Each solution was vortexed for approximately 1 minute and allowed to settle. The supernatant was transferred to a second vial and evaporated to solid residue under a stream of nitrogen at approximately 40°C. Each residue was reconstituted in 200 μL of the mobile phase, that is, 50:50 (vol:vol) acetonitrile:water with 0.1% formic acid, vortexed for 30 seconds, and transferred to a high-performance liquid chromatography vial with low volume insert for analysis. The calibration curve was generated using the same methodology, in which 8 different solutions of known concentrations ranging from 3.23 to 450 ng/mL were used to generate a linear calibration curve (R2 > 0.98). The LC-MS-MS analysis was performed on an Agilent 1100 high-performance liquid chromatography with API 4000 triple quad mass spectrometer, with a Thermo HyPurity C18 column (150 mm × 2.1 mm, 5-μm particle size) at a flow rate of 0.3 mL/min and an injection volume of 10 μL. For the mass spectrometry, multiple reaction monitoring of masses 289.2 and 275.1 was performed through the first quadrupole (representing bupivacaine and ropivacaine, respectively). Subsequently, second quadrupole fragmentation and third quadrupole monitoring of masses 140.2 and 126.0 (bupivacaine and ropivacaine, respectively) were performed. The data are expressed as the concentration of bupivacaine-free base equivalents in nanograms per milliliter. As in the published methodology,12 the lower limit of quantification was determined to be 3.23 ng/mL. This method does not resolve or differentiate between the R- and S-enantiomers of bupivacaine.
Male Sprague–Dawley rats, weighing 250 to 300 g, were injected subcutaneously with either substance-containing or control, drug-free materials in the same volumes (0.4 mL) and concentrations as used for the serum level assays. The same formulation of bupivacaine-releasing microspheres (MS-Bup) was delivered, either with suspending media containing carboxymethyl cellulose (CMC; Spectrum Chemical, Gardena, CA) at 0.75% (w/v) in phosphate-buffered saline (PBS), or with methyl cellulose (MC; R&D Systems, Minneapolis, MN) at 0.5% (w/v) in water for injection (B. Braun Medical, Irvine, CA). The reason for this comparison was the presence of high levels of lipopolysaccharides, that is, endotoxins (≥122.9 endotoxin units [EU]/mL) that had been detected in the CMC-containing suspending media using the US Pharmacopeia Chapter <85> Bacterial Endotoxins Test (Microbiology Research Associates, Inc., Acton, MA), with a Limulus amoebocyte lysate sensitivity of 0.03 EU/mL. MC had endotoxin levels below the detection limit (<0.96 EU/mL).
To determine whether there was any contribution to potential inflammation from bupivacaine per se, and to test the pathological effects of the differently formulated microspheres, other sets of rats were injected with the microspheres, suspended with either CMC or MC, but lacking the local anesthetic (microsphere-placebo).
In other “control” conditions, either pharmaceutical-grade bupivacaine solution or the vehicle for this bupivacaine, PBS-“vehicle,” was injected. Intact skin that had received no injections was taken from naive rats for comparison with all the other samples.
For each formulation of bupivacaine, 4 rats were injected bilaterally in 4 separate areas while under brief sevoflurane general anesthesia using the same technique of published behavioral studies10,12 and the serum assay portion investigated here. All procedures were approved by the Harvard Committee on Animals. At postinjection days 1, 3, 7, 14, and 30, skin samples were taken by punch biopsy (4 mm in diameter; Miltex, Inc., York, PA) and immediately placed in 10% neutral buffered formalin solution (StatLab, McKinney, TX). The punch skin samples were set in paraffin blocks, sectioned (5-μm thick), stained with hemotoxilin–eosin, and then viewed under an optical stereomicroscope at ×4 to ×20 magnification. Punch biopsies thus led to sections taken from 4 independent skin areas taken from 2 to 4 rats, providing 8 to 16 blocks of tissue, each receiving identical injections. From these tissue blocks, 4 sections with good tissue preservation from each treatment, in at least 2 different rats, were examined and scored for pathological signs (see Results). The histopathology was scored by a board-certified dermatopathologist who was blinded to the nature of injection or treatment.
Data are reported as semiquantitative observational findings, with infiltrations of macrophages (histiocytes), neutrophils, and lymphocytes, separately graded as: 0, no pathology; 1, mild; 2, moderate; or 3, severe. When any section contained a pathological sign, for example, lymphocyte infiltration, each such observation, with its intensity, was noted in Table 3.
Statistical Analysis of Serum Bupivacaine
A total of 56 rats were treated with bupivacaine microspheres (with and without SIE), and 42 rats were treated with bupivacaine solutions (with and without SIE). (The measurement obtained at 144 hours from the rat treated with bupivacaine microspheres with SIE was erroneous and was not included in the statistical analysis.) Descriptive statistics are presented for each treatment at each time point in Table 1. The data from the intact skin and postsurgery rats were pooled, and Wilcoxon rank sum tests were applied for comparing serum levels from BupHCl injections with those from MS-Bup injections. One-sided 95% upper prediction limit for the mean of k future observations was calculated using the CAPABILITY procedure with INTERVALS statement. A value of k was specified as 8 for MS-Bup. A value of k was specified as 6 for Bup.13 Wilcoxon rank sum test was also used to determine whether the maximal serum levels after bupivacaine injections differed from the maximal levels after MS-Bup. Analysis of variance with post hoc pairwise comparisons was used to determine which serum levels differed among all those taken at the different times after MS-Bup injections with or without surgery. All 28 possible pairwise comparisons were performed using Student t tests with pooled variance. The P values reported in Table 2 are Bonferroni adjusted. Statistics were conducted using SAS version 9.3 (SAS Institute, Cary, NC). No statistical analysis of the semiquantitative histological findings was conducted.
After subcutaneous injection of the bupivacaine solution (0.4 mL, 0.5%) into intact skin, the serum levels increased rapidly, reached peak value at 30 minutes, and had returned to barely detectable levels by 8 hours (Fig. 1A, Table 1). At 24 hours, serum bupivacaine was below the limit of detection, at 3.23 ng/mL. Maximal serum levels, occurring at 30 minutes, averaged at 374.9 ng/mL, with a 95% upper prediction limit calculated as 470.6 ng/mL.
When MS-Bup was injected, bupivacaine serum levels were different from those after bupivacaine solution injection (Fig. 1B). Serum concentrations were slower to increase, reaching the highest mean value at 8 hours, equal to 194.9 ng/mL, with a 95% upper prediction limit of 230.2 ng/mL. This value was significantly lower than the highest mean value of 374.9 ng/mL that followed 30 minutes after injection of bupivacaine solution (P = 0.0037; Wilcoxon rank sum test).
Serum samples were taken at 4 identical times from rats injected with bupivacaine or MS-Bup (Table 1). These values differed between the 2 injection formulations for the 3 times of 1, 8, and 24 hours (P < 0.001 for all; Wilcoxon rank sum test), but they were not significantly different at 3 hours (P = 0.12).
An overall test of analysis of variance indicated that there was a significant difference among all samples taken at different time points after MS-Bup injections with or without surgery (F = 6.29, df = 7, P < 0.0001). All 28 possible pairwise comparisons had made, of which 16 were P < 0.01 (Table 2). It is noteworthy that the pairs for which no significant difference was found include those at 8 to 24, 8 to 48, and 24 to 48 hours, corresponding to average serum levels of 194, 212, and 174 ng/mL, respectively, at 8, 24, and 48 hours.
The condition of the skin at different times after injection of the different treatments was determined by a blinded board-certified dermatopathologist. This qualitative analysis reports the presence of macrophages (histiocytes), neutrophils, and lymphocytes in tissues of and around the skin, with the extent of infiltration scored as absent (0), mild (1), moderate (2), or severe (3). All dermatopathology data are presented in Table 2.
Specific examples of pathological and normal conditions are presented in the photomicrographs of Figure 2. The naive skin shows no inflammatory cell migration and is uniform in dermis structure (Fig. 2A). Three days after injection of the bupivacaine solution, a moderate invasion of lymphocytes was detected in the subcutaneous skeletal muscle (Fig. 2B), a condition that was also present earlier, at 1 day, but not at 1 or 2 weeks (Table 3). No other signs of pathology resulted from bupivacaine solution that was unremarkably different from skin after vehicle (PBS) injection (not shown).
Injections of bupivacaine microspheres suspended with methyl cellulose (MS-Bup.MC) resulted in mild inflammation (macrophages, histiocytes in dermatopathology terminology, and lymphocytes) after 1, 3 (Fig. 2C) and 14 days (image not shown; cf. Table 3). The appearance of this tissue showed more inflammation than that of the “normal” skin 7 days after injection of MS-placebo, suspended with MC (Fig. 2E, Table 3), but was the same as that after bupivacaine solution (see above).
In contrast, when microspheres suspended with CMC were injected, a profound inflammation was observed (Fig. 2D) with a moderate to strong presence of macrophages and neutrophils, at days 1 to 7, and mild to moderate inflammation at later times (Table 3). A slightly milder pattern occurred when bupivacaine was absent (MS-placebo with CMC; Fig. 2F, Table 3), implying a small contribution of the anesthetic to the overall pathology.
The purpose of this study was to investigate the safety of the bupivacaine microsphere formulation, as defined by (1) the systemic levels of bupivacaine in the serum and (2) the local tissue histological response.
The maximal average serum level of bupivacaine reached 8 hours after subcutaneous injection of the microsphere formulation (195 ng/mL) is about half of the maximum from injections of bupivacaine solution (375 ng/mL), reached at 30 minutes, and is well within a safe range. The threshold plasma concentrations of bupivacaine associated with incidental toxicity in humans is in the range of 2.0 to 4.0 μg/mL,14,15 an observation borne out in subsequent, controlled (volunteer) human studies.16,17 A similar threshold toxic plasma concentration range (i.e., 2.0 μg/mL and higher) was found for preclinical test subjects.18 These systemic bupivacaine concentrations are also consistent with the lower end of the “safe range” identified during a human study with constant infusion and bolus local bupivacaine administration.19 In comparison, the rat serum concentration resulting from subcutaneous injections of the current bupivacaine microsphere formulation has a 95% upper prediction limit of 230 ng/mL, about 1 order of magnitude lower than the toxic concentration of 2 μg/mL (Fig. 1, Table 1). Importantly, because the extraction procedure removes all the bupivacaine in the serum (with alkalinization leading to a reduction in protein affinity,20 and bupivacaine dissociation from high-affinity α-1-acid glycoprotein occurring within a few minutes; Strichartz GR and Cogswel LP III, unpublished observation), the levels reported here are total bupivacaine. Such levels will be higher than unbound or “free” bupivacaine (i.e., not bound to proteins in plasma/serum), which is believed to be more closely related to toxic effects than the total (i.e., bound + unbound) concentrations.14,15,21
Although the measurement of unbound bupivacaine was beyond the scope of our current investigation, the literature estimates the unbound fraction of systemic bupivacaine at between 4.5% (for the R-enantiomer) and 6.6% (for the S-enantiomer) of the total bupivacaine, obviously depending on the plasma protein levels.15 Applying this estimated range to the data reported here, we calculate the 95% upper prediction limit for maximal systemic unbound bupivacaine concentration from subcutaneous administered bupivacaine microspheres to be circa 10.4 to 15.2 ng/mL, an order of magnitude below the toxic threshold of 240 ng/mL for unbound bupivacaine to induce central nervous system toxicity.21 Therefore, by both considerations (total and unbound bupivacaine), the obtained systemic levels are about one-tenth of the established thresholds for toxicity.
The highest measured rat serum concentration level corresponding to bupivacaine microspheres (276 ng/mL) is well below the corresponding maximal measured level from subcutaneous injection of bupivacaine-HCl solution (485 ng/mL), as are the average levels at the peaks (195 and 375 ng/mL, respectively). This difference in blood levels holds despite the very large opposing differences in drug dosage per animal: 2 mg of bupivacaine (HCl) in the injected solution (5 mg/mL × 0.4 mL) compared with 40 mg of bupivacaine (base) administered in the microspheres. In addition, the rat serum concentration levels corresponding to bupivacaine microsphere injections persist at elevated levels for 7 days compared with that from bupivacaine-HCl injections, for which levels are undetectable at 8 hours (Fig. 1). The serum concentration from the bupivacaine-HCl solution reaches peak levels within 30 to 60 minutes and decays rapidly thereafter, consistent with previous reports.22–24 In contrast, the serum concentration from injected bupivacaine microspheres is relatively constant at 8, 24, and 48 hours at 194, 212, and 174 ng/mL, respectively. The near steady-state concentration is reflective of the nearly zero-order sustained release profile of bupivacaine from these microspheres in vitro during the first 48 hours.11 There, approximately 90% of the bupivacaine had been released from the microspheres by 120 hours, comparable to the time when the serum concentration decreased to low levels in the present study (Fig. 1). Indeed, the declining phase of serum bupivacaine after injections of the 2 different forms indicates that the time when 100 ng/mL is reached is approximately 4 hours from the solution and 80 hours from the microspheres, times that are in almost exactly the same ratio as 20-fold difference in their relative dosing. This suggests that the rate-limiting step for the appearance of bupivacaine in blood is its transfer from the surrounding tissues rather than its direct release from the liquid depot or the microspheres.
Comparison of these time-dependent levels with the duration of pain mitigation achieved in preclinical experimentation may be instructive of mechanism.10,11,25 Percutaneous injection of MS-Bup (40 mg) at the sciatic nerve resulted in functional motor and nociceptive block lasting approximately 48 hours and an antihyperalgesic effect on mechano-hypersensitivity after incision of the innervated hindpaw lasting for 96 hours.11 When MS-Bup (40 mg) was injected subcutaneously, as in the present pharmacokinetic study, the subsequent anesthesia of the unoperated skin at that site lasted for 36 hours, but the antihyperalgesic effect after the SIE procedure endured for 3 to 5 days.10 In both scenarios, the therapeutic action to suppress postoperative hypersensitivity lasted several days longer than the anesthesia of intact nerve or skin, suggesting that the inhibition of processes that occur sooner after surgery is effective in suppressing later hyperalgesic states. Alternatively, for the subcutaneous injections used for the SIE procedure, the antihyperalgesic effect was matched by the presence of systemic bupivacaine, supporting the possibility that the IV drug provided the therapeutic activity.26
These data trends are consistent with the experience from other bupivacaine formulations, in which longer persistence in plasma concentration at elevated but subtoxic levels corresponded to longer durations of pain mitigation. An example is liposomal bupivacaine formulations, for which several published preclinical and clinical studies are available.21,27–29 In a comparison of human clinical data from 4 independent studies incorporating data from 4 separate patient populations (i.e., those undergoing hernia repair, hemorrhoidectomy, bunionectomy, and total knee arthroplasty), all 4 data sets showed that liposomal bupivacaine resulted in elevated (but subtoxic) levels of plasma bupivacaine, which persisted longer than the control groups receiving bupivacaine-HCl. In turn, this correlated with longer durations of postoperative pain mitigation for patients who received the liposomal bupivacaine. This correlation is consistent with other clinical studies,27,28 as well as preclinical studies.29 However, at this point, it is not possible to assign a direct antihyperalgesic action to the low systemic levels of bupivacaine, or other local anesthetics, present at >3 days after surgery versus an essential action that occurs from the actions of the drugs directly on local nerves and injured tissue during the perioperative and 1- to 2-day postoperative domain.26
These experiments were designed to compare, qualitatively, the local tissue responses to needle injection per se, to injection of vehicle (saline) and carriers (placebo drug-free microspheres, suspended with CMC or with methyl MC, i.e., respectively with and without endotoxins) and to injection of bupivacaine in solution or encapsulated in microspheres suspended in the different carriers. The summary results are listed as follows:
- Comparison of the tissue injected with MS-placebo (drug-free microspheres) formulated with MC with that formulated with CMC shows that the latter induces mild to moderate inflammation at 1, 3, and 7 days afterward. Macrophages and neutrophils are particularly evident, and these are found in subcutaneous fibrous tissue and skeletal muscle. In contrast, no signs of inflammation or tissue damage occurred after injection of plain microspheres formulated with MC. This difference is most likely attributable to the presence of endotoxins in the suspending media (see below).
- Comparison of tissue injected with MC-formulated MS-Bup and that with MC-formulated MS-placebo reveals the effects of the local anesthetic in microspheres. This difference consists of a primarily mild lymphocytic infiltration, at days 1 and 3, and 1 section with mild to moderate lymphocytic and histiocytic infiltration on day 14 in the bupivacaine-containing material, consistent with the similar difference between injected bupivacaine solution and vehicle, or naive skin (see below).
- Consistent with the effects from the presence of bupivacaine in microspheres, the injection of bupivacaine solution, at a common clinical dose and concentration, also caused mild to moderate lymphocyte infiltration at days 1 and 3, particularly in the subdermal skeletal muscle (the pannicular layer). This appearance contrasts with that of naive skin (or of vehicle-injected skin, not shown), for which no pathology was apparent. It is noteworthy that local anesthetics have long been known to produce local, reversible tissue damage when injected in vivo, particularly into skeletal muscle.29–31
- In none of the cases did any of the subcutaneously injected tissues show any pathology in the dermis or epidermis. There were no evident signs of induration, and the histology images did not suggest granulation tissue.
The inflammatory response elicited by the bupivacaine solution, both in terms of intensity and its resolution over time, is consistent with the literature.29–32 In addition, this response is mirrored (in both intensity and resolution over time) by the inflammatory response to bupivacaine-loaded microspheres suspended in MC, which suggests little or no inflammatory impact by other components of the formulation, namely, the encapsulating polymer and the suspending agent (MC). This inference is further strengthened by the absence of any significant inflammatory response elicited by placebo microspheres suspended with MC and the stronger inflammatory response elicited by microspheres (both placebo and with drug) suspended with endotoxin-containing CMC.
These findings suggest that the significant inflammatory response is likely to correlate to endotoxin content rather than any other inherent component of the microsphere formulation itself, namely, the bupivacaine or the PLGA polymer.33 In addition, the results, especially those obtained with the MC-based suspensions, suggest that it is possible to create bupivacaine microsphere-based formulations that elicit only a mild inflammatory response, no greater than that elicited by bupivacaine itself.
Previously developed microsphere formulations of bupivacaine with the same PLGA polymers have demonstrated effectiveness of pain mitigation in human studies.4–7 The local inflammatory side effects were reported in some of the human subjects,6,7 although we were unable to find any detailed histology micrographs or histopathology examination of these outcomes in the literature. Both induration and pruritus were observed in these studies, the former attributed to the PLGA polymer family on the basis of the characterization of the tissue response continuum to PLGA polymers.33 In one human study, >50% of the human subjects developed pruritus at the local site of administration.6 In this regard, it is interesting to note that unlike the present formulation, these previously developed bupivacaine microsphere formulations also incorporated dexamethasone,2 which has an established relationship with pruritus. Dexamethasone activates the histamine H4 receptor, which impacts allergic responses such as pruritus,34–36 and this, in turn, provides a mechanistic basis to better understand the clinical observations of perineal and vulvar pruritus occurring after administered dexamethasone.37–39 Although the current studies were not designed to investigate pruritus, the histology findings reported here suggest that a bupivacaine microsphere formulation (when coupled with the MC-suspending media) has the potential to minimize any inflammatory response and thus can avoid inclusion of an anti-inflammatory steroid, such as dexamethasone.
In summary, subcutaneous injections of bupivacaine-releasing microspheres, previously shown to be effective against experimental postoperative hyperalgesia, result in subtoxic serum levels of bupivacaine that remain detectable for 1 week. Incision and blunt dissection of the overlying skin after the injection does not alter the serum profile. No dermatopathological signs are evident in intact skin samples 1 to 14 days after the microsphere injection, unless the microspheres are suspended in endotoxin-containing CMC. In conclusion, the present slow-releasing formulation may be a safe and effective means for reducing acute postoperative pain.
Name: Birgitta Schmidt, MD.
Contribution: This author analyzed the data (dermatopathology) and helped write the manuscript.
Attestation: Birgitta Schmidt approved the final manuscript.
Conflicts of Interest: This author has no conflicts of interest to declare.
Name: Rachit Ohri, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Rachit Ohri approved the final manuscript.
Conflicts of Interest: Rachit Ohri has joined with a partnership to advance the commercialization of the bupivacaine microspheres described in this article.
Name: Jeffrey Chi-Fei Wang, MD.
Contribution: This author conducted the experiments to collect serum for the pharmacokinetic study and helped analyze the results.
Attestation: Jeffrey Chi-Fei Wang approved the final manuscript.
Conflicts of Interest: This author has no conflicts of interest to declare.
Name: Phillip Blaskovich, BS.
Contribution: This author helped write the manuscript.
Attestation: Phillip Blaskovich approved the final manuscript.
Conflicts of Interest: This author is employed by Covidien, Inc., which holds the rights to the material tested here. This author has no direct financial stake at this time in the development of the material described here.
Name: Allen Kesselring, PhD.
Contribution: This author analyzed the serum concentrations and helped write the manuscript.
Attestation: Allen Kesselring approved the final manuscript.
Conflicts of Interest: This author has no conflicts of interest to declare.
Name: Nelson Scarborough, PhD.
Contribution: This author helped design the studies and write the manuscript.
Attestation: Nelson Scarborough approved the final manuscript.
Conflicts of Interest: Although this author was employed by Covidien, Inc., at the time this research was conducted, at the time of manuscript submission, no authors were so employed. This author avers that he has no financial stake at this time in the development of the material described here.
Name: Clifford Herman, PhD.
Contribution: This author helped write the manuscript.
Attestation: Clifford Herman approved the final manuscript.
Conflicts of Interest: This author has no conflicts of interest to declare.
Name: Gary Strichartz, PhD.
Contribution: This author helped design the experiments, interpret the data, and write the manuscript.
Attestation: Gary Strichartz approved the final manuscript.
Conflicts of Interest: Gary Strichartz has joined with a partnership to advance the commercialization of the bupivacaine microspheres described in this article.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
The authors wish to thank Ms. Fannie Polcari for help with the preparation of this manuscript, and Xiaoxia Liu, Department of Anesthesiology, and Rie Maurer, Center for Clinical Investigation, Brigham and Women’s Hospital, for statistical analyses. Graphical assistance was provided by Mr. James Bell, also of the Department of Anesthesiology. The authors also wish to acknowledge Ms. Katie Grayson, CEO of EKG Life Science Solutions LLC, and the following coworkers at Covidien who advised and assisted with this work: Dr. Jeffrey Zaruby, Jason Fortier, Lan Pham, Daniel Costa, and Kreg Howk.
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