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Department of Anesthesiology, *Virginia Mason Clinic and
University of Washington, Seattle, Washington;
Medical Research and
Clinical Pharmacokinetics, Purdue Pharma L. P., Stamford;
||Magidom Discovery, LLC, Westport, Connecticut; and
¶Department of Anesthesia, University of Pennsylvania, Philadelphia, Pennsylvania
Address correspondence and reprint requests to Dr. Dan J. Kopacz, Department of Anesthesiology, Virginia Mason Clinic, 1100 Ninth Ave., B2-AN, PO Box 900, Seattle, WA 98111. Address e-mail to anedjk{at}vmmc.org
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Abstract |
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IMPLICATIONS: Microcapsules loaded with bupivacaine and dexamethasone and administered by subcutaneous injection produce prolonged cutaneous anesthesia and analgesia. Determination of local tissue pharmacokinetic variables of bupivacaine by microdialysis confirms that the prolonged duration of anesthesia is caused by the extended release characteristics of the microcapsules.
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Introduction |
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The injection of large amounts of bupivacaine could produce both systemic and local toxicity. Local anesthetics are neurotoxic when administered in large concentrations (4,5). Neurotoxicity seems to be related to both the perineural local anesthetic concentration and to the period that the nerve is exposed.
We previously used microdialysis methodology to demonstrate the effects of both epinephrine and clonidine on the elimination of lidocaine from the site of injection by continuous sampling over 5 h (6,7). This methodology is superior to others in which only blood concentrations are measured because pharmacokinetic (PK) variables derived by microdialysis sampling at the injection site are markedly different from those obtained by venous blood sampling. The former also correlate better with the pharmacodynamic (PD) behavior of these drugs. In vivo analysis of bupivacaine release from polymer microcapsules by microdialysis sampling has not been investigated, nor has microdialysis sampling of tissue local anesthetic concentrations been performed for longer than 5 h.
Microdialysis can determine whether a microcapsule formulation is releasing drug too fast or slow, to assess drug loading, and to determine how changes in microcapsule concentration and/or volume affect drug release and local tissue concentrations. Knowledge of the PK variables of bupivacaine release could lead to optimization of microcapsules and an improvement of their clinical anesthetic/analgesic characteristics.
The primary objective of this study was to use a robust in vivo microdialysis technique to confirm that subcutaneously injected polymer microcapsules produce a sustained release of bupivacaine into the surrounding tissue. Additionally, the study was designed to preliminarily explore the effect of bupivacaine dose and concentration on tissue PKs and PDs.
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Methods |
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The study was conducted in two parts. Polylactic-co-glycolic acid polymer microcapsules loaded with bupivacaine (approximately 72% free base with 0.04% dexamethasone; manufactured by Purdue Pharma L. P., Stamford, CT) was suspended to produce a concentration expressed as "% microcapsules" (microcapsules [mg]/mL). The microcapsule concentrations studied were 1.25% and 2.5%, which correspond to bupivacaine doses of 9 and 18 mg of bupivacaine base/mL, respectively. Part 1 (12 volunteers) was an open-label, randomized trial investigating whether the presence of a microdialysis catheter influences the onset or duration of cutaneous anesthesia. All subjects in Part 1 had microdialysis catheters placed in the left leg only. Injections were made into the left leg of subjects in 1A, and both legs of subjects in 1B and 1C. Plasma bupivacaine and dexamethasone concentrations were obtained from all subjects.
Part 1: Three subject groups (4 subjects each):
Microdialysis samples from Part 1 were used as "pilot" samples to determine the general range, timing, and variability of tissue bupivacaine concentrations to be expected for the formal PK analysis conducted in Part 2.
Part 2 (16 subjects) was a double-blinded, randomized, parallel trial investigating the relationship between dose (volume and concentration) and tissue bupivacaine and dexamethasone PKs, and on the duration of sensory anesthesia. All subjects had microdialysis catheters placed in both legs.
Part 2: Three subject groups:
Injections were performed on the anteromedial lower leg in a 6 x 6 cm diamond-shaped area. Opposite corners, and 2 interior points along the line connecting these 2 corners, were first anesthetized with 0.5 mL of 0.5% lidocaine. A 2-in., 18-gauge IV catheter and needle was inserted through the skin of one of the interior points, advanced subcutaneously for approximately 4 cm, exiting the skin at the second point. The needle was removed, leaving the "insertion" catheter tip protruding. A custom loop microdialysis catheter was inserted through the insertion catheter tip to span the middle of the diamond-shaped injection area. Custom microdialysis catheters were made by one of the authors (CMB) as previously described (6). The tip of the insertion catheter was then withdrawn to reside entirely in the subcutaneous tissue.
After microdialysis fluid was infused through the microdialysis catheter at 10 µL/min for a 10-min equilibration period, study drug was injected using the following standard sequence. The first 40% (3 or 6 mL) was injected fanwise (4 passes) from the distal corner. The second 40% was injected identically from the opposite proximal corner. The final 20% (1.5 or 3 mL) was injected through the "insertion" catheter as it was withdrawn through the subcutaneous tissue. This catheter was removed entirely at the end of the injection, ensuring that the previously placed microdialysis catheter resided in the center of this final 20%. Injections took approximately 4 min.
Dialysate collection commenced at the end of drug injection and was continuous for 3 h (10 µL/min, 20-min sampling periods, total sample = 200 µL). After this period, the microdialysis catheter was left in place, but was disconnected and capped. Additional microdialysis samples (20-min collection) were obtained 6, 12, 24, 48, 72, and 96 h after injection, with venous blood samples obtained simultaneously. The microdialysis catheter was removed after 96 h.
Sensory testing (pin-prick, thermal thresholds, and cold/wet sensation) were conducted after 30 min, 1, 3, 6, 12, 24, 48, 72, and 96 h. Pain to pinprick (dental needle) was rated on a 3-point scale: 0 = no sensation (anesthesia), 1 = pin sensed as dull (analgesia), 2 = sharp. Determination of warm and heat pain detection thresholds was performed with a custom-built thermode-thermocouple starting at 30°C, and increasing at 1.5°C/s, to a cutoff of 50°C. Cold/wet sensation was tested using an alcohol swab. Cutaneous blood flow velocity was tested using a laser Doppler flowprobe at the injection site and at an unblocked control site (left thigh). Adverse events at the injection sites were assessed through 96 h and at 2 wk, 6 wk, 2 mo, and 6 mo.
Venous blood samples for determination of bupivacaine and dexamethasone concentrations were obtained at 0.5, 1, 3, 6, 12, 24, 48, 72, and 96 h. Plasma was immediately separated by centrifugation and stored at -20°C.
Microdialysate and plasma bupivacaine and dexamethasone determinations and PK calculations were performed at Purdue Pharma L. P. Bupivacaine and dexamethasone concentrations were measured by liquid chromatography/mass spectrometry using multiple reaction monitoring, operating under positive electron spread mode. The calibration ranges were 5–300 (bupivacaine in plasma), 0.5–5 (dexamethasone in plasma), 50–80,000 (bupivacaine in dialysate), and 0.5–200 ng/mL (dexamethasone in dialysate). Blank plasma was used to prepare the calibrants and quality controls (QCs) for plasma sample analysis; saline was used to prepare the calibrants and QCs for dialysate sample analysis, after it was experimentally verified that saline was bioanalytically equivalent to dialysate. Sample preparation was performed on a 96-well solid-phase extraction plate, before instrument analysis. QC samples were inter-dispersed throughout the analysis, and all passed the acceptance criteria. The lower limits of quantitation for bupivacaine in plasma and tissue dialysate were 5 and 50 ng/mL, respectively, and for dexamethasone, 0.5 ng/mL, both plasma and tissue dialysate.
Concentration-time profiles of bupivacaine and dexamethasone were used to determine peak plasma/dialysate concentration (Cmax), time to reach Cmax (tmax), terminal elimination half-life (t1/2), and area under the concentration-time curve from injection to 96 h (AUCt). The t1/2 was calculated from the terminal slope by linear regression. The PK variables were estimated using a noncompartmental analysis technique.
Continuous data were expressed as mean ± SD or median (range). Because of the exploratory nature of this investigation, no power analysis was used to determine sample size. Postinvestigation analyses of clinical variables were conducted using repeated-measures analysis of variance, unpaired t-test, and 
2 analysis where appropriate, with P < 0.05 significant.
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Results |
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All subjects developed analgesia, but more subjects receiving 15-mL 2.5% microcapsules or 0.5% aqueous bupivacaine developed anesthesia at the injection site (Fig. 3A, 2 P = 0.01). Significantly more subjects receiving aqueous bupivacaine had analgesia present at 30 min (2 P < 0.04, 16 of 16 aqueous sites versus 25 of 36 microsphere sites [18 of 24, 2.5% 15 mL; 4 of 6, 2.5% 7.5 mL; 3 of 6, 1.25% 15 mL]). Onset of analgesia averaged 49 ± 41 min at microcapsule sites (P = 0.21 versus aqueous [30 ± 0 min]). In subjects who developed anesthesia, onset was faster for aqueous bupivacaine than for microcapsules (41 ± 16 versus 743 ± 874 min, respectively, P = 0.03) (Fig. 3A). Duration of analgesia was longer for subjects receiving microcapsules compared with aqueous (3.4 ± 0.8 days versus 0.9 ± 0.5 days, P < 0.001, Fig. 3B). The onset and duration of loss of cold/wet sensation paralleled analgesia to pinprick (Fig. 3C). Analgesia was still present in 78% (28 of 36) of the sites receiving microcapsules at the end of 96 h. These subjects reported this sensation persisting for an additional 48.9 ± 22 h.
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Because study infiltrations were into the area traversed by superficial branches of the saphenous nerve, anesthesia/hypesthesia also developed distal to the site of injection. Distal blockade developed in 19% of injections, tended to occur more frequently at sites of microcapsule injection (9 of 36 versus 1 of 16 for aqueous bupivacaine, P = 0.11), extended to the skin overlying the medial malleolus in some subjects (within the distribution of the saphenous nerve), but did not outlast anesthesia at the site of injection.
Pruritus occurred in 2 of 16 sites receiving aqueous bupivacaine and 20 of 36 sites injected with microcapsules. Mild induration occurred at 15 of 36 microcapsule sites. Pruritus tended to occur more frequently at sites receiving 2.5% microcapsules (18 of 30 = 60%), than at sites receiving 1.25% (2 of 6 = 33%, P = 0.23). Pruritus began from 1 to 32 days (median = 14 days, mean = 15 days) after injection and persisted for 1–125 days (median = 14 days, mean = 19 days). Induration started approximately 2 days after the onset of pruritus (range 1–27 days, median = 21 days, mean = 19 days), and lasted an average of 48 days (median = 37 days, range 21–120 days). No signs or symptoms of neurotoxicity were reported throughout the 6-mo study period.
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Discussion |
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Several release characteristics of this particular microcapsule formulation were detected by microdialysis sampling which document its safety, and which suggest modifications for future formulations. Although the peak tissue bupivacaine concentrations were significantly larger than for aqueous bupivacaine, they were attained much more gradually. Despite larger tissue bupivacaine concentrations, much smaller plasma bupivacaine concentrations were produced, affirming the safety of the microcapsules regarding systemic toxicity.
Ideally, one would design a sustained-release local anesthetic to rapidly achieve a minimally effective tissue concentration threshold, plateau just above this level for the desired period of time, and then decrease below that threshold such that sensory function returns to normal. Whereas microdialysis is the ideal technology for determining the minimally effective tissue concentrations for both anesthesia and analgesia, this study was not designed to conduct formal PK-PD analysis.
One might expect a larger concentration (or volume) of microcapsules to produce a longer duration of effect. The results of this study suggest otherwise, because the clinical characteristics of 1.25% and the small-volume (7.5 mL) 2.5% preparations were as effective as the large-volume 2.5% preparation. Furthermore, the 1.25% concentration produced a tissue bupivacaine "plateau" closer to the peak tissue concentration produced by aqueous bupivacaine. However, doubling the dose of microcapsules also did not double the peak bupivacaine concentration (tissue or plasma).
Presumably, there is also a tissue bupivacaine toxicity threshold for concentration and exposure time, such that when surpassed, irreparable damage to blood vessels, nerves, or nerve fibers is produced. Although tissue bupivacaine concentrations were much larger for a longer period in the microcapsule subjects, no damage attributable to bupivacaine was detected.
Different PK mechanisms are in effect for the aqueous and microcapsule preparations. Bupivacaine from the aqueous preparation is delivered as a bolus into the subcutaneous tissue. This is followed exclusively by elimination as a result of uptake into the systemic circulation. Bupivacaine delivered into the subcutaneous tissue from microcapsules is initially substantial, although not as much as with aqueous solution. This initial bupivacaine exposure is thought to be caused by the small amount of free drug that exists in solution after suspension of the microcapsules. In contrast to the aqueous injection, as elimination of this initial bupivacaine begins, additional bupivacaine is delivered to the tissue from the microcapsules. For the first 24–34 hours, the release of additional bupivacaine from the microcapsules exceeds elimination, and tissue concentrations continually increase (Fig. 1). Beyond that, either the bulk of bupivacaine has been released from the microcapsule, or the gradient between the microcapsule and its surrounding tissue is reduced, such that elimination exceeds further release of bupivacaine, and tissue bupivacaine declines. The t1/2ß only considers this final elimination phase, whereas elimination has actually been continuing for the previous 24–34-hour period. Elimination half-life may not be an appropriate PK variable for describing or comparing microcapsules. Furthermore, the extrapolation of AUC to infinity, which is often done after single bolus injection of other drugs, also is not appropriate in this study, because it is unlikely that bupivacaine release from the microcapsule remains constant, especially after 96 hours. Release of bupivacaine from microcapsules may be more analogous to a continuous drug infusion, where context-sensitive half-times are thought to be a more clinically relevant descriptor of drug behavior (8).
Finding different release rates of two drugs from the same microcapsule is new and unexplained. Dexamethasone concentrations peaked earlier in both tissue dialysate and plasma samples than bupivacaine. Previous studies have demonstrated a much longer duration with dexamethasone-containing microcapsules (3). This prolongation is likely attributable to a PD mechanism, because it would be difficult to pharmacokinetically explain an extension of bupivacaine anesthesia beyond 72–96 hours when tissue dexamethasone concentrations peak within 20 hours of injection.
We were also surprised to find no correlation of tissue bupivacaine with blood flow. Essentially, all sites exhibited vasodilation for the first 30 minutes with blood flow rapidly returning to baseline levels thereafter. Previous studies have shown that the addition of vasoconstrictive drugs can reduce skin blood flow for periods much longer than 30 minutes (6,7). Apparently, skin blood flow autoregulatory mechanisms can overcome the vasodilatory effects of bupivacaine, but are insufficient to offset the effects of supplementary vasoconstrictive drugs.
Because adverse effects seen in this study (mild pruritus and induration) occurred much later than peak tissue bupivacaine, we believe they are caused by the copolymer matrix and not bupivacaine. The induration response to implantable polysaccharide is well documented and described as a normal foreign body reaction appearing in parallel with polymer biodegradation (9). Because of the limited sample size, it could not be determined whether these effects are dose-related to either the microcapsule matrix and/or bupivacaine load.
In conclusion, the subcutaneous injection of dexamethasone-containing bupivacaine microcapsules produces a prolonged duration of skin anesthesia and analgesia. These effects are a result of the sustained release of bupivacaine from the microcapsules, a process in which PK mechanisms can be determine in vivo by microdialysis sampling.
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Acknowledgments |
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The authors thank Jormain Cady, ARNP, Jill Zeller, RN, Pearl Washburn, RN, Rose Lopez, RN, Joel Miller, and Steve Harris, MD, Purdue Pharma L. P., Stamford, CT, for their assistance and dedication during the conduct of this investigation.
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Footnotes |
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  J. L. Pedersen, J. Lilleso, N. A. Hammer, M. U. Werner, K. Holte, P. G. Lacouture, and H. Kehlet Bupivacaine in Microcapsules Prolongs Analgesia After Subcutaneous Infiltration in Humans: A Dose-Finding Study Anesth. Analg., September 1, 2004; 99(3): 912 - 918. [Abstract] [Full Text] [PDF]
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