POH and remdesivir combination for the treatment of CNS infections

Abstract

The present invention relates to the use of monoterpenes or sesquiterpenes to permeabilize the blood-brain barrier.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 092,607 (filed October 16, 2020), which is incorporated herein by reference in its entirety.

[0002] This invention relates to the use of monoterpenes or sesquiterpenes for delivering pharmaceuticals by enabling permeability across the blood-brain barrier. [Background technology]

[0003] The blood-brain barrier (BBB) ​​is a continuous boundary between the blood, interstitial fluid (IF), and the cerebrospinal fluid (CSF) of the brain. It is composed of a layer of endothelial cells, namely the brain capillary endothelium, which acts as an effective barrier against the entry of both high-molecular-weight and low-molecular-weight serum components into brain tissue. The restriction of such substances from entering the brain and CSF is due to the unique structure of the brain capillary endothelium. In other organs, the cells of the endothelial layer have gaps and channels that penetrate the layer between them, but such channels are absent in the brain capillary endothelium, which is unique both in terms of its anatomical tight junctions between cells and the rarity of phagocytic channels that can be frequently found in other endotheliums.

[0004] Under normal (healthy) conditions, only substances that can cross the blood-brain barrier (BBB) ​​can enter the brain, and such substances tend to be relatively hydrophobic (lipid-like). Hydrophilic (water-soluble) substances do not permeate the BBB very effectively, or not at all. Such water-soluble, low-permeability substances include the entire range of molecules, from large molecules like albumin to small ions like sodium, as well as chemotherapeutic agents, drugs, imaging compounds, and proteins with potential therapeutic applications. While some therapeutic agents have enough lipid solubility to permeate the BBB, most drugs (e.g., penicillin) and other therapeutically useful substances have limited lipid solubility and therefore cannot fully permeate the BBB. This insufficient BBB permeability by many potentially useful drugs poses serious limitations in the treatment of brain tissue and CSF diseases. Therefore, developing products and methods that "open" the BBB and allow access to brain tissue and CSF by drugs that are known to be effective in treating or diagnosing brain disorders but cannot cross the BBB on their own is of paramount clinical importance.

[0005] Malignant gliomas, the most common form of central nervous system (CNS) cancer, are currently considered essentially incurable. Among the various types of malignant gliomas, anaplastic astrocytoma (grade III) and glioblastoma multiforme (GBM; grade IV) have particularly poor prognoses due to their rapid growth and resistance to currently available treatments. Current standard care for malignant gliomas consists of surgery, ionizing radiation, and chemotherapy. Despite recent advances in medicine, no significant improvement in the prognosis of malignant gliomas has been observed in the past 50 years. (Wen et al. Malignant gliomas in adults. New England J Med. 359:492-507, 2008. Stupp et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England J Med. 352:987-996, 2005.) The main reason for the poor prognosis of malignant gliomas is the difficulty in delivering sufficient amounts of chemotherapeutic agents to the brain. Drug access to the brain is limited by the blood-brain barrier (BBB). The concentration of drugs that ultimately reach the brain is further reduced by first-pass metabolism in the liver and urinary excretion. Therefore, invasive surgical procedures such as tumor resection, stereotactic injection of antitumor drugs, or placement of catheters for convection-enhanced delivery of drugs are often required.

[0006] Intranasal drug delivery offers a novel, non-invasive therapy for bypassing the blood-brain barrier and rapidly delivering pharmaceuticals directly to the CNS. Drugs administered intranasally reach the parenchymal tissues of the brain, spinal cord, and / or cerebrospinal fluid (CSF) within minutes. In addition to delivery via the olfactory pathway and trigeminal nerve, animal studies suggest that therapeutic agents may also be delivered systemically via the nasal vascular system. Hashizume et al. New therapeutic approach for brain tumors: intranasal delivery of telomerase inhibitor GRN163. Neuro-oncology 10:112-120, 2008. Thorne et al. Delivery of insulin-like growth factor-1 to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience 127:481-496, 2004. Intranasal delivery of therapeutic agents may provide a systemic method for treating other types of cancer, such as lung cancer, prostate cancer, breast cancer, hematopoietic cancer, and ovarian cancer.

[0007] Despite decades of attempts, achieving curative immunotherapy for cancer remains extremely difficult, with the fundamental principle being the ability of antibodies or T cells (via T cell receptors) to recognize antigens (Cousin-Frankel, Science (2013) 342:1432). Antibody-based immunotherapy has been widely used for cancer when the target antigen is upregulated in tumor cells compared to normal cells (e.g., Her-2 in Her-2-amplifying breast cancer) or when tumor cells express antigens that can be recognized by antibodies or antibody-toxin conjugates (e.g., rituximab against CD20) (Baselga et al., Annals Oncology (2001) 12:S35). Clinical trials using antibody-based immunotherapy have shown improved patient survival rates in a limited number of cancer types (usually in combination with standard chemotherapy), but these effects are often accompanied by significant safety and efficacy concerns (Cousin-Frankel Cancer, Science (2013) 342:1432).

[0008] Effective T-cell therapy for cancer has been even more challenging to achieve clinically (Schmitt et al., Hum. Gene Ther. (2009) 20(11):1240). Effective T-cell therapy for cancer relies on T cells that have high affinity binding to antigens on cancer cells. Chimeric antigen receptor T cells (CAR T cells) are widely used to recognize antigens on cells with both high affinity and specificity without requiring accessory recognition molecules such as HLA antigens to "present" peptides. The T cell receptor of CAR T cells is "exchanged" with antigen-binding heavy and light chains, thereby eliminating the need for HLA accessory molecules. Recombinant CAR T receptors are fused to a signaling domain that results in T cell activation when the CAR T receptor binds to a target antigen.

[0009] Perillyl alcohol (POH), a naturally occurring monoterpene, has been suggested to be an effective agent against various cancers, including CNS cancers, breast cancer, pancreatic cancer, lung cancer, melanoma, and colon cancer. (Gould, M. Cancer chemoprevention and therapy by monoterpenes. Environ Health Perspect. 1997, 105(Suppl 4):977-979.) Hybrid molecules containing both perillyl alcohol and retinoids were prepared to increase apoptosis-inducing activity. (Das et al. Design and synthesis of potential new apoptosis agents: hybrid compounds containing perillyl alcohol and new constrained retinoids. Tetrahedron Letters 2010, 51, 1462-1466.) In the treatment of cancers such as malignant gliomas, as well as other brain disorders such as Parkinson's disease and Alzheimer's disease, it is still necessary to make the blood-brain barrier permeable for the delivery of various therapeutic agents. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Wen et al. Malignant gliomas in adults.New England J Med.359:492-507,2008 [Non-Patent Document 2] Stupp et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.New England J Med.352:987-996,2005 [Non-Patent Document 3] Hashizume et al. New therapeutic approach for brain tumors:intranasal delivery of telomerase inhibitor GRN163.Neuro-oncology 10:112-120,2008 [Non-Patent Document 4] Thorne et al. Delivery of insulin-like growth factor-1 to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration.Neuroscience 127:481-496,2004 [Non-Patent Document 5] Cousin-Frankel,Science(2013)342:1432 [Non-Patent Document 6] Baselga et al.,Annals Oncology(2001)12:S35 [Non-Patent Document 7] Schmitt et al.,Hum.Gene Ther.(2009) 20(11):1240 [Non-Patent Document 8] Gould, M. Cancer chemoprevention and therapy by monoterpenes.Environ Health Perspect.1997,105(Suppl4):977-979 [Non-Patent Document 9] Das et al. Design and synthesis of potential new apoptosis agents:hybrid compounds containing perillyl alcohol and new constrained retinoids.Tetrahedron Letters 2010,51,1462-1466 [Overview of the project] [Means for Solving the Problems]

[0011] The present invention provides a method of administering a therapeutic agent to the central nervous system of a mammal (e.g., a human), the method comprising administering a monoterpene before, after, or simultaneously with the therapeutic agent.

[0012] The central nervous system may be the brain.

[0013] The monoterpene may be perillyl alcohol.

[0014] The monoterpene (e.g., perillyl alcohol) may be administered (e.g., injected) into the vascular system of a mammal, for example into an artery. The monoterpene (e.g., perillyl alcohol) may be administered via inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular routes.

[0015] The monoterpene (e.g., perillyl alcohol) may be administered at a dose ranging from about 0.050 mg / kg to about 500 mg / kg of body weight.

[0016] The monoterpene (e.g., perillyl alcohol) may be administered about 0.2 minutes to about 60 minutes, or about 1 minute to about 15 minutes before the therapeutic agent is administered.

[0017] The monoterpene and the therapeutic agent may be administered separately.

[0018] The monoterpene and the therapeutic agent may be administered simultaneously. In one embodiment, the monoterpene and the therapeutic agent are administered together in a pharmaceutical composition (e.g., a solution).

[0019] The therapeutic agent may be a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, enzyme inhibitors, receptor antagonists, therapeutic antibodies, and combinations thereof.

[0020] The chemotherapeutic agent may be dimethyl celecoxib (DMC), irinotecan (CPT-11), temozolomide, or rolipram.

[0021] The treatment drug could be remdesivir.

[0022] The therapeutic agent may be an antibody or an antibody fragment.

[0023] The therapeutic agent may be immune cells expressing chimeric antigen receptors. The immune cells may be T cells. In one embodiment, the therapeutic agent is CAR-T cells.

[0024] Mammals can develop cancers, such as tumors of the nervous system (e.g., glioblastoma).

[0025] This method may further include a step of treating mammals with radiation. [Brief explanation of the drawing]

[0026] [Figure 1] Schematic diagrams of the Lym-1 CAR and CD19(FMC 63)CAR structures are shown. [Figure 2A] Figure 2A shows the accumulation of human CAR T cells within brain tumors. Immunohistochemical (IHC) staining is shown to detect the permeability of human CAR T cells into the brain and the resulting tumor (GL261 mouse glioma). Human-derived CD3-positive cells were identified using the primary antibody, anti-human CD3 antibody (CD3ε(D7A6E(trademark))XP(registered trademark) rabbit mAb (#85061)) (Cell Signaling, Boston, MA). [Figure 2B] This shows CD3 expression in cultured human CAR T cells. [Figure 2C] This shows CD3 staining in a normal C57 BL / 6 brain section. [Figure 2D] This shows CD3 expression in the brains of GL261 mice with gliomas after intravenous (IV) administration of Lym-1 human CAR T cells. [Figure 2E]This shows CD3 expression in the brains of GL261 mice with gliomas after intravenous injection of Lym-1 human CAR T cells following an IC injection of 3% NEO100. [Figure 2F] This shows CD3 expression in the brains of GL261 mice with gliomas after intravenous (IV) administration of anti-CD19 human CAR T cells. [Figure 2G] This shows CD3 expression in the brains of GL261 mice with gliomas after intravenous injection of anti-CD19 human CAR T cells following an intraconductive injection of 3% NEO100. [Figure 2H] This shows a comparison of CD3-positive cells in normal brain tissues with GL261 tumors. [Figure 3] This shows survival rates reflecting the efficacy of anti-mouse PD-1 antibody-mediated therapy in syngeneic GBM (GL261) mice from C57 BL / 6, in the absence or presence of periryl alcohol. [Figure 4A] We demonstrate that NEO100 can be applied across an in vitro BBB model, allowing labeled antibodies to temporarily cross it. Figure 4A shows an in vitro brain-barrier tight junction model. The labeled components are the Transwell chemotactic chamber; upper chamber; porous membrane; and lower chamber. Transwell culture chamber (pore size: 0.8 μm). Maidin Darby canine kidney (MDCK) cells are epithelial cells. TEER: transepithelial / transendothelial electrical resistance. Fluorescence Ab: Alexa Fluo® 488; donkey anti-rat IgG(H+L) fluorescence was measured in the lower chamber for approximately 120 minutes. [Figure 4B] We demonstrate that NEO100 can be applied across an in vitro BBB model, temporarily allowing labeled antibodies to cross it. Figure 4B shows enhanced permeability of the fluorescently labeled antibody through the upper chamber with increasing concentration. [Figure 4C]We demonstrate that NEO100 can be applied across an in vitro BBB model, temporarily allowing the labeled antibody to cross it. Figure 4C shows the decrease in TEER after application of 2 mM NEO100. [Figure 4D] We demonstrate that NEO100 can be applied across an in vitro BBB model, temporarily allowing the labeled antibody to cross it. Figure 4D shows the TEER recovery time after application of NEO100. [Figure 5A] This shows intracardiac injection (IC) of a mixture of NEO100 and 2% Evans Blue (EB) at different concentrations. [Figure 5B] This shows the penetration of EB into the brain after NEO100 has been administered by IC (intracardiac injection) or IV injection. [Figure 6] This indicates that a tight junction has been breached in the brain by NEO100. [Figure 7] This demonstrates NEO100-mediated dopamine delivery via the blood-brain barrier. [Figure 8] This shows the measured opening and closing times of the BBB (Ballpoint of Barrier). [Figure 9] This demonstrates anti-mouse IgG antibody delivery in the absence or presence of periryl alcohol. [Figure 10] This demonstrates the delivery of anti-PD-1 antibodies in the absence or presence of periryl alcohol. [Figure 11] This shows the Kaplan-Meier survival curves after NEO100-mediated human CAR T cell (Lym-1 CAR) delivery in NSG mice treated with intracranial Raji lymphoma xenografts. [Figure 12A] This shows the plasma concentration of remdesivir 30 to 240 minutes after intravenous (IV) delivery of remdesivir-cyclodextrin-H2O or intranasal (IN) delivery of remdesivir-cyclodextrin-POH. [Figure 12B] This shows the brain concentration of remdesivir 30 to 240 minutes after intravenous (IV) delivery of remdesivir-cyclodextrin-H2O or intranasal (IN) delivery of remdesivir-cyclodextrin-POH. [Figure 13] This demonstrates that POH was highly efficient in delivering remdesivir across the blood-brain barrier (BBB) ​​in an in vitro BBB model. [Modes for carrying out the invention]

[0027] As used herein, the term "NEO100" refers to periryl alcohol.

[0028] The present invention provides a method for using monoterpenes or sesquiterpenes or their derivatives (e.g., periryl alcohol i.e., POH, isoperiryl alcohol, or periryl alcohol derivatives) to permeate the blood-brain barrier. Thus, at least one therapeutic agent can be delivered across the BBB using monoterpenes or sesquiterpenes.

[0029] Monoterpenes (or sesquiterpenes) may have a purity of over approximately 98.5% (w / w), over approximately 99.0% (w / w), or over approximately 99.5% (w / w).

[0030] Monoterpenes (or sesquiterpenes) may be formulated into pharmaceutical compositions in or without the presence of (one or more) therapeutic agents, and the monoterpenes (or sesquiterpenes) may be present in amounts ranging from about 0.01% (w / w) to about 100% (w / w), about 0.1% (w / w) to about 80% (w / w), about 1% (w / w) to about 70% (w / w), about 10% (w / w) to about 60% (w / w), about 1% (w / w) to about 10% (w / w), about 1% (w / w) to about 5% (w / w), about 1% (w / w) to about 3% (w / w), about 3% (w / w) to about 10% (w / w), or about 0.1% (w / w) to about 20% (w / w).

[0031] Monoterpenes (e.g., periryl alcohol) can be administered in doses ranging from approximately 0.050 mg / kg to approximately 500 mg / kg body weight. Other ranges include approximately 0.1 mg / kg to approximately 100 mg / kg, approximately 1 mg / kg to approximately 50 mg / kg, approximately 5 mg / kg to approximately 25 mg / kg, and approximately 10 mg / kg to approximately 15 mg / kg.

[0032] Monoterpenes or sesquiterpenes may be used in combination with at least one therapeutic agent, including, but not limited to, chemotherapeutic agents, immunotherapeutic agents, immunomodulators, antibodies (e.g., monoclonal antibodies), immune cells (e.g., CAR-T cells), vaccines, antibody-drug conjugates, antiviral agents, anti-inflammatory agents, antibacterial agents, antimicrobial agents, antibiotics, and combinations thereof.

[0033] Anticancer agents that may be used in combination with purified monoterpenes or sesquiterpenes may have one or more of the following effects on cancer cells or subjects: cell death; reduced cell proliferation; reduced cell number; inhibition of cell growth; apoptosis; necrosis; mitotic cell death; cell cycle arrest; reduced cell size; reduced cell division; reduced cell viability; reduced cell metabolism; markers of cell damage or cytotoxicity; indirect indicators of cell damage or cytotoxicity such as tumor shrinkage; improved survival of the subject; or disappearance of markers associated with undesirable, unnecessary, or abnormal cell proliferation. U.S. Patent Application Publication No. 20080275057.

[0034] The therapeutic agent may be dissolved in periryl alcohol. This composition can be administered alone or concurrently with radiation or other drugs (e.g., chemotherapeutic agents) to treat diseases such as cancer.

[0035] In some embodiments, the drug is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an antigen-binding fragment and a toxin or drug that induces cytotoxicity in target cells. Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art and should be obvious to those skilled in the art. See, for example, Peters et al. Biosci. Rep. (2015) 35(4):e00225. In some embodiments, the antibody-drug conjugate may further include a linker (e.g., a peptide linker such as a cleavable linker) that conjugates the antibody and drug molecules.

[0036] Treatment may be sequential, with the monoterpene (or sesquiterpene) being administered before or after the administration of (one or more) therapeutic agents. The monoterpene (or sesquiterpene) and (one or more) therapeutic agents may be administered simultaneously.

[0037] Monoterpenes (or sesquiterpenes) and at least one therapeutic agent can be administered simultaneously, separately, or sequentially. They can exert advantageously combined effects (e.g., additive or synergistic effects).

[0038] In the case of sequential administration, the monoterpene (or sesquiterpene) is administered first, followed by (one or more) therapeutic agents, or (one or more) therapeutic agents are administered first, followed by the monoterpene (or sesquiterpene). In embodiments in which the monoterpene (or sesquiterpene) and therapeutic agents are administered separately, the administration of the monoterpene (or sesquiterpene) may precede or follow the administration of (one or more) therapeutic agents by a few seconds, minutes, hours, days, or weeks. The time difference in non-simultaneous administration may be greater than one minute, and may be, for example, exactly, at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 36 hours, or 48 hours, or less, or longer than 48 hours. Two or more drugs can be administered to each other within minutes, or within approximately 0.5, 1, 2, 3, 4, 6, 9, 12, 15, 18, 24, or 36 hours, or within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days, or within approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some cases, longer intervals are possible.

[0039] The disclosure also provides a pharmaceutical composition comprising (i) at least one monoterpene (or sesquiterpene) and (ii) at least one therapeutic agent.

[0040] The route of administration may vary and may include intra-arterial delivery, inhalation, intranasal delivery, oral delivery, transdermal delivery, intravenous delivery, subcutaneous delivery, or intramuscular delivery.

[0041] The present invention also provides a method for treating diseases such as cancer, comprising the step of delivering the composition to a patient.

[0042] The compositions of the present invention may contain one or more types of monoterpenes (or sesquiterpenes). Monoterpenes consist of two isoprene units and have the molecular formula C 10 H 16The invention includes terpenes having the following properties. Monoterpenes may be linear (acyclic) or may contain rings. Monoterpenoids produced by biochemical modifications such as oxidation or reconstitution of monoterpenes, and pharmaceutically acceptable salts of monoterpenes or monoterpenoids are also included in the present invention. Examples of monoterpenes and monoterpenoids include periryl alcohol (S(-) and R(+)), geranyl pyrophosphate, ocimene, myrcene, geraniol, citral, citronellol, citronellal, linalool, pinene, terpineol, terpinene, limonene, terpinenes, phellandrene, terpinolene, terpinen-4-ol (or tea tree oil), pinene, terpineol, terpinene; terpenoids such as p-cymene derived from monocyclic terpenes such as menthol, thymol, and carbochlor; and bicyclic monoterpenoids such as camphor, borneol, and eucalyptol.

[0043] Monoterpenes can be distinguished by the structure of their carbon skeleton and can be classified into acyclic monoterpenes (e.g., myrcene, (Z)- and (E)-ocimene, linalool, geraniol, nerol, citronellol, myrcenol, geranial, citral a, neral, citral b, citronellal, etc.), monocyclic monoterpenes (e.g., limonene, terpinene, phellandrene, terpinolene, menthol, carveol, etc.), bicyclic monoterpenes (e.g., pinene, myrtenol, myrtenal, verbanol, verbanone, pinocarveol, karene, sabinene, camphene, then, etc.), and tricyclic monoterpenes (e.g., tricyclene). See Encyclopedia of Chemical Technology, Fourth Edition, Volume 23, pages 834-835.

[0044] The sesquiterpene of the present invention consists of three isoprene units and has the molecular formula C 15 H 24The present invention includes terpenes having the following properties. Sesquiterpenes may be linear (acyclic) or may contain rings. Sesquiterpenoids produced by biochemical modifications such as oxidation or reconstitution of sesquiterpenes are also included in the present invention. Examples of sesquiterpenes include farnesol, farnesal, farnesylic acid, and nerolidol.

[0045] Derivatives of monoterpenes (or sesquiterpenes) include, but are not limited to, monoterpene (or sesquiterpene) esters, alcohols, aldehydes, and ketones. Monoterpene (or sesquiterpene) alcohols can be derivatized into esters, aldehydes, or acids.

[0046] The monoterpene (or sesquiterpene) alcohol esters of the present invention can be derived from inorganic or organic acids. Inorganic acids include, but are not limited to, phosphoric acid, sulfuric acid, and nitric acid. Organic acids include, but are not limited to, carboxylic acids, such as benzoic acid, fatty acids, acetic acid, and propionic acid. Examples of monoterpene (or sesquiterpene) alcohol esters include, but are not limited to, carboxylic acid esters (e.g., benzoic acid esters, fatty acid esters (e.g., palmitic acid esters and linoleic acid esters), acetates, propionates (or propanoates), and formates), phosphates, sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl). See Wikipedia - Esters. Search URL: http: / / en.wikipedia.org / wiki / Ester.

[0047] A specific example of a monoterpene that can be used in the present invention is periryl alcohol (commonly abbreviated as POH). The periryl alcohol composition of the present invention may contain (S)-periryl alcohol, (R)-periryl alcohol, or a mixture of (S)-periryl alcohol and (R)-periryl alcohol.

[0048] The terms “chimeric receptor,” “chimeric antigen receptor,” or “CAR” are used interchangeably throughout and refer to recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) which includes a functional signaling domain derived from a stimulating molecule as defined below. Lee et al., Clin. Cancer Res. (2012) 18(10):2780; Jensen et al., Immunol Rev. (2014) 257(1):127; www.cancer.gov / about-cancer / treatment / research / car-t-cells. In one embodiment, the stimulating molecule is a zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. The co-stimulatory molecule may also be 4-1BB (i.e., CD137), CD27 and / or CD28 or fragments of those molecules. In another embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In yet another embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulator and a functional signaling domain derived from a stimulator. Alternatively, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulators and a functional signaling domain derived from a stimulator. The antigen recognition portion of the CAR may contain any antigen-binding antibody fragment.The antibody fragment may contain one or more CDRs, variable regions (or parts thereof), constant regions (or parts thereof), or any combination thereof.

[0049] As used herein, a chimeric receptor refers to a molecule that can be expressed on the surface of a host cell and does not exist in nature, containing an antigen-binding fragment. Generally, a chimeric receptor comprises at least two domains derived from different molecules. In addition to the antigen-binding fragments described herein, a chimeric receptor may further comprise one or more hinge domains, transmembrane domains, at least one costimulatory domain, and cytoplasmic signaling domains. In some embodiments, a chimeric receptor comprises, from N-terminus to C-terminus, an antigen-binding fragment, a hinge domain, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric receptor further comprises at least one costimulatory domain.

[0050] In some embodiments, the chimeric receptors described herein include a hinge domain which may be located between the antigen-binding fragment and the transmembrane domain. A hinge domain is an amino acid segment commonly found between two domains of a protein that can enable flexibility of the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the antigen-binding fragment relative to another domain of the chimeric receptor can be used.

[0051] Any of the chimeric receptors described herein can be introduced into suitable immune cells for expression via conventional techniques. In some embodiments, the immune cells are T cells such as primary T cells or T cell lines. Alternatively, the immune cells may be NK cells such as established NK cell lines (e.g., NK-92 cells). In some embodiments, the immune cells express CD8 (CD8 + )T cells or those expressing CD8 and CD4 (CD8 + / CD4 + These are T cells. In some embodiments, the T cells are T cells from established T cell lines, such as 293T cells or Jurkat cells.

[0052] In some embodiments, immune cells expressing any of the chimeric receptors described herein are administered to a subject in an amount effective in reducing the number of target cells (e.g., cancer cells) by at least 20%, for example, 50%, 80%, 100%, 1 / 20, 1 / 5, 1 / 100, or more.

[0053] A typical amount of cells administered to a mammal (e.g., human), such as immune cells (e.g., CAR T cells), may range from, for example, 1 million to 100 billion cells, but amounts below or above this exemplary range are also within the scope of this disclosure. For example, a daily dose of cells may range from about 1 million to about 50 billion cells (e.g., a range defined by about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or any two of the aforementioned values), preferably about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 This could be 0 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the aforementioned values), more preferably approximately 100 million to approximately 50 billion cells (for example, approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, or a range defined by any two of the aforementioned values).

[0054] In one embodiment, a chimeric receptor (e.g., a nucleic acid encoding a chimeric receptor) is introduced into immune cells, and a subject (e.g., a human patient) receives an initial dose or initial administration of immune cells expressing the chimeric receptor. One or more subsequent doses of the drug (e.g., immune cells expressing the chimeric receptor) may be provided to the patient at intervals of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the initial dose. More than two doses of the drug per week, for example, two, three, four or more doses of the drug, may be administered to the subject. The subject may receive more than two doses of the drug (e.g., immune cells expressing the chimeric receptor) per week, then no further drug administration for one week, and finally one or more additional doses of the drug (e.g., more than two doses of immune cells expressing the chimeric receptor per week). Immune cells expressing chimeric receptors may be administered every other day for three doses per week for a period of 2, 3, 4, 5, 6, 7, 8 weeks or longer.

[0055] In the context of this disclosure, as far as it pertains to any of the disease conditions enumerated herein, terms such as “treat” and “cure” mean to reduce or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of this disclosure, the term “treat” also means to stop a disease, delay its onset (i.e., the period before the clinical manifestation of the disease), and / or reduce the risk of developing or worsening the disease. For example, in relation to cancer, the term “treat” may mean eliminating or reducing the tumor burden on a patient, or preventing, delaying, or inhibiting metastasis.

[0056] The methods and compositions described herein may be used to treat, but are not limited to, brain tumors, lung cancer, ear, nose and throat cancers, hematopoietic cancers, colon cancer, melanoma, pancreatic cancer, mammary cancer, prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancers; head and neck cancers; stomach cancers; carcinoma in situ; kidney cancer; laryngeal cancer; liver cancer; fibromas, neuroblastomas; oral cancers (e.g., lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; kidney cancer; respiratory system cancers; sarcomas; skin cancers; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancers, as well as other carcinomas and sarcomas.

[0057] Carcinoma is a cancer of epithelial origin. The carcinomas to which the methods described herein are intended to treat include, but are not limited to, acinar carcinoma, lobular carcinoma, alveolar adenocarcinoma (also known as adenomatous carcinoma, adenomyoepithelioina, cribriform carcinoma, and columnoma), adenomatous carcinoma (carcinoma adenomatosum), adenocarcinoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also known as bronchiololar carcinoma, alveolar cell tumor, and pulmonary adenomatosis), basal cell carcinoma, basal cell carcinoma (also known as basal cell carcinoma, basal cell tumor, and pilomatous carcinoma), basal cell carcinoma, basal squamous cell carcinoma, breast carcinoma, bronchoalveolar carcinoma, bronchiololar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma (also known as cholangiocarcinoma and cholangiocarcinoma), choriocarcinoma, and colloid carcinoma. Carcinoma, comedone carcinoma, corpus carcinoma, cribriform carcinoma, armory carcinoma, skin carcinoma, cylindrical carcinoma, cylindrical cell carcinoma, tubular carcinoma, carcinoma durum, embryonic carcinoma, cerebral carcinoma, epibulbar carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma (carcinoma epitheliale adenoides), carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma (gelatinous carcinoma), giant cell carcinoma, giant cell carcinoma (gigantocellulare), adenocarcinoma, granulosa cell carcinoma, piloma carcinoma, hematoid carcinoma Carcinoma, hepatocellular carcinoma (also called hepatocellular tumor, malignant hepatocellular carcinoma, liver carcinoma), Huirthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, pediatric embryonic carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ, Krompecher carcinoma, Kulchitzky cell carcinoma, lenticular carcinoma (carcinoma)Lenticulare, lipoma, lymphoepithelial carcinoma, mastitis carcinoma, medullary carcinoma (carcinoma medullare, medullary carcinoma), melanotic carcinoma (carcinoma melanodes, melanotic carcinoma), mucinous carcinoma (mucinous carcinoma, carcinoma muciparum), myxomatous carcinoma (carcinoma myxomatodes), nasopharyngeal carcinoma, melanotic carcinoma (carcinoma nigrum), oat cell carcinoma, ossificans carcinoma, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (also called renal adenocarcinoma and adrenal carcinoma), pre-cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma This includes carcinoma, sclerotic carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma (carcinoma tuberosum, tubeous carcinoma), verrucous carcinoma, and choriocarcinoma. In preferred embodiments, the methods of the present disclosure are used to treat subjects having cancer of the breast, cervix, ovaries, prostate, lung, colon and rectum, pancreas, stomach, or kidney.

[0058] Sarcomas are mesenchymal neoplasms that occur in bone and soft tissue. Various types of sarcomas are recognized, and these include: liposarcoma (including myxoid liposarcoma and pleomorphic liposarcoma), leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumors (also called malignant Schwann cell tumors, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including bone Ewing's sarcoma, extraosseous (i.e., non-osseous) Ewing's sarcoma, and primitive neuroectoderm tumors [PNETs]), synovial sarcoma, and angiosarcoma. Mangiosarcomas), lymphangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, tendonoid sarcoma (also known as invasive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), periangiocarcinoma, malignant mesenchymal sarcoma, hydatidiform soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, fibrogenic small cell tumor, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma) - skeletal and extraosseous, as well as chondrosarcoma.

[0059] In some embodiments, the cancer being treated may be a refractory cancer. As used herein, “refractory cancer” is a cancer that is resistant to prescribed standard care. These cancers may initially appear responsive to treatment (and then recur) or may be completely unresponsive to treatment. Typical standard care is likely to vary depending on the type of cancer and the stage of progression in the subject. It may be chemotherapy, surgery, radiation, or a combination thereof. Those skilled in the art are aware of such standard care. Thus, a subject being treated pursuant to this disclosure for a refractory cancer may have already been exposed to another treatment for that cancer. Or, if the cancer is likely to be refractory (for example, considering an analysis of the subject's cancer cells or medical history), the subject may not have already been exposed to another treatment. Examples of refractory cancers, but not limited to, include leukemia, melanoma, renal cell carcinoma, colon cancer, liver cancer, pancreatic cancer, non-Hodgkin lymphoma, and lung cancer.

[0060] Any of the chimeric receptor-expressing immune cells described herein may be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier or excipient.

[0061] When used in connection with the compositions and / or cells of this disclosure, the term “pharmaceutically acceptable” refers to molecular entities and other components of such compositions that are physiologically tolerable and, typically, do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, as used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, more specifically in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., nucleic acid, vector, cell or therapeutic antibody) and that the composition(s) do not adversely affect the subject to which they are administered. Any of the pharmaceutical compositions and / or cells used in this method may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.

[0062] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0063] Kits for therapeutic use Kits for using the agents / compositions of the present invention are also within the scope of this disclosure. Such a kit may comprise one or more containers comprising a first pharmaceutical composition comprising at least one monoterpene or sesquiterpene and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising at least one therapeutic agent and a pharmaceutically acceptable carrier. In another embodiment, the kit may comprise one or more containers comprising a pharmaceutical composition comprising at least one monoterpene or sesquiterpene, at least one therapeutic agent, and a pharmaceutically acceptable carrier.

[0064] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the first and second pharmaceutical compositions to a subject to achieve the intended activity in the subject. The kit may further include instructions for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions may include instructions for administering the pharmaceutical compositions to a subject in need of treatment.

[0065] Instructions for use of the pharmaceutical compositions described herein generally include information regarding the dosage, dosage schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions supplied with the kits of this disclosure are typically those written on the label or accompanying leaflet. The label or accompanying leaflet indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate, a disease or disorder in question.

[0066] The kits provided herein are in appropriate packaging. Appropriate packaging includes, but is not limited to, vials, bottles, flasks, flexible packaging, etc. Packaging for use in combination with specific devices such as inhalers, nasal infusion devices, or infusion devices is also intended. The kits may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). The container may also have a sterile access port.

[0067] The kit may include additional components such as buffers and interpretation information. Typically, the kit includes a container and a label or (one or more) accompanying documentation on or associated with the container. In some embodiments, this disclosure provides a product comprising the contents of the kit described above.

[0068] Examples of periryl alcohol derivatives include periryl alcohol esters, perillaldehyde, dihydroperylic acid, and perylic acid. Derivatives of periryl alcohol may also include their oxidizing and nucleophilic / electrophilic addition derivatives. U.S. Patent Application Publication No. 20090031455, U.S. Patents No. 6,133,324 and No. 3,957,856.

[0069] The present invention also provides a method of using a monoterpene (or sesquiterpene) and at least one therapeutic agent to treat diseases such as cancer or other neurological disorders. The monoterpene (or sesquiterpene) may be administered alone or in combination with a therapeutic agent. The monoterpene or sesquiterpene may also be administered concurrently with a therapeutic agent. The monoterpene (or sesquiterpene) may be administered or in combination with a therapeutic agent.

[0070] The drugs may be administered simultaneously or sequentially. Monoterpenes (or sesquiterpenes) may be administered before, during, or after the administration of the therapeutic agent.

[0071] Monoterpenes (or sesquiterpenes) can be used as solvents or permeabilisers for delivering therapeutic agents to lesion sites. Monoterpenes (or sesquiterpenes) can be used as solvents or permeabilisers for delivering chemotherapeutic agents to tumor cells. Monoterpenes or sesquiterpenes can also be used as solvents for vaccines that can be delivered via any suitable route.

[0072] This composition and method may be used to treat neurological cancers such as malignant gliomas (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), retinoblastoma, pilocytic astrocytoma (grade I), meningioma, metastatic brain tumors, neuroblastoma, pituitary adenoma, skull base meningioma, and skull base cancer. As used herein, the term “neurological tumor” refers to a condition in which the subject has malignant proliferation of nerve cells.

[0073] The cancers that can be treated with this composition and method are not limited to, but include, lung cancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma, pancreatic cancer, mammary cancer, prostate cancer, and breast cancer. Cancers including hematopoietic cancers, ovarian cancers, basal cell carcinomas, biliary tract cancers; bladder cancers; bone cancers; breast cancers; cervical cancers; choriocarcinomas; colorectal cancers; connective tissue cancers; digestive system cancers; endometrial cancers; esophageal cancers; eye cancers; head and neck cancers; stomach cancers; carcinomas in situ; kidney cancers; laryngeal cancers; leukemias including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia; liver cancers; lymphomas including Hodgkin lymphoma and non-Hodgkin lymphomas; myelomas; fibromas, neuroblastomas; oral cancers (e.g., lips, tongue, mouth, and pharynx); ovarian cancers; pancreatic cancers; prostate cancers; retinoblastomas; rhabdomyosarcomas; rectal cancers; kidney cancers; respiratory system cancers; sarcomas; skin cancers; stomach cancers; testicular cancers; thyroid cancers; uterine cancers; urinary system cancers, as well as other carcinomas and sarcomas. U.S. Patent No. 7,601,355.

[0074] The present invention also provides, but is not limited to, methods and compositions for treating CNS disorders, including primary degenerative neurological disorders such as Alzheimer's disease, Parkinson's disease, psychological disorders, psychosis, and depression.

[0075] This composition may be used in combination with radiotherapy.

[0076] The monoterpenes or sesquiterpenes of the present invention may be used in combination with at least one therapeutic agent, including, but not limited to, chemotherapeutic agents, immunotherapeutic agents, and antibodies (e.g., monoclonal antibodies). Anticancer agents that may be used in combination with purified monoterpenes or sesquiterpenes may have one or more of the following effects on cancer cells or subjects: cell death; reduced cell proliferation; reduced cell number; inhibition of cell growth; apoptosis; necrosis; mitotic cell death; cell cycle arrest; reduced cell size; reduced cell division; reduced cell viability; reduced cell metabolism; markers of cell damage or cytotoxicity; indirect indicators of cell damage or cytotoxicity such as tumor shrinkage; improved survival of the subject; or disappearance of markers associated with undesirable, unnecessary, or abnormal cell proliferation. U.S. Patent Application Publication No. 20080275057.

[0077] The present invention also includes mixtures and / or co-formulations of monoterpenes (or sesquiterpenes) and at least one therapeutic agent, including, but not limited to, a chemotherapeutic agent.

[0078] Chemotherapy agents include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilone, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, boron radiosensitizers (i.e., Velcade), and combination chemotherapy.

[0079] DNA alkylating agents are well known in the art and are used to treat various tumors. Non-limiting examples of DNA alkylating agents include nitrogen mustards such as mechloretamine, cyclophosphamide (ifosfamide, trophosfamide), chlorambucil (melphalan, prednimustine), bendamustine, uramustine, and estramustine; nitrosoureas such as carmustine (BCNU), lomustine (semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkyl sulfonates such as busulfan (mannosulfan, treosulfan); aziridines such as carbocone, thioTEPA, triadicone, and triethylenemelamine; triazenes such as hydrazine (procarbazine); dacarbazine, and temozolomide; and altretamine and mitobronitol.

[0080] Non-limiting examples of topoisomerase I inhibitors include CPT-11 (irinotecan), SN-38, APC, NPC, campotecan, topotecan, exatecan mesylate, 9-nitrocamptothecan, 9-aminocamptothecan, lulutotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT; Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer. Protoberberine alkaloids and their derivatives, including berberubin and cholalin, as described in Res.15(12):2795-2800; phenanthroline derivatives, including benzo[i]phenanthidine, nitidine, and fagaronine, as described in Makhey et al.(2003)Bioorg.Med.Chem.11(8):1809-1820; terbenzimidazole and its derivatives as described in Xu(1998)Biochemistry 37(10):3558-3566; and Foglesong et al.(1992)Cancer Chemother.Pharmacol.30(2):123-25, Crow et al.(1994)J.Med.Chem.37(19):31913194, and Crespi et al. These are anthracycline derivatives, including doxorubicin, daunorubicin, and mitoxantrone, as described in al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, etoposide and teniposide.Examples of dual topoisomerase I and II inhibitors include, but are not limited to, cyntopein and other naphthecenedions, DACA and other acridine-4-carboxamides, intoprisin and other benzopyridindoles, TAS-I03 and other 7H-indeno[2,1-c]quinoline-7-ones, pyrazoloacridines, XR 11576 and other benzophenazines, XR 5944 and other dimeric compounds, 7-oxo-7H-dibenz[f,ij]isoquinoline and 7-oxo-7H-benzo[e]perimidine, as well as anthracenyl-amino acid conjugates described in Denny and Baguley (2003) Curr. Top. Med. Chem. 3(3):339-353. Some drugs, for example, but not limited to, anthracyclines (acralubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, barurubicin, zorubicin) and anthracendions (mitoxantrone and pixantrone) inhibit topoisomerase II and have DNA intercalation activity.

[0081] Examples of endoplasmic reticulum stress inducers include, but are not limited to, dimethyl celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (i.e., Velcade (bortezomib)).

[0082] Platinum compounds are a subclass of DNA alkylating agents. Non-exclusive examples of such drugs include carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate, satraplatin, alloplatin, lovaplatin, and JM-216 (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic and Clinical Oncology, Angioli et al. Eds., 2004).

[0083] Non-limiting examples of antimetabolites include folate-based drugs, i.e., dihydrofolate reductase inhibitors, such as aminopterin, methotrexate, and pemetrexed; thymidylate synthase inhibitors, such as larcitrexed and pemetrexed; purine-based drugs, i.e., adenosine deaminase inhibitors such as pentostatin, thiopurines such as thioguanine and mercaptopurine, halogenated / ribonucleotide reductase inhibitors such as cladribine, clofarabine, and fludarabine, or guanine / guanosine:thiopurines such as thioguanine; or pyrimidine-based drugs, i.e., cytosine / cytidine:hypomethylating agents such as azacitidine and decitabine, DNA polymerase inhibitors such as cytarabine, ribonucleotide reductase inhibitors such as gemcitabine, or thymine / thymidine:thymidylate synthase inhibitors such as fluorouracil (5-FU). Equivalents of 5-FU include its prodrugs, analogs, and derivatives such as 5'-deoxy-5-fluorouridine (doxiflurosine) and 1-tetrahydrofuranyl-5-fluorouracil (Futraful), capecitabine (Xeloda), SI (MBMS-247616 consisting of tegafur and two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxoate), laricitrexed (Tomdex), noratexed (Thymitaq, AG337), and, for example, LY231514 and ZD9331 described in Papamicheal (1999) The Oncologist 4:478-487.

[0084] Examples of vincaloloids, though not limited to them, include vinblastine, vincristine, vinflunin, vindesine, and vinorelbine.

[0085] Examples of taxanes, though not limited to them, include docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel. An example of an epothilone is iabepilone.

[0086] Examples of enzyme inhibitors, though not limited to them, include farnesyltransferase inhibitors (tipifamib); CDK inhibitors (arbocidib, celecyclib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (anagrelide; rolipram); IMP dehydrogenase inhibitors (thiazofrin); and lipoxygenase inhibitors (masopropyl alcohol). Examples of receptor antagonists, though not limited to them, include ERA (atrasentan); retinoid X receptors (bexarotene); and sex steroids (testolactone).

[0087] Examples of therapeutic antibodies include, but are not limited to, anti-HER1 / EGFR (cetuximab, panitumumab); anti-HER2 / neu(erbB2) receptor (trastuzumab); anti-EpCAM (catumakisomab, edrecolomab); anti-VEGF-A (bevacizumab); anti-CD20 (rituximab, tositumomab, ibritumomab); anti-CD52 (aremtuzumab); and anti-CD33 (gemtuzumab). U.S. Patent Nos. 5,776,427 and 7,601,355.

[0088] Examples of tyrosine kinase inhibitors, though not limited to them, include inhibitors for the following: ErbB: HER1 / EGFR (erlotinib, gefitinib, lapatinib, vandetanib, sunitinib, neratinib); HER2 / neu (lapatinib, neratinib); RTK class III: C-kit (axitinib, sunitinib, sorafenib), FLT3 (restaurtinib), PDGFR (axitinib, sunitinib, sorafenib); and VEGFR (vandetanib, semaxanib, cedilanib, axitinib, sorafenib); bcr-abl (imatinib, nilotinib, dasatinib); Src (bosutinib); and Janus kinase 2 (restaurtinib).

[0089] Cetuximab is an example of an anti-EGFR antibody. It is a chimeric human / mouse monoclonal antibody that targets the epidermal growth factor receptor (EGFR). A bioequivalent antibody is identified herein as a modified antibody and one that binds to the same epitope of the EGFR antigen and produces a substantially equivalent biological response, e.g., prevention of ligand binding to EGFR, prevention of EGFR receptor activation, and blockade of downstream signaling in the EGFR pathway, resulting in disruption of cell growth.

[0090] Lapatinib (Tykerb®) is a dual inhibitor of EGFR and erbB-2. Lapatinib has been investigated in several clinical trials as a monotherapy and in combination with trastuzumab, capecitabine, letrozole, paclitaxel, and FOLFlR1 (irinotecan, 5-fluorouracil, and leucovorin). It is currently in a Phase III trial for the oral treatment of metastatic breast, head and neck, lung, gastric, renal, and bladder cancers. The chemical equivalents of lapatinib are small molecules or compounds that are tyrosine kinase inhibitors (TKIs), or HER-1 or HER-2 inhibitors. Several TKIs have been found to have effective antitumor activity and are either approved or in clinical trials. Such examples include, but are not limited to, Xactima (ZD6474), Iressa (gefitinib) and Tarceva (erlotinib), imatinib mesylate (STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib (CI 1033), semaxinib (SU5416), batalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), Sutent (SUI 1248) and leflutomamide (SU10l). Bioequivalents of lapatinib are peptides, antibodies or antibody derivatives that are HER-1 inhibitors and / or HER-2 inhibitors. Such examples include, but are not limited to, the humanized antibodies trastuzumab and Herceptin.

[0091] PTK / ZK is a broadly specific "small" molecule tyrosine kinase inhibitor that targets all VEGF receptors (VEGFR), platelet-derived growth factor (PDGF) receptors, c-KIT, and c-Fms. Drevs (2003) Idrugs 6(8):787-794. PTK / ZK is a targeted drug that blocks angiogenesis and lymphangiogenesis by inhibiting the activity of all known receptors that bind to VEGF, including VEGFR-I (Flt-1), VEGFR-2 (KDR / Flk-1), and VEGFR-3 (Flt-4). The chemical name of PTK / ZK is 1-[4-chloroanilino]-4-[4-pyridylmethyl]phthalazine succinate or 1-phthalazineamine, N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-butanedioate (1:1). Synonyms and analogues for PTK / TK are known as batalanib, CGP79787D, PTK787 / ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TK inhibitors, ZK 222584, and ZK.

[0092] Chemotherapy agents that can be used in combination with monoterpenes or sesquiterpenes may also include amsacrin, trabectedin, retinoids (alitretinoin, tretinoin), arsenic trioxide, asparagine depletion agents (asparaginase / pegaspargase), celecoxib, demecorsin, eruscromol, erusamitrusin, etogluside, ronidamine, lucanton, mitogwazon, mitotane, oblimersen, temsirolimus, and vorinostat.

[0093] The therapeutic agent may be remdesivir. Remdesivir is a broad-spectrum antiviral agent. In one embodiment, remdesivir may be administered by intravenous injection. In certain embodiments, remdesivir can be used to treat infections associated with RNA viruses. For example, remdesivir can be used to treat infections associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), or Middle East respiratory syndrome-associated coronavirus (MERS-CoV). In one embodiment, remdesivir can be used to treat COVID-19 in a subject.

[0094] Remdesivir may have the following structure: [ka]

[0095] remdesivir Other therapeutic agents that may be used in conjunction with the compositions and methods of the present invention include, for example, CAR-T cells, CAR-macrophages, or CAR-NK cells.

[0096] This composition and method may be used to increase paracellular permeability, for example, paracellular permeability of endothelial cells or epithelial cells. This composition and method may be used to increase blood-brain barrier permeability. The effect of administration on blood-brain barrier permeability is sustained from 5 minutes to 10 hours, and also includes other ranges, at least about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 24 hours, 48 ​​hours, or 72 hours.

[0097] This composition and method may be used to reduce or inhibit angiogenesis. This composition and method may, but may not be limited to, reduce or inhibit the production of pro-angiogenic cytokines, including vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8).

[0098] Monoterpenes or sesquiterpenes can be used in combination with angiogenesis inhibitors.

[0099] Examples of angiogenesis inhibitors, though not limited to these, include angiostatin, angiozyme, antithrombin III, AG3340, VEGF inhibitors (e.g., anti-VEGF antibodies), batimat, bevacizumab (Avastin), BMS-275291, CAI, 2C3, HuMV833 canstatin, captopril, cartilage-derived inhibitors (CDI), CC-5013, 6-O-(chloroacetyl-carbonyl)-fumagirol, COL-3, combretastatin, combretastatin A4 phosphate, dalteparin, and EMD. 121974 (Silengitide), Endostatin, Erlotinib, Gefitinib (Iressa), Genistein, Halofuginone Hydrobromide, Id1, Id3, IM862, Imatinib Mesylate, IMC-IC11 Inducing Protein 10, Interferon-α, Interleukin-12, Lavendastine A, LY317615 or AE-941, Marimast, Muspin, Medroxyprogesterone Acetate, Meth-1, Meth-2, 2-Methoxyestradiol (2-ME), Neovastat, Oteopontin Cleavage Product, PEX, Pigmented Epidermal Growth Factor (PEGF), Platelet Factor 4, Prolactin Fragment, Proliferin-Related Protein (PRP), PTK787 / ZK Examples include 222584, ZD6474, recombinant human platelet factor 4 (rPF4), lestin, squalamine, SU5416, SU6668, SU11248 squalamine, taxol, tecogalan, thalidomide, thrombospondin, TNP-470, troponin-1, vasostatin, VEG1, VEGF-Trap, and ZD6474.

[0100] Non-exclusive examples of angiogenesis inhibitors include tyrosine kinase inhibitors such as inhibitors of tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epidermal, fibroblast-derived, or platelet-derived growth factors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, pentosan polysulfate, angiotensin II antagonists, cyclooxygenase inhibitors (including nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, as well as selective cyclooxygenase-2 inhibitors such as celecoxib and lofecoxib), and steroidal anti-inflammatory drugs (e.g., corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone).

[0101] Other therapeutic agents that modulate or inhibit angiogenesis, and which may also be used in combination with monoterpenes or sesquiterpenes, include agents that modulate or inhibit the coagulation and fibrinolytic systems. Examples of such agents that modulate or inhibit the coagulation and fibrinolytic pathways include, but are not limited to, heparin, low molecular weight heparin, and carboxypeptidase U inhibitors (also known as inhibitors of activated thrombin-activated fibrinolysis [TAFIa]). U.S. Patent Application Publication No. 20090328239, U.S. Patent No. 7,638,549.

[0102] Immunomodulatory agents include, but are not limited to, cytokines such as interleukins, lymphokines, monokines, interferons, and chemokines.

[0103] Other permeation enhancers that may be used with monoterpenes (or sesquiterpenes) include, but are not limited to, fatty acid esters of glycerin, such as capric acid, caprylic acid, dodecylic acid, and oleic acid; fatty acid esters of isosorbide, sucrose, and polyethylene glycol; caproyl lactylic acid; laureth-2; laureth-2 acetate; laureth-2 benzoate; laureth-3 carboxylic acid; laureth-4; laureth-5 carboxylic acid; oleth-2; pyroglutamate; glyceryl oleate; oleate Examples include Lyceryl; N-Lauroyl Sarcosine; N-Myristoyl Sarcosine; Noctyl-2-Pyrrolidone; Lauraminopropionic Acid; Polypropylene Glycol-4-Laureth-2; Polypropylene Glycol-4-Laureth-5 Dimethyl Lauramide; Lauramide Diethanolamine (DEA), Lauryl Pyroglutamate (LP), Glyceryl Monolaurate (GML), Glyceryl Monocaprylate, Glyceryl Monocaprate, Glyceryl Monooleate (GMO), and Sorbitan Monolaurate. Polyols or ethanol may act as permeation enhancers or co-solvents. For additional permeation enhancers, see U.S. Patents 5,785,991, 5,843,468, 5,882,676, and 6,004,578.

[0104] Cosolvents are well known in the art and are not limited to these, but examples include glycerol, polyethylene glycol (PEG), glycol, ethanol, methanol, propanol, isopropanol, and butanol.

[0105] This composition can be administered by any method known in the art, but is not limited to, intra-arterial, intranasal, oral, ocular, intraperitoneal, inhalation, intravenous, intracardiac injection (IC), intraventricular (ICV), intracisional injection or infusion, subcutaneous, implant, vaginal, sublingual, urethral (e.g., urethral suppositories), subcutaneous, intramuscular, intravenous, percutaneous, rectal, sublingual, mucosal, ocular, spinal, intrathecal, intraarticular, intra-arterial, subarachnoid, bronchial, and lymphatic administration. Topical formulations may be in the form of gels, ointments, creams, aerosols, etc., intranasal formulations may be delivered as sprays or droplets, percutaneous formulations may be administered via percutaneous patches or ion electrophoresis, and inhalation formulations may be delivered using nebulizers or similar devices. The composition can also take the form of tablets, pills, capsules, semi-solids, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable composition.

[0106] To prepare such pharmaceutical compositions, one or more and / or at least one therapeutic agent of monoterpenes (or sesquiterpenes) can be mixed with a pharmaceutically acceptable carrier, adjuvant, and / or excipient, according to conventional pharmaceutical formulation techniques.

[0107] The pharmaceutically acceptable carriers that can be used in this composition include any of the standard pharmaceutical carriers such as phosphate-buffered saline, water, and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The composition may further contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, and dried skim milk. Liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Particularly suitable liquid carriers for injection include water, saline, aqueous dextrose, and glycol. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences, edited by EW Martin (Mack Publishing Company, 18th ed., 1990). The composition may also contain stabilizers and preservatives.

[0108] As used herein, the term “therapeutic dose” means an amount sufficient to treat a particular disorder or disease, or to produce a pharmacological response that treats a disorder or disease.

[0109] The most effective means of administration and method for determining the dosage may vary depending on the composition used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Therapeutic doses can generally be dose-set to optimize safety and efficacy. Single or multiple doses may be administered at dose levels and patterns selected by the treating physician. Appropriate dosage formulations and methods of administering the drug can be readily determined by those skilled in the art. For example, compositions may be administered at doses of approximately 0.01 mg / kg to approximately 200 mg / kg, approximately 0.1 mg / kg to approximately 100 mg / kg, or approximately 0.5 mg / kg to approximately 50 mg / kg. When the compounds described herein are administered concurrently with another drug or therapy, the effective dose may be less than when the drug is used alone.

[0110] This disclosure also provides the above-mentioned compositions for intranasal administration. The compositions may further include a permeation enhancer. (Southall et al. Developments in Nasal Drug Delivery, 2000). The compositions may be administered intranasally in liquid form, such as solutions, emulsions, suspensions, or drops, or in solid form, such as powders, gels, or ointments. Devices for delivering intranasal drugs are well known in the art. Nasal drug delivery can be carried out using devices including, but not limited to, intranasal inhalers, intranasal spray devices, atomizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, nebulizers, medium-dose inhalers (MDIs), pressurized inhalers, blowers, and bidirectional devices. Nasal delivery devices can be metered to administer an accurate effective dose into the nasal cavity. Nasal delivery devices may be for single-unit delivery or multiple-unit delivery. In certain cases, the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Washington) can be used in this invention (http: / / www.kurvetech.com). The compounds of this invention can also be delivered by tube, catheter, syringe, packtail, cotton ball, nasal tampon or submucosal injection. U.S. Patent Applications Publications No. 20090326275, 20090291894, 20090281522, and 20090317377.

[0111] The compositions of the present invention can be formulated as aerosols using standard procedures. Monoterpenes (or sesquiterpenes) and / or at least one therapeutic agent can be formulated with or without a solvent, with or without a carrier. The formulation may be a solution or an aqueous emulsion containing one or more surfactants. For example, an aerosol spray may be produced from a pressurized vessel containing a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen, carbon dioxide, or other suitable gas. Dosage units can be determined by providing a valve for delivering a fixed amount. A pump spray dispenser can dispense metered doses or doses having a specific particle or droplet size. As used herein, the term “aerosol” refers to a suspension of fine solid particles or droplets of a liquid solution in a gas. Specifically, an aerosol includes a gas-mediated suspension of droplets of monoterpenes (or sesquiterpenes), which may be produced in any suitable device such as an MDI, nebulizer, or mist atomizer. Aerosols also include a dry powder composition of the composition of the present invention suspended in air or other carrier gas. Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et al., (1992) Pharmacol. Toxicol. Methods 27:143-159.

[0112] The composition may be delivered into the nasal cavity as a powder, such as microspheres, delivered by a nasal inhaler. The composition may be absorbed onto a solid surface, such as a carrier. The powder or microspheres may be administered in an air-dispensable form. The powder or microspheres may be stored in the inhaler container. Alternatively, the powder or microspheres may be filled into capsules, such as gelatin capsules, or other single-dose units suitable for nasal administration.

[0113] Pharmaceutical compositions can be delivered to the nasal cavity by directly placing the composition into the nasal cavity, for example, in the form of a gel, ointment, nasal emulsion, lotion, cream, nasal tampon, dropper, or bioadhesive strip. In certain embodiments, it may be desirable to extend the residence time of the pharmaceutical composition in the nasal cavity, for example, to promote absorption. Therefore, pharmaceutical compositions may be formulated with bioadhesive polymers, gums (e.g., xanthan gum), chitosan (e.g., highly purified cationic polysaccharides), pectin (or any carbohydrate that thickens like a gel or emulsion when applied to the nasal mucosa), microspheres (e.g., starch, albumin, dextran, cyclodextrin), gelatin, liposomes, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and / or cellulose (e.g., methyl or propyl; hydroxyl or carboxy; carboxymethyl or hydroxylpropyl).

[0114] The composition can be administered by oral inhalation into the airways, i.e., the lungs.

[0115] Typical delivery systems for inhalable medications include nebulizers, dry powder inhalers (DPIs), and medium-dose inhalers (MDIs).

[0116] A nebulizer device generates a high-speed airflow that sprays a therapeutic agent in liquid form as a mist. The therapeutic agent is formulated in liquid form, such as a solution or suspension of particles of appropriate size. In one embodiment, the particles are micronized. The term "micronized" is defined as having about 90% or more particles with a diameter of less than about 10 μm. Suitable nebulizer devices are commercially available, for example, from PARI GmbH (Starnberg, Germany). Other nebulizer devices include those from Respimat (Boehringer Ingelheim) and those disclosed, for example, in U.S. Patents 7,568,480 and 6,123,068, and International Publication No. 97 / 12687. Monoterpenes (or sesquiterpenes) can be formulated for use in a nebulizer device as an aqueous solution or liquid suspension.

[0117] DPI devices typically administer therapeutic agents in the form of free-flowing powders that can be dispersed in the patient's airflow during inhalation. DPI devices using an external energy source can also be used in this invention. To achieve free-flowing powder, the therapeutic agent can be formulated with a suitable excipient (e.g., lactose). Dry powder formulations can be prepared, for example, by dry blending dry lactose having a particle size of about 1 μm to 100 μm with fine particles of a monoterpene (or sesquiterpene). Alternatively, the monoterpene can be formulated without an excipient. The formulation is loaded into a dry powder dispenser or into an inhalation cartridge or capsule for use in a dry powder delivery device. Examples of commercially available DPI devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Patent No. 6,378,519); Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Patent No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Patent No. 4,353,365). Further examples of suitable DPI devices are listed in U.S. Patents No. 5,415,162, No. 5,239,993, and No. 5,715,810, and their references.

[0118] MDI devices typically release a measured amount of therapeutic agent using a compressed propellant gas. Formulations for MDI administration include a solution or suspension of the active ingredient in a liquefied propellant. Examples of propellants include hydrofluoroalkanes (HFAs) such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), as well as chlorofluorocarbons such as CCl3F. Further components of HFA formulations for MDI administration include cosolvents such as ethanol, pentane, and water; and surfactants such as sorbitan trioleate, oleic acid, lecithin, and glycerin. (See, for example, U.S. Patent No. 5,225,183, European Patent No. 0717987, and International Publication No. 92 / 22286). The formulation is loaded into an aerosol canister that forms part of the MDI device. Examples of MDI devices specifically developed for use with HFA propellants are provided in U.S. Patents 6,006,745 and 6,143,227. For examples of processes for preparing appropriate formulations and devices suitable for inhalation administration, see U.S. Patents 6,268,533, 5,983,956, 5,874,063 and 6,221,398, as well as International Publications 99 / 53901, 00 / 61108, 99 / 55319 and 00 / 30614.

[0119] Monoterpenes (or sesquiterpenes) and / or at least one therapeutic agent may be encapsulated in liposomes or microcapsules for delivery by inhalation. Liposomes are vesicles consisting of a lipid bilayer membrane and an aqueous interior. The lipid membrane can be made from phospholipids, examples of which include phosphatidylcholine such as lecithin and lysolecithin; acidic phospholipids such as phosphatidylserine and phosphatidylglycerol; and sphingophospholipids such as phosphatidylethanolamine and sphingomyelin. Alternatively, cholesterol may be added. Microcapsules are particles coated with a coating material. For example, the coating material may consist of a mixture of film-forming polymers, hydrophobic plasticizers, surface activators, and / or lubricating nitrogen-containing polymers. U.S. Patents 6,313,176 and 7,563,768.

[0120] Due to their ability to easily penetrate the dermis, monoterpenes can also be used alone or in combination with at least one therapeutic agent via topical application. As transdermal delivery agents, monoterpenes can also be used in combination with anesthetics or analgesics for transdermal delivery of pain relievers.

[0121] The present invention also provides the above-mentioned compositions for ophthalmic use. Accordingly, the compositions may further contain a penetration enhancer. For ophthalmic use, the compositions described herein can be formulated as solutions, emulsions, suspensions, etc. Various vehicles suitable for administering the compounds to the eye are known in the art. Specific non-limiting examples are described in U.S. Patents 6,261,547, 6,197,934, 6,056,950, 5,800,807, 5,776,445, 5,698,219, 5,521,222, 5,403,841, 5,077,033, 4,882,150, and 4,738,851.

[0122] This composition can be administered for a short or long period of time. This composition can be administered to mammals, preferably humans. Examples of mammals, but not limited to, include mice, rats, rabbits, monkeys, cattle, sheep, pigs, dogs, cats, livestock, sporting animals, pets, horses, and primates.

[0123] Devices for intranasal administration may include intranasal spray devices, atomizers, nebulizers, medium-dose inhalers (MDIs), pressurized dose inhalers, blowers, intranasal inhalers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, or bidirectional devices.

[0124] The drugs may be administered simultaneously or sequentially.

[0125] The present invention also provides a method for inhibiting cell growth in vitro, ex vivo, or in vivo, comprising contacting cells such as cancer cells with an effective amount of a purified monoterpene (or sesquiterpene) as described herein. The composition and method may be used to inhibit the growth of cells that are resistant to chemotherapeutic agents. For example, the composition and method may be used to inhibit the growth of temozolomide-resistant cells.

[0126] Pathological cells or tissues, such as hyperproliferative cells or tissues, can be treated by contacting the cells or tissues with an effective amount of the composition of the present invention. Cells, such as cancer cells, may be primary cancer cells or cultured cells available from a tissue bank such as the American Type Culture Collection (ATCC). Pathological cells may be cells from systemic cancer, glioma, meningioma, pituitary adenoma, or CNS metastases from systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer, or ovarian cancer. Cells may be derived from vertebrates, preferably mammals, more preferably humans. U.S. Patent Application Publication No. 2004 / 0087651. Balassiano et al. (2002) Intern. J. Mol. Med. 10:785-788. Thorne, et al. (2004) Neuroscience 127:481-496. Fernandes, et al. (2005) Oncology Reports 13:943-947. Da Fonseca, et al. (2008) Surgical Neurology 70:259267. Da Fonseca, et al. (2008) Arch. Immunol. Ther. Exp. 56:267-276. Hashizume, et al. (2008) Neuroncology 10:112-120.

[0127] Cancer stem cells (CSCs), or tumor-initiating cells, are immature cells that possess stem cell characteristics such as self-regeneration. However, self-regeneration is intensified in CSCs. Reya et al., Stem cells, cancer, and cancer stem cells. Nature. 2001, 414(6859):105-11. Furthermore, glioma CSCs are resistant to chemotherapy and radiotherapy. Bao et al., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006, 444(7120):756-60. Rich et al., Chemotherapy and cancer stem cells. Cell Stem Cell. 2007;1(4):353-5. This composition and method can be used, but are not limited to, to inhibit the growth of cancer stem cells, including glioblastoma cancer stem cells.

[0128] The following examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0129] [Example 1] NEO100-mediated human CAR T cell delivery to the brain and tumors Preparation of human CAR T cells Human CAR T cells (CD19 and Lym-1) were provided by Dr. Epstein (USC). Chimeric antigen receptors (CARs) are synthetic molecules comprising three distinct modules: an extracellular antibody-based recognition site; a transmembrane module that immobilizes the molecule to the cell membrane; and a chimeric intracellular signaling domain that transmits an activation signal. Jensen et al., Designing chimeric antigen receptors to effectively and safely target tumors. Curr. Opin. Immunol. 2015, 33, 9-15. CAR T cells targeting CD19 have achieved remarkable results in the treatment of patients with relapsed or refractory (R / R) acute lymphoblastic leukemia (ALL). Ruella et al., Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J. Clin. Invest. 2016, 126, (10), 3814-3826. Maude et al., CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood 2015, 125, (26), 4017-23. Grupp et al., Durable Remissions in Children with Relapsed / Refractory ALL Treated with T Cells Engineered with a CD19-Targeted Chimeric Antigen Receptor (CTL019). Blood 2015, 126, (23), 681-681. Lym-1, a mouse IgG2a monoclonal antibody, was produced by immunizing mice with nuclei isolated from Raji lymphoma cells.Epstein et al., Two new monoclonal antibodies, Lym-1 and Lym-2, reactive with human B-lymphocytes and derived tumors, with immunodiagnostic and immunotherapeutic potential. Cancer Res. 1987, 47, (3), 830-40. Lym-1 binds to discontinuous conformational epitopes on several HLA-DR subtypes with greater binding affinity to malignant B cells than to normal B cells. Rose et al., Critical Lym-1 binding residues on polymorphic HLA-DR molecules. Mol Immunol 1999, 36, (11-12), 789-97. Schematic diagrams of Lym-1 CAR and CD19(FMC 63)CAR constructs are shown in Figure 1.

[0130] Two million CD19 and Lym-1 human CAR T cells suspended in 0.9% saline working solution for use in IV injection, with and without intracardiac NEO100.

[0131] NEO100 intracardiac puncture Preparation of the working solution for intracardiac injection of NEO100: Suspend 3% NEO100 in 0.9% physiological saline.

[0132] Standard procedure for ultrasound-guided intracardiac puncture In short, animals were anesthetized using 2% isoflurane gas and fixed on a platform for intracardiac puncture. The injection needle was inserted into the intercostal space and rapidly penetrated through the skin and muscle layers into the left ventricle under the guidance of ultrasound imaging.

[0133] An indicator of successful needle insertion into the left ventricle is the return of fresh arterial blood (pink in contrast to dark red venous blood) to the syringe. Intracardiac application was completed by slowly injecting 40 μl of 3% NEO100 in saline. Direct cell injection into the heart can cause local microinfarction if cells aggregate during injection, potentially leading to pericardial hematoma and death. Therefore, to minimize the potential for these adverse effects, ultrasound-guided injection using a small 30G needle is crucial, allowing (1) visualization of the needle tube to ensure the needle enters only the left ventricle, and (2) monitoring the subsequent cardiac progress through visualization of cardiac wall function as well as ECG after injection. A thin gauge needle ensures that cells do not aggregate when injected by intracardiac puncture.

[0134] Confirmation of intracardiac injection An indicator of successful needle insertion into the left ventricle is the return of fresh arterial blood (pink in color, as opposed to dark red venous blood) to the syringe.

[0135] Immediately after the completion of the NEO100 injection, 2 million human CAR T cells in 40 μl of PBS were injected through a tail vein catheter pre-primed with saline. To avoid the potential adverse effects of direct cell injection via intracardiac application, the inventors established a two-step procedure for the study.

[0136] Step 1: 40 μl of 3% NEO100 in physiological saline was slowly injected to complete intracardiac administration. This procedure allows NEO100 to exert its blood-brain barrier disrupting function.

[0137] Step 2: 2 million CAR T cells were injected intravenously through a tail vein catheter.

[0138] Evaluation of CAR T cell diffusion by IHC and confocal imaging Cerebral perfusion – To remove residual material remaining in the blood vessels after euthanasia, the test animals were perfused through the left ventricle with 10 ml of 0.9% saline to drain the blood. The brain was then removed, embedded in an OCT scanner, and stored at -80°C for further analysis.

[0139] Confocal imaging—fresh frozen sections with 8 μM imaging were prepared using a cryopreservation device and mounted on microslides. Before confocal imaging, coverslips were placed on the brain sections using DAPI mounting medium.

[0140] IHC staining—a standardized IHC staining procedure was applied to detect the permeability of human CAR T cells into the brain and the resulting tumors (GL261 mouse gliomas). Human-derived CD3-positive cells were identified using the primary antibody, anti-human CD3 antibody (CD3ε(D7A6E(trademark))XP(registered trademark) rabbit mAb (#85061)) (Cell Signaling, Boston, MA) (shown in Figure 2).

[0141] Study of a syngeneic mouse glioma animal model using C57 BL / 6 mice 100,000 GL261 mouse glioma cells were intracranially injected into immunocompetent C57 BL / 6 mice. Three weeks after tumor cell injection, mice with brain tumors were injected with 2 million human CAR T cells (anti-CD19 and Lym-1) via intravenous administration (IV) and intracardiac administration (IC) in combination with IV. The treated mice were euthanized 6 hours after the intervention.

[0142] Regarding intracardiac application: After intracardiac injection of 3% NEO100 in PBS, 2 million anti-CD19 or Lym-1 CAR T cells were administered by IV injection.

[0143] Regarding intravenous administration: 2 million anti-CD19 or Lym-1 CAR T cells were suspended in 40 µl PBS and injected via tail vein.

[0144] The brain was perfused with 0.9% saline solution, removed, and stored at -80°C for further analysis.

[0145] The antibodies applied to the test include a control antibody for negative staining: rabbit (DA1E) mAb IgG isotype, and an antibody used to detect CD3-positive cells in vitro and in vivo: CD3ε(D7A6E(trademark))XP(registered trademark) rabbit mAb (#85061).

[0146] conclusion No detectable CD3-positive cells were found in the brains of normal C57 BL / 6 mice.

[0147] Compared to conventional intravenous (IV) injection, intracardiac injection of human CAR T cells (anti-CD19 and Lym-1) mediated by NEO100 can significantly increase penetration into tumors formed inside the brain.

[0148] Intracardiac injection via 3% NEO100 does not cause any serious adverse effects or death in animals.

[0149] In normal brain tissue treated with intracardiac injection of NEO100, more CD3-positive cells were found than in samples treated with IV injection alone.

[0150] [Example 2] Anti-mouse PD-1 antibody-mediated therapeutic effect in C57 BL / 6 mice with intracranial syngeneic mouse glioma (GL261) 100,000 GL261 mouse glioma cells were injected intracranially into immunocompetent mice at C57 BL / 6. Seven days after injection, the mice were randomly divided into four experimental groups, and treatment began on the same day.

[0151] • Group 1. Control: IV and intracardiac injection of 40 μl physiological saline (5).

[0152] • Group 2. Antibody-treated mice: IV 40 μl of anti-mouse PD1 antibody at a dose of 2.5 mg / kg (5).

[0153] • Group 3. NEO100-treated mice: Intracardiac injection of 40 μl of 5% NEO100 (5).

[0154] • Group 4. Mice treated with a combination of NEO100 and antibody: 40 μl of 5% NEO100 intracardiac, followed by 40 μl of 1 anti-PD antibody intravenously at a dose of 2.5 mg / kg (6).

[0155] The results are shown in Figure 3. The inventors demonstrated that intracardiac injection of NEO100 (equivalent to intra-arterial injection in mice) can open the blood-brain barrier (BBB) ​​of antibodies. Subsequently, the inventors ran a syngeneic model using intracranially transplanted mouse GL26 glioma cells. Mice were treated intravenously with saline, NEO100 alone, anti-PD1 alone, or intracardiac NEO100 followed by intravenous anti-PD1. All mice administered anti-PD1 intravenously in combination with NEO100 remained alive, while all but one control mouse administered anti-PD1 intravenously died.

[0156] Periryl alcohol can be administered via the femoral artery using interventional neuroradiology (such as cerebral angiography).

[0157] statistical analysis Animal survival data were plotted using the Kaplan-Meier method. One-way ANOVA was used for the overall test of differences. Group comparisons were performed using Tukey's method, which adjusts for multiple comparisons. The log-rank (Mantel-Cox) test was applied to compare survival curves. A statistical evaluation result of p < 0.05 was considered significant.

[0158] • Control vs. IC NEO100+IV anti-mouse PD-1: *** P<0.0003 • Control vs. IV anti-mouse PD-1: ns, p=0.31 • IV anti-mouse PD-1 vs. IC NEO100+IV anti-mouse:** P<0.005 • Control pair IC NEO100: ns, p=0.397 [Example 3] The inventors demonstrated that NEO100 can be applied across an in vitro BBB model, allowing labeled antibodies to temporarily pass through it (Figures 4A-4D).

[0159] Experiments were conducted to investigate whether periryl alcohol (e.g., NEO100) could be used for intra-arterial delivery to temporarily disrupt the blood-brain barrier (BBB), allowing previously impermeable small or large molecules to penetrate the brain.

[0160] Administration of periryl alcohol (e.g., NEO100) may include intracardiac injection (intra-arterial injection in mice) and intravenous infusion.

[0161] The formulation contains 10% NEO100 (27.5 ml glycerol + 27.5 ml ethanol + 3.0 ml NEO100).

[0162] Cerebral perfusion – Before euthanasia, the test animals were perfused with 0.9% saline through the left ventricle. The brains were removed, embedded in an OCT scanner, and stored at -80°C for further analysis.

[0163] Ultrasound-guided intracardiac puncture—briefly, animals were anesthetized with 2% isoflurane gas and fixed on a platform for intracardiac puncture. The injection needle was guided by ultrasound imaging to penetrate the intercostal space and rapidly through the skin and muscle layers into the left ventricle. An indicator of successful needle insertion into the left ventricle is the return of fresh arterial blood (pink in color, as opposed to dark red venous blood) into the syringe.

[0164] Evans Blue is an azo dye with very high affinity for serum albumin. It allowed us to visualize extravasation of stained albumin from circulation.

[0165] NEO100 was delivered via intracardiac injection (left ventricle) to determine if there was increased uptake into the brain of Evans blue, BBB-impermeable small molecules (dopamine), or antibodies. Figure 5A shows intracardiac injection (IC) of a mixture of NEO100 and 2% Evans blue (EB). Different concentrations of NEO100 (40 μl in 0.9% saline) were tested by intracardiac puncture followed by immediate intravenous application of 2% Evans blue (40 μl by volume). The brain was removed after perfusion. The results indicate that NEO100 at a 1:1000 dilution (6.5 mM 40 μl) is still effective in interfering with the BBB.

[0166] Figure 5B shows the penetration of EB into the brain after NEO100 is administered by IC (intracardiac injection) or IV injection.

[0167] The experimental group includes the following: • IC 2% EB only • IC 20% Ethanol + 2% EB • IC 20% Ethanol + 2% EB + 5% NEO100 • IC: 20% ethanol + 5% NEO100, followed by 2% EB tail vein injection. • IV: 20% ethanol + 2% EB + 5% NEO100 IV 20% Ethanol + 2% EB Figure 6 demonstrates that in brains treated with 5% NEO100 intracardiac injection, tight junctions were dramatically breached compared to normal brains.

[0168] The pharmacological treatment of Parkinson's disease (PD) is primarily symptomatic, based on DA replacement therapy, because exogenous dopamine (DA) and other catecholamines cannot be administered due to insufficient blood-brain barrier (BBB) ​​permeability. Dopamine is a water-soluble, hydrophilic drug that does not meet the characteristics of a substance that can enter the brain through BBB permeability.

[0169] Figure 7 shows NEO100-mediated dopamine delivery via the blood-brain barrier.

[0170] Figure 8 shows the measured blood-brain barrier (BBB) ​​opening and closing times. Immunoenergetic C57 BL / 6 mice were injected with 5% NEO100(v / v) by intracardiac puncture (IC), followed by intravenous injection of 2% Evans Blue at different time points, including 0, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after IC injection.

[0171] The experimental procedure included the following: 1. Intracardiac injection (IC): 5% NEO100.

[0172] 2. Subsequently, 2% EB is administered intravenously (IV) at different times.

[0173] 3. The test animals were euthanized one hour after the IV injection.

[0174] Figure 9 shows the delivery of an anti-mouse IgG antibody (rabbit anti-mouse IgG H&L (Texas Red)-Ab6726) in the absence or presence of periryl alcohol.

[0175] Figure 10 shows the delivery of an anti-PD-1 antibody (Armenian hamster anti-mouse CD279 (PD-1) monoclonal antibody (J43)) in the absence or presence of periryl alcohol. PD-L1 binds to PD-1 and inhibits T cell killing in tumor cells. Blocking PD-L1 or PD-1 allows T cell killing in tumor cells.

[0176] Intra-arterial administration of NEO100 is safe.

[0177] [Example 4] NEO100-mediated human CAR T cell (Lym-1 CAR) delivery in the treatment of intracranial Raji lymphoma xenografts in NSG mice.

[0178] (a) Intracranial lymphoma xenograft: 50,000 (5×10 4 ) human B-cell lymphoma cells and Raji's-Luc / GFP were injected intracranially into NSG mice.

[0179] (b) Confirmation of tumor uptake: Five days after tumor cell injection, optical imaging was performed to confirm tumor uptake (100% tumor uptake).

[0180] (c) Initiation of CAR T infusion via tail vein catheter and intracardiac (IC)NEO100: There were three experimental groups: (1) control; (2) IV CAR T (5 × 10⁶); (3) IV CAR T (5 × 10⁶) + IC NEO100 (0.3% v / v = 492 μM). (d) Monitoring of NSG mice with IC lymphoma: The physical condition of the mice during treatment was monitored by monitoring their body weight. Tumor growth was monitored using optical imaging.

[0181] (e) Animal survival rate (Kaplan-Meier curve) As is clear from the survival curve (Figure 11), control mice, i.e., mice injected with human B-cell lymphoma cells, died within 15-20 days after injection, while mice injected with Lym-1 CAR T cells + NEO100 survived (P=0.0029).

[0182] [Example 5] The POH is placed in an intranasal inhaler (e.g., Kurve Technology's ViaNase Electronic Atomizer (Bethell, Washington)). Kurve Technology's intranasal delivery system can accurately deliver a predetermined drug volume (e.g., 0.2–6 mL). The device is loaded and cleaned in the same manner as a lung nebulizer. The device can deliver the drug to the olfactory region in bench tests in animals and humans.

[0183] Male athymic nu / nu mice (6 to 8 weeks old) are to be used in this study. A rodent subcutaneous / intracranial glioma model can be established as follows. Athymic nu / nu mice aged 6 to 8 weeks are anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (10 mg / kg). For the intracranial glioma model, mice are placed in a stereotaxic head frame (Harvard Apparatus), and a local anesthetic (0.2 cc of 0.25% xylocaine) is injected into the right frontal scalp. A knife blade is used to make a small incision, and a drill bit is used to form a small opening in the right frontal skull at the level of the coronal suture. Glioma cells (1×10 5 cells / 10 μl), for example, U-87 human glioma cells, are loaded into a calibrated Hamilton syringe. The needle tip is accurately placed into the right frontal lobe of the mouse, and the cells are injected slowly using a controlled push from the Hamilton syringe.

[0184] After the injection is completed, the syringe and needle are removed, and the wound is closed.

[0185] Two weeks after surgical implantation, the mice are divided into four groups (6 mice per group), and treated respectively with saline droplets alone (control), crude POH from Sigma (0.03%, 50 μl per droplet, 1 droplet per nostril), POH (purified to greater than 98.5% purity; 0.03%, 50 μl per droplet, 1 droplet per nostril), and TMZ (5 mg / kg, administered by gavage). TMZ serves as a positive control.

[0186] Brains are harvested, and tumor size is determined. Survival curves are constructed by tracking mice until the mice develop neurological deficits. In the experience of the present inventors, survival is approximately 4 weeks after implantation in untreated mice, and up to 8 weeks in mice treated with TMZ.

[0187] The inventors also have that RG2 rat glioma cells (1×10 5An immunocompetent syngeneic rat model is used in which cells (10µl) are transplanted into the right frontal lobe of Fisher 344 rats. The rats are divided into the same four groups as above. The inventors also intend to investigate the anti-invasion properties of POH using the rat RG2 model, since RG2 cells can migrate freely and therefore can invade rat parenchyma.

[0188] [Example 6] A recent clinical study in Brazil showed that intranasal delivery of perillyl alcohol in patients with recurrent malignant gliomas resulted in disease regression or stabilization. 50% of 140 treated patients achieved a 6-month progression-free survival period, and several patients enjoyed disease remission for as long as 3 years. Furthermore, there were virtually no side effects from the treatment. (Da Fonseca et al. Correlation of tumor topography and peritumoral edema of recurrent malignant gliomas with therapeutic response to intranasal administration of perillyl alcohol. Invest New Drugs 2009, Jan 13.) The inventors decided to deliver purified POH (purity exceeding 98.5%) into the nasal cavity of patients suffering from malignant glioma. To investigate whether POH can be directly delivered to brain tumor cells, the distribution of purified POH was investigated. 11 The study will involve delivering 14C-labeled POH to patients, followed by positron emission tomography (PET) imaging. Patients will then undergo a limited therapeutic trial using escalating doses of inhaled POH. Patients will be divided into three groups, each receiving 0.05% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), and 2.5% (w / v) of intranasal purified POH (purity >98.5%). The 2% (w / v) dose is the one currently used in Brazil. Delivery will be via a ViaNase nasal inhaler, administered three times daily.

[0189] PET imaging study. Ten patients with pathologically confirmed malignant gliomas were scanned using a Siemens Biograph TruePoint HD PET / CT scanner, with a range of 5–10 mCi. 11 Scanning will be performed after intranasal inhalation of the C-POH preparation. Static imaging will begin 30 minutes after inhalation, using a 10-minute acquisition in a single-bed position covering the skull. Subsequent continuous acquisitions will be performed at 30-minute intervals for 2 hours to assess progressive accumulation in brain and tumor tissue. Depending on patient compliance and the level of remaining and accumulated activity, imaging beyond 2 hours will be attempted. Overlayed PET / CT images will be compared to contrast-enhanced MRI studies for all patients to assess the correlation between activity accumulation and enhancement patterns.

[0190] [Example 7] In vivo administration of remdesivir to mice via intravenous (IV) or intranasal (IN) routes was investigated, either in the absence or in the presence of periryl alcohol (POH).

[0191] Remdesivir was mixed with ethanol (EtOH) (10%), cyclodextrin, and water (for IV delivery), or with ethanol (10%), cyclodextrin, and POH (for IN delivery). A mixture was prepared by dissolving cyclodextrin in EtOH, adding remdesivir once to the solution, adding water, then adding POH, and then homogenizing the mixture using a polytron. The administered dose of remdesivir was 60 mg / kg body weight. The concentration of POH was 3.4 mM.

[0192] Figure 12A shows the plasma concentrations of remdesivir 30 to 240 minutes after intravenous (IV) delivery of remdesivir-cyclodextrin-H2O or intranasal (IN) delivery of remdesivir-cyclodextrin-POH. Figure 12B shows the brain concentrations of remdesivir 30 to 240 minutes after intravenous (IV) delivery of remdesivir-cyclodextrin-H2O or intranasal (IN) delivery of remdesivir-cyclodextrin-POH.

[0193] [Example 8] In vitro, cells were seeded in the upper chamber of a Transwell. A mixture of POH-remdesivir or H2O-remdesivir was placed in the upper chamber. The POH concentration was 1.5 mM. Medium was collected from both the upper and lower chambers after 4 hours and remdesivir was quantified.

[0194] Figure 13 shows that POH was highly efficient in delivering remdesivir across the blood-brain barrier in an in vitro BBB model.

[0195] The scope of the present invention is not limited to what is specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the illustrated examples of materials, components, structures and dimensions. Numerous references, including patents and various publications, are cited and discussed in this description of the present invention. Such citations and discussions of references are provided solely to clarify the description of the present invention and do not constitute an admission that any reference is prior art to the present invention as described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Variations, modifications, and other practices of those described herein will be conceivable to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matters described herein and in the accompanying drawings are provided for illustrative purposes only and not as limitations.

Claims

1. A therapeutic agent for treating a disease of the central nervous system in a mammal, wherein the therapeutic agent comprises remdesivir, and the mammal is administered periryl alcohol before or simultaneously with the therapeutic agent, thereby improving the permeability of the blood-brain barrier and delivering the remdesivir to the central nervous system. A therapeutic agent.

2. The therapeutic agent according to claim 1, wherein the central nervous system is the brain.

3. The therapeutic agent according to claim 1, wherein periryl alcohol is administered intra-arterially.

4. The therapeutic agent according to claim 1, wherein periryl alcohol is administered in a dose in the range of 0.050 mg / kg to 500 mg / kg body weight.

5. The therapeutic agent according to claim 1, wherein the mammal is a human.

6. The therapeutic agent according to claim 1, wherein the periryl alcohol is administered 0.2 to 60 minutes before the remdesivir is administered.

7. The therapeutic agent according to claim 6, wherein the periryl alcohol is administered 1 to 15 minutes before the remdesivir is administered.

8. The therapeutic agent according to claim 1, wherein the periryl alcohol and the remdesivir are administered separately.

9. The therapeutic agent according to claim 1, wherein the periryl alcohol and the remdesivir are administered simultaneously.

10. The therapeutic agent according to claim 9, wherein the periryl alcohol and the remdesivir are administered together in the pharmaceutical composition.

11. The therapeutic agent according to claim 1, wherein the periryl alcohol is administered by inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular, and the remdesivir is administered by inhalation or intranasal.

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