Combination therapies for the central nervous system

Abstract

Methods and compositions for enhancing the potency and pharmacokinetics of a therapeutic agent that can cross the blood-brain barrier are provided. In some aspects, a cilia function inhibitor can be administered in combination with a chemotherapeutic such as temozolomide to treat a brain cancer such as glioblastoma (GBM). The cilia function inhibitor may be administered in combination with vorasidenib to treat a brain cancer such as a glioma. Pharmaceutical compositions are also provided.

Description

DESCRIPTIONCOMBINATION THERAPIES FOR THE CENTRAL NERVOUS SYSTEMBACKGROUND OF THE INVENTIONThis application claims the benefit of United States Provisional Patent Application No. 63 / 515,224, filed July 24, 2023, the entirety of which is incorporated herein by reference.1. Field of the Invention

[0001] The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns combination therapies that can the effectiveness of drugs that can target the brain or central nervous system to treat diseases including brain cancer.2. Description of Related Art

[0002] Temozolomide (TMZ) is the only drug approved for first-line treatment of glioblastoma multiforme, the most aggressive form of glioma. TMZ is an oral alkylating agent with good CNS penetration; it is one of the few drugs that has demonstrated a meaningful survival advantage in this population (Fernandes et al., 2017). For adjuvant treatment of glioblastoma (GBM) specifically, TMZ is the sole preferred chemotherapeutic agent (Jia etal., 2023). Although TMZ can provide benefits for the treatment of brain cancers, overall prognosis for patients with high-grade glioma are still very poor.

[0003] The current standard of treatment for newly diagnosed GBM was established in 2005, whereby the addition of TMZ to post-operative radiation demonstrated a 2.5-month improvement in median survival, leading to a median overall survival (OS) of 14.6 months or a two-year survival of 26% (Stupp et al., 2005). Despite over two decades of research, the standard of treatment for gliomas has not changed significantly. Although the use of tumourtreating fields has demonstrated a clinically significant improvement in median OS (Stupp et al., 2017), it is costly (USD 20,000 / month) and is an externally worn device that relies on high levels of treatment adherence (>18 h of wear per day). It has an evident impact on an individual’s quality of life (e.g., the stigma of wearing a visible scalp device, scalp irritation, and discomfort), making it an intervention that is not broadly implementable into practice. More importantly, most patients relapse within the first two years of diagnosis, and there have been no proven effective second-line therapies after disease relapse (Fazzari et al., 2022).

[0004] Patients with glioblastoma (GBM) face a poor prognosis with a median survival of less than two years. Escalating the dose of chemotherapy is often impossible due to patient comorbidities. Current strategies to increase the amount of therapeutic that reaches a GBM have achieved only modest improvements. Such efforts have typically focused on altering the drug formulation, the administration route, or modification for interaction with protein transporters. (9- 19) Unfortunately, these strategies only fractionally increase the amount of drug that reaches the tumor.(18, 20) Brain penetrating chemotherapies such as TMZ are already administered at the maximum tolerated dose, further limiting this approach.(3)

[0005] TMZ can also result in several toxic side effects, due at least in part to TMZ action at locations outside of the central nervous system (CNS). These side effects include severe and sometimes deadly liver problems. Liver toxicity has been observed based on serum aminotransferase elevations that occur during temozolomide therapy in up to 12% of patients, and severe temozolomide-induced liver injury has been observed (Grieco et al. 2015.). Other side effects include bleeding or a blood clot, high blood pressure, and abdominal pain.

[0006] Clearly, there is a need for methods to increase the effectiveness of drugs that target the brain or CNS. In particular, there is a need for compositions and methods to improve the effectiveness of TMZ for the treatment of brain cancers such as GBM.SUMMARY OF THE INVENTION

[0008] The present disclosure overcomes limitations in the prior art by providing combination therapies and methods for increasing the effectiveness of a therapeutic that targets the brain or central nervous system. In some aspects, it has been observed that inhibiting cilia function can synergistically improve retention of therapeutics that penetrate the blood-brain barrier in a mammalian subject. As shown in the below examples, a cilia function inhibitor (e.g. , lidocaine) can synergistically improve the effectiveness of temozolomide (TMZ). Without wishing to be bound by any theory, the results provided support the idea that inhibiting motile ependymal cilia can improve retention of blood-brain barrier penetrating compounds in the CNS. Compositions and methods of use of cilia function inhibitors in combination with TMZ, vorasidenib, or other chemotherapeutics to treat a brain cancer (e.g. , GBM or glioma) are also provided. The cilia function inhibitor may be administered in combination with vorasidenib to treat a brain cancer such as a glioma.

[0009] As shown in the below examples, multiple cilia inhibitors resulted in a synergistic when combined with temozolomide (TMZ) resulted in a synergistic improvement in vivo using a mouse model of brain cancer (mice bearing orthotopic GL261 glioblastoma). Five of the seven cilia inhibitors (lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, and MLN4924) tested significantly improved survival compared to mice treated with temozolomide alone. These results were particularly surprising as the cilia inhibitors that resulted in improved survival are not normally associated with any anti-cancer properties. Lidocaine resulted in 100% of animals surviving tumor free compared to controls (p<0.05, Log-rank test). Inhibition of ependymal cilia was validated ex vivo, and a 2.8 fold increase in temozolomide in the brains of mice treated with a combination of the cilia inhibitor and temozolomide was observed, as compared to control, with no observable adverse events (e.g., see FIGS. 3-4). Multiple in vivo models were tested, including testing orthotopic human xenograft PDX GBM108 cells and another orthotopic, human xenograft models (GBM 10) that has non-methylated MGMT. These results support the idea that inhibiting motile ependymal cilia improves response to temozolomide sensitive tumors. It is anticipated that administering a cilia inhibitor in combination with temozolomide may also sensitize temozolomide resistant tumors or increase exposure to temozolomide. By increasing the effectiveness of the temozolomide, combination therapies with temozolomide and a cilia inhibitor may allow for increased effectiveness of the temozolomide and / or for lower concentrations of temozolomideto be administered to a mammalian subject (e.g. , a human patient) which may reduce the incidence of adverse effects associated with temozolomide. The combination of either (ethanol and TMZ) or (ofloxacin and TMZ) were each shown to synergistically improve survival in vivo as observed in a mouse model of glioblastoma, as shown in FIG. 6 and FIG. 7, respectively.

[0010] An aspect of the present disclosure relates to a method of treating a disease in a mammalian subject comprising administering to the mammalian subject a pharmacologically relevant dose of: (i) a cilia function inhibitor, and (ii) a therapeutic compound that can cross the blood-brain barrier; wherein the disease is a cancer of the central nervous system (CNS) or other brain disease. The cilia function inhibitor may be lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, ethanol, ofloxacin, or MLN4924. In some preferred embodiments, the cilia function inhibitor is lidocaine. The cilia function inhibitor may preferably inhibit ependymal cilia motility. The cilia function inhibitor may be administered intraventricularly, intracranially, intravenously, orally, intra ventricularly, intrathecally, parenterally, or via injection into the cerebrospinal fluid (CSF). In some embodiments, the cilia function inhibitor is administered intraventricularly or by injection into the cerebrospinal fluid. In some embodiments, the cilia function inhibitor is injected into the lateral ventricle of the brain of the mammalian subject, or wherein the administration is intracerebroventricular. In some embodiments, the disease is a brain cancer. The brain cancer may be a glioma, medulloblastoma, oligodendroglioma, meningioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, primitive neuroectodermal cancer, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, or Diffuse intrinsic pontine gliomas (DIPG). In some embodiments, the brain cancer is a glioma, such as for example a glioblastoma (GBM). The glioblastoma may comprise methylation of the MGMT gene promoter. The therapeutic agent may be a chemotherapeutic agent, an antibiotic, or an antiviral drug. In some embodiments, the therapeutic agent is a chemotherapeutic agent and wherein the disease comprises a brain cancer. The chemotherapeutic agent may be temozolomide (TMZ), vorasidenib, lomustine, procarbazine, vincristine, vinblastine, carmustine, or methotrexate. In some embodiments, the chemotherapeutic agent is temozolomide. The method may comprise administering about 5-150 mg / m2temozolomide to the mammalian subject. The method may comprise administering about 50-140 mg / m2temozolomide to the mammalian subject. The method may further comprises administering a therapeutically relevant amount of bevacizumab to the subject. In some embodiments, an additional anticancer therapy is administered to the subject. The additional anti-cancer therapy may be aradiotherapy, an immunotherapy, a chemotherapy, or a surgery. In some embodiments, the method is further defined as a method of increasing the effectiveness of the therapeutic agent. The cilia function inhibitor may preferably be administered before the therapeutic agent.

[0011] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically relevant amount of: (i) a cilia function inhibitor, (ii) a therapeutic compound that can cross the blood-brain barrier, preferably temozolomide or vorasidenib; and an excipient. In some aspects, the pharmaceutical composition may contain (lomustine, procarbazine, vincristine, vinblastine, carmustine, or methotrexate) instead of the TMZ or vorasidenib. The cilia function inhibitor may be lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, ofloxacin, or MLN4924. In some embodiments, the cilia function inhibitor is lidocaine. The pharmaceutical composition may be formulated for intraventricular, intracranial, oral, intravenous, intrathecal administration or injection into the cerebrospinal fluid (CSF). The pharmaceutical composition may be formulated for oral or intravenous administration. In some embodiments, the pharmaceutical composition is formulated for oral administration, and wherein the cilia function inhibitor is ethanol, cotinine, or ofloxacin. In some embodiments, the pharmaceutical composition is formulated for intravenous administration, and wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, or MLN4924. In some embodiments, the pharmaceutical composition is formulated for intraventricular, intrathecal, or intravenous administration; and wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, or MLN4924. In some preferred embodiments, the cilia function inhibitor is lidocaine.

[0012] Yet another aspect of the present disclosure relates to a kit comprising the composition described above or herein in a container means.

[0013] Another aspect of the present disclosure relates to a medical device comprising the composition described above or herein. In some embodiments, the medical device is an intracerebroventricular (ICV) device, or an Alzet osmotic pump.

[0014] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0015] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although thedisclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0016] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0017] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0018] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0019] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0020] The term “immunotherapy” refers to treatment of disease (e.g., cancer) by modulating an immune response to a disease antigen.

[0021] The term “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth or development in an immunocompromised non-human animal model, or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell” or“cancerous cell” and encompasses cancer cells of a solid tumor and a liquid tumor. “Cancer” may be used interchangeably herein with “tumor”.

[0022] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0023] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression- free survival in a patient diagnosed with cancer.

[0024] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIGS. 1A-G: Therapeutics that inhibit respiratory tract motile cilia also impair the motility of ependymal cilia. FIGS. 1A-B, The overall scheme to enhance the accumulation of brain-penetrating therapeutics. A) During normal conditions, a gradient forms between the amount of drug in parenchymal fluid and cerebral spinal fluid (CSF) in the ventricles due to the rapid turnover of CSF. A brain-penetrating therapeutic in parenchymal fluid drains into CSF via glymphatic pathways and is quickly cleared from the CNS. FIG. IB, Reducing the concentration gradient of a brain-penetrating therapeutic between parenchymal fluid and CSF by impairing CSF flux slows the clearance of therapeutics from parenchymal fluid, thus increasing the overall amount of drug that interacts with a tumor over time. CSF flux is reduced by impairing the mechanical motion of motile ependymal cilia, which does not perturb the overall volume, secretion, or excretion of CSF. FIGS. 1C-E, One millimeter- thick brain sections are harvested from mice, and ependymal cilia are imaged using DIC microscopy every five minutes. At minute one, a known respiratory tract motile cilia inhibitor is added to the microscopy dish. Three fields are counted for each group with two independent replicates (*=p<0.01 using T-tests). FIG. 1C, Ependymal cilia beat frequency with lidocaine treatment. FIG. ID, Ependymal cilia beat frequency with cotinine treatment. FIG. IE, Ependymal cilia beat frequency with phentolamine mesylate treatment. FIG. IF, Mice are implanted with an osmotic pump loaded with lidocaine attached to a brain cannula to administer 0.5|J.L / hour intraventricularly. After seven days, brains are harvested, sectioned, and imaged via DIC microscopy to quantify cilia motility. FIG. 1G, The cilia beat frequency from mice treated in FIG. ID is quantified. A significant, 37% reduction in beat frequency, in both lateral ventricles, after seven days of lidocaine treatment compared to control animals (*=p<0.05 via ANOVA, n=3 mice / group with three fields counted in each group) was observed.

[0028] FIGS. 2A-G: Cilia inhibitors synergize with the brain-penetrating chemotherapy temozolomide (TMZ) to provide a dramatic survival benefit in a murine syngeneic glioblastoma model. FIG. 2A, C57BL / 6 mice bearing syngeneic, orthotopicGL261 glioblastoma (GBM) were administered a cilia inhibitor intraventricularly (I. Vtr.) using an osmotic pump attached to a brain cannula, in combination with systemically administered temozolomide (TMZ), the standard-of-care chemotherapeutic, which penetrates the brain. FIG. 2B, GL261 cells are implanted into the right striatum of C57BL / 6 mice. After two weeks, an osmotic pump is implanted and attached to a brain cannula to administer 0.5 pL / hr of cilia inhibitor into the right lateral ventricle for two weeks. Simultaneously mice receive daily intraperitoneal (I.P.) injections of TMZ (lOmg / kg) for two weeks. FIG. 2C, Survival curve depicting survival rate of mice treated with cilia inhibitor in combination with TMZ. Five of seven cilia inhibitors demonstrated a significant survival benefit compared to treatment with TMZ alone (*=p< 0.05 using log -rank tests. n=4-5 mice / group). All mice treated with a cilia inhibitor had at least one animal survive until the study endpoint; all mice treated with our lead candidate lidocaine survived tumor-free until the study endpoint. FIG. 2D, Survival curve of mice treated with I. Vtr. Lidocaine or control, as well as I.P. TMZ or control. As expected, TMZ alone conferred a significant but nondurable survival benefit in all groups (&=p<0.05, log-rank test). Intraventricular treatment with lidocaine. Treatment with only l.Vtr. lidocaine did not result in a significant change in survival. Combining intraventricular lidocaine with systemic TMZ resulted in a significant survival benefit from untreated animals and animals treated with TMZ alone (&,* = p<0.05 using log-rank tests of untreated control and TMZ control, respectively. n=4-5 mice / group). FIG. 2E, Similar studies as in A) using cotinine as the cilia inhibitor. FIG. 2F, Similar studies as in A) using phentolamine mesylate as the cilia inhibitor. These data demonstrate a synergy between cilia inhibitors and TMZ for treating glioblastoma. FIG. 2G, Representative H&E sections from groups described above. The tumor regions are outlined in dashed lines and labeled with a "T".

[0029] FIGS. 3A-G: Combining the cilia inhibitor lidocaine with brainpenetrating TMZ enhances the accumulation of TMZ in the brain with no apparent toxicity. FIG. 3A, C57BL / 6 mice receive 0.5mL / hour of 2% lidocaine l.Vtr. via an osmotic pump attached to a brain cannula. After seven days, mice are administered a single dose of 15 mg / kg TMZ I.P. After one hour, mice are perfused, and the tissue is harvested for analysis. FIG. 3B, The amount of TMZ in the brains of mice administered control PBS + TMZ as a control, or lidocaine + TMZ, is quantified from brain extracts using liquid chromatography plus tandem mass spectrometry (LC-MS). We observed a significant 2.8-fold increase in brain TMZ levels per dose in the combination-treated mice compared to controls (*=p<0.05 T-test, n=3 mice / group). FIG. 3C, TMZ serum levels (measured as in B) from the same mice as in B)did not significantly differ between groups. FIGS. 3D-E, The serum ALT and AST activity of mice treated in FIG. 3A is within normal ranges and not significantly different between groups (n=3 mice / group). FIG. 3F, Serum levels of GFAP (a marker of neural toxicity) were below detectable levels in both the combined and control groups (n=3 mice / group). FIG. 3G, The amount of water in the brains of mice is quantified as an indicator of hydrocephaly. We did not observe any significant differences in the percentage of water in any brains tested (n=3 mice / group p>0.05 via T-tests or ANOVA).

[0030] FIGS. 4A-F. Temozolomide (TMZ) synergizes with lidocaine to impart a significant survival benefit in both treatment -sensitive and -resistant xenograft models of glioblastoma. FIG. 4A, NOD-SCID mice bearing human PDX glioblastoma in the right striatum are administered lidocaine intraventricularly (I.Vtr.) via an osmotic pump attached to a brain cannula and temozolomide via intraperitoneal (I.P.) injection. FIG. 4B, NOD-SCID mice are implanted with either MGMT methylated human PDX GBM line GBM108 or unmethylated MGMT line GBM10, which is usually resistant to TMZ. Thirty days post GBM implantation, mice receive lidocaine or control I.Vtr. combined with TMZ I.P. or control for two weeks. FIG. 4C, NOD-SCID mice bearing GBM108 glioblastoma are treated as described in A) and B). We observed a small but significant difference in mice treated with temozolomide alone (&=p<0.05 Log-rank tests, n=4 mice / group); however, the combination of intraventricular lidocaine with TMZ resulted in a significant difference between both control and TMZ alone treated groups (&,*=p<0.05 via log-rank tests compared to non-treated and TMZ alone treated respectively, n=4 / group). Like the GL261 data above, 100% of mice treated with the combination survived tumor-free to the study endpoint. Additionally, treatment with intraventricular lidocaine alone had no effect, demonstrating the synergistic nature of this treatment. FIG. 4D, Representative whole brain tissue sections stained with hematoxylin and eosin are depicted for mice described above in C). The tumor regions are outlined in dashed lines and labeled with a "T". FIG. 4E, NOD-SCID mice bearing GBM10 mice were treated as described in FIG. 4 A and FIG. 4B. As expected with an unmethylated MGMT GBM, we did not observe a significant survival benefit in mice treated only with TMZ. We do observe a significant response to the combination of cilia inhibitor and TMZ compared to either nontreated or TMZ alone with -40% of animals surviving tumor-free to the study endpoint (&,*=p<0.05 via log-rank test, n=4-5 mice / group). FIG. 4F, Representative whole brain tissue sections stained with hematoxylin and eosin are depicted for mice described above in section E). The tumor regions are outlined in dashed lines and labeled with a "T".

[0031] FIG. 5. Ethanol inhibits ependymal cilia motility in vitro. One millimeterthick brain sections are harvested from mice, and ependymal cilia are imaged using DIC microscopy every five minutes. At minute one, 0.125% ethanol (final concentration) is added to the microscopy dish. Cilia motility in Hz for Ethanol and Control (PBS) is displayed. (*=p<0.01 using T-tests).

[0032] FIG. 6: Co-administration of ethanol and TMZ via I.P. injection impart a significant survival benefit to glioblastoma-bearing mice. Mice-bearing syngeneic, orthotopic GL261 tumors were administered 100 mL of 40% ethanol I.P. followed by 10 mg / Kg of TMZ I.P. twenty minutes later for two weeks. Mice co-administered ethanol and TMZ demonstrate a significant increase in survival (*=p<0.01 via Log-rank tests) compared to non, ethanol alone, or TMZ alone -treated mice with 100% of animals surviving to the study endpoint tumor free.

[0033] FIG. 7: Co-administration of ofloxacin and TMZ impart a significant survival benefit to glioblastoma-bearing mice. Mice-bearing syngeneic, orthotopic GL261 tumors were administered 200 mg / mL of ofloxacin (oral gavage) followed by 10 mg / Kg of TMZ I.P. forty-five minutes later for two weeks. Mice co-administered ofloxacin and TMZ demonstrate a significant increase in survival (*=p<0.05 via Log-rank tests) compared to non or TMZ alone -treated mice with 25% of animals surviving to the study endpoint tumor free.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] The present disclosure provides, in some aspects, methods and compositions for increasing the effectiveness of drugs that can cross the blood-brain barrier including drugs for the treatment of a brain cancer (e.g. , GBM or glioma). As shown in the below examples, a cilia function inhibitor can be administered in combination with a drug or chemotherapeutic that crosses the blood brain barrier (e.g., TMZ or vorasidenib) to synergistically improve the activity or effectiveness of the drug or chemotherapeutic. Without wishing to be bound by any theory, data provided herein supports the idea that modulating brain clearance can be altered to enhance drug accumulation in the brain or CNS, and enhancing drug concentration in the CSF also increases drug concentration in the parenchyma (e.g., where a tumor such as a GBM tumor may reside), possibly due to interconnectivity between brain parenchyma fluid and cerebral spinal fluid (CSF). By inhibiting the motility of ependymal cell cilia with a cilia function inhibitor, drug accumulation may be increased in the CSF. FDA-approved therapeutics that interact with cilia as a "side effect" can be used for this purpose, and it was observed that therapeutics that can inhibit airway cilia can also inhibit ependymal cilia. Multiple cilia-inhibiting drugs when administered in combination with GBM chemotherapy temozolomide (TMZ) significantly improved the overall survival of mice bearing orthotopic GBM. Combining TMZ with lidocaine results in 100% of animals surviving tumor-free to the study endpoint (e.g., see Figure 4). This treatment resulted in a ~40-fold increase in brain temozolomide levels and was well-tolerated. Mice bearing MGMT methylated, human PDX orthotopic GBM also responded with 100% of animals surviving tumor-free to the study endpoint. Thus, these approaches may be particularly beneficial for treating GBM in patients that have methylation of the MGMT gene promoter, as methylation of this gene can occur in approximately half of patients with GBM (Wick et al., 2014). Even mice bearing TMZ- resistant, orthotopic GBM responded to the combination treatment with 40% of animals surviving tumor free to the study endpoint implying this strategy can sensitize TMZ-resistant GBM. Malignant brain tumors can be treated with TMZ in combination with a cilia function inhibitor. Cilia function inhibitors can also be administered in combination with a brainpenetrating therapeutic or a therapeutic that can cross the blood-brain barrier to treat an encephalopathy. It is anticipated that modulating diffusion by inhibiting motile cilia may allow for enhanced accumulation of a therapeutic in the brain or CSF without altering the volume of CSF to avoid hydrocephaly. Acute and chronic encephalopathies may benefit fromadministering an ependymal cilia inhibitor to improve therapeutic windows by enhancing CNS accumulation of a brain-penetrating therapeutic.I. Cilia Function Inhibitors

[0036] Cilia function inhibitors can inhibit or decrease motility of cilia function. For example, the cilia function inhibitor may induce ciliostasis, decrease cilia beat frequency, reversibly induce ciliostasis, or by blocking formation of or primary cilia (e.g., by inhibiting cilia synthesis and / or promoting cilia disassembly). The cilia function inhibitor preferably inhibits motile cilia. In some preferred embodiments, the cilia function inhibitor is administered intraventricularly. In some preferred embodiments, the cilia function inhibitor does not induce cilia disassembly. It is anticipated that a variety of cilia inhibitors can be used to increase or synergistically increase the potency or effectiveness of a therapeutic that passes the blood-brain barrier (e.g., a chemotherapeutic or anti-cancer therapeutic targeting a brain cancer such as GBM). For example, the cilia function inhibitor may be lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, or MLN4924. In some embodiments, the cilia inhibitor is not MLN4924. MLN4924 may be administered in combination with TMZ and an additional cilia inhibitor. In some preferred embodiments, the cilia function inhibitor is lidocaine. The cilia inhibitor may be administered (e.g., orally, intravenously, intraventricularly) to a mammalian subject such as a human in combination with temozolomide, lomustine, procarbazine, vincristine, carmustine, methotrexate, and bevacizumab. In some preferred embodiments, the cilia function inhibitor is administered in combination with temozolomide (e.g., to treat a brain cancer such as GBM). By reducing the clearance rate of therapeutics out of the CNS that demonstrates high rates of fluid flux (Telano and Baker, 2018), significant or synergistic improvement in the therapeutic effectiveness of a drug that crosses the blood-brain barrier may be achieved. It is anticipated that any active salt of lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, or MLN4924 can be used to increase the function or effectiveness of a therapeutic that crosses the blood brain barrier as described herein.

[0037] The cilia function inhibitor may be lidocaine. Lidocaine is a local anesthetic that can be used to numb a region on the skin or mucous membranes, and lidocaine can be used to treat seizures or heart arrythmias. Lidocaine has also been approved for intravenous administration to treat neuropathic pain, hyperalgesia, or complex regional pain syndrome. Lidocaine was discovered in the 1940s and has been extensively used as a local anesthetic.Lidocaine can bind to voltage-gated sodium channels in a 1 : 1 fashion and inhibit or prevent the flow of sodium ions through the channel pore (Cummins, 2007). Lidocaine can reversibly induce ciliostasis in a dose-dependent manner (Hablitz and Nedergaard, 2021). Lidocaine has the structure:It is anticipated that a variety of dosages of lidocaine can be used to increase the effectiveness of a therapeutic as described herein (e.g. , TMZ). For example, about 0.5-7.5, 1-5, 1.5-2.5, 0.25, 0.5, 1, 2, 3, 4, 5, 6, or 7 mg / kg of lidocaine, or any range derivable, preferably about 1-3 or about 2 mg / kg can be administered to a mammalian subject such as a human. In some preferred embodiments, lidocaine is administered intravenously or intraventricularly, although other modes of administration can be used.

[0038] The cilia inhibitor may be ethanol. Ethanol can function as a cilia inhibitor and can be administered in combination with a therapeutic as described herein (e.g., TMZ) to increase the effectiveness of the therapeutic. Ethanol may be administered via a variety of routes including, e.g., orally, intravenously, and intraventricularly.

[0039] The cilia function inhibitor may be phentolamine mesylate. Phentolamine mesylate is an antihypertensive drug that has been approved for intravenous and intramuscular administration. Phentolamine mesylate is a nonselective alpha-adrenergic antagonist and has the structure:Phentolamine mesylate can reversibly induce ciliostasis (Satir and Christensen, 2007). A variety of dosages of phentolamine mesylate can be administered in combination with a therapeutic asdescribed herein (e.g., TMZ). For example, about 0.01-0.5, 0.05-0.2, or 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175, or 0.2 mg / kg, or any range derivable therein, can be administered to a mammalian subject such as a human.

[0040] The cilia function inhibitor may be cotinine (also called nicotine cotinine or urine). Cotinine is an alkaloid found in tobacco and is also the predominant metabolite of nicotine. The active isomer S(-)-cotinine accumulates in the body after tobacco consumption. Cotinine has the structure:Cotinine can partially induce ciliostasis, or inhibit cilia function without complete ciliostasis (Holstein-Ronsbo et al., 2023). A variety of dosages of cotinine can be administered in combination with a therapeutic as described herein e.g., TMZ). For example, about 1-10, 2.5- 7.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg, or any range derivable therein, of cotinine can be administered to a mammalian subject such as a human.

[0041] The cilia function inhibitor may be fluticasone propionate. Fluticasone propionate is a steroid that is used for long term management of asthma e.g., resulting from allergies or allergic rhinitis) and COPD. Fluticasone propionate is a selective agonist of the glucocorticoid receptor, with an absolute affinity (KD) of 0.5 nM. Fluticasone propionate shows little or no activity at other steroid receptors. Fluticasone propionate can be administered via intraventricular, inhalation, and other routes of administration. Fluticasone propionate has the structure:Fluticasone propionate can partially induce ciliostasis, or inhibit cilia function without complete ciliostasis (Holstein-Ronsbo et al., 2023; Al Omran et al., 2Q .5). A variety of dosages of fluticasone propionate can be administered in combination with a therapeutic as described herein (e.g., TMZ). For example, about 0.1-1, 0.25-0.75, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg / kg, or any range derivable therein, of fluticasone propionate can be administered to a mammalian subject such as a human.

[0042] The cilia function inhibitor may be MLN4924 (pevonedistat). MLN4924 is a selective NEDD8 that is being investigated as a treatment for mantle cell lymphoma (MCL). MLN4924 can be administered intravenously, intraventricularly, or via other routes. MLN4924, TMZ, and irinotecan (1RN) have been tested in pediatric patients with recurrent or refractory solid tumors (Foster, Jennifer, et al. "Phase 1 study of pevonedistat (MLN4924) a NEDD8 activating enzyme inhibitor, in combination with temozolomide (TMZ) and irinotecan (IRN) in pediatric patients with recurrent or refractory solid tumors (ADVL1615)." (2021): 10019-10019.). MLN4924 may preferably be administered to a mammalian subject such as a human patient in combination with both TMZ and another cilia function inhibitor such as lidocaine, phentolamine mesylate, cotinine, or fluticasone propionate. MLN4924 has the structure:MLN4924 may block formation of primary cilia (e.g., by inhibiting cilia synthesis assembly and by promoting cilia disassembly) (MacDonald et al., 2021). A variety of dosages of MLN4924 can be administered in combination with a therapeutic as described herein (e.g., TMZ), preferably further in combination with another cilia function inhibitor (e.g., lidocaine, phentolamine mesylate, cotinine, or fluticasone propionate). For example, about 0.25-5, 2.5- 4.5, 3.25-3.5, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25,4.5, or 4.75 mg / kg, or any range derivable therein, of MLN4924 can be administered to a mammalian subject such as a human.IL Therapeutic Agents

[0043] The cilia function inhibitor is preferably administered in combination with a drug that crosses the blood brain barrier or can be used to treat a brain cancer. In some preferred embodiments, the drug is a chemotherapeutic or anticancer agent that can be used to treat a brain cancer. For example, the therapeutic agent may be temozolomide (TMZ), lomustine, procarbazine, vincristine, carmustine, methotrexate, or bevacizumab. In some preferred embodiments, the therapeutic agent is TMZ. The therapeutic agent may be administered to a mammalian subject such as a human patient via a variety of routes including, e.g., direct administration to the brain (e.g., intraventricular administration, injection into a brain tumor, etc.) or administration to the subject peripherally or outside of the CNS (e.g., intravenously or orally). As shown in the below examples, the cilia function inhibitor can synergistically enhance the function of the therapeutic (e.g. , TMZ) and may be used to lower the dosage of the therapeutic used in order to reduce or eliminate one or more side effects of the therapeutic while maintaining a beneficial effect (e.g., treatment of a brain cancer) of the therapeutic.

[0044] In some preferred embodiments, the therapeutic agent is TMZ. TMZ the current standard-of-care chemotherapy for GBM in the central nervous system (CNS) (Stupp et al., 2017). TMZ has the structure:TMZ intercalates DNA causing double-stranded DNA breaks to selectively eradicate rapidly growing cells (Zhang et al., 2012). TMZ can penetrate the blood-brain barrier and enter the brain parenchyma where a GBM resides. Murine studies treating GBM with temozolomide have shown a significant but nondurable response (Pokorny et al., 2015; Kitange et al., 2009; Gao et al., 2015). TMZ can also cross the blood-brain barrier. Studies of the perivascular space surrounding brain blood vessels have revealed a mechanism where parenchymal fluid flows through these cavities and mixes with cerebral spinal fluid (CSF) of the ventricular system ina process termed glymphatics (Bedussi et al., 2017; Bakker et al., 2016; Iliff et al., 2012; Bedussi et al., 2018; Hablitz et al., 2020; Hablitz et al., 2021; Holstein-Ronsbo et al., 2023). These studies support the idea that TMZ extravasates from blood vessels into brain parenchyma fluid, then enters the glymphatic space(s) before draining into CSF in the ventricular system. CSF may be completely recycled four to six times daily (Telano and Baker, 2018). Without wishing to be bound by any theory, due to CSF flux and interconnectivity between the parenchymal fluid and CSF via glymphatics, the inventors propose that a concentration gradient can form between parenchymal fluid and CSF following the administration of a brain-penetrating therapeutic (figure la,b). Reducing the magnitude of this gradient by increasing the amount of TMZ in the CSF may subsequently enhance the drug concentration in parenchymal fluid surrounding a GBM. TMZ is preferably administered orally, although other routes of administration (e.g., intravenous) can be used if desired. A variety of dosages of TMZ can be administered to a mammalian subject in combination with a cilia function inhibitor. For example, about 1-500, 5-250, 1-140, 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425 mg, or any range derivable therein. Preferably the dose is from about 1 mg to 250 mg. In some embodiments, about 25-150, 25-125, 25-100, 10, 25, 50, 75, 100, 125, 150 mg / m2TMZ is administered to the mammalian subject. The TMZ may preferably be administered orally or intravenously but in some cases may be administered via other routes such as intratumoral injection and / or or intraventricular administration. In some preferred embodiments, the dosage of TMZ administered to the mammalian subject is less than 140 mg / m2or less than 75 mg / m2, e.g., about 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135 mg / m2, or any range derivable therein. Since the cilia function inhibitor can synergize with TMZ, administering the cilia function inhibitor to the subject in combination with the TMZ may allow for reduced amounts of TMZ to be used.

[0045] The therapeutic agent can be lomustine (also called INN or CCNU). Lomustine is a chemotherapeutic that is used to treat cancers such as Hodgkin’s disease and brain cancers. Lomustine is an alkylating nitrosourea compound and has the structure:The dosage of lomustine administered to a mammalian subject can vary and can include, e.g., about 10-250, 1-140, 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg / m2. Lomustine is preferably administered orally, but other routes of administration can be used including intraventricular. In some preferred embodiments, less than 130, more preferably less than 120, more preferably less than 100 mg / m2, or about 2.5-100, 10-100, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or any range derivable therein is administered to the subject.

[0046] In some embodiments, the therapeutic agent is procarbazine. Procarbazine is an alkylating agent that has been used to treat brain cancers such as gliomas and Hodgkin’s lymphoma. Procarbazine was approved for medical use in the United States in 1969. Procarbazine damages DNA by methylating guanine at the O-6 position. Non-proliferating and non-cancerous cells are more able to repair the DNA damage. Procarbazine has the structure:A variety of dosages of procarbazine can be used including, about 0.1-10, 0.5-5, 0.1-2, 0.5-2, 0.1-1.75, 0.5-1.75, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / kg / day, or any range derivable therein. The treatment may preferably be orally administered once or repeatedly, e.g., once per day for 1, 2, 3, 4, 5, 6, or 7 days. In some preferred embodiments, the procarbazine is administered in an amount of less than 2 mg / kg / day, e.g., about 0.1-2, 0.5- 2, 0.1-1.75, 0.5-1.75, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 mg / kg / day, or any range derivable therein.

[0047] The therapeutic agent may be vincristine (also called leurocristine) or vinblastine. Vincristine and vinblastine are chemotherapeutics that are classified as a vinca alkaloids. Vinca alkaloids are antimitotic drugs that inhibit microtubule assembly and induce tubulin self-association into coiled spiral aggregates. Vinblastine is a chemical derivative of vincristine that exhibits reduced neurotoxicity. Vincristine can be used to treat cancers including leukemias, Hodgkin’s disease, and brain cancers such as neuroblastoma. Vincristine and vinblastine have the structures:Vincristine may be administered in a dose of about 0.1-2.5 0.1-2, 0.5-1.5, 0.5-1.25, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.4, 1.5, 1.75, 2, 2.25, 2.5 mg / m2, or any range derivable therein. In some preferred embodiments, vincristine is administered at a dose of less than about 2 mg / m2, more preferably less than 1.5 mg / m2, or 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.4 mg / m2, or any range derivable therein Vinblastine can be administered at a dose of about 1-12.5, 2-12.5, or 0.5, 0.75, 1, 1.5, 1.75, 2, 2.5, 5, 7.5, 10, 10.5, 11, 11.5, 12, or 12.5 mg / m2, or any range derivable therein. Vincristine and vinblastine can preferably be administered intravenously, although other modes of administration including intraventricular can be used. Vincristine is preferably used over vinblastine since vincristine can more effectively cross the blood-brain barrier.

[0048] The therapeutic agent may be carmustine. Carmustine is a chemotherapeutic that can be used to treat cancers including brain cancer and multiple myeloma. Carmustine is a nitrogen mustard P-chloro-nitrosourea compound and can function as an alkylating agent that can form inter-strand crosslinks in DNA. Carmustine has the structure:Carmustine can be administered at a dose of about 25-225, 50-150, 50-140, 25, 50, 75, 100, 125, 150, 175, 200 mg / m2, or any range derivable therein. In some preferred embodiments, the dosage of carmustine is about 25-150, 25- 140, 25, 50, 75, 100, 125, 150 mg / m2, or any range derivable therein. Carmustine can preferably be administered intravenously, intratumorally (e.g., injection into a brain tumor), or via other modes of administration.

[0049] In some embodiments, the therapeutic agent is methotrexate. Methotrexate (also called amethopterin) is a chemotherapeutic drug and immunosuppressant that can be used to treat autoimmune diseases and a variety of cancers. Some evidence supports the idea that higher-dose methotrexate can also be used to treat brain cancers (Kiewe el al. , 2007). Methotrexate has the structure:A variety of dosages of methotrexate can be administered to a mammalian subject including, e.g., about 5-100, 10-50, 10-30, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / m2, or any range derivable therein. Methotrexate can be administered intravenously, orally, or via other routes of administration.

[0050] The therapeutic agent may be vorasidenib. Vorasidenib is a dual IDH1 / IDH2 inhibitor. Vorasidenib exhibited better brain permeability and target engagement than ivosidenib in clinical trial in patients with IDHl-mutant glioma (Mellinghoff et al., 2023b). Vorasidenib can be used to treat a glioma, such as a IDHl-mutant or IDH2-mutant grade 2 glioma (Mellinghoff et al. , 2023a). Vorasidenib can be orally administered. Vorasidenib may be administered at a dose of about 20-60 mg daily (e.g., 40 mg daily). It is anticipated that administration of a cilia inhibitor may allow dosages of less than 40 mg daily (e.g. , 10, 15, 20, 25, 30, 35 mg daily, or any range therein). Vorasidenib has the structure:

[0051] In some embodiments, the therapeutic agent is bevacizumab. Bevacizumab is a chemotherapeutic that can be used to treat cancers including colon cancer, lung cancer, glioblastoma, and renal-cell carcinoma. Bevacizumab is a monoclonal antibody that targets VEGF-A. Bevacizumab may be administered in combination with temozolomide and / or a radiotherapy to treat a glioblastoma (Chinot et al. 2014). Treatment with bevacizumab can affect brain tumor vasculature and may lead to a restoration of the blood-brain barrier (BBB), resulting in decreased contrast enhancement on MRI and reduced peritumoral edema (Stegmayr et al. , 2017). Bevacizumab is preferably administered intraarterially or intravenously (e.g., about 5-15 mg / kg, more preferably about 5-10 mg / kg every 2 weeks or about 7.5-15 mg / kg of body weight given once every 3 weeks) as a single agent or as a combined therapy. Bevacizumab may be administered in combination with a cilia function inhibitor and one or more of temozolomide (TMZ), vorasidenib, lomustine, procarbazine, vincristine, carmustine, or methotrexate.III. Combination Therapies

[0052] A cilia function inhibitor may be administered before, during, after, or in various combinations relative to an anti -cancer treatment or therapeutic that can cross the blood-brain barrier (e.g., temozolomide). The administrations may be in intervals ranging from concurrently to minutes to days. In embodiments where the cilia function inhibitor is provided to a patient separately from anti-cancer treatment or therapeutic that can cross the blood-brain barrier, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that onemay provide a patient with the cilia function inhibitor and the anti-cancer treatment or therapeutic that can cross the blood-brain barrier within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) lapse between respective administrations. Modulation of protein transporters (transferrin or LDL) or devices such as convection enhanced delivery or focus ultrasound can also be used to promote drug facilitation through the blood-brain barrier in combination with a cilia function inhibitor and a therapeutic agent (e.g., TMZ) as disclosed herein. In some preferred embodiments, TMZ is administered to the mammalian subject about 10-45 minutes, or more preferrably about 20-30 minutes, after ethanol administration. In some embodiments, TMZ is administered to the mammalian subject about 30-60 minutes e.g., about 45 minutes) after administration of ofloxcin to the mammalian subject. Intraventricular administration of a cilia inhibitor may performed continuously throughout chemotherapy administration of a therapeutic as described herein (e.g., TMZ ).

[0053] In certain embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and / or 1-5 weeks, or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. For example a TMZ cycle can be 5 days on, and 23 days off, for 6 cycles. The cilia inhibitor may be administered during the five days on portion using the timing described above.

[0054] Various combinations may be employed. For the example below a cilia function inhibitor is “A” and an an anti-cancer treatment or therapeutic that can cross the blood-brain barrier is “B”:A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0055] Administration of any anti-cancer treatment or therapeutic that can cross the blood-brain barrier to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. In some embodiments involving treating a cancer in a mammalian subject (e.g., a brain cancer in a human patient), an additional anti-cancer therapy can be administered to the mammalian subject, such as a further chemotherapy, a radiotherapy, an immunotherapy (e.g., a checkpoint inhibitor, such as for example an anti-PDl antibody or an anti-PDLl antibody), a gene therapy, an anti-inflammatory drug, an antibiotic, or a surgery.IV. Brain Cancers

[0056] In some preferred embodiments, a cilia function inhibitor and an anti- therapeutic agent that crosses the blood-brain barrier (e.g., TMZ) are administered to a mammalian subject such as a human to treat a brain cancer (e.g., a GBM). The brain cancer may be a glioma, glioblastoma (also called glioblastoma multiforme), medulloblastoma, oligodendroglioma, meningioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, primitive neuroectodermal cancer, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, or DIPG (diffuse pontine glioma). In some preferred embodiments, the brain cancer is GBM. The brain cancer may be malignant, metastatic, or non-metastatic.V. Pharmaceutical Compositions

[0057] Pharmaceutical compositions are provided herein that preferably comprise both a cilia function inhibitor and a therapeutic agent (e.g., preferably a brain chemotherapeutic such as TMZ), or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one cilia function inhibitor and therapeutic agent (e.g., preferably a brain chemotherapeutic such as TMZ), or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington - 23rd Edition, Elsevier, October 2020, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparationsshould typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0058] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington - 23rd Edition, Elsevier, October 2020). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0059] The pharmaceutical formulation may comprise TMZ and a therapeutically relevant amount of a cilia function inhibitor (e.g., using intraventricular administration via a canula) in an oral formulation or a formulation formulated for alimentary delivery. The TMZ may be present in the pharmaceutical formulation in an amount of about 1 -500, 5-250, 1-140, 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mg, or any range derivable therein. In some preferred embodiments, TMZ is present in an amount less than 140 mg, or 1- 135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 mg, or any range derivable therein. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. Ethanol may be formulated separately from TMZ to allow time between administration of the TMZ and the ethanol (e.g., about 20-30 minutes).

[0060] The pharmaceutical formulation may comprise TMZ and a therapeutically relevant amount of a cilia function inhibitor (e.g., preferably lidocaine, phentolamine mesylate, MLN4924) in a formulation formulated for parenteral (e.g., preferably intravenous) delivery. The TMZ may be present in the pharmaceutical formulation in an amount of about 1-500, 5- 250, 1-140, 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mg, or any rangederivable therein. In some preferred embodiments, TMZ is present in an amount less than 140 mg, or 1-135, 1-130, 1-120, 1-110, 1-100, 1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 mg, or any range derivable therein. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intraventricularly, intracranially, intratumorally, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0061] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0062] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations ofa preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.IV. Examples

[0063] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1Increasing the Therapeutic Potency ofGBM Chemotherapy via Altering Parenchymal / Cerebral Spinal Fluid Clearance RateCompounds that inhibit airway cilia motility also inhibit ependymal cilia motility:

[0064] Freshly harvested murine brain sections were imaged for cilia motility using DIC microscopy according to previous protocols (Al Omran et al., 2015; MacDonald et al., 2021). Lidocaine, cotinine, or phentolamine mesylate was added to the dish, and cilia were imaged over time (figure Ic-e). All three compounds significantly reduced cilia beat frequency after 15 minutes of incubation (p<0.05 via t-tests). Treatment with cilia inhibitors resulted in a variable reduction in mean beat frequency over thirty minutes ranging from 55% for lidocaine (66.0±2.8 to 29.7±2.1 hertz (Hz), n=3) to no change for the control PBS group (65.0±3.6 to 66.0+2.8 Hz, n=3).

[0065] The inhibition of ependymal motile cilia in vivo was quantified by implanting an osmotic pump delivering 0.5pL / hr of 2% lidocaine into the right ventricle for seven days. Brains were harvested, and cilia imaged (figure ld,e). We observed a 38% reduction in mean beat frequency between lidocaine and PBS -treated groups (42.8±2.9 versus 68.5±1.7 Hz,respectively. p<0.05 using ANOVA, n=3 mice / group). Representative videos are included in supplemental figure 2. Cilia motility is impaired in both the ipsilateral and contralateral ventricles, even with administration only into a single ventricle.Cilia inhibitors synergize with temozolomide (TMZ)

[0066] Literature was reviewed to identify FDA-approved compounds that can interact with cilia (table 1) (Kiseleva et al., 2019; Merkus et al., 2001; Rutland et al., 1982; Yaghi and Dolovich, 2016; Jiao and Zhang, 2019; Robson et al., 1992; Mao et al., 2019). Most compounds interact with either motile airway or primary cilia. We note the identified interaction with motile cilia and the primary use of these drugs in Figure la. Cilia interaction is considered a "side effect" of all these drugs, and only one of the therapeutics we tested, sunitinib, has any known tumoricidal properties. None of these drugs are in similar chemical families and were selected for broad use and activity.Table 1.DruaPhenotypic Interaction with Motile Cilia Typical Mechanism or Clinical UseLidocaine Reversibly induces ciliostasis (27) Anesthetic Phentolamine Mesylate Reversibly induces ciliostasis (30) Alpha-adrenergic antagonist vasodilatorCotinine Partially induces ciliostasis (28) Activate cholinergic receptors Fluticasone Propionate Partially induces ciliostasis (28,29) Second generation glucocorticoidMLN4924 Blocks formation of cilia (31 ) NEDD8-activating enzyme (NAE) InhibitorSunitinib Induces cilia disassembly (25) Targets PDGF and VEGF receptor familiesPropranolol Hydrochloride Partially induces ciliostasis (26) Nasal beta blocker

[0067] Figure 2a, b describes mice bearing syngeneic, orthotopic GBM that were administered a cilia inhibitor intraventricularly via an osmotic pump (0.5|lL / hour) attached to a brain cannula for two weeks. Temozolomide is administered via daily I.P. injections (lOmg / kg) for two weeks. We observed a significant increase in overall survival when mice were administered five of the seven cilia inhibitors in conjunction with temozolomide compared to mice receiving temozolomide alone (figure 2c and supplemental figures 3 and 4, p<0.05 via log-rank tests, n=4-5 mice / group). Additionally, every group had one mouse survive tumor-free until the study endpoint compared to none in the temozolomide alone treated group.

[0068] As demonstrated in figure 2d-f and supplemental figures 3 and 4, administering the cilia inhibitors lidocaine, cotinine, or phentolamine mesylate intraventricularly (I.Vtr.) without systemic temozolomide did not improve survival compared to control mice that received PBS I.Vtr. and vehicle control I.P. (p<0.05 via log-rank tests, n=4-5 mice / group). Administration of temozolomide I.P. even without a cilia inhibitor (PBS I.Vtr.) did result in a significant improvement in survival compared to mice treated with vehicle (p<0.05 via logrank tests, n=4-5 mice / group). Importantly, combining either lidocaine, cotinine, or phentolamine mesylate I.Vtr. with temozolomide I.P. significantly improved survival compared to untreated (PBS I.Vtr., Vehicle I.P.) and temozolomide (PBS I.Vtr., temozolomide I.P.) treated controls, demonstrating synergy between cilia-inhibiting compounds and temozolomide (p<0.05 via log-rank tests, n=4-5 animals / group).Combining lidocaine and temozolomide (TMZ) enhances the amount of TMZ in the brain

[0069] Similar to the above studies, an osmotic pump was implanted to administer 0.5pL / hr of 2% lidocaine into the right ventricle of a mouse for seven days. After day 7, a single dose of 15 mg / kg of temozolomide is administered I.P. One hour after injection, mice are sacrificed, perfused, and tissues harvested (figure 3a). Temozolomide is extracted from brain tissue and quantified using a previously published liquid chromatography-tandem mass spectrometry (LC-MS) protocol.(40) As demonstrated in Figure 3b and supplemental Figure 5, we observed a 2.8-fold increase in brain temozolomide concentration per dose in lidocaine + temozolomide compared to the PBS + temozolomide group (836.31±53.72 ng / mL versus 262.57±83.25 ng / mL, respectively, p<0.05 T-test, n=3-4 mice / group). These data demonstrate lidocaine synergizes with temozolomide by enhancing brain accumulation of temozolomide and results in at least a ~40-fold increase in TMZ concentration throughout a two-week study.Intraventricular administration of lidocaine is well tolerated

[0070] AST and ALT liver enzyme serum levels were quantified following seven days of I.Vtr. lidocaine; all levels were within normal ranges (Ibanez-Tallon et al., 2002). No significant differences were detected between the lidocaine and the control group (figure 3d,e). Serum levels of glial fibrillary acid protein (GFAP) are below detectable levels in treated mice, indicating no residual neural toxicity is associated with intraventricular administration of lidocaine (figure 3f) (Hiskens et al., 2020; Yang and Wang, 2015). Differences in the amount of water in the brains of lidocaine or control-treated mice were not observed, and the amount of water is within the normal range (figure 3g) (Sonnabend et al., 2021; Baskaya et al., 2000), indicating no apparent hydrocephaly.Validating the synergy between intraventricularly administered lidocaine and systemically administered temozolomide (TMZ)

[0071] To validate the synergy between I.Vtr. lidocaine and I.P. temozolomide as a GBM treatment strategy, human PDX GBM 108 cells were implanted into the right striatum of NOD-SCID mice (figure 4a) (Vaubel et al., 2020). Thirty days after GBM implantation, an osmotic pump was implanted that administered 0.5pL / hr of 2% lidocaine into the right ventricle for fourteen days (figure 4b). Simultaneously, mice were administered 10 mg / kg temozolomide I.P. for fourteen days. Similar to the GL261 data, the inventors observed a nondurable but significant benefit from treatment with temozolomide alone compared to negative controls (figure 4c and supplemental figures 6, p<0.05 via log-rank tests, n=4-5 mice / group); however, the combination of lidocaine I.Vtr. with systemic temozolomide resulted in a significant increase in survival between both the negative control (PBS I.Vtr., vehicle I.P.) and temozolomide alone (PBS I.Vtr., temozolomide I.P.) treated mice (figure 5c, p<0.05 via log-rank tests, n=4-5 mice / group). 100% of mice survived to the study endpoint tumor- free. These data mimiced the GL261 data above, validating the murine glioma data in a second human xenograft PDX glioma model.

[0072] In vitro studies treating GBM cell lines with methylated and un-methylated promoter regions of DNA repair enzyme MGMT with temozolomide often observe a 2-5 fold difference in IC50 (Kitange et al., 2009). Given the earlier data from this study indicating combining I.Vtr. lidocaine with I.P. temozolomide results in a 2.8-fold increase in temozolomide accumulation per dose in the brain (39.2-fold over a two week study), micebearing MGMT unmethylated human PDX GBM 10 tumors were treated using the groups and treatment regimen described above (Vaubel et al., 2020). As demonstrated in Figure 4d, a significant increase in the survival of mice bearing orthotopic GBM 10 treated with I.Vtr. lidocaine and I.P. temozolomide was observed compared to all controls (figure 4d and supplemental figure 7, p<0.05 via log-rank tests, n=4-5 mice / group). The inventors did not observe a significant difference in mice treated with temozolomide alone, as GBM 10 has nonmethylated MGMT and thus is normally resistant to TMZ. Despite MGMT methylation status, thirty-eight percent of mice survived tumor-free to the study endpoint, demonstrating the potential ability of this therapeutic scheme to expand the number of GBM patients that could benefit from temozolomide when combined with intraventricular cilia inhibitors.

[0073] These data provide a method to enhance the accumulation of brain-penetrating drugs within the CNS. Previous strategies have focused on improving the concentration of a drug in the CNS by increasing the dose reaching the pathology using chemistry, formulation, transporters, and devices (Sarkaria et al., 2018; Griffith et al., 2020; Liu et al., 2016; Fang et al., 2017; Nance et al., 2014). The approaches provided herein can utilize reducing clearance rates to enhance accumulation. The approaches provided herein that utilize TMZ support the idea that cilia function inhibitors can be used to improve the accumulation of any compound that penetrates the blood-brain barrier. Studies of glymphatics and brain ECM indicate particles under 100 nm in parenchymal fluid can migrate into the CSF; thus, reducing the concentration gradient between parenchyma and CSF is a generalizable method suitable for most therapeutic formulations (Mastorakos et al., 2015; Hablitz et al., 2020; Mestre et al., 2020). Additionally, reducing the gradient between these fluids could be achieved in multiple fashions.

[0074] The approaches provided herein can modulate the motility of ependymal cilia to affect or normalize the concentration between parenchymal fluid and CSF. Without wishing to be bound by any theory, the data provided herein supports the idea that reducing the mechanical energy in the system will reduce the overall diffusion rate, resulting in enhanced accumulation over time. The beating of ependymal cells can mimic the action of a fan or pump resulting in convection-assisted diffusion; thus, inhibiting the mechanical motion may return CSF clearance to a simple diffusion model. Modulating diffusion may allow for enhanced accumulation without altering the volume of CSF to avoid hydrocephaly. Studies have largely focused on the impact of compounds on airway cilia (Workman and Cohen, 2014). The above data supports the idea that select airway inhibitors can also inhibit ependymal cilia to a similardegree in vitro or in vivo. As demonstrated above, lidocaine functions as a cilia inhibitor and can impairs the motility of ependymal cilia both in vitro and in vivo.

[0075] In terms of tumor-free survival, the data provided herein for this combination therapy surpasses current state-of-art immunotherapies (Brown et al., 2015; Makkouk and Weiner, 2015; Jackson et al., 2011; Lim et al., 2018). Checkpoint and other immunotherapy studies also demonstrate tumor-free survival in murine models but are generally limited to highly immunogenic, syngeneic tumors such as GL261 that are not representative of the "normal" GBM patient (an exception is CAR-T cell studies that do use patient-derived sample but have demonstrated limited success in the clinic due to a myriad of issues) (Brown et al., 2015; Wang et al., 2018). We demonstrate tumor-free survival in syngenic and multiple PDX GBM murine models. Additionally, the drugs described in this study are FDA-approved and used daily in the clinic, further enhancing the relevance and potential power of this approach.

[0076] While lidocaine appears well-tolerated in our studies, it can cause seizures if administered to the brain cortex (DeToledo, 2000; De Jong and Walts, 1966). Surgeries in this study were performed using a method that prevents lidocaine from contacting the cortex. We did not observe any seizures when lidocaine is administered inside the lateral ventricle of our mice. Future studies can evaluate possible microseizures. A misplaced canula, or other accidental administration, should be avoided and could result in severe consequences. The data supports using cilia inhibitors to enhance the accumulation of TMZ and other brain-penetrating therapeutics.

[0077] Often when two therapeutics are administered in conjunction, the effect is additive or inconsistent due to patient variability (Kashif et al., 2014). The cilia inhibitors tested above were not tumoricidal alone; thus, the significant increase in survival observed with the combination treatment indicates these compounds synergize to generate a result greater than adding the outcome of the individual survival curves together. Synergy with temozolomide is present with multiple, diverse compounds with clinical applications varying from treating local pain to reducing blood pressure. These drugs have the ability to impair motile cilia as a "side effect." These studies demonstrate a correlation between cilia inhibition, TMZ accumulation, and the overall survival of animals bearing GBM.

[0078] Many genetic ciliopathy studies demonstrate hydrocephaly and other encephalopathic phenotypes associated with abnormal cilia (Albrahim et al., 2022); however, inour study, mice receiving <1.5% of their CSF volume of 2% lidocaine / hour appear normal. The inventors did not observe any external signs of hydrocephaly during the treatment regimen, including qualitative measurements made by observation and quantifying the amount of water in the brain described in Figure 3g. Additionally, systemic or neurological toxicity were not observed (figure 3d-f). Thus, administrating intraventricular lidocaine is well-tolerated and does not appear to cause hydrocephaly. In mice, mutations that prevent cilia formation are embryonically lethal; however, specific mutants with only non-motile cilia develop normally and do not exhibit signs of hydrocephaly (Norris and Grimes, 2012; Lucas et al., 2012). Thus, selective impairment of motile ependymal cilia might be a feasible therapeutic target.

[0079] Modest differences in ependymal cilia between rodents and humans exist, but the in vivo experiments support use of these approaches in mammals. Rodents have a significantly higher number of cilia relative to the volume of CSF, and the CSF recycling rate is significantly different for humans versus rodents (Telano and Baker, 2018). Additional cilia inhibitors with increased specific activity may also be used. The ex vivo microscopy studies of ependymal cells treated with lidocaine support the idea motile cilia could be further impaired (2% lidocaine only caused a 38% reduction in mean beat frequency) to further improve this treatment scheme.

[0080] This regimen may increase the duration a drug above the therapeutic concentration threshold in parenchymal fluid that surrounds a GBM to impart a survival benefit. These approaches may be used to benefit and even expand the GBM patient population benefiting from TMZ treatment. GBM cells that are resistant to TMZ via mechanisms that are not dependent on the overall dose of chemotherapy, such as loss of protein target, may affect responses. These studies combining cilia inhibitors with temozolomide dramatically enhanced outcomes in the in vivo studies provided herein.

[0081] Many normal neurological processes, such as circadian rhythm, long-term memory retention, and others rely on the concentration of metabolites and neural transmitters to function correctly (Hablitz et al. , 2020; Willie et al. , 2003; Rasmussen et al. , 2022). The inventors did not observe any gross neurological defects (mice ate, slept, and groomed typically). Enhanced CNS chemotherapy concentration could result in a greater incidence of "chemo brain" and other unpleasant side effects that are difficult to quantify using gross physiological measurements (Rao et al., 2022).

[0082] Studies to identify brain-penetrating, ependymal motile cilia inhibitors may prevent the need for implanting a ventricular catheter and injection pump, reducing both the risk of the procedure as well as patient discomfort. The relationship can be explored between the glymphatic fluid and ventricular cilia, the physical mechanics that account for changes in the rate of mass transfer, and the impacts on neural physiology / behavior would demonstrate the utility and translatability of this scheme for treating GBM patients. CSF tracer studies could further substantiate the correlation between cilia inhibition, TMZ accumulation, and overall survival benefit.Ethanol inhibits ependymal cilia

[0083] Literature searches indicated that ethanol both penetrates the blood-brain barrier (BBB) and can inhibit ependymal cilia (Al Omran et al. , 2015). Administering 0.125% ethanol inhibited ependymal cilia motility in vitro (FIG. 5).Ethanol enhances survival when co-administered with TMZ

[0084] Mice-bearing syngeneic, orthotopic GL261 tumors were co-administered ethanol (100 mL of 40% solution) and TMZ (lOmg / Kg) via l.P. injection for two weeks. Overall survival rate for mice treated with controls or the combination treatment of ethanol and TMZ is shown in FIG. 6. We observed a significant survival benefit in mice co-administered ethanol and TMZ compared to non-treated, ethanol alone, or TMZ alone, with 100% of treated mice surviving tumor- free to the study endpoint (*=p<0.01 using Log-rank tests). Interestingly, ethanol must be administered 20 minutes before TMZ to observe a significant survival benefit. These results demonstrate the utility of ethanol as a systemically administered cilia inhibitor that can be co-administered with TMZ to enhance the survival of mice bearing brain tumors.Ofloxacin enhances survival when co-administered with TMZ

[0085] Mice-bearing syngeneic orthotopic GL261 tumors were co-administered ofloxacin (oral gavage, 200 mg / mL), which is known to penetrate the blood-brain barrier and inhibit airway cilia, and TMZ (lOmg / Kg, l.P.) for two weeks. Results for overall survival for mice treated with controls or the combination treatment are shown in FIG. 7. A significant survival benefit was observed in mice co-administered ofloxacin and TMZ compared to nontreated mice or TMZ alone treated mice (*=p<0.05 using Log-rank tests), and 25% of mice coadministered ofloxacin and TMZ survived tumor-free to the study endpoint. Administrationof ofloxacin 45 minutes before TMZ was needed to observe a significant survival benefit; without wishing to be bound by any theory, it is anticipated that this effect may be due to pharmacokinetics of ofloxacin and ethanol. These results demonstrate the utility of ofloxacin as a systemically administered cilia inhibitor that can be co-administered with TMZ to enhance the survival of mice bearing brain tumors.

[0086] In conclusion, compounds that impair cilia motility can synergize with temozolomide, resulting in a dramatic survival benefit in mice bearing orthotopic GBM. Lidocaine impairs ependymal cilia in vitro and in vivo and is well-tolerated if administered inside a lateral ventricle. Administering intraventricular lidocaine in combination with TMZ improves brain accumulation of temozolomide. Combining intraventricular lidocaine with systemic TMZ can sensitize an unmethylated MGMT GBM to temozolomide, which may be used to broaden the patient population that can benefit from TMZ chemotherapy. The data demonstrates that cilia inhibition enhances the accumulation of blood-brain penetrating compounds to improve GBM treatment and other encephalopathies.Example 2Materials and Methods

[0087] The following materials and methods were used in the experiments described in Example 1.

[0088] Study Design: Power analysis - murine studies were powered to detect a 30% difference in mean survival with 95% confidence.

[0089] Rules for stopping data collection - murine studies were halted when euthanasia requirements set up by our IACUC were met, namely, >10% weight loss, neurological symptoms, or hunched posture. Studies were concluded after an approximately 3- fold increase in survival compared to controls. For GL261 studies, that is approximately 100 days (controls die at day 30) and 150 days (controls die between 50-60 days) for xenograft tumors. Due to these studies' invasive and labor-intensive nature, the murine studies were not blinded; however, the euthanization of each mouse presented in this study was done in consultation with a blinded veterinary technician.

[0090] Data inclusion / exclusion criteria - all mice in each study, including initial and duplicate independent replicates, are included in the data presented.

[0091] Outliers - no outliers were eliminated from the data presented.

[0092] Randomization - all mice are randomized before treatment (Post-GBM implantation) to eliminate surgical / cellular bias between groups.

[0093] Selection of endpoints - endpoints are selected based on IACUC guidance, and appropriate statical tests were applied to correct for differences in sample numbers and comparisons.

[0094] Replicates - the number and type of replicates and conditions are described for each assay below.

[0095] Statistical analysis - data is generally displayed as means and standard deviation. T-tests are used for pairwise comparisons, ANOVA for group analysis, and Logrank tests for survival analysis. Statistical details related to each specific assay are described below.

[0096] Murine Studies. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas at Austin. Mice used in this study were purchased from Jackson Laboratories (C57BL / 6 - 000664 and NOD-SCID - 001303) aged 4-6 weeks. Mice were housed under standard conditions and allowed food and water adlib.

[0097] Culture of GBM cells. GL261 cells were cultured via sterile technique as previously described (Umlauf et al., 2019). Human PDX GBM cell lines GBM 108 and GBM 10 were acquired from Dr. Jann Sarkaria, who runs the NIH PDX GBM core at the Mayo Clinic (Vaubel et al., 2020). Cells were cultured and maintained according to the Sarkaria Lab's protocols. All GBM cell lines underwent DNA fingerprinting and a pathology panel (Jackson Labs) to ensure authenticity and cleanliness for each cell line described. Cells are regularly subjected to mycoplasma detection using a kit from Lonza.

[0098] Implantation of GBM cells. For studies with GL261 tumors, 2* 103GL261 cells are implanted into the right striatum (2 mm right of bregma and 3.5 mm deep) using the standard stereotactic surgical technique previously described (Umlauf et al., 2019; Clark et al.,2016). For GBM 108 and GBM 10 studies, 2*105cells are implanted into the right striatum using the standard stereotactic surgical procedure (Vaubel et al., 2020).

[0099] Preparing osmotic pumps. Alzet model 2002 osmotic pumps, which deliver 0.5pL / hour for two weeks, were used as the osmotic pump in all studies described here. For cilia inhibitor studies, pumps were loaded with 200pL of: 20 mg / kg of sunitinib, 4 mg / kg propranolol hydrochloride, 2 mg / mL lidocaine, 5 mg / kg cotinine, 0.5 mg / kg fluticasone propionate, 0.1 mg / kg phentolamine mesylate, 3.75 mg / kg MLN4924, or PBS. Tubing is attached to the osmotic pump, and then pumps were covered in PBS and incubated overnight at 37°C before implantation according to the manufacturer's protocol.

[0100] Implantation of an osmotic pump and brain cannula. Intraventricular cilia inhibitors are all administered via an osmotic pump (Alzet - model 2002) attached to a brain cannula (Alzet - brain cannula 3) using the manufacturer's protocol. Briefly, the skull is exposed, and a bur hole is drilled within 1 mm to the right of the bregma. A subcutaneous pocket is created using a large hemostat on the animal’s back. A model 2002 pump loaded with 200 pL of cilia inhibitor or control is placed in the pocket. Plastic tubing attached to the pump is cut to size, and a cannula with 1 mm of spacers (so only 2 mm of the needle is exposed) is affixed to the tubing, placed into the bur hole, and bonded to the skull. Once the cannula is secured to the skull, the implant is covered via a skin flap and sutured closed.

[0101] Temozolomide administration. Temozolomide (TCI America) is suspended in DMSO at a lOx solution, then diluted to lx in PBS and filter sterilized before administration. For therapeutic studies, 10 mg / kg of temozolomide is administered daily via intraperitoneal injection for two weeks to match the dosing window from the osmotic pump. For accumulation studies, a single dose of 15 mg / kg of temozolomide via intraperitoneal injection.

[0102] In vitro cilia imaging studies. Cilia imaging studies were performed as previously described (Al Omran et al., 2015). C57BL / 6 mice were sacrificed and brains harvested into DMEM+10% FBS + pen / strep. One mm coronal sections through bregma were cut using a razor blade and a brain-cutting mold (Braintree Scientific). Sections were captured in 1.5 mm glass bottom dishes (Matek) and imaged on an upright Nikon microscope via DIC techniques. Videos of cilia beating were collected for analysis. A promising field is identified for lidocaine inhibition studies, and an initial video is collected. Next, 100 pL of 2% lidocaine is added to 2 mL of media in the imaging dish. A 15-second video of the same field of motilecilia is recorded every five minutes. Videos are analyzed as previously described and compared to controls that received 100 pL of PBS rather than cilia inhibitor.

[0103] Ex vivo cilia imaging studies. Osmotic pumps loaded with 2% lidocaine or PBS control are implanted into C57BL / 6 mice to administer 0.5 LtL / hr into the right ventricle for seven days. Brains were harvested, sectioned, imaged, and analyzed as described above. Three fields were collected for each brain in each ventricle. Three individual mice were used for each group.

[0104] Temozolomide accumulation in the brain. Osmotic pumps loaded with 2% lidocaine or PBS control are implanted into C57BL / 6 mice to administer 0.5 mL / hr into the right ventricle for seven days. A single dose of 15 mg / kg temozolomide is administered I.P. Exactly one hour after temozolomide administration, mice are anesthetized, perfused, brain harvested, and acidified to prevent temozolomide degradation. Temozolomide is quantified in brain extracts using ultra-precision liquid chromatograph + tandem mass spectrometry as previously described (Peer et al., 2016).

[0105] Quantifying the amount of water in the brain. Osmotic pumps loaded with 2% lidocaine or PBS control are implanted into C57BL / 6 mice to administer 0.5 pL / hr into the right ventricle for seven days. A single dose of 15 mg / kg temozolomide is administered I.P. Exactly one hour after temozolomide administration, mice are anesthetized, perfused, and brain harvested. Water content was measured as previously described (Baskaya et al., 2020). Briefly, brains were placed in a tared tube and weighed to establish baseline weight. Tubes are incubated at 60°C for 24 hours to remove water. The residual tissue is weighed, and the difference between the first and second measurements is used to quantify the amount of water in the brain.

[0106] Temozolomide toxicity panel. Osmotic pumps loaded with 2% lidocaine or PBS control are implanted into C57BL / 6 mice to administer 0.5 pL / hr into the right ventricle for seven days. A single dose of 15 mg / kg temozolomide is administered I.P. Exactly one hour after temozolomide administration, mice are anesthetized, and blood is collected via cardiac puncture. The serum is separated via micro serum collection tubes (B.D.). ALT and AST levels were quantified in serum using Sigma-Aldrich ALT and AST activity assays via the manufacturer's protocol. GFAP levels were quantified in serum using ELISA (R&D Systems).GFAP added into mouse serum is used as an additional positive control (in addition to the standard curve) to account for the serum background.* *

[0107] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating a disease in a mammalian subject comprising administering to the mammalian subject a pharmacologically relevant dose of:(i) a cilia function inhibitor, and(ii) a therapeutic compound that can cross the blood-brain barrier; wherein the disease is a cancer of the central nervous system (CNS).

2. The method of claim 1, wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, ethanol, ofloxacin, or MLN4924.

3. The method of claim 2, wherein the cilia function inhibitor is lidocaine.

4. The method of any of claims 1-3, wherein the cilia function inhibitor inhibits ependymal cilia motility.

5. The method of any one of claims 1-4, wherein the cilia function inhibitor is administered intra ventricularly, intracranially, intravenously, orally, intraventricularly, intrathecally, parenterally, or via injection into the cerebrospinal fluid (CSF).

6. The method of claim 5, wherein the cilia function inhibitor is administered intraventricularly or by injection into the cerebrospinal fluid.

7. The method of claim 6, wherein the cilia function inhibitor is injected into the lateral ventricle of the brain of the mammalian subject, or wherein the administration is intracerebroventricular.

8. The method of any one of claims 1 -7, wherein the disease is a brain cancer.

9. The method of claim 8, wherein the brain cancer is a glioma, medulloblastoma, oligodendroglioma, meningioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, primitive neuroectodermal cancer, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, or Diffuse intrinsic pontine gliomas (DIPG).

10. The method of claim 9, wherein the brain cancer is a glioma.

11. The method of claim 10, wherein the glioma is glioblastoma (GBM).

12. The method of claim 11, wherein the glioblastoma comprises methylation of the MGMT gene promoter.

13. The method of any one of claims 1-12, wherein the therapeutic agent is a chemotherapeutic agent, an antibiotic, or an antiviral drug.

14. The method of claim 13, wherein the therapeutic agent is a chemotherapeutic agent and wherein the disease comprises a brain cancer.

15. The method of claim 13, wherein the chemotherapeutic agent is temozolomide (TMZ), vorasidenib, lomustine, procarbazine, vincristine, vinblastine, carmustine, or methotrexate.

16. The method of claim 15, wherein the chemotherapeutic agent is temozolomide.

17. The method of claim 16, wherein the method comprises administering about 5-150 mg / m2temozolomide to the mammalian subject.

18. The method of claim 17, wherein the method comprises administering about 50-140 mg / m2temozolomide to the mammalian subject.

19. The method of any one of claims 1-18, wherein the method further comprises administering a therapeutically relevant amount of bevacizumab to the subject.

20. The method of any one of claims 1-19, wherein an additional anti-cancer therapy is administered to the subject.

21. The method of claim 20, wherein the additional anti-cancer therapy is a radiotherapy, an immunotherapy, a chemotherapy, or a surgery.

22. The method of any one of claims 1-21, wherein the method is further defined as a method of increasing the effectiveness of the therapeutic agent.

23. A pharmaceutical composition comprising a therapeutically relevant amount of:(i) a cilia function inhibitor,(ii) a therapeutic compound that can cross the blood-brain barrier, preferably temozolomide or vorasidenib; and an excipient.

24. The pharmaceutical composition of claim 23, wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, cotinine, fluticasone propionate, or MLN4924.

25. The pharmaceutical composition of claim 23, wherein the cilia function inhibitor is lidocaine.

26. The composition of any one of claims 23-25, wherein the pharmaceutical composition is formulated for intraventricular, intracranial, oral, intravenous, intrathecal administration or injection into the cerebrospinal fluid (CSF).

27. The composition of any one of claims 23-25, wherein the pharmaceutical composition is formulated for oral or intravenous administration.

28. The composition of claim 27, wherein the pharmaceutical composition is formulated for oral administration, and wherein the cilia function inhibitor is ethanol, cotinine, or ofloxacin.

29. The composition of claim 27, wherein the pharmaceutical composition is formulated for intravenous administration, and wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, or MLN4924.

30. The composition of claim 27, wherein the pharmaceutical composition is formulated for intraventricular, intrathecal, or intravenous administration; and wherein the cilia function inhibitor is lidocaine, phentolamine mesylate, or MLN4924.

31. The composition of claim 30, wherein the cilia function inhibitor is lidocaine.

32. A kit comprising the composition of any one of claims 23-31 in a container means.

33. A medical device comprising the composition of any one of claims 23-31.

34. The medical device of claim 33, wherein the medical device is an intracerebroventricular (ICV) device, or an Alzet osmotic pump.- M -

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