An investigation of in vivo performance of bupivacaine multivesicular liposomes in rats and the impacts from product qualities

An investigation of in vivo performance of bupivacaine multivesicular liposomes in rats and the impacts from product qualities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An investigation of in vivo performance of bupivacaine multivesicular liposomes in rats and the impacts from product qualities Ziyun Xia, Yayuan Liu, Ziyi Lu, Jingyao Gan, Minzhi Yu, Karl Olsen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5214719/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract A bupivacaine multivesicular liposomal injectable formulation, Exparel TM , is a nonopioid long-acting local analgesic indicated for pain management across and/or post surgeries. For such products, preclinical data is lacking to support bioequivalence determination for potential generic products. Therefore, in the present work, in vivo studies were set up in male Sprague-Dawley rats to understand the in vivo performance of bupivacaine multivesicular liposomes (MVLs), aiming to provide information on bioequivalence establishment between comparator products. Bupivacaine MVLs show a multiphasic release profile, and their pharmacokinetics (PK) may differ with different experimental conditions including doses, administration routes, and sample dilution factors. In this work, compromised bupivacaine MVLs were either generated in lab by freeze-thawing, mechanical agitation, and high-temperature incubation, or chosen from years-old expired batches of Exparel TM , for a preliminary investigation on the in vitro and in vivo association. The formulation attributes of different bupivacaine MVLs were characterized, including morphology, particle size distribution, formulation pH, free drug contents, in vitro release, and in vivo PK. In the rat study, even with an observation of inter- and intra-variability in PK, an association between product attributes and in vivo behaviors was demonstrated with bupivacaine MVLs. Overall, investigating the bupivacaine MVLs in vivo is beneficial not only to fill in gaps in preclinical data in the field of bupivacaine MVLs, but also to help pave the path for developing other MVL-related products. Multivesicular liposomes pharmacokinetics in vivo preclinical study liposomes bupivacaine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Exparel is a multivesicular liposomal formulation of bupivacaine utilizing the DepoFoam TM technology for sustained drug release from days to weeks [1,2]. An assembly of numerous nonconcentric liposomes within a large particle contributes to a unique honeycomb-like structure of multivesicular liposomes (MVLs), allowing for a better drug encapsulation efficiency as well as a longer drug release duration [3,4]. Exparel was approved by the US Food and Drug Administration (FDA) for the post-surgical local analgesia in 2011, and its label was extended for nerve-block pain relief after shoulder surgeries in 2018 [5,6]. As a long-acting nonopioid drug product, Exparel gained great market success in the past few years [7]. At the same time, its patents either have already or are going to expire soon, making Exparel an appealing target for many generic developers [8,9]. To facilitate the development and regulatory filing of liposomal drug product, FDA published a series of guidance documents, including Liposome Drug Products - Chemistry, Manufacturing and Controls, Human Pharmacokinetics and Bioavailability, as well as the Labeling Documentation [10]. However, the published guidance focuses more on the traditional liposomes such as unilamellar and multilamellar liposomes, instead of MVLs. A product-specific draft guidance (PSG) for bupivacaine liposomal injection was issued by FDA in 2018 [11], which recommends the evaluation of various parameters including drug product composition and the liposome characteristics between the ANDA and the reference listed drug (RLD) products. Clinical pharmacokinetic (PK) studies are also recommended for the demonstration of bioequivalence. Even though there have been some efforts in developing characterization methods and an accelerated in vitro release (IVR) assay for bupivacaine MVL formulation [12–14], the information regarding preclinical in vivo testing of MVL-based drug products is still lacking. There is also no waiver applicable for an in vivo testing so far [11]. The need for clinical trials presents challenges to generic developers as they are usually time and economic burdens. Meanwhile, even though a draft PSG was published for bupivacaine liposomal injection (i.e., Exparel), a lack of general guidelines on MVL drug products still challenges the industry to understand and meet the regulatory expectation in the development of MVL-based drug products [15]. Considering the high stake of clinical trials, a preclinical study is favorable in drug development over many aspects, such as its low cost and feasibility on conduction, as well as informative insights on in vivo performance and in vitro in vivo association. However, because of the complex formulation as well as unique release features of long acting injectables (LAIs) such as Exparel, preclinical study design can be challenging, and the lack of references due to a limitation on published preclinical studies has added more hurdles. Therefore, an investigation on the preclinical PK of bupivacaine MVLs is desired for understanding critical formulation attributes to facilitate formulation development and establishment of bioequivalence during generic development. Exparel and other MVL drug products exhibit characteristic drug release behaviors due to their unique non-lamellar honeycomb structures [2,3,16,17]. In general, three mechanisms are considered accountable for its release: drug molecule diffusion across the lipid membrane, internal lipids rearrangement causing vesicles coalescence, and the lipid bilayer decomposition [3]. In 2018, Manna et al., proposed a tri-phase release process for bupivacaine MVLs based on a reverse-dialysis IVRT method [14]. However, since these release behaviors were only examined with non-physiological relevant IVRT assays, whether the proposed release mechanisms can be applied in vivo remains to be investigated. Additionally, no PK studies were examined and compared. Since the physiological environment is far more complicated and variable than the in vitro settings, a lack of in vivo comparisons poses extra challenges on understanding the association between the in vitro and in vivo release of MVL formulation. A preclinical PK study can pave the first step on studying any in vitro in vivo relationship for this specific product, which can not only ease and fasten the process of generic development, but also reciprocally help understand the release process of MVL-related products. Therefore, in our present work, we set up several in vivo studies and developed an analytical method for the bupivacaine MVLs PK testing in Sprague-Dawley (SD) rats. Different experimental conditions such as drug doses, routes of administration, and sample dilution factors were investigated to understand their impacts on bupivacaine MVLs’ in vivo PK in SD rats. Commercial bupivacaine MVL samples with different manipulations were purposely varied and then characterized in terms of their in vitro and in vivo characteristics including morphology, sizes, formulation pH, free drug contents, in vitro release, and in vivo PK, to further understand how varied product quality attributes impact on the in vivo performances accordingly. Materials and Methods Materials Exparel (bupivacaine liposome injectable suspension) batch number of 17-4046, 20-P097 and 21-P080 were purchased from the Research Pharmacy at the University of Michigan Health System. United States Pharmacopeia (USP) grade bupivacaine hydrochloride hydrate was purchased from Fisher Scientific. 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) was purchased from NOF. Cholesterol and tricaprylin were purchased from Sigma. 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG-Na) was purchased from Avanti. Bupivacaine free base and ropivacaine hydrochloride hydrate were purchased from Cayman Chemical. Bupivacaine MVL formulations with compromised product qualities Commercial bupivacaine MVL formulations (Exparel, batch number of 20-P097 with expiration dated in July 2022) were stressed via 1) mechanical agitation, 2) freeze-thawing cycle, or 3) high-temperature incubation, to generate compromised formulations with different product quality attributes. In the scenario of mechanical agitation, Exparel was vortexed at a speed of 2500 rpm (Fisher Analog Vortex Mixer, 120V, 9454FIALUS) for 20 ss. In the freeze-thawing scenario, Exparel was first incubated at -20°C overnight and then thawed at 40°C for 5 min. In the scenario of high-temperature incubation, Exparel was incubated at 45°C for 13 h. Particle size distribution Particle size distribution of bupivacaine MVLs was analyzed by Malvern Mastersizer 2000 (Malvern Panalytical Ltd, Malvern, UK), equipped with a Hydro 2000S (AWA2001) dispersion unit. Undiluted bupivacaine MVLs were dispersed in filtered 0.9% saline in the instrument until they reached at least 10% obscuration level. Three measurements were conducted for each sample at a stirring speed of 3000 rpm, with a material refractive index of 1.5. Morphology The morphology of bupivacaine MVLs was observed via an optical microscope (Carl Zeiss Axiolab Re microscope). Briefly, bupivacaine MVL formulations diluted 10 times were added onto a glass slide and observed under the microscope immediately after the coverslip was placed on. Formulation pH measurement The pH of bupivacaine MVL formulations was measured using a 430 pH meter (Corning, USA) with MI-410 microelectrodes (Microelectrodes Inc., USA) at room temperature. Bupivacaine quantification in in vitro release study Ultra-performance liquid chromatography (UPLC) was used to quantify the released drug content from bupivacaine MVL samples. Samples collected from the in vitro release study were centrifuged at 600 rcf (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor) for 15 min to remove liposome particles. Then 200 µL of the supernatant was taken and diluted with 250 µL methanol and rigorously vortexed. Then 500 µL acetonitrile was added, followed by a rigorous vortex. The final mixture was syringe-filtered (0.45µm, PVDF) before being injected into a Waters ACQUITY UPLC H-Class System equipped with a Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm). The mobile phase was composed of 70% (A) water with 0.1% formic acid (FA); 15% (B) acetonitrile with 0.1% FA; 15% (C) methanol with 0.1% FA. The flow rate was 0.4 mL/min. The UV detection wavelength was 220 nm. The following equation was used to calculate the cumulative release of bupivacaine: Pharmacokinetics (PK) evaluation in rats Male Sprague-Dawley (SD) rats were used for the PK evaluation. Free bupivacaine solution and bupivacaine MVL samples were subcutaneously injected to the backs of healthy rats (n = 4 or 5) at a dose of 5, 15, or 25 mg/kg of bupivacaine. Blood was drawn from the jugular vein at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after dosing. Collected blood samples were immediately centrifuged at 15,000 rpm (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor) at 4°C for 5 min to separate the plasma. Plasma samples were stored at -80°C for future analysis. Bupivacaine quantification in plasma Bupivacaine was extracted from plasma samples via protein precipitation extraction. Briefly, 100 μL of plasma samples was spiked with 20 μL of internal standard (1 μg/mL ropivacaine) and then mixed with 50 μL of 1 M sodium hydroxide solution. Hexane-isopropanol-chloroform (v:v:v = 3:6:1) mixed solvent was used to extract the samples via a vortex-mix manner, following with a 10-minute centrifugation at 10,000 rpm (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor). The supernatants were collected and dried under nitrogen flow at 60℃. The dry residues were reconstituted in 200 μL of hydrochloride acid (0.5N) and syringe-filtered (0.45 μm, PVDF) before LC-MS analysis. Plasma bupivacaine concentration was determined by UPLC-MS (Waters ACQUITY system equipped with a QdaTM detector) using a Waters ACQUITY UPLC BEH300 C18 column (1.7 μm, 2.1 x 100 mm). The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in methanol (v:v = 60:40). The flow rate was 0.3 mL/min with a running time of 4 min. The injection volume was 10 μL. Samples were detected at m/z of 289.32 (+) for bupivacaine and 275.35 (+) for ropivacaine. Results and Discussion Investigation of PK of free bupivacaine solution and bupivacaine MVLs in rats An analytical method to quantify plasma bupivacaine concentration was developed with a linearity between 5 to 200 ng/mL (Supplementary information Fig. S-1 ). The analytical procedure was then applied to investigate the PK of and comparison between the free bupivacaine solution and bupivacaine MVL formulation at their clinical dosage strength (i.e., 4.43 mg/mL for free solution, and 13.3 mg/mL for Exparel, separately). Some unique characteristics with bupivacaine MVLs were observed in their PK profiles in our study. Compared to the free bupivacaine solution, bupivacaine MVLs featured a sustained drug plasma concentration until 48 h after administration, with a much lower C max around 100 ng/mL and a much longer mean residence time (MRT) up to 20 h, compared with the C max around 650 ng/mL and the MRT of 4 h in the situation of free drug solution ( Table 1 ). Specifically, a multiphasic profile with a second peak of plasma concentration after 12 h post administration was observed in bupivacaine MVLs’ PK, indicating the existence of multiple phases in the bupivacaine MVLs’s drug release process. A smaller area under the curve (AUC) was also featured in bupivacaine MVLs PK, suggesting a smaller systemic drug exposure compared to the free drug situation, which implies a hindered drug release and/or drug absorption after subcutaneous administration to the rats. The existence of MVLs particles themselves, and the lipids dissociated as MVLs decomposed along the release process, could also hinder drug absorption, resulting in a smaller AUC [18] . This multiphasic PK profile was also observed in many long acting injectable (LAI) formulations when administrated intramuscularly or subcutaneously [19–22] . For the LAIs, the first pharmacokinetic phase, featured with an early plasma spike, was attributed to fast initial absorption when the dissolution rate dominates in this period. At a later stage, LAIs progress to a slower release phase, resulting in the absorption rate slower than the elimination rate, featured with a flip-flop PK in which a second plasma peak may or may not show up in their profiles [23] . Notably with our observation in bupivacaine MVLs’s PK, the presence of a plasma peak in the second phase might further indicate an intermediate lag phase during the drug release and absorption process ( Fig. 1 ). Table 1 Pharmacokinetic parameters of free bupivacaine solution and Exparel (SC) in SD rats (n=4, mean ± SD) (Software: WinNonlin.) Formulation (n=4) Dose (mg/kg) Route C max (ng/mL) T max (h) a AUC 0→96h (ng/mL*h) b MRT (h) free bupivacaine solution 15 c SC 649.32 ± 235.13 1 3945.62 ± 419.89 4.13 ± 0.83** Exparel 15 c SC 104.27 ± 42.56 1-2 1724.41 ± 142.30 19.54 ± 2.94 a AUC: area under the curve b MRT: mean residence time c SC: subcutaneous Impacts of experimental conditions on bupivacaine MVLs PK in rats Experimental set-ups can impact the PK of long-acting drug products [24,25]. Therefore, different experimental conditions including doses, routes of administration, and dilution factors, were studied in our work to investigate their impacts on the PK of bupivacaine MVLs in rats. Bupivacaine doses ranging from 5 to 25 mg/kg were screened to investigate their impacts on bupivacaine MVLs PK in rats. The AUC and MRT were proportional to the administrated bupivacaine doses, suggesting a greater drug exposure with the higher doses ( Fig. 2 , Table 2 ). The C max was found to be consistent around 200 ng/mL for doses ranging from 5 to 15 mg/kg but rose as the dose went up to 25 mg/kg. Subcutaneous (SC) and intramuscular (IM) injection were investigated in our study as two of the most applied administration routes in animal study for LAIs., Administration routes were found to show impacts on the PK performances of bupivacaine MVLs in rats, indicating impacts from the physiological environment on drug dissolution and absorption in the case of bupivacaine MVLs. IM injection exhibited a greater AUC with a longer MRT but a lower C max , suggesting a more sustained systemic exposure of bupivacaine along the time, compared to the SC administration ( Table 2 ). Sample dilution factors were also investigated here as they usually relate to sink condition and thus can impact the drug dissolution. Notably, the commercial bupivacaine MVL product, Exparel, is also administrated with dilution in clinical practice [1]. Our results showed a higher C max and a shorter MRT with the more diluted MVL formulation, indicating a faster drug release and/or absorption. The greater AUC observed in the diluted condition further suggested a greater drug exposure with the dilution, which was similar to the situation observed with a higher dose. The biphasic PK of bupivacaine MVLs was also observed among different experimental conditions, but the second plasma peak was either shifted or less prominent as the dose, administration route, or dilution factor changed ( Fig. 2 ). The exact reasons for the second peak or the factors impacting the plasma concentration of the slow-release phase were not clear [20,23] . In the case of LAIs, their multiphasic profiles were considered to result from a complex interplay of formulation parameters and physiological elements: the initial fast-release phase is more likely associated with formulation factors impacting the drug dissolution which dominates in this period, and the physiological elements, especially at the injection or depot site, are usually assumed to be the major impactors of PK in the later slow-release phase when elimination plays a dominate role [23,26,27] . The similar biphasic PK observed in the case of bupivacaine MVLs highlighted the impacts from both the product attributes and physiology on their resulting in vivo performances, and the variation with the second peak might emphasize the impacts from an intermediate release phase specific with bupivacaine MVLs. Due to their nonconcentric structure, a lipid reorganization causing the coalescence of inner vesicles is unique with MVLs [3] . As there is no meaningful drug release outside the liposomes when the inner vesicles move and fuse with each other, the lipid rearrangement within MVLs might lead to an intermediate phase with sustained drug release, contributing to the second peak showing in their PK [14]. Furthermore, the other PK parameters, such as the time between the first and second peak, concentration in the secondary slow-release phase, and the slope of the terminal phase, would also be worth of future investigation in terms of their value of indicating underlying release processes of MVLs. Table 2 Pharmacokinetic parameters of bupivacaine MVLs evaluated with different experimental conditions in SD rats (n=4, mean ± SD) (Software: WinNonlin.) Formulation (n=4) Dose (mg/kg) Route dilution C max (ng/mL) T max (h) a AUC 0→96h (ng/mL*h) b MRT (h) Exparel 25 c SC / 351.99 ± 180.90 1-12 5569.42 ± 1747.34 23.15 ± 2.13 15 230.06 ± 172.06 0.5-2 2655.94 ± 571.00 16.56 ± 3.33 5 233.68 ± 37.64 0.5-1 930.56 ± 328.01 10.26 ± 6.31 15 SC / 230.06 ± 172.06 0.5-2 2655.94 ± 571.00 16.56 ± 3.33 d IM 132.37 ± 87.20 1-24 2835.94 ± 218.78 27.07± 2.41 15 SC / 104.27 ± 42.56 1-2 1724.41 ± 142.30 19.54 ± 2.94 3X 349.12 ± 111.76 1-2 2418.89 ± 608.49 12.16 ± 3.19 a AUC: area under the curve b MRT: mean residence time c SC: subcutaneous d IM: intramuscular Investigation of PK of commercial bupivacaine MVLs Besides the observation that various experimental conditions led to different PK of bupivacaine MVLs, variability in PKs was also found within the same experimental conditions. Four PK experiments were conducted with commercially available bupivacaine MVL products in SD rats from 2018 to 2021, with the same dose of 15 mg/kg administrated subcutaneously. Interestingly, even though the plasma profiles shared similar characteristics such as multiphases with multi-peaks, an inter-experimental variability was found nonnegligible in some of the PK parameters among the conducted PK experiments ( Table 3 ). To be specific, the value of AUC ranged from 2000 to 6000 ng/mL*h, and C max ranged from 100 to 350 ng/mL, showing a 3-fold difference. T max was also varied, which might have been due to the presence of multiple plasma peaks in their profiles. Furthermore, as discussed above, the different conditions such as doses, dilution, and administration routes exerted different impacts on the PK of bupivacaine MVLs in our study ( Fig. 2 , Table 2 ). However, such impacts were not indicated in the previous preclinical data with bupivacaine MVLs, and similar PK profiles were shown among different experimental settings [28] . This observed variability is important because the discrepancy between our study and the previous data supports concerns around inter-experimental variability. For example, further intra-experimental variability was also observed in other results. Specifically, C max exhibited a coefficient of variance (CV) over 40% and even up to 70%, and the difference between some of the PK parameters (e.g., Cmax and AUC) was up to 2- to 3-fold ( Table 2 , Table 3 ). This intra-experimental variability was also shown in the previous preclinical data with bupivacaine MVLs in rats [28] , with the CV of C max also up to 40% to 70% [28] . Furthermore, though not usually discussed, intra-experimental variability was also found relatively high (i.e., CV close to or over 20%) in the preclinical PK of various MVL formulations [29–32] . The acceptance for an inter- and intra-assay CV usually falls in the range of 10 to 15%, and the general recommendation of the CV for a preclinical study is usually down to 10% [33,34] . As the variability are often found with the MVL’s PK, acceptable range for %CV may need to rely on totality of evidence with justification from other supporting data. A variety of factors involved in in vivo drug exposure have been investigated with LAIs around aspects of 1) formulation properties, 2) administration site properties, and 3) the host response [19,23,35] . These aspects could also contribute to the PK variability in bupivacaine MVLs. Formulation dose strength is a major factor because dose determines injection volume, drug strength, and other factors and therefore impacts the drug dissolution and absorption accordingly. In the present study, with a same dose of bupivacaine administrated (e.g., 15 mg/kg), the injection volume was different among rats because of the different weight of each animal. A greater injection volume leading to particle aggregation and resulting in slower absorption has been observed in some LAIs [36,37] . The injection volume issue could also be the case with bupivacaine MVLs leading to different absorption of bupivacaine as the injection volume varied among individual rats. Even after the injection, the local drug movement was hard to control and potentially different among each injection, resulting in a different drug depot every time. Other injection techniques such as depth, site, speed, and pressure of injection can further affect the size and formation of drug depots, and thus the drug dissolution and absorption [23,24,38,39] . Batch-to-batch variability in the commercial products may also play a role and contribute to different formulation properties, causing PK variability accordingly. The physiological environment at administration site and how the matrix at the injection site interacts with formulations may be impactful in the case of bupivacaine MVLs since the MVLs are expected to stay in the local biological tissue (e.g., subcutaneous site) for a long time [40]. Finally, a local immune response initiated in the subcutaneous area after the bupivacaine MVLs injection can be another factor [19] . Injection site reactions were observed histologically with LAI suspensions in the intramuscular tissue, showing the formation of a dense inflammatory envelope which was considered to preclude drug dissolution and absorption [41] . As the host response may vary subject to subject and injection to injection, the discrepancy with the following inflammatory reactions could further impact the drug exposure and result in a PK variability. As discussed above, a variability in animal PK may be quite common with LAIs and multiple MVL formulations. Nevertheless, the observation of a variability with bupivacaine MVLs in our study, as well as the insufficient available comparable data, should be taken into account in future study designs. Table 3 Pharmacokinetic parameters of Exparel following SC administration at a dose of 15 mg/kg in SD rats (n=4, mean ± SD) (Software: WinNonlin) Formulation (n=4) Date of test (M/D/Y) C max (ng/mL) CV (%) T max (h) a AUC 0→96h (ng/mL*h) CV (%) b MRT (h) Exparel 09/24/18 351.03 ± 138.86 39.56 0.5-4 4515.24 ± 1776.49 39.34 18.44 ± 1.79 05/29/19 104.27 ± 42.56 40.82 1-2 1724.41 ± 142.30 8.25 19.54 ± 2.94 03/15/21 230.06 ± 172.06 74.79 0.5-2 2655.94 ± 571.00 21.50 16.56 ± 3.33 12/13/21 183.90 ± 31.30 17.02 1-24 5768.28 ± 554.45 9.61 20.49 ± 8.33 a AUC: area under the curve b MRT: mean residence time Comparison of the in vitro and in vivo characteristics between bupivacaine MVLs with different product qualities Several bupivacaine MVLs with different product qualities were compared in terms of their in vitro and in vivo characteristics, to investigate how the performances of MVL products can be impacted by their product attributes. Bupivacaine MVLs with compromised product quality were deliberately generated with commercial product (i.e., Exparel) in our lab based on some of the major issues considered in product manufacturing and distribution process: 1) freeze-thawing; 2) mechanical agitation; 3) elevated temperature; and 4) expiration. Compared to the intact product, all the compromised formulations showed differences in their morphology ( Fig. 4 ). Overall, freeze-thawing and high temperature led to the most prominent changes in the product qualities. Freeze-thawed bupivacaine MVLs not only lost the “honeycomb” structure, characterized with a much smaller particle size distribution, but also was the only formulation whose pH (i.e., 5.5) dropped outside the reported pH range of commercial product (i.e., 5.8 - 7.4) (Table 4). Much higher free bupivacaine content was also found after freeze-thawing, indicating a particle breakage following an inner phase leakage ( Table 4 ). High temperature, on the other hand, led to a much larger particle size distribution ( Table 4 ), which might be attributed to particle aggregation as observed in Fig. 4d . Particle size or formulation pH did not significantly change for bupivacaine MVLs being mechanically agitated or expired ( Table 4 ). However, unusual morphology such as broken outer layers and particle fusion was observed in the images taken of samples for these two compromised formulations ( Fig. 4 ), suggesting some extents of particle destruction. A higher free drug content found after mechanical agitation further suggests particle breakage occurred in this group ( Table 4 ). All the compromised formulations (i.e., freeze-thawed, mechanical agitated, and high-temperature incubated) except the expired one showed a different (i.e., faster) in vitro release (IVR) from the intact formulation, when estimated via an accelerated rotator-based IVRT method developed previously in our lab [13] ( Fig. 5 ). With a comparison to their in vivo PK, both the freeze-thawed and mechanical agitated groups also performed differently from the intact formulation, implying an association between their in vitro characteristics and in vivo behaviors ( Fig. 5 and Fig. 6 ). The freeze-thawed bupivacaine MVLs even lost the multiphasic PK characteristics completely, displaying a plasma profile similar to that of the free bupivacaine solution ( Fig. 1 and Fig. 6 ). Supported by the observation of complete broken particles in freeze-thawed group, this failing in observing a multiphasic PK or a second plasma peak may be linked to a disruption or lose of nonconcentric MVL structure in the formulation ( Fig. 4 and Fig. 6 ). Interestingly, while the expired bupivacaine MVL product exhibited a different PK from the intact product, the expired product’s IVR was similar to the unexpired group ( Fig. 5 and Fig. 6 ). On the other hand, while the high-temperature incubated bupivacaine MVLs exhibited a different (i.e., faster) IVR, the high temperature treated product’s PK was similar to the intact group ( Fig. 5 and Fig. 6 ). As both the expired and high-temperature incubated groups showed some particle aggregation and/or fusion in their morphology ( Fig. 4 ), this discrepancy found in their in vitro and in vivo features might suggest a more complicated interplay of particle aggregation on the drug release and absorption process of MVL formulation. Furthermore, because the IVRT method applied here was an accelerated testing method originally aiming for a quick quality check instead of presenting bio-relevance [13], some physiological features might not be captured with the in vitro settings, attributing to this discrepancy. Nevertheless, with a comprehensive analysis of and comparison between multiple in vitro characteristics (i.e., morphology, particle sizes, formulation pH, free drug contents, and IVR), an association between bupivacaine MVLs with different product qualities to their in vivo performances was observed. This association implies the potential of predicting in vivo performances by in vitro analytical formulation characterization of multiple product attributes. Table 4 Size distribution, pH value, and free drug contents of intact, expired, and compromised Exparel. All values are presented as Mean ± SD (n=3) Formulation d0.1 (μm) d0.5 (μm) d0.9 (μm) pH Free drug % a Intact 13.2 ± 0.2 26.0 ± 0.3 53.5 ± 0.1 6.3 5.26 Freeze-thawed 3.1 ± 0.1 13.0 ± 0.5 30.6 ± 1.7 5.5 80.04 High-temp incubated 16.6 ± 1.1 49.7 ± 1.7 95.1 ± 5.0 6.1 12.07 Mechanical agitated 13.3 ± 0.3 25.7 ± 0.3 50.6 ± 0.1 6.5 28.49 b Expired 13.5 ± 0.2 26.5 ± 0.2 54.0 ± 0.1 6.0 5.83 a Intact Exparel batch# 20-P097, with the expiration in July 2022 b Expired Exparel batch# 17-4046, with the expiration in April 2018 Table 5 Pharmacokinetic parameters of intact, expired, and compromised Exparel following SC administration at a dose of 15 mg/kg in SD rats (n=5, mean ± SD) (Software: WinNonlin.) Formulation (n=5) Conditions (dose/route/dilution) C max (ng/mL) T max (h) a AUC 0 → 96h (ng/mL*h) b MRT (h) Intact 15 mg/kg c SC undiluted 183.90 ± 31.30 1-24 5768.28 ± 554.45 20.49 ± 8.33 Freeze-thawed 2333.65 ± 450.10 0.5-1 6181.13 ± 781.34 3.96 ± 1.21 High-temp incubated 246.86 ± 62.27 0.5-1 4726.05 ± 1383.78 14.42 ± 2.95 Mechanical agitated 710.22 ± 94.68 1-12 13169.13 ± 2665.87 17.96 ± 2.72 Expired 1528.45 ± 484.63 2 12370.41 ± 4046.73 11.81 ± 1.97 a AUC: area under the curve b MRT: mean residence time c SC: subcutaneous Conclusion Preclinical data on bupivacaine MVLs is supportive for product class understanding and complements our knowledge on the in vitro and in vivo relationship of MVL based formulation [32,42]. In the present study, the PK of bupivacaine MVLs was investigated in SD rats, and compared under different experimental conditions (i.e., doses, administration routes, and dilution factors). A characteristic multiphasic PK with a second plasma peak was observed with the bupivacaine MVL formulations. Different experimental conditions exhibited impacts on not only the plasma profiles influencing the second peak, but also some of the key parameters such as C max and AUC, which was different from the previously reported preclinical results with bupivacaine MVLs [28]. ­Notably, both an inter- and intra-experimental variability was found to be nonnegligible in bupivacaine MVLs’ PK, calling for attention to MVL based sample handling and preparation in in vitro and/or in vivo studies for product development and bioequivalence. A comprehensive analysis of and comparison between bupivacaine MVLs with different product attributes in aspects of their morphology, particle sizes, formulation pH, free drug, IVR, and PK profiles was conducted. An association between the formulation characteristics and in vivo behaviors was identified in compromised bupivacaine MVL products in this study, suggesting a potential in vitro in vivo relationship. Overall, the present work investigating the in vivo behaviors of bupivacaine MVLs is beneficial not only to fill in the gap of preclinical studies in the field of bupivacaine MVLs, but also to help pave the path for developing future MVL-related products. Declarations Acknowledgement This study was funded in part by FDA 75F40120C00127. Minzhi Yu was supported in part by AHA postdoctoral fellowship 24POST1196020. Anna Schwendeman was supported in part by Duellman Graduate Student Research Fund. This author also wants to acknowledge the animal work contributed by Yayuan Liu and Karl Olsen. Funding This work was supported by the Broad Agency Announcement (BAA) Contract # 75F40120C00127 from the U.S. Food and Drug Administration (FDA). The content reflects the views of the authors and should not be construed to represent the views or policies of the U.S. FDA. Competing Interests Financial interests/personal relationships: Author Anna Schwendeman reports financial support was provided by US Food and Drug Administration. Author Anna Schwendeman reports a relationship with EVOQ Therapeutics that includes: board membership, consulting or advisory, equity or stocks, and funding grants. Author Contributions All authors contributed to the study conceptualization. Material preparation, data curation, and formal analysis were performed by Ziyun Xia, Yayuan Liu, Jingyao Gan, Ziyi Lu, and Karl Olsen. The original draft of the manuscript was written by Ziyun Xia. Previous versions of the manuscript were reviewed and edited by Minzhi Yu, Yan Wang, Xiaoming Xu, and Anna Schwendeman. Study supervision and funding acquisition were provided by Anna Schwendeman and Steve Schwendeman. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics Approval This study was performed in line with the Guide for the Care and Use of Laboratory Animals (Guide), NRC 2011. Approval was granted by the Institutional Animal Care & Use Committee (IACUC) of the University of Michigan (most recent AAALAC accreditation date: November 27, 2023/Unit number: 000285). References Exparel [package insert] Pacira Pharmaceuticals. 2018. Ye Q, Asherman J, Stevenson M, Brownson E, Katre N V. DepoFoam(TM) technology: A vehicle for controlled delivery of protein and peptide drugs. Journal of Controlled Release. Elsevier; 2000. p. 155–66. Mantripragada S. A lipid based depot (DepoFoam® technology) for sustained release drug delivery. Prog Lipid Res. 2002;41:392–406. Kim S, Turker MS, Chi EY, Sela S, Martin GM. Preparation of multivesicular liposomes. BBA - Biomembranes. 1983;728:339–48. Pacira Pharmaceuticals, Inc. Announces U.S. FDA Approval of Exparel For Postsurgical Pain Management [Internet]. [cited 2021 Apr 14]. Available from: https://www.drugs.com/newdrugs/pacira-pharmaceuticals-inc-announces-u-s-fda-approval-exparel-postsurgical-pain-management-2938.html FDA In Brief: FDA approves new use of Exparel for nerve block pain relief following shoulder surgeries | FDA [Internet]. [cited 2021 Feb 1]. Available from: https://www.fda.gov/news-events/fda-brief/fda-brief-fda-approves-new-use-exparel-nerve-block-pain-relief-following-shoulder-surgeries Tampa D, Newswire G, Biosciences P, Therapeutics F. Pacira BioSciences Reports Preliminary Unaudited Total Revenue for 2022 of $ 666 . 8 Million [Internet]. 2023. Available from: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://investor.pacira.com/node/16036/pdf Orange Book: Approved Drug Products with Therapeutic Equivalence Evaluations [Internet]. [cited 2023 Jan 8]. Available from: https://www.accessdata.fda.gov/scripts/cder/ob/patent_info.cfm?Product_No=001&Appl_No=022496&Appl_type=N Pacira BioSciences Notified of Abbreviated New Drug Application Filing for EXPAREL® | Pacira BioSciences, Inc. [Internet]. [cited 2023 Feb 2]. Available from: https://investor.pacira.com/news-releases/news-release-details/pacira-biosciences-notified-abbreviated-new-drug-application U.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Liposome Drug Products: Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation [Internet]. Available from: https://www.fda.gov/media/70837/download U.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Draft Guidance on Bupivacaine Liposomal Injection [Internet]. 2018. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070570.pdf Yu M, Yuan W, Xia Z, Liu Y, Wang Y, Xu X, et al. Characterization of exparel bupivacaine multivesicular liposomes. Int J Pharm. 2023;639:122952. Xia Z, Yu M, Liu Y, Yuan W, Wang Y, Xu X, et al. Development of an Accelerated Rotator-based Drug Release Method for the Evaluation of Bupivacaine Multivesicular Liposomes. Pharm Res. 2024; Manna S, Wu Y, Wang Y, Koo B, Chen L, Petrochenko P, et al. Probing the mechanism of bupivacaine drug release from multivesicular liposomes. Journal of Controlled Release. 2019;294:279–87. Chaurasiya A, Gorajiya A, Panchal K, Katke S, Singh AK. A review on multivesicular liposomes for pharmaceutical applications: preparation, characterization, and translational challenges. Drug Deliv Transl Res. 2022;12:1569–87. Mantripragada B.Sankaram, Sinil Kim. Preparation of Multivesicular Liposomes for Controlled Release of Encapsulated Biologically Active Substances. 1999. Sankaram MB, Kim S. Multivesicular liposomes with controlled release of encapsulated biologically active substances. 1998. Smith WC, Bae J, Zhang Y, Qin B, Wang Y, Kozak D, et al. Impact of particle flocculation on the dissolution and bioavailability of injectable suspensions. Int J Pharm. 2021;604:120767. Nguyen VTT, Darville N, Vermeulen A. Pharmacokinetics of Long-Acting Aqueous Nano-/Microsuspensions After Intramuscular Administration in Different Animal Species and Humans—a Review. AAPS J. 2022;25. Chamanza R, Darville N, van Heerden M, De Jonghe S. Comparison of the Local Tolerability to 5 Long-acting Drug Nanosuspensions with Different Stabilizing Excipients, Following a Single Intramuscular Administration in the Rat. Toxicol Pathol. 2018;46. Gautam N, Roy U, Balkundi S, Puligujja P, Guo D, Smith N, et al. Preclinical pharmacokinetics and tissue distribution of long-acting nanoformulated antiretroviral therapy. Antimicrob Agents Chemother. 2013;57:3110–20. D’aquino AI, Maikawa CL, Nguyen LT, Lu K, Hall IA, Jons CK, et al. Sustained Delivery of GLP-1 Receptor Agonists from Injectable Biomimetic Hydrogels Improves Treatment of Diabetes. 2023. Siemons M, Schroyen B, Darville N, Goyal N. Role of Modeling and Simulation in Preclinical and Clinical Long-Acting Injectable Drug Development. AAPS J. 2023;25. McDowell A, Medlicott NJ. Anatomy and Physiology of the Injection Site: Implications for Extended Release Parenteral Systems. Long Acting Injections and Implants. 2012;57–71. Ballard BE. Biopharmaceutical Considerations in Subcutaneous and Intramuscular Drug Administration. J Pharm Sci. 1968;57:357–78. Schwendeman SP, Shah RB, Bailey BA, Schwendeman AS. Injectable controlled release depots for large molecules. Journal of Controlled Release. 2014;190:240–53. Shah JC, Maniar M. pH-Dependent solubility and dissolution of bupivacaine and its relevance to the formulation of a controlled release system. Journal of Controlled Release. 1993;23:261–70. Hadzic A, Abikhaled JA, Harmon WJ. Impact of volume expansion on the efficacy and pharmacokinetics of liposome bupivacaine. Local Reg Anesth. 2015;8:105–11. Shen Y, Ji Y, Xu S, Chen DQ, Tu J. Multivesicular liposome formulations for the sustained delivery of ropivacaine hydrochloride: Preparation, characterization, and pharmacokinetics. Drug Deliv. 2011;18:361–6. Zhong H, Deng Y, Wang X, Yang B. Multivesicular liposome formulation for the sustained delivery of breviscapine. Int J Pharm. 2005;301:15–24. Mu H, Wang Y, Chu Y, Jiang Y, Hua H, Chu L, et al. Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization. Drug Deliv. 2018;25:1372–83. Abuzar SM, Park EJ, Seo Y, Lee J, Baik SH, Hwang S-J. Preparation and Evaluation of Intraperitoneal Long-Acting Oxaliplatin-Loaded Multi-Vesicular Liposomal Depot for Colorectal Cancer Treatment. Pharmaceutics 2020, Vol 12, Page 736. 2020;12:736. Little TA. Establishing Acceptance Criteria for Analytical Methods. Biopharm Int. 2016 Oct 1; U.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Bioequivalence Studies With Pharmacokinetic Endpoints for Drugs Submitted Under an Abbreviated New Drug Application [Internet]. 2021. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioequivalence-studies-pharmacokinetic-endpoints-drugs-submitted-under-abbreviated-new-drug Daublain P, Feng KI, Altman MD, Martin I, Mukherjee S, Nofsinger R, et al. Analyzing the Potential Root Causes of Variability of Pharmacokinetics in Preclinical Species. Mol Pharm. 2017;14:1634–45. Hirano K, Ichihashi T, Yamada H. Studies on the absorption of practically water-insoluble drugs following injection. III. Intramuscular absorption from aqueous nonionic surfactant solutions in rats. Chem Pharm Bull (Tokyo). 1981;29:834–43. Jucker BM, Fuchs EJ, Lee S, Damian V, Galette P, Janiczek R, et al. Multiparametric magnetic resonance imaging to characterize cabotegravir long‐acting formulation depot kinetics in healthy adult volunteers. Br J Clin Pharmacol. 2022;88:1655. Darville N, van Heerden M, Vynckier A, De Meulder M, Sterkens P, Annaert P, et al. Intramuscular Administration of Paliperidone Palmitate Extended-Release Injectable Microsuspension Induces a Subclinical Inflammatory Reaction Modulating the Pharmacokinetics in Rats. J Pharm Sci. 2014;103:2072–87. Schou J. Subcutaneous and Intramuscular Injection of Drugs. In: Brodie BB, Gillette JR, Ackerman HS, editors. Concepts in Biochemical Pharmacology: Part 1. Berlin, Heidelberg: Springer Berlin Heidelberg; 1971. p. 47–66. Kinnunen HM, Mrsny RJ. Improving the outcomes of biopharmaceutical delivery via the subcutaneous route by understanding the chemical, physical and physiological properties of the subcutaneous injection site. Journal of Controlled Release. 2014;182:22–32. Groseclose MR, Castellino S. Intramuscular and subcutaneous drug depot characterization of a long-acting cabotegravir nanoformulation by MALDI IMS. Int J Mass Spectrom. 2019;437:92–8. Alavi S, Mahjoob MA, Haeri A, Shirazi FH, Abbasian Z, Dadashzadeh S. Multivesicular liposomal depot system for sustained delivery of risperidone: development, characterization, and toxicity assessment. Drug Dev Ind Pharm. 2021;47:1290–301. Supplementary Files Graphicalabstract.docx SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Accept as is 18 Jan, 2025 Reviewers agreed at journal 18 Dec, 2024 Reviewers invited by journal 18 Dec, 2024 Editor assigned by journal 16 Dec, 2024 First submitted to journal 13 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5214719","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391733098,"identity":"79554563-0d2a-4abc-bfb1-c0d6c1acde22","order_by":0,"name":"Ziyun Xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYNACAwY5BgbGBmYGEEmsFmNStTAwJIJUEqdFfkaO2WOeArv0DdcON34uYLCR3XCAgBbGGTnmhjMMknM33E5slp7BkGZMUAuzRI6ZxAcDZpCWNmYehsOJBLWwgbQkGNSnG0C0/CeshQdiy+EEqJYDhLVI8Dwrk5xhcNxwJsgvPAbJxjMJaZFvT94mzfOnWp7vdvrDzzwVdrJ9hLQwCCQg8wwIKQcBfoKGjoJRMApGwYgHAPmOPiN1w5yPAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6409-1517","institution":"U-M: University of Michigan","correspondingAuthor":true,"prefix":"","firstName":"Ziyun","middleName":"","lastName":"Xia","suffix":""},{"id":391733099,"identity":"6c89f197-1a05-4df7-824a-0cb763a36f8f","order_by":1,"name":"Yayuan Liu","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Yayuan","middleName":"","lastName":"Liu","suffix":""},{"id":391733100,"identity":"af1e719c-731c-4517-a8d1-400fb0c7c66a","order_by":2,"name":"Ziyi Lu","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Ziyi","middleName":"","lastName":"Lu","suffix":""},{"id":391733101,"identity":"6a431656-7d6f-4e42-8903-0354b160f563","order_by":3,"name":"Jingyao Gan","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Jingyao","middleName":"","lastName":"Gan","suffix":""},{"id":391733102,"identity":"4c6cd344-ff2a-4803-8b80-d01c51436844","order_by":4,"name":"Minzhi Yu","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Minzhi","middleName":"","lastName":"Yu","suffix":""},{"id":391733103,"identity":"b46b7453-cb91-43b9-82ea-971374f2f794","order_by":5,"name":"Karl Olsen","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Karl","middleName":"","lastName":"Olsen","suffix":""},{"id":391733104,"identity":"2162914d-ae62-4d9e-8d64-2b0deefb66c0","order_by":6,"name":"Yan Wang","email":"","orcid":"","institution":"US FDA: US Food and Drug Administration","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wang","suffix":""},{"id":391733105,"identity":"f73331a7-ad46-4b0c-9e39-5f488e76f7b9","order_by":7,"name":"Xiaoming Xu","email":"","orcid":"","institution":"FDA: US Food and Drug Administration","correspondingAuthor":false,"prefix":"","firstName":"Xiaoming","middleName":"","lastName":"Xu","suffix":""},{"id":391733106,"identity":"81a55089-359c-44e3-a213-41025d2674ee","order_by":8,"name":"Steve Schwendeman","email":"","orcid":"","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Steve","middleName":"","lastName":"Schwendeman","suffix":""},{"id":391733107,"identity":"4eab4df6-5190-47df-a8d2-692f0c4fd6cd","order_by":9,"name":"Anna Schwendeman","email":"","orcid":"https://orcid.org/0000-0002-8023-8080","institution":"U-M: University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Schwendeman","suffix":""}],"badges":[],"createdAt":"2024-10-07 00:33:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5214719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5214719/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-025-01806-y","type":"published","date":"2025-03-04T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71938936,"identity":"7dcd309e-77c0-4443-b5bb-bc1a6b34ef83","added_by":"auto","created_at":"2024-12-20 00:59:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68126,"visible":true,"origin":"","legend":"\u003cp\u003eBupivacaine plasma concentration profiles of Exparel and free bupivacaine solution following subcutaneous (SC) administration at 15 mg/kg in SD rats (n=4, mean ± SD)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/78ccbcb612eaea6253659912.png"},{"id":71938937,"identity":"ad837b86-b737-481d-ab01-e7476fa7331e","added_by":"auto","created_at":"2024-12-20 00:59:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80158,"visible":true,"origin":"","legend":"\u003cp\u003eBupivacaine plasma concentration profiles of \u003cstrong\u003e(a)\u003c/strong\u003e bupivacaine MVLs following subcutaneous (SC) administration at 5, 15, and 25 mg/kg, \u003cstrong\u003e(b)\u003c/strong\u003e bupivacaine MVLs following subcutaneous (SC) or intramuscular (IM) administration at 15 mg/kg, and \u003cstrong\u003e(c)\u003c/strong\u003e undiluted and 3X diluted bupivacaine MVLs following subcutaneous (SC) administration at 15 mg/kg, in SD rats (n=4, mean ± SD)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/0f87b6b74cea5e6119c13fb6.png"},{"id":71938216,"identity":"6b6ca5f5-6220-4866-a372-7c616f661808","added_by":"auto","created_at":"2024-12-20 00:51:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78853,"visible":true,"origin":"","legend":"\u003cp\u003eBupivacaine plasma concentration profiles of commercially available bupivacaine MVL products tested at different dates following subcutaneous (SC) administration at a dose of 15 mg/kg in SD rats (n=4, mean ± SD\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/bd1c79c52dd1c4bc5340de21.png"},{"id":71938214,"identity":"f0d68d56-737d-4e05-b780-c7e6a61651ba","added_by":"auto","created_at":"2024-12-20 00:51:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":600963,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of (\u003cstrong\u003ea\u003c/strong\u003e) intact Exparel (batch# 20-P097), (\u003cstrong\u003eb\u003c/strong\u003e) freeze-thawed Exparel, (\u003cstrong\u003ec\u003c/strong\u003e) mechanical agitated Exparel, (\u003cstrong\u003ed\u003c/strong\u003e) high-temperature incubated Exparel, and (\u003cstrong\u003ee\u003c/strong\u003e) Exparel (batch# 17-4046) expired in April 2018\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/7f459189720bfaa1d4a91bf7.png"},{"id":71938217,"identity":"6787b79a-8729-4a30-9805-36faeeaa75d4","added_by":"auto","created_at":"2024-12-20 00:51:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro \u003c/em\u003edrug release profile of intact (20-P097), expired (17-4046), and compromised Exparel stressed via mechanical agitation, freeze-thawing, and high-temperature incubation. All values are presented as Mean ± SD (n=3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/57e05f6e029ea3f215cd8937.png"},{"id":71938938,"identity":"04b4be00-63b6-43c8-8254-0f74742434cf","added_by":"auto","created_at":"2024-12-20 00:59:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":101357,"visible":true,"origin":"","legend":"\u003cp\u003eBupivacaine plasma concentration profiles of subcutaneous injected intact, expired, and compromised Exparel stressed via freeze-thawing, mechanical agitation, and high-temperature incubation, at 15 mg/kg in SD rats (n=5, mean ± SD)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/08e5f1473e595401a10c0501.png"},{"id":78191341,"identity":"939a7058-9d0e-465f-b0cc-31c7403ccd47","added_by":"auto","created_at":"2025-03-10 19:56:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1796017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/4319de47-f5b2-424a-9ca9-b535cc107653.pdf"},{"id":71938209,"identity":"59b65b6b-a13a-48ba-a1dd-05bfcff2f48a","added_by":"auto","created_at":"2024-12-20 00:51:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":134514,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/41ac06dcfc50b4fb505974ba.docx"},{"id":71938206,"identity":"326da0ea-dfa7-4331-8817-c6d3dbcf4d83","added_by":"auto","created_at":"2024-12-20 00:51:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40559,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5214719/v1/bd7d0cdc26b2f0738e87204d.docx"}],"financialInterests":"","formattedTitle":"An investigation of in vivo performance of bupivacaine multivesicular liposomes in rats and the impacts from product qualities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExparel is a multivesicular liposomal formulation of bupivacaine utilizing the DepoFoam\u003csup\u003eTM\u003c/sup\u003e technology for sustained drug release from days to weeks [1,2]. An assembly of numerous nonconcentric liposomes within a large particle contributes to a unique honeycomb-like structure of multivesicular liposomes (MVLs), allowing for a better drug encapsulation efficiency as well as a longer drug release duration [3,4]. Exparel was approved by the US Food and Drug Administration (FDA) for the post-surgical local analgesia in 2011, and its label was extended for nerve-block pain relief after shoulder surgeries in 2018 [5,6]. As a long-acting nonopioid drug product, Exparel gained great market success in the past few years [7]. At the same time, its patents either have already or are going to expire soon, making Exparel an appealing target for many generic developers [8,9]. \u003c/p\u003e\n\n\u003cp\u003eTo facilitate the development and regulatory filing of liposomal drug product, FDA published a series of guidance documents, including Liposome Drug Products - Chemistry, Manufacturing and Controls, Human Pharmacokinetics and Bioavailability, as well as the Labeling Documentation [10]. However, the published guidance focuses more on the traditional liposomes such as unilamellar and multilamellar liposomes, instead of MVLs. A product-specific draft guidance (PSG) for bupivacaine liposomal injection was issued by FDA in 2018 [11], which recommends the evaluation of various parameters including drug product composition and the liposome characteristics between the ANDA and the reference listed drug (RLD) products. Clinical pharmacokinetic (PK) studies are also recommended for the demonstration of bioequivalence. Even though there have been some efforts in developing characterization methods and an accelerated \u003cem\u003ein vitro\u003c/em\u003e release (IVR) assay for bupivacaine MVL formulation [12\u0026ndash;14], the information regarding preclinical \u003cem\u003ein vivo\u003c/em\u003e testing of MVL-based drug products is still lacking. There is also no waiver applicable for an \u003cem\u003ein vivo\u003c/em\u003e testing so far [11]. The need for clinical trials presents challenges to generic developers as they are usually time and economic burdens. Meanwhile, even though a draft PSG was published for bupivacaine liposomal injection (i.e., Exparel), a lack of general guidelines on MVL drug products still challenges the industry to understand and meet the regulatory expectation in the development of MVL-based drug products [15]. Considering the high stake of clinical trials, a preclinical study is favorable in drug development over many aspects, such as its low cost and feasibility on conduction, as well as informative insights on \u003cem\u003ein vivo \u003c/em\u003eperformance and \u003cem\u003ein vitro in vivo\u003c/em\u003e association. However, because of the complex formulation as well as unique release features of long acting injectables (LAIs) such as Exparel, preclinical study design can be challenging, and the lack of references due to a limitation on published preclinical studies has added more hurdles. Therefore, an investigation on the preclinical PK of bupivacaine MVLs is desired for understanding critical formulation attributes to facilitate formulation development and establishment of bioequivalence during generic development. \u003c/p\u003e\n\n\u003cp\u003eExparel and other MVL drug products exhibit characteristic drug release behaviors due to their unique non-lamellar honeycomb structures [2,3,16,17]. In general, three mechanisms are considered accountable for its release: drug molecule diffusion across the lipid membrane, internal lipids rearrangement causing vesicles coalescence, and the lipid bilayer decomposition [3]. In 2018, Manna et al., proposed a tri-phase release process for bupivacaine MVLs based on a reverse-dialysis IVRT method [14]. However, since these release behaviors were only examined with non-physiological relevant IVRT assays, whether the proposed release mechanisms can be applied \u003cem\u003ein vivo \u003c/em\u003eremains to be investigated. Additionally, no PK studies were examined and compared. Since the physiological environment is far more complicated and variable than the \u003cem\u003ein vitro \u003c/em\u003esettings, a lack of \u003cem\u003ein vivo\u003c/em\u003e comparisons poses extra challenges on understanding the association between the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e release of MVL formulation. A preclinical PK study can pave the first step on studying any \u003cem\u003ein vitro in vivo\u003c/em\u003e relationship for this specific product, which can not only ease and fasten the process of generic development, but also reciprocally help understand the release process of MVL-related products.\u003c/p\u003e\n\n\u003cp\u003eTherefore, in our present work, we set up several \u003cem\u003ein vivo\u003c/em\u003e studies and developed an analytical method for the bupivacaine MVLs PK testing in Sprague-Dawley (SD) rats. Different experimental conditions such as drug doses, routes of administration, and sample dilution factors were investigated to understand their impacts on bupivacaine MVLs\u0026rsquo; \u003cem\u003ein vivo\u003c/em\u003e PK in SD rats. Commercial bupivacaine MVL samples with different manipulations were purposely varied and then characterized in terms of their \u003cem\u003ein vitro \u003c/em\u003eand\u003cem\u003e in vivo \u003c/em\u003echaracteristics including morphology, sizes, formulation pH, free drug contents, \u003cem\u003ein vitro \u003c/em\u003erelease, and \u003cem\u003ein vivo \u003c/em\u003ePK, to further understand how varied product quality attributes impact on the \u003cem\u003ein vivo \u003c/em\u003eperformances accordingly. \u003c/p\u003e\n"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eExparel (bupivacaine liposome injectable suspension) batch number of 17-4046, 20-P097 and 21-P080 were purchased from the Research Pharmacy at the University of Michigan Health System. United States Pharmacopeia (USP) grade bupivacaine hydrochloride hydrate was purchased from Fisher Scientific. 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) was purchased from NOF. Cholesterol and tricaprylin were purchased from Sigma. 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG-Na) was purchased from Avanti. Bupivacaine free base and ropivacaine hydrochloride hydrate were purchased from Cayman Chemical. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBupivacaine MVL formulations with compromised product qualities\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eCommercial bupivacaine MVL formulations (Exparel, batch number of 20-P097 with expiration dated in July 2022) were stressed via 1) mechanical agitation, 2) freeze-thawing cycle, or 3) high-temperature incubation, to generate compromised formulations with different product quality attributes. In the scenario of mechanical agitation, Exparel was vortexed at a speed of 2500 rpm (Fisher Analog Vortex Mixer, 120V, 9454FIALUS) for 20 ss. In the freeze-thawing scenario, Exparel was first incubated at -20\u0026deg;C overnight and then thawed at 40\u0026deg;C for 5 min. In the scenario of high-temperature incubation, Exparel was incubated at 45\u0026deg;C for 13 h.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eParticle size distribution\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eParticle size distribution of bupivacaine MVLs was analyzed by Malvern Mastersizer 2000 (Malvern Panalytical Ltd, Malvern, UK), equipped with a Hydro 2000S (AWA2001) dispersion unit. Undiluted bupivacaine MVLs were dispersed in filtered 0.9% saline in the instrument until they reached at least 10% obscuration level. Three measurements were conducted for each sample at a stirring speed of 3000 rpm, with a material refractive index of 1.5.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMorphology\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe morphology of bupivacaine MVLs was observed via an optical microscope (Carl Zeiss Axiolab Re microscope). Briefly, bupivacaine MVL formulations diluted 10 times were added onto a glass slide and observed under the microscope immediately after the coverslip was placed on. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFormulation pH measurement\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe pH of bupivacaine MVL formulations was measured using a 430 pH meter (Corning, USA) with MI-410 microelectrodes (Microelectrodes Inc., USA) at room temperature.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBupivacaine quantification in \u003cem\u003ein vitro\u003c/em\u003e release study\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eUltra-performance liquid chromatography (UPLC) was used to quantify the released drug content from bupivacaine MVL samples. Samples collected from the \u003cem\u003ein vitro \u003c/em\u003erelease study were centrifuged at 600 rcf (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor) for 15 min to remove liposome particles. Then 200 \u0026micro;L of the supernatant was taken and diluted with 250 \u0026micro;L methanol and rigorously vortexed. Then 500 \u0026micro;L acetonitrile was added, followed by a rigorous vortex. The final mixture was syringe-filtered (0.45\u0026micro;m, PVDF) before being injected into a Waters ACQUITY UPLC H-Class System equipped with a Waters ACQUITY UPLC BEH C18 column (2.1 \u0026times; 50 mm). The mobile phase was composed of 70% (A) water with 0.1% formic acid (FA); 15% (B) acetonitrile with 0.1% FA; 15% (C) methanol with 0.1% FA. The flow rate was 0.4 mL/min. The UV detection wavelength was 220 nm. The following equation was used to calculate the cumulative release of bupivacaine:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"536\" height=\"82\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetics (PK) evaluation in rats\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eMale Sprague-Dawley (SD) rats were used for the PK evaluation. Free bupivacaine solution and bupivacaine MVL samples were subcutaneously injected to the backs of healthy rats (n = 4 or 5) at a dose of 5, 15, or 25 mg/kg of bupivacaine. Blood was drawn from the jugular vein at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after dosing. Collected blood samples were immediately centrifuged at 15,000 rpm (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor) at 4\u0026deg;C for 5 min to separate the plasma. Plasma samples were stored at -80\u0026deg;C for future analysis.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBupivacaine quantification in plasma\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eBupivacaine was extracted from plasma samples via protein precipitation extraction. Briefly, 100 \u0026mu;L of plasma samples was spiked with 20 \u0026mu;L of internal standard (1 \u0026mu;g/mL ropivacaine) and then mixed with 50 \u0026mu;L of 1 M sodium hydroxide solution. Hexane-isopropanol-chloroform (v:v:v = 3:6:1) mixed solvent was used to extract the samples via a vortex-mix manner, following with a 10-minute centrifugation at 10,000 rpm (Axyspin Refrigerated Microcentrifuge, 120V, with 24 x 1.5/2.0 mL aluminum rotor). The supernatants were collected and dried under nitrogen flow at 60℃. The dry residues were reconstituted in 200 \u0026mu;L of hydrochloride acid (0.5N) and syringe-filtered (0.45 \u0026mu;m, PVDF) before LC-MS analysis. \u003c/p\u003e\n\n\u003cp\u003ePlasma bupivacaine concentration was determined by UPLC-MS (Waters ACQUITY system equipped with a QdaTM detector) using a Waters ACQUITY UPLC BEH300 C18 column (1.7 \u0026mu;m, 2.1 x 100 mm). The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in methanol (v:v = 60:40). The flow rate was 0.3 mL/min with a running time of 4 min. The injection volume was 10 \u0026mu;L. Samples were detected at m/z of 289.32 (+) for bupivacaine and 275.35 (+) for ropivacaine.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eInvestigation of PK of free bupivacaine solution and bupivacaine MVLs in rats\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn analytical method to quantify plasma bupivacaine concentration was developed with a linearity between 5 to 200 ng/mL (Supplementary information \u003cstrong\u003eFig. S-1\u003c/strong\u003e). The analytical procedure was then applied to investigate the PK of and comparison between the free bupivacaine solution and bupivacaine MVL formulation at their clinical dosage strength (i.e., 4.43 mg/mL for free solution, and 13.3 mg/mL for Exparel, separately). Some unique characteristics with bupivacaine MVLs were observed in their PK profiles in our study. Compared to the free bupivacaine solution, bupivacaine MVLs featured a sustained drug plasma concentration until 48 h after administration, with a much lower C\u003csub\u003emax\u003c/sub\u003e around 100 ng/mL and a much longer mean residence time (MRT) up to 20 h, compared with the C\u003csub\u003emax\u003c/sub\u003e around 650 ng/mL and the MRT of 4 h in the situation of free drug solution (\u003cstrong\u003eTable 1\u003c/strong\u003e). Specifically, a multiphasic profile with a second peak of plasma concentration after 12 h post administration was observed in bupivacaine MVLs\u0026rsquo; PK, indicating the existence of multiple phases in the bupivacaine MVLs\u0026rsquo;s drug release process. A smaller area under the curve (AUC) was also featured in bupivacaine MVLs PK, suggesting a smaller systemic drug exposure compared to the free drug situation, which implies a hindered drug release and/or drug absorption after subcutaneous administration to the rats. The existence of MVLs particles themselves, and the lipids dissociated as MVLs decomposed along the release process, could also hinder drug absorption, resulting in a smaller AUC \u003cspan class=\"MsoPlaceholderText\"\u003e[18]\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis multiphasic PK profile was also observed in many long acting injectable (LAI) formulations when administrated intramuscularly or subcutaneously \u003cspan class=\"MsoPlaceholderText\"\u003e[19\u0026ndash;22]\u003c/span\u003e. For the LAIs, the first pharmacokinetic phase, featured with an early plasma spike, was attributed to fast initial absorption when the dissolution rate dominates in this period. At a later stage, LAIs progress to a slower release phase, resulting in the absorption rate slower than the elimination rate, featured with a flip-flop PK in which a second plasma peak may or may not show up in their profiles \u003cspan class=\"MsoPlaceholderText\"\u003e[23]\u003c/span\u003e. \u0026nbsp;Notably with our observation in bupivacaine MVLs\u0026rsquo;s PK, the presence of a plasma peak in the second phase might further indicate an intermediate lag phase during the drug release and absorption process (\u003cstrong\u003eFig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003ePharmacokinetic parameters of free bupivacaine solution and Exparel (SC) in SD rats (n=4, mean \u0026plusmn; SD) (Software: WinNonlin.)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26.8041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.27835%;\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003cp\u003e(mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.21649%;\"\u003e\n \u003cp\u003eRoute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.5258%;\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.18557%;\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5567%;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eAUC\u003csub\u003e0\u0026rarr;96h\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL*h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eMRT\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26.8041%;\"\u003e\n \u003cp\u003efree bupivacaine solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.27835%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.21649%;\"\u003e\n \u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.5258%;\"\u003e\n \u003cp\u003e649.32 \u0026plusmn; 235.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.18557%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5567%;\"\u003e\n \u003cp\u003e3945.62 \u0026plusmn; 419.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.433%;\"\u003e\n \u003cp\u003e4.13 \u0026plusmn; 0.83**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26.8041%;\"\u003e\n \u003cp\u003eExparel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.27835%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.21649%;\"\u003e\n \u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.5258%;\"\u003e\n \u003cp\u003e104.27 \u0026plusmn; 42.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.18557%;\"\u003e\n \u003cp\u003e1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5567%;\"\u003e\n \u003cp\u003e1724.41 \u0026plusmn; 142.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.433%;\"\u003e\n \u003cp\u003e19.54 \u0026plusmn; 2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eAUC: area under the curve\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eMRT: mean residence time\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSC: subcutaneous\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpacts of experimental conditions on bupivacaine MVLs PK in rats\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental set-ups can impact the PK of long-acting drug products [24,25]. Therefore, different experimental conditions including doses, routes of administration, and dilution factors, were studied in our work to investigate their impacts on the PK of bupivacaine MVLs in rats. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBupivacaine doses ranging from 5 to 25 mg/kg were screened to investigate their impacts on bupivacaine MVLs PK in rats. The AUC and MRT were proportional to the administrated bupivacaine doses, suggesting a greater drug exposure with the higher doses (\u003cstrong\u003eFig. 2\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Table 2\u003c/strong\u003e). The C\u003csub\u003emax\u003c/sub\u003e was found to be consistent around 200 ng/mL for doses ranging from 5 to 15 mg/kg but rose as the dose went up to 25 mg/kg. Subcutaneous (SC) and intramuscular (IM) injection were investigated in our study as two of the most applied administration routes in animal study for LAIs., Administration routes were found to show impacts on the PK performances of bupivacaine MVLs in rats, indicating impacts from the physiological environment on drug dissolution and absorption\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the case of bupivacaine MVLs. IM injection exhibited a greater AUC with a longer MRT but a lower C\u003csub\u003emax\u003c/sub\u003e, suggesting a more sustained systemic exposure of bupivacaine along the time, compared to the SC administration (\u003cstrong\u003eTable 2\u003c/strong\u003e). Sample dilution factors were also investigated here as they usually relate to sink condition and thus can impact the drug dissolution. Notably, the commercial bupivacaine MVL product, Exparel, is also administrated with dilution in clinical practice [1]. Our results showed a higher C\u003csub\u003emax\u0026nbsp;\u003c/sub\u003eand a shorter MRT with the more diluted MVL formulation, indicating a faster drug release and/or absorption. The greater AUC observed in the diluted condition further suggested a greater drug exposure with the dilution, which was similar to the situation observed with a higher dose. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe biphasic PK of bupivacaine MVLs was also observed among different experimental conditions, but the second plasma peak was either shifted or less prominent as the dose, administration route, or dilution factor changed (\u003cstrong\u003eFig. 2\u003c/strong\u003e). The exact reasons for the second peak or the factors impacting the plasma concentration of the slow-release phase were not clear \u003cspan class=\"MsoPlaceholderText\"\u003e[20,23]\u003c/span\u003e. In the case of LAIs, their multiphasic profiles were considered to result from a complex interplay of formulation parameters and physiological elements: the initial fast-release phase is more likely associated with formulation factors impacting the drug dissolution which dominates in this period, and the physiological elements, especially at the injection or depot site, are usually assumed to be the major impactors of PK in the later slow-release phase when elimination plays a dominate role \u003cspan class=\"MsoPlaceholderText\"\u003e[23,26,27]\u003c/span\u003e. The similar biphasic PK observed in the case of bupivacaine MVLs highlighted the impacts from both the product attributes and physiology on their resulting \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eperformances, and the variation with the second peak might emphasize the impacts from an intermediate release phase specific with bupivacaine MVLs. Due to their nonconcentric structure, a lipid reorganization causing the coalescence of inner vesicles is unique with MVLs \u003cspan class=\"MsoPlaceholderText\"\u003e[3]\u003c/span\u003e. As there is no meaningful drug release outside the liposomes when the inner vesicles move and fuse with each other, the lipid rearrangement within MVLs might lead to an intermediate phase with sustained drug release, contributing to the second peak showing in their PK [14]. Furthermore, the other PK parameters, such as the time between the first and second peak, concentration in the secondary slow-release phase, and the slope of the terminal phase, would also be worth of future investigation in terms of their value of indicating underlying release processes of MVLs. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Pharmacokinetic parameters of bupivacaine MVLs evaluated with different experimental conditions in SD rats (n=4, mean \u0026plusmn; SD) (Software: WinNonlin.)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003cp\u003e(n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003cp\u003e(mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eRoute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003edilution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAUC\u003csub\u003e0\u0026rarr;96h\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL*h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eMRT\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" style=\"width: 13px;\"\u003e\n \u003cp\u003eExparel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003csup\u003ec\u003c/sup\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e351.99 \u0026plusmn; 180.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e5569.42 \u0026plusmn; 1747.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e23.15 \u0026plusmn; 2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e230.06 \u0026plusmn; 172.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2655.94 \u0026plusmn; 571.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e16.56 \u0026plusmn; 3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e233.68 \u0026plusmn; 37.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e930.56 \u0026plusmn; 328.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e10.26 \u0026plusmn; 6.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 9px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 9px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e230.06 \u0026plusmn; 172.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2655.94 \u0026plusmn; 571.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e16.56 \u0026plusmn; 3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003csup\u003ed\u003c/sup\u003eIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e132.37 \u0026plusmn; 87.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2835.94 \u0026plusmn; 218.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e27.07\u0026plusmn; 2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 9px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 8px;\"\u003e\n \u003cp\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e104.27 \u0026plusmn; 42.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1724.41 \u0026plusmn; 142.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e19.54 \u0026plusmn; 2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e3X\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e349.12 \u0026plusmn; 111.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2418.89 \u0026plusmn; 608.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e12.16 \u0026plusmn; 3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eAUC: area under the curve\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eMRT: mean residence time\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSC: subcutaneous\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u0026nbsp;\u003c/sup\u003eIM: intramuscular\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation of PK of commercial bupivacaine MVLs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBesides the observation that various experimental conditions led to different PK of bupivacaine MVLs, variability in PKs was also found within the same experimental conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFour PK experiments were conducted with commercially available bupivacaine MVL products in SD rats from 2018 to 2021, with the same dose of 15 mg/kg administrated subcutaneously. Interestingly, even though the plasma profiles shared similar characteristics such as multiphases with multi-peaks, an inter-experimental variability was found nonnegligible in some of the PK parameters among the conducted PK experiments (\u003cstrong\u003eTable 3\u003c/strong\u003e). To be specific, the value of AUC ranged from 2000 to 6000 ng/mL*h, and C\u003csub\u003emax\u0026nbsp;\u003c/sub\u003eranged from 100 to 350 ng/mL, showing a 3-fold difference. T\u003csub\u003emax\u003c/sub\u003e was also varied, which might have been due to the presence of multiple plasma peaks in their profiles. Furthermore, as discussed above, the different conditions such as doses, dilution, and administration routes exerted different impacts on the PK of bupivacaine MVLs in our study (\u003cstrong\u003eFig. 2\u003c/strong\u003e, \u003cstrong\u003eTable 2\u003c/strong\u003e). However, such impacts were not indicated in the previous preclinical data with bupivacaine MVLs, and similar PK profiles were shown among different experimental settings \u003cspan class=\"MsoPlaceholderText\"\u003e[28]\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis observed variability is important because the discrepancy between our study and the previous data supports concerns around inter-experimental variability. \u0026nbsp;For example, further intra-experimental variability was also observed in other results. Specifically, C\u003csub\u003emax\u003c/sub\u003e exhibited a coefficient of variance (CV) over 40% and even up to 70%, and the difference between some of the PK parameters (e.g., Cmax and AUC) was up to 2- to 3-fold (\u003cstrong\u003eTable 2\u003c/strong\u003e, \u003cstrong\u003eTable 3\u003c/strong\u003e). This intra-experimental variability was also shown in the previous preclinical data with bupivacaine MVLs in rats \u003cspan class=\"MsoPlaceholderText\"\u003e[28]\u003c/span\u003e, with the CV of C\u003csub\u003emax\u0026nbsp;\u003c/sub\u003ealso up to 40% to 70% \u003cspan class=\"MsoPlaceholderText\"\u003e[28]\u003c/span\u003e. Furthermore, though not usually discussed, intra-experimental variability was also found relatively high (i.e., CV close to or over 20%) in the preclinical PK of various MVL formulations \u003cspan class=\"MsoPlaceholderText\"\u003e[29\u0026ndash;32]\u003c/span\u003e. The acceptance for an inter- and intra-assay CV usually falls in the range of 10 to 15%, and the general recommendation of the CV for a preclinical study is usually down to 10% \u003cspan class=\"MsoPlaceholderText\"\u003e[33,34]\u003c/span\u003e. As the variability are often found with the MVL\u0026rsquo;s PK, acceptable range for %CV may need to rely on totality of evidence with justification from other supporting data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA variety of factors involved in \u003cem\u003ein vivo\u003c/em\u003e drug exposure have been investigated with LAIs around aspects of 1) formulation properties, 2) administration site properties, and 3) the host response \u003cspan class=\"MsoPlaceholderText\"\u003e[19,23,35]\u003c/span\u003e. These aspects could also contribute to the PK variability in bupivacaine MVLs. Formulation dose strength is a major factor because dose determines injection volume, drug strength, and other factors and therefore impacts the drug dissolution and absorption accordingly. In the present study, with a same dose of bupivacaine administrated (e.g., 15 mg/kg), the injection volume was different among rats because of the different weight of each animal. A greater injection volume leading to particle aggregation and resulting in slower absorption has been observed in some LAIs \u003cspan class=\"MsoPlaceholderText\"\u003e[36,37]\u003c/span\u003e. The injection volume issue could also be the case with bupivacaine MVLs leading to different absorption of bupivacaine as the injection volume varied among individual rats. Even after the injection, the local drug movement was hard to control and potentially different among each injection, resulting in a different drug depot every time. Other injection techniques such as depth, site, speed, and pressure of injection can further affect the size and formation of drug depots, and thus the drug dissolution and absorption \u003cspan class=\"MsoPlaceholderText\"\u003e[23,24,38,39]\u003c/span\u003e. Batch-to-batch variability in the commercial products may also play a role and contribute to different formulation properties, causing PK variability accordingly. The physiological environment at administration site and how the matrix at the injection site interacts with formulations may be impactful in the case of bupivacaine MVLs since the MVLs are expected to stay in the local biological tissue (e.g., subcutaneous site) for a long time [40]. Finally, a local immune response initiated in the subcutaneous area after the bupivacaine MVLs injection can be another factor \u003cspan class=\"MsoPlaceholderText\"\u003e[19]\u003c/span\u003e. Injection site reactions were observed histologically with LAI suspensions in the intramuscular tissue, showing the formation of a dense inflammatory envelope which was considered to preclude drug dissolution and absorption \u003cspan class=\"MsoPlaceholderText\"\u003e[41]\u003c/span\u003e. As the host response may vary subject to subject and injection to injection, the discrepancy with the following inflammatory reactions could further impact the drug exposure and result in a PK variability.\u003c/p\u003e\n\u003cp\u003eAs discussed above, a variability in animal PK may be quite common with LAIs and multiple MVL formulations. Nevertheless, the observation of a variability with bupivacaine MVLs in our study, as well as the insufficient available comparable data, should be taken into account in future study designs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003ePharmacokinetic parameters of Exparel following SC administration at a dose of 15 mg/kg in SD rats (n=4, mean \u0026plusmn; SD) (Software: WinNonlin)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"97%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003cp\u003e(n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eDate of test\u003c/p\u003e\n \u003cp\u003e(M/D/Y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAUC\u003csub\u003e0\u0026rarr;96h\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL*h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eMRT\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 13px;\"\u003e\n \u003cp\u003eExparel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e09/24/18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e351.03 \u0026plusmn; 138.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e39.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e4515.24 \u0026plusmn; 1776.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e39.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e18.44 \u0026plusmn; 1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e05/29/19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e104.27 \u0026plusmn; 42.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e40.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e1724.41 \u0026plusmn; 142.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e8.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e19.54 \u0026plusmn; 2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e03/15/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e230.06 \u0026plusmn; 172.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e74.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e2655.94 \u0026plusmn; 571.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e21.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e16.56 \u0026plusmn; 3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e12/13/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e183.90 \u0026plusmn; 31.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e17.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e5768.28 \u0026plusmn; 554.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e9.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e20.49 \u0026plusmn; 8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eAUC: area under the curve\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eMRT: mean residence time\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003echaracteristics between bupivacaine MVLs with different product qualities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral bupivacaine MVLs with different product qualities were compared in terms of their \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003echaracteristics, to investigate how the performances of MVL products can be impacted by their product attributes. Bupivacaine MVLs with compromised product quality were deliberately generated with commercial product (i.e., Exparel) in our lab based on some of the major issues considered in product manufacturing and distribution process: 1) freeze-thawing; 2) mechanical agitation; 3) elevated temperature; and 4) expiration. Compared to the intact product, all the compromised formulations showed differences in their morphology (\u003cstrong\u003eFig. 4\u003c/strong\u003e). Overall, freeze-thawing and high temperature led to the most prominent changes in the product qualities. Freeze-thawed bupivacaine MVLs not only lost the \u0026ldquo;honeycomb\u0026rdquo; structure, characterized with a much smaller particle size distribution, but also was the only formulation whose pH (i.e., 5.5) dropped outside the reported pH range of commercial product (i.e., 5.8 - 7.4) (Table 4). Much higher free bupivacaine content was also found after freeze-thawing, indicating a particle breakage following an inner phase leakage (\u003cstrong\u003eTable 4\u003c/strong\u003e). High temperature, on the other hand, led to a much larger particle size distribution (\u003cstrong\u003eTable 4\u003c/strong\u003e), which might be attributed to particle aggregation as observed in \u003cstrong\u003eFig. 4d\u003c/strong\u003e. Particle size or formulation pH did not significantly change for bupivacaine MVLs being mechanically agitated or expired (\u003cstrong\u003eTable 4\u003c/strong\u003e). However, unusual morphology such as broken outer layers and particle fusion was observed in the images taken of samples for these two compromised formulations (\u003cstrong\u003eFig. 4\u003c/strong\u003e), suggesting some extents of particle destruction. A higher free drug content found after mechanical agitation further suggests particle breakage occurred in this group (\u003cstrong\u003eTable 4\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the compromised formulations (i.e., freeze-thawed, mechanical agitated, and high-temperature incubated) except the expired one showed a different (i.e., faster) \u003cem\u003ein vitro\u003c/em\u003e release (IVR) from the intact formulation, when estimated via an accelerated rotator-based IVRT method developed previously in our lab [13] (\u003cstrong\u003eFig. 5\u003c/strong\u003e). With a comparison to their \u003cem\u003ein vivo\u003c/em\u003e PK, both the freeze-thawed and mechanical agitated groups also performed differently from the intact formulation, implying an association between their \u003cem\u003ein vitro\u003c/em\u003e characteristics and \u003cem\u003ein vivo\u003c/em\u003e behaviors (\u003cstrong\u003eFig. 5\u003c/strong\u003e and \u003cstrong\u003eFig. 6\u003c/strong\u003e). The freeze-thawed bupivacaine MVLs even lost the multiphasic PK characteristics completely, displaying a plasma profile similar to that of the free bupivacaine solution (\u003cstrong\u003eFig. 1\u003c/strong\u003e and \u003cstrong\u003eFig. 6\u003c/strong\u003e). Supported by the observation of complete broken particles in freeze-thawed group, this failing in observing a multiphasic PK or a second plasma peak may be linked to a disruption or lose of nonconcentric MVL structure in the formulation (\u003cstrong\u003eFig. 4\u003c/strong\u003e and \u003cstrong\u003eFig. 6\u003c/strong\u003e). Interestingly, while the expired bupivacaine MVL product exhibited a different PK from the intact product, the expired product\u0026rsquo;s IVR was similar to the unexpired group (\u003cstrong\u003eFig. 5\u003c/strong\u003e and \u003cstrong\u003eFig. 6\u003c/strong\u003e). On the other hand, while the high-temperature incubated bupivacaine MVLs exhibited a different (i.e., faster) IVR, the high temperature treated product\u0026rsquo;s PK was similar to the intact group (\u003cstrong\u003eFig. 5\u003c/strong\u003e and \u003cstrong\u003eFig. 6\u003c/strong\u003e). As both the expired and high-temperature incubated groups showed some particle aggregation and/or fusion in their morphology (\u003cstrong\u003eFig. 4\u003c/strong\u003e), this discrepancy found in their \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e features might suggest a more complicated interplay of particle aggregation on the drug release and absorption process of MVL formulation. Furthermore, because the IVRT method applied here was an accelerated testing method originally aiming for a quick quality check instead of presenting bio-relevance [13], some physiological features might not be captured with the \u003cem\u003ein vitro\u003c/em\u003e settings, attributing to this discrepancy. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNevertheless, with a comprehensive analysis of and comparison between multiple \u003cem\u003ein vitro\u003c/em\u003e characteristics (i.e., morphology, particle sizes, formulation pH, free drug contents, and IVR), an association between bupivacaine MVLs with different product qualities to their \u003cem\u003ein vivo\u003c/em\u003e performances was observed. This association implies the potential of predicting \u003cem\u003ein vivo\u003c/em\u003e performances by \u003cem\u003ein vitro\u003c/em\u003e analytical formulation characterization of multiple product attributes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eSize distribution, pH value, and free drug contents of intact, expired, and compromised Exparel. All values are presented as Mean \u0026plusmn; SD (n=3)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003ed0.1 (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003ed0.5 (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003ed0.9 (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eFree drug %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eIntact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e13.2 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e26.0\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e53.5 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e5.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eFreeze-thawed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e3.1 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e13.0 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e30.6 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e80.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eHigh-temp incubated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e16.6 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e49.7 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e95.1 \u0026plusmn; 5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e12.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003eMechanical agitated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e13.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e25.7\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e50.6 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e28.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eExpired\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e13.5 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e26.5\u0026nbsp;\u0026plusmn;\u0026nbsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e54.0 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e5.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eIntact Exparel batch# 20-P097, with the expiration in July 2022\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eExpired Exparel batch# 17-4046, with the expiration in April 2018\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Pharmacokinetic parameters of intact, expired, and compromised Exparel following SC administration at a dose of 15 mg/kg in SD rats (n=5, mean \u0026plusmn; SD) (Software: WinNonlin.)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003cp\u003e(n=5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003eConditions\u003c/p\u003e\n \u003cp\u003e(dose/route/dilution)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAUC\u003csub\u003e0\u003c/sub\u003e\u003csub\u003e\u0026rarr;\u003c/sub\u003e\u003csub\u003e96h\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(ng/mL*h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eMRT\u003c/p\u003e\n \u003cp\u003e(h)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eIntact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 19px;\"\u003e\n \u003cp\u003e15 mg/kg\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ec\u003c/sup\u003eSC\u003c/p\u003e\n \u003cp\u003eundiluted\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e183.90 \u0026plusmn; 31.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e5768.28 \u0026plusmn; 554.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e20.49 \u0026plusmn; 8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eFreeze-thawed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2333.65 \u0026plusmn; 450.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e6181.13 \u0026plusmn; 781.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e3.96 \u0026plusmn; 1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eHigh-temp incubated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e246.86 \u0026plusmn; 62.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e4726.05 \u0026plusmn; 1383.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e14.42 \u0026plusmn; 2.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eMechanical agitated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e710.22 \u0026plusmn; 94.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1-12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e13169.13 \u0026plusmn; 2665.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e17.96 \u0026plusmn; 2.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eExpired\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1528.45 \u0026plusmn; 484.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e12370.41 \u0026plusmn; 4046.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e11.81 \u0026plusmn; 1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eAUC: area under the curve\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eMRT: mean residence time\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSC: subcutaneous\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePreclinical data on bupivacaine MVLs is supportive for product class understanding and complements our knowledge on the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e relationship of MVL based formulation [32,42]. In the present study, the PK of bupivacaine MVLs was investigated in SD rats, and compared under different experimental conditions (i.e., doses, administration routes, and dilution factors). A characteristic multiphasic PK with a second plasma peak was observed with the bupivacaine MVL formulations. Different experimental conditions exhibited impacts on not only the plasma profiles influencing the second peak, but also some of the key parameters such as C\u003csub\u003emax\u003c/sub\u003e and AUC, which was different from the previously reported preclinical results with bupivacaine MVLs [28]. \u0026shy;Notably, both an inter- and intra-experimental variability was found to be nonnegligible in bupivacaine MVLs\u0026rsquo; PK, calling for attention to MVL based sample handling and preparation in \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand/or \u003cem\u003ein vivo\u003c/em\u003e studies for product development and bioequivalence. A comprehensive analysis of and comparison between bupivacaine MVLs with different product attributes in aspects of their morphology, particle sizes, formulation pH, free drug, IVR, and PK profiles was conducted. An association between the formulation characteristics and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003ebehaviors was identified in compromised bupivacaine MVL products in this study, suggesting a potential \u003cem\u003ein vitro in vivo\u003c/em\u003e relationship. Overall, the present work investigating the \u003cem\u003ein vivo\u003c/em\u003e behaviors of bupivacaine MVLs is beneficial not only to fill in the gap of preclinical studies in the field of bupivacaine MVLs, but also to help pave the path for developing future MVL-related products.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was funded in part by FDA 75F40120C00127. Minzhi Yu was supported in part by AHA postdoctoral fellowship 24POST1196020. Anna Schwendeman was supported in part by Duellman Graduate Student Research Fund. This author also wants to acknowledge the animal work contributed by Yayuan Liu and Karl Olsen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Broad Agency Announcement (BAA) Contract # 75F40120C00127 from the U.S. Food and Drug Administration (FDA). The content reflects the views of the authors and should not be construed to represent the views or policies of the U.S. FDA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinancial interests/personal relationships: Author Anna Schwendeman reports financial support was provided by US Food and Drug Administration. Author Anna Schwendeman reports a relationship with EVOQ Therapeutics that includes: board membership, consulting or advisory, equity or stocks, and funding grants. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conceptualization. Material preparation, data curation, and formal analysis were performed by Ziyun Xia, Yayuan Liu, Jingyao Gan, Ziyi Lu, and Karl Olsen. The original draft of the manuscript was written by Ziyun Xia. Previous versions of the manuscript were reviewed and edited by Minzhi Yu, Yan Wang, Xiaoming Xu, and Anna Schwendeman. Study supervision and funding acquisition were provided by Anna Schwendeman and Steve Schwendeman. All authors read and approved the final manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the Guide for the Care and Use of Laboratory Animals (Guide), NRC 2011. Approval was granted by the Institutional Animal Care \u0026amp; Use Committee (IACUC) of the University of Michigan (most recent AAALAC accreditation date: November 27, 2023/Unit number: 000285).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eExparel [package insert] Pacira Pharmaceuticals. 2018. \u003c/li\u003e\n\u003cli\u003eYe Q, Asherman J, Stevenson M, Brownson E, Katre N V. DepoFoam(TM) technology: A vehicle for controlled delivery of protein and peptide drugs. Journal of Controlled Release. Elsevier; 2000. p. 155\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eMantripragada S. A lipid based depot (DepoFoam\u0026reg; technology) for sustained release drug delivery. Prog Lipid Res. 2002;41:392\u0026ndash;406. \u003c/li\u003e\n\u003cli\u003eKim S, Turker MS, Chi EY, Sela S, Martin GM. Preparation of multivesicular liposomes. BBA - Biomembranes. 1983;728:339\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003ePacira Pharmaceuticals, Inc. Announces U.S. FDA Approval of Exparel For Postsurgical Pain Management [Internet]. [cited 2021 Apr 14]. Available from: https://www.drugs.com/newdrugs/pacira-pharmaceuticals-inc-announces-u-s-fda-approval-exparel-postsurgical-pain-management-2938.html\u003c/li\u003e\n\u003cli\u003eFDA In Brief: FDA approves new use of Exparel for nerve block pain relief following shoulder surgeries | FDA [Internet]. [cited 2021 Feb 1]. Available from: https://www.fda.gov/news-events/fda-brief/fda-brief-fda-approves-new-use-exparel-nerve-block-pain-relief-following-shoulder-surgeries\u003c/li\u003e\n\u003cli\u003eTampa D, Newswire G, Biosciences P, Therapeutics F. Pacira BioSciences Reports Preliminary Unaudited Total Revenue for 2022 of $ 666 . 8 Million [Internet]. 2023. Available from: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://investor.pacira.com/node/16036/pdf\u003c/li\u003e\n\u003cli\u003eOrange Book: Approved Drug Products with Therapeutic Equivalence Evaluations [Internet]. [cited 2023 Jan 8]. Available from: https://www.accessdata.fda.gov/scripts/cder/ob/patent_info.cfm?Product_No=001\u0026amp;Appl_No=022496\u0026amp;Appl_type=N\u003c/li\u003e\n\u003cli\u003ePacira BioSciences Notified of Abbreviated New Drug Application Filing for EXPAREL\u0026reg; | Pacira BioSciences, Inc. [Internet]. [cited 2023 Feb 2]. Available from: https://investor.pacira.com/news-releases/news-release-details/pacira-biosciences-notified-abbreviated-new-drug-application\u003c/li\u003e\n\u003cli\u003eU.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Liposome Drug Products: Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation [Internet]. Available from: https://www.fda.gov/media/70837/download\u003c/li\u003e\n\u003cli\u003eU.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Draft Guidance on Bupivacaine Liposomal Injection [Internet]. 2018. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070570.pdf\u003c/li\u003e\n\u003cli\u003eYu M, Yuan W, Xia Z, Liu Y, Wang Y, Xu X, et al. Characterization of exparel bupivacaine multivesicular liposomes. Int J Pharm. 2023;639:122952. \u003c/li\u003e\n\u003cli\u003eXia Z, Yu M, Liu Y, Yuan W, Wang Y, Xu X, et al. Development of an Accelerated Rotator-based Drug Release Method for the Evaluation of Bupivacaine Multivesicular Liposomes. Pharm Res. 2024; \u003c/li\u003e\n\u003cli\u003eManna S, Wu Y, Wang Y, Koo B, Chen L, Petrochenko P, et al. Probing the mechanism of bupivacaine drug release from multivesicular liposomes. Journal of Controlled Release. 2019;294:279\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eChaurasiya A, Gorajiya A, Panchal K, Katke S, Singh AK. A review on multivesicular liposomes for pharmaceutical applications: preparation, characterization, and translational challenges. Drug Deliv Transl Res. 2022;12:1569\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eMantripragada B.Sankaram, Sinil Kim. Preparation of Multivesicular Liposomes for Controlled Release of Encapsulated Biologically Active Substances. 1999. \u003c/li\u003e\n\u003cli\u003eSankaram MB, Kim S. Multivesicular liposomes with controlled release of encapsulated biologically active substances. 1998. \u003c/li\u003e\n\u003cli\u003eSmith WC, Bae J, Zhang Y, Qin B, Wang Y, Kozak D, et al. Impact of particle flocculation on the dissolution and bioavailability of injectable suspensions. Int J Pharm. 2021;604:120767. \u003c/li\u003e\n\u003cli\u003eNguyen VTT, Darville N, Vermeulen A. Pharmacokinetics of Long-Acting Aqueous Nano-/Microsuspensions After Intramuscular Administration in Different Animal Species and Humans\u0026mdash;a Review. AAPS J. 2022;25. \u003c/li\u003e\n\u003cli\u003eChamanza R, Darville N, van Heerden M, De Jonghe S. Comparison of the Local Tolerability to 5 Long-acting Drug Nanosuspensions with Different Stabilizing Excipients, Following a Single Intramuscular Administration in the Rat. Toxicol Pathol. 2018;46. \u003c/li\u003e\n\u003cli\u003eGautam N, Roy U, Balkundi S, Puligujja P, Guo D, Smith N, et al. Preclinical pharmacokinetics and tissue distribution of long-acting nanoformulated antiretroviral therapy. Antimicrob Agents Chemother. 2013;57:3110\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;aquino AI, Maikawa CL, Nguyen LT, Lu K, Hall IA, Jons CK, et al. Sustained Delivery of GLP-1 Receptor Agonists from Injectable Biomimetic Hydrogels Improves Treatment of Diabetes. 2023. \u003c/li\u003e\n\u003cli\u003eSiemons M, Schroyen B, Darville N, Goyal N. Role of Modeling and Simulation in Preclinical and Clinical Long-Acting Injectable Drug Development. AAPS J. 2023;25. \u003c/li\u003e\n\u003cli\u003eMcDowell A, Medlicott NJ. Anatomy and Physiology of the Injection Site: Implications for Extended Release Parenteral Systems. Long Acting Injections and Implants. 2012;57\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eBallard BE. Biopharmaceutical Considerations in Subcutaneous and Intramuscular Drug Administration. J Pharm Sci. 1968;57:357\u0026ndash;78. \u003c/li\u003e\n\u003cli\u003eSchwendeman SP, Shah RB, Bailey BA, Schwendeman AS. Injectable controlled release depots for large molecules. Journal of Controlled Release. 2014;190:240\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eShah JC, Maniar M. pH-Dependent solubility and dissolution of bupivacaine and its relevance to the formulation of a controlled release system. Journal of Controlled Release. 1993;23:261\u0026ndash;70. \u003c/li\u003e\n\u003cli\u003eHadzic A, Abikhaled JA, Harmon WJ. Impact of volume expansion on the efficacy and pharmacokinetics of liposome bupivacaine. Local Reg Anesth. 2015;8:105\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eShen Y, Ji Y, Xu S, Chen DQ, Tu J. Multivesicular liposome formulations for the sustained delivery of ropivacaine hydrochloride: Preparation, characterization, and pharmacokinetics. Drug Deliv. 2011;18:361\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eZhong H, Deng Y, Wang X, Yang B. Multivesicular liposome formulation for the sustained delivery of breviscapine. Int J Pharm. 2005;301:15\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eMu H, Wang Y, Chu Y, Jiang Y, Hua H, Chu L, et al. Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization. Drug Deliv. 2018;25:1372\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eAbuzar SM, Park EJ, Seo Y, Lee J, Baik SH, Hwang S-J. Preparation and Evaluation of Intraperitoneal Long-Acting Oxaliplatin-Loaded Multi-Vesicular Liposomal Depot for Colorectal Cancer Treatment. Pharmaceutics 2020, Vol 12, Page 736. 2020;12:736. \u003c/li\u003e\n\u003cli\u003eLittle TA. Establishing Acceptance Criteria for Analytical Methods. Biopharm Int. 2016 Oct 1; \u003c/li\u003e\n\u003cli\u003eU.S. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Bioequivalence Studies With Pharmacokinetic Endpoints for Drugs Submitted Under an Abbreviated New Drug Application [Internet]. 2021. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioequivalence-studies-pharmacokinetic-endpoints-drugs-submitted-under-abbreviated-new-drug\u003c/li\u003e\n\u003cli\u003eDaublain P, Feng KI, Altman MD, Martin I, Mukherjee S, Nofsinger R, et al. Analyzing the Potential Root Causes of Variability of Pharmacokinetics in Preclinical Species. Mol Pharm. 2017;14:1634\u0026ndash;45. \u003c/li\u003e\n\u003cli\u003eHirano K, Ichihashi T, Yamada H. Studies on the absorption of practically water-insoluble drugs following injection. III. Intramuscular absorption from aqueous nonionic surfactant solutions in rats. Chem Pharm Bull (Tokyo). 1981;29:834\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eJucker BM, Fuchs EJ, Lee S, Damian V, Galette P, Janiczek R, et al. Multiparametric magnetic resonance imaging to characterize cabotegravir long‐acting formulation depot kinetics in healthy adult volunteers. Br J Clin Pharmacol. 2022;88:1655. \u003c/li\u003e\n\u003cli\u003eDarville N, van Heerden M, Vynckier A, De Meulder M, Sterkens P, Annaert P, et al. Intramuscular Administration of Paliperidone Palmitate Extended-Release Injectable Microsuspension Induces a Subclinical Inflammatory Reaction Modulating the Pharmacokinetics in Rats. J Pharm Sci. 2014;103:2072\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eSchou J. Subcutaneous and Intramuscular Injection of Drugs. In: Brodie BB, Gillette JR, Ackerman HS, editors. Concepts in Biochemical Pharmacology: Part 1. Berlin, Heidelberg: Springer Berlin Heidelberg; 1971. p. 47\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eKinnunen HM, Mrsny RJ. Improving the outcomes of biopharmaceutical delivery via the subcutaneous route by understanding the chemical, physical and physiological properties of the subcutaneous injection site. Journal of Controlled Release. 2014;182:22\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eGroseclose MR, Castellino S. Intramuscular and subcutaneous drug depot characterization of a long-acting cabotegravir nanoformulation by MALDI IMS. Int J Mass Spectrom. 2019;437:92\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eAlavi S, Mahjoob MA, Haeri A, Shirazi FH, Abbasian Z, Dadashzadeh S. Multivesicular liposomal depot system for sustained delivery of risperidone: development, characterization, and toxicity assessment. Drug Dev Ind Pharm. 2021;47:1290\u0026ndash;301. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Multivesicular liposomes, pharmacokinetics, in vivo preclinical study, liposomes, bupivacaine","lastPublishedDoi":"10.21203/rs.3.rs-5214719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5214719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA bupivacaine multivesicular liposomal injectable formulation, Exparel\u003csup\u003eTM\u003c/sup\u003e, is a nonopioid long-acting local analgesic indicated for pain management across and/or post surgeries. For such products, preclinical data is lacking to support bioequivalence determination for potential generic products. Therefore, in the present work, \u003cem\u003ein vivo\u003c/em\u003e studies were set up in male Sprague-Dawley rats to understand the \u003cem\u003ein vivo\u003c/em\u003e performance of bupivacaine multivesicular liposomes (MVLs), aiming to provide information on bioequivalence establishment between comparator products. Bupivacaine MVLs show a multiphasic release profile, and their pharmacokinetics (PK) may differ with different experimental conditions including doses, administration routes, and sample dilution factors. In this work, compromised bupivacaine MVLs were either generated in lab by freeze-thawing, mechanical agitation, and high-temperature incubation, or chosen from years-old expired batches of Exparel\u003csup\u003eTM\u003c/sup\u003e, for a preliminary investigation on the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e association. The formulation attributes of different bupivacaine MVLs were characterized, including morphology, particle size distribution, formulation pH, free drug contents, \u003cem\u003ein vitro\u003c/em\u003e release, and \u003cem\u003ein vivo\u003c/em\u003e PK. In the rat study, even with an observation of inter- and intra-variability in PK, an association between product attributes and \u003cem\u003ein vivo\u003c/em\u003e behaviors was demonstrated with bupivacaine MVLs. Overall, investigating the bupivacaine MVLs \u003cem\u003ein vivo\u003c/em\u003e is beneficial not only to fill in gaps in preclinical data in the field of bupivacaine MVLs, but also to help pave the path for developing other MVL-related products.\u003c/p\u003e","manuscriptTitle":"An investigation of in vivo performance of bupivacaine multivesicular liposomes in rats and the impacts from product qualities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 00:51:18","doi":"10.21203/rs.3.rs-5214719/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-01-19T03:29:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-12-18T20:09:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-18T06:55:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-16T10:04:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2024-12-13T13:44:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9768972c-7c03-40e6-9b0e-92478ea08dbe","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T19:56:17+00:00","versionOfRecord":{"articleIdentity":"rs-5214719","link":"https://doi.org/10.1007/s13346-025-01806-y","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2025-03-04 15:57:09","publishedOnDateReadable":"March 4th, 2025"},"versionCreatedAt":"2024-12-20 00:51:18","video":"","vorDoi":"10.1007/s13346-025-01806-y","vorDoiUrl":"https://doi.org/10.1007/s13346-025-01806-y","workflowStages":[]},"version":"v1","identity":"rs-5214719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5214719","identity":"rs-5214719","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}