RU2844940C2 - Method of treating cancer using oral dosage form of inhibitor of estrogen receptor alpha - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutical industry, namely to oral dosage forms for treating breast cancer and a method of treating breast cancer. Oral dosage form for treating breast cancer which is a capsule containing: (i) an internal phase containing 150 mg of a compound of formula I or 150 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of the compound of formula I, where said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazole-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide of formula

67.74 mg of lactose monohydrate, 45 mg of low-substituted hydroxypropyl cellulose, 15 mg of microcrystalline cellulose, 9 mg of hydroxypropyl cellulose, 0.6 mg of colloidal anhydrous silicon dioxide and 1.5 mg of magnesium stearate; and (ii) an external phase containing 1.5 mg of magnesium stearate. Oral dosage form for treating breast cancer which is a tablet containing: (i) an internal phase containing 150 mg of a compound of formula I or 150 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of the compound of formula I, where said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazole-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide of formula

241.2 mg of lactose monohydrate, 90 mg of low-substituted hydroxypropyl cellulose, 18 mg of hypromellose and 1.2 mg of colloidal silicon dioxide; and (ii) an external phase containing 12 mg of magnesium stearate and 78 mg of microcrystalline cellulose. Oral dosage form for treating breast cancer which is a capsule containing: i) an internal phase containing 50 mg of a compound of formula I or 50 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of the compound of formula I, 22.58 mg of lactose monohydrate, 15 mg of low-substituted hydroxypropyl cellulose, 5 mg of microcrystalline cellulose, 3 mg of hydroxypropyl cellulose, 0.2 mg of colloidal anhydrous silicon dioxide and 0.5 mg of magnesium stearate, where said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazole-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, having structure

and ii) an external phase containing 0.5 mg of magnesium stearate. Oral dosage form for the treatment of breast cancer, which is a capsule containing: i) an internal phase containing 25 mg of a compound of formula I or 25 mg of a free base equivalent amount of a pharmaceutically acceptable salt of a compound of formula I, 11.29 mg of lactose monohydrate, 7.5 mg of low-substituted hydroxypropyl cellulose, 2.5 mg of microcrystalline cellulose, 1.5 mg of hydroxypropyl cellulose, 0.1 mg of colloidal anhydrous silicon dioxide and 0.25 mg of magnesium stearate, where said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazole-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, having structure

and ii) an external phase containing 0.25 mg of magnesium stearate. Method of treating breast cancer in a human subject, comprising administering to said subject the above-described oral dosage form in a single daily dose in range of 100 mg to 600 mg.

EFFECT: group of inventions described above is effective for treating breast cancer, dosage forms exhibit improved pharmacokinetic profiles.

25 cl, 7 dwg, 17 tbl, 7 ex

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/852,751, filed May 24, 2019, and U.S. Provisional Patent Application No. 62/853,498, filed May 28, 2019. These applications are incorporated herein by reference in their entireties.

Prerequisites for the creation of an invention

[0002] Altered estrogen receptor (ER) signaling is known to play a key role in the development of various types of breast cancer. Activation of ER signaling typically depends on the expression and ability of endogenous steroid hormones, such as estradiol, to enter the cell and interact with intracellular ERs. Once activated, ER signaling promotes a variety of cellular processes, such as proliferation, angiogenesis, metabolism, and cell survival (Toss A. et al. 2017, Int. J. Mol. Sci. , 18(1):85). Mutations in various ERs frequently arise during tumorigenesis, and these mutations confer on cancer cells the ability to activate ER signaling pathways in a ligand-independent manner. A specific ER known to be involved in breast cancer tumorigenesis, ERα, is a hormone-regulated transcription factor that is present in 50% of all breast cancers (Lumachi F, Curr. Med Chem. 2013; 20:596-604).

[0003] Expression of estrogen receptor alpha (ERα, ESR1), a hormone-regulated transcription factor, is found in approximately 70% of breast cancers. Lumachi F, Curr. Med. Chem . 2013; 20:596–604. A number of ERα-targeted therapies have been developed. Resistance to ERα antagonists, involving multiple mechanisms, is common in the clinic. One mechanism, ERα mutations, is found in 20–40% of endocrine-resistant metastases. Li S, Cell Rep . 2013; 4:1116–1130; Robinson DR, Nat Genet . 2013; 45:1446–1451; Toy W, Nat Genet . 2013; 45:1439–1445; Chandarlapaty S, JAMA Oncol . 2016; 2:1310–1315; Spoerke JM, Nat Commun . 2016; 7:11579. Mutations in ERα result in ligand-independent activation of the ERα pathway.

[0004] Endocrine therapy is at the forefront of treatment methods used to treat or cure breast cancer cases. Endocrine therapy is a treatment that involves stimulating or suppressing the expression or function of hormones in the body to treat a specific disease. Endocrine therapy targeting ERα has been shown to be ineffective in the long-term treatment of breast cancer cases, as 20% to 40% of all ERα-expressing breast cancers acquire a mutation in ERα after long-term endocrine therapy, with these mutations rendering the receptor refractory to traditional endocrine therapy (Robinson, DR, Nat. Genet . 2013; 45:1446-1451). Therefore, there is a need in the field of chemotherapy to develop new therapies that can effectively target mutated ERα isoforms that render cancers resistant to endocrine therapy.

During the development of new treatments for endocrine-resistant breast cancer, a new class of ERα inhibitors called selective estrogen receptor covalent antagonists (SERCAs) was discovered. SERCAs inactivate ER signaling by targeting a cysteine residue on the ER that is absent from other nuclear hormone receptors (Puyang, X., Cancer Discov . 2018, 8(9):1176-1193). One of the SERCAs that has emerged during this time is ( E )-N ,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, which is shown by formula I below:

( E )-N ,N -dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide

(Formula I)

[0005] The free base form of ( E ) -N,N -dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide is sometimes referred to as H3B-6545. H3B-6545 is a covalent small molecule inhibitor that inactivates both wild-type (ERα-WT) and mutant ERα (ERα-mut) without disrupting the receptor. H3B-6545 is able to interact with the Cys530 residue of ERα, which causes the receptor to adopt a unique conformation that inhibits the receptor’s ability to facilitate ligand-independent ERα signaling (Puyang, X., Cancer Discov . 2018, 8(9):1176–1193). H3B-6545 has been shown to exhibit potent chemotherapeutic properties in a variety of breast cancer cell lines and patient-derived xenograft models in nude mice (Smith, PG, et al ., Cancer Res . 2017).

[0006] Although H3B-6545 has been shown to be effective in in vitro and in vivo models, the manner in which H3B-6545 should be administered to a human breast cancer patient in need of treatment has yet to be determined. Therefore, there is a need to develop a formulation and dosing regimen of H3B-6545 that will allow the inhibitor to be administered more effectively and safely to patients in need of it. Characterization of the pharmacokinetic (PK) outcomes and PK profile will later enable their general use in human breast cancer treatments.

SUMMARY

[0007] Embodiments of the invention may relate, for example, to an oral dosage form comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E )-N ,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro- 1H- indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide; wherein said oral dosage form, when orally administered to a human once daily, is formulated to achieve a mean C _max of from about 1 ng/mL to about 4 ng/mL, as determined for each mg of the compound of Formula I in the said dosage. In some embodiments, said average C _max is from about 2 ng/mL to about 4 ng/mL for each mg of the compound of Formula I in said dosage. In some embodiments, said average C _max is from about 3 ng/mL to about 4 ng/mL for each mg of the compound of Formula I in said dosage. In some embodiments, said average C _max is in the range of 80%-125% for 3 ng/mL to 80%-125% for 3.5 ng/mL for each mg of the compound of Formula I in said dosage.

[0008] In other embodiments, the dosage form is formulated to achieve a mean t _max at said mean C _max of from about 2 hours to about 7 hours. In other embodiments, the dosage form is formulated to achieve a mean t _max at said mean C _max of from about 3 hours to about 7 hours. In other embodiments, the dosage form is formulated to achieve a mean t _max at said mean C _max of from about 3.5 hours to about 4.5 hours. In other embodiments, the dosage form is formulated to achieve a mean t _max at said mean C _max of from about 5.5 hours to about 6.5 hours.

[0009] In some embodiments, as described above, the dosage form contains a total equivalent of from about 100 mg to about 600 mg of the compound of Formula I. In some embodiments, the dosage form contains a total equivalent of from about 450 mg of the compound of Formula I.

[0010] In further embodiments, the invention provides an oral dosage form comprising a compound represented by Formula I, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E )-N ,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, and wherein said oral dosage form, when orally administered to a human once daily, is formulated to achieve a mean AUC _0-24 of from about 16 h ng/mL to about 44 h ng/mL for each mg of the compound of Formula I at said dosage. In some embodiments, said mean AUC _0-24 is between about 27 h ng/mL and about 44 h ng/mL for each mg of the compound of Formula I in said dosage. In some embodiments, said mean AUC _0-24 is between 80%-125% for 30 h ng/mL and 80%-125% for 44 h ng/mL for each mg of the compound of Formula I in said dosage. In some embodiments, the dosage form contains a total equivalent of about 100 mg to about 600 mg of the compound of Formula I. In some embodiments, the dosage form contains a total equivalent of about 450 mg of the compound of Formula I.

[0011] In further embodiments, the invention provides an oral dosage form comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound represented by Formula I is ( E ) -N,N -dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, and wherein said oral dosage form, when orally administered to a human once daily, is formulated to achieve a mean t _1/2 of the compound of Formula I at said dose of from about 8 hours to about 22 hours. In other embodiments, the mean t _1/2 is from about 8 hours to about 12 hours. In other embodiments, the average t _1/2 is from about 9 hours to about 11 hours. In some embodiments, the dosage form contains a total equivalent of from about 100 mg to about 600 mg of the compound of formula I. In some embodiments, the dosage form contains a total equivalent of from about 450 mg of the compound of formula I.

[0012] In further embodiments, the invention provides an oral dosage form comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound represented by Formula I is ( E ) -N,N -dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, and wherein said oral dosage form, when orally administered to a human once daily, is formulated to achieve a mean AUC _0-inf of from about 21 h ng/mL to about 67 h ng/mL for each mg of the compound of Formula I at said dosage. In some embodiments, the mean AUC _0-inf is from about 29 h ng/mL to about 67 h ng/mL for each mg of the compound of Formula I in a given dosage. In some embodiments, the mean AUC _0-inf is in the range of 80% to 125% for 36 h ng/mL to 80% to 125% for 57 h ng/mL for each mg of the compound of Formula I in a given dosage. In some embodiments, the dosage form contains a total equivalent of from about 100 mg to about 600 mg of the compound of Formula I. In other embodiments, the dosage form contains a total equivalent of from about 450 mg of the compound of Formula I.

[0013] Still further embodiments may include dosage forms described herein having means for achieving the pharmacokinetic values described herein.

[0014] In a further embodiment of the invention there is provided an oral dosage form comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide and wherein said oral dosage form, when orally administered to a human once daily, is formulated to achieve a mean AUC _0-t of from about 16 h ng/mL to about 41 h ng/mL for each mg of the compound of Formula I at said dosage. In another embodiment, said mean AUC _0-t is in the range of 80%-125% for 27 h ng/mL to 80%-125% for 36 h ng/mL for each mg of the compound of formula I in said dosage. In another embodiment, said dosage form contains a total equivalent of about 100 mg to about 600 mg of the compound of formula I. In another embodiment, said dosage form contains a total equivalent of about 450 mg of the compound of formula I.

[0015] In other embodiments, the dosage forms reported above may be a capsule comprising an inner phase that comprises a compound of formula I or a pharmaceutically acceptable salt, lactose monohydrate, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose, colloidal anhydrous silicon dioxide, and magnesium stearate; and an outer phase that comprises magnesium stearate. In some embodiments, the capsule is a hypromellose capsule. In some embodiments, the capsule comprises a mono-HCl salt form of the compound of formula I.

[0016] In some embodiments, as described above, the oral dosage form is a tablet comprising an internal phase that comprises a compound of Formula I or a pharmaceutically acceptable salt, lactose monohydrate, low-substituted hydroxypropyl cellulose, hypromellose, colloidal silicon dioxide, and purified water; an external phase that comprises microcrystalline cellulose and magnesium stearate; and a film coating that comprises hypromellose, talc, titanium dioxide, propylene glycol, iron oxide, and purified water. In other embodiments, the tablet comprises a mono-HCl salt form of the compound of Formula I.

[0017] In further embodiments, a method of treating a cancer in a human comprising administering to said subject an oral dosage form comprising a therapeutically effective amount of a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E ) -N,N- dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, wherein said therapeutically effective amount is a single daily dose in the range of about 100 mg to 600 mg and wherein said oral dosage form has a mean C _max of about 1 ng/mL to about 4 ng/mL in said subject's blood plasma for each mg of the compound of Formula I in a stated dosage. In some embodiments, said mean C _max is from about 2 ng/mL to about 4 ng/mL for each mg of the compound of Formula I in a stated dosage. In some embodiments, said mean C _max is from about 3 ng/mL to about 4 ng/mL for each mg of the compound of Formula I in a stated dosage. In some embodiments, said mean C _max is in the range of 80% to 125% for 3 ng/mL to 80% to 125% for 3.5 ng/mL for each mg of the compound of Formula I in a stated dosage. In some embodiments, the dosage form has a mean t _max at said mean C _max of the compound of Formula I from about 2 hours to about 7 hours. In some embodiments, the dosage form has a mean t _max at said mean C _max of the compound of Formula I from about 3 hours to about 7 hours. In some embodiments, the dosage form has a mean t _max at said mean C _max of the compound of formula I from about 3.5 hours to about 4.5 hours. In some embodiments, the dosage form has a mean t _max at said mean C _max of the compound of formula I from about 5.5 hours to about 6.5 hours.

[0018] Other embodiments may relate to a method of treating cancer in a human comprising administering to said subject an oral dosage form comprising a therapeutically effective amount of a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound represented by Formula I is (E) -N,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide and wherein said oral dosage form has a mean AUC _0-24 of from about 16 h ng/mL to about 44 h ng/mL for each mg of the compound of Formula I at said dosage. In other embodiments, said mean AUC _0-24 is from about 27 h ng/mL to about 44 h ng/mL for each mg of the compound of formula I in said dosage. In other embodiments, said mean AUC _0-24 is in the range of 80%-125% for 30 h ng/mL to 80%-125% for 44 h ng/mL for each mg of the compound of formula I in said dosage.

[0019] In further embodiments, a method of treating a cancer in a human comprising administering to said subject an oral dosage form comprising a therapeutically effective amount of a compound represented by Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E ) -N,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide and wherein said oral dosage form has a mean t _1/2 of from about 8 hours to about 22 hours. In some embodiments, said mean t _1/2 is from about 8 hours to about 12 hours. In some embodiments of the invention, said average t _1/2 value is from about 9 hours to about 11 hours.

[0020] In further embodiments, a method of treating a cancer in a human comprising administering to said subject an oral dosage form comprising a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E ) -N,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide and wherein said oral dosage form has a mean AUC _{0-inf of} from about 21 h ng/mL to about 67 h ng/mL for each mg of the compound of Formula I at said dosage. In some embodiments, said mean AUC _0-inf is from about 29 h ng/mL to about 67 h ng/mL for each mg of the compound of Formula I at said dosage. In some embodiments, said mean AUC _0-inf is in the range of 80%-125% for 36 h ng/mL to 80%-125% for 57 h ng/mL for each mg of the compound of Formula I at said dosage.

[0021] In further embodiments, a method of treating a cancer in a human comprising administering to said subject an oral dosage form comprising a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, wherein said compound of Formula I is ( E ) -N,N- dimethyl-4-[2-[5-[( Z )-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide and wherein said oral dosage form has a mean AUC _0-t of from about 16 h ng/mL to about 41 h ng/mL for each mg of the compound of Formula I at said dosage. In other embodiments, the mean AUC _0-t value is in the range of 80%-125% for 27 h ng/mL to 80%-125% for 36 h ng/mL for each mg of the compound of formula I at the stated dosage.

[0022] In some embodiments of the methods described herein, the dosage form comprises a total equivalent of about 100 mg to about 600 mg of the compound of Formula I. In other embodiments, the dosage form comprises a total equivalent of about 200 mg to about 600 mg of the compound of Formula I. In other embodiments, the dosage form comprises a total equivalent of about 300 mg to about 600 mg of the compound of Formula I. In other embodiments, the dosage form comprises a total equivalent of about 450 mg of the compound of Formula I.

[0023] In some embodiments of the methods described herein, the oral dosage form is a capsule comprising an inner phase that comprises a compound of Formula I or a pharmaceutically acceptable salt, lactose monohydrate, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose, colloidal anhydrous silicon dioxide, and magnesium stearate; and an outer phase that comprises magnesium stearate. In some embodiments, the capsule is a hypromellose capsule. In some embodiments, the capsule comprises a mono-HCl salt form of the compound of Formula I.

[0024] In some embodiments, as reported herein, the oral dosage form is a tablet comprising an internal phase that comprises a compound of Formula I or a pharmaceutically acceptable salt, lactose monohydrate, low-substituted hydroxypropyl cellulose, hypromellose, colloidal silicon dioxide, and purified water; an external phase that comprises microcrystalline cellulose and magnesium stearate; and a film coating comprising hypromellose, talc, titanium dioxide, propylene glycol, iron oxide, and purified water. In some embodiments, the tablet comprises a mono-HCl salt form of the compound of Formula I.

[0025] In some embodiments of the methods described herein, the malignancy is breast cancer. In some embodiments, the breast cancer is ERα-positive breast cancer. In some embodiments, the breast cancer expresses wild-type ERα. In some embodiments, the breast cancer expresses mutant ERα.

[0026] In some embodiments of the invention, as reported herein, said oral dosage form is administered once per day. In some embodiments, said oral dosage form is administered to a human on an empty stomach. In some embodiments, said oral dosage form is administered to a human in a fed state.

[0027] In embodiments of the invention, a method of treating a patient with compound H3B-6545 is provided:

[0028] (H3B-6545)

[0029] or a pharmaceutically acceptable salt thereof, comprising determining whether one or more reductions in mutant allele frequencies are observed in the patient's blood, selecting a patient if said reductions are observed, and administering to the selected patient a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof. The mutant allele frequency is the relative frequency of the mutant allele (gene variant) at a particular locus in the population, expressed as a fraction or percentage.

[0030] H3B-6545 is described in U.S. Patent No. 9,796,683 B2, incorporated herein by reference.

[0031] In some embodiments of the invention, as reported herein, the pharmaceutically acceptable salt of H3B-6545 is the HCl salt of H3B-6545.

[0032] In some embodiments, the reduction in mutant allele frequency is observed in one or more alleles selected from the group consisting of PIK3CA, ESR1, TSC1, TP53, FGFR1, CCND1, ARID1A, POLE, FGF19, MET, NOTCH3, FGF3, AKT1, KRAS, MYC, ERBB2, ERBB3, FGFR3, PMS2, PTEN, RB1, BRAF, MDM2, ATR, ATRX, BRCA2, SETD2, ATM, FANCA, JAK2, NF1, SLX4, and SMAD4.

[0033] In another embodiment, a decrease in mutant allele frequency is observed in AKT1. In another embodiment, the location of the AKT1 mutation causing the observed decrease in mutant allele frequency is E17. In another embodiment, the AKT1 mutation causing the observed decrease in mutant allele frequency is E17K.

[0034] In another embodiment, the reduction in mutant allele frequency is observed in ESR1. In yet another embodiment, the location(s) of the ESR1 mutation causing the observed reduction in mutant allele frequency is selected from one or more members of the group consisting of E380, D538, L536, S463, and Y537. In another embodiment, the ESR1 mutation(s) causing the observed reduction in mutant allele frequency is selected from one or more members of the group consisting of E380Q, D538G, L536H, L536P, L536R, S463P, Y537C, Y537N, and Y537S. In another embodiment, the ESR1 mutation causing the observed reduction in mutant allele frequency is S463P. In another embodiment of the invention, the ESR1 mutation causing the observed reduction in mutant allele frequency is Y537N.

[0035] In another embodiment, the reduction in mutant allele frequency is observed in PIK3CA. In another embodiment, the location(s) of the PIK3CA mutation causing the observed reduction in mutant allele frequencies is selected from one or more members of the group consisting of E542, E545, and H1047. In another embodiment, the PIK3CA mutation(s) causing the observed reduction in mutant allele frequencies is selected from one or more members of the group consisting of E542K, E545K, H1047L, and H1047R.

[0036] The following embodiments of the invention may relate to a method of discontinuing treatment with compound H3B-6545 or a pharmaceutically acceptable salt thereof in a patient undergoing treatment with compound 3B-6545:

[0037] (H3B-6545)

[0038] or a pharmaceutically acceptable salt thereof, comprising determining whether one or more increases in mutant allele frequencies are observed in the patient's blood and discontinuing treatment of the patient if said increase is observed.

[0039] In some embodiments, the increase in mutant allele frequencies is observed in one or more alleles selected from PIK3CA, ESR1, TSC1, TP53, FGFR1, CCND1, ARID1A, POLE, FGF19, MET, NOTCH3, FGF3, AKT1, KRAS, MYC, ERBB2, ERBB3, FGFR3, PMS2, PTEN, RB1, BRAF, MDM2, ATR, ATRX, BRCA2, SETD2, ATM, FANCA, JAK2, NF1, SLX4, and SMAD4.

[0040] In some embodiments, the increase in mutant allele frequencies is observed in ESR1. In yet further embodiments, the location of the ESR1 mutation causing the observed increase in mutant allele frequencies is selected from one or more members of the group consisting of E380, D538, L536, S463, and Y537. In other embodiments, the ESR1 mutation(s) causing the observed increase in mutant allele frequencies is selected from at least one or more members of the group consisting of E380Q, D538G, L536H, L536P, L536R, S463P, Y537C, Y537N, and Y537S.

[0041] In other embodiments, the increase in mutant allele frequencies is observed in PIK3CA. In yet further embodiments, the location(s) of the PIK3CA mutation causing said observed increase in mutant allele frequencies is selected from the group consisting of E542, E545, and H1047. In yet further embodiments, the PIK3CA mutations causing said observed increase in mutant allele frequencies are selected from the group consisting of E542K, E545K, H1047L, and H1047R. In other embodiments, the PIK3CA mutation is E545K.

[0042] In another embodiment, an increase in mutant allele frequencies is observed in AKT1. In another embodiment, the location of the AKT1 mutation causing the observed increase in mutant allele frequencies is E17. In other embodiments, the AKT1 mutation causing the observed increase in mutant allele frequencies is E17K.

[0043] In still further embodiments of the invention, the identification step is performed after the patient has received at least one treatment with the H3B-6545 compound:

(H3B-6545)

[0044] or a pharmaceutically acceptable salt thereof.

[0045] In other embodiments, said patient has breast cancer. In yet further embodiments, said breast cancer is an estrogen receptor alpha (ERα) positive breast cancer. In yet further embodiments, said ERα has a mutation.

[0046] In still further embodiments, an increase in the frequency of mutant alleles is observed in circulating tumor DNA. In other embodiments, a decrease in the frequency of mutant alleles is observed in circulating tumor DNA.

[0047] The documents presented herein are incorporated by reference. If these documents conflict with this document, this document shall control.

BRIEF DESCRIPTION OF DRAWINGS

[0048] FIG. 1 shows the mean plasma concentration-time profile of H3B-6545 (Cycle 1, Day 1) for Example 3. Preliminary PK analyses indicate that H3B-6545 exhibits an approximately dose-proportional increase in plasma exposure from 100 to 600 mg. T _max and half-life are independent of dose and time.

[0049] FIG. 2 shows the tumor response and duration of treatment with H3B-6545, as described in Example 3. Partial response, stable response, disease progression, and unassessable are shown for each tumor assessment. Arrows indicate treatment ongoing at the time of data cutoff.

[0050] FIG. 3 shows the percentage change in the sum of the diameters of the target lesions as described in Example 3.

[0051] FIG. 4 shows an overview of the liquid biopsy and tissue biopsy sample sets.

[0052] FIG. 5A-FIG. 5E show the baseline genomic profile of the patients of Example 7. FIG. 5A-5E show the BEAMing analysis. A. Baseline ESR1 and PIK3CA mutation status of the patients; B and C. Clonality of ESR1 and PIK3CA mutations; D and E. Amino acid distribution of PIK3CA and ESR1 mutations. FIG. 5F shows the oncoprint of mutations detected in the baseline tissue biopsy, as determined by the Oncomine Comprehensive Panel.

[0053] FIG. 6A and FIG. 6B show the correspondence of mutations detected in the tissue and liquid biopsy of Example 7. FIG. 6A shows the distribution of allele frequencies and the correspondence of mutations in the tissue and liquid biopsy. FIG. 6B is a summary table of the correspondence of mutations in the tissue and liquid biopsy.

[0054] FIGS. 7A-7C show that ctDNA dynamics correlate with overall best response to H3B-6545. AF=allele frequency, C=cycle, D=day, PD=disease progression, PR=partial response, SD=disease stable. The dotted line represents the LOD of the ddPCR assay. FIG. 8A shows the baseline AF/C2D1 ratio for each patient, stained individually. A single patient may have multiple mutations. FIGS. 7B and FIG. 7C show an example of ctDNA dynamics in a patient with disease progression (B) versus a partial response (C).

DETAILED DESCRIPTION

[0055] Definitions

[0056] The use of the singular forms herein shall be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. For example, the term "disintegrant" refers to one or more disintegrants included in or suitable for use in a formulation described herein. Similarly, the term "therapeutic amount" refers to one or more therapeutic amounts included in or suitable for use in a dosage form.

[0057] The terms "comprising,""having,""including," and "comprising" are to be construed as open terms (i.e., meaning "including, but not limited to") unless otherwise specified. Furthermore, whenever "comprising" or another open term is used in an embodiment of the invention, it is to be understood that the same embodiment may be claimed more narrowly using the intermediate term "consisting essentially of" or the closed term "consisting of."

[0058] The term "bioequivalent" or "bioequivalence" is a term of art and is intended to be defined in accordance with the Approved Drug Products with Therapeutic Equivalence Evaluations, 34th Edition, published by the U.S. Department of Health and Human Services and commonly known as the "Orange Book." Bioequivalence between different dosage forms of the same drug substance involves equivalence in the rate and extent of absorption of the drug. The extent and rate of absorption of the test formulation are compared with a reference formulation to determine whether the two formulations are bioequivalent. A standard bioequivalence study is conducted in a crossover fashion through a large study that involves administering single doses of the test and reference formulations to multiple volunteers, typically 12 to 24 healthy normal adults, and then measuring the blood or plasma levels of the drug over time. Detailed guidelines for establishing bioequivalence of a formulation with a reference formulation have been published by the FDA Office of Generic Drugs, Division of Bioequivalence.

[0059] As used herein, the term "mean value" refers to the geometric mean determined from a set of independent measurements. For example, the independent measurements may be collected from a statistically significant population. As further examples, when used to describe pharmacokinetic parameters (such as "mean C _max ", "mean AUC _0-x ", "mean AUC _0-t ", "mean AUC _0-inf ", "mean t _max ", or "mean t _1/2 " (or "mean half-life ")), the "mean value" refers to the geometric mean pharmacokinetic value obtained from the population from which the individual measurements were respectively collected. Therefore, as used herein, a dosage form may be administered to a human, wherein the dosage form has an average pharmacokinetic value obtained from a set of independently determined values.

[0060] A list of abbreviations and definitions of terms used in this application is as follows. AUC: area under the plasma concentration-time curve; AUC _0-x : area under the plasma concentration-time curve from zero to x hours post-dose (e.g., x may represent 12 or 24 hours); AUC _0-t : area under the plasma concentration-time curve from time zero to the last quantifiable concentration; AUC _0-inf : area under the plasma concentration-time curve from zero to infinity; ANCOVA: analysis of covariance; CI: confidence interval; C _max : maximum drug concentration; C _x : plasma concentration at x hours post-dose; CV: coefficient of variation; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; MAD: multiple ascending dose; MTD: maximum tolerated dose; PD: pharmacodynamics; PK: pharmacokinetics; RT: response time; SAD: single ascending dose; SD: standard deviation; t _1/2 : terminal half-life; t _max : time to reach maximum (peak) concentration after drug administration. As used herein, t _1/2 includes the terminal half-life of drug concentration, which may be the terminal half-life C _max . As used herein, C _max includes the maximum concentration of drug measured in human plasma.

[0061] Two dosage forms whose rate and extent of absorption differ by -20%/+25% or less are generally considered "bioequivalent." Another approach to average bioequivalence involves calculating a 90% confidence interval for the ratio of means (population geometric means) of the test and reference products. To establish bioequivalence, the calculated confidence interval should typically be in the range of 80-125% for the ratio of product means. In addition to this general approach, other approaches may be used to establish bioequivalence, including (1) log-transformation of pharmacokinetic data, (2) sequence effect assessment methods, and (3) outlier assessment methods. For example, in (1) above, the confidence interval should typically be in the range of 80-125% for the difference in log-transformed mean.

[0062] The term "about,""approximately," or "approximately," as used herein when referring to a measurable quantity such as a quantity, a time duration, and the like, is intended to cover deviations of ±20% or 10%, more preferably 5%, even more preferably 1%, and even more preferably 0.1%, from the stated value, as such variations are acceptable in the context.

[0063] When an amount or dosage range is indicated by weight or mass in the format "a compound of Formula I" or "H3B-6545" or "the free base of H3B-6545", one skilled in the art will appreciate that providing an equivalent molar amount of the active compound as a pharmaceutically acceptable salt will typically require administration of a greater mass of the salt than would be required if the compound were administered alone (i.e., the amount of free base and the amount of salt are in a 1:1 molar ratio). For example, the phrase "25 mg to 50 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof" means the free base of Formula I in an amount of 25 mg to 50 mg (including endpoints), as well as the monohydrochloride salt of the compound of Formula I in an amount of 26 mg to 53 mg (including endpoints). This conversion may be referred to, for example, as a "salt conversion factor", "salt correction factor", or "activity adjustment factor".

[0064] The efficiency control factor transformation also applies to crystalline forms of the compound of formula I existing as hydrates, solvates, or crystalline forms of the compound of formula I having both i) hydrate or solvate and ii) salt counterions. Moreover, such efficiency control factor transformations apply regardless of whether the co-crystallized solvent molecules and/or salt counterions exist in the crystalline form in integral or non-integer stoichiometric ratios. Therefore, one skilled in the art will appreciate that various potency adjustments can be made for the monohydrochloride salt of H3B-6545, the hemihydrochloride salt of H3B-6545, or unusual ratios such as 1:1.3, 1:1.25, etc.

[0065] In accordance with the preceding paragraphs above, as used herein, an "equivalent" amount (e.g., mass, weight, dosage, etc.) of a compound of Formula I (or H3B-6545, the free base of H3B-6545, or any other synonym thereof as used herein) refers to an amount of any salt and/or hydrate according to its activity control factor.

[0066] "H3B-6545 drug substance" refers to the monohydrochloride salt of H3B-6545 described in U.S. Patent No. 10,640,483, which is incorporated herein by reference.

[0067] As used herein, the term "person" is interchangeable with the term "person in need of treatment" or the term "person in need thereof," all of which refer to a person suffering from breast cancer or a person at increased risk of developing breast cancer relative to the general population. A person in need thereof may be a person who has previously been diagnosed or identified with breast cancer or a precancerous condition. Alternatively, a person in need thereof may be a person who has an increased risk of developing such a disorder relative to the general population (i.e., a subject who is predisposed to developing such a disorder relative to the general population). A person in need thereof may have a precancerous condition.

[0068] A person in need thereof may have refractory or resistant cancer (i.e., cancer that does not or has not yet responded to treatment). The subject may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, a subject in need thereof has experienced a relapse of cancer after remission in the most recent treatment. In some embodiments, a subject in need thereof has received all known effective cancer treatments but has failed. In some embodiments, a subject in need thereof has received at least one previous therapy. In a preferred embodiment, the subject has cancer or a cancer-related condition.

[0069] As used herein, the term "fasted state" describes a subject requiring it who has fasted overnight for at least 10 hours prior to administration. No food was allowed for at least 4 hours after dosing. Water was allowed ad libitum, except for one hour before and after dosing.

[0070] As used herein, the term "fed state" describes that after an overnight fast of at least 10 hours, a subject ate a high-fat breakfast within 30 minutes or less and then took a single dose of H3B-6545 30 minutes after the start of the meal. Water was given ad libitum, except for one hour before and after drug administration.

[0071] As used herein, "treating" or "treating" describes the management and care of a human being for the purpose of combating a disease, condition, or disorder and includes administering a dosage form of H3B-6545 or a pharmaceutically acceptable salt, polymorph, hydrate, or solvate thereof to alleviate the symptoms or complications of a disease, condition, or disorder or to eliminate the disease, condition, or disorder.

[0072] H3B-6545 or a pharmaceutically acceptable salt and/or solvate thereof may or may also be used to prevent a corresponding disease, condition, or disorder or used to identify suitable candidates for such purposes. As used herein, the terms "preventing,""preventing," or "protecting against" refer to reducing or eliminating the occurrence of symptoms or complications of such disease, condition, or disorder.

[0073] As used herein, "sample" means any biological sample obtained from a human being, including, but not limited to, cells, tissue samples, biological fluids (including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen), tumor cells, and tumor tissue. Preferably, the sample is selected from bone marrow, peripheral blood cells, blood, plasma, and serum. Samples may be provided by a subject undergoing treatment or research. Alternatively, samples may be obtained by a physician in accordance with routine practice in the art.

[0074] As used herein, the term "dosage form" refers to physically discrete units suitable as unitary dosages for the human to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Dosage forms are classified in terms of routes of administration and sites of administration, including, for example, oral, topical, rectal, vaginal, intravenous, subcutaneous, intramuscular, ophthalmic, nasal, optical, and inhalation administration. Alternatively, dosage forms are classified in terms of physical form, such as solid, semi-solid, or liquid. A dosage form is any of a variety of forms, including, for example, an intravenous bag, a tablet, a single-dose pump on an aerosol inhaler, or a vial. An "oral dosage form" refers to a dosage form that can be readily administered to a human by mouth. Non-limiting examples of oral dosage forms include capsules and tablets. The amount of active ingredient (e.g., a compound of the disclosed compound or a salt, hydrate, or solvate thereof) in a unit dosage form is an effective amount and varies depending on the particular treatment.

[0075] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.

[0076] As used herein, the phrase "pharmaceutically acceptable excipient" means an excipient that can be used in the manufacture of a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as pharmaceutical purposes. "Pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient. For example, a pharmaceutically acceptable excipient used in a formulation of the invention can be a diluent or inert carrier, a lubricant, a binder, or a combination thereof. A pharmaceutically acceptable excipient used in a formulation of the invention can further include a filler, an antimicrobial agent, an antioxidant, an anti-caking agent, a coating agent, or a mixture thereof.

[0077] The term "composition" as used herein includes a product containing a specified ingredient in a specified amount and any product obtained directly or indirectly by combining the specified ingredients in the specified amounts. Such term, when referring to pharmaceutical compositions, is intended to include a product containing an active ingredient (herein a compound of formula I or any pharmaceutically acceptable salts, hydrates and/or solvates thereof) and an inert ingredient constituting a carrier and includes any product formed directly or indirectly by the combination, complexation or aggregation of any two or more ingredients or by the dissociation, other types of reaction or interaction of one or more ingredients. Thus, a pharmaceutical composition of the present invention includes any composition obtained by mixing the compounds of formula I (or pharmaceutically acceptable salts, hydrates and/or solvates) with a pharmaceutically acceptable excipient.

[0078] As used herein, the term "therapeutically effective amount" refers to the amount of H3B-6545 that can produce a therapeutic effect in a human. A therapeutically effective amount is an amount that will treat, alleviate, or prevent an identified disease or condition, or that will exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay known in the art. The precise effective amount for a subject will depend on the weight, size, and health of the subject, the nature and extent of the disease, and the therapeutic agent selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of a clinician.

[0079] In a preferred aspect, the disease or condition being treated is cancer. In another aspect, the disease or condition being treated is a cell proliferative disorder. A therapeutically effective amount of H3B-6545 can be administered as a dosage form. A therapeutically effective amount of H3B-6545 can be in the form of a pharmaceutically acceptable salt, solvate, and/or hydrate.

[0080] As used herein, the term "therapeutic effect" is a consequence of medical treatment, the results of which are assessed as desirable and beneficial. This is true regardless of whether the result was expected, unexpected, or even an unintended consequence of the treatment. A desirable or beneficial result may be inhibition of altered cellular signaling pathways, inhibition of cell growth, preferably growth of cancer cells, stimulation of cell death, preferably death of cancer cells, or a reduction in tumor size, all observed without serious side effects. Slowing and, preferably, regression of tumor growth, and, preferably, complete regression of cancer, is another example of a therapeutic effect. A therapeutic effect may also be an objectively identifiable improvement noted by a clinician or other qualified observer. For example, tumor regression in a patient may be determined by the diameter of the tumor. A decrease in tumor diameter is indicative of regression. Regression is also indicated by the absence of recurrence of the tumor after cessation of treatment.

[0081] Serious side effects may include those that are life-threatening (such as liver failure, abnormal heart rhythms, and certain types of allergic reactions), those that result in permanent or significant disability or hospitalization, or those that cause a birth defect.

[0082] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of H3B-6545 wherein the parent compound is modified by forming acidic or basic salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids with basic moieties such as amines, alkali or organic salts with acidic moieties such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

[0083] As used herein, the term "quantifiable" means measurable, calculable, or expressible as a quantity or a numerical value. A quantifiable plasma concentration is the concentration of H3B-6545 that can be detected and measured in human plasma following administration. A quantifiable bioavailability AUC is the fraction of H3B-6545 that accesses the human systemic circulation, which can be calculated by analyzing plasma concentrations of H3B-6545 in plasma samples collected from the human over a given period of time. A quantifiable half-life is the determined or calculated time until the plasma concentration of H3B-6545 is 50% of the maximum quantifiable plasma concentration of H3B-6545. The methods and materials necessary for quantifying the aforementioned PK parameters are generally known to those skilled in the art. Specific methods for quantification are provided herein.

[0084] As used herein, the term "solvate" means solvent addition forms that contain stoichiometric or non-stoichiometric amounts of solvent.

[0085] As used herein, the term "tautomer" means one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomeric form to another.

[0086] Unless specifically stated or obvious from the context, as used herein, the term "or" shall be construed as inclusive.

[0087] In an aspect of the present invention, there is provided a dosage form with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient that can be administered to a human in need thereof, wherein the therapeutically effective amount provides a quantifiable plasma concentration after administration. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is a solid oral dosage form. In another embodiment, the solid oral dosage form can be an immediate-release oral solid dosage form. The solid oral dosage form can be a tablet or a capsule. These forms can have multiple phases, including, for example, an internal phase and an external phase.

[0088] In one embodiment of the invention, the dosage form is substantially free of water. In this context, "substantially" free of water means that the water content of the formulation during packaging is less than 7%, less than 5%, less than 1%, or less than 0.5% of the total weight of the formulation. In one embodiment, the amount of water is from 0.1 to 5% (e.g., 0.1-1% or 0.1-0.5%) of the total weight of the formulation. In one embodiment, the amount of water in a formulation of the present invention produced by a spray coating process is less than 0.5%.

[0089] At least one pharmaceutically acceptable excipient may be a diluent or inert carrier, a disintegrant, a lubricant, a binder, or a combination thereof. The pharmaceutically acceptable excipient may also include a filler, an antimicrobial agent, an antioxidant, an anti-caking agent, a coating agent, or a mixture thereof.

[0090] Examples of binders may include, but are not limited to, corn starch, potato starch, other starches, gelatin, natural and synthetic gums such as acacia, xanthan, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone (e.g., povidone, crospovidone, copovidone, etc.), methyl cellulose, methocel, pregelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM® sold by Colorcon, Ltd.), hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook, PA, USA), Emdex, Plasdone or mixtures thereof; fillers such as talc, calcium carbonate (e.g. granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g. granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, dextrose, fructose, honey, lactose anhydrate, lactose monohydrate, lactose and aspartame, lactose and cellulose, lactose and microcrystalline cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose & guar gum, molasses, sucrose or mixtures thereof.

[0091] Examples of disintegrating agents may include, but are not limited to: agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate (such as Explotab), potato or tapioca starch, other starches, pregelatinized starch, clays, other algins, other celluloses, gums (such as gellan), low-substituted hydroxypropyl cellulose, ployplasdone, or mixtures thereof.

[0092] Examples of lubricants may include, but are not limited to: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, compritol, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate (such as Pruv), vegetable-based fatty acid lubricant, talc, hydrogenated vegetable oil (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL 200, WR Grace Co., Baltimore, MD USA), coagulated synthetic silicon dioxide aerosol (Deaussa Co., Piano, TX USA), pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA) or mixtures thereof.

[0093] Examples of coating agents may include, but are not limited to: sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropyl methylcellulose phthalate, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, kamuba wax, microcrystalline wax, gellan gum, maltodextrin, methacrylates, microcrystalline cellulose and carrageenan or mixtures thereof.

[0094] In one embodiment of the invention, the dosage form is a solid oral dosage form that can optionally be treated with coating systems (e.g., Opadry® fx film coating system) to coat, for example, Opadry® blue (OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white (YS-1-7040), and black ink (S-1-8 106).

[0095] In one embodiment of the invention, the oral dosage form is made in the form of a capsule that has an internal phase that contains a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof, lactose monohydrate, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose, and colloidal anhydrous silicon dioxide. The capsule also has an external phase that contains magnesium stearate.

[0096] In one embodiment of the invention, the capsule is hypromellose.

[0097] In another embodiment of the invention, the capsule is hypromellose and further comprises red iron oxide and titanium dioxide.

[0098] In another embodiment of the invention, the oral dosage form is a capsule that contains, by weight of the total capsule, 25% to 30% of a therapeutically effective amount of H3B-6545, or a pharmaceutically acceptable salt, 10% to 15% lactose monohydrate, 5% to 10% low-substituted hydroxypropyl cellulose, 1% to 5% microcrystalline cellulose, 0.5% to 5% hydroxypropyl cellulose, 0.05% to 0.5% colloidal anhydrous silicon dioxide, 0.1% to 1% magnesium stearate, 40% to 45% hypromellose, 0.5% to 2% red iron oxide, and 0.5% to 2% titanium dioxide.

[0099] The pharmaceutically acceptable salt may include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, salts derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarboxylic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethanesulfonic, fumaric, glucoheptone, gluconic, glutamic, glycolic, glycollarsanilic, hexylresorcinol, hydrabaminic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, maleic, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic and commonly occurring amino acids such as glycine, alanine, phenylalanine, arginine, etc.

[0100] Other examples of pharmaceutically acceptable salts may include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present invention also includes salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. By salt form is meant that the ratio of the compound to the cation or anion of the salt may be 1:1 or any ratio other than 1:1, such as 3:1, 2:1, 1:2, or 1:3.

[0101] In another embodiment of the invention, the dosage form may also contain one or more active compounds (eg, H3B-6545 or a salt thereof) in combination with at least one pharmaceutically acceptable excipient or carrier.

[0102] Examples of solvates may include, if the solvent is water, a solvate formed as a hydrate; and, if the solvent is an alcohol, the solvate formed is an alcoholate. Hydrates are formed by combining one or more molecules of water with one molecule of a substance in which the water maintains its molecular state as _H2O . A hemihydrate is formed by combining one molecule of water with more than one molecule of a substance in which the water maintains its molecular state as _H2O .

[0103] An oral dosage form with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient that can be administered to a human in need thereof can achieve a quantitatively measurable concentration in plasma within a time of about 0.5 hour to about 12 hours, about 0.5 hour to about 5 hours, about 0.5 hour to about 3 hours, about 1 hour to about 5 hours, or about 1 hour to about 3 hours after administration.

[0104] A therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof, which can be in a dosage form for oral administration, is in the range of from about 50 mg to about 1000 mg, from about 100 mg to about 800 mg, from about 100 mg to about 600 mg, from about 200 mg to about 600 mg, or from about 400 mg to about 600 mg.

[0105] In a preferred embodiment of the invention, the therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof that may be present in an oral dose is 450 mg. In one embodiment of the invention, these aforementioned doses are daily doses.

[0106] An oral dosage form with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient that can be administered to a human in need thereof can achieve a maximum measurable plasma concentration of from about 170 ng/mL to about 2000 ng/mL, from about 200 ng/mL to about 1500 ng/mL, from about 500 ng/mL to about 2000 ng/mL, from about 500 ng/mL to about 1500 ng/mL, from about 1000 ng/mL to about 2000 ng/mL, or from about 1000 ng/mL to about 1500 ng/mL after administration.

[0107] A dosage form for oral administration with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient, which can be administered to a person in need thereof, 5 hours to about 24 hours, from about 8 hours to about 24 hours, from about 8 hours to about 15 hours, from about 10 hours to about 15 hours, or from about 15 hours to about 24 hours after administration.

[0108] An oral dosage form with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient that can be administered to a human in need thereof can achieve a quantitative bioavailability AUC of from about 2,000 ng*hr/mL to about 20,000 ng*hr/mL, from about 5,000 ng*hr/mL to about 20,000 ng*hr/mL, from about 10,000 ng*hr/mL to about 20,000 ng*hr/mL, from about 15,000 ng*hr/mL to about 20,000 ng*hr/mL, from about 5,000 ng*hr/mL to about 15,000 ng*hr/mL, or from about 10,000 ng*hr/mL to about 15000 ng*h/ml after administration.

[0109] An oral dosage form with a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient that can be administered to a human in need thereof can be administered orally as a single dose once a day for a 20-day cycle, a 21-day cycle, a 22-day cycle, a 23-day cycle, a 24-day cycle, a 25-day cycle, a 26-day cycle, a 27-day cycle, a 28-day cycle, or until a therapeutic effect is demonstrated in a subject in need thereof. The dosage form can also be administered in cycles of up to 20 days.

[0110] A. Oral dosage forms comprising from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

[0111] In some embodiments, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such embodiments, the dosage achieves a maximum quantifiable plasma concentration, C _max (in ng/mL), after administration to a subject in need of treatment of from about 150 ng/mL to about 2100 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 250 ng/mL to about 1700 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 1200 ng/mL to about 1500 ng/mL. In these embodiments, the claimed C _max can be achieved in a time of from about 1 hour to about 25 hours; from about 2 hours to about 12 hours; from about 2 hours to about 5 hours; or from about 4 hours.

[0112] In one embodiment, the oral dosage form comprises about 100 to 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the oral dosage form can achieve a C _max range of about 1.8 ng/mL to about 4.0 ng/mL per mg of H3B-6545. In another embodiment, the oral dosage form can achieve a C _max range of about 1.8 ng/mL to about 3.4 ng/mL per mg of H3B-6545. In another embodiment, the oral dosage form can achieve a C _max range of about 3.0 ng/mL to about 4.0 ng/mL per mg of H3B-6545.

[0113] In some embodiments, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In some of these embodiments, the oral dosage form achieves a half-life after administration to a human in need thereof of from about 8.0 hours to about 22.0 hours or a half-life of from about 10.0 hours to about 13.0 hours.

[0114] In one embodiment, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof of from about 1600 ng*hr/mL to about 23,000 ng*hr/mL, from about 2500 ng*hr/mL to about 18,500 ng*hr/mL, from about 4000 ng*hr/mL to about 20,000 ng*hr/mL, or from about 9000 ng*hr/mL to about 17,000 ng*hr/mL.

[0115] In one embodiment, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof, when measured on a per milligram H3B-6545 basis, of from about 17 ng*hr/mL/mg to about 40 ng*hr/mL/mg, from about 17 ng*hr/mL/mg to about 37 ng*hr/mL/mg, or from about 26 ng*hr/mL/mg to about 40 ng*hr/mL/mg.

[0116] In one embodiment, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (from 0 to infinity hours) after administration to a human in need thereof of from about 2,000 ng*hr/mL to about 40,000 ng*hr/mL, from about 5,000 ng*hr/mL to about 25,000 ng*hr/mL, or from about 20,000 ng*hr/mL to about 23,000 ng*hr/mL.

[0117] In one embodiment, the oral dosage form comprises from about 100 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0 to infinity) after administration to a human in need thereof, when measured on a per milligram H3B-6545 basis, of from about 21 ng*hr/mL/mg to about 66 ng*hr/mL/mg, from about 21 ng*hr/mL/mg to about 55 ng*hr/mL/mg, or from about 30 ng*hr/mL/mg to about 66 ng*hr/mL/mg.

[0118] B. Oral dosage forms comprising from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

[0119] In some embodiments, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such embodiments, the dosage achieves a maximum quantifiable plasma concentration, C _max (in ng/mL), after administration to a subject in need of treatment of from about 900 ng/mL to about 2100 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 1100 ng/mL to about 1900 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 1200 ng/mL to about 1700 ng/mL. In these embodiments, the claimed C _max can be achieved in a time of from about 1 hour to about 25 hours, from about 2 hours to about 10 hours, from about 2 hours to about 5 hours, or in about 4 hours.

[0120] In one embodiment, the oral dosage form comprises about 300 to 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the oral dosage form can achieve a C _max range of about 3.0 ng/mL to about 4.0 ng/mL per mg of H3B-6545. In another embodiment, the oral dosage form can achieve a C _max range of about 3.0 ng/mL to about 3.4 ng/mL per mg of H3B-6545.

[0121] In some embodiments, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In some of these embodiments, the dosage form achieves a half-life after administration to a human in need thereof of from about 9.5 hours to about 14.5 hours or a half-life of from about 10.0 hours to about 11.0 hours.

[0122] In one embodiment, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof of from about 9,000 ng*hr/mL to about 23,000 ng*hr/mL or from about 12,000 ng*hr/mL to about 19,000 ng*hr/mL.

[0123] In one embodiment, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof, measured on a per milligram H3B-6545 basis, of from about 30 ng*hr/mL/mg to about 40 ng*hr/mL/mg or from about 30 ng*hr/mL/mg to about 37 ng*hr/mL/mg.

[0124] In one embodiment, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0 to infinity hours) after administration to a human in need thereof of from about 11,000 ng*hr/mL to about 40,000 ng*hr/mL, from about 11,000 ng*hr/mL to about 33,000 ng*hr/mL, or from about 12,000 ng*hr/mL to about 40,000 ng*hr/mL.

[0125] In one embodiment, the oral dosage form comprises from about 300 mg to about 600 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0 to infinity) after administration to a human in need thereof, when measured on a per milligram H3B-6545 basis, of from about 37 ng*hr/mL/mg to about 66 ng*hr/mL/mg, from about 37 ng*hr/mL/mg to about 54 ng*hr/mL/mg, or from about 38 ng*hr/mL/mg to about 66 ng*hr/mL/mg.

[0126] C. Oral dosage forms comprising about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

[0127] In some embodiments, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such embodiments, the dosage achieves a maximum quantifiable plasma concentration, C _max (in ng/mL), after administration to a subject in need of treatment of from about 1000 ng/mL to about 1600 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 1100 ng/mL to about 1500 ng/mL. In other embodiments, the dosage achieves a maximum quantifiable plasma concentration after administration to a subject in need of treatment of from about 1200 ng/mL to about 1400 ng/mL. In these embodiments, the claimed C _max can be achieved in a time of from about 3 hours to about 6 hours or from about 4 hours to about 6 hours.

[0128] In one embodiment, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the oral dosage form can achieve a C _max range of about 2.4 ng/mL to about 3.5 ng/mL per mg of H3B-6545. In another embodiment, the oral dosage form can achieve a C _max range of about 3.0 ng/mL to about 3.5 ng/mL per mg of H3B-6545. In another embodiment, the oral dosage form can achieve a C _max range of about 3.3 ng/mL to about 3.5 ng/mL per mg of H3B-6545.

[0129] In some embodiments, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In some of these embodiments, the oral dosage form achieves a half-life after administration to a human in need thereof of about 8.0 hours to about 11.0 hours, or a half-life of about 9.0 hours to about 10.0 hours.

[0130] In one embodiment, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof of about 12,000 ng*hr/mL to about 20,000 ng*hr/mL; about 14,000 ng*hr/mL to about 16,000 ng*hr/mL; or about 12,000 ng*hr/mL to about 17,000 ng*hr/mL.

[0131] In one embodiment, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0-24 hours) after administration to a human in need thereof, measured on a per milligram H3B-6545 basis, of about 27 ng*hr/mL/mg to about 43 ng*hr/mL/mg, about 30 ng*hr/mL/mg to about 40 ng*hr/mL/mg, or about 30 ng*hr/mL/mg to about 35 ng*hr/mL/mg.

[0132] In one embodiment, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0 to infinity hours) after administration to a human in need thereof of about 16,000 ng*hr/mL to about 26,000 ng*hr/mL, about 16,000 ng*hr/mL to about 23,000 ng*hr/mL, or about 16,000 ng*hr/mL to about 18,000 ng*hr/mL.

[0133] In one embodiment, the oral dosage form comprises about 450 mg of H3B-6545 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In such an embodiment, the dosage can achieve a quantitative bioavailability AUC (0 to infinity) after administration to a human in need thereof, when measured on a per milligram H3B-6545 basis, of about 36 ng*hr/mL/mg to about 57 ng*hr/mL/mg, about 38 ng*hr/mL/mg to about 52 ng*hr/mL/mg, or about 35 ng*hr/mL/mg to about 40 ng*hr/mL/mg.

[0134] Embodiments of the invention may also relate to a method of treating breast cancer (i.e., a cancer that is ERα-positive and/or HER2-negative), wherein the method comprises administering to a human in need thereof an oral dosage form described herein to obtain the described PK results.

[0135] In all embodiments of the invention relating to the method of treatment, the person in need thereof may be in a fed or fasted state.

[0136] Preclinical data support dose-dependent inhibition of ERαWT and ERαmut-dependent transcription and subsequent reduction in cell proliferation. The safety, pharmacokinetics (PK), and pharmacodynamics (PD) of H3B-6545 in women with ER+, HER2-negative breast cancer (BC) are being considered to determine the recommended dose in the phase 2 trial (RP2D).

[0137] Constitutively activating mutations in the ESR1 gene, found in ~30% of MBC tumors, are located in the ligand-binding domain and can confer resistance to estrogen deprivation therapy (e.g., aromatase inhibition) and contribute to resistance to anti-ER therapy. Li S, et al . Cell Reports , 2013; 4:1116–1130; Robinson DR, Wu YM, Vats P, et al . Nature genetics , 2013; 45:1446–1451; Toy W, Shen Y, Won H, et al . Nature genetics . 2013; 45:1439–1445.

[0138] H3B-6545 is an orally available, selective, covalent ER antagonist that inhibits the activity of both mutant (MUT) and wild-type (WT) estrogen receptor (ERα) through irreversible interaction with a unique cysteine that is not conserved in other nuclear hormone receptors. H3B-6545 potently inhibits ERα function without destroying the receptor.

[0139] In embodiments, the invention provides a method of treating a patient with H3B-6545 or a pharmaceutically acceptable salt thereof, comprising determining whether one or more reductions in mutant allele frequencies are observed in the patient's blood, selecting a patient if said reduction is observed, and administering to the selected patient a therapeutically effective amount of H3B-6545 or a pharmaceutically acceptable salt thereof. The mutant allele frequency is the relative frequency of the mutant allele (gene variant) at a particular locus in a population, expressed as a fraction or percentage.

[0140] The authors report a dose escalation in Phase 1 of H3B-6545, a first-in-class highly selective covalent ERα antagonist (SERCA), in women with ER-positive, HER2-negative breast cancer (HR+ BC).

[0141] Examples

[0142] Example 1: Composition of capsules

[0143] Example 1 describes the composition of capsules with different amounts of the drug substance H3B-6545. The capsules used in the following examples were prepared using this method. Capsules with a dosage of 25, 50 and 150 mg were prepared according to the following table:

Table 1: Components and composition of H3B-6545 capsules

Component Compound Function Description 25 mg 50 mg 150 mg Quantity (mg) Internal phase (granules) Drug substance H3B-6545 ^a
(free base equivalent) 26.61
(25.0) 53.22
(50,0) 159.66
(150.0) Active ingredient No data Lactose monohydrate ^b 11.29 22.58 67.74 Diluent JP, NF, Ph. Eur. Low substituted hydroxypropyl cellulose 7.5 15.0 45.0 Baking powder JP, NF Microcrystalline cellulose 2.5 5.0 15.0 Diluent JP, NF, Ph. Eur. Hydroxypropyl cellulose 1.5 3.0 9.0 Binder JP, NF, Ph. Eur. Silicon dioxide, colloidal anhydrous 0,1 0.2 0.6 Glidant JP, NF, Ph. Eur. Magnesium stearate 0.25 0.5 1.5 Lubricant JP, NF, Ph. Eur. External phase (granules) Magnesium stearate 0.25 0.5 1.5 Lubricant JP, NF, Ph. Eur. Full capsule contents 50 100 300 - - Capsule Hypromellose capsules ^c 38 38 70 Capsule shell JP ^d Total weight of capsules 88 138 370 - -

NF=National Formulary (USA), Ph. Eur.=European Pharmacopoeia, JP=Japanese Pharmacopoeia

^a The amount of the drug substance H3B-6545 is adjusted according to the activity adjustment factor, a derivative value of the free-form assay value.

^b The amount of lactose monohydrate during mixing is adjusted depending on the amount of the drug substance H3B-6545 to maintain a constant powder weight.

^c The components and composition of hypromellose capsules are presented in Tables 2 and 3.

^d The official monograph JP16 "Capsules" or the official monograph JP17 "Hypromellose capsules" is used.

Table 2. Components and composition of the hypromellose capsule shell for H3B-6545 25 and 150 mg capsules

Component Description Composition (% w/w) Red iron oxide JPE, NF, EC regulation (E172) 1.1% Titanium dioxide JP, USP, Ph. Eur. 0.6% Hypromellose JP, USP, Ph. Eur. q.s. up to 100%

JPE=Japan Pharmaceutical Excipients, NF=National Formulary (US), JP=Japanese Pharmacopoeia,

USP=United States Pharmacopoeia, EC Regulation=European Commission Regulation, Ph. Eur.=European Pharmacopoeia, q.s.=as needed. E172 is the European standard for iron oxides and iron hydroxides.

Table 3. Components and composition of the hypromellose capsule shell for H3B-6545 50 mg capsules

Component Description Composition (% w/w) Lid Body Red iron oxide JPE, NF, EU regulation (E172) 1.1% - Titanium dioxide JP, USP, Ph. Eur. 0.6% 2.0% Hypromellose JP, USP, Ph. Eur. q.s. up to 100% q.s. up to 100%

JPE=Japan Pharmaceutical Excipients, NF=National Formulary (US), JP=Japanese Pharmacopoeia,

USP=United States Pharmacopoeia, EC Regulation=European Commission Regulation, Ph. Eur.=European Pharmacopoeia, q.s.=as needed. E172 is the European standard for iron oxides and iron hydroxides.

[0144] Briefly, capsules were prepared by preparing a master mixture of H3B-6545 drug substance, lactose monohydrate, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose, and colloidal anhydrous silicon dioxide in a high shear mixer. Magnesium stearate was then mixed with the master mixture using a high shear mixer. The resulting mixture was then compressed into ribbons using a roller compactor, passed through a screen (sizing), and mixed with magnesium stearate using a tumble mixer. The final mixtures were filled into capsules with hypromellose using an encapsulator.

[0145] Example 2: Making Tablets

[0146] Example 2 describes the preparation of tablets comprising the drug substance H3B-6545. The tablets discussed in the following examples were prepared using this method. Film-coated tablets of H3B-6545 containing 150 mg of the drug substance H3B-6545 as a free base were prepared according to the following formulation:

Table 4. Composition of H3B-6545 tablets

Component Description Function mg/tablet Tablet core Internal phase Drug substance H3B-6545 ^a
(free base equivalent) No data Active ingredient 159.6
(150.0) Lactose monohydrate ^b NF, Ph.Eur., JP Diluent 241.2 Low substituted hydroxypropyl cellulose NF, JP Baking powder 90.0 Hypromellose USP, Ph.Eur., JP Binder 18.0 Colloidal silicon dioxide NF, Ph.Eur., JP Glidant 1,2 Purified water ^c USP, Ph.Eur., JP Solvent q.s. External phase Microcrystalline cellulose ^d NF, Ph.Eur., JP Diluent 78.0 Magnesium stearate ^d NF, Ph.Eur., JP Lubricant 12.0 Tablet core weight (mg) - - 600,0 Film coating OPADRY® 03H420000 YELLOW ^e NC Covering agent 30.0 Purified water ^f USP, Ph.Eur., JP Solvent q.s. Gross weight (mg) - - 630,0

JP=Japanese Pharmacopoeia, NC=Non Pharmacopoeial, NF=National Formulary (US), Ph. Eur.=European Pharmacopoeia, q.s.=as needed, USP=United States Pharmacopoeia.

^a The amount of the drug substance H3B-6545 is adjusted according to the activity adjustment factor obtained from the free-form assay value.

^b The amount of lactose monohydrate during mixing is adjusted depending on the amount of the drug substance H3B-6545 to maintain a constant tablet weight.

^c Removed during drying.

^d Adjusted proportionally to the output of crushed granules.

^e The components and composition of OPADRY 03H420000 YELLOW are described in the table.

^f Removed during coating application.

Table 5. Components of OPADRY® 03H420000 YELLOW

Component Description Hypromellose USP, Ph.Eur., JP Talc USP, Ph.Eur., JP Titanium dioxide USP, Ph.Eur., JP Propylene glycol USP, Ph.Eur., JP Iron oxide (yellow) NF, JPE, EU Regulation

EU Regulation=European Commission Regulation, JP=Japanese Pharmacopoeia, JPE=Japanese Pharmaceutical Excipients, NF=National Formulary (USA), Ph. Eur.=European Pharmacopoeia, USP=United States Pharmacopoeia.

The tablets were prepared as follows:

[0148] Lactose monohydrate, low-substituted hydroxypropyl cellulose, hypromellose, and colloidal silicon dioxide were loaded into a high-shear wet granulator and mixed to form a first mixture. The H3B-6545 drug substance and the first mixture were then loaded into a convection mixer and mixed to form a second mixture.

[0149] The second mixture was continuously fed into a high shear wet granulator and granulated with purified water poured in using a pump to form wet granules. The wet granules were continuously dried using a fluid bed dryer at an elevated temperature until the loss on drying of the dried granules was no more than 1.5%.

[0150] The dried granules were then milled using a sifter mill. The milled granules, microcrystalline cellulose and magnesium stearate were then loaded into a convection mixer and mixed to form mixed granules, which were compressed into tablet cores. The coating suspension (prepared by suspending the coating agent in purified water) was then sprayed onto the tablet cores using a coating machine.

[0151] Blood samples were collected at scheduled time points from each patient and centrifuged. A portion of the plasma from each sample was then transferred to a K2EDTA tube, which was then sent to a bioanalytical laboratory for concentration determination using LC-MS/MS. The concentration data were then analyzed using Phoenix® WinNonlin software to obtain PK parameters. The obtained PK parameters were then summarized using analytical software.

[0152] Blood samples (approximately 5 mL each) were collected from human patients prior to dosing and at various hourly time points post-dose. Plasma H3B-6545 concentrations were determined using a validated high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay. The lower limit of quantification was 0.100 ng/mL.

[0153] The PK analysis population included subjects who received H3B-6545 capsule or tablet drug product and for whom sufficient plasma concentration data were available to obtain at least 1 primary PK parameter for each treatment. Plasma H3B-6545 concentrations (as free base) were tabulated and summarized at each nominal time point using descriptive statistics (number of subjects, arithmetic mean with standard deviation [SD], coefficient of variation [CV%], geometric mean, median, minimum, and maximum) as appropriate. Individual and mean (±SD) plasma concentration-time profiles were provided for each treatment.

[0154] The pharmacokinetic parameters of H3B-6545 were calculated using non-compartmental methods using a suitable model for plasma and extravascular data. PK parameters included, but were not limited to, area under the curve to last quantifiable concentration (AUC _0-t ), maximum concentration (C _max ), and time at which maximum concentration was reached (t _max ). When data permitted, area under the plasma concentration-time curve was extrapolated to infinity (AUC _0-inf ), and terminal half-life (t _1/2 ), apparent total body clearance (CL/F), total systemic clearance after oral administration at steady state (CLss/F), apparent volume of distribution during the terminal phase (Vz/F), apparent volume of distribution at steady state (Vss), accumulation ratio for C _max (RC _max ), and accumulation ratio for AUC _0-24 (RAUC) were also obtained.

[0155] Example 3: First interim pharmacokinetic (PK) analysis of capsules in fasting patients

[0156] Example 3 is a clinical and genomic analysis of a single trial. The study population in Example 3 was as follows:

Women ≥18 years with locally advanced or metastatic ER+ HER2- BC.

Progression on at least one hormonal therapy and at least one additional therapy/regimen; no maximum number of prior lines of therapy.

ECOG performance status 0 or 1.

Adequate bone marrow and organ function.

Excluded if there was only bone disease or inflammatory BC.

[0157] Methods were used to analyze whether H3B-6545 inactivates both wild-type and mutant ERα by targeting cysteine 530 and providing a unique antagonist conformation. Methods: Women with locally advanced or metastatic HR+ BC were treated (tx) with H3B-6545 administered orally once daily via capsule for 28-day cycles following progression on at least one hormonal therapy and at least one additional therapy/regimen. Dose escalation utilized a 3+3 design with the ability to revert to previously excluded doses and allow individual dose escalation. This example examined the safety, pharmacokinetics, and pharmacodynamics of H3B-6545 in women with HR+ BC to determine the recommended subsequent study dose.

[0158] Results: As of December 10, 2018, 32 patients had received H3B-6545 at doses ranging from 100 to 450 mg/day; 97% were previously treated with a CDK4/6 inhibitor, 56% had received ≥3 lines of prior anticancer therapy. No dose-limiting toxicities were observed, and only one grade 3 treatment-related adverse event (TRAE) (lymphocyte decrease) was observed.

[0159] The most common (≥10%) TRAEs included asymptomatic sinus bradycardia, diarrhea, nausea, fatigue, anemia, decreased appetite, and flushing. H3B-6545 was rapidly absorbed with a t _max of 2-4 hours. Plasma concentrations increased with dose from 100 to 450 mg and were similar for C1D1 and C1D15. Consistent with the mechanism of action of H3B-6545 and preclinical data, H3B-6545 inhibited ER target gene expression and showed a 50% reduction in Ki67 levels at all dose levels following treatment. ESR1 (60%) and PIK3CA (34%) mutations were detected in plasma at baseline, and changes in mutant allele frequencies show a correlation with treatment response. Disease stability was observed in 15 patients (47%), and 34% of patients completed at least 6 months of treatment. Partial responses (PRs) were observed in 3 patients: 1 patient (mutant) had received 2 prior lines of therapy and 2 patients (1 mutant and 1 wild-type) had received >5 prior lines of therapy, including fulvestrant and capecitabine; all 3 patients had previously received a CDK4/6 inhibitor.

[0160] H3B-6545 was well tolerated up to the 450 mg dose level with early evidence of single agent antitumor activity in the post-CDK4/6 setting. Dose escalation continued in patients with advanced HER2-negative breast cancer.

[0161] Taking medications and dosage:

H3B-6545 is administered orally once daily for a 28-day cycle.

Dose escalation using a standard 3+3 study design with cohorts of 100, 200, 300, 450 and 600 mg once daily.

Patients are allowed to return to previously excluded doses.

Individual dose escalation is permitted after completion of cycle 3.

[0162] Results (as of April 20, 2019)

[0163] Patient group:

46 patients received H3B-6545 at doses ranging from 100 to 600 mg once daily.

42 (91.3%) patients had previously received a CDK4/6 inhibitor and 32 (69.6%) patients had received fulvestrant.

30 (65.2%) patients had received ≥3 prior lines of anticancer therapy in advanced/metastatic cases.

[0164] Demographic and baseline characteristics are shown in Table 6.

Table 6

100 mg QD 200 mg QD 300 mg QD 450 mg QD 600 mg QD Total Category (N=6) (N=12) (N=11) (N=10) (N=7) (N=46) Age (years) ^a n 6 12 11 10 7 46 Average (SD) 67.3 (5.75) 55.6 (12.26) 56.4 (14.10) 57.5 (11.06) 62.3 (15.32) 58.7 (12.51) Min, max 62, 77 41, 81 31, 75 38, 72 33, 80 31, 81 Race, n (%) Whites 6 (100.0) 9 (75.0) 10 (90.9) 10 (100.0) 7 (100.0) 42 (91.3) Black or African American 0 (0,0) 1 (8.3) 1 (9.1) 0 (0,0) 0 (0,0) 2 (4.3) Other 0 (0,0) 2 (16.7) 0 (0,0) 0 (0,0) 0 (0,0) 2 (4.3) Number of previously used anticancer regimens, n (%) 1 0 (0,0) 3 (25.0) 2 (18.2) 3 (30.0) 1 (14.3) 9 (19.6) 2 2 (33.3) 3 (25.0) 1 (9.1) 0 (0,0) 1 (14.3) 7 (15.2) 3 0 (0,0) 1 (8.3) 2 (18.2) 1 (10.0) 0 (0,0) 4 (8.7) 4 0 (0,0) 3 (25.0) 2 (18.2) 2 (20.0) 0 (0,0) 7 (15.2) 5 1 (16.7) 0 (0,0) 0 (0,0) 2 (20.0) 3 (42.9) 6 (13.0) ≥6 3 (50.0) 2 (16.7) 4 (36.4) 2 (20.0) 2 (28.6) 13 (28.3) Previous groups of antitumor drugs, n (%) Fulvestrant 6 (100.0) 7 (58.3) 7 (63.6) 7 (70.0) 5 (71.4) 32 (69.6) Aromatase inhibitor 5 (83.3) 10 (83.3) 9 (81.8) 9 (90.0) 7 (100.0) 40 (87.0) CDK4/6 inhibitor 6 (100.0) 9 (75.0) 11 (100.0) 10 (100.0) 6 (85.7) 42 (91.3) Other 6 (100.0) 11 (91.7) 9 (81.8) 7 (70.0) 7 (100.0) 40 (87.0) Metastasis site ^b , n (%) Brain 0 (0,0) 0 (0,0) 1 (9.1) 0 (0,0) 0 (0,0) 1 (2,2) Bone 6 (100.0) 8 (66.7) 8 (72.7) 8 (80.0) 6 (85.7) 36 (78.3) Breast 2 (33.3) 3 (25.0) 2 (18.2) 2 (20.0) 1 (14.3) 10 (21.7) Liver 4 (66.7) 6 (50.0) 9 (81.8) 7 (70.0) 5 (71.4) 31 (67.4) Lung 1 (16.7) 2 (16.7) 6 (54.5) 5 (50.0) 2 (28.6) 16 (34.8) Bud 0 (0,0) 0 (0,0) 1 (9.1) 0 (0,0) 0 (0,0) 1 (2,2) Lymph node 5 (83.3) 5 (41.7) 3 (27.3) 5 (50.0) 3 (42.9) 21 (45.7) Peritoneum/omentum 1 (16.7) 1 (8.3) 2 (18.2) 2 (20.0) 2 (28.6) 8 (17.4) Other 3 (50.0) 6 (50.0) 3 (27.3) 4 (40.0) 2 (28.6) 18 (39.1)

^a Age represents the age of informed consent.

^b If an object has several plots of the same category, the object is counted in this category only once.

[0165] Security:

Two dose-limiting toxicities (DLTs) - grade 3 fatigue and grade 3 painful rash - were observed in the 600 mg once daily cohort.

The most common (≥10%) treatment-related adverse events (TRAEs) include sinus bradycardia, nausea, fatigue, anemia, diarrhea, and aspartate aminotransferase increased (Table 2).

Six grade 3 TRAEs were observed: syncope and prolongation of the QT interval on electrocardiogram at 450 mg; anemia, increased aspartate aminotransferase, fatigue, and painful rash were observed at 600 mg.

There was no TRAE grade ≥4.

The most common TRAE, sinus bradycardia, appears to be independent of dose or concentration and does not require dose reduction, interruption, or discontinuation.

[0166] TRAE are summarized in Table 7.

Table 7. ≥10% of treatment-related adverse events

Preferred term 100 mg QD 200 mg QD 300 mg QD 450 mg QD 600 mg QD Total (N=6) (N=12) (N=11) (N=10) (N=7) (N=46) Subjects with TRAE 4 6 9 9 5 33 Sinus bradycardia 0 2 7 3 0 12 Nausea 1 0 3 4 1 9 Fatigue 1 2 3 1 1 8 Anemia 0 1 2 3 1 7 Diarrhea 1 1 2 3 0 7 Increased aspartate aminotransferase levels 0 1 1 1 2 5

[0167] Pharmacokinetics:

t _max ranged from 2 to 4 hours

Plasma concentrations increased with increasing dose (100 to 600 mg) (FIG. 1).

Plasma concentrations were similar for C1D1 and C1D15.

[0168] Pharmacodynamics:

ESR1 (55%) and PIK3CA (39%) mutations were detected in baseline plasma.

Changes in mutant allele frequencies correlate with clinical response.

[0169] Case Study:

[0170] One patient, a 50-year-old woman, was newly diagnosed with ER+ HER2- breast cancer in 2006 and had previously received metastatic treatment including letrozole/palbociclib, entinostat/exemestane, capecitabine, eribulin, and carboplatin/gemcitabine. ESR1 Y537S and PI3KCA E545K mutations were detected in the baseline tumor and plasma. At enrollment, ECOG performance status was 1; disease sites included liver, bone, pleural effusion, pelvic ascites, and subcutaneous nodules. H3B-6545 was started at 450 mg once daily in June 2018.

[0171] The reduction in the sum of all target lesion diameters from baseline to C3D1 and C7D1 was -27.8% and -35.6%, respectively. After 4 cycles, the patient achieved a partial response and continued treatment in the C11 group.

[0172] Preliminary actions:

Disease stabilization was observed in 17 (37.0%) patients

At least 6 months of therapy were completed by 14 (30.4%) patients

Confirmed partial response was observed in 3 (6.5%) patients

[0174] Additional information on the preliminary activity is shown in FIG. 2. FIG. 3 shows the percentage change in the sum of the diameters of the target lesions.

Table 8 shows tumor response and progression-free survival.

Table 8

100 mg QD 200 mg QD 300 mg QD 450 mg QD 600 mg QD Total (N=6) (N=12) (N=11) (N=10) (N=7) (N=46) Best Overall Response (BOR), n (%) Complete Answer (CR) 0 (0,0) 0 (0,0) 0 (0,0) 0 (0,0) 0 (0,0) 0 (0,0) Partial response (PR) 1 (16.7) 1 (8.3) 0 (0,0) 1 (10.0) 0 (0,0) 3 (6.5) Disease stabilization (SD) 5 (83.3) 4 (33.3) 3 (27.3) 3 (30.0) 2 (28.6) 17 (37.0) Disease progression (PD) 0 (0,0) 2 (16.7) 5 (45.5) 3 (30.0) 1 (14.3) 11 (23.9) Not rated (NE) 0 (0,0) 4 (33.3) 3 (27.3) 3 (30.0) 4 (57.1) 14 (30.4) Unknown (UNK) 0 (0,0) 1 (8.3) 0 (0,0) 0 (0,0) 0 (0,0) 1 (2.2) Objective responses (CR+PR), n(%) 1 (16.7) 1 (8.3) 0 (0,0) 1 (10.0) 0 (0,0) 3 (6.5) 95% CI of objective response rate (ORR) ^a (0.4, 64.1) (0.2, 38.5) (0.0, 28.5) (0.3, 44.5) (0.0, 41.0) (1.4, 17.9) Progression-free survival (months) ^b Median (95% CI) 9.3 (7.4, NE) 5.5 (0.9, NE) 1.8 (1.6, 5.4) 7.2 (1.8, 7.3) NE(0.5,NE) 7.2 (1.9, 7.4) Q1 (95% CI) 7.5 (7.4, 11.0) 1.5 (0.9, 5.5) 1.8 (1.6, 1.8) 1.9 (1.8, 7.2) NE(0.5,NE) 1.8 (1.6, 2.1) Q3 (95% CI) NE (7.4, NE) NE (1.7, NE) 5.4 (1.8, NE) 7.3 (2.1, 7.5) NE(0.5,NE) 7.5 (7.3, NE)

[0175] Conclusions

Safety/tolerability: Most TRAEs were grade 1 or 2 and manageable; tolerated up to and including 450 mg QD.

Pharmacodynamics: Changes in mutant allele frequencies correlate with clinical response.

Preliminary Activity: An encouraging signal of antitumor activity was observed in the intensive pretreatment setting following CDK4/6i, including 3 confirmed partial responses and mPFS=7.2 months.

Phase 2: Initiated after RP2D is determined.

[0176] Example 4: Second interim analysis

[0177] The clinical trial of Example 3 was continued and PK estimates (in this Example 4) were subsequently made based on a larger group of patients. The data in Tables 1 in the Pharmacokinetic (PK) Analysis of Capsules in Fasted Patients and 2 show the PK parameters and profiles of human patients receiving capsules of the drug product H3B-6545 manufactured as in Example 1. Patients received a total equivalent dose of 100 mg, 200 mg, 300 mg, 450 mg, or 600 mg of the compound of Formula I as indicated in the table. Table 1 reflects the PK values for patients determined on day 1 of their first cycle. Table 2 reflects the PK values for patients determined on day 15 of their first cycle.

[0178] PK parameters were calculated after administration to individuals in need thereof, with individuals in the fasted state. QD indicates that the dose was administered "quaque die" or only once per day. The parameter n represents the number of individuals in need thereof who were administered the respective doses.

[0179] Tables 9 and 11 include geometric means and geometric mean coefficients of variation for all plasma PK parameters, except t _max and T _lag , associated with each dose, with these PK parameters calculated for individuals in need thereof (n). The t _max and T _lag values include the mean value calculated for individuals in need thereof, in addition to the minimum and maximum values. Tables 10 and 12 provide the normalized AUC _0-t , AUC _0-24 , AUC _0-inf , and C _max PK parameters shown in Table 9 and Table 11 to the plasma PK parameters per 1 mg of active pharmaceutical ingredient (API).

Table 9

PK plasma parameters ^a Units Cycle 1 Day 1 - Dose Increase H3B-6545 100mg QD H3B-6545 200mg QD H3B-6545 300mg QD H3B-6545 450 mg QD H3B-6545 600 mg QD AUC _0-t h·ng/ml 1679 (85.4); 6 4812 (71.8); 12 9525 (62.9); 11 16177 (43.2); 11 22302 (64.5); 7 AUC _0-24 h·ng/ml 1678 (85.2); 6 4761 (71); 12 9482 (60.8); 11 16170 (42.7); 11 22370 (63.3); 7 AUC _0-inf h·ng/ml 2132 (92.2); 6 5274 (68.3); 8 11077 (49.1); 10 22976 (42.4); 8 32853 (68.6); 5 C _max ng/ml 178 (85.3); 6 473 (72); 12 898 (66.2); 11 1501 (53.1); 11 2048 (62); 7 t _max ^b h 3.05 (2-6); 6 5 (2-25.1); 12 4 (2-24.8); 11 4 (2-10); 11 4 (2-23.37); 7 T _lag ^b h 0 (0-1); 6 0 (0-0.6); 12 0 (0-0.58); 11 0 (0-0.5); 11 0 (0-0.5); 7 t _1/2 h 10.2 (21.4); 6 9.73 (29.9); 8 9.58 (51.3); 10 11.0 (35.6); 8 13.1 (21.3); 5 CL/F l/h 46.9 (92.2); 6 37.9 (68.3); 8 27.1 (49.1); 10 19.6 (42.4); 8 18.3 (68.6); 5 Vz/F l 689 (86.2); 6 532 (70.4); 8 374 (61.3); 10 311 (43.7); 8 344 (77.9); 5 vsss l 713 (75.3); 6 567 (60.6); 8 416 (59.5); 10 343 (42.5); 8 366 (73.1); 5 ^a Geometric mean (GeoCV%); n ^b Median (min, max); n

Table 10

PK plasma parameters
(per 1 mg API) Units Cycle 1 Day 1 - Dose Increase H3B-6545 100mg QD H3B-6545 200mg QD H3B-6545 300mg QD H3B-6545 450 mg QD H3B-6545 600 mg QD AUC _0-t h·ng/ml/mg 16.8 24.1 31.8 35.9 37.2 AUC _0-24 h·ng/ml/mg 16.8 23.8 31.6 35.9 37.3 AUC _0-inf h·ng/ml/mg 21.3 26.37 36.9 51.1 54.8 C _max ng/ml/mg 1.78 2.4 3.0 3.3 3.4

Table 11

PK plasma parameters ^a Units Cycle 1 day 15 - Dose increase H3B-6545 100mg QD H3B-6545 200mg QD H3B-6545 300mg QD H3B-6545 450 mg QD H3B-6545 600 mg QD AUC _0-t h·ng/ml 2589 (57.4); 6 6910 (53.1); 10 12081 (51.7); 11 14053 (53.2); 10 18741 (80.9); 3 AUC _0-24 h·ng/ml 2559 (56.2); 6 6872 (52.8); 10 12009 (51); 11 14023 (52.6); 10 18366 (78.1); 3 AUC _0-inf h·ng/ml 2947 (55.4); 5 7953 (58); 8 12747 (55.6); 9 17391 (49.7); 9 39731 (101.6); 3 C _max ng/ml 287 (31.3); 6 605 (56); 10 1201 (38.2); 11 1415 (53.5); 10 1683 (98.7); 3 t _max ^b h 2 (2-8.03); 6 2.17 (2-6); 10 4 (2-5.95); 11 4 (1-8); 10 4 (4-6); 3 T _lag ^b h 0 (0-0); 6 0 (0-0); 10 0 (0-0); 11 0 (0-0); 10 0 (0-0); 3 t _1/2 h 11.5 (23.1); 5 10.1 (27.7); 8 8.52 (35.9); 9 9.81 (50.4); 9 21.8 (146.9); 3 CLSS/F l/h 39.1 (56.2); 6 29.1 (52.8); 10 26.2 (50.5); 10 32.1 (52.6); 10 32.7 (78.1); 3 Vz/F l 731 (57); 5 455 (69.4); 8 348 (37.1); 9 466 (98.2); 9 1030 (282.1); 3 vsss l 679 (49.3); 5 456 (61.2); 8 373 (39.3); 9 489 (86.7); 9 1100 (230.8); 3 RC _max 1.61 (69.3); 6 1.28 (118.2); 10 1.34 (38.3); 11 1.05 (42.1); 10 0.685 (69.2); 3 RAUC 1.52 (58.3); 6 1.43 (72.9); 10 1.27 (38.3); 11 0.920 (49); 10 0.712 (31.4); 3 ^a Geometric mean (GeoCV%); n ^b Median (min, max); n

Table 12

PK plasma parameters
(per 1 mg API) Units Cycle 1 day 15 - Dose increase H3B-6545 100mg QD H3B-6545 200mg QD H3B-6545 300mg QD H3B-6545 450 mg QD H3B-6545 600 mg QD AUC _0-t h·ng/ml/mg 25.9 34.6 40.3 31.2 31.2 AUC _0-24 h·ng/ml/mg 25.6 34.4 40.0 31.2 30.6 AUC _0-inf h·ng/ml/mg 29.5 39.8 42.5 38.6 66.2 C _max ng/ml/mg 2.9 3.0 4.0 3.1 2.8

[0180] Example 5: Pharmacokinetics (PK) of 450 mg capsules in fasted and fed subjects

[0181] Table 3 details the PK parameters and profiles of human patients receiving capsules prepared as in Example 1. The patients received a total equivalent dose of 450 mg of Formula I as indicated in Table 13. Table 14 provides the normalized AUC _0-t , AUC _0-24 , AUC _0-inf PK parameters, and C _max shown in Table 13 for the plasma PK parameters per 1 mg of active pharmaceutical ingredient (API).

Table 13

PK plasma parameters ^a Units H3B-6545 450 mg QD-dose escalation-fasting H3B-6545 450 mg QD-dose escalation-satiated AUC _0-t h·ng/ml 12464 (65.9); 18 16101 (97.3); 17 AUC _0-24 h·ng/ml 12252 (65.4); 18 19473 (61.2); 15 AUC _0-inf h·ng/ml 16457 (64.1); 16 25546 (51.9); 8 C _max ng/ml 1093 (81.6); 18 1561 (54.5); 17 t _max ^b h 4 (1-6,15); 18 6 (4-10); 17 T _lag ^b h 0 (0-0); 18 0 (0-0); 17 t _1/2 h 10.9 (37.9); 16 8.83 (30.8); 8 CLSS/F l/h 36.7 (65.4); 18 20.5 (42.7); 13 Vz/F l 566 (92.8); 16 279 (37.8); 8 vsss l 597 (89.5); 16 350 (38.9); 8 ^a Geometric mean (GeoCV%); n ^b Median (min, max); n

Table 14

PK plasma parameters
(per 1 mg API) Units H3B-6545 450 mg QD-dose escalation-fasting H3B-6545 450 mg QD-dose escalation-satiated AUC _0-t h·ng/ml/mg 27.7 35.8 AUC _0-24 h·ng/ml/mg 27.2 43.3 AUC _0-inf h·ng/ml/mg 36.6 56.8 C _max ng/ml/mg 2.4 3.5

[0182] Example 6: Pharmacokinetics (PK) of tablets in humans

[0183] The capsules described in Example 1 and the tablets described in Example 2 were studied in healthy humans. All doses were well tolerated. Similar exposure (both C _max and AUC) and t _max of H3B-6545 were noted for the capsules and tablets, as shown in Table 15.

Table 15

PK plasma parameters ^a 450 mg H3B-6545 (capsule)
n=16 450 mg H3B-6545 (tablet)
n=16 AUC _0-24 (h ng/ml) 12650 (58.8) 13320 (54.9) AUC _0-t (h ng/ml) 15580 (58.4) 16420 (57) AUC _0-inf (h ng/ml) 16560 (58.1) 17520 (57.6) C _max (ng/ml) 1069 (56.6) 1122 (46.1) t _max ^b (h) 3.03 (1.00-6.00) 4.00 (1.00-10.0) t _1/2 (h) 10.95 (21.4) 11.01 (21.7) CL/F (l/h) 27.17 (58.1) 25.69 (57.6) V _z /F (l) 429.0 (63.7) 408.0 (59.2) ^a Geometric mean (GeoCV%), ^b Median (min-max)

[0184] The relative bioavailability of H3B-6545 in capsules and tablets is shown in Table 16.

Table 16

Parameter Tablet
Geo.LSM (link) Capsule
Geo. LSM (test) Geo.LSM Ratio (%)
(test:link) 90% CI* Individual CV% AUC _0-24 (h ng/ml) 13320 12650 94.99 (79.27, 113.83) 29.7 AUC _0-t (h ng/ml) 16420 15580 94.86 (79.63, 113.00) 28.7 AUC _0-inf (h ng/ml) 17520 16560 94.56 (80.10, 111.64) 27.1 C _max (ng/ml) 1122 1069 95.28 (79.48, 114.21) 29.7

Geo. LSM: Geometric Least Square Mean.

CI: Confidence interval; *Column shows lower and upper bound.

CV: Coefficient of Variation

[0185] Example 7 - Genomics Research

[0186] In view of the lack of an effective treatment for endocrine-resistant metastatic breast cancer (MBC), the inventors developed H3B-6545, a novel selective covalent ERa antagonist capable of irreversibly inactivating both wild-type ERa and mutant ERa.

[0187] The objectives of this study are to 1) characterize hotspot mutation profiles in MBCs subjected to extensive pretreatment and correlate ESR1, PIK3CA, and AKT1 mutations in plasma with tumor tissue; 2) determine whether mutations in ESR1 or PIK3CA predict response to H3B-6545; and 3) assess whether longitudinal ctDNA tracking correlates with response to H3B-6545.

[0188] Methods

[0189] Fresh plasma samples were collected at baseline (pre-dose), day 15 of cycle 1 (C1D15), C2D1, C3D1, and every 8 weeks thereafter with final sampling at disease progression. At baseline, BEAMing digital PCR was used to assess hotspot mutations in ESR1, PIK3CA, and AKT1. Subsequently, patient-specific ctDNA mutations were monitored by ddPCR. Baseline tumor biopsies were subjected to a targeted next-generation sequencing (NGS) panel to identify hotspot mutations.

[0190] Results

[0191] In 77% of patients (30/39), mutations were detected at baseline by BEAMing, and 21/39, 16/39, and 3/39 patients had mutations in ESR1, PIK3CA, and AKT1, respectively. Co-mutations in ESR1 and PIK3CA were observed in 20% (9/39) of patients. In 60% (9/15) of patients, DNA mutations identified by BEAMing in plasma were also detected in tumor biopsy, whereas; DNA mutations detected in tissues were also detected in plasma in 86% (12/14) of cases. Serial ctDNA monitoring showed that patients with confirmed partial responses (3/3) had ctDNA levels that were undetectable by C2D1. In contrast, ctDNA levels increased from baseline in 3/4 of patients with disease progression. We present an investigation of ctDNA ratios (day 15/baseline and day 30/baseline) and the correlation of PIK3CA and ESR1 mutations with response to H3B-6545.

[0192] Conclusions for Example 7

[0193] ctDNA is a reliable sample type for assessing ESR1, PIK3CA and AKT1 mutations in MBC, overcoming the challenges of obtaining biopsies in metastatic settings. In addition, ctDNA dynamics appear to be a useful tool for monitoring the efficacy of H3B-6545.

[0194] Administration of the drug and dosage:

H3B-6545 is administered orally once daily (PO QD) over a 28-day cycle

Dose escalation using a standard 3+3 study design using dose cohorts of 100, 200, 300, 450, and 600 mg/day

[0195] Liquid biopsy assays: 10 mL of Strek plasma was collected on site and then converted to plasma at Sysmex Inostics. cfDNA was isolated from 2 mL of plasma for BEAMing and 3 mL for Biorad ddPCR.

[0196] Next-generation sequencing (NGS) analysis of tumors: DNA from 10÷5 µm slides was isolated using the Recoshops Kit, and then 10 ng of DNA was used as input for the Oncomine comprehensive assay.

[0197] Baseline study characteristics are shown in Table 17.

Table 17

Category Total (N=46) Number of previously used anticancer regimens, n (%) 1 9 (19.6) 2 7 (15.2) 3 4 (8.7) ≥4 26 (56.5) Previous groups of anticancer drugs, n (%) Fulvestrant 32 (69.6) Aromatase inhibitor 40 (87.0) CDK4/6 inhibitor 42 (91.3) Other 40 (87.0) Last previous anticancer drug, n (%) Fulvestrant 10 (21.7) Aromatase inhibitor 11 (23.9) CDK4/6 inhibitor 20 (43.5) Other 33 (71.7)

[0198] FIG. 4 shows an overview of the liquid biopsy and tissue biopsy sample sets.

[0199] FIG. 5A-FIG. 5E show the baseline genomic profile of patients in this example. AA=amino acid. *AKT1 mutation was observed in 3/49 patients (data not shown).

[0200] FIGS. 5A-5E show the BEAMing analysis. A. Baseline ESR1 and PIK3CA mutation status of patients; B and C. Clonality of ESR1 and PIK3CA mutations; D and E. Amino acid distribution of PIK3CA and ESR1 mutations.

[0201] FIG. 5F shows an oncoprint of mutations detected in the initial tissue biopsy as determined by the Oncomine Comprehensive Panel.

[0202] FIG. 6A and FIG. 6B show the correspondence of mutations detected in the tissue and liquid biopsy of Example 2. FIG. 6A shows the distribution of allele frequencies and the correspondence of mutations in the tissue and liquid biopsy. FIG. 6B is a summary table of the correspondence of mutations in the tissue and liquid biopsy.

[0203] FIGS. 7A-7C show that ctDNA dynamics correlate with overall best response to H3B-6545. AF=allele frequency, C=cycle, D=day, PD=disease progression, PR=partial response, SD=disease stable. The dotted line represents the LOD of the ddPCR assay. FIG. 7A. Baseline AF/C2D1 ratio for each patient, stained individually. A single patient may have multiple mutations. FIG. 7B and FIG. 7C. Example of ctDNA dynamics in a patient with disease progression (B) versus partial response (C).

[0204] Specific Patient Analysis - This is the same specific patient analysis described in Example 3.

A 50-year-old woman who was first diagnosed with ER+ HER2- breast cancer in 2006.

Previous treatment for metastases included letrozole/palbociclib, entinostat/exemestane, capecitabine, eribulin, and carboplatin/gemcitabine.

At the time of enrollment, ECOG performance status was 1; sites of disease included liver, bone, pleural effusion, pelvic ascites, and subcutaneous nodules.

ESR1 Y537S and PI3KCA E545K mutations were detected in the original tumor and plasma.

In June 2018, H3B-6545 was started at a dose of 450 mg once daily.

The reduction in the sum of the diameters of all target lesions from baseline to C3D1 and C7D1 was -27.8% and -35.6%, respectively.

After 4 cycles, the patient achieved a partial response and continued treatment in the C11 group.

[0205] Conclusions

In late ER+ MBC lines, PIK3CA and ESR1 mutations were detected in baseline plasma in 39% and 55%, respectively.

The tissue-plasma mutation level correspondence was 80%.

The concordance of liquid biopsy with tissue mutation level was 51%.

ctDNA levels were not detectable in C2D1 patients with confirmed partial response.

ctDNA dynamics prove to be a useful tool for monitoring the efficacy of H3B-6545 and characterizing baseline genomic profiles.

[0206] All publications and patent documents cited herein are herein incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. The citation of publications and patent documents does not constitute an admission that they are prior art, nor does it constitute an admission of their contents or date. The invention has now been disclosed by written description, and it will be understood by those skilled in the art that the invention may be practiced in a variety of embodiments and that the foregoing description and examples are intended to be illustrative and not limiting to the claims that follow.

[0207] The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be included therein.

Claims (65)

1. An oral dosage form for the treatment of breast cancer, which is a capsule containing: (i) an internal phase containing: 150 mg of a compound of formula I or 150 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of a compound of formula I, wherein said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, represented by the structure 67.74 mg lactose monohydrate, 45 mg low-substituted hydroxypropyl cellulose, 15 mg microcrystalline cellulose, 9 mg hydroxypropyl cellulose, 0.6 mg colloidal anhydrous silicon dioxide, and 1.5 mg magnesium stearate; and (ii) an external phase containing 1.5 mg magnesium stearate. 2. An oral dosage form according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula I is a monohydrochloride salt. 3. An oral dosage form according to claim 2, wherein the internal phase comprises 159.66 mg of the monohydrochloride salt. 4. An oral dosage form according to any one of claims 1 to 3, further comprising a hypromellose capsule shell comprising 70 mg of hypromellose. 5. The oral dosage form according to claim 4, wherein the hypromellose capsule shell contains red iron oxide and titanium dioxide. 6. An oral dosage form for the treatment of breast cancer, which is a tablet containing: (i) an internal phase containing: 150 mg of a compound of formula I or 150 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of a compound of formula I, wherein said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, represented by the structure 241.2 mg lactose monohydrate, 90 mg low-substituted hydroxypropyl cellulose, 18 mg hypromellose and 1.2 mg colloidal silicon dioxide; and (ii) an external phase containing 12 mg magnesium stearate and 78 mg microcrystalline cellulose. 7. An oral dosage form according to claim 6, wherein the pharmaceutically acceptable salt of the compound of formula I is a monohydrochloride salt. 8. An oral dosage form according to claim 7, wherein the internal phase comprises 159.6 mg of the monohydrochloride salt. 9. An oral dosage form according to any one of claims 6 to 8, further comprising a film coating comprising 30 mg of a coating agent, wherein the coating agent comprises hypromellose, talc, titanium dioxide, propylene glycol and iron oxide. 10. An oral dosage form for the treatment of breast cancer, which is a capsule containing: i) an internal phase containing: 50 mg of a compound of formula I or 50 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of a compound of formula I, 22.58 mg lactose monohydrate, 15 mg low-substituted hydroxypropyl cellulose, 5 mg microcrystalline cellulose, 3 mg hydroxypropyl cellulose, 0.2 mg colloidal anhydrous silicon dioxide and 0.5 mg magnesium stearate, wherein said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, represented by the structure and ii) an external phase containing 0.5 mg magnesium stearate. 11. An oral dosage form according to claim 10, wherein the pharmaceutically acceptable salt of the compound of formula I is a monohydrochloride salt. 12. An oral dosage form according to claim 11, wherein the internal phase comprises 53.22 mg of the monohydrochloride salt. 13. An oral dosage form according to any one of claims 10 to 12, further comprising a hypromellose capsule shell comprising 38 mg of hypromellose. 14. The oral dosage form according to claim 13, wherein said hypromellose capsule shell additionally contains red iron oxide and titanium dioxide. 15. An oral dosage form for the treatment of breast cancer, which is a capsule containing: i) an internal phase containing: 25 mg of a compound of formula I or 25 mg of an equivalent amount of the free base of a pharmaceutically acceptable salt of a compound of formula I, 11.29 mg lactose monohydrate, 7.5 mg low-substituted hydroxypropyl cellulose, 2.5 mg microcrystalline cellulose, 1.5 mg hydroxypropyl cellulose, 0.1 mg colloidal anhydrous silicon dioxide and 0.25 mg magnesium stearate, wherein said formula I is (E)-N,N-dimethyl-4-[2-[5-[(Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-enyl]pyridin-2-yl]oxyethylamino]but-2-enamide, represented by the structure and ii) an external phase containing 0.25 mg magnesium stearate. 16. An oral dosage form according to claim 15, wherein the pharmaceutically acceptable salt of the compound of formula I is a monohydrochloride salt. 17. An oral dosage form according to claim 16, wherein the internal phase comprises 26.61 mg of the monohydrochloride salt. 18. An oral dosage form according to any one of claims 15 to 17, further comprising a hypromellose capsule shell comprising 38 mg of hypromellose. 19. The oral dosage form according to claim 18, wherein said hypromellose capsule shell additionally contains red iron oxide and titanium dioxide. 20. A method of treating breast cancer in a human subject, comprising administering to said subject an oral dosage form according to any one of claims 1-19 in a single daily dose in the range of 100 to 600 mg. 21. The method of claim 20, wherein said breast cancer is ERα-positive breast cancer. 22. The method of claim 21, wherein said breast cancer expresses wild-type ERα. 23. The method of claim 21, wherein said breast cancer expresses mutant ERα. 24. The method according to any one of claims 20-23, wherein said oral dosage form is administered to a human subject on an empty stomach. 25. The method according to any one of claims 20-23, wherein said oral dosage form is administered to a human subject after a meal.