Compositions and methods for CNS disease

Abstract

The present invention describes agents, methods, compositions, kits and pharmaceutical uses for inhibiting or suppressing the expression of TGF-beta2 for treating or alleviating the symptoms of CNS disease, including diffuse midline glioma (DMG) and K27M GBM, in a human subject or animal. The agent may be used in combination with a cancer drug. These purposes are achieved by formulations of agents for inhibiting or suppressing the expression of TGF-beta2. More specifically, the present invention discloses compositions, methods and uses of antisense oligonucleotides directed against TGF-beta 2 in a CNS disease treatment regimen. The invention further describes novel devices and methods for delivery of pharmaceutical compositions by intracranial infusion.

Description

[0001] Sequence Listing

[0002] The application includes a sequence listing submitted electronically in the form of an ST.26 file, created on November 7, 2023, named 018988-006WO1_SL.xml, which is 120,186 bytes in size. Technical Field

[0003] The present invention relates to methods, agents, and uses for treating central nervous system (CNS) cancers by continuous intracranial infusion. More specifically, the present invention discloses methods, agents, and uses for inhibiting or suppressing TGF-β2, which provide improved clinical outcomes for such diseases. The present invention provides stable formulations of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents and methods of use for treating CNS cancers.

[0004] The present invention relates to devices and methods for delivering pharmaceutical compositions via intracranial infusion. More specifically, the devices of the present invention may 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 injections to continuous infusions, via an intraventricular access catheter into a target area of ​​the brain. Background Art

[0005] CNS diseases have been treated with a variety of agents via the intracranial route. For CNS tumors, a convection-enhanced delivery (CED) device can be used to dispense drugs directly to the tumor in the brain using multiple catheters placed close to the tumor.

[0006] However, disadvantages and complications of these infusion methods are: (a) positioning the catheter and its tip near the brain tumor is complex and often requires simultaneous imaging (e.g., x-ray-based imaging) to achieve this, (b) these methods require inserting multiple catheters into the tumor, which is difficult or impossible to achieve and may cause tissue destruction, (c) catheters cannot be permanently implanted because the brain grows larger, especially in pediatric patients, tumors migrate, and tumors metastasize, and (d) most tumors have already metastasized within or outside the CNS, making local treatment meaningless.

[0007] Furthermore, a disadvantage and complication of such approaches is the limited infusion and mobility of the drug throughout the CNS compartment.

[0008] Another disadvantage is that intratumorally implanted catheter tips may not provide adequate infusion and flow throughout the entire CNS compartment.

[0009] There is a need for a device for intracranial systemic, bolus, and / or continuous infusion of therapeutic agents for use in methods of treating central nervous system (CNS) disorders, including cancer.

[0010] What is needed are methods and agents for inhibiting or suppressing factors in the unpredictable pathology of central nervous system diseases, particularly cancer. For example, there is a pressing need for methods and agents for inhibiting TGF-β activity and / or suppressing TGF-β-related pathologies, which could improve the efficacy of treating central nervous system cancers. Summary of the Invention

[0011] The present invention provides therapies for treating or ameliorating the symptoms of CNS diseases, such as CNS cancers.

[0012] In some embodiments, the present invention includes agents and compositions for inhibiting or suppressing TGF-β2 to provide improved clinical outcomes for CNS diseases.

[0013] In a further embodiment, the present invention provides a stable formulation of an anti-TGF-β2 agent for use in various therapies for CNS diseases. Examples of anti-TGF-β2 agents include TGF-β2 inhibitors, such as antisense oligonucleotides, pharmaceutically acceptable salt forms, esters, polymorphs or stereoisomers thereof, and combinations thereof.

[0014] In a further aspect, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for use in therapies targeting CNS diseases. The stable formulations of the present invention provide surprisingly improved clinical outcomes. Stable formulations of agents that suppress TGF-β2 can be used to treat CNS diseases, particularly CNS cancers.

[0015] The present invention provides devices and systems for continuous intracranial, intrathecal or intraventricular infusion of therapeutic agents to treat 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.

[0016] The infusion system of the present invention offers significant advantages because it allows for the continuous delivery of an effective amount or dose of a pharmaceutical composition to a patient with greater safety and ease of insertion throughout the CNS. For example, compared to localized delivery via CED methods, the infusion system of the present invention will deliver a more effective amount of drug throughout the CNS / cerebral ventricles via the intraventricular route. This advantage is particularly important for the treatment of CNS cancers.

[0017] The device of the present invention can overcome the shortcomings and complexities of conventional methods. The infusion system of the present invention has significant advantages because placement of the catheter and its tip into the brain is relatively easy and does not require complete imaging. In addition, the infusion and flow of drugs to the brain utilizes the fluid circulation in the brain to effectively distribute the drug to a larger area and target tissue. Furthermore, the system of the present invention can be used in a novel mode of operation to provide continuous infusion to the target tissue.

[0018] The present invention also relates to devices and systems for the continuous intracranial, intrathecal, or intraventricular infusion of therapeutic agents for the treatment of central nervous system (CNS) diseases of the brain and spine. The system of the present invention will deliver compositions and agents for inhibiting or suppressing TGF-β2 to improve the clinical outcomes of such diseases. The system can use stable formulations of anti-TGF-β2 agents, including antisense oligonucleotide compositions and other agents for the treatment of CNS diseases (including CNS cancers).

[0019] The present invention also relates to methods of treating central nervous system (CNS) disorders of the brain and spine.

[0020] The present invention also provides a device for continuous intracranial, intrathecal or intraventricular infusion of therapeutic agents for treating central nervous system (CNS) diseases. Continuous infusion into brain tumors using the device and apparatus of the present invention is much easier than conventional CED methods and other similar or conventional methods.

[0021] The infusion device of the present invention offers significant advantages because it continuously delivers an effective amount or dose of a pharmaceutical composition to a patient with greater safety and ease of insertion throughout the CNS. For example, compared to localized delivery via CED methods, the infusion device and apparatus of the present invention will deliver more effective amounts of a drug throughout the CNS / cerebral ventricles via the intraventricular route. This advantage is particularly important for the treatment of CNS cancers.

[0022] The device of the present invention overcomes the shortcomings and complexities of known methods. The infusion device of the present invention offers significant advantages because placement of the catheter and its tip into the brain is relatively easy and does not require imaging. Furthermore, the infusion and flow of drugs into the brain utilizes the brain's fluid circulation, effectively distributing the drug 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.

[0023] The present invention also relates to a device for continuous intracranial, intrathecal, or intraventricular infusion of therapeutic agents for the treatment of central nervous system (CNS) diseases of the brain and spine. The device of the present invention delivers compositions and agents used to inhibit or suppress TGF-β2 to improve clinical outcomes of such diseases. The device uses a stable formulation of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents used to treat CNS diseases (including CNS cancers).

[0024] The present invention also relates to methods of treating central nervous system (CNS) disorders of the brain and spine.

[0025] Embodiments of the present invention include the following:

[0026] An agent for inhibiting or suppressing TGF-β2 expression, the agent is used to treat or improve the symptoms of CNS diseases in human subjects or animals.

[0027] Use of an agent for inhibiting or suppressing TGF-β2 expression in the preparation of a medicament for treating or ameliorating the symptoms of a CNS disease in a human subject or an animal.

[0028] A method for treating or ameliorating symptoms of a CNS disease in a human subject or animal in need thereof, the method comprising:

[0029] preparing a composition comprising an agent for inhibiting or suppressing TGF-β2 expression in a carrier; and

[0030] A therapeutically effective amount of the composition is administered to the subject.

[0031] The above-mentioned medicament, use or method, wherein the CNS disease is glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain cancer or spinal cancer, or CNS tumor.

[0032] The above-mentioned agents, uses or methods are combined with drugs comprising targeted cancer drugs, cancer growth inhibitors, EGFR inhibitors or combinations thereof.

[0033] The above-mentioned agent, use or method is combined with a drug selected from bevacizumab, everolimus, belzutifan, dabrafenib, trametinib and combinations thereof.

[0034] The above-mentioned agent, use or method is combined with a drug that is a cancer growth inhibitor, wherein the cancer growth inhibitor is selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0035] The above-mentioned agent, use or method is combined with a drug that is an EGFR inhibitor, wherein the EGFR inhibitor is selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and a combination thereof.

[0036] The above-mentioned medicament, use or method is combined with temozolomide.

[0037] The above-mentioned medicament, use or method, in combination with treatment of CNS diseases by radiation therapy or therapeutic electric fields.

[0038] The above-mentioned medicament, use or method, wherein the administration or use of the composition or medicament is combined with standard of care treatment for a CNS disease.

[0039] The above-mentioned medicament, use or method, wherein the medicament, drug, therapy, treatment and administration are each administered concurrently, simultaneously, sequentially or separately in time.

[0040] The above-mentioned medicament, use or method, wherein each medicament and drug are administered separately or in combination by infusion or injection.

[0041] The above-mentioned medicaments, uses or methods include administration or use by intracranial continuous infusion or bolus administration.

[0042] The above-mentioned medicament, use or method, wherein the intracranial continuous infusion comprises infusion using an ommaya-like reservoir with a partially-flexible top.

[0043] The above medicament, use or method, wherein the intracranial continuous infusion comprises a single access catheter placed into the target area of ​​the brain.

[0044] The above-mentioned medicament, use or method, wherein the subject has an improved TGF-β2 profile after administration or use.

[0045] The above-mentioned medicament, use or method, wherein the administration or use reduces the mortality rate at 6, 12, 18, 24, 30 or 36 months.

[0046] The above-mentioned medicament, use or method, wherein administration or use increases survival rate at 6, 12, 18, 24, 30 or 36 months.

[0047] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is selected from the following TGF-β2 specific antisense oligonucleotides complementary to TGF-β2 transcripts:

[0048] Table 2 SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0049] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide, which has no more than one or two mismatches compared to the human target TGF-β2.

[0050] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide, which reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80% or at least 90%.

[0051] The above-mentioned agent, use or method, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide, which reduces the level of any TGF-β1 transcript and any TGF-β3 transcript by less than 10%, or less than 5% or less than 1%.

[0052] The above-mentioned agents, uses or methods comprise TGF-β2-specific antisense oligonucleotides, which have one or more nucleotides chemically modified to be phosphorothioate internucleoside bonds, methoxypropylphosphonate internucleoside bonds, or aminophosphoric acid bonds connected 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.

[0053] The above-mentioned agent, use or method, wherein the agent is conjugated with polyethylene glycol, lipid or triantenarry N-acetyl-galactosamine.

[0054] The above-mentioned medicament, use or method comprises a carrier of sterile water for injection, saline, isotonic saline, phosphate-buffered saline or a combination thereof.

[0055] The above-mentioned medicament, use or method, wherein the medicament, drug or administration is substantially free of excipients.

[0056] The above-mentioned agent, use or method, wherein the agent, drug or administration is stable in the carrier for at least 14 days at 37°C when pumped as an intracranial continuous infusion, or has less than a 10% decrease in the concentration of the antisense active agent after 90 days of use.

[0057] The above-mentioned medicament, use or method, wherein the method comprises administering the composition by intracranial infusion at a rate of 2-8 μl / min and a drug concentration of 1-80 μM from day 1 to day 7, preferably by continuous intracranial infusion.

[0058] A kit comprising:

[0059] A medicament comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1-136 in Table 2, chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and

[0060] Omayeh-type reservoir with a partially flexible top.

[0061] A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0062] a reservoir

[117] containing a pharmaceutical composition;

[0063] a pump

[101] that forces the pharmaceutical composition through the infusion tube

[103] into the Omayr reservoir

[111] , the pump being in fluid communication with the Omayr reservoir, and wherein the pump is in fluid communication with the reservoir via a reservoir tube

[115] ;

[0064] a filter

[105] inline with the infusion tubing; and

[0065] An access catheter

[113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0066] The above device, wherein the access catheter

[113] is non-linear and has one or more bends to access a target area of ​​the brain within a cerebral ventricle.

[0067] A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0068] a reservoir

[117] containing a pharmaceutical composition;

[0069] a pump

[101] that forces the pharmaceutical composition through an infusion tube

[103] into an access port

[107] , the pump being in fluid communication with an Omayr reservoir

[111] , and wherein the reservoir is in fluid communication with the pump via a reservoir tube

[115] ;

[0070] a filter

[105] which is in line with the infusion tubing;

[0071] an indwelling tube

[109] in fluid communication with the access port and the Omayr reservoir

[111] ; and

[0072] An access catheter

[113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0073] The above device, wherein the access catheter

[113] is non-linear and has one or more bends to access a target area of ​​the brain within a cerebral ventricle.

[0074] The above device, wherein the Omayya reservoir

[111] includes a partially flexible top.

[0075] The above device, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to a target area.

[0076] The above device, wherein the distal end of the access catheter enters the target area of ​​the brain.

[0077] The above device, wherein the Omayr reservoir

[111] holds the pharmaceutical composition behind the membrane for a period of time to provide a sustained release of the pharmaceutical composition to the access catheter.

[0078] The above device, wherein the distal tip of the access catheter entering the brain is a step-down end, a recessed step end, a multi-port end, a microporous end or a balloon tipped end.

[0079] The above device, wherein the pump

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

[0080] The above device, wherein the infusion rate of the fluid drug 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.

[0081] The above device, wherein the infusion tube

[103] or the indwelling tube

[109] is a PEGA Line 100SF 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 an extension (#21-7106-24), or other tubes with similar specifications.

[0082] The above device, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β expression, can be used to treat or improve symptoms of CNS diseases in human subjects or animals.

[0083] The above device, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of a pharmaceutical agent, a drug or a delivery vehicle.

[0084] The above device, wherein the fluid pharmaceutical composition is a therapeutic agent for CNS disease or CNS cancer.

[0085] The above device, wherein the fluid drug composition is a therapeutic agent for glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cancer, or CNS tumor.

[0086] The above device, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β2 expression, the agent being selected from the group consisting of TGF-β2 specific antisense oligonucleotides complementary to TGF-β2 transcripts as described below:

[0087] Table 2 SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0088] The above device, wherein the device operates in combination with radiation therapy or electric field therapy.

[0089] A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0090] a reservoir

[402] containing a pharmaceutical composition, the reservoir comprising a hard shell

[403] , a flexible top

[401] , and a non-flexible mounting plate

[405] ;

[0091] a port

[407] in fluid communication with the reservoir; and

[0092] An access catheter

[413] is in fluid communication with the reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0093] A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0094] a reservoir

[502] containing a pharmaceutical composition, the reservoir comprising an upper hard shell

[503] , a lower hard shell

[504] , a flexible top

[501] , and a non-flexible mounting plate

[505] ;

[0095] a port

[507] in fluid communication with the reservoir and for connecting an infusion line; and

[0096] A port

[509] is in fluid communication with the reservoir and is used to connect an access catheter.

[0097] A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0098] a reservoir

[602] containing a pharmaceutical composition, the reservoir comprising an upper hard shell

[603] , a flexible top

[601] , and a non-flexible mounting plate

[605] ;

[0099] a port

[619] in fluid communication with the reservoir and for connecting an infusion line; and

[0100] A port

[607] is in fluid communication with the reservoir and is used to connect an access catheter.

[0101] A collar of a device for delivering a pharmaceutical composition by continuous intracranial infusion, the collar comprising:

[0102] A hard shell

[604] having an opening

[606] exposing the flexible top of the device.

[0103] A method of administering a pharmaceutical composition by continuous intracranial infusion, comprising:

[0104] Fitting the device according to above to the patient; and

[0105] The pharmaceutical composition is pumped into the device to provide a continuous intracranial infusion to the patient.

[0106] The method above, wherein the device is mounted and the access catheter is placed into the brain without simultaneous imaging of the head or brain.

[0107] The method above, wherein the device is mounted and a single access catheter is placed into the brain.

[0108] A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, the kit comprising:

[0109] a reservoir containing a pharmaceutical composition;

[0110] pumps;

[0111] Omayeh reservoir with partially flexible top;

[0112] infusion tubing for connecting the reservoir to the pump and the pump to the Omayeh reservoir;

[0113] Filters; and

[0114] Enter the catheter.

[0115] The above kit, wherein the access catheter is substantially linear and is used to access a target area of ​​the brain intraventricularly.

[0116] The above kit, wherein the access catheter is non-linear and has a bend to access a target area of ​​the brain intraventricularly.

[0117] The above kit, wherein the infusion tube is outside the subject's body.

[0118] The above kit, wherein a portion of the infusion tubing connecting the pump to the Omayr reservoir is left indwelling in the subject.

[0119] BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion is shown. Figure 1The device shown includes an external portion that advantageously allows the pump

[101] and reservoir

[117] to be located or connected to the outside of the patient's body or anywhere on the patient's body. The external portion delivers the pharmaceutical composition from the pump through an infusion tube

[103] and forces it into the Omayr reservoir

[111] . The device for delivering the pharmaceutical composition by continuous infusion intrathecally or intraventricularly includes a reservoir

[117] containing the pharmaceutical composition; a pump

[101] forcing the pharmaceutical composition through the infusion tube

[103] into the Omayr reservoir

[111] , the pump being in fluid communication with the Omayr reservoir, wherein the pump is in fluid communication with the reservoir through a reservoir tube

[115] ; a filter

[105] in line with the infusion tube; and an access catheter

[113] in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target area of ​​the brain intraventricularly.

[0121] Figure 2 A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion is shown. Figure 2 The device shown includes an indwelling portion that advantageously allows the patient to carry the device. The indwelling portion includes an indwelling tube

[109] in fluid communication with the access port

[107] and the Omayr reservoir

[111] , the indwelling tube being used to transfer fluid from the access port to the Omayr reservoir. The device for delivering a pharmaceutical composition by continuous infusion intrathecally or intraventricularly comprises: a reservoir

[117] containing a pharmaceutical composition; a pump

[101] for forcing the pharmaceutical composition through a pumping tube

[103] into the access port

[107] , the pump being in fluid communication with the Omayr reservoir, wherein the reservoir is in fluid communication with the pump via a reservoir tube

[115] ; a filter

[105] in line with the pumping tube; an indwelling tube

[109] connected from the access port to the Omayr reservoir

[111] ; and an access catheter

[113] in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target area of ​​the brain intraventricularly.

[0122] Figure 3 A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion is shown. Figure 3The device shown includes an external portion that advantageously allows the pump

[101] and reservoir

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

[103] and forces it into the Omayr reservoir

[111] . The device for delivering the pharmaceutical composition by continuous infusion intrathecally or intraventricularly comprises: a reservoir

[117] containing the pharmaceutical composition; a pump

[101] forcing the pharmaceutical composition through the infusion tube

[103] into the Omayr reservoir

[111] , the pump being in fluid communication with the Omayr reservoir, wherein the pump is in fluid communication with the reservoir through a reservoir tube

[115] ; a filter

[105] in line with the infusion tube; and an access catheter

[113] in fluid communication with the Omayr reservoir, wherein the access catheter is non-linear and has a bend to access a target area of ​​the brain intraventricularly.

[0123] Figure 4 Devices for delivering pharmaceutical compositions by intrathecal or intraventricular continuous infusion are shown. Figure 4 The device shown includes an indwelling portion that advantageously allows the patient to carry the device. The indwelling portion includes an indwelling tube

[109] in fluid communication with the access port

[107] and the Omayr reservoir

[111] , the indwelling tube being used to transfer fluid from the access port to the Omayr reservoir. The device for delivering a pharmaceutical composition by continuous infusion intrathecally or intraventricularly comprises: a reservoir

[117] containing a pharmaceutical composition; a pump

[101] for forcing the pharmaceutical composition into the access port

[107] through a pumping tube

[103] , the pump being in fluid communication with the Omayr reservoir, wherein the reservoir is in fluid communication with the pump through a reservoir tube

[115] ; a filter

[105] in line with the pumping tube; an indwelling tube

[109] connected from the access port to the Omayr reservoir

[111] ; and an access catheter

[113] in fluid communication with the Omayr reservoir, wherein the access catheter is non-linear and has a bend to access a target area of ​​the brain within the ventricle.

[0124] Figure 5 An Omayr delivery device is shown for use in a method of delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. Figure 5 The Omayyad delivery device shown can be used as e.g. Figure 1 and Figure 2The Omayyad delivery device comprises a non-flexible hard shell

[403] comprised of an inert material such as metal or hard plastic that encloses and defines a reservoir

[402] for a drug-containing fluid. The Omayyad delivery device further comprises a flexible top

[401] comprised of a flexible material such as rubber, elastomer, or plastic. The flexible top

[401] can be from 10% flexible to 50% flexible relative to the area of ​​the entire shell (

[401] plus

[403] ). The Omayyad delivery device further comprises a non-flexible mounting plate

[405] comprised of an inert material such as metal or hard plastic. The Omayyad delivery device further comprises a port

[407] for connecting an infusion line to fluidly communicate with the Omayyad delivery device. For example, the port

[407] can be connected to Figure 1 and Figure 2 infusion line

[103] in the shell

[403] . Fluid communication between the reservoir defined by the shell

[403] and the infusion line is provided by an internal passage

[421] . The Omayyeh delivery device also includes an access catheter

[413] in fluid communication with the Omayyeh reservoir, wherein the access catheter is substantially linear and enters a target area of ​​the brain intraventricularly. Fluid communication between the reservoir defined by the shell

[403] and the access catheter is provided by an internal passage

[423] . In another embodiment, the hard shell

[403] can be a separate protective collar that can be placed over a fully flexible top having an overall area of ​​

[401] plus

[403] to reduce the exposed area of ​​the flexible top to the area of ​​

[401] (see Figure 8 ).

[0125] Figure 6 An Omayr delivery device is shown for use in a method of delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. Figure 6 The Omayyad delivery device shown can be used as e.g. Figure 1 and Figure 2 The Omayyad delivery device comprises a non-flexible hard shell

[503] and

[504] composed of an inert material such as metal or hard plastic, which encloses and defines a reservoir

[502] for a drug-containing fluid. The Omayyad delivery device also comprises a flexible top

[501] composed of a flexible material such as rubber, elastomer or plastic. The flexible top

[501] can be from 10% flexible to 50% flexible relative to the area of ​​the entire shell (

[501] plus

[503] ), such as Figure 6 The Omayel delivery device further comprises a non-flexible mounting plate

[505] comprised of an inert material such as metal or hard plastic. The Omayel delivery device further comprises ports

[507] and

[509] for connecting an infusion line to fluidly communicate with the Omayel delivery device. For example, port

[507] may be connected to Figure 3 and Figure 4The indwelling tube

[109] in the housing

[503] . Fluid communication between the reservoir defined by the housing

[503] and the infusion line is provided by the internal passage

[521] . For example, the port

[509] can be connected to Figure 1 and Figure 2 In another embodiment, the hard shell

[503] can be a separate protective collar that can be placed over a fully flexible top having the total area of ​​

[501] plus

[503] to reduce the exposed area of ​​the flexible top to the area of ​​

[501] (see Figure 8 ).

[0126] Figure 7 An Omayr delivery device is shown for use in a method of delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. Figure 7 The Omayyad delivery device shown can be used as e.g. Figure 3 and Figure 4 The Omayyad delivery device includes a non-flexible hard shell

[603] composed of an inert material such as metal or hard plastic, which encloses and defines a reservoir

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

[601] composed of a flexible material such as rubber, elastomer, or plastic. The Omayyad delivery device also includes a non-flexible mounting plate composed of an inert material such as metal or hard plastic. The Omayyad delivery device also includes ports

[607] and

[619] for connecting an infusion line to communicate with the Omayyad delivery device fluid. For example, port

[607] can be connected to Figure 3 and Figure 4 The infusion line

[103] in the housing

[603] . Fluid communication between the reservoir defined by the housing

[603] and the infusion line is provided by the internal passage

[621] . For example, the port

[619] can be connected to Figure 3-4 The inlet conduit

[113] in the housing

[603] . Fluid communication between the reservoir defined by the housing

[603] and the inlet conduit is provided by the internal passage

[619] . In another embodiment, the hard housing

[603] can be a separate protective collar that can be placed over a fully flexible top having the total area of ​​

[601] plus

[603] to reduce the exposed area of ​​the flexible top to the area of ​​

[601] (see Figure 8 ).

[0127] Figure 8 An embodiment of a collar for an Omayr delivery device for use in a method for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion is shown. In this embodiment, the hard shell

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

[606] exposes only a portion of the flexible top of the Omayr reservoir.

[0128] Figure 9 The effects of delivering the pharmaceutical composition by intrathecal or intraventricular continuous infusion are shown. Figure 9 Shown is 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) for 7 days as indicated. Secreted TGF-β2 was measured in the cell supernatant by ELISA. Results represent the median, minimum, and maximum values ​​of three independent experiments.

[0129] Figure 10 Infusion from an Omayyad-type reservoir of the present invention is shown, the reservoir having a 339 mm 2 The surface area of ​​the catheter is 1.4 mm and the diameter of the opening in the catheter is 1.4 mm. In this example, the dark test solution is shown to slowly diffuse downward through the catheter opening. This diffusion from the Omayor-type reservoir of the present invention continues for several hours.

[0130] Figure 11 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from hours to days.

[0131] Figure 12 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from days to weeks.

[0132] Figure 13 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from weeks to months.

[0133] Figure 14 Shown are the mRNA levels of TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples and 29 normal pons samples.

[0134] Figure 15 A statistically significant positive correlation was shown with TGFB2 mRNA levels.

[0135] Figure 16 Shown are relative expression of specific transcription factors in tumor samples from 41 pediatric patients with DIPG (n=29) or H3K27M-mutant GBM.

[0136] Figure 17 It was shown that DIPG patients with high TGFB2 exhibited significantly worse OS outcomes than those with low TGFB2.

[0137] Figure 18 The PFS results were also significantly worse in the TGFB2-high subgroup.

[0138] Figure 19It was shown that patients in the TGFB2 high (N=29) and TGFB2 low (N=87) subgroups exhibited very similar OS outcomes.

[0139] Figure 20 Expression was shown to be selectively amplified in pediatric DIPG and associated with poor OS.

[0140] Figure 21 Shown is a waterfall plot depicting the maximum log10 reduction in tumor volume in high-grade glioma patients treated with OT-101 monotherapy and achieving a CR or PR.

[0141] Figure 22 Shown are semi-logarithmic plots of the combined 3-D tumor volume reduction curves for 19 high-grade glioma patients who received OT-101 monotherapy and achieved a CR or PR.

[0142] Figure 23 A swimmer plot shows the onset and duration of objective response in patients with high-grade glioma who received OT-101 monotherapy and achieved a CR or PR. The onset and duration of CR / PR, the end of OR, and the onset of PD are indicated by specific signals.

[0143] Figure 24 Overlaid Kaplan-Meier survival curves for both OT-101 and standard chemotherapy are shown.

[0144] Figure 25 Shown are chemotherapy-naive patients treated with temozolomide (TMZ).

[0145] Figure 26 It was shown that chemotherapy with the chemotherapy agents CCNU / BCNU was ineffective.

[0146] Figure 27 showed that TGF-β2 is a valid target for glioma therapy using three TGFB2 probe sets, which exhibited increased expression levels in DIPG patients.

[0147] Figure 28 showed that TGF-β2 is a valid target for pediatric GBM therapy using the TGFB2 probe panel, which exhibited increased expression levels in DIPG patients.

[0148] Figure 29 showed that low TGF-β2 expression confers an overall survival advantage in patients with glioma who received radiotherapy. Only TGF-β2 was predictive of survival.

[0149] Figure 30showed that low TGF-β2 expression confers an overall survival advantage in patients with glioma who received radiotherapy. Only TGF-β2 was predictive of survival.

[0150] Figure 31 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy (TMZ).

[0151] Figure 32 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy TMZ and radiation.

[0152] Figure 33 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with antiangiogenic therapy (bevacizumab). DETAILED DESCRIPTION

[0153] The present invention provides novel devices and methods of use for delivering and administering therapeutic agents ranging from bolus administration to fixed rate infusion and continuous infusion. The devices and methods of the present invention can be used to treat CNS diseases, such as CNS cancers, via an intracranial route.

[0154] The devices and methods of the present invention can deliver pharmaceutical agents to the brain or spinal region using a pump-based continuous infusion device / system.

[0155] The present invention provides therapies for treating or ameliorating the symptoms of CNS diseases, such as CNS cancers.

[0156] In some embodiments, the present invention includes agents and compositions for inhibiting or suppressing TGF-β2 to provide improved clinical outcomes for CNS diseases.

[0157] In other embodiments, the present invention provides stable formulations of anti-TGF-β2 agents for various therapies for CNS diseases. Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, pharmaceutically acceptable salts, esters, polymorphs or stereoisomeric forms thereof, and combinations thereof.

[0158] In other aspects, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for use in therapies targeting CNS diseases. The stable formulations of the present invention provide surprisingly improved clinical outcomes. Stable formulations of agents that suppress TGF-β can be used to treat CNS diseases, particularly CNS cancers.

[0159] The present invention also provides a novel device for continuous infusion to treat CNS diseases such as CNS cancer via intrathecal or intraventricular routes, whose operating parameters can be modified to extend the drug infusion release kinetics half-life of the agent from days to weeks.

[0160] The devices and methods of the present invention can deliver pharmaceutical agents to the brain or spinal region using a pump-based continuous infusion system.

[0161] In some embodiments, the present invention provides an Omayor reservoir device having conduits to the cerebrospinal fluid and the intraventricular space, the device being connected to a pump for incremental and continuous infusion.

[0162] In some embodiments, the present invention provides an Omayer-type reservoir device having conduits to the cerebrospinal fluid and the intraventricular space, which is connected to a pump for incremental and continuous infusion.

[0163] The present invention provides a device for enhancing delivery, in some cases a portable delivery device, by infusing fluids into specific locations within the body, particularly brain tissue and tumors, preferably the ventricular space.

[0164] The present invention also provides a device for continuous intrathecal or intraventricular infusion of a therapeutic agent, comprising an Omayer-type device (see Figure 1-8 and 10). The Omayer-type device allows for insertion of an access catheter into the ventricle. Alternatively, the access catheter can be placed within the ventricle to reach a brain tumor or other part of the brain to deliver a therapeutic agent. The Omayer-type device also advantageously acts as a reservoir to hold the therapeutic agent for long-term and sustained release into the underlying brain matter or ventricle. The Omayer-type device can be made of a non-collapsible material and have a flexible portion (see Figure 1-7 ). This structure and design can produce oscillatory compression and decompression to maintain fluid movement into and out of the Omayor-type device reservoir. In fact, this new Omayor-type device and the device of the present invention can advantageously allow the reservoir to breathe. In operational use, the Omayor-type device and method can include self-pulsating or self-oscillating operation due to the patient's physical and muscle activity, which stretches and relaxes the skin near the reservoir and maintains fluid movement and drug release. The flexible top enhances fluid movement in the chamber or internal reservoir. Without the flexible top, the stagnant compartment may not allow the fluid drug composition to fully flow out for patient infusion.

[0165] The flexible top can enhance fluid motion by acting as a shock absorber to produce a more uniform fluid flow rate in the conduit.

[0166] In some aspects, the Omayor-type devices and apparatuses of the present invention can include a pump for continuously pumping to drive fluid into and out of the Omayor-type device reservoir.

[0167] The present invention provides a device for use in therapy to treat or ameliorate the symptoms of a CNS disease, such as a CNS cancer.

[0168] In some aspects, the present invention provides devices for using pumps compatible with the infusion of agents and compositions that inhibit or suppress TGF-β2 to provide improved clinical outcomes for CNS diseases.

[0169] In a further aspect, the present invention provides a stable formulation of an anti-TGF-β2 agent for various therapies for CNS diseases, wherein the formulation can be pumped for continuous infusion. Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, modifications such as LNA / 2-MOE, pharmaceutically acceptable salts, esters, polymorphs or stereoisomeric forms thereof, and combinations thereof.

[0170] The device of the present invention is connected to a suitable pump to continuously deliver small amounts of the pharmaceutical composition over several days.

[0171] The devices of the present invention offer the advantage of using an Omayer intraventricular catheter to deliver pharmaceutical compositions through an externally mounted infusion line or through an indwelling infusion construct for long-term infusion.

[0172] In another example, the device of the present invention can use an Omayor reservoir catheter as an intratumoral catheter to deliver a pharmaceutical composition. This device has the advantage of being able to be used with an external pump for short-term infusion or with extended internal tubing and a subcutaneous tunneled catheter for long-term placement for infusion. The Omayor reservoir component of the device of the present invention can provide long-term access to the cerebrospinal fluid and intraventricular space.

[0173] The infusion device of the present invention also has the advantages of delivering the pharmaceutical composition to a tumor at any location in the brain and easily inserting a catheter from the Omayer reservoir into the intraventricular space, which does not require insertion under imaging.

[0174] The infusion device of the present invention also has the advantage of using a standardized intraventricular target.

[0175] 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.

[0176] The infusion device of the present invention also has the advantage that the pharmaceutical composition can be effectively delivered to the entire CNS, including the spinal cord.

[0177] The infusion device of the present invention also has the advantage of not requiring imaging such as X-ray imaging to guide the implantation of the delivery catheter. Because the device acts as a tap, when the catheter tip reaches the cerebrospinal fluid, the cerebrospinal fluid will flow back, indicating that the catheter has been successfully placed.

[0178] In some aspects, the infusion device of the present invention also has the advantage of being used with the access catheter tip placed a shorter distance into the patient's body. The access catheter can be shorter and can be placed more shallowly because the device takes advantage of the patient's natural fluid circulation (e.g., in the brain).

[0179] In another aspect, the infusion device of the present invention also has the advantage of being used with a single access catheter to provide infusion to a patient. For example, treatment of a glioma or large brain tumor may require the placement of multiple catheters near the cancer. However, the device of the present invention can provide improved infusion even with a single catheter near the tumor.

[0180] The infusion device of the present invention also has the advantage that implantation of the delivery catheter can be semi-permanent, or even permanent.

[0181] The infusion device of the present invention also has the advantage that the Omayr reservoir can have access for needle aspiration of fluid from the device or the brain or injection of fluid into the device or the brain.

[0182] The infusion device of the present invention also has the following advantages: the Omayor device can be placed and utilized as needed to effectively deliver drugs and treatments. For example, the Omayor device can be placed directly on the roof of the ventricle and a straight ventricular access catheter can be used

[113] , such as Figure 1 and Figure 2 In a further example, the Omayya device can be placed at a convenient location on the patient's skull and connected to a ventricular access catheter

[113] that is positioned to Figure 3 and Figure 4 The angle is bent to reach the target location in the brain.

[0183] Aspects of the present invention include the following:

[0184] A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion, the device comprising ( Figure 1 ): a reservoir

[117] containing a pharmaceutical composition; a pump

[101] that forces the pharmaceutical composition through an infusion tube

[103] into an Omayr reservoir

[111] , the pump being in fluid communication with the Omayr reservoir, and wherein the pump is in fluid communication with the reservoir via a reservoir tube

[115] ; a filter

[105] that is in line with the infusion tube; and an access catheter

[113] in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly. The access catheter

[113] may be non-linear and have a bend to enter a target region of the brain intraventricularly ( Figure 3 ).

[0185] A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion, the device comprising ( Figure 2): a reservoir

[117] containing a pharmaceutical composition; a pump

[101] that forces the pharmaceutical composition through a pumping tube

[103] into an access port

[107] , the pump being in fluid communication with the Omayer reservoir, wherein the reservoir is in fluid communication with the pump via a reservoir tube

[115] ; a filter

[105] in line with the pumping tube; an indwelling tube

[109] in fluid communication with the access port and the Omayer reservoir

[111] ; and an access catheter

[113] in fluid communication with the Omayer reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly. The access catheter

[113] may be non-linear and have a bend to enter a target region of the brain intraventricularly ( Figure 4 ).

[0186] The device can provide continuous infusion of a therapeutically effective amount of the pharmaceutical composition to a target area.

[0187] As used herein, a target region of the brain may be a region containing a tumor.

[0188] The distal end of the access catheter is advanced into the target area of ​​the brain.

[0189] The Omayya reservoir can hold the pharmaceutical composition behind the membrane for a period of time to continuously release the pharmaceutical composition to the access catheter. The distal tip of the access catheter entering the brain is a stepped end, a recessed stepped end, a multi-port end, a microporous end, or a balloon tip. In some aspects, the access catheter can be barium-impregnated silicone and have kink resistance and compression resistance. In other aspects, the access catheter can include an elongated surgically acceptable probe, such as a stainless steel probe, to allow the catheter to be guided during catheter placement.

[0190] The pump may be a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or another pump with similar specifications.

[0191] The infusion rate may 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.

[0192] Some examples of pumps and infusion rates for the devices of the present invention include those in Table 1 below:

[0193] Table 1: Pumps and Rates for Infusion Systems

[0194]

[0195]

[0196] The infusion line

[103] or indwelling line

[109] can be a PEGA Line 100SF 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 tubing with similar specifications.

[0197] In some aspects, the brain access catheter positioned 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 shorter in length and can be equipped with 24 inlet holes (e.g., 3 rows of 8 holes each) at the proximal end. In general, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The Omayya reservoir of the present invention can have an inner diameter of 300 to 400 mm. 2 The surface area of ​​the inner opening of the conduit can be 0.785mm 2 to 3.14mm 2 Therefore, the ratio of the reservoir surface area to the catheter surface area ranges from 96 to 509. With this ratio, the expected release time of the drug solution can be adjusted within a wide range, depending on the density of the test solution to water.

[0198] The Omayor-type device of the present invention allows for the delivery of drugs directly into the CSF and is particularly useful for the delivery of oligonucleotide and antisense oligonucleotide drugs such as OT-101, either alone or in combination with other cancer therapies.

[0199] In some aspects, the drug load can surprisingly be delivered as a single bolus infusion or as a short-term infusion of 15, 30 or 60 minutes and still achieve sustained delivery by a favorable ratio of reservoir volume to catheter opening and / or adjusting the ratio of CSF density to drug solution density.

[0200] For example, a typical reservoir surface area is 339 mm 2 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 reservoir surface area to the catheter surface area, which is related to the diffusion equation.

[0201] As described below, infusion data from rats was obtained using only a ventricular catheter without a reservoir, and the equation {Y = Span * exp(-K * X) + Plateau} governs the process, where a normalized value can be achieved when the span is 100 and the plateau is 0, with k being 7.571. Constraints were imposed on the drug reservoir, and rate changes related to the surface area ratio of the reservoir to the catheter were determined. By varying the reservoir-to-catheter ratio, drug infusion release kinetics can be slowed, resulting in favorable half-lives of days and weeks.

[0202] For example, Figure 11 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from hours to days.

[0203] For example, Figure 12 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from days to weeks.

[0204] For example, Figure 13 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from weeks to months.

[0205] One advantage of the device of the present invention is the design of the Omayor-type reservoir, which allows the device to maintain fluid flow in and out of the reservoir. The flexible top expands and contracts in response to changes in pressure, which allows the Omayor-type reservoir of the present invention to breathe properly to achieve continuous flow of solution, especially in the presence of an externally generated pumping action. Undesirable accidental or accidental release may also occur due to patient movement, such as movement of the patient's jaw or other muscles.

[0206] An advantage of the device of the present invention is that the Omayor-type device is not positioned as a mechanism for collecting CSF or for delivering a drug (drug composition) to the CSF, but rather as a reservoir for containing a drug to be infused into the CSF over a sustained period of time. To allow fluid to exit the Omayor device, a continuous flow of fluid from the reservoir is driven by a pump that also allows drug fluid to enter the reservoir. The structure of the device of the present invention advantageously prevents accidental Omayor device compression, which could suddenly deliver a large amount of drug from the reservoir into the CSF, because the Omayor delivery device portion of the device of the present invention has a rigid, non-flexible hard shell that is only partially collapsible (see Figure 1-7 ). In addition, the design of the device of the present invention advantageously maintains fluid movement in the reservoir because the reservoir includes a flexible top (see, for example, Figure 5

[401] ) that can generate oscillatory compression and decompression for fluid movement. For example, due to the flexible portion, movement of the skin or jaw muscles can slightly compress and decompress the Omayne device and maintain fluid flow in and out of the reservoir.

[0207] As used herein, a partially flexible top may refer to an Omayne-type reservoir made of a hard material with an integrated top, wherein only the uppermost portion of the top is flexible, or to an Omayne-type reservoir with a top made of a flexible material and fitted with a hard collar such that only the uppermost portion of the flexible top is exposed and can be compressed or bent.

[0208] The pharmaceutical composition may comprise an agent for inhibiting or suppressing TGF-β expression, which can be used to treat or improve the symptoms of a CNS disease in a human subject or animal. The CNS disease can be cancer. The cancer can be a glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cancer, or a CNS tumor.

[0209] In some embodiments, the compositions and methods of the invention can be used for diffuse midline glioma (DMG) and K27M GBM.

[0210] Methods and compositions for CNS diseases

[0211] Operational Parameters: The systems of the present invention can be modified to prolong the delivery of drugs for treating CNS diseases, such as CNS cancer, or ameliorating symptoms of CNS diseases in human subjects or animals in need thereof. The systems of the present invention can include pharmaceutical compositions with release kinetics half-lives of weeks to months for continuous infusion into the brain or spine.

[0212] The pharmaceutical composition for continuous infusion may comprise an agent for inhibiting or suppressing TGF-β expression, and a therapeutically sufficient amount of the composition is administered to the subject. The present invention provides systemic therapies for treating CNS diseases such as cancer or ameliorating symptoms of CNS diseases.

[0213] Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, artemisinin, pharmaceutically acceptable salts, esters, polymorphs or stereoisomeric forms thereof, and combinations thereof.

[0214] In some aspects, the present invention provides a system comprising a composition comprising an agent for inhibiting or suppressing TGF-β expression for use in treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0215] In another aspect, the present invention provides use of a composition of agents for inhibiting or suppressing TGF-β expression in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0216] 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 Omayer-type device with an access catheter.

[0217] As used herein, the term intracranial encompasses intrathecal and intraventricular. For example, intracranial infusion includes intrathecal infusion and intraventricular infusion. In addition, the term "intrathecal and intraventricular" is intended to encompass "intracranial".

[0218] Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide

[0219] Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapies can downregulate molecular targets by inducing RNase H endonuclease activity, which cleaves RNA-DNA heteroduplexes and significantly reduces target gene translation. Other ASO mechanisms may include inhibition of 5' cap formation, alterations in splicing processes (e.g., splice switching), and steric hindrance of ribosomal activity.

[0220] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating the catalytic degradation of target mRNA or by binding to sites on mRNA required for translation. Antisense oligonucleotides can be designed to target viral RNA genomes or viral transcripts. Antisense oligonucleotides can provide a method for identifying potential targets and therefore represent potential therapeutic agents.

[0221] 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 inhibit translation by inducing cleavage mechanisms or inhibiting mRNA maturation. ASOs can selectively inhibit gene expression with specificity. Chemical modification of DNA or RNA can be used to improve stability.

[0222] For example, modifications can be introduced into phosphodiester bonds, sugar rings, and backbones. ASO antiviral agents can block translation by (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage or (ii) by steric (non-bonding) blocking enzymes involved in target gene translation. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotides (OT-101; AP12009; Trabedersen), hereinafter referred to as OT-101 or AP 12009, are designed to reduce the level of TGF-β2 protein in malignant gliomas, thereby delaying the progression of the disease.

[0223] Antisense oligodeoxynucleotides are short strands of DNA designed to downregulate gene expression by interfering with the translation of specific coding proteins at the mRNA level. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' linkers are modified to phosphorothioates. The molecular formula is C 177 H 208 N 60 Na 17 O 94 P 17 S 17 The molecular weight is 6,143 g / mol. OT-101 is designed to be complementary to a specific sequence in human TGF-β2 mRNA after gene expression.

[0224] OT-101 is available as a lyophilized powder in three different amounts in 50 mL glass bottles. Dissolve the OT-101 lyophilized powder in an isotonic (0.9%) sodium chloride aqueous solution before use. The product can be prepared into the desired concentration for administration.

[0225] OT-101: Antisense oligodeoxynucleotides are short strands of DNA that are designed to downregulate gene expression by interfering with the translation of specific protein-coding proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and some 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 is designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression. It is a first-in-class RNA therapeutic designed to eliminate the immunosuppressive effects of TGF-β2 and reduce the levels of TGF-β2 in malignant gliomas, thereby delaying the progression of the disease.

[0226] The agent may be an antisense oligonucleotide or inhibitor specific for TGF-β1, TGF-β2 or TGF-β3. The agent for inhibiting or suppressing TGF-β expression may be selected from the TGF-β2 specific antisense oligonucleotides shown below.

[0227] The target TGF-β2 mRNA may be the NCBI reference sequence: NM_003238.3 with a sequence length of 5,882 bp. The target region of the TGF-β2 mRNA may be the protein coding sequence from references 1,369 to 2,613.

[0228] Examples of the agent for inhibiting or suppressing TGF-β2 expression disclosed herein include TGF-β2-specific antisense oligonucleotides given in SEQ ID NOs: 1-136 in Table 2.

[0229] Table 2: TGF-β2 specific antisense oligonucleotides

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] The sequences of 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 of Table 2 can be used as active agents in any combination, such as a pooling combination.

[0238] Examples of agents disclosed herein for inhibiting or suppressing TGF-β expression include artemisinin extracts, pharmaceutically acceptable salts, salt polymorphs, esters or isomers thereof, and any combination thereof. In some embodiments, the present disclosure includes substantially pure artemisinin having a purity of at least 60%, or 70%, or 80%, or 90%, or 95%.

[0239] In certain embodiments, the agent for inhibiting or suppressing TGF-β expression of the present disclosure can be prepared from a lyophilized powder of the agent.

[0240] In some embodiments, the TGF-β2-specific antisense oligonucleotides of the invention may have no more than one or two mismatches compared to the human target TGF-β2.

[0241] In certain embodiments, the TGF-β2-specific antisense oligonucleotides of the invention can reduce TGF-β2 transcript levels by at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0242] In other embodiments, the TGF-β2-specific antisense oligonucleotides of the invention may be selective for TGF-β2 and reduce any TGF-β1 transcript level and any TGF-β3 transcript level by less than 10%, or less than 5%, or less than 1%.

[0243] In other embodiments, a therapeutically effective amount of an antisense agent for inhibiting or suppressing TGF-β2 expression 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.

[0244] In certain embodiments, the concentration of antisense agent formulations used to inhibit or suppress TGF-β2 expression can be 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.

[0245] In certain embodiments, methods of using antisense agents for inhibiting or suppressing TGF-β2 expression may employ 1 to 1000 mg / m 2 / day, or 1 to 500 mg / m 2 / day, or 1 to 250 mg / m 2 / day, or 1 to 100 mg / m 2 / day or 1 to 50 mg / m 2 The average body surface area of ​​a human body is about 1.6 to 1.9 m 2 .

[0246] In other embodiments, methods of using antisense agents for inhibiting or reducing TGF-β2 expression may employ 0.05 to 40 mg / kg / day, or 0.1 to 30 mg / kg / day, or 0.2 to 20 mg / m 2 / day, or 0.3 to 10 mg / m 2 / day or 0.5 to 5 mg / m 2 The average human body weight may be about 60 kg.

[0247] In some examples and embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is administered or used at a dose level of 10 μM on days 1 to 7, or at a dose of 20 μM on days 1 to 7, or at a dose of 40 μM on days 1 to 7, or at a dose of 80 μM on days 1 to 7, at a dose of 4 μl / min by continuous intracerebroventricular or intrathecal or intracerebral administration.

[0248] In some examples and embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is administered or used at a dose level of 2 μM on days 1 to 7, or at a dose of 4 μM on days 1 to 7, or at a dose of 8 μM on days 1 to 7, or at a dose of 10 μM on days 1 to 7, at a dose of 4 μl / min or 2-8 μl / min by continuous intracerebroventricular or intrathecal or intracerebral administration.

[0249] In some embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and chemically modified variants thereof, and is administered by bolus injection into an Omayer-type 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.

[0250] In another embodiment, the agent can be a TGF-β2 specific antisense oligonucleotide selected from SEQ ID NO: 1-136 and chemically modified variants thereof, and is administered or used by continuous infusion alone or in combination with any form of artemisinin, which is taken orally on days 1 to 5 at a dose of 500 mg per day.

[0251] Embodiments of the present invention contemplate methods and uses comprising an agent that is a TGF-β2-specific antisense oligonucleotide that can be selected from SEQ ID NOs: 9-136.

[0252] Examples of the agent for inhibiting TGF-β of the present disclosure include agents for specifically inhibiting TGF-β1, TGF-β2, or TGF-β3, preferably TGF-β2.

[0253] Embodiments of the present invention involving the administration or use of pharmaceutical compositions can ameliorate or inhibit symptoms caused by TGF-β-induced proteins.

[0254] The agent used to inhibit or suppress TGF-β expression can be an artemisinin preparation comprising 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 comprise any one or more pharmaceutically acceptable excipients selected from a diluent, a stabilizer, a disintegrant and an anti-caking agent. In some embodiments, the excipients may comprise any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, cross-linked sodium carboxymethyl cellulose and magnesium stearate.

[0255] In other embodiments, the agent used to inhibit or suppress TGF-β expression can be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0256] As used herein, derivatives encompass chemical modifications that provide structural analogs of a compound. For example, substituents or substitutions of an alkyl group can provide structural analogs.

[0257] Embodiments of the present invention include methods or uses wherein the agent used to inhibit or suppress TGF-β expression is a compound or ligand, including a small molecule or polypeptide, that interacts with TGF-β site I comprising Trp30 and / or TGF-β site II comprising Arg15, Gln19, and Phe8, or a pharmaceutically acceptable salt, salt polymorph, ester, or isomer thereof.

[0258] In some embodiments, the agent used to inhibit or suppress TGF-β expression can be a polypeptide or peptide mimetic of TGF-β site I comprising residues Phe24-Lys37 and / or TGF-β site II comprising residues Cys7-Gln19, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0259] In other embodiments, the agent used to inhibit or suppress TGF-β expression can be a humanized or non-humanized antibody or antibody fragment that has affinity for TGF-β site I comprising residues Phe24-Lys37 and / or TGF-β site II comprising residues Cys7-Gln19.

[0260] In certain embodiments, the agent for inhibiting or suppressing TGF-β expression may be a compound comprising three isoprenyl groups and a lactone ring or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0261] Embodiments of the present invention also include pharmaceutical compositions for inhibiting or suppressing TGF-β expression in humans or animals, or for treating or ameliorating symptoms of CNS diseases in humans or animals. The pharmaceutical composition may contain a TGF-β inhibitor, artemisinin, a pharmaceutically acceptable salt, ester, polymorph or stereoisomer thereof, and any combination thereof, and a carrier. The TGF-β inhibitor may be selected from the group consisting of TGF-β2-specific antisense oligonucleotides SEQ ID NO: 1-136 and chemically modified variants thereof. The carrier may be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0262] Importantly, the compositions of the present disclosure can be substantially free of excipients. It has been found that compositions of the present disclosure that are substantially free of excipients are surprisingly stable in a carrier. In some embodiments, the compositions can be stable in a carrier at 37° C. for at least 14 days, or at least 21 days, or at least 28 days. In further embodiments, after 90 days in use, the reduction in antisense agent concentration can be less than 10%.

[0263] In further embodiments, the pharmaceutical composition for infusion may 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.

[0264]

[00146] Embodiments of the invention also include treatment regimens wherein the compositions of the invention are administered or used in combination with standard of care therapies for a disease. Examples of additional drugs that can be administered or used in combination with the compositions of the present invention include anti-inflammatory drugs, 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, pantoprazole, doxycycline, ivermectin, zinc, Foracort Rotacaps inhaler, injection ceftriaxone, paracetamol tablets, injection fragment protein Fragmin), Covifor Tablet, Azithromycin, Dexamethasone Injection, Ondansetron Injection, Multivitamin Tablet, Ascorbic Acid Tablet, Calcium Carbonate Tablet, and Zinc Sulfate Tablet.

[0265] The present invention also provides a kit comprising lyophilized powder of one or more TGF-β2 specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in vials, each containing 250 mg.

[0266] The present invention also provides a kit comprising lyophilized powder of the following substances in a vial at a content of 500 mg: artemisinin or its derivatives; or compounds or ligands that interact with TGF-β site II containing Arg15, Gln19 and Phe8, including small molecules or polypeptides; sesquiterpene lactones or their derivatives; or compounds containing three isoprenyl groups and one lactone ring and their derivatives, or pharmaceutically acceptable salts, salt polymorphs, esters or isomers thereof, or any combination of the foregoing.

[0267] Some TGF-β agents are given in US 9,963,703, US 9,758,786 and US 8,476,246.

[0268] Infusion devices and methods

[0269] The present invention provides novel devices and methods of use for the continuous infusion of pharmaceutical agents via the intracranial route for the treatment of CNS diseases, such as CNS cancers.

[0270] The devices and methods of the present invention can deliver pharmaceutical agents to the brain or spinal region using a pump-based continuous infusion system.

[0271] The device may be operated within a radiation therapy device and / or an electric field device, and the pharmaceutical composition may comprise the above-mentioned agents and any combination thereof.

[0272] In some examples and embodiments, the device of the present invention may include a portable extracorporeal pump with a fluid reservoir, which is connected to an infusion catheter that can be placed in any tissue or tumor (preferably the intraventricular space) via an infusion device. The fluid can be administered by high flow or continuous perfusion. The device of the present invention can infuse any type of fluid by continuous infusion or continuous convection-enhanced delivery. The device of the present invention can contain and deliver various 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.

[0273] The device of the present invention effectively delivers a therapeutically effective amount of a pharmaceutical composition to a subject, allowing for continuous infusion of the agent into a specific location within the subject's body, such as a specific tissue or tumor, preferably the ventricular space of the brain.

[0274] In some aspects ( Figure 1 and Figure 3 ), the device of the present invention can serve as a portable external convection-enhanced delivery device to infuse a liquid form of a medicament into a specific location in a subject's body. These aspects of the device can provide perfusion delivery in a hospital or inpatient center.

[0275] In other aspects ( Figure 2 and Figure 4 ), the device of the present invention can be used as a portable indwelling infusion device to deliver a pharmaceutical agent to a tissue or tumor, preferably an implantable ommaya in the ventricular space of a subject. These aspects of use can provide infusion delivery outside of an inpatient center, such as home infusion. These aspects of use can also provide infusion delivery outside of an inpatient center, such as home infusion, using, for example, an abdominally implanted pump.

[0276] The device of the present invention can inject fluids for continuous infusion.

[0277] The devices of the present invention can be used to deliver various therapeutic agents, such as drugs, proteins, protein toxins, imaging agents, antibodies for treatment or imaging, proteins for enzyme replacement therapy, growth factors, and / or viruses or oligonucleotides for gene therapy.

[0278] The devices of the present invention can advantageously improve the bioavailability and safety, as well as improve the pharmacokinetic and pharmacodynamic properties of the delivered therapeutic agents.

[0279] The device of the present invention can also be used for infusion delivery using a portable pump for outpatient treatment.

[0280] The device of the present invention ( Figure 2 and Figure 4 ) can be infused using an access port indwelling system, which has the advantage of easy replacement of the reservoir and pump and the pharmaceutical composition contained therein.

[0281] The device of the present invention ( Figure 2 and Figure 4 ) allows the infusion catheter to be precisely placed into the center of the tissue or tumor, preferably into the ventricular space, in a one-step surgical procedure. Advantageously, the access port can be positioned to minimize mechanical stress.

[0282] The devices of the present invention have the advantage of providing clinically useful constant flow rates for continuous infusion or convection-enhanced delivery.

[0283] The device of the present invention has the following advantages: it provides a small-step flow feature with a portable pump, with a flow rate of 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.

[0284] The device of the present invention may include an Omayer-type delivery device ( Figure 5 ). Figure 5 The Omayer-type delivery device shown can be used, e.g. Figure 1 and Figure 2The Omayyad delivery device includes a non-flexible hard shell

[403] composed of an inert material such as metal or hard plastic, which encloses and defines a reservoir for a fluid containing a drug. The Omayyad delivery device also includes a flexible top

[401] composed of a flexible material such as rubber, elastomer or plastic. The flexible top

[401] can be from 10% flexible to 50% flexible relative to the area of ​​the entire shell (

[401] plus

[403] ). The Omayyad delivery device also includes a non-flexible mounting plate

[405] composed of an inert material such as metal or hard plastic. The Omayyad delivery device also includes a port

[407] for connecting an infusion line to communicate with the Omayyad delivery device fluid. For example, the port

[407] can be connected to Figure 1 and Figure 2 infusion line

[103] . Fluid communication between the reservoir defined by the shell

[403] and the infusion line is provided by an internal passage

[421] . The Omayyeh delivery device also includes an access conduit

[413] in fluid communication with the Omayyeh reservoir, wherein the access conduit is substantially linear and enters a target area of ​​the brain intraventricularly. Fluid communication between the reservoir defined by the shell

[403] and the access conduit is provided by an internal passage

[423] . In another embodiment, the hard shell

[403] can be a separate protective collar that can be placed over a fully flexible top having an overall area of ​​

[401] plus

[403] to reduce the exposed area of ​​the flexible top to the area of ​​

[401] (see Figure 8 ).

[0285] In further examples or embodiments, the device of the present invention may comprise an Omayer-type delivery device ( Figure 6 ). Figure 6 An Omayer-type delivery device is shown for use in a method of delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. Figure 6 The Omayyad delivery device shown can be used as e.g. Figure 1 and Figure 2 The Omayr reservoir

[111] in the Omayr delivery device includes a non-flexible hard shell

[503] and

[504] composed of an inert material such as metal or hard plastic. The Omayr delivery device also includes a flexible top

[501] composed of a flexible material such as rubber, elastomer or plastic. The flexible top

[501] can be from 10% flexible to 50% flexible relative to the area of ​​the entire shell (

[501] plus

[503] ), such as Figure 6 The Omayel delivery device further comprises a non-flexible mounting plate

[505] comprised of an inert material such as metal or hard plastic. The Omayel delivery device further comprises ports

[507] and

[509] for connecting an infusion line to fluidly communicate with the Omayel delivery device. For example, port

[507] may be connected to Figure 3 and Figure 4 The indwelling tube

[109] in the housing

[503] . Fluid communication between the reservoir defined by the housing

[503] and the infusion line is provided by the internal passage

[521] . For example, the port

[509] can be connected to Figure 1 and Figure 2 In another embodiment, the hard shell

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

[501] plus

[503] to reduce the exposed area of ​​the flexible top to the area of ​​

[501] (see Figure 8 ).

[0286] In further examples or embodiments, the device of the present invention may comprise an Omayer-type delivery device ( Figure 7 ). Figure 7 An Omayer-type delivery device is shown for use in a method of delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion. Figure 7 The Omayyad delivery device shown can be used as e.g. Figure 3-4 The Omayyad delivery device includes an Omayyad reservoir

[111] in the Omayyad delivery device. The Omayyad delivery device includes a non-flexible hard shell

[603] composed of an inert material such as metal or hard plastic. The Omayyad delivery device also includes a flexible top

[601] composed of a flexible material such as rubber, elastomer, or plastic. The Omayyad delivery device also includes a non-flexible mounting plate composed of an inert material such as metal or hard plastic. The Omayyad delivery device also includes ports

[607] and

[619] for connecting an infusion line to fluidly communicate with the Omayyad delivery device. For example, port

[607] can be connected to Figure 3 and Figure 4 The infusion line

[103] in the housing

[603] . Fluid communication between the reservoir defined by the housing

[603] and the infusion line is provided by the internal passage

[621] . For example, the port

[619] can be connected to Figure 3-4 The inlet conduit

[113] in the housing

[603] . Fluid communication between the reservoir defined by the housing

[603] and the inlet conduit is provided by the internal passage

[619] . In another embodiment, the hard housing

[603] can be a separate protective collar that can be placed over a fully flexible top having the total area of ​​

[601] plus

[603] to reduce the exposed area of ​​the flexible top to the area of ​​

[601] (see Figure 8 ).

[0287] The devices of the present invention can deliver pharmaceutical agents, including any pharmaceutical agent suitable for delivery in a solvent system, or any pharmaceutical agent formulated for delivery in an aqueous solution, for example. Such pharmaceutical agents include, for example, analgesics, wound treatment agents, stimulants, anesthetics, anthelmintics, anticoagulants, antirheumatic agents, antiallergic agents, antiarrhythmic agents, antibiotics, antidementia agents, antidiabetic agents, antidotes, antiepileptics, antihemorrhagic agents, antihyperosmotic agents, antimigraine preparations, antifungals, antitumors, antiparkinsonian agents, anti-inflammatory drugs, antisense oligonucleotides, antituberculosis drugs, antiatherosclerotic drugs, biomaterials, blood flow stimulants, corticosteroids, cytokines, cytostatics, diagnostic agents, fibrinolytics, geriatric medicines, gonadotropins, hepatic drugs, stimulants, hormones and their inhibitors, hypnotic drugs, immunoglobulins, immunomodulators, immunotherapeutic agents, organ perfusion solvents, proteins, protein toxins, protective agents, sedatives, cardiac drugs, depressants and stimulants, minerals, muscle relaxants, neurotrophic agents, oligonucleotides, ophthalmic drugs, vaccines, antispasmodics, urological drugs, drugs, proteins, protein toxins, therapeutic antibodies, proteins in enzyme replacement therapy, growth factors, vectors, viruses in gene therapy and / or agents for diagnosis, agents or antibodies for imaging, X-ray contrast agents, oligonucleotides that inhibit expression.

[0288] The devices of the present invention can deliver pharmaceutical agents or active substances dissolved or suspended in physiological solvents or any other suitable solvent. The pharmaceutical agents can be in the form of free bases or salts, hydrates, esters, amides, enantiomers, isomers, tautomers, polymorphs, prodrugs or derivatives of these compounds. The above-mentioned pharmaceutical agents and combinations thereof can be used in the devices, methods, kits, combinations and compositions described herein.

[0289] The devices of the present invention may have an inner surface coated with a therapeutic agent.

[0290] The components of device of the present invention can be made of various materials, including for example metal, polymer and / or composite material, including materials such as titanium, high-grade steel, aluminum, alloy, polymer foam, plastic, stainless steel and / or metal and combination, mixture and modification thereof. The material intended to be implanted in the subject can be made of biocompatible material, for example polymer, for example polymer fragment of polystyrene, polyolefin, polyamide or polyurethane, and metal. The selection of such material depends on many factors, including porosity, surface properties or toxicity of required mechanical properties and material. The components of device of the present invention can be made of for example titanium, alloy, stainless steel, ceramic, silicon, Teflon, polypropylene, polyethylene, polystyrene, polyolefin, polyimide, polyamide, polyurethane, PET, PETG, PE, PIG, HDPE, PC, PVC, nylon, urethane and / or copolymer, and can be laminated with gold, silver and / or aluminum layer or otherwise include gold, silver and / or aluminum layer, to minimize the permeability of gas and liquid, sputtering or other ways deposit or incorporate therein. Fragmented polyurethane-urea, Polycarbonate urethane, Elasthane TM and Elasthane TM Polyether urethane can be used for long-term implantable medical devices. TM The chemical structure and properties of Similar to 2363. Thermoplastic silicone-urethane copolymers, such as PurSil TM Silicone Polyether Urethane and CarboSil TM Silicone polycarbonate urethanes may also be used in this device.

[0291] The devices of the present invention may include filters in any suitable location. The filters may include sterile filters for removing pathogens, biological filters for biological materials such as proteins and / or antibodies; particle filters for removing particulate matter; chemical filters for removing chemicals; and / or filters for removing air or gas from solvents. In one aspect, each filter may have a distal port and a proximal port and an inner lumen extending therethrough. Within the inner lumen of each filter may be a membrane or any other suitable device for removing air, particles, chemicals, and / or biological materials, such as pathogens, including bacteria, fungi, and / or viruses. The filters may have separate housings or a shared housing. In one aspect, the filter for removing air may be placed externally. In another aspect, the filter for removing particles may be placed upstream of the sterile filter. Sterile filters typically have a pore size of approximately 0.45μ or less, or approximately 0.22μ or less, or approximately 0.1μ or less. The particle filter may have a pore size greater than approximately 0.45μ, or greater than approximately 0.22μ.

[0292] The access port can be implanted subcutaneously, for example, on a rib. The access port chamber is in fluid communication with the Omayr reservoir.

[0293] The device of the present invention may contain a compound formulated as an injectable formulation to be infused into a subject, such as 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, for example, comprises an aqueous solution of a tonicity adjuster, buffered to a pH, for example, below 7, or, for example, below 6. Tonicity adjusters include, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other agents that make the osmotic pressure of the formulation isotonic with blood.

[0294] The device of the present invention may contain a preservative added to the formulation. Preservatives include benzalkonium chloride, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenol, sodium benzoate, or EDTA.

[0295] The device of the present invention may contain a pharmaceutically acceptable carrier. Carrier materials useful in preparing the compositions of the present invention are any of those commonly used excipients in pharmacy and should be selected based on compatibility with the pharmaceutical agent and the release profile properties of the desired dosage form.

[0296] The devices of the present invention may contain excipients such as those known from 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.

[0297] As used herein, the term medicament may refer to one or more active compounds, a combination of active compounds, or a composition comprising one or more active compounds with a carrier and / or solvent and / or any number of excipients. In some embodiments, the composition may be a pharmaceutical composition. In certain embodiments, the composition may be a pharmaceutical composition containing a therapeutically effective amount of one or more active compounds. Some examples of excipients are given in the following literature: Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0298] 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 subject's response to the administered agent and adjusting the dosage. See, for example, Remington, The Science and Practice of Pharmacy (Gennaro, ed., 20th ed.) (2000).

[0299] Device operation

[0300] During operation, the device can be adjusted relative to the patient by performing a computed tomography (CT) scan, brain MRI, or other suitable imaging method to determine the location of the ventricular space and the required catheter length. To place the device, a cranial burr hole is created and the brain access catheter is slowly inserted until fluid flows back out of the catheter. The ventricular catheter Omayer-type device is then connected to an external or indwelling infusion set.

[0301] For example, an approved silicone microcatheter is placed stereotactically into the ventricular space. The target position of the access catheter tip can be pre-calculated by CT scan or brain MRI. Intraventricular placement of the access catheter does not require simultaneous imaging. The external or indwelling infusion assembly can be pre-filled with normal saline. The postoperative position of the Omayer-type device and device of the present invention can be determined by native X-ray, computed tomography, brain MRI or other suitable imaging methods. The positioning of the catheter tip can be determined in the same manner. In some examples and embodiments, the infusion port and catheter can be impregnated with barium for imaging purposes.

[0302] The infusion line can be connected to the infusion set under sterile conditions. For example, an infusion line with a bacterial microporous filter (maximum 0.2 μm) can be used.

[0303] In some examples and embodiments, a special port puncture needle, such as a GRIPPER PORT-A-CATH TM The therapeutic agent solution can be passed through an external or indwelling infusion set until it reaches the tip of the port puncture needle. Air bubbles can be removed from this method.

[0304] For treatment, a therapeutic agent solution, such as the antisense oligonucleotide OT-101, can be infused into the ventricular space. The therapeutic agent solution can be prepared and used to fill an Omayor-type reservoir. At the patient's bedside, an automated pump is connected to the device. Before starting the infusion, the infusion set can be filled with the therapeutic agent solution and connected to the port by inserting a port puncture needle through its membrane under sterile conditions.

[0305] In some examples and embodiments, the therapeutic agent solution of OT-101 may be continuously infused at a flow rate of 4 μl / min or 2-8 μl / min.

[0306] In some examples and embodiments, a therapeutically effective amount of antisense (OT-101, etc.) 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 be performed in a subject on day 1-3, or day 1-7, or day 1-14, or day 1-21, or day 1-50 of a regimen or cycle.

[0307] Methods, compositions and combinations for CNS diseases

[0308] The device of the present invention can be used to treat a CNS disease such as CNS cancer or to ameliorate symptoms of a CNS disease in a human subject or animal in need thereof.The device of the present invention can contain a pharmaceutical composition for continuous infusion into the brain or spine.

[0309] The pharmaceutical composition for continuous infusion may comprise an agent for inhibiting or suppressing TGF-β expression, and a therapeutically effective amount of the composition is administered to a subject. The present invention provides therapies for treating CNS diseases such as cancer or ameliorating symptoms of CNS diseases.

[0310] Examples of anti-TGF-β2 agents include TGF-β inhibitors, such as antisense oligonucleotides, artemisinin, pharmaceutically acceptable salts, esters, polymorphs or stereoisomeric forms thereof, and combinations thereof.

[0311] In some aspects, the present invention provides kits and devices comprising compositions of agents for inhibiting or suppressing TGF-β expression for use in treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0312] In another aspect, the present invention provides use of a composition of agents for inhibiting or suppressing TGF-β expression in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

[0313] 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 Omayer-type device with an access catheter.

[0314] As used herein, the term intracranial encompasses intrathecal and intraventricular. For example, intracranial infusion includes intrathecal infusion and intraventricular infusion. In addition, the term "intrathecal and intraventricular" is intended to encompass "intracranial".

[0315] In certain embodiments, the agents, uses or methods of the present invention, upon administration to a subject, may reduce TGF-β2 levels in the subject, which may be referred to as an improved TGF-β2 profile.

[0316] In some aspects, the agents, uses or methods of the invention, when administered or used by a subject, can reduce mortality at 6, 12, 18, 24, 30 or 36 months.

[0317] In other aspects, the medicament, use or method of the present invention can increase the survival rate at 6, 12, 18, 24, 30 or 36 months after administration or use in a subject. Survival can be determined as overall survival or progression-free survival.

[0318] In some aspects, a substantially excipient-free pharmaceutical agent, medicament, or administration can include a carrier.

[0319] Embodiments of the present invention also include 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.

[0320] The present invention provides methods for selecting patients for therapy based on TGF-β2 levels as a biomarker for improved outcomes of radiation therapy in cancer.

[0321] Surprisingly, patients with low TGF-β2 expression experienced improved overall survival and survival after radiation therapy across a wide range of expression levels compared to patients with high TGF-β2 expression. Such differences were not observed for TGF-β-1 and TGF-β-3. Therefore, reduced TGF-β2 may serve as a successful biomarker for selecting patients who will benefit from such therapies.

[0322] In another study, using TGF-β2 as a selector, overall survival in pediatric gliomas treated with radiotherapy was unexpectedly improved. TGF-β-1 and TGF-β-3 did not predict survival.

[0323] In some embodiments, TGF-β2 levels can serve as a surprisingly effective biomarker for selecting patients who will benefit from cancer radiation therapy combined with chemotherapy (such as temozolomide (TMZ)) and / or TMZ plus radiation therapy and / or anti-angiogenic therapy (such as bevacizumab). No such predictive results were observed for TGF-β1 and TGF-β3.

[0324] In some embodiments, the present invention provides agents, uses, and methods for treating or ameliorating symptoms of CNS diseases in human subjects or animals by combining inhibition or suppression of TGF-β2 expression with drugs that are targeted cancer drugs, cancer growth inhibitors, or EGFR inhibitors.

[0325] In certain embodiments, the present invention provides agents, uses, and methods for treating or ameliorating symptoms of CNS diseases in human subjects or animals by combining inhibition or suppression of TGF-β2 expression with bevacizumab, everolimus, bezutifant, dabrafenib, trametinib, and combinations thereof.

[0326] In other embodiments, the present invention provides agents, uses and methods for treating or ameliorating symptoms of CNS diseases in human subjects or animals by combining inhibition or suppression of TGF-β2 expression with erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

[0327] In further embodiments, the present invention provides agents, uses, and methods for treating or ameliorating symptoms of CNS diseases in human subjects or animals by combining inhibition or suppression of TGF-β2 expression with temozolomide.

[0328] Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotide

[0329] Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapies can downregulate molecular targets by inducing RNase H endonuclease activity that cleaves RNA-DNA heteroduplexes and significantly reducing target gene translation. Other ASO mechanisms may include inhibition of 5' cap formation, alterations in splicing processes such as splice switching, and steric hindrance of ribosomal activity.

[0330] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating the catalytic degradation of target mRNA or by binding to sites on mRNA required for translation. Antisense oligonucleotides can be designed to target viral RNA genomes or viral transcripts. Antisense oligonucleotides can provide a method for identifying potential targets and therefore represent potential therapeutic agents.

[0331] Antisense oligonucleotides can be small synthetic fragments of single-stranded DNA, perhaps 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences via Watson-Crick hybridization, and once bound to the target RNA, they inhibit translation by inducing cleavage mechanisms or inhibiting mRNA maturation. ASOs can selectively and specifically inhibit gene expression. Chemical modification of DNA or RNA can be used to improve stability.

[0332] For example, modifications can be introduced into phosphodiester bonds, sugar rings, and backbones. ASO antiviral agents can block translation by (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage or (ii) by steric (non-bonding) blocking enzymes involved in target gene translation. Human TGF-β2-specific phosphorothioate antisense oligodeoxynucleotides (OT-101; AP12009; Trabedersen), hereinafter referred to as OT-101 or AP 12009, are designed to reduce the level of TGF-β2 protein in malignant gliomas, thereby delaying the progression of the disease.

[0333] Antisense oligodeoxynucleotides are short strands of DNA designed to downregulate gene expression by interfering with the translation of specific protein-coding genes at the mRNA level. OT-101 is a synthetic 18-mer phosphorothioate oligodeoxynucleotide (S-ODN) in which all 3'-5' linkers are modified to phosphorothioates. The molecular formula is C 177 H208 N 60 Na 17 O 94 P 17 S 17 The molecular weight is 6,143 g / mol. OT-101 is designed to be complementary to a specific sequence in human TGF-β2 mRNA after gene expression.

[0334] OT-101 is currently available in the form of a lyophilized powder in three different amounts in 50 mL glass bottles. Before use, the OT-101 lyophilized powder is dissolved in an isotonic (0.9%) sodium chloride aqueous solution.

[0335] Examples of the disclosed agents for inhibiting or suppressing TGF-β expression include antisense oligonucleotides specific for TGF-β1, TGF-β2, or TGF-β3.

[0336] Examples of the disclosed agents for inhibiting or suppressing TGF-β2 expression include TGF-β2-specific antisense oligonucleotides given in SEQ ID NOs: 1-136 in Table 2, including SEQ ID NO: 8: cggcatgtct attttgta (OT-101).

[0337] As is known in the art, the antisense oligonucleotides given in Table 2 herein may be chemically modified.

[0338] Examples of agents disclosed herein for inhibiting or suppressing TGF-β expression include artemisinin extracts, pharmaceutically acceptable salts, salt polymorphs, esters or isomers thereof, and any combination thereof. In some embodiments, the present disclosure includes substantially pure artemisinin having a purity of at least 60%, or 70%, or 80%, or 90%, or 95%.

[0339] In certain embodiments, the agent for inhibiting or suppressing TGF-β expression of the present disclosure can be prepared from a lyophilized powder of the agent.

[0340] In some examples and embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is administered or used at a dose level of 10 μM on days 1 to 7, or at a dose of 20 μM on days 1 to 7, or at a dose of 40 μM on days 1 to 7, or at a dose of 80 μM on days 1 to 7, at a dose of 4 μl / min by continuous intracerebroventricular or intrathecal or intracerebral administration.

[0341] In some examples and embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and is administered or used at a dose level of 2 μM on days 1 to 7, or at a dose of 4 μM on days 1 to 7, or at a dose of 8 μM on days 1 to 7, or at a dose of 10 μM on days 1 to 7, at a dose of 4 μl / min or 2-8 μl / min by continuous intracerebroventricular or intrathecal or intracerebral administration.

[0342] In some embodiments, the agent can be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 1-136 and chemically modified variants thereof, and is administered by bolus injection into an Omayer-type 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.

[0343] In another embodiment, the agent can be a TGF-β2 specific antisense oligonucleotide selected from SEQ ID NO: 1-136 and chemically modified variants thereof, and is administered or used by continuous infusion alone or in combination with any form of artemisinin, which is taken orally on days 1 to 5 at a dose of 500 mg per day.

[0344] Examples of the agent for inhibiting TGF-β of the present disclosure include agents for specifically inhibiting TGF-β1, TGF-β2, or TGF-β3, preferably TGF-β2.

[0345] Embodiments of the present invention relate to administering or using pharmaceutical compositions that can improve or inhibit symptoms caused by TGF-β-induced proteins.

[0346] The agent used to inhibit or suppress TGF-β expression can be an artemisinin preparation comprising 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.

[0347] In other embodiments, the agent used to inhibit or suppress TGF-β expression can be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0348] As used herein, derivatives encompass chemical modifications that provide structural analogs of a compound. For example, substituents or substitutions of an alkyl group can provide structural analogs.

[0349] Embodiments of the present invention include methods or uses wherein the agent for inhibiting or suppressing TGF-β expression is a compound or ligand, including a small molecule or polypeptide, that interacts with TGF-β site I comprising Trp30 and / or TGF-β site II comprising Arg15, Gln19 and Phe8; or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0350] In some embodiments, the agent used to inhibit or suppress TGF-β expression can be a polypeptide or peptide mimetic of TGF-β site I comprising residues Phe24-Lys37 and / or TGF-β site II comprising residues Cys7-Gln19, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0351] In other embodiments, the agent used to inhibit or suppress TGF-β expression can be a humanized or non-humanized antibody or antibody fragment that has affinity for TGF-β site I comprising residues Phe24-Lys37 and / or TGF-β site II comprising residues Cys7-Gln19.

[0352] Embodiments of the present invention also include pharmaceutical compositions for inhibiting or suppressing TGF-β expression in humans or animals, or for treating or ameliorating symptoms of CNS diseases in humans or animals. The pharmaceutical composition may contain a TGF-β inhibitor, artemisinin, a pharmaceutically acceptable salt, ester, polymorph or stereoisomer thereof, and any combination thereof, and a carrier. The TGF-β inhibitor may be selected from the group consisting of TGF-β2-specific antisense oligonucleotides SEQ ID NO: 1-136 and chemically modified variants thereof. The carrier may be sterile water for injection, saline, isotonic saline, or a combination thereof.

[0353] Importantly, the compositions of the present disclosure can be substantially free of excipients. It has been found that compositions of the present disclosure that are substantially free of excipients are surprisingly stable in a carrier. In some embodiments, the compositions can be stable in a carrier at 37°C for at least 14 days, or at least 21 days, or at least 28 days.

[0354] In further embodiments, the pharmaceutical composition for infusion may 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.

[0355]

[00146] Embodiments of the invention also include treatment regimens wherein the compositions of the invention are administered or used in combination with standard of care therapies for a disease. Examples of additional drugs that can be administered or used in combination with the compositions of the present invention include anti-inflammatory drugs, anti-inflammatory steroids, piperaquine, pyronaridine, curcumin, frankincense, remdesivir, Sompraz D, Zifi CV / Zac D, CCM, Broclear, budamate, Rapitus, Montek LC, low molecular weight heparin, prednisolone, acetaminophen, vitamin B complex, vitamin C, pantoprazole, doxycycline, ivermectin, zinc, Foracort Rotacaps inhaler, injection ceftriaxone, acetaminophen tablets (Tab Paracetamol), injection fragment protein (Fragmin), remdesivir tablets (Tablet Covifor), azithromycin, dexamethasone for injection, ondansetron for injection, multivitamin tablets, ascorbic acid tablets, calcium carbonate tablets and zinc sulfate tablets.

[0356] The present invention also provides a kit comprising lyophilized powder of any one or more TGF-β2 specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in a vial, each containing 3.75 mg.

[0357] The present invention also provides a kit comprising lyophilized powder of the following substances in a vial at a content of 500 mg: artemisinin or its derivatives; or compounds or ligands that interact with TGF-β site II containing Arg15, Gln19 and Phe8, including small molecules or polypeptides; sesquiterpene lactones or their derivatives; or compounds containing three isoprenyl groups and one lactone ring and their derivatives, or pharmaceutically acceptable salts, salt polymorphs, esters or isomers thereof, or any combination of the foregoing.

[0358] Delivery of therapeutic agents by infusion

[0359] Delivery of OT-101 (TGF-β antisense) to treat glioblastoma.

[0360] OT-101 is a TGF-β2 antisense drug that showed activity against recurrent glioblastoma in the 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).

[0361] OT-101 is delivered intratumorally via a brain-implanted catheter. To further expand the application of OT-101, intrathecal delivery of tritiated OT-101 was explored in Sprague-Dawley CD (albino) rats. Throughout the study, no gender differences were observed.

[0362] Surprisingly, OT-101 was similarly and extensively distributed in the cerebellum, the rest of the brain, and the cerebrospinal fluid (CSF) 1 hour after either intracerebral or intraventricular infusion in rats, indicating that intracerebral administration mimics intraventricular administration and reaches the entire CNS compartment via the CSF.

[0363] The concentration of OT-101 was stable within the first 4 hours after infusion and decayed biexponentially with a slow terminal half-life in tissues, but not in CSF, indicating rapid penetration into underlying tissues away from the CSF compartment. Minimal amounts of OT-101 were detected in the plasma compartment. Intrathecal bolus administration of 0.1 mL of 14, 30, 200, 300, and 500 μM OT-101 to cynomolgus monkeys did not result in any single-dose toxicity. Histopathological examination showed no substance-related histomorphological lesions in the subarachnoid space in the lumbar region. No changes were noted in the gray matter and white matter of the spinal cord, and nerve stems and neurons did not show any abnormalities. These data suggest that intrathecal administration of OT-101 is a potentially effective delivery route for delivering antisense therapeutics such as OT-101 to the midline, i.e., for diffuse midline glioma (DMG).

[0364] Targeting transforming growth factor β2 (TGF-β2) and OT-101 for post-radiation sclerosing of diffuse intrinsic pontine gliomas Solid treatment

[0365] Diffuse intrinsic pontine glioma (DIPG) in children has a poor prognosis, with a median overall survival (OS) of 10 months after standard radiotherapy and a 2-year OS rate of <10%.

[0366] Chemotherapy does provide clinically meaningful benefit. Therefore, innovative therapeutic agents for the treatment of DIPG in children are urgently needed. High-grade glioma cells, including pediatric glioblastoma and DIPG cells, have been shown to produce transforming growth factor β2 (TGF-β2).

[0367] TGFB2 is considered to be both a promoter of glioma cells and a key factor in the low T cell responsiveness of the tumor microenvironment (TME) to glioma cells. OT-101 is a first-in-class RNA therapeutic designed to eliminate the immunosuppressive and tumor-promoting effects of TGF-B2. At low micromolar concentrations, OT-101 can reduce TGFB2 secretion in human glioma cells, prevent their proliferation and migration, and restore the anti-glioma cell lytic function of patient-derived T cells. OT-101 administered intracerebrally has shown promising single-agent activity in recurrent / refractory (R / R) high-grade gliomas (HGGs) (Uckun et al., Cancers. 2019 28; 11(12): 1892). Direct intrathecal administration of antitumor drugs in CSF allows bypassing the selective filtration of the blood-brain barrier (BBB), achieving significant antitumor agent concentrations in CSF while reducing the possibility of systemic toxicity. Informed by the favorable safety profile of OT-101 delivered intrathecally in rabbits and primates, and encouraged by its single-agent activity in adult patients with HGG, pediatric patients with DIPG are being treated with OT-101. After completion of radiation therapy, multiple doses of OT-101 are administered as intrathecal bolus injections. The study is 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.

[0368] Numbered embodiments of the present invention may include:

[0369] 1) An agent for inhibiting or suppressing TGF-β2 expression, which is used to treat or improve the symptoms of CNS diseases in human subjects or animals.

[0370] 2) Use of an agent that inhibits or suppresses TGF-β2 expression in the preparation of a medicament for treating or ameliorating symptoms of a CNS disease in a human subject or an animal.

[0371] 3) A method for treating or ameliorating symptoms of a CNS disease in a human subject or animal in need thereof, the method comprising:

[0372] preparing a pharmaceutical composition comprising an agent for inhibiting or suppressing TGF-β2 expression in a carrier; and

[0373] A therapeutically effective amount of the composition is administered to the subject.

[0374] 4) The agent, use or method according to any one of embodiments 1-3, wherein the CNS disease is a glioma, a glioblastoma, a diffuse intrinsic pontine glioma (DIPG), a diffuse midline glioma (DMG), a leptomeningeal or brain metastasis, a brain or spinal cancer, or a CNS tumor.

[0375] 5) The agent, use or method according to any one of embodiments 1-4, in combination with drugs including targeted cancer drugs, cancer growth blockers, EGFR inhibitors and combinations thereof.

[0376] 6) The agent, use or method according to any one of embodiments 1-5, in combination with a drug selected from bevacizumab, everolimus, bezutifan, dabrafenib, trametinib and a combination thereof.

[0377] 7) The agent, use or method according to any one of embodiments 1-6, in combination with a drug that is a cancer growth inhibitor, wherein the cancer growth inhibitor is selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog factor blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0378] 8) The agent, use or method according to any one of embodiments 1-7, in combination with a drug that is an EGFR inhibitor, wherein the EGFR inhibitor is selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and a combination thereof.

[0379] 9) The medicament, use or method according to any one of embodiments 1-8, in combination with temozolomide.

[0380] 10) The agent, use or method according to any one of embodiments 1-9, in combination with treatment of CNS diseases by radiotherapy or therapeutic electric fields.

[0381] 11) The medicament, use or method according to any one of embodiments 1-10, wherein administration or use of the composition or medicament is combined with standard of care treatment for a CNS disease.

[0382] 12) The agent, use or method according to any one of embodiments 1-11, wherein the agent, drug, therapy, treatment and administration are each administered concurrently, simultaneously, sequentially or separately in time.

[0383] 13) The medicament, use or method according to any one of embodiments 1-12, wherein each medicament and drug is administered alone or in combination by infusion or injection.

[0384] 14) The agent, use or method according to any one of embodiments 1-13, comprising administration or use by intracranial continuous infusion or bolus administration.

[0385] 15) The agent, use or method of any one of embodiments 1-14, wherein the intracranial continuous infusion comprises infusion using an Omayer-type reservoir with a partially flexible top.

[0386] 16) The agent, use or method according to any one of embodiments 1-15, wherein the intracranial continuous infusion comprises a single access catheter placed into the target area of ​​the brain.

[0387] 17) An agent, use or method according to any one of embodiments 1-16, wherein the subject has an improved TGF-β2 profile following administration or use.

[0388] 18) The medicament, use or method according to any one of embodiments 1-17, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

[0389] 19) The agent, use or method according to any one of embodiments 1-18, wherein the administration or use increases survival at 6, 12, 18, 24, 30 or 36 months.

[0390] 20) The agent, use or method according to any one of embodiments 1-19, wherein the agent for inhibiting or suppressing TGF-β2 expression is selected from TGF-β2 specific antisense oligonucleotides complementary to TGF-β2 transcripts, as shown below:

[0391] Table 2 SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0392] 21) An agent, use or method according to any one of embodiments 1-20, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide that has no more than one or two mismatches compared to the human target TGF-β2.

[0393] 22) An agent, use or method according to any one of embodiments 1-21, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide, which reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80% or at least 90%.

[0394] 23) An agent, use or method according to any one of embodiments 1-22, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2-specific antisense oligonucleotide, which reduces the level of any TGF-β1 transcript and any TGF-β3 transcript by less than 10%, or less than 5% or less than 1%.

[0395] 24) The agent, use or method according to any one of embodiments 1-23 comprises a TGF-β2 specific antisense oligonucleotide, wherein the TGF-β2 specific antisense oligonucleotide has one or more nucleotides chemically modified to be a thiophosphate internucleoside bond, a methoxypropylphosphonate internucleoside bond, or an aminophosphoric acid bond connected 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.

[0396] 25) An agent, use or method according to any one of embodiments 1-24, wherein the agent is conjugated to polyethylene glycol, a lipid or tri-branched N-acetylgalactosamine.

[0397] 26) The medicament, use or method according to any one of embodiments 1-25 comprises a carrier, wherein the carrier is sterile water for injection, saline, isotonic saline, phosphate-buffered saline or a combination thereof.

[0398] 27) The agent, use or method according to any one of embodiments 1-26, wherein the agent, drug or administration is essentially free of excipients.

[0399] 28) The agent, use or method of any one of embodiments 1-27, wherein the agent, drug or administration is stable in the carrier at 37°C for at least 14 days when pumped as an intracranial continuous infusion, or the concentration of the antisense active agent decreases by less than 10% after 90 days of use.

[0400] 29) The agent, use or method according to any one of embodiments 1-28, wherein the method comprises administering the composition by intracranial infusion at a rate of 2-8 μl / min and a concentration of the agent of 1-80 μM on day 1 to day 7, preferably by continuous intracranial infusion.

[0401] 30) A kit comprising:

[0402] A medicament comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1-136 in Table 2, chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and

[0403] Omayeh-type reservoir with a partially flexible top.

[0404] 31) A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0405] a reservoir

[117] containing a pharmaceutical composition;

[0406] a pump

[101] that forces the pharmaceutical composition through the infusion tube

[103] into the Omayr reservoir

[111] , the pump being in fluid communication with the Omayr reservoir, and wherein the pump is in fluid communication with the reservoir via a reservoir tube

[115] ;

[0407] a filter

[105] inline with the infusion tubing; and

[0408] An access catheter

[113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0409] 32) The device of embodiment 31, wherein the access catheter

[113] is non-linear and has one or more bends to access the target area of ​​the brain within the ventricle.

[0410] 33) A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0411] a reservoir

[117] containing a pharmaceutical composition;

[0412] a pump

[101] that forces the pharmaceutical composition through an infusion tube

[103] into an access port

[107] , the pump being in fluid communication with an Omayr reservoir

[111] , and wherein the reservoir is in fluid communication with the pump via a reservoir tube

[115] ;

[0413] a filter

[105] which is in line with the infusion tubing;

[0414] an indwelling tube

[109] in fluid communication with the access port and the Omayr reservoir

[111] ; and

[0415] An access catheter

[113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0416] 34) The device of any one of embodiments 31-33, wherein the access catheter

[113] is non-linear and has one or more bends to access a target region of the brain within a cerebral ventricle.

[0417] 35) A device according to any of embodiments 31-34, wherein the Omayya reservoir

[111] includes a partially flexible top.

[0418] 36) A device according to any of embodiments 31-35, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to the target area.

[0419] 37) A device according to any of embodiments 31-33, wherein the distal end of the access catheter enters the target area of ​​the brain.

[0420] 38) A device according to any one of embodiments 31-37, wherein the Omayr reservoir

[111] retains the drug composition behind the membrane for a period of time to provide sustained release of the drug composition into the catheter.

[0421] 39) A device according to any of embodiments 31-38, wherein the distal tip of the access catheter entering the brain is a stepped end, a recessed stepped end, a multi-port end, a microporous end, or a balloon tip.

[0422] 40) The apparatus of any one of embodiments 31-39, wherein the pump

[101] is a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1, or other pumps of similar specifications.

[0423] 41) A device according to any one of embodiments 31-40, wherein the infusion rate of the fluid drug 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.

[0424] 42) The device of any one of embodiments 31-41, wherein the infusion tubing

[103] or the indwelling tubing

[109] is a PEGA Line 100SF 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 an extension (#21-7106-24), or other tubing with similar specifications.

[0425] 43) A device according to any one of embodiments 31-42, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β expression, which can be used to treat or ameliorate symptoms of a CNS disease in a human subject or animal.

[0426] 44) A device according to any one of embodiments 31-43, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of a pharmaceutical agent, drug or delivery vehicle.

[0427] 45) A device according to any one of embodiments 31-44, wherein the fluid drug composition is a therapeutic agent for a CNS disease or CNS cancer.

[0428] 46) A device according to any of embodiments 31-45, wherein the fluid drug composition is a therapeutic agent for glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cancer, or CNS tumor.

[0429] 47) A device according to any one of embodiments 31-46, wherein the fluid pharmaceutical composition comprises an agent that inhibits or suppresses TGF-β2 expression, the agent being selected from a TGF-β2-specific antisense oligonucleotide complementary to a TGF-β2 transcript as described below:

[0430] Table 2 SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0431] 48) A device according to any of embodiments 31-47, wherein the device is operated in combination with radiation therapy or electric field therapy.

[0432] 49) A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0433] a reservoir

[402] containing a pharmaceutical composition, the reservoir comprising a hard shell

[403] , a flexible top

[401] , and a non-flexible mounting plate

[405] ;

[0434] a port

[407] in fluid communication with the reservoir; and

[0435] An access catheter

[413] is in fluid communication with the reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

[0436] 50) A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0437] a reservoir

[502] containing a pharmaceutical composition, the reservoir comprising an upper hard shell

[503] , a lower hard shell

[504] , a flexible top

[501] , and a non-flexible mounting plate

[505] ;

[0438] a port

[507] in fluid communication with the reservoir and for connecting an infusion line; and

[0439] A port

[509] is in fluid communication with the reservoir and is used to connect an access catheter.

[0440] 51) A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0441] a reservoir

[602] containing a pharmaceutical composition, the reservoir comprising an upper hard shell

[603] , a flexible top

[601] , and a non-flexible mounting plate

[605] ;

[0442] a port

[619] in fluid communication with the reservoir and for connecting an infusion line; and

[0443] A port

[607] is in fluid communication with the reservoir and is used to connect an access catheter.

[0444] 52) A collar of a device for delivering a pharmaceutical composition by continuous intracranial infusion, the collar comprising:

[0445] A hard shell

[604] having an opening

[606] exposing the flexible top of the device.

[0446] 53) A method of administering a pharmaceutical composition by continuous intracranial infusion, comprising:

[0447] installing the device according to any one of embodiments 31-52 on a patient; and

[0448] The pharmaceutical composition is pumped into the device to provide a continuous intracranial infusion to the patient.

[0449] 54) A method according to embodiment 53, wherein the device is installed and the access catheter is placed into the brain without the need for simultaneous head or brain imaging.

[0450] 55) A method according to any of embodiments 53-54, wherein the device is mounted and a single access catheter is placed into the brain.

[0451] 56) A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, the kit comprising:

[0452] a reservoir containing the pharmaceutical composition;

[0453] pumps;

[0454] Omayeh reservoir with partially flexible top;

[0455] infusion tubing for connecting the reservoir to the pump and the pump to the Omayeh reservoir;

[0456] Filters; and

[0457] Enter the catheter.

[0458] 57) A kit according to any one of embodiments 30 and 56, wherein the access catheter is substantially linear and is used to access the target area of ​​the brain intraventricularly.

[0459] 58) A kit according to any one of embodiments 30 and 56-57, wherein the access catheter is non-linear and has a bend to access the target area of ​​the brain intraventricularly.

[0460] 59) A kit according to any one of embodiments 30 and 56-58, wherein the infusion tube is outside the subject's body.

[0461] 60) A kit according to any one of embodiments 30 and 56-59, wherein a portion of the infusion tubing connecting the pump to the Omayr reservoir is left in the subject's body.

[0462] All publications mentioned herein, including patents, patent application publications, and non-patent publications, as well as the Sequence Listing, are expressly incorporated by reference in their entirety for all purposes.

[0463] 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 will be understood based on the present disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented in an illustrative and non-restrictive manner. 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.

[0464] It is emphasized here that, according to common practice, the features of the drawings are to arbitrary scale and are intended to encompass similar features that may be arbitrarily enlarged or reduced.

[0465] Example

[0466] Example 1 - Examples of pumps for continuous infusion via the intrathecal or intraventricular routes are shown in Table 3.

[0467] Table 3: Examples of pumps used for continuous infusion

[0468]

[0469]

[0470] The infusion or indwelling line can be a PEGA Line 100SF 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 tubing with similar specifications.

[0471] In some aspects, the brain access catheter positioned 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 shorter in length and can be equipped with 24 inlet holes (e.g., 3 rows of 8 holes each) at the proximal end. In general, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The Omayya reservoir of the present invention can have an inner diameter of 300 to 400 mm. 2 The surface area of ​​the inner opening of the conduit can be 0.785mm 2 to 3.14mm 2 Therefore, the ratio of the reservoir surface area to the catheter surface area ranges from 96 to 509. With this ratio, the expected release time of the drug solution can be adjusted within a wide range, depending on the density of the test solution to water.

[0472] The system's Omayor-type device allows for direct drug delivery into the CSF and is particularly useful for delivering oligonucleotide and antisense oligonucleotide drugs (such as OT-101). It can be used alone or in combination with other cancer therapies.

[0473] In some aspects, the drug load can surprisingly be delivered as a single bolus infusion or as a short-term infusion of 15, 30 or 60 minutes and still achieve sustained delivery by a favorable ratio of reservoir volume to catheter opening and / or adjusting the ratio of CSF density to drug solution density.

[0474] Example 2 - Use of a device for continuous infusion via an intrathecal or intraventricular route is shown in Tables 4 and 5. This formulation in this example provides for efficient use of the agent because it is sufficiently stable to be delivered to the patient by continuous administration. The formulation of this example contains minimal or no excipients and is stable for at least 14 days without scaling. The formulation of the present invention is superior to conventional formulations using ingredients such as LNP particles and excipients such as lactose. Compared to such conventional formulations, the formulation of the present invention has improved stability and reduced bacterial growth.

[0475] Table 4: Compatibility study between OT-101 and the device

[0476]

[0477] Table 5: In-use stability studies conducted

[0478]

[0479] The following studies A) through C) were conducted as in-use tests under conditions simulating the clinical use of AP 12009(OT-101)-P002. This treatment utilized a pump and reservoir assembly similar to that used for OT-101-P002. The drug delivery system for this application consisted of the following medical device components:

[0480] Portable pump with corresponding drug reservoir and extension line

[0481] Gripper needle

[0482] Implantable port system with intravenous catheter

[0483] Three different drug delivery systems were tested in-use, with those components intended to be implanted for clinical use incubated at 37°C, while the non-implanted components were maintained at ambient temperature. The three drug delivery systems tested for the in-use stability of OT-101 drug solutions are described in Tables 6, 7, and 8 below.

[0484] Table 6: Drug delivery system 1

[0485]

[0486] Table 7: Drug delivery system 2

[0487]

[0488] *Not available

[0489] Table 8: Drug delivery system 3

[0490]

[0491]

[0492] * Unavailable

[0493] The conditions of the study are:

[0494] Drug solution concentration: 15 mg / mL (based on an average dose of 195 mg / mL 2 / d and an average body surface area of ​​1.85m 2 calculate)

[0495] Flow rate: 1 mL / h (equivalent to 24 mL / day)

[0496] The pump (including the drug reservoir) and non-implanted components of the drug delivery system are stored at ambient temperature.

[0497] live

[0498] Store implantable components of drug delivery systems at 37°C

[0499] Drug reservoir content: 120mL

[0500] Test duration: 5 days

[0501] 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 was considered satisfactory as long as it met the release specification of the AP 12009 (OT-101) 250 mg drug product. All samples met the release specification of the AP 12009 (OT-101) 250 mg drug product in terms of HPLC impurity profile. No relevant effects on the drug solution composition were observed after passage through the different drug delivery systems.

[0502] Table 9: Ion exchange HPLC analysis

[0503]

[0504]

[0505] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety (co-eluting with 3'N-2)

[0506] 3'N-2 = impurity with two missing 3'-terminal nucleotides (co-eluted with PO)

[0507] N-1 = impurity missing 3'- or 5'-terminal nucleotide

[0508] nd = Not Detected

[0509] Table 10: Reversed Phase HPLC Analysis

[0510]

[0511] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety (co-eluting with 5'N-1)

[0512] CNET = impurity with a cyanoethyl moiety added to one thymidine nucleotide

[0513] 3'N-2 = Impurity with two missing 3'-terminal nucleotides

[0514] 3'N-1 = Impurity with missing 3'-terminal nucleotide

[0515] 5'N-1 = impurity with missing 5'-terminal nucleotide (co-eluted with PO)

[0516] nd = Not Detected

[0517] Study B: The delivered drug solution was sampled once daily and analyzed by UV spectroscopy. The acceptance criteria were set as the reference sample UV 260nm The absorbance was between 90% and 110% of the initial sample concentration (reference value t=0 days).In addition, for all samples, UV spectra in the range of 230-320 nm were taken and compared with the spectrum of the reference sample.

[0518] The results are listed in Table 11, which show that all sample concentrations were within the range of 100.0%-102.7% of the reference solution, meeting the 90-100% acceptance criteria.

[0519] Table 11: UV analysis

[0520]

[0521] Study C: The delivered drug solution was sampled at the end of the in-use test. Leachable / extractable profiles were determined by a collaborating laboratory using a gas chromatography-based analysis method established for chemical analysis of medical devices according to ISO 10993 (i.e., quantification of released 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). Samples were sent to the collaborating laboratory, Medical Device Services, along with a reference sample (t-0d). Because saline is incompatible with gas chromatography, samples were extracted with tert-butyl ether and these extracts were then exposed to gas chromatography. No leachables / extractables were detected according to MDS report #111523-20.

[0522] Study D: Clinical study AP 12009-P002 will use two doses, 140 mg / m 2 / d and 250mg / m 2 / d. Assuming a body surface area of ​​1.75m 2 , the dose is 250mg / m 2 / d, calculate the concentration of representative drug solutions to be used in this test, as shown in Table 12.

[0523] Table 12: Drug parameters

[0524] The amount of trabedersen (250 mg / m 2 / dx 1.75m 2 x 4d): 1750mg

[0525] Volume required for 4-day continuous infusion: 96 mL

[0526] Drug solution concentration: 18.23 mg / mL

[0527] The representative drug solution was prepared in duplicate and aseptically filled into two separate Cadd cartridge reservoirs. The Cadd cartridge reservoirs were stored in an incubation chamber (non-sterile environment) at a temperature of 20° C.-25° C. The temperature of the incubation chamber was continuously monitored.

[0528] After 7 days of storage, the Cadd extension kit infusion line was connected to the Luer lock connector of the reservoir bag under sterile conditions. 20 mL of the incubated drug solution was moved to a separate sterility test device through an extension kit (including a 0.22 μm sterile filter). Another 20 mL of the incubated drug solution was moved directly from the reservoir bag to a separate sterility test device through a sterile cannula. The results of the sterility test are shown in Table 13.

[0529] Table 13: Sterility test results

[0530]

[0531]

[0532] The results of this study demonstrated that the Cadd cartridge reservoir maintained the sterility of aseptically filled drug solutions for at least 7 days.

[0533] The concentration of the dosing solution is 10 μM (61.43 μg / mL).

[0534] The compatibility of the drug used in the clinical study AP 12009(OT-101)–G005 with a 10 μM OT-101 drug solution was demonstrated by verifying the integrity of the drug solution after passage through the drug delivery system under conditions simulating its clinical use. The integrity of the drug solution was evaluated based on the following parameters: impurity profile, concentration, sterility, and leachable / extractable profile.

[0535] The following studies demonstrated the compatibility of OT-101 drug solution with the intended drug delivery system:

[0536] a) Determination of the impurity profile of OT-101 drug solution after passing through the drug delivery system

[0537] b) Verify the concentration of the drug solution after passing through the drug delivery system

[0538] c) Determination of leachable / extractable profile of OT-101 drug solution after passing through the drug delivery system

[0539] d) Sterility testing of the drug solution after incubation in the drug reservoir.

[0540] The drug delivery system suitable for this application consists of the following medical device components:

[0541] Portable pump with corresponding drug reservoir and extension line

[0542] Gripper needle

[0543] Implantable port system with intravenous catheter.

[0544] The drug delivery system was run for in-use testing, with components intended to be implanted for clinical use incubated at 37°C and non-implanted components maintained at ambient temperature.

[0545] The drug delivery systems that have been tested for in-use stability of OT-101 drug solutions are shown in Table 14 below.

[0546] Table 14: Drug delivery systems

[0547]

[0548]

[0549] Studies a) to c) were conducted as in-use tests under conditions simulating clinical use. These conditions were:

[0550] Drug solution concentration: 10 μM (61.43 μg / mL) OT-101 in isotonic saline solution

[0551] Flow rate: 4 μL / min (equivalent to 5.76 mL / day)

[0552] The pump (including the drug reservoir) and non-implanted components of the drug delivery system are stored at ambient temperature.

[0553] Store implantable components of drug delivery systems at 37°C

[0554] Drug reservoir content: 50mL

[0555] Due to the drug reservoir volume of 50 mL and the flow rate of 5.76 mL per day, the maximum duration of these tests was limited to approximately 8.5 days.

[0556] Study A: The delivered drug solution was sampled once daily and analyzed by two validated stability-indicating HPLC methods (ion exchange and reversed-phase HPLC). The acceptance criterion was set at twice the impurity area % of the corresponding impurity in the Tox batch (J982-15K9FP). In addition, a warning limit was set based on the release specification of the 7.37 mg drug product of OT-101 (AP 12009). Based on the impurity data shown in Table 15, the results indicate that only minor degradation was observed during use (under clinically relevant conditions) and that all samples met the drug product release specification. 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 seventeen phosphorothioate moieties is oxidized to a phosphodiester moiety. This impurity is considered to be a related product rather than an impurity (in terms of inactive degradation products).

[0557] Table 15: Impurity profile test results of drug solution after passing through drug delivery system

[0558]

[0559]

[0560] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety

[0561] CNET = impurity with a cyanoethyl moiety added to one thymidine nucleotide

[0562] 3'N-2 = Impurity with two missing 3'-terminal nucleotides

[0563] 3'N-1 = Impurity with missing 3'-terminal nucleotide

[0564] 5'N-1 = impurity with missing 5'-terminal nucleotide (co-eluted with PO)

[0565] 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 value t = 0 days). The test was performed in duplicate. The results are listed in Table 16, which shows that all sample concentrations were within the range of 98%-102% of the reference value. These results demonstrate that the drug solution concentration was not affected during passage through the drug delivery system (e.g., the drug was not adsorbed to the polymer surface of the drug delivery system).

[0566] Table 16: UV spectra of drug solutions after passing through the drug delivery system

[0567]

[0568] Study c: The delivered drug solution was sampled at the end of the in-use test. The leachable / extractable profile was determined by a collaborating laboratory using a gas chromatography-based analysis method established for the chemical analysis of medical devices according to ISO 10993 (i.e., quantification of released organic compounds using a flame ionization detector (GC-FID) and identification of potential leachables / extractables using gas chromatography-mass spectrometry (GC-MS), with comparison to the NIST / EPA / NIH 2005 mass spectral library, where applicable). No leachables / extractables were detected in this sample.

[0569] In all in-use stability studies, two HPLC data sets were collected over a period of 5 days, yielding the following regression results: Slope: -0.001065 ± 0.0009457, and the time to a 10% decrease in OT-101 concentration was 91 days. Therefore, the formulations of the present invention are surprisingly stable over long in-use periods.

[0570] Example 3 - Pharmacokinetics after intracerebral and intracerebroventricular administration in rats

[0571] After a single intracerebral injection into the prefrontal lobe of Sprague-Dawley rats, 3 Distribution, pharmacokinetics, and excretion of H-AP12009 (10 μCi / rat, 87 μM solution, flow rate: 0.4 μL / min, dosing time: 1 hour; Report GAS0002). Data from early time points (0 to 4 hours after completion of infusion) showed considerable inter-animal variability in both measured systemic radioactivity and tissue concentrations due to the small sample volume applied, the small size of the brain, and variability in the exact placement of the infusion device. Overall, trabedersen was readily distributed from the site of administration to other areas of the brain, particularly the cerebrospinal fluid, but also to the systemic circulation.

[0572] Intravenous administration of OT-101 resulted in minimal accumulation in the CNS. A single intravenous administration of OT-101 to male rats [ 3 H] OT-101, the concentrations of radioactivity in blood and tissues resulted in 0.1% OT-101 in the brain, as shown in Table 17.

[0573] Table 17: Radioactive material concentrations in blood and tissues

[0574]

[0575] To improve the use of OT-101 as a therapeutic agent, intrathecal delivery of tritiated OT-101 was studied in Sprague-Dawley CD (albino) rats. No sex differences were observed throughout the study. Following a one-hour intracerebral infusion in rats, OT-101 was confined to the infusion site. However, following a one-hour intraventricular infusion, OT-101 distribution was more widespread, with concentrations in the cerebellum, the rest of the brain, and the cerebrospinal fluid (CSF) reaching 35-fold, 19-fold, and 12-fold higher concentrations, respectively. For the first four hours after infusion, OT-101 concentrations were stable and exhibited a biexponential decay with a slow terminal half-life in tissues, but not in the CSF, indicating rapid penetration into underlying tissues distal to the CSF compartment. Minimal amounts of OT-101 were detected in the plasma compartment. Intrathecal bolus injections of 0.1 mL of 14, 30, 200, 300, and 500 μM OT-101 in cynomolgus monkeys did not result in any single-dose toxicity. Histopathological examination showed no material-related histomorphological changes in the subarachnoid space in the lumbar region. No changes were noted in the gray and white matter of the spinal cord, and the nerve stems and neurons did not show any abnormalities. These data suggest that intrathecal administration of OT-101 is a potentially effective delivery route for delivering antisense therapeutics such as OT-101 to the midline (i.e., for diffuse midline glioma (DMG)).

[0576] Diffuse midline glioma (DMG) is a highly prevalent pediatric central nervous system (CNS) tumor for which there is currently no effective treatment. DMGs account for 50% of all childhood HGGs. Due to their anatomical location and infiltrative nature, DMGs are not amenable to surgical resection and are typically diagnosed radiologically and treated with radiation therapy, which has no effect on survival. The median age at diagnosis is 5 to 11 years, with tumors arising in the pons occurring earlier (approximately 7 years) than in the thalamus (approximately 11 years). The median overall survival (OS) of patients with DMGs is very poor, at only 9-11 months, with <10% of patients with pontine tumors surviving two years after diagnosis. Radiation therapy remains the mainstay of treatment, although it is only palliative and is expected to extend survival by an average of 3 months.

[0577] In adult gliomas, OT-101 antisense was safe and effective during long-term (7-day) high-flow perfusion of the brain. As a single agent, it was as effective as the most effective drugs used in adult gliomas: TMZ (for chemotherapy-naive patients) and BCNU / CCNU (for chemotherapy-refractory patients).

[0578] TGF-β2 is highly expressed in both pediatric GBM (WHO grade IV) and pediatric DIPG (WHO grade IV) patients.

[0579] Expression analysis of pediatric brainstem cases from the TCGA database yielded a highly significant survival benefit across all four quartiles of TGF-β2 expression.

[0580] Expression analysis of all glioma cases treated with radiotherapy yielded a highly significant survival benefit across all quartiles of TGF-β2 expression.

[0581] Intracerebral infusion

[0582] Administration by intracerebral or intraventricular infusion resulted in the distribution of OT-101 throughout the CNS. 12 μg / rat was administered. The distribution profiles between cerebral and intraventricular administration were similar, with the difference being that direct administration into the CSF compartment via intraventricular administration resulted in higher drug concentrations in the cerebellum and CSF. Surprisingly, the similarities between intraventricular and intracerebral administration suggest that intraventricular administration is as effective as intracerebral administration, as shown in Table 18.

[0583] Table 18: Administration by Intracerebral Infusion or Intraventricular Infusion

[0584]

[0585] Cumulative distributions for the first 15 organs are shown below. At the end of the 1-hour infusion period, CSF-accessible, rapidly perfused portions of the CNS (administration site, cerebellum, CSF, pituitary gland) reached C max Although CSF is readily accessible, the pineal organ actively absorbs OT-101 and does not reach C until 4 hours. max Less perfused parts of the CNS reached C at 1 hour (rest of brain, rest of brain) and 4 hours (spinal cord). max .

[0586] A small portion of the administered OT-101 reaches the plasma compartment and reaches C max Organs that are easily perfused by blood reach C at the 4-hour time point (thyroid and liver). max , while those organs that actively absorbed OT-101 did not reach C until 24 hours (kidney, spleen, bone marrow) or 72 hours (thymus) max , see Table 19.

[0587] Table 19: Administration by Intracerebral Infusion or Intraventricular Infusion

[0588]

[0589]

[0590] The data from this distribution study clearly demonstrate that OT-101 reaches the entire CNS region, including the midline, via intracerebral (and therefore intraventricular) infusion. This profile is consistent with CSF flow and the location of the various compartments examined. Parts of the CNS with high OT-101 levels are CSF-accessible, including the pituitary and pineal glands.

[0591] OT-101 has been studied in a series of nonclinical safety and pharmacology studies. The salient features of the conclusions from these studies are as follows:

[0592] Long-term topical administration of OT-101 / AP 12009 may cause local tissue inflammation. Mild to moderate local toxicity was observed in animals following infusion of a 500 μM concentration, but without any macroscopic changes.

[0593] When administered as a bolus IT injection to 3 kg male or female rabbits at a dose level of 0.12 mg / kg (500 μM solution; 0.1 mL) (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.

[0594] When administered as a bolus IT injection to 6-7 kg cynomolgus monkeys at a dose level of 0.05 mg / kg (500 μM solution; 0.1 mL) (estimated CSF concentration 0.46 μM), OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes, consistent with subclinical toxicity.

[0595] When administered intracerebroventricularly to rats, radiolabeled OT-101 (0.18 mg / kg) was detected not only in the CSF but also in the cerebrum, cerebellum, and pineal gland within 1 hour of administration. The half-life in CSF and brain tissue was <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0596] Intratumoral placement of catheters carries potential risks that can be avoided by using an Omayor reservoir to access the ventricular space.

[0597] Omayor reservoirs can be used to treat leptomeningeal carcinoma (LM) caused by various malignancies.

[0598] Example 4- Figure 9 The effects of delivering the pharmaceutical composition by intrathecal or intraventricular continuous infusion are shown. Figure 9Shown is 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 / AP12009 (1 μM to 80 μM) for 7 days as indicated. Secreted TGF-β2 was measured in the cell supernatant by ELISA. Results represent the median, minimum, and maximum values ​​from three independent experiments.

[0599] Example 5 - Comparison of an example of continuous infusion using a partially collapsible Omayer-type reservoir of the present invention with a conventional direct injection method.

[0600] It was found that the apparatus and method for continuous infusion of therapeutic agent solutions of the present invention are advantageous compared to conventional methods ( Figure 10 ).

[0601] In conventional methods, a therapeutic agent solution is injected directly into the ventricular space of a patient's brain, or injected directly into the ventricular space via a catheter. In these conventional methods, the therapeutic agent solution rapidly disperses into the ventricular space within seconds to minutes. Furthermore, this rapidly injected therapeutic agent solution is rapidly cleared from brain tissue. Consequently, due to the limited exposure of brain tissue to the therapeutic agent solution, the therapeutic effect achieved with these conventional methods is very limited.

[0602] By comparison, the Omayne-type reservoir of the present invention having a partially collapsible top advantageously provides continuous infusion, substantially continuous infusion, over an extended period of time.

[0603] The Omayer-type reservoir of the present invention was filled with a test solution and placed on the water surface of a water tank to test the infusion rate, i.e., the rate at which the test solution flowed out of the distal tip of the catheter, as a model of continuous infusion into the brain.

[0604] In one embodiment, the surface area of ​​the Omayyad-type reservoir of the present invention is 339 mm 2 , and the area of ​​the inner opening of the catheter is 1.5386mm 2 , which provides a ratio of 339 / 1.5386 = 220. Based on this ratio, the expected release time of the test solution will be at least several hours, depending on the density of the test solution to water.

[0605] In this example, the test solution slowly diffused out through the catheter opening (see Figure 10 ). Diffusion from the Omayor-type reservoir of the present invention continued for more than 4 hours. This test shows that the test solution achieves continuous diffusion and that therapeutic agent solutions with adjusted or CSF-matched densities will provide continuous infusion over a period of at least one to several days.

[0606] This example demonstrates that the partially collapsible design of the Omayor-type reservoir of the present invention is advantageous because, when continuously infusing a therapeutic agent solution over an extended period of time, it is necessary to prevent accidental release of the therapeutic agent solution due to unintended or accidental pressure on the flexible top of the Omayor-type reservoir. For example, unintended or accidental pressure on the flexible top of the Omayor-type reservoir could result in the release of too much therapeutic agent solution in a very short period of time.

[0607] The partially collapsible design of the Omayor-type reservoir of the present invention prevents such undesirable accidental or inadvertent release of the therapeutic agent solution.

[0608] Furthermore, the partially collapsible design of the Omayor-type 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 pressure changes, which allows the Omayor-type reservoir of the present invention to properly breathe to achieve continuous flow of solution, especially in the presence of an externally generated pumping action. Undesirable accidental or incidental release may also occur due to patient movement, such as movement of the patient's jaw or other muscles.

[0609] In some examples and embodiments, the Omayer-type reservoirs of the present invention are prepared with a collar, which makes it impossible for the reservoir to collapse completely and prevents accidental or inadvertent release of the solution (see Figure 8 ).

[0610] In some examples and embodiments, the Omayyad-type reservoirs of the present invention are made partially collapsible by manufacturing a portion of the top of the Omayyad-type reservoir from a hard, non-flexible plastic.

[0611] Example 6 - Diffuse Intrinsic Pontine Glioma in Children.

[0612] In this application, methods for pediatric DMG / DIPG / or K27M GBM are disclosed.

[0613] In this example, new evidence suggests that enhanced expression of TGFB2 mRNA in pediatric DIPG and H3K27M-mutant GBM is associated with upregulation of 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 adverse treatment outcomes in DIPG. The reported results also support the view that further evaluation of the clinical potential of new strategies targeting TGFB2 mRNA in pediatric DIPG is warranted.

[0614] The median survival of 30 patients with high TGFB2 in the H3K27M mutation subgroup was 6.5 months (95% CI = 5-NA months, 6 events, N = 8), which was significantly shorter than the median survival of the remaining patients (median survival > 10 months, 7 events, N = 22);

[0615] Diffuse intrinsic pontine glioma (DIPG) in children is one of the most aggressive and lethal childhood brain tumors. The aim of this study was to evaluate the clinical significance of increased transforming growth factor beta 2 (TGFB2) expression in tumor tissue samples from patients with DIPG. Our findings provide the first evidence that high levels of TGFB2 expression are associated with adverse treatment outcomes in DIPG. The reported results also support the need for further evaluation of the clinical potential of novel strategies targeting TGFB2 in childhood DIPG.

[0616] Tumor samples from newly diagnosed pediatric diffuse intrinsic pontine glioma (DIPG) patients express significantly higher levels of transforming growth factor β2 (TGFB2, also known as TGF-β2) messenger RNA (mRNA) compared with control pontine samples, which is associated with enhanced expression of transcription factors that upregulate TGFB2 gene expression. Our study also demonstrated that high TGFB2 mRNA levels, as determined by RNA sequencing (RNAseq), are a poor prognostic indicator for patients with DIPG, but not for patients with pediatric glioblastoma (GBM) or pediatric diffuse midline glioma (DMG) tumors located outside the pons / brainstem. Notably, DIPG patients with high TGFB2 mRNA expression in their tumor samples had significantly worse overall survival (OS) and progression-free survival (PFS). In contrast, high levels of transforming growth factor β3 (TGFB3) mRNA expression in tumor samples were associated with significantly better survival outcomes in DIPG patients, whereas high levels of transforming growth factor β1 (TGFB1) expression did not predict prognosis. Our study fills a significant gap in our understanding of the clinical significance of elevated TGF expression in pediatric high-grade gliomas.

[0617] Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive and lethal pediatric brain tumors classified as diffuse midline gliomas (DMGs) with histone H3 lysine 27 (H3K27) alterations, including H3 K27 mutants as well as H3 wild-type subtypes with overexpression of EZH inhibitory protein (EZHIP). DIPG is the second most common pediatric malignant brain tumor, and complete resection is impossible due to its anatomical location in the brainstem and its rapidly infiltrative growth. Despite contemporary radiotherapy / chemoradiotherapy strategies, DIPG is associated with poor overall survival (OS) and is a driver of cancer-related mortality in children (median OS <1 year). Despite numerous clinical trials targeting chemotherapeutic agents, immuno-oncology agents, and specific targeted therapies to improve survival outcomes in children with DIPG, little progress has been made, and the prognosis for DIPG remains poor, with a median survival of approximately 10 months and a two-year survival rate of less than 10%.

[0618] We present novel evidence demonstrating that enhanced expression of TGFB2 mRNA in childhood DIPG and H3K27M-mutant GBM is associated with upregulation of 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 adverse treatment outcomes in DIPG. The reported results also support the need for further evaluation of the clinical potential of novel strategies targeting TGFB2 mRNA in childhood DIPG.

[0619] Furthermore, there is no standard treatment for progressive DIPG after failure of first-line radiotherapy, nor are there salvage regimens that have been shown to prolong OS. Effective therapeutic strategies that have the potential to improve the poor prognosis of these children are urgently needed, and their discovery represents a major focus of translational and clinical research in contemporary neuro-oncology. Multiple interventional strategies are currently being explored, such as immunotherapy with immune checkpoint inhibitors or T cells with chimeric antigen receptors (CARs) ("CAR-T cells"), inhibition of signal transduction pathways with small molecule drugs, and biologic therapy with fusion toxins administered via convection-enhanced delivery (CED) or oncolytic viruses.

[0620] Transforming growth factor-β (TGFB) is a disulfide-linked homodimeric cytokine with pleiotropic activities that are implicated in tumorigenesis and suppression of host antitumor immunity within the tumor microenvironment (TME). Enhanced TGFB signaling activity, mediated by autocrine or tumor-associated macrophage (TAM)-derived overproduction, has been implicated in the aggressive biology and poor overall survival of adult patients with high-grade glioma by promoting invasiveness and rapid glioma cell growth. The TGFB pathway has also been shown to contribute to the "cold" TME in high-grade gliomas through its immunosuppressive effects, characterized by suppression of CD8 antigen-positive cytotoxic T cells, natural killer (NK) cells, activated 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)-pyridinone, PFD), has been shown to inhibit TGFB expression in malignant glioma cells. A synthetic antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeting TGFB2 mRNA has demonstrated promising single-agent clinical activity associated with durable complete and partial responses in adult patients with recurrent or refractory glioblastoma and anaplastic astrocytoma when administered intratumorally via CED. In a study using a microarray-based gene expression platform, TGFB2 mRNA levels were selectively enhanced in primary tumor samples from 29 pediatric DIPG patients compared with normal samples and primary tumor samples from patients with low-grade gliomas.

[0621] This study evaluated the clinical prognostic significance of high tumor TGFB2 mRNA levels in pediatric DIPG, as measured by RNAseq. Pediatric DIPG patients with enhanced TGFB2 mRNA expression in brain tumor tissue may exhibit more aggressive disease and a worse prognosis. The present invention demonstrates that newly diagnosed pediatric DIPG patients with increased TGFB2 mRNA levels in their primary tumor samples, but not with increased transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 3 (TGFB3) mRNA levels, have significantly worse progression-free survival (PFS) and overall survival (OS) compared with other pediatric DIPG patients. High TGFB2 mRNA levels are a poor prognostic indicator for DIPG patients, but not for DMG patients or pediatric glioblastoma (GBM) patients whose tumors are located outside the pons / brainstem, such as in the cerebellum and thalamus.

[0622] A clinical study was conducted to determine clinical metadata and RNA sequencing (RNAseq)-based mRNA expression data for the TGFB subtypes TGFB1, TGFB2, and TGFB3 in 41 pediatric patients with DMG (mean age at diagnosis (months) = 7.02 ± 0.44; median = 6; range = 2-14) and 116 pediatric patients with GBM (mean age at diagnosis (months) = 60.01 ± 1.24; median = 60.4; range = 21-89.3) using genomic data obtained through the Cancer Genomics cBioPortal (https: / / pedcbioportal.kidsfirstdrc.org / ). An interactive web interface with full filtering capabilities provided by the portal was used. These data are compiled, coordinated, and annotated in multiple data consortia, such as 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 impact of TGFB2 mRNA expression levels on PFS and OS outcomes. Clinical trials related to the treatment of DIPG patients (https: / / clinicaltrials.gov / ct2 / show / NCT02274987) outline a general treatment strategy that includes standard radiation therapy followed by biomarker-guided specialized therapy with FDA-approved targeted therapies, guided by gene expression analysis, whole exome sequencing (WES), and predictive models.

[0623] 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 receptor 3 (TGFBR3) are reported using RNAseq V2 values ​​(appended to the expression profile data file "mRNA_expression_(RNA_Seq_V2_RSEM).txt"), normalized to "transcripts per million" ("TPM") values ​​calculated using RSEM, a software package for estimating gene and isoform expression levels from RNAseq data. The RSEM-based process involves two main steps: 1. generating a set of reference transcript sequences for gene-level mRNA abundance estimation; 2. aligning a set of RNAseq reads to these reference transcripts for TPM abundance estimation. This process allows for direct comparison and ranking of mRNA abundance between samples.

[0624] PFS and OS outcomes were compared among patient subgroups using the Kaplan-Meier (KM) method and the log-rank chi-square test, using the survival_3.2-13, survminer_0.4.9, and survMisc_0.5.5 packages implemented in the R environment. Graphical representations of treatment outcomes were generated using the graphics rendering 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 among the compared patient subgroups, and p-values ​​less than 0.05 were considered significant.

[0625] TBFB1, TGFB2, and TGFB3 mRNA expression values ​​for normal pons samples measured by mRNA sequencing (rna_tissue_hpa.tsv.zip) were downloaded from https: / / www.proteinatlas.org / about. mRNA expression values ​​for 29 different pontine regions of the brain were compiled (in TPM) by filtering the "Tissue Group" annotation in the accompanying description file (rna_tissue_hpa_description.tsv.zip). TGFB1 / TGFB2 / TGFB3 mRNA expression levels in normal pons samples were compared with those in brain tumor samples from 41 DIPG patients using a two-way analysis of variance (ANOVA) model. For each transcript, p-values ​​were adjusted for the false discovery rate (FDR) for comparisons between normal pons and DIPG samples. Calculations were performed using the multcomp_1.4-17 and emmeans_1.7.0 statistical packages in R version 4.1.2 and 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.

[0626] Log2 TPM of TGFB2 mRNA expression levels correlated with those of 11 transcription factors known to enhance TGFB2 expression, namely, activating transcription factor 1 (ATF1), activating transcription factor 2 (ATF2), cyclic AMP response 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) in 41 DIPG patients. Pairwise correlation coefficients were determined for all transcript combinations and visualized on a heat map color-coded from positive correlation (red = +1) to negative correlation (blue = -1). A clustering algorithm was used to identify co-regulated gene sets using the statistical package ggcorrplot_0.1.3 implemented in R. T-tests were used to test the null hypothesis that the Pearson correlation coefficient was equal to 0. Correlations were considered significant when the p-value was less than 0.05 and the FDR was less than 0.10.

[0627] Normalized archived transcriptome analysis datasets obtained from the Gene Expression Omnibus portal (https: / / www.ncbi.nlm.nih.gov / geo), including raw CEL files obtained using the Human Genome U133 Plus 2.0 array platform for DIPG (N = 29; GSE26576), normal control samples (N = 2; GSE26576), and pediatric GBM patients with 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 relative to brain tumor samples from 41 pediatric DIPG or pediatric GBM patients with H3K27M mutations. The normalization procedure for determining log2-transformed mRNA expression levels was performed using the Robust Multi-array Averaging (RMA) method as described previously. 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) running in the RStudio environment (R version 4.1.2, RStudio 2021.09.0Build 351). Statistical comparisons were performed using the ANOVA statistical model. FDR-adjusted p values ​​less than 0.05 were considered significant. As described previously, heat maps were used to visualize the mRNA expression levels of TGFB1, TGFB2, and TGFB3 in patients with DIPG / H3K27M mutant GBM. The expression level (log2 RMA) of TGFB2 mRNA was correlated with the mRNA in the two-way ANOVA model: 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.

[0628] result

[0629] Compared with normal pons samples, tumor samples from children with DIPG contained higher levels of TGFB2 mRNA, but not TGFB1 mRNA or TGFB3 mRNA.

[0630] Comparison of RNAseq-based mRNA levels of TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples and 29 normal pons samples ( Figure 14Notably, the mean (mean ± SE) TGFB2 mRNA level in primary DIPG samples was 1.5-fold higher than that in normal pons samples (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 those in normal pons samples (TGFB1 mRNA level was 1.7-fold lower, p = 0.0002, TGFB3 mRNA level was 2.7-fold lower, p < 0.001), as reflected by the blue color in the heat map of the hierarchical clusters ( Figure 14 ) and the average expression value was significantly decreased (expressed in log2 TPM) ( Figure 14 ).

[0631] Selective overexpression of TGFB2 mRNA in DIPG tumor samples is associated with enhanced expression of transcription factors that bind to multiple TGFB2 gene promoter sites.

[0632] The molecular mechanisms underlying the upregulation of TGFB2 mRNA expression levels in 41 DIPG tumor samples were determined, focusing on transcription factors / DNA-binding proteins known to enhance TGFB2 gene expression. 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. mRNA levels from 8 of these 11 transcription factors (i.e., SP1, RFX1, POLR2A, FOXO3, EP300, CREB1, ATF2, and ATF1) showed statistically significant positive correlations with TGFB2 mRNA levels ( Figure 15 SP1, FOXO3, and EP300 showed the most significant correlation with TGFB2 mRNA expression levels (p<0.0001).

[0633] Figure 14Selective upregulation of TGFB2 mRNA expression in DIPG tumor samples was shown. TGFB1 / 2 / 3 mRNA expression levels (log2-transformed TPM) in archived primary tumor samples from 41 DIPG patients (including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M-mutant DIPG patients, and 26 H3K27M-mutant DMG patients whose brain tumors were located in the pons / brainstem, obtained from cBioPortal) were compared with TGFB1 / 2 / 3 mRNA expression levels (log2 TPM) in normal pons samples from 29 pontine regions (downloaded from https: / / www.proteinatlas.org / about / download). TGFB2 expression levels in these 29 normal pontine regions were determined by averaging TPM values ​​from 2-8 independent samples / regions from 21 subjects. The log2-transformed TPM values ​​of TGFB1 / 2 / 3 mRNA levels in DIPG patient tumor samples were average-centered relative to mRNA expression levels in normal pons samples and depicted in a heat map (A), which shows overexpression (red) or underexpression (blue). (B) The bar graph shows that TGFB1 and TGFB3 mRNA expression levels are reduced, but TGFB2 mRNA expression levels are increased, in DIPG patient tumor samples (dark gray bars) compared to normal pons samples (light gray bars).

[0634] (C) The statistical significance of differences in TGFB1 / 2 / 3 mRNA expression levels (expressed as log2-transformed TPM values) was assessed using two-way ANOVA and linear comparisons using FDR-adjusted p-values. TGFB2 mRNA levels were found to be statistically significantly increased by 1.52-fold (p = 0.015), while TGFB1 mRNA levels (decreased by 1.72-fold; p = 0.002) and TGFB3 mRNA levels (decreased by 2.7-fold; p < 0.001) were statistically significantly decreased.

[0635] Figure 15The correlation matrix between TGFB2 and transcription factors in 41 DIPG patients is shown. Pairwise Pearson correlation analysis was performed on the mRNA levels of TGFB2 and 11 transcription factors using log2-transformed TPM values ​​obtained from the cBioPortal for CancerGenomics (https: / / pedcbioportal.kidsfirstdrc.org / ). Correlation coefficients were calculated for the 41 DIPG patients and are depicted on the heat map (A), ranging from positive correlation (red) to negative correlation (blue) and organized according to similarly expressed genes. The scale of the correlation range is shown in the color bar "Corr". Of the 66 pairwise correlations, 48 ​​were considered statistically significant (p < 0.05, FDR = 0.07; non-significant correlations are indicated by black crosses in the heat map). (B) mRNA levels of eight of the 11 transcription factors (i.e., SP1, RFX1, POLR2A, FOXO3, EP300 / transcription factor coactivator, CREB1, ATF2, and ATF1) showed statistically significant positive correlations with TGFB2 mRNA levels. SP1, FOXO3, and EP300 mRNA levels showed the most significant correlations with TGFB2 mRNA expression levels (p < 0.0001). (C) Transcription factor proteins (ovals) that bind to the binding sites of these transcription factors (rectangles) on the TGFB2 gene are depicted. Furthermore, recruitment of the transcription factor coactivator EP300 (E1A-binding protein P300) is demonstrated, which binds to the phosphorylated form of CREB1 / ATF1 / ATF2 and bridges to the basal transcription machinery via RNA polymerase II subunit A (POLR2A) to directly stimulate TGFB2 transcription.

[0636] Similar results were obtained in an independent validation dataset of microarray-based TGFB1, TGFB2, and TGFB3 mRNA levels and their correlation with specific transcription factors in tumor samples from 41 pediatric patients with DIPG (n = 29) or H3K27M-mutant GBM ( Figure 16 ). Three TGFB2 probe sets were observed to show significant increases in mRNA expression levels: 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, inset). Significant reductions in mRNA expression were observed for one TGFB1 probe set, TGFB1_203085_s_at (2.2-fold reduction, p=0.028), and one TGFB3 probe set, TGFB3_209747_at (2.3-fold reduction, p=0.026). Probe set TGFB2_220407_s_at exhibited the greatest number of positive correlations (21 significant positive correlations, p<0.05 (FDR=0.047)) with the probe sets for the 11 transcription factors and all four other TGFB2 probe sets. Figure 16 , inset). Eight 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 statistically significant positive correlations with TGFB2 mRNA levels ( Figure 16 , small picture).

[0637] Figure 16Showing the 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 is illustrated using a clustered representation of log2-transformed fold changes in 41 brain tumor samples from pediatric patients with DIPG or H3K27M-mutant GBM. Expression levels were mean-centered relative to normal samples and depicted as log2-transformed normalized RMA values. Blue to red colors in the heatmap indicate underexpression to overexpression of TGFB1, TGFB2, and TGFB3 mRNA levels, respectively. Co-regulated probe sets are organized and depicted using a dendrogram of probe sets (rows) and patients (columns). Significant increases in mRNA expression 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). Significant decreases in mRNA expression were observed for one TGFB1 probe set, TGFB1_203085_s_at (2.2-fold decrease, p = 0.028), and one TGFB3 probe set, TGFB3_209747_at (2.3-fold decrease, p = 0.026). (B) A total of 1640 correlation analyses were performed on 41 probe sets representing 11 transcription factors and TGFB2 mRNA. For 660 correlations, the p-value was less than 0.05 (FDR = 0.12), and for 324 correlations, the p-value was less than 0.001 (FDR = 0.005). The probe set TGFB2_220407_s_at showed the greatest number of positive correlations with the probe sets of 11 transcription factors and all four other TGFB2 probe sets. There were 21 significant positive correlations with p < 0.05 (FDR = 0.047). The color-coded Pearson correlation coefficients for the most significant positive correlations with TGFB2_220407_s_at are depicted on the heat map, ranging from positive correlation (red) to negative correlation (blue), organized according to similarly expressed mRNA levels. The scale of the correlation range is shown in the color bar "Corr". Non-significant correlations are indicated by black crosses in the heat map. 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 statistically significant positive correlations with TGFB2 mRNA levels.

[0638] Enhanced expression of TGFB2 mRNA, but not TGFB1 or TGFB3 mRNA, was associated with shorter OS and PFS in DIPG patients.

[0639] The survival outcomes of DIPG patients with TGFB2 mRNA expression levels greater than or equal to the upper quartile (TGFB2-high) were compared with the treatment outcomes of the remaining DIPG patients (TGFB2-low). The mean TGFB2 expression level in the subgroup of 11 TGFB2-high patients was 6.2 ± 0.2 (median, range = 5.8, 5.2–7.6). In contrast, the mean TGFB2 mRNA expression level in the subgroup of 30 TGFB2-low patients was 3.2 ± 0.2 (median, range = 3.5, -0.4–5.1). Patients with TGFB2-high DIPG showed significantly worse OS outcomes than those with TGFB2-low DIPG (median OS in the TGFB2-high subgroup: 5 months, 95% CI: 5-5 months, 11 events, N = 11); median OS in the TGFB2-low subgroup: 11.5 months, 95% CI: 10-14 months, 30 events, N = 30; log-rank chi-square = 16.2, p = 5.6 × 10 -5 )( Figure 17 The PFS results in the TGFB2 high subgroup were also significantly worse (median PFS in the TGFB2 high subgroup: 5 (95% CI: 5–NA) months, 11 events, N = 11); median PFS in the TGFB2 low subgroup = 11 months, 95% CI: 10–13 months, 30 events, N = 30 (log-rank chi-square = 14.7, p = 1.3 × 10 -4 )( Figure 18 In contrast to the adverse prognostic impact of high TGFB2 mRNA expression, high TGFB3 mRNA expression was associated with significantly better OS and PFS outcomes, whereas high TGFB1 mRNA expression had no statistically significant effect on OS or PFS outcomes ( Figure 17 and Figure 18 ).

[0640] OS outcome data for the 30 patients with H3K27M mutation DIPG were evaluated separately, excluding 11 patients with DIPG whose H3K27M mutation status was unknown. TGFB2-high patients in the 30-patient subgroup had a shorter time to death than the remaining patients, similar to the TGFB2-high patients in the full analysis set of 41 patients, which included 11 DIPG patients with unknown H3K27M mutation status (not shown). However, the differences did not reach statistical significance in the smaller subgroups, which may be due to a wider distribution of OS outcomes and a smaller sample size (not shown), resulting in reduced power to detect statistical differences. Notably, like the full analysis set of 41 patients (not shown), TGFB2-high patients in the 30-patient subgroup were characterized by early failure and significantly worse survival outcomes within 10 months (not shown). The median survival of 30 TGFB2-high patients in the H3K27M mutation subgroup was 6.5 months (95% CI = 5-NA months, 6 events, N = 8), which was significantly shorter than that of the remaining patients (median >10 months, 7 events, N = 22; log-rank chi-square = 5.5, p-value = 0.019) (not shown).

[0641] Figure 17 We show that increased TGFB2 expression is associated with shortened OS in patients with DIPG. Archival survival outcome data for 41 patients with DMG were obtained from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ), including 11 patients with DIPG of unknown H3K27M mutation status, 4 patients with H3K27M-mutant DIPG, and 26 patients with H3K27M-mutant DMG whose brain tumors were located in the pons / brainstem. These data were combined with RNAseq-based mRNA expression data for TGFB2 (A), TGFB3 (B), and TGFB1 (C) to assess the potential impact of various TGFB isoform levels on OS. Log2-transformed, TPM-normalized RNAseq values ​​were ranked according to the expression level of each TGFB isoform. Patients whose expression level of a given TGFB isoform was greater than or equal to the upper quartile (solid line) were compared with the remaining patients (dashed line). Figure 14Figure 3 shows the levels of TGFB2 isoforms in the analyzed patient subgroups. (A) The mean TGFB2 expression level in the subgroup of 11 TGFB2-high patients was 6.19 ± 0.24 (median, range = 5.81, 5.23–7.63). In contrast, the mean TGFB2 expression level in the subgroup of 30 TGFB2-low patients was 3.22 ± 0.23 (median, range = 3.48, -0.42–5.07). Patients with high TGFB2 mRNA expression (TGFB2-high) exhibited significantly worse OS outcomes than other patients (TGFB2-low) (median OS in the TGFB2-high subgroup: 5 months, 95% CI: 5-NA months, 11 events, N = 11); median OS in the TGFB2-low subgroup: 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 subgroup of 11 TGFB3-high patients was 4.7±0.2 (median, range = 4.6, 4–5.6). In contrast, the mean TGFB3 expression level in the subgroup of 30 TGFB3-low 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-NA months, 11 events, N = 11) showed significantly better OS outcomes than patients with TGFB3-low (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 subgroup of 11 TGFB1-high patients was 4.8±0.1 (median, range = 4.8, 4.5–5.4). In contrast, the mean TGFB1 expression level in the subgroup of 30 TGFB1-low 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 compared to other 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).

[0642] Figure 18Increased TGFB2 expression is associated with shortened PFS in patients with DIPG and DMG. Archival clinical outcome data from 41 DMG patients (including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M-mutant DIPG patients, and 26 H3K27M-mutant DMG patients whose brain tumors were located in the brainstem / pons) obtained from the cBioPortal for CancerGenomics (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 various TGFB isoform levels on PFS. For patients lacking information on disease progression, death was considered the first event in the evaluation of PFS outcomes. Log2-transformed, TPM-normalized RNAseq values ​​were ranked according to the expression level of each TGFB isoform. Patients with expression levels of a given TGFB isoform greater than or equal to the upper quartile (solid line) were compared with the remaining patients (dashed line). (A) Patients with high TGFB2 levels had significantly worse PFS than other patients (median PFS in the TGFB2-high subgroup: 5 (95% CI: 5–NA) months, 11 events, N = 11); median PFS in the TGFB2-low subgroup = 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).

[0643] TGFB2 expression levels did not affect OS or PFS in pediatric DMG patients whose tumors were not located in the pons / brainstem.

[0644] The survival outcomes of non-DIPG DMG patients whose tumors were located outside the pons / brainstem and whose TGFB2 mRNA expression levels were greater than or equal to the upper quartile (TGFB2 high) were compared with the treatment outcomes of the remaining non-DIPG DMG patients (TGFB2 low). Patients with TGFB2 high (median OS = 12.5 months, 95% CI: 9-NA months, 10 events) showed similar OS outcomes to those with TGFB2 low (median OS = 11 months, 95% CI: 2-NA months, 8 events, log-rank chi-square = 0.2, p = 0.6) (not shown). Non-DIPG DMG patients with TGFB2 high and TGFB2 low also had very similar PFS outcomes (not shown). Similarly, no statistically significant differences in OS or PFS outcomes were found in the comparison of non-DIPG DMG subgroups with TGFB1 high versus TGFB1 low or TGFB3 high versus TGFB3 low (not shown). Since activation of the TGFB signaling pathway requires TGFB binding to TGF-β receptor II (TGF-βRII), which in turn recruits TGF-βRI into a heterotetrameric complex, we determined whether the observed lack of prognostic effect of high TGFB2 status could be explained by reduced expression of the TGFB2 receptor. Tumor samples from non-DIPG DMG patients (N=19) did not exhibit lower expression of any TGFB2 receptor compared to tumor samples from DIPG patients (N=41), explaining the lack of prognostic significance of higher TGFB2 mRNA levels observed in this group of patients (not shown). DIPG patients (mean age at diagnosis (months) = 7.0 ± 0.4; median = 6, range = 2-14) were on average younger than non-DIPG DMG patients (mean age at diagnosis (months) = 10.5 ± 0.7; median = 11; range = 5-17) (p = 0.0003).

[0645] TGFB2 mRNA expression level did not affect OS or PFS in pediatric GBM patients.

[0646] Survival outcomes in pediatric GBM patients with TGFB2 mRNA expression levels greater than or equal to the upper quartile (TGFB2-high) were compared with treatment outcomes in the remaining pediatric GBM patients (TGFB2-low). In the 29 pediatric GBM subgroup with TGFB2-high, the mean TGFB2 expression level was 12.7 ± 0.1 (median, range = 12.6, 12–13.7). In contrast, the mean TGFB2 expression level in the 87 pediatric GBM subgroup with TGFB2-low was 10.4 ± 0.1 (median, range = 10.5, 8.2–12). Patients in the TGFB2 high (N=29) and TGFB2 low (N=87) subgroups showed very similar OS outcomes (median TGFB2 high: 12.6 (95% CI: 9.9–NA) months, 17 events; median TGFB2 low: 12.9 (95% CI: 11.2–15) months, 65 events; log-rank chi-square = 0.2, p = 0.7) ( Figure 19 ). Pediatric GBM patients with high TGFB2 and low TGFB2 also had very similar PFS outcomes (not shown). Similarly, no differences in OS or PFS outcomes were found in the subgroup comparisons of pediatric GBM patients with high TGFB1 versus low TGFB1 or high TGFB3 versus low TGFB3 ( Figure 18 None of the TGFB2 receptors showed lower expression levels in tumor samples from GBM patients compared with DIPG / DMP patients, explaining the lack of prognostic significance of higher TGFB2 mRNA levels observed in this patient population. Conversely, levels of all three receptors were significantly elevated in GBM patients compared with DIPG patients (not shown). GBM patients had higher TGFB2 mRNA levels than DIPG patients; even among the 11 DIPG patients with high TGFB2, 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 GBM patients with low TGFB2 (10.4 ± 0.1; median, range = 10.5, 8.2–12). The lack of a true TGFB2-low subgroup in GBM patients may have hindered the accurate assessment of the prognostic effect of high TGFB2 mRNA levels. The mean age of DIPG patients (mean age (months) at diagnosis = 7.0 ± 0.4; median = 6; range = 2–14) was younger than that of GBM patients (mean age (months) at diagnosis = 60.0 ± 1.2; median = 60.4; range = 21–89.3) (p < 0.0001).

[0647] Figure 19Figure 3: TGFB2 mRNA expression levels do not affect OS in pediatric GBM patients. Archival OS data from 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 various TGFB isoform levels on OS. Log2-transformed TPM-normalized RNAseq values ​​were ranked according to the expression level of each TGFB isoform. Patients with expression levels of a given TGFB isoform greater than or equal to the upper quartile (solid line) were compared with the remaining patients (dashed line). Figure 16Figure 5. TGFB2 isoform levels in the analyzed patient subgroups. (A) The mean TGFB2 expression level in the 29 TGFB2-high patient subgroup was 12.67 ± 0.1 (median, range = 12.63, 11.97–13.7). In contrast, the mean TGFB2 expression level in the 87 TGFB2-low patient subgroup 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) subgroups showed very similar OS outcomes (median TGFB2-high: 12.6 (95% CI: 9.9–NA) months, 17 events; median TGFB2-low: 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 29 TGFB3-high patient subgroup was 11.39 ± 0.1 (median, range = 11.25, 10.63–12.88). In contrast, the mean TGFB3 expression level in the 87 TGFB3-low patient subgroup was 9.4 ± 0.08 (median, range = 9.56, 7.14–10.58). Patients in the TGFB3-high and TGFB3-low subgroups showed very similar OS outcomes (median TGFB3-high: 12.9 (95% CI: 10.41–26.38) months, 20 events; median TGFB3-low: 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 29 TGFB1 high patient subgroup was 12.08 ± 0.08 (median, range = 11.92, 11.58–13.13). In contrast, the mean TGFB1 expression level in the 87 TGFB1 low patient subgroup 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).

[0648] In this clinical study, the prognostic significance of TGFB2-high status was examined in newly diagnosed pediatric DIPG patients. Notably, patients with high TGFB2 (but not high TGFB1 or high TGFB3) had significantly worse survival outcomes and significantly shortened OS times. High TGFB2 mRNA levels based on RNAseq were an indicator of poor prognosis in patients with DIPG, but not in pediatric GBM patients with tumors located outside the pons / brainstem or pediatric DMG patients. The lack of adverse prognostic effects of higher TGFB2 mRNA expression levels in the latter patient population was not due to lower expression levels of the TGFB2 receptor. Notably, TGFB2 mRNA levels were significantly higher in pediatric GBM patients than in DIPG patients. The lack of a true TGFB2-low subgroup in GBM patients may have prevented accurate assessment of the favorable prognostic effect of low TGFB2 mRNA levels.

[0649] This study provides important insights into the clinical significance of elevated TGFB2 expression in pediatric high-grade gliomas. TGFB2-promoted aggressive growth of DIPG cells, their TGFB2-associated radioresistance, and potentially TGFB2-mediated restriction of cellular anti-glioma immunity within the TME contribute to the observed adverse effects of elevated TGFB2 levels on survival outcomes in DIPG patients.

[0650] In a clinical study using a microarray-based gene expression platform, TGFB2 transcripts, but not TGFB1 or TGFB3 levels, were selectively increased in primary tumor samples from 29 pediatric patients with DIPG compared with normal samples and primary tumor samples from patients with low-grade gliomas. In this study, RNAseq-based TGFB2 mRNA levels were compared in primary DIPG tumor samples and normal control pons samples. Notably, TGFB2 mRNA expression levels were significantly higher in DIPG patient tumor samples than in control pons samples, whereas TGFB1 and TGFB3 mRNA levels were significantly lower in DIPG samples than in control pons samples. These results, generated using RNAseq-based mRNA data, confirm and significantly extend our previous findings using a microarray platform. The enhanced TGFB2 gene expression in DIPG samples is likely due to increased expression of transcription factors that upregulate TGFB2 mRNA expression. This hypothesis was supported by the finding of strong positive correlations (p < 0.0001) between mRNA levels of several such transcription factors in DIPG samples, including SP1, FOXO3, EP300, and TGFB2 mRNA levels. Similar results were obtained in an independent validation dataset of microarray-based TGFB1, TGFB2, and TGFB3 mRNA levels and their correlation with expression of specific transcription factors in tumor samples from 41 pediatric patients with DIPG (N = 29) or GBM with H3K27M mutations.

[0651] The TGFB gene superfamily contains three TGFB isoforms: TGFB1, TGFB2, and TGFB3. Despite sharing >70% sequence identity, the TGFB isoform TGFB3 differs in structure and biological function from TGFB1 and TGFB2. Notably, TGFB3 knockout mice exhibit significant differences from TGFB1 or TGFB2 knockout mice. Furthermore, TGFB3 exerts cancer-preventive effects in nonclinical models of tumor development and in human subjects. Furthermore, high TGFB3 expression has a favorable prognostic effect in breast, ovarian, and colon cancers. However, TGFB3 expression is associated with a poor prognosis in osteosarcoma. In this study, high TGFB3 expression was identified as a favorable prognostic indicator, in stark contrast to its reported adverse prognostic role in breast cancer. Our observations expand our current knowledge and provide new insights into the multifunctional roles of TGFB3.

[0652] Based on the results presented herein, enhanced TGFB2 mRNA expression is associated with poor prognosis and OS in DIPG ( Figure 20 ).

[0653] Figure 20They showed that TGFB2 mRNA expression is selectively enhanced and associated with poor OS in children with DIPG. TGFB2 mRNA levels, while TGFB1 and TGFB3 mRNA levels, were selectively enhanced in primary brain tumor samples from DIPG patients. High TGFB2 mRNA expression was associated with poor OS. In contrast, high TGFB1 mRNA expression had no prognostic value, while high TGFB3 mRNA expression was associated with good OS.

[0654] 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 catheter-directed endothelial growth factor receptor (CED). Among 77 patients with high-grade glioma in the response population, 26 achieved favorable responses, including 19 with a complete remission (CR) or partial response (PR), and seven with stable disease lasting longer than six months. The median progression-free survival (PFS) in this subgroup was 1109 days, and the overall survival (OS) was 1280 days. As reported here, the poor prognosis observed in newly diagnosed patients with TGFB2-high DIPG supports the need for further exploration of the clinical potential of TGFB2-targeted RNAi therapy in these patients. CED catheters have been used in patients with DIPG to deliver therapeutics intratumorally in an attempt to bypass the blood-brain barrier and achieve higher intratumoral concentrations while reducing the risk of systemic toxicity. However, results have been limited by the need for months-long intratumoral administration to achieve objective responses in high-grade adult glioma patients, coupled with the practical challenges of long-term use or repeated replacement of implanted CED catheters in difficult anatomical locations.

[0655] The present invention provides novel delivery methods and / or formulation strategies for TGFB2-targeted therapeutic agents that are effective for DIPG patients.

[0656] Example 7 - Single-agent activity of OT-101 in patients with recurrent / refractory high-grade glioma.

[0657] Phase 2 clinical data showed that OT-101 had significant single-agent activity in patients with recurrent / refractory high-grade glioma, with more than one-third of patients (26 of 77 patients) achieving durable complete remission, partial remission, or long-term disease stabilization after receiving the expected 4-11 cycles of therapy, with a median OS of 1280 days (95% CI: 1116->1743 days).

[0658] The median PFS of these 77 patients was significantly better than that of the 12 patients who received 1-3 cycles of OT-101 (86 days vs. 32 days, log-rank P value < 0.0001). Similarly, the median OS of the 77 patients who received 4-11 cycles of OT-101 was significantly better than that of the 12 patients who received 1-3 cycles of treatment (432 days vs. 128 days, log-rank P value < 0.0001).

[0659] Nineteen patients achieved durable objective responses (CR: 3, PR: 16). The median time to a 90% reduction in baseline tumor volume was 11.7 months (range: 4.9-57.7 months). The mean log reduction in tumor volume for these 19 patients was 2.2±0.4 (median=1.4; range: 0.4-4.5) logs.

[0660] Figure 21 Radiographic responses in patients with R / R high-grade glioma who achieved a CR or PR with OT-101 monotherapy. Figure 21 : Waterfall plot depicting the maximum log10 reduction in tumor volume.

[0661] Figure 22 Radiographic responses in patients with R / R high-grade glioma who achieved a CR or PR with OT-101 monotherapy. Figure 22 : Semi-logarithmic plot of the combined 3-D tumor volume reduction curves of 19 patients.

[0662] Figure 23 A swimmer's diagram showing the onset and duration of objective response. The onset and duration of CR / PR, the end of OR, and the onset of PD are indicated by specific signals.

[0663] OT-101 induced durable CRs and PRs in patients with R / R GBM and AA. Nineteen patients had an objective response. Sixteen patients had a partial response at 307 ± 159 days (mean ± SE). The median time to onset of PR was 287 days (range: 37-742). Three patients initially achieved a PR, which deepened to a CR at 917, 1120, and 1838 days, respectively. Six of these 16 patients achieved a PD at 970 ± 126.7 days (mean ± SE) (median = 1032 days, range: 374–1281 days).

[0664] Example 8 - Clinical Potential of OT-101 Targeting Transforming Growth Factor β2 for Post-Radiation Consolidation of Diffuse Intrinsic Pontine Glioma.

[0665] Children with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median overall survival (OS) of 10 months after standard radiotherapy and a 2-year OS rate of <10%. Chemotherapy fails to provide clinically meaningful benefit. Therefore, innovative therapeutic agents for the treatment of DIPG in children are urgently needed.

[0666] High-grade glioma cells, including pediatric glioblastoma and DIPG cells, have been shown to produce transforming growth factor β2 (TGF-β2), which is both a promoter of glioma cells and a key factor leading to the low T cell responsiveness of the tumor microenvironment (TME) to glioma cells.

[0667] 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 in human glioma cells, blocks their proliferation and migration, and restores the anti-glioma cytolytic function of patient-derived T cells.

[0668] Intrathecal / intracerebroventricular administration of antitumor drugs directly into the CSF allows bypassing the selectivity filter of the blood-brain barrier, achieving significant antitumor agent concentrations in the CSF while reducing the potential for systemic toxicity. Informed by the favorable safety pharmacology of intrathecal delivery of OT-101 in rabbits and primates, and encouraged by its single-agent activity in adult patients with HGG, a multicenter, two-part, randomized Phase 1-2 study of OT-101 in pediatric patients with DIPG was conducted. After completion of radiotherapy, multiple doses of OT-101 were administered by intrathecal (IT) / intracerebroventricular bolus injection. 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.

[0669] Antisense oligodeoxynucleotides are short strands of DNA that are designed to downregulate gene expression by interfering with the translation of specific protein-coding proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and some 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 is designed to be complementary to a specific sequence of human TGF-β2 mRNA after gene expression. It is a first-in-class RNA therapeutic designed to eliminate the immunosuppressive effects of TGF-β2 and reduce TGF-β2 levels in malignant gliomas, thereby delaying disease progression.

[0670] Functional in vitro assays showed that:

[0671] In the presence or absence of carrier liposomes In all cases, OT-101 was effectively and time-dependently taken up by human tumor cells.

[0672] OT-101 can reduce TGF-β2 secretion in human tumor cells without the use of any carrier.

[0673] In human A172 high-grade glioma cells, 10 μM was the most effective concentration for inhibiting TGF-β2 production at clinically used OT-101 concentrations up to 80 μM over 7 days.

[0674] OT-101 reduces the proliferation of human tumor cells while stimulating the proliferation of PBMCs. OT-101 does not affect the viability of human PBMCs.

[0675] OT-101 restored the immune function of human PBMCs derived from patients with high-grade gliomas, as demonstrated by immune cell-mediated cytotoxicity assays.

[0676] OT-101 inhibits human tumor cell migration.

[0677] OT-101 has been studied in a series of nonclinical safety and pharmacology studies. The salient features of the conclusions from these studies are as follows:

[0678] Long-term topical administration of OT-101 / AP 12009 may cause local tissue inflammation. Mild to moderate local toxicity was observed in animals following infusion of a 500 μM concentration, but without any macroscopic changes.

[0679] When administered as a bolus IT injection to 3 kg male or female rabbits at a dose level of 0.12 mg / kg (500 μM solution; 0.1 mL) (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.

[0680] When administered as a bolus IT injection to 6-7 kg cynomolgus monkeys at a dose level of 0.05 mg / kg (500 μM solution; 0.1 mL) (estimated CSF concentration 0.46 μM), OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes, consistent with subclinical toxicity.

[0681] When administered intracerebroventricularly to rats, radiolabeled OT-101 (0.18 mg / kg) was detected not only in the CSF but also in the cerebrum, cerebellum, and pineal gland within 1 hour of administration. The half-life in CSF and brain tissue was <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0682] Example 9 - Non-clinical in vitro studies of OT-101 / AP 12009.

[0683] Functional in vitro assays showed that:

[0684] In the presence and absence of carrier liposomes In all cases, OT-101 was effectively and time-dependently taken up by human tumor cells.

[0685] OT-101 can reduce TGF-β2 secretion in human tumor cells without the use of any carrier.

[0686] In human A172 high-grade glioma cells, 10 μM was the most effective concentration for inhibiting TGF-β2 production at clinically used OT-101 concentrations up to 80 μM over 7 days.

[0687] OT-101 reduces the proliferation of human tumor cells while stimulating the proliferation of PBMCs. OT-101 does not affect the viability of human PBMCs.

[0688] OT-101 restored the immune function of human PBMCs derived from patients with high-grade gliomas, as demonstrated by immune cell-mediated cytotoxicity assays.

[0689] OT-101 inhibits human tumor cell migration.

[0690] In existence or non-existence In the absence or presence of In the case of FITC-OT-101, the uptake of FITC-OT-101 was observed after 3 hours of incubation. After 48 hours, the fluorescence signal was detected in almost all cells and was not significantly different in the presence or absence of The intensity was comparable in cell preparations incubated in the absence of

[0691] Example 10 - Effect of OT-101 on TGF-β2 synthesis and secretion in human GBM cell lines.

[0692] Cells were incubated with OT-101 / AP 12009 at various concentrations (1 μM to 80 μM) for 7 days. Secreted TGF-β2 in cell supernatants was measured by ELISA. Results represent the median, minimum, and maximum values ​​of three independent experiments.

[0693] The ability of OT-101 to reduce the secretion of TGF-β2 by primary human glioma cells was determined by measuring the concentration of TGF-β2 in the cell culture supernatant using an enzyme-linked immunosorbent assay (ELISA). Glioma cells from 10 high-grade glioma patients were cultured for 72 hours (HTZ-209, -220, -243, -262, -349, -361, -378, -381) or 96 hours (A-172) in the presence or absence of OT-101 (5 or 10 μM). In 8 of the 10 glioma cell cultures, TGF-β2 secretion was reduced by up to 87%.

[0694] OT-101-mediated inhibition of human high-grade glioma cell proliferation.

[0695] Two human HGG cell cultures (HTZ-243 and HTZ-349, representing WHO grade III and IV) were incubated with OT-101 (1 μM to 10 μM). The results showed a concentration-dependent and time-dependent reduction in cell number over 6 days, as shown in Table 20.

[0696] Table 20: Effects of OT-101 on the proliferation of human high-grade glioma cells

[0697]

[0698] Two human glioma cell cultures (HTZ-243 and HTZ-349) were treated with OT-101 (1, 5 or 10 μM). Cell number was measured using a hemocytometer (as a % of the number of cells at the start of the experiment). Data show the mean of duplicate evaluations.

[0699] Example 11 - Administration of OT-101 (AP 12009).

[0700] This study evaluated two doses of AP 12009 in parallel treatment groups. AP 12009 at a concentration of 10 μM or 80 μM was administered intratumorally using continuous convection-enhanced delivery at a flow rate of 4 μL / min over a 7-day cycle every two weeks. The AP12009 treatment regimen was based on Phase I / II clinical data from three studies:

[0701] Concentration: AP 12009 was shown to be safe up to the maximum evaluated concentration of 80 μM. Flow Rate: Some results from Study G003 suggest that infusion volume may be an important safety issue, particularly in patients with larger tumors, who are at high risk for relatively rapid development of cerebral edema due to the potential depletion of the brain's ability to compensate for increased intracranial pressure. Although administration at a flow rate of 8 μL / min over four days was assessed as safe, more frequent and more severe AEs at a flow rate of 8 μL / min over seven days cannot be ruled out. Weighing safety and efficacy considerations, the decision was made to maintain the infusion cycle at 7 days, but reduce the flow rate to 4 μL / min (total volume administered per treatment cycle: 40.32 mL). This allowed for longer drug exposure, but reduced the infusion volume by half compared to the four-day infusion rate of 8 μL / min. The flow rate of isotonic saline infusion during the seven-day interval was set at 1 μL / min to reduce the risk of symptoms of increased intracranial pressure.

[0702] Due to the large dead space volume of the application system, the administration of the relevant concentration of AP 12009 to the brain was delayed at the beginning of each treatment cycle. It was determined that 95% of the nominal AP 12009 concentration was achieved after 10 hours. This delay at the start of each AP 12009 infusion had to be compensated to allow for the administration of the nominal AP 12009 dose within the prescribed infusion period. Therefore, after switching to isotonic saline infusion, the flow rate was set at 4 μL / min at the beginning of the interval period for the same 10 hours to flush any remaining AP 12009 from the dead space. The flow rate was then reduced to 1 μL / min.

[0703] Example 12 - Effects of OT-101 and chemotherapy on glioma.

[0704] A non-inferiority analysis of overall survival (OS) was performed for patients who received OT-101 or standard chemotherapy (TMZ, PCV, or BCNU). A total of 156 subjects were evaluated, including 101 subjects in the OT-101 test group (G004: 89 subjects, G005: 12 subjects) and 55 subjects in the standard chemotherapy control group (G004: 45 subjects, G005: 10 subjects).

[0705] Descriptive statistics were performed for the groups in the G004 and G005 studies. In 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, consisting of 45 subjects, the mean OS was 471.2 days; the median OS was 333 days, with a standard deviation of 373.0 days. In 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, consisting 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 form of the above data.

[0706] Table 21: Descriptive statistics for overall survival of patients receiving OT-101 or standard chemotherapy in studies G004 and G005.

[0707]

[0708]

[0709] For noninferiority studies, a two-sample noninferiority test was performed on the survival data using Cox regression. Specifically, the Wald test for noninferiority or the 100(1-2α)% confidence interval test was used to determine whether the 90.0% upper confidence limit (CL) of the hazard ratio (HR) was within the noninferiority hypothesis. The α level was set at 0.050, and the noninferiority margin for the hazard ratio (HR) (hazard ratio [HR] = risk [treatment group] / risk [reference group]) was 1.25. If the risk was greater than the noninferiority margin, a higher risk was considered worse. If H1: HR < noninferiority margin, the Efron Ties method was used.

[0710] For the alternative hypothesis of HR < 1.25, the calculated hazard ratio was 0.9168, with 90.0% confidence limits for the hazard ratio ranging from 0.6865 to 1.2245 and a p-value of 0.0390. The Wald-Z value was determined to be -1.7621. In Cox regression, the hazard ratio (HR) is often referred to as the risk and 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 described above, the hazard ratio (Risk Ratio or Hazard Ratio) was calculated as Exp(B) = 0.9168, with a mean of 0.6516.

[0711] Based on the above results, in G004 and G005, OT-101 was confirmed to be non-inferior to standard chemotherapy (TMZ, BCNU, or PCV) in terms of overall survival, as the hazard ratio was 0.9168 and the 90% upper limit of the confidence interval was 1.2245, both of which were smaller than the initial non-inferiority margin of 1.25.

[0712] In the control group, TMZ was the most common treatment, with 43 participants (36 in Study G004 and 7 in Study G005). The same two-sample noninferiority test was performed on the survival data for participants who received OT-101 or TMZ using Cox regression. Using the alternative hypothesis of noninferiority, a hazard ratio less than 1.25 was used, resulting in a hazard ratio of 0.7156 with a 90% confidence level between 0.5228 and 0.9794, and a p-value of 0.0017. The Wald's Z-score was determined to be -2.9231. Based on the obtained hazard ratio, OT-101 was concluded to be noninferior to TMZ.

[0713] The Kaplan-Meier curve (log-rank test) of NCSS software was used to obtain the survival curves of OT-101, standard chemotherapy and the combination of the two.

[0714] Figure 24 Overlaid Kaplan-Meier survival curves for OT-101 and standard chemotherapy are shown. The survival curves for the two drugs are completely within each other's confidence intervals, confirming the noninferiority of OT-101 compared with standard chemotherapy in overall survival.

[0715] Figure 25 Results showed that OT-101 showed non-inferiority in chemotherapy-naive patients treated with temozolomide (TMZ), confirming the non-inferiority of OT-101 after the addition of TMZ.

[0716] Figure 26 Chemotherapy with the chemotherapeutic agents CCNU / BCNU was shown to be ineffective, confirming the non-inferiority of OT-101 after the addition of CCNU / BCNU.

[0717] Example 13 - TGF-β2 is a valid target for glioma therapy.

[0718] Three TGFB2 probe sets showed increased expression levels in DIPG patients ( Figure 27 The average fold difference of probe group 228121_at was 2.48 (linear comparison P value = 3.40 x 10 -4); the mean fold difference for probe set 220407_s_at was 2.00 (linear comparison P value = 0.006); and the mean fold difference for probe set 209909_s_at was 1.81 (linear comparison P value = 0.0185). One of the TGFB2 probe sets also showed the highest mean expression in DIPG patients: TGFB2_228121_at (mean = 9.25 ± 0.18 log2 RMA); and YAP1_224894_at (mean = 8.55 ± 0.19 log2 RMA).

[0719] The results are summarized in Table 22.

[0720] Table 22: TGF-β2 is a valid target for glioma therapy

[0721]

[0722] The expression of the TGFB2 interactome represents the probe sets in pediatric DIPG patients compared with normal samples. The log2-transformed fold difference values ​​for each subject (column (N = 29)) and each probe set from the 4GEO archive dataset GSE26576 are depicted as the DIPG patient mean centered around the normal sample (N = 2). Subjects and probe sets were organized using a 2-way clustering algorithm that used an average distance metric to determine common regulation across all probe sets between patients and across all patients between probe sets. The heat map depicts the most significantly upregulated and downregulated probe sets, ranging from red to blue, representing higher than normal expression to lower than normal expression in DIPG samples, respectively. The fold difference and linear contrast p-value are depicted in the table, showing the three probe sets upregulated by TGFB2 in DIPG patients.

[0723] Example 14 - TGF-β2 is a valid target for pediatric GBM therapy.

[0724] Similar results were obtained for pediatric GBM patients ( Figure 28 Three TGFB2 probe sets were significantly upregulated more than 2-fold in pediatric GBM patients (probe set: 209909_s_at (fold change = 4.03, P = 1.78 x 10 -5 ); 228121_at (fold change = 3.57, P = 8.81 x 10 -5 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).

[0725] The results are summarized in Table 23.

[0726] Table 23: TGF-β2 is a valid target for pediatric GBM therapy

[0727]

[0728] Comparison of TGFB1 / 2 / 3 probeset expression in pediatric GBM patients with AO patients (control group). Log2-transformed fold difference values ​​for each of the nine probesets for each subject (columns (N = 82)) and from four GEO archived datasets (GSE19578 (N = 25); GSE32374 (N = 15); GSE34824 (N = 27); and GSE49822 (N = 15)) are plotted, centered on the pediatric GBM patient mean (N = 5; from GSE19578). Subjects and probesets were organized using a 2-way clustering algorithm that uses an average distance metric to determine co-regulation across all probesets between patients and across all patients. Heatmaps depict the expression changes for each probeset, ranging from red to blue, representing expression above control to expression below control in pediatric GBM samples, respectively. The table describes the fold difference and linear contrast p-values, showing that TGFB2 was significantly upregulated more than 2-fold in pediatric GBM patients for three probe sets (probe set: 209909_s_at (fold change = 4.03, P = 1.78 x 10 -5 ); 228121_at (fold change = 3.57, P = 8.81 x 10 -5 ); and 220407_s_at (fold change = 2.73, P = 0.0019).

[0729] Example 15 - TGF-β2 is a selective biomarker that predicts improved outcomes of cancer radiotherapy.

[0730] The inventors herein have discovered that TGF-β2 can be used as a biomarker to selectively predict improved outcomes of cancer radiotherapy.

[0731] A clinical study using the CTGA database demonstrated the impact of TGF-β2 expression on overall survival and survival after radiotherapy. Surprisingly, across all four quartiles of expression, a significant survival advantage was observed for low TGF-β2 expressers compared with high TGF-β2 expressers. No such differences were observed for TGF-β1 and TGF-β3.

[0732] The researchers studied overall survival in a pediatric brainstem subgroup and studied radiotherapy survival in all patients with glioma who received radiotherapy. Again, only TGF-β2 predicted survival. TGF-β1 and TGF-β3 did not.

[0733] Figure 29 Results showed that low TGF-β2 expression confers an overall survival advantage in patients with glioma who received radiotherapy. Only TGF-β2 predicted survival. Quartiles of TGF-β2 expression are represented as A, B, C, and D.

[0734] Figure 30 showed that for gliomas in patients receiving radiotherapy, low TGF-β2 expression conferred an overall survival advantage. Only TGF-β2 predicted survival.

[0735] Example 16 - Reduced TGF-β2 levels selectively predict improved overall survival (OS) when combined with chemotherapy (TMZ), chemotherapy (TMZ) and radiotherapy, or anti-angiogenic therapy (bevacizumab).

[0736] The inventors herein have found that TGF-β2 can surprisingly be used as a biomarker that selectively predicts improved outcomes in combination with chemotherapy (TMZ), TMZ and radiotherapy, or anti-angiogenic therapy (bevacizumab). No such predictive results were observed for TGF-β1 and TGF-β3.

[0737] A clinical study was conducted using 23 glioma datasets to determine TGF-β2 mRNA levels as a predictor of survival after treatment with a drug of interest.

[0738] Figure 31 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy (TMZ).

[0739] Figure 32 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy TMZ and radiation.

[0740] Figure 33 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with antiangiogenic therapy (bevacizumab).

Claims

An agent for inhibiting or suppressing TGF-β2 expression, for treating or ameliorating symptoms of a CNS disease in a human subject or an animal.

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

3. A method for treating or ameliorating the symptoms of a CNS disease in a human subject or animal in need thereof, the method comprising: preparing a composition comprising an agent for inhibiting or suppressing TGF-β2 expression in a carrier; as well as A therapeutically effective amount of the composition is administered to the subject.

4. The medicament, use or method of any one of claims 1 to 3, wherein the CNS disease is a glioma, a glioblastoma, a diffuse intrinsic pontine glioma (DIPG), a diffuse midline glioma (DMG), a leptomeningeal or brain metastasis, a brain or spinal cancer, or a CNS tumor.

5. The agent, use or method according to any one of claims 1 to 3, in combination with a drug comprising a targeted cancer drug, a cancer growth retardant, an EGFR inhibitor or a combination thereof.

6. The medicament, use or method according to any one of claims 1 to 3, in combination with a drug selected from bevacizumab, everolimus, bezutifan, dabrafenib, trametinib and combinations thereof.

7. The medicament, use or method according to any one of claims 1 to 3, in combination with a drug that is a cancer growth inhibitor selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

8. The medicament, use or method according to any one of claims 1 to 3, in combination with a drug that is an EGFR inhibitor selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

9. The medicament, use or method according to any one of claims 1 to 3, in combination with temozolomide.

10. The medicament, use or method according to any one of claims 1 to 3, in combination with treatment of a CNS disease by radiotherapy or therapeutic electric fields.

11. The medicament, use or method according to any one of claims 1 to 3, wherein administration or use of the composition or medicament is combined with standard of care treatment for a CNS disease.

12. The medicament, use or method of any one of claims 1 to 3, wherein the medicament, drug, therapy, treatment and administration are each administered concurrently, simultaneously, sequentially or separately in time.

13. The medicament, use or method according to any one of claims 1 to 3, wherein each medicament and drug is administered alone or in combination by infusion or injection.

14. The medicament, use or method according to any one of claims 1 to 3, comprising administering or using by intracranial continuous infusion or bolus administration.

15. The medicament, use or method of claim 14, wherein the intracranial continuous infusion comprises infusion using an Omayor-type reservoir having a partially flexible top.

16. The medicament, use or method of claim 15, wherein the intracranial continuous infusion comprises a single access catheter placed into the target area of ​​the brain.

17. The medicament, use or method of any one of claims 1-3, wherein the subject has an improved TGF-β2 profile following administration or use.

18. The medicament, use or method of any one of claims 1-3, wherein the administration or use reduces mortality at 6, 12, 18, 24, 30 or 36 months.

19. The medicament, use or method of any one of claims 1-3, wherein the administration or use increases survival at 6, 12, 18, 24, 30 or 36 months.

20. The agent, use or method according to any one of claims 1 to 3, wherein the agent for inhibiting or suppressing TGF-β2 expression is selected from TGF-β2-specific antisense oligonucleotides complementary to TGF-β2 transcripts, as shown below: Table 2 SEQ ID NO: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof and any combination thereof:

21. The agent, use or method according to any one of claims 1 to 3, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide having no more than one or two mismatches with the human target TGF-β2.

22. The agent, use or method according to any one of claims 1 to 3, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide that reduces the level of TGF-β2 transcript by at least 60%, or at least 70%, or at least 80% or at least 90%.

23. The agent, use or method of any one of claims 1 to 3, wherein the agent for inhibiting or suppressing TGF-β2 expression is a TGF-β2 specific antisense oligonucleotide that reduces the level of any TGF-β1 transcript and the level of any TGF-β3 transcript by less than 10%, or less than 5% or less than 1%.

24. The agent, use or method according to any one of claims 1 to 3, comprising a TGF-β2 specific antisense oligonucleotide having one or more nucleotides chemically modified to be phosphorothioate internucleoside linkages, methoxypropylphosphonate internucleoside linkages, or phosphoramidite linkages 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.

25. The agent, use or method of any one of claims 1-3, wherein the agent is conjugated to polyethylene glycol, a lipid or tri-branched N-acetylgalactosamine.

26. The medicament, use or method according to any one of claims 1 to 3, comprising a carrier selected from sterile water for injection, saline, isotonic saline, phosphate buffered saline or a combination thereof.

27. The medicament, use or method of any one of claims 1 to 3, wherein the medicament, drug or administration is substantially free of excipients.

28. The agent, use or method of any one of claims 1-3, wherein the agent, drug or administration is stable in the carrier for at least 14 days at 37°C when pumped as an intracranial continuous infusion, or has less than a 10% decrease in antisense active agent concentration after 90 days of use.

29. The medicament, use or method according to any one of claims 1 to 3, 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 day 1 to day 7, preferably by continuous intracranial infusion.

30. A kit comprising: A medicament 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, chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and Omayeh-type reservoir with a partially flexible top.

31. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising: a reservoir for containing a pharmaceutical composition[117]; a pump [101] for forcing the pharmaceutical composition through an infusion tube [103] into an Omayr reservoir [111], said pump being in fluid communication with said Omayr reservoir, and wherein said pump is in fluid communication with said reservoir via a reservoir tube [115]; A filter inline with the infusion line[105]; and An access catheter [113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

32. The apparatus of claim 31 , wherein the access catheter [1 13] is non-linear and has one or more bends to access a target region of the brain intraventricularly.

33. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising: a reservoir for containing a pharmaceutical composition[117]; a pump [101] for forcing the pharmaceutical composition through an infusion tube [103] into an access port [107], the pump being in fluid communication with an Omayr reservoir [111], wherein the reservoir is in fluid communication with the pump via a reservoir tube [115]; A filter inline with the infusion line[105]; an indwelling tube [109] in fluid communication with the access port and the Omayr reservoir [111]; and An access catheter [113] is in fluid communication with the Omayr reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

34. The apparatus of claim 33, wherein the access catheter [113] is non-linear and has one or more bends to access a target region of the brain intraventricularly.

35. The device according to any one of claims 31-34, wherein the Omayyad reservoir [111] comprises a partially flexible top.

36. The device of any one of claims 31-34, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to a target area.

37. The device of any one of claims 31-34, wherein the distal end of the access catheter enters a target area of ​​the brain.

38. The device according to any one of claims 31-34, wherein the Omayr reservoir [111] holds the pharmaceutical composition behind a membrane for a period of time to provide a sustained release of the pharmaceutical composition to the access catheter.

39. The device of any one of claims 31-34, wherein the distal tip of the access catheter into the brain is a stepped-down end, a concave stepped end, a multi-port end, a microbore end, or a balloon tip.

40. The apparatus according to any one of claims 31-34, wherein the pump [101] is a Pegasus Vario, PEGA PCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA, CADD-Legacy 1 or other pumps with similar specifications.

41. The device of any one of claims 31-34, wherein the infusion rate of the fluid drug 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.

42. The device according to any one of claims 31-34, wherein the infusion line [103] or the indwelling line [109] is a PEGA Line 100SF 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 tubing with similar specifications.

43. The device of any one of claims 31-34, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β expression, which is useful for treating or ameliorating symptoms of a CNS disease in a human subject or animal.

44. The device of any one of claims 31-34, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of a pharmaceutical agent, drug, or delivery vehicle.

45. The device of any one of claims 31-34, wherein the fluid pharmaceutical composition is a therapeutic agent for a CNS disease or CNS cancer.

46. ​​The device of any one of claims 31-34, wherein the fluid pharmaceutical composition is a therapeutic agent for a glioma, a glioblastoma, a diffuse intrinsic pontine glioma (DIPG), a diffuse midline glioma (DMG), a leptomeningeal or brain metastasis, a brain or spinal cancer, or a CNS tumor.

47. The device according to any one of claims 31 to 34, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β2 expression, the agent being selected from a TGF-β2-specific antisense oligonucleotide complementary to a TGF-β2 transcript, as shown below: Table 2 SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

48. The device of any one of claims 31-34, wherein the device operates in combination with radiation therapy or electric field therapy.

49. A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising: a reservoir [402] containing a pharmaceutical composition, the reservoir 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; as well as An access catheter [413] is in fluid communication with the reservoir, wherein the access catheter is substantially linear and enters a target region of the brain intraventricularly.

50. A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising: a reservoir [502] containing a pharmaceutical composition, the reservoir comprising an upper hard shell [503], a lower hard shell [504], a flexible top [501], and a non-flexible mounting plate [505]; a port [507] in fluid communication with the reservoir for connecting an infusion line; as well as A port [509] in fluid communication with the reservoir for connection to an access catheter.

51. A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising: a reservoir [602] containing a pharmaceutical composition, the reservoir comprising an upper hard shell [603], a flexible top [601], and a non-flexible mounting plate [605]; a port [619] in fluid communication with the reservoir for connecting an infusion line; and A port [607] in fluid communication with the reservoir for connection to an access catheter.

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

53. A method of administering a pharmaceutical composition by continuous intracranial infusion, comprising: attaching the device according to any one of claims 31-34 and 49-51 to a patient; and The pharmaceutical composition in the device is pumped to provide an intracranial continuous infusion to the patient.

54. The method of claim 53, wherein the device is mounted and the access catheter is placed into the brain without concurrent head or brain imaging.

55. The method of claim 54, wherein the device is mounted and a single access catheter is placed into the brain.

56. A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, the kit comprising: a reservoir containing a pharmaceutical composition; pumps; Omayeh reservoir with partially flexible top; infusion tubing for connecting the reservoir to the pump and the pump to the Omayya reservoir; Filters; and Enter the catheter.

57. The kit of claim 56, wherein the access catheter is substantially linear for intraventricular access to a target region of the brain.

58. The kit of claim 56, wherein the access catheter is non-linear and has a bend to access a target region of the brain intraventricularly.

59. The kit of any one of claims 56-58, wherein the infusion line is external to the subject.

60. The kit of any one of claims 56-58, wherein a portion of the infusion tubing connecting the pump to the Omayne reservoir resides in the subject.

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