Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: multi-spectroscopic and molecular modeling approaches.

This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.

Elucidation of urinary metabolites of fluoxymesterone by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.

The suitability of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) for the elucidation of fluoxymesterone metabolism has been evaluated. Electrospray ionization (ESI) and collision induced dissociation (CID) fragmentation in LC-MS/MS and electron impact spectra (EI) in GC-MS have been studied for fluoxymesterone and two commercially available metabolites. MS(n) experiments and accurate mass measurements performed by an ion-trap analyser and a QTOF instrument respectively have been used for the elucidation of the fragmentation pathway. The neutral loss scan of 20 Da (loss of HF) in LC-MS/MS has been applied for the selective detection of fluoxymesterone metabolites. In a positive fluoxymesterone doping control sample, 9 different analytes have been detected including the parent compound. Seven of these metabolites were also confirmed by GC-MS including 5 previously unreported metabolites. On the basis of the ionization, the CID fragmentation, the accurate mass of the product ions and the EI spectra of these analytes, a tentative elucidation as well as a proposal for the metabolic pathway of fluoxymesterone has been suggested. The presence of these compounds has also been confirmed by the analysis of five other positive fluoxymesterone urine samples.

Metabolism of anabolic steroids in humans: synthesis of 6 beta-hydroxy metabolites of 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, and metandienone.

Hydroxylation at position 6 beta testosterone I (17 beta-hydroxyandrost-4-en-3-one) and the anabolic steroids 17 alpha-methyltestosterone II (17 beta-hydroxy-17 alpha-methylandrost-4-en-3-one), metandienone III (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone IV (4-chloro-17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), and fluoxymesterone V (9-fluoro-11 beta, 17 beta-dihydroxy-17 alpha-methylandrost-4-en-3-one) was achieved via light-induced autooxidation of the corresponding trimethysilyl 3,5-dienol ethers dissolved in isopropanol or ethanol. The reaction further yielded the 6 alpha-hydroxy isomer in low amounts. The 6 beta-hydroxy isomer of I-V and the 6 alpha-hydroxy isomers of I, III, and IV were isolated and characterized by 1H and 13C NMR, high-performance liquid chromatography, gas chromatography, and mass spectrometry. Human excretion studies with single administered doses of boldenone (17 beta-hydroxyandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, metandienone, 17 alpha-methyltestosterone, and [16,16,17-2H3] testosterone showed that 6 beta-hydroxylation is the major metabolic pathway in the metabolism of 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, and metandienone, whereas for boldenone, 17 alpha-methyltestosterone, and testosterone, 6 beta-hydroxylation is negligible.

Effect of training and anabolic-androgenic steroids on drug metabolism in rat liver.

The effects of anabolic-androgenic steroid administration and exercise training on various aspects of hepatic function were investigated in sedentary and trained (treadmill for 12 wk) male and female rats treated orally with fluoxymesterone or methylandrostanolone (2 mg.kg-1 body weight, 5 d.wk-1 for 8 wk). The mean values of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total and direct bilirubin, and total- and high-density lipoprotein-cholesterol remained within normal range in all groups of male animals. The same is true for female rats, except for an increase in alkaline phosphatase activity in the steroid-treated groups. Hepatic microsomal aniline p-hydroxylase activity was reduced in male and increased in female rats by either steroid, whereas no significant effect was detected on 7-ethoxycoumarin deethylase activity. The levels of cytochrome P-450 and cytochrome b5 were markedly decreased by the anabolic-androgenic steroid treatment in male rat microsomes, but neither the steroid administration nor exercise training induced significant changes in the cytochrome levels of female rat livers. Taking into account the significant increase in microsomal protein yield elicited by fluoxymesterone or methylandrostanolone treatment both in males and females, it is noteworthy that the total monooxygenase activities and cytochrome P-450 content, expressed on a per gram liver basis, were significantly increased in female whereas they were apparently unchanged in male rats. In conclusion, the present data show that the prolonged ingestion of high doses of anabolic-androgenic steroids, either with or without concurrent exercise training, can modify in a sex-dependent manner the capacity of rat liver to metabolize drugs without affecting classical serum indicators of hepatic function.

Testing for fluoxymesterone (Halotestin) administration to man: identification of urinary metabolites by gas chromatography-mass spectrometry.

Fluoxymesterone, an anabolic steroid, is metabolized in man primarily by 6 beta-hydroxylation, 4-ene-reduction, 3-keto-reduction, and 11-hydroxy-oxidation. These pathways of metabolism are suggested by the positive identification of 4 metabolites and the tentative identification of 3 other metabolites. Detection of the drug in urine is possible for at least 5 days after a single 10 mg oral dose to previously untreated adult males, by monitoring the presence of 2 metabolites, since the parent drug is not detectable more than 1 day after the dose.

Effect of non aromatizable androgens on LHRH and TRH responses in primary testicular failure.

We have assessed the gonadotropin, TSH and PRL responses to the non aromatizable androgens, mesterolone and fluoxymestrone, in 27 patients with primary testicular failure. All patients were given a bolus of LHRH (100 micrograms) and TRH (200 micrograms) at zero time. Nine subjects received a further bolus of TRH at 30 mins. The latter were then given mesterolone 150 mg daily for 6 weeks. The remaining subjects received fluoxymesterone 5 mg daily for 4 weeks and 10 mg daily for 2 weeks. On the last day of the androgen administration, the subjects were re-challenged with LHRH and TRH according to the identical protocol. When compared to controls, the patients had normal circulating levels of testosterone, estradiol, PRL and thyroid hormones. However, basal LH, FSH and TSH levels, as well as gonadotropin responses to LHRH and TSH and PRL responses to TRH, were increased. Mesterolone administration produced no changes in steroids, thyroid hormones, gonadotropins nor PRL. There was, however, a reduction in the integrated and incremental TSH secretion after TRH. Fluoxymesterone administration was accompanied by a reduction in thyroid binding globulin (with associated decreases in T3 and increases in T3 resin uptake). The free T4 index was unaltered, which implies that thyroid function was unchanged. In addition, during fluoxymesterone administration, there was a reduction in testosterone, gonadotropins and LH response to LHRH. Basal TSH did not vary, but there was a reduction in the peak and integrated TSH response to TRH. PRL levels were unaltered during fluoxymesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of anabolic steroids with glucocorticoid receptor sites in rat muscle cytosol.

While glucocorticoid hormones act catabolically on skeletal muscle through their binding to glucocorticoid-specific receptors in the cytosol, androgens exert anabolic responses but no androgen-specific binding proteins could be detected in this responsive tissue. However, various nonradioactive androgens were effective in displacing labeled dexamethasone or cortisol from their respective cytoplasmic receptors in muscle, both in vitro and in vivo. The inhibition of glucocorticoid binding by androgens is competitive, and could be observed following a single or repeated administration of the androgens to adrenalectomized-castrated animals. The synthetic androgen fluoxymesterone and the hormone testosterone displayed Ki values of 7.5 X 10(-6) M and 1 X 10(-5) M, respectively, for the inhibition of [3H]dexamethasone binding in muscle cytosol. On the basis of competition experiments it is postulated that interaction of androgens with glucocorticoid receptors prevents the binding of glucocorticoids and might be responsible in part for the anabolic effects of pharmacologic doses of androgens in muscle.