Effect of a single intra-articular administration of stanozolol
This study describes the effect of a single intra-articular injection of stanozolol, showing that stanozolol had a significant impact on OA joints, improving weight distribution, pain, and function scores compared to the control group. The effect of stanozolol has been studies in different animal models. In horses with naturally occurring OA, a positive response to treatment has been described in 82.5% of cases22. The positive effect of stanozolol in this naturally occurring canine model was observed from 15–30 days up to 180 days after treatment, when considering the functional assessment based on weight distribution. Interestingly, this effect was observed even with a single administration, while in the remaining animal models, multiple administrations were carried out21,22,23. This effect is observable in the Kaplan Maier test results for SI, with results of the stanozolol group taking significantly longer to return to baseline values. SI is commonly used to assess lameness, but their calculation with pressure-sensitive walkways has some limitations in OA patients29. While it is still unknown if the same limitations apply to the static evaluation of weight-bearing, we looked at different weight-distribution compensation mechanisms by calculating SI and deviation values. It was reasonable to expected improvements in SG only after a relatively large period after the intra-articular administration since stanozolol acts by inducing transforming growth factor 1β synthesis. A further possible stanozolol mechanism of action may be related to its induction in aromatase expression30. It has been demonstrated that the human articular cartilage expresses aromatase and that reduced expression of aromatase could facilitate the development of OA31,32. Aromatase inhibitor therapy in humans to address other medical conditions might be associated with common musculoskeletal symptoms and with substantial functional disability33.
Pain is a hallmark of OA. Data from canine studies may translate to humans34,35,36. Results show that a single intra-articular stanozolol administration significantly improved pain and function scores compared with the control group, raging until the 90-day evaluation moment and, in some cases, until the last evaluation moment. For most of the considered scores, a significant difference was also observed with the Kaplan Meier test. Through the same period, control group scores worsened, as would be expected as the disease progresses. It is interesting to note that some patients in the control group still recorded better scores in follow-up evaluations. This may be due to OA's natural course, with patients sometimes showing spontaneous improvements through time, only to see symptoms reappear in the future. An additional possibility is based on the fact that placebo saline injections can produce an effect, reflected in functional improvements, described to last up to 6-month 37. Even though this is possible and may be reflected in some patients' scores, the control group as a whole showed the expected progression of the disease. Additional clinical improvements were observed in the stanozolol group, with improved range of motion during joint extension. A consistent finding with the thermographic evaluation was that higher values were registered in the control group throughout the study, particularly in the last evaluation moments. Digital thermography can assess inflammatory pain and identify osteoarthritic patients38,39. Our results seem to support this finding, with higher temperature values determined with this technique corresponding to patients with worse functional evaluation and clinical signs. During digital thermography of dogs, the coat's type and color must be taken into account40,41. All of the breeds represented in this study had short hair, some had a double coat, and breeds had similar distribution between groups.
IL-1 is commonly pointed out as a major proinflammatory cytokine responsible for the catabolism in OA in several species, dogs, horses, and humans included1,42,43. Therapeutic approaches targeting IL-1 have been developed and shown a positive effect in animal models44. The evaluation of synovial fluid can add important information regarding disease burden and progression45,46. A previous report has described an improvement in synovial fluid characteristics of animals treated with intra-articular stanozolol22. We only observed significant changes at eight days, with both groups showing a reduction from the values recorded at the initial evaluation, but the stanozolol group had higher values. Visual inspection of patients' synovial fluid in the control group at the 8-day evaluation point showed an easily noticeable increased turbidity. The amount of turbidity grossly relates to the amount of inflammation47. The stanozolol administration may cause a transient increase in joint inflammation, which may also account for functional improvements, measured with weight distribution, were only observed after this period. Also, since stanozolol acts by inducing transforming growth factor 1β synthesis and reducing nitric oxide, it may not significantly impact IL-1 levels. It is also important to keep in mind that exercise influences inflammatory arthropathies parameters, and increase joint loading adds to secondary inflammation in OA joints48,49. As these animals were working dogs, physical activity may also play a role in this finding. The injection of 0.9% NaCl, used in as the control, added to the removal of synovial fluid for analysis, thus removing pro-inflammatory cytokines, may have had a similar effect to that of a joint lavage, and therefore account for the lower IL-1 levels observed in the control group at 8 days.
Radiographic evaluation is still the staple of OA monitoring, with CCO and CFHO representing initial radiographic signs that predict the development of OA clinical signs50,51,52,53. There is a low relationship between radiographic changes, clinical signs, and limb function54. As expected, radiographic signs in the control group progress throughout the follow-up period, representing the natural evolution of OA. In the stanozolol group, the majority of considered radiographic signs did not progress, and some improved. This effect has been described in an ovine surgical induce model, with stanozolol reducing subchondral bone reaction and promoting articular cartilage regeneration21. Although the effect has been previously described, future studies have confirmed these changes as no histological samples were collected in this study.
Studies regarding the use of stanozolol in human OA are not performed due to its potential anabolic effects55,56. The dose demonstrated to produce anabolic effects is 10 mg twice a week, given through intramuscular administration57. In this model, we used the described 0.3 mg/kg dose for intra-articular use in dogs26,27. Even if the administered dose may have approached the 10 mg level in some patients, a single administration was used, thus not exceeding the dose needed to produce the anabolic effect. In a study aimed to determine the best intra-articular dose of stanozolol in horses, multiple administrations at the highest dose tested (5 mg) also did not produce any side effects23. It is known that after intra-articular administration of stanozolol, it passes rapidly from the joint space to systemic circulation, with maximal plasma concentration registered at 6 h post-administration. It is then eliminated rapidly and detected in plasm for no more than 36 h post local administration58. In an ovine model, no weight gain was attributed to the anabolic effect of stanozolol21. We also did not recorded significant increases in body weight, which could be attributed to stanozolol. In mice treated with a long-term, high-dose stanozolol regime did not produce significant changes in activity patterns and aggressiveness18. No event of aggressiveness or personality changes were reported in treated animals.
Side-effects of intra-articular stanozolol have been previously reported in horses. They include a transient post-injection swelling in the treated joint, which disappeared after a few days without intervention22. Similarly, we observed increased lameness in four cases, which spontaneously resolved within a few days. During the follow-up period, no additional medication was administered. The study presents some limitations, namely the fact that the majority of animals had mild OA. It would be of interest to include a larger proportion of animals representing the remaining hip grades. Altough we enroled in the study a similar number of animals to that of similar reports, including a formal sample size calculation and a larger number of patients also is of interest. It is also important to determine the biological significance and clinical relevance of the changes observed. This assessment was made with the Kaplan–Meier test, but the determination of what constitutes a meaningful improvement has not been yet made for some of the evaluations performed. For that reason, we evaluated how long did it take for the assessment to return or drop below the value of the initial presentation, as it was the point which motivated the need for medical assistance. We did not colletect joint histological samples, as this was clinical treatment experiment study. For that reason, the effect of stanozolol on actual disease progression could not be determined on this animal model, and only radiographic progression was evaluated. Further studies should also consider this drug's intra-articular effects, including cytotoxicity, different dose evaluations, and administration frequencies, effect on different parameters as TGF-β synovial levels, similar to what is described in other animal models.
Conclusions
We described the effect of a single intra-articular administration of stanozolol in a naturally occurring canine model, with a long follow-up period. The use of stanozolol was safe and produced significant improvements in weight-bearing, pain score, and clinical evaluations.
