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Clinical Evaluation of Intra-articular Administration of Stanozolol to Manage Lameness Associated With Acute and Chronic Osteoarthritis in Horses
Journal of Equine Veterinary Science 35 (2015) 105–110
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Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Clinical Evaluation of Intra-articular Administration of Stanozolol to Manage Lameness Associated With Acute and Chronic Osteoarthritis in Horses Alessandro Spadari, Riccardo Rinnovati*, Simona Babbini, Noemi Romagnoli Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (BO), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2014 Received in revised form 1 December 2014 Accepted 3 December 2014 Available online 9 December 2014
Poor data could be found in the literature on the effects of intra-articular (IA) administration of stanozolol in horses affected by osteoarthritis (OA). To verify the clinical effects of IA stanozolol in acute and chronic cases of OA in horses, a clinical double-blinded trial involving 60 client-owned horses was performed. Veterinary practitioners selected horses, all showing clinical signs of acute or chronic OA, diagnosed in a single joint. After lameness evaluation and synovial fluid collection, the clinicians administered one to four (acute cases) or one to six (chronic cases) weekly IA doses of 5 mg of stanozolol or placebo. Positive result was considered a grade 1 improvement in lameness scored according to the American Association of Equine Practitioner scale. The characteristics of the synovial fluid were also evaluated. Of the 60 horses included in the study, 31 were affected by acute OA and 29 by chronic OA. Horses were treated blindly with placebo. The overall outcomes were positive in 82.50% of cases. Lameness disappeared completely in 15 of 21 (acute) and 11 of 19 (chronic) animals, and there was a significant reduction after two and four treatments, in the acute and chronic cases, respectively. Improvement in the physical characteristics of the synovial fluid sampled was evident after the third treatment. Stanozolol injected IA at a dose of 5 mg significantly resolved lameness in affected joints. Stanozolol could be considered a safe option for the treatment of horses suffering from OA. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Stanozolol Lameness Horse Joint Osteoarthritis
1. Introduction Osteoarthritis (OA) is one of the most prevalent and debilitating diseases affecting horses, with a notable economic impact on the equine industry [1–3]. Numerous treatment methods (physical, biological, and pharmaceutical) have been advocated either to prevent OA or to minimize clinical signs of pain (lameness), reduce joint deterioration, and prolong the competitive career of athletes. Acute or chronic synovitis and capsulitis are
* Corresponding author at: Riccardo Rinnovati, Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano Emilia (BO), Italy. E-mail address: [email protected] (R. Rinnovati). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.12.003
commonly treated with intra-articular (IA) medications [4]. An increased number of IA medications and treatment options are available on the market today, with therapies focused on providing symptom-modifying and/or diseasemodifying effects [5,6], to decrease inflammation in the damaged joint by acting on both synoviocytes and chondrocytes [7,8]. Stanozolol is a synthetic derivative of testosterone. Its properties include anabolic and/or androgenic activity [9], probably associated with its affinity for androgenic and, at lower doses, glucocorticoid receptors [10]. It has been demonstrated that stanozolol induces in vitro upregulation of osteoblast proliferation, collagen synthesis through transforming growth factor-b1 activity [11], chondrocyte secretion of insulin-like growth factor-1 [12], and, in rats,
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growth plate chondrocyte proliferation [13]. Moreover, it reduces NO production [12]. By increasing the production of collagen and other fundamental elements of the cartilage matrix, stanozolol stimulates the cartilaginous tissue [14], as indicated by the latest histological studies and clinical reports. Stanozolol has been used to treat sheep with OA induced by bilateral surgical medial meniscectomy [15]. In that study, a protective effect of IA stanozolol was observed in animals treated with 1 mg of the drug after 3 and 9 months compared with animals not been treated at all or animals administered placebo. Published reports on the clinical IA use of stanozolol in horses for the treatment of articular diseases, to our knowledge, are limited to a recent citation concerning the benefits of its use for the treatment of subchondral bone pain [16] and a report on 60 clinical cases of articular diseases [17]. A product containing stanozolol has been recently licensed in Italy for IA use in horses. The dosage of the drug was established with a previous pharmacokinetic study (N. Romagnoli, personal communication, 2013). Anecdotal reports refer to positive results obtained in clinical practice in Italy and in the UK especially in some difficult cases. This report aims to assess the ability of stanozolol to produce clinical and/or disease-modifying effects in horses affected by acute or chronic OA when administered IA and to monitor for any adverse effects. This trial design is a field multicenter double-blinded study. Our hypothesis was that American Association of Equine Practitioner (AAEP) scale lameness scores would improve by at least one degree after adequate treatment. 2. Materials and Methods The care and handling of the animals were in accordance with the provisions of the European Economic Community Council Directive 86/609, adopted by the Italian Government (D.L. 27/01/1992 n 116). 2.1. Animals Client-owned horses were selected for the inclusion in a multicenter study by equine veterinarian practitioners across Italy. The practitioners involved contacted the owners and requested the participation of their horses in the trial. Horse owners signed an informed consent for the inclusion in the study and committed to strictly following the rules of the experimental design; they were asked to agree that the designated individual would be administered the treatment blindly as prescribed. Horses were selected and enrolled in this study on the basis of the following inclusion criteria. (1) Horses must not be destined to the food chain; they must be in activity and aged between 2 and 20 years, with no distinction in sex or breed and weighing 250–650 kg. (2) Horses were required to be lame and show signs of joint pain attributed to aseptic acute or chronic OA in one joint only. (3) Horses had to be
healthy other than the lameness from OA in one joint; no gross anatomic abnormalities must be detected on the affected limb. Horses were excluded from the study on the basis of several criteria. (1) Horses with lameness caused by joint disease in more than one joint. (2) Horses subjected to local or systemic administration of Nonsteroidal anti-inflammatory drugs, glycosaminoglycans, hyaluronic acid, corticosteroids, or other antiarthritic drugs in the 15 days preceding the study. (3) Horses that had received anabolic drugs in the 30 days before the inclusion visit. (4) Lactating or pregnant mares, stallions during the reproductive period, horses with systemic diseases or infectious septic arthritis, specifically those with signs of hepatic or renal pathologies or tumors. Postinclusion removal from the study cohort could be decided (1) for any horse that failed to conform to the inclusion criteria during the study period, specifically if the correct prescription and treatment duration were not respected; (2) occurrence of serious adverse effects caused by the drug; and (3) if the owner withdrew the horse from the study without any motivation necessary. Because of economic constraints, only 60 horses were enrolled in the study. 2.2. Tested Product Each phial contains 5 mg of micronized stanozolol, 1.50 mg of polysorbate, 3.70 mg of sodium chloride (NaCldsalt), 10 mg of NaH2PO4, and phosphoric acid in 1 mL of water to pH 6.6. The placebo phials contain the same preparation without the tested drug (1.50 mg of polysorbate, 3.70 mg of NaCl, 10 mg of NaH2PO4, phosphoric acid in 1 mL of water to pH 6.6). 2.3. Double-Blinded Procedure Stanozolol and placebo were formulated and packed with an identical physical appearance and distributed in identical code-labeled containers. A coin toss randomly assigned the horses to treatment, either with stanozolol or placebo. Blinding of both the veterinary investigators and the horse owners was maintained throughout the study. The coding information was confidentially stored until the conclusion of the trial. 2.4. Participating Veterinarians Selected veterinarians who agreed to participate in the study signed a bilateral endorsement letter in which, as investigators, they declared to follow the scheduled protocol of the random double-blinded study. The veterinarians must compile the required forms and log books as described in the protocol. They were provided with a package with phials containing stanozolol or placebo. 2.5. Procedures Every owner agreed with the conditions and signed the informed consent. The horses were clinically evaluated by
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general, hematological, and lameness examination, including radiology, ultrasonography, and synovial fluid assessment for morphologic and cytologic abnormalities in the affected joint. A veterinary lameness examination assessed limb, location, and severity of the lameness graded according to the AAEP scale. The veterinary investigators performed the examination as follows: the horse was evaluated while walking straight on a loose lead and then made to circle in left and right directions. Then, the horse was trotted on a loose lead in a straight line and a circle. After identification of the painful limb, the horse was examined for swelling, heat, and pain to localize and score the lameness. A passive flexion test was performed, and this involved flexing the joint suspected of OA for 60 seconds, after which the horse was immediately trotted off. The examination proceeded if the investigator considered the findings correspondent with joint disease. After this, 2% of lidocaine or mepivacaine was aseptically injected into the articular space of the affected joint. The horse was then reevaluated to assess any change in the degree of lameness. Ultrasonography and radiology assessed the affected joint recording the amount of swelling, the size of joint space, any changes in the subchondral bone, the presence and size of osteophytes, and any evidence of stress fractures. The horse was included in the study if diagnosed with OA with no complicating factors. The horses that satisfied the inclusion criteria were divided in two groups. The “acute” group included horses diagnosed with acute arthropathy (horses with OA from less than 1 month), treated IA with stanozolol, and 10 horses treated with placebo. The second “chronic” group included horses affected by chronic arthropathy (horses with OA for more than 1 month), treated IA with stanozolol, and 10 horses treated with placebo. Stanozolol or placebo distributed in identical code-labeled containers was used blindly by veterinary investigators. The coding information remained secure throughout the study until the conclusion of the trial. Depending on the clinical evolution of the lameness, horses in the acute OA group were subjected to one to four administrations, at 7-day intervals, for a maximum treatment period of 21 days. In the chronic OA group, the horses were subjected to one to six administrations, again depending on the clinical evaluation, one every 7 days, for a maximum treatment period of 35 days. Control visits were scheduled on days 7, 14, 21, and 28 for the acute cases and on days 7, 14, 21, 28, 35, 42, and 60 for the chronic cases before the IA administration. The evaluation consisted of the same physical examinations and lameness investigations as reported previously. Synovial fluid was sampled before every IA injection. Fluid was grossly evaluated as reported by Spadari et al, and a score was assigned to each sample. If the improvement was complete before scheduled treatment had ended, the IA injections were suspended, but the scheduled visits continued. The result was considered to be positive when there was at least a grade 1 reduction in lameness according to the AAEP score. To partially overcome subjectivity and differences between judgments, a veterinary inspector (S.B.)
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constantly shadowed the veterinary investigators during the visits. At the end of the blinded trial, placebo-treated animals that had not improved by the last scheduled control visit, could be treated in open with the tested drug. The outcomes of this second “open” trial were not included as results in this article. 2.6. Financial Aspects The investigations reported and medications were provided at no cost to the horse owners and warranted by the drug producer company. Additional diagnostic imaging to document the joint disease was not provided, and when performed, the cost was the responsibility of the owners. 2.7. Data Analysis Descriptive analysis included lameness scores recorded by veterinarians throughout the trial, the joint affected, and the overall outcome. Chi-square analyses were used to compare data in the two treatment groups; the primary variable in the assessment of efficacy was the clinical improvement in lameness. Significance was P < .05. Confidence intervals have also been calculated. 3. Results 3.1. Population The trial was run during 2010 and in the early months of 2011. Twenty-six veterinary investigators enrolled 94 horses, 62 of which satisfied the criteria for inclusion in the study. Of these, two were disqualified during the trial: within 1 week from the first injection, one horse included in the acute OA group sustained a distal fracture of the fourth left metatarsal bone unrelated to the stanozolol injection. The second horse in the chronic OA group was excluded because the veterinarian started simultaneous treatment with phenylbutazone. Horses ranged from 2 to 18 years of age (median, 12.0 years). Measured or estimated body weight recorded was 500 kg. Italian Saddle (32) and Thoroughbred (18) were the two most commonly represented breeds in our population. The remainder consisted of a variety of other breeds, which included Standardbred (6), Appaloosa (2), Quarter Horse (1), and crossbred (1). Of the 60 horses that qualified for the trial, 31 had an acute OAd21 cases were treated with stanozolol IA and 10 with placebo. Of the 31 animals, nine (29.03%) had starting lameness grade 1, 18 (58.06%) had starting lameness grade 2, three (9.68%) had starting lameness grade 3, and one (3.23%) had starting lameness grade 4. No horse had grade 5 lameness. The proximal interphalangeal joint was most frequently affected (21 of 31 horses [67.74%]), followed by the antebrachiocarpal and middle carpal joints (four of 31 horses [12.90%]), distal intertarsal and tarsometatarsal joints (three of 31 horses [9.68%]), femoropatellar, lateral femorotibial, and medial femorotibial joints (two of 31 horses [6.45%]), and distal interphalangeal joint (one of 31 horses [3.23%]).
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Twenty-nine horses were affected with chronic OA: 19 were treated with stanozolol and 10 with placebo. Of the 29 animals, 12 (41.38%) had starting lameness grade 1, 12 (41.38%) had starting lameness grade 2, and five (17.24%) had a starting lameness grade 3. No horses with lameness grades 4 and 5 were included in the study. In the chronic group, the proximal interphalangeal (16 of 29 horses [55.17%]) was the most frequently affected joint, followed by the distal interphalangeal joint (six of 29 horses [20.69%]), antebrachiocarpal and middle carpal joints (four of 29 horses [13.80%]), and distal intertarsal and tarsometatarsal joints (three of 29 horses [10.34%]). 3.2. Safety and Adverse Reactions No severe adverse effects were noted. In six horses treated with the drug, mild ectasia of the injected joint was observed for a few days after treatment with stanozolol, which regressed rapidly and spontaneously. 3.3. Lameness Of the 40 horses treated with stanozolol, there was an improvement in lameness in 33 horses (82.50%), with lameness disappearing completely in 26 (65.00%) of them. In the acute group, there was an improvement in lameness in 15 of 21 (71.43%) horses treated with stanozolol. In all of them, lameness disappeared completely. In this acute group, lameness improved significantly (P ¼ .001), with 95% confidence interval of 52.11%–90.75%, after two treatments with stanozolol. Of the 10 horses treated with placebo, only one showed an improvement in lameness with 95% confidence interval of 0%–29.00%. This horse had lameness grade 1 (AAEP scale), which disappeared after two treatments. Regarding the horses with chronic disease, lameness improved in 18 of 19 (95.73%) horses. In 11 of 19 (57.90%) horses, lameness disappeared completely; in seven of 19 (36.84%) horses, lameness improved by one grade (AAEP scale). The improvement was significant (P ¼ .000), with 95% confidence interval of 84.70%–100%, after the fourth treatment. In placebo-treated chronic cases, the lameness improved in only one horse by one degree on the AAEP scale, with 95% confidence interval 0%–29.00%.
3.4. Synovial Fluid Gross Examination (Table 1) The effectiveness of stanozolol on the quality of the synovial fluid was evident in acute cases only after the third treatment. Only one horse with acute arthropathy had normal synovial fluid at the moment of inclusion. The viscosity, appearance, and total protein quantity of the synovial fluid returned to physiological values after the third treatment in 13 of 21 acute horses. In chronic cases, only two horses had normal synovial fluid on baseline analysis; the quality of the synovial fluid had improved in four horses by the end of the treatment, whereas no significant improvement was observed in the placebo-treated horses with chronic disease.
Table 1 Synovial fluid gross evaluation (from Dondi 2008). Characteristic
Normal
Soft Changes
Hard Changes
Viscosity Transparency Color
Normal Clear Citrine
Decreased Turbid Amber
Very decreased Very turbid Red
4. Discussion This study aimed to evaluate the clinical effect of stanozolol after IA injection in horses affected by acute or chronic OA. The trial design is clinical field double blinded, performed on lame horses either treated with the drug or placebo. Positive results were considered to be a clinical improvement of one degree of lameness on the AAEP scale, obtained after a treatment with no expected effect. Frequently, the result of treatment with the tested drug was positive with improvement superior to the predetermined goal. The results of this clinical trial indicated that horses with OA in one joint, treated with 5 mg of IA injection of stanozolol, showed a significant reduction in the severity of lameness, and an improved quality of the synovial fluid, compared with the data from horses injected with the placebo. The clinical improvement of the animals persisted after the end of the therapeutic cycle scheduled in the trial, and diagnostic imaging privately obtained for some of the animals (radiology, magnetic resonance imaging, and scintigraphy), confirmed the clinical observations. In particular, subchondral bone sclerosis or lysis disappeared, and there was strong decrease of enthesophytes. Two horses were excluded after the inclusion in the trial: one horse, after the first IA injection because of a distal fracture of the fourth left metatarsal bone; a second horse because of simultaneous oral treatment with phenylbutazone. One horse included in the chronic OA group had a small skin wound in the treated limb after the fourth treatment; as lameness had disappeared, the veterinary investigator decided to stop the injections. The horse was still sound and was not excluded from the trial. In six horses, transient postinjection swelling was observed in the treated articulation. It did not require any intervention and disappeared in few days. In the definition of the experimental design, our main concerns were the subjective variability of lameness evaluations per se and the local environmental variables. Keegan et al [18] showed that there was 93.1% agreement in the clinical evaluation of lameness between experienced clinicians when the mean AAEP score for a specific limb was >1.5. Otherwise, veterinarians agreed that a forelimb was lame, in only about three of four times, reducing this value to two of three times in reference to hind limb lameness. We have assumed that the AAEP scale is well known and accepted in the standard practice by our veterinary investigators, and to partially overcome subjectivity, we decided to have an inspector shadowed the veterinary investigator during the visits. The same inspector (S.B.) observed the entire trial to give an assurance of uniformity in the examination protocol and to have uniformity of judgment for the horse population. The recommendation is
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to walk and trot horses along the same track length and on ground of similar characteristics, with the exception that the terrain was always concrete. The flexion tests had the same duration. Moreover, to record our lameness investigation for a subsequent assessment by other referees, the first cases included were filmed. The work did not produce the desired results, confirming the criticism reported in the literature for this method [18,19], in addition to a number of technical problems, including the inadequate perspective of some film clips and the consequently weak possibility of making a proper comparison of the serial tests. When the endorsement letters were signed, the owners were assured that, in the event of a negative response due to the blinded administration of placebo, the horses would be treated with the drug at a later stage, according to the same protocol used in the trial. The outcomes were also positive in these horses. They have been mentioned in this discussion to confirm the efficacy of the treatment but have not been included it the results of this article. Stanozolol is available as a commercial product, approved by the Italian Health Ministry for IA use in horses. Because of its strong anabolic effects, studies on the effect of stanozolol on OA have not been performed on humans [20,21]. Some promising results have recently led to renewed interest in the use of this drug in veterinary medicine for companion animals [9,22,23]. Systemic stanozolol has been used in horses as an anabolic steroid and is considered a doping agent [24,25]. In the unique experimental study present in the literature [15] in a model of OA induced in sheep, the authors observed that stanozolol did not produce any adverse joint reaction, even after repeated injections, and that it reduced morphologic changes associated with induced OA (osteophyte formation and subchondral bone reaction). The features observed in articular cartilage demonstrated a valid and organized articular chondral matrix regeneration with a normal morphologic structure. The recent data presented by Ramzan [17], obtained by the IA injection of stanozolol in the clinical practice in horses, are comparable with the results from this study. He used stanozolol after previous therapies failed, for cases of extreme pathologic severity or as postoperative medication after joint surgery, in horses affected by a variety of joint diseases including OA, subchondral bone diseases, and osteochondrosis. The results were very similar to ours, both in the perceived importance of the observed adverse reactions and in the efficacy of the treatment with 78.3% of positive results, after more than three treatments. “The strong impression that IA stanozolol was beneficial over and above previous intercurrent therapies” was indicated for 39.1%. Sixty-four percent of horses were healthy or required less IA therapies in subsequent seasons [17]. In our sample population, the results differed between acute and chronic cases in terms of the lameness score variation and with regard to the number of injections required to obtain lameness regression. In acute cases, only 15 of 21 patients improved; however, in these horses, lameness disappeared completely. In chronic cases, an improvement was observed in 18 of 19 horses; the lameness disappeared in only 11 of them.
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The reason for this difference is not easy to explain. On the basis of reported data, we can assume that a normalization of synovial fluid and a reduction in IA secretion of interleukins (ILs) or other inflammatory mediators with stimulation of synoviocyte anabolism could lead to shortterm reduction in joint inflammation and pain. If the insult had produced no severe structural lesions, the effect of the drug could be rapid and complete. Conversely, in chronic cases, the greatest positive effects of stanozolol could be expressed inside the joint, associated with articular cartilage repair and remineralization of bony structures involved in OA, as observed in sheep with experimentally induced OA after 9 months [15]. These delayed effects are not achieved with IA corticosteroid injection. After rapid pain release, this class of drugs normally does not improve bone rebuilding and interferes with cartilage repair. The prolonged effect observed could probably be associated with the onset time and the pharmacologic effects of stanozolol inside the joint. A possible explanation for this phenomenon could be found in the research performed by Capellino et al [26] concerning the osteoarthritic effects of rheumatoid arthritis; the inflammation and OA observed were attributed to an imbalance between androgens and estrogens in the articular space and in the synovia. This is essential in the development of inflammatory changes in the joints, also caused by the activation of ILs and other biochemical mediators of inflammation. In his experience, the androgenic activity associated with a high concentration of stanozolol in the articular space interferes with the androgen and/or estrogen imbalance and their inflammatory consequences and eliminates the aforementioned mechanisms, required for the induction and perpetuation of the inflammatory effects [26]. The use of stanozolol has its rationale in the affinity of this molecule for the androgen receptors located in cytoplasm or nucleus. More specifically, stanozolol modulates the enzymatic mechanisms of protein synthesis [14]. Like other anabolic steroids, stanozolol also stimulates osteoblast proliferation, the production of bone matrix and growth factor synthesis, and reduces in vitro apoptosis in equine chondrocytes, by reducing the production of NO and stimulating insulin-like growth factor-1 production [12]. It also has an antagonistic action toward parathyroid hormone and IL-1, and in vitro, it also prevents the degenerative effects on the mineral component [27]. The main side effects of anabolic steroids are masculine behavior, testicular atrophy, and liver dysfunction [12]. Nevertheless, the IA dosage used in the present trial is less than doses reported to produce side effects and far the below the 10 mg twice a week intramuscular dose that has been demonstrated to have anabolic effects in man [28]. However, considering the withdrawal implications associated with the use of anabolic steroids in competing horses, it should be take into account carefully to adopt the treatment during “out of season” periods. 5. Conclusions This study demonstrated that IA treatment with stanozolol, administered at a dosage of 5 mg for each joint on a
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weekly basis can be safely used without serious adverse effects; the product significantly reduces the severity of lameness and joint pain and improves response to flexion in affected joints. Clinical improvement of the lameness was evident in both acute and chronic cases as assessed by veterinary lameness investigations. Stanozolol therefore could be considered a viable option to prolong the athletic career of horses suffering from OA. Acknowledgments All authors contributed to the study design, article preparation, and data collection. References [1] Rossdale PD, Hopes R, Wingfield Digby NJ, Offord K. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985; 116:66–9. [2] Todhunter R, Lust G. Pathophysiology of synovitis: clinical signs and examination in horses. Comp Cont Educ Pract 1990;12: 980–92. [3] NAHMS. Lameness and laminitis in US horses. National Animal Health Monitoring System. Fort Collins: USDA APHIS, Veterinary Services-Centers for Epidemiology and Animal Health, http://www. aphis.usda.gov/animal_health/nahms/equine/downloads/equine98/ Equine98_dr_Lameness.pdf; 2000. Accessed March 29, 2013. [4] Frisbie DD. Synovial joint biology and pathobiology. In: Auer JA, Stick JA, editors. Equine surgery. 3rd edn. St. Louis: Saunders Elsevier; 2006. p. 1036–54. [5] McIlwraith CW. Licensed medications, “generic” medications, compounding, and nutraceuticalsdwhat has been scientifically validated, where do we encounter scientific mistruth, and where are we legally? Proc Am Assoc Equine Pract 2004;50:1482. [6] Frisbie DD. Principles of treatment of joint diseases. In: Auer JA, Stick JA, editors. Equine surgery. 3rd edn. St. Louis: Saunders Elsevier; 2006. p. 1055–72. [7] McIlwraith CW, Vachon A. Review of pathogenesis and treatment of degenerative joint disease. Equine Vet J Suppl 1988;(6):3–11. [8] Ghosh P, Smith M, Wells C. Second line agents in osteoarthritis. In: Dixon JS, Furst DE, editors. Second line agents in the treatment of rheumatic diseases. New York: Marcel Dekker Inc; 1992. p. 363–427. [9] Zannetti G. Impiego clinico classico dello stanazololo in terapia. Cavriago: ACME; 2004. [10] Fernandez L, Chirino R, Boada LD, Navarro D, Cabrera N, Del Rio I, et al. Stanozolol and danazol, unlike natural androgens, interact with low affinity glucocorticoid-binding sites from male rat live microsomes. Endocrinology 1994;134:1401–8. [11] Falanga V, Greenberg AS, Zhou L, Ochoa SM, Roberts AB, Falabella A, et al. Stimulation of collagen synthesis by the anabolic steroid stanozolol. J Invest Dermatol 1998;111:1193–7.
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Original Research
Clinical Evaluation of Intra-articular Administration of Stanozolol to Manage Lameness Associated With Acute and Chronic Osteoarthritis in Horses Alessandro Spadari, Riccardo Rinnovati*, Simona Babbini, Noemi Romagnoli Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (BO), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2014 Received in revised form 1 December 2014 Accepted 3 December 2014 Available online 9 December 2014
Poor data could be found in the literature on the effects of intra-articular (IA) administration of stanozolol in horses affected by osteoarthritis (OA). To verify the clinical effects of IA stanozolol in acute and chronic cases of OA in horses, a clinical double-blinded trial involving 60 client-owned horses was performed. Veterinary practitioners selected horses, all showing clinical signs of acute or chronic OA, diagnosed in a single joint. After lameness evaluation and synovial fluid collection, the clinicians administered one to four (acute cases) or one to six (chronic cases) weekly IA doses of 5 mg of stanozolol or placebo. Positive result was considered a grade 1 improvement in lameness scored according to the American Association of Equine Practitioner scale. The characteristics of the synovial fluid were also evaluated. Of the 60 horses included in the study, 31 were affected by acute OA and 29 by chronic OA. Horses were treated blindly with placebo. The overall outcomes were positive in 82.50% of cases. Lameness disappeared completely in 15 of 21 (acute) and 11 of 19 (chronic) animals, and there was a significant reduction after two and four treatments, in the acute and chronic cases, respectively. Improvement in the physical characteristics of the synovial fluid sampled was evident after the third treatment. Stanozolol injected IA at a dose of 5 mg significantly resolved lameness in affected joints. Stanozolol could be considered a safe option for the treatment of horses suffering from OA. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Stanozolol Lameness Horse Joint Osteoarthritis
1. Introduction Osteoarthritis (OA) is one of the most prevalent and debilitating diseases affecting horses, with a notable economic impact on the equine industry [1–3]. Numerous treatment methods (physical, biological, and pharmaceutical) have been advocated either to prevent OA or to minimize clinical signs of pain (lameness), reduce joint deterioration, and prolong the competitive career of athletes. Acute or chronic synovitis and capsulitis are
* Corresponding author at: Riccardo Rinnovati, Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano Emilia (BO), Italy. E-mail address: [email protected] (R. Rinnovati). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.12.003
commonly treated with intra-articular (IA) medications [4]. An increased number of IA medications and treatment options are available on the market today, with therapies focused on providing symptom-modifying and/or diseasemodifying effects [5,6], to decrease inflammation in the damaged joint by acting on both synoviocytes and chondrocytes [7,8]. Stanozolol is a synthetic derivative of testosterone. Its properties include anabolic and/or androgenic activity [9], probably associated with its affinity for androgenic and, at lower doses, glucocorticoid receptors [10]. It has been demonstrated that stanozolol induces in vitro upregulation of osteoblast proliferation, collagen synthesis through transforming growth factor-b1 activity [11], chondrocyte secretion of insulin-like growth factor-1 [12], and, in rats,
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growth plate chondrocyte proliferation [13]. Moreover, it reduces NO production [12]. By increasing the production of collagen and other fundamental elements of the cartilage matrix, stanozolol stimulates the cartilaginous tissue [14], as indicated by the latest histological studies and clinical reports. Stanozolol has been used to treat sheep with OA induced by bilateral surgical medial meniscectomy [15]. In that study, a protective effect of IA stanozolol was observed in animals treated with 1 mg of the drug after 3 and 9 months compared with animals not been treated at all or animals administered placebo. Published reports on the clinical IA use of stanozolol in horses for the treatment of articular diseases, to our knowledge, are limited to a recent citation concerning the benefits of its use for the treatment of subchondral bone pain [16] and a report on 60 clinical cases of articular diseases [17]. A product containing stanozolol has been recently licensed in Italy for IA use in horses. The dosage of the drug was established with a previous pharmacokinetic study (N. Romagnoli, personal communication, 2013). Anecdotal reports refer to positive results obtained in clinical practice in Italy and in the UK especially in some difficult cases. This report aims to assess the ability of stanozolol to produce clinical and/or disease-modifying effects in horses affected by acute or chronic OA when administered IA and to monitor for any adverse effects. This trial design is a field multicenter double-blinded study. Our hypothesis was that American Association of Equine Practitioner (AAEP) scale lameness scores would improve by at least one degree after adequate treatment. 2. Materials and Methods The care and handling of the animals were in accordance with the provisions of the European Economic Community Council Directive 86/609, adopted by the Italian Government (D.L. 27/01/1992 n 116). 2.1. Animals Client-owned horses were selected for the inclusion in a multicenter study by equine veterinarian practitioners across Italy. The practitioners involved contacted the owners and requested the participation of their horses in the trial. Horse owners signed an informed consent for the inclusion in the study and committed to strictly following the rules of the experimental design; they were asked to agree that the designated individual would be administered the treatment blindly as prescribed. Horses were selected and enrolled in this study on the basis of the following inclusion criteria. (1) Horses must not be destined to the food chain; they must be in activity and aged between 2 and 20 years, with no distinction in sex or breed and weighing 250–650 kg. (2) Horses were required to be lame and show signs of joint pain attributed to aseptic acute or chronic OA in one joint only. (3) Horses had to be
healthy other than the lameness from OA in one joint; no gross anatomic abnormalities must be detected on the affected limb. Horses were excluded from the study on the basis of several criteria. (1) Horses with lameness caused by joint disease in more than one joint. (2) Horses subjected to local or systemic administration of Nonsteroidal anti-inflammatory drugs, glycosaminoglycans, hyaluronic acid, corticosteroids, or other antiarthritic drugs in the 15 days preceding the study. (3) Horses that had received anabolic drugs in the 30 days before the inclusion visit. (4) Lactating or pregnant mares, stallions during the reproductive period, horses with systemic diseases or infectious septic arthritis, specifically those with signs of hepatic or renal pathologies or tumors. Postinclusion removal from the study cohort could be decided (1) for any horse that failed to conform to the inclusion criteria during the study period, specifically if the correct prescription and treatment duration were not respected; (2) occurrence of serious adverse effects caused by the drug; and (3) if the owner withdrew the horse from the study without any motivation necessary. Because of economic constraints, only 60 horses were enrolled in the study. 2.2. Tested Product Each phial contains 5 mg of micronized stanozolol, 1.50 mg of polysorbate, 3.70 mg of sodium chloride (NaCldsalt), 10 mg of NaH2PO4, and phosphoric acid in 1 mL of water to pH 6.6. The placebo phials contain the same preparation without the tested drug (1.50 mg of polysorbate, 3.70 mg of NaCl, 10 mg of NaH2PO4, phosphoric acid in 1 mL of water to pH 6.6). 2.3. Double-Blinded Procedure Stanozolol and placebo were formulated and packed with an identical physical appearance and distributed in identical code-labeled containers. A coin toss randomly assigned the horses to treatment, either with stanozolol or placebo. Blinding of both the veterinary investigators and the horse owners was maintained throughout the study. The coding information was confidentially stored until the conclusion of the trial. 2.4. Participating Veterinarians Selected veterinarians who agreed to participate in the study signed a bilateral endorsement letter in which, as investigators, they declared to follow the scheduled protocol of the random double-blinded study. The veterinarians must compile the required forms and log books as described in the protocol. They were provided with a package with phials containing stanozolol or placebo. 2.5. Procedures Every owner agreed with the conditions and signed the informed consent. The horses were clinically evaluated by
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general, hematological, and lameness examination, including radiology, ultrasonography, and synovial fluid assessment for morphologic and cytologic abnormalities in the affected joint. A veterinary lameness examination assessed limb, location, and severity of the lameness graded according to the AAEP scale. The veterinary investigators performed the examination as follows: the horse was evaluated while walking straight on a loose lead and then made to circle in left and right directions. Then, the horse was trotted on a loose lead in a straight line and a circle. After identification of the painful limb, the horse was examined for swelling, heat, and pain to localize and score the lameness. A passive flexion test was performed, and this involved flexing the joint suspected of OA for 60 seconds, after which the horse was immediately trotted off. The examination proceeded if the investigator considered the findings correspondent with joint disease. After this, 2% of lidocaine or mepivacaine was aseptically injected into the articular space of the affected joint. The horse was then reevaluated to assess any change in the degree of lameness. Ultrasonography and radiology assessed the affected joint recording the amount of swelling, the size of joint space, any changes in the subchondral bone, the presence and size of osteophytes, and any evidence of stress fractures. The horse was included in the study if diagnosed with OA with no complicating factors. The horses that satisfied the inclusion criteria were divided in two groups. The “acute” group included horses diagnosed with acute arthropathy (horses with OA from less than 1 month), treated IA with stanozolol, and 10 horses treated with placebo. The second “chronic” group included horses affected by chronic arthropathy (horses with OA for more than 1 month), treated IA with stanozolol, and 10 horses treated with placebo. Stanozolol or placebo distributed in identical code-labeled containers was used blindly by veterinary investigators. The coding information remained secure throughout the study until the conclusion of the trial. Depending on the clinical evolution of the lameness, horses in the acute OA group were subjected to one to four administrations, at 7-day intervals, for a maximum treatment period of 21 days. In the chronic OA group, the horses were subjected to one to six administrations, again depending on the clinical evaluation, one every 7 days, for a maximum treatment period of 35 days. Control visits were scheduled on days 7, 14, 21, and 28 for the acute cases and on days 7, 14, 21, 28, 35, 42, and 60 for the chronic cases before the IA administration. The evaluation consisted of the same physical examinations and lameness investigations as reported previously. Synovial fluid was sampled before every IA injection. Fluid was grossly evaluated as reported by Spadari et al, and a score was assigned to each sample. If the improvement was complete before scheduled treatment had ended, the IA injections were suspended, but the scheduled visits continued. The result was considered to be positive when there was at least a grade 1 reduction in lameness according to the AAEP score. To partially overcome subjectivity and differences between judgments, a veterinary inspector (S.B.)
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constantly shadowed the veterinary investigators during the visits. At the end of the blinded trial, placebo-treated animals that had not improved by the last scheduled control visit, could be treated in open with the tested drug. The outcomes of this second “open” trial were not included as results in this article. 2.6. Financial Aspects The investigations reported and medications were provided at no cost to the horse owners and warranted by the drug producer company. Additional diagnostic imaging to document the joint disease was not provided, and when performed, the cost was the responsibility of the owners. 2.7. Data Analysis Descriptive analysis included lameness scores recorded by veterinarians throughout the trial, the joint affected, and the overall outcome. Chi-square analyses were used to compare data in the two treatment groups; the primary variable in the assessment of efficacy was the clinical improvement in lameness. Significance was P < .05. Confidence intervals have also been calculated. 3. Results 3.1. Population The trial was run during 2010 and in the early months of 2011. Twenty-six veterinary investigators enrolled 94 horses, 62 of which satisfied the criteria for inclusion in the study. Of these, two were disqualified during the trial: within 1 week from the first injection, one horse included in the acute OA group sustained a distal fracture of the fourth left metatarsal bone unrelated to the stanozolol injection. The second horse in the chronic OA group was excluded because the veterinarian started simultaneous treatment with phenylbutazone. Horses ranged from 2 to 18 years of age (median, 12.0 years). Measured or estimated body weight recorded was 500 kg. Italian Saddle (32) and Thoroughbred (18) were the two most commonly represented breeds in our population. The remainder consisted of a variety of other breeds, which included Standardbred (6), Appaloosa (2), Quarter Horse (1), and crossbred (1). Of the 60 horses that qualified for the trial, 31 had an acute OAd21 cases were treated with stanozolol IA and 10 with placebo. Of the 31 animals, nine (29.03%) had starting lameness grade 1, 18 (58.06%) had starting lameness grade 2, three (9.68%) had starting lameness grade 3, and one (3.23%) had starting lameness grade 4. No horse had grade 5 lameness. The proximal interphalangeal joint was most frequently affected (21 of 31 horses [67.74%]), followed by the antebrachiocarpal and middle carpal joints (four of 31 horses [12.90%]), distal intertarsal and tarsometatarsal joints (three of 31 horses [9.68%]), femoropatellar, lateral femorotibial, and medial femorotibial joints (two of 31 horses [6.45%]), and distal interphalangeal joint (one of 31 horses [3.23%]).
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Twenty-nine horses were affected with chronic OA: 19 were treated with stanozolol and 10 with placebo. Of the 29 animals, 12 (41.38%) had starting lameness grade 1, 12 (41.38%) had starting lameness grade 2, and five (17.24%) had a starting lameness grade 3. No horses with lameness grades 4 and 5 were included in the study. In the chronic group, the proximal interphalangeal (16 of 29 horses [55.17%]) was the most frequently affected joint, followed by the distal interphalangeal joint (six of 29 horses [20.69%]), antebrachiocarpal and middle carpal joints (four of 29 horses [13.80%]), and distal intertarsal and tarsometatarsal joints (three of 29 horses [10.34%]). 3.2. Safety and Adverse Reactions No severe adverse effects were noted. In six horses treated with the drug, mild ectasia of the injected joint was observed for a few days after treatment with stanozolol, which regressed rapidly and spontaneously. 3.3. Lameness Of the 40 horses treated with stanozolol, there was an improvement in lameness in 33 horses (82.50%), with lameness disappearing completely in 26 (65.00%) of them. In the acute group, there was an improvement in lameness in 15 of 21 (71.43%) horses treated with stanozolol. In all of them, lameness disappeared completely. In this acute group, lameness improved significantly (P ¼ .001), with 95% confidence interval of 52.11%–90.75%, after two treatments with stanozolol. Of the 10 horses treated with placebo, only one showed an improvement in lameness with 95% confidence interval of 0%–29.00%. This horse had lameness grade 1 (AAEP scale), which disappeared after two treatments. Regarding the horses with chronic disease, lameness improved in 18 of 19 (95.73%) horses. In 11 of 19 (57.90%) horses, lameness disappeared completely; in seven of 19 (36.84%) horses, lameness improved by one grade (AAEP scale). The improvement was significant (P ¼ .000), with 95% confidence interval of 84.70%–100%, after the fourth treatment. In placebo-treated chronic cases, the lameness improved in only one horse by one degree on the AAEP scale, with 95% confidence interval 0%–29.00%.
3.4. Synovial Fluid Gross Examination (Table 1) The effectiveness of stanozolol on the quality of the synovial fluid was evident in acute cases only after the third treatment. Only one horse with acute arthropathy had normal synovial fluid at the moment of inclusion. The viscosity, appearance, and total protein quantity of the synovial fluid returned to physiological values after the third treatment in 13 of 21 acute horses. In chronic cases, only two horses had normal synovial fluid on baseline analysis; the quality of the synovial fluid had improved in four horses by the end of the treatment, whereas no significant improvement was observed in the placebo-treated horses with chronic disease.
Table 1 Synovial fluid gross evaluation (from Dondi 2008). Characteristic
Normal
Soft Changes
Hard Changes
Viscosity Transparency Color
Normal Clear Citrine
Decreased Turbid Amber
Very decreased Very turbid Red
4. Discussion This study aimed to evaluate the clinical effect of stanozolol after IA injection in horses affected by acute or chronic OA. The trial design is clinical field double blinded, performed on lame horses either treated with the drug or placebo. Positive results were considered to be a clinical improvement of one degree of lameness on the AAEP scale, obtained after a treatment with no expected effect. Frequently, the result of treatment with the tested drug was positive with improvement superior to the predetermined goal. The results of this clinical trial indicated that horses with OA in one joint, treated with 5 mg of IA injection of stanozolol, showed a significant reduction in the severity of lameness, and an improved quality of the synovial fluid, compared with the data from horses injected with the placebo. The clinical improvement of the animals persisted after the end of the therapeutic cycle scheduled in the trial, and diagnostic imaging privately obtained for some of the animals (radiology, magnetic resonance imaging, and scintigraphy), confirmed the clinical observations. In particular, subchondral bone sclerosis or lysis disappeared, and there was strong decrease of enthesophytes. Two horses were excluded after the inclusion in the trial: one horse, after the first IA injection because of a distal fracture of the fourth left metatarsal bone; a second horse because of simultaneous oral treatment with phenylbutazone. One horse included in the chronic OA group had a small skin wound in the treated limb after the fourth treatment; as lameness had disappeared, the veterinary investigator decided to stop the injections. The horse was still sound and was not excluded from the trial. In six horses, transient postinjection swelling was observed in the treated articulation. It did not require any intervention and disappeared in few days. In the definition of the experimental design, our main concerns were the subjective variability of lameness evaluations per se and the local environmental variables. Keegan et al [18] showed that there was 93.1% agreement in the clinical evaluation of lameness between experienced clinicians when the mean AAEP score for a specific limb was >1.5. Otherwise, veterinarians agreed that a forelimb was lame, in only about three of four times, reducing this value to two of three times in reference to hind limb lameness. We have assumed that the AAEP scale is well known and accepted in the standard practice by our veterinary investigators, and to partially overcome subjectivity, we decided to have an inspector shadowed the veterinary investigator during the visits. The same inspector (S.B.) observed the entire trial to give an assurance of uniformity in the examination protocol and to have uniformity of judgment for the horse population. The recommendation is
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to walk and trot horses along the same track length and on ground of similar characteristics, with the exception that the terrain was always concrete. The flexion tests had the same duration. Moreover, to record our lameness investigation for a subsequent assessment by other referees, the first cases included were filmed. The work did not produce the desired results, confirming the criticism reported in the literature for this method [18,19], in addition to a number of technical problems, including the inadequate perspective of some film clips and the consequently weak possibility of making a proper comparison of the serial tests. When the endorsement letters were signed, the owners were assured that, in the event of a negative response due to the blinded administration of placebo, the horses would be treated with the drug at a later stage, according to the same protocol used in the trial. The outcomes were also positive in these horses. They have been mentioned in this discussion to confirm the efficacy of the treatment but have not been included it the results of this article. Stanozolol is available as a commercial product, approved by the Italian Health Ministry for IA use in horses. Because of its strong anabolic effects, studies on the effect of stanozolol on OA have not been performed on humans [20,21]. Some promising results have recently led to renewed interest in the use of this drug in veterinary medicine for companion animals [9,22,23]. Systemic stanozolol has been used in horses as an anabolic steroid and is considered a doping agent [24,25]. In the unique experimental study present in the literature [15] in a model of OA induced in sheep, the authors observed that stanozolol did not produce any adverse joint reaction, even after repeated injections, and that it reduced morphologic changes associated with induced OA (osteophyte formation and subchondral bone reaction). The features observed in articular cartilage demonstrated a valid and organized articular chondral matrix regeneration with a normal morphologic structure. The recent data presented by Ramzan [17], obtained by the IA injection of stanozolol in the clinical practice in horses, are comparable with the results from this study. He used stanozolol after previous therapies failed, for cases of extreme pathologic severity or as postoperative medication after joint surgery, in horses affected by a variety of joint diseases including OA, subchondral bone diseases, and osteochondrosis. The results were very similar to ours, both in the perceived importance of the observed adverse reactions and in the efficacy of the treatment with 78.3% of positive results, after more than three treatments. “The strong impression that IA stanozolol was beneficial over and above previous intercurrent therapies” was indicated for 39.1%. Sixty-four percent of horses were healthy or required less IA therapies in subsequent seasons [17]. In our sample population, the results differed between acute and chronic cases in terms of the lameness score variation and with regard to the number of injections required to obtain lameness regression. In acute cases, only 15 of 21 patients improved; however, in these horses, lameness disappeared completely. In chronic cases, an improvement was observed in 18 of 19 horses; the lameness disappeared in only 11 of them.
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The reason for this difference is not easy to explain. On the basis of reported data, we can assume that a normalization of synovial fluid and a reduction in IA secretion of interleukins (ILs) or other inflammatory mediators with stimulation of synoviocyte anabolism could lead to shortterm reduction in joint inflammation and pain. If the insult had produced no severe structural lesions, the effect of the drug could be rapid and complete. Conversely, in chronic cases, the greatest positive effects of stanozolol could be expressed inside the joint, associated with articular cartilage repair and remineralization of bony structures involved in OA, as observed in sheep with experimentally induced OA after 9 months [15]. These delayed effects are not achieved with IA corticosteroid injection. After rapid pain release, this class of drugs normally does not improve bone rebuilding and interferes with cartilage repair. The prolonged effect observed could probably be associated with the onset time and the pharmacologic effects of stanozolol inside the joint. A possible explanation for this phenomenon could be found in the research performed by Capellino et al [26] concerning the osteoarthritic effects of rheumatoid arthritis; the inflammation and OA observed were attributed to an imbalance between androgens and estrogens in the articular space and in the synovia. This is essential in the development of inflammatory changes in the joints, also caused by the activation of ILs and other biochemical mediators of inflammation. In his experience, the androgenic activity associated with a high concentration of stanozolol in the articular space interferes with the androgen and/or estrogen imbalance and their inflammatory consequences and eliminates the aforementioned mechanisms, required for the induction and perpetuation of the inflammatory effects [26]. The use of stanozolol has its rationale in the affinity of this molecule for the androgen receptors located in cytoplasm or nucleus. More specifically, stanozolol modulates the enzymatic mechanisms of protein synthesis [14]. Like other anabolic steroids, stanozolol also stimulates osteoblast proliferation, the production of bone matrix and growth factor synthesis, and reduces in vitro apoptosis in equine chondrocytes, by reducing the production of NO and stimulating insulin-like growth factor-1 production [12]. It also has an antagonistic action toward parathyroid hormone and IL-1, and in vitro, it also prevents the degenerative effects on the mineral component [27]. The main side effects of anabolic steroids are masculine behavior, testicular atrophy, and liver dysfunction [12]. Nevertheless, the IA dosage used in the present trial is less than doses reported to produce side effects and far the below the 10 mg twice a week intramuscular dose that has been demonstrated to have anabolic effects in man [28]. However, considering the withdrawal implications associated with the use of anabolic steroids in competing horses, it should be take into account carefully to adopt the treatment during “out of season” periods. 5. Conclusions This study demonstrated that IA treatment with stanozolol, administered at a dosage of 5 mg for each joint on a
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weekly basis can be safely used without serious adverse effects; the product significantly reduces the severity of lameness and joint pain and improves response to flexion in affected joints. Clinical improvement of the lameness was evident in both acute and chronic cases as assessed by veterinary lameness investigations. Stanozolol therefore could be considered a viable option to prolong the athletic career of horses suffering from OA. Acknowledgments All authors contributed to the study design, article preparation, and data collection. References [1] Rossdale PD, Hopes R, Wingfield Digby NJ, Offord K. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985; 116:66–9. [2] Todhunter R, Lust G. Pathophysiology of synovitis: clinical signs and examination in horses. Comp Cont Educ Pract 1990;12: 980–92. [3] NAHMS. Lameness and laminitis in US horses. National Animal Health Monitoring System. Fort Collins: USDA APHIS, Veterinary Services-Centers for Epidemiology and Animal Health, http://www. aphis.usda.gov/animal_health/nahms/equine/downloads/equine98/ Equine98_dr_Lameness.pdf; 2000. Accessed March 29, 2013. [4] Frisbie DD. Synovial joint biology and pathobiology. In: Auer JA, Stick JA, editors. Equine surgery. 3rd edn. St. Louis: Saunders Elsevier; 2006. p. 1036–54. [5] McIlwraith CW. Licensed medications, “generic” medications, compounding, and nutraceuticalsdwhat has been scientifically validated, where do we encounter scientific mistruth, and where are we legally? Proc Am Assoc Equine Pract 2004;50:1482. [6] Frisbie DD. Principles of treatment of joint diseases. In: Auer JA, Stick JA, editors. Equine surgery. 3rd edn. St. Louis: Saunders Elsevier; 2006. p. 1055–72. [7] McIlwraith CW, Vachon A. Review of pathogenesis and treatment of degenerative joint disease. Equine Vet J Suppl 1988;(6):3–11. [8] Ghosh P, Smith M, Wells C. Second line agents in osteoarthritis. In: Dixon JS, Furst DE, editors. Second line agents in the treatment of rheumatic diseases. New York: Marcel Dekker Inc; 1992. p. 363–427. [9] Zannetti G. Impiego clinico classico dello stanazololo in terapia. Cavriago: ACME; 2004. [10] Fernandez L, Chirino R, Boada LD, Navarro D, Cabrera N, Del Rio I, et al. Stanozolol and danazol, unlike natural androgens, interact with low affinity glucocorticoid-binding sites from male rat live microsomes. Endocrinology 1994;134:1401–8. [11] Falanga V, Greenberg AS, Zhou L, Ochoa SM, Roberts AB, Falabella A, et al. Stimulation of collagen synthesis by the anabolic steroid stanozolol. J Invest Dermatol 1998;111:1193–7.
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