Aromatase

The pathway of E biosynthesis is summarized in Figure 1. The first step of cholesterol side-chain cleavage is rate-limiting but the last step catalyzed by aromatase is unique to E biosynthesis. Aromatase removes the methyl group between the steroid A and B rings, and the A ring becomes aromatic. NADPH is required as a cofactor, being generated by a NADP reductase and a transfer of electrons via a specific CYP 19 p450 arom (9).

C27 Sterol (Cholesterol) | Choiesteral desmolase

C19 Steroid (Androgens)

| Aromatas

Cis Steroid (Estrogens)

biosynthesis from cholesterol

E production may be reduced by inhibiting any step in the biosynthetic pathway but blockade of aromatase (the last step in the sequence) is less likely to affect other steroid classes. Consequently, there has been an impetus to develop selective and potent inhibitors of the aromatase enzyme.

Aromatase inhibitors can be subdivided into steroidal (Type I) and nonsteroidal (Type II) agents (10) (Figure 2). Type I agents are androgen analogues that bind to the enzyme, competing with the natural substrate. Inhibitors such as formestane (11) and exemestane (12) are thought to be metabolized by the aromatase enzyme into reactive intermediates resulting in tight or irreversible binding. Because the enzyme is inhibited as an outcome of its own metabolism, the drugs have been termed "suicide inhibitors" (13) or "inactivators". A consequence of such "inactivation" is that the inhibition is usually specific and long-term. Unlike other reversible agents, continued presence of drug is not required for inhibition (14), and duration of inhibitory effects is dependent upon de novo synthesis of aromatase.

The development of Type II inhibitors followed the recognition that the anti-epileptic drug, aminoglutethimide, inhibited aromatase (15). Aminoglutethimide lacks specificity but newer second and third generation non-steroidal aromatase inhibitors have greater selectivity and efficacy and less toxicity. The lead third generation type II inhibitors are anastrozole (16) and letrozole (17). Both are triazoles that bind reversibly to the aromatase enzyme, interacting with the heme iron in the cytochrome p450 and occluding the substrate binding site (18).

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Figure 1. Classical pathway of E Aromatase Inhibitors

Steroidal - Type I Androgen Substrate

CH; OH Formestan« And rosten edkine

Non-steroidal — Type II

Anastrozole

Figure 2. Different classes of aromatase inhibitors. Type I, steroidal inhibitors are androgen analogues. Type II, non-steroidal inhibitors, i.e., aminoglutethimide, letrozole, and anastrozole are azoles.

Potency has been tested in a variety of model systems (19) including placental microsomes, particulate fractions of BC and whole-cell systems such as cultured fibroblasts. The effects of anti-aromatase agents in these three types oftest systems are summarized in Table 1. Aminoglutethimide, anastrozole, letrozole, formestane, and exemestane all inhibit aromatase activity in each ofthe systems, but the newer agents are orders of magnitude more potent than aminoglutethimide. Thus, whereas concentrations are required for aminoglutethimide, only nanomolar concentrations are needed with letrozole, anastrozole, exemestane, and formestane. In general, the newer Type II inhibitors (anastrozole and letrozole) are more potent than Type I inhibitors (formestane and exemestane). Amongst Type II agents, letrozole appears most effective. Letrozole and exemestane are more active in whole-cell cultures than in disrupted-cell preparations (20). The newer anti-aromatase agents also have great specificity. For example, exemestane only affects other steroid hydroxylases at concentrations that are at least 2,500 times higher than those affecting aromatase.

Table 1. Inhibition of Aromatase Activity in Whole- and Disrupted-cell Preparations.

Mammary

Placental Breast Cancer Fibroblast Microsomes Homogenates Cultures

Mammary

Placental Breast Cancer Fibroblast Microsomes Homogenates Cultures

ic50

Relative

fC,o

Relative

ic50

Relative

(nM)

Potency

(nM)

Potency

(nM)

Potency

Aminoglutethimide

3,000

1

4500

1

8,000

1

Anastrozole

12

250

10

450

14

570

Letrozole

12

250

2.5

1,800

0.8

10,000

Formestane

50

60

30

150

45

180

Exemestane

50

60

15

300

5

1,600

0 0

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