Chimeric antigen receptor (CAR) T cell adjuvant therapy

Abstract

Methods, compositions, and kits that can be used to treat cytokine release syndrome, ICANS, or both, associated with CAR T cell administration are described herein. For example, a pharmaceutical composition comprising beraprost, a beraprost isomer, or a salt thereof can be used as an adjuvant therapy for chimeric antigen receptor T cells (CAR T cells) for treating cancer while reducing or eliminating undesirable potentially dangerous side effects.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 347,221, filed on May 31, 2022, and titled "Chimeric Antigen Receptor (CAR) T Cell Adjuvant Therapy," and U.S. Provisional Application No. 63 / 482,448, filed on January 31, 2023, and titled "Chimeric Antigen Receptor (CAR) T Cell Adjuvant Therapy," the contents of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Chimeric antigen receptor T cells (CAR T cells) are genetically engineered T cells that produce receptors capable of binding to specific proteins. CAR T cell therapy is attractive for use in immunotherapy because it can be designed and engineered to specifically target and recognize cancer cells.

[0003] T cells are collected, genetically engineered before being infused into a patient, and attack cancer cells. T cells can be collected from a patient's blood ("autologous") or from another donor ("allogeneic"). Chimeric antigen receptors can target antigens present on the surface of cancer cells in a tumor and / or antigens present on non-cancer cells, such as B cells.

[0004] One of the common but serious side effects of CAR T cell therapy is cytokine release syndrome ("CRS"). CAR-T cells cause activation of immune cells (T cells, macrophages, dendritic cells). CAR T cells and related cells release large amounts of cytokines into the bloodstream. The released cytokines can cause hyperactivation of cytokine signaling, which may lead to immune hyperactivity. As a result, it can cause high fever, low blood pressure (hypotension), fatigue, headache, muscle pain, nausea, capillary leakage, tachycardia, and multiple organ failure, such as liver failure and kidney damage, and even death. CRS is seen in 50-100% of patients. Discussions of side effects are described in the NCCN Patient Guide "Immunotherapy Side Effects": CAR T Cell Therapy - English (2020) and the NCCN Guidelines Version 4.2021 "Management of CAR T Cell-Related Toxicities" (2021). Immune effector cell-associated neurotoxicity syndrome (ICANS) effects can occur in more than 25% of patients who receive CAR T cell therapy. ICANS presents as headache, confusion, speech disorder, altered consciousness, seizures, and cerebral edema. Almost all patients who develop ICANS may have previously had CRS.

[0005] For example, in Novartis' Kymriah product (Kymriah is a registered trademark of Novartis AG; Basel, Switzerland), CRS was observed in 79% of patients with acute lymphoblastic leukemia and 74% of patients with relapsed / refractory diffuse large B-cell lymphoma, among which grades 3 and above were observed in 49% of patients with relapsed / refractory acute lymphoblastic leukemia and 23% of patients with relapsed / refractory diffuse large B-cell lymphoma. The median time to onset was 3 days, and the median time to resolution was 8 days (range: 1 - 36 days). Similarly, in Kite / Gilead's Yescarta product (Yescarta is a registered trademark of Kite Pharma, Inc.; Santa Monica, California, USA), CRS developed in more than 90% of patients, and 9% were grade 3 or above. Among the patients who died after receiving Yescarta, 4 patients had CRS continuing at the time of death. The median time to onset was 2 days (range: 1 - 12 days), and the median duration was 7 days (range: 2 - 58 days).

[0006] From a certain perspective, the occurrence of CRS is an indicator or diagnostic marker that CAR T cells are functioning to attack cancer cells. CRS develops late after the initiation of CAR T cell administration. This delay time is very predictable and consistent, but the length of the delay cannot be predicted. Thus, it is difficult to predict when a patient will develop CRS.

[0007] In the case of CRS or ICANS, currently approved treatments include supportive care, corticosteroids, and administration of tocilizumab, an IL-6 antagonist. Corticosteroids are effective for both CRS and ICANS, but can cause very serious side effects such as severe hypertension, impaired glucose tolerance, susceptibility to severe infections, delayed wound healing, gastrointestinal bleeding, sepsis, and heart failure. There is also concern that corticosteroids may impair the function of infused CAR T cells. For this reason, the use of corticosteroids is often limited to the most severe cases of CRS or ICANS. Tocilizumab has been approved by the FDA as a treatment for CRS, but it is not clear whether blocking IL-6 impairs the proliferation of CAR T cells in vivo, as it has been clearly demonstrated that IL-6 promotes the proliferation of T cells. Furthermore, serum IL-6 concentrations may actually increase after administration of tocilizumab, and such an increase in circulating cytokines may increase the risk of vascular leakage and BBB disruption, which are thought to contribute to the development of ICANS. Therefore, tocilizumab is generally not recommended for the treatment of ICANS, and there are opinions that its use may worsen the condition. Therefore, although useful in some populations, the mechanisms of action of drugs for dealing with CRS and ICANS in this population may be limited. New treatment strategies are needed to prevent or reduce the use of tocilizumab and / or steroids.

[0008] CAR T cell therapy is a powerful and effective "last resort", but new and improved therapies are needed to reduce or eliminate the unwanted and potentially fatal side effect of CRS.

[0009] Disclosed herein are methods and compositions for administering CAR T cell therapy while reducing or eliminating the side effects of cytokine release syndrome (CRS) and / or the effects of immune effector cell-associated neurotoxicity syndrome (ICANS). CAR T cell therapy can be used to treat cancer or other diseases.

[0010] Methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration generally involve administering, as an adjuvant / combination therapy approach, CAR T cells and a first pharmaceutical composition to a subject. The first pharmaceutical composition can include an analog selected from the group consisting of an effective amount of a prostacyclin / prostaglandin analog, such as carbaprostacyclin, beraprost, taprostene, niloprost, iloprost, cicaprost, ciprostene, treprostinil, bosentan, vapiprost, eptaloprost, or an isomer thereof, and pharmaceutically acceptable salts thereof. In some embodiments, the prostacyclin / prostaglandin analog is beraprost or a beraprost salt. The salt can be a pharmaceutically acceptable salt of beraprost, such as beraprost sodium. Beraprost can be an isomer of beraprost, such as beraprost CTO1681. The first pharmaceutical composition causes a negligible to no decrease in CAR T cell-mediated killing; for example, the decrease in CAR T cell-mediated killing is about 5% or less.

[0011] Another method of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration to a subject can involve administering CAR T cells, a first pharmaceutical composition, and a second pharmaceutical composition to the subject, where the first pharmaceutical composition includes an effective amount of at least beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the second pharmaceutical composition includes at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof.

[0012] Additional methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration may include administering to a subject CAR T cells and a first pharmaceutical composition, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the subject experiences a reduction in ICANS effects, parkinsonism effects, or both, as compared to a similar subject who receives the administered CAR T cells but does not receive the administered pharmaceutical composition.

[0013] Further methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering to the subject CAR T cells and a first pharmaceutical composition, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the subject experiences a decrease in severity measurements as compared to a similar subject who is administered the administered CAR T cells but not the administered pharmaceutical composition. In some embodiments, the subject experiences a reduction in the severity of CRS and / or ICANS effects.

[0014] Additional methods for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering CAR T cells and a first pharmaceutical composition to the subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition is administered at a time when the onset of CRS is detected by an increase in the level of one or more of the cytokines MIF, IL-5, IL-17A, IL-23, IFN-γ, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β. Alternatively or additionally, the onset of CRS can be detected by an increase in the level of one or more of the cytokines CCL2, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, CXCL9, CXCL-10, VEGF, CCL3, GCSF, and GMCSF. Alternatively, the onset of CRS can be detected by detecting an increase in the level of one or more inflammatory markers associated with CRS, such as CRP (C-reactive protein) or ferritin. Alternatively, the onset of CRS can also be detected by the manifestation of symptoms such as one or more of fever, hypotension, and hypoxemia.

[0015] Additional methods for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering CAR T cells and a first pharmaceutical composition to the subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, the subject experiences CRS, ICANS, parkinsonism effects, or a combination thereof, and the subject needs to reduce treatment with at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof as compared to a similar subject who receives the administered CAR T cells but does not receive the administered first pharmaceutical composition.

[0016] A kit for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject generally includes a first container containing a first pharmaceutical composition comprising at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, a second container containing a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof, a third container containing CAR T cells, and instructions regarding administration of the first pharmaceutical composition, the second pharmaceutical composition, and the CAR T cells to the subject.

[0017] An additional kit may include a first container containing a first pharmaceutical composition comprising at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, a second container containing a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof, and instructions for administering the first pharmaceutical composition, the second pharmaceutical composition, and the CAR T cells to a subject.

Brief Description of the Drawings

[0018] The foregoing and other objects, features, and advantages will become apparent from the following description of particular embodiments of the disclosure shown in the accompanying drawings. The drawings are not necessarily to scale, and instead, emphasis is placed on illustrating the principles of the various embodiments of the disclosure.

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DETAILED DESCRIPTION OF THE INVENTION

[0019] As used herein, the term "about" immediately preceding a numerical value means a range of plus or minus 10% of that numerical value. For example, "about 50" means from 45 to 55, and "about 25,000" means from 22,500 to 27,500, etc., unless the context of the present disclosure indicates otherwise or such an interpretation is inconsistent.

[0020] As used herein, the term "analog" refers to a compound that produces the biological activity of a receptor, the presence of which is the same as the biological activity that occurs when a ligand that occurs naturally for the receptor is present.

[0021] As used herein, the terms "administer", "administering", or "administration" refer to the direct administration of a compound or composition to a subject.

[0022] As used herein, the term "effective amount" refers to an amount that results in a measurable inhibition of at least one symptom or parameter of a particular disorder or pathological process. As used herein, the term "therapeutically effective amount" of the compositions of the present application is an amount that provides a therapeutic effect to a subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows or feels an effect, or the physician observes a change).

[0023] As used herein, the term "immediate release" refers to a pharmaceutical composition that releases the active ingredient in a short time.

[0024] As used herein, the term "modified release" refers to a pharmaceutical composition that does not release the active ingredient immediately, for example, releases the active ingredient at a sustained or controlled rate over a long period of time, releases the active ingredient after a lag time after administration, or can optionally be used in combination with an immediate release composition. Modified release includes sustained release, extended release, and delayed release. As used herein, the terms "sustained release" or "extended release" are dosage forms that make the drug available over a long period of time after administration. As used herein, the term "delayed release" is a dosage form that releases the drug at a time other than immediately after administration.

[0025] As used herein, the term "pharmaceutically acceptable salt" includes salts of the compounds of the present application that are safe and effective for use in mammals and have the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in the compounds of the present application or the compounds identified according to the methods of the present application. Pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucarate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds of the present application may form pharmaceutically acceptable salts with various amino acids. Suitable basic salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron, and diethanolamine salts. Pharmaceutically acceptable base addition salts are also formed with amines such as organic amines. Examples of suitable amines include Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, procaine, and the like.

[0026] The term "prevent" is considered to mean preventing a particular disorder, disease, or condition and / or preventing the recurrence of a particular disorder, disease, or condition.

[0027] The term "substantially pure isomer" refers to a formulation or composition in which a single isomer is present at about 70% or more, or about 80% or more, or about 90% or more, or about 95% or more, or about 98% or more, or about 99% or more of the various isomers of a compound, or a formulation or composition in which the compound or composition consists of only a single isomer of the compound.

[0028] As used herein, the terms "treat," "treated," or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to (partially or wholly) protect from, or delay, an undesirable physiological condition, disorder, or disease (e.g., reducing or delaying onset), or to obtain a beneficial or desired clinical result such as partial or total restoration or suppression of abnormal or aberrant parameters, values, functions, or outcomes. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the degree, severity, or rate of onset of a condition, disorder, or disease, stabilization of the state of a condition, disorder, or disease (i.e., not getting worse), delay in the onset or progression of a condition, disorder, or disease, improvement in the state of a condition, disorder, or disease, and remission (whether partial or total), whether or not it leads to an immediate reduction in actual clinical symptoms, or enhancement or improvement of a condition, disorder, or disease. Treatment is aimed at eliciting a clinically significant response without undue side effects.

[0029] "Unit dosage form" refers to physically discrete units suitable as unitary quantities for administration to humans and other animals, each unit containing a predetermined quantity of an active substance calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.

[0030] "Weight %" as disclosed herein may be weight to weight % or weight to volume % depending on the composition.

[0031] As used herein, the term "disease" is generally synonymous with, and intended to be used interchangeably with, the terms "disorder," "syndrome," and "condition" (in the medical context). That is, they all reflect an abnormal condition of the body or part thereof of a human or animal that impairs normal function and is typically manifested by characteristic signs and symptoms, and that reduces the length or quality of life of a human or animal.

[0032] The term "combination therapy" means administering two or more therapeutic agents to treat a medical condition or disorder. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a certain ratio of active ingredients, or in a dosage form, or in multiple separate capsules for each active ingredient. Further, such administration also includes sequentially using each type of therapeutic agent in the same patient with the delivery of the individual therapeutic agents separated by about 1 to 24 hours, about 1 to 7 days, or about one week or more. In any case, the treatment regimen provides a beneficial effect of the combination of agents in the treatment of the condition or disorder described herein.

[0033] The present disclosure is not limited in terms of the specific embodiments described in this application, which are intended as examples of various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims, together with the full scope of equivalents to which such claims are entitled. It should be understood that the present disclosure is not limited to a particular method, reagent, compound, composition, or biological system. Also, it should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0034] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that any of the content of this disclosure is prior art to any invention described herein. As used herein, the term "comprising" means "including but not limited to".

[0035] Various compositions, methods, and apparatuses are described in terms of the term "comprising" various components or steps (interpreted to mean "including but not limited to"), but the compositions, methods, and apparatuses can also "consist essentially of" or "consist of" the various components and steps, and such terms should be interpreted as defining an essentially closed group of members.

[0036] With respect to the use of substantially any plural and / or singular terms herein, one of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be explicitly set forth herein for clarity.

[0037] Generally, it will be understood by those skilled in the art that the terms used in this specification, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be open terms (e.g., the term "comprising" should be interpreted as "comprising but not limited to", the term "having" should be interpreted as "having at least", the term "including" should be interpreted as "including but not limited to", etc.). Those skilled in the art will further understand that where a specific number of introductions of a claim is intended, such intent is explicitly recited in the claim, and where there is no such recitation, such intent does not exist. For example, for the sake of understanding, in the following appended claims, introductory phrases such as "at least one" and "one or more" may be used to introduce the recitation of the claims. However, the use of such phrases should not be construed to mean that the repeated introduction of a claim by the indefinite article "a" or "an" limits a particular claim that includes such repeated introduction of the claim to an embodiment that includes only one such repetition, even if the same claim includes an introductory phrase such as "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); the same is true for the use of the definite article at the beginning of the claim. Further, even where a specific number of repetitions of an introduced claim is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a bare recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations).Furthermore, when conventions similar to “at least one of A, B, and C, etc.” are used, generally, such a configuration is intended in the sense that those skilled in the art understand the convention (e.g., “a system having at least one of A, B, and C” includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When conventions similar to “at least one of A, B, or C, etc.” are used, generally, such a configuration is intended in the sense that those skilled in the art understand the convention (e.g., “a system having at least one of A, B, or C” includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It should also be understood by those skilled in the art that substantially any conjunctive word and / or phrase presenting two or more alternative terms in any of the specification, claims, or drawings is intended to contemplate the possibility of including one of the terms, any of the terms, or both terms. For example, the phrase “A or B” is understood to include the possibilities of “A” or “B,” or “A and B.”

[0038] In addition, when a feature or aspect of the present disclosure is described from the perspective of a Markush group, those skilled in the art will recognize that the present disclosure is thereby also described from the perspective of any individual member or subgroup of members of the Markush group.

[0039] As will be understood by those skilled in the art, all ranges disclosed herein include all possible sub-ranges and combinations thereof for any purpose, such as for the purpose of providing a written description. Any recited range can be readily recognized as being fully described and enabled to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range. By way of non-limiting example, each range discussed herein can be readily decomposed into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," etc. includes the recited number and refers to a range that can then be decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, a range includes individual members. Thus, for example, a group having from about 1-3 cells refers to a group having about 1, 2, or 3 cells. Similarly, a group having from about 1-5 cells refers to a group having about 1, 2, 3, 4, or 5 cells, etc.

[0040] The various features and functions described above, or alternatives thereof, may be combined in many other different systems or applications. Various alternatives, modifications, variations, or improvements not presently foreseen or expected may subsequently be made by those skilled in the art, but each of these is also intended to be encompassed by the disclosed embodiments.

[0041] The present disclosure is not limited to the specific systems, devices, and methods described, as these may vary. The terms used herein are for the purpose of describing particular versions or embodiments only and are not intended to limit the scope.

[0042] Cytokine release syndrome ("CRS") and / or ICANS are common but serious side effects of CAR T cell therapy. CAR T cells can cause the release of large amounts of inflammatory cytokines into the bloodstream of subjects undergoing CAR-T cell therapy. This can cause high fever, low blood pressure (hypotension), fatigue, headache, muscle pain, nausea, capillary leakage, tachycardia, and potentially liver and kidney failure. CRS occurs in approximately 50-100% of patients. To enhance the therapeutic value of CAR T cell therapy, compositions and methods for overcoming CRS are needed. Various methods, compositions, and kits are described herein for performing CAR T cell therapy with reduced or eliminated CRS effects, immune effector cell-associated neurotoxicity syndrome (ICANS) effects, parkinsonism effects, or combinations thereof in a subject. The methods can include administering to the subject a CAR T cell and at least one pharmaceutical composition such as a first pharmaceutical composition and / or a second pharmaceutical composition. The T cells and T cell therapy can be autologous or allogeneic.

[0043] In the present disclosure, the inventors have demonstrated that a first pharmaceutical composition comprising beraprost, an isomer of beraprost, or a pharmaceutically acceptable salt of beraprost can mitigate the cytokine response associated with CRS. In particular, the inventors observed a decrease in inflammatory cytokines such as TNF-α and INF-γ. Importantly, the inventors have found that beraprost, an isomer of beraprost, or a pharmaceutically acceptable salt of beraprost does not inhibit the killing of CAR T cells in both in vitro and in vivo settings.

[0044] In some embodiments, administration of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof alleviates the over-release of inflammatory cytokines, thereby reducing or eliminating damage due to CRS, and can reduce or eliminate the subsequent immune effector cell-associated neurotoxicity syndrome (ICANS) effect or parkinsonism effect. ICANS usually occurs after CRS, but may also occur simultaneously. When it occurs after CRS, it may occur several days later (e.g., about 1 day later, about 2 days later, about 3 days later, about 4 days later, about 5 days later or more), and sometimes weeks later. Even among subjects with the same genetic background, there is some variation among subjects.

[0045] Current standard of care (SOC) for CRS includes treatment with tocilizumab, treatment with corticosteroids, and supportive therapy based on observed CRS symptoms. The standard of care (SOC) for ICANS does not include the use of tocilizumab, but more intensive corticosteroid treatment and supportive therapy based on observed ICANS symptoms are performed. Tocilizumab is used to block the IL-6 receptor, but more IL-6 circulates. The use of corticosteroids has significant limitations due to immunosuppression and other adverse downstream effects. Corticosteroids can be administered prophylactically, but this is not typical or common. Patients receiving corticosteroids usually have to be hospitalized for monitoring, which lengthens the hospital stay and increases associated costs.

[0046] Due to the side effects of CAR T cell therapy, the hospital stay is usually an average of 7 to 14 days. In particular, patients with symptoms of CRS or ICANS require long-term hospitalization. Since the immunosuppressed patient population has a high risk of infection, the use of outpatient treatment is not recommended.

[0047] The use of a first pharmaceutical composition provided orally, such as one containing veraprost, an isomer thereof or a salt, will enable more patients to access CAR T cell and bispecific antibody therapies for life-saving.

[0048] The onset of CRS can be detected in multiple ways. For example, the onset can be detected by measuring an increase in one or more cytokines and / or one or more inflammatory markers. Alternatively, the onset of CRS can also be measured by the manifestation of clinical symptoms. Common symptoms include fever, hypotension, hypoxia, etc.

[0049] The methods, compositions, and kits can be used in therapies that reduce or eliminate the effects of immune effector cell-associated neurotoxicity syndrome (ICANS), parkinsonism, or both. The methods, compositions, and kits can also be used in therapies in which severity measurement is reduced or eliminated. The methods, compositions, and kits can also be used in treatments that reduce or eliminate the need for corticosteroid treatment, IL-6 receptor blocker therapeutics, and other biological therapies. The veraprost compound CTO1681 may replace tocilizumab as an option for early treatment.

[0050] CAR T cell therapy CAR T cell therapy reprograms T cells to recognize antigens expressed on the surface of target cells, such as tumor cells, by injecting T cells genetically engineered to express a CAR. The specificity of the CAR is derived from the extracellular domain of a genetically recombinant receptor that is derived from the antigen-binding site of an antibody. The intracellular domain can be designed to recapitulate a series of events by which T cells are activated, and stimulatory and costimulatory domains such as CD28 and / or 4-1BB or CD3z can be incorporated to enhance the activation, survival, and proliferation of CAR T cells. Furthermore, CAR T cells can proliferate and differentiate into central memory cells and effector memory cells, and have been observed to persist in the body for up to 4 years after administration as a "living drug". Once activated, the clonally expanded CAR T cells release cytokines and other soluble mediators and directly kill target cells and normal cells expressing the antigen.

[0051] The CAR-T cells can be any CAR T cells such as, but not limited to, CD19 CAR T cells, BCMACAR T cells and / or CD19-CD3 bispecific CAR-T cells.

[0052] Cancer type In some embodiments, the CAR-T cell therapy is administered to a patient having cancer. The cancer can generally be any cancer suitable for treatment by CAR T cell therapy. In some embodiments, the cancer can be a hematological malignancy. For example, the cancer can be B cell lymphoma, advanced, recurrent, or refractory diffuse large B cell lymphoma (DLBCL), primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma. In some examples, the cancer is B cell lymphoma. In some embodiments, the cancer can be a solid tumor. Cancers suitable for treatment by CAR T cell therapy include brain tumors, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, and liver metastases.

[0053] Methods for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration

[0054] The compounds and pharmaceutical compositions described herein can be administered in therapeutically effective dosages to treat the aforementioned conditions, disorders, and diseases.

[0055] The compounds and pharmaceutical compositions described herein can be administered in prophylactically effective dosages to reduce or prevent the recited conditions, disorders, and diseases.

[0056] In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to treat CRS, ICANS, or both. In some embodiments, CRS or ICANS can be associated with CAR T cell administration.

[0057] In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof does not interfere with cell death mediated by CAR T cells. Cell death can occur when CAR T cells engage or interact with a corresponding antigen on a cancer cell that can result in the death of the cancer cell. In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof does not reduce or inhibit CAR T cell-mediated cell death. In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof does not reduce CAR T cell-mediated killing by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 1% to about 10%, about 10% to about 20%, about 0.5% to about 5%, about 5% to about 15%, about 15% to about 25%, or more. In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof does not inhibit or reduce the activation or proliferation of CAR T cells.

[0058] In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to treat grade 1 CRS, grade 2 CRS, grade 3 CRS, or grade 4 CRS. Treatment with veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can reduce the severity of CRS such that treatment results in a higher grade of CRS, such as grade 4 CRS, becoming a lower grade of CRS, such as grade 1. In some embodiments, treatment with veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can eliminate or prevent CRS. In some embodiments, grade 1 CRS can include a fever of about 38°C or higher. In some embodiments, grade 1 can include nausea, fatigue, headache, and may require hospitalization. In some embodiments, grade 2 CRS can include a fever of about 38°C or higher and hypotension that does not require vasopressors. Grade 2 CRS can further include hypoxemia, or a decrease in oxygen that requires a low-flow nasal cannula or blow-by oxygen. In some embodiments, grade 2 CRS can include hypotension and / or organ toxicity. In some embodiments, grade 3 can include a fever of about 38°C or higher and hypotension that requires vasopressin, with or without vasopressin treatment. Grade 3 can further include hypoxemia that requires a high-flow nasal cannula, face mask, non-rebreather mask, or Venturi mask. In some embodiments, grade 4 can include a fever of about 38°C or higher and hypotension that requires multiple vasopressors (excluding vasopressin) and / or organ toxicity. Grade 4 can further include hypoxemia that requires positive pressure (CPAP, BiPAP, intubation, mechanical ventilation) and / or organ toxicity.

[0059] In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to treat grade 1 ICANS, grade 2 ICANS, grade 3 ICANS, or grade 4 ICANS. Treatment with veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can reduce the severity of CRS such that, upon treatment, a higher grade of ICANS, such as grade 4 ICANS, becomes a lower grade of ICANS, such as grade 1 ICANS. In some embodiments, treatment with veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can eliminate or prevent ICANS. In some embodiments, grade 1 ICANS can have an immune effector cell-associated encephalopathy (ICE) score of about 7-9. In some embodiments, grade 1 ICANS can include: consciousness: wakes up spontaneously; seizures: none; motor findings: none; ICP elevation / brain edema: none. In some embodiments, grade 2 ICANS can include an ICE score of about 3-6. In some embodiments, grade 2 ICANS can include: consciousness: wakes up to voice; seizures: none; motor findings: none; ICP elevation / brain edema: none. In some embodiments, grade 3 ICANS can include an ICE score of about 0-2. In some embodiments, grade 3 ICANS can include: consciousness: wakes up only to tactile stimuli; seizures: non-convulsive seizures on electroencephalogram that disappear with any clinical seizure or intervention that rapidly disappears; motor findings: none; ICP elevation / brain edema: local / focal edema on neuroimaging. In some embodiments, grade 4 ICANS can include an ICE score of about 0 (i.e., the patient or subject is unable to perform ICE).Grade 4 ICANS includes consciousness: the subject is unarousable or requires strong or repeated tactile stimuli to arouse, coma or stupor; seizures: life-threatening prolonged seizures (more than 5 minutes); or repeated clinical or electrical seizures that do not return to baseline during that time; motor findings: increased ICP / brain edema: may include diffuse brain edema on neuroimaging, decerebrate or decorticate postures, or cranial nerve VI palsy, or papilledema, or parameters of Cushing's triad.

[0060] In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can reduce the level of one or more cytokines. Non-limiting examples of cytokines whose levels can be reduced include IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and / or GM-CSF. In some embodiments, the level of one or more cytokines can be reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, the level of one or more cytokines can be reduced by about 1-10%, about 5-15%, about 10-20%, about 15-25%, about 20-30%, about 25-35%, about 30-40%, about 35-45%, about 40-50%, about 45-55%, about 50-60%, about 55-65%, about 60-70%, about 65-75%, about 70-80%, about 75-85%, about 80-90%, about 85-95%, or about 90-100%.

[0061] Veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to reduce the level of one or more cytokines that are known to increase about 0 to 36 hours after CAR T cell therapy. For example, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to reduce the levels of IL-2, IL-15, and / or MCP-1. In some embodiments, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used to reduce the level of one or more cytokines that are known to increase up to about 2 to 5 days after CAR T cell therapy. For example, the cytokine can be IL-6, IL-8, IL-10, IFN-γ, or TNF-α.

[0062] In some methods, veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be used in a combination therapy approach with other active pharmaceutical ingredients. For example, a method of treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject can include administering to the subject a CAR T cell, a first pharmaceutical composition, and a second pharmaceutical composition, wherein the first pharmaceutical composition includes at least an effective amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof, and the second pharmaceutical composition includes at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof.

[0063] Additional methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration may include administering CAR T cells and a first pharmaceutical composition to a subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the subject experiences a reduction in ICANS effects, parkinsonism effects, or both, compared to a similar subject who has received the administered CAR T cells but not the administered pharmaceutical composition. ICANS can be evaluated and graded using a cognitive assessment tool called the "Immune Effector Cell-associated Encephalopathy (ICE) Assessment" tool, level of consciousness, presence and severity of seizures, movement control disorders, and presence of cerebral edema. The effects of parkinsonism can be measured by various metrics such as tremors, muscle rigidity, neurological problems, psychomotor retardation, changes in handwriting, changes in gait, etc.

[0064] Further methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering CAR T cells and a first pharmaceutical composition to the subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the subject experiences a decrease in severity measurements compared to a similar subject who receives the administered CAR T cells but not the administered first pharmaceutical composition. Severity measurements may include grade of the event, duration of the event, incidence of the event, incidence of ICU or hospitalization, duration of ICU or hospitalization, timing of onset, mortality, interference with antibiotics or other supportive medications, or combinations thereof. Further severity measurements include use of supportive therapy, use of medical interventions, use of intensive medical interventions such as intubation.

[0065] Additional methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering CAR T cells and a first pharmaceutical composition to the subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition is administered at a time when the onset of CRS is detected by an increase in the level of one or more of the cytokines MIF, IL-5, IL-17A, IL-23, IFN-γ, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β. Alternatively or additionally, the onset of CRS can be detected by an increase in the level of one or more cytokines such as CCL2, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, CXCL9, CXCL-10, VEGF, CCL3, GCSF, GMCSF. For example, the onset can be detected by measuring an increase in one or more cytokines, one or more inflammatory markers. Alternatively, the onset of CRS can also be measured by the manifestation of symptoms. Common symptoms include fever, hypotension, and hypoxia.

[0066] Additional methods of treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject may include administering CAR T cells and a first pharmaceutical composition to the subject, where the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, the subject experiences CRS, ICANS, a parkinsonism effect, or a combination thereof, and the subject needs to reduce treatment with at least one corticosteroid compared to a similar subject who receives the administered CAR T cells but does not receive the administered first pharmaceutical composition. In some embodiments, the subject does not need treatment with at least one corticosteroid.

[0067] The described methods, kits, and uses of pharmaceutical compositions can result in a reduction or elimination of CRS, ICANS, parkinsonism, or combinations thereof. The reduction can be an improvement or elimination of undesirable physiological symptoms experienced by a patient subject, a quantifiable reduction in the concentration of one or more cytokines, or both. The reduction can generally be reduced by any amount. For example, the reduction can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and in an ideal situation, about 100% reduction (complete removal of the disease, symptom, or other undesirable characteristic). The reduction can be relative to the effect observed upon administration of CAR T cells, but without administration of the first pharmaceutical composition.

[0068] CAR T cells are typically administered once by infusion, but multiple administrations are also possible. CRS does not occur in all patients, but approximately 50 - 100% of patients receiving CAR T cell therapy develop CRS. CRS typically develops within the first week and can typically develop over the first two weeks after CAR T cell administration. The first pharmaceutical composition can be started simultaneously with (i.e., without a delay period) or after a delay period relative to the CAR T cells. In some embodiments, the first pharmaceutical composition can be administered one or more additional times before administration of the CAR T cells. The delay period can be a predetermined period after administration of the CAR T cells (e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more, or a range between any two of these values). Exemplary ranges of the delay period include about 3 days to 7 days, about 2 days to 5 days, about 3 days to 5 days, about 2 days to 5 days, about 4 days to 8 days, etc. Alternatively, the delay period may continue until the onset of CRS is detected.

[0069] The administration of the first pharmaceutical composition can be carried out at various times and in various sequences. For example, the first pharmaceutical composition can be administered before the administration of CAR T cells, on the same day as the administration of CAR T cells, after the administration of CAR T cells, and in combinations thereof. For example, the first pharmaceutical composition can be administered before the administration of CAR T cells, on the same day as the administration of CAR T cells, and after the administration of CAR T cells. In one specific example, the first pharmaceutical composition can be administered 1 day before the administration of CAR T cells, and then administration can be continued for at least about 14 more days.

[0070] The onset of CRS can generally be detected by any means, such as fever, headache, loss of appetite, nausea, fatigue, muscle pain, hypoxia, low blood pressure (hypotension), coagulation disorders, capillary leakage, tachycardia, and organ damage. For example, a simple way to detect the onset of CRS is to detect fever. Alternatively, the onset of CRS can be detected by monitoring an increase in the level of one or more cytokines such as IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, GM-CSF. Alternatively or additionally, the onset of CRS can be detected by an increase in the level of one or more of the cytokines CCL2, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, CXCL9, CXCL-10, VEGF, CCL3, GCSF, and GMCSF. In some embodiments, the onset of CRS can be detected by monitoring an increase in the level of one or more cytokines such as IL-6, IFN-γ, TNF-α, IL-10. Alternatively or additionally, the onset of CRS can be detected by monitoring an increase in the level of one or more cytokine levels such as MIF, IL-5, IL-17A, IL-23, IFN-γ, CXCL9 / MIG, GCSF, VEGF-A, TGF-β. The onset of CRS can be detected by multiple methods. For example, the onset can be detected by measuring an increase in one or more cytokines or one or more inflammatory markers. Alternatively, the onset of CRS can also be measured by the appearance of symptoms. Common symptoms include fever, hypotension, and hypoxia. In some embodiments, the first pharmaceutical composition can reduce the level of one or more cytokines such as IL-1, IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF, but is not limited thereto.

[0071] For example, after a delay period or upon detection of the onset of CRS, the first pharmaceutical composition can be administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more, or within a range between any two of these values. CRS typically resolves within about one week, but has been recorded to last about 30 days or more. In some examples, the first pharmaceutical composition can be administered for about 14 days or more, such as for about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or more, or within a range between any two of these values. Exemplary ranges include about 1 day to about 7 days, about 1 day to about 14 days, about 1 day to about 21 days, about 1 day to about 30 days, about 7 days to about 14 days, about 7 days to about 21 days, about 7 days to about 30 days, about 14 days to about 21 days, and about 21 days to about 30 days. In one specific example, the first pharmaceutical composition can be administered starting 1 day prior to CAR T cell administration and continue for at least about 14 days.

[0072] Treatment can generally be carried out on any effective schedule. For example, the first pharmaceutical composition disclosed herein can be administered once, or as needed, about once a day, about twice a day, about three times a day, about four times a day, about once a week, about twice a week, about three times a week, about four times a week, about five times a week, about six times a week, about seven times a week, about once every other week, about once every other day, etc., over one or more dosing cycles. It will be understood that the specific dosage levels and dosing frequencies for a particular subject can vary and depend on various factors including the species, age, weight, general health, sex and diet of the subject, the mode and time of administration, the rate of excretion, the combination of drugs, and the severity of the particular condition.

[0073] Administration can generally be performed by any method. Exemplary administration methods include local delivery, subcutaneous delivery, intravenous (IV) injection delivery, intramuscular (IM) injection delivery, intrathecal (IT) injection delivery, intraperitoneal (IP) injection delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, and combinations thereof. In some embodiments, administration includes oral administration, subcutaneous administration, inhalation administration, intravenous administration, or intramuscular administration.

[0074] In some embodiments, by administering at least an effective amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof, the administered CAR T cells are compared to similar subjects that have received the administered CAR T cells but not the administered first pharmaceutical composition. Compared to similar subjects who have received the administered CAR T cells but not the administered first pharmaceutical composition, the need for treatment with at least one corticosteroid, tocilizumab, an IL-6 receptor blocking therapeutic agent, or combinations thereof can be reduced or eliminated. In an ideal embodiment, the subject does not require treatment with at least one corticosteroid, tocilizumab, or an IL-6 receptor blocking therapeutic agent. In some embodiments, the subject experiences CRS, ICANS, a parkinsonism effect, or combinations thereof, but in order to achieve a similar (or superior) clinical effect compared to similar subjects who have received the administered CAR T cells but not the administered first pharmaceutical composition, it is necessary that the dosage or administration of corticosteroids, tocilizumab, IL-6 receptor blocking therapeutic agents, or combinations thereof be reduced or eliminated.

[0075] The CAR T cells can generally be any CAR T cells. Examples of currently commercially available CAR T cell formulations include Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), and the like.

[0076] In some embodiments, beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof can reduce or eliminate hospitalizations associated with the onset of CRS, ICANS, or combinations thereof. In some embodiments, the methods of the present disclosure using beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof can change CAR-T therapy from mainly in-patient treatment to outpatient treatment.

[0077] Pharmaceutical composition The pharmaceutical composition described herein can be a first pharmaceutical composition or a second pharmaceutical composition. The first pharmaceutical composition can include at least one prostacyclin / prostaglandin analog. The second pharmaceutical composition can include at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof.

[0078] Examples of prostacyclin or prostaglandin analogs include carbaprostacyclin, beraprost, taprostene, niloprost, iloprost, cicaprost, ciprostene, treprostinil, bosentan, vapiprost, eptaloprost, or isomers thereof, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutical composition includes an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof. Beraprost has the chemical formula CH 24 H30 It has O5 and has a single carboxylic acid group. In some embodiments, the prostacyclin analog is a beraprost salt such as sodium beraprost (CH 24 H 29 NaO5; 2,3,3a,8b-tetrahydro-2-hydroxyl-1-(3-hydroxyl-4-methyl-1-octen-6-ynyl)-1H-cyclopenta[b]benzofuran-5-butanoic acid, sodium salt). Sodium beraprost (BPS; GP1001) is a mixture of four isomers - two diastereomers (BPS-314 and BPS-315) and two enantiomers of each of them, BPS-314d (CTO1681; GP1681, (+)-[1R,2R,3aS,8bS]-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4S)-3-hydroxy-4-methyl-1-octen-6-ynyl)-cyclopenta[b]benzofuran-5-butanoic acid, monosodium salt; also called esberaprost sodium salt) and BPS-314l (GP1684), as well as BPS-315d (GP1683) and BPS-315l (GP1682). The beraprost isomers are further described in U.S. Patent Publication No. US2014-0275237A1. In some examples, the first pharmaceutical composition can contain 1, 2, 3, or all 4 isomers of beraprost. In some embodiments, the isomer of beraprost is BPS-314d (CTO1681; esberaprost sodium salt).

[0079] Beraprost and its preparation method are shown in U.S. Patent No. 7,345,181 and PCT Publication No. WO2004 / 026224 "Preparation Method of Prostaglandin Derivatives and Their Starting Materials". Beraprost is commercially available from Yonsung Fine Chemicals (Gyeonggi-do, South Korea). Beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof can generally be present in the first pharmaceutical composition in any effective amount or concentration. Different pharmaceutical forms may have different amounts or concentrations of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the first pharmaceutical composition can include all one, two, three, or four isomers of beraprost. An isomer of beraprost refers to a beraprost molecule that has the same molecular formula as another beraprost molecule but a different arrangement of atoms in space. In some embodiments, the isomers of beraprost can be structural isomers or stereoisomers. In some embodiments, structural isomers can include isomers that have the same molecular formula but different bonds between atoms. In some embodiments, the isomers can be stereoisomers of beraprost where the bonds between atoms are the same but the relative positions of the atoms are different. In some embodiments, the stereoisomers of beraprost can be diastereomers of beraprost or enantiomers of beraprost. The isomers can be different stereoisomers arising from one or more chiral centers in their chemical structure. Different isomers of beraprost can have different biological activities and sometimes may have no activity or even undesirable activity compared to other desired isomers.

[0081] In some embodiments, the first pharmaceutical composition can include BPS-314d (which may include CTO1681; GP1681, (+)-[1R,2R,3aS,8bS]-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4S)-3-hydroxy-4-methyl-1-octen-6-ynyl)-1H-cyclopenta[b]benzofuran-5-butanoic acid, monosodium salt; also called sodium esberaprost). In some embodiments, the first pharmaceutical composition can include BPS-314l (GP1684). In some embodiments, the first pharmaceutical composition can include BPS-315d (GP1683). In some embodiments, the first pharmaceutical composition can include BPS-315l (GP1682).

[0082] In some embodiments, the first pharmaceutical composition may comprise one or more enantiomers of beraprost. A common purity measurement is "enantiomeric excess" or "ee". A racemic mixture has an ee of about 0%, while a completely pure enantiomer has an ee value of about 100%. The ee value of the first pharmaceutical composition is desirably at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and ideally 100%.

[0083] As an example, a first pharmaceutical composition comprising the beraprost isomer BPS-314d (CTO1681; GP1681; esberaprost sodium salt) can have an ee value of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and ideally 100%.

[0084] In some embodiments, one or more isomers of beraprost can be separated from other isomers so that only the selected isomer is included in the first pharmaceutical composition. In one embodiment, BPS-314d (CTO1681) can be separated from other isomers in beraprost. Separation of the isomers from beraprost can be achieved using a commercially available chiral chromatography column. Additional purification steps can also be performed. In some embodiments, a single isomer can be obtained by chiral synthesis methods.

[0085] In some embodiments, synthetic methods can be used to prepare a desired isomer (e.g., an enantiomer or a stereoisomer) at a high concentration relative to an undesired enantiomer or stereoisomer.

[0086] In some embodiments, the first pharmaceutical composition can have a high purity both during manufacture and at later times such as during use. In some embodiments, the first pharmaceutical composition can have a low or no level of degradation products of beraprost, beraprost isomers, or pharmaceutically acceptable salts thereof.

[0087] Impurities are undesirable in the first pharmaceutical composition and can be derived from different sources. Impurities can be starting materials, residual solvents, or synthetic components that have not been completely removed or purified from the final desired product. Contaminants can be substances unintentionally included in the final desired product due to the manufacturing environment or other sources. Impurities and contaminants can be harmful or harmless and can be identified or unidentified. In some examples, the first pharmaceutical composition contains about 0.1 wt% or less of impurities or contaminants, about 0.2 wt% or less of impurities or contaminants, or about 0.5 wt% or less of impurities or contaminants. In an ideal example, the composition contains no detectable impurities or contaminants.

[0088] One or more degradation products can arise from various pathways such as the instability, degradation, or oxidation of the first pharmaceutical composition itself, or the incompatibility or reaction of the first pharmaceutical composition with another component of the composition (such as one or more excipients), moisture, or the packaging of the composition. In some examples, the first pharmaceutical composition contains about 0.1 wt% or less of degradation products, about 0.2 wt% or less of degradation products, or about 0.5 wt% or less of degradation products. In one example, the composition contains no detectable degradation products.

[0089] The daily dosage (mass) of a prostacyclin / prostaglandin analog or beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof can generally be any effective amount or dosage. For example, a therapeutically effective amount (micrograms) can include from about 0.1 μg to about 100 μg, from about 10 μg to about 90 μg, or from about 15 μg to about 90 μg, or from about 0.1 μg to about 5000 μg. The mass value is the weight of the salt combining the anion and the cation. Specific examples of a therapeutically effective amount include about 0.1 μg, about 1 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, and ranges between any two of these values. When administered in two or more daily dosages, the amounts of each dosage can be summed to obtain the total daily dosage. For example, CTO1681 can be administered at a dosage of about 15 - 90 μg / day divided into about 3 doses, with individual doses being about 5 - 30 μg.

[0090] In some embodiments, an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof is at least about 1 microgram, from about 1 microgram to about 100 micrograms, from about 1 microgram to about 80 micrograms, from about 1 microgram to about 60 micrograms, from about 1 microgram to about 50 micrograms, from about 1 microgram to about 40 micrograms, from about 51 micrograms to about 30 micrograms, from about 1 microgram to about 20 micrograms, from about 1 mg to about 10 micrograms, or from about 1 microgram to about 5 micrograms, or any value between these ranges, in the unit dosage (mass) of the first pharmaceutical composition. Specific examples include about 1 microgram, about 5 micrograms, about 10 micrograms, about 25 micrograms, about 50 micrograms, about 75 micrograms, about 100 micrograms, from about 60 micrograms to about 360 micrograms, or ranges between any two of these values.

[0091] In some embodiments, the amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof can be calculated based on the presence of a single desired isomer. For example, if a single isomer such as BPS-314d (CTO1681; also sodium esveraprost) is desired in an amount of about 15 micrograms to about 90 micrograms, this corresponds to an amount of about 60 micrograms to about 360 micrograms of a racemic mixture of four isomers (where the amount of a single isomer is one quarter of the mass).

[0092] In some embodiments, the first pharmaceutical composition comprising veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof achieves a Cmax of about 0.01 nanomolar to about 10 nanomolar, about 0.01 nanomolar to about 5 nanomolar, about 0.01 nanomolar to about 3 nanomolar, about 0.01 nanomolar to about 2 nanomolar, about 0.01 nanomolar to about 1 nanomolar, about 0.01 nanomolar to about 0.5 nanomolar, or any value between these ranges. Specific examples include about 0.01 nanomolar, about 0.05 nanomolar, about 0.075 nanomolar, about 0.1 nanomolar, about 0.5 nanomolar, about 1 nanomolar, about 2 nanomolar, about 5 nanomolar, or about 10 nanomolar.

[0093] In some embodiments, the first pharmaceutical composition comprising veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof has a T max achieved at about 0.1 hour to about 5 hours, about 0.1 hour to about 4 hours, about 0.1 hour to about 3 hours, about 0.1 hour to about 2 hours, about 0.1 hour to about 1 hour, or any specific value between these ranges. Specific examples include about 0.1 hour, about 0.5 hour, about 1 hour, about 1.5 hour, about 1.7 hour, about 2 hours, or about 5 hours.

[0094] In some embodiments, the first pharmaceutical composition comprising veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof is from about 0.01 ng.hr / mL to about 30 ng.hr / mL over 48 hours, from about 0.01 ng.hr / mL to about 20 ng.hr / mL over 48 hours, from about 0.01 ng.hr / mL to about 10 ng.hr / mL over 48 hours, from about 0.01 ng.hr / mL to about 5 ng.hr / mL over 48 hours, from about 0.01 ng.hr / mL to about 3 ng.hr / mL over 48 hours, from about 0.01 ng.hr / mL to about 2 ng.hr / mL over 48 hours, or from about 0.01 ng.hr / mL to about 1 ng.hr / mL over 48 hours. Specific examples include about 0.01 ng.hr / mL, about 0.05 ng.hr / mL, about 0.1 ng.hr / mL, about 0.5 ng.hr / mL, about 1 ng.hr / mL, about 2 ng.hr / mL, about 5 ng.hr / mL, about 10 ng.hr / mL, or about 30 ng.hr / mL.

[0095] In some examples, the first pharmaceutical composition may further comprise at least one anti-inflammatory component such as at least one corticosteroid or at least one therapeutic monoclonal antibody.

[0096] In some embodiments, the first pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients that may be present in the composition include, but are not limited to, fillers / vehicles, solvents / co-solvents, preservatives, antioxidants, suspending agents, surfactants, defoaming agents, buffers, chelating agents, sweeteners, flavoring agents, binders, bulking agents, disintegrants, diluents, lubricants, fillers, wetting agents, lubricants, and combinations thereof.

[0097] In some embodiments, the first pharmaceutical composition can further comprise one or more exemplary fillers. Examples of exemplary fillers include celluloses and cellulose derivatives such as microcrystalline cellulose; starches such as dried starch, hydrolyzed starch, and corn starch; starches such as starch derivatives; cyclodextrin; saccharides such as powdered sugar, and sugar alcohols such as lactose, mannitol, sucrose, and sorbitol; inorganic fillers such as aluminum hydroxide gel, precipitated calcium carbonate, carbonates, magnesium aluminosilicate, dibasic calcium phosphate; and sodium chloride, silicon dioxide, titanium dioxide, titanium oxide, dicalcium phosphate dihydrate, calcium sulfate, alumina, kaolin, talc, or combinations thereof. The filler can be present in the composition at about 20 wt% to about 65 wt%, about 20 wt% to about 50 wt%, about 20 wt% to about 40 wt%, about 45 wt% to about 65 wt%, about 50 wt% to about 65 wt%, or about 55 wt% to about 65 wt%, or any value between these ranges, based on the total weight of the composition.

[0098] In some embodiments, the first pharmaceutical composition further comprises one or more disintegrants. Examples of disintegrants include starches, alginic acid, cross-linked polymers such as cross-linked polyvinylpyrrolidone, croscarmellose sodium, potassium starch glycolate, sodium starch glycolate, clay, cellulose, starch, gum, or combinations thereof. The disintegrant can be present in the composition at about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 7 wt%, about 1 wt% to about 6 wt%, or about 1 wt% to about 5 wt%, or any value between these ranges, based on the total weight of the composition.

[0099] In some examples, the first pharmaceutical composition further comprises one or more binders including, but not limited to, celluloses such as hydroxypropylcellulose, methylcellulose, and hydroxypropylmethylcellulose; starches such as corn starch, pregelatinized starch, and hydroxypropyl starch; waxes, natural and synthetic gums such as acacia, tragacanth, and sodium alginate; synthetic polymers such as polymethacrylate, polyvinylpyrrolidone; and povidone, dextrin, pullulan, agar, gelatin, tragacanth, macrogol, or combinations thereof. The binder can be present in the composition at about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 4 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, or about 0.5 wt% to about 1 wt%, or any value between these ranges, based on the total weight of the composition.

[0100] In some embodiments, the first pharmaceutical composition further comprises one or more wetting agents including, but not limited to, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, poloxamer, poloxamer 188, polyoxyethylene ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polysorbate, cetyl alcohol, glycerin fatty acid ester (such as triacetin, glyceryl monostearate, etc.), polyoxymethylene stearate, sodium lauryl sulfate, sorbitan fatty acid ester, sucrose fatty acid ester, benzalkonium chloride, polyethoxylated castor oil, and combinations thereof. The wetting agent can be present in the composition at about 0.1 wt% to about 1 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 4 wt%, or about 0.1 wt% to about 5 wt%, or any value between these ranges, based on the total weight of the composition.

[0101] In some embodiments, the first pharmaceutical composition further comprises one or more lubricants including, but not limited to, stearic acid, magnesium stearate, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali metal salts and alkaline earth metal salts, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (PEG), methoxypolyethylene glycol, propylene glycol, sodium oleate, glyceryl, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (PEG), methoxypolyethylene glycol, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof. The lubricant can be present in the composition at about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, or about 0.1 wt% to about 1 wt%, or any value between these ranges, based on the total weight of the composition.

[0102] In some embodiments, the first pharmaceutical composition further comprises one or more lubricants including, but not limited to, colloidal silicon dioxide, talc, sodium lauryl sulfate, native starch, and combinations thereof. The lubricant can be present in the composition at about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 0.9 wt%, about 0.05 wt% to about 0.8 wt%, about 0.05 wt% to about 0.5 wt%, or about 0.05 wt% to about 0.1 wt%, or any value between these ranges, based on the total weight of the composition.

[0103] In some embodiments, the first pharmaceutical composition is a tablet and further includes a top coat such as a hydroxypropyl methylcellulose coating or a polyvinyl alcohol coating, and is available under the trade name of Opadry such as Opadry White, Opadry II (Opadry is a registered trademark of BPSI Holdings LLC, Wilmington, DE, USA). The top coat can be present in the composition at about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 7 wt%, about 1 wt% to about 6 wt%, or about 1 wt% to about 5 wt%, or any value between these ranges, based on the total weight of the composition.

[0104] In some embodiments, the first pharmaceutical composition may further include one or more preservatives. Examples of preservatives include sodium benzoate, paraoxybenzoic acid esters, methyl, ethyl, butyl, and propyl parabens, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenylmercuric nitrate, thimerosal, or combinations thereof. The preservative can be included in the liquid dosage form. The preservative can be in an amount sufficient to extend the shelf life or storage stability, or both, of the liquid dosage form. The preservative can be present in the composition at about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 0.9 wt%, about 0.05 wt% to about 0.8 wt%, about 0.05 wt% to about 0.5 wt%, or about 0.05 wt% to about 0.1 wt%, or any value between these ranges, based on the total weight of the composition.

[0105] In some embodiments, the first pharmaceutical composition can further comprise one or more flavoring agents. Examples of flavoring agents include synthetic flavor oils and flavoring fragrances and / or natural oils, extracts from plant leaves, flowers, fruits, etc., or any combination thereof. Further examples include cinnamon oil, wintergreen oil, peppermint oil, clove oil, bay oil, anise oil, eucalyptus, thyme oil, sassafras oil, nutmeg oil, sage oil, bitter almond oil, cassia oil, etc., or combinations thereof. Also useful as flavorings are citrus oils such as vanilla, lemon, orange, grape, lime, grapefruit, fruit essences such as apple, banana, pear, peach, strawberry, raspberry, cherry, plum, pineapple, apricot, strawberry flavor, tutti-frutti flavor, mint flavor, etc., or combinations thereof. The flavoring agent can be present in the composition at about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, or about 0.1 wt% to about 1 wt%, or any value between these ranges, based on the total weight of the composition.

[0106] In some embodiments, the first pharmaceutical composition can further comprise one or more antioxidants. Examples of antioxidants include flavonoids, anthocyanidins, proanthocyanidins, or combinations thereof. The antioxidant can be present in the composition at about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 0.9 wt%, about 0.05 wt% to about 0.8 wt%, about 0.05 wt% to about 0.5 wt%, or about 0.05 wt% to about 0.1 wt%, or any value between these ranges, based on the total weight of the composition.

[0107] Physical form of the pharmaceutical composition The first and / or second pharmaceutical composition can generally be in any physical form suitable for use in treating a subject. These forms can be referred to as unit dosage forms such as individual tablets or pills. In some embodiments, the first and / or second pharmaceutical composition can be formulated as tablets, capsules, granules, powders, solutions, suspensions, gels, syrups, slurries, suppositories, patches, nasal drops, aerosols, injections, implantable sustained-release formulations, or mucoadhesive films. In some embodiments, the first and / or second pharmaceutical composition can be formed as tablets, bilayer tablets, capsules, multiparticulates, drug-coated beads, matrix tablets, or multi-core tablets. The physical form can be selected according to the desired method of treatment. In some embodiments, the physical form can be a liquid, for example, for oral or intravenous, IP, IM, or IT administration.

[0108] The first and / or second pharmaceutical composition can be manufactured by various conventional methods such as conventional mixing, dissolving, granulating, dragee-making, suspending, emulsifying, encapsulating, enclosing, or lyophilizing processes. The first and / or second pharmaceutical composition can be formulated by conventional methods using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of the active agent into a pharmaceutically usable formulation. Suitable formulations can be selected according to the selected route of administration.

[0109] For local administration, the first and / or second pharmaceutical compositions described herein can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Systemic compositions include those designed for administration by injection, for example, subcutaneous injection, intravenous injection (IV), intramuscular injection (IM), intrathecal injection (IT), intraperitoneal injection (IP), as well as those designed for transdermal, subcutaneous, transmucosal oral, or pulmonary administration, but are not limited thereto. For injection, the first and / or second pharmaceutical compositions can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer, and / or in a specific emulsion. The solution can contain one or more formulating agents such as suspending agents, stabilizers, and / or dispersing agents. In certain examples, the first and / or second pharmaceutical compositions can be provided in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, for example, prior to use. For transmucosal administration, one or more penetration enhancers suitable for the permeation barrier can be used in the formulation. Such penetration enhancers are generally known in the art.

[0110] For oral administration, the first and / or second pharmaceutical composition can combine beraprost, its isomers or pharmaceutically acceptable salts with one or more pharmaceutically acceptable carriers well-known in the art. Such carriers facilitate formulation as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. For example, in the case of oral solid formulations such as powders, capsules, tablets, etc., suitable excipients include saccharides such as lactose, sucrose, mannitol, sorbitol; starches such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or cellulose formulations such as polyvinylpyrrolidone (PVP); granulating agents; and fillers such as saccharides as binders. Optionally, disintegrants such as cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts such as sodium alginate may be added. Optionally, the solid dosage form may be sugar-coated or enteric-coated using standard techniques.

[0111] For example, in the case of oral liquid formulations such as suspensions, elixirs, solutions, etc., suitable carriers, excipients, or diluents include water, glycols, oils, alcohols, etc. Furthermore, flavoring agents, preservatives, coloring agents, etc. can be added. For oral administration, the composition can take the form of tablets, lozenges, etc. formulated by conventional methods.

[0112] For administration by inhalation, the first and / or second pharmaceutical composition can be delivered in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

[0113] In some embodiments, the first and / or second pharmaceutical composition is an immediate release first and / or second pharmaceutical composition, a modified release first and / or second pharmaceutical composition, or a combination thereof. In some embodiments, the immediate release first pharmaceutical composition releases veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof within a short time after administration, typically less than about 4 hours, less than about 3.5 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 90 minutes, less than about 60 minutes, less than about 45 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes.

[0114] In some embodiments, the first pharmaceutical composition is an immediate release first pharmaceutical composition comprising microcrystalline cellulose, hydroxypropyl cellulose, lactose monohydrate, pregelatinized starch, magnesium stearate, and / or purified water. In some embodiments, the composition can include a coating prepared using an Opadry® film coating process.

[0115] In some embodiments, the modified release composition can release veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof, either continuously or at a controlled rate over an extended period of time, or after a lag time following administration. For example, it can be released from the composition 4 hours after administration, 8 hours after administration, 12 hours after administration, 16 hours after administration, or 24 hours after administration. Modified release compositions include sustained release compositions, sustained release compositions, and delayed release compositions. In some embodiments, the modified release composition can release about 10% in about 2 hours, about 20% in about 2 hours, about 40% in about 2 hours, about 50% in about 2 hours, about 10% in about 3 hours, about 20% in about 3 hours, about 40% in about 3 hours, about 50% in about 3 hours, about 10% in about 4 hours, about 20% in about 4 hours, about 40% in about 4 hours, about 50% in about 4 hours, about 10% in about 6 hours, about 20% in about 6 hours, about 40% in about 6 hours, or about 50% in about 6 hours.

[0116] In some embodiments, the modified release composition can include a matrix selected from microcrystalline cellulose, sodium carboxymethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl methylcellulose and hydroxypropyl cellulose, polyethylene oxide, alkyl celluloses such as methylcellulose and ethylcellulose, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, methylcellulose, ethylcellulose, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, polyalkyl methacrylate, polyvinyl acetate, and mixtures thereof.

[0117] The sustained release composition can also be formulated as a depot formulation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or, for example, as a poorly soluble salt or a poorly soluble derivative.

[0118] Subject to be treated The subject is generally a mammal. Examples of subjects include non-human primates, humans, dogs, cats, mice, rats, cows, goats, sheep, rabbits, horses, monkeys, pigs, etc. In some embodiments, the subject is a human. The terms "subject", "individual" or "patient" are used interchangeably and are intended to include humans and non-human animals as used herein. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, monkeys, sheep, dogs, rats, cats, cows, horses, chickens, amphibians, reptiles, etc. Examples of mammals include non-human primates, monkeys, sheep, dogs, cats, cows, horses, etc. In some examples, the subject is a human or a human. The method is suitable for treating humans having cancer. The subject may be symptomatic or asymptomatic.

[0119] Kit In further embodiments, a kit is provided for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject. The kit includes a first container comprising a first pharmaceutical composition comprising at least an effective amount of a prostacyclin / prostaglandin analog or beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof; a second container comprising a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof; a third container comprising CAR T cells; and can include instructions for administering the first pharmaceutical composition, the second pharmaceutical composition, and the CAR T cells to a subject. Any of the above pharmaceutical compositions can be included in the kit. The kit can further include a fourth container, such as a fourth container comprising an additional pharmaceutical composition or other active ingredient. In some embodiments, the first container can comprise the first pharmaceutical composition, the second container can comprise the second pharmaceutical composition, the third container can comprise CAR T cells, and the fourth container can comprise at least one solvent or solvent for mixing with the first pharmaceutical composition prior to administering to the subject according to the instructions. In one embodiment, the kit can include a first container comprising beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, a second container comprising a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof, a third container comprising CAR T cells, and a fourth container comprising an aqueous solvent. In some examples, the beraprost isomer is BPS-314d (CTO1681).

[0120] In other embodiments, the kit comprises a first container containing a first pharmaceutical composition comprising at least an effective amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof; a second container containing a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof; and instructions for administration of the first pharmaceutical composition, the second pharmaceutical composition, and the CAR T cells to a subject. The kit can further comprise a third container containing CAR T cells. The kit can further comprise a fourth container containing water or an aqueous solution.

[0121] Further aspects of the invention are also described in the numbered sections below: 1. A method of treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject, the method comprising administering to the subject CAR T cells, a first pharmaceutical composition, and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of veraprost or a pharmaceutically acceptable salt thereof, and the second pharmaceutical composition comprises at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof. 2. The method of claim 1, wherein the pharmaceutically acceptable salt is the sodium salt of veraprost. 3. The method of claim 1, wherein veraprost comprises at least one of BPS-314d, BPS-314l, BPS-315d, and BPS-315l. 4. The method of claim 1, wherein veraprost is BPS-314d (sodium esveraprost). 5. The method of claim 1, wherein the CAR T cell administration is performed to treat cancer, and A method wherein the cancer is B-cell lymphoma, progressive, recurrent or refractory diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, high-grade B-cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma. 6. In the method according to claim 1, the CAR T cell administration is performed to treat the cancer, and the cancer is a brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, or liver metastasis. 7. In the method according to claim 1, the CAR T cell administration is performed to treat the cancer, and here, the cancer is B-cell lymphoma. 8. In the method according to claim 1, the subject is a mammal. 9. In the method according to claim 1, the subject is a non-human primate, monkey, cat, dog, pig, cow, goat, horse, sheep, or rabbit. 10. In the method according to claim 1, the subject is a human, the method according to claim 1. 11. In the method according to claim 1, before the CAR T cells are administered to the subject, a first pharmaceutical composition is administered to the subject, and the second pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject. 12. In the method according to claim 1, before and after the CAR T cells are administered to the subject, a first pharmaceutical composition is administered to the subject, and the second pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject. 13. In the method according to claim 1, the CAR T cells, the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject simultaneously. 14. The method according to claim 1, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject after the CAR T cells have been administered to the subject. 15. In the method according to claim 1, after the CAR T cells have been administered to the subject, the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject, and the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. 16. The method according to claim 1, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject before the CAR T cells are administered to the subject. 17. The method according to claim 1, wherein the first pharmaceutical composition is administered to the subject starting from about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the CAR T cells have been administered to the subject. 18. The method according to claim 1, wherein the first pharmaceutical composition is administered to the subject starting from about 3 days to about 7 days after the CAR T cells have been administered to the subject. 19. The method according to claim 1, wherein the first pharmaceutical composition is administered to the subject after the CAR T cells have been administered to the subject and when the onset of CRS is detected. 20. The method according to claim 1, wherein after the CAR T cells have been administered to the subject, the first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the level of one or more of the cytokines IL-1α, IL-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF. 21. The method according to claim 1, wherein after the CAR T cells have been administered to the subject, the first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the level of one or more of the cytokines IL-6, IL-10, IFN-γ, TNF-α. 22. The method according to claim 1, wherein the first pharmaceutical composition is administered for a period of about 1 day to about 30 days. 23. The method according to claim 1, wherein the first pharmaceutical composition is administered for about 7 days to about 14 days. 24. The method according to claim 1, wherein the first pharmaceutical composition is administered starting 1 day before the administration of CAR T cells and continued for at least about 14 days. 25. The method according to claim 1, further comprising administering the first pharmaceutical composition to the subject before administering the CAR T cells. 26. The method according to claim 1, wherein the subject experiences a reduction in CRS as compared to a similar subject who receives the administered CAR T cells but does not receive the administered first pharmaceutical composition. 27. The method according to claim 1, wherein the subject does not experience CRS. 28. The method according to claim 1, wherein the CAR T cells are autologous. 29. The method according to claim 1, wherein the CAR T cells are allogeneic. 30. The method according to claim 1, wherein the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof. 31. The method according to claim 1, wherein the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, solution, suspension, gel, syrup, slurry, suppository, patch, nasal drop, aerosol, injection, implantable sustained-release formulation, or mucoadhesive film. 32. The method according to claim 1, wherein the administration comprises local delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof. 33. The method according to claim 1, wherein the administration comprises oral administration or intravenous injection (IV) administration. 34. The method according to claim 1, wherein veraprost or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms. 35. The method according to claim 1, wherein the administration comprises delivering the first pharmaceutical composition to a subject in an amount of at least about 0.1 microgram of veraprost or a pharmaceutically acceptable salt thereof. 36. A method comprising delivering a first pharmaceutical composition to a subject in an amount of about 1 microgram to about 5000 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 37. The method according to claim 1, wherein the administration comprises delivering the first pharmaceutical composition to a subject in an amount of about 15 micrograms to about 90 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 38. The method according to claim 1, wherein the administration comprises delivering the first pharmaceutical composition to a subject in an amount of about 60 micrograms to about 360 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 39. The method according to claim 1, wherein the CAR T cells are Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), Blincyto (blinatumomab), or a combination thereof. 40. A method of treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject, the method comprising administering a CAR T cell and a first pharmaceutical composition to the subject, wherein the first pharmaceutical composition comprises at least an effective amount of veraprost or a pharmaceutically acceptable salt thereof, and A method in which the subject receives the administered CAR T cells and the ICANS effect, the parkinsonism effect, or both are reduced as compared to a similar subject who does not receive the administered first pharmaceutical composition. 41. The method according to item 40, wherein the pharmaceutically acceptable salt is beraprost sodium salt. 42. The method according to item 40, wherein beraprost comprises at least one of BPS-314d, BPS-314l, BPS-315d and BPS-315l. 43. The method according to item 40, wherein beraprost is BPS-314d (esberaprost sodium salt). 44. In the method according to item 40, the CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, progressive, recurrent or refractory diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma. 45. In the method according to item 40, the CAR T cell administration is performed to treat cancer, and the cancer is a brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, or liver metastasis. 46. In the method according to item 40, the CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma. 47. The method according to item 40, wherein the subject is a mammal. 48. The method according to item 40, wherein the subject is a non-human primate, monkey, cat, dog, pig, cow, goat, horse, sheep, or rabbit. 49. The method according to item 40, wherein the subject is a human. 50. A method wherein a first pharmaceutical composition is administered to a subject before the CAR T cells are administered to the subject. 51. In the method according to item 40, the first pharmaceutical composition is administered to the subject before the CAR T cells are administered, and 50. A method wherein a first pharmaceutical composition is administered to a subject after the CAR T cells are administered to the subject. 52. In the method according to item 40, a method of simultaneously administering CAR cells and a first pharmaceutical composition to a subject. 53. In the method according to item 40, a method wherein the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject. 54. In the method according to item 40, a method wherein the first pharmaceutical composition is administered to the subject about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the CAR T cells are administered to the subject. 55. In the method according to item 40, a method wherein the first pharmaceutical composition is administered to the subject about 3 days to about 7 days after the CAR T cells are administered to the subject. 56. In the method according to item 40, a method of administering a first pharmaceutical composition to a subject after the CAR T cells are administered to the subject and when the onset of CRS is detected. 57. In the method according to item 40, after the CAR T cells are administered to the subject, a first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the level of one or more of the cytokines IL-1α, IL-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF. 58. In the method according to item 40, after the CAR T cells are administered to the subject, a first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the value of one or more of the inflammation markers CRP (C-reactive protein) and ferritin related to CRS. 59. The method according to item 40, wherein the first pharmaceutical composition is administered for a period of about 1 day to about 30 days. 60. The method according to item 40, wherein the first pharmaceutical composition is administered for a period of about 7 days to about 14 days. 61. The method according to item 40, wherein the first pharmaceutical composition is administered starting 1 day before the administration of CAR T cells and continued for at least about 14 days. 62. The method according to item 40, further comprising administering the first pharmaceutical composition to the subject before administering the CAR cells. 63. The method according to item 40, wherein the subject experiences a reduction in the ICANS effect as compared to a similar subject who receives the administered CAR T cells but does not receive the administered first pharmaceutical composition. 64. The method according to item 40, wherein the subject does not experience the ICANS effect. 65. The method according to item 40, wherein the subject experiences a reduction in parkinsonism as compared to a similar subject who receives the administered CAR T cells but does not receive the administered first pharmaceutical composition. 66. The method according to item 40, wherein the subject does not experience the parkinsonism effect. 67. The method according to item 40, wherein the CAR-T cells are autologous. 68. The method according to item 40, wherein the CAR-T cells are allogeneic. 69. The method according to item 40, wherein the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof. 70. The method according to item 40, wherein the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, solution, suspension, gel, syrup, slurry, suppository, patch, nasal drops, aerosol, injection, implantable sustained-release formulation, or mucoadhesive film. 71. The method according to item 40, wherein the administration comprises local delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof. 72. The method according to item 40, wherein the administration comprises oral administration or intravenous injection (IV) administration. 73. The method according to item 40, wherein veraprost or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms. 74. The method according to item 40, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of at least about 0.1 microgram of veraprost or a pharmaceutically acceptable salt thereof. 75. The method according to item 40, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 0.1 microgram to about 5000 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 76. The method according to item 40, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 15 micrograms to about 90 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 77. The method according to item 40, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 60 micrograms to about 360 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 78. The method according to item 40, wherein the CAR T cells are Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), Blincyto (blinatumomab), or a combination thereof. 79. A method for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject, comprising administering to the subject CAR T cells and a first pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of beraprost or a pharmaceutically acceptable salt thereof, and the subject has a reduced severity measurement as compared to a similar subject who receives the administered CAR T cells and does not receive the administered first pharmaceutical composition. 80. The method according to item 79, wherein the pharmaceutically acceptable salt is beraprost sodium salt. 81. The method according to item 79, wherein beraprost comprises at least one of BPS-314d, BPS-314l, BPS-315d and BPS-315l. 82. The method according to item 79, wherein beraprost is BPS-314d (esberaprost sodium salt). 83. The method according to item 79, wherein the CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, progressive, recurrent or refractory diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma. 84. The method according to item 79, CAR T cell administration is performed to treat cancer, and the cancer is a brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, or liver metastasis, method. 85. In the method according to item 79, CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, method. 86. In the method according to item 79, the subject is a mammal, method. 87. In the method according to item 79, the subject is a non-human primate, monkey, cat, dog, pig, cow, goat, horse, sheep, or rabbit, method. 88. In the method according to item 79, the subject is a human, method. 89. In the method according to item 79, the first pharmaceutical composition is administered to the subject before the CAR T cells are administered to the subject, method. 90. In the method according to item 79, the first pharmaceutical composition is administered to the subject before the CAR T cells are administered, and the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject, method. 91. In the method according to item 79, the CAR cells and the first pharmaceutical composition are co-administered to the subject, method. 92. In the method according to item 79, the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject, method. 93. In the method according to item 79, the first pharmaceutical composition is administered to the subject about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the CAR T cells are administered to the subject, method. 94. In the method according to item 79, the first pharmaceutical composition is administered to the subject about 3 days to about 7 days after the CAR T cells are administered to the subject, method. 95. The method according to item 79, wherein after the CAR T cells are administered to a subject and when the onset of CRS is detected, a first pharmaceutical composition is administered to the subject. 96. The method according to item 79, wherein after the CAR T cells are administered to a subject, a first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the level of one or more of the cytokines IL-1α, IL-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF. 97. The method according to item 79, wherein after the CAR T cells are administered to a subject, a first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the value of one or more of the CRS-related inflammatory markers CRP (C-reactive protein) and ferritin. 98. The method according to item 79, wherein the first pharmaceutical composition is administered for a period of about 1 day to about 30 days. 99. The method according to item 79, wherein the first pharmaceutical composition is administered for a period of about 7 days to about 14 days. 100. The method according to item 79, wherein the first pharmaceutical composition is administered starting 1 day before the CAR T cell administration and continues for at least about 14 days. 101. The method according to item 79, further comprising administering a first pharmaceutical composition to the subject before administering the CAR cells. 102. The method according to item 79, wherein the severity measurement is the grade of the event, the duration of the event, the incidence of the event, the incidence of ICU or hospitalization, the duration of ICU or hospitalization, the timing of onset, the mortality rate, interference by antibiotics or other supportive drugs, or a combination thereof. 103. The method according to item 79, wherein the CAR-T cells are autologous. 104. The method according to item 79, wherein the CAR-T cells are allogeneic. 105. In the method according to item 79, the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof. 106. In the method according to item 79, the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, solution, suspension, gel, syrup, slurry, suppository, patch, nasal drop, aerosol, injection, implantable sustained-release preparation, or mucoadhesive film. 107. In the method according to item 79, administration includes local delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof. 108. In the method according to item 79, administration includes oral administration or intravenous injection (IV) administration. 109. In the method according to item 79, beraprost or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms. 110. In the method according to item 79, administration includes delivering the first pharmaceutical composition to a subject in an amount of at least about 0.1 microgram of beraprost or a pharmaceutically acceptable salt thereof. 111. In the method according to item 79, administration includes delivering the first pharmaceutical composition to a subject in an amount of about 0.1 microgram to about 5000 micrograms of beraprost or a pharmaceutically acceptable salt thereof. 112. In the method according to item 79, administration includes delivering the first pharmaceutical composition to a subject in an amount of about 15 micrograms to about 90 micrograms of beraprost or a pharmaceutically acceptable salt thereof. 113. The method according to item 79, wherein the administration comprises delivering a first pharmaceutical composition to a subject in an amount of about 60 micrograms to about 360 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 114. The method according to item 79, wherein the CAR T cells are Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), Blincyto (blinatumomab), or a combination thereof. 115. A method of treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject, comprising administering CAR T cells and a first pharmaceutical composition to a subject, the first pharmaceutical composition comprising at least an effective amount of veraprost or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition is administered at a time when an increase in the level of one or more of the cytokines MIF, IL-5, IL-17A, IL-23, IFN-γ, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β, or one or more of the inflammatory biomarkers C-reactive protein (CRP) and ferritin detects the onset of CRS. 116. The method according to item 115, wherein the pharmaceutically acceptable salt is veraprost sodium salt. 117. The method according to item 115, wherein the veraprost comprises at least one of BPS-314d, BPS-314l, BPS-315d, and BPS-315l. 118. The method according to item 115, wherein the veraprost is BPS-314d (esveraprost sodium salt). 119. The method according to item 115, wherein CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, progressive, recurrent, or refractory diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma, method. 120. In the method according to item 115, CAR T cell administration is performed to treat cancer, and the cancer is a brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, or liver metastasis, method. 121. In the method according to item 115, CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, method. 122. In the method according to item 115, the subject is a mammal, method. 123. In the method according to item 115, the subject is a non-human primate, monkey, cat, dog, pig, cow, goat, horse, sheep, or rabbit, method. 124. In the method according to item 115, the subject is a human, method. 125. In the method according to item 115, simultaneously administer CAR cells and a first pharmaceutical composition to the subject, method. 126. In the method according to item 115, the first pharmaceutical composition is administered to the subject after the CAR T cells have been administered to the subject, method. 127. In the method according to item 115, the first pharmaceutical composition is administered to the subject about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the CAR T cells have been administered to the subject, method. 128. In the method according to item 115, the first pharmaceutical composition is administered to the subject about 3 days to about 7 days after the CAR T cells have been administered to the subject, method. 129. The method according to item 115, wherein the first pharmaceutical composition is administered for a period of about 1 day to about 30 days. 130. The method according to item 115, wherein the first pharmaceutical composition is administered for a period of about 7 days to about 14 days. 131. The method according to item 115, wherein the first pharmaceutical composition is administered starting 1 day before the administration of CAR T cells and continues for at least about 14 days. 132. The method according to item 115, further comprising administering the first pharmaceutical composition to the subject before administering CAR T cells. 133. The method according to item 115, wherein the CAR-T cells are autologous. 134. The method according to item 115, wherein the CAR T cells are allogeneic. 135. The method according to item 115, wherein the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof. 136. The method according to item 115, wherein the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, liquid, suspension, gel, syrup, slurry, suppository, patch, nasal drop, aerosol, injection, implantable sustained-release formulation, or mucoadhesive film. 137. The method according to item 115, wherein the administration comprises local delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof. 138. The method according to item 115, wherein the administration comprises oral administration or intravenous injection (IV) administration. 139. The method according to item 115, wherein veraprost or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms. 140. The method according to item 115, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of at least about 0.1 microgram of veraprost or a pharmaceutically acceptable salt thereof. 141. The method according to item 115, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 0.1 microgram to about 5000 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 142. The method according to item 115, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 15 micrograms to about 90 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 143. The method according to item 115, wherein the administration comprises the step of delivering the first pharmaceutical composition to a subject in an amount of about 60 micrograms to about 360 micrograms of veraprost or a pharmaceutically acceptable salt thereof. 144. The method according to item 115, wherein the CAR T cells are Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), Blincyto (blinatumomab), or a combination thereof. 145. A method for treating cytokine release syndrome, ICANS, or both, associated with CAR T cell administration in a subject, the method comprising the step of administering CAR T cells and a first pharmaceutical composition to the subject, wherein the first pharmaceutical composition comprises at least an effective amount of veraprost or a pharmaceutically acceptable salt thereof, The subject experiences CRS, ICANS, Parkinsonism effects, or a combination thereof, and the subject has received the administered CAR T cells and has received the administered first pharmaceutical composition, the subject needs to reduce treatment with at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof, compared to a similar subject who has received the administered CAR T cells and has not received the administered first pharmaceutical composition. 146. The method according to item 145, wherein the pharmaceutically acceptable salt is the sodium salt of beraprost. 147. The method according to item 145, wherein beraprost comprises at least one of BPS-314d, BPS-314l, BPS-315d, and BPS-315l. 148. The method according to item 145, wherein beraprost is BPS-314d (sodium salt of esberaprost). 149. The method according to item 145, the CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, progressive, recurrent or refractory diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma. 150. The method according to item 145, the CAR T cell administration is performed to treat cancer, and the cancer is brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, or liver metastasis. 151. The method according to item 145, the CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma. 152. The method according to item 145, wherein the subject is a mammal. 153. The method according to item 145, wherein the subject is a non-human primate, monkey, cat, dog, pig, cow, goat, horse, sheep, or rabbit. 154. The method according to item 145, wherein the subject is a human. 155. The method according to item 145, further comprising the step of administering a first pharmaceutical composition to the subject before administering the CAR-T cells. 156. The method according to item 145, the first pharmaceutical composition is administered to the subject before the CAR T cells are administered, and the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject. 157. The method according to item 145, wherein the CAR cells and the first pharmaceutical composition are administered to the subject simultaneously. 158. The method according to item 145, wherein the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject. 159. The method according to item 145, wherein the first pharmaceutical composition is administered to the subject about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after the CAR T cells are administered to the subject. 160. The method according to item 145, wherein the first pharmaceutical composition is administered to the subject about 3 days to about 7 days after the CAR T cells are administered to the subject. 161. The method according to item 145, wherein the first pharmaceutical composition is administered to the subject after the CAR T cells are administered to the subject and when the onset of CRS is detected. 162. The method according to item 145, wherein after the CAR T cells are administered to the subject, the first pharmaceutical composition is administered to the subject, and the onset of CRS is detected by an increase in the level of one or more of the cytokines IL-1α, IL-β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF. 163. In the method according to item 145, after the CAR T cells are administered to the subject, the first pharmaceutical composition is administered to the subject, and The onset of CRS is detected by an increase in the level of one or more of the cytokines IL-6, IL-10, IFN-γ, TNF-α. 164. In the method according to item 145, the first pharmaceutical composition is administered for a period of about 1 day to about 30 days. 165. In the method according to item 145, the first pharmaceutical composition is administered for a period of about 7 days to about 14 days. 166. In the method according to item 145, the first pharmaceutical composition is administered starting 1 day before the administration of the CAR T cells and continues for at least about 14 days. 167. In the method according to item 145, the CAR-T cells are autologous. 168. In the method according to item 145, the CAR-T cells are allogeneic. 169. In the method according to item 145, the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof. 170. In the method according to item 145, the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, solution, suspension, gel, syrup, slurry, suppository, patch, nasal drop, aerosol, injection, implantable sustained-release formulation, or mucoadhesive film. 171. In the method according to item 145, the administration includes local delivery, subcutaneous delivery, intravenous injection (IV) delivery, intramuscular injection (IM) delivery, intrathecal injection (IT) delivery, intraperitoneal injection (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof. 172. In the method according to item 145, the administration includes the step of oral delivery or intravenous injection (IV) delivery. 173. The method according to item 145, wherein beraprost or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms. 174. The method according to item 145, wherein the administration comprises delivering the first pharmaceutical composition to the subject in an amount of at least about 0.1 microgram of beraprost or a pharmaceutically acceptable salt thereof. 175. The method according to item 145, wherein the administration comprises delivering the first pharmaceutical composition to the subject in an amount of about 0.1 microgram to about 5000 micrograms of beraprost or a pharmaceutically acceptable salt thereof. 176. The method according to item 145, wherein the administration comprises delivering the first pharmaceutical composition to the subject in an amount of about 15 micrograms to about 90 micrograms of beraprost or a pharmaceutically acceptable salt thereof. 177. The method according to item 145, wherein the administration comprises delivering the first pharmaceutical composition to the subject in an amount of about 60 micrograms to about 360 micrograms of beraprost or a pharmaceutically acceptable salt thereof. 178. The method according to item 145, wherein the subject does not require treatment with at least one corticosteroid. 179. The method according to item 145, wherein the subject does not require treatment with tocilizumab. 180. The method according to item 145, wherein the subject does not require treatment with an IL-6 receptor antagonist. 181. The method according to item 145, wherein the CAR T cells are Abecma (idecabtagene vicleucel), Breyanzi (isocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel), Blincyto (blinatumomab), or a combination thereof. 182. A kit comprising: a first container containing a first pharmaceutical composition comprising at least an effective amount of beraprost or a pharmaceutically acceptable salt thereof; a second container containing a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof; and instructions for administering the first pharmaceutical composition, the second pharmaceutical composition, and the CAR T cells to a subject. 183. The kit according to item 182, further comprising a third container containing CAR-T cells. 184. The kit according to item 182, further comprising a fourth container containing water or an aqueous solution. 185. The method or kit according to any of the preceding items, wherein the beraprost is BPS-314d.

[0122] Further aspects of the invention are described in the following numbered sections: 1A. A method of treating cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or both, associated with CAR T cell administration in a subject, comprising administering to the subject a population of CAR T cells and a first pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and A method in which the first pharmaceutical composition does not reduce the killing cells mediated by a population of CAR T cells by about 5% or more. 2A. A method for treating cytokine release syndrome, ICANS, or both, associated with administration of CAR T cells in a subject, the method comprising administering to the subject CAR T cells, a first pharmaceutical composition, and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of beraprost or a pharmaceutically acceptable salt thereof, and the second pharmaceutical composition comprises at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof. 3A. The method according to 2A, wherein the corticosteroid is dexamethasone. 4A. The method according to 2A - 3A, wherein the subject needs to reduce the treatment with the second pharmaceutical composition as compared to a subject not administered the first pharmaceutical composition. 5A. A method for treating cytokine release syndrome, ICANS, or both, associated with administration of CAR T cells in a subject may comprise administering to the subject CAR T cells and a first pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition is administered when the onset of CRS is detected by an increase in the level of one or more of the cytokines MIF, IL-5, IL-17A, IL-23, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β, CCL2, CXCL9, CXCL-10, VEGF, CCL3, GCSF, CRP (C-reactive protein), and ferritin. 6A. A method for treating cytokine release syndrome and ICANS associated with CAR T cell administration in a subject, or ICANS, comprises administering to the subject CAR T cells and a first pharmaceutical composition, the first pharmaceutical composition comprising at least an effective amount of beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof, and wherein the first pharmaceutical composition is administered at a time when the onset of CRS is detected by an increase in the level of one or more of the cytokines MIF, IL-5, IL-17A, IL-23, IFN-γ, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β, CCL2, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, CXCL9, CXCL-10, VEGF, CCL3, GCSF, and GMCSF, CRP (C-reactive protein), and ferritin. 7A. A method for treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject, the method comprising administering to the subject CAR T cells and a first pharmaceutical composition, wherein the first pharmaceutical composition comprises at least an effective amount of beraprost or a pharmaceutically acceptable salt thereof, and wherein the subject has a reduced ICANS effect, parkinsonism effect, or both, as compared to a similar subject that receives the administered CAR T cells but does not receive the administered first pharmaceutical composition. 8A. The method according to any one of 1A - 7A, wherein the beraprost is BPS-314d (sodium esberaprost). 9A. A method for treating cytokine release syndrome, ICANS, or both associated with CAR T cell administration in a subject, the method comprising administering to the subject CAR T cells and a first pharmaceutical composition, the first pharmaceutical composition comprising at least an effective amount of BPS-314d (sodium esberaprost) or a pharmaceutically acceptable salt thereof, and wherein the first pharmaceutical composition is administered at a time when the onset of CRS is detected by an increase in the level of one or more of the cytokines Il-6, IFN-Y, and TNF-α. 10A. In the method according to any one of 1A to 9A, CAR T cell administration is performed to treat cancer, and the cancer is B cell lymphoma, progressive, recurrent or refractory diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high-grade B cell lymphoma, transformed follicular lymphoma, recurrent or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, brain tumor, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonal sarcoma, prostate cancer, sarcoma, liver metastasis, method. 11A. In the method according to any one of 1A to 10A, the CAR T cells are autologous CAR T cells or allogeneic CAR T cells, method.

Example

[0123] Example 1: Evaluation of CRS model mice This study includes three groups: a placebo control group, a positive control group (tocilizumab, or dexamethasone), and a CTO1681 treatment group. The CTO1681 treatment group has multiple arms to determine the dose response in a mouse model of CRS. These doses provide information necessary for a secondary in vivo model of CRS treatment related to CAR-T therapy.

[0124] Humanized mice (expressing human PBMC-HU-PBMC NSG™; commercially available from The Jackson Laboratory, Bar Harbor, Maine, USA) are administered either a control treatment or one of five doses of CTO1681 prior to CRS induction. To induce CRS, the treated mice are administered an OKT3 antibody (anti-CD3 monoclonal antibody) intraperitoneally (IP). Mice are sacrificed 24 - 48 hours after CRS induction, and cytokine production is quantified (peripheral and tissue).

[0125] As a result, it is shown that mice administered with CTO1681 before CRS induction had less undesirable cytokine production than the mice in the control group. The results will also show a reduction in the acute symptoms that occur within the first 48 hours in the mouse model. In a longer-term model, a survival advantage would be shown, but this particular example is not carried out that far.

Example

[0126] Example 2: Mouse model of CRS by CAR T cell therapy This test includes three groups (placebo control, dexamethasone control, CTO1681 dosage determined from the primary CRS mouse test in Example 1). Raji-luc tumor cells are injected (IP) into SCID mice or humanized mice (expressing human PBMC - HU-PBMC NSG (trademark)), and tumor growth is observed for about 3 weeks. Tumor burden is evaluated by bioluminescence.

[0127] CAR-T cell therapy (IP) is performed to induce CRS (performed about 2 - 3 days after CAR-T injection). Control and CTO1681 treatment (IP, BID, for 7 days) are started about 5 hours before CAR-T cell introduction. At the end of the 7-day treatment, the tumor volume is evaluated by bioluminescence, the mice are sacrificed for gross histopathological examination, and cytokine concentrations in the periphery and tissues are measured.

[0128] As a result, it is shown that mice administered with dexamethasone or CTO1681 had less undesirable cytokine production than the mice in the control group, indicating that CTO1681 is superior to dexamethasone. Both the dexamethasone control group and CTO1681 show a decrease in cytokine levels, but CTO1681 is expected to have a better survival rate than dexamethasone.

Example

[0129] Example 3: Human treatment without delay period Fifty to one hundred human subjects with B-cell lymphoma are divided into two groups: a control group and a CTO1681 treatment group. CAR T cells are infused into both groups. In the CTO1681 treatment group, CTO1681 is administered daily for 7 to 15 days starting the day before CAR T cell administration or simultaneously with CAR T cell administration. The clinical symptoms and cytokines of CRS are monitored daily in both groups. As a result, it is shown that the subjects administered with CTO1681 had reduced CRS symptoms and decreased undesirable cytokine production compared to the subjects in the control group.

Example

[0130] Example 4: Treatment with a predetermined delay time Eighty human subjects with mantle cell lymphoma are divided into two groups: a control group and a CTO1681 treatment group. CAR T cells are infused into both groups. In the CTO1681 treatment group, CTO1681 is administered daily for 11 days starting 3 days after CAR T cell administration. Cytokines are monitored daily in both groups. As a result, it is shown that the subjects administered with CTO1681 had reduced CRS symptoms and decreased undesirable cytokine production compared to the subjects in the control group.

Example

[0131] Example 5: Treatment with monitored delay time Forty subjects with acute myeloid leukemia are divided into two groups: a control group and a CTO1681 treatment group. CAR T cells are infused into both groups. Cytokines are monitored daily in both groups. In the CTO1681 treatment group, administration of CTO1681 is started when an increase in any of the cytokines IL-6, IFN-γ, or IL10 is detected. Treatment with CTO1681 is continued daily for 10 days. As a result, it is shown that the subjects administered with CTO1681 had reduced CRS symptoms and decreased undesirable cytokine production compared to the subjects in the control group.

Example

[0132] Example 6: Kit for cancer treatment The box is composed of a first container containing an effective amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof, and written instructions for administering the first pharmaceutical composition and CAR T cells to a subject. The instructions may be printed on paper and placed in the box, or may be a hyperlinked website with the instructions. The box is combined with a second container containing autologous or allogeneic CAR T cells. The box can optionally include a third container containing water or an aqueous solution for dissolving veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof.

Example

[0133] Example 7: In vitro evaluation of cytokine release after CTO1681 treatment To analyze the cytokine production inhibition by CTO1681, a cytokine release assay using normal human PBMC was developed and performed according to the parameters evaluated in previous pilot studies. In this assay, PBMC were obtained from 5 healthy donors.

[0134] The assay began with pretreatment of resting cells with a positive control drug (dexamethasone) or CTO1681. After pretreatment, the cells were stimulated with LPS or Poly(I:C). The supernatant was collected 24 - 48 hours after stimulation, and both viability (48 hours) and 29 cytokine concentrations (24 hours) were examined. Viability assessment confirmed no cytotoxicity associated with CTO1681. Statistical comparisons in the results of individual donors were performed between all test groups (individual stimulant - treatment pairs) within each donor group to determine not only the potential CTO1681 effect on cytokine production but also the effectiveness of the stimulation. Additionally, statistical analysis was performed on the pooled donor data for each cytokine stimulant - treatment pair to comprehensively determine the cytokine inhibition effect of CTO1681 on each cytokine.

[0135] Both LPS and poly(I:C) stimulated PBMCs from five donors, but some variability was observed. Neither stimulant was able to induce significant stimulation (stimulated cells vs. unstimulated control cells) in various cytokines from various donors. Similarly, there was donor-specific variability in the level of stimulation, particularly evident in donor 5, where the level of stimulation was low for all cytokines.

[0136] Such marked variability in cytokine responses was an unpredictable and unintentional result, but this phenomenon adequately represents the variability naturally observed in the human immune response. Considering both individual donors and pooled responses, administration of CTO1681 showed a significant decrease in 21 cytokines, as detailed in Table 1 below.

Table 1

[0137] These 21 cytokines include 10 cytokines that have not been previously identified in the studies of Gemmus Pharma, Inc. Furthermore, this study showed that the signals of IL-2, IL-6, IL-8, and IL-10 did not significantly decrease in this ex vivo assay. It is important to note that these four cytokines have been confirmed to decrease upon CTO1681 treatment in Gemmus Pharma's in vivo influenza (H5N1) treatment study. Additionally, IL-2, IL-6, and IL-10 decreased upon CTO1681 administration in the current in vivo influenza (H1N1) trial of CytoAgents. The differences in the decrease of IL-2, IL-6, and IL-10 observed between in vivo and ex vivo tests highlight an important aspect of the differences inherent in the assays, along with the caveat that a negative result in an ex vivo test is not necessarily a negative result in an in vivo test. Such results can sometimes be unexpected and unanticipated.

[0138] Although not analyzed in previous Gemmus Pharma, Inc. studies, the last cytokine measured in these assays was IFN-α. IFN-α is a type I interferon and is closely related to the antiviral response of the immune system but is generally not associated with suppression by inhibition of NFkB induction. CTO1681 significantly decreased the very low levels of IFN-α produced in this ex vivo system. This is not directly related to the suppression of IFN-α antiviral activity (IFN-α was not suppressed in previous in vivo CTO1681 influenza administration studies). Combining the Gemmus Pharma, Inc. studies with the current study, it was confirmed that administration of CTO1681 suppressed the production of approximately 25 cytokines.

Example

[0139] Example 8: Cell target - normal human PBMC Cytokine release assays were performed using normal human PBMC obtained from 5 donors of different ages, ethnicities, and genders. Normal human PBMC were obtained from Lonza's extensive catalog of cell reagents. The cells were thawed according to the manufacturer's instructions, washed with complete growth medium, and viability was assessed using trypan blue staining. A cell stock solution of 2×10 6 cells / mL was suspended in complete growth medium and dispensed into black-walled plate cell wells (2X10 5 / well) for the assay. The cells were incubated at 37 °C, 5% CO2 for 1 hour.

[0140] Treatment, stimulation, cell collection, and cell viability measurement After resting the cells, 100 μl of CTO1681 was added to the appropriate wells at a final administration concentration of 750 μM. Vehicle and dexamethasone (final administration concentration 1 μM) were also each added to the appropriate wells at 100 μl each, and all were incubated at 37 °C and 5% CO2 for 15 minutes. For cell stimulation, 20 μL of a 1.0 ng / mL LPS solution or a 250 μg / ml Poly(I:C) solution was added to the appropriate wells according to the group designation. After 24 hours, 100 μL of supernatant was collected from all wells and stored for cytokine analysis. Approximately 4 hours before the final harvest, alamar blue dye was added to all wells. At 48 hours after stimulation, all samples were collected and the cell viability of the supernatant was examined. When added to cells, AlamarBlue is modified by the reducing environment of viable cells and comes to emit strong fluorescence. Therefore, an increase in fluorescence after cell staining indicates an increase in viability. In the viability assessment with alamar blue, no cytotoxicity was observed when the cells of donors 1 to 5 were treated with CTO1681. Similarly, no signs of cytotoxicity were observed even when the cells were stimulated with LPS or Poly(I:C).

[0141] Cytokine measurements, final results, and conclusions Cytokines were evaluated in samples collected 24 hours after stimulation using the MAGPIX device with Luminex multiplex technology and a Millipore multiplex kit. The concentration of each cytokine was evaluated based on a standard curve. Sample values collected by multiplex analysis below the detection limit were not included in the final determination. Since the undiluted donor IL-6 concentration exceeded the linearity of the assay, it was too high to be evaluated with a typical standard curve. Therefore, the IL-6 samples were re-run at a 1:5 dilution. The results of the cytokine concentration (pg / ml) for each cytokine were plotted as stimulant-treatment pairs for individual donors. Statistical comparisons of the results for individual donors for all cytokines were performed between control groups via one-way ANOVA with multiple comparisons. ANOVA analysis was performed between the stimulated groups (LPS only, Poly(I:C) only) and the control groups (media only, dex, vehicle) to determine the effectiveness of the stimulation and dexamethasone positive control, and the possibility of a vehicle effect. Next, a normal one-way t-test was used to compare the vehicle group with the CTO1681 stimulation to determine if there was an effect by the test item. To evaluate the overall effectiveness of CTO1681 cytokine suppression, the stimulant-treatment pairs of pooled five donors were plotted. Statistical analysis (Wilcoxon matched-pairs signed rank test) was performed on the results of the pooled donors for each cytokine plotted similarly. The final result of cytokine suppression was determined by evaluating both the pooled results and the results of individual donors. CTO1681 decreased 21 out of 29 cytokines analyzed in this study.

[0142] Observations from in vitro treatment results The effective stimulation of PBMC by LPS or Poly(I:C) was determined by comparison between the medium control group and the stimulated (untreated) group. Both mitogens stimulated most cytokines, but there were variations among donors and among cytokines. VEGF and IL-7 could not be consistently stimulated to levels exceeding the lower limit of the standard curve by either LPS or Poly(I:C), so they were not calculated in the pooled analysis. The failure to stimulate was likely an artifact of the ex vivo assay, which had to be performed with a relatively short observation time and lacked the complexity of a complete in vivo system. Additional cytokines were successfully stimulated, but no significant level of stimulation was obtained compared to unstimulated medium wells. Statistically significant increases with LPS or Poly(I:C) were detected in the results for TGF-β and IL-2 in a few donors. For GM-CSF, IL-8, IL-17A, CXCL10 / IP-10, and CCL5 / RANTES, no statistical significance was obtained in the Poly(I:C) group. The variation in the general stimulation levels among donors was also a notable observation. Overall, donor 5 had significantly lower stimulation levels for all cytokines when treated with both Poly(I:C) and LPS. Due to this insufficient stimulation, the data from donor 5 were not included in the pooling and final analysis for IL-2, IL-4, IL-5, IL-9, IL-15, IL-12, IL-13, IL-17, IL-18, GM-CSF, and PDGF. The stimulation of donor 5 for the cytokines IL-10 and CXCL10 / IP-10 exceeded the lower limit of detection, but the values were much lower (1 - 3 logs) than those of the other four donors. Therefore, the data from donor 5 were not included in the pooled analysis for these two cytokines, but the individual results were considered in the final determination of the efficacy of CTO1681. Similarly, the results of TGF-β for donor 1 were all below the lower threshold from the standard curve, so the data for this donor regarding this cytokine were not included in the overall pooled analysis of the efficacy of CTO1681. Donor 4 had significantly lower overall stimulation levels for several cytokines (but not below the threshold for inclusion in the calculations).Taking these differences together, it is emphasized that the general immune response to mitogens shows natural variation within the population that appears to be due to genetic differences between subjects.

[0143] Similar to the observations noted regarding the variability seen between groups and donors that were successfully stimulated, similar unpredictability was also observed in the results of the treatment groups in both the stimulation method and the donors. CTO1681 was able to reduce the concentrations of several cytokines in which dexamethasone showed no effect. Overall, CTO1681 suppressed the production of 21 out of the 29 cytokines evaluated. Only three cytokines, GCSF, TGF-β, and IL-8, were not suppressed in production by both CTO1681 and dexamethasone, despite successful stimulation in both LPS- and Poly(I:C)-induced cells. Similarly, there were examples where dexamethasone was unable to suppress cytokine production while CTO1681 was successful in reducing the cytokine concentrations produced (Poly(I:C)-stimulated cells - IL-4, CCL2 / MCP1), and conversely, examples where CTO1681 was unable to suppress cytokine production while dexamethasone was successful in reducing the cytokine concentrations (Poly(I:C)-stimulated cells - IL-17, IL-9). Furthermore, as observed with FGF and CCL3 / MIP1a, there were also examples where both dexamethasone and CTO1681 significantly reduced cytokine production with one stimulatory mitogen (LPS), but not with the other (Poly(I:C)). The complexity of evaluating the entire study, which involved comparing control and CTO1681 in two different stimulants, five donors, and 29 cytokines, cannot be denied. Ultimately, the conclusions based on the evaluation of the pooled data and the data of individual donors showed suppression of cytokine production by CTO1681 in the following cytokines - IL-1-α, IL-1β, IL-4, IL-5, IL-12,p70, IL-13, IL-15, IL-18, TNF-α, CCL2 / MCP-1, CCL5 / RANTES, CXCL9 / MIG, CXCL10 / IP-10, IFN-γ, IFN-α, and PDGF-, and suppression of cytokines - IL-9, IL-17, CCL3 / MIP1a, GM-CSF, FGF- by LPS stimulation only was observed.

[0144] There are many operational factors to consider in the interpretation of the mechanisms and actions underlying the results of these tables. Each stimulant can elicit a response via different cell receptors and can take different timings in the subsequent cell cascade. Furthermore, although the 24-hour analysis time is necessary due to the nature of in vitro studies in this research, it is highly likely that it is not sufficient time for the production of all the cytokines to be evaluated to start or reach significant expression levels. Additionally, due to the lack of a complete biological system, the recruitment of immunological factors that may be involved in various cytokine reactions also does not exist in a complete state as in an intact physiological system. Therefore, negative results should be observed with such knowledge, and it is necessary to recognize that the in vivo results may be somewhat different. For example, in this ex vivo study, IL-2, IL-6, IL-8, and IL-10 were all negative for CTO1681 inhibition. However, in simultaneous data from in vivo lethal influenza H1N1 tests (and previous Gemmus Pharma Inc. H5N1 in vivo tests), CTO1681 significantly suppressed the production of these cytokines.

[0145] The suppression of ex vivo IFN-α activity by CTO1681 was unexpected and unanticipated. IFN-α is a type I interferon and is not normally associated with the same pathways or patterns as standard inflammatory cytokines. Type I interferons are an important part of the antiviral immune response. Furthermore, since IFN-α is produced from different promoters and transcriptional elements than standard inflammatory cytokines, type I interferons are not normally associated with NFκB production. The pathway of IFN-α from the cell receptor to the feedback loop is completely different from that of inflammatory cytokines. IFN-α was incorporated into this study initially not as an efficacy metric but as a safety metric. The results seen here are the reverse of the above-described ex vivo / in vivo phenomena for IL-2, IL-6, etc. The suppression of IFN-α by CTO1681 is clear from the data of these ex vivo studies, but our in vivo data show the opposite, namely, that there is no significant suppression of IFN-α concentration by CTO1681 treatment during lethal H1N1 influenza infection. What is important when evaluating these two contrasting results is to recognize that there is a large difference between the amount of INF-α produced by ex vivo-stimulated PBMCs and the amount of INF-α induced by active viral replication in vivo. The amount of INF-α produced by LPS-stimulated PBMCs was less than 101 pg / ml, while the level stimulated by Poly(I:C) was approximately 101 pg / ml. In H1N1 lethally infected mice, approximately 103 pg / ml of INF-α was detected in the BALF fluid. The nominal decrease in in vitro production of INF-α (although statistically significant) was less than 2-fold. At the levels of INF-α produced during active viral replication, this slight decrease would not result in a significant change in BALF INF-α. There are two stages of INF-α production, an early and a late stage, and the late-stage production correlates with high levels of INF-α associated with viral infection. One of the transcription factors responsible for a significant part of the early-stage INF-α production is IRF7. IRF7 has an NFkB response element in its promoter.Mechanistically, this would be that CTO1681 indirectly affects INF-α production and has a significant but minimal impact on the type 1 interferon produced, as observed in this ex vivo assay.

[0146] In summary, this data and information demonstrate the broad efficacy of CTO1681 cytokine suppression against 25 cytokines affected by new cytokines (10 types compared to previous Gemmus Pharma, Inc. data) by treating PBMCs stimulated with this molecule ex vivo.

Example

[0147] Example 9: Kit containing veraprost and tocilizumab The box is configured to contain a first container housing a first pharmaceutical composition containing an effective amount of veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof, a second container housing a second pharmaceutical composition containing an effective amount of tocilizumab, and written instructions for administering the first pharmaceutical composition, the second pharmaceutical composition, and CAR T cells to a subject. The instructions may be printed on paper and placed in the box, or may be a hyperlinked website with the instructions. The box is combined with a third container containing autologous or allogeneic CAR T cells. The box may optionally include a fourth container containing water or an aqueous solution for dissolving veraprost, a veraprost isomer, or a pharmaceutically acceptable salt thereof and tocilizumab.

Example

[0148] Example 10: Combined therapy of veraprost and tocilizumab Subjects suffering from cancer are treated, one day before the administration of CAR T cells, with orally administered beraprost, a beraprost isomer, or a pharmaceutically acceptable salt thereof. Beraprost, a beraprost isomer or a pharmaceutically acceptable salt thereof is further administered continuously for at least 14 days. Tocilizumab and / or corticosteroids can be added according to a physician's instructions, but the required amount is reduced compared to similar subjects not receiving beraprost, a beraprost isomer or a pharmaceutically acceptable salt thereof. An improvement in clinical outcome is observed by combination compared to other subjects. It is observed that the ICANS effect is reduced and the average incidence of hospitalization and the average duration of hospitalization are reduced in the subjects.

Example

[0149] Example 11: Phase 1b trial regarding prevention or reduction of CAR T cell - induced toxicity The Phase 1b trial of this study is a multi-center, non-blind, dose-escalation, safety and pharmacokinetics (PK) study of CTO1681 in B-NHL patients who received a commercially available CD19-directed CAR T cell therapy. This study is conducted using a Bayesian optimal interval (BOIN) design to provide dose-escalation information in the B-NHL patient cohort. Screening, enrollment, pre-treatment with lymph node dissection (LD) chemotherapy, initiation of the test drug (CTO1681), CAR T cell infusion, continuous treatment with CTO1681 (for a total of 15 days), and safety follow-up. Patients in whom CRS or ICANS has not resolved by day 15 may continue treatment for an additional 13 days (for a total of 28 days) at the discretion of the treating physician, subject to the approval of the medical monitor and the sponsor. The timing of treatment with CTO1681 is carried out according to the following schedule in relation to LD and CAR T cell infusion:

[0150] Days 12 to 3: LD chemotherapy (if there is a legitimate reason based on the prescribing information [PI] of the CAR T cell manufacturer)

[0151] Day 1: Initiate the test drug (CTO1681) approximately 24 hours before CAR T cell infusion. The test drug is administered orally three times a day (TID) for 15 days.

[0152] The total daily dose of CTO1681 will be 30, 60, or 90 μg, depending on the cohort in which each patient is enrolled. The investigational drug must be taken within 30 minutes after a meal or a snack.

[0153] Day 1: CAR T cell infusion (by the PI of the CAR T cell manufacturer). Patients will be monitored for up to 6 months to record tumor responses.

[0154] Introduce the BOIN algorithm and provide advice on dose escalation. This algorithm aims to identify the maximum tolerated dose (MTD) with a toxicity occurrence probability of less than 33%. The observation period for dose-limiting toxicity (DLT) is 42 days after the first administration of the investigational drug.

[0155] Examine three dose cohorts for up to 27 patients. The first dose level (30 μg) will be enrolled in a cohort of 3 patients. At the 60 μg and 90 μg dose levels, 6 patients will be enrolled first. The total number of enrolled patients per cohort at each dose level will be a maximum of 9.

[0156] The MTD is defined as the maximum dose achieved until a toxicity occurrence rate of 33% or more is observed. The recommended phase 2 dose (RP2D) is the MTD or the highest dose at which no MTD discontinuation occurred. The target toxicity rate is 33% or less. See Table 2.

Table 2

Example

[0157] Example 12: Phase 2a trial Phase 2a Trial: The Phase 2a trial is conducted as a double-blind, randomized, placebo-controlled trial, and approximately 104 patients are enrolled. Patients are treated with the RP2D of CTO1681 established in the Phase 1b trial or with placebo. Patients are randomly assigned to the CTO1681 group and the placebo group at a 1:1 ratio before receiving LD chemotherapy. If multiple commercially available CAR T cell products are carried over from the Phase 1b trial, efforts are made to balance as many CAR T cell products as possible. Each patient follows the same trial procedures as in the above-mentioned Phase 1b trial portion. The treatment duration is determined based on the results and recommendations of the interim analysis after the completion of the Phase 1b trial (e.g., average number of treatment days, number of clinician-led requests for extension, recommendations of the Safety Review Committee (SRC), etc.). This part of the trial includes a PK sub-study at selected sites. Patients enrolled in this part of the trial have their tumor responses monitored for up to 12 months after CAR T cell infusion.

[0158] In both the Phase 1b trial and the Phase 2a trial, safety, efficacy, and biomarker evaluations are performed at intervals specified in the protocol according to the Schedule of Events.

[0159] Trial patients are hospitalized for the observation and management of CAR T cell-related toxicities based on the facility's guidelines. All assessments of CRS and ICANS follow the criteria of the American Society for Transplant and Cellular Therapy (ASTCT). Detailed management guidelines for CRS and ICANS are described in the protocol and are consistent with standard treatment.

[0160] Axicabtagene ciloleucel [YESCARTA (registered trademark)], tisagenlecleucel [KYMRIAH (registered trademark)], and lisocabtagene maraleucel [Breyanzi (registered trademark)], which are currently commercially available CD19-directed CAR T cell therapies for B-NHL, are included in the Phase 1b trial portion of this trial. Based on the interim analysis results of the Phase 1b trial, one or more of these CAR T cell therapies are carried over to the Phase 2a trial.

Example

[0161] Example 13: Management of CRS and ICANS For patients who develop CRS, CTO1681 should be continued, except as described in the "Management of DLT" section of the protocol. Detailed management guidelines for CRS and ICANS are provided to the principal investigators at participating trial sites to standardize care across sites. These guidelines are consistent with the standard treatments outlined in the prescribing information for the CAR T cell product, except for the use of prophylactic steroids or tocilizumab.

Example

[0162] Example 14: Main dose - limiting toxicity The main DLT criteria are as follows: Hypotension not attributable to the underlying CAR T cell therapy and defined as a documented sustained (≥ 2 hours) systolic blood pressure < 90 mmHg that does not respond to basic medical therapy (i.e., fluid challenge, vasopressors). Bleeding: Clinically significant bleeding (excluding accidental cuts, periodontal bleeding, menstrual bleeding, hemorrhoids, and easily controllable epistaxis). Hepatic dysfunction: Manifestation of transaminase elevations > 5 × upper limit of normal (ULN; Common Terminology Criteria for Adverse Events [CTCAE] Grade 3). QTc prolongation: Corrected QT interval (QTcF) ≥ 501 msec by Fridericia's formula, or a change from baseline of ≥ 60 msec that persists for ≥ 5 minutes. Ventricular tachycardia requiring urgent medical intervention (CTCAE Grade 3).

Example

[0163] Example 15: Management of dose - limiting toxicity DLT of hypotension or hepatic dysfunction: CTO1681 is interrupted until the DLT resolves. If the DLT recurs, administration of CTO1681 is discontinued. DLT of bleeding: CTO1681 is discontinued.

[0164] DLT of QTc prolongation or ventricular tachycardia: If the patient develops grade 3 or 4 CRS, CTO1681 is interrupted.

Example

[0165] Example 16: Objectives Phase 1b trial: Determine the RP2D of CTO1681 and, if necessary, the MTD in B-NHL patients receiving CAR T cell therapy.

[0166] Determine the preliminary safety profile of CTO1681 in B-NHL patients receiving CAR T cell therapy.

[0167] Phase 2a trial: Preliminarily determine the CRS prevention and reduction effect of CTO1681 compared with placebo.

[0168] Determine the preliminary efficacy of CTO1681 for ICANS prevention or reduction compared with placebo.

[0169] Determine the expanded safety profile of CTO1681 in B-NHL patients receiving CAR T cell therapy.

[0170] Examine the potential impact of CTO1681 on the antitumor activity of CAR T cell therapy, comparing with past data and placebo.

Example

[0171] Example 17: Endpoints Phase 1b trial: Primary Adverse events (AEs) and abnormal clinical laboratory values, including DLTs characterized by type, frequency, timing, severity (graded according to CTCAE v5.0), severity, and relationship to CTO1681. Exploratory: Incidence, severity, onset, and duration of CRS (incidence, severity, onset, and duration of CRS from the start of CAR T-cell therapy to 30 days after the start of CAR T-cell therapy, as measured by the area under the curve (AUC) of the CRS grade curve) Incidence, severity, onset, and duration of ICANS measured by the ICANS grade AUC from the start of CAR T-cell therapy to 30 days after the start of CAR T-cell therapy Number of treatment days with CTO1681 Incidence of grade 3 or higher CRS and grade 3 or higher ICANS Antitumor activity including overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS) by the treating investigator Frequency and duration of hospitalization and intensive care unit (ICU) admission due to CRS or ICANS Levels and persistence of CAR-T cells Use of tocilizumab, steroids, and other anti-cytokine therapies in the treatment of CRS or ICANS PK concentration profile of CTO1681 Mechanism of action (MOA) of CTO1681 and biomarkers related to immune response Immune reconstitution Phase 2a trial: Primary Incidence, severity, onset, and duration of CRS (measured by the CRS grade AUC from the start of CAR T-cell therapy to 30 days after the start of CAR T-cell therapy). Secondary: Additional evaluation of the incidence and severity of CRS: Maximum grade CRS Incidence of grade 2 or higher CRS Incidence of grade 3 or higher CRS Duration of grade 1 or higher CRS Incidence, severity, onset, and duration of ICANS measured by the ICANS grade AUC from the start of CAR T-cell therapy to 30 days after the start of CAR T-cell therapy Additional evaluation of the onset rate and severity of ICANS: Maximum grade ICANS Incidence of grade 2 or higher ICANS Incidence rate of ICANS of grade 3 or higher Duration of ICANS of grade 1 or higher Frequency and duration of hospitalization and ICU admission due to CRS or ICANS Use of tocilizumab, steroids, and other anti-cytokine therapies in the treatment of CRS or ICANS Antitumor activity (including ORR, DOR, PFS, OS reviewed by the principal investigator of the clinical trial) Exploratory: Levels and persistence of CAR-T cells PK concentration profile of CTO1681 Biomarkers related to the MOA and immune response of CTO1681 Immune reconstitution

Example

[0172] Example 18: Number of patients (planned) Phase 1b trial: 15 - 27 Phase 2a trial: 104

Example

[0173] Example 19: Inclusion criteria 1. 18 years of age or older. 2. Patients who have received leukapheresis and are scheduled to receive protocol-specified commercially available CD19-directed CAR T cell therapy for B NHL without prophylactic administration of corticosteroids for CRS. Eligible patients for this trial must have relapsed or refractory DLBCL after at least 2 lines of systemic therapy. 3. Meet all eligibility criteria for CAR T cell therapy according to institutional guidelines. 4. Adequate organ function: A. Serum creatinine ≤ 1.5 × ULN, or estimated glomerular filtration rate (eGFR) ≥ 60 mL / min / 1.73m by the Cockroft-Gault formula 2 . B. Serum alanine aminotransferase (ALT) / aspartate aminotransferase (AST) ≤ 2.5 × ULN. Except for patients with Gilbert's syndrome with total bilirubin level ≥ 3.0 × ULN and indirect bilirubin level ≤ 1.5 × ULN, bilirubin level ≤ 2.0 mg / dL. D. Left ventricular ejection fraction (LVEF) ≥ 40% as determined by echocardiogram (ECHO) or MUGA, and no clinically significant pericardial fluid accumulation. E. Platelets ≥ 50,000 / mm3. F. Absolute neutrophil count > 1000 / μL G. Absolute lymphocyte count > 100 / μL 5. Measurable lesions sufficient to be evaluated according to the Lugano criteria are recorded. 6. Eastern Cooperative Oncology Group performance status is 0 - 1. 7. Female participants with the potential for pregnancy and all male participants must agree to use contraception approved by the principal investigator during the administration of the investigational drug and for 30 days thereafter. 8. Patients who can provide written informed consent before pre - treatment, or patients who have a legal representative who can provide informed consent on their behalf.

Examples

[0174] Example 20: Exclusion criteria 1. Cytotoxic chemotherapy within 14 days after leukapheresis. 2. Clinically significant malabsorption syndrome and dysphagia (e.g., dysphagia, gastroesophageal reflux disease) as evaluated by the principal investigator. 3. Clinically significant electrolyte abnormalities (e.g., hypokalemia, hypomagnesemia) as evaluated by the principal investigator. 4. If there are abnormalities or clinically important abnormalities on electrocardiogram at the time of screening and baseline (day 1). This includes, but is not limited to, cases where the QTcF value is ≥ 450 msec in men and ≥ 470 msec in women. Patients with QTcF values in the borderline range, or patients with difficult - to - interpret conditions such as bundle - branch block, or patients with overlapping T and U waves are excluded. 5. Have a history of arrhythmia (e.g., bradycardia, congenital long QT syndrome, atrial fibrillation), and / or require therapeutic anticoagulation / antiplatelet therapy. 6. Clinically significant (i.e., active) cardiovascular diseases including cerebrovascular disorder / stroke (within less than 6 months before registration), myocardial infarction (within less than 6 months before registration), or unstable angina, congestive heart failure of New York Heart Association classification class III or higher. 7. Uncontrolled thromboembolic events or recent severe bleeding. 8. Need to continue therapeutic anticoagulation therapy, antiplatelet therapy, or fibrinolytic therapy. 9. Patients who, in the judgment of the principal investigator of the clinical trial, are unlikely to follow the trial procedures or are considered ineligible for registration.

Example

[0175] Example 21: Investigational drug, dosage, and administration method Phase 1b: CTO1681 is administered as 10 μg tablets. In cohort 1, CTO1681 tablets are orally administered at a total daily dose of 30 μg for 15 days or more but within 28 days. In cohorts 2 and 3, CTO1681 tablets are orally administered at total daily doses of 60 μg and 90 μg, respectively, for at least 15 days and for a period not exceeding 28 days. Dose changes within a cohort and dose modifications are not permitted. Phase 2a: CTO1681 is administered as 10 μg tablets and may be administered as multiple tablets at a time depending on the dose determined by the results of Phase 1b. The treatment period is also determined by the results of the Phase 1b study. Note: Since CTO1681 has a food effect, it should be administered within 30 minutes after a meal or a snack.

Example

[0176] Example 22: Trial period (per patient) Phase 1b: Approximately 7 months (including screening) Phase 2a: Approximately 13 months (including screening)

Example

[0177] Example 23: Reference therapy, dosage, and administration method Phase 1b: None Phase 2a: The placebo tablets are orally administered in the same manner as the active treatment. The placebo tablets are identical in size, shape, color, and packaging to the CTO1681 10 μg tablets.

Example

[0178] Example 24: Safety review The SRC consists of the principal investigator of the trial, the medical staff of the sponsor of the trial (or a qualified surrogate), and, if necessary, ad hoc members (e.g., a statistician, a PK specialist). The SRC monitors the course and safety of the patients throughout the entire duration of the Phase 1b trial. The regular systematic review of AEs serves as the basis for temporarily suspending or prematurely terminating the trial. Unanticipated SAEs related to CTO1681 serve as the main criterion for temporarily suspending or terminating the trial. If DLTs related to CTO1681 occur in more than 33% of the patients within a cohort (excluding fevers, hematological toxicities, CRS or ICANS, or events not related to the underlying disease or CAR T cell therapy), this indicates that the MTD has been reached and the trial is terminated.

[0179] During the Phase 1b trial period of this study, the SRC reviews the safety data after the end of the DLT evaluation period (42 days after the first administration of CTO1681). To determine whether dose escalation can occur in the cohort at each dose level according to the BOIN algorithm, the SRC bases its decision on the safety data at the earlier of 42 days after the first administration of the investigational drug if the treatment period of the last-treated patient in the current cohort is at most 28 days, or 14 days after the last administration if the treatment period of the last-treated patient in the current cohort is less than 28 days. Dose increases, additional enrollment of subjects (up to a maximum of 9 per dose level), or termination of the trial are possible. The cumulative safety data are reviewed at each SRC meeting. The cohort is suspended or terminated, and if any of the suspension or termination rules are met, the SRC reviews and determines the relationship between the AE and CTO1681.

[0180] After at least 1 month of follow-up from the first administration of CTO1681 in the Phase 1b trial is completed, or after the trial of all patients enrolled in the Phase 1b trial is prematurely terminated, an interim analysis summarizing the results of the Phase 1b trial is conducted. The SRC advises on the progression to Phase 2a of this trial and the selection of the RP2D based on safety, including available anti-tumor efficacy data. The SRC also advises on the dosing schedule and the recommended CAR T cell product to include in the Phase 2a trial.

[0181] The SRC can also advise on any protocol modifications necessary regarding patient safety through the Phase 1b trial.

[0182] An Independent Data Monitoring Committee (IDMC) consisting of 2 CAR T / hematology-oncology experts and 1 independent biostatistician is established to monitor safety data during the Phase 2a trial. The IDMC conducts a planned review of safety data after approximately 20, 40, 60, 80, and 104 patients have been followed for at least 1 month after their first exposure to the investigational product, or after the trial has been prematurely terminated. The trial is suspended or terminated if any of the pre-specified stopping or termination rules are met. If a death is determined by the treating investigator and confirmed by the sponsor, and the death is consistent with known side effects of CTO1681, such as a DLT identified as a stopping criterion during the Phase 1b trial, an ad hoc meeting of the IDMC is convened. Additionally, the IDMC meets more frequently as needed to consider serious adverse events (SAEs) and make recommendations regarding the timing of suspension of enrollment or termination of the trial.

[0183] IDMC will review the complete response (CR) rate according to a pre-specified set of non-binding assessments of the impact on anti-tumor efficacy, which are planned to be conducted at the 40th, 60th, and 80th patient safety data reviews. This assessment includes patients who achieved CR according to the Lugano Response Criteria, patients with the best anti-tumor efficacy in progressive disease, patients who discontinued the trial early, or patients who were followed for at least 3 months from the first administration of CAR T cell therapy. For each assessment of the impact on anti-tumor efficacy, the CR rate and the exact Clopper-Pearson 95% CI are calculated for patients randomly assigned to CTO1681, and the upper limit of the CI is compared with the benchmark rate derived from the lower limit of the 95% CI of the 6-month CR rate obtained from the prior literature. The lower limit value is determined at the time when the sponsor selects the CAR T cell therapy to be incorporated into the Phase 2a trial at the end of the Phase 1b trial. For the planned safety data reviews or anti-tumor efficacy impact analyses, the trial registration will not be suspended unless justified according to the safety stopping rules.

[0184] Develop a formal charter that specifies the rules and scope of responsibilities of the SRC and IDMC.

Example

[0185] Example 25: Criteria for trial suspension or termination If any of the following events occur within 42 days after the first treatment with the investigational drug and the treating physician determines that they are not related to the underlying disease, CAR T cell therapy, or events or factors unrelated to trial participation, they will be reported immediately to the SRC (for the Phase 1b trial) or IDMC (for the Phase 2a trial), and a special meeting will be scheduled to review all available safety data. Thereafter, the committee will provide recommendations on the next steps. Criteria for suspension Phase 1b trial: The DLT rate of CTO1681 exceeds 33%: Bleeding: Clinically significant bleeding events (excluding accidental cuts, periodontal disease, menstruation, hemorrhoids, and easily controllable epistaxis). Hypotension: Systolic blood pressure less than 90 mmHg for a prolonged symptomatic period (over 2 hours) and unresponsive to basic medical treatment. QTc prolongation: QTcF ≥ 501 msec or a change from baseline of 60 msec lasting for 5 minutes or more. Ventricular tachycardia (CTCAE grade 3) Elevated transaminases > 5 × ULN (CTCAE grade 3) In the assigned cohort, two or more patients experience CTCAE grade 4 toxicity (excluding events considered to be due to disease progression / underlying disease or CAR T cell therapy, such as fever, hematological toxicity, CRS / ICANS, etc.). Death related to the trial, not related to underlying disease or CAR T cell therapy. Phase 2a trial: The principal investigator of the trial determines death and the agent of the trial sponsor confirms that it is consistent with known side effects of CTO1681 (such as DLT identified as a stopping criterion in the Phase 1b trial). After safety review, if it is determined that the cohort and / or trial can proceed safely, enrollment and treatment will proceed as planned. If there are unresolved issues, the trial can proceed according to the recommendations of the SRC or IDMC. Stopping criteria Phase 1b trial: The DLT rate of CTO1681 exceeds 33%. CTCAE grade 4 toxicity (unmanageable, unexpected) evaluated by the principal investigator of the trial and confirmed by the SRC and sponsor to be likely, probably, or definitely related to CTO1681 (excluding CAR T cell therapy-related toxicities such as fever, hematological toxicity, CRS / ICANS, etc.). Death due to CTO1681-related DLT not related to underlying disease, CAR T cell therapy, or exogenous events (unrelated to the investigational drug or trial procedure). Specifically Fatal bleeding. Refractory hypotension leading to death. Refractory ventricular tachycardia leading to death. Phase 2a trial: CTCAE grade 4 toxicities (unmanageable, unexpected) that occurred in more than 33% of the patients administered, and that are likely, probably, or definitely related to CTO1681 (excluding fever, hematological toxicities, CRS / ICANS). Death due to CTO1681-related DLT not related to underlying disease, CAR T cell therapy, or exogenous events (unrelated to the investigational drug or the test procedure). Specifically Fatal bleeding. Refractory hypotension leading to death. Refractory ventricular tachycardia leading to death. The sponsor can decide to discontinue or modify the trial based on the recommendations of the SRC / IDMC and the overall evidence. The SRC can recommend discontinuation of the trial if it determines that the safety of the patients may be compromised by continuing the trial. The IDMC can recommend discontinuation of the trial based on safety or futility.

Example

[0186] Example 26: Statistical methods Consideration of sample size: Phase 1b trial:: A maximum sample size of 27 (maximum 9 per dose level) was selected to support dose escalation of CTO1681 and selection of dose levels by RP2D according to the BOIN algorithm. The safety results of the Phase 1b trial contribute to the treatment duration and the decision on CAR T cell therapy to be continued in the Phase 2a trial.

[0187] Phase 2a trial: The sample size was selected so that sufficient power would be obtained for the selection of primary and secondary evaluation items. Although the CRS grade AUC is the primary evaluation item, there is also interest in evaluating the impact of CTO1681 on other indicators of CRS severity in order to better understand the sensitivity to capture the treatment effect.

[0188] When calculating the CRS grade AUC for each patient as the sum of the daily maximum (non-zero) grade CRS over the efficacy observation period (30 days after the start of CAR T cell therapy), with a sample size of 104 (52 in each of the CTO1681 group and the placebo group), a one-sided type I error rate of 0.05 using a t-test for the difference in means gives a power of 85% or more to detect a true between-group difference in mean AUC of 4.5 CRS grade days (standard deviation 8).

[0189] With a sample size of 104 (52 in each group), a power of 85% or more, and a one-sided type I error rate of 0.05, using the Wilcoxon-Mann-Whitney test, assuming that in the case of no treatment, 35% of CAR T cell patients experience a maximum grade 1 CRS, 20% experience a maximum grade 2, 12% experience a maximum grade 3, and 8% experience a maximum grade 4, and in the case of treatment with CTO1681, 30% of patients experience a maximum grade 1, 10% experience a maximum grade 2, and 10% experience a maximum grade 3, the difference in ordinal variables was established.

[0190] Since the sample size is 104 (52 in each group), the power to detect the difference in proportions where the control group experiencing CRS of Grade≧2 was 40% while the CTO1681 group was 20% is 65% or more with a one-sided type I error rate of 0.05. Analysis population: The modified Intent-to-Treat (mITT) population includes all patients who receive both CAR T cell therapy and at least one dose of the investigational drug (CTO1681 or placebo). Patients are randomly grouped. The standard ITT population including all randomized patients may be analyzed as a robustness test of the mITT.

[0191] The safety population includes all patients who receive at least one dose of the investigational drug (CTO1681 or placebo). Patients are classified into treatment groups. The PP (Per-Protocol) Population includes all patients in the mITT Population who received CAR T cell therapy 5 or more times and had no major protocol violations. Patients are classified into treatment groups.

[0192] The dose-limiting toxicity (DLT) Population includes all Phase 1b patients who received both CAR T cell therapy and at least one dose of CTO1681. Patients who discontinued the study for reasons unrelated to the study procedure, conduct, or treatment before experiencing DLT and before the end of the DLT observation period (42 days from the first dose of the investigational drug) are not included in this population.

[0193] Data Presentation / Descriptive Statistics All statistical analyses are conducted in accordance with a detailed Statistical Analysis Plan (SAP) described prior to unblinding of the Phase 2a data. In case of a conflict between the protocol and the SAP, the SAP is used to determine the correct analysis and methodology. Demographic data, safety, PK, and efficacy parameters are summarized for each phase of the study, presented overall for Phase 1b and by treatment group for Phase 2a, and supplemented with graphs as appropriate. Descriptive summaries include n, mean, standard deviation, minimum, maximum for continuous parameters, and frequency, percentage for categorical parameters. Longitudinal data are presented at appropriate time intervals depending on the endpoint being summarized. Phase 1b Study: In accordance with the exploratory nature of the Phase 1 study, no inferential hypothesis testing is conducted. All endpoint summaries are presented as descriptive statistics. Aggregates are summarized by dose level and overall. Summaries are done for the mITT Population and the DLT Population. At the end of the Phase 1b study and before the start of the Phase 2a study, an end-of-Phase 1b study analysis is conducted. This analysis provides information for the RP2D, the dosing schedule of CTO1681, and SRC recommendations and sponsor decisions regarding the CAR T cell product to be included in the Phase 2a study. Phase 2a Study: Analyses of primary and secondary endpoints are conducted in the mITT Population and the PP Population. Analyses of safety parameters are conducted in the safety population. The incidence, severity, onset, and duration of CRS, which is the endpoint of the 2a phase trial, are measured by the CRS grade AUC for 30 days starting immediately after the initiation of CAR T cell therapy. The AUC is a single numerical value representing the occurrence, duration, and severity of CRS. The mean AUC is compared between treatment groups using analysis of covariance (ANCOVA) considering the randomized strata. A comparison of the difference in means is applied, and a one-sided p-value of the difference in AUC is calculated. The details of this method are described in the SAP.

[0194] The incidence, severity, onset, and duration of ICANS are measured by the ICANS grade AUC for 30 days starting immediately after the initiation of CAR T cell therapy. Other secondary evaluation items (including ICANS grade AUC) are analyzed using the Cochran Mantel-Haenszel chi-square statistic for categorical variables and ANCOVA considering randomized strata for continuous variables. Ordinal variables including the maximum severity of CRS are analyzed using the Wilcoxon-Mann-Whitney test overall and by stratum. Time-event endpoints are compared using the stratified logrank test.

[0195] The robustness of the CRS grade AUC is established through an evaluation of the correlation between the CRS grade AUC and each component. Specifically, a graph of the duration and severity against the AUC is displayed. Tests for relevance include the t-test for the difference in means and the receiver operating characteristic curve, for example, to determine whether there are appropriate criteria for establishing the likelihood of CRS events of grade 3 or higher using the AUC. Furthermore, the AUC is evaluated in relation to the frequency and duration of hospitalizations and ICU admissions due to CRS, and the use of concomitant medications and anti-cytokine therapies for CRS treatment, to determine the relative association with medical utilization. The same analysis is performed for the ICANS grade AUC. Additional information is described in the SAP.

[0196] The analysis of safety parameters includes AEs, clinical laboratory values, vital signs, electrocardiogram results, and a summary of concomitant medications.

Example

[0197] Example 27: Methodology of Phase 1b / Phase 2a Trial: The Phase 1b / Phase 2a trial was designed to examine the safety and efficacy of CTO1681 in the prevention or mitigation of CAR T cell-induced toxicity. This trial is divided into two parts: the Phase 1b trial and the Phase 2a trial. After obtaining written informed consent for the Phase 1b or Phase 2a trial of this study, the patient is considered enrolled. During the screening period, the eligibility of the trial participants is evaluated. For all participants, the 19-day screening period ends two days before the baseline (-3 days).

[0198] Phase 1b Trial: The Phase 1b trial is a multi-center, non-blind, dose-escalation, safety, and pharmacokinetics (PK) study that administers multiple escalating doses of CTO1681 to DLBCL patients who have received a commercially available axicabtagene ciloleucel CD19-directed CAR T cell therapy. This part of the study is conducted using a rolling 6 design to inform dose escalation among cohorts of DLBCL patients. In the Phase 1b trial of this study, for each cohort of patients, the following procedures are sequentially implemented: screening, treatment enrollment, pre-treatment with lymph node dissection (LD) chemotherapy, initiation of the study drug (CTO1681), CAR T cell infusion, continuation of treatment with CTO1681 (for a total of 15 days), and safety follow-up. The trial eligibility is determined after leukapheresis and before LD chemotherapy. The timing of treatment with CTO1681 is carried out according to the following schedule in relation to CAR T cell infusion (CAR T cell infusion is "Day 0"): Days -12 to -3: LD chemotherapy (if justified according to the prescribing information [PI] of YESCARTA). Day -1: Initiate the study drug (CTO1681) approximately 24 hours before CAR T cell infusion. The study drug is administered orally three times a day (TID) for a total of 15 days. The total daily dose of CTO1681 is 30, 60, or 90 μg according to the cohort in which each patient is enrolled (administered in TID units of 10, 20, or 30 μg / dose respectively). The study drug should be taken within 30 minutes after a meal or a snack. Day 0: CAR T cell infusion (by YESCARTA PI).

[0199] Patients are monitored for up to 6 months to record tumor response using the Lugano criteria for malignant lymphoma. Additionally, efficacy and safety are monitored throughout the study period. A rolling 6 design is implemented to aid dose escalation. The dose-limiting toxicity (DLT) observation period is defined as 43 days after the first dose of the study drug (day -1), i.e., 42 days after the start of CAR T cell therapy (day 0), i.e., day +41. Up to 3 dose cohorts are considered, and up to 18 patients are enrolled during the rolling 6 dose escalation. A minimum of 3 and a maximum of 6 patients are enrolled at each dose level.

[0200] Before data review for dose escalation, at least 3 patients must complete the DLT observation period (up to Day 41). If 0 / 3, 0 / 4, 0 / 5, or 1 / 6 DLTs are recorded, escalation to the next higher dose level is possible. The recommendation for the next dose level is made by the Safety Review Committee (SRC) according to the rolling 6 design, based on safety, clinical tests, and all available PK and pharmacodynamic (PD) data available at the time of review. If sufficient safety data cannot be obtained from more than 1 patient in a certain cohort, additional enrollment can be made in that cohort, and only patients with sufficient safety data can be included in the safety assessment. If 1 DLT is observed, new patients are enrolled at the same dose level until 6 patients are enrolled and the DLT observation period ends, or until at least 2 DLTs are observed and the maximum tolerated dose (MTD) is exceeded. The MTD is defined as the maximum CTO1681 dose at which patients can be safely treated. If 0 DLTs are observed in 3 - 5 patients, or 1 DLT is not observed in 6 patients, escalation to the next dose level can be made. If a certain dose level is too toxic (2 or more DLTs are observed in 3 - 6 patients), the MTD stopping rule for that cohort / dose level can be applied. According to the dose escalation procedure, the Safety Review Committee (SRC) can backfill up to 12 patients in part or all of the cohort determined to be safe by the medical monitor and the sponsor's judgment. Backfill samples can provide additional information on safety, anti - tumor effect, PK, and PD, and can more appropriately select the recommended Phase 2 dose (RP2D) below the MTD for the Phase 2a trial. The maximum sample size for the Phase 1b trial is 36 patients. The dose escalation and stopping rules are shown in Table 3 below.

Table 3

[0201] Phase 2a trial: The Phase 2a trial will be conducted as a randomized double-blind placebo-controlled trial, and approximately 100 patients are planned to be enrolled. Patients will be treated with the RP2D of CTO1681 established in the Phase 1b trial or with placebo during the Phase 2a trial period. Patients will be randomly assigned to the CTO1681 group and the placebo group at a ratio of 1:1. Patients will receive a commercially available axicabtagene ciloleucel CD19-directed CAR T cell therapy. Each patient follows the same trial process as the above-mentioned Phase 1b trial part. Patients enrolled in the Phase 2a part of this trial will have their tumor responses monitored for up to 12 months after CAR T cell infusion (using the Lugano criteria for lymphoma). Sparse PK samples will be collected from all patients in the Phase 2a trial for population PK and exploratory exposure-response analysis.

[0202] In both the Phase 1b and Phase 2a trials of this study, the evaluations of safety, efficacy, PK, and PD are performed at the intervals specified in the protocol. The study patients will be hospitalized for the observation and management of CAR T cell-related toxicities based on the facility guidelines. All assessments of CRS and ICANS will follow the criteria of the American Society for Transplant and Cellular Therapy (ASTCT). The detailed management guidelines for CRS and ICANS are described in the full protocol and are consistent with the standard of care (SOC) management.

[0203] An independent Data Monitoring Committee (IDMC) consisting of at least two CAR T cell / hematology-oncology experts and one independent biostatistician is established to monitor safety data during the Phase 2a trial. The IDMC will conduct planned reviews of safety data after approximately 20, 40, 60, and 80 patients have been followed up to Day 41, or after the trial has been prematurely terminated. Each review will include data available from the Phase 1b trial. This trial will be paused or terminated if any of the pre-specified stopping or discontinuation rules are met.

[0204] Phase 1b and Phase 2a Trials In both the Phase 1b and Phase 2a trials, evaluations of safety, efficacy, PK, and PD will be conducted at intervals specified in the protocol for the Phase 1b trial and at intervals specified in the protocol for the Phase 2a trial. From baseline (-1 day) to +6 days, patients are planned to be treated as inpatients for appropriate monitoring. From +7 days to +13 days, in the absence of CRS / ICANS, at the discretion of the treating investigator and with the approval of the treating investigator, patients may be discharged.

Example

[0205] Example 28: Medical monitor and sponsor If a patient is discharged, a minimum of two follow-up visits (including the visit on Day +13) are required during that week (+7 days to +13 days) for AE collection and status evaluation during the treatment period. Additionally, for the observation and management of CAR T cell-related toxicities, study patients will be admitted to the hospital based on the facility's guidelines. Axicabtagene ciloleucel (YESCARTA), a CD19-directed CAR T cell therapy for currently marketed B-NHL (including DLBCL), will be used in both the Phase 1b and Phase 2a trials of this study.

Example

[0206] Example 29: Phase 1b trial - Safety review committee The SRC consists of one or more principal investigators, medical monitors, the medical staff of the sponsor (or a qualified surrogate), and, as needed, ad hoc members (e.g., statisticians, PK, pharmaceutical experts). The SRC monitors the patient's course and safety during the Phase 1b trial. Regular systematic reviews of AEs serve as the basis for temporarily halting or prematurely terminating the trial. Dose-limiting toxicity is the main criterion for halting or terminating the trial in accordance with the operating characteristics of the rolling 6 design. The SRC carefully considers any observed adverse events that may be considered DLTs that occur more than 5 months after the discontinuation of the study drug administration and evaluates whether such adverse events are reasonably considered related to the study drug. The SRC reviews the available safety, clinical laboratory, electrocardiogram (ECG), and PK data up to the end of the DLT assessment period (i.e., day +41, 43 days after the first dose of CTO1681). The SRC may recommend increasing the dose or enrolling additional patients (up to 6 per dose level), or may determine that further increases are not appropriate because the MTD has been reached. Cumulative safety data are reviewed at each SRC meeting. After the dose level is determined to be safe according to the dose escalation procedure and the SRC review of safety and PK data (if available), backfill patients can be enrolled (up to 12 per cohort). If any of the rules for halting or terminating (based on the DLT criterion) are met, the cohort is halted or terminated. The SRC advises on the progression of this trial to the Phase 2a trial and the selection of the RP2D based on the available safety, anti-tumor efficacy data, PK, and PD results. An interim analysis summarizing the results of the completed Phase 1b trial is conducted. The SRC may also advise on any protocol amendments necessary regarding patient safety throughout the Phase 1b portion of this trial. Details are described in a separate Safety Review Committee charter.

Example

[0207] Example 30: Phase 2a trial - Independent data monitoring committee Constitute a minimal IDMC with two CAR T cell / hematology oncology experts and one independent biostatistician to monitor safety data during the Phase 2a trial. The IDMC will conduct a planned review of safety data after approximately 20, 40, 60, and 80 patients have been followed up to Day 41 or after the trial has been prematurely terminated. The trial will be paused or terminated if either of the pre-specified stopping or discontinuation rules are met. The IDMC will carefully consider any observed AEs that may be considered DLTs and first manifest more than 5 months after discontinuation of the study drug and evaluate whether it is reasonably likely to be related to the study drug. In the event of patient death, an ad hoc meeting of the IDMC will be held. Additionally, the IDMC will meet more frequently as needed to review SAEs and provide recommendations regarding the timing of suspension of enrollment or end of the trial. The IDMC will also review available data on the proportion of CRs according to a pre-specified set of non-binding anti-tumor effect impact assessments that are planned to be conducted during the review of patient safety data at 40, 60, and 80 (20, 30, 40 for each treatment group) patients. Patients who received Phase 1b trial treatment with R2PD are included in these assessments. Anti-tumor effect assessments include all patients who achieved CR according to the Lugano Criteria, patients who demonstrated the best anti-tumor effect in progressive disease, patients who had the trial prematurely terminated, or patients who were followed for at least 3 months from the first dose of the study drug. Patients who have not achieved CR, are not progressive, and have not been followed for at least 3 months after treatment initiation are not included in the analysis. This distinction is made to ensure sufficient follow-up to account for the possibility of response or disease progression after treatment initiation and to avoid artificial underreporting of response rates.

[0208] For each anti-tumor effect evaluation, for patients randomly assigned to CTO1681, the CR rate and the exact Clopper-Pearson 95% CI are calculated and compared with the lower limit of 41% benchmark minimum rate and the upper limit of the CI derived from the lower limit of the 95% CI of the CR rate described in the YESCARTA label. The first anti-tumor effect evaluation is scheduled to be performed after 20 patients are enrolled and treated with CTO1681, but at least 12 eligible patients are required for the analysis to be carried out. For the examination of anti-tumor effect data, the registration of the trial may be temporarily suspended as necessary.

Example

[0209] Example 31: Management of CRS and ICANS CTO1681 should be continued in patients who develop CRS and / or ICANS, except as described in the section on DLT management. Detailed management guidelines for CRS and ICANS are provided to the principal investigators of the participating trial sites to standardize care among them. These guidelines are consistent with the SOC outlined by the PI for the selected CAR T cell therapy, but the discretionary use of prophylactic corticosteroids or tocilizumab for Grade 1 CRS or ICANS is not permitted. For Grade 2 or higher CRS and ICANS, treatment with corticosteroids and tocilizumab, as well as other supportive therapies, are considered.

Example

[0210] Example 32: Main dose - limiting toxicity DLT refers to an AE related to the drug, which is considered severe in nature, intolerable, or is considered to pose a medical risk to the patient when administering a high dose of CT01681 according to the opinion of the SRC / IDMC. The main DLT criteria are as follows: Hypotension: Defined as a documented long-term (more than 2 hours) systolic blood pressure < 80 mmHg that does not respond to basic medical treatment (i.e., fluid challenge). Bleeding: Clinically significant bleeding events (Common Terminology Criteria for Adverse Events v5.0 [CTCAE]) Grade 3 or higher (excluding accidental cuts, periodontal bleeding, menstrual bleeding, hemorrhoids, and easily controllable epistaxis). Liver function impairment: Transaminase elevation exceeding 5 times the upper limit of normal (ULN) (CTCAE Grade ≥ 3). QTc prolongation: QTc interval corrected by Fridericia's formula (QTcF) ≥ 550 msec, or a change of 60 msec or more from baseline, confirmed by repeat 12-lead electrocardiogram (ECG). Ventricular tachycardia: Requiring urgent treatment with CTCAE Grade 3 or higher. Other safety data evaluate other non-hematological toxicities of CTCAE Grade 3 or higher in treatment groups where the dose may need to be restricted: Toxicities of Grade 3 or 4 that result in severe and irreversible organ damage and / or test values involving vital organs (e.g., heart or lungs). Non-hematological toxicities of CTCAE Grade ≥ 3 that do not improve to CTCAE Grade ≤ 2 within 72 hours. Death not due to the underlying malignancy (Note: All CTCAE Grade 5 events will cause the trial to be interrupted temporarily to collect and evaluate the events for DLT determination. Immediate safety reports to the Food and Drug Administration [FDA] are included). Exclusions from DLT criteria are based on the high prevalence of confounding expected in specific study patient populations and include the following. Grade 3 or 4 anemia, lymphopenia, thrombocytopenia. Grade 3 or 4 fever or febrile neutropenia lasting for more than 14 days. Grade 3 or 4 tumor lysis syndrome lasting for more than 14 days. Other laboratory findings that are not clinically significant and can rapidly recover to baseline Grade 2 or less within 14 days. Grade 3 peripheral sensory neuropathy is not considered a DLT in subjects with a history of peripheral sensory neuropathy. Grade 3 nephrotoxicity and hepatotoxicity improve to Grade 2 or lower within 7 days. Grade 3 fatigue and anorexia that do not lead to hospitalization, tube feeding, or total parenteral nutrition.

Example

[0211] Example 33: Objectives Phase 1b trial: Determine the preliminary safety profile of CTO1681 in DLBCL patients receiving CAR T cell therapy. Determine the RP2D of CTO1681 and, if necessary, the MTD in DLBCL patients receiving CAR T cell therapy. Phase 2a trial: Preliminarily examine the efficacy of CTO1681 in preventing CRS or reducing the severity and duration of CRS compared to placebo. Determine the preliminary efficacy of CTO1681 in preventing ICANS or reducing the severity and shortening the duration of ICANS compared to placebo. Determine the expanded safety profile of CTO1681 in DLBCL patients receiving CAR T cell therapy. Examine the potential impact of CTO1681 on the antitumor activity of CAR T cell therapy, comparing with past data and placebo.

Example

[0212] Example 34: Endpoints: Phase 1b trial Primary Adverse events (AEs) including DLT, clinical laboratory values, and electrocardiogram abnormalities characterized by type, frequency, timing, severity (graded according to CTCAE v5.0), severity, and relationship with CTO1681. Exploratory: Incidence of CRS (regardless of grade). Duration until disappearance of CRS (regardless of grade). Incidence of ICANS (regardless of grade). The period until ICANS disappears (regardless of grade). Biomarkers related to the mechanism of action (MOA) of CTO1681 and immune response. Frequency and duration of hospitalization and intensive care unit (ICU) admission due to CRS or ICANS. Use (amount and duration) of tocilizumab, steroids, and other anti-cytokine therapies for the treatment of CRS or ICANS. PK concentration profile of CTO1681. Levels and persistence of CAR T cells. Immune reconstitution (i.e., B cell aplasia). Antitumor activity including complete response (CR), overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Phase 2a trial (active vs placebo): Primary Incidence of grade 2 or higher CRS. Secondary: Additional evaluation of the incidence and severity of CRS: Incidence of CRS (regardless of grade). Incidence of grade 3 or higher CRS. Time to resolution of CRS. Additional evaluation of the incidence and severity of ICANS: Incidence of ICANS (regardless of grade). Incidence of grade 2 or higher ICANS. Incidence of grade 3 or higher ICANS. Time to resolution of ICANS. AEs, clinical laboratory value abnormalities, and electrocardiogram abnormalities characterized by type, frequency, timing, severity (graded according to CTCAE v5.0), severity, and relationship to CTO1681. Frequency and duration of hospitalization and ICU admission due to CRS or ICANS. Use of tocilizumab, steroids, and other anti-cytokine therapies for the treatment of CRS or ICANS. Antitumor activity (including CR rate, ORR, DOR, PFS, OS). Exploratory: Levels and persistence of CAR T cells. PK concentration profile of CTO1681. Biomarkers related to the MOA and immune response of CTO1681. Immune reconstitution.

Examples

[0213] Example 35: Study completion and study discontinuation The end of the trial is defined as the time when all patients have completed or discontinued treatment with CTO1681 (placebo in the Phase 2a trial) and have been followed up for 6 months or 12 months (Phase 1b trial or Phase 2a trial respectively) to evaluate the efficacy of CAR T, or the time of death, loss to follow-up, or withdrawal of consent.

[0214] If there are sufficiently reasonable reasons, the sponsor can decide to end this trial at its discretion. If such measures are taken, a notice documenting the reason for the end of the study will be provided to each researcher. Situations that can justify the early termination of the trial include, but are not limited to, the following: Determination of unexpected, serious, or unacceptable risks to patients. Failure to fully comply with the protocol requirements. Plans to change, interrupt, or discontinue the development of the investigational drug. When patients cannot be enrolled at an acceptable rate despite all efforts to improve the enrollment rate. Other administrative reasons. If this trial ends early, all trial materials must be returned to the sponsor or the person designated by the sponsor.

Examples

[0215] Example 36: Exclusion criteria: 1. 18 years of age or older. 2. Patients who are scheduled to receive protocol-specified commercially available axicabtagene ciloleucel CD19-directed CAR T cell therapy for DLBCL without prophylactic administration of corticosteroids for CRS and / or ICANS after receiving leukocyte therapy. Patients eligible for this study must have relapsed or refractory DLBCL that has received at least two or more lines of systemic therapy. 3. Meet all eligibility criteria for CAR T cell therapy according to institutional guidelines. 4. Adequate organ function: Serum creatinine ≤ 1.5 × ULN and estimated glomerular filtration rate (eGFR) ≥ 60 mL / min × BSA m2 / 1.73 m2 by the Cockroft-Gault formula. Serum alanine aminotransferase / aspartate aminotransferase ≤ 2.5 × ULN. Total bilirubin ≤ 1.5 × ULN. Left ventricular ejection fraction ≥ 40% on echocardiogram or multigated scan, and no clinically significant pericardial effusion. Platelet count ≥ 50,000 / mm3. Absolute neutrophil count ≥ 1000 / μL. Absolute lymphocyte count > 100 / μL. 5. Measurable lymphoma lesions are recorded, sufficient for determination by Lugano Criteria. 6. Eastern Cooperative Oncology Group performance status is 0 - 1. 7. Female participants of childbearing potential and all male participants must consent to use contraception approved by the principal investigator during and for 30 days after administration of the investigational drug. 8. Patients who are willing to provide written informed consent before pre-treatment, or patients who have a legal representative who can provide informed consent on their behalf.

Example

[0216] Example 37: Exclusion criteria: Patients who meet any of the following criteria will be excluded from the study: 1. Those who received cytotoxic chemotherapy within 14 days before leukocyte therapy. 2. Clinically significant malabsorption syndrome and dysphagia that cannot be adequately controlled by drug therapy (e.g., pica, dysphagia, gastroesophageal reflux disease), as evaluated by the principal investigator of the clinical trial. 3. The electrolyte imbalance is unstable or unresponsive to treatment. 4. Clinically significant electrocardiogram abnormalities at the time of screening or baseline (-1 day). This includes, but is not limited to, cases where the QTcF value is confirmed to exceed 470 msec. Excluded patients include those with a QTcF value in the borderline range, or patients with conditions such as bundle branch block that are difficult to interpret, or patients in whom it is difficult to measure the end of the T wave. 5. A history of clinically significant arrhythmia and / or a history of requiring anticoagulation / antiplatelet therapy at therapeutic doses. 6. Clinically important (i.e., active) cardiovascular diseases, including cerebrovascular accident / stroke (within less than 6 months before registration), myocardial infarction (within less than 6 months before registration), or unstable angina, and congestive heart failure of New York Heart Association classification class III or higher. 7. Thromboembolic events that have not been controlled within the past 6 months or recent severe bleeding. 8. A history of hemorrhagic disease. 9. Requirement for continuous administration of therapeutic doses of anticoagulant therapy, antiplatelet drugs, or fibrinolytic drugs (prophylactic administration of low molecular weight heparin is acceptable). 10. Baseline systolic blood pressure less than 100 mmHg. 11. A history of autoimmune diseases / graft-versus-host disease that required immunosuppressive therapy within the past 2 years. However, physiological steroids (equivalent prednisone amount) can be administered if it is 5 mg or less. 12. Patients who, in the judgment of the principal investigator of the clinical trial, are unlikely to follow the trial procedures or are considered ineligible for registration.

Example

[0217] Example 38: Study treatment Lymph node dissection chemotherapy Lymph node dissection chemotherapy is carried out in accordance with the PI of the CAR T cell manufacturer and the guidelines of the facility. Lymph node dissection chemotherapy is not necessary for patients with a white blood cell count of 1,000 cells / μL or less within 1 week before the scheduled CAR T cell injection, but may be carried out at the discretion of the principal investigator of the clinical trial.

[0218] Unless otherwise specified, lymph node dissection chemotherapy is provided by the medical institution conducting the clinical trial. Guidance on packaging, storage, preparation, administration, and toxicity management related to the administration of LD chemotherapy should be followed as described in the current product label.

[0219] Investigational drug In the Phase 1b trial of this study, the investigational drug will be CTO1681-IR-OSD-10 μg immediate-release tablets.

[0220] In the Phase 2a trial, CTO1681-IR-OSD-10 μg immediate-release tablets or placebo tablets will be used. In the Phase 1b trial, CTO1681 will be orally administered as 10 μg tablets. In Cohort 1, CTO1681 tablets will be orally administered at a total daily dose of 30 μg for 15 days. In Cohorts 2 and 3, CTO1681 tablets will be orally administered at total daily doses of 60 μg and 90 μg, respectively, for 15 days. In the Phase 2a trial of this study, CTO1681 will be orally administered as 10 μg tablets, and multiple tablets may be administered per dose depending on the RP2D dose determined based on the results of the Phase 1b trial. The placebo tablets are the same size, shape, color, and packaging as the CTO1681 10 μg tablets.

Example

[0221] Example 39: Investigational drug, usage / dosage, and administration method: Administration of CTO1681 must be initiated at baseline (-1 day) approximately 24 hours (±1 hour) before CAR T cell infusion and administered TID within 30 minutes after starting a meal or snack. Treatment compliance is measured on all dosing days (-1 day to +13 days). Between day +7 and day +13, a dosing diary is distributed to outpatients, and any relevant information regarding the investigational drug is recorded. The diary is checked by the facility staff for compliance during the inpatient's examination, and the required dates are recorded in the patient's eCRF.

[0222] Phase 1b trial: CTO1681 is administered orally as 10 μg tablets. In cohort 1, CTO1681 tablets are orally administered at a total daily dose of 30 μg for 15 days. In cohorts 2 and 3, CTO1681 tablets are orally administered at total daily doses of 60 μg and 90 μg, respectively, for 15 days. Except as specified in the management of DLT, dose changes or dose modifications within a cohort are not permitted.

[0223] Phase 2a trial: CTO1681 is administered orally as 10 μg tablets, and multiple tablets may be administered per dose depending on the RP2D dose determined by the results of the Phase 1b trial.

[0224] Note: Since CTO1681 has a diet effect, it should be administered within 30 minutes after the end of a meal or snack. Study period (per patient): Phase 1b trial: Approximately 9 months (including screening). Phase 2a trial: Approximately 15 months (including screening).

Example

[0225] Example 40: Reference therapy, dosage, and administration pattern : No Phase 1b trial Phase 2a: Placebo tablets are administered orally in the same manner as the active treatment. The placebo tablets are identical in size, shape, color, and packaging to the 10 μg CTO1681 tablets.

Example

[0226] Example 41: CAR T cell therapy Patients are administered a commercially available axicabtagene ciloleucel CD19-directed CAR T cell therapy, administered in accordance with the manufacturer's PI and institutional guidelines.

Example

[0227] Example 42: Packaging and labeling CTO1681-IR-OSD-10 μg immediate release tablets and placebo tablets are provided in plastic bottles containing 45 tablets each. The study number is printed on the individual drug labels. Since only CTO1681-IR-OSD-10 μg immediate release tablets are distributed to patients in the open-label portion of this study on the label of the Phase 1b study, the identification of the contents is included. In Phase 2a, which is the randomized portion of this study, the drug or placebo bottles are appropriately masked. The patient identification number, initials, and date the bottle was dispensed for each patient are entered by the staff or pharmacist at the study site. The packaging meets all regulatory requirements.

Example

[0228] Example 43: Storage The tablets of the investigational drug are placed in the original container and stored at room temperature (20°C to 25°C) in accordance with the accompanying documents. All investigational drugs are stored in a secure location with restricted access, and can only be dispensed by the principal investigator of the clinical trial or staff specifically authorized by the principal investigator of the clinical trial.

Example

[0229] Example 44: Patient participation period The patient participation period in the Phase 1b study is approximately 9 months including screening. The patient participation period in the Phase 2a study of this trial is approximately 15 months including screening.

Example

[0230] Example 45: Treatment compliance Patients participating in the Phase 1b trial of this study will be assigned 1, 2, and 3 tablets of CTO1681 10 μg tablets at doses of 30, 60, and 90 μg / day, respectively. In the Phase 2a trial, patients assigned CTO1681 or placebo will be administered 1, 2, or 3 tablets of CTO1681 10 μg tablets or placebo according to randomization assignment and the RP2D carried forward in this part of the study. Each bottle contains 45 tablets of CTO1681 10 μg tablets or placebo (only in the Phase 2a trial).

Example

[0231] Example 46: Prior medications, concomitant medications, and treatment Previous and concomitant medications and treatments All medications and treatments administered within 20 days before the start of the trial (-1 day) and up to the end of the trial (6 months for Phase 1b and 12 months for Phase 2a) will be recorded in the eCRF. Additionally, record all previous treatment histories for DLBCL. During the study period, LD chemotherapy and CAR T cell therapy will be recorded on the designated eCRF pages. All other medications being taken at the time of signing the informed consent should be recorded as concomitant medications on the appropriate eCRF pages.

[0232] Prohibited concomitant therapies The following concomitant therapies are prohibited during the study: Bridging chemotherapy is permitted, but no additional therapy should be administered within 7 days after the start of LD chemotherapy.

[0233] After leukapheresis, additional steroids exceeding physiological amounts should not be administered. For 3 months after CAR T cell infusion, systemic administration of steroids and other immunosuppressive agents is prohibited, except when used for the management of CAR T cell-related toxicities.

[0234] Systemic administration of steroids or tocilizumab for the prevention of CRS / ICANS (prophylactic steroids or tocilizumab) or for the treatment of grade 1 CRS / ICANS. Steroids and / or tocilizumab should only be administered for grade 2 or higher CRS / ICANS.

[0235] Chemotherapy, immunotherapy, targeted agents, radiation therapy, high-dose corticosteroids, and investigational drugs other than LD chemotherapy are not permitted in this protocol, except when necessary for the treatment of disease progression after CAR T cell therapy.

[0236] Granulocyte macrophage colony-stimulating factor (GM-CSF) may exacerbate the symptoms of CRS and should be avoided.

[0237] Short-acting granulocyte colony-stimulating factor within 3 days after CAR T cell infusion or long-acting granulocyte colony-stimulating factor within 10 days after CAR T cell infusion.

[0238] Prophylactic agents for the central nervous system (e.g., intrathecal administration of methotrexate) must be discontinued more than 1 week before CAR T cell infusion. Herbs and natural therapies. Agents that may interact with CTO1681 (CTO1681 [Investigators Brochure]).

Example

[0239] Example 47: Permissible combined therapies Medications or inactivated vaccines (including over-the-counter or prescription drugs, excluding vitamins) administered at registration or during the trial and considered for the supportive care and safety of the patient must be recorded in the eCRF along with the reason for use, administration dates (including start and end dates), and administration information (including dose and frequency). This includes, but is not limited to: Routine prophylactic agents for CAR T cell infusion (e.g., diphenhydramine, acetaminophen / paracetamol). Systemic administration of steroids based on test guidelines: Dexamethasone, prednisone, or other corticosteroids used for the treatment of CRS or ICANS. Physiological steroid replacement (prednisone 5 mg / day or less or equivalent), topical and inhaled steroids. Symptomatic treatment of tumor-related symptoms (e.g., pain and nausea). Oxygen therapy, blood products, or transfusions. Therapeutic agents for chronic diseases (e.g., hypertension and hypercholesterolemia). For concomitant medications not described in the trial protocol, the trial responsible physician can consider them on a case-by-case basis in consultation with the trial sponsor or their agent as necessary.

Example

[0240] Example 48: Management of major dose - limiting toxicity DLTs of hypotension, liver dysfunction, bleeding, QTc prolongation, and ventricular tachycardia are managed as follows: DLT of hypotension or liver dysfunction: CTO1681 is interrupted until the DLT resolves. If the DLT recurs, the administration of CTO1681 is discontinued.

[0241] DLT of bleeding: CTO1681 is interrupted until the DLT disappears. If the DLT recurs, the administration of CTO1681 is discontinued.

[0242] DLT of QTc prolongation or ventricular tachycardia: In the case of QTc prolongation, the administration of CTO1681 is interrupted until the DLT resolves. If the DLT recurs, the administration of CTO1681 is discontinued.

[0243] In the case of ventricular tachycardia, the administration of CTO1681 to that patient should be permanently discontinued. All other DLTs are managed according to the facility's SOC.

Example

[0244] Example 49: Infection prevention Patients should receive prophylaxis for Pneumocystis pneumonia, herpes virus, and fungal infections according to standard institutional care or National Comprehensive Cancer Network guidelines.

[0245] Tumor lysis syndrome All patients with a high burden of malignancy and no contraindications such as allergies should initiate prophylaxis (e.g., allopurinol) according to institutional guidelines prior to CAR T cell infusion. Prophylaxis should be discontinued when the risk of tumor lysis has passed.

[0246] Depletion of B cells The effects of CAR T cell therapy on normal B cells can lead to depletion of B cells and hypogammaglobulinemia. For hypogammaglobulinemia, gamma globulin is administered according to institutional guidelines. At a minimum, the trough concentration of immunoglobulin G (IgG) should be maintained at 400 mg / dL or higher, especially in cases of infection (Hill, J.A., et al. Blood Rev 38:100596.).

Example

[0247] Example 50: Cytokine Release Syndrome The goal of CRS management is to prevent life-threatening conditions while maintaining the benefits of the antitumor effect. The CRS grading system created by ASTCT and published by Lee, et al. (Lee, et al: 625 - 638) and the treatment guidelines for CRS are detailed in Table 4. General recommendations and monitoring for CRS are as follows: Patients require adequate hydration before and during CTO1681 administration. CTO1681 should be administered according to the study schedule and dosing instructions. However, at the discretion of the principal investigator with approval from the medical monitor and sponsor, except for discharge from day +7 to day +13 if CRS / ICANS is not recognized.

[0248] If symptoms (fever, hypotension, hypoxia) occur, evaluate the patient to rule out other causes (e.g., infections). For other causes of symptoms, appropriate treatment should be administered.

[0249] Neither steroids nor tocilizumab should be administered prophylactically. Table 4 shows the management of CRS.

Table 4

[0250] Fever is defined as a body temperature of 38°C or higher that is not due to other causes. In patients who develop CRS and receive anti-cytokine therapy such as antipyretics, tocilizumab, or steroids, fever is not required for subsequent CRS severity assessment. In this case, the severity of CRS is determined by hypotension and / or hypoxia.

[0251] In Table 4, ASTCT = American Society for Transplantation and Cellular Therapy, BiPAP = bilevel positive airway pressure, C = Celsius, CPAP = continuous positive airway pressure, CRS = cytokine release syndrome, h = hour, IV = intravenous, PRN = as needed.

[0252] The grade of CRS is determined by more severe events such as hypotension or hypoxia not caused by other factors. For example, a patient with a body temperature of 39.5°C, hypotension requiring one dose of vasopressor, and hypoxia requiring a low-flow nasal cannula is classified as having grade 3 CRS.

[0253] A low-flow nasal cannula is defined as an oxygen supply of 6 L / min or less. Low flow includes blow-by oxygen supply, which may also be used in pediatrics. A high-flow nasal cannula is defined as oxygen supplied at a rate exceeding 6 L / min.

Example

[0254] Example 51: Immune Cell-Related Neurotoxicity Syndrome Similar to CRS, ICANS is a life-threatening toxic syndrome that is often caused by CAR T cell therapy and often occurs within one week after CAR T cell therapy. ICANS gradually worsens and progresses. The evaluation system and treatment guidelines for ICANS developed by ASTCT are detailed in Table 5. If the co-occurrence of CRS is suspected, treat CRS according to the recommendations in Table 4.

Table 5

[0255] Patients with an ICE score of 0 may be classified as grade 3 ICANS if they are awake and have global aphasia, but patients with an ICE score of 0 may be classified as grade 4 ICANS if they are not awake.

[0256] The decrease in the level of consciousness must not be due to other causes (e.g., not using sedatives).

[0257] The tremors and myoclonus associated with immune effector cell therapy can be evaluated according to CTCAE v5.0 but do not affect the evaluation of ICANS.

[0258] Intracranial hemorrhage with or without edema is not considered a feature of neurotoxicity and is excluded from the evaluation of ICANS. It can be evaluated according to CTCAE v5.0.

[0259] The evaluations of CRS and ICANS are performed daily starting from day 0 (also performed when clinically indicated according to the ASTCT criteria).

[0260] For days +7 to +13, if the patient is not hospitalized, regardless of the administration timing of CTO1681, the CRS / ICANS evaluation is only performed during medical consultations.

[0261] Consensus evaluation of CRS In the evaluation of CRS, a CRS severity scale related to antibody therapeutics was published by researchers at the National Cancer Institute of the United States. Considering the need to define mild, moderate, severe, and life-threatening events, take into account overlapping symptoms, and adapt this scale to other treatment methods for guiding treatment recommendations, a revised CRS evaluation system was created and published by Lee, et al. at a meeting held with the support of ASTCT (Table 4).

[0262] ICANS grading Immune effector cell-associated neurotoxicity syndrome is a clinical neuropsychiatric syndrome that can occur after immunotherapy, especially in relation to T cell-mediated therapies, and can often occur simultaneously or immediately after CRS. ASTCT created and published the ICANS grading system (Table 5).

Example

[0263] Example 52: Fever and Neutropenia The evaluation of the infection source is performed according to the facility's guidelines. Fever is treated with acetaminophen and the patient is kept at rest. Non-steroidal anti-inflammatory drugs and corticosteroids should be avoided. Patients with fever and neutropenia are administered broad-spectrum antibiotics. In particular, when oral intake is poor or there is tachycardia, maintenance infusion (normal saline) should be initiated for most patients with high fever. For patients without hypotension and with inactive tumor lysis syndrome, it is necessary to equalize the daily fluid balance.

[0264] Filgrastim should be used in accordance with published guidelines (e.g., Infectious Diseases Society of America).

Example

[0265] Example 53: Support of Blood Products for Anemia and Thrombocytopenia The treatment of anemia and thrombocytopenia should be carried out in accordance with the facility's guidelines. All blood products should be irradiated. Platelets and red blood cells should be administered as needed, using the complete blood count as a guide. Efforts should be made to maintain a hemoglobin level of 8.0 gm / dL or higher and a platelet count of 20,000 / mm3 or higher. Leukocyte filters should be used for all blood and platelet transfusions to reduce sensitization to transfused leukocytes and to reduce the risk of cytomegalovirus infection.

Example

[0266] Example 54: Prevention of Deep Vein Thrombosis The prevention of deep vein thrombosis should be carried out in accordance with the facility's guidelines. The prevention of deep vein thrombosis should be carried out in accordance with the facility's guidelines for all patients with reduced motor function during hospitalization. Low molecular weight heparin is recommended if there are no contraindications (e.g., recent surgery, bleeding disorder, platelet count less than 50,000 / μL) based on benefit / risk. Non-invasive mechanical intermittent pneumatic compression devices for the prevention of deep vein thrombosis should be used in patients who cannot receive anticoagulants due to an increased risk of bleeding or other concerns (Lyman, Carrier et al. 2021 Blood Adv 5(4):927-974).

Example

[0267] Example 55: Criteria for Temporary Suspension or Termination of the Trial If any of the events mentioned below occurs within 43 days (i.e., the 41st day) after the first exposure to the investigational drug, the SRC (in the case of a Phase 1b trial) or the IDMC (in the case of a Phase 2a trial) shall be notified immediately, and a special meeting shall be scheduled to review all available safety data. At that point, the suspension of trial enrollment shall be initiated. Thereafter, the committee shall provide recommendations on the future course of action.

[0268] Criteria for Suspension If any of the DLT criteria occur, an ad hoc meeting of the SRC (Phase 1b) or IDMC (Phase 2a) shall be convened.

[0269] Criteria for Termination In the following cases, the SRC (Phase 1b) or IDMC (Phase 2a) shall recommend termination of the trial: CTCAE Grade 4 toxicities (unmanageable, unexpected) related to CTO1681, probably related, or definitely related, have occurred in more than 25% of the treated patients (excluding fever, hematological toxicities, CRS / ICANS).

[0270] Deaths due to CTO1681-related DLTs not related to underlying diseases, CAR T cell therapy, or exogenous events (unrelated to the investigational drug or trial procedures). Specifically, Fatal bleeding. Refractory hypotension leading to death. Refractory ventricular tachycardia leading to death. Note: The safety termination criteria for the Phase 2a trial may also consider data from the Phase 1b trial subjects treated at the RP2D and low doses. The sponsor may decide to suspend, terminate, or continue the trial based on the recommendations of the SRC / IDMC and a comprehensive assessment of the evidence. The SRC may recommend suspension or termination of the trial if it determines that the safety of the patients may be compromised by continuing the trial. The IDMC may recommend termination of the trial based on a decline in safety or anti-tumor efficacy.

[0271] In the following cases, an ad hoc meeting of the SRC (Phase 1b trial) or IDMC (Phase 2a trial) is held: Hypotension: Systolic blood pressure less than 80 mmHg that is recorded and defined as having persisted for more than 2 hours and does not respond to basic medical treatment (i.e., fluid challenge). Bleeding: Clinically significant bleeding events (graded by CTCAE v5.0) Grade > 3 (i.e., excluding accidental cuts, gingival bleeding, menstrual bleeding, hemorrhoids, or easily controllable epistaxis), including episodes of hemoptysis. Liver dysfunction: Transaminase elevation > 5 x upper limit of normal (ULN) (CTCAE Grade ≥ 3). QTc prolongation: Manifestation of QTcF > 550 msec or change from baseline > 60 msec, confirmed by repeated 12-lead electrocardiogram (ECG). Ventricular tachycardia: (CTCAE Grade > 3) Requires urgent medical intervention. Other safety data evaluate other CTCAE Grade > 3 non-hematological toxicities in treatment groups where the dose may need to be restricted: Grade 3 or 4 toxicities and / or test values including major organs (e.g., heart or lungs) that result in major and irreversible organ damage. Grade 3 non-hematological toxicities that do not improve to Grade 2 or less within 72 hours. Death not due to the underlying malignancy (Note: All Grade 5 events will temporarily halt the trial to collect and evaluate events for DLT determination. Immediate reporting to the FDA is also included). In the following cases, the SRC (Phase 1b trial) or IDMC (Phase 2a trial) recommends halting the trial: CTCAE Grade 4 toxicities (unmanageable, unexpected) possibly related, probably related, or definitely related to CTO1681 have occurred in more than 25% of the treated patients (excluding fever, hematological toxicities, CRS / ICANS). Death due to CTO1681-related DLT not related to underlying disease, CAR T cell therapy, or exogenous events (unrelated to the investigational drug or procedure). Specifically Fatal bleeding Refractory hypotension leading to death Refractory ventricular tachycardia leading to death Note: The safety stopping criteria for the Phase 2a trial can also consider data from subjects in the Phase 1b trial treated at the RP2D as well as at the low dose. The sponsor can decide to suspend, terminate, or adapt the trial based on the recommendations of the SRC / IDMC and the overall evidence. The SRC can recommend a temporary suspension or termination of the trial if it determines that patient safety may be compromised by continuing the trial. The IDMC can recommend termination of the trial based on a decrease in safety or anti-tumor efficacy.

Example

[0272] Example 56: Statistical Methods Consideration of sample size: Phase 1b trial: To support the dose escalation of CTO1681 and the selection of the RP2D according to the rolling 6-dose escalation procedure and to ensure sufficient safety data for the Phase 2a trial dose selection, a maximum sample size of 36 (maximum 12 at each dose level) was selected. Phase 2a trial: The sample size was selected so that sufficient power could be obtained for the primary evaluation item. When the sample size was 100 cases (50 cases in each group), the power to detect the difference in the ratio of 40% in the control group experiencing CRS grade 2 or higher versus 16% in the CTO1681 group was 80% with a one-sided type I error rate of 0.05. Analysis population: Modified Intent-to-treat (mITT) population: The mITT population includes all patients who received both CAR T cell therapy and at least one dose of the investigational drug (CTO1681 / placebo). Patients are grouped by treatment group in the Phase 1b analysis and by randomized group in the Phase 2a analysis. Safety population: The safety population includes all patients who received at least one dose of the investigational drug (CTO1681). Patients are classified into treatment groups. DLT cohort: The DLT cohort includes all patients in the Phase 1b who received at least one administration of CAR T cell therapy and CTO1681. Patients who discontinued the study for reasons unrelated to the study procedure, conduct, or treatment before experiencing DLT and before the end of the DLT observation period (up to +41 days, 43 days after the first administration of CTO1681 or placebo) are not included in this cohort. An additional analysis cohort can be defined in the Statistical Analysis Plan (SAP). A standard intent-to-treat (ITT) cohort including all randomized patients can be analyzed as a robustness assessment of the mITT.

Example

[0273] Example 57: Rationale for the Study CTO1681 affects a plurality (20 or more) of cytokines widely (CTO1681) in order to alleviate hypercytokinemia. By administering CTO1681 early before inflammatory cytokines (such as IFN-γ, TNF-α, IL-1, IL-2, IL-6) are first released, it may be possible to reduce the risk of CRS associated with CAR T cell therapy. Due to excessive cytokine suppression, CTO1681 may prevent the expression or exacerbation of immune-mediated toxicity induced by CAR T cells and reduce related morbidity and hospitalization. The purpose of this trial is to evaluate the safety and tolerability of CTO1681 and determine the preliminary efficacy of CTO1681 measured by the reduction of CAR T cell-induced toxicity mainly including CRS and ICANS and other efficacy indicators. This Phase 1b trial / Phase 2a trial enrolls adult B-NHL (DLBCL subtype) patients scheduled to receive axicabtagene ciloleucel (i.e., YESCARTA®) CD19-directed CAR T cell therapy and manages potential variability in response rates and side effect rates between CAR T cell therapies. However, CRS and ICANS are not specific to axicabtagene ciloleucel, and in the future, other CAR T cell therapies may be included in CTO1681 trials. In the Phase 1b trial, an initial non-blind, dose-setting, safety, and toxicity assessment is conducted on a small number of patients (n = 9 to 36). The Phase 2a trial (n = 100) is a randomized proof-of-concept trial to examine the utility of CTO1681 versus placebo, which is initiated prior to CAR T cell therapy, and alleviates or reduces the toxicity induced by CAR T cell therapy.

[0274] In B-NHL with CRS and / or ICANS associated with CAR T cell therapy, treatment options alternative to tocilizumab and steroids are medically needed. CTO1681 showed a tolerable safety profile in healthy volunteers, similar to that reported for veraprost sodium. The results of the Phase 1a trial in healthy volunteers support further investigation of CTO1681 to alleviate the toxicity of CAR T cell therapy in the DLBCL patient population.

[0275] Basis for Dose Selection and Schedule The doses in the Phase 1b trial of this study are scheduled to be administered three times a day (TID) at 10, 20, 30 μg (30, 60, 90 μg per day). The doses for the Phase 2a trial will be selected based on the results of the Phase 1b trial.

[0276] From the non-clinical and clinical use history of veraprost sodium and immediate-release formulations of the same active isomer as CTO1681, there is substantial evidence that these doses are well-tolerated and may be effective in the prevention and treatment of CRS. Veraprost sodium has four stereoisomers, and one of them (314d) contains almost all of the biological activity and is chemically identical to CTO1681. The clinical experience of veraprost sodium is extensive worldwide, and the safe and effective doses for indications other than CRS are well-documented. In Japan, the approved dose of the immediate-release formulation of veraprost sodium is 60 - 180 μg / day (equivalent to 15 - 45 μg / day of CTO1681), which is administered 3 - 4 times with meals and is often titrated upward according to tolerance. In studies of patients with pulmonary arterial hypertension, the safety of administering up to 480 μg / day (120 μg / day of CTO1681) of veraprost sodium for up to 36 months has been reported (Vizza, Sciomer et al, 2001 Heart 86(6):661 - 665, Galie, Humbert et al, 2002 J Am Coll Cardiol 39(9):1496 - 1502, Nagaya, Shimizu et al:340 - 345, Barst, McGoon et al, 2003 J Am Coll Cardiol 41(12):2119 - 2125, Ono, Nagaya et al.2003 Circ J 67(5):375 - 378, Ono, Nagaya et al:1583 - 1588, Durongpisitkul, Laoprasitiporn et al.2005 Circ J 69(1):61 - 64:264 - 271, Vizza, Badagliacca et al, 2006 Cardiology 106(3):168 - 173.).

[0277] In the recently completed first-phase multiple repeat ascending-dose study of CTO1681, healthy volunteers were given doses of 15 - 60 μg / day for 7 days. As a result, this generally mild safety profile was confirmed. No SAEs, particularly no treatment-emergent adverse events (TEAEs) (defined as clinically significant changes in coagulation parameters or platelets, or persistent / recurrent symptomatic orthostatic hypotension), or TEAEs leading to study discontinuation or dose reduction occurred. Treatment-emergent adverse events were only mild (62 cases) or moderate (4 cases) in severity, and were completely consistent with the previously reported safety profiles for veraprost sodium and 314d.

[0278] The dose of veraprost sodium is within the dose equivalence of CTO1681 and has been shown to cause a significant decrease in TNF-α in diabetic patients (Fujiwara, Nagasaka et al. 2004 Exp Clin Endocrinol Diabetes 112(7):390 - 394, Xu, Pan et al., 2020 Exp Ther Med 19(1):639 - 645). By similarly reducing TNF-α and other inflammatory cytokines, it is thought that CRS, often observed in CAR T cell therapy, may be alleviated. Importantly, no clinically significant decrease in cytokine levels (a panel of 27 cytokines) was observed in healthy volunteers treated with CTO1681. This suggests that CTO1681 does not completely abolish cytokine responses, but rather moderates excessive responses, further supporting the safety of CTO1681 and justifying the continuation of clinical evaluation.

Example

[0279] Example 58: Measurement of Efficacy The incidence rate of grade 2 or higher CRS, the primary evaluation item in the phase 2a study, is summarized as the frequency and proportion of patients who developed it and is compared between treatment groups using Fisher's exact test. An exact 90% confidence interval is shown. The incidence rates of CRS (regardless of grade), CRS with grade ≥ 3, ICANS (regardless of grade), ICANS with grade ≥ 2, and ICANS with grade ≥ 3 are summarized in the same manner as CRS with grade ≥ 2. The comparison between treatment groups is performed exploratorily using Fisher's exact test.

[0280] The ordinal variable including the maximum severity of CRS is analyzed using the Wilcoxon-Mann-Whitney test as appropriate. The periods until the disappearance of CRS and the disappearance of ICANS are analyzed using the Kaplan-Meier method and compared between treatment groups using the logrank test. The frequencies and periods of hospitalization and ICU admission due to CRS or ICANS, and the use of tocilizumab, steroids, and anti-cytokine therapy for the treatment of CRS or ICANS are summarized using descriptive statistics.

Example

[0281] Example 59: Pharmacokinetic Measurement During the 1b and 2a phases of this trial, serial blood sampling for PK evaluation will be performed. These samples are used to determine the PK concentration profile of CTO1681.

[0282] In the 1b phase trial, blood samples for preparing plasma are collected and analyzed: - Day 1 (first [morning] dose): time = 0 (within 1 hour before meal and before morning dose), and 0.5 (± 10 minutes), 1 (± 10 minutes), 1.5 (± 10 minutes), 2 (± 10 minutes), 3 (± 10 minutes), 4 (± 10 minutes) hours after the first (morning) dose of CTO1681. Day 1 (second and third doses): 0.5 hour (± 10 minutes) before and 1 hour after the second and third doses of CTO1681. - Day 0, +2 days, +4 days, +6 days, and +13 days: 1 hour (± 10 minutes) after the morning single dose of CTO1681. In the Phase 2a trial, sparse blood samples for plasma preparation are collected from all patients to conduct population PK and exploratory exposure-response analysis. Samples are collected approximately 1 hour (±10 minutes) after the first morning dose. If PK samples are scheduled to be taken simultaneously with electrocardiogram evaluation and cytokine blood sampling, the evaluations are performed in the following order: (1) electrocardiogram measurement, (2) PK blood sampling, (3) cytokine blood sampling. Pharmacokinetic specimens should be processed within 1 hour after collection and frozen at -80°C until shipment. Details regarding the collection, handling, and processing of pharmacokinetic specimens are described in a separate test manual.

Example

[0283] Example 60: Biomarker and Immunoreconstitution Measurement For the patients in the Phase 1b and Phase 2a trials of this study, biomarkers related to the mechanism of action (MOA) of CTO1681 and immune response and immune reconstitution are collected by blood sampling.

[0284] Biomarkers related to the MOA of CTO1681 and immune response include cytokines (IFN-γ, TNF-α, IL-1, IL-2, IL-6, GM-CSF), C-reactive protein (CRP), and ferritin. Additionally, blood samples for IgG, lymphocyte subset enumeration (TBNK) panel, and immunophenotyping are collected to determine immune reconstitution.

[0285] The purpose, evaluation, and analysis criteria of biomarkers and reconstitution analysis are defined below (Table 6):

Table 6

Example

[0286] Example 61: Cytokine Sampling Patients should avoid strenuous exercise starting 24 hours before cytokine collection. In the Phase 1b and Phase 2a trials, cytokine blood sampling is performed before CTO1681 administration and approximately 1 hour after morning administration on Days 1 to 6 after administration. For screening, LD chemotherapy, Day -1, Day 0, Days +7 to +13, Follow-up 1, 1 month, 3 months, 6 months, and 12 months (Phase 2a trial only), sampling is carried out during the inpatient treatment period at approximately the same time as sampling after morning CTO1681 administration. On the days when PK sampling is also scheduled in this time period (Day 0, Day +2, Day +4, Day +6, and Day +13), cytokine samples should be collected immediately after the PK samples. Specimens are processed within 1 hour after collection and cryopreserved at -80°C until shipment. Details regarding the collection, handling, and processing of PK specimens are described in a separate study manual.

Example

[0287] Example 62: Disease Evaluation Disease evaluation is performed by computed tomography (CT) scan or positron emission tomography (PET) scan. Screening tests are acceptable if they were performed within 30 days before screening visit. Magnetic resonance imaging as additional imaging is performed only if determined necessary by the study physician's evaluation or facility guidelines. If bridging therapy is performed, reimaging is required before LD chemotherapy.

Example

[0288] Example 63: Lugano Criteria for Malignant Lymphoma The Lugano criteria are the standard response criteria currently used for lymphoma. These criteria are based on two-dimensional tumor measurements by positron emission tomography (PET) or CT in cases of non-fluorodeoxyglucose (FDG)-positive lymphoma or when PET images are not available (Cheson, Fisher et al: 3059-3068). These criteria are a revised version of the Cheson criteria published in 2014 (Barrington, Mikhaeel et al: 3048-3058). The most important changes to the classification criteria for imaging diagnosis (Barrington, Mikhaeel et al. 2014, Cheson, Fisher et al. 2014) are as follows:

[0289] Replacement of PET binary evaluation using the Deauville 5-point scale (5-PS). Introduction of interim PET examinations during treatment.

[0290] The 5-PS is a purely visual and qualitative evaluation, enabling more differentiated classification than the conventional method of evaluating FDG uptake. The criteria for the 5-PS are summarized in Table 6.

[0291] Interim PET examinations can, in some cases, enable early adjustment of treatment. This allows for a rapid response to the lack of treatment effect and initiation of an appropriate increase in treatment, or, if a treatment effect is obtained early, reduction of treatment to minimize toxicity and secondary diseases (Spaepen, Stroobants et al: 1356-1363, Hutchings, Mikhaeel et al: 1160-1168, Hutchings, Loft et al: 52-59, Cerci, Pracchia et al. 2010 J Nucl Med 51(9): 1337-1343). These response criteria are used to evaluate the response of patients who have received CAR T cell therapy (Table 7). Table 6: Lugano criteria for malignant lymphoma

Table 7

[0292] Overall survival The overall survival is defined as the period from the start of treatment until death due to any cause. Levels and persistence of CAR T cells The levels of CAR+ T cells in serum after CAR T cell infusion are measured from day 0 to day 180 in the Phase 1b trial and from day 0 to day 360 in the Phase 2a trial.

Example

[0293] Example 64: In Vitro Analysis of CTO1681 Activity in CAR T Cell Assays The effect of CTO1681 on the efficacy of CD19-targeted CAR T cells in vitro and the ability of CTO1681 to reduce inflammatory cytokine levels that induce CRS in the presence of CAR T cells and tumor cells in vitro were investigated. The aim of the study was to determine whether CTO1681 exhibits an anti-CRS phenotype while maintaining the antitumor function of CAR T cells. CD3 T cells were sorted from PBMC of one healthy donor, stimulated briefly, and transduced with a CD19 CAR-T lentivirus (LV) containing the CD28 and CD3ζ domains. CD19 CAR T cells were expanded for 6 days and used in the assay.

[0294] CD3 T cells targeting CD19 were treated with five concentrations of CTO1681 (0.36 nM, 1.8 nM, 9 nM, 45 nM, 225 nM) or vehicle for 30 minutes before and during co-culture with CD19+ Raji lymphoma target cells. Wells containing medium alone, vehicle, and a positive control (dexamethasone) were also included. After the first treatment, CAR T cells (effectors) were co-cultured with fluorescently labeled CD19+ Raji tumor cells (target cells) at three effector ratios (10:1): effector:target cell ratios were three (10:1, 5:1, 1:1) and treated with five concentrations of CTO1681 or vehicle for 24 hours. Target cells (Raji cells) were fluorescently labeled with CPD (eBioscience™ Cell Proliferation Dye eFluor™) before co-culture to distinguish Raji cells from effector cells and enable analysis of target cell viability by flow cytometry. After culture, the supernatant was collected and the levels of the inflammatory cytokines IL-6 and TNF-α were measured by multiplex Luminex assay, and the level of INF-γ was measured by time-resolved fluorescence resonance energy transfer (TR-FRET) assay. These cytokines are early cytokines released after CAR T cell injection in vivo and were selected for quantification because they cause overactivation of the immune system, which can lead to acute systemic inflammation, CRS, multiple organ failure, and death. Additionally, to measure the effect of CTO1681 on the efficacy of CAR T cells, the killing level of Raji tumors / target cells after CAR T treatment was measured by flow cytometry with varying conditions of CTO1681. The co-culture was stained with the viability dye 7-AAD to measure viability.

[0295] The results when the ratio of effector to target cells was 10:1 are shown in Figures 1A, 1B, and 1C. The maximum reduction rates reached approximately 50% for TNF-α, 26% for IFN-γ, and 23% for IL-6, respectively. In this study, it was found that CTO1681 did not affect or interfere with the tumor / target cell killing effect of CD19-directed CAR T cells in vitro (for example, CTO1681 did not interfere with the efficacy of CAR T cells). All concentrations of CTO1681 showed target cell killing. As expected, CTO1681 decreased the levels of inflammatory cytokines and CRS-inducing cytokines IL-6, TNF-α, and INF-γ in a dose-dependent manner. Similar results were obtained when the ratio of effector to target cells was 5:1 and 1:1 (Figures 1D, 1E, 1F, and 1G). It has been shown that dozens of cytokines increase after the injection of CAR T cells in patients, and CTO1681 has been shown to decrease the levels of each cytokine in a non-CAR T model. The current results showing that CTO1681 significantly decreased the levels of IL-6, TNF-α, and IFN-γ are consistent with the previous finding that CTO1681 can decrease these cytokine levels in in vitro and in vivo infectious disease models. This result also further verifies that CTO1681 targets the underlying cause of inflammatory cytokine release from immune cells. Therefore, CTO1681 is an optimal therapeutic candidate for CAR T cell therapy. Summarizing the results of this study, it was proven in vitro that CTO1681 does not inhibit the efficacy of CAR T cells. Also, strong evidence was obtained that CTO1681 acts on CAR T cells and significantly reduces the release of inflammatory cytokines, which are important for the onset of life-threatening conditions including enhanced immune responses and CRS.

Example

[0296] Example 65: In Vivo Effects of CTO1681 on CAR T Cell Efficacy in NSG Tumor-Bearing Mice If CTO1681 has an impact on CAR T cell activity in vivo, it was measured. Human T cells expressing the CD19-CD28-CD3Z CAR construct were used in this experiment.

[0297] Six- to seven-week-old NSG (trademark) (NOD SCIDγ) mice were each transplanted with 1×10 6 luciferase (luc)-expressing Raji tumor cells (referred to herein as "Raji-luc" tumor cells) on day 1, allowing tumor growth over a 7-day period prior to the start of treatment. CAR T cell injection was performed intravenously via the lateral tail vein (5x106 CD19 CAR T cells generated from healthy donors). Since NSG mice are severely immunodeficient, tumor engraftment is easy and subsequent clearance by CAR T cells can be measured.

[0298] Post-engraftment tumor growth and subsequent clearance were monitored once a week (starting on day 7) using IVIS imaging to detect luciferase signals in live mice. Administration of CTO1681 was initiated on day 8, 2 days prior to CAR T cell injection, and performed as twice-daily injections for 14 days. The total daily dose of CTO1681 was 0.3 mg / kg / day, 0.2 mg / kg / day, or 0.15 mg / kg / d, injected twice (BID). The dose of CTO1681 was selected based on the pharmacokinetic profile in mice. Control groups included mice administered Raji-luc tumor cells without CAR T cell treatment, mice receiving Raji-luc tumor cells and CAR T cell injection, and mice receiving vehicle treatment and administered Raji-luc tumor cells. Mice were monitored during the study period for body temperature and health assessment (body weight, movement, posture, fur condition, etc.).

[0299] Tumor burden was confirmed before treatment initiation, and the CTO1681 treatment group started BID administration 48 hours before CAR T cell infusion. In the 2-way ANOVA for the measurements on day 15, a statistical difference (p < 0.0001) was observed between group 1 (without CAR-T treatment) and all other groups. CD19 CAR-T cells significantly inhibited the growth of Raji tumor cells. All animals administered CD19 CAR-T cells, including those co-administered with CTO1681, showed a significant decrease in tumor burden with an increase of 3.9-fold to 9.5-fold. Therefore, treatment with CTO1681 did not affect CD19 CAR T cell activity at any of the tested doses. No statistical differences (p = 0.17 - 0.99) were observed at any dose level between the groups administered only CAR-T cells (groups 2 and 3) and the animals administered both CAR-T cells and CTO1681. IVIS images and luciferin signal quantification are shown on days 7 and 15 considering appropriate tumor growth after transplantation, and the values on day 7 represent the pre-treatment baseline (Figure 2). By day 6 of CAR T cell treatment (day 15 of the study start), tumor burden was significantly reduced without interference with the decrease associated with CAR T cell treatment at all three CTO1681 doses (Figure 2). In the follow-up at subsequent time points, disease progression was observed in all experimental groups, including the group not administered CTO1681. This is a phenomenon sometimes seen in the NSG tumor model, indicating that replication and proliferation of Raji-luc cells not removed by CAR-T treatment were observed again. These data clearly show that co-administration of CTO1681 with CAR T cell therapy does not inhibit the therapeutic effect in vivo. Furthermore, CTO1681 did not cause an increase in clinical pathological conditions including weight loss and limb movement. In conclusion, regardless of the presence or absence of CTO1681, CAR T cell treatment caused a dramatic reduction in tumor burden compared to mice that received no treatment.

Example

[0300] Example 66: CTO1681 Cytokine Regulation Peripheral blood mononuclear cells (PBMCs) from five donors obtained from AllCells (Emeryville, CA, catalog number: PB001) were activated through TLR-3 using Poly r(I:C) expression. Next, the cells were incubated with veraprost (a mixture of four isomers), or the isomers BPS-314d (CTO1681, isomer A in this example), BPS-315l (isomer B in this example), BPS-315d (isomer C in this example), BPS-314l (isomer D in this example) at concentrations ranging from 0.3 nM to 750 nM. Samples were analyzed for TNFα production using a commercially available TNFα sandwich ELISA, and EC50 values were calculated for each isomer. The following data clearly show that isomer BPS-314d (isomer A) is superior to the other three isomers tested.

[0301] CTO1681 (BPS-314d or isomer A) decreased TNFα production in activated PBMCs compared to isomer B, isomer C, isomer D, or a mixture of the four isomers in three repeated experiments (Figure 3). The EC50 of CTO1681 (BPS-314d or isomer A) was significantly lower than that of the other isomers, with an EC50 of 5.7 nM, while it was 33.59 nM for isomer C in the first repeated experiment (Table 7).

Table 8

[0302] The EC of the second replicate 50 values were consistent with those of the first replicate, with an EC of isomer A 50 of 4.141 nM and an EC of isomer C 50 of 21.71 nM (Table 8).

Table 9

[0303] The EC of the third replicate 50 values also show that the EC of isomer A 50 is lower than that of the other isomers (Table 9). [Table 10] EXAMPLES

[0304] Example 67: Pharmacokinetics in African Green Monkeys This example shows plasma concentrations of CTO1681 in African green monkeys (AGM) after dosing three times a day (TID) at various drug concentrations, where increasing dose concentrations of CTO1681 correlate with increasing plasma CTO1681 concentrations over multiple dose intervals. Taken together, the results of this study demonstrate that oral administration of CTO1681 in AGMs is a viable method of delivering CTO1681 systemically. Furthermore, the results of this study support the tolerability of oral administration of CTO1681, which is important for efficacy testing of CTO1681 in reducing cytokine levels and ARDS in this non-human primate model.

[0305] Data collected in this study demonstrated that plasma concentrations of CTO1681 in AGM increased with respect to the TID administered concentration / dose of CTO1681 administered orally. Overall, the maximum observed concentration (Cmax) measured after dosing and the area under the concentration vs. time curve from the start of dosing to the calculated last observed concentration (AUC0-t) increased as the dose of CTO1681 increased in rounds 1-3 (0.008, 0.04, and 0.2 mg / kg / day administered TID, respectively).

[0306] Trial Design- In the single-dose PK study design of CTO1681 in mice (BALB / C), the T1 / 2 was approximately 1 to 2 hours. Similarly, in the PK study (CAS-2201) of mice (NSG) with a twice-daily (BID) dosing design, the T1 / 2 of the BID exposure was approximately 5 hours, while in the calculation of the elimination half-life using the first-dose and measured exposure on the first day, it was shown to be 1.8 hours using a range of 6 to 2 hours (Rsq = 0.40). Therefore, the estimated half-life values of the BID study and the single-dose study are well-matched PK parameters between individual studies. Knowing that the half-life of CTO1681 is so short, a study design with more frequent blood sampling early after injection was implemented in the study plan. Therefore, considering the number and frequency of blood sampling throughout the planned sampling schedule, and also due to the limitation of the allowable number of blood samplings within a 24-hour frame, a "9 vs 3" animal sampling design (i.e., three sampling cohorts of three animals each) was adopted over the blood sampling time frame to maximize the blood sampling volume within the 24-hour observation period.

[0307] Measurement Criteria for Individual Animals - Nine African green monkeys (about 16 years old, wild-caught) were used in this PK study. In accordance with the requirements of the CDC, all animals were isolated at the CDC facility for 30 days before the start of the study, and then a 90-day isolation / acclimation period was provided at the TNPRC. The animals were administered orally three times a day (TID), and the study was conducted in three divided doses (the dosing concentration increased). The body weight of each individual animal was tracked throughout the entire study period. The changes in the body weight of each individual animal during the study period are reported in Table 10. The average body weight of the entire cohort decreased by 5.5%, and there was no individual whose body weight decreased by more than 8.7% of the initial body weight during the study period. There was no difference in the average value of body weight changes between males and females. Overall, the individual body weights of the animals suggest that the TID oral administration of CTO1681 does not cause significant weight loss. Finally, the animals successfully completed the schedule, and all animals maintained good health without losing more weight than expected by the veterinary staff at the intensity of the schedule. CTO1681 was demonstrated to be generally tolerable for all animals in this study.

Table 11

[0308] Pharmacokinetics of CTO1681 in AGMPharmacokinetic parameters were determined using a continuous sampling plan based on the individual plasma concentrations of each animal. Individual animal profiles were evaluated after each administration in single-day dosing. Single-day TID dosing was performed using the same animals after a minimum 7-day washout between rounds of increasing dose. Round 1 was TID 0.008 mg / kg / day (0.00267 mg / kg / dose), Round 2 was TID 0.04 mg / kg / day (0.0133 mg / kg / dose), and Round 3 was TID 0.2 mg / kg / day (0.0667 mg / kg / dose). Washout (negative) plasma concentrations of CTO1681 between dosing rounds were confirmed. To measure the parent drug exposure of CTO1681, blood samples were collected from monkeys by femoral venipuncture under anesthesia. Samples were collected at the following target time points related to the first TID dose of the day: 0.25, 0.5, 1, 2, 4, and 8 hours later (before the second single-day dose), 8.25, 8.5, 9, 10, 12, and 16 hours later (before the third single-day dose), 16.25, 16.5, 17, 18, 20, 24, 28, and 40 hours later (after TID dosing of the test item by oral gavage). Plasma concentrations of CTO1681 were measured over 40 hours from the first TID dose administered at t = 0 hours. Plasma CTO1681 was below the lower limit of detection level at t = 0 for all animals across all dosing rounds. Mean plasma CTO1681 concentrations over time are reported in Figure 4 for dosing rounds 1, 2, and 3 independently (Figure 4A, Figure 4B, Figure 4C), and combined (Figure 4D), corresponding to doses of 0.008, 0.04, and 0.2 mg / kg / day, respectively. Plasma concentrations at the lowest dose level (0.008 mg / kg / day) significantly depressed the lower limit of quantification of CTO1681. However, from the concentration data over time, it was suggested that plasma CTO1681 concentrations increased with each oral administration of the drug (indicated by arrows in Figure 4). Variability in CTO1681 concentrations among animals was observed, but the level of variability was within the range of norms expected for the test design, particularly in animals captured in the wild, and there was a clear dose-dependent increase in plasma drug concentrations, as shown by the increase in plasma CTO1681 concentrations from Round 1 to Round 3.During the third dosing period (t = 16 - 40 hours), greater variability was observed among individual animals, which was hypothesized to be due to differences in drug administration by oral gavage from different technicians that occurred at the late shift (11 PM).

[0309] As part of this study, standard PK parameters including Cmax and AUC0-t were measured (Figure 5). The increase in mean Cmax was measured across each dosing interval in all dosing rounds except for the dosing interval 2 in the third round (Figure 5A). The mean Cmax levels at the lowest dose were more affinity with the Cmax levels detected in the human PK analysis of CTA-1901 and increased with the dose level. There was variability among individual animals within the dosing groups across rounds, but dose-dependent increases in both Cmax and AUC0-t were calculated at the last dosing interval (dosing interval 3; Figure 5B). The highest dose of CTO1681 (0.2 mg / kg / day) resulted in a maximum plasma Cmax of approximately 3 ng / mL after TID dosing. Furthermore, the mean time (Tmax) to reach the maximum plasma CTO1681 concentration was positively correlated with the dose concentration. The Tmax in rounds 1, 2, and 3 were calculated to be 1.5 hours, 2.0 hours, and 2.1 hours, respectively. No correlation of Tmax with the dosing interval was observed regardless of the dose administered.

[0310] Collectively, these data demonstrate that oral administration of CTO1681 to African green monkeys results in detectable and quantifiable dose-dependent CTO1681 measurements and, on average, after TID dosing of 0.2 mg / kg / day, the maximum plasma concentration of CTO1681 reaches approximately 3 ng / mL. Furthermore, the mean time to reach the highest plasma CTO1681 concentration is positively correlated with the drug dose administered.

[0311] Summary- This study demonstrated that CTO1681 could be detected and quantified in plasma following oral administration of CTO1681 by oral gavage. This trial confirmed that plasma CTO1681 increased in a dose-dependent manner with increasing administered concentration. After TID administration of CTO1681 at 0.2 mg / kg / day (the highest test dose), the mean Cmax of CTO1681 measured in plasma was ~3 ng / mL, while the lowest dose was comparable to that detected following administration of 60 μg in healthy humans. Importantly, no toxicity or adverse health effects, including significant weight loss, were observed following CTO1681 administration. Overall, this study demonstrated that oral administration of CTO1681 in African green monkeys is a feasible model for studying the efficacy of CTO1681 in non-human primate models in subsequent studies.

Example

[0312] Example 68: Cytokine Measurement after CTO1681 Administration in African Green Monkeys Serum samples were collected longitudinally during the observation period. At the end of all three cohorts, the circulating concentrations of 12 cytokines were measured using serum samples. Representative data for TNFα, the major central cytokine of CRS, are shown below (Figure 6). AGMs treated with the highest dose of CTO1681 had significantly and consistently decreased cytokine concentrations compared to the other two cohorts. Overall, there appears to be a dose-dependent trend in the decrease of cytokines.

[0313] In the foregoing detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, the drawings, and the claims are not meant to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the present disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations as generally described herein and illustrated in the figures, all of which are readily contemplated herein.

Claims

1. The use of BPS-314d (esveraprost sodium salt) for the treatment of cytokine release syndrome (CRS), immunoeffector cell-associated neurotoxicity syndrome (ICANS), or both, associated with CAR T cell administration in a subject, wherein the use comprises administering to a population of CAR T cells and a first pharmaceutical composition. The first pharmaceutical composition comprises at least an effective amount of BPS-314d (esveraprost sodium salt), or a pharmaceutically acceptable salt thereof, and The first pharmaceutical composition does not reduce cytotoxicity mediated by a population of CAR T cells by more than approximately 5%.

2. The use according to claim 1, wherein the CRS is a Grade 1 CRS, a Grade 2 CRS, a Grade 3 CRS, or a Grade 4 CRS.

3. The use described in claim 1, wherein the ICANS is Grade 1 ICANS, Grade 2 ICANS, Grade 3 ICANS, or Grade 4 ICANS.

4. The use according to claim 1, wherein the first pharmaceutical composition reduces the level of one or more cytokines of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IP-10, MCP-1, MIP-1, RANTES, and GM-CSF in the subject.

5. A use according to claim 1, further comprising administering a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof.

6. The use according to claim 5, wherein the subject requires a reduction in treatment with the second pharmaceutical composition compared to a subject not administered with the first pharmaceutical composition.

7. The use described in claim 1, wherein the subject experiences a reduced parkinsonist effect upon administration of the first pharmaceutical composition compared to a subject not administered the first pharmaceutical composition.

8. The use according to claim 1, wherein the subject experiences a reduction in a severity measure related to CRS, ICANS, or both, upon administration of the first pharmaceutical composition compared to a subject not administered the first pharmaceutical composition.

9. The use according to claim 1, wherein the first pharmaceutical composition is administered when the onset of CRS is detected by an increase in the level of one or more cytokines from among IL-6, IL-10, IFN-γ, TNF-α, MIF, IL-5, IL-17A, IL-23, CXCL9 / MIG, GCSF, VEGF-A, and TGF-β, or one or more inflammatory biomarkers from among C-reactive protein (CRP) and ferritin.

10. In the use described in claim 1, The aforementioned CAR T cell administration is performed to treat cancer, and The aforementioned cancers are B-cell lymphoma, progressive, relapsed, or refractory diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, high-grade B-cell lymphoma, transformed follicular lymphoma, relapsed or refractory mantle cell lymphoma, acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, or multiple myeloma.

11. In the use described in claim 1, The aforementioned CAR T cell administration is performed to treat cancer, and The aforementioned cancers include brain tumors, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, multiple myeloma, ovarian cancer, neuroblastoma, colorectal cancer, biliary tract cancer, pancreatic cancer, mesothelioma, hepatoblastoma, embryonic sarcoma, prostate cancer, sarcoma, or liver metastases.

12. In the use described in claim 1, The aforementioned CAR T cell administration is performed to treat cancer, and The aforementioned cancer is B-cell lymphoma.

13. The use according to claim 1, wherein the subject is a mammal.

14. The use according to claim 1, wherein the subject is a non-human primate, monkey, cat, dog, pig, cattle, goat, horse, sheep, or rabbit.

15. The use described in claim 1, wherein the subject is a human.

16. The use according to claim 1, wherein the first pharmaceutical composition is administered to the subject before the population of CAR T cells is administered to the subject.

17. The use according to claim 5, wherein the second pharmaceutical composition is administered to the subject after the population of CAR T cells has been administered to the subject.

18. The use according to claim 5, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject after the population of CAR T cells has been administered to the subject.

19. The use described in claim 18, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously.

20. The use according to claim 5, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject before the population of CAR T cells is administered to the subject.

21. The use described in claim 1, wherein the first pharmaceutical composition is administered to the subject approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the CAR T cells are administered to the subject.

22. The use described in claim 1, wherein the first pharmaceutical composition is administered to the subject approximately 3 to 7 days after the population of CAR T cells is administered to the subject.

23. The use described in claim 1, wherein the first pharmaceutical composition is administered for a period of about 1 to about 30 days.

24. The use described in claim 1, wherein the first pharmaceutical composition is administered for about 7 to about 14 days.

25. The use described in claim 1, wherein the first pharmaceutical composition is administered starting one day before the administration of the CAR T cells and continued for at least about 14 days.

26. The use described in claim 1, wherein the CAR T cells are autologous CAR T cells.

27. The use described in claim 1, wherein the CAR T cells are allogeneic CAR T cells.

28. The use according to claim 1, wherein the first pharmaceutical composition further comprises at least one excipient, at least one filler, at least one disintegrant, at least one binder, at least one wetting agent, at least one lubricant, at least one lubricant, at least one preservative, at least one flavoring agent, at least one antioxidant, or a combination thereof.

29. The use described in claim 1, wherein the first pharmaceutical composition is formulated as a tablet, capsule, granule, powder, liquid, suspension, gel, syrup, slurry, suppository, patch, nasal spray, aerosol, injection, implantable sustained-release formulation, or mucoadhesive film.

30. The use described in claim 1, wherein the administration includes local delivery, subcutaneous delivery, intravenous (IV) delivery, intramuscular (IM) delivery, intrathecal (IT) delivery, intraperitoneal (IP) delivery, transdermal delivery, subcutaneous delivery, oral delivery, transmucosal oral delivery, pulmonary delivery, inhalation delivery, intranasal delivery, buccal delivery, rectal delivery, vaginal delivery, or a combination thereof.

31. The use according to claim 1, wherein the administration includes oral administration or intravenous injection (IV) administration.

32. The use according to claim 1, wherein the BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable salt thereof is present in a unit dose of the first pharmaceutical composition in an amount of about 1 microgram to about 100 micrograms.

33. The use according to claim 1, wherein the administration includes the step of delivering the first pharmaceutical composition to the subject in an amount of at least about 0.1 micrograms of BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable amount thereof.

34. The use according to claim 1, wherein the administration includes the step of delivering the first pharmaceutical composition to the subject in an amount of about 0.1 micrograms to about 5000 micrograms of BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable salt thereof.

35. The use according to claim 1, wherein the administration includes the step of delivering the first pharmaceutical composition to the subject in an amount of about 15 micrograms to about 90 micrograms of BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable salt thereof.

36. The use according to claim 1, wherein the administration includes the step of delivering the first pharmaceutical composition to the subject in an amount of about 60 micrograms to about 360 micrograms of BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable salt thereof.

37. The use according to claim 1, wherein the population of CAR T cells includes a population of BCMA CAR-T cells, a population of CD19 CAR-T cells, a population of CD19-CD3 bispecific CAR-T cells, or a combination thereof.

38. This is a kit for treating a target with CAR T cells. The kit comprises a first container containing a first pharmaceutical composition comprising at least an effective amount of BPS-314d (esveraprost sodium salt) or a pharmaceutically acceptable salt thereof, A second container comprising a second pharmaceutical composition comprising at least one corticosteroid, tocilizumab, an IL-6 receptor blocker, or a combination thereof, and A kit comprising a first pharmaceutical composition, a second pharmaceutical composition, and instructions for administering CAR T cells to the subject.

39. The kit according to claim 38, further comprising a third container containing CAR T cells.

40. A kit according to claim 38, further comprising a fourth container containing water or an aqueous solution.

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