Patent Application
20240316056
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: one 12,619 Byte ASCII (Text) file named “743502_ST25.txt,” dated May 13, 2019.
Metastasis is the cellular mechanism used by disease to spread from an organ to another non-adjacent part of the organism. This process may be involved in the development of solid tumors and may be responsible for the majority of deaths associated with disease. Treatment of a tumoral lesion may have a better prognosis if started in a pre-metastatic stage. In the last decade, although understanding of the underlying mechanisms involved in metastasis has advanced, the therapeutic tools impacting specifically the metastatic process are very limited. Accordingly, there is a need for new formulations and methods of treating subjects suffering from metastatic disease.
An embodiment of the invention provides pharmaceutical formulations comprising a compound of formula (1):
in which R1, R2, R3, and R4 are as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable surfactant comprising one or more caprylocaproyl polyoxylglycerides and PEG-8 caprylic/capric glycerides.
Another embodiment of the invention provides pharmaceutical formulations comprising a compound of formula (I):
in which R1, R2, R3, and R4 are as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable surfactant comprising one or more polyoxyethylene esters of 12-hydroxystearic acid.
A further embodiment of the invention provides methods for treating pancreatic adenocarcinoma in a mammal, comprising administering to a mammal in need thereof a compound of formula (I):
in which R1, R2, R3, and R4 are as described herein, or a pharmaceutically acceptable salt thereof, in an amount effective to treat pancreatic adenocarcinoma in the mammal.
Another embodiment of the invention provides methods of detecting the change in expression levels of one or both of FoxA1 and FoxO6 in a pancreatic adenocarcinoma tumor sample from a mammal, wherein the mammal has been administered a compound of formula (I):
in which R1, R2, R3, and R4 are as described herein, or a pharmaceutically acceptable salt thereof, the method comprising providing a first pancreatic adenocarcinoma tumor sample from the mammal, assaying the tumor sample to determine the expression levels of one or both of Forkhead box protein A1 (“FoxA1”) and Forkhead box protein O6 (“FoxO6”), providing a second pancreatic adenocarcinoma tumor sample from the mammal, assaying the second tumor sample to determine the expression levels of one or both of FoxA1 and FoxO6, and comparing one or both of (i) the first determined expression level of FoxA1 to the second determined level of FoxA1 and (ii) comparing the first determined expression level of FoxO6 and the second determined level of FoxO6, to detect a change in expression levels of one or both of FoxA1 and FoxO6, wherein the first tumor sample is removed from the mammal before the second tumor sample is removed from the mammal.
Formulations comprising inhibitors of the perinucleolar compartment (“PNC”), a subnuclear body characterized by its location to the periphery of the nucleolus and which may be associated with malignancy both in vitro and in vivo, are disclosed as a solution to the unmet need for treating cancer, particularly the metastatic cancers. Compounds of formula (I) are PNC inhibitors. Additionally, methods of treating pancreatic adenocarcinoma by administering a compound of formula (I) are disclosed as a solution to the unmet need for treating pancreatic adenocarcinoma, especially metastatic carcinoma. Further, methods of detecting the change in expression levels of one or both of FoxA1 and FoxO6 in a pancreatic adenocarcinoma tumor sample are disclosed as a solution to the unmet need of determining which mammals will likely respond positively to administration of a compound of formula (I) as a treatment for pancreatic adenocarcinoma.
As seen in the Examples, a compound of formula (I), metarrestin, unexpectedly was selective against metastasis across different preclinical cancer histologies, with high intratumoral exposure and without appreciable toxicity. Metarrestin inhibits Pol I transcription, induces nucleolar segregation, reduces nucleolar volume, and disrupts PNCs, in part by interfering with eEF1A2 function. Further, detecting the change in expression levels of FoxA1 and Fox06 in pancreatic adenocarcinoma tumor samples unexpectedly provided valuable insight into which mammals should continue to receive metarrestin.
An embodiment of the invention provides formulations comprising a compound of formula (I),
wherein R1 is alkyl, hydroxyalkyl, thioalkyl, alkoxyalkyl, alkylthioalkyl, cycloalkyl, hydroxycycloalkyl, hydroxycycloalkylalkyl, thiocycloalkyl, alkoxycycloalkyl, alkylthiocycloalkyl, dialkylaminoalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, arylalkyl, arylalkylpiperidin-4-yl, arylpiperazinylalkyl, or heteroarylalkyl, R2 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halo, alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino, and alkylcarbonyl, R3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halo, alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino, and alkylcarbonyl, and R4 is alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, or a pharmaceutically acceptable salt thereof, wherein R1 and R4 are optionally substituted on the aryl and/or alkyl portion with one or more substituents selected from the group consisting of halo, alkyl, hydroxyalkyl, thioalkyl, alkoxy, alkylthioalkyl, alkoxycarbonyl, alkylthiocarbonyl, amino, alkylamino, dialkylamino, aminosulfonyl, hydroxyl, perfluoroalkoxy, alkylenedioxy, and alkylcarbonyl.
In an embodiment, R1 is a 5 or 6-membered heterocyclyl group having at least one hetero atom selected from the group consisting of O, N, and S; a hydroxy C1-C7 cycloalkyl group; a hydroxy C1-C6 alkyl group; a N,N-di(C1-C6 alkyl)amino C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkyl group; a heteroaryl C1-C6 alkyl group; a heterocyclyl C1-C6 alkyl group; phenyl C1-C6 alkyl group where the phenyl ring is substituted with one or more C1-C6 alkoxy groups; N-benzyl piperazinyl; N-phenyl piperazinylalkyl; a phenyl C1-C6 alkyl group where the alkyl is substituted with a hydroxy group; or a 5 or 6 membered heteroarylamino C1-C6 alkyl group wherein the heteroaryl group has at least one hetero atom selected from the group consisting of O, N, and S.
In an embodiment, R1 is selected from the following:
In an embodiment, R2 is phenyl, R3 is phenyl, R4 is benzyl, and R1 can be any of the following structures:
In an embodiment, R2 is phenyl, R3 is phenyl, R4 is benzyl, and R1 can be any of the following structures:
In an embodiment, R4 is 4-methoxybenzyl, R2 is phenyl, R3 is phenyl, and R1 can be any one of the following structures:
In an embodiment, R4 is phenylethyl, R2 is phenyl, R3 is phenyl, and R1 can be any one of the following structures:
In an embodiment, R4 is 4-aminosulfonylbenzyl, 4-trifluoromethoxybenzyl, 4-methoxybenzyl, or cyclopropylmethyl, and R1 can be any one of the following structures:
In an embodiment, R4 is heteroaryl C1-C6 alkyl.
In an embodiment, R2 is phenyl, R3 is phenyl, R4 is
and R1 is selected from the following:
In an embodiment, R is phenyl, R3 is phenyl, R4 is benzyl, and R1 is
In an embodiment, the compound of formula (I) is metarrestin (see U.S. Pat. No. 9,663,521, incorporated herein in its entirety).
An embodiment of the invention provides a formulation comprising a pharmaceutically acceptable surfactant comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides. In an embodiment, the formulation is administered by any one of the following methods: oral, aerosol, nasal, pulmonary, parenteral (e.g., intravenously [“IV” ], subcutaneously, intradermally, or intramuscularly), subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginal. Preferably, the formulation is suitable for oral administration.
A suitable source of one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides is LABROSOL™ (available from Gattefosse, Lyon, France). Preferably, the source of one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides is LABROSOL™ ALF (Gattefosse).
In an embodiment, the oral formulation comprises LABROSOL™ (available from Gattefosse). In an embodiment, the oral formulation comprises LABROSOL™ ALF (Gattefosse).
The formulation can contain from about 0.1 to about 90 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 1 to about 90 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 50 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 65 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 75 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises about 80 wt % of the pharmaceutically acceptable surfactant.
The oral formulation can contain from about 0.1 to about 90 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 1 to about 90 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 50 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 65 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 75 to about 85 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises about 80 wt % of the pharmaceutically acceptable surfactant.
The formulation can contain from about 0.1 to about 75 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 1 to about 70 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 25 to about 65 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 30 to about 60 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises from about 40 to about 50 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the formulation comprises about 45 wt % of the pharmaceutically acceptable surfactant.
The oral formulation can contain from about 0.1 to about 75 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 1 to about 70 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 25 to about 65 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 30 to about 60 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises from about 40 to about 50 wt % of the pharmaceutically acceptable surfactant. In an embodiment, the oral formulation comprises about 45 wt % of the pharmaceutically acceptable surfactant.
The formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides can also contain caprylic acid. In an embodiment, the formulation comprises from about 0.1 to about 50 wt % of caprylic acid. In an embodiment, the formulation comprises from about 1 to about 50 wt % of caprylic acid. In an embodiment, the formulation comprises from about 3 to about 40 wt % of caprylic acid. In an embodiment, the formulation comprises from about 5 to about 30 wt % of caprylic acid. In an embodiment, the formulation comprises from about 5 to about 15 wt % of caprylic acid. In an embodiment, the formulation comprises about 10 wt % of caprylic acid.
In an embodiment, the formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides also comprises from about 15 to about 25 wt % of caprylic acid. In an embodiment, the formulation comprises about 20 wt % of caprylic acid.
The oral formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides can also contain caprylic acid. In an embodiment, the oral formulation comprises from about 0.1 to about 50 wt % of caprylic acid. In an embodiment, the oral formulation comprises from about 1 to about 50 wt % of caprylic acid. In an embodiment, the oral formulation comprises from about 3 to about 40 wt % of caprylic acid. In an embodiment, the oral formulation comprises from about 5 to about 30 wt % of caprylic acid. In an embodiment, the oral formulation comprises from about 5 to about 15 wt % of caprylic acid. In an embodiment, the oral formulation comprises about 10 wt % of caprylic acid.
In an embodiment, the oral formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides also comprises from about 15 to about 25 wt % of caprylic acid. In an embodiment, the oral formulation comprises about 20 wt % of caprylic acid.
The formulations comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides can also contain water. The formulation can contain from about 0.1 to about 95 wt % water. In an embodiment, oral formulation comprises from about 0.1 to about 90 wt % water. In an embodiment, the formulation comprises from about 0.1 to about 85 wt % water. In an embodiment, the formulation comprises from about 0.1 to about 80 wt % water. In an embodiment, the formulation comprises from about 0.1 to about 75 wt % water. In an embodiment, the formulation comprises from about 1 to about 70 wt % water. In an embodiment, the formulation comprises from about 25 to about 65 wt % water. In an embodiment, the formulation comprises from about 30 to about 60 wt % water. In an embodiment, the formulation comprises from about 40 to about 50 wt % water. In an embodiment, the formulation comprises about 45 wt % water.
The oral formulations comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides can also contain water. The oral formulation can contain from about 0.1 to about 95 wt % water. In an embodiment, the oral formulation comprises from about 0.1 to about 90 wt % water. In an embodiment, the oral formulation comprises from about 0.1 to about 85 wt % water. In an embodiment, the oral formulation comprises from about 0.1 to about 80 wt % water. In an embodiment, the oral formulation comprises from about 0.1 to about 75 wt % water. In an embodiment, the oral formulation comprises from about 1 to about 70 wt % water. In an embodiment, the oral formulation comprises from about 25 to about 65 wt % water. In an embodiment, the oral formulation comprises from about 30 to about 60 wt % water. In an embodiment, the oral formulation comprises from about 40 to about 50 wt % water. In an embodiment, the oral formulation comprises about 45 wt % water.
In addition to the pharmaceutically acceptable surfactant, the formulations suitable for oral administration can include (a) liquid solutions, such as a therapeutically effective amount of the compound dissolved in diluents (e.g., water, saline, or juice), (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions. Liquid formulations may include diluents, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, additional surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
Preferably, the formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides is an oral formulation and is a suspension.
Preferably, the formulation comprising one or more caprylocaproyl polyoxylglycerides and one or more PEG-8 caprylic/capric glycerides is an oral formulation and is a pill. A pill can be a tablet or capsule (hard or soft).
An embodiment of the invention provides a formulation comprising a pharmaceutically acceptable surfactant comprising one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation is administered by any one of the following methods: oral, aerosol, nasal, pulmonary, parenteral (e.g., IV, subcutaneously, intradermally, or intramuscularly), subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginal. Preferably, the formulation is suitable for intravenous administration.
A suitable source of one or more polyoxyethylene esters of 12-hydroxystearic acid is Solutol HS 15.
In an embodiment, the formulation comprises from about 1 to about 60 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises from about 5 to about 55 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises from about 10 to about 50 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises from about 15 to about 45 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises from about 20 to about 40 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises from about 25 to about 35 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the formulation comprises about 30 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid.
In an embodiment, the IV formulation comprises from about 1 to about 60 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises from about 5 to about 55 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises from about 10 to about 50 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises from about 15 to about 45 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises from about 20 to about 40 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises from about 25 to about 35 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid. In an embodiment, the IV formulation comprises about 30 wt % of one or more polyoxyethylene esters of 12-hydroxystearic acid.
In an embodiment, the formulation comprises from about 1 to about 60 wt % Solutol HS 15. In an embodiment, the formulation comprises from about 5 to about 55 wt % Solutol HS 15. In an embodiment, the formulation comprises from about 10 to about 50 wt % Solutol HS 15. In an embodiment, the formulation comprises from about 15 to about 45 wt % Solutol HS 15. In an embodiment, the formulation comprises from about 20 to about 40 wt % Solutol HS 15. In an embodiment, the formulation comprises from about 25 to about 35 wt % Solutol HS 15. In an embodiment, the formulation comprises about 30 wt % Solutol HS 15.
In an embodiment, the IV formulation comprises from about 1 to about 60 wt % Solutol HS 15. In an embodiment, the IV formulation comprises from about 5 to about 55 wt % Solutol HS 15. In an embodiment, the IV formulation comprises from about 10 to about 50 wt % Solutol HS 15. In an embodiment, the IV formulation comprises from about 15 to about 45 wt % Solutol HS 15. In an embodiment, the IV formulation comprises from about 20 to about 40 wt % Solutol HS 15. In an embodiment, the IV formulation comprises from about 25 to about 35 wt % Solutol HS 15. In an embodiment, the IV formulation comprises about 30 wt % Solutol HS 15.
Preferably, the IV formulation comprises water.
Preferably, the IV formulation comprises saline.
A embodiment of the invention provides methods for treating pancreatic adenocarcinoma in a mammal, comprising administering to a mammal in need thereof a compound of formula (I):
in which R1, R2, R3, and R4 are as described herein by aspects of the invention, or a pharmaceutically acceptable salt thereof, in an amount effective to treat pancreatic adenocarcinoma in the mammal.
In an embodiment, the method comprises administering the compounds of formula (I) with another treatment. The additional treatment can be a radiation treatment. The radiation treatment can be any suitable radiation treatment used in the treatment of pancreatic adenocarcinoma.
The additional treatment can be a chemotherapeutic agent. The chemotherapeutic agent can be any suitable chemotherapeutic agent, for example, the chemotherapeutic agent can be selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and vincristine. Preferably, the chemotherapeutic agent is gemcitabine.
The chemotherapeutic agent can be administered sequentially with a compound of formula (I). The chemotherapeutic agent can be administered before a compound of formula (1). The chemotherapeutic agent can be administered after a compound of formula (1).
Gemcitabine can be administered sequentially with a compound of formula (I). Gemcitabine can be administered before a compound of formula (I). Gemcitabine can be administered after a compound of formula (I).
The chemotherapeutic agent can be administered simultaneously with a compound of formula (I).
Gemcitabine can be administered simultaneously with a compound of formula (I).
When administered, the compounds of formula (I) reduce the likelihood that pancreatic cancer will metastasize. If the tumor has already metastasized, then administration of the compounds of formula (I) may reduce the number or volume of metastatic tumors. In an embodiment, the mammal has stage I pancreatic adenocarcinoma. In an embodiment, the mammal has stage II pancreatic adenocarcinoma. In an embodiment, the mammal has stage III pancreatic adenocarcinoma. In an embodiment, the mammal has stage IV pancreatic adenocarcinoma.
In an embodiment, the mammal has metastatic pancreatic adenocarcinoma.
In a further embodiment, administration of the compound of formula (I) reduces or delays further metastasizing of established metastases. Further, administration of the compounds of formula (I) may reduce the amount or size of metastases (e.g., by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%).
The compounds of formula (I) may be administered before one or more pancreatic adenocarcinoma tumor(s) has been removed from the mammal. The compounds of formula (I) may be administered after one or more pancreatic adenocarcinoma tumor(s) has been removed from the mammal. The tumor(s) may be removed, for example, by surgery.
In an embodiment, the methods of treatment result in the disrupting of a perinucleolar compartment in a cell in the mammal.
In an embodiment, the methods of treatment result in reducing the prevalence of perinucleolar compartment in a cell in the mammal.
In an embodiment, the methods of treatment result in reducing adenosine triphosphate (ATP) levels produced by metastatic cancer cells in the mammal.
In an embodiment, the methods of treatment result in reducing the colony formation of cancer cells in the mammal.
In an embodiment, the methods of treatment result in reducing the migration of cancer cells in the mammal.
The term “treat,” as well as words stemming therefrom, as used herein, does not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment of the cancer in a mammal. Furthermore, the treatment provided by the inventive method can include treatment of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment can include promoting the regression of a tumor.
The therapeutically effective amount of the compound administered can vary depending upon the desired effects and the factors noted above. In an embodiment, the dosages will be between 0.1 mg/kg and 80 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 70 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 60 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 50 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 40 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 30 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 20 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 10 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 9 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 8 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 7 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 6 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 5 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 4 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 3 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 2 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.1 mg/kg and 1 mg/kg of the subject's body weight.
In an embodiment, the dosages will be between 0.5 mg/kg and 1.5 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 0.8 mg/kg and 1.2 mg/kg of the subject's body weight. In an embodiment, the dosages will be about 1 mg/kg of the subject's body weight.
In an embodiment, the dosages will be between 1.5 mg/kg and 2.5 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 1.8 mg/kg and 2.2 mg/kg of the subject's body weight. In an embodiment, the dosages will be about 2 mg/kg of the subject's body weight.
In an embodiment, the dosages will be between 4.5 mg/kg and 5.5 mg/kg of the subject's body weight. In an embodiment, the dosages will be between 4.8 mg/kg and 5.2 mg/kg of the subject's body weight. In an embodiment, the dosages will be about 5 mg/kg of the subject's body weight.
The doses disclosed herein can be administered daily. The dose can be administered at one time per day or more than one time per day. The dose can be administered simultaneously or sequentially with other treatments.
The methods herein comprise administering an effective amount of the compound of formula (I) to an animal afflicted with pancreatic adenocarcinoma. Preferably, the animal is a mammal. More preferably, the mammal is a human.
The term “mammal” includes, but is not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Camivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. Furthermore, the subject can be the unborn offspring of any of the forgoing hosts, especially mammals (such as, humans), in which case any screening of the subject or cells of the subject, or administration of compounds to the subject or cells of the subject, can be performed in utero.
An embodiment of the invention provides methods for detecting the change in expression levels of one or both of FoxA1 and FoxO6 in a pancreatic adenocarcinoma tumor sample from a mammal, wherein the mammal has been administered a compound of formula (I). The method involves the steps of providing a first pancreatic adenocarcinoma tumor sample from the mammal, assaying the tumor sample to determine the expression levels of one or both of FoxA1 and FoxO6, providing a second pancreatic adenocarcinoma tumor sample from the mammal, assaying the second tumor sample to determine the expression levels of one or both of FoxA1 and FoxO6, and comparing one or both of (i) the first determined expression level of FoxA1 to the second determined level of FoxA1 and (ii) comparing the first determined expression level of FoxO6 and the second determined level of FoxO6, to detect a change in expression levels of one or both of FoxA1 and FoxO6, wherein the first tumor sample is removed from the mammal before the second tumor sample is removed from the mammal.
FoxA1 is also referred to as hepatocyte nuclear factor 3-alpha (HNF-3A). FoxA1 is encoded by the gene FOXA1 in humans. The sequence of FOXA1 is publicly available (see, for example, NCBI's database, Gene ID: 3169).
FoxO6 is encoded by the gene FOXO6 in humans. The sequence of FOXO6 is publicly available (see, for example, NCBI's database, Gene ID: 100132074).
In an embodiment, the method comprises obtaining a first sample from the subject. In an embodiment, the first sample is a pancreatic adenocarcinoma tissue sample. Obtaining a first sample from the subject may be carried out in any suitable manner known in the art, and the sample may be from any suitable source, for example, from tumor resection material or a tumor biopsy (i.e., gross biopsy or fine needle biopsy).
In an embodiment, the method comprises obtaining a second sample from the subject. In an embodiment, the second sample is a pancreatic adenocarcinoma tissue sample. Obtaining a second sample from the subject may be carried out in any suitable manner known in the art, and the sample may be from any suitable source, for example, from tumor resection material or a tumor biopsy (i.e., gross biopsy or fine needle biopsy).
The first and second samples may be obtained by different methods. The first and second samples may be obtained from different parts of the tumor, or different parts of the organ.
In an embodiment, the method comprises assaying the first sample and the second sample to detect the levels of FoxA1 in the samples. For example, the FoxA1 protein can be purified from the samples (either partially or substantially) and assayed via immunohistological techniques (e.g., Western blotting, ELISA, immunoprecipitation, etc.) using one or more antibodies recognizing FoxA1 protein. In this regard, the assaying may comprise contacting the samples with an antibody that specifically binds to FoxA1 protein and thereby forming a complex, and detecting the complex. In an embodiment, the first sample and second sample are purified and assayed via the same or similar techniques.
In an embodiment, the FoxA1 protein comprises an amino acid sequence of SEQ ID NO: 19. In an embodiment, the FoxA1 protein can be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 19.
In an embodiment, the method comprises assaying the first sample and the second sample to detect the levels of FOXO6 in the samples. For example, the FoxO6 protein can be purified from the samples (either partially or substantially) and assayed via immunohistological techniques (e.g., Western blotting, ELISA, immunoprecipitation, etc.) using one or more antibodies recognizing FoxO6 protein. In this regard, the assaying may comprise contacting the sample with an antibody that specifically binds to FoxO6 protein and thereby forming a complex, and detecting the complex. In an embodiment, the first sample and second sample are purified and assayed via the same or similar techniques.
In an embodiment, the FoxO6 protein comprises an amino acid sequence of SEQ ID NO: 20. In an embodiment, the FoxO6 protein can be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 20.
By assaying the first sample and second sample, the change in the level of the biomarker expression can be determined. In an embodiment, the first sample is taken from the mammal before a compound of formula (I) is administered to the mammal. In an embodiment, the level of one or both biomarkers in the second sample is compared to the mammal's pre-treatment level of each biomarker (which is known by assaying the first sample).
In an embodiment, the first sample is taken from the mammal within 1 week of the first administration of a compound of formula (I). In an embodiment, the first sample is taken from the mammal within 2 weeks of the first administration of a compound of formula (I).
In an embodiment, the first sample is taken from the mammal at least 1 week before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 2 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 3 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 4 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 5 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 6 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 7 weeks before the second sample is taken from the mammal. In an embodiment, the first sample is taken from the mammal at least 8 weeks before the second sample is taken from the mammal.
If the mammal's level of FoxA1 expression in the second sample is below the level of FoxA2 expression in the first sample, then the mammal is predicted to have a favorable response to the compound of formula (I) (fewer or smaller metastatic tumors) compared to not being administered the compound of formula (I). If the mammal's level of FoxO6 expression in the second sample is above the level of FoxO6 expression in the first sample, then the mammal is predicted to have a favorable response to the compound of formula (I) (fewer or smaller metastatic tumors) compared to not being administered the compound of formula.
In an embodiment, the methods of treating pancreatic adenocarcinoma comprise receiving a determination of the mammal as having a lower level of FoxA1 and/or a higher level of FoxO6 expression in the second sample compared to the first sample, wherein the lower level of FoxA1 or the higher level of FoxO6 expression has been determined by a method according to an embodiment of the invention, predicting the clinical response to the administration of the compound of formula (I), and treating the mammal for pancreatic adenocarcinoma by administering a compound of formula (I) to the mammal if the mammal has a lower level of FoxA1 and/or a higher level of FoxO6 expression in the second sample as compared to the first sample.
As used herein, the phrase “predicting the clinical response” refers to determining whether the number or size or volume of metastatic tumors would likely decrease in a subject following administration of a compound of formula (I).
When claimed herein, the term “biomarker” refers to FoxA1 or FoxO6.
Referring now to terminology used generically herein, the term “alkyl” means a straight-chain or branched alkyl substituent containing from, for example, about 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from about 1 to about 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.
The term “alkenyl,” as used herein, means a linear alkenyl substituent containing at least one carbon-carbon double bond and from, for example, about 2 to about 6 carbon atoms (branched alkenyls are about 3 to about 6 carbons atoms), preferably from about 2 to about 5 carbon atoms (branched alkenyls are preferably from about 3 to about 5 carbon atoms), more preferably from about 3 to about 4 carbon atoms. Examples of such substituents include vinyl, propenyl, isopropenyl, n-butenyl, sec-butenyl, isobutenyl, tert-butenyl, pentenyl, isopentenyl, hexenyl, and the like.
The term “alkynyl,” as used herein, means a linear alkynyl substituent containing at least one carbon-carbon triple bond and from, for example, 2 to about 6 carbon atoms (branched alkynyls are about 3 to about 6 carbons atoms), preferably from 2 to about 5 carbon atoms (branched alkynyls are preferably from about 3 to about 5 carbon atoms), more preferably from about 3 to about 4 carbon atoms. Examples of such substituents include ethynyl, propynyl, isopropynyl, n-butynyl, sec-butynyl, isobutynyl, tert-butynyl, pentynyl, isopentynyl, hexynyl, and the like.
The term “cycloalkyl,” as used herein, means a cyclic alkyl substituent containing from, for example, about 3 to about 8 carbon atoms, preferably from about 4 to about 7 carbon atoms, and more preferably from about 4 to about 6 carbon atoms. Examples of such substituents include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The cyclic alkyl groups may be unsubstituted or further substituted with alkyl groups such as methyl groups, ethyl groups, and the like. The term “cycloalkylalkyl,” as used herein, refers to an alkyl group linked to a cycloalkyl group and further linked to a molecule via the alkyl group.
The term “heterocyclyl,” as used herein, refers to a monocyclic or bicyclic 5- or 6-membered ring system containing one or more heteroatoms selected from the group consisting of O, N, S, and combinations thereof. The heterocyclyl group can be any suitable heterocyclyl group and can be an aliphatic heterocyclyl group, an aromatic heterocyclyl group, or a combination thereof. The heterocyclyl group can be a monocyclic heterocyclyl group or a bicyclic heterocyclyl group. Suitable bicyclic heterocyclyl groups include monocylic heterocyclyl rings fused to a C6-C10 aryl ring. When the heterocyclyl group is a bicyclic heterocyclyl group, both ring systems can be aliphatic or aromatic, or one ring system can be aromatic and the other ring system can be aliphatic as in, for example, dihydrobenzofuran. Non-limiting examples of suitable aromatic heterocyclyl groups include tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopheneyl, pyrrolidinyl, piperidinyl, and morpholinyl. Non-limiting examples of suitable aromatic heterocyclyl groups include furanyl; thiopheneyl; pyrrolyl; pyrazolyl; imidazolyl; 1,2,3-triazolyl; 1,2,4-triazolyl; isoxazolyl; oxazolyl; isothiazolyl; thiazolyl; 1,3,4-oxadiazol-2-yl; 1,2,4-oxadiazol-2-yl; 5-methyl-1,3,4-oxadiazole; 3-methyl-1,2,4-oxadiazole; pyridinyl; pyrimidinyl; pyrazinyl; triazinyl; benzofuranyl; benzothiopheneyl; indolyl; quinolinyl; isoquinolinyl; benzimidazolyl; benzoxazolinyl; benzothiazolinyl; and quinazolinyl. The heterocyclyl group is optionally substituted with 1, 2, 3, 4, or 5 substituents as recited herein such as with alkyl groups such as methyl groups, ethyl groups, and the like, or with aryl groups such as phenyl groups, naphthyl groups and the like, wherein the aryl groups can be further substituted with, for example halo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino, substituted amino, alkylcarbonyl, alkoxycarbonyl, arylcarbonyl, aryloxycarbonyl, thio, alkylthio, arylthio, and the like, wherein the optional substituent can be present at any open position on the heterocyclyl group.
The term “heterocyclylalkyl,” as used herein, refers to an alkyl group linked to a heterocyclyl group and further linked to a molecule via the alkyl group.
The term “arylalkyl,” as used herein, refers to an alkyl group linked to a C6-C10 aryl ring and further linked to a molecule via the alkyl group. The term “alkylaryl,” as used herein, refers to a C6-C10 aryl ring linked to an alkyl group and further linked to a molecule via the aryl group.
The term “alkylcarbonyl,” as used herein, refers to an alkyl group linked to a carbonyl group and further linked to a molecule via the carbonyl group, such as alkyl-C(═O)—. The term “alkoxycarbonyl,” as used herein, refers to an alkoxy group linked to a carbonyl group and further linked to a molecule via the carbonyl group, such as alkyl-O—C(═O)—.
Whenever a range of the number of atoms in a structure is indicated (such as a C1-C12, C1-C8, C1-C6, C1-C4, or C2-C12, C2-C8, C2-C6, C2-C4 alkyl, alkenyl, alkynyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-8 carbon atoms (such as C1-C8), 1-6 carbon atoms (such as C1-C6), 1-4 carbon atoms (such as C1-C4), 1-3 carbon atoms (such as C1-C3), or 2-8 carbon atoms (such as C2-C8) as used with respect to any chemical group (such as alkyl, alkylamino, etc.) referenced herein encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, and combinations thereof, as appropriate, as well as any sub-range thereof (such as 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11 carbon atoms, and/or 4-12 carbon atoms, etc., as appropriate). Similarly, the recitation of a range of 6-10 carbon atoms (such as, C6-C10) as used with respect to any chemical group (such as, aryl) referenced herein encompasses and specifically describes 6, 7, 8, 9, and/or 10 carbon atoms, as appropriate, as well as any sub-range thereof (such as, 6-10 carbon atoms, 6-9 carbon atoms, 6-8 carbon atoms, 6-7 carbon atoms, 7-10 carbon atoms, 7-9 carbon atoms, 7-8 carbon atoms, 8-10 carbon atoms, and/or 8-9 carbon atoms, etc., as appropriate).
The term “halo” or “halogen,” as used herein, means a substituent selected from Group VIIA, such as, for example, fluorine, bromine, chlorine, and iodine.
The term “aryl” refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and the term “C6-C10 aryl” includes phenyl and naphthyl. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 π electrons, according to Hickel's Rule.
The phrase “pharmaceutically acceptable salt” is intended to include non-toxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 22nd ed., Pharmaceutical Press, (2012).
Suitable bases include inorganic bases such as alkali and alkaline earth metal bases, such as those containing metallic cations such as sodium, potassium, magnesium, calcium and the like. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. Preferred pharmaceutically acceptable salts of compounds having an acidic moiety include sodium and potassium salts. Preferred pharmaceutically acceptable salts of compounds having a basic moiety (such as a dimethylaminoalkyl group) include hydrochloride and hydrobromide salts. The compounds containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.
It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
It is further understood that the above compounds and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term “solvate” refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.
In any of the above embodiments, the compound or salt of formula (I) can have at least one asymmetric carbon atom. When the compound or salt has at least one asymmetric carbon atom, the compound or salt can exist in the racemic form, in the form of its pure optical isomers, or in the form of a mixture wherein one isomer is enriched relative to the other. In particular, in accordance with the present invention, when the compounds have a single asymmetric carbon atom, the compounds may exist as racemates, that is as mixtures of equal amounts of optical isomers, that is equal amounts of two enantiomers, or in the form of a single enantiomer. As used herein, “single enantiomer” is intended to include a compound that comprises more than 50% of a single enantiomer (that is enantiomeric excess up to 100% pure enantiomer).
When the compound or salt has more than one chiral center, the compound or salt can therefore exist as a mixture of diastereomers or in the form of a single diastereomer. As used herein, “single diastereomer” is intended to mean a compound that comprises more than 50% of a single diastereomer (that is diastereomeric excess to 100% pure diastereomer).
Embodiments of the present subject matter described herein may be beneficial alone or in combination, with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure numbered 1-44 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below:
(1) A pharmaceutical formulation comprising
(2) The formulation of embodiment (1), further comprising caprylic acid.
(3) The formulation of embodiment (1) or (2), wherein the formulation is a suspension formulation.
(4) The formulation of embodiment (1) or (2), wherein the formulation is a pill.
(5) A pharmaceutical formulation comprising
(6) The formulation of any one of embodiments (1)-(3) and (5), wherein the formulation comprises water.
(7) The formulation of embodiment (5), wherein the formulation comprises saline.
(8) The formulation of any one of embodiments (1)-(7), wherein R1 is a 5 or 6-membered heterocyclyl group having at least one hetero atom selected from the group consisting of O, N, and S; a hydroxy C1-C7 cycloalkyl group; a hydroxy C1-C6 alkyl group; a N,N-di(C1-C6 alkyl)amino C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkyl group; a heteroaryl C1-C6 alkyl group; a heterocyclyl C1-C6 alkyl group; phenyl C1-C6 alkyl group where the phenyl ring is substituted with one or more C1-C6 alkoxy groups; N-benzyl piperazinyl; N-phenyl piperazinylalkyl; a phenyl C1-C6 alkyl group where the alkyl is substituted with a hydroxy group; or a 5 or 6 membered heteroarylamino C1-C6 alkyl group wherein the heteroaryl group has at least one hetero atom selected from the group consisting of O, N, and S.
(9) The formulation of any one of embodiments (1)-(8), wherein R1 is selected from the group consisting of the following:
(10) The formulation of embodiment (9), wherein R2 is phenyl, R3 is phenyl, R4 is benzyl, and R1 is selected from the group consisting of the following:
(11) The formulation of any one of embodiments (1)-(10), wherein R4 is 4-methoxybenzyl, R2 is phenyl, R3 is phenyl, and R1 is selected from the group consisting of the following:
(12) The formulation of any one of embodiments (1)-(10), wherein R4 is phenylethyl, R2 is phenyl, R3 is phenyl, and R1 is selected from the group consisting of the following:
(13) The formulation of any one of embodiments (1)-(11), wherein R4 is selected from 4-aminosulfonylbenzyl, 4-trifluoromethoxybenzyl, 4-methoxybenzyl, and cyclopropylmethyl and wherein R1 is selected from the following:
(14) The formulation of any one of embodiments (1)-(11), wherein R4 is heteroaryl C1-C6 alkyl.
(15) The formulation of embodiment (14), wherein R2 is phenyl, R3 is phenyl, R4 is
and R1 is selected from the group consisting of the following:
(16) A method for treating pancreatic adenocarcinoma in a mammal, comprising administering to a mammal in need thereof a compound of formula (I):
(17) The method of embodiment (16), wherein R1 is a 5 or 6-membered heterocyclyl group having at least one hetero atom selected from the group consisting of O, N, and S; a hydroxy C1-C7 cycloalkyl group; a hydroxy C1-C6 alkyl group; a N,N-di(C1-C6 alkyl)amino C1-C6 alkyl group; a C1-C6 alkoxy C1-C6 alkyl group; a heteroaryl C1-C6 alkyl group; a heterocyclyl C1-C6 alkyl group; phenyl C1-C6 alkyl group where the phenyl ring is substituted with one or more C1-C6 alkoxy groups; N-benzyl piperazinyl; N-phenyl piperazinylalkyl; a phenyl C1-C6 alkyl group where the alkyl is substituted with a hydroxy group; or a 5 or 6 membered heteroarylamino C1-C6 alkyl group wherein the heteroaryl group has at least one hetero atom selected from the group consisting of O, N, and S.
(18) The method of embodiment (16), wherein R1 is selected from the group consisting of the following:
(19) The method of embodiment (16), wherein R2 is phenyl, R3 is phenyl, R4 is benzyl, and R1 is selected from the group consisting of the following:
(20) The method of embodiment (16), wherein R4 is 4-methoxybenzyl, R2 is phenyl, R3 is phenyl, and R1 is selected from the group consisting of the following;
(21) The method of embodiment (16), wherein R4 is phenylethyl, R2 is phenyl, R3 is phenyl, and R1 is selected from the group consisting of the following:
(22) The method of embodiment (16), wherein R4 is selected from 4-aminosulfonylbenzyl, 4-trifluoromethoxybenzyl, 4-methoxybenzyl, and cyclopropylmethyl and wherein R1 is selected from the following:
(23) The method of embodiment (16), wherein R4 is heteroaryl C1-C6 alkyl.
(24) The method of embodiment (23), wherein R2 is phenyl, R3 is phenyl, R4 is
and R1 is selected from the group consisting of the following;
(25) The method of any one of embodiments (16)-(24), further comprising administering to the mammal a chemotherapeutic agent or subjecting the mammal to a radiation treatment.
(26) The method of any one of embodiments (16)-(25), wherein the treating results in any one or more of (I)-(V):
(27) The method of any one of embodiments (16)-(26), wherein the mammal has stage I pancreatic adenocarcinoma.
(28) The method of any one of embodiments (16)-(26), wherein the mammal has stage II pancreatic adenocarcinoma.
(29) The method of any one of embodiments (16)-(26), wherein the mammal has stage III pancreatic adenocarcinoma.
(30) The method of any one of embodiments (16)-(26), wherein the mammal has stage IV pancreatic adenocarcinoma.
(31) The method of any one of embodiments (16)-(26), wherein the mammal has metastatic pancreatic adenocarcinoma.
(32) The method of embodiment (25), comprising administering to the mammal a chemotherapeutic agent.
(33) The method of embodiment (32), wherein the chemotherapeutic agent is gemcitabine.
(34) The method of embodiment (32) or (33), wherein the chemotherapeutic agent is administered sequentially in any order with the compound of formula (I).
(35) The method of embodiment (32) or (33), wherein the chemotherapeutic agent is administered simultaneously with compound of formula (I).
(36) The method of any one of embodiments (16)-(26) and (29)-(35), wherein the compound of formula (I) reduces or delays further metastasizing of established metastases.
(37) The method of any one of embodiments (16)-(36), wherein the compound of formula (I) is administered after one or more pancreatic adenocarcinoma tumor(s) has been removed from the mammal.
(38) The method of embodiment (37), wherein the one or more pancreatic adenocarcinoma tumor(s) has been removed from the mammal by surgery.
(39) The method of embodiment (38), wherein the administration of the compound of formula (I) reduces the amount or size of metastases.
(40) The method of any one of embodiments (16)-(26) and (29)-(39), wherein the administration of the compound of formula (I) reduces or delays the growth of metastases.
(41) A method of detecting the change in expression levels of one or both of FoxA1 and FoxO6 in a mammal, wherein the mammal has been administered a compound of formula (1):
42. A method of treating pancreatic adenocarcinoma in a mammal, which method comprises:
(43) The method of any one of embodiments (16)-(42), wherein from about 0.1 to about 80 mg/kg of the compound of formula (I) is administered to the mammal.
(44) The method of any one of embodiments (16)-(43), wherein the mammal is a human.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
This example explains how the compounds of formula (I) were identified using PNC as a phenotypic marker and surrogate of metastatic behavior.
A metastatic prostate cancer cell line, PC3M, was developed in order to screen small molecules that effectively disassemble PNCs (see Pettaway, et al., “Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice,” Clin. Cancer Res., (2): 1627-36 (1996), incorporated herein in its entirety). PC3M has a PNC prevalence of 75-85% to stably express GFP-PTB (polypyrimidine track binding protein) (
24-well Transwell Permeable MATRIGEL gelatinous protein mixture Invasion assay: The effect of metarrestin on invasion activity of PANC1 and PC3M cells was measured using 24-well Transwell Permeable MATRIGEL gelatinous protein mixture Invasion Chambers with 8 μm pores (Corning, Cat #354480). Membranes were rehydrated with 500 μL of serum-free DMEM (Thermo Fisher, Cat #11965084) both inside the chamber and inside the well and incubated for two hours at 37° C., 5% CO2. Cells were detached, washed, resuspended in serum-free DMEM, counted, and diluted to a final concentration of 1×105 cells/mL in six separate 15 mL conical tubes. Metarrestin or vehicle was added to each tube in decreasing concentrations, as indicated in
A compound, named MLS000556915, was selected for further study based on its potency as a PNC inhibitor, soft agar growth inhibitor, ability to block invasion, and lack of cytotoxicity.
Based on the screening results, additional medicinal chemistry studies were then used to design a compound of formula (I), metarrestin (structure below).
Throughout the examples and figures, data are presented as means+/−SD. Student's t tests were performed for all the experiments, except where indicated. Mean nucleolar areas were analyzed using Mann Whitney U test, 2-tailed.
This example shows that a compound of formula (I), metarrestin, disrupts the PNC in cancer cell types.
In summary, several cell types (see list below in Table 1) were treated with 1 μM (IC100 for PC3M cells) of metarrestin for 24 hours. Specifically, PC-3M cells were maintained in RPMI 1640 medium. Other cell lines including HeLa, Panc1, PACADD159, PACADD183, PA-TU-8988T, KP-3, PK-8, NOR-P1, L3.6sl, Colo357, Suit-2, HPAC were maintained in DMEM with 10% FBS (GEMINI™ Bio Products) and 100 units/mL of penicillin and streptomycin. Cells were generally plated approximately 24 h before treatment with metarrestin for 5 h or 24 h at 1 μM.
Surprisingly, it was found that metarrestin reduced PNC prevalence in different human cancer cell lines (
This example demonstrates that a compound of formula (I), metarrestin, suppresses metastasis and extends survival in mouse models of human pancreatic cancer xenografts.
Because pancreatic cancer patients suffer particular high mortality from metastasis, the in vivo efficacy of metarrestin against metastasis in an orthotopic pancreatic cancer metastasis model was evaluated. The 3D PANC1 cell spheres model, deployed in NOD/interleukin 2 receptor common gamma chain (Il2rγ)null (NSG) PANC-1 mice (see Suemizu et al., “Identification of a key molecular regulator of liver metastasis in human pancreatic carcinoma using a novel quantitative model of metastasis in NOD/SCID/gammacnull (NOG) mice,” Int. J Oncol., (31): 741-51 (2007), incorporated herein in its entirety), recapitulates human cancer progression and metastatic phenotype of the disease without the limitations of early death due to complications of local invasion, such as gastric outlet obstruction or impingement of the common bile duct. Sixty thousand 3D luciferase-expressing PANC1-luc cell spheres were injected orthotopically into the pancreas of NSG mice. Histopathological examination revealed that measurable metastasis, in the form of occasional peri-portal infiltrates and micrometastatic deposits, had developed in livers approximately 4 weeks after implantation in the form of parenchymal infiltration. By 8 weeks, macrometastasis on the liver surface were visible. Mice had a lifespan of 10-14 weeks (see
The PNC prevalence was measured in a panel of pancreatic cancer cell lines derived from primary tumors and metastatic lesions from various organs in NSG PANC1 mice to determine whether the preclinical model used retained the features of PNCs observed previously in clinical specimens. The results showed that PNCs were more numerous in human cancer lineages from a metastatic origin and in metastatic lesions compared to those from primary tumors (
This example demonstrates that a compound of formula (I), metarrestin, has a favorable PK profile and that administration of metarrestin successfully suppresses metastasis-related death in metarrestin-treated mice.
This study (and those that follow) were randomized, unblinded, and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The animals were sacrificed at the predetermined time point or upon signs of animal distress (immobility or weight loss greater than 15%). Animal data were excluded as outliers in cases of unspecified death or sacrifice prior to the predetermined endpoint, which affected less than 10% of initially enrolled mice.
BALB/c mice, 17-19 g, female, n=66, were purchased and had free access to food and water. They were dosed with metarrestin at 5 or 25 mg/kg (10 ml/kg) in 5% o NMP [NMP (Sigma-Aldrich cat #270458), 20% PEG400 (Sigma-Aldrich cat #202398), and 75% 10% HPBCD (Sigma-Aldrich cat #H107)] via lower left abdominal quadrant injection (n=33 each dose). The animals were anesthetized with isoflurane and restrained manually at the designated time points. Approximately 120 μL of blood were taken from the animals into K2EDTA tubes via retro-orbital puncture. Blood samples were placed on ice and centrifuged (2,000 g, 5 min under 4° C.) to obtain plasma samples within 15 minutes. An aliquot of 20 μL plasma sample was protein-precipitated with 200 μL acetonitrile that contained 50 ng/mL IS (dexamethasone). The mixture was vortexed for 2 min, centrifuged at 14,000 rpm for 5 min, and an aliquot of 30 μL supernatant was diluted with 70 μL MeOH:H2O (1:1,v/v), then vortexed for 2 min. A 2 μL aliquot of supernatant was injected into UPLC-MS/MS. Analysis of samples was done with an LC-MS/MS-02 (API4000), Waters-BEH-C18 (2.1×50 mm, 1.7 μm) column, flow rate 0.6 mL/min, with a mobile phase consisting of solvent A (H2O-0.025% FA-1 mM NH4OAc) and solvent B (MeOH-0.025% FA-1 mM NH4OAc). The gradient was 10, 90, 90, 10% B at 0.30, 0.60, 1.20, 1.21, 1.80 (stop) min. Retention times for metarrestin and dexamethasone were 1.09 and 1.08 min, respectively. Calibration curves were established with known serially diluted concentrations of metarrestin before analysis. SCID Beige mice were purchased and dosed with metarrestin at 5 and 25 mg/kg, once daily at 24 hour intervals for 4 weeks as above. Twenty-four hours after the final dose, the concentrations of metarrestin in plasma were analyzed.
PK studies using single and multiple daily dosing via IP administration of metarrestin in mice (
Four weeks after inoculation, mice were treated once daily with metarrestin (5 mg/kg or 25 mg/kg) or vehicle via IP injections, continuing for six weeks. At the end of the tenth week after initial inoculation, the cohort exposed to daily administration of 25 mg/kg metarrestin displayed a decrease in metastatic burden in both liver (p<0.01) and lung (p<0.05) compared to vehicle-treated control as measured by Xenogen photon organ/tumor ratios (
To determine whether PNCs in treated animals were impacted by metarrestin, PNC prevalence in primary tumors and metastatic lesions from control and metarrestin-treated animals (25 mg/kg) were examined 12 weeks after tumor inoculation. The results demonstrated a significant reduction of PNC prevalence (
To evaluate whether metarrestin treatment can provide a survival advantage by preventing metastatic progression, the above experiment was repeated, but mice were followed until death or when animals reached study endpoint. NSG mice were injected with 60,000 3D PANC1 cells (details of assay below), and daily treatment with metarrestin-infused chow (10 mg/kg; 70 ppm, micronized particles added to NIH 31 Haslan rodent diet) began 6 weeks after tumor cell implantation, when metastasis was limited to micrometastasis by histopathological examination (
To evaluate whether metarrestin treatment impacts survival of mice with further evolved metastasis, NSG mice were injected with 60,000 3D PANC1 cells. After macrometastasis was visible on the liver surface, animals were randomized to receive metarrestin-infused chow (10 mg/kg; 70 ppm) or vehicle diet. Mice on the vehicle diet began to die within the first week of the treatment course (
Metarrestin was tested in PC3M xenograft mice to determine whether metarrestin's anti-metastasis activity (observed in the NSG PANC1 mice and in vitro pan-cancer PNC suppression) translates into anti-metastasis activity in additional cancer models. Two weeks after subcutaneous implantation of PC3M cells, mice were started on 5 mg/kg or 25 mg/kg metarrestin or vehicle by daily IP injection for 4 additional weeks. Tumor progression was tracked by both in vivo imaging systems (“IVIS”) spectroscopy and tumor volume. Metastasis to the lungs was evaluated ex vivo through IVIS spectroscopy and by histopathology at the experimental endpoint. Metarrestin treatment at 25 mg/kg (P<0.05) decreased development of lung metastasis compared to vehicle-treated mice (
The results show that metarrestin effectively inhibited the PDX growth, which formed entirely from metastatic cells from a patient without passing through culture in vitro (
Pancreatic cancer model: 60,000 luciferase-tagged PANC1 spheres were injected into the pancreas of Nod/IL2gamma (null) mice (obtained from NCI Mouse Repository, Frederick, MD; on ACUC-approved animal protocol SB-211-2). At 4 weeks after inoculation, mice were treated once daily with metarrestin (5 mg/kg or 25 mg/kg) or vehicle via IP injections, which extended out for 6 more weeks. Mice were injected with luciferin 5 minutes before being sacrificed by CO2 euthanasia. Organs were then individually dissected, subjected to quantitative xenogene imaging, and fixed with 4% non-buffered formaldehyde. Impact of metastatic burden was calculated and graphed as liver and lung/primary tumor ratios based on a five-minute photon count for the liver normalized to liver weight (g), which was then compared to the average photon count per weight (g) of the primary pancreatic tumor of the same animal. This calculation corrects for variations in luciferin injection, mouse heart rate, and perfusion. For overexpression of eEF1A2, 6×10Λ4 PANC1 3D spheres transduced with eEF1A2 (eEF1A2 OE) were injected into the tail of the pancreas of NSG mice. Mice of both groups were harvested at 6 weeks after implantation and subjected to necropsy. Liver metastasis/mm2 was calculated as the sum of the volumes of the individual metastases. Tumor volume (mm3) was calculated as (L×W2), where L=length (mm) and W=width (mm). Animal survival was measured from the first day of treatment until death. Animals in control and metarrestin treatment cohorts were allowed to progress under continuous treatment conditions until they reached a predetermined study end-point (15% weight loss, recognizable signs of morbidity, general lack of reflexes, abnormal posture, loss of ability to ambulate, labored respiration, inability to drink or feed) where, in order to prevent animal suffering, animals were euthanized in accordance with local animal care guidelines. Only “warm” necropsy specimens (blood, primary tumor, liver, and lungs) were used for PK analysis. All applicable institutional guidelines for the care and use of animals were followed.
Breast cancer model: The model derived from 0.2 liter of pleural effusion from a breast cancer patient (model #373342). These cancer cells had metastasized to the lung pleura from the initial tumor detected in the breast. The effusion was centrifuged at 5000 RPM for 10 minutes. The supernatant was removed, and the cells were inoculated with MATRIGEL gelatinous protein mixture into a mouse mammary fat pad. After the size of the tumor reached 1 cm, a 2×2 mm fragment of mammary fat pad was retransplanted into a mammary fat pad of another mouse. The tumors used in this experiment went through 4 passages. NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac strain of mice from Taconic was used in the experiment. The animals were acclimated up to 7 days before tumor inoculation. The 2×2 mm tumor was inoculated into the mammary fat pad. The study started after the tumor reached 150-200 mm3. Doses of 25 mg/kg metarrestin in 5% NMP, 20% PEG400, and 75% of 10% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in water were injected IP 5 days a week for 4 weeks. Tumor size and total body weight were measured twice a week.
Prostate cancer model: Mice were implanted in the flank with 3×106 human PC-3M-luc-C6 pancreatic tumor cells (Caliper Life Sciences) and split into 4 groups of 10 mice. Dosing with 5 or 25 mg/kg drug or vehicle alone (negative control) was initiated after 2 weeks and continued until the experimental endpoint, 6 weeks after implantation. Primary tumor size (measured with calipers), live tumor cell content (measured by biophotonic imaging), and total body weight were determined each week. At the endpoint, animals were euthanized, metastatic organs removed, and metastases quantitated by biophotonic imaging of the organs.
This example demonstrates that metarrestin treatment disrupts nucleolar structure and inhibits Pol I transcription.
To address the mechanisms by which metarrestin inhibits metastasis, cellular changes after metarrestin treatment were observed. Primary antibodies: 1:300 dilution for SH54 (anti-PTB antibody); 1: 300 for Nopp140 (anti-human Nopp140 rabbit polyclonal serum RS8); 1:50 for UBF (F-9) (Santa Cruz Biotechnology); 1:100 for RPA194 (Santa Cruz Biotechnology); 1:100 for CUG-BP1 (3B1) (Santa Cruz Biotechnology); 1:10 for fibrillarin (Sigma Cat #: ANA-N); 1:1000 for SC35; 1:300 for paxillin (ab32084); 1:600 for vinculin (Sigma V9131). The fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch) and ALEXA FLUOR antibodies (Thermofisher Scientific) were used at a dilution of 1:200. Samples were visualized on a Nikon ECLIPSE Ti-E inverted fluorescence microscope using NIS-Elements AR 3.2 software (Nikon).
At 1 μM concentration, metarrestin induced collapse of the nucleolus from the typically integrated three substructures (arrows in
To examine whether the disruption of the nucleolar structure impacts ribosome synthesis, a cell line expressing inducible GFP-RPL29 (a ribosomal subunit) was used. GFP-RPL29 induced by the addition of tetracycline (
This example demonstrates that metarrestin treatment reduces pre-RNA synthesis and Pol I occupancy at rDNA promoters.
To determine which steps in ribosomal biogenesis that are disrupted by metarrestin, rDNA transcription was evaluated using a BrU incorporation assay, an in situ run-on assay that detects the localization pattern of newly synthesized RNA. Cells were rinsed once in glycerol buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.5 mM EGTA) and followed by Br-U incorporation assay protocol. The cells were co-immunolabeled with C23 (Santa Cruz Biotechnology) at a 1:100 dilution and anti-BrdU that also recognizes Br-U (Sigma) at a 1:50 dilution.
A five-minute incubation of semi-permeabilized cells with a transcription cocktail containing BrU (
ChIP-qPCR: 2-5×107 cells were used for each ChIP assay according to previously described protocols. Briefly, HeLa cells with or without metarrestin treatment were crosslinked with 1% formaldehyde for 10 min at room temperature with rotation, and then crosslinking was quenched by the addition of glycine. Fixed chromatin was sonicated by Covaris and used for immunoprecipitation with the indicated antibody. Isolated DNA was analyzed by qPCR using SYBR green on CFX Connect Real-Time PCR Detection System (BioRad). The comparative cycle threshold method was applied to evaluate occupancy from replicate PCR reactions relative to the level of input. The primer sets used in the study included: rDNA promoter (F: 5′-GCT GCG ATG GTG GCG TTT TTG GGG (SEQ ID NO: 1) and R: 5′-ATA TAA CCC GGC GGC CCA AAA TTG CC) (SEQ ID NO: 2), 5′ETS (F: 5′-CGTGCCTGAGGTTTCTCC (SEQ ID NO: 3) and R: 5′-CCACCAACGGACGTGAAG (SEQ ID NO: 4)), 5.8S (F: 5′-GCA GGA CAC ATT GAT CAT CGA CAC (SEQ ID NO: 5) and R: 5′-GCG CGG CGG CAA GAG GAG (SEQ ID NO: 6)), 28S (F: 5′ GGAGGAAAAGAAACTAACCAGGAT (SEQ ID NO: 7) and R: 5′ GCCTCGATCAGAAGGACTTG (SEQ ID NO: 8)), and U12 (F: 5′GATCTGCCCGACCTTATTCA (SEQ ID NO: 9) and R 5′AAACGCATTCACCACCTACC (SEQ ID NO: 10)).
RT-PCR: Total RNA was isolated from cells using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with Random Hexamer (IDT DNA Technologies) or gene-specific primers using M-MLV Reverse Transcriptase (Invitrogen 28025-013). The DNA was PCR-amplified with gene-specific primers. 5′ETS-F: CCTCCAGTGGTTGTCGACTT (SEQ ID NO: 11); 5′ETS-R: GAACGACACACCACCGTTC (SEQ ID NO: 12); eEF1A1-F: AACATTGTCGTCATTGGACA (SEQ ID NO: 13; eEF1A1-R: TTGATCTTTCCCTTTCTGGT (SEQ ID NO: 14); eEF1A2-F: 5′CCATGTGTGTGGAGAGCTTCTC (SEQ ID NO: 15); eEF1A2-R: 5′ TCTCCACGTTCTTGATGACGCC (SEQ ID NO: 16; GAPDH-F: 5′ACCACAGTCCATGCCATCAC (SEQ ID NO: 17); GAPDH-R: 5′TCCACCACCCTGTTGCTGTA (SEQ ID NO: 18). The PCR products were resolved on a 1-2% agarose gel. Real time PCR was performed with ABI PRISM 7900HT instrument and Power SYBR Green PCR Master Mix (Life Technologies Cat #4367659) at the Northwestern University Core Facility.
To examine the mechanisms by which metarrestin disrupts Pol I transcription, the overall state of the transcription machinery and rDNA chromatin structure in treated cells was evaluated.
Western blots were performed according to the manufacturer's protocols (Li-COR). Transfer membrane was from Millipore (cat. no. IPFL00010). The primary antibodies used were phospho-γ H2AX (Upstate 07-164, rabbit, 1:1000); p53 (Santa Cruz Biotechnology sc-6243 rabbit 1:500); UBF (F-9) (Santa Cruz Biotechnology, mouse, 1:500); RPA194 (Santa Cruz Biotechnology, sc-48385, mouse, 1:500); p53BP1 (Novus, NB100-304, rabbit 1:10,000); EF1a (Millipore 05-235, mouse, 1:1000), HA-Tag (C29F4, Cell Signaling, rabbit 1:1000) and actin (Sigma A5060, rabbit, 1:3000, A4700, mouse, 1:1000) as a loading control. LI-COR IRDye secondary antibodies (1:10,000), goat anti-mouse-680RD (926-68070), goat anti-mouse-800CW (926-32210), goat-anti-rabbit-680RD (926-68071), goat-anti-rabbit-800CW (926-32211) were used for visualization. Protein bands were detected using LI-COR Odyssey Image Studio (LI-COR Biosciences).
Western blot demonstrated that the amounts of Pol I large subunit RPA194 and UBF did not change significantly after 24 hours of treatment with metarrestin at 1 μM concentration in the three cell lines, PANC1, PC3M, and HeLa (
Genotoxic agents that intercalate into or alkylate DNA, such as actinomycin D, induce similar nucleolar segregation to that observed with metarrestin treatment. To determine whether metarrestin induces nucleolar changes through genotoxic effects or general cytotoxicity, the impact of metarrestin on DNA damage repair, cell cycle, and general Pol II transcription status in three cell lines, PANC1, PC3M, and HeLa, was evaluated. Cells were treated with 1 μM metarrestin or DMSO for 24 hours before fixation for flow cytometry, immunolabeling, or Western blot analyses. Evaluation of DNA damage response signature factors in treated cells (
To further determine whether metarrestin interferes with Pol II transcription in general, CUGBP was immunolabeled. The steady state nuclear distribution of CUGBP, a multifunctional hnRNP protein highly enriched in the PNC, is dependent upon Pol II transcription. Selective inhibition of Pol II by α-amanitin induces a localization shift to the cytoplasm except for PNC-localized CUGBP (
This example demonstrates that reduction of RPA194 partially phenocopies the disruption of nucleoli and PNCs by metarrestin.
RNA interference: 75 nM Pol Il Stealth siRNA (Invitrogen Cat #: 10620318) or Stealth negative control siRNA (Invitrogen Cat #: 12935-300) were transfected into cells with Lipofectamine RNAiMAX Transfection Reagent (Thermofisher Scientific) according to the manufacturer's instructions. The experiments were performed after 72 hours transfection. For eEF1A2 knockdown, 50 nM eEF1A2 DsiRNA (IDT Duplex pool HSC.RNAI.N001958.12/HSC.RNAI.N001958.12.2) or DS Scramble Negative Control siRNA (IDT) were transfected with Lipofectamine RNAiMAX as described above for 3 days. pcDNA3.1-HA-eEF1A2 plasmid was transfected after DsiRNA RNAi for 1 day with Lipofectamine 2000. The cells were then fixed for immunofluorescent labeling.
It was next determined whether metarrestin disassembles PNCs by inhibiting Pol I function and disrupting nucleoli. The large subunit of Pol I, RPA194, was knocked down by a specific siRNA, as evident by the reduction of the protein 72 hours after transfection with the siRNA oligos in cell lines PANC1, HeLa, and PC3M (
This example demonstrates that metarrestin binds the translation elongation factor eEFIA.
To identify the molecular target(s) of metarrestin and upstream factors involved in ribosomal and Pol I function, proteins that bind metarrestin through affinity purification using biotin-conjugated metarrestin (for structure and synthesis, see
eEF1As are multi-functional proteins, implicated in translation elongation, actin bundling, nuclear transport, and tRNA export. To evaluate whether eEF1A mediates the effect of metarrestin on cancer cells, eEF1A was either overexpressed or reduced through siRNA silencing. Whereas overexpression of HA-eEF1A1 or HA-EFF1A2 did not significantly increase total PNC prevalence (
To determine whether the reduction of eEF1A2 phenocopies metarrestin's impact on PNCs and nucleolar structure, eEF1A2 expression was reduced using siRNA oligos. Seventy-two hours after transfection, qRT-PCR showed a selective reduction of eEF1A2 (
The above examples demonstrate, through in vitro assays and in vivo xenograft modeling, that metarrestin effectively inhibits PNC, cancer cell growth, cell invasion, tumor growth, and metastasis in pancreatic cancer models.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This patent application claims the benefit of U.S. Provisional Patent Application No. 62/671,964, filed May 15, 2018, which is incorporated by reference in its entirety herein.
This invention was made with Government support under project number 1ZIABC011267-08 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62671964 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17055256 | Nov 2020 | US |
| Child | 18735506 | US |
