Martin Perreault

Effects of androgens on adipocyte differentiation and adipose tissue explant metabolism in men and women.

Objective  To examine the effects of aromatizable or nonaromatizable androgens on abdominal subcutaneous (SC) and omental (OM) adipose tissue lipid metabolism and adipogenesis in men and women.

Profiling Serum Bile Acid Glucuronides in Humans: Gender Divergences, Genetic Determinants, and Response to Fenofibrate

Glucuronidation, catalyzed by uridine 5′‐diphospho‐glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic bile acids (BAs). We aimed to (i) characterize the circulating BA‐glucuronide (BA‐G) pool composition in humans, (ii) determine how sex and UGT polymorphisms influence this composition, and (iii) analyze the effects of the lipid‐lowering drug fenofibrate on the circulating BA‐G profile in 300 volunteers and 5 cholestatic patients. Eleven BA‐Gs were determined in pre‐ and postfenofibrate samples. Men exhibited higher BA‐G concentrations, and various genotype/BA‐G associations were discovered in relevant UGT genes. The chenodeoxycholic acid‐3G (CDCA‐3G) concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA‐3G formation. Fenofibrate exposure increased the serum levels of five BA‐G species, including CDCA‐3G, and upregulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrated that fenofibrate stimulates BA glucuronidation in humans and thus reduces BA toxicity in the liver.

Regulation of endobiotics glucuronidation by ligand-activated transcription factors: physiological function and therapeutic potential.

Recent progresses in molecular pharmacology approaches have allowed the identification and characterization of a series of nuclear receptors (NR) which efficiently control the level UDP-glucuronosyltransferase (UGT) genes expression. These regulatory processes ensure optimized UGT expression in response to specific endogenous and/or exogenous stimuli. Interestingly, numerous endogenous activators of these NRs are conjugated by the UGT enzymes they regulate. In such a case, the NR-dependent regulation of UGT genes corresponds to a feedforward/feedback mechanism by which a bioactive molecule controls its own concentrations. In the present review, we will discuss i) how bilirubin reduces its circulating levels by activating AhR in the liver; ii) how bile acids modulate their hepatic glucuronidation via PXR- and FXR-dependent processes in enterohepatic tissues; and iii) how androgens inhibit their cellular metabolism in prostate cancer cells through an AR-dependent mechanism. Subsequently, with further discussion of the same examples (bilirubin and bile acids), we will illustrate how NR-dependent regulation of UGT enzymes may contribute to the beneficial effects of pharmacological activators of nuclear receptors, such as CAR and PPARa.

The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C23-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

Role of Glucuronidation for Hepatic Detoxification and Urinary Elimination of Toxic Bile Acids during Biliary Obstruction

Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.

The Human UDP-Glucuronosyltransferase UGT2A1 and UGT2A2 Enzymes Are Highly Active in Bile Acid Glucuronidation

Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.

Impact of structural modifications at positions 13, 16 and 17 of 16β-(m-carbamoylbenzyl)-estradiol on 17β-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16β-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17β-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1μM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1μM, not at all at 0.1μM, and showed an interesting inhibitory potency on 17β-HSD1 (90, 59 and 22%, at 10, 1 and 0.1μM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17β-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies.

Design of a Mestranol 2‐N‐Piperazino‐Substituted Derivative Showing Potent and Selective in vitro and in vivo Activities in MCF‐7 Breast Cancer Models

Anticancer structure–activity relationship studies on aminosteroid (5α‐androstane) derivatives have emerged with a promising lead candidate: RM‐133 (2β‐[1‐(quinoline‐2‐carbonyl)pyrrolidine‐2‐carbonyl]‐N‐piperazine‐5α‐androstane‐3α,17β‐diol), which possesses high in vitro and in vivo activities against several cancer cells, and selectivity over normal cells. However, the relatively weak metabolic stability of RM‐133 has been a drawback to its progression toward clinical trials. We investigated the replacement of the androstane backbone by a more stable mestranol moiety. The resulting compound, called RM‐581 ({4‐[17α‐ethynyl‐17β‐hydroxy‐3‐methoxyestra‐1,3,5(10)‐trien‐2‐yl]piperazin‐1‐yl}[(2S)‐1‐(quinolin‐2‐ylcarbonyl)pyrrolidin‐2‐yl]methanone), was synthesized efficiently in only five steps from commercially available estrone. In comparison with RM‐133, RM‐581 was found to be twice as metabolically stable, retains potent cytotoxic activity in breast cancer MCF‐7 cell culture, and fully blocks tumor growth in a mouse xenograft model of breast cancer. Advantageously, the selectivity over normal cells has been increased with this estrane version of RM‐133. In fact, RM‐581 showed a better selectivity index (15.3 vs. 3.0) for breast cancer MCF‐7 cells over normal breast MCF‐10A cells, and was found to be nontoxic toward primary human kidney proximal tubule cells at doses reaching 50 μm.

Explorative study on the anticancer activity, selectivity and metabolic stability of related analogs of aminosteroid RM-133

RM-133 is a key representative of a new family of aminosteroids reported as potent anticancer agents. Although RM-133 produced interesting results in 4 mouse xenograft cancer models when injected subcutaneously, it needs to be improved to increase its in vivo potency. Thus, to obtain an analog of RM-133 with a better drug potential, a structure-activity relationship study was conducted by synthesizing eleven RM-133-related compounds and addressing their antiproliferative activity on 3 human cancer cells (HL-60, OVCAR-3 and PANC-1) and 3 human normal cell lines (primary ovary, pancreas and renal proximal tubule) as well as their metabolic stability in human liver microsomes. When the 2β-tertiary amine of RM-133 was transformed into a salt or moved to position 3β, the anticancer activity was lost. Modifying the orientation of the side chain of RM-133 increased anticancer activity and selectivity, but led to a drastic loss of stability. The protection of the 3α-hydroxyl of RM-133 by the formation of an ester or a carbamate stabilized the molecule against the phase I metabolic enzymes without affecting its anticancer activity. In comparison to RM-133, the 3-dimethylcarbamate derivative 3 is more selective for cancer cells over normal cells and is much more stable in liver microsomes. Those results support the use of a pro-drug strategy targeting the 3α-hydroxyl of RM-133 as an approach to improve its drug properties. The work presented will enable the development of an optimized anticancer drug of the aminosteroid family that is suitable for a future phase I clinical trial.

Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1

ABSTRACT 17&bgr;‐Hydroxysteroid dehydrogenase type 1 (17&bgr;‐HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen‐dependent diseases. A steroidal derivative, 3‐{[(16&bgr;,17&bgr;)‐3‐(2‐bromoethyl)‐17‐hydroxyestra‐1,3,5(10)‐trien‐16‐yl]methyl} benzamide (PBRM), has recently been described as a non‐estrogenic, irreversible inhibitor of 17&bgr;‐HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off‐targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki = 368 nM) and the enzyme inactivation rate (kinact = 0.087 min−1) values for PBRM were determined with purified 17&bgr;‐HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3–5 days) is required to recover 17&bgr;‐HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17&bgr;‐HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non‐estrogenic and steroidal covalent irreversible inhibitor of 17&bgr;‐HSD1 warrant its further development as a potential drug candidate for estrogen‐dependent disorders.