HK1164698B - Crystalline forms of genistein - Google Patents

Abstract

The disclosure relates to new crystalline forms of genistein. The disclosed crystalline forms include crystalline genistein sodium salt dihydrate; crystalline genistein potassium salt dihydrate; crystalline genistein calcium salt; crystalline genistein magnesium salt; crystalline genistein L-iysine salt; crystalline genistein N -methylglucamine salt; crystalline genistein N - ethylglucamine salt; crystalline genistein diethylamine salt; and crystalline genistein monohydrate. The disclosure also relates to the novel genistein salts represented by these crystalline forms. Therapeutic compositions containing at least one of these crystalline forms of genistein and/or a genistein salt and a pharmaceutically acceptable carrier are described. The disclosure also relates to methods of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition containing the compounds of the disclosure, of a crystalline form of genistein, or of a genistein salt.

Description

Crystalline forms of genistein

Reference to related applications

This application claims priority to U.S. provisional application 61/121,778 filed on 11/12/2008 and U.S. provisional application 61/121,787 filed on 11/12/2008, both of which are incorporated herein by reference.

Background

Cancer is characterized by uncontrolled cell growth that occurs when normal regulation of cell proliferation is lost. This loss is often manifested as the result of dysregulation of cellular pathways involved in cell growth and division, apoptosis, angiogenesis, tumor invasion and metastasis.

Genistein, 4', 5, 7-trihydroxyisoflavone-5, 7-dihydroxy-3- (4-hydroxyphenyl) -4H-1-benzopyran-4-one (shown below) is a natural compound present in plants such as soybean. The potential role of genistein in the prevention and treatment of a number of human diseases including cancer has been extensively studied.

Genistein

Genistein is a BCS class II isoflavone that is commercially available from a number of sources including LC laboratories of Woburn, MA. Cellular targets for and signaling pathways modulated by genistein have been identified, and those associated with cancer include targets and pathways important for cell growth and division, apoptosis, angiogenesis, tumor invasion and metastasis. In addition to the inherent anti-tumor effects of genistein itself, studies have shown that genistein potentiates or highlights the anti-tumor effects of several clinically used chemotherapeutic agents, both in vitro human cancer cell lines and in vivo animal models of cancer. From a therapeutic point of view, these data are interesting, since chemotherapy is the cornerstone in the treatment of most solid tumors.

Genistein is practically insoluble in water, but has high cell membrane permeability. The low water solubility and slow dissolution rate are often limiting factors responsible for the low bioavailability of the pharmaceutical compounds, thereby limiting their use.

Despite the long-known fact that genistein has specific properties of anticancer drugs, no or no successful genistein treatment regimen has been used in the treatment of cancer. A plausible explanation for this could be the poor solubility and bioavailability of the known forms of genistein and the rapid phase II metabolism.

Due to the development of drug discovery strategies over the last 20 years, the physicochemical properties of drug development candidates have changed significantly. Development candidates are generally more oleophilic and less water soluble, which poses a significant problem for the industry. Studies have shown that some drug candidates fail in the clinical phase due to poor human bioavailability and formulation problems. Traditional approaches to solving these problems without completely redesigning the molecule include salt selection, making amorphous materials, particle size reduction, prodrugs and different formulation approaches. Recently, crystalline forms of Active Pharmaceutical Ingredients (APIs) have been used to alter the physicochemical properties of the APIs.

Although therapeutic efficacy is a major concern for therapeutic agents, the salt and solid state forms (i.e., crystalline or amorphous forms) of drug candidates can be critical to their pharmacological properties as viable APIs and their development. For example, each salt or each crystal form of a drug candidate may have different solid state (physical and chemical) properties. The differences in physical properties exhibited by the novel solid forms of the API (such as co-crystals, salts or polymorphs of the original compound) affect pharmaceutical parameters such as storage stability, compressibility and density (important in formulation and product manufacture) as well as solubility and dissolution rate (important factors in determining bioavailability). Because these actual physical properties are influenced by the solid state properties of the crystalline form of the API, they can significantly affect the choice of compound as an API, the final pharmaceutical dosage form, optimization of the manufacturing process, and absorption in vivo. Moreover, finding the most appropriate polymorph for further drug development can reduce the time and expense of such development.

Obtaining a crystalline form of an API is extremely useful in drug development. Which allows better characterization of the chemical and physical properties of the drug candidate. The desired properties of a particular API may also be obtained by forming a salt of the API and/or a crystalline salt of the API. Crystalline forms and crystalline salts often have better chemical and physical properties than the free base in its amorphous state. Such salts and crystalline forms may have more favorable pharmaceutical and pharmacological properties or be easier to process than amorphous polymorphs, as with the present invention. They may also have better storage stability.

One such physical property that can affect processability is the flowability of the solid before and after grinding. Flowability affects the ease with which the material is handled during processing into a pharmaceutical composition. When the particles of the powdered compound cannot flow past each other easily, the formulation specialist must take this fact into account when developing a tablet or capsule formulation, which may require the use of glidants such as colloidal silicon dioxide, talc, starch or tribasic calcium phosphate.

Another potentially important solid state property of an API is its dissolution rate in aqueous fluids. The rate of dissolution of the active ingredient in the patient's stomach fluids may be of therapeutic importance as it affects the rate at which the orally administered active ingredient can reach the patient's bloodstream.

By forming and/or crystallizing the API salt, the new solid state form of the API may have unique properties compared to the existing solid form of the API or salt thereof. For example, the crystalline salt may have different dissolution and solubility properties than the API itself and may be used to deliver the API therapeutically. The new pharmaceutical formulation comprising the crystalline salt of the API may have superior properties over existing pharmaceutical formulations.

Crystalline salts or other crystalline forms of APIs generally have particular crystalline and spectroscopic properties when compared to other forms having the same chemical composition. The crystalline and spectroscopic properties of a particular form are typically measured by single crystal X-ray crystallography in X-ray powder diffraction (XRPD) and other techniques. Particular crystalline forms often also exhibit special thermal behavior. Thermal behavior is measured in the laboratory by such techniques as capillary melting point, thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC).

Disclosure of Invention

The present invention relates to crystalline forms of genistein, including crystalline genistein salts and crystalline genistein hydrates. Therapeutic compositions comprising crystalline forms of genistein according to the invention represent another embodiment of the invention, as do methods for treating or preventing cancer and other hyperproliferative diseases using those crystalline forms of the invention or therapeutic compositions comprising them. Therapeutic compositions of crystalline genistein may also be used to treat or prevent chronic inflammation, infection, cystic fibrosis and amyloidosis. As used herein and as known in the art, the term "ambient temperature" refers to the temperature within an enclosed space to which a human being is adapted, i.e., room temperature. For example, the ambient temperature may be in the range of, for example, about 20 ℃ to about 25 ℃.

As used herein and as known in the art, the term "about" means close in number or amount.

As used herein and as known in the art, the term "slurry" refers to a suspension of solids in a liquid.

As used herein and as known in the art, the terms "° 2 θ" are interchangeable with [ degrees-2 θ ], [ ° 2Th. ] and variants thereof.

Drawings

The following drawings, which are described below and incorporated in and constitute a part of this specification, illustrate exemplary embodiments in accordance with the disclosure and should not be construed as limiting the scope of the invention, as the invention may admit to other equally effective embodiments. The figures are not necessarily to scale and certain features and certain views of the figures may be shown exaggerated in scale or in schematic for clarity and conciseness.

FIG. 1 depicts the XRPD pattern of crystalline genistein sodium salt dihydrate.

FIG. 2 depicts the DSC curve of dried crystalline genistein sodium salt dihydrate.

FIG. 3 depicts crystalline genistein sodium salt dihydrateGravimetric vapor adsorption(GVS) curve.

Figure 4 depicts a TGA profile of a sample from a prepared crystalline genistein sodium salt dihydrate dried at ambient temperature for about 24 hours.

FIG. 5 depicts a TGA curve of a sample from crystalline genistein sodium salt dihydrate prepared by drying at 80 ℃ overnight.

Figure 6 depicts four XRPD patterns of crystalline genistein sodium salt dihydrate obtained after a stability study at 80 ℃ for 7 days and at 40 ℃/75 Relative Humidity (RH)% for 7 days.

FIG. 7 is a crystalline genistein sodium salt dihydrate¹H Nuclear Magnetic Resonance (NMR) spectrum.

Figure 8 depicts the XRPD pattern after hydration studies of crystalline genistein sodium salt dihydrate.

FIG. 9 is a molecular model of crystalline genistein sodium salt dihydrate showing two sodium cations that are centrosymmetric in the dimeric structure of crystalline genistein sodium salt dihydrate, wherein intramolecular hydrogen bonds are shown as dashed lines.

Fig. 10 is a molecular model showing the layer formation of crystalline genistein sodium salt dihydrate.

FIG. 11 is a molecular model showing the packing of crystalline genistein sodium salt dihydrate.

Figure 12 depicts a calculated XRPD pattern based on single crystal data for crystalline genistein sodium salt dihydrate.

Figure 13 depicts the plasma concentration of total genistein (mean, n-3) after intraduodenal administration of genistein and crystalline genistein sodium salt dihydrate.

Figure 14 depicts XRPD patterns for crystalline genistein sodium salt dihydrate from large scale synthesis.

Figure 15 depicts the XRPD pattern for amorphous genistein potassium salt.

Figure 16 depicts the XRPD pattern for crystalline genistein potassium salt dihydrate.

Figure 17 depicts the TGA profile of crystalline genistein potassium salt dihydrate.

FIG. 18 depicts DSC curves of crystalline genistein potassium salt dihydrate.

FIG. 19 depicts the GVS curves for crystalline genistein potassium salt dihydrate.

FIG. 20 depicts crystalline genistein potassium salt dihydrate¹H NMR。

Figure 21 depicts an XRPD pattern from a stability study of crystalline genistein potassium salt dihydrate.

Figure 22 depicts an XRPD pattern for hydration studies of crystalline genistein potassium salt dihydrate.

Figure 23 depicts XRPD patterns for crystalline genistein calcium salt.

FIG. 24 depicts a TGA curve for crystalline genistein calcium salt.

Figure 25 depicts an XRPD pattern of 1 equivalent for the crystalline magnesium genistein.

FIG. 26 depicts a TGA curve for 1 equivalent of crystalline genistein magnesium salt.

Figure 27 depicts an XRPD pattern of 2 equivalents for the crystalline magnesium genistein.

FIG. 28 depicts a TGA curve of 2 equivalents for crystalline genistein magnesium salt.

Figure 29 depicts an XRPD pattern for crystalline genistein.

FIG. 30 depicts an XRPD pattern for crystalline genistein L-lysine salt from toluene.

Figure 31 depicts an XRPD pattern for crystalline genistein L-lysine salt from isopropanol.

Figure 32 depicts a TGA trace for a crystalline genistein/genistein mixture from isopropanol.

Figure 33 depicts an XRPD pattern for crystalline genistein N-methylglucamine (meglumine) salt.

FIG. 34 depicts an XRPD pattern for the crystalline genistein N-ethylglucamine (glucethylamine) salt prepared from acetone.

FIG. 35 depicts an XRPD pattern for the crystalline genistein N-ethylglucamine (glucethylamine) salt prepared from isopropanol.

FIG. 36 depicts the TGA profile of crystalline genistein N-ethylglucamine salt from acetone.

Figure 37 depicts an XRPD pattern for crystalline genistein diethylamine salt.

Figure 38 depicts an XRPD pattern for crystalline genistein monohydrate.

FIG. 39 depicts a TGA curve of crystalline genistein monohydrate.

Detailed Description

The present invention relates to an improvement in the physicochemical properties of genistein, whereby this compound is suitable for use in drug development. Disclosed herein are several novel crystalline forms of genistein, including, for example, crystalline genistein sodium, potassium, magnesium, N-methylglucamine (meglumine) salts, calcium, L-lysine, N-ethylglucamine (glufosinate) and diethylamine salts, as well as crystalline monohydrate of genistein. These crystalline forms of genistein are described below. Although crystalline forms of genistein are described herein, the present invention also relates to novel chemical compositions comprising the disclosed crystalline forms of genistein. Therapeutic applications of those crystalline forms are described, as well as therapeutic compositions comprising them. Methods for characterizing the crystalline forms are also described below.

One embodiment of the present invention relates to crystalline genistein sodium salt dihydrate. Crystalline genistein sodium salt dihydrate may have suitable characteristics for drug development. The only possible disadvantage may be its needle-like morphology, which is not necessarily ideal for flowability or compaction during manufacture. The needle morphology was observed using a polarizing microscope (PLM). Milling of such crystalline needle-like materials, or similar techniques known in the art, may be used to obtain a more uniform particle morphology that may be used to prepare materials for the manufacture of pharmaceutical compositions thereof. The ordinarily skilled artisan can determine a suitable particle size for use in a desired pharmaceutical composition. A particle size of, for example, about 5 μm may be used. It should be noted, however, that continuous grinding may dehydrate the material due to the high temperatures involved during such a process. On the other hand, storage tests at 80 ℃ have indicated that, at elevated temperatures, the material is able to exist as a hydrate with only slight changes over a period of 7 days. This mitigates the risk of dehydration upon grinding.

As shown in fig. 9, the crystalline genistein sodium salt dihydrate of the present invention has two sodium cations that are centrosymmetric in a dimeric structure in which two genistein molecules and four water molecules are bound. Crystalline genistein sodium salt dihydrate can be prepared from, for example, the common solvent IPA (isopropanol or 2-propanol) at ambient temperature without any special treatment such as temperature cycling, sonication or rapid evaporation. As shown in the following example 1, the crystalline genistein sodium salt dihydrate of the present invention has excellent stability. Compared to genistein itself, it is more soluble in water, aqueous solvent systems and organic solvents. In addition, crystalline genistein sodium salt dihydrate showed superior early and late intrinsic kinetic solubility curves compared to genistein. It was also shown that the crystalline genistein sodium dihydrate of the present invention has a higher bioavailability compared to genistein.

Another embodiment of the present invention is crystalline genistein potassium salt dihydrate. Crystalline genistein potassium salt dihydrate can also be prepared from e.g. the common solvent IPA (isopropanol or 2-propanol) at ambient temperature without any special treatment like temperature cycling, ultrasound or flash evaporation. Crystalline genistein potassium salt dihydrate is readily formed from solid genistein potassium salt. Near the time of recovery, the potassium genistein salt appears to be an unstable anhydrous amorphous salt which then rapidly absorbs water from the environment to crystallize into a dihydrate material. As discussed in example 2 below, the crystalline genistein potassium dihydrate salt has good stability. The potassium genistein salt dihydrate is crystalline and has a needle-like morphology (but thicker needles than the corresponding crystalline genistein sodium salt dihydrate).

In addition to the crystalline genistein sodium and potassium salts according to the invention, a further independent embodiment of the invention relates to crystalline salts of genistein with magnesium, N-methylglucamine (meglumine), calcium, L-lysine, N-ethylglucamine (glufosamine) and diethylamine. Yet another embodiment of the present invention relates to a crystalline monohydrate form of genistein. Each of these crystalline forms of genistein, their preparation and characterization are described in the following examples.

Therapeutic application of genistein crystal form

The present invention relates to the therapeutic use of at least one crystalline form of genistein, for example at least one crystalline genistein salt. The term "treatment" or "treating" refers to any treatment of a disease or disorder in a mammal, including: preventing or protecting against a disease or disorder, i.e., such that no clinical symptoms are produced; inhibiting the disease or disorder, i.e., arresting or inhibiting the development of clinical symptoms; and/or relieving the disease or disorder, i.e., allowing clinical symptoms to recover. It will be appreciated by those skilled in the art that in human medicine it is not always possible to distinguish between "prevention" and "inhibition" because the event or events ultimately induced may be unknown, latent, or the patient may be determined long after the event or events have occurred. Thus, as used herein, the term "prevention" is intended as an element of "treatment" to encompass both "prevention" and "inhibition" as defined herein. As used herein, the term "protection" is intended to include "prevention".

The crystalline genistein forms according to the present invention are useful as pharmaceutical agents, which can be used for the treatment of hyperproliferative diseases such as, for example, various cancers including, for example, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, prostate cancer, bladder cancer, brain cancer, kidney cancer, ovarian cancer, liver cancer, skin cancer, thyroid cancer and pancreatic cancer, as well as leukemia or lymphoma. The leukemias and lymphomas mentioned herein may be tumors of the myeloid system, such as, for example, acute myeloid leukemia of the lymphoid system.

In addition, the crystalline forms of genistein disclosed herein may also be used in methods of treating warm-blooded animals such as, for example, humans by therapy. For example, the crystalline genistein salt according to the present invention may be used in a method for treating hyperproliferative diseases such as various cancers including, for example, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, prostate cancer, bladder cancer, brain cancer, kidney cancer, ovarian cancer, liver cancer, skin cancer, thyroid cancer and pancreatic cancer, as well as leukemia or lymphoma. The leukemias and lymphomas mentioned herein may be tumors of the myeloid system, such as, for example, acute myeloid leukemia of the lymphoid system.

Furthermore, the crystalline form of genistein according to the present invention may be used in a method for the treatment of a human suffering from hyperproliferative diseases, such as for example various cancers including, for example, intestinal, gastric, esophageal, breast, lung, prostate, bladder, brain, kidney, ovary, liver, skin, thyroid and pancreatic cancers, as well as leukemias or lymphomas. In another embodiment, the crystalline genistein forms according to the present disclosure may be used for the prevention of hyperproliferative diseases such as, for example, various cancers including, for example, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, prostate cancer, bladder cancer, brain cancer, kidney cancer, ovarian cancer, liver cancer, skin cancer, thyroid cancer and pancreatic cancer, as well as leukemia or lymphoma. The leukemias and lymphomas mentioned herein may be tumors of the myeloid system, such as, for example, acute myeloid leukemia of the lymphoid system. Comprising the step of administering to a human in need thereof a therapeutically effective amount of at least one crystalline form of genistein. The use of at least one crystalline form of genistein in any of the above methods of treating humans also forms an aspect of the invention.

The treatment as defined herein may be used as monotherapy or may involve, in addition to at least one compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following classes of antineoplastic agents: (i) antiproliferative/antineoplastic agents and combinations thereof for use in medical oncology, such as alkylating and alkylating like agents (e.g., cisplatin, carboplatin, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, and nitrosourea), antimetabolites (e.g., gemcitabine hydrochloride, 5-fluorouracil, tegafur, raltitrexed, methotrexate, cytarabine, and hydroxyurea), antitumor antibiotics (e.g., anthracyclines such as doxorubicin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, and mithramycin), antimitotics (e.g., vinca alkaloids such as vincristine, vinblastine, vindesine, and vinorelbine, and taxanes such as violaxane and taxotere) and topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and teniposide), Amsacrine, topotecan, and camptothecin); (ii) cytostatic agents such as antiestrogens (e.g. tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), estrogen receptor antagonists (e.g. fulvestrant), antiandrogens (e.g. bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (e.g. goserelin, leuprolide and buserelin), progestogens (e.g. megestrol acetate), aromatase inhibitors (e.g. anastrozole, letrozole, vorozole (vorazole) and exemestane) and inhibitors of 5-alpha-reductase (e.g. finasteride); (iii) agents that inhibit cancer cell invasion (e.g., metalloproteinase inhibitors such as marimastat and inhibitors of urokinase plasminogen activator receptor function); (iv) inhibitors of growth factor function, for example, include growth factor antibodies, such inhibitors of growth factor receptor antibodies (e.g., anti-ErbB 2 antibodies trastuzumab (herceptin) and anti-ErbB 1 antibody (cetuximab)), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine-threonine kinase inhibitors such as inhibitors of the epidermal growth factor family (e.g., EGFR family tyrosine kinase inhibitors such as N- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZDI839), N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N- (3-chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (CI1033)), inhibitors of the platelet-derived growth factor family and inhibitors of the hepatocyte growth factor family; (v) anti-angiogenic agents such as those that inhibit the action of vascular endothelial growth factor (e.g., the anti-vascular endothelial growth factor antibody bevacizumab (avastin) as well as compounds such as those disclosed in international patent applications WO97/22596, WO97/30035, WO97/32856 and WO 98/13354) and compounds that act by other mechanisms (e.g., linoamine, inhibitors of integrin function and angiostatin); (vi) vascular disrupting agents such as combretastatin A4 and the compounds disclosed in International patent applications WO99/02166, WO00/40529, WO00/41669, WO01/92224, WO02/04434 and WO 02/08213; (vii) antisense therapies, such as those directed against the targets listed above, e.g., ISIS2503, anti-antisense oligonucleotides (anti-ras antisense); (viii) gene therapy methods, including, for example, methods of replacing abnormal genes such as abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) methods such as those using cytosine deaminase, thymidine kinase, or bacterial nitroreductase, and methods of increasing patient tolerance to chemotherapy or radiation therapy such as multi-drug resistance gene therapy; and (ix) immunotherapy approaches, including for example in vitro and in vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.

In the above discussed treatments, at least one crystalline form of genistein according to the invention may also be used in combination with more than one cell cycle inhibitor, for example with a cell cycle inhibitor inhibiting cyclin-dependent kinases (CDKs), or with imatinib mesylate (gleevec). Such combination therapy may be achieved by way of the simultaneous, sequential or separate administration of the individual therapeutic ingredients. Such a combination product may employ at least one compound of the present invention within the dosage ranges described herein and at least one other pharmaceutically active agent within its approved dosage range. The combination product may be formulated into a single dosage form.

The present invention also provides a combination which may be suitable for use in the treatment of a cell proliferative disorder, such as cancer, comprising at least one crystalline form of genistein, such as at least one crystalline genistein salt as described hereinbefore, and at least one further anti-tumour agent as described hereinbefore. Such combinations may be used as medicaments for the combined treatment of cell proliferative disorders, such as cancer.

In addition to its use in therapeutic medicine, at least one crystalline form of genistein according to the present invention may be used as a pharmacological tool in the development and standardization of in vitro and in vivo test systems for evaluating the effect of inhibitors of cell cycle activity in experimental animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.

Another aspect of the present invention relates to the therapeutic use of at least one crystalline form of genistein according to the invention for the preparation of a medicament for the treatment of diseases in which inhibition of inflammation is beneficial, such as for example chronic inflammation, inflammatory bowel disease, crohn's disease, sjogren's disease, rheumatoid arthritis, atopic dermatitis, vasculitis, psoriasis, benign prostatic hyperplasia, wound healing, end-stage renal disease, chronic kidney disease, chronic obstructive pulmonary disease or asthma.

In addition, at least one crystal form of genistein according to the invention may also be used for the preparation of a medicament for the treatment of diseases in which inhibition of infection is beneficial, such as e.g. local infections, systemic infections, sepsis, systemic fungal infections or local fungal infections.

Yet another aspect of the present invention relates to the use of at least one crystal form of genistein for the treatment of diseases in which restoration of normal chloride and salt (water) mobilization in body organs and human glands is beneficial, such as for example the stimulation of cystic fibrosis transmembrane conductance regulator.

A further aspect of the invention relates to the use of at least one crystal form of genistein for the treatment of diseases in which inhibition of soluble protein by formation of insoluble extracellular fibril precipitates responsible for organ dysfunction is beneficial, such as for example the inhibition of transthyretin (TTR) amyloidosis caused by a change in the amino acid sequence of the TTR gene product. In another embodiment of the present disclosure, at least one crystalline form of genistein as described herein may be used for the treatment of familial amyloidogenic polyneuropathy.

Pharmaceutical composition containing genistein crystal form

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one crystalline form of genistein according to the invention and a pharmaceutically acceptable carrier (also referred to as pharmaceutically acceptable excipient). As discussed above, the crystalline forms of genistein according to the invention may be therapeutically useful for the treatment or prevention of disease states such as those discussed above, including for example those associated with abnormal angiogenesis.

Pharmaceutical compositions for treating those disease states may comprise a therapeutically effective amount of at least one crystalline form of genistein according to the invention in order to down-regulate the transcription of genes involved in the control of angiogenesis, thereby treating patients with specific diseases. The pharmaceutical composition of the present invention may be any pharmaceutical form comprising at least one crystalline form of genistein according to the present invention. The pharmaceutical compositions may be, for example, tablets, capsules, liquid suspensions, injectable, topical or transdermal. The pharmaceutical compositions typically comprise, for example, from about 1% to about 99% by weight of at least one crystalline form of genistein according to the invention and, for example, from 99% to 1% by weight of at least one suitable pharmaceutical excipient. In one embodiment, the composition may be between about 5% to about 75% by weight of at least one crystalline form of genistein of the invention, the remainder being at least one suitable pharmaceutical excipient or at least one other adjuvant, as discussed below.

A "therapeutically effective amount of at least one crystalline form of genistein according to the invention" is typically in the range of about 0.05 to about 500 mg/kg. The actual amount required to prevent or treat any particular patient may depend on various factors including, for example, the disease state being treated and its severity; the specific pharmaceutical composition used; the age, weight, general health, sex, and diet of the patient; a mode of administration; the time of administration; the route of administration; and the excretion rate of the genistein crystal form; the duration of the treatment; any drug used in combination or concomitantly with the specific mixture used; and other such factors well known in the medical arts. These factors are discussed in Goodman and Gilman, "The Pharmacological Basis of Therapeutics", Tenth Edition, A.Gilman, J.Hardman and L.Limbird, eds., McGraw-Hill Press, 155-. The crystalline forms of genistein according to the invention and the pharmaceutical compositions containing them can be used in combination with anticancer agents or other agents, which are generally administered to patients treated for cancer. They may also be co-formulated with more than one such agent in a single pharmaceutical composition.

Depending on the type of pharmaceutical composition, the pharmaceutically acceptable carrier may be selected from any one or a combination of carriers known in the art. The choice of a pharmaceutically acceptable carrier depends on the form of drug to be used and the desired method of administration. For the pharmaceutical composition of the present invention, i.e. a pharmaceutical composition having at least one crystalline form of genistein according to the present invention, a carrier should be selected which retains the crystalline form. In other words, the carrier should not substantially change the crystalline form of genistein. Nor should the carrier be otherwise incompatible with the crystalline form of the genistein salt used, such as by producing any undesirable biological effects, or otherwise interacting in a deleterious manner with any other ingredient of the pharmaceutical composition.

The Pharmaceutical compositions of the present invention may be prepared by methods known in the art of Pharmaceutical formulation, see, for example, Remington's Pharmaceutical Sciences, 18th ed., incorporated herein by reference (Mack Publishing Company, Easton, Pa., 1990). In solid dosage forms, at least one crystalline form of genistein may be mixed with at least one pharmaceutically acceptable excipient such as, for example, sodium citrate or dicalcium phosphate, or: (a) fillers or extenders such as, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as, for example, cellulose derivatives, starch, alginates (alignates), gelatin, polyvinylpyrrolidone, sucrose and gum arabic, (c) humectants such as, for example, glycerol, (d) disintegrating agents such as, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates and sodium carbonate, (e) solution retarders such as, for example, paraffin, (f) absorption promoters such as, for example, quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, magnesium stearate and the like, (h) adsorbents such as, for example, kaolin and bentonite, and (i) lubricants such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Pharmaceutically acceptable adjuvants known in the art of pharmaceutical formulation may also be used in the pharmaceutical compositions of the present invention. These include, but are not limited to, preservatives, wetting agents, suspending agents, sweetening, flavoring, perfuming, emulsifying and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. If desired, the pharmaceutical compositions of the present invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene and the like.

Coatings and shells, such as enteric coatings and others well known in the art, may be utilized to prepare solid dosage forms as described above. They can comprise soothing agents and can also be compositions which release one or more active compounds in a delayed manner in specific parts of the intestinal tract. Non-limiting examples of embedding compositions that can be used are polymers and waxes. The active compound may also be in the form of microcapsules, if desired with more than one of the above-mentioned excipients.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Compositions for rectal administration are, for example, suppositories which can be prepared by mixing at least one crystalline form of genistein according to the present disclosure with, for example, a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which may be solid at ordinary temperatures and liquid at body temperature and therefore melt and release the active ingredient therein when in a suitable body cavity.

Solid dosage forms are preferred for the pharmaceutical compositions of the present invention because the crystalline form of genistein is maintained during preparation. Solid dosage forms for oral administration may be used, including capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be admixed with at least one inert pharmaceutical excipient (also referred to as a pharmaceutical carrier). The crystalline forms of genistein according to the invention may also be used as precursors in the formulation of liquid pharmaceutical compositions. The crystalline genistein forms may be administered in pure form or in a suitable pharmaceutical composition by any recognized mode of administration or agents for providing similar uses. Thus, administration may be in the form of a solid, semi-solid, lyophilized powder or liquid dosage form such as, for example, a tablet, suppository, pill, soft elastic hard capsule, powder, solution, suspension, or aerosol, etc., as, for example, in a unit dosage form suitable for simple administration of precise dosages, e.g., oral, buccal, nasal, parenteral (intravenous, intramuscular, or subcutaneous), topical, transdermal, intravaginal, intravesical, systemic, or rectal administration. One route of administration may be oral administration using a convenient daily dosage regimen which may be adjusted depending on the severity of the disease state to be treated.

The present invention also relates to the preparation of pharmaceutical agents using at least one crystalline form of genistein for the treatment of various diseases. These include, but are not limited to: diseases in which inhibition of more than one protein tyrosine kinase is beneficial, such as for example kinases affected by genistein are possible targets; hyperproliferative diseases such as various cancers, such as, for example, intestinal, breast, lung, prostate, bladder, kidney or pancreatic cancer, or leukemias or lymphomas or proliferative inflammatory atrophy; diseases in which inhibition of inflammation is beneficial, such as, for example, chronic inflammation, inflammatory bowel disease, crohn's disease, sjogren's disease, rheumatoid arthritis, atopic dermatitis, vasculitis, psoriasis, benign prostatic hyperplasia, wound healing, end stage renal disease, chronic kidney disease, chronic obstructive pulmonary disease, asthma; diseases in which inhibition of infection is beneficial, such as, for example, topical infection, systemic infection, sepsis, systemic fungal infection, topical fungal infection; diseases in which restoration of normal chloride and salt (water) mobilization in human body organs and glands is beneficial, such as, for example, stimulation of cystic fibrosis transmembrane conductance regulators and diseases and conditions involving post-menopausal conditions such as hot flashes and osteoporosis and diseases in which inhibition of soluble proteins by formation of insoluble extracellular protofibrillar precipitates that trigger organ dysfunction is beneficial, such as amyloidosis, e.g., those in which the fibrillar precipitates are composed of transthyretin (TTR), such as familial amyloidosis polyneuropathy.

Examples

The following analytical techniques were used in the following examples:

x-ray powder diffraction (XRPD): x-ray powder diffraction studies were performed on a Bruker D8-Discover diffractometer. Approximately 5mg of sample was gently compressed on a single 96-well plate sample holder with zero background XRPD. The samples were then loaded into a Bruker D8-Discover diffractometer by transport and analyzed using the experimental conditions shown in Table 1.

Table 1: XRPD measurement conditions

Differential Scanning Calorimetry (DSC): about 2mg of the sample was weighed into a DSC aluminum pan and closed with an aluminum lid (non-hermetically). The sample pan was then loaded into a Pyris 1 Perkin Elmer (Perkin-Elmer) DSC (equipped with a liquid nitrogen cooling unit) cooled and maintained at 25 ℃. Once a stable heat flow response was obtained, the sample was then heated to 300 ℃ at a scan rate of 10 ℃/minute and the resulting heat flow response was monitored. Using 20cm³A/minute helium purge to prevent thermally induced oxidation of the sample during heating and also to reduce thermal lag through the sample to improve device sensitivity. Prior to analysis, the device was temperature and heat flow calibrated using an indium reference standard.

Gravimetric Vapor Sorption (GVS): approximately 15mg of sample was placed in a wire mesh vapor sorption balance pan and loaded into a provided SMS intrinsic vapor sorption balance (surface measurement system instrument). The sample was then dried by maintaining a 0% humidity environment until no further weight change was indicated. Subsequently, the sample was then subjected to a temperature ramp profile of 0-90% RH at 10% RH increments, holding the sample at each step until equilibrium has been achieved (99.5% step complete). When equilibrium is reached, the% RH in the device is raised to the next step and the equilibration procedure is repeated. After the adsorption cycle is complete, the same procedure is then used for the samplesDrying is carried out. The weight change during the adsorption/desorption cycle is then monitored, thereby allowing the hygroscopicity of the sample to be determined.

Thermogravimetric analysis (TGA): about 5mg of the sample was accurately weighed into a TGA platinum pan and loaded into a perkin elmer TGA7 thermogravimetric analyzer maintained at room temperature. The sample was then heated from 25 ℃ to 300 ℃ at a rate of 10 ℃/min, during which time the change in weight was monitored. The cleaning gas used was 20cm³Nitrogen per minute flow. Prior to analysis, the equipment was weight calibrated using a 100mg reference weight and temperature calibrated using a nickel aluminium alloy reference standard.

Polarizing microscope (PLM): the presence of crystallinity (birefringence) was determined using a Leica Leitz (Leica Leitz) DMRB polarization microscope equipped with a high resolution Leica camera and image acquisition software (Firecam v.1.0). All images were recorded using a 10x objective unless otherwise noted.

¹ H Nuclear Magnetic Resonance (NMR)：¹H NMR was performed on a Bruker AC 200200 MHz spectrometer and NMR of each sample was performed in deuterated methanol. Each sample was prepared at a concentration of about 5 mg.

Example 1-crystalline genistein sodium salt dihydrate

1.1 preparation of genistein sodium salt dihydrate: placing about 300mg of genistein into 6cm³(20 volumes) of IPA. Upon addition of 1M sodium hydroxide (NaOH), the reaction was rapidly evident (color change from light yellow to bright yellow). The mixture was shaken at ambient temperature for about 3 hours and then allowed to stand for about 2 days (weekend). The solid was isolated by filtration and then allowed to dry at ambient temperature for about 24 hours. The genistein sodium salt prepared according to this method is crystalline genistein sodium salt dihydrate, which has been characterized by the following method.

1.2 XRPD of crystalline genistein sodium salt dihydrate

The XRPD pattern as shown in figure 1 was obtained using the procedure described above. As shown in fig. 1, XRPD analysis showed solid-type impurities that could be IPA solvates of the sodium salt. The material was dried overnight at 80 ℃ to remove impurities. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 2. A full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein sodium salt dihydrate. A subset of the peaks from figure 1 that can be used alone or in combination to characterize crystalline genistein sodium salt dihydrate include 5.9, 11.6, 11.8, 15.2, 24.8, 28.2, 28.9 and 28.9 ° 2 θ ± 0.2 ° 2 θ.

TABLE 2

1.3 DSC of dried crystalline genistein sodium salt dihydrate

Samples were prepared by drying crystalline genistein sodium dihydrate salt, according to the procedure described in 1.1 above, at 80 ℃ overnight. Figure 2 shows the DSC of a sample of dried crystalline genistein sodium salt dihydrate. DSC indicates dehydration at about 91 ℃ followed by melting at about 132 ℃. Other peaks may be associated with degradation (also indicated by the TGA curves shown in figures 4 and 5 discussed below).

1.4 GVS crystalline genistein sodium salt dihydrate

As shown in figure 3, GVS studies of crystalline genistein sodium dihydrate indicated hydrate formation (GVS cycle dehydrated material before analysis) and adsorption of up to 45 wt% water. However, between 20 and 70 RH% (typical working range of materials), a humidity change of only about 2% is observed.

1.5 TGA of crystalline genistein sodium salt dihydrate

Figure 4 shows a TGA profile of a sample of crystalline genistein sodium salt dihydrate from 1.1 above dried at ambient temperature for about 24 hours. Figure 5 is TGA of a sample from a prepared crystalline genistein sodium dihydrate salt dried overnight at 80 ℃. TGA indicates that sodium salts hydrate and begin water loss at about 75 ℃ and are suitable for further development. The weight loss corresponds to one mole of water to one mole of sodium.

1.6 crystalline genistein sodium salt dihydrate PLM

PLM of crystalline genistein sodium salt dihydrate shows a needle-like morphology.

1.7 solubility measurement of crystalline genistein sodium salt dihydrate

Water solubility: the following protocol was used to measure water solubility. Slurries of genistein and crystalline genistein sodium dihydrate salt were prepared in an aqueous medium with pH set at 4.5, 6.7 and 7.5, each slurry was shaken at ambient temperature for about 24 hours and then filtered into clean vials using a 0.2 μm filter. The saturated solution was then diluted and purified on a Chirobiotic T High Performance Liquid Chromatography (HPLC) column set at lambda_{Maximum of}API (genistein) content was analyzed on a uv detector at 270nm using N-Ac-DL-methionine. The mobile phase was acetonitrile/water flowing in an isocratic mode over a period of 30 minutes. The results are shown in table 3 (BDL ═ below the detection limit). The API peak is not evident from the HPLC curve using genistein flow (should appear at about 6-7 minutes) indicating that genistein is very insoluble in aqueous media and at a level lower than the sensitivity of the HPLC technique used (sensitivity of the technique mg to μ g level). Genistein was reported to show water solubility in the range of 10-40 nM.

TABLE 3

Solubility in different solvents: the solubility in different organic solvents was measured using the following protocol. Approximately 25mg parts of genistein and crystalline genistein sodium dihydrate salt were placed in 48 different vials, respectively. To the vial was added 5 volume aliquots of each solvent exclusively. Between each addition, the mixture was checked for dissolution and if dissolution was not evident, the procedure was continued until dissolution was observed or when 50 volumes had been added. The results are shown in table 4.

TABLE 4

1.8 stability Studies of crystalline genistein sodium salt dihydrate

The samples were tested for stability at 80 ℃ for 7 days and at 40 ℃/75 RH% for 7 days. Observations such as color changes were noted after 7 days, and XRPD of the samples was performed after 7 days to investigate any solid form changes. Figure 6 shows XRPD patterns of the original sample of crystalline genistein sodium salt dihydrate and the samples at 80 ℃ for 7 days and at 40 ℃/75 RH% for 7 days. The 40 ℃/75 RH% study indicated no change over the 7 day period. Storage of the material at 80 ℃ for a period of 7 days indicated a slight loss of crystallinity, which suggests slow dehydration. The light stability test for 7 days revealed no change in either color or solid form.

1.9 preparation of crystalline genistein sodium salt dihydrate ¹ H NMR Spectrum

FIG. 7 shows crystalline genistein sodium salt dihydrate¹H NMR spectrum. Table 5 shows¹Peaks in the H NMR spectrum. In FIG. 8¹Aromatic at about 5.9 in genistein in H NMRChemical shifts of the group protons shifted to 6.1ppm confirmed salt formation.

TABLE 5

Chemical shift	Multiplicity (mulitlpicity)	Range (ppm)
			7.952	s	7.932-7.919
7.372	m	7.429-7.306
			6.861	m	6.927-6.791
6.101	dd	6.187-6.028
			4.936	s	5.256-4.723
3.34	q	3.577-3.096
			1.085	s	1.213-0.934

s is singlet, m is multiplet, dd is two doublets, q is quartet (quadruplet)

1.10 disproportionation study of crystalline genistein sodium salt dihydrate

A50 mg sample of crystalline genistein sodium salt dihydrate was slurried in 250. mu.L of distilled water for about 48 hours and then checked for disproportionation by XRPD. The pH of the supernatant was also measured using a Corning (Corning)240pH meter. No evidence of disproportionation was observed. The pH of the supernatant after slurrying was 7.1.

1.11 hydration Studies of crystalline genistein sodium salt dihydrate

At the water level, about 100mg of crystalline genistein sodium salt dihydrate was placed in about 500 μ L of IPA/water mixture (3%, 5% and 10%). The various mixtures were stirred at ambient temperature for about 48 hours and then filtered to recover the solids for XRPD and TGA studies. As shown in fig. 8, hydration is indicated by the change in the XRPD pattern of the original material, consistent with weight loss from TGA (material dependence). Hydration studies revealed no further hydration; but the IPA solvate impurities were removed.

1.12 Single Crystal X-ray diffraction of crystalline genistein sodium salt dihydrate

Preparing a single crystal:from crystalline genistein sodium salt dihydrate (about 48mg) dissolved in 50: 50 IPA/water (3 cm)³) Crystal growth in the solution of (1). The solution was then slowly evaporated through a punctured parafilm (parafilm). After about 2 weeks of evaporation, needle-shaped crystals were clearly obtained.

Single crystal X-ray diffraction:the sample's slatted needle was selected for data collection. Diffraction data were collected using a Bruker Smart Apex CCD diffractometer equipped with an Oxford Cryosystem cryodevice operating at 150K, using Mo-K α radiation.

When indexing the data set, the crystal structure was determined to be pseudo-symmetric. The sizes of a, b, c and c can be 3.76, 30.23 and 12.12 respectivelyβ＝106.2°，V＝1324 The original, measurably monoclinic cell of (a) indexes the strong data. A full index of all data can only be used with a size of 7.52 for a, 11.65 for b, and 30.46 for cα＝89.8°，β＝82.9°，γ＝88.1°，V＝2647 To a larger triclinic cell. The cell itself can be varied to a size of 7.52, 60.46, 11.65β＝91.9°，V＝5295 Pseudo monoclinic C-centered unit cell of (a).

The diffraction data was normalized and reduced (SAINT) and system errors were corrected using the multiple scanning procedure SADABS. The structure was solved in P-1 by direct method (SHELXS) using a data set normalized on the triclinic cell described above. Using all data (SHELL) to align the structure to | F²And (7) performing fine modification. The incorporation of bimorphs is necessary for the completion of the structure. The bimorph law used is about [ -102 ]]Double rotation of directions, said pair of directionsCorresponding to the b-axis direction of the above-described monoclinic cell.

In addition to being bimorph, the structure is also pseudo-symmetric. This means that the atomic coordinates within the organic fragment are interrelated and it leads to correlations and mathematical instability that goes into least squares refinement. To address these similarities, constraints are imposed on all chemically related bond lengths and bond angles. The molecular pairs (1 and 2, and 3 and 4) are related by the a/2 transformation, thus forcing equivalent anisotropic displacement parameters to be equal. Some dampening (damming) is required to achieve convergence. For example, correlation also causes equivalent bond lengths to become artificially different, and it should be noted that no significance can be attributed to a significant difference in chemical equivalent bond lengths. A more refined refinement model would be required to address these effects.

The hydrogen atom attached to the carbon is placed in the calculated position. Some of the hydrogen atoms attached to oxygen may be arranged in different figures. In particular, the H atom is attached to the O atoms (O141 and O144) of the coordinating sodium ion. The positions for the ligand-water H atoms are set in fourier maps calculated for the location of the possible H positions; included in the model are those that constitute geometrically sound H-bonds and avoid short contacts. The H atoms attached to O8 were placed in different figures, then the entire molecule was initially refined as a rotationally rigid group, after which the H atoms were treated with a riding model (training model). The remaining H atoms (H7A and H142) are placed along the short O. There is no evidence of hydrogen atoms on O42 and O43 in the fourier map, and attempts to place them resulted in the development of unreasonably short H.

The final 'regular' R factor [ based on F and 7355 data with F > 4 σ (F) ] was 0.0616. Other crystal and refinement parameters are listed in table 6.

TABLE 6Single crystal data and structural refinements on crystalline genistein sodium salt dihydrate.

A crystal data

B data Collection

C answering and refining

Discussion:the single crystal structure of crystalline genistein sodium salt dihydrate shows that said compound has [ Na₂(H₂O)₄(μ-H₂O)₂(LH)₂]L₂·2H₂O, wherein LH ═ is the fully protonated genistein ligand C₁₅H₁₀O₅And μ -H₂O is a bridging water molecule between Na ions (i.e., Na ions are each bound to two terminal waters and two bridging waters (designated. mu. -H)₂O), with the addition of an LH ligand-see figure 9). This conclusion depends on the model for H atom placement described above. The placement of hydrogen atoms using X-ray data is generally considered to be tentative because of the problems encountered during structural analysis, and more so here. That is, the proposed H atom positions do form an apparent set of H bonds with all H atoms involved in the geometric standard hydrogen bonds.

As shown in fig. 9, the cationic sodium complex consists of dimeric units formed by crossing the inversion center. The sodium ions are penta-coordinated, the coordination sphere consisting of two terminal and two bridging water ligands and one LH ligand. Hydrogen bonds are formed between the coordinated alcohol moieties and one terminal water molecule (h141.. O1 and h141.. O4). L is^-The anion is deprotonated at the O42 and O43 sites of the phenol. C-O^-The distance is rather short (average 1.34)). Internal hydrogen bonds H6 and O8 coordinate LH and L^-Anions are formed between them.

FIG. 9 shows two sodium cations that are centrosymmetric in the dimeric structure of crystalline genistein sodium salt dihydrate, with intramolecular hydrogen bonds shown as dashed lines.

The stacking in the crystal is dominated by hydrogen bonds. The cations are linked to the anions through water molecules to form a layer, which is also characteristic of stacking interactions between the cations and the anions. Fig. 10 shows one such layer involving O11-based cations and O12-based anions. Water molecules are shown in blue-green. The figure is along [010 ].

Similar layers consisting of molecules based on O13 and O14 were also formed, the two types of layers alternating along the b-axis, connected by H bonds. Fig. 11 shows the overall image as a three-dimensional network. FIG. 11 shows the packing of crystalline genistein sodium salt dihydrate as viewed along the [100] direction.

Analysis using the PLATON/MISSYM program indicates that small (1324) can be used) Unit cell and space group P2₁The organic fragment is described independently and it is simply sodium ions and water molecules that break this symmetry, explaining the pattern of strong and weak data in the diffraction pattern and the pseudo-symmetry problem experienced in refinement.

A calculated XRPD pattern based on single crystal data and structure for crystalline genistein sodium salt dihydrate is shown in fig. 12. The peaks of the calculated XRPD pattern are listed in table 7. A full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein sodium salt dihydrate. A subset of the peaks from figure 12 that can be used alone or in combination to characterize crystalline genistein sodium salt dihydrate include 5.8, 11.6, 15.2, 17.6, 25.1, 28.4, 28.8 and 29.2 ° 2 θ ± 0.2 ° 2 θ.

TABLE 7

1.13 bioavailability of genistein alone and from crystalline genistein sodium salt dihydrate after intraduodenal and intravenous administration in male Steiner rats.

Preparation of dosing solutions for in vivo studies:the genistein and crystalline genistein sodium salt dihydrate were stored in a desiccant and in the shade at room temperature. The dosing solution was freshly prepared from the powder on the day of dosing. Dosing solutions for intravenous administration (IV) were prepared at 1mg/mL (free acid) in 50: 50 DMSO: saline. Dosing solutions for intraduodenal administration (ID) were prepared at 2mg/mL (genistein free acid) in a 0.2% solution of sodium carboxymethylcellulose (NaCMC) in water.

Animal dosing: the pharmacokinetics of genistein were evaluated in fasted male Si-O rats. Each animal was fitted with a Jugular Vein Cannula (JVC) for blood sampling. Animals for intravenous dosing were equipped with an additional JVC for dosing. Animals for intraduodenal dosing were fitted with a duodenal sleeve (IDC) for dosing. Each cage contains a surgically modified animal. All animals were provided with commercial Rodent chow ad libitum prior to study initiation (labdie, Certified Rodent die # 5002). Then, animals were deprived of food for a minimum of 12 hours before and during the study, up to 8 hours post-dose when food was returned. Water is optionally provided.

On the dosing day, intraduodenal dosing solutions were given as a single bolus dose at time zero. Intravenous doses were given as slow IV injections over about 1 minute. The blood sampling times started at the end of the infusion. A blood sample is collected. The study design is shown in table 8.

Table 8:summary of comparative pharmacokinetic studies of genistein and crystalline genistein sodium salt dihydrate in rats.

Each blood sample from the rat was collected through a jugular vein cannula and placed in a frozen polypropylene tube containing sodium heparin as an anticoagulant. The samples were centrifuged at 13,000rpm for 5 minutes at a temperature of 4 ℃. The samples were kept frozen throughout the treatment. Each plasma sample was divided into two portions. The first portion contained 50 μ L of plasma. All remaining plasma volumes were used for the second portion. The samples were then placed on dry ice and stored in a refrigerator set to maintain-60 ℃ to-80 ℃. The total concentration of genistein in the plasma samples was analyzed by LC-MS/MS after overnight incubation with the glucuronidase/arylsulfatase mixture. Pharmacokinetic parameters were calculated using WinNonlin software.

Analysis of plasma samples:an LC-MS/MS analytical method for the determination of genistein in rat plasma was developed. Prior to sample analysis, the standard curve was analyzed to determine the characteristics, range, and linearity of the method. Prior to analysis, total genistein in plasma samples was determined by pre-treatment and incubation of all samples with β -glucuronidase/arylsulfatase. Incubation with the enzyme mixture is used to bind any glucuronide or sulfate metabolites of genistein back to the parent form.

Acceptance criteria for LC-MS/MS analysis:one standard curve was dispersed throughout each analysis run. In order to qualify a process, the criteria of at least 5/8 must be precisely within + -20% except at + -25% acceptable LLOQ.

Pharmacokinetic analysis:non-compartmental model analysis (non-metabolic analysis) of plasma concentrations alone versus time data on genistein was performed using the pharmacokinetic program WinNonlin v.4.1. Plasma concentrations below the limit of quantitation (10ng/mL) were assigned as zero values for PK analysis only.

As a result:as shown in fig. 13, mean plasma concentrations and PK profiles of genistein were significantly different compared to crystalline genistein sodium salt dihydrate after ID dose administration. Mean peak plasma concentration (C) of genistein from crystalline genistein sodium dihydrate salt_{Maximum of}) Compared with the peak plasma concentration of genistein, the concentration is 4.2 times higher, and the two concentrations are 8330 +/-2176 ng/mL and 1983 +/-1130 ng/mL respectively. Maximum plasma concentrations (C) of genistein have been observed within 15 minutes after ID dosing of crystalline genistein sodium salt dihydrate_{Maximum of}) And C of genistein_{Maximum of}Observed 2 hours after dosing (figure 13 and table 10). The bioavailability of genistein from the crystalline genistein sodium salt dihydrate was 55 + -16% compared to 16 + -4.4% for genistein (Table 9).

Table 9:pharmacokinetic parameters after intraduodenal administration of 20mg/kg of the various forms (mean ± SD, n ═ 3).

PK parameters	Genistein	Crystalline genistein sodium salt dihydrate
			CMaximum of(ng/ml)	1983±1130	8330±2176
tMaximum of(h)	2.0±0	0.83±1.0
			AUCFinally, the(h·kg·ng/ml/mg)	414±111	1161±358
Bioavailability (%)	16±4.4	55±16

As shown in table 10, the pharmacokinetic profiles of genistein and crystalline genistein sodium salt dihydrate after IV dosing did not differ significantly between the two forms.

Table 10:pharmacokinetic parameters after intravenous administration of 1mg/kg of each form (mean ± SD, n ═ 3).¹Extrapolate to t-0.

PK parameters	Genistein	Crystalline genistein sodium salt dihydrate
			C0(ng/ml)1	6617±1059	6640±1223
T1/2(h)	1.4±0.3	1.6±0.9
			CL(L/h/kg)	0.40±0.09	0.47±0.08
Vss(L/kg)	0.40±0.06	0.36±0.09
			AUCFinally, the(h·kg·ng/ml/mg)	2533±638	2129±331
AUC∞(h·kg·ng/ml/mg)	2584±639	2189±356

1.14 comparison of physicochemical characteristics and kinetic and equilibrium solubilities between genistein and crystalline genistein sodium salt dihydrate.

Crystalline genistein sodium salt dihydrate in EtOH/dH as compared to genistein₂The excellent early and late intrinsic kinetic solubility curves are shown in O solution. The low late intrinsic kinetic solubility of crystalline genistein sodium salt dihydrate in 100% EtOH has lower practical implications for preclinical development given the non-physiological nature of the solvent.

Experimental:genistein and crystalline genistein sodium salt dihydrate were run in a SuperSol 1000 (preventator Gmbh) solubility assay and the concentration of the compound was measured over time in a closed system by measuring the absorbance flowing through the measurement chamber at a wavelength of 250 nm. Because the two compounds are in pure deionization H₂O, so according to the European pharmacopoeia guidelines 01/2008, section 2.9.3, Table 2.9.3.5, from a solution of 100% EtOH and dH₂O and EtOH, in particular EtOH50/50 (vol/vol) and EtOH/dH₂O75/25 (volume/volume) mixture to evaluate physicochemical properties.

The following parameters were measured:

t _[MSS] is defined as: time from start of analysis to maximum dissolution rate (minutes)

C _[MSS] Is defined as: early kinetic solubility (mg × 1) expressed as concentration at maximum dissolution rate^-1)

C _[Eq] Is defined as: late kinetic solubility (mg × 1) expressed as concentration at equilibrium kinetic solubility^-1)

t _[Eq] Is defined as: time from start of analysis to equilibrium kinetic solubility (min)

ΔC[C _Eq -C _MSS ]Is defined as: concentration difference between early and late kinetic solubilities as described above (mg × 1)^-1)

Δt[C _Eq -C _MSS ]Is defined as: time difference between early and late kinetic solubility endpoints (minutes)

MSS is defined as: from C_[MSS]/t_[MSS]Defined maximum rate of solubility (mg × 1)^-1X minutes^-1)

ISI is defined as: from Δ C [ C ]_Eq-C_MSS]/Δt[C_Eq-C_MSS]Defined intrinsic solubility coefficient

The higher the ISI value, the faster the dissolution, and the later inherent kinetic equilibrium solubility C_[Eq]The stronger the relative contribution of.

KSR is defined as: from C_[MSS]/C_[Eq]Resulting kinetic solubility ratio

KSR is a numerical ratio indicator of the relative contribution of early kinetic solubility to the solubility of the overall late kinetic equilibrium. The higher the KSR value, the early kinetic solubility C_[MSS]The stronger the relative contribution of.

As a result:the thermodynamic, kinetic and equilibrium solubility data for genistein and crystalline genistein sodium salt dihydrate were evaluated under the conditions reported in tables 11, 12 and 13.

As shown in table 11, genistein showed (a) good MSS, (b) acceptable KSR and (c) good late solubility curve, while crystalline genistein sodium salt dihydrate showed (a) excellent MSS, (b) excellent KSR and (c) good to acceptable ISI. For EtOH/dH₂O50/50 (v/v), crystalline genistein sodium salt dihydrate showed the best late intrinsic kinetic solubility curve.

Table 11:EtOH/dH₂o50/50 (volume/volume)

For EtOH/dH₂O75/25 (v/v), genistein showed (a) good MSS, (b) good KSR and (c) good late solubility curve as shown in table 13. Crystalline genistein sodium salt dihydrate exhibits (a) excellent MSS, (b) excellent KSR and (c) excellent ISI, which are the best early and late intrinsic kinetic solubility curves.

Table 12:EtOH/dH₂o75/25 (volume/volume)

As reported in table 13, at 100% EtOH, genistein showed (a) good MSS, (b) acceptable KSR and (c) good late solubility curve, while crystalline genistein sodium salt dihydrate showed (a) excellent MSS, (b) excellent KSR and (c) poor ISI, compared. Crystalline genistein sodium salt dihydrate shows the best early intrinsic kinetic solubility curve, but contributes little to the overall curve.

Table 13:EtOH 100％

1.15 Large Scale Synthesis of crystalline genistein sodium salt dihydrate

Synthesizing:crystalline genistein sodium salt dihydrate was prepared on a kilogram scale using the following procedure:

1. 5.2kg of 2-propanol (IPA) and 320g of neutral dye-wood flavonoids were fed into a 15L glass reactor.

2. The temperature of the mixture was adjusted to 22. + -. 3 ℃ and 632g of 2M aqueous NaOH solution was added dropwise over a period of about 40 minutes at 22. + -. 4 ℃.

3. The mixture was stirred at 22 ± 4 ℃ for about 19 hours and cooled to about 15 ℃ and stirred for 4 hours.

4. The mixture was stirred under temperature cycling (15 ± 3 ℃ → 35 ± 3 ℃, during 1 hour → 35 ± 3 ℃, held for 4 hours → 15 ± 3 ℃, during 1 hour → 15 ± 3 ℃, held for 4 hours) for about 90 hours and finally at 15 ± 3 ℃ for about 4.5 hours.

5. The precipitated product was filtered and washed with 1.2kg of pre-cooled 2-propanol.

6. In a vacuum tray dryer, the filtered product is not dried under vacuum, first at a set temperature of 30 ℃ for about 19 hours, then at a set temperature of 40 ℃ for about 20 hours, then at a set temperature of 50 ℃ for about 24 hours, then at a set temperature of 60 ℃ for about 16 hours, and finally at a set temperature of 70 ℃ for about 10 hours, until the water content measured by KF titration meets the set specifications.

7. Finally, the product (0.24kg) was ground and filled into PE bags.

Optional recrystallization procedure: the crystalline genistein sodium salt dihydrate was recrystallized using the following procedure:

1. 24g of crystalline genistein sodium salt dihydrate prepared as described above were added to 240ml of ethanol.

2. The mixture was stirred at 250rpm and heated at 45 ℃ for about 30 minutes.

3. The resulting solution was allowed to cool to room temperature.

4. Heptane was then added in 1 part multiple (as detailed below) per 1 minute addition. Intermittent 40rpm stirring was used between each addition.

4.151ml of heptane were added and stirred intermittently at 40 rpm.

3.272ml of heptane were added and stirred intermittently at 40 rpm.

5.209ml of heptane were added and stirred intermittently at 40 rpm.

3.505ml of heptane were added and stirred intermittently at 40 rpm.

3.885ml of heptane were added and stirred intermittently at 40 rpm.

5.465ml of heptane were added and stirred intermittently at 40 rpm.

6.314ml of heptane were added and stirred intermittently at 40 rpm.

6.656ml of heptane were added and stirred intermittently at 40 rpm.

8.258ml of heptane were added and stirred intermittently at 40 rpm.

6.969ml of heptane were added and stirred intermittently at 40 rpm.

11.115ml of heptane were added and stirred intermittently at 40 rpm.

10.750ml of heptane were added and stirred intermittently at 40 rpm.

14.219ml of heptane were added and stirred intermittently at 40 rpm.

9.261ml of heptane were added and stirred intermittently at 40 rpm.

14.913ml of heptane were added and stirred intermittently at 40 rpm.

13.471ml of heptane were added and stirred intermittently at 40 rpm.

15.753ml of heptane were added and stirred intermittently at 40 rpm.

19.172ml of heptane were added and stirred intermittently at 40 rpm.

23.441ml of heptane were added and stirred intermittently at 40 rpm.

25.503ml of heptane were added and stirred intermittently at 40 rpm.

26.856ml of heptane were added and stirred intermittently at 40 rpm.

28.126ml of heptane were added and stirred intermittently at 40 rpm.

28.070ml of heptane were added and stirred intermittently at 40 rpm.

36.738ml of heptane were added and stirred intermittently at 40 rpm.

35.989ml of heptane were added and stirred intermittently at 40 rpm.

49.677ml of heptane were added and stirred intermittently at 40 rpm.

50.145ml of heptane were added and stirred intermittently at 40 rpm.

32.579ml of heptane were added and stirred intermittently at 40 rpm.

61.538ml of heptane were added and stirred intermittently at 40 rpm.

57.143ml of heptane were added and stirred intermittently at 40 rpm.

51.948ml of heptane were added and stirred intermittently at 40 rpm.

90.909ml of heptane were added and stirred intermittently at 40 rpm.

5. Then, the sample was left to stand overnight at room temperature to crystallize (about 18 hours).

6. The crystalline product was collected by vacuum filtration.

7. The crystalline product was then dried for about 21 hours while monitoring the water content by karl fischer titration to avoid the risk of dehydration.

Figure 14 shows the XRPD pattern of recrystallized crystalline genistein sodium salt dihydrate. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 14. A full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein sodium salt dihydrate. A subset of the peaks from figure 14 that can be used alone or in combination to characterize crystalline genistein sodium salt dihydrate include 6.0, 7.1, 11.8, 11.9, 15.3, 17.8, 21.3, 25.0, 28.3, 28.6 and 29.1 ° 2 θ ± 0.2 ° 2 θ. Preferred subsets of peaks include 6.0, 7.1, 15.3, 25.0 and at least two of the three peaks 28.3, 28.6, and 29.1 ° 2 θ ± 0.2 ° 2 θ, and 6.0, 7.1, 15.3, 25.0, and 28.3 ° 2 θ ± 0.2 ° 2 θ.

TABLE 14

Example 2 crystallization of genistein Potassium salt dihydrate

2.1 preparation of potassium genistein salt: placing about 300mg of genistein into 6cm³(20 volumes) of IPA. The reaction of the slurry was evident upon addition of 1M potassium hydroxide (KOH) (i.e., slurry to clear solution). The mixture was shaken at ambient temperature for about 3 hours, during which time precipitation was evident. Then, it was allowed to stand at ambient temperature for about 2 days (weekend). The solid was isolated by filtration and then allowed to dry at ambient temperature for about 24 hours.

Crystalline genistein potassium salt dihydrate is formed from the amorphous potassium salt upon standing when opened to the air at ambient room conditions. It may also be prepared from an amorphous potassium salt when the potassium genistein salt is slurried in an IPA/water mixture as described in the hydration study to form crystalline potassium genistein salt dihydrate by water absorption.

Thus, it appears that the potassium genistein salt is an unstable anhydrous amorphous salt near recovery, which then rapidly absorbs water from the environment to crystallize into a dihydrate material. This finding was subjected to all the light stability tests described hereinafter; hydration studies; 40 ℃/75 RH% study; storage at 80 ℃ study and TGA test support. The 80 ℃ storage study is particularly noteworthy because the material appears to absorb water at this elevated temperature; thus indicating that the hydrate is stable at 80 ℃. The GVS data also indicate that genistein potassium salt dihydrate is the most stable form and therefore exploitable. Although the above synthetic procedure does not directly produce a dihydrate material, it can be produced well by further treating or changing the solvent system to contain a higher water content (i.e., 3% water/IPA). Similar to the crystalline genistein sodium salt dihydrate, the risk of dehydration during milling was somewhat reduced by the 80 ℃ storage test.

2.2 XRPD of amorphous genistein Potassium salt

As shown in fig. 15, XRPD analysis revealed that the solid genistein potassium salt produced as described in 2.1 was amorphous (i.e. without peaks).

2.3 XRPD of crystalline genistein Potassium salt dihydrate

Figure 16 shows the XRPD pattern of crystalline genistein potassium salt dihydrate. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 15. A full list of peaks or a subset thereof may be sufficient to characterize crystalline genistein potassium salt dihydrate. A subset of the peaks from figure 16 that can be used alone or in combination to characterize crystalline genistein potassium salt dihydrate include 11.6, 14.5, 14.8, 24.5, 25.2, 27.6, 28.0 and 28.4 ° 2 θ ± 0.2 ° 2 θ. A preferred subset of the peaks includes 11.6, 14.5, 24.5, 25.2 and at least two of the three peaks 27.6, 28.0 and 28.4 ° 2 θ ± 0.2 ° 2 θ.

Watch 15

2.4 crystalline genistein Potassium salt dihydrate PLM

PLM analysis of genistein potassium salt dihydrate showed that the potassium salt was crystalline and had a needle-like morphology. The needles were thicker than those of crystalline genistein sodium salt dihydrate.

2.5 TGA of crystalline genistein Potassium salt dihydrate

As shown in figure 17, TGA indicated that the potassium salt was hydrated and started water loss at about 75 ℃, thus being suitable for further development. The weight loss corresponds to 2 moles of water to 1 mole of potassium.

2.6 DSC of crystalline genistein Potassium salt dihydrate

As shown in fig. 18, DSC indicated dehydration without melting at about 91 ℃. Other peaks may be associated with degradation (also indicated by TGA of figure 16).

2.7 GVS crystalline Genistein Potassium salt dihydrate

As shown in figure 19, the GVS study indicated hydrate formation (GVS cycle dehydrated material before analysis) and adsorbed up to 16 wt% water. However, between 20 and 70 RH% (typical working range of materials), a humidity change of only about 3% is observed. This is a valuable property for drug development.

2.8 solubility Studies of crystalline genistein Potassium salt dihydrate

The water solubility of crystalline genistein potassium salt dihydrate was measured using the protocol described in example 1.7. Table 16 compares the water solubility of crystalline genistein potassium salt dihydrate with genistein.

TABLE 16

2.9 preparation of crystalline genistein potassium salt dihydrate¹H NMR

FIG. 20 shows crystalline dye woodProcess for preparing flavone sylvite dihydrate¹H NMR spectrum. Table 17 shows¹Peaks in the H NMR spectrum. In FIG. 20¹Salt formation was confirmed by chemical shift of the aromatic proton at about 5.9 in genistein to 6.1ppm in H NMR.

TABLE 17

Chemical shift	Multiplicity of properties	Range of
			7.959	S	8.009-7.941
7.375	m	7.437-7.322
			6.861	m	6.932-6.800
6.154	dd	6.246-6.068
			4.949	s	5.148-4.714
3.34	q	3.444-3.137
			1.182	d	1.228-1.149

S is singlet, m is multiplet, d is doublet, dd is doublet, q is quartet

2.10 stability Studies of crystalline genistein Potassium salt dihydrate

The samples were tested for stability at 80 ℃ for 7 days and at 40 ℃/75 RH% for 7 days. Observations such as color changes were noted after 7 days, and XRPD of the samples was performed after 7 days to investigate any solid form changes. Figure 21 shows XRPD patterns of crystalline genistein potassium salt dihydrate at 80 ℃ for 7 days and at 40 ℃/75 RH% for 7 days. The 40 ℃/75 RH% study indicated that the potassium genistein salt crystallized to form the potassium genistein salt dihydrate. The crystallization of the potassium genistein dihydrate was indicated by storing the crystalline potassium genistein dihydrate at 80 ℃ for a period of 7 days.

2.11 hydration Studies of crystalline genistein Potassium salt dihydrate

At the water level, about 100mg of crystalline genistein sodium salt dihydrate was placed in about 500 μ L of IPA/water mixture (3%, 5% and 10%). The various mixtures were stirred at ambient temperature for about 48 hours and then filtered to recover the solids for XRPD and TGA studies. As shown in fig. 22, hydration studies revealed hydration consistent with crystalline genistein potassium salt dihydrate.

2.12 disproportionation study of crystalline genistein Potassium salt dihydrate

A sample of crystalline genistein potassium salt dihydrate was slurried in distilled water for about 48 hours and then checked for disproportionation by XRPD. The pH of the supernatant was also measured using a Corning (Corning)240pH meter. No evidence of disproportionation was observed. The pH of the supernatant was 7.3 indicating no disproportionation.

Example 3 crystallization of genistein calcium salt

3.1 preparation of crystalline genistein calcium salt

About 25mg of genistein was put into the same container as about 7mg of solid calcium hydroxide. To the solid mixture, 500 μ L of IPA/water (50: 50) was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

3.2 characterization of crystalline genistein calcium salt

Figure 23 shows the XRPD pattern of crystalline genistein calcium salt. The peaks in the experimental XRPD pattern at 2 θ ± 0.2 ° 2 θ are listed in table 18. The full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein calcium salt. A subset of the peaks from figure 23 that can be used alone or in combination to characterize the crystalline genistein calcium salt include 8.0, 15.3, 25.1 and 25.6 ° 2 θ ± 0.2 ° 2 θ. TGA of crystalline genistein calcium salt is shown in figure 24. PLM images of crystalline genistein calcium salt showed needle-like crystals.

Watch 18

Example 4 crystalline magnesium genistein, 1 equivalent preparation

4.1 preparation of crystalline genistein magnesium salt, 1 equivalent

About 25mg of genistein was placed in the same container as about 5.5mg of solid magnesium hydroxide. To the solid mixture, 500 μ L of IPA/water (50: 50) was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

4.2 characterization of crystalline genistein magnesium salt, 1 equivalent

Figure 25 shows the XRPD pattern of the crystalline magnesium genistein salt prepared from 1 equivalent. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 19. A full list of peaks or a subset thereof may be sufficient to characterize the crystalline magnesium genistein salt. A subset of the peaks from figure 25 that can be used alone or in combination to characterize the crystalline magnesium genistein salt include 9.0, 18.6, 23.7, 25.7 and 38.0 ° 2 θ ± 0.2 ° 2 θ. TGA prepared from crystalline genistein magnesium salt, 1 equivalent is shown in fig. 26. Crystalline genistein magnesium salt, PLM image prepared at 1 equivalent showed genistein magnesium salt as crystals.

Watch 19

Example 5 crystalline magnesium genistein, 2 equivalents preparation

5.1 preparation of crystalline genistein magnesium salt, 2 equivalents

About 25mg of genistein was placed in the same container as about 11mg of solid magnesium hydroxide. To the solid mixture, 500 μ L of IPA/water (50: 50) was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

5.2 characterization of crystalline genistein magnesium salt, 2 equivalents preparation

Figure 27 shows the XRPD pattern for the 2 equivalent preparation of crystalline magnesium genistein. The peaks in the XRPD pattern at ° 2 θ ± 0.2 ° 2 θ of the experiment are listed in table 20. A full list of peaks or a subset thereof may be sufficient to characterize the crystalline magnesium genistein salt. The TGA profile of 2 equivalents of crystalline genistein magnesium salt is shown in fig. 28.

Watch 20

Similar XRPD patterns and TGA curves for the crystalline magnesium genistein salt from both 1 equivalent preparation and 2 equivalent preparation indicate that the same crystalline magnesium genistein salt was obtained from both preparations. A subset of XAPD peaks that can be used alone or in combination to characterize the magnesium salt of crystalline genistein includes 9.0, 18.6, 23.7, 25.7 and 38.0 ° 2 θ ± 0.2 ° 2 θ.

Example 6-crystalline genistein L-lysine salt

6.1 preparation of crystalline genistein L-lysine salt

About 25mg of genistein was put into the same container as about 15mg of solid L-lysine monohydrate. To the solid mixture, 500 μ L of IPA or toluene was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

6.2 characterization of crystalline genistein L-lysine salt/genistein mixture

Samples of crystalline genistein L-lysine salt from toluene and IPA were analyzed by XRPD and resulted in the XRPD patterns shown in figures 30 and 31. The XRPD pattern for crystalline genistein is also shown below. Both methods produced a mixture of genistein and genistein L-lysine salt as indicated by XRPD. Figure 29 shows the XRPD pattern of crystalline genistein. The peaks in the XRPD pattern of fig. 29 at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 21.

TABLE 21

6.3 characterization of crystalline genistein L-lysine salt from toluene

Figure 30 shows the XRPD pattern of crystalline genistein L-lysine salt from toluene. The peaks in the XRPD pattern at ° 2 θ ± 0.2 ° 2 θ of the experiment are listed in table 22.

TABLE 22

6.4 characterization of crystalline genistein L-lysine salt from IPA

Figure 31 shows the XRPD pattern of crystalline genistein L-lysine salt from IPA. The peaks in the XRPD pattern at ° 2 θ ± 0.2 ° 2 θ of the experiment are listed in table 23. The TGA of the crystalline genistein L-lysine/genistein mixture is shown in FIG. 32. As was done for PLM images from crystalline mixtures of toluene, PLM images of genistein L-lysine/genistein mixtures from IPA show crystalline material.

TABLE 23

Similar XRPD patterns for crystalline genistein L-lysine salt from both isopropanol and toluene indicate that the same crystalline genistein L-lysine salt was obtained from both preparations. A full list of peaks from tables 21 or 22, or a subset thereof, may be sufficient to characterize the crystalline genistein L-lysine salt. By comparing the XRPD patterns of crystalline genistein L-lysine salt in fig. 30 and 31 with the XRPD pattern of crystalline genistein in fig. 29, a subset of the peaks that can be used alone or in combination to characterize crystalline genistein L-lysine salt include 5.2, 18.6, 19.7, 20.6 and 21.0 ° 2 θ ± 0.2 ° 2 θ.

Example 7-crystalline genistein N-methylglucamine (meglumine) salt

7.1 preparation of crystalline genistein N-methylglucamine salt

About 25mg of genistein was put into the same container as about 20mg of solid N-methylglucamine. To the solid mixture, 500 μ L of acetone was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

7.2 characterization of crystalline genistein N-methylglucamine salt

FIG. 33 shows the XRPD pattern of crystalline genistein N-methylglucamine salt. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 24. The full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein N-methylglucamine salt. A subset of the peaks from figure 33 that can be used alone or in combination to characterize the crystalline genistein N-methylglucamine salt include 7.5, 7.8, 12.3, 14.8, 16.5, 17.1, 17.6, 18.8, 19.4, 20.0, 20.8 and 29.1 ° 2 θ ± 0.2 ° 2 θ. Preferred subsets include peaks at 12.3, 14.8, 17.6, and 19.4 ° 2 θ ± 0.2 ° 2 θ.

Watch 24

Example 8 crystalline genistein N-Ethyl glucosamine (Glutamine) salt

8.1 preparation of crystalline genistein N-ethylglucamine (Glutamine) salt

About 25mg of genistein was put into the same container as about 19mg of solid N-ethylglucamine. To the solid mixture, 500 μ L of acetone or IPA was added and the mixture was shaken at ambient temperature for about 24 hours. After stirring, the slurry was then temperature cycled (40 ℃ to ambient temperature over a4 hour period) for about 72 hours with shaking. The solid was then isolated by filtration and allowed to dry at ambient temperature for about 24 hours.

8.2 characterization of the crystalline genistein N-Ethyl glucosamine (Glutamine) salt

A sample of the crystalline genistein N-ethylglucamine (glucethylamine) salt prepared above was analyzed by XRPD and resulted in the patterns shown in fig. 34 and 35. Unstable crystalline salts were identified from both acetone and IPA. Figure 34 shows an XRPD pattern of crystalline genistein N-ethylglucamine (glucethylamine) salt from acetone. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 25. Figure 35 shows an XRPD pattern of crystalline genistein N-ethylglucamine (glucethylamine) salt from IPA. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 26. A full listing of peaks in any one table, or a subset thereof, may be sufficient to characterize the crystalline genistein N-ethylglucamine (glucethylamine) salt. A subset of the peaks based on fig. 34 and 35 that can be used alone or in combination to characterize the crystalline genistein N-ethylglucamine (glucamine) salt include 7.4, 12.7, 14.7, 16.0, 18.1, 19.0, 19.2, 21.7, 22.1 and 26.3 ° 2 θ ± 0.2 ° 2 θ. Preferred subsets of peaks include 7.4, 12.7, 14.7, 16.0, 18.1, and 26.3 ° 2 θ ± 0.2 ° 2 θ. The TGA profile of crystalline genistein N-ethylglucamine salt from acetone is shown in FIG. 36. PLM images of crystalline genistein N-ethylglucamine salt from acetone show that the material is crystalline.

TABLE 25

Watch 26

Example 9 crystallization of genistein diethylamine salt

9.1 preparation of crystalline genistein diethylamine salt

Stock solutions of genistein in THF (520.2mg in 19.25mL of THF) and diethylamine in THF: ETOH (1: 1) were prepared. Stock solutions of genistein and diethylamine were added together in stoichiometric amounts and the solution was filtered through a 0.2 μm nylon filter into a clean vial and allowed to evaporate at ambient conditions.

9.2 characterization of crystalline genistein diethylamine salt

XRPD analysis of the solid material isolated above was performed using the following equipment: an Inel XRG-3000 diffractometer equipped with a CPS (bending position sensitive) detector with a2 θ range of 120 °; shimadzu (Shimadzu) XRD-6000X-ray powder diffractometer using Cu ka radiation; and a Bruker D-8Discover diffractometer equipped with a Bruker's General Area Diffraction Detection System (GADDS, v.4.1.19) two-dimensional Diffraction Detection System. In the data section, specific acquisition parameters are listed on the profile for each sample. Figure 37 shows the XRPD pattern of crystalline genistein diethylamine salt. The peaks in the XRPD pattern at the experimental ° 2 θ ± 0.2 ° 2 θ are listed in table 27. The full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein diethylamine salt. A subset of the peaks from figure 37 that can be used alone or in combination to characterize the crystalline genistein diethylamine salt include 7.4, 8.2, 15.3, 25.3 and 28.4 ° 2 θ ± 0.1 ° 2 θ.

Watch 27

Example 10 crystalline genistein monohydrate

10.1 preparation of crystalline genistein monohydrate

Stock solutions of genistein were prepared in THF (472mg in 17.47mL THF). The genistein stock solution (1mL) was added to a glass vial followed by 1mL of D-glucuronic acid solution (84.1mg in 4.33mL of water). The solution was allowed to evaporate at ambient conditions. After 1 day, the solid was separated by decanting the remaining solution, which was then blotted dry with filter paper.

10.2 characterization of crystalline Genistein monohydrate

XRPD analysis of crystalline genistein monohydrate samples was performed using the following equipment: an Inel XRG-3000 diffractometer equipped with a CPS (bending position sensitive) detector with a2 θ range of 120 °; shimadzu (Shimadzu) XRD-6000X-ray powder diffractometer using Cu ka radiation; and a Bruker D-8Discover diffractometer equipped with a Bruker's General Area Diffraction Detection System (GADDS, v.4.1.19). Figure 38 shows the XRPD pattern of crystalline genistein monohydrate. The peaks in the XRPD pattern at ° 2 θ ± 0.2 ° 2 θ of the experiment are listed in table 28. The full list of peaks or a subset thereof may be sufficient to characterize the crystalline genistein monohydrate. A subset of the peaks from figure 38 that can be used alone or in combination to characterize crystalline genistein monohydrate include 9.0, 11.3, 13.4, 14.8, 23.1, 25.0, 26.8 and 28.5 ° 2 θ ± 0.1 ° 2 θ.

Watch 28

Thermogravimetric analysis (TGA) of crystalline genistein monohydrate was performed using a TA Instruments 2950 thermogravimetric analyzer. Figure 39 shows a TGA trace of a crystalline genistein monohydrate sample. Thermogravimetric analysis indicated that the sample contained 6% by weight of volatile components, which is comparable to the monohydrate.

Claims (13)

1. Crystalline genistein sodium salt dihydrate characterized by an XRPD pattern having peaks at 6.0, 7.1, 15.3 and 25.0 ° 2 Θ ± 0.2 ° 2 Θ, and at least two of the three peaks 28.3, 28.6 and 29.1 ° 2 Θ ± 0.2 ° 2 Θ.

2. Crystalline genistein sodium salt dihydrate characterized by an XRPD pattern having peaks at 6.0, 7.1, 15.3, 25.0 and 28.3 ° 2 Θ ± 0.2 ° 2 Θ.

3. A therapeutic composition comprising a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 and at least one pharmaceutically acceptable carrier.

4. Use of a therapeutically effective amount of the therapeutic composition of claim 3 in the manufacture of a medicament for treating or preventing cancer.

5. Use of a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 for the preparation of a medicament for the treatment of cancer.

6. Use of a therapeutically effective amount of the therapeutic composition of claim 3 in the manufacture of a medicament for treating chronic inflammation.

7. Use of a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 for the preparation of a medicament for the treatment of chronic inflammation.

8. Use of a therapeutically effective amount of the therapeutic composition of claim 3 in the manufacture of a medicament for treating transthyretin amyloidosis.

9. Use of a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 for the preparation of a medicament for the treatment of transthyretin amyloidosis.

10. Use of a therapeutically effective amount of the therapeutic composition of claim 3 in the manufacture of a medicament for the treatment of cystic fibrosis.

11. Use of a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 for the preparation of a medicament for the treatment or prevention of cystic fibrosis.

12. Use of a therapeutically effective amount of the therapeutic composition of claim 3 in the manufacture of a medicament for treating an infection.

13. Use of a therapeutically effective amount of at least one crystalline genistein sodium salt dihydrate according to claim 1 or 2 for the preparation of a medicament for the treatment of infections.