Main Research Findings

1) Researchers detected the presence of metabolites in equine hair, plasma, and urine samples as well as in vitro. Additional analysis occurred in order to determine the structure of the metabolites that were detected in vitro.

2) The selective androgen receptor modulator, AC 262,563 exhibits a partial agonist effect on testosterone while also acting as an antagonist against the effects of dihydrotestosterone.

 

Selected Data

1) Researchers Cutler et. Al examined the metabolism of AC 262, 536 in the bodies of two equine test subjects. Two Thoroughbred horses were obtained for experimentation purposes; they were fed a typical racehorse diet, exercised in a similar manner to British train yards, and housed at the British Horseracing Authority’s Centre for Racehorse Studies in Newmarket UK. The first subject, a 6-year-old gelding, weighed in at 518 kg and was referred to as Horse 1, while the second subject, the 5-year-old mare, weighed 498 kg and was referred to as Horse 2 [1].

All naturally excreted urine was collected for samples after administration of the first dose. Only 2 urine samples were collected after the second dosing. After the third dose was administered all excreted urine was collected over the following 48 hours. Urine samples were obtained from the subjects once daily for 13 days after the final dose of AC 262,536. In addition to urine samples collected once daily for 4 days, blood samples were gathered from both horses daily for 3 days. Initially, an IV catheter was placed into the left jugular veins of the horse the first day of the study, pre-administration, in order to collect plasma samples before and throughout the study.

After the pre-administration plasma samples were collected, a 50 mg dose of AC 262,536 was administered to the horses at 9 am with food, every day for 3 days. Plasma samples after oral administration of AC 262,536 were collected at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14, and 18 hours. Following the final dose, plasma samples were collected from both subjects consistently over the course of 12 days. For further metabolic testing the researchers gathered hair samples from multiple sections of the mane by pulling out the hair at the root. Analysis of the hair samples took place 0.1, 168, 629, 1805, and 5065 hours after the final dose of AC 262,536 was administered to the horses. The hair samples were gathered from the subjects post-exercise and post-washing, however, the 0.1 hour measurement took place pre-washing and drying due to time constraints [1].

The researchers collected the hair samples in accordance with methodology published regarding substances observed in mane hair. Dry hair samples were compared to post-wash hair samples in order for the research team to gain a full understanding of how AC 262,536 is both internally and externally deposited in the hair. The hair samples were segmented and washed a total of three times. The first two washes were completed with the use of 4 mL of DCM and were followed by the final wash with 4 mL 10% IPA. This procedure was followed in order to compare the amount of drug found in the hair versus the amount observed in the wash. The hair samples were dried at 40 degree Celsius and ground into a fine powder in order for the weight to be measured and analyzed [1].

All of the completed washes were retained and prepared for evaporation so the washes could be compared to the hair samples. The preparation was adjusted to account for the amount of hair washed in compared to how much hair was weighed out for analysis. Blank hair samples were spiked so researchers could compare extraction recovery to the extracted spikes.

In order to carry out in vitro incubations, 11 ug/mL of AC 262,563, liver microsomes, 2.5 mM of NADPH regenerating cofactor solution, and 50 mM, pH 7.4 TRIS buffer were combined to make up an incubation volume of 0.34 mL. The samples were incubated for 180 minutes in a 37 degree Celsius water bath along with positive and negative control samples. After proper preparation the samples were transferred to different tubes so the researchers could observe the compound’s evaporation to dryness. The dried samples were prepared one of two ways before analysis: reconstituted in 10 uL of MeOh and 90 uL of RG H2O with BDPA injection markers, or dissolved in 100 uL of MO derivatizing solution and incubated for one hour at 80 degrees Celsius [1].

Metabolites were identified in vitro through a two-step process. First, the FS mass and data mining for postulated metabolites occurred. Second, targeted MS^2 analysis was performed on any metabolites that were identified. In vitro samples underwent FS mass analysis, carried out in positive ionization mode. All accurate mass estimations of potential metabolites were determined by the expected mass shifts from the parent compound, AC 262,536. MS^2 analysis was further used in order to identify and characterize metabolic structures. In order to make interpretations related to the general rules of fragmentation and identification of mass shifts in key fragments, the researchers had to first investigate the fragmentation of MS^2 in order to label key ions for comparison to the MS^2 metabolite spectra [1].

2) Male Sprauge-Dawley rats were utilized by the research team of Piu et. Al in order to examine how the levator ani muscle, seminal vesicles, and prostate were affected by AC 262,536. Castration or sham-operations were performed on each subject; the rats were given 5 days to assimilate to their surroundings and recover from surgery. The experiment took place over the course of two weeks where the subjects were randomly split into testing groups and administered a vehicle compound, a 1 mg/kg dose of testosterone propionate (TP), or an active dose of AC 262,536 of 3, 10, or 30 mg/kg [2].

In addition to measuring weight changes of the prostate, vesicles, and levator ani, all observed differences in receptor responses were measured through receptor selection and amplification technology (R-SAT). R-SAT is a cell based assay that assists in the identification of receptor-dependent proliferative responses that are found throughout a multitude of different receptor classes. Luciferase reporter gene assays were also conducted using human breast carcinoma MDA-kb2 cells. These cells are linked to luciferase by transfection with the mouse mammary tumor virus (MMTV) promoter. These cells were grown in DMEM charcoal-stripped 10% FBS and plated onto luciferase assay plates. After plating, the medium was replaced with one containing various concentrations of the ligand. After 24 hours the extracted cells were removed from the plate, lyzed, and luciferase activity was measured [2].

Furthermore, hamster DDT cells were grown in DMEM 5% charcoal-stripped FBS in the presence of 10 nM of testosterone. The cells were plated and grown to 80% confluency typically over the course of three days. Once this point was reached the previous medium was replaced with ^3H-DHT at a 2 nM concentration, in addition to various concentrations of test ligands. The cells were incubated for 24 hours, washed with ice-cold salt solution, and resuspended in 100 ul/well of 100% ethanol. The plates were sealed and shaken for 6 hours.

Human prostate cancer LNCaP cells were plated overnight; the medium was replaced the next day with DMEM 2% charcoal-stripped FBS in the presence of 10 nM concentration of DHT. From there, AC 262,536 was added to the medium at indicated doses. After 5 days the medium was removed in order for researchers to record cell proliferation. Cell proliferation was measured through the incubation of tetrazolium salt WST-1. A microplate reader was used 1 hour after incubation at 37 degrees Celsius for the purpose of measuring absorbance rates at a concentration of 405 nM [2].

Both the R-SAT and the luciferase data was normalized in a manner similar to the response typically seen as a result of testosterone. The minimum and maximum efficacy values measured throughout the study were defined by the dose-response curve the researchers developed. Following the 2-week rat study, all data was normalized to testosterone and analyzed via one-way ANOVA as well as an unpaired-t test. The researchers noted that the effects elicited in the vehicle castrated rats were defined as 0% while the effects of the 1 mg/kg dose of TP in castrated rats were marked as 100% [2].

 

Discussion

1) Two Thoroughbred horses, one gelding and one mare, were orally administered AC 262,536. After the final dose was given to the subjects, urination samples were collected once a day for 13 days in order for the researchers to identify different metabolites found in the sample. Urine samples taken prior to AC 262,536 administration did not include any identifiable traces of metabolites or the parent compound. On the other hand, the post-administration samples contained significant levels of M2, M3b, M3c, and M4d in the urine samples collected from both horses. Furthermore, the researchers noticed elevated levels of the metabolites M1, M4b, and M4c, however, the peak intensity elicited by each compound was not increased to a significant level [1].

Figure 1: Signal intensities of AC 262,536 and the primary metabolites found in post-administration urine samples collected up to 120 hours after final administration

Pre-administration plasma samples were collected from the two equine test subjects for up to 12 days following the final administration of AC 262,536. Results of analysis reported that there was no trace of AC 262,536 or any related metabolites in the pre-administration plasma samples. However, the samples gathered after the final dosing found that there was AC 262,536, the M2 metabolite, and the M3c metabolite present in the plasma. The researchers noted that the signal intensity of the metabolites were very low in the plasma samples; without the use of enzyme hydrolysis, only M3c and Ac 262,563 were observed at very low levels. With the use of enzyme hydrolysis the metabolites were detected at a slightly better rate: detection of M3c increased 3-fold while detection of AC 262,536 increased 5-fold [1].

Figure 2: Signal intensities of AC 262,536 and primary metabolites found in post-administration plasma samples

Like the urine and plasma sample analysis, pre- and post-administration hair samples were collected from the test subjects in an attempt to identify the presence of metabolites in the samples. AC 262,536 or any related metabolites were not found in the pre-administration samples of the negative controls. The researchers estimated that extraction recovery of AC 262,536 was approximately 70% based on the spikes observed before and after dosing [1].

Preliminary analysis performed on the post-administration hair samples indicated that the parent compound, AC 262, 536, was present in the samples. These samples were collected 5 minutes, 7 days, and 26 days after the final dose was administered to the subjects. A final post-administration sample was collected from both horses 75 days after the final dose was administered. After 3 SRM transitions AC 262,563 was identified in the sample provided by Horse 1, however after only 1 SRM transition, AC 262,563 was found in the hair sample from Horse 2 [1].

Figure 3: SRM chromatogram displaying concentrations of AC 262,563 present in a) the preadministration hair sample, b) spiked hair sample, c) sample collected 5 minutes after final administration, and d) sample collected 7 days after the last dose was given.

In initial analysis of the in vitro samples of AC 262,536 identified nine potential phase I metabolites. The metabolites were then grouped based on the biotransformations observed by the researchers; metabolites were further labeled based on their t_R. The most abundant metabolites found in vitro were a mono-oxidized metabolite of AC 262,563, referred to as M2; a possible epimer of AC 262,563, M2, was also identified. Both the M1 and M2 metabolites were detected in vitro with levels of chromatographic peak signal intensity higher than the parent AC 262,536.

M1 was detected with a mass difference of 2.015 amu, in comparison to AC 262,536, most likely due to a mono-oxidation reaction. The ion spectrum for M1 at t_R of 3.67 mins identified fragments of AC 262,563 at m/z 235.1228, 219.0916, 193.0761, 179.0604, and 169.0759. These findings indicate the metabolite has a tropane structure, and ultimately remains unchanged in respect to the parent compound. M2 was suspected to be epi-AC-262563 and was detected with a t_R of 2.62 minutes. This results in a chromatographic baseline separation of the isomers. Differences in the stereochemistry leads to variations between isomer fragments that indicate lower stability levels [1].

Figure 4: MS^2 spectra of in vitro metabolites

Metabolites M3a-3c were also identified in vitro, representing a mass increase of 19.995 amu. Following M1 and M2, M3b and M3c were considered the most abundantly found metabolites with t_R recorded at 3.32 and 3.54 minutes, respectively. Based on FS signal intensity, M3a was found to be the lowest abundant metabolite observed in vitro. Additionally, the metabolites M4a-4d were also detected. Researchers believed these metabolites were produced by successive mono-oxidation and mono-hydroxylation transformation. It’s important to note that researchers are unclear which transformation occurs first. All four metabolites represented a mass increase of 13.891 amu from the parent compound [1].

Due to the fragment ions found at m/z 179.0604m 181.0758, and 195.0916, the metabolites M4a and M4b were thought to be hydroxylated within the tropane moiety. These ions were also observed in the parent compound, a fact that was determined by the maintenance of the napthonitrile ring. M4a and M4b represented a mass shift of 15.995 amu from the product ion of the parent compound, AC 262,536. Furthermore, metabolites M4c and M4d produced significantly different Ms^2 spectra results than those observed for M4a and M4b. The respective spectra indicate an additional occurrence of hydroxylation in the napthonitrile ring rather than within the tropane ring [1].

2) Initial R-SAT data reported that AC 262,536 resulted in increased activity at the androgen receptors in a manner similar to the natural ligand testosterone. It is important to note that AC 262,536 was also found to act as a partial agonist to testosterone. Through the use of cell extracts, binding assays observed the physical interaction between AC 262,536 and the androgen receptors with an affinity of 5 nM. Further evaluation of the agonistic properties of AC 262,536 took place by generating androgen receptor mutants. The mutant receptors were compared to wild-type (WT) receptors in order to display enhanced or impaired activity.

The R-SAT data determined that the agonistic actions of AC 262,536 is improved when the mutant receptors exhibit impaired activity. The agonistic behavior of AC 262,536 was observed in the WT androgen receptors at a comparison level to testosterone, measured at 64% and 100%, respectively. Between the three mutants, T877A, G743A, and A748T, the T877A mutant experienced the greatest improvement in testosterone activity, measured at 122%. The G743A mutant saw an 87% improvement, while the A748T mutant only increased by 27%. The research team notably mentioned that the T877A mutant displayed very little AC 262,536 activity, however, this was in comparison to the G743A and A748T mutants that experienced no AC 262,536 activity at all. The reported results related to the mutant androgen receptors allowed the researcher to conclude that AC 262, 536 successfully acts as a partial agonist to testosterone [2].

Figure 5: Comparing the efficacy of the mutant androgen receptors in comparison to the control group.

In addition to the R-SAT assay, the luciferase reporter gene assay, and a binding assay reported that activity levels of AC 262,536 were comparable to the natural ligand testosterone. The binding assay report exhibited the most statistically significant results in terms of the effects of AC 262,536 on dihydrotestosterone (DHT) in the human prostate cancer cell line, LNCaP. Cell proliferation was induced with 10 nM concentration of DHT while AC 262,536 acted as a dose-dependent antagonist against DHT-induced proliferation of LNCaP cells. The antagonist effects were significant with doses of 100 nM and experienced a plateau at 1 uM of AC 262,536. These results allowed researchers to conclude that in addition to acting as a partial agonist to testosterone, AC 262,536 acts as a functioning antagonist in prostate cells [2].

Figure 6: Changes in absorbance units elicited by the partial inhibition of DHT-proliferation by AC 262,536.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Cutler C, Viljanto M, Taylor P, Habershon-Butcher J, Muir T, Biddle S, Van Eenoo P. Equine metabolism of the selective androgen receptor modulator AC-262536 in vitro and in urine, plasma and hair following oral administration. Drug Test Anal. 2021 Feb;13(2):369-385. doi: 10.1002/dta.2932. Epub 2020 Oct 15. PMID: 32959959.

[2] Piu F, Gardell LR, Son T, Schlienger N, Lund BW, Schiffer HH, Vanover KE, Davis RE, Olsson R, Bradley SR. Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator. J Steroid Biochem Mol Biol. 2008 Mar;109(1-2):129-37. doi: 10.1016/j.jsbmb.2007.11.001. Epub 2007 Nov 22. PMID: 18164613.

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