Combination of angubindin-1 ABD chemotherapy for the treatment of CNS malignancies

Abstract

The invention provides a method for treating a central nervous system (CNS) malignancy in a subject in need of treatment thereof. The method comprises administering angubindin-1 and a chemotherapeutic agent to the subject. Thus, through the use of angubindin-1 in combination with chemotherapy, the invention provides an improvement in the chemotherapeutic treatment of CNS malignancies.

Description

COMBINATION OF ANGUBINDIN-1 ABD CHEMOTHERAPY FOR THE TREATMENT OF CNS MALIGNANCIESSTATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with Government support by the National Institutes of Health, National Institute of Neurological Disorders and Stroke. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 2,418 Byte XML file named " 769827. xml," dated November 13, 2023.BACKGROUND OF THE INVENTION

[0003] The blood-brain barrier (BBB) endothelium is held together by tight junctions (bicellular and tricellular), which maintain BBB integrity. Specifically, tricellular tight junction endothelial proteins, angulin-1 (Lipolysis-stimulated lipoprotein receptor, LSR) and tri cellulin, cooperate to limit penetration of many substances into the central nervous system (CNS). Among these are many drug substances, notably chemotherapeutic agents. Thus, the relative impermeability of the BBB presents a technical hurdle, which limits the use of such agents in the treatment of CNS diseases, particularly malignancies of the brain and spinal cord. Surmounting this hurdle represents a significant unmet need in the field of chemotherapy, given the severity and prevalence of such cancers. The present invention addresses these technical concerns.BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a method for treating a CNS malignancy in a subject in need of treatment thereof. The method comprises administering angubindin-1 and a chemotherapeutic agent to the subject. Thus, through the inventive use of angubindin-1 in combination withchemotherapy, the invention provides an improvement in the chemotherapeutic treatment of CNS malignancies, which can be in accordance with known therapeutic regimens, via the administration of angubindin-1 to the subject.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0005] Figure 1 graphically presents a survival curve depicting data that demonstrate that angubindin-1 significantly prolongs survival of subjects carrying gliomas when combined with chemotherapy.

[0006] Figure 2 graphically depicts data demonstrating that both angubindin-1 and combination treatment decreased tumor volume.

[0007] Figure 3 graphically depicts data demonstrating that, as opposed to plasma concentration (top panel), measurement of doxil concentration within brain tumors was significantly increased by co-treatment with angubindin-1 (bottom panel).

[0008] Figure 4 is a Western blot displaying levels of angulin-1 expression following treatment with 600 pg / mL angubindin-1 .

[0009] Figure 5 graphically depicts data concerning the minimum normalized protein expression for angulin-1.

[0010] Figure 6 graphically depicts data concerning endothelial cell-cell integrity (cell index) for angubindin-1, C-CPE, and C-CPE-mt. Control data are a solid line at 100%, and the lowest curve in the figure represents treatment with 600 pg / mL angubindin-1.

[0011] Figure 7 graphically depicts data concerning the PGP efflux of rhodamine in glioma cells.

[0012] Figure 8 graphically depicts data concerning the dose-dependent migratory effect (cell index) of angubindin-1 on rat glioma cells. The top panel depicts data concerning cell index, and the bottom panel contains data concerning the cell index normalized to the vehicle.

[0013] Figure 9 graphically depicts data demonstrating that downregulation of angulin-1 induces downstream signaling activation of phospho-JNK to regulate the endothelial barrier. The top panel depicts data concerning angulin-1 expression, and the bottom panel depicts data concerning angulin-1 expression of pJNK / JNK.DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention provides a method for treating a CNS malignancy in a subject in need of treatment thereof. The method comprises administering both angubindin-1 and a chemotherapeutic agent to the subject. The use of angubindin-1 in this context (i.e., in the treatment of a CNS malignancy in combination with the chemotherapeutic agent), thus, represents an improvement over standard methods of chemotherapy for CNS malignancies.

[0015] The subject can be any animal (typically a mammal), which is in need of treatment of a CNS malignancy. Thus, the subject can be, for example, animals of veterinary importance (e.g., cats, dogs, horses, etc.), agricultural importance (e.g., cattle, sheep, goats, fowl, and the like), zoological importance (e.g., amphibians, reptiles, birds, mammals, such as induvial members of threatened or endangered species), laboratory specimens (e.g., mice, rats, etc.), and primates, including humans. In this respect, the inventive method and use can be employed in the context of veterinary or medical treatment involving human or animal patients.

[0016] The CNS malignancy to be treated in accordance with the inventive method and use can be any malignant or cancerous growth or tumor within the CNS (brain or spinal cord). The experimental Examples herein concern treatment of glioma, but the invention is not limited to glioma. Other examples of many such CNS malignancies that can be treated in accordance with the present invention (such as embryonal brain tumors, metastatic brain cancer, primary brain cancer, and the like) are documented by competent authorities and known to those of skill in the art. See, e.g., Louis et al., Neuro Oncol. 23(8): 1231-1251 (2021) (the entire contents of which are incorporated herein). However, as gliomas (and particularly glioblastoma multiforme) represent a significant public health threat, certainly the invention can be used in the treatment of such CNS malignancies.

[0017] In connection with the inventive method and use, the subject also is one which is undergoing or is to undergo chemotherapy for the treatment of the CNS malignancy (such as a patient who has been diagnosed with a CNS malignancy). Such treatment involves administration of a chemotherapeutic agent to the subject, which can be any suitable chemotherapeutic agent approved or under investigation for treatment of the particular type of CNS malignancy. Thus, for example, for treatment of gliomas, the inventive method can be used in conjunction with the administration of doxil to the subject. However, for use in the inventivemethod, the chemotherapeutic agent is not limited to doxil; for example, agents such as doxorubicin, etoposide, taxanes, platinum agents, temozolomide, anthracyclines, and the like, also are contemplated (as are other chemotherapeutic agents). Of course, by “agent” in this context, multiple agents are not excluded; thus, the invention can be employed wherein multiple (combined) agents are administered to the subject for the treatment of the CNS malignancy. Also, in connection with the inventive method, the chemotherapeutic agent can be administered via routes and at dosages and regimens known to those of ordinary skill in the art.

[0018] For the inventive use, i.e., in the performance of the inventive method, angubindin-1 is administered to the subject in need of treatment of the CNS malignancy in combination with the chemotherapeutic agent. As demonstrated in the Examples that follow, the combined use of angubindin-1 and a chemotherapeutic agent can increase the odds of survival of the subject treated in accordance with the inventive method and use of angubindin-1.

[0019] In the context of the present invention, angubindin-1 can consist of, consist essentially of, comprise, or be derived from the Clostridium perfringens iota-toxin lb polypeptide fragment corresponding to amino acids 421-664 of such protein (see e.g., Krug et al., J. Controlled Release 260, 1-11 (2017) (the entire contents of which are incorporated herein), which is an angulin-l / LSR binding peptide. The amino acid sequence of this exemplary angubindin-1 is set forth at SEQ ID NO:1. However, in the context of the present invention, the angubindin-1 also can be a functional derivative of such polypeptide fragment, such as comprising a sequence of amino acids having at least 75%, or at least 85% or at least 95%, or at least 99% identity with SEQ ID NO: 1. The angubindin-1 for use in the context of the present invention also can be derived by conjugation with other agents or substrates, such as liposomes, microparticles, or other polypeptides, as desired.

[0020] In the context of the present invention, the angubindin-1 can be administered to the subject via any suitable route and in any suitable dose to achieve the therapeutic effects. Moreover, either or both of the angubindin-1 and the chemotherapeutic agent can be administered once or several times over a treatment regimen. Also, as with the chemotherapeutic agent, the angubindin-1 can be administered to the subject within a pharmaceutical preparation including angubindin-1 and a pharmaceutically active carrier. Of course, the pharmaceutical preparation can include both the angubindin-1 and the chemotherapeutic agent, if desired, or thetwo can be administered in separate pharmaceutical preparations. In this respect, the angubindin-1 can be administered prior to, following, or concurrently with the chemotherapeutic agent.

[0021] The pharmaceutically active carrier used in the pharmaceutical preparation can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active agent(s) (i.e., the angubindin-1 and optionally the chemotherapeutic agent, if compounded together), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically and physiologically inert to such active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0022] The choice of carrier and manner of formulation of the inventive composition will be determined in part by the particular method used to administer such active agent(s). A variety of suitable formulations of the pharmaceutical composition, thus, can be employed in carrying out the inventive methods described herein. Formulations for parenteral, subcutaneous, intravenous, intramuscular, and intraperitoneal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering active agents are known and that more than one route can be used to administer a particular compound.

[0023] Injectable formulations are among those formulations that are preferred in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238 250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). The pharmaceutical preparation can be formulated for injection by any desired route, such as via intravenous, intraperitoneal, intratumoral, or peritumoral injection.

[0024] Formulations suitable for injection include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. The active agent(s) can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methyl cellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0025] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral and other injectable formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0026] Suitable soaps for use in injectable formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl -b-aminopropi onates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0027] Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophiledipophile balance of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

[0028] Injectable formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0029] The dosage of angubindin-1 to be administered in the context of the present invention can be any suitable dosage to achieve therapeutic effectiveness. The suitable dosage will be determined by the treating physician or veterinarian in the exercise of professional judgment. However, in the experimental Examples that follow, angubindin-1 was administered to experimental subjects (rats) at dosages of either 10 mg / kg or 30 mg / kg, based on the weight of the subject, and such dosages (including dosages falling between these values) are certainly contemplated. Also, lower dosages of angubindin-1 are contemplated, such as 5 mg / kg or 1 mg / kg, or even lower dosages. Higher dosages of angubindin-1 are contemplated as well, such as 40 mg / kg, 50 mg / kg, 75 mg / kg, or even higher dosages.EXAMPLES

[0030] The following experimental working Examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. In brief, the results of the experiments discussed in the Examples demonstrate that angubindin-1 treatment transiently disrupts BBB tricellular junctions, thereby increasing chemotherapy permeability to brain tissues, leading to enhanced survival outcomes for subjects carrying gliomas.EXAMPLE 1

[0031] This Example demonstrate that angubindin-1 improves delivery of chemotherapy and that combined angulin-1 inhibition and chemotherapy improves survivability of glioma subjects.

[0032] The experiments involved injecting S635 rat glioma cells into 5-week-old female F344 rats. On days 7 and 14, the subjects were treated with doxil (3 mg / kg), angubindin-1 (10 mg / kg or 30 mg / kg), or combination therapy for assessments on tumor volume, BBB permeability, plasma and intratumoral doxil concentration, and survival.

[0033] Angubindin-1 significantly increased survival rat glioma models to 21 days, compared to 15.5 days (control), and combination therapy of angubindin-1 and doxil increased rat survival to 24 days. Combined angubindin-1 and doxil also increased survival in rat glioma models compared with doxil alone (24 days vs. 18 days, p < 0.0001). (Figure 1).

[0034] Day 14 tumor volume also was significantly decreased with angubindin-1 and combination treatment respectively (77.5 % vs 81.6 %, p<0.05). Additionally, when tumor volume was measured, it was revealed that both angubindin-1 and combination treatment decreased tumor volume (Figure 2). Also, as opposed to plasma concentration, which appeared unaffected, measurement of doxil concentration within brain tumors was significantly increased by co-treatment with angubindin-1 (Figure 3).

[0035] These results demonstrate that angubindin-1 significantly prolongs survival of subjects carrying gliomas, when combined with chemotherapy.EXAMPLE 2

[0036] This Example demonstrates that angubindin-1 transiently decreases BBB junctional integrity.

[0037] Rat brain endothelial cells were treated with TJ and BJ inhibitors against angulin-1 (angubindin-1 600pg / mL), claudin-3 (C-CPE 200pg / mL) or claudin-5 (C-CPE-MT 200pg / mL). Endothelial integrity was assessed by immunoblotting and cell-cell electrical impedance. Effects of angubindin-1 on efflux transporter P-gly coprotein (PGP) and migration were studied using rat and human derived glioma cells.

[0038] Decreased angulin-1 expression five hours after angubindin-1 treatment was observed, with return to baseline by 24 hours (p<0.05) (Figure 4). The minimum normalized protein expression for angulin-1 occurred at 6 hours (Figure 5). Angubindin-1, CCPE, and CCPE-mt globally reduced endothelial cell-cell integrity, maximally at four hours with a return to baseline by twelve hours; with angubindin-1 demonstrating the largest decreased cell-adhesion (angubindin-1 vs control, p< 0.0001) (Figure 6).

[0039] Angubindin-1 also decreased PGP efflux of rhodamine in both endothelial and, as demonstrated in Figure 7, glioma cells, as well as demonstrating a pro-migratory dose-dependent effect on rat glioma cells (Figure 8).

[0040] Collectively, these results demonstrate that angubindin-1 transiently decreases BBB junctional integrity, which can increase chemotherapy permeability.EXAMPLE 3

[0041] This example demonstrates that angubindin-1 treatment transiently disrupts BBB tricellular junctions.

[0042] Junctional disruption was evaluated with drug proteins: angubindin-1 (600pg / mL), C- CPE (200pg / mL), and C-CPEmt (200pg / mL) on rat brain endothelium. Endothelial junctional integrity studies were assessed by immunoblotting and cell-cell electrical impedance assays. Treatment effects on rat malignant glioma (S635) were measured via migration and rat glioma models.

[0043] Overall, a time-dependent effect of drug proteins on junctional expression and function was observed. Immunoblotting demonstrated a significant decrease (p< 0.05) in angulin-1 expression five hours after angubindin-1 treatment, while claudin-3 and claudin-5 expression barely decreased between two and 24-hours after C-CPE and C-CPEmt treatment, respectively. Cell-cell integrity was disrupted by 73%, 52%, and 69% compared with control, three hours after angubindin-1, C-CPE, and C-CPEmt treatment, respectively. Additionally, experiments revealed that downregulation of angulin-1 induces downstream signaling activation of phospho-JNK to regulate the endothelial barrier (Figure 9). Also, glioma cells expressed high angulin-1, and interestingly, migration was decreased 50% with angubindin-1 treatment, yet no migration changes were evident with C-CPE or C-CPEmt treatment.

[0044] Thus, these results demonstrate (a) that angubindin-1 treatment transiently disrupts BBB tricellular junctions and (b) that overexpression of angulin-1 inhibits migration of gliomas.SEQUENCE

[0045] The sequence of an exemplary angubindin-1 for use in the context of the present invention, which represents amino acids 421-664 (Ib421-664) of Clostridium perfringens iotatoxin (lb), is set forth as follows (SEQ ID NO: 1):MRSHHHHHHGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSGSDITSLYKKVGENLYFQGAQDNQIGNNLSPNETYPKKGLSPLALNTMDQFSSKLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNNQGQIVTEGNSWSDYISQIDSISASLILDTGNETFERRVAAKDSSNPEDKTPELTIGEAIEKAFGATKNGGLLYFNEIPIDESCVELIFDDNTANIIKNSLKTLDDKKIYNVKLERGMNILIKTPSYFTNFDGHNTFPKSWSNINTQNKDGLQGTANEVNGETKITLPMSNLKPYK*(SEQ ID NO: !)

[0046] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0047] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0048] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method for treating a central nervous system (CNS) malignancy in a subject in need of treatment thereof, the method comprising administering angubindin-1 and a chemotherapeutic agent to the subject.

2. In a method comprising chemotherapeutic treatment of a CNS malignancy in a subject comprising administering a chemotherapeutic agent to the subject, wherein the improvement comprises administering angubindin-1 to the subject.

3. Use of angubindin-1 in the treatment of a CNS malignancy in a subject comprising administering angubindin-1 in combination with chemotherapy.

4. The method of 1 or 2 or the use according to claim 3, wherein the CNS malignancy comprises an embryonal brain tumor, a metastatic brain cancer, a primary brain cancer, or glioma.

5. The method or use of claim 4, wherein the CNS malignancy comprises glioma.

6. The method or use of claim 5, wherein the glioma comprises glioblastoma multiforme.

7. The method or use of any one of claims 1-6, wherein the chemotherapeutic agent comprises doxorubicin, etoposide, a taxane, a platinum agent, temozolomide, an anthracycline, or a combination of two or more of these.

8. The method or use of claim 7, wherein the chemotherapeutic agent comprises Doxil.

9. The method or use of any one of claims 1-8, wherein the angubindin-1 is a polypeptide comprising a sequence of amino acids having at least 95% identity with SEQ ID NO: 1.

10. The method or use of any one of claims 1-9, wherein the angubindin-1 is a polypeptide comprising a sequence of amino acids consisting of SEQ ID NO: 1.

11. The method or use of any one of claims 1-10, wherein the angubindin-1 is administered prior to the chemotherapeutic agent.

12. The method or use of any one of claims 1-11, wherein the angubindin-1 is administered following the chemotherapeutic agent.

13. The method or use of any one of claims 1-12, wherein the angubindin-1 is administered concurrently with the chemotherapeutic agent.

14. The method or use of any one of claims 1-13, wherein the angubindin-1 is administered via intravenous injection.

15. The method or use of any one of claims 1-14, wherein the angubindin-1 is administered at a dose of from 10 mg / kg to 30 mg / kg, based on the weight of the subject.

16. The method or use of any one of claims 1-15, wherein the subject is a human patient.

Images