The Safety of Adiponectin Receptor Agonist AdipoRon in a Rabbit Model of Arthrofibrosis

Abstract

AdipoRon is an adiponectin receptor 1, 2 (ADIPOR1 and ADIPOR2) agonist with numerous reported physiological benefits in murine models of human disease, including a proposed reduction in fibrosis. However, AdipoRon has never been investigated in rabbits, which provide a robust model for orthopedic conditions. We examined the safety of intravenous (IV) AdipoRon in New Zealand White (NZW) female rabbits surgically stressed by a procedure that mimics human arthrofibrosis. Fifteen female NZW rabbits were prospectively studied using increasing AdipoRon doses based on established literature. AdipoRon was dissolved in dimethyl sulfoxide (DMSO), diluted in normal saline, and administered IV preoperatively and for 5 subsequent days postoperatively. The primary outcome was overall toxicity to rabbits, whereas secondary outcomes were change in rabbit weights and hemodynamics and defining acid–base characteristics of the drug formulation. Two rabbits expired during preoperative drug administration at 25 mg/kg. Remaining rabbits received preoperative doses of DMSO (vehicle), 2.5, 5, or 10 mg/kg of AdipoRon without complications. On postoperative day 1, one rabbit sustained a tonic–clonic seizure after their second dose of 10 mg/kg AdipoRon. The remaining 12 rabbits (4 in each group) received six serial doses of vehicle, 2.5, or 5 mg/kg of AdipoRon without adverse effects. All formulations of AdipoRon were within safe physiological pH ranges (4–5). We are the first to report the use of IV AdipoRon in a surgically stressed rabbit model of orthopedic disease. AdipoRon doses of 5 mg/kg or less appear to be well-tolerated in female NZW rabbits.

Impact statement

We provided the first in vivo toxicity assessment and dose optimization of a new antifibrotic experimental medication, AdipoRon, in a surgically stressed rabbit model of knee arthrofibrosis.

Introduction

Adiponectin (ADIPOQ) is a hormone with multiple physiological functions including lipid and glucose metabolism, energy regulation, and mediation of inflammation and fibrosis.¹ ADIPOQ potentiates its downstream effects through its two primary receptors, ADIPOR1 and ADIPOR2, which are expressed in numerous tissues including brain, liver, kidney, spleen, and skeletal muscle.^2,3 Given these pleiotropic effects, ADIPOQ represents an attractive remedy for multiple disease states.

Despite its appeal, exogenously administered ADIPOQ is structurally unstable and has poor bioavailability.⁴ Thus, direct agonists of its downstream receptors that can be delivered using feasible instillation methods may be more clinically impactful. AdipoRon is a synthetic and orally bioavailable ADIPOQ derivative developed in 2013 as a direct agonist of ADIPOR1 and ADIPOR2.⁵ In the first reported use of AdipoRon by Okada-Iwabu et al.,⁵ 50 mg/kg of orally administered AdipoRon improved insulin and glucose profiles in diabetic knockout mice.

Numerous studies have since emerged highlighting the therapeutic role of AdipoRon across a wide array of diseases including effects on cardiac lipotoxicity, aging skeletal muscle, treatment of pancreatic cancer, hepatic and renal protection, bone health and healing, neurocognition, and anti-inflammatory/antifibrotic effects.^4,6–16 Despite these benefits, AdipoRon has not been tested in larger animal models of musculoskeletal (MSK) disease, such as rabbits, and few have reported its use intravenously (IV) limiting its current translatability to orthopedic pathologies.^14,17,18

As such, the primary purpose of this study was to investigate the safety of AdipoRon in a surgically stressed rabbit model of arthrofibrosis (knee stiffness), which is a relatively common orthopedic condition for which rabbit models are considered the gold standard animal model.^19–27 Importantly, our study addressed a critical gap in the literature to inform future investigative efforts into the safe use and efficacy of AdipoRon in translatable animal models of orthopedic diseases.

Methods

Experimental animals

Fifteen skeletally mature female New Zealand White (NZW) rabbits were included in this study. All animals were housed in a professionally maintained animal housing room with their own cage space (1 m³). All rabbits had continuous access to rabbit chow and water. Institutional animal care and use committee (IACUC) approval was obtained before this study was conducted and the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines were followed to perform this investigation. All animal cares were supervised by a board-certified consultant veterinarian.

Dosing selection

A literature review was conducted to estimate safe dose ranges for AdipoRon in animal models given that AdipoRon had never been tested in rabbits. Literature on AdipoRon in animal models is limited mainly to murine studies in mice and rats.^4,6–16 Among these studies, safe weight-based dose ranges of up to 50 mg/kg in serial dosing were identified, with one study reporting neurotoxicity (memory deficits) after 14 days of 50 mg/kg dosing intraperitoneal in mice.²⁸

As such, initial dose ranges of 2.5, 5, 10, and 25 mg/kg were selected for this experiment as 25 mg/kg represented the middle of the effective dose ranges reported in multiple other small animal models.^4,6–16 Once daily dosing was selected based on efficacy demonstrated from these established studies. Treatments were not blinded to the investigative team, to permit dynamic reallocation of dose groups as indicated by any observed toxicities.

Solvent selection and drug preparation

AdipoRon was purchased from Tocris (Bio-Techne, Minneapolis, MN) in powdered form. Its industry-recommended solvent, dimethyl sulfoxide (DMSO), was selected as the primary solvent to bring the drug into a sterile stock solution. Stock AdipoRon in DMSO solution was then aliquoted into isolated tubes for each rabbit and stored in a −20°C freezer for use on the day of the intended rabbit surgery and thereafter. Normal saline was selected as the final carrier/dilutant after an assessment of safe acid–base characteristics (i.e., physiological pH) using multiple dosing combinations of both buffered and nonbuffered saline.

Safety assessment

Fifteen female NZW rabbits were prospectively included with the intention to treat these animals with DMSO (vehicle) or escalating doses of IV AdipoRon (2.5, 5, 10, and 25 mg/kg) before and after a surgical intervention that induced an arthrofibrosis phenotype (Fig. 1).¹⁹ In brief, this procedure involved an arthrotomy of the joint, hyperextension of the knee to disrupt the posterior knee capsule, drilling of two cortical windows in the femoral condyles, and finally transtibial to perifemoral Kirschner wire fixation to fix knees in maximum flexion.

FIG. 1.

Study timeline (A) reflecting scheduled dosing with AdipoRon (●). Experimental design (B) including 15 rabbits that were distributed into four dose groups to test the safety of AdipoRon in New Zealand White rabbit models of arthrofibrosis. Toxicity resulting in removal from the study marked (X).

Within 1 h of surgery, AdipoRon stock solution (or vehicle) was thawed (a total of one thaw per sample) and mixed with normal saline (1:1 volume mixture) to bring it up to weight-based dosing (amounts of AdipoRon and DMSO normalized to total body weight).

After sedation with weight-based ketamine and xylazine, cefazolin and buprenorphine were administered followed by intubation by a trained veterinary technologist. Vehicle and AdipoRon in saline were administered before the surgical stressor on postoperative day (POD) 0, and once daily for 5 days on PODs 1–5. Rabbits were followed throughout these 6 days for evidence of toxicity and were weighed after the final dose to monitor for clinically significant weight loss (defined as weight loss >20% of body weight) and measured for cardiovascular effects at the time of initial drug dosing.

Study outcomes

The primary outcome was clinical evidence of toxicity observed in rabbits that were administered AdipoRon solution or vehicle alone. Secondary endpoints included weight loss after six daily doses, observation for clinically significant effects on the cardiovascular system during instillation, and acid–base characteristics of various combinations of AdipoRon in DMSO with normal saline or buffered saline solution.

Statistical methods

Descriptive statistics were used to describe differences in the continuous variables of weight and vital signs.

Experiment

Of the 15 female NZW rabbits that were studied, 2 died during initial AdipoRon drug instillation preoperatively at a dose of 25 mg/kg. Both mortalities were sudden, but both rabbits had received weight-based dosing of ketamine, xylazine, cefazolin, and intramuscular instant release buprenorphine before instillation of AdipoRon IV through the marginal ear vein. Warm necropsy by a board-certified veterinarian followed by gross pathology by a board-certified animal pathologist was conducted to assess for gross tissue evidence of drug-induced toxicity. The impression from that pathology report was inconclusive.

Dose groups were reduced to vehicle, 2.5, 5, and 10 mg/kg for the remaining rabbits, and long-acting buprenorphine was administered to account for potential drug synergy or potentiation with AdipoRon or DMSO (Fig. 1). The index surgical procedures occurred over the course of 3 days. The first operative day included a single rabbit that received vehicle alone that was well tolerated. The second operative day added four rabbits with one each in the vehicle, 2.5, 5, and 10 mg/kg dose groups, respectively.

On POD 1, the single rabbit that received 10 mg/kg sustained a tonic–clonic seizure after their second dose of AdipoRon. This rabbit received no further doses of AdipoRon and was removed from the study to a training protocol. As such, this observation led to the decision to reduce all remaining doses to no more than 5 mg/kg.

Twelve rabbits received all six serial doses of AdipoRon or vehicle with four each in the vehicle, 2.5 and 5 mg/kg dose groups, respectively. These 12 rabbits had no observed toxicity throughout the dosing period. Furthermore, these rabbits had a mean weight loss of 8.9% after final drug administration (Fig. 2) and no clinically significant effects on their heart rates during initial drug administration (Fig. 3). Finally, drug pH with or without buffered saline solution was consistently 4–5, reflecting safe use in vivo (Fig. 4). At the conclusion of the safety analysis, all rabbits were humanely transferred to another protocol.

FIG. 2.

Effect of AdipoRon on rabbit weight by dose group with DMSO as the vehicle control group. DMSO, dimethyl sulfoxide.

FIG. 3.

Effect of AdipoRon on the heart rate of rabbits by dose group with DMSO as the vehicle control group.

FIG. 4.

Acid–base characteristics of AdipoRon stock solution in DMSO with and without buffered saline.

Discussion

We report the first use of the direct ADIPOR1 and ADIPOR2 agonist, AdipoRon, instilled IV in a surgically stressed rabbit model of arthrofibrosis. Despite 8 rabbits having successfully received six daily doses of either 2.5 or 5 mg/kg AdipoRon, unfortunately, 2 of the 12 expired at 25 mg/kg, and 1 sustained a tonic–clonic seizure on POD 1 after its second dose of 10 mg/kg, suggesting a potential dose-dependent toxicity in rabbits. This contradicted broader previously published dosing ranges observed in studies of murine species.^4,6–16 Finally, of the 12 rabbits that received 2.5–5 mg/kg injections, clinically significant weight loss and hemodynamic changes were not observed.

Existing in vivo studies of AdipoRon are limited to murine species that seldom reported dose-dependent toxicity in the animals under study. Of the available literature on AdipoRon in vivo, only one article suggested mild memory impairment in mice administered 50 mg/kg of AdipoRon for 14 days.²⁸ The remaining studies concluded that the drug is safe when administered both orally (through gavage or intraperitoneal injection) or IV.^4,6–18 AdipoRon is thought to cross the blood–brain barrier after both systemic and oral administration.^29,30

In our study, although two rabbits died acutely, another was removed on POD 1 after a tonic–clonic seizure after the second dose of 10 mg/kg AdipoRon. This suggested the potential for cumulative dosing-related neurotoxicity at 10 mg/kg in rabbits. However, only one rabbit was tested at 10 mg/kg at the recommendation of our consultant veterinarian, and future investigations would be useful to understand whether AdipoRon is tolerated at 10 mg/kg serial dosing in rabbits. Although other studies in smaller animal models have determined that AdipoRon is safe in sequential dosing up to 50 mg/kg, our experience suggested that AdipoRon IV in rabbits was well tolerated at 5 mg/kg or lower.

DMSO was selected as the solvent for AdipoRon given its capacity to distribute to tissues and its well-described use and safety profile in animal research.³¹ Although we deliberately minimized the amount of weight-based DMSO used in each rabbit injection to ensure we were below toxic limits, DMSO is recognized as a strong potentiator of other agents, particularly sedatives.³² Notably, of the two rabbits that expired after instillation of 25 mg/kg AdipoRon, both had received instant acting buprenorphine rather than sustained release, whereas the 12 that survived received sustained release narcotic.

Although the seizure on POD 1 suggested toxicity of AdipoRon, our findings could not exclude the possibility that potentiation of narcotics with DMSO and/or AdipoRon did not contribute to sudden death as well. Based on our results, in addition to reducing the dose to 5 mg/kg or less in rabbits, we recommend mitigating DMSO use to that which is minimally necessary to completely dissolve AdipoRon, utilizing non-narcotic analgesics as permitted by one's study protocol, and, when narcotics are necessary, administering sustained release formulations at least 1 h before the intended administration of AdipoRon with DMSO. These measures are recommended to mitigate all possibilities for the acute toxicity observed in our study.

Our study was not without its limitations. Although our series was the first to report on AdipoRon in a rabbit model of arthrofibrosis, providing critical insights that will help reduce the need for additional rabbits for safety testing in future investigations, we needed to adapt our methodology to react to changing clinical conditions observed after the first two rabbits died. As such, although our findings suggested a dose-dependent cumulative toxicity of the drug, that variable was not assessed in isolation as buprenorphine regimens were also adjusted as previously described. Thus, our ability to directly imply causation rather than association is limited.

However, our findings provided meaningful grounds to inform future decision making when administering AdipoRon IV using DMSO solvent in rabbit models of MSK diseases. Second, our study did not assess efficacy at the prescribed dose groups or collect serological parameters assessing pharmacokinetics as these aims were outside of the study scope. Thus, future investigations are needed to determine whether 5 mg/kg is both a safe and effective dose range in rabbit models of disease translatable to humans. Despite these limitations, this is the first investigation of AdipoRon in the most translatable animal model of arthrofibrosis, providing essential information that will inform future investigation into translating AdipoRon into clinically impactful uses, from the bench to the bedside.

In conclusion, contrary to the existing literature studying AdipoRon in murine species, our findings suggested that there was a dose-dependent toxicity over 10 mg/kg of IV AdipoRon in rabbits, with no adverse events observed in rabbits that received up to 5 mg/kg of AdipoRon. Future study is warranted to evaluate the efficacy of AdipoRon at this reduced dose range. Lastly, investigators should be mindful of reducing the volume of DMSO to the minimum that is necessary to appropriately dissolve AdipoRon into solution and adjust analgesics to long-acting formulations accordingly.

Authors' Contributions

H.I.S. contributed to conceptualization, design, data acquisition, investigation, writing—original draft and editing, and analysis. C.G., A.K.L., J.W.B., M.F.C., A.N.P., M.E.M., J.S.S., D.J.B., and A.D. were involved in data acquisition, investigation, and writing—review and editing. M.P.A. carried out design, data acquisition, investigation, writing—review and editing, and funding acquisition.

Disclaimer

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Disclosure Statement

The authors declare no conflicts of interest in relation to this study.

Funding Information

This study was pursued with the generous philanthropic support of Anna-Maria and Stephen Kellen Foundation (to M.P.A.). This study was also supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R01 AR072597 (to M.P.A.) and Regenerative Medicine Minnesota under award number RMM 091620 TR 010 (to M.P.A.). D.J.B. is funded by grants from the National Institutes of Health (R01AR73147, R01HL147155), NIAMS (P30AR76312).