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Mechanisms of antiestrogen resistance in breast cancer

Abstrict

The present invention involves a method for early detection of developing tamoxifen resistance in breast cancer. Tamoxifen is the drug of choice for hormonal therapy of a first recurrence of breast cancer, but its use is always associated eventually with emergence of resistant tumors. Whereas initial treatment is usually followed by tumor regression, resistant tumors may actually resume growth under continued tamoxifen treatment. Because such growth may actually be augmented by the tamoxifen, it is essential to identify the onset of resistance as early as possible so alternative therapy may be promptly instituted.

Claims

What is claimed is:

1. A method for detecting in vivo development of tamoxifen-resistant breast tumors comprising:

obtaining ratios of cis-4-hydroxy-tamosifen concentration to trans-4-hydroxy-tamoxifen concentration in tissue samples from a breast cancer patient being subjected to a course of tamoxifen treatment; and

following said ratios during the course of tamoxifen treatment to determine onset of treatment resistance as characterized by an increase in the ratio of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen concentration.

2. The method according to claim 1 wherein said tissue is breast tumor.

3. The method according to claim 1 wherein said tissue is blood.

4. A method for detecting in vivo development of tamoxifen-resistant breast tumors comprising:

obtaining breast tumor tamoxifen concentration in a patient during a course of tamoxifen treatment; and

following said concentration during the course of tamoxifen treatment to determine onset of treatment resistance as characterized by a decrease in said tamoxifen concentration.

5. A method for detecting in vivo development of tamoxifen-resistant breast tumors comprising:

obtaining breast tumor tamoxifen concentrations and tissue ratios of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen concentration in a patient being subjected to a course of tamoxifen treatment; and

following said tamoxifen concentrations and ratios during the course of tamoxifen treatment to determine onset of treatment resistance as characterized by a decrease in tamoxifen concentration and an increase in the ratio of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen concentration.

6. The method according to claim 5 wherein said tissue is breast tumor.

7. The method according to claim 5 wherein said tissue is blood.

8. A method for detecting in vivo development of resistance in breast tumors to treatment by a triphenylethylene estrogen comprising:

obtaining concentration ratios tissue of cis to trans forms of triphenylethylene antiestrogen in a patient being subjected to a course of treatment with a triphenylethylene antiestrogen; and

following said ratios during the course of treatment to determine onset of treatment resistance as characterized by an increase in the ratio of concentration of cis to trans forms of said triphenylethylene antiestrogen.

9. The method according to claim 8 wherein said tissue is breast tumor.

10. The method according to claim 8 wherein said tissue is blood.

11. The method according to claim 1, 5 or 8 wherein said obtaining step comprises photoactivation of a tissue sample extract.

12. The method of claim 11 wherein the photoactivation involves irradiation with ultraviolet light.

13. The method of claim 1, 5 or 8 wherein the obtaining step comprises high performance liquid chromatography.

14. A method for detecting in vivo development of tamoxifen-resistant breast tumors in a breast cancer patient being subjected to a course of tamoxifen treatment, the method comprising:

obtaining tissue ratios of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen concentration in said patient; and

following said ratios during the course of tamoxifen treatment to determine onset of treatment resistance as characterized by an increase in the ratio of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen concentration.

Description

BACKGROUND OF THE INVENTION

The present invention relates to methods for monitoring the effectiveness of tamoxifen (2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine) therapy in the treatment of breast cancer, and more particularly to methods of detecting the emergence of tamoxifen-resistant tumors and the resulting treatment resistance by means of an analytical assay for antiestrogenic and other forms of tamoxifen and its metabolites.

Breast cancer is the most common form of malignant disease among women of the Western world, and it is the most common cause of death among those between 40 and 45 years of age. This disease will develop in about six to seven percent of women in the United States, and at the present time about one half of this group can be cured. The treatment of breast cancer involves surgery, radiation, chemotherapeutics and hormonal therapy, the last category including consideration of antiestrogens for treatment of endocrine-responsive tumors.

Tumors sensitive to estrogen stimulation may regress following competitive inhibition of estrogen receptors by tamoxifen (an antiestrogen), and response is currently predicted based on the stage of disease and on the basis of assays for estrogen receptors (ER) and progesterone receptors (PR) in the tissue. Many breast cancers and all normal estrogen-responsive tissues contain these labile cytoplasmic proteins which bind estrogen and progesterone. Patients with positive assays for these proteins have an objective response to hormone therapy of about 65%, while those with negative assays have an objective response rate of <10%. For postmenopausal women having a first recurrence of breast cancer with an ER+ or PR+ assay, tamoxifen therapy is the treatment of choice.

Notwithstanding strong interest in the use of antiestrogens in breast cancer treatment, however, incomplete knowledge of their basic pharmacology persists. Substituted triphenylethylenes (including tamoxifen) have antiestrogen effects which appear to be dependent on geometric isomerism. For example, trans-tamoxifen (the isomer used in tamoxifen therapy) is an antiestrogen, whereas the cis isomer is a weak estrogen. The present invention makes use of newly acquired knowledge about in vivo interconversion of geometric isomers to improve treatment of breast cancer.

Although tamoxifen is the most widely used antiestrogen for treating breast cancer, development of tamoxifen resistance and subsequent tumor progression during tamoxifen therapy represents a major reason for treatment failures. The mechanism of tamoxifen resistance has been unknown, but an estrogenic metabolite of tamoxifen which would promote growth in ER+ tamoxifen-resistant tumors has been identified.

SUMMARY OF THE INVENTION

The present invention relates to the use of tamoxifen metabolites as early indicators of tamoxifen resistance (prior to clinical treatment failure). Tamoxifen is representative of a group of compounds called triphenylethylene antiestrogens, whose effect is to slow or stop the growth of estrogen-dependent tumors. While it is the most commonly used drug for treatment of breast cancer today, tamoxifen is associated with the development of drug resistance in virtually all patients who take it. With the onset of resistance, tumor growth resumes or accelerates and tamoxifen therapy should be discontinued. Renewed tumor growth will eventually become obvious, of course, but an objective of the present invention is to provide early warning of tamoxifen resistance so that therapy can be changed promptly as needed.

The mechanism of tamoxifen resistance is unknown, but it is known that tamoxifen exists as two geometric isomers, the trans form (an antiestrogen) and the cis form (a weak estrogen). The trans form, of course, is the therapeutic drug for breast cancer. Further, however, it has been shown that there can be interconversion from one isomer to the other in tissue culture cells and also, presumably, in patients (though this has not been demonstrated).

The model with which the present invention was developed consists of human breast cancer cells growing subcutaneously in athymic nude mice. Treatment of the mice with tamoxifen results in tumor growth inhibition for four to six months, followed by the onset of tumor resistance and regrowth (much as in humans). Further, tumor regrowth is actually stimulated by tamoxifen. Even when transplanted into different mice, the resistant tumors will not grow unless stimulated by tamoxifen or estrogen. Tamoxifen, in these cases, appears to mimic the action of estrogen.

One of the possible mechanisms for development of tamoxifen resistance is hypothesized to be conversion over time of both tamoxifen and its metabolites from trans (antiestrogen) forms to cis forms which have estrogen-like activity. Assays for the presence of both trans and cis forms can be accomplished with a high performance liquid chromatography (HPLC) system. In three separate blinded experiments using such a system, it has been shown that:

1) tumors from tamoxifen-resistant mice have significantly lower concentrations of tamoxifen than sensitive tumors (whose growth is still arrested); and

2) there is an increase in the cis-4-hydroxytamoxifen/trans-4-hydroxytamoxifen (C/T-OH-TAM) ratio in resistant tumors.

Actual values of the C/T-OH-TAM ratio are about 0.4-0.5 in sensitive tumors and 0.8-0.9 in resistant tumors. While it can not be confirmed that the increased estrogen effect resulting from an elevated C/T-OH-TAM ratio is the reason for development of resistance, the association appears to be consistent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC tracings of 400 ng/ml stock solutions of trans and cis 4-hydroxytamoxifen (4-OH-TAM), respectively.

FIG. 2 shows HPLC chromatographs indicating that the cis/trans ratio of 4-OH-TAM is lower in sensitive than in resistant tumors isolated from athymic nude mice.

FIG. 3 shows the approximately linear inverse relationship which exists between the logarithm of total tumor tamoxifen concentration and the cis/trans 4-OH-TAM ratio (N=32,R=0.83, linear regression analysis).

FIG. 4 shows a scatter plot of tamoxifen concentrations in sensitive and resistant MCF-7 tumors (N=32).

FIG. 5 shows a scatter plot of 4-OH-TAM ratios (cis/trans) in sensitive and resistant MCF-7 tumors (N=32).

FIG. 6 shows a scatter plot of tamoxifen versus cis/trans 4-OH-TAM ratios in resistant (open symbols) and sensitive (solid symbols) tumors.

FIG. 7 shows HPLC chromatographs of a resistant tumor analyzed for total tumor tamoxifen, cytosol tamoxifen, and tamoxifen found in nuclear pellets extracted with KC1 (from top to bottom, respectively).

FIG. 8 shows example HPLC chromatographs demonstrating cis >trans concentrations of 4-OH-TAM extracted from tumor nuclear pellets representing patients with clinically-evident tumor resistance. These findings must be contrasted with analogous measurements for tamoxifen-sensitive tumors in the mouse model (showing trans >cis 4-OH-TAM) because sensitive tumors in human patients are not biopsied and the information is not available.

FIG. 9 shows example HPLC chromatographs demonstrating detection of tamoxifen, des-tamoxifen, and cis and trans 4-OHTAM following treatment with 20 mg tamoxifen per day. These measurements demonstrate the capability to measure tamoxifen and its metabolites in the serum.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Those skilled in the art will recognize that changes analogous to those detected in the C/T-OH-TAM ratio might occur in cis-tamoxifen or other (monophenol and bisphenol) tamoxifen metabolites or in any of the triphenylethylene antiestrogens related to tamoxifen. Nevertheless, the best mode of implementing the present invention now appears to be in conjunction with treatment of estrogen-responsive breast cancer with tamoxifen and detection of the onset of tamoxifen resistance.

Those skilled in the art will also recognize that if geometric isomerization or metabolism to other estrogenic metabolites proves to be the mechanism of antiestrogen resistance, synthesis of antiestrogens which preclude isomerization may be a fruitful path to improved treatment. At the present time, however, tamoxifen is the drug of choice for hormonal treatment of recurrent breast cancer, and its invariable association with the onset of tumor resistance and tamoxifen-enhanced tumor growth makes the present invention a valuable aid to therapy.

Claims are directed to the two main indicators which have been discovered to be closely associated with the onset of tumor tamoxifen resistance. Elevation of the C/T ratio for 4-OH-TAM in tumor or blood, and depression of the tamoxifen concentration in tumor are both shown by experimental data to be useful predictors of recrudescence of tumor growth despite (and perhaps in-part because of) tamoxifen treatment. Either indicator can be used alone, or they may be used together.

For each indicator, small changes (about a few percent) in the direction indicated by this specification and claims are believed to indicate the early stages of tumor resistance. Larger indicator changes (up to about 80 to 100 percent) can be expected when tumor resistance is well established. Examples of in vitro and in vivo tests are provided to illustrate the specific utility of the claimed invention and to support both the approximate magnitude of indicator changes and the extrapolations suggested in this specification.

These examples are presented to describe preferred embodiments and utilities of the present invention and are not meant to limit the present invention unless otherwise stated in the claims appended hereto. Taken together, the examples illustrate the best mode of implementing the invention as it is currently understood.

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