HK1250007B - Method of treating a brain tumor - Google Patents

Description

Treatments for brain tumors

Incorporation by reference into any priority application

This application claims the benefit of U.S. Provisional Application No. 62/129,623, filed March 6, 2015, and U.S. Provisional Application No. 62/249,807, filed November 2, 2015, the disclosures of which are incorporated herein by reference in their entireties.

Background Art

Technical Field

The present invention relates to the fields of chemistry and medicine. More particularly, the present invention relates to a method for treating brain tumors using Plinabulin.

Description of related art

Cancers of the brain and nervous system are among the most difficult to treat. The prognosis for patients with these cancers depends on the type and location of the tumor, as well as its stage of development. For many types of brain cancer, the average life expectancy after symptom onset may be a few months or a year or two. Treatment primarily consists of surgical resection and radiation therapy. Chemotherapy is also used, but the range of suitable chemotherapeutic agents is limited, likely because most therapeutic agents cannot adequately penetrate the blood-brain barrier to treat brain tumors. The use of known chemotherapy combined with surgery and radiation rarely prolongs survival far beyond that achieved by surgery and radiation alone. Therefore, there is a need for improved treatment options for brain tumors, and this condition represents an extremely unmet medical need.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, known for its lethality and lack of response to current treatments. For patients with GBM, treatment options have not improved substantially in recent years, and there has been minimal improvement in survival prospects. For GBM, the average life expectancy after symptom onset is approximately 6-12 months. In addition, there are no drugs approved for the treatment of metastatic brain tumors, which have an average life expectancy of 4-6 months after symptom onset. Therefore, there is an urgent need to improve the treatment of brain cancer.

SUMMARY OF THE INVENTION

Some embodiments relate to a method of treating a brain tumor comprising administering an effective amount of Plinabulin to a subject in need thereof.

Some embodiments relate to a method of inhibiting proliferation of brain tumor cells comprising contacting the brain tumor cells with Plinabulin.

Some embodiments relate to a method of inducing apoptosis in brain tumor cells comprising contacting the brain tumor cells with Plinabulin.

Some embodiments relate to a method of inhibiting brain tumor progression comprising administering to a subject in need thereof an effective amount of Plinabulin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1a-1d show a proneural genetically engineered mouse model (GEMM) of glioblastoma (GBM) that mimics human pathology. Figure 1a shows a T2 MRI image of a human GBM exhibiting peritumoral edema; Figure 1b shows a T2 MRI image of a mouse GBM exhibiting peritumoral edema; Figure 1c shows an H&E-stained human GBM micrograph image showing hallmark pseudo-palisading necrosis and microvascular proliferation; Figure 1d shows an H&E-stained mouse GBM micrograph image showing hallmark pseudo-palisading necrosis and microvascular proliferation.

Figures 2A and 2B show T2-weighted MRI images. Figure 2A shows tumor size in mice with PDGF-induced gliomas treated with vehicle, temozolomide, or fractionated radiation; and Figure 2B shows survival of mice with PDGF-induced gliomas treated with vehicle, temozolomide, or fractionated radiation.

FIG3 shows the survival of mice bearing glioblastoma tumors treated with control and Plinabulin.

FIG4 shows the survival of mice bearing PDGF-induced gliomas characterized by expression of KRAS mutations treated with a combination of plinabulin, temozolomide and radiation, and a combination of temozolomide and radiation.

Detailed description of preferred embodiments

Plinabulin, i.e., (3Z,6Z)-3-benzylidene-6-{[5-(2-methyl-2-propyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione, is a synthetic analog of the natural compound Phenylahistin. Plinabulin can be readily prepared according to the methods and steps described in detail in U.S. Patent Nos. 7,064,201 and 7,919,497, which are incorporated herein by reference in their entireties. Some embodiments relate to the use of Plinabulin to treat brain cancer, including but not limited to metastatic brain tumors, anaplastic astrocytomas, glioblastomas multiforme, oligodendrogliomas, ependymomas, and mixed gliomas. Some embodiments relate to the use of Plinabulin to inhibit the proliferation of brain tumor cells. Some embodiments relate to the use of Plinabulin to induce apoptosis in brain tumor cells. Some embodiments relate to the use of Plinabulin to inhibit the progression of brain tumors. Some embodiments relate to the use of Plinabulin in combination with another therapeutic agent or radiation to inhibit the progression of brain tumors.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If multiple definitions of a term are used herein, the definition in this section will prevail unless otherwise stated.

As used herein, "subject" means a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird (such as chicken), as well as any other vertebrate or invertebrate.

The term "mammal" is used in its usual biological sense and thus specifically includes, but is not limited to, primates (including apes (chimpanzees, apes, monkeys) and humans), cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of therapeutic agent effective to alleviate or reduce the likelihood of onset of one or more symptoms of a disease or condition to an extent, and includes curing the disease or condition.

As used herein, "treat," "treatment," or "treating" refers to administering a compound or pharmaceutical composition to a subject for preventive and/or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who does not yet exhibit symptoms of a disease or condition but is susceptible to or otherwise at risk for a particular disease or condition, thereby reducing the likelihood that the patient will develop the disease or condition in the future. The term "therapeutic treatment" refers to treating a subject who already has a disease or condition.

The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compound and is not biologically or otherwise undesirable for pharmaceutical use. In many cases, the compounds disclosed herein are capable of forming acid salts and/or basic salts due to the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable salts can also be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, bases containing sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. In some embodiments, treatment of the compounds disclosed herein with an inorganic base results in the loss of labile hydrogen from the compound to provide salt forms comprising inorganic cations such as Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , and Ca ²⁺ . Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, specifically, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described in WO 87/05297 to Johnston et al., published September 11, 1987 (incorporated herein by reference in its entirety).

In some embodiments, the composition may further comprise one or more pharmaceutically acceptable diluents. In some embodiments, the pharmaceutically acceptable diluent may comprise (polyethylene glycol (15)-hydroxystearate). In some embodiments, the pharmaceutically acceptable diluent may comprise propylene glycol. In some embodiments, the pharmaceutically acceptable diluent may comprise kolliphor and propylene glycol. In some embodiments, the pharmaceutically acceptable diluent may comprise kolliphor and propylene glycol, wherein the kolliphor is approximately 40% by weight and the propylene glycol is approximately 60% by weight, based on the total weight of the diluent. In some embodiments, the composition may further comprise one or more other pharmaceutically acceptable excipients.

Standard pharmaceutical formulation techniques, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), which is incorporated herein by reference in its entirety, can be used to prepare the pharmaceutical compositions described herein. Accordingly, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of plinabulin or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof.

Other embodiments include co-administering Plinabulin and the additional therapeutic agent in separate compositions or in the same composition. Thus, some embodiments include a first pharmaceutical composition comprising: (a) a safe and therapeutically effective amount of Plinabulin or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof; and a second pharmaceutical composition comprising: (a) a safe and therapeutically effective amount of the additional therapeutic agent, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. Some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of Plinabulin or a pharmaceutically acceptable salt thereof; (b) a safe and therapeutically effective amount of the additional therapeutic agent; and (c) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

The pharmaceutical compositions described herein can be administered via any acceptable mode of administration for agents suitable for similar uses, including but not limited to oral, sublingual, buccal, subcutaneous, intravenous, intranasal, topical, transdermal, intradermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular. In the treatment of indications (which are the subject of preferred embodiments), oral and parenteral administration are commonly used.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except in cases where any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. In addition, various adjuvants, such as those commonly used in the art, may be included. Considerations for inclusion of various components in pharmaceutical compositions are described below, for example, in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which are incorporated herein by reference in their entirety.

Some examples of substances that can serve as pharmaceutically acceptable carriers or components thereof are: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as TWEENS; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline and phosphate buffered saline.

The compositions described herein are preferably provided in unit dosage form. As used herein, "unit dosage form" is a composition comprising a certain amount of compound or composition, which is suitable for use in a single dose to an animal (preferably a mammalian subject) according to good medical practice. However, a single or unit dosage formulation does not mean that the formulation is administered once a day or once per course of treatment. It is expected that such formulations are administered once a day, twice, three times or more, and can be administered for a period of time (e.g., about 30 minutes to about 2-6 hours) by infusion, or as a continuous infusion, and can be administered more than once during the course of treatment, although single administration is not particularly excluded. It will be appreciated by those skilled in the art that the formulation does not specifically anticipate the entire course of treatment, and that these decisions are left to the technical staff of the therapeutic field rather than the formulation field.

Useful compositions as described above can be any of a variety of suitable forms for a variety of routes of administration, for example, for oral, sublingual, oral, nasal, rectal, topical (including transdermal and intradermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular administration, or other parenteral administration routes. It will be appreciated by those skilled in the art that oral and nasal compositions include compositions administered by inhalation and prepared in available methods. Depending on the specific route of administration required, a variety of pharmaceutically acceptable carriers well known in the art can be used. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropes, surfactants, and encapsulating materials. Optional pharmaceutically active substances can be included that do not substantially interfere with the activity of the compound or composition. The amount of the carrier used in conjunction with the compound or composition is sufficient to provide the actual amount of material for the compound administered per unit dose. Techniques and compositions for preparing dosage forms useful in the methods described herein are described in the following (incorporated herein by reference in their entirety): Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including solid forms such as tablets, capsules (e.g., liquid gel capsules and solid gel capsules), granules, and bulk powders. Tablets can be compressed tablets, tablet grinds, enteric-coated, sugar-coated, film-coated tablets, or multiple compressed tablets containing suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings.

Pharmaceutically acceptable carriers suitable for preparing unit dosage forms for oral administration are well known in the art. Tablets typically contain conventional pharmaceutically compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; binders, such as starch, gelatin, and sucrose; disintegrants, such as starch, alginic acid, and croscarmellose; and lubricants, such as magnesium stearate, stearic acid, and talc. Glidants such as silicon dioxide can be used to improve the flow characteristics of the powder mixture. Colorants, such as FD&C dyes, can be added for appearance. Sweeteners and flavorings, such as aspartame, saccharin, menthol, mint, sucrose, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically contain one or more of the solid diluents disclosed above. The choice of carrier component depends on secondary considerations, such as taste, cost, and storage stability, which is not critical and can be easily made by those skilled in the art.

Oral compositions may also include liquid solutions, emulsions, suspensions, and the like. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions, and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth gum, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methylparaben and sodium benzoate. Oral liquid compositions may also contain one or more of the sweeteners, flavorings, and coloring agents disclosed above.

Such compositions may also be coated by conventional methods, typically using pH or time-dependent coatings, so that the subject composition is released in the gastrointestinal tract near the desired topical application, or released at different times to prolong the desired effect. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethylcellulose, Eudragit coatings, waxes, and shellac.

The compositions described herein may optionally include other pharmaceutically active substances.

Other compositions for achieving systemic delivery of the subject compounds include sublingual, oral, and nasal dosage forms. Such compositions typically contain one or more soluble filler materials, such as sucrose, sorbitol, and mannitol; and binders, such as gum arabic, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Glidants, lubricants, sweeteners, colorants, antioxidants, and flavorings disclosed above may also be included.

Liquid compositions are formulated (formulated for topical ophthalmic applications) so that they can be topically applied to the eye. Comfort should be maximized as much as possible, but sometimes optimal comfort may not be achieved due to formulation considerations (e.g., drug stability). Where comfort cannot be maximized, the liquid can be formulated so that the liquid is tolerable for patients using the topical ophthalmic application. Additionally, ophthalmologically acceptable liquids can be packaged for single use or contain preservatives to prevent contamination from repeated use.

For ophthalmic applications, solutions or medicines are typically prepared using physiological saline solutions as the primary vehicle. Suitable buffer systems may be used to maintain a comfortable pH for ophthalmic solutions. The formulations may also contain conventional pharmaceutically acceptable preservatives, stabilizers, and surfactants.

Preservatives that can be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. Useful surfactants are, for example, Tween 80. Similarly, a variety of useful vehicles can be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethyl cellulose, hydroxyethyl cellulose, and purified water.

Tonicity adjusting agents may be added as needed or convenient. These include, but are not limited to, salts (especially sodium chloride, potassium chloride), mannitol and glycerol, or any other suitable ophthalmologically acceptable tonicity adjusting agent.

A variety of buffers and methods for adjusting pH can be used, as long as the resulting formulation is ophthalmologically acceptable. For many compositions, the pH is between 4 and 9. Thus, buffers include acetate buffers, citrate buffers, phosphate buffers, and borate buffers. The pH of these formulations can be adjusted with acids or bases as needed.

Ophthalmically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

Other excipient components that may be included in ophthalmic formulations are chelating agents. A useful chelating agent is disodium ethylenediaminetetraacetate (EDTA), but other chelating agents may be used in place of or in combination with it.

For topical application, creams, ointments, gels, solutions or suspensions, etc. containing the compositions disclosed herein may be used. Topical formulations may generally include a pharmaceutical carrier, cosolvents, emulsifiers, penetration enhancers, preservative systems, and emollients.

For intravenous administration, compositions as herein described can be dissolved or dispersed in a pharmaceutically acceptable diluent (such as saline or glucose solution). Suitable excipients can also be included to reach the pH of expectation, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl and citric acid. In various embodiments, the pH of the final composition is 2 to 8, or preferably 4 to 7. Antioxidant excipients can include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea and EDTA. Other non-limiting examples of suitable excipients that appear in the final intravenous composition can include sodium phosphate or potassium phosphate, citric acid, tartaric acid, gelatin and carbohydrates such as glucose, mannitol and dextran. Other acceptable excipients are described in Powell et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entireties. Antimicrobial agents may also be included to obtain a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

The composition for intravenous administration can be provided to the caregiver in one or more solid forms and can be reconstituted with a suitable diluent such as sterile water, saline or dextrose in water immediately prior to administration. In other embodiments, the composition is provided as a ready solution for parenteral administration. In other embodiments, the composition is provided as a solution that requires further dilution prior to administration. In embodiments comprising administering a combination of a compound described herein with other agents, the combination is provided to the caregiver as a mixture, or the caregiver mixes the two agents prior to administration, or the two agents can be administered separately.

The actual dosage of the active compounds described herein will depend on the specific compound and the condition to be treated; the selection of appropriate dosages is within the skill of the art. In some embodiments, a single dose of plinabulin or other therapeutic agent can be about 5 mg/ ^m2 to about 150 mg/ ^m2 of body surface area, about 5 mg/ ^m2 to about 100 mg/ ^m2 of body surface area, about 10 mg/ ^m2 to about 100 mg/ ^m2 of body surface area, about 10 mg/ ^m2 to about 80 mg/ ^m2 of body surface area, about 10 mg/ ^m2 to about 50 mg/ ^m2 of body surface area, about 10 mg/ ^m2 to about 40 mg/ ^m2 of body surface area, about 10 mg/ ^m2 to about 30 mg/ ^m2 of body surface area, about 13.5 mg/ ^m2 to about 100 mg/ ^m2 of body surface area, about 13.5 mg/ ^m2 to about 80 mg/ ^m2 of body surface area, about 13.5 mg/ ^m2 to about 50 mg/ ^m2 of body surface area, about 13.5 mg/m2 to about 1 ... In ^{some embodiments} , the single dose of Plinabulin or ^{other therapeutic agent may be about 13.5 mg/m 2 to} about 30 mg/m ² of body surface area, about 13.5 mg/m ² to about 30 mg/m ² of body surface area, about 15 mg/m ² to about 80 mg/m ^{2 of body surface area, about 15 mg/m 2} to about 50 mg/m ² of body surface area, or about 15 mg/m ² to about 30 mg/m 2 of body surface area. In some embodiments, a single dose of Plinabulin or other therapeutic agent may be about 13.5 mg/m ² to about 30 mg/m ² of body surface area. In some embodiments, a single dose of Plinabulin or other therapeutic agent can be about 5 mg/ ^m2 , about 10 mg/ ^m2 , about 12.5 mg/ ^m2 , about 13.5 mg/ ^m2 , about 15 mg/ ^m2 , about 17.5 mg/ ^m2 , about 20 mg/ ^m2 , about 22.5 mg/ ^m2 , about 25 mg/ ^m2 , about 27.5 mg/ ^m2 , about 30 mg/ ^m2 , about 40 mg/ ^m2 , about 50 mg/ ^m2 , about 60 mg/ ^m2 , about 70 mg/ ^m2 , about 80 mg/ ^m2 , about 90 mg/ ^m2 , or about 100 mg/ ^m2 of body surface area.

In some embodiments, a single dose of Plinabulin or other therapeutic agent can be about 5 mg to about 300 mg, about 5 mg to about 200 mg, about 7.5 mg to about 200 mg, about 10 mg to about 100 mg, about 15 mg to about 100 mg, about 20 mg to about 100 mg, about 30 mg to about 100 mg, about 40 mg to about 100 mg, about 10 mg to about 80 mg, about 15 mg to about 80 mg, about 20 mg to about 80 mg, about 30 mg to about 80 mg, about 40 mg to about 80 mg, about 10 mg to about 60 mg, about 15 mg to about 60 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, or about 40 mg to about 60 mg. In some embodiments, a single dose of Plinabulin or other therapeutic agent can be about 20 mg to about 60 mg, about 27 mg to about 60 mg, about 20 mg to about 45 mg, or about 27 mg to about 45 mg. In some embodiments, a single dose of Plinabulin or other therapeutic agent can be about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg.

As long as the tumor remains under control and the regimen is clinically tolerated, the administration cycle can be a multi-week treatment cycle. In some embodiments, a single dose of Plinabulin or other therapeutic agent can be administered once a week, and preferably on day 1 and day 8 of a three-week (21-day) treatment cycle. In some embodiments, a single dose of Plinabulin or other therapeutic agent can be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily over a one-week, two-week, three-week, four-week, or five-week treatment cycle. Administration can be on the same day or on different days of each week in the treatment cycle.

The treatment cycle can be repeated as long as the regimen is clinically tolerated. In some embodiments, the treatment cycle is repeated n times, where n is an integer from 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a new treatment cycle can occur immediately after the previous treatment cycle is completed. In some embodiments, a new treatment cycle can occur within a period of time after the previous treatment cycle is completed.

In some embodiments, the compositions described herein can be used in combination with other therapeutic agents.In some embodiments, the compositions described herein can be administered or used in combination with treatments such as chemotherapy, radiation, and biotherapy.

Treatment

Some embodiments relate to a method of treating a brain tumor comprising administering to a subject in need thereof an effective amount of Plinabulin.

In some embodiments, the brain tumor can be selected from the group consisting of metastatic brain tumors, anaplastic astrocytomas, glioblastoma multiforme, oligodendrogliomas, ependymomas, meningiomas, mixed gliomas, and combinations thereof. In some embodiments, the brain tumor is glioblastoma multiforme. In some embodiments, the brain tumor is a metastatic brain tumor.

In some embodiments, the brain tumor can be selected from anaplastic astrocytoma, central neurocytoma, choroid plexus carcinoma, choroid plexus papilloma, choroid plexus tumor, dysembryoplastic neuroepithelial tumor, ependymal tumor, fibrillary astrocytoma, giant cell glioblastoma, glioblastoma multiforme, gliomatosis cerebri, gliosarcoma, hemangiopericytoma, medulloblastoma, medullary epithelioma, meningeal carcinomatosis, neuroblastoma, neurocytoma, oligoastrocytoma, oligodendroglioma, optic nerve sheath meningioma, pediatric ependymoma, pilocytic astrocytoma, pineoblastoma, pineocytoma, pleomorphic anaplastic neuroblastoma, pleomorphic xanthoastrocytoma, primary central nervous system lymphoma, sphenoid ridge meningioma, subependymoma giant cell astrocytoma, subependymoma, central nervous system myeloma, and trilateral retinoblastoma.

In some embodiments, the methods described herein may include administering another therapeutic agent. In some embodiments, another therapeutic agent may be temozolomide, bevacizumab, everolimus, carmustine, lomustine, procarbazine, vincristine, irinotecan, cisplatin, carboplatin, methotrexate, etoposide, vinblastine, bleomycin, dactinomycin, cyclophosphamide or ifosfamide. In some embodiments, another therapeutic agent may be temozolomide. In some embodiments, another therapeutic agent may be lomustine.

In some embodiments, the methods described herein may further comprise subjecting the subject to radiation therapy. In some embodiments, the radiation therapy may be whole brain irradiation, fractionated radiation therapy, and radiosurgery.

In some embodiments, the brain tumor is characterized by expression of a mutant form of KRAS. In some embodiments, the brain tumor is characterized by expression of a mutant gene that is not KRAS.

In some embodiments, the methods described herein may further comprise identifying a patient with a cancer characterized by mutant KRAS expression. In some embodiments, the methods described herein may further comprise identifying a patient with a cancer characterized by wild-type KRAS expression. In some embodiments, identifying a patient may comprise determining whether the patient has a KRAS mutation. Some embodiments relate to a method for treating cancer in a patient identified as having a KRAS mutation, the method comprising administering a pharmaceutically effective amount of Plinabulin to the patient, wherein the patient has been identified by: (i) collecting a sample from the patient; (ii) isolating DNA from the sample; (iii) amplifying a KRAS gene or a fragment thereof in the isolated DNA; and (iv) detecting the presence or absence of a mutation in the amplified KRAS gene, thereby determining whether the patient has a cancer characterized by a KRAS mutation.

Some embodiments relate to a method of inhibiting the proliferation of brain tumor cells, comprising contacting the brain tumor cells with Plinabulin. In some embodiments, contacting comprises administering an effective amount of Plinabulin to a subject having the brain tumor cells. In some embodiments, the brain tumor is glioblastoma multiforme.

Some embodiments relate to a method of inducing apoptosis in brain tumor cells, comprising contacting the brain tumor cells with Plinabulin. In some embodiments, the contacting comprises administering an effective amount of Plinabulin to a subject having the brain tumor cells. In some embodiments, the brain tumor is glioblastoma multiforme.

Some embodiments relate to a method of inhibiting brain tumor progression, comprising administering to a subject in need thereof an effective amount of Plinabulin.

The following examples are included to further illustrate the present invention. The examples should not be construed as specifically limiting the present invention. Variations of these examples within the scope of the claims are within the purview of those skilled in the art and are considered to fall within the scope of the present invention as described and claimed herein. The reader will recognize that those skilled in the art and those skilled in the art, having grasped this disclosure, will be able to prepare and use the present invention without the need for exhaustive examples.

Example

Example 1

The mouse glioma model used is a GEMM of PDGF-driven gliomas, which mimics the proneural molecular subset of glioblastoma (GBM). The model is based on somatic cell-specific gene transfer; the replication-competent ALV splice acceptor (RCAS) retroviral system allows the infusion of specific genetic alterations in a cell type-specific manner within a tightly regulated differentiation window. The RCAS/tv-a system uses RCAS retroviral vectors to infect genetically engineered mice to express the RCAS receptor (tv-a) in a specific cell population. Here, gliomas are generated by RCAS-mediated PDGF transfer to cells expressing nestin in the brain. Nestin is expressed in stem/progenitor cell populations of the brain and has been shown to be a marker for cancer stem cells located in the perivascular zone (PVN) of human and mouse brain tumors. When combined with Ink4a-arf-/- deletion, PDGF-driven gliomas show complete penetrance 4-5 weeks after infection. These tumors accurately mimic the "pro-neural" subtype of GBM, in which loss of CDKN2A (encoding p16INK4A and p14ARF) was observed in 56% of "pro-neural" human gliomas. As shown in Figures 1a-1d, tumor cell structures that define human gliomas, such as Scherer structures, microvascular proliferation, and pseudo-palisading necrosis, were recapitulated in this GEMM. Specifically, Figure 1a shows a T2MRI image of a human GBM showing peritumoral edema; Figure 1b shows a T2MRI image of a mouse GBM showing peritumoral edema; Figure 1c shows an H&E-stained human micrograph image of a GBM with hallmark pseudo-palisading necrosis and microvascular proliferation; and Figure 1d shows an H&E-stained mouse micrograph image of a GBM with hallmark pseudo-palisading necrosis and microvascular proliferation.

Glioma cells migrate along white matter trajectories, encircle neurons and blood vessels, and accumulate at the brain margins in the subpial space. In this respect, PDGF-driven glioma GEMMs closely resemble PVN-GBMs and represent an excellent experimental system to define the interactions between tumor and non-neoplastic cells within the tumor microenvironment.

As shown in Figures 2A and 2B, the PDGF-induced glioma model was used to determine the response to radiation and temozolomide. Figure 2A shows the tumor size of mice with PDGF-induced gliomas treated with vehicle, temozolomide, or fractionated radiation; and Figure 2B shows the survival rate of mice with PDGF-induced gliomas treated with vehicle, temozolomide, or fractionated radiation.

The mice with glioma were identified by symptoms and verified by T2-weighted MRI. Mice were treated with vehicle, 25mg/kg temozolomide every day for 12 days, or treated with 2Gy of dose-graded radiation for 5 days a week for 2 weeks (20Gy in total). The two top images in Fig. 2A show the growth of the tumor of untreated (vehicle), and by contrast, the tumor processed by temozolomide and irradiated was reduced in volume in the same time period. As seen in the survival curves of these corresponding mouse groups, these tumors recurred after treatment, and all animals died of recurrent tumors. Data show: 1) tested in this mouse model, 2) these treatments reflect the human condition on the impact of mouse survival, 3) all mice died of disease, and 4) the relatively uniform results of these mouse groups supported the use of this experimental paradigm to detect the survival differences in this study.

Produce mice with PDGF-induced gliomas using RCAS/tv-a. Mice are transgenic, for expressing the RCAS receptor (tv-a) from the nestin promoter and having ink4a/arf-/- and lox-stop-lox luciferase background, and they are infected with a combination of RCAS-PDGF or RCAS-PDGF and the RCAS-KRAS of the KRAS expressing G12D mutation. For independent PDGF, under this background, the tumor produced occurs in the first 4-5 weeks, while the tumor caused by the combination of PDGF and RCAS occurs as early as one week. The tumor has the histological characteristics of GBM and is identified by lethargy and poor grooming symptoms, MRI scans using T2-weighted sequences, or bioluminescent imaging with an IVIS system. For radiotherapy, mice are treated with 10Gy in the skull with a single dose every day. As shown in Figure 2B, the treatment extends the median survival of the mouse group with GBM by approximately 3 weeks. These treated mice began to gain weight, showed improved symptoms within a few days, and their MRI imaging characteristics showed stabilization or reduction in tumor size before recurring and dying.

Plinabulin was tested in mice with PDGF-induced gliomas of KRAS expressing G12D mutations. Nestin-tv-a/ink4a-arf-/- mice aged 4-6 weeks were anesthetized with isoflurane and injected with Df-1 cells transfected with RCAS-PDGF-B-HA, RCAS-KRAS. A stereotactic frame was used to inject 1 microliter of 2X10 ⁵ of RCAS-PDGF-B-HA/RCAS-KRAS into mice via a 26G (26-gauge) needle connected to a Hamilton syringe: a 1:1 mixture. The cells were injected into the right frontal cortex at a coordinate of 1.75mm for the anterior bregma, lat-0.5mm, and a depth of 2mm. The weight loss of mice was carefully monitored, and when a total of>0.3 grams was lost or external signs of a tumor were shown for 2 consecutive days, the mice were studied. In the KRAS group, mice were injected with 7.5mg/kg Plinabulin ip twice a week for 10 weeks. In a control group, mice were injected with only the Plinabulin diluent (40 wt% Kolliphor and 60 wt% propylene glycol). Mice were monitored for lethargy, hunched posture, loss of appetite, external signs of tumor growth, restlessness, weight loss, and overall failure to thrive. Mice were sacrificed when they lost more than 20% of their body weight, lost mobility, were unable to eat, or weighed less than 14 grams for males/12 grams for females. Mice were sacrificed using _CO2 , and brains were harvested and stored overnight in 10% neutral buffered formalin, then replaced with Flex 80 and stored at 4 degrees.

Figure 3 shows the survival rate of mice with glioblastoma having G12D Kras mutation.As shown in Figure 3, mice with PDGF-induced glioblastoma model generally had significantly better survival rate in the Plinabulin-treated group compared with the control group (p=0.001).

Example 2.

Mice with PDGF-induced gliomas expressing KRAS with the G12D mutation were prepared using the protocol according to Example 1 and used in this experiment. Nestin-tv-a/ink4a-arf-/- mice aged 4-6 weeks were anesthetized with isoflurane and injected with Df-1 cells transfected with RCAS-PDGF-B-HA and RCAS-KRAS. Using a stereotactic frame, mice were injected with 1 microliter of a 1:1 mixture of 2X10 ⁵ of RCAS-PDGF-B-HA/RCAS-KRAS via a 26G needle connected to a Hamilton syringe. The cells were injected into the right frontal cortex at coordinates of 1.75mm anterior bregma, 0.5mm lat and 2mm deep. The mice were carefully monitored for weight loss and studied when they lost >0.3 grams in total for 2 consecutive days or showed external signs of tumors.

Mice were enrolled in two study groups. One group was treated with a combination of temozolomide (TMZ), radiation, and plinabulin: radiation was administered at 10 gy/1, and TMZ and 7.5 mg/kg of plinabulin in plinabulin diluent were administered intraperitoneally twice weekly on Mondays and Thursdays for 10 weeks. The other group, the control group, was treated with a combination of TMZ and radiation: radiation was administered at 10 gy/1, and TMZ was administered intraperitoneally twice weekly on Mondays and Thursdays for 10 weeks. Mice were monitored for lethargy, hunched posture, decreased appetite, external signs of tumor growth, restlessness, weight loss, and overall failure to thrive. Mice were sacrificed when they lost more than 20% of their body weight, lost mobility, were unable to eat, or weighed less than 14 g for males and less than 12 g for females. Mice were sacrificed using _CO₂ ; brains were harvested and stored overnight in 10% neutral buffered formalin, then replaced with Flex 80 and stored at 4°C. As shown in FIG4 , mice with the PDGF-induced glioblastoma model generally had significantly better survival rates in the Plinabulin plus TMZ plus radiation treatment group compared to the control group that received TMZ plus radiation (p=0.0149).

Claims (6)

1. Use of punabulin in the preparation of a medicament for treating a brain tumor in a subject, wherein the brain tumor is a glioblastoma multiforme and the brain tumor is characterized by the expression of a mutated form of KRAS. 2. The use as described in claim 1, wherein the medicament further comprises additional therapeutic agents. 3. The use as described in claim 2, wherein the additional therapeutic agent is temozolomide. 4. The use as claimed in claim 1, wherein the subject undergoes radiotherapy. 5. The use as described in claim 2, wherein the subject undergoes radiotherapy. 6. Use of punabulin in the preparation of a medicament for inhibiting the progression of a brain tumor, wherein the brain tumor is a glioblastoma multiforme and the brain tumor is characterized by the expression of a mutated form of KRAS.