CN117295522A

CN117295522A - Compositions and methods for megalin inhibitors and photodynamic therapy for the treatment of cancer

Info

Publication number: CN117295522A
Application number: CN202280034093.2A
Authority: CN
Inventors: 汪卫平; 李亚霏
Original assignee: University of Hong Kong HKU
Current assignee: University of Hong Kong HKU
Priority date: 2021-05-14
Filing date: 2022-05-12
Publication date: 2023-12-26
Also published as: WO2022237864A1

Abstract

Inhibition of megacytosis in cancer cells has been shown to enhance the anticancer efficacy of photodynamic therapy (PDT). A method for treating cancer in a subject in need thereof comprises administering to a subject having cancer an effective amount of a combination of one or more inhibitors of megacell potion and PDT. Also provided are pharmaceutical compositions comprising a combination of an inhibitor of megaloblastic actions and a photosensitizer. The method is particularly effective for treating PDT resistant cancers. An exemplary inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA). An exemplary photosensitizer is chlorin 6 (Ce 6).

Description

Compositions and methods for megalin inhibitors and photodynamic therapy for the treatment of cancer

The international patent application claims the benefit of U.S. provisional patent application No. 63/188,803 filed on day 2021, 5, 14, the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The present invention relates generally to combination therapies for enhancing anti-cancer therapies, and in particular to inhibiting megaloblastic effects to enhance photodynamic therapy for the treatment of cancer.

Background

Photodynamic therapy (PDT) is a cancer therapy that uses photosensitizers and light irradiation to generate Reactive Oxygen Species (ROS) to kill cancer cells (Castano, et al Mechanisms in photodynamic therapy: parts one-photodetectors, photochemistry and cellular localization, photodiagnosis and Photodynamic Therapy 1 (4) (2004) 279-293). It induces cell death by several mechanisms, including mitochondrial injury, cell membrane disintegration, and depletion of oxygen and nutrients (Mroz, et al Cell deathpathways in photodynamic therapy of cancer, cancers (Basel) 3 (2) (2011) 2516-39). PDT has been widely studied in various biomedical applications (van Straten, et al Oncologic Photodynamic Therapy: basic Principles, current Clinical Status and Future Directions, cancers (Basel) 9 (2) (2017)). However, although single mode PDT can induce rapid cell death, single mode PDT is still limited in clinical practice due to tumor heterogeneity. Moreover, PDT does not sufficiently inhibit tumor recurrence and metastasis (Castano, et al Photodynamic therapy and anti-tur immunity, nat. Rev. Cancer 6 (7) (2006) 535-45). Thus, the development of a combination therapy with PDT participation is an emerging trend (Xie, et al Emerging combination strategies with phototherapy in cancer nanomedicine, chem. Soc. Rev. (2020)). The combination therapy of autophagy inhibition with PDT treatment was found to improve anticancer efficacy (Niu, et al Inhibition of Autophagy Enhances Curcumin United light irradiation-induced Oxidative Stress and Tumor Growth Suppression in Human Melanoma Cells, sci Rep 6 (2016) 31383). Liu et al propose a strategy combining PDT with immune system activation, which successfully reduces tumor metastasis (Liu, et al, redox-Activated Porphyrin-Based Liposome Remote-Loaded with Indoleamine 2,3-Dioxygenase (IDO) Inhibitor for Synergistic Photoimmunotherapy through Induction of Immunogenic Cell Death and Blockage of IDO Pathway, nano Lett.19 (10) (2019) 6964-6976). However, there remains a need to enhance the mechanism of PDT treatments to selectively target cancer cells and reduce side effects and toxicity to non-cancer cells.

Megapotion is a process of extracellular fluid solute internalization that plays an important role in tumor cell survival and proliferation. Up-regulated megacell potion is an important marker for Ras mutated cancer cells and has been shown to confer therapeutic resistance associated with cancer cell anabolism. It has been demonstrated that living cells can internalize necrotic cell debris by megapinocytosis to support cell survival, particularly when the living cells are under the pressure of radiation or chemotherapy. (Jayashankar et al macropinocytossis confers resistance to therapies targeting cancer anabolism, nat Commun 11 (1) (2020) 1121). Other studies have demonstrated that activation of megaloblastic actions in therapies based on starvation or mTOR inhibition to arrest the cell cycle can provide additional nutrition from the cell matrix to help relieve these stresses. (Michloopoulou, et al Macropinocytosis Renders a Subset of Pancreatic Tumor Cells Resistant to mTOR Inhibition, cell Rep 30 (8) (2020) 2729-2742e 4).

Inhibition of megaloblastic action has proven to be a potential strategy for inhibiting tumor progression, as it can limit nutrient uptake by tumor cells (Caro-Maldonado anddying for something to eat: how cells respond to starvation, the Open Cell Signaling Journal 3 (1) (2011)). However, few examples of inhibition of megaloblastic effects are used in cancer therapy, because inhibition of megaloblastic effects is insufficient to inhibit tumor growth, as it only partially limits nutritional supplements.

Although some studies emphasize the importance of megacell effects in cancer treatment, they do not provide a practical anti-tumor strategy from a clinical point of view. In one study, the giant pinocytosis inhibitor 5- (N-ethyl-N-isopropyl) amiloride (EIPA) was used as an anticancer drug for the treatment of giant pinocytosis tumors. (Commisso, et al Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells, nature 497 (7451) (2013) 633-7). However, this is an inefficient method because it only partially limits extracellular nutritional support (file, et al Nutrient scavenging in cancer, nat. Rev. Cancer 18 (10) (2018) 619-633). Inhibition by megacytosis alone does not sufficiently inhibit tumor progression, as there are other nutrient endocytic pathways, such as phagocytosis and receptor-mediated endocytosis.

Summary of The Invention

It is an object of the present invention to provide combination therapies for cancer treatment and methods of use thereof.

It is another object to provide compositions and methods for enhancing anticancer efficacy of photodynamic therapy (PDT).

It is a further object of the present invention to provide compositions and methods for treating cancers that are resistant to conventional photodynamic therapy (PDT).

It has been determined that anticancer efficacy of photodynamic therapy (PDT) is enhanced by inhibiting or reducing nutrient uptake in cancer cells, for example by inhibiting megaloblastic action, glucose transport pathway or glutamine transport pathway. Inhibitors of megacytosis such as 5- (N-ethyl-N-isopropyl) amiloride (EIPA) have been demonstrated to enhance the efficacy of photosensitizers such as chlorin e6 (Ce 6) for PDT-mediated reduction of viability and proliferation of cancer cells. Glucose transporter inhibitors such as Bay876 have been demonstrated to enhance the efficacy of photosensitizers such as chlorin e6 (Ce 6) for PDT-mediated reduction of viability and proliferation of cancer cells. Glutamine transporter inhibitors such as O-benzyl-L-serine have been demonstrated to enhance the efficacy of photosensitizers such as chlorin e6 (Ce 6) for PDT-mediated reduction of viability and proliferation of cancer cells. Combination therapies containing a nutrient uptake inhibitor (e.g., a megalin inhibitor, a glucose transporter inhibitor, and a glutamine transporter inhibitor) and a photosensitizer may be used to increase the efficacy of PDT, or to re-sensitize cancers that develop resistance when administered alone to a dose (e.g., a maximum dose) of one or more photosensitizers. Compositions and methods for reducing and/or inhibiting nutrient uptake, such as megaloblastic effects, glucose transport pathways, or glutamine transport pathways, in combination with PDT, for treating and/or preventing the occurrence and progression of cancer are provided.

A pharmaceutical composition for enhancing the efficacy of photodynamic therapy in a subject is provided comprising an effective amount of a combination of a nutrient uptake inhibitor and a photosensitizer. The nutrient intake inhibitor is selected from the group consisting of megaloblastic inhibitors, glucose transporter inhibitors, and glutamine transporter inhibitors. Exemplary inhibitors of megapotion include 5- (N-ethyl-N-isopropyl) amiloride (EIPA), calcipolid, amiloride, phellodendrine hydrochloride, wortmannin, BKM120, ZSTK474, EHT1864, EHop-016, TBOPP, FRAX597, GCS-100, cytochalasin D, LY294002, terfenadine, itraconazole, phentermine, vincristine, auranofin, imipramine, MLS000394177, MLS000730532, and MLS000733230. In a preferred form, the inhibitor of megacell potion is 5- (N-ethyl-N-isopropyl) amiloride (EIPA), a pharmaceutically acceptable salt of EIPA, a prodrug, analog or derivative of EIPA, or a pharmaceutically acceptable salt of a prodrug, analog or derivative of EIPA. Exemplary amounts of EIPA are between about 0.1mg/kg body weight and about 1,000mg/kg body weight, inclusive. Exemplary glucose transporter inhibitors include Bay876, KL-11743, STF-31, WZB117, faentin and SW157765. In a preferred form, the glucose transporter inhibitor is Bay876. Exemplary glutamine transporter inhibitors include O-benzyl-L-serine, V-9302, and GPNA hydrochloride. In a preferred form, the glutamine transporter inhibitor is O-benzyl-L-serine. Exemplary photosensitizers include chlorin e6 (Ce 6), aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenyl chlorin (mTHPC), mono-L-aspartyl chlorin e6 (NPe 6), porphin sodium and benzoporphyrin derivatives (BPD verteporfin), photofrin, temoporphyrin, hematoporphyrin, chlorophyll a, tookad, allumera, visudyne, metvix, hexvix, cysview, laserphyrin, antrin, photolor, photosens, photrex, lumacan, cevira, visonac, BF-200ALA, amphinex and Azadipyrromethenes. In a preferred form, the photosensitizer is chlorin e6 (Ce 6). Exemplary amounts of Ce6 are between about 1mg/kg body weight and about 250mg/kg body weight.

Also provided are methods of treating cancer comprising administering to a subject having cancer an effective amount of a nutrient uptake inhibitor, including, for example, a megalin effect inhibitor, a glucose transporter inhibitor, and a glutamine transporter inhibitor, and photodynamic therapy (PDT) to the subject. Typically, performing PDT includes one or more steps of administering an effective amount of a photosensitizer to a subject, and exposing one or more cancer cells in the subject to light, wherein the light interacts with the photosensitizer to reduce cancer cell proliferation and/or viability in the subject. The combined administration of a nutrient uptake inhibitor (e.g., a megaloblastic inhibitor, a glucose transporter inhibitor, and a glutamine transporter inhibitor) and PDT is effective to reduce proliferation or viability of cancer cells in a cancer patient to a greater extent than administration of the same amount of PDT alone to the subject.

The nutrient uptake inhibitor (e.g., megalin inhibitor, glucose transporter inhibitor, or glutamine transporter inhibitor) and the photosensitizer may be part of the same mixture or administered as separate compositions. In some forms, the separate compositions are administered by the same route of administration. For example, in some forms, both the macropolysaccharide inhibitor (e.g., EIPA) and the photosensitizer (e.g., ce 6) are administered intravenously. In some forms, both the glucose transporter inhibitor (e.g., bay 876) and the photosensitizer (e.g., ce 6) are administered intravenously. In some forms, both the glutamine transporter inhibitor (e.g., O-benzyl-L-serine) and the photosensitizer (e.g., ce 6) are administered intravenously. In other forms, the separate compositions are administered by different routes of administration.

In some forms, the method comprises administering to the subject a nutrient uptake inhibitor (e.g., a megacell effect inhibitor, a glucose transporter inhibitor, or a glutamine transporter inhibitor) 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, 1, 2, 3, 4, 5, 6, 10, 12, 18, or 24 hours prior to administering PDT to the subject. In certain forms, the method comprises administering a nutrient uptake inhibitor (e.g., a megaloblastic inhibitor, a glucose transporter inhibitor, or a glutamine transporter inhibitor) to the subject concurrently with administration of the photosensitizer to the subject. In some forms, the method further comprises one or more steps of chemotherapy, surgery, or radiation therapy to the subject. In some forms, the method comprises treating the subject with one or more additional active agents. Exemplary additional active agents include one or more additional chemotherapeutic agents, such as Gemcitabine (Gemcitabine), oxaliplatin (Oxaliplatin), cisplatin (cispratin), doxorubicin, capecitabine (Capecitabine), and/or Mitoxantrone (Mitoxantrone).

The compositions and methods are particularly effective for treating cancers characterized by increased nutrient uptake, such as upregulation of one or more of megaloblastic effects, glucose transport, and glutamine transport. As used herein, "featuring …" refers to an object being characterized as having or displaying the features it is characterized as. Methods of characterizing the gene expression profile of cancer cells and/or tumor microenvironments have also been developed to assess the sensitivity of cancer cells or tumor-associated cells to treatment with a combination of a nutrient uptake inhibitor (e.g., a megacell potion inhibitor, a glucose transporter inhibitor, or a glutamine transporter inhibitor) and PDT. The method is useful for diagnosis, prognosis, patient selection, and cancer treatment. In some forms, the method is effective to treat a cancer characterized by one or more mutations in the KRAS, HRAS and NRAS genes. Exemplary cancers that may be treated include breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular tumor, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, and colorectal cancer. In certain forms, the cancer to be treated is characterized by resistance to PDT in the absence of a nutrient uptake inhibitor (e.g., a megaloblastic inhibitor, a glucose transporter inhibitor, or a glutamine transporter inhibitor). In some forms, when PDT is performed on a subject, the method administers to the subject an effective amount of one or more nutrient uptake inhibitors (e.g., megacell potion inhibitors, glucose transporter inhibitors, or glutamine transporter inhibitors) and one or more photosensitizers to reduce tumor size, reduce cancer cell viability, prevent metastasis, reduce or prevent one or more symptoms of cancer, or a combination thereof.

The following preferred embodiments are provided herein:

1. a pharmaceutical composition for enhancing the efficacy of photodynamic therapy in a subject comprising an effective amount of a combination of an inhibitor of macropolytics and a photosensitizer.

2. A pharmaceutical composition of embodiment 1, wherein the inhibitor of megalin is selected from the group consisting of 5- (N-ethyl-N-isopropyl) amiloride (EIPA), californicol, amiloride, phellodendrine hydrochloride, wortmannin, BKM120, ZSTK474, EHT1864, EHop-016, TBOPP, FRAX597, GCS-100, cytochalasin D, LY294002, terfenadine, itraconazole, phentermine, vinca alkali, auranofin, imipramine, MLS000394177, MLS000730532, and MLS000733230.

3. A pharmaceutical composition of embodiment 1 or 2, wherein the inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA), a pharmaceutically acceptable salt of EIPA, a prodrug, analog or derivative of EIPA.

4. The pharmaceutical composition of embodiment 3, wherein the dose of EIPA is between about 0.1mg/kg body weight and about 1,000mg/kg body weight, inclusive.

5. The pharmaceutical composition of any one of embodiments 1 to 4, wherein the photosensitizer is selected from chlorin e6 (Ce 6), aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenyl chlorin (mTHPC), mono-L-aspartyl chlorin e6 (NPe 6), porphin sodium and benzoporphyrin derivatives (BPD verteporfin), photofrin, temoporphyrin, hematoporphyrin, chlorophyll a, tookad, allumera, visudyne, metvix, hexvix, cysview, laserphyrin, antrin, photolor, photosens, photrex, lumacan, cevira, visonac, BF-200ALA, amphinex and Azadipyrromethenes.

6. The pharmaceutical composition of any one of embodiments 1 to 5, wherein the photosensitizer is chlorin e6 (Ce 6).

7. The pharmaceutical composition of embodiment 6, wherein the dosage of Ce6 is between about 1mg/kg body weight and about 250mg/kg body weight.

8. A method of treating cancer comprising

(a) Administering to a subject suffering from cancer an effective amount of an inhibitor of megaloblastic, and

(b) Photodynamic therapy (PDT) of a subject,

wherein administration of the megapotion inhibitor enhances the efficacy of the PDT for reducing proliferation and/or viability of cancer cells in the subject relative to photodynamic therapy (PDT) of the subject without administration of the megapotion inhibitor.

9. The method of embodiment 8, wherein performing PDT comprises administering to the subject an effective amount of a photosensitizer and exposing one or more cancer cells in the subject to light, wherein the light interacts with the photosensitizer to reduce proliferation and/or viability of the one or more cancer cells in the subject.

10. The method of embodiment 8 or 9, wherein the megacell inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, before or after the PDT is administered to the subject.

11. The method of embodiment 9, wherein the inhibitor of megalin potion is administered to the subject concurrently with the administration of the photosensitizer to the subject.

12. The method of any one of embodiments 8 to 11, further comprising surgery or radiation therapy.

13. The method of any one of embodiments 8 to 12, wherein the cancer to be treated is characterized by upregulation of megaloblastic effects.

14. The method of any one of embodiments 8 to 13, wherein the cancer to be treated is characterized by up-regulation of megaloblastic effects, and/or mutation of one or more RAS genes.

15. The method of embodiments 8 to 14, wherein the one or more genes are selected from KRAS, HRAS and NRAS.

16. The method of any of embodiments 8 to 15, wherein the cancer is selected from breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular tumor, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, and colon cancer.

17. The method of any one of embodiments 8 to 16, wherein the cancer is resistant to PDT.

18. The method of any one of embodiments 8 to 17, further comprising administering one or more additional active agents or procedures (procedures) to the subject.

19. The method of any one of embodiments 8 to 18, wherein the effective amount is effective to reduce tumor size.

20. A method of treating cancer comprising

(a) Administering to a subject having cancer an effective amount of 5- (N-ethyl-N-isopropyl) amiloride (EIPA), an

(b) Photodynamic therapy (PDT) of a subject,

wherein photodynamic therapy (PDT) of the subject comprises administering chlorin e6 (Ce 6) to the subject.

Drawings

FIG. 1 is a bar graph of quantitative megaloblastogenesis measured by flow cytometry using FITC-dextran as a marker of megaloblastogenesis. For each of the control and PDT treated A549 cells, fluorescence signals of FITC-dextran (0-100,000) are shown. FTIC signal correlates with megapotive activity. P < 0.0001).

FIGS. 2A-2B are bar graphs showing analysis of cell viability of A549 cells under co-treatment of PDT and megaloblastic inhibition. FIG. 2A shows the cell viability (0-150%) of A549 cells treated with Ce6 (0-3. Mu.M), each A549 cell alone, and A549 cells co-treated with the macropolytics inhibitor EIPA. Fig. 2B shows total intracellular glutathione (% GSH) of each of control a549 cells, a549 cells treated with EIPA alone, and a549 cells treated with EIPA and PDT. (n=4, < P <0.01, < P < 0.0001)

Figures 3A-3D are graphs showing viability of a549cells treated in combination with/without a megacell-down inhibitor after Ce 6-mediated PDT treatment. FIG. 3A is a graph showing A549cells treated with Ce6 (0-6. Mu.M) respectivelyAnd PDT-resistant A549cells (rA 549cells,)>) Line graphs of cell viability (0-150%). n=4. FIG. 3B is a graph showing that A549cells treated with EIPA (0-100. Mu.M) respectively>And rA549 cells->Line graphs of cell viability (0-150%). FIG. 3C is a bar graph showing measurement of cellular uptake (0-5 fold change) by flow cytometry in control groups (A549 cells and rA549 cells) and PDT-treated groups (A549 cells and rA549 cells), respectively. FIG. 3D is a bar graph showing measurement of SDC1 protein expression (0-2.0SDC1/GAPDH) of A549cells and rA549cells, respectively, by Western blotting. (n=3) (/ p)<0.001，****p<0.0001)。

Fig. 4 is a line graph (n=4, p < 0.0001) showing viability (0-150%) of rA549cells in different concentrations Ce6 (0-10 μm), respectively, in the control group (·), EIPA treated group (■), and in the amino acid free medium group (+d).

FIGS. 5A-5B are bar graphs showing cell viability of A549cells under PDT and glucose or glutamine transporter inhibitor co-treatment. Fig. 5A shows cell viability (0-120%) of control a549cells, a549cells treated with Ce 6-mediated PDT alone, a549cells treated with Bay876 alone, and a549cells treated with Ce 6-mediated PDT and Bay876, respectively (n=4, P < 0.0001). Fig. 5B shows the cell viability (0-120%) of control a549cells, a549cells treated with Ce 6-mediated PDT alone, a549cells treated with O-benzyl-L-serine alone, and a549cells treated with Ce 6-mediated PDT and O-benzyl-L-serine, respectively (n=4, P < 0.0001).

Detailed Description

I. Definition of the definition

The term "inhibitor of megaloblastic action (macropinocytosis inhibitor)" or "inhibitor of megaloblastic action (inhibitor of macropinocytosis)" or "antagonist of megaloblastic action" refers to a pharmaceutical substance that, when administered to a cell, can specifically prevent or reduce the course of cellular megaloblastic action in the cell. An exemplary inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA).

The term "photodynamic therapy" or "PDT" refers to cancer treatment by administering one or more photosensitizers and then exposing the cancer to light having a wavelength that causes the photosensitizers to kill cells. The wavelength of the light source needs to be suitable for exciting the photosensitizer to generate free radicals and/or reactive oxygen species in the tissue. In some forms, performing PDT includes one or more steps of administering an effective amount of a photosensitizer to the subject, and exposing one or more cancer cells in the subject to light, wherein the photosensitizer is stimulated to reduce proliferation and/or viability of the cancer cells.

The term "photosensitizing agent (photosensitizing agent)" or "photosensitizer" refers to a drug substance that when administered into a tissue or organ, under excitation of light of an appropriate wavelength, generates free radicals and/or reactive oxygen species in the tissue or organ. The generated excited state oxygen then selectively degrades the surrounding tissue. Photosensitizers for treating diseases by photodynamic therapy are known in the art. An exemplary photosensitizer for treating tumors or cancerous tumors is chlorin e6 (Ce 6).

In the context of inhibition, the term "inhibition" or "reduction" refers to a reduction or decrease in activity and amount. This may be a complete inhibition or reduction, or a partial inhibition or reduction, of the activity or amount. Inhibition or reduction can be compared to a control or standard level. Inhibition may be measured as a% value, for example from 1% up to 100%, such as 5%, 10, 25, 50, 75, 80, 85, 90, 95, 99 or 100%. For example, a composition comprising an inhibitor of megacell action may inhibit or reduce megacell action in a recipient cancer cell by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95% or 99% of its megacell action in the same cancer cell prior to treatment or not treated with the composition. In some forms, inhibition and reduction are compared according to the level of mRNA, protein, cell, or tissue in the recipient.

The term "treat" or "preventing" means to ameliorate, reduce or otherwise prevent the occurrence and progression of a disease, disorder and/or condition in an animal that may be susceptible to the disease, disorder and/or condition but has not yet been diagnosed with the disease, disorder and/or condition; inhibiting a disease, disorder, or condition, e.g., impeding its progression; and alleviating the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and/or condition. Treating a disease or disorder includes ameliorating at least one symptom of the disease or disorder, even without affecting underlying pathophysiology, e.g., treating pain in a subject by administration of an analgesic, even if such agent does not treat the cause of the pain. Desirable effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with HCC are reduced or eliminated, including, but not limited to, reducing and/or inhibiting the rate of tumor cell proliferation/growth, improving the quality of life of those suffering from the disease, reducing the dosage of other drugs required to treat the disease, slowing the progression of the disease, and/or prolonging survival of the individual.

The term "combination therapy" refers to a method of treating a disease or symptom thereof, or achieving a desired physiological change, comprising administering to an animal, such as a mammal, especially a human, an effective amount of two or more chemical agents or components to treat the disease or symptom thereof, or to produce a physiological change, wherein the chemical agents or components are administered together, e.g., as part of the same composition, or are administered separately or independently at the same time or at different times (i.e., the administration of each agent or component is separated from each other by a limited period of time).

The term "dosing regimen" refers to administration of a drug with respect to formulation, route of administration, drug dosage, dosing interval, and duration of treatment.

The terms "individual," "host," "subject," and "patient" are used interchangeably to refer to mammals, including, but not limited to, rats, apes, humans, mammalian farm animals, mammalian sports animals, and mammalian pets.

The term "effective amount" or "therapeutically effective amount" refers to an amount capable of treating, reversing progression of, preventing progression of, or preventing the occurrence of one or more symptoms of cancer in a subject to whom the formulation is administered, e.g., as compared to a matched subject that does not receive the compound. The actual effective amount of the compound may vary depending on the particular compound or combination thereof being used, the particular composition being formulated, the mode of administration, and the age, weight, condition of the individual, and severity of the symptoms or conditions being treated.

The term "pharmaceutically acceptable" or "biocompatible" refers to compositions, polymers and other materials and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, solvent, or encapsulating material, that participates in carrying or transporting any subject composition from one organ, or part of the body, to another organ, or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient. The term "pharmaceutically acceptable salts" is art-recognized and includes compounds that are relatively non-toxic, inorganic and organic acid addition salts. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid. Examples of suitable inorganic bases for salt formation include hydroxides, carbonates and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For purposes of illustration, classes of such organic bases may include mono-, di-and tri-alkylamines, such as methylamine, dimethylamine and triethylamine; monohydroxyalkylamines, dihydroxyalkylamines or trihydroxyalkylamines such as monoethanolamine, diethanolamine and triethanolamine; amino acids such as arginine and lysine; guanidine; n-methyl glucamine; meglumine; l-glutamine; n-methylpiperazine; morpholine; ethylenediamine; and N-benzylphenethylamine.

II composition

Megapotion has been shown to play an important role in the nutrient uptake of cancer cell self-repair after PDT. Based on this finding, combination therapies involving inhibition of megacytosis together with PDT have been developed to overcome PDT resistance and achieve high anticancer efficiency.

Combination therapy comprises administering to a subject in need thereof an effective amount of at least two active agents, one being a megacell potion inhibitor and the other being a photosensitizer, prior to exposing the subject to a PDT light source.

A. Inhibitors of megalopathy

Combination therapies include one or more agents that inhibit or reduce the course of a cell megalin effect.

1. Giant pinocytosis

Megapotion is a form of endocytosis involving the nonspecific uptake of extracellular substances, such as soluble molecules, nutrients, and antigens. It is a non-selective extracellular protein internalization pathway that provides amino acids and nutrients for cell survival and proliferation, and is a widely conserved process in cells that exhibit amoeboid morphology. Warren Lewis was first observed in 1931 when rat macrophages were studied and megapotion was described as the folding of some cell surface folds inward to fuse with the basement membrane to form a vesicle structure called megapotions. Megapotions are large, uncoated vesicles that vary widely in size, with their diameters between 0.2 and 5 microns. Giant pinocytophage formation is an actin-dependent process that is initiated under stimulation by growth factors (colony stimulating factor (CSF-1), epidermal Growth Factor (EGF), or Platelet Derived Growth Factor (PDGF)) (Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol. Cell biol.2011;89 (8): 836-43.[ PMID:21423264 ]). Activation of the signal-induced receptor results in an increase in actin filament polymerization at the cell membrane, which continually pushes the cell membrane forward to form a hem. While most platelike pseudopodia-induced ruffles retract into the membrane, some of them fold inward and fuse with the basal membrane to form large membrane vesicles called megapinocytosis, in which large amounts of extracellular fluid are encapsulated.

Megapotion helps to combat starvation induced cell cycle arrest, whereas tumor microenvironments are often starved (Recouvreux, et al, macropinocytosis: A Metabolic Adaptation to Nutrient Stress in Cancer, front Endocrinol (Lausanne) 8 (2017) 261;Muranen,et al, starved epithelial cells uptake extracellular matrix for survival, nat Commun 8 (2017) 13989). During amino acid starvation, megacytosis and autophagy are both up-regulated. By macropolytics, extracellular proteins and other nutrients are encapsulated within the macropolytics and then fused with lysosomes to digest the proteins and restore amino acid homeostasis. In cancer cells, particularly in Ras-mutated cancer cells, megapinocytosis activity is often up-regulated to support rapid growth of tumors (Palm, et al The Utilization of Extracellular Proteins as Nutrients Is Suppressed by mTORC1, cell162 (2) (2015) 259-270; nofal, et al mTOR Inhibition Restores Amino Acid Balance in Cells Dependent on Catabolism of Extracellular Protein, mol. Cell 67 (6) (2017) 936-946e 5). In chemotherapy, which blocks the Cell cycle based on starvation or mTOR inhibition, activation of megaloblastic offers additional nutrition from the Cell matrix to help relieve these stresses (michloopoulou, et al Macropinocytosis Renders a Subset of Pancreatic Tumor Cells Resistant to mTOR Inhibition, cell Rep 30 (8) (2020) 2729-2742e 4).

2. Inhibiting megacytosis

Inhibitors of megacell potion inhibit tumor progression by limiting nutrient uptake by tumor cells and overcome resistance to therapy associated with anabolism of cancer cells, particularly cancer cells under the pressure of radiation or chemotherapy. In some forms, the inhibitor of megacell function reduces or inhibits the function of one or more cellular receptors associated with megacell function, or reduces or inhibits endogenous ligands including a combination of amino acids, carbohydrates, and lipids. In some forms, the inhibitor of megacell function is a competitive blocker of one or more cellular receptors associated with megacell function. In other forms, the inhibitor of megacell potion indirectly inhibits one or more cellular receptors associated with megacell potion. In other forms, the inhibitor of megacell action is a non-competitive or non-competitive blocker of a cellular receptor associated with megacell action. In other forms, the inhibitor of megapotion is an antagonist that disrupts the formation of megapotoid vesicle structures.

In a preferred form, the inhibitor of megacell potion reduces or inhibits the passage of nutrients across the cell membrane into the cell. For example, inhibitors of megacell potion may reduce or inhibit the amount of intracellular nutrients.

In other forms, the mechanism or region of giant cell potion inhibitor activity is not a receptor or receptor binding site associated with giant cell potion. In some forms, the mechanism of inhibitors of megacell potion has not been established. In some forms, the inhibitor of megacell action is a calcium sensitive receptor (CaSR) antagonist. In some forms, the inhibitor of megacell potion is a sodium-hydrogen exchanger (NHE) antagonist. A preferred inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA).

5- (N-ethyl-N-isopropyl) amiloride (EIPA)

In some forms, the combination therapy comprises 5- (N-ethyl-N-isopropyl) amiloride (EIPA); CAS number 1154-25-2, experimental formula C ₁₁ H ₁₈ ClN ₇ O, molecular weight 299.76g/mol.

5- (N-ethyl-N-isopropyl) -amiloride (EIPA) is a potent inhibitor of various NHE isoforms. Sodium hydrogen exchanger (NHE) is involved in maintaining sodium and pH balance in a variety of tissues. They are also known as sodium hydrogen antiporters and members of solute carrier family 9. EIPA inhibited NHE1, NHE2, NHE3 and NHE5, with Ki values of 0.02, 0.5, 2.4 and 0.42 μm, respectively. It has low inhibiting effect on NHE4 (IC 50 is more than or equal to 10 mu M). EIPA is typically used at a concentration of 5 to 10 μm to inhibit cellular HNE activity.

EIPA is available from a number of sources, including Sigma-Aldrich catalog No. a3085.

The chemical structure of EIPA is shown below.

In some forms, the inhibitor of megacell potion is a pharmaceutically acceptable salt of EIPA, a prodrug, analog, or derivative of EIPA, or a pharmaceutically acceptable salt of a prodrug, analog, or derivative of EIPA.

b. Other inhibitors of megacytosis

In some forms, the combination therapy includes one or more inhibitors of megacell potion, which are proteins, polypeptides, or small molecule drugs.

Exemplary inhibitors of megapotion include cariipolide, amiloride, phellodendrine chloride, wortmannin, BKM120, ZSTK474, EHT1864, EHop-016, TBOPP, FRAX597, GCS-100, cytochalasin D, LY294002, terfenadine, loratadine, itraconazole, phentermine, vinca alkaloid, auranofin, imipramine, NPS-3, MLS000394177, MLS000730532 and MLS000733230.

B. Photosensitizers

Combination therapy includes one or more agents acting as photosensitizers for killing cells by photodynamic therapy (PDT). In some forms, the combination therapy includes one or more photosensitizers that are proteins, polypeptides, or small molecule drugs. Photosensitizers are molecules that absorb light (hν) and transfer energy from incident light to another molecule nearby. This light (preferably near infrared frequency, since it can penetrate the skin without acute toxicity) excites the electrons of the photosensitizer into a triplet state. Upon excitation, the photosensitizer begins to transfer energy to the adjacent ground state triplet oxygen to produce excited singlet oxygen. The resulting excited state oxygen then selectively degrades the tumor or cancerous tumor.

1. Photodynamic therapy (PDT)

Photodynamic therapy (PDT) is a form of phototherapy, which involves light and photosensitive chemicals, used in combination with molecular oxygen to induce cell death (phototoxicity). PDT applications involve three components, a photosensitizer, a light source, and tissue oxygen. The wavelength of the light source needs to be suitable for exciting the photosensitizer to generate free radicals and/or reactive oxygen species. PDT is a multi-stage process. First, in the absence of light, the photosensitizer is applied systemically or locally with negligible dark toxicity. When a sufficient amount of photosensitizer is present in the diseased tissue, the photosensitizer is activated by exposure to light for a certain period of time. The light dose provides enough energy to stimulate the photosensitizer but is insufficient to damage adjacent healthy tissue. The reactive oxygen species kills the target cells. (Josefsen, et al, photodynamic Therapy and the Development of Metal-Based photosensitizers. Metal-Based drugs.2008: 276109). Photodynamic therapy is capable of selectively killing cells through tissue oxygenation, which is mediated by light penetrating tissue at different wavelengths. In order to calculate the efficiency spectrum of PDT on human tissue, it is necessary to know the excitation spectrum of the photosensitizer of interest and the relative fluence rate as a function of tissue depth. These values can be measured and calculated by means of a precise radiation transmission algorithm. The efficiency spectrum may be determined as a function of the depth of different types of cancers (BCC). In some cases, PDT using different photosensitizers is most effective at different wavelengths, depending on the type and location of the cancer. Exemplary wavelengths of light required for excitation are 300 to 700nm, e.g., 630nm or 390nm. Those skilled in the art are familiar with reagents and procedures required for PDT. For example, in some forms, light penetration into tumors at the 630nm band is largely dependent on oxygenation of the blood. In some forms, light penetration into tumors is independent of blood oxygenation at the 390nm band.

2. Photosensitizers

Many PDT photosensitizers have heterocyclic structures similar to chlorophyll or heme in hemoglobin. After the photosensitizer captures the light energy, the light energy is transferred and converted into chemical reaction to generate singlet oxygen in the presence of molecular oxygen ¹ O ₂ ) Or super oxide (O) _2- ) And induces cell damage by direct and indirect cytotoxicity. Photosensitizers can be categorized by their chemical structure and origin. Photosensitizers for clinical PDT should have low dark toxicity but strong photocytotoxicity, good selectivity for target cells, long wavelength absorption, rapid excretion from the body, and ease of administration by a variety of routes. In general, they can be divided into three general classes, (i) porphyrin-based photosensitizers (e.g., photofrin, ALA/PpIX, BPD-MA), (ii) chlorophyll-based photosensitizers (e.g., chlorides, purprins, chlorins), and (iii) dyes (e.g., phthalocyanine)Naphthalocyanines).

A variety of photosensitizers for anticancer PDT are known in the art, and the corresponding photodynamic fz irradiation wavelength required for the anticancer activity of each photosensitizer is also known in the art. Exemplary photosensitizers include chlorin e6 (Ce 6), aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenyl chlorin (mTHPC), mono-L-aspartyl chlorin e6 (NPe 6), porphin sodium and benzoporphyrin derivatives (BPD verteporfin), photofrin, temoporphyrin, hematoporphyrin, chlorophyll a, tookad, allumera, visudyne, metvix, hexvix, cysview, laserphyrin, antrin, photolor, photosens, photrex, lumacan, cevira, visonac, BF-200ALA, amphinex and Azadipyrromethenes. In a preferred form, the photosensitizer is chlorin e6 (Ce 6).

a. Chlorin e6 (Ce 6)

In some situations, the combination therapy includes chlorin e6 (Ce 6). CAS number: 19660-77-6, experimental formula C ₃₄ H ₃₆ N ₄ O ₆ Molecular weight 596.7g/mol.

Chlorin e6 (Ce 6) is a naturally occurring chlorin commonly used as a photosensitizer. Ce6 is a second generation photosensitizer that has anti-tumor activity when used in combination with irradiation. In the mouse model of implantation type fibrosarcoma, ce6 (2.5-10 mg/kg, intravenous injection, irradiation of 50-200J/cm after various degrees of irradiation ² ) The tumor is completely eliminated. (Furukawa et al T.A phase I clinical study of photodynamic therapy for early stage lung carcinoma using ME2906 and a diode laser system. Porphyrins.7,199-206 (1998)). In a phase I clinical study for patients with early stage bronchogenic superficial squamous cell carcinoma, ce 6-containing formulations were tested with positive results (40 mg/m ² Intravenous injection, laser irradiation of 100J/cm ² ). In phase II clinical trials for early lung cancer patients, the same mode of administration resulted in complete remission in 82.9% of patients. (Kato, et al phase II clinical study of photodynamic therapy using mono-L-aspartyl chlorin e6 and diode laser for early superficial squamous cell carcinoma of the lung.Lung Cancer42 (1), 103-111 (2003) ))。

Ce6 is commercially available from several sources, including Cayman Chemical project No. 21684. In some forms, when exposed to 650nm (6J/cm ² ) Ce6 exhibits anticancer activity under photodynamic irradiation.

The chemical structure of chlorin e6 is shown below.

C. Formulations

Formulations and pharmaceutical compositions comprising inhibitors of megalopathy and photosensitizers are provided. The formulations may include the active agent in the same mixture, or in separate formulations. Thus, the pharmaceutical composition may comprise one or more inhibitors of megaloblastic action and one or more photosensitizers, or a combination of more than one inhibitor of megaloblastic action and more than one photosensitizer. In some forms, the pharmaceutical composition may include one or more additional active agents. Thus, in some forms, the pharmaceutical composition includes two, three, or more active agents. The pharmaceutical compositions may be formulated in pharmaceutical dosage units known as unit dosage forms.

The formulation typically comprises an effective amount of a mixture of an inhibitor of macropolytics and a photosensitizer, or an effective amount of a mixture of one or more inhibitors of macropolytics and one or more photosensitizers. Effective amounts of the combined active agents are discussed in detail below. It will be appreciated that in some forms, the effective amount of the photosensitizer used in combination with the macropolybdenuresis inhibitor in combination therapy is different from the effective amount of the macropolybdenuresis inhibitor or photosensitizer when administered alone to achieve the same result. For example, in some forms, the effective amount of the inhibitor of megacell or photosensitizer is that amount of the inhibitor of megacell or photosensitizer in combination therapy that is lower than the amount that would be effective if one of the agents were administered alone without the other agent. In other forms, the dosage of one agent is higher and the dosage of the other agent is lower than when one agent is administered but the other agent is not administered. In some cases, the agents are not effective when administered alone, and are only effective when administered in combination.

1. Delivery vehicle

The active agent may be administered with or without the aid of a delivery vehicle and taken up into the cells of the subject. Suitable delivery vehicles for the disclosed active agents are known in the art and may be selected to suit a particular agent. For example, in some forms, the active agent is incorporated or encapsulated in nanoparticles, microparticles, micelles, synthetic lipoprotein particles, or carbon nanotubes. For example, the composition may be incorporated into a carrier such as polymeric microparticles that provide controlled release of the active agent. In some forms, the release of the drug is controlled by diffusion of the active agent from the microparticles and/or degradation of the polymer particles by hydrolysis and/or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers that slowly dissolve in an aqueous environment and form gels, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials containing particulate drugs. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxyacids, such as Polylactide (PLA), polyglycolide (PGA), poly (lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4 HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. In some forms, the two agents are incorporated into the same particle and formulated for release at different times and/or over different periods of time. For example, in some forms, one agent is released completely from the particle before the second agent begins to release. In other forms, the first medicament is initially released and then the second medicament is released before all of the first medicament is released. In other forms, both agents are released simultaneously during the same time period or different time periods.

The active agent may be incorporated into a delivery vehicle made of a material that is insoluble or slowly soluble in aqueous solution but is capable of degrading in the gastrointestinal tract by means including enzymatic degradation, surfactants of bile acids, and/or mechanical erosion. As used herein, the term "slowly soluble in water" refers toA material that is insoluble in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, waxy substances, and mixtures thereof. Suitable fats and fatty materials include fatty alcohols (e.g., lauryl, myristyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di-and triglycerides), and hydrogenated fats. Specific examples include, but are not limited to, hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, commercially available under the nameHydrogenated oils of (2), stearic acid, cocoa butter and stearyl alcohol. Suitable waxes and waxy materials include natural or synthetic waxes, hydrocarbons and conventional waxes.

Specific examples of waxes include beeswax, sugar wax, castor wax, carnauba wax, paraffin wax, and candelilla wax. As used herein, a waxy material is defined as any material that is generally solid at room temperature and has a melting point of about 30 to 300 ℃. The release point and/or release period may vary as discussed above.

2. Pharmaceutical composition

Pharmaceutical compositions comprising an active agent with or without a delivery vehicle are provided. The pharmaceutical compositions may be administered by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration or using bioerodible inserts, and may be formulated into dosage forms suitable for each route of administration.

In certain forms, the composition is administered topically, e.g., by injection directly into the site to be treated (e.g., into a tumor). In some forms, the composition is injected or otherwise administered directly into the vasculature on the vascular tissue at the intended treatment site. In some forms, the composition is injected or otherwise directly administered into the vasculature of vascular tissue adjacent to the intended treatment site. Typically, topical administration results in an increase in the local concentration of the composition that is greater than that achievable by systemic administration. Targeting of molecules or formulations can be used to achieve more selective delivery.

a. Formulations for parenteral administration

The active agents and pharmaceutical compositions thereof may be administered in the form of aqueous solutions by parenteral injection. The formulation may also be in the form of a suspension or emulsion. Generally, pharmaceutical compositions are provided that include an effective amount of an active agent and optionally include a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and/or carrier. Such compositions include diluent sterile water, various buffer levels (e.g., tris-HCl, acetate, phosphate), pH and ionic strength buffered saline; optionally, additives such as detergents and solubilizers (e.g., 20、/>80 also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of nonaqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulation may be lyophilized and re-dissolved/resuspended immediately prior to use. The formulation may be sterilized by, for example, filtration through a bacterial-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

b. Enteral formulation

Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups and throat drops. Tablets may be prepared using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules may be prepared as hard capsule shells or soft capsule shells using techniques well known in the art, which may encapsulate liquid, solid and semi-solid fill materials. The formulations may be prepared using pharmaceutically acceptable carriers. As generally used herein, "carrier" includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers and combinations thereof. For example, inhibitors of megalin action such as amiloride and photosensitizers such as ALA may be administered enterally.

c. Sustained release formulations and other formulations

Alternatively, the sustained release formulation may be prepared using an osmotic system or by applying a semipermeable coating to the dosage form. In the latter case, the desired drug release profile may be achieved by combining low permeability and high permeability coating materials in the appropriate proportions.

The devices described above with different drug release mechanisms may be combined into a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multi-layered tablets and capsules containing tablets, beads or granules. The immediate release portion may be added to the extended release system by applying an immediate release layer on top of the slow release core using a coating or compression process, or in a multi-unit system such as a capsule containing slow release and immediate release beads.

Sustained release tablets containing hydrophilic polymers are prepared by techniques well known in the art, such as direct compression, wet granulation or dry granulation methods. Their formulations generally comprise polymers, diluents, binders, lubricants and active pharmaceutical ingredients. Common diluents include inert powdered substances such as starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, cereal flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin, and sugars such as lactose, fructose, and glucose. Natural and synthetic gums including gum arabic, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose, and waxes may also be used as binders. Lubricants in the tablet formulation are necessary to prevent sticking of the tablet and punch to the die. The lubricant is selected from the group consisting of slippery solids such as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Sustained release tablets containing wax are typically prepared using methods known in the art, such as direct blending, coagulation and aqueous dispersion. In the coagulation method, the drug is mixed with wax, spray coagulated or coagulated, and then subjected to screening processing.

Delayed release formulations may be prepared by coating a solid dosage form with a polymer film that is insoluble in the acidic environment of the stomach but soluble in the neutral environment of the small intestine.

Delayed release dosage units may be prepared, for example, by coating a drug or drug-containing composition with a selected coating material. The drug-containing composition may be, for example, a tablet for incorporation into a capsule, a tablet used as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, microparticles, or granules for incorporation into a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble and/or enzymatically degradable polymers, and may be conventional "enteric" polymers. As will be appreciated by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon. Coating materials suitable for achieving delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate phthalate, cellulose acetate trimellitate, sodium carboxymethyl cellulose; acrylic polymers and copolymers, preferably made of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and are available under the trade name (Rohm Pharma; westerstadt, germany) other methacrylic resins commercially available are formed, including +.>L30D-55 and L100-55 (dissolved at pH 5.5 and above),L-100 (dissolved at pH 6.0 and above),>s (dissolved at pH 7.0 and above, due to higher degree of esterification), and +.>NE, RL and RS (water insoluble polymers with varying degrees of permeability and swelling); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate phthalate, vinyl acetate crotonic acid copolymers, ethylene-vinyl acetate copolymers; enzymatically degradable polymers such as azo polymers, pectins, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multiple layers of coatings using different polymers may also be applied.

The person skilled in the art can easily determine the preferred coating weight of a particular coating material by evaluating the individual release profiles of tablets, beads and granules prepared with different amounts of the various coating materials. It is a combination of materials, methods and forms of application that yields the desired release profile, which can only be determined by clinical studies.

The coating composition may include conventional additives such as plasticizers, pigments, colorants, stabilizers, glidants, and the like. Plasticizers are typically present to reduce the brittleness of the coating, typically about 10wt.% to 50wt.% relative to the dry weight of the polymer. Typical examples of plasticizers include polyethylene glycol, propylene glycol, glyceryl triacetate, dimethyl phthalate, diethyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, acetyl triethyl citrate, castor oil, and acetylated monoglycerides. Preferably, a stabilizer is used to stabilize the particles in the dispersion. Typical stabilizers are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are suggested to reduce the sticking effects during film formation and drying, typically from about 25wt.% to 100wt.% of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glyceryl monostearate may also be employed. Pigments such as titanium dioxide may also be used. Small amounts of defoamers, such as silicones (e.g., simethicone), may also be added to the coating composition.

Preferably, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts and/or buffers, such as Phosphate Buffered Saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to those skilled in the art and include, but are not limited to, distilled, deionized, pure or ultrapure water, brine, phosphate Buffered Saline (PBS). Other suitable aqueous carriers include, but are not limited to, ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl parahydroxybenzoate.

In another form, solvents such as ethanol, acetone, ethyl acetate, tetrahydrofuran, diethyl ether, and propanol that are low toxicity organic (i.e., anhydrous) 3-type residual solvents may be used in the formulation. The choice of solvent is based on its ability to readily atomize the formulation. The solvent should not react deleteriously with the compound. Suitable solvents should be used to dissolve the compound or to form a suspension of the compound. The solvent should be sufficiently volatile to be able to form an aerosol of the solution or suspension. Additional solvents or atomizing agents, such as freon, may be added as needed to increase the volatility of the solution or suspension.

In one form, the composition may contain minor amounts of polymers, surfactants, or other excipients well known to those skilled in the art. Herein, "small amount" means that no excipient is present that may affect or mediate the uptake of the compound in the lung, and that the excipient is present in an amount that does not adversely affect the uptake of the compound in the lung.

Due to its hydrophobic nature, the dry lipid powder can be directly dispersed in ethanol. For lipids stored in an organic solvent, such as chloroform, a desired amount of the solution is placed in a vial and the chloroform is evaporated under a stream of nitrogen to form a dry film on the surface of the glass vial. When reconstituted with ethanol, the film swells easily. In order to sufficiently disperse the lipid molecules in the organic solvent, the suspension is subjected to ultrasonic treatment. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC jet+ atomizer (PARI Respiratory Equipment, montrey, calif.).

Dry powder formulations ("DPFs") having large particle sizes have improved flowability characteristics, such as less aggregation, easier atomization, and possibly less phagocytosis. Dry powder aerosols for inhalation therapy generally produce a dry powder aerosol having an average diameter predominantly in the range of less than 5 microns, although the preferred aerodynamic diameter range is between 1 micron and 10 microns. Large "carrier" particles (without drug) have been co-delivered with therapeutic aerosols to help achieve effective aerosolization and other possible benefits.

The polymer particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods known to those of ordinary skill in the art. Microparticles may be prepared using methods known in the art for preparing microspheres or microcapsules. Preferred methods of manufacture are spray drying and freeze drying, which require the use of a surfactant-containing solution, spraying to form droplets of the desired size, and removal of the solvent.

D. Auxiliary and additional treatments and procedures

The combination therapy may be administered to the subject in combination with one or more adjuvant therapies or procedures, or may be an adjuvant therapy to one or more primary therapies or procedures. Additional treatments or procedures may be performed simultaneously or sequentially with the combination therapy. In some forms, the additional treatment is performed between drug cycles or during drug holidays as part of a combination therapeutic dosage regimen. In a preferred form, the additional treatment is conventional treatment of cancer. For example, in some forms, the additional treatment or procedure is surgery, transplant surgery, radiation therapy, or chemotherapy. For example, in a particular form, a combination therapy such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin (pegylated liposomal Doxorubicin), capecitabine (Xeloda), mitoxantrone (Novantrone), docetaxel, or cabazitaxel is used concurrently or sequentially with a chemotherapeutic regimen. As discussed in more detail below, in some forms, the adjuvant or additional therapy is part of a combination therapy.

III methods of treatment

Inhibition of megacytosis has been shown to be useful in enhancing the anti-tumor efficacy of photodynamic therapy (PDT). PDT involves administering one or more photosensitizers to a subject and then exposing the cancer to light to excite the photosensitizers and kill cells.

Methods of treating cancer by administering one or more inhibitors of megalopathy or derivatives, analogs or prodrugs thereof, or pharmacologically active salts thereof, in combination with one or more photosensitizers, may sensitize the cancer to photodynamic therapy (PDT). Thus, the compositions and methods described herein enable photodynamic therapy to kill cancer to a significantly greater extent. Therapeutic treatment involves administering to a subject a therapeutically effective amount of an inhibitor of megacell potion, or a pharmaceutically acceptable salt thereof, and subsequently PDT the subject. In some forms, the method comprises administering to a subject in need thereof an effective amount of an inhibitor of megaloblastic action or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof in combination with one or more photosensitizers to enhance anticancer efficacy of photodynamic therapy (PDT) in the subject.

Methods of treating one or more symptoms of cancer in a subject are provided. In some forms, the method comprises administering to a subject having cancer an effective amount of an inhibitor of megacell or a derivative, analog or prodrug thereof, or a pharmacologically active salt thereof, and performing photodynamic therapy (PDT) on the subject to reduce or inhibit one or more symptoms of the cancer. In some forms, the method administers a megaloblastic inhibitor in combination with one or more photosensitizers to provide enhanced PDT. For example, the method administers a macropolysaccharide inhibitor and a photosensitizer to a subject having cancer prior to exposing the cancer to a light source for PDT. In a preferred form, the method administers the giant cell potion inhibitor 5- (N-ethyl-N-isopropyl) amiloride (EIPA) and the photosensitizer chlorin e6 (Ce 6) to a subject suffering from cancer prior to exposing the cancer to light having a wavelength between about 650nm and about 670nm, inclusive. In a preferred form, the macropolytics inhibitor and photosensitizer are used in combination to provide enhanced PDT anti-tumor efficacy compared to either agent used alone. Generally, the method reduces or inhibits proliferation and/or viability of cancer cells compared to untreated control cancer cells.

Also provided are compositions for use in methods of enhancing PDT treatment of cancer. For example, a composition comprising an inhibitor of megacell action is disclosed for use in a method of treating a subject having cancer, wherein the subject is a subject who has previously or is concurrently administered a composition comprising a photosensitizer, and wherein the response obtained after administration of PDT to the subject is greater than the response obtained by administration of the inhibitor of megacell action alone or PDT alone.

Many tumors have been shown to be resistant to PDT, with important clinical effects on tumor treatment and prognosis. In certain forms, the methods are effective in treating cancer in a subject having cancer cells that exhibit resistance to PDT in the absence of inhibition of megacytosis. In some forms, the method is effective to treat cancer in a subject having cancer cells that exhibit one or more mutations in the RAS gene relative to non-cancer cells. In some forms, the method is effective to treat cancer in a subject having cancer cells that exhibit increased megacell potion relative to non-cancer cells. Generally, the method administers an effective amount of an inhibitor of megacell potion to reduce or prevent nutrient intake by cancer cells relative to untreated cancer cells. The macropolytics inhibitor and photosensitizer may be administered to the subject systemically or locally, or coated or incorporated onto or into the device. Thus, in some forms, the inhibitor of megacell potion is administered to the subject locally or systemically, either simultaneously with, or before, or after, the photosensitizer is administered to the same subject locally or systemically.

A. Patients with selection of combination therapy

In some forms, the method comprises one or more steps of identifying a subject in need of cancer therapy. Thus, methods of selecting patients for megaloblastic inhibition in combination with PDT treatment are provided. Typically, the subject to be treated is a subject having one or more solid tumors. Solid tumors are abnormal masses of tissue, usually without cysts or areas of fluid. Solid tumors may be benign (not cancer) or malignant (cancer). Examples of solid tumors are sarcomas, carcinomas and lymphomas.

In some forms, the method characterizes a tumor and/or characterizes a tumor microenvironment in a subject with a cancer to assess the extent to which tumor cells and/or tumor-infiltrating cells or tumor-associated cells express genes related to sensitivity of combination therapy combining megaloblastic inhibition with PDT. For example, in some forms, tumor cells and/or tumor infiltrating cells or tumor-associated cells that are sensitive to the more than additive effects of combination therapies that inhibit megalin potion in combination with PDT, express genes associated with enhanced nutrient uptake, and/or exhibit resistance to PDT prior to combination therapy. Thus, in a particular form, the method comprises one or more steps for determining whether a tumor cell expresses one or more genes and/or signaling pathways involved in enhanced nutrient uptake, increased megaloblastic function, and/or resistance to PDT. In other forms, the method comprises determining whether the tumor cell comprises one or more mutations in one or more RAS genes, such as HRAS mutations, NRAS mutations, and KRAS mutations. Suitable detection methods are known in the art. For example, in a specific form, the method comprises the step of contacting tumor cells with a molecule that immunospecifically or physiologically specifically binds to a protein involved in or associated with enhanced megapotion, or examining RNA expression of a gene involved in megapotion using qPCR, microarray methods, or RNA sequencing.

Also disclosed are methods for assessing the anticancer efficacy of a megaloblastic inhibition in combination with PDT in a subject in need thereof. In some forms, the method comprises the step of assessing the tumor size and viability of the subject before and after administering the combination therapy to the subject. For example, in some forms, tumor samples from cancer patients are characterized before and after treatment with megaloblastic inhibition in combination with PDT to monitor changes in tumor size and viability as well as phenotypic and/or genetic changes within the tumor microenvironment.

In some forms, the subject is a subject with cancer and has been currently or previously administered PDT treatment for cancer, and wherein the anti-cancer response achieved after administration of the macropolysaccharide inhibitor in combination with PDT is greater than the response achieved by PDT alone. In other forms, the subject has previously been administered a megacell inhibitor, but not in combination with PDT, and the anti-cancer response achieved after administration of the megacell inhibitor in combination with PDT is greater than that achieved by administration of the megacell inhibitor alone. In some forms, the cancer may already be resistant to PDT when administered without combination with a megaloblastic inhibitor. Thus, in some forms, a population of subjects being treated is defined as a population in which the cancer being treated is resistant or insensitive to the administration of PDT or a megaloblastic inhibitor alone.

B. Methods of administration and dosage regimens

Combination therapies and treatment regimens typically include treatment of cancer, including administration of an effective amount of a megaloblastic inhibitor in combination with PDT to an animal, such as a mammal, especially a human, to treat the cancer, wherein the megaloblastic inhibitor and photosensitizer are administered together, e.g., as part of the same composition, or separately and independently at the same time or at different times (i.e., administration of the megaloblastic inhibitor and photosensitizer/PDT are separated from each other by a time). Thus, the term "combination" or "combined" is used to refer to the concomitant, simultaneous or sequential administration of a macropolytics inhibitor and photosensitizer/PDT. The macropolytics inhibitor and photosensitizer may be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., into the same subject through separate intravenous infusion lines; one agent administered orally and the other administered by infusion or injection, etc.), or sequentially (e.g., one agent administered first followed by the second). PDT is accomplished by subsequently exposing the cancer to light having a wavelength that induces the photosensitizer to kill the cells.

The amount of the inhibitor of megacell potion present in the pharmaceutical dosage unit or otherwise administered to the subject may be an amount that reduces proliferation, viability, or a combination thereof of cancer cells when administered in combination with PDT. Likewise, the amount of photosensitizer present in a pharmaceutical dosage unit or otherwise administered to a subject may be an amount effective to reduce proliferation, viability, or a combination thereof of cancer cells by PDT when administered in combination with a megacell potion inhibitor. Thus, in some forms, the amount of active agent is effective to reduce or inhibit proliferation and/or viability of the cancer cells as compared to untreated control cancer cells.

In some forms, the amount of active agent is effective to reduce, slow or prevent tumor progression, or reduce tumor burden of one or more tumors in the recipient, or a combination thereof. In some forms, the amount of active agent is effective to alter a measurable biochemical or physiological marker. In a preferred form, administration of the megakaryocyte potentiation inhibitor in combination with PDT achieves better results than administration of the megacell potentiation inhibitor and PDT alone or independently. For example, in some forms, the result obtained by combining is a partial or complete sum of the results obtained from the individual components alone. In the most preferred form, the result obtained by combining is more than the sum of the results obtained from the individual components alone. In some forms, the effective amount of one or both agents in combination is less than the effective amount of each drug when administered alone. In some forms, the amount of one or both agents when used in combination therapy is sub-therapeutic when used alone.

The effect of the combination therapy or its individual agents may depend on the cancer to be treated or its progression. For example, as shown in the examples below, agents such as the photosensitizer chlorin e6 (Ce 6) can be used for one-wire or two-wire PDT to treat cancer. However, over time, cancer may develop resistance to Ce 6-based PDT. The cancer is then treated with a Ce 6-based PDT combined megacytosis inhibitor, such as 5- (N-ethyl-N-isopropyl) amiloride (EIPA), making the cancer "re-sensitized" to PDT for Ce6 treatment. Thus, in some forms, the anti-tumor effect of a giant pinocytosis inhibitor in combination with PDT may be compared to the effect of PDT alone or a giant pinocytosis inhibitor alone on cancer.

The treatment regimen of the combination therapy may include one or more administrations of an inhibitor of megalin potion, and one or more administrations of PDT. In some forms, the inhibitor of megacell potion may be administered simultaneously with the photosensitizer. When the inhibitor of megaloblastic actions and the photosensitizer are administered simultaneously, the inhibitor of megaloblastic actions and the photosensitizer may be in the same pharmaceutical composition. In other forms, the inhibitor of megacell and the photosensitizer are administered sequentially, e.g., in two or more different pharmaceutical compositions. In some forms, the inhibitor of megalin is administered prior to the first administration of the photosensitizer. In other forms, the photosensitizer is administered prior to the first administration of the megaloblastic inhibitor. Thus, in some forms, the inhibitor of megacell and the photosensitizer are administered to the subject on the same day. In other forms, the inhibitor of megacell and the photosensitizer are administered to the subject on different days. For example, in some forms, the photosensitizer is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 30 hours or days before or after administration of the macropolytics inhibitor. In other forms, the inhibitor of megacytosis is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 30 hours or days before or after administration of the photosensitizer. In certain forms, the additive or more additive effects of the combined administration of one or more macropolytics inhibitor and one or more photosensitizers are apparent when the tumor is exposed to light having a wavelength that induces the photosensitizers to kill cells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more than 1 week after administration.

The dosing schedule or cycle of the agents may overlap entirely or partially, or may be continuous. For example, in some forms, administration of all such inhibitors of megacytosis occurs prior to administration of one or more PDT cycles (i.e., administration of a photosensitizer followed by exposure to light for PDT). Alternatively, the administration of one or more doses of the megakaryocytosis inhibitor may be staggered in time from the administration of the photosensitizer to form a uniform or non-uniform course of treatment, whereby one or more doses of the megakaryocytosis inhibitor are administered, followed by one or more PDT cycles. In some forms, one or more doses of an inhibitor of megaloblastic action are administered after one or more PDT cycles; subsequent one or more PDT; etc., all according to any schedule selected or desired by the researcher or clinician performing the treatment.

An effective amount of one or both of the inhibitor of megaloblastic effect and the photosensitizer may be administered as a single unit dose (e.g., as a dosage unit), or as a sub-therapeutic dose administered over a limited time interval. Such unit doses may be administered daily for a limited period of time, for example up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days, all of which are particularly contemplated.

Inhibition of cancer growth capacity may be measured using biochemical assays, for example measuring the decline of one or more biomarkers of cancer in blood, or by morphometric analysis, for example by Computed Tomography (CT), magnetic Resonance Imaging (MRI) or ultrasound. Thus, in some forms, the method comprises the step of measuring the concentration of one or more biomarkers in the blood of the recipient. The measurement may be performed before and after administration of the combination of the megalin potion inhibitor and PDT to the subject.

In some forms, the method treats cancer that regains growth capacity after prior treatment. For example, in certain forms, cancer has acquired resistance to PDT. Cancers that regain growth capacity may be an increase in tumor growth, the appearance of symptoms, reappearance or exacerbation, or a new metastatic site.

In a further form, the method is for prophylactic use, i.e., delaying onset, alleviating, eradicating, or delaying exacerbation of a sign or symptom after onset, and preventing recurrence. For prophylactic use, a therapeutically effective amount of an inhibitor of megacell action is administered to a subject along with PDT during early onset (e.g., at the time of initial signs and symptoms of cancer) or after the cancer has been established to progress. Prophylactic administration can be used, for example, in chemopreventive treatment of subjects presenting with pre-cancerous lesions and subjects diagnosed with early stage malignancy.

C. Cancer to be treated

Methods of administering megaloblastic inhibitors prior to or in combination with PDT are effective in enhancing the anti-tumor efficacy of PDT for the treatment of various types of cancer. In some forms, the method treats cancer characterized by upregulation of expression of one or more genes involved in megaloblastic or PDT resistance. In some forms, the method treats cancer characterized by mutations in one or more RAS genes. Cancers characterized by mutations in one or more of the RAS genes include pancreatic cancer, lung cancer, and colorectal cancer. In some forms, the cancer is characterized by having one or more KRAS mutations, HRAS mutations, NRAS mutations, EGFR mutations, ALK mutations, RB1 mutations, HIF mutations, KEAP mutations, NRF mutations, or other metabolism-related mutations, or a combination thereof.

In preferred forms, the compositions and methods are effective for treating one or more symptoms of skin cancer, lung cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, breast cancer, prostate cancer, colon and rectal cancer, bladder cancer, and the like. In a further form, the tumor is a focal lymphoma or follicular lymphoma.

Exemplary tumor cells treated according to the described methods include, but are not limited to, tumor cells of cancer, including leukemia, including but not limited to acute leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, Such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia and myelodysplastic syndrome, chronic leukemia, such as but not limited to chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, hodgkin's disease, non-hodgkin's disease; multiple myeloma such as, but not limited to, smoky multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasmacytic leukemia, isolated plasmacytoma and extramedullary plasmacytoma;macroglobulinemia; monoclonal gammaglobinopathy of unknown significance; benign monoclonal gammaglobinopathy; heavy chain disease; osteo and connective tissue sarcomas such as, but not limited to, osteosarcoma (bone sarcomas), osteosarcoma (osteosacoma), chondrosarcoma, ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglioma, auditory neuroma, craniopharyngeal tube tumor, medulloblastoma, meningioma, pineal tumor, primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, paget's disease, and inflammatory breast cancer; adrenal cancer including, but not limited to, pheochromocytoma and adrenal cortical cancer; thyroid cancer, such as, but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and thyroid undifferentiated cancer; pancreatic cancers, including but not limited to insulinomas, gastrinomas, glucagon tumors, vasoactive intestinal peptide tumors, somatostatin secreting tumors, and carcinoid or islet cell tumors; pituitary cancers, including but not limited to cushing's disease, prolactin Secretory tumors, acromegaly and diabetes insipidus; eye cancers, including but not limited to ocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and paget's disease; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancers, including but not limited to endometrial cancer and uterine sarcoma; ovarian cancers, including but not limited to ovarian epithelial cancers, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancers, including but not limited to squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, myxoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, warty carcinoma, and oat cell (small cell) carcinoma; gastric cancer including, but not limited to, adenocarcinoma, mycoid carcinoma (polypoid carcinoma), ulcerative carcinoma, superficial diffuse carcinoma, diffuse carcinoma, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinomatous sarcoma; colon cancer; rectal cancer; liver cancer including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer including but not limited to adenocarcinoma; bile duct cancers, including but not limited to papillary, nodular and diffuse bile duct cancers; lung cancer includes, but is not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer, including but not limited to germinal tumors, seminomas, anaplastic cancers, classical (classical) cancers, seminomas, non-seminomas, embryonic cancers, teratoma cancers, choriocarcinomas (yolk sac tumors), prostate cancers, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancers, including but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancers, including but not limited to squamous cell carcinoma and wart cell carcinoma; skin cancers, including but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial diffuse melanoma, nodular melanoma, freckle-like malignant melanoma, acro-freckle-like melanoma; renal cancers, including but not limited to renal cell carcinoma, adenocarcinoma, adrenoid tumor, fibrosarcoma, transitional cell Cell carcinoma (renal pelvis and/or ureter); nephroblastoma; bladder cancer includes, but is not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinomatosis. Cancers that may be prevented, treated, or otherwise reduced by the compositions include myxosarcoma, osteogenic sarcoma, endothelial sarcoma, lymphatic endothelial sarcoma, mesothelioma, synovioma, angioblastoma, epithelial cancer, cystic carcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, and gastric cancer (for reviews of such diseases, see fisheman et al, 1985,Medicine,2d Ed, j.b. lippincott co., philadelphia and Murphy et al, 1997,Informed Decisions:The Complete Book of Cancer Diagnosis,Treatment,and Recovery,Viking Penguin,Penguin Books U.S.A, inc., united States of America).

IV. kit

Medical kits are also disclosed. The medical kit may include a dosage supply of, for example, a macropolytics inhibitor, a photosensitizer, or a combination thereof, alone or together in the same mixture. The active agents may be provided alone (e.g., lyophilized), or in the form of a pharmaceutical composition. The active agent may be in unit dosage form or in stock solution which should be diluted prior to administration. In some forms, the kit includes a supply of pharmaceutically acceptable carrier. The kit may also include a device, such as a syringe, for administering the active agent or composition. The kit may include printed instructions for administering the compounds in the above-described methods.

The disclosed compositions and methods may be further understood by the following numbered paragraphs.

2. The pharmaceutical composition of paragraph 1, wherein the inhibitor of megalin is selected from the group consisting of 5- (N-ethyl-N-isopropyl) amiloride (EIPA), calcipol, amiloride, phellodendrine hydrochloride, wortmannin, BKM120, ZSTK474, EHT1864, EHop-016, TBOPP, FRAX597, GCS-100, cytochalasin D, LY294002, terfenadine, itraconazole, phentermine, vinca-alkali, auranofin, imipramine, MLS000394177, MLS000730532, and MLS000733230.

3. The pharmaceutical composition of paragraph 1 or 2, wherein the inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA), a pharmaceutically acceptable salt of EIPA, a prodrug, analog or derivative of EIPA.

4. The pharmaceutical composition of paragraph 3, wherein the dose of EIPA is between about 0.1mg/kg body weight and about 1,000mg/kg body weight, inclusive.

5. The pharmaceutical composition of any one of paragraphs 1-4, wherein the photosensitizer is selected from chlorin e6 (Ce 6), aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenyl chlorin (mTHPC), mono-L-aspartyl chlorin e6 (NPe 6), porphin sodium and benzoporphyrin derivatives (BPD verteporfin), photofrin, temoporphyrin, hematoporphyrin, chlorophyll a, tookad, allumera, visudyne, metvix, hexvix, cysview, laserphyrin, antrin, photolor, photosens, photrex, lumacan, cevira, visonac, BF-200ALA, amphinex and Azadipyrromethenes.

6. The pharmaceutical composition of any one of paragraphs 1-5 wherein the photosensitizer is chlorin e6 (Ce 6).

7. The pharmaceutical composition of paragraph 6, wherein the dosage of Ce6 is between about 1mg/kg body weight and about 250mg/kg body weight.

8. A method of treating cancer comprising

(b) Photodynamic therapy (PDT) of a subject,

wherein administration of the megapotion inhibitor enhances the efficacy of the PDT to reduce proliferation and/or viability of cancer cells in the subject relative to photodynamic therapy (PDT) of the subject without administration of the megapotion inhibitor.

9. The method of paragraph 8, wherein performing PDT comprises administering to the subject an effective amount of a photosensitizer and exposing one or more cancer cells in the subject to light, wherein the light interacts with the photosensitizer to reduce proliferation and/or viability of the one or more cancer cells in the subject.

10. The method of paragraph 8 or 9, wherein the megacell inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, before or after the PDT is administered to the subject.

11. The method of paragraph 9, wherein the inhibitor of megalin potion is administered to the subject concurrently with the administration of the photosensitizer to the subject.

12. The method of any one of paragraphs 8-11, further comprising surgery or radiation therapy.

13. The method of any one of paragraphs 8-12, wherein the cancer to be treated is characterized by upregulation of megaloblastic effects.

14. The method of any one of paragraphs 8-13, wherein the cancer to be treated is characterized by upregulation of megaloblastic function, and/or mutation of one or more RAS genes.

15. The method of any one of paragraphs 8-14, wherein the one or more genes are selected from KRAS, HRAS and NRAS.

16. The method of any of paragraphs 8-15, wherein the cancer is breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular tumor, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, and colon cancer.

17. The method of any one of paragraphs 8-16, wherein the cancer is resistant to PDT.

18. The method of any of paragraphs 8-17, further comprising administering one or more additional active agents or procedures to the subject.

19. The method of any one of paragraphs 8-18, wherein the effective amount is effective to reduce tumor size.

20. A method of treating cancer comprising

(b) Photodynamic therapy (PDT) of a subject,

The disclosed compositions and methods may be further understood by the following non-limiting examples.

Examples

Example 1: up-regulation of megacytosis following PDT treatment

It was demonstrated that in Ras mutated megapotion cancer cell line a549, megapotion activity was elevated to reestablish damaged organelles and restore nutritional balance after PDT treatment.

FITC-dextran was used as a fluorescent label to indicate megaloblastic activity. As shown in fig. 1, confocal microscopy images and results of flow cytometry analysis showed that the uptake of FITC-dextran in cells was higher than control cells after PDT treatment with chlorin 6 (Ce 6), indicating that macropolytics were potentially up-regulated for cell reconstitution after PDT treatment.

Example 2: inhibition of megacytosis to increase PDT efficiency

The megacell viability of the megacell potion inhibitor 5- (N-ethyl-N-isopropyl) amiloride (EIPA) co-treated with Ce 6-mediated PDT was evaluated.

As shown in fig. 2A, a549 was more sensitive to PDT after inhibition of megapotion, demonstrating synergistic effects of megapotion inhibition and PDT. Intracellular antioxidants, glutathione (GSH), which plays a key role in the elimination of ROS, were quantified using a commercial kit (Abcam, ab 138881). EIPA treatment induced a significant decrease in intracellular GSH concentration (fig. 2B), presenting a redox-deregulated cellular state. Giant pinocytosis activity and intracellular ROS were examined with confocal microscopy. Lower tetramethylrhodamine-dextran (TMR-dextran) signals were observed in EIPA treated cells, indicating successful inhibition of megacytosis (data not shown). At the same time, a significant increase in ROS levels was observed in these cells, which may be a direct cause of cell death. Thus, inhibition of megaloblastic action reduces GSH content and increases ROS levels in cancer cells, resulting in cells that are sensitive to PDT treatment.

Example 3: improved megaloblastic activity and high EIPA sensitivity in PDT resistant cell lines.

After repeated single mode PDT treatments, the cells may acquire PDT resistance (a.casas, g.di Venosa, T.Hasan, A.Batlle, mechanisms of resistance to photodynamic therapy, curr.med.chem.18 (16) (2011) 2486-2515.).

After 20 cycles of PDT treatment, PDT resistant a549 cell lines (rA 549) were screened to assess whether combined treatment with megapotion inhibition and PDT could overcome or reduce PDT resistance. rA549 cells exhibited high tolerance to PDT treatment (FIG. 3A) and had higher sensitivity to EIPA treatment than A549 cells (FIG. 3B). Macropolysaccharide activity was also measured by flow cytometry (fig. 3C), and expression of protein multi-ligand glycan 1 (SDC 1), a mediator of macropolysaccharide formation, was also shown by western blotting (fig. 3D). Up-regulation of megaloblastic activity and SDC1 protein expression in rA549 cells was observed, indicating that PDT resistance mechanisms are involved in improvement of megaloblastic activity. The high sensitivity of rA549 to EIPA treatment may also be due to an upregulation of megaloblastic activity, which provides the possibility to utilize megaloblastic inhibition to kill PDT resistant cell lines.

Example 4: inhibition of megacytosis leads to an increase in PDT efficiency in PDT resistant cell lines.

Viability of rA549 cells was measured after three different treatments: single mode PDT, EIPA and PDT, and amino acid starvation medium PDT.

As shown in fig. 4, the latter two groups showed significantly reduced viability compared to the single mode PDT treatment group. As with amino acid starvation, EIPA reduces extracellular nutrient supply for cancer cell reconstitution following PDT treatment, providing synergy for higher antitumor efficacy.

Example 5 inhibition of glucose or Glutamine transport may increase PDT efficiency

In addition, the correlation between PDT and other endocytosis or nutrient transport pathways, including glucose transport and glutamine transport pathways, has been further investigated. It is speculated that these pathways may also play an important role in reconstructing damaged organelles and restoring nutritional balance after PDT to aid cancer cell survival. Inhibiting these pathways may also provide strategies to sensitize cancer cells to PDT.

To verify the effect of inhibition of glucose transport pathway on PDT efficiency, cell viability of a549 cells was measured after four different treatments: control, ce6 mediated PDT, glucose transporter inhibitor Bay876, and Ce6 mediated PDT in combination with Bay 876.

To verify the effect of inhibition of glutamine transport pathway on PDT efficiency, cell viability of a549 cells was measured after four different treatments: control, ce 6-mediated PDT, glutamine transporter inhibitor O-benzyl-L-serine, and Ce 6-mediated PDT in combination with O-benzyl-L-serine.

The results are shown in FIGS. 5A and 5B. The results indicate that the glucose transporter inhibitor Bay876 sensitizes a549 cells to PDT treatment (fig. 5A), and the glutamine transporter inhibitor O-benzyl-L-serine sensitizes a549 cells to PDT treatment (fig. 5B), indicating that blocking the glucose or glutamine transport pathway increases PDT treatment efficiency.

In summary, studies of the relationship between nutrient uptake and PDT resistance reveal the mechanism of PDT resistance and provide a synergistic strategy involving inhibition of nutrient uptake, including inhibition of megaloblastic action, inhibition of glucose transport, and inhibition of glutamine transport in combination with PDT, to overcome PDT resistance and have high anticancer efficacy.

Claims

1. A pharmaceutical composition for enhancing the efficacy of photodynamic therapy in a subject comprising an effective amount of a combination of a nutrient uptake inhibitor and a photosensitizer.

2. The pharmaceutical composition of claim 1, wherein the nutrient uptake inhibitor is selected from the group consisting of a megaloblastic inhibitor, a glucose transporter inhibitor, and a glutamine transporter inhibitor.

3. The pharmaceutical composition of claim 2, wherein the inhibitor of megacell potion is selected from the group consisting of 5- (N-ethyl-N-isopropyl) amiloride (EIPA), casipolide, amiloride, phellodendrine hydrochloride, wortmannin, BKM120, ZSTK474, EHT1864, EHop-016, TBOPP, FRAX597, GCS-100, cytochalasin D, LY294002, terfenadine, itraconazole, phenoxybenzamine, vinca alkaloid, auranofin, imipramine, MLS000394177, MLS000730532, and MLS000733230.

4. The pharmaceutical composition of claim 3, wherein the inhibitor of megalin is 5- (N-ethyl-N-isopropyl) amiloride (EIPA), a pharmaceutically acceptable salt of EIPA, a prodrug, analog or derivative of EIPA.

5. The pharmaceutical composition of claim 4, wherein the dose of EIPA is between about 0.1mg/kg body weight and about 1,000mg/kg body weight, inclusive.

6. The pharmaceutical composition of claim 2, wherein the glucose transporter inhibitor is selected from the group consisting of Bay876, KL-11743, STF-31, WZB117, fasentin, and SW157765.

7. The pharmaceutical composition of claim 6, wherein the glucose transporter inhibitor is Bay876.

8. The pharmaceutical composition of claim 2, wherein the glutamine transporter inhibitor is selected from the group consisting of O-benzyl-L-serine, V-9302, and GPNA hydrochloride.

9. The pharmaceutical composition of claim 8, wherein the glutamine transporter inhibitor is O-benzyl-L-serine.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the photosensitizer is selected from chlorin e6 (Ce 6), aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenyl chlorin (mTHPC), mono-L-aspartyl chlorin e6 (NPe 6), porphin sodium and benzoporphyrin derivatives (BPD verteporfin), photofrin, temopofen, hematoporphyrin, chlorophyll a, tookad, allumera, visudyne, metvix, hexvix, cysview, laserphyrin, antrin, photolor, photosens, photrex, lumacan, cevira, visonac, BF-200ALA, amphinex and Azadipyrromethenes.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the photosensitizer is chlorin e6 (Ce 6).

12. The pharmaceutical composition of claim 11, wherein the dosage of Ce6 is between about 1mg/kg body weight and about 250mg/kg body weight.

13. A method of treating cancer comprising

(a) Administering an effective amount of a nutrient uptake inhibitor to a subject having cancer, an

(b) Photodynamic therapy (PDT) of the subject,

wherein administration of the nutrient uptake inhibitor enhances the efficacy of PDT for reducing proliferation and/or viability of cancer cells in the subject relative to photodynamic therapy (PDT) of the subject without administration of the nutrient uptake inhibitor.

14. The method of claim 13, wherein performing PDT comprises administering an effective amount of a photosensitizer to the subject and exposing one or more cancer cells in the subject to light, wherein the light interacts with the photosensitizer to reduce proliferation and/or viability of the one or more cancer cells in the subject.

15. The method of claim 13 or 14, wherein the nutrient uptake inhibitor is selected from the group consisting of a megaloblastic inhibitor, a glucose transporter inhibitor, and a glutamine transporter inhibitor.

16. The method of any one of claims 13 to 15, wherein the nutrient uptake inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, before or after the PDT is administered to the subject.

17. The method of claim 14, wherein the nutritional intake inhibitor is administered to the subject concurrently with administration of the photosensitizer to the subject.

18. The method of any one of claims 13 to 17, further comprising surgery or radiation therapy.

19. The method of any one of claims 13 to 18, wherein the cancer to be treated is characterized by increased nutrient uptake.

20. The method of claim 19, wherein the cancer to be treated is characterized by upregulation of one or more of megaloblastic, glucose transport, and glutamine transport.

21. The method of any one of claims 13 to 20, wherein the cancer to be treated is characterized by up-regulation of megaloblastic effects, and/or mutation of one or more RAS genes.

22. The method of claim 21, wherein the one or more genes are selected from KRAS, HRAS, and NRAS.

23. The method of any one of claims 13 to 22, wherein the cancer is selected from breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular tumor, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, and colon cancer.

24. The method of any one of claims 13 to 23, wherein the cancer is resistant to PDT.

25. The method of any one of claims 13 to 24, further comprising administering to the subject one or more additional active agents or procedures (procedures).

26. The method of any one of claims 13 to 25, wherein the effective amount is effective to reduce tumor size.

27. A method of treating cancer comprising

(b) Photodynamic therapy (PDT) of the subject,

wherein performing photodynamic therapy (PDT) on the subject comprises administering chlorin e6 (Ce 6) to the subject.

28. A method of treating cancer comprising

(a) Administering an effective amount of Bay876 to a subject having cancer, an

(b) Photodynamic therapy (PDT) of the subject,

29. A method of treating cancer comprising

(a) Administering to a subject suffering from cancer an effective amount of O-benzyl-L-serine, an

(b) Photodynamic therapy (PDT) of the subject,