KR20200059188A - Composition, packaged medicament, and method using posaconazole for sensitization of resistant tumors - Google Patents

Abstract

Compositions, packaged medicaments and methods of treatment by sensitization of resistant tumors are provided. The composition includes posaconazole and a chemotherapeutic agent. Tumor cells of mammalian subjects treated with posaconazole are sensitized to the effect of the chemotherapeutic agent, increasing the therapeutic index of the agent and reducing toxicity to the subject.

Description

Composition, packaged medicament, and method using posaconazole for sensitization of resistant tumors

Cross reference to related applications

This application claims the benefit of U.S. Provisional Application No. 62 / 484,852, filed on April 12, 2017, the disclosure of which is incorporated herein by reference in its entirety.

Most primary chemotherapy drugs are capable of destroying large tumor cells, but do not remove cancer stem cells, cells that contribute to the tumor's recurrence or relapse, further progression, metastasis, and subsequent chemotolerance. . This indicates that cancer stem cells may be “intrinsically” resistant to chemotherapy, or that resistance is induced during primary therapy through the acquisition of a mutation and that the mutation is carried to a secondary therapy and is present during secondary therapy. Therefore, targeting cancer stem cells as a primary therapy can eliminate cancer cells of intrinsic resistance, prevent the acquisition of resistant mutations, limit further progression and metastasis of cancer, and also present reactive cancer cells. Can be applied to secondary treatment. Cancer cells, and more particularly cancer stem cells, can express one or multiple ATP binding cassette (ABC) transporters as a mechanism of resistance to chemotherapy drugs. ABC transporter proteins can promote the outflow of drugs from cancer cells, making them resistant. The outflow of drugs from cancer cells means that higher concentrations of drugs are required to achieve apoptosis, and at these concentrations the drugs can be toxic to the patient and essentially reduce the therapeutic index of the drugs. . Many known inhibitors of ABC transporters, such as verapamil, reserpine and cyclosporin, directly reduce or prevent the removal of chemotherapeutic drugs from the cells, when used sequentially or in combination with other drugs, such that the drug Make it more effective at lower concentrations. By increasing the intracellular concentration of the drug and by reducing the initial therapeutic concentration required to achieve cancer cell death, the therapeutic window of the drug is improved and toxicity to the patient is alleviated. However, the concentration of the ABC transporter inhibitor required to block the transporter is too toxic and cannot be used in patients, therefore, the inhibitor is not effective for use in combination therapy. Gottesman et al. (1993) Annu. Rev. Biochem. 62: 385-427].

In some instances, ABC transporter activity is tightly regulated by sequestering the transporter into intracellular compartments. Rocchi et al. (2000) Biochem. Biophys. Res. Commun. 271: 42-6]. For example, translocation of the ABC transporter, ABCG2, to the cell membrane depends on post-translational modification through phosphorylation by Akt kinase. Takada et al. (2005) Drug Metab. Dispos. 33: 905-9]. In cells expressing ABCG2, Akt inhibitors such as Gleevec, LY294002 or LY335979 reduce or completely eliminate translocation of the transporter to the cell membrane, reduce transporter activity or completely discard, thereby It has been shown to sensitize resistant cells to drugs. See Shepard et al. (2003) Int. J. Cancer 103: 121-5; Nakanishi et al. (2006) Blood 108: 678-84; Burger et al. (2005) Cancer Biol. Ther. 4: 747-52; Ozvegy-Laczka et al. (2004) Mol. Pharmacol. 65: 1485-95; Houghton et al. (2004) Cancer Res. 64: 2333-7]. However, this treatment strategy has led to a compensatory increase in transporter expression to maintain tolerance, and thus has been shown to be insufficient for effective therapeutic application.

Another strategy for overcoming ABC transporter-related drug resistance is to inhibit the pathways that control ABC transporter expression in resistant cancer cells, including cancer stem cells. Combination of Smo (smoothed) antagonist, cyclopamine with chemotherapy drugs has been shown to reduce ABCG2 and ABCB1 / MDR1 activity and increase cancer cell death compared to in vitro drug alone, by a mechanism that has not yet been identified. . See Singh et al. (2011) Oncogene 30: 4874-86; Zhang et al. (2009) Neoplasia 11: 96-101; Sims-Mourtada et al. (2007) Oncogene 26: 5674-9; Lou et al. (2007) Oncogene 26: 1357-60]. However, cyclopamine is a toxic alkaloid that is lethal to humans without viable therapeutic application.

Itraconazole is a prescription-only antifungal agent that has been used to treat fungal infections such as nail fungus, aspergillosis, candidiasis, cryptococcosis, and histoplasmosis. Hardin et al. (1988) Antimicrob. Agents Chemother. 32: 1310-3]. Itraconazole has also been shown to directly inhibit P-gp / MDR-1 / ABCB1 activity. See Miyama et al. (1998) Antimicrob. Agents Chemother. 42: 1738-44]. It has also been shown to be a strong CYP3A4, cytochrome P450 3A4 inhibitor. Tapaninen et al. (2011) J. Clin Pharmacol. 51: 359-67]. Recently, itraconazole, arsenic trioxide, vitamin D3 and various other agents have been shown to inhibit the hedgehog pathway. See Kim et al. (2010) Cancer Cell. 17: 388-99]. It has been shown that these compounds can be used as a single agent that inhibits the growth of tumors containing a deregulated hedgehog pathway or mutation in Ptc, Smo or Gli proteins or induces cell death thereof. See Kim et al. (2013) Cancer Cell. 23: 23-34]. Itraconazole is currently in clinical trials for the treatment of several tumor types caused by deregulation of the hedgehog pathway. Itraconazole has been shown to inhibit ABCG2 and ABCB1 / MDR1 in cells artificially engineered to replicate acquired chemoresistance, or in patients pretreated with severely pretreated or in vitro secondary therapy. However, these experiments were performed using cytotoxic and non-therapeutic dosages in combination with dye substrates as readouts. Gupta et al. (1991) J. Clin. Invest. 87 (4): 1467-1469; Kurosawa et al. (1996) Ann. Hematol. 72 (1): 17-21]. In this context, acquired chemoresistance can be defined as when the cells are exposed to chemotherapy drugs until the cancer cells “acquire” a mutation that activates the mechanism and makes the cancer cells resistant to chemotherapy.

Itraconazole also includes AML (acute myelogenous leukemia), ALL (acute lymphocytic leukemia); Vreugdenhil et al. (1993) Ann. Hematol. 67 (3): 107-109], pancreatic cancer; Tsubamoto et al. (2015) Anticancer. Res. 35 (7); 4191-4196], biliary tract cancer; Tsubamoto et al. (2015) Anticancer. Res. 35 (9): 4923-4927], triple-negative breast cancer; Tsubamoto et al. (2014) Anticancer. Res. 34 (7): 3839-3844], ovarian cancer; Tsubamoto et al. (2014) Anticancer. Res. 34 (5): 2481-2487, and non-squamous NSCLC (non-small cell lung carcinoma), Rudin et al. (2013) J. Thorac. Oncol. 8 (5): 619-623 has been shown to increase patient survival when administered in combination with secondary therapy. However, in this context, itraconazole was severely pre-treated in a second line therapy. Since these patients are prior to treatment with a chemotherapeutic agent, they may have acquired mutations that confer resistance to the chemotherapy.

Posaconazole is a prescription antifungal agent that is used to treat fungal infections such as aspergillosis, candidiasis, hairy mildew, and spores. See Schiller et al. (2007) Clinical Therapeutics 29: 1862-1886]. Similar to itraconazole, posaconazole is known to be a potent inhibitor of CYP3A4 (cytochrome P450 subunit 3A4). However, unlike itraconazole, posaconazole has not been shown to directly inhibit P-gp / MDR-1 / ABCB1. Posaconazole has also not been used in combination with chemotherapeutic agents, but has been used as a single agent to inhibit the growth of tumors containing a deregulated Hedgehog pathway or mutation in Ptc, Smo or Gli proteins. See Chen et al. (2016) Mol. Cancer Therap. 15 (5): 866-876].

US Patent Publication No. 2007/0281040 A1 discloses the use of a highly toxic hedgehog inhibitor, cyclopamine, for the treatment of cells with an activated hedgehog pathway, wherein the hedgehog pathway of the cell is directed to the chemotherapeutic agent. It is targeted directly by the inhibitor, not by sensitization of the cells to the cells.

Accordingly, there is a need for improved compounds, compositions, packaged medicaments and methods to overcome chemoresistance in tumor cells, particularly tumor cells expressing ABC transporters, as a primary treatment.

The present invention hedges to reduce or eliminate MYC expression, or to modulate the activity of other regulators that can lead to down-regulation of ABC transporter expression and mitigate chemical resistance in cancer cells. These and other problems are addressed by providing compositions, packaged medicaments and methods for modulating the hog pathway in various aspects. Modulation of the hedgehog pathway is used in combination with chemotherapy drugs, increasing the patient's therapeutic index and reducing the associated side effects of these drugs for several cancer types. The composition, packaged medicament and method can be used to determine the type, formulation, dosage or treatment schedule of any chemotherapy drug determined by preclinical and clinical trials for each cancer type as a primary treatment or for reactive cells in secondary treatment. It can contain.

In certain embodiments, the present invention reduces or eliminates ABC transporter expression, thereby reducing or eliminating ABC transporter activity in resistant cancer cells, thereby increasing the therapeutic index of chemotherapy drugs, posaconazole, itraconazole, The use of hedgehog pathway modulators, including arsenic trioxide, vitamin D3 and various other agents. In a preferred embodiment, the hedgehog pathway modulator is posaconazole.

In another aspect, the present invention aims to inhibit molecules or pathways not related to the hedgehog pathway, but of experimental and FDA approved therapeutic compounds that can have an inhibitory effect on the hedgehog pathway for inactivation of ABC transporters. Provide a change of use. These compounds are less toxic and more resistant to patients when used in combination with drugs that improve the therapeutic index of the drug and reduce the related side effects of the drug compared to when the drug is used alone.

Degree. 1. Diagram of hedgehog pathway regulation of ABC transporters, showing exemplary points of inhibition to which the present invention can be applied to reduce ABC transporter expression.
Degree. 2. Illustrating aspects of the invention using competitive inhibitors or scavengers of the ligands Sonic and Indian hedgehog proteins to reduce MYC expression or other modulators and subsequently reduce downstream ABC transporter expression.
Degree. 3a-3b. Of the present invention, including induction of patching or stabilization of the Ptc: Smo complex to inhibit smoothed receptor signaling to reduce MYC expression or other modulators and subsequently reduce downstream ABC transporter expression. A diagram illustrating an aspect.
Degree. 4a-4b. A diagram illustrating aspects of the invention that utilize inhibitors of the cholesterol synthesis pathway or sterolization or specifically prevention of cholesterol in Smo.
Degree. 5. Illustrative aspects of the invention, including direct inhibition of smoothed receptor signaling to reduce MYC expression or other modulators and subsequently reduce downstream ABC transporter expression.
Degree. 6. Illustrative aspects of the invention, including suppression of effectors that signal SUFU for activation of MYC expression or other modulators and activate SUFU and subsequently reduce downstream ABC transporter expression.
Degree. 7. Illustrative aspects of the invention, including inhibition of Glil, Gli2 and induction of Gli3, to reduce MYC expression or other modulators and subsequently reduce downstream ABC transporter expression.
Degree. 8. Illustrative aspects of the invention using inhibition or reduction of MYC to down-regulate ABC transporter expression directly or indirectly.
Degree. 9. Itraconazole or fossa for inhibiting Smo signaling and thereby reducing MYC expression or other modulators and subsequently reducing downstream ABC transporter expression thereby sensitizing resistant cancer cells to chemotherapy drugs. Drawings illustrating preferred embodiments of the invention demonstrating the use of conazole.
Degree. 10a-10l. Vinca alkaloids in the H295 (adrenal cortical carcinoma), Kelly (neuroblastoma (boy brain cancer)), HeLa (uterine cervical cancer) and Caco-2 (colon or colorectal cancer) cell lines; Vincristine, and taxane; A series of preclinical studies demonstrating preferred embodiments of the present invention demonstrating a “dose de-escalation” strategy by use of cyclopamine in combination with docetaxel (positive control and toxicant antagonist of the hedgehog pathway), and itraconazole. data.
Degree. 11. Itraconazole, vincristine, itraconazole, which was tested in phased reduction in combination with vincristine; A series of preclinical data demonstrating a preferred embodiment of the present invention demonstrating a “dose stepwise reduction” strategy by H295 cell injected tumors treated in vivo with 10 times less vincristine and itraconazole in combination with 10 times less vincristine.
Degree. 12a-12d and 13a-13d. Various colon cancer cell lines: Caco-2 (12a, 12b), DLD-1 (12c, 12d), HT-29 (13a, 13b) and HCT-15 (13c, 13d), tomatetidine (a structural analog of cyclopamine And negative control), cyclopamine (experimental positive control and toxic antagonist of the hedgehog pathway), itraconazole (strong hedgehog pathway antagonist, positive control), and posaconazole (experimental formulation) (12a, 12c, 13a and 13c) A series of preclinical data demonstrating a preferred embodiment of the present invention demonstrating cytotoxicity measurements on vinca alkaloids; A series of demonstrating preferred embodiments of the present invention demonstrating a “dose stepwise reduction” strategy by use of posaconazole (strong hedgehog pathway antagonist, sensitizer) in combination with vincristine (12b, 12d, 13b and 13d) Preclinical data.
Degree. 14a-14d. Two small cell lung cancer cell lines: in H69 (14a, 14b) and H146 (14c, 14d), tomatidine (a structural analog and negative control of cyclopamine), cyclopamine (experimental positive control and toxic antagonist of the hedgehog pathway) , A series of preclinical data demonstrating preferred embodiments of the invention demonstrating cytotoxicity measurements for itraconazole (strong hedgehog pathway antagonist, positive control), and posaconazole (experimental formulations) (14a, 14c), and vinca alkaloids ; A series of preclinical data demonstrating preferred embodiments of the invention demonstrating a “dose stepwise reduction” strategy by the use of posaconazole (strong hedgehog pathway antagonist, sensitizer) 14b, 14d in combination with vincristine.
Degree. 15a-15d and 16a-16d. Various neuroblastoma cell lines: Kelly (15a, 15b), SK-N-SH (15c, 15d), SH-5YSY (16a, 16b), and Lan-5 (16c, 16d), tomatidine (of cyclopamine Structural analogs and negative controls), cyclopamine (experimental positive control and toxic antagonist of the hedgehog pathway), itraconazole (strong hedgehog pathway antagonist, positive control) and posaconazole (experimental formulations) (15a, 15c, 16a and 16c) A series of preclinical data demonstrating preferred embodiments of the invention demonstrating cytotoxicity measurements for), and vinca alkaloids; A series of demonstrating preferred embodiments of the invention demonstrating a “dose stepwise reduction” strategy by using posaconazole (strong hedgehog pathway antagonist, sensitizer) (15b, 15d, 16b, and 16d) in combination with vincristine Preclinical data.

The present invention relates generally to the field of cancer treatment. In particular, the present invention relates to sensitization of chemoresistant cancer cells using the modulator of the hedgehog pathway. Specifically, the present invention relates to sensitizing chemoresistant cancer cells through reduction, elimination and / or inactivation of pumps responsible for the removal of chemotherapy drugs from cancer cells. More specifically, the present invention relates to the modulation of signaling pathways that regulate pump expression in cancer cells. Specifically, the present invention relates to the modulation of any component of the hedgehog pathway that can down-regulate MYC activators or other modulators of the ABC transporter and render chemoresistant cancer cells susceptible to chemotherapy drugs. The present invention can also be further applied to experiments and to altering the use of FDA-approved compounds that can modulate the hedgehog pathway and sensitize chemoresistant cancer cells to chemotherapy drugs.

The present invention provides a method of achieving enhanced synergistic killing of chemoresistant cancer cells when compounds, first of all, are modulators of the hedgehog pathway and second are chemotherapy drugs, known substrates of the ABC transporter. . The combination reduces the toxicity to the patient while making the cells vulnerable to lower concentrations of chemotherapy drugs.

The following aspects of the invention describe several methods of using the Hedgehog Path Modulator in combination with chemotherapy drugs to achieve improved therapeutic indices in patients. The compositions and methods can include any chemotherapy drugs, formulations, dosages, and treatment schedules determined in preclinical and clinical trials for each cancer type as a primary therapy or for reactive cells in a secondary therapy.

The hedgehog pathway includes several control points, outlined in Figure 1, that can be used as targets for downregulation of ABC transporters. Compounds or biologics are used to modulate the hedgehog pathway at any regulatory point shown in Figure 1, which results in downregulation of MYC or other modulators of the ABC transporter. When the MYC or other modulator is downregulated, the ABC transporter is downregulated. Down-regulation of ABC transporters allows the cells to be sensitized to lower concentrations of chemotherapy drugs.

As shown in Figure 2, the binding of Sonic or Indian hedgehog (SHH or IHH) to Ptc can be disrupted using competitive inhibitors (compounds or biological agents) that occupy the binding sites of SHH and IHH on Ptc. (Figure 2, left). In addition, binding of SHH and IHH on Ptc can be prevented by using therapeutic agents (compounds or biological agents) that act as scavengers and directly bind SHH and IHH, where they normally bind Ptc (Figure 2, right). ). This prevents activation of the pathway or inhibits the active pathway, which reduces the activity of MYC expression or other modulators and subsequently decreases ABC transporter expression.

In another embodiment, the compound or biologic is used to induce Ptc activity that inhibits Smo activity, or to stabilize the Ptc: Smo complex that results in inhibition of the hedgehog pathway (FIGS. 3A-3B). . Induction of Ptc activity is achieved using compounds or biologicals that cause molecular / morphological / structural changes to the receptor (FIG. 3A). Stabilization of the Ptc: Smo complex is achieved by using a compound or a biological agent having the ability to crosslink the two receptors or by stabilizing the combined state of the two receptors (FIG. 3B). These compounds or biologics down regulate or prevent activation of the hedgehog pathway, thereby reducing the activity of MYC expression or other modulators and subsequently reducing ABC transporter expression.

In another embodiment, the compound or biologic is used to prevent cholesterol-dependent activation of Smo by inhibition of enzymes in the cholesterol synthesis pathway. Inhibition of any enzyme that produces cholesterol precursors results in a decrease in intracellular cholesterol required for Smo signaling activity (FIG. 4A). In addition, induction of Ptc pump activity may increase the removal of intracellular cholesterol required for Smo signaling activity (FIG. 4B). Reduction of cholesterol by the point of inhibition or induction prevents activation of this pathway or inhibits the active pathway, thereby reducing the activity of MYC expression or other modulators and subsequently reducing ABC transporter expression.

In another embodiment, the compound or biologic is used to inhibit the activity of Smo. This is accomplished using compounds or biologicals that bind and antagonize Smo, which reduces the activity of MYC expression or other modulators and subsequently reduces downstream ABC transporter expression (Figure 5).

In another embodiment, the compound or biologic is used to inhibit a signaling molecule that induces SUFU activity (eg, FIG. 6, “A” position on the right). The inactive state of SUFU sequesters downstream effector proteins such as Glil and Gli2, which stops downstream pathway activation. In addition, compounds and biologics can be used to inhibit SUFU activity (eg, FIG. 6, “B” position on the right) or SUFU: Gli complex (eg, FIG. 6, “C” position on the right). Directly stabilizing and inactivating this pathway, reducing the activity of MYC expression or other modulators and subsequently reducing downstream ABC transporter expression.

In another embodiment, the compound or biologic is used to inhibit a molecule that induces Glil or Gli2 transcription factor activity (eg, the “A” position on the left side of FIG. 7,). In addition, the compound or biologic is intended to directly inhibit Glil or Gli2 transcription factor activity (eg, position “B” on the right in FIG. 7,) or activity of Gli3 (eg, “C on the right in FIG. 7,” "Position), reducing the activity of MYC expression or other modulators and subsequently reducing downstream ABC transporter expression.

In another embodiment, the compound or biologic is used to inhibit the activity of the MYC transcription factor for reducing ABC transporter expression. This can be done through the inhibition of MYC (eg, FIG. 8, top), or by preventing the formation of a MYC: MAX complex that prevents activation of the target gene, specifically the ABC transporter (eg, FIG. 8, bottom) DNA This is achieved by using compounds or biologicals that interfere with the binding of MYC to the binding site.

In a preferred embodiment, synergistic killing of resistant cancer cells is achieved by using an FDA approved drug, such as posaconazole, itraconazole, arsenic trioxide, vitamin D3, or various other hedgehog pathway modulators (see Table 1), in combination with chemotherapy drugs. Is achieved. Posaconazole, itraconazole and other hedgehog pathway modulators can make resistant cancer cells vulnerable to lower concentrations of chemotherapy drugs while reducing toxicity to the patient. FIG. 9 shows how inhibition of Smo signaling by posaconazole or itraconazole reduces the activity of MYC expression or other modulators and subsequently reduces downstream ABC transporter expression, thereby allowing resistant cancer cells to chemotherapy drugs. Show if you are sensitizing.

Chemotherapy drugs for use in all aspects of the invention include, but are not limited to, vinca alkaloids, taxanes, platinum, anthracyclines, topoisomerase inhibitors, kinase inhibitors, mTOR inhibitors, PARP inhibitors, Bcl-2 or Bcl-XL antagonists , BET bromodomain / BRD4 inhibitors, HDAC inhibitors, SGLT antagonists and statins. It should be understood that chemotherapeutic drugs include all types of anti-cancer agents, conventional chemotherapy drugs, as well as more recently developed targeted cancer therapeutics. See, eg, Baudino (2015) Curr. Drug Disc. Tech. 12: 3-20] and the My Cancer Genome website (www.mycancergenome.org/content/molecular-medicine/anticancer-agents). Also, see Lin et al. (2014) Int'l J. Endocrin. Article ID 719578; Perez-Salvia et al. 2017 Epigenetics 12: 323-339; Hindler et al. 2006 The Oncologist 11: 306-315; and Boudreau et al. 2010 Expert Opin. Drug. Saf. 9: 603-621. In some cases, the drug is released from the cell by the ABC transporter, and the use of posaconazole, itraconazole, arsenic trioxide, vitamin D3 or other hedgehog pathway modulators in combination with one or more chemotherapeutic drugs is the drug or It provides an improved therapeutic index for drugs.

Any agent (biological or small molecule) that modulates an immunological target for therapeutic benefit in cancer, such as PD-1, PD-L1, PD-L2, CTLA-4, CD4, CD28, CD38, CD80, CD86 , CD47, CD19, CD20, CD27, CD137, LAG-3, GITR, CD40, OX40, 4-IBB, GR-1, Ly6G, NKG2D, BTLA, ICOS, KIR, TIM-3, IL-2, IL-4 , IL-5, IL-6, IL-10, IL-12, IL-17, IFN-gamma, IDO / TDO, ADC and CAR T-cells should also be considered chemotherapeutic agents for the purposes of the present invention. . See, eg, Oiseth et al. (2017) J. Cancer Metastasis Treat. 3: 250-261; Meiliana et al. (2016) Cancer Immunother, 8: 1-20; Forkona et al. (2016) BMC Medicine 14:73; And Hanoteau et al. (2016) Oncoimmunol. 5: el190061. The combination of posaconazole with any of these immuno-modulating chemotherapeutic agents can also result in additional therapeutic benefits.

The compositions, packaged medicaments and methods of the invention are not a single agent for treating tumors with tumorigenic mutations in the hedgehog pathway, but inhibit the hedgehog signaling to reduce MYC expression or activity of other modulators and subsequent It is advantageous in the use of FDA approved anti-tumor agents, posaconazole, itraconazole, arsenic trioxide, vitamin D3, or another hedgehog pathway modulator, as a single agent that reduces the expression of ABC transporters that confer tumor resistance. In addition, the dosage, toxicity and ADME information is well documented for posaconazole, itraconazole, arsenic trioxide, vitamin D3 and other hedgehog pathway modulators, making them more resistant to the patient compared to cyclopamine or new experimental drugs. Can be. Dosage required for humans to down-regulate hedgehog-controlled chemical resistance in cancer cells is readily determined during clinical trials as will be understood by those skilled in the art. In addition, the minimum effective dose (MED) and maximum tolerated dose (MTD) of posaconazole, itraconazole, arsenic trioxide, vitamin D3 and other hedgehog pathway modulators provide parameters for more effective treatment of these tumors. Thus, these parameters enable physicians to prescribe and evaluate efficacy with familiarity with high toxicity hedgehog inhibitors such as cyclopamine, and experimental drugs with unknown dosages and toxicity. This effect can also be achieved by reducing the concentration of the hedgehog pathway modulator, which reduces toxicity or side effects to the patient in primary therapy, and does not exclude secondary therapy. Reducing the mechanism of resistance can increase the therapeutic index of chemotherapy drugs required to kill tumor cells, which can reduce or avoid known toxicity affecting the patient during treatment.

Exemplary hedgehog pathway modulators useful in the compositions, packaged medicaments and methods of the present invention include, but are not limited to, the formulations listed in Table 1.

In some embodiments, the hedgehog pathway modulator can be provided as a solid dispersion of a modulator in a polymer, for example, to improve absorption of drugs in the gastrointestinal tract and thereby achieve bioavailability compared to conventional formulations. Such dispersions can, for example, improve the dissolution of poorly soluble drugs compared to their normal crystalline form. Itraconazole was formulated with this dispersion in combination with HP-50 (Reference; SUBA Itraconazole, MaynePharma, Raleigh, NC 27609, USA). Posaconazole can be formulated in a similar manner.

Accordingly, the present invention, in some aspects, administering a hedgehog pathway modulator to a mammalian subject; And administering a chemotherapeutic agent to the subject, wherein the subject suffers from cancer and the hedgehog pathway modulator is administered in an effective amount to sensitize the tumor cells of the subject to a chemotherapeutic agent. to provide.

In some embodiments, the chemotherapeutic agent is administered in a dose less than required in the absence of the hedgehog pathway modulator. In some embodiments, the hedgehog pathway modulator is administered at less than the maximum tolerated dose, and the chemotherapeutic agent is administered at a dose less than that required in the absence of the hedgehog pathway modulator. In some embodiments, the hedgehog pathway modulator and the chemotherapeutic agent are administered simultaneously or almost simultaneously.

In some embodiments, the hedgehog pathway modulator is administered prior to administration of the chemotherapeutic agent. Modifications to this method include a single step of administering a chemotherapeutic agent to a mammalian subject, the subject suffering from cancer, and the subject, in an effective amount, sensitizing the tumor cells of the subject to the chemotherapeutic agent, the hedgehog pathway. Modulators have been previously administered. In these methods, as will be appreciated by those skilled in the art, prior administration of the hedgehog pathway modulator may be performed at any suitable time point prior to administration of the chemotherapeutic agent, as long as sufficient sensitizing effects from the hedgehog pathway modulator administration remain in the subject. Can be performed on.

According to another method aspect, the hedgehog pathway modulator is administered after administration of the chemotherapeutic agent.

In a preferred method embodiment, the mammalian subject has not been previously treated with a chemotherapeutic agent prior to treatment with the Hedgehog Path Modulator.

In some method embodiments, the hedgehog pathway modulator and chemotherapeutic agent are each administered orally, intramuscularly, subcutaneously, intranasally, or intravenously.

In some method embodiments, the subject has adrenal cortical carcinoma, neuroblastoma, cervical cancer, colon cancer, colorectal cancer or small cell lung cancer.

In certain embodiments, the hedgehog pathway modulator is posaconazole or itraconazole. Preferably, the hedgehog pathway modulator is posaconazole.

In another aspect, the present invention provides compositions, packaged medicaments and methods of treatment according to the following numbered paragraphs:

1. As a composition,

Hedgehog path modulator; And

A composition comprising a chemotherapeutic agent.

2. The composition of paragraph 1, wherein the hedgehog pathway modulator sensitizes tumor cells to the chemotherapeutic agent.

3. The composition of paragraph 1, further comprising a pharmaceutically acceptable carrier.

4. As a packaged drug,

A packaged medicament comprising a hedgehog pathway modulator, chemotherapeutic agent, and instructions for using a composition for treating cancer in a mammalian subject.

5. As a treatment method,

Administering a hedgehog pathway modulator to a mammalian subject; And

And administering a chemotherapeutic agent to the subject, wherein the subject suffers from cancer and the hedgehog pathway modulator is administered in an effective amount to sensitize the tumor cells of the subject to the chemotherapeutic agent.

6. The method of paragraph 5, wherein the chemotherapeutic agent is administered in a dose less than required in the absence of the hedgehog pathway modulator.

7. The method of paragraph 5, wherein the hedgehog path modulator is administered at less than the maximum tolerated dose, and the chemotherapeutic agent is administered at a dose less than is required in the absence of the hedgehog path modulator.

8. The method of paragraph 5, wherein the hedgehog pathway modulator and the chemotherapeutic agent are administered simultaneously or nearly simultaneously.

9. The method of paragraph 5, wherein the hedgehog pathway modulator is administered prior to administration of the chemotherapeutic agent.

10. The method of paragraph 5, wherein the hedgehog pathway modulator is administered after administration of the chemotherapeutic agent.

11. The method of paragraph 5, wherein the mammalian subject has not been previously treated with a chemotherapeutic agent.

12. The method of paragraph 5, wherein the hedgehog pathway modulator and the chemotherapeutic agent are each administered orally, intramuscularly or intravenously.

13. The method of paragraph 5, wherein the hedgehog pathway modulator is posaconazole, itraconazole, arsenic trioxide or vitamin D3.

14. The method of paragraph 13, wherein the hedgehog pathway modulator is posaconazole.

 It will be readily apparent to those skilled in the art that other suitable modifications and variations of the methods and applications described herein can be made without departing from the scope of the invention or any aspect thereof. The invention has been described in detail below, and the invention will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to limit the invention.

Example

The following examples demonstrate the application of the compositions and methods of the present invention both in vitro and in vivo.

Example 1

Cancer cells grown in vitro are treated with chemotherapy drugs alone or concurrently with a hedgehog pathway modulator, including itraconazole. The difference in cell death between cells treated with chemotherapy drug alone or cells co-treated by the hedgehog pathway modulator determines the degree of synergistic killing effect of the combination therapy. A two-dimensional dose response that results in a continuous 10-fold dose step reduction of cyclopamine and itraconazole is a naive cell line that has never been exposed to chemotherapy in the patient prior to isolation and establishment as a primary treatment model: H295, Kelly; And sensitization to vincristine and docetaxel in resistant cell lines that have been exposed to chemotherapy prior to isolation and establishment as secondary treatment models: Hela, and Caco-2 cells. As cyclopamine and itraconazole decreased from 10 micromolar to 1 micromolar and 0.1 micromolar, the concentration of chemotherapy (IC50) required to stop proliferation and kill half of the amount of cells increased, but the concentration of chemotherapy alone Lower than (Figures 10a-10h). The HeLa and Caco-2 cell lines did not respond (Figures 10i-10l). This clearly demonstrates the broad sensitization of cancer to chemotherapy with itraconazole, and suggests that other hedgehog modulators may have similar effects. It also indicates that the use of fewer sensitizers or hedgehog modulators, including itraconazole, can improve the efficacy of chemotherapy in primary therapy while minimizing toxicity and side effects. The data provided herein acquires resistant modifications that can prevent cancer cells from responding to a proposed sensitization strategy using a hedgehog pathway modulator, including itraconazole, for cancer pre-exposed to chemotherapeutic drugs, thus further in primary therapy. It may be useful, but demonstrates that residual cancer cells may be useful in secondary treatments that may be responsive to hedgehog pathway modulation.

Materials and methods (in vitro):

Cell culture: H295 cell lines were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with insulin, transfer agent and selenium, 10% fetal bovine serum and gentamicin. Kelly cell lines were grown in DMEM supplemented with 10% fetal bovine serum and gentamicin. Cell-based experiments were performed in a 37 degree incubator supplemented with 5% carbon dioxide.

In vitro pharmacology: H295 cells were plated in a 96-well dish at a density of 10000 cells per well, and Kelly cells were plated at a density of 2000 cells per well. After 16 hours, cells were treated with a 2-fold dilution of vincristine (VCR) or docetaxel (DTX). For sensitization experiments, tomatidine, cyclopamine or itraconazole was added to all of the wells at a concentration of 10 micromolar, 1 micromolar or 0.1 micromolar. The cells were incubated until untreated wells reached maximum confluency where the cells did not divide rapidly.

MTT assay: The drug containing medium was discarded from all of the dishes and the MTT solution was added at a concentration of 5 micrograms per ml. The dish was placed in the incubator again for 4 hours. The excess MTT substrate was discarded and a solubilizing solution of acid treated isopropanol and Triton X-100 was added to the dish and shaken for 10 minutes. The dish was read on a plate reader equipped to read based on wavelengths of 570 nanometers and 690 nanometers.

Data analysis: Raw data was normalized to untreated wells to determine the percentage of cell death and plotted on a log scale using Graphpad Prism 6 software.

Example 2

A hedgehog pathway modulator comprising posaconazole and itraconazole was used as a pretreatment to block the hedgehog pathway, thereby downregulating ABC transporter expression. It also reduces the outflow of chemotherapy drugs. The difference in cell death between cells treated with chemotherapy drugs and cells pretreated with the hedgehog pathway modulator determines the degree of synergistic killing effect of the combination therapy.

Example 3

The stepwise reduction of the drug provides a typical determined dose of the hedgehog pathway modulator comprising posaconazole and itraconazole, but the dose of chemotherapy determines the synergistic killing effect of the combination therapy, which gradually decreases in each study. A phased reduction study demonstrates whether to maintain effective killing of the tumor while reducing side effects of the hedgehog path modulator and known toxicity of the chemotherapy drug in patients.

Example 4

The efficacy of an in vitro study is tested in vivo using a genetically engineered mouse model, xenograft model, or simultaneous xenograft model. Dose gradual reduction of vincristine is performed to demonstrate sensitization by itraconazole in H295 cell-derived tumors. Mice containing 0.5 cm tumors were administered in 6 cohort, untreated or tolerated doses of itraconazole or vincristine alone, itraconazole in combination with vincristine, 10 times less vincristine, and 10 times less vincristine in combination with itraconazole. Divided. Tumor growth in itraconazole in combination with vincristine and 10 times less vincristine cohort ceased to become necrotic 6 weeks later, demonstrating that the tumor also passed through cell death, and less chemotherapy was in the presence of itraconazole It can be used under (Fig. 11). The data provided here demonstrates that cancer sensitization to chemotherapy with a hedgehog pathway modulator, including itraconazole, requires less chemotherapy, which will reduce toxicity and side effects without reducing the efficacy of the chemotherapy. do. This approach can be used in primary therapy as well as in secondary methods where tumor cells have been found to be responsive.

Materials and methods (in vivo):

Cells: H295 cells were cultured as indicated above.

Xenografts in vivo: 1 million H295 cells were injected into the subcutaneous portion of the skin on the left flank of NOD-SCID mice in combination with matrigel. After 6 weeks of tumor growth to 0.5 centimeters, the animals were randomized and saline alone (untreated control), vincristine, 10-fold less vincristine (dose reduction control in dose), itraconazole, vincristine in combination with itraconazole, and Treated with 10 times less vincristine (stepwise reduction in dose) in combination with itraconazole. Tumors were measured once a week, and size was determined using the formula (1 / 2W x L) / 2. Tumor growth for each cohort was plotted using Graphpad Prism 6.

Example 5

Cancer cells grown in vitro were treated with the inactive structural analogs of the hedgehog modulator cyclopamine, tomatidine, cyclopamine, itraconazole and posaconazole. The cells were treated with a 2-fold diluted compound, and the difference between cell death between cells treated with each compound and untreated cells was colon cancer cell lines: Caco-2, DLD-1, HT-29, HCT-15; Small cell lung cancer cell line: H69, H146; Neuroblastoma cell lines: Cell cytotoxicity and cell death were determined in Kelly, SK-N-SH, SH-5YSY, and Lan-5 (FIGS. 12A, 12C, 13A, 13C, 14A, 14C, 15A, 15C, 16A) And 16c). The data provided here demonstrates that posaconazole has less cytotoxicity than itraconazole and that posaconazole has a significantly higher therapeutic effect than itraconazole. Thus, compared to itraconazole, the wider therapeutic range of posaconazole can be used to minimize side effects in patients and sensitize cells to chemotherapeutic agents that have a stronger impact.

Cancer cells grown in vitro were treated with chemotherapy drugs alone or with a hedgehog pathway modulator containing posaconazole. The difference in cell killing between cells treated with chemotherapy drug alone or concurrently treated with the hedgehog pathway modulator determined the degree of synergistic killing effect of the combination therapy. Cell lines resistant to vincristine; Colon cancer: Caco-2, DLD-1, HT-29, HCT-15; Small cell lung cancer: H69, H146; Neuroblastoma: Two-dimensional dose response assay with 10-, 2-, or 3-fold dose step reduction of posaconazole to demonstrate sensitization in Kelly, SK-N-SH, SH-5YSY, and Lan-5 Was performed. As the posaconazole decreased from 10, 5 or 3 micromolar to 1 micromolar to 0.1 micromolar, the concentration of chemotherapy (IC50) required to kill half of the cell amount increased, but lower than that of chemotherapy alone. (Fig. 12b, 12d, 13b, 13d, 14b, 14d, 15b, 15d, 16b and 16d). These results clearly demonstrate the broad sensitization of cancer to chemotherapy with posaconazole, and suggest that other hedgehog modulators may have similar effects. In addition, these results indicate that the efficacy of chemotherapy in primary therapies can be enhanced while minimizing toxicity and side effects using lower concentrations of sensitizers or hedgehog modulators containing posaconazole. The data provided herein acquires resistance modifications that can prevent cancer cells from responding to the proposed sensitization strategy using a hedgehog pathway modulator, including posaconazole, for cancer pre-exposed to chemotherapy drugs, Thus, while it may be more useful in primary therapy, it demonstrates that residual cancer cells may also be useful in secondary therapy that can respond to hedgehog pathway modulation.

Materials and methods (in vitro)

Cell culture: colon cancer cell line; Caco-2, DLD-1, HT-29, HCT-15 and neuroblastoma cell lines; Kelly, SK-N-SH, SH-5YSY, Lan-5 were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum and gentamicin. The small cell lung cancer cell line; H69, H146 were grown in RPMI-1640 supplemented with 10% fetal bovine serum and gentamicin. Cell-based experiments were performed in a 37 degree incubator supplemented with 5% carbon dioxide.

In vitro pharmacology: Colon cancer and neuroblastoma cell lines were plated in 96-well dishes at a density of 2000 cells per well and small cell lung cancer cells were plated in a 96-well dish at a density of 5000 cells per well. After 16 hours, the cells were treated with a 2-fold dilution of vincristine (VCR) or docetaxel (DTX). For sensitization experiments, posaconazole was added to all of the wells at a concentration of 10 micromolar, 5 micromolar, 3 micromolar, 1 micromolar or 0.1 micromolar. The cells were incubated until untreated wells reached maximal confluence, the point at which the cells did not divide rapidly.

All patents, patent publications, and other published documents mentioned herein are incorporated herein by reference in their entirety, individually and specifically as incorporated herein.

Although specific embodiments have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described aspects can be combined in any manner with one or more features of any other aspect of the invention. In addition, many variations of the present invention will become apparent to those skilled in the art upon review of this specification. Therefore, the scope of the present invention should be determined with reference to the appended claims, along with their equivalent overall scope.

Claims (25)

As a composition,
Posaconazole; And a chemotherapeutic agent. The composition of claim 1, wherein posaconazole sensitizes the tumor cells to the chemotherapeutic agent. The composition of claim 1, further comprising a pharmaceutically acceptable carrier. The method of claim 1, wherein the chemotherapeutic agent is vinca alkaloid, taxane, platinum drug, anthracycline, topoisomerase inhibitor, kinase inhibitor, mTOR inhibitor, PARP inhibitor, Bcl-2 or Bcl-. A composition that is an XL antagonist, BET bromodomain / BRD4 inhibitor, HDAC inhibitor, SGLT antagonist or statin. The composition of claim 4, wherein the chemotherapeutic agent is vinca alkaloid or taxane. The composition of claim 5, wherein the chemotherapeutic agent is vincristine or docetaxel. As a packaged drug,
Packaged medicament comprising posaconazole, chemotherapeutic agent, and instructions for using a composition for treating cancer in a mammalian subject. The chemotherapeutic agent according to claim 7, wherein the chemotherapeutic agent is vinca alkaloid, taxane, platinum drug, anthracycline, topoisomerase inhibitor, kinase inhibitor, mTOR inhibitor, PARP inhibitor, Bcl-2 or Bcl-XL antagonist, BET bromodomain. A packaged drug that is a / BRD4 inhibitor, HDAC inhibitor, SGLT antagonist or statin. The composition of claim 8, wherein the chemotherapeutic agent is vinca alkaloid or taxane. 10. The composition of claim 9, wherein said chemotherapeutic agent is vincristine or docetaxel. As a treatment method,
Administering posaconazole to a mammalian subject; And
And administering a chemotherapeutic agent to the subject, wherein the subject suffers from cancer and the posaconazole is administered in an effective amount to sensitize the tumor cells of the subject to the chemotherapeutic agent. 12. The method of claim 11, wherein the chemotherapeutic agent is administered in a lower dose than required in the absence of posaconazole. 12. The method of claim 11, wherein the posaconazole is administered at less than the maximum tolerated dose, and the chemotherapeutic agent is administered at a lower dose than is required in the absence of the posaconazole. 12. The method of claim 11, wherein the posaconazole and the chemotherapeutic agent are administered simultaneously or almost simultaneously. The method of claim 11, wherein posaconazole is administered prior to administration of the chemotherapeutic agent. The method of claim 11, wherein posaconazole is administered after administration of the chemotherapeutic agent. 12. The method of claim 11, wherein the mammalian subject has never been previously treated with the chemotherapeutic agent. The method of claim 11, wherein the posaconazole and the chemotherapeutic agent are each administered orally, intramuscularly, subcutaneously, intranasally, or intravenously. The method of claim 11, wherein the subject has adrenal cortical carcinoma, neuroblastoma, cervical cancer, colon cancer, colorectal cancer or small cell lung cancer. 12. The method of claim 11, wherein the chemotherapeutic agent is vinca alkaloid, taxane, platinum drug, anthracycline, topoisomerase inhibitor, kinase inhibitor, mTOR inhibitor, PARP inhibitor, Bcl-2 or Bcl-XL antagonist, BET bromodomain / BRD4 inhibitor, HDAC inhibitor, SGLT antagonist or statin. 12. The method of claim 11, wherein the chemotherapeutic agent is vinca alkaloid or taxane. 22. The method of claim 21, wherein the chemotherapeutic agent is vincristine or docetaxel. The method of claim 11, wherein the posaconazole is administered at a concentration of 10 micromolar or less. 24. The method of claim 23, wherein the posaconazole is administered at a concentration of 1 micromolar or less. 12. The method of claim 11, wherein said tumor cells express ABC transporter protein and / or MYC family genes.

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