Device for delivery of fluid pharmaceutical composition by intracranial continuous infusion

Abstract

There is described a device for delivering a fluid pharmaceutical composition by intracranial continuous infusion, the device comprising: a reservoir [117] containing a pharmaceutical composition; a pump [101] forcing the pharmaceutical composition through an infusion tube [103] into an OMARY reservoir [111], the pump in fluid communication with the OMARY reservoir, and wherein the pump in fluid communication with the reservoir through a reservoir tube [115]; a filter [105] in line with the infusion tube; and an access conduit [113] in fluid communication with the OMYER reservoir, wherein the access conduit is substantially linear and accesses a target region of the brain within the ventricle.

Description

Technical Field

[0001] The present disclosure relates to methods, agents and uses for treating central nervous system (CNS) cancers by continuous intracranial infusion. More specifically, the present disclosure discloses methods, agents and uses for inhibiting or suppressing TGF-β2, which provide improved clinical outcomes for such diseases. The present disclosure provides stable formulations of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents and methods of use for treating CNS cancers.

[0002] The present disclosure relates to devices and methods for delivering a pharmaceutical composition by intracranial infusion. More specifically, the device of the present disclosure may include an ommaya-like reservoir into which the pharmaceutical composition is pumped. The ommaya-like reservoir may provide a series of intracranial infusions from bolus to continuous via an access catheter that enters a target area of ​​the brain from within the cerebral ventricle. Background Art

[0003] 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 distribute drugs directly to tumors in the brain using multiple catheters close to the tumor.

[0004] However, disadvantages and complications of these infusion methods are that: (a) placement of the catheter and its tip into position near a brain tumor is complicated and often requires simultaneous imaging (e.g., x-ray-based imaging) to achieve, (b) these methods require insertion of multiple catheters into the tumor, which is difficult or impossible to achieve and may result in 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.

[0005] Furthermore, a disadvantage and complication of such approaches is that the infusion and mobility of the drug throughout the CNS compartment is limited.

[0006] Other disadvantages are that intratumorally implanted catheter tips may not provide adequate infusion and flow throughout the entire CNS compartment.

[0007] 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.

[0008] What is needed is a method and medicament for inhibiting or suppressing factors in the unpredictable pathology of central nervous system diseases, particularly cancer. For example, there is an urgent need for methods and medicaments for inhibiting TGF-β activity and / or suppressing TGF-β related pathology, which can improve the efficacy of treating central nervous system cancer. Utility Model Content

[0009] The utility model provides a therapy for treating or ameliorating the symptoms of CNS diseases such as CNS cancer.

[0010] In some embodiments, the present invention includes agents and compositions for inhibiting or suppressing TGF-β2 to provide improved clinical outcomes for CNS diseases.

[0011] In further embodiments, the present disclosure provides stable formulations of anti-TGF-β2 agents for 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.

[0012] In a further aspect, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for therapies against CNS diseases. The stable formulations of the present disclosure provide surprisingly improved clinical results. Stable formulations of agents for suppressing TGF-β2 can be used to treat CNS diseases, particularly CNS cancers.

[0013] The present disclosure provides devices and systems for intracranial, intrathecal or intraventricular continuous infusion of therapeutic agents to treat central nervous system (CNS) diseases. Continuous infusion into brain tumors using the system of the present disclosure is much easier than conventional CED systems and other similar or conventional methods.

[0014] The infusion system of the present disclosure has significant advantages because it continuously delivers an effective amount or effective dose of a pharmaceutical composition to a patient in a manner that is safer and easier to insert throughout the CNS. For example, compared to local delivery via a CED approach, the infusion system of the present disclosure will deliver a more effective amount of a drug via an intraventricular route and throughout the CNS / cerebral ventricles. This advantage is particularly important for the treatment of CNS cancers.

[0015] The device disclosed herein can overcome the shortcomings and complexity of conventional methods. The infusion system disclosed herein has significant advantages because it is relatively easy to place the catheter and its tip into the brain and imaging does not need to be completed. In addition, the infusion and flow of drugs to the brain utilizes the fluid circulation in the brain to effectively distribute the drugs to larger areas and target tissues. In addition, the system disclosed herein can be used in a novel operating mode to provide continuous infusion to the target tissue.

[0016] The present disclosure also relates to devices and systems for intracranial, intrathecal or intraventricular continuous infusion of therapeutic agents to treat central nervous system (CNS) diseases of the brain and spine. The disclosed system 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 treating CNS diseases (including CNS cancers).

[0017] The present disclosure also relates to methods of treating central nervous system (CNS) diseases of the brain and spine.

[0018] The present disclosure 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 infusion device and apparatus of the present disclosure is much easier than conventional CED methods and other similar or conventional methods.

[0019] The infusion device of the present disclosure has significant advantages because it continuously delivers an effective amount or effective dose of a pharmaceutical composition to a patient with greater safety and ease of insertion throughout the CNS. For example, compared to local delivery via a CED approach, the infusion device and apparatus of the present disclosure will deliver more effective amounts of a drug via an intraventricular route and throughout the CNS / cerebral ventricles. This advantage is particularly important for the treatment of CNS cancers.

[0020] The device of the present disclosure overcomes the shortcomings and complexity of the known methods. The infusion device of the present disclosure has significant advantages because it is relatively easy to place the catheter and its tip into the brain and imaging does not need to be completed. In addition, the infusion and flow of drugs to the brain takes advantage of the fluid circulation in the brain, effectively distributing the drugs to a larger area and target tissue. In addition, the device of the present disclosure can be used in a novel operating mode to provide continuous infusion to the target tissue.

[0021] The present disclosure also relates to a device for intracranial, intrathecal or intraventricular continuous infusion of therapeutic agents for the treatment of central nervous system (CNS) diseases of the brain and spine. The device of the present disclosure will deliver compositions and agents for inhibiting or suppressing TGF-β2 to improve the clinical outcomes of such diseases. The device uses a stable formulation of anti-TGF-β2 agents (including antisense oligonucleotide compositions) and other agents for the treatment of CNS diseases (including CNS cancers).

[0022] The present disclosure also relates to methods of treating central nervous system (CNS) diseases of the brain and spine.

[0023] Embodiments of the present disclosure include the following:

[0024] An agent for inhibiting or suppressing the expression of TGF-β2, which is used to treat or improve the symptoms of CNS diseases in human subjects or animals.

[0025] Use of a composition comprising 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.

[0026] A method for treating or ameliorating symptoms of a CNS disease in a human subject or animal in need thereof, the method comprising:

[0027] preparing a pharmaceutical composition comprising an agent for inhibiting or suppressing TGF-β2 expression; and

[0028] A therapeutically effective amount of the composition is administered to the subject.

[0029] 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.

[0030] The above-mentioned medicaments, uses or methods may comprise any one or more additional drugs, wherein the additional drugs comprise targeted cancer drugs, cancer growth blockers, EGFR inhibitors and combinations thereof.

[0031] The above-mentioned medicament, use or method comprises any one or more additional drugs selected from bevacizumab, everolimus, belzutifan, dabrafenib, trametinib and combinations thereof.

[0032] The above-mentioned medicament, use or method comprises any one or more additional drugs that are targeted cancer drugs.

[0033] The above-mentioned medicament, use or method comprises any one or more additional drugs that are cancer growth inhibitors, wherein the cancer growth inhibitor is selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0034] The above-mentioned medicament, use or method comprises any one or more additional drugs that are EGFR inhibitors, wherein the EGFR inhibitor is selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitininb and a combination thereof.

[0035] The above-mentioned medicament, use or method comprises another drug temozolomide or irinotecan.

[0036] The above-mentioned medicament, use or method comprises additional treatment of CNS diseases by radiation therapy or electric field therapy.

[0037] 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 CNS disease.

[0038] 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.

[0039] The above-mentioned medicament, use or method, wherein each medicament and drug is administered separately or in combination by infusion or injection.

[0040] The above-mentioned medicament, use or method includes administration or use by intracranial continuous infusion or bolus administration.

[0041] The above-mentioned medicament, use or method, wherein the intracranial continuous infusion comprises infusion using an ommaya reservoir with a partially-flexible top.

[0042] The above medicament, use or method, wherein the intracranial continuous infusion comprises a single access catheter placed into the target area of ​​the brain.

[0043] The above-mentioned medicament, use or method, wherein the subject has improved TGF-β2 characteristics after administration or use.

[0044] 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.

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

[0046] 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:

[0047] Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0048] The above-mentioned agent, use or method comprises a TGF-β2-specific antisense oligonucleotide, which has one or more nucleotides, and the nucleotides are chemically modified to be a thiophosphate internucleoside linker, a methoxypropylphosphonate internucleoside linker, an aminophosphoro linker connected to a morpholine group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' restricted methoxyethyl bicyclic ribose group, a 2'-4' restricted ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group or a 5-methylcytosine base.

[0049] The above-mentioned agent, use or method, wherein the agent is conjugated with polyethylene glycol, lipid or triantenarry N-acetyl-galactosamine.

[0050] 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.

[0051] The above-mentioned medicament, use or method, wherein the medicament, drug or administration is substantially free of excipients.

[0052] The above-mentioned medicament, use or method, wherein the medicament, 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 in use.

[0053] The above-mentioned medicament, use or method, wherein the method comprises administering the composition by intracranial continuous infusion at a rate of 2-8 μl / min and a concentration of 1-20 μM of the agent from day 1 to day 7. 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 concentration of 1-80 μM of the agent from day 1 to day 7, preferably by intracranial continuous infusion.

[0054] A kit comprising:

[0055] A medicament comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and

[0056] Omayeh reservoir with partially flexible top.

[0057] A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0058] a reservoir

[117] containing a pharmaceutical composition;

[0059] 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] ;

[0060] a filter

[105] inline with the infusion line; and

[0061] An access catheter

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

[0062] 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.

[0063] A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0064] a reservoir

[117] containing a pharmaceutical composition;

[0065] 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] ;

[0066] a filter

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

[0067] an indwelling tube

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

[111] ; and

[0068] An access catheter

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

[0069] 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.

[0070] The above device, wherein the Omayya reservoir

[111] includes a partially flexible top.

[0071] The above device, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to a target area.

[0072] The above device, wherein the distal end of the access catheter enters the target area of ​​the brain.

[0073] The above device, wherein the Omayne reservoir

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

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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 the symptoms of CNS diseases in human subjects or animals.

[0079] The above device, wherein the fluid drug composition comprises microparticles or nanoparticles of a pharmaceutical agent, a drug or a delivery vehicle.

[0080] The above device, wherein the fluid drug composition is a therapeutic agent for CNS disease or CNS cancer.

[0081] 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.

[0082] 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:

[0083] Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0084] The above device, wherein the device operates in combination with radiation therapy or electric field therapy.

[0085] A device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0086] 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] ;

[0087] a port

[407] in fluid communication with the reservoir; and

[0088] An access catheter

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

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

[0090] 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] ;

[0091] a port

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

[0092] A port

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

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

[0094] 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] ;

[0095] a port

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

[0096] A port

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

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

[0098] A hard shell

[604] having an opening

[606] exposing the flexible top of the device.

[0099] A method of administering a pharmaceutical composition by intracranial continuous infusion, comprising:

[0100] installing a device as described above on a patient; and

[0101] The pharmaceutical composition is pumped in a device to provide continuous intracranial infusion to the patient.

[0102] The method above, wherein the device is mounted and the access catheter is placed into the brain without simultaneous head or brain imaging.

[0103] The method above, wherein the device is mounted and a single access catheter is placed into the brain.

[0104] A kit for continuous intracranial infusion of a pharmaceutical composition into a subject, the kit comprising:

[0105] a reservoir containing a pharmaceutical composition;

[0106] Pumps;

[0107] Omayeh reservoir with partially flexible top;

[0108] infusion tubing for connecting the reservoir to the pump and the pump to the Omayeh reservoir;

[0109] Filters; and

[0110] Enter the catheter.

[0111] The above kit, wherein the access catheter is substantially linear and is used to access a target area of ​​the brain intraventricularly.

[0112] The above kit, wherein the access catheter is non-linear and has a bend to access a target area of ​​the brain intraventricularly.

[0113] The above kit, wherein the infusion tube is outside the subject's body.

[0114] The above kit, wherein a portion of the infusion tube connecting the pump to the Omayne reservoir is left indwelling in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion is shown. Figure 1 The 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 drug composition from the pump through an infusion tube

[103] and forces it into the Omayr reservoir

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

[117] containing the drug composition; a pump

[101] that forces the drug 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.

[0116] Figure 2 A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous 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 intrathecal or intraventricular infusion includes: a reservoir

[117] containing a pharmaceutical composition; a pump

[101] 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] ; 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.

[0117] Figure 3 A device for delivering a pharmaceutical composition by intrathecal or intraventricular continuous infusion is shown. Figure 3 The 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 drug composition from the pump through an infusion tube

[103] and forces it into the Omayr reservoir

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

[117] containing the drug composition; a pump

[101] forcing the drug 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.

[0118] 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 an access port

[107] and an 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 intrathecal or intraventricular infusion includes: a reservoir

[117] containing a pharmaceutical composition; a pump

[101] 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.

[0119] 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 Omayr delivery device shown can be used as 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

[402] for a drug-containing fluid. 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 fluid of 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 Omayya delivery device also includes an access catheter

[413] in fluid communication with the Omayya 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 entire area of ​​

[401] plus

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

[401] (see Figure 8 ).

[0120] 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 Omayr delivery device shown can be used as e.g. Figure 1 and Figure 2 The Omayr delivery device includes 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 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 Omayr delivery device also includes a non-flexible mounting plate

[505] comprised of an inert material such as metal or hard plastic. The Omayr delivery device also includes ports

[507] and

[509] for connecting an infusion line to fluidly communicate with the Omayr 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 ).

[0121] 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 Omayr 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 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 hard shell

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

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

[619] . In another embodiment, the hard shell

[603] can be a separate protective collar that can be placed on the 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 ).

[0122] Figure 8 An embodiment of a collar for an Omayr delivery device for use in a method of 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 so that the top is partially flexible. The opening

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

[0123] Fig. 9 The effects of delivering the pharmaceutical composition by intrathecal or intraventricular continuous infusion are shown. Fig. 9 The effect of OT-101 treatment on TGF-β2 secretion from the human GBM cell line A-172 is shown. Cells were incubated for 7 days with different concentrations of OT-101 / AP12009 (1 μM to 80 μM) as shown. Secreted TGF-β2 in the cell supernatant was measured by ELISA. The results represent the median, minimum, and maximum values ​​of 3 independent experiments.

[0124] Fig.10 Infusion from an Omayor-type reservoir of the present disclosure is shown, the reservoir having a 339 mm 2 The surface area of ​​the 1.4 mm and the diameter of the opening in the conduit of 1.4 mm. In this example, the dark test solution is shown to diffuse slowly downward through the conduit opening. This diffusion from the Omayor-type reservoir of the present disclosure continued for several hours.

[0125] Fig.11 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from hours to days.

[0126] Fig.12 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from days to weeks.

[0127] Fig.13 showed that continuous infusion operating parameters can be modified to extend the drug infusion release kinetic half-life from weeks to months.

[0128] Fig.14 Shown are the mRNA levels of TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples and 29 normal pons samples.

[0129] Fig.15 A statistically significant positive correlation was shown with TGFB2 mRNA levels.

[0130] Fig.16 Shown are relative expression of specific transcription factors in tumor samples from 41 pediatric patients with DIPG (n=29) or H3K27M-mutant GBM.

[0131] Fig.17 It was shown that DIPG patients with high TGFB2 exhibited significantly worse OS outcomes than those with low TGFB2.

[0132] Fig.18 The PFS results were also significantly worse in the subgroup showing high TGFB2.

[0133] Fig.19It was shown that patients in the TGFB2 high (N=29) and TGFB2 low (N=87) subgroups exhibited very similar OS results.

[0134] Fig. 20 Expression was shown to be selectively amplified in pediatric DIPG and correlated with poor OS.

[0135] Fig.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.

[0136] Fig. 22 Shown are semi-logarithmic plots of the combined 3-D tumor volume reduction curves for 19 high-grade glioma patients who were treated with OT-101 monotherapy and achieved a CR or PR.

[0137] Fig.23 A swimmer plot showing the onset and duration of objective response in high-grade glioma patients who received OT-101 monotherapy and achieved a CR or PR is shown. The onset and duration of CR / PR, the end of OR, and the onset of PD are indicated by specific signals.

[0138] Fig.24 Superimposed Kaplan-Meier survival curves for both OT-101 and standard chemotherapy are shown.

[0139] Fig.25 Indicated for chemotherapy-naive patients treated with temozolomide (TMZ).

[0140] Fig.26 Chemotherapy with the chemotherapeutic agents CCNU / BCNU was shown to be ineffective.

[0141] Fig. 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.

[0142] Fig.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.

[0143] Fig.29 showed that for gliomas in patients treated with radiation therapy, low TGF-β2 expression conferred an overall survival advantage. Only TGF-β2 predicted survival.

[0144] Fig.30showed that for gliomas in patients treated with radiation therapy, low TGF-β2 expression conferred an overall survival advantage. Only TGF-β2 predicted survival.

[0145] Fig.31 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy (TMZ).

[0146] Fig.32 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy TMZ and radiation.

[0147] Fig.33 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with antiangiogenic therapy (bevacizumab). DETAILED DESCRIPTION

[0148] The present disclosure 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 disclosure can be used to treat CNS diseases, such as CNS cancers, via an intracranial route.

[0149] The devices and methods of the present disclosure may deliver agents to the brain or spinal region using a pump-based continuous infusion device / system.

[0150] The present disclosure provides therapies for treating or ameliorating symptoms of CNS diseases, such as CNS cancer.

[0151] In some embodiments, the present disclosure includes agents and compositions for inhibiting or suppressing TGF-β2 to provide improved clinical outcomes for CNS diseases.

[0152] In other embodiments, the present disclosure 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 stereoisomers thereof, and combinations thereof.

[0153] In other aspects, the present disclosure provides highly stable formulations of anti-TGF-β2 agents for therapies for CNS diseases. The stable formulations disclosed herein provide surprisingly improved clinical results. Stable formulations of agents for suppressing TGF-β can be used to treat CNS diseases, particularly CNS cancers.

[0154] The present disclosure 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 kinetic half-life of the agent from days to weeks.

[0155] The devices and methods of the present disclosure may deliver agents to the brain or spinal region using a pump-based continuous infusion system.

[0156] In some embodiments, the present disclosure provides an Omayr reservoir device having conduits to the cerebrospinal fluid and the intraventricular space, the device being connected to a pump for incremental and continuous infusion.

[0157] In some embodiments, the present disclosure provides an Omayer-type reservoir device having conduits to the cerebrospinal fluid and the intraventricular space, the device being connected to a pump for incremental and continuous infusion.

[0158] The present disclosure provides a device for enhancing delivery, in some cases a portable delivery device, which provides delivery by infusing fluids into specific locations within the body, particularly brain tissue and tumors, preferably the ventricular space.

[0159] The present disclosure also provides a device for continuous intrathecal or intraventricular infusion of a therapeutic agent, comprising an Omayor-type device (see Figure 1-8 and 10). The Omayer-type device allows an access catheter to be inserted into the ventricle. Alternatively, the access catheter can be 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 non-collapsible material and have a flexible portion (see Figure 1-7 ). This structure and design can produce oscillating 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 disclosure 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.

[0160] The flexible top can enhance fluid motion by acting as a shock absorber to produce a more uniform fluid flow rate in the conduit.

[0161] In some aspects, the Omayne-type devices and apparatuses of the present disclosure can include a pump for continuously pumping to drive fluid into and out of the Omayne-type device reservoir.

[0162] The present disclosure provides a device for therapy to treat or ameliorate symptoms of CNS diseases, such as CNS cancer.

[0163] In some aspects, the present disclosure 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.

[0164] In a further aspect, the present disclosure provides a stable formulation of an anti-TGF-β2 agent for various therapies for CNS diseases, wherein the formulation can be pumped as a 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 stereoisomers thereof, and combinations thereof.

[0165] The device of the present disclosure is connected to a suitable pump to continuously deliver small amounts of the pharmaceutical composition over several days.

[0166] The devices of the present disclosure have the advantage of using an Omayer intraventricular catheter to deliver a pharmaceutical composition through an externally mounted infusion line or through an indwelling infusion structure for long-term infusion.

[0167] In another embodiment, the device of the present disclosure can use an Omayr reservoir catheter as an intratumoral catheter to deliver the drug composition. An advantage of this device is that it can 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 Omayr reservoir component of the device of the present disclosure can provide long-term access to the cerebrospinal fluid and intraventricular space.

[0168] The infusion device of the present disclosure also has the advantages of delivering the drug 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.

[0169] The infusion device of the present disclosure also has the advantage of using a standardized intraventricular target.

[0170] The infusion device of the present disclosure also has the advantage that the intraventricular pathway to the target is well understood and safe to use.

[0171] The infusion devices of the present disclosure also have the advantage that drug compositions can be effectively delivered to the entire CNS, including the spinal cord.

[0172] The infusion device of the present disclosure also has the advantage of not requiring imaging such as X-ray imaging to guide the implantation of the delivery catheter. Since 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.

[0173] In some aspects, the infusion device of the present disclosure 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).

[0174] In additional aspects, the infusion device of the present disclosure also has the advantage of being used with a single access catheter placed to provide an infusion to a patient. For example, treatment of a glioma or large brain tumor may require placement of multiple catheters near the cancer. However, the device of the present disclosure can provide improved infusion even with a single catheter near the tumor.

[0175] The infusion devices of the present disclosure also have the advantage that implantation of the delivery catheter may be semi-permanent, or even permanent.

[0176] The infusion devices of the present disclosure also have the advantage that the Omayne 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.

[0177] The infusion device of the present disclosure 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 top 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 Omayne device can be placed at a convenient location on the patient's skull and connected to a ventricular access catheter

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

[0178] Aspects of the present disclosure include the following:

[0179] A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion, the device comprising: Figure 1 ): a reservoir

[117] containing a drug composition; a pump

[101] that forces the drug 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 ).

[0180] A device for delivering a pharmaceutical composition by continuous intrathecal or intraventricular infusion, the device comprising: Figure 2): a reservoir

[117] containing a drug composition; a pump

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

[103] into an access port

[107] , 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 catheter

[109] in fluid communication with the access port and 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 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 ).

[0181] The device can provide continuous infusion of a therapeutically effective amount of a pharmaceutical composition to a target area.

[0182] As used herein, a target region of the brain may be a region containing a tumor.

[0183] The distal end of the access catheter is advanced into the target area of ​​the brain.

[0184] The Omayr 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 a silicone impregnated with barium and has anti-kink and anti-compression properties. 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.

[0185] The pump may be a PegasusVario, PEGAPCA, CADDSolisVIP, CADD-Legacy PLUS, CADD-LegacyPCA, CADD-Legacy1 or another pump with similar specifications.

[0186] 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.

[0187] Some examples of pumps and infusion rates for the devices of the present disclosure include those in Table 1 below:

[0188] Table 1: Pumps and Rates for Infusion Systems

[0189]

[0190] 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.

[0191] In some aspects, the brain access catheter positioned into 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 per row) at the proximal end. In general, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The Omayor-type reservoir of the present disclosure 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 Up 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.

[0192] The Omayor-type devices of the present disclosure allow for the delivery of drugs directly into the CSF and are particularly useful for the delivery of oligonucleotide and antisense oligonucleotide drugs such as OT-101, either alone or in combination with other cancer therapies.

[0193] 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.

[0194] 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.

[0195] As described below, infusion data for rat animals were 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 reached 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 changing the reservoir to catheter ratio, drug infusion release kinetics can be slowed down, such that the half-life can be advantageously changed to days and weeks.

[0196] For example, Fig.11 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from hours to days.

[0197] For example, Fig.12 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from days to weeks.

[0198] For example, Fig.13 As shown, drug infusion release kinetics can be prolonged, such that the half-life of a single reservoir changes from weeks to months.

[0199] One advantage of the device of the present disclosure is the design of the Omayne-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 pressure changes, which allows the Omayne-type reservoir of the present disclosure to breathe properly to achieve continuous flow of solution, especially in the presence of externally generated pumping action. Undesirable accidental or accidental release may also occur due to patient movement, such as movement of the patient's jaw and other muscles.

[0200] An advantage of the device of the present disclosure is that the Omayor-type device is placed not as a mechanism for collecting CSF or for delivering a drug (drug composition) to the CSF, but rather as a reservoir to hold a drug to be infused into the CSF over a sustained period of time. In order for fluid to leave the Omayor device, a continuous flow of fluid from the reservoir is driven by a pump, which also causes the drug fluid to enter the reservoir. The structure of the device of the present disclosure 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 disclosure 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 disclosure advantageously maintains fluid movement in the reservoir because the reservoir includes a flexible top (see, for example, Figure 5

[401] ), which can produce oscillating 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.

[0201] 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 having a top made of a flexible material and equipped with a hard collar so that only the uppermost portion of the flexible top is exposed and can be compressed or bent.

[0202] The pharmaceutical composition may include 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 an animal. The CNS disease may be cancer. The cancer may be 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.

[0203] In some embodiments, the compositions and methods of the present disclosure may be used for diffuse midline glioma (DMG) and K27M GBM.

[0204] Methods and compositions for CNS diseases

[0205] Operational Parameters: The disclosed system can be modified to prolong the drug for treating a CNS disease such as CNS cancer or improving the symptoms of a CNS disease in a human subject or animal in need thereof. The disclosed system can comprise a drug composition with a release kinetic half-life of weeks to months for continuous infusion into the brain or spine.

[0206] The pharmaceutical composition for continuous infusion may include an agent for inhibiting or suppressing TGF-β expression, and a therapeutically sufficient amount of the composition is administered to a subject. The present disclosure provides systemic therapies for treating CNS diseases such as cancer or ameliorating symptoms of CNS diseases.

[0207] 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.

[0208] In some aspects, the present disclosure provides a system comprising a composition of 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 an animal.

[0209] In a further aspect, the present disclosure provides use of a composition of an agent 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 an animal.

[0210] 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.

[0211] 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".

[0212] Human TGF-β2 specific phosphorothioate antisense oligodeoxynucleotide

[0213] Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapy can downregulate molecular targets, which can be achieved 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, alteration of splicing processes (e.g., splice switching), and steric hindrance of ribosomal activity.

[0214] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating 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.

[0215] Antisense oligonucleotides can be small synthetic fragments of single-stranded DNA, which can be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization, and once bound to the target RNA, they inhibit the translation process by inducing a cleavage mechanism or inhibiting mRNA maturation. ASOs can selectively inhibit gene expression and have specificity. Chemical modification of DNA or RNA can be used to improve stability.

[0216] For example, modifications can be introduced into phosphodiester bonds, sugar rings, and backbones. ASO antiviral agents can block the translation process by (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage or (ii) by steric (non-bonding) blocking enzymes involved in the translation of target genes. 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.

[0217] 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 of human TGF-β2 mRNA after gene expression.

[0218] OT-101 is available in the form of 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.

[0219] OT-101: Antisense oligodeoxynucleotides are short strands of DNA designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and 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.

[0220] The agent may be an antisense oligonucleotide or inhibitor specific for TGF-β1, TGF-β2 or TGF-β3. The agent used to inhibit or suppress TGF-β expression may be selected from the TGF-β2 specific antisense oligonucleotides shown below.

[0221] 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.

[0222] Table 2: TGF-β2 specific antisense oligonucleotides

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] The sequences of Table 2 may 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 may be used as active agents in any combination, such as a pooling combination.

[0230] 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%.

[0231] 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.

[0232] 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 intraventricular or intrathecal or intracerebral administration.

[0233] 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 intraventricular or intrathecal or intracerebral administration.

[0234] In some embodiments, 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 by bolus injection into an Omayor-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.

[0235] 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.

[0236] Embodiments of the present disclosure contemplate methods and uses comprising an agent that may be a TGF-β2-specific antisense oligonucleotide selected from SEQ ID NOs: 9-136.

[0237] 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.

[0238] Embodiments of the present disclosure relate to administration or use of pharmaceutical compositions that can improve or inhibit symptoms caused by TGF-β-induced proteins.

[0239] The agent used to inhibit or suppress TGF-β expression can be an artemisinin preparation, which contains 90-95% pure artemisinin extract or its pharmaceutically acceptable salt, salt polymorph, ester or isomer, and one or more pharmaceutically acceptable excipients. The excipient may include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants and anti-caking agents. In some embodiments, the excipient may include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, cross-linked carboxymethyl cellulose sodium and magnesium stearate.

[0240] In other embodiments, the agent used to inhibit or suppress TGF-β expression may be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0241] As used herein, derivatives encompass chemical modifications that provide structural analogs of a compound. For example, a substituent or substitution of an alkyl group can provide a structural analog.

[0242] Embodiments of the present disclosure include methods or uses, wherein the agent used to inhibit or suppress TGF-β expression is a compound or ligand that interacts with TGF-β site I comprising Trp30 and / or TGF-β site II comprising Arg15, Gln19 and Phe8, including a small molecule or a polypeptide, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0243] 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.

[0244] 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.

[0245] In certain embodiments, the agent that inhibits or suppresses TGF-β expression may be a compound comprising three isoprenyl groups and one lactone ring or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0246] Embodiments of the present disclosure also include pharmaceutical compositions for inhibiting or suppressing TGF-β expression in humans or animals, or for treating or improving the 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 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.

[0247] Importantly, the compositions of the present disclosure may be substantially free of excipients. It has been found that the compositions of the present disclosure substantially free of excipients are surprisingly stable in a carrier. In some embodiments, the compositions may 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 active agent concentration may be less than 10%.

[0248] 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.

[0249] Embodiments of the present disclosure also include treatment regimens in which the compositions of the present disclosure are administered or used in conjunction 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 disclosure 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, pantoprozol, doxycycline, ivermectin, zinc, Foracort Rotacaps inhalation, injection Ceftriaxone, tab paracetamol, injection fragment protein Fragmin), Covifor Tablet, Azithromycin, Dexamethasone Injection, Ondansetron Injection, Multivitamin Tablet, Ascorbic Acid Tablet, Calcium Carbonate Tablet, and Zinc Sulfate Tablet.

[0250] The present disclosure 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.

[0251] The present disclosure also provides a kit comprising a lyophilized powder of the following substances in a vial at a content of 500 mg: artemisinin or its derivatives; or a compound or ligand that interacts with TGF-β site II comprising Arg15, Gln19 and Phe8, including a small molecule or a polypeptide; a sesquiterpene lactone or its derivatives; or a compound comprising three isoprenyl groups and one lactone ring and its derivatives, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof, or any combination of the foregoing.

[0252] Some TGF-β agents are given in US 9,963,703, US 9,758,786 and US 8,476,246.

[0253] Infusion devices and methods

[0254] The present disclosure provides novel devices and methods of use for the treatment of CNS diseases, such as CNS cancers, by continuous infusion of pharmaceutical agents via an intracranial route.

[0255] The devices and methods of the present disclosure may deliver agents to the brain or spinal region using a pump-based continuous infusion system.

[0256] The device may be operated within a radiation therapy device and / or an electric field device, and the pharmaceutical composition may include the above-mentioned agents and any combination thereof.

[0257] In some examples and embodiments, the device of the present disclosure 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 disclosure can infuse any type of fluid by continuous infusion or continuous convection enhanced delivery. The device of the present disclosure 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.

[0258] The devices of the present disclosure effectively deliver therapeutically effective amounts of a pharmaceutical composition to a subject, allowing for continuous infusion of the agent into a specific location within the subject, such as a specific tissue or tumor, preferably the ventricular space.

[0259] In some aspects ( Figure 1 and Figure 3 ), the device of the present disclosure can act as a portable external convection-enhanced delivery device to infuse a medicament in liquid form into a specific location in a subject's body. These aspects of the device can provide perfusion delivery in a hospital or inpatient center.

[0260] In other aspects ( Figure 2 and Figure 4 ), the device of the present disclosure can act as a portable indwelling infusion device to deliver the agent to a tissue or tumor of a subject, preferably an implantable ommaya in the ventricular space. 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.

[0261] The devices of the present disclosure can inject fluids for continuous infusion.

[0262] The devices of the present disclosure can be used to deliver various therapeutic agents, such as drugs, proteins, protein toxins, imaging agents, antibodies for treatment or imaging, proteins in enzyme replacement therapy, growth factors, and / or viruses or oligonucleotides in gene therapy.

[0263] The devices of the present disclosure can advantageously improve the bioavailability and safety, as well as improve the pharmacokinetic and pharmacodynamic properties of the delivered therapeutic agents.

[0264] The devices of the present disclosure may also be used for infusion delivery using a portable pump for outpatient treatment.

[0265] The device disclosed herein ( 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 drug composition contained therein.

[0266] The device disclosed herein ( 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.

[0267] The devices of the present disclosure have the advantage of providing clinically useful constant flow rates for continuous infusion or convection enhanced delivery.

[0268] The device of the present disclosure 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.

[0269] The devices of the present disclosure may include an Omayor-type delivery device ( Figure 5 ). Figure 5 The Omayor-type delivery device shown can be used, for example, 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 fluid of the Omayyad delivery device. For example, 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 Omayya delivery device also includes an access catheter

[413] in fluid communication with the Omayya 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 entire area of ​​

[401] plus

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

[401] (see Figure 8 ).

[0270] In further examples or embodiments, the devices of the present disclosure may include an Omayor-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 Omayr 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 also includes a non-flexible mounting plate

[505] comprised of an inert material such as metal or hard plastic. The Omayel delivery device also includes 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 an overall area of ​​

[501] plus

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

[501] (see Figure 8 ).

[0271] In further examples or embodiments, the devices of the present disclosure may include an Omayor-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 Omayr delivery device shown can be used as e.g. Figure 3-4 The Omayyeh delivery device includes a non-flexible hard shell

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

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

[607] and

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

[607] may 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 hard shell

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

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

[619] . In another embodiment, the hard shell

[603] can be a separate protective collar that can be placed on the 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 ).

[0272] The devices of the present disclosure can deliver agents, including any agent suitable for delivery in a solvent system, or any agent, for example, formulated for delivery in an aqueous solution. Such 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, corticoids, cytokines, cytostatics, diagnostic agents, fibrinolytics, geriatric medicines, gonadotropins, liver drugs, stimulants, hormones and their inhibitors, hypnotic drugs, immunoglobulins, immunomodulators, immunotherapeutic agents, organ perfusion solvents, proteins, protein toxins, protective agents, sedatives, cardiac drugs, inhibitors and stimulants, minerals, muscle relaxants, neurotrophic agents, oligonucleotides, ophthalmic drugs, vaccines, spasmolytics, 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.

[0273] The devices disclosed herein can deliver agents or active substances dissolved or suspended in physiological solvents or any other suitable solvent. The 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 agents and combinations thereof can be used in the devices, methods, kits, combinations and compositions described herein.

[0274] The devices of the present disclosure may have an interior surface coated with a therapeutic agent.

[0275] The components of the device of the present disclosure can be made of a variety of materials, including, for example, metals, polymers and / or composite materials, including materials such as titanium, high-grade steel, aluminum, alloys, polymer foams, plastics, stainless steel and / or metals and combinations, mixtures and modifications thereof. The material intended to be implanted in the subject can be made of biocompatible materials, such as polymers, such as polymer fragments of polystyrene, polyolefins, polyamides or polyurethanes, and metals. The selection of such materials depends on many factors, including the required mechanical properties and the porosity, surface properties or toxicity of the material. The components of the device of the present disclosure can be made of, for example, titanium, alloys, stainless steel, ceramics, silicon, Teflon, polypropylene, polyethylene, polystyrene, polyolefins, polyimides, polyamides, polyurethanes, PET, PETG, PE, PIG, HDPE, PC, PVC, nylon, urethane and / or copolymers, and can be laminated with gold, silver and / or aluminum layers or otherwise include gold, silver and / or aluminum layers to minimize the permeability of gases and liquids, sputtering or other methods of deposition or incorporation therein. Fragmented polyurethane-urea, Polycarbonateurethane, Elasthane TM and Elasthane TM Polyether urethane can be used for long-term implanted medical devices. TM The chemical structure and properties of Similar to 2363. Thermoplastic silicone-urethane copolymers, such as PurSil TM Silicone Polyether Urethanes and CarboSil TM Silicone polycarbonate urethanes may also be used in the present device.

[0276] The device of the present disclosure may include a filter in any suitable position. The filter may include a sterile filter for removing pathogens, a biological filter for biological materials such as proteins and / or antibodies; a particle filter for removing particulate matter; a chemical filter for removing chemicals; and / or a filter for removing air or gas from a solvent. In one aspect, each filter may have a distal port and a proximal port and an inner cavity passing therethrough. In the inner cavity of each filter, there 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 filter may have a separate housing, or a shared housing. In one aspect, the filter for removing air may be placed outside the body. In another aspect, the filter for removing particles may be placed upstream of the sterile filter. The sterile filter typically has a pore size of about 0.45 μm or less, or about 0.22 μm or less, or about 0.1 μm or less. The particle filter may have a pore size greater than about 0.45 μm, or greater than about 0.22 μm.

[0277] The access port can be implanted subcutaneously, for example, on a rib. The access port chamber is in fluid communication with the Omayne reservoir.

[0278] The device of the present disclosure 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, as an example, the continuous phase 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.

[0279] The device of the present disclosure may contain a preservative added to the formulation. Preservatives include benzalkonium chloride, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenol, sodium benzoate or EDTA.

[0280] The devices of the present disclosure may contain a pharmaceutically acceptable carrier. Carrier materials useful in preparing the compositions of the present disclosure are any of those commonly used excipients in pharmacy and should be selected based on compatibility with the medicament and the release profile properties of the desired dosage form.

[0281] The devices of the present disclosure 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.

[0282] Device Operation

[0283] In operation, the device can be adjusted relative to the patient by performing a computed tomography (CT), brain MRI, or other suitable imaging method on the patient to determine the location of the ventricular space and the length of catheter required. 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 Omayor-type device is then connected to an external or indwelling infusion set.

[0284] 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 saline. The postoperative position of the Omayer-type device and device disclosed herein 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.

[0285] 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.

[0286] In some examples and embodiments, a special port puncture needle may be used, such as the 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 process.

[0287] For treatment, a therapeutic agent solution, such as 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 automatic pump is connected to the device. Before starting the infusion, the infusion device 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.

[0288] 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.

[0289] In some examples and embodiments, a therapeutically effective amount of antisense (OT-101, etc.) preparation 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 concentration of the agent 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 subjects on days 1-3, or 1-7, or 1-14, or 1-21, or 1-50 of a regimen or cycle.

[0290] Methods, compositions and combinations for CNS diseases

[0291] The devices of the present disclosure can be used to treat CNS diseases such as CNS cancer or to ameliorate symptoms of CNS diseases in a human subject or animal in need thereof.The devices of the present disclosure can contain a pharmaceutical composition for continuous infusion into the brain or spine.

[0292] The pharmaceutical composition for continuous infusion may include an agent for inhibiting or suppressing TGF-β expression, and a therapeutically effective amount of the composition is administered to a subject. The present disclosure provides therapies for treating CNS diseases such as cancer or ameliorating symptoms of CNS diseases.

[0293] 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.

[0294] In some aspects, the present disclosure provides kits and devices comprising compositions of agents for inhibiting or suppressing TGF-β expression for use in treating or ameliorating symptoms of CNS diseases in human subjects or animals.

[0295] In a further aspect, the present disclosure provides use of a composition of an agent 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 an animal.

[0296] 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.

[0297] 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".

[0298] In certain embodiments, the agents, uses or methods of the present disclosure, upon administration to a subject, may reduce TGF-β2 levels in a subject, which may be referred to as an improved TGF-β2 profile.

[0299] In some aspects, the agents, uses or methods of the present disclosure, when administered or used by a subject, can reduce mortality at 6, 12, 18, 24, 30 or 36 months.

[0300] In a further aspect, the medicament, use or method of the present disclosure, after administration or use in a subject, can increase the survival rate at 6, 12, 18, 24, 30 or 36 months. Survival can be determined as overall survival or progression-free survival.

[0301] In some aspects, a substantially excipient-free medicament, medicament, or administration may comprise a carrier.

[0302] Embodiments of the present disclosure also include the use of TGF-β2 as a selective biomarker for providing improved outcomes for cancer therapy using the agents of the present disclosure in combination with radiation therapy.

[0303] The present disclosure provides methods for selecting patients for therapy based on TGF-β2 levels as a biomarker for improved outcomes of radiation therapy in cancer.

[0304] Surprisingly, it was found that patients with low TGF-β2 expression had improved overall survival and survival after radiation therapy over 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 therapy.

[0305] In another study, overall survival in pediatric gliomas treated with radiotherapy was unexpectedly improved using TGF-β2 as a selector. TGF-β-1 and TGF-β-3 did not predict survival.

[0306] In some embodiments, TGF-β2 levels can serve as unexpectedly effective biomarkers 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). Such predictive results were not observed for TGF-β1 and TGF-β3.

[0307] In some embodiments, the present disclosure 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 a drug that is a targeted cancer drug, a cancer growth blocker, or an EGFR inhibitor.

[0308] In certain embodiments, the present disclosure 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, bezutifan, dabrafenib, trametinib and combinations thereof.

[0309] In additional embodiments, the present disclosure 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.

[0310] In further embodiments, the present disclosure 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 or irinotecan.

[0311] Human TGF-β2 specific phosphorothioate antisense oligodeoxynucleotide

[0312] Antisense oligonucleotides (ASOs) can be single-stranded deoxyribonucleotides that are complementary to mRNA targets. Antisense therapy can downregulate molecular targets, which can be achieved 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, alteration of splicing processes such as splice switching, and steric hindrance of ribosomal activity.

[0313] Antisense therapeutic strategies can utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating 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.

[0314] Antisense oligonucleotides can be small synthetic fragments of single-stranded DNA, which may be 15-30 nucleotides in length. ASOs can specifically bind to complementary DNA / RNA sequences through Watson-Crick hybridization, and once bound to the target RNA, they inhibit the translation process by inducing a cleavage mechanism or inhibiting mRNA maturation. ASOs can selectively inhibit gene expression and have specificity. Chemical modification of DNA or RNA can be used to improve stability.

[0315] For example, modifications can be introduced into phosphodiester bonds, sugar rings, and backbones. ASO antiviral agents can block the translation process by (i) ribonuclease H (RNAse H) or RNase P-mediated mRNA cleavage or (ii) by steric (non-bonding) blocking enzymes involved in the translation of target genes. 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.

[0316] 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 of human TGF-β2 mRNA after gene expression.

[0317] OT-101 is currently available in the form of a lyophilized powder in three different amounts in 50 mL glass vials. OT-101 lyophilized powder is dissolved in an isotonic (0.9%) sodium chloride aqueous solution before use.

[0318] Examples of the agent for inhibiting or suppressing TGF-β expression disclosed herein include antisense oligonucleotides specific for TGF-β1, TGF-β2 or TGF-β3.

[0319] 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).

[0320] As is known in the art, the antisense oligonucleotides given in Table 2 herein may be chemically modified.

[0321] 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%.

[0322] 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.

[0323] 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 intraventricular or intrathecal or intracerebral administration.

[0324] 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 intraventricular or intrathecal or intracerebral administration.

[0325] In some embodiments, 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 by bolus injection into an Omayor-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.

[0326] 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.

[0327] 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.

[0328] Embodiments of the present disclosure relate to administration or use of pharmaceutical compositions that can improve or inhibit symptoms caused by TGF-β-induced proteins.

[0329] The agent used to inhibit or suppress TGF-β expression can be an artemisinin preparation, which contains 90-95% pure artemisinin extract or its pharmaceutically acceptable salt, salt polymorph, ester or isomer, and one or more pharmaceutically acceptable excipients. The excipient may include any one or more pharmaceutically acceptable excipients selected from diluents, stabilizers, disintegrants and anti-caking agents. In some embodiments, the excipient may include any one or more of microcrystalline cellulose, polysorbate 80, crospovidone, cross-linked carboxymethyl cellulose sodium and magnesium stearate.

[0330] In other embodiments, the agent used to inhibit or suppress TGF-β expression may be an artemisinin compound or a derivative thereof, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof.

[0331] As used herein, derivatives encompass chemical modifications that provide structural analogs of a compound. For example, a substituent or substitution of an alkyl group can provide a structural analog.

[0332] Embodiments of the present disclosure include methods or uses, wherein the agent for inhibiting or suppressing TGF-β expression is a compound or ligand, including a small molecule or a 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.

[0333] 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.

[0334] 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.

[0335] Embodiments of the present disclosure also include pharmaceutical compositions for inhibiting or suppressing TGF-β expression in humans or animals, or for treating or improving the 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 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.

[0336] Importantly, the compositions of the present disclosure can be substantially free of excipients. It has been found that the 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.

[0337] 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.

[0338] Embodiments of the present disclosure also include treatment regimens in which the compositions of the present disclosure are administered or used in conjunction 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 disclosure include anti-inflammatory drugs, anti-inflammatory steroids, piperaquine, pyronaridine, curcumin, frankincense, remdesivir, SomprazD, ZifiCV / ZacD, CCM, Broclear, budamate, Rapitus, MontekLC, 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 Fragmin, remdesivir tablets (Tablet Covifor), azithromycin, dexamethasone for injection, ondansetron for injection, multivitamin tablets, ascorbic acid tablets, calcium carbonate tablets and zinc sulfate tablets.

[0339] The present disclosure also provides a kit comprising lyophilized powder of any one or more of the TGF-β2-specific antisense oligonucleotides selected from SEQ ID NOs: 1-136 in vials, each containing 3.75 mg.

[0340] The present disclosure also provides a kit comprising a lyophilized powder of the following substances in a vial at a content of 500 mg: artemisinin or its derivatives; or a compound or ligand that interacts with TGF-β site II comprising Arg15, Gln19 and Phe8, including a small molecule or a polypeptide; a sesquiterpene lactone or its derivatives; or a compound comprising three isoprenyl groups and one lactone ring and its derivatives, or a pharmaceutically acceptable salt, salt polymorph, ester or isomer thereof, or any combination of the foregoing.

[0341] Delivery of therapeutic agents by infusion

[0342] Delivery of OT-101 (TGF-β antisense) to treat glioblastoma.

[0343] OT-101 is a TGF-β2 antisense drug that was active against recurrent glioblastoma in the Phase 2 clinical trial G004 (UckunFM, QaziS, HwangL, TrieuVN. 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 followup. Cancers. 2019, 11: 1892).

[0344] OT-101 is delivered intratumorally via a brain-implanted catheter. To further expand the application of OT-101, intrathecal delivery of tritiated OT-101 into Sprague-Dawley CD (albino) rats was explored. Throughout the study, there was no gender difference.

[0345] Surprisingly, OT-101 was similarly widely 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 is similar to intraventricular administration and enters the entire CNS compartment via the CSF.

[0346] 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. The smallest amount of OT-101 was 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 material-related histomorphological lesions in the subarachnoid space in the lumbar region. No changes were noted in the gray and white matter of the spinal cord, and no abnormalities were shown in nerve stems and neurons. 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 gliomas (DMG)).

[0347] Targeting transforming growth factor β2 (TGF-β2) and OT-101 for post-radiation therapy of diffuse intrinsic pontine gliomas Solid treatment

[0348] 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%.

[0349] Chemotherapy does provide clinically meaningful benefit. Therefore, innovative therapeutic agents for the treatment of childhood DIPG are urgently needed. High-grade glioma cells, including childhood glioblastoma and DIPG cells, have been shown to produce transforming growth factor β2 (TGF-β2).

[0350] 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 the first RNA therapeutic agent in a class, designed to eliminate the immunosuppressive and tumor-promoting effects of TGF-B2. At low micromolar concentrations, OT-101 can reduce TGFB2 secretion of human glioma cells, prevent their proliferation and migration, and restore the anti-glioma cell lysis function of patient-derived T cells. OT-101 applied intracerebrally was administered and showed promising single-agent activity in recurrent / refractory (R / R) high-grade gliomas (HGG) (Uckun et al., Cancers. 201928; 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 pharmacology studies of intrathecal delivery of OT-101 in rabbits and primates, and encouraged by its single-agent activity in adult HGG patients, pediatric patients with DIPG are 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.

[0351] Numbered embodiments of the present disclosure may include:

[0352] 1) An agent for inhibiting or suppressing the expression of TGF-β2, which is used for treating or improving the symptoms of CNS diseases in human subjects or animals.

[0353] 2) Use of a composition comprising 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.

[0354] 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:

[0355] preparing a pharmaceutical composition comprising an agent for inhibiting or suppressing TGF-β2 expression; and

[0356] A therapeutically effective amount of the composition is administered to the subject.

[0357] 4) The agent, use or method according to any one of embodiments 1-3, wherein the CNS disease is glioma, glioblastoma, diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), leptomeningeal or brain metastasis, brain or spinal cancer, or a CNS tumor.

[0358] 5) The agent, use or method according to any one of embodiments 1-4, comprising any one or more additional drugs, wherein the additional drugs include targeted cancer drugs, cancer growth blockers, EGFR inhibitors and combinations thereof.

[0359] 6) The medicament, use or method according to any one of embodiments 1-5, comprising any one or more additional drugs selected from bevacizumab, everolimus, bevacizumab, dabrafenib, trametinib and a combination thereof.

[0360] 7) The agent, use or method according to any one of embodiments 1-6, comprising any one or more additional drugs that are cancer growth inhibitors, wherein the cancer growth inhibitor is selected from angiogenesis inhibitors, histone deacetylase inhibitors, hedgehog blockers, mTOR inhibitors, p53 inhibitors, PARP inhibitors, proteasome inhibitors, tyrosine kinase inhibitors and combinations thereof.

[0361] 8) The medicament, use or method according to any one of embodiments 1-7, comprising any one or more additional drugs that are EGFR inhibitors, wherein the EGFR inhibitors are selected from erlotinib, gefitinib, afatinib, osimertinib, dacomitinib and combinations thereof.

[0362] 9) The medicament, use or method according to any one of embodiments 1-8, comprising the additional drug temozolomide or irinotecan.

[0363] 10) An agent, use or method according to any one of embodiments 1-9, comprising additional treatment of the CNS disease by radiation therapy or electric field therapy.

[0364] 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.

[0365] 12) The medicament, use or method according to any one of embodiments 1-11, wherein the medicament, drug, therapy, treatment and administration are each administered concurrently, simultaneously, sequentially or separately in time.

[0366] 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.

[0367] 14) The medicament, use or method according to any one of embodiments 1-13, comprising administration or use by intracranial continuous infusion or bolus administration.

[0368] 15) A medicament, use or method according to embodiments 1-14, wherein the intracranial continuous infusion comprises infusion with an Omayr reservoir having a partially flexible top.

[0369] 16) The medicament, 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.

[0370] 17) An agent, use or method according to any one of embodiments 1-16, wherein the subject has improved TGF-β2 characteristics after administration or use.

[0371] 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.

[0372] 19) An agent, use or method according to any one of embodiments 1-18, wherein the administration or use increases the survival rate at 6, 12, 18, 24, 30 or 36 months.

[0373] 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:

[0374] Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0375] 21) An agent, use or method according to any one of embodiments 1-20, comprising a TGF-β2-specific antisense oligonucleotide, wherein the TGF-β2-specific antisense oligonucleotide has one or more nucleotides, which are chemically modified to be a thiophosphate internucleoside linker, a methoxypropylphosphonate internucleoside linker, an aminophosphoro linker linked to a morpholine group, a 2'-OMe ribose group, a 2'-MOE methoxyethyl ribose group, a 2'-4' restricted methoxyethyl bicyclic ribose group, a 2'-4' restricted ethyl bicyclic ribose group, an LNA ribose group, a 2'-F ribose group or a 5-methylcytosine base.

[0376] 22) An agent, use or method according to any one of embodiments 1-21, wherein the agent is conjugated to polyethylene glycol, a lipid or triantennary N-acetyl-galactosamine.

[0377] 23) The medicament, use or method according to any one of embodiments 1-22, comprising a carrier, which is sterile water for injection, saline, isotonic saline, phosphate-buffered saline or a combination thereof.

[0378] 24) A medicament, use or method according to any one of embodiments 1-23, wherein the medicament, drug or administration is substantially free of excipients.

[0379] 25) The agent, use or method according to any one of embodiments 1-24, 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 the concentration of the antisense active agent decreases by less than 10% after 90 days of use.

[0380] 26) The agent, use or method according to any one of embodiments 1-25, wherein the method comprises administering the composition by intracranial continuous infusion at a rate of 2-8 μl / min and a concentration of 1-20 μM of the agent from day 1 to day 7. The above-mentioned agent, use or method, wherein the method comprises administering the composition by intracranial infusion at a rate of 2-8 μl / min and a concentration of 1-80 μM of the agent from day 1 to day 7, preferably by intracranial continuous infusion.

[0381] 27) A kit comprising:

[0382] A medicament comprising a total of 250 mg of one or more TGF-β2-specific antisense oligonucleotides selected from Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof; and

[0383] Omayeh reservoir with partially flexible top.

[0384] 28) A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0385] a reservoir

[117] containing a pharmaceutical composition;

[0386] 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] ;

[0387] a filter

[105] inline with the infusion line; and

[0388] An access catheter

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

[0389] 29) A device according to embodiment 28, 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.

[0390] 30) A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0391] a reservoir

[117] containing a pharmaceutical composition;

[0392] 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] ;

[0393] a filter

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

[0394] an indwelling tube

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

[111] ; and

[0395] An access catheter

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

[0396] 31) A device according to embodiment 30, 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.

[0397] 32) A device according to any of embodiments 28-31, wherein the Omayya reservoir

[111] includes a partially flexible top.

[0398] 33) A device according to any of embodiments 28-32, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to a target area.

[0399] 34) A device according to any of embodiments 28-33, wherein the distal end of the access catheter enters a target area of ​​the brain.

[0400] 35) A device according to any one of embodiments 28-34, wherein the Omayr reservoir

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

[0401] 36) A device according to any of embodiments 28-35, wherein the distal tip of the access catheter entering the brain is a stepped end, a recessed step end, a multi-port end, a microporous end or a balloon tip.

[0402] 37) An apparatus according to any of embodiments 28-36, 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.

[0403] 38) A device according to any one of embodiments 28-37, 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.

[0404] 39) A device according to any of embodiments 28-38, 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.

[0405] 40) A device according to any one of embodiments 28-39, wherein the fluid pharmaceutical composition comprises an agent for inhibiting or suppressing TGF-β expression, which can be used to treat or improve symptoms of CNS diseases in human subjects or animals.

[0406] 41) A device according to any of embodiments 28-40, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of a pharmaceutical agent, drug or delivery vehicle.

[0407] 42) A device according to any of embodiments 28-41, wherein the fluid drug composition is a therapeutic agent for CNS disease or CNS cancer.

[0408] 43) A device according to any of embodiments 28-42, 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.

[0409] 44) A device according to any one of embodiments 28-43, wherein the fluid drug 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:

[0410] Table 2 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof.

[0411] 45) A device according to any of embodiments 28-44, wherein the device is operated in combination with radiation therapy or electric field therapy.

[0412] 46) A device according to any one of embodiments 28-45, comprising a device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0413] 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] ;

[0414] a port

[407] in fluid communication with the reservoir; and

[0415] An access catheter

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

[0416] 47) A device according to any one of embodiments 28-46, comprising a device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0417] 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] ;

[0418] a port

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

[0419] A port

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

[0420] 48) A device according to any one of embodiments 28-47, comprising a device for delivering a pharmaceutical composition by continuous intracranial infusion, the device comprising:

[0421] 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] ;

[0422] a port

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

[0423] A port

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

[0424] 49) A device according to any one of embodiments 28-48, comprising a collar of a device for delivering a pharmaceutical composition by continuous intracranial infusion, the collar comprising:

[0425] A hard shell

[604] having an opening

[606] exposing the flexible top of the device.

[0426] 50) A method of administering a pharmaceutical composition by continuous intracranial infusion, comprising:

[0427] installing the device according to any one of embodiments 28-49 to a patient; and

[0428] The pharmaceutical composition is pumped in a device to provide continuous intracranial infusion to the patient.

[0429] 51) A method according to embodiment 50, wherein the device is installed and the access catheter is placed into the brain without the need for simultaneous head or brain imaging.

[0430] 52) A method according to embodiment 50 or 51, wherein the device is installed and a single access catheter is placed into the brain.

[0431] 53) A device according to any one of embodiments 28-49, comprising a kit for continuous intracranial infusion of a pharmaceutical composition into a subject, the kit comprising:

[0432] a reservoir containing the pharmaceutical composition;

[0433] Pumps;

[0434] Omayeh reservoir with partially flexible top;

[0435] infusion tubing for connecting the reservoir to the pump and the pump to the Omayeh reservoir;

[0436] Filters; and

[0437] Enter the catheter.

[0438] 54) A kit according to embodiment 53, wherein the access catheter is substantially linear and is used to access a target area of ​​the brain intraventricularly.

[0439] 55) A kit according to any of embodiments 53-54, wherein the access catheter is non-linear and has a bend to access a target area of ​​the brain intraventricularly.

[0440] 56) A kit according to any of embodiments 53-55, wherein the infusion tube is outside the subject's body.

[0441] 57) A kit according to any of embodiments 53-56, wherein a portion of the infusion tubing connecting the pump to the Omayne reservoir is left indwelling in the subject.

[0442] All publications mentioned herein, including patents, patent application publications, and non-patent publications and Sequence Listing, are expressly incorporated by reference in their entirety for all purposes.

[0443] Although the foregoing disclosure has been described in detail by way of example for the purpose of clear understanding, it is apparent to the skilled person that certain changes and modifications based on the disclosure may be understood and practiced within the scope of the appended claims without undue experimentation, and the claims are presented in an exemplary and non-restrictive manner. The disclosure includes all such additional embodiments, equivalents, and modifications. The disclosure includes any combination or mixture of features, materials, elements, or limitations of the various exemplary components, examples, and claimed embodiments.

[0444] It is emphasized here that, according to common practice, the features of the drawings are in arbitrary scale and are intended to encompass similar features that may be arbitrarily enlarged or reduced.

[0445] Example

[0446] Example 1 - Examples of pumps for continuous infusion via the intrathecal or intraventricular route are shown in Table 3.

[0447] Table 3: Examples of pumps used for continuous infusion

[0448]

[0449] The infusion or indwelling line can be PEGALine 100SF 100cm with a 0.2μm sterile filter, or 200cm infusion line with a 0.2μm sterile filter, or Port-a-Cath (#21-4034-24) with a 22G needle (#21-2737-24) and extension (#21-7106-24), or other tubing with similar specifications.

[0450] In some aspects, the brain access catheter positioned into 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 per row) at the proximal end. In general, the inner diameter of the catheter can determine the diffusion of the drug into the ventricular space. The Omayor-type reservoir of the present disclosure can have an inner diameter of 300 to 400 mm. 2 The surface area of ​​the inner opening of the conduit can be 0.785mm2 Up 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.

[0451] The Omayor-type device of this system allows for the delivery of drugs directly into the CSF and is particularly useful for the delivery of oligonucleotide and antisense oligonucleotide drugs (e.g., OT-101). It can be used alone or in combination with other cancer therapies.

[0452] 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.

[0453] Example 2 - Use of a device for continuous infusion via an intrathecal or intraventricular route Examples are shown in Tables 4 and 5. This formulation in this example provides an effective use of the agent because the formulation is sufficiently stable to be delivered to the patient by continuous administration. The formulation of this example contains minimal or no excipients and can be stable for at least 14 days without scaling. The formulation of the present disclosure 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 disclosure has improved stability and reduced bacterial growth.

[0454] Table 4: Compatibility study of OT-101 and the device

[0455]

[0456] Table 5: In-use stability studies conducted

[0457]

[0458] The following studies A) to C) were conducted as in-use tests under conditions that simulate the clinical use of AP12009(OT-101)-P002. The treatment used a pump and reservoir assembly similar to OT-101-P002. The drug delivery system suitable for this application consisted of the following medical device components:

[0459] Portable pump with appropriate drug reservoir and extension line

[0460] Gripper needle

[0461] Implantable port system with intravenous catheter

[0462] 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 kept at ambient temperature. The three drug delivery systems that were tested for in-use stability of OT-101 drug solutions are described in Tables 6, 7, and 8 below.

[0463] Table 6: Drug delivery system 1

[0464]

[0465] Table 7: Drug delivery system 2

[0466]

[0467] * Unavailable

[0468] Table 8: Drug delivery system 3

[0469]

[0470] * Unavailable

[0471] The conditions of the study are:

[0472] Drug solution concentration: 15 mg / mL (average dose is 195 mg / mL 2 / d and an average body surface area of ​​1.85m 2 calculate)

[0473] Flow rate: 1 mL / h (equivalent to 24 mL / day)

[0474] Pumps (including drug reservoirs) and non-implanted components of drug delivery systems stored at ambient temperature

[0475] Implantable components of drug delivery systems should be stored at 37°C

[0476] Drug reservoir content: 120mL

[0477] Duration of test: 5 days 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 to meet the requirements as long as it met the release specifications of the AP12009 (OT-101) 250 mg drug product (DrugProduct). All samples met the release specifications of the AP12009 (OT-101) 250 mg drug product in terms of HPLC impurity profile. No relevant effects on the composition of the drug solution were observed after passing through the different drug delivery systems.

[0478] Table 9: Ion exchange HPLC analysis

[0479]

[0480] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety (co-eluted with 3'N-2)

[0481] 3'N-2 = impurity with two missing 3'-terminal nucleotides (co-eluted with PO)

[0482] N-1 = Impurity with missing 3'- or 5'-terminal nucleotide

[0483] nd = Not Detected

[0484] Table 10: Reversed Phase HPLC Analysis

[0485]

[0486] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety (co-eluted with 5'N-1)

[0487] CNET = impurity with a cyanoethyl moiety added to one thymidine nucleotide

[0488] 3'N-2 = Impurity with two missing 3'-terminal nucleotides

[0489] 3'N-1 = Impurity with missing 3'-terminal nucleotide

[0490] 5'N-1 = impurity with missing 5'-terminal nucleotide (co-eluted with PO)

[0491] nd = Not Detected

[0492] 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 Absorbance 90%-110% of initial sample concentration (reference value t=0 day) 。 In addition, for all samples, UV spectra in the range of 230–320 nm were taken and compared with the spectra of the reference samples.

[0493] The results are listed in Table 11, which shows that all sample concentrations were within the range of 100.0%-102.7% of the reference solution, meeting the acceptance criteria of 90-110%.

[0494] Table 11: UV analysis

[0495]

[0496] 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 established for chemical analysis of medical devices according to ISO 10993 (i.e., quantification of released organic compounds using a hydrogen flame ionization detector (GC-FID) and identification of potential leachables / extractables using a gas chromatography-mass spectrometer (GC-MS), and comparison with the NIST / EPA / NIH2005 mass spectral library when applicable). The samples were sent to the collaborating laboratory Medical Device Services along with a reference sample (t-0d). Since saline is incompatible with gas chromatography, the samples were extracted with tert-butyl ether and these extracts were then exposed to gas chromatography. According to MDS report #111523-20, no leachables / extractables were detected.

[0497] Study D: Clinical study AP12009-P002 will use two doses, 140 mg / m 2 / d and 250mg / m 2 / d. Assuming the body surface area is 1.75m 2 , the dose is 250mg / m 2 / d, calculate the representative drug solution concentration to be used in this test, as shown in Table 12.

[0498] Table 12: Drug parameters

[0499] The amount of trabedersen (250 mg / m 2 / dx1.75m 2 x4d): 1750mg

[0500] Volume required for 4-day continuous infusion: 96 mL

[0501] Drug solution concentration: 18.23mg / mL

[0502] The representative drug solution was prepared in duplicate and filled into two separate Cadd cartridge reservoirs under sterile conditions. 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.

[0503] 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 sterile 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 sterile test device through a sterile cannula. The results of the sterility test are shown in Table 13.

[0504] Table 13: Sterility test results

[0505]

[0506] The results of this study demonstrated that the Cadd cartridge reservoir maintained the sterility of aseptically filled drug solutions for at least 7 days.

[0507] The concentration of the dosing solution is 10 μM (61.43 μg / mL).

[0508] The compatibility of the drug substance used in the clinical study AP12009(OT-101)-G005 with the 10 μM OT-101 drug solution was demonstrated by verifying the integrity of the drug solution after passing 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.

[0509] The following studies demonstrated the compatibility of OT-101 drug solution with the intended drug delivery system:

[0510] a) Determination of the impurity profile of OT-101 drug solution after passing through the drug delivery system

[0511] b) Verify the concentration of the drug solution after passing through the drug delivery system

[0512] c) Determination of leachable / extractable profile of OT-101 drug solution after passing through the drug delivery system

[0513] d) Sterility testing of the drug solution after incubation in the drug reservoir.

[0514] The drug delivery system suitable for this application consists of the following medical device components:

[0515] Portable pump with appropriate drug reservoir and extension line

[0516] Gripper needle

[0517] Implantable port system with intravenous catheter.

[0518] 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.

[0519] The drug delivery systems that have been tested for in-use stability of OT-101 drug solutions are shown in Table 14 below.

[0520] Table 14: Drug delivery systems

[0521]

[0522] Studies a) to c) were conducted as in-use tests under conditions simulating clinical use. These conditions were:

[0523] Drug solution concentration: 10 μM (61.43 μg / mL) OT-101 in isotonic saline solution

[0524] Flow rate: 4 μL / min (equivalent to 5.76 mL / day)

[0525] Pumps (including drug reservoirs) and non-implanted components of drug delivery systems stored at ambient temperature

[0526] Implantable components of drug delivery systems should be stored at 37°C

[0527] Drug reservoir content: 50mL

[0528] 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.

[0529] Study A: The delivered drug solution was sampled once a day and analyzed by two validated stability-indicating HPLC methods (ion exchange and reverse phase HPLC). The acceptance criteria were set to 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 7.37mg drug product of OT-101 (AP12009). Based on the impurity data shown in Table 15, the results show that only slight degradation was observed during use (under clinically relevant conditions) and all samples met the drug product release specifications. The main trend observed was a slight increase in PO impurities. This is consistent with the forced degradation results, which identified PO as the most obvious degradation product. PO is an impurity in which one of the seventeen thiophosphate moieties is oxidized to a phosphodiester moiety. This impurity is considered to be a related product, not an impurity (in terms of inactive degradation products).

[0530] Table 15: Impurity profile test results of drug solution after passing through drug delivery system

[0531]

[0532] *) PO = impurity with one phosphorothioate moiety replaced by a phosphate moiety

[0533] CNET = impurity with a cyanoethyl moiety added to one thymidine nucleotide

[0534] 3'N-2 = Impurity with two missing 3'-terminal nucleotides

[0535] 3'N-1 = Impurity with missing 3'-terminal nucleotide

[0536] 5'N-1 = impurity with missing 5'-terminal nucleotide (co-eluted with PO)

[0537] Study b: The delivered drug solution was sampled once a day 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 are within the range of 98%-102% of the reference value. These results demonstrate that the drug solution concentration is not affected during passage through the drug delivery system (e.g., the drug is not adsorbed to the polymer surface of the drug delivery system).

[0538] Table 16: UV spectra of drug solutions after passing through the drug delivery system

[0539]

[0540] 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 established for chemical analysis of medical devices according to ISO 10993 (i.e., quantification of released organic compounds using a hydrogen flame ionization detector (GC-FID) and identification of potential leachables / extractables using gas chromatography-mass spectrometry (GC-MS), and comparison to the NIST / EPA / NIH2005 mass spectral library when applicable). No leachables / extractables were detected in this sample.

[0541] Taking two HPLC data for 5 days in all in-use stability studies, the following regression results were obtained: Slope: -0.001065 ± 0.0009457, and the time to reach a 10% reduction in OT-101 concentration was 91 days. Therefore, the formulations of the present disclosure are unexpectedly stable over a long in-use period.

[0542] Example 3 - Pharmacokinetics after intracerebral and intracerebroventricular administration in rats

[0543] After a single intracerebral injection into the prefrontal lobe of Sprague-Dawley rats, 3Distribution, pharmacokinetics, and excretion of H-AP12009 (10 μCi / rat, 87 μM solution, flow rate: 0.4 μL / min, dosing time: 1 h; Report GAS0002). Data from early time points (0 to 4 h 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 location of the infusion set. 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.

[0544] Intravenous administration of OT-101 resulted in minimal accumulation in the CNS. A single intravenous administration of [ 3 H] OT-101, the concentrations of radioactive substances in blood and tissues resulted in 0.1% OT-101 in the brain, as shown in Table 17.

[0545] Table 17: Radioactive material concentrations in blood and tissues

[0546]

[0547] To improve the use of OT-101 therapeutics, intrathecal delivery of tritiated OT-101 to Sprague-Dawley CD (albino) rats was investigated. Throughout the study, there were no sex differences. After 1 hour intracerebral infusion in rats, OT-101 was confined to the infusion site. Whereas for 1 hour intraventricular infusion, OT-101 was more widely distributed, with concentrations in the cerebellum, the rest of the brain, and the cerebrospinal fluid (CSF) being 35-fold, 19-fold, and 12-fold higher, respectively. For the first 4 hours after infusion, OT-101 concentrations were stable 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 injections 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 material-related histomorphological lesions in the subarachnoid space of the lumbar region. No changes were noted in the gray matter and white matter of the spinal cord, and the nerve stems and nerve cells did not show any abnormalities. These data indicate that intrathecal administration of OT-101 is a potentially effective delivery route for delivering antisense therapeutic agents such as OT-101 to the midline (i.e., for diffuse midline glioma (DMG)).

[0548] 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 pediatric 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 (approximately 7 years) occurring earlier than those arising 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 remains the mainstay of therapy, although it is only palliative and is expected to prolong survival by an average of 3 months.

[0549] In adult gliomas, OT-101 antisense was safe and effective during long-term (7 days) 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).

[0550] TGF-β2 is expressed at high levels in both pediatric GBM (WHO grade IV) and pediatric DIPG (WHO grade IV) patients.

[0551] Expression analysis of pediatric brainstem cases from the TCGA database yielded a highly significant survival benefit across all four quartiles of TGF-β2 expression.

[0552] Expression analysis of all glioma cases treated with radiotherapy yielded a highly significant survival benefit across all quartiles of TGF-β2 expression.

[0553] Intracerebral infusion

[0554] Administration by intracerebral infusion or intraventricular infusion results in the distribution of OT-101 throughout the CNS. Administration was performed at 12 μg / rat. The distribution profiles between brain administration and ventricular administration were similar, except that administration directly into the CSF chamber by intraventricular administration resulted in higher drug concentrations in the cerebellum and CSF. Surprisingly, the similarity between intraventricular administration and intracerebral administration indicates that intraventricular administration is as effective as intracerebral administration, as shown in Table 18.

[0555] Table 18: Administration by Intracerebral Infusion or Intraventricular Infusion

[0556]

[0557]

[0558] Cumulative distributions for the first 15 organs are shown below. At the end of the 1-hour infusion period, CSF-accessible, rapidly perfused parts 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 The less perfused CNS parts reached C at 1 hour (rest of brain, rest of brain) and 4 hours (spinal cord). max。

[0559] 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.

[0560] Table 19: Administration by Intracerebral or Intraventricular Infusion

[0561]

[0562]

[0563] The data from this distribution study clearly demonstrate that, by intracerebral infusion (and therefore intraventricular infusion), OT-101 reaches the entire CNS region, including the midline. This feature is consistent with CSF flow and the location of the various chambers examined. Parts of the CNS where OT-101 levels are high are CSF accessible, including the pituitary and pineal glands.

[0564] A series of nonclinical safety and pharmacology studies have been conducted on OT-101. The salient features of the conclusions of these studies are as follows:

[0565] Chronic topical administration of OT-101 / AP 12009 may result in local tissue inflammation. Mild to moderate local toxicity was observed in animals following infusion of a 500 μM concentration, but without any macroscopic changes.

[0566] 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 4.16 μM), OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes, consistent with subclinical toxicity.

[0567] 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.

[0568] When administered to rats via intracerebroventricular administration, radiolabeled OT-101 (0.18 mg / kg) was detected not only in CSF but also in the cerebrum, cerebellum, and pineal gland within 1 hour after administration. The half-life in CSF and brain tissue was <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0569] Intratumoral placement of catheters carries potential risks that can be avoided by using an Omayr reservoir to access the ventricular space.

[0570] Omayor reservoirs can be used to treat leptomeningeal cancer (LM) arising from a variety of malignancies.

[0571] Example 4- Fig. 9 The effects of delivering the pharmaceutical composition by intrathecal or intraventricular continuous infusion are shown. Fig. 9 The effect of OT-101 treatment on TGF-β2 secretion from the human GBM cell line A-172 is shown. Cells were incubated for 7 days with different concentrations of OT-101 / AP12009 (1 μM to 80 μM) as shown. Secreted TGF-β2 in the cell supernatant was measured by ELISA. The results represent the median, minimum, and maximum values ​​of 3 independent experiments.

[0572] Example 5 - Comparison of an example of continuous infusion using a partially collapsible Omayne-type reservoir of the present disclosure with a conventional direct injection method.

[0573] It was found that the disclosed apparatus and method for continuous infusion of therapeutic agent solution is advantageous compared to conventional methods ( Fig.10 ).

[0574] In conventional methods, a therapeutic agent drug solution is injected directly into the ventricular space of the patient's brain, or is injected directly into the ventricular space through a catheter. In these conventional methods, the therapeutic agent solution is rapidly dispersed into the ventricular space within a few seconds to a few minutes. In addition, this rapidly injected therapeutic agent solution is quickly cleared by brain tissue. Therefore, due to the limited exposure of brain tissue to the therapeutic agent solution, the therapeutic effect obtained by this conventional method is very limited.

[0575] By comparison, the Omayne-type reservoir with a partially collapsible top of the present disclosure advantageously provides continuous infusion, substantially continuous infusion, over a longer period of time.

[0576] The Omayor-type reservoir of the present disclosure was filled with a test solution. The device was placed on the water surface of a water tank to test the infusion rate, ie, the rate at which the test solution flows out of the distal tip of the catheter as a model of continuous infusion into the brain.

[0577] In one embodiment, the surface area of ​​the Omayne-type reservoir of the present disclosure 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.

[0578] In this example, the test solution diffused slowly through the catheter opening (see Fig.10 ). Diffusion from the Omayor-type reservoir of the present disclosure continued for more than 4 hours. This test shows that the test solution achieves continuous diffusion and that a therapeutic agent solution with a density adjusted or matched to CSF ​​will provide continuous infusion over a period of at least one to several days.

[0579] This example shows that the partially collapsible design of the Omayor-type reservoir of the present disclosure is advantageous because when the therapeutic agent solution is continuously infused over an extended period of time, it is necessary to prevent accidental release of the therapeutic agent solution due to accidental or incidental pressure on the flexible top of the Omayor-type reservoir. For example, accidental or incidental pressure on the flexible top of the Omayor-type reservoir may result in the release of too much therapeutic agent solution in a very short period of time.

[0580] The partially collapsible design of the Omayne-type reservoir of the present disclosure prevents such undesirable accidental or inadvertent release of the therapeutic agent solution.

[0581] In addition, the partially collapsible design of the disclosed Omayne-type reservoir allows the device to maintain flow and movement of fluid into and out of the reservoir. The flexible top expands and contracts in response to pressure changes, which allows the disclosed Omayne-type reservoir to properly breathe to achieve continuous flow of solution, especially in the presence of externally generated pumping action. Unwanted accidental or incidental releases may also occur due to patient movement, such as movement of the patient's jaw and other muscles.

[0582] In some examples and embodiments, the Omayne-type reservoir of the present disclosure is 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 ).

[0583] In some examples and embodiments, the Omayne-type reservoirs of the present disclosure are made partially collapsible by making a portion of the top of the Omayne-type reservoir out of a hard, non-flexible plastic.

[0584] Example 6 - Diffuse intrinsic pontine glioma in children.

[0585] In the present application, methods for DMG / DIPG / or K27MGBM in children are disclosed.

[0586] In this example, new evidence shows that the enhanced expression of TGFB2 mRNA in childhood DIPG and H3K27M mutant GBM is associated with up-regulation 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 poor treatment outcomes in DIPG. The reported results also support the view that it is necessary to further evaluate the clinical potential of new strategies targeting TGFB2 mRNA in childhood DIPG.

[0587] 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 that of the rest of the patients (median survival > 10 months, 7 events, N = 22);

[0588] Diffuse intrinsic pontine glioma (DIPG) in children is one of the most aggressive and lethal brain tumors in children. The aim of this study was to evaluate the clinical significance of enhanced transforming growth factor beta 2 (TGFB2) expression levels in tumor tissue samples from patients with DIPG. Our findings provide the first evidence that high levels of TGFB2 expression are associated with poor treatment outcomes in DIPG. The reported results also support the idea that further evaluation of the clinical potential of novel strategies targeting TGFB2 in childhood DIPG is warranted.

[0589] Tumor samples from newly diagnosed pediatric diffuse intrinsic pontine glioma (DIPG) patients expressed significantly higher levels of transforming growth factor β2 (TGFB2, also known as TGF-β2) messenger RNA (mRNA) compared with control pons samples, which was associated with enhanced expression of transcription factors that upregulate TGFB2 gene expression. Our study also demonstrated that high TGFB2 mRNA levels based on RNA sequencing (RNAseq) were an indicator of poor prognosis in patients with DIPG, but not in patients with pediatric glioblastoma (GBM) or pediatric diffuse midline glioma (DMG) with 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 were not prognostic. Our study fills a significant gap in our understanding of the clinical significance of elevated TGF expression in pediatric high-grade gliomas.

[0590] Diffuse intrinsic pontine glioma (DIPG) of childhood is among the most aggressive and lethal of the diffuse midline gliomas (DMGs) of childhood brain tumors classified as histone H3 lysine 27 (H3K27) alterations, including H3 K27 mutants as well as H3 wild-type subtypes with overexpressed EZH inhibitory protein (EZHIP). DIPG is the second most common childhood malignant brain tumor, and complete resection is impossible due to its anatomical location in the brainstem and its rapid infiltrative growth. Despite contemporary radiotherapy / chemoradiotherapy strategies, DIPG is still associated with poor overall survival (OS) and is one of the driving factors for childhood cancer-related mortality (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 of DIPG remains poor, with a median survival of approximately 10 months and a two-year survival rate of less than 10%.

[0591] We present novel evidence 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 notion that further evaluation of the clinical potential of novel strategies targeting TGFB2 mRNA in childhood DIPG is warranted.

[0592] Furthermore, there is no standard treatment for progressive DIPG after failure of first-line radiation therapy, 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 biotherapy with fusion toxins administered via convection-enhanced delivery (CED) or oncolytic viruses.

[0593] Transforming growth factor-β (TGFB) is a disulfide-linked homodimeric cytokine with pleiotropic properties that are associated with 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 high-grade glioma patients by promoting invasive 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, and activated regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs). The TGFB pathway has emerged as a possible therapeutic target for high-grade gliomas. The FDA-approved TGFB inhibitor pirfenidone (5-methyl-1-phenyl-2(1H)-pyridone, PFD) has been shown to inhibit TGFB expression in malignant glioma cells. When administered intratumorally via CED, a synthetic antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeting TGFB2 mRNA showed promising single-agent clinical activity associated with durable complete and partial responses in adult patients with relapsed or refractory glioblastoma and anaplastic astrocytoma. In a study using a microarray-based gene expression platform, TGFB2 mRNA levels were found to be 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.

[0594] 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 worse prognosis. The present disclosure demonstrates that newly diagnosed pediatric DIPG patients with increased TGFB2 mRNA levels in their primary tumor samples, but not pediatric DIPG patients 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.

[0595] A clinical study was completed to determine clinical metadata and RNA sequencing (RNAseq)-based mRNA expression data for TGFB subtypes TGFB1, TGFB2, TGFB3 in 41 pediatric DMG patients (mean age at diagnosis (in months) = 7.02 ± 0.44; median = 6; range = 2-14) and 116 pediatric GBM patients (mean age at diagnosis (in months) = 60.01 ± 1.24; median = 60.4; range = 21–89.3) using genomic data obtained through the Cancer Genomics cBioPortal (https: / / pedcbioportal.kidsfirstdrc.org / ). An interactive web interface was used with full filtering capabilities provided by the portal. 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 therapeutics guided by gene expression analysis, whole exome sequencing (WES), and predictive models.

[0596] The downloaded mRNA expression levels of 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 ​​(expression profile data files attached after "mRNA_expression_(RNA_Seq_V2_RSEM).txt"), which are 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 consists of two main steps: 1. generating a set of reference transcript sequences for gene-level mRNA abundance assessment; 2. aligning a set of RNAseq reads to these reference transcripts for TPM abundance estimation. This process allows for direct comparison of mRNA abundance and ranking between samples.

[0597] The Kaplan-Meier (KM) method, log-rank chi-square test, and packages survival_3.2-13, survminer_0.4.9, and survMisc_0.5.5 running in the R environment were used to compare PFS and OS results in patient subgroups. Graphical representations of treatment outcomes were generated using graphics drawing 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 the differences in the results of the compared patient subgroups, and p values ​​less than 0.05 were considered significant.

[0598] TBFB1, TGFB2, and TGFB3 mRNA expression values ​​for normal pons samples measured by mRNAseq (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 of 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. The p-values ​​were adjusted by the false discovery rate (FDR) for the comparison between normal pons and DIPG samples for each transcript. 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 graphs were constructed using the ggplot2_3.3.5 R package.

[0599] The mRNA expression levels of TGFB2 in log2 TPM were 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 for all transcript combinations were determined and visualized on a heat map that was color-coded from positive correlation (red = +1) to negative correlation (blue = -1). The clustering algorithm identified 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 p-value was less than 0.05 and FDR was less than 0.10.

[0600] 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 H3K27M-mutated pediatric GBM patients. 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 AromaAffymetrix statistical package (aroma.affymetrix_3.2.0, aroma.core_3.2.2, and aroma.light_3.24.0) running in an 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. Heat maps were used to visualize the mRNA expression levels of TGFB1, TGFB2, and TGFB3 in DIPG / H3K27M mutant GBM patients as described previously. The expression level (log2 RMA) of TGFB2 mRNA was correlated with that of mRNA in a 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.

[0601] result

[0602] Compared with normal pons samples, tumor samples from pediatric patients with DIPG contained higher levels of TGFB2 mRNA, but not TGFB1 mRNA or TGFB3 mRNA.

[0603] Comparison of RNAseq-based mRNA levels of TGFB1 / 2 / 3 isoforms in 41 primary DIPG samples and 29 normal pons samples ( Fig.14Notably, the mean (mean ± SE) TGFB2 mRNA levels in primary DIPG samples were 1.5-fold higher than those in normal pons samples (4 ± 0.3 vs. 3.4 ± 0.1, p = 0.015) ( Fig.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 ( Fig.14 ) and the mean expression value was significantly decreased (expressed in log2 TPM) ( Fig.14 ).

[0604] Selective overexpression of TGFB2 mRNA in DIPG tumor samples was associated with enhanced expression of transcription factors that bind to multiple TGFB2 gene promoter sites.

[0605] The molecular mechanisms underlying the upregulated TGFB2 mRNA expression levels in 41 DIPG tumor samples were determined, with a focus on transcription factors / DNA-binding proteins known to enhance TGFB2 gene expression. The transcript-level 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, were examined in relation to TGFB2 mRNA levels. The mRNA levels from eight 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 ( Fig.15 SP1, FOXO3, and EP300 showed the most significant correlation with TGFB2 mRNA expression levels (p<0.0001).

[0606] Fig.14Selective upregulation of TGFB2 mRNA expression in DIPG tumor samples was shown. TGFB1 / 2 / 3 mRNA expression levels (in the form of log2-transformed TPM) of 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 expression of 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) showing overexpression (red) or underexpression (blue). (B) The bar graph shows that TGFB1 and TGFB3 mRNA expression levels were reduced, but TGFB2 mRNA expression levels were increased, in DIPG patient tumor samples (dark gray bars) compared to normal pons samples (light gray bars).

[0607] (C) Two-way ANOVA and linear contrast using FDR-adjusted p-values ​​were used to assess the statistical significance of differences in TGFB1 / 2 / 3 mRNA expression levels (expressed as log2-transformed TPM 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.

[0608] Fig.15The correlation matrix of 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 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) The mRNA levels of 8 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 had 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. In addition, the recruitment of the transcription factor coactivator EP300 (E1A binding protein P300) is also shown, which binds to the phosphorylated form of CREB1 / ATF1 / ATF2 and bridges to the basal transcription machinery through RNA polymerase II subunit A (POLR2A) to directly stimulate TGFB2 transcription.

[0609] 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-mutated GBM ( Fig.16 Significant increases in mRNA expression levels were observed for three TGFB2 probe sets: TGFB2_228121_at (2.7-fold increase, p=0.006); TGFB2_209909_s_at (2.4-fold increase, p=0.019); and TGFB2_220407_s_at (2.2-fold increase, p=0.032) ( Fig.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 showed the greatest number of positive correlations (21 significant positive correlations, p<0.05 (FDR=0.047) with the probe sets of 11 transcription factors and all four other TGFB2 probe sets ( Fig.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 ( Fig.16 , small picture).

[0610] Fig.16Figure 2 shows 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-mutated 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 under- to over-expression of TGFB1, TGFB2, and TGFB3 mRNA levels, respectively. Co-regulated probe sets are organized and depicted by 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 for 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 exhibited the greatest number of positive correlations with the probe sets for 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 correlations (red) to negative correlations (blue), organized according to similarly expressed mRNA levels. The scale of the range of correlations is shown in the color bar "Corr". Non-significant correlations are indicated by black crosses in the heat map. The mRNA levels of 8 out of 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 correlation with TGFB2 mRNA levels.

[0611] Enhanced expression of TGFB2 mRNA, but not TGFB1 or TGFB3 mRNA, was associated with shorter OS and PFS in DIPG patients.

[0612] 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 expression level of TGFB2 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). TGFB2-high DIPG patients showed significantly worse OS outcomes than TGFB2-low DIPG patients (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 )( Fig.17 The PFS results of the TGFB2 high subgroup were also significantly worse (median PFS of the TGFB2 high subgroup: 5 (95% CI: 5–NA) months, 11 events, N = 11); median PFS of 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 )( Fig.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 ( Fig.17 and Fig.18 ).

[0613] OS outcome data for the 30 H3K27M-mutant DIPG patients were evaluated separately, excluding 11 DIPG patients with unknown H3K27M mutation status. The 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 the reduced power to detect statistical differences due to the wider distribution of OS outcomes and the smaller sample size (not shown). Notably, like the full analysis set of 41 patients (not shown), the 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 the 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).

[0614] Fig.17 Figure 3: Enhanced TGFB2 expression was shown to be associated with shortened OS in DIPG patients. Archival survival outcome data of 41 DMG patients were obtained from the cBioPortal for Cancer Genomics (https: / / pedcbioportal.kidsfirstdrc.org / ), 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, and these data were combined with RNAseq-based mRNA expression data of TGFB2 (A), TGFB3 (B), and TGFB1 (C) to evaluate 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 rest of the patients (dashed line). Fig.14The levels of TGFB isoforms in the analyzed patient subgroups are shown. (A) The mean expression level of TGFB2 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) showed 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 TGFB3-low patients (median OS: 8 months, 95%CI: 7-11 months, 30 events, N=30; log-rank chi-square=5.6, p=0.018). (C) The mean TGFB1 mRNA expression level in the 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).

[0615] Fig.18Figure 1: Enhanced TGFB2 expression was shown to be associated with shortened PFS in patients with DIPG and DMG. Archived clinical outcome data of 41 DMG patients (including 11 DIPG patients with unknown H3K27M mutation status, 4 H3K27M mutant DIPG patients, and 26 H3K27M mutant DMG patients) obtained from the cBioPortal for CancerGenomics (https: / / pedcbioportal.kidsfirstdrc.org / ) whose brain tumors were located in the brainstem / pons were combined with RNAseq-based mRNA expression data of TGFB2 (A), TGFB3 (B), and TGFB1 (C) to evaluate the potential impact of various TGFB isoform levels on PFS. For patients with missing disease progression information, 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 rest of the patients (dashed line). (A) The PFS results of TGFB2-high patients were significantly worse than those of 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 results 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 results (median PFS = 8.5 months, 95% CI: 7-13 months, 30 events, N = 30; log-rank chi-square = 0.2, p = 0.7).

[0616] TGFB2 expression level did not affect OS or PFS in pediatric DMG patients whose tumors were not located in the pons / brainstem.

[0617] The survival outcomes of non-DIPG DMG patients with tumor location outside the pons / brainstem and TGFB2 mRNA expression levels 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 results 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 results (not shown). Similarly, no statistically significant differences in OS or PFS results 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 to bind to TGF-β receptor II (TGF-βRII), which in turn recruits TGF-βRI to a heterotetrameric complex, it was determined whether the observed lack of prognostic effect of TGFB2 high status could be explained by reduced expression levels of the TGFB2 receptor. Tumor samples from non-DIPG DMG patients (N=19) did not show lower expression levels 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).

[0618] TGFB2 mRNA expression level did not affect OS or PFS in pediatric GBM patients.

[0619] The survival outcomes of pediatric GBM 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 pediatric GBM patients (TGFB2 low). In the 29 pediatric GBM subgroup with TGFB2 high, the mean expression level of TGFB2 was 12.7 ± 0.1 (median, range = 12.6, 12–13.7). In contrast, the mean expression level of TGFB2 in the 87 pediatric GBM subgroup with TBFB2 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 results (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) ( Fig.19 ). Pediatric GBM patients with TGFB2-high and TGFB2-low 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 with TGFB1-high versus TGFB1-low or TGFB3-high versus TGFB3-low ( Fig.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 subgroup patients with high TGFB2, the mean expression level of TGFB2 mRNA was 6.19 ± 0.24 (median, range = 5.81, 5.23–7.63), which was lower than that of 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 hampered the accurate assessment of the prognostic effect of high TGFB2 mRNA levels. The mean age of DIPG patients (mean age at diagnosis (months) = 7.0 ± 0.4; median = 6; range = 2–14) was younger than that of GBM patients (mean age at diagnosis (months) = 60.0 ± 1.2; median = 60.4; range = 21–89.3) (p < 0.0001).

[0620] Fig.19Figure 3: TGFB2 mRNA expression levels do not affect OS in pediatric GBM patients. Archived 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 rest of the patients (dashed line). Fig.16is the level of TGFB2 isoforms in the analyzed patient subgroups. (A) The mean expression level of TGFB2 in the subgroup of 29 TGFB2-high patients was 12.67 ± 0.1 (median, range = 12.63, 11.97–13.7). In contrast, the mean expression level of TGFB2 in the subgroup of 87 TBFB2-low patients was 10.37 ± 0.1 (median, range = 10.49, 8.17–11.94). Patients in the TGFB2-high (N = 29) and TGFB2-low (N = 87) subgroups showed very similar OS results (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 expression level of TGFB3 in the subgroup of 29 TGFB3-high patients was 11.39 ± 0.1 (median, range = 11.25, 10.63–12.88). In contrast, the mean expression level of TGFB3 in the subgroup of 87 TBFB3-low patients 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 expression level of TGFB1 in the subgroup of 29 TGFB1 high patients was 12.08 ± 0.08 (median, range = 11.92, 11.58–13.13). In contrast, the mean expression level of TGFB1 in the subgroup of 87 TGFB1 low patients was 10.53 ± 0.09 (median, range = 10.71, 7.5–11.56). TGFB1 high patients (median: 12.6 (95% CI: 8.8–15.8) months, 22 events; N = 29) and TGFB1 low patients (median: 13.6 (95% CI: 11.7–15.4) months, 60 events) showed similar OS results (log-rank chi-square = 0.5, p = 0.5).

[0621] In this clinical study, the prognostic significance of TGFB2-high status was examined in newly diagnosed pediatric DIPG patients. Notably, patients with TGFB2-high (but not TGFB1-high or TGFB3-high) had significantly worse survival outcomes and significantly shorter 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 tumor location 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, pediatric GBM patients had significantly higher TGFB2 mRNA levels than 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.

[0622] This study provides important insights into the clinical significance of high TGFB2 expression in pediatric high-grade gliomas. The aggressive growth of DIPG cells promoted by TGFB2, their TGFB2-associated radiation resistance, and the possible TGFB2-mediated restriction of cellular anti-glioma immunity within the TME account for the observed adverse effects of high TGFB2 levels on survival outcomes in DIPG patients.

[0623] In a clinical study using a microarray-based gene expression platform, TGFB2 transcripts were found to be 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, but TGFB1 or TGFB3 levels were not selectively enhanced. 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 tumor samples from DIPG patients than in control pons samples, but 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 results obtained using a microarray platform. The enhanced TGFB2 gene expression in DIPG samples may be due to enhanced expression of transcription factors that upregulate TGFB2 mRNA expression. This hypothesis was supported by the finding of strong positive correlations (p<0.0001) between the 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 associated expression with specific transcription factors in tumor samples from 41 pediatric patients with DIPG (N=29) or H3K27M-mutant GBM.

[0624] There are three isoforms of TGFB in the TGFB gene superfamily, namely TGFB1, TGFB2, and TGFB3. Despite having >70% sequence homology, the structure and biological function of the TGFB isoform TGFB3 are different from those of TGFB1 and TGFB2. Notably, TGFB3 knockout mice are significantly different from TGFB1 knockout or TGFB2 knockout mice. In addition, TGFB3 plays a cancer preventive role in nonclinical models of tumor development as well as in human subjects. In addition, high TGFB3 expression has a favorable prognostic effect in breast cancer, ovarian cancer, and colon cancer. However, TGFB3 expression is associated with a poor prognosis in osteosarcoma. In this study, high TGFB3 expression was identified as a favorable prognostic indicator, which is in sharp contrast to its reported poor prognostic role in breast cancer. Our observations extend our existing knowledge and provide new insights into the multifunctional roles of TGFB3.

[0625] Based on the results presented herein, enhanced TGFB2 mRNA expression is associated with poor prognosis and OS in DIPG ( Fig. 20 ).

[0626] Fig. 20showed that TGFB2 mRNA expression was selectively enhanced and associated with poor OS in pediatric DIPG. TGFB2 mRNA levels were selectively enhanced in primary brain tumor samples from DIPG patients, while TGFB1 and TGFB3 mRNA levels were not. High levels of TGFB2 mRNA expression were associated with poor OS. In contrast, high TGFB1 mRNA expression had no prognostic value, while high TGFB3 mRNA expression was associated with good OS.

[0627] OT-101 is a TGFB2-targeting S-ODN that has demonstrated single-agent clinical activity in adult patients with recurrent or refractory glioblastoma and anaplastic astrocytoma when administered intratumorally via CED. Of the 77 high-grade glioma patients in the response population, 26 achieved a good response, including 19 patients who achieved CR or PR, and 7 patients had stable disease lasting more than 6 months. The median PFS for this subgroup was 1109 days, and its OS was 1280 days. As reported in this article, the poor prognosis observed in newly diagnosed TGFB2-high DIPG patients supports the view that it is necessary to further explore the clinical potential of TGFB2-targeted RNAi therapy in TGFB2-high DIPG patients. CED catheters have been used in DIPG patients to deliver therapeutic drugs intratumorally and 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 of implanted CED catheters in difficult anatomical locations or repeated replacement of CED catheters.

[0628] The present disclosure provides novel delivery methods and / or formulation strategies for TGFB2-targeted therapeutics that are effective for DIPG patients.

[0629] Example 7 - Single-agent activity of OT-101 in patients with relapsed / refractory high-grade glioma.

[0630] Phase 2 clinical data showed that OT-101 had significant single-agent activity in patients with relapsed / 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).

[0631] 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).

[0632] 19 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.

[0633] Fig.21 Showing radiographic responses in patients with R / R high-grade glioma who received OT-101 monotherapy and achieved CR or PR. Fig.21 : Waterfall plot depicting the maximum log10 reduction in tumor volume.

[0634] Fig. 22 Showing radiographic responses in patients with R / R high-grade glioma who received OT-101 monotherapy and achieved CR or PR. Fig. 22 : Semi-logarithmic plot of combined 3-D tumor volume reduction curves for 19 patients.

[0635] Fig.23 Swim chart 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.

[0636] OT-101 induced durable CR and PR in R / RGBM and AA patients. 19 patients had an objective response. 16 patients had a partial response at 307±159 days (mean±SE). The median time to PR onset was 287 days (range: 37-742). Three patients first achieved PR and deepened to CR at 917, 1120, and 1838 days, respectively. Six of these 16 patients had PD at 970±126.7 days (mean±SE) (median=1032 days, range: 374–1281 days).

[0637] Example 8 - Clinical potential of OT-101 targeting transforming growth factor β2 for post-radiation consolidation of diffuse intrinsic pontine gliomas.

[0638] 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%. Chemotherapy does not provide clinically meaningful benefits. Therefore, innovative therapeutic agents for the treatment of DIPG in children are urgently needed.

[0639] 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.

[0640] 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 cell lytic function of patient-derived T cells.

[0641] Intrathecal / intraventricular administration of antineoplastic drugs directly in the CSF allows bypassing the selectivity filter of the blood-brain barrier, achieving significant antineoplastic agent concentrations in the CSF while reducing the potential for systemic toxicity. Informed by the favorable safety pharmacology studies of intrathecal delivery of OT-101 in rabbits and primates, and encouraged by its single-agent activity in adult HGG patients, a multicenter, two-part, randomized, Phase 1-2 study of OT-101 in pediatric patients with DIPG was conducted. After completion of radiation therapy, multiple doses of OT-101 were administered by intrathecal / intraventricular 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.

[0642] Antisense oligodeoxynucleotides are short strands of DNA that are designed to downregulate gene expression by interfering with the translation of specific encoded proteins at the mRNA level. Several RNA therapeutics, including antisense oligonucleotides, have been evaluated in clinical trials, and 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 the first RNA therapeutic in a class and is 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.

[0643] Functional in vitro assays showed that:

[0644] In the presence or absence of carrier liposomes In all cases, OT-101 was shown to be effectively and time-dependently taken up by human tumor cells.

[0645] OT-101 can reduce TGF-β2 secretion in human tumor cells without the use of any carrier.

[0646] 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.

[0647] 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.

[0648] OT-101 restored the immune function of human PBMCs derived from patients with high-grade gliomas, as demonstrated by immune cell-mediated cytotoxicity assays.

[0649] OT-101 inhibits human tumor cell migration.

[0650] A series of nonclinical safety and pharmacology studies have been conducted on OT-101. The salient features of the conclusions of these studies are as follows:

[0651] Long-term topical administration of OT-101 / AP12009 may result in local tissue inflammation. Mild to moderate local toxicity was observed in animals following infusion of a 500 μM concentration, but without any macroscopic changes.

[0652] 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 4.16 μM), OT-101 did not cause any clinical toxicity or drug-related macroscopic / microscopic changes, consistent with subclinical toxicity.

[0653] 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.

[0654] When administered to rats via intracerebroventricular administration, radiolabeled OT-101 (0.18 mg / kg) was detected not only in CSF but also in the cerebrum, cerebellum, and pineal gland within 1 hour after administration. The half-life in CSF and brain tissue was <24 hours, with <15% residual OT-101 remaining at 72 hours.

[0655] Example 9 - Non-clinical in vitro studies of OT-101 / AP12009.

[0656] Functional in vitro assays showed that:

[0657] In the presence and absence of carrier liposomes In all cases, OT-101 was shown to be effectively and time-dependently taken up by human tumor cells.

[0658] OT-101 can reduce TGF-β2 secretion in human tumor cells without the use of any carrier.

[0659] 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.

[0660] 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.

[0661] OT-101 restored the immune function of human PBMCs derived from patients with high-grade gliomas, as demonstrated by immune cell-mediated cytotoxicity assays.

[0662] OT-101 inhibits human tumor cell migration.

[0663] In existence or non-existence In the absence or presence of In the case of 5% paraformaldehyde, the uptake of FITC-OT-101 was observed after 3 h of incubation. After 48 h, 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

[0664] Example 10 - Effects of OT-101 on TGF-β2 synthesis and secretion in human GBM cell lines.

[0665] Cells were incubated with different concentrations of OT-101 / AP12009 (1 μM to 80 μM) as indicated for 7 days. Secreted TGF-β2 in cell supernatants was measured by ELISA. Results represent the median, minimum and maximum values ​​of 3 independent experiments.

[0666] 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%.

[0667] OT-101-mediated inhibition of proliferation of human high-grade glioma cells.

[0668] 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.

[0669] Table 20: Effects of OT-101 on proliferation of human high-grade glioma cells

[0670]

[0671]

[0672] Two human glioma cell cultures (HTZ-243 and HTZ-349) were treated with OT-101 (1, 5 or 10 μM). Cell numbers were measured using a hemacytometer (% of the number of cells at the start of the experiment). Data show the mean of duplicate assessments.

[0673] Example 11 - Administration of OT-101 (AP12009).

[0674] This study evaluated two doses of AP12009 in parallel treatment groups. AP12009 was administered intratumorally at a concentration of 10 μM or 80 μM using continuous convection-enhanced delivery at a flow rate of 4 μL / min over a 7-day cycle every two weeks. The treatment regimen of AP12009 was based on clinical Phase I / II data from three studies:

[0675] Concentration: AP12009 was shown to be safe up to the maximum evaluated concentration of 80 μM. Flow rate: Some results from study G003 suggest that the infusion volume may constitute an important safety aspect, especially for patients with larger tumor sizes, who are at high risk of developing cerebral edema quite rapidly, as their brain's ability to compensate for increased intracranial pressure may be exhausted. Although administration at a flow rate of 8 μL / min over four days was assessed as safe, AEs with higher severity that occurred more frequently at a flow rate of 8 μL / min over 7 days cannot be excluded. Weighing safety and efficacy considerations, it was decided to maintain the infusion cycle at 7 days, but the flow rate was reduced to 4 μl / min (total volume applied per treatment cycle: 40.32 mL). Thus, a longer drug exposure time can be achieved, but the infusion volume is reduced to half compared to an infusion rate of 8 μL / min over four days. The flow rate of isotonic saline infusion during the 7-day interval was set to 1 μL / min to reduce the risk of symptoms of increased intracranial pressure.

[0676] Due to the large deadspace volume of the application system, the administration of the relevant concentration of AP12009 to the brain was delayed at the beginning of each treatment cycle. It was determined that 95% of the nominal concentration of AP12009 was reached after 10 hours. This delay in the start of each AP12009 infusion had to be compensated to allow the administration of the nominal dose of AP12009 to be completed within the prescribed infusion period. Therefore, after switching to isotonic saline infusion, at the beginning of the interval period, the flow rate was 4 μL / min for the same 10 hours to flush the remaining AP12009 out of the deadspace. The flow rate was then reduced to 1 μL / min.

[0677] Example 12 - Effects of OT-101 and chemotherapy on glioma.

[0678] 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).

[0679] Descriptive statistics were performed for the groups in the G004 and G005 studies. Among the 89 subjects in the test group of the G004 study, the mean overall survival (OS) was 507.5 days; the median OS was 364 days, with a standard deviation of 411.5 days. In the G004 control group, 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. Among the 12 subjects in the G005 test group, the mean OS was 507.8 days; the median OS was 368.4 days, with a standard deviation of 461.1 days. In the G005 control group, 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.

[0680] Table 21: Descriptive statistics of overall survival for patients receiving OT-101 or standard chemotherapy in studies G004 and G005.

[0681]

[0682] For the non-inferiority study, a two-sample non-inferiority test was performed on the survival data using Cox regression. Specifically, the Wald test or 100 (1-2α)% confidence interval test for non-inferiority was used to see whether the 90.0% upper confidence limit (CL) of the hazard ratio (HR) was within the non-inferiority hypothesis. The α level was set to 0.050, and the non-inferiority margin of the hazard ratio (hazard ratio [HR] = risk [treatment group] / risk [reference group]) was 1.25. If it is greater than the margin of the non-inferiority hypothesis, the higher the risk, the worse it is. H1: HR < non-inferiority margin, then the EfronTies method is used.

[0683] For the alternative hypothesis of HR < 1.25, the calculated hazard ratio was 0.9168, the 90.0% confidence limits of the hazard ratio were 0.6865-1.2245, and the p-value was 0.0390. The Wald-Z value was determined to be -1.7621. In Cox regression, the hazard ratio (HR) is often referred to as 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 (RiskRatio or HazardRatio) was calculated as Exp(B) = 0.9168 with a mean of 0.6516.

[0684] 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.

[0685] In the control group, subjects receiving TMZ were the most common, with 43 subjects (36 subjects in the G004 study and 7 subjects in the G005 study). The same two-sample noninferiority test was performed on the survival data using Cox regression for subjects receiving OT-101 or TMZ. Using the alternative hypothesis of noninferiority as a hazard ratio less than 1.25, the hazard ratio was determined to be 0.7156 with a 90% confidence level between 0.5228 and 0.9794 and a p-value of 0.0017. The Wald's Z value was determined to be -2.9231, and based on the hazard ratio obtained, it was concluded that OT-101 was noninferior to TMZ.

[0686] 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.

[0687] Fig.24 Overlaid Kaplan-Meier survival curves for both OT-101 and standard chemotherapy are shown. The survival curves for both drugs are well within each other's confidence intervals, confirming the non-inferiority of OT-101 compared with standard chemotherapy in terms of overall survival.

[0688] Fig.25 Patients who had not received chemotherapy were shown to have non-inferiority to OT-101 after the addition of TMZ.

[0689] Fig.26 Chemotherapy with the chemotherapeutic agents CCNU / BCNU was shown to be ineffective, which confirmed the non-inferiority of OT-101 after the addition of CCNU / BCNU.

[0690] Example 13 - TGF-β2 is a valid target for glioma therapy.

[0691] Three TGFB2 probe sets showed increased expression levels in DIPG patients ( Fig. 27 The average fold difference of probe group 228121_at was 2.48 (linear contrast P value = 3.40x10 -4); the mean fold difference of probe set 220407_s_at was 2.00 (linear contrast P value = 0.006); the mean fold difference of probe set 209909_s_at was 1.81 (linear contrast P value = 0.0185). One of the TGFB2 probe sets also showed the highest level of average expression in DIPG patients; TGFB2_228121_at (mean = 9.25 ± 0.18 log2RMA); YAP1_224894_at (mean = 8.55 ± 0.19 log2RMA).

[0692] The results are summarized in Table 22.

[0693] Table 22: TGF-β2 is a valid target for glioma therapy

[0694]

[0695] The expression of the TGFB2 interactome represents the probe sets in pediatric DIPG patients compared to normal samples. The log2-transformed fold difference values ​​for each subject (column (N=29)) and each probe set from the GEO archived dataset GSE26576 are depicted as the DIPG patient average centered on 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 co-regulation across all probe sets between patients and across all patients between probe sets. The heat map depicts the most significantly up- and down-regulated 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-values ​​are depicted in the table, showing the 3 probe sets upregulated by TGFB2 in DIPG patients.

[0696] Example 14 - TGF-β2 is a valid target for pediatric GBM therapy.

[0697] Similar results were obtained for pediatric GBM patients ( Fig.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 = 1.78x10 -5 ); 228121_at (fold change = 3.57, P = 8.81x10 -5 ); and 220407_s_at (fold change = 2.73, P = 0.0019). The three probe sets with the highest expression levels in pediatric GBM were: TGFB2_228121_at (mean = 9.24 ± 0.15); TGFB3_209747_at (mean = 6.99 ± 0.086); and TGFB1_203085_s_at (mean = 6.80 ± 0.16).

[0698] The results are summarized in Table 23.

[0699] Table 23: TGF-β2 is a valid target for pediatric GBM therapy

[0700]

[0701] Comparison of the expression of the TGFB1 / 2 / 3 probe set in pediatric GBM patients with AO patients (control group). The log2-transformed fold difference values ​​for each subject (column (N = 82)) and each of the 9 probe sets from 4 GEO archived datasets (GSE19578 (N = 25); GSE32374 (N = 15); GSE34824 (N = 27); and GSE49822 (N = 15)) depict the pediatric GBM patient mean centered on the control sample (N = 5; from GSE19578). Subjects and probe sets were organized using a 2-way clustering algorithm that used an average distance metric to determine co-regulation across all probe sets between patients and across all patients between probe sets. The heat map depicts the expression changes for each probe set, 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 3 probe sets were significantly upregulated more than 2-fold in pediatric GBM patients (probe set: 209909_s_at (fold change = 4.03, P = 1.78x10 -5 ); 228121_at (fold change = 3.57, P = 8.81x10 -5 ); and 220407_s_at (fold change = 2.73, P = 0.0019).

[0702] Example 15 - TGF-β2 is a selective biomarker that predicts improved outcomes of cancer radiation therapy.

[0703] It has been discovered herein that TGF-β2 can be used as a biomarker to selectively predict improved outcomes of cancer radiation therapy.

[0704] A clinical study using the CTGA database showed the effect of TGF-β2 expression on overall survival and survival after radiotherapy. Surprisingly, a significant survival advantage was observed for low TGF-β2 expressers compared with high TGF-β2 expressers in all four quartiles of expression. No such difference was observed for TGF-β1 and TGF-β3.

[0705] studied overall survival in a pediatric brainstem subgroup and studied radiation therapy survival in all patients with glioma who received radiation therapy. Again, only TGF-β2 predicted survival. TGF-β1 and TGF-β3 did not predict survival.

[0706] Fig.29 Results showed that low TGF-β2 expression conferred an overall survival advantage for gliomas in patients treated with radiotherapy. Only TGF-β2 predicted survival. The quartiles of TGF-β2 expression were represented as A, B, C, and D.

[0707] Fig.30 showed that for glioma in patients treated with radiation therapy, low TGF-β2 expression conferred an overall survival advantage. Only TGF-β2 predicted survival.

[0708] Example 16 - Reduction of TGF-β2 levels selectively predicts improved overall survival (OS) when combined with chemotherapy (TMZ), chemotherapy (TMZ) and radiotherapy, or anti-angiogenic therapy (bevacizumab).

[0709] It has been found herein 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). Such predictive results were not observed for TGF-β1 and TGF-β3.

[0710] 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.

[0711] Fig.31 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy (TMZ).

[0712] Fig.32 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with chemotherapy TMZ and radiation.

[0713] Fig.33 TGF-β2 levels were shown to selectively predict improved overall survival (OS) when combined with antiangiogenic therapy (bevacizumab).

Claims

1. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: 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], 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 inline with the infusion line [105]; and An access catheter [113] is in fluid communication with the Omayr reservoir, wherein the access catheter is linear and enters a target area of ​​the brain intraventricularly.

2. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: 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], 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 inline with the infusion line [105]; and An access catheter [113] is in fluid communication with the Omayr reservoir, wherein the access catheter [113] is non-linear and has one or more bends to access a target region of the brain within the cerebral ventricle.

3. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: a reservoir for containing a pharmaceutical composition[117]; a pump [101] for forcing a 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]; 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 linear and enters a target area of ​​the brain intraventricularly.

4. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: a reservoir for containing a pharmaceutical composition[117]; a pump [101] for forcing a 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]; 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 [113] is non-linear and has one or more bends to access a target region of the brain within the cerebral ventricle.

5. The device according to any one of claims 1-4, wherein the Omayyad reservoir [111] comprises a partially flexible top.

6. The device of any one of claims 1-4, wherein the device provides continuous infusion of a therapeutically effective amount of a fluid drug composition to the target area.

7. The device of any one of claims 1-4, wherein the distal end of the access catheter enters a target area of ​​the brain.

8. The device according to any one of claims 1-4, wherein the Omayr reservoir [111] retains the drug composition behind a membrane for a period of time to provide a sustained release of the drug composition to the access catheter.

9. The device of any one of claims 1-4, wherein the distal tip of the access catheter entering the brain is a stepped end, a concave stepped end, a multi-port end, a micro-bore end, or a balloon tip.

10. The device according to any one of claims 1-4, wherein the pump [101] is a Pegasus Vario, PEGAPCA, CADD Solis VIP, CADD-Legacy PLUS, CADD-Legacy PCA or CADD-Legacy 1.

11. The device according to any one of claims 1-4, 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.

12. The device according to any one of claims 1-4, wherein the infusion tube [103] 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).

13. The device according to any one of claims 3-4, wherein 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).

14. The device according to any one of claims 1-4, 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 an animal.

15. The device of any one of claims 1-4, wherein the fluid pharmaceutical composition comprises microparticles or nanoparticles of a pharmaceutical agent, drug, or delivery vehicle.

16. The device of any one of claims 1-4, wherein the fluid drug composition is a therapeutic agent for a CNS disease or CNS cancer.

17. The device of any one of claims 1-4, wherein the fluid drug 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.

18. The device according to any one of claims 1 to 4, 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: SEQ ID NOs: 1-136 and chemically modified variants thereof, LNA variants thereof, gapmer variants thereof, and any combination thereof are as follows:

19. The device of any one of claims 1-4, wherein the device operates in combination with radiation therapy or electric field therapy.

20. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: 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; and An access catheter [413] is in fluid communication with the reservoir, wherein the access catheter is linear and is directed intraventricularly into a target region of the brain.

21. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: 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; and A port [509] is in fluid communication with the reservoir for connection to an access catheter.

22. A device for delivering a fluid pharmaceutical composition by continuous intracranial infusion, characterized in that: The device comprises: 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.

23. The device of any one of claims 1-4 and 20-22, wherein the device comprises a collar comprising: A hard shell [604] having an opening [606] exposing the flexible top of the device.

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