Pharmaceutical composition of a chemotherapeutic agent based on a β-substituted β-amino acid derivative

Abstract

A pharmaceutical composition comprising a chemotherapeutic agent based on a β-substituted β-amino acid derivative is disclosed. The pharmaceutical composition comprises a β-substituted β-amino acid derivative and a cyclodextrin derivative. The pharmaceutical composition is useful for treating cancer.

Description

Technical Field

[0001] This application claims the benefit of PCT International Application No. PCT / CN2021 / 070782, filed on January 8, 2021, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to a pharmaceutical composition of a chemotherapeutic agent based on a β-substituted β-amino acid derivative. The pharmaceutical composition includes a β-substituted β-amino acid derivative and a cyclodextrin derivative. The pharmaceutical composition is useful for the treatment of cancer.

Background Art

[0003] The ability to selectively target chemotherapy has great value in clinical practice. Cancer is the leading cause of death in developed countries, and one in three people will develop cancer in their lifetime. There are many treatment options for cancer, including surgery, chemotherapy, radiotherapy, immunotherapy, and monoclonal antibody therapy. Unfortunately, for many patients, the options for cancer treatment are limited and the response rate remains low.

[0004] β-Substituted β-amino acid derivatives can be used as LAT1 transport chemotherapeutic agents. Certain β-substituted β-amino acid derivatives are unstable in aqueous buffer solutions suitable for intravenous administration.

Summary of the Invention

[0005] According to the present invention, the guest-host inclusion complex is a compound of formula (I):

Chemical Formula

[0006] According to the present invention, a pharmaceutical composition comprises a compound of formula (I): [Chemical formula] or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, wherein R 1 is selected from C 1-6 alkyl and C 1-6 alkoxy, a compound of formula (I), or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, and a cyclodextrin derivative of formula (2): [Chemical formula] or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, wherein n is selected from 4, 5, and 6, R 1 ~R 9 each independently is hydrogen, C 1-8 alkanediylsulfonate, C 1-6 alkyl, and substituted C 1-6selected from alkyl, R 1 ~R 9 at least one of which is C 1-8 a cyclodextrin derivative of formula (2), or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, wherein the group is an alkanediylsulfonate.

[0007] According to the present invention, a pharmaceutical kit comprises a guest-host inclusion complex according to the present invention and an aqueous solution.

[0008] According to the present invention, a method of treating cancer in a patient comprises administering to a patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] For the purposes of the following detailed description, it is to be understood that the embodiments provided by this disclosure may assume various alternative variations and step sequences unless explicitly specified to the contrary. Further, except where otherwise indicated by way of an example of an operation or otherwise, all numbers expressing amounts of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0010] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0011] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, the range "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, i.e., having a minimum value of 1 or more and a maximum value of 10 or less.

[0012] "Alkoxy" refers to a radical -OR where R is alkyl. Examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy. The alkoxy group can be, for example, C 1-6 alkoxy, C 1-5 alkoxy, C 1-4 alkoxy, C 1-3 alkoxy, ethoxy, or methoxy.

[0013] "Alkyl" refers to a saturated, branched, or straight-chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane. The alkyl group can be, for example, C 1-6 alkyl, C 1-5 alkyl, C 1-4 alkyl, or C 1-3 alkyl. The alkyl group can be methyl, ethyl, n-propyl, iso-propyl, or tert-butyl.

[0014] "Alkandiyl" refers to a saturated, branched-chain, or straight-chain divalent hydrocarbon radical derived by removing two hydrogen atoms from one or two carbon atoms of a parent alkane. The alkandiyl group can be, for example, C 1-8 alkandiyl, C 1-6 alkandiyl, C 1-5 alkandiyl, C 1-4 alkandiyl, or C 1-3 alkandiyl. The alkandiyl can be, for example, methane-diyl, ethane-diyl, n-propane-diyl, iso-propane-diyl, or butane-diyl.

[0015] "Alkane diyl sulfonate" refers to an alkane diyl group in which one of the carbon atoms is bonded to a sulfonate, -SO 3 - X + group (wherein X + is a counter cation). In alkane diyl sulfonate, the sulfonate can associate with the terminal carbon of the alkane diyl group. Alkane diyl sulfonate can be, for example, C 1-8 alkane diyl sulfonate, C 1-6 alkane diyl sulfonate, C 1-5 alkane diyl sulfonate, C 1-4 alkane diyl sulfonate, or C 1-3 alkane diyl sulfonate. In alkane diyl sulfonate, the terminal carbon atom can be substituted with a sulfonate. For example, C 1-4 alkane diyl sulfonate has the structure -CH 2 -SO 3 - X + , -CH 2 -CH 2 -SO 3 - X + , or -CH 2 -CH 2 -CH 2 -SO a - X + , or -CH 2 -CH 2 -CH 2 -CH 2 -SO a - X + . The counter cation can be, for example, Na + and C 1-8 alkane diyl sulfonate.

[0016] "Substituted" refers to a group in which one or more hydrogen atoms are independently substituted with the same or different substituents. Each substituent is independently a halogen, -OH, -CN, -CF 3 , -OCF 3 , =O (oxo), -NO 2 , C1-6 Alkoxy, C 1-6 alkyl, -COOR, -NR 2 , and -CONR 2 may be selected from, and each R is independently hydrogen and C 1-6 alkyl. Each substituent is independently halogen, -NH 2 , -OH, C 1-3 alkoxy, and C 1-3 alkyl, trifluoromethoxy, and trifluoromethyl. Each substituent may be independently selected from -OH, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, and trifluoromethoxy. Each substituent may be selected from C 1-3 alkyl, =O (oxo), C 1-3 alkyl, C 1-3 alkoxy, and phenyl. Each substituent may be selected from -OH, -NH 2 , C 1-3 alkyl, and C 1-3 alkoxy.

[0017] "Average degree of substitution" (ADS) refers to the average number of substituents per cyclodextrin molecule. The concept of the average degree of substitution of cyclodextrin derivatives is described in PCT International Publication No. WO2009 / 018069. As an example, the following notations may be used to describe cyclodextrin derivatives. Substituents are abbreviated with subscripts indicating the ADS of the substituent. For example, sulfobutyl ether-derivatized β-cyclodextrin having an ADS of 6.5 is denoted as SBE 6.5 -β-CD (wherein SBE is an abbreviation for the sulfobutyl ether group). As another example, β-cyclodextrin derivatized with both a sulfobutyl ether group and a hydroxypropyl group is denoted as SBE 4.2 -HP 2.5 -β-CD (wherein the ADS of the sulfobutyl ether (SBE) group is 4.2 and the ADS of the hydroxypropyl (HP) group is 2.5).

[0018] The "compounds" disclosed herein include any specific compound within the disclosed formula. Compounds can be identified by either their chemical structure and / or chemical name. Compounds are named using the ChemBioDraw Ultra 14.0.0.117 (CambridgeSoft, Cambridge, MA) naming program. If there is a conflict between the chemical structure and the chemical name, the chemical structure determines the identity of the compound. The compounds described herein may contain one or more asymmetric centers and / or double bonds, and thus can exist as stereoisomers such as double bond isomers (i.e., geometric isomers), enantiomers, diastereomers, tautomers, or atropisomers. Accordingly, any chemical structure having the relative configuration within the scope shown herein includes, in whole or in part, stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as all possible enantiomers and stereoisomers of the exemplified compounds, including mixtures of enantiomers and (diastereomer) stereoisomers. Mixtures of enantiomers and (diastereomer) stereoisomers can be separated into their component enantiomers or (diastereomer) stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art.

[0019] The compounds of formula (1) include the compounds of formula (1a), the compounds of formula (1b), the compounds of formula (1c), and any combination of the foregoing.

[0020] "Cyclodextrin derivative" refers to a cyclic oligosaccharide containing five or more α-D-glucopyranoside units linked in a cyclic arrangement and containing substituents attached to one or more glucopyranoside units at the 2, 3, and / or 6 positions via γ-1,4-glycosidic linkages.

[0021] The "nominal concentration" of the compounds of formula (1) refers to the concentration of the complex compounds of formula (1) and the non-complex compounds of formula (1) in a composition or solution.

[0022] "Patient" refers to a mammal, e.g., a human.

[0023] "Pharmaceutically acceptable" refers to approved or approvable substances that are described in the regulations of the federal or state government regulatory agencies or in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more specifically, in humans.

[0024] "Pharmaceutically acceptable salts" refer to salts of compounds that possess the desired pharmacological activity of the parent compound. Such salts include inorganic acids, as well as acid addition salts formed with one or more protonable functional groups such as primary, secondary, or tertiary amines within the parent compound. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. The salts can be formed with organic acids, for example, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucic acid, and the like. The salts can be formed when one or more acidic protons present in the parent compound are replaced by coordination with metal ions, such as alkali metal ions, alkaline earth metal ions, or aluminum ions, or combinations thereof, or with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. The pharmaceutically acceptable salts can be hydrochloride salts. The pharmaceutically acceptable salts can be sodium salts. In the case of compounds having two or more ionizable groups, the pharmaceutically acceptable salts can contain one or more counterions such as dibasic salts, for example, dihydrochloride salts. Examples of pharmaceutically acceptable salts are disclosed, for example, in Stahl and Wermuth (Eds), Handbook of Pharmaceutical Salts, Properties, Selection and Use, First Edition, Wiley-VCH, 2008.

[0025] "Pharmaceutically acceptable salts" include hydrates and other solvates, as well as salts in crystalline or amorphous form. When a particular pharmaceutically acceptable salt is disclosed, the particular salt (e.g., hydrochloride) is an example of a salt, and it should be understood that other salts can be formed using techniques known to those skilled in the art. In addition, using techniques generally known in the art, those skilled in the art will be able to convert pharmaceutically acceptable salts into the corresponding compounds, free bases, and / or free acids.

[0026] "Pharmaceutically acceptable vehicle" refers to a pharmaceutically acceptable diluent, pharmaceutically acceptable adjuvant, pharmaceutically acceptable excipient, pharmaceutically acceptable carrier, or any combination of the foregoing, with which the compounds provided by the present disclosure can be administered to a patient, which does not destroy its pharmacological activity and is non-toxic when administered in a dosage sufficient to provide a therapeutically effective amount of the compound.

[0027] "Pharmaceutical composition" refers to a β-substituted β-amino acid derivative provided by the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable vehicle administered to a patient together with the β-substituted β-amino acid derivative or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable vehicles are known in the art.

[0028] "Curing" a disease means eliminating the disease or disorder, or eliminating the symptoms of the disease or disorder.

[0029] "Disease" refers to any of the foregoing diseases, disorders, conditions or symptoms.

[0030] "Treating" or "treatment" of a disease or disorder means reducing the severity of one or more clinical symptoms of the disease or disorder, delaying the onset of one or more clinical symptoms of the disease or disorder, and / or alleviating one or more clinical symptoms of the disease or disorder.

[0031] "Treating" or "treatment" of a disease or disorder refers to inhibiting the disease or disorder or one or more clinical symptoms thereof, preventing the onset of the disease or disorder or one or more clinical symptoms thereof, alleviating the disease or disorder or one or more clinical symptoms thereof, causing regression of the disease or disorder or one or more clinical symptoms thereof, and / or stabilizing the disease or disorder or one or more clinical symptoms thereof, and "treating" or "treatment" refers to producing a clinically beneficial effect without curing the underlying disease or disorder.

[0032] "Therapeutically effective amount" refers to the amount of a compound, such as a pharmaceutically active ingredient, which is sufficient to affect the treatment of a disease or one of its clinical symptoms when administered to a patient for treating at least one of the disease or its clinical symptoms. The "therapeutically effective amount" can vary, for example, depending on the compound, the disease and / or its symptoms, the severity of the disease, and / or the symptoms of the disease or disorder, the age, weight, and / or health of the patient being treated, and the judgment of the prescribing physician. The therapeutically effective amount in any given case can be ascertained by one of ordinary skill in the art or can be determined by routine experimentation.

[0033] "Therapeutically effective dosage" refers to the dosage that provides effective treatment of a disease or disorder in a patient. The therapeutically effective dosage can vary depending on the compound and can vary among patients and can depend on factors such as the condition of the patient and the route of delivery. The therapeutically effective dosage can be determined according to routine pharmacological procedures known to those of ordinary skill in the art.

[0034] "Vehicle" refers to a diluent, excipient, or carrier that is administered to a patient together with a compound. The vehicle can be a pharmaceutically acceptable vehicle. Pharmaceutically acceptable vehicles are known in the art.

[0035] "About" refers to within 5% of a particular value, for example, within 5% of a stated concentration range or within 5% of a stated time frame.

[0036] Reference is now made to pharmaceutical compositions and methods of using the pharmaceutical compositions. The disclosed pharmaceutical compositions and methods of using the pharmaceutical compositions are not intended to limit the claims. On the contrary, the claims are intended to cover all alternatives, modifications, and equivalents.

[0037] The guest-host inclusion complex provided by the present disclosure can include a β-substituted β-amino acid derivative and a cyclodextrin derivative. The guest-host inclusion complex can be in the form of a lyophilized product.

[0038] The pharmaceutical composition provided by the present disclosure can include a β-substituted β-amino acid derivative and a cyclodextrin derivative. The pharmaceutical composition can include the guest-host inclusion complex provided by the present disclosure reconstituted in an aqueous formulation. The pharmaceutical composition provided by the present disclosure can be an aqueous solution for intravenous injection. The pharmaceutical composition provided by the present disclosure can be useful in the treatment of cancer.

[0039] Cyclodextrins have been used to improve the solubility and stability of chemotherapeutic agents such as melphalan. Cyclodextrins are a family of cyclic oligosaccharides consisting of a macrocyclic ring of glucose subunits linked by α-1,4 glycosidic bonds. Cyclodextrins are produced from starch by enzymatic conversion. They are used in food, pharmaceuticals, drug delivery, the chemical industry, agriculture, and environmental engineering. Cyclodextrins contain five or more α-D-glucopyranoside units, like amylose, which is a fragment of starch. Typical cyclodextrins contain a number of glucose monomers in the range of 6 to 8 units in the ring and form a toroidal shape. For example, α-cyclodextrin contains six glucose subunits, β-cyclodextrin contains seven glucose subunits, and γ-cyclodextrin contains eight glucose subunits.

[0040] Sulfoalkyl-substituted cyclodextrins are water-soluble and characterized by a hydrophilic outer surface surrounding an internal lipophilic cavity. Cyclodextrins and lipophilic therapeutic agents can form guest-host inclusion complexes and enhance the physicochemical properties of the drug.

[0041] The guest-host inclusion complex or pharmaceutical composition provided by the present disclosure can include a β-substituted β-amino acid derivative or a combination of β-substituted β-amino acid derivatives.

[0042] The β-substituted β-amino acid derivative has the structure of formula (1) or a combination of β-substituted β-amino acid derivatives of formula (1)

Chemical formula

[0043] In the compound of formula (1), R 1 can be C 1-6 alkyl.

[0044] In the compound of formula (1), R 1 can be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl.

[0045] In the compound of formula (1), R 1 can be C 1-6 alkoxy

[0046] In the compound of formula (1), R 1 can be selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

[0047] In the compound of formula (1), the carbon atom to which the amino group is attached may have the (S) absolute configuration

[0048] In the compound of formula (1), the carbon atom to which the amino group is attached may have the (R) absolute configuration

[0049] The compound of formula (1) can be a mixture having both (S) and (R) enantiomers, such as a racemic mixture having (S) and (R) enantiomers in a ratio of 1:1, or a non-racemic mixture such as a mixture having about 75% (S) enantiomer and about 25% (R) enantiomer. The compound of formula (1) can contain, for example, X% (S) enantiomer and 100% - X% (R) enantiomer (where X is from 0 to 100).

[0050] The compound of formula (1) can be 3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (1a), or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof.

Chemical formula

[0051] The compound of formula (1) can be (R)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (1b), or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof.

Chemical formula

[0052] The compound of formula (1) can be (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (1c), or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof.

Chemical formula

[0053] In the compound of formula (1), the pharmaceutically acceptable salt can be the monohydrochloride.

[0054] In the compound of formula (1), the pharmaceutically acceptable salt can be the bis(hydrochloride) salt.

[0055] The salt form of the compound of formula (1) can depend on the pH of an aqueous solution containing the compound of formula (1).

[0056] The compound of formula (1) can be a free base, zwitterion, or inner salt.

[0057] The compound of formula (1) can have an enantiomeric purity of, for example, greater than about 90%, greater than about 95%, greater than about 98%, greater than about 99%, greater than about 99.5%, or greater than about 99.9%.

[0058] The compound of formula (1) is a substrate of LAT1 / 4F2hc (large amino acid 1 transporter).

[0059] The method for synthesizing the compound of formula (1) is disclosed in U.S. Patent No. 9,394,237 and U.S. Patent No. 9,783,487, each of which is incorporated herein by reference in its entirety.

[0060] The guest-host inclusion complex or pharmaceutical composition provided by the present disclosure can include a cyclodextrin derivative or a combination of cyclodextrin derivatives.

[0061] The cyclodextrin derivative has the structure of formula (2):

Chemical formula

[0062] In the cyclodextrin derivative of formula (2), n can be 4, 5, or 6.

[0063] In the cyclodextrin derivative of formula (2), C 1-8 alkanediyl sulfonate can be selected, for example, from sulfonates of sulfoethyl, sulfopropyl, 1-methyl-sulfopropyl, sulfobutyl, 1-methyl-sulfobutyl, 2-methyl-sulfobutyl, 1-methyl-sulfobutyl-3-yl, 2-ethyl-sulfobutyl, 3-ethyl-sulfobutyl, sulfopentyl, 1-sulfopent-3-yl, sulfhexyl, sulfheptyl, and sulfooctyl.

[0064] In the cyclodextrin derivative of formula (2), C 1-8 alkanediyl sulfonate is, for example, -(CH 2 ) 1 -SO 3 - X + -, -(CH 2 ) 2 -SO 3 - X + -, -(CH 2 ) 3 -SO 3 - X + -, -(CH 2 ) 4 -SO 3 - X + -, -(CH 2 ) 5 -SO 3 - X + -, -(CH 2 ) 6 -SO3 - X + 、 -(CH 2 ) 7 -SO 3 - X + 、 and -(CH 2 ) 8 -SO 3 - X + (wherein X + can be selected from, for example, Li + , Na + , K + , Mg 2+ , and Ca 2+ ) can be selected.) can be selected.

[0065] C 1-6 The alkyl group can be selected from, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0066] Substituted C 1-6 The alkyl group can be selected from, for example, substituted methyl, substituted ethyl, substituted n-propyl, substituted isopropyl, substituted n-butyl, substituted iso-butyl, substituted tert-butyl, substituted n-pentyl, and substituted n-hexyl.

[0067] Substituted C 1-6 The alkyl group can be a hydroxyl-substituted C 1-6 alkyl group (for example, 2-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 3-oxobutyl, or 2-ethoxy-ethyl).

[0068] The cyclodextrin derivative of formula (2) can have, for example, an average degree of substitution (ADS) of about 4 to about 8, about 5 to about 8, about 5.5 to about 7.5, about 6 to about 7.5, about 6 to about 7, or about 6.5 to about 7.

[0069] In the cyclodextrin derivative of formula (2), each R 1 ~ R 9 is independently hydrogen and C1-8 It may be selected from alkanediyl sulfonates, and can have an ADS of, for example, about 4 to about 8, about 5 to about 8, about 5.5 to about 7.5, about 6 to about 7.5, about 6 to about 7, or about 6.5 to about 7.

[0070] In the cyclodextrin derivative of formula (2), R 1 ~R 9 At least one of them may be a hydroxy-substituted C 3 alkyl such as hydroxy-substituted C 1-6 alkyl group, and can have an ADS of, for example, about 1 to about 8, about 2 to about 8, about 3 to about 7, or about 4 to about 7.

[0071] In the cyclodextrin derivative of formula (2), the (counter) cation can be, for example, Li + , Na + , K + , Mg 2+ , Ca 2+ , a quaternary ammonium cation (for example, C 1-8 tetraalkylammonium), and an amine cation (for example, C 1-6 alkylamine, C 4-8 cycloalkylamine, C 1-6 alkanolamine, and C 4-8 cycloalkylamine).

[0072] In the cyclodextrin derivative of formula (2), the cation can be Na + . The cyclodextrin derivative of formula (2) can be a polynatrium salt or a mixture of salts.

[0073] In the cyclodextrin derivative of formula (2), each of the (counter) cations can be, for example, sodium, and the number of sodium cations is equal to the number of sulfonic acid groups.

[0074] The cyclodextrin derivative of formula (2) can have the structure of formula (2a),

Chemical formula

[0075] The cyclodextrin derivative of formula (2a) can have an ADS of about 6 to about 7.5, about 6.2 to about 7.3, or about 6.5 to about 7.1.

[0076] The cyclodextrin derivative of formula (2a) can be, for example, an SBE-β-CD (sulfobutylether-β-cyclodextrin) derivative having an ADS of about 6 to about 7.5, about 6.2 to about 7.3, or about 6.5 to about 7.1.

[0077] The cyclodextrin derivative of formula (2a) can be, for example, an SBE-β-CD derivative having an ADS of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0078] Examples of suitable sulfobutylether-β-cyclodextrin derivatives of formula (2a) having an ADS of about 6 to about 7 are available, for example, as Captisol®. Captisol® cyclodextrin is a polyanionic β-cyclodextrin derivative having a sodium sulfonate salt separated from the lipophilic cyclodextrin cavity by a butyl ether spacer group. The Captisol® cyclodextrin derivative is available from CyDex Pharmaceuticals, Inc and Ligand Pharmaceuticals. The Dexolve® cyclodextrin derivative is also available from CycloLab Cyclodextrin Research and Development Laboratory Ltd.

[0079] The cyclodextrin derivative of formula (2) is SBE 6.5It can contain -β-CD.

[0080] The guest - host inclusion complex provided by the present disclosure can contain a β - substituted β - amino acid derivative of formula (1) and a cyclodextrin derivative of formula (2).

[0081] The guest - host inclusion complex can contain a weight ratio of the compound of formula (1) to the cyclodextrin of formula (2), for example, about 1:1 to about 1:100, about 1:10 to about 1:90, about 1:20 to about 1:80, about 1:30 to about 1:70, or about 1:40 to about 1:60. The guest - host inclusion complex can contain a weight ratio of the compound of formula (1) to the cyclodextrin of formula (2), for example, about 1:48 to about 1:60, about 1:50 to about 1:58, or about 1:52 to about 1:56.

[0082] The guest - host inclusion complex can contain a molar ratio of the compound of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:6 to about 1:11, about 1:7 to about 1:10, about 1:7.5 to about 1:9.5, about 1:8 to about 1:9, or about 1:8.2 to about 1:8.8.

[0083] The guest - host inclusion complex can contain a molar ratio of the compound of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:7.4 to about 1:9.25.

[0084] The guest - host inclusion complex can be a lyophilized product. The lyophilized product can be prepared by dissolving a β - substituted β - amino acid derivative of formula (1) and a cyclodextrin derivative of formula (2) in water adjusted to a suitable pH, and then lyophilizing the solution to provide the corresponding lyophilized guest - host inclusion complex.

[0085] The pharmaceutical composition provided by the present disclosure can contain a guest - host inclusion complex of a β - substituted β - amino acid derivative of formula (1) and a cyclodextrin derivative of formula (2).

[0086] The pharmaceutical composition provided by the present disclosure can include a β-substituted β-amino acid derivative of formula (1) and a cyclodextrin derivative of formula (2).

[0087] The pharmaceutical composition provided by the present disclosure can include a mass ratio of the compound of formula (1) to formula (2), for example, about 1:1 to about 1:100, about 1:10 to about 1:90, about 1:20 to about 1:80, about 1:30 to about 1:70, or about 1:40 to about 1:60. The pharmaceutical composition can include a weight ratio of the compound of formula (1) to formula (2), for example, about 1:48 to about 1:60, about 1:50 to about 1:58, or about 1:52 to about 1:56. A mass ratio of 1:1 means 50 mg of compound (1): 50 mg of cyclodextrin derivative. SBE 6.5 When the average MW of SBE-β-CD is 2,163 g / mol, this corresponds to 150 μmol of compound (1): 23.1 μmol of SBE 6.5 -β-CD in molar ratio. A 1:1 molar ratio corresponds to 50 mg of compound (1): 324.5 mg of SBE 6.5 -β-CD in mass ratio or a mass ratio of 1:6.49.

[0088] The pharmaceutical composition provided by the present disclosure can include a molar ratio of the compound of formula (1) to the compound of formula (2), for example, about 1:6 to about 1:11, about 1:7 to about 1:10, about 1:7.5 to about 1:9.5, about 1:8 to about 1:9, or about 1:8.2 to about 1:8.8.

[0089] The pharmaceutical composition can include a molar ratio of the compound of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:7.4 to about 1:9.25.

[0090] The pharmaceutical composition can include an aqueous formulation.

[0091] The reconstituted aqueous formulation can have a nominal concentration of the β-substituted β-amino acid derivative of formula (1), for example, from about 1 mg / mL to about 9 mg / mL, from about 2 mg / mL to about 8 mg / mL, from about 3 mg / mL to about 7 mg / mL, from about 4 mg / mL to about 6 mg / mL, or from about 4.5 mg / mL to about 5.5 mg / mL.

[0092] The aqueous dosing formulation can have a nominal concentration of the β-substituted β-amino acid derivative of formula (1), for example, from about 0.1 mg / mL to about 2.0 mg / mL, from about 0.2 mg / mL to about 1 mg / mL, from about 0.2 mg / mL to about 0.8 mg / mL, from about 0.3 mg / mL to about 0.7 mg / mL, or from about 0.4 mg / mL to about 0.6 mg / mL.

[0093] The aqueous formulation can contain a combination of a β-substituted β-amino acid derivative of formula (1) and a cyclodextrin derivative of formula (2) suspended in a sodium chloride solution such as a sodium chloride solution from about 0.4% w / v to about 1.5% w / v, a sodium chloride solution from about 0.6% w / v to about 1.3% w / v, or a sodium chloride solution from about 0.8% w / v to about 1.1% w / v. The aqueous solution can be a sodium chloride solution such as a physiological saline aqueous solution of about 0.9% w / v.

[0094] The pharmaceutical composition provided by the present disclosure can include an aqueous solution having a nominal concentration of the compound of formula (1), for example, from about 3.0 mg / mL to about 7.0 mg / mL, for example, from about 4.0 mg / mL to about 6.0 mg / mL, or from about 4.5 mg / mL to about 5.5 mg / mL. The pharmaceutical composition can include an aqueous solution having a nominal concentration of the compound of formula (1) of about 5.0 mg / mL. The aqueous solution can be a sodium chloride solution, for example, a physiological saline aqueous solution of about 0.9% w / v, for example, a sodium chloride solution of about 154 mM.

[0095] In the case of intravenous administration, the pharmaceutical composition can include, for example, an aqueous solution having a nominal concentration of the compound of formula (1) of about 0.3 mg / mL to about 0.7 mg / mL, for example about 0.4 mg / mL to about 0.6 mg / mL, or about 0.45 mg / mL to about 0.55 mg / mL. The pharmaceutical composition can include an aqueous solution having a nominal concentration of the compound of formula (1) of about 0.5 mg / mL. The aqueous solution can be a sodium chloride solution, for example about 0.9% aqueous physiological saline solution.

[0096] The aqueous formulation can have a pH, for example, of about 3 to about 7, about 3.5 to about 6.5, about 4 to about 6, or about 4 to about 5.

[0097] The pharmaceutical composition can include a lyophilized product. The lyophilized product can include, for example, a lyophilized product of the aqueous formulation provided by the present disclosure. The lyophilized product can be storage-stable. For example, it can be included in a kit containing an aqueous diluent so that the lyophilized product can be reconstituted in the aqueous diluent to provide an aqueous formulation (for example, an aqueous injectable formulation). For example, the lyophilized product provided by the present disclosure can be stable at 5°C for 6 months, 12 months, or 18 months. For example, the lyophilized product provided by the present disclosure can be stable at 25°C / 60% RH for 6 months, 3 months, or 6 months in form. For example, the lyophilized product provided by the present disclosure can be photo-stable as determined using the ICH photo-stability test method.

[0098] The pharmaceutical composition provided by the present disclosure can include a mass ratio of the β-substituted β-amino acid derivative of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:1 to about 1:100, about 1:10 to about 1:90, about 1:20 to about 1:80, about 1:30 to about 1:70, or about 1:40 to about 1:60. The pharmaceutical composition provided by the present disclosure can include a mass ratio of the β-substituted β-amino acid derivative of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:48 to about 1:60, about 1:50 to about 1:58, or about 1:52 to about 1:56.

[0099] The pharmaceutical composition provided by the present disclosure can include a mass ratio of the β-substituted β-amino acid derivative of formula (1) to the cyclodextrin derivative of formula (2) of about 1.54, for example, about 1:50 to about 1:58, about 1:51 to about 1:57, about 1:52 to about 1:56, or about 1:53 to about 1:55.

[0100] The pharmaceutical composition provided by the present disclosure can include a molar ratio of the β-substituted β-amino acid derivative of formula (1) to the cyclodextrin derivative of formula (2), for example, about 1:6 to about 1:11, about 1:7 to about 1:10, about 1:7.5 to about 1:9.5, about 1:8 to about 1:9, or about 1:8.2 to about 1:8.8.

[0101] The pharmaceutical composition provided by the present disclosure can include a nominal concentration of the compound of formula (1), for example, about 0.25 mg / mL to about 2.0 mg / mL, for example, about 0.25 mg / mL, about 0.5 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL.

[0102] The pharmaceutical composition provided by the present disclosure can include, for example, a sodium chloride solution, for example, an aqueous physiological saline solution of about 0.9% w / v.

[0103] The pharmaceutical composition can include one or more pharmaceutically acceptable excipients. The pharmaceutical composition can include a pharmaceutically acceptable buffer and / or a pH adjuster (e.g., an acidifying agent or an alkalizing agent). After dilution with an aqueous diluent, the pharmaceutical composition can have a pH of, for example, about 3 to about 6, for example, about 4 to about 6, or about 4.5 to about 5.5.

[0104] The pharmaceutical composition can include a pH adjuster in an amount sufficient to provide a diluted composition having a pH of about 4 to about 6. The pharmaceutical composition can include, as a pH adjuster, an aqueous solution of NaHCO 3 or an aqueous solution of NaOH. The pharmaceutical composition can include an aqueous solution of HCl as a pH adjuster.

[0105] Pharmaceutical compositions provided by the present disclosure, such as an aqueous sodium chloride solution, can be stable, for example, at a temperature of about 25°C for about 2 hours, about 4 hours, or about 8 hours.

[0106] Pharmaceutical compositions provided by the present disclosure, such as an aqueous sodium chloride solution, can be stable, for example, at a temperature of about 0°C for about 12 hours, about 24 hours, or about 36 hours.

[0107] A stable pharmaceutical composition refers to a composition in which, for example, after storage under the described storage conditions including the indicated time and indicated temperature, more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 98% of the initial amount of the compound of formula (1), or its pharmaceutically acceptable zwitterion, inner salt, or salt remains.

[0108] For example, an approximately 0.9% aqueous saline solution containing a concentration of the compound of formula (1) / cyclodextrin inclusion-host complex (e.g., the HCl salt of the compound of formula (1c) / SBE 6.5 -β-CD inclusion-host complex) in a concentration of about 0.125 mg / mL to about 2 mg / mL can be storage-stable at about 25°C for about 2 to about 12 hours and at about 0°C for about 12 hours to about 48 hours. For example, an approximately 0.9% aqueous saline solution containing a concentration of the compound of formula (1) / cyclodextrin inclusion-host complex (e.g., the HCl salt of the compound of formula (1c) / SBE 6.5 -β-CD inclusion-host complex) in a concentration of about 0.125 mg / mL to about 2 mg / mL can be storage-stable at about 25°C for about 2 hours and at about 0°C for about 12 hours to about 48 hours.

[0109] For example, an approximately 0.9% aqueous saline solution containing a concentration of the compound of formula (1) / cyclodextrin inclusion-host complex (e.g., the free base of the compound of formula (1c) (compound (1c)) / SBE 6.5 -β-CD inclusion-host complex) in a concentration of about 0.5 mg / mL to about 2 mg / mL can be storage-stable at about 25°C for about 5 to about 19 hours and at about 0°C for about 31 hours to about 46 hours.

[0110] After the lyophilized product containing the compound of formula (1) and the cyclodextrin derivative of formula (2) could be reconstituted in an aqueous solution (for example, an aqueous solution such as about 0.9% physiological saline solution), the aqueous pharmaceutical solution may be suitable for intravenous administration, for example, for about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, or about 240 minutes.

[0111] The pharmaceutical kit provided by the present disclosure can include a lyophilized product of the pharmaceutical composition provided by the present disclosure and an aqueous solution for reconstituting the lyophilized product to provide a formulation suitable for intravenous administration. The kit can be used to treat cancer (for example, cancer of the central nervous system, brain cancer, metastatic cancer, metastatic cancer of the central nervous system, or metastatic cancer of the brain) in a patient.

[0112] The lyophilized product containing the β-substituted β-amino acid derivative of formula (1) and the cyclodextrin derivative of formula (2) can be provided in a glass vial equipped with a butyl rubber stopper. For example, the lyophilized product can contain about 50 mg of the β-substituted β-amino acid derivative of formula (1) and about 2,700 mg of the cyclodextrin derivative of formula (2).

[0113] The kit can optionally include an aqueous solution such as about 0.9% physiological saline solution (for example, 0.9% physiological saline of about 8.0 mL to about 9.0 mL, about 8.2 mL to about 8.6 mL, or about 8.3 mL to about 8.5 mL). After mixing the aqueous solution such as about 0.9% physiological saline solution with the lyophilized product that can be added to the vial containing the lyophilized product of the compound / cyclodextrin guest-host inclusion complex of formula (1), the vial can contain about 10 mL of an aqueous solution having a nominal concentration of about 5 mg / mL of the compound of formula (1), such as about 3 mg / mL to about 7 mg / mL, about 3.5 mg / mL to about 6.5 mg / mL, about 4 mg / mL to about 6 mg / mL, or about 4.5 mg / mL to about 5.5 mg / mL.

[0114] The kit can include, for example, a lyophilized guest-host inclusion complex of about 1 mg to about 100 mg, a lyophilized guest-host inclusion complex of about 5 mg to about 80 mg, about 10 mg to about 70 mg, or about 20 mg to about 60 mg.

[0115] Any suitable vial size can be used, for example, about 10 mL to about 50 mL, about 10 mL to about 40 mL, or about 20 mL to about 30 mL. The vial size can be, for example, about 10 mL, about 20 mL, about 30 mL, about 40 mL, or about 50 mL.

[0116] This solution can be further diluted either by adding an additional 0.9% aqueous saline solution or by diluting an aliquot with an appropriate amount of about 0.9% aqueous saline solution in a suitable container such as another vial or bag to a final theoretical or nominal concentration of the compound of formula (1) of about 0.2 mg / mL to about 1.0 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.4 mg / mL to about 0.6 mg / mL, or about 0.5 mg / mL.

[0117] The pH of the solution of the reconstituted lyophilized product can be in the range of, for example, about 4 to about 6, about 3 to about 5, about 3.5 to about 4.5, or the pH can be about 4.0.

[0118] The reconstituted lyophilized product solution can contain the compound of formula (1) as a free base, HCl salt, or a combination thereof. For example, the reconstituted lyophilized product solution of formula (1) can contain more than about 0%, more than about 20%, more than about 40%, more than about 60%, or more than about 80%, or about 100% of the HCl salt of the compound of formula (1) (where the percentages are based on the relative molar amount of the compound of formula (1)). For example, about 50 mg of the compound of formula (1c) corresponds to 0.150038 mmol. For example, the reconstituted solution of formula (1) can contain from about 0% to about 100%, from about 10% to about 90%, from about 20% to about 80%, from about 40% to about 60%, or from about 30% to about 70% of the HCl salt of the compound of formula (1) (where the percentages are based on the relative molar amount of the compound of formula (1)).

[0119] The methods provided by the present disclosure include methods of administering a pharmaceutical composition provided by the present disclosure to a patient.

[0120] The pharmaceutical composition can be administered intravenously, for example, by bolus injection, intravenous infusion, limb perfusion, normothermic isolated limb perfusion, percutaneous hepatic perfusion, etc. Intravenous administration includes administration by injection using a cannula, central line, peripherally inserted central catheter line, and / or by a drip line.

[0121] The pharmaceutical composition can be administered as an infusion for an appropriate period.

[0122] The pharmaceutical composition provided by the present disclosure is useful for the treatment of cancer, including the treatment of solid tumors and metastases.

[0123] The amount of the compound of formula (1) effective for the treatment of cancer depends at least in part on the nature of the disease and can be determined by standard clinical techniques known in the art. Additionally, in vitro or in vivo assays may be used to help identify the optimal dosage range. The dosing regimen and dosing interval can also be determined by methods known to those skilled in the art. The amount of the β-substituted β-amino acid derivative / cyclodextrin derivative administered can depend, inter alia, on the patient being treated, the patient's body weight, potential co-existing diseases, the patient's clinical condition, the severity of the disease, the route of administration, and the judgment of the prescribing physician.

[0124] For systemic administration, a therapeutically effective amount can initially be estimated from in vitro assays. The initial dose can also be estimated from in vivo data, such as animal models, using techniques known in the art. Such information can be used to more accurately determine the useful dosage in humans. One skilled in the art may optimize the administration to humans based on the animal data.

[0125] The pharmaceutical compositions provided by the present disclosure can be administered, for example, once a day, twice a day, and in certain embodiments, at intervals greater than once a day. The dosing can be provided alone or in combination with other drugs and can continue as long as necessary for effective treatment of the disease. The dosing can also be carried out using continuous or semi-continuous administration over a certain period. Dosing includes administering the pharmaceutical composition to a mammal, such as a human, in a fed or fasting state.

[0126] To maintain a continuous therapeutically effective concentration of the compound of formula (1) in the patient's blood, the dosage of the compound of formula (1) and an appropriate dosing interval can be selected.

[0127] The pharmaceutical compositions provided by the present disclosure can be administered using a suitable dosing regimen. The dosing regimen can be adjusted, discontinued, or extended as appropriate to achieve the treatment objective.

[0128] A pharmaceutical composition comprising the compound of formula (1) can be administered to treat cancer in a patient so as to provide a therapeutically effective concentration of the compound of formula (1) in the patient's plasma. The therapeutically effective concentration of the compound of formula (1) in the patient's plasma can be less than an amount that causes unacceptable adverse effects including adverse effects on homeostasis. The therapeutically effective concentration of the compound of formula (1) in the patient's plasma is an amount sufficient to treat cancer in the patient.

[0129] The pharmaceutical composition provided by the present disclosure can be administered to treat cancer in a patient to provide a therapeutically effective concentration of the compound of formula (1) in the patient's blood or plasma for a long period of time, for example, at least about 0.5 hour, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, or at least about 12 hours.

[0130] The amount of the compound of formula (1) administered can vary in the treatment regimen.

[0131] The method provided by the present disclosure includes a method of treating cancer in a patient, and includes administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical composition provided by the present disclosure.

[0132] The cancer can be a cancer in the brain or a cancer of the central nervous system, regardless of the origin of the cancer. The cancer can be a metastatic cancer in the brain or central nervous system. The cancer can be a solid tumor derived from the brain or central nervous system.

[0133] The pharmaceutical composition can be used to treat breast cancer and metastatic breast cancer.

[0134] The pharmaceutical composition provided by the present disclosure can be used to treat central nervous system cancer.

[0135] The pharmaceutical composition provided by the present disclosure can be used to treat brain tumors.

[0136] Cancer can be a cancer of the central nervous system or a metastasis of cancer originating from tissues outside the central nervous system.

[0137] Cancer can be primary brain cancer or metastatic brain cancer. Cancer can be a primary cancer of the central nervous system or a metastatic central nervous system cancer.

[0138] The pharmaceutical composition provided by the present disclosure can be used to treat metastatic cancer (for example, metastatic cancer of any origin that highly expresses LAT1 / 4F2hc).

[0139] The pharmaceutical composition provided by the present disclosure can be administered together with one or more compounds effective in treating cancer treated with the compound of formula (1) and / or treating side effects caused by administering the compounds of formula (1) to (1c).

[0140] The pharmaceutical composition provided by the present disclosure can be administered simultaneously with the administration of another therapeutic agent that can be part of the same pharmaceutical composition or a different pharmaceutical composition containing the compound of formula (1). The compound of formula (1) can be administered before or after the administration of another therapeutic agent. The combination therapy can include, for example, alternating the administration of the compound of formula (1) and a composition containing another therapeutic agent in order to minimize drug adverse effects associated with a specific drug. When the compound of formula (1) is administered simultaneously with another therapeutic agent that can potentially cause harmful drug effects including toxicity, the other therapeutic agent can be administered at a dose below the threshold at which a harmful drug reaction is induced.

[0141] The pharmaceutical composition provided by the present disclosure can be administered in combination with an agent known or believed to be effective in treating cancer in a patient.

Example

[0142] Embodiments provided by the present disclosure are further illustrated by reference to the following examples, which describe the pharmaceutical compositions provided by the present disclosure and the properties of the pharmaceutical compositions. It will be apparent to those skilled in the art that many changes can be made to both the materials and methods without departing from the scope of the present disclosure.

[0143] Example 1 Solubility of Compound (1c) Free Base Mannitol (500 mg; MW 182.17 g / mol; 2.744 mmol) was added to 10 mg (0.030 mmol) of (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (Compound (1c)) (free base; MW 333.25 g / mol) and dissolved by vortexing in 5 mL, 10 mL, or 15 mL of distilled water for about 30 seconds to obtain a solution having a pH of 3.8 - 4.2.

[0144] Compound (1c) did not completely dissolve in 5 mL or 10 mL of the mannitol solution. Compound (1c) completely dissolved in 15 mL of the mannitol solution.

[0145] Example 2 Solubility of Compound (1c) Free Base Lactose (500 mg; MW 342.30 g / mol; 1.461 mmol) was added to 10 mg (0.030 mmol) of (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (Compound (1c)) (free base) and dissolved by vortexing in 5 mL, 10 mL, or 15 mL of distilled water for about 30 seconds to obtain a solution having a pH of 3.8 - 4.2.

[0146] Compound (1c) did not completely dissolve in 5 mL or 10 mL of the lactose solution. Compound (1c) completely dissolved in 15 mL of the lactose solution.

[0147] Example 3 Stability of Compound (1c) Free Base The stability assay was performed in a 96-well microtiter plate. (S)-3-Amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (compound (1c) free base) was incubated at 37 °C in phosphate buffered saline (PBS buffer) at pH 7.4. The reaction mixture (30 μL) contained compound (1c) at a final concentration of 1 μM. The percentage of compound (1c) remaining in the mixture was determined using LC / MS / MS.

[0148] At each sampling time point, 300 μL of quenching solution (50% by volume of acetonitrile, 50% by volume of methanol containing 1.3% (v / v) of 6N HCl) with an internal standard (bucetin in the case of positive ESI mode or warfarin in the case of negative ESI mode) was transferred to each well. The plate was sealed and centrifuged at 4,000 rpm for 15 minutes at 4 °C. The supernatant was transferred to a new plate for LC / MS / MS analysis.

[0149] All samples were analyzed by LC / MS / MS using an AB Sciex API4000® instrument coupled to a Shimadzu LC-20AD LC pump system. The analytical samples were separated at a flow rate of 0.4 mL / min using a Waters Atlantis T3 dC18 reversed-phase HPLC column (20 mm × 2.1 mm). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B). Table 1 shows the details regarding the gradient of the eluent used. Table 2 shows the results of the stability of the test compound in PBS buffer.

Table 1

Table 2

[0150] Example 4 Stability of compound (1c) free base Cyclodextrin derivative (270 mg, SBE 6.5-β-CD, Captisol (registered trademark, average MW 2,163 g / mol), was added to 5.5 mg (0.0165 mmol) of (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid (Compound (1c)) (free base) in a 12 mL glass vial and dissolved by vortexing in 3 mL to 4 mL of redistilled water for about 30 seconds to obtain a clear, colorless solution. The solution was filtered through a 0.45 μm nylon syringe filter into a 12 mL glass vial. The filter was washed twice with redistilled water (about 0.5 mL each) to obtain a clear, colorless solution. The filtered solution was frozen at -78 °C (dry ice / acetone bath), and the solvent was lyophilized at about 100 mTorr for about 16 hours to obtain 5.5 mg (0.0165 mmol) of Compound (1c) free base and 270 mg of SBE 6.5 A colorless powdery solid consisting of -β-CD and Captisol (registered trademark) was obtained. According to this method, several vials having cyclodextrin derivative / Compound (1c) guest-host inclusion complexes were prepared.

[0151] The cyclodextrin derivative / Compound (1c) guest-host inclusion complex was reconstituted by dissolving the guest-host inclusion complex in 0.86 mL of 0.9% saline to obtain a 1.0 mL solution having a nominal concentration of 5.5 mg / mL of the cyclodextrin derivative / Compound (1c) guest-host inclusion complex.

[0152] A 1.0 mL aliquot of the reconstituted solution was diluted with 10.0 mL of 0.9% saline, and the pH was adjusted with about 5 μL of saturated NaHCO 3 aqueous solution to obtain an 11.0 mL clear, colorless solution having a pH of 5.8 to 6.1 and a nominal concentration of 0.5 mg / mL of Compound (1c).

[0153] A 1.0 mL aliquot of the reconstituted solution was diluted with 4.5 mL of 0.9% saline, and the pH was adjusted with about 5 μL of saturated NaHCO 3Adjusted with an aqueous solution to obtain a 5.5 mL clear, colorless solution with a nominal concentration of 1.0 mg / mL of compound (1c) and a pH of 6.1.

[0154] A 1.0 mL aliquot of the reconstituted solution was diluted with 1.75 mL of 0.9% saline, and the pH was adjusted to approximately 6.1 with about 10 μL of saturated NaHCO 3 Adjusted with an aqueous solution to obtain a 2.75 mL clear, colorless solution with a nominal concentration of 2.0 mg / mL of compound (1c) and a pH of 6.1.

[0155] The solution was placed in glass vials and stored either at room temperature (about 25 °C) or refrigerated (about 0 °C), and the amount of compound (1c) was measured at regular intervals using analytical high-performance liquid chromatography (HPLC / UV) connected to a UV detector.

[0156] HPLC / UV analysis was performed using an Agilent 1200 HPLC system equipped with a G1379B degasser, a G1312A binary pump, G1367B Hip-ALs, a G1316A TCC, a G1315B DAD, Phenomenex® Kinetex 5μm column, C18 (4.6×150 mm), and a Zorbax® Eclipse XDB C18 column (2.1×150 mm), as well as a personal computer for data calculation. For the analytical HPLC / UV analysis, a gradient of water (solvent A) (Arrowhead, Nestle North America, Inc.) containing 0.1% (v / v) trifluoroacetic acid (TFA) (Oakwood Chemical, 001271) and acetonitrile (MeCN; solvent B) (EMD AX0145-1 or Aldrich Chromasolv® 439134) was used. The HPLC / UV method used a flow rate of 1.0 mL / min, a runtime of 15 minutes, a gradient from 5% (v / v) solvent B to 100% (v / v) solvent B, and UV detection at λ = 220 nm and λ = 254 nm. Table 3 shows the details of the eluent gradient. The percentage of the remaining compound (1c) was determined based on the AUC determined at the absorption wavelength λ = 254 nm using linear extrapolation to time zero (set as 100% AUC). Table 4 shows the results.

Table 3

Table 4

[0157] Example 5 Stability of Compound (1c) Hydrochloride The stock solution was prepared by dissolving 68.8 mg (0.206 mmol) of compound (1c) (free base) in a mixture of approximately 20.0 mL of acetonitrile / water (1:1, v / v) with vortexing in a clean glass volumetric cylinder (25 mL). 0.407 mL of 0.5072 M aqueous HCl (0.206 mmol, 1.0 equivalent; Fluka, 318957) was added with gentle vortexing, and the resulting clear solution was further diluted to 25.0 mL with a mixture of acetonitrile / water (1:1, v / v) with gentle vortexing to obtain a stock solution of compound (1c) as the mono-HCl salt at a concentration of 2.75 mg / mL. This solution was filtered through a 0.45 μm nylon syringe filter into a clean 50 mL Erlenmeyer flask. 2.0 mL aliquots (2 × 1.0 mL; 2 × 2.75 mg) of the stock solution were quickly pipetted into clean 12 mL scintillation vials. The aliquots were frozen at -78 °C (dry ice / acetone bath) and lyophilized at approximately 100 mTorr for approximately 16 hours to obtain test vials each containing 6.1 mg (0.0165 mmol) of compound (1c) monohydrochloride (mono-HCl salt) (corresponding to 5.5 mg of compound (1c)) as a colorless powdery solid.

[0158] Cyclodextrin derivative (270 mg, SBE 6.5-β-CD, Captisol (registered trademark, average MW 2,163 g / mol), was added to 6.1 mg (0.0165 mmol) of (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid monohydrochloride (Compound (1c) monohydrochloride) in a 12 mL glass vial and dissolved by vortexing in 3 mL to 4 mL of redistilled water for about 30 seconds to obtain a clear, colorless solution. The solution was filtered through a 0.45 μm nylon syringe filter into a 12 mL glass vial. The filter was washed twice with redistilled water (about 0.5 mL each) to obtain a clear, colorless solution having a pH of 4.4 - 4.7. The filtered solution was frozen at -78 °C (dry ice / acetone bath), and the solvent was lyophilized at about 100 mTorr for about 16 hours to obtain 6.1 mg (0.0165 mmol) of Compound (1c) monohydrochloride (corresponding to 5.5 mg of Compound (1c) as the free base) and 270 mg of SBE 6.5 A colorless powdery solid consisting of -β-CD and Captisol (registered trademark) was obtained. According to this method, several vials having cyclodextrin derivative / Compound (1c) monohydrochloride guest-host inclusion complexes were prepared.

[0159] The cyclodextrin derivative / Compound (1c) monohydrochloride guest-host inclusion complex was reconstituted by dissolving the guest-host inclusion complex in 0.86 mL of 0.9% saline to obtain a 1.0 mL solution having a nominal concentration of 6.1 mg / mL of Compound (1c) monohydrochloride guest-host inclusion complex (which corresponds to a nominal concentration of 5.5 mg / mL of Compound (1c) guest-host inclusion complex).

[0160] An aliquot of 0.5 mL of this solution was diluted with 5.0 mL of 0.9% saline, and the pH was adjusted with 5 μL of saturated NaHCO 3 aqueous solution to obtain a 5.5 mL clear, colorless solution having a pH of 5.3 - 5.5 and a nominal concentration of 0.5 mg / mL of Compound (1c).

[0161] A 1.0 mL aliquot of the reconstituted solution was diluted with 4.5 mL of 0.9% saline, and the pH was adjusted with approximately 10 μL of saturated NaHCO 3 aqueous solution to obtain a 5.5 mL clear, colorless solution with a nominal concentration of 1.0 mg / mL of compound (1c) and a pH of 5 - 6.

[0162] A 1.0 mL aliquot of the reconstituted solution was diluted with 1.75 mL of 0.9% saline, and the pH was adjusted with approximately 10 μL of saturated NaHCO 3 aqueous solution to obtain a 2.75 mL clear, colorless solution with a nominal concentration of 2.0 mg / mL of compound (1c) and a pH of 5 - 6.

[0163] A 0.25 mL aliquot of the reconstituted solution was diluted with 11.75 mL of 0.9% saline, and the pH was adjusted with approximately 10 μL of saturated NaHCO 3 aqueous solution to obtain an 11.0 mL clear, colorless solution with a nominal concentration of 0.125 mg / mL of compound (1c) and a pH greater than 6.

[0164] The solutions were placed in glass vials and stored either at room temperature (about 25 °C) or refrigerated (about 0 °C). The amount of compound (1c) was measured at regular intervals using analytical high - performance liquid chromatography (HPLC / UV) coupled to a UV detector as described in Example 4. The results are shown in Table 5.

Table 5

[0165] Example 6 At different pH values Compound (1c): SBE 6.5 -β - CD guest - host inclusion complex in the stability of compound (1c) in formulations Compound (1c) at different pH: SBE 6.5 -β - CD guest - host inclusion complex in the stability of compound (1c) in formulations was determined by analytical HPLC / UV. Compound (1c) and SBE 6.5The mass ratio of -β-CD was either 1:54 or 1:50.

[0166] To prepare the formulation, SBE 6.5 -β-CD was dissolved in water for injection (WFI) by continuous stirring until a clear solution was obtained. The solution was acidified by the addition of 1N HCl. Compound (1c) was dissolved in the acidified SBE 6.5 -β-CD aqueous solution, and an appropriate amount of water was added to the final volume to prepare a solution containing compound (1c) at a nominal concentration of 5.0 mg / mL. The pH was adjusted to the values shown in Table 7 with 1N HCl. This corresponds to the pH of the final formulation after reconstitution with 0.9% saline. The solution was filtered through a 0.22 μm syringe filter and then lyophilized to obtain a colorless powdery solid. The lyophilized lyophilizate was reconstituted with 0.9% saline to a nominal target concentration of 5.0 mg / mL of compound (1c).

[0167] The total AUC of impurities and the AUC of impurities at relative retention times (RRT) of 0.42 and 1.27 were also determined by analytical HPLC / UV. The RRT of 0.42 corresponds to the mono-hydrolysis product of compound (1c). The identity of the impurity at an RRT of 1.27 has not been determined.

[0168] Analytical HPLC / UV was performed on an HPLC chromatograph equipped with a UV detector, a Waters Xbridge® C18 column (4.6 × 150 mm; 5 μm) operating at 30 °C, and a personal computer for data calculation. For the analytical HPLC / UV analysis, a 0.01 M potassium dihydrogen phosphate solution (KH 3 PO 4 ): acetonitrile = 850:150 (v / v) adjusted to pH 3.0 with phosphoric acid (H 2 PO 4)A gradient of solution (solvent A) and acetonitrile (MeCN; solvent B) was used. The needle was washed with 50% aqueous acetonitrile solution. Before injecting into the HPLC / UV system (10 μL injection volume), the sample was diluted to a nominal concentration of compound (1c) of 0.5 mg / mL using a dilution solution of 0.9 M sodium chloride (NaCl) and 0.1 N hydrochloric acid (HCl). The dilution solution was prepared from 5.26 g (90.0 mmol) of sodium chloride and 850 μL of concentrated hydrochloric acid (HCl) (36 - 38 wt%, d 1.184, 1.00 g, 10.2 mmol) and diluted to 100 mL solution.

[0169] The HPLC / UV method used had a flow rate of 1.5 mL / min, a runtime of 36 minutes, a gradient from 10% by volume of solvent B to 100% by volume of solvent B, and UV detection at λ = 220 nm. Table 6 shows the details of the eluent gradient used. Table 7 shows the results of the HPLC / UV analysis.

Table 6

Table 7

[0170] Example 7 Different compound (1c): SBE 6.5 Stability of compound (1c) in a formulation containing the guest - host inclusion complex of -β-CD Compound (1c): SBE in various ratios at pH 4.5 6.5 The stability of compound (1c) in a formulation containing the -β-CD guest - host inclusion complex was determined by analytical HPLC / UV. The mass ratio of compound (1c) to SBE 6.5 -β-CD was either 1:54 or 1:70.

[0171] A formulation was prepared as described in Example 6, except that a solution containing compound (1c) at a nominal concentration of 3.3 mg / mL was prepared and the pH of the solution was adjusted to 4.5 with 1N HCl. The solution was filtered through a 0.22 μm syringe filter and then lyophilized to obtain a colorless powdery solid. The lyophilized lyophilizate was reconstituted with 0.9% saline to a nominal target concentration of 0.5 mg / mL of compound (1c) and a pH of 4.6 and subjected directly to HPLC / UV analysis. Both a nominal target concentration of 0.5 mg / mL of compound (1c) and a pH of 4.6 represent useful concentrations and pH for intravenous injection.

[0172] As described in Example 6, the total AUC of impurities and the AUC of impurities at relative retention times (RRT) of 0.42 and 1.27 were also determined by analytical HPLC. The RRT of 0.42 corresponds to the mono-hydrolysis product of compound (1c). The identity of the impurity at an RRT of 1.27 has not been determined.

[0173] The analytical HPLC / UV equipment and analytical HPLC / UV analysis method used were the same as those for Example 6. The results of the HPLC / UV analysis are shown in Table 8.

Table 8

[0174] Example 8 Compound (1c): SBE 6.5 -β-CD And the stability of compound (1c) in a formulation containing a mannitol guest-host inclusion complex The stability of a formulation containing a compound (1c):SBE 6.5 -β-CD:mannitol guest-host inclusion complex in a weight ratio of 1:50:30 was compared with the stability of a formulation containing a compound (1c):SBE 6.5 -β-CD guest-host inclusion complex in a weight ratio of 1:50.

[0175] As described in Example 6, a formulation was prepared. A solution containing compound (1c) at a nominal concentration of 5.0 mg / mL was prepared, and the pH of the solution was adjusted to 5.5 with 1N HCl. The solution was filtered through a 0.22 μm syringe filter and then lyophilized to obtain a colorless powdery solid. The lyophilized lyophilizate was reconstituted with 0.9% saline to a nominal target concentration of 5.0 mg / mL of compound (1c) and a pH of 5.5, and after dilution to a nominal concentration of 0.5 mg / mL with a 0.9M NaCl / 0.1N HCl diluent as described in Example 6, it was directly analyzed using HPLC / UV.

[0176] As described in Example 6 and Example 7, the total AUC of impurities and the AUC of impurities at relative retention times (RRT) of 0.42 and 1.27 were also determined by analytical HPLC / UV at temperatures of 25°C and 2°C to 8°C. The RRT of 0.42 corresponds to the mono-hydrolysis product of compound (1c). The identity of the impurity at an RRT of 1.27 has not been determined.

[0177] The analytical HPLC / UV instrument and analytical HPLC / UV analysis method used were the same as those in Example 6 and Example 7. Table 9 shows the results of the HPLC / UV analysis.

Table 9

[0178] Example 9 Comparative Stability of Compound (1c), Melphalan, and Bendamustine in Injectable Formulations The stability of compound (1c) as the HCl salt or as the free base was compared with the stability of melphalan HCl and / or bendamustine HCl in various pharmaceutical formulations, at different concentrations, and at temperatures of 0°C or 25°C.

[0179] The stability of the compounds in the pharmaceutical formulations was determined at concentrations in the ranges of 0.4 mg / mL to 0.5 mg / mL, 0.9 mg / mL to 1.1 mg / mL, and 1.8 mg / mL to 2.1 mg / mL.

[0180] The amounts of the active pharmaceutical ingredient (API) (compound (1c), melphalan, or bendamustine) in the pharmaceutical formulation at different times during storage were determined using HPLC / UV as described in detail for Examples 4 and 5.

[0181] For the measurement of stability, four pharmaceutical compositions were included: Na 3 -citrate buffer formulation (Formulation 1), formulation based on Alkeran® (Formulation 2), formulation based on Evomela® (Formulation 3), and formulation based on Treanda® (Formulation 4).

[0182] Formulation 1. The first formulation contained a mixture of 57.5% by volume of 1,2-propylene glycol (1,2-PG), 30% by volume of Kolliphor® HS15 (Solutol®, Macrogol®, BASF AG / SigmaAldrich), and 12.5% by volume of ethanol (EtOH). The resulting solution was further diluted and adjusted to pH 6.8 using Na 3 -citrate buffer (51 mM of Na 3 -citrate·2H 2 O and 73.6 mM of NaCl).

[0183] The Na 3 -citrate buffer at pH 8.1 - 8.3 was prepared by dissolving Na 3 -citrate·2H 2 O (Na 3 C 6 H 5 O 7 ·2H 2 O; MW 294.10 g / mol; J.T. Baker, 3650 - 01) and 0.43 g (7.36 mmol) of sodium chloride (NaCl; MW 58.44 g / mol; SigmaAldrich, S9888) in 100 mL of redistilled water (dd-H 2 O) (Arrowhead). The solution was filtered through a 0.45 μm nylon syringe filter, and the pH of the solution was adjusted with sodium bicarbonate (NaHCO3 ) was adjusted to 8.1 - 8.3 with a saturated aqueous solution of

[0184] The excipient mixture was prepared from 4.77 g (62.7 mmol; 4.6 mL) of 1,2 - propylene glycol (1,2 - PG; MW 76.09 g / mol; d 1.036; Acros Organics, 220870010), 2.52 g (2.40 mL, d 1.05) of melted (15 - second microwave treatment) Kolliphor® HS15 (BASF AG / SigmaAldrich, 42966), and 0.798 g (17.32 mmol, 1.0 mL) of ethanol (EtOH, MW 46.06 g / mol, d 0.798, KOPTEC, V1016). The clear viscous mixture was warmed to about 40 °C (water bath) before use.

[0185] A series of 12 - mL glass vials each containing 6.1 mg (0.0318 mmol) of compound (1c) monohydrochloride (equivalent to 5.5 mg of compound (1c) as the free base) were prepared by lyophilizing an aliquot of the acidified stock solution as described in Example 5.

[0186] 660 μL (6% by volume) of the pre - warmed excipient mixture was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was gently heated and dissolved while vortexing. The viscous solution was further diluted with 10,340 μL (94% by volume) of pH 8.1 - 8.3 Na 3 - citrate buffer to obtain an 11.0 - mL clear, colorless formulation containing compound (1c) at a concentration of 0.5 mg / mL, as the monohydrochloride and with a pH of 6 - 7.

[0187] 330 μL (6% by volume) of the pre - warmed excipient mixture was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was gently heated and dissolved while vortexing. The viscous solution was further diluted with 5,170 μL (94% by volume) of pH 8.1 - 8.3 Na 3 - citrate buffer to obtain a 5.5 - mL clear, colorless formulation containing compound (1c) at a concentration of 1.0 mg / mL, as the monohydrochloride and with a pH of 6 - 7.

[0188] 165 μL (6% by volume) of pre-warmed excipient mixture was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was gently heated and dissolved with vortexing. The viscous solution was further diluted with 2,585 μL (94% by volume) of Na 3 -citrate buffer at pH 8.1 - 8.3 to obtain a 2.75 mL clear and colorless formulation containing the compound (1c) at a concentration of 2.0 mg / mL, as the monohydrochloride and at a pH of 6 - 7.

[0189] The solution was placed in a glass vial, stored at room temperature (about 25 °C), and the amount of the compound (1c) derived from the monohydrochloride of the compound of formula (1c) (mono HCl salt) was measured at regular intervals using analytical high performance liquid chromatography (HPLC / UV) connected to a UV detector as described in Example 4. The results are shown in Table 10.

[0190] Formulation 2. Alkeran® is an injectable formulation of melphalan-HCl approved by the FDA. Injectable Alkeran® is supplied as a sterile, non-pyrogenic, lyophilized powder. Each vial contains melphalan hydrochloride corresponding to 50 mg of melphalan and 20 mg of povidone (polyvinylpyrrolidone, PVP). The solid powder is reconstituted in a total of 10 mL of sterile diluent containing 0.2 g of sodium citrate, 6.0 mL of propylene glycol, and 0.52 mL of ethanol (96%), as well as water for injection. This provides a nominal 5 mg / mL solution of melphalan (mixture). The dose to be administered is immediately diluted in 0.9% sodium chloride injection (USP) to a concentration of 0.45 mg / mL or less. Injectable Alkeran® is administered intravenously.

[0191] The stock solution was prepared by dissolving 68.8 mg (0.206 mmol) of compound (1c) (free base) and 25.0 mg of Plasdone® C-12 (Povidone; Ashland, 830796) in approximately 20.0 mL of a mixture of acetonitrile / water (1:1, v / v) while vortexing in a clean glass volumetric cylinder (25 mL). 0.407 mL of a 0.5072 M HCl solution (0.206 mmol, 1.0 equivalent; Fluka, 318957) was added while gently vortexing, and the resulting clear solution was further diluted to 25.0 mL with a mixture of acetonitrile / water (1:1, v / v) while gently vortexing to obtain a stock solution of compound (1c) as the mono HCl salt at a concentration of 2.75 mg / mL and Plasdone® at a concentration of 1.0 mg / mL. This solution was filtered through a 0.45 μm nylon syringe filter into a clean 50 mL Erlenmeyer flask. 2.0 mL aliquots (2 × 1.0 mL; 2 × 2.75 mg) of this stock solution were quickly pipetted into clean 12 mL scintillation vials. The aliquots were frozen at -78 °C (dry ice / acetone bath) and lyophilized at approximately 100 mTorr for approximately 16 hours to obtain test vials each containing 6.1 mg (0.0165 mmol) of compound (1c) monohydrochloride (mono HCl salt) (corresponding to 5.5 mg of compound (1c) as the free base) as a colorless powdery solid.

[0192] The stock solution was prepared by dissolving 62.5 mg (0.205 mmol) of melphalan (free base; Sigma Aldrich, M2011) and 25.0 mg of Plasdone® C-12 (Povidone; Ashland, 830796) in a mixture of approximately 20.0 mL of acetonitrile / water (1:1, v / v) while vortexing in a clean glass graduated cylinder (25 mL). 0.404 mL of 0.5072 M aqueous HCl (0.205 mmol, 1.0 equivalent; Fluka, 318957) was added while gently vortexing, and the resulting clear solution was further diluted to 25.0 mL with a mixture of acetonitrile / water (1:1, v / v) while gently vortexing to obtain a stock solution of melphalan as the mono HCl salt at a concentration of 2.50 mg / mL and Plasdone® at a concentration of 1.0 mg / mL. This solution was filtered through a 0.45 μm nylon syringe filter into a clean 50 mL Erlenmeyer flask. 2.0 mL aliquots (2 × 1.0 mL; 2 × 2.50 mg) of this stock solution were quickly pipetted into clean 12 mL scintillation vials. The aliquots were frozen at -78 °C (dry ice / acetone bath) and lyophilized at approximately 100 mTorr for approximately 16 hours to obtain test vials each containing 5.6 mg (0.0164 mmol) of melphalan monohydrochloride (mono HCl salt) (corresponding to 5.0 mg of melphalan as the free base).

[0193] 12.43 g (163.4 mmol; 12.0 mL) of 1,2 - propylene glycol (1,2 - PG; MW 76.09 g / mol; d 1.036; Acros Orgaincs, 220870010), 0.40 g (1.36 mmol) of Na 3 - citrate·2H 2 O (Na 3 C 6 H 5 O 7 ·2H 2O; MW 294.0 g / mol; J.T. Baker, 3650-01), and 0.821 g (17.82 mmol, 1.04 mL) of ethanol (EtOH, MW 46.07 g / mol, d 0.798, KOPTEC, V1016) were dissolved to 20.0 mL in a 25.0 mL volumetric flask. The solution was filtered through a 0.45 μm nylon syringe filter into a clean 20 mL glass vial.

[0194] 1.0 mL of Alkeran® type diluent was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was dissolved by gently shaking and vortexing. The solution was further diluted with 9.0 mL of 0.9% saline to obtain a 10.0 mL clear colorless formulation containing the compound (1c) at a concentration of 0.55 mg / mL as the monohydrochloride at a pH of 5.3 - 5.7.

[0195] 0.5 mL of Alkeran® type diluent was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was dissolved by gently shaking and vortexing. The solution was further diluted with 4.5 mL of 0.9% saline to obtain a 5.0 mL clear colorless formulation containing the compound (1c) at a concentration of 1.1 mg / mL as the monohydrochloride and at a pH of 5.3.

[0196] 0.25 mL of Alkeran® type diluent was added to the compound (1c) monohydrochloride in the vial. The compound (1c) monohydrochloride was dissolved by gently shaking and vortexing. The solution was further diluted with 2.25 mL of 0.9% saline to obtain a 2.5 mL clear colorless formulation containing the compound (1c) at a concentration of 2.2 mg / mL as the monohydrochloride at a pH of 5.3.

[0197] 1.0 mL of Alkeran® diluent was added to melphalan monohydrochloride in the vial. The melphalan monohydrochloride was gently shaken and dissolved while vortexing. The solution was further diluted with 9.0 mL of 0.9% saline to obtain a 10.0 mL clear, colorless formulation containing melphalan at a concentration of 0.50 mg / mL as the monohydrochloride at a pH of 5.5 - 5.8.

[0198] 0.5 mL of Alkeran® diluent was added to melphalan monohydrochloride in the vial. The melphalan monohydrochloride was gently shaken and dissolved while vortexing. The solution was further diluted with 4.5 mL of 0.9% saline to obtain a 5.0 mL clear, colorless formulation containing melphalan at a concentration of 1.0 mg / mL as the monohydrochloride at a pH of 5.3 - 5.5.

[0199] 0.25 mL of Alkeran® diluent was added to melphalan monohydrochloride in the vial. The melphalan monohydrochloride was gently shaken and dissolved while vortexing. The solution was further diluted with 2.25 mL of 0.9% saline to obtain a 2.5 mL clear, colorless formulation containing melphalan at a concentration of 2.0 mg / mL as the monohydrochloride at a pH of 5.0 - 5.3.

[0200] The solution was placed in a glass vial and stored at room temperature (about 25 °C). As described in Example 4, the amount of compound (1c) derived from melphalan monohydrochloride (1c) or monohydrochloride (single HCl salt) of the melphalan compound derived from the monohydrochloride (single - HCl salt) was measured at intervals using analytical high - pressure liquid chromatography coupled to a UV detector (HPLC / UV). The results are shown in Table 10.

[0201] Formulation 3. Evomela (registered trademark) is an injectable formulation of melphalan hydrochloride, 4-[bis(2-chloroethyl)amino]-L-phenylalanine hydrochloride, and is approved by the FDA. Evomela (registered trademark) is supplied as a sterile white to off-white lyophilized powder in single-dose vials for intravenous use. Each vial contains 50 mg of melphalan free base (equivalent to 56 mg of melphalan hydrochloride) and 2,700 mg of beta-cyclodextrin sulfobutyl ether (SBE 6.5 -β-CD; sulfobutyl ether β-cyclodextrin sodium), NF. Evomela (registered trademark) is reconstituted with sterile aqueous physiological saline solution (0.9% sodium chloride injection, USP) (8.6 mL according to the instructions) to a total of 10 mL having a nominal concentration of 5 mg / mL of melphalan. The required amount of Evomela (registered trademark) needed for the patient's dose is removed from the vial and added to an appropriate amount of 0.9% sodium chloride injection (USP) to give a final nominal concentration of 0.45 mg / mL of melphalan. This solution is then infused via an injection port or a central venous catheter.

[0202] A series of 12 mL glass vials each containing 5.5 mg (0.0165 mmol of the free base of compound (1c) and 270 mg of SBE 6.5 -β-CD, Captisol (registered trademark)) were prepared as described in Example 4.

[0203] The cyclodextrin derivative / compound (1c) free base guest-host inclusion complex was reconstituted by dissolving in 0.86 mL of 0.9% saline to give a 1.0 mL solution having a nominal concentration of 5.5 mg / mL of the free base of compound (1c).

[0204] An aliquot of 1.0 mL of this solution was diluted with 10.0 mL of 0.9% saline and the pH was adjusted with 5 μL of saturated NaHCO 3 aqueous solution to give an 11.0 mL clear colorless solution having a nominal concentration of 0.50 mg / mL of the free base of compound (1c) from compound (1c) and a pH of 5.8 - 6.1.

[0205] An aliquot of 1.0 mL of this solution was diluted with 4.5 mL of 0.9% saline, and the pH was adjusted with 5 μL of saturated NaHCO 3 aqueous solution to obtain a 5.5 mL clear, colorless solution of compound (1c) from compound (1c) free base at a nominal concentration of 1.0 mg / mL and having a pH of about 6.1.

[0206] An aliquot of 1.0 mL of this solution was diluted with 1.75 mL of 0.9% saline, and the pH was adjusted with 5 μL of saturated NaHCO 3 aqueous solution to obtain a 2.75 mL clear, colorless solution of compound (1c) from compound (1c) free base at a nominal concentration of 2.0 mg / mL and having a pH of about 6.1.

[0207] 6.1 mg (0.0165 mmol of compound (1c) monohydrochloride (corresponding to 5.5 mg of compound (1c) free base) and 270 mg of SBE 6.5 -β-CD, Captisol® were each included in a series of 12 mL glass vials prepared as described in Example 5.

[0208] The cyclodextrin derivative / compound (1c) monohydrochloride guest-host inclusion complex was reconstituted by dissolving the guest-host inclusion complex in 0.86 mL of 0.9% saline to obtain a 1.0 mL solution having a nominal concentration of 6.1 mg / mL of compound (1c) monohydrochloride (corresponding to a nominal concentration of 5.5 mg / mL of compound (1c) free base).

[0209] An aliquot of 0.5 mL of this solution was diluted with 5.0 mL of 0.9% saline, and the pH was adjusted with 5 μL of saturated NaHCO 3 aqueous solution to obtain a 5.5 mL clear, colorless solution of compound (1c) from compound (1c) monohydrochloride at a nominal concentration of 0.50 mg / mL and having a pH of 5.3 - 5.5.

[0210] An aliquot of 1.0 mL of this solution was diluted with 4.5 mL of 0.9% saline, and the pH was adjusted with 10 μL of saturated NaHCO 3 aqueous solution to obtain a 5.5 mL clear colorless solution of compound (1c) from compound (1c) monohydrochloride with a nominal concentration of 1.0 mg / mL and having a pH of 5 - 6.

[0211] An aliquot of 0.5 mL of this solution was diluted with 0.875 mL of 0.9% saline, and the pH was adjusted with 10 μL of saturated NaHCO 3 aqueous solution to obtain a 1.375 mL clear colorless solution of compound (1c) from compound (1c) monohydrochloride with a nominal concentration of 2.0 mg / mL and having a pH of 5 - 6.

[0212] An aliquot of 0.25 mL of this solution was diluted with 10.75 mL of 0.9% saline, and the pH was adjusted with 10 μL of saturated NaHCO 3 aqueous solution to obtain an 11.0 mL clear colorless solution of compound (1c) from compound (1c) monohydrochloride with a nominal concentration of 0.125 mg / mL and having a pH greater than 6.

[0213] A series of 12 mL glass vials each containing 5.0 mg (0.0164 mmol of melphalan free base and 270 mg of SBE 6.5 -β-CD, Captisol (registered trademark)) were prepared as described in Example 4.

[0214] The cyclodextrin derivative / melphalan free base guest - host inclusion complex was reconstituted by dissolving the guest - host inclusion complex in 0.86 mL of 0.9% saline to obtain a 1.0 mL solution with a nominal concentration of 5.0 mg / mL of melphalan free base.

[0215] An aliquot of 1.0 mL of this solution was diluted with 10.0 mL of 0.9% saline, and the pH was adjusted with 5 μL of saturated NaHCO 3Adjusted with an aqueous solution to obtain 11.0 mL of a clear, colorless solution having a nominal concentration of 0.45 mg / mL of melphalan and a pH of 5.8 - 6.1 from melphalan free base.

[0216] 5.6 mg (0.0164 mmol of melphalan monohydrochloride (equivalent to 5.0 mg of melphalan free base) and 270 mg of SBE 6.5 A series of 12 mL glass vials each containing -β-CD (Captisol®) were prepared as described in Example 5.

[0217] The cyclodextrin derivative / melphalan monohydrochloride guest - host inclusion complex was reconstituted by dissolving the guest - host inclusion complex in 0.86 mL of 0.9% saline to obtain 1.0 mL of a solution having a nominal concentration of 5.6 mg / mL of melphalan monohydrochloride (equivalent to a nominal concentration of 5.0 mg / mL of melphalan free base).

[0218] An aliquot of 0.5 mL of this solution was diluted with 5.0 mL of 0.9% saline and the pH was adjusted with 7 μL of saturated NaHCO 3 Adjusted with an aqueous solution to obtain 5.5 mL of a clear, colorless solution having a nominal concentration of 0.45 mg / mL of melphalan hydrochloride and a pH of 5.8 - 6.1 from melphalan hydrochloride.

[0219] The solution was placed in a glass vial and stored at room temperature (about 25 °C), and a certain amount of the compound (1c) obtained from the monohydrochloride (mono HCl salt) of the compound of formula (1c) or melphalan obtained from the monohydrochloride (mono HCl salt) of melphalan was measured at regular intervals using analytical high - performance liquid chromatography (HPLC / UV) connected to a UV detector as described in Example 4. The results are shown in Table 10.

[0220] Formulation 4. Treanda (registered trademark) is an injectable formulation of bendamustine hydrochloride, 1H-benzimidazole-2-butanoic acid, 5-[bis(2-chloroethyl)amino]-1-methyl-, 4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid monohydrochloride, which has been approved by the FDA. Injectable Treanda (registered trademark) is provided as a white to off-white lyophilized powder in vials containing either 25 or 100 mg of bendamustine HCl. Each vial contains either 25 mg or 100 mg of bendamustine hydrochloride and either 42.5 mg or 170 mg of mannitol, USP, which is reconstituted with either 0.9% Sodium Chloride Injection, USP, or 2.5% Dextrose / 0.45% Sodium Chloride Injection, USP to a final concentration of 0.2 mg / mL to 0.6 mg / mL and adjusted to a pH of 2.5 to 3.5.

[0221] A series of 12 mL glass vials each containing 12.5 mg (0.0317 mmol) of bendamustine monohydrochloride (C 16 H 21 Cl 2 N 3 O 2 ·HCl, 394.72 g / mol; MedKoo, 200470) (corresponding to 11.3 mg of bendamustine as the free base) were prepared by weighing the commercial compound into the vials. To each vial containing either bendamustine monohydrochloride, 21.3 mg (0.117 mmol) of D-mannitol (C 6 H 14 O 6 , MW 182.17 g / mol; Sigma Aldrich, M1902) was added. The mixture was dissolved in 2.0 mL of redistilled water (dd-H 2 O (Arrowhead)) to give a clear colorless solution, which was frozen at -78 °C (dry ice / acetone bath) and the solvent was lyophilized at approximately 100 mTorr within about 16 hours to give a colorless solid.

[0222] 85.0 mg (0.255 mmol) of compound (1c) (free base) and 170.4 mg of D-mannitol (0.935 mmol) (C 6 H 14 O 6 (MW 182.17 g / mol; Sigma Aldrich, M1902), and 0.503 mL of 0.5072 M aqueous HCl solution (0.255 mmol, 1.0 eq) (Fluka, 318957) in a mixture of about 16.0 mL of acetonitrile / water (1:1, v / v) were dissolved by vortexing in a clean glass volumetric cylinder (25 mL) to obtain a stock solution of compound (1c) as the mono-HCl salt at a concentration of 5.313 mg / mL and D-mannitol at a concentration of 10.65 mg / mL. This solution was filtered through a 0.22 μm PTFE syringe filter into a clean 25 mL Erlenmeyer flask. A 2.0 mL aliquot (2 × 1.0 mL; 2 × 5.313 mg) of this stock solution was quickly pipetted into a clean 12 mL scintillation vial. The aliquot was frozen at -78 °C (dry ice / acetone bath) and lyophilized at about 100 mTorr for about 16 h to obtain a test vial containing 11.8 mg (0.0319 mmol) of compound (1c) monohydrochloride (mono-HCl salt) (equivalent to 10.6 mg of compound (1c) as the free base) and 21.3 mg (0.117 mmol) of D-mannitol as a colorless powdered solid.

[0223] The material in the vial was dissolved in 2.5 mL of redistilled water (dd-H 2 O) (Arrowhead) to obtain a clear, colorless, non-viscous stock solution of compound (1c) as the monohydrochloride at a concentration of 4.24 mg / mL or bendamustine as the monohydrochloride at a concentration of 4.54 mg / mL, respectively.

[0224] A 1.0 mL stock solution containing compound (1c) as the monohydrochloride at 4.24 mg / mL was diluted with 9.0 mL of 0.9% saline to obtain a 10.0 mL test solution of compound (1c) as the monohydrochloride having a concentration of 0.424 mg / mL and a pH of about 4. A 1.0 mL stock solution containing compound (1c) as the monohydrochloride at 4.24 mg / mL was diluted with 4.0 mL of 0.9% saline to obtain a 5.0 mL test solution of compound (1c) as the monohydrochloride having a concentration of 1.06 mg / mL and a pH of 3 - 4. A 1.0 mL stock solution containing compound (1c) as the monohydrochloride at 4.24 mg / mL was diluted with 2.0 mL of 0.9% saline to obtain a 3.0 mL test solution of compound (1c) as the monohydrochloride having a concentration of 2.12 mg / mL and a pH of about 3.

[0225] A 1.0 mL stock solution containing bendamustine as the monohydrochloride at 4.54 mg / mL was diluted with 9.0 mL of 0.9% saline to obtain a 10.0 mL test solution of bendamustine as the monohydrochloride having a concentration of 0.454 mg / mL and a pH of 4 - 5. A 1.0 mL stock solution containing bendamustine as the monohydrochloride at 4.54 mg / mL was diluted with 4.0 mL of 0.9% saline to obtain a 5.0 mL test solution of bendamustine as the monohydrochloride having a concentration of 1.135 mg / mL and a pH of about 4 - 4.5. A 1.0 mL stock solution containing bendamustine as the monohydrochloride at 4.54 mg / mL was diluted with 2.0 mL of 0.9% saline to obtain a 3.0 mL test solution of bendamustine as the monohydrochloride having a concentration of 2.27 mg / mL and a pH of about 4.

[0226] The solutions were placed in glass vials and stored at room temperature (about 25 °C) or refrigerated (about 0 °C), and a certain amount of compound (1c) obtained from the monohydrochloride (mono HCl salt) of the compound of formula (1c) or bendamustine obtained from the monohydrochloride (mono HCl salt) was measured at regular intervals using analytical high - performance liquid chromatography (HPLC / UV) connected to a UV detector as described in Example 4. The results are shown in Table 10.

Table 10

[0227] Finally, it should be understood that there are alternative ways of implementing the embodiments disclosed herein. Accordingly, this embodiment should be regarded as illustrative and not restrictive, and the claims are not limited to the details provided in this disclosure.

Claims

1. A guest-host inclusion complex comprising: (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof; and Sulfobutylether-β-cyclodextrin (SBE 6-8-β-CD) having an average degree of substitution (ADS) of 6 to 8, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof; wherein the mass ratio of the (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, to the SBE 6-8-β-CD, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, is 1:50 to 1:60; The guest-host inclusion complex.

2. The guest-host inclusion complex according to claim 1, wherein the (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, is (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid hydrochloride.

3. The guest-host inclusion complex according to claim 1, wherein the sulfobutylether-β-cyclodextrin, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, has an average degree of substitution of 6.5 to 7.

4. The guest-host inclusion complex according to claim 1, wherein the sulfobutylether-β-cyclodextrin, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, has an average degree of substitution of 6.5 (SBE 6.5-β-CD).

5. The guest-host inclusion complex according to claim 1, wherein the molar ratio of the (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, to the SBE 6-8-β-CD, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof, is 1:7 to 1:

10.

6. The guest-host inclusion complex according to claim 1, wherein the guest-host inclusion complex comprises a lyophilized product.

7. A pharmaceutical composition comprising the guest-host inclusion complex according to claim 1.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition comprises an aqueous solution containing (S)-3-amino-4-(5-(bis(2-chloroethyl)amino)-2-methylphenyl)butanoic acid at a concentration of 0.1 mg / mL to 1.0 mg / mL, or a pharmaceutically acceptable zwitterion, inner salt, or salt thereof.

9. The pharmaceutical composition according to claim 8, wherein the aqueous solution contains a sodium chloride solution.

10. A pharmaceutical kit comprising the guest-host inclusion complex according to claim 1 and an aqueous solution.

11. The pharmaceutical kit according to claim 10, wherein the aqueous solution is a sodium chloride solution.

12. The pharmaceutical composition according to claim 7 for treating cancer.

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