EA040062B1 - METHOD FOR TREATMENT OF GLIOBLASTOMA WITH COMBINED THERAPY WITH BROMODOMAIN AND EXTRATERMINAL PROTEIN INHIBITOR - Google Patents

Description

Related Applications

The application for this invention claims the priority of a provisional application for a US patent

62/387359, filed Dec. 24, 2015, and U.S. Provisional Application No. 62/413,763, filed Oct. 27, 2016, the entire contents of which are incorporated herein by reference.

The field of technology to which the invention belongs

The embodiments described herein provide compositions, formulations, and methods for the treatment of cancer and neoplastic disease; however, such treatments involve combination therapies comprising the administration of a bromodomain and extraterminal protein (BET) inhibitor and a chemotherapeutic agent such as temozolomide or paclitaxel.

Background Art Information

There remains a need for compositions, formulations, and methods for the treatment of subjects with cancer, such as, for example, basal cell carcinoma, relapsed or refractory non-Hodgkin's lymphoma (NHL), glioblastoma multiforme, anaplastic astrocytoma, or other common solid tumors.

For example, basal cell carcinoma (BCC) is a common form of cancer worldwide and its incidence is increasing. In the United States alone, more than 3.5 million new patients with non-melanoma skin cancer are diagnosed each year. Most basal cell carcinomas (BCCs) are treatable with topical therapy, surgery, radiation therapy, or a combination of both. However, widespread SCD often leads to significant disfigurement and complications, along with associated physical and psychological consequences, as SCD usually occurs in sun-exposed areas such as the face. In addition, a small proportion of these types of cancer are metastatic and cannot be treated with standard therapy. Almost all basal cell carcinomas are associated with the aberrant Hedgehog (Hh) signaling pathway, which stimulates unregulated cell growth, and several therapeutic Hh inhibitors have proven effective in the treatment of SCD. Unfortunately, about 20% of basal cell carcinomas (BCCs) develop resistance to existing Hh inhibitors, typically through reactivation of the Hh pathway through mutations that affect the drug-binding pocket, increase Hh signaling activity, or act through concurrent gene copy number changes. suppressors. Patients will benefit from the development of well tolerated agents that overcome these resistance pathways, for example by targeting downstream proteins in the appropriate signaling pathways.

The essence of the invention

A method for treating glioblastoma, comprising administering to a human patient a therapeutically effective amount of at least one inhibitor of bromodomain and extraterminal protein (BET) and temozolomide, wherein the BET inhibitor is 4-[2-(cyclopropylmethylamino)-5methylsulfonylphenyl]-2-methylisoquinoline-1 -one or a pharmaceutically acceptable salt thereof. In one embodiment, administration of the BET inhibitor and temozolomide results in a synergistic reduction in cell proliferation in the patient's tumor or a synergistic increase in apoptosis in the patient's tumor compared to either the BET inhibitor or temozolomide alone. In one embodiment, the therapeutically effective amount for both the BET inhibitor and temozolomide when co-administered can be at least 50% lower than when the BET inhibitor and temozolomide are used alone. In one embodiment, the glioblastoma is resistant to temozolomide. In one embodiment, the BET inhibitor is present in an amount that enhances the therapeutic effect of temozolomide.

Description of drawings

In FIG. 1 is a graph showing dose-dependent tumor growth inhibition measured by tumor volume in the TNBC PDX model, COH70, following dosing of Compound A, (4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one).

Compound A was administered orally (RO) once a day for three consecutive days followed by four days of drug holidays (3x/week); --- carrier;

- Compound A, 12.5 mg/kg, PO, 3x/week; — — Compound A, 16 mg/kg, PO, 3x/week; --- Compound A, 20 mg/kg, PO, 3x/week; SEM is the standard error of the mean.

In FIG. 2 is a graph showing dose-dependent tumor growth inhibition measured by tumor volume in the GBM PDX model, GBM15, following dosing of Compound A. ---vehicle; --- Compound A, 15 mg/kg, PO, once daily for 5 consecutive days followed by 2 days of drug holiday (5/2); - - Compound A, 25 mg/kg, PO, once a day for 3 consecutive days followed by 4 days of drug holidays; ____ Compound A,

37.5 mg/kg, PO, once daily for 2 consecutive days followed by 5 days of drug holiday (2/5); SEM is the standard error of the mean.

In FIG. 3 is a graph showing xenograft tumor growth inhibition.

- 1 040062

GBM3 (GBM PDX) by administering Compound A, temozolomide (TMZ) or a combination of Compound A and TMZ.

---carrier; - Compound A, 12 mg/kg, PO, once a day; ---- Compound A, 6 mg/kg, PO, twice a day; - - Compound A, 6 mg/kg, PO, twice a day in combination with TMZ, 50 mg/kg, IP (intraperitoneal injection), administered on days 7-9 and 22-24; ---TMZ, 50 mg /kg, IP administered on days 7-9, 22-24; SEM is the standard error of the mean.

In FIG. 4 shows a schematic representation of the entire study design useful for demonstrating the safety or efficacy of pharmaceutical compositions.

Fig. 5 refers to the probability of developing dose-limiting toxicity (DLT) according to the prior distribution. □SE; about SM; δSL; +FM; x FL.

In FIG. 6 shows dose toxicity curves useful for simulations.

In FIG. 7 is a diagram showing published guidelines for the management of patients with treatment-induced diarrhea (Benson et al., 22, J. Clin. Oncol. 2918 (2004)), modified to conform to the study protocol.

In FIG. 8 is a graph showing tumor growth inhibition of PA0165 xenografts by administration of Compound A, romidepsin, or a combination of Compound A and romidepsin.

3/4 denotes 3 days of administration and 4 days of medicinal holidays; Q4D means once every 4 days; Q7D stands for once every 7 days; --- control; - Compound A, 25 mg/kg, 3/4;

----romidepsin, 1.5 mg/kg, Q4Dx3; — — Compound A, 25 mg/kg, 3/4, in combination with romidepsin, 1.5 mg/kg, Q7D; --- Compound A, 25 mg/kg, 3/4, in combination with romidepsin, 0, 75 mg/kg, Q7D. Tumor volumes are plotted as mean ± standard error of the mean (SEM).

In FIG. 9 is a graph showing the survival curve of PA0165 xenografts with Compound A, romidepsin, or a combination of Compound A and romidepsin.

3/4 denotes 3 days of administration and 4 days of medicinal holidays; Q4D stands for once every 4 days. --- control; - Compound A, 25 mg/kg, 3/4; romidepsin, 1.5 mg/kg, Q4Dx3;

- - Compound A, 25 mg/kg, 3/4, in combination with romidepsin, 1.5 mg/kg, Q7D; --- Compound A, 25 mg/kg, 3/4, in combination with romidepsin, 0.75 mg/kg, Q7D.

In FIG. 10 is a graph showing tumor growth inhibition of PA0165 xenografts by Compound A, abraxane, or a combination of Compound A and abraxane.

control; - Compound A, 25 mg/kg; ---- abraxane, 10 mg/kg; — — Compound A, mg/kg, in combination with abraxane, 10 mg/kg; --- Compound A, 12.5 mg/kg, in combination with abraxane, 10 mg/kg. Tumor volumes are plotted as mean ± standard error of the mean (SEM).

In FIG. 11 is a graph showing the survival curve of PA0165 xenografts with Compound A, abraxane, or a combination of Compound A and abraxane.

control; - Compound A, 25 mg/kg; ---- abraxane, 10 mg/kg; — — Compound A, mg/kg, in combination with abraxane, 10 mg/kg; --- Compound A, 12.5 mg/kg, in combination with abraxane, 10 mg/kg.

Detailed description of the invention

Aspects and embodiments of the present invention provide methods and pharmaceutical compositions for the treatment of patients with cancer and neoplastic disease; for example, patients with advanced solid tumors, relapsed or refractory forms of non-Hodgkin's lymphoma, glioblastoma multiforme, anaplastic astrocytoma, basal cell carcinoma, or other types of cancer. In at least one embodiment, a method of treating cancer and neoplastic disease is provided, comprising administering to a subject in need thereof a therapeutically effective amount of at least one BET inhibitor and a therapeutically effective amount of at least one chemotherapeutic agent. The chemotherapeutic agent may be an alkylating agent such as temozolomide or a mitotic inhibitor such as paclitaxel or paclitaxel in the form of protein-bound particles.

A typical BET inhibitor is 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2methylisoquinolin-1-one. According to the method, the administration of the BET inhibitor and the chemotherapeutic agent may be simultaneous or sequential.

In at least one embodiment, the BET inhibitor and the combination therapy chemotherapeutic agent are administered in a single pharmaceutical composition. In some embodiments, a composition is provided comprising a pharmaceutically effective amount of a BET inhibitor and temozolomide, formulated in a pharmaceutically acceptable carrier. In some embodiments, a composition is provided comprising a pharmaceutically effective amount of a BET inhibitor and protein-bound paclitaxel formulated in a pharmaceutically acceptable carrier. In one embodiment, the BET inhibitor and the combination therapy chemotherapeutic agent may exist as separate pharmaceutical compositions administered simultaneously or sequentially. In another embodiment, the BET inhibitor and chemotherapeutic agent

- 2 040062 are independent pharmaceutical compositions that are mixed before administration (ie mixed in a pharmaceutically acceptable solution for injection or infusion). In yet another embodiment, the BET inhibitor and the chemotherapeutic agent are presented as separate pharmaceutical compositions packaged together for administration (eg, a blister pack containing oral formulations or a pack containing an oral dosage form and an injectable dosage form).

In at least one embodiment, the administration of the BET inhibitor and the chemotherapeutic agent results in a synergistic inhibition of cell proliferation or an increase in cell death (eg, tumor cell death) as compared to the separate administration of the BET inhibitor or the chemotherapeutic agent. The chemotherapeutic agent may be an antiproliferative or proapoptotic compound, and may be selected to exhibit a synergistic antiproliferative or proapoptotic effect when co-administered with a BET inhibitor. Combination treatment with a BET inhibitor and a chemotherapeutic agent may result in a synergistic anticancer effect or overcome developed resistance. Synergistic effects or overcoming the developed resistance may allow to reduce doses, significantly reduce the cost of therapy in a significant population of patients.

It should be understood that this invention is not limited to specific methodology, protocols and reagents, as they may vary. The terminology used herein is only intended to describe specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

Used in the present description and the attached claims, the terms in the singular include plural designations, unless the context clearly indicates otherwise. The term or is inclusive unless it is modified, for example, by the term either. Except for working examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term approximately. The term roughly when used together with percentages can mean ±1%. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as generally understood by a person skilled in the art to which the present invention pertains.

All identified patents and other publications are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that can be used in the present invention, but should not contain definitions of terms that are inconsistent with the terms presented in this document. . These publications are provided solely for their disclosure prior to the filing date of the application for this invention. Nothing in this connection should be construed as an admission that applicants are not entitled to backdate such disclosure on the basis of prior invention or otherwise. All statements regarding the date or statement regarding the content of these documents are based on information available to the applicant and do not constitute any assumption as to the correctness of the dates or the content of these documents.

In at least one embodiment, a method of treating cancer with a combination therapy comprising administering a bromodomain and extraterminal (BET) protein inhibitor and a chemotherapeutic agent is provided. For example, the BET inhibitor may be a bromodomain inhibitor such as 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2methylisoquinolin-1-one (compound A); and the chemotherapeutic agent may be temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-trien-9-carboxamide) protein bound paclitaxel (eg ABRAXANE®) or romidepsin, (1S,4S,7Z,10S,16E,21R)-7-ethylidene4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23- tetraazabicyclo[8.7.6]tricos-16-en-3,6,9,19,22-pentone).

Thus, in an exemplary embodiment, a combination therapy is provided comprising Compound A and temozolomide. In another exemplary embodiment, a combination therapy is provided comprising Compound A and protein-bound paclitaxel. And in yet another exemplary embodiment, a combination therapy is provided comprising Compound A and romidepsin. As described in more detail herein, Compound A is a potent and reversible inhibitor of BET epigenetic proteins. Surprisingly, combination therapy comprising the administration of a BET inhibitor (eg Compound A) and a chemotherapeutic agent (eg paclitaxel protein-bound temozolomide or romidepsin) has been found to exhibit synergistic therapeutic effects.

In at least one embodiment, the treatment of subjects with cancer, in particular advanced solid tumors or relapsed/refractory forms of NHL, is provided, comprising administering a pharmaceutical composition containing a BET inhibitor and a chemotherapeutic agent such as an alkylating agent (temozolomide) or a mitotic inhibitor (such like connected

- 3 040062 with paclitaxel protein). For example, the BET inhibitor may be a bromodomain inhibitor such as Compound A. A specific example relates to evaluating the safety, tolerability, pharmacokinetics, and preliminary efficacy of Compound A in human subjects.

The present embodiments provide methods and compositions, such as pharmaceutical formulations, that provide therapeutic benefit in the treatment of types of cancer such as advanced solid tumors or relapsed/refractory forms of NHL, such as DLBCL or iNHL. Additional examples of cancer types associated with solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nose cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, small cell carcinoma lung, bladder carcinoma , lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma.

The terms subject or patient as used herein refer to any subject, in particular a mammalian subject, to which the diagnosis, prognosis, or therapy of a cancer, such as a solid tumor or relapsed/refractory form of NHL (e.g., diffuse large B-cell lymphoma) pertains. (DLBCL) or unexpressed NHL (iNHL)). The terms subject or patient may include any non-human human or animal as the context indicates.

As used herein, the terms treat, relieve, relieve, treat, or treat... (e.g., in terms of treating a patient with advanced solid tumor or relapsed/refractory NHL) are used interchangeably and refer generally to therapeutic or prophylactic benefit, such as reducing the likelihood of a disease, reducing the occurrence of a disease, or reducing the severity of a disease. For example, a treatment may refer to the ability of a therapy, if administered to a subject, to prevent further growth of a tumor or malignancy, or to cure or alleviate, at least in part, a symptom, sign, or cause of a disease. Treatment also refers to alleviating or reducing at least one clinical symptom, or inhibiting or delaying the progression of a condition, or preventing or delaying the onset of a disease or disease state. Thus, the terms treat, treat, or therapy... (or grammatically equivalent terms) refer to both prophylactic and therapeutic regimens. These terms refer to an approach to obtain beneficial or desired results, including, but not limited to, therapeutic benefit or prophylactic benefit. By therapeutic benefit is meant the elimination or reduction of the underlying disorder being treated. Also, therapeutic benefit is achieved by eliminating or reducing the intensity of one or more physiological symptoms associated with the underlying disorder, such that the patient is improved despite the fact that the patient may still be affected by the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient with confirmed one or more physiological symptoms of a disease, even if the disease has not been diagnosed.

Accordingly, a therapeutic agent as used herein refers to any therapeutically active substance that is administered to a subject to produce a desired, usually beneficial effect. The term therapeutic agent includes, for example, classical small molecule therapeutic agents commonly referred to as small molecule drugs; and biologicals, including, but not limited to, antibodies or functionally active fragments thereof, peptides, lipids, protein-based drugs, drug-protein conjugates, fusion proteins, enzymes, nucleic acids, ribozymes, genetic material, viruses, bacteria, eukaryotic cells and vaccines. The therapeutic agent may also be a prodrug. The therapeutic agent may also be a radioactive isotope. The therapeutic agent may be an agent activated by a form of energy such as light or ultrasound energy, or activated by other circulating molecules that can be administered systemically or locally. In addition, the therapeutic agent may be formulated pharmaceutically.

References to pharmaceutical agent, therapeutic agent, pharmaceutically active, pharmaceutical, drug, medicinal product, active agent, active

- 4 040062 drug, active pharmaceutical ingredient, etc. refers in a general sense to substances useful in medical and scientific fields, including, for example, drugs, biologicals, diagnostic agents (for example, dyes or contrast agents) or other substances used in therapeutic, diagnostic or prophylactic (for example, vaccines) , or research purposes. Examples of pharmaceutical agents include small molecules, chemotherapeutic agents, contrast agents, anesthetics, interfering RNAs, gene vectors, biologics, immunogens, antigens, interferons, polyclonal antibody preparations, monoclonal antibodies, insulins, or combinations of any of these. As noted, the pharmaceutical composition or pharmaceutical composition may contain one or more active therapeutic agents, or a combination of active and diagnostic agents, etc., usually additionally containing suitable excipient(s).

Inactive substances refer to carriers, excipients, diluents, and the like, well known in the art, and these substances can perform a useful function in a mixed injectable preparation, for example, as a surfactant, inorganic or organic salt, stabilizer , a diluent, a solubilizer, a reducing agent, an antioxidant, a chelating agent, a preservative, an adjuvant, an isotonic or buffering agent, or any excipient commonly used in pharmaceutical compositions (i.e., a pharmaceutically acceptable excipient), and the like. These active or inactive substances may also include substances with immediate, delayed, controlled or sustained release characteristics.

Pharmaceutical formulation, formulation or pharmaceutical composition refers to a drug product that includes at least one active agent and may further include at least one pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other material well known to those skilled in the art. areas. For example, a typical pharmaceutical formulation for injection includes a suitable parenteral aqueous solution that is free of pyrogens and has a suitable pH, isotonicity, and stability. Pharmaceutical compositions may be of diagnostic, therapeutic or research use in various species, such as human patients or subjects. In at least one embodiment, the pharmaceutical composition comprises a BET inhibitor and a chemotherapeutic agent such as temozolomide, protein-bound paclitaxel, or romidepsin. For example, the BET inhibitor may be 4-[2-(cyclopropylmethylamino)-5methylsulfonylphenyl]-2-methylisoquinolin-1-one (compound A). The agents and compositions described herein may be formulated by any conventional method using one or more pharmaceutically acceptable carriers or excipients as described in the conventional literature. See, for example, Remington - Science & Practice of Pharmacy, ^22nd edition (Lloyd, ed., Pharmaceutical Press, London, UK, 2012). Such formulations contain a therapeutically effective amount of the active agent(s) described herein, preferably in purified form, together with a suitable amount of carrier, to thereby provide a form for proper administration to a subject.

A prodrug means a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound as described herein. Thus, the term prodrug refers to a precursor of a biologically active compound that is pharmaceutically acceptable. The prodrug may be inactive when administered to a subject, but converted in vivo to the active compound, for example by hydrolysis. The prodrug often has advantages in solubility, tissue compatibility, or sustained release in mammals. The term prodrug also includes any covalently linked carriers that release the active compound in vivo upon administration of such a prodrug to a mammalian subject. Prodrugs of the active compound can be prepared by modifying functional groups present on the active compound such that such modifications are cleaved either by routine manipulation or in vivo to provide the parent active compound. Prodrugs include compounds in which a hydroxy, amino, or mercapto group is bonded to any group such that when the prodrug of the active compound is administered to a mammalian subject, it is cleaved off to form a free hydroxyl, free amino, or free mercapto group. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol or amino functional groups in the active compounds. See, for example, Design of Prodrugs, at 7-9, 21-24 (Bundgaard, Ed., Elsevier, Amsterdam, 1985). For example, temozolomide is a prodrug imidazotetrazine derivative of the alkylating agent dacarbazine.

The pharmaceutical composition may include a therapeutically effective amount of at least one active agent. Such effective amounts can be readily determined by one of skill in the art based, in part, on the effect of the administered dosage form, or the combined effect of the agent and one or more additional active agents, if more than

- 5 040062 than one agent. The therapeutically effective amount of the active agent may also vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the agent (and one or more additional active agents) to elicit a desired response in the individual, such as improving at least one state parameter. For example, a therapeutically effective amount of the dosage form may inhibit (reduce the severity or prevent occurrence), prevent the onset of a particular disorder, or reduce any of the symptoms of a particular disorder known in the art or described herein. A therapeutically effective amount can also be an amount such that any toxic or deleterious effects of the active agent or dosage form are outweighed by the therapeutically beneficial effects.

Thus, the active agent may be administered to the subject as monotherapy, or as combination therapy with another active agent in a combination dosage form, or as an adjunctive treatment, such as another treatment for the same, associated or additional disorder. For example, a BET inhibitor can be combined with a chemotherapeutic agent such as temozolomide or protein-bound paclitaxel in the same formulation or in a different formulation administered simultaneously or sequentially. In addition, combination therapy may include administering to a subject (eg, a human patient) one or more agents (eg, antibiotics, anticoagulants, antihypertensives, or anti-inflammatory drugs) that provide a therapeutic benefit to the subject. In another example, combination therapy may include administering to a subject a BET inhibitor, temozolomide, or a combination containing a BET inhibitor and temozolomide and one or more additional agents that provide a therapeutic benefit to a subject having a cancer such as advanced solid tumor or relapsed/refractory NHL. Similarly, in another example, the combination therapy may include administering to the subject a BET inhibitor bound to the paclitaxel protein, or a combination comprising a BET inhibitor and paclitaxel and one or more additional agents that provide a therapeutic benefit to the subject having cancer. Similarly, in yet another example, combination therapy may include administering to a subject a BET inhibitor, romidepsin, or a combination containing a BET inhibitor and romidepsin and one or more additional agents that provide a therapeutic benefit to a subject having cancer. In some embodiments, the active agent and one or more additional active agents are administered in a single dosage form, such as a pharmaceutical composition containing a BET inhibitor and temozolomide, paclitaxel, or romidepsin. In other embodiments, the active agent is administered first in time and the additional active agent(s) is administered second in time. In some embodiments, one or more additional active agents are administered at the same time, but using different drug delivery devices or delivery modes, such as combination therapy, including the administration of a BET inhibitor and temozolomide, or comprising the administration of a BET inhibitor and paclitaxel or involving the administration of a BET inhibitor and romidepsin. In at least one embodiment, the BET inhibitor is 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one (compound A).

The administration of a BET inhibitor or a BET inhibitor and a chemotherapeutic agent as a combination therapy described herein may replace or enhance previous or current therapy. For example, in the treatment of one pharmaceutical composition, the introduction of additional active agent(s) can be stopped or reduced, for example administered at lower concentrations or with longer intervals between injections. In some embodiments, the administration of the prior therapy may be retained. In some embodiments, the prior therapy is maintained until the level of the active agent reaches a level sufficient to provide a therapeutic effect. Thus, the two therapies can be administered in combination, sequentially or simultaneously.

In at least one embodiment, a combination therapy comprising administration of a BET inhibitor and a chemotherapeutic agent has an additive effect compared to therapy comprising a separate administration of the BET inhibitor or chemotherapeutic agent. In other embodiments, the administration of the BET inhibitor and the chemotherapeutic agent in combination therapy has a synergistic effect compared to the therapy comprising the separate administration of the BET inhibitor or the chemotherapeutic agent. In some embodiments, a combination therapy comprising the administration of a BET inhibitor and a chemotherapeutic agent reduces side effects compared to a therapy comprising the administration of the BET inhibitor or chemotherapeutic agent alone, or the administration of one or more other agents alone. For example, combination therapy involving the administration of Compound A and temozolomide, paclitaxel, or romidepsin resulted in a synergistic therapeutic outcome.

Therapeutic benefit does not necessarily mean a cure for a particular cancer (eg, advanced solid tumor or relapsed/refractory NHL), but rather means an outcome, most often including relief; increased survival; elimination of the tumor; amelioration of a symptom associated with cancer; preventing or ameliorating a secondary disease, disorder, or condition associated with cancer; or prevention of metastases.

Common solid tumors include inoperable solid tumors. Relapsed or refractory forms of NHL include DLBCL and iNHL.

In at least one embodiment described herein, the disease state of the treated subject (eg, advanced solid tumor or relapsed/refractory NHL) is related to the subject's epigenetics or epigenetic state. Epigenetics refers, in general, to variations in cellular and physiological phenotypic traits in which external or environmental factors influence gene expression to a greater extent than changes in DNA sequence per se. In other words, unlike genetics based on changes in the DNA sequence (genotype), changes in gene expression or the cellular phenotype of epigenetics have other causes. For example, DNA methylation and post-translational modifications of nucleosomal histone proteins alter chromatin organization and gene expression without altering the original DNA sequence. Thus, epigenetic modification may have an effect if or where specific genes are expressed, allowing the cell to regulate differential gene expression reversibly and selectively. Chaidos et al., 6, Ther. Adv. Hematol. 128 (2015). Epigenetic modification is a dynamic and reversible process recorded, removed, and read by enzyme families: recorders covalently attach acetyl or methyl groups; prospectors remove these groups; and readers recognize and associate with these groups. Arrowsmith et al., 11, Nature Rev. drug discov. 384 (2012). The initiation and progression of cancer is increasingly associated with misreading, miswriting, or miserasing of these modifications. Chi et al., 10, Nature Rev. Cancer, 457 (2010).

Bromodomain and extraterminal (BET) proteins represent a group of epigenetic readers that play a key role in the epigenetic process and can indeed control the expression of genes involved in cell growth and tumorigenesis. Wyce, 4, Oncotarget 2419 (2013a). Post-translational acetylation of the N-terminal tails of nucleosomal histones is a fundamental epigenetic mark of open chromatin structure and active gene transcription. Members of the BET protein family are distinguished by highly homologous tandem bromodomains (BD-1 and BD-2) that recognize and bind these acetylated lysines at the histone tails. BET proteins then act as scaffolds that recruit transcription factors and chromatin organizers that are essential for transcription. For example, through a set of hydrogen-bonding interactions between highly conserved asparagine and tyrosine residues and acetylated lysine, BET bromodomains bind chromatin to the CDK9-containing P-TEFb complex, which phosphorylates the large subunit of RNA polymerase II and promotes pause release and transcript elongation. Chaidos et al., 2015.

The BET family includes four proteins: BRD2, BRD3, BRD4, and BRDT. Dawson et al., New Engl. J. Med. 367 (2012); Jenuwein & Allis, 293, Science, 1074 (2001). BRDT is found exclusively in germ cells, but BRD2, BRD3 and BRD4 are ubiquitous in germ and somatic cells. Chaidos et al., 2015. BRD4 (bromodomain-containing protein-4) acts as a transcriptional coregulator that binds to s-N-lysine acetylation pockets on the H3 and H4 histone tails; where it can regulate gene expression by recruiting additional proteins to its chromatin binding sites, thereby influencing chromatin structure and function. Jacobson et al., 288, Science, 1422 (2000). In addition, BRD4 binds preferentially to hyperacetylated regions of the super enhancer promoter and regulates the transcription of target genes by recruiting coactivator or coexpressor complexes. Jung et al., 12, J. Neuroinflammation, 1 (2015); Junwei & Vakoc, 54 Molec. Cell 728 (2014); Jenuwein & Allis, 2001.

In addition, deregulation of the BET protein has been observed in several neoplastic diseases. For example, a rare aggressive epithelial tumor (nuclear protein in testicular midline carcinoma (NUT)) is driven by NUT protein fusions with BRD3 or BRD4; and BET inhibitors have shown preclinical activity in this tumor. Filippakopoulos & Knapp, 2010; French, 203, Cancer Genet. & Cytogenet. 16 (2010). BRD4 deregulation also occurs in leukemia, hepatocellular carcinoma, and breast cancer. Zuber et al., 478, Nature, 524 (2011); Li et al., 7, Oncotarget, 2462 (2015). In addition, overexpression of BRD2 and BRD4 was demonstrated in glioblastoma cells, and inhibition of BET I-BET-151 (GSK1210151A) showed activity in glioblastoma multiforme (GBM) xenografts comparable to temozolomide. Pastori et al., 9, Epigenetics, 611 (2014). Separately, BET inhibition suppressed the oncogenic transcription factor FOSL1 and its targets in a lung adenocarcinoma cell line. Lockwood et al., 109, PNAS, 19408 (2012).

BRD4 has also been shown to control the expression of genes involved in cell growth and tumorigenesis, such as MFC, FOSL1, and GLI1. Shi et al., 25, Cancer Cell, 210 (2014); Filippakopoulos & Knapp, 13, Nature Rev. 337 (2014). Binding of BRD-containing complexes at super-enhancer sites is often localized to the promoter regions of key transcription factors, such as the c-MYC oncogene, which is activated in almost 70% of all cancers. Nilsson & Cleveland, 22, Oncogene, 9007 (2003); Whyte et al., 153, Cell, 307 (2013); Loven et al., 153, Cell, 320 (2013). BET inhibitors degrade these complexes, reduce MYC expression, and have been shown to be active in xenografts of human MYC-driven hematologic and solid tumors. Mertz et al., 108, PNAS, 16669 (2011); Puissant et al., 3, Cancer Discov. 308 (2013); Shimamura et al., 19, Clin. Cancer Res. 6183 (2013); Wyce et al., 8, PLoS One e72967 (2013b); Bandopadhayay et al., 20, Clin. Cancer Res. 912 (2014); Hu et al. 16, Int. J. Mol. sci. 1928 (2015); Li et al., 2015; Mazur et al., 21, Nat. Med. 116 (2015).

Moreover, activity has been observed in clinical studies of a BET inhibitor in refractory/resistant lymphoma and leukemia. Dombret et al., ASH 2014, Abstract 117. Thus, BRD4 may play a role in the transcription of many genes, and BRD4 inhibition could potentially reduce the expression of these transcribed genes, including genes involved in drug resistance such as drug pumps. Examples of genes involved in drug/therapy resistance are multidrug resistance protein (P-glycoprotein, MDR1), multidrug resistance transporter protein (MRP1, ABCC1), breast cancer resistance protein (BCRP, MXR, ABCG2), and glutathione (GSH).

BET proteins also appear to play a role in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) development. The epithelial-mesenchymal transition is associated with the progression and metastasis of many carcinomas, and there appears to be a correlation between EMT, chemotherapy resistance, and the occurrence of CSC. Thiery, 2 Nat. Rev. Cancer 442 (2002); Thiery, 15 Curr. Opin. Cell biol. 740 (2003); Huber et al., 17 Curr. Opin. Cell biol. 548 (2005); Mani et al., 133 Cell 704 (2008); Castellanos et al., 6 OncoTargets Ther. 1261 (2013); Satoh et al., 50 J. Gastroenterol. 140 (2015). CSCs have unlimited proliferation and can self-renew, differentiate into other cell types, and form tumors in immunodeficient mice. Castellanos et al., 2013. Indeed, CSCs may be responsible for tumor initiation, progression, recurrence, and metastasis, as well as tumor heterogeneity and treatment resistance. Sheridan et al., 8 Breast Cancer Res. R59 (2006); Campbell & Polyak, 6 Cell Cycle 2332 (2007); Li et al., 100 J. Natl. Cancer Inst. 672 (2008); Zhu et al., 32 Clin. Translat. Med. 1 (2014); Dawood et al., 28 Oncol. J. 1101 (2014). CSCs have been identified in leukemias, carcinomas of the breast (especially basal-like breast cancer), colon, GBM, head and neck, liver, lung, melanoma, pancreatic and prostate carcinomas. Fang et al., 65 Cancer Res 9328 (2005); Ma et al., 132 Gastroenterol. 2542 (2007); Tang et al., 21 FASEB J. 3777 (2007); Eppert et al., 17 Nature Med. 1086 (2011); Lathia et al., 29 Genes & Devel. 120 (2015).

In addition, with regard to EMT, the transcription factor Twist has been identified as a key activator of EMT. Wu & Donohoe, 2 RNA Dis. 1 (2016). Twist is found at high levels in both aggressive pancreatic cancer cells with high metastatic potential and breast cancer CSCs. Mani et al., 2008; Von Burstin et al., 137 Gastroenterol. 361 (2009). Importantly, BRD4 binds to Twist and this Twist/BRD4 interaction causes oncogenicity and invasion into BLBC. Shi, (2014). However, BET inhibitors can block this Twist-BRD4 interaction and inhibit growth in a basal-like breast cancer xenograft model. Work regarding colorectal carcinoma supports a key role for BRD4 in EMT: the BRD4 inhibitor, MS417, inhibited colon cell proliferation, migration, and invasion; disrupted growth in the CRC xenograft model; and inhibited the development of liver metastases. Hu et al., 16 Intl. J. Mol. sci. 1928 (2015).

In addition, BET proteins are important regulators of the Hedgehog (Hh) pathway, which is activated in cSc. Varnat et al., 1 EMBO Mol. Med. 338 (2009); Amakye, 19 Nature Med. 1410 (2013); Tang et al., 2014; Infante et al., 36 Trends Pharma. sci. 54 (2015). The Hh pathway is a key regulator of cell growth and differentiation during embryogenesis, but is usually inactive in adult tissues. Ingham & McMahon, 15 Genes & Devel. 3059 (2001); Von Hoff et al., 361 New Engl. J. Med. 1164 (2009). Aberrant activation of this pathway has been implicated in the oncogenesis of various cancers such as medulloblastoma, rhabdomyosarcoma, and nearly all SCD. Xie et al., 391 Nature 90 (1998); Epstein, 8 Nature Rev. 743 (2008); Teglund & Toftgard, 1805 Biochim. Biophys. Acta 181 (2010). Hh ligand overexpression has also been observed in breast, colorectal, esophageal, lung, gastric, pancreatic, and prostate tumors. Teglund & Toftgard, 2010.

In addition, aberrant Hh signaling activates the Smoothened receptor (SMO), which in turn increases the transcriptional activity of glioma-associated oncogenic homologue 1 (GLI1). GLI transcription is otherwise independent of Hh signaling, driven by growth factor-beta and KRAS. GLI1-driven transcription contributes to the progression of pancreatic cancer. Nolan-Stevaux et al., 23 Genes & Devel. 24 (2009). BRD4 and other BET proteins regulate GLI1 transcription downstream of SMO. In particular, BRD4 directly occupies the GLI1 and GLI2 promoters. Tang et al., 20 Nature 732 (2014). This filling can be inhibited by BET inhibitors, thus offering a target in Hh-driven tumors despite being dependent on SMO activation. Of note, the BET inhibitor, JQ1, reduced tumor cell proliferation in vitro and in vivo in Hh-driven tumors, including tumors resistant to SMO antagonists. Tang et al., 2014.

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Another BET inhibitor, I-BET-151, suppressed Hh-dependent growth of medulloblastoma in vitro and in vivo and suppressed SMO-independent activation of the Hh pathway in vitro. Long et al., 289 J. Biol. Chem. (2014).

Aberrant Hh signaling also occurs in 95% of basal cell carcinomas (BCCs). Migden et al., 16 Lancet Oncol. 716 (2015). BCC is a common cancer worldwide and its incidence is increasing. Rubin, 353 New Engl. J. Med. 2262 (2005); Am. cancer. Soc, Skin Cancer Facts, via ACS website, 2015. It is estimated that two to three million non-melanoma cancers appear worldwide each year and approximately 80% are BCC. World Health Organization, Ultraviolet radiation & the INTERSUN Program website, (2015); ACS, 2015. This is probably underestimated because in the United States, where the registry is better documented than in most countries, it is estimated that more than 3.5 million new patients are diagnosed with non-melanoma skin cancer every year. In addition, the incidence in Europe is increasing by 100,000 people a year. ACS, 2015; Rubin et al., 2005; Lomas et al., 166 Br. J. Dermatol. 1069 (2012).

Most BCCs can be treated with topical therapy, surgery, or radiation therapy, or a combination of both. NCCN guidelines; Trakatelli et al., 24 Eur. J. Dermatol. 312 (2014). However, a small proportion are progressive or present, locally advanced, or less than 1% of metastatic BCC that is not amenable to such therapy. Alonso et al., 20 JEADV 735 (2006); Danial et al., 169 Br. J. Dermatol. 673 (2013); Sekulic et al., 366 New Engl. J. Med. 2171 (2013); Bassett-Seguin et al., 16 Lancet Oncol. 729 (2015). Widespread BCC often causes significant malformation and morbidity, along with associated physical and psychological problems, as it most often appears in sun-exposed areas such as the head. Wong et al., 327 Br. J. Med. 794 (2003). Treatment of advanced and metastatic cases was difficult before the advent of Hh inhibitors.

In BCC, an aberrant Hh signaling pathway is initiated when the extracellular Hh protein binds to the Patched transmembrane receptor (PTCH1) and releases the SMO transmembrane protein. Ingham, 15 Genes & Devel. 3059 (2001); Rubin et al., 2006. Signaling by the SMO protein mobilizes the normally latent zinc finger transcription factor GLI2, which transactivates the GLI1 promoter. Huangfu & Anderson, 102 PNAS 11325 (2005); Haycraft et al., 1 PLoS Genet 48 (2005); Liu et al., 132 Devel. 3103 (2005). GLI1 and GLI2 directly activate transcription of Hh target genes, including some involved in cell growth such as MYCN and CCND1. Daya-Grosjean & Couve-Privat, 225 Cancer Lett. 181 (2005); Scales, 30 Trends Pharma Sci. 303 (2009); Oliver et al., 100 PNAS 7331 (2003); Tang et al., 2014. In addition, GLI1 amplifies Hh signaling by activating GLI2 transcription in a positive feedback loop. Regl et al., 21 Oncol. 5529 (2002).

In addition, PTCH1 and SMO mutations have been identified in basal cell nevus syndrome and sporadic BCC. Hahn, 1996; Gailani, 1996; Unden, 1997; Xie, 1998. In 80-90% of cases, BCC mutations cause loss of function of PTCH1, which normally inhibits SMO signaling activity. Alcedo, 1996; Hahn et al., 85 Cell 841 (1996); Johnson et al., 272 Science 1668 (1996); Bassett-Seguin, 2015. Another 10% of BCC cases are due to constitutive SMO activation. Xie, 1998; Bassett-Seguin et al., 16 Lancet Oncol. 729 (2015); Reifenberger et al., 152 Br. J. Dermatol. 43 (2005). These mutations cause constitutive Hh signaling and the ultimate expression of GLI1 in basal cells is associated with the development of BCC. Dahman et al. 389 Nature 876 (1997); Von Hoff et al., 361 New Engl. J. Med. 1164 (2009). Accordingly, agents capable of inhibiting SMO have been developed.

Erivedge® (vismodegib) directly binds to and inhibits SMO and therefore reduces GLI1 formation. LoRusso et al., 17 Cancer Res 2502 (2011); Sekulic et al., 2012; Von Hoff et al., 2009. See, for example, Erivedge (vismodegib) Eur. PAR (Grenzach-Wyhlen, Germany, Roche Pharma AG, 2015) available online from the EMA Europa website. Vismodegib targets BCCs associated with constitutively activated SMO mutations and PTCH1 mutations. Although an independent analysis reported a response rate of 30.3% for metastatic BCC to vismodegib and 42.9% for locally advanced BCC in subjects for whom surgery or radiotherapy was unacceptable, the mean duration of response was only 7 6 months and two-thirds of treated subjects did not respond. A recent safety analysis with a follow-up period of at least 12 months showed that 36% of subjects discontinued vismodegib treatment due to adverse events and an additional 10% at the subject's request. Bassett-Seguin et al., 2015.

Odomzo® (sonidegib), another SMO inhibitor, has a response rate of 58% in locally advanced BCC in an independent study, and responses appear to be longer lasting, with the investigator estimating that a response of 60% of locally advanced BCC lasts at least six months. Migden et al., 2015. However, 28% of subjects discontinued treatment and 32% of subjects had dose adjustments due to adverse reactions. Currently, due to the duration of responses and tolerance to SMO inhibitors, a significant number of subjects remain with an unmet need for medical care. See, for example, Odomzo (sonidegib), European PAR (Nuremberg, Germany, Novartis Pharma GmbH, 2015), available online from the EMA website

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Europa.

Importantly, about 20% of BCC cancer types develop resistance. Ridky & Cotsarelis, 27 Cancer Cell 315 (2015). Typically, this is due to reactivation of the Hh pathway through SMO mutations present in only 15-33% of untreated BCCs compared to 69%-77% of resistant BCCs. SMO mutations interfere with the drug binding pocket, increase the basal activity of SMO, or act by simultaneously changing the copy number of the fusion protein suppressor (SUFU) and GLI2. Atwood et al., 27 Cancer Cell 342 (2015); Sharpe et al., 27 Cancer Cell 327 (2015). A well-tolerated agent capable of overcoming these resistance pathways through targeting mechanisms below the SMO would be useful.

BRD4 and other BET bromodomain proteins regulate the transcription of GLI1 downstream of SMO, with BRD4 directly occupying the GLI1 and GLI2 promoters. This filling can be inhibited by BET inhibitors; and the BET inhibitor, JQ1, reduces tumor cell proliferation both in vitro and in vivo in Hh-driven tumors - even those that are resistant to SMO inhibition. Tang et al., 2014. Therefore, a clinical trial of a BET inhibitor is required in subjects with locally advanced or metastatic BCC with de novo or acquired resistance.

Thus, certain substituted heterocyclic derivatives based on isoquinolinones and related heterocyclic structures have proven to be useful for epigenetic regulation because they inhibit bromodomain-mediated recognition of acetyl-lysine regions in proteins such as histones; and therefore are useful for the treatment of cancer and neoplastic disease. Examples of cancers for which these compounds and pharmaceutical compositions are useful include NUT midline carcinoma, Burkitt's lymphoma, prostate cancer, breast cancer, bladder cancer, lung cancer, melanoma, glioblastoma, and the like. These substituted heterocyclic derivatives are based on isoquinolinones and related heterocyclic structures, and are usually substituted at the 4-position with a group such as aryl, heteroaryl, etc., and on the nitrogen atom of the isoquinolinone, or a related heterocyclic structure with a small alkyl group, such as a methyl group. An example of such compounds, 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2methylisoquinolin-1-one, which is further discussed herein, is a potent and reversible inhibitor of BET epigenetic target proteins, including BRD. As a rule, substituted heterocyclic derivatives according to the present invention belong to the class of compounds having structures represented by, for example, formula 1, formula 2 or their salts. See WO 2015/058160; U.S. Patent Publication US 20150111885; US patent No. 9034900.

Specifically, an embodiment of a substituted heterocyclic derivative having BET inhibitory activity is shown in Formula 1:

R ^A

O

Formula 1 wherein R ² is CH3, CH2CH3, CH2CF3, CH2F, CHF2, CF3, CH2D, CHD2 or CD3;

X5 is C-R ⁵ or N, where R ⁵ is hydrogen, halogen, OH, CN, OR ⁶¹ , NHR ⁶¹ , N(R ⁶¹ ) ₂ , alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, where each R ⁶¹ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl;

X6 is CR ⁶ or N, where R ⁶ is hydrogen, halogen, OH, CN, alkyl, cycloalkyl, cycloalkylalkyl, amino, alkylamino, dialkylamino, cycloalkylalkylamino, alkoxy, or cycloalkylalcoxy;

X7 is C-R ⁷ or N, where R ⁷ is hydrogen, halogen, OH, CN, OR ⁶¹ , NHR ⁶¹ , N(R ⁶¹ ) ₂ , alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl;

X8 is CR ⁸ or N, where R ⁸ is hydrogen, halogen or alkyl;

wherein no more than two of X5, X6, X7 or X8 may be N; And

^Ra is:

X2^R ^1S where X2 is N or CR ¹² where R ¹² is hydrogen, halogen, alkyl or alkoxy;

R ¹³ is YZ, where Y is selected from a bond, CH2, CH((C ₁ -C4) alkyl) and Z is selected from SO2R ²¹ , N(R ²² )SO2R ²¹ , SO2N(R ²² )2, N(R ²² )SO2N(R ²² )2, CON(R ²² )2, N(R ²² )CO2R ²¹ , N(R ²² )CON(R ²² )2, N(R ²² )COR ²¹ , COR ²¹ , OC(O) N(R ²² )2, OSO2N(R ²² )2 or N(R ²² )SO3R ²¹ ;

- 10 040062 each R ²¹ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl;

each R ²² is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl;

X3 is N or CR ¹⁴ where R ¹⁴ is hydrogen, halogen, -CN, alkyl, cycloalkyl or alkoxy;

X4 is N or CR ¹⁵ where R ¹⁵ is hydrogen, halogen, alkyl, CN or alkoxy;

R ¹⁶ is hydrogen, halogen, or WX, where W is a bond, O, S, or NH, and X is selected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkynyl, cycloalkylalkynyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl.

Another embodiment of a substituted heterocyclic derivative having BET inhibitory activity is shown as Formula 2:

JC.

X5 ^Xb

DD

R ⁶ 1< R ²

ABOUT

Formula 2 wherein R ² is alkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, aralkyl, or heteroarylalkyl;

X5 is C-R ⁵ or N, where R ⁵ is hydrogen, halogen, OH, CN, OR ⁶¹ , NHR ⁶¹ , N(R ⁶¹ ) ₂ , alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, where each R ⁶¹ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl;

X6 is C-H or N, provided that if X6 is N then X5 is C-R ⁵ and if X5 is N then X6 is CH;

R ⁶ is hydrogen, halogen, OH, CN, alkyl, cycloalkyl, cycloalkylalkyl, amino, alkylamino, dialkylamino, cycloalkylalkylamino, alkoxy, S-alkyl, cycloalkylalkoxy, heterocyclyl, aralkoxy, heteroaryloxy, aryloxy, alkynyloxy, or N(H)COalkyl;

^Ra is:

in which X2 represents N or CR ¹² where R ¹² represents hydrogen, halogen, alkyl or alkoxy;

R ¹³ is -YZ where Y is selected from a bond, -CH ₂ - or -CH((C ₁ -C ₄ )alkyl)- and Z is selected from SO2R ²¹ , -N(R ²² )SO2R ²¹ , -SO2N( R ²² )2, -N(R ²² )SO2N(R ²² )2, -CON(R ²² )2, -N(R ²² )CO2R ²¹ , -N(R ²² )CON(R ²² )2, -N (R ²² )COR ²¹ , -COR ²¹ , -OC(O)N(R ²² )2, -OSO2N(R ²² )2 or -N(R ²² )SO3R ²¹ ;

each R ²¹ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl;

each R ²² is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl. heteroaryl or heteroarylalkyl;

X3 is N or CR ¹⁴ where R ¹⁴ is hydrogen, halogen, -CN, alkyl, cycloalkyl or alkoxy;

X4 is N or CR ¹⁵ where R ¹⁵ is hydrogen, halogen, alkyl, -CN or alkoxy; And

R ¹⁶ is hydrogen, halogen, N(H)COX or WX, where W is a bond, O, S or NH and X is selected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkynyl, cycloalkylalkynyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl;

with the proviso that when X6 is N, then R ⁵ and R ⁶ are not hydrogen.

A specific example of a heterocyclic derivative having BET inhibitory activity is 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one; which has the chemical formula C ₂₁ H ₂₁ NO ₄ S, molecular weight 383 and the structure shown by formula 3:

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Formula 3.

See WO 2015/058160; U.S. Patent Publication US 20150111885; US patent No. 9034900.

4-[2-(Cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one (Compound A) is a potent reversible inhibitor of BET family members including BRD2, BRD3, BRD4 and BRDT. It exhibits dose- and time-dependent inhibition of GLI1 and thus plays an important role in the treatment of Hh-driven and GLI-driven transcription tumors. As discussed in more detail below, Compound A reduced tumor cell inoculation in the BLBC in vivo model and showed superior activity compared to the current clinical standard, temozolomide, in the GBM3 xenograft model. Interestingly, Compound A showed additive or synergistic effects in combination with temozolomide, suggesting that it may be useful in CSC and MYC-driven tumors. As noted and exemplified here, Compound A may be formulated for oral administration.

Alkylating agents are an example of chemotherapeutic agents that can be used in combination with BET inhibitors to treat cancer. For example, temozolomide is a prodrug and an imidazotetrazine derivative of the alkylating agent dacarbazine. The chemical name of temozolomide is 3,4-dihydro-3-methyl-4-oxoimidazo[5,1^]-1,2,3,5-tetrazine-8-carboxamide, which has the following structure/formula:

h ₂ noc

temozolomide

Temozolomide hydrolyzes rapidly to the active 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC) at neutral and alkaline pH, with hydrolysis occurring even faster at alkaline pH. See US patent No. 5260291; WO 1997/027202; WO2002/057269; WO2008/038031; EP 0252682; US 2006/183898.

Temozolomide is used as an alkylating agent in the treatment of certain types of brain cancer, as a second-line therapy for astrocytoma, and as a first-line therapy for glioblastoma multiforme. See NICE Guidance (2001); Stevens, in Cancer Drug Design & Discovery (Neidle, Ed., Academic Press, New York, 2008). The therapeutic benefit of temozolomide depends on its ability to alkylate/methylate DNA, most commonly occurring at the N-7 or O-6 positions of guanine residues. This methylation damages DNA and initiates the death of tumor cells. Unfortunately, some tumor cells are able to repair this type of DNA damage by expressing O ⁶ -alkylguanine-DNA alkyltransferase (AGT), encoded in humans by the O ⁶ -methylguanine-DNA methyltransferase (MGMT) gene, thus weakening the therapeutic efficacy of temozolomide. In some tumors, epigenetic silencing of the MGMT gene prevents the synthesis of this enzyme and therefore these tumors are more susceptible to killing by temozolomide. In contrast, the presence of the AGT protein in brain tumors predicts a poor response to temozolomide. See Sitruk et al., 38 Gynecologie Obstetrique & Fertilite 660 (2010); Jacinto & Esteller, 6 DNA Repair 1155 (2007); Hegi et al., 352 New Eng. J. Med. 997 (2005); Hegi et al., 10 Lancet Oncol. 459 (2009).

Temozolomide can be formulated as an oral capsule, with each capsule containing 5, 20, 100, 140, 180, or 250 mg of temozolomide. Temozolomide can also be formulated for injection by intravenous infusion, the infusion dose being the same as the oral capsule formulation. For example, for newly diagnosed glioblastoma, administration is at a dose of 75 mg/m ² for 42 days (simultaneously with focal radiotherapy) followed by 150 mg/m ² for days 1 to 5 of a 28-day cycle. For refractory anaplastic astrocytoma, the initial dose is 150 mg/m ² times a day for five consecutive days of a 28-day cycle.

Taxanes (paclitaxel and docetaxel) are another example of a chemotherapeutic agent,

- 12 040062 which can be used in combination therapy with BET inhibitors. See, for example, U.S. Patent No. 814,470. Originally isolated as a natural diterpene from Taxus brevifolia (Pacific yew tree), the alkaloid paclitaxel binds to microtubule beta-tubulin subunits, thereby stabilizing microtubules from disassembly that should occur during cell division: blocking normal progression of cell division by inhibition of spindle function eventually triggers apoptosis. Currently, paclitaxel, obtained inter alia by extraction by plant cell fermentation, purification by chromatography and crystallization, is used in the treatment of ovarian, breast, lung, pancreatic and other cancers. The full chemical name of paclitaxel is ]hydroxy }-1,7-dihydroxy-9oxo-5,20-epoxytax-11-en-2-yl benzoate; and paclitaxel has the following structure:

paclitaxel.

In some embodiments, the taxane is a nanoparticulate albumin-bound Abraxane® (Protein-bound paclitaxel particles for injectable suspension) (also referred to as nab-paclitaxel). See, for example, WO 2001/089522A1. This protein-bound paclitaxel is indicated as first-line or combination therapy for several types of cancer, including non-small cell lung cancer, pancreatic cancer, and breast cancer. See, for example, WO 2008/057562. This composition utilizes the natural properties of albumin to reversibly bind to paclitaxel, transport it across the endothelial cell, and concentrate paclitaxel in tumor regions. More specifically, the drug delivery mechanism includes, but is not limited to, glycoprotein-60 mediated endothelial cell transcytosis of paclitaxel-associated albumin and accumulation at the tumor site by binding of albumin to a secreted protein, acidic, rich in cysteines (SPARC), also known as osteonectin, a glycoprotein , predominantly expressed in tissues undergoing remodeling during normal development or in response to injury. Clinical studies have shown that nab-paclitaxel is significantly more effective than other paclitaxel formulations, with nab-paclitaxel nearly doubling the response rate, increasing time to disease progression, and increasing survival in second-line patients. See WO 2010/06595.

Romidepsin acts as a prodrug, with the disulfide bond undergoing reduction within the cell to release the zinc-binding thiol. The thiol reversibly interacts with the zinc atom in the binding pocket of the Zn-dependent histone deacetylase to block its activity. Thus, it is an HDAC inhibitor. Many HDAC inhibitors are potential cancer treatment regimens due to their ability to epigenetically restore normal expression of tumor suppressor genes, which can lead to cell cycle arrest, differentiation, and apoptosis. Romidepsin is indicated for the treatment of patients with cutaneous T-cell lymphoma (CTCL) who have received > one previous systemic therapy and patients with peripheral T-cell lymphoma (PTCL) who have received > one previous therapy.

In at least one embodiment, a combination therapy is provided comprising one of the heterocyclic derivative BET inhibitors and temozolomide. In at least one embodiment, the heterocyclic derivative is 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one of Formula 3 (Compound A). In particular, synergistic effects have been observed with Compound A and temozolomide in a temozolomide-resistant glioblastoma multiforme (GBM) xenograft model. In particular, O ⁶ -methylguanyl methyltransferase (MGMT) is involved in the resistance of GBM to the alkylating DNA damage of temozolomide. GBM3 is a GBM, patient-derived xenograft (PDX) mouse tumor xenograft model with high MGMT expression, an unmethylated MGMT promoter, and a temozolomide-resistant phenotype. In previous studies of neurospheres cultured from GBM3, RT-PCR showed that Compound A reduced MGMT expression in a dose-dependent manner. When mice bearing GBM3 were given a single dose of Compound A, qRT-PCR showed a decrease in MGMT expression in the extracted tumor. In an efficacy experiment, it was investigated whether Compound A could sensitize temozolomide-resistant GBM to temozolomide and whether the combination had synergistic effects. Briefly, cohorts of mice carrying GBM3 were treated with temozolomide, Compound A, or a combination of Compound A and tezozolomide. Tumor growth inhibition (TGI) has been observed following dosing of single

- 13 040062 Compound A or in combination with temozolomide. Although temozolomide, when administered alone, did not give a significant TGI (3%), Compound A, when administered alone, resulted in a significant TGI (63%) (12 mg/kg QD) and 76% (6 mg/kg BID). See fig. 3. These data support the use of a BET inhibitor such as Compound A as a sensitizer to a chemotherapeutic agent such as temozolomide, possibly by reducing the expression of genes responsible for resistance (eg, drug pumps).

Surprisingly, the combination of Compound A and temozolomide was found to be significantly superior to all other treatment regimens and demonstrated synergy. See fig. 3. Thus, lower doses of both Compound A and temozolomide can be effectively used. This, in turn, reduces the toxicity and side effects, if any, associated with the administration of Compound A or temozolomide without compromising efficacy.

Other in vitro and in vivo studies have been conducted to characterize Compound A. For example, tumor growth inhibition (TGI) by Compound A has been demonstrated in TNBC and GBM tumor xenograft models. In a triple negative breast cancer (TNBC) PDX model, COH7, treatment with Compound A showed significant tumor growth inhibition (TGI) in NOD/SCID/IL2Ryc'/· (NSG) mice. See fig. 1. In the PDX GBM model, GBM15, the efficacy of Compound A has been demonstrated using several treatment regimens. See fig. 2. Compound A has shown dose and time dependent inhibition of GLI1 and may be of value in the treatment of Hh-driven or GLI-driven transcription tumors such as BCC. Compound A also reduced tumor cell inoculation in an in vivo basal-like breast cancer (BLBC) model and showed higher activity than temozolomide in a GBM3 xenograft model, and also showed synergistic effects in combination with temozolomide, thus suggesting that Compound A in combination with temozolomide is useful against cancer stem cell or MYC driven tumors.

For example, regulation of MYC gene expression by BRD4 has been shown in models of Burkitt's lymphoma with BRD4 inhibition leading to growth arrest. Mertz, 2011. Similarly, in a lung adenocarcinoma model, inhibition of BRD4 was found to be antiproliferative; but this effect was attributed to a decrease in FOSL1 expression. Lockwood, 2012. BRD4 has also been shown to regulate GLI1 gene expression, thereby modulating the hedgehog signaling pathway, which is known to be deregulated in several types of cancer. Tang, 2014. Compound A treatment inhibited MYC gene expression in Raja Burkitt's lymphoma cells with a mean IC ₅₀ of 0.06 μM; expression of the FOSL1 gene in glioblastoma U 87 astrocytoma cells with an IC ₅₀ value of 0.03 μM; and expression of the GLI1 gene in MIA-PaCa-2 pancreatic adenocarcinoma cells with an IC ₅₀ value of 0.24 μM. Treatment of mice bearing COH7 (a patient-derived xenograft (PDX) tumor xenograft with triple-negative breast cancer (TNBC)) with Compound A resulted in a down-regulation of MYC, and modulation of MYC expression levels correlated with intratumoral concentrations of Compound A In addition to regulating gene expression in a dose-dependent manner, tumor cell growth was inhibited in vitro.

Several other in vitro and in vivo studies were conducted to characterize the absorption, PK, distribution, metabolism and elimination of Compound A. The pharmacokinetics and oral bioavailability of Compound A were evaluated in Sprague-Dawley rats and Beagle dogs. In vivo treatment of tumor-bearing mice recreated in vitro data and provided dosage, schedule, and plasma exposure information. Reliable and reproducible bioanalytical methods for quantifying Compound A levels have been developed and used in PK and toxicokinetic studies. Human PK parameters and exposure were predicted using allometric scaling.

The metabolism of Compound A was assessed in vitro using human hepatocytes and the N-desmethyl derivative was identified as one metabolite. This metabolite has also been observed in rat, dog and monkey hepatocytes. No unique human metabolites have been found. Studies using recombinant CYP enzymes have shown that a variety of CYP enzymes can metabolize Compound A. In vitro, Compound A does not inhibit CYP1A2 and CYP3A4; but may inhibit CYP2C9, CYP2C19 and CYP2D6. In hepatocytes, Compound A does not induce CYP1A2, CYP2B6 or CYP3A4. Therefore, at clinically relevant concentrations, Compound A has minimal potential to cause drug-drug interactions with co-administered drugs that are CYP substrates.

The clinical study evaluated the safety and tolerability of combination therapy comprising Compound A and temozolomide in humans, as well as biological and clinical activity. For this purpose, preclinical studies of Compound A are useful. Based on the doses and exposures at which major treatment-related effects occurred in studies according to good laboratory practice (GLP) in 4-week-old rats and dogs, both species are considered equally sensitive to toxicity associated with with Compound A administration. The suggested starting dose for humans is 15 mg of Compound A, once daily for three consecutive days, followed by four consecutive days of rest each week (3/7 day dosing schedule). Since Compound A and temozolomide exhibit a synergistic effect, the dose of either or both in combination therapy was investigated.

In embodiments, the present invention provides a method for treating cancer, comprising administering a BET inhibitor and a chemotherapeutic agent; for example Compound A and temozolomide. Thus, embodiments further provide pharmaceutical compositions that include a BET inhibitor as an active ingredient, or a BET inhibitor and temozolomide as active ingredients. Such pharmaceutical compositions may take any desired physical form depending on a number of factors, including the desired route of administration, and the physicochemical and stereochemical form taken by these agents or their pharmaceutically acceptable salts. Such physical forms include a solid, liquid, gas, sol, gel, aerosol, or any other physical form known or not yet disclosed. The term pharmaceutical composition comprising one or both of these agents also encompasses these agents without any other additive. The physical form of a pharmaceutical composition can influence the route of administration, and one skilled in the art knows how to choose a route of administration that takes into account both the physical form of the composition and the disorder being treated. Pharmaceutical compositions that include a BET inhibitor or a BET inhibitor and temozolomide can be prepared using techniques well known in the pharmaceutical art. A pharmaceutical composition that includes Compound A or Compound A and temozolomide may include an additional active agent. This additional active agent may have the same or similar molecular target as Compound A, or a similar molecular target such as temozolomide or paclitaxel bound to albumin, or it may act upstream or downstream of the molecular target(s) to one or more biochemical pathways.

Routes of administration include, but are not limited to, oral administration and parenteral administration. Parenteral administration includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical administration to the ears, nose, eyes or skin application. Other modes of administration include, but are not limited to, infusion methods, including infusion or bolus injection, by absorption through epithelial or mucosal surfaces such as the oral mucosa, rectal mucosa, and intestinal mucosa. Compositions for parenteral administration may be enclosed in an ampoule, disposable syringe or multi-dose vial made of glass, plastic or other material. The combination therapy described herein encompasses BET inhibitors and temozolomide, paclitaxel, or romidepsin formulated for the same or different routes of administration. For example, Compound A may be formulated for oral administration while temozolomide is formulated for infusion.

Determining the effective amount of the BET inhibitor (such as Compound A) and the chemotherapeutic agent (such as temozolomide, paclitaxel, or romidepsin) is within the skill of the art in light of the disclosure provided herein. The effective amount of the pharmaceutical composition used for a particular purpose, as well as the pharmacologically acceptable dose, determined by toxicity, excretion and general tolerability, can be determined in cell cultures or experimental animals using pharmaceutical and toxicological procedures currently known to those skilled in the art or in any other way not yet disclosed. One example is the determination of _IC50 (half the maximum inhibitory concentration) of pharmaceutical compositions in vitro in cell lines or target molecules. Another example is the determination of the LD ₅₀ (lethal dose causing death in 50% of test animals) of a pharmaceutical composition in experimental animals. The precise methods used in determining the effective amount depend on factors such as the type and physico-chemical properties of the pharmaceutical composition, the property being tested, and whether the test is to be performed in vitro or in vivo. Determination of the effective amount of a pharmaceutical composition is well known to the person skilled in the art, who uses the data obtained in any tests when making this determination. Determination of an effective amount of a combination of agents, for example, Compound A and temozolomide, paclitaxel, or romidepsin, to add to a cancer cell also includes determining an effective therapeutic amount, including drawing up a range of effective doses for in vivo use, including in humans.

Treatment is considered in living organisms, including, but not limited to, mammals (in particular, humans), as well as other mammals of economic or social importance, including endangered ones. Other examples include farm animals or other animals commonly raised for human consumption and companion animals. The toxicity and therapeutic efficacy of the pharmaceutical composition(s) can be determined using standard pharmaceutical procedures in cell cultures or animals. Examples include determination of IC ₅₀ and LD ₅₀ for combination therapy under consideration

- 15 040062 connections. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range for human use. The dose may vary depending on the dosage form used and the route of administration used.

Effective amounts of active agents in combination therapy with Compound A and temozolomide result in slowing down the spread of cancer cells or tumor growth inhibition (TGI), but may have minimal effects on non-cancerous cells. The concentrations that produce these effects can be determined using, for example, markers of apoptosis such as apoptotic index and/or in vitro or in vivo caspase activity.

A method of treating cancer using a combination of Compound A and temozolomide, paclitaxel or romidepsin comprises a therapeutically effective amount of these substances and encompasses any method of dosing one or both of these compounds. Dosing may include single or multiple administrations of any of a number of pharmaceutical compositions that include Compound A, temozolomide, paclitaxel or romidepsin or Compound A and temozolomide, paclitaxel or romidepsin as the active ingredient(s). Examples include a single administration of a slow release formulation, a course of treatment comprising multiple treatments on a regular or irregular basis, multiple administrations over a period of time until relief is achieved, prophylactic therapies given prior to the onset of symptoms, or any other dosing regimen known to in the art, or still to be disclosed, which one of ordinary skill in the art recognizes as a potentially effective regimen. The final dosage regimen, including regularity and route of administration, depends on any of numerous factors, including the subject being treated; biomarkers that determine a particular pathological condition or the effectiveness of an agent; the severity of the disease; method of administration; the stage of development of the disease; the presence of one or more other conditions, such as pregnancy, infancy; the presence of one or more additional diseases; or any other factor currently known or not yet disclosed that influences the choice of route of administration, the dose to be administered, and the period of time over which the dose is administered.

Pharmaceutical compositions that include Compound A may be administered before, simultaneously with, or after administration of a pharmaceutical composition that includes temozolomide, paclitaxel, or romidepsin. If the compositions are administered simultaneously, they are administered at the same time or 1 minute apart from each other. In case of non-simultaneous administration, a pharmaceutical composition containing tomozolomide, paclitaxel or romidepsin and Compound A may be administered over a period of 1 or more minutes, hours, days, weeks or months before or after administration of a pharmaceutical composition that includes another agent. Alternatively, the combination of pharmaceutical compositions may be administered cyclically. Cyclic therapy involves the administration of one or more pharmaceutical compositions over a period of time, followed by the administration of one or more different pharmaceutical compositions over a period of time, and the repetition of this sequential administration in order to reduce the development of resistance to one or more compositions, avoid or reduce side effects of one or more compositions, or improve the effectiveness of treatment.

In addition, a set of genes has been identified that is downregulated by Compound A treatment ex vivo in peripheral blood mononuclear cells (PBMCs) and in whole blood. In the present study, changes in the expression of these genes in whole blood or other genes in a biopsy sample of tumor tissue of the tumor can provide confirmation that the dose is pharmacologically active, and can help distinguish which dose shows the highest pharmacological activity. Predictive biomarkers allow for the prospective identification of patients who are most likely to benefit clinically from Compound A alone, in combination with temozolomide, paclitaxel, or romidepsin, or in combination with other agents. While the predictive diagnostic assays in the current study are exploratory in nature, they identify associations between biomarkers and responses that serve as a basis for future diagnostic studies.

These embodiments further include methods of treating cancer that include the combination therapy described herein and further include another method of treatment. Such treatments include, but are not limited to, radiation therapy, chemotherapy, surgery, immunotherapy, cancer vaccines, radioimmunotherapy, treatment with pharmaceutical compositions other than those described herein, or any other method that effectively treats cancer in combination with a disclosed compound known to currently or not yet disclosed. The combination therapy according to the present invention works synergistically: the combination of Compound A and temozolomide, paclitaxel or romidepsin is more effective than any therapy administered alone. Another treatment may be additive or synergistic in efficacy. Thus, lower doses of both treatments can be effectively used. This, in turn, reduces the toxicity and side effects, if any, associated with the use of any method without compromising effectiveness.

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In another aspect, combination therapy comprising Compound A and temozolomide is administered in combination with a therapeutically effective amount of radiation therapy. Radiation therapy can be used simultaneously, before or after therapy with Compound A and temozolomide, paclitaxel or romidepsin. Radiation therapy may act additively or synergistically with combination therapy. This particular aspect of the invention will be most effective against cancers known to respond to radiation therapy. Cancers known to respond to radiation therapy include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, laryngeal cancer, cervical cancer, cancer nasopharynx, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small cell lung cancer, non-small cell lung cancer, brain tumors, other tumors of the central nervous system, or any other such tumor known to currently or not yet disclosed.

In another aspect, a patient with glioblastoma is treated with a bromodomain inhibitor, such as Compound A, in combination with temozolomide, paclitaxel, or romidepsin. An effective dose of the agents in a combination therapy is amounts effective to prevent the onset of symptoms of a disorder or to treat some of the symptoms of a disorder suffered by a patient. An effective dose also includes an effective amount, a therapeutic amount, or any amount sufficient to produce the desired pharmacological or therapeutic effects, thereby effectively preventing or treating the disorder. Thus, in the treatment of a patient with glioblastoma, an effective amount of combination therapy provides an amount of Compound A and temozolomide, paclitaxel or romidepsin sufficient to slow or stop the progression, migration, metastasis, growth or development of the tumor. The result can be life extension. A pharmacologically acceptable dose or maximum acceptable dose includes a dose that can be administered to a patient that is not lethal to the patient or does not cause effects that threaten the health or life of the patient.

In particular, patients include a human, non-human primate, pet or mammal suffering from the disease. In one aspect, the patient has symptoms that indicate the presence of a tumor or other growth in the brain. Such symptoms include headache, seizures, mental or personality changes, syndrome of a mass in the cranial cavity, or one of a number of focal or localized systems, including ringing or buzzing sounds, hearing loss, loss of coordination, decreased sensation, weakness or paralysis, difficulty with walking or talking, difficulty maintaining balance, decreased muscle control, or double vision. Patients may present with one or more different types of brain tumors, including acoustic neuroma, astrocytoma, ependyoma, glioblastoma multiforme, meningioma, metastatic tumors derived from another tumor type, glioblastoma mixed, oligodendroglioblastoma, or a tumor of the pinel region of the brain.

Thus, a clinical trial of an exemplary BET inhibitor, Compound A, particularly in combination with temozolomide, paclitaxel or romidepsin, is required for antitumor activity in various cancers. The human study is designed to evaluate drug safety and pharmacokinetic profiles across different dose levels/dosing regimens, and reflects initial drug efficacy signals to accelerate the development of Phase 2 clinical trials. All human studies were conducted in accordance with the Good Clinical Practice rules initiated by the International Conference on Harmonization Good Clinical Practices.

In particular, the study of a BET inhibitor in combination with a chemotherapeutic agent is an open-label, first-in-human (First-In-Human, FIH) phase 1a clinical trial with dose escalation and cohort expansion, in subjects e.g. with advanced solid tumors or recurrent /refractory forms of NHL. A study can consist of two parts: dose escalation (Part A) and cohort expansion (Part B). An exemplary suggested starting human dose of Compound A is 15 mg administered once daily for three consecutive days followed by four consecutive days of drug holiday, each week (3/7 day dosing regimen). The main goal of the study is to identify a dose of a BET inhibitor and a chemotherapeutic agent that is not only safe, but also exhibits pharmacological activity. For example, the intended starting dose of temozolomide, paclitaxel, or romidepsin and Compound A in combination therapy can be determined by referring to existing dosing regimens, typically with additional pharmacokinetic, pharmacological, and toxicological studies.

The dose escalation part of the study (Part A) examines increasing oral doses of combination therapy to evaluate the maximum tolerated dose (MTD) and/or RPTD of a BET inhibitor and a chemotherapeutic agent. The cohort expansion part of the study (Part B) further assesses the safety and efficacy of combination therapy,

- 17 040062 conducted at MTD or lower dose in selected expanded cohorts. One or more dosing regimens or disease subsets may be selected to expand cohorts. Parts A and

B consists of three periods: screening, treatment and follow-up periods (see Fig. 4). The objectives of the study are summarized in Table. 1, and the endpoints of the study are summarized in Table. 2.

Table 1 _________________________ Research objectives _________________________ Primary research objectives

The primary objectives of the study are as follows:

- Determining the safety and tolerability of combination therapy containing a BET inhibitor.

- Determination of the Maximum Tolerated Dose (MTD) or Dosage (recommended for Phase 2 Clinical Trial (RP2D) for ____________ Combination Therapy ___________________________________________ Secondary Study Objectives_______________________________________________ Secondary Study Objectives are as follows:

- Obtaining information on the preliminary efficacy of combination therapy.

- Characteristics of the pharmacokinetics (PK) of each component of ____________ combination therapy.

- Evaluation of the pharmacodynamic (PD) effects of combination therapy on gene expression in peripheral blood and, if present, in tumor samples.

- Investigation of the relationship between combination therapy dose, plasma levels and selected study endpoints (eg toxicity, pre-activity or biomarkers).

- Characterization of the main metabolites of combination therapy in plasma, subject to the availability of sufficient data,

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table 2

Study Endpoints

end point Name Description Time range Primary Security endpoints DLT and MTD were assessed using the NCI CTCAE test version 4.03 Dose escalation Secondary Preliminary Efficiency Determined by response rate using disease-specific response criteria Dose Escalation and Cohort Expansion Disease control rate (DCR), objective response rate (ORR), duration of disease response or stabilization, and progression-free survival (PFS) Dose Escalation and Cohort Expansion Overall survival From randomization to death from any cause Dose Escalation and Cohort Expansion Pharmacokinetic (PK) Endpoints Peak (maximum) plasma concentration of drugs (Cmax), area under the plasma concentration-time curve (AUC), time to peak (maximum) plasma concentration (tmax), terminal half-life (ΐι/ ₂ ), apparent clearance (CL/F), apparent volume of distribution (Vz/F), and accumulation index of each component of combination therapy Dose escalation search engine Pharmacodynamic endpoints (PD) • Gene expression in cellular components of peripheral blood • Gene expression in tumor tissue, if present Dose escalation and cohort expansion

DLT = dose-limiting toxicity; MTD = maximum tolerated dose; NCI CTCAE = National Cancer Institute Common Terminology Criteria for Adverse Events; NTD = intolerable dose; RNA = ribonucleic acid.___________

During the treatment period, formulations containing a BET inhibitor may initially be administered orally once a day (QD) for three consecutive days followed by four days of drug holiday (dosing regimen of three days on/seven days of drug holiday) in each four-week cycle. Alternative dosing regimens (eg, two days on/five days of drug holiday, each week) are being explored based on the Science Review Committee (SRC) analysis of available data on safety, PK, pharmacodynamics (PD), and efficacy. During the combination treatment period, formulations containing a BET inhibitor may initially be administered orally, once a day for three consecutive days followed by four consecutive days of drug holidays, every week (three/seven day dosing regimen) in each four-week cycle; and formulations containing temozolomide can be administered on days 79 and 22-24 of a four-week cycle. Alternative dosing regimens (eg, two days of dosing/five days of medication holidays, each week) are being explored based on SRC analysis of available safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy data.

A decision regarding the evaluation of additional subjects within a cohort of subjects receiving a given dose; the cohort of subjects receiving the higher dose; cohorts receiving an intermediate dose; a smaller dose increase step; alternative dosing regimens (eg, two days on/five days of drug holiday, each week) or MTD ads, also accepted by SRC based on BLRM assessment and analysis of available safety information (i.e. DLT and non-DLT data), PK , PD and efficiency.

Following the first dose in any cohort during dose escalation, subjects in each cohort are observed for 28 days before moving on to the next dose cohort. No more than one subject per day is selected for this dose escalation cohort. Subjects not evaluable for DLT are replaced.

For Part B, Cohort Expansion, after completion of the dose escalation (Part A), cohorts of subjects with selected tumors are selected for the expansion phase (Part B), each consisting

- 19 040062 out of approximately 20 measurable entities. Expansion may occur at the MTD and dosage regimen established in the dose escalation phase, or at an alternative tolerated dose and dosage regimen based on an analysis of the safety, PK, PD, and efficacy data obtained in Part A of the combination therapy. One or more dosing regimens may be chosen to expand cohorts. The SRC continues to review safety data regularly throughout the study and makes recommendations for continuation of the study and dose modification as needed.

For example, Compound A may be formulated as tablets for oral administration; and temozolomide can be formulated as capsules for oral administration. Alternatively, Compound A and temozolomide are formulated together as a single tablet or capsule for oral administration. In another alternative example, Compound A is formulated as tablets for oral administration and temozolomide is formulated for infusion. As another example, since albumin-bound paclitaxel is formulated for infusion, Compound A can be formulated for oral administration. Alternatively, Compound A can be adapted for infusion with protein bound paclitaxel. The labeling is appropriate, for example, for research use in accordance with the regulations of the relevant country health authority.

For baseline efficacy evaluations, subjects were assessed for efficacy after every two cycles until cycle 6, and every three cycles thereafter. All subjects who discontinued treatment were followed up until progression or initiation of new systemic anticancer therapies. During the follow-up period, all subjects were monitored for safety after the last dose of any component of the combination therapy. After a safety follow-up visit, all subjects were followed up every three months thereafter to assess survival for up to two years or until death, loss of contact for follow-up, or study termination.

The tumor response was determined. For solid tumors, the score is based on the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Eisenhauer et al., 45 Eur. J. Cancer 228 (2009). For the NHL, the score is based on the International Working Group Revised Response Criteria for Malignant Lymphoma. Cheson et al., 25 J. Clin. oncol. 579 (2007). Positron emission tomography (PET) with [ ¹⁸ F]-fluorooxyglucose (FDG) or FDG PET/CT imaging is required to confirm a complete response in subjects with tumors with increased FDG accumulation.

During part A of the dose escalation, approximately 30-40 subjects were selected. During part B of the expansion of the cohorts, at least 14 evaluable subjects were initially selected for each cohort of subjects having a particular tumor type. If the response rate is 20% or more, there is a greater than 95% chance that one or more responders will be observed among the first 14 subjects, which is subject to statistical update based on the change in DCR as the main indicator of effectiveness. Gehan, 13 J. Chronic Dis. 346 (1961). If no responder is observed among the 14 subjects, then recruitment for that cohort of subjects with a particular tumor type is stopped due to lack of efficacy. Otherwise, the cohort of subjects with a particular tumor type is expanded to approximately 20 subjects in case a responder is observed.

At all decision times, the BLRM allows for dose increment changes based on observed DLT values; however, the dose for the next cohort will not exceed a 100% increase from the previous dose. The maximum tolerated dose (MTD) is the highest dose that is unlikely (post hoc probability <25%) to cause DLT y>33% of treated subjects in the first cycle of treatment with the active agent.

With respect to part B of cohort expansion, once the dose escalation (Part A) is completed, cohorts of subjects with the selected tumor type are entered into the expansion phase (Part B), with each cohort consisting of approximately 20 evaluable subjects. Expansion may occur at the MTD and dosage regimen established in the dose escalation phase, or at an alternative tolerated dose and dosage regimen based on an analysis of the safety, PK, PD and efficacy data obtained in Part A. One or more dosing regimens may be chosen to expand cohorts.

The end of study is defined as the date of the last visit of the last subject to complete post-treatment follow-up, or the date of receipt of the latest data for the last subject required for primary, secondary, and/or exploratory analysis, as previously specified in the protocol, whichever of these dates is the later.

Examples

Example 1 Synthesis of 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1one (compound A).

Unless otherwise indicated, reagents and solvents were used as received from commercial suppliers. Anhydrous solvents and oven dried glassware have been used for moisture and/or oxygen sensitive synthetic transformations. The outputs have not been optimized. Response times are approximate and have not been optimized. Column chromatography and thin layer chromatography (TLC) were performed on silica gel unless otherwise indicated.

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Spectra are in ppm (δ) and coupling constants (J) are in hertz. For spectra

1H NMR peak was used as a reference peak of the solvent.

4-(Methylsulfonyl)phenol was mixed with N-bromosuccinimide (NBS) and H ₂ SO ₄ (catalytic amount) in tetrahydrofuran (TFH) to form 2-bromo-4-(methylsulfonyl)phenol, which was then reacted with cyclopropylmethyl bromide and K2CO3 in acetone to give 2-bromo-N-(cyclopropylmethyl)-4-methylsulfonylaniline. A mixture of 2-bromo-1h[-(cyclopropylmethyl)-4methylsulfonylaniline, K ₃ PO ₄ and Pd(dppf)Cl ₂ in 5:1 dioxane:H ₂ O was purged three times with nitrogen and then stirred at 70°C for 18 h in an N2 atmosphere. The mixture was filtered and concentrated. The residue was purified by preparative HPLC to give the title compound. See US Pat. No. 9,034,900.

Characterization of 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1one:

1Н NMR (CDCl ₃ , 400 MHz) δ 8.54 (d, J=7.6 Hz, 1H), 7.80 (dd, J1=8.8 Hz, J2=2.4 Hz, 1H), 7.67 (d, J=2.4 Hz, 1H) , 7.60-7.55 (m, 2H), 7.17 (d, J=8.0 Hz, 1H), 7.15 (s, 1H), 6.77 (d, J=8.8 Hz, 1H), 4.24-4.23 (m, 1H), 3.66 (s, 3H), 3.06 (s, 3H), 3.03-2.99 (m, 2H), 0.93-0.91 (m, 1H), 0.45-0.37 (m, 2H), 0.12-0.054 (m, 2H).

LCMS (M+H)+=383.1 (M+H) ⁺ .

Example 2 BET and temozolomide inhibition assay in vitro and cell assay in vitro.

The IC ₅₀ value was determined for the heterocyclic derivatives described herein used as inhibitors of BRD4 (see US Pat. No. 9,034,900), including Compound A. His-tagged BRD4 was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (final concentration 2 nM) was mixed with peptide (final concentration 15 nM) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA and 0.005% (w/v) Brij-35 in the presence of DMSO (final DMSO concentration 0.4%) or serial dilution of the compound in DMSO. After a 20-minute incubation at room temperature, alpha-streptavidin donor beads and nickel chelate acceptor beads were added to a final concentration of 5 μg/ml. After 2 hours of equilibration, plates were read on an Envision plate reader and IC ₅₀ was calculated by fitting a four parameter non-linear curve. The ability of Compound A to inhibit BRD4 activity and the corresponding IC ₅ o value was quantified.

A colorimetric cell proliferation assay (Cell-MTS assay) was performed to evaluate the ability of the heterocyclic derivatives described herein as inhibitors of BRD4 (see US Pat. No. 9,034,900), including Compound A, to influence the proliferation of known cancer cell lines. The Cell-MTS assay is a 7-day colorimetric plate assay assessing the amount of newly formed NADH in the presence or absence of a test compound. The NADH level is used to quantify the proliferation of cancer cells. Known cancer cell lines with various master mutations were obtained from the American Type Culture Collection (ATCC) and subcultured according to the ATCC protocols according to common practice. For routine analysis, these cells were seeded at densities that provided about 90% confluency after 7 days of culture. Human Burkitt Raji lymphoma cells (cMYC) were seeded in a 96-well plate at a density of 15,000 cells per well. Human promyelocytic leukemia HL-60 cells (NRAS, p16, p53, c-Myc amplified) were seeded in a 96-well plate at a density of 5000 cells per well. NCI-H460 human non-small cell lung cancer cells (KRAS, PIK3CA, STLK11, p16) were seeded in a 96-well plate at a density of 3000 cells per well.

Then, 24 hours after seeding, the test compound was added to the cells at an 11-point dilution, with the final concentration ranging from 100 μM to 2.0 nM. Cells were incubated in the presence of the compound for 168 h at 37°C and in an atmosphere of 5% CO ₂ . At the end of this incubation period, 80 µl of medium was removed and 20 µl of CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay solution (Promega) was added. Cells were incubated until reaching an optical density OD ₄₉₀ >0.6. IC ₅₀ values were calculated using the IDBS XLfit software package and included OD ₄₉₀ values with background subtraction and normalization to DMSO controls. Cell proliferation IC ₅₀ values were downloaded and archived using the Chem Biography Platform.

IC ₅₀ data for 4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinoline-1-

it in these in vitro assays are as follows: HL-60 IC50 H460 1C ₅₀ BRD4 ICso Raji_______IC50 (µM) (µM) (µM) (µM) < 5 µM < 5 µM < 5 µM > 5 µM

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Example 3 Pharmacology in vitro.

The regulation of MYC gene expression by bromodomain-containing protein 4 (BRD4) has been shown in models of Burkitt's lymphoma with inhibition of BRD4 leading to growth arrest (Mertz, 2011). Similarly, in a lung adenocarcinoma model, BRD4 inhibition was found to be antiproliferative; however, this effect was attributed to a decrease in FOSL1 expression (Lockwood, 2012). BRD4 has also been shown to regulate GLI1 gene expression, thereby modulating the Hh signaling pathway, which is known to be deregulated in some types of cancer. (Tang, 2014). The effect of Compound A therapy on MYC, FOSL1 and GLI1 gene expression was assessed by reverse transcription polymerase chain reaction (qRT PCR). Therapy with Compound A inhibited MYC gene expression in Raji cells of Burkitt's lymphoma with a mean half-maximal inhibitory concentration (IC ₅₀ ) of 0.06 μM; expression of the FOSL1 gene in U 87 glioblastoma cells with an IC ₅₀ value of 0.03 μM; and expression of the GLI1 gene in MIA-PaCa-2 pancreatic adenocarcinoma cells with an IC ₅₀ value of 0.24 μM.

Compound A demonstrated in vitro inhibition of tumor cell growth using antiproliferative two-dimensional (2-D) cultures with cell lines and inhibition of colony formation using three-dimensional (3-D) organoid cultures with cells from PDX GBM tumor models and PDX breast cancer models .

The effect of Compound A on colony formation in fourteen PDX GBM tumor models was evaluated using in vitro neurosphere assay. Compound A was tested at concentrations ranging from 0.0003 to 20 μM in 3-fold increments. Colony formation was assessed after seven days of treatment by counting the number of colonies by microscopy. Compound A inhibited colony formation in a dose-dependent manner with mean half-maximal inhibitory concentration (IC ₅₀ ) ± standard error of the mean (SEM) ranging from 0.11 ± 0.04 to 2.00 ± 0.40 μM and spanning a range of 18-fold activity. The overall mean for the GBM models was 0.62±0.13 µM.

The effect of Compound A on colony formation in four PDX models of breast cancer was evaluated using a 3-D in vitro culture system based on Matrigel. Compound A was tested at concentrations ranging from 0.008 to 5 μM or from 0.0016 to 1 μM in 5-fold increments. Colony formation was assessed after 7 days or 14 days of treatment by counting the number of colonies by microscopy. Compound A inhibited colony formation in a dose-dependent manner resulting in a mean IC ₅₀ for BR0869f estrogen receptor negative (ER), progesterone receptor (PR) negative and HER2/neu positive (ER-PR-Her2+) tumor models of 0.12 ±0.01 µM, and IC ₅₀ values for COH69, COH71, and TNBR3 triple negative breast cancer (TNBC) models of 0.07 µM, 0.18±0.02 µM, and 0.08±0.00 µM, respectively . The overall mean value for the three TNBC models was 0.11±0.04 µM.

Example 4 Pharmacology in vivo.

In murine studies, Compound A demonstrated dose-dependent tumor growth inhibition (TGI) in patient-derived xenografts (PDX) of TNBC and GBM tumors. In addition, using cut-off assays, a reduction in the incidence of tumor initiating cells (TICs) was demonstrated after treatment with Compound A (performed using a daily dosing regimen and not included in the Clinical Trial Application).

Various doses and dosing regimens of Compound A were evaluated preclinically. Compound A, dosed on a 3 day on/4 day drug holiday regimen, showed TGI efficacy equivalent to that seen with continuous dosing regimens, as well as improved tolerability compared to continuous dosing regimens. Body weight, gastrointestinal (GI) and bone marrow (BM) toxicities were completely reversible with lower frequency dosing regimens and recovery was appropriate for weekly repeat dosing.

Treatment of mice bearing COH70, a TNBC PDX tumor, with Compound A at 2 mg/kg or 10 mg/kg resulted in a decrease in MYC expression. Compound A at 2 mg/kg maximally suppressed MYC expression by 51.3% after 2 hours, with MYC expression restored to control levels 8 hours post-dose. Compound A at a dose of 10 mg/kg maximally suppressed MYC expression by 63.4% after 4 hours; however, MYC expression did not recover to control levels 24 hours post-dose. Corresponding concentrations of Compound A in the tumor were determined in the COH70 model at 2, 4 and 8 hours post-dose. The maximum measured levels of Compound A in the tumor were observed 2 hours after administration and amounted to 1.3±0.3 and 6.7±1.7 μM at 2 and 10 mg/kg, respectively. Modulation of MYC expression levels correlated with intratumoral concentrations of Compound A.

It was noted that there was a significant TGI in the subcutaneous PDX model of TNBC in NOD/SCID gamma (NSG) mice at Compound A doses of 12.5, 16 and 20 mg/kg. The introduction was carried out orally

- 22 040062 by gavage once a day (QD) for three consecutive days followed by four days of drug holidays (indicated as 3x/week in Figure 1) (3x/week = three consecutive days of dosing Compound A once a day followed by 4 days of drug holiday; PO = oral, SEM = standard error of the mean), every week for six weeks. Compound A was well tolerated up to a daily dose of 25 mg/kg. When tumor volumes were measured on Day 38 compared to vehicle control, the mean percentage of TGI treated tumors was 64% for the 12.5 mg/kg/dose group, 68% for the 16 mg/kg/dose group, and 72% for the 20 mg /kg/dose. Average body weight increased in all groups. Steady state pharmacokinetic parameters were determined after final dose administration for dose levels of 12.5 and 16 mg/kg. The area under the plasma concentration-time curve from 0 to 24 h (AUC ₀ - _{24 h} ) Compound A at 12.5 mg/kg was 12.003 ng-h/mL and at 16 mg/kg was 15.174 ng-h /ml

In the subcutaneous PDX GBM model, GBM15, the efficacy of Compound A was demonstrated across several dosing regimens, from dosing 5 times QD per week to twice per week for 4 weeks (FIG. 2) (PO = oral, SEM = standard error of the mean) . Tumor-bearing mice were dosed orally QD according to several dosing regimens, with a cumulative weekly dose of Compound A for each dosing regimen being 75 mg/kg. Dosing regimens are as follows:

mg/kg Compound A for 5 consecutive days and 2 drug holiday days (5/2), 25 mg/kg Compound A for 3 consecutive days and 4 drug holiday days (3/4), 37.5 mg/kg Compounds A for 2 consecutive days and 5 days of medicinal holidays (2/5). When tumor volumes were measured on Day 29 and compared with vehicle controls, the mean TGI percentage of tumors treated was 65% for the 15 mg/kg dose group (5/2), 65% for the 25 mg/kg dose group ( 3/4) and 70% for the 37.5 mg/kg group (2/5). Minimal weight loss was observed in all groups (vehicle group = -1.2%, 15 mg/kg dose group = -6.6%, 25 mg/kg group = -3.7%, and receiving 37.5 mg/kg = -3.1%).

Models of xenografts of NUT-carcinoma (NUT Midline Carcinoma, NMC) in mice were studied. Matched cohorts of mice with advanced tumors were randomized to treatment with test compound (Compound A or temozolomide or a formulation containing Compound A and temozolomide) or vehicle administered by daily intraperitoneal injection. Before randomization and after 4 days of therapy, mice were assessed by PET positron emission tomography imaging using the 18F-fluorodeoxyglucose (FDG) radiopharmaceutical. Tumor volume, toxicity or weight loss was measured.

Tumors were harvested, dissected and examined immunohistochemically for BRD4-NUT oncoprotein, cell proliferation, keratin expression, nuclear Ki67 staining and TUNEL. Paired samples from treated and untreated mice were prepared and analyzed using standardized protocols and commercially available software (ie ImageScopt; Aperio Technologies).

Example 5 Antitumor Efficacy in MCF-7 Breast Cancer Xenograft Model.

Sustained release beads containing 0.72 mg of 17-b-estradiol were subcutaneously implanted in nu/nu mice. MCF-7 cells were grown in RPMI medium containing 10% FBS in 5% CO2, 37°C. Cells were centrifuged and resuspended in 50% RPMI (no serum) and 50% Matrigel at 1 x 10 ⁷ cells/ml. MCF-7 cells were injected subcutaneously (100 μl/animal) in the right flank 2-3 days after pellet implantation, and tumor volume ((length x width)/2) was monitored biweekly. When the tumors reached an average volume of ~200 mm ³ , the animals were randomized and treated. Animals were treated with test compound or vehicle daily for 4 weeks. Tumor volume and body weight were monitored biweekly throughout the study. At the end of the treatment period, plasma and tumor samples were taken for pharmacokinetic and pharmacodynamic analyses, respectively.

Example 6 Antitumor Efficacy in the Raji Cell Line Model, Burkitt's Human Lymphoma Cell Line.

Procedure: Female SCID CB17 mice (6-8 weeks, Charles River Laboratories) were inoculated subcutaneously into the right flank of Raji cells (at 3.5 x 10 ⁶ cells/mouse) and the tumor was allowed to grow to about 150 mm ³ . Mice were then randomized into treatment cohorts (N=8) and treated orally once daily with vehicle control or test compound for 21 days. The test compound was administered as a suspension in 1% Tween 80, 40% PEG400 and either: 59% 0.5% HPMC or 9% DMSO + 50% 0.5% HPMC at doses of 5 to 50 mg/kg . The length and width of the tumors were measured in millimeters three times a week. Tumor volumes were calculated using the formula V=LxWxW/2. Tumor growth inhibition (TGI) was calculated as TGI=100-(mean tumor volume of treatment group/mean tumor volume of vehicle control group)x100. TGI measurements were performed until

- 23 040062 until the tumor volume in the control group reached 3000 mm ³ . Statistical analysis was performed using a two-tailed Student's t-test. P values<0.05 were considered statistically significant. TGI has been found to range from 42 to 80%.

Example 7 Synergistic Effects of Compound A and temozolomide in a temozolomide-resistant GBM xenograft model.

O ⁶ -Methylguanyl methyltransferase (MGMT) is implicated in the resistance of GBM (glioblastoma multiforme) to the alkylating DNA damaging agent temozolomide (TMZ). GBM3 is a subcutaneous GBM PDX model with high PCR expression of MGMT, an unmethylated MGMT promoter and a TMZ resistant phenotype. In previous studies of neurospheres cultured from GBM3, RT-PCR analysis showed that Compound A reduced MGMT expression in a dose-dependent manner. When GBM3-bearing mice were dosed with a single dose of Compound A at 20 mg/kg, qRT-PCR revealed a decrease in MGMT expression in the taken tumor. This led to an efficacy experiment to see if Compound A could sensitize TMZ-resistant GBM to TMZ and show synergistic effects compared to each of the compounds administered alone.

Cohorts of NSG mice bearing GBM3 were treated with TMZ at 50 mg/kg by intraperitoneal injection (IP)x3 Q2 weeks; Compound A 6 mg/kg po twice a day (BID) or 12 mg/kg po once a day or a combination of Compound A 6 mg/kg po BID and TMZ 50 mg/kg IPx3 Q2 weeks. Significant amounts of tumor growth inhibition, as measured by tumor volumes, were observed after dosing Compound A alone or in combination with TMZ (FIG. 3). TMZ alone did not induce significant TGI when administered alone (3%). Taken alone, Compound A induced significant tumor growth inhibition (TGI) of 63% (12 mg/kg QD) and 76% (6 mg/kg BID). However, the combination of Compound A and TMZ showed a synergistic effect and significantly outperformed all other regimens in terms of TGI. Moderate weight loss was observed during part of the study (lowest loss -5.1%) in the combination treatment group; but weight loss rebounded and all treatment groups showed a net gain in mean body weight at the end of the study.

Example 8 Oral dosage form.

A tablet was made by mixing 48% by weight of Compound A or a pharmaceutically acceptable salt thereof, 45% by weight of microcrystalline cellulose, 5% by weight of low substituted hydroxypropyl cellulose and 2% by weight of magnesium stearate.

Tablets were made by direct compression. The total weight of the compressed tablets was maintained at 250-500 mg.

Example 9 Non-Clinical Pharmacokinetics and Drug Metabolism.

As described here, a series of in vitro and in vivo studies were conducted to characterize the absorption, PK, distribution, metabolism, and elimination of Compound A. Reliable and reproducible bioanalytical methods for quantifying Compound A levels have been developed and applied in PK and toxicokinetic studies. Human and exposure PK parameters were predicted using allometric scaling.

The pharmacokinetics and oral bioavailability of Compound A were evaluated in Sprague-Dawley rats and Beagle dogs. Systemic clearance was low (approximately 5 to 13% of hepatic blood flow) in male and male rats, but males showed approximately 2-fold higher clearance than females. The volume of distribution varied from about 1-3 times the total body water, indicating the distribution of Compound A in the tissue. The mean oral bioavailability of Compound A was 40% in rats and 76% in dogs. Due to sex differences in systemic clearance between male and female rats and to determine comparable systemic exposure in toxicology studies, doses of Compound A administered to male rats were 3 times higher than to female rats. The toxicokinetics of Compound A in rats and dogs showed no sex differences in systemic exposure, showed a dose-proportional increase in systemic exposure, no accumulation in rats, and up to 3-fold accumulation in dogs after repeated dosing. Compound A showed limited distribution in the brain, with brain to plasma ratios ranging from 0.14 to 0.16 in NSG tumor-bearing mice.

Based on the allometrically derived PK parameters and the assumption of 62% oral _{bioavailability} (mean observed in preclinical species), it can be assumed that the predicted steady state systemic exposure (AUC _{0.24 M} ) of Compound A in humans after a weekly (3 days of intake/4 days of drug holidays) administration of an oral dose of 15 mg can vary from 731 to 2263 ng-h / ml.

In plasma obtained from preclinical species (89.9-93.3%) and human sources (90.2%), no significant differences in plasma protein binding to Compound A were observed.

The metabolism of Compound A was assessed in vitro using human hepatocytes and one metabolite was identified, namely the N-desmethyl derivative.

This metabolite has been observed in hepatocytes of rats, dogs and monkeys. No unique human metabolites have been found. Studies using recombinant cytochrome P450 (CYP) enzymes suggest that a variety of CYP enzymes (CYP2C9, CYP2C19 and CYP3A4) are capable of metabolizing Compound A; however, the relative contribution of individual enzymes is unknown.

In vitro, Compound A does not inhibit CYP1A2 and CYP3A4. Compound A caused inhibition of CYP2C9, CYP2C19 and CYP2D6 with IC ₅₀ 1 values of 3.9, 26.7 and 54.3 μM, respectively. In hepatocytes, Compound A (up to 10 μM) is not an inducer of CYP1A2, CYP2B6 and CYP3A4. Therefore, at clinically relevant concentrations, there is a minimal likelihood that Compound A will cause drug-drug interactions with co-administered drugs that are CYP substrates.

In rats following intravenous (IV) administration of non-radiolabeled Compound A, an average of 0.9% of the dose was excreted unchanged in the bile or urine, indicating that unchanged drug excretion is not the major route of elimination and that metabolism may play an important role in the distribution of Compound A.

Example 10 Non-Clinical Toxicology

Compound A was evaluated in non-GLP exploratory toxicology and genetic toxicology studies and in GLP repeated dose toxicity studies (<4 weeks nonclinical toxicology study). 4-week GLP-compliant oral toxicity studies (with 4-week recovery period) were performed in rats (0, 5, 10, or 20 mg base/kg/dose in females and 0, 15, 30, or 60 mg base/kg /dose for males) and Beagle dogs (0, 1.75, 3.75 or 7.5 mg base/kg/dose). The dosing regimen consisted of once daily administration for three consecutive days followed by four consecutive days of drug holidays, each week for a total of four weeks.

In rats, the main target tissues for toxic effects are the tissues that form the gastrointestinal (GI) tract, bone marrow, lymphoid organs, testicles, and bone. In dogs, the main target tissues for toxic effects are the tissues that form the GI tract, bone marrow, lymphoid organs, and testicles.

In a four week study in rats, a dose of >20 mg base/kg/dose was a dangerous toxic dose. This dose resulted in death or euthanasia of dying animals as early as Day 6, which eventually led to dosing cessation and euthanization on Day 11 of the group of surviving animals (males) treated with 60 mg base/kg/dose; and stopping dosing and killing on Day 11 a group of surviving animals (females) (N=9) treated with 20 mg base/kg/dose, or starting the recovery phase for (N=4). There were no deaths associated with Compound A at doses below 20 mg base/kg/dose. There were no adverse events at the low dose level (5 mg base/kg/dose [females], 15 mg base/kg/dose [males]).

With regard to toxicity, based on the results of a number of clinical, laboratory, macropathological and histopathological studies, the dangerous toxic dose in 10% of rats (STD10) was 20 mg base/kg/dose in females and 30 mg base/kg/dose in males. For any clinical study, the main dose causing toxicity in 10% of animals (STD10) should be considered to be 20 mg base/kg/dose. Due to the absence of adverse results, the maximum no apparent adverse effects (NOAEL) dose in females was 5 mg/kg/dose and in males 15 mg/kg/dose. For any clinical study, the baseline NOAEL should be considered to be 5 mg base/kg/dose. These values apply to a Compound A dosing regimen of three days of administration/four days of medication holidays. Animal recovery evaluation showed that all test drug related results were reversible 4 weeks after dosing was discontinued (with the exception of testis related results, which could not be assessed due to the killing of a group of dying males treated with 60 mg base/kg/dose , originally intended to evaluate reversibility).

Pharmacological safety assessments were also carried out, i.e. standard test battery (FOB) to determine the potential effects of Compound A on the central nervous system as part of a four-week GLP repeated-dose toxicity study in rats. FOB effects associated with Compound A were not observed.

In a four-week study in Beagle dogs, the dangerous toxic dose was 7.50 mg base/kg/dose. This dose resulted in the death of moribund animals (four males and one female) as early as Day 11, which eventually led to discontinuation of dosing in the surviving male group treated with 7.50 mg base/kg/dose and initiation of the recovery phase of the surviving male group. treated with 7.50 mg base/kg/dose. Compound A related deaths were not observed at doses below 7.50 mg base/kg/dose, but Compound A related results were obtained at all doses evaluated.

Based on the results of a number of clinical, laboratory, macropathological and histopathological studies, a dose of 3.75 mg base/kg/dose has been established as the highest non-hazardous toxic dose (HNSTD); NOAEL was not identified. These values were applied to a dosing regimen of three days of intake/four days of medication holidays. At the lowest dose (1.75 mg base/kg/dose), adverse effects were limited to reduced thymus weight and testicular/epididymal toxicity. Evaluation of recovered animals showed that all test drug related results were reversible four weeks after dosing was discontinued, with the exception of testicular and epididymis related results.

Safety pharmacology evaluations were performed to determine the potential effects of Compound A on the cardiovascular and respiratory system in awake Beagle dogs in a four-week GLP repeated dose toxicity study. Compound A-related effects on electrocardiograms, heart rate, or respiration rate were not observed.

In an in vitro study of the human heart specific potassium channel (hERG) gene, an IC ₅₀ value of 24.3 μM was identified.

Compound A was determined to be non-mutagenic in the non-GLP bacterial Ames backmutation test.

In general, Compound A exhibits a non-clinical safety profile suitable for use as a candidate in cancer clinical trials, and the toxicology program for Compound A adequately supports clinical trials in patients with cancer.

Example 11 Human Safety and Tolerability of Compound A.

Compound A is a new investigational drug that has a strong biological case for treating subjects with solid tumors and NHL. The safety and tolerability of Compound A in humans, as well as the biological and clinical activity, were evaluated in a clinical study.

Since no clinical studies have been conducted with Compound A, the efficacy and safety profiles of Compound A in humans are unknown. Potential toxicities for Compound A have been identified based on preclinical studies using Compound A. The safety profiles of the two BET inhibitors tested in Phase I First Time Human Studies (FIH) have been shown to be well tolerated at continuous daily dosing for 14 days at every 21- diurnal cycle, with thrombocytopenia being the main DLT (Abramson, 2015; Herait, 2015), or GI tract toxicity (mainly diarrhea) was observed as DLT (Dombret, 2014; Herait, 2015).

The frequency and extent of the safety assessments proposed for Compound A-ST-001 are representative of the FIH study and are consistent with Compound A toxicology studies in rats and dogs. In rats and dogs, the main target tissues for toxic effects are the GI tract, bone marrow, lymphoid organs, and testicles. General data from preclinical and histopathological studies suggest that the GI system may be a major target of Compound A-mediated toxicity.

Frequent early monitoring of weight, hydration status, serum electrolytes, frequency and severity of diarrhea and vomiting, and episodes of abdominal pain (gastric, intestinal) in subjects is an important part of a plan to monitor safety and implement aggressive symptomatic therapy for nausea, vomiting, or diarrhea with early onset (eg, grade 1). Based on morphological changes, flattening of intestinal villi, and mucosal erosions observed in the GI tract of rats and dogs, subjects with malabsorption syndromes, active ulcer/gastritis, or recurrent episodes of GI bleeding will be disqualified from enrollment in the study. At the discretion of the investigator, mucosal coating agents will be recommended to protect the esophageal/gastric mucosa, as well as monitoring subjects for GI bleeding. Subjects will be asked to report instances of GI discomfort or pain, loss of appetite, or blood in stools.

The detection of bone marrow hypocellularity and depletion of lymphoid tissue (thymus, spleen, lymph nodes) emphasize the importance of frequent monitoring of complete blood count, with platelet count and leukocyte differentiation (WBC). Subjects will be monitored for possible toxicity using standard and specialized laboratory tests, including complete blood count, prothrombin time (PT)/activated partial thromboplastin time (APTT)/international normalized ratio (INR), and serum chemistry.

Temporary changes in blood glucose levels were observed in only a few cases in preclinical toxicology studies using Compound A. In addition, preliminary data from a clinical study of a new experimental drug BETi, OTX015, reported that 7 of 37 patients with non-leukemic forms of hematological malignancies - 26 040062 patients had grade 1-2 hyperglycemia and 1 patient experienced grade 3 hyperglycemia (Thieblemont,

2014). It is not known whether hyperglycemia can occur with Compound A in humans, and standard laboratory tests will include fasting glucose measurements. General guidelines for the management of possible hyperglycemia are presented in FIG. 7.

The results of histopathological examination of the testes and epididymis of male rats and dogs will warrant a ban on sperm donation and adoption of children for the duration of the clinical study and for at least 3 months after the last dose of the study. Histological lesions in the reproductive organs of female animals were not observed in preclinical studies. The significance of this preclinical finding and the potential and relative clinical risk remains unknown at this time. Long-term toxicity and reproductive toxicity studies have not been conducted for Compound A. Subjects will need to follow the pregnancy prevention guidelines described herein.

Because this study is an FIH study, subjects with a history of heart failure, coronary artery disease, uncontrolled hypertension, severe cardiac arrhythmias, or long QT interval on ECG will be excluded from enrollment. All study subjects will require confirmation of adequate left ventricular ejection fraction (>45%) at baseline.

As detailed herein, the study is conducted in two parts: dose escalation (Part A) and cohort expansion (Part B).

In Part A, a Bayesian logistic regression model (BLRM) using dose escalation with overdose control (EWOC) controls dose escalations to the estimated MTD for Compound A. Babb 1998, Neuenschwander 2008. Traditional dose escalation designs (e.g., 3+3, rolling six , accelerated titration) have been developed for cytotoxic agents, and dose escalation decisions are based on toxicity rates, with the assumption that efficacy and toxicity increase with dose. More recent molecularly targeted drugs may have different dose-toxicity and dose-effect curves, and design based on more than just toxicity data may be more effective in determining the recommended dose. Tourneau et al., 101 J. Natl. Cancer Inst. 708 (2009); Ivy et al., 16 Clin. Cancer Res. 1726 (2010).

A statistical model-based approach (BLRM with EWOC) allows the use of preclinical data in conjunction with observed clinical data (e.g., toxicity, pharmacodynamics, pharmacokinetics, efficacy, etc.) when assigning each subject a dose level and can potentially reduce the number of subjects treated at subtherapeutic or intolerable doses. Tourneau et al., 7 PLoS ONE e51039 (2012). The use of the EWOC provides rules or restrictions to avoid dosing outside of the MTD. Additional design details are provided below. One or more dosing regimens and/or disease subsets may be selected to expand the Part B cohorts to provide additional safety and efficacy information for larger cohorts of subjects (up to about 20 subjects in each cohort).

Based on the doses and exposures at which the major treatment-related effects occurred, in the GLP studies in 4-week-old rats and dogs, both species were found to be equally susceptible to the toxic effects associated with the administration of Compound A. The suggested starting dose of Compound A in humans is 15 mg given once daily for three consecutive days followed by four consecutive days of drug holidays, each week (3/7 day dosing regimen). This dose of Compound A was calculated using the approach described in ICH Harmonised Tripartite Guideline S9, Nonclinical evaluation for anticancer pharmaceuticals (2009), and is summarized in Table 3:

Table 3

Suggested Clinical Starting Dose of Compound A based on 10% acute toxic toxic dose in rats and the highest non-hazardous toxic dose in dogs in a 1-month toxicity study

Kinds rat STD10 or dog HNSTD (mg base/ kg) HED (mg base/ kg) HED (mg base/ kg) safety factor. HED/Safety Factor (mg base) ³ The proposed clinical initial dose (mg base)b Rat 20 3.2 194 10 19 15

- 27 040062

Dog 3.75 2.1 125 6 21 HED = human equivalent dose; HNSTD = highest non-hazardous toxic dose; STD10 = dangerous toxic dose causing acute toxicity in 10% of animals. ^a Based on the HED conversion factor for a 60 kg individual from the FDA Guidance for Industry “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” (FDA, 2005) and ICH S9 Guideline “Nonclinical Evaluation for Anticancer Pharmaceuticals .” ICH, 2009. ^b Using allometric estimates of plasma clearance (mL/h/kg) and volume of distribution (L/kg), and assuming X% oral bioavailability (based on mean from preclinical species), predicted Cmax and AUC24h at estimated baseline human dose of 15 mg are approximate.

See also CDER, Guidance for Industry: Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers (July 2005).

The suggested human starting dose is below 1/10th of STD10 in rats, below 1/6th of HNSTD in dogs, and is considered safe based on repeated exposures (as measured by AUC) in rats and dogs compared to predicted human exposure at dose 15 mg Compound A as base. As indicated in Table. 1, human exposure to Compound A as a base at 15 mg is predicted to range from 736 to 2263 ng-h/mL; these values are about 23-272 times lower than the average exposure corresponding to STD10 for rats (52800 ng-h/ml) and about 4-14 times lower than the average exposure corresponding to HNSTD for dogs (10000 ng-h/ml). Based on these toxicokinetic data, the suggested human starting dose of 15 mg of Compound A as base would be considered safe.

A key exploratory goal of this study is to identify a dose of Compound A that is not only safe, but exhibits pharmacological activity. A set of genes has been identified whose expression is reduced by ex vivo treatment with Compound A in peripheral blood mononuclear cells (PBMCs) and in whole blood. In the present study, changes in the expression of these genes in whole blood or other genes in tumor biopsies can provide confirmation that the dose is pharmacologically active and help to recognize which dose shows the highest pharmacological activity.

Predictive biomarkers allow for the prospective identification of patients who are most likely to benefit clinically from the use of Compound A alone or in combination with other agents. Although the predictive diagnostic assays in the current study are exploratory in nature, they identify associations between biomarkers and responses, which may provide a basis for future diagnostically guided studies.

Different tumor types were chosen to expand the Compound A dose cohorts in Part B, depending on the results obtained in Part A of the study, preclinical efficacy, and supporting literature. With respect to the reversible inhibitor of BET family members, subjects with locally advanced basal cell carcinoma (BCC) were selected for part B of the expansion cohorts.

BRD4 and other BET bromodomain proteins regulate GLI1 transcription downstream of SMO, with BRD4 directly occupying the GLI1 and GLI2 promoters. Tang, 2014. This uptake can be inhibited by BET inhibitors, and the BET inhibitor, JQ1, reduces tumor cell proliferation in vitro and in vivo in Hh-driven tumors, even those that are resistant to SMO inhibition. Tang, 2014. Therefore, a clinical trial of a BET inhibitor in subjects with locally advanced or metastatic BCC with de novo or acquired resistance is required. Likewise, a clinical study of the BET inhibitor, Compound A, is required for antitumor activity in various cancers. This example proposes a study of Compound A in humans designed to evaluate the safety and pharmacokinetic profiles of the drug at various dose levels/dosing regimens, as well as to detect initial signals of drug efficacy to accelerate the development of phase 2 clinical trials.

More specifically, the Compound A study includes an open-label, phase 1a, first time human (FIH) dose escalation, cohort expansion study of Compound A in subjects with advanced solid tumors, or relapsed or refractory NHL. The dose escalation part of the study (Part A) examines increasing oral doses of Compound A to assess the MTD and/or RPTD of Compound A. The BLRM model using EWOC (see Babb, 1998; Neuenschwander 2008) facilitates final decisions about dose escalation of Compound A by the Scientific Review Committee (SRC). The cohort expansion part of the study (Part B) further evaluates the safety and efficacy of Compound A administered at or below MTD in selected expanded cohorts of approximately 20 evaluable subjects to further determine RP2D. One or more dosing regimens or disease subsets may be selected to expand cohorts. Parts A and B consist of three periods: screening, therapy and follow-up period (see Fig. 4). The objectives of the study are summarized in Table. 1, and the study endpoints are summarized in Table 1. 2.

Typically, the screening period begins 28 days prior to the first dose of Compound A. An Informed Consent Document (ICD) is signed and dated by the subject and administrative staff prior to any other study procedures. All screening tests and procedures are completed within 28 days prior to the first dose of Compound A.

During the treatment period, formulations containing Compound A are first administered orally once a day for three consecutive days followed by four consecutive days of drug holidays, each week (three/seven day dosing regimen) in each four week cycle. Alternative dosing regimens (eg, two days of dosing/five days of medication holiday, each week) are being explored based on SRC analysis of available data on safety, PK, pharmacodynamics (PD), and efficacy. In Part A below, the Dose Limiting Toxicity (DLT) window will be 28 days (four weeks) during Cycle 1.

During the follow-up period, all subjects are observed for 28 days (±2 days) for safety after the last dose of Compound A. Subjects who discontinue treatment for reasons other than disease progression (or relapse), initiate new anticancer therapy, or withdraw informed consent for participation in the entire study, are subject to disease assessment conducted in accordance with an established tumor assessment scheme, prior to progression or initiation of new systemic anticancer therapies. After a safety follow-up visit, all subjects are followed up every subsequent three months (± two weeks) to evaluate survival up to two years or until death, loss of contact for follow-up, or completion of the study, whichever occurs first.

For Part A, dose escalation, a minimum of three subjects are selected for each dose level. The starting dose of Compound A is 15 mg. The BLRM with EWOC combines the available a priori safety information and updates the model parameters after each new cohort of subjects complete cycle 1. The decision on the next dose is made by the SRC based on the risk assessment using the BLRM and the available safety information (i.e. DLT safety data). and non-DLT), PK, PD and efficiency. In addition, relevant non-clinical data (eg, GLP toxicity studies, in vivo pharmacology from xenograft models, etc.) can be used in the evaluation. Details of the statistical methodology are provided below.

At all decision times, the BLRM allows for changes in the dose escalation step based on observed DLT values. However, the dose for the next cohort will not exceed 100% of the increase from the previous dose. The maximum tolerated dose (MTD) is the highest dose that is unlikely (posterior probability <25%) to cause DLT in y>33% of treated subjects in the first cycle of Compound A treatment. The SRC makes the final decision on Compound A dose for each cohort .

During a dose escalation, the dose of Compound A may be declared as the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) when the following conditions are met:

at least six evaluable subjects were treated at this dose;

the posterior probability of target toxicity at the specified dose is greater than 60% and is the highest among increasing doses, or at least 21 subjects were treated in the study; and the dose is recommended according to the BLRM and the SRC approves it.

The SRC includes the investigators (or designated representatives), the sponsoring investigator, the physician responsible for drug safety, the trial statistician, and the trial director. Specially for this case, the involved participants may include a specialist in pharmacokinetics and additional specialists in clinical studies. Other in-house and outside experts may seek advice from the SRC as needed.

A decision regarding the evaluation of additional subjects within a cohort of subjects receiving a given dose; the cohort of subjects receiving the higher dose; cohorts receiving an intermediate dose; a smaller dose increase step; alternative dosing regimens (e.g., two days on/five days of drug holiday, each week) or announcement of an MTD is also accepted by the SRC based on BLRM assessment and analysis of available safety information (i.e., DLT and non-DLT data), PK, PD and efficiency. The final decision is made by the SRC.

Following the first dose in any cohort during dose escalation, subjects in each cohort are observed for 28 days (cycle 1, DLT window) before moving on to the next dose cohort. No more than one subject per day is selected for this dose escalation cohort. Subjects not evaluable for DLT are replaced. A subject eligible for evaluation for DLT is defined as one who received at least 10 of the 12 doses (or >80% of the total planned dose rate) of Compound A during Cycle 1, without experiencing DLT; or experienced DLT after receiving at least one dose of Compound A.

Dose escalation in the same subject is not allowed during the DLT evaluation period. However, in >3 cycles, subjects with no evidence of disease progression who tolerate their prescribed dose of Compound A may (at the discretion of the investigator and after consultation with the study medical supervisor) increase the dose to the highest level that has been shown to be adequately tolerated at least one cohort of subjects in this study (i.e. when the risk of overdose is less than 25% as assessed by BLRM).

With regard to cohort expansion part B, after completion of the dose escalation (Part A), cohorts of subjects with the selected tumor type are entered into the expansion phase (Part B), with each cohort consisting of approximately 20 evaluable subjects. Expansion may occur at the MTD and dosage regimen established in the dose escalation phase, or at an alternative tolerated dose and dosage regimen based on an analysis of the safety, PK, PD and efficacy data obtained in Part A. The SRC selects doses and regimens of interest to expand cohorts. One or more dosing regimens may be chosen to expand cohorts. The SRC continues to review safety data regularly throughout the study and makes recommendations for continuation of the study and dose modification as needed.

In terms of study population recruitment, the study includes men and women aged 18 years or older with advanced or inoperable solid tumors and relapsed or refractory NHL (DLBCL and iNHL). Recruitment is expected to take approximately 30 months (12 to 18 months for dose escalation and nine to twelve months for expansion). Completion of active treatment and follow-up is expected to take an additional 4 to 28 months. It is expected that the duration of the entire study will be about four years. The end of the study is defined as the date of the last visit of the last subject to complete follow-up after the end of treatment, or the date of receipt of the last data for the last subject required for primary, secondary and/or exploratory analysis, as previously specified in the protocol.

Study treatment may be terminated if there is evidence of clinically significant disease progression, unacceptable toxicity, or subject/physician decision to withdraw from the study. Subjects may continue to receive study drug after disease progression at the discretion of the investigator in consultation with a medical supervisor.

In at least one embodiment, Compound A is formulated as tablets for oral administration. The labeling is appropriate, for example, for research use in accordance with the regulations of the relevant country health authority.

For baseline efficacy evaluations, subjects were assessed for efficacy after every two cycles until cycle 6 and every three cycles thereafter. All subjects who discontinue treatment for reasons other than disease progression, initiate new anticancer therapy, or withdraw informed consent to participate in the entire study are followed up until progression or initiation of new systemic anticancer therapies.

The response of the tumor is determined by the researcher. For solid tumors, the score is based on the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Eisenhauer et al., 45 Eur. J. Cancer 228 (2009). For the NHL, the score is based on the International Working Group Revised Response Criteria for Malignant Lymphoma. Cheson et al., 25 J. Clin. oncol. 579 (2007). Positron emission tomography (PET) with [ ¹⁸ F]-fluorooxyglucose (FDG) or FDG PET/CT imaging is required to confirm a complete response in subjects with tumors with increased FDG accumulation.

Safety variables for this study include adverse events, clinical laboratory safety variables, 12-lead electrocardiogram, Eastern Joint Oncology Group General Status score, left ventricular ejection fraction assessment, physical exams, vital signs, study treatment impact, assessment concomitant medications and a pregnancy test for women of childbearing potential. The PK profiles of Compound A are determined from collected serial blood samples.

Since no clinical studies have been conducted with Compound A, the efficacy and safety profiles of Compound A in humans are unknown. Potential toxicities for Compound A have been identified based on preclinical studies using Compound A. The frequency and extent of safety assessments proposed for Compound A-ST-001 are specific to the FIH study and are consistent with Compound A toxicology studies in rats and dogs. In rats and dogs, the main target tissues for toxic effects are the GI tract, bone marrow, lymphoid organs, and testicles. General data from preclinical and histopathological studies suggest that the gastrointestinal system may be a major target of Compound A-mediated toxicity.

Frequent early monitoring of weight, hydration status, serum electrolytes, frequency and severity of diarrhea and vomiting, and episodes of abdominal pain (gastric, intestinal) in subjects is an important part of a plan to monitor safety and implement aggressive symptomatic therapy for nausea, vomiting, or diarrhea with early onset (eg, grade 1). Based on morphological changes, flattening of intestinal villi, and mucosal erosions observed in the GI tract of rats and dogs, subjects with malabsorption syndromes, active ulcer/gastritis, or recurrent episodes of GI bleeding will be excluded from the study. At the discretion of the investigator, mucosal coating agents will be recommended to protect the esophageal/gastric mucosa, as well as monitoring subjects for GI bleeding. Subjects will be asked to report episodes of GI discomfort or pain, loss of appetite, or blood in the stool.

Because this study is an FIH study, subjects with a history of heart failure, coronary artery disease, uncontrolled hypertension, severe cardiac arrhythmias, or long QT interval on ECG will be excluded from enrollment. All study subjects will require confirmation of adequate left ventricular ejection fraction (>45%) at baseline. Deviations from the protocol are not permitted during this test under any circumstances.

The detection of bone marrow hypocellularity and depletion of lymphoid tissue (thymus, spleen, lymph nodes) emphasize the importance of frequent monitoring of complete blood count, with platelet count and leukocyte differentiation (WBC). Subjects will be monitored for possible toxicity using standard and specific laboratory tests, including complete blood count, prothrombin time (PT)/activated partial thromboplastin time (APTT)/international normalized ratio (INR), and serum chemistry.

The results of histopathological examination of the testes and epididymis of male rats and dogs will warrant a ban on sperm donation and adoption of children for the duration of the clinical study and for at least 3 months after the last dose of the study. No histological lesions were observed in the reproductive organs of female animals in preclinical studies, although the significance of this was unknown. Long-term toxicity and reproductive toxicity studies have not been conducted with Compound A. Subjects will need to follow pregnancy prevention guidelines.

Pharmacodynamic (PD) evaluations are described below. The primary purpose of this study is to evaluate the safety and tolerability of treatment with pharmaceutical formulations containing Compound A, including the determination of MTD or RP2D. The analysis method for estimating MTD is the BLRM model based on the EWOC principle (Babb, 1998; Neuenschwander, 2008).

Statistical analyzes will be performed by dose level (Part A) and cohort of patients with a particular tumor type (Part B) as needed or applicable. All analyzes will be descriptive. All summary safety data will be obtained using subjects receiving Compound A (treated population). Study data are summarized by distributional characteristics, demographic and baseline characteristics, impact, efficacy, safety, PK and PD. Categorical data are summarized by distribution density (number and percentage of patients), and continuous data are summarized by descriptive statistics (mean, standard deviation, median, minimum and maximum).

Treatment-related adverse events (TEAE) were summarized by severity according to the National Cancer Institute Common Terminology Criteria for Adverse Event grades. TEAE frequency is tabulated by system organ class and preferred term according to the Medical Dictionary for Regulatory Activities. Grade 3 or 4 TEAE adverse events include TEAE leading to discontinuation of Compound A; TEAE associated with the investigational medicinal product; and serious adverse events (SAE) are grouped separately. Changes from baseline are summarized from laboratory analytes, vital signs, lead ECG, and ECHO/MUGA scans. All data are presented in lists by subjects.

The primary efficacy variable is DCR. The mode of action (MoA) of the compound may result in SD and disease control, however PFS and OS may serve as additional measures of efficacy. Although OOS and PFS are not normally assessed in FIH, administration of Compound A may result in Sds and responses (eg, in cases of NHL). Disease control is defined as tumor responses CR, PR and SD (assessed by investigators). Point estimates and 95% confidence intervals for DCR are reported. Objective response rates (defined as the percentage of patients whose best response is a complete response or partial response), duration of response/stabilization of disease, progression-free survival, and overall survival are summarized using repetition rate tables for categorical variables or

- 31 040062 descriptive statistics for continuous variables. Efficacy analyzes are repeated for the Treated Population and the Efficacy Evaluable Population (subjects who received a disease score at baseline, at least one treatment cycle within the study, and one disease score during the study period ), while the result using the treated population was considered the main one.

During Dose Escalation Part A, approximately 30-40 subjects are selected. During part B of cohort expansion, at least 14 subjects evaluable for efficacy are selected first for each cohort of subjects having a particular tumor type. If the response rate is 20% or more, there is a greater than 95% chance that one or more responders will be observed among the first 14 subjects, which is subject to statistical update based on the change in DCR as the main indicator of effectiveness. Gehan, 13 J. Chronic Dis. 346 (1961). If no responder is observed among the 14 subjects, then recruitment for that cohort of subjects with a particular tumor type is stopped due to lack of efficacy. Otherwise, the cohort of subjects with a particular tumor type is expanded to approximately 20 subjects in case a responder is observed.

More specifically, Compound A is being evaluated in an open-label, phase 1a, first-time human (FIH) dose escalation and cohort expansion clinical trial in subjects with advanced solid tumors, or relapsed or refractory NHL. The dose escalation part of the study (Part A) examines increasing oral doses of Compound A to assess the MTD and/or RPTD of Compound A. The BLRM model using EWOC (see Babb, 1998; Neuenschwander 2008) facilitates final decisions about dose escalation of Compound A by the Scientific Review Committee (SRC). The cohort expansion part of the study (Part B) further evaluates the safety and efficacy of Compound A administered at or below MTD in selected expanded cohorts of approximately 20 evaluable subjects to further determine RP2D. One or more dosing regimens and/or disease subsets may be selected to expand cohorts. Parts A and B consist of three periods: screening, therapy and follow-up period (see Fig. 4).

As noted, the screening period begins 28 days prior to the first dose of Compound A. An Informed Consent Document (ICD) is signed and dated by the subject and administrative staff prior to the commencement of any other study procedures. All screening tests and procedures must be completed within 28 days prior to the first dose of Compound A. During the treatment period, Compound A is first administered orally once a day for three consecutive days followed by four consecutive days of drug holidays, each week (dosing regimen three/ seven days) in each four-week cycle. Alternative dosing regimens (eg, two days on/five days of drug holiday, each week) may be considered based on SRC analysis of available safety, PK, PD, and efficacy data. In Part A, the dose-limiting toxicity (DLT) window will be 28 days (four weeks) during Cycle 1. During the follow-up period, all subjects are observed for 28 days (±2 days) for safety after the last dose of Compound A. Subjects who discontinue treatment for reasons other than disease progression (or relapse), initiate a new anticancer therapy, or withdraw informed consent to participate in the entire study, undergo disease evaluation according to an established tumor evaluation scheme prior to progression or initiation of new systemic anticancer therapies . After a safety follow-up visit, all subjects are followed up every subsequent three months (± two weeks) to evaluate survival up to two years or until death, loss of contact for follow-up, or completion of the study, whichever occurs first.

For Part A, dose escalation, a minimum of three subjects are selected for each dose level. The starting dose of Compound A is 15 mg. The BLRM with EWOC combines the available a priori safety information and updates the model parameters after each new cohort of subjects complete cycle 1. The decision on the next dose is made by the SRC based on the risk assessment using the BLRM and the available safety information (i.e. DLT safety data). and non-DLT), PK, PD and efficiency. In addition, relevant non-clinical data (eg, GLP toxicity studies, in vivo pharmacology from xenograft models, etc.) can be used in the evaluation. Detailed information on the statistical methodology is provided in Annex H.

At all decision times, the BLRM allows for dose increment changes based on observed DLT values; however, the dose for the next cohort will not exceed a 100% increase from the previous dose. The maximum tolerated dose (MTD) is the highest dose that is unlikely (posterior probability <25%) to cause DLT y >33% of treated subjects in the first cycle of Compound A treatment. A for each cohort.

During a dose escalation, the dose of Compound A may be declared as the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) when the following conditions are met:

at least six evaluable subjects were treated at this dose;

the posterior probability of target toxicity at the specified dose is greater than 60% and is the highest among increasing doses or at least 21 subjects were treated in the study; and the dose is recommended according to the BLRM and the SRC approves it.

The SRC includes the investigators (or designated representatives), the sponsoring investigator, the physician responsible for drug safety, the trial statistician, and the trial director. Specially for this case, the involved participants may include a specialist in pharmacokinetics and additional specialists in clinical studies. Other in-house and outside experts may seek advice from the SRC, as appropriate.

The decision concerns the evaluation of additional subjects within the cohort of subjects receiving a given dose; cohorts of subjects receiving the higher dose; cohorts receiving an intermediate dose; a smaller dose increase step; alternative dosing regimens (eg, two days on/five days of drug holiday, each week) or MTD announcements are also determined by SRC based on BLRM assessment and analysis of available safety information (i.e., DLT and non-DLT data), PK , PD and efficiency.

Following the first dose in any cohort during dose escalation, subjects in each cohort are observed for 28 days (cycle 1, DLT window) before moving on to the next dose cohort. No more than one subject per day is selected for this dose escalation cohort. Subjects not evaluable for DLT are replaced. A DLT-evaluable entity is defined as an entity that:

received at least 10 of 12 doses (or >80% of the total planned dose intensity) of Compound A during cycle 1, without DLT testing; or experienced DLT after receiving at least one dose of Compound A.

Dose escalation in the same subject is not allowed during the DLT evaluation period. However, in >3 cycles, subjects with no evidence of disease progression who tolerate their prescribed dose of Compound A may (at the discretion of the investigator and after consultation with the study medical supervisor) increase the dose to the highest level that has been shown to be adequately tolerated at least one cohort of subjects in this study (i.e. when the risk of overdose is less than 25% as assessed by BLRM).

With regard to cohort expansion part B, after completion of the dose escalation (Part A), cohorts of subjects with the selected tumor type are entered into the expansion phase (Part B), with each cohort consisting of approximately 20 evaluable subjects. Expansion may occur at the MTD and dosage regimen established in the dose escalation phase, or at an alternative tolerated dose and dosage regimen based on an analysis of the safety, PK, PD and efficacy data obtained in Part A. The SRC selects doses and regimens of interest to expand cohorts. One or more dosing regimens may be chosen to expand cohorts. The SRC continues to review safety data regularly throughout the study and makes recommendations for continuation of the study and dose modification as needed.

The schedule of estimates is shown in Table. 4 and the evaluations are described below. Safety variables for this study include adverse events, clinical laboratory safety variables, 12-lead electrocardiogram, Eastern Joint Oncology Group General Status score, left ventricular ejection fraction assessment, physical exams, vital signs, study treatment impact, assessment concomitant medications and a pregnancy test for women of childbearing potential. Subjects are evaluated for efficacy after every two cycles until cycle 6, and every three cycles thereafter. All subjects who discontinue treatment for reasons other than disease progression, initiate new anticancer therapy, or withdraw informed consent to participate in the entire study are followed up until progression or initiation of new systemic anticancer therapies.

Blood is collected at the indicated time points to determine the PK profiles of Compound A and for exploratory PD scores. Paired tumor biopsies for analysis of biomarkers of treatment activity are optional in the dose escalation phase but mandatory in the cohort expansion phase.

The study is conducted in accordance with the International Conference on Harmonization (ICH) of the technical requirements for the registration of medicinal products for human use / Rules for conducting good clinical trials (GCP) and the requirements of regulatory authorities.

- 33 040062

Recruitment is expected to take approximately 30 months (12 to 18 months for dose escalation and 9 to 12 months for expansion). Completion of active treatment and follow-up is expected to take an additional 4 to 28 months. It is expected that the duration of the entire study will be about four years.

The end of the study is defined as the date of the last visit of the last subject to complete post-treatment follow-up or as the date of receipt of the latest data for the last subject required for primary, secondary and/or exploratory analysis, as previously specified in the protocol, whichever these dates is later.

This example proposes a multicenter, open-label study that will enroll approximately 30-40 patients in Part A (dose escalation). In Part B (expanding cohorts), up to 20 evaluable subjects may be recruited into each of the selected expanding cohorts. Recruitment will take place at approximately 4-6 centers in Europe for Part A. Recruitment for Part B may include additional centers in the US and Europe.

Regarding the inclusion criteria, subjects must meet the criteria below to be included in the dose escalation phase (Part A) of this study:

1. Men and women aged >18 years at the time of signing the Informed Consent Document (ICD).

2. The subject must understand and voluntarily sign the ICD prior to undertaking any assessments/procedures related to the study.

3. The subject is willing and able to comply with the visit schedule and other requirements of the study protocol.

4. Subjects with histological or cytological evidence of advanced unresectable solid tumors or iNHL (DLBCL and iNHL), including those who progress (or are intolerant due to comorbid medical conditions or unacceptable toxicity) on standard cancer therapy, or for whom there is no other approved conventional therapy.

5. At least one lesion of measurable disease (>1.5 cm in long axis or >1.0 cm in both long and short axis) must be present in subjects with solid tumors and iNHL.

6. Subject agrees to mandatory tumor biopsies (screening and cycle 1) in Part B. Tumor biopsies are optional for Part A.

7. General condition on the ECOG scale from 0 to 1.

8. Subjects must have the following laboratory values at screening:

absolute neutrophil count (ANC) >1.5 x 10 ⁹ /L without growth factor support for 7 days (14 days if the subject received pegfilgrastim);

hemoglobin (Hgb) >9 g/dl (>8 g/dl for subjects with NHL);

platelet count (plt) >75x10 ⁹ /l (>50x10 ⁹ /l without transfusion within 7 days for subjects with NHL);

serum potassium concentration in the normal range or correctable with dietary supplements;

serum levels of aspartate aminotransferase (AST/SGOT) and alanine aminotransferase (ALT/SGPT) <3.0x upper limit of normal (ULN) or <5.0xULN if liver metastases are present;

serum total bilirubin <1.5xULN or <2xULN if liver metastases are present;

serum creatinine level <1.5xULN or 24-hour creatinine clearance >50 ml/min, measured using the Cockcroft-Gault equation;

subjects with documented liver metastases must have serum albumin >3 g/dL;

INR <1.5xULN and PTT <1.5xULN.

9. Women of reproductive potential (FCBP) should:

completely refrain from heterosexual intercourse (which is subject to monthly review and should be recorded in primary documents) or agree to use and are able to perform at least two effective methods of contraception (oral, injectable or implantable hormonal contraceptive; tubal sterilization, intrauterine device, barrier contraceptive with spermicidal agent or vasectomy partner), one of which must serve as a barrier, from the time of signing the ICD throughout the study and up to 28 days or up to three months after the last dose of Compound A; and have two negative pregnancy tests certified by the investigator prior to starting Compound A:

a negative pregnancy blood test (sensitivity of at least 25 mIU/mL) at screening;

- 34 040062 negative blood and urine test for pregnancy (at the discretion of the investigator) within hours before cycle 1 Day -1 of the study;

avoid conception within three months after the last dose of Compound A;

agree to continue the diagnosis of pregnancy during the course of the study and after the end of treatment within the study. This applies even if the subject practices total abstinence ² from heterosexual contact.

10. Men must practice total abstinence (subject to monthly review) or be willing to use a condom (a latex condom is recommended) during sexual contact with a pregnant woman or FCBP and avoid conception from signing an ICD, during study participation, during interruptions doses and for at least 3 months after discontinuation of Compound A, even if he has been subjected to a successful vasectomy.

A woman of reproductive potential is a mature woman who:

1) has not undergone a hysterectomy (surgical removal of the uterus) or bilateral oophorectomy (surgical removal of both ovaries), or

2) has not been in a natural postmenopausal period for at least 24 consecutive months (eg, has had a period at any time during the previous 24 consecutive months).

Complete abstinence is acceptable when it is consistent with the subject's preferred and habitual lifestyle. Intermittent abstinence (eg, calendar, ovulation, symptomothermal, postovulation methods) and withdrawal are not acceptable methods of contraception.

The presence of any of the following will preclude the subject from participating in the enrollment phase of the study:

1) the patient received anti-cancer therapy (approved or investigational) for <4 weeks or 5 half-lives, whichever is shorter, prior to signing the ICD;

2) toxic events due to prior systemic cancer therapies must resolve to < NCI CTCAE Grade 1 prior to initiation of Compound A treatment. Peripheral neuropathy > NCI CTCAE Grade 2;

3) the subject received autologous hematological stem cell transplantation (HSCT) <3 months or allogeneic HSCT <6 months prior to Compound A treatment;

The 6-month exclusion period for recovery from HSCT-related toxicity applies regardless of whether autologous or allogeneic transplantation was performed;

4) the subject has undergone major surgery <4 weeks or minor surgery <2 weeks prior to signing the ICD, or who has not recovered from surgery;

5) the subject completed any radiation therapy <4 weeks prior to signing the ICD;

6) the subject has persistent diarrhea due to a malabsorption syndrome (eg, celiac sprue or inflammatory bowel disease) > NCI CTCAE Grade 2 despite treatment, or any other significant GI disorder that may interfere with absorption of Compound A;

7) subjects with symptomatic or uncontrolled ulcers (gastric or duodenal), especially with a history and/or risk of GI tract perforation and bleeding;

8) Symptomatic or unstable central nervous system metastases Subjects recently undergoing whole brain irradiation or stereotactic radiosurgery for CNS metastases should have completed therapy at least 4 weeks prior to Cycle 1, Day 1, and have follow-up CT or MRI of the brain done on a follow-up period demonstrating stable or improving metastases 4 or more weeks after completion of radiotherapy (the latter should be obtained as part of the screening examination);

9) rapidly proliferating high-grade solid tumors (eg, small cell lung cancer, embryonic cell tumors, neuroblastoma) with a significant tumor burden (>10 cm for the sum of measurable lesion diameters) and LDH > ULN;

10) known symptomatic acute or chronic pancreatitis;

11) impaired cardiac function or clinically significant heart disease, including any of the following:

left ventricular ejection fraction (LVEF) <45%, determined by radionuclide angiography (MUGA) or echocardiogram (ECHO);

complete blockade of the left leg of the atrioventricular bundle or two-bundle blockade;

congenital long QT syndrome;

persistent or clinically significant ventricular arrhythmias or atrial fibrillation;

QTcF >470 ms on ECG screening (mean of triplicate records);

- 35 040062 unstable angina or myocardial infarction <6 months before starting Compound A;

other clinically significant heart disease such as congestive heart failure requiring treatment or uncontrolled hypertension (blood pressure > 160/95 mm Hg);

12) pregnant or lactating women;

13) known HIV infection;

14) known viral infection of chronic active hepatitis B or C (HBV, HCV):

patients who are seropositive due to HBV vaccination are eligible to participate in the study;

patients who do not have an active viral infection and are receiving adequate prophylaxis against HBV reactivation are eligible to participate in the study;

HCV clearance for HCC may be considered;

15) current treatment with long-term therapeutic dosing of anticoagulants (eg, warfarin, low molecular weight heparin, factor Xa inhibitors). Low molecular weight heparin is allowed to maintain catheter patency;

16) history of concurrent presence of other types of cancer requiring active, ongoing systemic treatment;

17) subjects with a history of clinically significant cognitive impairment(s) or active cognitive impairment(s);

18) the subject has any significant medical condition (e.g., active or uncontrolled infection or kidney disease), laboratory abnormality, or psychiatric illness that would preclude patient participation (or compromise treatment adherence) in the study, or would expose the subject to unacceptable risk if she/he participates in the study;

19) subjects with a history of clinically significant cognitive impairment(s) or active cognitive impairment(s); And

20) the subject has any condition that prevents the interpretation of the data obtained during the study.

Regarding procedures, questions regarding the protocol should be directed to the medical supervisor or designated person. The procedures performed for each subject included in the study are set out in Table. 4.

Table 4

Events

Treatment period Follow-up period Cycle 1 Cycles 2-4 Cycles 5+ EOT ^Security Duration ^With Screening Week 1 Week 2 Week 3 Ned 4 Week 1 Week 2 Not DZ Week4 Week 1 Week 3 Events ³ Day -28 to -1 D1 D 2 DZ D8 D15 D22 D1 D8 D1 5 D22 D1 D15 - 28 D 28 D ±2 D q3 m (±2 weeks) Inclusion in the study Informed consent and advice on contraceptives X Informed consent for optional exploratory analyzes/collection of samples for RK X Criteria inclusion/exclusion X Medical/cancer history and therapies X Demographics X IRT registration X X X Prior/concomitant drugs, procedures X X X X X X X X X X X X X X X

- 36 040062

Investigational drug Oral administration of Compound A according to the prescribed dosing regimen ^a X X X X X X X X X X X X Keeping/analyzing the patient's diary Safety ratings X X X X X X X X X X X X X X X X X X X X (D17 only) X X X X X X X X X X X X (C2 only ±7d) X X X X X X X X X Adverse Event Assessment X Height X Weight X Vital Signs X Physical Examination X ECOG PS X X (>72 h before 12-lead ECG (Single D1) or Triple ECG) ⁶ LVEF (ECHO/MUGA) X XX X X X X XXXXX XX XX X X X X X (C5 only ± X 7d) (±7d) Pregnancy test (FCBP only) X X X X X General blood analysis X (D -14 to -1) X X X X X XC2 ONLY o X X (C2 only) X X Chem. laboratory with analyzes of LDH, uric acid X (D -14 on -1) X X X X X XC2 ONLY ABOUT X X (C2 only) X X PT, INR, PTT X (D -14 on -1) X X X X X XC2 ONLY X X (C2 only) X X Amylase, lipase, subsets T cells (CD4+ and CD8+), TSH X X (C2 Only) X(q3 su cl C5, C8, Clletc.) X Analysis of urine X (D-14 to -1) X X X X

RK and PD scores

Blood, RK X X X D17- 18 CSF, PKf X D17 Blood (whole), pharmacogenomics X Blood (whole), PAX gene for RNA X X Tumor biopsy ⁸ X (D -7 to -1) X D16 or D17 Archival Tissue Sample (FFPE) X Efficacy X (D28 ± 7d at C6, then q3 X (D28 cycles, i.e. Solid tumor/NHL scores: ±7d _; C2 end C9, C12, KT/MPT imaging ^h X &C4), etc.) X NHL-specific bone marrow assessment if known χ _ONLY with or suspected involvement χί X D28 bone marrow confirmation ±7d in C2 and CR X NHL-specific: FDG PET or PET/CT scan (not required if X, tumor is FDG-confirm negative) X and CR Additional follow-up Follow-up after anticancer therapies Follow-up for SAE assessment — X X X Follow-up to assess survival X

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Abbreviations:

β-hCG = beta human chorionic gonadotropin;

BMMC = bone marrow mononuclear cells;

C = cycle;

CK = creatine kinase;

CSF = cerebrospinal fluid;

CT = computed tomography;

D = day(s);

ECHO = echocardiogram;

ECOG Eastern Joint Oncology Group;

FCBP = women of reproductive potential;

FFPE = formalin-fixed, paraffin-embedded;

fT4 = free T4;

INR = international normalized ratio;

IRT = interactive communication technology;

LVEF = left ventricular ejection fraction;

mo = months;

MUGA = radionuclide angiography;

PK = pharmacokinetics;

PT = prothrombin time;

PTH = parathyroid hormone;

PTT = partial thromboplastin time;

q = every;

TSH = thyroid stimulating hormone;

WK(s) = week(s).

^a All study visits/procedures will have a window of ±2 days and all blood samples for laboratory testing will be taken prior to dosing unless otherwise noted.

^b This follow-up safety assessment can be done by telephone.

^c Follow-up to assess survival up to 2 years or until death or end of study, whichever occurs first. May be done by reviewing records (including public records) and/or by telephone contact with the patient, family, or treating physician.

^d Not all dosing days of Compound A are shown. The dosing regimen includes first 3 consecutive days of Compound A administration and 4 consecutive days of drug holidays, each week (3/7 day schedule). Alternative dosing regimens may be implemented based on the decisions of the SRC.

^e Triple ECG screening must be performed >72 h prior to dosing on Day 1 so that central readings are available for analysis.

^f Optional for subjects with primary or metastatic CNS involvement and a shunt or reservoir. The recommended time for collecting CSF is 4 hours (±1 hour) after dosing on Day 17 (or the day of the last dose of Compound A in cycle 1). Other times for CSF collection are allowed if the CSF collection occurs on PK day and coincides with one of the scheduled PK blood sample collections 1-8 hours after dosing.

^g Paired tumor biopsies are mandatory for Part B and highly recommended for Part A. Screening biopsies should be obtained after all inclusion/exclusion criteria have been met. A biopsy during Cycle 1 may be obtained on Day 16 or 17 provided that 2 consecutive doses of Compound A are administered.

^h All subjects who discontinue treatment for reasons other than disease progression, initiate new anticancer therapy, or withdraw informed consent to participate in the entire study, are observed until progression or initiation of new systemic anticancer therapies.

¹ May be omitted if results are normal on the patient's most recent bone marrow biopsy. In addition, this analysis may be skipped if the previous analysis is within 90 days before Cycle 1 Day 1.

All study visits will have a window of ±2 days unless otherwise noted below or in the event table (see Table 4). All blood samples for laboratory analysis should be taken prior to dosing, unless otherwise specified (eg PK samples). Study procedures should be recorded in the primary document and in electronic individual registration cards (eCRF). In the event that subjects are not screened, the minimum information will be recorded in the eCRF in accordance with the database's instructions.

Laboratory analyzes for safety assessment can be performed on site. The results of the screening laboratory tests should confirm the suitability of the subject, but may be repeated during the screening period as needed. ICD signing is done at the screening visit for all patients by qualified research staff. It must be signed and dated by the patient and administrative staff before starting any other examination procedures and kept in the completed form in the primary documents and eCRF. All screening tests and procedures must be completed within 28 days prior to the first dose of Compound A in accordance with the schedule shown in table. 4.

After screening and obtaining informed consent, the following will be done:

inclusion and exclusion criteria are assessed at screening and recorded in primary documents and eCRF;

counseling on contraception;

medical, oncological and surgical history, as well as demographic data (including date of birth, gender, race and ethnicity of each patient) are collected at the time of screening in accordance with state regulations. The cancer history includes a detailed history of the initial diagnosis and date, treatments, and responses.

Collection of information on prior and concomitant medications and procedures.

Registration in the Interactive Communication Technology (IRT) system.

Monitoring of adverse events.

Measurement of height and weight.

Assessment of the main indicators of the state of the body.

Physical examination (registration only in the primary document) and general condition according to the ECOG scale.

A 12-lead triplicate ECG is performed >72 hours prior to the first dose of Compound A, with results obtained prior to dosing to meet study entry criteria.

Assessment of the left ventricular ejection fraction (LVEF).

Pregnancy testing for all women of reproductive potential.

Appropriate methods of contraception and possible risks of fetal exposure will be discussed with subjects at the time of screening. Dual methods of contraception (one of which must be a barrier method) for women of childbearing potential (eg, oral, injectable, or implantable hormonal contraception, an intrauterine device; a spermicidal barrier contraceptive; or a vasectomy partner) and one method of contraception for men (condom) must be used by subjects from the time of signing the ICD throughout the study, and for 28 days after the last dose of Compound A. This is recorded in the primary documents:

clinical laboratory tests must be completed within the time frame indicated in Table. 2; and efficacy/tumor scores.

Qualified health care workers are trained in the requirements specific to counseling subjects on contraceptives. After training, these healthcare professionals will counsel subjects prior to Compound A administration to ensure that the subject is following all requirements, including the use of contraceptives, and that the subject is aware of the risks associated with taking Compound A.

During the treatment period, all concomitant medications and procedures taken or performed after the subject signed the ICD, throughout the study and within 28 days after the last dose of Compound A, are recorded in the primary documents and eCRF.

Adverse events and serious adverse events (SAEs) are recorded from the time the subject signs the ICD up to 28 days after the last dose of Compound A. Subjects experiencing adverse events are followed up with appropriate clinical assessments and laboratory tests as determined by the investigator. Dates of resolution of current adverse events are recorded. Adverse events (AEs) are recorded on the AE page of the eCRF and in the subjects' primary documents. Photographs of the skin rash are obtained whenever possible, anonymized and stored appropriately for future retrieval.

The weight of the subjects is recorded in the primary document and eCRF during the visits indicated in table. 4. The vital signs include body temperature, blood pressure, pulse rate, and respiratory rate, and are recorded at screening and during the study at various time points to monitor safety, as described in Table 1. 4. Recorded measurement readings are captured in the primary document and eCRF. A complete physical examination and an assessment of the general condition according to the scale of the Eastern United Oncology Group (ECOG PS, see Appendix D) are performed during the visits indicated in Table. 4. The results of both assessments are recorded in the primary document. The results for ECOG PS are also recorded in the eCRF. Physical examination findings are classified as normal or abnormal. In the case of abnormalities, a description of the abnormalities and clinical significance is included in the primary documents. Clinically significant changes from baseline are recorded in the adverse events section of the eCRF. Standard electrocardiograms at 12

- 39 040062 leads (ECG) in three repetitions are recorded during the visits indicated in the table. 4. ECG in 12 leads (12 leads at 25 mm/s recorded rhythm, ventricular velocity, PR interval, complex

QRS, QT interval, and QTc interval) are performed after the subject has been in the supine position for at least 5 minutes. ECG in three repetitions (three records with an interval of 2 ± 1 min) is performed during:

screening;

cycle 1;

on Day 1: before dosing (30 min before dosing) and 2 hours (±10 min) after dosing;

on Day 17: before dosing (30 min before dosing) and 2 hours (±10 min) after dosing;

cycle 2 and subsequent cycles:

on Day 1: before dosing (30 minutes before dosing).

One ECG is performed at the End of Treatment (EOT) visit. For alternative dosing regimens, ECG during Cycle 1 on Day 17 is performed on the last day of Compound A dosing in Cycle 1. Investigators make immediate clinical decisions based on their interpretation of the ECG results and enter their overall ECG score into the eCRF. Clinically significant changes from baseline are recorded in the adverse events (AE) section of the eCRF. The ECG results are also uploaded to the central ECG laboratory for final analysis and interpretation. Left ventricular ejection fraction (LVEF) (multi-entry arteriography [MUGA] or echocardiogram [ECHO]) is measured at screening in all subjects. Follow-up assessments are required, as indicated in Table 1. 4. Evaluations at follow-up should follow the same procedure used at screening. A clinically significant decrease is defined as an absolute decrease of >20% in LVEF or a fall below 45%. A woman of reproductive potential (FCBP) is defined as a sexually mature woman who:

has not undergone a hysterectomy or bilateral oophorectomy and is not naturally postmenopausal (amenorrhea after cancer therapy does not rule out reproductive potential) for at least 24 consecutive months (eg, has menstruated at any time in the previous 24 consecutive months).

The researcher classifies the female subject as FCBP according to this definition. Pregnancy testing is not required for non-FCBP subjects, but justification must be included in the eCRF and source document. Pregnancy testing will be done in a local laboratory.

The results of pregnancy tests are entered into the primary document and eCRF. For women of reproductive potential (FCBP), pregnancy testing is performed during the visits listed in Table. 4:

A blood pregnancy test with a sensitivity of at least 25 mIU/mL must be performed at screening, and a blood or urine pregnancy test (at the discretion of the investigator) within 72 hours prior to cycle Day 1-1 of study treatment. The subject may not receive Compound A until the investigator is satisfied that two pregnancy screening tests are negative.

A blood or urine pregnancy test (at the discretion of the investigator and minimum test sensitivity [25 mIU/mL]) should be performed within 72 hours prior to Day 1 of each cycle and at the End of Treatment (EOT) visit. The subject may not receive Compound A until the investigator is satisfied that the pregnancy test is negative.

A woman of reproductive potential (FCBP) or a male subject whose partner is a FCBP should avoid activities that could lead to conception for 3 months after the last dose of Compound A.

The following laboratory tests will be performed during the screening visit and during the study at the time points shown in Table. 4. All samples must be taken prior to dosing unless otherwise noted. Laboratory studies are recorded in the primary document and eCRF and include the following:

Hematology: complete blood count (CBC) including hemoglobin, hematocrit, white blood cell count (WBC) with absolute differentiation (including blast count) and platelet count.

Serum chemistry: albumin, total protein, bicarbonate or _CO2 , magnesium, phosphorus, calcium, creatinine, urea/BUN, glucose (fasting >4 h), potassium, sodium, chloride, total bilirubin (fractionation if out of range) outside of the normal range), alkaline phosphatase, AST or serum glutamic oxaloacetic transaminase (SGOT), ALT or serum glutamate pyruvate transaminase (SGPT), LDH and uric acid; baseline hemoglobin A1c in case hyperglycemia is significant based on other BETi in the clinic.

Special chemical analysis: amylase, lipase, subsets of T cells (CD4+ and CD8+), thyroid stimulating hormone (TSH, in case of abnormal reaction to free T4).

Coagulation: RT, INR and PTT.

- 40 040062

Urinalysis: test strip:

microscopy in case of positive (1+ or more) blood or protein;

collection of a 24-hour urine sample to quantify creatinine and protein clearance in case of 2+ or more protein.

Screening creatinine clearance must meet the study inclusion criteria.

An EOT assessment (see Table 4 for procedures) is performed for subjects who refuse treatment for any reason as soon as possible (<28 days) after the decision to permanently discontinue treatment is made. All subjects are observed for 28 days after the last dose of Compound A for registration of AE and information on concomitant medicinal products. Follow-up after 28 days (± 2 days) to assess safety can be done by phone. In addition, any SAEs subsequently reported to the investigator that are suspected to be related to Compound A are recorded. death, loss of contact for follow-up, or termination of the study, whichever occurs first. Therapies for new diseases should be carried out simultaneously. Follow-up to assess survival may be performed by reviewing records (including public information) and/or telephone contact with the patient, the patient's family, or the patient's primary care physician.

With respect to efficacy evaluation, tumor evaluations are performed at screening and include computed tomography (CT) of the chest, abdomen, and pelvis and brain scan (CT or MRI) for subjects with known or suspected brain involvement. After screening, x-ray evaluations of the tumor are performed at the end (Day 28 ± 7 days) of cycles 2, 4 and 6, and then every 3 cycles using the same CT/MRI scan methods used at screening. An EOT scan is not required if the previous scan was within 28 days.

In addition, subjects with NHL are screened for FDG PET or FDG PET/CT unless the tumors are FDG-avid negative. The next scan is performed to confirm CR.

For NHL patients with known or suspected bone marrow involvement, bone marrow evaluation by flow cytometric immunophenotyping is performed at screening, after 2 cycles (end of cycle 2) and to confirm CR.

All subjects who discontinue treatment for reasons other than disease progression, initiate new anticancer therapy, or withdraw informed consent to participate in the entire study are observed according to the specified tumor evaluation schedule until progression or initiation of new systemic anticancer therapies. Tumor response at each post-screening evaluation will be determined by the investigator based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 as described in Appendix B for solid tumors and the revised criteria for evaluating outcome in malignant lymphomas as described in Appendix C for NHL.

The PK scores are described below. To assess the PK of Compound A in plasma, blood samples are collected from all subjects at the time points indicated in table. 5. The actual time of collection of each sample is recorded in primary documents and in electronic individual registration cards (eCRF). A sample with PK at baseline may include a Day 1 collection in Part B.

Table 5

PK Blood Sampling Schedule for Cycle 1______

Time in hours relative dose Part A, cycle 1 Part B, cycle 1 Compounds A Collection period Days 1 and 17 ^a Day 17 ^a 0 Within 30 X X min to 0.5 ± 5 min X X 1 ± 5 min X X E5 ± 5 min X X 2 ± 5 min X X 3 ±10 min X X 4 ± 10 min X X 6 ±10 min X X 8 ± 10 min X X X(before 24 ± 1 h dosing per X (Day 18) Day 2) ^a For alternative dosing regimens, blood sampling on Day 17 for PK will be performed on the last day of dosing in Cycle 1 at the indicated time points.

- 41 040062

Exploratory analysis of Compound A concentrations in CSF can be performed on subjects who have a primary or metastatic CNS lesion with a shunt or reservoir and who consent to optional sampling. CSF sampling is recommended before dosing, then 4 h (±1 h) after dosing on Day 17 (or on the last day of Compound A dosing in cycle 1 if alternative dosing regimens are used). Other time points for CSF collection are allowed if CSF collection occurs on PK day and coincides with one of the scheduled PK blood sample collections 1-8 hours after dosing (see Table 4). The Sponsor may perform additional tests on PK samples to monitor the safety of study treatments or to better understand disease progression or disease response to treatment. Collection, manipulation and processing of samples is carried out in accordance with the standard instructions for good laboratory practice.

For biomarkers, pharmacodynamics, and pharmacogenomics, archival tumors in the form of formalin-fixed, paraffin-embedded (FFPE) blocks or embedded sections (15 slides recommended) are retrieved after eligible subjects are recruited into the IRT system, unless there is only one exception. not provided by the sponsor. For blood samples for pharmacogenomics, a whole blood sample is collected after eligible subjects are included in the IRT system to evaluate potential pharmacogenomic markers for safety, potency, or exposure to Compound A. See Lab Instructions and Appendix G for instructions on collection, manipulation, and processing. samples.

Schemes for pharmacodynamic and predictive biomarkers are presented below.

Whole blood for PD biomarker studies:

Cycle 1 Day 1: before dosing (<3 h) and 2, 4, 8 (every ±15 min) and 24 h (±1 h) after dosing Compound A;

Tumor tissue for PD biomarker studies:

Screening: Day -7 to Day -1 (after all inclusion and exclusion criteria have been met);

Cycle 1 Days 16 or 17: 2 hours (± 1 hour) after dosing Compound A;

Optionally any other time prior to the EOT visit.

The Sponsor may perform additional tests on PD specimens to monitor the safety of study treatments or to better understand disease progression or disease response to treatment.

Tumor biopsies are mandatory in Part B and optional (but desirable) in Part A. Biopsy specimens are obtained by excision of the tumor (preferred) or by punch biopsy (four passages recommended) at Screening and Cycle 1 on Day 16 or 17. Fine needle aspiration is insufficient as a source of tumor biopsy material. Samples may be presented as fresh frozen, paraffin-embedded (FFPE) samples. Optimally, tumor tissue samples are obtained from the same site of the tumor. If Compound A was interrupted prior to completion of dosing during Cycle 1 on Day 16 or 17, it is recommended that tumor biopsy be delayed until at least two consecutive doses have been administered. In addition, an optional tumor biopsy may be performed in both Part A and Part B during subsequent treatment cycles or after discontinuation of treatment (at any time during the 28-day follow-up period) to elucidate the effects of long-term treatment or resistance mechanisms, respectively. . See Lab Instructions and Appendix G for sample collection, handling, and processing.

The investigational drug(s) (Investigational Product) is Compound A having a molecular weight expressed in g/mol. The drug for clinical use, Compound A, is presented as a formulation. The clinical medicinal product, Compound A, should be stored in accordance with the conditions indicated on the packaging label.

Compound A is administered once daily in the morning on an empty stomach (i.e. >1 h before breakfast) with at least 240 ml of water after fasting at night for >6 h in both parts A and B. Subjects must fast or other medicinal products for >1 hour after each dose. Subjects will take Compound A starting on Day 1 for 3 consecutive days followed by four consecutive days of drug holidays, each week (3/7 day dosing schedule) in each 4 week cycle. Alternative dosing regimens may be implemented based on clinical safety analysis and SRC laboratory data.

On study days when PK assessments are required, Compound A is administered in the clinic after any pre-dosing assessments have been completed. On all other study days, subjects will self-administer their prescribed doses at home and record the time of dosing in a diary.

Study treatment may be terminated if there is evidence of clinically significant disease progression, unacceptable toxicity, or subject/physician decision to withdraw from the study. Subjects may continue to receive investigational drug beyond disease progression at the discretion of the investigator in consultation with the sponsor's medical supervisor.

For dose escalation decisions, at least three subjects are recruited into consecutive cohorts. The first cohort is treated with an initial dose of 15 mg. Subjects must complete at least one treatment cycle with a minimum safety and drug effect rating or have a DLT during the first treatment cycle that is considered eligible for dose escalation decisions. Dose escalation decisions will be made when a cohort of subjects meets these criteria. Dose escalation decisions will be made by the SRC. Decisions will be based on a summary of all relevant data from all dose levels evaluated in the current study, including safety information, DLT values, all data on CTCAE grade >2 treatment-related toxicity during Cycle 1, and PK data from measurable entities. PK data from subjects will be available on an ongoing basis throughout the study and dosing will be adapted accordingly. The recommended dose for the next cohort of subjects will be determined by the BLRM with the EWOC principle.

The adaptive Bayesian methodology provides an estimate of Compound A dose levels that do not exceed the MTD and includes all DLT information at all dose levels for this estimate. Generally, the next recommended dose will have the highest probability of dropping DLT in the target range (16-33%) and will always satisfy the EWOC principle. In all cases, the recommended dose for the next cohort will not exceed a 100% increase from the previous dose. Smaller dose increases may be recommended by the SRC after considering all available clinical data.

The procedure for enrolling study subjects in each dose cohort and the conditions for making dose escalation/de-escalation decisions are as follows:

1. To limit the number of subjects treated at a sub-therapeutic dose, this study begins by evaluating Compound A in cohorts of at least 3 evaluable subjects at each dose level. Initially, the dose escalation step between cohorts will be 100%. When two subjects (who may be in different cohorts) experience NCI CTCAE Grade 2 treatment-related toxicity, or one subject experiences DLT or Grade >3 toxicity, cohort size may be increased to at least 6 measurable subjects for current and subsequent cohorts. Dose escalation of Compound A will be <50% for each subsequent dose escalation cohort.

2. After completion of cycle 1, for all evaluable subjects in the cohort, a two-parameter BLRM model is applied using the EWOC principle to make recommendations to the SRC regarding the next dose level with the following exceptions:

If the first two subjects in a cohort experience DLT, no additional subjects will be included in that cohort until the Bayesian model is updated with this new information. Similarly, the model will be re-evaluated if two subjects in the cohort experience DLT before recruiting any additional subject.

If a decision is made to escalate the dose to a higher level, but one or more additional subjects treated at the previous level doses (see item 4 below) experience DLT in cycle 1, the BLRM model will be updated before enrolling any additional subject in the current ( higher) dose level.

3. After each cohort, the SRC will receive and analyze the results of the BLRM assessment and available information on safety (ie DLT and non-DLT data), PK, PD and efficacy. Final decisions on dose escalation will be made by the SRC.

After repeating the above steps, the dose of Compound A may be declared MTD and/or RP2D after the following conditions are met:

at least six evaluable subjects were treated at this dose;

the posterior probability of target toxicity at the specified dose is greater than 60% and is the highest among escalating doses or at least 21 subjects were treated in the study; and the dose is recommended according to the BLRM and the SRC approves it.

At the discretion of the SRC, in order to better understand the safety, tolerability, and PK of Compound A, additional cohorts of subjects may be recruited at prior dose levels or to intermediate dose levels prior to or concurrent with further dose escalation.

Tentative dose levels to be administered to individual cohorts of subjects are described herein. However, dose decisions made during an escalation are not limited to these doses. Based on the BLRM recommendation for the highest dose that cannot be exceeded at any time during an escalation decision and the maximum dose increase allowed by the protocol, intermediate doses may be administered at subsequent new doses.

- 43 040062 cohorts of subjects. The decision to evaluate additional subjects in a fixed-dose cohort, higher-dose cohort, intermediate-dose cohorts, smaller dose escalation steps, alternative dosing regimens, or MTD announcement will also be determined by the SRC based on an analysis of clinical and laboratory safety data. .

All subjects receiving at least one dose of Compound A will be evaluated for safety. Following the first dose in any cohort during dose escalation, subjects in each cohort are observed for 28 days (cycle 1, DLT window) before moving on to the next dose cohort. No more than one subject per day may be recruited into a given dose escalation cohort. A subject being evaluated for DLT is defined as a patient who:

received at least 10 of 12 doses (or >80% of the total planned dose intensity) of Compound A during cycle 1 without experiencing DLT; or experienced DLT after receiving at least one dose of Compound A.

Subjects not evaluable for DLT are replaced. Additional subjects in any given dose cohort may be recruited at the discretion of the SRC. Dose escalation in the same subject is not allowed during the DLT evaluation period. The MTD is defined as the highest dose that results in <33% of subjects experiencing DLT during their first treatment cycle. The MTD score is described in this document. A variable dose cohort (eg, less frequent dosing) may be assessed to accurately determine the MTD at the discretion of the SRC.

During dose escalation, the period for evaluating DLT is cycle 1 (28 days). The National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.03, is used as a guideline for assessing adverse events by severity. DLT is defined as any of the following toxic reactions occurring within the limits of the DLT score, unless the event is clearly identified as not related to Compound A.

Dose-limiting toxicities are described below:

any grade 4 non-hematological toxicity of any duration;

any grade 3 non-hematologic toxicity, except grade 3 diarrhea, nausea, or vomiting lasting <3 days (with optimal treatment);

Grade 3 acneiform, pustular, or maculopapular type rash that resolves to grade <2 within 7 days of discontinuation of study drug and does not recur at the same level when study drug is restarted at the same dose (with optimal treatment);

grade 3 morbid fatigue that resolves to grade <2 within 7 days of drug discontinuation and does not recur at the same level when study drug is resumed at the same dose (with optimal treatment);

the following hematological toxicities: febrile neutropenia;

Grade 4 neutropenia lasting >7 days;

Grade 4 thrombocytopenia lasting >7 days, Grade 3 thrombocytopenia with clinically significant bleeding;

any adverse event (AE), unless it is clearly determined that it is not related to the drug, requiring a dose level reduction during cycle 1; and possibly prolonged grade 3 hyperglycemia (X2 at least 24 h) or symptomatic grade 3 fasting or higher grade hyperglycemia.

Isolated laboratory changes without associated clinical signs or symptoms (eg, hypomagnesemia, hypermagnesemia, hypoalbuminemia, hypophosphatemia, lymphocyte count, elevated or decreased) cannot be included in this definition. These findings will be discussed and analyzed by the SRC.

Criteria for dose escalation in the next cohort of subjects are evaluated as follows. Cohorts consist of at least three measurable subjects. The SRC will make all final decisions on dose escalation. Dose escalation decision criteria include:

if no more than 0 out of 3 or 1 out of 6 evaluable subjects experience DLT within the DLT measurement window in the dose cohort, dose escalation to a higher dose in the next cohort may occur. Additional patients will be recruited to expand the cohort to six evaluable subjects if there are fewer than six evaluable subjects at the time of DLT follow-up;

if two or more up to six evaluable subjects experience DLT within the DLT measurement window in a cohort of subjects receiving a particular dose, any additional set will be terminated and that dose will be determined as NTD;

The SRC will determine whether additional subjects will be recruited into the lower dose cohorts so that there are 6 evaluable subjects to determine the MTD, or whether an intermediate dose cohort or an alternative regimen of up to six will be considered.

- 44,040,062 new recruits;

the number of cohorts depends on the frequency of occurrence of DLT. The subject may experience more than one

DLT. Dose escalation decisions are based on the number of subjects experiencing DLT events.

This document describes the rules for stopping dose escalation during Part A. Dose reductions are permitted in any cycle, including Cycle 1. Dose reductions that occur in Cycle 1 during dose escalation will be determined by the DLT as indicated, but subjects are allowed to continue taking the Compound A at a reduced dose. In the event that dose reduction is indicated, the next lower dose cohort or less frequent dosing regimen will be selected. Two dose reductions are allowed. After reduction, the dose may be increased when toxicity reaches grade <1. If toxicity recurs at a higher dose, the dose is reduced a second time, but in this case, a second increase is not allowed. If any subject continues to experience unacceptable toxicity after two dose reductions (one for the initial dose), Compound A is permanently discontinued. Dose escalation in the same subject is not permitted during the DLT evaluation period.

In addition, with regard to dose reduction, any adverse event that meets the definition of DLT requires interruption of dosing. Dosing should be deferred if any Grade >2 toxicity does not resolve to Grade <1 by the time of the next dose. Grade 3 toxicity or Grade 2 chronic toxicity may require a dose reduction of Compound A. Such cases should be discussed with the sponsor (medical supervisor and investigator) before making changes in dosage.

In addition, with respect to the dose escalation criteria in Part A (escalation phase), dose escalation in the same patient above the doses originally given to the patient is not permitted in Cycle 1. Those who continue to take Compound A after Cycle 2 may after SRC approval, take an increased dose, provided that the alternative dose was proven to be well tolerated by at least one cohort of subjects in this study (i.e. risk of overdose is less than 25% as assessed by BLRM). In the event of a dose escalation in the same subject and with the (optional) consent of the subject, blood will be drawn for PK assessments according to the PK schedule in Cycle 1 on Day 1 for Part A. Sampling for pharmacokinetic (PK) analysis is performed after administration of at least 2 doses of Compound A at a higher dose to evaluate the pharmacokinetics (PK) of Compound A in the same subject. In Part B (expansion phase), dose escalation above the MTD is not allowed.

Treatment may be interrupted for up to four weeks until toxicity (excluding alopecia) reaches grade >1 or baseline. Treatment may be restarted at the same or reduced dose at the discretion of the investigator, or as described herein. Any such treatment interruptions should be discussed with the sponsor's designated medical supervisor.

During the dose escalation phase DLT evaluation, interruption of treatment with a missed >2 doses of Compound A for reasons other than DLT will render the subject ineligible for DLT, and necessitate replacement of that subject in the dose cohort. Any such treatment interruptions should be discussed with the sponsor's designated medical supervisor.

Regarding the management of selected adverse events such as neutropenia, thrombocytopenia and anemia, hematopoietic growth factors or other hematological agents such as erythropoietin, darbepoetin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), transfusions erythrocytes (RBC) or platelets are allowed in a therapeutic study. Therapeutic use of G-CSF is permitted at any time for subjects experiencing grade 3/4 neutropenia or any grade of febrile neutropenia. Prophylactic use of granulocyte (or granulocyte-macrophage) growth factors is not allowed during cycle 1. Subjects with grade 3 or 4 neutropenia should be monitored regularly with laboratory tests until resolution to grade <1. Antimicrobial, antifungal and antiviral prophylaxis should be considered. With regard to pain, pain in the area of the tumor or pain caused by treatment can be controlled with opioid and opioid-related analgesics such as codeine, meperidine, propoxyphene or morphine administered at the discretion of the physician and as dictated by medical necessity. The risk of bleeding, especially if thrombocytopenia occurs, should be considered prior to the use of non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin.

With respect to gastrointestinal effects, muco-coating agents to protect the esophageal/gastric mucosa and monitoring of subjects for GI bleeding are recommended at the discretion of the investigator. Subjects are encouraged to report all episodes of GI discomfort or pain, loss of appetite, or blood in the stool. It is recommended that subjects suffering from diarrhea be treated according to the guidelines presented in FIG. 7. Antidiarrheal drugs such as loperamide should be started as soon as grade 1-2 diarrhea occurs. Antidiarrheal drugs may be prescribed to prevent and treat diarrhea. Dehydration and fluid and electrolyte imbalances must be corrected promptly. General measures to stop diarrhea should be considered, such as a low-fiber diet and increased fluid intake.

Changes in blood glucose levels were not observed in preclinical toxicology studies using Compound A. However, preliminary data from a clinical study of a new experimental drug BETi, OTX015, reported that 7 of 37 patients with non-leukemic forms of hematological malignancies had grade 1-2 hyperglycemia and 1 The patient experienced grade 3 hyperglycemia. Thieblemont, 2014. It is not known whether hyperglycemia may occur with Compound A and general recommendations for the management of possible hyperglycemia are shown in Appendix E.

Overdose, as defined for this protocol, refers only to dosing of Compound A. On a per-dose basis, overdose is defined as the following amount in excess of the protocol-specified dose of Compound A administered to that patient, regardless of any associated adverse events or effects:

RO any amount in excess of the dose indicated in the protocol.

Based on the mode or frequency of administration, an overdose is defined as anything more frequent than the mode or frequency required by the protocol. Complete data regarding the administration of the medicinal product, including any overdose, whether the overdose was accidental or intentional, must be recorded in the individual registration card.

With regard to the method of allocation to treatment groups, subjects eligible for participation in the study will be recruited sequentially in Part A (dose escalation). Recruitment for Part B (Cohort Expansion) will be stratified by disease group and dosing regimen as appropriate. An Interactive Communications Technology (IRT) system will be used to track the distribution of subjects by dose level in Part A and tumor groups in Part B.

Label(s) for Compound A include name of sponsor, address and telephone number, protocol number, Compound A, dosage form and dosage (if required), amount of Compound A per container, lot number, expiration date (if necessary), drug identification number agent/kit number, dosing instructions, storage conditions and required precautions and/or regulatory authority statements, as appropriate. Additional information may be included on the label as required in accordance with national regulations.

Investigator and relevant research site staff receive training on procedures for documenting the acceptance of Compound A, as well as procedures for accounting, reconciliation of Compound A, disposal of Compound A, and documentation of these processes, for example, review by the investigator and appropriate personnel of the return, disposal or destruction of Compound A , including responsibility for the center or investigator's designee.

Only the pharmacist or the investigator's designee dispenses the Compound A dosage form. A record of the number of Compound A capsules/tablets dispensed to and received by each subject must be kept. The pharmacist or investigator's designee will document the doses dispensed/administered in the appropriate study documentation. Subjects use diaries to record daily self-administration of Compound A at home. The person completing the diary signs/initials and dates the diary in accordance with good documentation practice. They are analyzed by research staff at each patient visit. Where necessary, entries are updated so that relevant information can be entered into the eCRF. Research site staff oversees Compound A administration and enters this information into the subject's primary documentation and the corresponding electronic individual registration card (eCRF).

All medicinal products (excluding prior anticancer therapy for the tumor being evaluated) initiated after the subject signs the ICD and all concomitant therapy during the study up to 28 days after treatment cessation, as well as dose, dosing frequency, and reasons for the use of therapy, will be documented in primary documents and on the page of concomitant drugs in eCRF. All prior cancer therapy for the tumor being evaluated, including chemotherapy, biological, immunological, radiation therapy, and surgery, will be documented on the eCRF's Cancer Prior Treatment page. Investigator instructs subjects to notify Investigational Personnel of any new drugs taken after signing the ICD. All medications and important non-drug therapies (herbal medicines, physiotherapy, etc.) and any changes in the dosage of current medications will be documented in the eCRF. Subject to precautionary measures, the use of any concomitant medications/therapies considered necessary for the treatment of the patient should be used. Reassessments of PK may be performed if changes are made to concomitant medicinal products that may affect absorption or metabolism. The following are authorized concomitant medicinal products and procedures:

Subjects with grade 1 diarrhea should promptly begin treatment with diphenyloxylate/atropine (Lomotil) or loperamide (Imodium) or an alternative over-the-counter remedy for diarrhea. Premedication with an antidiarrheal drug before subsequent doses

Compound A may be appropriate and should be discussed with a medical supervisor.

Antiemetics will be delayed until subjects experience CTCAE Grade 1 nausea or vomiting. Subjects may then receive prophylactic antiemetics at the investigator's discretion.

Subjects may receive prophylactic mucosal protection at the discretion of the investigator.

Therapeutic use of granulocyte growth factors is permitted at any time for subjects experiencing febrile or grade 3/4 neutropenia. Regular prophylaxis with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor is at the discretion of the investigator from cycle 2 onwards.

Subjects receiving stable doses of recombinant erythropoietin or darbepoetin alfa for at least 4 weeks prior to initiation of Compound A may continue at the same doses throughout the study. Subjects may begin treatment with de novo erythropoietin-stimulating agents (ESA) in cycle 2 for hypoproliferative anemias that occurred during prior chemotherapy, provided there is no clinical suspicion of a concurrent cause of anemia (eg, induced by Compound A).

Parenteral influenza vaccination is permitted.

Standard infectious disease prophylaxis is not required. However, prophylaxis with antibiotics, antivirals, antipneumocystic agents, antifungal agents, or other prophylaxis may be administered during the study at the discretion of the investigator.

Treatment with bisphosphonates (eg, pamidronate, zolendronate) or other agents (eg, denosumab) is permitted to prevent or slow the progression of bone metastases. It is recommended to maintain a stable dosing regimen throughout the study.

Focal palliative radiotherapy to treat cancer-related symptoms (eg, localized bone pain) is permitted during study treatment at the discretion of the investigator.

Subjects may receive physiological replacement doses of glucocorticoids (up to the equivalent of 10 mg daily prednisone) as maintenance therapy in case of adrenal insufficiency.

Hormone maintenance therapy is permitted in patients with a history of breast or prostate cancer.

Other investigational therapies should not be used while the subject is participating in the study. Anticancer therapies (chemotherapy, biological or research therapies, and surgery) other than study treatments should not be administered to subjects while they are participating in the study. If such treatment is required, the subject must discontinue participation in the study. Treatment by long-term therapeutic dosing of anticoagulants (eg, warfarin, low molecular weight heparin, factor Xa inhibitors) is not allowed. Short-term prophylactic administration of anticoagulants may be considered in subjects if medically indicated (eg, hospitalized subjects, after surgery).

In terms of statistical considerations, the primary goals of this study are to determine the safety, tolerability, and MTD of Compound A when administered orally on a 3/7 day schedule in adult subjects with advanced solid tumors and relapsed/refractory NHL, and to determine its PK characteristics. A secondary goal is to provide a preliminary assessment of the antitumor activity of Compound A. Summary analyzes/statistical analyzes are performed by study part (Part A or B), dose level (Part A) and tumor group (Part B), as appropriate.

Study population definitions include the following:

population included in the study - all subjects who are assigned a number at inclusion and who meet the inclusion / exclusion criteria;

treated population - all subjects who are included in the study and receive at least one dose of Compound A;

Efficacy Evaluable (EE) Population - all subjects included in the study meet the inclusion criteria, complete at least one cycle of Compound A (taking at least 80% of prescribed doses) and have a tumor score at baseline and at least one valid score after baseline;

- 47 040062 Pharmacokinetic (PK) Evaluable Population all subjects included in the study receive at least one dose of Compound A and have at least one measurable concentration of Compound A;

Biomarker Evaluable (BE) Population - All subjects included in the study receive at least one dose of investigational medicinal product and have an assessment of at least one biomarker, except for assessments that are deemed unsuitable.

During part A of the study, an adaptive Bayesian logistic regression (BLR) model (with two parameters) using dose escalation with overdose control (EWOC) was used. For this study, no formal power calculations were performed to determine the sample size. The actual number of subjects will depend on the number of dose levels/cohorts tested. The estimated number of subjects is approximately 40. After determining the MTD or RPTD from Part A to Part B, approximately 14-20 additional subjects will be recruited for each predefined tumor type.

For Part B, sample sizes are determined not on the basis of study power calculations, but on clinical, empirical and practical considerations commonly used for exploratory studies of this kind. Recruitment to the tumor-specific cohort will be stopped for no prospects if there are no objective responses or there are fewer than three subjects with disease stabilization lasting for at least 4 months (i.e. two or more tumor evaluation time points after baseline) , among the first 14 subjects within tumor type. If at least one objective response or three subjects with disease stabilization lasting >4 months are seen among the first recruited 14 evaluable subjects, up to six additional subjects will be recruited for a total of 20 evaluable subjects in the cohort . If the response rate is 20%, there is a 4.4% chance that the first 14 subjects will not respond. If the rate of disease stabilization lasting at least 4 months is 40%, there is a 4% chance that there will be fewer than three subjects with disease stabilization lasting at least 4 months. If there are more SD than objective responses, the disease control rate may be assessed rather than ORR.

In Part A, the baseline characteristics of the subjects will be summarized by the cohort of subjects receiving a given dose for the study population. In Part B, baseline characteristics of subjects will be summarized by tumor type. Age, weight, height, and other continuous demographic and baseline variables will be summarized using descriptive statistics. General condition, gender, race and other categorical variables will be summarized using frequency tables. History data will be summarized using frequency tables by system organ class and preferred term.

The distribution of subjects (distribution of the analyzed population, subjects participating in the study, who discontinued participation in the study with an indication of the underlying reason) during treatment and study will be summarized using frequency and percentage. Provides summary information about the subjects included in the study by research center. Protocol violations are summarized using frequency tables. An auxiliary list of eligible subjects is also provided.

Efficacy analyzes are based on the treated population and include pooled data on disease control rate (DCR), objective response rate (ORR), duration of response or disease stabilization, progression-free survival (PFS), and OS by dose cohort, and dosing regimen (Part A), or by tumor type and dosing regimen (Part B). Tumor response (CR, PR, SD, PD, or not assessable) will be assessed by investigators in accordance with the Criteria for Evaluating Response in Solid Tumors (RECIST) version 1.1 and the International Working Group (IWG) criteria. DCR is defined as the percentage of subjects whose best response is complete response (CR), partial response (PR), or stable disease (SD). ORR is defined as the percentage of subjects whose best response is CR or PR. In cases where SD is the best response, it should be radiographically documented at least once after entry into the study at least 7 weeks later (i.e. coincides with the first response score after baseline minus the evaluation window). If the minimum time to achieve the best response, which is disease stabilization (SD), is not met, the subject's best response will depend on the results of subsequent assessments. For example, a subject who demonstrates SD during the first assessment (when the first assessment does not meet the minimum time to reach SD criterion) and PD during the second assessment will be classified as having the best PD response. A subject with whom follow-up contact is lost after the first SD assessment will be considered unassessable if the minimum time to reach SD criterion is not met.

Two-sided 95% confidence intervals according to the Klopper-Pearson method are used for

- 48 040062 ORR and DCR ratings. Similar analyzes will be performed to include those subjects with validated responses as well as for the population that can be assessed for efficacy. For subjects with the best response, which is either CR or PR, the duration of response is measured from when the criteria for CR/PR are first met (whichever is reported first) to the first date that disease progression is objectively documented. For subjects with the best response, which is SD, the duration of SD is measured from the date of the first dose until the progression criteria are met. If progression is not documented prior to discontinuation of Compound A, overall response duration and SD duration will be censored at the date of the last adequate tumor evaluation.

Duration of response/SD based on investigator estimates will be summarized using descriptive statistics (mean, standard deviation, median, minimum and maximum) for the treated population. With the exception of medians, which will be calculated from both observed and censored rates using the Kaplan-Meier method, all other statistics (mean, standard deviation, minimum and maximum) will be calculated from observed values only.

Progression-free survival (PFS) is defined as the time elapsed from the first dose of Compound A to the first manifestation of disease progression or death from any cause. Subjects who are not progressing or who are not dying by the end date of data collection will be censored by the date of their last adequate tumor evaluation. PFS data will be summarized using descriptive statistics (mean, standard deviation, median, minimum and maximum) for the treated population. With the exception of medians, which will be calculated from both observed and censored rates using the Kaplan-Meier method, all other statistics (mean, standard deviation, minimum and maximum) will be calculated from observed values only.

Overall survival (OS) is measured as the time elapsed from administration of the first dose of Compound A to death from any cause and will be analyzed in a manner similar to that described for PFS.

Adverse events, including adverse events occurring during treatment (TEAE), laboratory tests, vital signs, ECG results, ECOG general health, LVEF scores, physical examinations, vital signs, study treatment exposure, assessment concomitant medications and pregnancy testing for women of reproductive potential will be summarized for the treated population (by dose cohort in Part A and tumor type in Part B).

Observed adverse events are classified using the Medical Regulatory Terminology Dictionary (MedDRA), version 17.1 or higher, Organ System Class (SOC), and Preferred Term (PT). In an analysis performed on the same subject, a subject having the same AE more than once is counted only once. All adverse events are summarized by SOC, PT and NCI CTCAE grade (version 4.0 or higher). Study discontinuation adverse events, those classified as grade 3 or 4, study drug related adverse events, and serious adverse events (SAEs) (including deaths) are tabulated separately. For each subject, a list of all AEs, TEAEs, SAEs (including deaths) and their attribution is provided.

Clinical laboratory results are summarized descriptively by dose cohort (Part A) or by tumor type (Part B) and visit, which also reflect change from baseline. Change tables showing change (low/normal/high) from baseline to worst laboratory value after baseline will be displayed in cross-tab tables by dose cohort (Part A) or by tumor type (Part B). Similar change tables showing the change in NCI CTCAE grades from baseline to highest severity after baseline during the treatment period are presented by dose cohort (Part A) or by tumor type (Part B) for eligible analytes. Lists of abnormal clinical laboratory findings according to NCI CTCAE severity (if applicable), indicators of abnormality (low or high) and their clinical significance are provided.

Graphical representations (eg spaghetti plots or block plots) are provided for the main laboratory analytes. Descriptive statistics for key body parameters, both observed values and change from baseline, will be summarized by dose cohort (Part A) or by tumor type (Part B) and visit. Change tables showing change from baseline to worst laboratory value after baseline will be displayed in cross tabulation tables by dose cohort (Part A) or by tumor type (Part B). The results of measurements of the main indicators of the state of the body are given for the subject and for the visit. ECG parameters and change from baseline will be summarized by dose cohort (Part A) or by tumor type (Part B) and visit using descriptive statistics. Abnormal QTc values after baseline (both QTcF and QTcB) are summarized using frequency tables for the following five categories:

QTc >450 ms,

QTc >480 ms,

QTc >500 ms, QTc increase from baseline >30 ms, QTc increase from baseline >60 ms.

The change from baseline to the worst quality deviation score after baseline (i.e., Normal, Clinically Significant Abnormal Score, and Clinically Significant Abnormal Score, or Normal and Abnormal) will be displayed in the cross-tab tables by dose cohort (Part A) or by tumor type (Part B). A list of ECG parameters for each subject and visit will be provided.

No formal interim review is planned. The data is analyzed on an ongoing basis.

With regard to the statistical method for dose escalation, an adaptive BLRM model using the principle of dose escalation with overdose control (EWOC) will be used to make dose recommendations and estimate MTD during the escalation phase of the study (see Appendix H). The relationship with DLT in the dose escalation part of the study is described by the following Bayesian logistic regression model:

log р, ] [ d, 1 = loga + /Mog -2- La >ΰ,β >0

1-p I (d in which each value pj represents the frequency of occurrence of dose-limiting toxicity (DLT) at each dose;

each value dj represents dose levels;

d*=90 mg is the reference dose;

α represents the odds of DLT at d*.

Regarding prior specifications, prior information for (log(a), log(e)): undefined bivariate normal priors for model parameters (log(a),log(3)) were extracted based on prior assumptions (medians) from preclinical data and wide confidence intervals for DLT probabilities at each dose. The a priori MTD is assumed to be 180 mg based on preclinical data. The probability of DLT for the first dose is considered low. The parameters of the prior distributions of the model parameters are chosen based on the method of constructing a weakly informative prior distribution, as described in Neuenschwander et al. (2015) and is presented in Table. 6. In FIG. 5 shows the final prior distribution of the frequency of occurrence of DLT, obtained from the prior probability distribution given in table. 6.

Table 6

Prior Distribution Parameters for Bivariate Normal Distribution of Model Parameters

Options Averages Standard deviation Correlation log(a),log(3) (-0.693, 0.3936) (2.1) 0

The provisional dose levels are as follows: 15, 30, 60, 90, 120, 150, 180 and 200 mg. It is possible that some doses will be skipped or additional doses will be added during the study based on new emerging safety information. Then, for each cohort of subjects, posterior distributions are obtained for the probabilities of DLT frequencies at various dose levels. The results of this analysis are summarized in terms of estimated probabilities that the true frequency of DLT at each dose level will fall within each of the following ranges:

[0, 0.16] dose decrease,

[0.16, 0.33] target toxicity,

[0.33, 1.00] excessive toxicity.

Following the principle of escalation using EWOC, for each cohort of subjects, the recommended dose is the dose that has the highest posterior probability that the incidence of DLT will be in the target range (16%, 33%) among doses that meet the EWOC, i.e. it is unlikely (<25% posterior probability) that the incidence of DLT at the indicated dose would be in the excess toxicity range.

It should be noted that the dose that maximizes the posterior probability of the target

- 50 040062 toxicity is the best estimate of the MTD, but may not be a valid dose under the overdose criterion if the amount of data is insufficient. If uncertain priors are used for DLT probabilities, this escalation procedure will reflect a conservative strategy early in the study.

The dose recommended by the adaptive bayesian logistic model can be considered as guidance and information that should be combined with a clinical assessment of the toxicity profiles observed during the analysis in order to determine the next dose level to be investigated.

Regarding pharmacokinetic evaluation, plasma PK parameters such as AUC _24h , C _max , T _max , t _1/2 , CL/F and VZ/F of Compound A are calculated by a non-compartmental analysis method from the plasma concentration profiles of Compound A over time . Additional PK parameters can be calculated if data permit. Summary statistics, including number of subjects (N), mean, standard deviation (SD), coefficient of variation (CV%), geometric mean, geometric CV%, median, minimum and maximum, are presented for Compound A concentration at nominal time point, the day of the study and the cohort receiving the specific dose. Mean and individual plasma concentration plots are presented on original and semi-log scales. Summary statistics are shown for Compound A PK parameters by study day and dose cohort and are presented in tabular form. The relationship between Compound A dose, plasma levels, and selected endpoints (eg, toxicity, efficacy, and/or biomarkers) can be studied.

For pharmacodynamic assessment, descriptive statistics (N, mean, standard deviation (SD), median, minimum and maximum) will be presented for baseline, post-baseline values, and change from baseline or percentage change from baseline of each biomarker for the cohort receiving specific dose (Part A), or by tumor type (Part B) and visit. The results of the assessment of biomarkers in subjects will be plotted against time. Comparison of biomarker levels before and during treatment will be performed using the Wilcoxon rank test. If sufficient and reliable results are obtained from the analysis of biomarkers, the relationship between percentage changes in biomarker levels and endpoints, including ORR and DCR, is studied.

In addition, with regard to adverse events, in particular, monitoring, recording and reporting of adverse events, AE is any harmful, unintended or undesirable medical event that may occur or worsen in a subject during the study. This may be a new intercurrent disease, worsening comorbidity, trauma, or any comorbid health condition of the subject, including laboratory test results, regardless of etiology. Any worsening (i.e., any clinically significant adverse change in the frequency or intensity of the preexisting condition) should be considered an AE. The AE page of the CRF must record the diagnosis or syndrome, not the individual signs or symptoms of the diagnosis or syndrome. Misuse, withdrawal from a clinical trial, sensitivity or toxicity to an investigational drug should be reported as an AE. Overdose, whether accidental or intentional, whether AE related or not, must be reported on the CRF Overdose page. Any consequences of accidental or intentional overdose of study drug must be reported as AE on the AE page of the CRF. If the consequence of the overdose is an SAE, then the consequences must be reported on the SAE report and on the AE page of the CRF. An overdose resulting in an SAE should be identified as the cause of the event in the SAE report and in the CRF, but should not be reported as the SAE itself.

In the event of an overdose, the subject should be under appropriate supervision and receive support as needed. In the event of an overdose of Compound A, there is no known specific antidote. Treatment, in fact, will depend on the severity of the clinical situation, the judgment and experience of the attending physician.

All subjects will be monitored for the occurrence of adverse events (AEs) during the study. The assessments may include monitoring any or all of the following: the subject's clinical symptoms, laboratory, pathological, radiological, or surgical findings, physical examination results, or results of other tests and/or procedures.

The investigator records all AEs from the time the subject signed the informed consent up to 28 days after the last dose of Compound A, as well as those SAEs known to the investigator at any time thereafter that are suspected to be related to Compound A. AEs and SAEs are recorded on the page AE in the CRF and in the subject's primary documents. All SAEs must be reported to the Office of Drug Safety within 24 hours of the event being reported to the investigator, by facsimile or other appropriate means, using the SAE Report Form or an approved equivalent form. .

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A qualified investigator will rate all adverse events by severity. SAE is any AE occurring at any dose that:

leads to death;

poses a threat to life (i.e., in the opinion of the investigator, the subject has an immediate risk of death from AE);

requires hospitalization in a hospital or an extension of an ongoing hospitalization (hospitalization is defined as an admission to a hospital, regardless of the length of stay);

leads to permanent or significant incapacity/incapacity for work (significant impairment of the subject's ability to perform normal life functions);

is a congenital anomaly/birth defect;

represents an important medical phenomenon.

Significant medical events are defined as events that may not be directly life-threatening or result in death, hospitalization, or disability, but may pose a threat to the subject or require medical or surgical intervention to prevent one of the other consequences listed above. Medical and scientific judgment should be taken into account when deciding whether such an AE should be considered serious.

Events that are not considered SAEs are hospitalizations for:

carrying out a standard therapy procedure according to the protocol. However, hospitalization or prolonged hospitalization due to a post-treatment complication will be reported as SAE;

carrying out standard treatment or monitoring of the studied disease, not associated with any deterioration in the condition;

receiving blood or platelet transfusions as standard treatment for the disease studied. However, hospitalization or prolonged hospitalization due to the occurrence of a complication after such a transfusion is reported as SAE;

conducting protocol/disease-related examination procedures (eg, surgery, scanning, endoscopy, sampling for laboratory tests, bone marrow sampling). However, hospitalization or prolonged hospitalization due to the occurrence of a complication following such procedures is reported as SAE;

hospitalization or prolongation of hospitalization for technical, practical or social reasons in the absence of AE;

performing a planned procedure (i.e. scheduled before the start of study treatment); must be documented in the primary document and CRF. Hospitalization or extended hospitalization due to a complication is reported as SAE.

selective treatment or elective procedure for pre-existing disease, not related to the disease under study, which has not worsened from baseline;

emergency outpatient treatment or observation that does not result in hospitalization unless the other severity criteria above are met.

If the AE is considered serious, the AE page/CRF screen and the SAE report form must be completed. For each SAE, the investigator will provide information on severity, start and end dates, relationship to IP, actions taken on IP, and result.

For AE and SAE, the investigator should assess the severity/intensity of the event. The severity/intensity of the AE will be rated based on the subject's symptoms according to the current active minor version of the Common Terminology Criteria for Adverse Events (CCAE version 4.03); http://ctep.cancer.goV/protocolDevelopment/electronic_applications/ctc.htm#ctc_40 AUs that are not defined in the CTCAE should be rated for severity/intensity according to the following scale:

Grade 1 = Mild toxicity - temporary or mild discomfort; no restrictions on activities; medical intervention/therapy is not required.

Grade 2 = Moderate toxicity - mild to moderate activity limitation, some assistance may be required; no or minimal medical intervention/therapy required.

Grade 3 = Severe toxicity - a marked limitation in activity, some assistance is usually required; medical intervention/therapy is required, hospitalization is possible.

Grade 4 = Life-threatening condition - significant activity limitation, usually requires significant assistance; medical intervention/therapy is required, hospice care may be required, a program to alleviate the suffering of the terminally ill.

Grade 5 = Death due to toxicity.

The term severe is often used to describe the intensity of a particular event (as in mild, moderate, or severe myocardial infarction); however, the event itself may be of relatively little medical significance (eg, severe headache). This criterion is not identical to the term serious, which is based on outcome or action criteria.

- 52 040062 subject/phenomena associated with phenomena that pose a threat to the life or functioning of the subject. Severity, not severity, serves as a guide for defining regulatory obligations.

Causality assessment. The investigator should determine the relationship between the administration of Compound A and the occurrence of AE/SAE as unsuspected or suspected, as defined below:

Unsuspected: A causal relationship of an adverse event with Compound A administration is unlikely or remote, or other drugs, therapeutic interventions, or underlying diseases adequately explain the observed event.

Suspected: There is a suspected possibility that the administration of Compound A causes an adverse event. Estimated probability means that there is reason to believe that there is a causal relationship between IP and an adverse event.

Causality must be assessed and presented for each AE/SAE based on information currently available. Causality should be reassessed and provided as additional information becomes available. If the phenomenon is assessed as suspect, i.e. associated with a comparator, auxiliary or complementary drug to Compound A that was not manufactured or provided by the sponsor, the name of the manufacturer must be indicated when reporting the event.

As regards duration, for both AE and SAE, the researcher records the start and end dates of the event. Investigator reports decisions made regarding IP following the onset of AE or SAE, as appropriate (e.g., stopping, interrupting, or reducing the dose of IP, as appropriate), and whether concomitant and/or additional treatment for this phenomena. The researcher reports the outcome of the event for both AE and SAE. All SAEs that do not resolve upon discontinuation of the subject's participation in the study should be observed until recovery (return to baseline), recovery with consequences, or death (due to SAE).

With regard to Abnormal Laboratory Values, an abnormal laboratory value is considered to be an AE if the deviation is:

leads to termination of participation in the study;

requires treatment, dose modification/dosing interruption of Compound A, or any other therapeutic intervention; or is considered to be of significant clinical importance, eg indicative of a new pathological process and/or organ toxicity; or represents an exacerbation or worsening of a current condition.

Regardless of severity, only laboratory abnormalities that meet the criteria for severity should be documented as a serious adverse event. If a laboratory abnormality is one of the components of a diagnosis or syndrome, then only the diagnosis or syndrome should be recorded on the AE page/CRF screen. If the abnormality was not part of the diagnosis or syndrome, then the laboratory abnormality should be recorded as an AE. If possible, laboratory abnormalities should be reported as a medical term and not simply as an abnormal laboratory result (eg, reported as thrombocytopenia rather than low platelets).

All pregnancies or suspected pregnancies occurring in a female subject of reproductive potential or in a partner of reproductive potential in a male subject are immediately reported events. Exposure of any pregnant woman (eg, caregiver, pharmacist, clinical trial coordinator or observer) to Compound A is also an immediately reported event. Pregnancies and suspected pregnancies (including elevated β-hCG or a positive pregnancy test in a female subject of childbearing potential, regardless of pathological condition) occurring while the subject is taking Compound A, or within three months (to be determined) after the subject's last intake doses of Compound A are considered to be immediately reported events. The study drug should be discontinued immediately. Pregnancy, suspected pregnancy, or a positive pregnancy test must be reported to Sponsor Drug Safety immediately by email, telephone, facsimile, or other appropriate means using the Pregnancy Initial Report Form or an approved equivalent.

The female subject should be referred to an ob/gyn, preferably with experience in reproductive toxicity, for further evaluation and counselling. The Investigator observes the female subject until the pregnancy is completed and must promptly notify Sponsor Drug Safety of the results of the pregnancy (normal and abnormal) using the Pregnancy Follow-up Report Form or an approved equivalent form. If the outcome of pregnancy

- 53 040062 was abnormal (eg spontaneous abortion), the investigator will report the abnormal outcome as AE. If an abnormal outcome meets any of the major criteria, it must be reported as SAE Sponsor Drug Safety by facsimile or other appropriate means within 24 hours of the event being reported to the investigator using the SAE Report Form or an approved equivalent form. All neonatal deaths that occur within 28 days of birth are reported, regardless of causality, as SAE. In addition, any infant death after 28 days that the Investigator believes may be related to in utero exposure to Compound A must also be reported to Sponsor Drug Safety by facsimile or other appropriate means within 24 hours of the Investigator being informed of the event using the SAE form. Report Form or an approved equivalent.

For male subjects, if the female partner of a male subject taking Compound A becomes pregnant, the male subject taking Compound A should notify the investigator and the pregnant female partner will be advised to call their physician immediately. If appropriate, the male subject should discontinue Compound A, but may be restarted at a later time at the discretion of the investigator and medical supervisor.

Any AE that meets any of the SAE criteria is required to complete the SAE Report Form in addition to registering on the AE page/CRF screen. All SAEs are reported to Sponsor Drug Safety within 24 hours of the event being reported to the investigator via facsimile or other appropriate means (eg, email) using the SAE Report Form or an approved equivalent form. This instruction applies to initial SAE messages as well as any subsequent messages. The researcher must ensure the accuracy and consistency of the data in these forms. This requirement applies to all SAEs (regardless of association with Compound A) that occurred during the study (from the time the subject signed the informed consent up to 28 days after the last dose of Compound A), or to any SAE that became known to the investigator at any time after this, which may be related to Compound A. Serious adverse events occurring before treatment (after signing the ICD) will be recorded. The SAE report must contain a detailed description of the SAE and include a summary of the entries in the inpatient record and other relevant documents. In the event of the death of the subject and an autopsy being performed, copies of the autopsy report and death certificate must be sent to Sponsor Drug Safety as soon as they become available. Any additional information must be detailed on the SAE Report Form or approved equivalent and submitted to Sponsor Drug Safety. Where required by law, the investigator is responsible for informing the Institutional Review Board/Ethics Committee (IRB/EC) about the SAE and providing all relevant initial and subsequent information about the phenomenon. The investigator must keep copies of all SAE information on file, including correspondence with sponsor and IRB/EC.

Inquiries related to SAE are transmitted from Drug Safety to the research center via facsimile or email. Response time is expected to be no more than five (5) business days. Urgent requests (for example, no causality assessment) can be handled over the phone.

For regulatory reporting purposes, Drug Safety defines the expected events associated with Compound A based on the Investigator Brochure. In the US, all suspected unanticipated serious adverse reactions (SUSARs) are reported on an expedited basis in accordance with 21 CFR 312.32. For countries within the European Economic Area (EEA), the Authorized Representative shall report suspected Unanticipated Serious Adverse Reactions (SUSAR) in accordance with Directive 2001/20/EC and the Detailed Collection, Testing Guidelines to regulatory authorities and relevant ethics committees on an expedited basis. and reporting of adverse reactions arising from clinical trials of investigational products for use in humans (ENTR/CT3), as well as in accordance with the requirements of a particular country. Adverse events such as disease progression, disease progression-related death (in the absence of Compound A related serious events), and serious events due to relapse of the studied disease will not be subject to expedited regulatory sponsor reporting.

The authorized representative must notify the investigator of the following.

Any AE that is suspected to be related to the use of Compound A in this study or in other studies that is serious and unanticipated (eg SUSAR).

Any finding in tests performed on laboratory animals that suggests a significant risk to humans, including reports of mutagenicity, teratogenicity, or carcinogenicity.

Where required by national law, the investigator should promptly notify the IRB/EC of these new serious and unanticipated AEs or significant risks to subjects. The investigator should keep copies of all relevant safety information,

- 54 040062 including correspondence with the drug supplier, Compound A, the responsible party, and

IRB/EC.

The following events are considered sufficient grounds for discontinuation of the study medication by the subject:

Undesirable event.

Termination of participation in a clinical trial by a subject.

Lack of efficiency.

Doctor's decision.

Protocol violation.

progressive disease.

Death.

Loss of contact for follow-up.

Other (specified in CRF).

The reason for discontinuing treatment must be recorded in the CRF and in the primary documents. The decision to terminate a subject's treatment remains the responsibility of the treating physician and will not be delayed or overruled by the sponsor. However, prior to discontinuing the subject's treatment, the investigator may contact the medical supervisor and forward appropriate supporting documents for review and discussion.

The following events are considered sufficient grounds for termination of the subject's participation in the study:

Didn't get screened.

Undesirable event.

Termination of participation in a clinical trial by a subject.

Lack of efficiency.

Doctor's decision.

Protocol violation.

progressive disease.

Death.

Loss of contact for follow-up.

Other (specified in CRF).

The reason for discontinuing treatment must be recorded in the CRF and in the primary documents.

This is an open study, so Compound A is listed on the package label.

Subjects enrolled in the study are issued a study participant identification card, which contains the name of the study and an emergency contact number. This can be used by medical professionals in an emergency to obtain information regarding a subject's participation in a study.

The procedures set out in this study protocol regarding the conduct, evaluation and documentation of this study shall be designed to ensure that the sponsor, its authorized representative and the investigator comply with Good Clinical Practice (GCP) as described in the International Conference on Harmonization (ICH) guidelines. and in accordance with the general ethical principles laid down in the Declaration of Helsinki. The study will be approved by the IRB/EC before it starts. The Investigator will direct all aspects of this study in accordance with applicable national, state, and local laws of the relevant regulatory authorities.

Investigator responsibilities are set out in the ICH Guideline for Good Clinical Practice and government regulations. The staff or authorized representative evaluates and approves all investigators, who in turn select their own staff. The Investigator must ensure that all staff involved in the clinical investigation are properly aware of the protocol, amendments, investigational product, and their responsibilities and functions related to the investigation, including confidentiality obligations regarding sponsor information. The investigator shall maintain a list of junior investigators and other suitably qualified persons to whom he or she has delegated important research-related responsibilities. The investigator is responsible for compiling a list of all subjects who have signed an Informed Consent Form (ICF) and are screened for participation in the study. Subjects not screened must have the cause(s) recorded on the subject's primary records. The investigator or a designated member of the investigator's staff must be on site during monitoring visits to verify data, resolve queries, and gain direct access to subjects' records (e.g., medical records, office records, hospital records, and study-related records) to verify primary data. The investigator must ensure that CRFs and requests are completed in a timely and accurate manner.

The investigator obtains the informed consent of the subject and/or the subject's legal representative prior to any investigation-related procedures. Documentary proof that

- 55 040062 informed consent is signed prior to the subject's enrollment in the study, and the process of signing informed consent must be recorded in the subject's primary records, including the date. The original ICF, signed and dated by the research participant and the person who gave consent to the research participant before the subject was included in the study, must be kept in the investigator's files, and a copy must be given to the research participant. In addition, if the protocol is amended and this affects the content of the informed consent, the ICF should be reviewed. Study participants participating in the study after the implementation of the amended protocol must re-sign the revised ICF. The revised ICF is signed and dated by the study participant and must be kept in the investigator's files, with a copy given to the study participant.

Any change to the study protocol must be approved by the clinical investigator/medical observer. Amendments are submitted to the IRB/EC for written approval. Written approval must be obtained prior to the implementation of the modified version. The written, signed IRB/EC approval must state the investigator's name, protocol number, study name, and the amendment number(s) that is applicable. Amendments that are of an administrative nature do not require IRB/IEC approval but will be submitted to the IRB/IEC for information purposes.

Before the start of the study, the study protocol, ICF, and any other relevant documents are submitted to the IRB/IEC with a cover letter or form that lists the documents to be submitted, their dates of issue, and the location (or region or area of jurisdiction, as appropriate) for which approval is sought. If required, the documents will also be submitted to government authorities in accordance with the requirements of local legislation. An IP may only be supplied to an investigator by the sponsor or its authorized representative after the sponsor or its authorized representative has received documentation of all ethical and legal requirements to start the study. This documentation must also include a list of IRB/EC members and their titles and qualifications. If the IRB/EC does not disclose the names, titles and qualifications of committee members, the IRB/EC will be asked to issue a statement confirming that the composition of the committee is GCP compliant. For example, the IRB General Assurance Number may be accepted as a replacement for this list. Formal IRB/EC approval must state the name of the protocol, number, amendment number (if applicable), study location (or region or area of jurisdiction, as appropriate) and any other documents considered. It must include the date the decision was made and be formally signed by the member of the committee. Before the first subject is included in the study, all ethical and legal requirements must be met. The IRB/EC and, where appropriate, government authorities must be informed of any subsequent changes to the protocol as required by local law. Amendments should be evaluated for the need to seek formal approval and updates from the ICF. The investigator must record all correspondence with the IRB/EC and, as appropriate, between the investigator coordinator and the IRB/EC. This also applies to any correspondence between the investigator (or Investigator Coordinator, as appropriate) and regulatory authorities.

Where required by law or the IRB/EC, the investigator must submit to the IRB/EC:

Information about serious or unexpected adverse events as soon as possible.

Periodic progress reports.

Deviations from the protocol, or anything that may pose additional risk to subjects.

Sponsor reserves the right to terminate this study early at any time for reasonable medical or administrative reasons. Any early termination of a study is properly documented in accordance with local requirements (eg IRB/EC, regulators, etc.). In addition, the investigator or sponsor has the right to close one study site at any time during the study for medical or administrative reasons, such as:

Unsatisfactory set.

Non-compliance with GCP.

Inaccurate or incomplete data collection.

Falsified records.

Non-compliance with the study protocol.

With regard to the processing and recording of data, the investigator must ensure that records and documents relating to the conduct of the study and the distribution of the investigational product are complete, accurate, recorded and maintained. Examples of primary documentation include an inpatient record; clinical and office cards; laboratory records; notes; subjects' diaries or questionnaires; medicine dispensing logs; records of automatic devices; verified and certified copies or extracts; microfiche; x-rays and reports and any records, including those held by the pharmacy and laboratories, as well as copies of the CRF or CD-ROM.

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Data is collected by CRF and entered into the clinical database according to the sponsor's SOP. These data are electronically verified using editors specified by the clinical investigation team. Discrepancies in data will be brought to the attention of the clinical investigation team and clinical investigation site staff, as appropriate. Resolutions on these issues will be reflected in the database. The audit trail in the system will keep track of all changes made to the data.

Key documents must be kept by the investigator for the period of time specified in the agreement to conduct the clinical trial. The investigator should keep these records for the period of time described above, or in accordance with local laws or requirements, whichever is longer. Key documents include, but are not limited to, the following:

Signed ICF for all subjects.

List of subject identification codes, list of subjects included in the screening (if necessary) and registration log.

A record of all communications between the investigator and the IRB/EC.

Composition of the IRB/EC.

A record of all communications between investigator, sponsor and their authorized representative(s).

A list of junior investigators and other suitably qualified individuals to whom the investigator has delegated important research-related responsibilities, along with their roles in the study, professional backgrounds, and their signatures.

Copies of the CRF (in case of hard copy) and correction documentation for all subjects.

Accounting records for Compound A.

A record of any retained samples of body fluids or tissues.

All other primary documents (subject records, inpatient record, laboratory records, etc.).

All other documents listed in Section 8 of the ICH Consolidated Guide to GCP (Essential Documents for the Conduct of a Clinical Trial).

The investigator must notify the sponsor if he/she wishes to transfer the core documents to someone else, move them to another location, or is unable to keep them for a specified period of time. The investigator must obtain written approval from the sponsor before destroying any records. If the investigator cannot fulfill this obligation, he must ask the sponsor for permission to take alternative measures. Details of these measures should be documented. All study-related documents must be provided upon request by the relevant health authorities. The investigator or institution must take steps to prevent accidental or premature destruction of these documents.

All aspects of the study will be closely monitored by the sponsor or its authorized representative for compliance with applicable regulations regarding current GCPs and SOPs. The sponsor shall ensure that appropriate monitoring procedures are in place before, during and after the study. All aspects of the study are reviewed by the investigator and staff during the study start visit and/or at the investigators' meeting. Before enrolling subjects in a study, the representative reviews the protocol, CRT, informed consent procedures, record keeping, and AE/SAE reporting with the investigator. Monitoring includes visits to study sites with the investigator and his/her staff, and any relevant interactions by mail, e-mail, fax, or telephone. During laboratory monitoring visits, study storage space, CRFs, subject primary documents, and all other study documentation are reviewed/reviewed by the sponsor's representative in accordance with the Study Monitoring Plan.

Accuracy is verified by performing a primary data check, which consists of a direct comparison of the records made on the CRT with the corresponding primary documentation. Any discrepancies that arise are reviewed with the investigator and/or his/her staff. Any necessary corrections will be made directly to the CRT or through requests from the investigator and/or his/her staff. Monitoring procedures require that informed consents, compliance with inclusion/exclusion criteria, and documentation of SAEs and their proper registration be verified. Additional monitoring activities may be specified in the monitoring plan for the specific study.

In addition to standard monitoring procedures, the sponsor maintains a Good Clinical Practice Quality Assurance unit. Representatives of this service will conduct audits of clinical research activities in accordance with the sponsor's SOP to assess compliance with the guidelines and provisions of Good Clinical Practice.

The investigator must provide direct access to the laboratories where the study was performed, primary documents, CRFs, and relevant supporting records of the subjects taking

- 57 040062 participation in research, for audits and inspections by IRB/EC, regulatory authorities (e.g. FDA, EMA,

Health Canada) and authorized representatives of the company. The investigator should make every effort to be present during audits and/or inspections. If the investigator is contacted by any regulatory authority regarding an inspection, the sponsor should be contacted immediately.

Appendix A. Table of abbreviations.________________________________________________

Abbreviations or special terms Explanation AD Antibodies to the drug ADCC Antibody dependent cellular cytotoxicity ADL Daily self care activities AE Adverse event ALL Acute lymphoid leukemia ALT Alanine aminotransferase (SGPT) AML Acute myeloid leukemia ANC Absolute neutrophil count Ara-C Cytarabine AST Aspartate aminotransferase (SGOT) AUC Area under the curve β-hCG β-subunit of human chorionic gonadotropin BID Twice a day BM Bone marrow BMI Body mass index BSA body surface area BUN The concentration of urea nitrogen in the blood C Cycle CBC General blood analysis CD Cluster of differentiation CEBPa CCAAT/enhancer-binding protein alpha CI Confidence interval c-kit Mast and stem cell growth factor receptor CL Clearance Stakh The maximum concentration of the drug in blood plasma CNS central nervous system CR Complete remission CRc Cytogenetic complete remission CRi Complete remission with incomplete recovery of neutrophil count CRp Complete remission with incomplete recovery of platelet count CRP C-reactive protein CRR Percentage of patients in complete remission

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CRO Contract Research Organization CRF Individual registration card CRP Clinical Research Specialist CRS Scientific Advisor for Clinical Research CRT Calreticulin CT CT scan CTCAE Criteria for assessing the severity of the most frequent adverse events cv% The coefficient of variation DAT Direct antiglobulin test DCR Disease control frequency DIC Syndrome of disseminated intravascular coagulation DLT Dose limiting toxicity DMC Data Monitoring Committee DOR Response duration EU ethics committee ECG Electrocardiogram ECHO Echocardiogram ECOGPS General condition according to the Eastern joint oncology group eCRF Electronic individual registration card EEA European Economic Area ELISA Linked immunosorbent assay EOI End of infusion EOT End of treatment ESR Erythrocyte sedimentation rate FACS Sorting of fluorescent activated cells FCBP Women with reproductive potential FDA Food and Drug Administration medicines FISH Fluorescent in-situ hybridization FLT3 Fms-like tyrosine kinase-3 FLT3-ITD Fms-like tyrosine kinase-3-intrinsic tandem duplication FOXP3 Forkhead box R3 (transcription factor FOXP3 GCP Good Clinical Practice GVHD Graft-versus-host disease HBV Hepatitis B virus HCV Hepatitis C virus HGB Hemoglobin HIV AIDS virus HLA human leukocyte antigen HNSTD Highest non-hazardous toxic dose HSCT Hematopoietic stem cell transplantation huCD Cluster of human differentiation ICD Informed consent document

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ICF Informed Consent Form ICH International Conference on Harmonization ICSH International Council for Standardization in Hematology IFN Interferon IgE Immunoglobulin subclass E IgG Immunoglobulin subclass G IL Interleukin IL-Ιβ Interleukin-1 beta IND Investigational new drug INR International normalization factor IP Investigational drug IPSS-R Updated International Predictive Index IRB Expert Council of the organization IRR infusion reaction IRT Interactive communication technology IV Intravenous administration IVIG Intravenous immunoglobulin IWG International working group KC-GRO Growth-regulating oncogene LDH lactate dehydrogenase LSC Leukemic stem cells LVEF Ejection fraction of the left ventricle mCR Complete molecular remission MCP-1 Monocytic chemoattractant protein-1 MDR Multidrug resistance MDS myelodysplastic syndrome MedDRA Medical glossary of terminology for the regulation of activities MIP la Macrophage inflammatory protein-alpha 1 MM multiple myeloma MRI Magnetic resonance imaging MTD Maximum tolerated dose MUGA Radionuclide angiography N Quantity NCI National Cancer Institute NHL Non-Hodgkin's lymphoma NOD-SCID Non-obese diabetic, severe-combine immunodeficiency, NOD-SCID mice NOAEL Maximum dose that does not cause detectable adverse effects on human health NOEL The maximum dose that does not lead to the development of observed effects NPM1 Nucleophosmin 1 NSG NSG mice (Non-obese diabetic, severe-combine immunodeficiency gamma) NTD Intolerable dose _O2 Oxygen

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ORR Objective response rate OS Overall survival RVMS Mononuclear cells in peripheral blood PCR Polymerase ch PD Pharmacodynamics PFS Progression-free survival RK Pharmacokinetics PLT Platelet PR Partial remission RT prothrombin time PTT Partial thromboplastin time Q2W Every two weeks QD Once a day QW Once a week QWx2 Once a week for 2 weeks QWx4 Once a week for 4 weeks RAEB Refractory anemia with excess blasts RBC Platelet count RFS Disease free survival RP2D Dose Recommended for Phase 2 Clinical Study SAE Serious adverse event SAP Statistical analysis plan SC Study Oversight Committee SD Standard deviation SE standard error SGOT Serum glutamate oxaloacetate transaminase SGPT Serum glutamate-pyruvate transaminase SIRPa Signaling regulatory protein alpha SOP Standard Operating Procedures SRC Safety Committee SUSAR Suspected unexpected serious adverse reaction tl/2 Half-life tmax Time to reach maximum plasma concentration TLS Tumor lysis syndrome TNBC Triple negative breast cancer TNFa tumor necrosis factor alpha ULN Upper limit of normal US United States USP U.S.P Vss Volume of distribution WBC White blood cell count WHO WHO (World Health Organization) wks weeks

Appendix B. RECIST Version 1.1.

The following information is derived/summarized from Eisenhauer, 2009, New Response Evaluation Criteria in Solid Tumors: Revised RECIST Guideline (Version 1.1). See the main link for more information.

Definitions.

On screening, tumor foci/lymph nodes will be classified as measurable or non-measurable.

measurable disease.

Foci of the tumor. Must be accurately measured in at least one direction (the longest diameter in the measuring plane must be recorded) with a minimum dimension of:

mm according to computed tomography (CT) (thickness of the tomographic slice is not more than 5 mm);

mm when measured with a caliper during clinical examination (lesions that cannot be accurately measured with calipers should be recorded as unmeasurable);

mm on chest x-ray.

Malignant lymph nodes.

To be considered pathologically enlarged and measurable, the size of a lymph node must be >15 mm in the short axis as assessed by computed tomography (tomographic slice thickness should not exceed 5 mm). At baseline and during the follow-up period, the measurement will be carried out only along the short axis.

An unmeasurable disease.

All other lesions, including small lesions (longest diameter <10 mm or short axis abnormal lymph nodes 10 to <15 mm) and truly unmeasurable lesions.

Lesions considered truly non-measurable include meningeal disease, ascites, pleural or pericardial effusion, inflammatory disease of the breast, lymphogenous involvement of the skin or lung, abdominal mass organomegaly/abdominal organomegaly identified by physical examination that is not measurable by reproducible methods visualization.

Evaluation of tumor response.

Target defeats.

When more than one measurable tumor lesion is present at baseline, all lesions, up to a maximum of five lesions in total (and a maximum of two lesions per organ) representing all organs involved, should be identified as target lesions and will be recorded and measured at the original level. Target lesions should be selected based on their size (lesions with the longest diameter), representative of all organs involved, but in addition should be one that can be reproducibly measured repeatedly. It should be noted that pathological nodules must meet the measurable short axis criterion of >15 mm by CT scan, and only the short axis of these nodules will affect the baseline sum. All other lesions (short axis >10 mm but less than 15 mm) should be considered non-target lesions. Nodules with a short axis <10 mm are considered non-pathological and should not be reported or observed. At baseline, the sum of target lesions should be recorded (longest diameter of tumor lesions plus short axis of lymph nodes: a maximum of 5 in total).

After baseline, a value must be entered into the eCRF for all identified target lesions for each assessment, even very small ones. If the lesions are extremely small and weak, and cannot be accurately measured, but are considered to be present, the default value of 5 mm can be used. If the lesions are too small and are believed to be virtually absent, the default value of 0 mm can be used.

Non-target lesions.

All non-measurable foci (or disease foci) and any measurable foci in addition to those specified as target foci are considered non-target foci. Measurements are not required, but these lesions should be reported at baseline and should be considered present, absent, or obvious progression.

response criteria.

Target and non-target lesions are assessed for response separately, and then the tumor burden as a whole is assessed as an overall response.

Target lesion response: Target lesions are assessed as follows:

Complete response (CR). Disappearance of all target lesions. Any pathological lymph nodes (target or non-target) should show short-axis reduction to <10 mm.

Partial response (PR). At least a 30% reduction in the sum of diameters of the target lesions using the sum of diameters at baseline as a comparison.

Disease progression (PD). Increase by at least 20% the sum of the target lesion diameters, using the smallest sum in the study as a comparison (this includes the original sum if it is the smallest in the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression).

Disease stabilization (SD). Neither a sufficient decrease in tumor mass to qualify as PR, nor a sufficient increase to qualify as PD, using the smallest sum of diameters during the study for comparison.

Non-target lesion response: Non-target lesions will be scored as follows:

Complete response (CR). Disappearance of all non-target lesions and normalization of the level of the tumor marker. All lymph nodes should be non-pathological in size (<10 mm on the short axis).

He-CR/He-PD. Persistence of one or more off-target lesions and/or retention of a tumor marker level above normal limits.

Progressive disease (PD). Obvious progression (see comments below) of existing non-target lesions. (Note: the appearance of one or more new lesions is also considered progression).

In the case where the subject also has a measurable disease: Under these conditions, for

- 62 040062 achieve apparent progression based on non-target disease, there should be an overall level of significant worsening of the non-target disease such that even in the presence of SD or PR in the target disease, the total tumor burden increases sufficiently to stop therapy. A modest increase in the size of one or more off-target lesions is usually not sufficient for the status of clear progression. Representing overall progression status solely on the basis of change in non-target disease in terms of SD or PR of the target disease would therefore be extremely rare.

In the case where the subject only has a non-measurable disease: This circumstance occurs in some Phase 3 trials when the presence of a measurable disease is not a criterion for inclusion in the study. The same general concepts apply in this document as noted above; however, in this case there is no measurable disease score that could influence the interpretation of the increase in non-measurable disease burden. Since worsening of a non-target disease cannot be easily quantified (by definition: if all lesions are truly unmeasurable), a useful test that can be applied in evaluating subjects for apparent progression is to assess whether an increase in overall burden is disease based on a change in non-measurable disease comparable in magnitude to the increase that would be required to declare a PD for a measurable disease: i.e. an increase in tumor burden representing an additional 73% increase in volume (equivalent to a 20% increase in diameter in a modifiable lesion). Examples include an increase in pleural effusion from trace to significant, an increase in lymphangitis from localized to widespread, or may be described in protocols as sufficient to require a change in therapy. In the event that obvious progression is observed, the subject will be considered as having a total PD at that point. While it would be ideal to have objective criteria to apply to an unmeasurable disease, the very nature of that disease makes this impossible: hence the increase must be significant.

The overall response should be evaluated in accordance with Table. 7 for subjects with target tumor lesions and Table. 8 for subjects with non-target lesions only

Table 7

Time Point Response: Subjects with Target (± Non-Target) Lesion

Target foci response Response of non-target foci New outbreaks General response CR CR No CR CR He-CR/ non-PD No PR CR Inestimable No PR PR He-PD or not all evaluated No PR SD He-PD or not all evaluated No SD Not everyone is rated HePD No NE PD Any Yes or no PD Any PD Yes or no PD Any Any Yes PD CR = complete response, PR = partial response, SD = disease stabilization, PD = disease progression, NE = not measurable.

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Table 8

Time Point Response: Subjects with non-target lesion only

Response of non-target foci New outbreaks General response CR No CR He-CR/ non-PD No Non-CR/ He-PD ^1} Not everyone is rated No NE Apparent PD Yes or no PD Any Yes PD CR = complete response, PR = partial response, SD = stable disease, PD = disease progression, NE = not assessable. ^a He-CR/He-PD” is preferred over “disease stabilization” for non-target lesions, as SD is more commonly used as an endpoint for efficacy in some studies, thus assigning this category when no tumor lesion can be measured, Not recommended.

Symptomatic worsening.

Subjects with a global deterioration in health requiring discontinuation of treatment, without objective evidence of disease progression at that point, should be reported as having a symptomatic deterioration. Every effort should be made to record objective progression, even after cessation of treatment. Symptomatic deterioration is not indicative of an objective response: it is the reason for discontinuation of study therapy. The objective response status of such subjects should be determined by assessing the target and non-target disease.

Appendix C.

Updated response criteria for malignant lymphoma International Working Group Revised Response Criteria for Malignant Lymphoma (Cheson, 2007) are available online at: http://jco.ascopubs.Org/cgi/reprint/25/5/579 (click download manuscripts in PDF format).

Appendix D. General Condition Criteria.

score Description 0 The patient is fully active, able to do everything as before the disease without limitation. 1 The patient is unable to do heavy work, but can do light or sedentary work, such as light housework or office work. 2 The patient is treated as an outpatient and is capable of self-care, but is unable to perform work. He spends more than 50% of his waking time actively. 3 The patient is capable of only limited self-care, spends more than 50% of his waking time in a chair or bed. 4 Disabled, completely incapable of self-care. Chained to a chair or bed. 5 Death

Appendix E. General guidelines for the management of hyperglycemia.

Fasting glucose is defined as the level observed >4 h after the last meal to assess dose-limiting toxicity and clinical management decisions. Subjects should be instructed on how to recognize hypo- and hyperglycemia. Any subject who experiences hyperglycemia or symptoms associated with hyperglycemia should be subjected to standard treatment with interruptions/reductions in the administration of Compound A. Additional guidelines are described below.

In the case of persistent fasting hyperglycemia (>126 mg/dl or 14 mmol/l) or equal to grade 2 or greater, at any time deemed appropriate by the investigator, treatment with an oral antidiabetic agent (OAD) is recommended.

In case of fasting hyperglycemia grade >3, follow-up in the clinic should continue until the hyperglycemia is resolved to grade <2.

In the case of persistent grade 3 fasting hyperglycemia (>250 mg/dL or 27.8 mmol/L), insulin therapy should be considered either in combination with OAD or alone. Long-acting insulin should only be used if the subject is hospitalized. Glucose control should continue for at least 6 hours after insulin administration (rapid or long acting) due to possible withdrawal effects. The medical supervisor must be notified.

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In the case of a grade 4 fasting blood glucose (>50 mg/dl or 27.8 mmol/l), Compound A will be delayed when insulin therapy is initiated.

The medical supervisor must be notified. Treatment interruptions for >4 weeks will require removal of the subject from the study.

At the discretion of the investigator, daily home monitoring by taking blood from a finger (abstaining from food in the morning) can be initiated. Subjects will be provided with a glucometer and trained in how to test and record results in a diary to be reviewed at each clinic visit. They will also be instructed to immediately contact the study staff in the event of high fasting glucose (>160 mg/dL or 8.9 mmol/L), in which case prompt evaluation in the clinic is needed; or call the clinic and indicate when visiting the clinic if grade 3 or higher is present. In such cases, the opinion of the endocrinologist regarding adequate management of the subject is appropriate.

Glucophage and other biguanide therapy should be temporarily suspended during scheduled tumor radiological evaluations (eg, CT) that include an iodinated contrast agent. Goldberg, 2005; and Turina, 2006 offer resources for the management of hyperglycemia.

Appendix G. Management of Biological Specimens (Appendix to the Laboratory Manual).

Sample Handling and Storage: All blood and tissue samples collected for the biomarker expression analysis and genetic study that is part of this study that are not depleted after analysis will be stored for study use for up to 5 years after study completion. With the consent of the subject, the retention period will be extended to 20 years after study completion for use in future studies to learn more about cancer and other diseases. Samples will be stored in a secure laboratory facility designed for long-term storage of samples, with appropriate access control, monitoring and back-up systems.

Sample Coding: All samples for biomarker expression analysis and genetic testing will be identified by code only (subject identification number). No other personal information will be imprinted on these samples. The research doctor will keep the code key. Samples and code key will be kept confidential and separate. Researchers who perform analyzes on samples will only see the code and will not see any information that specifically identifies the subject.

Testing of Blood and Tissue Samples: Samples for biomarker expression analysis and genetic testing will be tested by the sponsor or companies employed by the sponsor to determine the effects of Compound A on the subject and the subject's cancer. This includes determining whether biomarkers in blood cells or tumor cells demonstrate that Compound A is biologically active. In addition, DNA samples from whole blood and tumor tissue will be analyzed for genetic changes that may correlate with the subject's response to the drug.

Reporting and Availability of Results of Biomarker Expression Analysis and Genetic Testing: Results of biomarker expression testing and genetic testing of samples will not be shared with the subject, insurance companies, or other third parties not involved in the sample analysis described above. The results are not entered into the subject's medical records. The results are for research purposes only and will not be used to make decisions about the subject's daily medical care.

Subject names and identifiers will not be mentioned in publications or reports, which minimizes the possibility of psychological or social risks that may arise from knowledge of the results of biomarker expression analysis and genetic research, such as employment or insurance risk or discrimination risk.

Mechanism for requesting destruction of a sample after withdrawal of informed consent: If subjects withdraw their informed consent to participate in a study, they may additionally request the destruction of their samples for biomarker expression analysis and genetic testing. In such cases, the subject will inform the investigator that informed consent has been withdrawn and ask that any stored, unused specimens be destroyed. All unused samples will then be destroyed by the sponsor. If the samples were analyzed before the informed consent was withdrawn, in this case the sponsor may use the data already available.

If subjects have agreed to store specimens for biomarker expression and genetic studies for 20 years for future research, they may also revoke this decision at any time. The subject will inform the investigating physician that the authorization has been withdrawn for specimens intended to be used for future studies. All unused samples will then be destroyed by the sponsor. If the samples were analyzed before the consent was withdrawn, then the sponsor may use the data already available.

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Appendix H. Characteristics of the Bayesian logistic regression model.

An adaptive Bayesian logistic regression model (BLRM, Neuenschwander, et al., 2008) for dose escalation with overdose control (EWOC, Babb et al., 1988) can be used to manage dose escalation in this study.

This annex provides performance measures (performance) that illustrate the accuracy of the design in relation to MTD estimation under various dose-toxicity relationships through computer simulations. In addition, recommendations for the next dose level of BLRM with an overdose control approach are provided under various hypothetical outcome scenarios in the early cohorts (assuming three evaluable patients in each cohort for simplicity) to show how this facilitates dose escalation decisions in research.

With respect to the specifications and results of the simulation study, performance characteristics can be considered that illustrate the accuracy of the design in relation to the MTD estimate under various hypothesized true dose-toxicity relationships. The simulation (see Fig. 6) is performed for BLRM in five true dose-DLT scenarios:

a) the dose-DLT relationship is steep and the MTD is achieved at an early dose level (SE);

b) the dose-DLT relationship is a steep curve and the MTD is achieved at the average dose level (SM);

c) the dose-DLT relationship is steep and the MTD is achieved at the late dose level (SL);

d) the dose-DLT relationship is flat and the MTD is achieved at the average dose level (FM);

e) the dose-DLT relationship is flat and the MTD is achieved at the late dose level (FL).

Table 10 _________________P(DLT) for 5 simulated scenarios_________________

P(DLT) at different dose levels (mg) Scenario 15 thirty 60 90 120 150 180 200 SE 0.0879 0.1647 0.2874 0.3800 0.4520 0.5094 0.5563 0.5829 SM 0.0015 0.008 0.0418 0.1045 0.1901 0.2874 0.3857 0.4478 SL 0.000 0.0004 0.0049 0.0224 0.0635 0.1362 0.2389 0.3184 FM 0.0295 0.0677 0.1477 0.2239 0.2928 0.3539 0.4079 0.4402 FL 0.0026 0.0106 0.0428 0.0935 0.1574 0.2284 0.3013 0.349

Performance is analyzed to examine the overall performance of the BLRM under each true scenario. In table. 11 summarizes the results of the conducted simulations

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Table 11

Summary of simulation scores for BLRM and comparison with 3+3

Scenario/ Way Average number of subjects Share of subjects with DLT Dose recommendation probability with true P(DLT) value 0.16-0.33 >0.33 <0.16 SE, N-CRM 20.14 0.23 0.73 0.13 0.15 SE, 3+3 15.35 0.23 0.60 0.11 0.29 SM, N-CRM 24.20 0.13 0.68 0.10 0.22 SM, 3+3 23.76 0.13 0.53 0.12 0.35 SL, N-CRM 26.33 0.10 0.53 0.00 0.47 SL, 3+3 26.81 0.09 0.51 0.00 0.49 FM, N-CRM 22.90 0.16 0.51 0.11 0.38 FM, 3+3 20.26 0.17 0.39 0.12 0.49 FL, N-CRM 25.13 0.12 0.48 0.14 0.47 FL, 3+3 24.77 0.12 0.42 0.11 0.53

In general, the BLRM model with the established prior probability performs satisfactorily. With a similar or slightly larger sample size, the BLRM may be more likely to select an MTD in the target range, especially for scenarios a, b, and d.

With respect to hypothetical dose escalation scenarios in early cohorts, in addition to the general performance characteristics discussed above, design should make reasonable decisions during the study based on observed toxicity. After completion of a given cohort, the decision on dose escalation and the actual dose selected for the subsequent cohort will depend on the recommendation of the BLRM with EWOC model and the medical review of available clinical and laboratory data.

Some scenarios illustrating dose escalation to the third dose cohort are listed in Table 1. 12 using a 2-parameter BLRM model. It is assumed that there are at least three evaluable patients in each cohort. If any patient experiences DLT, the dose increase will be no more than 50% for any subsequent dose escalation. The BLRM model performs satisfactorily for hypothetical dose escalation scenarios.

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Table 12

Likely scenarios up to the third cohort, with three patients per cohort

Scenario Dosing history (mg) #DLT/#Patient Next dose by N-CRM(Mr) 1 15 0/3 thirty 15 0/3 thirty 2 thirty 0/3 60 15 0/3 thirty 3 thirty 1/3 thirty 15 0/3 thirty 4 thirty 2/3 15 15 0/3 thirty 5 thirty 0/3 60 60 0/3 90 15 0/3 thirty 6 thirty 0/3 60 60 1/3 60

A Bayesian logistic regression model allows preclinical information to be included as well as updating the recommended dose based on all safety data in the study. When analyzing the metrics presented in the table, it can be seen that the model is not sensitive to different truth scenarios. Overall, this model is conservative due to overdose control criteria. In all scenarios, the probability of recommending a dose with a true P(DLT) is >33%, since the MTD is much lower than the probabilities of recommending a dose with a true P(DLT) between 16 and 33%, as is the MTD.

Recommendations during the study period based on this model are consistent with the clinical decision-making process and should be considered in conjunction with other available clinical information by the Sponsor Clinical Trial Team and Study Investigators when deciding on dose levels. to be tested to determine the MTD.

Example 12 Synergistic effects of Compound A and the histone deacetylase (HDAC) inhibitor, romidepsin, in the PA0165 mouse pancreas xenograft model.

The bromodomain-containing protein BRD4 of the BET family is involved in the regulation of metabolic pathways in the pancreas. BRD4 expression is significantly increased in pancreatic ductal adenocarcinoma cell lines compared to expression in human pancreatic ductal epithelial cells. In addition, studies have shown that BRD4 promotes the proliferation of pancreatic ductal adenocarcinoma cells and increases resistance to certain chemotherapeutic agents such as gemcitabine. Therefore, inhibition of BRD4 is promising in terms of the treatment of pancreatic cancer. As a result, an in vivo experiment was performed to understand whether Compound A-mediated inhibition of BRD4 could sensitize pancreatic tumor cells to treatment with the HDAC inhibitor, romidepsin.

Cohorts of 4-6 week old NSG mice bearing PA0165 were treated with intravenous (IV) x3 Q4D 1.5 mg/kg romidepsin; oral administration of 25 mg/kg of Compound A according to the scheme QD 3 days of admission, then 4 days of drug holidays; or a combination of Compound A at 25 mg/kg, orally, on a QD schedule for 3 days on, followed by a 4-day drug holiday, and romidepsin at a dose of 1.5 or 0.75 mg/kg, IV, on a Q7D schedule. Treatment continued for 21 days. Significant inhibition of tumor growth as measured by tumor volumes was observed for all treatment groups (FIG. 8). Taken alone, romidepsin produced a significant tumor growth inhibition (TGI) of 45%. Taken alone, Compound A caused a significant tumor growth inhibition (TGI) of 38%. The combination of Compound A and romidepsin showed synergy and significantly outperformed all other regimens in terms of TGI (68% with Compound A in combination with romidepsin 1.5 mg/kg, 65% with Compound A in combination with 0.75 mg /kg romidepsin). All treatment groups experienced significant weight loss between measurements on day 10 and day 15 and then regained. The treatment groups receiving Compound A alone or the combination showed a significantly higher survival rate than the treatment group receiving romidepsin alone (FIG. 9). At day 30 after initial treatment, the survival rate for the romidepsin-only treatment group was about 10%. In contrast, the survival rates for treatment groups receiving Compound A alone or the combination were about 70%. There was no significant difference in survival between treatment groups treated with Compound A and the combination alone.

Example 13 Synergistic effects of Compound A and the protein-bound paclitaxel, abraxane, in the mouse pancreas xenograft model PA0165.

The bromodomain-containing protein BRD4 of the BET family is involved in the regulation of metabolic pathways in the pancreas. BRD4 expression is significantly increased in pancreatic ductal adenocarcinoma cell lines compared to expression in human pancreatic ductal epithelial cells. In addition, studies have shown that BRD4 promotes the proliferation of pancreatic ductal adenocarcinoma cells and increases resistance to certain chemotherapeutic agents such as gemcitabine. Therefore, inhibition of BRD4 is promising in terms of the treatment of pancreatic cancer. As a result, an in vivo experiment was performed to understand whether Compound A-mediated inhibition of BRD4 could sensitize pancreatic tumor cells to treatment with the protein-bound paclitaxel, abraxane.

Cohorts of NSG mice bearing PA0165 were treated with abraxane by intravenous administration (IV) at a dose of 10 mg/kg according to schedule 3 Q4D; Compound A at 25 mg/kg, orally, QD regimen 3 days on, then 4 days off; or a combination of abraxane 10 mg/kg, IV, Q7D, and Compound A 25 or 12.5 mg/kg, orally, on a QD regimen of 3 days on, followed by a 4 day drug holiday. Treatment continued for 21 days. Significant tumor growth inhibition as measured by tumor volumes was observed for all treatment groups (FIG. 10). Taken alone, abraxane produced a significant tumor growth inhibition (TGI) of 55%. Taken alone, Compound A caused a significant tumor growth inhibition (TGI) of 49.3%. The combination of Compound A and abraxane showed synergy and significantly outperformed all other regimens in terms of TGI (78.1% for abraxane in combination with 25 mg/kg of Compound A; 79.1% for abraxane in combination with 12.5 mg/kg Compound A). Moderate weight loss was observed during part of the study in all groups; body weight loss was observed in mice bearing larger tumors. Treatment groups receiving the combination showed significantly higher survival rates compared to individual treatment groups (FIG. 11). On day 41 after initial treatment, the survival rate for the treatment group treated with abraxane alone was 0% and for the treatment group treated with Compound A was about 20%. In contrast, the survival rate for the combination groups was about 60% when treated with abraxane in combination with 25 mg/kg of Compound A and 70% when treated with abraxane in combination with 12.5 mg/kg of Compound A, respectively.

Claims (5)

CLAIM 1. A method of treating glioblastoma, comprising administering to a human patient a therapeutically effective amount of at least one inhibitor of bromodomain and extraterminal protein (BET) and temozolomide, wherein the BET inhibitor is 4-[2-(cyclopropylmethylamino)-5methylsulfonylphenyl]-2-methylisoquinoline -1-one or a pharmaceutically acceptable salt thereof. 2. The method of claim 1, wherein administration of the BET inhibitor and temozolomide results in a synergistic reduction in cell proliferation in the patient's tumor or a synergistic increase in apoptosis in the patient's tumor compared to either the BET inhibitor or temozolomide alone. 3. The method of claim 1, wherein the therapeutically effective amount for both the BET inhibitor and temozolomide when administered together is at least 50% lower than when the BET inhibitor and temozolomide are administered alone. 4. The method according to claim 1, characterized in that the glioblastoma is resistant to temozolomide. 5. The method according to claim 1, characterized in that the BET inhibitor is present in an amount that enhances the therapeutic effect of temozolomide.