Reagents and chemicals
Benzyl alcohol and benzyl benzoate were purchased from Merck Chemical Co., Ltd., Germany. Soybean oil was produced in Tieling Pharmaceutical Co., Ltd., China. Ropivacaine aqueous injection (RAI, 9 mg/mL) was obtained from Libang Pharmaceutical Co., Ltd., China. The ropivacaine reference was obtained from USP; and bupivacaine hydrochloride was obtained from the National Institutes for Food and Drug Control, China.
Preparation of RODD and solvent
Ropivacaine 36.5 g was added to benzyl alcohol 100.8 g and benzyl benzoate 405.5 g, then heated, and stirred. After the ropivacaine was dissolved, soybean oil 651.3 g were added to the ropivacaine solution. Then, the mixture was stirred until the solution clear. After sterile filtration with filter, the bulk solution of RODD 30 mg/mL was obtained. The RODD was diluted with solvent to 9 mg/mL. The solvent was prepared with the same procedure without ropivacaine.
Thirty New Zealand rabbits, male, 2.5 ± 0.5 kg, provided by Slakey Experimental Animal Center, Shanghai, China (SCXK (Hu).2007-0005). Specific-pathogen-free (SPF) Sprague Dawley (SD) rats, 6⁓8 weeks and 180⁓220g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., China (No.11400700175820). The animal were allowed to freely consume water. The houses were well ventilated, the temperature was 20°C⁓26°C, and the humidity was 40%⁓70%. All animals were conducted in compliance with the guidelines of ethical animal research. Procedures used in this study were approved by the Institutional Animal Care and Use Committee at Joinn Laboratories (No.ACU13-722). Animals were euthanized by intravenous injection of sodium pentobarbital after the study.
Rabbits were anesthetized with 5% isoflurane at beginning and 2% isoflurane in the process of the surgery. Five cm incision was cut at the right hind leg to expose the sciatic nerve. As shown in Fig. 1, three acupuncture needles marked as No 1, 2 and 3, as stimulating electrodes, were respectively bent, hooked to the sciatic nerve, and fixed on muscle by sutures. Needle 1 and 2 formed the proximal stimulating electrode of heart, while the needles 2 and 3 formed the distal stimulation electrode of heart. The distance between the needles around was 1 cm. After the surgery, ampicillin 50 mg was injected intramuscularly to prevent infection . The No. 4 needle, a reference electrode, was inserted into the ankle. No. 5 and No. 6 needles, as receiving electrode, were inserted into the sole and the muscle between toes, respectively.
Grouping and measurement
Rabbits were randomly divided into 5 groups with 6 in each group as follows: saline (model) group; solvent group, RAI 0.9 mg group; RODD 0.9 mg and 3 mg groups. The exposed sciatic nerve on needles 2 and 3 were dropped 0.1 mL of drugs, respectively. The animal hip was raised to keep infiltration status for 10 min, then the wound was sutured. Voltage stimulus in the electrode 2 and 3 was applied until waveform became stable. The stimulate voltage, the time and peak of electrical signal in the electrode 5 and 6 were recorded. The nerve conduction velocity were calculated at 0, 1, 2, 3, 4, 5, 30 min; 1, 2, 4, 6, 8, 10, 12, 16, 20, 24 h after administrations. Nerve conduction velocity (m/s) = distance between proximal electrode of heart and telecentric electrode/ (conduction time between proximal and distal electrodes).
Grouping and administration
In toxicology experiment 150 rats were divided into 5 groups with 30 rats per group: saline group, solvent group and RODD 75, 150 and 300 mg/kg groups, half male and half female. After fasting 12 h, the rats were subcutaneously injected with RODD (30 mg/mL) at multiple points, and the volume was not more than 1 mL per point. Rats of saline or solvent group were given 10 mL/kg of saline or solvent, respectively.
Daily observations were performed to evaluate the external signs and behavioral activities of the rats, including mental condition, hair color and oral, nasal, eye, ear and external genital secretion. The analysis of toxicity included the evaluation of the central and motor nerve systems (behavior, movement, response to stimulation, neural reflex and muscular tension), vegetative nervous system (pupil and secretion), respiratory system (nasal and respiratory rate), urogenital system (labia, mammary gland and perineum), skin and hair (color and integrity), eye (eyelid and eyeball), ear (auditory meatus), etc.
Weight and food consumption
The rats were raised in individual cages and their body weights were measured before the experiment and on days 1, 4, 7, 11 and 14 of the experiment. Additionally, the food consumption of the rats within the first 24 h of the experiment were measured by the weight loss method.
After the rats were anesthetized, blood was gathered from the abdominal aorta and the plasma was separated. Blood cell characteristics and blood biochemical parameters, including alanine amino transferase, albumin, albumin/globulin ratio, total protein, triglyceride and so on, were evaluated. Coagulation function were analyzed. Electrolytes such as total calcium, inorganic phosphorus, sodium, potassium ion concentration and chloride ion concentration were detected by an easylyte electrolyte analyzer.
Anatomy and pathological examination
The rats were euthanized on days 3 and 14. The body surface, orifice, cranial cavity, thorax, abdominal cavity, pelvic cavity and their contents were observed. The organs were separated and fixed in 10% neutral formalin solution, and the testicles were preserved with Davidson's solution. The body and visceral organs were weighed, and visceral indexes were calculated, including the brain, thymus, thyroid gland, parathyroid gland, hepatic, kidney, adrenal gland, spleen, lung, heart, prostate, testis, epididymis, uterus, ovary, etc. The organs of the rats were treated with routine histological operations, including paraffin embedding, sectioning, preparation, HE staining, etc., and examined under a microscope.
Detection of blood ropivacaine concentration
The chromatographic conditions and methods of LC-MS/MS in the study were from the revious study as follows: chromatographic column, Ultimate XB-C18 (4.6×150 mm, 5 µm); mobile phase, aqueous solution of 5 mM ammonium acetate:acetoni-trile (5:95 (v/v)); flow rate, 1.0 mL/min; injection volume, 10 µL; and run time, 4 min. The electrospray ionization source was an API 4000 Qtrap, ion trap mass spectrometry was used as the detector and a positive ion detection method was applied. The capillary voltage was 0.5 kV, the ion source temperature was 120°C, the atomization temperature was 350°C, the conical hole gas velocity was 150 L/h, and the atomized gas velocity was 650 L/h. Methodological validation included specificity, accuracy, limit of quantitation, precision, matrix effects and recovery, etc.
Grouping and testing
Fifty four rats were divided into RODD 75, 150 and 300 mg/kg groups with 18 rats per group, half male and half female. After fasting 12 h, RODD was injected subcutaneously on the back neck of rats at multiple points, and the volume was not more than 1 mL per point. Blood (0.5 mL) was extracted from the orbital veins at 0, 0.25, 0.5, 1, 2, 4, 8, 24, 48 and 72 h after injection. Plasma (50 µL) was added into 950 µL of biological matrix (containing 30 ng/mL internal standard). The protein in plasma was precipitated with methanol. Then, the samples were vortexed for approximately 1 min and centrifuged at 12000 RPM for 10 min, the supernatant (20 µL) was accurately transferred and diluted with 380 µL of water. Finally, the solution was vortexed and analyzed by LC-MS/MS. The toxicokinetic parameters, including AUC, Cmax, Tmax, etc., were calculated in WinNonlin (V6.2) software.
Data are presented as mean ± standard deviation (SD). The pharmacodynamics data were analyzed by one-way ANOVA. The toxicological data were tested for homogeneity of variance by Levene’s test and when the variance was homogeneous, one-way ANOVA was performed. If Levene’s test showed heterogeneity of variance, the Kruskal-Wallis nonparametric test was performed. The part of statistical analyses were conducted using GraphPad prism 6.0 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered significant.