Importance of Measuring Testosterone in Enzyme-Inhibited Plasma for Oral Testosterone Undecanoate Androgen Replacement Therapy Clinical Trials

Figures & data

Figure 1.  Molecular structures of testosterone undecanoate, dihydrotestosterone undecanoate, testosterone and dihydrotestosterone.

Figure 2.  Testosterone and dihydrotestosterone NaF/Na₂ ethylenediaminetetraacetate plasma versus serum mean concentrations.

DHT: Dihydrotestosterone.

Table 1.  Conversion of testosterone undecanoate into testosterone according to the testosterone undecanoate concentrations and the incubation time.

TU Concentration Fortified (ng/dl)	Duration of incubation (min)	Concentration testosterone measured (ng/dl)	% difference vs TU = 0 (%)	% difference 30 vs 60 min (%)
0	0	23.75	–	–
1500	30	36.76	54.8	24.6
	60	45.81	92.9
10,000	30	152.66	542.8	65.8
	60	253.18	966.0
30,000	30	306.02	1188.5	73.9
	60	532.19	2140.8
70,000	30	732.49	2984.2	72.4
	60	1262.81	5217.1

TU: Testosterone undecanoate.

Table 2.  Conversion of testosterone undecanoate into testosterone in whole blood collected with different collection tubes and endogenous level of testosterone in different collection tubes.

Matrix	Anticoagulant	Comparison sample^†	Testosterone concentration (ng/dl)
			Subject 1					Subject 2
			Stability sample	Comparison sample	% Change vs unfortified serum	Stability sample	% change	Endogenous level^‡	% change vs unfortified serum
Serum	Without anticoagulant not fortified	30 min at RT	NA	370.61	Reference	NA	NA	287.21	Reference
Serum	Without anticoagulant	30 min at RT	60 min at RT	1270.19	242.7	1967.80	54.9	282.51	-1.6
Serum	Rapid serum tube without anticoagulant	10 min at RT	40 min at RT	814.41	119.7	1228.29	50.8	247.77	-13.7
Serum	NaF 0.43%	30 min at RT	60 min at RT	992.38	167.8	1592.15	60.4	285.29	-0.7
Plasma	EDTA K2	10 min at 4°C	60 min at 4°C	441.91	19.2	528.99	19.7	262.77	-8.5
Plasma	NaF (0.15%)-Na2 EDTA	10 min at 4°C	60 min at 4°C	392.77	6.0	452.53	15.2	247.89	-13.7
Plasma	NaF (0.25%)-K2C2O4	10 min at 4°C	60 min at 4°C	409.15	10.4	461.07	12.7	233.78	-18.6
Plasma	BD™ P800 containing protease and esterase inhibitor	10 min at 4°C	60 min at 4°C	479.57	29.4	720.30	50.2	283.29	-1.4

Samples were fortified with 60,000 ng/dl of testosterone undecanoate.

† Samples are incubated for 30 min when blood is collected to allow the coagulation and to mimic the clinical sample handling of serum samples and for plasma samples, whole blood is incubated for 10 min at 4°C as handled in clinic.

‡ Endogenous level was determined in matrices from a different donor than the testosterone undecanoate conversion tests.

EDTA: Ethylenediaminetetraacetate; NA: Not applicable; RT: Room temperature.

Table 3.  Comparison of testosterone endogenous levels between serum and plasma with NaF/Na₂ ethylenediaminetetraacetate and NaF/K₂C₂O₂ in different donors.

Donors	Testosterone concentration in serum (ng/dl)	Testosterone concentration in NaF/Na2 EDTA (ng/dl)	% change vs serum	Testosterone concentration in NaF/K2C2O4 (ng/dl)	% change vs serum
1	301.62	273.63	-9.3	254.22	-15.7
2	309.23	273.18	-11.7	280.06	-9.4
3	479.70	424.16	-11.6	399.71	-16.7
4	555.39	485.15	-12.7	457.20	-17.7
5	511.13	467.45	-8.6	507.68	-0.7
6	576.13	487.55	-15.4	483.81	-16.0
7	490.22	432.20	-11.8	421.20	-14.1
8	263.80	230.30	-12.7	211.26	-19.9
Mean	435.90	384.20	-11.7	376.89	-13.8
SD (±)	124.31	106.86		112.97
CV (%)	28.5	27.8		30.0

EDTA: Ethylenediaminetetraacetate.

Table 4.  Testosterone and phosphate buffer saline concentrations in serum or plasma harvested from blood spiked with testosterone undecanoate working solution prepared in phosphate buffer saline or methanol; spiking level 60,000 ng/dl.

Experiments	Endogenous level in serum	Endogenous level in plasma	30 min serum RT	60 min serum RT	Plasma 10 min 4°C	Plasma 30 min 4°C	Plasma 60 min 4°C
Mean testosterone concentration (ng/dl)
Whole blood spiked with TU in PBS	255.4	221.7	295.4	349.0	236.0	232.9	231.1
Whole blood spiked with TU in methanol	255.4	221.7	854.8	1255.4	322.5	339.9	320.5
Mean TU Concentration (ng/dl)
Whole blood spiked with TU in PBS	N/A	N/A	3991.0	5892.3	74408.7	54088.0	26618.7
Whole blood spiked with TU in methanol	N/A	N/A	97110.3	96132.3	97235.0	95628.0	93816.3

PBS: Phosphate buffer saline; RT: Room temperature; TU: Testosterone undecanoate.

Table 5.  Pharmacokinetic results obtained from serum and NaF-Na₂ ethylenediaminetetraacetate plasma.

Analyte	Parameter	Ratio (%)	90% geometric CI (%)		Intra-subject CV (%)
			Lower	Upper
Testosterone	AUC0–24	130.02	128.06	132.01	2.34
	Cmax0–24	134.01	128.66	139.58	6.29
Dihydrotestosterone	AUC0–24	122.40	120.33	124.50	2.63
	Cmax0–24	130.61	125.38	136.05	6.30