Detection rate of IGF-1 variants and their implication to protein binding: study of over 240,000 patients.

OBJECTIVES: To determine the detection rate of IGF-1 variants in a clinical population and assess their implications. METHODS: IGF-1 variants were detected based on their predicted mass-to-charge ratios. Most variants were distinguished by their isotopic distribution and relative retention times. A67T and A70T were distinguished with MS/MS. Patient specimens with a detected variant were de-identified for DNA sequencing to confirm the polymorphism. RESULTS: Of the 243,808 patients screened, 1,099 patients containing IGF-1 variants were identified (0.45 %, or 4,508 occurrences per million). Seven patients were identified as homozygous or double heterozygous. Majority of variants (98 %) had amino acid substitutions located at the C-terminus (A62T, P66A, A67S, A67V, A67T, A70T). Isobaric variants A38V and A67V were detected more frequently in children than in adults. Six previously unreported variants were identified: Y31H, S33P, T41I, R50Q, R56K, and A62T. Compared with the overall population, z-score distribution of patients with IGF-1 variants was shifted toward negative levels (median z-score -1.4); however, it resembled the overall population when corrected for heterozygosity. Chromatographic peak area of some variants differed from that of the WT IGF-1 present in the same patient. CONCLUSIONS: In the IGF-1 test reports by LC-MS, the concentrations only account for half the total IGF-1 for patients with heterozygous IGF-1 variants. An IGF-1 variant may change the binding to its receptor and/or its binding proteins, affecting its activity and half-life in circulation. Variants located in or close to the C-domain may be pathogenic. Cross-species sequence comparison indicates that A38V and A70T may have some degree of pathogenicity.

Isotopic Peak Index, Relative Retention Time, and Tandem MS for Automated High Throughput IGF-1 Variants Identification in a Clinical Laboratory.

Measuring insulin-like growth factor-1 (IGF-1) is useful for assessing and managing growth-related disorders, such as acromegaly and growth hormone deficiency. High-resolution liquid chromatography-mass spectrometry (LC-MS) is used for measuring IGF-1 due to its molecular specificity, quantitative performance, well-characterized reference materials, and detailed age/sex-specific reference intervals. However, polymorphisms in the IGF1 gene may cause mass shifts in the polypeptide, which can impede quantitation and cause errors in clinical interpretation. We (1) developed a concept of "isotopic peak index", which allows simultaneous monitoring of 15 IGF-1 variants by using only four m/z ratios; (2) developed a "relative retention time" parameter that allows distinction of previously unresolved variants; and (3) utilized tandem mass spectrometry (MS/MS) to distinguish between the most common pair of variants: isobaric A67T and A70T. All methods were validated with DNA sequencing. This approach identified six variants from the ExAC database, P66A, A67S, S34N, A38 V, A67T, and A70T; two previously reported V44M and A67V variants; and discovered six unreported variants, Y31H, S33P, R50Q, R56K, T41I, and A62T. Major improvements in our workflow include enhanced automation, avoiding detailed manual calculations that are prone to human error, and the ability to monitor more, and discover new, IGF-1 variants. The workflow provides a profile of a patient's IGF-1 status and can be used to explore genotype-phenotype relationships in IGF-1 variants.

Discordance between mass spectrometry and immunometric IGF-1 assay in pituitary disease: a prospective study.

PURPOSE: Measuring IGF-1, a biomarker for GH activity, is critical to evaluating disordered hypothalamic-pituitary GH axis. Inconsistent IGF-1 measurements among different immunoassays are well documented. We switched from Immulite 2000 immunoassay to narrow-mass-extraction, high-resolution liquid chromatography mass-spectrometry (LC-MS) compliant with recent consensus recommendations on assay standardization. Comparability of these two assays in patients with pituitary disease in a clinical practice setting is not known. We sought to compare IGF-1 levels on Immulite 2000 and LC-MS in samples from naïve and treated patients with secretory and non-secretory pituitary masses. METHODS: We prospectively collected serum samples from 101 patients treated at the Cedars-Sinai Pituitary Center between February 2012 and March 2014. We intentionally recruited more patients with acromegaly or GH deficiency to ensure a clinically representative cohort. Samples were classified as in or out of the respective reference ranges. Bland-Altman analysis was used to assess agreement between assays. RESULTS: Twenty-four percent of samples were classified differently as below, in, or above range. Agreement between the assays was poor overall, with a significant bias for immunoassay reporting higher values than LC-MS. This pattern was also observed in patients with acromegaly and those with ≥ 2 pituitary hormone deficiencies. CONCLUSIONS: IGF-1 results may differ after switching from an older immunoassay to a consensus-compliant assay such as LC-MS. Clinicians should consider the potential impact of assay switching before altering treatment due to discrepant results, particularly in patients monitored over time, such as those with acromegaly and GH deficiency.

Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography-High-Resolution Mass Spectrometry in a Clinical Laboratory.

Protein and peptide hormones often exist as sequence variants with different molecular mass. Monitoring these variants of different molecular mass by mass spectrometry using mass-to-charge (m/z) ratio that is indicative of the wild type may lead to inaccurate quantitative results. However, liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based techniques can capture these differences and provide an opportunity to resolve, or partially resolve, variant complexity. In this chapter, we describe a general approach for monitoring a set of peptide variants with similar m/z ratios and isotopic envelopes, but different in amino acid sequences. As an example, we use insulin-like growth factor-1 (IGF-1) to demonstrate a DNA database-guided approach to monitor protein variants by LC-HRMS in a clinical laboratory. The workflow is automated and therefore avoids manual calculations that are prone to human error. The method can also monitor multiple IGF-1 variants and discover new ones. It can also provide a profile of a patient's IGF-1 status and be used to explore genotype-phenotype relationships in IGF-1 variants.

Quantitation of Free Thyroxine by Equilibrium Dialysis and Liquid Chromatography-Tandem Mass Spectrometry.

A method for free thyroxine measurement in human serum using equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) is described. Free thyroxine in serum is first separated from protein-bound thyroxine by equilibrium dialysis and then measured by LC-MS/MS.

High prevalence of subnormal testosterone in obese adolescent males: reversal with bariatric surgery.

OBJECTIVE: Obesity in adolescent males is associated with the lowering of total and free testosterone concentrations. Weight loss may increase testosterone concentrations. DESIGN AND METHODS: We evaluated the changes in sex hormones following bariatric surgery in 34 males (age range: 14.6-19.8 years) with obesity. These participants were part of a prospective multicenter study, Teen-Longitudinal Assessment of Bariatric Surgery. The participants were followed up for 5 years after surgery. Total testosterone, total estradiol, luteinizing hormone, follicle-stimulating hormone, sex hormone-binding globulin, C-reactive protein, insulin and glucose were measured at baseline, 6 months and annually thereafter. Free testosterone, free estradiol and HOMA2-IR were calculated. RESULTS: Study participants lost one-third of their body weight after bariatric surgery, with maximum weight loss achieved at 24 months for most participants. Free testosterone increased from 0.17 (95% CI: 0.13 to 0.20) at baseline to 0.34 (95% CI: 0.30 to 0.38) and 0.27 nmol/L (95% CI: 0.23 to 0.32) at 2 and 5 years (P < 0.001 for both), respectively. Total testosterone increased from 6.7 (95% CI: 4.7 to 8.8) at baseline to 17.6 (95% CI: 15.3 to 19.9) and 13.8 (95% CI: 11.0 to 16.5) nmol/L at 2 and 5 years (P < 0.001), respectively. Prior to surgery, 73% of the participants had subnormal free testosterone (<0.23 nmol/L). After 2 and 5 years, only 20 and 33%, respectively, had subnormal free testosterone concentrations. Weight regain was related to a fall in free testosterone concentrations. CONCLUSIONS: Bariatric surgery led to a robust increase in testosterone concentrations in adolescent males with severe obesity. Participants who regained weight had a decline in their testosterone concentrations.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19.

Importance: Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective: To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants: This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures: Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures: Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results: Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (ß = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (ß = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (ß = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (ß = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (ß = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. Conclusions and Relevance: In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Free Thyroxine Concentrations in Sera of Individuals with Familial Dysalbuminemic Hyperthyroxinemia: A Comparison of Three Methods of Measurement.

Background: Euthyroid individuals with familial dysalbuminemic hyperthyroxinemia (FDH) have often falsely elevated serum free thyroxine (fT4) concentrations determined by different automated immunoassays. Methods: We measured serum fT4 using direct dialysis coupled with tandem mass spectrometry (fT4 DDMS) in individuals with the common albumin gene mutation (ALB R218H) from 14 FDH families and compared them with results obtained by direct immunometric assay (fT4 DIMM) and free thyroxine index (fT4I). Results: While all 14 individuals with FDH had elevated total serum T4, the fT4 measured by DIMM was elevated in 12, by fT4I in 5, and by DDMS in 1. Conclusion: The latter method greatly reduced the discordance of fT4 results relative to thyrotropin in FDH.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Correlation of Insulin-Like Growth Factor-I and -II Concentrations at Birth Measured by Mass Spectrometry and Growth from Birth to Two Months.

BACKGROUND: Immunoassays used to measure insulin-like growth factor (IGF)-I and -II concentrations are susceptible to interference from IGF-binding proteins. The aim of this study was to investigate the association of IGF-I and -II concentrations at birth with neonatal anthropometry using a liquid chromatography/mass spectrometry (LCMS) assay. METHODS: LCMS was used to measure IGF-I and -II concentrations in umbilical cord blood of term, healthy infants enrolled in the Cork BASELINE Birth Cohort Study. Weight, length, and occipitofrontal head circumference (OFC) were measured at birth and 2 months. RESULTS: Cord blood IGF-I and -II concentrations were measured in 1,100 infants. Mean (SD) IGF-I and -II concentrations were 52.5 (23.9) ng/mL and 424.3 (98.2) ng/mL, respectively. IGF-I and -II concentrations at birth were associated (p < 0.05) with weight (R2 = 0.19, R2 = 0.01), length (R2 = 0.07, R2 = 0.004), and OFC (R2 = 0.03, R2 = 0.04) at birth. Low IGF-I concentrations at birth were associated with increases in weight (p < 0.001) and OFC (p < 0.01) Z-scores in the first 2 months. CONCLUSION: Using an LCMS assay, we have shown that anthropometric parameters at birth are associated with IGF-I and weakly with IGF-II concentrations. This indicates that, at the time of birth, IGF-I is the more important growth factor for regulating infant growth.

Synthesis and antifungal activities of alkyl N-(1,2,3-thiadiazole-4-carbonyl) carbamates and S-alkyl N-(1,2,3-thiadiazole-4-carbonyl) carbamothioates.

A series of alkyl N-(1,2,3-thiadiazole-4-carbonyl) carbamates and S-alkyl N-(1,2,3-thiadiazole-4-carbonyl) carbamothioates with unsubstituted or monobrominated straight chain alkyl groups were synthesized and evaluated as fungistatic agents against Gibberella zeae and Alternaria kikuchiana. These compounds showed variable antifungal activities at concentrations of 5 and 50 microg/mL. The results showed that antifungal activities depended on the length of the alkyl chain with the optimal chain length of 6-11 carbons. Carbamic acid, (1,2,3-thiadiazole-4-ylcarbonyl)-, hexyl ester (4) showed a strong fungistatic activity against A. kikuchiana at both concentrations, with 90.7 and 54% growth inhibition at 50 and 5 microg/mL, respectively. Carbamic acid, (1,2,3-thiadiazole-4-ylcarbonyl)-, heptyl ester (5); Carbamic acid, (1,2,3-thiadiazole-4-ylcarbonyl)-, octyl ester (6); and Carbamic acid, (1,2,3-thiadiazole-4-ylcarbonyl)-, undecyl ester (9) showed strong fungistatic activity against G. zeae at both concentrations. Their growth inhibitions against G. zeae at the concentration of 5 microg/mL were 78, 63, and 59%, respectively.

Serum vitamin D metabolites in colorectal cancer patients receiving cholecalciferol supplementation: correlation with polymorphisms in the vitamin D genes.

Cholecalciferol (D(3)) supplementation results in variable increases in serum 25(OH)D(3) levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D(3), 24,25(OH)(2)D(3), 1,25(OH)2D(3) and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D(3) supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D(3) supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D(3) metabolites levels before and after D(3) supplementation was analyzed. The mean baseline serum 25(OH)D(3) level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D(3) supplementation, serum levels of 25(OH)D(3) increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)(2)D(3), 1,25(OH)(2)D(3), VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D(3) and 1,25(OH)(2)D(3) levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D(3) and 1,25(OH)(2)D(3) levels both before and after D(3) supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D(3) levels after supplementation but not with baseline 25(OH)D(3). Our results show that D(3) supplementation increased 25(OH)D(3) levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D(3) insufficiency and suboptimal increase in 25(OH)D(3) levels after D(3) supplementation. Individuals with these genotypes may require higher D(3) supplementation doses to achieve vitamin D(3) sufficiency.

Pharmacokinetics, pharmacodynamics and metabolism of a novel anticancer agent for prostate cancer.

The objective of these studies was to examine the murine pharmacokinetics, pharmacodynamics and metabolism of (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (Indole 15), a novel tubulin inhibitor for the treatment of cancer. We developed HPLC and LC/MS/MS assays to quantitate Indole 15 and characterize its metabolites in vivo. Pharmacokinetic studies were performed after intravenous (IV), oral (PO) and subcutaneous (SC) administration of 10 mg/kg doses to male ICR mice. Urine and fecal samples were also collected over a 72-h period to identify metabolites. Pharmacodynamic studies were conducted by monitoring the tumor size change during a period of two weeks in PC-3 tumor bearing mice after daily IV administration of Indole 15 at doses of 0, 10, 50, 100 and 150 mg/kg. The pooled plasma concentration data after administration via different dose routes was simultaneously fitted by a two-compartmental model. Indole 15 was moderately well absorbed after PO and SC administration, with a bioavailable fraction of 0.27 and 0.72, respectively. The steady state volume distribution (Vss) and clearance (CL) were estimated to be 7.0 l/kg and 4.36 l/h/kg, respectively. The mean data of PC-3 tumor growth in mice was fitted well by a transduction model using fixed plasma pharmacokinetics as a driving function. Analysis of the metabolites in mice indicated that the compound undergoes extensive oxidative metabolism with subsequent sulfation. These studies demonstrate that favorable pharmacokinetic and pharmacodynamic properties of Indole 15 offer hope for achieving pharmacological activity in early clinical trials.

Pharmacokinetics, biodistribution and metabolism of a novel selective androgen receptor modulator designed for prostate cancer imaging.

Knowledge of the presence and extent of disease plays a major role in clinical management of prostate cancer, as it provides meaningful information as to which therapy to choose and who might benefit from this therapy. The wide expression of androgen receptor (AR) in primary and metastatic prostate tumors offers a cellular target for receptor-mediated imaging of prostate cancer. In our previous study, a non-steroidal AR ligand, S-26 [S-3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-iodophenyl)-propionamide] showed promising in vitro pharmacological properties as an AR-mediated imaging agent, with high AR binding affinity and AR specificity. The overall goal of this study was to characterize the in vivo metabolic and biodistribution profile of S-26 in rats. Non-compartmental pharmacokinetic analysis of S-26 in rat plasma showed that clearance (CL), volume of distribution (Vd(ss)), and half-life (T(1/2)) of S-26 were 0.30 + or - 0.07 l/h/kg, 1.44 + or - 0.33 l/kg, and 4 h, respectively, after intravenous (i.v.) administration. Dose proportionality (1, 10 and 30 mg/kg) studies suggested that the pharmacokinetics of S-26 are dose-independent. The plasma concentrations of all 3 doses were further simultaneously fitted with a two-compartmental model and the results were similar to those obtained from non-compartmental analysis. Biodistribution studies using (125)I-labeled S-26 indicated that it did not specifically target AR-rich tissue (e.g. prostate). A substantial amount of radioactivity recovered from thyroid gland indicated the release of free iodine. In metabolism studies, unchanged S-26 and its metabolites were detected in rat urine and fecal samples. Oxidation, de-iodination, hydrolysis, and sulfate conjugation were the major metabolic pathways of S-26 in rats, with de-iodination representing a unique metabolic pathway of S-26 among other selective androgen receptor modulators. In conclusion, the extensive plasma clearance and de-iodination of S-26 likely contribute to its lack of AR tissue selectivity in vivo. Future studies using metabolically stable ligands with less lipophilicity and higher AR binding affinity may represent a promising and rational approach for AR-mediated imaging.

Crystal structure of the T877A human androgen receptor ligand-binding domain complexed to cyproterone acetate provides insight for ligand-induced conformational changes and structure-based drug design.

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8A in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor.drug complex. The structure demonstrates that bulk from the 17alpha-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Pharmacokinetics and metabolism of a selective androgen receptor modulator in rats: implication of molecular properties and intensive metabolic profile to investigate ideal pharmacokinetic characteristics of a propanamide in preclinical study.

S-1 [3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide] is one member of a series of potent selective androgen receptor modulators (SARMs) that are being explored and developed for androgen-dependent diseases. Recent studies showed that S-1 holds great promise as a novel therapeutic agent for benign hyperplasia [W. Gao, J. D. Kearbey, V. A. Nair, K. Chung, A. F. Parlow, D. D. Miller, and J. T. Dalton (2004) Endocrinology 145:5420-5428]. We examined the pharmacokinetics and metabolism of S-1 in rats as a component of our preclinical development of this compound and continued interest in structure-activation relationships for SARM action. Forty male Sprague-Dawley rats were randomly assigned to treatment groups and received either an i.v. or a p.o. dose of S-1 at a dose level of 0.1, 1, 10, or 30 mg/kg. S-1 demonstrated a low clearance (range, 3.6-5.2 ml/min/kg), a moderate volume of distribution (range, 1460-1560 ml/kg), and a terminal half-life ranging from 3.6 to 5.2 h after i.v. doses. The oral bioavailability of S-1 ranged from 55% to 60%. Forty phase I and phase II metabolites of S-1 were identified in the urine and feces of male Sprague-Dawley rats dosed at 50 mg/kg via the i.v. route. The two major urinary metabolites of S-1 were a carboxylic acid and a sulfate-conjugate of 4-nitro-3-trifluoromethylphenylamine. Phase I metabolites arising from A-ring nitro reduction to an aromatic amine and B-ring hydroxylation were also identified in the urinary and fecal samples of rats. Furthermore, a variety of phase II metabolites through sulfation, glucuronidation, and methylation were also found. These studies demonstrate that S-1 is rapidly absorbed, slowly cleared, moderately distributed, and extensively metabolized in rats.

Characterization of the in vitro metabolism of selective androgen receptor modulator using human, rat, and dog liver enzyme preparations.

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km = 16.1 microM, Vmax = 1.6 pmol/(pmol x min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation.