Epidural anesthesia/analgesia is a technique commonly used for control of acute and chronic pain. Clinically, there is need for a long-acting drug and/or formulation which provides prolonged sensory block with minimal coincidental side effects on motor function . Prolongation of analgesia can be achieved readily by drug infusion via indwelling epidural catheters. However, these infusions require careful monitoring and the catheters may become infected, kinked, fibrosed, or dislodged from their intended site [2,3].
The development of new chemical entities, such as long-acting local anesthetics, is complex and expensive due to the rigorous pathologic, morphologic, and neurologic assessment required of compounds destined for spinal administration [4,5]. Consequently, considerable formulation work has been done to identify approaches which prolong the duration of action of drugs now available. For example, recent reports describe biocompatible liposomes containing alfentanil for spinal analgesia , lecithin-coated microdroplets of methoxyflurane for intradermal administration , complexes of hydroxypropyl-beta-cyclodextrin with fentanyl for epidural and intrathecal administration , polyanhydride implants of bupivacaine for sciatic nerve blockade , and lipid carriers of amide local anesthetics for epidural administration . Unfortunately, many of these potentially useful formulations may be inappropriate for spinal administration, as they are not readily injectable or may not meet the stringent biocompatability requirements for administration into the spinal column [4,5].
Hyaluronic acid is a naturally occurring high molecular weight glycosaminoglycan composed of a repeating disaccharide unit consisting of N-acetylglucosamine and D-glucuronic acid Figure 1. The clinical profile of hyaluronic acid is well established, and its nonimmunogenicity, biocompatibility, and viscous properties make it an attractive formulation component. Viscous formulations of high molecular weight hyaluronic acid prolong the duration of local anesthetics in the rat infraorbital nerve block model  and the rat spinal model , although subsequent studies investigating ulnar nerve blockade in humans have failed to demonstrate an advantage of viscous formulations over a solution .
Previous studies with hyaluronic acid have utilized a high molecular weight fraction (106 d) in which drug release was controlled only by viscosity due to the high intrinsic viscosity of the polymer. Recently, purified medium molecular weight hyaluronic acid (105 d) has become available and, due to its lower intrinsic viscosity, larger masses of polymer are required to produce viscous formulations. Using the higher polymer loads of the medium molecular weight hyaluronates offers the potential to prepare viscous formulations where cationic drugs are ionically complexed with the anionically charged carboxylate groups of each repeating disaccharide unit in hyaluronic acid. The advantage of ionically linking a cationic local anesthetic (e.g., lidocaine) to the anionic medium molecular weight hyaluronic acid is that drug release would be controlled by both viscous and electrostatic forces.
This study evaluated the utility of medium molecular weight hyaluronic acid for the prolongation of the local anesthetic activity of lidocaine in a rabbit model of epidural analgesia . Equiviscous formulations were prepared as either a physical mixture of lidocaine hydrochloride and sodium hyaluronate (where drug release was expected to occur only via diffusion) or as a lidocaine-hyaluronate complex (where drug release was expected to occur via diffusion and electrostatic processes). The formulations were functionally evaluated in an intact conscious rabbit model, relative to lidocaine solution, and the release characteristics and pharmacokinetic profiles of lidocaine were assessed.
All experimental procedures were approved and performed in accordance with the guidelines of the Institutional Animal Experimentation Ethics Committee. Adult, New Zealand White, cross-strain rabbits (3-5 kg) were obtained from CSL Limited (Parkville, Victoria, Australia) and housed individually in standard cages with free access to food and water. A standard 12-h light-dark cycle was maintained.
Lidocaine HCl (L.HCl) was obtained from David Craig Laboratories (Rocklea, Queensland, Australia). The free base was obtained by alkalinization of an aqueous solution of L.HCl (20 mg/mL), followed by filtration and recrystallization of the precipitate from toluene. Sterile 2% L.HCl solution was obtained from Astra Pharmaceuticals (North Ridge, New South Wales, Australia). Sodium hyaluronate (Na-HA, [eta] = 3.4 dL/g, 165 kd average molecular weight) was kindly provided by Fidia S.p.A. (Abano Terme, Italy). The free acid form of hyaluronic acid was prepared by ion exchange and characterized as previously described . All other chemicals were at least analytical reagent grade and water was obtained from a Milli-Q (Millipore, Milford, MA) purification system.
The physical mix formulation contained 2% (w/w) L.HCl and 2.96% (w/w) Na-HA and was prepared by the addition of the respective components to sterile water for injection. The formulation was allowed to hydrate at 4 degrees C for at least 12 h to attain maximum viscosity, after which the pH was adjusted to 4.0 prior to administration.
The lidocaine-hyaluronate (L-HA) complex was formed by addition of an appropriate quantity of lidocaine free base to an equimolar solution (based on the repeating disaccharide subunit) of the free acid form of hyaluronic acid. The solution was agitated overnight to ensure complete dissolution of the lidocaine base, sterile filtered under aseptic conditions, lyophilized, and then stored at 4 degrees C under desiccation. The L-HA complex was reconstituted by addition of water to produce a formulation containing the equivalent of 2% (w/w) L.HCl. The final pH was 4.0 +/- 0.1 and the equivalent concentration of Na-HA was 2.96% (w/w).
The rheologic profile of each viscous formulation was measured using a Rheometrics Fluid Spectrometer RFS II (Rheometrics, NJ) as previously described . A viscosity of 0.6 Pa centered dot s, determined at a frequency of 100 rad/s, was arbitrarily chosen for each of the hyaluronate formulations as it allowed ready passage through a 20-gauge needle used for drug administration.
The release of lidocaine from the hyaluronate formulations was assessed in a side-by-side apparatus where the donor and receptor chambers were separated by a 10,000 molecular weight cutoff dialysis membrane. The entire apparatus was submerged in a water bath maintained at 38 degrees C. The donor chamber was filled with 1.9 mL of the formulation and was unstirred. The receptor chamber contained 4.5 mL of deionized water and was stirred to provide sink conditions. Aliquots of the receptor phase were withdrawn at selected time intervals and the lidocaine content determined by high-performance liquid chromatography (HPLC).
The concentration of lidocaine in all formulations was verified prior to dosing by HPLC , and the chemical and physical stability of the formulations was monitored for the duration of the study. Administration of the formulations to conscious rabbits was by injection into the epidural space (identified by a modified loss-of-resistance technique) via the readily identified lumbosacral space . The administered dose volume was 0.2 mL/kg which had previously been shown to produce consistent pharmacodynamic responses. The latency of onset and the duration of three simple end-points was used to assess the degree of epidural block as previously described . Briefly, sensation was assessed using a tail clamp, where a rubber-covered 6-in. surgical clamp was closed over the base of the tail and rotated in its long axis. Any sign of discomfort was noted and the stimuli ceased. Loss of weight-bearing was noted when the rabbit could no longer spontaneously support its hindquarters, and recovery was readily discernible when the animal was again steady on its hind limbs. Flaccid paresis was defined as no discernible tone in either hind limb, and recovery was readily apparent when the animal demonstrated recovery of any muscle tone. Testing of each end-point was assessed at 1-min intervals until onset was identified, and at 5-min intervals thereafter.
Two separate studies were performed using the medium molecular weight hyaluronic acid formulations. The first study was a three-treatment, randomized, cross-over study conducted in four rabbits with a 7-day washout period between each treatment. The formulations evaluated were a solution, a viscous physical mix of L.HCl and Na-HA, and an equiviscous formulation of the L-HA complex (administration of the viscous formulations was blinded). Pharmacodynamic end-points were assessed as described above, and venous blood samples (0.6 mL) were taken from the marginal ear vein prior to drug administration, and at 5, 10, 15, 20, 30, 45, 60, and 90 min postadministration. The second study, which was a three-way randomized cross-over study conducted in three rabbits, was undertaken to characterize the absorption characteristics of lidocaine after epidural administration in the rabbit. The treatments consisted of epidural administration of 2% lidocaine as either a solution or the L-HA complex, and intravenous (IV) administration of a 2% lidocaine solution via the marginal ear vein. There was a 7-day washout period between each treatment leg. The blood sampling schedule was as described above, except an additional sample was taken at 2 min after IV administration. Analysis of plasma samples for lidocaine concentrations was performed using a validated HPLC assay .
Pharmacokinetic variable values were estimated using standard compartmental analysis. The plasma concentration-time data after IV administration were analyzed according to a biexponential Equation asdescribed in Equation 1 where Ct is the plasma concentration at time t, A and B are preexponential constants, and alpha and beta are first-order hybrid rate constants describing the biphasic nature of the concentration profile. A weighting factor of 1/C1.5 was used to assist in fitting the data .
The plasma concentration-time data after epidural administration were fitted to a standard one-compartment model with first-order drug absorption as described in Equation 2 where Ct is the plasma concentration at time t, F is the bioavailability, D is the administered dose, ka is the first-order absorption rate constant, ke is the first-order elimination rate constant, and V is the apparent volume of distribution. As the bioavailability after epidural administration was essentially quantitative, the term FD/V was treated as a constant to obtain variable estimates of ka and ke. In some instances, the maximum observed plasma concentration (Cmax) after epidural administration of lidocaine solution occurred at the first measured time point (5 min). This resulted in Equation 2 estimating unreasonably high values of ka. In these situations, Equation 3 was used to provide a minimum estimate of ka where Tmax was assumed to have occurred at the first measured time point, and ke was estimated from the terminal phase of the plasma concentration-time profile. Equation 3
The area under the plasma concentration-time profile (area under the curve [AUC0 right arrow infinity]), was determined by summing the area calculated using the linear trapezoidal rule from time zero to the last measured concentration, and adding to that the tail area calculated by dividing the last measured plasma concentration by the terminal rate constant.
Statistical significance of the formulation effects was determined using one-way analysis of variance and the Student-Newman-Keuls multiple comparison test.
The rank order release rate of lidocaine into the aqueous receptor phase of the dissolution apparatus was solution > physical mix > L-HA complex Figure 2. The decreased rate of drug release from the physical mix relative to the solution reflected the higher viscosity of the hyaluronate formulation. Although the viscosity of the physical mix and L-HA complex were the same (0.6 Pa centered dot s), the rate of drug release from the L-HA complex was further decreased by a factor of four.
The hyaluronate-based formulations and the lidocaine solution were administered epidurally to rabbits in a cross-over study. There were no procedural, formulation, or side-effects observed in any of the rabbits, and motor and sensory function always returned to normal. Table 1 presents the onset and duration of the measured pharmacodynamic end-points after administration of the formulations. All three measured end-points (sensory loss, loss of weight-bearing, and flaccid paresis) were observed after administration of the solution, whereas only loss of sensation and loss of weight-bearing occurred after administration of the hyaluronate formulations. The L-HA complex significantly increased the duration of the loss of sensation, and both the hyaluronate formulations increased the duration of the loss of weight-bearing ability relative to lidocaine solution. However, neither hyaluronate formulation produced a bilateral flaccid paresis. In terms of onset time, the only significant increase (relative to lidocaine solution) was in the loss of weight-bearing ability after administration of the L-HA complex. The duration and character of blocks obtained with subsequent epidural administration of lidocaine solution (after completion of the cross-over study) were indistinguishable from control indicating that the medium molecular weight hyaluronates caused no major or lasting effect on normal spinal column function.
A representative plasma concentration-time profile of lidocaine after epidural administration of the different formulations is presented in Figure 3, and the pertinent mean pharmacokinetic data are listed in Table 2. The rank order of the Tmax values was L-HA > physical mix > solution, and the rank order effect on the magnitude of the Cmax was solution > physical mix = L-HA complex. The AUC values, which are a surrogate for the extent of drug release from the formulations, were similar. Pharmacokinetic modeling of the plasma concentration-time data enabled estimation of the apparent absorption rate constant (ka). The rank order of the magnitude of the absorption rate constants was solution > physical mix > L-HA complex, with the apparent rate of drug absorption into the systemic circulation being approximately fivefold slower after administration of the L-HA complex formulation compared to lidocaine solution.
A subsequent group of rabbits was studied in which an IV dose of lidocaine was administered in a cross-over study with epidurally administered solution and L-HA complex formulations. In this study, the pharmacodynamic end-points after administration of the solution and L-HA complex were consistent with the values reported in Table 1 (data not shown). Figure 4 presents a representative plasma concentration-time profile and Table 3 lists the relevant pharmacokinetic variables after epidural administration. After IV administration of lidocaine, the mean (+/-SD, n = 3) disposition (alpha) and terminal elimination (beta) rate constants were 0.240 +/- 0.040 min-1 and 0.017 +/- 0.001 min-1, respectively, and the mean calculated AUC0 right arrow infinity value was 99.5 +/- 10.3 micro gram centered dot min centered dot mL-1. The fraction of the administered dose which was absorbed after epidural administration of the solution or L-HA complex was essentially quantitative (relative to the IV dose), and the rate of drug release from the L-HA complex was approximately fourfold slower than from the solution. The terminal elimination rate constants were similar after epidural or IV administration.
The epidural space is a potential space which contains adipose tissue, spinal nerves, and many thin-walled blood vessels . The latter traverse the epidural space and present a large surface area for the absorption of local anesthetics. For an epidurally administered local anesthetic to produce clinically relevant analgesia, it must reach and maintain sufficient drug concentrations in the neural tissue, despite clearance from the injection site, via uptake into the blood, lymphatics, and epidural adipose tissue .
Hyaluronic acid enabled the preparation of viscous formulations for epidural administration with the expectation of formulation biocompatability due to subsequent clearance of hyaluronic acid fragments via the lymph and blood . In the current study, use of a novel medium molecular weight hyaluronate fraction provided the opportunity for ionically complexing lidocaine with the carboxylate groups of the repeating glucuronic acid residues present in the polymer.
The in vitro release of lidocaine was markedly slower from the L-HA complex compared with either the equiviscous physical mix or the solution formulation Figure 2. The decrease in the rate of drug release from the L-HA complex, over and above the effect of viscosity observed with the physical mix, reflected the electrostatic nature of the L-HA complex as drug release would require a counter cation to displace the positively charged lidocaine from the ionic complex (after which the lidocaine would be free to diffuse through the viscous matrix of the formulation). Although ionic interactions between lidocaine and hyaluronic acid could potentially occur in the physical mix formulation, the higher ionic strength of the physical mix formulation precludes this form of interaction.
The rank order effect of the formulations on the duration of action Table 1 paralleled the respective release-rate profiles. Although both hyaluronatebased formulations increased the duration of the pharmacodynamic end-points relative to solution, the duration of the loss of weight-bearing ability was further prolonged by the L-HA complex relative to the physical mix. Although neither hyaluronate formulation produced a bilateral flaccid paresis (equivalent to a Bromage category 1 block ), the motor tone present in the hind limbs was diminished (relative to control) and was broadly equivalent to a Bromage category 2-3 block. Unfortunately, the rabbit does not allow categorization of the degree of motor block as the only motor end-point which could be reliably assessed and interpreted was flaccid paresis. The dissociation of effects on sensory and motor function with the viscous formulations probably reflects the development of concentration gradients of drug within the formulation (due to absorption) in the immediate vicinity of the neural tissue.
Pharmacokinetic analysis of the individual plasma concentration-time data confirmed the slow release of lidocaine from the hyaluronate formulations Table 2 and Figure 3, although estimation of the apparent first-order absorption rate constant was complicated by the rapidity of drug uptake. In some instances, the maximum observed plasma concentration occurred at the first measured time point (5 min) which precluded accurate compartmental modeling of the data to estimate ka. In these cases, Equation 3 was used to provide a minimum estimate of ka. Notwithstanding the difficulties associated with estimating ka, it was readily apparent that the reduction in the rate of drug absorption after administration of the L-HA complex was the basis for the prolongation of action. In absolute terms, drug absorption from the epidural space in the rabbit was rapid as the approximate absorption half-lives after administration of the solution and L-HA complex were 1 and 5 min, respectively. This rapid absorption most likely reflects the inherent anatomic and physiologic characteristics of the epidural space of the rabbit (large ratio of the absorptive surface area to the volume of the epidural space).
The absorption and disposition of lidocaine after epidural administration of the L-HA complex was further investigated by studying its pharmacokinetics relative to an epidural solution and IV dose Table 3 and Figure 4. Drug absorption from the epidural space was complete, and the rate of absorption from the L-HA complex was similar between the two studies. The absorption of lidocaine was rapid and a single phase of absorption was adequate to describe the data. These observations are further supported by separate IV and epidural studies conducted with 0.5% bupivacaine in rabbits where drug absorption was also rapid and occurred via a single phase (unpublished data). The rapidity of drug absorption from the epidural space in rabbits does not reflect what is typically observed in higher species where absorption is slower and generally biphasic in nature [22-24]. The basis for these differences is unknown, although it is likely that anatomic and physiologic factors such as the absorptive surface area-to-volume relationships and the relative amounts of epidural adipose tissue are contributing factors.
In conclusion, the duration of sensory analgesia and the loss of weight-bearing achieved with the L-HA complex was increased approximately twofold relative to a solution, and in absolute terms, this duration is comparable to that observed with solution formulations of lidocaine-epinephrine (1:200,000) or bupivacaine . A potential advantage of the L-HA complex over solutions of lidocaine-epinephrine or bupivacaine is the dissociation of complete motor block from the prolonged sensory analgesia. These comparative data suggest that viscous L-HA complexes may have utility in prolonging the duration of sensory analgesia after epidural administration of lidocaine. Further studies investigating the effect of local anesthetichyaluronic acid complexes in a larger animal model of epidural analgesia are underway.
We thank Mr. Ian McLeod for his enthusiastic support of this research, and Professor David Mainwaring (Swinburne University of Technology) for the rheologic measurements.
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