Aryl-Propionamide-Derived Selective Androgen Receptor Modulators: Liquid Chromatography-Tandem Mass Spectrometry Characterization of the in Vitro Synthesized Metabolites for Doping Control Purposes

Abstract

ABSTRACT:Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H]؊ . The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.For decades, steroidal androgens have been clinically used in the treatment of diseases related to androgen deficiency, including muscle-wasting, osteoporosis, and benign prostate hyperplasia, but recently their suitability for hormone replacement therapy of aging men and regulation of male fertility has been under investigation (NegroVilar, 1999;Gao and Dalton, 2007). Traditional anabolic-androgenic therapies, applying steroid-structured compounds such as testosterone, are often limited due to low oral bioavailability, cross-reactivity with steroid receptors other than the androgen receptor, hepatic toxicity, and other undesirable side effects on the prostate and cardiovascular system (Bhasin and Bremner, 1997). To overcome these drawbacks, a series of nonsteroidal selective androgen receptor modulators (SARM) have been developed. SARM represent enhanced tissue selectivity, binding to the androgen receptor (AR) with affinity similar to testosterone, but exhibiting only partial agonist properties in androgenic tissue. Based on their chemical structure, most prominent pharmacophores of SARM can be categorized at least in four classes: 1) aryl-propionamide, 2) bicyclic hydantoin, 3) quinoline, and 4) tetrahydroquinoline analogs, which all have also entered different stages of clinical studies (Chen et al., 2005a;Thevis and Schänzer, 2007). Among the published data on SARM research, the series of aryl-pr...

Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high‐resolution mass spectrometry

“…Blood samples were collected at time 0 (immediately prior to SARM administration) and at 5, 10, 15, 30, and 45 min, and 1, 1.5, 2, 2.5, 3,4,5,6,8,12,18,24,48,72 and 96 h post SARMs administration. Prior to drawing each sample of blood for analysis, 10 mL of blood was aspirated and discarded from the catheter and T-Port extension set Figure 1.…”

“…[7] Furthermore, the identification of metabolites also corroborates that a substance has passed through a metabolic system and that an adverse finding is not caused by contamination of the sample. The metabolism of different SARMs of the arylpropionamide class has been studied in vitro in a variety of systems such as human liver microsomes, [8] equine liver microsomes, [9] bovine liver microsomes [10] and fungal incubates, [11] as well as in vivo in both human [12,13] and equine [14] urine. The three aforementioned in vivo studies showed that the SARMs were metabolized rapidly and extensively.…”

In vitro metabolism and bioavailability tests for endocrine active substances: What is needed next for regulatory purposes?

“…A two-step approach whereby the first step (Phase I) used a mixture of human liver microsomal and S9 fractions in a phosphate buffer with NADPH, and for the second step (Phase II) either UD-PGA or PAPS were added for glucuronidation or sulfation, respectively. In this example, the application was for the bioactivation and biodegradation of selective AR modulators (Kuuranne et al, 2008 The method of S9 incubation S9 may be co-incubated with the cells and chemical (Li et al, 2011) or a chemical may be needs to be considered pre-incubated with S9 and then the extract applied to the assay (Taxvig et al, 2011). In general, co-incubation is a time-saving approach but may lead to cytotoxicity in the assay system.…”

“…For example, Kuuranne et al (2008) successfully used human S9 liver mixes on selective androgen receptor modulators (SARMs) to predict the in vivo marker metabolites. Similarly, bovine S9 mixes have been used with SARMs and showed that the metabolites formed also were found in vivo (De Rijke et al, in press).…”

Pharmacokinetics and pharmacodynamics of a novel Acetylcholinesterase Inhibitor, DMNG-3

“…Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials (Kuuranne et al 2008). Thereby, the objective of this study was to develop a step-down passive avoidance test to investigate whether DMNG-3 could modulate impairment of learning and memory induced by scopolamine.…”