Design, synthesis and characterization of a PEGylated stanozolol for potential therapeutic applications.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signaling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH2)10kDa, (MeO-PEG-NH)10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, 1H NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.

Population pharmacokinetics of ciclosporin in Chinese children with aplastic anemia: effects of weight, renal function and stanozolol administration.

AIM: To develop a population pharmacokinetic model for the immunosuppressant ciclosporin in Chinese children with aplastic anemia and to identify covariates influencing ciclosporin pharmacokinetics. METHODS: A total of 102 children with either acquired or congenital aplastic anemia aged 8.8±3.6 years (range 0.9-17.6 years) were included. Therapeutic drug monitoring (TDM) data for ciclosporin were collected. The population pharmacokinetic model of ciclosporin was described using the nonlinear mixed-effects modeling (NONMEM) VI software. The final model was validated using bootstrap and normalized prediction distribution errors. RESULTS: A one-compartment model with first-order absorption and elimination was developed. The estimated CL/F was 15.1, which was lower than those of children receiving stem cell or kidney transplant reported in the West (16.9-29.3). The weight normalized CL/F was 0.45 (range: 0.27-0.70) Lh(-1)·kg(-1). The covariate analysis identified body weight, serum creatinine and concomitant administration of the anabolic steroid stanozolol as individual factors influencing the CL/F of ciclosporin. CONCLUSION: Our model could be used to optimize the ciclosporin dosing regimen in Chinese children with aplastic anemia.

Prolonged treatment with the anabolic–androgenic steroid stanozolol increases antioxidant defences in rat skeletal muscle

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Testosterone and its synthetic derivatives anabolic–androgenic steroids have been shown to increase skeletal muscle work capacity and fatigue resistance, but the molecular basis for these effects remains uncertain. Since muscle performance has been related to redox status of exercising muscles, this investigation was aimed at testing whether a treatment with suprapharmacological doses of the anabolic–androgenic steroid stanozolol, (2 mg/kg body weight, 5 days/week, for 8 weeks), either alone or in conjunction with treadmill training (12 weeks), enhanced antioxidant defences in rat muscles. Stanozolol treatment did not modify thiobarbituric acid reactive substances and glutathione content in soleus and extensor digitorum longus (EDL) homogenates. In soleus from sedentary rats, superoxide dismutase and glutathione reductase activities were increased by 25% (P < 0.05) and by 40% (P < 0.01) after stanozolol administration, whereas catalase and glutathione peroxidase activities were not modified. This response was similar to that induced by training alone. In EDL from sedentary rats, stanozolol increased only superoxide dismutase activity (20%, P < 0.05). In no case, the effects of steroid administration and training were additive. HSP72 levels were up-regulated in soleus (1.5-fold, P < 0.01) and EDL (threefold, P < 0.001) following training but remained unchanged after stanozolol treatment. Endurance capacity, assessed in a treadmillendurance test, was similar for treated and control rats. We conclude that stanozolol treatment increases antioxidant capacity in selected skeletal muscles from sedentary rats. However, the steroid was not effective in improving endurance capacity or enhancing the training effects on muscle antioxidant defence systems (AU)

Metabolism of stanozolol: chemical synthesis and identification of a major canine urinary metabolite by liquid chromatography-electrospray ionisation ion trap mass spectrometry.

The canine phase I and phase II metabolism of the synthetic anabolic-androgenic steroid stanozolol was investigated following intramuscular injection into a male greyhound. The major phase I biotransformation was hydroxylation to give 6alpha-hydroxystanozolol which was excreted as a glucuronide conjugate and was identified by comparison with synthetically derived reference materials. An analytical procedure was developed for the detection of this stanozolol metabolite in canine urine using solid phase extraction, enzyme hydrolysis of glucuronide conjugates and analysis by positive ion electrospray ionisation ion trap LC-MS.

Detection of mono-hydroxylated metabolites of stanozolol by HPLC-ESI (+) MS/MS in Indian sports persons.

The abuse of stanozolol is quite widespread in Indian sport. Its analysis is challenging and this has led to the development of new methods to improve its detection. A method was developed and validated for the detection of the three main monohydroxylated metabolites of stanozolol. The excretion profile of these metabolites was studied in four healthy male volunteers. The excretion study samples, after a single oral dose of drug, showed that 3'-OH-stanozolol was excreted at the highest concentration, followed by 16beta-OH stanozolol, with 4beta-OH stanozolol as the least excreted. Ninety-eight old doping samples with adverse analytical findings for 3'-OH-stanozolol were reanalysed using this method. This showed 3'-OH-stanozolol and 16beta-OH stanozolol in all the 98 samples whereas 4beta-OH-stanozolol was identified in 90 samples. The percentage of positive identifications of stanozolol in Indian sportspeople has increased markedly in the last five years, from 31.9% in 2004 to 81.8% in 2009; however, this may be due to the more effective detection of stanozolol metabolites. It can thus be concluded that the marked increase in percent positive of stanozolol in Indian sportspersons in 2009 may be due to the improved detection by a more effective LCMS/MS method.

Pharmacokinetics of boldenone and stanozolol and the results of quantification of anabolic and androgenic steroids in race horses and nonrace horses.

Anabolic steroids (ABS) boldenone (BL; 1.1 mg/kg) and stanozolol (ST; 0.55 mg/kg) were administered i.m. to horses and the plasma samples collected up to 64 days. Anabolic steroids and androgenic steroids (ANS) in plasma were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of detection of all analytes was 25 pg/mL. The median absorption (t1/2 partial differential) and elimination (t1/2e) half-lives for BL were 8.5 h and 123.0 h, respectively, and the area under the plasma concentration-time curve (AUCho) was 274.8 ng.h/mL. The median t1/2e for ST was 82.1 h and the was 700.1 ng.h/mL. Peak mean (X+/-SD) plasma concentrations (Cmax) for BL and ST were 1127.8 and 4118.2 pg/mL, respectively. Quantifiable concentrations of ABS and ANS were found in 61.7% of the 988 plasma samples tested from race tracks. In 17.3% of the plasma samples two or more ABS or ANS were quantifiable. Testosterone (TES) concentrations mean (X+/-SE) in racing and nonracing intact males were 241.3+/-61.3 and 490.4+/-35.1 pg/mL, respectively. TES was not quantified in nonracing geldings and female horses, but was in racing females and geldings. Plasma concentrations of endogenous 19-nortestosterone (nandrolone; NA) from racing and nonracing males were 50.2+/-5.5 and 71.8+/-4.6 pg/mL, respectively.

Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry.

The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC-MS.

Photoaffinity labeling identification of a specific binding protein for the anabolic steroids stanozolol and danazol: an oligomeric protein regulated by age, pituitary hormones, and ethinyl estradiol.

We have demonstrated previously that both rat and human liver microsomes contain a highly specific binding protein for the anabolic steroids stanozolol (ST) and danazol (DA). In this study we solubilized the male rat liver ST-binding protein (STBP) and investigated the following parameters: 1) pharmacological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17alpha-ethinyl estradiol. We found that STBP is an integral protein bound to the endoplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) provided its optimal solubilization without changes in its pharmacological properties, i.e. high specificity for ST and danazol, between natural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STBP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently labeled STBP under nonreducing conditions showed that [3H]ST binds to a 110-kDa protein. The STBP was resolved under reducing conditions into three peptides of 55, 31, and 22 kDa, respectively. STBP increased from immature to adult rats, and it dramatically decreased after hypophysectomy. Unlike the 22-kDa peptide, both the 55- and 31-kDa peptides drastically decreased in both immature and hypophysectomized rats. 17alpha-Ethinyl estradiol administration to immature or hypophysectomized rats induced the 55- and 31-kDa [3H]STBP to a greater extent than the 22-kDa peptide. Thus, STBP appears as an oligomeric protein composed of hormone-regulated peptides. The availability of solubilized STBP and the fact that it can be induced in vivo represent major steps toward the purification and functional significance of this unique steroid-binding protein.

The effects of probenecid on the excretion kinetics of stanozolol, an anabolic steroid, in rats.

The pharmacokinetic behaviour and the mechanism of renal excretion of stanozolol (STZ), as affected by co-treatment with probenecid, were studied in male Sprague-Dawley rats. Pharmacokinetic parameters following intravenous (i.v.) administration of STZ (20 mg kg-1 body wt.) were measured in both STZ-treated (control) and STZ plus probenecid-treated (treatment) groups. In order to assess the renal clearance of STZ, bolus doses of STZ and inulin (40 mg kg-1 body wt.) were injected i.v. either in the presence or absence of probenecid (40 mg kg-1 body wt.). The blood and urine concentrations of STZ were determined by capillary gas chromatography-mass spectrometry (GC-MS). In the probenecid treatment group, the area under the plasma disappearance and urinary excretion curves (AUC) of STZ were significantly decreased (P < 0.01) and the volume of distribution (Vd) and total clearance (CLt) were significantly increased statistically (P < 0.05 and P < 0.01, respectively). No remarkable differences in the urine flow rate, urine pH values, glomerular filtration rate (GFR) or renal clearance were observed in the treatment group. However, the clearance ratio in the treatment group was significantly increased from 11.72 to 17.27. From these results, it is suggested that the significant decrease of AUC, i.e. increase of disappearance of STZ in plasma by co-administration with probenecid, is due to the increase of the clearance ratio.

Studies on anabolic steroids. III. Detection and characterization of stanozolol urinary metabolites in humans by gas chromatography-mass spectrometry.

The metabolism of stanozolol (17 beta-hydroxy-17 alpha-methyl-5 alpha-androstano[3,2-c]pyrazole), an androgenic-anabolic steroid widely used in sport for the purpose of enhancing performance, was investigated in humans. The analysis method was based on the use of solid-phase extraction on the Sep-Pak C18 cartridge, enzymic hydrolysis of steroid conjugates and high-resolution gas chromatograph-mass spectrometric (GC-MS) analysis of trimethylsilylated steroid extracts. After administration of a single 20-mg oral dose, twelve metabolites including unchanged stanozolol were recovered predominantly from the conjugated steroid fraction and characterized by GC-MS. In the unconjugated fraction, 16 alpha-hydroxystanozolol, 17-epistanozolol, stanozolol and 3'-hydroxy-17-epistanozolol were the most abundant metabolites. In the aglycone fraction, 16 alpha- and 16 beta-hydroxystanozolol, stanozolol and 3'-hydroxystanozolol were the most abundant metabolites. Other metabolites resulted from regioselective hydroxylation of stanozolol at C-4, whereas other were 17-epimers of 3'- and 16 alpha-hydroxystanozolol. Further hydroxylation leading to the formation of four isomeric dihydroxylated metabolites was also observed. They were tentatively assigned the structures of 3',16 alpha-, 4 beta,16 alpha-, 3',16 beta- and 4 beta,16 beta-dihydroxystanozolol. The mass spectral features of their bis-N,O-trimethylsilyl derivatives obtained under electron-impact ionization are presented. The effect of pH on the relative recovery of some of these metabolites is also presented. The usefulness of this analytical methodology for the detection and identification of stanozolol urinary metabolites in doping-control situations is demonstrated. The metabolism of stanozolol is also discussed, and metabolic pathways accounting for the formation of its biotransformation products are proposed.