Drug Release from Microspheres
Release of lidocaine or bupivacaine from 50 mg of 10% (w/w) or 50% (w/w) microspheres (Fig. 3) was slowed compared with unencapsulated drug (1 mL of 0.5% [w/v] drug, or 5 mg). Bupivacaine release was slower than that of lidocaine. For example, at 215 hours, the 50% (w/w) bupivacaine particles had released 53.4% (50.6%–57.2%) of their content compared with 74.2% (70.1%–78.5%) for 50% (w/w) lidocaine particles (P = 0.032). Similar trends were seen with the 10% (w/w) formulations.
Effect of Drug Selection on Duration of Sciatic Nerve Block
Animals received injections with 75 mg of particle at the sciatic nerve (Fig. 4). Maximal sensory block (i.e., a thermal latency of 12 seconds) was achieved in 6 of 8 animals injected with 50% (w/w) lidocaine particles and 8 of 8 animals injected with 50% (w/w) bupivacaine particles (P = 0.13, χ2). By 210 minutes, sensory block had resolved completely in the 50% (w/w) lidocaine group, whereas the 50% (w/w) bupivacaine group remained fully blocked. However, the median duration of thermal nociceptive block from 50% (w/w) lidocaine particles was 255 (90–540) minutes versus 840 (277–1215) minutes from 50% (w/w) bupivacaine particles; the difference was not statistically significant (P = 0.056; n = 8).
To evaluate whether 50% (w/w) drug loading was so high that differences in quality of nerve blockade were obscured, 8 animals were injected with 10% (w/w) loaded particles. The resulting duration of sensory block for rats injected with 10% (w/w) lidocaine was 15 (0–38) minutes (n = 4). Only 2 met our definition of sensory nerve block (one lasting 30 minutes, the other 60 minutes). Of the 4 animals injected with 10% (w/w) bupivacaine particles, 3 developed sensory block lasting from 90 to 180 minutes with a median duration 105 (23–180) minutes. The difference in median durations of block between lidocaine and bupivacaine groups was not statistically significant (P = 0.37).
The durations of sensory and motor blockade were compared to determine whether the formulations were sensory or motor selective. For 50% (w/w) bupivacaine particles, the duration of sensory blockade was 840 (277–1215) minutes versus motor blockade of 570 (300–1140) minutes (P = 0.63). Fifty percent (w/w) lidocaine particles produced sensory blockade lasting 255 (90–540) minutes compared with motor blockade of 540 (420–870) minutes (P = 0.025). Ten percent (w/w) bupivacaine particles resulted in 105 (23–180) minutes of sensory blockade versus 105 (60–165) minutes of motor blockade (P = 0.32), and for 10% (w/w) lidocaine particles, sensory duration was 15 (0–38) minutes compared with 30 (0–98) minutes (P = 0.51). The durations of sensory and motor blockade of these formulations were not significantly different, except that in 50% (w/w) lidocaine particles, motor blockade outlasted sensory blockade by 450 (330–728) minutes (P = 0.025) (Fig. 4).
Groups of rats were injected at the sciatic nerve with 50% (w/w) lidocaine particles (n = 8) or 50% (w/w) bupivacaine particles (n = 8). The sciatic nerves were removed 4 days (n = 4) or 2 weeks (n = 4) after injection and processed for histology. Animals were also injected with 10% (w/w) lidocaine particles (n = 4) or 10% (w/w) bupivacaine particles (n = 4) to determine whether the high loading of drug in the 50% (w/w) loaded groups obscured differences in anesthetic effect or tissue toxicity. This was only done at 4 days, the time of maximal injury in the 50% (w/w) drug groups.
All injected rats had firm, white, globular deposits of particle in discrete pockets directly adjacent to the sciatic nerve (Fig. 5), without spread to distant sites. The particle deposits appeared similar at 4 days and 2 weeks. The tissues immediately adjacent to the particle mass were adherent to the residue, but the latter was clearly separate from both muscle and nerve, i.e., there was no evidence of intraneural or intramuscular injection (on gross dissection or on subsequent microscopic examination), and tissue planes were easily identified and separated. Gross appearance was similar for all particle formulations and durations of exposure. On histologic examination, there was evidence of inflammation and myotoxicity in all animals (Table 2, Fig. 6).
Tissue Reaction at 4 Days
Tissue reaction was evaluated in all groups at 4 days after injection, when inflammation and tissue injury is the most intense.21 On necropsy, gross appearance was similar between groups, as reflected in the lack of difference in dissection scores irrespective of drug or drug loading. On histologic examination, the cellular response for all formulations was characterized by inflammatory cells consisting of neutrophils, lymphocytes, and macrophages. PLGA microspheres were visible as round structures 40 to 60 µm across. There was evidence of myotoxicity in all animals (Fig. 6, A–D), characterized by shrunken myofibers with basophilic cytoplasm, in the cell layers proximal to the microspheres. Muscle injury occurred in a perifascicular pattern, worse in areas adjacent to pockets of particle residue, indicating extrinsic tissue injury originating from the particles. Deeper layers demonstrated edema and cells with centralized nuclei (i.e., a lesser degree of myotoxicity). There was no statistically significant difference in the inflammatory or myotoxicity scores between particles loaded with lidocaine or bupivacaine at either concentration (Fig. 7, Table 2).
Particle drug loading did not influence inflammation or myotoxicity scores (Fig. 7, A and C). The median inflammation and myotoxicity scores for 10% (w/w) lidocaine particles were 1.0 (1.0–1.2) and 1.2 (0.9–1.7) respectively, compared with 3.0 (2.1–3.5) and 3.4 (2.1–4.2) for 50% (w/w) lidocaine particles (smallest P = 0.30). For 10% (w/w) bupivacaine particles, the inflammation and myotoxicity scores were 1.4 (1.2–1.6) and 1.7 (1.5–1.9), respectively, compared with 2.7 (2.5–2.9) and 3.3 (2.9–3.5) for 50% (w/w) bupivacaine particles (smallest P = 0.029). These differences were not significant by our predetermined α of P < 0.013.
Tissue Reaction 2 Weeks After Injection
For the 50% loaded particles, in which tissue reaction had been more severe, we followed tissue reaction out to 2 weeks after injection. The acute inflammatory reaction had been replaced by a chronic reaction with macrophages and lymphocytes. The microspheres were surrounded by multinucleated foreign-body giant cells. In all samples, adjacent muscle showed myofiber regeneration in more advanced stages than was observed in the 4-day cohort. In the layers close to the microspheres, regenerating myofibers remained shrunken, but demonstrated normal chromicity. Deeper myofibers had normal morphology with nuclei located at the periphery of the cell (Fig. 6, E–H).
The cellular inflammatory infiltrate resulting from both drug formulations was less severe and did not involve as much of the muscle fascicle as noted on day 4 (Fig. 6, G and H). For 50% (w/w) bupivacaine particles, the median inflammation scores decreased from 2.7 (2.5–2.9) on day 4 to 0.7 (0.5–1.1) at 2 weeks, and the myotoxicity scores decreased from 3.3 (2.9–3.5) to 1.7 (1.3–1.9) (smallest P < 0.029). For 50% (w/w) lidocaine particles, the median inflammation scores decreased from 3.0 (2.1–3.5) on day 4 to 1.4 (1.3–1.6) at 2 weeks, and the myotoxicity scores decreased from 3.3 (2.9–3.5) to 1.7 (1.3–1.9) (smallest P = 0.34). These differences were not significant by our predetermined α of P < 0.013 (Fig. 7, B and D).
There were no statistically significant differences in the low inflammation or myotoxicity scores between the lidocaine and bupivacaine formulations at 2 weeks (Table 2). The inflammation scores for 50% (w/w) lidocaine particles were 1.4 (1.3–1.6) vs 0.7 (0.5–1.1) for 50% (w/w) bupivacaine particles (P = 0.10). The myotoxicity scores were 1.9 (1.8–2.4) compared with 1.7 (1.3–1.9) for bupivacaine particles (P = 0.23).
The key question addressed in this work, from a practical pharmaceutical point of view, was whether the selection of amino-amide (and by inference amino-ester) local anesthetic with respect to myotoxicity affects tissue injury from a controlled-release system. Our results, using a very common, well-established drug delivery vehicle, suggest that it does not. One might have expected the lidocaine microspheres to result in less-severe tissue injury than bupivacaine, based on previous in vivo research documenting differences in the myotoxic potential of these 2 drugs,30 and based on some reports using sustained-release vehicles.25 Furthermore, our myotoxicity experiments with a mouse myoblast cell line (C2C12) demonstrated bupivacaine to be more toxic than lidocaine. These findings were similar to previous research, in which bupivacaine was documented to be approximately 7-fold more cytotoxic than lidocaine.46 In contrast, in PLGA microspheres, the choice of drug had little influence on the severity of inflammation and myotoxicity. This lack of difference might be attributable to the relatively high concentrations that may have been obtained in the first few hours, and/or from long duration of exposure, both of which can have marked effects on toxicity.6 (Note that the concentrations used for in vitro toxicity were well below those used clinically [0.25%–0.75% for bupivacaine and 0.5%–2% for lidocaine] because clinical concentrations caused immediate cell death [data not shown].) Such high concentrations might have been sufficiently toxic with both drugs that no difference could be seen. However, this lack of difference in toxicity was noted even in the 10% (w/w) loaded particles, in which there was relatively minimal nerve blockade (and therefore presumably not very high drug levels). By way of comparison, 0.5% (w/v) bupivacaine reliably yields a nerve block lasting approximately 150 minutes,19,47 with minimal tissue toxicity.42,48 These observations are not consistent with the lack of difference in toxicity being simply due to excessive levels of local anesthetic.
We have previously noted that the mere presence of a sustained-release vehicle can exacerbate local tissue toxicity19,42 in vivo, even though the vehicle might have no effect on local anesthetic cytotoxicity in cell culture.6 The mechanism for this effect is unknown. It is possible that the inflammation caused by the particles, or the particles themselves, worsens the severity of myotoxicity from agents that would otherwise be mild.6,21,37 Particles alone can also cause inflammation at the nerve that can considerably outlast the duration of blockade.21,41,43 The specific nature of the delivery vehicle itself may therefore have an impact on local tissue injury from local anesthetics. As with myotoxicity, the severity of inflammation seemed to correlate with drug loading (Fig. 7A) and decrease over time.
Conventional local anesthetics are also neurotoxic.32,38,39,49 There are reports of bupivacaine-containing PDLA formulations for which no neurotoxicity was documented.1,32 However, as noted in the introductory text, neurotoxicity was a major factor leading to withdrawal of the investigational new drug application (IND #53,441)44 of a sustained-release bupivacaine-dexamethasone formulation.3 The hematoxylin & eosin staining used here was not sufficiently sensitive to detect any but severe nerve injury. Nonetheless, because amino-amide (and amino-ester) local anesthetics cause injury to both tissues in a concentration-dependent manner, the presence of myotoxicity suggests the potential for neurotoxicity.
As noted in the Results section, dissection revealed the presence of microparticulate residue immediately adjacent to the sciatic nerve. This observation would suggest that block durations as presented here are likely reflections of the local anesthetic capabilities of the microparticulate formulations rather than operator error (failed blocks). The lack of statistically significant difference in the durations of block from bupivacaine and lidocaine microspheres may seem surprising given that lidocaine solution provides shorter nerve blocks than bupivacaine.6,49,50 This lack of difference may, in part, have arisen from the relatively short duration of effect of the drugs used here compared with the timeframes over which the microspheres can release drugs. (Note the extremely long durations of block that can be achieved with these microspheres using synergistic drug combinations.37) The great variability in nerve block durations from polymeric microsphere formulations may also have made differences hard to detect. That variability was consistent with our prior experience with PLGA microspheres for nerve blockade.6,20,37,43
It is interesting to consider the risk of PDLA-related local toxicity in the context of ultrasound-guided regional anesthesia, which is now common practice. In many circumstances, ultrasound guidance may allow PDLA formulations to be deposited in locales where they are adjacent to nerves but remote from major muscle groups. There will also be circumstances whereby that is not possible. It would remain to be seen how far muscle groups need to be from such devices to be unaffected. Of course, ultrasound guidance is not perfect: there is always the possibility of inadvertent injection into or near muscle. Many PDLA formulations could be used for infiltration anesthesia or field blocks, in which case ultrasound guidance would likely not be used, even in areas near muscles (perianal procedures, hernia repairs, etc.). It also bears emphasizing that one principal virtue of ultrasound guidance, that it can deposit anesthetics right on the nerve, also has the potential to increase the probability of one of the most serious potential complications of PDLA: nerve injury.
We have observed myotoxicity with a wide range of delivery systems with very different compositions of matter, including PLGA,6,21,37,51 lipid-sugar-protein particles,21,41,43,51 polysaccharide-based gels,19,52 and thermosensitive nanogels.53 This has raised the concern that myotoxicity may be an unavoidable consequence of increased concentrations or prolonged exposures to conventional local anesthetics, regardless of drug selection.6 The observation that even particles with low drug loading (10% in this study) and little anesthetic benefit generate myotoxicity is consistent with that view. Given the potentially severe tissue toxicity (muscle and nerve injury) that has been documented with sustained release of local anesthetics,44 these results suggest that caution is warranted in the application of sustained-release formulations of amino-amide and amino-ester local anesthetics, particularly around muscle and nerve.
Name: J. Brian McAlvin, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: J. Brian McAlvin has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Gally Reznor, BS, MBS.
Contribution: This author helped conduct the study.
Attestation: Gally Reznor has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Sahadev A. Shankarappa, MPH, MBBS, PhD.
Contribution: This author helped conduct the study.
Attestation: Sahadev A. Shankarappa has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Cristina F. Stefanescu, BS.
Contribution: This author helped conduct the study.
Attestation: Cristina F. Stefanescu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Daniel S. Kohane, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Daniel S. Kohane has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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