after each injection. For detailed description of behavioral criteria, see (14).
After completion of dosing, dogs were anesthetized with pentobarbital (35–50 mg/kg IV). The chest was opened and the dog was perfused via the aorta with 0.9% saline then 10% neutral buffered formalin. The dura was exposed by laminectomy. The location of the catheter tip and the condition of the dura and spinal cord in the vicinity of the catheter tip was noted by close visual inspection by JWA or TLY at necropsy.
For the formalin test, analyses were performed on the cumulative flinches measured for each rat during Phase I (0–9 min), Phase II (10–60 min), Phase IIA (10–30 min), and Phase IIB (40–60 min). One-way analysis of variance was used to compare differences in means across controls and dosages. When a significant effect was found (P < 0.05), a Bonferroni post hoc test was used where P < 0.05 was defined to be significant. To determine equi-effective doses, flinching totals for each phase were divided by the mean flinching for that phase in the respective saline-treated group to obtain a percentage of control. This percentage was then used to compute percent of control for each treatment group with mean and sem and to calculate dose-effect curves. Using linear regression analysis, the ED50 with 95% confidence intervals was calculated using an Excel template on a Macintosh Computer.
For dogs, physiological variables after dosing were assessed by analysis of variance as described above at the time of peak drug effect. Skin twitch latency was computed as percentage maximal effect (%MPE) using the following formula %MPE = (skin twitch latency – baseline latency)/(maximal latency – baseline latency). If %MPE was negative, a value of zero was assigned. One-way analysis of variance was used to compare differences in means across controls and dosages and, where significant (P < 0.05), a Bonferroni post hoc test was used.
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RESULTS |
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Epidural Contulakin-G also significantly decreased flinching behavior in rats (Fig. 2A). All doses of Contulakin-G tested (10, 30, 89 nmol) decreased Phase IIA flinching (Fig. 2B), whereas the effect was only significant for the two highest doses (30 and 89 nmol) when analyzing the entire Phase II (Fig. 2C).
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The calculated intrathecal and epidural ED50 values for each phase of the formalin test are presented in Figure 3. Phase II ED50 was 0.07 nmol for intrathecal Contulakin-G, whereas the epidural ED50 was 45.3 nmol. The epidural:intrathecal ED50 ratio for Contulakin-G was 647.
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No changes in motor function (symmetrical ambulation, placing and stepping), pinnae or corneal reflexes were noted at any dose. The lack of disruption of motor function indicates that rats were able to make appropriate responses to the stimulus. A larger dose of intrathecal Contulakin-G in a single rat (31 nmol, data not shown) did produce a transient catalepsy.
Intrathecal Contulakin-G (500 nmol) produced a dose-dependent increase in the thermally evoked skin twitch latency at 30 min after dosing in dogs, which reached statistical difference as compared to vehicle at 500 nmol. (Fig. 4A-B). At this dose, the analgesic effect was significant at 30 min. One-way analysis of variance across time for the large dose revealed a statistically significant main effect (P < 0.05). Bonferroni multiple comparisons showed that the increase in latency was greater than preinjection control at 30, 60 and 240 min. At all intrathecal Contulakin-G doses, including the largest, arousal and motor tone/coordination scores were 0 or 1 for all animals. There were no differences between saline control and any dose of intrathecal Contulakin-G with regard to peak changes in arterial blood pressure (Fig. 4 C, D) or heart rate (Fig. 4 E, F). Over time, saline animals displayed a gradual decline in body temperature. This decline was not noted in the large dose Contulakin-G treatment. Thus, body temperature at 240 min was significantly greater after 500 nmol Contulakin-G as compared with saline (Fig. 4 G, H).
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Intrathecal MSO4 (450 nmol) significantly increased skin twitch within 30 min in dogs and reached the maximal latency (6 s) within 60 min and remained at maximal latency up to 8 h after dosing (Fig. 5A-B). A smaller dose of MSO4 (150 nmol) significantly increased skin twitch at all time points from 60 min to 8 h. By 24 h, skin twitch latency had returned to approximately baseline levels (data not shown). Although no change in motor function was noted in vehicle or small dose morphine animals (motor tone and coordination scores ranging from 0–1), mild hindlimb paresis was observed at the largest dose of intrathecal morphine (motor coordination scores ranging from 1–2). There were no differences between saline control and any dose of intrathecal morphine with regard to peak changes in arterial blood pressure (Fig. 5 C, D) or heart rate (Fig. 5 E, F). Over time, all animals displayed a small numerical decrease in body temperature that did not reach statistical significance (Fig. 5, G,H).
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Epidural Contulakin-G was examined at 1000 nmol and had no effect on skin twitch latency or any other measured variable in dogs (Fig. 6A-B). In contrast, 7500 nmol MSO4 increased skin twitch to the maximal latency at 120 min (Fig. 6C-D), whereas 750 nmol MSO4 had a more modest effect (Fig. 6E-F). Further epidural studies were not undertaken because of the large epidural-to-intrathecal ratio seen in rats (more than 600) and the limited availability of the compound. Because of the small sample size (n = 2) statistical analysis was not possible in the dog epidural arm of the study; however, in the dogs tested it was apparent that although MSO4 was effective, 1000 nmol Contulakin-G given epidurally displayed no antinociceptive properties.
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Necropies performed by JWA or TLY found that all intrathecal catheters terminated in the vicinity of the L2 spinal level. All catheters were free in the intrathecal space and no discoloration or cellular accumulations were noted on gross dissection. For the epidural catheters, tips were located at approximately the L4-6 spinal level. We noted a minimum degree of fibrotic investment of the epidural catheter at death, consistent with previous work with this catheter model (15).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Rat: Formalin Test
Flinching during Phase I of the formalin test is thought to represent an acutely nociceptive event in response to the injection of formalin, as the flinching behavior occurs temporally and with a magnitude correlated with increased afferent traffic from the injected limb (16,17). The ability of Contulakin-G to reduce Phase I flinching suggests an acute inhibitory effect on peripheral nociceptive input, either by decreasing input to the dorsal horn or by a direct spinal inhibitory process.
Phase II flinching in the formalin test is a spinally mediated sensitization phenomenon that is decreased by a number of analgesic and antihyperalgesic drug classes including opioids, Ca+2 blockers, nonsteroidal antiinflammatory drugs, and NMDA antagonists (18). Some of the actions of Contulakin-G may be explained by a decrease in Phase I afferent traffic such that the input into the dorsal horn is attenuated, thus preventing development of spinal sensitization.
Dog: Skin Twitch
In the thermally evoked skin twitch model, intrathecal Contulakin-G significantly increased twitch latency in dogs, which indicates an acute antinociceptive effect. This antinociceptive effect appeared to be dose-dependent and lasted up to 4 hours. This duration of action is consistent with the intrathecal kinetics in dogs, which are described in the companion manuscript (6). MSO4 also significantly decreased skin twitch; however, there were decreases in motor function associated with MSO4 not seen with Contulakin-G. The thermally evoked skin twitch is a spinally mediated reflex and is at least partially mediated by C-fibers, as capsaicin treatment decreases the reflex (19). Thus, the ability of Contulakin-G to inhibit afferent input, as also suggested by the decrease in Phase I of the formalin test in rats, may explain at least part of the antinociceptive effects. Importantly, these antinociceptive effects occurred over a range of intrathecal Contulakin-G doses that had no effect on arousal, motor coordination or tone, heart rate, mean arterial blood pressure, or body temperature.
Epidural Versus Intrathecal Efficacy
A striking observation was the large difference in potency between epidural and intrathecal delivery. In rats, the epidural dose required was 600 times larger than that required intrathecally. In dogs, the intrathecal ED50 for Contulakin-G was approximately 150 nmol. A dose as large as 1000 nmol was without effect after epidural delivery. Larger doses were not used because of limited drug availability. Nevertheless, this lack of effect suggests that the intrathecal/epidural ratio was at least 8, and the lack of effect is consistent with the marked difference of intrathecal and epidural activity observed with the rat model. In the dog, we did not observe any change in thermal escape latencies, even when twice the maximally effective intrathecal Contulakin-G dose was given epidurally. This large epidural-to-intrathecal dose ratio suggests there is minimal diffusion occurring across the dura, arachnoid, and pial layers.
It is appreciated that one component of this barrier relates to the behavior of the dura acting as an ultrafilter. Early work suggested that there was an inverse relationship between meningeal penetration and the molecular weight of a compound (20). The molecular weight of Contulakin G is approximately 1.6 kDa. Nevertheless, previous work in humans has shown relatively large peptides such as the 31 amino acid peptide β endorphin to be active when given epidurally (21). An alternative possibility as to the lack of epidural efficacy is that Contulakin-G transdural distribution may be hindered by passage through the arachnoid or metabolized as it passes through the meninges. Previous work has shown that the arachnoid layer is metabolically active, and there is a possibility that the diffusion of Contulakin-G from the epidural space to the intrathecal space is diminished by meningeal enzyme activity (22,23).
Therapeutic Ratio
No behavioral or motor deficits were noted in any rats receiving spinal Contulakin-G at the doses producing antinociception. Although not systematically studied, larger doses of intrathecal Contulakin-G (31 nmol) produced catalepsy. Given that the ED50 on Phase II for intrathecal Contulakin-G was 0.07 nmol, these data suggest an approximate therapeutic ratio of 400. In the dog, no untoward behavioral or physiological effects were observed at an intrathecal dose that was prominently analgesic (500 nmol), though larger doses were not examined. These data jointly suggest a potent Contulakin-G analgesia in two species with an evident therapeutic ratio.
In summary, these data jointly suggest, in two species, a potent Contulakin-G analgesia at doses that have minimal side effects. This activity and the associated favorable therapeutic ratio suggest its clinical potential as an intrathecally delivered analgesic.
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Footnotes |
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Accepted for publication February 17, 2006.
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  S. E. Kern, J. Allen, J. Wagstaff, S. L. Shafer, and T. Yaksh The Pharmacokinetics of the Conopeptide Contulakin-G (CGX-1160) After Intrathecal Administration: An Analysis of Data from Studies in Beagles Anesth. Analg., June 1, 2007; 104(6): 1514 - 1520. [Abstract] [Full Text] [PDF]
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