Accurate measurement of androgen after androgen esters: problems created by ex vivo esterase effects and LC‐MS/MS interference

Abstract

Background Ex vivo androgen prodrug conversion by blood esterases after oral androgen ester administration may result in an overestimation of the measured blood androgens. Objective We investigated whether blood collection tubes with esterase inhibitors decreased the conversion of testosterone undecanoate (TU) and dimethandrolone undecanoate (DMAU) to their active metabolites, testosterone (T), and dimethandrolone (DMA), providing a more accurate assessment of circulating T/DMA levels. Methods Blood was collected in tubes with/without esterase inhibitors from: (i) four healthy and four hypogonadal men receiving no androgens and spiked ex vivo with TU/DMAU; (ii) four men taking oral TU (Andriol®); and (iii) eight hypogonadal men dosed with oral 316 mg TU and 15 healthy men with 200 mg DMAU. T/DMA levels were measured by LC‐MS/MS. Results Sodium fluoride (NaF, an esterase inhibitor) decreased measured T levels by 14.2% in men not receiving TU. Increasing amounts of TU/DMAU added to blood collected into plain tubes resulted in a concentration‐dependent overestimation of T/DMA that was reduced by collecting blood into NaF tubes (by 30–85%), and keeping samples at 4 °C and minimizing time prior to centrifugation. After oral TU/DMAU administration to men, when TU/DMAU levels were >15/10 ng/mL, respectively, blood collected in NaF tubes yielded lower measured T concentrations by 15–30% and DMA by 22% due to an additional inhibitory effect of NaF on blood esterases. Conclusion NaF directly lowers plasma T/DMA levels measured by LC‐MS/MS and also inhibits blood esterase activity. Overestimation of T/DMA in blood collected in tubes without NaF after oral TU/DMAU administration is important for pharmacokinetics studies in drug development clinical trials but may have limited impact in clinical practice/utilization because the differences between measured and true androgen values are modest and the wide therapeutic androgen efficacy ranges obviate the need for highly accurate androgen measurements during therapy.

A new oral testosterone undecanoate therapy comes of age for the treatment of hypogonadal men

“…The C avg serum T on days 90 and 365 in trial I were 628 ± 343 and 524 ± 215 ng/dl, respectively [Based on data generated after completion of CLAR-09007, it became apparent that the most accurate measure of circulating T for determination of PK efficacy in men dosed with oral TU necessitated that post-collection conversion of TU to T during blood samples processing be accounted/corrected for (see Ceponis et al . 12 and Lachance et al . 13 ).…”

“…Efficacy in the topical T arms of each study were similar in magnitude [79.0% (trial I) and 87.3% (trial II)] and not statistically different from oral TU. The Cavg serum T on days 90 and 365 in trial I were 628 ± 343 and 524 ± 215 ng/dl, respectively [Based on data generated after completion of CLAR-09007, it became apparent that the most accurate measure of circulating T for determination of PK efficacy in men dosed with oral TU necessitated that post-collection conversion of TU to T during blood samples processing be accounted/corrected for (see Ceponis et al 12 and Lachance et al 13 ). We now know that the serum T values in CLAR-09007 overestimated the actual circulating level of T at the time blood was drawn by about 20% on average].…”

“…Additionally, in trial II, we evaluated T responses based on T concentrations in NaF-EDTA plasma, while serum T measurements were used in trial I that, in retrospect, yielded T concentrations that were artefactually elevated (by approximately 20%) due to post-collection of TU to T during bloodsample processing. 12,13 Materials and methods The phase III clinical trials detailed herein were approved by a central or site-specific institutional review boards before study initiation at each clinical site and were conducted in accordance with the Declaration of Helsinki and/or all relevant federal regulations, including good clinical practice guidelines. Written informed consent was obtained from trial participants before any studyrelated procedures were conducted.…”

Effects of the oral testosterone undecanoate Kyzatrex™ on ambulatory blood pressure in hypogonadal men

“…quantitation was by liquid chromatography-mass spectrometry. 5,6 A single pre-treatment (Day 1) pre-dose sample for total testosterone had a mean value of 231.5 ng/dl.…”

Androgen Treatment in Adolescent Males With Hypogonadism

“…Nonetheless, systemic absorption remains variable (McBride et al, 2015;Shoskes et al, 2016). A new formulation of testosterone undecanoate has recently been approved and is available in capsules of 158, 198, or 237 mg. An advantage of this new formulation is its better lipophilicity and more consistent absorption less dependent on a lipid meal (Ceponis et al, 2019). No experience has been reported yet in boys and adolescents.…”