Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

Figures & data

Figure 1.  Metabolic pathways for the biosynthesis of DHT in the prostate. The steps that do not require the presence of T are indicated in bold lines.

Table 1.  DHT and 5α-dione production from different concentrations of labeled T and 4-dione by human prostate homogenates. Effect of increasing concentrations of unlabeled DHT and 5α-dione on the prostatic 5α-reductase activity.

Substrate	Concentration of the substrate (nM)					Vmax	Km nM (pmol/mg of protein/h)
1. Labeled testosterone	2	55	108	148	203
Production of labeled DHT (pmol/mg of protein/h)	0.35 ± 0.06	21.7 ± 7.2	48.9 ± 14.1	88.2 ± 14.1	122 ± 19.5	96.29	59.6
2. 2 nM of labeled T plus increasing concentrations of DHT
Production of labeled DHT (pmol/mg of protein/h)	0.57 ± 0.23	39 ± 12.32	90.3 ± 30.7	153.3 ± 57.7	194.6 ± 70.79	89.2	103.9
3. Labeled 4-dione
Production of labeled 5α-dione (pmol/mg of protein/h)	0.075 ± 0.02	5.96 ± 0.20	9.9 ± 1.3	30.6 ± 9.2	40.3 ± 6.2	176	26.10
4. 2 nM of labeled 4-dione plus increasing concentrations of 5α-dione
Production of labeled 5α-dione (pmol/mg of protein/h)	0.76 ± 0.06	21.4 ± 4.2	45.2 ± 9.1	48.1 ± 10.1	52.1 ± 2.3	58.9	67.5

Table 2.  T and DHT production from different concentrations of labeled 4-dione by human prostate homogenates. Effect of increasing concentrations of unlabeled 5α-dione.

SUBSTRATE	Concentration of the substrate (nM)					Vmax	Km nM (pmol/mg of protein/h)
1. Labeled 4- dione	2	55	108	148	203
Production of labeled T (pmol/mg of protein/h)	0.07 ± 0.01	1.01 ± 0.025	2.35 ± 0.01	4.03 ± 0.35	4.03 ± 0.2	331	9.7
Production of labeled DHT (pmol/mg of protein/h)	0.095 ± 0.06	7.025 ± 0.95	14.14 ± 10.4	25.77 ± 16.35	28.0 ± 11.79	86.45	18
2. 2 nM of labeled 4-dione plus increasing concentrations of 5α-dione
Production of labeled T (pmol/mg of protein/h)	0.01 ± 0.001	0.67 ± 0.02	0.69 ± 0.06	2.01 ± 0.4	2.1	188.3	1.9
Production of labeled DHT (pmol/mg of protein/h)	0.067 ± 0.08	8.6 ± 0.02	15.2 ± 5.6	22.8 ± 0.09	33.6 ± 2.5	69	19.2

Figure 2.  Enzymatic efficiency (V_max/Km) was obtained from the rate between maximal velocities (V_max) of DHT or 5α-dione produced, using different substrates and the Michaelis constants (Km). V_max/Km was higher when [³H]T+DHT were used as substrate than when T was used as a substrate. Furthermore, V_max/Km was higher also when [³H]4-dione+5α-dione were used as substrate than when 4-dione was used. However, human prostate 5α-reductase enzyme converts 4-dione to 5α-dione in a similar manner as compared to T to DHT.

Table 3.  Effect of different compounds as inhibitors of the activity of 5α-reductase.

Steroid structure	IC50 (nM)
Finasteride	1.3 ± 0.4
4	500 ± 30
5	2 ± 0.6
6	NA
7a	63 ± 1.2
7b	1.6 ± 0.4
8a	73 ± 9
8b	50 ± 6

5α-reductase was obtained from the pellet fraction of homogenates of human prostate centrifuged at 1500g. The homogenates were incubated with labeled 4-dione plus 125 nM of unlabeled 5α-dione and increasing concentration of the synthesized compounds.

The results show the concentration of the steroids necessary for inhibiting 50% of the activity of 4-dione 5α-reductase (IC₅₀). NA, non-active compound.

Figure 3.  Inhibition plots using different concentrations of the tested steroids against the percentage of activity of 4-dione 5α-reductase. These plots were used for the determination of the concentrations of finasteride as well as the progesterone derivatives 4, 5, 6, 7a, 7b, 8a and 8b required for inhibiting 4-dione 5α-reductase activity by 50%.