Combination Agents and Their Use in the Treatment of Cancer

Abstract

The present invention discloses a pharmaceutical product comprising L-2,4-diaminobutyric acid (DAB) and prazosin for use in the treatment of cancer by administration to a tumor. The present invention also discloses the use of said at least two active agents in the manufacture of a medicament, and a method for treating cancer comprising intratumoral administration of the pharmaceutical formulation using a novel delivery method.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to combinations, compositions thereof, and their use in the treatment of cancer, particularly when delivered by novel methods.

[0002] Cancer is a disease in which a group of cells exhibit the trait of uncontrolled growth. This means that the cells grow and divide beyond normal limits. The uncontrolled division (mitosis) of cancer cells means the supply of physiological amino acids, which are the building blocks of proteins; that is, without physiological amino acids, cell division (mitosis) cannot occur. Cells can also invade and destroy surrounding tissues. In addition, cancer cells can metastasize, which means that cancer cells spread to other parts of the body via the blood or lymph.

[0003] Most cancers are caused by abnormalities in the genetic material of cells. These abnormalities may be due to the effects of carcinogens. Other cancer-promoting genetic abnormalities may be acquired randomly through epigenetic factors or errors in DNA replication, or they may be inherited and therefore present in cells from birth.

[0004] Genetic abnormalities found in cancer usually affect two general classes of genes: cancer-promoting oncogenes are often activated in cancer cells and confer new properties to those cells, such as hyperactive growth and division, protection against programmed cell death, disregard for normal tissue boundaries, and the ability to establish and grow in diverse tissue environments.

[0005] Tumor suppressor genes are often inactivated in cancer cells, resulting in the loss of normal functions in cancer cells, such as accurate DNA replication, cell cycle control, orientation and adhesion within tissues, and interaction with defensive cells of the immune system.

[0006] There are many different types of cancer, and cancers are usually classified according to the type of tissue from which they originate.

[0007] Cancer is usually treated with one or more of the following: surgery, chemotherapy, radiation therapy, and immunotherapy, including monoclonal antibody therapy. The type of treatment depends on the location and grade of the tumor and the stage of the disease. The goal of treatment is to completely remove the cancer without damaging the rest of the body. Sometimes this can be achieved through surgery, but the effectiveness of surgery is often limited by the tendency of cancer to invade adjacent tissues or spread to distant sites through microscopic metastasis. The effectiveness of chemotherapy is often limited by the different ways in which cancer cells evade its mechanism of action and by toxicity to other tissues in the body. Radiation can also cause damage to normal tissues.

[0008] Cancer is known to affect many areas of the body, the most common types of cancer, for example, in order of incidence (Source: Estimated new cases 2018 by cancer type (both sexes combined) American Cancer Society - https: / / cancerstatisticscenter.cancer.org / #! / ): breast cancer; lung cancer; prostate cancer; colon melanoma; bladder cancer; non-Hodgkin's lymphoma; kidney cancer; uterine cancer; leukemia; pancreatic cancer; thyroid cancer; oral and pharyngeal cancer; liver cancer; multiple myeloma; stomach cancer; cancers of the brain, spinal cord and other nervous system; ovarian cancer; esophageal cancer; cervical cancer; laryngeal cancer; cancers of soft tissue (including the heart); gallbladder and other biliary tract (biliary gland). Cancers of the rectum, anus, anal canal, and anorectum, Hodgkin's lymphoma, vulvar cancer, vaginal, ureter, and other urinary tract cancers, eye and orbit cancer, bone cancer, and penile cancer, as well as (without ranking by incidence): neuroendocrine tumors (including carcinoid and other types), head and neck cancer, Kaposi's sarcoma, lymph node cancer, mesothelioma, skin cancer, soft tissue sarcoma, adrenal gland cancer, rhabdomyosarcoma, salivary gland cancer, and thymic carcinoma.

[0009] Tumors that develop in the brain can destroy or damage brain cells by causing inflammation, putting pressure on other parts of the brain, and inducing cerebral edema (swelling), which can lead to increased intracranial pressure (pressure inside the skull).

[0010] Approximately 4,300 people are diagnosed with brain tumors in the UK each year. Primary brain tumors are masses that result from the proliferation or uncontrolled growth of cells in the brain. Malignant primary brain tumors are most likely to cause problems by spreading into the normal brain tissue surrounding the malignant primary brain tumor, causing pressure and damage to surrounding areas of the brain. These tumors rarely spread to other parts of the body outside the brain. However, secondary brain tumors occur when cancer cells from other parts of the body, such as the lung or breast, spread to the brain.

[0011] Surgery is the treatment option of choice for many brain tumors. Some can be completely removed, but those that are deep or have infiltrated brain tissue may be debulked rather than removed.

[0012] Depending on the type of tumor involved, radiation therapy and chemotherapy may be recommended.

[0013] Glioma cell tumors often have a poor prognosis. Because of their characteristic diffuse infiltrative tumor growth, surgical removal of gliomas is often not possible, which greatly complicates the clinical management of glioma patients.

[0014] Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor, accounting for 52% of all primary brain tumor cases and 20% of all intracranial tumors. The median survival time is 12-18 months, with only 25% of glioblastoma patients surviving beyond one year and only 5% of patients surviving beyond five years.

[0015] Different approaches are being investigated to improve mortality rates in patients diagnosed with glioma. These include treatments that target glioma cells but leave normal cells unharmed, methods to limit the spread of cancer cells, and treatments that block molecules necessary for tumor maintenance.

[0016] However, despite all efforts to develop cancer treatments, there remains a need to realize new approaches for the management of cancer, particularly brain tumors.

[0017] More specifically, there remains a need for new, efficient and less toxic approaches for the treatment of cancer.

[0018] L-2,4 diaminobutyric acid (DAB) is a synthetic (non-physiological) cationic amino acid analogue that exhibits antitumor activity in vitro against human glioma cells (Ronquist G et al., Anticancer Res 4 (1984) 225-228; Panasci L et al., Cancer Chemother Pharmacol 21 (1988) 143-144). However, clinical use of L-2,4 diaminobutyric acid (DAB) has been limited to a small number of patient series (Ronquist et al., (1992) Acta Neurochirurgica 114, 8-11; Bergenheim et al., (2006) Journal of Neuro-Oncology 80, 285-293). Two clinical trials failed to demonstrate significant clinical benefit.

[0019] Prazosin is a clinically approved drug that has been used in clinical practice for over 40 years to treat hypertension (Cavero & Roach, 1980). Its use has expanded to the treatment of sleep disorders associated with benign prostatic hyperplasia, congestive heart failure, pheochromocytoma, post-traumatic stress disorder, and Raynaud's disease. For the treatment of hypertension, prazosin hydrochloride is administered orally at a recommended total daily dose of 20–40 mg, administered in divided doses. Prazosin has been reported to inhibit glioblastoma growth in mouse xenografts (Assad Kahn et al., 2016, EMBO Mol. Med. 8, 511–526), ​​but there have been no reports of its clinical use as an anticancer agent. Summary of the Invention

[0020] The present disclosure provides combinations, pharmaceutical products and methods for the treatment of cancer that overcome at least some of the problems outlined above. The combinations of the present invention are effective in inhibiting tumors, particularly brain tumors, developing in humans.

[0021] The present invention is based, at least in part, on data demonstrating a synergistic cytotoxic effect of L-2,4 diaminobutyric acid (DAB) in combination with prazosin against human glioma cell lines. Furthermore, the cytotoxicity is selective for malignant cells compared to non-malignant cells.

[0022] According to a first aspect, the present invention is a product comprising at least L-2,4 diaminobutyric acid (DAB) and prazosin as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of cancer.

[0023] According to a second aspect, the present invention is a composition comprising at least L-2,4 diaminobutyric acid (DAB) and prazosin for use in the treatment or prevention of cancer.

[0024] The combination of the present invention is effective in inhibiting tumor development, particularly in reducing the growth of brain tumors in a subject and limiting the spread of cancer cells. Advantageously, the combination of the present invention targets brain tumor cells while leaving normal cells unharmed. This is particularly important in the brain. The combination of two active agents according to the present invention may have one or more of the following advantageous properties: reduced adverse side effects; stability in an aqueous environment; selective cytotoxicity against cancer cells; and reduced cytotoxicity against non-malignant cells. In addition, the product may further comprise an additional therapeutic agent or may be administered to a subject together with an additional therapeutic agent.

[0025] Accordingly, the present disclosure provides a product comprising the active agents L-2,4-diaminobutyric acid (DAB) and prazosin for use in the treatment of cancer by intratumoral administration to tumors. Intratumoral administration of the combination allows for active drug concentrations in tumors that are not possible with systemic administration. Intratumoral administration also reduces adverse side effects resulting from systemic exposure to the active agents. Furthermore, use of the combination of the present invention results in fewer side effects than treatment with other anticancer drugs. Common adverse effects of chemotherapeutic agents include, for example, fatigue, hair loss, easy bruising and bleeding, infection, anemia (low red blood cell count), nausea and vomiting, appetite changes, constipation, diarrhea, mouth, tongue, and throat problems, such as sores and pain with swallowing, peripheral neuropathy or other nerve problems, such as numbness, tingling, and pain, skin and nail changes, such as dry skin and color changes, urinary and bladder changes and kidney problems, weight changes, chemo-brain, which can affect focus and concentration, mood changes, changes in libido and sexual function, and fertility issues.

[0026] Accordingly, the present disclosure provides a product comprising the active agents L-2,4-diaminobutyric acid (DAB) and prazosin for use in the treatment of cancer by intratumoral administration to a tumor.

[0027] The cancer to be treated may be any cancerous tumor or malignant tumor. The malignant tumor may be a tumor in the central nervous system or anywhere in the body suitable for intratumoral administration. Preferably, the cancer to be treated is a brain tumor.

[0028] Brain tumors are usually classified according to the location of the tumor and the type of cell in which the cancer develops.For example, different types of brain tumors include acoustic neuroma, astrocytoma, CNS lymphoma, ependymoma, hemangioblastoma, medulloblastoma, meningioma, glioma, mixed glioma, oligodendroglioma, pineal tumor and pituitary tumor.Glioma is a tumor of glial cells, and glial cells support and protect nerve cells in the brain.Glioma accounts for nearly half of all primary brain tumors and one-fifth of all primary spinal tumors.

[0029] The present disclosure further provides the use of a combination of at least L-2,4-diaminobutyric acid (DAB) and prazosin in the manufacture of a medicament for intratumoral administration to treat cancer.

[0030] According to another aspect, the combination for use according to the invention is administered intratumorally using novel drug delivery methods, which enhance the therapeutic effect of the combination.

[0031] In one embodiment, the method involves infusion of the combination of the present invention through one or more catheters left inside the tumor, tumor resection cavity, or biopsy site, with periodic interruptions for drainage and infusion.

[0032] Typically, the method involves infusion of the combination of the present invention through one or more catheters left inside the tumor, tumor resection cavity, or biopsy site at a specific flow rate (e.g., a total flow rate of about 3 ml / hour), with periodic interruptions for drainage and infusion of physiological isotonic solution and / or isotonic Tris buffer. Preferably, the buffer has a pH of 7.2 to 9.1. Preferably, the volume of isotonic solution injected is 1 to 2 ml. In a preferred embodiment, the volume of isotonic solution injected is equal to or less than the volume of drained fluid. Preferably, the interruptions include passive drainage of 1 to 2 ml for a few seconds or manual aspiration with a syringe, followed by infusion of 1 to 2 ml, or an interruption for a period of time, e.g., 5 minutes. In another preferred method, an Irraflow or any other fluid exchange catheter or catheter combination is left in place after surgery and activated periodically (e.g., every 2 hours) for about 5 minutes to exchange the intratumoral fluid environment as described above. In an additional preferred method for inoperable cases or patients where only a biopsy is planned, multiple infusion and fluid exchange catheters can be inserted through the craniotomy, positioned and planned to serve different tumor areas, resulting in optimal drug and buffer distribution.

[0033] The present disclosure further provides a method for treating cancer, comprising the step of intratumorally administering to a subject in need thereof a product described herein comprising L-2,4-diaminobutyric acid (DAB) and prazosin. The product is administered directly, i.e., intratumorally, to the tumor by a novel drug delivery method comprising infusion of the combination of the present invention (preferably at a total flow rate of about 3 ml / hr) through one or more catheters left within the tumor, tumor resection cavity, or biopsy site, preferably with periodic interruptions for drainage and infusion of 1-2 ml of isotonic Tris buffer (pH 7.2-9.1).

[0034] Periodic drainage of the extracellular fluid from the surgical wound and infusion of isotonic Tris buffer (pH 7.2-9.1) enhances the therapeutic effect.

[0035] Without being bound by theory, it is believed that intratumoral administration of a combination of DAB and prazosin using this delivery method results in increased interaction of the drug with the target cells. Thus, it is hypothesized that this method increases the interaction of the active drug molecules with the malignant cells by eliminating competing physiological amino acids (see below), inactive drug metabolites, and cellular debris from previous treatments that now act as a protective shield for the malignant cells, preventing new drug penetration.

[0036] Additionally, the effect of DAB may be enhanced if the concentration of competing extracellular amino acids is reduced by periodic "washing" of the wound, and the penetration of DAB and prazosin into the peritumoral tissue area where glioma-initiating cells responsible for tumor recurrence reside may be enhanced by the diffusion and convection forces that arise during the periodic drainage and infusion of the drug, buffer, and physiological fluids. Administration of this active drug in a Tris-buffered solution with a pH of 7.2 to 9.1, in conjunction with the neutral, preferably slightly alkaline, washout of the buffer, may alter the tumor microenvironment and enhance the efficacy of the active agent. It is well known that a lower extracellular pH in the tumor microenvironment correlates with better cancer survival and immune evasion (Calcinotto A et al., Cancer Res 72 (2012) 2746-2756; Azzarito T et al., Cancer Lett 356 (2015) 697-703). Acidic conditions also increase the release of so-called "exosomes" from tumor cells, which enhance tumor proliferation in a paracrine manner.

[0037] Various intratumoral methods of drug administration have been described for the treatment of gliomas. However, all of these experimental treatment methods described involve continuous or intermittent drug infusion, rather than the regularly alternating drainage or infusion of physiological fluids and / or slightly alkaline Tris-buffered solution, as in the present method.

[0038] The treatment method may also include the administration of an additional therapeutic agent.

[0039] Although the preferred subject is a human, the invention has application in veterinary medicine and the animal husbandry industry and therefore extends to non-human animals. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows the dose-response effect of DAB on the viability of cultured HMC3 cells. [Figure 2] FIG. 2 shows the dose-response effect of DAB on the viability of cultured LN-18 cells. [Figure 3] FIG. 3 shows the dose-response effect of prazosin on the viability of cultured HMC3 cells. [Figure 4] FIG. 4 shows the dose-response effect of prazosin on the viability of cultured LN-18 cells. [Figure 5] FIG. 5 shows the effects of DAB, prazosin, and the combination of DAB and prazosin on LN-18 cell viability. [Figure 6] FIG. 6 shows a comparison of the observed and predicted (if additive) effects on cell viability for the combination of DAB and prazosin. [Figure 7] FIG. 7 shows phase contrast micrographs of LN-18 cells before and after treatment with the DAB / prazosin combination. Detailed Description of the Invention

[0041] A pharmaceutical product comprising L-2,4-diaminobutyric acid (DAB) and prazosin as a combined preparation for separate, simultaneous, or sequential use is used in the treatment of cancer by intratumoral administration.

[0042] In the context of the present invention, unless otherwise specified, the term "treatment" may refer to curing or ameliorating the cancer condition, i.e., completely eliminating or reducing cancer recurrence, including delaying cancer recurrence (progression-free period, PFS), completely eliminating the tumor, inhibiting tumor growth, extending patient survival or improving quality of life.

[0043] The cancer to be treated may be, for example, bile duct cancer, bladder cancer, bone cancer, intestinal cancer (including colon and rectal cancer), brain cancer, breast cancer, cancer of the neuroendocrine system (commonly known as carcinoid), cervical cancer, eye cancer, esophageal cancer, head and neck cancer (this group includes carcinomas that begin in cells that form the lining of the mouth, nose, throat, ear canals or the surface layer covering the tongue), Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymph node cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

[0044] In certain embodiments, the cancer being treated is a brain tumor.

[0045] Brain tumors include acoustic neuromas, astrocytomas, CNS lymphomas, ependymomas, hemangioblastomas, medulloblastomas, meningiomas, gliomas, mixed gliomas, oligodendrogliomas, pineal tumors, and pituitary tumors. Gliomas are tumors of glial cells; these cells support and protect nerve cells in the brain. Gliomas account for nearly half of all primary brain tumors and one-fifth of all primary spinal tumors.

[0046] The treatment methods of the present invention are particularly useful when the brain tumor is a glial tumor, more specifically glioblastoma multiforme (GBM).

[0047] The terms "tumor" and "cancer" may be used interchangeably herein. Reference to "glioma" includes related brain cancers, including GBM, astrocytoma, anaplastic astrocytoma, mixed glioma, oligodendroglioma, or glioblastoma-initiating cell (GIC).

[0048] The "pharmaceutical product" is as defined in claim 1. In certain embodiments, the product comprises a combination of L-2,4-diaminobutyric acid (DAB) and prazosin.

[0049] "(Pharmaceutical) product" in the context of this invention also includes multi-dose compositions, such as two-dose compositions, in which the active agents L-2,4-diaminobutyric acid (DAB) and prazosin are provided separately and given or dispensed separately, or mixed together before dispensing. For example, a multi-dose pharmaceutical package may have two or three active agents maintained separately.

[0050] The product may also include at least one additional therapeutic agent.

[0051] The term "additional therapeutic agent," as used herein, is used in the broadest sense and includes any substance or mixture of substances other than L-2,4-diaminobutyric acid (DAB) and prazosin, which provides a clinical use or alters normal physical or cellular function when taken up or introduced into the body of an organism. An additional therapeutic agent may be any agent that provides a therapeutic or prophylactic effect, a compound that affects or is involved in tissue growth, cell proliferation, cell differentiation, a compound that can cause a biological effect such as an immune response, or a compound that can play any other role in one or more biological processes. In certain embodiments, the therapeutic agent may be selected from the group consisting of, for example, amino acids, peptides, polypeptides, proteins, polysaccharides, muteins, immunoglobulins, antibodies, cytokines (e.g., lymphokines, monokines, chemokines), blood clotting factors, hematopoietic factors, interleukins (1-18), interferons (e.g., β-IFN, α-IFN, and γ-IFN), erythropoietin, nucleases, tumor necrosis factors, colony stimulating factors (e.g., GCSF, GM-CSF, MCSF), insulin, anti-tumor agents and tumor suppressors, blood proteins, fibrin, thrombin, fibrinogen, synthetic thrombin, synthetic fibrin, synthetic fibrinogen, gonadotropins (e.g., FSH, LH, CG, etc.), hormones, and the like. growth factors (e.g., nerve growth factor, insulin-like growth factor); bone morphogenetic proteins, TGF-B, protein inhibitors, protein antagonists and protein agonists; nucleic acids, e.g., antisense molecules, DNA, RNA, RNAi; oligonucleotides; polynucleotides; cells, viruses, anti-tumor antibodies, antigens and antigen fragments, tumor cells and tumor cell fragments (fragments of tumors that can generate an immune response against the tumor), stem cells, exosomes and ribosomes.

[0052] Also, non-limiting examples of suitable additional therapeutic agents include analgesics / antipyretics (e.g., aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine, oxycodone, codeine, dihydrocodeine bitartrate, pentazocine, hydrocodone bitartrate, levorphanol, dihydrogen phosphate, benzodiazepine, benzophenone, benzocaine ... Flunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol, choline salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine citrate, methotrimeprazine, cinnamurene hydrochloride, and meprobamate; antiasthmatics (e.g., ketotifen and traxanox); antibiotics (e.g., neomycin, streptomycin, chloramphenicol, cephalosporins, ampicillin, penicillin, antidepressants (e.g., nefopam, oxypertine, doxepin, amoxapine, trazodone, amitriptyline, maprotiline, phenelzine, desipramine, nortriptyline, tranylcypromine, fluoxetine, imipramine, imipramine pamoate, isocarboxazid, trimipramine, and protriptyline); antidiabetic agents (e.g., biguanides and sulfonylurea derivatives); antifungals antihypertensives (e.g., propranolol, propafenone, oxprenolol, nifedipine, reserpine, trimethaphan, phenoxybenzamine, pargyline hydrochloride, deserpidine, diazoxide, guanethidine monosulfate, minoxidil, resinamine, sodium nitroprusside, rauwolfia serpentina), alseroxiron, and phentolamine); anti-inflammatory agents (e.g., (non-steroidal) indomethacin, ketoprofen, flurbiprofen, naproxen, ibuprofen, ramifenazone, piroxicam, (steroidal) cortisone, dexamethasone, fluazacort, deflazacort, celecoxib, rofecoxib, hydrocortisone, prednisolone, and prednisone);Antineoplastic drugs (e.g., cyclophosphamide, actinomycin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, gemcitabine, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, camptothecin and its derivatives, phenesterine, paclitaxel and its derivatives, docetaxel and its derivatives, vinblastine, vincristine, gossereline antianxiety agents (e.g., lorazepam, buspirone, prazepam, chlordiazepoxide, oxazepam, clorazepate dipotassium, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, chlormezanone, and dantrolene); immunosuppressants (e.g., cyclosporine, azathioprine, Thiopurines, mizoribine, and FK506 (tacrolimus); antimigraine medications (e.g., ergotamine, propranolol, isomethopene mucate, and dichloralphenazone); sedatives / hypnotics (e.g., barbiturates such as pentobarbital, pentobarbital, and secobarbital; and benzodiazepines such as flurazepam hydrochloride, triazolam, and midazolam); antianginal medications (e.g., beta-adrenergic blockers; calcium phosphate blockers such as nifedipine and diltiazem). channel blockers; and nitrates such as nitroglycerin, isosorbide dinitrate, pentaerythritol tetranitrate, and erythrityl tetranitrate); antipsychotics (e.g., haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride, thiothixene, fluphenazine, fluphenazine decanoate, fluphenazine enanthate, trifluoperazine, chlorpromazine, perphenazine, lithium citrate, and prochlorperazine); antimanic agents (e.g., lithium carbonate);antiarrhythmics (e.g., bretylium tosylate, esmolol, verapamil, amiodarone, encainide, digoxin, digitoxin, mexiletine, disopyramide phosphate, procainamide, quinidine sulfate, quinidine gluconate, quinidine polygalacturonate, flecainide acetate, tocainide, and lidocaine); antiarthritic drugs (e.g., phenylbutazone, sulindac, penicillamine, salsalate, piroxicam, azathioprine, indomethacin, meclofenamic acid, gold sodium thiomalate, ketoprofen, auranofin, aurothioglucose, and tolmetin sodium; antigout drugs (e.g., colchicine and allopurinol); anticoagulants (e.g., heparin, heparin sodium, and warfarin sodium); thrombolytic agents (e.g., urokinase, streptokinase, and alteplase); antifibrinolytic agents (e.g., aminocaproic acid); hemorheological agents (e.g., pentoxifylline); antiplatelet agents (e.g., aspirin, thrombus dissolving agent ... pyridine); anticonvulsants (e.g., valproic acid, divalproex sodium, phenytoin, phenytoin sodium, clonazepam, primidone, phenobarbital, carbamazepine, amobarbital sodium, methsuximide, metharbital, mephobarbital, mephenytoin, phensuximide, paramethadione, ethotoin, phenacemide, secobarbital sodium, clorazepate dipotassium, and trimethadione); antiparkinsonian agents (e.g., antihistamines / antipruritics (e.g., hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine maleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate, triprolidine, carbinoxamine, diphenylpyraline, phenindamine, azatadine, tripelennamine, dexchlorphenirnine maleate, methdilazine, and others); agents useful in calcium regulation (e.g., calcitonin and parathyroid hormone);antibacterial agents (e.g., amikacin sulfate, aztreonam, chloramphenicol, chloramphenicol palmitate, ciprofloxacin, clindamycin, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, lincomycin hydrochloride, tobramycin sulfate, vancomycin hydrochloride, polymyxin B sulfate, colistimethate sodium, and colistin sulfate); antiviral agents (e.g., interferon alpha, beta, or gamma, zidovudine antimicrobials (e.g., cefazolin sodium, cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium, cefoperazone sodium, cefotetan disodium, cefuroxime axetil, cefotaxime sodium, cefadroxil monohydrate, cephalexin, cephalothin sodium, cephalexin hydrochloride monohydrate, cefamandole nafate, cefoxitin sodium, cefonicid sodium, ceforanide cephalosporins such as ceftriaxone sodium, ceftazidime, cefadroxil, cephradine, and cefuroxime sodium; ampicillin, amoxicillin, penicillin G benzathine, cyclacillin, ampicillin sodium, penicillin G potassium, penicillin V potassium, piperacillin sodium, oxacillin sodium, bacampicillin hydrochloride, cloxacillin sodium, ticarcillin disodium, azlocillin sodium, carbenicillin indanyl sodium, and penicillin G procaine penicillins such as methicillin sodium, and nafcillin sodium; macrolides such as azithromycin, clarithromycin, and erythromycins such as erythromycin ethylsuccinate, erythromycin, erythromycin estolate, erythromycin lactobionate, erythromycin stearate, and erythromycin ethylsuccinate; and tetracyclines such as tetracycline hydrochloride, doxycycline hydrate, and minocycline hydrochloride; anti-infectives (e.g., GM-CSF);Bronchodilators (e.g., sympathomimetics such as epinephrine hydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetharine, isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate, albuterol, bitolterol mesylate, isoproterenol hydrochloride, terbutaline sulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine, and epinephrine bitartrate); anticholinergics such as ipratropium bromide; xanthines such as aminophylline, dyphylline, metaproterenol sulfate, and theophylline; Mast cell stabilizers such as cromolyn sodium; inhaled corticosteroids such as beclomethasone dipropionate (BDP) and beclomethasone dipropionate monohydrate; salbutamol; ipratropium bromide; budesonide; salmeterol; xinafoic acid; triamcinolone; nedocromil sodium; flunisolide; fluticasone propionate; steroidal compounds and hormones (e.g., danazol, testosterone cypionate, fluoxymesterone, ethyltestosterone, testosterone enanthate, methyltestosterone) androgens such as progesterone; estrogens such as estradiol, etropipate, and conjugated estrogens; progestins such as methoxyprogesterone acetate and norethindrone acetate; triamcinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate thyroid hormones such as flucloxin, hydrocortisone sodium succinate, triamcinolone hexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tebutate, prednisolone acetate, prednisolone sodium phosphate, and levothyroxine sodium; hypoglycemic agents (e.g., human insulin, purified beef insulin, purified pork insulin, glyburide, metformin, chlorpropamide, glipizide, tolbutamide, and tolazamide);Lipid-lowering drugs (e.g., clofibrate, dextrothyroxine sodium, probucol, pravastatin, atorvastatin, lovastatin, and niacin); proteins (e.g., DNase, arginase, superoxide dismutase, and lipase); nucleic acids (e.g., sense or antisense nucleic acids encoding therapeutically useful proteins, including any of the proteins described herein); agents useful for stimulating erythropoiesis (e.g., erythropoietin); antiulcer / antireflux agents (e.g., famotidine, cimetidine, and ranitidine hydrochloride); antiemetic / antiemetic agents (e.g., meclizine hydrochloride, nabituric acid, riboflavin ... fluprexa, fluprexa, fluprexa (promethazine, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, and scopolamine), and other drugs useful in the compositions and methods described herein include mitotane, halonitrosourea, anthrocyclines, ellipticine, ceftriaxone, ketoconazole, ceftazidime, oxaprozin, valacyclovir, urofollitropin, famciclovir, flutamide, enalapril, itraconazole, buspirone, gabapentin, fosinopril, tramadol, acarbose, lorazepam, follitropin, omeprazole; , fluoxetine, lisinopril, tramadol, levofloxacin, zafirlukast, interferon, growth hormone, interleukin, erythropoietin, granulocyte-stimulating factor, nizatidine, bupropion, perindopril, erbumine, adenosine, alendronate, alprostadil, benazepril, betaxolol, bleomycin sulfate, dexfenfluramine, diltiazem, fentanyl, flecainide, gemcitabine, glatiramer acetate, granisetron, lamivudine, mangafodipir trisodium, mesalamine, metoprolol fumarate, metronidazole, miglitol, moexipril, monteleukast, octreotide acetate, olopatadine, paclitaxel ricalcitol, somatropin, sumatriptan succinate, tacrine, verapamil, nabumetone, trovafloxacin, dolasetron, zidovudine, finasteride, tobramycin, isradipine, tolcapone, enoxaparin, fluconazole, lansoprazole, terbinafine, pamidronate, didanosine, diclofenac, cisapride, venlafaxine, troglitazone, fluvastatin, losartan, imiglucerase, donepezil, olanzapine, valsartan, fexofenadine, calcitonin, and ipratropium bromide, including combinations thereof, and alternate forms such as alternate salt forms, free acid forms, free base forms, prodrugs, and hydrates.

[0053] In some embodiments, the additional therapeutic agent may be water soluble. In some embodiments, the additional therapeutic agent may not be water soluble.

[0054] In some embodiments, the therapeutic agent may be selected from corticosteroids such as triamcinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate, hydrocortisone sodium succinate, triamcinolone hexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tebutate, prednisolone acetate, and prednisolone sodium phosphate.

[0055] Various forms of other therapeutic agents can be used, including but not limited to uncharged molecules, molecular complexes, salts, ethers, esters, amides, etc., which become biologically active upon injection or insertion into the tumor.

[0056] The terms "effective amount," "effective concentration," and "therapeutically effective amount" of at least two active agents, as used herein, refer to a sufficient amount of at least two agents to provide a desired therapeutic or physiological effect or outcome. Such effects or outcomes include inhibiting the proliferation or viability of cancer-related cells. Undesirable effects, such as side effects, may occur along with the desired therapeutic effect. Therefore, a physician must balance the potential benefits and potential risks when determining what an appropriate "effective amount" is. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, mode of administration, etc. Therefore, it may not be possible to specify an exact "effective amount" or "effective concentration." However, an appropriate "effective amount" or "effective concentration" in any individual case can be determined by one of ordinary skill in the art using only routine experimentation.

[0057] An effective amount is considered to be the amount of at least two active agents required to inhibit the proliferation or viability of cells associated with cancer. An effective concentration (e.g., when the product is administered intratumorally) is considered to be the concentration of at least two active agents of the product required to inhibit the proliferation or viability of cells associated with cancer.

[0058] According to certain embodiments, for intratumoral delivery, effective concentrations of the combination of L-2,4-diaminobutyric acid (DAB) and prazosin include about 50 mM to about 300 mM L-2,4-diaminobutyric acid (DAB) and about 1 μM to about 500 μM (500 μM) prazosin, respectively.

[0059] The combinations for use according to the invention are administered by a novel drug delivery method, which involves infusion of the combination through one or more catheters left inside the tumor, tumor resection cavity, or biopsy site at a total flow rate of about 3 ml / hour, with periodic interruptions for repeated drainage and infusion of 1-2 ml of physiological isotonic solution and / or isotonic Tris buffer, pH 7.2-9.1. Typically, the number of drainage and infusion interruptions is based on the individual parameters of the patient.

[0060] Administration may be continuous or periodically interrupted. Active agent administration may be simultaneous or separate in any order. Doses may be administered intratumorally through one or more catheters at different flow rates over a specific period, e.g., several days. To maximize the active drug-target cell interface, periodic irrigation of the tumor bed with alkaline Tris buffer (pH 7.2-9.1) to flush out cellular debris and inactive metabolites through one of the indwelling catheters is recommended.

[0061] The flow rate of administration may vary within a wide range. Lower flow rates are used for continuous administration, while intermittent administration usually requires higher to much higher flow rates. Typically, flow rates of 1 μl / min to 3 ml / sec, particularly 1 μl / min to 2 ml / sec, or 5 μl / min to 1 ml / sec, or 10 μl / min to 0.5 ml / sec may be used.

[0062] "Treating" a subject includes altering the natural course or biological behavior of cancer in an affected subject, as well as treating clinically asymptomatic subjects with possible tumors or biochemical or immunological markers of developing tumors for the benefit of the subject. In a specific embodiment, the present invention contemplates reducing the proliferation or viability of cells associated with cancer.

[0063] As used herein, "subject" refers to an animal, preferably a mammal, more preferably a primate, including lower primates, and even more preferably a human, that can benefit from the products and methods of the present invention. A subject, whether human or non-human, may be referred to as an individual, patient, animal, host, or recipient. The agents and methods of the present invention have applications in human medicine, veterinary medicine, and general husbandry of domestic or wild animals. For convenience, "animal" includes avian species such as poultry (including ducks, chickens, turkeys, and geese), domestic birds, or game birds. Conditions in non-human animals do not occur naturally, but may be induced, such as in animal models.

[0064] As indicated above, preferred animals are humans, non-human primates such as marmosets, baboons, orangutans, lower primates such as tupaia, livestock animals, laboratory test animals, pet animals, or captive wild animals. Humans are the most preferred target. However, non-human animal models may also be used. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs, and hamsters. Rabbits and rodents, such as rats and mice, provide convenient test systems or animal models, as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses, and donkeys. Non-mammals such as avian species, zebrafish, amphibians (including cane toads), and Drosophila species such as Drosophila melanogaster are also contemplated. Instead of live animal models, test systems may also include tissue culture systems.

[0065] In certain embodiments, the subject is preferably a human with suspected brain tumor, a treatment-naive human, or a previously treated human who has undergone a standard biopsy or surgical craniotomy and decompression under local or general anesthesia, with or without subsequent radiation therapy and / or chemotherapy. More preferably, the subject is a subject with suspected glioblastoma, a treatment-naive human, or a previously treated human who has undergone a standard biopsy or surgical craniotomy and decompression under local or general anesthesia.

[0066] In some embodiments, the subject is preferably a human who is suitable for tumor removal or has had a brain tumor completely resected.

[0067] In some other embodiments, subjects suitable for treatment according to the present invention may present with tumors deep within the brain or may have tumors that have infiltrated brain tissue and therefore have been debulked rather than removed.

[0068] Pharmaceutical preparations The formulations described herein, which include L-2,4-diaminobutyric acid (DAB) and prazosin, are for use in the treatment of cancer.

[0069] The formulation may further comprise one or more pharmaceutically acceptable carriers in which the active agent is mixed, dissolved, suspended, emulsified, etc.

[0070] According to certain embodiments, the formulations of the invention comprise one or more additional therapeutic agents.

[0071] "Pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, buffers, etc., preferably compatible with intratumoral administration. Particularly useful examples of such carriers include, but are not limited to, water, saline, buffers (Tris, Hanks, Sorenson), surfactants (Tween, Pluronic, etc.), dispersants, lactated Ringer's solution, and dextrose solution. For intratumoral injection, Tris-buffered water or phosphate-buffered saline may be suitable carriers for the compositions described herein. It is contemplated that the volume of the pharmaceutical carrier may vary according to the particular active agent selected and the solubility of the active agent in the carrier.

[0072] The formulations described herein are formulated to be compatible with intratumoral administration. Solutions, suspensions, dispersions, emulsions, mixtures, etc. may be used for such administration and may contain sterile diluents such as water for injection, saline solution, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as tris (tris(hydroxymethyl)aminomethane), acetate, citrate, or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH of the formulations may vary between 7.2 and 8.1, preferably between 7.5 and 7.7, and more preferably between 7.55 and 7.60. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

[0073] An additional therapeutic agent can be incorporated into the formulation of the present invention for intratumoral administration along with L-2,4-diaminobutyric acid (DAB) and prazosin in some instances. The therapeutic agent can be incorporated into the formulation in any physical form suitable for intratumoral delivery. Some non-limiting examples include powders, particles, fibers, beads, microspheres, solutions, suspensions, and the like. In other embodiments, the additional therapeutic agent is not incorporated into the formulation of the present invention but is administered intratumorally in a separate manner, e.g., from a separate formulation.

[0074] The formulations of the present invention may be prepared by methods well known in the art of pharmacy and may conveniently be presented in unit dosage form for intratumoral administration, for example, in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0075] Thus, some preparation methods involve separately associating each of the active agents, L-2,4-diaminobutyric acid (DAB) and prazosin, with one or more carriers.

[0076] In some other methods, the two active ingredients, L-2,4-diaminobutyric acid (DAB) and prazosin, are associated with one or more carriers.

[0077] Typically, the formulation is prepared by a manufacturing process well known to those skilled in the art. The manufacturing process may include a step of mixing, dissolving, emulsifying, suspending, etc., the active agent in a liquid carrier, optionally containing other excipients such as pH buffers and preservatives, followed by a step of sterilization. Any suitable sterilization method can be used, including chemical sterilization such as steam sterilization, sterile filtration, exposure to ethylene oxide, and radiation sterilization such as gamma irradiation, electron beam treatment, and ultraviolet irradiation. The formulation is then packaged in a unit dosage form for intratumoral administration, for example, in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0078] L-2,4-diaminobutyric acid (DAB) is soluble in aqueous media. However, prazosin is not. For this reason, to prepare the formulations of the present invention, these active agents may first be dissolved in an appropriate amount of organic solvent, and then the resulting solution may be diluted with an aqueous carrier or DAB solution. Examples of organic solvents used to dissolve prazosin include DMSO, dimethylformamide, ethanol, methanol, etc. Therefore, organic solvents may be present in the formulation in reduced amounts, typically between 0.01% and 2% by weight, and optionally between 0.01% and 1% by weight, based on the total weight of the formulation.

[0079] According to some embodiments, the method for preparing the formulation of the present invention comprises: a) dissolving L-2,4-diaminobutyric acid (DAB) in an aqueous carrier; b) dissolving prazosin in an organic solvent and diluting the resulting solution with an aqueous carrier; Includes.

[0080] In some embodiments, the method comprises steps a) and b) to obtain a formulation comprising L-2,4-diaminobutyric acid (DAB) and prazosin. Optionally, the aqueous carrier used in b) is the product obtained in step a), e.g., an aqueous solution comprising L-2,4-diaminobutyric acid (DAB).

[0081] The formulations may contain different concentrations of each active agent.

[0082] There is no critical upper limit to the amount of active agent incorporated into the formulation, except for the amount of solution or dispersion that is acceptable for maintaining the desired physical properties of the composition.The lower limit of the active agent incorporated into the delivery system depends on the activity of the drug and the length of time required for treatment.Therefore, the amount of active agent should not be so small that it cannot produce the desired therapeutic effect, and should not be so large that the active agent is released in an uncontrollable manner.

[0083] The concentrations of each active agent may vary over a wide range and are not particularly limited. However, typically, the concentration of L-2,4-diaminobutyric acid (DAB) is 50 mM to 300 mM, and the concentration of prazosin is 1 μM to 500 μM to maintain the physiological amino acid concentration in the tumor microenvironment.

[0084] In some embodiments, the concentration of L-2,4-diaminobutyric acid (DAB) is between 75 mM and 145 mM, and the concentration of prazosin is between 10 μM and 100 μM.

[0085] In some other embodiments, the concentration of L-2,4-diaminobutyric acid (DAB) is between 80 and 140 mM, preferably 130 mM, and the concentration of prazosin is between 20 and 80 μM, preferably 50 μM.

[0086] As mentioned above in the context of the present invention, the products of the present invention also include multi-dose compositions, such as two-dose compositions in which the active agents L-2,4-diaminobutyric acid (DAB) and prazosin are provided separately and given or dispensed separately, or mixed together before dispensing. For example, a multi-dose pharmaceutical pack may have two or three active agents maintained separately.

[0087] Additionally, L-2,4-diaminobutyric acid (DAB) and prazosin may be administered to a subject by different routes.

[0088] The products and compositions of the present invention may be included in a kit, container, pack, or dispenser along with a medical device suitable for delivering the composition intratumorally. The compositions included in the kit may be provided in any type of container, ensuring that the shelf life of the different components is preserved and that they are not adsorbed or altered by the container material. For example, a sealed glass ampoule or vial may contain the compositions described herein packaged under a neutral, non-reactive gas, such as nitrogen. The ampoule may be made of any suitable material, such as glass, an organic polymer (e.g., polycarbonate, polystyrene, etc.), ceramic, metal, or any other material commonly used to hold reagents. Other examples of suitable containers include bottles made from materials similar to ampoules and foil-lined jackets, such as aluminum or alloy foils. Other containers include test tubes, vials, flasks, bottles, syringes, etc. Some containers may have a sterile, resealable access port, such as a bottle with a stopper that may be repeatedly pierced by a hypodermic needle.

[0089] The pharmaceutical product according to the present invention can be administered intratumorally at a therapeutically effective dose.The effective amount for this use depends, of course, on the severity of the disease and the subject's weight and general condition.For the treatment of a subject, different daily doses of a combination of at least two active ingredients selected from L-2,4-diaminobutyric acid (DAB) and prazosin can be administered depending on the activity of the active agent, the nature and severity of the disorder, and the subject's age and weight.However, under certain circumstances, higher or lower daily doses may be appropriate.

[0090] The product may be administered by a variety of methods, for example, the product may be injected directly into the solid tumor being treated with a needle such as a Turner Biopsy Needle or a Chiba Biopsy Needle, or may be injected directly through a catheter with different flow rates after craniotomy and decompression, or through a burr hole craniotomy, depending on the condition of the subject being treated.

[0091] In some embodiments, the product is administered intratumorally via a catheter previously inserted into the subject, with the rate of administration being set taking into account the subject's condition, such as the subject's intracranial, intraperitoneal, or intrathoracic pressure, as well as other factors such as the positioning of the catheter tip and the subject's general or neurological condition.

[0092] Typically, the flow rate for administration of the product may vary within wide limits and is usually between 1 μl / min and 3 ml / sec, in particular between 50 μl / min and 2 ml / sec, or between 100 μl / min and 1 ml / sec, or between 250 μl / min and 0.5 ml / sec.

[0093] Further provided is a method of treating cancer comprising the step of intratumorally administering to a subject in need thereof a product comprising L-2,4-diaminobutyric acid (DAB) and prazosin.

[0094] According to some embodiments, the method of treatment comprises intratumoral administration of L-2,4-diaminobutyric acid (DAB) and prazosin.

[0095] The administration of the different combinations of active agents in the treatment method of the present invention may be in any order.

[0096] The treatment method of the present invention is administered to a subject in need thereof for a period of time, e.g., several days, which may vary depending on the activity of the active agent, the nature and severity of the disorder, the age and weight of the subject, or different daily doses of L-2,4-diaminobutyric acid (DAB) and prazosin. Typically, treatment is administered for a period of between 2 and 20 days, e.g., between 4 and 16 days, or between 7 and 14 days.

[0097] The same or different combinations of the two active agents may be given during any given treatment period.

[0098] The aforementioned "subject" is preferably a mammal, more preferably a human, who can benefit from the products and methods of the present invention, also referred to as a patient.

[0099] In certain embodiments, the patient is a human suspected of having glioblastoma, a treatment-naive human, or a human who has previously been treated for cancer, who undergoes a standard biopsy after craniotomy under local or general anesthesia.

[0100] After the biopsy sample is taken, a catheter(s) is implanted along the same path as the biopsy stylet to minimize trauma. Instead, the catheter is implanted so that it is biased to create a pressure gradient when used for injection and drainage. After confirmation of the diagnosis by frozen section, the combination drug is administered at a flow rate based on the patient's intracranial pressure, clinical course, and neuroradiological imaging of tumor necrosis and cerebral edema.

[0101] In some other embodiments, the patient is a human suitable for tumor removal undergoing standard surgery under general anesthesia, with the catheter(s) left in the tumor resection bed. After confirmation of the diagnosis by frozen section, the therapeutic solution is administered at a flow rate based on the patient's intracranial pressure, clinical course, and neuroradiological imaging of residual tumor necrosis and cerebral edema.

[0102] In certain embodiments, the patient is a human with suspected somatic cancer, a treatment-naive human, or a human who has previously been treated for cancer, who undergoes a standard, ultrasound- or CT-guided percutaneous biopsy under local or general anesthesia.

[0103] After the biopsy sample is taken, the catheter(s) are implanted along the same path as the biopsy stylet to minimize trauma. Alternatively, the catheters are implanted so that they are biased to create a pressure gradient when used for infusion and drainage. After confirmation of the diagnosis by frozen section, product administration is initiated at a flow rate based on the patient's individual parameters.

[0104] In some other embodiments, the patient is a human suitable for bodily tumor removal undergoing standard surgery under general anesthesia, with the catheter(s) left in the tumor resection bed. After confirmation of the diagnosis by frozen section, the therapeutic solution is administered at a flow rate based on the patient's individual parameters.

[0105] By adopting the product and delivery method described herein, at least two active agents can be initially localized within the borders of tumor, thereby enhancing the local effect of the active agent contained in tumor, while the blood level of the active agent outside the tumor periphery remains low.In this way, enhanced therapeutic effect can be achieved.The therapeutic effect on tumor cells is greater, and in addition, sensitive normal cells far from the administration site can be more or less affected.Therefore, the active agent is used in a less systemic way, that is, a way that is more specifically directed to cancerous cells, but simultaneously protects sensitive normal cells both near and far from the cancerous cell site.

[0106] The treatment methods of the present invention may also include the step of administering one or more additional therapeutic agents.

[0107] An additional therapeutic agent may, in some instances, be incorporated into the formulation of the present invention for intratumoral administration. The therapeutic agent may be incorporated into the product in any physical form suitable for intratumoral delivery. Some non-limiting examples include powders, particles, fibers, beads, microspheres, solutions, suspensions, etc. In other embodiments, the additional therapeutic agent is not incorporated into the formulation of the present invention, but is also administered intratumorally from a separate formulation, either simultaneously or sequentially.

[0108] In other examples, the additional therapeutic agents may be administered simultaneously or sequentially by any other different route of administration, eg, oral, topical, parenteral, intravenous, inhalation, etc.

[0109] Examples of therapeutic agents that may be utilized in accordance with the present disclosure are provided above.

[0110] Generally, the additional therapeutic agent may be administered throughout the entire course of treatment, at intervals during treatment, or even at some point during treatment.

[0111] The additional therapeutic agent may be used in a therapeutically effective amount, which will vary widely depending largely on the particular additional therapeutic agent used.

[0112] In some embodiments, additional therapeutic agents can be administered intratumorally through the same or different lumens of the catheter(s). This depends, for example, on the number of catheters, the positioning of the catheter tips, the subject's intracranial pressure, and other factors. The flow rate of administration can vary within a wide range, typically 1 μl / min to 3 ml / sec, particularly 5 μl / min to 2 ml / sec, or 100 μl / min to 1 ml / sec, or 250 μl / min to 0.5 ml / sec.

[0113] Without wishing to be bound by any theory, the inventors believe that a major reason that previous attempts to deliver local chemotherapy to gliomas have lacked clinical efficacy is that previous drug action generates large amounts of inactive drug metabolites and cellular debris that protect malignant cells; i.e., the absence of transport proteins in the extracellular space of the central nervous system, and the elevated intracranial pressure and disorganized cytoarchitecture of glioblastomas contribute to the maintenance of this inert fluid between the tip of the infusion catheter and the adjacent malignant cell layer.

[0114] Thus, in some embodiments, the therapeutic method of the present invention may include one or more steps of draining and washing fluid around the tip(s) of the catheter(s) to achieve maximum contact of the active agent with the target cells (also enhanced by diffusive and convective forces arising during the periodic drainage and infusion of drug and physiological fluids) and to reduce local pressure and acidity—generated from cellular debris and inactive products of previous drug-tissue interactions. In this way, the concentration of the active agent in contact with the membrane of malignant cells remains high, and the interaction of the active agent with the cells will occur in an improved physicochemical environment, especially when an isotonic Tris buffer (pH 7.2-9.1) is used to reverse the acidity of the extracellular space of tumors.

[0115] These steps of the drainage and irrigation period are especially recommended in subjects where surgical resection of the tumor is not possible or is suboptimal.

[0116] The present invention is based at least in part on the following research.

[0117] 1. Preparation of the formulation: 130 mM L-2,4-diaminobutyric acid (DAB) and 50 μM prazosin L-2,4-diaminobutyric acid and prazosin are commercially available from several approved vendors.

[0118] L-2,4-diaminobutyric acid (DAB, dihydrochloride salt form, MW 191.06) was dissolved in water to a final concentration of 130 mM. Prazosin was first dissolved in DMSO to a concentration of 25 mM, and to the resulting solution, 130 mM L-2,4-diaminobutyric acid (DAB) solution was added under stirring to achieve a final concentration of 50 μM prazosin. The pH of the prazosin and DAB solution was then adjusted to pH 7.55 (37°C) with pH buffer Tris base.

[0119] To enhance the effect of the above solution, an isotonic Tris buffer solution at pH 8.1 was also prepared for intratumoral injection of several ml at regular intervals (every 2 hours).

[0120] This periodic injection is performed after waiting 1-2 minutes for gravity drainage or manual aspiration of a few ml to maintain the same intracranial pressure despite the injection. This procedure, involving aspiration of 1-2 ml and injection of 1-2 ml, is preferably repeated gently several times for about 5 minutes every 2 hours, with or without interruption in the drug infusion (if there is more than one catheter in the tumor).

[0121] 2. Study: In vitro efficacy of the combination drug against glioma and normal cell lines The ability of a combination of two drugs, L-2,4 diaminobutyric acid (DAB) and prazosin, to inhibit the proliferation in vitro of two cell lines: i) LN-18, a human line derived from a patient with grade IV glioblastoma, and ii) HMC3, a human brain nonmalignant cell line that retains properties of primary microglial cells.

[0122] Two colorimetric assays were established to monitor cytotoxicity in response to the tested drugs. One measured the release of lactate dehydrogenase (LDH) from cells, a well-established marker of cytotoxicity. The other measured mitochondrial lactate dehydrogenase activity, an indicator of cell viability, via reduction of the soluble tetrazolium dye MTT. Because both assays yielded similar results in terms of their ability to measure cytotoxicity, the LDH assay was selected for further experiments. Cells were also monitored by direct microscopic observation. Controls included cells treated with drug-free medium and cells treated with camptothecin, a known cytotoxic agent, as a positive control. Incubation with the drug concentrations was performed at 37°C for 24 hours.

[0123] DAB was tested in vitro under ideal conditions, in the absence of any competing physiological amino acids, allowing concentrations as low as 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 16 mM. As shown in Figures 1 and 2, DAB exhibited weak toxicity against nonmalignant HMC3 cells (IC50 could not be determined) and strong cytotoxic effects against the glioma cell line LN-18 (IC50 = 3.8 mM).

[0124] Prazosin was tested at concentrations of 0.1, 0.5, 1, 2, 5, 10, 20, 30, and 50 μM. Similar to DAB, this compound exhibited little cytotoxicity against the nonmalignant HMC3 cell line (Figure 3; IC50 could not be determined), but exhibited a strong cytotoxic effect against the glioma LN-18 cell line (Figure 4; IC50 = 3.4 μM).

[0125] Based on the above results with single drugs, we investigated the effectiveness of combinations to elucidate possible synergistic effects. DAB was used at concentrations of 1, 2, or 4 mM. These concentrations were tested alone or in combination with 1, 2, or 4 μM prazosin, i.e., the following combinations were tested: 1 mM DAB + 1 μM prazosin 1 mM DAB + 2 μM prazosin 1 mM DAB + 4 μM prazosin 2 mM DAB + 1 μM prazosin 2 mM DAB + 2 μM prazosin 2 mM DAB + 4 μM prazosin 4 mM DAB + 1 μM prazosin 4 mM DAB + 2 μM prazosin 4 mM DAB + 4 μM prazosin

[0126] Figure 5 compares the effect of either drug alone (white for DAB, gray for prazosin) with the effect of the combination (black). The results show that the combination of the two drugs has a stronger effect than the additive effect of the two drugs alone. For clarity, this is further illustrated in Figure 6, which compares the expected cell viability for the additive effect, i.e., simply adding the effects of the two drugs alone (white) with the observed effect (gray), showing that the latter is clearly stronger, indicating a synergistic effect.

[0127] The results were further confirmed by direct microscopy, as shown in FIG.

[0128] The data presented show that the combination of DAB and prazosin exerts a cytotoxic effect on human glioma cell lines but not on non-malignant glioma cell lines, and a synergistic effect exists.

[0129] 3. Clinical study: Intratumoral administration of DAB to five glioma patients The clinical course of five patients with grade IV glioma was studied. All patients underwent craniotomy and gross tumor removal. They were treated with postoperative intratumoral administration of DAB for 2 to 5 days via conventional drainage or an extraventricular catheter (EVD) left in the resected tumor cavity. The catheter was connected to an intravenous drug delivery pump (SMITHS), with a flow rate of 1 to 3 ml / h (depending on the patient's condition and lesion volume). Every 2 hours, the on-duty neurosurgeon interrupted treatment to aspirate necrotic fluid from the injection site by gravity and / or manually and manually inject physiological fluid (Ringer's solution).

[0130] Patients will be followed up both clinically and with MRI imaging.

[0131] 4. Clinical study: Intratumoral administration of DAB and prazosin to one glioma patient using a novel drug delivery method This 36-year-old woman, who had undergone surgery for a right frontoparietal high-grade lesion (glioblastoma multiforme), developed a large recurrence in the surgical area within 3 months. She had symptoms of elevated intracranial pressure with left hemiplegia. After preoperative evaluation and imaging, she underwent surgery on November 8, 2017. Craniotomy was performed with re-exploration and gross removal of the tumor. Because of the short-term recurrence, on October 26, 2017, immunohistochemistry revealed glioblastoma, IDH1(R132H) mutant, which is WHO grade IV glioma. During surgery, a catheter was placed in the cavity. After appropriate explanation and consent from the spouse, intratumoral infusion of an isotonic drug solution (containing 130 mM L-2,4-diaminobutyric acid (DAB) and 50 μM prazosin, with a pH of 7.55 at 37°C) was administered inside the postoperative cavity at 3 ml / h for 96 hours (November 13–16, 2017). Every 2 hours, there was a 5-minute break, with multiple manual alternating injections and aspirations through a 5 ml syringe: 1–2 ml of isotonic Tris-buffered solution at pH 8.1 was injected, and 1–2 ml of necrotic fluid generated from the interaction of the drug with the malignant cells was aspirated. The patient was continuously monitored for any adverse events throughout the treatment.

[0132] She tolerated treatment very well and was discharged in stable condition on November 20, 2017.

[0133] She was serially followed up both clinically and with MRI imaging. Follow-up report with visit dates: On December 8, 2017, she was clinically improved and asymptomatic. Her left hemiparesis improved. She experienced occasional left-sided focal seizures involving the upper extremities. She was receiving temozolomide chemotherapy. On January 1, 2018, she fully recovered. She had left hemiplegia with the ability to ambulate unassisted. There was a marked reduction in focal seizures in the left upper extremity. Adjuvant temozolomide chemotherapy was continued. As of February 6, 2018, she had fully recovered with no neurological deficits and was able to independently perform activities of daily living. Adjuvant temozolomide chemotherapy was continued. As of March 7, 2018, she remained in the same stable condition and continued adjuvant temozolomide chemotherapy. On May 7, 2018, she reported mild instability, no neurologic deficits, and occasional focal seizures involving the left upper extremity. MRI showed no evidence of tumor. June 14, 2018: Occasional focal seizures continued. No new neurological deficits. Adjuvant TMZ chemotherapy continued. On July 17, 2018, the condition was the same except for two episodes of focal seizures. On August 24, 2018, he had the same neurological condition. October 25, 2018, no neurological deficits. January 15, 2019: No focal or motor deficits. Independent. Repeat MRI showed no evidence of tumor. MRI reports and images dated July 17, 2020, and July 14, 2020. Asymptomatic with no focal neurological deficits. A separate, contrast-enhanced MRI shows no evidence of tumor.

[0134] 5. A pilot study of intratumoral administration of DAB and prazosin in 10 patients with glioma The clinical study has received ethical and scientific approval (delayed due to COVID-19) and the protocol is described below.

[0135] Patients and methods Number of patients: 10 patients with glioma grade III-IV, treatment-naive or previously treated. Age: 20-70 years old for both men and women.

[0136] Other than general health status, there are no other exclusion criteria that make the patient refractory to craniotomy and / or burr hole craniotomy.

[0137] After full informed consent, patients undergo either a standard craniotomy for tumor resection or, if the tumor is deemed inoperable, a standard craniotomy followed by a biopsy, both of which are performed under general anesthesia.

[0138] After tumor resection or biopsy, one microdialysis catheter, one EVD catheter, and one IRRA flow catheter are left in the resection cavity or implanted along the same path as the biopsy stylet to minimize trauma.

[0139] After confirmation of the diagnosis by frozen section, the EVD catheter is connected to an intravenous drug pump, and the drug solution is administered at 3 ml / h. An IRRA flow catheter is used to monitor intracranial pressure, periodically infuse an isotonic Tris-buffered solution at pH 8.1, and drain the necrotic fluid generated from the interaction of the drug with the malignant cells.

[0140] Fluid exchange rate and treatment duration are individualized based on the patient's intracranial pressure, clinical course, and neuroradiological imaging (first MRI 48 hours after treatment initiation, expected total treatment time 5 days, with a maximum of 7 days for large, inoperable tumors).

[0141] Patients are monitored for side effects during treatment, including seizures, drowsiness, worsening of neurological deficits, and any other clinical signs.

[0142] Before, during, and after treatment, patients undergo neurological examinations, and their general condition and quality of life are assessed according to the Karnofsky Scale every two months for at least 12 months. The clinical follow-up records described above are complemented by simultaneous MRI scans performed according to the latest RANO guidelines (https: / / radiopaedia.org / articles / rano-criteria-for-glioblastoma). These guidelines are applied to define each patient's tumor: location, size, extent of resection, cavity size, residual volume, edema compared to preoperative MRI; and neuroradiological progression.

[0143] If the patient shows any signs of recurrence on MRI according to RANO guidelines, adjuvant standard radiotherapy will be administered.

[0144] The objectives of the study are as follows: a) extending the time until tumor recurrence; b) reducing morbidity; c) improving the quality of life of patients; d) Prolong overall survival.

[0145] L-2,4-Diamino-n-butyric acid (dihydrochloride) (DAB) and prazosin are commercially available from different licensed vendors. The hospital pharmacy prepared a solution containing 100 mmol / L DAB and 100 μmol / L prazosin at pH 7.8, to which Tris base (Sigma) and sodium hydroxide were added to provide 50 mmol / L sodium ions (Na + The concentration will be adjusted to achieve a concentration of 310-390 mOsmol / L and an osmolality of 310-390 mOsmol / L. There will be no changes from the published literature regarding the origin of the drug.

Claims

1. A pharmaceutical product comprising L-2,4-diaminobutyric acid (DAB) and prazosin as a combined preparation for simultaneous, sequential or separate use in the treatment of cancer.

2. 10. The pharmaceutical product for use according to claim 1 for the treatment of cancer by intratumoral administration to a tumor.

3. Amino acids, peptides, polypeptides, proteins, polysaccharides, muteins, immunoglobulins, antibodies, cytokines (e.g., lymphokines, monokines, chemokines), blood clotting factors, hematopoietic factors, interleukins (1-18), interferons (e.g., β-IFN, α-IFN, and γ-IFN), erythropoietin, nucleases, tumor necrosis factors, statins (such as lovastatin, simvastatin, atorvastatin, etc.), colony stimulating factors (e.g., GCSF, GM-CSF, MCSF), insulin, antitumor agents and tumor suppressors, blood proteins, fibrin, thrombin, fibrinogen, synthetic thrombin, synthetic fibrin, synthetic fibrinogen, gonadotropins (e.g., FSH, LH, CG, etc.

3. The pharmaceutical product for use according to claim 1 or 2, comprising at least one additional therapeutic agent selected from the group consisting of: hormones and hormone analogues (e.g. growth hormone, luteinizing hormone releasing factor), vaccines (e.g. tumor, bacterial and viral antigens); somatostatin; antigens; blood clotting factors; growth factors (e.g. nerve growth factor, insulin-like growth factor); bone morphogenetic proteins, TGF-B, protein inhibitors, protein antagonists and protein agonists; nucleic acids, e.g. antisense molecules, DNA, RNA, RNAi; oligonucleotides; polynucleotides; cells, viruses, anti-tumor antibodies, antigens and antigen fragments, tumor cells and tumor cell fragments (fragments of tumors that can generate an immune response against the tumor) and ribosomes.

4. 4. The pharmaceutical product for use according to claim 3, wherein the additional therapeutic agent is selected from the group consisting of corticosteroids such as triamcinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate, hydrocortisone sodium succinate, triamcinolone hexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tebutate, prednisolone acetate, and prednisolone sodium phosphate.

5. The pharmaceutical product for use according to any one of claims 1 to 4, wherein the cancer to be treated is a brain tumor.

6. 6. The pharmaceutical product for use according to claim 5, wherein the brain tumor is selected from acoustic neuroma, astrocytoma, CNS lymphoma, ependymoma, hemangioblastoma, medulloblastoma, meningioma, glioma, mixed glioma, oligodendroglioma, pineal tumor and pituitary tumor.

7. Use of L-2,4-diaminobutyric acid (DAB) and prazosin in the manufacture of a drug for intratumoral administration to treat cancer.

8. 8. Use of L-2,4-diaminobutyric acid (DAB) and prazosin according to claim 7, and at least one additional therapeutic agent.

9. A pharmaceutical product for use according to any one of claims 1 to 6 for the treatment of cancer and / or for preventing or delaying recurrence.

10. 10. The pharmaceutical product for use according to any one of claims 1 to 6 and 9, wherein the subject is a human suspected of having glioma, a treatment-naive human, or a human who has previously been treated for cancer, who undergoes a standard biopsy after craniotomy under local or general anesthesia.

11. The pharmaceutical product for use according to any one of claims 1 to 6, 9 and 10, wherein the subject is a human undergoing standard surgery suitable for tumor removal.

12. 12. The pharmaceutical product for use according to any one of claims 1 to 6 and 9 to 11, administered by at least one or more steps of regular irrigation and drainage of the tumor bed with physiological fluid and / or isotonic Tris buffer through one or more indwelling catheters.

13. 13. The pharmaceutical product for use according to claim 12, wherein the isotonic Tris buffer has a pH of 7.2 to 9.

1.

14. 14. The pharmaceutical product for use according to any one of claims 1 to 6 and 9 to 13, administered by infusion through one or more catheters left inside the tumor, tumor resection cavity or biopsy site, with periodic interruptions for drainage and infusion of physiological isotonic fluid and / or isotonic Tris buffer.

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