Background Effective postoperative pain management plays a key role in enhancing recovery of patients after surgery. Bupivacaine hydrochloride is one of the most commonly local anesthetics used for the postoperative pain control. However, the relatively short anesthesia duration of bupivacaine preparations limited their clinical application.
Methods Both guinea pig pin-prick study and rat tail-flick test were performed to evaluate the local anesthesia efficacy of HYR-PB21-LA, a new microparticle suspension injection of bupivacaine pamoate.
Results In the pin-prick test, the complete cutaneous trunci muscle reflex inhibitions were observed at 30 min in all treatment groups containing bupivacaine. In comparison with 6.7 mg/mL HYR-PB21-LA, both 10 and 20 mg/mL HYR-PB21-LA groups had significantly higher area under effect time curve (AUEC) values (p<0.001 and p<0.0001) and slower offset time (p<0.0001). Significantly higher AUEC (p<0.0001) and slower offset time (p<0.0001) were also found in 10 mg/mL HYR-PB21-LA treatment group compared with bupivacaine liposome injectable suspension (liposomal bupivacaine). In the rat tail-flick test, significantly increased local anesthesia effect was lasted for 5 hours after 2.5 mg/mL HYR-PB21-LA administration, which was fivefold longer than bupivacaine hydrochloride. The longer lasted efficacy of significantly increased local anesthesia was also observed in 5 mg/mLHYR-PB21-LA than those in liposomal bupivacaine (8 hour vs 1 hour).
Conclusions The results demonstrated that the HYR-PB21-LA produced longer local anesthesia effect than current clinical preparations of bupivacaine in two animal models. These findings raise the potential clinical value of HYR-PB21-LA as a long-lasting local anesthesia for controlling postsurgical pain in humans.
- animal studies
- pharmacology: local anesthetics
- postoperative pain
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Long-lasting intensive postoperative pain caused by variously surgical procedures, such as hemorrhoidectomy, hallux valgus, total knee arthroplasty, inguinal herniorrhaphy and colectomy, is a key disadvantage factor associated with the functional recovery, healing or cost of hospitalization for patients.1–5 More effective and appropriate control of postsurgical pain is necessary for shortening hospital stays and improving life quality of patients with local surgeries.6–10 Standard methods of postsurgical systematic analgesia include administration of parenteral and oral non-steroidal anti-inflammatory drugs and opioids,11–13 but these can cause adverse effects such as vomiting, central nervous system depression, respiratory depression and constipation.14 15 Local analgesics have been employed for the postoperative pain control to avoid these side effects from systemic analgesia.16–19
Previous studies indicated that bupivacaine hydrochloride (bupivacaine HCl), one of the longest-acting local anesthetics,20 is an effective analgesic for controlling postoperative pain, and avoiding opioids-related side effects. However, the relatively short duration of the local analgesic action of bupivacaine HCl, commonly less than 8 hours, limited its clinical usage for postoperative pain control, which usually lasting more than 72 hours postsurgically. Therefore, new formulations of bupivacaine with long-lasting local analgesic action are needed for patients with variously local surgeries.
Bupivacaine pamoate freeze-dried powder for injection (HYR-PB21-LA), a developing new extended-release formulation of bupivacaine, is a microparticle suspension injection, prepared by bupivacaine pamoate cocrystal. The single crystal X-ray diffraction data indicated that HYR-PB21-LA was a supermolecular complex, comprised eight bupivacaine, four pamoic acid and eight water molecules, which was stabilized by an intricate network of hydrogen bonds and hydrophobic bonds. In comparison with bupivacaine HCl, HYR-PB21-LA has very low solubility in simulated body fluid in vitro (Solubility of HYR-PB21-LA was shown in online supplementary table 1). It can slowly dissolve the active ingredient, that is, the bupivacaine molecule, into the local site of injection to achieve long-acting local anesthesia. In present study, we have investigated the anesthesia effects in two common in vivo local anesthetic animal models, the guinea pig intradermal weal/pin-prick study to test for peripheral nociceptive blockade and the rat tail-flick test to evaluate the nerve sensory blockade. To further investigate the pharmacodynamics/pharmacokinetics (PD/PK) relationship of HYR-PB21-LA in rat tail-flick test, satellite groups of animals were employed for the concomitant PK study.
Materials and methods
Animal care and housing
Male adult guinea pigs and Sprague Dawley rats (SD rats), supplied by Beijing Vital River Laboratory Animal Technology, were used in dermal weal/pin-prick model and the rat tail-flick test, respectively. All animals were housed in temperature and humidity controlled (temperature: 20°C–26℃; humidity: 40%–70%) rooms with a 12-hour light-dark cycle. Food and water were accessed freely by all animals.
Test article and reference drugs
HYR-PB21-LA, 100 mg/vial and 300 mg/vial, were provided by Hefei Co-source Pharmaceutical, Hefei, China. Both 10 mg/mL and 30 mg/mL working suspensions of HYR-PB21-LA were prepared by adding 10 mL sterile water into 100 mg/vial and 300 mg/vial, respectively. Bupivacaine liposome injectable suspension (liposomal bupivacaine), 13.3 mg/mL, was manufactured by Pacira Pharmaceuticals, San Diego, California, USA. Bupivacaine HCl injection, 6.7 mg/mL, was manufactured by Shanghai Zhaohui Pharmaceutical, Shanghai, China. The contents of the three drugs were expressed as bupivacaine free base. All other concentrations of HYR-PB21-LA, liposomal bupivacaine or bupivacaine HCl injection could be obtained by further diluting with normal saline.
Guinea pig dermal weal/pin-prick assay
Male adult guinea pigs weighted 300–335 g were employed in the dermal weal/pin-prick study. A total of 48 animals were randomly divided into six experimental groups (ie, vehicle group, low-dose HYR-PB21-LA group (0.3 mL of a 6.7 mg/mL suspension), moderate dose HYR-PB21-LA group (0.3 mL of 10 mg/mL suspension), high-dose HYR-PB21-LA group (0.3 mL of a 20 mg/mL suspension), liposomal bupivacaine group (0.3 mL of a 10 mg/mL solution) and bupivacaine HCl group (0.3 mL of a 6.7 mg/mL solution)).
A modified dermal weal/pin-prick model21 was used to evaluate the local anesthetic efficacy of intradermal administrations of HYR-PB21-LA, liposomal bupivacaine, bupivacaine HCl, or vehicle in male guinea pigs. In brief, the left backs of male guinea pigs were shaved 1 day before treatment. A volume of 0.3 mL injection was administrated intradermally on the up flank of left back of each animal using 25-gage hypodermic needle. A weal with diameter of about 1.5 cm was produced by injection. Nine pin-pricks were applied for the anesthetic effect evaluations using 30-gage needles. Eight of them were evenly distributed along a 0.8 cm diameter circle drawn with the origin at the center of the weal, and the left one was at the center of the weal. The number of pin-pricks without cutaneous trunci reflex was recorded by operator who was blinded to the treatment groups at 0.5, 3, 6, 12, 18, 24 and 48 hours, respectively, after injection.
Rat tail-flick test and concomitant PK study
Rat tail-flick test
Nerve sensory blockade efficacy of HYR-PB21-LA was evaluated using a modified tail-flick test model22 in male SD rats. In brief, four 0.1 mL experimental solutions were injected into the both sides of the tail subcutaneously with 23-gage needles. Needles were inserted with a 30° angle until their tips contacted the caudal vertebrae, and then needles were withdrawn by 1 mm for injection. A tail-flick test apparatus meter (Shanghai Yu Yan Scientific Instrument, Shanghai, China) was used to measure the tail-flick latency of the rats before injection as baseline value and animals with baseline latencies less than 2 s or more than 4 s were excluded. After injection, the tail-flick latency was evaluated by assessor who was blinded to the treatment groups at 0.5, 1, 3, 5, 8, 12, 16, 24 and 48 hours and a cut-off time of 12 s was set to avoid the thermal damage to the tail of experimental animals.
A total of 48 male SD rats weighted 189–213 g were randomly divided into six groups (vehicle group, low-dose HYR-PB21-LA group (0.4 mL of a 2.5 mg/mL suspension), moderate dose HYR-PB21-LA group (0.4 mL of a 5 mg/mL suspension), high-dose HYR-PB21-LA group (0.4 mL of a 10 mg/mL suspension), liposomal bupivacaine group (0.4 mL of a 5 mg/mL solution), and bupivacaine HCl group (0.4 mL of a 2.5 mg/mL solution)) for the dose–response efficacy study.
Concomitant PK study
A total of 30 male SD rats were randomly assigned to five satellite groups (six rats per group) for concomitant PK study, which included low-dose HYR-PB21-LA group (0.4 mL of a 2.5 mg/mL suspension), moderate-dose HYR-PB21-LA group (0.4 mL of a 5 mg/mL suspension), high-dose HYR-PB21-LA group (0.4 mL of a 10 mg/mL suspension), bupivacaine HCl group (0.4mL of a 2.5 mg/mL solution) and liposomal bupivacaine group (0.4 mL of a 5 mg/mL solution). The process of drug administration was exactly same with the tail-flick test. A volume of 0.3 mL vein blood sample with ethylene diamine tetraacetic acid anticoagulation was collected from each experimental animal at 0 hour (predose) and 0.5, 1, 3, 5, 8, 12, 16, 24, 48 hours (postdose). Plasma samples were obtained by centrifugation (TGL-18C-C desk centrifuge, Shanghai Anting Scientific Instrument Factory, Shanghai, China) at 1500 g for 15 min at room temperature and stored at −80°C for further analysis. A prevalidated liquid chromatography–tandem mass spectrometry (LC-MS/MS) analytical method was used to determine plasma bupivacaine concentrations. In brief, plasma samples were vortex-mixed for 30 s after being defrosted at room temperature. A 20 µL aliquot of plasma and 400 µL of the internal standard (10 ng/mL of ropivacaine methanol solution) was pipetted into a 2 mL 96 deep-well plate, vortex-mixed for 5 min and centrifuged (H2050R high-speed freezing centrifuge, Hunan XiangYi Laboratory Instrument Development, Hunan, China) at 3000 g for 15 min. A 200 µL aliquot of supernatant was mixed with the same volume of pure water, and then 10 µL of mixture was loaded into the LC-MS/MS system (Shimadzu LCMS-8045, Shimadzu, Kyoto, Japan) for the concentration measurement of bupivacaine. Standard curves were constructed using a series of standard working solutions (ie, 2, 5, 10, 20, 50, 100, 250 and 500 ng/mL). The lower and upper limits of quantifications were 2 and 500 ng/mL, respectively. WinNonlin version 6.4 (Pharsight, St. Louis, Missouri, USA) was used for all PK calculations.
Microscopic evaluation of local toxicity
Tail tissues of injection sites (approximately 1 cm in length) from SD rats, and skin and subcutaneous tissues of injection sites (approximately 1.5 cm in length) from guinea pigs, were collected at necropsy at 48 hours postdose in concomitant local subcutaneously injection toxic study and local intradermally injection toxic study, respectively. Tail tissues were fixed with 10% neutral formalin fix solution and followed by decalcification in Clayden’s solution for about 6 hours. Formalin-fixed, decalcified, paraffin-embedded tail tissues of injection sites (approximately 0.3 cm in length) and formalin-fixed, paraffin-embedded skin and subcutaneous tissues of injection sites were sectioned and stained with hematoxylin and eosin-stain using standard procedures.
The microscopic evaluation of local toxicity performed by blinded examiners. The severity of histological findings was graded on a 4-point scoring system (ie, 0=normal, 1=minimal, 2=slight, 3=moderate and 4=severe). The description and definition of the 4-point scoring system, modified from the report of Mann et al,23 was illustrated in table 1.
The nociceptive blockade score of pin-prick test was converted from the number of negative cutaneous trunci reflex responses out of nine of 100%. The area under the nociceptive blockade score-time curve (area under effect time curve (AUEC)) was calculated using the trapezoidal rule for the estimation of the overall efficacy of various treatments. The onset time of effective inhibition was defined as the first time point with a nociceptive blockade score more than 50%, and the offset time of effective inhibition was set as the first time point with a nociceptive blockade score less than 50%. Differences of AUEC and offset time among different treatments were tested using the generalized linear model with adjustment for predrug weight. Post hoc multiple comparisons were further analyzed with Bonferroni test.
To analyze the tail nerve sensory conduct block effects of the various treatments on rat tail flick test, tail flick latencies were converted to the percentage of maximal possible effect (MPE%) using following formula:
Differences in MPE% between various treatment groups and vehicle group were analyzed at each time points employing the generalized linear model (Bonferroni method) with adjustment for predose weight. Statistically significant difference was considered as p<0.05. Data analyses were performed with SPSS Statistics V.23 (IBM) and Microsoft Excel 2010 software (Microsoft, Redmond, Washington, USA).
In guinea pig pin-prick test, the complete inhibitions (100% effect) of cutaneous trunci muscle reflex were observed at 30 min in all treatment groups containing bupivacaine. In contrast, no inhibitions were observed at any time point in all the animals of the vehicle group (figure 1 and table 2). HYR-PB21-LA dose dependently inhibited the cutaneous trunci muscle reflex in guinea pig pin-prick model. In comparison with 6.7 mg/mL HYR-PB21-LA group, both 10 mg/mL and 20 mg/mL HYR-PB21-LA groups had significantly higher mean AUECs (1483(83) vs 1050(111), p<0.001; 2063(88) vs 1050(111), p<0.0001) and slower setoff time (1080(192) min vs 765(231) min, p<0.0001; 1395(127) min vs 765(231) min, p<0.0001). Similarly, compared with 10 mg/mL group, 20 mg/mL HYR-PB21-LA also produced higher AUEC (p<0.0001) and slower offset time (p<0.0001). Significantly higher AUEC (1483(83) vs 915(78), p<0.0001) and slower offset time (1080 (192) min vs 720 (0) min, p<0.0001) were also found in 10 mg/mL HYR-PB21-LA treatment group compared with equivalent dose of liposomal bupivacaine (figure 1 and table 2). The durations of effective inhibition of the cutaneous trunci muscle reflex observed in 10 mg/mL HYR-PB21-LA and liposomal bupivacaine groups were 690 (192) min and 330 (0) min, respectively, suggesting more than twofold increase in duration for HYR-PB21-LA than liposomal bupivacaine.
In rat tail-flick test, significantly elevated mean MPEs% were observed in all five treatment groups containing bupivacaine at the first and second time point (30 min and 1 hour) after drug administration compared with the vehicle group. At 3-hour following drug injection, only all the three HYR-PB21-LA groups produced significantly higher mean MPEs% than those in the vehicle group. In contrast, no significant differences of mean MPE% were found between bupivacaine HCl group and vehicle group (−0.5 (12.7) vs 2.7 (7.7), p=1.000) as well as liposomal bupivacaine group and vehicle group (29.6 (36.2) vs 2.7 (7.7), p=0.113). At 5-hour time point, significantly higher mean MPEs% was still observed in three HYR-PB21-LA groups and the tail nerve sensory conduct block effects (MPEs%) were dose dependently increased among three HYR-PB21-LA groups (35.0±32.1 (low dose) vs 68.7±33.3 (moderate dose) vs 88.6±18.1 (high dose)). In addition, the significantly greater mean MPE% was sustained for 8 hours and 12 hours after drug administration in moderate and high-dose HYR-PB21-LA groups, respectively, compared with the vehicle group (figure 2A and online supplementary table 2).
In concomitant PK study, as shown in figure 2B, the mean plasma bupivacaine maximum concentration (Cmax) was 123 ng/mL for 2.5 mg/mL HYR-PB21-LA group. This value was about 30% of the bupivacaine Cmax of 2.5 mg/mL bupivacaine HCl group (409 ng/mL). In comparison with 5 mg/mL liposomal bupivacaine treatment group (420 ng/mL), a greater than 50% decrease in plasma bupivacaine Cmax (191 ng/mL) was produced in 5 mg/mL HYR-PB21-LA group. In contrast, the similar plasma bupivacaine area under curves0-∞ (AUCs0-∞) were observed at the equivalent concentration groups (ie, 2.5 mg/mL bupivacaine HCl (929 ng.hour/mL) vs 2.5 mg/mL HYR-PB21-LA (879 ng.hour/mL), and 5 mg/mL liposomal bupivacaine (1570 ng.hour/mL) vs 5 mg/mL HYR-PB21-LA (1700 ng.hour/mL)). The detail information of all PK parameters for plasma bupivacaine from this study is shown in online supplementary table 3.
Among various HYR-PB21-LA treatment groups, the dose dependently increase in plasma bupivacaine AUCs (2.5 mg/mL group, AUC0-∞=879 ng.hour/mL; 5 mg/mL group, AUC0-∞=1700 ng.hour/mL; 10 mg/mL group, AUC0-∞=2780 ng.hour/mL) was detected. However, similar increase in plasma bupivacaine Cmax was not found from 5 mg/mL HYR-PB21-LA group (Cmax=191 ng/mL) to 10 mg/mL HYR-PB21-LA group (Cmax=194 ng/mL) in the present concomitant PK study.
As shown in figure 2A,B, the patterns of MPE% change over time for various groups in the tail flick test were consistent with the plasma bupivacaine concentration-time curves in the concomitant PK study. The plasma bupivacaine concentrations in liposomal bupivacaine group were twofold greater than those in HYR-PB21-LA groups while they had similar MPE% levels at 30 min and 1 hour time points.
As shown in table 3, the pathological changes of minimal to moderate monocytic inflammation were found in injection sites in SD rats at 48 hours after a single injection of HYR-PB21-LA in concentration groups of 2.5, 5 and 10 mg/mL. The representative histopathological changes were shown in figure 3. The incidences and total scores of subcutaneous monocytic inflammation of 2.5, 5 and 10 mg/mL HYR-PB21-LA groups were 1/8︱1 score, 5/8︱9 scores and 8/8︱15 scores, respectively. The findings indicated that there was a trend of dose dependently increasing for incidence and severity of subcutaneous monocytic inflammation in injection sites of tails in SD rats injected with HYR-PB21-LA suspension in concentration scope of 2.5–10 mg/mL. No similar trend was found in the injection sites in guinea pigs at 48 hours after a single injection of HYR-PB21-LA intradermally in concentration scope of 6.7–20 mg/mL (incidences︱total scores: 6.7 mg/mL group, 7/8︱13 scores; 10 mg/mL group, 7/8︱15 scores; 20 mg/mL group, 5/8︱10 scores). Additionally, the incidence and severity of local inflammation in injection sites from HYR-PB21-LA groups were comparable with liposomal bupivacaine at equivalent doses in both animal species (incidences︱total scores: SD rats, 5/8︱9 scores vs 5/8︱9 scores (5 mg/mL HYR-PB21-LA group vs 5 mg/mL liposomal bupivacaine group); guinea pigs, 7/8︱15 scores vs 7/8︱19 scores (10 mg/mL HYR-PB21-LA group vs 10 mg/mL liposomal bupivacaine group)).
In the present study, we have demonstrated that HYR-PB21-LA, a developing new extended-release microparticle suspension injection of bupivacaine pamoate cocrystal, could produce robust dose dependently long-lasting local anesthesia efficacy in two in vivo local anesthetic animal models, the guinea pig pin-prick study and rat tail-flick test, compared with the same dose of clinical preparations (ie, bupivacaine HCl and liposomal bupivacaine). Compared with liposomal bupivacaine, more than twofold longer local anesthesia was founded in HYR-PB21-LA treatment in both guinea pig intradermal weal assay and rat tail sensory conduction blockade test. The local anesthesia onsets were similar among HYR-PB21-LA, bupivacaine HCl and liposomal bupivacaine treatment groups. In general, fivefold-longer local anesthesia was observed in HYR-PB21-LA treatment than bupivacaine HCl treatment, in both guinea pig intradermal weal study and rat tail sensory conduction blockade test. The results indicated that HYR-PB21-LA has substantially long-lasting local anesthesia than bupivacaine HCl and liposomal bupivacaine. Furthermore, the findings provided strong preclinically PD support for the development of HYR-PB21-LA, as a useful long-acting local anesthetic for clinically postoperative pain medicine.
To develop the new bupivacaine formulation with more safety and long duration of local analgesia, several innovative long-acting delivery methods for local anesthetics have been employed in previous studies.24 25 The typical formulation technology is DepoFoam, developed by Pacira Pharmaceuticals, which uses the multivesicular liposomes to encapsulate the bupivacaine for more prolonged drug release.26 Another formulation technology is SABER-Bupivacaine from DURECT (Cupertino, California, USA), which mixes bupivacaine, sucrose acetate isobutyrate and benzyl alcohol as a solution to produce sustained-release effect.27 Unlike to the forgoing two preparations, the prolonged local anesthesia action of HYR-PB21-LA is derived from its unique physicochemical properties, that is, the very low solubility in simulated body fluid (solubility≈0.25 mM (0.072 mg/mL), shown in online supplementary table 1) which is approximately 0.2% of the solubility of Bupivacaine HCl (≈40 mg/mL).28 The micron sized crystal particles depot plus very low solubility produced the prolonged release of bupivacaine in the local area of HYR-PB21-LA injection. The mechanism of the drug extended release in local injection site is similar to some depot intramuscular formulations, such as olanzapine pamoate monohydrate,29 which also employed the pamoate to formulate the crystalline salt formulation for slowing down the drug release.
In the tail-flick test and concomitant PK study, HYR-PB21-LA at an equivalent bupivacaine concentration could produce longer tail sensory conduction blockade effect and lower plasma bupivacaine Cmax than those in liposomal bupivacaine and bupivacaine HCl treatment groups. Previous studies reported that the systemic toxicity reactions of bupivacaine, including CNS toxicity reactions and cardiovascular collapse, occurred as a result of the high plasma drug concentration.30 Hence, the risk of systemic toxicity reactions of bupivacaine may be decreased through lowering the systemic plasma bupivacaine peak concentration of local anesthetic. Our findings indicated that HYR-PB21-LA, which possessed longer local anesthetic efficacy along with the obviously lower plasma bupivacaine Cmax in the present tail-flick test and concomitant PK study, had better characteristics of systemic safety while using in local anesthesia compared with currently clinical relevant preparations.
The findings of the local toxicity studies indicated that minimal to moderate local inflammation reaction (mainly presented as monocytic inflammation) was observed in the injection sites of experimental animals at 48 hours after HYR-PB21-LA-dosing in concentrations scope of 2.5–10 mg/mL. The local reaction to injection of HYR-PB21-LA was consistent with that expected as a response to a foreign material and similar to the histological changes of liposomal bupivacaine and bupivacaine HCl. Furthermore, the incidence and severity of local inflammation of HYR-PB21-LA was comparable with liposomal bupivacaine groups at equivalent doses in both species.
In summary, we have demonstrated that HYR-PB21-LA, a developing new microparticle suspension injection of bupivacaine pamoate cocrystal, could exert dose dependently longer lasting, recoverable local anesthesia in two in vivo animal models compared with clinical preparations of bupivacaine at equivalent dosages. Additionally, in comparison with bupivacaine HCl and liposomal bupivacaine, the potential safety advantage from lower systemic plasma peak concentration also had been found from the tail-flick test and concomitant PK study in HYR-PB21-LA treatment. These findings raise the potential clinical value of HYR-PB21-LA, which may be used as a more effective long-lasting local anesthesia for controlling postsurgical pain in humans. Further clinical trials are warranted to evaluate the clinical delay-action local anesthesia efficacy and the clinical safety.
JP and CL are joint first authors.
Contributors All authors make substantial contributions to this manuscript.
Competing interests Competing Interests statement:SZ reports grants from Development and Reform Committee of Anhui Province, grants from Talent Office of Anhui Province, during the conduct of the study; In addition, JP, YW, XL, HS, SG and SZ have a patent PCT patent application number: WO2018177232 pending. All other authors have declared no conflict of interests for this manuscript.
Patient consent for publication Not required.
Ethics approval The guinea pig pin-prick study and rat tail-flick test were approved by the Animal Care and Use Ethics Committee of Hefei Blooming Drug Safety Evaluation Company.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request. Data relevant to this article are avialable on reasonalbe request.