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REGIONAL ANESTHESIA AND PAIN MEDICINE

The Effects of Intrathecal Morphine Encapsulated in L- and D-Dipalmitoylphosphatidyl Choline Liposomes on Acute Nociception in Rats

Tomoki Nishiyama, MD, PhD^*,, Rodney J. Y. Ho, PhD, Danny D. Shen, PhD^,§, and Tony L. Yaksh, PhD^*

*Department of Anesthesiology, University of California, San Diego, California; Department of Anesthesiology, the University of Tokyo, Faculty of Medicine, Tokyo, Japan; Department of Pharmaceutics, University of Washington; and §Pain Research, Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, Washington

Address correspondence and reprint requests to Tomoki Nishiyama, MD, PhD, 3-2-6-603, Kawaguchi, Kawaguchi-shi, Saitama, 332-0015, Japan.

	Abstract

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Liposomes can serve as a sustained-release carrier system, permitting the spinal delivery of large opioid doses restricting the dose for acute systemic uptake. We evaluated the antinociceptive effects of morphine encapsulated in liposomes of two isomeric phospholipids, L-dipalmitoylphosphatidyl choline (L-DPPC) and D-dipalmitoylphosphatidyl choline (D-DPPC), in comparison with morphine in saline. Sprague-Dawley rats with chronic lumbar intrathecal catheters were tested for their acute nociceptive response using a hindpaw thermal escape test. Their general behavior, motor function, pinna reflex, and corneal reflex were also examined. The duration of antinociception was longer in both liposomal morphine groups than in the free morphine group. The peak antinociceptive effects were observed within 30 min after intrathecal morphine, L-DPPC or D-DPPC morphine injection. The rank order of the area under the effect-time curve for antinociception was L-DPPC morphine > D-DPPC morphine > morphine. The 50% effective dose was: 2.7 µg (morphine), 4.6 µg (L-DPPC morphine), and 6.4 µg (D-DPPC morphine). D-DPPC morphine had less side effects for a given antinociceptive AUC than morphine. In conclusion, L-DPPC and D-DPPC liposome encapsulation of morphine prolonged the antinociceptive effect on acute thermal stimulation and could decrease side effects, compared with morphine alone.

Implications: Two isomers of liposome (L-dipalmitoylphosphatidyl choline and D-dipalmitoylphosphatidyl choline) encapsulation of morphine prolonged the analgesic effect on acute thermal-induced pain when administered intrathecally and could decrease side effects, compared with morphine alone.

	Introduction

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A single intrathecal injection of morphine produces a potent analgesia that has a duration of action limited by the clearance of the drug (1). To increase the duration of action associated with a bolus injection requires delivery of a larger dose. Larger doses, however, have an increased extraspinal side effect secondary to redistribution along the concentration gradient. One possibility is that the pharmacokinetics of morphine may be altered by incorporating it into a system that alters the diffusibility of the active agent. Various delivery systems have been evaluated, such as iophendylate (2), ß-cyclodextrins (3), and liposomes (4–6).

Liposomes are phospholipid membrane vesicles that may be used to alter drug kinetics (7). Encapsulation by a liposome reduces the amount of drug available for redistribution, and presumably serves to increase the amount of drug that may be injected as a bolus without inducing a high-dose–related extraspinal side effect. In previous work, we have examined liposome encapsulated µ-opioid alfentanil (4,5,8). Such liposomal encapsulation had only a modest effect on duration of action, presumably because of the ability of the lipophilic alfentanil to rapidly diffuse through the membrane of the liposomes (5,8). In addition, we demonstrated that intrathecal delivery of the natural lipid L-dipalmitoylphosphatidyl choline (L-DPPC) resulted in a steroid-sensitive allodynia, whereas the D-isomer, D-dipalmitoylphosphatidyl choline (D-DPPC), had no such effect. In the present study, we examined the dose-effect curves of the intrathecal delivery of free morphine or morphine encapsulated in liposomes prepared from L-DPPC or D-DPPC.

	Methods

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Animal Preparation
Experiments were performed according to a protocol approved by the Institutional Animal Care Committee of the University of California, San Diego. Sprague-Dawley rats (250–300 g; Harlan Industries, Indianapolis, IN) were implanted with chronic lumbar intrathecal catheters under halothane (2%) anesthesia, according to a modification of the method described by Yaksh and Rudy (9). Briefly, an 8.5-cm polyethylene (PE-10; Clay Adams, Parsippany, NJ) catheter was advanced caudally through an incision in the atlanto-occipital membrane to the thoracolumbar level of the spinal cord. The external part of the catheter was tunneled subcutaneously to exit on top of the skull and plugged with a steel wire. Rats with normal motor function and behavior were used 5–7 days after surgery. Each rat was used only once, and 122 rats were used in total.

Drugs and Injection
Drugs for intrathecal injection were delivered in a volume of 10 µL. Lipids were obtained from Genzyme (Cambridge, MA) and Sigma (St. Louis, MO). Morphine (morphine sulfate, Merck, Sharpe and Dohme, West Point, PA) was dissolved in normal saline. L-DPPC, D-DPPC, and L-DPPC– and D-DPPC–encapsulated morphine (at a concentration of 10 mg/mL and 12 mg/mL, respectively) were delivered from Washington University. Liposomal encapsulation of morphine was performed according to the previous method (8). Briefly, the DPPCs, in chloroform, were dried under a stream of N₂ gas. The dried gases were desiccated to remove the residual organic solvents. Then, morphine was added and incubated in a 55°C water bath. The suspensions were vortexed and flash frozen with methanol dry ice, then thawed at room temperature, and warmed to 55°C. This procedure was repeated, and they were dialized at 4°C. Both isomers of liposomal morphine were stored at 4°C (not frozen). Before injection, they were incubated at 37°C for 5–10 min. They were suspended in normal saline just before injection. After intrathecal drug injection, the catheter was flushed with 10 µL of normal saline. Microinjector syringes were used for all injections. In each treatment group, 6–10 randomly selected rats received one of the following doses of morphine: 1 µg, 3 µg, 10 µg, 30 µg, 50 µg, or 100 µg L-DPPC-morphine; 1 µg, 10 µg, 50 µg, or 100 µg D-DPPC-morphine; 1 µg, 10 µg, 50 µg, or 100 µg L-DPPC liposomes without morphine, and D-DPPC liposomes without morphine or saline. The latter three treatment groups are the controls.

Nociceptive Test
All animals were tested for an acute nociceptive response using a commercially available hindpaw thermal escape test (10). The rats were placed in a clear plastic cage on an elevated floor of clear glass. A radiant heat source (halogen projector lamp CXL/CXP 50 W 8 V, Ushio, Tokyo, Japan) was contained in a movable holder placed beneath the glass floor. The radiant heat source’s aperture was 4 mm in diameter, and bulb intensity was controlled by a constant voltage source. The interior of the box under the animal was heated with a heat source to regulate the under plate temperature to 30°C. The under floor heat source was then positioned so that it focused at the plantar surface of one hindpaw, where it was in contact with the glass. The calibration of the thermal test system was such that the average response latency in normal untreated rats, measured before the initiation of an experiment, was approximately 6 s. To initiate a test, the rat was placed in the box and allowed to adapt for approximately 20 min. The light was then activated. The time interval between the application of the light beam and the brisk hindpaw withdrawal response was measured. Cutoff time in the absence of a response to avoid tissue injury was 20 s.

Behavioral and Motor Function Assessment (Side Effects)
The general behavior (including agitation, allodynia, and catalepsy), motor function, pinna reflex, and corneal reflex of each animal were examined. They were judged as present (positive in side effect) or absent. The presence of allodynia was determined by looking for agitation (escape or vocalization) evoked by lightly stroking the flank of the rat with a pencil. Catalepsy was assessed by noting movement after the forepaws were placed on a surface 4 cm higher than the hindpaw. Lack of movement was judged as a positive response (drug effect). Pinna reflex was tested by a paper string inserted into the ear, which normally induces ear or head shaking. The lack of these reflexes were judged as positive side effects. Motor function was evaluated by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hindpaw across the edge of the table. This normally evokes a lifting and placing of the paw. The latter was assessed by placing the rat horizontally with its back on the table, which normally results in an immediate, coordinated twisting of the body to an upright position. Each side effect was scored as positive or negative. The number of rats with each side effect was assessed.

Experimental Paradigm
To determine the dose-response and time course of the antinociceptive actions and side effects of intrathecally administered liposomal encapsulated morphine and free morphine on acute thermal nociception, the hot-box test, behavioral and motor function tests were performed before and at 15, 30, 60, 90 min, and at 2, 3, 4, and 5 h after the injection.

Data Analysis and Statistics
Response latency data from hot box measurements were converted to % MPE (maximum possible effect) according to the formula: % MPE = [(postdrug latency - baseline latency)/(cutoff time - baseline latency)] x 100. Area under the time-effect curve (AUC) from 0 to 5 h was calculated as % MPE x time by trapezoidal method. The 50% effective dose (ED₅₀) values (effective dose resulting in a 50% prolongation of the control hot box latency; 50% MPE) were determined for each drug using a computer program based on the analysis published by Tallarida and Murray (11).

Effect-time and dose-response data are graphically presented as mean ± standard error. The latencies were analyzed by a two-way, repeated-measures analysis of variance, followed by pair-wise contrast. One-way factorial analysis of variance was used to analyze AUC and ED₅₀. As a post-hoc test, the Fisher’s Protected Least Significant Difference was used. A P value less than 0.05 was considered statistically significant.

	Results

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Antinociceptive Effect
Saline, L-DPPC, and D-DPPC without morphine did not induce any apparent antinociceptive effects. The peak antinociceptive effect was observed within 30 min after intrathecal administration of morphine, L-DPPC or D-DPPC morphine administrations (Fig. 1). L-DPPC morphine had a larger peak effect at 10 µg than the corresponding doses of free morphine (P = 0.037) and D-DPPC morphine (P = 0.041), whereas at 1 µg free morphine had a larger peak effect than liposomal morphine (Fig. 2). The analgesic data of morphine, 50 µg and 100 µg, were not used because of a large incidence rate of motor disturbance. There were clear differences in the duration of action. The duration of antinociception was longer in both of the liposomal morphine groups than in the morphine group (Fig. 1). The rank ordering of the AUC for the thermal escape was: L-DPPC morphine > D-DPPC morphine > morphine (Fig. 3). The ED₅₀ was 2.7 µg (95% confidence interval: 1.5 µg–5.1 µg) in the morphine group, which was smaller than those in both liposomal morphine groups (4.6 µg; 1.9 µg–11.4 µg in the L-DPPC morphine group [P = 0.029] and 6.4 µg; 3.4 µg–12.0 µg in the D-DPPC morphine group [P = 0.014]).

View larger version (24K): [in this window] [in a new window]   Figure 1. Time course of peak % MPE for intrathecal morphine, 10 µg; L-DPPC– and D-DPPC–encapsulated morphine, 10 µg. Each point presents the results of 6–10 animals. Bars indicate mean ± standard error. *P < 0.05 vs morphine, +P < 0.05 vs D-DPPC-morphine. MPE = maximum possible effect, L-DPPC = L-dipalmitoylphosphatidyl choline, D-DPPC = D-dipalmitoyl-phosphatidyl choline.

View larger version (20K): [in this window] [in a new window]   Figure 2. Dose-response of % MPE for intrathecal morphine, L-DPPC– and D-DPPC–encapsulated morphine. Each point presents the results of 6–10 animals. Bars indicate mean ± standard error. *P < 0.05 vs morphine, +P < 0.05 vs D-DPPC-morphine. MPE = maximum possible effect, L-DPPC = L-dipalmitoylphosphatidyl choline, D-DPPC = D-dipalmitoylphosphatidyl choline.

View larger version (16K): [in this window] [in a new window]   Figure 3. Dose-response of AUC (area under the time-effect curve in units of % MPE x minutes for 5 h) for intrathecal morphine, L-DPPC– and D-DPPC–encapsulated morphine. Each point presents the results of 6–10 animals. Bars indicate mean ± standard error. *P < 0.05 vs morphine, +P < 0.05 vs D-DPPC-morphine. MPE = maximum possible effect, L-DPPC = L-dipalmitoylphosphatidyl choline, D-DPPC = D-dipalmitoylphosphatidyl choline.

View larger version (20K): [in this window] [in a new window]   Figure 4. Relation between antinociceptive effect (area under the time-effect curve [AUC]) and side effect (loss of pinna reflex) for several doses of intrathecal morphine, L-DPPC–, and D-DPPC–encapsulated morphine. Each point presents the mean of 6–10 animals. Bars indicate mean ± standard error. *P < 0.05 vs morphine, +P < 0.05 vs D-DPPC-morphine. MPE = maximum possible effect, L-DPPC = L-dipalmitoylphosphatidyl choline, D-DPPC = D-dipalmitoylphosphatidyl choline.

	Discussion

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The spinal delivery of large-dose opioids can evoke a dose-dependent blockade of the pinna reflex, the corneal reflex, a reduction in spontaneous motor activity (catalepsy), and a depression of respiration (13). In the present study, these side effects were observed less with L-DPPC and D-DPPC morphine, compared with free morphine. Encapsulation of alfentanil in the liposomes also decreased loss of corneal, paw step, and righting reflexes and slightly decreased catalepsy and loss of the pinna response (8), thus indicating less systemic redistribution of alfentanil to supraspinal sites (4). Thus, liposomal preparations can significantly enhance the therapeutic ratio of opioids after spinal delivery (5). The side effects of not only opioids, but also of local anesthetics can be reduced with liposomal encapsulation. Boogaerts et al. (14) showed a reduction in neural and cardiac toxicity with IV bupivacaine encapsulated in multilamellar liposomes.

The extended duration of analgesia is explained by the slow passage of the poorly lipid-soluble morphine across the liposomal membrane (15). Prolonged neur-axial residence time of the liposome has been attributed to the inability of the liposomal particle to pass through the subarachnoid villi (16). Because only free morphine can pass into the systemic circulation, the rate-limiting factor in morphine removal from the neuraxis was likely to be the rate of opioid release from the liposomal depot. Studies with epidurally administered liposomes (DepoFoam^TM) in dogs confirm these altered kinetics. The t_1/2 of morphine increases about three times, and the residency time increased about 14 times in the lumbar cerebrospinal fluid (CSF) by liposomal encapsulation of morphine (17). Moreover, the peak concentration of morphine in lumbar CSF was significantly decreased after liposomal delivery than administration with saline (17).

Liposomal preparation of alfentanil prolonged the spinal antinociceptive effect, but did not affect the peak time or the intensity of analgesia, compared with free alfentanil using hot-plate and paw pressure tests in rats (4). Isackson et al. (8) reported that both peak effect and duration of antinociception were not significantly different between intrathecally administered alfentanil encapsulated in liposome and that dissolved in saline. In the present study, L- and D-liposomal encapsulation prolonged the analgesic effect of morphine, but the earliest peak effect was obtained with L-DPPC morphine, followed by free morphine then D-DPPC morphine. The D-isomer phospholipids are resistant to hydrolysis; therefore, the peak effect might come in the slowest. The preparation of L-DPPC morphine in this study might have contained more free morphine than D-DPPC morphine, thus enabling the earliest peak effect. In contrast to opioids, liposomal encapsulation of local anesthetics results in a pronounced sustained effect (18,19). The differences between these results, including the present study, are not clear, but may have to do with the physiochemistry of the respective liposomes.

In our earlier alfentanil study, L-DPPC produced allodynia in 100% of the animals, whereas significantly less allodynia was observed with the D-DPPC preparations (8). Allodynia was reported to be observed in L-DPPC liposome preparations containing no alfentanil and was not produced by saline nor saline plus alfentanil, which indicates that allodynia was induced by L-DPPC liposomes per se (5). The touch-evoked agitation does not appear to be related to a direct action of the L-DPPC, but rather to a product of phospholipase hydrolysis (20). The liposomes we used were the same as in the previous studies (5,8), but L-DPPC liposome itself did not induce allodynia. Agitation or allodynia seen with morphine decreased by both liposomal encapsulation. Thus, liposome seemed to inhibit agitation and allodynia. The reason for the different results among the studies is not clear. One difference among the studies was that the solution was kept at 37°C before administration in our study. This might have inhibited allodynia or something was different during preparation. Further studies are necessary to fully elucidate what factors induce allodynia.

Since liposomes are synthesized from phosphocholine and cholesterol, constituents of human tissue, they might be inherently nontoxic (21). However, those composed of lecithin-cholesterol-diacetyl phosphate or lecithin-cholesterol-stearylamine produce generalized epileptic seizures and some deaths caused by respiratory failure immediately after injection, followed by widespread tissue necrosis. However, liposomes composed of lecithin-cholesterol-phosphatidic acid, or dipalmitoyl lecithin only, produced minimal morphologic changes. By the sixth day postinjection, the pathology was limited to the mechanical trauma caused by the injection (22). After intracerebroventricular (22) or spinal (4) delivery, no behavioral pathologic change has been reported with L--phosphatidylcholine liposomes. The liposomes used in this study may not produce tissue toxicity, although we did not investigate this.

The peak plasma concentration of alfentanil was larger in saline preparation than in liposome preparation, indicating a rapid movement of alfentanil from the CSF to the plasma in saline preparation (4). The route by which a spinally administered drug reaches the supraspinal sites is controversial. Direct movement by bulk redistribution rostrally through the cerebrospinal axis may occur, as evidenced by the time-dependent evolution of a rostrocaudal drug gradient (23) and the delayed appearance of drug in the cisterna (24). Alternately, as drugs move into the spinal cord, they are cleared into the parenchymal vasculature; from there, they may enter the brain and evoke effects proportional to systemic plasma concentrations. Plasma concentrations of morphine were not measured in this study. However, from the results of prolonged effect, larger ED₅₀, and decreased side effects of liposomal morphine, morphine is suggested to be released slowly from liposomes.

In conclusion, intrathecal administration of L-DPPC and D-DPPC liposome-encapsulated morphine prolonged the antinociceptive effect and produced fewer side effects, compared with free morphine.

	Acknowledgments

 
This work was funded by NIH Grant DA 07313 (to DDS, RJYH, TLY).

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Nishiyama, T. Articles by Yaksh, T. L. Search for Related Content PubMed PubMed Citation Articles by Nishiyama, T. Articles by Yaksh, T. L.