thesafetyofexparel (bupivacaineliposome ...2.1.2. description of depofoam bupivacaine. depofoam...

Hindawi Publishing CorporationJournal of Drug DeliveryVolume 2012, Article ID 962101, 10 pagesdoi:10.1155/2012/962101

Research Article

The Safety of EXPAREL � (Bupivacaine LiposomeInjectable Suspension) Administered by Peripheral NerveBlock in Rabbits and Dogs

Brigitte M. Richard,1 Paul Newton,2 Laura R. Ott,2 Dean Haan,2 Abram N. Brubaker,2

Phaedra I. Cole,2 Paul E. Ross,2 Marlon C. Rebelatto,2 and Keith G. Nelson2

1 Clinical Research and Drug Safety Assessment, Pacira Pharmaceuticals, Inc., 10450 Science Center Drive, San Diego,CA 92121, USA

2 MPI Research Laboratories, North Main Street, Mattawan, MI 49071, USA

Correspondence should be addressed to Brigitte M. Richard, [email protected]

Received 20 July 2011; Accepted 4 October 2011

Academic Editor: Guru V. Betageri

Copyright © 2012 Brigitte M. Richard et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

A sustained-release DepoFoam injection formulation of bupivacaine (EXPAREL, 15 mg/mL) is currently being investigated forpostsurgical analgesia via peripheral nerve block (PNB). Single-dose toxicology studies of EXPAREL (9, 18, and 30 mg/kg),bupivacaine solution (Bsol, 9 mg/kg), and saline injected around the brachial plexus nerve bundle were performed in rabbitsand dogs. The endpoints included clinical pathology, pharmacokinetics, and histopathology evaluation on Day 3 and Day 15(2/sex/group/period). EXPAREL resulted in a nearly 4-fold lower Cmax versus Bsol at the same dose. EXPAREL was well toleratedat doses up to 30 mg/kg. The only EXPAREL-related effect seen was minimal to mild granulomatous inflammation of adiposetissue around nerve roots (8 of 24 rabbits and 7 of 24 dogs) in the brachial plexus sites. The results indicate that EXPAREL was welltolerated in these models and did not produce nerve damage after PNB in rabbits and dogs.

1. Introduction

Bupivacaine is a local anesthetic/analgesic widely used inthe perioperative and postsurgical settings. Bupivacaine so-lutions have been used for many years by multiple routesfor the relief of postoperative pain [1]. With technology nowallowing for directly visualizing a peripheral nerve prior toinjection, perineural nerve block, including brachial plexusnerve block, has become increasingly popular.

The brachial plexus is a large, complex bundle of nerves(arising from the nerve roots C5-T1). A single injection oflocal anesthetic around the brachial plexus nerve bundleresults in block of arm tissue innervated by several peripheralnerves. Several approaches to the brachial plexus blockadehave been described (i.e., the axillary, infraclavicular, supr-aclavicular, and interscalene) and each have advantages incertain situations. Stabilization of the needle for catheterinsertion after brachial plexus blockade is localized and is achallenging aspect of this technique [2].

Brachial plexus blockade may require dispersion of a rel-atively large volume of bupivacaine in solution to accomplishblockade of the entire plexus. Complications may includeinfection, hematoma, vascular puncture, toxicity, injury,and total spinal anesthesia [3]. After performing the blockprocedure, peripheral nerves may be damaged from pro-longed contact with concentrated formulations [4, 5]. Froma systemic standpoint, high doses of bupivacaine may beassociated with a wide range of systemic complications, suchas central nervous and cardiovascular effects [6].

A formulation of bupivacaine that provides prolongedrelease of the active ingredient after a single administrationwould simplify pain management in the postoperative periodand eliminate the undesired peak plasma concentrations as aresult of excessively high concentrations and reduce the riskof local and systemic reactions [7].

EXPAREL (bupivacaine liposome injectable suspension)is a sterile suspension of multivesicular liposomes using

2 Journal of Drug Delivery

proprietary DepoFoam formulation technology to releasebupivacaine over several days. EXPAREL, the proposed pro-prietary name, was designed to provide prolonged analgesiafor 72 hours after wound infiltration in patients [8].

Although different liposomal formulations have beenadministered to humans without toxicity [9], the in vivotolerability of liposomes continues to be investigated. Ourgoal was to evaluate the potential local and systemic toxicityof EXPAREL after a bolus injection into the brachial plexus(i.e., a large, complex bundle of nerves in the shoulder).Specifically, the study was designed to assess whetherEXPAREL did not produce nerve damage in the setting ofperipheral nerve block by comparison with unencapsulatedbupivacaine or saline control.

2. Materials and Methods

2.1. Materials

2.1.1. Description of DepoFoam Technology. The DepoFoamdrug-delivery system is a proprietary, injectable technologythat provides a sustained release of therapeutic compounds.The DepoFoam system consists of multivesicular liposomescomposed of hundreds to thousands of chambers perparticle, resembling a “honeycomb-like” matrix [10].

Such particles are to be distinguished from multilamellarvesicles, also known as a multilamellar liposome, which con-tain multiple concentric membranes within each liposomeparticle [11, 12].

The DepoFoam particle components are naturally occur-ring or synthetic analogues of common lipids, includingphospholipids (e.g., dierucoylphosphatidylcholine and di-palmitoylphosphatidylglycerol), cholesterol, and triglyc-erides (e.g., triolein and tricaprylin). The particles typicallyconsist of >97% water (with dissolved drug) and 1% to3% lipids, and are expected to be fully biodegradable. TheDepoFoam particles are typically suspended in isotonicsolutions containing sodium chloride 0.9% in water forinjection. The DepoFoam drug-delivery system is alreadyused in two marketed products, DepoDur and DepoCyt,which are produced by Pacira Pharmaceuticals, Inc.

2.1.2. Description of DepoFoam Bupivacaine. DepoFoam bu-pivacaine (bupivacaine liposome injectable suspension),was supplied by Pacira Pharmaceuticals, Inc., San Diego,California, USA. This formulation was previously designatedSKY0402.

The manufacture of DepoFoam particles has been pre-viously described [12]. Briefly, the process involves a doubleemulsification process where the bupivacaine is added as partof the initial emulsification process. The amount of unencap-sulated bupivacaine is controlled as part of the process and isgenerally less than 10%. In DepoFoam Bupivacaine, one ofthe specific lipids in the final formulation is dierucoylphos-phatidylcholine, EXPAREL was initially formulated at twodifferent dose concentrations (15 and 25 mg/mL in 0.9%saline, expressed as anhydrous bupivacaine HCl equivalent).The 15-mg/mL formulation is intended for commercial use.The 15 mg/mL of bupivacaine is the bupivacaine salt HCl;

it is chemically equivalent to 13.3 mg/mL bupivacaine freebase. The 25-mg/mL formulation is a concentrated versionand was intended to increase exposure of local tissues torelatively higher concentrations of both the active drug andDepoFoam matrix.

2.1.3. Reference Product. Sensorcaine-MPF (methyl parabenfree; bupivacaine HCl injection, USP) 0.75% bupivacainesolution, is manufactured by AstraZeneca, Wilmington,Delaware, USA.

2.1.4. Control Article. Saline (0.9% sodium chloride injec-tion, USP) is manufactured by Abbott Laboratories, NorthChicago, lllinois, USA.

2.1.5. Animals. New Zealand White rabbits and beagle dogswere ordered from Covance Research Products, Philadelphia,Pennsylvania, and Kalamazoo, Michigan, USA, respectively.The animals were 5 and half months (rabbit) and 5 to 6months (dog) of age on arrival.

A total of 40 rabbits (20 males and 20 females) weighing2.6 to 3.7 kg and 40 dogs (20 males and 20 females) weighing6.2 to 9.7 kg, were used. Individual body weights were within20% of the mean body weight for each gender.

2.2. Methods

2.2.1. Study Protocols. These pivotal studies were conductedaccording to International Conference on Harmonisationguidelines in accordance with Good Laboratory Practicesprinciples [13] as set forth by the United States Food andDrug Administration 21 CFR Part 58 and in accordancewith single-dose acute toxicity testing for pharmaceuticals[14]. All protocols were reviewed and approved by theInstitutional Animal Care and Use Committee of MPIResearch for compliance with regulations prior to studyinitiation.

The brachial nerve block procedure was performed in anMPI Research surgical suite using aseptic technique. Generalanesthesia was induced and maintained with isoflurane toeffect delivered in oxygen through a facemask. Each dosewas administered by nerve block once on Day 1. A 22 g3.5 inch needle was used to perform a brachial plexusnerve block in the left thoracic limb [15]. After placementthe needle was slowly withdrawn while each treatmentwas injected. The animals were closely monitored duringanesthetic recovery for physiological disturbances includ-ing cardiovascular/respiratory depression, hypothermia, andexcessive bleeding from the injection site.

Using a standard, by weight, block randomization proce-dure, 20 males and 20 females were assigned to five groupsin each study. Each group consisted of 4 males and 4 females.Groups 1, 2, and 3 received EXPAREL 9, 18, and 25 mg/kg(or 30 mg/kg), respectively. Group 4 received bupivacainesolution 9 mg/kg, Group 5 received saline.

In rabbits, EXPAREL was administered at a dose volumeof 0.6, 1.2, and 1.2 mL/kg, respectively. In dogs, EXPARELwas administered at a dose volume of 0.6, 1.2, and 1.0 mL/kg,

Journal of Drug Delivery 3

respectively. (The dogs intended for the 30 mg/kg groupwere erroneously administered only 1.0 mL/kg (25 mg/mL)resulting in a dose level of 25 mg/kg.) Bupivacaine solutionand saline control was administered at a dose volume of1.2 mL/kg in each species.

Following dose administration, animals were observedwithout further treatment. Four animals (2 males and 2females) in each group were sacrificed on Day 3, and theremainder (designated as recovery animals, 2 males and 2females) were observed for an additional two weeks until Day15 sacrifice.

Endpoints included physical examinations, clinical signs,clinical pathology, hematology, organ weight, and histopa-thology of a standard list of tissues (including injection sites)on Day 3 and Day 15, as well as pharmacokinetics (PK) onDay 1 through 96 hours postdosing.

2.2.2. Pharmacokinetic Data Analysis. Plasma bupivacaineconcentrations were measured using a validated LC/MS/MSassay in rabbit and dog K3EDTA plasma in the concen-trations ranging from 10.0 to 10,000 ng/mL. The lowerquantitation limit was 10 ng/mL.

The PK parameters were evaluated by a noncompart-mental model using WinNonlin, version 5.0 (PharsightCorp., Mountain View, California). The PK parameterswere maximum plasma concentration (Cmax), time atwhich the Cmax occurred (tmax), and area under the plasmaconcentration-time data from time 0 to selected time point(t) (AUC0−t). The appropriate group mean values andstandard deviation were calculated from the individual data(combined sexes). Statistical results of pairwise comparisonsbetween EXPAREL and bupivacaine solution groups werereported at the 0.05 significance levels using Student’s two-tailed t-test.

2.2.3. Tissue Processing and Microscopic Evaluation. All ani-mals had a complete necropsy examination. Organ weightswere recorded for the following organs prior to fixation:adrenal glands, brain, heart, kidneys, liver, lungs (withbronchi), ovaries, spleen, testes, and thyroid. Paired organswere weighed together.

A selection of routine tissues (approximately 70) includ-ing gross lesions, injection sites and surgical wound tissueswere collected at necropsy from 2 males and 2 femalesper group sacrificed on Day 3 and remaining 2 malesand 2 females on Day 15 (recovery group). Tissues weretrimmed, embedded, sectioned, and hematoxylin- andeosin-stained using standard procedures. All pathology slideswere prepared by MPI Research Laboratories. The severityof histological findings was graded on a scale of one tofour with 0 = none, 1 = minimal, 2 = mild, 3 = moderate,and 4 = severe. All protocol-specified tissues were examined,and grading/interpretations of findings were made by apathologist certified by the American College of VeterinaryPathology.

The nerve plexus site was excised, and histopathologicalpreparations were prepared across the complete site. Nerveplexus sites examined microscopically at the three sampling

sites with as much nerve and connective tissue as possible(proximal, middle, and distal to the injection sites).

All changes in the skin and underlying muscle tissues andother organs were recorded.

Neurotoxicity was assessed primarily on a histopatholog-ical level using light microscopic evaluation of hematoxylin-and eosin-stained injection sites. Any neural changesobserved at the injection sites would typically be listed asseparate findings (such as degeneration or inflammation).

3. Results

3.1. Toxicology Results. In both rabbits and dogs, a single-dose administration of EXPAREL was well tolerated even at alarge dose and concentration (up to 30 mg/kg, 25 mg/mL).There were no discernable EXPAREL-related effects onhematology, clinical chemistry, or urinalysis parameters(data not shown). Few sporadic changes were noted attermination or recovery, but these effects were considerednot toxicologically relevant, may be the results of biologicalvariability, and were not considered treatment-related.

Microscopic findings at the brachial plexus sites in maleand female rabbits and dogs (combined sexes) are shown inTables 1 and 2. Microscopic findings on Day 3 consisted ofgranulomatous inflammation and hemorrhage; females alsohad minimal subacute inflammation. On Day 15, brachialplexus lesions included granulomatous inflammation andhemorrhage; females also had minimal fibrosis; males alsohad subacute inflammation and mineralization.

The only EXPAREL-related effect seen was minimal tomild granulomatous inflammation of adipose tissue aroundnerve roots (8 of 24 rabbits and 7 of 24 dogs) in brachialplexus sites. Granulomatous inflammation was present in4/12 rabbits on Day 3 or Day 15, and in only 1/12 dog(Day 3) and 6/12 dogs (Day 15). Apart from granulomatousinflammation observed at the injection sites, there was nooverall incidence or severity of lesions in the brachial plexusbetween animals receiving EXPAREL and the saline controlor Bupivacaine solution groups. All other microscopic find-ings were considered incidental and unrelated to EXPAREL.

This change was characterized by aggregates of macro-phages with abundant vacuolated cytoplasm (Figures 1(a)and 1(b)). With the low incidence and severity of theseeffects, this reaction was considered a normal response to theliposomes and not adverse. There was no other difference inthe incidence or severity of lesions between groups.

3.2. Pharmacokinetic Results. In rabbits, Cmax values weredose dependent, averaging 106± 67.9, 363 ± 478 and 205 ±60.4 ng/mL for the three EXPAREL dose levels (9, 18, and30 mg/kg, resp.) (Figure 2(a)). As a result of the relativelyflat nature of the concentration-time profile over the first 48hours, the mean time to maximum plasma concentration,tmax, varied considerably: 10.3±10.3, 20.0±20.1, and 36.5±23.0 hours for the three doses (Figure 2(b)). The AUC0–96 h

values determined for each of the three doses were 2700 ±781,5540 ± 2520, and 9370 ± 1170 ng·h/mL indicating doseproportionality.


Table 1: Injection site microscopic findings in rabbits (combined sexes).

Day 3 ObservationsSeverity

gradeSaline

EXP9 mg/kg

EXP18 mg/kg

EXP30 mg/kg

Bsol9 mg/kg

Brachial plexus,distal

Hemorrhageminimal 0 0 1 0 1

moderate 0 1 1 0 0

Inflammation,granulomatous

minimal 0 3 1 1 0

Inflammation, subacute minimal 0 0 0 0 1

Brachial plexus,middle


moderate 0 1 0 0 0


minimal 0 1 1 3 0


Brachial plexus,proximal


mild 0 1 0 0 0

moderate 0 1 0 0 0


minimal 0 0 1 1 0


Day 15



mild 0 0 1 1 0

moderate 0 0 0 1 0



Fibrosis minimal 0 0 0 0 1


mild 0 0 0 0 1


mild 0 0 1 0 0

Inflammation subacute minimal 0 1 0 0 0

Mineralization minimal 0 0 1 0 0



mild 0 1 1 1 0

moderate 0 0 0 0 1

EXP, EXPAREL (bupivacaine liposome injectable suspension); Bsol, bupivacaine solution.Note: number of animals examined was 4/group on Day 3 or Day 15.

(a) Rabbit (b) Dog

Figure 1: Injection site findings (Day 3) in a female rabbit (a) or dog (b) of the EXPAREL 18 mg/kg (a) and 25 mg/kg (b) showinggranulomatous inflammation of adipose tissue. H&E 20X.


Table 2: Injection site findings (Day 3 and Day 15) in dogs (combined sexes).

Day 3 ObservationsSeverity

gradeSaline

EXP9 mg/kg

EXP18 mg/kg

EXP25 mg/kg

Bsol9 mg/kg


Degeneration/regenerationmyofibers

mild 0 0 0 0 1

Hemorrhage, adipose tissuemild 0 0 0 1 0

minimal 0 3 1 3 1

Inflammation, granulomatous,adipose tissue

minimal 0 0 1 1 0



Degeneration/regenerationmyofiber

mild 0 0 0 0 1


mild 0 1 2 1 0

moderate 0 0 0 0 0

Inflammation, granulomatous minimal 0 1 0 0 0


Hemorrhage mild 0 1 2 1 0

Inflammation, adipose tissue,subacute

mild 0 0 0 1 0

Day 15


Degeneration/regenerationmyofibers

mild 0 0 0 0 1

Hemorrhage minimal 2 0 1 0 2


Cyst minimal 0 0 1 0 0

Fibrosis minimal 0 0 0 0 0


mild 0 0 0 0 1

Inflammation, granulomatous minimal 0 0 1 2 0


Degeneration/regeneratonmyofibers

minimal 0 0 1 0 1

Hemorrhage minimal 0 1 1 0 1

Inflammation, granulomatousminimal 0 0 1 1 0

mild 0 0 1 0 0

Refer to Table 1 footnote.

These results can be compared with the PK values foundfor the bupivacaine solution administered at the lowest dose,9 mg/kg. The plasma bupivacaine concentration peakedquickly and fell below the limit of detection by 48 hours. TheCmax, tmax, and AUC0–96 h were 433± 26.2 ng/mL, 2.25± 2.50hours and 1670± 249 ng·h/mL, respectively.

In dogs receiving bupivacaine solution (9 mg/kg), plasmabupivacaine concentrations peaked quickly (tmax of 1.00 ±0.00 hour, Cmax of 1490 ± 131 ng/mL) and declined rapidlythereafter (Figure 3(a)). Half-life was estimated to be 5.92 ±2.51 hours. The AUC0–96 h value was 6100± 1520 ng·h/mL.

Detectable plasma bupivacaine concentrations wereobserved in most animals with the EXPAREL formulation(9 mg/kg) over the entire 96-hour study period (Figure 3(b)).The bupivacaine plasma concentrations declined slowly overtime, and the Cmax values were dose dependent, averaging402 ± 513, 715 ± 747, and 1130 ± 604 ng/mL for the threedose levels (9, 18, and 25 mg/kg, resp.). The correspondingAUC0–96 h values were 7460 ± 1370, 18200 ± 8640, and

22600±13700 ng·h/mL. When comparing the results in bothspecies, the peak concentrations were reduced and elevatedplasma drug concentrations were maintained for longerperiods with EXPAREL compared to bupivacaine solutionat the same dose (9 mg/kg) in both species. Occasionaland expected differences in individual PK parameters werepresent although not observed across all groups. Particularly,due to the known variability in the absorption of bupiva-caine,Cmax was found to be higher in one of the three animalsreceiving the intermediate formulations (18 mg/kg). In thisdose group, the individual plasma Cmax values were 1080,162, 108, and 103 ng/mL achieved at 4, 48, 24, and 4 hoursand 1790, 648, 239, and 181 ng/mL at 1, 24, 24, and 2 hours,in rabbits and dogs, respectively.

The attenuation of Cmax with EXPAREL (9 mg/kg) was4.1 and 3.7 fold in both rabbits and dogs (combinedsexes), respectively. The difference was statistically significantcompared to bupivacaine solution at the same dose (P <0.05).


0

250

500

750

1000

0 6 12 18 24

Time (h)

Pla

sma

bupi

vaca

ine

(ng/

mL)

EXPAREL, 9 mg/kg

EXPAREL, 18 mg/kg

EXPAREL, 30 mg/kgBupivacaine HCl solution, 9 mg/kg

(a)

0

250

500

750

1000

0 24 48 72 96

Time (h)

Pla

sma

bupi

vaca

ine

(ng/

mL

)

EXPAREL, 9 mg/kg

EXPAREL, 18 mg/kg


(b)

Figure 2: Mean pharmacokinetic profile of EXPAREL in rabbits from 0–24 hours (a) and 0–96 hours (b).

0

500

1000

1500

0 6 12 18 24

Time (h)

Pla

sma

bupi

vaca

ine

(ng/

mL)

EXPAREL, 9 mg/kgEXPAREL, 18 mg/kg


(a)

0

500

1000

1500

0 24 48 72 96

Time (h)

Pla

sma

bupi

vaca

ine

(ng/

mL)

EXPAREL, 9 mg/kgEXPAREL, 18 mg/kg


(b)

Figure 3: Mean pharmacokinetic profile of EXPAREL in dogs from 0–24 hours (a) and 0–96 hours (b).

The AUC0–24 h was statistically significantly lower in dogswith EXPAREL compared to bupivacaine solution (2860 ±1400 versus 6020 ± 1380 ng·h/mL, 2-fold difference, P <0.05), while the corresponding values in rabbits were notsignificantly different (1230 ± 536 versus 1620 ± 288 ng·h/mL).

The AUC0–96 h was statistically significantly greater inrabbits with EXPAREL compared to bupivacaine solutionat the same dose (9 mg/kg, 2700 ± 781 versus 1670 ±249 ng·h/mL (1.6 fold difference P < 0.05), whereas thecorresponding values in dogs were not significantly different(7460± 1370 versus 6100± 1520 ng·h/mL) in dogs.

4. Discussion

The ultimate goal is to design a liposomal bupivacaine for-mulation which would produce maximum prolongationof analgesia without causing local or systemic toxicity. Inour studies, we evaluated the local and systemic toxicityproduced by EXPAREL in comparison with bupivacainesolution and saline after a single bolus injection around thebrachial plexus nerve bundle. Since the local and systemictoxicity of bupivacaine solution is well known, our experi-ment focuses on showing that EXPAREL did not cause overtirritation or local tissue damage even when used at high doseor concentration.


This is the first reported toxicology evaluation ofEXPAREL using brachial plexus block. We used both a clini-cal concentration of 15 mg/mL and a higher concentration of25 mg/mL for a total dosing up to 30 mg/kg to demonstratea wider safety margin for both concentration and totaldosing of bupivacaine and lipid components. Brachial plexusblockade was selected as the large network of nerve fiberswhich distributes the innervation of the upper extremity isclinically relevant.

Neurological damage is a well-recognized side effect oflocal analgesics applied in high concentrations close to neu-ronal structures such as peripheral nerves, nerve plexuses,or the spinal cord [16]. Local analgesics do not cause anydirect nerve damage unless they are injected intraneurallyor given in higher concentrations than that which is com-mercially available. Several different laboratory models haveproven that all local analgesics can be neurotoxic but thatlidocaine and tetracaine are potentially more neurotoxic thanbupivacaine [17].

The pathogenesis of local analgesics-induced local tis-sue toxicity is poorly understood. There appears to be arelationship between concentration and neurotoxicity. In1985, Ready et al. [18] evaluated the neurotoxic effects ofsingle injections of local analgesics in rabbits. They reportedthat spinal cord histopathology remained normal and thatpersistent neurologic deficits were not seen with clinicallyused concentrations of tetracaine, lidocaine, bupivacaine,or chloroprocaine. However, histopathologic changes andneurologic deficits did occur with higher concentrationsof tetracaine (1%, up to 8%) and lidocaine (8%, up to32%). It was found that high concentrations of lidocaine(and tetracaine) caused neural injury. Notably, in thismodel, extensive neurologic impairment was not necessarilyaccompanied by equally extensive lesions in the spinal cordand nerve roots, thus demonstrating the need for multiplemodels to fully assess neurotoxicity. Particularly, the highestconcentration of bupivacaine (3.3%) was not consistentlyassociated with comparable neural damage.

Peripheral nerve injury is a rare complication of regionalanesthesia. The pathogenesis of local analgesics-inducedlocal tissue toxicity is poorly understood. The mechanismof this enhanced toxicity remains to be established, but itmay be related to an effect of diverse vasoconstriction onanesthetic exposure [19]. Ischemia is one of the possiblecausative mechanisms which may result from changes inperipheral blood flow caused by a vasopressor adjuvant suchas epinephrine.

Some believe that this neurological damage is a result ofspinal cord ischemia either due to prolonged hypotensionduring surgery or as a consequence of arterial constrictionresulting from the use of epinephrine in the local anestheticssolution [20]. The use of additives in the solution also hasbeen implicated as contributing factors. The pressure of theinjected agent may cause nonspecific pressure-related nervedamage.

An immune-mediated mechanism may be possible assuggested by others [4, 16]. In Brummett’s study, rat sciaticnerves were harvested at either 24 hours or 14 days afterinjection and analyzed for perineural inflammation and

nerve damage. When compared with the saline controlgroup, the bupivacaine group had significantly higher peri-neural inflammation scores at 24 hours. Nerves in the bupi-vacaine and dexmedetomidine group showed less perineuralinflammation at 24 hours when compared to the bupivacainegroup.

The response to liposomes is well documented and de-scribed as a normal foreign body reaction appearing inparallel with liposome deposition. It is generally well ac-cepted that liposomes containing natural phospholipids,triglycerides, and cholesterol should not present any riskof antigenicity, presumably due to their similarities withbiological membranes [21]. Natural phospholipids such asphosphatidylcholines with neutral net charge in physiologicconditions are used to construct liposomes that closelyresemble biologic membranes. This type of liposomes madeof naturally occurring phospholipids is generally consideredsafe for parenteral use.

Certain types of liposomes may cause extensive tissuedamage. Particularly, those composed of lecithin-choles-terol-dicetyl phosphate or lecithin-cholesterol-stearylaminehave been reported to cause widespread tissue necrosis,epilepsy, and some deaths due to respiratory failure imme-diately after injection in mice whereas liposomes composedof phosphatidylcholine cholesterol-phosphatidic acid, ordipalmitoyl phosphatidylcholine only, produced minimalmorphological changes and by the sixth day post-injection;the histopathology was limited to the mechanical traumacaused by the injection [22]. Published studies with Lipo-Spheres containing tristearin and egg phosphatidylcholinein rats have shown no evidence of nerve damage and verylittle perineural inflammation or foreign body response[23]. Similarly, multilamellar vesicles liposomes made ofegg yolk phosphatidylcholine and cholesterol-containingbupivacaine have not been shown to produce histologiclesions on peripheral nerves after either brachial plexusinjection [24] or intracerebral administration [25]. Mali-novsky et al. has found that the incorporation of bupivacaineinto multivesicular liposomes devoid of phosphatidylcholinehydrolysis products or oxidation compounds produce spinalcord histopathologic changes not significantly different frombupivacaine solution after intracisternal administration inrabbits [26].

More recently, drug carriers such as cyclodextrins haveshown that the inclusion of bupivacaine 0.5% in hydrox-ypropyl-[beta]-cyclodextrin in equal amounts producedminimal histological alterations of the rat sciatic nerve 48hours after intraneural injection [27].

During an investigation of the pharmacological activity,cytotoxicity and local effects of ropivacaine 0.125%, 0.25%,and 0.5% concentrations encapsulated into large unilamellarvesicles composed of egg phosphatidylcholine, cholesterol,and alpha-tocopherol (4 : 3 : 0.07, mole %) compared withdrug solution showed that there was no morphological tissuechanges in the area of injection and sparse inflammatory cellswere observed in only one of the animals treated with plainsolution or ropivacaine at 0.5% [28].

In sciatic-nerve block experiments in rats, Soderberg etal. [29] showed that after two weeks following perineural


injection of various formulations containing 2.0%, 10%,20%, 60%, or 80% of lidocaine:prilocaine 1 : 1 mixtures inmedium chain triglycerides compared to saline, vehicle, 2.0%lidocaine : prilocaine solution and ethanol, pathologicalchanges in the sciatic nerves revealed that the formulations60% or greater (and ethanol) had neurotoxic effects, thatis, axonal swelling and neuronal degeneration, demyeliza-tion, and myelin degeneration associated with moderate tomarked diffuse inflammation in both the epineural andneuronal tissues. The cellular infiltrate was mainly composedof macrophages, lymphocytes, fibroblasts/fibrocytes, and oc-casional giant cells.

In a similar study in rats, formulations containing 2%,4%, 8%, 16%, 32%, or 64% of a mixture of bupivacaineand lidocaine base 4 : 1 in medium-chain triglyceride wereevaluated, together with 0.5%, 1.0%, and 2.0% bupivacaineHCl solutions, bupivacaine 4.2% or 7.0% in medium-chaintriglyceride, and 20% lidocaine base in a polar lipid vehicle[30]. Histopathologic examination of sciatic nerves by lightmicroscopy revealed slight to moderate signs of neurotoxicityonly after administration of the 64% formulation, a weekafter dosing.

With regard to pathological effects of EXPAREL on pe-ripheral nerves, no remarkable findings were observedusing the standard hematoxylin- and eosin-staining method.The brachial plexus sites analyzed for histopathologicalchanges were normal on Day 3 and Day 15. There wasno evidence of adverse local reactions even at the high-est concentration, 25 mg/mL (30 mg/kg dose). With theexception of granulomatous inflammation, there were noobservations of abnormal gross pathology findings at thesite of drug administration or elsewhere, and no significantchanges in blood chemistry or animal behavior beyondthose observed with Bupivacaine solution or saline. It ispostulated that macrophages phagocytosed liposome mate-rial, as they would any other foreign material in tissues.The increased presence of these cells was therefore notunexpected; the transient local inflammatory response toEXPAREL is a normal foreign body reaction appearing inparallel with liposome deposition. In Boogaerst’s study inrabbits, the systemic bupivacaine concentration were lowerduring the first 10 minutes (P < 0.05) and higher after24 hours (P < 0.05) after brachial plexus injection of2.5 mg bupivacaine in 1 mL of 0.25% multilamellar liposo-mal bupivacaine made of PC and cholesterol (ratio 4 : 3)compared to bupivacaine solution, while the Cmax was notvery different between the two formulations (∼0.2 µg/mL)[31].

In our studies, the PK profile displays an initial rise(reflective of unencapsulated drug present in the aqueousphase of EXPAREL) (i.e., outside of the particles) followedby a curve typical of a slow release delivery system (asafforded by the DepoFoam delivery system). In both rabbitsand dogs, the peak plasma concentrations of bupivacainewith EXPAREL were significantly attenuated, that is, up toapproximately fourfold (9 mg/kg; 106 versus 433 and 402versus 1490 ng/mL, resp.) compared with equivalent doses ofbupivacaine solution (9 mg/kg, P < 0.05).

Occasional spurious high Cmax values were observedwhich accounted for the relatively larger deviation seen withEXPAREL compared with bupivacaine solution. Althoughthe precise nature of the mechanism(s) involved are un-known, there was no compelling evidence that this findingwas related to the specific formulation of bupivacaine used(EXPAREL).

The twofold lower AUC0–24 h with EXPAREL comparedto bupivacaine solution reflects a slower absorption in dogs(2860 versus 6020 ng·h/mL, P < 0.05 while in rabbits, theAUC0–24 h values were not significantly different (1230 versus1620 ng·h/mL). Plasma concentrations of bupivacaine wereapproximately similar or even more prolonged in rabbits(1.6 fold difference in AUC0–96 h P < 0.05) consistent withsustained release of EXPAREL. As the toxicity of bupivacaineis known to be generally associated with its Cmax, the lowerCmax observed with EXPAREL as compared to bupivacainesolution at the same dose demonstrates potential safetyadvantages with this liposomal formulation.

The rate of systemic absorption of local anestheticsis dependent upon the total dose and concentration ofdrug administered, the route of administration, and thevascularity of the administration site. Absorption from thesite of injection depends on the blood flow, the morerapid the rate at which plasma concentrations increase andthe greater the peak concentration of the drug [7]. It isalso possible that the interanimal variability in the PKresponse may be the consequence of unequal dispersion ofthe dosing material through the injection site resulting inpacked material as well as drug-induced vasodilation so thatvaried amounts of drug were absorbed.

In summary, a depot formulation with bupivacaine asthe active component and DepoFoam lipid carrier was testedafter PNB in rabbits and dogs. In the present studies, therewere no local signs of toxicity, including no histologicalevidence for any increase in local reactions or generalexacerbations of bupivacaine toxicity after peripheral nerveblock. Particularly, there was no evidence of nerve damageat doses up to 30 mg/kg bupivacaine (more than threefoldhigher doses of EXPAREL versus bupivacaine solution).Bupivacaine did not impact directly on neural tissue, andthe findings of granulomatous inflammation were moreconsistent with a nonspecific foreign—body type reactionmost likely mediated by the DepoFoam particles.

Importantly, the DepoFoam delivery system leads to aslower release of the drug allowing a longer duration ofaction and, from a toxicological standpoint, to a sloweruptake into the systemic circulation avoiding high plasmaconcentrations.

5. Conclusions

In conclusion, a single administration of EXPAREL wasdemonstrated to be safe by peripheral nerve block in rabbitsand dogs when tested in comparison with bupivacaine HCland saline. EXPAREL did not cause overt irritation or localtissue damage even when injected at high dose or concentra-tion around the brachial plexus nerve bundle.


Additional studies are ongoing to further examine theutility of this novel formulation by various routes (e.g., localinfiltration, epidural, and intra-articular).

Abbreviations

Bsol: Bupivacaine HCl solutionEXP: EXPAREL (bupivacaine liposome

injectable suspension using multivesicularDepoFoam technology)

PK: PharmacokineticsPNB: Peripheral nerve block.

Conflict of Interests

B. M. Richard is a consultant for Pacira Pharmaceuticals, Inc.

Acknowledgment

The authors wish to thank Dr. Doug Rickert for pharmacoki-netic evaluation of the results.

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