Double-blinded, randomized tolerance study of a biologically enhanced Nanogel with endothelin-1 and bradykinin receptor antagonist peptides via intra-articular injection for osteoarthritis treatment in horses

Abstract

Background

Osteoarthritis is a leading cause of pain and retirement in athletic horses. Hydro-expansive functionalized nanogels, acting as Drug Delivery Systems, constitute one of the current therapeutic prospects. These nanogels have the potential to combine mechanical benefits through polymers with the biological effect of prolonged release of bioactive molecules. The purpose of this double-blinded randomized tolerance study versus negative control was to evaluate the response of healthy joints to a single injection of the expected efficient dose (further referred to as the trial dose) and overdose of nanogels composed of chitosan and hyaluronic acid and featuring a type A endothelin receptor antagonist and a type B1 bradykinin receptor antagonist. The metacarpophalangeal joints of 8 healthy horses were randomly injected with 2.4 mL of functionalized nanogels and 2.4 mL of saline as control on the contralateral limb. Injections were repeated twice at one-week intervals, followed by injection of a triple dose of nanogel on week four. Clinical, ultrasonographic and synovial fluid cellular and biochemical follow-ups were performed up to three months following the first injection.

Results

No change in general clinical parameters, lameness or sensitivity to passive flexion of the fetlocks was noted. Mild to moderate synovitis was noted on the day following injection in the treated group, with a significant difference (p < 0.05) compared to the control group. It spontaneously resolved on day 3 following the injections and did not increase with repeated injections. Similar effects were noted after injection of the triple dose but lasted for a week. Synovial fluid markers of inflammation also showed a transient significant increase in the treated group one week after each injection, but no differences were detected at the end of the study.

Conclusions

Injections of the expected therapeutic dose of functionalized nanogel in healthy joints induced a mild transient inflammatory response in the joint. Three injections of the trial dose at one-week intervals and injection of thrice the trial dose induce a mildly greater inflammation without harmful effects on joints. Functionalized nanogels are well tolerated prospects for the treatment of osteoarthritis in horses. Their beneficial effects on arthritic joints have yet to be evaluated to determine their therapeutic potential.

Background

Osteoarthritis (OA) is one of the leading causes of poor performance, lameness and early retirement in athletic horses [1]. It is characterized by periarticular bone remodeling, subchondral bone failure, cartilage breakdown and synovitis [2, 3]. Although it is responsible for major financial losses in the equine industry, there is currently no curative treatment for OA, and medical treatment remains mostly palliative. This difficulty arises from the lack of self-regenerative capacity of cartilage, which is an avascular tissue. To control the irreversible process of OA, the use of disease-modifying drugs is now commonly favored over symptom-modifying ones for long-term management. Disease-modifying drugs advantageously counteract the pathophysiological process to restore joint homeostasis to curb the catabolic process and provide a suitable environment for anabolic activity. However, when the joint shows advanced signs of OA and its structural organization is altered, this simple restoration of physiological balance is insufficient. In such cases, it is worthwhile to combine OA disease-modifying drugs with those that improve the mechanical properties of the damaged joint to promote its functional recovery [4]. Ideally, these therapies should also be able to act in the long term and persist despite repeated mechanical stress, to avoid the need for repeated injections.

This is the promising interest offered by hybrid functionalized hydro-expansive nanogels. These are true nanobiotechnology platforms made of a combination of biopolymers, used as drug delivery systems (DDSs) employed as carriers for bioactive molecules and administered directly into the joint [5,6,7]. The selection and combination of bioactive molecules can be tailored to specific therapeutic targets. In the context of OA management, molecules designed to address pain, mitigate inflammation, or prevent cartilage degradation can be strategically chosen.

This study focused on functionalized nanogels composed of a combination of 2 biopolymers, chitosan (CHI) and hyaluronic acid (HA), each of which has excellent viscoelastic properties and is capable of improving the viscoelastic properties of injured joints [8,9,10]. In addition, chitosan has structural and biochemical similarities to the glycosaminoglycans contained within the cartilage matrix. Chondroprotective and anti-inflammatory effects have been reported, as well as its ability to promote the synthesis of hyaluronan and other cartilage components [10]. Hyaluronan is further known for promoting chondrogenic differentiation of stem cells, supporting chondrocyte growth [11] and, like chitosan, for stimulating the synthesis of cartilage matrix components [12].

In the present study, these two biopolymers were used as DDSs for the progressive release of two grafted bioactive peptides, BQ-123 and R-954. These molecules are receptor antagonists of bradykinin (R-954) and endothelin-1 (BQ-123) respectively, two peptides involved in the inflammatory process of OA [13, 14]. Endothelin is a vasoconstrictor that activates the production of nitric oxide and metalloproteinases after binding to its receptors on chondrocytes and inhibits proteoglycan synthesis [15,16,17]. Bradykinin activities are mediated by type 1 and type 2 receptors. Bradykinin B2 receptors are responsible for the physiological activities of bradykinin, whereas bradykinin B1 receptors are induced during inflammatory reactions and are mostly absent under physiological conditions[14, 18]. In vivo studies in a rat model of OA showed that type A endothelin receptor antagonist (BQ-123) and type B1 bradykinin receptor antagonist (R-954) decreased cartilage degradation and nociception [14]. An in vitro study on an equine cartilage organoid model of OA revealed that the combination of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA) induced a decrease in inflammatory and catabolic markers [19]. These results reinforce the potential therapeutic value of this type of functionalized nanogels in the management of chronic joint disorders involving painful and inflammatory components. However, before assessing its clinical efficacy, its in vivo safety must be validated. The biocompatibility of the abovementioned functionalized nanogels has already been validated in a previous in vitro study in which nanogels showed no cytotoxic effects but rather sustained metabolic activity and proliferation of equine articular chondrocytes [19].

The aim of the present study was to investigate the potential adverse effects of an intra-articular injection of a hyaluronic acid–chitosan hybrid nanogel functionalized with a type A endothelin receptor antagonist (BQ-123) and a type B1 bradykinin receptor antagonist (R-954) on sound horses at the expected therapeutic dose, further referred to as trial dose. Three scenarios were evaluated by monitoring clinical, imaging and synovial fluid parameters: injection of the trial dose, overdose by three repeated injections of the trial dose, and overdose by injection of a dosage three times higher than the trial dose. To assess the effects of the nanogels alone, we hypothesized firstly that a single intra-articular injection of the nanogels would not produce more side effects than injection of saline as control during the week following injection, and secondly that overdose injections of the nanogels would trigger a transient inflammatory response in the joints compared to saline injections. To provide this therapy to client-owned horses, we hypothesized thirdly that there would be no side effects from the injection of the nanogels one week after injection of the trial dose or in the medium-term following overdose compared with baseline parameters of the same joint.

Results

Horses

Freeze-dried nanogels were reconstituted with 2.4 mL of 5% glucose solution and injections were successfully performed in all joints within 30 min of the nanogels preparation. Rectal temperature, heart and respiratory rates, appetite and comfort (general attitude, habitus, and willingness to move) remained within normal limits in every horse for the duration of the study, and no major inflammatory reactions occurred. Horse 4 sustained a hind fetlock dorsal skin injury on day (D) 14 and had to remain confined to a stall and bandaged for one week (until D21) during the study. In addition, horse 8 showed grade 3/5 lameness on the control limb on D3, which was significantly improved by D7 and resolved on D21; this lameness was localized to the foot by a palmar digital nerve block. During those corresponding periods, these horses were temporarily excluded from the lameness evaluations. Mild and transient forelimb lameness (grade 1/5) occurred for 6 nanogel-treated limbs and 5 control limbs during the study period (Fig. 1; Additional Dataset 1). The data regarding the median (Q1, Q3) lameness grades throughout the study are available in Additional Table 1.

Fig. 1

Degree of forelimb lameness observed during the study. The number of forelimbs per lameness grade in the negative control (saline) (S)- and nanogel (N)-treated groups respectively, at each time point of the study, expressed in days (D). Horses 4 and 8 had to be temporarily excluded from the lameness evaluations for a hind fetlock dorsal skin injury and a front foot lameness respectively

No horse showed sensitivity to passive flexion of any of the fetlocks for the duration of the study (Additional Dataset 1). Mild to moderate local swelling occurred in 1 to 3 fetlocks per group on the days following the second, third and fourth (triple-dose) injections (D8, D15 and D22), which returned to normal within 2 to 6 days after each injection, except for one horse that showed substantial (grade 3) swelling on D15 on the treated limb (Fig. 2, Additional Dataset 1). Similarly, mild to moderate subcutaneous edema was visible on ultrasound in 2 to 6 fetlocks in the nanogel group versus 0 to 3 in the control group on D8, D15 and D22, and one horse showed substantial (grade 3) swelling on D15 in its nanogel-treated fetlock (Fig. 3, Additional Dataset 1). The curves illustrating the evolution of the circumferences of the joint and of the more proximal metacarpal region are shown in Fig. 4. The data regarding the mean (± SD) evolution of the perimeters and median (Q1, Q3) swelling and edema grades throughout the study are available in Additional Table 1 and Additional Dataset 1.

Fig. 2

Local swelling scores observed during the study. The number of fetlocks per score category regarding physical swelling in the negative-control (saline) (S)- and nanogel (N)-treated groups respectively, at each point of the study, expressed in days (D)

Fig. 3

Scores of subcutaneous oedema measured by ultrasound observed during the study. The number of fetlocks per score category in the negative-control (saline) (S) - and nanogel (N)-treated groups respectively, at each point of the study, expressed in days (D)

Fig. 4

Fetlock (a) and metacarpal (b) circumferences. Evolution of the circumferences (cm) of the saline-treated (blue) and nanogel-treated (orange) front limbs patients throughout the study, expressed as mean ± SD

Primary outcome

The primary outcome was the clinical sign of acute joint inflammation evaluated by joint effusion. Pictures illustrating the different grades that occurred throughout the study are shown in Fig. 5. The evolution of the median grades of joint effusion throughout the study is presented in Fig. 6. The data regarding the evolution of the joint effusion grades throughout the study are available in Additional Table 1 and Additional Dataset 1.

Fig. 5

Joint effusion grades measured during the study. Photographs of the lateral aspect of the fetlock joint showing examples of the different grades of joint effusion observed during the study. See the proximo-palmar recess of the metacarpophalangeal joint (light arrow). Also see the moderate (grade 2) local swelling observed (arrowhead)

Fig. 6

Joint effusion grade evolution of treated (orange) and control (blue) fetlock joints. The results are expressed as the median ± first and third quartiles. Significant differences (p < 0.05) between limbs are represented by black asterisks

Nanogel-injected fetlocks were first compared to contralateral saline-injected fetlocks from the same horses. The first single injection of the trial dose of nanogel induced significantly more joint effusion than an identical injection of saline control the following day (D1: p = 0.004). All horses exhibited grade 1 to grade 3 joint distension in the treated joint, with three horses displaying grade 1 distension, four horses displaying grade 2 distension, and one horse displaying grade 3 distension. The distension resolved by day 3 following injection. Only half of the horses showed grade 1 distension of the control joints on the day following injection.

Three repeated injections of the trial dose at one-week intervals induced significantly more joint effusion than did an identical injection of saline control on the day after the last injection of the trial dose (D15: p = 0.008). All horses showed grade 1 to grade 3 joint distension of the treated joint, with a greater number of horses showing higher grades than following the first injection (two horses had grade 1 distension, three horses had grade 2 distension, and three horses had grade 3 distension). This distension resolved by day 7 following injection. By comparison, only 2 out of the 8 fetlocks injected with saline displayed grade 1 joint distension on D15, and 2 others injected with saline displayed a grade 3 joint distension on D15. This distension resolved by day 7 following injection except for horse 2 which went from a grade 3 on D15 to a grade 1 on D21.

Injecting a triple dose the following week also led to a significant increase in joint effusion compared to control fetlocks the day after injection (D22: p = 0.002), with all horses showing grade 1 to grade 3 joint distension of the treated joint. A greater number of horses showed higher grades than following the third injection of the trial dose, with two horses having grade 1 distension, two horses having grade 2 distension and four horses having grade 3 distension. This distension lasted for up to 2 weeks following injection. By comparison, only half of the horses showed distension of the control joints on D22 (one horse had grade 3 distension, one horse grade 2 and two horses grade 1, that also resolved within the 2 weeks following injection).

Nanogel-injected fetlocks were then compared to their baseline status (D0). No difference in joint effusion grade was noted in the treated limbs between baseline (D0) and D7 or D84. Detailed results from the statistical analysis are available in Additional Table 2.

Secondary outcomes

Synovitis evaluated on ultrasound

A moderate synovitis in the treated joint was noted on the day following the injections, with a significant difference between treated and control joints on D8 (p < 0.001), D15 (p < 0.001) and D22 (p = 0.002). Synovitis was also transient as a return to baseline was noted within 7 days following injection, except following the injection of the triple dose, where it remained slightly higher (median grade 1) for up to two weeks following injection. This increase did not persist on longer term, and no ultrasonographic evidence of synovitis was noted 3 months after the first injection.

The ultrasonographic appearance of the evolution of the synovitis throughout the study is presented in Fig. 7. The evolution of the median grade of synovitis over the course of the study is presented in Fig. 8. Data regarding the evolution of synovitis grades throughout the study are available in Additional Table 1 and Additional Dataset 1.

Fig. 7

Transverse ultrasonographic images of both metacarpophalangeal joints laterally of one horse during the study. The nanogel-treated joint (bottom images) and negative control (saline)-treated joint (top images) were followed on D0, D1, D15, D22, D28 and D84. See the skin surface (arrow) becoming transiently convex in the nanogel-treated joint and the emergence of a moderate subcutaneous oedema (arrowhead). The synovial fluid quantity (yellow asterisk) transiently increases in both fetlocks

Fig. 8

Evolution of secondary outcomes during the study period of treated (orange) and control (blue) fetlock joints. Synovitis grade (a), viscosity grade (b) and MCP-1/CCL2 concentration (f) are expressed as the median ± first and third quartiles. Total protein concentration (c), total nucleated cell count (d) and PGE2 concentration (e) in synovial fluid samples are expressed as the mean ± standard deviation. Significant differences (p < 0.05) between limbs are represented by black asterisks. Significant differences (p < 0.05) in the nanogel-treated limbs compared to day 0 are represented by black latin crosses

No difference in synovitis grade was noted in the treated limbs between D7 and D0, or between D84 and D0 (Additional Table 2).

Synovial fluid analysis

Synovial fluid was obtained from all sampled joints, with minimal blood contamination in some samples. A significantly lower viscosity was noted in the treated joints one week following injection of the third trial dose (D21; p = 0.005) and one week following injection of the triple dose (D28; p < 0.001). Data regarding the evolution of synovial fluid parameters throughout the study are available in Additional Table 1 and Additional Dataset 1. The evolution of the median (IQR) viscosity grades is presented in Fig. 8. No differences from D0 were observed one week after the first injection of the trial dose or 3 months after injection in the treated joints (Additional Table 2).

One week after the third injection of the trial dose (D21; p = 0.03) and one week after the injection of the triple dose (D28; p < 0.001), significantly higher protein levels were observed in the treated joints compared to those in the control joints. Similarly, a significantly greater total nucleated cell count in the treated joints than in the control joints was noted one week following the first injection of the trial dose (D7; p = 0.01), one week following injection of the third trial dose (D21; p < 0.001) and one week following injection of the triple dose (D28; p < 0.001). Additionally, a significantly greater total nucleated cell count was observed in the treated joint one week after the first injection of the trial dose compared to day 0 (p = 0.02). The evolution of the mean (± SD) protein levels and total nucleated cell count throughout the study are presented in Fig. 8.

No significant differences were noted between limbs, treated with either saline or nanogels, in terms of prostaglandin E2 (PGE2), Monocyte Chemoattractant Protein-1 / Chemokine Ligand-2 (MCP1/CCL2) and type 1β interleukin (IL-1) levels throughout the study (Additional Table 2). Compared to baseline, only a significant (p < 0.001) decrease in PGE2 was detected in the treated limbs 3 months after the first injection of the trial dose. The evolution of the mean (± SD) levels of PGE2 and median (IQR) levels of MCP-1/CCL2 throughout the study are presented in Fig. 8.

Discussion

This study evaluated whether a single or an overdosed intra-articular administration of a functionalized nanogel in horses’ healthy joints could induce adverse events that would prevent a clinical trial from being carried out safely. When injected in the metacarpophalangeal joints, the single, repeated and the overdose (corresponding to a triple dose) of biopolymeric nanogels functionalized with BQ-123 and R-954 were well tolerated. This was clinically confirmed by transitory mild acute joint inflammation, also visible on ultrasound examination and in synovial fluid analyses. The study design was elected to test the most strenuous conditions while optimizing and reducing the number of animals in line with the 3Rs’ statement. The two overdosing conditions were therefore tested back-to-back, surmising that if such conditions were tolerated, a single injection of a trial dose would be as well. Indeed, this protocol allowed us to evaluate the joint response to the trial dosage during the week following the first injection, but the study design prevented proper medium-term evaluation of that single dose. Similarly, the medium-term effect of three repeated injections alone and the effect of a triple dose alone were not assessed in the present study. In this protocol, we chose to use each horse as its own control to avoid inter-individual variability in inflammatory reactions.

The results were divided into primary and secondary outcomes. The primary outcome was the clinical pattern of joint inflammation. It was expected to be the most reactive, as it is one of the most sensitive indicators of early joint stress [20]. This choice was supported by previously published in vivo biocompatibility studies after intra-articular injection of a combination of chitosan and hyaluronic acid hydrogels in a rabbit model, resulting in mild swelling with no discharge, edema or pain palpation [10]. Joint effusion was also noted as part of the inflammatory response to injection of other types of hydrogels or bioactive substances in horses [20,21,22,23]. In addition, this outcome might be a concern for owners and thus constitute a limiting factor for the use of this therapy on privately owned animals.

We found that most horses exhibited grade 1 and 2 joint distension in the treated joint after a single injection of the trial dose, and only one horse showed grade 3. That was resolved within the third day post-injection. A greater number of horses showed higher grades of joint effusion following three consecutive injections (at one-week intervals) of trial dose and following single injection of a triple dose, that were resolved 7 and 14 days respectively.

Overall, the response to expected therapeutic use (single dose injection) suggested good tolerance with low inflammation and rapid resolution. Even with repeated doses and overdosing, the inflammatory response remained mild to moderate and transient. This is in contrast with the findings of other safety studies [21,22,23] that reported more significant joint effusion. For instance, the study by Johnston [22] reported a mild joint effusion in 15 out of 24 fetlocks but lasted for up to 7 to 28 days following intra-articular injection of hyaluronic acid. Similarly, mild to substantial effusion has been found in 17 out of 32 fetlocks and lasted for up to 14 days following injection of mesenchymal stem cells [20] or mild to substantial effusion on the day following the injection of polyacrylamide gel that decreased to baseline 7 days following injection [23].

Our study focused on an important aspect of pre-requirement for clinical trial, to ensure the safety of a local injection into the joint. It is well known from the literature that intra-articular injection has certain limits and that the adverse events, such as pain or swelling depend on the product injected [3, 23]. Indeed, the use of intra-articular injection, i.e. for analgesic/anti-inflammatory drugs administration, remains controversial both in human and veterinary medicines, because of their risk/benefit profile regarding the potential chondrotoxicity [3, 24].

The intra-articular administration of drugs is a common practice in equine orthopedics for both lameness diagnosis and management of treatments that promote joint health in horses. However, evidence regarding the safety of each drug is limited, and controversies persist in these topics, including the popular injections of HA (viscosupplementation) that was introduced in equine practice in the 1990s [3]. There are still limited data from randomized, placebo-controlled clinical studies in horses regarding HA risk/benefit [25]. The recent study evaluating two injectable hyaluronic acid formulations (of low and high molecular weight of hyaluronic acid) concluded that both formulations displayed inflammatory responses in healthy equine joints and were often associated with lameness [21]. Along in the lines of pro-inflammatory effects of HA, small fragments of HA resulting from its depolymerization in the synovial fluid, have been reported as associated with catabolic and pro-inflammatory effects on the cartilage matrix [26]. In contrast, in a pilot study by Sladek et al. [27], chitosan (CS) and hyaluronic acid (HA) were combined and tested for both their anti-inflammatory properties and toxicity. This combination had no detectable toxicity and in addition, clear benefit was evidenced by the decrease of inflammatory biomarkers in synovial fluid [27].

The functionalized nanogel that we tested in this study is a biomaterial composed of two peptides, BQ-123 and R-954, conjugated to CS and HA and manufactured into nanogels. They were produced in aseptic conditions to ensure sterile, pyrogen-free nanogels of specific physicochemical characteristics. A similar formulation was previously investigated in vitro [19] and was proven to be biocompatible with no cytotoxic effect and benefice metabolic activity and proliferation of equine chondrocytes [19].

In our study, nanogel-injected fetlocks were compared to their baseline status to assess both the cumulative effect of the nanogel and arthrocentesis. Our results showed no differences one week after a single injection of the trial dose, demonstrating the transient nature of the clinical sign of acute joint inflammation. In addition, no differences were observed three months after repeated injections and an overdose. All together, these results suggested that nanogels were well tolerated in the medium term, even after repeated doses and overdosing.

The secondary outcome variables confirmed the transitional characteristics of the acute joint inflammation (effusion). Notably, all secondary outcome variables, including imaging, cellular and biochemical analyses, served as indicators of joint inflammation. MCP-1/CCL-2, recognized as a sensitive biomarker of early joint inflammation is also indicative of osteoarthritis, as this chemokine is pivotal in mediating the recruitment of monocytes and the destruction of cartilage [28, 29]. Ultrasonographic scores of synovitis revealed a similar pattern of changes compared to clinical scores of joint effusion. One week after injection of the single trial dose, synovial markers of inflammation did not significantly differ in the elevation of total protein, PGE2, IL-1 or MCP-1/CCL2 concentrations compared to those in the control fetlocks. Moreover, these parameters did not differ from the basal joint values. The number of nucleated cells was significantly greater, but only by one limited order of magnitude [23]. These results confirmed the good tolerance of the nanogel one week after injection of the trial dose. In contrast, three repeated injections of the trial dose and injection of the triple dose led to a significant increase in proteins and nucleated cells and a significant decrease in viscosity compared to those of the control one week after the last injection (D21 and D28). However, no differences were observed in the IL-1, PGE2 and MCP-1/CCL2 levels.

We acknowledge that our study presents limitations inherent to its design. First, we elected to inject both front fetlocks in each horse, which complicated the objective evaluation of the degree of lameness of each limb individually in response to the injections. However, this approach allowed each horse to serve as its own control, reducing inter-individual variation, and was in line with the 3Rs principles to minimize the use of animals. Second, the absence of a washout period between the first trial dose injection and the two overdosing conditions was motivated by the limited study time due to the inherent cost of husbandry in large animal research. Cost efficiency and animal welfare also implied returning the horses to paddocks between injections, where no control of their activity was possible. Nevertheless, the low level and short duration of inflammation, despite back-to-back overdosing and with free-roaming activity, suggest good tolerance to the nanogels. Additionally, the tolerance to injection was evaluated in healthy joints, and the alteration of the synovial environment in diseased joints might cause different inflammatory reactions. Finally, evaluation of joint health did not include arthroscopic or histologic assessment of cartilage or synovial membrane, despite them being the gold standard for macroscopic and microscopic cartilage assessment. Arthroscopy is an invasive procedure as it is performed under general anesthesia and only allows partial evaluation of the articular surface, which can lead to under- or overestimation of the cartilage damage [30]. Standing arthroscopy has been described for fragment removal in the metacarpo-/tarsophalangeal joints [31]. However, this technique allows a smaller field of view, a lower image quality is reported and reliability for cartilage assessment for diagnostic purposes has not been evaluated. Collection of samples for histologic examination is also invasive as it requires, at the least, surgical collection under general anesthesia and is more generally performed post-mortem. Additionally, synovial markers have been demonstrated to be good to excellent predictors of joint disease in the horse [32]. In this context, although the expected therapeutic application envisioned involves a single dose, it would have been beneficial to evaluate not only acute inflammation markers but also chronic inflammation markers such as TGF-ß (mainly produced by macrophages) and lactate dehydrogenase [33], as well as cartilage degradation markers like CTX-II (cross-linked C-telopeptide fragments of type II collagen) or metalloproteinases. This broader study of biomarkers would be particularly valuable under the planned trial conditions. With consideration for animal welfare and respect of the 3R rules, we aimed to replace by testing the innocuity of the nanogels in vitro on organoid models of cartilage developed from chondrocytes obtained from cartilage biopsies performed on cadavers sent for necropsy [19]. Additionally, we aimed to refine by limiting invasiveness of our procedures and not submitting the horses to general anesthesia.

Conclusion

In conclusion, injections of the expected therapeutic dose of functionalized nanogels in healthy joints resulted in a significant increase in joint effusion on the day after injection compared to saline injections. However, the clinical, ultrasonographic and synovial fluid parameters of the nanogel-injected fetlocks did not differ from those at baseline one week after injection, except for the total nucleated cell count, indicating a transient inflammatory response of the joint. Similarly, three repeated injections of trial dose appear to induce greater, albeit moderate, levels of inflammation without significantly different effect on any of the evaluated biomarkers of inflammation (IL-1, PGE2), including MCP-1/CCL2 which is additionally a marker of OA. Injecting an additional triple dose the following week led to similar significant changes of greater magnitude. The absence of differences between limbs by the end of the study suggests that there are no greater midterm repercussions of injection of a trial dose than of saline. These findings highlight the promising clinical prospects of the nanogels, suggesting a manageable inflammatory response and supporting their further exploration for therapeutic applications in osteoarthritis.

Methods

Horses and study design

The study protocol was approved by the ComEth Anses/ENVA/UPEC (protocol code 21–037#30,112 OA-ACTIVE). A power analysis was performed considering an alpha of 0.05 and an actual power of 0.95. Since no detailed information on the primary outcome of this treatment in horses was available, the power analysis was based on synovial effusion score data obtained from a previous study evaluating the effects of another biological therapy in normal equine joints [20]. Based on the analysis, seven fetlocks were required per treatment group. To ensure the exact number in case of a major health problem causing exclusion of a horse from the study, 8 clinically healthy horses owned by the Center of Imaging and Research on Equine Locomotor Pathology (CIRALE) were included. There were 5 geldings and 3 mares, aged from 12 to 28 years old. There were 6 French Trotters, one Warmblood and one Thoroughbred with a mean weight of 540 kg (459–598 kg). General clinical and locomotor evaluations were performed before inclusion on each horse, and all underwent ultrasonographic and radiographic examinations of both front fetlocks to rule out the presence of preexisting disease.

The timeline of the study design is represented in Fig. 9. On day (D) 0 (D0; baseline), all horses included in the study were randomly administered 2.4 mL of functionalized nanogels in one metacarpophalangeal (fetlock) joint and 2.4 mL of 0.9% saline solution in the contralateral metacarpophalangeal joint as a negative control. A uniform distribution of the nanogel and saline as negative control was split between left and right fetlocks. A trained operator blinded to the contents of the injected substance (LB) performed all injections after aseptic preparation of the skin and sedation (intravenous administration of a combination of 0.01 mg/kg detomidine and 0.01 mg/kg butorphanol). Two overdosing conditions were tested: overdose by repeated weekly injections of the trial dose and overdose by a single injection of thrice the trial dose. Trial dose injections were performed once weekly for 3 weeks (D0, D7, D14) using a lateral approach on the flexed limb, with a 20-gauge needle inserted between the metacarpal condyle and the articular surface of the lateral proximal sesamoid bone. Injection of the triple dose was performed on D21 using the same approach.

Fig. 9

Timeline of the study design. Injections of trial doses were performed on day (D) 0, D7 and D14, and injection of thrice the treating dose on D21. Follow-up examinations were performed prior to each injection

All horses were confined to 3.5 × 4 m stalls from the day of each injection until 3 days after injection and then returned to the paddock. No bandages or medications were used to avoid any interference with the inflammatory response. The vital parameters and appetite of the horses were monitored twice daily to check for signs of discomfort.

Clinical and ultrasonographic follow-up of the front fetlocks were performed on each injection day prior to injection, one, three and seven days after each injection, and 14 days after the last joint injection (D35) for short-term evaluation. A medium-term follow-up was finally performed three months after the first injection (D84). Synovial fluid samples (1 mL) were obtained from each fetlock joint on D0 (before injection), 7 days after each injection and on D84 using the same technique used for injections and during the same procedure on the corresponding days (D0, D7, D14, D21): the same needle was used but the syringes were changed and synovial samples were collected before injection. The samples were divided into EDTA and dry tubes.

In the case of a major inflammatory reaction leading to clinical grades ≥ 3/4 (or ≥ 3/5 for lameness, investigators were allowed to administer non-steroidal inflammatory drugs to relieve pain and excessive inflammation. The grading system for clinical parameters is detailed in Table 1 and grading of lameness was performed in accordance with the lameness scale of the American Association of Equine Practitioners [34]. This deviation from the protocol excluded the horse from the study, from the time point of drug administration onward. All methods performed in this study were carried out in accordance with the ARRIVE guidelines.

Preparation of functionalized nanogels for injection

Two formulations of functionalized nanogels were combined according to the protocol described in our previous study [19]. The first formulation (BQ123-CS) was composed of chitosan (CS) conjugated to the peptide BQ-123 (ChinaPeptides Co., Ltd., Shanghai, China), and the second (R954-HA = R) was composed of HA conjugated to the peptide R-954 (kind gift from Pr. Sirois, Québec, Canada). Briefly, peptide grafting was performed according to the EDC/NHS (Sigma‒Aldrich Canada Co., Oakville, ON, Canada) coupling chemistry. The concentration and grafting rates were determined by spectrofluorometry (Hitachi F-2710 spectrophotometer, Hitachi High Technologies America, Inc., Pleeasanton, CA, USA).

An ionic gelation process under aseptic conditions was performed for nanogel synthesis as previously described [9]. A combination of a chitosan solution (CS, 2.5 mg/mL), sodium tripolyphosphate (TPP, 1.2 mg/mL) (Alfa Aesar, Ward Hill, MA, USA) and 60 kDa sodium hyaluronate solution (HA, 0.8 mg/mL) was used. Nanogel suspensions were then purified by dialysis and lyophilized with 8% sucrose (Sigma‒Aldrich Canada Co., Oakville, ON, Canada) as a cryoprotective agent before storage at -20 °C.

Two to three hours before injection, the frozen nanogel powder was placed at room temperature. One to two hours before injection, 0.05 mL of sterile 5% glucose solution (Laboratories Chaix et du Marais, La Chaussee Saint-Victor, France) were injected under aseptic conditions with an insulin syringe and a 23G needle into the vial containing the freeze-dried nanogels. The vial was vortexed for one minute and allowed to rest for 14 min at room temperature. The injection of 0.05 mL of 5% glucose, vortexing and resting was repeated three more times. The diluted vial was then added with 0.2 mL of 5% glucose solution with a sterile insulin syringe. The vial was vortexed for 20 s and allowed to rest for 5 min at room temperature. These steps were repeated another 2 times, adding 1 mL of 5% glucose solution each time. A final volume of 2.4 mL was obtained. The single dose of the functionalized nanogels contained 1.2 μM BQ-123 and 0.54 μM R-954. The triple dose contained 3.6 μM BQ-123 and 1.6 μM R-954. All injection vials were sterile and endotoxin-free. The trial dose was calculated based on findings from in vitro studies [19], for an average horse weight of 500 kg.

Assessments and outcomes

The primary outcome was joint effusion as a clinical sign of joint inflammation. This outcome was assessed blindly by the same experienced operator (SJ) using a five-point scale [20] from normal to severe (Table 1). The secondary outcomes included one ultrasonographic outcome and six outcomes related to synovial fluid analysis.

Table 1 Detailed grading criteria for each clinical and imaging parameter

Ultrasonographic examinations were performed by the same blinded veterinary specialist (SJ) using a 7.5 MHz linear transducer (Aloka Prosound Alpha 10, Hitachi Healthcare, Twinsburg, OH, USA) and a systematic protocol including transverse and longitudinal scans from the dorsal to the collateral aspects of each fetlock [20]. This operator assigned a synovitis score according to the scoring system previously described [20] as the secondary ultrasonographic outcome (Table 1).

Synovial fluid outcomes included measurements of total protein concentration, total nucleated cell count (ADVIA® 120 Hematology System, Siemens) and viscosity scoring [35]. Viscosity was evaluated subjectively by observing a drop of synovial fluid placed on the thumb and then touched with the index finger, with separation of the thumb and finger. The viscosity grade was then determined according to the criteria in Table 2. The concentrations of PGE2, IL-1β and MCP-1/CCL-2 were also quantified by ELISA (IL-1β: DuoSet ELISA Equine IL-1β/IL-1F2, R&D Systems; PGE2: Prostaglandin E2 Assay, R&D Systems; MCP-1/CCL-2: Equine MCP-1/CCL-2 ELISA Kit, Thermo Fisher Scientific) to assess joint inflammation and cartilage degradation.

Table 2 Viscosity grading system

Five other clinical parameters and one ultrasonographic parameter were monitored to document the response to the injections but were not included as variables in the statistical analysis. The fetlock circumference at the mid-level of the proximal sesamoid bones and mid-metacarpal circumference (identified by shaved skin landmarks) were measured by the same operator (AT). Both measurements were taken in triplicate and averaged. Second, sensitivity to digital flexion tests and local swelling were evaluated by the same operator (SJ) using the same five-point scale from normal to severe [20] (Table 1). Finally, the degree of forelimb lameness was graded after two 30-m round trips at the trot on a straight line on a scale of 0 to 5 in accordance with the lameness scale of the American Association of Equine Practitioners [34] by the same veterinarian specialist (SJ). The presence of subcutaneous edema was also recorded on ultrasound and graded using a five-point scale from normal to severe (Table 1). This complementary ultrasound parameter was evaluated to distinguish a true subcutaneous edema from a subcutaneous thickening or fibrosis which was not possible using the physical parameter “swelling”.

Statistical analysis

Statistical analysis was performed after the data had been visually assessed for normality and homogeneity. The mean and standard deviation of normally distributed outcomes and the median and quartiles of the other variables were calculated at each time point for each treatment group. To test the first two hypotheses, a linear mixed model with treatment and date as fixed effects and horse as a random effect was used. The treatment groups were then compared at selected dates using a Dunnett's test or its nonparametric equivalent for outcomes for which normality and/or homogeneity were not met. Comparisons were made on D1 (first hypothesis), D15 and D22 (second hypothesis) for the primary outcome and the secondary ultrasonographic outcome and on D7 (first hypothesis), D21 and D28 (second hypothesis) for secondary synovial outcomes. To test the last hypothesis, a linear mixed model with treatment and date as fixed effects and horse as a random effect was also used. D7 and D84 were compared to D0 for each treatment separately using Dunnett's test or its nonparametric equivalent for outcomes for which normality and/or homogeneity were not met. All the statistical analyses were performed using R software (version 3.4.3; R Foundation for Statistical Computing, Vienna, Austria) or Excel 2016 for Windows (Microsoft, Redmond, DC, USA). The alpha type 1 error was set at 5%.