WO2012119010A9 - Substances inhibitrices d'aromatase et procédé d'utilisation desdites substances pour le diagnostic, le traitement et la surveillance du cancer du sein - Google Patents

Substances inhibitrices d'aromatase et procédé d'utilisation desdites substances pour le diagnostic, le traitement et la surveillance du cancer du sein Download PDF

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WO2012119010A9
WO2012119010A9 PCT/US2012/027346 US2012027346W WO2012119010A9 WO 2012119010 A9 WO2012119010 A9 WO 2012119010A9 US 2012027346 W US2012027346 W US 2012027346W WO 2012119010 A9 WO2012119010 A9 WO 2012119010A9
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aromatase
group
inhibitor
tamoxifen
independently selected
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PCT/US2012/027346
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WO2012119010A2 (fr
WO2012119010A3 (fr
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David A. Flockhart
Wenjie Lu
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Indiana University Research And Technology Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates generally to producing and. using aromatase inhibitors to treat, diagnose and evaluate the progress of various diseases and conditions including breast cancer.
  • the aromatase pathway is the primary pathway for the production of estrogen in women after ovarian function subsides during the menopause or after another physiological change or medical intervention that reduces or eliminates ovarian function.
  • the enzyme aromatase is a target of many therapies that are designed to reduce estrogen in postmenopausal women, and thereby reduce their risk for recurrent breast cancer.
  • the enzyme can also be valuably targeted as part of treatments for infertility in which reduction of systemic estrogen is used to stimulate pituitary function and increase ovarian function.
  • An object of the present invention is to provide benefit to patients who arc treated with aromatase inhibitors for breast cancer and other conditions by cxpaiHlmg their treatment options and enhancing the efficacy of their treatment.
  • aromatase inhibitors include using aromatase inhibitor to diagnose and/or characterize a particular form of disease mat is characterized by aberrant aromatase activity.
  • aromatase inhibitors including the compoundsdisclosed herein, are used to track a given disease and/or to assess the effectiveness of a given therapy used to treat diseases such as breast cancer.
  • Still other embodiments include treating humans or animals with aromatase inhibitors such as those disclosed herein to treat diseases such as breast cancer.
  • Some embodiments of the invention include at least one aromatase inhibitor having structures according to Formu
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 " , CH 3 -CH 2 -, CH 3 -CH 2 -CH 2 -;
  • Still other embodiments of the invention include methods of inhibiting aromatase; comprising the step of: providing at least one compound according to Formula A
  • the contacting step occurs in vitro, while in still other embodiments the contacting step occurs in vivo.
  • Some embodiments include, at least one aromatase inhibitor; according to Formula A;
  • the oxyalkylamine side chain on the phenyl group may have structural variants including: length in -(CH 2 ) n -; position on the phenyl group; position of oxygen in relation to -(CH 2 ) n - chain; and oxyalkene composition of the side chain;
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 " , CH 3 -CH 2 -, CH 3 -CH 2 -CH 2 -;
  • R4 is selected from the group consisting of: H, CH 3 " , CH 3 - (CH 2 ) n -, hydoxy, methoxy, ethoxy; and
  • n 1 , 2, 3, 4 or 5 such as CH 2 -CH 2 -, CH 2 -CH 2 - CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -CH 2
  • R 2 and R 3 consisting of H, and OH; R 2 and R 3 are independently selected from the group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me.
  • Some embodiments include methods of inhibiting an aromatase; comprising the step of: providing at least one compound of Formula A, or an ester of pharmaceutically acceptable salt thereof; and contacting said compound with aromatase.
  • the contacting step occurs in vitro. While in other embodiments, the contacting step occurs in vivo.
  • Some embodiments of the invention include methods of screening patients for treatment with aromatase inhibitors, comprising the steps of: providing at least one compound according to Formula A, measuring the level of aromatase activity in both the presence and in absence of said aromatase inhibitor in a sample of tissue, blood, cells and/or fluid from a patient; and attributing the change in aromatase activity measured in the presence and in the absence of said aromatase inhibitor to the level of aromatase in the sample that is sensitive to inhibition with at least one aromatase inhibitor.
  • Some embodiments further include the step of comparing the level of aromatase activity in the sample that can be inhibited with the aromatase inhibitor with an amount of aromatase activity that is diagnostic for a given pathology. Some embodiments include the step of correlating the level of aromatase activity measured in the sample from the patient with the patient's likelihood of responding to a treatment regime.
  • the disease diagnosed, monitored or treated with at least one compound according to Formula A is breast cancer.
  • Some embodiments of the invention include methods of treating a patient, comprising the steps of: providing at least on
  • the oxyalkylamine side chain on the phenyl group may have structural variants including: length in - (CH 2 ) n -; position on the phenyl group; position of oxygen in relation to -(CH 2 ) n - chain; and oxyalkene composition of the side chain;
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 ⁇ , CH 3 -CH 2 -, CH 3 -CH 2 -CH 2 -;
  • the method treating a patient with at least one compound according to Formula A further includes the step of: administering at least a therapeutically effective dose of at least one compound according to formula A.
  • the patient is either an animal or a human being, in some embodiments the patient is symptomatic for breast cancer.
  • Some methods of treating a disease with at least once compound according to Formula A further include the step of monitoring the course of the diseasing by obtaining at least one more sample of tissue, cells, blood or fluid from the patient after said patient is treated with at least one therapeutic dose of the compound for Formula A.
  • a patient treated with at least one compound of Formula A is also treated with at least one other therapy selected from the group consisting of: radiation, convention chemotherapy and surgery.
  • Some embodiments of the invention include methods treating a patient, comprising the step: of providing a compound according to:
  • the treated patient is symptomatic for breast cancer.
  • the patient is also treated with at least one other therapeutic compound.
  • the patient is also treated with at least one other therapy selected from the group consisting of:
  • Some aspects of the invention include inhibiting a aromatase; comprising the step of:
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 " , CH 3 -CH 2 -, CH 3 - CH 2 -CH 2 -;
  • the aromatase inhibitor is selected from the group consisting of norendoxifen and endoxifen.
  • the contacting step occurs in vitro while in some it occurs in vivo.
  • the aromatase inhibitor is a better inhibitor of aromatase CYP19 than it is an inhibitor of the aromatases selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.
  • Some aspects of the invention include methods of screening patients for treatment with aromatase inhibitors, comprising the steps of: contacting at least one aromatase inhibitor of Formula A:
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 " , CH 3 -CH 2 -, CH 3 -CH 2 -CH 2 -;
  • Some aspects of the invention include methods of treating a patient, comprising the steps of: identifying a patient in need of an aromatase inhibitor; and administering a therapeutically effective amount of a compound according to Formula A, or a pharmaceutically acceptable salt thereof:
  • Ri may be independently selected from the group consisting of H, CH 3 and OH;
  • R 2 and R 3 are independently selected from the group consisting of H, CH 3 " , CH 3 -CH 2 -, CH 3 - CH 2 -CH 2 -;
  • R4 is selected from the group consisting of: H, or Me.
  • Therapeutic doses are in the range of 10 to 40 mg per day, generally not to exceed more than 20 mg at a time.
  • the aromatase inhibitor is endoxifen or a pharmaceutically acceptable salt thereof or norendoxifen or pharmaceutically acceptable salt thereof.
  • the patient is an human or another mammal.
  • the method of treating a patient further includes the step of: monitoring the course of the diseasing by obtaining at least one more sample of tissue, cells, blood or fluid from the patient after said patient is treated with at least one therapeutic dose of the compound for Formula A.
  • the patient treated with at least one aromatasc inhibitor of Formula A is also treated with at least one other compound and or with at least one other therapy.
  • co-therapies can be selected from the group consisting of: radiation, convention chemotherapy and surgery.
  • the treated patient is symptomatic for breast cancer.
  • the aromatase inhibitor is a better inhibitor of aromatasc CYP19 than it is an inhibitor of the aromatascs selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.
  • Some aspects of the invention include at least one aromatase inhibitor; comprising: an aromatase inhibitor of Formul
  • i may be independently selected from the group consisting of H, CHj and OH;
  • R 2 and 3 ⁇ 4 are independently selected from the group consisting of H, C3 ⁇ 4 ⁇ , CH3-CH4-, CH3-CH2-CH2-;
  • R4 is selected from the group consisting of: H, CH 3 ', CHHCH-dn-, hydoxy, methoxy, ethoxy;
  • n 1, , 3, 4 or an w ?e ormu a nm > tS 8t castone
  • n 2; R ( is independently selected from the group consisting of H, and OH; R2 and R 3 arc independently selected from fee group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me.
  • the aromatasc inhibitor is a better inhibitor of aromatase CYP1 man it is an inhibitor of the aromatascs selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.
  • FIG. 1 Relative potency of tamoxifen and its primary and secondary metabolites in the inhibition of aromatase.
  • Test compounds (10 uM) were incubated with 7.5 oM recombinant human aromatasc at 37°C for 30 min. Letrozole (0.1 uM) and vehicle (acetonitrile) were used as positive and negative controls respectively. Data are plotted as means of triplicate incubation with standard deviations. The dotted line represents 100 percent activity that was observed with the vehicle control.
  • FIG. 2 Inhibition of aromatase by tamoxifen and its metabolites. Curves represent percent aromatase activity remaining in the presence of a range of concentrations of endoxifen (o), tamoxifen (T), NDMT ( ⁇ ) and 4HT ( ⁇ ). Individual points represent the mean of four independent incubations.
  • FIG. 3A Non-competitive inhibition of MFC metabolism by endoxifen.
  • FIG. 3B Eadie-Hofstee plot of inhibition of aromatase by endoxifen with increasing inhibitor concentrations: 0 (A), 1.56 (o), 3.13 ( ⁇ ), 6.25 ( ⁇ ) and 12.5 ( ⁇ ) ⁇ .
  • a range of MFC concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 1.56, 3.13, 6.25 or 12.5 ⁇ endoxifen.
  • the rates of HFC generation were determined by measuring fluorescence response as described in the methods section. Individual points represent the mean of duplicate incubations
  • FIG. 4 A Non-competitive inhibition of MFC metabolism by NDMT.
  • FIG. 4B Eadie-Hofstee plot of inhibition of aromatase by NDMT with increasing inhibitor concentrations: 0 (A), 3.13 (o), 6.25 ( ⁇ ), 12.5 ( ⁇ ) and 25 ( ⁇ ) ⁇ .
  • a range of MFC concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 3.13, 6.25, 12.5 or 25 ⁇ NDMT.
  • the rates of HFC generation were determined by measuring fluorescence response as described in the methods section. Individual points represent the mean of duplicate incubations
  • FIG. 5A Non-competitive inhibition of testosterone metabolism by endoxifen.
  • FIG. 5B Eadie-Hofstee plot of inhibition of aromatase by endoxifen with increasing inhibitor concentrations: 0 ( ⁇ ), 50 (A), 100 (o), 200 ( ⁇ ), 400 ( ⁇ ) and 600 ( ⁇ ) ⁇ .
  • a range of testosterone concentrations was incubated with 50 nM recombinant human aromatase for 10 min in the absence and presence of 0, 50, 100, 200, 400 or 600 ⁇ endoxifen.
  • the rates of estradiol generation were determined by HPLC with UV detection as described in the methods section. Individual points represent the mean of duplicate incubations.
  • FIG. 6 A Competitive inhibition of aromatase by norendoxifen.
  • a range of substrate (MFC) concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of norendoxifen.
  • Dixon plot of inhibition of aromatase by norendoxifen with substrate (MFC) concentrations set at 10 (o), 20 ( ⁇ ), 30 ( ⁇ ), 50 ( ⁇ ) and 100 ( ⁇ ) ⁇ .
  • FIG. 6B Lineweaver-Burke plot of inhibition of aromatase with increasing norendoxifen concentrations: 0 ( ⁇ ), 10 ( ⁇ ), 25 (o), 50 ( ⁇ ) and 100 ( ⁇ ) nM. Individual points represent the mean of duplicate incubations
  • FIG. 7 Selective inhibition of CYP450 isoforms by norendoxifen.
  • the remaining activity of human placental aromatase ( ⁇ ), human liver CYP3A (o), human liver CYP2C9 ( ⁇ ) and human liver CYP2C19 (A) were determined by measuring the formation rates of metabolites from specific probe drugs and were expressed as percentage of control. Individual points represent the mean of three to four independent incubations
  • FIG. 8 Structure-function relationships: stepwise hydroxylation and demethylation of tamoxifen progressively increase the potency of aromatase inhibition.
  • the horizontal open arrows represent the addition of a hydroxyl group.
  • the vertical dark arrows represent the removal of a methyl group.
  • FIG. 9 Hypothetical binding mode of E-norendoxifen in the human aromatase active site (PDB ID 3eqm).
  • the ligands are gray, with oxygen depicted in red and nitrogen in blue.
  • the protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors.
  • the stereo view is programmed for walleyed (relaxed) viewing
  • FIG. 10 Hypothetical binding mode of Z-norendoxifen in the human aromatase active site (PDB ID 3eqm).
  • the ligands are gray, with oxygen depicted in red and nitrogen in blue.
  • the protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors.
  • the stereoview is programmed for wall-eyed (relaxed) viewing
  • FIG. 11 Hypothetical binding mode of 4,4'-dihydroxytamoxifen in the human aromatase active site (PDB ID 3eqm).
  • the ligands are gray, with oxygen depicted in red and nitrogen in blue.
  • the protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors.
  • the stereoview is programmed for wall-eyed (relaxed) viewing.
  • the terms 'therapeutically effective dose,' 'therapeutically effective amounts,' and the like refers to a portion of a compound that has a net positive effect on the health and well being of a human or other animal.
  • Therapeutic effects may include an improvement in longevity, quality of life and the like these effects also may also include a reduced susceptibility to developing disease or deteriorating health or well being.
  • the effects may be immediate realized after a single dose and/or treatment or they may be cumulative realized after a series of doses and/or treatments.
  • Pharmaceutically acceptable salts include salts of compounds of the invention that are safe and effective for use in mammals and that possess a desired therapeutic activity.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-
  • Certain compounds of the invention may form pharmaceutically acceptable salts with various amino acids.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • endoxifen 4-hydroxy-N-desmethyl-tamoxifen
  • Z-4-hydroxy-tamoxifen 4HT
  • endoxifen 4-hydroxy-N-desmethyl-tamoxifen
  • HT Z-4-hydroxy-tamoxifen
  • aromatase inhibitors are generally used to treat post-menopausal women who have tumors that are "estrogen receptor positive" by widely used clinical assays. Nearly 80% of all newly diagnosed breast cancers are positive for the estrogen receptor (ER).
  • AIs aromatase inhibitors
  • aromatase inhibitors There are three commercially available aromatase inhibitors: the azoles, anastrozole (ArimidexTM) and letrozole (FemaraTM), which are potent competitive inhibitors, and the steroidal compound, exemestane (AromasinTM), which is a mechanism-based inhibitor. All three of these compounds result in decreased circulating estrogen concentrations by at least 10-fold compared to concentrations before treatment in untreated postmenopausal women. Despite their efficacy, it is clear that many patients with ER positive breast cancer do not benefit from an adjuvant aromatase inhibitor, even if their tumor is destined to recur, and more than half of women with ER positive breast cancer still develop incurable metastastic breast cancer. It follows that there remains a need for better therapies or improved administration of existing therapies.
  • AIMSS syndrome consists of a constellation of musculoskeletal symptoms, including generalized arthralgias, trigger finger, digital stiffness, carpal tunnel syndrome, or tendinitis/tendinopathy that occur in the absence of any alternative reason for development of these symptoms, such as trauma, pre-existing rheumatoid arthritis, or other definable causes.
  • Previously studies suggest that AIMSS is the reason for discontinuation in 10-20% of all patients taking an aromatase inhibitor and in the majority of those who discontinue therapy.
  • AIMSS was the reason for discontinuation in 70%> of those who stopped the drug.
  • tamoxifen may be mediated in part by actions of tamoxifen or its metabolites as aromatase inhibitors.
  • tamoxifen and its primary human metabolites were tested.
  • the data disclosed herein suggests that some tamoxifen metabolites are able to inhibit aromatase with potencies in the nanomolar range, entirely consistent with the potency of the known aromatase inhibitors.
  • the compounds describe herein have the potential to provide alternative treatment options, or to be more effective medications than the current aromatase inhibitors especially if they are more tolerable.
  • Suitable aromatase inhibitors for use in the present invention are a series of compounds that have triphenylalkene structure with side chain(s) on the phenyl group(s) that are oxyalkanes or oxyalkenes and that terminate in an unsubstituted or mono-substituted amine, including a geometric isomer, a stereoisomer, a pharmaceutically acceptable salt, an ester thereof or a metabolite thereof.
  • Such structures include, but are not limited to the N-demethylated metabolites of tamoxifen, such as N-desmethyltamoxifen and N,N-didesmethyl-4- hydroxytamoxifen.
  • 4-hydroxylated derivatives such as N-desmethyl- 4-hydroxytamoxifen (endoxifen) and norendoxifen may be used as drugs with combined effects involving both aromatase inhibition and selective estrogen receptor modulation.
  • endoxifen N-desmethyl- 4-hydroxytamoxifen
  • norendoxifen may be used as drugs with combined effects involving both aromatase inhibition and selective estrogen receptor modulation.
  • This range of structures and their attendant pharmacologic potencies provides a reasonable pharmacophore upon which to build novel aromatase inhibitors that operate via this new biochemical mechanism.
  • the activity of aromatase was determined by measuring the conversion rate of a fluorometric substrate, 7-methoxy-4- trifluoromethylcoumarin (MFC), to its fluorescent metabolite, 7- hydroxytrifluoromethylcoumarin (HFC), in the presence and absence of multiple concentrations of tested inhibitors as described in methods section below.
  • MFC 7-methoxy-4- trifluoromethylcoumarin
  • HFC 7- hydroxytrifluoromethylcoumarin
  • N,N-didesmethyl-4-hydroxytamoxifen, endoxifen and N- desmethyltamoxifen were further characterized for their mechanisms of action on aromatase.
  • a range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 10, 25, 50 or 100 nM N,N-didesmethyl-4-hydroxytamoxifen. Details of incubation conditions and quantitative assays are provided in the methods section. Individual points represent the mean of duplicate incubations.
  • a range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 1.56, 3.13, 6.25 or 12.5 ⁇ endoxifen. Details of incubation conditions and quantitative assays are provided in the methods section. [0059] A range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 3.13, 6.25, 12.5 or 25 ⁇ N- desmethyltamoxifen. Details of incubation conditions and quantitative assays are provided in the methods section.
  • FIGs. 1 and 8 The basic triphenylalkene structure of tamoxifen is shown as an example, as it undergoes progressive demethylation or hydroxylation.
  • the horizontal open arrows represent the addition of a hydroxyl group, while the vertical, dark arrows represent the removal of a methyl group.
  • inhibitory potency appears to increase, while the addition of a single hydroxyl group also increases potency to some extent.
  • a generic formula for a family of effective aromatase inhibition may be brought about by compounds based upon a triphenylalkene structure with side chain(s) on the phenyl group(s) that are oxyalkanes or oxyalkenes, and that terminate in an unsubstituted or mono- substituted amine.
  • This basic structure can be modified to provide a series of potential aromatase inhibitors for use in various conditions, and could provide a wider range of therapeutic choices than those that are currently available.
  • FIG. 1 shows that endoxifen and NDMT inhibited aromatase with higher potency than tamoxifen or 4HT.
  • substrate concentration was set at 25 ⁇
  • endoxifen and NDMT exhibited IC50S of 6.1 ⁇ and 20.7 ⁇ , respectively, while tamoxifen and 4HT were estimated to have IC50S of 986 ⁇ and 531 ⁇ , respectively (Table 4).
  • letrozole was used as a positive control and had an IC50 of 5.4 nM.
  • the endogenous substrate of aromatase, testosterone was included as a competing substrate, an IC50 of 0.33 ⁇ was observed.
  • IC50 values were determined when MFC and aromatase concentrations were set at 25 ⁇ and 7.5 nM respectively, as described in the methods section.
  • Table 5 Effect of endoxifen and NDMT on Ks app and V ma5a - of recombinant human aromatase
  • Ks app the apparent Michaelis constant.
  • V maxz - the apparent maximum reaction rate.
  • Ks app and m a5 u values were determined using Lineweaver-Burke plot as described in the methods section.
  • the data were plotted as Dixon and Eadie-Hofstee plots (FIGs. 2 to 5).
  • the parallel relationship of the lines in the Eadie-Hofstee plots is consistent with decreasing maximum enzyme activity V maxz and unchanged substrate equilibrium dissociation constant Ks app as inhibitor concentration was increased (FIGs. 3B and 4B), observations that were also consistent with a non-competitive mechanism.
  • the data in these assays indicate a K; for endoxifen of 4.0 ⁇ , and a K ; for NDMT of 15.9 ⁇ .
  • Tissue concentrations of endoxifen are higher, especially in breast tumors, where they appear to be 10 to 100 times more, i.e. above 10 ⁇ . Furthermore, in rats the ratio of endoxifen concentrations in uterus to those in serum has been reported to be at least 20: 1 , and to be at least 500: 1 between lung and serum. These high tissue concentrations are consistent with the large apparent distribution volume for tamoxifen, the parent drug, which is about 50 to 60 liters/kg in humans, indicating that most of the drug (99.9%) is present in peripheral compartments, and suggesting extensive tissue binding.
  • FIG. 1 shows the relative potency of tamoxifen and its available metabolites as AIs.
  • N-desmethyl-tamoxifen, ⁇ -hydroxy- tamoxifen and tamoxifen-N-oxide were all relatively weak inhibitors.
  • the inhibitory potency order of the tested compounds was as follows: norendoxifen » 4,4'-dihydroxy-tamoxifen > endoxifen > N-desmethyl-tamoxifen, N-desmethyl-4'-hydroxy- tamoxifen, tamoxifen-N-oxide, 4'-hydroxy-tamoxifen, N-desmethyl-droloxifene > 4-hydroxy- tamoxifen, tamoxifen.
  • CYP 19 Three experimental systems were used to test inhibition of aromatase (CYP 19), CYP2C9, CYP2C19 and CYP3A by norendoxifen: drug incubations with recombinant CYP isoforms, pooled placental microsomes or pooled human liver microsomes. Initially, when recombinant CYP isoforms were used, norendoxifen inhibited CYP 19, CYP2C9 and CYP2C19 with IC 50 values of 30, 95 and 61 nM respectively. These data did not suggest obvious CYP isoform selectivity.
  • CYP3 A was not tested in this system. Instead, the selectivity of norendoxifen was further characterized using pooled placental and pooled human liver microsomes under more
  • Norendoxifen inhibited placental aromatase with an IC 50 value of 90 nM, while it inhibited human liver CYP2C9 and CYP3A with IC 50 values of 990 and 908 nM respectively (FIG. 7). Inhibition of human liver CYP2C19 by norendoxifen appeared even weaker, with less than 25% inhibition observed at concentrations up to 5 ⁇ (FIG. 7).
  • the docking and energy minimization procedure was validated by reproducing the published crystal structure of aromatase-androstenedione complex by extracting the ligand structure and then docking it back into the aromatase active site, merging the highest- scored binding pose with the protein, and then minimizing the complex energy following the same protocol used with other tamoxifen metabolites.
  • the root mean standard deviation between the structure of the newly generated complex derived from molecular modeling and the original crystal structure (PDB ID 3eqm) was 1.73 A.
  • the unsubstituted phenyl ring and the ethyl moiety in both E and Z forms are surrounded by hydrophobic residues including Phe221, Leu477, Val370, Ile70, and the benzene ring of Trp224.
  • the phenyl ring that contains the hydroxyl group is calculated to from a possible side-to-face stacking interaction with Phel34 in both isomeric forms. A comparison of the two complexes reveals that the ethyl and phenyl groups switch locations, but the two remaining phenyl rings that contain hydrogen bonding substituents maintain their positions.
  • Aromatase inhibition was observed to occur via a non-competitive mechanism, which is consistent with an allosteric interaction with aromatase. This may explain why it was possible for endoxifen to effectively inhibit testosterone metabolism, although the observed IC 50 value for inhibition of MFC metabolism by testosterone was 19-fold lower than that of endoxifen (Table 4).
  • the structure of the active catalytic site of aromatase and its interactions with androgens has been well studied. However, potential interactions at other drug binding sites have not been considered until now. It is possible that the allosteric inhibition occurs via a site remote from the catalytic site, or that it occurs via interaction of two drugs that bind differently within the active site.
  • aromatase inhibition by its metabolites is more prominent.
  • the data may help explain the inconsistency in observed associations between CYP2D6 genotype and outcomes in patients with breast cancer. If aromatase inhibition contributes to the action of tamoxifen, then it is possible that this inhibition may confound simple associations between endoxifen
  • an estrogen receptor modulator that is not an aromatase inhibitor may inadequately represent tamoxifen action in vivo.
  • Tamoxifen metabolites including endoxifen and N-desmethyl-tamoxifen can act as AIs in vitro with K z values of 4 and 15.9 ⁇ respectively.
  • norendoxifen is a potent and selective inhibitor of human aromatase with a IQ value in the nanomolar range, close to the potency of the positive control used: letrozole (IC 50 of 5.3 nM), which is the most potent AI that is available for clinical use.
  • Norendoxifen also appears to be a selective AI.
  • norendoxifen is a known metabolite of tamoxifen in humans, little is known about its tissue concentrations or its contribution to tamoxifen effects. It is a minor metabolite of tamoxifen that exists at notably lower concentrations than the parent drug or its major metabolites, but these data make clear that it is a much more potent inhibitor of aromatase than the other known inhibitory tamoxifen metabolites, endoxifen and N-desmethyl-tamoxifen. In as much as these two metabolites may contribute to tamoxifen action via aromatase inhibition, it is equally possible that norendoxifen contributes significantly to the clinical effects of tamoxifen. In addition, endoxifen itself is being developed as a drug. Accordingly, the role of norendoxifen, the demethylated metabolite, in endoxifen action may be even more important.
  • norendoxifen is the demethylated metabolite of endoxifen, a widely recognized and potent estrogen receptor modulator. It follows that norendoxifen may also act as an estrogen receptor ligand, that is able to modulate estrogen receptor signalling. Norendoxifen or its derivatives may therefore be valuable as alternative AIs that are able to mitigate the debilitating musculoskeletal toxicities experienced by breast cancer patients via tissue specific mechanisms involving estrogen receptor signalling. This possibility deserves further investigation.
  • tamoxifen and related molecules such as norendoxifen and endoxifen and may have multiple pharmacologic effects in the treatment of breast cancer that are mediated by their active metabolites.
  • These data also illustrate the effects of multiple tamoxifen metabolites on aromatase.
  • norendoxifen which is a potent and selective inhibitor.
  • the structure-function relationships characterized and the molecular modelling carried out suggest that norendoxifen merits further investigation as a clinical aromatase inhibitor, and may be able to serve as a lead compound for the rational design of novel aromatase inhibitors.
  • Tamoxifen, N-desmethyltamoxifen, Z-4-hydroxytamoxifen, endoxifen, and letrozole were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). 17- ⁇ - estradiol, testosterone, ⁇ -NADP, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from Sigma-Aldrich (St. Louis, MO). Magnesium chloride was purchased from Fisher Scientific (Pittsburgh PA). All drug solutions were prepared by dissolving each compound in methanol or acetonitrile, and were stored at -20 °C. Tamoxifen and its metabolites were prepared under dim light and in brown tubes to minimize photodegradation.
  • HLMs human liver microsomes
  • CYP cytochrome P450
  • Baculo virus-insect cell-expressed human CYP 19 (with oxidoreductase) and the CYP19/MFC high throughput inhibitor screening kit were purchased from BD Biosciences (San Jose, CA). All microsomal preparations were stored at -80°C.
  • the activity of aromatase is determined by measuring the conversion rate of a fluorometric substrate, 7-methoxy-4-trifluoromethylcoumarin (MFC), to its fluorescent metabolite, 7-hydroxytrifluoromethylcoumarin (HFC).
  • MFC 7-methoxy-4-trifluoromethylcoumarin
  • HFC 7-hydroxytrifluoromethylcoumarin
  • MFC and inhibitors are prepared in acetonitrile. A series of concentrations of inhibitor in a volume of 4 ⁇ are mixed with 96 ⁇ of NADPH-Cofactor Mix (16.3 ⁇ NADP, 828 ⁇ glucose-6-phosphate, 828 ⁇ MgCl 2 , and 0.4 U/ml glucose 6-phosphate dehydrogenase), and prewarmed for 10 min at 37°C. MFC and recombinant human CYP 19 are mixed with 0.1 M potassium phosphate buffer (pH 7.4), and then added to an Enzyme/Substrate Mix.
  • Reactions are initiated by adding 100 ⁇ of Enzyme/Substrate Mix to bring the incubation volume to 200 ⁇ .
  • Final MFC concentrations 10 15, 20 and 25 ⁇ are tested.
  • the final recombinant CYP19 concentration is 7.5 nM.
  • aromatase inhibition was tested using human placental microsomes, experimental conditions were the same as described above except that the final total protein concentration was 0.12 mg/ml.
  • HFC The generation of HFC is determined immediately by measuring the fluorescence response (excitation 400 nm, emission 540 nm) using a BioTek (Winooski, VT) Synergy 2 fluorometric plate reader. Standard curves are constructed using fluorescent metabolite HFC standard. Quantification of metabolite generation is carried out by applying the linear regression equation of the standard curve to the fluorescence response from each sample. The limit of quantification for HFC is 0.02 ⁇ in a final volume of 200 ⁇ , with intra- and inter-day coefficients of variation of 6.2% and 8.4% respectively.
  • aromatase was determined by measuring the rate of conversion of testosterone to estradiol. All incubations were carried out using incubation times and protein concentrations that were within the linear range for reaction velocity. Testosterone and the tested inhibitors were prepared in methanol. All experiments were performed under dim light and in brown, gall tubes to minimize photodegradation of tamoxifen and its metabolites.
  • the final recombinant CYP19 concentration was 50 nM.
  • Final testosterone concentrations of 1, 2, 4 and 8 ⁇ were tested. All reactions were terminated by the addition of 20 ⁇ of 60%> (w/v) perchloric acid, followed by immediate vortexing and placement of the tubes on ice.
  • experiments were carried out as previously described. Lu WJ, Bies R, Kamden LK, Desta Z, Flockhart DA (2010) Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase).
  • the fluorometric substrate, MFC was tested under these same conditions in order to compare IC 50 values of tested inhibitors with a different substrate.
  • estradiol concentrations were analyzed immediately using high performance liquid chromatography (HPLC) assays with ultraviolet (UV) detection as previously described.
  • HPLC high performance liquid chromatography
  • UV ultraviolet
  • Peak areas for each peak were obtained from an integrator, and peak area ratios with internal standard were calculated. Standard curves were constructed by linear regression of peak area ratios. Quantification of samples was carried out by applying the linear regression equation of the standard curve to the peak area ratio.
  • the limit of quantification for estradiol was 2.5 pmol on column, with intra- and inter-day coefficients of variation of 2.4% and 5.3% respectively.
  • Rats are treated with a single intraperitoneal injection of a representative aromatase inhibitor, for example, ⁇ , ⁇ -didesmethyl -4-OH-tamoxifen or vehicle control.
  • a representative aromatase inhibitor for example, ⁇ , ⁇ -didesmethyl -4-OH-tamoxifen or vehicle control.
  • a period of time after administering the compounds the animals are biopsied or sacrificed and tissues of the animals with high aromatase activity including ovary, brain, and adipose tissue are sampled.
  • Aromatase activity in the samples is measured. Standard assays that can be used include following the conversion of testosterone to ⁇ -estradiol.
  • the measured activity may be expressed as aromatase activity per unit of protein e.g., per mg of protein and/or of tissue.
  • Aromatase activity in the treated and untreated (control) animals is compared; the level of aromatase inhibition determined maybe expressed as percent of the aromatase activity measured in animals that are dosed with the vehicle only (control). Inhibition of Aromatase using Placental Microsomes
  • the activity of aromatase was determined by measuring the rate of conversion of testosterone to estradiol. The incubation conditions and the quantification methods were as previously described.
  • the separation column used was Chiral-AGP (150 x 4.60 mm; 5 ⁇ ; Phenomenex).
  • a gradient elution profile was used: initial mobile phase: 95% (v/v) 20 mM ammonium acetate (adjusted to pH 6.5) and 5% acetonitrile; secondary mobile phase: 10% 20 mM ammonium acetate (adjusted to pH 6.5) and 90% acetonitrile.
  • the secondary mobile phase was increased from 0% to 40% linearly between 0 and 8 min; the initial mobile phase was resumed after 9 min and remained constant for an additional 6 min, allowing the column to equilibrate.
  • the elute was introduced, without splitting, at 0.5 ml/min to the turbo ion source.
  • R-hydroxyomeprazole and R-lansoprazole were detected using multiple reactions monitoring at m/z values of 362.13/214.10 and 370.25/252.30, respectively. Formation rates of metabolites from their respective probe substrates were quantified by using the appropriate standard curve. Intra- and inter-day coefficients of variation of the assays were less than 15%.
  • the rates of metabolite formation from substrate probes in the presence of the test inhibitors are compared with those for control in which the inhibitor is replaced with vehicle.
  • the extent of aromatase inhibition is expressed as percent enzyme activity remaining compared to control.
  • the percent of aromatase activity remaining at different inhibitor concentrations is used to estimate IC 50 values when the substrate concentration is set at 25 ⁇ .
  • IC 5 o values are determined as the inhibitor concentration that brought about a 50% reduction in enzyme activity by fitting all the data to a one-site competition equation using Prism version 5.01 for Windows (GraphPad Software Inc., San Diego, CA).
  • v is the velocity of reaction
  • [S] is the substrate concentration
  • [I] is the inhibitor concentration
  • K m is the Michaelis constant
  • V max is the maximum reaction rate.
  • the equilibrium dissociation constant of the inhibitor K z is determined by estimating the intercept using linear regression.
  • Ks app is the apparent Michaelis constant and V ma5a is the apparent maximum reaction rate in the presence of the inhibitor.
  • V ma5a is the apparent maximum reaction rate in the presence of the inhibitor.
  • intercepts on the X-axis are used to determine the apparent K m values and intercepts on the Y-axis are used to determine the apparent V max values.
  • Fresh frozen breast tumor tissue is obtained anonymously from the Indiana University School of Medicine tumor bank.
  • the tissue samples are homogenized under conditions selected to preserve any aromatase activity present in the samples.
  • aromatase activity is measured in either the homogenates or in at least partially purified samples of the homogenates.
  • Aromatase activity is normalized to the amount of protein in the sample analyzed, and maybe expressed in units such as aromatase activity per mg of protein.
  • Methods for measuring aromatase activity include, for example, incubating a portion of the homogenate with testosterone, or another suitable substrate for aromatase and measuring the amount of product produced, for example, ⁇ -estradiol generated or substrate consumed.
  • the aromatase activity assay is repeated in the presence of an inhibitory level of at least one aromatase inhibitor, the substrate testosterone or another suitable substrate and with the homogenized tissue.
  • Aromatase inhibitors that can be used include, for example, N,N- didesmethyl -4-OH-tamoxifen.
  • the levels of aromatase activity measured in the presence and absence of the inhibitor are compared to one another. And the difference, if any, in aromatase activity is calculated; a statistically significant drop in aromatase activity indicates that the sample included a detectable level of at least one aromatase that can be inhibited by the type of aromatase inhibitor used in the assay.
  • the amino side chain was rotated manually to place the nitrogen atom within hydrogen bonding distance to the Ala306 carbonyl oxygen, which ultimately resulted in a more favorable calculated binding energy after energy minimization.
  • the structures of the new ligand-protein complexes were subsequently subjected to energy minimization using the Amber force field with Amber charges.
  • the structures of the compounds of interest and a surrounding 10 A sphere of the protein were allowed to move.
  • the structure of the remaining protein was kept frozen.
  • the energy minimizations were performed using the Powell method with a 0.05 kcal/(mol A) energy gradient convergence criterion and a distance-dependent dielectric function.

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Abstract

La présente invention concerne des triphénylalcènes pouvant être utilisés pour inhiber les aromatases. L'ordre du pouvoir inhibiteur des composés analysés est le suivant : norendoxifène » 4,4'-dihydroxy- tamoxifène > endoxifène > N-desméthyl-tamoxifène, N-desméthyl-4'-hydroxy-tamoxifène, tamoxifène-N-oxyde, 4'-hydroxy-tamoxifène, N-desméthyl-droloxifène > 4-hydroxy-tamoxifène, tamoxifène. Le norendoxifène inhibe l'aromatase recombinante par le biais d'un mécanisme concurrent avec un K i de 35 nM. Le norendoxifène inhibe l'aromatase placentaire avec un IC50 de 90 nM, tandis qu'il inhibe CYP2C9 et CYP3A du foie humain avec des valeurs d'IC50 respectives de 990 et de 908 nM. L'inhibition de CYP2C19 du foie humain par le norendoxifène semble encore plus faible. Aucune inhibition sensible de CYP2B6 et de CYP2D6 par le norendoxifène n'est observée. Ces composés, et les formulations pharmaceutiquement acceptables de ces composés, peuvent être utilisés pour le traitement de maladies, le diagnostic et la surveillance d'affections, y compris certaines formes de cancer du sein. Ces composés peuvent être utilisés seuls ou associés à d'autres thérapies et composés thérapeutiques pour le traitement de maladies telles que le cancer.
PCT/US2012/027346 2011-03-01 2012-03-01 Substances inhibitrices d'aromatase et procédé d'utilisation desdites substances pour le diagnostic, le traitement et la surveillance du cancer du sein WO2012119010A2 (fr)

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