Methods of penetrating blood brain barrier

Abstract

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

Description

[0001] This application is a divisional application of Chinese patent application 201980023885.8 (PCT / US2019 / 017076), whose application date is February 7, 2019 and whose invention name is “Method for penetrating the blood-brain barrier”. Technical Field

[0002] The present invention relates to the use of monoterpenes or sesquiterpenes to penetrate the blood-brain barrier. Background Art

[0003] The blood-brain barrier (BBB) ​​is a continuous boundary between the blood, interstitial fluid (IF), and cerebrospinal fluid (CSF) of the brain. It is composed of a layer of endothelial cells, the brain capillary endothelium, which is an effective barrier to prevent high- and low-molecular-weight serum components from entering the brain tissue. The restriction of such substances into the brain and CSF is due to the unique structure of the brain capillary endothelium. Although there are gaps and channels between cells of the endothelium in other organs that run throughout the endothelium, such channels are lacking in the brain capillary endothelium, which is unique in its anatomical tight junctions between cells and the rare presence of pinocytic vesicles (common in other endothelia).

[0004] Under normal (healthy) conditions, only substances that can cross the BBB can enter the brain, and such substances tend to be relatively hydrophobic (lipid-like). Hydrophilic (water-soluble) substances penetrate the BBB much less effectively, or even not at all. Such substances with poor water solubility and penetrability include a range of molecules, from molecules as large as albumin to ions as small as sodium, as well as chemotherapeutic agents, drugs, diagnostic imaging compounds, and proteins with potential therapeutic uses. Although some therapeutic agents have sufficient lipid solubility to penetrate the BBB, the vast majority of drugs (such as penicillin) and other therapeutically useful substances have limited lipid solubility and therefore cannot penetrate the BBB well. Many potentially useful drugs have poor penetration of the BBB, which severely limits the treatment of brain tissue and cerebrospinal fluid diseases. Therefore, it will be of extremely important clinical significance to develop products and methods that can "open" the BBB to allow drugs that are known to be effective in treating or diagnosing brain disorders but cannot cross the BBB themselves to enter brain tissue and cerebrospinal fluid.

[0005] Malignant gliomas are the most common form of central nervous system (CNS) cancer and are currently considered largely incurable. Among the various malignant gliomas, anaplastic astrocytomas (grade III) and glioblastoma multiforme (GBM; grade IV) have particularly poor prognoses due to their aggressive growth and resistance to currently available treatments. Existing standard treatments for malignant gliomas include surgery, ionizing radiation therapy, and chemotherapy. Despite recent advances in medicine, no significant improvement has been seen in the prognosis of malignant gliomas over the past 50 years. Wen et al. Malignant gliomas in adults. NewEngland JMed. 359:492-507, 2008. Stupp et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New England J Med. 352:987-996,2005.

[0006] One of the main reasons for the poor prognosis of malignant glioma is the difficulty in delivering sufficient amounts of chemotherapeutic agents to the brain. Drug entry into the brain is limited by the blood-brain barrier (BBB). The drug concentration that ultimately reaches the brain is further reduced by first-pass metabolism in the liver and urinary excretion. Therefore, invasive surgery is often required, such as tumor resection, stereotactic injection of anti-tumor drugs, or placement of catheters for convection-enhanced delivery of drugs.

[0007] Intranasal delivery of drugs offers a new non-invasive treatment approach that can bypass the blood-brain barrier and rapidly deliver drugs directly to the CNS. Intranasally administered drugs reach the brain parenchyma, spinal cord, and / or cerebrospinal fluid (CSF) within minutes. In addition to delivery via the olfactory tract and trigeminal nerve, animal studies have shown that therapeutic drugs are also delivered systemically via the nasal vasculature. 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 cordal olfactory and trigeminal pathways following intranasal administration. Neuroscience127:481-496, 2004. Intranasal delivery of therapeutic agents may provide systemic treatment for other types of cancer, such as lung cancer, prostate cancer, breast cancer, blood cancer, and ovarian cancer.

[0008] Despite decades of attempts, curative immunotherapy for cancer has been difficult to achieve, and its fundamental basis is the ability to recognize antigens, whether by antibodies or by T cells (through T cell receptors) (Cousin-Frankel, Science (2013) 342: 1432). Antibody-based immunotherapy has been widely used in anticancer when the target antigen is upregulated in tumor cells compared to normal cells (for example, Her-2 in Her-2 amplified breast cancer), or when tumor cells express antigens that can be recognized by antibodies or antibody-toxin conjugates (for example, rituximab (Rituximab) against CD20) (Baselga et al., Annals Oncology (2001) 12: S35). Although clinical trials using antibody-based immunotherapy have shown that patient survival has been improved in a limited number of cancer types (usually in combination with standard chemotherapy), these effects are often accompanied by significant safety and efficacy issues (Cousin-Frankel Cancer, Science (2013) 342: 1432).

[0009] Effective T cell therapy for cancer is more difficult to achieve clinically (Schmitt et al., Hum. Gene Ther. (2009) 20 (11): 1240). Effective anti-cancer T cell therapy 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 high affinity and specificity, and do not require auxiliary recognition molecules such as HLA antigens to "present" peptides. The T cell receptors of CAR T cells are "exchanged" by the heavy and light chains of the bound antigen, so HLA auxiliary molecules are not required. The recombinant CAR T receptor is fused with a signaling domain, resulting in the activation of T cells when the CAR T receptor binds to the target antigen.

[0010] Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to be an effective agent for the treatment of a variety of cancers including CNS cancer, 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 perillyl alcohol and retinoids were prepared to enhance 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.

[0011] In the treatment of cancers such as malignant gliomas as well as other brain disorders such as Parkinson's disease and Alzheimer's disease, there is still a need to penetrate the blood-brain barrier to deliver a variety of therapeutic agents. SUMMARY OF THE INVENTION

[0013] The present invention provides a method of administering a therapeutic agent to the central nervous system of a mammal (eg, a human), the method comprising administering a monoterpene before, after, or concurrently with the therapeutic agent.

[0014] The central nervous system may be the brain.

[0015] The monoterpene may be perillyl alcohol.

[0016] The monoterpene (eg, perillyl alcohol) can be administered into the vascular system of a mammal, such as intra-arterially (eg, injected into an artery). The monoterpene (eg, perillyl alcohol) can be administered by inhalation, intranasally, orally, intravenously, subcutaneously, or intramuscularly.

[0017] The monoterpene (eg, perillyl alcohol) may be administered at a dosage ranging from about 0.050 mg / kg to about 500 mg / kg body weight.

[0018] The monoterpene (eg, perillyl alcohol) can be administered from about 0.2 minutes to about 60 minutes, or from about 1 minute to about 15 minutes prior to administration of the therapeutic agent.

[0019] The monoterpene and the therapeutic agent may be administered separately.

[0020] The monoterpene and the therapeutic agent may be administered concurrently. In one embodiment, the monoterpene and the therapeutic agent are administered together in a pharmaceutical composition (eg, a solution).

[0021] The therapeutic agent can 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. Chemotherapeutic agents can be dimethyl celecoxib (DMC), irinotecan (CPT-11), temozolomide or rolipram.

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

[0023] The therapeutic agent can be an immune cell expressing a chimeric antigen receptor. The immune cell can be a T cell. In one embodiment, the therapeutic agent is a CAR-T cell.

[0024] The mammal may have cancer, such as a nervous system tumor (eg, glioblastoma).

[0025] The method may further comprise the step of treating the mammal with radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram showing Lym-1 CAR and CD19 (FMC 63) CAR constructs.

[0028] Figure 2A Showing accumulation of human CAR T cells within brain tumors. Figure 2A Immunohistochemistry (IHC) staining is shown to detect the infiltration of human CAR T cells into the brain and the formed tumor (GL261 mouse glioma). TM ) Rabbit mAb (#85061) (Cell Signaling, Boston, MA) primary antibody recognizes human CD3 positive cells.

[0029] Figure 2B Shown is CD3 expression on cultured human CAR T cells.

[0030] Figure 2C CD3 staining in normal C57 BL / 6 brain sections is shown.

[0031] Figure 2D Shown is the expression of CD3 in the brains of GL261 mouse gliomas when Lym-1 human CAR T cells were given by intravenous (IV) injection.

[0032] Figure 2E Shown is the expression of CD3 in the brains bearing GL261 mouse gliomas following IC injection of 3% NEO100 followed by IV injection of Lym-1 human CAR T cells.

[0033] Figure 2F Shown is the expression of CD3 in the brains of GL261 mouse gliomas when anti-CD19 human CAR T cells were given by intravenous (IV) injection.

[0034] Figure 2G Shown is the expression of CD3 in the brains of GL261 mouse gliomas following IC injection of 3% NEO100 followed by IV injection of anti-CD19 human CAR T cells.

[0035] Figure 2H Comparison of CD3 positive cells in normal sections of brains bearing GL261 tumors is shown.

[0036] Figure 3 Shown are survival rates reflecting the therapeutic effects mediated by anti-mouse PD-1 antibody in C57 BL / 6 bearing syngeneic mouse GBM (GL261) in the absence or presence of perillyl alcohol.

[0037] Figures 4A-4D It was shown that NEO100 can be applied to cross the BBB model in vitro and transiently allow labeled antibodies to cross the model. Figure 4A In vitro brain barrier tight junction model is shown. The labeled components are Transwell chemotaxis chamber, upper chamber, porous membrane and lower chamber. Transwell culture chamber (pore size: 0.8 μm). Madin-Darby canine kidney (MDCK) cells are epithelial cells. TEER: transepithelial / transendothelial electrical resistance. Fluorescent Ab: Alexa 488; Donkey anti-rat IgG (H+L). Fluorescence in the lower chamber was measured over approximately 120 minutes. Figure 4B It was shown that the permeation of fluorescently labeled antibodies through the upper chamber increased with increasing concentration. Figure 4C Shown is the reduction in TEER after using NEO100 at a concentration of 2 mM. Figure 4D Shown is the recovery time of TEER after application of NEO100.

[0038] Figure 5A Intracardiac injection (IC) of a mixture of NEO100 (different concentrations) and 2% Evans blue (EB) is shown.

[0039] Figure 5B Shown is the penetration of EBs into the brain after application of NEO100 by IC (intracardiac injection) or IV injection.

[0040] Figure 6 NEO100 was shown to disrupt tight junctions in the brain.

[0041] Figure 7 showed that NEO100 mediates dopamine delivery across a disrupted blood-brain barrier.

[0042] Figure 8 Displays measurements of the BBB opening and closing times.

[0043] Fig. 9 Shown is the delivery of anti-mouse IgG antibody in the absence or presence of perillyl alcohol.

[0044] Fig.10 Delivery of anti-PD-1 antibody in the absence or presence of perillyl alcohol is shown.

[0045] Fig.11 Shown are Kaplan-Meier survival curves after NEO100-mediated delivery of human CAR T cells (Lym-1 CAR) to treat intracranial Raji lymphoma xenografts in NSG mice.

[0046] The term "NEO100" as used herein refers to perillyl alcohol. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides methods for penetrating the blood-brain barrier using monoterpenes or sesquiterpenes or derivatives thereof (e.g., perillyl alcohol or POH, isoperillyl alcohol, or perillyl alcohol derivatives). Thus, monoterpenes or sesquiterpenes can be used to deliver at least one therapeutic agent across the BBB.

[0049] The purity of the monoterpene (or sesquiterpene) may be greater than about 98.5% (w / w), greater than about 99.0% (w / w), or greater than about 99.5% (w / w).

[0050] The monoterpene (or sesquiterpene) may be formulated as a pharmaceutical composition in the presence or absence of a therapeutic agent, wherein the monoterpene (or sesquiterpene) is present in an amount of 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).

[0051] Monoterpenes (e.g., perillyl alcohol) can be administered at a dosage ranging from about 0.050 mg / kg to about 500 mg / kg body weight. Other ranges include about 0.1 mg / kg to about 100 mg / kg, about 1 mg / kg to about 50 mg / kg, about 5 mg / kg to about 25 mg / kg, and about 10 mg / kg to about 15 mg / kg.

[0052] The monoterpenes or sesquiterpenes can be used in combination with at least one therapeutic agent, including but not limited to chemotherapeutic agents, immunotherapeutic agents, immunomodulatory agents, 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.

[0053] Anticancer agents that can be used in combination with purified monoterpenes or sesquiterpenes can have one or more of the following effects on cancer cells or individuals: cell death; reduced cell proliferation; reduced cell number; cell growth inhibition; apoptosis; necrosis; mitotic catastrophe; cell cycle arrest; reduced cell size; reduced cell division; reduced cell survival; reduced cell metabolism; markers of cell damage or cytotoxicity; indirect indicators of cell damage or cytotoxicity, such as tumor shrinkage; improved survival of an individual; or disappearance of markers associated with undesirable, unwanted or abnormal cell proliferation. U.S. Patent Publication No. 20080275057.

[0054] The therapeutic agent may be dissolved in perillyl alcohol.The compositions of the invention may be administered alone, or may be co-administered with radiation or another active agent (eg, a chemotherapeutic agent) to treat a disease such as cancer.

[0055] In some embodiments, the active agent 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 a target cell. Toxins or drugs that can be used for antibody-drug conjugates are well known in the art and are obvious to those of ordinary skill in the art. See, for example, Peters et al. Biosci. Rep. (2015) 35 (4): e00225. In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker, such as a cleavable linker) connecting the antibody and the drug molecule.

[0056] Treatment may be sequential, the monoterpene (or sesquiterpene) may be administered before or after administration of the therapeutic agent. Alternatively, the monoterpene (or sesquiterpene) and the therapeutic agent may be administered concurrently.

[0057] The monoterpene (or sesquiterpene) and at least one therapeutic agent may be administered simultaneously, separately or sequentially. They may exert a beneficial combined effect (eg, additive or synergistic effect).

[0058] For sequential administration, the monoterpene (or sesquiterpene) may be administered first and then the therapeutic agent, or the therapeutic agent may be administered first and then the monoterpene (or sesquiterpene). In embodiments where the monoterpene (or sesquiterpene) and the therapeutic agent are administered separately, the monoterpene (or sesquiterpene) may be administered seconds, minutes, hours, days or weeks before or after the therapeutic agent is administered. The time difference in non-simultaneous administration may be greater than 1 minute, and may be, for example, precisely at least, at most or less than 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 more than 48 hours. The two or more active agents can be administered within a few minutes of each other, or within about 0.5, about 1, about 2, about 3, about 4, about 6, about 9, about 12, about 15, about 18, about 24, or about 36 hours of each other, or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other, or within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases, the intervals can be longer.

[0059] The present disclosure also provides pharmaceutical compositions comprising (i) at least one monoterpene (or sesquiterpene); and (ii) at least one therapeutic agent.

[0060] The route of administration may vary and may include intra-arterial administration, inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection.

[0061] The present invention also provides a method for treating a disease such as cancer, comprising the step of delivering a composition of the present invention to a patient.

[0062] The composition of the present invention may contain one or more types of monoterpenes (or sesquiterpenes). Monoterpenes include terpenes having two isoprene units and having a molecular formula of C 10 H 16Monoterpenes can be linear (acyclic) or contain rings. Monoterpenoids produced by biochemical modifications such as oxidation or rearrangement of monoterpenes, as well as pharmaceutically acceptable salts of monoterpenes or monoterpenoids, are also encompassed by the present invention. Examples of monoterpenes and monoterpenoids include perillyl alcohol (S(-) and R(+)), geranyl pyrophosphate (geranyl pyrophosphate), ocimene, myrcene, geraniol, citral, citronellol, citronellal, linalool, pinene, terpineol, terpinen, limonene, terpinenes, phllandrenes, terpinolene, terpinen-4-ol (or tea tree oil), pinene, terpineol, terpinene; terpenoids such as p-cymene, which are derived from monocyclic terpenes such as menthol, thymol and carvocrol; bicyclic monoterpenoids such as camphor, borneol and eucalyptol.

[0063] Monoterpenes can be distinguished by carbon skeleton structure and can be grouped 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, verbanon, pinocarveol, carene, sabinene, camphene, thujene, etc.), and tricyclic monoterpenes (e.g., tricyclene). See Encyclopedia of Chemical Technology , 4th edition, Volume 23, Pages 834-835.

[0064] The sesquiterpenes of the present invention include terpenes having three isoprene units and having a molecular formula of C 15 H 24Terpenes. Sesquiterpenes can be linear (acyclic) or contain rings. Sesquiterpenoids produced by biochemical modifications such as oxidation or rearrangement of sesquiterpenes are also encompassed by the present invention. Examples of sesquiterpenes include farnesol, farnesal, farnesylic acid, and nerolidol.

[0065] Derivatives of monoterpenes (or sesquiterpenes) include, but are not limited to, esters, alcohols, aldehydes and ketones of monoterpenes (or sesquiterpenes). Monoterpene (sesquiterpene) alcohols can be derived into esters, aldehydes or acids.

[0066] The esters of the monoterpene (or sesquiterpene) alcohol 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 esters of monoterpene (or sesquiterpene) alcohols include, but are not limited to, carboxylic acid esters (such as benzoates, fatty acid esters (such as palmitate and linoleate), acetates, propionates (propionates or propanoates), and formates), phosphates, sulfates, and carbamates (such as N, N-dimethylaminocarbonyl). Wikipedia - Ester, available from URL: http: / / en.wikipedia.org / wiki / Ester.

[0067] A specific example of a monoterpene useful in the present invention is perillyl alcohol (commonly abbreviated as POH). The perillyl alcohol composition of the present invention may contain (S)-perillyl alcohol, (R)-perillyl alcohol, or a mixture of (S)-perillyl alcohol and (R)-perillyl alcohol.

[0068] The terms "chimeric receptor", "chimeric antigen receptor" or alternatively "CAR" are used interchangeably herein and refer to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") containing a functional signaling domain of a stimulatory molecule 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 stimulatory molecule is a ζ chain that binds to a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule defined below. The co-stimulatory molecule may also be 4-1BB (i.e., CD137), CD27 and / or CD28 or fragments of these molecules. On the other hand, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule. CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. Alternatively, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. CAR can also comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. The antigen recognition portion of CAR may comprise any antigen-binding antibody fragment. The antibody fragment may comprise one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination of any of the foregoing.

[0069] As used herein, a chimeric receptor refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an antigen binding fragment. In general, a chimeric receptor comprises at least two domains derived from different molecules. In addition to the antigen binding fragments described herein, the chimeric receptor may further comprise one or more of a hinge domain, a transmembrane domain, at least one co-stimulatory domain, and a cytoplasmic signaling domain. In some embodiments, the chimeric receptor comprises an antigen binding fragment, a hinge domain, a transmembrane domain, and a cytoplasmic signaling domain from the N-terminus to the C-terminus. In some embodiments, the chimeric receptor also comprises at least one co-stimulatory domain.

[0070] In some embodiments, the chimeric receptors described herein comprise a hinge domain, which may be located between the antigen binding fragment and the transmembrane domain. A hinge domain is an amino acid stretch that is typically found between two domains of a protein and that allows for flexibility of the protein and movement of one or both of the two domains relative to each other. Any amino acid sequence that provides this flexibility and mobility of the antigen binding fragment relative to another domain of the chimeric receptor may be used.

[0071] Any of the chimeric receptors described herein can be introduced into suitable immune cells for expression by conventional techniques. In some embodiments, the immune cell is a T cell, such as a primary T cell or a T cell line. Alternatively, the immune cell can be a NK cell, such as an established NK cell line (e.g., NK-92 cells). In some embodiments, the immune cell is a cell expressing CD8 (CD8 + ) or CD8 and CD4 (CD8 + / CD4 + ). In some embodiments, the T cells are T cells of an established T cell line, such as 293T cells or Jurkat cells.

[0072] In some embodiments, immune cells expressing any of the chimeric receptors described herein are administered to an individual in an amount effective to reduce the number of target cells (e.g., cancer cells) by at least 20%, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more.

[0073] A typical amount of cells (e.g., immune cells (such as CAR T cells)) administered to a mammal (e.g., a human) can be, for example, in the range of 1 million to 100 billion cells; however, amounts below or above this exemplary range are also within the scope of the present disclosure. For example, a daily dose of cells can be about 1 million to about 50 billion cells (e.g., 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 a range defined by any two of the above values), preferably about 10 million to 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 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the above values), more preferably about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the above values).

[0074] In one embodiment, a chimeric receptor (e.g., a nucleic acid encoding a chimeric receptor) is introduced into an immune cell, and an individual (e.g., a human patient) receives an initial administration or dose of an immune cell expressing the chimeric receptor. One or more subsequent administrations of an active agent (e.g., an immune cell expressing a chimeric receptor) may be provided to the patient at intervals of 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days after the previous administration. One or more doses of an active agent may be administered to an individual per week, such as 2, 3, 4, or multiple doses of an active agent. An individual may receive one or more doses of an active agent (e.g., an immune cell expressing a chimeric receptor) per week, followed by no active agent for one week, and finally one or more additional doses of an active agent (e.g., multiple doses of an immune cell expressing a chimeric receptor per week). Immune cells expressing chimeric receptors may be administered every other day, 3 times per week, for 2, 3, 4, 5, 6, 7, 8, or more weeks.

[0075] In the present invention, as it relates to any disease condition described herein, the term "treat" and the like means to alleviate or relieve at least one symptom associated with such condition, or to slow or reverse the progression of such condition. In the present invention, the term "treat" also means to prevent, delay the onset (i.e., the period before the clinical manifestation of the disease) and / or reduce the risk of the disease developing or worsening. For example, the term "treat" related to cancer can refer to eliminating or reducing the patient's tumor burden, or preventing, delaying or inhibiting metastasis, etc.

[0076] The methods and compositions described herein can be used to treat, but are not limited to, brain tumors, lung cancer, ear, nose and throat cancer, blood cancer, colon cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; intraepithelial neoplasia; kidney cancer; laryngeal cancer; liver cancer; fibroma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; kidney cancer; respiratory system cancer; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancer, and other epithelial cancers and sarcomas.

[0077] Carcinomas are cancers of epithelial origin. The epithelial carcinomas intended to be treated with the disclosed methods include, but are not limited to, acinar carcinoma, acinous carcinoma, alveolar adenocarcinoma (also known as adenocystic carcinoma, adenomyoepithelioina, cribriform carcinoma, and cylindroma), carcinoma adenomatosum, adenocarcinoma, carcinoma ofadrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also known as bronchiolar carcinoma, alveolar cell tumor, and pulmonary adenomatosis), basal cell carcinoma, basal cell carcinoma, carcinoma basocellulare (also known as basaloma or basal cell carcinoma and hair matrix carcinoma), basaloid carcinoma, basosquamous cell carcinoma, breast carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma (also known as cholangioma and cholangioadenoma), chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse), carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epibulbar carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoides, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma carcinoma, hepatocellular carcinoma (also known as hepatoma, malignant hepatoma, hepatocarcinoma), Huirthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinomamastitoides, carcinoma medullare, medullary carcinoma, carcinoma melanodes, melanotic carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinomamyxomatodes, nasopharyngeal carcinoma, carcinoma nigrum, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (also called adenocarcinoma of kidney and hypemephoroid carcinoma), reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous cell carcinomacarcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma vilosum. In a preferred embodiment, the method of the present disclosure is used to treat an individual suffering from breast cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, colon and rectal cancer, pancreatic cancer, stomach cancer or kidney cancer.

[0078] Sarcomas are mesenchymal tumors that arise in bone and soft tissue. Different types of sarcomas are recognized and include: liposarcomas (including myxoid liposarcomas and pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal (ie, nonbone) Ewing's sarcoma, and primitive neuroectodermal tumors [PNET]), synovial sarcomas, and ulcerative colitis. sarcoma), angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, desmoid tumor (also called aggressive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor), gastrointestinal stromal tumortumor (GIST)) (also called GI stromal sarcoma), osteosarcoma (also called osteogenic sarcoma) - skeletal and extraskeletal, and chondrosarcoma.

[0079] In some embodiments, the cancer to be treated may be a refractory cancer. "Refractory cancer" as used herein refers to a cancer that tolerates the prescribed standard treatment. These cancers may initially respond to treatment (and then relapse), or may not respond to treatment at all. Common standard treatments will vary depending on the type of cancer and the degree of progression of the individual. It may be chemotherapy, or surgery, or radiotherapy, or a combination thereof. Those of ordinary skill in the art understand such standard treatments. Therefore, an individual treated for a refractory cancer according to the present disclosure may have received another treatment for their cancer. Alternatively, if the cancer may be refractory (e.g., based on analysis of cancer cells or individual medical history), the individual may not have received another treatment. Examples of refractory cancers include, but are not limited to, leukemia, melanoma, renal cell carcinoma, colon cancer, liver cancer, pancreatic cancer, non-Hodgkin's lymphoma, and lung cancer.

[0080] Any immune cell expressing the chimeric receptor described herein can be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier or excipient.

[0081] The phrase "pharmaceutically acceptable" used in conjunction with the compositions and / or cells of the present disclosure means that the molecular entities and other components of the composition are physiologically tolerable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" as used herein refers to approved by a regulatory agency of a federal or state government, or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, especially humans. "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the individual to whom the composition is administered. Any pharmaceutical composition and / or cell used in the method of the present invention may include a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formation or an aqueous solution.

[0082] Pharmaceutically acceptable carriers (including buffers) are well known in the art and may include: phosphoric acid, citric acid 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 sugars; 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.

[0083] Treatment kit

[0084] Also included within the scope of the present invention are kits for the active agents / compositions of the present invention. Such kits may include 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 include 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.

[0085] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include a description of administering the first and second pharmaceutical compositions to an individual to achieve a desired activity in the individual. The kit may also include a description of selecting an individual suitable for treatment based on identifying whether the individual needs treatment. In some embodiments, the instructions include a description of administering the pharmaceutical composition to an individual in need of treatment.

[0086] Instructions related to the use of pharmaceutical compositions described herein typically contain information about the dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the medicine box of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in an individual.

[0087] The medicine box provided herein adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, soft packaging, etc. It is also considered to be used in combination with a specific device (such as an inhaler, a nasal administration device, or an infusion device). The medicine box can have a sterile interface (for example, the container can be an intravenous infusion bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container can also have a sterile interface.

[0088] The kit may optionally provide additional ingredients, such as buffer and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0089] Perillyl alcohol derivatives include perillyl alcohol esters, perillic aldehyde, dihydroperillic acid and perillic acid. Perillyl alcohol derivatives may also include its oxidized derivatives and nucleophilic / electrophilic addition derivatives. U.S. Patent Publication No. 20090031455. U.S. Patent Nos. 6,133,324 and 3,957,856.

[0090] The present invention also provides methods for treating diseases such as cancer or other nervous system disorders using a monoterpene (or sesquiterpene) and at least one therapeutic agent. The monoterpene (or sesquiterpene) can be administered alone or in combination with a therapeutic agent. The monoterpene or sesquiterpene can also be co-administered with a therapeutic agent. The monoterpene or sesquiterpene can also be administered in combination with a therapeutic agent. The therapeutic agent can be administered concurrently or sequentially. The monoterpene (or sesquiterpene) can be administered before, during, or after administration of the therapeutic agent.

[0091] Monoterpenes (or sesquiterpenes) can be used as solvents or penetration enhancers to deliver therapeutic agents to the lesion site. For example, monoterpenes (or sesquiterpenes) can be used as solvents or penetration enhancers to deliver chemotherapeutic agents to tumor cells. Monoterpenes or sesquiterpenes can also be used as solvents for vaccines, which can be delivered by any suitable route.

[0092] The compositions and methods of the invention can be used to treat nervous system cancers, such as malignant gliomas (e.g., astrocytomas, anaplastic astrocytomas, glioblastoma multiforme), retinoblastomas, pilocytic astrocytomas (grade I), meningiomas, brain metastases, neuroblastomas, pituitary adenomas, skull base meningiomas, and skull base cancers. The term "nervous system tumor" as used herein refers to a condition in which an individual has a malignant proliferation of nervous system cells.

[0093] Cancers that may be treated by the compositions and methods of the present invention include, but are not limited to, lung cancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, breast cancer, blood cancer, ovarian cancer, basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemia, including acute myeloid leukemia disease, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia; liver cancer; lymphomas, including Hodgkin and non-Hodgkin lymphomas; myeloma; fibroids, neuroblastomas; oral cancers (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; kidney cancer; respiratory system cancers; sarcomas; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancers, and other cancers and sarcomas. U.S. Patent No. 7,601,355.

[0094] The present invention also provides methods and compositions for treating CNS disorders including, but not limited to, primary degenerative neurological disorders (eg, Alzheimer's disease, Parkinson's disease), psychological disorders, psychosis, and depression.

[0095] The composition of the present invention may be used in combination with radiotherapy.

[0096] 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 the purified monoterpenes or sesquiterpenes may have one or more of the following effects on cancer cells or individuals: cell death; reduced cell proliferation; reduced cell number; cell growth inhibition; apoptosis; necrosis; mitotic catastrophe; cell cycle arrest; reduced cell size; reduced cell division; reduced cell survival; reduced cell metabolism; markers of cell damage or cytotoxicity; indirect indicators of cell damage or cytotoxicity, such as tumor shrinkage; improved survival of an individual; or disappearance of markers associated with undesirable, unwanted, or abnormal cell proliferation. U.S. Patent Publication No. 20080275057.

[0097] The present invention also encompasses mixtures and / or co-formulations of a monoterpene (or sesquiterpene) and at least one therapeutic agent (including but not limited to a chemotherapeutic agent).

[0098] Chemotherapeutic agents include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vinca alkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, boron radiosensitizers (i.e., Velcade), and chemotherapy combination therapies.

[0099] DNA alkylating agents are well known in the art and are used to treat a variety of tumors. Non-limiting examples of DNA alkylating agents are nitrogen mustards such as mechlorethamine, cyclophosphamide (Ifosfamide, Trofosfamide), chlorambucil (Melphalan, Prednimustine), bendamustine, uracil mustard, and estramustine; nitrosoureas such as carmustine (BCNU), lomustine (Semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkyl sulfonic acids such as thiamethoxam, oxadiazine, dapoxet ... sulfonates, such as busulfan (Mannosulfan, Treosulfan); Aziridines, such as Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine); Triazenes, such as Dacarbazine and Temozolomide; Altretamine and Mitobronitol.

[0100] Non-limiting examples of topoisomerase I inhibitors include camptothecin derivatives, including for example Pommier Y. (2006) Nat.Rev.Cancer6(10):789-802 and CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, exatecan, BN-80927, DX-8951f and MAG-CPT described in U.S. Patent Publication No. 200510250854; Protoberberine alkaloids and their derivatives, including, for example, Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. Berberrubine and coralyne as described in 15(12):2795-2800; Phenanthroline derivatives, including those described in Makhey et al. (2003) Bioorg.Med.Chem Benzo[i]phenanthridine, nitidine and fagaronine as described in .11(8):1809-1820; as described in Xu (1998) Biochemistry Terbenzimidazole and its derivatives as described in 37(10):3558-3566; and anthracycline derivatives, including those described in 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. (1986) Biochem.Biophys.Res.Commun. Doxorubicin, daunorubicin, and mitoxantrone are described in 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, etoposide and teniposide. Dual topoisomerase I and II inhibitors include, but are not limited to, Denny and Baguley (2003). Curr.Top.Med.Chem.3(3):339-353, Saintopin and other naphthecenediones, DACA and other acridine-4-carboxamides, Intoplicine and other benzopyridoindoles, TAS-I03 and other 7H-indeno[2,1-c]quinolin-7-ones, Pyrazoloacridine, XR 11576 and other benzophenazines, XR 5944 and other dimeric compounds, 7-oxo-7H-dibenzo[f,ij]isoquinoline and 7-oxo-7H-benzo[e]perimidines and anthracenyl-amino acid conjugates. Some agents inhibit topoisomerase II and have DNA intercalation activity, such as, but not limited to, anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and anthracenediones (Mitoxantrone and Pixantrone).

[0101] Examples of ER stress inducers include, but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (ie, Velcade (bortezomib)).

[0102] Platinum-based compounds are a subclass of DNA alkylating agents. Non-limiting examples of such agents include Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216. (See McKeage et al. (1997) J. Clin. Oncol. 201: 1232-1237, and generally, see CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVELAPPROACHES, in the Series Basic and Clinical Oncology, Angioli et al., eds., 2004).

[0103] Non-limiting examples of antimetabolite agents include folate-based agents, i.e., dihydrofolate reductase inhibitors such as Aminopterin, Methotrexate, and Pemetrexed; thymidylate synthase inhibitors; inhibitors, such as Raltitrexed, Pemetrexed; purine-based agents, i.e., adenosine deaminase inhibitors, such as Pentostatin, thiopurines, such as Thioguanine and Mercaptopurine, halogenated / ribonucleotide reductase inhibitors, such as Cladribine, Clofarabine, Fludarabine, or guanine / guanosine: mercaptopurines, such as Thioguanine; or pyrimidine-based agents, i.e., cytosine / cytidine: hypomethylating agents agents, such as azacitidine and decitabine, DNA polymerase inhibitors, such as cytarabine, ribonucleotide reductase inhibitors, such as gemcitabine, or thymidine / thymidine:thymidylate synthase inhibitors, such as 5-fluorouracil (5-FU). Equivalents of 5-FU include its prodrugs, analogs and derivatives thereof, such as 5'-deoxy-5-fluorouridine (doxifluroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), capecitabine (Xeloda), SI (MBMS-247616, comprising tegafur and two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, as described in Papamicheal (1999) The Oncologist 4:478-487.

[0104] Examples of vinca alkaloids include, but are not limited to, Vinblastine, Vincristine, Vinflunine, Vindesine, and Vinorelbine.

[0105] Examples of taxanes include, but are not limited to, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel. An example of an epothilone is iabepilone.

[0106] Examples of enzyme inhibitors include, but are not limited to, farnesyl transferase inhibitors (tipifamib; CDK inhibitors (Alvocidib, Seliciclib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (Anagrelide; rolipram); IMP dehydrogenase inhibitors (Tiazofurine); and lipoxygenase inhibitors (Masoprocol). Examples of receptor antagonists include, but are not limited to, ERA (Atrasentan); retinoid X receptor (Bexarotene); and sex steroids (Testolactone).

[0107] Examples of therapeutic antibodies include, but are not limited to, anti-HER1 / EGFR (Cetuximab, Panitumumab); anti-HER2 / neu(erbB2) receptor (Trastuzumab); anti-EpCAM (Catumaxomab, Edrecolomab); anti-VEGF-A (Bevacizumab); anti-CD20 (Rituximab, Tositumomab, Ibritumomab); anti-CD52 (Alemtuzumab); and anti-CD33 (Gemtuzumab). U.S. Pat. Nos. 5,776,427 and 7,601,355.

[0108] Examples of tyrosine kinase inhibitors include, but are not limited to, inhibitors against ErbB: HER1 / EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2 / neu (Lapatinib, Neratinib); Class III RTK: C-kit (Axitinib, Sunitinib, Sorafil The present invention relates to the inhibition of glioma (glioma, ...

[0109] Cetuximab is an example of an anti-EGFR antibody. It is a human / mouse chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR). Bioequivalent antibodies are identified herein as modified antibodies and those that bind to the same epitope of the EGFR antigen and produce substantially equivalent biological responses, such as preventing ligand binding of EGFR, preventing activation of EGFR receptors, and blocking downstream signaling of EGFR pathways (causing cell growth disruption).

[0110] Lapatinib It is EGFR and erbB-2 dual inhibitor. Lapatinib has been studied in many clinical trials as anticancer monotherapy, and combined with trastuzumab, capecitabine, letrozole, paclitaxel and FOLF1R1 (irinotecan, 5-fluorouracil and leucovorin). Currently, the oral treatment for metastatic breast cancer, head and neck cancer, lung cancer, gastric cancer, kidney cancer and bladder cancer is being tested in phase III. The chemical equivalent of lapatinib is a small molecule or compound, which is a tyrosine kinase inhibitor (TKI) or alternatively a HER-1 inhibitor or a HER-2 inhibitor. It has been found that several TKIs have effective anticancer activity, and have been approved or in clinical trials. Such examples include, but are not limited to, Zactima (ZD6474), Iressa (gefitinib) and Tarceva (erlotinib), imatinib mesylate (STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY43-9006), sutent (SUI1248) and leflunomide (SU101). The bioequivalent of lapatinib is a peptide, an antibody or an antibody derivative thereof, which is a HER-1 inhibitor and / or a HER-2 inhibitor. Such examples include, but are not limited to, the humanized antibodies trastuzumab and Herceptin.

[0111] PTK / ZK is a "small" molecule tyrosine kinase inhibitor with broad specificity 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-1 (Flt-1), VEGFR-2 (KDR / Flk-1) and VEGFR-3 (Flt-4). The chemical name of PTK / ZK is 1-[4-chloroaniline]-4-[4-pyridylmethyl]phthalazine succinate or 1-phthalazineamine, N-(4-chlorophenyl)-4-(4-pyridylmethyl)-succinate (1:1). Synonyms and analogs of PTK / TK are called Vatalanib, CGP79787D, PTK787 / ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TK inhibitor, ZK 222584 and ZK.

[0112] Chemotherapeutic agents that can be used in combination with monoterpenes or sesquiterpenes can also include amsacrine, Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide, asparagine deplete (Asparaginase / Pegaspargase), Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and Vorinostat.

[0113] Other therapeutic agents that can be used with the compositions and methods of the invention include, for example, CAR-T cells, CAR-macrophages, or CAR-NK cells.

[0114] The compositions and methods of the invention can be used to increase paracellular permeability, such as paracellular permeability of endothelial cells or epithelial cells. The compositions and methods of the invention can be used to increase blood-brain barrier permeability. The effect of administration on blood-brain barrier permeability can last from 5 minutes to 10 hours; other ranges include at least about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 24 hours, 48 ​​hours, or 72 hours.

[0115] The compositions and methods of the present invention can be used to reduce or inhibit angiogenesis. The compositions and methods of the present invention can reduce or inhibit the production of pro-angiogenic cytokines, including but not limited to vascular endothelial growth factor (VEGF), and interleukin 8 (IL8).

[0116] Monoterpenes or sesquiterpenes can be used in combination with angiogenesis inhibitors. Examples of angiogenesis inhibitors include, but are not limited to, angiostatin, angiozyme, antithrombin III, AG3340, VEGF inhibitors (e.g., anti-VEGF antibodies), batimastat, bevacizumab (Avastin), BMS-275291, CAI, 2C3, HuMV833 Canstatin, Captopril, carboxyamidotriazole, cartilage inhibitor (CDI), CC-5013, 6-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin, combretastatin A4 phosphate, Dalteparin, EMD 121974 (Cilengitide), endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginone hydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11 inducible protein 10, interferon alpha, interleukin 12, lavendustin A, LY317615 or AE-941, marimastat, mspin, medroxpregesterone acetate, Meth-1, Meth-2, 2-methoxyestradiol (2-ME), neovastat, oteopontin cleaved products product), PEX, pigment epithelial growth factor (PEGF), platelet factor 4, prolactin fragment, proliferin-related protein (PRP), PTK787 / ZK222584, ZD6474, recombinant human platelet factor 4 (rPF4), restin, squalamine, SU5416, SU6668, SU11248 suramin, Taxol, Tecogalan, thalidomide, thrombospondin, TNP-470, troponin-1, vasostatin, VEG1, VEGF-Trap and ZD6474.

[0117] Non-limiting examples of angiogenesis inhibitors also include tyrosine kinase inhibitors, such as tyrosine kinase receptor Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2) inhibitors, epidermal derived growth factor inhibitors, fibroblast derived growth factor inhibitors, or platelet derived growth factor inhibitors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, pentosan polysulfate, angiotensin II antagonists, cyclooxygenase inhibitors (including nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, and selective cyclooxygenase-2 inhibitors, such as celecoxib and rofecoxib), and steroidal anti-inflammatory drugs (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone).

[0118] Other therapeutic agents that regulate or inhibit angiogenesis and can also be used in combination with monoterpenes or sesquiterpenes include agents that regulate or inhibit the coagulation and fibrinolysis systems. Examples of such agents that regulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin, low molecular weight heparin, and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin-activatable fibrinolysis inhibitor [TAFIa]). U.S. Patent Publication No. 20090328239. U.S. Patent No. 7,638,549.

[0119] Immunomodulators include, but are not limited to, cytokines, such as interleukins, lymphokines, monokines, interferons, and chemokines.

[0120] Other penetration enhancers that can be used with monoterpenes (or sesquiterpenes) include, but are not limited to: fatty acid esters of glycerol, such as capric glyceride, caprylic glyceride, lauric glyceride, oleic glyceride; fatty acid esters of isosorbide, sucrose, polyethylene glycol; caproyl lactylic acid (caproyllactylic acid); acid); laureth-2; laureth-2 acetate; laureth-2 benzoate; laureth-3 carboxylic acid; laureth-4; laureth-5 carboxylic acid; oleth-2; glyceryl pyroglutamate oleate; glyceryl oleate; N-lauroyl sarcosine; N-tetradecanoyl sarcosine; N-octyl-2-pyrrolidone; lauryl aminopropionic acid; polypropylene glycol-4-laureth-2; polypropylene glycol-4-laureth-5-dimethyl lauramide; lauramide diethanol (DEA), dodecyl pyroglutamate (LP), glyceryl monolaurate (GML), glyceryl monocaprylate, glyceryl monocaprate, glyceryl monooleate (GMO), and sorbitan monolaurate. The polyol or ethanol may act as a penetration enhancer or cosolvent. Other penetration enhancers are described in US Pat. Nos. 5,785,991; 5,843,468; 5,882,676; and 6,004,578.

[0121] Co-solvents are well known in the art and include, but are not limited to, glycerol, polyethylene glycol (PEG), ethylene glycol, ethanol, methanol, propanol, isopropanol, butanol, and the like.

[0122] The compositions of the present invention can be administered by any method known in the art, including but not limited to intra-arterial, intranasal, oral, ocular, intraperitoneal, inhalation, intravenous, intracardiac injection (IC), intracerebroventricular (ICV), intracisternal injection or infusion, subcutaneous, implanted, intravaginal, sublingual, urethral (e.g., urethral plug), subcutaneous, intramuscular, intravenous, transdermal, rectal, sublingual, mucosal, intraocular, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial and lymphatic administration. Topical preparations can be in the form of gels, ointments, creams, aerosols, etc.; intranasal preparations can be delivered in the form of sprays or drops; transdermal preparations can be administered by transdermal patches or iontophoresis; inhalation preparations can be administered using a nebulizer or similar device. The composition can also be in the form of tablets, pills, capsules, semisolids, powders, sustained-release preparations, solutions, suspensions, elixirs, aerosols, or any other suitable composition.

[0123] In order to prepare such pharmaceutical compositions, one or more monoterpenes (or sesquiterpenes) and / or at least one therapeutic agent may be mixed with a pharmaceutically acceptable carrier, adjuvant and / or excipient according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers that can be used for the compositions of the present invention encompass any standard pharmaceutical carrier, 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 additionally contain solid pharmaceutical excipients, such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, skimmed milk powder, and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and a variety of oils, including those of petroleum, animal, plant or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Liquid carriers, particularly liquid carriers for injection solutions, include water, saline, glucose water and glycols. Examples of carriers, stabilizers and adjuvants are given in Remington's Pharmaceutical Sciences , EW Martin, ed. (Mack Publishing Company, 18th ed., 1990). The composition may also contain stabilizers and preservatives.

[0124] The term "therapeutically effective amount" as used herein is an amount sufficient to treat a particular condition or disease, or alternatively, an amount sufficient to obtain a pharmacological response for treating a condition or disease. The method for determining the most effective mode of administration and dosage can vary with the composition used for treatment, the purpose of treatment, the target cells for treatment, and the individual for treatment. The therapeutic dose can usually be titrated to optimize safety and effectiveness. Single or multiple administrations can be performed according to the dosage level and mode selected by the treating physician. Those skilled in the art can easily determine the appropriate dosage formulation and method for administering an active agent. For example, the composition is administered with about 0.01 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, or about 0.5 mg / kg to about 50 mg / kg. When the compound described herein is administered together with another active agent or therapy, the effective amount may be less than the effective amount when the active agent is used alone.

[0125] The present disclosure also provides compositions for intranasal administration as described above. Likewise, the compositions may further comprise a penetration enhancer. Southall et al. Developments in Nasal Drug Delivery, 2000. The compositions of the present invention may be administered intranasally in liquid form such as solutions, emulsions, suspensions, drops, or in solid form such as powders, gels or pastes. Devices for delivering intranasal drugs are well known in the art. Nasal drug delivery may be performed using devices including but not limited to: intranasal inhalers, intranasal spray devices, atomizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, sprayers, metered dose inhalers (MDI), pressure metered dose inhalers, insufflators, and bidirectional devices. The nasal delivery device may be metered to administer an accurate effective dose to the nasal cavity. The nasal delivery device may be used for single unit delivery or multi-unit delivery. In a specific example, the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Washington) may be used in the present invention (http: / / www.kurvetech.com). The compounds of the invention may also be delivered via a tube, catheter, syringe, packtail, cotton wool, nasal plug or by submucosal infusion. U.S. Patent Publication Nos. 20090326275, 20090291894, 20090281522 and 20090317377.

[0126] The composition of the present invention can be formulated into an aerosol using standard methods. The monoterpene (or sesquiterpene) and / or at least one therapeutic agent can be formulated with or without a solvent, and with or without a carrier. The preparation can be a solution, or can be an aqueous emulsion containing one or more surfactants. For example, an aerosol spray can be prepared by a pressurized container with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen, carbon dioxide or other suitable gases. The dosage unit can be determined by providing a valve to deliver a metered amount. A pump spray dispenser can dispense a metered dose or a dose with a specific particle size or droplet size. The term "aerosol" used herein refers to a suspension of fine solid particles or liquid solution drops in a gas. Specifically, an aerosol includes an airborne suspension of monoterpene (or sesquiterpene) droplets, which can be generated in any suitable device such as an MDI, a nebulizer or a misting machine. Aerosols also include dry powder compositions of the composition of the present invention suspended in air or other carrier gases. Gonda(1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et al. (1992) Pharmacol.Toxicol.Methods 27:143-159.

[0127] The composition of the present invention can be delivered to the nasal cavity in the form of a powder, such as a microsphere, through a nasal insufflator. The composition of the present invention can be adsorbed to a solid surface, such as a carrier. The powder or microsphere can be applied in a dry, air-dispensable form. The powder or microsphere can be stored in a container of an insufflator. Alternatively, the powder or microsphere can be filled into a capsule, such as a gelatin capsule, or other single-dose unit suitable for nasal administration.

[0128] Pharmaceutical composition can be delivered to the nasal cavity by directly placing the composition in the nasal cavity, for example, in the form of gel, ointment, nasal emulsion, lotion, cream, nasal congestion, dropper or bioadhesive strip. In certain embodiments, for example, in order to enhance absorption, it may be desirable to extend the residence time of the pharmaceutical composition in the nasal cavity. Therefore, the pharmaceutical composition can be optionally prepared together with bioadhesive polymers, gums (for example xanthan gum), chitosan (for example high-purity cationic polysaccharides), pectin (or any carbohydrates that can be thickened like gel or emulsified when applied to nasal mucosa), microspheres (for example starch, albumin, dextran, cyclodextrin), gelatin, liposomes, carbomer (carbamer), polyvinyl alcohol, alginate, gum arabic, chitosan and / or cellulose (for example methyl or propyl; Hydroxyl or carboxyl; Carboxymethyl or hydroxypropyl).

[0129] The compositions may be administered by oral inhalation into the respiratory tract (ie, lungs).

[0130] Typical delivery systems for inhalable active agents include nebulizers, dry powder inhalers (DPIs), and metered dose inhalers (MDIs).

[0131] The nebulizer device generates a high-speed air stream to spray a liquid therapeutic agent in the form of a mist. The therapeutic agent is formulated into a liquid form, such as a solution or suspension of particles of suitable size. In one embodiment, the particles are micronized. The term "micronized" is defined as particles with a diameter of about 90% or more less than about 10 microns. Suitable nebulizer devices are commercially available, for example, by PARI GmbH (Starnberg, Germany). Other nebulizer devices include Respimat (Boehringer Ingelheim), and those disclosed in, for example, U.S. Patent Nos. 7,568,480 and 6,123,068 and WO97 / 12687. Monoterpenes (or sesquiterpenes) can be formulated into aqueous solutions or liquid suspensions for use in nebulizer devices.

[0132] DPI devices typically administer therapeutic agents in the form of free-flowing powders that can be dispersed in the airflow during the patient's inspiration. DPI devices using external energy sources can also be used in the present invention. In order to obtain free-flowing powders, the therapeutic agent can be formulated with a suitable excipient (e.g., lactose). For example, a dry powder formulation can be prepared by mixing micronized particles of dry lactose and monoterpenes (or sesquiterpenes) having a particle size of about 1 micron to 100 microns. Alternatively, monoterpenes can be formulated without excipients. The preparation is loaded into a dry powder dispenser, or loaded into an inhalation box or capsule used together with a dry powder delivery device. Examples of commercially available DPI devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519); Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365). Other examples of suitable DPI devices are described in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810, and references therein.

[0133] MDI devices generally utilize compressed propellant gas to release a quantitative therapeutic agent. The preparation for MDI administration includes a solution or suspension of the active ingredient in a liquefied propellant. Examples of propellants include hydrofluoroalkanes (HFA), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA227), chlorofluorocarbons, such as CCl3F. Other components of the HFA preparation for MDI administration include cosolvents, such as ethanol, pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin and glycerol. (See, for example, U.S. Patent No. 5,225,183, EP 0717987 and WO92 / 22286). The preparation is loaded into an aerosol cartridge, which constitutes a part of the MDI device. Examples of MDI devices specifically developed for use with HFA propellants are provided in U.S. Pat. Nos. 6,006,745 and 6,143,227. Examples of methods for preparing suitable formulations and devices suitable for inhalation administration are found in U.S. Pat. Nos. 6,268,533, 5,983,956 and 6,221,398, and WO 99 / 53901, WO 00 / 61108, WO 99 / 55319 and WO 00 / 30614.

[0134] For delivery by inhalation, the monoterpene (or sesquiterpene) and / or at least one therapeutic agent can be encapsulated in liposomes or microcapsules. Liposomes are vesicles consisting of a bilayer lipid membrane and an aqueous interior. The lipid membrane can be composed of phospholipids, examples of which include phosphatidylcholines, such as lecithin and lysolecithin; acidic phospholipids, such as phosphatidylserine and phosphatidylglycerol; sphingomyelins, such as phosphatidylethanolamine and sphingomyelin. Alternatively, cholesterol can be added. Microcapsules are particles coated with a coating material. For example, the coating material can include a mixture of a film-forming polymer, a hydrophobic plasticizer, a surfactant, or / and a lubricant nitrogen-containing polymer. U.S. Patent Nos. 6,313,176 and 7,563,768.

[0135] Due to their ability to readily penetrate the dermis, monoterpenes may also be used by topical application alone or in combination with at least one therapeutic agent. As transdermally delivered active agents, monoterpenes may also be combined with anesthetics or analgesics for transdermal delivery of analgesics.

[0136] The present invention also provides a composition for ocular administration as described above. Likewise, the composition may further comprise a penetration enhancer. For ocular administration, the composition described herein may be formulated into a solution, emulsion, suspension, etc. A variety of carriers suitable for administering the compound to the eye are known in the art. Specific non-limiting examples are described in U.S. Patent Nos. 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; 4,882150 and 4,738,851.

[0137] The composition of the present invention can be administered short-term or long-term. The composition of the present invention can be administered to mammals, preferably humans. Mammals include, but are not limited to, mice, rats, rabbits, apes, cattle, sheep, pigs, dogs, cats, livestock, sport animals, pets, horses, and primates.

[0138] The device for intranasal administration can be an intranasal spray device, a nebulizer, a sprayer, a metered dose inhaler (MDI), a pressurized metered dose inhaler, an insufflator, an intranasal inhaler, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle or a two-way device.

[0139] The active agents may be administered concurrently or sequentially.

[0140] The present invention also provides a method of inhibiting cell growth in vitro, ex vivo, or in vivo, wherein a cell, such as a cancer cell, is contacted with an effective amount of a purified monoterpene (or sesquiterpene) described herein. The compositions and methods of the present invention can be used to inhibit the growth of cells that are resistant to chemotherapeutic agents. For example, the compositions and methods of the present invention can be used to inhibit the growth of cells that are resistant to temozolomide.

[0141] Pathological cells or tissues, such as highly proliferative cells or tissues, can be treated by contacting the cells or tissues with an effective amount of the composition of the present invention. The cells, such as cancer cells, can be primary cancer cells, or can be cultured cells that can be obtained from a tissue bank, such as the American Type Culture Collection (ATCC). The pathological cells can be systemic cancer, glioma, meningioma, pituitary adenoma, or cells from CNS metastasis of systemic cancer, lung cancer, prostate cancer, breast cancer, blood cancer, or ovarian cancer. The cells can be from vertebrates, preferably mammals, more preferably humans. U.S. Patent 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 Reports13:943-947. DaFonseca 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.

[0142] Cancer stem cells (CSCs) or tumor initiating cells are immature cells that have stem cell properties such as self-renewal. However, self-renewal is exacerbated in CSCs. Reya et al., Stem cells, cancer, and cancer stem cells. Nature .2001,414(6859):105-11. In addition, 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. CellStem Cell. 2007; 1(4):353-5. The compositions and methods of the present invention can be used to inhibit the growth of cancer stem cells, including but not limited to glioblastoma cancer stem cells.

[0143] The following examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0144] Example 1 NEO100 mediates human CAR T cell delivery to the brain and tumors

[0145] Preparation of human CAR T cells

[0146] Human CAR T cells (CD19 and Lym-1) were provided by Dr. Epstein (USC). Chimeric antigen receptors (CARs) are synthetic molecules that contain 3 different modules: an antibody-based extracellular recognition site; a transmembrane module that anchors the molecule in the cell membrane; and a chimeric intracellular signaling domain that transmits the 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 efficacy in the treatment of 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 aCD19-Targeted Chimeric Antigen Receptor (CTL019). Blood 2015, 126, (23), 681-681. Mouse IgG2a monoclonal antibody Lym-1 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, and has a higher binding affinity for malignant B cells than for normal B cells. Rose et al., Critical Lym-1 binding residues on polymorphic HLA-DR molecules. Mol Immunol 1999, 36, (11-12), 789-97. E.g. Figure 1 Shown are schematic diagrams of Lym-1 CAR and CD19 (FMC63) CAR constructs.

[0147] Two million CD19 and Lym-1 human CAR T cells suspended in 0.9% saline working solution were injected IV with and without intracardiac NEO100.

[0148] NEO100 intracardiac puncture

[0149] Preparation of NEO100 working solution for intracardiac injection: 3% NEO100 was suspended in 0.9% saline.

[0150] Standard operating procedures for ultrasound-guided intracardiac puncture

[0151] Briefly, animals were anesthetized with 2% isoflurane gas and fixed on a platform for intracardiac puncture. Under ultrasound imaging guidance, a syringe needle was inserted into the intercoastal space and rapidly penetrated through the skin and muscle layers into the left ventricle.

[0152] The return of fresh arterial blood (pink in contrast to the deep red venous blood) into the syringe is a sign of successful needle insertion into the left ventricle. Intracardiac application is completed by slowly injecting 40 μl of 3% NEO100 in saline. Direct injection of cells into the heart can cause local microobstruction and, if cells clump during injection, can lead to pericardial effusion and death. This is why injections with a 30G needle under ultrasound guidance are important to minimize these potential adverse reactions: (1) visualization of the needle tract to ensure that the needle enters only the left ventricle and (2) cardiac monitoring after injection, not only by ECG but also by visualization of cardiac wall function. The fineness of the needle ensures that the cells will not clump when injected through the intracardiac puncture.

[0153] Intracardiac injection confirmation

[0154] The return of fresh arterial blood (pink in contrast to the dark red venous blood) into the syringe is a sign of successful needle insertion into the left ventricle.

[0155] Immediately after the NEO100 injection, 2 million human CAR T cells in 40 μl of PBS were injected through the tail vein catheter pre-treated with saline. To avoid the above-mentioned adverse reactions that may be caused by direct injection of cells through intracardiac application, we established a two-step procedure for this study.

[0156] Step 1: Slowly inject 40 μl of saline containing 3% NEO100 to complete the intracardiac application. This procedure allows NEO100 to exert its BBB-disrupting function.

[0157] Step 2: Two million CAR T cells are injected IV via the tail vein catheter.

[0158] Evaluation of CAR T cell spreading by IHC and confocal imaging

[0159] Brain perfusion - To eliminate the residual material remaining in the blood vessels after euthanasia, the experimental animals were perfused through the left ventricle with 10 ml of 0.9% normal saline solution to flush out the blood. Then, the brain was removed, embedded in OCT, and stored at -80°C for further analysis.

[0160] Confocal imaging - 8 μM fresh frozen sections were cut using a cryostat and mounted on slides. Coverslips were mounted on brain sections using DAPI mounting medium prior to confocal examination.

[0161] IHC staining - A standardized IHC staining procedure was used to detect the infiltration of human CAR T cells into the brain and the established tumors (GL261 mouse glioma). Anti-human CD3 antibody (CD3ε (D7A6E TM ) Rabbit mAb (#85061) (Cell Signaling, Boston, MA) primary antibody recognized human CD3 positive cells (as shown in Figure 2).

[0162] Syngeneic mouse glioma animal model in C57 BL / 6 mice

[0163] 100,000 GL261 mouse glioma cells were injected intracranially into immune competent 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) by intravenous application (IV) and intracardiac (IC) combined with IV application. Treated mice were sacrificed 6h after intervention. For intracardiac application: 2 million anti-CD19 or Lym-1 CAR T cells were injected IV after intracardiac injection of PBS containing 3% NEO100. For intravenous application: 2 million anti-CD19 or Lym-1 CAR T cells were suspended in 40ul PBS and injected via tail vein.

[0164] The brains were perfused with 0.9% saline, removed, and stored at -80°C for further analysis.

[0165] Antibodies used for detection included: control antibody for negative staining: rabbit (DA1E) mAb IgG isotype; and antibodies for in vitro and in vivo detection of CD3-positive cells: CD3ε (D7A6E TM ) Rabbit mAb (#85061).

[0166] in conclusion

[0167] No detectable CD3-positive cells were found in the brain of normal C57 BL / 6 mice.

[0168] Intracardiac injection of NEO100-mediated human CAR T cells (anti-CD19 and Lym-1) significantly increased the penetration into tumors established in the brain compared with conventional intravenous (IV) injection.

[0169] The intracardiac injection of 3% NEO100 did not cause any serious adverse reactions or animal deaths.

[0170] In the normal part of the brain treated by intracardiac injection of NEO100, more CD3 positive cells were seen than in the samples injected only IV.

[0171] Example 2 Anti-mouse PD-1 antibody-mediated therapeutic effects in C57 BL / 6 mice bearing intracranial syngeneic mouse glioma (GL261)

[0172] 100,000 GL261 mouse glioma cells were injected intracranially into immune competent C57 BL / 6 mice. Seven days after injection, mice were randomly divided into four experimental groups and treatments were started on the same day.

[0173] Group 1. Control: IV and intracardiac injection of 40 μl of physiological saline solution (5).

[0174] Group 2. Antibody-treated mice: 40 μl of anti-mouse PD1 antibody (5) was administered IV at a dose of 2.5 mg / kg.

[0175] Group 3. NEO100-treated mice: 40 μl of 5% NEO100 (5) were injected intracardially.

[0176] Group 4. Mice treated with NEO100 and antibody combination: 40 μl of 5% NEO100 was injected intracardially, followed by 40 μl of anti-PD1 antibody (6) at a dose of 2.5 mg / kg IV.

[0177] The results are as follows Figure 3 We demonstrated that intracardiac injection of NEO100 (equivalent to intra-arterial injection in mice) can open the BBB for antibodies. We then established a syngeneic model with intracranially implanted mouse GL26 glioma cells. Mice were treated with saline, NEO100 alone, intravenous anti-PD1 alone, or intracranially NEO100 followed by intravenous anti-PD1. All mice treated with intravenous anti-PD1 combined with NEO100 remained alive, while all controls except one mouse that received intravenous anti-PD1 died.

[0178] Perillyl alcohol can be administered via the femoral artery for interventional neuroradiology (eg, cerebral angiography).

[0179] Statistical analysis

[0180] Animal survival data were plotted using the Kaplan-Meier method. One-way analysis of variance was used for overall testing of differences. Tukey's method adjusted for multiple comparisons was used for group comparisons. Logrank (Mantel-Cox) test was used to compare survival curves. Statistical evaluation results were considered significant when p < 0.05.

[0181] Control vs. IC NEO100 + IV anti-mouse PD-1: ***P < 0.0003

[0182] Control vs. IV anti-mouse PD-1: ns, p=0.31

[0183] IV anti-mouse PD-1 vs. IC NEO100 + IV anti-mouse: **P<0.005

[0184] Control vs. IC NEO100: ns, p=0.397

[0185] Example 3

[0186] We demonstrate that NEO100 can be used to cross an in vitro BBB model and transiently allows labeled antibodies to cross the model ( Figures 4A-4D ).

[0187] Experiments were conducted to investigate whether intra-arterial delivery of perillyl alcohol (eg, NEO100) could be used to transiently disrupt the BBB, allowing previously impenetrable small or large molecules to penetrate the brain.

[0188] Administration of perillyl alcohol (eg, NEO100) can include intracardiac injection (intrapratrial injection in mice) and intravenous infusion.

[0189] The formulation included 10% NEO100 (27.5 ml glycerol + 27.5 ml ethanol + 3.0 ml NEO100).

[0190] Brain perfusion-Before euthanasia, the experimental animals were perfused via the left ventricle with 0.9% normal saline solution. The brains were removed, embedded in OCT, and stored at -80°C for further analysis.

[0191] Ultrasound-guided intracardiac puncture - Briefly, animals were anesthetized with 2% isoflurane gas and secured on a platform for intracardiac puncture. Under ultrasound imaging guidance, the syringe needle was inserted into the intercostal space and rapidly passed through the skin and muscle layers into the left ventricle. Fresh arterial blood (pink in contrast to the dark red venous blood) returning to the syringe was a sign of successful needle insertion into the left ventricle.

[0192] Evans blue is an azo dye with a high affinity for serum albumin. Extravasation of albumin from the circulation can be visualized after staining.

[0193] NEO100 was delivered via intracardiac injection (left ventricle) to determine whether brain uptake of Evans blue, BBB-impermeant small molecules (dopamine), or antibodies was increased. Figure 5A Intracardiac injection (IC) of a mixture of NEO100 and 2% Evans blue (EB) is shown. Different concentrations of NEO100 (40 μl in 0.9% saline) were tested by intracardiac puncture, followed immediately by intravenous application of 2% Evans blue (volume 40 μl). The brain was removed after perfusion. The results show that NEO100 can still effectively disrupt the BBB at a 1:1000 dilution (6.5 mM 40 μl).

[0194] Figure 5B EB penetration into the brain is shown after application of NEO100 by IC (intracardiac injection) or IV injection.

[0195] The experimental groups included:

[0196] IC 2% EB only

[0197] IC 20% ethanol + 2% EB

[0198] IC 20% ethanol + 2% EB + 5% NEO100

[0199] IC 20% ethanol + 5% NEO100, followed by 2% EB tail vein injection

[0200] IV 20% ethanol + 2% EB + 5% NEO100

[0201] IV 20% ethanol + 2% EB

[0202] Figure 6 It was shown that tight junctions in the brain treated with intracardiac injection of 5% NEO100 were significantly disrupted compared with the normal brain.

[0203] The drug treatment of Parkinson's disease (PD) is mainly based on symptomatic replacement therapy of dopamine (DA), but exogenous DA and other catecholamines cannot be administered because of their poor BBB permeability. Dopamine is a water-soluble hydrophilic drug that does not meet the characteristics of substances that can penetrate through the BBB and enter the brain.

[0204] Figure 7 showed that NEO100 mediates dopamine delivery across a disrupted blood-brain barrier.

[0205] Figure 8 Measurement of BBB opening and closing time is shown. Immunocompetent C57 BL / 6 mice were injected with 5% NEO100 (v / v) via intracardiac puncture (IC) and then injected with 2% Evans blue intravenously at different time points such as 0, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h after IC injection.

[0206] The experimental process includes:

[0207] 1. Intracardiac injection (IC): 5% NEO100.

[0208] 2. Then 2% EB was injected intravenously (IV) at different times.

[0209] 3. Experimental animals were euthanized 1 hour after IV injection.

[0210] Fig. 9 Delivery of anti-mouse IgG antibody (rabbit anti-mouse IgG H&L (Texas Red)—Ab6726) in the absence or presence of perillyl alcohol is shown.

[0211] Fig.10 The delivery of an anti-PD-1 antibody (Armenian hamster anti-mouse CD279 (PD-1) monoclonal antibody (J43)) in the absence or presence of perillyl alcohol was shown. PD-L1 binds to PD-1 and inhibits T cells from killing tumor cells. Blocking PD-L1 or PD-1 allows T cells to kill tumor cells.

[0212] NEO100 is safe when administered intra-arterially.

[0213] Example 4 NEO100-mediated human CAR T cell (Lym-1 CAR) delivery to treat intracranial Raji lymphoma xenografts in NSG mice

[0214] (a) Intracranial lymphoma xenografts:

[0215] 50,000 (5×10 4 ) Human B cell lymphoma cells Raji's-Luc / GFP were injected intracranially into NSG mice.

[0216] (b) Tumor uptake confirmation:

[0217] Five days after tumor cell injection, optical imaging was performed to confirm tumor uptake (100% tumor uptake).

[0218] (c) Start CAR T infusion and intracardiac (IC) NEO100 via tail vein catheter:

[0219] 3 experimental groups: (1) control; (2) IV CAR T (5x10e6); (3) IV CAR T (5x10e6) + IC NEO100 (0.3% v / v = 492 μM)

[0220] (d) Monitoring of NSG mice bearing IC lymphoma:

[0221] During the treatment, the physiological status of the mice was monitored by monitoring the body weight of the mice and the tumor growth was monitored by optical imaging.

[0222] (e) Animal survival rate (Kaplan Meier curve)

[0223] From the survival curve ( Fig.11 ) It can be clearly seen that the control mice, i.e. mice injected with human B cell lymphoma cells, died within 15-20 days after injection, while the mice injected with Lym-1 CAR T cells plus NEO100 survived (P=0.0029).

[0224] Example 5

[0225] The POH is placed into an intranasal inhaler (e.g., ViaNase Electronic Atomizer from Kurve Technology (Bethell, Washington)). The intranasal delivery system from Kurve Technology is capable of accurately delivering a predetermined volume of drug (e.g., 0.2-6 mL). The device is loaded and cleaned in the same manner as a lung nebulizer. The device can deliver drugs to the olfactory region of animals and humans in bench tests.

[0226] Male athymic nu / nu mice (6-8 weeks old) were used for this study. A rodent subcutaneous / intracranial glioma model was established as follows. 6 to 8 week old athymic nu / nu mice were anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (10 mg / kg). For the intracranial glioma model, mice were placed in a head stereotaxic frame (Harvard Apparatus) and local anesthetic (0.2cc of 0.25% lidocaine) was injected into the right frontal scalp. A small incision was made with a blade and a small opening was made in the right frontal skull at the level of the coronal suture with a drill. Glioma cells (1×10 5 Cells / 10 μl) such as U-87 human glioma cells are loaded into a graduated Hamilton syringe. The needle tip is precisely placed in the right frontal lobe of the rat, and the cells are slowly injected from the Hamilton syringe using a controlled push. After the injection is completed, the syringe and needle are removed and the wound is sutured.

[0227] Two weeks after surgical implantation, mice were divided into 4 groups (6 mice / group) and treated with the following drugs: saline drops alone (control), crude POH from Sigma (0.03%, 50ul / drop, one drop in each nostril), POH (purified to a purity greater than 98.5%; 0.03%, 50ul / drop, one drop in each nostril) and TMZ (5mg / kg, oral gavage). TMZ was used as a positive control.

[0228] The brains were harvested and the tumor size was determined. Survival curves were drawn by following the mice until they developed neurological deficits. Our experience is that untreated mice survived about 4 weeks after implantation and mice treated with TMZ survived up to 8 weeks.

[0229] We also used an immune-competent syngeneic rat model in which RG2 rat glioma cells (1 × 10 5 Cells / 10ul) were implanted into the right frontal lobe of Fisher 344 rats. The rats were divided into the same 4 groups as above. Because RG2 cells can migrate freely and thus invade into the rat parenchyma, we also used this rat RG2 model to examine the anti-invasive properties of POH.

[0230] Example 6

[0231] In a recent clinical study conducted in Brazil, intranasal delivery of perillylalcohol in patients with recurrent malignant gliomas resulted in regression or stabilization of the disease, with 50% of the 140 treated patients achieving a 6-month progression-free period and several patients achieving remission for up to 3 years. In addition, side effects of treatment were almost non-existent. Da Fonseca et al. Correlation of tumor topography and peritumoraledema of recurrent malignantgliomas with therapeutic response to intranasal administration of perillylalcohol. Invest in New Drugs 2009,Jan 13.

[0232] We delivered purified POH (with a purity greater than 98.5%) intranasally to patients with malignant gliomas. To investigate whether POH could be delivered directly to brain tumor cells, we delivered POH to patients intranasally. 11 C-labeled POH and subsequent positron emission tomography (PET) imaging were used to study the distribution of purified POH. The patients then underwent a limited treatment trial using increasing doses of inhaled POH. Three groups were used to increase the dose of patients, each receiving 0.05% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), 2.5% (w / v) of intranasal purified POH (with a purity greater than 98.5%). Currently used in Brazil is 2% (w / v). Delivery will be carried out via the ViaNase intranasal inhaler and administered three times a day.

[0233] PET imaging analysis. Intranasal inhalation of 5-10 mCi 11 After C-POH preparation, ten patients with pathologically confirmed malignant gliomas were scanned using Siemens Biograph TruePoint HD PET / CT scanners. Static imaging will begin 30 minutes after inhalation, with 10-minute acquisitions in a single bed position covering the skull. Subsequent series acquisitions will be at 30-minute intervals for 2 hours to assess progressive accumulation in brain and tumor tissues. Depending on the patient's compliance and the level of residual and cumulative activity, we will attempt imaging for more than 2 hours. In all patients, the co-recorded PET / CT images were compared with contrast-enhanced MRI studies to assess the correlation of activity accumulation with enhancement patterns.

[0234] Some embodiments of the present invention:

[0235] 1. A method of administering a therapeutic agent to the central nervous system of a mammal, the method comprising administering a monoterpene prior to or concurrently with the therapeutic agent.

[0236] 2. The method of embodiment 1, wherein the central nervous system is the brain.

[0237] 3. The method of embodiment 1, wherein the monoterpene is perillyl alcohol.

[0238] 4. The method of embodiment 3, wherein perillyl alcohol is administered intra-arterially.

[0239] 5. The method of embodiment 3, wherein perillyl alcohol is administered at a dose ranging from about 0.050 mg / kg to about 500 mg / kg body weight.

[0240] 6. The method of embodiment 1, wherein the mammal is a human.

[0241] 7. The method of embodiment 1, wherein the monoterpene is administered from about 0.2 minutes to about 60 minutes prior to administration of the therapeutic agent.

[0242] 8. The method of embodiment 7, wherein the monoterpene is administered from about 1 minute to about 15 minutes prior to administration of the therapeutic agent.

[0243] 9. The method of embodiment 1, wherein the monoterpene and the therapeutic agent are administered separately.

[0244] 10. The method of embodiment 1, wherein the monoterpene and the therapeutic agent are administered concurrently.

[0245] 11. The method of embodiment 10, wherein the monoterpene and the therapeutic agent are administered together in a pharmaceutical composition.

[0246] 12. The method of embodiment 1, wherein the therapeutic agent is a chemotherapeutic agent.

[0247] 13. The method of embodiment 12, wherein the chemotherapeutic agent is selected from the group consisting of DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, enzyme inhibitors, receptor antagonists, therapeutic antibodies, and combinations thereof.

[0248] 14. The method of embodiment 12, wherein the chemotherapeutic agent is dimethyl-celecoxib (DMC), irinotecan (CPT-11), temozolomide or rolipram.

[0249] 15. The method of embodiment 1, wherein the therapeutic agent is an antibody or antibody fragment.

[0250] 16. The method of embodiment 1, wherein the therapeutic agent is an immune cell expressing a chimeric antigen receptor.

[0251] 17. The method of embodiment 16, wherein the immune cell is a T cell.

[0252] 18. The method of embodiment 16, wherein the therapeutic agent is CAR-T cells.

[0253] 19. The method of embodiment 1, wherein the monoterpene is administered by inhalation, intranasally, orally, intravenously, subcutaneously or intramuscularly.

[0254] 20. The method of embodiment 1, wherein the mammal suffers from cancer.

[0255] 21. The method of embodiment 20, wherein the cancer is a tumor of the nervous system.

[0256] 22. The method of embodiment 21, wherein the tumor is a glioblastoma.

[0257] 23. The method of embodiment 1, further comprising treating the mammal with radiation.

[0258] The scope of the present invention is not limited to the contents specifically given and described above. Those skilled in the art will recognize that there are suitable alternatives to the examples of materials, constructions, structures and sizes described. Multiple references, including patents and multiple publications, are cited and discussed in the detailed description of the invention. The citation and discussion of these references are provided only to clarify the description of the present invention, and it is not admitted that any reference is the prior art of the present invention described herein. All references cited and discussed in this specification are incorporated by reference in their entirety. Those of ordinary skill in the art will understand that deformation, modification and other embodiments of the contents described herein may occur without departing from the spirit and scope of the present invention. Although certain embodiments of the present invention have been shown and described, it is obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the present invention. The subject matter stated in the above description and the accompanying drawings is provided only for illustration and not as a limitation.

Claims

1. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering an antibody or antibody fragment to a mammal for the treatment of central nervous system (CNS) cancer.

2. The use of claim 1, wherein the antibody or antibody fragment is delivered across the BBB to the brain of a mammal.

3. The use of claim 1, wherein the medicament is formulated for administering the POH to the mammal prior to or concurrently with the antibody or antibody fragment.

4. The use of claim 1, wherein the POH is administered by intracardiac injection and the antibody or antibody fragment is administered intravenously.

5. The use of claim 1, wherein the POH and the antibody or antibody fragment are administered by intra-arterial injection.

6. The use of claim 1, wherein the medicament is formulated for administration of perillyl alcohol at a dosage of about 0.050 mg / kg to about 500 mg / kg body weight.

7. The use of claim 1, wherein the mammal is a human.

8. The use of claim 1, wherein the medicament is formulated for administration of the POH 0.2 minutes to 60 minutes prior to administration of the antibody or antibody fragment.

9. The use of claim 8, wherein the medicament is formulated for administration of the POH 1 minute to 15 minutes prior to administration of the antibody or antibody fragment.

10. The use of claim 1, wherein the medicament is formulated for separate administration of the POH and the antibody or antibody fragment.

11. The use of claim 1, wherein the medicament is formulated for concurrent administration of the POH and the antibody or antibody fragment.

12. The use of claim 11, wherein the medicament is formulated as a pharmaceutical composition comprising the POH and the antibody or antibody fragment.

13. The use of claim 1, wherein the mammal suffers from malignant glioma, retinoblastoma, pilocytic astrocytoma, meningioma, brain metastasis, neuroblastoma, pituitary adenoma, skull base meningioma or skull base cancer.

14. The use according to claim 13, wherein the pilocytic astrocytoma is a grade I pilocytic astrocytoma.

15. The use according to claim 13, wherein the medicament is used for treating malignant glioma.

16. The use of claim 15, wherein the malignant glioma is a glioblastoma, an astrocytoma or an anaplastic astrocytoma.

17. The use according to claim 16, wherein the malignant glioma is a glioblastoma.

18. The use of claim 1, wherein the mammal is also receiving radiation therapy.

19. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering an antibody or antibody fragment to a mammal for the treatment of central nervous system (CNS) cancer, wherein the POH and the antibody or antibody fragment are administered by intra-arterial injection.

20. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering an antibody or antibody fragment to a mammal for treating a brain tumor, wherein the POH and the antibody or antibody fragment are administered by intra-arterial injection.

21. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering chimeric antigen receptor T cells (CAR-T cells) to a mammal for the treatment of central nervous system (CNS) cancer, wherein the POH and the CAR-T cells are administered by intra-arterial injection.

22. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering chimeric antigen receptor T cells (CAR-T cells) to a mammal for the treatment of brain tumors, wherein the POH and the CAR-T cells are administered by intra-arterial injection.

23. Use of perillyl alcohol (POH) in the preparation of a medicament for delivering chimeric antigen receptor T cells (CAR-T cells) to a mammal for the treatment of central nervous system (CNS) cancer.

24. The use of claim 23, wherein the CAR-T cells are delivered across the BBB into the brain of a mammal.

25. The use of claim 23, wherein the medicament is formulated to administer the POH to the mammal before or in parallel with the CAR-T cells.

26. The use of claim 23, wherein the POH is administered by intracardiac injection and the CAR-T cells are administered intravenously.

27. The use of claim 23, wherein the POH and the CAR-T cells are administered by intra-arterial injection.

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