Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
  • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/32—Antioestrogens

Definitions

• the present invention relates to methods for the treatment of cancer based on the coadministration HDAC inhibitors.
• Cancer, tumors, tumor-related disorders, and neoplastic disease states are serious and often times life-threatening conditions. These diseases and disorders, which are characterized by rapidly- proliferating cell growth, continue to be the subject of research efforts directed toward the identification of therapeutic agents which are effective in the treatment thereof. Such agents prolong the survival of the patient, inhibit the rapidly-proliferating cell growth associated with the neoplasm, or effect a regression of the neoplasm.
• EGFR epidermal growth factor receptor
• erlotinib erlotinib
• gefitinib erlotinib
• additional conventional treatment as currently employed, offers limited benefit.
• aromatase inhibitor chemotherapy regimens such as the use of letrozole, anastrozole or exemestane, are employed. If patients fail to respond to an aromatase inhibitor treatment, additional conventional treatment offers limited benefit.
• EGFR inhibitor therapy encounters limitations, such as side-effects resulting from its use.
• limitations such as side-effects resulting from its use.
• EGFR inhibitors for the treatment of tumors may initially shrink the size of the tumor, the tumor may eventually enlarge in size, indicating, among other things, the development of resistance.
• Erlotinib a widely used EGFR inhibitor, may be representative of the types of therapeutic agents being used for cancer treatment in that its use has an effect on cancer, but because of other factors, which are not entirely known, the tumor develops resistance and progresses.
• aromatase inhibitors for the treatment of early and late stage breast cancer
• side-effects result from its use.
• common side effects include hot flashes, vasodilation and nausea.
• aromatase inhibitors for the treatment of tumors may initially shrink the size of the tumor, the tumor may eventually enlarge in size, indicating, among other things, the development of resistance.
• Letrozole a widely used aromatase inhibitor, may be representative of the types of therapeutic agents being used for cancer treatment; in that its use has an effect on cancer, but because of other factors, which are not entirely known, the tumor develops resistance and progresses.
• Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutic agents that promote differentiation and apoptosis in hematologic and solid malignancies through chromatin remodeling and gene expression regulation.
• HDAC inhibitors include benzamides (entinostat), short-chain fatty acids (i.e., Sodium phenylbutyrate);
• hydroxamic acids i.e. , suberoylanilide hydroxamic acid and thrichostatin A
• cyclic tetrapeptides containing a 2-amino-8-oxo-9, 10-epoxy-decanoyl moiety i.e., trapoxin A
• cyclic peptides without the 2-amino-8-oxo-9, 10-epoxy-decanoyl moiety i.e., FK228).
• Entinostat is a benzamide HDAC inhibitor undergoing clinical investigation in multiple types of solid tumors and hematologic cancers. Entinostat is rapidly absorbed and has a half-life of about 100 hours and, importantly, changes in histone acetylation persist for several weeks following the administration of entinostat.
• compositions and/or methods of treatment for cancer which take advantage of the synergy found in a therapeutic combination that could increase the effectiveness of the agents and reduce and/or eliminate the side effects typically associated with conventional treatments.
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the Cmax of entinostat is increased when the entinostat is administered under fasting conditions, compared to when entinostat is administered under fed conditions.
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the Tmax of entinostat is increased when the entinostat is administered under fed conditions, compared to when entinostat is administered under fasting conditions.
• One embodiment provides a method of treating breast cancer in a patient in need thereof, comprising oral administration of exemestane and entinostat, wherein the entinostat is administered to a fasting patient.
• One embodiment provides a method of treating non-small cell lung cancer in a patient in need thereof, comprising oral administration of erlotinib and entinostat, wherein the entinostat is administered to a fasting patient.
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the administration of entinostat under fasting conditions results in an increase of the Cmax as compared to the administration of entinostat under fed conditions, and wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 2: 1.
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the administration of entinostat under fed conditions results in an increase of the Tmax as compared to the administration of entinostat under fasting conditions, and wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 2: 1.
• Figure 1 provides a pharmacokinetic analysis of the food effect study described in Example 1.
• HDAC histone deacetylase
• the methods include administering the HDAC inhibitor without food.
• the methods of treatment may incorporate patient selections based on levels of protein lysine acetylation observed during treatment.
• the methods may further include treatments wherein the administration of the HDAC inhibitor and the aromatase inhibitor are supplemented with one or more therapeutic agents or therapies.
• kits for treating lung cancer based on the administration of an HDAC inhibitor and an epidermal growth factor receptor (EGFR) inhibitor.
• the methods include administering the HDAC inhibitor without food.
• the methods may further include treatments wherein the administration of the HDAC inhibitor and the EGFR inhibitor are supplemented with one or more therapeutic agents or therapies.
• EGFR epidermal growth factor receptor
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the administration of entinostat under fasting conditions results in an increase of the Cmax as compared to the administration of entinostat under fed conditions, and wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 2: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 3: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 4: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 5: 1. Another embodiment provides the method of treating cancer wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 6: 1. Another embodiment provides the method of treating cancer wherein the ratio of Cmax following administration under fasting conditions to Cmax following administration under fed conditions is at least about 7: 1. Another embodiment provides the method of treating cancer wherein the cancer is lung cancer. Another embodiment provides the method of treating cancer wherein the lung cancer is non-small cell lung cancer.
• Another embodiment provides the method of treating cancer wherein the cancer is breast cancer. Another embodiment provides the method of treating cancer further comprising oral administration of an EGFR inhibitor. Another embodiment provides the method of treating cancer wherein the EGFR inhibitor is erlotinib. Another embodiment provides the method of treating cancer wherein the erlotinib is administered at a different time of day than entinostat. Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 2 hours prior to administration or erlotinib. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 1 hour after administration of erlotinib. Another embodiment provides the method of treating cancer wherein about 150 mg of erlotinib is administered.
• Another embodiment provides the method of treating cancer wherein the erlotinib is administered once daily. Another embodiment provides the method of treating cancer further comprising oral administration of an aromatase inhibitor. Another embodiment provides the method of treating cancer wherein the aromatase inhibitor is exemestane. Another embodiment provides the method of treating cancer wherein the exemestane is administered at a different time of day than entinostat. Another embodiment provides the method of treating cancer wherein exemestane is administered after a meal. Another embodiment provides the method of treating cancer wherein exemestane is administered with a meal. Another embodiment provides the method of treating cancer wherein about 25 mg of exemestane is administered. Another embodiment provides the method of treating cancer wherein the exemestane is administered once daily.
• Another embodiment provides the method of treating cancer wherein the patient is administered about 10 mg of entinostat. Another embodiment provides the method of treating cancer wherein the patient is administered about 5 mg of entinostat. Another embodiment provides the method of treating cancer wherein the patient is administered from about 1 mg to about 20 mg of entinostat. Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 2 hours prior to administration of entinostat under fasting conditions.
• Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 1 hour prior to administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 2 hours after administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 30 minutes after administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient consumes a high fat meal under fed conditions.
• One embodiment provides a method of treating cancer in a patient in need thereof, comprising oral administration of entinostat, wherein the administration of entinostat under fed conditions results in an increase of the Tmax as compared to the administration of entinostat under fasting conditions, and wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 2: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is from about 2: 1 to about 5: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is from about 5: 1 to about 8: 1.
• Another embodiment provides the method of treating cancer wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is from about 8: 1 to about 12: 1. Another embodiment provides the method of treating cancer wherein the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is from about 12: 1 to about 15: 1. Another embodiment provides the method of treating cancer wherein the cancer is lung cancer. Another embodiment provides the method of treating cancer wherein the lung cancer is non-small cell lung cancer. Another embodiment provides the method of treating cancer wherein the cancer is breast cancer. Another embodiment provides the method of treating cancer further comprising oral administration of an EGFR inhibitor. Another embodiment provides the method of treating cancer wherein the EGFR inhibitor is erlotinib.
• Another embodiment provides the method of treating cancer wherein the erlotinib is administered at a different time of day than entinostat. Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 2 hours prior to administration or erlotinib. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 1 hour after administration of erlotinib. Another embodiment provides the method of treating cancer wherein about 150 mg of erlotinib is administered. Another embodiment provides the method of treating cancer wherein the erlotinib is administered once daily. Another embodiment provides the method of treating cancer further comprising oral administration of an aromatase inhibitor. Another embodiment provides the method of treating cancer wherein the aromatase inhibitor is exemestane.
• Another embodiment provides the method of treating cancer wherein the exemestane is administered at a different time of day than entinostat. Another embodiment provides the method of treating cancer wherein exemestane is administered after a meal. Another embodiment provides the method of treating cancer wherein exemestane is administered with a meal. Another embodiment provides the method of treating cancer wherein about 25 mg of exemestane is administered. Another embodiment provides the method of treating cancer wherein the exemestane is administered once daily. Another embodiment provides the method of treating cancer wherein the patient is administered about 10 mg of entinostat. Another embodiment provides the method of treating cancer wherein the patient is administered about 5 mg of entinostat. Another embodiment provides the method of treating cancer wherein the patient is administered from about 1 mg to about 20 mg of entinostat.
• Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 2 hours prior to administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient has not consumed food within 1 hour prior to administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 2 hours after administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient does not consume food within 30 minutes after administration of entinostat under fasting conditions. Another embodiment provides the method of treating cancer wherein the patient consumes a high fat meal under fed conditions.
• abnormal cell growth refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including the abnormal growth of normal cells and the growth of abnormal cells.
• Neoplasia as described herein, is an abnormal, unregulated and disorganized proliferation of cells that is distinguished from normal cells by autonomous growth and somatic mutations. As neoplastic cells grow and divide they pass on their genetic mutations and proliferative
• a neoplasm, or tumor is an accumulation of neoplastic cells.
• the neoplasm can be benign or malignant.
• Metastasis refers to the dissemination of tumor cells via lymphatics or blood vessels. Metastasis also refers to the migration of tumor cells by direct extension through serous cavities, or subarachnoid or other spaces. Through the process of metastasis, tumor cell migration to other areas of the body establishes neoplasms in areas away from the site of initial appearance.
• angiogenesis is prominent in tumor formation and metastasis.
• Angiogenic factors have been found associated with several solid tumors such as
• Tumors in which angiogenesis is important include solid tumors such as renal cell carcinoma, hepatocellular carcinoma, and benign tumors such as acoustic neuroma, and neurofibroma.
• Angiogenesis has been associated with blood-born tumors such as leukemias. It is believed that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia. Prevention of angiogenesis could halt the growth of cancerous tumors and the resultant damage to the subject due to the presence of the tumor.
• subject refers to an animal, including, but not limited to, a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse.
• primate e.g., human
• cow, sheep, goat horse
• dog cat
• rabbit rat
• patient are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject.
• treat means to include alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
• terapéuticaally effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated.
• therapeutically effective amount also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.
• pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.
• pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.
• Each component must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
• composition refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers.
• the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.
• Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• fasting are defined to mean, in general, the condition of not having consumed food during the period between from at least about 30 minutes prior to the administration of a therapeutic agent described herein to at least about 30 minutes after the administration of a therapeutic agent described herein.
• food is not consumed from at least about 2 hours prior to the administration of a therapeutic agent described herein to at least about 1 hour after the administration of a therapeutic agent described herein.
• food is not consumed from at least about 1 hours prior to the administration of a therapeutic agent described herein to at least about 1 hour after the administration of a therapeutic agent described herein.
• food is not consumed from at least about 1 hours prior to the administration of a therapeutic agent described herein to at least about 2 hour after the administration of a therapeutic agent described herein.
• the term "fed condition" refers to the condition of having eaten a meal.
• the food is a high fat or a high calorie meal.
• a high calorie meal can include, but is not limited to, a meal comprising 500 calories or more, from about 300 to about 800 calories, from about 500 calories to about 1,000 calories, and from about 800 calories to about 1,500 calories.
• a high fat meal includes, but is not limited to, a calorie from fat percentage of a daily caloric intake from about 20% to about 50%, from about 30 to about 60%, and from about 40 to about 70%.
• the meal is not high fat.
• the meal is not high calorie.
• bioavailability generally means the rate and extent to which an active ingredient is absorbed from a therapeutic agent and becomes available at the site of action.
• bioavailability relates to the processes by which the active ingredient is released from the oral dosage form and moves to the site of action.
• oral bioavailability or “%F” is defined as AUC 0 r a i/AUC wherein AUC ora i is the AUC determined after oral
• AUCi v is the AUC determined after iv administration.
• AUC refers to the area under the drug-concentration curve.
• AUC 0 1
• AUClast refers to the area under the drug-concentration curve from zero to last data point of drug-concentration curve.
• AUC 0-00 or "AUCinf ' refers to the area under the drug-concentration curve from zero to infinite time.
• ti/2 refers to the elimination half-life of the indicated species.
• t max refers to the time of the maximum concentration of the indicated species.
• C max refers to the maximum concentration of the indicated species.
• the HDACs are a family including at least eighteen enzymes, grouped in three classes (Class I, II and III).
• Class I HDACs include, but are not limited to, HADCs 1, 2, 3, and 8.
• Class I HDACs can be found in the nucleus and are believed to be involved with transcriptional control repressors.
• Class II HDACs include, but are not limited to, HDACS 4, 5, 6, 7, and 9 and can be found in both the cytoplasm as well as the nucleus.
• Class III HDACs are believed to be NAD dependent proteins and include, but are not limited to, members of the Sirtuin family of proteins. Non-limiting examples of sirtuin proteins include SIRT1-7.
• selective HDAC refers to an HDAC inhibitor that does not interact with all three HDAC classes.
• HDAC inhibitors can be classified broadly into pan HDAC inhibitors and selective HDAC inhibitors. Although there is a large structural diversity of known HDAC inhibitors, they share common features: a part that interacts with the enzyme active site and a side-chain that sits inside the channel leading to the active site. This can be seen with the hydroxamates such as SAHA, where the hydroxamate group is believed to interact with the active site. In the case of the depsipeptides, it is believed that an intracellular reduction of the disulphide bond creates a free thiol group (which interacts with the active site) attached to a 4-carbon alkenyl chain.
• HDAC inhibitors A difference between the HDAC inhibitors is in the way that they interact with the rim of the HDAC channel, which is at the opposite end of the channel to the active site. It is this interaction, between the HDAC inhibitor and the rim of the channel, which is believed to account, at least in part, for some observed differences in HDAC selectivity between pan-HDAC inhibitors, such as SAHA and selective HDAC inhibitors such as the depsipeptides.
• a particularly preferred HDAC inhibitor is entinostat. Entinostat has the chemical name N-(2-aminophenyl)-4-[N-(pyridine-3- yl)methoxycarbonylamino-methyl]-benzamide and the chemical structure shown below.
• Estrogen is one of the female sex hormones and has many functions in the body. It has been found that about 80% of breast cancer tumors overexpress the estrogen receptor and respond positively to the presence of estrogen. In postmenopausal women, ovarian estrogen production is reduced and plasma estrogen levels are generally lower than in premenopausal women.
• a residual source of estrogen in post-menopausal women is the synthesis of estrogens from androgens, which is catalyzed by aromatase. Inhibition of aromatase activity should lead to a reduction in the levels of estrogen and therefore a reduction in the growth of breast cancer tumors which respond positively to the presence of estrogen.
• Aromatase is an enzyme of the cytochrome P450 family and a product of the CYP19 gene.
• the chemical function of aromatase is to convert testosterone to estradiol and androstenedione to estrone.
• Aromatase inhibitors decrease the body's estrogen by blocking the enzyme aromatase from turning androgen into estrogen.
• certain aromatase inhibitors may be used as adjuvant therapy instead of tamoxifen or after 2 or more years of tamoxifen.
• aromatase inhibitors are being tested in clinical trials to compare them to hormone therapy with tamoxifen.
• an "aromatase inhibitor” is a molecule which inhibits the activity of the aromatase enzyme.
• Compounds which are inhibitors of aromatase can be readily identified by one skilled in the art using methods such as, for example, standard pharmacological test procedures which measure the inhibition of the conversion of l,2- 3 H-androstenedione to estrone.
• a microsomal fraction is prepared from human placenta by the method as described by Thompson and Siiteri (J. Biol. Chem., Vol. 249, p. 5364 (1974)).
• the microsomal preparation so obtained is lyophilized and stored at -40 °C.
• the human placental microsomes are added to 1,2- 3 H- androstenedione and incubated for 20 minutes at 37 °C.
• the amount of aromatization of the labelled substrate is detected by the loss of 3 H 2 0 into the incubation medium.
• the substrate is removed by chloroform extraction, followed by adsorption to charcoal in suspension.
• compositions are tested for aromatase inhibitory activity by adding them to the incubation medium prior to the addition of the microsomes.
• the relative cpm obtained with and without the composition is used to calculate the percent inhibition of the aromatization of androstenedione to estrone.
• IC5 0 values can be determined graphically as the concentration of test composition at which the aromatization of androstenedione to estrone is reduced to 50% of control value.
• Subcutaneous fat is a major site of aromatase activity and it has been suggested that plasma estrogen levels correlate with body-mass index (Longcope et al , Metabolism 1986, 35, 235-7). It has been suggested that at menopause, plasma estrogen levels fall from about 110 pg/mL to a much lower level of about 7 pg/mL. However, in post-menopausal women, the intra-tumoral concentration of estradiol has been found to be about 10 times higher than in the plasma, probably due to aromatase activity within the tumor.
• aromatase as a treatment option for breast cancer has been studied with some success.
• three aromatase inhibitors are approved for marketing in the US for the treatment of breast cancer, at various stages, in post-menopausal women.
• Letrozole (Femara®) is indicated for several treatment options including, extended adjuvant treatment of early breast cancer in postmenopausal women with 5 years prior tamoxifen treatment, treatment of post menopausal women with hormone receptor positive (or unknown) locally advanced or metastatic breast cancer and advanced breast cancer treatment in postmenopausal women with disease progression following antiestrogen therapy.
• Anastrozole (Arimidex®) is indicated for several treatment options including, adjuvant treatment of postmenopausal women with hormone receptor-(+) early breast cancer, first-line treatment of post menopausal women with hormone receptor-(+) (or unknown) locally advanced or metastatic breast cancer and advanced breast cancer in postmenopausal women with disease progression following tamoxifen therapy.
• Exemestane (Aromasin®) is indicated for several treatment options including, adjuvant treatment of postmenopausal women with estrogen-receptor-(+) early breast cancer who have received 2-3 years of tamoxifen treatment and advanced breast cancer in postmenopausal women with disease progression following tamoxifen therapy.
• Type 1 exemestane is based on a steroid chemical structure
• type 2 letrozole and anastrozole are based on a non-steroidal chemical structure.
• Clinical trials have shown letrozole to be superior to tamoxifen in the treatment of advanced ER(+) disease. In early disease, adjuvant therapy with anastrozole appears to be superior to therapy with tamoxifen in reducing risk of relapse. Recent clinical trial results have led to aromatase inhibitors replacing tamoxifen as the standard of care for breast cancer treatment.
• Cancers associated with overexpression of HER2/neu include breast, ovarian, endometrial, prostate, gastric, salivary gland, pancreatic, colorectal, oral and non-small cell lung cancers.
• Breast cancer has been a focus of anti-HER2/neu treatments.
• compositions and therapies described herein may be combined with other therapeutic agents.
• agents include, by way of example only, cetuximab, paclitaxel, docetaxel, taxane formulations, for example, Abraxane® (ABI-007), Paclitaxel-Cremophor EL, Paclitaxel poliglumex, and Paclitaxel injectable emulsion (PIE).
• BCI-007 Abraxane®
• Paclitaxel-Cremophor EL Paclitaxel-Cremophor EL
• Paclitaxel poliglumex Paclitaxel injectable emulsion
• PIE Paclitaxel injectable emulsion
• Hormonal therapies are the mainstay of treatment of estrogen receptor positive (ER+) breast cancer (BC). Due to both the clinical activity and the overall favorable side effect profile and tolerance of hormonal agents, the standard of care typically involves sequencing of hormonal agents until either the development of resistance and/or visceral crises necessitate switching to chemotherapy.
• the aromatase inhibitors are a preferred class of anti-estrogen therapy that functions by blocking endogenous estrogen synthesis.
• Exemestane is a steroidal AI which irreversibly binds and inactivates the aromatase enzyme with demonstrated efficacy in the metastatic setting after progression on a non-steroidal AI, NSAI; i.e.
• letrozole or anastrozole (Chia S, Gradishar W, Mauriac L, et al: Double-blind, randomized placebo controlled trial of fulvestrant compared with exemestane after prior nonsteroidal aromatase inhibitor therapy in postmenopausal women with hormone receptor-positive, advanced breast cancer: results from EFECT. J Clin Oncol 26: 1664-1670, 2008).
• Entinostat is a novel, oral inhibitor of histone deacetylases (HDAC), with high specificity towards class 1 HDACs and a unique pharmacological profile allowing for weekly dosing. HDAC inhibition leads to elevated protein lysine acetylation in tumor and peripheral blood cells serving as a surrogate potential pharmacodynamic marker of activity.
• HDAC histone deacetylases
• Entinostat' s class 1 specificity distinguishes it from the United States (US) Food and Drug Administration (FDA)-approved HDAC inhibitors (HDACi) vorinstat (Zolinza®) and romidepsin (Istodax®).
• HDACi HDAC inhibitors
• Vorinstat Zolinza®
• romidepsin Istodax®
• entinostat has demonstrated inhibition of ERa positive tumor growth and restoration of hormone sensitivity as a result of down-regulation of estrogen- independent growth factor signaling pathways, normalization of ERa levels and increases in aromatase enzyme levels.
• Available additional treatments for breast cancer include, without limitation, radiation therapy, chemotherapy, antibody therapy, and tyrosine kinase inhibitors as adjuvant therapy.
• Radiotherapy is a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing.
• Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
• chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy).
• systemic chemotherapy When chemotherapy is placed directly into the spinal column, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.
• Cytoxic agents used for treating breast cancer include doxorubicin, cyclophosphamide, methotrexate, 5- fluorouracil, mitomycin C, mitoxantrone, paclitaxel, taxane formulations such as by way of example only, Abraxane® (ABI-007), Paclitaxel-Cremophor EL, Paclitaxel poliglumex, and Paclitaxel injectable emulsion (PIE), gemcitabine, docetaxel, capecitabine and epirubicin.
• Other chemotherapy against breast cancer includes treatment with one or more of bendamustine, carboplatin (for example, Paraplatin®), carmustine (for example, BCNU®), chlorambucil (for example, Leukeran®), cisplatin (for example, Platinol®), cyclophosphamide injection (for example, Cytoxan®), oral cyclophosphamide (for example, Cytoxan®), dacarbazine (for example, DTIC®), ifosfamide (for example, ifex®), lomustine (for example, CCNU®), mechlorethamine (for example, nitrogen mustard, Mustargen®), melphalan (for example,
• Alkeran® procarbazine (for example, Matulane®), bleomycin (for example, Blenoxane®), doxorubicin (for example, Adriamycin®, Rubex®), epirubicin, Idarubicin (for example,
• Idamycin® mitoxantrone (for example, Novantrone®), gemcitabine (for example, Gemzar®), oral mercaptopurine (for example, Purinethol®).
• methotrexate pentostatin IV (for example, Nipent®), oral thioguanine (for example, Lanvis®), oral etoposide (for example, VP- 16, VePesid®,
• Etopophos - etoposide IV (for example, VP- 16, VePesid®, Etopophos), vinblastine (for example, Velban®), vincristine (for example, Oncovin®), vinorelbine (for example, Navelbine®), dexamethasone (for example, Decadron®), methylprednisolone (for example, Medrol®), and prednisone (for example, Deltasone®).
• vinblastine for example, Velban®
• vincristine for example, Oncovin®
• vinorelbine for example, Navelbine®
• dexamethasone for example, Decadron®
• methylprednisolone for example, Medrol®
• prednisone for example, Deltasone®
• Monoclonal antibody therapy is a cancer treatment that uses antibodies made in the laboratory, from a single type of immune system cell. These antibodies can identify substances on cancer cells or normal substances that may help cancer cells grow. The antibodies attach to the substances and kill the cancer cells, block their growth, or keep them from spreading. Monoclonal antibodies are given by infusion. They may be used alone or to carry drugs, toxins, or radioactive material directly to cancer cells. Monoclonal antibodies are also used in combination with chemotherapy as adjuvant therapy.
• Trastuzumab (Herceptin®) is a monoclonal antibody that blocks the effects of the growth factor protein HER2, which transmits growth signals to breast cancer cells.
• trastuzumab leads to clinical responses as a single agent and improves survival when added to chemotherapy for advanced HER2 -positive breast cancer.
• some patients do not respond to trastuzumab, and most eventually develop clinical resistance.
• Mechanisms of intrinsic and acquired trastuzumab resistance are poorly understood.
• One study which utilized a cell line- based approach to delineate genetic and protein alterations associated with resistance has been reported (D. Tripathy et al Journal of Clinical Oncology, 2005 Vol 23, No 16S, 3121). These researchers studied two HER2 -positive breast cancer cell lines (BT474 and SKBR3) that were serially passaged in the presence of trastuzumab until in vitro resistance was documented.
• Resistant cell lines emerged after 12 months and exhibited a 3-fold more rapid growth rate in the absence of trastuzumab. Following trastuzumab exposure, Go/Gi arrest was observed in sensitive compared to resistant cells (84 vs. 68%), with fewer cells in S-phase (3 vs. 14%). Resistant cell lines exhibited fewer changes in gene expression with trastuzumab as well as upregulation of the chemokine receptor CXCR4 and mitotic checkpoint regulators, and downregulation of PTEN compared to sensitive cells.
• Additional, illustrative, treatments that may be advantageously combined with the compositions and therapies disclosed herein may include, without limitation, administration of agents including, but not limited to lapatinib, alone or in combination with capecitabine, docetaxel, epirubicin, epothilone A, B or D, goserelin acetate, paclitaxel, pamidronate, bevacizumab, or trastuzumab.
• agents including, but not limited to lapatinib, alone or in combination with capecitabine, docetaxel, epirubicin, epothilone A, B or D, goserelin acetate, paclitaxel, pamidronate, bevacizumab, or trastuzumab.
• the additional therapy comprises chemotherapy comprising administering to the subject one or more of doxorubicin, cyclophosphamide, paclitaxel, lapatinib, capecitabine, trastuzumab, bevacizumab, gemcitabine, eribulin, or nab-paclitaxel.
• One embodiment provides a method of treating breast cancer in a patient comprising (i) measuring the level of protein lysine acetylation prior to administration of entinostat-aromatase inhibitor combination therapy, (ii) administering entinostat-aromatase inhibitor combination therapy, (iii) measuring the level of protein lysine acetylation after administration of entinostat- aromatase inhibitor combination therapy, (iv) comparing the level of protein lysine acetylation after administration of entinostat-aromatase inhibitor combination therapy with the level of protein lysine acetylation prior to administration of entinostat-aromatase inhibitor combination therapy, and (v) continuing treatment with entinostat-aromatase inhibitor combination therapy if the level of protein lysine acetylation after administration of entinostat-aromatase inhibitor combination therapy is greater than the level of protein lysine acetylation prior to administration of
• One embodiment provides a method of treating breast cancer in a patient comprising (i) administring entinostat-aromatase inhibitor combination therapy, and (ii) determining the change in protein lysine acetylation levels during the course of said therapy compared to pre-therapy protein lysine acetylation levels.
• entinostat is administered to a fasting patient.
• One embodiment provides a method of treating breast cancer in a patient comprising (i) determining the level prior to administration of protein lysine acetylation, (ii) administring entinostat-aromatase inhibitor combination therapy, and (iii) determining the level of protein lysine acetylation during the course of therapy.
• entinostat is administered to a fasting patient.
• entinostat it is desirable to increase the oral bioavailability of therpeutic agents, such as entinostat, to increase the extent of the therapeutic effect on the patient.
• food has a variable effect on the bioavailability of a therapeutic agent. Interactions between a therapeutic agent and food may result in reduced, delayed or increased systemic drug availability.
• Food may interact with a therapeutic agent at the following phases: (i) before and during gastrointestinal absorption; (ii) during distribution; (iii) during metabolism; and (iv) during elimination.
• entinostat bioavailability decreases when administered with food.
• Food can affect peak exposure (Cmax) and time to peak exposure (Tmax) by delaying gastric emptying and prolonging intestinal transit time. In some instances, food affects the total exposure, or area under the concentration-time curve (AUC). In some embodiments, the Cmax is higher when entinostat is administered without food as compared to the Cmax when entinostat is administered with food. In some embodiments, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 2: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 3 : 1.
• the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 4: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 5: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 6: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 7: 1.
• the Tmax is lower when entinostat is administered without food as compared to the Tmax when entinostat is administered with food.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 2: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 3: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 4: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 5: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 6: 1.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 7: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 8: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 9: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 10: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 1 1 : 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 12: 1.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 13 : 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 14: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 15: 1.
• exemestane is administered a different time of day than entinostat. In one embodiment, exemstane is administerd after a meal. In one embodiemt, exemstane is administerd with a meal.
• Another embodiment provides the method wherein determining the change in protein lysine acetylation level during the course of said therapy occurs after about 2 days of therapy, about 5 days of therapy, about 7 days of therapy, about 15 days of therapy, or about 21 days of therapy.
• Another embodiment provides the method wherein the protein lysine acetylation levels are obtained from a tissue sample selected from B-cells, T-cells, or monocytes.
• Another embodiment provides the method wherein the aromatase inhibitor is exemestane. Another embodiment provides the method wherein the aromatase inhibitor is anasrozole. Another embodiment provides the method wherein the aromatase inhibitor is letrozole. Another embodiment provides the method wherein the aromatase inhibitor is administered daily. Another embodiment provides the method wherein the aromatase inhibitor is exemestane and is
• etinostat is administered daily.
• etinostat is administered every 7 days of a 28-day cycle.
• etinostat is administered every 14 days of a 28-day cycle.
• the entinostat-aromatase inhibitor combination therapy comprises oral administration of entinostat every 7 days of a 28-day cycle, and oral administration of exemestane every day.
• the entinostat-aromatase inhibitor combination therapy comprises oral administration of entinostat every 14 days of a 28-day cycle, and oral administration of exemestane every day.
• Another embodiment provides the method wherein etinostat is administered to a fasting patient every 7 days of a 28-day cycle. Another embodiment provides the method wherein etinostat is administered to a fasting patient every 14 days of a 28-day cycle. Another embodiment provides the method wherein the entinostat-aromatase inhibitor combination therapy comprises oral administration of entinostat to a fasting patient every 7 days of a 28-day cycle, and oral administration of exemestane every day. Another embodiment provides the method wherein the entinostat-aromatase inhibitor combination therapy comprises oral administration of entinostat to a fasting patient every 14 days of a 28-day cycle, and oral administration of exemestane every day.
• Another embodiment provides the method wherein the step of determining the protein lysine acetylation level during the course of therapy is performed more than once. Another embodiment provides the method wherein the step of determining the protein lysine acetylation level during the course of therapy is performed once.
• Another embodiment provides the method further comprising selecting the patient for further treatment if the level of protein lysine acetylation level increases during the course of therapy.
• Another embodiment provides the method further comprising selecting the patient for further treatment if the level of protein lysine acetylation level increases during the first week of the course of therapy. Another embodiment provides the method further comprising selecting the patient for further treatment if the level of protein lysine acetylation level increases during the first and second week of the course of therapy.
• One embodiment provides a method of selecting a patient for further entinostat-aromatase inhibitor combination therapy comprising comparing the protein lysine acetylation level in a tissue sample obtained after initiating therapy to the protein lysine acetylation levels determined prior to initiating therapy.
• One embodiment provides a method of selecting a patient for further entinostat-aromatase inhibitor combination therapy comprising comparing the protein lysine acetylation level in a tissue sample obtained after initiating therapy to the protein lysine acetylation levels determined prior to initiating therapy, wherein an increase in protein lysine acetylation level after initiating therapy indicates the patient will benefit from further therapy.
• Another embodiment provides the method wherein the protein lysine acetylation level in a tissue sample obtained after initiating therapy is determined more than once. Another embodiment provides the method wherein increase in protein lysine acetylation level after initiating therapy occurs over a time period of one week. Another embodiment provides the method wherein the protein lysine acetylation level after initiating therapy is determined on days 2, 8 and 15.
• Another embodiment provides the method wherein the increase is from about 10 % to about 500 %. Another embodiment provides the method wherein the increase is from about 10 % to about 400 %. Another embodiment provides the method wherein the increase is from about 10 % to about 300 %. Another embodiment provides the method wherein the increase is from about 10 % to about 200 %. Another embodiment provides the method wherein the increase is from about 10 % to about 100 %. Another embodiment provides the method wherein the increase is about 10%, about 20%, about 30%, about 40%, about 50% or about 60%. Another embodiment provides the method wherein the increase is about 25%, about 50%, about 75%, about 100%, about 125% or about 150%.
• tissue sample is selected from B- cells, T-cells, or monocytes.
• tissue sample obtained after initiating therapy is obtained at least 2 days after initiating therapy.
• tissue sample obtained after initiating therapy is obtained between day 2 and day 28 after initiating therapy.
• tissue sample obtained after initiating therapy is obtained on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 after initiating therapy.
• One embodiment provides a method of selecting a patient for further entinostat-aromatase inhibitor combination therapy comprising comparing the percent change in protein lysine acetylation levels in a tissue sample obtained after initiating therapy to the protein lysine acetylation levels determined prior to initiating therapy, wherein a percent decrease in protein lysine acetylation levels after initiating therapy of about 5 percent to about 50 percent indicates the patient will not benefit from further therapy.
• One embodiment provides a method of treating breast cancer which displays resistance to prior aromatase inhibitor therapy, the method comprising administering to a patient a combination comprising entinostat and an aromatase inhibitor, wherein the patient did not demonstrate a complete response, a partial response or stable disease for greater than six months during prior treatment with an aromatase inhibitor.
• entinostat is administered to a fasting patient.
• Another embodiment provides the method wherein the patient relapsed during treatment on or within 6 months of completion of prior non-steroidal aromatase inhibitor given as adjuvant therapy.
• Another embodiment provides the method wherein the patient demonstrated progressive disease after at least 3 months treatment on prior non-steroidal aromatase inhibitor.
• Another embodiment provides the method wherein the breast cancer is ER-positive.
• Another embodiment provides the method wherein the aromatase inhibitor administered in combination with entinostat is letrozole. Another embodiment provides the method wherein the aromatase inhibitor administered in combination with entinostat is anastrozole. Another embodiment provides the method wherein the aromatase inhibitor administered in combination with entinostat is exemestane. [0088] Another embodiment provides the method wherein entinostat and the aromatase inhibitor are administered sequentially in either order or simultaneously. Another embodiment provides the method wherein entinostat and the aromatase inhibitor are administered simultaneously. Another embodiment provides the method wherein the aromatase inhibitor is administered first. Another embodiment provides the method wherein the aromatase inhibitor is administered daily and the entinostat is administered periodically. Another embodiment provides the method wherein entinostat is adminsistered weekly and the aromatase inhibitor is administered daily. Another embodiment provides the method wherein entinostat is introduced to an ongoing aromatase inhibitor course of therapy.
• Another embodiment provides the method further comprising administering to the subject one or more therapies in addition to the combination of entinostat and the aromatase inhibitor selected from the group consisting of: letrozole, anastrozole or exemestane, or their
• the one or more therapies comprise one or more of radiation therapy, chemotherapy, high dose chemotherapy with stem cell transplant, and monoclonal antibody therapy.
• radiation therapy comprises internal and/or external radiation therapy.
• the chemotherapy comprisies administering to the subject one or more of doxorubicin, cyclophosphamide, paclitaxel, lapatinib, capecitabine, trastuzumab, bevacizumab, gemcitabine, eribulin, or nab-paclitaxel.
• One embodiment provides a method of treating breast cancer in a patient in need thereof, comprising oral administration of exemestane and entinostat, wherein the entinostat is administered to a fasting patient. Another embodiment provides the method of treating breast cancer wherein the entinostat Tmax is less than 1 hour post administration. Another embodiment provides the method of treating breast cancer wherein the entinostat Tmax is less than 90 minutes post administration. Another embodiment provides the method of treating breast cancer wherein the entinostat Tmax is less than 2 hours post administration. Another embodiment provides the method of treating breast cancer wherein the entinostat Tmax is between 30 minutes and 2 hours post administration.
• Another embodiment provides the method of treating breast cancer wherein the entinostat Cmax is at least 150 ng/mL following oral administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the entinostat Cmax is at least 125 ng/mL following oral administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the entinostat Cmax is at least 100 ng/mL following oral administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the entinostat Cmax is at least 80 ng/mL following oral administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the entinostat Cmax is at least 50 ng/mL following oral administration of entinostat.
• Another embodiment provides the method of treating breast cancer wherein about 5 mg of entinostat is administered. Another embodiment provides the method of treating breast cancer wherein about 10 mg of entinostat is administered. Another embodiment provides the method of treating breast cancer wherein from about 1 mg to about 20 mg of entinostat is administered. Another embodiment provides the method of treating breast cancer wherein entinostat is administered once per week. Another embodiment provides the method of treating breast cancer wherein entinostat is administered for a 28-day cycle. Another embodiment provides the method of treating breast cancer wherein the patient has not consumed food within 2 hours prior to administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the patient has not consumed food within 1 hour prior to administration of entinostat.
• Another embodiment provides the method of treating breast cancer wherein the patient does not consume food within 2 hours after administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the patient does not consume food within 30 minutes after administration of entinostat. Another embodiment provides the method of treating breast cancer wherein the exemestane is administered at a different time of day than entinostat. Another embodiment provides the method of treating breast cancer wherein exemestane is administered after a meal. Another embodiment provides the method of treating breast cancer wherein exemestane is administered with a meal. Another embodiment provides the method of treating breast cancer wherein about 25 mg of exemestane is administered once daily.
• EGFR also known as ErbB l or Herl
• ErbB l is a transmembrane glycoprotein encoded by a gene located on chromosome 7 (7pl2.1-12.3).
• EGFR comprises 1186 amino acids (a.a.) and 26 exons.
• Exons 1-14 encode the extracellular domain, exon 15 encodes the transmembrane region and exons 16-26 the intracellular domain.
• This glycoprotein belongs to the ErbB receptor family, which also consists of: ErbB2 (HER2/neu), ErbB3 (HER3) and ErbB4 (HER4).
• Each of these proteins is structurally composed of an extracellular domain, a hydrophobic transmembrane domain and an intracellular domain with intrinsic tyrosine kinase (TK) activity (except ErbB3).
• TK tyrosine kinase
• These receptors exist as inactive monomers, being activated by their interaction, through the extracellular domain, with growth factors of the EGF family.
• the binding of ErbB receptor molecules to one of these ligands leads to its interaction with other monomers of the same family (receptor dimerization). This dimerization can occur between two identical receptors (homodimerization, e.g., ErbB l- ErbB l) or between two different receptors (heterodimerization, e.g., ErbB l-ErbB3).
• the stimulation caused by a specific ligand triggers a unique pattern of dimerization, which is also specific to the tissue/tumor in which the phenomenon occurs. Dimerization of the receptors leads to their autophosphorylation with activation of TK and activation of a cascade of intracellular biochemical processes that regulate such diverse activities, like proliferation, differentiation, apoptosis and cell migration.
• Epithelial cadherin also known as cadherin-1, CAM 120/80 or uvomorulin, is a protein that in humans is encoded by the CDH1 gene.
• E-cadherin is a classical member of the cadherin superfamily.
• E-cadherin is a calcium-dependent cell-cell adhesion glycoprotein composed of five extracellular cadherin repeats (EC1-EC5) in the extracellular domain, a transmembrane domain, an intracellular domain that binds pl20-catenin and beta-catenin, and a highly conserved cytoplasmic tail.
• the intracellular domain contains a highly-phosphorylated region vital to beta- catenin binding and, therefore, to E-cadherin function.
• Beta-catenin can also bind to alpha-catenin.
• Alpha-catenin participates in regulation of actin-containing cytoskeletal filaments. In epithelial cells, E-cadherin-containing cell-to-cell junctions are often adjacent to actin-containing filaments of the cytoskeleton.
• Mutations in this gene are correlated with gastric, breast, colorectal, thyroid, and ovarian cancers. Loss of function or expression is thought to contribute to progression in cancer and metastasis. E-cadherin downregulation decreases the strength of cellular adhesion within a tissue, resulting in an increase in cellular motility. This in turn may allow cancer cells to cross the basement membrane and invade surrounding tissues.
• E-cadherin protein levels can be quantitatively measured by ELISA.
• E-cadherin ELISA kits such as the E-cadherin EIA kit provided by TaKaRA, are a solid phase sandwich EIA that utilizes two mouse monoclonal E-cadherin antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of human E-cadherin using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled E-cadherin antibody.
• sample soluble E-cadherin is determined by measuring absorbance using an EIA plate reader. Accurate soluble E-cadherin sample concentrations can be determined by comparing their specific absorbances with the absorbance obtained for the Standard plotted on a standard curve. In some embodiments, E-cadherin protein levels are quantitatively measured by ELISA.
• E-cadherin protein levels can be detected by immunohistochemistry.
• To detect E-cadherin levels in immersion fixed cells cells are incubated with Human E-Cadherin Antigen Affinity- purified Polyclonal Antibody (R&D Systems® Catalog # AF648) at 10 ⁇ g/mL for 3 hours at room temperature. Cells are then stained using the NorthernLightsTM 557-conjugated Anti-Goat IgG Secondary Antibody (R&D Systems® Catalog # NL001) and counterstained with DAPI. E- cadherin and DAPI can be visualized using a fluorescence microscope and filter sets appropriate for the label used. In some embodiments, E-cadherin protein levels are detected by
• coverslips for immunocytochemistry can be prepared using gelatin.
• a method for preparing coverslips for ICC includes a) placing sterilized coverslips into the wells of a 24-well plate, b) adding 400 ⁇ , of the gelatin-coating solution and c) incubating the coverslips for 10 minutes at room temperature. Then the gelatin-coating solution is removed and the coverslips are air-dried for 15 minutes. The dried coverslips can be stored at room temperature until use. Once the coverslips have been prepared, the cells can be prepared and fixed as follows.
• Culture cells by adding 500 ⁇ , of culture media containing approximately 5000 cells to the wells of a cell culture plate containing gelatin-coated coverslips. When cells have reached the desired density/age, remove the culture media from each well and wash twice with PBS. Add 300-400 ⁇ ⁇ of 2-4%
• Formaldehyde Fixative Solution to each well, and incubate for 20 minutes at room temperature. Wash the wells twice with PBS and cover with 400 ⁇ , of wash buffer. The coverslips can be stored at 2-8 °C for up to 3 months or they may be stained immediately. Once the cells have been prepared, the cells can be stained for ICC as follows. Wash the coverslips containing the fixed cells two times in 400 ⁇ , of wash buffer. Block non-specific staining by adding 400 ⁇ , of blocking buffer and incubate for 45 minutes at room temperature. Remove blocking buffer. No rinsing is necessary. Dilute the unconjugated primary antibody (or fluorescence-conjugated primary) in dilution buffer according to the manufacturer's instructions.
• DAPI binds to DNA and is a convenient nuclear counterstain. It has an absorption maximum at 358 nm and fluoresces blue at an emission maximum of 461 nm. Rinse once with PBS and once with water. Carefully remove the coverslips from the wells and blot to remove any excess water. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip. Mount the coverslip with the cells facing towards the microscope slide. Visualize using a fluorescence microscope and filter sets appropriate for the label used. Slides can also be stored in a slide box at ⁇ -20 °C for later examination. In some embodiments, E-cadherin protein levels are detected by
• E-cadherin gene expression can be determined by measuring E-cadherin methylation.
• E-cadherin methylation kits such as the CpG WIZ® E-cadherin amplification kit provided by Millipore®, determine the methylation of status of the E-cadherin promoter by methylation-specific PCR (MSP).
• MSP methylation-specific PCR
• the kit contains primers targeted to regions of the promoter where the sequences are most divergent after bisulfite treatment. PCR parameters have been identified so that all primer sets in the kit amplify under the same conditions. Control genomic DNA samples (methylated and unmethylated) for E-cadherin are also included.
• E-cadherin gene expression is determined by measuring E-cadherin methylation.
• One embodiment provides a method of treating cancer in an EGFR inhibitor-nai ' ve patient progressed on prior therapy, wherein said patient exhibits high E-cadherin expression levels, the method comprising administering to the patient a combination comprising entinostat and an EGFR inhibitor.
• Another embodiment provides the method wherein high E-cadherin expression levels are characterized by ELISA, immunohistochemistry, immunocytochemistry or determination of E-cadherin methylation levels.
• Another embodiment provides the method wherein high E- cadherin expression levels are determined by immunohistochemistry.
• Another embodiment provides the method wherein the high E-cadherin expression levels are scored as +3 as determined by immunohistochemistry.
• Lung cancer is the leading cause of cancer deaths in women and men both in the
• lung cancer has surpassed breast cancer as the leading cause of cancer deaths in women.
• 157,300 people were projected to die from lung cancer, which is more than the number of deaths from colon and rectal, breast, and prostate cancer combined.
• Only about 2% of those diagnosed with lung cancer that has spread to other areas of the body are alive five years after the diagnosis, although the survival rates for lung cancers diagnosed at the earliest stage are higher, with approximately 49% surviving for five years or longer.
• Cancer occurs when normal cells undergo a transformation that causes them to grow and multiply without control.
• the cells form a mass or tumor that differs from the
• Tumors are dangerous because they take oxygen, nutrients, and space from healthy cells and because they invade and destroy or reduce the ability of normal tissues to function.
• lung tumors are malignant. This means that they invade and destroy the healthy tissues around them and can spread throughout the body. The tumors can spread to nearby lymph nodes or through the bloodstream to other organs. This process is called metastasis.
• metastasis When lung cancer metastasizes, the tumor in the lung is called the primary tumor, and the tumors in other parts of the body are called secondary tumors or metastatic tumors.
• Some tumors in the lung are metastatic from cancers elsewhere in the body.
• the lungs are a common site for metastasis. If this is the case, the cancer is not considered to be lung cancer.
• prostate cancer spreads via the bloodstream to the lungs, it is metastatic prostate cancer (a secondary cancer) in the lung and is not called lung cancer.
• Lung cancer comprises a group of different types of tumors. Lung cancers usually are divided into two main groups that account for about 95% of all cases. The division into groups is based on the type of cells that make up the cancer.
• the two main types of lung cancer are characterized by the cell size of the tumor when viewed under the microscope. They are called small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
• SCLC small cell lung cancer
• NSCLC non-small cell lung cancer
• SCLC includes several subtypes of tumors. SCLCs are less common, but they grow more quickly and are more likely to metastasize than NSCLCs. Often, SCLCs have already spread to other parts of the body when the cancer is diagnosed. About 5% of lung cancers are of rare cell types, including carcinoid tumor, lymphoma, and others.
• the term "lung cancer” includes, but is not limited to, SCLC, NSCLC, carcinoid tumor, lymphoma, and their various subtypes.
• NSCLC is a cancer of the lung which is not of the small cell carcinoma (oat cell carcinoma) type.
• the term "non-small cell lung cancer” applies to the various types of
• bronchogenic carcinomas (those arising from the lining of the bronchi).
• specific types of NSCLC include, but are not limited to, adenocarcinoma, squamous cell carcinoma, and large cell cancer (i.e., large cell undifferentiated carcinoma).
• Adenocarcinoma is a cancer that develops in the lining or inner surface of an organ. Adenocarcinoma is the most common type of lung cancer, making up 30%-40% of all cases of lung cancer. A subtype of adenocarcinoma is called bronchoalveolar cell carcinoma, which creates a pneumonia-like appearance on chest X-rays.
• Squamous cell carcinoma is a cancer that begins in squamous cells.
• Squamos cells are thin, flat cells that look under the microscope like fish scales.
• Squamous cells are found in the tissue that forms the surface of the skin, the lining of hollow organs of the body, and the passages of the respiratory and digestive tracts. Squamous cell carcinomas may arise in any of these tissues.
• Squamous cell carcinoma is the second most common type of lung cancer, making up about 30% of all cases.
• tumors are often diagnosed by default, when all other possibilities have been excluded. These tumors lack any diagnostic features to suggest their diagnosis prior to biopsy. They tend to grow rapidly, metastasize early, and are strongly associated with smoking. Large cell tumors are usually large, bulky, well-circumscribed, pink-grey masses with extensive hemorrhage and necrosis. Although they commonly have central necrosis, they rarely cavitate. They tend to present in the mid to peripheral lung zones. They may extend locally to involve the segmental or subsegmental bronchi. A variant of large cell carcinoma is giant cell carcinoma. This subtype is particularly aggressive and carries a very poor prognosis. These tumors generally present as a large peripheral mass with a focal necrotic component. They do not involve the large airways, unless by direct extension. Large cell cancer makes up 10%-20% of all cases of lung cancer.
• SCLC is also called oat cell lung cancer and is a type of lung cancer in which the cells appear small and round under the microscope. SCLC is considered distinct from other lung cancers because of their clinical and biologic characteristics. Small cell lung cancer exhibits aggressive behavior, with rapid growth, early spread to distant sites, extraordinar sensitivity to chemotherapy and radiation, and frequent association with distinct paraneoplastic syndromes. Small cell carcinomas arise in peribronchial locations and infiltrate the bronchial submucosa. Widespread metastases occur early in the course of the disease, with common spread to the mediastinal lymph nodes, liver, bones, adrenal glands, and brain.
• SIADH antidiuretic hormone
• ACTH ectopic adrenocorticotropic hormone
• SCLC makes up 20% of all cases.
• Carcinoid tumor is a tumor which secretes large amounts of the hormone serotonin.
• Carcinoid tumor is also called an argentaffinoma.
• the tumor usually arises in the gastrointestinal tract, anywhere between the stomach and the rectum (the favorite spot is in the appendix) and from there may metastasize to the liver. In the liver the tumor produces and releases large quantities of serotonin into the systemic bloodstream. The consequences are called the carcinoid syndrome.
• Carcinoid tumors are considered a type of endocrine tumor since they secrete a hormone (serotonin). They can occur as part of certain genetic disorders such as the multiple endocrine neoplasia (MEN) type 1 and neurofibromatosis type 1 (NF1 or von Recklinghausen disease). Carcinoid tumors account for 1% of all cases.
• MEN multiple endocrine neoplasia
• NF1 or von Recklinghausen disease neurofibromatosis type 1
• Lymphoma is a type of cancer involving cells of the immune system, called lymphocytes, and primarily represents cells involved in the lymphatic system of the body.
• Lymphoma is a malignant transformation of either B or T cells or their subtypes. Lymphomas fall into one of two major categories: Hodgkin's lymphoma (HL, previously called Hodgkin's disease) and all other lymphomas (non-Hodgkin's lymphomas or NHLs). These two types occur in the same places, may be associated with the same symptoms, and often have similar appearance on physical examination. However, they are readily distinguishable via microscopic examination. Hodgkin's disease develops from a specific abnormal B lymphocyte lineage. NHL may derive from either abnormal B or T cells and are distinguished by unique genetic markers. There are five subtypes of Hodgkin's disease and about 30 subtypes of non-Hodgkin's lymphoma.
• lymphomas Because there are so many different subtypes of lymphoma, the classification of lymphomas is complicated (it includes both the microscopic appearance as well as genetic and molecular markers). Many of the NHL subtypes look similar, but they are functionally quite different and respond to different therapies with different probabilities of cure. HL subtypes are microscopically distinct, and typing is based upon the microscopic differences as well as extent of disease.
• EGFR inhibitors interrupt signaling through the epiderdermal growth factor receptor
• EGFR EGFR in target cells.
• Certain EGFR inhibitors such as erlotinib, have been approved for the treatment of metastatic NSCLC.
• EGFR inhibitors such as gefitinib
• Several more EGFR inhibitors are being tested in clinical trials for the treatment of NSCLC and additional lung cancers.
• an "EGFR inhibitor” is a molecule which inhibits the activity of the EGF receptor.
• Compounds which are inhibitors of EGFR can be readily identified by one skilled in the art using methods such as, for example, an EGFR kinase assay which measures ADP formed from a kinase reaction.
• Inhibition of EGFR as a treatment option for lung cancer has been studied with some success. Currently three EGFR inhibitors, erlotinib, gefinitib, and cetuximab, are approved for marketing in the US for the treatment of lung cancer.
• Erlotinib (Tarceva ®) is approved to treat metastatic non-small cell lung cancer and pancreatic cancer that cannot be removed by surgery or has metastasized. This small-molecule drug inhibits the tyrosine kinase activity of EGFR.
• Gefitinib (Iressa®) is approved to treat patients with advanced non-small cell lung cancer. This small-molecule drug is restricted to use in patients who, in the opinion of their treating physician, are currently benefiting, or have previously benefited, from gefitinib treatment. Gefitinib inhibits the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), which is overproduced by many types of cancer cells.
• EGFR epidermal growth factor receptor
• Cetuximab (Erbitux ®) is a monoclonal antibody that is approved for treating some patients with squamous cell carcinoma of the head and neck or colorectal cancer.
• the therapy binds to the external portion of EGFR, thereby preventing the receptor from being activated by growth signals, which may inhibit signal transduction and lead to antiproliferative effects.
• EGFR inhibitors include, but are not limited to,
• Panitumumab (Vectibix ®) is approved to treat some patients with metastatic colon cancer. This monoclonal antibody attaches to EGFR and prevents it from sending growth signals.
• Vandetanib (Caprelsa ®) is approved to treat patients with metastatic medullary thyroid cancer who are ineligible for surgery. This small-molecule drug binds to and blocks the growth-promoting activity of several tyrosine kinase enzymes, including EGFR, several receptors for vascular endothelial growth factor receptor (VEGF), and RET.
• VEGF vascular endothelial growth factor receptor
• Lapatinib (Tykerb ®) is approved for the treatment of certain types of advanced or metastatic breast cancer. This small-molecule drug inhibits several tyrosine kinases, including the tyrosine kinase activity of HER-2. Lapatinib treatment prevents HER-2 signals from activating cell growth.
• Canertinib is an orally bioavailable irreversible pan-ErbB tyrosine kinase inhibitor, targeting EGFR, HER-2, ErbB-3 and ErbB-4. It effectively inhibits the growth of esophageal squamous cell carcinoma which co-expresses both EGFR and HER2 with the inhibition of phosphorylation of both MAPK and AKT. In vitro studies of human cancer cell lines indicate that canertinib results in prompt, potent, and sustained inhibition of tyrosine kinase activity. [00125] Afatinib is an irreversible EGFR/HER2.
• afatinib shows potent activity against wild-type and mutant forms of EGFR and HER2, similar to gefitinib in potency for L858R EGFR, but about 100-fold more active against the gefitinib resistant L858R- T790M EGFR double mutant.
• Afatinib was effective in inhibiting survival of lung cancer cell lines harboring wild-type (HI 666) or L858R/T790M ( CI-H1975) EGFR. Assessed in a standard xenograft model of the epidermoid carcinoma cell line A431.
• afatinib is a next generation tyrosine kinase inhibitor (TKI) that irreversibly inhibits human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) kinases.
• TKI tyrosine kinase inhibitor
• Afatinib is not only active against EGFR mutations targeted by first generation TKIs like erlotinib or gefitinib, but also against those not sensitive to these standard therapies. Because of its additional activity against Her2, it is investigated for breast cancer as well as other EGFR and Her2 driven cancers.
• Necitumumab is a fully human IgGl monoclonal antibody directed against the epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Necitumumab binds to and blocks the ligand binding site of EGFR, thereby preventing the activation and subsequent dimerization of the receptor. This may lead to an inhibition of EGFR-dependent downstream pathways and so inhibition of EGFR-dependent tumor cell proliferation and metastasis.
• EGFR epidermal growth factor receptor
• Nimotuzumab is a humanized monoclonal antibody directed against the epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Nimotuzumab binds to and inhibits EGFR, resulting in growth inhibition of tumor cells that overexpress EGFR. This agent may act synergistically with radiation therapy.
• EGFR epidermal growth factor receptor
• PF299804 is a potent, irreversible inhibitor of human epidermal growth factor receptor (HER)-1/EGFR, -2, and -4 tyrosine kinases (TK), is active in E-sensitive and -resistant preclinical models.
• PF299804 had clinical activity in phase VII trials in EGFR TK inhibitor (TKI)- refractory NSCLC.
• RO5083945 is a glycoengineered anti EGFR IgGl mAb exhibiting increased binding affinity for all FcyRIIIa variants expressed on immune effector cells.
• RO5083945 demonstrates significantly improved cell killing in ADCC-based assays and greater activity in in vivo models compared to cetuximab and panitumumab.
• RO5083945 has the potential to show clinical activity in patients with solid tumors, including KRAS mutant CRC.
• ABT-806 is a humanized monoclonal antibody (MoAb) against human epidermal growth factor receptor (EGFR) with antineoplastic activity.
• MoAb ABT-806 targets the EGFR deletion variant, de2-7 EGFR as well as wild-type EGFR expressed in cells overexpressing the receptor, thereby preventing the activation and subsequent dimerization of the receptor; the decrease in receptor activation and dimerization result in an inhibition in signal transduction and anti-proliferative effects.
• This MoAb targets cells expressing aberrant EGFR, hence making it an ideal candidate for generation of radioisotope or toxin conjugates.
• AP261 13 is an orally available inhibitor of receptor tyrosine kinases anaplastic lymphoma kinase (ALK) and the epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Dual ALK/EGFR inhibitor AP261 13 binds to and inhibits ALK kinase and ALK fusion proteins as well as EGFR and mutant forms. This leads to the inhibition of ALK kinase and EGFR kinase, disrupts their signaling pathways and eventually inhibits tumor cell growth in susceptible tumor cells. In addition, AP261 13 appears to overcome mutation-based resistance. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development; ALK dysregulation and gene rearrangements are associated with a series of tumors. EGFR is overexpressed in a variety of cancer cell types.
• Radiotherapy is a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing.
• Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
• chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy).
• systemic chemotherapy When chemotherapy is placed directly into the spinal column, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.
• Cytoxic agents used for treating lung cancer include carboplatin (for example, Paraplatin®, Paraplat®), cisplatin (for example, Platinol®, Platinol-Aq®), crizotinib (for example Xalkori®), etoposide (for example Toposar®, VePesid®), etoposide Phosphate (for example Etopophos®), gemcitabine hydrochloride (for example Gemzar®), gemcitabine-cisplatin, methotrexate (for example Abitrexate®, Folex®, Folex Pfs®, Methotrexate Lpf®, Mexate®, Mexate-Aq®), paclitaxel (for example Taxol®), pemetrexed Disodium (for example Alimta®), and topotecan Hydrochloride (for example Hycamtin®) [00135] Monoclonal antibody therapy is a cancer treatment that uses antibodies made in the laboratory, from a single
• Monoclonal antibodies can identify substances on cancer cells or normal substances that may help cancer cells grow.
• the antibodies attach to the substances and kill the cancer cells, block their growth, or keep them from spreading.
• Monoclonal antibodies are given by infusion. They may be used alone or to carry drugs, toxins, or radioactive material directly to cancer cells. Monoclonal antibodies are also used in combination with chemotherapy as adjuvant therapy.
• Bevacizumab (Avastin®) is a recombinant humanized monoclonal antibody directed against the vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine. Bevacizumab binds to VEGF and inhibits VEGF receptor binding, thereby preventing the growth and
• VEGF vascular endothelial growth factor
• Bevacizumab is used currently to treat several types of cancer, including certain types of colorectal, lung, breast, and kidney cancers and glioblastoma.
• Additional, illustrative, treatments that may be advantageously combined with the compositions and therapies disclosed herein may include, without limitation, administration of agents including, but not limited to lapatinib, alone or in combination with capecitabine, docetaxel, epirubicin, epothilone A, B or D, goserelin acetate, paclitaxel, pamidronate, bevacizumab, or trastuzumab.
• agents including, but not limited to lapatinib, alone or in combination with capecitabine, docetaxel, epirubicin, epothilone A, B or D, goserelin acetate, paclitaxel, pamidronate, bevacizumab, or trastuzumab.
• the additional therapy comprises chemotherapy comprising administering to the subject one or more of doxorubicin, cyclophosphamide, paclitaxel, lapatinib, capecitabine, trastuzumab, bevacizumab, gemcitabine, eribulin, or nab-paclitaxel.
• One embodiment provides a method of treating cancer in an EGFR inhibitor-nai ' ve patient progressed on prior therapy, wherein the method comprises: (1) determining the E-cadherin expression level in the patient; (2) selecting the patient exhibiting a high E-cadherin expression level scored as +3; and (3) administering to the patient a combination comprising entinostat and an EGFR inhibitor. In some instances, entinostat is administered to a fasting patient.
• the Cmax is higher when entinostat is administered without food as compared to the Cmax when entinostat is administered with food.
• the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 2: 1.
• the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 3 : 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 4: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 5: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 6: 1. In one embodiment, the ratio of Cmax following administration of entinostat under fasting conditions to Cmax following administration of entinostat under fed conditions is at least about 7: 1.
• the Tmax is lower when entinostat is administered without food as compared to the Tmax when entinostat is administered with food.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 2: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 3: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 4: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 5: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 6: 1.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 7: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 8: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 9: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 10: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 1 1 : 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 12: 1.
• the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 13 : 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 14: 1. In one embodiment, the ratio of Tmax following administration under fed conditions to Tmax following administration under fasting conditions is at least about 15: 1.
• the EGFR inhibitor is administered a different time of day than entinostat. In one embodiment, the EGFR inhibitor is administerd to a fasting patient. [00143] Another embodiment provides the method wherein the prior therapy was one prior chemotherapy.
• Another embodiment provides the method wherein the prior therapy was two or more prior chemotherapies.
• Another embodiment provides the method wherein high E-cadherin expression levels are determined by ELISA, immunohistochemistry, immunocytochemistry or determination of E-cadherin methylation levels. Another embodiment provides the method wherein high E- cadherin expression levels are determined by immunohistochemistry. Another embodiment provides the method wherein the high E-cadherin expression levels are scored as +3 as determined by immunohistochemistry.
• Another embodiment provides the method wherein the cancer is lung cancer.
• Another embodiment provides the method wherein the lung cancer is non-small cell lung cancer.
• Another embodiment provides the method wherein the EGFR inhibitor
• erlotinib administered in combination with entinostat is erlotinib.
• Another embodiment provides the method wherein entinostat and the EGFR inhibitor are administered sequentially in either order or simultaneously. Another embodiment provides the method wherein entinostat and the EGFR inhibitor are administered simultaneously. Another embodiment provides the method wherein the EGFR inhibitor is administered first.
• Another embodiment provides the method wherein the EGFR inhibitor is administered daily and the entinostat is administered periodically. Another embodiment provides the method wherein the EGFR inhibitor is administered daily and the entinostat is administered weekly.
• Another embodiment provides a method of treating cancer in an EGFR inhibitor- naive patient progressed on prior therapy, wherein said patient exhibits high E-cadherin expression levels, the method comprising administering to the patient a combination comprising entinostat and an EGFR inhibitor.
• Another embodiment provides the method of treating cancer in an EGFR inhibitor- naive patient progressed on prior therapy, wherein said patient exhibits high E-cadherin expression levels, wherein the method further comprises administering to the subject one or more additional therapies in addition to the combination of entinostat and the EGFR inhibitor.
• the one or more therapies comprise one or more of radiation therapy, chemotherapy, high dose chemotherapy with stem cell transplant, and monoclonal antibody therapy.
• radiation therapy comprises internal and/or external radiation therapy.
• the chemotherapy comprises administering to the subject one or more of doxorubicin,
• cyclophosphamide paclitaxel, lapatinib, capecitabine, trastuzumab, bevacizumab, gemcitabine, eribulin, or nab-paclitaxel.
• chemotherapy comprises administering to the subject one or more IGF-1R inhibitors.
• IGF-1R inhibitor is AEW541.
• One embodiment provides a method of treating non-small cell lung cancer in a patient in need thereof, comprising oral administration of erlotinib and entinostat, wherein the entinostat is administered to a fasting patient.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Tmax is less than 1 hour post administration.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Tmax is less than 90 minutes post administration.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Tmax is less than 2 hours post administration.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Tmax is between 30 minutes and 2 hours post administration.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Cmax is at least 150 i g/mL following oral administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Cmax is at least 125 njg/mL following oral administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Cmax is at least 100 i g/mL following oral administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Cmax is at least 80 njg/mL following oral administration of entinostat.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat Cmax is at least 50 njg/mL following oral administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein about 10 mg of entinostat is administered. Another embodiment provides the method of treating non-small cell lung cancer wherein from about 1 mg to about 20 mg of entinostat is administered. Another embodiment provides the method of treating non-small cell lung cancer wherein entinostat is administered every 14 days. Another embodiment provides the method of treating non-small cell lung cancer wherein the entinostat is administered for a month. Another embodiment provides the method of treating non-small cell lung cancer wherein the patient has not consumed food within 2 hours prior to administration of entinostat.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the patient has not consumed food within 1 hour prior to administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the patient does not consume food within 1 hour after administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the patient does not consume food within 30 minutes after administration of entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the erlotinib is administered at a different time of day than entinostat. Another embodiment provides the method of treating non-small cell lung cancer wherein the erlotinib is administered once daily to the fasting patient.
• Another embodiment provides the method of treating non-small cell lung cancer wherein the patient has not consumed food within 2 hours prior to administration of erlotinib. Another embodiment provides the method of treating non-small cell lung cancer wherein the patient does not consume food within 1 hour after administration of erlotinib. Another embodiment provides the method of treating non-small cell lung cancer wherein about 150 mg of erlotinib is administered.
• Oral formulations containing the active pharmaceutical ingredients described herein may comprise any conventionally used oral forms, including: tablets, capsules, pills, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, granules, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, solutions, wafers, sprinkles, elixirs, syrups, buccal forms, and oral liquids.
• Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g.
• Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,
• surface modifying agents which include nonionic and anionic surface modifying agents.
• surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.
• Oral formulations herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s).
• the oral formulation may also consist of administering the active ingredient in water or a fruit juice, containing appropriate solubilizers or emulsifiers as needed.
• the combination therapy described herein can be given simultaneously or can be given in a staggered regimen, with entinostat being given at a different time during the course of chemotherapy than the EGFR inhibitor.
• This time differential may range from several minutes, hours, days, weeks, or longer between administrations of the two
• the term combination does not necessarily mean administered at the same time or as a unitary dose, but that each of the components are administered during a desired treatment period.
• the agents may also be administered by different routes. As is typical for chemotherapeutic regimens, a course of chemotherapy may be repeated several weeks later, and may follow the same timeframe for administration of the two compounds, or may be modified based on patient response.
• the pharmaceutical compositions provided herein may be provided in solid, semisolid, or liquid dosage forms for oral administration.
• oral administration also include buccal, lingual, and sublingual administration.
• Suitable oral dosage forms include, but are not limited to, tablets, capsules, pills, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, granules, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, solutions, wafers, sprinkles, elixirs, and syrups.
• the pharmaceutical compositions may contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, and flavoring agents.
• pharmaceutically acceptable carriers or excipients including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, and flavoring agents.
• Binders or granulators impart cohesiveness to a tablet to ensure the tablet remaining intact after compression.
• Suitable binders or granulators include, but are not limited to, starches, such as corn starch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500); gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, alginic acid, alginates, extract of Irish moss, Panwar gum, ghatti gum, mucilage of isabgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powdered tragacanth, and guar gum; celluloses, such as ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose, methyl cellulose, hydroxye
• Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
• the binder or filler may be present from about 50 to about 99% by weight in the pharmaceutical compositions provided herein.
• Suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar.
• Certain diluents, such as mannitol, lactose, sorbitol, sucrose, and inositol when present in sufficient quantity, can impart properties to some compressed tablets that permit disintegration in the mouth by chewing. Such compressed tablets can be used as chewable tablets.
• Suitable disintegrants include, but are not limited to, agar; bentonite; celluloses, such as methylcellulose and carboxymethylcellulose; wood products; natural sponge; cation-exchange resins; alginic acid; gums, such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses, such as croscarmellose; cross-linked polymers, such as crospovidone; cross-linked starches;
• microcrystalline cellulose such as sodium starch glycolate; polacrilin potassium; starches, such as corn starch, potato starch, tapioca starch, and pre-gelatinized starch; clays; aligns; and mixtures thereof.
• the amount of disintegrant in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
• the pharmaceutical compositions provided herein may contain from about 0.5 to about 15% or from about 1 to about 5% by weight of a disintegrant.
• Suitable lubricants include, but are not limited to, calcium stearate; magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol; mannitol; glycols, such as glycerol behenate and polyethylene glycol (PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetable oil, including peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyl laureate; agar; starch; lycopodium; silica or silica gels, such as AEROSIL ® 200 (W.R. Grace Co., Baltimore, MD) and CAB-O-SIL ® (Cabot Co. of Boston, MA); and mixtures thereof.
• the pharmaceutical compositions provided herein may contain about 0.1 to about 5% by weight of a lubricant.
• Suitable glidants include colloidal silicon dioxide, CAB-O-SIL ® (Cabot Co. of
• Coloring agents include any of the approved, certified, water soluble FD&C dyes, and water insoluble FD&C dyes suspended on alumina hydrate, and color lakes and mixtures thereof.
• a color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye.
• Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation, such as peppermint and methyl salicylate.
• Sweetening agents include sucrose, lactose, mannitol, syrups, glycerin, and artificial sweeteners, such as saccharin and aspartame.
• Suitable emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitan monooleate (TWEEN ® 20), polyoxyethylene sorbitan monooleate 80 (TWEEN ® 80), and triethanolamine oleate.
• Suspending and dispersing agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum, acacia, sodium carbomethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrolidone.
• Preservatives include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol.
• Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.
• Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
• Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium
• the pharmaceutical compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets.
• Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach.
• Enteric -coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates.
• Sugar- coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation.
• Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium
• Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press- coated or dry-coated tablets.
• the tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, dis integrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.
• the pharmaceutical compositions provided herein may be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate.
• the hard gelatin capsule also known as the dry- filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient.
• the soft elastic capsule is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol.
• the soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl- and propyl-parabens, and sorbic acid.
• the liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545.
• the capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
• the pharmaceutical compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups.
• An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil.
• Emulsions may include a pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative.
• Suspensions may include a pharmaceutically acceptable suspending agent and preservative.
• Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di(lower alkyl) acetal of a lower alkyl aldehyde (the term "lower” means an alkyl having between 1 and 6 carbon atoms), e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol.
• Elixirs are clear, sweetened, and hydroalcoholic solutions.
• Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative.
• a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.
• liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) provided herein, and a dialkylated mono- or poly-alkylene glycol, including, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350- dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol.
• a dialkylated mono- or poly-alkylene glycol including, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350- dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol.
• formulations may further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.
• antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.
• antioxidants such as but
• compositions provided herein for oral administration may be also provided in the forms of liposomes, micelles, microspheres, or nanosystems.
• Miccellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
• the pharmaceutical compositions provided herein may be provided as non- effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form.
• Pharmaceutically acceptable carriers and excipients used in the non- effervescent granules or powders may include diluents, sweeteners, and wetting agents.
• Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.
• Coloring and flavoring agents can be used in all of the above dosage forms.
• compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.
• compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.
• Example 1 A Phase 1, Randomized, Open-Label Study to Assess the Food Effect on the
• Patients will be assessed at screening and at pre-prescribed times during study enrollment using standard clinical and laboratory assessments. Patents will also be assessed for tumor response after each 2 cycles. Tumor progression will be assessed by CT, MRI or other appropriate radiologic study. Patients will continue receiving their appropriate cycles of study treatment until tumor progression or adverse events occur which necessitate discontinuing therapy as determined by the Investigator.
• AUCinf area under the plasma concentration-time curve from time zero
• Sample Size Up to 28 patients (approximately 14 patients with breast cancer and 14 patients with NSCLC, with a minimum of 4 male patients) will be enrolled to ensure that 24 patents (approximately 12 per treatment sequence) complete Cycle 1 of study treatment.
• the study will enroll postmenopausal women with histologically or cytologically confirmed estrogen receptor positive (ER+) breast cancer at initial diagnosis whose disease has progressed to where the investigator determines that the patient is a candidate to receive exemestane. In addition, it will enroll adults with cytologically or
• Entinostat is a synthetic small molecule with a molecular formula C21H20N4O3 and a molecular weight of 376.41. Entinostat is classified as an antineoplastic agent, specifically functioning as an inhibitor of histone deacetylases by promoting hyperacetylation of nucleosomal histones. Entinostat is orally bioavailable and will be supplied as yellow coated tablets containing 5.0 mg of active ingredient.
• One cycle will be defined as 28 days of study treatment.
• Group A Entinostat 10 mg po on Day 1 under test fasted conditions and Day 15 under test fed conditions
• Group B Entinostat 10 mg po on Day 1 under test fed conditions and Day 15 under test fasted conditions
• Entinostat will be administered at a dose of 10 mg po on Days 1 and 15 at least 2 hours after breakfast, followed by at least a 1 -hour fast.
• Erlotinib 150 mg will be administered once daily starting on Cycle 2 Day 1.
• Entinostat will be administered at a dose of 10 mg po on Days 1 and 15, at least 2 hours after breakfast, followed by at least a 1 -hour fast.
• Patients will be administered one of two treatments according to their randomization: 10 mg entinostat under test fed conditions or 10 mg entinostat under test fasted conditions. All treatments will be given as a single dose with 240 mL of water. Water will be allowed in all treatment groups as desired for up to 2 hours prior to dosing, then restricted up until 2 hours post-dose except for the fluid taken during breakfast in the fed treatment group. Blood will be obtained for determination of entinostat concentrations at the following times: pre-dose (within 60 minutes of dosing), and then at .25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 72, 120, 168, 240, and 336 hours post dose. ECGs will be obtained in triplicate at -60 and -45 minutes pre-dose and then at the same time points as the PK blood samples. Holier monitor will be used for Cycle 1 Day 1 and Day 15 from pre-dose through the 12-hour post dose.
• Blood will be obtained for determination of entinostat acetylation at the following times: pre-dose (within 60 minutes of dosing), and then at 12, 24, 168 and 336 hours post dose.
• Blood will be obtained for determination of protein acetylation at the final study visit.
• Patients will be considered evaluable for PK analysis if they receive entinostat in each treatment period according to the conditions defined by the randomization (ie, fed or fasted). Patients must also have sufficient plasma concentration-time data from each treatment period in order to provide for meaningful assessment of the PK parameters (eg, Cmax, AUCiast).
• concentration-time data will be listed and displayed graphically on the linear and log scale.
• concentration-time data will be summarized descriptively in tabular and graphical format (linear and log scale).
• PK parameters estimated using noncompartmental methods will be calculated using WinNonlin version 5.1 or higher. Such estimates will be listed and summarized descriptively in tabular and graphical format.
• PK parameters eg, Cmax, AUCiast
• comparisons between the fed and fasted conditions will be made using a linear mixed effects ANOVA model.
• the model will include terms for treatment (fed, fasted), period, and sequence as fixed effects, and patient within sequence as the random effect.
• the assessment of bioequivalence will be based on the classical (shortest) confidence interval approach which is operationally equivalent to Schuirmann's two one-sided test procedure for interval hypotheses.
• the estimates from the ANOVA model will be used to calculate 90% confidence limits for the ratio of the true mean AUC for the fed and fasted conditions.
• Tmax Time to maximum observed plasma concentration of entinostat
• Safety data analysis will be conducted on all patients receiving at least one dose of entinostat. Analyses will consist of data summaries for clinical and laboratory parameters, and for adverse events. Unless otherwise specified, the safety analyses will be performed by primary diagnosis. The number and percentage of patients with one or more adverse events will be summarized by relationship to the individual study treatments and by severity grade. Severity grade will be determined using the NCI-CTCAE (version 4.0). Adverse events will be coded using the Medical Dictionary for Regulatory Activities Terminology (MedDRA). Laboratory parameters will be summarized using descriptive statistics, by shifts relative to baseline, and data listings of clinically significant abnormalities. Vital signs and ECG data will be summarized by changes from baseline values using descriptive statistics.
• Figure 1 displays the mean concentration time profiles following administration of 10 mg entinostat under fasted or fed conditions.
• a summary of the pharmacokinetic parameters is presented in Table 1.
• Secondary Outcome Measures are to compare objective response rate (ORR) and clinical benefit rate (CBR), and to evaluate the safety and tolerability of entinostat in combination with exemestane as measured by adverse events and laboratory safety parameters.
• Metastatic disease must be measurable
• Patient may have had one prior chemotherapy as part of first line therapy as long as it was received before initiation of prior AI
• At least 1 measurable lesion > 20mm by conventional CT scan or > 10mm by spiral CT scan
• Hemoglobin > 9.0 g/dL; platelets > 75 x 10 9 /L; ANC > 1.0 x 10 9 /L without the use of hematopoietic growth factors
• Patient or legally acceptable representative has granted written informed consent before any study-specific procedure (including special screening tests) is performed
• Another serious or uncontrolled medical condition within 3 months of enrollment such as hypertension, diabetes mellitus, or suppressed immune system
• Patient has any kind of medical, psychiatric, or behavioral disorder that places the patient at increased risk for study participation or compromises the ability of the patient to give written informed consent and/or to comply with study procedures and requirement
• Table 1 Summary of pharmacokinetic parameters following administration of 10 mg entinostat under fasted and fed conditions
• AUClast ratio AUClast Fed/AUClast Fasted
• AUCinf ratio AUCinf Fed/AUCinf Fasted

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