Effectiveness of two extended-release buprenorphine formulations during postoperative period in neonatal rats.

Information on the effectiveness of a new long-lasting buprenorphine formulation, extended-release buprenorphine, in the neonatal rat is very limited. This study compares whether a high dose of extended-release buprenorphine (XR-Hi) attenuates thermal hypersensitivity for a longer period than a low dose of extended-release buprenorphine (XR-Lo) in a neonatal rat incisional pain model. Two experiments were performed. Experiment one: Male and female postnatal day-5 rat pups (n = 38) were randomly assigned to 1 of 4 treatment groups and received a subcutaneous administration of one of the following: 1) 0.9%NaCl (Saline), 0.1 mL; 2) sustained release buprenorphine (Bup-SR), 1 mg/kg; 3) XR-Lo, 0.65 mg/kg; and 4) XR-Hi, 1.3 mg/kg. Pups were anesthetized with sevoflurane in 100% O2 and a 5 mm long skin incision was made over the left lateral thigh and underlying muscle dissected. The skin was closed with surgical tissue glue. Thermal hypersensitivity testing (using a laser diode) and clinical observations were conducted 1 hour (h) prior to surgery and subsequently after 1, 4, 8, 24, 48, 72 h of treatment. Experiment two: The plasma buprenorphine concentration level was evaluated at 1, 4, 8, 24, 48, 72 h on five-day-old rat pups. Plasma buprenorphine concentration for all treatment groups remained above the clinically effective concentration of 1 ng/mL for at least 4 h in the Bup-SR group, 8 h in XR-Lo and 24 h in XR-Hi group with no abnormal clinical observations. This study demonstrates that XR-Hi did not attenuate postoperative thermal hypersensitivity for a longer period than XR-Lo in 5-day-old rats; XR-Hi attenuated postoperative thermal hypersensitivity for up to 4 h while Bup-SR and XR-Lo for at least 8 h in this model.

Introduction

Postoperative analgesia is a critical component of laboratory animal medicine as untreated pain causes distress [1] and can impact research results [2]. Neonatal rodents are used for variety of survival surgical procedures, including stereotaxic [3-5], spinal [6, 7], and thoracic [8, 9] surgeries which are known to evoke pain responses. Untreated pain in neonates can lead to long-term behavioral changes [10, 11], such as altered development of pain perception, neurodevelopmental functioning, and social-emotional functioning [12]. Providing analgesia to neonates is challenging as there are significant physiological differences in neonates from adults. They have immature vital organs which can lead to rapid changes in their renal and hepatic systems [13]. This results in differing pharmacodynamics and an increased incidence of adverse effects from analgesic drugs, leading to urinary retention, sedation, and respiratory depression [14-17]. Because of this, additional consideration is needed prior to the use of analgesic drugs in neonates.

Opioid analgesics, including buprenorphine HCL (Bup-HCL, Hospira, Lake Forest, IL) and sustained-released buprenorphine (Bup-SR, Zoopharm, Fort Collins, CO), are the most commonly used analgesic drugs for managing post-operative pain in laboratory rodents [18]. In neonatal rats, a single dose of Bup-HCL (0.025 or 0.05 mg/kg) was found to effectively attenuate post-operative thermal hypersensitivity for 4 h [19], while a single dose Bup-SR (either 0.5 or 1 mg/kg) was found to effectively attenuate hypersensitivity for 8 h [20]. Recently, a new FDA-indexed, lipid-bound extended-release buprenorphine (XR-Hi, Ethiqa®, Fidelis Animal Health, North Brunswick, NJ) was introduced as an alternative buprenorphine analgesic for rodents. Bup-SR is a polymeric formulation that contains a water-insoluble, biodegradable lipid-bound and suspended in medium-chain fatty acid triglyceride (MCT) oil that is degraded over time with lipase and esterase activity [21-25]. In a previous study, our group found that a single dose of XR-Lo 0.65 mg/kg or XR-Hi 1.3 mg/kg, effectively attenuated post-operative mechanical hypersensitivity for 2 days in adult rats. However, the safety and efficacy of XR-Hi in neonatal rats is currently unknown.

In this study, we investigate the effectiveness of XR-Lo and high dose of extended-release buprenorphine (XR-Hi). We hypothesized that a high dose (1.3 mg/kg) of XR-Hi would attenuate post-operative thermal hypersensitivity longer than a low dose (0.65 mg/kg) of extended-release buprenorphine (XR-Lo, Fidelis Animal Health, North Brunswick, NJ) in a neonatal rat incisional pain model.

Materials and methods

Animals

One-day-old male and female Sprague Dawley rat pups [(Crl: CD (SD) IGS, (n = 94), Charles River Laboratories, Hollister, CA], housed in litters of eight to ten with the dam, arrived at the facility on day 0. The rats were free of rat coronavirus, rat Theiler virus, Kilham rat virus, rat parvovirus, Toolan H1 virus, rat minute virus, lymphocytic choriomeningitis virus, murine adenovirus type 1 and 2, reovirus type 3, Sendai virus, pneumonia virus of mice, Mycoplasma pulmonis, mites, lice, and pinworms. All animals were housed in static microisolator cages under a 12:12 light:dark cycle (lights on at 7:00 am). Rat dams were provided with Teklad Global 18% Protein Rodent Diet 2018 (Harlan Laboratories, Madison, WI) and water filtered by reverse osmosis ad libitum. All experiments in this study were approved by the Stanford University Administration Panel for Laboratory Animal Care. All animals were handled and housed according to the Guide for the Care and Use of Laboratory Animals [26] in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International. At the conclusion of the study, rat pups and dams were euthanized with carbon dioxide asphyxiation followed by decapitation as a secondary method of euthanasia.

Experimental groups

Five-day-old pups of either sex was randomly assigned into one of four treatment groups (n = 8–10 per group) and observers were blinded to the treatment group assignment. Treatment groups consisted of: 1) Saline (Saline; 0.9% NaCl, Hospira, Lake Forest, IL) – 0.1 ml subcutaneous (SC) once (n = 8), 2) Bup-SR (1 mg/mL)– 1mg/kg SC once (n = 10), 3) XR-Lo (1.3 mg/mL) – 0.65 mg/kg SC once (n = 10), 4) XR-Hi 1.3 mg/kg SC once (n = 10). Doses of buprenorphine chosen were based on previous studies: 1) Bup-SR at 1 mg/kg was based on Blaney et al. [20]; 2) XR-Hi at 0.65 and 1.3 mg/kg was based on doses in adult Sprague Dawley rats by Levinson et al. [27] and Alamaw et al. [28] (note that doses of XR-Hi in rat pups are not known).

Anesthesia and surgical model

Anesthesia was induced with sevoflurane (5–8%) in 100% O2 (0.5–1 L/minute) via nose cone until a surgical plane of anesthesia was reached (absence of paw withdrawal reflex). Anesthesia was maintained with 4–7% sevoflurane in 100% O2 (0.5 L/minute) via nose cone. The pups were placed on a warm water circulating blanket set to 38°C (Stryker T/Pump, Portage, Michigan) for the duration of the anesthetic procedure. All drugs (Bup-SR, XR-Lo, and XR-Hi) were administered SC at the left shoulder. All rat pups were administered supplemental fluid (0.9% NaCl, 5 ml/kg, SC) at the right shoulder. XR-Lo and XR-Hi were administered with 29-gauge 0.3 ml insulin syringes (UltiMed, Inc., Excelsior, MN) while Bup-SR was administered with 22-gauge 1 ml Leur Lock syringe (Becton, Dickinson and Company, Franklin Lakes, NJ). All injection sites were pinched post-injection for 5 seconds (sec) to prevent leakage. All drug administrations were performed by the same personnel, who did not participate in subsequent testing or pathological evaluations, to ensure the tester is blinded to the treatment groups. The surgical method was adapted from Blaney et al. [20] and was performed aseptically as required by the Stanford IACUC Guidelines for Rodent Survival Surgery. Briefly, the pups were positioned in right (contralateral) recumbency, and the left (ipsilateral) thigh was aseptically prepared with three alternating betadine solution swab sticks (10% povidone-odine, Dynare Corporation, Orangeburg, NY) and 70% Isopropyl Alcohol USP (Henry Schein, Melville, NY). After the animal reached a surgical plane of anesthesia (as determined by a lack of withdrawal response to toe pinch), a 0.5-cm longitudinal skin incision was made on the ipsilateral thigh, 0.5-cm above the hock, using #15 blade (VetOne®, Boise, ID), and the superficial biceps femoris muscle was incised 0.3-cm longitudinally without disturbing the muscle attachment. The incision was closed with sterile surgical tissue glue (Covetrus, Dublin, OH). After surgery, antibiotic ointment (triple antibiotic ointment with bacitracin zinc, neomycin sulfate, and polymyxin B sulfate, Johnson & Johnson Consumer Inc., Skillan, NJ) was applied to the surgical area. Pups were then recovered in a warm recovery cage and placed back with the dam once fully recovered.

Thermal hypersensitivity

Thermal hypersensitivity testing was performed by the same person, who is adequately trained in laser stimulation, throughout the experiment. Prior to the laser stimulation, skin temperatures of both the left (ipsilateral) and right (contralateral) thigh of each pup was measured with an infrared thermometer (Extech instruments, Nashua, NH). The skin temperature of both ipsilateral and contralateral side was 33.8 ± 0.1˚C. Baseline thermal hypersensitivity of 5-day-old pups was determined at -1 h with laser stimulation adapted from Blaney et al. [20]. During stimulation and testing, two to three pups were evaluated at a time by maintaining them on a warm water circulating blanket (set at 38 ˚C) (Stryker T/Pump, Portage, Michigan, USA). Then, pups were tested with an infrared diode laser stimulator (LASMED, Mountain View, CA, USA, 490 mA, 5 mm diameter, 3.5 inch from skin) with a cutoff time of 19-sec to prevent skin burn. Thermal hypersensitivity was measured in sec as the time from when the laser was started until a purposeful paw movement away from the laser. The right and left thighs were tested twice, at three-minute intervals, and these two measurements were averaged. Thermal hypersensitivity was defined as a significant decrease in withdrawal latency following focal thermal stimuli.

Clinical observations and gross pathology

For the duration of the study, all pups were: 1) weighed daily at -1, 24, 48, 72 h(s); 2) observed for abnormal behaviors (i.e., general activity, mobility, maternal acceptance, and milk spots); 3) monitored for abnormalities at the injection site and incisional site. Gross pathology was performed at the end of the experiment.

Plasma collection

In a separate experiment, pups (n = 56) were assigned to the same treatment groups as the surgery experiment. Pups were anesthetized with 5–8% sevoflurane and injected with either Saline 0.1 ml SC (n = 2); Bup-SR 1 mg/kg SC (n = 18); XR-Lo 0.65 mg/kg SC (n = 18), or XR-Hi 1.3 mg/kg SC (n = 18). All pups then recovered in a warm cage and were placed back with the dam once recovered. For blood collection, pups were deeply anesthetized with 5–8% sevoflurane in 100% O2. After a surgical plane of anesthesia was confirmed by toe pinch, whole blood was collected by cardiac puncture using heparinized 1 mL tuberculin syringes and 25G needles. Blood was collected at 1, 4, 8, 24, 48, and 72 h(s) post- drug administration. For Saline group, blood collection was performed only at 1 h after injection. At the completion of blood collection, euthanasia was confirmed by cervical dislocation. Whole blood was collected (50 μL and up) in lithium heparinized microtainers and spun in a microcentrifuge at 2,500 rpm for 20 minutes. The plasma was collected into cryogenic tubes and stored in -80°C prior to shipment for analysis.

Plasma concentration analysis

Plasma samples were shipped on dry ice through overnight delivery to the McWhorter School of Pharmacy Pharmaceutical Sciences Research Institute (Samford University, Birmingham, AL) to analyze the plasma concentration of buprenorphine. The plasma concentrations of buprenorphine are obtained through liquid chromatography-tandem mass spectrometry (HPLC MS/MS). Buprenorphine standard spiking solutions were prepared by mixing deionized water and acetonitrile in 50:50 ratio to allow concentrations in plasma to range from 0.2–200 ng/mL. The buprenorphine plasma samples and standards (100 μL) were fortified with internal standard (50 ng/mL terfenadine). To precipitate the plasma proteins, 1 mL acetonitrile was added. The mixture was then vortexed and centrifuged followed by organic layer transferred to clean test tube and evaporated under nitrogen in a water bath at 50˚C until dry. The samples were then reconstituted in dilution solvent and analyzed by HPLC MS/MS. Blank control plasma was used to prepare matrix matched standards and QC sample. The lower limit of detection for this assay was 0.1 ng/ml. Due to the small volume of whole blood collected from pups, not all samples could be analyzed for buprenorphine concentration. Each sample analyzed had a minimum volume of 50 μL. The sample size for most timepoints analyzed was n = 3 but was n = 2 in XR-Lo 48 h, Bup-SR 1 h, Bup-SR 24 h, and n = 1 in XR-Lo 72 h and Bup-SR 72 h.

Statistical analysis

To assess significances of differences in skin temperature, thermal hypersensitivity, and body weight, over time, an F test in ANOVA with repeated measures, followed by Bonferroni correction to examine differences within groups over time and between groups at the same time point. Data was also tested for normality using R software (R Core Team, 2021). The data for plasma concentration was not statistically analyzed. Data is presented as mean ± standard error of the mean (SEM). A p-value of less than 0.05 was considered significant.

Results

Body weight

There were no differences between sexes for weight, therefore data was combined. The body weight statistically increased in all treatment groups from -1 h (Saline = 14.6 ± 0.3 g; Bup-SR = 15 ± 0.3 g; XR-Lo = 15.2 ± 0.3 g; XR-Hi = 14.9 ± 0.4 g) to 72 h (Saline = 22.8 ± 0.3 g; Bup-SR = 21.4 ± 0.3 g; XR-Lo = 21.7 ± 0.4 g; XR-Hi = 21.8 ± 0.4 g). Baseline (-1 h) body weight of rat pups in Saline (14.6 ± 0.3 g), Bup-SR (15 ± 0.3 g), XR-Lo (15.2 ± 0.3 g) and XR-Hi (14.9 ± 0.4 g) groups was not significantly different (Fig 1). The weight of the Saline group at 24 h (17.9 ± 0.3 g) was significantly higher than that of the Bup-SR group (16.7 ± 0.3 g) and the XR-Hi group (16.8 ± 0.4 g) but was not significantly different between the four treatment groups at any other time point.

Fig 1

Body weights of rat pups throughout the course of the study.

Mean body weight ± SEM. Arrow indicates the time of surgery. *Value significantly (P<0.05) increased compared to -1 h (baseline).

There was no thermal hypersensitivity difference between sexes; therefore, data was combined. Thermal hypersensitivity did not differ at -1 h (before surgery, baseline) between groups in the ipsilateral or contralateral thigh (Fig 2).

Fig 2

Thermal hypersensitivity results.

Thermal hypersensitivity of the (A) ipsilateral thigh measured in sec; mean ± SEM; * = Values significantly (P<0.05) different from that of -1 h (baseline) for the same treatment group. # = Values significantly (P<0.05) different compared to the Saline group at the same time point. (B) Contralateral thigh measured in sec; mean ± SEM. * = Values significantly (P<0.05) different from that of -1 h (baseline) for the same treatment group. # = Values significantly (P<0.05) different compared to the Saline group at the same time point. Arrow indicates the time of surgery.

Comparison within a drug group to baseline value (-1 h)

Ipsilateral thigh. Rat pups in the Saline group had significantly decreased (more sensitive) thermal latency at 1 (5.3 ± 0.4 sec), 4 (7.1 ± 0.7 sec), and 8 (6.9 ± 0.8 sec) h compared to –1 h (16.8 ± 1.4 sec; baseline). No significant differences were observed in the Bup-SR group at any time point compared to -1 h (15.5 ± 1.2 sec, baseline) in the ipsilateral thigh. Compared to its baseline values at -1 h (15.6 ± 0.7 sec), XR-Lo’s thermal latency significantly decreased (more sensitive) at 48 (10.6 ± 0.8 sec), and 72 (10.6 ± 1.0 sec) h. Compared to its baseline values -1 h (17.4 ± 0.5 sec), XR-Hi’s thermal latency significantly decreased (more sensitive) at 8 (11.6 ± 0.9 sec), 24 (11.3 ± 1.1 sec), 48 (10.7 ± 0.5 sec), 72 (11.1 ± 0.9 sec) h.

Contralateral thigh. No significant differences in thermal latency were observed between any group at any time points from their baseline measurements (Saline = 14.2 ± 1.2 sec; Bup-SR = 15.9 ± 1.1 sec; XR-Lo = 16.7 ± 0.8 sec; XR-Hi = 16.3 ± 0.8 sec).

Comparison between drug groups at the same time point

Ipsilateral thigh. Compared to the Saline group, the thermal latency for the Bup-SR, XR-Lo and XR-Hi groups were significantly increased (less sensitive) at 1 (Saline = 5.3 ± 0.4 sec; Bup-SR = 15.7 ± 1.1 sec; XR-Lo = 15.2 ± 1.1 sec; XR-Hi = 13.8 ± 0.9 sec), 4 (Saline = 7.1 ± 0.7 sec; Bup-SR = 11.8 ± 1.2 sec; XR-Lo = 11.0 ± 0.8 sec; XR-Hi = 11.6 ± 1.2 sec), 8 (Saline = 6.9 ± 0.8 sec; Bup-SR = 10.6 ± 0.7 sec; XR-Lo = 12.5 ± 1.0 sec; XR-Hi = 11.6 ± 0.9 sec), and decreased (more sensitive) at 72 (Saline = 14.0 ± 0.7 sec; Bup-SR = 11.2 ± 1.0 sec; XR-Lo = 10.6 ± 1.0 sec; XR-Hi = 11.1 ± 1.0 sec) h. The thermal latency for the Bup-SR, XR-Lo and XR-Hi groups was not significantly different between groups at any time point throughout the study.

Contralateral thigh. Compared to the Saline group, the thermal latency for the Bup-SR, XR-Lo and XR-Hi group was significantly increased (less sensitive) at 1 (Saline = 11.2 ± 1.4 sec; Bup-SR = 18.1 ± 0.6 sec; XR-Lo = 18.5 ± 0.5 sec; XR-Hi = 18.6 ± 0.4 sec), 4 (Saline = 11.0 ± 0.7 sec; Bup-SR = 17.4 ± 0.6 sec; XR-Lo = 16.8 ± 0.8 sec; XR-Hi = 17.5 ± 0.7 sec), 8 (Saline = 10.5 ± 1.4 sec; Bup-SR = 16.8 ± 0.4 sec; XR-Lo = 17.3 ± 0.8 sec; XR-Hi = 15.8 ± 0.8 sec) and 24 (Saline = 10.7 ± 1.2 sec; Bup-SR = 17.4 ± 0.4 sec; XR-Lo = 13.3 ± 1.2 sec; XR-Hi = 15.9 ± 0.8 sec) h. The thermal latency for the Bup-SR, XR-Lo and XR-Hi groups was not significantly different between groups at any time point throughout the study.

Plasma drug concentration analysis

Buprenorphine plasma concentration remained above 1 ng/mL at 1 and 4 h(s) after drug administration for all three treatment groups (1 h: Bup-SR = 3.375 ± 1.905 ng/mL, XR-Lo = 9.8 ± 1.3 ng/mL, XR-Hi = 17.0 ± 8.1 ng/mL; 4 h: Bup-SR = 4.1 ± 2.0 ng/mL, XR-Lo = 3.8 ± 0.5 ng/mL, XR-Hi = 5.0 ± 1.7 ng/mL) (Fig 3). The plasma concentration decreased to below 1 ng/mL in Bup-SR group at 8 h, XR-Lo group at 24 h, and the XR-Hi group at 48 h post drug administration (Bup-SR = 8 h: 0.7 ± 0.2 ng/mL; XR-Lo = 24 h: 0.5 ± 0.1 ng/mL; XR-Hi = 48 h: 0.5 ± 0.2 ng/mL). Saline injected pups were used as negative controls at 1 h (0 ng/mL).

Fig 3

Plasma concentration results of rat pups.

Plasma concentration (ng/mL, mean ± SEM) of Bup-SR, XR-Lo, and XR-Hi in treated rat pups (n = the number pups sampled at each time point). Samples were analyzed at 1, 4, 8, 24, 48, 72 h(s) after administration. n = 3 for all groups except n = 2 in XR-Lo at 48 h, Bup-SR at 1 h and Bup-SR at 24 h and n = 1 for XR-Lo at 72 h and Bup-SR at 72 h due to insufficient sample collected. Dotted line indicates clinically effective plasma concentration (1.0 ng/mL). Arrow indicates the time of drug administration.

Clinical observation and gross pathology

Decreased overall activity was observed for the first h after surgery in the Bup-SR (40%; 4/10 pups), XR-Lo (70%; 7/10 pups), and XR-Hi (70%; 7/10 pups) groups. Gross pathologic examination was performed at the end of the study at 72 h. At the drug injection sites (left shoulder), mass lesions characterized by firm ellipsoid nodules in the subcutis (0.4–1 cm) were observed in rat pups in the Bup-SR (40%; 4/10 pups), XR-Lo (40%; 4/10 pups), and XR-Hi (50%; 5/10 pups) treatment groups. No lesions were observed in the Saline group.

Discussion

The aim of this study was to evaluate if XR-Hi attenuates post-operative thermal hypersensitivity longer than XR-Lo in an incisional pain model in 5-day-old neonatal rats. This study demonstrates that XR-Hi did not attenuate postoperative thermal hypersensitivity longer than XR-Lo in 5-day-old rats. In short, a single dose of XR-Lo (0.65 mg/kg SC) attenuated thermal hypersensitivity for up to 8 h and XR-Hi (1.3 mg/kg SC) attenuated thermal hypersensitivity for 4 h in a modified incisional pain model of neonatal rats. This is the first study evaluating the efficacy of extended-release buprenorphine (XR-Lo and XR-Hi) in a neonatal rat model of incisional pain. As a result, we recommend XR-Lo at a dose of 0.65 mg/kg for analgesic management of an incisional pain model in neonatal rodents.

We adopted a modified incisional pain model because our lab has extensive experience with evaluating analgesic efficacy using this model representing a minor pain surgical procedure in rodents. Post-operative thermal hypersensitivity has been observed for up to four days in adult rats when using the plantar incisional pain model [29-32]. However, due to the small hind paw size of neonatal rats, we modified the incisional pain model and performed a skin incision with dissection of the underlying muscle on the left thigh to increase the area for post-operative hypersensitivity testing. Previous studies by our lab have indicated that the thigh incisional pain model induces a post-operative thermal hypersensitivity response for 4 to 8 h in neonatal rats [19, 20]. Previous work indicated that a diode laser provides sufficient stimuli to cause a withdrawal reflex to evaluate thermal hypersensitivity in adult rats [33]. Using this technique, we found that post-operative thermal hypersensitivity lasted for 8 h in the Saline group, indicating that this thigh incisional model produced thermal hypersensitivity for at least 8 h in rat pups. The contralateral thigh was also tested to serve as an uninjured control during testing. In this current study, differences in thermal hypersensitivity testing on the contralateral thigh found in the Bup-SR, XR-Lo, and XR-Hi groups as compared to Saline group, at 1, 4, 8, and 24 h are likely due to sedation from the treatment drugs. Although sedation was not clinically evident in this study, it has been previously noted in the literature when administering Bup-SR or XR-Hi to rats [28, 29, 34].

Sustained release formulations of buprenorphine offer refinement to laboratory animal analgesia practices. Bup-SR has been frequently used to manage post-operative pain as it requires less frequent dosing and provides a sustained duration of analgesia [20, 25]. Bup-SR was found to attenuate post-surgical thermal hypersensitivity (with a laser diode test) in neonatal rats for at least 8 h using the thigh incisional pain model [20]. In this current study, we confirmed these results. Recently, a new long-lasting buprenorphine formulation (XR-Hi) that is FDA-indexed and cGMP-compliant, became commercially available. Although both Bup-SR and XR-Hi have an identical active ingredient, buprenorphine, differences in their formulations and release technology may affect the drug’s pharmacokinetics. Therefore, we decided to evaluate XR-Hi as compared to Bup-SR in neonatal rodents. Our group has found that XR-Hi effectively attenuates post-operative pain for 48 h in adult mice [25] and adult rats [28] using the plantar incisional model. Such a difference between Bup-SR and XR-Hi in the duration of attenuating pain may be attributed to the technological differences between the two drugs. Bup-SR dissolves liquid polymer, with buprenorphine encapsulated inside, in a biocompatible organic solvent [25]. After injection, the polymer undergoes biodegradation, hydrolysis, and drug diffusion, resulting in the release of buprenorphine [24]. XR-Hi is contained in a lipid capsule, suspended in medium train fatty acid triglyceride. Unlike Bup-SR, buprenorphine in XR-Hi is released due to lipase and esterase activity [22]. This difference between Bup-SR and XR-Hi regarding how buprenorphine is released is one of the reasons for the different dosing adopted in this paper.

Due to developmental differences between neonates and adults, neonatal rodent pups are known to be more sensitive to anesthetics and analgesics. Analgesic dosages for neonatal pups should be adjusted to reflect the appropriate clinically effective dose for their age. Therefore, in this current study, to evaluate the safety, dosing, and efficacy of this new analgesic drug in neonatal pups, we also evaluated the low and high dose of XR-Hi which are efficacious for adult rats [28]. The XR-Lo group received the manufacturer’s recommended dose for rats (0.65 mg/kg) and the XR-Hi group received two times (1.3 mg/kg) the recommended dose. We found that XR-Lo attenuated thermal hypersensitivity up to 8 h while XR-Hi only attenuated thermal hypersensitivity until 4 h.

In the present study, both the XR-Lo and XR-Hi groups achieved peak plasma concentration at 1 h (XR-Lo = 9.8 ± 1.3 ng/mL; XR-Hi = 17.0 ± 8.1 ng/mL) and the Bup-SR group achieved peak concentration at 4 h (4.1 ± 2.0 ng/mL). At 8 h, the plasma concentration of Bup-SR was 0.7 ± 0.2 ng/mL and XR-Lo was 2.9 ± 0.5 ng/mL. During hypersensitivity testing at the 8 h timepoint, we found that both Bup-SR and XR-Lo groups attenuated thermal hypersensitivity. At 0.7 ng/mL, the buprenorphine concentration of Bup-SR at 8 h is near the clinically effective plasma concentration (1 ng/mL) and previous work has indicated that a lower effective plasma concentration of opioids may provide sufficient analgesia for neonates [35]. Although the clinically effective plasma buprenorphine for adult rats is reported to be at least 1 ng/mL [36, 37], in this modified incisional pain model, we found the effective plasma buprenorphine for neonatal rat pups to be at least 0.7 ± 0.2 ng/mL. To our knowledge, this is the first study determining the effective plasma buprenorphine concentration for attenuation of thermal hypersensitivity in five-day-old Sprague Dawley rats.

In this study, we did not find a clear correlation between the plasma buprenorphine level and hypersensitivity attenuation observed at each timepoint. For example, at 8 h the XR-Hi group had plasma concentration of 2.0 ± 0.9 ng/mL and thermal hypersensitivity was also observed on the ipsilateral thigh for this treatment group. Thermal hypersensitivity was also observed in XR-Hi treatment groups on ipsilateral thigh at 8 (XR-Hi) and 24 (XR-Hi) h despite plasma buprenorphine levels measuring above 1 ng/mL for these time points. One possible explanation for this discrepancy is that buprenorphine can have a low receptor binding rate leading to an unequal correlation between the plasma concentration of buprenorphine and the degree of analgesia [38]. Other possible reasons are that high levels of plasma buprenorphine may result in nociceptin induced hyperalgesia [39, 40] and/or opioid induced hyperalgesia [41]. Nociception induced hyperalgesia may be the result of buprenorphine interacting with the opioid receptor-like (ORL-1) receptor [39, 42, 43]. Nociceptin is structurally similar to the endogenous opioid peptide, is the endogenous ligand for ORL-1 receptor, and is also known to block the antinociceptive effects induced through μ, δ, and κ opioid receptors [44]. Opioid induced hyperalgesia (OIH), is a paradox where the ongoing or increased administration of an opioid leads to an unexpected increased pain perception and sensitivity [45]. Neonates in particular are known to have increased sensitivity to the effect of opioids [46] and OIH has been described in human neonates [45] and rat neonates [47]. Additionally, hyperalgesia has been previously associated with administration of low or very high opiate dosage such as 10 times higher than recommended dose [41, 48, 49]. We found this level of buprenorphine in the plasma for the XR-Lo (9.8 ± 1.3 ng/mL) and XR-Hi (17.0 ± 8.1 ng/mL) group at 1 h. OIH is also known to have dosage-dependent effects: the higher the dose administered, the more hyperalgesia observed [50, 51]. The administration of a large dose of opioid can increase the frequency and onset time of OIH [52, 53]. We suspect the fast onset of OIH in XR-Hi group at 8 h post-operatively is due to the high dosage administered, and that at the lower dosage (XR-Lo group), OIH was not observed until 48 h after drug administration.

While generally safe, administration of opioid drugs has been reported to produce nausea [54, 55], constipation [56, 57], vomiting [54, 58], body weight change [59, 60], and impaired gastrointestinal motility [61] in a variety of species which can lead to changes in body weight. Body weight is commonly used as a measurement of the post-operative well-being of research animals and can be used to access adverse effects following drug administration [60, 62]. After surgery and drug administration, all pups were accepted by the dam as evidenced by the presence of a milk spot and increased weight gain. Additional clinical signs that may be associated with buprenorphine include respiratory depression [63], pica [64], cardiovascular depression [65], and sedation [66]. In this study, cardiovascular or respiratory depression and pica were not observed. However, at 1 h post-surgery, decreased general activity was observed in 40% of pups in the Bup-SR group, 70% of the pups in XR-Lo and XR-Hi groups, and 10% of pups in the Saline group. The decreased general activity in the Saline group at 1 h might be due to residual anesthetic effects. A small number of pups still had decreased activity at 4 h post-drug administration (10% of Bup-SR and 20% of XR-Lo). This observation indicates that buprenorphine was likely responsible for some sedation after administration.

Sustained release formulations of analgesic drugs have been reported to lead to skin irritation and injection site reactions to both rats and mice [67-69]. Although in a previous study by our lab evaluating XR-Hi in adult mice, we did not find any gross pathologic abnormalities or injection site reactions [25]. A study by our lab investigated the effect of XR-Hi in adult rats and found subcutaneous nodules at the site of administration of XR-Hi and Bup-SR [28]. In this current study, although it was not clinically evident, we also found subcutaneous nodules at the time of gross pathological examination in the Bup-SR, XR-Lo, and XR-Hi groups. We suspect that the nodules can be attributed to the polymer matrix that is used in sustained release formulations [69]. In addition, previous studies have shown prolonged hyperalgesia to be observed up to 5 to 12 days after administration of very high dose and repeated subcutaneous injection of opioids in adult rats [41, 70, 71].

A limitation of this study is that only thermal and not mechanical hypersensitivity was evaluated, even though mechanical hypersensitivity is known to be present in neonatal rats [72, 73]. Von Frey monofilament test is commonly used for mechanical hypersensitivity [74]. Despite von Frey monofilament had been used to test for pain

(-like) hypersensitivity in neonatal rats [75], previous work from our lab indicated that this test was impractical and had inconsistent results with neonatal rats [19]. During a pilot study, the Randall-Selitto analgesiometer (Ugo Basile, Comerio, Italy) was also performed. This method aims to quantify the mechanical hypersensitivity threshold using an electronic device that quantifies the force applied. This method is commonly used in adult [76, 77] and neonatal rats [78]. However, we found that it was difficult to perform this test on the thigh and tail of neonatal rats, to identify painful behaviors with pups restrained, and to obtain consistent results. Future studies should also look at the effect of lower doses of XR-Hi in alleviating pain in neonatal rats to avoid OIH.

From a practical standpoint, assuming a 15 g pup, the current cost of XR-Hi ($0.65 mg/kg) is US $0.94, or for XR-Hi (1.3 mg/kg) US $1.88, based on list price. For comparison, the current cost of Bup-SR (1 mg/kg) is US $0.35. The cost estimated excludes human labor and clinical supplies required for the injection. Although Bup-SR is less expensive compared to XR, some institutions may elect to use XR-Hi because of requirements to use of cGMP compliant and FDA approved drugs. Furthermore, some readers may prefer XR-Hi since it is slightly less viscous than Bup-SR and is easier to draw up.

In summary, we found that a single dose of XR-Lo was safe and effective at attenuating post-operative thermal hypersensitivity in 5-day-old rat pups for at least 8 h for this model. XR-Hi was effective at attenuating thermal hypersensitivity until 4 h but resulted in opioid induced hypersensitivity, therefore, this dose is not recommended. Future studies should continue to evaluate the dosing, duration, and effectiveness of sustained release analgesics in neonatal rodents using a model that causes sustained hypersensitivity.

(XLSX)

Click here for additional data file.

11 Aug 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear author.

If the authors consider the following points, the manuscript will become more valuable:

1. The title is inconsistent; if possible, rewrite it to make it more clear.

2. The abstract is vague and inconsistent; the goal, experiment protocol, and groups are unclear; it should be rewritten. Abbreviations such as XR-Hi, XR-Lo, and XR should be placed appropriately. Introduction: from lines 50-53, these are results, that must be put in the result section.

Add some lines for the differences between Bup-SR and Bup-XR and why the author chooses these doses.

3. Experimental groups: lines 84-85 the dose of the group is 0.65 mg/ kg low dose extended-release buprenorphine (XR-Lo; ), but write 1.3 mg/kg, please confirm which dose is correct. Additionally, this section needs rewriting to be consistent. Why does the author use these doses?

4. Anesthesia and surgical section: lines 93-95, Then all experimental treatments and supplemental fluid (0.9% NaCl, 5 ml/kg) were administered SC at the left and right shoulders, respectively. This section is not obvious, please rewrite this section.

5. Thermal Hypersensitivity Testing: in line 116, write 700 AM, what are the authors mean here? Furthermore, this section is vague.

6. In plasma collection: this part of the study why separated from the first part?

7. Plasma Concentration Analysis: abbreviation of DI to what refer?

8. Results: In line 214, the authors write there is a significant decrease in the thermal hypersensitivity in times 1,4, and 8 hrs, but in figure 2 A there is an increase in the thermal hypersensitivity, please give an explanation for this increment. Please, all results need checking.

9. Discussion:

a. The aim written in line 264 differs from that in the introduction and abstract. Confirm which one is correct.

b. Write whole word before abbreviation of Ethiqa-xr.

c. Many sentences are repeated frequently in the manuscript, so, revisions are required. See lines 273- 275 and the paragraph before these lines as an example.

d. The authors write the tested drug is safely and effectively attenuated the thermal hypersensitivity, please how measure these parameters?

e. Lines 285-287 are related to the materials and method section.

f. The word Saline should be used in uniform, because some times write with capital letter and other times with small letter.

g. From line 296 to 310 are related to the introduction section.

h. Lines 325- 337 are repeated and related to results section.

With best regards,

Reviewer #2: Report on manuscript

�  Extended-release buprenorphine effectively attenuates thermal hypersensitivity in an incisional model in neonatal rats (Rattus norvegicus)

�  Manuscript ID: PONE-D-22-06559

�  Journal: PLOS ONE

�  Article type: Original Article

Thank you for the opportunity to review this manuscript. Current manuscript by Zhang et al, reports the analgesic effect of extended-release (XR) buprenorphine on thermal hypersensitivity in neonatal rats. Overall, the article addresses an interesting question: effectiveness of analgesic techniques in neonatal rats to manage post-operative pain. The authors demonstrate that low dose of XR-buprenorphine attenuate post-operative thermal hypersensitivity for longer than a high dose XR-buprenorphine.

Abstract is written in a precise way.

Introduction part is well-structured making sufficient connection between literature survey and research question.

Methodology is feasible and provides sufficient details. Study design is appropriate.

Results and discussion portions are described in an appropriate fashion.

Major Concern:

However, I have some concerns. Authors have already published their research data on “buprenorphine”/ “sustained-released buprenorphine” as an analgesic drug in rats/neonatal rats/mice (Reference # 19, 20, 22, 24, 30, 31). Further, analgesic effect of “XR-buprenorphine” on mechanical hypersensitivity in rats, was investigated and published (Reference 29). Current study is the continuation of the same research strategy involving XR-buprenorphine in neonatal rat model. Limiting point of this study is that this work is lacking novelty. Most of the work is repetitive based on the parameters of the previous publications and data. Can authors justify the novelty of current work in a more elaborating way? Another limitation (as described in discussion) is mechanical hypersensitivity which is not evaluated. This should be considered to reach a conclusive argument about the analgesic effect of the drug. This work is interesting and after improvement this can be helpful to have an insight into the improved analgesic techniques, in lab animal models.

Minor concerns:

• Abbreviation should be spelled out in the first instance. For example: Abstract Line 16: “Bup SR”.

• Line 24: Re-phrasing. “Subsequently after 1,4,8,24,48, 72 hr of treatment or subsequently post-treatment of….”

• Line 403: For comparison, the current “cost” not “cause”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Waleed K. Abdulsahib

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Dear author.docx

Click here for additional data file.

12 Sep 2022

Reviewer #1: Dear author.

If the authors consider the following points, the manuscript will become more valuable:

1. The title is inconsistent; if possible, rewrite it to make it more clear.

Response: The new title is renamed to make it clearer, “Effectiveness of Two Extended-Release Buprenorphine Formulations during Postoperative Period in Neonatal Rats”.

2. The abstract is vague and inconsistent; the goal, experiment protocol, and groups are unclear; it should be rewritten. Abbreviations such as XR-Hi, XR-Lo, and XR should be placed appropriately. Introduction: from lines 50-53, these are results, that must be put in the result section. Add some lines for the differences between Bup-SR and Bup-XR and why the author chooses these doses.

Response: Abstract is rewritten to make the goal, experiment protocol and groups consistent. The abbreviation for extended-release buprenorphine has been modified to Bup-XR to be consistent with previous published studies done by our institution. The abbreviations are now listed when the drug was first mentioned. .

Line 58-61 “Our group found that a single dose of 0.65 or 1.3 mg/kg extended-release

buprenorphine (XR), effectively attenuated post-operative mechanical hypersensitivity for 2 days in adult rats (data not shown). However, the safety and efficacy of extended-release buprenorphine in neonatal rats is currently unknown” are results of previous study or known information. “In a previous study,” has been added to the sentence to clarify this.

Some lines for the differences between Bup-SR and Bup-XR were added in the Introduction, “Bup-SR is a polymeric formulation that contains a water-insoluble, biodegradable polymer encapsulating buprenorphine and a biocompatible organic solvent. Bup-XR is lipid-bound and suspended in medium chain fatty acid triglyceride (MCT) oil that is degraded overtime with lipase and esterase activity”

“Doses of buprenorphine chosen were based on previous studies: 1) Bup-SR at 1 mg/kg was based on Blaney et al. (PMID 33534864) study; 2) Bup-XR at 0.65 and 1.3 mg/kg was based on doses in adult Sprague Dawley rats by Levinson et al (PMID 34183094) and Alamaw et al. (PMID 34903316) (note that doses of Bup XR in rat pups are not known).” This was also added into Materials and Methods section.

3. Experimental groups: lines 84-85 the dose of the group is 0.65 mg/ kg low dose extended-release buprenorphine (XR-Lo; ), but write 1.3 mg/kg, please confirm which dose is correct. Additionally, this section needs rewriting to be consistent. Why does the author use these doses?

Response: Lines 89-90, 1.3 mg/ml, a concentration (mg/ml) of the drug, was correct. This section was rewritten to be consistent. Doses used were explained in previous questions and added into “Materials and Methods”

4. Anesthesia and surgical section: lines 93-95, Then all experimental treatments and supplemental fluid (0.9% NaCl, 5 ml/kg) were administered SC at the left and right shoulders, respectively. This section is not obvious, please rewrite this section.

Response: This part was rewritten “All drugs (Bup-HCL, XR-Lo, and XR-Hi) were administered (SC) at the left shoulder. All rat pups were administered supplemental fluid (0.9% NaCl, 5 ml/kg, SC at the right shoulder).”

5. Thermal Hypersensitivity Testing: in line 116, write 700 AM, what are the authors mean here? Furthermore, this section is vague.

Response: We have revised accordingly

6. In plasma collection: this part of the study why separated from the first part?

Response: In plasma collection, this part of the study was separated from the first part because, to collect sufficient plasma (blood) volume for plasma buprenorphine concentration analysis in rat pups (approximately 15 grams), a terminal blood collection must be performed. Therefore, this part of the study was separated from the first part.

7. Plasma Concentration Analysis: abbreviation of DI to what refer?

Response: DI is “deionized” water. This is revised.

8. Results: In line 214, the authors write there is a significant decrease in the thermal hypersensitivity in times 1,4, and 8 hrs, but in figure 2 A there is an increase in the thermal hypersensitivity, please give an explanation for this increment. Please, all results need checking.

Response: Line 214 was revised to make it clearer, “there was a significant decrease in the thermal latency time at 1, 4, and 8 h”. The explanation is included in the discussion (line 218-220). In short, this incisional pain model decreased thermal latency (more painful and shorter response time) at 1, 4 and 8 h post-surgery in Saline (control) group which was similar to a study reported by Blaney et al. (PMID 33534864).

9. Discussion:

a. The aim written in line 264 differs from that in the introduction and abstract. Confirm which one is correct.

Response: The aim written in line 264, introduction and abstract were revised.

b. Write whole word before abbreviation of Ethiqa-xr.

Response: Ethiqa-xr was spelled out (revised)

c. Many sentences are repeated frequently in the manuscript, so, revisions are required. See lines 273- 275 and the paragraph before these lines as an example.

Response: Repeated sentences and lines 273-275 were revised.

d. The authors write the tested drug is safely and effectively attenuated the thermal hypersensitivity, please how measure these parameters?

Response: The tested drugs were evaluated for safety and efficacy through the determination that: 1) Thermal latency times of treatment drug groups on the ipsilateral thigh was not different from baseline (D-1) values at 4 and 8 (except for XR-Hi) h; 2) thermal latency times of treatment drug groups were higher than those of the Saline group at 4 and 8 h; 3) treatment drugs did not alter latency times on non-injured (contralateral) thighs; 4) maternal acceptance and the presence of a milk spot was evident in all pups (maternal acceptance including the presence of a milk spot is key for pup survival) (PMID 33534864); 5) All pups gained weight throughout the study (weight has been used to measure well-being PMID 31896391).; 5) Gross necropsies (in internal organs such as liver, kidneys etc) did not find significant or abnormal changes.

e. Lines 285-287 are related to the materials and method section.

Response: Lines 285-287 were moved to Materials and Methods section.

f. The word Saline should be used in uniform, because sometimes write with capital letter and other times with small letter.

Response: “Saline” was revised (uniformed).

g. From line 296 to 310 are related to the introduction section.

Response: Although line 296-310 is related to introduction, it is also appropriate to be present in the discussion section. Therefore, the authors decided to keep this section for the discussion.

h. Lines 325- 337 are repeated and related to results section.

Response: For lines 325-337, although this is similar to the result section, in the discussion these results are scientifically and analytically further summarized by addressing what new knowledge was found in the current study and what is previously known from other studies.

Reviewer #2: Report on manuscript

However, I have some concerns. Authors have already published their research data on “buprenorphine”/ “sustained-released buprenorphine” as an analgesic drug in rats/neonatal rats/mice (Reference # 19, 20, 22, 24, 30, 31). Further, analgesic effect of “XR-buprenorphine” on mechanical hypersensitivity in rats, was investigated and published (Reference 29). Current study is the continuation of the same research strategy involving XR-buprenorphine in neonatal rat model. Limiting point of this study is that this work is lacking novelty. Most of the work is repetitive based on the parameters of the previous publications and data. Can authors justify the novelty of current work in a more elaborating way? Another limitation (as described in discussion) is mechanical hypersensitivity which is not evaluated. This should be considered to reach a conclusive argument about the analgesic effect of the drug. This work is interesting and after improvement this can be helpful to have an insight into the improved analgesic techniques, in lab animal models.

Response: Although this current study is the continuation of the same research strategy, the novelty of the current study includes: 1) this is the first study demonstrating a newly marketed rodent FDA-indexed (pharmaceutical grade) extended-release buprenorphine (XR) in rat pups. This current study firstly showed that thermal hypersensitivity was attenuated for 4 h in XR-Hi and 8 h in XR-Lo groups. There is limited knowledge regarding the use of any extended-release formulation of buprenorphine in neonatal rodents. Different formulations of drugs can affect dosing regimen and effectiveness of the drug (PMID 25787030; 26854975). Although Bup-SR is an effective analgesic in adult rodents or other species, an effectiveness of XR (a different extended-release formulation of buprenorphine) in neonatal rats is not known; 2) this current study is the first to show that XR-Lo is as effective as commonly used sustained-release buprenorphine, Bup-SR, in rat pups; 3) this current study showed that the new formulation, XR, did not cause any skin reactions as have been observed with the use of Bup-SR (PMID 23294889; 21439213), hyperactivity observed was also observed in adult rodents (PMID 24459403; 34179720) and did not affect maternal acceptance (the key for pups survival and not evaluated in adult rodent studies). Although the parameters seemed to be repetitive, in pups, reliable and short duration hypersensitivity testing modalities were still not known. In addition, testing duration was crucial in pups because pups should not be removed too long from the dam due to potential complications of hypothermia and stress for both the pups and dam which can affect maternal acceptance or potentially lead to cannibalism. Therefore, the testing method chosen was based on what our group and others have published. Moreover, different surgical pain models cause different hypersensitivity responses; therefore, to be able to compare results to previous studies, a similar surgical model was selected. We are among the first groups to perform buprenorphine extended release testing in rat pups.

• Abbreviation should be spelled out in the first instance. For example: Abstract Line 16: “Bup SR”.

Response: Abbreviations were spelled out.

• Line 24: Re-phrasing. “Subsequently after 1,4,8,24,48, 72 hr of treatment or subsequently post-treatment of….”

Response: Line 24, re-phrasing was revised “subsequently after 1,4,8,24,48, 72 h of treatment”

• Line 403: For comparison, the current “cost” not “cause”.

Response: Line 403, “cause” was revised to “cost”.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

26 Sep 2022

30 Sep 2022

ACADEMIC EDITOR:

I will suggest to increase the clarity of discussion by incorporating scientific explanations of the research question and correct minor grammatical mistakes. The resolution of figures shall be increased to enhance clarity.

Response: We revised our Discussion section accordingly. The manuscript was proofread, and grammatical mistakes are corrected. The resolution of figures has been increased.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Response: We have revised accordingly.

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Response: Our statistical analysis was performed by Dr. Katechan Jampachaisri, a statistician, from the Department of Mathematics, Naresuan University.

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Response: We revised the manuscript and typographical or grammatical errors were corrected.

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear author,

Many sections of the manuscript are still vague and uninformative. The abstract is still vague and lacks a conclusion.

with regards,

Response: We revised the Abstract and Discussion sections.

Reviewer #2: Authors have made the required changes in the manuscript as suggested. Abstract is looking better now. I am satisfied with the explanation of authors regarding novelty of the research question. I will suggest to include this explanation in discussion to reveal its novelty and mentioning the limitations. I will also suggest to improve the resolution of the figures.

Response: We revised Discussion section accordingly. We also improved the resolution of the figures.

Submitted filename: Zhang et al. rebuttal letter.docx

Click here for additional data file.

5 Oct 2022

Effectiveness of Two Extended-Release Buprenorphine Formulations during Postoperative Period in Neonatal Rats

PONE-D-22-06559R2

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Oct 2022

PONE-D-22-06559R2

Effectiveness of Two Extended-Release Buprenorphine Formulations during Postoperative Period in Neonatal Rats

Dear Dr. Zhang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Sairah Hafeez Kamran

Academic Editor

PLOS ONE