HK1235009B - Oral preparation and preparation method therefor - Google Patents

Description

Oral preparation and preparation method thereof

Technical Field

The present invention belongs to the field of pharmaceuticals, and in particular relates to an oral preparation and a preparation method thereof.

Background Art

KX2-361 is a small molecule compound developed by Kinex Pharmaceuticals in the United States that can be used to treat cell proliferative diseases, particularly cancer. This compound may treat cell proliferative diseases through tyrosine kinase inhibition (see patent document WO2006/071960). KX2-361's Chinese chemical name is N-(3-fluorobenzyl)-2-(5-(4-morpholinophenyl)pyridin-2-yl)acetamide, and its English chemical name is N-(3-Fluorobenzyl)-2-(5-(4-Morpholinophenyl)pyridin- ₂ -yl)-acetamide. Its molecular formula is _C24H24FN3O2 , _{and its} molecular weight is 405.46 g/mol. Its structural formula is shown in Formula 1:

KX2-361 is in the form of a free base, and its pharmaceutically acceptable salts include benzenesulfonate, hydrochloride, and phosphate. KX2-361 is poorly soluble in water, and its pharmaceutically acceptable salts also have unsatisfactory water solubility and are also poorly soluble in water. When a pharmaceutically active molecule is poorly soluble in water, oral administration can lead to problems such as low bioavailability and poor absorption. To formulate it into a suitable injection, its solubility needs to be increased to allow for intravenous administration. Therefore, in order to effectively administer KX2-361 or its pharmaceutically acceptable salts to patients, there is an urgent need to find ways to increase its solubility.

For poorly soluble drugs, commonly used solubilization methods in this field include: adding surfactant solubilizers, adding cosolvents, using mixed solvents, making soluble salts, introducing hydrophilic groups into the molecular structure of the main drug, making solid dispersions, making cyclodextrin inclusion compounds, adding block copolymer solubilizers, making liposomes, making microemulsions, making microspheres and nanoparticles, adding dendrimer solubilizers, etc. (See scientific literature: "Wu Peiying, Xu Lianying, Tao Jiansheng. Research progress on solubilization methods for poorly soluble drugs [J]. Chinese Patent Medicine, 2005, 09: 1126-1129.").

When formulating drugs into cyclodextrin inclusion complexes, a variety of cyclodextrins and their derivatives are available. Three known cyclodextrins are α, β, and γ, composed of six, seven, and eight glucose groups, respectively. X-ray diffraction and nuclear magnetic resonance studies have shown that cyclodextrins have a three-dimensional structure: a hollow cylinder with a wide top and a narrow bottom, open at both ends. The molecules of the included substance enter the cylinder, forming an inclusion complex. Typically, in inclusion complexes formed by cyclodextrin and drugs, the molar ratio of drug to cyclodextrin is 1:1, but other ratios are also common. Currently, a series of derivatives based on the above-mentioned cyclodextrins have been developed, including pharmaceutically suitable ones such as methylated β-cyclodextrin (RM-β-CD), hydroxypropyl β-cyclodextrin (HP-β-CD), sulfobutyl ether β-cyclodextrin (SBE-β-CD), maltose β-cyclodextrin, and hydroxypropyl γ-cyclodextrin (HP-γ-CD). The scientific literature "Loftsson T, Brewster ME. Pharmaceutical applications of cyclodextrins: basic science and product development. J Pharm Pharmacol. 2010 Nov; 62(11): 1607-21" lists some of the marketed preparations containing cyclodextrins worldwide, such as alprostadil injection and ceftriaxone hydrochloride tablets containing α-cyclodextrin; cephalosporin tablets and benacetate hydrochloride capsules containing β-cyclodextrin; Tc-99 technetium injection containing γ-cyclodextrin; voriconazole injection and ziprasidone mesylate injection containing SBE-β-CD; itraconazole oral solution and injection, mitomycin injection containing HP-β-CD. However, different cyclodextrins and their derivatives are suitable for different drugs. At present, it is difficult to infer which cyclodextrin or its derivative is suitable in terms of solubilization ability for a given compound based on its specific structure.

The aforementioned patent document WO2006/071960 discloses a series of general compounds, including KX2-361, which can be administered in a variety of ways, such as oral administration, injection, rectal administration, pulmonary administration, intranasal administration, transient administration, and pulsed-release administration. The patent document further discloses that different solid and liquid dosage forms can be used for different administration methods. The patent document also discloses that the injectable solution can be prepared using solvents such as sesame or peanut oil and aqueous propylene glycol. Alternatively, the injectable solution can be prepared using an aqueous solution of a pharmaceutically acceptable salt of KX2-361 that is soluble in water. In specific examples, the dispersible phase can be glycerol, liquid polyethylene glycol, or mixtures thereof in oil. The patent document also discloses dosage forms (e.g., liquid dosage forms) containing cyclic or aliphatic encapsulating agents or dissolving agents, such as cyclodextrins, polyethers, and polysaccharides; preferred examples include methylcellulose or (i.e., sulfobutyl ether-β-cyclodextrin). In other words, the patent document discloses some commonly used solubilization methods and reagents for poorly soluble drugs in the art.

However, the inventors of the present invention discovered during the pharmaceutical development of KX2-361 that the specific solubilization methods and agents disclosed in WO2006/071960 were not ideal and were not suitable for further development of KX2-361 into a cost-effective pharmaceutical formulation that could be effectively administered to patients. Therefore, there remains an urgent need to identify more pharmaceutically suitable solubilization methods and agents for KX2-361 or a pharmaceutically acceptable salt thereof.

Summary of the Invention

In order to solve the problems existing in the above-mentioned prior art, the present invention provides an oral preparation and a preparation method thereof.

Specifically, the present invention provides:

(1) An oral preparation for treating a cell proliferative disease, comprising hydroxypropyl-β-cyclodextrin and an active ingredient, wherein the active ingredient is KX2-361 or a pharmaceutically acceptable salt thereof, wherein KX2-361 is represented by the following formula 1:

(2) The oral preparation according to (1), wherein the molar ratio of the active ingredient to hydroxypropyl-β-cyclodextrin is 1:(4-59).

(3) The oral preparation according to (1), wherein the active ingredient is KX2-361, KX2-361·monobenesulfonate, KX2-361·dihydrochloride, KX2-361·monophosphate and/or KX2-361·diphosphate.

(4) The oral preparation according to (1), wherein at least a portion of the hydroxypropyl-β-cyclodextrin encapsulates at least a portion of the active ingredient to form a drug inclusion complex.

(5) The oral preparation according to (1), further comprising a pharmaceutically acceptable excipient.

(6) The oral preparation according to (5), wherein the pharmaceutically acceptable excipient is selected from one or more of a filler, a disintegrant, and a lubricant.

(7) The oral preparation according to (6), wherein the filler is selected from one or more of microcrystalline cellulose, lactose, starch, and mannitol, and the amount of the filler is 0% to 69% (w/w) based on the total weight of the oral preparation.

(8) The oral preparation according to (6), wherein the disintegrant is selected from one or more of cross-linked carboxymethyl cellulose sodium, cross-linked polyvinylpyrrolidone, and sodium starch glycolate, and the amount of the disintegrant is 0% to 5% (w/w) based on the total weight of the oral preparation.

(9) The oral preparation according to (6), wherein the lubricant is selected from one or more of magnesium stearate, micronized silica gel, and talc, and the amount of the lubricant is 0.5% to 3% (w/w) based on the total weight of the oral preparation.

(10) The oral preparation according to (1), wherein the oral preparation is in the form of a tablet, wherein the content of the active ingredient in a single-dose tablet is 0.5% to 6.5% (w/w).

(11) A method for preparing the oral preparation according to any one of (1) to (10), comprising the following steps:

A) providing a first solution in which hydroxypropyl-β-cyclodextrin is dissolved, wherein the pH of the first solution is 1-2;

B) mixing KX2-361 or a pharmaceutically acceptable salt thereof with the first solution to prepare a second solution in which KX2-361 or a pharmaceutically acceptable salt thereof is dissolved;

C) drying the second solution to obtain a dried product.

(12) The method according to (11), wherein the concentration of hydroxypropyl-β-cyclodextrin in the first solution is 10% (w/v) to 50% (w/v).

(13) The method according to (11), wherein the concentration of KX2-361 or a pharmaceutically acceptable salt thereof in the second solution is 0.5 mg/ml to 15 mg/ml, preferably 0.5 mg/ml to 10 mg/ml, calculated as KX2-361.

(14) The method according to (11), wherein, in the second solution, the molar ratio of KX2-361 or a pharmaceutically acceptable salt thereof to hydroxypropyl-β-cyclodextrin is 1:(4-59).

(15) The method according to (11), wherein, before step C) and after step B), it further includes I) adjusting the pH of the second solution to 3-7.

(16) The method according to (11), wherein after step C), the method further comprises D) tableting the dried product.

(17) The method according to (16), wherein step D) comprises mixing the dried product with one or more excipients selected from fillers, disintegrants, and lubricants, and then performing the tableting.

(18) The method according to (17), wherein the filler is selected from one or more of microcrystalline cellulose, lactose, starch, and mannitol, and the amount of the filler is 0% to 69% (w/w) based on the total weight of the oral preparation.

(19) The method according to (17), wherein the disintegrant is selected from one or more of cross-linked carboxymethyl cellulose sodium, cross-linked polyvinylpyrrolidone, and sodium starch glycolate, and the amount of the disintegrant is 0% to 5% (w/w) based on the total weight of the oral preparation.

(20) The method according to (17), wherein the lubricant is selected from one or more of magnesium stearate, micronized silica gel, and talc, and the amount of the lubricant is 0.5% to 3% (w/w) based on the total weight of the oral preparation.

(21) The method according to (11), wherein the pharmaceutically acceptable salt of KX2-361 is KX2-361·monobenesulfonate, KX2-361·dihydrochloride, KX2-361·monophosphate and/or KX2-361·diphosphate.

Compared with the prior art, the present invention has the following advantages and positive effects:

1. The present invention mixes hydroxypropyl-β-cyclodextrin with KX2-361 or a pharmaceutically acceptable salt thereof to form an inclusion complex, which greatly improves the solubility of the poorly soluble drug KX2-361. In addition, compared with other cyclodextrin and its derivative excipients, injectable surfactant solutions, injectable solvents or composite solvents, hydroxypropyl-β-cyclodextrin exhibits stronger solubilization ability.

2. The present invention forms an inclusion complex by mixing hydroxypropyl-β-cyclodextrin with KX2-361 or a pharmaceutically acceptable salt thereof, thereby enabling the poorly soluble KX2-361 or a pharmaceutically acceptable salt thereof to be formulated into a stable liquid preparation, which can then be made into a lyophilized preparation, and thereby various pharmaceutical preparations that can be clinically administered to patients, including intravenous preparations (such as intravenous injections or intravenous drips), oral preparations (such as tablets), and the like.

3. The present invention further optimizes the inclusion process and the drug loading formulas for both intravenous and oral formulations, resulting in pharmaceutical formulations for direct intravenous injection, intravenous drip formulations that can be diluted up to 10-fold, and oral formulations that meet all criteria (including drug reconstitution, shelf stability, and solubility), while achieving maximum optimization of drug loading. These formulations exhibit excellent stability, high safety, good absorbability, high bioavailability, and relatively low cost.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the changes in blood drug concentration over time for the oral tablet of Formulation B4 according to Example 7 of the present invention. The horizontal axis represents time in hours, and the vertical axis represents the plasma concentration of KX2-361 in ng/ml following oral administration of 51 mg/dog (calculated as KX2-361) to male beagle dogs.

DETAILED DESCRIPTION

The present invention will be further illustrated below through the description of specific implementation methods and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but as long as they do not deviate from the basic idea of the present invention, they are all within the scope of the present invention.

In this article, the term "injection" refers to: a sterile solution (including true solution, emulsion and suspension) made of medicine for injection into the body, as well as a lyophilized powder or concentrated solution prepared into the sterile solution (including true solution, emulsion and suspension) before use.

In this article, the term "intravenous drip" refers to the method of administering a large amount of liquid containing medication through a vein via an infusion tube. It is also known as "infusion," "drip," "intravenous drip," or "hanging water."

As used herein, the term "intravenous preparation" is intended to encompass "intravenous injections" and "intravenous drips," including "lyophilized powders" that can be used to prepare intravenous solutions and intravenous drip solutions.

In this article, the term "oral preparation" refers to a preparation form that is administered orally and the drug is absorbed into the blood in the gastrointestinal tract, including tablets, granules, capsules, oral solutions, etc.

As used herein, the term "cell proliferative disorder" includes the disorders described in WO 2006/071960, which refers to a state of uncontrolled and/or abnormal cell proliferation, including both cancerous and non-cancerous conditions. "Cancer" includes solid tumors, such as lung cancer, breast cancer, colon cancer, ovarian cancer, brain cancer, liver cancer, pancreatic cancer, prostate cancer, malignant melanoma, non-melanoma skin cancer, hematological malignancies, such as childhood leukemia and lymphoma, myeloma, Hodgkin's disease, lymphocytic and primary cutaneous lymphomas, acute and chronic leukemias, such as acute lymphoblastic, acute myeloid, or chronic myeloid leukemia, plasma cell neoplasms, lymphoid neoplasms, and AIDS-related cancers. Non-cancerous conditions include, for example, psoriasis, epithelial and dermoid cysts, lipomas, adenomas, hairy and cutaneous hemangiomas, lymphangiomas, spider angiomas, teratomas, nephromas, myofibromas, osteoblasts, and other dysplastic aggregates, and the like.

In this article, the term "inclusion compound" refers to a unique complex formed by the complete or partial inclusion of a drug molecule within the molecular cavity of another substance. Inclusion compounds are an active area of pharmaceutical formulation research. As early as the 1950s, researchers recognized that inclusion compounds can affect drug properties, such as increasing drug solubility and stability, and influencing drug absorption, distribution, and onset of action.

As used herein, the term "active ingredient" refers to a drug molecule that has a therapeutic effect on cell proliferative diseases, such as KX2-361 described herein or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts of KX2-361 described herein include, for example, KX2-361 benzenesulfonate, KX2-361 hydrochloride, KX2-361 phosphate, etc. The pharmaceutically acceptable salts of KX2-361 described herein can be in the form of a monosalt or a disalt of KX2-361, including, for example, KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate, and/or KX2-361 diphosphate.

Herein, the term "inclusion component" refers to a substance that encapsulates all or part of the structure of a drug molecule into its molecular cavity, such as the hydroxypropyl-β-cyclodextrin described herein.

In this article, the term "hydroxypropyl-β-cyclodextrin" refers to the etherified product of beta-cyclodextrin and 1,2-ethylene oxide; calculated on anhydrous basis, the hydroxypropoxy (-OCH ₂ CHOHCH ₃ ) content is 19.6% to 26.3% (see Part IV of the 2015 edition of the Chinese Pharmacopoeia).

1. Pharmaceutical Composition

KX2-361 and its pharmaceutically acceptable salts are poorly soluble in water. The inventors of the present invention sought to find an effective method for solubilizing KX2-361 and its pharmaceutically acceptable salts, thereby enabling their formulation into clinically useful pharmaceutical preparations. As described above, various methods for solubilizing drugs are known. Specifically for KX2-361 and its pharmaceutically acceptable salts, patent document WO2006/071960 also lists a variety of solubilization methods. However, the inventors of the present invention have found that these solubilization methods for KX2-361 and its pharmaceutically acceptable salts are not ideal. Furthermore, it is currently difficult to determine which solubilization method or solubilization agent is most suitable for the specific structure of a given compound. Therefore, the inventors of the present invention conducted extensive pharmaceutical research and screening on different solubilization methods and different solubilization agents for KX2-361 and its pharmaceutically acceptable salts. Ultimately, they discovered that inclusion technology can effectively solubilize KX2-361 or its pharmaceutically acceptable salts, and surprisingly found that the most suitable inclusion agent is hydroxypropyl-β-cyclodextrin.

Therefore, the present invention provides a pharmaceutical composition comprising hydroxypropyl-β-cyclodextrin and an active ingredient, wherein the active ingredient is KX2-361 or a pharmaceutically acceptable salt thereof, and the KX2-361 is represented by the following formula 1:

Preferably, in the pharmaceutical composition, the molar ratio of the active ingredient to hydroxypropyl-β-cyclodextrin is 1:(1-250), preferably 1:(4-59), such as 1:(9-59), or such as 1:(4-17).

Preferably, in the pharmaceutical composition, the mass ratio of KX2-361 to hydroxypropyl-β-cyclodextrin contained in the active ingredient is (0.1-29):100, more preferably (0.5-5):100, for example: (2-2.5):100, (0.5-1.5):100, or (0.5-0.7):100, etc.

Preferably, the active ingredient is KX2-361, KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate and/or KX2-361 diphosphate, with KX2-361 monobenzenesulfonate being preferred. In this document, unless otherwise specified, "KX2-361 benzenesulfonate" refers to KX2-361 monobenzenesulfonate, which has a molecular weight of 563.64.

In the pharmaceutical composition of the present invention, at least a portion of the hydroxypropyl-β-cyclodextrin can encapsulate at least a portion of the active ingredient to form a drug inclusion complex. In this drug inclusion complex, the hydroxypropyl-β-cyclodextrin can form a hollow cylindrical structure with open ends, and the KX2-361 or a pharmaceutically acceptable salt thereof can be fully or partially encapsulated in the cylindrical structure.

Preferably, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient.

The excipient can be selected according to the desired dosage form. For example, when the dosage form is an intravenous preparation, the excipient can be one or more selected from mannitol, lactose, dextran, xylitol, sorbitol, glucose and sodium chloride. In addition, when the dosage form is an oral preparation, the excipient can also be one or more selected from fillers, disintegrants, and lubricants. Fillers can be selected from one or more of microcrystalline cellulose, lactose, starch, mannitol, etc., disintegrants can be selected from one or more of cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, etc., and lubricants can be selected from one or more of magnesium stearate, micropowdered silica gel, talc, etc.

The present invention also provides use of the pharmaceutical composition in preparing a drug for treating cell proliferative diseases.

2. Drug inclusion complex

On the other hand, the present invention also provides a drug inclusion compound comprising an active ingredient and an inclusion component encapsulating the active ingredient, wherein the inclusion component is hydroxypropyl-β-cyclodextrin, the active ingredient is KX2-361 or a pharmaceutically acceptable salt thereof, and the KX2-361 is represented by the following formula 1:

In the drug inclusion compound, the hydroxypropyl-β-cyclodextrin can form a hollow cylindrical structure with both ends open, and the KX2-361 or a pharmaceutically acceptable salt thereof can be fully or partially encapsulated in the cylindrical structure.

Preferably, the active ingredient is KX2-361, KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate and/or KX2-361 diphosphate, with KX2-361 monobenzenesulfonate being preferred. In this document, unless otherwise specified, "KX2-361 benzenesulfonate" refers to KX2-361 monobenzenesulfonate, which has a molecular weight of 563.64.

3. Intravenous Preparations

On the other hand, the present invention also provides an intravenous preparation for treating cell proliferative diseases, which comprises the pharmaceutical composition of the present invention.

Preferably, in the intravenous preparation, the molar ratio of the active ingredient to hydroxypropyl-β-cyclodextrin is 1:(1-250), more preferably 1:(9-59), for example 1:(9-15), 1:(12-59), 1:(16-53), 1:(31-56), etc.

Preferably, in the intravenous preparation, the mass ratio of KX2-361 to hydroxypropyl-β-cyclodextrin contained in the active ingredient is (0.1-29):100, more preferably (0.5-2.5):100, for example: (2-2.5):100, (0.5-2):100, (0.5-1.5):100, or (0.5-0.7):100, etc.

The intravenous preparation of the present invention may contain an excipient, which is preferably selected from one or more of mannitol, lactose, dextran, xylitol, sorbitol, glucose and sodium chloride.

The intravenous preparation may be in the form of a lyophilized powder or a liquid preparation. In the case of a liquid preparation, the concentration of hydroxypropyl-β-cyclodextrin in the liquid preparation may be 100 mg/ml to 500 mg/ml, preferably 200 mg/ml to 400 mg/ml; and the concentration of KX2-361 or a pharmaceutically acceptable salt thereof, calculated as KX2-361, may be 0.5 mg/ml to 5 mg/ml, preferably 1.4 mg/ml to 4.3 mg/ml.

The lyophilized powder can be reconstituted with an injection solution to prepare a liquid preparation. The injection solution can be any injection solution known in the art, such as a buffered saline solution, a glucose solution, a sodium chloride solution, or a lactated Ringer's solution. Buffered saline solutions can include, for example, citrate buffer, acetic acid-sodium acetate buffer, or phosphate buffer; glucose solutions can include, for example, a 5% (w/v) glucose solution; and sodium chloride solutions can include, for example, a 0.9% (w/v) sodium chloride solution. Preferably, the buffered saline solution is a pH 4.0 buffered saline solution, such as a pH 4.0 citrate buffer.

In another aspect, the present invention further provides a method for preparing the intravenous preparation of the present invention, comprising the following steps:

1) providing a first solution in which hydroxypropyl-β-cyclodextrin is dissolved, wherein the first solution is an aqueous solution with a pH of 1-2;

2) mixing KX2-361 or a pharmaceutically acceptable salt thereof with the first solution to prepare a second solution in which KX2-361 or a pharmaceutically acceptable salt thereof is dissolved.

Preferably, the concentration of hydroxypropyl-β-cyclodextrin in the first solution is 10% (w/v) to 50% (w/v), more preferably 20% (w/v) to 40% (w/v), and even more preferably 30% (w/v) to 40% (w/v). If the concentration of hydroxypropyl-β-cyclodextrin is lower than 10% (w/v), the amount of drug included is too low; if it is higher than 50% (w/v), the viscosity of the resulting solution is too high and freeze-drying is impossible. The first solution may contain a pH adjuster, which may be a strongly acidic solution such as hydrochloric acid.

Preferably, in the second solution, the concentration of KX2-361 or a pharmaceutically acceptable salt thereof, calculated as KX2-361, is 0.5 mg/ml to 5 mg/ml, preferably 1.4 mg/ml to 4.3 mg/ml. If the concentration of KX2-361 is lower than 0.5 mg/ml, the amount of drug included is too low; if it is higher than 5 mg/ml, the stability of the resulting lyophilized powder after reconstitution, as well as the stability of the resulting reconstituted solution and the solution formed with other dilution media, is poor, and the drug is easily precipitated. The pharmaceutically acceptable salt of KX2-361 can be KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate, and/or KX2-361 diphosphate, with KX2-361 monobenzenesulfonate being preferred.

Preferably, in the second solution, the molar ratio of KX2-361 or a pharmaceutically acceptable salt thereof to hydroxypropyl-β-cyclodextrin is 1:(1-250), more preferably 1:(9-59).

Regarding the pH of the first solution, since KX2-361 or a pharmaceutically acceptable salt thereof is acid-labile, a pH below 1 can significantly increase acid-degradable impurities. A pH above 2 can slow the dissolution of the drug and significantly increase dissolution time, causing significant production inconvenience and increasing the amount of acid-degradable impurities. More preferably, the pH of the first solution is between 1.2 and 2.0.

In step 2) of the method of the present invention, the drug inclusion compound of the present invention is obtained.

In step 2) of the method of the present invention, the mixing includes mixing by stirring or mixing by ultrasound.

Preferably, the method of the present invention further comprises 3) freeze-drying the second solution to obtain a freeze-dried powder. Freeze-drying can be performed using conventional methods in the art.

It is also preferred that, before step 3) and after step 2), the step of i) adjusting the pH of the second solution to 3-7, more preferably to 4-6, is further included. This step of adjusting the pH back is beneficial for avoiding drug degradation caused by the long subsequent freeze-drying process and acid residues in the freeze-dried sample. Optional pH adjusters include alkaline solutions such as aqueous NaOH solutions. In the step of adjusting the pH, if the pH is adjusted to more than 7, the encapsulation effect of hydroxypropyl-β-cyclodextrin on KX2-361 or its pharmaceutically acceptable salt is poor, and KX2-361 or its pharmaceutically acceptable salt is easily precipitated. In the step of adjusting the pH, if the pH is adjusted to less than 3, since the structure of KX2-361 is easily degraded by ring opening in acid, degradation impurities will increase significantly, and the resulting preparation will be unstable.

Preferably, the step ii) of sterile filtration is further included before step 3) and after step 2). Sterile filtration methods are well known in the art, for example, filtration using a filter membrane, such as a 0.22 μm filter membrane, can be used to physically retain live microorganisms.

The solution obtained after sterile filtration can be directly filled into a small-volume injection or freeze-dried to obtain a lyophilized powder. The lyophilized powder can be reconstituted to obtain an injection solution for injection or diluted with a large-volume diluent to obtain an infusion solution for infusion.

It is also preferred that the step of removing pyrogens is further included before step ii) and after step i). Methods for removing pyrogens are well known in the art, such as adding activated carbon to remove pyrogens and filtering to remove the activated carbon.

In a specific embodiment of the present invention, the method for preparing the intravenous preparation of the present invention comprises the following steps performed in sequence:

a) Add an appropriate amount of concentrated hydrochloric acid to water for injection to prepare a hydrochloric acid solution with a pH of 1.2 to 2.0;

b) adding a prescribed amount of hydroxypropyl-β-cyclodextrin to the hydrochloric acid solution obtained in a) and stirring to dissolve; then adding a prescribed amount of KX2-361 or a pharmaceutically acceptable salt thereof and stirring or ultrasonically dissolving it;

c) adjusting the pH of the solution obtained in step b) to 4.0-6.0 with an aqueous sodium hydroxide solution;

d) Add activated carbon to remove pyrogens by adsorption and filter to remove carbon;

e) sterile filtration;

f) Freeze drying.

As a preferred embodiment of the present invention, the freeze-drying process in the above step f) is carried out under conditions of controlling the vacuum degree and temperature. The temperature control process can be (for example): keeping warm at -40°C to -50°C for 2-5 hours; heating to -20°C in 5-7 hours and keeping warm for 5-7 hours; heating to 0°C in 5-7 hours and keeping warm for 5-7 hours; heating to 10°C in 5-7 hours and keeping warm for 5-8 hours until the sample is dry.

In another preferred embodiment of the present invention, when the concentration of hydroxypropyl-β-cyclodextrin is 10% (w/v), the molar ratio of KX2-361 or its pharmaceutically acceptable salt to hydroxypropyl-β-cyclodextrin is 1:(31-56); when the concentration of hydroxypropyl-β-cyclodextrin is 20% (w/v), the molar ratio of KX2-361 or its pharmaceutically acceptable salt to hydroxypropyl-β-cyclodextrin is 1:(16-53); when the concentration of hydroxypropyl-β-cyclodextrin is 30% (w/v), the molar ratio of KX2-361 or its pharmaceutically acceptable salt to hydroxypropyl-β-cyclodextrin is 1:(12-59); when the concentration of hydroxypropyl-β-cyclodextrin is 40% (w/v), the molar ratio of KX2-361 or its pharmaceutically acceptable salt to hydroxypropyl-β-cyclodextrin is 1:(9-15).

When preparing a liquid formulation for intravenous administration, the method further includes the step of reconstituting the lyophilized powder with an injectable solution. Any injectable solution known in the art can be used, such as a buffered saline solution, an aqueous glucose solution, an aqueous sodium chloride solution, or a lactated Ringer's solution. The buffered saline solution may be, for example, a citrate buffer, an acetic acid-sodium acetate buffer, or a phosphate buffer; the aqueous glucose solution may be, for example, a 5% (w/v) aqueous glucose solution; and the aqueous sodium chloride solution may be, for example, a 0.9% (w/v) aqueous sodium chloride solution. Preferably, the buffered saline solution is a pH 4.0 buffered saline solution, such as a pH 4.0 citrate buffer.

In the research on intravenous preparations, the present invention optimized the inclusion process, thereby optimizing the drug-loaded formula. Ultimately, a formula with qualified indicators was obtained that can be used for direct intravenous injection and a formula that can be diluted within 10 times for intravenous infusion. After intravenous injection or intravenous infusion, the behavior of the drug in the blood vessels can be tested through dynamic and static simulation tests, and the inventors found that no precipitation was observed. Therefore, the present invention provides a formulation that can be safely administered by injection. The present invention also conducted preliminary stability tests on the lyophilized powder, and the test results showed that the formulation was stable after being stored at 40°C and 25°C for 3 months.

4. Oral Preparations

On the other hand, the present invention also provides an oral formulation for treating cell proliferative diseases, comprising hydroxypropyl-β-cyclodextrin and an active ingredient, wherein the active ingredient is KX2-361 or a pharmaceutically acceptable salt thereof, and the KX2-361 is represented by the following formula 1:

Preferably, in the oral preparation, the molar ratio of the active ingredient to hydroxypropyl-β-cyclodextrin is 1:(1-250), more preferably 1:(4-59), and even more preferably 1:(4-17).

Also preferably, in the oral preparation, calculated as KX2-361, the mass ratio of KX2-361 to hydroxypropyl-β-cyclodextrin contained in the active ingredient is (0.1-29):100, more preferably (0.5-5):100, and even more preferably (1.7-5):100.

Preferably, the active ingredient is KX2-361, KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate and/or KX2-361 diphosphate, with KX2-361 monobenzenesulfonate being preferred. In this document, unless otherwise specified, "KX2-361 benzenesulfonate" refers to KX2-361 monobenzenesulfonate, which has a molecular weight of 563.64.

In the oral formulation of the present invention, at least a portion of the hydroxypropyl-β-cyclodextrin can encapsulate at least a portion of the active ingredient to form a drug inclusion complex. In this drug inclusion complex, the hydroxypropyl-β-cyclodextrin can form a hollow cylindrical structure with open ends, and the KX2-361 or a pharmaceutically acceptable salt thereof can be fully or partially encapsulated in the cylindrical structure.

Preferably, the oral formulation of the present invention further comprises a pharmaceutically acceptable excipient, which may be selected from one or more of a filler, a disintegrant, and a lubricant.

The filler can be selected from one or more of microcrystalline cellulose (MCC), lactose, starch, mannitol, etc., wherein lactose is preferred; based on the total weight of the oral preparation, the amount of the filler is preferably 0% to 69% (w/w). The disintegrant can be selected from one or more of cross-linked carboxymethyl cellulose sodium (CC-Na), cross-linked polyvinylpyrrolidone (PVPP), sodium carboxymethyl starch, etc., wherein cross-linked carboxymethyl cellulose sodium is preferred; based on the total weight of the oral preparation, the amount of the disintegrant is preferably 0% to 5% (w/w). The lubricant can be selected from one or more of magnesium stearate, micropowdered silica gel, talc, etc., wherein magnesium stearate is preferred; based on the total weight of the oral preparation, the amount of the lubricant is 0.5% to 3% (w/w).

Preferably, the oral preparation is in the form of a tablet, wherein the content of the active ingredient in a single-dose tablet is 0.5% to 6.5% (w/w).

In a specific embodiment of the present invention, the tablet of the present invention contains: about 1% (w/w) KX2-361·monobenzenesulfonate, greater than 28.6% (w/w) and less than 29% (w/w) hydroxypropyl-β-cyclodextrin, about 64% (w/w) lactose, about 5% (w/w) cross-linked sodium carboxymethylcellulose, about 1% (w/w) magnesium stearate, and greater than 0 and less than 0.4% (w/w) NaCl.

In another aspect, the present invention further provides a method for preparing the oral formulation of the present invention, comprising the following steps:

A) providing a first solution in which hydroxypropyl-β-cyclodextrin is dissolved, wherein the pH of the first solution is 1-2;

B) mixing KX2-361 or a pharmaceutically acceptable salt thereof with the first solution to prepare a second solution in which KX2-361 or a pharmaceutically acceptable salt thereof is dissolved;

C) drying the second solution to obtain a dried product.

Preferably, in the first solution, the concentration of hydroxypropyl-β-cyclodextrin is 10% (w/v) to 50% (w/v), more preferably 20% (w/v) to 40% (w/v), and even more preferably 30% (w/v) to 40% (w/v). If the concentration of hydroxypropyl-β-cyclodextrin is lower than 10% (w/v), the amount of drug included is too low; if it is higher than 50% (w/v), the viscosity of the resulting solution is too high and drying (e.g., freeze-drying) is impossible. The first solution may contain a pH adjuster, which may be a strongly acidic solution such as hydrochloric acid.

Preferably, in the second solution, the concentration of KX2-361 or a pharmaceutically acceptable salt thereof, calculated as KX2-361, is 0.5 mg/ml to 15 mg/ml, preferably 3.6 mg/ml to 15 mg/ml, and more preferably 5 mg/ml to 10 mg/ml. If the concentration of KX2-361 is lower than 0.5 mg/ml, the amount of drug included is too low; if it is higher than 15 mg/ml, the inclusion system is unstable and the drug is prone to precipitation. The pharmaceutically acceptable salt of KX2-361 can be KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate, and/or KX2-361 diphosphate, with KX2-361 monobenzenesulfonate being preferred.

Preferably, in the second solution, the molar ratio of KX2-361 or a pharmaceutically acceptable salt thereof to hydroxypropyl-β-cyclodextrin is 1:(1-250), more preferably 1:(4-59), and even more preferably 1:(4-17).

Regarding the pH of the first solution, since KX2-361 or a pharmaceutically acceptable salt thereof is acid-labile, a pH below 1 can significantly increase acid-degradable impurities. A pH above 2 can slow the dissolution of the drug and significantly increase dissolution time, causing significant production inconvenience and increasing the amount of acid-degradable impurities. More preferably, the pH of the first solution is between 1.2 and 2.0.

In step B) of the method of the present invention, the mixing comprises mixing by stirring or mixing by ultrasound.

In step C) of the method of the present invention, examples of the drying include freeze drying and spray drying, etc. Freeze drying and spray drying can be performed using conventional methods in the art.

It is also preferred that, before step C) and after step B), the method further comprises: I) adjusting the pH of the second solution to 3-7, more preferably to 4-6. This pH adjustment step helps to avoid drug degradation caused by the long subsequent freeze-drying process and acid residues in the freeze-dried sample. Optional pH adjusters include alkaline solutions such as aqueous NaOH solutions. In the pH adjustment step, if the pH is adjusted to above 7, the encapsulation effect of hydroxypropyl-β-cyclodextrin on KX2-361 or its pharmaceutically acceptable salt is poor, and KX2-361 or its pharmaceutically acceptable salt is easily precipitated. If the pH is adjusted to below 3, since the structure of KX2-361 is easily degraded by ring opening in acid, degradation impurities will increase significantly, and the resulting preparation will be unstable.

Preferably, before step C) and after step B), the method further comprises the step III) of filtration. Filtration methods are well known in the art, for example, a clear solution can be obtained by filtration using a filter membrane, such as a 0.8 μm filter membrane.

Preferably, after step C), the method further comprises D) tableting the dried product. Specifically, step D) comprises mixing the dried product with one or more excipients selected from fillers, disintegrants, and lubricants, and then tableting.

The filler can be selected from one or more of microcrystalline cellulose (MCC), lactose, starch, mannitol, etc., wherein lactose is preferred; based on the total weight of the oral preparation, the amount of the filler is preferably 0% to 69% (w/w). The disintegrant can be selected from one or more of cross-linked carboxymethyl cellulose sodium (CC-Na), cross-linked polyvinylpyrrolidone (PVPP), sodium carboxymethyl starch, etc., wherein cross-linked carboxymethyl cellulose sodium is preferred; based on the total weight of the oral preparation, the amount of the disintegrant is preferably 0% to 5% (w/w). The lubricant can be selected from one or more of magnesium stearate, micropowdered silica gel, talc, etc., wherein magnesium stearate is preferred; based on the total weight of the oral preparation, the amount of the lubricant is 0.5% to 3% (w/w).

In a specific embodiment of the present invention, the method for preparing the oral formulation of the present invention comprises the following steps performed in sequence:

① Take an appropriate amount of concentrated hydrochloric acid and add water for injection to prepare a hydrochloric acid solution with a pH of 1.2-2.0;

② Add the prescribed amount of hydroxypropyl-β-cyclodextrin to the hydrochloric acid solution obtained in step ① and stir to dissolve; then add the prescribed amount of KX2-361 or a pharmaceutically acceptable salt thereof and stir or ultrasonicate to dissolve;

③ Adjusting the pH value of the solution obtained in step ② to 4.0-6.0 with an aqueous sodium hydroxide solution;

④ Filter;

⑤ Freeze drying;

⑥ Sieve the freeze-dried powder obtained in step ⑤;

⑦ mixing the sieved material obtained in step ⑥ with one or more excipients selected from fillers, disintegrants, and lubricants;

⑧Press into tablets.

As a preferred embodiment of the present invention, the freeze-drying process in the above step ⑤ is carried out under the conditions of controlling the vacuum degree and temperature, and the temperature control process can be (for example): keeping warm at -40°C to -50°C for 2-5 hours; heating to -20°C in 5-7 hours and keeping warm for 5-7 hours; heating to 0°C in 5-7 hours and keeping warm for 5-7 hours; heating to 10°C in 5-7 hours and keeping warm for 5-8 hours, until the sample is dry.

In another preferred embodiment of the method for preparing the oral preparation of the present invention, when the concentration of hydroxypropyl-β-cyclodextrin is 10% (w/v)-30% (w/v), the concentration of KX2-361 or a pharmaceutically acceptable salt thereof is 0.5 mg/ml to 6 mg/ml, calculated as KX2-361; when the concentration of hydroxypropyl-β-cyclodextrin is 40% (w/v)-50% (w/v), calculated as KX2-361, the concentration of KX2-361 or a pharmaceutically acceptable salt thereof is 0.5 mg/ml to 10 mg/ml.

The oral preparation of the present invention is a tablet further developed based on the freeze-dried powder of the present invention. The present invention optimizes the inclusion process and uses the shelf stability, related substances, dissolution rate, etc. of the inclusion solution as evaluation indicators to achieve maximum optimization of the drug loading of the drug-loaded formula. Finally, a process of including KX2-361 or its pharmaceutically acceptable salt (active ingredient) in a solution with a pH of 1-2 and then adjusting the pH was adopted, and freeze-drying technology was used to prepare the freeze-dried powder, so that the inclusion concentration of the active ingredient can reach 10 mg/ml based on KX2-361. The oral tablets of the present invention have achieved high bioavailability. In addition, the tablets were also tested for key indicators such as dissolution rate and related substances, and all were qualified.

example

Unless otherwise specified, the percentage concentration (%) of each reagent refers to the percentage concentration (% (w/v)) of the reagent by weight (g)/volume (100 mL). For example, a 40% concentration of HP-β-CD means a 40% (w/v) concentration of HP-β-CD.

When ethanol is mentioned in the following examples, it refers to a 95% (v/v) ethanol aqueous solution unless the concentration is otherwise specified.

The abbreviations are as follows:

KX2-361·BSA: KX2-361·benzenesulfonate

HP-β-CD: Hydroxypropyl-β-cyclodextrin

SBE-β-CD: sulfobutyl ether-beta-cyclodextrin

API: Active Pharmaceutical Ingredient (Unless otherwise specified, this refers to KX2-361·BSA)

PTFE: polytetrafluoroethylene

The materials and equipment used in the following examples are as follows:

Example 1: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection and Reconstituted Solution (HP-β-CD Concentration of 30%, High Drug Loading)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 1.8 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of approximately 1.2 (concentration 0.1 mol/L, the same below).

2) Weigh HP-β-CD according to the above recipe and add it to three 50 mL volumetric flasks. Add the solution obtained in 1) and dissolve it by ultrasonication (ultrasonic frequency 59 kHz). Then, dilute the volume to approximately 46 mL with the above hydrochloric acid solution.

3) API (KX2-361·benzenesulfonate) was weighed according to the above prescription amount, added to the above HP-β-CD solution, and dissolved by ultrasound (ultrasonic frequency 59 kHz).

4) Adjust the pH of the solution obtained in step 3 to approximately 6.0 (±0.05) with 5N NaOH and 0.1N NaOH, respectively. Dose the solution to 50 mL with distilled water.

5) Filter through a 0.45 μm polyethersulfone filter membrane and dispense the three prescription solutions into 10 mL vials (in 2 mL volumes) for freeze-drying.

The freeze-drying curve is as follows:

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample status after freeze drying:

The samples have good formability. The freeze-dried samples are all light yellow loose solids.

The freeze-dried samples of the above three formulations (formulations A1, A2, and A3) were reconstituted, and their reconstitution, insoluble particles, and dilution stability were evaluated.

1. Reconstitute with water and buffered saline solution

Reconstitution was performed by adding 2 mL of the following reconstitution medium to each vial of each lyophilized sample. After addition, the sample was inverted 30 times. If the sample did not dissolve, the sample was shaken until dissolved. The timer was started after the addition of the buffered saline solution, and two replicates were performed.

The buffer salt composition is as follows:

Note: c represents the molar concentration of the solute used to prepare the buffer salt.

Reconstitution time is as follows:

Note: The reconstitution time of each prescription is the average of 2 repetitions.

Insoluble particles (the detection method refers to the first method of the light obscuration method in Part IV 0903 of the Chinese Pharmacopoeia 2015 edition, the same below) are as follows:

Note: The insoluble particles are the average of the results obtained by testing two samples separately, measuring the insoluble particles after 0 hour and 4 hours respectively.

The above results show that when samples containing 30% HP-β-CD were reconstituted using different pH buffers, the reconstitution time was shortened to approximately 2.5 minutes, compared to approximately 4 minutes when reconstituted using pure water. The 30% HP-β-CD samples were significantly less likely to have insoluble particles when reconstituted using pH 4.0 buffer, with Formulation A2 showing the best results in all three buffers.

Furthermore, according to the Chinese Pharmacopoeia, the number of insoluble particles ≥10 μm in any container smaller than 100 mL must not exceed 6,000, and the number of insoluble particles ≥25 μm must not exceed 600. Since the insoluble particle test results shown in the table above are for 1 mL of solution, formulations A1-A3 are suitable for further development into finished pharmaceutical preparations with scaled-up volumes (considering their specifications for efficacy and safety) that can achieve a larger single dose and comply with the Chinese Pharmacopoeia standards.

2. Redissolve with 5% glucose aqueous solution and 0.9% sodium chloride aqueous solution

Reconstitution was performed by adding 2 mL of the following reconstitution medium to each vial of each lyophilized sample. After addition, the sample was inverted 30 times. If the sample did not dissolve, the sample was shaken until dissolved. The timer was started after the addition of the reconstitution medium, and two replicates were performed.

Reconstitution time is as follows:

Reconstitution medium Prescription A1 Prescription A2 Prescription A3 5% glucose aqueous solution About 3 minutes About 3 minutes About 3 minutes 0.9% sodium chloride aqueous solution About 3 minutes About 3 minutes About 3 minutes

Note: The reconstitution time of each prescription is the average of 2 repetitions.

The insoluble particles are as follows:

Note: The insoluble particles are the average of the results obtained by testing two samples separately, measuring the insoluble particles after 0 hour and 4 hours respectively.

The above results indicate that the reconstitution effect of pH 4.0 buffered saline solution is better than that of 5% glucose aqueous solution and 0.9% sodium chloride aqueous solution.

Example 2: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection and Reconstituted Solution (HP-β-CD Concentration of 30%, Low Drug Loading)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 1.8 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of approximately 1.2.

2) Weigh HP-β-CD according to the above prescription and add it to three 25 mL volumetric flasks. Add the solution obtained in 1) and dissolve it by ultrasonication (ultrasonic frequency 59 kHz). Then, dilute the volume to approximately 23 mL with the above hydrochloric acid solution.

3) Weigh the API according to the above prescription amount and add it to the above HP-β-CD solution. For prescription A6, add 50.7 mg of citric acid and dissolve it by ultrasound (ultrasonic frequency 59 kHz).

4) Adjust the pH of the solution obtained in 3) to the values shown in the table above (±0.05) using 5N NaOH and 0.1N NaOH, respectively. Dose the solution to 25 mL with distilled water.

5) Filter through a 0.45 μm polyethersulfone filter membrane and dispense the three prescription solutions into 10 mL vials (2 mL per vial) and freeze-dry.

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample state after freeze drying: The sample has good formability. The freeze-dried samples are all fluffy snow-like solids.

The freeze-dried samples of the above three formulations (formulations A4, A5, and A6) were reconstituted, and their reconstitution and insoluble particles in 5% glucose aqueous solution and normal saline were evaluated.

Reconstitution method: Add 2 mL of the following reconstitution medium to each vial of each lyophilized sample. After addition, invert 30 times. If the sample does not dissolve, shake until dissolved. Start timing after the reconstitution medium is added. Repeat the measurement for two samples. The reconstitution time is as follows:

Note: The reconstitution time of each prescription is the average of the test results of two samples.

The insoluble particles are as follows:

Note: The insoluble particles are the average of the results obtained from two samples tested separately.

The freeze-dried samples of the three prescriptions (prescriptions A4, A5, and A6) were reconstituted with the reconstitution media described in the table below. The reconstituted drug solutions were diluted in the same media at a certain ratio (volume of reconstituted solution: volume of dilution medium) of 1:2, 1:5, and 1:10, respectively. The diluted solutions were tested for insoluble particles at 0 and 4 hours. The insoluble particles after dilution are as follows:

Note: The insoluble particles are the average of the results obtained by testing two samples separately, measuring the insoluble particles after 0 hour and 4 hours respectively.

Observation of the dilution profile of the above samples revealed no precipitation of the API within 4 hours. All three formulations have the potential to be used for intravenous infusion of diluted solutions using a filter.

The high drug loading formulation A2 with a 30% HP-β-CD concentration and the low drug loading formulation A5 were selected for the following experiments.

Test Example 1: Measurement of insoluble particles and osmotic pressure after 8 days

Formulations A2 and A5 were re-prepared in 200 mL batches, respectively, according to the methods described in Examples 1 and 2. Lyophilized samples of Formulations A2 (30% HP-β-CD + 6 mg/mL API) and A5 (30% HP-β-CD + 2 mg/mL API) were stored at 4°C for 8 days and then assayed for insoluble particulate matter. The results are as follows:

Note: n=10 means that 10 samples were tested for each formulation, and the insoluble particles are the average of the results obtained from the 10 samples tested separately.

The results in the table above show that after the freeze-dried powders of the two formulas were placed at 4°C for 8 days, the insoluble particles after reconstitution were still qualified.

Isotonic infusions in elderly patients and infants with poor renal and cardiopulmonary function can easily lead to electrolyte retention and serious complications such as edema. Therefore, isotonic infusions are often referred to as "hazardous infusions" in pediatric clinical practice. Because the human body continuously loses water through evaporation from the body surface and exhalation from the lungs, the safety margin for hypotonic infusions is generally wider, and the safe range for hypotonic infusion volumes is larger, leading to a wider range of hypotonic infusions. Therefore, dilution stability tests and osmotic pressure measurements using hypotonic solutions were conducted. Lyophilized samples of prescription A5 (30% HP-β-CD + 2 mg/mL API) were reconstituted with 2 mL of distilled water per vial. The samples were then diluted in various proportions with hypotonic solutions (0.45% sodium chloride in water and 2.5% glucose in water), and insoluble particulate matter and osmotic pressure were measured.

Here are the results:

Note: The insoluble particles are the average of the results obtained by testing two samples separately, measuring the insoluble particles after 0 hour and 4 hours respectively.

The results of osmotic pressure determination are as follows:

Note: Osmotic pressure is the average of the results obtained by testing two samples separately.

The osmotic pressure molar concentration of a general isotonic solution is 280-320 mOsm. It can be seen that after prescription A5 is diluted with a hypotonic solution in different proportions, the osmotic pressure of the solution is lower and the insoluble particle detection results are also better.

Test Example 2: 3-month stability study

Lyophilized samples of Formulations A2 and A5 were placed in thermostats at 25°C and 40°C for three months for stability testing. Two samples were collected for each condition and analyzed by HPLC for API content, related substances (i.e., substances resulting from API degradation), and insoluble particulate matter. See Tables 1 and 2.

The HPLC determination conditions for content and related substances are as follows:

Table 1 Three-month stability study of freeze-dried preparations - content % and total impurities %

As can be seen from the above table, the content of the two freeze-dried powders remained basically unchanged within 3 months. From the impurity spectrum, 3 new degradation impurities appeared and the total impurity content increased slightly, but all were within an acceptable range.

Samples of prescription A2 and prescription A5 were taken at 25℃ and 40℃ for three months at the time points shown in Table 2 and tested for insoluble particles. See the table below for details:

Table 2 Three-month stability study of lyophilized formulations - insoluble particles

Note: n=2 means that 2 samples were tested for each formulation, and the insoluble particles are the average of the results obtained by testing the 2 samples separately.

It can be seen that the two prescriptions were placed at 25℃ and 40℃ for 3 months respectively, and the results of the insoluble particle test were both good.

Test Example 3: Dynamic adsorption test

The freeze-dried samples of prescription A2 and prescription A5 were reconstituted with water for injection, and the drug concentration of prescription A2 was prepared to 6 mg/mL, and the drug concentration of prescription A5 was prepared to 2 mg/mL. Then, the state of their injection into the blood vessel was simulated, and their physical stability under physiological conditions was examined, and the possibility of precipitation after intravenous administration was speculated.

1. Experimental Procedure: Use a peristaltic pump to pass 5% BSA (bovine serum albumin) through a 1.6 mm inner diameter flexible tube (Longer pump, #14) at a flow rate of 5 ml/min. Inject the reconstituted drug solution into the flexible tube via a needle inserted 30 cm from the end of the flexible tube. Use a syringe pump to control the sample injection rate, which ranges from 0.2 to 5 ml/min (see Table 3). After the drug solution and 5% BSA solution are mixed at a constant rate, collect the outflow at the end of the flexible tube:

(1) Take 4 ml of the effluent and measure the absorbance at 540 nm using a UV spectrophotometer to detect the presence of precipitates. A blank preparation (30% hydroxypropyl-β-cyclodextrin aqueous solution) and a mixed solution of 5% BSA were used as a control.

(2) Take 1 ml of the effluent, add 9 ml of acetonitrile, vortex for 5 minutes, filter through a 0.45 μm nylon filter membrane, and take the filtrate as a 100% concentration sample for HPLC detection.

Filter 4 mL of the effluent through a 0.45 μm polyethersulfone membrane. Take 1 mL of the filtrate, add 9 mL of acetonitrile, vortex for 5 minutes, and filter through a 0.45 μm nylon membrane. Take 1 mL of the filtrate as the test sample and analyze by HPLC. The ratio of the test sample to the 100% concentration sample is the proportion of unprecipitated API in the sample.

2. Test results:

Table 3 Dynamic precipitation test results (n=2)

Note: n=2 means that 2 samples were tested for each prescription, and the test results are the average of the results obtained by testing the 2 samples separately.

The results of precipitate detection and drug content testing are shown in the table above. As can be seen from the results, the UV absorbance values for each group were very low, indicating that no significant precipitation occurred after injection of 5% BSA at different rates in all three formulations. After filtration of the sample effluent, virtually all of the drug was present in the filtrate, indicating no precipitation.

The results of this experiment show that the prescriptions A2 and A5 investigated in the experiment have good compatibility with simulated plasma, and the risk of precipitation after intravenous drip or push injection is relatively low.

Test Example 4: Static adsorption test

In order to more fully verify the conclusion of the dynamic adsorption experiment, a static adsorption experiment was conducted.

The diluent 1 is a PBS (phosphate buffer solution, pH 7.4) solution containing 50 mmol/L NaH ₂ PO _4. BSA (bovine serum albumin) is dissolved in the diluent 1 to prepare a 5% BSA diluent 2.

Lyophilized samples of Formulation A5 (30% HP-β-CD + 2 mg/mL API, final pH = 4.0) and Formulation A2 (30% HP-β-CD + 6 mg/mL API, final pH = 6.0) were reconstituted with 2 mL of distilled water. The samples were diluted at different volume ratios using the two diluents 1 and 2, and the samples were observed for precipitation. The dilution test was repeated once for each dilution ratio. The results are as follows:

The experimental results showed that no significant solid precipitation occurred when formula A5 was diluted with PBS (pH=7.4) and PBS containing 5% BSA (pH=7.4) at different ratios. This result further confirmed that formula A5 can be administered by intravenous drip after dilution within 10 times.

When prescription A2 was diluted 1:5 and 1:10 with PBS (pH = 7.4) (diluent 1), solids precipitated within half an hour. However, when diluted with PBS (pH = 7.4) containing 5% BSA (diluent 2) at the same ratio, no significant precipitation occurred within 2 hours. After 16 hours, a large amount of solids precipitated in the diluent 1 dilution group, while less solids precipitated in the diluent 2 dilution group. The experimental results showed that BSA bound to part of the drug, preventing drug precipitation. The results of the dynamic adsorption test showed that prescription A2 did not precipitate drug at different infusion rates. Therefore, it is believed that this prescription is feasible when administered at a dose of 69.5 mg by intravenous push (11.6 mL push, which can be pushed over 2 to 3 minutes).

Example 3: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection and Reconstituted Solution (HP-β-CD Concentration of 20%, Higher Drug Loading)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 1.8 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of approximately 1.2.

2) Weigh HP-β-CD according to the above prescription and add it to two 25 mL volumetric flasks. Add the solution obtained in 1) and dissolve it by ultrasonication (ultrasonic frequency 59 kHz). Then, dilute the volume to approximately 23 mL with the above hydrochloric acid solution.

3) Weigh the API according to the above prescription amount, add it to the above HP-β-CD solution, and dissolve it by ultrasound (ultrasonic frequency 59 kHz).

4) Adjust the pH of the solution obtained in step 3 to approximately 6.0 (±0.05) with 5N NaOH and 0.1N NaOH, respectively. Dose the solution to 25 mL with distilled water.

5) Filter through a 0.45 μm polyethersulfone filter membrane and dispense the two prescription solutions into 10 mL vials (2 mL in volume) for freeze-drying.

The freeze-drying curve is as follows:

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample state after freeze drying: The sample has good formability. The freeze-dried samples are white, fluffy, snow-like solids.

The freeze-dried samples of the above two formulations (formulations A7 and A8) were reconstituted, and their reconstitution and insoluble particulates were evaluated.

Reconstitution was performed by adding 2 mL of water and the following buffered saline solution to each vial of each lyophilized sample. After addition, the sample was inverted 30 times. If the sample did not dissolve, the sample was shaken until dissolved. A timer was started after the addition of the reconstitution medium, and two replicates were performed.

The buffer salt composition is as follows:

pH (theoretical/actual) Element Theoretical weighing/actual weighing concentration 4.0/4.00 Citric acid monohydrate 210.14mg/211.4mg c＝0.01mmol/L 5.0/5.01 Sodium acetate trihydrate 136.08mg/137.4mg c＝0.01mmol/L 6.0/6.00 Sodium dihydrogen phosphate dihydrate 156.06mg/156.6mg c＝0.01mmol/L

Reconstitution time is as follows:

Note: The reconstitution time of each prescription is the average of 2 repetitions.

The insoluble particles are as follows:

Note: The insoluble particles of each prescription are measured by combining two bottles, and the insoluble particles are measured after 0 hour and 4 hours respectively.

The above results show that when samples containing 20% HP-β-CD were reconstituted in different pH buffer solutions, the reconstitution time was shortened to approximately 1.5 minutes, compared to approximately 3 minutes when reconstituted in pure water. Furthermore, the insoluble particle test results for Formulation A8 after reconstitution in pH 4.0 and pH 5.0 buffer solutions were superior to those for Formulation A7.

Example 4: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection and Reconstituted Solution (HP-β-CD Concentration of 20%, Low Drug Loading, Inclusion pH of 1.2)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 1.8 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of 1.21.

2) Weigh HP-β-CD according to the above prescription and place it in a 25 mL volumetric flask. Add the solution obtained in 1) and dissolve it by ultrasonication (ultrasonic frequency 59 kHz). Then, dilute the volume to approximately 23 mL with the above hydrochloric acid solution.

3) Weigh the API according to the above prescription, add it to the above HP-β-CD solution, and dissolve it under ultrasound (ultrasonic frequency 59 kHz).

4) Adjust the pH of the solution obtained in 3) to approximately 4.0 (±0.05) using 5N NaOH aqueous solution and 0.1N NaOH aqueous solution. Dose the solution to 25 mL with distilled water.

5) Filter through a 0.45 μm polyethersulfone filter membrane and dispense the solution into 10 mL vials (2 mL per vial) and freeze-dry.

The freeze-drying curve is as follows:

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample state after freeze drying: The sample has good formability. The freeze-dried sample is a light yellow fluffy snow-like solid.

Two vials of freeze-dried sample of prescription A9 were reconstituted with 2 mL of normal saline (i.e., 0.9% sodium chloride aqueous solution) and 5% glucose aqueous solution, respectively. The sample was then diluted with the corresponding solution and tested for insoluble particles. The dilution test was repeated once at each dilution factor. The results are as follows:

Note: The insoluble particles are the average of the results obtained by testing two samples separately, measuring the insoluble particles after 0 hour and 4 hours respectively.

As shown in the table above, after reconstitution, a two-fold internal dilution of Prescription A9 in the same medium yields a stable solution. However, multiple dilutions lead to instability and the precipitation of solids. Therefore, Prescription A9 can be administered directly after reconstitution or after a two-fold internal dilution.

Example 5: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection (HP-β-CD Concentration of 20%, Low Drug Loading, Inclusion pH of 2)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 0.18 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of 2.03.

2) Weigh the HP-β-CD according to the above prescription and add each into a 25 mL volumetric flask. Add the solution obtained in 1) and dissolve it by ultrasonication (ultrasonic frequency 59 kHz). Then, dilute to approximately 23 mL with the above hydrochloric acid solution.

3) Weigh the API according to the above prescription amount and add it to the above HP-β-CD solution respectively. Stir at 40°C for about 1.5 hours (prescription 10) and 1 hour (prescription 11) to dissolve.

4) Adjust the pH of the solution obtained in step 3 to approximately 4.0 (±0.05) with 5N NaOH and 0.1N NaOH, respectively. Dose the solution to 25 mL with distilled water.

5) Filter through a 0.45 μm polyethersulfone filter membrane and dispense the two prescription solutions into 10 mL vials (2 mL per vial) and freeze-dry.

The freeze-drying curve is as follows:

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample state after freeze drying: The sample has good formability. The freeze-dried sample is a light yellow to white fluffy snow-like solid.

Test Example 5: Study on the properties of prescriptions A9-A11

1. Redissolution test and inspection of insoluble particles

This test example investigated the re-dissolution and insoluble particles of prescriptions A9-A11 in 5% glucose aqueous solution, distilled water and normal saline (i.e., 0.9% sodium chloride injection).

Reconstitution method: Add the following volume of reconstitution medium to each vial of each lyophilized sample. After addition, invert the vial 30 times. If the vial does not dissolve, shake until dissolved. Start timing after the reconstitution medium is added.

Reconstitution time is as follows:

The insoluble particles are as follows:

Note: The reconstitution time of each prescription is the average value of 2 bottles. The insoluble particles are measured by combining the two bottles, and the insoluble particles are measured after 0 hour and 4 hours respectively.

The experimental observations and results indicate that increasing the inclusion pH to 2.03 increases the complexity of inclusion (for example, requiring stirring at 40°C for more than an hour), and the number of insoluble particles in the reconstituted solution after lyophilization increases compared to the inclusion pH of 1.2. However, the insoluble particles in formulations A9-A11 all meet the requirements for preparing intravenous injections.

2. Stability of inclusion samples at different pH values:

Take the freeze-dried samples of prescriptions A9-A11 and prepare two bottles of solutions with a concentration of 0.1 mg/mL with water. The related substances are measured by HPLC (for details, see the content and HPLC determination conditions of related substances described in Test Example 2). The results are as follows.

As can be seen from the table above, the total impurities in formulations A10 and A11 were not significantly reduced compared to formulation A9. Considering that the inclusion time at pH 2 is significantly increased and the inclusion process is more difficult compared to the pH 1.2 inclusion process, the most preferred inclusion process is at pH 1.2.

Example 6: Preparation and Evaluation of KX2-361·Benzenesulfonate Lyophilized Injection and Reconstituted Solution (HP-β-CD Concentration of 10%, Low Drug Loading)

Prepare a prescription consisting of:

Note: The molecular weight of HP-β-CD is 1431-1806, so the upper and lower limits of the molecular weight are used for calculation when calculating the molar ratio.

Prepare the above prescription in the following steps:

1) Place 1.8 mL of concentrated hydrochloric acid in a 200 mL volumetric flask and dilute to 200 mL with distilled water to obtain a hydrochloric acid solution with a pH of approximately 1.2.

2) Weigh HP-β-CD according to the above recipe and add it to two 25 mL volumetric flasks. Add the solution obtained in 1) to each flask, dissolve it by ultrasonication (ultrasonic frequency 59 kHz), and then adjust the volume to approximately 25 mL with the above hydrochloric acid solution.

3) Weigh the API according to the above prescription amount, add it to the above HP-β-CD solution, and dissolve it by ultrasonication (ultrasonic frequency 59 kHz).

4) Adjust the pH of the solution obtained in step 3 to approximately 4.0 (±0.05) with 5N NaOH and 0.1N NaOH, respectively. Dose the solution to 50 mL with distilled water.

5) Add 0.1 g of activated carbon for injection to the two solutions obtained in 4), stir at room temperature for half an hour, filter using a 0.45 μm polyethersulfone filter membrane, and dispense the resulting solution into 10 mL vials (2 mL per vial) and freeze-dry.

The freeze-drying curve is as follows:

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser pre-freeze (℃) 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure 3 -40 20±2 360 Open 4 -20 20±2 360 Open 5 -20 10±2 360 Open 6 0 10±2 360 Open 7 0 10±2 360 Open 8 10 No control 360 Open 9 10 No control 120 Open

Sample state after freeze drying: The sample has good formability. The freeze-dried samples are all white loose solids.

The freeze-dried samples of the above two formulations (formulations A12 and A13) were reconstituted, and their reconstitution and insoluble particulates in physiological saline, distilled water, and buffered saline solutions of different pH values (prepared in the same manner as the buffered saline solutions of pH 4, 5, and 6 in Example 3) were evaluated.

Reconstitution was performed by adding 2 ml of the following reconstitution medium to each vial of each lyophilized sample. After addition, the sample was inverted 30 times. If the sample did not dissolve, the sample was shaken until dissolved. A timer was started after the addition of the reconstitution medium. The reconstitution test was repeated once for each medium.

The reconstitution time for both prescriptions is about 1 minute.

The insoluble particles are as follows:

Note: All values in the table are the average of 3 measurements.

The above results indicate that both A12 and A13 meet the requirements for intravenous injections. Compared to A13, A12 has fewer insoluble particulates, but higher than A9 (20% HP-β-CD + 2 mg/mL API). This indicates that when the concentrations of hydroxypropyl-β-cyclodextrin and drug are proportionally reduced, the drug inclusion capacity of the low cyclodextrin concentration is weakened. Therefore, further selecting the appropriate hydroxypropyl-β-cyclodextrin concentration and drug inclusion concentration based on the present invention is also important for the development of KX2-361 injection.

Comparative Example 1: Drug Solubility Test

KX2-361 and its salts are both poorly soluble drugs. This comparative example tested the solubility of KX2-361 benzenesulfonate in the following media.

(1) Aqueous solution containing surfactant

The drug (KX2-361 benzenesulfonate) (equivalent to 10 mg of KX2-361) was added to 50 ml each of 10% Cremophor EL aqueous solution, 2% Tween 80 aqueous solution, 8% Solutol HS15 aqueous solution, and 0.6% Poloxamer 188 aqueous solution, and magnetically stirred for 1 hour, but the drug was not completely dissolved.

(2) Single solubilizer (anhydrous)

Using a sufficient amount of solubilizer alone can dissolve the drug, but the solubility is generally very low; and none of them can prevent the drug solution from precipitating during dilution with water. The amounts of each component in the system when the drug precipitates are shown in Table 4.

Table 4 Water precipitation critical point of drug solution of each solubilizer

(3) Single non-aqueous solvent or composite solvent

This study used ethanol, a single non-aqueous solvent, for dissolution testing and found that the solubility of KX2-361 benzenesulfonate in ethanol was 1.12-2.38 mg/mL, meaning that approximately 50-100 ml of ethanol was required to dissolve 100 mg of the drug. Obviously, large amounts of ethanol cannot be used for intravenous administration.

This study also used composite solvents composed of PEG400 or PEG200 and anhydrous ethanol for dissolution tests, and found that the required solvent volume was too large and it was impossible to dilute with an aqueous solution system.

This study also conducted dissolution tests using the non-aqueous solvent propylene glycol alone, or a combination of propylene glycol and other non-aqueous solvents. It was found that 20 ml of propylene glycol alone could substantially dissolve 50 mg of KX2-361 (KX2-361 benzenesulfonate), but trace amounts of small particles remained in the clear solution. Combining the non-aqueous solvent propylene glycol with other solvents, such as ethanol and PEG-400, made little contribution to drug dissolution.

(4) The solubilizer and other solvents form a composite solvent

The drug was solubilized using binary or ternary systems composed of a solubilizer (Cremophor EL or Solutolo HS 15) and a solvent (including water and propylene glycol). Two solvent systems were screened for drug solubility: ① 5 ml of propylene glycol:Cremophor EL (50:50) (v/v) dissolved 50 mg of KX2-361 and exhibited good solution stability; ② 11.5 ml of a combined solvent (10 ml of propylene glycol, 1 ml of Cremophor EL, and 0.5 ml of water) also dissolved the drug. The dilution stability of these two solvent systems was then tested.

(5) Dilution stability test

The above-mentioned drug-containing solution that can be dissolved was subjected to a dilution stability test. It was diluted with equal volumes of aqueous solutions containing different surfactants, 0.9% sodium chloride injection or 5% glucose injection as described in (1) above. It was found that the solutions became turbid, that is, the drug precipitated.

The above experiments show that the drug described in the present invention cannot achieve a high solubility in the solvents, solubilizers or mixed systems currently available for injection, or even after being dissolved, it cannot be diluted, that is, it is difficult to ensure that the drug solution does not precipitate after entering the blood vessels.

Comparative Example 2: Determination of API inclusion capacity of cyclodextrins of different concentrations and types

Different cyclodextrin types were dissolved in a 0.1N hydrochloric acid solution at pH 1.2 to determine the saturation concentration of each cyclodextrin. KX2-361 benzenesulfonate was dissolved in cyclodextrin solutions of varying concentrations and types to investigate the inclusion capacity and efficacy of the API with different cyclodextrin types and concentrations.

(1) Weigh a certain amount of α-, β-, and γ-cyclodextrin into a 10 mL volumetric flask. Gradually add pH 1.2 hydrochloric acid to the mark. Vortex and sonicate to dissolve the cyclodextrin. Record the maximum amount of cyclodextrin that can be dissolved in 10 mL of solution. The experimental results show that the maximum solubility of α-cyclodextrin in pH 1.2 hydrochloric acid is approximately 8%, β-cyclodextrin is approximately 1%, and γ-cyclodextrin is approximately 20%.

(2) 8% α-cyclodextrin solution, 1% β-cyclodextrin solution, 20% γ-cyclodextrin solution, 20%, 30% and 40% sulfobutyl ether beta-cyclodextrin solution, and 20%, 30% and 40% hydroxypropyl beta-cyclodextrin solution were prepared with a pH 1.2 hydrochloric acid aqueous solution.

Weigh a certain amount of API and add an appropriate volume of the above-mentioned cyclodextrin solution. If the API completely dissolves, filter the solution through a 0.45μm PTFE membrane. Then slowly adjust the pH to 4.0 using 5N NaOH and 0.1N NaOH solutions, observing the pH as you adjust. If the solution becomes cloudy during pH adjustment, try reducing the API concentration. Let the clarified solution stand after pH adjustment and observe the state after 2, 4, and 24 hours. The experimental process and results are shown in Table 5 below:

Table 5 Detection of API inclusion capacity of different types and concentrations of cyclodextrin

/: Terminate observation

The experimental phenomena and results show that HP-β-CD has a significantly better inclusion effect on API than SBE-β-CD, while α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin have poor inclusion effects on API.

Example 7: Preparation and evaluation of oral tablets

(1) Preparation of lyophilized powder

Preparation of pH 1.2 hydrochloric acid aqueous solution: Place 0.9 ml of concentrated hydrochloric acid in a 100 ml volumetric flask and dilute to 100 ml with distilled water.

Preparation of 30% HP-β-CD solution and 7 mg/ml drug-containing solution (HP-β-CD:API molar ratio is 13-17:1, HP-β-CD:KX2-361 mass ratio is 100:1.68):

Add 70 ml of pH 1.2 hydrochloric acid solution to a 250 ml beaker. Weigh 30 g of HP-β-CD and add it to the hydrochloric acid solution while stirring magnetically until it is completely dissolved.

Weigh 700 mg of KX2-361 benzenesulfonate and add the API to the prepared HP-β-CD solution while magnetically stirring. Dissolve the drug completely by sonication (59 kHz). Adjust the pH of the drug-containing solution to approximately 4.0 with 5N and 0.1N aqueous NaOH solutions. Add appropriate amount of distilled water to 100 ml. The final pH is 4.0. Alternatively, the solution can be directly diluted without pH adjustment (i.e., the pH of the drug-containing solution is 1.2).

Filtration: Filter the above drug-containing solution through a 0.45 μm PES filter membrane and place the filtrate in a stainless steel tray with a liquid level of approximately 0.3 cm.

Freeze drying: freeze drying curve is shown in the table

stage Temperature (℃) Vacuum control (Pa) Time (minutes) Vacuum pump status Condenser prefreeze 1 -40 Not enabled 5 closure -40 2 -40 Not enabled 120 closure - 3 -40 20±2 360 Open - 4 -20 20±2 360 Open - 5 -20 10±2 360 Open - 6 0 10±2 360 Open - 7 0 10±2 360 Open - 8 10 No control 360 Open - 9 10 No control 60 Open -

(2) Prescription process and tableting process

The freeze-dried powder (pH 1.2) and the freeze-dried powder (pH 4.0) were respectively passed through a 40-mesh sieve.

Weigh the raw and auxiliary materials according to the proportion designed in the prescription and mix them evenly.

Tablets were compressed using a 19×8 shaped punch, each containing 14 mg of API. During the compression process, tablet thickness was adjusted based on the hardness, maintaining a hardness of 50-100 N.

The dissolution characteristics of tablets with acceptable hardness and tablet weight were investigated.

Table 5 Prescription design (API content 14 mg)

(3) Dissolution evaluation

When the tableting scale was increased, the formulations B1 and B2 containing 0.5% magnesium stearate experienced sticking during tableting.

Dissolution test method:

Preparation of dissolution medium (pH 4.0, containing 1.0% SDS): Dissolve 1.22 g of sodium acetate trihydrate and 10 g of SDS in 950 ml of pure water, adjust the pH to 4.0 with acetic acid, and make up to 1 L.

The test used the rotating basket method (referring to the first method of dissolution and release determination in Part IV of the Chinese Pharmacopoeia 2015 edition, 0903). Each tablet was dissolved using 900 ml of dissolution medium at a rotation speed of 50 rpm and a temperature of 37°C. Sampling was performed at 5, 10, 15, 20, 30, 45, 60, and 90 minutes. After 60 minutes, the rotation speed was increased to 250 rpm, and the 90-minute sample was used as the terminal dissolution value for each tablet.

The amount of dissolved API was determined by HPLC (refer to the HPLC determination conditions for the content and related substances described in Test Example 2), and the dissolution rate was then calculated by the external standard method.

According to the above method, the dissolution rates of prescriptions B1, B3, and B4 were tested and compared, as shown in the following table:

Table 6 Dissolution results

Comparison of formulations B3 and B4 showed that whether the pH value of the drug-containing solution was adjusted from 1.2 to 4.0 before lyophilization had almost no effect on the dissolution results.

Comparison of prescriptions B1 and B4 shows that increasing the magnesium stearate content from 0.5% (w/w) to 1% (w/w) slowed down the API dissolution rate slightly within 15 minutes, but still reached more than 95% at 15 minutes.

Comparative Example 3: Comparative bioavailability test of tablets

The tablets of Prescription B4 in Example 7 were orally administered to dogs. For comparison, the test substance was intravenously injected into dogs at a dose equivalent to 0.5 mg/kg of KX2-361. The absolute bioavailability of the oral tablets was calculated.

How to do it:

1. Oral Administration: Administer five tablets orally to three healthy male beagle dogs (weight range 8-12 kg, dog numbers 201M, 201M, and 203M). Fast overnight before administration and resume feeding 4 hours after administration. Do not drink water for 1 hour before or after administration.

2. Intravenous injection:

Preparation of the test substance for intravenous administration: Accurately weigh 19.34 mg of KX2-361·benzenesulfonate and place it in a glass bottle. Add 5.565 mL of ethanol, vortex and sonicate at 24°C for 3 minutes, then add 33.393 mL of PEG400, vortex and sonicate at 24°C for 4 minutes, then add 16.696 mL of normal saline, vortex and sonicate at 24°C for 4 minutes to obtain a colorless, transparent solution with a theoretical concentration of 0.25 mg/mL.

The dogs were fasted overnight before administration and fed four hours after administration. The test substance was administered by slow intravenous injection (approximately 8 minutes) and no water was allowed before and within 1 hour after administration.

3. Bioavailability Determination: For both the oral and intravenous administration groups, blood (approximately 0.5 mL) was collected via cephalic puncture of the forelimb vein at 0.083 (for the intravenous administration group only), 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing into anticoagulant tubes containing 5 μL of 20% EDTA-K2. Blood samples were centrifuged at 8000 rpm for 6 minutes within 1 hour (placed on wet ice before centrifugation). The supernatant, i.e., plasma, was frozen at -20°C and analyzed by LC-MS/MS using methylsulfonamide as the internal standard, APCI ionization mode, MRM detection. Pharmacokinetic parameters are shown in the table below, and the time-dependent changes in plasma concentration are plotted in Figure 1. The area under the curve _(0-t) (AUC _(0-t) ) for the intravenous administration group was 164.3. The absolute bioavailability (F%) was calculated as follows: F% = (oral AUC _(0-t) / oral dose) ÷ (intravenous AUC _(0-t) / intravenous dose) × 100%.

Table 7

It can be seen that the tablets had a high bioavailability (average F% was 75.7%) when orally administered to dogs.

Other formulations were used to test animal absorption, such as formulations 1 and 2 (formulations and processes are shown in the table below). Oral administration was performed on dogs, and pharmacodynamic studies were conducted (using the same method as above). The absolute bioavailability was calculated, and the results are as follows:

It can be seen that compared with the other two formulation schemes, the inclusion complex of KX2-361·benzenesulfonate and hydroxypropyl-β-cyclodextrin and then tablet preparation achieved higher absorption (about 20 times), reflecting the significant effect of the excipient hydroxypropyl-β-cyclodextrin in improving the in vivo bioavailability of the drug in the formulation of the present invention.

Test Example 6: Stability Investigation

Tablets of prescription B3 (containing freeze-dried powder at pH 1.2) and prescription B4 (containing freeze-dried powder at pH 4.0) were placed in high-density polyethylene (HDPE) bottles with a desiccant (color-changing silica gel) and placed in a stability chamber at 40°C/75% RH for high-temperature accelerated testing. Samples were taken at the end of 1 month, 2 months, and 3 months, and the related substances were determined (refer to the content and HPLC determination conditions of related substances described in Test Example 2).

Table 8 Tablet stability test results of prescriptions B3 and B4

serial number sample Total impurities (%) 1 Prescription B3-0 days 0.36 (n=2) 2 Prescription B3-1 month 2.28 3 Prescription B3-2 months 3.88 4 Prescription B3-3 months 9.04 5 Prescription B4-0 days 0.30 (n=2) 6 Prescription B4-1 month 0.39 7 Prescription B4-2 months 0.26 8 Prescription B4-3 months 0.65

Note: n=2 means 2 samples were taken and measured separately. The result is the average of the two samples.

During the accelerated January-March inspection, impurities in Formulation B3 increased, likely due to its highly acidic environment. While impurities in Formulation B4 increased slightly, the change was not significant. This suggests that a pH adjustment process after inclusion is preferred for preparing oral tablets.

Example 8: Preparation and evaluation of tablets

(1) Freeze-drying process

Preparation of pH 1.2 hydrochloric acid aqueous solution: Place 9 ml of concentrated hydrochloric acid in a 1000 ml volumetric flask and make up to volume with distilled water.

To prepare a 30% HP-β-CD solution: Add 700 ml of pH 1.2 hydrochloric acid to a 1000 ml beaker. Weigh 300 g of HP-β-CD and add it to the pH 1.2 hydrochloric acid solution while magnetically stirring until completely dissolved. Add distilled water to 1000 ml. Reserve a sufficient amount for future use.

(1) Preparation of a drug-containing preparation at approximately 7 mg/ml (HP-β-CD:API molar ratio of 13-17:1, HP-β-CD:KX2-361 mass ratio of 100:1.68): 1750 mg of KX2-361 benzenesulfonate was weighed and added to 250 ml of the above HP-β-CD solution with stirring. The solution was completely dissolved by ultrasonication (ultrasonic frequency 59 kHz). The pH was adjusted to 4.0 with 5N aqueous NaOH and 0.1N aqueous NaOH. The solution was then filtered through a 0.45 μm PES filter and the filtrate was placed in a stainless steel tray to a liquid level of approximately 0.5 cm. After lyophilization, the solution was designated as lyophilized powder I (lyophilization method was the same as in Example 7, hereinafter the same).

(2) Preparation of a drug-containing preparation at approximately 14 mg/ml (HP-β-CD:API molar ratio of 7-8:1, HP-β-CD:KX2-361 mass ratio of 100:3.36); 4200 mg of KX2-361 benzenesulfonate was weighed and added to 300 ml of the above HP-β-CD solution with stirring. Ultrasonication (ultrasonic frequency 59 kHz) was performed to completely dissolve the drug. The drug was filtered through a 0.45 μm PES filter and the filtrate was placed in a stainless steel tray to a liquid level of approximately 0.5 cm. After lyophilization, the product was designated as lyophilized powder II.

(3) Preparation of a 21 mg/ml drug-containing preparation (HP-β-CD:API molar ratio of 4-6:1, HP-β-CD:KX2-361 mass ratio of 100:5.03): 6300 mg of KX2-361 benzenesulfonate was weighed and added to 300 ml of the above HP-β-CD solution with stirring. Ultrasonication (ultrasonic frequency 59 kHz) was performed for 30 minutes. The API was not completely dissolved, and a certain amount of insoluble particles were still visible. The preparation was filtered using a 0.45 μm PES filter membrane, and the filtrate was placed in a stainless steel tray with a liquid level of approximately 0.5 cm. The concentration of the 21 mg/ml drug-containing preparation after filtration was calibrated by HPLC and was 20.66 mg/ml. After lyophilization, it was named lyophilized powder III.

(2) Prescription composition and tableting process

The prepared freeze-dried powders were respectively passed through a 40-mesh sieve.

Weigh the raw and auxiliary materials according to the ratio designed in the following prescription.

A 19×8 shaped punch was used to compress the tablets, each containing 600 mg of HP-β-CD. During the tableting process, the tablet thickness was adjusted according to the hardness, and the hardness was controlled within 50-100N.

The dissolution characteristics of tablets with acceptable hardness and tablet weight were investigated according to the method described in Example 7. See Table 9.

Table 9 Dissolution test results (n=3)

Note: n=3 means 3 samples were taken and measured separately. The result is the average value of the 3 samples.

The dissolution test results are as follows:

All the prescriptions can be dissolved by more than 95% within 20 minutes.

As the API content increased, the dissolution rate decreased. There was little difference between prescriptions B5 and B6, but both were slower than prescription B4.

Experimental Example 7: Study on the dissolution effect of dissolution media containing different concentrations of SDS on high-dose tablets

In order to make the dissolution medium more able to demonstrate its ability to distinguish prescriptions, the dissolution behavior of the following prescriptions in different concentrations of SDS was investigated.

The dissolution rates of Formulation B3 of Example 7 and Formulations B5 and B6 of Example 8 were investigated in dissolution media containing 0.5% SDS, 0.2% SDS, and no SDS. The data are shown in Tables 10-12.

Dissolution testing was performed using a rotating basket method, 900 mL of dissolution medium, a rotation speed of 50 rpm, and a temperature of 37°C. Sampling was performed at 5, 10, 15, 20, 30, 45, and 60 minutes. The pH 4.0 dissolution medium was prepared as described in Example 7, except that 1 L of the dissolution medium containing 0.5% SDS, 0.2% SDS, and no SDS, respectively, was prepared using a pH 4.0 buffered saline solution containing 5 g, 2 g, or 0 g of SDS.

Table 10 Dissolution of three formulations in pH 4.0 dissolution medium without SDS

Table 11 Dissolution of three formulations in pH 4.0 dissolution medium containing 0.5% SDS

Table 12 Dissolution of three formulations in pH 4.0 dissolution medium containing 0.2% SDS

The observed phenomenon was that when the dissolution medium contained no SDS, a small amount of flocs were present in the dissolution cup of formulation B5, while a large amount of flocs were present in the dissolution cup of formulation B6. When the dissolution medium contained 0.2% or 0.5% SDS, flocs were present in the dissolution cup of formulation B6, but the flocs decreased after 30 minutes.

Conclusion: The absence of SDS significantly impacted sample dissolution, resulting in poor dissolution for all three formulations. Compared to the dissolution medium containing 0.5% SDS, the dissolution rate of formulation B3 in the presence of 0.2% SDS was similar to that in the medium containing 0.5% SDS. However, the dissolution rates of formulations B5 and B6 slowed, and the terminal dissolution rates decreased, indicating that 0.2% SDS is not suitable for the dissolution of these two formulations. When the dissolution medium contained 0.5% SDS, the dissolution of all three formulations was excellent and very similar to that in the medium containing 1% SDS. There was no significant difference in the dissolution characteristics of formulations B5 and B6.

Example 9: Preparation and evaluation of tablets

Tablets of prescription B16 were prepared by encapsulating 14 mg/ml of the drug in 40% HP-β-CD. The lyophilized powder was prepared using the same process as in Example 7 (pH adjusted to 4.0). The lyophilized powder was passed through a 40-mesh sieve, and the lyophilized powder and magnesium stearate (magnesium stearate was 1.0% (w/w) of the lyophilized powder) were weighed and mixed. Tablets were punched into 19×8 shaped tablets, weighing approximately 600 mg and with a hardness of 50-100 N. The disintegration time was 8 minutes. Dissolution results are as follows:

Table 15 Comparison of dissolution results between prescription B16 and prescription B3

Conclusion: Formulation B16 dissolves slightly slower than Formulation B3, but more than 85% of the drug has dissolved by 15 minutes. The drug loading (API content) per tablet of Formulation B16 is approximately 20.29 mg, a 45% increase compared to Formulation B3, achieving the goal of increasing drug loading.

Example 10: Preparation and evaluation of tablets

As shown in Experiment 6, the stability of the tablet containing the freeze-dried powder at pH 1.2 was poor, while the stability of the tablet containing the freeze-dried powder at pH 4.0 was significantly improved. Therefore, an attempt was made to adjust the pH to 6.0 to ensure its stability.

(1) Freeze-drying process

1. Preparation of pH 1.2 hydrochloric acid aqueous solution: Place 0.9 ml of concentrated hydrochloric acid in a 100 ml volumetric flask and dilute to 100 ml with distilled water.

2. Preparation of 40% HP-β-CD solution: Weigh 10 g of HP-β-CD, dissolve it in an appropriate amount of pH 1.2 hydrochloric acid solution, transfer it to a 25 ml volumetric flask and make up to volume with the above hydrochloric acid solution.

3. Preparation of a 14 mg/ml drug-containing preparation: Weigh 56 mg of KX2-361 benzenesulfonate and add it to 4 ml of the above HP-β-CD solution. Ultrasonicate (ultrasonic frequency 59 kHz) to completely dissolve the drug.

4. pH Adjustment: Adjust the pH of the drug-containing solution to approximately 6.0 using 5N NaOH and 0.1N NaOH. At this point, the solution is nearly colorless. No noticeable turbidity or precipitation is observed after standing for 30 minutes. A small amount of precipitation occurs overnight, and the liquid portion remains in solution.

5. Preparation of lyophilized powder: After adjusting the pH to approximately 6.0, filter through a 0.22 μm PES filter and lyophilize to obtain a white lyophilized powder. The lyophilization process is the same as in Example 7.

Reconstitution investigation: The freeze-dried powder was reconstituted with water under gentle shaking in about 10 minutes, and no obvious abnormalities were found.

(2) Prescription composition and tableting process

1. Pass the freeze-dried powder and each filler through a 40-mesh sieve respectively.

2. Weigh the API and fillers according to the designed dosage (see Tables 16 and 17), mix them, and then add equal amounts of the lubricant magnesium stearate in successive amounts. Pass the mixture through a 40-mesh sieve three times.

3. Use a Φ8mm die and compress the tablets to a weight of approximately 200mg (containing approximately 2mg of API). During the tableting process, adjust the tablet thickness according to the hardness, controlling the hardness between 50-100N.

Experimental results: All prescription tablets were successfully compressed.

Table 16 Prescription design 1

Table 17 Prescription design 2

prescription Prescription B11 Prescription B12 Prescription B13 Prescription B14 Prescription B15 Lyophilized powder (mg) 60 60 60 60 60 Spray dried lactose (mg) 128 128 134 128 Mannitol SD200 (mg) 134 PVPP XL (mg) 10 CC-Na (mg) 10 6 Magnesium stearate (mg) 2(1%) 2(1%) 6(3%) 6(3%) 6(3%)

The disintegration time and dissolution rate of the above-mentioned tablets were tested using a disintegration time tester (the method was based on the disintegration time test method in Appendix 0921 of Part IV of the Chinese Pharmacopoeia 2015 edition). The results are shown in Tables 18 and 19.

Table 18 Disintegration time results of each prescription tablet

prescription B7 B8 B9 B10 B11 B12 B13 B14 B15 Disintegration time (minutes) ~0.5 ~1 ~11 ~6 ~4 ~4 ~14 ~6 ~8

The results show that the disintegration time of tablets of prescriptions B9 and B13 exceeds 10 minutes, while the disintegration time of other prescriptions is shorter, which is more conducive to the rapid dissolution of the drug.

Table 19 Dissolution test results of each prescription tablet

The dissolution data show that formulations B9, B10, B13, B14, and B15 all dissolve slowly. The dissolution rates of the other formulations after 15 minutes are all greater than 90%, demonstrating the rapid dissolution of the drug from the immediate-release tablet.

Each tablet formulation was placed under conditions of high temperature (60°C), high humidity (92.5% RH), and -20°C. Samples were collected and tested at specific time points (referring to the HPLC determination conditions for content and related substances described in Experimental Example 2). The results were compared with the impurity growth of the API. The results are shown in Table 20.

Table 20 Stability test results of each prescription tablet

As shown in the table above, formulation B11 is less stable than the other formulations, and formulations B7 and B8 also perform poorly. The remaining formulations exhibited no abnormalities after 20 days of storage under various conditions. Based on the comprehensive dissolution data, formulation B12 is the superior formulation.

Claims (22)

1. An oral formulation for treating cell proliferative disorders, comprising hydroxypropyl-β-cyclodextrin and an active ingredient, said active ingredient being KX2-361 or a pharmaceutically acceptable salt thereof, said KX2-361 being represented by Formula 1: 2. The oral formulation according to claim 1, wherein the molar ratio of the active ingredient to hydroxypropyl-β-cyclodextrin is 1:(4-59). 3. The oral formulation according to claim 1, wherein the active ingredient is KX2-361, KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate and/or KX2-361 diphosphate. 4. The oral formulation according to claim 1, wherein at least a portion of the hydroxypropyl-β-cyclodextrin encapsulates at least a portion of the active ingredient to form a drug inclusion complex. 5. The oral formulation according to claim 1 further comprises a pharmaceutically acceptable excipient. 6. The oral formulation according to claim 5, wherein the pharmaceutically acceptable excipient is selected from one or more of fillers, disintegrants, and lubricants. 7. The oral formulation according to claim 6, wherein the filler is selected from one or more of microcrystalline cellulose, lactose, starch, and mannitol, and the amount of the filler is 0% to 69% (w/w) based on the total weight of the oral formulation. 8. The oral formulation according to claim 6, wherein the disintegrant is selected from one or more of croscarmellose sodium, croscarmellose, and carboxymethyl starch sodium, and the amount of the disintegrant is 0% to 5% (w/w) based on the total weight of the oral formulation. 9. The oral formulation according to claim 6, wherein the lubricant is selected from one or more of magnesium stearate, micronized silica gel, and talc, and the amount of the lubricant is 0.5% to 3% (w/w) based on the total weight of the oral formulation. 10. The oral formulation according to claim 1, wherein the oral formulation is in the form of a tablet, wherein the active ingredient is present in a concentration of 0.5% to 6.5% (w/w) in a single tablet. 11. A method for preparing an oral formulation according to any one of claims 1-10, comprising the following steps: A) Provide a first solution containing dissolved hydroxypropyl-β-cyclodextrin, wherein the pH of the first solution is 1-2; B) Mix KX2-361 or its pharmaceutically usable salt with the first solution to obtain a second solution containing KX2-361 or its pharmaceutically usable salt; C) Dry the second solution to obtain a dried product. 12. The method of claim 11, wherein the concentration of hydroxypropyl-β-cyclodextrin in the first solution is from 10% (w/v) to 50% (w/v). 13. The method according to claim 11, wherein, in the second solution, the concentration of KX2-361 or its pharmaceutically acceptable salt is from 0.5 mg/ml to 15 mg/ml. 14. The method according to claim 11, wherein, in the second solution, the molar ratio of KX2-361 or its pharmaceutically acceptable salt to hydroxypropyl-β-cyclodextrin is 1:(4-59). 15. The method of claim 11, wherein, before step C) and after step B), it further comprises I) adjusting the pH of the second solution to 3-7. 16. The method according to claim 11, wherein, after step C), it further includes D) compressing the dried material into tablets. 17. The method of claim 16, wherein step D) comprises mixing the dried material with one or more excipients selected from fillers, disintegrants, lubricants, and then performing the tableting. 18. The method of claim 17, wherein the filler is selected from one or more of microcrystalline cellulose, lactose, starch, and mannitol, and the amount of the filler is 0% to 69% (w/w) based on the total weight of the oral formulation. 19. The method of claim 17, wherein the disintegrant is selected from one or more of croscarmellose sodium, croscarmellose, and carboxymethyl starch sodium, and the amount of the disintegrant is 0% to 5% (w/w) based on the total weight of the oral formulation. 20. The method of claim 17, wherein the lubricant is selected from one or more of magnesium stearate, micronized silica gel, and talc, and the amount of the lubricant is 0.5% to 3% (w/w) based on the total weight of the oral formulation. 21. The method according to claim 11, wherein the pharmaceutically acceptable salt of KX2-361 is KX2-361 monobenzenesulfonate, KX2-361 dihydrochloride, KX2-361 monophosphate and/or KX2-361 diphosphate. 22. The method according to claim 13, wherein, in the second solution, the concentration of KX2-361 or its pharmaceutically acceptable salt is from 0.5 mg/ml to 10 mg/ml.