WO2011072244A1

WO2011072244A1 - Method of treatment of breast cancer with tamoxifen

Info

Publication number: WO2011072244A1
Application number: PCT/US2010/059927
Authority: WO
Inventors: Robert J. Desnick; Myra F. Barginear; George Raptis; Chunli Yu; Malgorzata Jaremko
Original assignee: Mount Sinai School Of Medicine Of New York University
Priority date: 2009-12-10
Filing date: 2010-12-10
Publication date: 2011-06-16

Abstract

The present invention encompasses methods of optimizing the dose of tamoxifen in the prevention and treatment of breast cancer based upon tamoxifen uptake, metabolism and excretion. A primary use for such methods is in guiding therapeutic decisions for individual cancer patients.

Description

METHOD OF TREATMENT OF BREAST CANCER WITH TAMOXIFEN

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Application Ser. No. 61/285,533 filed December 10, 2009, the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Breast cancer is the most common non-cutaneous cancer in women in the Western world; the lifetime risk of developing invasive breast cancer in the United States is 12.6 percent (one in eight women). Jemal et al. (2008) CA Cancer J Clin 58:71-96. Two-thirds of breast cancer patients are estrogen receptor (ER)-positive and candidates for tamoxifen therapy, the drug used most worldwide for the prevention and treatment of hormone receptor- positive breast cancer. Aromatase inhibitors have become the first choice for adjuvant therapy in post-menopausal women with ER-positive breast cancer. Yet, the continued importance of tamoxifen is measured by the fact that it is the only hormonal agent approved by the United States Food and Drug Administration (FDA) for the prevention of breast cancer, the treatment of ductal carcinoma in situ, and the treatment of pre-menopausal breast cancer. Fisher et al.(2005) J Natl Cancer Inst 97: 1653-62; Fisher et al. (2002) J Clin Oncol 20:4141-9; Colleoni et al. 2006 J Clin Oncol 24: 1332-41. Tamoxifen is also the hormonal agent most commonly employed in the treatment of early and advanced male breast cancer. Fentiman et al. (2006) Lancet 367:595-604. The recommended and uniformly prescribed dose of tamoxifen is 20 mg daily.

Tamoxifen almost halves the risk of recurrence and reduces the mortality rate by one- third in women with ER-positive breast cancer. Early Breast Cancer Trialists' Collaborative Group (2005) Lancet 365: 1687-1717. Nevertheless, the efficacy of tamoxifen varies widely. Despite initial successful responses, 50% of patients on adjuvant tamoxifen therapy experience relapse and subsequently die of the disease. Early Breast Cancer Trialists' Collaborative Group (2005) Lancet 365: 1687-1717; Early Breast Cancer Trialists'

Collaborative Group (1998) Lancet 351 : 1451-1467.

NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV

Tamoxifen (Z-l-(p-Dimethylaminoethoxyphenyl)-l,2-diphenyl-l-butene) is a prodrug that is converted to its therapeutically active metabolites, E-, Z- and Z' -4-hydroxy-N- desmethyltamoxifen (endoxifen) isomers and E-, Z- and Z' -4-hydroxy-tamoxifen (4-OH- Tam) isomers. Over 90% of tamoxifen is demethylated by the cytochrome (CYP) P450 enzyme CYP3A4/5 to N-desmethyl-Tam, which is then hydroxylated to Z-endoxifen by CYP2D6 and to Z' -endoxifen by an unknown hepatic enzyme (Figure 1). Compared to tamoxifen itself, Z-endoxifen and Z-4-OH-Tam both have an approximately 100-fold greater affinity for the ER and a 30- to 100-fold greater potency in suppressing estrogen-dependent cell proliferation. Jordan (1982) Breast Cancer Res Treat 2: 123-138; Lim et al (2005) Cancer Chemother Pharmacol 55:471-478. Compared to tamoxifen, the anti-estrogenic activities of the Z isomers of endoxifen and 4-OH-Tam are equivalent, while the Z'~ endoxifen and Z' -4-OH-Tam are estimated to have about 10% of their Z isomer activity based upon in vitro studies. The anti-estrogenic effects of endoxifen and 4-OH-Tam and presumably their Z and Z' isomers are suppression of ER-dependent proliferation of breast cancer cells, modulation of ER-mediated global gene expression, and ER degradation Lim et al. (2006) J Pharmacol Exp Ther 318:503-512. In vitro studies have shown that endoxifen's anti-estrogenic effects are concentration-dependent; Z-endoxifen levels of 40 nM or greater prevent ER-mediated transcriptional activation and optimally inhibit ER-induced breast cancer cell proliferation. Wu et al. (2009) Cancer Res 69: 1722-1727.

The CYP2D6 gene is highly polymorphic, with more than 100 alleles having been identified to date (htt ://www .cypalleles .ki.se; Algeciras-Schimnich et al., 2008, Clin. Lab. Med. 28:553-567). For example, normal functional alleles include CYP2D6*1 , *2 and *35. Homozygous normal individuals are classified phenotypically as extensive metabolizers (EM). Duplicated normal alleles are classified as ultra-rapid metabolizers (UM). Inactive alleles include CYP2D6*3-*8, * 11-* 16, *18-*21, *31, *38, *40, *42 and *44. Individuals having a genotype with no active alleles have a poor metabolizer (PM) phenotype. Reduced function alleles include CYP2D6*9, *10, *17, *29, *41 and *69. Individuals having genotypes with one active and one inactive allele, or one inactive and one reduced function allele, or two reduced function alleles are classified phenotypically as intermediate metabolizers (IM), although those with one inactive and one reduced function allele also may be considered poor metabolizers (PM).

An individual's CYP2D6 alleles predict his or her metabolizer phenotype activity (MP A) score. Gaedigk et al. (20080 Clin Pharmacol Ther 83:234-242. According to this

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV system each CYP2D6 allele is assigned a value that reflects its expected enzymatic activity. Fully functional CYP2D6 alleles (*1, *2, *2A) have a score of 1, and duplication of any of these alleles are given a score of 2.0. Alleles associated with reduced enzyme activity (*9, *10, *17 and *41) are scored as 0.5, and CYP2D6 null alleles (*3-*8 ,*11, *12, *14 and *15) and their duplications are scored a 0. Thus, the MPA score for and individual is the sum of their scores for both CYP2D6 alleles, which ranges between 0 and 3.0. The MPA score related to the traditional metabolic phenotype classification as follows: UM have an MPA score >2.0, EM have a score of 1.5-2.0, IM have a score of 0.5-1.0, and PM have a score of 0.

Tamoxifen efficacy and clinical outcome are associated with CYP2D6

polymorphisms, and plasma concentrations of the active metabolites of tamoxifen are linked with metabolizer status and clinical outcome. Brauch et al. (2009) Clinical Chemistry 55:1770-1782. The presence of nonfunctional or reduced function alleles is associated with worse outcomes. For example, Schroth et al. report a study in which patients lacking CYP2D6 function (PM) and treated with tamoxifen had an almost two-fold increased risk of recurrence compared with patients having two functional alleles (EM), and conclude that alternatives to tamoxifen should be considered for patients whose genotype is indicative of the PM phenotype. Schroth et al. (2009) JAMA 302:1429-1436. Similarly, Goetz et al. report a study in which CYP2D6 metabolism status was predictive of outcome in women treated with tamoxifen for breast cancer, and also conclude that alternative therapy should be considered for patients having PM and IM status. Goetz et al. (2008) Clin Pharmacol Ther 83:160-16.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for the prevention and treatment of breast cancer with tamoxifen comprising the steps of determining the patient' s genotypic profile for one or more genes predictive of tamoxifen activity, and administering to the patient a dosage of tamoxifen based on the patient' s profile.

In another embodiment, the present invention provides a method for treating a patient with tamoxifen, comprising the steps of: determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity; administering tamoxifen to the patient at an initial dosage based on the patient's genotypic profile; performing a measurement of steady state plasma levels of one or more active metabolites of tamoxifen; and administering tamoxifen at an optimized dosage based on levels of active metabolites.

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In another embodiment, the present invention provides a method for treating a patient with tamoxifen, comprising the steps of: determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity; administering tamoxifen to the patient at an initial dosage based on the patient's genotypic profile; performing a first measurement of steady state plasma levels of one or more active metabolites of tamoxifen; administering tamoxifen at a first optimized dosage based on levels of active metabolites in the first measurement; performing one or more subsequent measurements of steady state plasma levels of one or more active metabolites; and administering tamoxifen at an optimized dosage based upon the subsequent measurements of levels of active metabolites.

In another embodiment, the present invention provides a method of optimizing treatment of breast cancer in a patient in need thereof comprising administering tamoxifen to the patient at an initial dosage of 20 mg/day, obtaining a sample of blood from the patient, measuring steady state plasma levels of one or more active tamoxifen metabolites in the sample, and administering an optimized dosage of tamoxifen to the patient based upon the level of one or more active tamoxifen metabolites.

In yet another embodiment, tamoxifen dosage may be optimized using an Estrogen Receptor Activity Score based on the plasma levels of tamoxifen and its active metabolite isomers, and their relative anti-estrogenic activities.

Other embodiments are apparent from the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the pathways for conversion of tamoxifen to its therapeutically active metabolites. Part A depicts the pathway in CYP2D6 extensive and ultra-rapid metabolizers and part B depicts the pathway in CYP2D6 poor metabolizers.

Figures 2A-E show 60-day changes in plasma tamoxifen concentration of the women who received higher tamoxifen doses. A, Endoxifen; B, 4-OH-Tamoxifen; C, Z-Endoxifen, and D, Z'-Endoxifen. E. ER activity scores. P- values indicate the effect of time and metabolic phenotype activity (MP A) score on the rate of change based on mixed models.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly discovered in accordance with the present invention that tamoxifen therapy can be optimized in breast cancer patients whose pharmacogenetic profile

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV is predictive of poor outcome when treated with the standard 20 mg/day dosage of tamoxifen. In particular, in one aspect of the present invention it has been determined that therapeutic levels of tamoxifen's active metabolites can be achieved in breast cancer patients

characterized as IM or PM of tamoxifen as determined by pharmacogenetic profiles. A therapeutic level of tamoxifen' s active metabolites is defined herein as a plasma endoxifen level of greater than or equal to about 40 nM or combined 4-OH-TAM and endoxifen levels of greater than or equal to about 50 nM.

In patients receiving the standard dosage of 20 mg/day of tamoxifen, the presence of genetic variants may alter uptake, metabolism, binding and excretion relative to wild-type such that therapeutic levels of tamoxifen's active metabolites are not achieved. For example, after treatment with the standard 20 mg/day of tamoxifen, levels of endoxifen may be less than the accepted therapeutic level of greater than or equal to about 40 nM. It has been discovered in accordance with one aspect of the present invention that therapeutic levels of endoxifen can be achieved in patients having genetic variants associated with altered tamoxifen metabolism by increasing the standard dosage of tamoxifen to more than 20 mg/day, for example about 21 to about 50 mg/day, or about 25 to about 45 mg/day, or about 30 mg/day, or about 40 mg/day, or more preferably 30 mg/day or 40 mg/day. The term tamoxifen as used in the present methods is defined as tamoxifen or its pharmaceutically acceptable salts such as tamoxifen citrate. The terms endoxifen and 4-OH-Tam include the Z and Z' isomers thereof.

It has further been discovered in accordance with the present invention that some patients whose genotype includes a functional CYP2D6 allele fail to achieve therapeutic levels of tamoxifen's active metabolites when treated with the standard tamoxifen dosage of 20 mg/day. The present invention provides methods of optimizing dosage in such patients.

Accordingly, in one embodiment, the present invention provides a method for the prevention and treatment of breast cancer in a patient in need of such treatment comprising the steps of determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity, and administering to the patient a dosage of tamoxifen based on the patient's profile. The genes predictive of tamoxifen activity include the genes encoding CYP2D6, CYP3A5, CYP3A4, CYP2C19, UGT2B7, UGT2B15, UGT1A4 and SULTIAI. In a preferred embodiment, genotypic profile is determined for one gene and the gene encodes CYP2D6. If the patient's genotype includes at least one null CYP2D6 allele, for example

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV

CYP2D6*3-*8, *11, *12, *14 or *15, or at least one reduced function allele, for example CYP2D6*9, *10 , *17 or *41, and does not include a functional CYP2D6 allele, for example CYP2D6*1, *2, or *2A, then the recommended initial dosage of tamoxifen is more than 20 mg/day. In preferred embodiments, the dosage of tamoxifen is from 21 to 50 mg/day, or from 25 to 45 mg/day. In other preferred embodiments, the dosage of tamoxifen is 30 mg/day or 40 mg/day. If the patient' s genotype includes at least one functional CYP2D6 allele, the dosage of tamoxifen is preferably 20 mg/day.

The CYP allele nomenclature used herein is the art-recognized nomenclature of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee.

The patient's genotypic profile can be determined by obtaining a nucleic acid- containing sample from the patient and performing genotyping methods known in the art. The nucleic acid-containing sample may be, for example, whole blood, saliva, buccal cells, skin, hair, biopsies, and other nucleic acid-containing samples. In a preferred embodiment, the nucleic acid-containing sample is whole blood. Genotyping methods are known in the art and include, for example, multiplex allele specific primer extension and hybridization of extended primers to a solid support. Genotyping methods are disclosed, for example, by de Leon et al. (2006) Mol Diagn Ther 10:135-151 and in U.S. Patent Application Publications 2009/0215637 and 2010/0105041, the disclosures of which are incorporated herein by reference in their entireties. Kits for genotyping are also commercially available.

In another embodiment, the present invention provides a method for treating a patient with tamoxifen comprising the steps of: determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity; administering tamoxifen to the patient at an initial dosage based on the patient's genotypic profile; performing a measurement of steady state plasma levels of one or more active metabolites of tamoxifen; and administering tamoxifen at an optimized dosage based on levels of active metabolites and their relative individual anti-estrogenic activities. The genes predictive of tamoxifen activity include the genes encoding CYP2D6, CYP3A5, CYP3A4, CYP2C19, UGT2B7, UGT2B15, UGT1A4 and SULTIAI. In a preferred embodiment, the genotypic profile is determined for one gene and the gene encodes CYP2D6. If the patient's genotype includes at least one null CYP2D6 allele, for example CYP2D6*3-*8, *11, *12, *14 or *15, or at least one reduced function allele, for example CYP2D6*9, *10 , *17 or *41, and does not include a functional CYP2D6 allele, for example CYP2D6*1, *2, or *2A, then the initial dosage of tamoxifen is more than

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20 mg/day. In preferred embodiments, the initial dosage of tamoxifen is from 21 to 50 mg/day, or from 25 to 45 mg/day. In other preferred embodiments, the initial dosage of tamoxifen is 30 mg/day or 40 mg/day. If the patient's genotype includes at least one functional CYP2D6 allele, the initial dosage of tamoxifen is preferably 20 mg/day. The patient' s genotypic profile can be determined by methods known in the art as described above.

After several days of treatment at the initial dosage, the steady state plasma levels of one or more active metabolites of tamoxifen in the patient are measured. Preferably the plasma levels are measured after at least about 30 days so that steady state levels of the metabolites have been reached. For example, the metabolite isomer levels may be measured after about 30 or more days, or after about 30 days of treatment at the initial dosage. In a preferred embodiment, the tamoxifen active metabolite isomers are Z and Z' 4-OH-TAM and Z and Z' endoxifen. The metabolites can be measured by obtaining a blood sample from the patient, and measuring the metabolite isomers by newer methods known in the art, including for example liquid chromatography/mass spectrometry. Such methods are disclosed, for example, by Jaremko et al., Anal Chem 2010 Nov. 18 [Epub ahead of print], the disclosure of which is incorporated herein by reference in its entirety. If the levels of active metabolite isomers are less than therapeutic levels as defined hereinabove, the optimized dosage of tamoxifen is greater than the initial dosage, for example 30 mg/day or 40 mg/day. If the levels of active metabolite isomers are greater than or equal to therapeutic levels as defined hereinabove, the optimized dosage of tamoxifen is the same as or less than the initial dosage. The levels of active metabolite isomers may optionally be monitored every month to annually and dosage optimized accordingly until steady state therapeutic levels of active metabolites are obtained.

The foregoing method may further comprise the steps of performing one or more subsequent measurements of steady state plasma levels of one or more active metabolite isomers, and administering tamoxifen at anoptimized dosage based upon the subsequent measurement of levels of active metabolite isomers. Preferably the plasma levels are measured after at least about 30 days after treatment at the first optimized dosage so that steady state plasma levels of the active metabolite isomers have been reached. For example, the active metabolite isomer levels may be measured after about 30 or more days, or after 30 thirty days of treatment at the initial dosage. If the levels of active metabolite isomers are greater than or equal to therapeutic levels as defined hereinabove, the subsequent optimized

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV dosage is the same as or lower than the first or previous optimized dosage. If the levels of active metabolite isomers are less than therapeutic levels as defined hereinabove, the subsequent optimized dosage of tamoxifen is greater than the first or previous optimized dosage, for example up to 40 mg/day.

In another embodiment, the present invention provides a method of optimizing treatment of breast cancer in a patient in need thereof comprising administering tamoxifen to the patient at an initial dosage of 20 mg/day, obtaining a sample of blood from the patient, measuring plasma levels of one or more active tamoxifen metabolite isomers in the sample, and administering an optimized dosage of tamoxifen to the patient based upon the level of one or more active tamoxifen metabolite isomers. If the levels of active metabolites are less than therapeutic levels as defined hereinabove, the optimized dosage of tamoxifen is greater than the initial dosage, for example 30 mg/day or 40 mg/day. If the levels of active metabolite isomers are greater than or equal to therapeutic levels as defined hereinabove, the optimized dosage of tamoxifen is the same as or less than the initial dosage.

In another embodiment, tamoxifen dosage may be optimized based upon the steady state plasma levels of tamoxifen and the isomers of its active metabolites. An anti-estrogenic activity estimate is calculated as Tam*0.01+Z-Endoxifen+Z'-Endoxifen*0.1+Z-4OH_Tam_+Z'-4OH- Tam*0.1 based on their individual anti-estrogenic activity and using steady state plasma concentrations (nM) of tamoxifen and its metabolites. An anti-estrogenic activity estimate is considered therapeutically effective in a range preferably from about 30 to 50, more

preferably from about 30 to 40, and most preferably about 30. Tamoxifen dosage may be increased if the anti-estrogenic activity estimate is below the effective range, and conversely, tamoxifen dosage may be decreased if the anti-estrogenic activity estimate is higher than the effective range.

In the foregoing methods, tamoxifen may be administered orally or parentally. Oral administration may be in solid dosage forms or liquid dosage forms. Parenteral administration forms include, for example, emulsions and aqueous solutions of liposomes containing the active ingredient. In a preferred embodiment, tamoxifen is administered orally in a solid dosage form such as a tablet.

In preferred embodiments of the foregoing methods, tamoxifen is administered in the absence of administration of an agent that inhibits CYP2D6 or that produces an inhibitor through metabolism. Inhibitors of CYP2D6 are known in the art and include, for example,

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV selective serotonin reuptake inhibitor antidepressants, bupropion, duloxetine, cimetidine, diphenylhydramine, and others.

The following examples serve to illustrate the present invention further.

EXAMPLE 1

Varying Tamoxifen Dosage in Setting of Low Serum

Endoxifen Levels and CYP2D6 Varients

Subjects

Eligible women and men were prospectively recruited from The Tisch Cancer Institute at The Mount Sinai School of Medicine and Queens Cancer Center at Mount Sinai Queens Hospital Center. Pre-menopausal and menopausal women, defined as age > 55 and no menses for 12 months, who were taking tamoxifen (Tarn) 20 mg a day for at least 90 days were included in this study. Patients were excluded if they had started Tarn therapy concurrently with adjuvant chemotherapy and/or adjuvant radiation therapy or if they were taking other adjuvant endocrine therapy. Other reasons for exclusion included elevated plasma alanine aminotransferase, aspartate aminotransferase, bilirubin, or alkaline phosphatase, defined as higher than 2.5-fold greater than the respective upper limit of normal. Patients who were pregnant or lactating were excluded. Study participants were allowed to take herbal remedies or potential CYP2D6 inhibitors, provided that the participant had been taking the agent for at least four weeks and intended to continue taking the agent for the duration of the study.

Medications leading to CYP2D6 inhibition were recorded according to their potency and defined as 1) weak, causing >1.25-fold, but <2-fold increase in the plasma area under the curve (AUC), of endoxifen or 20-50% decrease in clearance, 2) moderate, causing a >2-fold increase in the plasma AUC values or 50-80% decrease in clearance, and 3) strong, causing a >5-fold increase in the plasma AUC values or more than 80% decrease in clearance. For each patient, medical histories, including self -reported race, a comprehensive list of current medications, and the results of clinical laboratory examinations were obtained. Peripheral blood samples were collected in heparinized tubes and plasma was separated within 1 hour of collection. Genotyping and tamoxifen metabolite levels were determined in the New York State and Clinical Laboratory Improvement Act (CLIA)- approved Genetic Testing

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Laboratory of the Department of Genetics and Genomic Sciences at The Mount Sinai School of Medicine. Samples from the Queens Cancer Center at the Mount Sinai Queens Hospital were delivered daily to the laboratory on ice.

The study protocol was approved by the Institutional Review Boards at both study sites and all patients provided written informed consent.

Genotyping Analysis

DNA was isolated from whole blood using Puregene® DNA purification kits (Centra, Minneapolis, MN). The CYP2D6 allele designations refer to those defined by the

Cytochrome P450 Allele Nomenclature Committee (http://cypalleles.ki.se). Genotyping of functional (* 1, *2, *2A), reduced function (*9, *10, * 17 and *41) and non-functional (e.g., *3-*6, *8, *11, *12, * 14 and * 15) CYP2D6 alleles including their possible duplications was performed using Tag-It™ Mutation Detection Kit P450-2D6 Version 2 (GeneMark). Briefly, the regions surrounding the mutations were multiplex polymerase chain reaction (PCR)- amplified, subjected to allele- specific primer extension, hybridized to specific Luminex^® beads via GeneMark Universal Tags, and sorted on a Luminex 100 xMAP™ platform (Luminex Corporation, Austin, TX).

The CYP2D6 genotype for each participant, was classified by the number of functional alleles to predict the individual's metabolizer phenotype. Individual's CYP2D6 alleles predicted his/her MPA score. Gaedigk et al. (2008 Clin Pharmacol Ther 83:234-242. Each CYP2D6 allele was assigned a value that reflected its expected enzymatic activity. Fully functional CYP2D6 alleles (*1, *2, *2A) were assigned a score of 1, alleles associated with reduced enzyme activity (*9, * 10, * 17 and *41) were scored as 0.5, and CYP2D6 null alleles (*3-*6, *8,*11, *12, * 14 and * 15) and their duplications were scored a 0. Thus, the MPA score for each patient was the sum of their scores for both of their CYP2D6 alleles, which ranged between 0 and 2.0.

Measurement of Plasma Concentrations of Tarn and Its Metabolites

Initially, plasma Tarn and its active metabolites, 'total' endoxifen and 4-OH-Tam, were measured by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) using an isocratic gradient that separated Tarn and its active metabolites. In addition, a method to separate and characterize the E, Z and Z' isomers of endoxifen and 4-OH-Tam was developed. Jaremko et al. Anal. Chem. 2010 Nov. 18 [EPub ahead of print]. Since all patient

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV plasma samples were stored at -80°C in the dark, the samples were reassayed by the new LC- MS/MS method to determine active metabolite isomer concentrations. Stable isotope labeled internal standards D5- Z-4-OH-Tam and D5-E, Z-endoxifen and a series of calibrators spiked with Z-4-OH-Tam and Z- and Z' -endoxifen in blank plasma were used in the improved assay for verification and quantification. The ERAS was calculated from the plasma Tarn and active metabolite isomer concentrations as 0.01x[Tam] + lx[Z-endoxifen + Z-4-OH-Tam] + 0. lx[Z' -endoxifen + Z'-4-OH-Tam] based on their respective metabolite activity toward ER affinity.

Dose Increase Study

Patients who met inclusion/exclusion criteria and had a CYP2D6 MPA score of 0 and/or baseline plasma endoxifen concentrations < 40 nM were prescribed a Tarn dose of 30 mg/day and were followed every two weeks for up to 90 days. During each follow-up visit, the subject's medical history, weight, current medications, Tarn compliance, and new symptoms, including hot flashes, night sweats, vaginal discharge and/or bleeding, were recorded and graded as per the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. To assess compliance, the patients were asked at each clinic visit whether they were taking the 30 mg dose daily. Also, only a month supply of the 30 mg dose was provided to assure that each patient requested a new supply.

Statistical Analysis

Continuous variables were summarized using means and standard deviations or medians and quartiles as appropriate, while categorical variables were summarized by frequencies. Genotype frequencies for all studied varients were tested for Hardy- Weinberg equilibrium using the chi-square test. Intra-individual variation in metabolite levels was assessed using one-way ANOVA. Baseline metabolite concentrations were compared with the patients' genotype scores, body mass index (BMI) categories, and the number of concomitant medications using a Kruskal-Wallis test. A paired t-test was applied to compare changes in Tarn metabolite concentrations between baseline and day 60 following the dose increase. In addition, mixed models were used to estimate the effect of the CYP2D6 genotype on the rate of change in endoxifen, 4-OH-Tam, and their respective isomer levels. Metabolite levels were logarithmically transformed due to their skewed distribution. All statistical analyses were performed using SAS/STAT software (SAS Institute, Inc., Cary, NC).

Statistical significance was assigned at < 0.05.

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Results

Patient Characteristics

Of the 121 enrolled patients, 117 (97%) completed the study. The characteristics of the study population are shown in Table 1 below. The mean age was 53 years and about half (56%) of the female patients were pre-menopausal. Just over half of the patients were Caucasian, with the other half divided almost equally among African Americans, Asians, and Hispanic/Latinos. The majority of patients (88%) were not taking concomitant medications known to inhibit CYP2D6.

Table 1: Patient Characteristics at Baseline.

See text for specific drugs

The frequency of CYP2D6 alleles and genotypes varied significantly among the ethnic and racial groups as shown in Tables 2 and 3 below. However, the median MPA score was similar among all ethnicities (Table 2); in the pooled sample, 61.5% had MPA scores of 2 or 1.5, 26.5% were 1.0, 8.5% were 0.5 and 2.5% were 0.

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Table 2: CYP2D6 Frequencies of Ethnicity/Race

Overall African American Asian Caucasian Hispanic/Latino

CYP2D6 Allele American Indian

(n=117) (N=21) (N=2) (N=18) (N=60) (N=16)

*1 0.34 0.26 0.75 0.47 0.33 0.31

*10 0.05 0.10 - 0.21 0.008 -

*17 0.04 0.14 - - 0.008 0.06

*2 0.05 0.26 - - - 0.03

*2A 0.19 0.02 0.25 0.12 0.23 0.31

*2AxN(duplication) 0.004 - - - 0.008 -

*4 0.13 0.05 - 0.09 0.17 0.15

HI 0.013 0.10 - 0.06 0.18 0.09

*5 0.03 0.05 - 0.06 0.03 -

*6 0.009 - - - 0.02 -

«9 0.02 0.02 - - 0.03 0.03

MPA ScoreMedian 1.5 1.5 2.0 1.4 1.4 1.5

(25^th %-tile, 75^th (1.0,2.0) (1.0,2.0) (2.0,2.0) (1.0,2.0) (1.0,2.0) (1.0,2.0)

%-tile)

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Table 3. Metabolizer Phenotype Activity (MPA) Scores and Genotype Frequencies by Race/Ethnicity

Frequency, % (N)

MPA CYP2D6 African American Hispanic/

Overall Asian Caucasian

Score Genotype American Indian Latino

(N=117) (N=18) (N=60)

(N=21) (N=2) (N=16)

0 *4/*4 0.03 (3) - - - 0.05 (3) -

0.5 *17/*4 0.09 (1) 0.05 (1) - - - -

0.5 *17/*6 0.09 (1) - - - 0.02 (1) -

0.5 *4/*9 0.02 (2) - - - 0.02 (1) 0.06 (1)

0.5 *4/*41 0.06 (7) 0.05 (1) - - 0.10 (6) -

0.5 *41/*5 0.09 (1) 0.05 (1) - - - -

1 *]y*4 0.07 (8) - - 0.12 (2) 0.08 (5) 0.06 (1)

1 *l/*5 0.009(1) - - 0.06 (1) - -

1 *l/*6 0.009(1) - - - 0.02 (1) -

1 *10/*10 0.02 (2) - - 0.12 (2) - -

1 no/* 17 0.09 (1) 0.05 (1) - - - -

1 *10/*41 0.09 (1) 0.05 (1) - - - -

1 * 17/41 0.02 (2) 0.05 (1) - - - 0.06 (1)

1 *2A/*4 0.04 (5) - - 0.06 (1) 0.02 (1) 0.19 (3)

1 *2A/*5 0.04 (5) 0.05 (1) - 0.06 (1) 0.05 (3) -

1 *41/*41 0.03 (3) - - - 0.05 (3) -

1.5 *1/*10 0.03 (4) 0.05 (1) - 0.18 (3) - -

1.5 *jy*9 0.009(1) 0.05 (1) - - - -

1.5 *1/*41 0.05 (6) - - - 0.08 (5) 0.06 (1)

1.5 *10/*2 0.09 (1) 0.05 (1) - - - -

1.5 *10/*2A 0.09 (1) - - - 0.02 (1) -

1.5 *17/*2 0.03 (3) 0.14 (3) - - - -

1.5 *17/2A 0.09 (1) - - - - 0.06 (1)

1.5 *2A/*9 0.02 (2) - - - 0.03 (2) -

1.5 *2A/*41 0.07 (8) - - 0.12 (2) 0.08 (5) 0.06 (1)

2 *1/*1 0.16(19) 0.14 (3) 0.50 (1) 0.29 (5) 0.13 (8) 0.13 (2)

2 *l/*2 0.03 (3) 0.14 (3) - - - -

2 *1/*2A 0.15(17) - 0.50 (1) - 0.20 (12) 0.25 (4)

2 *2/*2 0.02 (2) 0.10 (2) - - - -

2 *2/*2A 0.09 (1) - - - - 0.06 (1)

2 *2A/*2A 0.02 (2) - - - 0.03 (2) -

2 *2AxN/*4 0.09 (1) - - - 0.02 (1) -

Plasma Concentrations of Tam and Its Metabolites at Baseline

To evaluate the steady-state variation of Tam and its metabolites prior to the study, plasma levels were determined on two to five samples collected over five weeks from 10

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV patients with a total of 34 observations. No significant inter- individual differences for any metabolite were found (P>0.06). BMI did not affect the steady-state levels of Tarn or its metabolites (data not shown). Concomitant medications which have been reported to inhibit CYP2D6 also did not significantly affect Tarn's steady-state or endoxifen concentrations (Table 4). Of the 117 patients, three were each taking two moderate CYP2D6 inhibitors (duloxetine and/or cimetidine and/or sertraline), and their baseline endoxifen concentrations were 40 nM, 40nM, and 52 nM, respectively.

Table 4: Baseline Tamoxifen and Tamoxifen Metabolite Concentrations by

Concomitant CYP2D6 Inhibitors.

Strength of Concomitant CYP2D6 Inhibitors⁺

Median (range) P*

Metabolites (nM) No Weak (N=8) Moderate Strong (N=4)

inhibitors (N=2)

(N=103)

Tarn 294 242 308 360 0.88

(36-648) (78-751) (289-327) (126-393)

Endoxifen 50 42 43 51 0.48

(23-113) (32-79) (42-45) (40-56)

4-OH-Tam 13 14 10 16 0.54

(2-48) (9-29) (6-14) (12-18)

ER-activity estimate 30 25 27 22 0.24

(7-111) (16-52) (26-28) (15-23)

Z and Z'-Metabolites

Z-Endoxifen 21 14 17 10 0.13

(3-87) (9-33) (17-17) (6-17)

Z' -Endoxifen 17 17 22 28 0.23

(5-42) (11-35) (18-26) (16-39)

Z-4-OH-Tam 4 3 4 3 0.82

(1-23) (2-9) (4-4) (2-4)

Z' -4-OH-Tam 6 6 6 8 0.39

(1-14) (4-17) (6-7) (4-10)

^Kruskal Wallis test

patients may have been on more than 1 medication known to inhibit CYP2D6, see text.

The baseline mean and range of plasma levels for Tarn, 4-OH-Tam, endoxifen, Z- and Z'-endoxifen, and Z - and Z'-4-OH-Tam for all 117 patients and their respective MPA scores

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV are shown in Table 5 below. While no significant differences in Tarn and 4-OH-Tam levels were observed across different MPA scores, the MPA score (ranging between 0 and 2) was linearly associated with higher endoxifen (P=0.0009), Z-endoxifen (P<0.0001) and the

calculated ER-activity estimate (P=0.0002), as well as lower Z' -endoxifen concentrations

(P=0.0004). The range for total endoxifen in each MPA group extended below 40 nM, the selected endoxifen concentration cut off for increasing the oral Tarn dose.

Table 5: Baseline Tamoxifen and Tamoxifen Metabolite Concentrations by Metabolizer

Phenotype Activity (MPA) Score

Median (range)

Metabolites

MPA Score

(nM) Overall

(N=117) 0 (N=3) 0.5 (N=10) 1.0 (N=31) 1.5 (N=27) 2 (N=45) p*

Tarn 289 195 232 308 271 284 0.32

(36-751) (108-432) (84-370) (54-648) (126-581) (36-751)

Endoxifen 49 31 38 48 49 52 0.001

(23-113) (23-42) (26-52) (23-113) (32-79) (25-109)

4-OH-Tam 14 12 12 13 12 15 0.27

(2-48) (6-13) (5-18) (4-48) (5-24) (2-37)

Anti-estrogenic 28 13 18 28 30 35 0.0004 activity estimate** (7-113) (10-21) (8-26) (7-96) (17-61) (9-111)

Z and Z'-Metabolites:

Z-Endoxifen 19 6 9 18 20 27 <0.0001

(3-87) (5-9) (4-15) (3-66) (10-45) (6-87)

Z' -Endoxifen 18 27 24 21 19 15 0.0004

(5-42) (14-33) (13-37) (6-42) (9-39) (5-35)

Z+ -Endoxifen 39 33 32 39 42 43 0.12

(11-107) (19-42) (18-50) (11-86) (25-79) (13-107)

Z-4-OH-Tam 4 2 3 4 4 5 0.06

(1-23) (2-4) (1-5) (1-23) (2-7) (1-15)

Z' -4-OH-Tam 6 7 6 6 6 5 0.43

(1-17) (3-8) (3-11) (2-14) (3-11) (1-17)

*Kruskal Wallis test. **, calculated as [Tam*0.01+Z-endoxifen+Z'-endoxifen*0.1+Z-OH-Tam+Z'- OH-Tam*0.1];

Plasma Concentrations of Tarn and Its Metabolites Following Increased Dose

Based on the selected plasma endoxifen level < 40 nM and/or MPA score of 0, 26

participants were eligible of which 24 patients (92%) agreed to increase their Tarn dose to 30 mg/day. Two individuals (2/24, 8%) had an MPA score of 0 ; their baseline plasma

endoxifen levels were 23 and 43 nM, the latter slightly exceeding the therapeutic goal (Table

6). The remaining participants (22/24, 92%) had MPA scores > 0.5, but endoxifen levels <

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV

40 nM. Of the 24 patients, 18 (75%) continued on the increased dose for 86 to 90 days when their last plasma metabolite concentrations were determined. The last plasma metabolite concentrations were obtained from two subjects at 60 days and from one subject each at 56, 30, 16 and 14 days of increased dose.

Tables 6A and B. Baseline Levels and up to 90-Day Plasma Tamoxifen Metabolite Concentration Changes of the 25 Women who Received Higher Dose

Table 6A

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Table 6B

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At 60 days of the increased doses, both the endoxifen and 4-OH-Tam levels increased in 18/21 individuals (90%) compared to baseline showing an average increase of 54% and 84%, respectively (paired t-test's P=0.005 and P<0.0001). These subjects included patients who were CYP2D6 homozygous null (genotype*4/*4, no enzyme activity, MPA score of 0) (Patients #1 and #2; Table 6 and Figure 2). They received the higher dose for at least 60 days and increased their mean endoxifen and 4-OH-Tam levels by 24% from 32.5 to 40.4 nM and 45% from 9.5 to 13.8 nM, respectively.

Five patients with MPA scores of 0.5 (designated low intermediate metabolizers) received the higher dose for an average of 61 days and increased their mean endoxifen and 4- OH-Tam levels by 33% from 32.4 to 43.1 nM and 71% from 9.4 to 16.1 nM, respectively. This group included patients #4 and #7whose last measurements were at 30 and 14 days. Despite the shorter follow-up, their levels increased by 30% from 26.4 to 34.2nM and 28% from 32.7 to 42.0 nM for endoxifen and 155% from 5.3 to 13.5nM and 20% from 10.4 to 12.5 nM. for 4-OH-Tam, respectively (Table 6; *MPA score, Metabolizer Genotype Activity score; **, calculated as [Tam*0.01+Z-endoxifen+Z'-endoxifen*0.1+Z-OH-Tam+Z'-OH- Tam*0.1];^†experienced side effects including hot flashes and/or night sweats; -, no measurement at 60 days. Negative values refer to percent decrease.).

Nine patients with MPA scores of 1.0 (intermediate metabolizers) received the higher dose for an average of 84 days and increased their mean endoxifen and 4-OH-Tam levels by 44% from 30.8 to 44.3 nM and 30% from 9.6 to 12.5 nM, respectively. Patients #12 and #13 in this score group increased their endoxifen and patient #12 increased her 4-OH-Tam level after day 60 and then had a 10%-34% decrease at 86 days. Two additional patients (#15 and #16) decreased their endoxifen levels, but increased their 4-OH-Tam levels, compared to baseline at both 60 and 86 days.

Four patients with the MPA scores of 1.5 (extensive metabolizers) received the higher dose for an average of 61 days and increased their mean endoxifen and 4-OH-Tam levels both by 30% from 34.0 to 44.3 nM and from 11.5 to 15.0 nM, respectively. That includes patient #17 whose last measurement was at 16 days. While her endoxifen levels increased 4% from 32.5 to 33.7 nM, her 4-OH-Tam levels decreased by 13% from 11.7 to 10.2 nM.

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Four patients with MPA scores of 2.0 (extensive metabolizers) received the higher dose for an average of 87 days. Three of the four patients increased their mean total endoxifen and 4-OH-Tam levels by 29% from 33.8 to 43.7 nM and 79% from 7.0 to 12.5 nM, respectively. One patient had a much greater increase in both active metabolite levels (patient #21; a 261% increase to 80.6 nM for total endoxifen and a 426% increase to 17.0 nM for 4-OH-Tam at 86 days) compared to other participants with the same MPA score (Table 6). These levels were repeated to rule out random measurement errors.

Significant increases were also observed for the independently determined endoxifen and 4-OH-Tam Z-isomer levels. On average, 60-day metabolite levels 15% and 103% for Z- endoxifen, 48% and 69% for Z' -endoxifen, 10% to 122% for Z-4-OH-Tam, and 36% to 134% for Z' -4-OH-Tam (Table 6, Figure 2). The Z-endoxifen to Z' -endoxifen ratio did not significantly change in response to the dose increase (data not shown).

The MAP score was significantly associated with the rate of change for Z and Z'- endoxifen levels, but not other metabolites. In extensive and ultra-rapid metabolizers (MPA score of 1.5 or 2.0, respectively), while Z-endoxifen levels increased more gradually than in other CYP2D6 carriers, their Z' -endoxifen levels remained the lowest compared to other genotype groups (Figure 2C and 2D). An opposite trend was observed in intermediate (MPA score of 1.0) and low intermediate and poor (MPA score of 0.5 and 0, respectively) metabolizers showing a significant effect of MPA score on the rate of change of Z (p=0.002) and Z' (p=0.02) endoxifen.

The separation of the endoxifen and 4-OH-Tam isomers, which have different antiestrogenic activities, provided the ability to estimate the composite therapeutic effectiveness of these isomers by calculating the ERAS. At baseline, the mean ERAS for the 117 enrolled patients was 28. The ERAS increased with increasing MPA scores from 13 to 35 (p=0.0002). Among the 24 women in whom Tarn dose was increased, the ERAS ranged from 7 to 27 at baseline. When the dose was increased, the mean ERAS of patients in each MPA score increased. The two patients with an MPA score of 0 increased from 14.4 to 18.8, or 24.5%; the five patients with a 0.5 MPA score increased from 14.2 to 36.4, or 122%; the nine with 1.0 MPA scores increased from 15.1 to 24.2, or 75.1%; the four patients with 1.5 MPA scores increased from 22.0 to 39.1, or 72.2%; and the four women with MPA scores of 2.0 increased from 14.4 to 40.2, or 110% (Table 6, Figure 2E).

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None of the 24 patients in the Tarn cohort at 30 mg/day were taking concomitant medications reported to inhibit CYP2D6. Side effects, considered to be from the increased drug dose, were noted in 21% (5/24) of patients by the 14^th day of the higher dose. Four patients reported moderate hot flashes and one reported mild hot flashes. Two of the four patients reported that their hot flashes had improved to mild by the 90^th day of the higher dose. The same four patients reported grade 1 diaphoresis and another reported grade 2 diaphoresis. One patient went off the study on day 16 of the higher dose secondary to hot flashes and vaginal dryness.

Conclusion

The foregoing results demonstrated that sub-therapeutic steady-state plasma endoxifen concentrations (<40 nM) occurred in over 20% of patients treated with Tarn for up to 90 days. Significantly higher circulating endoxifen and total active Tarn levels, calculated as the ER-activity estimate, were associated with higher CYP2D6 activity as assessed by the MPA score.

The fact that sub-therapeutic plasma endoxifen concentrations <40 nM occurred in all CYP2D6 genotypes indicates that Tarn's active plasma metabolite levels were not entirely predicted by the CYP2D6 metabolizer phenotype.

The median baseline plasma Z-endoxifen level was significantly increased with increasing MPA score (P<0.0001), indicating that the conversion of Tarn to Z-endoxifen was proportionate to the CYP2D6 activity. In contrast, the median baseline plasma levels of Z'- endoxifen decreased with increasing CYP2D6 activity (P=0.0004). This finding is consistent with the fact that the decreased CYP2D6 activity results in the increased conversion of N- desmethyl-Tam to Z' -endoxifen, which has a 10-fold less potent estrogen receptor potency in suppressing estrogen-dependent cell proliferation (see Figure 1).

Based on sub-therapeutic steady-state endoxifen levels and/or poor CYP2D6 metabolizer phenotype, 24 patients had their Tarn dose increased to 30 mg/day for up to 90 days. After increasing the dose for at least 60 days, both the total endoxifen and 4-OH-Tam levels increased in 18/20 (90%) individuals with complete follow-up, including the two CYP2D6 homozygous null patients (MPA score of 0) (Table 6 and Figure 2), showing an overall average increase of 54% and 84%, respectively. The rate and magnitude of increase did not seem to be affected by the CYP2D6 MPA score. When the Tarn dose increased, the Z

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV and Z' concentrations also increased for patients with all MPA scores. The CYP2D6 extensive metabolizers had a greater increase in Z-endoxifen levels compared to other CYP2D6 groups, whereas their Z'-endoxifen levels remained the lowest.

Increased plasma endoxifen levels in the two subjects with MPA scores of 0.5 and 1.5 followed for 14-16 days were observed (Table 6), indicating that plasma levels can increase rapidly. Similarly, side effects were noted in the first days/weeks of increased dose.

One individual with an MPA score of 2 (extensive metabolizer) had a much higher increase in all metabolite concentrations compared to her counterparts, going up from 24.7 nM at baseline to 80.6 nM at 86 days. Such a steady increase may indicate impaired drug excretion resulting in active metabolite accumulation. In addition, two patients were observed whose endoxifen levels decreased following the higher Tarn dose. Two additional patients had their levels decreased following an initial increase. It is possible that poor compliance may have been a significant factor in these two cases.

These results demonstrate that the plasma concentrations of endoxifen and 4-OH-Tam can be elevated more by increasing the Tarn dose, independent of the patient' s CYP2D6 genotype.

All references cited herein are incorporated herein in their entirety.

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Claims

Atty. Docket No.: 102756.62156PV What is claimed is:

1. A method for the prevention and treatment of breast cancer in a patient in need of such treatment comprising the steps of determining the patient' s genotypic profile for one or more genes predictive of tamoxifen activity, and administering to the patient a dosage of tamoxifen based on the patient's genotypic profile.

2. The method of claim 1 wherein the genotypic profile is determined for one gene and the gene encodes CYP2D6.

3. The method of claim 2 wherein the patient's genotype includes at least one null CYP2D6 allele, or at least one reduced function allele, and does not include a functional CYP2D6 allele, and the dosage of tamoxifen is more than 20 mg/day.

4. The method of claim 3 wherein the dosage of tamoxifen is 30 mg/day.

5. The method of claim 3 wherein the dosage of tamoxifen is 40 mg/day.

6. The method of claim 2 wherein the patient's genotype includes at least one functional CYP2D6 allele, and the dosage of tamoxifen is 20 mg/day.

7. A method for treatment of breast cancer in a patient in need of such treatment comprising the steps of: determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity; administering tamoxifen to the patient at an initial dosage based on the patient's genotypic profile; performing a measurement of steady state plasma levels of one or more active metabolites of tamoxifen; and administering tamoxifen at an optimized dosage based on levels of active metabolites.

8. The method of claim 7 wherein the genotypic profile is determined for one gene and the gene encodes CYP2D6.

9. The method of claim 8 wherein the patient's genotype includes at least one null CYP2D6 allele, or at least one reduced function allele, and does not include a functional CYP2D6 allele, and the initial dosage of tamoxifen is more than 20 mg/day.

10. The method of claim 9 wherein the initial dosage of tamoxifen is 30 mg/day.

11. The method of claim 9 wherein the initial dosage of tamoxifen is 40 mg/day.

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV

12. The method of claim 8 wherein the patient's genotype includes at least one functional CYP2D6 allele, and the initial dosage of tamoxifen is 20 mg/day.

13. The method of claim 7 wherein the tamoxifen metabolites are 4-OH-TAM and endoxifen.

14. The method of claim 13 wherein the levels of one or more active metabolites are less than therapeutic levels and the optimized dosage of tamoxifen is greater than 20 mg/day.

15. The method of claim 13 wherein the optimized dosage of tamoxifen is 30 mg/day.

16. The method of claim 13 wherein the optimized dosage of tamoxifen is 40 mg/day.

17. The method of claim 13 wherein the levels of one or more active metabolites are equal to therapeutic levels and the optimized dosage of tamoxifen is 20 mg/day.

18. The method of claim 7 further comprising the steps of performing a

second measurement of the steady state levels of one or more active metabolite isomers, and administering tamoxifen at a second optimized dosage based upon the second measurement of levels of active metabolite isomers.

19. The method of claim 7 wherein the levels of active metabolite isomers in the second measurement are greater than or equal to therapeutic levels, and the second optimized dosage is the same as or lower than the first optimized dosage.

20. The method of claim 7 wherein the levels of active metabolite isomers in the second measurement are less than therapeutic levels, and the second optimized dosage is greater than the first optimized dosage.

21. A method of optimizing treatment of breast cancer in a patient in need thereof comprising administering tamoxifen to the patient at an initial dosage of 20 mg/day, obtaining a sample of blood from the patient, measuring steady state plasma levels of one or more active tamoxifen metabolite isomers in the sample, and administering an optimized dosage of tamoxifen to the patient based upon the level of one or more active tamoxifen metabolite isomers.

22. The method of Claim 21 further comprising determining the patient's genotypic profile for one or more genes predictive of tamoxifen activity, and administering an

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NYl 486845vl 12/10/10 Atty. Docket No.: 102756.62156PV optimized dosage of tamoxifen to the patient based upon the level of one or more active tamoxifen metabolites and the patient's genotypic profile.

23. The method of claim 21 wherein the levels of one or more active metabolites are less than therapeutic levels, and the optimized dosage of tamoxifen is greater than 20 mg/day.

24. The method of claim 21 wherein the levels of one or more active metabolite isomers are greater than or equal to therapeutic levels, and the optimized dosage of tamoxifen is 20 mg/day.

25. A method of optimizing treatment of breast cancer in a patient in need thereof comprising obtaining a sample of blood from the patient undergoing treatment with tamoxifen, measuring steady state plasma levels of one or more active tamoxifen metabolites in the sample, determining the anti-estrogenic activity score, and administering an optimized dosage of tamoxifen to the patient based upon the anti-estrogenic activity score.

26. Tamoxifen for use in treatment of breast cancer in a patient having a CYP2D6 genotype that includes at least one null CYP2D6 allele, or at least one reduced function allele, and does not include a functional CYP2D6 allele, wherein tamoxifen is administered at a dosage of greater than 20 mg/day.

27. Tamoxifen for use in treatment of breast cancer in a patient having steady state plasma levels of endoxifen of less than 40 nM after treatment with tamoxifen at a dosage of 20 mg/day, wherein tamoxifen is administered at a dosage of greater than 20 mg/day.

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