All-trans retinoic acid liposome and application thereof in tumor treatment

Abstract

Described herein is a method of treating cancer by administering an effective concentration of liposomes of all-transretinoic acid.

Description

Background Art

[0001] All trans retinoic acid (ATRA) is a metabolite of vitamin A in the body. ATRA is used as a drug to treat acne, and is also an important drug used in the clinic to treat acute promyelocytic leukemia (APL). ATRA plays a gene regulation function by binding to specific nuclear receptors (RARs, RXR and ROR) in cells. In addition, ATRA can also promote the differentiation of APL cells by promoting the degradation of PML / RAR fusion protein in acute promyelocytic leukemia, thereby achieving the effect of inducing cell differentiation and treating and alleviating the disease.

[0002] However, in clinical treatment, the application of ATRA is limited by the following: 1. ATRA has extremely low water solubility; 2. ATRA has a short plasma half-life. In order to achieve the desired therapeutic effect, a certain level of drug concentration needs to be maintained in the target organ. Therefore, it is particularly important to choose a suitable drug delivery method for ATRA.

[0003] Liposomes have been used as nano drug delivery carriers for more than 30 years, and a variety of anti-tumor drugs with liposomes as carriers have been widely developed and used in clinical practice. Among them, doxorubicin liposomes are a typical liposome technology drug. As a drug delivery system, liposome drug carriers can effectively change the original biodistribution characteristics of drugs, reduce the systemic toxicity of drugs, and achieve long-term circulation and targeted delivery of drugs. Ultimately, it will effectively increase the drug concentration in the target tissue.

[0004] Previously, almost all liposome-based ATRA liposome drugs encapsulated ATRA into the phospholipid bilayer of liposomes. However, due to the physicochemical properties of ATRA, this drug encapsulation method was not ideal for the drug loading of ATRA and its stability in the body. Technical Summary

[0005] The present disclosure relates to a method for treating cancer by administering a drug-effective concentration of all-trans retinoic acid to a cancer subject. The administered retinoic acid is encapsulated in a liposome structure, and the liposome includes an intraliposome aqueous phase environment containing all-trans retinoic acid and calcium acetate, and the pH range is between 7 and 10. The phospholipid components of the liposome mainly include hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000). In certain embodiments, at least a portion of the all-trans retinoic acid in the aqueous phase within the liposome is in the form of crystals, and the phase transition temperature and / or melting point of the all-trans retinoic acid is lower than that of solid all-trans retinoic acid. This patent provides important evidence for the existence of a crystal structure, and can be used to further deduce more possible situations. Preferably, the pH range of the aqueous phase within the liposome should be about 8.5 to about 9.5. Preferably, the pH within the aqueous phase within the liposome should be about 9.

[0006] In certain embodiments, the cancer is brain cancer. In certain embodiments, the brain cancer is a glioma. In certain embodiments, the glioma is a glioblastoma or an oligodendroglioma. In certain embodiments, the glioma is a recurrent or progressive glioma. In certain embodiments, the recurrent or progressive glioma is a recurrent or progressive glioblastoma.

[0007] The invention discloses a method for treating cancer, comprising administering an all-trans retinoic acid liposome preparation with a certain drug effective concentration to a patient, wherein the liposome comprises an inner aqueous phase containing all-trans retinoic acid and a calcium acetate solution, and having a pH of about 7 to about 10; wherein the lipid components of the liposome include hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), and at least a portion of the all-trans retinoic acid in the aqueous phase within the liposome is crystalline.

[0008] In certain embodiments, the average particle size of the liposomes is from about 50 nanometers to about 100 nanometers.

[0009] In certain embodiments, the liposomes have an average particle size of about 70 nanometers.

[0010] In certain embodiments, the pH of the aqueous solution of calcium acetate is about 9.

[0011] In certain embodiments, the administration method is intravenous injection.

[0012] In certain embodiments, the intravenous infusion time is about 60 to about 90 minutes.

[0013] In certain embodiments, the dose of ATRA is increased during treatment from 45 mg ATRA per square meter of patient body surface area to 160 mg ATRA per square meter of patient body surface area.

[0014] In certain embodiments, the subject receives an initial dose of about 45 mg of all-trans retinoic acid per square meter of body surface area.

[0015] In certain embodiments, the subject receives a subsequent dose of about 90 mg of all-trans retinoic acid per square meter of body surface area.

[0016] In certain embodiments, the subject receives a subsequent dose of about 120 mg of all-trans retinoic acid per square meter of body surface area.

[0017] In certain embodiments, the subject receives a subsequent dose of about 160 mg of all-trans retinoic acid per square meter of body surface area.

[0018] In certain embodiments, the course of treatment comprises at least one 3-week treatment cycle, each cycle comprising 2 weeks of all-trans retinoic acid treatment every other day, followed by 1 week off.

[0019] In certain embodiments, the course of treatment comprises at least 17 treatment cycles.

[0020] In certain embodiments, the course of treatment comprises at least 9 treatment cycles.

[0021] In certain embodiments, the course of treatment comprises at least 4 treatment cycles.

[0022] In certain embodiments, the course of treatment comprises at least 2 treatment cycles.

[0023] In certain embodiments, ATRA is loaded into liposomes by a method comprising: Incubating a composition comprising all-trans retinoic acid, a buffer solution and blank liposomes (i.e., liposomes without all-trans retinoic acid), wherein the blank liposomes include an inner phase comprising an aqueous calcium acetate solution having a pH in the range of about 7 to about 10, and an outer phase outside the liposomes comprising an aqueous calcium acetate solution having a pH in the range of about 6 to about 7 and having a lower pH than that of the inner phase of the blank liposomes; and Remove free all-trans retinoic acid that is not loaded into blank liposomes.

[0024] In certain embodiments, the pH of the aqueous calcium acetate solution in the liposome internal phase is 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The test results of nanoDSC of all-trans retinoic acid (ATRA) liposomes with a drug-lipid ratio of 0.1 at different drug loading times are shown.

[0026] Figure 2 The test results of nanoDSC of all-trans retinoic acid (ATRA) liposomes with a drug-lipid ratio of 0.2 at different drug loading times are shown.

[0027] Figure 3 The nanoDSC test results of ATRA (all-trans retinoic acid) liposomes with a drug-lipid ratio of 0.25 at different drug loading times are shown.

[0028] Figure 4 The test results of nanoDSC of all-trans retinoic acid (ATRA) liposomes with a drug-lipid ratio of 0.3 at different drug loading times are shown. Detailed description

[0029] The protection scope of this application is not limited to the specific embodiments described below; the terms used in this application are intended to describe specific embodiments rather than to limit the protection scope of this application. The test methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or under conditions recommended by each manufacturer.

[0030] All technical and scientific terms used in this application have the same meaning as commonly understood by technicians, unless otherwise defined. In addition to the specific methods, equipment and materials used in the examples, any prior art similar to or equivalent to the methods, equipment and materials described in this application can also be used to implement this application based on the prior art mastered by technicians and the description of this application.

[0031] When numerical values ​​are given in the examples, it should be understood that both ends and any endpoint of the numerical range can be selected to be included unless otherwise described in the present application.

[0032] Unless otherwise indicated, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture and recombinant DNA technology. These techniques are well described in the literature, and for details, please refer to Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0033] As used in the description and appended claims, phrases such as "a," "an," "the," and "the" do not refer to singular forms unless the context clearly indicates otherwise. Thus, for example, reference to "a composition" includes a mixture of two or more such compositions, reference to "an inhibitor" includes mixtures of two or more such inhibitors, and so forth.

[0034] Unless otherwise indicated, the term "about" means within 10% above or below the particular value that "about" modifies (eg, within 5%, 2%, or 1% above or below the value).

[0035] The transitional term "comprising" is used herein as a synonym for "including," "containing," or "representing," and has an inclusive or open-ended meaning that does not exclude additional, unlisted elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics of the invention."

[0036] This patent discloses a liposome preparation containing all-trans retinoic acid, wherein all-trans retinoic acid is encapsulated inside the liposome. The lipid material constituting the liposome may form a phospholipid bilayer structure due to its special physicochemical properties. Among them, the phospholipid material includes but is not limited to phosphatidylcholine (such as hydrogenated soybean phosphatidylcholine (HSPC), stearic acid diphosphatidylcholine (DSPC) or stearic acid dodecyl phosphatidylcholine (DPPC)), cholesterol (CHOL) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0037] In certain embodiments, the molar ratio between phosphatidylcholine (e.g., HSPC), CHOL, and DSPE-PEG2000 is (30-80): (0.1-40): (0.1-30). In certain embodiments, the molar ratio between HSPC, CHOL, and DSPE-PEG2000 is about 57:38:5. In certain embodiments, the molar ratio between HSPC, CHOL, and DSPE-PEG2000 is about 55:40:5.

[0038] In certain embodiments, the molar ratio between DSPC, CHOL and DSPE-PEG2000 is (30-80):(0.1-40):(0.1-30). In certain embodiments, the molar ratio between DPSC, CHOL and DSPE-PEG2000 is about 55:40:5. In certain embodiments, the molar ratio between DSPC, CHOL and DSPE-PEG2000 is about 55:40:5.

[0039] In certain embodiments, the molar ratio of DPPC, CHOL and DSPE-PEG2000 is (30-80): (0.1-40): (0.1-30). In certain embodiments, the molar ratio of DPPC, CHOL and DSPE-PEG2000 is about 57:38:5. In certain embodiments, the molar ratio of DPPC, CHOL and DSPE-PEG2000 is about 55:40:5. The aqueous phase in the liposome contains all-trans retinoic acid and a calcium acetate solution at a pH of about 7 to about 10.

[0040] All-trans retinoic acid, also known as all-trans retinoic acid, is represented by (I):

[0041] At least a portion of the all-trans retinoic acid exists in the internal liposome aqueous phase of the liposome in the form of crystals. For example, when the all-trans retinoic acid forms crystals with calcium ions, the structure of the portion of the crystals is shown in (II). Therefore, all-trans retinoic acid in this form may be present in aqueous solutions of calcium acetate.

[0042] In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is at least about 2 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 30 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 25 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 20 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 15 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 10 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml to about 5 mg / ml. In some embodiments, the concentration of all-trans retinoic acid in the internal liposome aqueous phase is about 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml or even higher. These concentrations do not include other all-trans retinoic acid that may be present in the liposomes other than the all-trans retinoic acid in the internal liposome aqueous phase.

[0043] In certain embodiments, the molar ratio of all-trans retinoic acid to lipid component is about 1 to 20. In certain embodiments, the molar ratio of all-trans retinoic acid to lipid component is about 1 to 5.

[0044] In certain embodiments, the concentration of calcium acetate in the aqueous phase is 100 mM to 500 mM. In certain embodiments, the concentration of calcium acetate in the aqueous phase is 120 mM to 400 mM.

[0045] The pH of the aqueous calcium acetate solution in the intraliposomal aqueous phase is from 7 to about 10. In certain embodiments, the pH of the aqueous calcium acetate solution is from about 8 to about 10. In certain embodiments, the pH of the aqueous calcium acetate solution is from about 8.5 to about 9.5. In certain embodiments, the pH of the aqueous calcium acetate solution is about 7.5. In certain embodiments, the pH of the aqueous calcium acetate solution is about 8. In certain embodiments, the pH of the aqueous calcium acetate solution is about 8.5. In certain embodiments, the pH of the aqueous calcium acetate solution is about 9. In certain embodiments, the pH of the aqueous calcium acetate solution is about 9.5. In certain embodiments, the pH of the aqueous calcium acetate solution is about 10.

[0046] In some embodiments, the average particle size of the liposome is about 30 nanometers to 200 nanometers. In some embodiments, the average particle size of the liposome is about 50 nanometers to 150 nanometers. In some embodiments, the average particle size of the liposome is about 70 nanometers to 130 nanometers. In some embodiments, the average particle size of the liposome is about 50 nanometers to 100 nanometers. In some embodiments, the average particle size of the liposome is about 80 nanometers.

[0047] ATRA can be encapsulated into blank liposomes preformed through a calcium acetate gradient, which is an active drug loading method. Generally, this method involves the use of the following components: a) ATRA, sometimes with a certain cosolvent, b) buffer solution, c) blank liposomes (i.e., liposomes without ATRA), whose internal liposome aqueous phase is a calcium acetate aqueous solution in the pH range of 7-10.

[0048] In certain embodiments, the calcium acetate gradient method comprises the following operations or steps: 1) weighing all raw materials (lipid materials) for preparing liposomes according to the required quantity and concentration, and adding ethanol to obtain an ethanol mixture; 2) adding the ethanol mixture produced in operation 1 to a calcium acetate aqueous solution having a pH of about 7 to about 10 by injection or addition, thereby producing blank liposome vesicles containing a calcium acetate solution (i.e., the aqueous phase inside the liposome vesicles contains calcium acetate); 3) extruding the blank liposome vesicles obtained in operation (2) through a polycarbonate membrane to obtain a blank (i.e., not containing all-trans retinoic acid) liposome composition containing a calcium acetate aqueous solution in the aqueous phase inside the liposomes; 4) dialyzing the liposome composition obtained in operation (3) to remove the calcium acetate in the aqueous phase outside the liposomes by dialysis. In this step, the dialysis medium used is an isotonic solution (e.g., 7.5% sucrose buffer, 10 mM histidine, 5% vitamin C (w / w) and 0.01% EDTA-NA or 0.9% NaCl (w / w) and 10 mM HEPES), and the pH of these dialysis solutions is between about 6.0 and about 8.0. In this way, blank liposomes with a calcium acetate gradient are obtained (e.g., the pH in the inner aqueous phase is greater than that in the outer aqueous phase, wherein the pH concentration in the inner aqueous phase ranges from about 7 to about 10); 5) using a buffer solution to prepare a solution containing all-trans retinoic acid (ATRA), sometimes, the buffer solution may contain some cosolvent; 6) adding the buffer solution containing ATRA to the blank liposome solution with a calcium acetate gradient obtained in operation (4); 7) incubating the blank liposome / ATRA buffer / suspension formed in operation 6), and removing free ATRA that is not encapsulated into the liposome. Finally, all-trans retinoic acid liposomes were obtained.

[0049] In certain embodiments, in operation (2), the pH value of the calcium acetate aqueous solution in the aqueous phase within the liposome is about 8.5 to 9.5. In certain embodiments, in operation (2), the pH value of the calcium acetate solution in the aqueous phase within the liposome is about 9. In certain embodiments, in operation (2), the pH value of the calcium acetate solution in the aqueous phase within the liposome is about 9.5. In certain embodiments, in operation (2), the concentration of the calcium acetate aqueous solution is 100 mM to 500 mM. In certain embodiments, in operation (2), the concentration of the calcium acetate aqueous solution is 200 mM to 400 mM.

[0050] In certain embodiments, the buffer solution in (5) above further includes a solubilizer to increase the solubility of all-trans retinoic acid. In certain embodiments, the solubilizer is any one or more of dimethyl sulfoxide (DMSO), polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), cyclodextrin, and polyethylene glycol (PEG). In certain embodiments, the cyclodextrin is hydroxypropyl-β-cyclodextrin (HP-β-CD). Typically, the weight ratio of the solubilizer to all-trans retinoic acid ranges from about 5:1 to about 50:1 (w:w). In certain embodiments, the weight ratio of cyclodextrin (e.g., hydroxypropyl-β-cyclodextrin) to all-trans retinoic acid is 5:1 to 50:1 (w:w).

[0051] In some embodiments, the incubation process in step (7) lasts about 15 minutes to 1 hour. In some embodiments, the incubation process lasts about 45 minutes. In some embodiments, the incubation process lasts about 30 minutes.

[0052] In some embodiments, the incubation process in step (7) is performed at about 20° C. to 40° C. In some embodiments, the incubation process is performed at about 25° C. to 37° C. In some embodiments, the incubation process is at a temperature of about 25° C. and the incubation process lasts no longer than one hour. Drug ingredients

[0053] All-trans retinoic acid liposomes can be further used as active substances in pharmaceutical products, and pharmaceutical products should include all-trans retinoic acid liposomes covering effective concentrations of drugs and other carriers that meet the requirements of drugs. In the art, "other carriers that meet the requirements of drugs" generally refer to materials, compounds or carriers that meet the requirements for all-trans retinoic acid liposomes for mammalian administration. Suitable carriers may include liquids (including aqueous and non-aqueous liquids and combinations thereof), which function to transport all-trans retinoic acid liposomes from one organ or body part to another. The carrier is physiologically inert to other formulation ingredients and has good biocompatibility, and will not cause harm to the subject or patient. Depending on the type of the composition, the composition may also include one or more pharmaceutically acceptable excipients.

[0054] In general, all-trans retinoic acid liposomes can be prepared into specific types of formulations according to conventional pharmaceutical practices (e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds., J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation depends on its intravenous injection (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im) and intracardiac injection) route of administration. Preferably, intravenous injection (e.g., cerebrospinal fluid, subcutaneous, intravenous, intraventricular, intramuscular or intraarterial injection, including bolus or infusion, which can be continuous or discontinuous) is used because injection of all-trans retinoic acid liposomes as a therapeutic intervention can be given relatively quickly, such as single dose administration and / or emergency administration.

[0055] In certain embodiments, the all-trans retinoic acid liposomes are formulated for intravenous injection (e.g., systemic intravenous injection). In certain embodiments, the intravenous injection time is about 60 to 90 minutes. In certain embodiments, the all-trans retinoic acid liposomes are formulated for intrathecal injection.

[0056] Injectable preparations for in vivo injection can include sterile aqueous solutions or oily suspensions. Pharmaceutical preparations can also be prepared using appropriate dispersants or wetting agents and suspending agents according to standard techniques. Sterile injectables can also be sterile injectable solutions, suspensions or emulsions in nontoxic parent injection diluents or solvents, for example, solutions in isotonic saline or glucose solutions. Adoptable carriers and solvents include water, Ringer's solution, isotonic sodium chloride solution, etc. In addition, sterile oily solutions are also commonly used as solvents or suspension media. Injectable preparations should be filterable, such as by means of a filter, or sterilized by adding a soluble sterilizing agent. How to use

[0057] In certain aspects, the present invention relates to a method for treating cancer. The treatment regimen comprises administering a pharmaceutically effective concentration of all-trans retinoic acid liposomes to a subject.

[0058] The term "subject" (or "patient") as used herein includes all members of the animal kingdom who are susceptible to or suffering from the disease or disorder. In certain embodiments, the subject is a mammal, such as a human or a non-human mammal. The method is also applicable to companion animals, such as dogs and cats, and livestock, such as cattle, horses, sheep, goats, pigs and other domestic and wild animals. A subject who is in need of treatment according to the present disclosure may "suffer from or be suspected of having" a particular disease or disorder, may have been clearly diagnosed, or may have a sufficient number of risk factors or a sufficient number or combination of signs or symptoms so that medical personnel can diagnose or suspect that the subject is suffering from the disease or disorder. Therefore, subjects suffering from a particular disease or disorder and subjects suspected of having a particular disease or disorder are not necessarily two different groups.

[0059] The cancer described herein may be thymic epithelial carcinoma, ovarian adenocarcinoma, duodenal cancer, bile duct cancer or small cell lung cancer, etc.

[0060] In certain embodiments, the cancer is brain cancer, which can be a glioma, which can be an astrocytoma, a glioblastoma, an oligodendroglioblastoma, or an ependymoma, etc. In certain embodiments, the glioma is a recurrent or progressive glioma. In certain embodiments, the recurrent or progressive glioma is a recurrent or progressive glioblastoma.

[0061] All-trans retinoic acid can be given to cancer patients as a single therapeutic dose or in combination therapy, either as a front-line therapy or as a follow-up therapy for patients who fail to respond to the original front-line therapy. The treatment can be a "first-line therapy", that is, the initial treatment for patients who have not received any anti-cancer treatment regimen, whether used alone or in combination with other treatment regimens; or a "second-line therapy", that is, a treatment in patients who have previously received an anti-cancer treatment regimen, whether used alone or in combination with other treatment regimens, and the patient cannot tolerate the treatment or the cancer has recurred; or as a "third-line therapy", "fourth-line therapy" and other treatment methods, whether used alone or in combination with other treatment regimens. The treatment can also be used for patients who have undergone partial successful treatment, but they can no longer tolerate a particular treatment method. In addition, the treatment can also be used as an adjuvant therapy, that is, in patients who have not found current disease or to prevent cancer recurrence after tumor resection. Therefore, in some embodiments, all-trans retinoic acid can be injected into patients who have received other treatments, such as chemotherapy, radiation therapy, surgery, immunotherapy, radioimmunotherapy, targeted therapy, or any combination of treatments.

[0062] In certain embodiments, the treatment methods of the present application involve at least one 3-week cycle, during which the patient is administered all-trans retinoic acid liposomes, with a dosing frequency of once every other day, for a 2-week dosing period, followed by a 1-week break. The number of treatment cycles a patient receives is variable and can also be continued after the patient achieves complete remission. In certain embodiments, the treatment cycle can be from 1 to about 10 3-week cycles. In certain embodiments, the treatment method includes at least two 3-week treatment cycles. In certain embodiments, the treatment method includes at least four 3-week treatment cycles. In certain embodiments, the treatment method includes at least nine 3-week treatment cycles. In certain embodiments, the treatment method includes at least 17 3-week treatment cycles. In certain embodiments, the treatment method includes at least 26 3-week treatment cycles. In certain embodiments, the treatment method includes at least 34 3-week treatment cycles.

[0063] In this application, the term "pharmaceutically effective concentration" refers to the amount of all-trans retinoic acid that produces the desired response in treating a particular patient suffering from cancer. Thus, the term "pharmaceutically effective concentration" includes an amount of all-trans retinoic acid that, when administered, can cause a desired positive change in the cancer, or is sufficient to prevent or at least inhibit the progression of the cancer, or at least alleviate one or more symptoms of the cancer in the subject being treated, or simply kill or inhibit the growth of cancer cells.

[0064] The single daily dosage and method of use of all-trans retinoic acid can be determined according to standard medical practice, for example, by a physician using reasonable medical judgment. The specific effective dose for any particular subject may depend on a variety of factors, including the severity of the disease or disease being treated (e.g., its current state); the age, weight, general health, sex and diet of the subject; the administration time, route of administration and excretion rate of the applied all-trans retinoic acid; the duration of treatment; drugs used simultaneously or in combination with all-trans retinoic acid; and similar factors well known in the medical field (e.g., see Goodman and Gilman's "Basics of Therapeutic Pharmacology" 10th edition, edited by A. Gilman, J. Hardman and L. Limbird, McGraw-Hill Publishing House, 155-173 pages, 2001).

[0065] In certain embodiments, the dosage may range from about 45 mg to about 160 mg per square meter of body surface area. In certain embodiments, all-trans retinoic acid liposomes are administered at 45 mg / m 2 In certain embodiments, the all-trans retinoic acid liposomes are administered at a dose of 90 mg / m2 of the body surface area. 2 In certain embodiments, the all-trans retinoic acid liposome is administered at a dose of 120 mg / m 2In certain embodiments, all-trans retinoic acid is administered at a dose of 160 mg / m 2 The dosage should be given.

[0066] In some embodiments, the dosage is a fixed dose. In some embodiments, the dosage range may be between about 60 mg and about 350 mg. In some embodiments, the dosage range may be between about 80 mg and about 300 mg. In some embodiments, the dosage range may be between about 120 mg and about 240 mg. In some embodiments, the dosage range may be between about 160 mg and about 200 mg. In some embodiments, the dosage range may be between about 65 mg and about 90 mg. In some embodiments, the dosage range may be between about 135 mg and about 180 mg. In some embodiments, the dosage range may be between about 180 mg and about 240 mg. In some embodiments, the dosage range may be between about 240 mg and about 320 mg. In some embodiments, the dosage is about 280 mg. In some embodiments, the dosage is about 210 mg. In some embodiments, the dosage is about 180 mg. In some embodiments, the dosage is about 160 mg, for example, about 157.5 mg. In some embodiments, the dosage is about 80 mg, for example, about 78.75 mg. Combination therapy

[0067] All-trans retinoic acid liposomes can be used simultaneously or in combination with at least one other active anticancer drug or treatment. In this article, the words "combined use" and "simultaneous use" can be used interchangeably, meaning that these drugs are used together, including almost simultaneous administration by the same or different forms of administration, and the same or different routes of administration, or continuous administration according to the same treatment regimen. This order and time interval can be determined as they can work together (for example, synergy provides greater benefits than when used alone). For example, therapeutic drugs can be given simultaneously or sequentially at different time points, but if not administered simultaneously, they can be close enough in time to provide the desired therapeutic effect, which may be synergistic. Therefore, these terms are not limited to giving active drugs at exactly the same time.

[0068] The additional anticancer therapeutic dose may be the same as or lower than the known or recommended dose. See Hardman et al., eds., Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference 60th ed., 2006. Anticancer drugs that can be used in combination with bispecific compounds are known in the art. See, for example, U.S. Pat. No. 9,101,622 (Section 5.2 therein) and U.S. Pat. No. 9,345,705 B2 (Columns 12-18 therein). Representative examples of additional active agents and treatment regimens include radiation therapy, chemotherapeutic agents (e.g., mitotic inhibitors, angiogenesis inhibitors, anti-hormones, autophagy inhibitors, alkylating agents, intercalated antibiotics, growth factor inhibitors, anti-androgens, signal transduction pathway inhibitors, anti-tubulin agents, platinum coordination complexes, HDAC inhibitors, proteasome inhibitors, and topoisomerase inhibitors), immunomodulators, therapeutic antibodies (e.g., monospecific and bispecific antibodies), and CAR-T therapy.

[0069] In certain embodiments, all-trans retinoic acid liposomes are used in combination with one or more of the following drugs: programmed cell death protein (PD-1) inhibitors, programmed cell death ligand 1 (PD-L1) inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, chimeric antigen receptor (CAR) immunity (e.g., T cells) or cancer vaccines.

[0070] Representative examples of PD-1 inhibitors include pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMP-224, AMP-514, and acrixolimab.

[0071] Representative examples of PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170 and BMS-986189. Representative examples of CTLA-4 inhibitors include ipilimumab and tremelimumab.

[0072] Considering that readers may prefer specific examples to better understand various aspects of the present disclosure, some examples are provided below, which are intended to illustrate certain specific embodiments of the present disclosure but are not meant to limit the scope of the present disclosure, which is defined by the claims. DETAILED DESCRIPTION

[0073] Example 1: All-trans retinoic acid liposomes

[0074] The raw materials of each lipid component in the liposome can be obtained commercially. For example, hydrogenated soybean phosphatidylcholine (HSPC) can be purchased from NOF Corporation, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and cholesterol can be purchased from Avanti Polar Lipids;

[0075] All-trans retinoic acid can be purchased from Sigma. Preparation of all-trans retinoic acid liposomes

[0076] (1) Hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and cholesterol (CHOL) were mixed in ethanol at a weight ratio of 3:1:1.

[0077] (2) adding the ethanol mixture obtained in step (1) to a solution containing 400 mM calcium acetate buffer (pH 9.0) to obtain a liposome solution containing 10-15% (mass concentration) ethanol.

[0078] (3) The liposome solution obtained in step (2) is extruded through a double-layered polycarbonate membrane (100 nm + 100 nm) with pore sizes of 100 nm each. Subsequently, it is further extruded through a double-layered polycarbonate membrane (100 nm + 50 nm) with pore sizes of 100 nm and 50 nm each. The process of extrusion through the polycarbonate membrane can also be performed by extrusion through a single layer each time.

[0079] (4) The extruded liposomes were sent to a tangential flow dialysis apparatus and dialyzed in 8X volume of 10 mM HEPES buffer (pH 7.6).

[0080] (5) All-trans retinoic acid (ATRA) was added to a solution containing 100 mg / mL hydroxypropyl-β-cyclodextrin (HP-β-CD) and 10 mM HEPES, and stirred at 53° C. The pH of the suspension was then adjusted to 7.6 to obtain an ATRA solution with a concentration of 4 mg / mL.

[0081] (6) The all-trans retinoic acid solution containing HP-β-CD and the liposome solution were mixed in equal amounts at a lipid concentration of 40 mg / mL. The mixture was incubated at room temperature for 30-45 minutes for drug encapsulation, and then the encapsulated drug was dialyzed through a tangential flow dialysis device, the dialysis solution was a buffer solution containing 10% sucrose and 10 mM histidine, and the dialysis volume was 9 times the volume of the drug.

[0082] (7) The prepared liposome solution is filtered through 0.8 μm, 0.65 μm and 0.45 μm filters in sequence and stored after filtration.

[0083] Example 2: Preparation of all-trans retinoic acid liposomes

[0084] (1) Weigh 121.742 g of hydrogenated soybean phosphatidylcholine (HSPC, molecular weight 783.8), 38.22 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 40.04 g of cholesterol (CHOL), dissolve them in 1.6 ml of ethanol, and place them in a water bath at 65° C. to dissolve and mix, thereby obtaining an ethanol mixture;

[0085] (2) adding 6.4 ml of calcium acetate buffer (pH 9.0, composed of 400 mM calcium acetate and water) to the ethanol mixture obtained in step (1), and placing the mixture in a water bath at 65 degrees Celsius for 30 minutes to obtain liposome vesicles;

[0086] (3) extruding the liposome vesicles obtained in step (2) through polycarbonate membranes with pore sizes of 200 nm, 100 nm, 80 nm, and 50 nm, respectively, 8 times each, to finally obtain liposomes with an average particle size of about 75 nm and a water phase containing calcium acetate;

[0087] (4) The liposomes prepared in step (3) are dialyzed in a 10% by mass aqueous sucrose solution with a pH of 6 to 7 through a 10,000 pore size dialysis membrane, and the outer aqueous phase of the liposomes is replaced with a 10% by mass aqueous sucrose solution with a pH of 6 to 7, to obtain calcium acetate liposomes having a phospholipid bilayer membrane, and the inner and outer aqueous phases of the double-sided membrane have a certain pH gradient and ion gradient. Specifically, the inner aqueous phase of the bilayer membrane is a calcium acetate aqueous solution (pH 9.0, concentration 400 mM), and the outer aqueous phase of the bilayer membrane is a sucrose aqueous solution (pH 6 to 7, mass fraction 10%).

[0088] (5) Prepare 0.5 ml of 400 mM carbonate buffer solution with a pH of about 9 and add 3.5 g of all-trans retinoic acid thereto. Specifically, the mass ratio of sodium carbonate to sodium bicarbonate in the buffer solution is 2.82:39.76) and 10% volume (10% of the volume of the carbonate buffer solution, the same below) of DMSO, and heat at 65 degrees Celsius until the drug is completely dissolved.

[0089] (6) Blank liposomes were mixed with the drug solution, and the drug was loaded by shaking at room temperature for 5 hours to obtain all-trans retinoic acid liposomes. After the incubation, the free all-trans retinoic acid that was not loaded into the liposomes was removed again using a 10,000 pore size dialysis membrane, and the obtained liposome sample was filtered through a 0.22 um filter membrane to finally obtain all-trans retinoic acid liposomes.

[0090] (7) The encapsulation efficiency (EE) of all-trans retinoic acid was calculated according to the following formula: EE = (Wi / Wtotal) * 100%, where Wi is the mass of all-trans retinoic acid in the complex destroyed by methanol after purification, and Wtotal is the mass of all-trans retinoic acid before dialysis to separate free drugs and the same volume of all-trans retinoic acid after dialysis. The encapsulation efficiency of the obtained all-trans retinoic acid liposomes reached 94%-100%. In addition, we also prepared all-trans retinoic acid liposomes with different drug-lipid ratios and different encapsulation times. As shown in the following table:

[0091] Table 1: Information on the prescriptions of different drug loading conditions in Example 2 As shown in Table 1, it was found through comparison that the all-trans retinoic acid liposomes prepared by the preparation method of the present application have a higher drug-lipid ratio, and at the same time, as the drug loading time increases, the drug release stability is also significantly improved.

[0092] Example 3: Preparation of all-trans retinoic acid liposomes using the dropwise addition method

[0093] (1) Weigh 121.742 g of hydrogenated soybean phosphatidylcholine (HSPC, molecular weight 783.8), 38.22 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 40.04 g of cholesterol (CHOL), dissolve them in 1.6 ml of ethanol, and place them in a water bath at 65° C. to dissolve and mix, thereby obtaining an ethanol mixture;

[0094] (2) adding 6.4 ml of calcium acetate buffer (pH 9.0, composed of 400 mM calcium acetate and water) to the ethanol mixture obtained in step (1), and placing the mixture in a water bath at 65 degrees Celsius for 30 minutes to obtain liposome vesicles;

[0095] (3) extruding the liposome vesicles obtained in step (2) through polycarbonate membranes with pore sizes of 200 nm, 100 nm, 80 nm, and 50 nm, respectively, 8 times each, to finally obtain liposomes with an average particle size of about 75 nm and a water phase containing calcium acetate;

[0096] (4) The liposomes prepared in step (3) are dialyzed in a 10% by mass aqueous sucrose solution with a pH of 6 to 7 through a 10,000 pore size dialysis membrane, and the outer aqueous phase of the liposomes is replaced with a 10% by mass aqueous sucrose solution with a pH of 6 to 7, to obtain calcium acetate liposomes having a phospholipid bilayer membrane, and the inner and outer aqueous phases of the double-sided membrane have a certain pH gradient and ion gradient. Specifically, the inner aqueous phase of the bilayer membrane is a calcium acetate aqueous solution (pH 9.0, concentration 400 mM), and the outer aqueous phase of the bilayer membrane is a sucrose aqueous solution (pH 6 to 7, mass fraction 10%).

[0097] (5) 2 mg of all-trans retinoic acid was dissolved in DMSO, and blank liposomes were heated to 65 degrees Celsius. The drug solution was added dropwise to the blank liposomes and incubated for 30 minutes. After the incubation, the free all-trans retinoic acid, DMSO and carbonate buffer that were not loaded into the liposomes were removed again using a 10,000 pore size dialysis membrane. The obtained liposome sample was filtered through a 0.22 um filter membrane to finally obtain all-trans retinoic acid liposomes.

[0098] (6) The encapsulation efficiency (EE) of ATRA was calculated by the following formula: EE = (Wi / Wtotal) * 100%, where Wi is the mass of ATRA in the purified methanol-destroyed complex, and Wtotal is the mass of ATRA before dialysis to separate free drug and the same volume of ATRA after dialysis. The ATRA liposomes with drug-lipid ratio of 0.1 to 0.3 were prepared, and the encapsulation efficiency was too low, ranging from 50% to 80%.

[0099] Example 4: In vitro release of all-trans retinoic acid liposomes at different drug loading times

[0100] With 40mg / ml bovine serum albumin (BSA) solution as the release medium, put the 1:50 volume of all-trans retinoic acid liposome and BSA solution (60μL liposome + 2940μL BSA solution) into a 300KD dialysis bag, and put 400ml BSA solution as the medium outside the dialysis bag. The total release system of all-trans retinoic acid liposome: BSA solution volume ratio is about 1:6700 times. The protein binding rate of all-trans retinoic acid is 95%. After the all-trans retinoic acid drug is released, it binds to BSA and enters and exits the dialysis bag with BSA, and the drug is taken out of the dialysis bag.

[0101] 0.5h, 2h, 5h, 10h, and 24h were selected as sampling points, and the unreleased drug content in the dialysis bag was measured each time to obtain the in vitro release of all-trans retinoic acid liposomes. By this method, the drug release results of different prescriptions prepared in Example 2 can be obtained.

[0102] The experimental results show that all-trans retinoic acid liposomes with a drug-lipid ratio of 0.35 can release the drug uniformly and stably in vitro, and the stability of samples with drug loading time of 5h and 24h is better than that of samples with drug loading time of 1h.

[0103] Example 5: Nano-DSC Characterization of All-trans Retinoic Acid Liposomes

[0104] We used nano-DSC to study the thermodynamic behavior of all-trans retinoic acid liposomes and investigate the drug presence form within the liposomes. Enthalpy is a state function. The state of the system is constant, the enthalpy value is constant, and the enthalpy change is the difference in enthalpy between the product and the reactant. If the enthalpy change is positive, the reaction is endothermic. At the same time, in the liposome formulation, the added cholesterol can adjust the fluidity of the lipid bilayer. Adding 2-6% cholesterol to the bilayer composed of C14-C20 phosphatidylcholine can eliminate the pre-phase transition and sub-phase transition of phospholipids, and will cause the DSC absorption peak to become wider and the phase change enthalpy value to decrease. The Tm value is an important parameter for determination. It can analyze the phase transition temperature range of the sample to undergo the highest heat capacity change. During the heating process of the lipid membrane, the Tm value of the lipid membrane is the critical temperature for the change from the gel state to the liquid crystal state. The Tm value of the drug precipitation is the temperature at which the drug crystals melt during the heating process. Generally speaking, in Figure 1 The peaks at 45°C-55°C are the lipid phase transition peaks of liposomes, while the peaks at 70°C-80°C are the characteristic peaks of drug crystals (in the form of microcrystals / liquid crystals / crystaloids) formed by all-trans retinoic acid and calcium acetate.

[0105] Liposomes with a drug concentration of 1.75 mg / ml were prepared, and all-trans retinoic acid liposomes with drug-lipid ratios of 0.1, 0.2, 0.25, and 0.3 were prepared while keeping the drug concentration constant. DSC was used to investigate the drug loading at loading times of 1 h, 5 h, and 24 h. Figures 1 to 4 shown.

[0106] Under the same drug loading, as the drug loading time increases, the larger the Tm value of the drug crystallization, the more stable the drug crystallization. As the drug-lipid ratio increases, the lipid peak will undergo phase separation. We believe that lipid phase separation will reduce the stability of the liposome. This may be due to the excessive drug loading. Due to the insertion of the drug, the lipids in the lipid membrane are unevenly arranged, and local accumulation occurs, resulting in phase separation. However, as the drug loading time increases, the lipid phase separation phenomenon has been significantly improved. Therefore, we believe that increasing the drug loading time helps to improve the stability of liposome drugs. When the drug loading is high, stable drug-loaded liposomes can be obtained by increasing the drug loading time.

[0107] Example 6: Regulation of Myeloid-derived suppressor cells (MDSCs)

[0108] Immature myeloid cells or myeloid-derived suppressor cells (MDSCs) have been found to accumulate in peripheral blood mononuclear cells (PBMCs) and tumor tissues of cancer patients. All-trans retinoic acid is one of the active metabolites of vitamin A in the body, which can induce the maturation and differentiation of immature myeloid cells or myeloid-derived suppressor cells.

[0109] method

[0110] The tolerability and safety of all-trans retinoic acid liposomes in patients with recurrent and refractory solid tumors were investigated using a "3+3" dose escalation regimen using the all-trans retinoic acid liposome preparation prepared by the above method. In each treatment cycle, the subjects received all-trans retinoic acid treatment every other day for two weeks, followed by a 1-week break. This treatment cycle was repeated until the end of treatment. Safety and tolerability records, repeated dose pharmacokinetic (PK) parameters, and exploratory pharmacodynamic (PD) analysis of PBMC samples were evaluated during the first treatment cycle. Efficacy evaluations were performed every 6 weeks or less until the end of treatment.

[0111] result

[0112] This study recruited 11 patients who received 45 mg / m 2 , 90mg / m 2 and 120 mg / m 2 The patients’ diagnosis and treatment information are detailed in Table 2. When used as monotherapy, all-trans retinoic acid liposomal formulations were given at 45 mg / m 2 and 90 mg / m 2The dose level was well tolerated. According to RECIST 1.1 criteria, the measurable lesion area of ​​one of the subjects decreased from 70 mm to 23.7 mm, which was a significant response to the drug. The PFS of four patients exceeded 2 months. Five of the patients included in the study were in the subgroup of glioblastoma patients who had relapsed and were refractory after multiple treatments, and the disease of three of them was controlled and they are still receiving treatment.

[0113] Table 2. Subject information and treatment information

[0114] All patent and non-patent publications reflect the level of skill of professionals in the art and are incorporated herein by reference in the same manner as if each individual publication was specifically and expressly indicated to be incorporated by reference.

[0115] Although the embodiments disclosed herein have been described in considerable detail, it should be understood that these embodiments are only used to illustrate the principles and applications of this invention. Therefore, various modifications may be made to these embodiments, and other different schemes may be designed, and these modifications and designs do not exceed the spirit and scope of the present invention defined by the attached claims.

[0116] Example 7: Use of a fixed dose to modulate myeloid-derived suppressor cells

[0117] method

[0118] To evaluate the efficacy and safety of a fixed dose of ATRA in a liposomal formulation for patients with recurrent or progressive gliomas. The fixed dose is given by intravenous infusion over approximately 60 to 90 minutes.

[0119] The subjects will be monitored for tolerability, dose-limiting toxicities, pharmacokinetic characteristics, and preliminary efficacy during the study.

[0120] ATRA was infused intravenously every other day for 2 weeks, followed by 1 week of rest, for a total of 3-week cycles, and the 3-week cycles were repeated until the end of treatment (including complete remission, death, or the judgment of the treating physician).

Claims

1. A method for treating cancer, comprising administering to a patient a certain pharmaceutically effective concentration of an all-trans retinoic acid liposome preparation, wherein: The liposomes comprise an inner aqueous phase containing all-trans retinoic acid and calcium acetate solution at a pH of about 7 to about 10; wherein The lipid components of the liposome include hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), and at least a part of the all-trans retinoic acid in the aqueous phase of the liposome is crystal.

2. The method of claim 1, wherein the cancer is brain cancer.

3. The method of claim 2, wherein the brain cancer is glioma.

4. The method of claim 3, wherein the glioma is an oligodendroglioma.

5. The method of claim 3, wherein the glioma is a glioblastoma.

6. The method of any one of claims 1-5, wherein the molar ratio of all-trans retinoic acid to lipid component is about 1 to about 20.

7. The method of any one of claims 1-5, wherein the molar ratio of all-trans retinoic acid to lipid component is about 1 to about 5.

8. The method of any one of claims 1-7, wherein the molar ratio between HSPC, CHOL and DSPE-PEG2000 is in the range of (30-80):(0.1-40):(0.1-30).

9. The method of any one of claims 1 to 8, wherein the amount of all-trans retinoic acid in the liposomes is at least 2 mg / mL based on the volume of the calcium acetate solution in the liposomes.

10. The method of claim 9, wherein the concentration of all-trans retinoic acid in the interior of the liposomes is about 2 mg / ml to about 5 mg / ml.

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