Pharmacokinetics and local tissue response to local instillation of vocacapsaicin, a novel capsaicin prodrug, in rat and rabbit osteotomy models

Abstract Vocacapsaicin is a novel prodrug of trans‐capsaicin (trans‐8‐methyl‐N‐vanillyl‐6‐nonenamide) being developed as a nonopioid, long‐lasting, site‐specific treatment for postsurgical pain management. The objective of these studies was to examine the safety and tolerability of vocacapsaicin in an osteotomy model in two animal species and to evaluate bone healing parameters. Rats undergoing unilateral femoral osteotomy received a single perioperative administration (by instillation) of vocacapsaicin (vehicle, 0.15, 0.3, and 0.6 mg/kg). Rabbits undergoing unilateral ulnar osteotomy received a single perioperative administration (by infiltration and instillation) of vocacapsaicin (vehicle, 0.256 and 0.52 mg) alone or in combination with 0.5% ropivacaine. Clinical signs, body weights, food consumption, radiography, histopathologic examinations, ex vivo bone mineral density measurements (rats only), and biomechanical testing were evaluated at 4 and 8 weeks in rats and at 2 and 10 weeks in rabbits. Plasma samples were also collected in rabbits. There were no vocacapsaicin‐related effects on mortality, clinical observations, body weight, or food consumption in either species. Systemic exposure to vocacapsaicin and its metabolites, including capsaicin, was transient. In rats, vocacapsaicin was devoid of deleterious effects on bone healing parameters, and there was a trend for enhanced bone healing in rats treated with the mid‐dose. In rabbits, vocacapsaicin administered alone or in combination with ropivacaine did not adversely affect bone healing parameters. In conclusion, a single perioperative administration of vocacapsaicin in unilateral osteotomy models was well tolerated, locally and systemically, supporting its continued development as a novel, nonopioid treatment for postsurgical pain management.


| INTRODUCTION
Enhanced recovery protocols have improved postsurgical outcomes in a wide array of surgeries. A cornerstone of this approach is multimodal analgesia, which utilizes a range of mechanistically distinct pharmacological treatments to inhibit nociceptive processing at multiple levels of the neuroaxis. 1 By using various combinations of acetaminophen, nonsteroidal anti-inflammatory drug, gabapentinoids, and local anesthetics, it is possible to decrease postsurgical opioid requirements, and, in turn, opioid-related adverse events and risk of persistent opioid use in the outpatient setting. 2 Opioid-sparing strategies have been shown to reduce acute postoperative pain, the risk of developing chronic pain, length of hospital stay, and per patient costs. [3][4][5][6] Despite these advances, up to 75% of patients report significant pain in the postsurgical setting. 7,8 There remains an unmet Once released from vocacapsaicin, capsaicin produces agonist activity at TRPV1 receptors located on primary afferent C-fibers. [9][10][11] This neuronal excitation initially causes a painful, burning sensation; however, with continued depolarization, the nociceptors become reversibly desensitized, leading to durable analgesia. 12 Capsaicin has been used for decades as a topical analgesic, but its utility for other routes of administration has been limited by its poor water solubility.
Prodrugs afford one approach for altering the physicochemical properties of existing molecules to enhance their delivery to the desired tissue. 13 Vocacapsaicin was developed to improve upon the solubility profile of capsaicin and allow for an aqueous formulation that could be simply administered in the wound site to achieve a long-lasting analgesic effect. Vocacapsaicin has little or no pharmacologic activity as the intact prodrug and is rapidly converted to capsaicin. As vocacapsaicin is administered and designed to release capsaicin rapidly at the surgical site, in contrast to historical topical uses, it is important to examine its overall safety and tolerability profile, including any systemic exposure and evaluation of any effects on healing at the surgical site. The objective of these studies was to examine the safety and tolerability of vocacapsaicin in an osteotomy model in two animal species (rats and rabbits), and to evaluate radiographic, histopathologic, and biomechanical bone healing parameters in both species. While analgesia was not formally measured in these studies, concentrations of vocacapsaicin were equivalent to or higher than those associated with postsurgical analgesia in randomized, controlled clinical trials in patients following bunionectomy and total knee arthroplasty. 14 Rats were anesthetized with isoflurane gas and prepared for surgery. An incision was made on the lateral aspect of the right femur. The fascia, separating the tensor fascia lata and biceps femoris muscles, was identified and incised. The vastus lateralis muscle was elevated off the lateral border of the femur, from the greater trochanter to the lateral femoral trochanter condyle, with care to preserve the overlying periosteum. A fixation kit that included a connecting polyether ether ketone plate and four metallic screws was used. An osteotomy was performed at the midshaft using an oscillating saw. The osteotomy site was thoroughly irrigated with sterile fluids during the osteotomy to avoid any microdamage caused by heat. A cerclage wire was used to stabilize the bone and the site was irrigated with saline to remove bone debris. The soft tissues were meticulously closed in layers. At least 30 min before surgery, subcutaneous injections of buprenorphine (0.1 mg/kg), meloxicam (2 mg/kg), and trimethoprim sulfa (30 mg/kg) were administered to each rat. Following surgery, three additional injections of buprenorphine and trimethoprim sulfa were administered at 12-h intervals F I G U R E 1 Mechanism of conversion of vocacapsaicin to capsaicin and CA-101 (for a total of 4 injections), and two additional injections of meloxicam were administered at approximate 24-h intervals (for a total of 3 injections). Additional doses of buprenorphine and meloxicam could be administered thereafter at the discretion of the study director.
Within 2 days of arriving at the animal facility and before surgery, rats were assigned to dosing groups in a blinded process by a stratified randomization scheme designed to achieve similar body weights among groups. Rats were assigned to one of four dosing groups (n = 35-36/group): 0 (vehicle control-saline for Injection, USP), 0.15, 0.3, or 0.6 mg/kg vocacapsaicin. Each dosing group was comprised of a 4-week and an 8-week subset of animals (n = 17-18/ subset) ( Table 1). Animals in poor health or at extremes of body weight range were not assigned to groups. Immediately after surgery, vehicle or vocacapsaicin was administered once (in a volume of 0.5 ml/kg), before wound closure, by instillation directly on the sur-  All animals received 120 g per day of a standard commercial laboratory diet (PMI Certified Rabbit 5325: PMI Nutrition International Inc.) and water was available ad libitum. The study plan was reviewed and approved by Charles River MTL IACUC.
Rabbits were anesthetized with ketamine and dexmedetomidine before surgical preparation and were maintained under isoflurane anesthesia for the entire surgery. An incision of approximately 4 cm in length was performed on the lateral aspect of the distal ulna of the right forelimb and the ulna was exposed extraperiosteally. The periosteum was cut longitudinally and detached from the bone along the incision. A cut the width of the saw blade was performed in the midulna using a pendular saw. The soft tissues were meticulously closed in layers. Each animal received subcutaneous injections of trimethoprim-sulfa 24% (30 mg/kg), buprenorphine (0.05 mg/kg), and carprofen (4 mg/kg) 30 min before surgery. Following surgery, additional doses of trimethoprim-sulfa 24% (q12 h for 2 days), buprenorphine SR (0.1 mg/kg, 2 injections, 3 days apart), and carprofen (qd for 7 days) were administered.
Vehicle, vocacapsaicin, ropivacaine, and vocacapsaicin/ropivacaine were administered with a syringe needle on the day of surgery (Day 1) as described in Table 2. For Groups 1-3, Vehicle Food consumption was measured daily from Day −1 to the end of the study. Body weights were measured weekly starting at Week −3.
T A B L E 1 Dosing for rat unilateral femoral osteotomy model a Rats were assigned to dosing groups using a stratified randomization scheme designed to achieve similar mean body weights.
b Several animals were replaced by spare animals (as established a priori) due to radiographic observations (e.g., incomplete fracture), death during surgery, or poor condition following surgery. c One animal was euthanized during surgery and not replaced. PK parameters were estimated for CA-008, CA-101, and capsaicin using noncompartmental methods with Phoenix PK software.
The following parameters were calculated for each analyte: time after dosing at which the maximum concentration was observed (T max ), the maximum observed concentration (C max ), the area under the concentration versus time curve from dosing to time of last quantifiable concentration (AUC 0-t ), AUC 0-t /dose, C max /dose, and half-life (t 1/2 ).

| Radiography
Rats  Note:represents not applicable.
Abbreviations: F, females; M, males. a Rabbits were assigned to dosing groups using a stratified randomization scheme designed to achieve similar mean body weights.
b On the basis of a 2.5 kg body weight.
c Several animals were replaced by spare animals (as established a priori) due to radiographic observations (e.g., incomplete fracture) or poor condition following surgery. d Scheduled for termination on Week 2 for histopathological evaluation.
e Scheduled for termination on Week 10, 2 animals/sex/group for histopathological evaluation and 8/sex/group for biomechanical testing.

Rabbits
For the histopathological population, the right ulna bone and the surrounding soft tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Histopathological evaluation was performed by a board-certified veterinary pathologist.

| Ex vivo bone densitometry (rats only)
Peripheral quantitative computed tomography (pQCT) scans of the osteotomy site were performed using an XCT Research SA bone scanner (Stratec Medizintechnik). The first slice was performed proximally and adjacent to the osteotomy site and four subsequent slices were then acquired distally to image as much of the osteotomy site as possible.
The three sequential slices determined to most accurately span the osteotomy site were selected for analysis. The area (mm 2 ), bone mineral content (mg/mm), and bone mineral density (mg/cm 3 ) were reported for the total osteotomy site, the mature callus area, and the immature callus area, using averaged values from analyses of the three approved slices.

Rat study
A stock solution of vocacapsaicin (2.4 mg/ml) at pH 3 was prepared and used to prepare individual dosing solutions. The appropriate amount of vocacapsaicin item was dissolved in pH 3.0 saline to meet necessary volume requirements, and the pH of the resulting solution was adjusted to 4.5 using 0.01 N NaOH. Vehicle control was saline for injection, USP.

Rabbit study
Vocacapsaicin was dissolved in citrate buffer and diluted with saline, with a final pH of 3.6-3.8. Ropivacaine (0.5%, 5.0 mg/ml) was filtered, aliquoted, and then dispensed as received on the day of dosing for administration. Vehicle control consisted of 0.5 mM citrate buffer and 0.4 mg/ml mannitol, in saline, with a final pH of 3.5-3.8.     on Day 29 because of large cutaneous ulceration on the scapular region, which was unrelated to vocacapsaicin administration.

| Pharmacokinetics
In males and females, systemic exposure to CA-008 was transient, with T max observed at the first sampling time (15 min) and concentrations decreasing below the limit of quantitation by 2 h postdose ( Figure 4). Similarly, for CA-101 and capsaicin in both males and females, mean T max was observed at the first sampling time and concentrations were below the limit of quantitation by 4 h postdose. Derived PK parameters for all three analytes indicate dose-dependent increases in exposure that were generally linear (Table 5). For all three analytes, exposure was slightly lower when administered with ropivacaine. There were no sex differences in exposure.   17,18 In rabbits treated with ropivacaine alone, there were also no adverse effects on bone healing. Some in vitro studies have demonstrated the cytotoxic potential of local anesthetics, particularly bupivacaine and mepivacaine, and a bupivacaine collagen-matrix implant delayed bone healing in a rat osteotomy model. [19][20][21] The lack of effect of ropivacaine on bone healing in the current studies is generally consistent with preclinical and clinical observations for ropivacaine and other local anesthetics, including liposomal bupivacaine, in both bone and wound healing studies. [22][23][24][25][26][27][28] Interestingly, in rats treated with the mid-dose of vocacapsaicin, 0.3 mg/kg (0.6 mg/ml), there was a trend for enhanced bone healing relative to control rats, as evidenced by increases in radiographic bone healing scores, percentage of animals with mature or nearly mature calluses, and AUCs (+78%) for the femur in 4-point bending.

| In vivo radiographic evaluation
Such trends were not observed; however, for the low or high dose in rats or for either dose in rabbits. A potential role of TRPV1 receptors in bone healing has been previously documented in rodent models. were no clinically relevant findings and the no-adverse-effect level was equal to or higher than the highest concentrations tested in the current study (unpublished data). When vocacapsaicin was administered in combination with ropivacaine, systemic exposure to intact vocacapsaicin, CA-101, and capsaicin were somewhat lower relative to vocacapsaicin administration alone. Due to the small number of animals tested, it is not possible to ascertain whether this is due to interanimal variability or to a modest effect of ropivacaine on exposure to these analytes.
In summary, a single perioperative administration of vocacapsaicin in rat and rabbit unilateral osteotomy models was well tolerated, locally and systemically, as demonstrated by the absence of mortality directly related to vocacapsaicin, adverse clinical observations, or effect on body weight and food consumption. Systemic exposure to vocacapsaicin and its metabolites, including capsaicin, was transient. In rats, vocacapsaicin was devoid of deleterious effects on bone healing as assessed microscopically and by radiography and biomechanical testing, and there was a trend for enhanced bone healing in rats treated with the mid-dose, but not low or high doses, of vocacapsaicin. In rabbits, vocacapsaicin was also devoid of adverse effects on bone healing parameters and coadministration of ropivacaine did not alter clinical observations or bone healing findings. The collective findings are consistent with a product that could potentially provide a well-tolerated, nonopioid, site-specific treatment for longlasting postsurgical pain management.