Establishment of physiological and supra-physiological testosterone dosing regimen in mice

The testicular feminised (Tfm) mouse exhibits a non-functional androgen receptor due to a single base-pair deletion in the gene encoding the androgen receptor. Tfm mice are also deficient in the steroidogenic enzyme 17α-hydroxylase, which contributes to a reduced circulating level of testosterone (T). We aim to investigate the role of T in the development of atherosclerosis and examine whether genomic or non-genomic pathways are involved in the anti-atherogenic action of T. The aim of this study was therefore to determine dosing regimen to replace T to physiological levels in the T deficient Tfm mouse, and also to produce sustainable supraphysiological T concentrations in Tfm and XY littermates. As T is converted to 17β-oestradiol (E2), via the enzyme aromatase, we also need to quantify any changes in E2 post dosing.

Eight-week-old Tfm (n=66) and XY littermate controls (n=24) received a single 10µl intramuscular (i.m.) injection of Sustanon100 (S100: testosterone propionate 20mg, testosterone phenylpropionate 40mg, and testosterone isocaproate 40mg/mL, dose = 50 mg/kg: human physiological replacement dose = 3.5 mg/kg), or Sustanon250 (S250: testosterone propionate 30mg, testosterone phenylpropionate 60mg, testosterone isocaproate 60mg, and testosterone decanoate 100mg/mL, dose=125mg/mL). Mice were sacrificed at serial intervals at 1, 2, 4, 7, 10 or 14 days post injection and whole blood collected. Whole blood was centrifuged at 3000rpm for 10 minutes. Quantification of serum levels of T and E2 were made via ELISA. To ensure reproducibility of this pharmacokinetic profile additional Tfm (n=38) and XY (n=22) mice received a second injection at day 14 and sacrificed at day 15, 16, 21 or 28 and T quantified.

Concentrations of T and E2 rose significantly at each end point (Table 1). The mean area under the curve (AUC) for Tfm mice receiving 10µl of S100 was 17.5nM (range in men 10-30nM) with statistically similar T levels seen following second cycle analysis (AUC=18.8nM). The mean AUC for Tfm mice and XY littermates receiving S250 were 287.9nM and 259.1nM respectively. Second cycle analysis revealed statistically similar levels of T generating an AUC of 250.2nM and 260.7nM respectively. Interestingly, XY littermates had significantly lower levels of E2 compared to Tfm mice, following 10 µl i.m. injection of S250 with significance was observed at day 7 and 14.

Table 1. Hormone profile showing serum concentrations of T and E2 at baseline and following 10 µl i.m. injection of S100 or S250 in Tfm and XY littermates. Results are expressed as mean (S.E.M). * p<0.05 compared to baseline, † p<0.05 compared to Tfm mice receiving S250, ‡ p<0.0001 compared to Tfm mice at baseline. All via Students unpaired t-test.

VEW TABLE

Reproducible physiological T replacement is achieved by a fortnightly 10 µl i.m. injection of S100 in Tfm mice. Reproducible supra-physiological T concentrations were produced by once fortnightly 10 µl i.m. injection of S250 in Tfm and XY littermates. The high level of E2 observed in Tfm mice compared to XY littermates post injection, may be due to differences in aromatase expression.