Methods of pharmacological evaluation

22.3.1 Androgen receptor transactivation and binding

Steroid receptor activity is dependent on three factors: the concentration of the ligand, the receptor, and co-regulatory proteins. The composition and presence of steroid receptors in the cell determine the response of the ligand and the transactivation of target genes by ligand-occupied receptors is modulated by the presence of nuclear co-activators and co-repressors (Katzenellenbogen et al. 1996). For the determination of the androgenic activity of compounds, androgen receptor binding assays and androgen receptor transactivation assays can be used. With the binding assay the affinity of a compound for the androgen receptor can be determined but

Experimental conditions:

ClCH2PPh3Cl, i-BuONa, THF, 0°C RT, 53 %(E:Z = 3 «-BuLi, THF, hexane, -15°C RT, 63 %. HC1, acetone, RT, 89 %.

Fig. 22.5 Preparation of nandrolone derivative 4e.

the biological effect of that compound cannot be predicted. The biological effect can be estimated in agonistic and antagonistic transactivation assays. To study the agonistic activity of androgens, Chinese hamster ovary (CHO) cells were trans-fected with the human androgen receptor and the mouse mammary tumor virus (MMTV) promotor in front of the firefly luciferase reporter gene. This cell line is named CHO-AR-MMTV-LUC (clone 1G12-A5-CA). The agonistic activity for the androgen receptor was determined according to the procedure described by Schoonen etal. (1998). In brief, 5 x 104 cells/well were seeded into a 96-well plate and incubated with test compounds (final ethanol content: 1% v/v) for 16 h in medium with 5% charcoal-treated bovine calf serum supplement at 37°C in a humidified atmosphere of air supplemented with 5% CO2. Thereafter, of the total 250 |l incubation volume, 200 |l was removed,and 50 |lLucLite was added for cell lysis and luciferase measurement. Luciferase activity was measured in a Topcount luminescence counter (Canberra Packard, Meridan, USA). Relative agonistic activity (RAA) studies were carried out with various concentrations (1:2:4 dilutions) of the standard and compounds of interest. From these curves the EC50 values were determined. The RAA of the different compounds tested was expressed as a percentage of the EC50 value of the reference compound 5a-dihydrotestosterone (EC50 of 5a-DHT=100%).

The binding affinity to the androgen receptor was determined according to the procedure described by Bergink et al. (1983). For displacement analysis CHO-AR-MMTV-LUC (clone 1G12-A5-CA) cells were used. Cells were cultured and harvested, and cytosolic preparations prepared as described previously (Bergink et al. 1983). Before use, cytosol equivalent to 1 g of cells was diluted with buffer at a ratio of 1:15. Ethanolic solutions of compounds were pipetted (10 |l) into 96-well plates, the ethanol was evaporated and the residue was dissolved in buffer containing radiolabeled 5a-DHT (50 |l). After mixing thoroughly, 50 |lofice cold cytosolic preparation containing human androgen receptor was added, thoroughly mixed and incubated over night at 4°C. The free and receptor bound 5a-DHT was separated by a dextran-coated charcoal precipitation. Finally, 100 |l supernatant was counted in a Topcount microplate scintillation counter (Canberra Packard, Meridan, USA). Specific binding was determined by subtracting nonspecific from total binding. Relative binding affinity (RBA) studies were carried out with various concentrations of the standards (1:2:4 dilutions) and compounds of interest. The RBA was calculated relative to 5a-DHT of which the EC50 was set at 100%.

22.3.2 Determination of metabolic stability with human hepatocytes

Compounds used for oral application can be converted during first-pass metabolism in the intestine and the liver. In contrast to testosterone the androgen 17a-methyltestosterone is an orally active androgen. To predict the oral availability of nandrolone derivatives the metabolic stability was assessed in cryopreserved human male hepatocytes and compared to the metabolically stable reference 17a-methyltestosterone (MT, compound 2 in Fig. 22.1) and the unstable reference testosterone (compound 1, in Fig. 22.1). The nandrolone derivatives and reference compounds were tested at a concentration of 10nmol/l. Cryopreserved human hepatocytes from healthy young men (25-45 years) were obtained from In Vitro Technologies (Baltimore, MD, USA). The hepatocytes were thawed at 37°C and immediately placed on ice. The cells were washed twice in cold medium (William's medium E without phenol red, supplemented with glutamax I, gentamycin (50 |xg/ml), insulin (1 |xmol/l) and hydrocorticosterone (10 |xmol/l)). The hep-atocytes were seeded at a density of 5 x 105 viable cells/well in a 12-well plate. The incubations were started by the addition of a test compound in medium. The incubations were performed at 37°C in an atmosphere of air/O2/CO2 (55/40/5) for different interval times (0, 30, 60, and 180 minutes). The incubations were terminated by pipetting the incubation mixture into one volume of acetone on ice. The acetone was evaporated under a stream of nitrogen, the volume was adjusted to 1.5 ml and centrifuged at 10,000 g during 30 min at 4°C. The supernatants were collected for LC-MS/MS analysis.

The parent compound was determined using a Supelcosil LC-8-DB (33 x 4.0 mm) column and agradient of methanol in 20 mmol/l ammonium acetate dissolved in formic acid (0.1 mol/l) at a flow-rate of 1 ml/min. All mass measurements were performed at an API-3000 mass spectrometer (Perkin-Elmer Sciex, Toronto, Canada).

22.3.3 Aromatase susceptibility of new androgens

The experiments were performed as described by de Gooyer etal. (2003). In brief, the nandrolone derivatives, testosterone and 17a-methyltestosterone were incubated with supersomes containing human aromatase (Gentest, Woburn, MA, USA) for 0, 15, 30, 60, and 120 min. The final reaction mixture in a phosphate buffer (pH 7.4) contained 100 nmol/l compound, 20 nmol/l cytochrome P450 and 1 mmol/l NADPH. The incubations were terminated by extraction with ethylacetate. The ethylacetate was evaporated and the residue was dissolved in 1 ml 100% ethanol. The ethanolic solution was added to CHO cells stably transfected with either the human androgen receptor (AR) or human estrogen receptor a (ERa) and a promotor gene in front of a firefly luciferase reporter gene. The final concentration of test compounds in the receptor transactivation assay was maximally 1 nmol/l. After an incubation of 18h the luciferase activity was measured using the LucLite assay kit (Canberra Packard) and the luminescence signal was measured on a Topcount 96-well plate scintillation counter (Canberra Packard).

22.3.4 Effects on bone, LH/FSH, ventral prostate and muscle in castrated male rats

Screening for oral in vivo activity was performed in male castrated rats which were treated once daily for 4 days with gelatin/mannitol suspension or arachis oil solution, as has been reported in a restoration study by Kumar etal. (1992). Three hours after the last oral application, blood was collected from the tail and serum LH was determined with a sensitive immunofluometrix method (van Casteren etal. 2000). The minimal active dose (MAD) was defined as the dose that results in a statistically significant suppression of 65% (±10%) of serum LH. For effects on bone, rats were castrated and directly treated for 6 weeks. Effects on trabecular bone mineral density (BMD) in the distal femur were determined as described by (Ederveen and Kloosterboer 2001). Androgenic/anabolic effects were determined by the increase of the wet weight of the ventral prostate (VP) and musculus levator ani (MLA), respectively (Herschberger etal. 1953).

22.3.5 Effects on plasma testosterone of intact male monkeys

Intact male Macaca arctoides were treated orally once daily for seven successive days with different doses of Org X in arachis oil solution. In 2-3 pre-treatment samples plasma testosterone was determined on day 7 and 15 with a commercial immunoassay (Immunolite, DPC). Plasma levels of Org X were determined in blood sampled before each oral administration (trough levels) and determined by LCMSMS.

22.3.6 Pharmacokinetic evaluation in different animals

Single-dose pharmacokinetic (SD-PK) evaluation of the different androgens was performed in castrated male rats, castrated rabbits and intact female monkeys (Macaca arctoides) after a single oral administration of 10, 10, and 5 mg/kg, respectively. Blood was collected at eight different time points. The amount of applied androgen was determined after sample pre-treatment (C18 separation) and detected with a Sciex API 4000 MSMS detector as reported by de Gooyer et al. (2003). Most important PK parameters such as elimination half-life (tV2), area under curve (AUC) expressed as nmol*hours after complete elimination (infinitive, called AUCinf) and maximal concentration (Cmax) are reported. Values were normalised to 1 mg/kg for the dose given, therefore nAUCinf and nCmax will be used throughout the text.

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