HK1113971B - Oral formulations for delivery of catecholic butanes including ndga compounds - Google Patents

Abstract

The present invention provides for compositions, kits and methods for treatment of diseases, where the compositions contain catecholic butanes, including NDGA compounds, such as NDGA derivatives, for example tetro-O-methyl NDGA. The present invention also provides for solubilizing agents and excipients that are suitable for administration of the present compounds into animals via an oral route, whether in a liquid, semi-solid or solid form.

Description

Oral formulations for delivery of catecholic butanes including NDGA compounds

Cross Reference to Related Applications

This application claims priority from U.S. patent provisional applications 60/647,495 and 60/647,648, filed on 27/1/2005, which are incorporated herein by reference in their entirety. This application is also related to international patent application, filed concurrently herewith, entitled "injectable animal formulations of catecholic butanes including NDGA compounds" having attorney docket No. 682714-10WO, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present application relates to compositions of catecholic butanes, such as NDGA derivatives, and methods of administering them to animals, such as humans, for the treatment of diseases, such as cancer or other proliferative or inflammatory diseases, metabolic diseases, or neurodegenerative diseases.

Background

The compounds were tested by intratumoral injection or therapeutic administration of topical catecholic butanes such as nordihydroguaiaretic acid ("NDGA") in experimental animals. For example, Jordan et al in US5,008,294 describe the effect of NDGA on human breast cancer cells MX-1, which are subcutaneously implanted into mice on day 0 (example 2). On day 1, tumor-containing mice were injected with different doses of NDGA as separate intratumoral injections. In another experiment, Jordan et al injected human mammary cells MX-1 into mouse skin and treated the animals by topical administration of NDGA after day 23 (example 7).

Certain derivatives of NDGA (i.e., "NDGA derivatives") are described by Huang et al in US 6,214,874. In one experiment, mice were injected with an immortalized mouse cell line, C3 cells, and M was injected intratumorally alone₄N or with G₄N-binding for treatment, both NDGA derivatives.

It would be desirable if oral formulations of these anti-tumor compounds could be obtained that could be more conveniently administered without the need for hospitalization, particularly self-administration. The present invention provides these desirable benefits.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide oral formulations of one or more novel catecholic butane and/or NDGA compounds, such as NDGA derivatives, for treating a disease by oral administration of the formulations to a subject in need thereof.

It is another object to provide a formulation as described above which is safe and has little adverse side effects when administered to animals including humans.

It is another object to provide a formulation of one or more of the above with a commercially reasonable stability period.

It is another object to provide one or more of the above formulations which have a commercially reasonable half-life in the circulation of administration to an animal.

In accordance with one or more of the objects of the invention set forth above, there is provided embodiments of the invention, exemplified by the following:

a composition for oral administration to an animal comprising an active pharmaceutical ingredient and a pharmaceutically acceptable carrier, wherein the active pharmaceutical ingredient comprises catecholic butane and the carrier comprises at least one solubilizing agent and an excipient selected from the group consisting of: (a) a water-soluble organic solvent other than DMSO, if the water-soluble organic solvent is propylene glycol, the propylene glycol is present in the absence of white petrolatum, the absence of xanthan gum (also known as xantham gum and xanthum gum), the absence of at least one of glycerol or chicle, when the water-soluble organic solvent is polyethylene glycol, the polyethylene glycol is present in the absence of ascorbic acid or butylated hydroxytoluene ("BHT"), and when the polyethylene glycol is polyethylene glycol 400, the polyethylene glycol 400 is present in the absence of polyethylene glycol 8000; (b) a cyclodextrin; (c) ionic, nonionic or amphoteric molecular surfactants, if the surfactant is a nonionic surfactant, the nonionic surfactant is present in the absence of xanthan gum; (d) a modified cellulose; (e) a water-insoluble lipid, if the water-insoluble lipid is castor oil, the castor oil is present in the absence of beeswax or carnauba wax; and (a) to (e) a carrier.

A method of treating a disease in a subject, the method comprising: (a) providing a composition of the invention; and (b) orally administering to the subject, wherein the composition comprises an effective dose of the active pharmaceutical ingredient.

A kit for the treatment of a disease, said kit comprising a composition of the invention and instructions for its use.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 includes FIGS. 1A and 1B and depicts the C-33A cell line and the Hela cell line via M₄Results of cell proliferation experiments after N treatment. FIG. 1A shows M₄The number of cells present after N treatment is equal to that of cells not subjected to M₄The ratio of the number of N-treated cells present, wherein M is provided₄The amount of N ranges from 0 μ M to 80 μ M in DMSO formulations. FIG. 1B shows M₄The number of cells present after N treatment is equal to that of cells not subjected to M₄The ratio of the number of N-treated cells present, wherein M is provided₄The amount of N is in the range of 0. mu.M to 80. mu.M in the HP-beta-CD/PEG formulation ("CPE" formulation).

FIG. 2 includes FIGS. 2A and 2B and shows the absence of M₄N, or the presence of different concentrations of M₄N in DMSO (FIG. 2A) or HP- β -CD/PEG (FIG. 2B) based on the percentage of cell death assays of C-33A cells and Hela cells. M₄The concentration of N varies from 0. mu.M to 80μM。

FIG. 3 is a single 500mg/kg dose of M orally administered to Spira-dawley rats in various liquid solubilizers and/or excipients ("carriers")₄M at 0.5, 1, 2 or 3 hour time points after N₄N absorption histogram. In all vectors M₄N is present at a concentration of about 60 mg/mL. The carrier comprises: (a) HP-beta-CD (500mg/mL) + HPMC (5 mg/mL); (b) HP-beta-CD (500mg/mL) + CMC (5 mg/mL); (c) TPGS (200 mg/mL); (d) TPGS (100mg/mL) + PEG400 (50% v/v); (e) tween _ 20; (f) PEG400 (50% v/v) + Tween _20 (50% v/v); (g) PEG400 (33% v/v) + Tween _20 (33% v/v) + peppermint oil (33%); (h) peppermint oil (50%) + PEG400 (50% v/v); (h) peppermint oil (50%) + Tween _20 (50% v/v); (i) peppermint oil (50%) + sesame oil (50%).

FIG. 4 shows a single dose of 100mg/kg M in beagle dog (beagle dog) oral powder form or in different solid carriers₄M at 0.5, 1, 1.5, 2, 4, 6, or 8 hour time points after N₄N uptake, the dosage form was as follows: (a) m₄N powder; (b) lyophilized HP-beta-CD (81%) + M₄N(185mg/g)；(c)TPGS(20％)+M₄N (133 mg/g); (d) soybean oil (95%) + beeswax (5%) + M₄N (200 mg/g); and (d) olive oil (95%) + beeswax (5%) + M₄N(200mg/g)。

Figure 5 includes figures 5A and 5B and shows non-micronized M in glycerol monooleate at 0.5 hour, 1 hour, 1.5 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 36 hour time points for beagle dogs following oral administration of a single 100mg/kg dose₄And (4) absorbing N. Fig. 5A is a non-logarithmic stage. Fig. 5B is a logarithmic scale.

Figure 6 includes figures 6A and 6B and shows micronized M in glycerol monooleate at 0.5 hour, 1 hour, 1.5 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 36 hour time points for beagle dogs following oral administration of a single 100mg/kg dose₄And (4) absorbing N. Fig. 6A is a non-logarithmic stage. Fig. 6B is a logarithmic scale.

FIG. 7 includes FIGS. 7A and 7B and shows M in different vehicles at 0.5, 1, 1.5, 2, 4,8, 12, 16, 24, or 36 hour time points for male beagle dogs after oral administration of a single 75mg/kg dose₄Serum levels of different concentrations of N. FIG. 7B shows in detail the abbreviation, M, of the vector used₄N, and instructions for whether to administer or feed fasted dogs. Fig. 7A shows data at non-logarithmic scale. Fig. 7B shows data at logarithmic level.

FIG. 8 includes FIGS. 8A and 8B and shows M in different vehicles at 0.5 hour, 1 hour, 1.5 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours, or 36 hours time points for female beagle dogs following oral administration of a single 75mg/kg dose₄Serum levels of different concentrations of N. FIG. 8B shows in detail the abbreviation, M, of the vector used₄N, and instructions for whether to administer or feed fasted dogs. Fig. 8A shows data in non-logarithmic scale. Fig. 8B shows data at logarithmic level.

Detailed Description

The present invention provides novel compositions, kits and methods for treating diseases including tumors and psoriasis, hypertension, obesity, type I or II diabetes, central nervous system diseases or neurodegenerative diseases including but not limited to pain, alzheimer's disease, shock, inflammatory diseases, precancerous lesions, dysplasias, including infectious diseases such as HIV, HTLV, HPV, HSV, HBV, EBV, varicella-zoster, adenovirus, parvovirus, JC virus or other viral infections. The present invention provides novel compositions, kits and methods for treating diseases, including: proliferative diseases including tumors and psoriasis, hypertension, obesity, type I or II diabetes, central nervous system diseases or neurodegenerative diseases, including but not limited to pain, alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, dementia, shock, and inflammatory diseases, precancerous lesions, dysplasia, infectious diseases, including viral infections such as human immunodeficiency virus ("HIV"), human T-lymphotropic virus ("HTLV"), human papilloma virus ("HPV"), herpes simplex virus ("HSV"), hepatitis b virus ("HBV"), epstein-barr virus ("EBV"), varicella-zoster, adenovirus, parvovirus, jacobb-creutzfeldt-jakob virus ("JC virus"), or others.

The present invention provides novel compositions comprising catecholic butanes, including NDGA compounds, such as NDGA derivatives, e.g., M dissolved in a pharmaceutically acceptable solubilizer₄N, the solubilizing agent may be formulated with other diluents, excipients, and the like (collectively "carriers") suitable for administration to a subject, e.g., a human, for the treatment of a disease. Such formulations are suitable for oral administration. Suitable pharmaceutically acceptable carriers include at least one of the following: (a) water-soluble organic solvents other than DMSO, e.g., polyethylene glycol ("PEG"), such as PEG300, PEG400, or PEG400 monolaurate, propylene glycol ("PG"), polyvinylpyrrolidone ("PVP"), ethanol, benzyl alcohol, or dimethylacetamide; (b) cyclodextrins or modified cyclodextrins, for example, hydroxypropyl- β -cyclodextrin ("HP- β -CD") or sulfobutyl ether- β -cyclodextrin ("SBE- β -CD"); (c) ionic, nonionic, or amphiphilic surfactants, such as polyoxyethylene sorbitan laurate (also known as polysorbate), which is a nonionic surfactant, such as polysorbate 20 and polysorbate 80 commercially available as Tween 20 or Tween 80, vitamin E polyethylene glycol 1000 succinate ("TPGS"), glycerol monooleate (also known as glycerol-based monooleate), a 35:1 molar ratio reaction product of esterified fatty acids or ethylene oxide with castor oil, commercially available as Cremophor EL; (d) modified celluloses, such as ethyl cellulose ("EC"), hydroxypropylmethyl cellulose ("HPMC"), methyl cellulose ("MC"), or carboxymethyl cellulose ("CMC"); and (e) water-insoluble lipids, e.g., waxes, oils, fats, oils, etc,Or fat emulsions, for example, Intellipide (Intralipid). Preferably, when PG is used, PG is used in the absence of white petrolatum, in the absence of xanthan gum, and in the absence of at least one of glycerol or glycogelatin. Preferably, when PEG is used, in the absence of vitamin C or BHT, PEG is used; when a nonionic surfactant is used, the nonionic surfactant is used in the absence of xanthan gum; when the oil is castor oil, castor oil is used in the absence of beeswax or carnauba wax.

In one embodiment of the invention, the compounds herein are dissolved or dissolved and diluted in different carriers to form liquid compositions for administration in animals. For example, in one aspect of this embodiment, the active pharmaceutical ingredient ("APT") compounds herein are dissolved in a water-soluble organic solvent such as PEG300, PEG400, or PEG400 monolaurate ("PEG compound") or in PG. In another embodiment, the compounds herein are solubilized in a modified cyclodextrin, e.g., HP- β -CD or SBE- β -CD. In another embodiment, the compounds herein are dissolved and/or diluted in a combined preparation comprising a PEG compound and HP- β -CD. In a further embodiment, the compounds herein are dissolved in a modified cellulose such as HPMC, CMC or EC. In another embodiment, the compounds herein are dissolved in another combined preparation containing both modified cyclodextrin and modified cellulose, such as HP- β -CD and HPMC, or HP- β -CD and CMC.

In another embodiment, the compounds herein are dissolved in an ionic, nonionic or amphiphilic surfactant such as Tween _20, Tween _80, TPGS or esterified fatty acids. For example, the compounds herein may be solubilized in only TPGS, or only Tween _20, or in a combination such as TPGS and PEG400, or Tween _20 and PEG 400.

In another embodiment, the compounds herein are dissolved in a water-soluble lipid such as a wax, a fat emulsion, e.g., Intralipid @, or an oil. For example, the compounds herein may be dissolved in peppermint oil alone, or in a combination of peppermint oil with Tween _20 and PEG400, or in a combination of peppermint oil with PEG400, in a combination of peppermint oil with Tween _20, or in a combination of peppermint oil with sesame oil.

Of course, in the above-described implementation, ethylcellulose may be substituted or added instead of HPMC or CMC; in the above-described implementation, PEG300 or PEG400 monolaurate may be replaced or PEG300 or PEG400 monolaurate added in place of PEG 400; in the above implementation, Tween _80 may be replaced or Tween _80 may be added in place of Tween _ 20. In the above-described implementation, other oils such as corn oil, olive oil, soybean oil, mineral oil or glycerin may be substituted or added in place of the peppermint oil or sesame oil.

Further heating may be carried out at a temperature of from about 30 ℃ to about 90 ℃ during the preparation of any of the compositions in order to achieve complete dissolution of the compositions herein or to obtain a uniform distribution of the composition in suspension.

In a further embodiment, the API compounds herein may be administered orally without a carrier or in solid form using a carrier. In one embodiment, as in the above examples, the compounds herein are first dissolved in a liquid carrier and then formulated into a solid composition for administration as an oral composition. For example, the compounds herein are dissolved in a modified cyclodextrin such as HP- β -CD, and the composition is lyophilized to produce a powder suitable for oral administration.

In another embodiment, the compounds herein are dissolved or suspended in TPGS solution with heating suitable to obtain a uniformly distributed solution or suspension. Upon cooling, the composition becomes creamy and suitable for oral administration.

In another embodiment, the compounds herein are dissolved in an oil and beeswax is added to produce a waxy solid composition.

In another embodiment of the invention, the compounds herein are dissolved in a modified cellulose such as EC. Modified cellulose, such as ethyl cellulose, may be diluted with ethanol ("EtOH") prior to use.

The present invention also provides water-insoluble lipids as solvents for the compounds herein. The water-insoluble lipids include: such as oils and mixed fat emulsion compositions such as Intralipid (Pharmacia & Upjohn, now Pfizer), according to the manufacturer's recommendations. For example, an adult dose of not more than 2g fat/kg body weight/day (Intralipid-10%, 20% and 30% at 20mL, 10mL and 6.7mL/kg, respectively) is recommended. It can be ensured that the content in 1,000mL Intralipid _ 10% contains: 100g of purified soybean oil, 12g of purified lecithin, 22g of anhydrous glycerol and the balance of water for injection to 1,000 mL. The pH was adjusted to about 8 with sodium hydroxide. 1,000mL Intralipid-20% contained: 200g of purified soybean oil, 12g of purified lecithin, 22g of anhydrous glycerol and the balance of water for injection to 1,000 mL. The pH was adjusted to about 8 with sodium hydroxide. 1,000mL Intralipid _ 30% contained: 300g of purified soybean oil, 12g of purified lecithin, 16.7g of anhydrous glycerol and the balance of water for injection to 1,000 mL. The pH was adjusted to about 7.5 with sodium hydroxide. These Intralipid products were stored at room temperature controlled below 25 ℃ and were not frozen.

In summary, to prepare an oral formulation, the compounds herein are first dissolved to yield a highly stable composition before the addition of other excipients. Unstable formulations are undesirable. Unstable liquid formulations continue to form crystalline precipitates or biphasic solutions. Unstable solid formulations continue to present particles and agglomerates, and sometimes contain dilute liquids. The optimized solid formulation appeared smooth, uniform, and had a small melting temperature range. In summary, the proportion of excipients in a formulation can affect stability. For example, too little of a hardening agent such as beeswax may result in a formulation that is too soft.

Thus, in general, for liquid formulations of the invention, the excipient used should be an API or a compound herein, such as M₄A good solvent for N. Alternatively, the excipients should be able to be dispensed withThe heating dissolves the API, and the excipients should also be compatible with each other independently of the API so that they can form stable solutions, suspensions or emulsions. In addition, in general, for the solid formulations of the present invention, the excipients used should be good solvents for the API to avoid caking or formation of inhomogeneous formulations. To avoid excessive softness or non-uniform texture of the solid formulation, which is undesirable, the excipients should be compatible with each other so that they can form a smooth, uniform solid, even in the absence of an API.

The invention also provides "placebo" similar to the API-containing formulation but without the API, which are used to determine the compatibility of the different components or excipients in the formulation.

Terms used herein have their dictionary meanings and are used by those skilled in the art unless other meanings are provided. In particular, the invention may be better understood in light of the following definitions, which are used in conjunction with terms defined elsewhere herein.

The term "about" as used herein in reference to concentration or dosage means that the specified value is as high as ± 10% to 20%.

The terms "active pharmaceutical ingredient," "API," or "compound" as referred to herein, mean one or more catecholic butanes of formula I or NDGA compounds such as NDGA derivatives, which are present in the pharmaceutical compositions herein.

As used herein, "dioxyalkylene" refers to methylene or substituted dioxymethylene or ethylene or substituted dioxyethylene.

"unsubstituted or substituted amino acid residue or salt thereof" in reference to an R-group of formula I or formula II as used herein refers to an amino acid or substituted amino acid that is linked to an aromatic group of formula I or formula II through at least one H at the C-terminus, wherein the amino acid or substituted amino acid includes, but is not limited to: glycine, arginine, asparagine, aspartate, cysteine, glutamic acid, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5-hydroxylysine, 4-hydroxyproline, thyronine, 3-methylhistidine, epsilon-N-methyllysine, epsilon-N, N, N-trimethyllysine, aminoadipic acid, gamma-carboxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine, N-methylarginine, N-acetyl lysine, and N, N-dimethyl-substituted amino acids, or a salt of any of the foregoing, such as a halide salt.

Suitable for the use of the "buffer" includes any conventional buffer in the field, for example, Tris, phosphate, imidazole, and bicarbonate.

As used herein, "carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, vehicle, excipient, solubilizer, encapsulating material, or formulation excipient of any conventional type, and includes all compositional components of the inactive pharmaceutical ingredient. The carrier may include additional factors such as wetting or emulsifying agents, or pH buffering agents. Other materials such as antioxidants, humectants, viscosity stabilizers, or approximation factors may be added as needed.

As used herein, "catecholic butane" refers to compounds of formula I:

wherein R is₁And R₂Each independently represents-H, lower alkyl, lower acyl, lower alkylene; or-R₁O and-R₂Each O independently represents an unsubstituted or substituted amino acid residue or a salt thereof; r₃、R₄、R₅、R₆、R₁₀、R₁₁、R₁₂And R₁₃Each independently represents-H or lower alkyl; and R is₇、R₈And R₉Each independently represents-H, -OH, lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or a salt thereof, or any two adjacent groups together may be a dioxyalkylene group.

As used herein, "cyclodextrin" refers to unmodified cyclodextrins or modified cyclodextrins, including but not limited to alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and modified cyclodextrins containing modifications thereto, such as HP-beta-CD or SBE-beta-CD. Cyclodextrins typically have 6 (. alpha. -cyclodextrin), 7 (. beta. -cyclodextrin), and 8 (. gamma. -cyclodextrin) sugars, up to 3 substituents per sugar, and may have from 0 to 24 primary substituents (a primary substituent is defined as a substituent directly attached to the cyclodextrin ring). The modified or unmodified cyclodextrins used in the present invention may have primary substitutions or other modifications of appropriate numbers and positions.

As used herein, a "derivative" of NDGA refers to a "NDGA derivative" (see below).

The term "disease" as used herein includes all diseases, conditions, infections, syndromes or disorders for which the use of the compositions of the present invention has a therapeutic effect. Such diseases include, for example, but are not limited to, cancer, psoriasis and other proliferative diseases, inflammatory diseases including rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, atherosclerosis, chronic obstructive pulmonary disease ("COPD"), hypertension, obesity, diabetes, pain, shock and/or other neurological or neurodegenerative diseases or conditions including alzheimer's disease, parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis ("ALS"), and precancerous lesions such as intraepithelial neoplasia or dysplasia, and infectious diseases.

"G" as used herein₄N "or" tetra-N, N-dimethylglycinyl NDGA "or" tetra-dimethylglycinyl NDGA "is an NDGA derivative in formula II, wherein R is₁₄、R₁₅、R₁₆And R₁₇Each independently represents-O (C ═ O) CH in solid or liquid form₂N(CH₃)₂or-O (C ═ O) CH₂N⁺(CH₃)₂·Cl^-(ii) a And R is₁₈And R₁₉Each represents-CH₃。

As used herein, "lower acyl" refers to C₁-C₆Acyl, preferably C₁-C₃An acyl group.

As used herein, "lower alkyl" refers to C₁-C₆A hydrocarbon group, preferably C₁-C₃A hydrocarbyl group.

"M" as used herein₄N' or "tetra-O-methyl NDGA" is a NDGA derivative in formula II, wherein R is₁₄、R₁₅、R₁₆And R₁₇Each independently represents-OCH₃And R is₁₈And R₁₉Each is-CH₃。

As used herein, "modified cellulose" is meant to include one or more modifications to the cellulose molecule and includes, for example, EC, HPMC, CMC, and MC.

As used herein, "NDGA" refers to nordihydroguaiaretic acid and has the following structural formula:

as used herein, "NDGA compound" means NDGA and/or any one or more NDGA derivatives, individually or collectively.

As used herein, "NDGA derivative" refers to an NDGA derivative having the structural formula II:

wherein R is₁₄、R₁₅、R₁₆And R₁₇Each independently represents-OH, lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or a pharmaceutically acceptable salt thereof; and R is₁₈And R₁₉Each independently represents-H, or lower alkyl, wherein R₁₄、R₁₅、R₁₆And R₁₇Not simultaneously being-OH. Thus, the term includes compounds which are methylated derivatives of NDGA, e.g., tetra-O-methyl NDGA (M)₄N), tri-O-methyl NDGA (M)₃N), bis-O-methyl NDGA (M)₂N) and mono-O-methyl NDGA (M)₁N). Alternatively, the NDGA derivative may be a compound in which one or more hydrogens in the hydroxyl or methyl group of NDGA are substituted, for example, wherein R is₁₄、R₁₅、R₁₆And R₁₇Each independently represents a lower alkoxy group, an acyloxy group, or an amino acid, or a substituted amino acid or a salt thereof; and R is₁₈And R₁₉Each independently represents-H or a hydrocarbyl group such as a lower hydrocarbyl group. The term includes, for example, where R is₁₄、R₁₅、R₁₆And R₁₇Each independently represents-OCH₃or-O (C ═ O) CH₃Or disubstituted amino acid residues, e.g. N, N-dimethyl substituted amino acid residues, e.g., -O (C ═ O) CH₂N(CH₃)₂or-O (C ═ O) CH₂N⁺(CH₃)₂·Cl^-(ii) a And R is₁₈And R₁₉Each independently represents-H, or lower alkyl, e.g., -CH₃or-CH₂CH₃。

As used herein, "percentage," "percentage," or the symbol "%" refers to the percentage of the component indicated in the composition based on the amount of carrier present in the composition, referring to weight/weight (w/w), weight/volume (w/v), or volume/volume (v/v), as indicated for any particular component, all based on the amount of carrier present in the composition. Thus, different types of carriers may be present as shown in amounts up to 100%, which carriers do not exclude the presence of API, which amounts may be shown as% or as a specific value present in the composition, or as a specific value of mg/g present, or as a specific value of mg/mL present, wherein% or mg/g or mg/mL is based on the amount of total carrier present in the composition. Certain types of carriers may be present in combination making up 100% of the carrier.

As used herein, a "pharmaceutically acceptable carrier" is a non-toxin that is directed to the recipient in an amount or concentration used and is compatible with the other components of the formulation. For example, the formulation carrier comprising the catecholic butane, NDGA or NDGA derivative preferably does not include oxidizing agents and other known compounds deleterious thereto. The pharmaceutically acceptable carrier includes a solubilizing agent. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, dextrose, glycerol, salts, ethanol, buffers, Cremaphor EL, phosphate buffered saline, PEG300, PEG400, modified cyclodextrins, and combinations of any of the foregoing.

The term "pharmaceutically acceptable excipient" includes vehicles, adjuvants, or diluents or other auxiliary substances, such as are conventional in the art, readily available to the public, and not deleterious to the dosage or concentration employed by the recipient, and compatible with the other components of the formulation. For example, pharmaceutically acceptable auxiliary substances include pH adjusting and buffering factors, osmotic pressure adjusting factors, stabilizing factors, wetting factors, and the like.

The term "solubilizer" as used herein refers to a compound having dissolved therein one or more catecholic butanes or NDGA compounds, such as NDGA derivatives. The solubilizing agent may also be a carrier or a pharmaceutically acceptable carrier.

The terms "subject," "host," and "patient" are used interchangeably herein to refer to an animal treated with the present compositions, including, but not limited to, apes, humans, cats, dogs, horses, cows, pigs, sheep, goats, farm mammals, sports mammals, and pet mammals.

As used herein with reference to a "substantially purified" compound as used with reference to a catecholic butane or NDGA derivative, refers to a material that is substantially free of non-catecholic butanes, NDGA compounds, or NDGA derivatives (collectively referred to as "non-NDGA materials"). By substantially free is meant at least about 50% free of non-NDGA material, preferably at least about 70%, more preferably at least about 80%, even more preferably at least 90%, and even more preferably at least about 95% free of non-NDGA material.

As used herein, the terms "treat," "treating," and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially arresting the condition or disease or a syndrome thereof, or may be therapeutic in terms of a partial or complete cure for the condition or disease and/or due to a side effect of the condition or disease. "treatment" of a subject includes, for example, any treatment of a disease in a mammal, in part a human, and includes: (a) preventing the onset of the disease in a subject susceptible to the disease but not yet diagnosed when the disease is present; (b) inhibiting the development or progression of a disease, e.g., inhibiting its development; and (c) alleviating, moderating or ameliorating the disease, e.g., causing regression or removal of the disease.

Before the present invention is described further, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to 1/10 where the lower limit is stated, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of the smaller ranges may each be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

It must be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an" includes a plurality of such compounds, and reference to "the NDGA compound" includes reference to one or more NDGA compounds, such as NDGA derivatives, and equivalents thereof known to those skilled in the art.

All publications, including patents, patent applications, and journal articles, referred to herein are incorporated by reference in their entirety and the documents cited therein are also incorporated by reference in their entirety.

Each of the publications discussed herein is disclosed before the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such prior invention by virtue of prior publication. In addition, the dates of publication provided may be different from the actual publication dates which may need to be independently ensured. The references cited in the present application document are explained in detail in the references preceding the claims.

The invention is described below by way of example only and is not to be construed as being in any way limiting.

Preparation of catecholic butanes

The catecholic butanes of the present invention may be prepared by any method known in the art. Such compounds may be prepared, for example, with reference to that described in US5,008,294.

Preparation of NDGA derivatives

NDGA may be purchased from any commercial source, for example, Alexis biochemicals corp. Company, san diego, galileo, usa (catalog: LKT-N5669), or a.g. scientific, Inc, san diego, galileo, usa (catalog: N1071), or Cayman Chemical Company, anaba, michigan, usa (catalog: 70300).

NDGA derivatives and formulations thereof may be prepared by any conventional method. For example, the present invention may be used in accordance with U.S. patent 5,008,294; us patent 6,291,524; hwu, J.R et al (1998) or McDonald, R.W. et al (2001).

In one embodiment of the invention, the NDGA derivative, tetra-O-methyl NDGA, also known as meso-1, 4-bis (3, 4-dimethoxybenzene) -2, 3-dimethylbutane, or M, is prepared as follows₄N: a methanol solution containing NDGA and potassium hydroxide was prepared in a reaction beaker. Dimethyl sulfate was then added to the reaction flask and the reaction was allowed to proceed. Finally, the reaction is quenched with water to precipitate the product. The precipitate was isolated by filtration and dried in a vacuum oven. The compound was then dissolved in a solution of dichloromethane and toluene and then purified by an alumina column. The solvent was removed by rotary evaporation and the solid was resuspended in isopropanol and isolated by filtration. The filter cake was dried in a vacuum oven. The resulting tetra-O-methyl NDGA (M) was prepared by refluxing the filter cake in isopropanol₄N) crystallization and re-isolation of the crystals by filtration.

In some embodiments of the invention, certain NDGA derivatives of the invention, e.g., G₄N, also known as meso-1, 4-bis (3, 4-dimethoxybenzene) -2R, 3S-dimethylbutane or tetra-dimethylglycinoyl NDGA, or the hydrochloride salt thereof, and similar compounds with amino acid substitutions, can also be prepared according to conventional methods, for example, according to the teachings of U.S. patent 6,417,234.

Preparation of therapeutic compositions

The present invention provides compositions comprising a pharmaceutical composition comprising a catecholic butane, the catecholic butane comprising an NDGA compound and an NDGA derivative as an active pharmaceutical ingredient ("API"), and a pharmaceutically acceptable carrier or excipient. Generally, the compositions of the present invention will comprise from less than about 0.1% up to about 99% of an API, namely catecholic butanes, which herein include NDGA compounds and NDGA derivatives; alternatively, the present invention will comprise from about 2% to about 90% API.

The invention also provides compositions comprising catecholic butanes in the compositions, which include NDGA compounds, e.g., NDGA derivatives, such as M₄N, the catecholic butane is present at a concentration of about 1mg/mL to about 200mg/mL, or about 10mg/mL to about 175mg/mL, or about 20mg/mL to about 150mg/mL, or about 30mg/mL to about 125mg/mL, or about 40mg/mL to about 100mg/mL, or about 50mg/mL to about 75 mg/mL. In one embodiment, the NDGA compound is present in the compositions herein at a concentration of about 1mg/mL, about 2mg/mL, about 2.5mg/mL, about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35 mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 75mg/mL, about 80mg/mL, about 90mg/mL, about 100mg/mL, about 120mg/mL, about 125mg/mL, about 150mg/mL, about 175mg/mL, or about 200 mg/mL.

The invention further provides compositions comprising catecholic butanes in the compositions, which include NDGA compounds, e.g., NDGA derivatives, such as M₄N, the catecholic butane being present at a concentration in the range of about 1mg/g to about 250mg/g, or alternatively, about 20mg/g to about 200mg/g, or about 40mg/g to about 180mg/g, or about 60mg/g to about 160mg/g, or about 80mg/g to about 130mg/g, or about 50mg/g to about 100 mg/g. In one embodiment, the compound is present in the compositions herein at a concentration of about 133mg/g, about 185mg/g, about 200mg/g, and about 250 mg/g. Exemplary dosages of the API in the composition include, but are not limited to, 20mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 120mg/g, about 130mg/g, about 140mg/g, about 150mg/g, about 175mg/g, or about 200 mg/g.

Alternatively stated, other embodiments of the compositions of the present invention may include less than about 0.1mg to about 200mg or more of the API, for example about 10mg, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 75mg, about 100mg or about 200mg of the API.

In one embodiment of the invention, a composition is provided comprising an API and a carrier, wherein the API is catecholic butane, and the carrier comprises a solubilizing agent and/or an excipient that solubilizes the API, wherein the solubilizing agent and/or excipient comprises at least one, but may comprise a plurality of the following: (a) a water-soluble organic solvent, e.g., a PG (propylene glycol) or PEG (polyethylene glycol) compound, wherein the PEG compound is, e.g., PEG300, PEG400, or PEG400 monolaurate; (b) cyclodextrins, such as modified cyclodextrins; (c) ionic, nonionic or amphoteric surfactants such as Tween _20, Tween _80 or TPGS, glycerol monooleate and esterified fatty acids; (d) modified celluloses, such as EC, HPMC, MC, or CMC; and (e) a water-insoluble lipid, e.g., an oil, wax, or fat emulsion, e.g., Intralipid (r), if the composition contains castor oil, it does not contain beeswax or palm wax; and mixtures of the foregoing factors (a) - (e).

In one embodiment, the present invention provides a composition as above, which is a liquid composition suitable for oral administration. In another embodiment, the present invention provides a composition as above, which is a solid composition suitable for oral administration.

In another embodiment, the present invention provides a composition, such as above, which, when the composition contains propylene glycol, also does not contain (a) one of glycerol or glycine; or (b) white petrolatum; or (c) xanthan gum.

In another embodiment, the present invention provides a composition as above that also does not contain xanthan gum when the composition contains a nonionic surfactant.

In another embodiment, the present invention provides a composition as above that, when containing PEG400, also does not contain PEG8000 or BHT.

Among the preferred water-soluble organic solvents are ethanol, benzyl alcohol, dimethylacetamide, PVP, PG, and PEG compounds, such as PEG300, PEG400, or PEG400 monolaurate.

The PEG compound in the compositions of the invention is provided in an amount of about 5% to about 100%, or in an amount of about 5% to about 60%, or about 10% to about 90%, or about 20% to about 80%, or 30% to about 70%, or about 40% to about 60%, all concentrations being volume/volume (v/v) percent. PG may be present at a concentration of about 2.5% to about 100% (v/v).

The concentration of the PEG compound in the composition of the invention may vary depending on the solubilizer or diluent also present. For example, the concentration of PEG300, PEG400, or PEG400 monolaurate of the present invention may be about 5%, about 10%, about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, all concentrations given as a percentage of volume/volume (v/v).

The invention also provides compositions of a di-theaphenolbutane or NDGA compound in a cyclodextrin, the cyclodextrin comprising a modified cyclodextrin. The cyclodextrin herein can be alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and modified cyclodextrins can include, for example, HP-beta-CD and SBE-beta-CD. In one embodiment, the compositions of the present invention contain concentrations in the range of about 5% to about 80%, or about 10% to about 70%, or about 20% to about 60%, or about 30% to about 50%, all concentrations being given as weight/volume (w/v).

In another embodiment, the modified cyclodextrin, e.g., HP- β -CD, is present in the composition at a concentration of about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%, all concentrations being given as a weight/volume (w/v) percentage.

Another pharmaceutically acceptable carrier or excipient which may be used alone or in combination with other components of the composition of the invention is an ionic, non-ionic or amphoteric surfactant, e.g., Cremophor EL, polysorbate, which are non-ionic surfactants, e.g., polysorbate 20 and polysorbate 80, commercially available as Tween 20 or Tween 80, TSGS, amphoteric in many other classes. Further examples of suitable surfactants include, but are not limited to, glycerol monooleate, and esterified fatty acids, such as are commonly made by transesterification of vegetable oils, several varieties and grades available from Gattefose Corp, Paramus, N.J., such as Labrafil, Labrasol, and Gelucire. The surfactant may be present in any desired effective dose, for example, from about 1% (v/v) to about 100% (v/v), preferably from about 9% (v/v) to about 80% (v/v), and more preferably, from about 10% (v/v) to about 50% (v/v). As a specific example, the preferred concentration of the nonionic surfactant is Tween _20 in a range of about 9% (v/v) to about 100% (v/v), and Tween _80 in a range of about 33% (v/v) to about 100% (v/v). All percentages of surfactant are volume percentages (v/v).

Another pharmaceutically acceptable carrier or excipient that may be used alone or in combination with other components of the compositions of the invention is modified cellulose, such as EC, HPMC, MC and CMC. The modified cellulose may be present in any desired effective amount, for example, in a concentration of about 0.1% to about 25%, or about 0.5% to about 7.5%, or about 1.0% to about 5%. As particular examples, the EC may be present at a concentration of about 5% to about 20%; HPMC may be present at a concentration of about 0.5% to about 1%; MC may be present at a concentration of about 1% to about 3% and CMC may be present at a concentration of about 1% to about 4%. The percentage of modified cellulose is weight per volume (w/v).

In another embodiment, the present invention provides a composition as above, comprising a PEG compound, e.g., PEG400, e.g., and a surfactant, e.g., Tween _20, as a solubilizing agent and/or excipient.

Another pharmaceutically acceptable carrier or excipient which may be used alone or in combination with other components of the composition of the invention is a water-insoluble lipid, such as an oil, a fat emulsion or a wax. The water-insoluble lipid carrier can be present in any desired effective amount, for example, in a concentration of from about 10% to about 100%, or from about 15% to about 85%, or from about 25% to about 75%.

Non-limiting examples of oils include corn oil, olive oil, peppermint oil, soybean oil, sesame oil, mineral oil, and glycerol. In one embodiment, the oil is present at a concentration of about 10% (v/v) to about 100% (v/v). Mixed fat emulsion compositions are available, such as the Intralipid _ emulsions described above. In various embodiments, the mixed fat milk may be present at a concentration of about 10% (w/v) to about 30% (w/v), preferably 20% (w/v). Non-limiting examples of suitable waxes include beeswax and carnauba wax. In one embodiment, the wax is present at a concentration of about 5% (w/w) to about 50% (w/w).

The water-insoluble carrier may be used in combination with any or more water-soluble carriers, such as PEG compounds, and surfactants, such as Tween 20 or Tween 80. As a further example, the compositions of the invention include a combination of solubilizing agents and/or excipients, such as oils and surfactants, such as peppermint oil and Tween 20 or oils and PEG compounds, such as peppermint oil and PEG 400.

In another embodiment, the compositions of the invention comprise an oil, a surfactant, and a PEG compound, for example, peppermint oil, Tween _20, and a PEG400 compound.

As a further example, the compositions of the present invention comprise a combination of oils or a combination of oils and waxes, such as peppermint and sesame oil, soybean oil and beeswax or olive oil and beeswax. Beeswax may be present in a concentration range of about 5% to 50% (w/w).

Alternatively, in another embodiment, Tween 80 may be substituted for Tween 20, and PEG300 or PEG400 monolaurate may be substituted for PEG400, and any oil may be substituted for peppermint, soybean and olive oils.

Other excipients include glycerol monooleate and various types of esterified fatty acids, such as Labrafil, Labrasol and Gelucire products. These materials are generally classified as surfactants or emulsions, but Labrasol (R), and Gelucire (R) products are also used as bioavailability enhancers.

Labrafil _, and in particular Labrafil _ M1944 CS, may be used as an API, e.g., M₄N, wherein the concentration of dissolved API is from about 5mg/mL to about 500 mg/mL. The final product may be a solution, suspension or solid.

Labrasol-can be used as API, e.g.M₄N, wherein the concentration of dissolved API is from about 1mg/mL to about 500 mg/mL. The final product may be a solution, suspension or solid.

Gelucire _, and in particular Gelucire _44/14, may be used as an API, e.g., M₄N, wherein the concentration of dissolved API is from about 0.1mg/mL to about 500 mg/mL. The final product may be a solution, suspension or solid.

Combinations of various carrier components may be used as APIs, as described above. A non-limiting example of such an embodiment is 10mg/ml M₄N is in 25% (w/v) PEG300, 30% (w/v) HP- β -CD, and the balance of the carrier is "water suitable for injection" for injection into animals ("WFI", meaning a grade approved in the pharmaceutical industry). In this preferred embodiment, HP- β -CD has 6-8 substitutions, but other substitutions in other embodiments are within the scope of the invention, as described above.

Moreover, other excipients and additives, such as bioavailability enhancers, may be used in combination with any of the carriers in the compositions of the present invention, and suitable bioavailability enhancers include, for example, but are not limited to, eugenol, cinnamaldehyde, lecithin, naringenin, naringin, and piperine (also known as piperine), as well as those already mentioned above.

It is to be understood that one or more solubilizing agents or diluents or excipients herein may be added to the composition to optimize delivery of the composition to a subject in need of such treatment, so long as the catecholic butanes herein are dissolved and maintained in solution, suspension, or in a semi-solid or solid form of the composition.

Other pharmaceutically acceptable carriers or excipients suitable for use are described in numerous publications herein. Examples of useful carriers or excipients are described, for example, in Gennaro, A.R (2003); ansel, h.c. et al, (2004); rowe, r.c. et al. (2003); and Garg, s. et al (2001).

The liquid form of the composition may include a buffer, which may be selected according to the desired use of the catecholic butane or the NDGA compound, e.g., NDGA derivative, and the liquid form of the composition may also include other materials suitable for the desired use. One skilled in the art can readily select an appropriate buffer, many of which are known in the art, suitable for the desired use.

Method of treatment

Compositions containing catecholic butanes, including NDGA compounds, such as NDGA derivatives, have use as therapeutic agents in a number of diseases in which the catecholic butanes may be used or for treating a subject in need of such treatment.

The present invention provides methods and compositions for treating diseases, such as proliferative diseases, e.g., benign or malignant tumors, psoriasis, precancerous lesions and neoplastic tumors, e.g., intraepithelial tumors, or dysplasia. The invention also provides for the treatment of diabetes, including type I and type II diabetes, obesity and complications arising therefrom, including cardiovascular disease, shock and hypertension. The invention further provides for the treatment of inflammatory diseases, including rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, crohn's disease, Chronic Obstructive Pulmonary Disease (COPD) and other immune system related diseases. In addition, the invention provides for the treatment of neurological disorders, including central nervous system disorders or neurodegenerative diseases, such as Alzheimer's disease, dementia, Amyotrophic Lateral Sclerosis (ALS) and Parkinson's disease. In another embodiment, the invention provides for the treatment of infectious diseases, such as viral infections, including viruses that require Sp1 binding for transcription or replication. Examples of such viruses that require Sp1 binding include: HIV, HTLV, HPV, HSV, EBV, varicella-zoster virus, adenovirus, parvovirus and JC virus.

Depending on the method tested, a variety of animal hosts may be treated, including human and non-human animals. Typically such hosts are "mammals" (mammals) "or" mammals "(mammalian)," where these terms are used broadly to describe organisms within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodents (e.g., guinea pigs, and rats), and other mammals, including cows, goats, horses, sheep, rabbits, pigs, and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host may be a human. Animal models are relevant for experimental studies, for example providing models for the treatment of human diseases. In addition, the present invention is applicable to veterinary diseases as well.

Formulation, dosage, and route of administration

In one embodiment of the invention, an effective amount of a composition of the invention is administered to a host, wherein "effective amount" refers to an amount sufficient to produce the desired result. In some embodiments, for example, the desired result is at least inhibition of a neoplastic tumor or dysplasia.

The invention further provides compositions in which the active pharmaceutical ingredient, e.g., catecholic butane, comprises an NDGA compound, e.g., an NDGA derivative, e.g., M₄N, in smallAn oral dose in the range of 1mg/kg animal body weight to about 600mg/kg animal body weight is administered to an animal, such as a human. Alternatively, the animal may be treated at a dose of 1mg/kg, or 50mg/kg, or 100mg/kg, or 150mg/kg, or 200mg/kg, or 250mg/kg, or 300mg/kg, or 350mg/kg, or 400mg/kg, or 450mg/kg, or 500mg/kg, or 550 mg/kg. The administration to the animal may be repeated once or over a period of days or weeks or months. Alternatively, such a dose may be extended over a period of time depending on the health of the subject, the susceptibility of the subject, the degree of the disease to be treated, the age of the subject, and the like.

In one embodiment, the therapeutic compositions herein are prepared by first dissolving the active pharmaceutical ingredient in the solubilizing agent, with stirring and heating if necessary. Additional excipients are added to the dissolved mixture to produce the desired properties based on texture and stability requirements. In another embodiment, the active pharmaceutical ingredient may not be actually dissolved in the solubilizing agent, but may simply be homogeneously dispersed in the suspension. In another embodiment, the dissolved composition may be frozen and used in powder form. The final oral composition may be a liquid solution or suspension, or may be a solid powder, tablet, or capsule.

As indicated above, the appropriate dose to be administered depends on the subject to be treated, e.g. the general health of the subject, the age of the subject, the state or condition of the disease, the weight of the subject, the extent of the disease, e.g. the size of the tumor. Typically about 0.1mg to about 500mg or less may be administered to a child, and about 0.1mg to about 5 grams or less may be administered to an adult. The active factors can be administered individually, or more usually, in multiples. The preferred dosage of a given factor can be readily determined by one skilled in the art by various methods. Other effective dosages can be readily determined by one of ordinary skill in the art by routine experimentation to establish dose response curves. The dosage of the factor should of course vary depending on the particular factor used.

The frequency of administration of the active factors, in combination with the dosage, will be determined by the caregiver based on age, weight, disease state and response of the patient. Thus, the factor may be used one or more times per day, week, month, or as appropriate according to convention. The factor may be administered intermittently, for example over a period of days, weeks or months, after which it is not administered until a period of time has elapsed, for example 3 or 6 months, and then administered again over a period of days, weeks or months.

In pharmaceutical dosage forms, the active agent may be used alone or in appropriate combination or conjugate with other pharmaceutically active agents or therapeutic substances including other small molecules, antibodies or proteins.

In addition, if desired, the carrier or excipient may include minor amounts of auxiliary substances such as pH adjusting and buffering agents, osmotic pressure adjusting agents, stabilizing agents, wetting agents or emulsifying agents. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 20th ed (remmington Pharmaceutical science, 20th edition), Mack Publishing Co, (Mack Publishing company) Rawlins EA, (1997). The composition or formulation to be applied will in any case comprise an amount of API sufficient to achieve the desired state in the subject being treated.

The invention includes kits containing multiple or unit doses of active factors. In this kit, the container will be an information package containing instructions describing the use and concomitant benefits of the drug in treating the associated pathological condition, in addition to a multiple or unit dose composition comprising catecholic butanes, which include NDGA compounds, such as NDGA derivatives. Optionally, a kit for administering the composition of the present invention is also included in each kit.

The embodiments set forth below are exemplary in nature and are not to be construed as limiting the invention.

Example 1M contained in HP-beta-CD and/or PEG300₄And (4) preparing a preparation of N.

In this example, M was prepared as described in PCT/US2004/016117₄N and solubilized in a solubilizer. The resulting solution may optionally be mixed with excipients and/or diluents. Solubilizers and excipients may be used interchangeably or in combination with each other. One solubilizing agent or excipient used was endotoxin-controlled hydroxypropyl-beta-cyclodextrin ("HP-beta-CD") obtained from Research Diagnostics, Inc (catalog number RDI-82004HPB, lot number H3N188P) (Flanders, new jersey, usa). Another solubilizing agent or excipient used was PEG300, obtained from Spectrum Chemicals, Inc. (Cat. No. P0108, batch No. TB1228) (Gardena, Calif., USA).

In one embodiment of the invention, the HP- β -CD and PEG300 are present in a single formulation. To prepare these formulations, M₄N is first dissolved in PEG300 to form M₄PEG300 solution of N ("M)₄N/PEG 300”)。M₄The N/PEG300 solution was then added to the pre-prepared HP-beta-CD solution to form M in HP-beta-CD and PEG300₄N solution (hereinafter, referred to as "CPE" preparation).

The volume expansion must be taken into account when preparing the HP-beta-CD solution. For example, for a 40% (w/v) HP- β -CD solution, 0.7mL/g should be considered (i.e., increasing the removal of 0.7mL of water per gram of HP- β -CD).

100mL of a 40% HP- β -CD solution used as solubilizer and/or excipient was prepared as follows: 65 mL of WFI was placed in a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. About 40 grams of HP- β -CD was slowly added to the agitated WFI, using a spatula to guide the HP- β -CD to the center of the beaker to prevent HP- β -CD crystals from sticking to the walls of the beaker. The HP- β -CD solution was stirred for 24 hours until complete dissolution of HP- β -CD was observed. The resulting solution was measured to be approximately 93 mL. Approximately 7mL of WFI was added to the resulting solution to reach 100mL, resulting in a final solution of approximately 40% HP- β -CD. The final solution was stirred for 1 hour, stored at room temperature and protected from light. The method of preparing the modified cyclodextrin solution can be scaled up or down to obtain the desired volume or concentration. Other concentrations or other modified cyclodextrin solutions can also be similarly prepared, for example, by replacing HP- β -CD with other modified cyclodextrins, adjusted to the appropriate concentration in the steps described above.

10mL of M at a concentration of 10mg/mL in 40% HP-beta-CD was prepared as follows₄And (4) N solution. About 10mL of 40% HP- β -CD was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. With the aid of a spatula, about 10mg M₄N was slowly added to the center of the beaker in 40% HP-beta-CD. M₄The N/40% HP-beta-CD mixture was stirred for 2 hours, or until all M was present₄N was suspended uniformly without any clumping. M₄The N/40% HP-beta-CD mixture may alternatively be heated at 80 ℃ for about 30 minutes. (or longer, if a large volume of solution is desired, e.g., 100mL of M₄N/HP-beta-CD, 1 hour at 80 ℃), or longer, if necessary to ensure M₄Complete dissolution of N. Observe the beaker against a white background M₄N/HP-beta-CD mixture or solution of any undissolved M₄N, and the presence of particles was checked against a black background. Final M₄The N/40% HP-beta-CD solution was stored at room temperature and protected from light. The method can be scaled up or down to obtain the desired M₄Volume or concentration of N. Solutions containing M in other cyclodextrins can also be prepared analogously₄N, for example, by replacing HP-beta-CD with another cyclodextrin in the above steps. The results shown in Table 1 show the M of the solutions at concentrations of 1mg/mL and 10mg/mL in the 40% HP-beta-CD cyclodextrin formulation after cooling for more than 7 days₄N。

M at a concentration of 25mg/mL in PEG300 was prepared as follows₄100mL of N. About 100mL of PEG300 was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stirring rod was provided toStirring at moderate speed. With the aid of a spatula to stop M₄N adhered to the beaker wall, about 2.5g M₄N was added slowly to PEG300 in the center of the beaker. M₄The N/PEG300 mixture was stirred for 24 hours, or until all M was present₄N dissolved or suspended uniformly without any lumps. M₄The N/PEG300 mixture may alternatively be heated at about 60 ℃ for about 30 minutes. (or longer, if a larger volume is desired, e.g., 500mL of M₄N/PEG300 mixture, heated at 60 deg.C for 1 hour), or longer, if necessary to ensure M₄Complete dissolution of N. Observe the beaker against a white background M₄Any undissolved M of the N/PEG300 mixture or solution₄N, and the presence of particles was checked against a black background. When all M's are observed₄After N has dissolved, the M produced is immediately applied₄The N/PEG300 solution may otherwise form additional crystals or other precipitates over a 48 hour useful life. If crystals are formed, M₄The N/PEG300 solution can be heated again on the magnetic disk at 60 ℃ with stirring for about 1 hour until all M₄The N redissolved back into solution. Last M₄The N/PEG300 solution was stored at room temperature and protected from light. The method can be scaled up or down to obtain the desired M₄Volume and concentration of N. M contained in other PEGs can be similarly prepared₄N, for example, by replacing PEG300 with PEG400 or PEG400 monolaurate in the above-described method.

Containing 50% PEG300 (v/v), 20% HP-beta-CD (w/v) and 12.5mg M₄The preparation of 100mL stock of formulation of N was: 50mL of a 40% pre-prepared HP-beta-CD solution (prepared according to the method described above) was added to a glass beaker placed on a magnetic disk, containing a stir bar that was stirred at a medium speed, and 50mL of pre-prepared M was slowly added₄The N/PEG300 solution (prepared according to the methods described previously) is, for example, at a rate of about 10mL per minute. Using a pipette₄N/PEG300 was added to the center of the beaker to prevent it from adhering to the beaker walls and to ensure complete dissolution. Will M₄Adding N/PEG300 to the HP-beta-CD solutionInitially appear as a white solution, but eventually become clear after continued mixing. To prepare the desired M₄The volume and concentration of N/PEG300 and HP- β -CD, the formulation can be scaled up or down as appropriate. The stock solution can be filter sterilized using a 0.22 μm PVDF membrane, for example, a pre-sterilized, vacuum-driven set sterile vial top filtration membrane obtained from Millipore corporation (catalog number SCGV T05 RE) (Billerica, Mass., USA). The filtration process was driven by vacuum force and the filtrate was collected in a pre-sterilized 250mL glass vial. The bottles were then tightly sealed, stored at room temperature and protected from light. M can be similarly prepared by substituting PEG300 for PEG400 or PEG400 monolaurate in the methods described previously₄Storage solution of N/PEG400 or M4N/PEG 400 monolaurate in HP-beta-CD.

M prepared in the foregoing manner₄N/PEG300/HP-β-CD，M₄N/PEG 400/HP-beta-CD or M₄N/PEG400 monolaurate/HP-beta-CD stock solutions may be diluted prior to use in vitro or administration to an animal. If dilution is required, for example, the diluent may be preferably diluted in WFI, in place of brine, to reduce osmotic pressure. To prepare 100mL of a 1: 1 stock diluted in WFI, about 50mL of the stock was added to a glass vial. About 50mL of WFI was added to 50mL of stock solution in a vial to form a diluted solution. The glass vial was sealed and the diluent mixed by shaking and inverting the vial several times. The dilutions were filter sterilized using a 0.22 μm PVDF membrane, e.g., a pre-sterilized vacuum-driven set sterile vial top filter membrane obtained from Millipore corporation (catalog number SCGVT05 RE) (Billerica, ma, usa). The filtration process was driven by vacuum force and the filtrate was collected in a pre-sterilized 250mL glass vial. The bottles were then tightly sealed, stored at room temperature and protected from light. The method can be scaled up or down to obtain the desired M₄Volume and dilution of N, for example 1: 2 or 1: 4 dilution.

Formulations suitable for use as placebo controls, including 50% PEG300 and 20% HP- β -CD, can be prepared as follows. To prepare 100mL of a placebo or control formulation, approximately 50mL of 40% HP- β -CD was added to a glass beaker placed on a magnetic disk containing a stir bar, which was set to stir the HP- β -CD solution at a moderate speed. About 50mL of PEG300 was slowly added by pipette to the center of a 50mL beaker of HP- β -CD in a glass beaker to prevent PEG300 from sticking to the beaker walls. The mixture was stirred for about 1 hour until completely mixed. The placebo was filter sterilized using a 0.22 μm PVDF membrane by vacuum force-driven filtration. The filtrate was collected in a pre-sterilized 250mL glass vial. The bottles were tightly sealed, stored at room temperature and protected from light. The formulation can be scaled up or down as necessary to achieve the desired concentration and volume amounts. In addition, PEG300 may be replaced with PEG400 or PEG400 monolaurate, if desired.

M₄The results of the solubility of N in formulations containing HP- β -CD and/or PEG300, PEG400, prepared according to the foregoing or similar methods, as well as in formulations containing HP- β -CD and propylene glycol ("PG"), Hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), or Tween 80, and the characteristics of the resulting formulations are shown in tables 1-5, where N stands for "no" and Y stands for "yes".

TABLE 1 modified Cyclodextrins as solubilizers and/or excipients

[0144]

[0145]

[0146]

[0147]

[0148] All formulations were kept at 4 ℃ for 24 hours and then centrifuged at 5000rpm for 5 minutes without formation of visible precipitates. Contains 50% PEG300, 20% HP-beta-CD, 12.5mg/mL M₄The formulation of the N stock solution was kept at 4 ℃ for at least 4 months. Dilutions stored identically at 1: 1 or 1: 2 were kept at 4 ℃ for at least 4 months.

TABLE 2M in PEG300 and HP-beta-CD₄N formulations

TABLE 3M in PEG400 and HP-beta-CD₄N formulations

[0153]

TABLE 4M in PEG300₄Stability of N formulations

TABLE 5M in PEG400₄Stability of N formulations

Approximately, M₄N may be dissolved in other solubilizing agents, for example, water-soluble organic solvents including ethanol, PVP (polyvinylpyrrolidone), propylene glycol or glycerol.

Example 2M in formulations of DMSO or PEG 300/HP-beta-CD conjugate₄Effects of N on proliferation and death of tumors in culture.

M in 10% (w/v) HP-beta-CD and 25% (v/v) PEG300₄N (hereinafter, referred to as "CPE preparation"), M in 30% (w/v) HP-beta-CD and 25% (v/v) PEG300₄N (hereinafter, referred to as "CPE 25/30 preparation"), and M in 27% (w/v) HP-beta-CD and 33% (v/v) PEG300₄N (hereinafter, referred to as "CPE 33/27 preparation") was tested for its effect on two different tumor cell lines: HeLa cells of HPV-18-positive human cervical cancer cell lines, and C-33A cells of HPV-negative human cervical cancer cell lines. M in DMSO₄N was also tested in parallel. Two tumor cell lines with M₄N escalated doses were treated with DMSO or CPE preparations for 72 hours: 0. mu.M, 20. mu.M, 40. mu.M, 60. mu.M and 80. mu.M. Each formulation was added to growth medium (minimal essential medium containing L-glutamine supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, 1 × non-essential amino acid solution, and 1,000 IU/mL penicillin/1,000 μ g/mL streptomycin solution) to 1%. Control cells were grown under the same conditions and were not treated. After 72 hours of treatment or non-treatment, the total number of cells and the number of viable cells in each sample were counted using trypan blue exclusion. The cell proliferation rate and percentage of dead cells in each sample were analyzed. The experimental results are shown in the figure1. Fig. 2 and tables 6 to 12.

FIG. 1 is a graph showing the ratio of the number of treated cells to the number of untreated cells to M in a DMSO preparation or a CPE preparation in which C-33A cells and HeLa cells were treated₄Plot of increasing concentration of N. FIG. 2 is a graph showing the M in DMSO or CPE preparation representing the percentage of dead cells corresponding to the treatment of C-33A cells and HeLa cells of two tumor cell lines in culture₄Plot of increasing concentration of N.

The results show that, based on the% dead cells determined, the control is compared to untreated control in the absence of M₄DMSO alone at N has a significant antiproliferative effect and some toxic effects on both tumor cell lines tested. In contrast, CPE preparation alone, had a significant antiproliferative effect and a minimal toxic effect on both tumor cell lines compared to the untreated control.

As non-M in the same formulation₄N treatment control comparison (i.e. M)₄N is 0. mu.g/mL or 0. mu.M), by adding M in DMSO or CPE preparation₄After N treatment, the cell proliferation rate decreased for both cell lines. Indeed, the antiproliferative effect showed M in CPE preparations₄Dose dependence of N. In CPE preparations, for example, M of about 20. mu.M or 7.2. mu.g/mL is found₄N is sufficient to cause about 50% inhibition of cell proliferation for both tumor cell types. M in CPE formulation₄Further increases in the concentration of N lead to further increases in the antiproliferative effect on both cell types.

In summary, higher concentrations of M in DMSO or CPE preparations₄The N dose induced a higher percentage of cell death for both C-33A cells and HeLa cells. However, M in DMSO formulations₄N to M at the corresponding concentration in CPE preparation₄N is more toxic to cells. M when in DMSO₄The highest concentration detected of N (80 μ M or 28.7 μ g/mL) contributed to about 40% of cell death in the cell population. In this experiment, M in CPE preparation₄The same concentration of N promotes only about 20% of the cells in the cell populationThe cell dies.

It was found that these results can be repeated in both cell lines. Data from this study show M in CPE preparation₄N has the ability to prevent cell proliferation, and M in DMSO preparations₄N is similar while inducing less cytotoxicity than DMSO formulations.

Data was collected from successive time points to check the effectiveness of the CPE formulation over time. The data show that after a period of 12 months of storage at 2-8 ℃, the CPE formulation is as effective as the freshly prepared. The cells maintained approximate viability during the 12 months that CPE was stored. The cell death and proliferation rates remained in the same range.

Data was collected to compare the original CPE preparation with the freshly prepared CPE25/30 preparation. Studies were conducted at 0 and 3 months to examine the effectiveness of the formulations over time, as well as to examine how new formulations correlate with old formulations. The data show that the CPE25/30 preparation was effective in limiting tumor cell growth with the original CPE preparation. Between HeLa cells treated with CPE preparation or CPE25/30 preparation at different drug concentrations, the viability of the cells was similar. Cell death and proliferation remained in the same range even throughout the entire period.

Information was collected comparing the effect of the original CPE preparation to the CPE 33/27 preparation on HeLa cells at time point 0. The data show that CPE 33/27 has the same effect on HeLa cells in terms of cell viability, percentage of dead cells and proliferation rate.

TABLE 6M in DMSO or CPE preparations₄Effect of N on C-33A cells

[0171]

TABLE 7M in DMSO or CPE preparations₄Effect of N on Hela cells at time point 0

TABLE 8M in DMSO or CPE preparations₄Effect of N on Hela cells at month 9

[0176]

TABLE 9M in DMSO or CPE preparations₄Effect of N on Hela cells at month 12

TABLE 10 comparison of CPE preparation and CPE25/30 preparation in Hela cells

[0181]

TABLE 11 comparison of CPE preparation and CPE25/30 preparation in Hela cells at month 3

TABLE 12 comparison of CPE preparation and CPE33/25 preparation in Hela cells at time point 0

[0186]

Example 3 multiple batches (lot) of M can be used₄N and produce the same result

Detection of M from different batches₄N to show the potency of different batches of drug. By M₄N ascending doses: HeLa cells were treated with CPE preparation for 72 hours at 0. mu.M, 20. mu.M, 40. mu.M, 60. mu.M and 80. mu.M. Each formulation was added to growth medium (minimal essential medium containing L-glutamine supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, 1 × non-essential amino acid solution, and 1,000 IU/mL penicillin/1,000 μ g/mL streptomycin solution) to 1%. Control cells were grown under the same conditions and were not treated. After 72 hours of treatment or non-treatment, the total number of cells and the number of viable cells in each sample were counted using trypan blue exclusion. The cell proliferation rate and percentage of dead cells in each sample were analyzed. The results of the experiments are shown in tables 13 and 14. These results show that M is used regardless of the type₄N, the potency of the drug remained the same.

TABLE 13M with different batches₄N-treated HeLa cells (lot EM1001)

TABLE 14M with different batches₄N-treated HeLa cells (lot EM1002)

[0193]

Example 4M in modified cellulose₄Solubility of N

A10 mL solution of 50% HP- β -CD (w/v) and 0.5% hydroxypropyl methylcellulose ("HPMC") (w/v) used as a solubilizing agent and/or excipient was prepared as follows: 5.9mL of WFI was placed in a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. 5 grams of HP- β -CD was slowly added to the agitated WFI, and the HP- β -CD was directed to the center of the beaker using a spatula. The HP- β -CD solution was stirred for 24 hours until complete dissolution of HP- β -CD was observed. The resulting solution was measured to be approximately 9.4 mL. About 0.6mL of WFI was added to the resulting solution to 10mL to produce a 50% HP- β -CD (w/v) solution. 50mg of HPMC was added to a solution of 50% HP- β -CD and stirred for about 1 hour, or until complete dissolution of HPMC was observed. The final solution was stirred for about 1 hour, then stored at room temperature and protected from light. The process of preparing the modified cyclodextrin solution with the modified cellulose may be scaled up or down to obtain the desired volume or concentration of HP- β -CD/HPMC solution. Other concentrations or other modified cyclodextrin solutions can also be similarly prepared, for example, by replacing HP- β -CD with other modified cyclodextrins, adjusted to the appropriate concentration in the steps described above. Other modified cyclodextrin/modified cellulose solutions may also be similarly prepared, for example, by replacing HP- β -CD with other modified cyclodextrins, or HPMC with modified cellulose, in the foregoing methods.

M in 50% HP-beta-CD/0.5% HPMC₄A10 mL solution having an N concentration of about 10mg/mL was prepared as follows. Approximately 10mL of a 50% HP- β -CD/0.5% HPMC solution was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. With the aid of a spatula, about 100mg of M is added₄N was slowly added to 50% HP- β -CD/0.5% HPMC in the center of the beaker. M₄The N/50% HP-beta-CD/0.5% HPMC mixture was stirred for 24 hours, or until all M was present₄N was completely suspended with no lumps present. M₄The N/50% HP- β -CD/0.5% HPMC blend was heated at about 90 ℃ for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/50% HP-beta-CD/0.5% HPMC mixture, heated at 90 deg.C for 1 hour), or longer, if necessary to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄N/50% HP-beta-CD/0.5% HPMC mixture of any undissolved M₄N, and the presence of particles was checked against a black background. Final M₄N/50% HP- β -CD/0.5% HPMC solution was stored at room temperature and protected from light. When heated at 90 ℃, M₄N was dissolved in the 50% HP- β -CD/0.5% HPMC formulation at concentrations of 1mg/mL and 10mg/mL and remained stable in solution for more than 7 days at room temperature after cooling. Even at 90 ℃ M₄N was not dissolved in the same preparation at a concentration of 50 mg/mL.

The foregoing method may be scaled up or down to obtain the desired M₄Volume and concentration of N. M can be prepared analogously in other cyclodextrin/cellulose solutions₄N, for example, by replacing HP-beta-CD in the foregoing process with other cyclodextrins, or HPMC with other modified celluloses.

A10 mL solution of 5% ethyl cellulose ("EC") (w/v) in ethanol used as a solubilizing agent and/or excipient was prepared as follows: 10mL of 100% ethanol ("EtOH") was placed in a glass beaker containing a stir bar, covered with a circular Teflon _ lid. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. 500mg of EC was slowly added to the agitated ethanol and the EC was directed to the center of the beaker with a spatula to prevent the EC powder from sticking to the walls of the beaker. The EC solution was stirred for about 2 hours, or until complete dissolution of the EC was observed. The final solution was stored at room temperature and protected from light.

The manufacturing process to make the modified cellulose solution can be scaled up or down to achieve the desired volume or concentration. Other modified celluloses can also be similarly prepared, for example, by replacing EC with other modified celluloses in the foregoing methods.

M in 5% EC (w/v)₄A10 mL solution ("EC formulation") with an N concentration of about 20mg/mL was prepared as follows. Approximately 10mL of a 5% EC formulation prepared as previously described was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a medium speed and covered with a Teflon _ lid in a circular shape. To prevent adhesion to the beaker wall, about 200mg of M₄N was slowly added to the 5% EC formulation in the center of the beaker with the aid of a spatula. M₄The N/EC mixture was stirred for 2 hours until all M was present₄N was completely dissolved, or suspended uniformly without any lumps present. M₄The N/EC mixture was heated at about 60 ℃ for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/EC mixture, heated at 60 ℃ for 1 hour), or longer, if necessary to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄Any undissolved M of the N/EC mixture₄N, and the presence of particles was checked against a black background. Final M₄The N/EC solution was stored at room temperature and protected from light.

The foregoing method may be scaled up or down to obtain the desired M₄N volume and concentration, and the heating temperature can be increased or decreased to reach M₄And (4) dissolving N. Can be similarly madePreparation containing M in other modified celluloses₄Formulations of N, for example, HPMC, MC, and CMC. Table 15 shows M in modified cellulose₄Solubility of N.

Results display M₄N was not soluble in 2.3% (w/v) HPMC at any concentration tested or heated to 90 ℃. M₄N was suspended in 1% (w/v) HPMC at a concentration of 10 mg/mL. The suspension is stable at room temperature for a period of less than 2 days.

M in the presence of HP-beta-CD (50% w/v) and HPMC (0.5% w/v)₄N was dissolved at 90 ℃ and was kept in solution at concentrations of 1mg/mL and 10mg/mL by cooling. The solution is stable at room temperature for a period longer than 7 days. M₄N was not soluble in the same HP- β -CD (50% w/v) and HPMC (0.5% w/v) compositions at a concentration of 50mg/mL even when heated to 90 ℃.

In another experiment, M was performed without heating₄N in the HP- β -CD (50% w/v) and HPMC (0.5% w/v) compositions at all concentrations tested, i.e., 1mg/mL, 10mg/mL, 20mg/mL and 50mg/mL, formed suspensions that were stable at room temperature for longer than 7 days.

The results in Table 15 also show M₄N is soluble at 1mg/mL in EC formulations without heating, and the solution is stable at room temperature for longer than 3 days. M₄N is soluble at 40 ℃, at a concentration of 10mg/mL, and remains in solution after cooling, which is stable for longer than 3 days at room temperature. M₄N is soluble in EC formulations at 60 ℃, at a concentration of 20mg/mL, and remains in solution after cooling, which is stable for longer than 3 days at room temperature. M₄N is soluble in EC formulations at 60 ℃, at a concentration of 30mg/mL, but is not kept in solution after cooling. M₄Higher concentrations of N in the EC formulation, for example, levels of 50mg/mL or 100mg/mL, are soluble at 90 ℃, but are not kept in solution upon cooling.

M₄N in 1% low viscosity CMCSuspensions were also formed at 10mg/mL and 20mg/mL levels. These suspensions are stable at room temperature for a period of less than 2 days.

TABLE 15.M₄Solubility of N in modified cellulose-containing formulations

[0209]

Example 5.M₄Solubility of N in water-insoluble lipids and in water-soluble organic solvents.

In sesame oil M₄A10 mL solution having an N concentration of about 50mg/mL was prepared as follows. About 10mL of sesame oil was added to a glass beaker containing a stir-bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. With the aid of a spatula, about 500mg of M will be added₄N was slowly added to the sesame oil in the center of the beaker to prevent sticking to the beaker wall. M₄The N/sesame oil mixture is stirred for about 2 hours, or until all M is present₄N dissolved, or suspended homogeneously, with no lumps present. M₄The N/sesame oil mixture was heated at about 60 ℃ for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/sesame oil mixture, heated at 60 ℃ for 1 hour), or longer, if necessary, to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄Any undissolved M of the N/sesame oil mixture₄N, and the presence of particles was checked against a black background. If crystals are formed, M₄The N/sesame oil mixture can be heated again at 60 deg.C for 1 hour with stirring on a hot magnetic plate until all M is present₄The N redissolved back into solution. Final M₄The mixture of N/sesame oil is stored inRoom temperature and protected from light.

The foregoing method may be scaled up or down to obtain the desired M₄N by volume or concentration, and increasing or decreasing the heating temperature to reach M₄And (4) dissolving N. M in other water-insoluble lipids can be similarly prepared₄N, for example, sesame oil is substituted for corn oil, olive oil, soybean oil, peppermint oil, or other solubilizing agents, and combinations thereof, in the foregoing methods. The results are shown in Table 16.

M in 95% Olive oil and 5% beeswax₄A10 mL mixture with an N concentration of about 200mg/g was prepared as follows. About 7.6mL of olive oil was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. With the aid of a spatula, about 2g of M are added₄N was slowly added to the olive oil in the center of the beaker to prevent sticking to the beaker walls. M₄The N/olive oil mixture is stirred for about 2 hours, or until all M is present₄N dissolved, or suspended homogeneously, with no lumps present. M₄The N/olive oil mixture may optionally be heated at about 60 c for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/olive oil mixture, heating at 60 deg.C for 1 hr), or longer, if necessary to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄N/Olive oil mixture any undissolved M₄N, and the presence of particles was checked against a black background. If crystals are formed, M₄The N/olive oil mixture may be heated again at 60 ℃ for about 1 hour with stirring on a hot magnetic plate until all M is present₄The N redissolved back into solution. Approximately 400mg of white beeswax was added to a glass beaker with a stir bar. The beaker was also placed on a magnetic disk and a stir bar was set to stir at a moderate speed. The beeswax is heated at about 50 deg.C for about 30 minutes. (or longer if a greater amount is desired), or until all of the wax is melted. Then M₄The N/olive oil solution is added to about 400mg of melted beeswax, stirred, and heated at about 50 deg.C for about 30 minutesClocks, or up to all M₄The N/olive oil/beeswax mixture is dissolved or mixed evenly. Remove the stir bar from the beaker and let M₄The N/olive oil/beeswax mixture was cooled. Last M₄The N/olive oil/beeswax mixture was stored at room temperature and protected from light.

The method can be scaled up or down to obtain the desired M₄Volume or concentration of N. M in other water-insoluble lipids can be similarly prepared₄N, for example, in the foregoing methods, by replacing olive oil with corn oil, sesame oil, soybean oil, lecithin, or other solubilizing agents or combinations thereof, and beeswax with paraffin, PEG 3350, or other hardening agents or combinations thereof. Also, if desired, combinations with any carriers, bioavailability enhancers, such as eugenol, cinnamaldehyde, lecithin, naringenin, naringin, and piperine (also known as piperine) may be included. The results are shown in Table 16.

M in 85% Olive oil and 15% Tween _20₄A10 mL solution having an N concentration of about 60mg/mL was prepared as follows. About 8.5mL of sesame oil was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. About 1.5mL of Tween _20 was slowly added to the center of the beaker. With the aid of a spatula, about 600mgM₄N was added slowly to the sesame oil/Tween _20 mixture in the center of the beaker to prevent M₄N adheres to the beaker wall. M₄The N/sesame oil/Tween _20 mixture was stirred for about 2 hours, or until all M was present₄N dissolved, or suspended homogeneously, with no lumps present. M₄The N/sesame oil/Tween _20 mixture was heated at about 60 ℃ for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/sesame oil/Tween-20 mixture, heated at 60 ℃ for 1 hour), or longer, if necessary to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄N/Olive oil mixture any undissolved M₄N, and the presence of particles was checked against a black background. Last M₄The N/sesame oil/Tween _20 mixture was stored at room temperature and protected from light. If crystals form during storage, M₄The N/sesame oil/Tween _20 mixture can be heated again at 60 ℃ with stirring on a hot magnetic disk until all M is present₄The N redissolved back into solution.

The method can be scaled up or down to obtain the desired M₄N volume or concentration, and the heating temperature may be increased or decreased to reach M₄And (4) dissolving N. Can be similarly prepared to contain M in other water-insoluble lipid-bound nonionic surfactants, ionic surfactants or water-soluble organic solvents₄N, for example, by replacing sesame oil or Tween _20 with corn oil, olive oil, soybean oil, peppermint oil, Tween _80, TPGS, lecithin, PEG300, PEG400 monolaurate, glycerol, PVP, PG, or other solubilizing agents or combinations thereof in the foregoing methods. M₄The results of the solubility of N in the water-insoluble lipid are shown in table 16.

M can be prepared analogously to the presence of other water-insoluble lipids in combination with nonionic, ionic or amphoteric surfactants or water-soluble organic solvents₄N, for example, in the foregoing methods, by replacing sesame oil with corn oil, olive oil, soybean oil, peppermint oil, or mineral oil, and replacing Tween _20 with Tween _80, TPGS, lecithin, PEG300, PEG400 monolaurate, glycerol, PVP, PG, or other solubilizing agents or combinations thereof. M₄The results of the solubility of N in the water-insoluble lipid and the stability of the composition are shown in table 16. Stability for a clear liquid solution herein refers to the time it takes to form a crystalline precipitate in solution. The stable solution was clear over a long period of time without particles.

Table 16 shows, for example, that M when heated at 60 deg.C₄N is soluble in corn oil at concentrations up to 100mg/mL, and, except at 100mg/mL, after cooling, M₄N is kept in solution at low concentrations, at 1 and 10mg/mLStability at concentration greater than 3 days, stability at levels of 20, 40, and 50mg/mL less than 3 days, and at 60mg/mL less than 1 day. In addition, M₄N is soluble in olive oil at a level of 30mg/mL at 60 ℃, but does not remain in solution after cooling.

In sesame oil, M₄N was soluble at a level of 10mg/mL at room temperature. At 60 ℃ M₄N concentrations up to 50mg/mL were soluble and remained in solution after cooling. The 10mg/mL and 20mg/mL solutions were more than 3 days stable at room temperature, and the 50mg/mL solution was less than 1 day stable at room temperature.

In peppermint oil, M₄N is soluble between 1mg/mL and 125 mg/mL. At a concentration of up to 20mg/mL, M₄N is soluble without heating. These compositions have a stability at room temperature of more than 3 days. M₄N is soluble at higher concentrations up to levels of 125mg/mL, heated at 40 ℃, and remains in solution after cooling. These M₄N higher concentration in peppermint oil, 40mg/mL of the composition had stability at room temperature for more than 3 days.

M₄N was also dissolved in soybean oil at concentrations up to 50mg/mL when heated at 60 ℃. Therein, only a 10mg/mL concentration remains in solution upon cooling, which remains stable for more than 7 days.

When heated at 60 ℃, M₄N is also soluble in mineral oil at concentrations up to 200 mg/mL. The 10mg/mL and 50mg/mL compositions remained in solution upon cooling. Therein, a 10mg/mL solution is stable at room temperature for more than 7 days.

In a combination of 50% peppermint oil and 50% PEG300, M when heated at 35 deg.C₄N is soluble at concentrations up to 125 mg/mL. When heated at 35 ℃ at a level between 40mg/mL and 60mg/mL, M₄N remains in solution after cooling and is stable at room temperature for more than 7 days. In a combination of 60% peppermint oil and 40% PEG300, M when heated at 40 deg.C₄N was soluble at 60mg/mL and was also kept in solution upon cooling. The composition is prepared byStability at room temperature was over 3 days.

When peppermint oil and PEG400 are 50% bound to each other, M is when heated to 40 deg.C₄N may be dissolved to 125 mg/mL. At M₄At concentrations of N between 40mg/mL and 60mg/mL, the compound remained in solution upon cooling, and the composition was stable at room temperature for more than 7 days. Heating at 40 deg.C in a combination of 60% peppermint oil and 40% PEG400, M₄N may be dissolved at 60 mg/mL.

Heating in 50% peppermint oil and 50% Tween-20 at 40 deg.C, M₄N was soluble up to 125mg/mL as detected. In which M is₄N was maintained at a concentration of 40mg/mL and 60mg/mL in the cooled solution.

In another combination, for example, peppermint oil and sesame oil are each combined at 50%, M₄N was soluble at concentrations up to 60mg/mL as measured. M₄N is soluble at 20mg/mL at room temperature and the composition is stable at room temperature for more than 3 days. M₄Solutions of N at 40mg/mL and 60mg/mL were kept in solution by cooling and were stable for more than 3 days at room temperature.

In another combination containing 33% peppermint oil, 33% Tween _20, and 33% PEG400, M was heated at 40 deg.C₄N was soluble at 60mg/mL and remained in solution upon cooling. The stability of these compositions was over 3 days.

Table 16 also shows, for example, M₄N is soluble in soybean oil and, when combined with beeswax, can be prepared into waxy solids. In addition, M₄N may be dissolved in olive oil and beeswax may be added to convert the composition to a waxy solid.

TABLE 16.M₄Solubility of N in Water-insoluble lipids

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

Table 17 shows M₄Solubility of N in water-soluble organic solvents, ethanol, PG, PEG300, PEG400 monolaurate, glycerol, PVP, and certain combinations thereof.

TABLE 17.M₄Solubility of N in Water-soluble organic solvent

[0240]

[0241]

Example 6.M₄Solubility of N in nonionic surfactant amounts

A10 mL solution of 20% TPGS (w/v) used as solubilizer and/or excipient was prepared as follows: 8mL of WFI was placed in a glass beaker containing a stir bar. The beaker was placed on a hot magnetic plate and a stir bar was set to stir at a moderate speed. The WFI was heated at about 95 deg.C for 5 minutes. Add 2 grams of TPGS to another glass beaker and heat at about 40 ℃ for 15 minutes, or until all TPGS melts. The melted TPGS was slowly added to the near boiling WFI and was guided to the center of the beaker using a spatula to prevent any TPGS from sticking to the beaker walls. The 20% TPGS solution was stirred for 24 hours or until complete dissolution of the TPGS was observed. The final solution was stored at room temperature and protected from light.

The process for making TPGS can be scaled up or down to obtain a desired volume or concentration of TPGS solution. Other TPGS solutions can be similarly prepared in combination with other nonionic surfactants, ionic surfactants, amphoteric surfactants, water-soluble organic solvents, or other solubilizing agents in the foregoing processes. The results are shown in Table 18.

A10 mL solution of M4N in Tween _20 at a concentration of about 60mg/mL was prepared as follows. About 10mL of Tween _20 was slowly added to the glass beaker containing the stir bar. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. With the aid of a spatula, about 600mg of M are added₄N was slowly added to Tween-20 in the center of the beakerTo prevent M₄N adheres to the beaker wall. M₄The N/Tween _20 mixture was stirred for about 2 hours, or until all M was present₄N dissolved, or suspended homogeneously, with no lumps present. M₄The N/Tween _20 mixture was heated at about 60 ℃ for about 30 minutes. (or longer, if a larger volume of solution is desired, e.g., 500mL M₄N/Tween-20 mixture, heated at 60 ℃ for 1 hour), or longer, if necessary to ensure M₄N was completely dissolved. Observe the beaker against a white background M₄N/Tween-20 mixture any undissolved M₄N, and the presence of particles was checked against a black background. Last M₄The N/Tween _20 mixture was stored at room temperature and protected from light. If crystals are formed, M₄The N/Tween-20 mixture may be heated again at 60 ℃ for about 1 hour, with stirring on a hot magnetic plate, or until all M is present₄The N redissolved back into solution.

The method can be scaled up or down to obtain the desired volume or concentration of M₄N, the heating temperature can be increased or decreased to obtain M₄And (4) dissolving N. M can be similarly prepared containing moieties in other nonionic, ionic or amphoteric surfactants₄N, for example, by replacing Tween _20 with Tween _80, other solubilizing agents, or a combination thereof in the foregoing methods. M₄The results of the solubility of N in Tween _20, Tween _80, and the conjugate of Tween _20 and PEG400 are shown in table 18.

Table 18 shows M₄N is soluble in Tween-20 or Tween-80 at room temperature at a concentration of 1 mg/mL. M₄Solutions of N in Tween-20 ("M)₄N/Tween-20') over 7 days at room temperature, with M₄N in Tween-80 solution ("M)₄N/Tween _80 ") over 3 days of observation. Higher temperature M₄N is soluble at 50 ℃ in Tween-20 or Tween-80. Furthermore, when the concentration of Tween _20 reached 60mg/mL and that of Tween _80 reached 50mg/mL, after cooling, M was added₄N remains in solution while, in Tween-20, at 80mg/mL and 100mg/mLAt the concentration level, upon cooling, M₄N becomes insoluble. M of 10mg/mL and 20mg/mL was observed₄The stability of the N/Tween _20 solution at room temperature was over 7 days. 40mg/mL and 80mg/mL M were observed₄The stability of the N/Tween _20 solution at room temperature is less than 3 days. For M₄N/Tween-80 solution, stability of 10mg/mL solution at room temperature was observed to be longer than 3 days, while stability of 50mg/mL solution at room temperature was observed to be shorter than 1 day.

The results also show M₄N in the conjugate of 50% Tween-20 and 50% PEG400 as M₄N reached the concentration of 60mg/mL as detected and was soluble upon heating at 65 ℃. Upon cooling, M₄N is maintained in solution in these formulations, which is stable at room temperature for more than 3 days.

The conjugates of PEG400 and Tween _20 were tested at different heating temperatures, increased heating times, methods and times of cooling, and increased concentrations of the drug.

Prepare 10mL of solution: first, 10mL of glycerol monooleate ("Glymo") was added to the glass beaker. The beaker was placed on a magnetic disk and a stir bar was set to stir at a moderate speed. Heat to 60 ℃ for 30 minutes to dissolve all the drug. The beaker was observed against a white background for any undissolved M in the mixture₄N, and the presence of particles was checked against a black background. The Glymo mixture was cooled to room temperature with heating and the mixture became a suspension. It was stored at room temperature and protected from light. The suspension remained stable for two weeks, after which time separation began. Shaking may cause it to recombine.

TABLE 18.M₄Solubility of N in nonionic surfactants

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

Example 7

The melting temperatures of 47.5% (v/v) Tween-20, 47.5% (Wv) PEG400, 2.5% (w/v) PEG 3350, 2.5% (w/v) beeswax from example 6 (see Table 18) were varied by several M₄And (3) determining the N concentration:

example 8M contained in HP-beta-CD₄Lyophilization of formulations of N

120mg M in HP-beta-CD at a concentration of about 185mg/g (w/w)₄Lyophilized powder of N was prepared as follows. Equimolar amounts of HP-beta-CD and M₄N was used to increase HP-beta-CD and M₄Complexation ratio between N. About 98mg of HP- β -CD and about 22.2mg of M in a 1.5mL size polypropylene tube₄N are mixed together. Adding about 0.2ml of LWFI to the solution containing HP-beta-CD/M₄N powder mixture in polypropylene tube and vortexed for 1 minute to generate HP-beta-CD/M₄A suspension of N in water. HP-beta-CD/M₄The N suspension was frozen at-20 ℃ for 24 hours. Postero HP-beta-CD/M₄The N suspension was centrifuged at 1,400rpm for about 2 hours at 60 ℃ under vacuum to remove all water from the suspension. HP-beta-CD/M₄The dry powder of the N blend weighed about 120 mg. HP-beta-CD/M₄The N powder blend may then be solubilized or resuspended in water or other solubilizing agent. Most preferablyFinal M₄The N/HP-beta-CD powder mixture was stored at room temperature and protected from light. The method can be scaled up or down to obtain the desired M₄N volume or concentration. Can be similarly prepared to contain M₄Formulations of N with other cyclodextrins, for example, by replacing HP- β -CD in the foregoing methods with other cyclodextrins. The results shown in Table 1 describe a composition of about 81.5% HP-beta-CD and 18.5% M₄HP-beta-CD/M of N composition₄The suspension of N is lyophilized to obtain HP-beta-CD/M₄And (4) mixing the N powder.

400mg M at a concentration of about 150mg/g (w/w) in HP-beta-CD/HP₄Lyophilized powder of N was prepared as follows. Approximately 1mL of 50% HP- β -CD/0.5% HPMC solution prepared as described above was added to a 1.5mL size polypropylene tube. About 60mg M₄N was added to polypropylene tubes containing HP-beta-CD/HPMC suspension and vortexed for 1 minute to generate HP-beta-CD/HPMC/M₄And (4) N suspending liquid. HP-beta-CD/HPMC/M₄The N suspension was frozen at-20 ℃ for 24 hours. Then HP-beta-CD/HPMC/M₄The N suspension was centrifuged at 1,400rpm for about 5 hours at 60 ℃ under vacuum to remove all water from the suspension. HP-beta-CD/HPMC/M₄The dry powder weight of the N blend was about 400 mg. HP-beta-CD/HPMC/M₄The N powder blend may then be redissolved or resuspended in water or other solubilizing agent. The final M4N/HP- β -CD/HPMC powder mix was stored at room temperature and protected from light. The method can be scaled up or down to obtain the desired M₄N volume or concentration. Can be similarly prepared to contain M₄Formulations of N with other cyclodextrins, for example, by replacing HP- β -CD in the foregoing process with other cyclodextrins, or replacing HPMC with other modified celluloses. The results shown in Table 15 describe a composition of approximately 84% HP- β -CD, 1% HPMC and 15% M₄HP-beta-CD/HPMC/M composed of N₄The suspension of N is lyophilized to obtain HP-beta-CD/HPMC/M₄And (4) mixing the N powder.

Example 9 oral administration of liquid formulations₄N uptake in Spela-dawn rats

10 groups of male rats (5 per group) (age 8-10 weeks) were given oral gavageAt a single dose of 500mg/kg of M in the vehicle₄N to define the preferred oral liquid excipient. Animals were fasted overnight prior to dosing. As shown in table 19, using the formulations prepared as described previously, the excipients/formulations tested were: (a) HP-beta-CD + HPMC; (b) HP-beta-CD + CMC; (c) TPGS; (d) TPGS + PEG 400; (e) tween _ 20; (f) PEG400 + Tween — 20; (g) PEG400 + Tween _20+ peppermint oil; (h) peppermint oil + PEG 400; (i) peppermint oil + Tween _ 20; (j) peppermint oil + sesame oil. Blood was collected from the jugular vein of each animal at the following time points: before administration, 0.5, 1, 2 and 3 hours after administration. M₄The concentration of N in serum was determined by LC/MS, LC means liquid chromatography and MS means mass spectrometry. The method separates M by LC₄N, after which analysis was performed with two consecutive rounds of MS detection and quantification.

As shown in FIG. 3 and Table 20, M₄The absorption of N varies between excipient formulations. PEG400 + Tween _20 absorbed most, which was higher than PEG400 + Tween _20+ peppermint oil, which was higher than peppermint oil + Tween _20, which was higher than peppermint oil + PEG400, which was higher than peppermint oil + sesame oil, which was higher than or equal to Tween _20, which was higher than or equal to HP- β -CD + CMC, which was higher than HP- β -CD + HPMC, which was higher than TPGS + PEG 400.

In table 20, 4 formulations are shown as stable suspensions: HP-beta-CD + HPMC, HP-beta-CD + CMC, TPGS, and TPGS + PEG 400. The criterion for determining that the suspension is "stable" is that the contents of the suspension do not exhibit precipitation (components do not precipitate from the suspension to the bottom of the beaker). Other formulations in the rat study were clear liquid solutions, not suspensions, and without any floating ingredients.

TABLE 19 absorption study of oral formulations

[0276]

[0277]

TABLE 20.M₄N is administered orally to rats^aIn (1) absorption

^an-5 males/group

^bValues are expressed as mean. + -. standard error

Example 10.M₄Absorption of N orally administered in beagle dogs

Groups 5 dogs (n ═ 2 per group, 1 male, 1 female) (age ═ about 6-9 months) were given a single dose of 100mg/kg of M in vehicle₄N to define the preferred oral solid excipient. Before oral administration, M₄N composition in vehicle was encapsulated as a size 12 hard gel capsule. Animals were fasted prior to dosing. The excipients/formulations tested are listed in Table 19 and are (a) excipient free, where M is₄N is filled into a bag; (b) HP-beta-CD; (c) TPGS; (d) soybean oil + beeswax; and (e) olive oil + beeswax. Blood was collected from the jugular vein of each animal at the following time points: before administration, 0.5 hour, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours and 8 hours after administration. M₄The concentration of N in serum was determined by liquid chromatography coupled with tandem mass spectrometry (LC/MS).

As shown in FIG. 4, M₄The absorption of N varies depending on the excipient/formulation. The formulations containing olive oil + beeswax had the highest absorption, which was higher than the formulations containing HP-beta-CDFormulation with TPGS, which is higher than formulation with soybean oil + beeswax, which is higher than M without excipients₄N。

Although the foregoing embodiments incorporate M₄N is illustrated, but the weight and volume of other NDGA derivatives or other catecholic butanes are suitably adjusted to achieve the appropriate concentration.

Example 11 samples were taken to determine micronized and non-micronized M₄Pharmacokinetics of N after oral administration to dogs.

The objective of this study was to evaluate the oral administration of M to beagle dogs when using micronized and non-micronized forms via gavage₄Pharmacokinetic profile of N. The items tested were prepared in glycerol monooleate at a concentration of 60 mg/mL. The study design is summarized in table 21.

TABLE 21 non-micronized and micronized M measured in dogs₄Study design of N

M Male sex

F female

Note that: there was an elution period of about 7 days between the phases.

Jugular blood (approximately 2mL) was collected into Monoject 2mL red push tubes without anticoagulants before administration, and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 36 hours after administration.

After the two agents are applied, M appears₄And (4) absorbing N. The mean pharmacokinetic parameters are listed in Table 22, where C_maxIs M₄The maximum concentration of N absorption, and AUC represents the area under the curve, vs. M₄The N total absorption is relevant. With non-micronised M₄N phase ratio, micronized M₄N has a higher absorption. However, in the case of non-micronized M₄After N dosing, the concentration curves tended to be more consistent (fig. 5A and 5B, non-logarithmic and logarithmic scale, each showing non-micronized M₄Absorbing N; and, FIGS. 6A and 6B, non-logarithmic and logarithmic scale, each showing micronized M₄And (4) absorbing N.

TABLE 22 the following M₄Mean pharmacokinetic parameters of N administration

Example 11 two species M₄Comparison of pharmacokinetics of oral dosing/intravenous dosing/oral dosing of N formulations in beagle dogs

The aim of this study was to evaluate the administration of M to be pre-prepared orally (by gavage or capsule) in fed or fasted conditions, or intravenously₄N serum levels achieved after administration of 75mg/kg in a single dose. 3 dogs/gender were given EM-1421 in Glycerol monooleate ("Glymo"), M in Tween-20/PEG 400/Naringin ("TPN")₄N, or M in 20% hydroxypropyl-beta-cyclodextrin (HP. beta. CD), 50% PEG300 ("CPE")₄And N is added. Glymo and TPN were also administered as non-concentrated (60mg/mL) or concentrated (300mg/g, w/w) as shown in the legends in FIGS. 7B and 8B.

All animals survived the entire study. Clinical observations included diarrhea, vomiting, and ataxia (ataxia appeared and persisted for about 1 hour after intravenous administration in 1 female dog).

M₄Serum levels of N vary greatly depending on formulation and condition. Table 23 summarizes these findings. Serum levels were lowest after concentrated Glymo or TPN administration. After administration of TPN (non-concentrated, fasted conditions), C_maxHighest (Table 23 and FIGS. 7A and 8A, non-log scale showing oral administration of male and female dogs, respectivelyAdministration of M₄Serum levels after N). AUCs were highest after Glymo administration (non-condensed, fasted condition) (Table 23 and FIGS. 7B and 8B, logarithmic scale showing oral administration of M to male and female dogs, respectively₄Serum levels after N). After oral administration of Glymo (non-condensed, fasted condition), the AUCs reached 35% (male) and 47% (female) of the intravenously administered AUCs, assuming 100% (table 23).

TABLE 23 formulated M₄Pharmacokinetics of N in dogs following single dose administration of 75mg/kg

[0303]

Example 12.M₄N pharmacokinetic Studies on oral (gavage) repeat dosing in rats

The aim of this study was to provide M in 10 different excipient formulations₄Pharmacokinetics of 500mg/kg of N administered. 50 Crl: (CD) SD male and female rats were divided into 10 dose groups of 5 rats per sex. Samples with the following carriers were prepared for administration to subjects of the following groups.

[0307]

Each M₄The N preparation is taken orally in 3 cases by strong feeding methodThe medicine is taken orally. M₄The concentration of N was 60mg/mL, and the administration was carried out at a dose volume of 8.33mL/kg depending on the most recent body weight. After a 72 hour elution (recovery) period, overnight fasting, rats were dosed for the first time (day of study). On study day 5, non-fasted rats that passed the 1 week elution (recovery) period were dosed 2 of the test item. On day 6, rats were given a high fat meal (about 10% fat content, whereas the standard meal was about 5%). On day 12, non-fasted mice were dosed a third time. The viability of the rats was observed at least twice daily and clinical observations and general characterization were made weekly during the acclimation period. Clinical observations and deaths of rats were examined prior to dosing, and at approximately hourly intervals during the first 4 hours after dosing, as well as at the end of the normal working day on the first day of dosing, and approximately 2 hours during the days following dosing. These observations were recorded once daily during the non-dosed days and during the days after dosing. Body weight was recorded at least weekly during the acclimation period, at least daily during the dosing period, and weighed at the final sacrifice. Feed consumption values (feed remaining values) were recorded during dosing and at sacrifice. Blood samples (approximately 0.5mL each) were collected from the caudal vein of each rat studied on days 1,5, and 12 of the study. The time of each blood collection was recorded in the original record. At the following time points on study days 1,5 and 12: blood samples were collected from each rat prior to dosing, 15 minutes after dosing, 1 and 4 hours after dosing. The specimen was transferred to a serum separation tube and spun down for centrifugation. The resulting sera were transferred to polypropylene tubes labeled with experimental number, sponsored study number, rat number, group number, medication level, study days, collection interval, collection date, seed name, generation number, and storage conditions. All samples were immediately frozen on dry ice and kept frozen (-68 ℃ -78 ℃) until removed for analysis.

All rats were alive at the scheduled time of sacrifice. Urine contaminating abdominal fur was present in 4 of 5 female rats dosed with PEG400/Tween _ 20/peppermint oil (group IX). This indication appears on days 2-3 and does not persist. In addition to PEG400/Tween _ 20/peppermint oil (group IX) rats, only soft rats appeared in at least some of the rats in each groupOr clinical observation of liquid feces. All groups had an average weight gain in the study. The weight gain of female and male rats fed the high fat diet on days 6 to 13 was always lower than that of the standard diet on days 1 to 6. The weight gain was substantially comparable in each group, except that female rats in the groups of PEG400/Tween _20 and PEG400/Tween _20/PEG 3350/beeswax (groups I and X, respectively) had a decrease in weight gain on days 6 to 13. The feed consumption values of each group were both absolute and relative equivalent throughout the study. No gross weight loss was observed in cadaver examinations and there was no correlation between the project reagents tested. M occurred from all vectors₄And (4) absorbing N. After fasting, the blood levels in rats on the high fat diet were always higher than those achieved in rats on the standard diet. The highest absorption occurred after the PEG400/Tween _ 20/naringin group on the high fat diet. The highest absorption occurred after the PEG400/Tween _ 20/naringin group was used on the standard diet. The highest absorption occurred after fasting with PEG400/Tween _ 20/peppermint oil. The highest exposure level was generally reached within one hour after dosing, which showed a continued rise 4 hours after dosing, except for the glycerol monooleate formulation.

In summary, all tested project preparations had no mortality and all groups gained weight. The usual clinical observation is soft and liquid stools, but there is no effect on the feed consumption that occurs with any vehicle. On a high fat diet in rats, M₄Blood levels of N are always higher. With the exception of the glycerol monooleate formulation, a peak exposure occurred within 1 hour after dosing.

Example 13 of 8 healthy male subjects, in stage 0 of the human,¹⁴c-labelled M₄Triple crossover microdose pharmacokinetic study of N

The study was designed to evaluate M in humans at sub-therapeutic doses, either as oral doses alone or as intravenous doses alone (formulas A-C, respectively, in Table 24) under fed or fasted conditions₄And (4) absorbing N. The study was conducted in a target population of healthy male subjectsAnd consists of 3 study phases of about 35 hours duration, each phase being separated by a minimum period of at least 7 days between administrations. At each stage of the study period, pharmacokinetic blood samples were collected at specific time points after drug administration, and urine was collected at preset time intervals. The subject may leave the clinical unit 24 hours after drug administration after completion of the study specific procedure.

In this study, M₄N was administered to humans in an amount of 100. mu.g. M₄N is slightly used¹⁴C (3.3 kBq per 100. mu.g) and administered to healthy volunteers. Each M₄N is administered orally from 0.1mg in size 0 gelatin capsules¹⁴C-labelled M₄N and 376.8mg of glycerol monooleate. M₄N intravenous infusion alone was diluted with water to 1mL of formulation for intravenous injection from 0.1mg/mL¹⁴C-labelled M₄N, 30% (w/v) HP- β -CD, and 25% (v/v) PEG. After blood collection from each subject, samples were analyzed using Accelerator Mass Spectrometry (AMS)¹⁴C content was analyzed to determine at T_maxTime M₄Maximum concentration of N (C)_max) And time-to-detection (AUC)_o-t) And total (AUC)_0-∞) M of (A)₄Total area under the curve (AUC) related to total absorption of N, final half-life of each specimen, and₄comparison of N intravenous administration, M₄Relative and Total bioavailability of N oral administration (F)_relAnd F). Table 24 shows¹⁴Median of pharmacokinetic parameters for C ± SD. After the period between doses, any residual M remaining in the subject₄N to M₄Correction of baseline level of N to not elevate any subsequent doses of M₄N level.

Table 24.¹⁴Median ± SD values (corrected baseline) of pharmacokinetic parameters of C

^aMedian value

The formula A is as follows: 100 μ g administered in a single oral dose after a high fat breakfast¹⁴C-labelled M₄N(3.3 kBq)。

And the formula B is as follows: 100 μ g administered in separate oral doses after overnight fasting¹⁴C-labelled M₄N(3.3kBq)

And a formula C: 100 μ g administered as 1mL intravenous solution after overnight fasting¹⁴C-labelled M₄N(3.3kBq)。

For oral administration, 100 μ g is given after a high fat breakfast¹⁴C-labelled M₄After N administration, the total¹⁴C of C_maxValue (C)_max5.94 ± 1.33pmol/L) was lower than oral administration after fasting overnight (C)_max9.29 ± 1.40 pmol/L). In a fed subject T_maxThe appearance time tends to be later than in fasted subjects. In a subject who is fed C_maxTime of occurrence T_maxHighly variable, ranging from 1.5 to 24 hours after administration. In fasted subjects, T_maxTypically occurring 1 hour after administration (range of 0.50 to 2.00 hours). AUC in subjects with food_0-tThe values are usually lower than in fasted subjects.

Maximum concentration (C) after intravenous administration in 8 of 10 subjects_max10.31 ± 1.77pmol/L) as expected, occurred at the first sampling time (0.08 hour post-dose). In two subjects, T_maxIs 0.17 hour after administration. In fed subjects, plasma total after oral administration alone¹⁴AUC of C concentration_0-tThe values were slightly lower than AUC after intravenous administration_0-tThe value is obtained. In contrast, in fasted subjects, the corresponding AUC after oral administration alone_0-tThe values were slightly higher than AUC after intravenous administration_0-tThe value is obtained.

The formulations of this study are well suited for oral and intravenous administration. There were no serious or acute side effects, nor were there discontinuations of study treatments associated with side effects. No clinical vital signs or significant changes in the electrocardiogram were found.

In summary, for this study, M was administered orally in both fed and fasted states after administration₄N is absorbed very significantly. In the presence of food, the absorption rate and degree are lower than in the fasted state, and in the fed state C_maxThe time of occurrence is prolonged. These conclusions are based on the assumption that prior to absorption,¹⁴c-labelled M₄N does not degrade upon oral administration.

Example 14.M₄Additional study of solubility of N in Water-soluble organic solvent

Evaluation M as described in Table 25₄The solubility of N in water-soluble organic solvent combinations is as long as 48 hours. After 2, 24 and 48 hours of incubation at room temperature, the samples were analyzed by reverse phase-HPLC ("RP-HPLC") to quantify M₄Solubility of N. To prepare M₄N sample, 200. mu.L of 100mg/mL M dissolved in acetone₄N was placed in 1.5mL polypropylene microtubes. The solvent was allowed to evaporate at room temperature for 48 hours until the sample was completely dried.

The water soluble organic solvent formulation was prepared in a 15mL polypropylene centrifuge tube. 10mL of each formulation was prepared. Each solvent added was based on the weight of the respective solvent at a density of 25 ℃. After brief mixing, each formulation was filtered through 0.45 μm surfactant-free cellulose acetate ("SFCA") into a fresh 15mL tube. The preparation is stored at room temperature for later use.

400 μ L of each solvent combination (table 25, where Benz ═ benzyl alcohol; Crem ═ Cremophor _ EL; DMA ═ dimethylacetamide; T80 ═ Tween _80) were added to the microtubes to give M₄The maximum solubility of N is 50 mg/Ml. Evaluation of M by RP-HPLC after 2, 24 and 48 hours incubation at RT₄Solubility of N. At each time point, the samples were centrifuged at 13,000rpm for 2 minutes to pellet any solidsM of (A)₄And N is added. As indicated in Table 25, more than half of the conditions of the tested formulations dissolved M₄N is greater than 10 mg/Ml. Can not detect M₄Solubility of N in glycerol at 2 and 48 hours.

TABLE 25.M₄Solubility of N in water-soluble organic solvents for up to 48 hours

[0330]

[0331] Example 15.M₄Solubility of N in hydration solution

Evaluation M was made as in the foregoing example 14₄Solubility of N in a hydration solution containing hydroxypropyl HP-beta-CD or sulfobutyl ether beta-cyclodextrin (SE-beta-CD) (Captisol, CyDex, Inc., Lenexa, Kansas, USA). Based on the weight volume ratio to prepare 10 HP-beta-CD and SE-beta-CD 50% solution. Preparation of M to be used in the samples, as described in example 14₄And N is added. Between 1.0g and 5.0g of either compound was weighed into a 10mL volumetric flask on an OHAUS Analytical Plus Balance. Each sample was accompanied by an appropriate amount to 10mL of water for injection (WFI). After incubation for 1 hour at 40 ℃, the formulation was filtered through 0.45 μm SFCA into fresh 15mL tubes. The preparation is stored at room temperature for later use.

As shown in Table 26, M₄The solubility of N in WFI, 0.9% saline, 5% dextrose (D5W) was below the detection limit for RP-HPLC throughout the study. For M as a function of concentration and time of HP-beta-CD and Captisol-₄The increased solubility of N was recorded.

TABLE 26.M₄N in hydration solution for up to 48 hoursSolubility in water

[0336]

More than 20 kinds of preparations using water-soluble organic solvent can dissolve M₄N to a concentration greater than 10 mg/mL. M in WFI, 0.9% brine, D5W₄The solubility of N is below the detection limit of the RP-HPLC method. M₄The solubility of N increases as a function of HP- β -CD and Captisol _ concentration and time.

Example 16.M₄Solubility of N in hydroxypropyl beta-cyclodextrin

M was evaluated at different HP-beta-CD concentrations according to the method reported by Higuchi and Connors (1965)₄Hydration solubility of N. In short, M is precisely weighed₄N, and added in an amount exceeding its hydration solubility, and gently swirled (. about.12 rpm) with increasing concentrations of HP- β -CD hydration solution (0-350mmol/L) at room temperature for 48 hours. Then, M₄The N/HP-beta-CD solution was filtered through 0.45 μm SFCA and analyzed by RP-HPLC.

Although most drug/cyclodextrin complexes are considered inclusion complexes, and complex aggregation is capable of solubilizing the drug in a micelle-like structure. The phase-solubility curve does not confirm the formation of inclusion complexes, but only how increasing concentrations of cyclodextrin affect the solubility of the drug. M₄The formation of the N/HP- β -CD complex is non-linear, but the precise determination of stoichiometry (and stability constants) has not been investigated in the experiments of this example, but can be determined by other methods such as nuclear magnetic resonance or potentiometry.

Example 17.M₄Stability of N in HP-beta-CD/PEG 300 buffer solution

After incubation at 60 ℃, 10mg/mL M in 15mM buffer was evaluated₄N(40％HP-β-CD：40mg/mL M₄N PEG300 formulated at a ratio of 75: 25).

A40% solution of buffered HP- β -CD was prepared according to weight to volume ratio. Preparation of M to be used in the samples, as described in example 14₄And N is added. 2.0g of HP- β -CD was weighed into a 5mL volumetric flask on an OHAUS Analytical PlusBalance balance. 1mL of 100mM buffer was added to each beaker. Each sample was accompanied by an appropriate amount to 5mL of WFI. After incubation for 1 hour at 40 ℃, the formulation was filtered through 0.45 μm SFCA into fresh 15mL tubes. The preparation is stored at room temperature for later use.

750 μ L of 40% HP- β -CD buffer was placed in a 1.5mL polypropylene microtube. 250 μ L of 40mg/mL M₄N PEG300 was added to each microtube to have a M of 10mg/mL₄Solubility of N. After gentle inversion of the sample tubes, the pH of each solution was measured using an Orion, Model 420ApH meter. The original aliquot was removed for RP-HPLC analysis. The test sample was then placed in a Precision 60 ℃ incubator. Evaluation of M by RP-HPLC₄Stability of N. At each time point, the samples were centrifuged at 13,000rpm for 2 minutes to remove any solid M₄N。

As shown in Table 27, after 14 days of incubation at 60 ℃ different M's were observed₄The concentration of the N solution decreased slightly. RP-HPLC data did not show an increase in sample impurity levels, which could indicate M₄The N concentration is greatly reduced. However, the degree of impurities in the sample was observed to vary depending on the pH, but these degrees of impurities accounted for less than 0.1% of the total peak area. During the incubation period, little, if any, change in pH of the samples was observed (table 27).

TABLE 27.M₄Stability of N in HP-beta-CD/PEG solution at 60 deg.C for up to 14 days

[0348] After 14 days of incubation at 60 ℃ M was not considered₄A drop in stability was observed at the apparent sample pH or buffer indicated by the loss of N in recovery.

Example 18.M₄Stability of N in 11-14mg/mL PEG 300/HP-beta-CD solution.

To maintain production specifications, this study examined PEG 300/HP-beta-CD conjugate in M₄Stability at room temperature for 24 hours when the target concentration of N was changed. M was prepared in 100% PEG300 at a drug concentration of 33-56mg/mL at 60 ℃ as follows₄Stock samples of N feedstock.

After at least 2 hours of incubation at 60 ℃, M was incubated using the methods set forth in examples 14 and 17₄The N block drug was dissolved in PEG300 to 33, 44, 48, 52, 55 and 56mg/mL (w/w). Intense swirling and mixing is required to ensure M₄The dissolution of N was higher than 44 mg/mL. M₄The N stock solution was filtered through 0.45 μm SFCA and used within 30 minutes of preparation. Separately, a solution of 40% (w/v) HP- β -CD was prepared in sterile WFI and filtered. 40% HP-beta-CD stock solution and M₄The conjugate of N/PEG300 stock solution was conjugated in 1.5mL polypropylene microtubes. Evaluation of M at 2 and 24 hours of incubation at Room temperature₄Solubility of N.

Will M₄The required amount of N stock solution was added to 40% HP- β -CD to produce the final concentrations of drugs and excipients listed in table 28. The sample was rotated slightly at room temperature (-12 rpm). At 2 nd and 24 th hours of incubation, the samples were centrifuged at 13,000rpm for 2 minutes and 50 μ L aliquots were removed for RP-HPLC analysis. Irrespective of the target M₄N or formulation, little if any change was observed after 24 hours incubation (table 28).

TABLE 28M₄Stability of N in 11-14mg/mL PEG 300/HP-beta-CD solution

[0355]

After 24 hours incubation at room temperature, M was prepared to 25% PEG300 and 30% HP-beta-CD to a final concentration of between 11-14mg/mL₄The test sample of N was stable.

Example 19.40mg/mL M₄Stability of N/PEG300

Incubation at 30 deg.C, 45 deg.C and 60 deg.C to evaluate 40mg/mLM solubilized in 100% PEG300₄Stability of N up to 24 hours. Preparation of 40mg/mL M in PEG300 at 60 ℃ according to the procedure set forth in example 18₄And N storage solution. Thereafter, an aliquot is removed and incubated at an appropriate temperature. Throughout the incubation, the sample was spun at 450 rpm. Data for eye observations and RP-HPLC were collected throughout. As shown in Table 29, after 6 hours of incubation at 30 ℃ M was at 40mg/mL₄Micro-crystallization was observed in the N/PEG300 formulation, with more appearing after 24 hours. Formation of crystals and soluble M₄The loss of N is consistent (Table 30). 40mg/mL M incubated at 45 ℃ and 60 ℃ as assessed by ocular observation and RP-HPLC analysis₄The N/PEG300 samples were stable after 24 hours incubation (tables 29 and 30). No changes in the amount and type of impurity peaks were observed at any incubation temperature.

TABLE 29.40mg/mL M₄Visual appearance of N/PEG300 stability samples

TABLE 30.40mg/mL M₄RP-HPLC analysis of N/PEG300 stability samples

[0363]

After 6 hours of incubation at 30 ℃, micro-crystallization was observed in the 40mg/mL M4N/PEG300 formulation. After 24 hours, more crystallization was observed, and the dissolved M was determined according to RP-HPL analysis₄There is a loss of more than 5% of N.

According to eye observation and RP-HPLC analysis, the 40mg/mL M4N/PEG300 sample was stable after incubation at 45 ℃ and 60 ℃ for 24 hours.

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Reference to the literature

Ansel, H.C. et al, (2004). Pharmaceutical document Forms and drug delivery Systems eds, 8^thed. (pharmaceutical dosage forms and drug delivery systems, 8 th edition), Lippincott Williams and Wilkins.

Garg, s. et al, (2001) complex of Pharmaceutical Excipients for Pharmaceutical formulations.

Pharmaceutical technical drug Delivery sept.1, 2001, pp.14-24.

Gennaro，A.R.(2003).Remington：The Science and Practice of Pharmacy，20^thed. with features and Comparisons: DrugfactsPlus (Remington: science and practice of pharmacy, 20th edition, argumentation and comparison: pharmaceutical supplements).

Higuchi T and comparators KA (1965) "Phase solubility technologies", Adv anti-Chem Instr (analytical chemistry progress) 4, 117-212.Hwu, j.r. (1998) anti-viral activities of methyl dihydroguaiaretic acids.1.synthesis, structure identification, inhibition of Tat-regulated HIV activation j.med.chem (journal of medical chemistry) 41: 2994-3000.

McDonald, r.w. et al, (2001) Synthesis and anticancer activity of nordihydroguaiaretic acid (NDGA) and analogs (Synthesis and anticancer activity of nordihydroguaiaretic acid (NDGA) and analogs), Anti-Cancer Drug Design 16: 261-270.

Rowe, R.C. et al, eds. (2003) Handbook of Pharmaceutical excipients.4^thedition (handbook of Pharmaceutical excipients, 4 th edition.) Pharmaceutical Press and Pharmaceutical Association (Pharmaceutical Press and American society for pharmacy).

Claims (40)

1. A composition for oral administration to an animal comprising an active pharmaceutical ingredient and a pharmaceutically acceptable carrier, wherein the active pharmaceutical ingredient is tetra-O-methyl NDGA and the carrier comprises a cyclodextrin and at least one of a solubilizing agent and an excipient selected from the group consisting of: (a) a water-soluble organic solvent other than DMSO; (ii) no white petrolatum, no xanthan gum, no glycerin or no sugar gum if the water-soluble organic solvent is propylene glycol, polyethylene glycol being present in the absence of ascorbic acid or butylated hydroxytoluene if the water-soluble organic solvent is polyethylene glycol, and polyethylene glycol 400 being present in the absence of polyethylene glycol 8000 if the polyethylene glycol is polyethylene glycol 400; (b) ionic, nonionic or amphoteric molecular surfactants, if the surfactant is a nonionic surfactant, the nonionic surfactant is present in the absence of xanthan gum; (c) a modified cellulose; (d) a water-insoluble lipid, if the water-insoluble lipid is castor oil, the castor oil is present in the absence of beeswax or carnauba wax; and any combination of (a) - (d) vectors.

2. The composition of claim 1, wherein the composition comprises from 0.1mg to 200mg of the active pharmaceutical ingredient.

3. The composition of claim 2, wherein the composition comprises 10mg, 20mg, 25mg, 30mg, 40mg, 50mg, 60mg,75mg, 100mg or 200mg of the active pharmaceutical factor.

4. The composition of claim 1, wherein the active pharmaceutical ingredient is present at a concentration of 1mg/mL to 200mg/mL or 1mg/g to 250 mg/g.

5. The composition of claim 4, wherein the active pharmaceutical ingredient is present at a concentration of 1mg/mL, 2mg/mL, 2.5mg/mL, 5mg/mL, 10mg/mL, 12.5mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 75mg/mL, 100mg/mL, 125mg/mL, 150mg/mL or 175 mg/mL.

6. The composition of claim 4, wherein the active pharmaceutical ingredient is present at a concentration of 20mg/g, 50mg/g, 75mg/g, 100mg/g, 120mg/g, 130mg/g, 140mg/g, 150mg/g, 175mg/g or 200 mg/g.

7. The composition of claim 1, wherein the water-soluble organic solvent is selected from the group consisting of polypropylene glycol, polyethylene glycol, polyvinylpyrrolidone, ethanol, benzyl alcohol and dimethylacetamide.

8. The composition of claim 7, wherein the water-soluble organic solvent is polyethylene glycol.

9. The composition of claim 8, wherein the polyethylene glycol is present at a concentration of 5% (v/v) to 100% (v/v).

10. The composition of claim 9, wherein the polyethylene glycol is present at a concentration of 20% (v/v) to 80% (v/v).

11. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 50% (v/v).

12. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 40% (v/v).

13. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 33% (v/v).

14. The composition of claim 8, wherein the polyethylene glycol is PEG 300.

15. The composition of claim 14, wherein the PEG300 is present at a concentration of 10% (v/v), 20% (v/v), 30% (v/v),40% (v/v), or 50% (v/v).

16. The composition of claim 8, wherein the polyethylene glycol is PEG 400.

17. The composition of claim 16, wherein the PEG400 is present at a concentration of 10% (v/v), 20% (v/v), 30% (v/v),40% (v/v), or 50% (v/v).

18. The composition of claim 8, wherein the polyethylene glycol is PEG400 monolaurate.

19. The composition of claim 18, wherein the PEG400 monolaurate is present at a concentration of 20% (v/v) to 50% (v/v).

20. The composition of claim 1, wherein the cyclodextrin comprises an unmodified cyclodextrin or a modified cyclodextrin.

21. The composition of claim 20, wherein the modified cyclodextrin is hydroxypropyl- β -cyclodextrin or sulfobutyl ether- β -cyclodextrin.

22. The composition of claim 20, wherein the modified cyclodextrin is present at a concentration of 5% (w/v) to 80% (w/v).

23. The composition of claim 22, wherein the modified cyclodextrin is present at a concentration of 15% (w/v), 20% (w/v), 25% (w/v), 30% (w/v), 35% (w/v), 40% (w/v), or 50% (w/v).

24. The composition of claim 1, wherein the surfactant is selected from the group consisting of polysorbates, vitamin E polyethylene glycol 1000 succinates, esterified fatty acids, and reaction products of ethylene oxide and castor oil in a molar ratio of 35: 1.

25. The composition of claim 24, wherein the surfactant is present at a concentration of 5% (v/v) to 100% (v/v).

26. The composition of claim 25, wherein the surfactant is present at a concentration of 30% (v/v),40% (v/v), or 50% (v/v).

27. The composition of claim 24, wherein the surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80.

28. The composition of claim 27, wherein the surfactant is polysorbate 20.

29. The composition of claim 24, wherein the surfactant is an esterified fatty acid.

30. The composition of claim 1, wherein the modified cellulose is selected from the group consisting of ethyl cellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose.

31. The composition of claim 30, wherein the modified cellulose is present at a concentration of 0.1% (w/v) to 10% (w/v).

32. The composition of claim 1, wherein the water-insoluble lipid is selected from the group consisting of at least one of water-insoluble lipid oils, waxes, and fat emulsions.

33. The composition of claim 32, wherein the water-insoluble lipid is a fat emulsion.

34. The composition of claim 33, wherein the fat emulsion is present at a concentration of 10% to 30%.

35. The composition of claim 34, wherein the fat emulsion is present at a concentration of 20%.

36. The composition of claim 32, wherein the water-insoluble lipid is an oil.

37. The composition according to claim 36, wherein the oil is at least one selected from the group consisting of corn oil, olive oil, peppermint oil, soybean oil, sesame oil, mineral oil and glycerol.

38. The composition of claim 36, wherein the oil is present at a concentration of 10% to 100%.

39. The composition according to claim 37, wherein the oil is peppermint oil.

40. The composition of claim 39, further comprising sesame oil.