3β-hydroxyandrost-5-en-17-one acetate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

High bioavailability of dehydroepiandrosterone administered percutaneously in the rat

Author:

Labrie C, Flamand M, Belanger A and Labrie F

Year:

1996

Bibliographic source:

J Endocrinol 150: S107-S118

Materials and methods

Principles of method if other than guideline:

Dehydroepiandrosterone (DHEA) was administered percutaneously by twice daily application for 7 days to the dorsal skin of orchiectomized (ORCH) male and ovariectomized (OVX) female Sprague-Dawley rats to compare the bioavailability of DHEA after percutaneous and oral administration. The subcutaneous route of administration was used as a reference of maximal bioavailability. The doses of DHEA used for all three exposure routes were 0, 0.3, 1.0, 3.0 10 and 30 mg. DHEA was dissolved in 50% ethanol/50% propylene glycol and applied twice daily (0-5 ml) on a shaved dorsal skin area (3 cm x 3 cm) for 7 days starting on the day of OVX or ORCH. For subcutaneous and oral administration, DHEA was dissolved in 0-5 ml 10% ethanol/1% gelatin/0-9% NaCl and injected twice daily in the dorsal area or given in the same volume (0-5 ml) orally for 7 days starting on the day of OVX and ORCH. A separate experiment compared the efficiency of absorption of DHEA in 50% ethanol/50% propylene glycol with specific cream and gel formulations. The animals were sacrified 7 days after the start of treatment. Blood samples were collected and serum was frozen at -20 °C until assayed for the most relevant steroids (DHEA, DHEA-Sulfate, androstenediol, androstenediol sulfate, androstenedione, testosterone, dihdyrotestosterone and estrone). Uteri, ventral prostates, dorsal prostates and seminal vesicles were excised, weighed, and stored at -80 °C for further analyses.

GLP compliance:

not specified

Test material

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (Dehydroepiandrosterone; DHEA)
- Source: Steraloids, Wilton, New Hampshire, USA
- Purity: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of the test material: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified

FORM AS APPLIED IN TEST
- Percutaneous route: solution, cream or gel
- Oral route: solution
- Subcutaneous route: solution

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats (Crl:CD(SD)Br)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles-River Canada Inc. (St-Constant, Quebec, Canada)
- Age: Not specified
- Weight: Males (225-250 g); Females (175-200 g)
- Housing: Animals were housed two per cage.
- Diet: Purina rat chow ad libitum.
- Water: Available ad libitum.
- Acclimation: Not specified.
- Ovariectomy (OVX) / Orchiectomy (ORCH): Bilaterally performed under ether general anesthesia on day of first dose.
- Other: Studies were conducted according to the Guideline for Care and Use of Experimental Animals.

ENVIRONMENTAL CONDITIONS
- Temperature: Not specified
- Humidity (%): Not specified
- Photoperiod (hrs dark / hrs light): 12-hrs dark, 12-hrs light cycle (lights on at 0715)

Administration / exposure

Route of administration:

other: Group 1: Subcutaneous. Group 2: Percutaneous. Group 3: Oral.

Vehicle:

other: Gr 1 (SC): 10% EtOH/1% gelatin/0-9% NaCl. Gr 2 (Percutaneous): Exp 1 (50% EtOH/50% propylene glycol); Exp 2 (5% EtOH and 95% glaxal base (cream) or in 49% EtOH, 49% propylene glycol and 2% Klucel (gel)). Gr 3 (Oral): 10% EtOH 1% gelatin/0-9% NaCl.

Remarks:

EtOH = ethanol. Gr = Group.

Details on exposure:

GROUP 1 (SUBCUTANEOUS): DHEA was dissolved in 0.5 ml 10% ethanol/1% gelatin/0-9% NaCl and injected twice daily in the dorsal area for 7 days starting on the day of OVX and ORCH.

GROUP 2 (PERCUTANEOUS): DHEA was dissolved in 50% ethanol/50% propylene glycol and applied twice daily (0.5 ml) on a shaved dorsal skin area (3 cm x 3 cm) for 7 days starting on the day of OVX or ORCH. One experiment compared the efficiency of absorption of DHEA in 50% ethanol/50% propylene glycol with the following cream and gel formulations: DHEA was dissolved in 5% ethanol and 95% glaxal base (cream) or in 49% ethanol, 49% propylene glycol and 2% Klucel (gel).

GROUP 3 (ORAL): DHEA was dissolved in 0.5 ml 10% ethanol/1% gelatin/0-9% NaCl and given in the same volume (0.5 ml) orally for 7 days starting on the day of OVX and ORCH.

Duration and frequency of treatment / exposure:

GROUP 1 (SUBCUTANEOUS): DHEA was injected twice daily for 7 days.

GROUP 2 (PERCUTANEOUS): DHEA was applied twice daily (0-5 ml) for 7 days.

GROUP 3 (ORAL): DHEA was administered orally (0-5 ml) twice daily for 7 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg

Remarks:

Control Group. A separate Control Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral)

Doses / concentrations 2

Dose / conc.:

0.3 other: mg

Remarks:

A 0.3 mg Dose Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral).

Doses / concentrations 3

Dose / conc.:

1 other: mg

Remarks:

A 1.0 mg Dose Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral).

Doses / concentrations 4

Dose / conc.:

3 other: mg

Remarks:

A 3.0 mg Dose Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral).

Doses / concentrations 5

Dose / conc.:

10 other: mg

Remarks:

A 10 mg Dose Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral).

Doses / concentrations 6

Dose / conc.:

30 other: mg

Remarks:

A 30 mg Dose Group was used for each route of administration (Subcutaneous, Percutaneous, and Oral).

No. of animals per sex per dose / concentration:

10

Control animals:

yes

Positive control reference chemical:

No

Details on study design:

- Animals were randomly divided into the indicated groups (ten rats per group). The female and male animals were bilaterally ovariectomized (OVX) or orchiectomized (ORCH), respectively, under ether anesthesia. Some rats were used as intact controls.
- The animals were sacrified 7 days after the start of treatment.
- Blood samples were collected and serum was frozen at -20 °C until assayed for the most relevant steroids (DHEA, DHEA-Sulfate, androstenediol, androstenediol sulfate, androstenedione, testosterone, dihdyrotestosterone and estrone).
- Uteri, ventral prostates, dorsal prostates and seminal vesicles were excised, weighed, and stored at -80 °C for further analyses.
- Steroid analysis in serum: The most relevant steroids (DHEA, DHEA-Sulfate, androstenediol, androstenediol sulfate, androstenedione, testosterone, dihdyrotestosterone, luteinizing hormone and estrone) were extracted from 1 ml of serum using serial additions of 5 ml ethanol and centrifugation at 2000 g. Pellets were resuspended in 5 ml hexane then recentrifuged. Ethanol extracts were combined with the hexane extract. The organic solvent was evaporated under nitrogen. The residue was dissolved in 1 ml water/ethanol; this solution was loaded onto C-18 columns and steroid fractions were collected. Chromatography was performed on Sephadex LH-20 columns. Fractions were collected and evaporated, and the concentration of the various steroids was determined by radioimmunoassays.

Details on dosing and sampling:

No additional details

Statistics:

Radioimmunoassay data were analyzed using a program based on model II of Rodbard and Lewald (Rodbard 1974). Plasma steroid levels were shown as the means ± s.e.m. of duplicate determinations of individual samples. Statistical significance was measured according to the Duncan-Kramer multiple range test (Kramer 1956).

Results and discussion

Main ADME results

Type:

absorption

Results:

Assuming the bioavailability obtained by the subcutaneous route to be 100%, it was estimated based on relative pharmacodynamic responses that the potencies of DHEA in rats by the percutaneous and oral routes were approximately 33% and 3%, respectively.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

MALE RAT RESULTS
- DHEA administered percutaneously by twice daily application for 7 days to the dorsal skin of the rat stimulated an increase in ventral prostate weight with approximately one third the potency of the compound given by subcutaneous injection. The doses required to achieve a 50% reversal of the inhibitory effect of orchiectomy were approximately 3 and 1 mg respectively.
- By the oral route, DHEA had only 10-15% of the activity of the compound given percutaneously.
- Similar ratios of activity were obtained when dorsal prostate and seminal vesicle weight were used as parameters of androgenic activity.

FEMALE RAT RESULTS
- When examined on an estrogen-sensitive parameter, namely uterine weight in ovariectomized rats, the stimulatory effect of DHEA was much less potent than its androgenic activity measured in the male animal, a 50% reversal of the inhibitory effect of ovariectomy on uterine weight being observed at the 3 and 30 mg doses of DHEA administered by the subcutaneous and percutaneous routes

Any other information on results incl. tables

STEROID ANALYSIS IN SERUM (FEMALE OVX RATS):

The serum levels of the most relevant steroids, as hypothesized by the study authors, were measured in female OVX rats to better understand the mechanism of how DHEA affects uterine weight and serum luteinizing hormone in OVX females. At the high dose of DHEA used, namely 100 mg applied to the skin twice daily for 7 days, serum DHEA increased from undetectable levels to 140 ±12.5 nM. Serum DHEA-Sulfate increased to 7.25 ± 0.5 µM. The addition of the antiandrogen, flutamide, decreased serum DHEA levels and increased serum DHEA-Sulfate levels. Serum androstenediol increased to 8.0 ± 1.25 nM after treatment with DHEA, while serum androstenediol sulfate increased to 16.2 ± 1.4 nM. Treatment with flutamide did not significantly affect the serum androstenediol concentration achieved after administration of DHEA while a tendency towards an increase in serum androstenediol sulfate levels was observed after the same treatment.

Serum androstenedione increased to 17.3 ± 1.5 nM after treatment with DHEA while serum testosterone reached 15.8 ± 0.6 nM. Serum estrone, a direct aromatization product of androstenedione, reached a value of 410 ± 80 pM after treatment with DHEA, while the change in serum DHT was parallel to those of serum DHEA, androstenedione and testosterone, and reached a value of 2.50 ± 0.23 nM. Changes in serum 17ß-estradiol were parallel to those observed with estrone, attaining a value of 70 ± 12 pM after treatment with DHEA.

DHEA decreased serum luteinizing hormone levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. However, flutamide had no significant effect on the increase in uterine weight caused by DHEA, suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an androgenic effect on the feedback control of luteinizing hormone secretion.

COMPARISON OF PERCUTANEOUS ABSORPTION OF THREE DIFFERENT DHEA PREPARATIONS

In the investigation of the bioavailability of three different formulations of DHEA applied to dorsal rat skin, ventral prostate weight was increased to the same extent when 30 mg of DHEA was applied in 50% ethanol/50% propylene glycol or in a cream or gel formulation.

DHEA EFFECT ON LUTEINIZING HORMONE VERSUS UTERINE WEIGHT IN OVX FEMALE RATS

DHEA decreased serum luteinizing levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. On the other hand, flutamide had no significant effect on the increase in uterine weight caused by DHEA, suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an androgenic effect on the feedback control of luteinizing hormone secretion.

Applicant's summary and conclusion

Executive summary:

Dehydroepiandrosterone (DHEA) was administered percutaneously by twice daily application for 7 days to the dorsal skin of orchiectomized (ORCH) male and ovariectomized (OVX) female Sprague-Dawley rats to compare the bioavailability of DHEA after percutaneous and oral administration. The subcutaneous route of administration was used as a reference of maximal bioavailability. The animals were sacrified 7 days after the start of treatment. Uteri, ventral prostates, dorsal prostates and seminal vesicles were excised and weighed. Blood samples were collected and serum was frozen at -20 °C until assayed for the most relevant steroids. Based on changes in parameters of androgenic and estrogenic action, namely ventral prostate, dorsal prostate, seminal vesicle and uterine weight, the results showed that the bioavailability of DHEA administered by the percutaneous route was approximately one-third of that observed after subcutaneous administration and significantly more than that obtained by the oral route.  Assuming the bioavailability obtained by the subcutaneous route to be 100%, it was estimated based on relative pharmacodynamic responses that the potencies of DHEA in rats by the percutaneous and oral routes were approximately 33% and 3%, respectively.