Compositions and methods for pretreatment of cancer

Abstract

The invention relates to a pharmaceutical composition for oral administration. The pharmaceutical composition comprises: a) immediate release particles comprising i. An active ingredient selected from the group consisting of valproic acid, semi-sodium valproate, sodium valproate and magnesium valproate, and ii. A filler, b) sustained release pellets comprising: i. A pellet core comprising (1) an active ingredient selected from the group consisting of valproic acid, semi-sodium valproate, sodium valproate and magnesium valproate, and (2) a filler, ii. A sub-coating disposed on the pellet core, the content of the sub-coating layer is 10-20 wt% based on the weight of the pellet core and the sub-coating layer comprises a film-forming agent, and iii. A slow-release coating layer disposed on the sub-coating layer, the content of the slow-release coating layer is 25-100 wt% based on the weight of the pellet core coated with the sub-coating layer and the slow-release coating layer comprises a film-forming agent, and the content of the slow-release coating layer is 25-100 wt% based on the weight of the pellet core coated with the sub-coating layer. Wherein the amount of the active ingredients in the quick-release particles accounts for 70-80 wt% of the total weight of the active ingredients in the pharmaceutical composition, and the amount of the active ingredients in the slow-release pellets accounts for 20-30 wt% of the total weight of the active ingredients in the pharmaceutical composition. In further aspects, the present invention relates to methods of preparing pharmaceutical compositions and pharmaceutical compositions for use in methods of pre-treating cancer.

Description

Technical Field

[0001] This invention relates to the field of compositions and methods for pretreatment of cancer, namely, applying one or more compositions to an individual requiring cancer treatment as a pretreatment prior to other treatments to enhance the effectiveness of the treatment or reduce its side effects. Background Technology

[0002] Cancer can be defined as the abnormal growth of cells, exhibiting signs of uncontrolled proliferation and disordered programmed cell death. From a classical perspective, a series of genetic events leads to malignant transformation, causing cell clones to disregard the integrity of other cells and tissues, and potentially eventually metastasize. Cancer can affect any tissue in the body, and there are many different forms in each region. Malignant lymphoma can be defined as the malignant transformation of lymphocytes in the hematopoietic system. Lymphomas can be divided into aggressive lymphomas and indolent lymphomas. Aggressive lymphomas are characterized by a rapid growth pattern and may have striking clinical features. However, aggressive lymphomas can be completely cured with chemotherapy, radiotherapy, and monoclonal antibody therapy. In contrast, indolent lymphomas (such as follicular lymphomas) have a slow growth pattern and generally have a milder clinical presentation. However, although indolent lymphomas cannot be completely cured with standard lymphoma treatment, they can sometimes be cured with allogeneic stem cell transplantation. The median survival for patients with follicular lymphoma is 8–10 years. Diffuse large B-cell lymphoma and Hodgkin's lymphoma are aggressive lymphomas, while follicular lymphoma and chronic lymphocytic leukemia are indolent lymphomas. Myeloma is composed of malignantly transformed plasma cells. They are associated with indolent lymphomas but are generally considered a separate entity. The prognosis is pessimistic, with a median survival of 5-7 years. One of the most common subtypes of malignant lymphoma is diffuse large B-cell lymphoma (DLBCL), with an incidence of approximately 500 cases per year in Sweden. DLBCL accounts for 60-70% of aggressive lymphomas. The median age at diagnosis is 70 years, and the incidence of DLBCL is slightly higher in men than in women. The standard first-line treatment for DLBCL is chemotherapy, including combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). In recent years, the addition of the CD20 antibody rituximab has become the international clinical standard (R-CHOP), thereby improving progression-free, event-free, disease-free, and overall survival rates (Morrison, Expert Rev Anticancer Ther, 2008; 8(10): pp. 1651-1658). Nevertheless, since up to 45% of patients die from the disease, there is a clear clinical need to improve progression-free survival in DLBCL patients.

[0003] A crucial area of ​​cancer research is the regulation of DNA transcription. This is a complex process, and the mechanisms involved are only partially understood. Histone deacetylases (HDACs) can regulate the expression of tumor suppressor genes and the activity of transcription factors involved in cancer development and progression. HDACs function by altering the structural components of DNA or chromatin through histone deacetylation, thereby affecting the three-dimensional conformation of DNA (epigenetic modifications) without altering or disrupting its sequence. It has also been suggested that they may modify sensitivity to DNA-damaging chemotherapy by regulating chromatin structure. Following these lines of thought, several in vitro studies have shown that inhibiting HDACs can synergize with chemotherapy. Therefore, many HDAC inhibitors have been developed in recent years. They can be divided into four classes: hydroxamic acid / carbamic acid, cyclic peptides, fatty acids, and benzamides. Examples of HDAC inhibitors that have been approved for the treatment of cancer include vonosta and romidipexole, which have been approved by the FDA (Food and Drug Administration) for the treatment of cutaneous T-cell lymphoma and are currently being evaluated for the treatment of other malignancies.

[0004] The most well-known HDAC inhibitor in clinical practice is the anticonvulsant valproic acid, which has been used to treat epilepsy since the 1970s. Valproic acid belongs to the fatty acid inhibitor class.

[0005] The inventors have previously demonstrated that combining HDAC inhibitors with steroids as a pretreatment for patients requiring cancer treatment is useful, resulting in enhanced treatment efficacy, see WO2012 / 128709.

[0006] Despite the promise of more effective cancer treatments made possible by the use of HDAC inhibitors as taught in WO2012 / 128709, further development is still needed.

[0007] One challenge is how to effectively administer HDAC inhibitors to meet the requirements of HDAC inhibitor pretreatment. One HDAC inhibitor, valproic acid, has been used clinically since the 1970s, but this clinical use has been associated with a different medical field—the treatment of epilepsy—and not with the emerging field of cancer pretreatment. Therefore, when administering HDAC inhibitors (such as valproic acid or its derivatives) as pretreatment for cancer, earlier methods of administering HDAC inhibitors (such as valproic acid and its derivatives) and the subsequent compositions used are not necessarily the most effective.

[0008] Therefore, the object of the present invention is to provide a pharmaceutical composition that provides an effective pretreatment for cancer.

[0009] Another object of the present invention is to provide a pharmaceutical composition that can be produced in a simple and effective manner.

[0010] Another object of the present invention is to provide a method for preparing a pharmaceutical composition.

[0011] Another object of the present invention is to provide a pharmaceutical composition for a method of pretreatment of cancer.

[0012] Another object of the present invention is to provide a method for pretreating cancer by administering a pharmaceutical composition. Summary of the Invention

[0013] According to corresponding first and second aspects of the present invention, at least one of the above-mentioned objectives is achieved by an orally administered pharmaceutical composition and a method for preparing a pharmaceutical composition according to the first aspect, said pharmaceutical composition comprising: a) Immediate-release granules, which contain: i. Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and ii. Filler, b) Sustained-release pellets, which contain: i. Pellet core, comprising: (1) Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and (2) Filler, ii. A sub-coating disposed on the pellet core, wherein the sub-coating content is 10-20 wt% based on the weight of the pellet core, and the sub-coating comprises a film-forming agent, and iii. A slow-release coating disposed on the sub-coating, wherein the content of the slow-release coating is 25-100 wt% based on the weight of the pellet core coated with the sub-coating, and the slow-release coating contains a film-forming agent. The amount of active ingredient in the immediate-release granules accounts for 70-80 wt% of the total weight of active ingredients in the pharmaceutical composition, and the amount of active ingredient in the sustained-release pellets accounts for 20-30 wt% of the total weight of active ingredients in the pharmaceutical composition. The film-forming agent of the sustained-release coating is different from the film-forming agent of the sub-coating, and The method includes the following steps: i. Production of immediate-release granules ii. Production of pellet cores, iii. Apply a sub-coating to the pellet core by performing the following sub-steps: a. Suspend or dissolve the sub-coating in an aqueous medium to form an aqueous solution or dispersion of the sub-coating. b. Contact the pellet core with an aqueous solution or dispersion of the sub-coating to prepare pellet cores with a sub-coating. iv. Further coat the pellet core with a sub-coating using a slow-release coating by performing the following sub-steps: a. Suspending or dissolving the slow-release coating in an aqueous medium to form an aqueous solution or dispersion of the slow-release coating, and b. Contact the pellet core coated with a sub-coating with an aqueous solution or dispersion of a sustained-release coating to prepare sustained-release pellets.

[0014] Therefore, the present invention is based on the discovery that when a sub-coating is applied to the pellet core, it ensures that the subsequently applied sustained-release coating (which is advantageously applied using an aqueous solution or dispersion) does not interfere with the structure of the pellet core, and thus does not cause the active ingredient to be accidentally released from the sustained-release pellet.

[0015] In other words, as shown in Example 1, applying the sustained-release coating directly to the pellet core may result in an unexpected release rate of the active ingredient. As discovered, this unexpected release rate can be attributed to the aqueous solution or dispersion used to apply the sustained-release coating. Specifically, the high solubility of the API and the swelling properties of the MCC, combined with the porosity of the sustained-release coating, are found to allow water to permeate the coating into the pellet core. This causes the core to swell and rupture, thereby providing a rapid release of all active ingredients.

[0016] The inventors solved the problem of unintended release rates by applying a sub-coating to the pellet core before applying the sustained-release coating. Therefore, the sub-coating appears to protect the pellet core from aqueous solutions or dispersions, allowing the sustained-release coating to be applied using either aqueous solutions or dispersions.

[0017] Furthermore, as shown in Example 2, the pharmaceutical composition according to the first aspect of the present invention has several advantages of known compositions. These advantages include the maximum plasma concentration (C0) of the active ingredient. max The C4 concentration was lower, with smaller differences in plasma concentration distribution when administered under fed and fasted conditions, resulting in a lower risk of active ingredient accumulation. Even with lower frequency of administration of the drug composition, such as twice daily, more stable exposure to the maximum concentration of the active ingredient was maintained. This differs from the reference composition, which appears to produce higher C4 concentrations when taken twice daily at the corresponding dose. max .

[0018] Finally, as shown in Example 3, the pharmaceutical composition according to the first aspect of the invention provides a higher degree of histone deacetylation inhibition than known compositions.

[0019] A third aspect of the invention relates to a pharmaceutical composition according to a first aspect of the invention for a method of pretreating cancer, wherein the pharmaceutical composition is administered to a person suffering from cancer. Attached Figure Description

[0020] Figure 1A ,1B 1C shows the dissolution results of sustained-release pellets containing different amounts of sub-coating, sustained-release coating, pore-forming agent and drying method.

[0021] Figure 1D The dissolution profile of the finished pharmaceutical composition is shown.

[0022] Figure 2A The relationship between the mean (± standard deviation) plasma concentration of total valproate (µM) and nominal time (h) is shown. Figure 2B Displayed in the form of a semi-logarithmic graph Figure 2A The data in Figure 3A The mean (± standard deviation) plasma concentration (µM) of free (unbound) valproate is shown as a function of nominal time (h). Figure 3B Displayed in the form of a semi-logarithmic graph Figure 3A The data in Figure 4A The mean (± standard deviation) plasma concentration of total sodium valproate (µM) after twice-daily administration of 30 mg sodium valproate / kg body weight is shown as a function of nominal time (h). Figure 4B The mean (± standard deviation) plasma concentration of free sodium valproate (µM) administered three times daily at 20 mg / kg body weight is shown as a function of nominal time (h). Figure 5 Box plot showing lysine 9-acetylation of lysine H3 (acH3K9) in peripheral blood mononuclear cells (PBMCs) compared to baseline expression. Detailed Implementation

[0023] Corresponding first and second aspects of the present invention relate to orally administered pharmaceutical compositions and methods for preparing pharmaceutical compositions according to the first aspect, said pharmaceutical compositions comprising: a) Immediate-release granules, which contain: i. Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and ii. Filler, b) Sustained-release pellets, which contain: i. Pellet core, comprising: (1) Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and (2) Filler, ii. A sub-coating disposed on the pellet core, wherein the sub-coating content is 10-20 wt% based on the weight of the pellet core, and the sub-coating comprises a film-forming agent, and iii. A slow-release coating disposed on the sub-coating, wherein the content of the slow-release coating is 25-100 wt% based on the weight of the pellet core coated with the sub-coating, and the slow-release coating contains a film-forming agent. The amount of active ingredient in the immediate-release granules accounts for 70-80 wt% of the total weight of active ingredients in the pharmaceutical composition, and the amount of active ingredient in the sustained-release pellets accounts for 20-30 wt% of the total weight of active ingredients in the pharmaceutical composition. The film-forming agent of the sustained-release coating is different from the film-forming agent of the sub-coating, and The method includes the following steps: i. Production of immediate-release granules ii. Production of pellet cores, iii. Apply a sub-coating to the pellet core by performing the following sub-steps: a. Suspend or dissolve the sub-coating in an aqueous medium to form an aqueous solution or dispersion of the sub-coating. b. Contact the pellet core with an aqueous solution or dispersion of the sub-coating to prepare pellet cores with a sub-coating. iv. Further coat the pellet core with a sub-coating using a slow-release coating by performing the following sub-steps: a. Suspending or dissolving the slow-release coating in an aqueous medium to form an aqueous solution or dispersion of the slow-release coating, and b. Contact the pellet core coated with a sub-coating with an aqueous solution or dispersion of a sustained-release coating to prepare sustained-release pellets.

[0024] This pharmaceutical composition is intended for oral administration, meaning it is suitable for oral administration. In other words, the pharmaceutical composition is formulated for oral administration by a subject. The pharmaceutical composition can be packaged to be suitable for oral administration, allowing the subject to swallow or ingest immediate-release granules and sustained-release pellets. The pharmaceutical composition may include capsules containing immediate-release granules and sustained-release pellets. The capsules may be swallowable and may be made of a material that corrodes in the stomach or intestines to release the immediate-release granules and sustained-release pellets. As an alternative to capsules, the pharmaceutical composition may include sachets or pouches containing immediate-release granules and sustained-release pellets. Sachets or pouches may be made of a material that corrodes in the stomach or intestines to release the immediate-release granules and sustained-release pellets. Alternatively, the capsules, sachets, or pouches may be opened to release the immediate-release granules and sustained-release pellets, which may then be ingested directly.

[0025] Immediate-release granules and sustained-release pellets may alternatively be packaged in individual capsules, sachets, or pouches, each of which may be swallowed or ingested, or opened to release the immediate-release granules or sustained-release pellets, as described above.

[0026] The pharmaceutical composition is suitable for pretreatment of cancer, wherein the cancer is preferably selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, T-cell lymphoma, myeloma and Hodgkin's lymphoma.

[0027] Immediate-release granules are granules formulated to release the active ingredient immediately or at least shortly after ingestion. This provides a rapid or sudden release of the active ingredient and, consequently, rapid absorption of the active ingredient into the subject's bloodstream. This reduces the time required for the active ingredient to reach an effective concentration in the subject's blood after administration of the pharmaceutical composition.

[0028] Preferably, the immediate-release granules release at least 75 wt%, for example at least 80 wt%, preferably at least 95 wt%, such as 100 wt%, of the active ingredient contained therein within 15 minutes in a pH 1 medium.

[0029] The release of the active ingredient in pH 1 medium was tested as follows: Immediate-release granules were placed in a basket, with 0.1N HCl medium placed beneath the basket. Samples were taken at 5, 15, 30, 45, and 60 minutes to determine the amount of active ingredient released from the immediate-release granules. During the test, the medium was stirred using a paddle or by rotating the basket. The stirring speed (paddle or basket) was 100 rpm.

[0030] The number of immediate-release particles in a pharmaceutical composition depends on the amount of active ingredient in each particle, the total amount of active ingredients in the pharmaceutical composition, and the ratio of the amount of active ingredient in the immediate-release particles to the amount of active ingredient in the sustained-release pellets. Typically, a pharmaceutical composition contains multiple immediate-release particles. Immediate-release particles are usually not spherical.

[0031] The active ingredients are selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate.

[0032] Valproic acid is a histone deacetylation inhibitor, i.e., an HDAC inhibitor. As shown in WO2012 / 128709, HDAC inhibitors (especially in combination with steroids) are useful as a pretreatment for subjects requiring cancer treatment prior to other therapies.

[0033] Chemically, valproic acid (VPA) is a weak organic acid. Its conjugate base is valproate. The sodium salt of this acid is sodium valproate, and the magnesium salt is magnesium valproate. The coordination complex of valproic acid and sodium valproate is called sodium valproate hemisodium salt. The latter is marketed under various brand names such as Depakote, Depakote ER, Depaakene, Depacon, Depaakine, Valparin, Stavzor, ​​and Ergenyl. Sodium valproate is marketed in Sweden under the names Absenor, Depaakine, and Orfiril. Valproic acid is marketed in Sweden under the names Ergenyl and Depaakine.

[0034] The purpose of a filler is to provide a matrix to which the active ingredient can be mixed. The filler further helps to provide a compressible substance, thereby enabling the production of immediate-release granules. The filler may, for example, comprise or consist of cellulose, such as preferably microcrystalline cellulose and / or calcium phosphate.

[0035] Sustained-release pellets are granules formulated to release active ingredients over a sustained period of time. Preferably, at pH 6.8, the release rate of the active ingredient contained in the sustained-release pellet is 95-100% within 8 hours, preferably 100%. Furthermore, the sustained-release pellets preferably release 55-65% of their active ingredients, more preferably 60%, within 3 hours at pH 6.8.

[0036] In addition, the sustained-release pellets preferably release 25-35% of their active ingredients, more preferably 30%, within 2 hours.

[0037] The release of the active ingredient in pH 6.8 medium was tested as follows: 100 mg of sustained-release pellets were placed in a basket, which was then immersed in 0.1 N HCl medium for 15 minutes before removal. A container containing pH 1 medium was cleaned and filled with a pH 6.8 phosphate buffer solution equilibrated at 37°C. Samples were taken at 30 minutes, 1, 2, 3, 5, 8, and 12 hours. During the test, the medium was stirred using a paddle or by rotating the basket. At both stages of the test, the stirring speed (paddle or basket) was 100 rpm, and finally, the basket was rotated indefinitely at 250 rpm for 15 minutes to conclude the test. The term "sustained" should be understood to include the term "extended". In the context of this invention, the terms granules and pellets are used to distinguish between the immediate-release portion and the sustained-release portion of a pharmaceutical composition. Therefore, sustained-release pellets can have the same size and shape as granules and can be formed by the same process. However, generally, immediate-release granules are smaller than sustained-release pellets. Furthermore, immediate-release granules may not be as spherical as sustained-release pellets. Additionally, immediate-release granules can be formed by processes other than those used for sustained-release pellets.

[0038] The pellet core is the innermost layer of a sustained-release pellet. The pellet core is formed from the active ingredient and filler, and therefore typically allows for rapid or immediate release of the active ingredient. The pellet core becomes a sustained-release pellet primarily through the application of a sustained-release coating, as the coating slows the release of the active ingredient from the pellet core.

[0039] As described above, the active ingredient in the pellet core is selected from the same active ingredient found in the immediate-release granules. Preferably, the active ingredient is the same in the pellet core of both the immediate-release and sustained-release granules, but the active ingredient may also be different. Furthermore, the active ingredient may include a mixture of valproic acid in the listed forms, and the pellet core of both the immediate-release and sustained-release granules may include different active ingredients or different mixtures of active ingredients.

[0040] However, preferably, the active ingredient in the core of both immediate-release granules and sustained-release pellets is sodium valproate.

[0041] Similar to the fillers in immediate-release granules, the purpose of the fillers in pellet cores is to provide a matrix that can be mixed with the active ingredients. The fillers further contribute to providing a quality suitable for processing, thereby enabling the production of pellet cores. The fillers may, for example, comprise or consist of cellulose, such as preferably microcrystalline cellulose and / or calcium phosphate.

[0042] A sub-coating is applied to the pellet core. In other words, the surface of the pellet core is at least partially, and preferably completely, covered by the sub-coating. Therefore, the sub-coating is applied between the surface of the pellet core and the sustained-release coating. As shown in the embodiments, the purpose of this sub-coating is to ensure that the sustained-release coating can be applied using an aqueous solution or dispersion. It is not desirable to be bound by theory, but when the sustained-release coating is applied by contacting the pellet core with an aqueous solution or dispersion containing the sustained-release coating, the presence of the sub-coating appears to prevent over-wetting, swelling, and breakage of the pellet core, and subsequently, the release of the active ingredient. Therefore, the sub-coating is specifically configured to limit or reduce the wetting of the pellet core during the application of the sustained-release coating.

[0043] Therefore, a sub-coating can be used to apply a sustained-release coating with an aqueous solution or dispersion. Another effect obtained by the sub-coating is to smooth the surface of the pellet core. This also helps to ensure a good coating of the sustained-release coating onto the pellet core, as the sustained-release coating has a smoother surface to adhere to. Furthermore, the smoother surface provided by the sub-coating allows the sustained-release coating to be applied with a more uniform layer thickness, which further improves the uniformity of the sustained-release coating and the release rate of the sustained-release pellets. The sub-coating content is 10-20 wt% based on the weight of the pellet core. It has been found that this range provides the necessary protection against wetting of the pellet core while maintaining a sufficiently low total sub-coating content so as not to unnecessarily reduce the content of the active ingredient in the pellet core and / or unnecessarily increase the size of the sustained-release pellets. The film-forming agent can be any reagent that forms a film or coating on the pellet core when mixed with an aqueous solution and in contact with the pellet core. Film-forming agents can include, for example, cellulose, such as hydroxypropyl cellulose and / or hydroxypropyl methylcellulose. Preferably, the sub-coating and / or film-forming agent are more or less hydrophobic. Increased hydrophobicity can be achieved by increasing the number of aliphatic groups in the film-forming agent, i.e., by replacing the polar hydroxyl groups of cellulose with nonpolar substituents, as shown by the increased number of aliphatic groups in hydroxypropyl cellulose and hydroxypropyl methylcellulose compared to cellulose. Alternatively, increased hydrophobicity can be achieved by providing hydrophobic compounds or substances, such as minerals like talc (magnesium silicate).

[0044] Preferably, the subcoating comprises hydroxypropyl cellulose and / or hydroxypropyl methylcellulose as a film-forming agent. Preferably, the subcoating consists of one or more film-forming agents and one or more hydrophobic compounds or substances.

[0045] A sustained-release coating is provided on the sub-coating. This means that the sustained-release coating at least partially, and preferably completely, covers the sub-coating, thereby covering the pellet core. The purpose of the sustained-release coating is to slowly erode in the small intestine, allowing the active ingredient to be slowly released from the sustained-release pellet. This ensures the stable and slow release of the active ingredient, thus allowing for stable and slow absorption of the active ingredient into the subject's bloodstream. This helps maintain the concentration of the active ingredient in the blood at or above the effective concentration between administrations of the pharmaceutical composition.

[0046] Based on the weight of the pellet core coated with the sub-coating, the content of the sustained-release coating is 25-100 wt%. This range has been found to provide the desired release profile for the sustained-release pellets. The film-forming agent can be any reagent that, when mixed with an aqueous solution and contacted with the pellet core coated with the sub-coating, forms a film or coating on the sub-coating. The film-forming agent can, for example, include cellulose, such as ethyl cellulose. The film-forming agent of the sustained-release coating differs from the film-forming agent of the sub-coating. Preferably, the film-forming agent in the sustained-release coating is more hydrophobic, i.e., has a higher content of aliphatic groups compared to the film-forming agent in the sub-coating. For example, the sustained-release coating may preferably contain ethyl cellulose, which has a higher aliphatic group content compared to the preferred hydroxypropyl cellulose and hydroxypropyl methyl cellulose in the sub-coating. It is specified that the film-forming agent of the sustained-release coating differs from the film-forming agent of the sub-coating. In other words, the sub-coating is chemically different from the sustained-release coating, for example, containing different compounds. Further, in other words, the sub-coating is not a sustained-release coating, such as the first sustained-release coating applied to the pellet core. In other words, the sustained-release coating is not a sub-coating, such as a second sub-coating applied on top of a first sub-coating applied to the pellet core. Furthermore, the sub-coating and the sustained-release coating are neither two sub-coatings nor two sustained-release coatings.

[0047] The sustained-release coating may also contain 0-10%, preferably 0-5%, more preferably 1-4%, for example 2-3% of a pore-forming agent. By providing a pore-forming agent in the sustained-release coating, the release rate of the active ingredient from the sustained-release pellets can be further modulated.

[0048] A pore-forming agent is a more hydrophilic reagent than the film-forming agent in a slow-release coating. The pore-forming agent is dispersed in the slow-release coating, and when the slow-release coating comes into contact with an aqueous medium, pores are formed on the slow-release coating due to its higher hydrophilicity.

[0049] Preferably, the sustained-release coating comprises ethyl cellulose as a film-forming agent. Preferably, the sustained-release coating consists of one or more film-forming agents and optionally one or more pore-forming agents.

[0050] The active ingredient in immediate-release granules accounts for 70-80 wt% of the total weight of the active ingredients in the pharmaceutical composition, while the active ingredient in sustained-release pellets accounts for 30-20 wt% of the total weight of the active ingredients in the pharmaceutical composition. The sum of these percentages is 100 wt%. In other words, 70-80 wt% of the total weight of the active ingredients in the pharmaceutical composition is present in the immediate-release granules, and the remaining active ingredients are present in the sustained-release pellets.

[0051] The inventors have discovered that distributing the active ingredient into immediate-release granules and sustained-release pellets provides a good balance between rapidly achieving an effective concentration of the active ingredient in the bloodstream and maintaining the concentration of the active ingredient at or above the effective concentration between the administration of the pharmaceutical composition.

[0052] Step i in producing immediate-release granules may include a sub-step of mixing the active ingredient with a filler, followed by a sub-step of compacting the mixture to form granules. Compaction may preferably be performed by hammering and milling, but more preferably by rolling. The resulting granules may be further dried.

[0053] Step ii. of producing pellet cores may include a sub-step of mixing the active ingredient with a filler, followed by a sub-step of compacting the mixture to form pellets. The pellets may be produced, particularly, by extrusion and spheronization. The resulting pellets may preferably be dried. Preferably, the pellets are produced and / or dried to a moisture content of less than 5%, preferably less than 3.5%.

[0054] Step iii involves coating the produced pellet core with a sub-coating. This coating step includes suspending or dissolving the sub-coating in an aqueous medium to form an aqueous solution or dispersion of the sub-coating, followed by a sub-step of contacting the pellet core with the aqueous solution or dispersion of the sub-coating. Contact can be achieved by mixing the pellet core with the aqueous solution or dispersion of the sub-coating. A drying step can be performed after contact to dry the pellet core. The result of coating the pellet core with a sub-coating is a pellet core with a sub-coating. Step iv further involves coating the pellet core with a sustained-release coating. This step includes a sub-step of suspending or dissolving the sustained-release coating in an aqueous medium to form an aqueous solution or dispersion, and a sub-step of contacting the sub-coated pellet core with the aqueous solution or dispersion of the sustained-release coating to prepare sustained-release pellets. As described above, contact can be achieved by mixing the sub-coated pellet core with the aqueous solution or dispersion of the sustained-release coating. The aqueous medium preferably contains at least 90%, preferably at least 95%, more preferably 99-100% water. Typically, the aqueous solution or dispersion is an aqueous dispersion.

[0055] Following step iv, the sustained-release pellets are preferably dried to a moisture content of less than 5%, more preferably less than 3.5%. This can be carried out in a fluidized bed at 55-65°C, preferably 50-60°C, for 60-150 minutes, more preferably 80-150 minutes, and even more preferably 90-150 minutes. However, it is more preferable to dry the sustained-release pellets in an oven at 35-45°C, more preferably 40°C, for 18-30 hours, preferably 22-26 hours, such as 24 hours. Typically, sustained-release pellets do not require an enteric coating on top of the sustained-release coating. Further preferably, the sustained-release pellets are not re-wetted in an aqueous medium after production, as this may cause the active pharmaceutical ingredient to migrate through the sustained-release coating.

[0056] Preferably, the amount of active ingredient in the immediate-release granules accounts for 72-77 wt% of the total weight of active ingredients in the pharmaceutical composition, more preferably 75 wt%, and the amount of active ingredient in the sustained-release pellets accounts for 23-27 wt% of the total weight of active ingredients in the pharmaceutical composition, more preferably 25 wt%.

[0057] These ranges provide a shorter time between administration of the drug composition and the achievement of effective plasma concentrations. Furthermore, these ranges offer lower maximum plasma concentrations. Preferably, the maximum diameter of the immediate-release granules is 710-1000µm, and the maximum size of the sustained-release pellets is 1250-1700µm. Here, the maximum diameter corresponds to the maximum size of the immediate-release granules and the sustained-release pellets, such as length, width, and height.

[0058] This maximum diameter range helps particles pass through the stomach in a liquid manner, i.e., without being hindered by the activity of the pyloric sphincter.

[0059] Sieves can be used to produce immediate-release granules and sustained-release pellets with these maximum diameters. A first sieve with an opening corresponding to the upper limit of the range can be used to remove immediate-release granules and sustained-release pellets with a maximum diameter exceeding that limit, respectively. A second sieve with an opening corresponding to the lower limit of the range can be used to remove immediate-release granules and sustained-release pellets with a maximum diameter below that limit, respectively.

[0060] Preferably, based on the weight of the immediate-release granules, the content of active ingredients in the immediate-release granules is 75-95 wt%, more preferably 77-83 wt%, and based on the weight of the pellet core, the content of active ingredients in the pellet core is 35-45 wt%, more preferably 37-43 wt%.

[0061] This is advantageous because a high content of active ingredient reduces the amount of pharmaceutical composition that must be applied to achieve an effective concentration compared to other components such as fillers and other excipients.

[0062] Further investigation revealed that these ratios and ranges provide a good balance between maintaining high levels of active ingredients in the pharmaceutical composition and good compressibility and formability during the production of immediate-release granules and sustained-release pellets.

[0063] Preferably, the content of filler in the immediate-release particles is at least 5 wt% based on the weight of the particles, and the content of filler in the pellet core is at least 50 wt% based on the weight of the pellet core.

[0064] These filler values ​​are preferred because they help maintain good compatibility and formability during the production of immediate-release granules and sustained-release pellets.

[0065] Preferably, the filler in the immediate-release granules is dicalcium phosphate, and the filler in the pellet core is microcrystalline cellulose.

[0066] Dicalcium phosphate has been shown to be an effective filler in immediate-release granules, where the filler content is typically low. In contrast, microcrystalline cellulose has been shown to be an effective filler in pellet cores, where the filler content is high, and the wettability of microcrystalline cellulose may help control the release rate of the active ingredient during the dissolution of sustained-release pellets.

[0067] Preferably, based on the weight of the immediate-release particles, the immediate-release particles further contain 0.3-1 wt%, preferably 0.5 wt%, of lubricant, and based on the weight of the pellet core, the pellet core further contains 0.5-2 wt%, preferably 1 wt%, of lubricant.

[0068] Adding these amounts of lubricant further facilitates the production of immediate-release granules and sustained-release pellets.

[0069] In both cases, magnesium stearate is the preferred lubricant.

[0070] Preferably, based on the weight of the pellet core, the content of the sub-coating in the sustained-release pellet is 14-18 wt%, more preferably 15 wt%. This is advantageous because, when the sustained-release coating is applied, this amount of sub-coating achieves the purpose of preventing pellet core wetting, while being low enough not to interfere with the release rate determining function of the sustained-release coating. The referenced content corresponds to coating the pellet core until a corresponding weight increase is achieved.

[0071] Preferably, the sub-coating comprises, by weight, the following: i. 35-40 wt%, preferably 37.5 wt% hydroxypropyl cellulose, ii. 35-40 wt%, preferably 37.5 wt%, of hydroxypropyl methylcellulose, and iii. 20-30 wt%, preferably 25 wt% talc.

[0072] This composition has been shown to provide a sub-coating with good hydrophobic properties, partly due to the addition of talc (chemical formula Mg3Si4O). 10 The magnesium silicate hydrate (OH)2 provides this, which further protects the pellet core during the application of the sustained-release coating. In these embodiments, hydroxypropyl cellulose and hydroxypropyl methylcellulose are film-forming agents.

[0073] Preferably, the slow-release coating comprises, by weight, the slow-release coating: i. 97-100wt% film-forming agent, and ii. 0-3 wt% of a pore-forming agent comprising or consisting of hydroxypropyl methylcellulose.

[0074] These ranges provide a sustained-release coating that offers a favorable release profile. In addition, a pore-forming agent content of 0-3 wt% helps to regulate the release rate of sustained-release pellets.

[0075] If multiple film-forming agents are present, the 97-100wt% film-forming agent range refers to the total content of film-forming agents.

[0076] Preferably, the film-forming agent is ethyl cellulose. Preferably, the pore-forming agent includes hydroxypropyl methylcellulose.

[0077] Preferably, based on the weight of the pellet core with the sub-coating, the content of the sustained-release coating in the sustained-release pellet is 65-80 wt%, more preferably 68-75 wt%, and more preferably 70 wt%.

[0078] These ranges have been shown to provide favorable release rates of the active ingredient. These amounts of sustained-release coating correspond to a corresponding weight increase during the application of the sustained-release coating to the sub-coated pellet core.

[0079] More preferably: a) Immediate-release granules consist of the following: i. 89-91 wt% valproic acid or valproate, ii. 8.5-10.5 wt% anhydrous dicalcium phosphate, and iii. 0.4-0.6 wt% magnesium stearate, b) The pellet core consists of the following: i. 39-41 wt% valproic acid or valproate, ii. 58-60 wt% microcrystalline cellulose, and iii. 0.5-1.5wt% magnesium stearate, c) Sub-coating: i. Set to an amount equivalent to 16-17% of the pellet core weight, and ii. By weight of the sub-coating, it includes: 1) 36.5-38.5 wt% hydroxypropyl cellulose, 2) 36.5-38.5 wt% hydroxypropyl methylcellulose, and 3) 24-26wt% talc, and d) Slow-release coating: i. Set to an amount equivalent to 69-71% of the weight of the pellet core coated with the sub-coating, and ii. By weight of the slow-release coating, it includes: 1) 97-99% ethyl cellulose, 2) 1-3% pore-forming agent, which contains hydroxypropyl methylcellulose. in: The amount of active ingredient in the immediate-release granules accounts for 74-76 wt% of the total weight of active ingredients in the pharmaceutical composition, and The amount of active ingredient in sustained-release pills accounts for 24-26 wt% of the total weight of active ingredients in the pharmaceutical composition.

[0080] More preferably: a) Immediate-release granules consist of the following: i. 90wt% valproic acid or valproate, ii. 9.5 wt% anhydrous dicalcium phosphate, and iii. 0.5wt% magnesium stearate, b) The pellet core consists of the following parts: i. 40wt% valproic acid or valproate, ii. 59wt% microcrystalline cellulose, and iii. 1wt% magnesium stearate, c) Sub-coating: i. Set to an amount corresponding to 15% of the pellet core weight, and ii. By weight of the sub-coating, it includes: 1) 37.5wt% hydroxypropyl cellulose, 2) 37.5 wt% hydroxypropyl methylcellulose, and 3) 25wt% talc, and d) Slow-release coating: i. Set to an amount corresponding to 70% of the weight of the pellet core coated with the sub-coating, and ii. By weight of the slow-release coating, it includes: 1) 98% ethyl cellulose, 2) 2% pore-forming agent, which contains hydroxypropyl methylcellulose. in: The amount of active ingredient in the immediate-release granules accounts for 75 wt% of the total weight of active ingredients in the pharmaceutical composition, and The active ingredient in the sustained-release pills accounts for 25 wt% of the total weight of the active ingredients in the pharmaceutical composition.

[0081] This implementation typically corresponds to the formulation derived in Example 1.

[0082] Preferably, the pharmaceutical composition further includes a capsule for containing immediate-release granules and sustained-release pellets, the capsule defining a unit dose of the pharmaceutical composition.

[0083] When the capsules are size 0, each unit dose of the pharmaceutical composition may contain 250-285 mg of the active ingredient. As initially discussed, the capsules are preferably made of materials such as hydroxypropyl methylcellulose, which are eroded in the stomach or intestine to release immediate-release granules and sustained-release pellets. The content of the active ingredient in each capsule is typically selected such that, in twice-daily administration, each dose comprises 5-12 capsules, such as 7-10 capsules.

[0084] The third aspect of the invention relates to a pharmaceutical composition according to the first aspect of the invention for a method of pretreating cancer, wherein the pharmaceutical composition is administered to a person suffering from cancer.

[0085] Another third aspect of the invention relates to a method for pretreating cancer, the method comprising administering a pharmaceutical composition according to a first aspect of the invention to a person suffering from cancer.

[0086] In the context of this invention, cancer pretreatment includes treatments that make the cancer more receptive to further treatment. Therefore, cancer pretreatment includes applying one or more compositions to an individual requiring cancer treatment as a pretreatment prior to other treatments to enhance the effectiveness of the treatment or reduce its side effects.

[0087] The typical dose of the active ingredient may be up to 60 mg per kilogram of body weight per day, for example, 30-60 mg per kilogram of body weight per day.

[0088] Typical doses of the active ingredient can be 1,500 to 2,500 mg per day, more preferably 1,800 to 2,200 mg, and most preferably 2,000 mg.

[0089] Preferably, the cancer is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, T-cell lymphoma, myeloma, and Hodgkin's lymphoma.

[0090] As shown in WO2012 / 128709, these cancer types are particularly well-suited for pretreatment with active ingredients.

[0091] Preferably, the pharmaceutical composition is administered before the person is treated with chemotherapy and / or immunotherapy.

[0092] Chemotherapy and / or immunotherapy should be selected based on the type of cancer a person is suffering from.

[0093] Chemotherapy may include the administration of CHOP, a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone, at doses of 750+ / -10% mg / m2 cyclophosphamide, 50+ / -10% mg / m2 doxorubicin, 1.4+ / -10% mg / m2 vincristine, and 50+ / -10% mg / m2 prednisone, where m2 refers to the human body surface area.

[0094] Alternatively, chemotherapy may include the administration of R-CHOP, which is a combination of the antibody rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone.

[0095] CHOP and R-CHOP can each contain etoposide, so the corresponding chemotherapy is called CHOEP or R-CHOEP.

[0096] Immunotherapy may include the administration of antibodies, monoclonal antibodies, or functional fragments thereof, such as rituximab, olfatumab, GA101, tosimomab, ibrutinumab, occrazumab, vetuzumab, ipazumab, FTBA05, AME-133V, or R603. All of the above antibodies bind to CD20 present on B cells. Antibodies may be administered at a dose of 375 + / - 10% mg / m². Other possible treatments following pretreatment include surgery, radiation therapy, and gene therapy. Preferably, the pharmaceutical composition is administered twice daily. Since the active ingredient is preferably taken with or in combination with food, it is advantageous to take the pharmaceutical composition twice a day, as this provides more possibilities for taking it with meals.

[0097] Preferably, a steroid selected from prednisone, prednisolone, dexamethasone, and betamethasone is also administered to the person. The steroid may be administered concurrently with the pharmaceutical composition or between administrations of the pharmaceutical composition.

[0098] Prednisone or prednisolone can be administered in doses ranging from 20 to 200 mg daily, such as 50-200, 100-150, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg daily. This dose can be administered once or multiple times.

[0099] Betamethasone can be administered in doses ranging from 4 to 32 mg daily, such as 10-25, 10-20, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 mg daily. This dose can be administered once or multiple times.

[0100] Dexamethasone can be administered in doses of 10 to 80 mg daily, such as 20-70, 10, 20, 30, 40, 50, 60, 70, or 80 mg. This dose can be administered once or multiple times.

[0101] Another aspect of the present invention relates to a reagent kit comprising: - Multiple unit doses of the pharmaceutical composition, - Multiple doses of steroids selected from prednisone, prednisolone, dexamethasone, and betamethasone.

[0102] This kit can be used to provide and maintain effective concentrations of the active ingredient during a course of cancer pretreatment. A course of cancer pretreatment may include, for example, a time period of at least 12 hours, such as 24 hours, or at least 36 hours or at least 48 hours, and a time period of less than 96 hours, such as less than 84 hours, such as less than 72 hours. In addition, the kit makes cancer pretreatment easier and increases adherence to pretreatment.

[0103] The kit preferably includes multiple unit doses sufficient to administer two or three doses every 24 hours during cancer pretreatment. The number of unit doses in the kit can be from 2 to 8 doses.

[0104] Pretreatment for cancer is preferably initiated 48 hours before the start of cancer treatment. It typically involves a dose of 6 or 9 units. At least one dose may also contain a steroid selected from prednisone, prednisolone, dexamethasone, and betamethasone. In this case, the steroid does not need to be present in each dose.

[0105] In some embodiments of the kit, the quantity of unit doses is provided in a suitable container and / or suitable packaging, and the kit preferably also includes instructions on how and when to administer the dose.

[0106] Therefore, the kit may include, for example, one or more blister packs for maintaining the dose. Preferably, the kit includes blister packaging comprising multiple collapsible blister packs sealed with fragile seals, each blister pack containing one or more doses.

[0107] In some implementations, the kit includes multiple unit doses sufficient to perform multiple (e.g., 6) cancer pretreatments. Thus, a single kit can provide all the unit doses required for a typical cycle of cancer treatment, which comprises six cycles spaced 14-21 days apart, wherein in each cycle, the pharmaceutical composition is administered as a pretreatment prior to other chemotherapy or immunotherapy.

[0108] Example 1 - Development of Valproate Sustained-Release Compositions 1.1 Development of Immediate-Release Granules The immediate-release granules are developed to contain the following ingredients, as shown in Table 1.

[0109] Table 1

[0110] Immediate-release granules are formed by mixing materials in a planetary mixer or high-shear mixer. The resulting mixture is then rolled into a solid belt and ground into granules using a 1 mm sieve.

[0111] 1.2 Development of pellet cores The pellet core is developed to contain the following ingredients, as shown in Table 2: Table 2

[0112] Pellet cores are made by mixing materials in a planetary mixer or high-shear mixer. Water is added to the mixture, the resulting wet paste is extruded to form a portion of the material, which is then rounded and dried to form pellet cores.

[0113] 1.3 Development of Sub-coatings In the initial development of sustained-release pellets, the pellet core was coated with various sustained-release water-based coating polymers, such as Surelease, Eudragit NM30D, and AcryCoat. These polymers were designed to provide release over 8 to 12 hours under alkaline pH conditions. However, the combination of the highly porous sustained-release coating and the swelling properties of the highly soluble API and microcrystalline cellulose in the aqueous medium posed a challenge. This porosity allowed water to permeate through the coating into the core, causing the core to swell, which was visually observed as the expansion and rupture of the coated core, allowing for the complete release of the API within one hour.

[0114] Various attempts have been made to solve this problem. Surprisingly, the introduction of a sub-coating has yielded a successful response in limiting the dissolution of pellet cores in acidic media. Adding a 10% weight increase in the sub-coating (i.e., 10% by weight based on the weight of the pellet core) not only enabled the slow-release coating to function as expected, but also smoothed out irregularities on the pellet surface. The initial sub-coating is applied as an aqueous formulation containing the following ingredients, as shown in Table 3A: Table 3A

[0115] A sub-coating was applied until the weight of the pellet core increased by 10 wt%. The sub-coating was further optimized to make it more hydrophobic by using hydroxypropyl cellulose and hydroxypropyl methylcellulose and adding mineral talc (magnesium silicate). The optimized sub-coating contains the following components, as shown in Table 3B.

[0116] Table 3B

[0117] The amount of the optimized sub-coating provided corresponds to 14-18% of the pellet core weight, preferably 15%. These amounts of sub-coating are particularly advantageous in preventing the active ingredient from migrating from the pellet core and also provide a smooth surface for the sustained-release coating.

[0118] 1.4 Development of Slow-Release Coatings The sustained-release coating is administered in an aqueous formulation containing the following components, as shown in Table 4: Table 4

[0119] Apply the sustained-release coating until a weight increase of at least 25 wt% is achieved relative to the weight of the sub-coated pellet core. Increasing the weight increase of the sustained-release coating from 25% to 35%, 60%, and 100% shows a further decrease in the release rate.

[0120] Furthermore, reducing the concentration of the pore-forming agent from 20% to 10%, 5%, and 0% significantly slowed the release rate, resulting in minimal release in acidic media and dissolution for several hours in phosphate buffer medium at pH 6.8.

[0121] In addition, it is worth noting that the drying of the sustained-release pellets can also affect the release rate.

[0122] Various kinds of pellets were made. Figure 1A , Figure 1B and Figure 1C The dissolution results of sustained-release pellets with different amounts of sustained-release coating, pore-forming agent, and drying / curing methods are shown. As previously mentioned, a two-stage dissolution was used to simulate in vivo conditions where appropriate, with 0.1N HCl as the acidic phase and phosphate buffer at pH 6.8 as the basic phase. Symbols and figures are shown in the table below: Table 5A shows the comparison with Figure 1A The symbols and legends in the diagram correspond to the following parameters:

[0123] Table 5B shows the comparison with Figure 1B The symbols and legends in the diagram correspond to the following parameters:

[0124] Table 5C shows the comparison with Figure 1C The symbols and legends in the diagram correspond to the following parameters:

[0125] Two-stage dissolution data for the pellets showed that application of a 10% w / w Opadry sub-coating, including no pore-forming agent and a sustained-release Surelease coating up to 60% w / w, provided sustained-release properties exceeding 12 hours, while a thinner coating (35% w / w) resulted in complete release within 6 hours. However, after drying / curing the coated pellets at 40°C for 24 hours, the coating became smoother, and in some cases, the release profile changed significantly (68% release at 12 hours with a 35% weight increase, and 31% release at 12 hours with a 60% weight increase). This indicates that drying / curing affects the release profile by allowing the coating to coalesce over time, forming increasingly smaller pores through the coating. Pelletizing with 10% and 5% pore-forming agents resulted in faster release rates than without pore-forming agents. For pellets coated with Surelease containing 10% pore-forming agent, 100% release was achieved within 6 hours after a 60% weight increase. With a 100% weight increase, the release time extended to over 12 hours; however, this would result in coated pellets that were too large, given the limitations of capsule size. For pellets coated with Surelease containing 5% pore-forming agent, 100% release occurred within two hours, with a 35% weight increase. For pellets with a 60% weight increase after oven drying / curing, complete release was not achieved after 12 hours. This data suggests that 10% pore-forming agent can provide a suitable dissolution profile, but the coated pellets would be too large (>1.4 mm). Using 5% pore-forming agent results in a 60% weight increase, but an ideal dissolution profile can be achieved with the correct drying / curing time.

[0126] Version 1.5 further optimized The completed slow-release pellets were fluidized bed dried / cured at 55°C for 1 hour, and then oven dried / cured at 40°C for 24 hours. Significant differences were observed in the release characteristics between the oven-dried and fluidized bed-dried pellets. The oven-dried pellets, exhibiting a slower release rate over 8 or 12 hours, performed significantly better. The maximum coating weight gain (60% or 100%) of each batch of pellets was further achieved by coating with the enteric polymer AcrylEZE (methacrylic acid copolymer type C). However, the addition of enteric coating did not further improve release characteristics.

[0127] 1.6 Development of unit-dose capsules Immediate-release granules and sustained-release pellets are filled into capsules to provide a total active ingredient content of 250 or 285 mg, of which 75 wt% of the active ingredient is provided by the immediate-release granules and 25 wt% by the sustained-release pellets.

[0128] 1.7 Final drug composition and dissolution test Mass production of the final pharmaceutical composition. The pharmaceutical composition is as follows: Sub-coating of the pellet cores in Table 2 was performed using the optimized sub-coating method in Table 3B to increase the weight by 15%. The sub-coated particles were further coated using the sustained-release coating method in Table 4 to increase the weight by 70% to produce sustained-release pellets (850-1400µm), which were then encapsulated.

[0129] Immediate-release granules were prepared according to Table 1 and 710-1000µm and then encapsulated.

[0130] HPMC capsules (size 0EL) are filled with 208.3 mg of immediate-release granules and 301.5 mg of sustained-release pellets, providing 187.5 mg (75 wt%) of immediate-release active ingredient and 62.5 mg (25 wt%) of sustained-release active ingredient. The total amount of active ingredient in the capsules is 250.0 mg.

[0131] Figure 1D The two-stage dissolution curves of the final capsule are shown. As shown in the figure, the capsule initially releases the drug composition rapidly, with approximately 70% released at 1 hour, followed by slow release, with approximately 90% released at 4 hours and 95% released at 6 hours.

[0132] The final capsule samples were further stored under various conditions, such as at different temperatures (5, 25, and 40°C) and humidity levels (60% and 75% RH) for 1 or 3 months. Storage of the pharmaceutical formulation under these different conditions did not significantly affect the dissolution profile. Further storage tests showed no significant deterioration after 24 months of storage.

[0133] Example 2 - A phase 1 open-label randomized trial investigating drug use in healthy subjects under fasting and feeding conditions. Pharmacokinetics of the composition VAL001 In the trial, 25 subjects took either the final pharmaceutical composition described in 1.7 above (N=12), named VAL001, or the reference composition from Orion Pharma (N=12), named Absenor.

[0134] 2.1 Single dose (N=12) In the first part of the trial, healthy subjects were administered a single dose of the final drug composition (VAL001) described in section 1.7 above (equivalent to 30 mg of valproate per kilogram of body weight) and a single dose of the reference composition (Absenor) under both fasting and feeding conditions. Blood samples were then drawn at fixed time points to determine plasma valproate concentrations. The results are shown in Table 6 below. Figure 2A-2B As shown in 3A-3B.

[0135] Table 6. Single-dose administration under fasting conditions

[0136] As shown in the table, the VAL001 composition provides similar exposure, but with a maximum concentration (C0).max The peak concentration is lower. This means a lower peak concentration, and therefore a lower risk of side effects, considering the high concentrations required to achieve the desired effect. Therefore, the lower peak concentration is particularly advantageous, given that high doses of valproic acid are typically needed for effective pretreatment of cancer.

[0137] Figure 2A and 3A and the corresponding semilogarithm Figure 2B and 3B This also demonstrates this behavior. Specifically, Figure 2A The relationship between the mean (± standard deviation) plasma concentration (µM) of total valproate and nominal time (h) is shown. Figure 2A and 2B As shown, the VAL001 composition reaches C4 faster than the reference composition. max Especially during fasting. Accordingly, Figure 3A The mean (± standard deviation) plasma concentration (µM) of free (unbound) valproate is shown as a function of nominal time (h).

[0138] Table 6 and Figure 2A-2B , Figures 3A-3B Together, the results show that the VAL001 composition provides more effective plasma properties for cancer pretreatment. In other words, the VAL001 composition is particularly suitable for achieving high concentrations of sodium valproate relatively quickly, without excessively high C60 levels. max The concentration was kept high during administration to achieve a good synergistic effect between sodium valproate pretreatment and subsequent cancer treatment.

[0139] 2.2 Two-dose and three-dose (N=13) In the second part of the trial, the composition / formulation was administered to healthy subjects who were eating, either in two doses of 30 mg valproate (VAL001) per kilogram of body weight or in three doses of 20 mg valproate (Absenor) per kilogram of body weight.

[0140] Blood samples were then collected at fixed time points to determine the plasma concentration of valproate. The results are shown in Table 7 below. Figures 4A-4B As shown.

[0141] Table 7. Repeated administration under feeding conditions

[0142] Figure 4A The mean (± standard deviation) plasma concentration of total sodium valproate (µM) administered twice daily at a dose of 30 mg / kg body weight for 3 days was shown as a function of nominal time (h). Figure 4BThe mean (± standard deviation) plasma concentration of free sodium valproate (μM) after three days of administration of 20 mg potassium valproate three times daily was shown to be relative to its nominal time (hours).

[0143] Figure 4A It is clearly demonstrated that VAL001 provides a more stable concentration distribution and valproate exposure compared to the reference composition Absenor, even when taken twice daily instead of three times, at the same total daily dose. Figure 4B Similar results were shown, and the figure further shows that the VAL001 composition resulted in less valproate accumulation in plasma compared to the reference Absenor composition. Example 3 - Semi-quantitative protein analysis of acetylated histone (K9) in isolated peripheral blood mononuclear cell lysates Imprint Assessment The Bioanalytical Laboratory at the University of Turku, Finland, conducted an analysis to determine the extent of histone acetylation in peripheral blood mononuclear cell lysates obtained from subjects who had been repeatedly administered the final pharmaceutical composition described in 1.7 above (referred to as VAL001) or the reference composition (Absenor). Histone acetylation was measured by the acetylation of lysine 9 (acH3K9) of histone H3 compared to baseline expression.

[0144] As described in Example 1, samples were obtained from subjects participating in the clinical trial. In the second part of the clinical trial, PBMC samples were collected before administration and three times daily during treatment. A total of 96 samples were analyzed, including four time points from 24 subjects.

[0145] For each sample, the experimental signal of acH3K9 was normalized to the loaded control GAPDH to correct for loading variations and to confirm that the observed changes represented actual differences between samples. The fold change in relative acH3K9 expression of the study samples compared to pre-drug baseline expression was comparable across different blots.

[0146] 3.1 Sample materials and control samples Sample material consists of frozen cell particles of human peripheral blood mononuclear cells delivered at the bottom of a cryotube, which is stored vertically. Cell preparation tubes are used. TM (CPT, BD Biosciences) PBMCs were isolated from whole blood samples. Cell particles were washed and diluted with phosphate-buffered saline (PBS) and aliquoted into cryovials to equal cell concentrations (0.75–1.0 x 10⁻⁶ cells per tube). 6 (1 cell), PBS has been discarded, and the cell particles were cryopreserved at nominal -80°C before being delivered (on dry ice) to the bioanalytical laboratory. Prior to the commencement of the treatment trial, PBMC samples were collected from each study subject. These samples served as control samples to determine baseline acH3K9 protein expression in each study subject. On September 15, 2022, CRST Oy received the frozen study samples on dry ice, three copies of each sample (3 x 96 = 288). Subsequently, the study samples were stored in the cryostat of the bioanalytical laboratory at -80°C.

[0147] 3.2 Summary of Trial Treatment The experimental treatment of the subjects from whom the samples were obtained included repeated administration of the sustained-release composition of the present invention or the reference composition.

[0148] 3.3 Methods Cell pellets were lysed in 200 μl of Laemmli sample buffer containing SDS, thiol reducing agent β-mercaptoethanol, protease inhibitor (Roche), and phosphatase inhibitor (Roche). Samples were sonicated in TPX tubes (Diagenode) using a Diagenode Bioruptor system for 10 min, heated (95°C, 5 min), and centrifuged at high speed (4°C, 5 min). The supernatant was then transferred to a new tube.

[0149] SDS-PAGE (saturated polyacrylamide gel electrophoresis) is used to separate soluble proteins by molecular weight. Before loading, the sample is heated at 95°C for 5 minutes. 15 μl of sample, representing approximately 75,000 cells, is loaded into each well into Any kD well. TM MiniPROTEAN® TGX TM On a pre-made protein gel (Bio-Rad).

[0150] All analyzed sample gels included two protein standards (Precision Plus protein dual-color standards) for molecular weight reference. One protein standard channel on each gel overlapped with the ECL channel on the raw data.

[0151] Following electrophoresis, proteins were transferred to PVDF membranes using a semi-dry transfer turbine cell (Bio-Rad) procedure with a transfer time of 30 minutes and a constant voltage of 25 V, following the "standard SD" procedure. The transfer quality of each PVDF membrane was ensured by Ponceau S S staining and visual inspection. After destaining, the membranes were blocked for 1 hour at room temperature (RT) on a shaker with 5% skim milk (Valio) in TBST buffer.

[0152] The primary antibody was incubated overnight on a roller at +4°C. A 1:1000 (0.042 μg / ml) dilution of rabbit anti-acH3K9 antibody (Cell Signaling Technologies) and a 1:10000 (0.1 μg / ml) dilution of rabbit anti-GAPDH antibody (Abcam) were used. For primary antibody detection, anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technologies) were used: a 1:10000 (0.006 μg / ml) dilution for acH3K9 detection and a 1:3000 (0.020 μg / ml) dilution for GAPDH detection. All antibodies used were diluted in 1% skim milk in TBST buffer.

[0153] Chemiluminescence signals were generated using the extended-duration substrate ECL reagent from the SuperSignal West Dura and captured using a CCD-based sapphire imaging system (Azure Biosystems). Signals were quantified using Image J version 1.53c densitometrics, and the analyzed lanes, signal density maps, and measured peak areas were recorded.

[0154] Import the measured signals (peak areas) into an Excel file. Normalize the acH3 (K9) signal with the load-controlled GAPDH signal to mathematically correct for unavoidable inter-sample and inter-channel variations in protein loading. Calculate the lane normalization factor for each lane by dividing the observed GAPDH signal in the lane by the highest observed GAPDH signal in that blot. To calculate the normalized experimental signal for acH3K9, divide the observed experimental signal by the lane normalization factor.

[0155] 3.4 Results The study samples were analyzed in twelve (12) separate 10-well SDS-PAGE gels.

[0156] All protein blotting was of good quality and did not require retesting. Save and print the digital ECL image with protein markers as a reference, signal density map, and measured signal (peak area) as raw data, and visually check its quality.

[0157] acH3K9 signal was normalized based on the expression of the housekeeping protein GAPDH. Cell / protein load and antibody dilution were optimized to ensure successful signal normalization. However, normalization was considered optimal when the difference between protein load and GAPDH signal was small. Therefore, results should be combined with visual examination of ECL images. Furthermore, it should be noted that normalized acH3K9 signal values ​​are only comparable within samples on the same blot and should not be compared with signals on other blots. However, reported fold changes (representing relative acH3K9 expression in the study sample compared to baseline expression before administration) are likely to be compared between different blots. Following administration of sodium valproate (VAL001 or Absenor), the average fold change in relative acH3K9 expression was 1.38 (change +38%) at time point 23.5 h, 1.49 (change +49%) at time point 47.5 h, and 1.12 (change +12%) at time point 80 h. The standard deviation (SD) and coefficient of variation (CV%) of the fold change were 0.50 and 36% at time point 23.5h, 0.64 and 43% at time point 47.5h, and 0.59 and 53% at time point 80h, respectively.

[0158] In the VAL001 treatment group, the mean fold change was 1.44 (+44%) at time 23.5h, 1.69 (+69%) at time 47.5h, and 1.26 (+26%) at time 80h. The standard deviation (SD) and coefficient of variation (CV%) were 0.51 and 36% at time 23.5h, 0.79 and 47% at time 47.5h, and 0.74 and 59% at time 80h, respectively.

[0159] In the Absenor treatment group, the mean fold change was 1.32 at time 23.5 h (change +32%), 1.29 at time 47.5 h (change +29%), and 0.98 at time 80 h (change -2%). The standard deviation (SD) and coefficient of variation (CV%) of fold change were 0.48 and 36% at time 23.5 h, 0.34 and 26% at time 47.5 h, and 0.33 and 34% at time 80 h, respectively.

[0160] Calculate the mean, percentage of change, standard deviation (SD), and coefficient of variation (CV)% of fold change in Excel, and report them separately for each time point. The standard deviation and coefficient of variation (CV)% of fold change represent biological differences among study subjects and variations caused by assay changes, and should be carefully considered when reporting differences between experimental and control samples.

[0161] Figure 5A block diagram showing the lysine 9-acetylation in histone H3 is displayed.

[0162] 2.5 Discussion As the results show, the final pharmaceutical composition (VAL001) according to section 1.7 above provides a higher degree of histone acetylation, and therefore exhibits a more effective inhibition of histone deacetylation than the reference composition (Absenor). The pharmaceutical compositions of the present invention thus provide a more effective pretreatment for cancer.

[0163] The present invention is further explained by the following points.

[0164] 1. A pharmaceutical composition for oral administration, said pharmaceutical composition comprising: a) Immediate-release granules, which contain: i. Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and ii. Filler, b) Sustained-release pellets, which contain: i. The pellet core includes: (1) Active ingredients selected from valproic acid, hemisodium valproate, sodium valproate, and magnesium valproate, and (2) Filler, ii. A sub-coating disposed on the pellet core, wherein the sub-coating content is 10-20 wt% based on the weight of the pellet core, and the sub-coating comprises a film-forming agent, and iii. A slow-release coating disposed on the sub-coating, wherein the content of the slow-release coating is 25-100 wt% based on the weight of the pellet core coated with the sub-coating, and the slow-release coating contains a film-forming agent. The amount of active ingredient in the immediate-release granules accounts for 70-80 wt% of the total weight of active ingredients in the pharmaceutical composition, and the amount of active ingredient in the sustained-release pellets accounts for 20-30 wt% of the total weight of active ingredients in the pharmaceutical composition.

[0165] 2. The pharmaceutical composition according to point 1, wherein: The amount of active ingredient in the immediate-release granules accounts for 72-77 wt% of the total weight of active ingredients in the pharmaceutical composition, preferably 75 wt%, and The amount of active ingredient in the sustained-release pellets accounts for 23-27 wt% of the total weight of active ingredients in the pharmaceutical composition, preferably 25 wt%.

[0166] 3. A pharmaceutical composition according to any of the foregoing points, wherein: Based on the weight of the immediate-release granules, the content of active ingredients in the immediate-release granules is 75-95 wt%, preferably 77-83 wt%, and Based on the weight of the pellet core, the content of active ingredients in the pellet core is 35-45 wt%, preferably 37-43 wt%.

[0167] 4. The pharmaceutical composition according to any of the foregoing points, wherein: Based on the weight of the pellet core, the content of the sub-coating in the sustained-release pellet is 14-18 wt%, preferably 15 wt%. 5. A pharmaceutical composition according to any of the foregoing points, wherein: By weight, the sub-coating comprises: i. 35-40 wt%, preferably 37.5 wt% hydroxypropyl cellulose, ii. 35-40 wt%, preferably 37.5 wt%, of hydroxypropyl methylcellulose, and iii. 20-30 wt%, preferably 25 wt% talc.

[0168] 6. A pharmaceutical composition according to any of the foregoing points, wherein Based on the weight of the sustained-release coating, the sustained-release coating comprises: 1) 97-100wt% film-forming agent, and 2) 0-3wt% pore-forming agent.

[0169] 7. A pharmaceutical composition according to any of the foregoing points, wherein Based on the weight of the pellet core with a sub-coating, the content of the sustained-release coating in the sustained-release pellet is 65-80 wt%, preferably 68-75 wt%, and more preferably 70 wt%.

[0170] 8. A pharmaceutical composition according to any of the foregoing points, wherein a) Immediate-release granules consist of the following: i. 90wt% valproic acid or valproate, ii. 9.5 wt% anhydrous dicalcium phosphate, and iii. 0.5wt% magnesium stearate, b) The pellet core consists of the following: i. 40wt% valproic acid or valproate, ii. 59wt% microcrystalline cellulose, and iii. 1wt% magnesium stearate, c) Sub-coating: i. Set to an amount corresponding to 15% of the pellet core weight, and ii. By weight of the sub-coating, it includes: 1) 37.5wt% hydroxypropyl cellulose, 2) 37.5 wt% hydroxypropyl methylcellulose, and 3) 25wt% talc, and d) Slow-release coating: i. Set to an amount corresponding to 70% of the weight of the pellet core coated with the sub-coating, and ii. By weight of the slow-release coating, it includes: 1) 98% ethyl cellulose, 2) 2% pore-forming agent, which contains hydroxypropyl methylcellulose. in: The amount of active ingredient in the immediate-release granules accounts for 75 wt% of the total weight of active ingredients in the pharmaceutical composition, and The active ingredient in the sustained-release pills accounts for 25 wt% of the total weight of the active ingredients in the pharmaceutical composition.

[0171] 9. The pharmaceutical composition according to any of the foregoing points further includes a capsule for containing immediate-release granules and sustained-release pellets, said capsule defining a unit dose of the pharmaceutical composition.

[0172] 10. A method for preparing a pharmaceutical composition according to any of the foregoing points, comprising the following steps: i. Production of immediate-release granules ii. Production of pellet cores, iii. Apply a sub-coating to the pellet core by performing the following sub-steps: a. Suspend or dissolve the sub-coating in an aqueous medium to form an aqueous solution or dispersion of the sub-coating. b. Contact the pellet core with an aqueous solution or dispersion of the sub-coating to prepare pellet cores with a sub-coating. iv. Further coat the pellet core with a sub-coating using a slow-release coating by performing the following sub-steps: a. Suspending or dissolving the slow-release coating in an aqueous medium to form an aqueous solution or dispersion of the slow-release coating, and b. Contact the pellet core coated with a sub-coating with an aqueous solution or dispersion of a sustained-release coating to prepare sustained-release pellets.

[0173] 11. A method of pretreating cancer with a pharmaceutical composition according to any one of points 1-10, wherein the pharmaceutical composition is administered to a person suffering from cancer.

[0174] 12. The pharmaceutical composition according to point 11, wherein the cancer is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, T-cell lymphoma, myeloma and Hodgkin lymphoma.

[0175] 13. A pharmaceutical composition according to any one of points 11-12, wherein the pharmaceutical composition is administered prior to treatment of a person with chemotherapy and / or immunotherapy.

[0176] 14. A pharmaceutical composition according to any one of points 11-13, wherein the pharmaceutical composition is administered twice daily.

[0177] 15. A pharmaceutical composition according to any of points 11-14, wherein a steroid selected from the group consisting of prednisone, prednisolone, dexamethasone and betamethasone is administered to a human.

Claims

1. A pharmaceutical composition for oral administration comprising: a) immediate release granules comprising: i. an active ingredient selected from the group consisting of valproic acid, valproic acid hemi-sodium, sodium valproate and magnesium valproate, and ii. a filler, b) sustained release pellets comprising: i. a pellet core comprising: (1) an active ingredient selected from the group consisting of valproic acid, valproic acid hemi-sodium, sodium valproate and magnesium valproate, and (2) a filler, ii. a sub-coat disposed on the pellet core in an amount of 10-20 wt% based on the weight of the pellet core and comprising a film-forming agent, and iii. a sustained release coating disposed on the sub-coat in an amount of 25-100 wt% based on the weight of the pellet core coated with the sub-coat and comprising a film-forming agent, wherein the amount of active ingredient in the immediate release granules is 70-80 wt% of the total weight of active ingredient in the pharmaceutical composition and the amount of active ingredient in the sustained release pellets is 20-30 wt% of the total weight of active ingredient in the pharmaceutical composition, and wherein the film-forming agent of the sustained release coating is different from the film-forming agent of the sub-coat.

2. The pharmaceutical composition according to claim 1, wherein: the amount of active ingredient in the immediate release granules is 72-77 wt%, preferably 75 wt%, of the total weight of active ingredient in the pharmaceutical composition, and the amount of active ingredient in the sustained release pellets is 23-27 wt%, preferably 25 wt%, of the total weight of active ingredient in the pharmaceutical composition.

3. The pharmaceutical composition according to any one of the preceding claims, wherein: the content of active ingredient in the immediate release granules is 75-95 wt%, preferably 77-83 wt%, based on the weight of the immediate release granules, and the content of active ingredient in the pellet core is 35-45 wt%, preferably 37-43 wt%, based on the weight of the pellet core.

4. The pharmaceutical composition according to any one of the preceding claims, wherein: the content of sub-coat in the sustained release pellets is 14-18 wt%, preferably 15 wt%, based on the weight of the pellet core.

5. The pharmaceutical composition according to any one of the preceding claims, wherein: the sub-coat comprises, based on the weight of the sub-coat: i. 35-40 wt%, preferably 37.5 wt%, of hydroxypropylcellulose, ii. 35-40 wt%, preferably 37.5 wt%, of hydroxypropylmethylcellulose, and iii. 20-30 wt%, preferably 25 wt%, of talc.

6. The pharmaceutical composition according to any one of the preceding claims, wherein the sustained release coating comprises, based on the weight of the sustained release coating: 1) 97-100 wt% of a film-forming agent, and 2) 0-3 wt% of a pore-forming agent.

7. The pharmaceutical composition according to any one of the preceding claims, wherein the content of sustained release coating in the sustained release pellets is 65-80 wt%, preferably 68-75 wt%, more preferably 70 wt%, based on the weight of the pellet core provided with the sub-coat.

8. The pharmaceutical composition according to any of the preceding claims, wherein a) the immediate release granules consist of: i. 90 wt% valproic acid or valproate salt, ii. 9.5 wt% anhydrous dicalcium phosphate, and iii. 0.5 wt% magnesium stearate, b) the pellet core consists of: i. 40 wt% valproic acid or valproate salt, ii. 59 wt% microcrystalline cellulose, and iii. 1 wt% magnesium stearate, c) the sub-coating: i. is provided in an amount corresponding to 15% of the weight of the pellet core, and ii. comprises, by weight of the sub-coating: 1) 37.5 wt% hydroxypropyl cellulose, 2) 37.5 wt% hydroxypropyl methylcellulose, and 3) 25 wt% talc, and d) the sustained release coating: i. is provided in an amount corresponding to 70% of the weight of the pellet core coated with the sub-coating, and ii. comprises, by weight of the sustained release coating: 1) 98% ethyl cellulose, 2) 2% of a pore former comprising hydroxypropyl methylcellulose, wherein: the amount of active ingredient in the immediate release granules represents 75 wt% of the total weight of active ingredient in the pharmaceutical composition, and the amount of active ingredient in the sustained release pellets represents 25 wt% of the total weight of active ingredient in the pharmaceutical composition.

9. The pharmaceutical composition according to any of the preceding claims, further comprising a capsule for containing the immediate release granules and the sustained release pellets, the capsule defining the unit dose of the pharmaceutical composition.

10. A method of preparing the pharmaceutical composition according to any of the preceding claims, comprising the steps of: i. producing the immediate release granules, ii. producing the pellet core, iii. coating the pellet core with the sub-coating by performing the following sub-steps: a. suspending or dissolving the sub-coating in an aqueous medium to form an aqueous solution or dispersion of the sub-coating, b. contacting the pellet core with the aqueous solution or dispersion of the sub-coating to produce a pellet core having the sub-coating, iv. further coating the pellet core provided with the sub-coating with the sustained release coating by performing the following sub-steps: a. suspending or dissolving the sustained release coating in an aqueous medium to form an aqueous solution or dispersion of the sustained release coating, and b. contacting the pellet core coated with the sub-coating with the aqueous solution or dispersion of the sustained release coating to produce the sustained release pellets.

11. The pharmaceutical composition according to any of claims 1-10, for use in a method of pre-treating a cancer, wherein the pharmaceutical composition is administered to a person having a cancer.

12. The pharmaceutical composition according to claim 11, wherein the cancer is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, T-cell lymphoma, myeloma and Hodgkin lymphoma.

13. The pharmaceutical composition according to any of claims 11-12, wherein the pharmaceutical composition is administered prior to the treatment of the person with chemotherapy and / or immunotherapy.

14. The pharmaceutical composition of any one of claims 11-13, wherein the pharmaceutical composition is administered twice a day.

15. The pharmaceutical composition of any one of claims 11-14, wherein a steroid selected from the group consisting of prednisone, prednisolone, dexamethasone, and betamethasone is also administered to the human.

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