Compositions and methods for CNS disorders

Abstract

The present invention describes the use of agents, methods, compositions, kits, and medicaments for inhibiting or suppressing the expression of TGF-β2 to treat or ameliorate symptoms of CNS diseases, including diffuse midline glioma (DMG) and K27M GBM, in human subjects or animals. The agents can be used in combination with anticancer drugs. These objectives can be achieved using formulations of agents for inhibiting or suppressing the expression of TGF-β2. More specifically, the present invention discloses compositions, methods, and uses related to antisense oligonucleotides against TGF-β2 in regimens for CNS diseases. The present invention further describes novel devices and methods for delivering pharmaceutical compositions via intracranial injection. TIFF2025538189000035.tif130133

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that was filed electronically as an ST.26 file named 018988-006WO1_SL.xml, created on November 7, 2023, and is 120,186 bytes in size.

[0002] Technical Field The present invention relates to methods, agents, and uses for treating central nervous system (CNS) cancer by continuous intracranial infusion. More specifically, the present invention discloses methods, agents, and uses for inhibiting or suppressing TGF-β2, which result in improved clinical outcomes of such diseases. The present invention provides stable formulations and methods of use of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents for treating CNS cancer.

[0003] The present invention relates to devices and methods for delivering pharmaceutical compositions by intracranial infusion. More specifically, the devices of the present invention can include an Ommaya-like reservoir into which the pharmaceutical composition is pumped. The Ommaya-like reservoir can provide a range of intracranial infusions, from bolus to continuous, via an intraventricular entry catheter into a target region of the brain. [Background technology]

[0004] background CNS diseases have been treated with various agents through the intracranial route. For CNS tumors, convection-enhanced delivery (CED) devices can be used to distribute drugs directly to the tumor in the brain using several catheters placed near the tumor.

[0005] However, drawbacks and complications of these injection methods are: (a) placement of the catheter and its tip within the brain near the tumor is complicated and typically requires simultaneous imaging, such as X-ray-based imaging, to be achieved; (b) these methods require the insertion of multiple catheters into the tumor, which can be difficult or impossible to achieve and can cause tissue destruction; (c) catheters cannot be permanently implanted, particularly in pediatric patients, because the brain is likely to grow, tumors are likely to migrate, and tumors are likely to metastasize; and (d) the majority of tumors are likely to have already metastasized within or outside the CNS, making local treatment impractical.

[0006] Furthermore, a drawback and complication of such methods is the limited infusion and flux of drugs throughout the CNS compartments.

[0007] A further drawback is that the catheter tip implanted within the tumor may not provide sufficient infusion and flow of the drug throughout the CNS compartment.

[0008] What is needed is a device for intracranial systemic, bolus, and / or continuous infusion of therapeutic agents for use in methods for treating central nervous system (CNS) diseases, including cancer.

[0009] What is needed are methods and agents for use in inhibiting or suppressing factors contributing to the unpredictable pathology of diseases of the central nervous system, particularly cancer. For example, there is an urgent need for methods and agents for use in inhibiting the activity of TGF-β and / or suppressing TGF-β-associated pathologies that can improve the effectiveness of treating central nervous system cancers. Summary of the Invention

[0010] overview The present invention provides therapeutic methods for treating or ameliorating the symptoms of CNS diseases, such as CNS cancers.

[0011] In some embodiments, the present invention comprises agents and compositions for inhibiting or suppressing TGF-β2 to result in improved clinical outcomes of CNS disorders.

[0012] In a further aspect, the present invention provides stable formulations of anti-TGF-β2 agents for various treatments for CNS disorders. Examples of anti-TGF-β2 agents include TGF-β2 inhibitors, such as antisense oligonucleotides, their pharmaceutically acceptable salt forms, esters, polymorphs, or stereoisomers, and combinations thereof.

[0013] In a further aspect, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for therapy against CNS diseases. The stable formulations of the present invention surprisingly improve clinical outcomes. The stable formulations of agents for inhibiting TGF-β2 can be used to treat CNS diseases, particularly CNS cancers.

[0014] The present invention provides a device and system for intracranial, intrathecal, or intraventricular continuous infusion of therapeutic agents for treating central nervous system (CNS) diseases. Continuous infusion into brain tumors using the system of the present invention is much easier than conventional CED systems and other similar or conventional methods.

[0015] The infusion system of the present invention has a significant advantage because it continuously delivers effective amounts or doses of pharmaceutical compositions to patients with much greater safety and ease of insertion throughout the CNS.For example, it is believed that the infusion system of the present invention delivers more effective amounts of drugs through intraventricular route throughout the CNS / brain compartment compared with the local delivery by CED method.This advantage may be particularly important for the treatment of CNS cancer.

[0016] The device of the present invention can overcome the drawbacks and complications of conventional methods. The injection system of the present invention has considerable advantages because the placement of the catheter and its tip into the brain is relatively easy and imaging is not required. Furthermore, the injection and flow of drugs into the brain utilizes the fluid circulation in the brain to distribute the drug effectively over a larger area and to the target tissue. Furthermore, the system of the present invention can be used in a novel mode of operation to provide continuous injection into the target tissue.

[0017] The present invention also relates to devices and systems for intracranial, intrathecal, or intraventricular continuous infusion of therapeutic agents for treating central nervous system (CNS) diseases of the brain and spine. The systems of the present invention are believed to deliver compositions and agents for inhibiting or suppressing TGF-β2, improving clinical outcomes for such diseases. The systems can use stable formulations of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents to treat CNS diseases, including CNS cancers.

[0018] The present invention further relates to methods for treating central nervous system (CNS) disorders of the brain and spine.

[0019] The present invention further provides a device for intracranial, intrathecal, or intraventricular continuous infusion of a therapeutic agent for treating central nervous system (CNS) diseases. Continuous infusion into brain tumors using the device and device of the present invention is much easier than conventional CED methods and other similar or conventional methods.

[0020] The infusion device of the present invention has a significant advantage because it continuously delivers effective amount or dose of pharmaceutical composition to patients with much greater safety and ease of insertion throughout the CNS.For example, the infusion device and device of the present invention are believed to deliver more effective amount of drug through intraventricular route throughout the CNS / brain compartment compared with the local delivery by CED method.This advantage may be particularly important for the treatment of CNS cancer.

[0021] The device of the present invention overcomes the drawbacks and complications of known methods. The infusion device of the present invention has significant advantages because the placement of the catheter and its tip into the brain is relatively easy and imaging is not required. Furthermore, the infusion and flow of drugs into the brain utilizes the fluid circulation in the brain to distribute the drug effectively over a larger area and to the target tissue. Furthermore, the device of the present invention can be used in a novel mode of operation to provide continuous infusion to the target tissue.

[0022] The present invention also relates to a device for intracranial, intrathecal, or intraventricular continuous infusion of therapeutic agents for treating central nervous system (CNS) diseases of the brain and spine. The device of the present invention delivers compositions and agents for inhibiting or suppressing TGF-β2, which is believed to improve clinical outcomes for such diseases. The device uses a stable formulation of an anti-TGF-β2 agent (including an antisense oligonucleotide composition) and other agents to treat CNS diseases, including CNS cancers.

[0023] The present invention further relates to methods for treating central nervous system (CNS) disorders of the brain and spine.

[0024] Aspects of the present invention include the following.

[0025] An agent for inhibiting or suppressing the expression of TGF-β2 to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

[0026] 20. Use of an agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0027] A method for treating or ameliorating symptoms of a CNS disorder in a human subject or animal in need thereof, comprising: preparing a composition comprising an agent for inhibiting or suppressing the expression of TGF-β2 in a carrier; and administering a therapeutically effective amount of the composition to the subject.

[0028] The above agent, use, or method, wherein the CNS disease is glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal metastasis or brain metastasis, brain or spinal cord cancer, or CNS tumor.

[0029] The above agent, use or method in combination with a medicament comprising a targeted anti-cancer agent, a cancer growth blocker, an EGFR inhibitor, or a combination thereof.

[0030] The above agent, use or method in combination with a medicament selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0031] The above agent, use, or method in combination with a pharmaceutical agent that is a cancer growth blocker selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0032] The above agent, use, or method in combination with a medicament which is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0033] Any of the above agents, uses or methods in combination with temozolomide.

[0034] The above agent, use or method in combination with the treatment of a CNS disease by radiation therapy or electric field therapy.

[0035] The agent, use or method as described above, wherein the administration or use of the composition or agent is in combination with a standard treatment for the CNS disease.

[0036] The above agents, uses or methods, wherein the agents, medicaments, therapies, treatments and administrations are administered concurrently, simultaneously, sequentially or temporally separately, respectively.

[0037] The above agents, uses or methods, wherein each agent and medicament is administered by infusion or injection, separately or in combination.

[0038] Any of the above agents, uses or methods, including administration or use by intracranial continuous infusion or bolus administration.

[0039] The agent, use or method as described above, wherein the continuous intracranial infusion comprises infusion using an Ommaya-like reservoir having a partially flexible top.

[0040] An agent, use or method as described above, wherein the continuous intracranial infusion comprises a single entry catheter placed into the target region of the brain.

[0041] The above agent, use or method, wherein the subject has an improved TGF-β2 signature upon administration or use.

[0042] Any of the above agents, uses or methods, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0043] An agent, use or method as described above, wherein the administration or use improves survival at 6, 12, 18, 24, 30 or 36 months.

[0044] The agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2 specific antisense oligonucleotide complementary to the TGF-β2 transcript: SEQ ID NOs: 1-136 in Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof. The agent, use or method as defined above, selected from the group consisting of:

[0045] The agent, use, or method described above, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide having one or two or fewer mismatches compared to the target human TGF-β2.

[0046] The above agent, use, or method, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0047] The above agent, use, or method, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0048] The agent, use, or method described above, comprising a TGF-β2-specific antisense oligonucleotide having one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0049] An agent, use or method as described above, wherein the agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

[0050] The agent, use, or method as described above, comprising a carrier which is sterile water for injection, saline, isotonic saline, phosphate buffered saline, or a combination thereof.

[0051] The agent, use or method as described above, wherein the agent, medicament or administration is substantially free of excipients.

[0052] The above agent, use, or method, wherein the agent, medicament, or administration is stable for at least 14 days in a carrier at 37°C while being pumped via intracranial continuous infusion, or the concentration of the antisense active agent decreases by less than 10% after 90 days of use.

[0053] The agent, use or method as described above, wherein the method comprises administering the composition by intracranial infusion at a rate of 2-8 μl / min and an agent concentration of 1-80 μM on days 1-7, preferably for continuous intracranial infusion.

[0054] The kit includes: A composition comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and Ommaya-like reservoir with partially flexible top.

[0055] 1. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir containing a pharmaceutical composition; a pump

[0101] for pumping the pharmaceutical composition into an Ommaya reservoir

[0111] through an inlet tube

[0103] , the pump

[0101] being in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube

[0115] ; a filter in line with the injection tube; and An entry catheter

[0113] in fluid communication with an Ommaya reservoir, the entry catheter being substantially straight and entering the ventricles into a target region of the brain.

[0056] The above device, wherein the entry catheter

[0113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

[0057] 1. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir containing a pharmaceutical composition; a pump

[0101] for pumping a pharmaceutical composition through an infusion tube

[0103] into an access port

[0107] , the pump being in fluid communication with an Ommaya reservoir

[0111] , the reservoir being in fluid communication with the pump through a reservoir tube

[0115] ; Filter in line with the injection tube

[0105] ; an indwelling tube

[0109] in fluid communication with the access port and the Onmaya reservoir

[0111] ; and An entry catheter

[0113] in fluid communication with an Ommaya reservoir, the entry catheter being substantially straight and entering the ventricles into a target region of the brain.

[0058] The above device, wherein the entry catheter

[0113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

[0059] The above device, wherein the Ommaya reservoir

[0111] comprises a partially flexible top portion.

[0060] The above device provides continuous infusion of a therapeutically effective amount of a fluid pharmaceutical composition to a target area.

[0061] The above device, wherein the distal end of the entry catheter enters the target region of the brain.

[0062] The above device, wherein the Ommaya reservoir

[0111] holds the pharmaceutical composition behind a membrane for a period of time for sustained release of the pharmaceutical composition into the entry catheter.

[0063] The above device, wherein the distal end of the entry catheter that enters the brain has a step-down end, a recessed step end, a multi-port end, a micro-hole end, or an end with a balloon at its tip.

[0064] The above device, wherein the pump

[0101] is a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or other pump with similar specifications.

[0065] The above device, wherein the infusion rate of the fluid pharmaceutical composition is 0.01 to 3000 ml / hour, or 0.01 to 100 ml / hour, or 0.01 to 2 ml / hour, or 0.01 to 1 ml / hour, or 0.05 to 0.5 ml / hour.

[0066] The above device, wherein the infusion tube

[0103] or indwelling tube

[0109] is PEGA Line 100 SF 100cm with a 0.2μm sterile filter, or a 200cm infusion line with a 0.2μm sterile filter, or a Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tube with similar specifications.

[0067] The above device, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing the expression of TGF-β, which can be used to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

[0068] The above device, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of an agent, drug, or delivery vehicle.

[0069] The above device, wherein the fluid pharmaceutical composition is for treating a CNS disease or CNS cancer.

[0070] The above device, wherein the fluid pharmaceutical composition is for the treatment of glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cord cancer, or CNS tumor.

[0071] The above device, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing expression of TGF-β2 selected from TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts, such as SEQ ID NOs:1-136 in Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0072] The above devices operating in combination with radiation therapy or electric field therapy.

[0073] 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir

[0402] containing a pharmaceutical composition, comprising a hard shell

[0403] , a flexible top

[0401] , and a non-flexible mounting plate

[0405] ; a port in fluid communication with the reservoir; and An entry catheter

[0413] that is substantially straight and in fluid communication with a reservoir for intraventricular entry into a target region of the brain.

[0074] 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir

[0502] containing a pharmaceutical composition, comprising an upper rigid shell

[0503] , a lower rigid shell

[0504] , a flexible top

[0501] , and a non-flexible mounting plate

[0505] ; a port in fluid communication with the reservoir for attaching an infusion line; and A port in fluid communication with the reservoir for attaching an entry catheter

[0509] .

[0075] 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir

[0602] containing a pharmaceutical composition, comprising an upper rigid shell

[0603] , a flexible top

[0601] , and a non-flexible mounting plate

[0605] ; a port in fluid communication with the reservoir for attaching an infusion line; and A port in fluid communication with the reservoir for attaching an entry catheter

[0607] .

[0076] 1. A collar for a device for delivering a pharmaceutical composition by intracranial continuous infusion, comprising: A hard shell

[0604] with an opening

[0606] exposing the flexible top of the device.

[0077] 1. A method for administering a pharmaceutical composition by continuous intracranial infusion, comprising: Mounting the device on the patient; and Pumping the pharmaceutical composition within the device to provide continuous intracranial infusion to the patient.

[0078] The above method, wherein the device is mounted and the entry catheter is placed into the brain without concurrent imaging of the head or brain.

[0079] The above method, wherein the device is mounted and a single entry catheter is placed into the brain.

[0080] 1. A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, comprising: a reservoir containing a pharmaceutical composition; pump; Ommaya reservoir with partially flexible top; an infusion tube for connecting the reservoir to the pump and the pump to the Onmaya reservoir; filters; and Entry catheter.

[0081] The above kit, wherein the entry catheter is substantially straight for intraventricular entry into the target region of the brain.

[0082] The above kit, wherein the entry catheter is non-linear and has a bend for intraventricular entry into the target region of the brain.

[0083] The above kit, wherein the injection tube is outside the subject.

[0084] The above kit, wherein a portion of the infusion tubing connecting the pump to the Ommaya reservoir is placed indwelling in the subject. [Brief explanation of the drawings]

[0085] [Figure 1] 1 shows a device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. The device shown in FIG. 1 conveniently includes an external portion that allows the pump

[0101] and reservoir

[0117] to be placed or attached anywhere outside or on the patient's body. The external portion delivers the pharmaceutical composition from the pump through an infusion tube

[0103] and into the Ommaya reservoir

[0111] . A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion includes a reservoir

[0117] containing the pharmaceutical composition, a pump

[0101] for pumping the pharmaceutical composition through an infusion tube

[0103] into an Ommaya reservoir

[0111] , the pump

[0101] being in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube

[0115] , a filter

[0105] collinear with the infusion tube, and an entry catheter

[0113] in fluid communication with the Ommaya reservoir, the entry catheter

[0113] being substantially straight and entering intraventricularly into a target region of the brain.

[0086] [Figure 2]2 shows a device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. The device shown in FIG. 2 includes an indwelling portion that conveniently allows the device to be carried by the patient. The indwelling portion includes an indwelling tube

[0109] that is in fluid communication with an access port

[0107] and an Ommaya reservoir

[0111] , and the indwelling tube is used to transfer fluid from the access port to the Ommaya reservoir. A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion includes a reservoir

[0117] containing the pharmaceutical composition, a pump

[0101] that pumps the pharmaceutical composition through a pumping tube

[0103] into an access port

[0107] , the pump

[0101] being in fluid communication with an Ommaya reservoir, the reservoir being in fluid communication with the pump through a reservoir tube

[0115] , a filter

[0105] collinear with the pumping tube, an indwelling tube

[0109] fluidly connected from the access port to the Ommaya reservoir

[0111] , and an entry catheter

[0113] in fluid communication with the Ommaya reservoir, the entry catheter

[0113] being substantially straight and entering intraventricularly into a target region of the brain.

[0087] [Figure 3]A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion is shown. The device shown in Figure 3 conveniently includes an external portion that allows the pump

[0101] and reservoir

[0117] to be placed or attached anywhere outside or on the patient's body. The external portion delivers the pharmaceutical composition from the pump through an infusion tube

[0103] and into the Ommaya reservoir

[0111] . A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion includes a reservoir

[0117] containing the pharmaceutical composition, a pump

[0101] for pumping the pharmaceutical composition through an infusion tube

[0103] into an Ommaya reservoir

[0111] , the pump

[0101] being in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube

[0115] , a filter

[0105] collinear with the infusion tube, and an entry catheter

[0113] in fluid communication with the Ommaya reservoir, the entry catheter

[0113] being non-linear and having a bend for intraventricular entry into a target region of the brain.

[0088] [Figure 4]4 shows a device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. The device shown in FIG. 4 includes an indwelling portion that conveniently allows the device to be carried by the patient. The indwelling portion includes an indwelling tube

[0109] that is in fluid communication with an access port

[0107] and an Ommaya reservoir

[0111] , and the indwelling tube is used to transfer fluid from the access port to the Ommaya reservoir. A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion includes a reservoir

[0117] containing the pharmaceutical composition, a pump

[0101] that pumps the pharmaceutical composition through a pumping tube

[0103] into an access port

[0107] , the pump

[0101] in fluid communication with an Ommaya reservoir, the reservoir being in fluid communication with the pump through a reservoir tube

[0115] , a filter

[0105] collinear with the pumping tube, an indwelling tube

[0109] fluidly connected from the access port to the Ommaya reservoir

[0111] , and an entry catheter

[0113] in fluid communication with the Ommaya reservoir, the entry catheter

[0113] being non-linear and having a bend for intraventricular entry into a target region of the brain.

[0089] [Figure 5]An Ommaya delivery device for use in a method for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion is shown. The Ommaya delivery device shown in Figure 5 can be used, for example, as the Ommaya reservoir

[0111] in Figures 1-2. The Ommaya delivery device includes a rigid, inflexible shell

[0403] made of an inert material, such as metal or hard plastic, which surrounds and defines a reservoir

[0402] for a drug-containing fluid. The Ommaya delivery device also includes a flexible top

[0401] made of a flexible material, such as rubber, elastomer, or plastic. The area of ​​the flexible top

[0401] relative to the overall shell (

[0401] +

[0403] ) can be 10% to 50% flexible. The Ommaya delivery device further includes a rigid mounting plate

[0405] made of an inert material, such as metal or hard plastic. The Ommaya delivery device further includes a port

[0407] for connecting an infusion line in fluid communication with the Ommaya delivery device. For example, the port

[0407] can be connected to the infusion tube

[0103] of FIGS. 1-2. Fluid communication between the reservoir defined by the shell

[0403] and the infusion line is provided by an internal channel

[0421] . The Ommaya delivery device further includes an entry catheter

[0413] in fluid communication with the Ommaya reservoir, the entry catheter

[0413] being substantially straight and entering the ventricle into the target region of the brain. Fluid communication between the reservoir defined by the shell

[0403] and the entry catheter is provided by an internal channel

[0423] . In another embodiment, the hard shell

[0403] may be a separate protective collar that can be placed over a fully flexible tip having the total area of ​​

[0401] +

[0403] so as to reduce the exposed area of ​​the flexible tip to the area of ​​

[0401] (see Figure 8).

[0090] [Figure 6]An Ommaya delivery device for use in a method for delivering a pharmaceutical composition via intrathecal or intraventricular continuous infusion is shown in Figure 6. The Ommaya delivery device shown in Figure 6 can be used, for example, as the Ommaya reservoir

[0111] in Figures 1-2. The Ommaya delivery device includes rigid, inflexible shells

[0503] and

[0504] made of an inert material such as metal or hard plastic, which surround and define a reservoir

[0502] for a drug-containing fluid. The Ommaya delivery device also includes a flexible top

[0501] made of a flexible material such as rubber, elastomer, or plastic. The area of ​​the flexible top

[0501] relative to the entire shell (

[0501] +

[0503] ) can be 10% to 50% flexible, as shown in the bottom diagram (Figure A-A) of Figure 6. The Ommaya delivery device further includes a non-flexible mounting plate

[0505] made of an inert material such as metal or hard plastic. The Ommaya delivery device further includes ports

[0507] and

[0509] for connecting an infusion line in fluid communication with the Ommaya delivery device. For example, port

[0507] can be connected to the indwelling tube

[0109] of FIGS. 3-4. Fluid communication between the reservoir defined by the shell

[0503] and the infusion line is provided by an internal channel

[0521] . For example, port

[0509] can be connected to the entry catheter

[0113] of FIGS. 1-2. In another embodiment, the hard shell

[0503] can be a separate protective collar that can be placed over a fully flexible apex having the entire area of ​​

[0501] +

[0503] to reduce the exposed area of ​​the flexible apex to the area of ​​

[0501] (see FIG. 8).

[0091] [Figure 7]An Ommaya delivery device for use in a method for delivering a pharmaceutical composition via intrathecal or intraventricular continuous infusion is shown in Figure 7. The Ommaya delivery device shown in Figure 7 can be used, for example, as the Ommaya reservoir

[0111] of Figures 3-4. The Ommaya delivery device includes a rigid, inflexible shell

[0603] made of an inert material, such as metal or hard plastic, which surrounds and defines a fluid-containing drug reservoir

[0602] . The Ommaya delivery device also includes a flexible top

[0601] made of a flexible material, such as rubber, elastomer, or plastic. The Ommaya delivery device further includes a rigid mounting plate made of an inert material, such as metal or hard plastic. The Ommaya delivery device further includes ports

[0607] and

[0619] for connecting an infusion line for fluid communication with the Ommaya delivery device. For example, the port

[0607] can be connected to the infusion tube

[0103] of Figures 3-4. Fluid communication between the reservoir defined by the shell 0603 and the infusion line is provided by the internal channel 0621. For example, the port 0619 can be connected to the entry catheter 0113 of FIGS. 3-4. Fluid communication between the reservoir defined by the shell 0603 and the entry catheter is provided by the internal channel 0619. In another embodiment, the rigid shell 0603 can be a separate protective collar that can be placed over a fully flexible apex having the total area of ​​0601 + 0603 so as to reduce the exposed area of ​​the flexible apex to the area of ​​0601 (see FIG. 8).

[0092] [Figure 8] 1 shows an embodiment of a collar for an Ommaya delivery device for use in a method for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. In this embodiment, the hard shell

[0604] is a separate protective collar that can be placed over the fully flexible top of the Ommaya reservoir to make the top partially flexible. The opening

[0606] exposes only a portion of the flexible top of the Ommaya reservoir.

[0093] [Figure 9] Figure 9 shows the effect of delivering pharmaceutical compositions by intrathecal or intracerebroventricular continuous infusion. Figure 9 shows the effect of OT-101 treatment on TGF-β2 secretion from human GBM cell line A-172. Cells were incubated with various concentrations of OT-101 / AP 12009 (1 μM to 80 μM) as indicated for 7 days. Secreted TGF-β2 was measured in cell supernatants by ELISA. Results represent the median, minimum, and maximum values ​​from three independent experiments.

[0094] [Figure 10] This example shows injection from an Ommaya-like reservoir of the present invention, which has a surface area of ​​339 mm and a catheter with an inner opening diameter of 1.4 mm. This example shows a dark test solution slowly diffusing downward through the catheter opening. This diffusion from the Ommaya-like reservoir of the present invention occurred continuously over several hours.

[0095] [Figure 11] We demonstrate that the operating parameters of continuous infusion can be modified to extend the drug infusion release kinetic half-life from hours to days.

[0096] [Figure 12] We demonstrate that the operating parameters of continuous infusion can be modified to extend the drug infusion release kinetic half-life from days to weeks.

[0097] [Figure 13] We demonstrate that the operating parameters of continuous infusion can be modified to extend the drug infusion release kinetic half-life from weeks to months.

[0098] [Figure 14] Shows the mRNA levels of TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples versus 29 normal pontine specimens.

[0099] [Figure 15]A statistically significant positive correlation with TGFB2 mRNA levels is shown.

[0100] [Figure 16] Correlated expression of specific transcription factors in tumor samples from 41 pediatric patients with DIPG (N=29) or H3K27M-mutated GBM is shown.

[0101] [Figure 17] We show that TGFB2-high DIPG patients had significantly worse OS outcomes than TGFB2-low DIPG patients.

[0102] [Figure 18] We show that PFS outcomes were also significantly worse for the TGFB2-high subset.

[0103] [Figure 19] We show that patients in the TGFB2-high (N=29) and TGFB2-low (N=87) subsets had very similar OS outcomes.

[0104] [Figure 20] We show that expression is selectively amplified and associated with poor OS in pediatric DIPGs.

[0105] [Figure 21] 1 shows a waterfall plot showing the maximum log10 reduction in tumor volume in high-grade glioma patients treated with OT-101 monotherapy who achieved CR or PR.

[0106] [Figure 22] 1 shows semi-log plots of composite 3-D tumor volume reduction curves for 19 high-grade glioma patients treated with OT-101 monotherapy who achieved CR or PR.

[0107] [Figure 23]1 shows swimmer plots for the onset and duration of objective response in patients with high-grade gliomas treated with OT-101 monotherapy who achieved CR or PR. The onset and duration of CR / PR, the end of OR, and the onset of PD are indicated with specific symbols.

[0108] [Figure 24] Overlaid Kaplan-Meier survival curves for both OT-101 and standard chemotherapy are shown.

[0109] [Figure 25] Chemotherapy naive subjects treated with temozolomide (TMZ) are shown.

[0110] [Figure 26] Chemotherapy failures treated with the chemotherapy agents CCNU / BCNU are shown.

[0111] [Figure 27] Using three TGFB2 probe sets that showed increased expression levels in DIPG patients, we demonstrate that TGF-β2 is a valid target for therapeutic approaches against glioma.

[0112] [Figure 28] Using the TGFB2 probe set, which showed increased expression levels in DIPG patients, we demonstrate that TGF-β2 was a valid target for therapeutic treatment of pediatric GBM.

[0113] [Figure 29] We show that low TGF-β2 expression conferred an overall survival advantage in gliomas treated with radiation. Only TGF-β2 predicted survival.

[0114] [Figure 30] We show that low TGF-β2 expression conferred an overall survival advantage in gliomas treated with radiation. Only TGF-β2 predicted survival.

[0115] [Figure 31] We show that TGF-β2 levels selectively predict improved overall survival (OS) in combination with chemotherapy (TMZ).

[0116] [Figure 32] We show that TGF-β2 levels selectively predict improved overall survival (OS) in combination with chemotherapy TMZ and radiation.

[0117] [Figure 33] We show that TGF-β2 levels selectively predict improved overall survival (OS) in combination with antiangiogenic therapy (bevacizumab). DETAILED DESCRIPTION OF THE INVENTION

[0118] Detailed Description of the Disclosure The present invention provides novel devices and methods for the delivery and administration of therapeutic agents ranging from bolus administration to fixed infusion and continuous infusion. The devices and methods of the present invention can be used to treat CNS diseases, such as CNS cancers, via the intracranial route.

[0119] The devices and methods of the present invention can deliver agents to the brain or spinal cord region using a pump-based continuous infusion device / system.

[0120] The present invention provides therapeutic methods for treating or ameliorating the symptoms of CNS diseases, such as CNS cancers.

[0121] In some embodiments, the present invention comprises agents and compositions for inhibiting or suppressing TGF-β2 to result in improved clinical outcomes of CNS disorders.

[0122] In a further aspect, the present invention provides stable formulations of anti-TGF-β2 agents for various treatments for CNS disorders. Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, their pharmaceutically acceptable salt forms, esters, polymorphs, or stereoisomers, and combinations thereof.

[0123] In a further aspect, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for therapy against CNS diseases. The stable formulations of the present invention surprisingly improve clinical outcomes. The stable formulations of agents for suppressing TGF-β can be used to treat CNS diseases, particularly CNS cancers.

[0124] The present invention further provides a novel device that can modify the operating parameters of continuous infusion to extend the drug infusion release kinetic half-life from days to weeks for agents for treating CNS diseases such as CNS cancers via intrathecal or intraventricular routes.

[0125] The devices and methods of the present invention can deliver agents to the brain or spinal cord region using a pump-based continuous infusion system.

[0126] In some aspects, the present invention provides an Ommaya reservoir device with catheter access to the cerebrospinal fluid and intraventricular spaces, connected to a pump for increased and continuous infusion.

[0127] In some embodiments, the present invention provides an Ommaya-like reservoir device with catheter access to the cerebrospinal fluid and intraventricular space, connected to a pump for increased and continuous infusion.

[0128] The present invention provides a device for enhanced delivery, optionally a portable delivery device, that provides delivery by injection of fluids to specific locations within the body, particularly brain tissue and tumors, preferably the ventricular space.

[0129] The present invention also provides devices for intrathecal or intraventricular continuous infusion of therapeutic agents, including Ommaya-like devices (see Figures 1-8 and 10). Ommaya-like devices allow access of an entry catheter into the ventricles of the brain. An entry catheter can otherwise reach a brain tumor or other part of the brain via the intraventricular cavity for delivery of the therapeutic agent. Ommaya-like devices advantageously also serve as reservoirs to hold therapeutic agents for long-term and sustained release into the underlying brain matter or ventricles. Ommaya-like devices can be made of non-collapsible materials and can have flexible sections (see Figures 1-7). This structure and design can generate oscillating pressure and decompression to maintain fluid movement in and out of the Ommaya-like device reservoir. Indeed, the novel Ommaya-like devices and devices of the present invention can advantageously allow for reservoir breathing. In operational applications, Ommaya-like devices and methods can include a self-pulsating or self-oscillating motion due to the patient's physical and muscular activity, stretching and opening the skin near the reservoir, maintaining fluid movement and drug release. The flexible tip enhances fluid movement in the chamber or internal reservoir. Without the flexible tip, a stagnant compartment may not allow the fluid drug composition to flow out sufficiently for patient infusion.

[0130] The flexible tip can enhance fluid transfer by acting as a shock arrestor and creating a more uniform fluid flow rate through the catheter.

[0131] In some aspects, the Ommaya-like devices and devices of the present invention can include a pump for continuous pumping to drive fluid flow in and out of the Ommaya-like device reservoir.

[0132] The present invention provides devices for therapy to treat or ameliorate the symptoms of CNS diseases, such as CNS cancers.

[0133] In some aspects, the present invention describes a device that uses a pump adapted for infusion of agents and compositions for inhibiting or suppressing TGF-β2 to result in improved clinical outcomes of CNS disorders.

[0134] In a further aspect, the present invention provides stable formulations of anti-TGF-β2 agents that can be pumped for continuous infusion for various treatments for CNS disorders. Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, modified forms such as LNA / 2-MOE, pharmaceutically acceptable salt forms, esters, polymorphs, or stereoisomers thereof, and combinations thereof.

[0135] The device of the present invention is coupled to a suitable pump for continuous delivery of small amounts of pharmaceutical composition over several days.

[0136] The device of the present invention has the advantage of delivering pharmaceutical compositions by chronic infusion using an Ommaya intraventricular catheter, either with an externally mounted infusion line, or with an indwelling infusion structure.

[0137] In another example, the device of the present invention can deliver pharmaceutical compositions using an Ommaya reservoir catheter as an intratumoral catheter. This device has the advantage of being used with an external pump for short-term infusion or with an internal extended tube and catheter that passes under the skin for long-term placement for infusion. The Ommaya reservoir component of the device of the present invention can provide long-term access to cerebrospinal fluid and intraventricular space.

[0138] The injection device of the present invention also has the advantage of delivering pharmaceutical compositions to tumors located anywhere in the brain by simple insertion of a catheter from the Ommaya reservoir into the intraventricular space, without the need for insertion under imaging.

[0139] The infusion device of the present invention also has the advantage of using a single standardized intraventricular target.

[0140] The infusion device of the present invention also has the advantage that the intraventricular route to the target is well understood and safe to use.

[0141] The infusion device of the present invention also has the advantage of allowing effective delivery of pharmaceutical compositions to the entire CNS, including the spinal cord.

[0142] The infusion device of the present invention also has the advantage that imaging, such as x-ray imaging, is not required to guide implantation of the delivery catheter. Because the device acts as a tap, once the catheter tip reaches the CSF fluid, the CSF fluid will flow back, indicating successful placement of the catheter.

[0143] In some aspects, the infusion devices of the present invention also have the advantage of being used with entry catheter tips positioned shorter distances within the patient. Because the devices take advantage of the natural fluid circulation within the patient, for example, within the brain, the entry catheter can be shorter and used at a shallower position.

[0144] In a further aspect, the infusion device of the present invention has the advantage of being used with the placement of a single entry catheter for infusion into a patient. For example, treatment of a glioma or large brain tumor may require the placement of several catheters near the cancer. However, the device of the present invention can provide improved infusion even with a single catheter near the tumor.

[0145] The infusion device of the present invention also has the advantage that the implantation of the delivery catheter can be semi-permanent or even permanent.

[0146] The infusion device of the present invention also has the advantage that the Ommaya reservoir is available for needle aspiration of fluid from the device or brain, or injection of fluid into the device or brain.

[0147] The infusion device of the present invention also has the advantage of allowing for the placement and utilization of an Ommaya device as needed for effective delivery of drugs and treatments. For example, as shown in Figures 1 and 2, the Ommaya device can be placed directly above the ventricles of the brain, and the Ommaya device can utilize a straight ventricular entry catheter. In a further example, the Ommaya device can be placed in a convenient location on the patient's skull and connected to an angled ventricular entry catheter similar to Figures 3 and 4 to reach a target location within the brain.

[0148] Aspects of the invention include the following.

[0149] A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion, comprising (FIG. 1): a reservoir containing the pharmaceutical composition; a pump for pumping the pharmaceutical composition through an infusion tube into an Ommaya reservoir, the pump being in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube; a filter collinear with the infusion tube; and an entry catheter in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain. The entry catheter is non-straight and has a bend for intraventricular entry into the target region of the brain (FIG. 3).

[0150] A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion, comprising (Figure 2): a reservoir

[0117] containing the pharmaceutical composition; a pump

[0101] that pumps the pharmaceutical composition through a pumping tube

[0103] into an access port

[0107] , the pump

[0101] in fluid communication with an Ommaya reservoir, the reservoir being in fluid communication with the pump through a reservoir tube

[0115] ; a filter

[0105] collinear with the pumping tube; an indwelling tube

[0109] in fluid communication with the access port and the Ommaya reservoir

[0111] ; and an entry catheter

[0113] in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and entering intraventricularly into a target region of the brain. The entry catheter

[0113] may be non-linear and have bends to allow intraventricular entry into the target region of the brain (Figure 4).

[0151] The device is capable of providing a continuous infusion of a therapeutically effective amount of a pharmaceutical composition to a target area.

[0152] As used herein, a target region of the brain may be a region containing a tumor.

[0153] The distal end of the entry catheter enters the target region of the brain.

[0154] The Ommaya reservoir can hold the pharmaceutical composition behind the membrane for a period of time for sustained release of the pharmaceutical composition into the entry catheter. The distal tip of the entry catheter that enters the brain can be a step-down end, a recessed step end, a multi-port end, a microporous end, or a balloon-tipped end. In some aspects, the entry catheter can be barium-impregnated silicone and can be resistant to kinking and pressure. In a further aspect, the entry catheter can include an elongated, surgically acceptable probe, such as a stainless steel probe, to allow for orientation of the catheter during catheter placement.

[0155] The pump can be a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or other pump with similar specifications.

[0156] The infusion rate can be 0.01 to 3000 ml / hour, or 0.01 to 100 ml / hour, or 0.01 to 2 ml / hour, or 0.01 to 1 ml / hour, or 0.05 to 0.5 ml / hour.

[0157] Some examples of pumps and infusion rates for the devices of the present invention include the following in Table 1:

[0158] Table 1. Pumps and speeds for the injection system TIFF2025538189000002.tif68128

[0159] The infusion tube

[0103] or indwelling tube

[0109] may be a PEGA Line 100 SF 100cm with a 0.2um sterile filter, or a 200cm infusion line with a 0.2um sterile filter, or a Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tube with similar specifications.

[0160] In some aspects, the brain entry catheter placed in the ventricular space can be a non-specific ventricular catheter. The ventricular catheter can have an inner diameter of 1.0 to 2.0 mm. For example, the ventricular catheter can have an inner diameter of 1.4 mm and an outer diameter of 2.7 mm. The catheter can be 14 cm or less in length and can have 24 inlet holes (e.g., 3 rows of 8 holes) at the proximal end. Generally, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The omma-like reservoir of the present invention is 300 to 400 mm. 2 and the area of ​​the inner opening of the catheter is 0.785 mm 2~3.14mm 2 Therefore, the ratio of the reservoir surface area to the catheter surface area may range from 96 to 509. This ratio can be used to adjust the expected release time of the drug solution over a wide range, depending on the density of the test solution relative to water.

[0161] The Ommaya-like device of the present invention allows for the delivery of drugs directly to the CSF and is particularly useful for delivering oligonucleotide and antisense oligonucleotide drugs, such as OT-101, either alone or in combination with other cancer therapies.

[0162] In some aspects, the drug load can surprisingly be delivered as a single bolus injection or as a short infusion of 15, 30, or 60 minutes, yet achieve sustained delivery through an advantageous ratio of reservoir volume to catheter opening and / or a tailored ratio of CSF density to drug solution density.

[0163] For example, the typical surface area of ​​a reservoir is 339mm 2 and the inner diameter of the catheter opening is 1.4 mm. The release profile of the device can be controlled by varying the ratio of the surface area of ​​the reservoir to the surface area of ​​the catheter, in relation to the diffusion equation.

[0164] As described below, infusion data for rat animals were obtained with only a ventricular catheter and no reservoir, and the equation {Y = span * exp(-K * X) + plateau} governed the process, with a normalized value of k of 7.571 to achieve a span of 100 and a plateau of 0. Constraints were placed on the drug reservoir to determine the change in rate associated with the reservoir-to-catheter surface area ratio. By varying the reservoir-to-catheter ratio, the release kinetics of the drug infusion can be slowed so that half-lives can be advantageously measured in days and weeks.

[0165] For example, as shown in Figure 11, the release kinetics of a drug infusion can be extended such that the half-life varies from hours to days for a single reservoir.

[0166] For example, as shown in Figure 12, the release kinetics of a drug infusion can be extended such that the half-life varies from days to weeks for a single reservoir.

[0167] For example, as shown in Figure 13, the release kinetics of a drug infusion can be extended such that the half-life varies from weeks to months for a single reservoir.

[0168] An advantage of the device of the present invention is the design of the Ommaya-like reservoir, which allows the device to maintain fluid flow and movement into and out of the reservoir. The flexible top expands and contracts in response to changes in pressure, allowing the Ommaya-like reservoir of the present invention to properly brace for continuous flow of solution, especially in the presence of an externally generated pumping action. Patient movement, such as movement of the patient's jaw and other muscles, can also cause unwanted accidental or inadvertent release.

[0169] An advantage of the present device is its positioning as an Ommaya-like device, not as a mechanism for collecting CSF or for delivering drugs (pharmaceutical compositions) to the CSF, but as a reservoir for holding drugs to be infused into the CSF over an extended period of time. To allow fluid to exit the Ommaya, a pump causes continuous flow out of the reservoir, which also carries the drug fluid into the reservoir. The structure of the present device advantageously prevents accidental pressurization of the Ommaya, which could suddenly deliver large amounts of drug from the reservoir into the CSF, because the Ommaya delivery device portion of the present device has a rigid, inflexible, hard shell and can only be partially crushed (see Figures 1-7). Furthermore, the design of the present device advantageously maintains fluid movement within the reservoir because the reservoir includes a flexible top (see, for example, Figure 5), which allows for oscillating pressure and pressure changes for fluid movement. For example, movement of the skin or jaw muscles can slightly pressurize and depressurize the Ommaya device through the flexible portion, keeping fluid moving in and out of the reservoir.

[0170] As used herein, a partially flexible top can refer to an Ommaya-like reservoir made of a rigid material with an integrated top where only the top of the top is flexible, or an Ommaya-like reservoir with a top made of a flexible material and a rigid collar attached so that only the top of the flexible top is exposed and can be depressed or bent.

[0171] The pharmaceutical composition can include an agent for inhibiting or suppressing the expression of TGF-β, which can be used to treat or alleviate the symptoms of a CNS disease in a human subject or animal. The CNS disease can be cancer. The cancer can be glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal metastasis or brain metastasis, brain or spinal cord cancer, or CNS tumor.

[0172] In some embodiments, the compositions and methods of the present invention can be used against diffuse midline glioma (DMG) and K27M GBM.

[0173] Methods and compositions for CNS disorders Operating Parameters. The systems of the present invention can be modified to expand the range of drugs used to treat or ameliorate symptoms of CNS diseases, such as CNS cancers, in human subjects or animals in need. The systems of the present invention can contain pharmaceutical compositions for sustained infusion release kinetic half-lives of weeks to months to the brain or spinal cord.

[0174] The pharmaceutical composition for continuous infusion can include an agent that inhibits or suppresses the expression of TGF-β and administers a therapeutically sufficient amount of the composition to a subject. The present invention provides a systems therapy for treating CNS diseases, such as cancer, or ameliorating the symptoms of CNS diseases.

[0175] Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, artemisinin, pharmaceutically acceptable salt forms, esters, polymorphs, or stereoisomers thereof, and combinations thereof.

[0176] In some aspects, the present invention provides a system containing a composition of agents for inhibiting or suppressing the expression of TGF-β for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0177] In a further aspect, the present invention provides the use of a composition of agents for inhibiting or suppressing expression of TGF-β in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0178] As used herein, "intraventricular" infusion can be used for continuous infusion of pharmaceutical compositions to treat CNS diseases, including cancer. An example of intraventricular administration is an Ommaya-like device with an entry catheter.

[0179] As used herein, the term intracranial includes intrathecal and intraventricular. For example, intracranial injection includes intrathecal and intraventricular injection. Furthermore, the terms "intrathecal and intraventricular" are intended to include "intracranial."

[0180] Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides and can be complementary to mRNA targets. Antisense therapy can downregulate molecular targets by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes and significantly reduces the translation of target genes. Other ASO mechanisms include inhibiting 5' cap formation, altering splicing processes such as splice switching, and steric hindrance of ribosomal activity.

[0181] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating catalytic degradation of target mRNA or by binding to the site on mRNA that is required for translation.Antisense oligonucleotides can be designed to target the RNA genome of viruses or viral transcription products.Antisense oligonucleotides can provide an approach to identify potential targets and therefore represent potential therapeutic agents.

[0182] Antisense oligonucleotides are small, synthetic fragments of single-stranded DNA that can be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization. Once bound to the target RNA, they can inhibit the translation process either by inducing cleavage mechanisms or by inhibiting mRNA maturation. ASOs can selectively inhibit gene expression with specificity. Chemical modifications of DNA or RNA can be used to enhance stability.

[0183] For example, modifications can be introduced into the phosphodiester bond, sugar ring, and backbone. ASO antiviral agents can block the translation process either by (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage, or (ii) steric (non-binding) blockage of enzymes involved in the translation of target genes. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide (OT-101; AP 12009; travedersen), hereafter referred to as OT-101 or AP 12009, is intended to reduce TGF-β2 protein levels in malignant gliomas, thereby slowing disease progression.

[0184] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' linkages are modified to phosphorothioate. The molecular formula is C 177 H 208 N 60 Na 17 O 94 P 17 S 17 and has a molecular weight of 6,143 g / mol. OT-101 was designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression.

[0185] OT-101 can be supplied as a lyophilized powder in three different amounts in 50 mL glass vials. The OT-101 lyophilized powder is dissolved in isotonic (0.9%) aqueous sodium chloride solution before use. The product can be prepared for administration at the desired concentration.

[0186] OT-101: Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and several have been approved. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which the non-bridging oxygen of each phosphate moiety is replaced by a sulfur atom. OT-101 was designed to be complementary to a specific sequence in human TGF-β2 mRNA after gene expression. It is a first-in-class RNA therapeutic designed to block the immunosuppressive effects of TGF-β2 in malignant gliomas, reduce TGF-β2 levels, and thereby slow disease progression.

[0187] The agent can be an antisense oligonucleotide or inhibitor specific for TGF-β1, TGF-β2, or TGF-β3. Agents for inhibiting or suppressing TGF-β expression can be selected from the TGF-β2-specific antisense oligonucleotides described below.

[0188] The target TGF-β2 mRNA can be the NCBI reference sequence: NM_003238.3, which is 5,882 bp in length. The target region of the TGF-β2 mRNA can be the protein-coding sequence from 1,369 to 2,613 of the reference.

[0189] Examples of agents of the present disclosure for inhibiting or suppressing the expression of TGF-β2 include TGF-β2-specific antisense oligonucleotides shown in SEQ ID NOs: 1 to 136 in Table 2.

[0190] Table 2. TGF-β2-specific antisense oligonucleotides TIFF2025538189000003.tif207147TIFF2025538189000004.tif235147TIFF2025538189000005.tif235147TIFF2025538189000006.tif129147

[0191] The sequences in Table 2 can be chemically modified to provide active variants thereof, LNA variants thereof, and gapmer variants thereof, as known in the art. The sequences in Table 2 can be used in any combination, e.g., pooled combinations, as active agents.

[0192] Examples of the agents of the present disclosure for inhibiting or suppressing the expression of TGF-β include artemisinin extracts, its pharmaceutically acceptable salts, salt polymorphs, esters, or isomers, and any combination thereof. In some embodiments, the present disclosure includes substantially pure artemisinin having a purity of at least 60%, 70%, 80%, 90%, or 95%.

[0193] In certain embodiments, the agent of the present disclosure for inhibiting or suppressing the expression of TGF-β can be prepared from a lyophilized powder of the agent.

[0194] In some embodiments, the TGF-β2-specific antisense oligonucleotides of the present invention can have no more than one or two mismatches compared to the target human TGF-β2.

[0195] In certain embodiments, the TGF-β2-specific antisense oligonucleotides of the invention are capable of reducing TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0196] In additional embodiments, the TGF-β2-specific antisense oligonucleotides of the present invention may be selective for TGF-β2 and may reduce any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0197] In further embodiments, the therapeutically effective amount of an antisense agent for inhibiting or suppressing expression of TGF-β2 can be 0.1 to 3000 mg per day, or 1 to 1000 mg per day, or 2 to 500 mg per day, or 2 to 200 mg per day.

[0198] In certain embodiments, a formulation of an antisense agent for inhibiting or suppressing expression of TGF-β2 can have a concentration of 0.05 to 50 μM, or 0.1 to 25 μM, or 0.1 to 10 μM, or 0.1 to 7.5 μM, or 0.1 to 5 μM.

[0199] In certain embodiments, methods for using antisense agents to inhibit or suppress expression of TGF-β2 include administering a dose of 1 to 1000 mg / m 2 / day, or 1-500 mg / m 2 / day, or 1-250 mg / m 2 / day, or 1-100 mg / m 2 / day, or 1-50 mg / m 2 A dosage of 1 / day can be used. The average human body surface area is approximately 1.6 to 1.9 m 2 It could be.

[0200] In additional embodiments, methods for using antisense agents to inhibit or suppress expression of TGF-β2 include administering doses of 0.05 to 40 mg / kg / day, or 0.1 to 30 mg / kg / day, or 0.2 to 20 mg / m2 / day, or 0.3-10 mg / m 2 / day, or 0.5-5 mg / m 2 A dosage of 100 mg / day can be used. The average human weight can be about 60 kg.

[0201] In some examples and embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, and may be administered or used by continuous intracerebroventricular or intrathecal or intracerebral administration at a dose of 4 μl / min, at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7.

[0202] In some examples and embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and may be administered or used by continuous intracerebroventricular or intrathecal or intracerebral administration at a dose of 4 μl / min or 2-8 μl / min at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7.

[0203] In some embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, as well as chemically modified variants thereof, and may be administered as a bolus injection into an omma-like reservoir at a concentration of 61.43 mg / ml (10 μM), 1 mg / ml, 7.35 mg / ml, 15 mg / ml, or 18.23 mg / ml.

[0204] In a further embodiment, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, and chemically modified variants thereof, administered or used by continuous infusion, either alone or in combination with any form of artemisinin, at a dose of 500 mg per day given orally on days 1-5.

[0205] Embodiments of the present invention contemplate methods and uses that include an agent that may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs:9-136.

[0206] Examples of agents of the present disclosure for inhibiting TGF-β include agents for specifically inhibiting TGF-β1, TGF-β2, or TGF-β3, preferably TGF-β2.

[0207] Embodiments of the invention that involve the administration or use of compositions of agents can ameliorate or suppress symptoms caused by TGF-β-induced proteins.

[0208] The agent for inhibiting or suppressing TGF-β expression may be an artemisinin formulation containing a 90-95% pure artemisinin extract or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, and one or more pharmaceutically acceptable excipients. The excipients may include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants, and anti-caking agents. In some embodiments, the excipients may include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, croscarmellose sodium, and magnesium stearate.

[0209] In a further embodiment, the agent for inhibiting or suppressing the expression of TGF-β can be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0210] As used herein, derivative encompasses chemical modifications that provide structural analogs of a compound. For example, substitution or replacement of an alkyl group can provide a structural analog.

[0211] Embodiments of the present invention include processes or uses in which the agent for inhibiting or suppressing expression of TGF-β is a compound, or a ligand comprising a small molecule or polypeptide, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, that interacts with site I of TGF-β comprising Trp30 and / or site II of TGF-β comprising Arg15, Gln19, and Phe8.

[0212] In some embodiments, the agent for inhibiting or suppressing expression of TGF-β can be a polypeptide or peptidomimetic of site I of TGF-β comprising residues Phe24 to Lys37 and / or site II of TGF-β comprising residues Cys7 to Gln19, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0213] In a further aspect, the agent for inhibiting or suppressing expression of TGF-β can be a humanized or non-humanized antibody or antibody fragment having affinity for site I of TGF-β, which includes residues Phe24 to Lys37, and / or site II of TGF-β, which includes residues Cys7 to Gln19.

[0214] In certain embodiments, the agent for inhibiting or suppressing the expression of TGF-β may be a compound containing three isoprene groups and one lactone ring or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0215] Embodiments of the present invention further include pharmaceutical compositions for inhibiting or suppressing TGF-β expression or treating or ameliorating symptoms of CNS disorders in humans or animals. The pharmaceutical compositions can include a TGF-β inhibitor, artemisinin, a pharmaceutically acceptable salt form, ester, polymorph, or stereoisomer thereof, or any combination thereof, and a carrier. The TGF-β inhibitor can be selected from TGF-β2-specific antisense oligonucleotides SEQ ID NOs: 1-136, and chemically modified variants thereof. The carrier can be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0216] Importantly, the composition of the present disclosure can be substantially free of excipients.It has been found that the composition of the present invention that is substantially free of excipients is surprisingly stable in carrier.In some embodiments, the composition can be stable in carrier at 37 ℃ for at least 14 days, or at least 21 days, or at least 28 days.In another embodiment, the concentration of antisense active agent can decrease by less than 10% after 90 days of use.

[0217] In additional embodiments, pharmaceutical compositions for injection can contain less than 1% by weight of excipients, or less than 0.5% by weight of excipients, or less than 0.1% by weight of excipients.

[0218] Aspects of the present invention further contemplate treatment modalities in which the compositions of the present invention are administered or utilized in conjunction with standard of care therapy for a disease. Examples of additional pharmaceutical agents that may be administered or utilized in combination with the compositions of the present invention include anti-inflammatory agents, anti-inflammatory steroids, piperiquine, pyronaridine, curcumin, frankincense, remdesivir, Sompraz D, Zifi CV / Zac D, CCM, Broclear, Budamate, Rapitus, Montek LC, low molecular weight heparin, prednisolone, paracetamol, vitamin B complex, vitamin C, pantoprozol, doxycycline, ivermectin, zinc, Foracort Rotacaps inhalation, ceftriaxone injection, paracetamol tablets, Fragmin injection, Covifor tablets, azithromycin, dexamethasone injection, ondansetron injection, multivitamin tablets, ascorbic acid tablets, calcium carbonate tablets, and zinc sulfate tablets.

[0219] The present invention further provides a kit comprising a lyophilized powder of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in vials, each containing 250 mg.

[0220] The present invention also provides a kit containing, in a vial, a 500 mg lyophilized powder of artemisinin or a derivative thereof, or a ligand comprising a compound, small molecule, or polypeptide that interacts with site II of TGF-β comprising Arg15, Gln19, and Phe8, a sesquiterpene lactone or a derivative thereof, or a compound comprising three isoprene groups and one lactone ring and its derivative, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, or any combination of the foregoing.

[0221] Some TGF-β agents are listed in US 9,963,703, US 9,758,786, and US 8,476,246.

[0222] Infusion Devices and Methods The present invention provides novel devices and methods of use for the continuous infusion of agents via an intracranial route to treat CNS diseases, such as CNS cancers.

[0223] The devices and methods of the present invention can deliver agents to the brain or spinal cord region using a pump-based continuous infusion system.

[0224] The device can operate within a radiation therapy device and / or an electric field device, and the pharmaceutical composition can include the above agents and any combination thereof.

[0225] In some examples and embodiments, the device of the present invention can include a portable extracorporeal pump having a fluid reservoir connected via an infusion device to an infusion catheter that can be placed in any tissue or tumor, preferably in the intraventricular space. Fluids can be administered by high flow or continuous perfusion. The device of the present invention allows for the infusion of any type of fluid by continuous infusion or continuous convection-enhanced delivery. The device of the present invention can contain and deliver a variety of pharmaceutical compositions, drugs, proteins, protein toxins, antibodies for treatment or imaging, proteins in enzyme replacement therapy, growth factors, and viruses or oligonucleotides in gene therapy.

[0226] The devices of the present invention effectively deliver a therapeutically effective amount of a pharmaceutical composition to a subject and allow for sustained infusion of an agent to a specific location within a subject, for example, a specific tissue or tumor, preferably the ventricular space.

[0227] In some aspects (FIGS. 1 and 3), the device of the present invention can serve as a portable, external, convection-enhanced delivery device for injecting a liquid form of a pharmaceutical agent to a specific location within a subject. Such aspects of the device can provide perfusion delivery in a hospital or inpatient center.

[0228] In a further aspect (FIGS. 2 and 4), the device of the present invention can serve as a portable indwelling infusion device for the target tissue or tumor, preferably an onmaya implantable in the ventricular space. Such an aspect of use can provide perfusion delivery outside of a patient center, for example, for home infusion. Such an aspect of use can also provide perfusion delivery outside of a patient center, for example, for home infusion, for example, using an abdominally implanted pump.

[0229] The device of the present invention is capable of injecting fluid for continuous infusion.

[0230] The devices of the present invention can be used to deliver a variety of therapeutic agents, such as, for example, drugs, proteins, protein toxins, imaging agents, antibodies for treatment or imaging, proteins in enzyme replacement therapy, growth factors, and / or viruses or oligonucleotides in gene therapy.

[0231] The devices of the present invention can advantageously improve the bioavailability and safety, as well as the pharmacokinetics and pharmacodynamics of the therapeutic agents delivered.

[0232] The device of the present invention may further allow for outpatient treatment with infusion delivery using a portable pump.

[0233] The device of the present invention (FIGS. 2 and 4) can use an access port placement system for infusion, which has the advantage that the reservoir and pump, as well as the pharmaceutical composition contained therein, can be easily changed.

[0234] The access port of the device of the present invention (FIGS. 2 and 4) allows precise placement of the infusion catheter into the center of the tissue or tumor, preferably the ventricular space, in a single surgical step. The access port can be conveniently positioned to minimize mechanical stress.

[0235] The device of the present invention has the advantage of providing a clinically useful constant flow rate for continuous infusion or convection-enhanced delivery.

[0236] The device of the present invention has the advantage of providing small-step flow characteristics using a portable pump at flow rates of 0.01 to 3000 ml / hr, or 0.01 to 100 ml / hr, or 0.01 to 2 ml / hr, or 0.01 to 1 ml / hr, or 0.05 to 0.5 ml / hr.

[0237] The devices of the present invention can include an Ommaya-like delivery device for use in a method for delivering a pharmaceutical composition by intracranial continuous infusion (Figure 5). The Ommaya-like delivery device shown in Figure 5 can be used, for example, as the Ommaya reservoir

[0111] of Figures 1-2. The Ommaya delivery device includes a rigid, inflexible shell

[0403] made of an inert material, such as metal or hard plastic, which surrounds and defines a reservoir for a fluid-containing drug. The Ommaya delivery device also includes a flexible top

[0401] made of a flexible material, such as rubber, elastomer, or plastic. The area of ​​the flexible top

[0401] relative to the entire shell (

[0401] +

[0403] ) can be 10% to 50% flexible. The Ommaya delivery device further includes a rigid mounting plate

[0405] made of an inert material, such as metal or hard plastic. The Ommaya delivery device further includes a port

[0407] for connecting an infusion line in fluid communication with the Ommaya delivery device. For example, the port

[0407] can be connected to the infusion tube

[0103] of FIGS. 1-2. Fluid communication between the reservoir defined by the shell

[0403] and the infusion line is provided by an internal channel

[0421] . The Ommaya delivery device further includes an entry catheter

[0413] in fluid communication with the Ommaya reservoir, the entry catheter

[0413] being substantially straight and entering the ventricle into the target region of the brain. Fluid communication between the reservoir defined by the shell

[0403] and the entry catheter is provided by an internal channel

[0423] . In another embodiment, the hard shell

[0403] may be a separate protective collar that can be placed over a fully flexible tip having the total area of ​​

[0401] +

[0403] so as to reduce the exposed area of ​​the flexible tip to the area of ​​

[0401] (see Figure 8).

[0238] In a further example or embodiment, the device of the present invention may include an Ommaya-like delivery device for use in a method for delivering a pharmaceutical composition by intracranial continuous infusion (Figure 6). Figure 6 illustrates an Ommaya-like delivery device for use in a method for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. The Ommaya delivery device shown in Figure 6 can be used, for example, as the Ommaya reservoir

[0111] in Figures 1 and 2. The Ommaya delivery device includes rigid, inflexible shells

[0503] and

[0504] made of an inert material such as metal or hard plastic. The Ommaya delivery device also includes a flexible apex

[0501] made of a flexible material such as rubber, elastomer, or plastic. The area of ​​the flexible apex

[0501] relative to the entire shell (

[0501] +

[0503] ) can be 10% to 50% flexible, as shown in the lower diagram (Figure A--A) of Figure 6. The Ommaya delivery device further includes a non-flexible mounting plate

[0505] made of an inert material such as metal or hard plastic. The Ommaya delivery device further includes ports

[0507] and

[0509] for connecting an infusion line in fluid communication with the Ommaya delivery device. For example, port

[0507] can be connected to the indwelling tube

[0109] of FIGS. 3-4. Fluid communication between the reservoir defined by the shell

[0503] and the infusion line is provided by an internal channel

[0521] . For example, port

[0509] can be connected to the entry catheter

[0113] of FIGS. 1-2. In another embodiment, the hard shell

[0503] can be a separate protective collar that can be placed over a fully flexible apex having the entire area of ​​

[0501] +

[0503] to reduce the exposed area of ​​the flexible apex to the area of ​​

[0501] (see FIG. 8).

[0239] In a further example or embodiment, the device of the present invention may include an Ommaya-like delivery device for use in a method for delivering a pharmaceutical composition by intracranial continuous infusion (FIG. 7). FIG. 7 illustrates an Ommaya-like delivery device for use in a method for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. The Ommaya delivery device shown in FIG. 7 can be used, for example, as the Ommaya reservoir

[0111] in FIGS. 3-4. The Ommaya delivery device includes a non-flexible, hard shell

[0603] made of an inert material such as metal or hard plastic. The Ommaya delivery device also includes a flexible top

[0601] made of a flexible material such as rubber, elastomer, or plastic. The Ommaya delivery device further includes a non-flexible mounting plate made of an inert material such as metal or hard plastic. The Ommaya delivery device further includes ports

[0607] and

[0619] for connecting an infusion line for fluid communication with the Ommaya delivery device. For example, port 0607 can be connected to the infusion tube 0103 of FIGS. 3-4. Fluid communication between the reservoir defined by the shell 0603 and the infusion line is provided by the internal channel 0621. For example, port 0619 can be connected to the entry catheter 0113 of FIGS. 3-4. Fluid communication between the reservoir defined by the shell 0603 and the entry catheter is provided by the internal channel 0619. In another embodiment, the rigid shell 0603 can be a separate protective collar that can be placed over a fully flexible apex having the total area of ​​0601 + 0603 so as to reduce the exposed area of ​​the flexible apex to the area of ​​0601 (see FIG. 8).

[0240] The devices of the present invention can deliver pharmaceutical agents, including any agent suitable for delivery in a solvent system or formulated for delivery in, for example, an aqueous solution, including, for example, analgesics, wound care agents, stimulants, anesthetics, anthelmintics, anticoagulants, antirheumatics, antiallergics, antiarrhythmics, antibiotics, antidementia drugs, antidiabetics, antidotes, antiepileptics, hemostatics, antihypertonics, antimigraine preparations, antifungals, antineoplastics, antiparkinsonian agents, anti-inflammatory agents, antisense oligonucleotides, antituberculous agents, antiarteriosclerotic agents, biological substances, blood flow stimulants, corticoids, cytokines, cytostatics, diagnostic agents, fibrinolytics, geriatrics, gonadotropins, hepatics, hormones, and the like. and inhibitors thereof, hypnotics, immunoglobulins, immunomodulators, immunotherapeutics, organ perfusion media, proteins, protein toxins, protective agents, sedatives, cardiology drugs, depressants and stimulants, minerals, muscle relaxants, neurotropic agents, oligonucleotides, ophthalmics, vaccines, antispasmodics, urologics, drugs, proteins, protein toxins, therapeutic antibodies, proteins in enzyme replacement therapy, growth factors, vectors, viruses in gene therapy and / or diagnostic agents as agents or antibodies for imaging, x-ray contrast agents, oligonucleotides inhibiting expression.

[0241] The device of the present invention can deliver pharmaceutical agents or active substances dissolved or suspended in a physiological solvent or any other suitable solvent. The agents can be in the form of a free base or a salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, prodrug, or derivative of these compounds. The above-mentioned agents and combinations thereof can be used in the devices, methods, kits, combinations, and compositions described herein.

[0242] The devices of the present invention can have an interior surface coated with a therapeutic agent.

[0243] Components of the devices of the present invention can be fabricated from a variety of materials, including, for example, metallic materials, polymeric materials, and / or composites, such as titanium, high-grade steel, aluminum, alloys, polymer foams, plastics, stainless steel, and / or metals, as well as combinations, mixtures, and modifications thereof. Materials intended for implantation into a subject can be made of biocompatible materials, such as polymers, polystyrene, polyolefins, polyamides, or polyurethane polymer segments, and metals. The selection of such materials depends on several factors, including the desired mechanical properties and the porosity, surface properties, or toxicity of the material. Components of the devices of the present invention can be made of, for example, titanium, alloys, stainless steel, ceramics, silicon, Teflon, polypropylene, polyethylene, polystyrene, polyolefins, polyimides, polyamides, polyurethanes, PET, PETG, PE, PIG, HDPE, PC, PVC, nylon, urethanes, and / or copolymers, and can be laminated with or contain, sputtered, or otherwise deposited or incorporated with layers of gold, silver, and / or aluminum to minimize permeability to gases and liquids. Bio-Span® segmented polyurethane-urea, Bionate® polycarbonate urethane, Elasthane™, and Elasthane™ polyether urethane can be used in chronically implanted medical devices. Elasthane™ has a chemical structure and properties similar to Pellethane® 2363. Thermoplastic silicone-urethane copolymers, such as PurSil™ silicone polyether urethane and CarboSil™ silicone polycarbonate urethane, can also be used in the devices.

[0244] The device of the present invention can include filters in any suitable location. The filters can include a sterile filter for removing pathogens, a biological filter for biological substances such as proteins and / or antibodies, a particle filter for removing microparticles, a chemical filter for removing chemicals, and / or a filter for removing air or gas from a solvent. In one aspect, each filter can have a distal port and a proximal port and a lumen therethrough. Within the lumen of each filter can be a membrane or any other suitable device for removing air, particles, chemicals, and / or biological substances, such as pathogens including bacteria, fungi, and / or viruses. The filters can have separate casings or a common casing. In one aspect, the filter for removing air can be placed outside the body. In another aspect, the filter for removing particles can be placed upstream of the sterile filter. The sterile filter typically has a pore size of about 0.45 μm or less, or about 0.22 μm or less, or about 0.1 μm or less. The particle filter can have a pore size of greater than about 0.45 μm or greater than about 0.22 μm.

[0245] The access port can be implanted subcutaneously, for example above a rib, and the access port chamber is in fluid communication with the Ommaya reservoir.

[0246] The device of the present invention can include a compound to be injected into a subject, formulated as an injectable formulation, for example, an aqueous solution or suspension of the compound suitable for intravenous delivery. When preparing a composition for injection, particularly for intravenous delivery, the continuous phase illustratively comprises an aqueous solution of a tonicity adjusting agent, buffered, for example, to a pH of less than 7, or, for example, less than 6. Tonicity adjusting agents include, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that make the osmotic pressure of the formulation isotonic with blood.

[0247] The device of the present invention may include a preservative added to the formulation, such as benzalkonium chloride, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenol, sodium benzoate, or EDTA.

[0248] The device of the present invention can comprise a pharmaceutically acceptable carrier. The carrier material that can be used in preparing the composition of the present invention is any of the excipients commonly used in pharmaceutics, and should be selected based on the compatibility with the pharmaceutical agent and the properties of the release profile of the desired dosage form.

[0249] The devices of the present invention may contain excipients such as those known in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975 and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0250] As used herein, the term agent can refer to one or more active compounds, a combination of active compounds, or a composition comprising one or more active compounds and a carrier, and / or a solvent, and / or any number of excipients.In some embodiments, the composition can be a pharmaceutical composition.In certain embodiments, the composition can be a pharmaceutical composition comprising a therapeutically effective amount of one or more active compounds.Some examples of excipients are listed in Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0251] Methods for determining a therapeutically effective amount of a compound are known in the art. A therapeutically effective amount can also be determined by routine experimentation, for example, by monitoring the response of an animal to administration of an agent and adjusting the dosage. See, for example, Remington, The Science and Practice of Pharmacy (Gennaro ed. 20th edition) (2000).

[0252] Device Operation In operation, the device can be tailored to the patient by performing a computed tomography scan (CT), brain MRI, or other appropriate imaging procedure on the patient to determine the location of the ventricular space and the length of the catheter required. To place the device, a burr hole is created and the brain entry catheter is slowly inserted until fluid emerges from the catheter. The ventricular catheter-on-the-maya device is then connected to an external or indwelling infusion component.

[0253] For example, an approved silicone microcatheter is stereotactically placed into the ventricular space. The target location of the entry catheter tip can be calculated in advance from a CT scan or brain MRI. Simultaneous imaging is not required for intraventricular placement of the entry catheter. The external or indwelling infusion component can be pre-filled with normal saline. The postoperative location of the Ommaya-like device and device of the present invention can be determined by native X-ray, computed tomography scan, brain MRI, or other appropriate imaging method. The location of the catheter tip can also be determined in the same manner. In some examples and embodiments, the injection port and catheter can be barium-impregnated for imaging purposes.

[0254] An infusion line can be connected to the infusion device under sterile conditions, for example, an infusion tube + bacterial micropore filter (up to 0.2 μm) can be used.

[0255] In some examples and embodiments, a special port puncture needle, such as a GRIPPER PORT-A-CATH™ needle, can be used. The treatment solution can be passed through an external or indwelling infusion component until it reaches the tip of the port puncture needle. This method can remove air bubbles.

[0256] For treatment, a therapeutic solution, such as the antisense oligonucleotide OT-101, can be infused into the ventricular space. The therapeutic solution can be prepared and used to fill the omma-like reservoir. At the patient's bedside, an automated pump is connected to the device. Before infusion begins, the infusion device can be filled with the therapeutic solution and connected to the port by inserting the port puncture needle through its membrane under sterile conditions.

[0257] In some examples and embodiments, the therapeutic solution of OT-101 can be continuously infused at a flow rate of 4 μl / min or 2-8 μl / min.

[0258] In some examples and embodiments, a therapeutically effective amount of an antisense (such as OT-101) formulation can be continuously infused at a flow rate of 0.5-20 μl / min, or 1-20 μl / min, or 2-10 μl / min, or 2-8 μl / min, or 1 μl / min, or 2 μl / min, or 3 μl / min, or 4 μl / min, or 5 μl / min, or 6 μl / min, or 7 μl / min, or 8 μl / min, and the agent concentration can be 1-100 μM, or 1-80 μM, or 1-50 μM, or 1-20 μM, or 1-10 μM, or 1 μM, or 2 μM, or 3 μM, or 4 μM, or 5 μM, or 6 μM. Such administration can occur in a subject on days 1 to 3, or days 1 to 7, or days 1 to 14, or days 1 to 21, or days 1 to 50 of a regimen or cycle.

[0259] Methods, Compositions, and Combinations for CNS Disorders The devices of the present invention can be used to treat or ameliorate the symptoms of CNS diseases, such as CNS cancer, in human subjects or animals in need thereof. The devices of the present invention can contain pharmaceutical compositions for continuous infusion into the brain or spinal cord.

[0260] The pharmaceutical composition for continuous infusion can include an agent that inhibits or suppresses the expression of TGF-β and administers a therapeutically effective amount of the composition to a subject. The present invention provides therapeutic methods for treating CNS disorders, such as cancer, or ameliorating the symptoms of CNS disorders.

[0261] Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, artemisinin, pharmaceutically acceptable salt forms, esters, polymorphs, or stereoisomers thereof, and combinations thereof.

[0262] In some aspects, the present invention provides kits and devices containing compositions of agents for inhibiting or suppressing the expression of TGF-β for treating or ameliorating symptoms of CNS disorders in human subjects or animals.

[0263] In a further aspect, the present invention provides the use of a composition of agents for inhibiting or suppressing expression of TGF-β in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0264] As used herein, "intraventricular" infusion can be used for continuous infusion of pharmaceutical compositions to treat CNS diseases, including cancer. An example of intraventricular administration is an Ommaya-like device with an entry catheter.

[0265] As used herein, the term intracranial includes intrathecal and intraventricular. For example, intracranial injection includes intrathecal and intraventricular injection. Furthermore, the terms "intrathecal and intraventricular" are intended to include "intracranial."

[0266] In certain embodiments, the agents, uses, or methods of the present invention, when administered to a subject, can reduce the level of TGF-β2 in the subject, which can be referred to as improving the TGF-β2 signature.

[0267] In some aspects, the agent, use or method of the invention can reduce mortality at 6, 12, 18, 24, 30 or 36 months upon administration or use by a subject.

[0268] In a further aspect, the agent, use, or method of the present invention, when administered or used by a subject, can improve survival at 6, 12, 18, 24, 30, or 36 months. Survival can be determined by overall survival or progression-free survival.

[0269] In some aspects, an agent, medicament, or administration that is substantially free of excipients can include a carrier.

[0270] Embodiments of the present invention further contemplate the use of TGF-β2 as a selective biomarker for providing improved outcomes for cancer therapy using the agents of the present invention in combination with radiation therapy.

[0271] The present invention provides methods for treatment by selecting patients based on TGF-β2 levels as a biomarker for improved radiation therapy outcomes in cancer.

[0272] Surprisingly improved overall survival and survival after radiation therapy were found in patients with low TGF-β2 expression compared with patients with high TGF-β2 expression across a wide range of expression levels. No such differences were observed for TGF-β1 and TGF-β3. Thus, decreased TGF-β2 can be used as a successful biomarker to select patients who are likely to benefit from such therapy.

[0273] In a further study, overall survival in pediatric gliomas treated with radiation therapy was significantly improved when TGF-β2 was used as a selector; TGF-β1 and TGF-β3 did not predict survival.

[0274] In some embodiments, TGF-β2 levels can be used as a surprisingly effective biomarker to select patients who are likely to benefit from cancer radiotherapy in combination with chemotherapy, such as temozolomide (TMZ), and / or TMZ plus radiotherapy, and / or anti-angiogenic therapy, such as bevacizumab. Such predictive results were not observed for TGF-β1 and TGF-β3.

[0275] In some embodiments, the present invention provides agents, uses, and methods that combine inhibiting or suppressing the expression of TGF-β2 with a pharmaceutical agent that is a targeted anti-cancer agent, a cancer growth blocker, or an EGFR inhibitor to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

[0276] In certain embodiments, the present invention provides agents, uses, and methods that combine inhibiting or suppressing the expression of TGF-β2 with bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof to treat or ameliorate symptoms of CNS diseases in human subjects or animals.

[0277] In additional aspects, the present invention provides agents, uses, and methods that combine inhibiting or suppressing expression of TGF-β2 with erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof to treat or ameliorate symptoms of CNS disorders in human subjects or animals.

[0278] In a further aspect, the present invention provides agents, uses, and methods that combine inhibiting or suppressing the expression of TGF-β2 with temozolomide to treat or ameliorate symptoms of CNS diseases in human subjects or animals.

[0279] Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides and can be complementary to mRNA targets. Antisense therapy can downregulate molecular targets by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes and significantly reduces the translation of target genes. Other ASO mechanisms include inhibiting 5' cap formation, altering splicing processes such as splice switching, and steric hindrance of ribosomal activity.

[0280] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating catalytic degradation of target mRNA or by binding to the site on mRNA that is required for translation.Antisense oligonucleotides can be designed to target the RNA genome of viruses or viral transcription products.Antisense oligonucleotides can provide an approach to identify potential targets and therefore represent potential therapeutic agents.

[0281] Antisense oligonucleotides are small, synthetic fragments of single-stranded DNA that can be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization. Once bound to the target RNA, they can inhibit the translation process either by inducing cleavage mechanisms or by inhibiting mRNA maturation. ASOs can selectively inhibit gene expression with specificity. Chemical modifications of DNA or RNA can be used to enhance stability.

[0282] For example, modifications can be introduced into the phosphodiester bond, sugar ring, and backbone. ASO antiviral agents can block the translation process either by (i) cleavage of mRNA mediated by ribonuclease H (RNAse H) or RNase P, or (ii) steric (non-binding) blocking of enzymes involved in the translation of target genes. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide (OT-101; AP 12009; travedersen), hereafter referred to as OT-101 or AP 12009, is intended to reduce the level of TGF-β2 protein in malignant gliomas, thereby slowing disease progression.

[0283] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' linkages are modified to phosphorothioate. The molecular formula is C 177 H 208 N 60 Na 17 O 94 P 17 S 17 and has a molecular weight of 6,143 g / mol. OT-101 was designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression.

[0284] OT-101 is currently supplied as a lyophilized powder in 50 mL glass vials in three different aliquots. The OT-101 lyophilized powder is dissolved in isotonic (0.9%) aqueous sodium chloride solution prior to use.

[0285] Examples of agents of the present disclosure for inhibiting or suppressing the expression of TGF-β include antisense oligonucleotides specific for TGF-β1, TGF-β2, or TGF-β3.

[0286] Examples of agents of the present disclosure for inhibiting or suppressing the expression of TGF-β2 include the TGF-β2-specific antisense oligonucleotides shown in SEQ ID NOs: 1 to 136 in Table 2, including SEQ ID NO: 8, cggcatgtct attttgta (OT-101).

[0287] The antisense oligonucleotides shown in Table 2 herein can be chemically modified as is known in the art.

[0288] Examples of the agents of the present disclosure for inhibiting or suppressing the expression of TGF-β include artemisinin extracts, its pharmaceutically acceptable salts, salt polymorphs, esters, or isomers, and any combination thereof. In some embodiments, the present disclosure includes substantially pure artemisinin having a purity of at least 60%, 70%, 80%, 90%, or 95%.

[0289] In certain embodiments, the agent of the present disclosure for inhibiting or suppressing the expression of TGF-β can be prepared from a lyophilized powder of the agent.

[0290] In some examples and embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, and may be administered or used by continuous intracerebroventricular or intrathecal or intracerebral administration at a dose of 4 μl / min, at a dose level of 10 μM on days 1-7, or at a dose of 20 μM on days 1-7, or at a dose of 40 μM on days 1-7, or at a dose of 80 μM on days 1-7.

[0291] In some examples and embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and may be administered or used by continuous intracerebroventricular or intrathecal or intracerebral administration at a dose of 4 μl / min or 2-8 μl / min at a dose level of 2 μM on days 1-7, or at a dose of 4 μM on days 1-7, or at a dose of 8 μM on days 1-7, or at a dose of 10 μM on days 1-7.

[0292] In some embodiments, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, as well as chemically modified variants thereof, and may be administered as a bolus injection into an omma-like reservoir at a concentration of 61.43 mg / ml (10 μM), 1 mg / ml, 7.35 mg / ml, 15 mg / ml, or 18.23 mg / ml.

[0293] In a further embodiment, the agent may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136, and chemically modified variants thereof, administered or used by continuous infusion, either alone or in combination with any form of artemisinin, at a dose of 500 mg per day given orally on days 1-5.

[0294] Examples of agents of the present disclosure for inhibiting TGF-β include agents for specifically inhibiting TGF-β1, TGF-β2, or TGF-β3, preferably TGF-β2.

[0295] Embodiments of the invention that involve the administration or use of compositions of agents can ameliorate or suppress symptoms caused by TGF-β2-induced proteins.

[0296] The agent for inhibiting or suppressing TGF-β expression may be an artemisinin formulation containing a 90-95% pure artemisinin extract or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, and one or more pharmaceutically acceptable excipients. The excipients may include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants, and anti-caking agents. In some embodiments, the excipients may include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, croscarmellose sodium, and magnesium stearate.

[0297] In a further embodiment, the agent for inhibiting or suppressing the expression of TGF-β can be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0298] As used herein, derivative encompasses chemical modifications that provide structural analogs of a compound. For example, substitution or replacement of an alkyl group can provide a structural analog.

[0299] Embodiments of the present invention include processes or uses in which the agent for inhibiting or suppressing expression of TGF-β is a compound, or a ligand comprising a small molecule or polypeptide, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, that interacts with site I of TGF-β comprising Trp30 and / or site II of TGF-β comprising Arg15, Gln19, and Phe8.

[0300] In some embodiments, the agent for inhibiting or suppressing expression of TGF-β can be a polypeptide or peptidomimetic of site I of TGF-β comprising residues Phe24 to Lys37 and / or site II of TGF-β comprising residues Cys7 to Gln19, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0301] In a further aspect, the agent for inhibiting or suppressing expression of TGF-β can be a humanized or non-humanized antibody or antibody fragment having affinity for site I of TGF-β, which includes residues Phe24 to Lys37, and / or site II of TGF-β, which includes residues Cys7 to Gln19.

[0302] Embodiments of the present invention further include pharmaceutical compositions for inhibiting or suppressing TGF-β expression or treating or ameliorating symptoms of CNS disorders in humans or animals. The pharmaceutical compositions can include a TGF-β inhibitor, artemisinin, a pharmaceutically acceptable salt form, ester, polymorph, or stereoisomer thereof, or any combination thereof, and a carrier. The TGF-β inhibitor can be selected from TGF-β2-specific antisense oligonucleotides SEQ ID NOs: 1-136 and chemically modified variants thereof. The carrier can be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0303] Importantly, the composition of the present disclosure may be substantially free of excipients.The composition of the present disclosure that is substantially free of excipients has been found to be surprisingly stable in a carrier.In some embodiments, the composition may be stable in a carrier at 37°C for at least 14 days, or at least 21 days, or at least 28 days.

[0304] In additional embodiments, pharmaceutical compositions for injection can contain less than 1% by weight of excipients, or less than 0.5% by weight of excipients, or less than 0.1% by weight of excipients.

[0305] Embodiments of the present invention further contemplate treatment modalities in which the compositions of the present invention are administered or utilized in combination with standard care therapy for a disease. Examples of additional pharmaceuticals that may be administered or utilized in combination with the compositions of the present invention include anti-inflammatory agents, anti-inflammatory steroids, piperiquine, pyronaridine, curcumin, frankincense, remdesivir, Sonpraz D, Zifi CV / Zac D, CCM, Broclear, Budamet, Rapitas, Montec LC, low molecular weight heparin, prednisolone, paracetamol, vitamin B complex, vitamin C, pantoprozole, doxycycline, ivermectin, zinc, Folacort Rotacap inhalation, ceftriaxone injection, paracetamol tablets, Fragmin injection, Covifor tablets, azithromycin, dexamethasone injection, ondansetron injection, multivitamin tablets, ascorbic acid tablets, calcium carbonate tablets, and zinc sulfate tablets.

[0306] The present invention further provides a kit comprising a lyophilized powder of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in a vial, each containing 3.75 mg.

[0307] The present invention also provides a kit containing, in a vial, a 500 mg lyophilized powder of artemisinin or a derivative thereof, or a ligand comprising a compound, small molecule, or polypeptide that interacts with site II of TGF-β comprising Arg15, Gln19, and Phe8, a sesquiterpene lactone or a derivative thereof, or a compound comprising three isoprene groups and one lactone ring and its derivative, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof, or any combination of the foregoing.

[0308] Delivery of therapeutic agents by injection Delivery of OT-101-TGF-β antisense for the treatment of glioblastoma OT-101 is a TGF-β2 antisense antibody that was active against recurrent glioblastoma in a phase 2 clinical trial, G004 (Uckun FM, Qazi S, Hwang L, Trieu VN. Recurrent or refractory high-grade gliomas treated by convection enhanced delivery of a TGF-β2 targeting RNA therapeutic: a post-hoc analysis with long-term follow-up. Cancers. 2019, 11:1892).

[0309] OT-101 was delivered intratumorally through a catheter implanted in the cerebrum. To further expand the application of OT-101, we explored intrathecal delivery of tritium-labeled OT-101 to Sprague-Dawley CD (albino) rats. No gender differences were observed throughout the study.

[0310] Surprisingly, after 1 hour of intracerebral or intracerebroventricular infusion in rats, OT-101 was equally widespread in the cerebellum, the remaining cerebrum, and the cerebrospinal fluid (CSF), with intracerebral administration being similar to intracerebroventricular administration, indicating access to the entire CNS compartment via the CSF.

[0311] OT-101 concentrations remained stable for the first 4 hours after injection and then decayed biexponentially, with a slow terminal half-life in tissues but not in CSF, suggesting rapid penetration away from the CSF compartment into underlying tissues. Minimal amounts of OT-101 were detected in the plasma compartment. Intrathecal bolus administration of 0.1 mL of OT-101 at 14, 30, 200, 300, and 500 μM to cynomolgus monkeys did not result in any single-dose toxicity. Histopathological examination revealed no substance-related histomorphological lesions in the lumbar subarachnoid space. No changes were observed in the gray and white matter of the spinal cord or in nerve trunks, and neurons did not exhibit any abnormalities. These data suggest that intrathecal administration of OT-101 is a potentially effective delivery route for antisense therapeutics, such as OT-101, to the midline, i.e., diffuse midline glioma (DMG).

[0312] Targeting transforming growth factor beta 2 (TGF-β2) with OT-101 for post-radiation consolidation in diffuse intrinsic pontine glioma Diffuse intrinsic pontine glioma (DIPG) in children has a poor prognosis, with a median overall survival (OS) of 10 months and a 2-year overall survival rate of <10% after standard radiotherapy.

[0313] Chemotherapy does indeed provide clinically meaningful benefits. Therefore, therapeutic innovations for the treatment of pediatric DIPG are urgently needed. High-grade glioma cells, including pediatric glioblastoma and DIPG cells, have been shown to produce transforming growth factor beta 2 (TGF-β2).

[0314] TGF-B2 (TGFB2) is implicated as both a glioma cell promoter and a key contributor to T cell hyporesponsiveness in the tumor microenvironment (TME) toward glioma cells. OT-101 is a first-in-class RNA therapeutic designed to abrogate the immunosuppressive and tumor-promoting effects of TGF-B2. At low micromolar concentrations, OT-101 reduces TGFB2 secretion by human glioma cells, blocks their proliferation and migration, and restores the anti-glioma cytolytic function of patient-derived T cells. Intracerebrally administered OT-101 has shown promising single-agent activity in recurrent / refractory (R / R) high-grade glioma (HGG) (Uckun et al., Cancers. 2019 28;11(12):1892). Intrathecal administration of antineoplastic agents directly into the CSF allows bypassing the selective filter of the blood-brain barrier (BBB), achieving significant concentrations of antineoplastic agents in the CSF while reducing the potential for systemic toxicity. Based on favorable safety pharmacology studies of intrathecally delivered OT-101 in rabbits and primates, and encouraged by its single-agent activity in adult patients with HGG, pediatric patients with DIPG will be treated with OT-101. Multiple doses of OT-101 will be administered as intrathecal bolus injections after completion of radiation therapy. The study was designed to determine: 1) the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of OT-101, and 2) its efficacy in children with DIPG.

[0315] Numbered aspects of the invention may include the following.

[0316] 1) Agents for inhibiting or suppressing the expression of TGF-β2 to treat or ameliorate symptoms of CNS disorders in human subjects or animals.

[0317] 2) Use of an agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0318] 3) A method for treating or ameliorating symptoms of a CNS disorder in a human subject or animal in need thereof, comprising: preparing a composition comprising an agent for inhibiting or suppressing the expression of TGF-β2 in a carrier; and administering a therapeutically effective amount of said composition to said subject. A method comprising:

[0319] 4) The agent, use, or method of any one of aspects 1 to 3, wherein the CNS disease is glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cord cancer, or CNS tumor.

[0320] 5) The agent, use, or method of any of aspects 1 to 4 in combination with a medicament comprising a targeted anti-cancer agent, a cancer growth blocking agent, an EGFR inhibitor, or a combination thereof.

[0321] 6) The agent, use, or method of any of aspects 1 to 5 in combination with a pharmaceutical agent selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

[0322] 7) The agent, use, or method of any of aspects 1 to 6 in combination with a pharmaceutical agent that is a cancer growth inhibitor selected from an angiogenesis inhibitor, a histone deacetylase inhibitor, a hedgehog blocker, an mTOR inhibitor, a p53 inhibitor, a PARP inhibitor, a proteasome inhibitor, a tyrosine kinase inhibitor, and combinations thereof.

[0323] 8) The agent, use, or method of any of aspects 1 to 7 in combination with a pharmaceutical agent that is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

[0324] 9) The agent, use, or method of any of aspects 1-8 in combination with temozolomide.

[0325] 10) The agent, use, or method of any of aspects 1 to 9 in combination with treatment of a CNS disease with radiation therapy or electric field therapy.

[0326] 11) The agent, use, or method of any of aspects 1 to 10, wherein the administration or use of said composition or agent is in combination with a standard treatment for said CNS disease.

[0327] 12) The agent, use, or method of any one of aspects 1 to 11, wherein the agents, medicaments, therapies, treatments, and administrations are administered in parallel, simultaneously, sequentially, or temporally separately, respectively.

[0328] 13) The agent, use, or method of any of aspects 1 to 12, wherein each agent and medicament is administered by infusion or injection, separately or in combination.

[0329] 14) The agent, use, or method of any of aspects 1 to 13, comprising administration or use by intracranial continuous infusion or bolus administration.

[0330] 15) The agent, use, or method of any one of aspects 1 to 14, wherein the continuous intracranial infusion comprises infusion using an Ommaya-like reservoir having a partially flexible top.

[0331] 16) The agent, use, or method of any one of aspects 1 to 15, wherein the continuous intracranial infusion comprises a single entry catheter placed into the target region of the brain.

[0332] 17) The agent, use, or method of any one of aspects 1 to 16, wherein the subject has an improved TGF-β2 signature due to the administration or use.

[0333] 18) The agent, use, or method of any one of aspects 1 to 17, wherein said administration or use reduces mortality at 6, 12, 18, 24, 30, or 36 months.

[0334] 19) The agent, use, or method of any one of aspects 1 to 18, wherein said administration or use improves survival at 6, 12, 18, 24, 30, or 36 months.

[0335] 20) The agent for inhibiting or suppressing the expression of TGF-β2 is the following TGF-β2-specific antisense oligonucleotide complementary to the TGF-β2 transcript: The agent, use, or method of any of embodiments 1-19, wherein the antibody is selected from SEQ ID NOs: 1-136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0336] 21) The agent, use, or method of any one of aspects 1 to 20, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide having one or two or fewer mismatches compared to the target human TGF-β2.

[0337] 22) The agent, use, or method of any one of aspects 1 to 21, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0338] 23) The agent, use, or method of any of aspects 1 to 22, wherein the agent for inhibiting or suppressing expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces any TGF-β1 transcript levels and any TGF-β3 transcript levels by less than 10%, or less than 5%, or less than 1%.

[0339] 24) The agent, use, or method of any of aspects 1-23, comprising a TGF-β2-specific antisense oligonucleotide having one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

[0340] 25) The agent, use, or method of any of aspects 1-24, wherein the agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

[0341] 26) The agent, use, or method of any one of aspects 1 to 25, comprising a carrier that is sterile water for injection, saline, isotonic saline, phosphate buffered saline, or a combination thereof.

[0342] 27) The agent, use, or method of any one of aspects 1 to 26, wherein the agent, medicament, or administration is substantially free of excipients.

[0343] 28) The agent, use, or method of any of aspects 1-27, wherein the agent, medicament, or administration is stable in a carrier at 37°C for at least 14 days while pumped via intracranial continuous infusion, or the concentration of the antisense active agent decreases by less than 10% after 90 days of use.

[0344] 29) The agent, use, or method of any of embodiments 1-28, wherein the method comprises administering the composition by intracranial infusion at a rate of 2-8 μl / min and an agent concentration of 1-80 μM on days 1-7, preferably for continuous intracranial infusion.

[0345] 30) A composition comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1 to 136 in Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and Ommaya-like reservoir with partially flexible top Includes a kit.

[0346] 31) A device for delivering a fluid pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir containing the pharmaceutical composition; a pump

[0101] for pumping the pharmaceutical composition into an Ommaya reservoir

[0111] through an infusion tube

[0103] , the pump

[0101] being in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube

[0115] ; a filter collinear with the injection tube; and an entry catheter in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain; Including, the device.

[0347] 32) A device as described in aspect 31, wherein the entry catheter

[0113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

[0348] 33) A device for delivering a fluid pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir containing the pharmaceutical composition; a pump

[0101] for pumping the pharmaceutical composition through an infusion tube

[0103] into an access port

[0107] , the pump being in fluid communication with an Ommaya reservoir

[0111] , the reservoir being in fluid communication with the pump through a reservoir tube

[0115] ; a filter collinear with the injection tube; an indwelling tube

[0109] in fluid communication with the access port and the Onmaya reservoir

[0111] ; and an entry catheter in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain; Including, the device.

[0349] 34) The device of any of aspects 31 to 33, wherein the entry catheter

[0113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

[0350] 35) The device of any of aspects 31-34, wherein the Ommaya reservoir

[0111] comprises a partially flexible top.

[0351] 36) The device of any of aspects 31-35, wherein the device provides continuous infusion of a therapeutically effective amount of the fluid pharmaceutical composition into the target area.

[0352] 37) The device of any one of aspects 31-36, wherein the distal end of the entry catheter enters the target region of the brain.

[0353] 38) The device of any of aspects 31-37, wherein the Ommaya reservoir

[0111] holds the pharmaceutical composition behind a membrane for a period of time for sustained release of the pharmaceutical composition into the entry catheter.

[0354] 39) The device of any one of aspects 31 to 38, wherein the distal end of the entry catheter that enters the brain has a step-down end, a recessed step end, a multi-port end, a micro-hole end, or a balloon-tipped end.

[0355] 40) The device of any one of aspects 31 to 39, wherein the pump

[0101] is a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or other pump with similar specifications.

[0356] 41) The device of any of aspects 31-40, wherein the infusion rate of the fluid pharmaceutical composition is 0.01 to 3000 ml / hour, or 0.01 to 100 ml / hour, or 0.01 to 2 ml / hour, or 0.01 to 1 ml / hour, or 0.05 to 0.5 ml / hour.

[0357] 42) The device of any one of aspects 31 to 41, wherein the infusion tube

[0103] or the indwelling tube

[0109] is a PEGA Line 100 SF 100 cm with a 0.2 μm sterile filter, or a 200 cm infusion line with a 0.2 μm sterile filter, or a Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tube with similar specifications.

[0358] 43) The device of any of aspects 31-42, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing expression of TGF-β, which can be used to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

[0359] 44) The device of any of aspects 31-43, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of an agent, drug, or delivery vehicle.

[0360] 45) The device of any one of aspects 31 to 44, wherein the fluid pharmaceutical composition is for treating a CNS disease or CNS cancer.

[0361] 46) The device of any of aspects 31-45, wherein the fluid pharmaceutical composition is for treating glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cord cancer, or CNS tumor.

[0362] 47) The fluid pharmaceutical composition comprises a TGF-β2-specific antisense oligonucleotide complementary to the TGF-β2 transcript: SEQ ID NOs: 1-136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof. The device of any of embodiments 31 to 46, comprising an agent for inhibiting or suppressing expression of TGF-β2 selected from the group consisting of:

[0363] 48) The device of any of aspects 31-47, which operates in combination with radiation therapy or electric field therapy.

[0364] 49) A device for delivering a pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir

[0402] containing the pharmaceutical composition, comprising a hard shell

[0403] , a flexible top

[0401] , and a non-flexible mounting plate

[0405] ; a port in fluid communication with the reservoir; and an entry catheter in fluid communication with the reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain; Including, the device.

[0365] 50) A device for delivering a pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir

[0502] containing the pharmaceutical composition, comprising an upper rigid shell

[0503] , a lower rigid shell

[0504] , a flexible top

[0501] , and a non-flexible mounting plate

[0505] ; a port in fluid communication with the reservoir for attaching an infusion line; and a port in fluid communication with the reservoir for attaching an entry catheter. Including, the device.

[0366] 51) A device for delivering a pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir

[0602] containing the pharmaceutical composition, comprising an upper rigid shell

[0603] , a flexible top

[0601] , and a non-flexible mounting plate

[0605] ; a port in fluid communication with the reservoir for attaching an infusion line; and a port in fluid communication with the reservoir for attaching an entry catheter. Including, the device.

[0367] 52) A collar for a device for delivering a pharmaceutical composition by intracranial infusion, comprising: a hard shell having an opening exposing the flexible top of the device; Including color.

[0368] 53) A method for administering a pharmaceutical composition by continuous intracranial infusion, comprising: Mounting the device of any of aspects 31-52 on a patient; and pumping the pharmaceutical composition within the device to provide continuous intracranial infusion to the patient. A method comprising:

[0369] 54) The method of embodiment 53, wherein the device is mounted and the entry catheter is placed in the brain without concurrent imaging of the head or brain.

[0370] 55) The method of embodiments 53 to 54, wherein the device is mounted and a single entry catheter is placed in the brain.

[0371] 56) A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, comprising: a reservoir containing the pharmaceutical composition; pump; Ommaya reservoir with partially flexible top; an inlet tube for connecting the reservoir to the pump and the pump to the Ommaya reservoir; filters; and Entry catheter Includes a kit.

[0372] 57) The kit of embodiments 30 and 56, wherein the entry catheter is substantially straight for intraventricular entry into the target region of the brain.

[0373] 58) The kit of any one of aspects 30 and 56 to 57, wherein the entry catheter is non-linear and has a bend for intraventricular entry into the target region of the brain.

[0374] 59) The kit of any of aspects 30 and 56-58, wherein the infusion tube is outside the subject.

[0375] 60) The kit of any of aspects 30 and 56-59, wherein a portion of the infusion tubing connecting the pump to the Ommaya reservoir is placed indwelling in the subject.

[0376] All publications, including patents, published patent applications, and non-patent publications, and the sequence listing, referred to in this description are each expressly incorporated herein by reference in their entirety for all purposes.

[0377] Although the foregoing disclosure has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications are included in this disclosure and can be made without undue experimentation within the scope of the appended claims, which are presented by way of example and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the features, materials, elements, or limitations of the various exemplary components, examples, and claimed embodiments.

[0378] It is emphasized herein that, according to common practice, the features of the drawings are of any scale and are intended to cover similar features which may be arbitrarily enlarged or reduced. [Example]

[0379] Example 1 - Examples of pumps for continuous infusion via intrathecal or intraventricular routes are shown in Table 3.

[0380] Table 3: Examples of pumps for continuous infusion TIFF2025538189000007.tif67157

[0381] The infusion or indwelling tube can be PEGA Line 100 SF 100cm with a 0.2um sterile filter, or a 200cm infusion line with a 0.2um sterile filter, or a Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tube with similar specifications.

[0382] In some aspects, the brain entry catheter placed in the ventricular space can be a non-specific ventricular catheter. The ventricular catheter can have an inner diameter of 1.0 to 2.0 mm. For example, the ventricular catheter can have an inner diameter of 1.4 mm and an outer diameter of 2.7 mm. The catheter can be 14 cm or less in length and can have 24 inlet holes (e.g., 3 rows of 8 holes) at the proximal end. Generally, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The omma-like reservoir of the present invention is 300 to 400 mm. 2 and the area of ​​the inner opening of the catheter is 0.785 mm 2 ~3.14mm 2 Therefore, the ratio of the reservoir surface area to the catheter surface area may range from 96 to 509. This ratio can be used to adjust the expected release time of the drug solution over a wide range, depending on the density of the test solution relative to water.

[0383] The Ommaya-like device of the present system allows for the delivery of drugs directly to the CSF and is particularly useful for delivering oligonucleotide and antisense oligonucleotide drugs, such as OT-101, either alone or in combination with other cancer therapies.

[0384] In some aspects, the drug load can surprisingly be delivered as a single bolus injection or as a short infusion over 15, 30, or 60 minutes, yet achieve sustained delivery through advantageous ratios of reservoir volume to catheter opening and / or tailored ratios of CSF density to drug solution density.

[0385] Example 2 - An example of the use of the device for continuous infusion via intrathecal or intraventricular routes is shown in Tables 4 and 5. The formulation in this example provided effective use of the agent because the formulation was stable enough to be delivered to patients by continuous administration. The formulation in this example contained minimal to no excipients and was stable for at least 14 days without fouling. The formulation of the present invention was superior to conventional formulations that use components such as LNP particles and excipients such as lactose. The formulation of the present invention exhibited improved stability and reduced bacterial growth compared to such conventional formulations.

[0386] Table 4. OT-101 and device compatibility studies TIFF2025538189000008.tif140166

[0387] Table 5. In-use stability studies performed TIFF2025538189000009.tif54166

[0388] The following studies A)-C) were conducted as in-use tests under conditions mimicking clinical use for AP 12009 (OT-101)-P002. Treatments follow a pump and reservoir setup similar to that of OT-101-P002. A drug delivery system suitable for this application consists of the following medical device components: Portable pump with corresponding drug reservoir and extension line Gripper needle Implantable port system with venous catheter

[0389] Three different drug delivery systems were subjected to in-use testing, with the components intended to be implanted for clinical use incubated at 37° C. and the non-implanted components maintained at ambient temperature. The three drug delivery systems tested for in-use stability of OT-101 drug solution are described below in Tables 6, 7, and 8.

[0390] Table 6: Drug Delivery System 1 TIFF2025538189000010.tif38167

[0391] Table 7: Drug Delivery System 2 TIFF2025538189000011.tif42167 * Not available

[0392] Table 8: Drug Delivery System 3 TIFF2025538189000012.tif57167 * Not available

[0393] The conditions for this study are as follows: Concentration of drug solution: 15 mg / mL (195 mg / mL) 2 / day average dosage and 1.85m 2 (calculated based on the average body surface of Flow rate: 1 mL / hour (equivalent to 24 mL / day) Storage of the pump (including the drug reservoir) and non-implanted parts of the drug delivery system at ambient temperature Storage of the implanted portion of the drug delivery system at 37°C Drug reservoir content: 120mL Test duration: 5 days

[0394] Study A: The delivered drug solution was sampled once daily and analyzed by two validated stability-indicating HPLC methods (ion exchange and reverse-phase HPLC). The results are summarized in Tables 9 and 10. The impurity profile of the pumped solution is considered adequate if it meets the release specifications for AP 12009 (OT-101) 250 mg drug product. All samples meet the release specifications for AP 12009 (OT-101) 250 mg drug product with respect to HPLC impurity profile. No relevant effects on the drug solution composition were observed after passage through the different drug delivery systems.

[0395] Table 9. Ion-exchange HPLC analysis TIFF2025538189000013.tif98167*) PO = impurity in which one phosphorothioate moiety is replaced by a phosphate moiety (coeluting with 3'N-2) 3'N-2 = impurity lacking two 3' terminal nucleotides (coeluting with PO) N-1 = impurity lacking the 3' or 5' terminal nucleotide nd = not detected

[0396] Table 10. Reverse-phase HPLC analysis TIFF2025538189000014.tif90170 *) PO = impurity in which one phosphorothioate moiety is replaced by a phosphate moiety (coeluting with 5'N-1) CNET = an impurity in which a cyanoethyl moiety has been added to one of the thymidine nucleotides 3'N-2 = impurity lacking the two 3' terminal nucleotides 3'N-1 = impurity lacking the 3' terminal nucleotide 5'N-1 = impurity lacking the 5' terminal nucleotide (coeluting with PO) nd = not detected

[0397] Study B: The delivered drug solution was sampled once daily and analyzed by UV spectroscopy. The acceptance criteria were: UV spectroscopy of the reference sample 260nm The absorbance was set to 90%–110% of the initial sample concentration (reference t = day 0). Furthermore, for all samples, UV spectra were taken in the range of 230–320 nm and compared with the spectrum of the reference sample.

[0398] The results are listed in Table 11, and the results show that all sample concentrations were within the range of 100.0%-102.7% of the reference solution and met the acceptance criteria of 90-100%.

[0399] (Table 11) UV analysis TIFF2025538189000015.tif88157

[0400] Study C: The delivered drug solution was sampled at the end of point-of-use testing. Leachable / extractable profiles were determined by a contract laboratory using gas chromatography-based analysis established for chemical analysis of medical devices according to ISO 10993 (i.e., quantification of emitted organic compounds using a flame ionization detector (GC-FID) and identification of potential leachables / extractables using gas chromatography-mass spectrometry (GC-MS) and comparison to the NIST / EPA / NIH 2005 Mass Spectral Library, if applicable). Samples along with a reference sample (day t-0) were sent to the contract laboratory, Medical Device Services. Due to incompatibility between saline and gas chromatography, samples were extracted with tert-butyl ether, and these extracts were then subjected to gas chromatography. No leachables / extractables were detected according to MDS report #111523-20.

[0401] Study D: Clinical Study AP 12009-P002 was conducted at 140 mg / m 2 / day and 250 mg / m 2 Two dosages are used: 1.75 ml / day. Representative drug solution concentrations used in this study are 1.75 ml / day as shown in Table 12. 2 of body surface and 250 mg / m 2 Calculations were based on an assumed daily dosage.

[0402] Table 12. Drug parameters TIFF2025538189000016.tif15128

[0403] This representative drug solution was prepared in duplicate and loaded under sterile conditions into two separate Cadd drug cassette reservoirs. The Cadd drug cassette reservoirs were stored in an incubation chamber (non-sterile setting) at a temperature of 20°C to 25°C. The temperature of this incubation chamber was continuously monitored.

[0404] After 7 days of storage, the Cadd extension set infusion line was connected under sterile conditions to the luer lock connector of the reservoir bag. 20 mL of the incubated drug solution was drawn through the extension set (containing a 0.22 μm sterile filter) into a separate sterility test apparatus. An additional 20 mL of the incubated drug solution was drawn via cannula directly from the reservoir bag into a separate sterility test apparatus. The sterility test results are shown in Table 13.

[0405] Table 13: Sterility test results TIFF2025538189000017.tif50128

[0406] The results of this study demonstrate that the Cadd drug cassette reservoir ensures sterility of the sterile filled drug solution for at least 7 days.

[0407] Dosing solution at a concentration of 10 μM (61.43 μg / mL).

[0408] The compatibility of the drug delivery system with a 10 μM OT-101 drug solution for clinical study AP 12009(OT-101)-G005 was demonstrated by examining the integrity of the drug solution after passing the drug delivery system under conditions that mimic its clinical use. The integrity of the drug solution was evaluated based on the following parameters: impurity profile, concentration, sterility, and leachable / extractable profile.

[0409] The compatibility of the OT-101 drug solution with the intended drug delivery system was demonstrated by the following studies: a) Determination of the impurity profile of OT-101 drug solution after passing through the drug delivery system b) Verification of the concentration of the drug solution after passing through the drug delivery system c) Determination of the leachability / extractability profile of the OT-101 drug solution after passage through the drug delivery system d) Sterility testing of the drug solution after incubation in the drug reservoir

[0410] A drug delivery system suitable for this application consists of the following medical device components: Portable pump with corresponding drug reservoir and extension line Gripper needle Implantable port system with venous catheter

[0411] The drug delivery systems were subjected to in-use testing, with components intended to be implanted for clinical use being incubated at 37°C and non-implanted components being maintained at ambient temperature.

[0412] The drug delivery systems tested for in-use stability of OT-101 drug solutions are listed below in Table 14.

[0413] Table 14. Drug delivery systems TIFF2025538189000018.tif56166

[0414] Studies a)-c) were conducted as in-use tests under conditions that mimic clinical use. These conditions were as follows: Drug solution concentration: 10 μM (61.43 μg / mL) OT-101 in isotonic saline Flow rate: 4 μL / min (equivalent to 5.76 mL / day) Storage of the pump (including the drug reservoir) and non-implanted parts of the drug delivery system at ambient temperature Storage of the implanted portion of the drug delivery system at 37°C Drug reservoir content: 50mL These studies were limited to a maximum duration of approximately 8.5 days due to the 50 mL drug reservoir volume and 5.76 mL per day flow rate.

[0415] Study a: The delivered drug solution was sampled once daily and analyzed by two validated stability-indicating HPLC methods (ion-exchange and reverse-phase HPLC). Acceptance criteria were set at twice the impurity area % for each impurity in Tox-Batch (J982-15K9FP). Additionally, a warning limit was set based on the release specifications for OT-101 (AP 12009) 7.37 mg drug product. Based on the impurity data shown in Table 15, the results demonstrate that only minor degradation was observed during the in-use period (under clinically relevant conditions) and that all samples met the drug product release specifications. The main trend observed was a slight increase in the PO impurity. This is consistent with the forced degradation results, which identified PO as the most significant degradation product. PO is an impurity in which one of the 17 phosphorothioate moieties has been oxidized to a phosphate diester moiety. This impurity is discussed as a related product rather than an impurity (in terms of inactive degradation products).

[0416] Table 15. Results of impurity profile testing of drug solutions after passing through the drug delivery system TIFF2025538189000019.tif76170 *) PO = impurity in which one phosphorothioate moiety is replaced by a phosphate moiety CNET = an impurity in which a cyanoethyl moiety has been added to one of the thymidine nucleotides 3'N-2 = impurity lacking the two 3' terminal nucleotides 3'N-1 = impurity lacking the 3' terminal nucleotide 5'N-1 = impurity lacking the 5' terminal nucleotide (coeluting with PO)

[0417] Study b: The delivered drug solution was sampled once daily and analyzed by UV spectroscopy. The acceptance criteria were set at 90%-110% of the initial sample concentration (reference t = day 0). This test was performed in duplicate. The results are listed in Table 16 and show that all sample concentrations were within 98%-102% of the reference. These results demonstrate that the concentration of the drug solution was not affected during its passage through the drug delivery system (e.g., there was no adsorption of the drug onto the polymer surface of the drug delivery system).

[0418] Table 16. Results of UV spectroscopy of drug solutions after passing through the drug delivery system TIFF2025538189000020.tif77166

[0419] Study c: The delivered drug solution was sampled at the end of point-of-use testing. The leachable / extractable profile was determined by a contract laboratory using gas chromatography-based analysis established for chemical analysis of medical devices in accordance with ISO 10993, i.e., quantification of emitted organic compounds using a flame ionization detector (GC-FID) and identification of potential leachables / extractables using gas chromatography-mass spectrometry (GC-MS) and comparison to the NIST / EPA / NIH 2005 Mass Spectral Library, where applicable. No leachables / extractables were detected in this sample.

[0420] By acquiring both 5-day HPLC data across all in-use stability studies, the following regression was obtained: slope: -0.001065±0.0009457, time to 10% decline in OT-101 concentration was 91 days. Thus, the formulations of the present invention were surprisingly stable over extended use.

[0421] Example 3 - Pharmacokinetics after intracerebral and intracerebroventricular administration in rats.

[0422] 3The distribution, pharmacokinetics, and excretion of H-AP12009 were investigated after a single intravenous injection into the frontal lobe of the brain of Sprague-Dawley rats (10 μCi / rat, 87 μM solution, flow rate: 0.4 μl / min, administered over 1 h; report GAS0002). Due to the small sample volume added, small brain size, and variability in the exact location of the injection device, there was considerable interanimal variability in the data for early time points (0–4 h after the end of the infusion) in terms of both measured whole-body radioactivity and tissue concentrations. Overall, travedelsen distributed readily from the dosing site to other regions of the brain, particularly the cerebrospinal fluid, but also into the systemic circulation.

[0423] Intravenous administration of OT-101 resulted in minimal accumulation in the CNS. 3 Radioactivity concentrations in blood and tissues after a single intravenous administration of [H]OT-101 resulted in 0.1% OT-101 in the brain, as shown in Table 17.

[0424] Table 17: Radioactivity concentrations in blood and tissues TIFF2025538189000021.tif34166

[0425] To improve the use of OT-101 as a therapeutic agent, we studied intrathecal delivery of tritium-labeled OT-101 to Sprague-Dawley CD (albino) rats. There were no gender differences throughout the study. After 1-hour intracerebral infusion in rats, OT-101 was confined to the infusion site. However, after 1-hour intracerebroventricular infusion, OT-101 was more widespread, with 35×, 19×, and 12× higher concentrations found in the cerebellum, remaining cerebrum, and cerebrospinal fluid (CSF), respectively. OT-101 concentrations remained stable for the first 4 hours after infusion and decayed biexponentially, with a slow terminal half-life in tissues but not in CSF, suggesting rapid penetration away from the CSF compartment into underlying tissues. Minimal amounts of OT-101 were detected in the plasma compartment. Intrathecal bolus administration of 0.1 mL of OT-101 at 14, 30, 200, 300, and 500 μM to cynomolgus monkeys did not result in any single-dose toxicity. Histopathological examination revealed no substance-related histomorphological lesions in the lumbar subarachnoid space. No changes were observed in the gray and white matter of the spinal cord or nerve trunks, and neurons did not show any abnormalities. These data indicate that intrathecal administration of OT-101 is a potentially effective route of delivery of antisense therapeutics such as OT-101 to the midline, i.e., diffuse midline glioma (DMG).

[0426] Diffuse midline gliomas (DMGs) are highly morbid pediatric central nervous system (CNS) tumors for which no effective treatment currently exists. DMGs are involved in 50% of all pediatric HGGs. Due to their anatomical location and infiltrative nature, DMGs are not amenable to surgical resection and are most often diagnosed radiologically and treated with radiation therapy, which has no effect on survival. The median age at diagnosis is 5–11 years, with tumors arising in the pons occurring at a younger age (approximately 7 years) than those arising in the thalamus (approximately 11 years). Patients with DMGs face an extremely poor median overall survival (OS) of only 9–11 months, and only <10% of patients with pontine tumors survive 2 years after diagnosis. Radiation remains the mainstay of treatment, but it is only palliative and is expected to extend survival by an average of 3 months.

[0427] The OT-101 antisense drug is safe and effective during prolonged (7 days) high-flow perfusion of the brain in adult gliomas. As a single agent, it is as effective as the most active drugs in adult gliomas, namely TMZ in chemotherapy-naive patients and BCNU / CCNU in chemotherapy-failure patients.

[0428] TGF-β2 is expressed at high levels in both pediatric GBM (WHO grade IV) and pediatric DIPG (WHO grade IV) patients.

[0429] Expression analysis of pediatric brainstem cases in the TCGA database found highly significant survival benefit across all four quartiles of TGF-β2 expression.

[0430] Expression analysis of all glioma cases treated with radiation yielded a highly significant survival benefit across all quartiles of TGF-β2 expression.

[0431] Intracerebral injection Dosing via intracerebral or intracerebroventricular infusion distributed OT-101 throughout the CNS. Dosing was at 12 μg / rat. The distribution profiles between cerebral and ventricular administration were similar, except that intracerebroventricular administration directly into the CSF compartment resulted in higher drug concentrations in the cerebellum and CSF. The similarities between intracerebroventricular and intracerebral administration surprisingly demonstrated that intracerebroventricular administration was as effective as intracerebral administration, as shown in Table 18.

[0432] Table 18. Medication by intracerebral or intraventricular injection TIFF2025538189000022.tif113166

[0433] The accumulation profile of the top 15 organs is shown below. At the end of the 1-hour infusion period, rapidly perfused CNS components readily accessible by CSF (administration site, cerebellum, CSF, pituitary) were found to be C maxAlthough readily accessible to the CSF, the pineal gland actively takes up OT-101 and maintains C for up to 4 hours. max The less perfused CNS components were CNS at 1 hour (rest of cerebrum, rest of brain) and 4 hours (spinal cord). max was achieved.

[0434] A small fraction of the administered OT-101 was max As shown in Table 19, organs readily perfused by blood reached the plasma compartment at C at 4 hours. max OT-101 was achieved (thyroid and liver), and organs that actively took up OT-101 maintained C for 24 hours (kidney, spleen, bone marrow) or 72 hours (thymus). max was not achieved.

[0435] Table 19. Medication by intracerebral or intraventricular injection TIFF2025538189000023.tif73166TIFF2025538189000024.tif233166TIFF2025538189000025.tif36166

[0436] The data from this distribution study clearly demonstrate that intracerebral (and therefore intracerebroventricular) infusion provides access to OT-101 throughout the CNS, including the midline. The profile is consistent with CSF flow and the location of the various compartments examined. CNS components with high levels of OT-101 are readily accessible to the CSF, including the pituitary and pineal glands.

[0437] OT-101 was subjected to a series of non-clinical safety and pharmacology studies. Salient features of the conclusions of these studies are as follows:

[0438] Long-term local administration of OT-101 / AP 12009 may result in local tissue inflammation. Mild to moderate local toxicity was observed in animals after injection of a concentration of 500 μM, without any macroscopic changes.

[0439] When administered to 3 kg male or female rabbits as a bolus IT injection at the 0.12 mg / kg dose level (500 μM solution; 0.1 mL) with an estimated CSF concentration of 4.16 μM, OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes consistent with subclinical toxicity.

[0440] When administered to 6-7 kg cynomolgus monkeys as a bolus IT injection at a 0.05 mg / kg dose level (500 μM solution; 0.1 mL) with an estimated CSF concentration of 0.46 μM, OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes consistent with asymptomatic toxicity.

[0441] When administered intracerebroventricularly to rats, radiolabeled OT-101 (0.18 mg / kg) was detected in the CSF as well as the cerebrum, cerebellum, and pineal gland within 1 hour of administration. CSF and brain tissue half-lives were <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0442] Intratumoral placement of the catheter posed a potential risk that could be avoided by using an Ommaya reservoir to access the ventricular space.

[0443] The Ommaya reservoir can be used in the treatment of leptomeningeal carcinoma (LM), which is derived from multiple malignancies.

[0444] Example 4 - Figure 9 shows the effect of delivering a pharmaceutical composition by intrathecal or intracerebroventricular continuous infusion. Figure 9 shows the effect of OT-101 treatment on TGF-β2 secretion from the human GBM cell line A-172. Cells were incubated with various concentrations of OT-101 / AP 12009 (1 μM to 80 μM) as indicated for 7 days. Secreted TGF-β2 was measured in the cell supernatant by ELISA. Results represent the median, minimum, and maximum values ​​from three independent experiments.

[0445] Example 5 - Example of continuous infusion using a partially collapsible Ommaya-like reservoir of the present invention compared to conventional direct injection methods.

[0446] The device and method of the present invention for continuous infusion of therapeutic solution has been found to be advantageous compared to conventional methods (FIG. 10).

[0447] In conventional methods, therapeutic solutions are injected directly into the ventricular space of a patient's brain or directly into the ventricular space via a catheter. In these conventional methods, the therapeutic solution rapidly disperses throughout the ventricular space within seconds to minutes. Furthermore, such rapidly injected therapeutic solutions are rapidly eliminated by brain tissue. Therefore, such conventional methods achieve minimal therapeutic effect due to limited exposure of brain tissue to the therapeutic solution.

[0448] By comparison, the Ommaya-like reservoir of the present invention, having a partially collapsible top, advantageously provides continuous infusion, essentially continuous infusion, over an extended period of time.

[0449] The Ommaya-like reservoir of the present invention was filled with the test solution, and the device was placed on the surface of the tank to test the rate of infusion, i.e., the rate at which the test solution exited the tip of the catheter, as a model for continuous infusion into the brain.

[0450] In one embodiment, the Ommaya reservoir of the present invention is 339 mm 2 and the area of ​​the inner opening of the catheter is 1.5386 mm 2 which gives a ratio of 339 / 1.5386=220. Using this ratio, the expected release time of the test solution is expected to be at least several hours, depending on the density of the test solution relative to water.

[0451] In this example, the test solution diffused slowly through the catheter opening (see Figure 10). Diffusion from the Ommaya-like reservoir of the present invention occurred continuously over a 4-hour period. This study demonstrated that continuous diffusion can be achieved with the test solution and that a therapeutic solution with a density adjusted or matched to CSF ​​will provide continuous infusion for at least one to several days.

[0452] This example demonstrates that the partially collapsible design of the Ommaya-like reservoir of the present invention is advantageous because, when infusion of therapeutic solution is sustained over a long period of time, it is necessary to prevent accidental or unintended pressure on the flexible apex of the Ommaya-like reservoir from accidentally releasing the therapeutic solution. For example, accidental or unintended pressure on the flexible apex of the Ommaya-like reservoir may release too much therapeutic solution in a very short period of time.

[0453] The partially collapsible design of the present invention's omma-like reservoir prevents such unwanted accidental or accidental release of therapeutic solution.

[0454] Additionally, the partially collapsible design of the Ommaya-like reservoir of the present invention allows the device to maintain fluid flow and movement into and out of the reservoir. The flexible top expands and contracts in response to changes in pressure, allowing the Ommaya-like reservoir of the present invention to properly brace for sustained flow of solution, particularly in the presence of an externally generated pumping action. Patient movement, such as movement of the patient's jaw and other muscles, can also cause unwanted accidental or inadvertent release.

[0455] In some examples and embodiments, the Ommaya-like reservoirs of the present invention are prepared with a collar that makes it impossible to completely collapse the reservoir and prevents accidental or accidental release of solution (see Figure 8).

[0456] In some examples and embodiments, the Ommaya-like reservoirs of the present invention are prepared to be partially crushable by making a portion of the top of the Ommaya-like reservoir from a hard, non-flexible plastic.

[0457] Example 6 - Pediatric diffuse intrinsic pontine glioma.

[0458] Disclosed in this application are methods for pediatric DMG / DIPG / or K27M GBM.

[0459] This example presents new evidence that amplified TGFB2 mRNA expression in pediatric DIPG and H3K27M-mutated GBM correlates with upregulated mRNA expression of several transcription factors / DNA-binding proteins known to enhance TGFB2 gene expression. This example provides the first evidence that high levels of TGFB2 mRNA expression are associated with poor treatment outcomes in DIPG. The reported results also support the idea that further evaluation of the clinical potential of new strategies targeting TGFB2 mRNA in pediatric DIPG is warranted.

[0460] The median survival for TGFB2-high patients within the H3K27M-mutated subset of 30 patients was 6.5 months (95% CI = 5-NA months, 6 events, N = 8), which was significantly shorter than the median survival for the remaining patients (median > 10 months, 7 events, N = 22).

[0461] Pediatric diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive and deadly pediatric brain tumors. The purpose of this study was to evaluate the clinical significance of amplified expression levels of transforming growth factor beta 2 (TGFB2) in tumor tissue specimens from patients with DIPG. Our findings provide the first evidence that high levels of TGFB2 expression are associated with poor treatment outcomes in DIPG. The reported results also support the idea that further evaluation of the clinical potential of novel strategies targeting TGFB2 in pediatric DIPG is warranted.

[0462] Tumor samples from newly diagnosed pediatric diffuse intrinsic pontine glioma (DIPG) patients expressed significantly higher levels of transforming growth factor beta 2 (TGFB2, also known as TGF-β2) messenger ribonucleic acid (mRNA) than control pontine samples, which correlated with enhanced expression of transcription factors that upregulate TGFB2 gene expression. Our study also demonstrated that high TGFB2 mRNA levels based on RNA sequencing (RNA-seq) are an indicator of poor prognosis for DIPG patients, but not for pediatric glioblastoma (GBM) patients or for pediatric diffuse midline glioma (DMG) patients whose tumors are located outside the pons / brainstem. Notably, DIPG patients with high levels of TGFB2 mRNA expression in their tumor samples had significantly worse overall survival (OS) and progression-free survival (PFS). By comparison, high levels of transforming growth factor beta 3 (TGFB3) mRNA expression in tumor samples were associated with significantly better survival outcomes in patients with DIPG, whereas high levels of transforming growth factor beta 1 (TGFB1) expression were not prognostic. Our study fills a significant gap in understanding the clinical significance of high TGF expression in pediatric high-grade gliomas.

[0463] Pediatric diffuse intrinsic pontine gliomas (DIPGs) are classified as histone H3 lysine 27 (H3K27)-mutated diffuse midline gliomas (DMGs), including H3 wild-type subtypes with H3K27 mutants and EZH inhibitor protein (EZHIP) overexpression. Complete resection of DIPGs, the second most common malignant pediatric brain tumor, is impossible due to their anatomical location in the brainstem and their rapid infiltrative growth. Despite modern radiotherapy / chemoradiotherapy strategies, DIPGs are associated with poor overall survival (OS) and are one of the driving contributors to cancer-related mortality in children (median OS <1 year). Despite numerous clinical trials of chemotherapy agents, immuno-oncology drugs, and specific targeted therapies aimed at improving survival outcomes for pediatric patients with DIPG, little progress has been achieved, and the prognosis for DIPG remains poor, with a median survival of approximately 10 months and a 2-year survival rate of less than 10 percent.

[0464] We present new evidence that amplified TGFB2 mRNA expression in pediatric DIPG and H3K27M-mutated GBM correlates with upregulated mRNA expression of several transcription factors / DNA-binding proteins known to enhance TGFB2 gene expression. Our findings provide the first evidence that high levels of TGFB2 mRNA expression are associated with poor treatment outcomes in DIPG. The reported results also support the idea that further evaluation of the clinical potential of novel strategies targeting TGFB2 mRNA in pediatric DIPG is warranted.

[0465] Furthermore, there is no standard treatment for advanced DIPG after failure of state-of-the-art radiation therapy, and no salvage regimens have been shown to prolong OS. Effective treatment strategies that may improve the poor prognosis of these children are urgently needed, and their discovery represents a major focus of translational and clinical research in modern neuro-oncology. Several intervention strategies are being explored, including immunotherapy using immune checkpoint inhibitors or chimeric antigen receptor (CAR)-bearing T cells ("CAR-T cells"), inhibition of signaling pathways using small molecule drugs, and biotherapy using fusion toxins or oncolytic viruses administered via convection-enhanced delivery (CED).

[0466] Transforming growth factor-beta (TGFB) is a disulfide-linked homodimeric cytokine with a pleiotropic activity profile implicated in carcinogenesis and suppression of host antitumor immunity within the tumor microenvironment (TME). Enhanced TGFB signaling pathway activity, mediated by autocrine or tumor-associated macrophage (TAM)-derived overproduction, promotes the invasive and rapid proliferation of glioma cells, contributing to the aggressive biology and poor overall survival of adult high-grade glioma patients. The TGFB pathway has also been shown to contribute to a "cold" TME in high-grade gliomas through its immunosuppressive effects, characterized by the inhibition of CD8 antigen-positive cytotoxic T cells, natural killer (NK) cells, and the activation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The TGFB pathway has emerged as a potential therapeutic target for high-grade gliomas. The FDA-approved TGFB inhibitor, pirfenidone (5-methyl-1-phenyl-2(1H)-pyridone, PFD), has been shown to inhibit TGFB expression in glioma cells. A synthetic antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeting TGFB2 mRNA, when administered intratumorally via CED, has shown promising single-agent clinical activity associated with durable complete and partial responses in adult patients with recurrent or refractory glioblastoma and anaplastic astrocytoma. A study using a microarray-based gene expression platform found that TGFB2 mRNA levels were selectively amplified in primary tumor samples from 29 pediatric DIPG patients compared with normal samples and primary tumor samples from patients with low-grade glioma.

[0467] This study evaluated the clinical prognostic significance of high tumor TGFB2 mRNA levels in pediatric DIPG, as measured by RNA sequencing. Pediatric DIPG patients with amplified TGFB2 mRNA expression in brain tumor tissue may indicate a poor prognosis and more aggressive disease. This study demonstrated that newly diagnosed pediatric DIPG patients with elevated TGFB2 mRNA levels in their primary tumor samples, but not those with elevated mRNA levels of transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 3 (TGFB3), had significantly worse progression-free survival (PFS) and overall survival (OS) than other pediatric DIPG patients. High TGFB2 mRNA levels were an indicator of poor prognosis for DIPG patients, but not for DMG patients or pediatric glioblastoma (GBM) patients whose tumors were located outside the pons / brainstem, such as the cerebellum and thalamus.

[0468] A clinical study was conducted to determine clinical metadata and RNA sequencing (RNAseq)-based mRNA expression data for 41 pediatric DMG patients (mean age at diagnosis in months = 7.02 ± 0.44; median = 6; range = 2-14) and 116 pediatric GBM patients (mean age at diagnosis in months = 60.01 ± 1.24; median = 60.4; range = 21-89.3) for the TGFB isoforms TGFB1, TGFB2, and TGFB3 using genomic data obtained through the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ). An interactive web interface with full filtering capabilities provided by the portal was used. Data were compiled, harmonized, and annotated across multiple data consortia, including the Open Pediatric Brain Tumor Atlas (PBTA) project (project ID=openpbta) and the Pacific Pediatric Neuro-Oncology Consortium Clinical Genomics Atlas (project ID=pbta_pnoc). This clinical study examined the effect of TGFB2 mRNA expression levels on PFS and OS outcomes. A general treatment strategy was outlined in a clinical trial (https: / / clinicaltrials.gov / ct2 / show / NCT02274987) related to the treatment of patients with DIPG, which included standard radiation therapy followed by biomarker-guided specific therapy with FDA-approved targeted agents guided by gene expression analysis, whole exome sequencing (WES), and predictive modeling.

[0469] Downloaded mRNA expression levels for TGFB1, TGFB2, TGFB3, transforming growth factor beta receptor 1 (TGFBR1), transforming growth factor beta receptor 2 (TGFBR2), and transforming growth factor beta receptor 3 (TGFBR3) were reported using RNAseq V2 values ​​(expression profile data files appended with "mRNA_expression_(RNA_Seq_V2_RSEM).txt") normalized to "transcripts per million" ("TPM") values ​​calculated using RSEM, a software package that estimates gene and isoform expression levels from RNAseq data. The RSEM-based process consists of two main steps: 1. Generate a set of reference transcript sequences for assessment of mRNA abundance at the gene level; 2. Align a set of RNAseq reads to these reference transcripts for estimation of TPM abundance. This process allows for direct comparison and ranking of mRNA abundance across samples.

[0470] PFS and OS outcomes of patient subsets were compared using the Kaplan-Meier (KM) method, the log-rank chi-square test, and the software packages survival_3.2-13, survminer_0.4.9, and survMisc_0.5.5, which run in the R environment. Graphical representations of treatment outcomes were generated using the following graphical representation packages implemented in the R programming environment: dplyr_1.0.7, ggplot2_3.3.5, and ggthemes_4.2.4. The log-rank chi-square test was used to examine the statistical significance of differences in outcomes of compared patient subsets, and a p-value of less than 0.05 was considered significant.

[0471] TBFB1, TGFB2, and TGFB3 mRNA expression values ​​for normal pontine specimens measured by RNA sequencing (rna_tissue_hpa.tsv.zip) were downloaded from https: / / www.proteinatlas.org / about. mRNA expression values ​​were compiled (in TPM) for 29 separate pontine brain regions by filtering the "tissue group" annotation in the accompanying description file ("rna_tissue_hpa_description.tsv.zip"). A two-way analysis of variance (ANOVA) model was used to compare TGFB1 / TGFB2 / TGFB3 mRNA expression levels in normal pontine specimens with those in brain tumor specimens from 41 DIPG patients. For each transcript, contrasts were made between normal pontine and DIPG samples, and p-values ​​were adjusted for the false discovery rate (FDR). Calculations were performed using the multcomp_1.4-17 and emmeans_1.7.0 statistical packages in R version 4.1.2 with the RStudio front end (RStudio 2021.09.0 + 351 "Ghost Orchid" release). Bar chart graphics were constructed using the ggplot2_3.3.5 R package.

[0472] TGFB2 mRNA expression levels at log2 TPM were correlated with the mRNA expression levels of 11 transcription factors known to enhance TGFB2 expression across 41 DIPG patients: activating transcription factor 1 (ATF1), activating transcription factor 2 (ATF2), cyclic AMP-responsive element-binding protein 1 (CREB1), E1A-binding protein P300 (EP300), forkhead box protein O3 (FOXO3), polymerase II subunit A (POLR2A), regulatory factor X1 (RFX1), specificity protein 1 transcription factor (SP1), TATA box-binding protein (TBP), upstream transcription factor 1 (USF1), and upstream transcription factor 2 (USF2). Pairwise correlation coefficients were determined for all transcript combinations and visualized in a heatmap, color-coded from positive correlation (red = +1) to negative correlation (blue = -1). A clustering algorithm identified co-regulated gene sets using the statistical package ggcorrplot_0.1.3 implemented in R. A t-test was used to test the null hypothesis that the Pearson correlation coefficient equals zero. A significant correlation was confirmed by a p-value of less than 0.05 and an FDR of less than 0.10.

[0473] Normalized archived transcriptome profiling datasets obtained from the Gene Expression Omnibus web portal (https: / / www.ncbi.nlm.nih.gov / geo), including raw CEL files obtained using the Human Genome U133 Plus 2.0 Array platform for pediatric GBM patients with DIPG (N=29; GSE26576), normal control samples (N=2; GSE26576), and H3K27M mutations (N=5, GSE34824; N=7, GSE49822), were also used as independent validation datasets to compare the mRNA expression levels of TGFB1, TGFB2, and TGFB3 in normal control samples versus brain tumor specimens from 41 patients with pediatric DIPG or H3K27M mutations. The normalization procedure to determine log2-transformed mRNA expression levels was performed using the Robust Multi-array Averaging (RMA) method, as previously described. mRNA expression levels were calculated using the Aroma Affymetrix statistical package (aroma.affymetrix_3.2.0, aroma.core_3.2.2, and aroma.light_3.24.0) implemented in the RStudio environment (R version 4.1.2, RStudio 2021.09.0 Build 351). Statistical comparisons were performed using an ANOVA statistical model. A FDR-adjusted p-value of less than 0.05 was considered significant. The mRNA expression levels of TGFB1, TGFB2, and TGFB3 in DIPG / H3K27M-mutated GBM patients were visualized using heatmaps, as previously described. The expression level of TGFB2 mRNA (log2 RMA) was correlated with the expression levels of 11 transcription factor genes known to enhance TGFB2 expression: ATF1, ATF2, CREB1, EP300, FOXO3, POLR2A, RFX1, SP1, TBP, USF1, and USF2 using a two-way mRNA ANOVA model.

[0474] result Tumor samples from pediatric patients with DIPG contain high levels of TGFB2 mRNA, but not high levels of TGFB1 or TGFB3 mRNA, compared with normal pontine samples.

[0475] We compared RNA-seq-based mRNA levels for TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples and 29 normal pontine specimens (Figure 14). Notably, the mean (mean ± SE) TGFB2 mRNA level in primary DIPG samples was 1.5-fold higher than that in normal pontine specimens (4 ± 0.3 vs. 3.4 ± 0.1, p = 0.015) (Figure 14). In contrast, both TGFB1 and TGFB3 mRNA levels in DIPG samples were significantly lower than the corresponding levels in normal pontine samples (1.7-fold lower TGFB1 mRNA level, p = 0.0002 and 2.7-fold lower TGFB3 mRNA level, p < 0.001), as reflected by the blue color in the hierarchical cluster heatmap (Figure 14) and the significantly lower mean expression values ​​(log2 TPM) (Figure 14).

[0476] Selective overexpression of TGFB2 mRNA in DIPG tumor specimens is associated with enhanced expression of transcription factors that bind to multiple TGFB2 gene promoter sites.

[0477] We focused on transcription factors / DNA-binding proteins known to enhance TGFB2 gene expression to identify the molecular mechanisms underlying the upregulation of TGFB2 mRNA expression in 41 DIPG tumor samples. The transcript expression of 11 transcription factors with enhancing activity on TGFB2 gene expression, namely, ATF1, ATF2, CREB1, EP300, FOXO3, POLR2A, RFX1, SP1, TBP, USF1, and USF2, was examined in relation to TGFB2 mRNA levels. The mRNA levels of eight of these 11 transcription factors (i.e., SP1, RFX1, POLR2A, FOXO3, EP300, CREB1, ATF2, and ATF1) showed a statistically significant positive correlation with TGFB2 mRNA levels (Figure 15). SP1, FOXO3, and EP300 showed the most significant correlation with TGFB2 mRNA expression levels (p<0.0001).

[0478] Figure 14. Selective upregulation of TGFB2 mRNA expression in DIPG tumor samples. Archived TGFB1 / 2 / 3 mRNA expression levels (log2-transformed TPM) for primary tumor samples obtained from cBioPortal from 41 DIPG patients, including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M-mutated DIPG patients, and 26 H3K27M-mutated DMG patients, whose brain tumors were confined to the pons / brainstem, were compared with TGFB1 / 2 / 3 mRNA expression levels (log2 TPM) in normal pontine samples from 29 pontine regions (downloaded from https: / / www.proteinatlas.org / about / download). TGFB2 expression levels for these 29 normal pontine regions were determined by averaging the TPM values ​​of 2 to 8 independent samples / regions from 21 subjects. The log2-transformed TPM values ​​of TGFB1 / 2 / 3 mRNA levels in tumor samples from DIPG patients were mean-centered relative to the mRNA expression levels in normal pons samples and shown in a heatmap (A) representing overexpression (red) or underexpression (blue). (B) The bar chart illustrates the decreased expression levels for TGFB1 and TGFB3 mRNA, but the increased expression levels for TGFB2 mRNA, in tumor specimens from DIPG patients (dark gray bars) compared to normal pons samples (light gray bars).

[0479] (C) The statistical significance of differences in TGFB1 / 2 / 3 mRNA expression levels (log2-transformed TPM values) was assessed using two-way ANOVA with linear contrasts using FDR-adjusted p-values. A statistically significant 1.52-fold increase in TGFB2 mRNA levels (p=0.015) was found, along with a statistically significant decrease in TGFB1 mRNA (1.72-fold decrease; p=0.002) and TGFB3 (2.7-fold decrease; p<0.001) mRNA levels.

[0480] Figure 15. Correlation matrix of TGFB2 and transcription factors in 41 DIPG patients. Pairwise Pearson correlations were performed between TGFB2 and the mRNA levels of 11 transcription factors using log2-transformed TPM values ​​obtained from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ). Correlation coefficients were calculated across the 41 DIPG patients and are shown in a heatmap (A), ranging from positive correlation (red) to negative correlation (blue) and organized according to similarly expressed genes. The scale of the correlation range is indicated by the color bar "Correlation (Corr)." Of the 66 pairwise correlations, 48 ​​were considered statistically significant (p<0.05 and FDR=0.07; non-significant correlations are indicated by black crosses in the heatmap). (B) The mRNA levels of eight of the 11 transcription factors (i.e., SP1, RFX1, POLR2A, FOXO3, EP300 / transcription factor coactivator, CREB1, ATF2, and ATF1) showed a statistically significant positive correlation with TGFB2 mRNA levels. The mRNA levels of SP1, FOXO3, and EP300 showed the most significant correlation with TGFB2 mRNA expression levels (p<0.0001). (C) Transcription factor proteins (ovals) bound to the binding sites on the TGFB2 gene for these transcription factors (rectangles) are shown. Additionally, we show recruitment of the transcription factor coactivator EP300 (E1A-binding protein P300) to the phosphorylated form of CREB1 / ATF1 / ATF2 and its cross-linking to the basal transcriptional apparatus via RNA polymerase II subunit A (POLR2A) to directly stimulate TGFB2 transcription.

[0481] Similar results were obtained in an independent validation dataset of microarray-based mRNA levels of TGFB1, TGFB2, and TGFB3 and their correlated expression with specific transcription factors in tumor samples from 41 pediatric patients with DIPG (N=29) or H3K27M-mutated GBM (Figure 16). Significant increases in mRNA expression levels were observed for three TGFB2 probe sets: TGFB2_228121_at (2.7-fold increase, p=0.006); TGFB2_209909_s_at (2.4-fold increase, p=0.019); and TGFB2_220407_s_at (2.2-fold increase, p=0.032) (Figure 16, panel). Significant decreases were observed in mRNA expression of one of the TGFB1 probe sets, TGFB1_203085_s_at (2.2-fold decrease, p=0.028), and one of the TGFB3 probe sets, TGFB3_209747_at (2.3-fold decrease, p=0.026). The probe set TGFB2_220407_s_at showed the greatest number of positive correlations with 11 transcription factor probe sets and all four other TGFB2 probe sets (21 significant positive correlations at p<0.05 (FDR=0.047)) (Figure 16, panel A). The mRNA levels of 8 of the 11 transcription factors (i.e., SP1, USF1, POLR2A, FOXO3, EP300 (2 probe sets), CREB1 (6 probe sets), ATF2 (2 probe sets), and ATF1 (3 probe sets)) showed a statistically significant positive correlation with TGFB2 mRNA levels (Figure 16, panel).

[0482] Figure 16. Correlated expression of TGFB2 mRNA and specific transcription factor mRNAs in an independent validation dataset. (A) Differential expression of TGFB1, TGFB2, and TGFB3 mRNA levels in 41 brain tumor specimens from pediatric patients with DIPG or H3K27M-mutated GBM is illustrated using a cluster display of log2-transformed fold changes. Expression levels are shown as mean-centered, log2-transformed, normalized RMA values ​​relative to normal samples. Blue to red in the heatmap indicates under- to overexpression for TGFB1, TGFB2, and TGFB3 mRNA levels, respectively. Co-regulatory probe sets were organized and displayed by dendrogram for both probe sets (rows) and patients (columns). A significant increase in mRNA expression was observed for three TGFB2 probe sets: TGFB2_228121_at (2.7-fold increase, p = 0.006); TGFB2_209909_s_at (2.4-fold increase, p = 0.019); and TGFB2_220407_s_at (2.2-fold increase, p = 0.032). A significant decrease in mRNA expression was observed for one of the TGFB1 probe sets, TGFB1_203085_s_at (2.2-fold decrease, p = 0.028), and one of the TGFB3 probe sets, TGFB3_209747_at (2.3-fold decrease, p = 0.026). (B) A total of 1,640 correlations were performed for 41 probe sets corresponding to 11 transcription factors and TGFB2 mRNA. For 660 correlations, the p-value was less than 0.05 (FDR = 0.12), and 324 correlations showed a p-value less than 0.001 (FDR = 0.005). The probe set TGFB2_220407_s_at showed the greatest number of positive correlations with 11 transcription factor probe sets and all four other TGFB2 probe sets. There were 21 significant positive correlations with p < 0.05 (FDR = 0.047). Color-coded Pearson correlation coefficients for the most significant positive correlations with TGFB2_220407_s_at are shown in a heatmap ranging from positive correlations (red) to negative correlations (blue), organized according to similarly expressed mRNA levels.The scale of the range of correlation is shown in the color bar "Correlation"; non-significant correlations are indicated by black crosses in the heatmap. The mRNA levels of 8 of the 11 transcription factors (i.e., SP1, USF1, POLR2A, FOXO3, EP300 (2 probe sets), CREB1 (6 probe sets), ATF2 (2 probe sets), and ATF1 (3 probe sets)) showed a statistically significant positive correlation with TGFB2 mRNA levels.

[0483] Amplification of TGFB2 mRNA expression, but not TGFB1 or TGFB3 mRNA, is associated with shorter OS and PFS in patients with DIPG.

[0484] We compared survival outcomes of DIPG patients with TGFB2 mRNA expression levels above the upper quartile (TGFB2-high) with treatment outcomes of the remaining DIPG patients (TGFB2-low). The mean TGFB2 expression level in the TGFB2-high subset of 11 patients was 6.2 ± 0.2 (median, range = 5.8, 5.2 to 7.6). By comparison, the mean TGFB2 mRNA expression level in the TGFB2-low subset of 30 patients was 3.2 ± 0.2 (median, range = 3.5, -0.4 to 5.1). TGFB2-high DIPG patients showed significantly worse OS outcomes than TGFB2-low DIPG patients (median OS for TGFB2-high subset: 5 months, 95% CI: 5-NA months, 11 events, N=11); median OS for TGFB2-low subset: 11.5 months, 95% CI: 10-14 months, 30 events, N=30; log-rank chi-squared = 16.2, p=5.6×10-5) (Figure 17). PFS outcomes were also significantly worse for the TGFB2-high subset (median PFS for TGFB2-high subset: 5 (95% CI: 5-NA) months, 11 events, N=11) vs. TGFB2-low subset: 11 months, 95% CI: 10-13 months, 30 events, N=30 (log-rank chi-squared = 14.7, p=1.3×10-4) (Figure 18). In contrast to the poor prognostic impact of high TGFB2 mRNA expression, high TGFB3 mRNA expression was associated with significantly better OS and PFS outcomes, and high TGFB1 mRNA expression had no statistically significant effect on either OS or PFS outcomes (Figures 17 and 18).

[0485] We evaluated the OS outcome data for 30 H3K27M-mutated DIPG patients separately, excluding 11 DIPG patients with unknown H3K27M mutation status. TGFB2-high patients within the 30-patient subset had a shorter time to death than the remaining patients, as did TGFB2-high patients within the full analysis set of 41 patients, including 11 DIPG patients with unknown H3K27M mutation status (not shown). However, this difference did not reach statistical significance in the smaller subset, likely due to reduced power to detect statistical differences resulting from the broad distribution of OS outcomes and smaller sample sizes (not shown). Notably, TGFB2-high patients within the 30-patient subset, similar to the full analysis set of 41 patients (not shown), were characterized by early failure and significantly worse survival outcomes within 10 months (not shown). The median survival for TGFB2-high patients within the H3K27M-mutated subset of 30 patients was 6.5 months (95% CI = 5-NA months, 6 events, N = 8), which was significantly shorter than the median survival for the remaining patients (median > 10 months, 7 events, N = 22; log-rank chi-square = 5.5, p-value = 0.019) (not shown).

[0486] Figure 17. Amplification of TGFB2 expression is associated with shorter OS in DIPG patients. Archived survival outcome data from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ) for 41 DMG patients, including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M-mutated DIPG patients, and 26 H3K27M-mutated DMG patients whose brain tumors were confined to the pons / brainstem, were combined with RNA-seq-based mRNA expression data for TGFB2 (A), TGFB3 (B), and TGFB1 (C) to assess the potential impact of each TGFB isoform level on OS. Log-transformed, TPM-normalized RNA-seq values ​​were ranked according to the expression level of each TGFB isoform. Patients whose expression levels of a given TGFB isoform were in the upper quartile or above (solid line) were compared with the remaining patients (dashed line). See legend to Figure 14 for TGFB isoform levels in the analyzed patient subsets. (A) The mean TGFB2 expression level in the TGFB2-high subset of 11 patients was 6.19 ± 0.24 (median, range = 5.81, 5.23 to 7.63). By comparison, the mean TGFB2 expression level in the TGFB2-low subset of 30 patients was 3.22 ± 0.23 (median, range = 3.48, -0.42 to 5.07). Patients with high TGFB2 mRNA expression (TGFB2-high) showed significantly worse OS outcomes than the remaining patients (TGFB2-low) (median OS in the TGFB2-high subset: 5 months, 95% CI: 5-NA months, 11 events, N=11); median OS in the TGFB2-low subset: 11.5 months, 95% CI: 10-14 months, 30 events, N=30; log-rank chi-square = 16.2, p=5.6 × 10-5). (B) The mean TGFB3 expression level in the TGFB3-high subset of 11 patients was 4.7 ± 0.2 (median, range = 4.6, 4-5.6). By comparison, the mean TGFB3 expression level in the TGFB3-low subset of 30 patients was 3 ± 0.1 (median, range = 3.1, 1.5-4).Patients with high TGFB3 expression (TGFB3-high) (median OS: 14 months, 95% CI: 12-11 months, 11 events, N=11) had significantly better OS outcomes than TGFB3-low patients (median OS: 8 months, 95% CI: 7-11 months, 30 events, N=30; log-rank chi-square = 5.6, p=0.018). (C) The mean TGFB1 mRNA expression level in the TGFB1-high subset of 11 patients was 4.8 ± 0.1 (median, range = 4.8, 4.5-5.4). By comparison, the mean TGFB1 expression level in the TGFB1-low subset of 30 patients was 3.8 ± 0.1 (median, range = 3.9, 1-4.5). Patients with high TGFB1 expression (TGFB1 high) (median OS = 10 months, 95% CI: 9-NA months, 11 events, N = 11) showed similar OS outcomes when compared with the remaining patients (TGFB1 low) (median OS = 10 months, 95% CI: 8-13 months, 30 events, N = 30; log-rank chi-square = 0.1, p = 0.8).

[0487] Figure 18. Amplification of TGFB2 expression is associated with shorter PFS in DIPG and DMG patients. Archived clinical outcome data from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ) for 41 DMG patients, including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M-mutated DIPG patients, and 26 H3K27M-mutated DMG patients whose brain tumors were confined to the brainstem / pons, were combined with RNAseq-based mRNA expression data for TGFB2 (A), TGFB3 (B), and TGFB1 (C) to assess the potential impact of each TGFB isoform level on PFS. For patients lacking information on disease progression, death was used as the first event when assessing PFS outcomes. Log2-transformed, TPM-normalized RNAseq values ​​were ranked according to the expression level of each TGFB isoform. Patients whose expression levels of a given TGFB isoform were in the upper quartile or higher (solid line) were compared with the remaining patients (dashed line). (A) TGFB2-high patients had significantly worse PFS outcomes than the remaining patients (median PFS in the TGFB2-high subset: 5 (95% CI: 5-NA) months, 11 events, N=11; median PFS in the TGFB2-low subset = 11 months, 95% CI: 10-13 months, 30 events, N=30; log-rank chi-square = 14.7, p=1.3 × 10-4). (B) TGFB3-high patients (median PFS = 13 months, 95% CI: 11-NA months, 11 events, N = 11) showed significantly better PFS outcomes than TGFB3-low patients (median PFS = 8 months, 95% CI: 7-11 months, 30 events, N = 30; log-rank chi-square = 4.1, p = 0.043). (C) TGFB1-high patients (median PFS = 10 months, 95% CI: 9-NA months, 11 events, N = 11) and TGFB1-low patients showed similar OS outcomes (median PFS = 8.5 months, 95% CI: 7-13 months, 30 events, N = 30; log-rank chi-square = 0.2, p = 0.7).

[0488] TGFB2 expression levels do not affect OS or PFS in pediatric DMG patients whose tumors are not located in the pons / brainstem.

[0489] Survival outcomes of patients with non-DIPG DMG whose tumors were located outside the pons / brainstem (TGFB2-high) and had TGFB2 mRNA expression levels above the upper quartile were compared with the treatment outcomes of the remaining non-DIPG DMG patients (TGFB2-low). TGFB2-high patients (median OS = 12.5 months, 95% CI: 9-NA months, 10 events) demonstrated similar OS outcomes when compared with TGFB2-low patients (median OS = 11 months, 95% CI: 2-NA months, 8 events; log-rank chi-square = 0.2, p = 0.6) (not shown). TGFB2-high and TGFB2-low non-DIPG DMG patients also had very similar PFS outcomes (not shown). Similarly, no statistically significant differences in OS or PFS outcomes were found when comparing the TGFB1-high vs. TGFB1-low or TGFB3-high vs. TGFB3-low non-DIPG DMG subsets (not shown). Because activation of the TGFB2 signaling pathway requires binding of TGFB2 to TGF-β receptor II (TGF-βRII), which recruits TGF-βRI into a heterotetrameric complex, we determined whether the observed lack of prognostic benefit of high TGFB2 status could be due to reduced expression levels of the TGFB2 receptor. None of the TGFB2 receptors showed lower expression levels in tumor samples from non-DIPG DMG patients (N=19) compared with DIPG patients (N=41), explaining the observed lack of prognostic significance of higher TGFB2 mRNA levels in this patient population (not shown). DIPG patients were, on average, younger (mean age at diagnosis in months = 7.0±0.4; median = 6; range = 2-14) than non-DIPG DMG patients (mean age at diagnosis in months = 10.5±0.7; median = 11; range = 5-17) (p=0.0003).

[0490] TGFB2 mRNA expression levels have no effect on OS or PFS in pediatric GBM patients.

[0491] Survival outcomes of pediatric GBM patients with TGFB2 mRNA expression levels in the upper quartile or higher (TGFB2-high) were compared with treatment outcomes of the remaining pediatric GBM patients (TGFB2-low). In the TGFB2-high pediatric GBM subset of 29 patients, the mean TGFB2 expression level was 12.7 ± 0.1 (median, range = 12.6, 12-13.7). By comparison, in the TGFB2-low pediatric GBM subset of 87 patients, the mean TGFB2 expression level was 10.4 ± 0.1 (median, range = 10.5, 8.2-12). Patients in the TGFB2-high (N=29) and TGFB2-low (N=87) subsets had very similar OS outcomes (TGFB2-high median: 12.6 (95% CI: 9.9-NA) months, 17 events; TGFB2-low median: 12.9 (95% CI: 11.2-15) months, 65 events; log-rank chi-square = 0.2, p = 0.7) (Figure 19). TGFB2-high and TGFB2-low pediatric GBM patients also had very similar PFS outcomes (not shown). Similarly, no differences in OS or PFS outcomes were found when comparing TGFB1-high vs. TGFB1-low or TGFB3-high vs. TGFB3-low pediatric GBM subsets (Figure 18). Compared with DIPG / DMP patients, tumor samples from GBM patients did not show lower expression levels of any of the TGFB2 receptors, explaining the observed lack of prognostic significance of higher TGFB2 mRNA levels in this patient population. Conversely, GBM patients showed significantly higher levels of all three receptors compared with DIPG patients (not shown). TGFB2 mRNA levels in GBM patients were higher than those in DIPG patients; even in the TGFB2-high DIPG subset of 11 patients, the mean TGFB2 mRNA expression level was 6.19 ± 0.24 (median, range = 5.81, 5.23–7.63), which was lower than the TGFB2 mRNA level in TGFB2-low GBM patients (10.4 ± 0.1; median, range = 10.5, 8.2–12). The lack of a truly TGFB2-low subset of GBM patients may have precluded accurate assessment of the prognostic effect of high TGFB2 mRNA levels.DIPG patients were on average younger (mean age at diagnosis in months = 7.0 ± 0.4; median = 6; range = 2–14) than GBM patients (mean age at diagnosis in months = 60.0 ± 1.2; median = 60.4; range = 21–89.3) (p<0.0001).

[0492] Figure 19. TGFB2 mRNA expression levels do not affect OS in pediatric GBM patients. Archived OS data for 116 GBM patients obtained from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ) were combined with RNAseq-based mRNA expression data for TGFB2 (A), TGFB3 (B), and TGFB1 (C) to assess the potential impact of each TGFB isoform level on OS. Log2-transformed, TPM-normalized RNAseq values ​​were ranked according to the expression level of each TGFB isoform. Patients whose expression levels of a given TGFB isoform were in the upper quartile or higher (solid line) were compared with the remaining patients (dashed line). See legend to Figure 16 for TGFB isoform levels in the analyzed patient subset. (A) The mean TGFB2 expression level in the TGFB2-high subset of 29 patients was 12.67 ± 0.1 (median, range = 12.63, 11.97-13.7). By comparison, the mean TGFB2 expression level in the TGFB2-low subset of 87 patients was 10.37 ± 0.1 (median, range = 10.49, 8.17-11.94). Patients in the TGFB2-high (N = 29) and TGFB2-low (N = 87) subsets showed very similar OS outcomes (TGFB2-high median: 12.6 (95% CI: 9.9-NA) months, 17 events; TGFB2-low median: 12.9 (95% CI: 11.24-14.95) months, 65 events; log-rank chi-square = 0.2, p = 0.7). (B) The mean TGFB3 expression level in the TGFB3-high subset of 29 patients was 11.39 ± 0.1 (median, range = 11.25, 10.63-12.88). By comparison, the mean TGFB3 expression level in the TGFB3-low subset of 87 patients was 9.4 ± 0.08 (median, range = 9.56, 7.14-10.58).Patients in the TGFB3-high and TGFB3-low subsets had very similar OS outcomes (TGFB3-high median: 12.9 (95% CI: 10.41-26.38) months, 20 events; TGFB3-low median: 13 (95% CI: 11-14.95) months, 62 events; log-rank chi-square = 0, p = 0.9). (C) The mean TGFB1 expression level in the TGFB1-high subset of 29 patients was 12.08 ± 0.08 (median, range = 11.92, 11.58-13.13). By comparison, the mean TGFB1 expression level in the TGFB1-low subset of 87 patients was 10.53 ± 0.09 (median, range = 10.71, 7.5-11.56). TGFB1-high patients (median: 12.6 (95% CI: 8.8-15.8) months, 22 events; N=29) and TGFB1-low patients (median: 13.6 (95% CI: 11.7-15.4) months, 60 events) showed similar OS outcomes (log-rank chi-square = 0.5, p = 0.5).

[0493] This clinical study investigated the prognostic significance of TGFB2-high status in newly diagnosed pediatric patients with DIPG. Notably, patients with TGFB2-high (but not TGFB1- or TGFB3-high) had significantly worse survival outcomes, with substantially shorter OS times. RNA-seq-based high TGFB2 mRNA levels were an indicator of poor prognosis in DIPG patients, but not in pediatric GBM patients or pediatric DMG patients with tumors located outside the pons / brainstem. The lack of an adverse prognostic effect of higher TGFB2 mRNA expression levels in the latter patient population was not due to lower expression levels of the TGFB2 receptor. It is noteworthy that TGFB2 mRNA levels in pediatric GBM patients were significantly higher than those in DIPG patients. The lack of a truly TGFB2-low subset of GBM patients may have precluded accurate assessment of the favorable prognostic effect of low TGFB2 mRNA levels.

[0494] This study provides important understanding of the clinical significance of high TGFB2 expression in pediatric high-grade gliomas. The TGFB2-promoted invasive growth of DIPG cells, their TGFB2-associated radioresistance, and possibly TGFB2-mediated restriction of cellular anti-glioma immunity within the TME contributed to the observed adverse impact of high TGFB2 levels on survival outcomes in DIPG patients.

[0495] In a clinical study using a microarray-based gene expression platform, we found that TGFB2 transcripts, but not TGFB1 or TGFB3, were selectively amplified in primary tumor samples from 29 pediatric DIPG patients compared with normal samples and primary tumor samples from patients with low-grade glioma. In this study, we compared RNA-seq-based TGFB2 mRNA levels in primary DIPG tumor samples and normal control pontine samples. Notably, tumor samples from DIPG patients expressed significantly higher levels of TGFB2 mRNA than control pontine samples, whereas TGFB1 and TGFB3 mRNA levels in DIPG samples were significantly lower than control pontine samples. These results, obtained using RNA-seq-based mRNA data, confirm and significantly expand upon our previous results obtained using a microarray platform. Enhanced TGFB2 gene expression in DIPG samples may be due to enhanced expression of transcription factors that upregulate TGFB2 mRNA expression. This hypothesis was supported by the finding of a strong positive correlation (p<0.0001) between the mRNA levels of several such transcription factors, e.g., SP1, FOXO3, and EP300, and TGFB2 mRNA levels in DIPG samples. Similar results were obtained in an independent validation dataset of microarray-based mRNA levels of TGFB1, TGFB2, and TGFB3 and their correlated expression with specific transcription factors in tumor specimens from 41 pediatric patients with DIPG (N=29) or H3K27M-mutated GBM.

[0496] There are three isoforms of TGFB within the TGFB gene superfamily: TGFB1, TGFB2, and TGFB3. Despite >70% sequence homology, the structure and biological function of the TGFB isoform TGFB3 differ from those of TGFB1 and TGFB2. Notably, TGFB3 knockout mice are uniquely different from TGFB1 or TGFB2 knockout mice. Furthermore, TGFB3 exerts cancer preventive effects in preclinical models of tumorigenesis and in human subjects. Furthermore, high TGFB3 expression has a favorable prognostic effect in breast, ovarian, and colon cancer. However, TGFB3 expression correlates with poor prognosis in osteosarcoma. In this study, high TGFB3 expression was identified as a favorable prognostic indicator, in contrast to its reported adverse prognostic role in breast cancer. Our findings advance our current knowledge and provide new insights into the multifunctional role of TGFB3.

[0497] Based on the results presented herein, amplification of TGFB2 mRNA expression is associated with poor prognosis and OS in DIPG (Figure 20).

[0498] Figure 20. TGFB2 mRNA expression is selectively amplified and associated with poor OS in pediatric DIPG. TGFB2 mRNA levels, but not TGFB1 and TGFB3 mRNA levels, were selectively amplified in primary brain tumor specimens from patients with DIPG. High levels of TGFB2 mRNA expression were associated with poor OS. In contrast, high TGFB1 mRNA expression had no prognostic value, and high TGFB3 mRNA expression was associated with favorable OS.

[0499] OT-101, a TGFB2-targeting S-ODN, demonstrated single-agent clinical activity in adult patients with recurrent or refractory glioblastoma and anaplastic astrocytoma when administered intratumorally via CED. Of 77 high-grade glioma patients in the efficacy population, 26 had a favorable response, including 19 patients with CR or PR and 7 patients with stable disease for more than 6 months. The median PFS and OS in this subset were 1,109 and 1,280 days, respectively. The poor prognosis observed in newly diagnosed TGFB2-high DIPG patients, as reported herein, supports the notion that further exploration of the clinical potential of TGFB2-targeting RNAi therapeutics in TGFB2-high DIPG patients is warranted. CED catheters have been used in DIPG patients for intratumoral delivery of therapeutic agents, bypassing the blood-brain barrier and achieving higher intratumoral concentrations while reducing the risk of systemic toxicity. However, results are limited by the need for several months of intratumoral drug administration to achieve objective responses in adult patients with high-grade glioma, combined with the practical challenges of long-term use of implanted CED catheters or repeated CED catheter exchange procedures in this difficult anatomical location.

[0500] The present invention provides novel delivery methods and / or formulation strategies for therapeutic agents targeting TGFB2 that are effective in patients with DIPG.

[0501] Example 7 - Single-agent activity of OT-101 in patients with recurrent / refractory high-grade glioma.

[0502] Phase 2 clinical data demonstrated remarkable single-agent activity of OT-101 in patients with recurrent / refractory high-grade glioma, with more than one-third of patients (26 of 77) who received the intended 4 to 11 cycles of therapy achieving durable complete response, partial response, or long-term stable disease, and a median OS of 1280 days (95% CI: 1116 to >1743 days).

[0503] The median PFS of these 77 patients was significantly better than that of the 12 patients treated with 1 to 3 cycles of OT-101 (86 days vs. 32 days, log-rank P<0.0001). Similarly, the median OS of the 77 patients treated with 4 to 11 cycles of OT-101 was significantly better than that of the 12 patients treated with 1 to 3 cycles (432 days vs. 128 days, log-rank P<0.0001).

[0504] Nineteen patients achieved durable objective responses (CR: 3, PR: 16). The median time to 90% reduction in baseline tumor volume was 11.7 months (range: 4.9-57.7 months). The mean log reduction in tumor volume in these 19 patients was 2.2 ± 0.4 (median = 1.4, range: 0.4-4.5) logs.

[0505] Figure 21. Imaging responses in R / R high-grade glioma patients treated with OT-101 monotherapy who achieved CR or PR. Figure 21: Waterfall plot showing maximum log10 reduction in tumor volume.

[0506] Figure 22. Imaging responses in R / R high-grade glioma patients treated with OT-101 monotherapy who achieved CR or PR. Figure 22: Semi-log plot of composite 3-D tumor volume reduction curves for 19 patients.

[0507] Figure 23 shows swimmer plots for the onset and duration of objective response. The onset and duration of CR / PR, censoring of OR, and onset of PD are indicated with specific symbols.

[0508] OT-101 induces durable CR and PR in patients with R / R GBM and AA. Nineteen patients had an objective response. Sixteen had a partial response, with onset at 307 ± 159 days (mean ± SE). The median time to onset of PR was 287 days (range: 37-742). Three patients had an initial PR that progressed to CR at 917, 1120, and 1838 days, respectively. Six of these 16 patients developed PD at 970 ± 126.7 days (mean ± SE) (median 1032 days, range: 374-1281 days).

[0509] Example 8 - Clinical potential of targeting transforming growth factor-β2 with OT-101 for post-radiation consolidation in diffuse intrinsic pontine glioma.

[0510] Diffuse intrinsic pontine glioma (DIPG) in children has a poor prognosis, with a median overall survival (OS) of 10 months and a 2-year OS rate of <10% after standard radiation therapy. Chemotherapy does not provide clinically meaningful benefit. Therefore, therapeutic innovations for the treatment of pediatric DIPG are urgently needed.

[0511] High-grade glioma cells, including pediatric glioblastoma and DIPG cells, have been shown to produce transforming growth factor beta 2 (TGF-β2), which has been implicated both as a promoter of glioma cells and as an important contributor to T cell hyporesponsiveness of the tumor microenvironment (TME) to glioma cells.

[0512] OT-101 is a first-in-class RNA therapeutic designed to abrogate the immunosuppressive and tumor-promoting effects of TGF-β2. At low micromolar concentrations, OT-101 reduces TGF-β2 secretion by human glioma cells, blocks their proliferation and migration, and restores the anti-glioma cytolytic function of patient-derived T cells.

[0513] Intrathecal / intracerebroventricular administration of antineoplastic agents directly into the CSF allows bypassing the selective filter of the BBB, achieving significant concentrations of antineoplastic agents in the CSF while reducing the potential for systemic toxicity. Based on favorable safety pharmacology studies of intrathecally delivered OT-101 in rabbits and primates, and encouraged by its single-agent activity in adult patients with HGG, we conducted a multicenter, two-part, randomized phase 1-2 study of OT-101 in pediatric patients with DIPG. Multiple doses of OT-101 were administered as intrathecal (IT) / intracerebroventricular bolus injections after the completion of radiation therapy. The study was designed to determine: 1) the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of OT-101, and 2) its efficacy in children with DIPG.

[0514] Antisense oligodeoxynucleotides are short strings of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and several have been approved. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which the non-bridging oxygen of each phosphate moiety is replaced by a sulfur atom. OT-101 was designed to be complementary to a specific sequence in human TGF-β2 mRNA after gene expression. It is a first-in-class RNA therapeutic designed to block the immunosuppressive effects of TGF-β2 in malignant gliomas, reduce TGF-β2 levels, and thereby slow disease progression.

[0515] Functional in vitro assays demonstrated the following:

[0516] OT-101 exhibits efficient, time-dependent uptake into human tumor cells in the presence and absence of the liposomal carrier Lipofectin®.

[0517] OT-101, without any carrier, reduces the secretion of TGF-β2 by human tumor cells.

[0518] At clinically used OT-101 concentrations up to 80 μM over 7 days in A172 human high-grade glioma cells, 10 μM is the most effective concentration for inhibition of TGF-β2 production.

[0519] OT-101 reduces the proliferation of human tumor cells while simultaneously stimulating the proliferation of PBMCs, without affecting the viability of human PBMCs.

[0520] OT-101 restores immune function of human PBMCs derived from high-grade glioma patients, as demonstrated by immune cell-mediated cytotoxicity assays.

[0521] OT-101 inhibits the migration of human tumor cells.

[0522] OT-101 was subjected to a series of non-clinical safety and pharmacology studies. Salient features of the conclusions of these studies are as follows:

[0523] Long-term local administration of OT-101 / AP 12009 may result in local tissue inflammation. Mild to moderate local toxicity was observed in animals after injection of a concentration of 500 μM, without any macroscopic changes.

[0524] When administered to 3 kg male or female rabbits as a bolus IT injection at the 0.12 mg / kg dose level (500 μM solution; 0.1 mL) with an estimated CSF concentration of 4.16 μM, OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes consistent with subclinical toxicity.

[0525] When administered to 6-7 kg cynomolgus monkeys as a bolus IT injection at a 0.05 mg / kg dose level (500 μM solution; 0.1 mL) with an estimated CSF concentration of 0.46 μM, OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes consistent with asymptomatic toxicity.

[0526] When administered intracerebroventricularly to rats, radiolabeled OT-101 (0.18 mg / kg) was detected in the CSF as well as the cerebrum, cerebellum, and pineal gland within 1 hour of administration. CSF and brain tissue half-lives were <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0527] Example 9 - Non-clinical in vitro studies of OT-101 / AP 12009.

[0528] Functional in vitro assays demonstrated the following:

[0529] OT-101 exhibits efficient, time-dependent uptake into human tumor cells in the presence and absence of the liposomal carrier Lipofectin®.

[0530] OT-101, without any carrier, reduces the secretion of TGF-β2 by human tumor cells.

[0531] At clinically used OT-101 concentrations up to 80 μM over 7 days in A172 human high-grade glioma cells, 10 μM is the most effective concentration for inhibition of TGF-β2 production.

[0532] OT-101 reduces the proliferation of human tumor cells while simultaneously stimulating the proliferation of PBMCs, without affecting the viability of human PBMCs.

[0533] OT-101 restores immune function of human PBMCs derived from high-grade glioma patients, as demonstrated by immune cell-mediated cytotoxicity assays.

[0534] OT-101 inhibits the migration of human tumor cells.

[0535] The fluorescent signal increased up to 48 hours in human A-172 glioblastoma cells incubated with FITC-OT-101 both with and without Lipofectin®. FITC-OT-101 uptake was already observed after 3 hours of incubation with or without Lipofectin®. After 48 hours, the fluorescent signal was detectable in almost all cells, and the intensity of the cell preparations incubated with or without Lipofectin® was comparable.

[0536] Example 10 - Effect of OT-101 on the synthesis and secretion of TGF-β2 by human GBM cell lines.

[0537] Cells were incubated with various concentrations of OT-101 / AP 12009 (1 μM to 80 μM) for 7 days. Secreted TGF-β2 was measured in cell supernatants by ELISA. Results represent the median, minimum, and maximum values ​​from three independent experiments.

[0538] The ability of OT-101 to reduce TGF-β2 secretion by primary human glioma cells was determined by measuring TGF-β2 concentrations in cell culture supernatants using an enzyme-linked immunosorbent assay (ELISA). Glioma cells from 10 patients with high-grade gliomas were cultured in the presence or absence of OT-101 (5 or 10 μM) for 72 hours (HTZ-209, -220, -243, -262, -349, -361, -378, and -381) or 96 hours (A-172). TGF-β2 secretion was reduced by up to 87% in 8 of the 10 glioma cell cultures.

[0539] OT-101-mediated inhibition of human high-grade glioma cell proliferation Two human HGG cell cultures (HTZ-243 and HTZ-349, corresponding to WHO grades III and IV) were incubated with OT-101 (1 μM to 10 μM). The results showed a concentration- and time-dependent reduction in cell number within 6 days, as shown in Table 20.

[0540] Table 20: Effect of OT-101 on the proliferation of human high-grade glioma cells TIFF2025538189000026.tif48166

[0541] Two human glioma cell cultures (HTZ-243 and HTZ-349) were treated with OT-101 (1, 5, or 10 μM). Cell numbers (% of the cell number at the start of the experiment) were determined using a hemocytometer. Data represent the average of duplicate evaluations.

[0542] Example 11 - Administration of OT-101 (AP 12009).

[0543] This study evaluated two doses of AP12009 in parallel treatment groups. AP12009 was administered intratumorally at concentrations of 10 μM or 80 μM using continuous convection-enhanced delivery at a flow rate of 4 μL / min for 7 days every 2 weeks. The AP12009 regimen was based on Phase I / II clinical data from three studies.

[0544] Concentration: AP 12009 was found to be safe up to the maximum evaluated concentration of 80 μM. Flow rate: Some results from Study G003 indicated that infusion rate may constitute an important safety aspect, especially in patients with large tumors, who are at high risk of developing cerebral edema rather rapidly due to a depletion of the brain's ability to compensate for increased intracranial pressure. Administration at a flow rate of 8 μL / min for 4 days was evaluated as safe, but it could not be excluded that AEs of higher severity occurred more frequently at 8 μL / min for 7 days. Considering the balance between safety and efficacy, it was decided to maintain the infusion period for 7 days but reduce the flow rate to 4 μL / min (total volume added in each treatment cycle: 40.32 mL). Thus, although longer drug exposure could be achieved, the infusion volume was reduced by half compared to the 8 μL / min infusion rate for 4 days. The flow rate of isotonic saline infusion during the 7-day rest period was set at 1 μL / min to reduce the risk of developing symptoms of increased intracranial pressure.

[0545] Due to the significant dead space volume of the application system, administration of the appropriate concentration of AP 12009 in the brain was delayed at the beginning of each treatment cycle. After 10 hours, it was determined that 95% of the nominal AP 12009 concentration had been achieved. This delay at the beginning of each AP 12009 infusion had to be compensated for to allow complete administration of the nominal dose of AP 12009 within the specified infusion period. Therefore, after switching to isotonic saline infusion, the flow rate was increased to 4 μL / min for the equivalent of 10 hours at the beginning of the rest period to flush any remaining AP 12009 out of the dead space. The flow rate was then reduced to 1 μL / min.

[0546] Example 12 - Effect of OT-101 and chemotherapy in glioma.

[0547] A non-inferiority analysis was performed on overall survival (OS) for patients who received either OT-101 or standard chemotherapy (TMZ, PCV, or BCNU).A total of 156 subjects were evaluated: 101 subjects in the OT-101 study group (G004: 89 subjects, G005: 12 subjects) and 55 subjects in the standard chemotherapy group (G004: 45 subjects, G005: 10 subjects).

[0548] Descriptive statistics were performed for both the G004 and G005 studies. Among the 89 subjects in the test group of the G004 study, the mean overall survival (OS) was 507.5 days; the median OS was 364 days, with a standard deviation of 411.5 days. In the G004 control group of 45 subjects, the mean OS was 471.2 days; the median OS was 333 days, with a standard deviation of 373.0 days. Among the 12 subjects in the G005 test group, the mean OS was 507.8 days; the median OS was 368.4 days, with a standard deviation of 461.1 days. In the G005 control group of 9 subjects, the mean OS was 397.1 days; the median OS was 417.1 days, with a standard deviation of 216.0 days. Table 21 is a tabular representation of the data presented above.

[0549] Table 21. Descriptive statistics of overall survival for patients receiving OT-101 or standard chemotherapy in the G004 and G005 studies TIFF2025538189000027.tif34166

[0550] For the noninferiority study, a two-sample noninferiority test was performed on survival data using Cox regression. Specifically, to determine whether the upper 90.0% confidence limit (CL) of the hazard ratio (HR) was within the noninferiority hypothesis, a Wald test or a 100 (1-2α)% CI test for noninferiority was used. The α level was set at 0.050 for the hazard ratio (hazard ratio [HR] = hazard [treatment group] / hazard [reference group]), with a noninferiority limit of 1.25. A higher hazard was considered worse if it exceeded the limit of the noninferiority hypothesis, i.e., H1:HR < noninferiority limit, and the Efron Ties Method was used.

[0551] For the alternative hypothesis of HR < 1.25, the hazard ratio was calculated to be 0.9168, with a 90.0% confidence limit of the hazard ratio of 0.6865 to 1.2245, and a p-value of 0.0390. The Wald Z score was determined to be -1.7621. In Cox regression, the hazard ratio (HR), commonly referred to as the risk, is equal to Exp(B), where B is the estimated regression coefficient. For the regression coefficient of the independent variable, B1 (treatment = "treatment"), the regression coefficient (B) was determined to be -0.086823, with a standard error of 0.175912. As listed above, the risk ratio or hazard ratio was calculated to be Exp(B) = 0.9168, with a mean of 0.6516.

[0552] Based on the above results, both the hazard ratio, which was 0.9168, and the 90% upper limit of the confidence interval, which was 1.2245, were smaller than the first non-inferiority limit of 1.25, thus confirming the non-inferiority of OT-101 in terms of overall survival when compared with standard chemotherapy (TMZ, BCNU, or PCV) in both G004 and G005.

[0553] Among the control groups, the largest number of subjects received TMZ, with 43 subjects (36 subjects in the G004 study and 7 subjects in the G005 study). For subjects receiving either OT-101 or TMZ, an identical two-sample noninferiority test was performed on survival data using Cox regression. With the noninferiority alternative hypothesis determined as a hazard ratio of less than 1.25, the hazard ratio was determined to be 0.7156, with a 90% confidence level of 0.5228–0.9794, and a p-value of 0.0017. The Wald Z value was determined to be -2.9231. Based on the obtained hazard ratio, it was concluded that OT-101 was noninferior to TMZ.

[0554] Kaplan-Meier curves (log-rank test) in NCSS software were used to obtain survival curves for OT-101, standard chemotherapy, and the combination of both.

[0555] Figure 24 shows the overlaid Kaplan-Meier survival curves for both OT-101 and standard chemotherapy. The survival curves for both drugs are well within each other's confidence intervals, confirming the non-inferiority of OT-101 when compared to standard chemotherapy with respect to overall survival.

[0556] Figure 25 shows chemotherapy naive subjects treated with temozolomide (TMZ), confirming that the non-inferiority of OT-101 persists even with the addition of TMZ.

[0557] Figure 26 shows chemotherapy failers treated with the chemotherapy agents CCNU / BCNU, confirming the non-inferiority of OT-101 with the addition of CCNU / BCNU.

[0558] Example 13 - TGF-β2 is a valid target for therapy against glioma.

[0559] Three TGFB2 probe sets showed increased expression levels in DIPG patients (Figure 27: the mean fold difference for probe set 228121_at was 2.48 (linear contrast P-value=3.40×10-4); probe set 220407_s_at was 2.00 (linear contrast P-value=0.006); and probe set 209909_s_at was 1.81 (linear contrast P-value=0.0185). One of the TGFB2 probe sets also showed the highest level of mean expression in DIPG patients: TGFB2_228121_at (mean=9.25±0.18 log2 RMA); YAP1_224894_at (mean=8.55±0.19 log2 RMA).

[0560] The results are summarized in Table 22.

[0561] Table 22. TGF-β2 is a valid target for glioma therapy TIFF2025538189000028.tif41170

[0562] TGFB2 interactome expression representative of probe sets in pediatric DIPG patients compared to normal samples. Log2-transformed fold-difference values ​​for each subject (column (N = 29)) and each probe set from the GEO Archive dataset GSE26576 are shown for mean-centered DIPG patients relative to normal samples (N = 2). Subjects and probe sets were organized using a two-way clustering algorithm using an average distance metric to determine co-regulation of all probe sets across patients and all patients across probe sets. Heat maps show the most significantly up- and down-regulated probe sets, ranging from red to blue, representing higher-than-normal to lower-than-normal expression in DIPG samples, respectively. Fold differences and linear contrast p-values ​​are shown in a table showing three probe sets for TGFB2 that were up-regulated in DIPG patients.

[0563] Example 14 - TGF-β2 is a valid target for therapy in pediatric GBM.

[0564] Similar results were obtained for pediatric GBM patients (Figure 28). Three probe sets for TGFB2 were significantly upregulated by more than two-fold in pediatric GBM patients (probe sets: 209909_s_at (fold change = 4.03, p = 1.78 × 10-5); 228121_at (fold change = 3.57, p = 8.81 × 10-5); and 220407_s_at (fold change = 2.73, p = 0.0019)). The three probe sets with the highest expression levels in pediatric GBM were TGFB2_228121_at (mean = 9.24 ± 0.15); TGFB3_209747_at (mean = 6.99 ± 0.086); and TGFB1_203085_s_at (mean = 6.80 ± 0.16).

[0565] The results are summarized in Table 23.

[0566] Table 23. TGF-β2 is a valid target for therapy in pediatric GBM TIFF2025538189000029.tif41166

[0567] Expression of TGFB1 / 2 / 3 probe sets in pediatric GBM patients compared to AO patients (control group). Log2-transformed fold-difference values ​​for each subject (column (N = 82)) and each of nine probe sets from four GEO Archive datasets (GSE19578 (N = 25); GSE32374 (N = 15); GSE34824 (N = 27); and GSE49822 (N = 15)) are shown for mean-centered pediatric GBM patients relative to control samples (N = 5; from GSE19578). Subjects and probe sets were organized using a two-way clustering algorithm using an average distance metric to determine co-regulation of all probe sets across patients and all patients across probe sets. Heat maps show the expression change for each probe set, ranging from red to blue, representing expression higher than controls to expression lower than controls in pediatric GBM samples, respectively. Fold differences and linear contrast p-values ​​are shown in the table showing three probe sets for TGFB2 that were significantly upregulated by more than two-fold in pediatric GBM patients (probe sets: 209909_s_at (fold change = 4.03, p = 1.78 × 10-5); 228121_at (fold change = 3.57, p = 8.81 × 10-5); and 220407_s_at (fold change = 2.73, p = 0.0019).

[0568] Example 15 - TGF-β2 is a selective biomarker predicting improved outcome of cancer radiotherapy.

[0569] It has been discovered herein by the inventors that TGF-β2 can be used as a biomarker to selectively predict improved outcome of radiation therapy in cancer.

[0570] A clinical study using the CTGA database demonstrated the impact of TGF-β2 expression on overall survival and survival after radiation therapy. Surprisingly, a significant survival advantage was observed in low TGF-β2 expressers compared with high TGF-β2 expressers across all four quartiles of expression. No such differences were observed for TGF-β1 and TGF-β3.

[0571] Overall survival was studied for the pediatric brainstem subset, and radiation therapy survival was performed for all glioma patients treated with radiation. Again, only TGF-β2 predicted survival; TGF-beta-1 and TGF-beta-3 did not.

[0572] Figure 29 shows that for gliomas in patients treated with radiation, there was an overall survival advantage with low TGF-β2 expression. Only TGF-β2 predicted survival. Quartiles of TGF-β2 expression are shown as A, B, C, and D.

[0573] Figure 30 shows that for gliomas in patients treated with radiation, there was an overall survival advantage with low TGF-β2 expression. Only TGF-β2 predicted survival.

[0574] Example 16 - Decreased TGF-β2 levels selectively predict improved overall survival (OS) in combination with chemotherapy (TMZ), chemotherapy (TMZ) and radiation, or anti-angiogenic therapy (bevacizumab).

[0575] It has been surprisingly discovered herein by the inventors that TGF-β2 can be used as a biomarker to selectively predict improved outcome in combination with chemotherapy (TMZ), TMZ and radiation, or anti-angiogenic therapy (bevacizumab). No such predictive results were observed for TGF-β1 and TGF-β3.

[0576] A clinical study was conducted using 23 datasets for glioma to determine TGF-β2 mRNA levels as predictive of survival after treatment with an agent of interest.

[0577] FIG. 31 shows that TGF-β2 levels selectively predict improved overall survival (OS) in combination with chemotherapy (TMZ).

[0578] FIG. 32 shows that TGF-β2 levels selectively predict improved overall survival (OS) in combination with chemotherapy TMZ and radiation.

[0579] FIG. 33 shows that TGF-β2 levels selectively predict improved overall survival (OS) in combination with anti-angiogenic therapy (bevacizumab).

Claims

1. A medicament comprising an agent for inhibiting or suppressing the expression of TGF-β2 for use in a method of treating or ameliorating symptoms of a CNS disease in a human subject or animal.

2. 10. Use of an agent for inhibiting or suppressing the expression of TGF-β2 in the preparation of a medicament for use in a method of treating or ameliorating symptoms of a CNS disease in a human subject or animal.

3. 3. The pharmaceutical or use according to claim 1 or 2, wherein the CNS disease is glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal metastasis or brain metastasis, brain or spinal cord cancer, or CNS tumor.

4. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a pharmaceutical comprising a targeted anticancer drug, a cancer growth blocking drug, an EGFR inhibitor, or a combination thereof.

5. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a pharmaceutical selected from bevacizumab, everolimus, velzutifan, dabrafenib, trametinib, and combinations thereof.

6. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a pharmaceutical that is a cancer growth inhibitor selected from an angiogenesis inhibitor, a histone deacetylase inhibitor, a hedgehog inhibitor, an mTOR inhibitor, a p53 inhibitor, a PARP inhibitor, a proteasome inhibitor, a tyrosine kinase inhibitor, and combinations thereof.

7. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with a pharmaceutical that is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib, and combinations thereof.

8. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with temozolomide.

9. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with the treatment of a CNS disease by radiation therapy or electric field therapy.

10. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is used in combination with standard treatment for the CNS disease.

11. 3. The pharmaceutical or use according to claim 1 or 2, wherein said agents, medicines, therapies and treatments are administered in parallel, simultaneously, sequentially or temporally separately, respectively.

12. 3. The medicament or use according to claim 1 or 2, wherein each agent and medicament is administered by infusion or injection, separately or in combination.

13. A pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is administered or used by continuous intracranial infusion or bolus administration.

14. 14. The method or use according to claim 13, wherein the continuous intracranial infusion comprises infusion using an Ommaya-like reservoir having a partially flexible top.

15. 15. The method or use of claim 14, wherein the continuous intracranial infusion comprises a single entry catheter placed into the target region of the brain.

16. The pharmaceutical or use according to claim 1 or 2, wherein the subject has an improved TGF-β2 signature as a result of said use.

17. 3. The medicament or use according to claim 1 or 2, wherein the use of the medicament reduces mortality at 6, 12, 18, 24, 30, or 36 months.

18. 3. The medicament or use according to claim 1 or 2, wherein the use of the medicament improves survival at 6, 12, 18, 24, 30, or 36 months.

19. The agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide complementary to a TGF-β2 transcript, as follows: SEQ ID NOs: 1 to 136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; The pharmaceutical or use according to claim 1 or 2, selected from the group consisting of:

20. The pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide having one or two or fewer mismatches compared to the target human TGF-β2.

21. The pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

22. The pharmaceutical or use according to claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 is a TGF-β2-specific antisense oligonucleotide that reduces any TGF-β1 transcript level and any TGF-β3 transcript level by less than 10%, or less than 5%, or less than 1%.

23. A pharmaceutical or use as described in claim 1 or 2, wherein the agent for inhibiting or suppressing the expression of TGF-β2 comprises a TGF-β2-specific antisense oligonucleotide having one or more nucleotides chemically modified as a phosphorothioate internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, an aminophosphoro linkage to a morpholino group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' constrained methoxyethyl bicyclic ribose group, a 2'-4' constrained ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group, or a 5-methylcytosine base.

24. 3. The method or use of claim 1, wherein the agent is conjugated to polyethylene glycol, a lipid, or triantenarry N-acetyl-galactosamine.

25. The pharmaceutical or use of claim 1 or 2, wherein the agent comprises a carrier that is sterile water for injection, saline, isotonic saline, phosphate buffered saline, or a combination thereof.

26. 3. The pharmaceutical or use according to claim 1 or 2, wherein the agent or pharmaceutical is substantially free of excipients.

27. The pharmaceutical or use of claim 1 or 2, wherein the agent or pharmaceutical is stable for at least 14 days in a carrier at 37°C while being pumped by intracranial continuous infusion, or the concentration of the antisense active agent decreases by less than 10% after 90 days of use.

28. 3. The pharmaceutical or use according to claim 1 or 2, wherein the method comprises administering the agent for inhibiting or suppressing expression of TGF-β2 by intracranial infusion, preferably for continuous intracranial infusion, on days 1 to 7 at a rate of 2 to 8 μl / min and at a concentration of the agent of 1 to 80 μM.

29. an agent comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and Ommaya-like reservoir with partially flexible top Includes a kit.

30. 1. A device for delivering a fluid pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir containing the pharmaceutical composition [117]; a pump [101] for pumping the pharmaceutical composition into an Ommaya reservoir [111] through an infusion tube [103] in fluid communication with the Ommaya reservoir and in fluid communication with the reservoir through a reservoir tube [115]; a filter [105] collinear with the injection tube; and an entry catheter [113] in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain; Including, the device.

31. 31. The device of claim 30, wherein the entry catheter [113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

32. 1. A device for delivering a fluid pharmaceutical composition by intracranial continuous infusion, comprising: a reservoir containing the pharmaceutical composition [117]; a pump [101] for pumping the pharmaceutical composition through an infusion tube [103] into an access port [107], the pump being in fluid communication with an Ommaya reservoir [111], the reservoir being in fluid communication with the pump through a reservoir tube [115]; a filter [105] collinear with said injection tube; an indwelling tube [109] in fluid communication with the access port and the Ommaya reservoir [111]; and an entry catheter [113] in fluid communication with the Ommaya reservoir, the entry catheter being substantially straight and adapted for intraventricular entry into a target region of the brain; Including, the device.

33. 33. The device of claim 32, wherein the entry catheter [113] is non-linear and has one or more bends for intraventricular entry into the target region of the brain.

34. 34. The device of any one of claims 30 to 33, wherein the Ommaya reservoir [111] comprises a partially flexible top.

35. 34. The device of any one of claims 30 to 33, wherein the device provides continuous infusion of a therapeutically effective amount of the fluid pharmaceutical composition to the target area.

36. 34. The device of any one of claims 30 to 33, wherein a distal end of the entry catheter enters the target region of the brain.

37. 34. The device of any one of claims 30-33, wherein the Ommaya reservoir [111] holds the pharmaceutical composition behind a membrane for a period of time for sustained release of the pharmaceutical composition into the entry catheter.

38. The device of any one of claims 30 to 33, wherein the distal end of the entry catheter that enters the brain has a step-down end, a recessed step end, a multi-port end, a micro-hole end, or a balloon-tipped end.

39. 34. The device of any one of claims 30 to 33, wherein the pump [101] is a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or other pump with similar specifications.

40. 34. The device of any one of claims 30-33, wherein the infusion rate of the fluid pharmaceutical composition is from 0.01 to 3000 ml / hr, or from 0.01 to 100 ml / hr, or from 0.01 to 2 ml / hr, or from 0.01 to 1 ml / hr, or from 0.05 to 0.5 ml / hr.

41. The device according to any one of claims 30 to 33, wherein the infusion tube [103] or the indwelling tube [109] is a PEGA Line 100 SF 100cm with a 0.2μm sterile filter, or a 200cm infusion line with a 0.2μm sterile filter, or a Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tube with similar specifications.

42. 34. The device of any one of claims 30-33, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing expression of TGF-β, which can be used to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

43. 34. The device of any one of claims 30-33, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of an agent, medicament, or delivery vehicle.

44. 34. The device of any one of claims 30 to 33, wherein the fluid pharmaceutical composition is for the treatment of a CNS disease or CNS cancer.

45. 34. The device of any one of claims 30-33, wherein said fluid pharmaceutical composition is for the treatment of glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cord cancer, or CNS tumor.

46. The fluid pharmaceutical composition comprises a TGF-β2 specific antisense oligonucleotide complementary to the TGF-β2 transcript: SEQ ID NOs: 1-136 of Table 2, and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof. The device of any one of claims 30 to 33, comprising an agent for inhibiting or suppressing expression of TGF-β2 selected from:

47. 34. The device of any one of claims 30 to 33, which operates in combination with radiation therapy or electric field therapy.

48. 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir [402] containing the pharmaceutical composition, comprising a hard shell [403], a flexible top [401], and a non-flexible mounting plate [405]; a port [407] in fluid communication with the reservoir; and an entry catheter that is substantially straight and in fluid communication with the reservoir for intraventricular entry into the target region of the brain [413] Including, the device.

49. 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir [502] containing the pharmaceutical composition, comprising an upper rigid shell [503], a lower rigid shell [504], a flexible top [501], and a non-flexible mounting plate [505]; a port [507] in fluid communication with the reservoir for attaching an infusion line; and a port in fluid communication with said reservoir for attaching an entry catheter [509] Including, the device.

50. 1. A device for delivering a pharmaceutical composition by continuous intracranial infusion, comprising: a reservoir [602] containing the pharmaceutical composition, comprising an upper rigid shell [603], a flexible top [601], and a non-flexible mounting plate [605]; a port [619] in fluid communication with the reservoir for attaching an infusion line; and a port in fluid communication with said reservoir for attaching an entry catheter [607] Including, the device.

51. 1. A collar for a device for delivering a pharmaceutical composition by intracranial infusion, comprising: A hard shell [604] having an opening [606] exposing the flexible top of the device. Including color.

52. 1. A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, comprising: a reservoir containing the pharmaceutical composition; pump; an Ommaya reservoir having a partially flexible top; an inlet tube for connecting the reservoir to the pump and the pump to the Ommaya reservoir; filters; and Entry catheter Includes a kit.

53. 53. The kit of claim 52, wherein the entry catheter is substantially straight for intraventricular entry into a target region of the brain.

54. 53. The kit of claim 52, wherein the entry catheter is non-linear and has bends for intraventricular entry into the target region of the brain.

55. The kit of any one of claims 52 to 54, wherein the infusion tube is external to the subject.

56. 55. The kit of any one of claims 52-54, wherein a portion of the infusion tubing connecting the pump to the Ommaya reservoir is placed indwelling in the subject.

Images