2.
Medizinische Klinik der Universität Düsseldorf1),
Dermatologische Abteilung des Evangelischen Krankenhauses Düsseldorf2), Germany and Organon Scientific Development Group, Oss, The Netherlands3)
PLASMA ANDROGEN LEVELS IN MEN AFTER ORAL ADMINISTRATION OF TESTOSTERONE
OR TESTOSTERONE UNDECANOATE
By E. Nieschlag,
J. Mauss, A. Coert and P. Ki\l=c'\ovi\l=c'\ ABSTRACT
Plasma testosterone and androstenedione levels in men were measured after oral administration of free testosterone and testosterone undecanoate. Both androgens were determined by simultaneous, specific radioimmunoassays after separation and isolation by thin layer chromatography. While free unesterified testosterone had no effect on plasma androgen levels, a striking increase of both testosterone and androstenedione levels was noted after administration of testosterone undecanoate, which is otherwise only achieved by parenteral testosterone application. This effect of testosterone undecanoate is probably due to absorption via the lymph rather than via the portal vessels so that peripheral circulation is reached before metabolism in the liver. Testosterone undecanoate promises to be an effective medication for oral androgen replacement.
The therapy of the deficient endocrine function of the testes is impeded by the ineffectiveness of orally administered genuine testosterone (Foss 1939), which should be the first choice for substitution purposes. Since the ineffec¬ tiveness is probably due to rapid metabolism in the intestinal wall (Harri et al. 1970) and in the liver, testosterone must either be injected or implanted in order to reach target organs before liver inactivation, or the molecule must be modified by introducing a methyl group at C 17 to become orally effective. While injections and implantations are inconvenient for the patient, altered testosterone molecules may have untoward effects on his health. Therefore, an orally effective preparation releasing genuine testosterone to the organism would be a desirable addition to the available medication.
In this respect it was of interest to investigate the bioavailability (Dittert et al. 1972) of testosterone after oral administration of testosterone undecanoate in men. This compound had shown potent androgenic activity upon oral ad¬ ministration to rats, especially when administered as a solution in arachis oil, and had no deleterious effects on the liver (Coert et al, in preparation). Plasma testosterone levels in men after oral administration of two different pharmaceutical preparations of testosterone undecanoate (a) solution in arachis oil, b) crystalline form) were compared with testosterone levels after oral administration of free unesterified testosterone. Androstenedione was measured in the same plasma aliquot by a simultaneous radioimmunoassay.
MATERIALS AND METHODS
Steroid
preparations
Testosterone undecanoate (17/?-hydroxy-4-androsten-3-one ß- undecanoate, C^qH^Ojj) has a molecular weight of 457 so that 100 mg are equivalent to 63 mg free unesterified testosterone. Therefore, for the comparative study 63 mg free tes¬ tosterone was administered in capsules with arachis oil or 100 mg testosterone un¬ decanoate either in capsules with arachis oil or in capsules in dry crystalline form.
Study protocol From a first group of 8 convalescent male patients, 16 to 73 years of age free from liver or kidney dysfunction, heparinized blood was obtained at 8.00, 12.00 and 18.00 h on the control day. These times were chosen in order to account for diurnal variations of plasma testosterone (Nieschlag 1974). On the following day 63 mg free unesterified testosterone was given orally at 8.05 h and blood was obtained at 8.00, 10.00, 11.00, 12.00, 13.00, 14.00, 15.00, 16.00 and 18.00 h. In a second group of 8 male patients, 31 to 73 years old, suffering from skin diseases and free from signs of liver or kidney dysfunction, a cross-over study with testosterone undecanoate in two different pharmaceutical preparations was performed. On the control days (day 1 and 9) blood was obtained at 8.00, 12.00 and 18.00 h. At 8.05 h on the test days (day 2 or 10) 100 mg testosterone undecanoate either in capsules in dry crystalline form or in capsules in arachis oil was administered orally. On the test days blood was obtained at the same times as in patients of the first group. All plasma samples were stored at -20°C until analysis could be performed.
Radioimmunoassay General outline. In all plasma samples testosterone and androstenedione were meas¬ ured by simultaneous radioimmunoassay, whereby both steroids were separated and isolated by thin layer chromatography on silica gel. The method is an extension of the previously described radioimmunoassay for testosterone (Nieschlag 8c Loriaux 1972) and outlined in Fig. 1. In principle, the assay for androstenedione is the same as for testosterone, only that standards, tracers and antisera for testosterone were replaced by those for androstenedione. -
Simultaneous
radioimmunoassay for testosterone (T) and androstenedione (A): Flow sheet .
1 ml Plasma - and H -A recovery trace
+
Ether extraction
Evaporation Thin layer chromatography on Silica GF Elution of
Aliquot for
Elution of A
recovery )
Aliquot for recovery
Aliquot for RIA:
Aliquot for RIA: Incubation with
Incubation with H3-TandT-3-BSA
H^-AandA-3-BSA antiserum.
antiserum. Standards from 25 to 10,000 pg
Standards from 10 to 5,000 pg.
Separation of bound and free
by dextran/globulin coated charcoal
_and centrifugation_ Supernatant liquid scintillation counting Calculation of results by logit _transformation
Simultaneous
radioimmunoassay
Fig.
1.
for testosterone (T) and androstenedione in plasma: Flow sheet.
(A)
Antisera. Testosterone-3-bovine serum albumin and androstenedione-3-bovine serum albumin were prepared by a mixed anhydride reaction (Erlanger et al. 1957). Antisera were raised in rabbits by the one-time, multiple site, intradermal immunization -
(Vaitukaitis
et al.
1971; Nieschlag 1975).
Method in detail. To 1 ml of all unknown plasma samples, to 1 ml standard and to 1 ml HoO (serving as blank value) 1000 cpm of each [ 1,2,6,7-3H] testos¬ terone and [1,2-3H] androstenedione (NEN, Dreieichenhain) in 50 µ\ methanol (Merck, Darmstadt) are pipetted, mixed and left at room temperature for 15-30 min for equilibration, followed by an ether extraction with 2 5 ml (ether pro narcosi, Hoechst, Frankfurt). The ether phases are dried under nitrogen at 35°C in a water bath. The residues are applied with 2 75 µ\ methanol to ready-made silica thin layer plates with fluorescent indicator (Merck). Standards are spotted on the outer lanes of the plates, which are then developed in chloroform/ethyl acetate/petroleum ether (60°C) 50:45:5 (v/v). The Rf values for testosterone, androstenedione and other steroids
plasma
-
shown in Table 2. The standards are located by ultraviolet light and the areas corresponding to testosterone and androstenedione are aspirated to separate glass wool stoppered Pasteur pipettes by means of a suction pump. The steroids are eluted with 5 ml methanol. After evaporation the residues are dissolved in 1.0 ml methanol, so that the final volume after chromatography is identical to the initial plasma volume. 0.5 ml of the methanol phases are taken for recovery and two 0.1 ml aliquots from the testosterone tubes for the testosterone assay and two 0.2 ml aliquots from the are
androstenedione tubes for the androstenedione assay. The actual assay is performed in disposable 10 75 mm glass tubes. Standards of 0.025, 0.05, 0.1, 0.2, 0.5, 0.7, 1.0, 2.0, 5.0 and 10.0 ng testosterone (Merck) and standards of 0.005, 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 0.7, 1.0 and 2.0 ng androstenedione (Steraloids) in methanol are pipetted in duplicate to two sets of tubes and all tubes with standards and unknowns are taken to dryness. Two empty tubes are added to each assay for the zero binding (B(l) and two for the non-specific binding (Bn). Each tube then receives 0.5 ml of diluent (0.1 % bovine /-globulin in sterilized saline) containing 25 000 cpm [l,2,6,7-3H]testosterone or 20 000 cpm [1,2-3H]androstenedione, respec¬ tively. The tracers are purified routinely by chromatography every 3 months. This is followed by 0.5 ml of the respective antiserum dilution in diluent. The testosterone antiserum is used in a final dilution of 1/80 000 and the androstenedione antiserum of 1/50 000. Instead of antiserum, 0.5 ml plain diluent is added to the Bn-tubes. After overnight incubation at 4°C bound and free steroids are separated by dextran/globulin coated charcoal. The radioactivity in the supernatant (= antibody bound steroid) is counted by liquid scintillation counting as previously described (Nieschlag 8c Loriaux 1972). The results are calculated by logit transformation (Rodbard et al. 1969) and corrected for losses. The standard curves are usually linear over the entire dose range.
Table 1. Simultaneous
radioimmunoassay
for testosterone androstenedione (A). Reliability criteria.
(T)
and
Precision
Intraassay (c. v. %) Interassay (c. v. Vo) Accuracy Recovery from water (Vo) Recovery from plasma (%) Overall recovery
(%) Sensitivity (pg/tube) Practicability
samples/technician/week
8 11
9 12
99 ± 4 100 ± 6 73 ± 7
99 ± 3 100 ± 4 76 ± 4
25
10
40-50
40-50
Table 2. Cross-reaction of various steroids with the antisera and in the TLC system. Per cent
("lo)
Dihydrotestosterone Epitestosterone Androstenedione Androstenediol
Dehydroepiandrosterone Pregnenolone 17-OH-Pregnenolone Progesterone 17-OH-Progesterone Deoxycorticosterone Oestrone Oestradiol Oestriol
Reliability
values of these steroids
cross-reaction
Steroid
Testosterone
R¡.-
T-3-BSA
/HA-3-BSA
antiserum
antiserum
100 60 0.1 0.3 1.6 0.002 < 0.001
< 0.001 0.05 0.03 < 0.001 0.003 0.003 0.001
2 0.8 100 0.006 3.0 0.003 < 0.001 0.05 0.05 < 0.001 < 0.001 < 0.001 < 0.001
RF
values
0.45 0.57 0.45 0.65 0.36 0.57 0.57 0.36 0.70 0.52 0.77 0.56 0.09
criteria
In addition to the criteria shown in Table 1, the specificity of the assays is deter¬ mined by the specificity of the antisera, which was established by displacement studies according to Abraham (1969) and is shown in Table 2. Both testosterone and an¬ drostenedione are separated by thin layer chromatography from the cross-reacting steroids, i. e. testosterone is separated from dihydrotestosterone as well as from an¬ drostenedione and androstenedione is separated from dehydroepiandrosterone, testos¬ terone and dihydrotestosterone. The blank values of both methods are usually in the range of the Bn values, but may occasionally rise to 5 pg in the androstenedione assay and 10 pg in the testosterone assay and are then not subtracted from the values. When testosterone undecanoate was investigated for interference with the antisera, it showed a cross-reaction of 0.2 "lo with the testosterone antiserum and indiscernible cross-reaction with the androstenedione antiserum. This apparent cross-reaction is probably due to contamination with free testosterone, since testosterone undecanoate, when subjected to thin layer chromatography and taken through the assay, appeared to contain 0.2 °/o free testosterone.
RESULTS
The results are shown in Figs. 2-4. While only slight and statistically insig¬ nificant increases of plasma testosterone and androstenedione were observed after free testosterone, both steroids were greatly elevated after oral adminis-
tration of testosterone undecanoate in oily or crystalline form. Peak levels occurred in most cases 4 to 5 h after ingestion. While the increase over the 12.00 h values of the control days were highly significant with both prepara¬ tions (P
Medizinische Klinik der Universität Düsseldorf1),
Dermatologische Abteilung des Evangelischen Krankenhauses Düsseldorf2), Germany and Organon Scientific Development Group, Oss, The Netherlands3)
PLASMA ANDROGEN LEVELS IN MEN AFTER ORAL ADMINISTRATION OF TESTOSTERONE
OR TESTOSTERONE UNDECANOATE
By E. Nieschlag,
J. Mauss, A. Coert and P. Ki\l=c'\ovi\l=c'\ ABSTRACT
Plasma testosterone and androstenedione levels in men were measured after oral administration of free testosterone and testosterone undecanoate. Both androgens were determined by simultaneous, specific radioimmunoassays after separation and isolation by thin layer chromatography. While free unesterified testosterone had no effect on plasma androgen levels, a striking increase of both testosterone and androstenedione levels was noted after administration of testosterone undecanoate, which is otherwise only achieved by parenteral testosterone application. This effect of testosterone undecanoate is probably due to absorption via the lymph rather than via the portal vessels so that peripheral circulation is reached before metabolism in the liver. Testosterone undecanoate promises to be an effective medication for oral androgen replacement.
The therapy of the deficient endocrine function of the testes is impeded by the ineffectiveness of orally administered genuine testosterone (Foss 1939), which should be the first choice for substitution purposes. Since the ineffec¬ tiveness is probably due to rapid metabolism in the intestinal wall (Harri et al. 1970) and in the liver, testosterone must either be injected or implanted in order to reach target organs before liver inactivation, or the molecule must be modified by introducing a methyl group at C 17 to become orally effective. While injections and implantations are inconvenient for the patient, altered testosterone molecules may have untoward effects on his health. Therefore, an orally effective preparation releasing genuine testosterone to the organism would be a desirable addition to the available medication.
In this respect it was of interest to investigate the bioavailability (Dittert et al. 1972) of testosterone after oral administration of testosterone undecanoate in men. This compound had shown potent androgenic activity upon oral ad¬ ministration to rats, especially when administered as a solution in arachis oil, and had no deleterious effects on the liver (Coert et al, in preparation). Plasma testosterone levels in men after oral administration of two different pharmaceutical preparations of testosterone undecanoate (a) solution in arachis oil, b) crystalline form) were compared with testosterone levels after oral administration of free unesterified testosterone. Androstenedione was measured in the same plasma aliquot by a simultaneous radioimmunoassay.
MATERIALS AND METHODS
Steroid
preparations
Testosterone undecanoate (17/?-hydroxy-4-androsten-3-one ß- undecanoate, C^qH^Ojj) has a molecular weight of 457 so that 100 mg are equivalent to 63 mg free unesterified testosterone. Therefore, for the comparative study 63 mg free tes¬ tosterone was administered in capsules with arachis oil or 100 mg testosterone un¬ decanoate either in capsules with arachis oil or in capsules in dry crystalline form.
Study protocol From a first group of 8 convalescent male patients, 16 to 73 years of age free from liver or kidney dysfunction, heparinized blood was obtained at 8.00, 12.00 and 18.00 h on the control day. These times were chosen in order to account for diurnal variations of plasma testosterone (Nieschlag 1974). On the following day 63 mg free unesterified testosterone was given orally at 8.05 h and blood was obtained at 8.00, 10.00, 11.00, 12.00, 13.00, 14.00, 15.00, 16.00 and 18.00 h. In a second group of 8 male patients, 31 to 73 years old, suffering from skin diseases and free from signs of liver or kidney dysfunction, a cross-over study with testosterone undecanoate in two different pharmaceutical preparations was performed. On the control days (day 1 and 9) blood was obtained at 8.00, 12.00 and 18.00 h. At 8.05 h on the test days (day 2 or 10) 100 mg testosterone undecanoate either in capsules in dry crystalline form or in capsules in arachis oil was administered orally. On the test days blood was obtained at the same times as in patients of the first group. All plasma samples were stored at -20°C until analysis could be performed.
Radioimmunoassay General outline. In all plasma samples testosterone and androstenedione were meas¬ ured by simultaneous radioimmunoassay, whereby both steroids were separated and isolated by thin layer chromatography on silica gel. The method is an extension of the previously described radioimmunoassay for testosterone (Nieschlag 8c Loriaux 1972) and outlined in Fig. 1. In principle, the assay for androstenedione is the same as for testosterone, only that standards, tracers and antisera for testosterone were replaced by those for androstenedione. -
Simultaneous
radioimmunoassay for testosterone (T) and androstenedione (A): Flow sheet .
1 ml Plasma - and H -A recovery trace
+
Ether extraction
Evaporation Thin layer chromatography on Silica GF Elution of
Aliquot for
Elution of A
recovery )
Aliquot for recovery
Aliquot for RIA:
Aliquot for RIA: Incubation with
Incubation with H3-TandT-3-BSA
H^-AandA-3-BSA antiserum.
antiserum. Standards from 25 to 10,000 pg
Standards from 10 to 5,000 pg.
Separation of bound and free
by dextran/globulin coated charcoal
_and centrifugation_ Supernatant liquid scintillation counting Calculation of results by logit _transformation
Simultaneous
radioimmunoassay
Fig.
1.
for testosterone (T) and androstenedione in plasma: Flow sheet.
(A)
Antisera. Testosterone-3-bovine serum albumin and androstenedione-3-bovine serum albumin were prepared by a mixed anhydride reaction (Erlanger et al. 1957). Antisera were raised in rabbits by the one-time, multiple site, intradermal immunization -
(Vaitukaitis
et al.
1971; Nieschlag 1975).
Method in detail. To 1 ml of all unknown plasma samples, to 1 ml standard and to 1 ml HoO (serving as blank value) 1000 cpm of each [ 1,2,6,7-3H] testos¬ terone and [1,2-3H] androstenedione (NEN, Dreieichenhain) in 50 µ\ methanol (Merck, Darmstadt) are pipetted, mixed and left at room temperature for 15-30 min for equilibration, followed by an ether extraction with 2 5 ml (ether pro narcosi, Hoechst, Frankfurt). The ether phases are dried under nitrogen at 35°C in a water bath. The residues are applied with 2 75 µ\ methanol to ready-made silica thin layer plates with fluorescent indicator (Merck). Standards are spotted on the outer lanes of the plates, which are then developed in chloroform/ethyl acetate/petroleum ether (60°C) 50:45:5 (v/v). The Rf values for testosterone, androstenedione and other steroids
plasma
-
shown in Table 2. The standards are located by ultraviolet light and the areas corresponding to testosterone and androstenedione are aspirated to separate glass wool stoppered Pasteur pipettes by means of a suction pump. The steroids are eluted with 5 ml methanol. After evaporation the residues are dissolved in 1.0 ml methanol, so that the final volume after chromatography is identical to the initial plasma volume. 0.5 ml of the methanol phases are taken for recovery and two 0.1 ml aliquots from the testosterone tubes for the testosterone assay and two 0.2 ml aliquots from the are
androstenedione tubes for the androstenedione assay. The actual assay is performed in disposable 10 75 mm glass tubes. Standards of 0.025, 0.05, 0.1, 0.2, 0.5, 0.7, 1.0, 2.0, 5.0 and 10.0 ng testosterone (Merck) and standards of 0.005, 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 0.7, 1.0 and 2.0 ng androstenedione (Steraloids) in methanol are pipetted in duplicate to two sets of tubes and all tubes with standards and unknowns are taken to dryness. Two empty tubes are added to each assay for the zero binding (B(l) and two for the non-specific binding (Bn). Each tube then receives 0.5 ml of diluent (0.1 % bovine /-globulin in sterilized saline) containing 25 000 cpm [l,2,6,7-3H]testosterone or 20 000 cpm [1,2-3H]androstenedione, respec¬ tively. The tracers are purified routinely by chromatography every 3 months. This is followed by 0.5 ml of the respective antiserum dilution in diluent. The testosterone antiserum is used in a final dilution of 1/80 000 and the androstenedione antiserum of 1/50 000. Instead of antiserum, 0.5 ml plain diluent is added to the Bn-tubes. After overnight incubation at 4°C bound and free steroids are separated by dextran/globulin coated charcoal. The radioactivity in the supernatant (= antibody bound steroid) is counted by liquid scintillation counting as previously described (Nieschlag 8c Loriaux 1972). The results are calculated by logit transformation (Rodbard et al. 1969) and corrected for losses. The standard curves are usually linear over the entire dose range.
Table 1. Simultaneous
radioimmunoassay
for testosterone androstenedione (A). Reliability criteria.
(T)
and
Precision
Intraassay (c. v. %) Interassay (c. v. Vo) Accuracy Recovery from water (Vo) Recovery from plasma (%) Overall recovery
(%) Sensitivity (pg/tube) Practicability
samples/technician/week
8 11
9 12
99 ± 4 100 ± 6 73 ± 7
99 ± 3 100 ± 4 76 ± 4
25
10
40-50
40-50
Table 2. Cross-reaction of various steroids with the antisera and in the TLC system. Per cent
("lo)
Dihydrotestosterone Epitestosterone Androstenedione Androstenediol
Dehydroepiandrosterone Pregnenolone 17-OH-Pregnenolone Progesterone 17-OH-Progesterone Deoxycorticosterone Oestrone Oestradiol Oestriol
Reliability
values of these steroids
cross-reaction
Steroid
Testosterone
R¡.-
T-3-BSA
/HA-3-BSA
antiserum
antiserum
100 60 0.1 0.3 1.6 0.002 < 0.001
< 0.001 0.05 0.03 < 0.001 0.003 0.003 0.001
2 0.8 100 0.006 3.0 0.003 < 0.001 0.05 0.05 < 0.001 < 0.001 < 0.001 < 0.001
RF
values
0.45 0.57 0.45 0.65 0.36 0.57 0.57 0.36 0.70 0.52 0.77 0.56 0.09
criteria
In addition to the criteria shown in Table 1, the specificity of the assays is deter¬ mined by the specificity of the antisera, which was established by displacement studies according to Abraham (1969) and is shown in Table 2. Both testosterone and an¬ drostenedione are separated by thin layer chromatography from the cross-reacting steroids, i. e. testosterone is separated from dihydrotestosterone as well as from an¬ drostenedione and androstenedione is separated from dehydroepiandrosterone, testos¬ terone and dihydrotestosterone. The blank values of both methods are usually in the range of the Bn values, but may occasionally rise to 5 pg in the androstenedione assay and 10 pg in the testosterone assay and are then not subtracted from the values. When testosterone undecanoate was investigated for interference with the antisera, it showed a cross-reaction of 0.2 "lo with the testosterone antiserum and indiscernible cross-reaction with the androstenedione antiserum. This apparent cross-reaction is probably due to contamination with free testosterone, since testosterone undecanoate, when subjected to thin layer chromatography and taken through the assay, appeared to contain 0.2 °/o free testosterone.
RESULTS
The results are shown in Figs. 2-4. While only slight and statistically insig¬ nificant increases of plasma testosterone and androstenedione were observed after free testosterone, both steroids were greatly elevated after oral adminis-
tration of testosterone undecanoate in oily or crystalline form. Peak levels occurred in most cases 4 to 5 h after ingestion. While the increase over the 12.00 h values of the control days were highly significant with both prepara¬ tions (P
