CR1447 in Endocrine Responsive-HER2neg and TN-ARpos Breast Cancer

Breast cancer is the most frequently diagnosed cancer and a heterogeneous disease and tumor categorization based on molecular characteristics beyond the currently used markers such as the estrogen receptor (ER), progesterone receptor (PR), and HER2, makes tumor categorization and treatment a demanding task.

The treatment and prevention of relapses of breast cancer is mainly based on I) surgery of primary tumors, II) radiotherapy, III) chemotherapy, IV) endocrine therapy, and V) specific interventions. Although there have been advances in breast cancer therapy with three out of four women with breast cancer now being cured, one out of four patients will relapse or present with metastatic disease at the time of first diagnosis and hence be incurable and die of their disease.

The endocrine responsive breast cancer is defined by the immunohistochemical detection of the ER (estrogen receptor) and/or the PR (progesterone receptor) on at least 1% of tumor cells on the tissue sample to a variable degree and offers the possibility of treatment using endocrine strategies in about 80% of patients. Endocrine responsive breast cancers may also express HER2. The triple negative breast cancer (TNBC) is defined by the lack of expression of ER, PR, and HER2. The analysis is made on the pathological tumor tissue sample. While for ER and PR, the threshold is less than 1% of tumor cells expressing both receptors, HER2 negativity is defined as immunohistochemical scores of 0/1+ or tumors with scores of 0/1+ or 2+ that are lacking HER2 gene amplification after in situ hybridization. Approximately 15% of all breast cancers are found to be triple-negative. However, due to the biologically more aggressive behavior of these tumors, the patient group suffering from this type of disease are overrepresented within the first five years after initial diagnosis.

Recently, androgen receptor (AR) targeted therapies gained new interest due to the fact that the AR is the most abundantly expressed sex hormone receptor in breast cancer (approx. 70% of all breast tumors) and, importantly, in about 20% (range 10% to 50% depending on the respective reference) of triple-negative breast tumors. The threshold for ARpos breast cancer is that >0% of tumor cells expresses the AR. In addition, a significant association was found between AR expression and longer overall survival of breast cancer patients. Androgens can induce proliferative changes in breast cancer cell lines, and testosterone acts as a tumor promoter in several animal models of breast cancer.

Endocrine responsive breast cancer Anti-estrogen therapy includes ovariectomy, to remove the primary source of female sex hormone in premenopausal women, and the application of luteinizing hormone releasing hormone (LHRH) agonists, to decrease the release of LH and prevent gonadal estrogen synthesis. These therapies induce a postmenopausal status. The hormonal treatment of choice for premenopausal women with ER positive metastatic breast tumors is the use of ER antagonists such as tamoxifen. Tamoxifen and aromatase inhibitors, preventing the final conversion of androgens to estrogens, are the main endocrine treatment options for postmenopausal women with metastatic breast cancer. The persisting estrogen levels in postmenopausal women are mainly due to aromatase activity in extragonadal sources such as breast tissue, skeletal muscle and adipose tissue. Thus, both ER blockade by tamoxifen and inhibition of estrogen synthesis inhibit the growth signal for breast cancer cells expressing ER.

Aromatase, the key enzyme of estrogen production in women, is expressed in and around breast tumors, in ovaries, and in skeletal muscle and adipose tissue in postmenopausal women. Several aromatase inhibitors (AI) have been developed. The first generation aromatase inhibitors include testolactone and aminogluthetimide that were replaced by the more potent second generation inhibitors fadrazole and formestane (4-hydroxy-4-androstenedione, 4-OHA), and later on by the even more potent third generation inhibitors exemestane, letrozole and anastrozole. 4OHA and exemestane are steroidal AI and irreversibly block aromatase while the non-steroidal AI letrozole and anastrozole are reversible inhibitors. Importantly, there seems to exist no cross-resistance between the two aromatase inhibitor classes, and 4-OHA or exemestane may still show efficacy after relapse upon letrozole/anastrozole treatment.

In summary, there is a great demand for novel and improved therapeutic strategies, especially to overcome resistance to chemotherapy or presently available endocrine therapy in postmenopausal women.

While patients with ER and/or PR positive tumors can be treated with endocrine therapies and patients with HER2pos disease with treatments directed against HER2 such as trastuzumab, lapatinib, and trastuzumab/pertuzumab combination therapy, no endocrine or targeted anti-HER2 directed therapy exists for patients suffering from triple negative disease.

For these patients, only chemotherapy is a therapeutic option. Unfortunately, these tumors tend to be aggressive and quickly growing with a high relapse rate even after intensive adjuvant chemotherapy. Due to the absence of estrogen receptors, endocrine therapy based on anti-estrogen intervention strategies must fail.

There are currently no suitable treatment options for patients with metastatic triple-negative breast tumors whose disease has failed to respond to chemotherapy. Thus, there is a great demand for novel and improved therapeutic strategies in this patient group and in all patients with acquired resistance to combat breast cancer.

Testosterone was already used extensively between the 1930s and 1960s for breast cancer treatment, with anecdotal tumor responses (especially bone metastases) seen in up to 20% of treated women. Side effects such as hirsutism and aggressive behavior as signs of virilization in treated women and the evidence that testosterone may be easily be converted to estrogens in the body led to the discontinuation of its use.

CR1447 (4-hydroxytestosterone [4-OHT]) is a steroidal small molecule which has two distinct properties, acting as a steroidal aromatase inhibitor (AI) and binding to the AR with high affinity (IC50 4.4 nM). In vitro studies indicate that human breast cancer cell lines are inhibited by CR1447 in their growth if they express the androgen receptor, while knock out of the AR abolishes this effect. A significant proportion of 4-OHT is converted to 4-OHA (4-hydroxyandrostenedione, formestane). In vivo 4-OHT and 4-OHA, form a redox system. 4-OHA is the 17-beta-oxidized isoform of CR1447. 4-OHA had previously been approved as an aromatase inhibitor for the treatment of BC via intramuscular (i.m.) injection (Formestane, Lentaron®) injection, but was discontinued by Novartis due to the development of oral aromatase inhibitors. Unlike testosterone, both compounds, 4-OHA and 4-OHT, are not converted to estrogens in vivo. Due to a high first pass effect after oral dosing, 4-OHA had to be administered parentally e.g. as an intramuscular injection. While this regimen was clinically effective, local side effects were a dose limiting issue.

A clinical feasibility study of transdermally applied 4-OHA showed that significant amounts of 4-OHA are absorbed by the skin. Intramuscular and transdermally (twice daily) applied 4-OHA achieved comparable the same plasma levels from day 3 on.