CN101150956B - Formulations for injection of catecholic butanes, including NDGA compounds, into animals - 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 tetra-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

Be used for being injected into the catecholic butanes prescription that comprises the NDGA chemical compound of animal

The cross reference of related application

The application has required to be filed in the U.S. Provisional Patent Application 60/647,495 on January 27th, 2005 and 60/647,648 priority, and described application is all incorporated into herein by reference.The application is also relevant with the international patent application of submitting to simultaneously with the application, the attorney docket of this international application is 682714-9WO, name is called " formula of oral that conveying comprises the catecholic butanes (catecholic butanes) of NDGA chemical compound ", and the disclosure of this application is incorporated into herein by reference in full.

Background technology

The application relates to the compositions and the method that comprise the injection prescription such as the mankind's animals administer catecholic butanes, described catecholic butanes comprises that the NDGA chemical compound is such as the NDGA derivant, EM-1421 for example, to be used for the treatment of disease, described disease is cancer for example, psoriasis or other hypertrophy or inflammatory diseases are such as the metabolic disease of diabetes or comprise such as Alzheimer (Alzheimer ' s disease), apoplexy, amyotrophic lateral sclerosis, the neuronal disease of the neurodegenerative disease of Parkinson's disease (Parkinson ' s disease).

Tested the treatment application of nordihydroguaiaretic acid (" NDGA ") in some laboratory animal.For example, the people such as Jordan are at United States Patent (USP) 5,008, have put down in writing NDGA to human breast carcinoma in 294, the effect of MX-1, and this cancerous cell is in the 0th day subcutaneous implantation rat (embodiment 2).At first day, the rat that suffers from tumor is injected the NDGA of various dosage in the mode of injecting in the single tumor.The in this embodiment not openly solubilizing agent of NDGA.In another embodiment, with 10 ^-2Reserve liquid and the 6mL distilled water of M NDGA in 4mLDMSO (that is, dimethyl sulfoxine) carries out cells in vitro test (embodiment 5).

The people such as Huang, such as United States Patent (USP) 6,214, record in 874 has been tested the treatment of the derivant (being the NDGA derivant) of some NDGA and has been used.In one embodiment, the NDGA derivant is dissolved in (embodiment 5) among the DMSO.

Using DMSO is controversial to the human body administration.In addition, use DMSO can cause undesirable side effect, such as calm, headache, nauseating, dizzy, fever or ophthalmalgia and obvious breath odor.(for example, seeing Brobyn, R.D. " The human toxicology ofdimethyl sulfoxide (human toxicity of dimethyl sulfoxine) " Ann.N.Y. Acad.Sci.243:497-506, on January 27th, 1975).If catecholic butanes, comprise that (nominal is for NDGA or NDGA derivant, " NDGA chemical compound ") be useful on the therapeutic agent as people or other animals, for example the international publication number published in 29th of December in 2004 is that the PCT/US2004/016117 of WO 04/112696 is described, be starved of the new formulation that exploitation is different from the prescription that contains DMSO, comprise the NDGA chemical compound such as the NDGA derivant to dissolve these, for example, M ₄The catecholic butanes of N.In addition, people wish not only safety but also stable of these prescriptions, have minimum side effect behind animals administer.People also wish to develop a kind of prescription for these chemical compounds, can make these chemical compounds of effective dose be delivered to required people or the interior destination organization of other animal bodies.The present invention has required advantage.

Summary of the invention

Therefore, one of purpose of the present invention provides one or more new formulation that dissolving comprises the catecholic butanes of NDGA chemical compound such as NGGA derivant herein, and wherein this kind prescription does not contain DMSO and is suitable for injection into animal.

Another object of the present invention provides when when comprising people's animals administer, safety and the as above prescription of almost being free from side effects.

Another object of the present invention provides one or more aforementioned formula, and described prescription has the stability of commercial reasonable time.

Another purpose of the present invention provides one or more aforementioned formula, and described prescription can be to the other places administration of animal the intestines and stomach.

Another object of the present invention provides one or more aforementioned formula, and described prescription has the commercial rational half-life in the circulation behind animals administer.

According to aforesaid one or more purposes of the present invention, provide the specific embodiment of the present invention as follows:

A kind of compositions be used to being injected into animal, said composition comprises acceptable carrier in active pharmaceutical ingredient and the pharmacy, wherein said active pharmaceutical ingredient comprises catecholic butanes, and described carrier comprises at least a in the solubilizing agent that is selected from the group that is comprised of following substances and the excipient: (a) water-miscible organic solvent except dimethyl sulfoxine; If when described water-miscible organic solvent is propylene glycol, described propylene glycol do not contain white vaseline, do not contain xanthan gum (being also referred to as xantham gum and xanthumgum) and do not contain at least a in glycerol or the glycine, when described water-miscible organic solvent is Polyethylene Glycol, described Polyethylene Glycol is not containing existence under ascorbic acid or the butylated hydroxytoluene (butylatedhydroxytoluene) (" BHT "), and when described Polyethylene Glycol was PEG400, described PEG400 existed not containing under the PEG 8000; (b) cyclodextrin; (c) ion, nonionic or amphoteric surfactant, if when described surfactant is non-ionic surface active agent, described non-ionic surface active agent exists not containing under the xanthan gum; (d) modified cellulose; (e) the water-insoluble lipid except Oleum Ricini; And the combination in any of described carrier (a)-(e).

The present invention also comprises the method for the treatment of experimenter disease, comprising: compositions of the present invention (a) is provided; And (b) by described compositions is injected into the experimenter and described compositions is carried out administration, wherein said compositions comprises the described active pharmaceutical ingredient of effective dose.

In addition, the present invention includes the test kit for the treatment of disease, comprise compositions of the present invention and operation instruction thereof.

Description of drawings

When read in conjunction with the accompanying drawings, can understand better aforementioned summary of the invention, and detailed description of the present invention subsequently.In order to play explanation purpose of the present invention, at present preferred embodiment shown in the drawings.Yet should be understood that the embodiment shown in the invention is not restricted to.

In the accompanying drawings:

Fig. 1 comprises Figure 1A and Figure 1B, shows at M ₄After the N treatment, the analysis of cell proliferation result that C-33A cell line and HeLa cell line are carried out.Figure 1A is M ₄The cell quantity that exists after N processes is relatively without M ₄The diagram of the ratio of the cell quantity that N processes, the M that wherein provides ₄The N amount changes from 0 μ M to 80 μ M in the DMSO prescription.Figure 1B is M ₄The cell quantity that exists after N processes is relatively without M ₄The diagram of the ratio of the cell quantity that N processes, the M that wherein provides ₄The N amount changes from 0 μ M to 80 μ M in HP-β-CD/PEG prescription (" CPE " prescription).

Fig. 2 comprises Fig. 2 A and Fig. 2 B, is based on the M that there are or do not exist various concentration in (Fig. 2 B) in (Fig. 2 A) or HP-β in the DMSO prescription-CD/PEG prescription ₄During N, the diagram of the cell death measurement result of the percentage ratio of the dead cell of C-33A cell and HeLa cell.M ₄The concentration of N is between 0 μ M to 80 μ M.

Fig. 3 comprises Fig. 3 A and Fig. 3 B, is to an IV administration of Canis familiaris L. M ₄Behind the N, concentration effect diagram in time in the dog serum in the 1st day, described M ₄N is in the prescription that comprises 30% (w/v) hydroxypropylβ-cyclodextrin (" HP-β-CD ") and 25% (v/v) PEG 300.Fig. 3 A adopts non-logarithmic scale, and Fig. 3 B adopts the logarithmic scale of concentration.

The specific embodiment

The invention provides a kind of new compositions for disease treatment, test kit and method, described disease comprises such as cancer and psoriasic proliferative disease, hypertension, obesity, I type or type ii diabetes, include but not limited to pain, Alzheimer, amyotrophic lateral sclerosis, Parkinson's disease, dull-witted, the central nervous system disease of apoplexy or neurodegenerative disease, and inflammation, neoplasia (premalignant neoplasia) or abnormal development comprise such as HIV (human immunodeficiency virus) (" HIV ") before the canceration, the mankind have a liking for T lymphocyte virus (" HILV "), human papillomavirus (" HPV "), herpes simplex virus (" HSV "), hepatitis B virus (" HBV "), Epstein-Barr virus (" EBV "), varicella zoster virus, adenovirus, parvovirus, the infectious disease of Jakob Creutafeldt virus (" JC virus ") or other viral viral infections.

The invention provides a kind of novel compositions, described compositions comprises derivant, for example M such as NDGA of containing that is dissolved in some pharmacy in the acceptable solubilizing agent ₄The catecholic butanes of the NDGA chemical compound of N, described catecholic butanes and other diluent, excipient and analogous components (nominal is " carrier ") form the prescription be suitable for injecting to treat to the experimenter such as the people disease.This prescription is suitable for injection, for example intravenous injection.In the pharmacy that is fit to acceptable carrier comprise be selected from following at least a: (a) water-miscible organic solvent except DMSO, such as Polyethylene Glycol (" PEG "), for example PEG300, PEG400 or PEG400 monolaurate, propylene glycol (" PG "), polyvinylpyrrolidone (" PVP "), ethanol, benzylalcohol or dimethyl acetylamide; (b) cyclodextrin of cyclodextrin or modification is such as HP-β-CD (" HP-β-CD ") or sulfobutyl ether-beta-schardinger dextrin-(" SBE-β-CD "); (c) ion, nonionic or amphoteric surfactant are as being the Tween-20 of non-ionic surface active agent (being also referred to as polysorbate), for example as Tween

20 or Tween

80 can commercial polysorbate20 and the polysorbate80 that obtains, polyethylene glycol 1000 vitamin E succinic acid ester (d-alpha-tocopheryl polyethyleneglycol 1000 succinate) (" TPGS "), glyceryl monooleate (being also referred to as glycerin mono-fatty acid ester) is with Cremophor

EL is can be commercial that obtain, ethylene oxide,1,2-epoxyethane and Oleum Ricini be with esterified fatty acid or the product of 35: 1 molar ratio; (d) cellulose of modification, such as ethyl cellulose (" EC "), hydroxypropyl emthylcellulose (" HPMC "), methylcellulose (" MC ") or carboxy methyl cellulose (" CMC "); And (e) water-insoluble lipid, such as oil or fat emulsions, for example Intralipid

Preferably, when using PG, it does not contain xanthan gum not containing white vaseline, and does not contain at least a situation in glycerol or the glycine and use.Preferably, when using PEG, it uses in the situation that does not contain ascorbic acid or BHT; When using non-ionic surface active agent, it uses in the situation that does not contain xanthan gum; And described oil is not Oleum Ricini.

In one embodiment of the invention, described compound dissolution is at PEG300, in PEG400 or the PEG400 monolaurate (" PEG " chemical compound).Preferably, when using PEG400, it exists not containing under the PEG8000.In another embodiment, described compound dissolution is in the cyclodextrin of modification, in HP-β-CD.In another embodiment, compound dissolution of the present invention and being diluted in the combined formulation that contains one or more PEG chemical compounds and HP-β-CD.In another embodiment, cellulose that can modification at the chemical compound of PEG described in the combined formulation substitutes or with it combination.In order to reach goal of the invention, the dissolving of the compounds of this invention can be carried out in room temperature or through heating.When using heating in course of dissolution, those solubilizing agents that chemical compound of the present invention is remained in the solution are particularly useful.

In again another embodiment of the present invention, described catecholic butanes or NDGA compound dissolution are in the cellulose such as EC or HPMC of modification.Before use, EC can be diluted in the ethanol (" EtOH ").

The present invention also provides the solubilizing agent of water-insoluble lipid as the compounds of this invention.Described water-insoluble lipid comprises, for example oil and the fat emulsions compositions of mixing are such as Intralipid

(Pharmacia ﹠amp; Upjohn is Pfizer now), use according to manufacturer's recommendation.For example, adult's dosage of recommendation is no more than 2g fat/kg body weight/day and (is respectively 20mL, the Intralipid of 10mL and 6.7mL/kg

10%, 20% and 30%).Intralipid

10% is considered to contain in 1000mL: purification Oleum Glycines 100g, and purification lecithin 12g, anhydrous glycerol 22g, water for injection is supplied 1000mL.PH is adjusted to about pH8 with sodium hydroxide.Intralipid

20% contains in 1000mL: purification Oleum Glycines 200g, and purification lecithin 12g, anhydrous glycerol 22g, water for injection is supplied 1000mL.PH is adjusted to about pH8 with sodium hydroxide.Intralipid

30% is considered to contain in 1000mL: purification Oleum Glycines 300g, and purification lecithin 12g, anhydrous glycerol 16.7g, water for injection is supplied 1000mL.PH is adjusted to about pH7.5 with sodium hydroxide.These Intralipid

Product is stored under the controlled room temperature that is lower than 25 ℃ and should be not freezing.

In another embodiment, the present invention separately or with other oil in conjunction with or with any one or more PEG chemical compound and Tween

20 or Tween

80 in conjunction with providing Semen Maydis oil, Oleum sesami, Oleum menthae, Oleum Glycines, mineral oil, glycerol.

The present invention also comprises the solubilizing agent material, such as the combination in any of propylene glycol and aforementioned substances.

In addition, the present invention includes the injection of described compositions or be infused into suitable material in the animal body, vein (" IV ") pipe for example, it is suitable for the conveying of the present invention's prescription.The pipe that is fit to comprise by such as the polymer of politef (" PTFE ") separately or with fluorubber, polyethylene, polypropylene, PEP (" FEP ") such as CHEM-Sure (Barnant company), Teflon And platinum is processed the pipe that the polymer of silicone rubber (small size) combination (Cole-Parmer) etc. is made.

The present invention can more clearly be understood according to following definition, and described definition is used with the term of other local definition of this paper:

This paper use the term " about " relate to concentration or dosage refer to concrete numerical value at the most ± 10% to 20%.

Term used herein " active pharmaceutical ingredient ", " API " or relate to catecholic butanes or the NDAG chemical compound that " chemical compound " refers to be present in any formula I in the pharmaceutical composition described herein is such as the NDGA derivant.

" alkylenedioxy (alkylene dioxy) " used herein refers to methylene dioxy base (methylene dioxy) or the methylene dioxy base that replaces, perhaps the ethylidine dioxy base of ethylidine dioxy base (ethylenedioxy) or replacement.

" amino acid residue or its salt that do not replace or replace " of one of used herein relate among formula I or the formula II-R base refers to the amino acid residue of amino acid residue or replacement, includes but not limited to: alanine, arginine, agedoite, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5-hydroxylysine, 4-Hydroxyproline, desiodothyroxine, 3-Methyl histidine, ε-N-methyllysine, ε-N, N, the N-trimethyl lysine, amino adipic acid, Gla, phosphoserine, phosphothreonine, phosphotyrosine, the N-methylarginine, N-acetyllysine, and N, the amino acid residue that the N-dimethyl replaces, or aforementioned any salt are such as villaumite.

" buffer agent " that herein is suitable for using comprises conventional in the art any buffer agent, as, Tris, phosphate, imidazoles, and heavy carbonate.

" carrier " used herein refers to capsule material or the prescription adjuvant of non-toxic solid, semisolid or liquid filler, diluent, excipient (vehicle), excipient (excipient), solubilizing agent, any general type, and comprises all composition components except described active pharmaceutical ingredient.Described carrier can contain other reagent, such as moistening or emulsifying agent, or the pH buffer agent.If necessary, can add other material, such as antioxidant, wetting agent, viscosity stabiliser, and similar reagent.

" catecholic butanes " used herein refers to the chemical compound of formula I:

R wherein ₁And R ₂Expression-H independently of one another, low alkyl group, lower acyl, alkylene; Or-R ₁O and-R ₂O represents the amino acid residue or its salt that do not replace or replace independently of one another; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Independently of one another expression-H or low alkyl group; And R ₇, R ₈And R ₉Expression-H independently of one another ,-OH, lower alkoxy, low-grade acyloxy, the amino acid residue or its salt that do not replace or replace perhaps can be any two adjacent groups of alkylenedioxy altogether.

" cyclodextrin " used herein refers to unmodified cyclodextrin or modified cyclodextrin, comprise and be not limited to alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin with and on contain the cyclodextrin of any modification of modification, such as HP-β-CD or SEB-β-CD.Cyclodextrin has 6 (alpha-cyclodextrins) usually, 7 (beta-schardinger dextrin-s), with 8 (gamma-cyclodextrin) sugar, every sugar is 3 substituent groups the most nearly, and 0-24 elementary substituent group (elementary substituent group is defined as the direct substituent group that links to each other with the cyclodextrin ring) therefore can be arranged.The modification that the present invention uses or unmodified cyclodextrin can have elementary substituent group or other modifications that is fit to arbitrarily quantity and location.

" derivant " of NDGA used herein refers to " NDGA derivant " (seeing below).

" disease " used herein comprises disease, discomfort, infection, syndrome or the disorder that all are used compositions of the present invention and can produce therapeutic effect.These " diseases " include but not limited to cancer, psoriasis and other proliferative diseases, struvite disorder comprises rheumatic arthritis, osteoarthritis, ulcerative colitis, Crohn disease, arteriosclerosis, chronic obstructive pulmonary disease (" COPD "), hypertension, fat, diabetes, pain, apoplexy and/or other neurological disorders or neurodegenerative disease or disease, comprise Alzheimer, Parkinson's disease, multiple sclerosis, the front disease of amyotrophic lateral sclerosis (" ALS ") and canceration is such as intraepithelial neoplasia or abnormal development, and infectious disease.

" G used herein ₄N " or " four-N, the sweet amine acyl of N-dimethyl NDGA " or " the sweet amine acyl of four-dimethyl NDGA " be the NDGA derivant of formula II (see lower), wherein R ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-O (C=O) CH independently of one another ₂N (CH ₃) ₂Or O (C=O) CH ₂N ⁺(CH ₃) ₂Cl ^-, with solid form or in solution; And R ₁₈And R ₁₉Expression-CH separately ₃

" lower acyl " used herein refers to C ₁-C ₆Acyl group, preferably, C ₁-C ₃Acyl group.

" low alkyl group " used herein refers to C ₁-C ₆Alkyl, preferably, C ₁-C ₃Alkyl.

" M used herein ₄N " or " EM-1421 " be the NDGA derivant of formula II (see lower), wherein R ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-OCH independently of one another ₃, and R ₁₈And R ₁₉Expression-CH separately ₃

" modified cellulose " used herein refers to contain at cellulosic molecule the cellulose of one or more modification groups, comprise, as, EC, HPMC, CMC and MC.

" NDGA " used herein refers to nordihydroguaiaretic acid and has following formula:

" NDGA chemical compound " used herein refer to individually or the NDGA that gathers and/or NDGA derivant any one or multiple.

" NDGA derivant " used herein refers to the NDGA derivant of formula II

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-OH independently of one another, lower alkoxy, low-grade acyloxy, or the amino acid residue or its pharmaceutically acceptable salt that do not replace or replace; And R ₁₈And R ₁₉Independently of one another expression-H or low alkyl group, wherein R ₁₄, R ₁₅, R ₁₆And R ₁₇Be not simultaneously-OH.Therefore, this formula comprises the chemical compound of the derivant that methylates of NDGA, such as EM-1421 (M ₄N), three-O-methyl N DGA (M ₃N), two-O-methyl N DGA (M ₂N) and list-O-methyl N DGA (M ₁N).Alternatively, the NDGA derivant can be the hydroxyl of NDGA or the substituted chemical compound of one or more hydrogen atoms on the methyl, such as, R wherein for example ₁₄, R ₁₅, R ₁₆And R ₁₇Represent independently of one another lower alkoxy, low-grade acyloxy, the perhaps aminoacid of aminoacid or replacement or its salt; And R ₁₈And R ₁₉Independently of one another expression-H or alkyl are such as low alkyl group.This term comprises, for example, and a chemical compound, the R of described chemical compound ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-OCH independently of one another ₃Or-O (C=O) CH ₃Or disubstituted amino acid residue, such as N, the two methyl substituted amino acid residues of N-, such as-O (C=O) CH ₂N (CH ₃) ₂Or-O (C=O) CH ₂N ⁺(CH ₃) ₂Cl ^-And R ₁₈And R ₁₉Independently of one another expression-H or low alkyl group, as-CH ₃Or-CH ₂CH ₃

" percentage ratio " used herein, " percent " or symbol " % " refer to the composition shown in the compositions based on vector contg in the compositions about w/w (w/w), the percentage ratio of weight/volume (w/v) or volume/volume (v/v), with respect to any specific component, all based on the vector contg that exists in the compositions.Therefore, shown in the content that exists of dissimilar carrier reach 100%, it does not get rid of the existence of API, and the scale of API is shown % or is present in the compositions several milligrams or be present in several mg/ml in the compositions, and wherein % or mg/ml are based on the carrier total amount that is present in the compositions.The carrier of some type can reach in conjunction with existence 100% carrier.

" pharmaceutically acceptable carrier " used herein is nontoxic to the receiver on the dosage that uses and concentration, and can be compatible with other compositions of prescription.For example, be used for containing catecholic butanes of the present invention, the carrier of the prescription of NDGA chemical compound or NDGA derivant preferably do not comprise oxidant and other known to its disadvantageous chemical compound.Pharmaceutically acceptable carrier comprises solubilizing agent.The pharmaceutically acceptable carrier that is fit to includes but not limited to water, dextrose, glycerol, saline, ethanol, buffer agent, Cremaphor

EL, phosphate-buffered saline, PEG 300, and PEG 400, modified cyclodextrin, and combination, all substances see above described.

Term " pharmaceutically acceptable excipient " comprises excipient, adjuvant, or diluent or other adjuvants, as conventional in this area, the public can obtain, and nontoxic to the receiver on the dosage that uses and concentration, and can be compatible with other compositions of prescription these.For example, pharmaceutically acceptable adjuvant comprises pH regulator and buffer agent, soaks into the pressure regulator, stabilizing agent, wetting agent etc.

Term used herein " solubilizing agent " refers to wherein one or more catecholic butanes or NDGA chemical compound, such as the compositions of NDGA derivant dissolving.Solubilizing agent also can be carrier or pharmaceutically acceptable carrier.

Term " experimenter ", " host " and " patient " is used interchangeably herein, refer to the animal treated with compositions of the present invention, include but not limited to ape, the mankind, felid, Canis animals, equine species, cattle, pig, sheep, goat, suckling farm animal, suckling match poultry, and the suckling house pet.

What be used for administration herein relates to catecholic butanes or NDGA chemical compound or derivant " substantially pure " chemical compound for substantially not containing the chemical compound of the material (hereinafter referred to as " non-NDGA material ") that is not catecholic butanes, NDGA chemical compound or NDGA derivant.Substantially do not contain and refer to not contain non-NDGA material at least about 50%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% and also more preferably do not contain non-NDGA material at least about 95%.

Term used herein " treatment (treatment) ", " processing (treating) " etc. refer to obtain required pharmacology and/or physiologic effect.Described effect is preventative according to completely or partially preventing disease or disease or its symptom can be, and/or can be curative according to completely or partially treating the adverse effect that disease or disease and/or disease or disease cause." treatment " therefore, for example, comprises situation that mammal is especially human or any treatment of disease, and it comprises: (a) prevention disease or disease occur in subject, and described experimenter easily suffers from described disease or disease but also do not diagnose ill; (b) suppress described disease or disease, such as, its development stoped; And (c) remove, alleviate or improve described disease or disease, such as described disease or disease are disappeared.

Before further setting forth the present invention, should be understood that the present invention is not limited to described specific embodiment, because these embodiment can change certainly.It is to be further understood that term used herein to be intended to describe specific embodiment and and be not intended to restriction because scope of the present invention is limited by appended claim only.

When the scope of the value of providing, should be understood that each value between two parties, clearly point out in addition except in the literary composition, to 1/10th of the unit that hangs down limit value, in this scope between the higher and low limit value or in described scope any other described or value between two parties, be included in the present invention.Except the boundary of clearly getting rid of in described scope, these higher and low limit values more among a small circle can be included in the described less scope independently, and are also contained among the present invention.When described scope comprises one or more described boundaries, the scope of getting rid of any one or two kinds of boundaries that those are included is also included among the present invention.

Must be noted that as used herein singulative " (a) ", " one (an) ", and " should (the) " comprise plural thing, unless know expression in the literary composition is not.Therefore, for example, mention " chemical compound " and comprise many this chemical compounds, mention " this NDGA chemical compound " and comprise and mention one or more NDGA chemical compounds, such as NDGA derivant and its equivalent known to persons of ordinary skill in the art.

All publications of herein mentioning comprise patent, patent application, and magazine article by reference integral body incorporate into herein, described integral body comprises the list of references of wherein quoting, described list of references is also quoted by integral body and is incorporated into herein.

Publication discussed herein only is provided at the application and submits day disclosure before to.This not should be understood to the present invention since in first to file disqualification prior to these publications.In addition, the date of the publication that provides may be different from the actual publication date, and it needs to confirm respectively.The quoted passage of the list of references of mentioning in the literary composition in this application was listed in the bibliography before claim in detail.

Following description of the present invention is only provided in the mode of embodiment, do not mean any limitation of the invention.

The preparation of catecholic butanes

Catecholic butanes of the present invention can be prepared by any known method in this area.For example, such as United States Patent (USP) 5,008,294 described these chemical compounds that make.

The preparation of NDGA derivant

NDGA can buy from any obtainable commercial source, Alexis Biochemicals company (catalog number (Cat.No.) LKT-N5669) such as San Diego, CA, USA, or the A.G.Scientific company of San Diego, CA, USA (catalog number (Cat.No.) N1071), or the Cayman Chemical company (catalog number (Cat.No.) 70300) of Michigan, USA Ann Arbor.

NDGA derivant and prescription thereof can be prepared by the method for any routine in this area.For example, the NDGA derivant can be such as United States Patent (USP) 5,008,294; United States Patent (USP) 6,291,524; Hwu, the people such as J.R. (1998); Or McDonald, described being prepared of the people such as R.W. (2001).

In one embodiment of the invention, a NDGA derivant, EM-1421 also is called as meso-1, two (3, the 4-Dimethoxyphenyls)-2 of 4-, 3-dimethylbutane, or M ₄N, following making: preparation contains the methanol solution of NDGA and potassium hydroxide in reaction flask.Then, in this reaction flask, add dimethyl sulfate and allow reaction to carry out.Final water quenching reaction makes the product precipitation.By isolated by filtration precipitation and dry in vacuum drying oven.Then, this chemical compound dissolves in the solution of dichloromethane and toluene, and carries out purification by alumina chromatographic column subsequently.By the rotary evaporation desolventizing, described solid is separated in isopropyl alcohol and by filtration by Eddy diffusion.Filter cake is dry in vacuum drying oven., described filter cake reaches the EM-1421 (M that makes gained by filtering again fractional crystallization by being refluxed in isopropyl alcohol ₄N) crystallization.

In some embodiments of the invention, some NDGA derivant of the present invention is such as G ₄N, be also referred to as meso-1, two [3,4-(dimethylamino acetoxyl group) phenyl]-(2R, 3S)-dimethylbutane or the sweet amine acyl of the four-dimethyl NDGA of 4-, perhaps its hydrogen chlorate, and have the similar compound of aminoacid replacement base, also can be prepared by the method for routine, such as for example United States Patent (USP) 6,417,234 is described.

The preparation of therapeutic combination

The invention provides the compositions that comprises pharmaceutical composition, it comprises the catecholic butanes as active pharmaceutical ingredient (" API ") that contains NDGA chemical compound such as NDGA derivant, and pharmaceutically acceptable carrier or excipient.Usually, compositions of the present invention contains less than about 0.1% (w/v) to active pharmaceutical ingredient or the API of about 99% (w/v), namely described catecholic butanes, comprise herein NDGA chemical compound and NDGA derivant; Alternatively, the present invention can contain about 2% (w/v) to the API of about 90% (w/v).

The present invention also provides compositions, and in described compositions, described catecholic butanes is such as NDGA derivant, for example M ₄The concentration that N exists is that about 1mg/ml is to about 200mg/ml, perhaps about 10mg/ml is to about 175mg/ml, and perhaps about 20mg/ml is to about 150mg/ml, and perhaps about 30mg/ml is to about 125mg/ml, perhaps about 40mg/ml is to about 100mg/ml, and perhaps about 50mg/ml is to about 75mg/ml.In one embodiment, the about 1mg/ml of the concentration of NDGA chemical compound herein, 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 35mg/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 200mg/ml.

Alternatively, the compositions of other embodiments of the present invention can contain less than about 0.1mg to about 200mg or more API, 10mg according to appointment, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 75mg, the API of about 100mg or about 200mg.

Pharmaceutically acceptable carrier or excipient can contain one or more solubilizing agents, and described API solvent is in described solubilizing agent.Described pharmaceutically acceptable carrier or excipient also can contain diluent.

In one embodiment, the invention provides a kind of solubilizing agent, described solubilizing agent contains one or more water-miscible organic solvents except DMSO.Preferably water-miscible organic solvent has ethanol, benzylalcohol, dimethyl acetylamide, PVP, PG and such as: PEG 300, and PEG 400, or the PEG chemical compound of PEG 400 monolaurates.The amount that PEG chemical compound in the present composition provides is about 5% to about 100%, or about 5% to about 60%, or about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%, and all concentration is volume/volume (v/v) percentage ratio.The concentration about 2.5% that PG exists is to about 100% (v/v).

The concentration of the PEG chemical compound in the present composition can change according to other solubilizing agents that exist or diluent or excipient.For example, PEG 300 of the present invention, PEG400 or PEG 400 mono laurate ester concentrations can 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 concentration that provide are volume/volume (v/v) percentage ratio.

The present invention also provides catecholic butanes or the compositions of NDGA chemical compound in cyclodextrin, and described cyclodextrin comprises modified cyclodextrin.For example, cyclodextrin herein can be alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, and modified cyclodextrin comprises HP-β-CD and SEB-β-CD.In one embodiment, the concentration of the modified cyclodextrin that compositions of the present invention contains is about 5% to about 80%, or about 10% to about 70%, or about 20% to about 60%, or about 30% to about 50%, and all concentration that provide are weight/volume (w/v) percentage ratio.

In another embodiment again, the cyclodextrin of modification is such as HP-β-CD, the concentration that exists in compositions is 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 concentration that provide are weight/volume (w/v) percentage ratio.

Wherein, can be separately or other pharmaceutically acceptable carriers that other materials use in compositions of the present invention or excipient as ion, nonionic or amphoteric surfactant, such as Cremophor

EL, for the polysorbate of non-ionic surface active agent for example as Tween

20 or Tween

80 can commercial polysorbate20 and the polysorbate80 that obtains, and is the TPGS of amphoteric surfactant.The other example of the surfactant that is fit to includes, without being limited to the fatty acid of glyceryl monooleate and esterification, such as usually made by the ester exchange reaction of vegetable oil these, can multiple kind and grade obtain, such as N.J., para female this, the Labrafil of Gattefosse company , Labrasol

, and Gelucire

Surfactant can exist by any required effective dose, as take concentration as about 1% (v/v) to about 100% (v/v), be preferably about 9% (v/v) to about 80% (v/v), and more preferably, about 10% (v/v) is to about 50% (v/v).In a specific embodiment, the concentration of preferred non-ionic surface active agent is that concentration is that about 9% (v/v) is to the Tween of about 100% (v/v)

20, and concentration is that about 33% (v/v) is to the Tween of about 100% (v/v)

80.The percentage ratio of all surface activating agent is percent by volume (v/v).

Acceptable carrier or excipient are modified celluloses in another pharmacy of using separately in compositions of the present invention or being used, such as EC, HPMC, MC and CMC.Modified cellulose can be any required effective dose exist, be about 0.1% to about 25% such as concentration, or about 0.5% to about 7.5%, or about 1.0% to about 5%.In a specific embodiment, the concentration that EC exists can be about 5% to about 20%; The concentration that HPMC exists can be about 0.5% to about 1%; The concentration that MC exists can be about 1% to about 3%; And the concentration that CMC exists can be about 1% to about 4%.The percentage ratio of modified cellulose is in the every volume of weight (w/v).

Acceptable carrier or excipient are the water-insoluble lipids in other pharmacy of using separately in compositions of the present invention or being used, such as oil or mixed lipomul.The example of oil comprises Semen Maydis oil, Oleum sesami, Oleum menthae, soybean oil, mineral oil and glycerol.Mixed lipomul can obtain as previously mentioned, such as Intralipid Emulsion.

Described water insoluble carrier can be combined with any or multiple water-solubility carrier, such as PEG chemical compound and surfactant, such as Tween

20 or Tween

80.

Described water-insoluble lipid carrier can exist by any required effective dose, as take concentration as about 10% (v/v) to about 100% (v/v), or about 15% (v/v) be to about 85% (v/v), or about 25% (v/v) is to about 75% (v/v).The concentration that oil exists can be about 9% (v/v) to about 100% (v/v).The concentration that mixed lipomul exists can be about 10% (w/v) to about 30% (w/v); And preferred about 20% (w/v).

As previously mentioned, the combination of various carrier components can be used with API.A preferred non-limiting example is 10mg/ml M at present ₄N is at 25% (w/v) PEG 300,30% (w/v) HP-β-CD, the compositions in the carrier of the water that is suitable for injection into animal of surplus (" WFI ", it represents the checking rank of water in pharmaceuticals industry).In this preferred embodiment, described HP-β-CD has the replacement of 6 to 8 degree, but as previously mentioned, other replacements in other embodiments also fall within the scope of the present invention.

Should be understood that, as long as described catecholic butanes dissolves and is kept in the solution, just can add one or more described solubilizing agents or diluent or excipient to described solution, with the conveying of this solution of optimization to the patient of this kind of needs treatment.

In another embodiment, the invention provides a kind of diluent, described diluent is saline or the water that is suitable for injecting.In one aspect of the invention, when the osmolarity of described pharmaceutical composition is higher, use water for injection as diluent.

Being suitable in the pharmacy used herein acceptable carrier or diluent describes in multiple publication.Useful carrier or the example of excipient are described in, Gennaro for example, A.R. (2003); Ansel, the people such as H.C. (2004); Rowe, the people such as R.C. (2003); And Garg, among the people such as S. (2001).

The compositions of liquid form can comprise buffer, and described buffer is selected such as the required application of NDGA derivant according to catecholic butanes or NDGA chemical compound, and also can comprise other materials that are suitable for required use.Those of ordinary skills can easily select a kind of suitable buffer that is suitable for required application, and various buffer are by well known in the art.

Therapeutic Method

It is found that, contain catecholic butanes, the compositions that comprises the NDGA chemical compound as therapeutic agent or be used for the treatment of, in these treatments, can be used these catecholic butanes or NDGA chemical compound the patient who suffers from numerous disease, needs treatment.

The invention provides the method and composition for disease treatment, described disease comprises that for example, proliferative disease is such as optimum and malignant tumor, and disease and neoplasia before psoriasis and the cancer form such as intraepithelial neoplasia, or abnormal development.The present invention also provides diabetes to comprise I type and type ii diabetes, obesity and comprises the treatment of cardiovascular disease, apoplexy and hypertensive complication.The present invention further provides the treatment that inflammation comprises rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, Crohn disease, chronic obstructive pulmonary disease (" COPD ") and other immune system relevant diseases.In addition, the invention provides nervous system disease, comprise the treatment of central nervous system disease and neurodegenerative disease such as Alzheimer, dementia, amyotrophic lateral sclerosis and Parkinson's disease.In another embodiment, the invention provides infectious disease, such as the treatment that comprises the viral infection of transcribing or copying that needs the Sp1 combination.Need the example of the virus of Sp1 combination to comprise: HIV, HTLV, HPV, HSV, HBV, EBV, varicella zoster virus, adenovirus, parvovirus and JC virus.

The method according to this invention can be treated various animal reservoirs, and described animal reservoir comprises the mankind or non-human animal.Usually, these hosts are " mammals " or " mammal ", these terms that are widely used in describing the organism that belongs to Class Mammalia comprise carnivore order (such as Canis familiaris L. and cat), Rodents (Cavia porcellus for example, and home mouse), and other mammals, comprise cattle, goat, horse, sheep, rabbit, pig, and primates (for example, the mankind, chimpanzee and monkey).In many examples, described host is human.Animal model is used for experimentation, as the model for the treatment of human diseases is provided.In addition, the present invention also can be applicable to the veterinary.

Prescription, dosage, and route of administration

In one embodiment of the invention, the present composition of effective dose is carried out administration to the host, wherein " effective dose " refers to be enough to produce the dosage of results needed.In certain embodiments, required result is at least the inhibition to neoplasia or abnormal development process.

The appropriate dose of administration depends on the host who is treated, such as the state of this host's health condition, host's age, disease or disease, host's body weight, the size of tumor.Usually, for the child can about 0.1mg to about 500mg or still less carry out administration, and for the adult can about 0.1mg about 5g or still less carry out administration extremely.Usually dosage is in the every kg experimenter's body weight of about 10mg active pharmaceutical ingredient extremely in the wide region of the every kg experimenter's body weight of about 600mg active pharmaceutical ingredient.Described activating agent can carry out administration with compound dosage separately or more generally.Those of ordinary skills can easily determine the preferred dose of the reagent of giving by the whole bag of tricks.By the dose response curve of setting up with routine test, those of ordinary skills can easily determine other effective doses.Certainly, the amount of reagent will change with the reagent of concrete use.

The administration frequency of described active agent and dosage will be determined based on replying of age, body weight, morbid state, health status and patient by the nursing staff.Therefore, described reagent can every day, weekly, one or many per month, perhaps as the conventional suitable number of times of determining carry out administration.Described reagent can carry out administration off and on, such as carrying out several days, administration phase in a few week or several months, then until after a while as 3 or continued in 6 months, then carries out several days, the administration in a few week or several months again again.

In pharmaceutical dosage form, described active agent can suitably be united separately or with other drug activating agent or therapeutic agent, and in conjunction with administration, described other drug activating agent or therapeutic agent comprise micromolecule, antibody or protein therapeutic agent.Following method and embodiment are not limitation of the present invention for example only.

In addition, if necessary, described carrier or excipient can contain a small amount of auxiliary substance, such as pH regulator and buffer agent, soak into and press regulator, stabilizing agent, wetting agent or emulsifying agent.The practical methods for preparing described medicine type is known, perhaps those of ordinary skills is now easily seen.Can referring to, Remington ' s Pharmaceutical Science for example, the 20th edition, Mack PublishingCo.Rawlins EA, (1997).The compositions or the prescription that are used for administration contain the API amount that is enough to obtain required state in the treatment host in any situation.

The unit dosage forms that is used for injection or intravenously administrable can comprise the API of compositions, such as the solution of acceptable carrier in sterilized water, normal saline or other pharmacy.

Term used herein " unit dosage forms " refers to be suitable for the physical dispersion unit as human or animal host's single dose, constituent parts contains the API of the present invention of scheduled volume, and described scheduled volume is to be enough to produce in conjunction with acceptable diluent, carrier or excipient in the pharmacy amount calculating of required effect.The specification of new unit dosage forms of the present invention depends on employed specific compound and the effect that will reach, and the pharmacodynamics relevant with each chemical compound among the host.

The present invention includes the test kit of the many or single dose of described active agent.In this test kit, contain the container of the compositions of catecholic butanes such as NDGA chemical compound except depositing many or single dose, the data bag of interpolation description is arranged, what described description had been described in the morbid state that treatment is concerned about this medicine uses and follows advantage.Alternatively, the administrator of the present composition is included in each test kit.

Injectable compositions of the present invention can be to be suitable for the mode parenterai administration of disease to be treated and this area routine, comprise vein ground, intra-arterial ground, intraperitoneal ground, hypodermically with vesicle in (intravesicularly) administration.Although compositions of the present invention is defined as drug administration by injection, it also is suitable for by other administrations, for example by local, intranasal, suction or drug delivery implant.

The example that the below lists is exemplary, can not be interpreted as limitation of the present invention.

Embodiment 1. contains M ₄HP-β-CD of N and/or PEG 300 prescriptions

In this embodiment, M ₄Description among N such as the PCT/US2004/016117 is prepared, and dissolves in solubilizing agent.Gained solution mixes with excipient and/or diluent alternatively.Described solubilizing agent and excipient are used interchangeably, or the use that mutually combines.One of the solubilizing agent of using or excipient are the HP-β-CD (" HP-β-CD ") of endotoxin control, it obtains from (the catalog number (Cat.No.) RDI-82004HPB of Research Diagnostics company, lot number H3N188P) (Flanders this, New Jersey).The solubilizing agent that other use or excipient are PEG300, obtain from Spectrum Chemicals company (catalog number (Cat.No.) P0108, lot number TB1228) (Gardner, California, USA).

In one embodiment of the invention, HP-β-CD and PEG300 exist with single prescription.In order to make this prescription, at first with M ₄N is dissolved among the PEG300 to form M ₄PEG300 the solution (" M of N ₄N/PEG 300 ").Subsequently, with M ₄N/PEG 300 solution are added in pre-prepared HP-β-CD solution, to form the M among PEG 300 and the HP-β-CD ₄N solution (after this being called " CPE prescription ").

When preparation described HP-β-CD solution, must consider volumetric expansion.For example, for 40% (w/v) HP-β-CD solution, (that is, the water of 0.7mL has been replaced every gram HP-β of adding-CD) must to consider the volumetric expansion of 0.7mL/g.

The 100mL solution that is used as the 40%HP-β-CD of solubilizing agent and/or excipient is prepared as follows: 65mLWFI is placed the glass beaker that contains stirring rod.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.About 40gHP-β-CD is added to WFI in the stirring lentamente, uses spatula with the central authorities of HP-β-CD guiding beaker, be bonded on the walls of beaker to prevent HP-β-CD crystallization.This HP-β-CD solution is stirred about 24 hours, perhaps until under the visualization HP-β-CD dissolve fully.The solution of gained records about 93mL.About 7mL WFI is added to this gained solution to obtain 100mL, makes the final solution of about 40%HP-β-CD.With described final solution stir about 1 hour, at room temperature store, and avoid sunlight.This method for preparing modified cyclodextrin solution can be zoomed in or out to obtain required volume or concentration in proportion.Other concentration or other modified cyclodextrin solution can make similarly, for example, by HP-β-CD is replaced by other modified cyclodextrins, are adjusted to suitable concentration in above-mentioned method.

The 10mL M of about 10mg/mL concentration among 40%HP-β-CD ₄N solution is prepared as follows: 40%HP-β that will about 10mL-CD solution is added to and contains in the stirring rod glass beaker.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Will about 10mg M ₄N is added to 40%HP-β-CD lentamente, is added to the central authorities of beaker under the help of spatula.This M ₄N/40%HP-β-CD mixture is stirred about 2 hours, perhaps until all M ₄N evenly suspends and does not have any caking.Alternatively, this M ₄N/40%HP-β-CD mixture can be at 80 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 100mL ₄N/HP-β-CD mixture is at 80 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By beaker being faced toward white background, seeking whether have granule, the described M of observable facing to black background afterwards ₄Whether N/HP-β-CD mixture or solution exist any undissolved M ₄N.Final M ₄N/40%HP-β-CD solution is in room temperature storage, and keeps in Dark Place.This method can zoom in or out to obtain M in proportion ₄Volume required or the concentration of N.Contain M ₄The prescription of other cyclodextrin solutions of N can prepare similarly, for example, and by the HP-β-CD in the said method is replaced with other cyclodextrin.At the presentation of results shown in the table 1, the M after the cooling in solution ₄N remained on the concentration of 1mg/mL in the 40%HP-β-CD prescription and 10mg/mL greater than 7 days.

The 100mL M of about 25mg/mL concentration among the PEG 300 ₄N solution is prepared as follows: will about 100mL PEG 300 be added to and contain in the stirring rod glass beaker.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Will about 2.5g M ₄N is added to PEG 300 lentamente, is added to the central authorities of beaker under the help of spatula.This M ₄N/PEG 300 mixture are stirred about 24 hours, perhaps until all M ₄N evenly suspends and does not have any caking.Alternatively, this M ₄The N/PEG300 mixture can be at 60 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄N/PEG 300 mixture are at 60 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By beaker being faced toward white background, facing toward black background afterwards, observe the existence of microgranule, the described M of observable ₄Whether N/PEG 300 mixture or solution exist any undissolved M ₄N.Observing all M ₄After the N dissolving, the M of gained ₄N/PEG300 solution uses immediately or used before the effect duration of 48hr, otherwise can form crystallization or other precipitations.If crystallization forms, M ₄N/PEG 300 solution can be followed stirring, reheat 1hr at 60 ℃ on the thermomagnetion platform, until all M ₄N dissolves back in the solution.Final M ₄N/PEG 300 solution are in room temperature storage, and keep in Dark Place.This method can zoom in or out to obtain M in proportion ₄Volume required or the concentration of N.Contain M ₄The prescription of other PEGs solution of N can prepare similarly, for example, and by the PEG 300 usefulness PEG 400 in the said method or PEG 400 monolaurates are replaced.

By adding the previously prepared HP-β of 50mL40%-CD solution (as above preparation) to being positioned at the beaker that contains stirring rod on the magnetic force platform, follow stirring rod to stir with middling speed, and slowly add the previously prepared M of 50ml ₄N/PEG 300 solution (as above preparation), for example with the speed of about 10mL per minute, preparation contains 50%PEG 300 (v/v), 20%HP-β-CD (w/v) and 12.5mg M ₄The 100mL prescription stock solution of N.By using pipette, with M ₄N/PEG 300 is added to the central authorities of beaker, thereby avoids on its wall that is bonded at beaker and guarantee abundant dissolving.With M ₄N/PEG 300 is added to HP-β-CD solution and begins to show as white solution, but finally becomes clarification under constantly stirring.This method for making can be zoomed in or out in proportion, to be suitable for producing the M of volume required or concentration ₄N/PEG 300 and HP-β-CD.Pvdf membrane with 0.22 μ m carries out filtration sterilization to described stock solution, such as the vacuum driving Disposable bottle top filter membrane that obtains from the pre-degerming of Millipore (catalog number (Cat.No.) SCGV T05 RE) (blocking Massachusetts, United States in the Bill).This filter process is driven by vacuum power, and filtrate collection is in pre-sterilized 250mL vial.Subsequently, this bottle is tightly sealed, in room temperature and keep in Dark Place.M among HP-β-CD ₄N/PEG 400 or M ₄The stock solution of N/PEG 400 monolaurates can similarly prepare by substitute PEG 300 with PEG 400 or PEG 400 monolaurates in said method.

M with the preceding method preparation ₄N/PEG 300/HP-β-CD, M ₄N/PEG 400/HP-β-CD or M ₄The stock solution of N/PEG 400 monolaurates/HP-β-CD can or be used in external use diluting before animals administer.If dilution is necessary, described stock solution preferably with WFI but not for example saline dilute, to reduce osmotic pressure.In order to prepare the 100mL solution of stock solution dilution in 1: 1 in WFI, the stock solution of about 50mL is added in the vial.The WFI of about 50mL is added in the 50mL stock solution in the bottle to form the solution of dilution.With the sealing of described vial, and by rocking and reversing these vial several times the solution after the dilution is fully mixed.Pvdf membrane with 0.22 μ m carries out filtration sterilization to this dilute solution, such as the vacuum driving Disposable bottle top filter membrane that obtains from the pre-degerming of Millipore (catalog number (Cat.No.) SCGV T05 RE) (blocking Massachusetts, United States in the Bill).This filter process is driven by vacuum power, and filtrate collection is in pre-sterilized 250mL vial.Subsequently, this bottle is tightly sealed, in room temperature and keep in Dark Place.This method can be zoomed in or out to obtain required volume or dilution factor in proportion, such as the dilution factor of 1: 2 or 1: 4.

The prescription that is suitable for use as placebo that contains 50%PEG 300 and 20%HP-β-CD can be prepared as follows.In order to prepare 100mL placebo or control formula, about 50mL 40%HP-β-CD is added to is arranged in the beaker that contains stirring rod on the magnetic force platform.Set this magnetic force platform and stir HP-β-CD solution with middling speed.By moving liquid to the central authorities of beaker, avoid PEG 300 to be bonded on the wall of beaker, 50mL PEG 300 slowly is added among 50mL HP-β-CD in the glass beaker.This mixture is stirred about 1hr or until mixes fully.Pvdf membrane with the power-actuated 0.22 μ m of vacuum carries out filtration sterilization to the placebo prescription.Filtrate collection is in pre-sterilized 250mL vial.This bottle is tightly sealed, in room temperature and keep in Dark Place.This method can be zoomed in or out to produce required concentration and volume as required in proportion.In addition, as required, PEG 300 can be replaced with PEG 400 or PEG 400 monolaurates.

According to the prescription that contains HP-β-CD and/or PEG 300, PEG 400 aforementioned or that similar approach makes, and contain HP-β-CD and propylene glycol (" PG "), hydroxypropyl emthylcellulose (HPMC), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP) or Tween

M in 80 the prescription ₄The solubility results of N, and the character of gained prescription is shown in the table 1-5, wherein N represents that "No" and Y represent "Yes".

Table 1 modified cyclodextrin is as solubilizing agent and/or excipient

All prescriptions stand to place 24hr and with the centrifugal 5min of 5000rpm, do not form visible precipitate at 4 ℃.Contain 50%PEG 300,20%HP-β-CD, 12.5mg/mL M ₄The prescription of N stock solution can keep 4 months at 4 ℃ at least.Also can be 4 ℃ of maintenances at least 4 months with the diluent that 1: 1 or 1: 2 dilution factor make identical storing solution.

Table 2 M ₄The PEG 300 of N and HP-β-CD prescription

Table 3 M ₄The PEG 400 of N and HP-β-CD prescription

Table 4 M ₄The stability of N prescription in PEG300

Table 5 M ₄The stability of N prescription in PEG400

Similarly, M ₄N may be dissolved in other solubilizing agents, such as water miscible organic solvent, comprises ethanol, polyvinylpyrrolidone (PVP), propylene glycol or glycerol.

Embodiment 2.M ₄The DMSO of N or PEG 300/HP-β-CD combined formulation are on propagation and the dead impact of tumor cell in cultivating

M ₄N is (hereinafter referred to as " CPE " prescription) in 10% (w/v) HP-β-CD and 25% (v/v) PEG 300, M ₄N is (hereinafter referred to as " CPE25/30 " prescription) in 30% (w/v) HP-β-CD and 25% (v/v) PEG 300, and M ₄N is (hereinafter referred to as " CPE 33/27 " prescription) in 27% (w/v) HP-β-CD and 33% (v/v) PEG 300, be used to test on the cell death of two kinds of different tumor cell lines and the impact of propagation: Hela, it is HPV-18 positive human cervical cancer tumer line, and C-33A, it is the negative Human cervical cancer cell lines of HPV-, M ₄N is also tested in DMSO abreast.Two kinds of tumor cell line M to increase progressively ₄The N amount is processed: 0 μ M, and 20 μ M, 40 μ M, 60 μ M and 80 μ M process 72hr with DMSO or CPE prescription.Add each prescription and reach 1% of growth medium (minimal essential medium that contains L-glutaminate has been added 10% hyclone, 1mM Sodium Pyruvate, the nonessential Freamine Ⅲ of 1x and 1000 IU/mL penicillins/1000 μ g/mL streptomycin solution) total amount.Control cells is cultivated under the same conditions, but does not process.After the processing or non-processing of 72hr, count total cellular score and living cells quantity in each sample by using trypanblue exclusion method.The cell proliferation rate of analysis in each sample and the percentage rate of dead cell.The result of this experiment at Fig. 1, Fig. 2 and table 6 to shown in 12.

Fig. 1 is that the ratio of treated cell quantity/untreated cell quantity is with respect to for the treatment of ever-increasing M in the DMSO prescription of C-33A cell and Hela cell or the CPE prescription ₄The presentation graphs of N concentration mapping.Fig. 2 be the percentage ratio of dead cell with respect to for the treatment of two kinds of cancerous cell lines in culture medium, ever-increasing M in the DMSO of C-33A cell and Hela cell prescription or the CPE prescription ₄The presentation graphs of N concentration mapping.

The result shows, than untreated contrast, is not containing M ₄During N, with the % measurement of dead cell, single DMSO has significant anti-proliferative effect and some toxic actions for two kinds of tested tumor cell lines.On the contrary, when than untreated contrast, single CPE prescription has anti-proliferative effect and does not almost have toxicity for two kinds of tumor cell lines.

When with same recipe in non--M ₄Contrast (that is, 0 μ g/mL or 0 μ M M that N processes ₄When N) comparing, with M ₄After the DMSO prescription of N or CPE prescription were processed, cell proliferation rate descended in two kinds of cell lines.In fact, in the CPE prescription, this anti-proliferative effect shows as M ₄The N dose dependent.In the CPE prescription, for example, the M of about 20 μ M or 7.2 μ g/mL ₄The cell proliferation that N is found to be enough to two kinds of tumor cell types causes about 50% to suppress.In the CPE prescription, M ₄The further increase of N concentration will produce further at anti-proliferative effect two kinds of cellular types to be increased.

Usually, the M in DMSO prescription or CPE prescription ₄N dosage is higher, and two kinds of C-33A cells of inducing and the cell death percentage ratio of HeLa cell are higher.Yet, M ₄The M of the DMSO recipe ratio respective concentration of N ₄The CPE prescription of N is larger to the toxicity of cell.Yet, the M of test in the DMSO prescription ₄N Cmax (80 μ M or 28.7 μ g/mL) causes cell death in about 40% cell mass, in this experiment, and the M of same concentrations ₄The CPE of N prescription is only causing cell death in about 20% the cell mass.

These results are repeatably in two kinds of cell lines.The data declaration that obtains from this research is with the M in the DMSO prescription ₄N is similar, the M in the CPE prescription ₄N can stop cell proliferation, yet causes lower cytotoxicity than the DMSO prescription.

Collect the data of continuous time point, with test CPE prescription effectiveness in time.These data show were stored in 2-8 ℃ after 12 months, and the CPE prescription is still effective as new.The survival rate of cell keeps similar in 12 middle of the month that the CPE prescription is stored.Cell death is with in the rate of increase remains on identical scope.

Collect data, more former CPE prescription and new CPE 25/30 prescription.Research is the 0th and carry out March, and with test formulations effectiveness in time, and the new prescription of test is than the effect of old prescription.Data show CPE 25/30 prescription is the same effective with former CPE prescription in the growth of inhibition tumor cell.Using CPE prescription or CPE 25/30 prescription, to process in the HeLa cell with various drug level, the survival rate of cell is similar.Cell death is with propagation even remain in time in the identical scope.

Gather information is with more former CPE prescription and CPE 33/27 prescription effect to the HeLa cell in the 0th time.Data show, CPE 33/27 has identical effect at the cell survival rate of HeLa cell, percentage ratio and the rate of increase of dead cell.

Table 6 M ₄The DMSO of N or CPE prescription are on the impact of C-33A cell

Table 7 M ₄The DMSO of N or CPE prescription when the 0th time on the impact of HeLa cell

Table 8 M ₄The DMSO of N or CPE prescription when JIUYUE on the impact of HeLa cell

Table 9 M ₄The DMSO of N or CPE prescription when December on the impact of HeLa cell

Table 10 CPE prescription and CPE 25/30 comparison of prescription in the HeLa cell

Table 11 CPE prescription and the comparison in HeLa cell during March of CPE 25/30 prescription

Table 12 CPE prescription and the comparison in HeLa cell during the 0th time of CPE 33/27 prescription

Embodiment 3. can use multiple batch M ₄N, and reach same effect

M to multiple batch ₄N tests, to show the effect of different batches medicine.With the CPE prescription, with the M that increases progressively ₄The N amount: 0 μ M, 20 μ M, 40 μ M, 60 μ M and 80 μ M process 72hr to the HeLa cell.Add each prescription and reach 1% of culture medium (minimal essential medium that contains L-glutaminate has been added 10% hyclone, 1mM Sodium Pyruvate, the nonessential Freamine Ⅲ of 1x and 1000 IU/mL penicillins/1000 μ g/mL streptomycin solution) total amount.Control cells is cultivated under the same conditions, but does not process.After the processing or non-processing of 72hr, count total cellular score and living cells quantity in each sample by using trypanblue exclusion method.The cell proliferation rate of analysis in each sample and the percentage rate of dead cell.The result of this experiment is shown in table 13 and 14.These results show, no matter use which kind of M ₄N batch, the effect of medicine is consistent.

Table 13 is with various batches M ₄N processes HeLa cell (1001 crowdes of EM)

Table 14 is with various batches M ₄N processes HeLa cell (1002 crowdes of EM)

Embodiment 4. intravenous injection toxicity tests

The purpose of this test is to measure when by intravenous injection during to male and female beagle administration EM-1421 (M ₄N) maximum tolerated dose, wherein beagle is provided by ConvinceResearch Products (Cumberland, Virginia, USA).Article two, about 6 to 9 months large beagles when first time administration, one male one is female, contain the cyclodextrin excipient of 20% (w/v) HP-β-CD and 50%PEG 300 in test day (" SD ") 1 administration, subsequently to be prepared in the M of the gradually increase in the cyclodextrin excipient ₄N (that is, trial drug) administration.The cyclodextrin excipient that is prefabricated into and trial drug are in room temperature preservation.As the sterilized water of diluent from Baxter HealthCare company (Deerfield, Michigan, USA) or Abbott Laboratories (northern Chicago, Illinois, USA) obtains and in room temperature preservation.

This trial drug, cyclodextrin excipient and diluent are considered to prescription 100% sterling.By with asepsis injector an amount of trial drug or cyclodextrin excipient being added in the glass container, prepare dosage formulation in each administration sky.For each dosage, the sterilized water of equivalent is added to M ₄In N prescription or the cyclodextrin excipient, with form 50: 50 dilution of (v/v).200mg/kg dosage need not dilute.Mix all prescriptions by soft inversion, to guarantee to form suitable solution.Prescription is packed in the medicine box, and at room temperature store until need to use.

At SD3, M ₄N carries out administration with 25mg/kg, at SD5, and M ₄N carries out administration with 50mg/kg, and at SD8, M ₄N carries out administration with 100mg/kg.Only jenny is accepted full 100mg/kg dosage; Because the mechanical accident that precipitation causes in infusion tube, male dog is accepted the script dosage of about 72mg/kg (that is, 72%).The two other beagle carries out administration with the dosage of 200mg/kg; Yet mechanical accident occurs in succession, and the script dosage of the dog about 180mg/kg of acceptance (90%), and the amount that bitch is accepted can not be measured.

At SD13, because the mechanical breakdown of pump can not be finished dosed administration, this mechanical breakdown may be relevant with the viscosity of the prescription of 12.5mg/mL.Untapped prescription is transferred in the brown reagent bottle and in room temperature preservation, until again be made into prescription at SD15 by diluting with sterilized water 50: 50 (v/v).Then, the 6.25mg/mL diluent is changed in the medicine box.

Before the first dosage, make the animal of test adapt to laboratory condition at least 7 days, and by veterinary work personnel release quarantine.During this period, each animal is identified by ear tag, and puts at each cage and to record interim numbering.In a conventional manner, and according to USDA (United States Department of Agriculture (USDA)) animal welfare bill (Animal Welfare Act), about the PHS policy (the PHS Policy on Humane Care and Use of Laboratory Animals) that humanity is looked after and laboratory animal uses, and the U.S. is inter-sectional zoologizes committee about using and looking after the principle (U.S.Interagency Research Animal Committee Principles for the Utilization andCare of Research Animals) of zoologizeing, and animal is taken care of.

Except specified otherwise, foodstuff and water are unrestrictedly supplied.In diet and water, there is not the pollutant level of can impact obtaining this test objective.Provide good exchanging with the mutual of the mankind to animal, such as during the administration and when carrying out physical examination, stroke, wiping, and speak.Because the vein blood vessel catheterization, the test Canis familiaris L. activity of can not coming out of steamer behind the surgical procedures.Rubber Kong toy or nylon toy are provided in cage.

Use sterile surgical technique, inlying catheter is inserted in the jugular vein of animal, and the same day animal is carried out Prevention Processing in conjunction with analgesic and antibiotic at surgical operation, and after surgical operation, processed animal 8 days with antibiotic and/or analgesic.Result of study is summed up and is listed in the table 15,16 and 17.

Table 15 in the PEG of toxicity research 300/HP-β-CD prescription to the M of beagle administration ₄N dosage

Table 16 infusion M ₄The whose body weight of the beagle of N (in kg)

SD=tests the sky

M after the table 17 infusion various dose (in mg/kg) in the individual beagle body ₄N serum-concentration (in mg/mL)

ND=does not detect;＜LLOQ=is lower than lower limit of quantitation

Dosage 1,2 and 3 smooth delivery of power.At SD8 days, during 100mg/kg dosage, syringe pump quit work in advance, and only the 88.6mL of former 123.2mL dosage is transfused in the dog (No.10529).Manually restart as required pump, but only bitch (No.10530) has been accepted full dosage.

At SD15 days, with tester prescription and the 8hr target infusion time of dilution, attempt 200mg/kg dosage is inputted in No. 10567 Canis familiaris L. and No. 10568 Canis familiaris L. body.Behind dosed administration 7hr45min, the pump of dog stops.Therefore, dog (No. 10567) is only accepted the 289mL of expection 320mL dosage.Behind dosed administration 2hr12min, find that infusion needle breaks away from from the infusion line of bitch (No. 10568), dosed administration stops.M in the vein input bitch body ₄The N amount can not be measured.

After finishing each venoclysis, use the 1mL Sterile Saline slowly to inject, thereby test material is poured from infusion line in the animal body, input subsequently heparin-saline solution to fill infusion line to keep open.Animal is observed.The cage side is observed and is comprised death, condition of illness (morbundity), comprehensive health and poisoning sign.Clinical observation comprises skin and fur characteristic, surgical procedures site, eye and mucosa, respiratory system, blood circulation, certainly advocate peace central nervous system and the evaluation of body power and way of act.Gather blood, and blood is placed the 2.5mL serum separator tube, to carry out serum M ₄The N concentration analysis.These pipes are inverted for several times, are stored on ice wet, at 4 ℃ with the centrifugal about 10min of about 3000rpm.Serum transfers is stored to micro-centrifuge tube and under-75 ± 10 ℃.

Sample in dry ice is transported to the Experiment of Analytical Chemistry chamber (Genelogic, Maryland, USA) of Genelogic and carries out the tester analysis.The method relates to the acetonitrile treatment dog serum of the equal portions of 0.5mL at least, filters, and filtrate is injected the HPLC post (LC-MS/MS) that detects with tandem mass spectrum.Analyte carries out quantitatively with the outer standard curve of ALprazolanic as internal standard substance.

Dog No.10529 and bitch No.10530 are respectively in SD9 and eliminating from test in 17 days.At SD17 days dog No.10567 and bitch No.10568 are carried out euthanasia and blood drawing.All animals are by intravenous injection Nembutal

Carry out euthanasia.

After death, as quickly as possible all Canis familiaris L.s are carried out obduction.Carry out all sidedly obduction, comprised outer surface, surgical procedures site, all perforates, skull, breast and abdominal cavity and the content thereof of having a physical examination.Prepare bone marrow smear by femur marrow.Air drying is carried out in section, fix with methanol, and preserve to be used for carrying out further possible evaluation.Tissue is kept in the formalin (" NBF ") of 10% neutral buffered.In addition, collect with blood vessel and obtain the conveyance conduit that end (" VAP ") links, in the two ends knotting, be stored among the NBF.Kidney, lung, bladder and any gross lesion are reached the veterinary pathologist that is authenticated by committee by embedding, section, dyeing and detect.In detected tissue, do not find the test substances with disease association.

The result shows, with M ₄N treats not impact of mortality rate.On not significantly relevant clinical observation or the impact for the treatment of of body weight.Find or microscopic variation without the macroscopic pathology that test substances is relevant.M ₄The serum level of N is being right after the infusion after date for the highest, in all measuring intervals of TIMEs, and the M of bitch ₄The N level is higher than dog.At infusion 16hr after the phase, M ₄N still can detect, and detection limit increases along with the increase of dosage.

In a word, reaching under the dosage of about 200mg/kg, do not observing the relevant side effect for the treatment of.

The venoclysis toxicity test that embodiment 5 replenishes

A. the M in beagle ₄7 days venoclysis toxicity research of N (CPE)

This research is in order to measure when being formulated in the M among 30%HP-β-CD and the 25%PEG 300 as single venoclysis dosage successive administration in beagle in the time of 7 days ₄The toxicity kinetic curve of N.This test has consisted of M ₄N should specifically fill a prescription first research in Canis familiaris L., with the evaluate toxicity toleration of comparison diluent (10mg/mL is to 5mg/mL) also.

With the venoclysis of 54 minutes (group 2) or 108 minutes (group 1,3 and 4), to 12 beagles (8 heros/4 are female) administration M altogether ₄N.7 dosage of all animals received, once a day, continuous 7 days.

Estimate following parameter: clinical observation and body weight.The trier analysis is carried out in the test days 1 and 7, at following time point from the suitable blood vessel blood sample collection of each animal (index is the 2.0mL volume):

Roughly dissect organizing 1 and 3 animal, and all injection site are processed for the histology.Detailed clinical observation has disclosed following discovery: it is uncomfortable to observe of short duration reversible hind leg in group 2,3 and 4.Most of these observe phenomenas are observed in group 3 and 4.In group 1 and 3, observe the urine of variable color.Because these discoveries are found in vehicle group, so this may be relevant with excipient.Observe the enlargement in three routine forelimb zones in 1,2 and 3 in group, start from the 3rd day, the 4th day finally.This discovery may represent because a plurality of syringe needle inserts the inflammation that same dose administered area (cephalic vein) causes.In group 3 jennies, observe rapid breathing of an example into isolated event, and after this do not observe again.At last, observe fragmentary diarrhoea, vomiting, mucus feces and soft excrement.These observed results are comparatively general in beagle, and it is relevant to be considered to because pressure produces tester.

The analysis of serum shows, the ebb (being nominally 18759ng/mL) of tester concentration is found in accepting group 4 animals of 45mg/kg (5mg/mL).Higher peak concentration (being nominally 33420ng/mL) is found in accepting group 2 animals of 45mg/kg (10mg/mL).The same with expection, maximum concentration (being nominally 46704ng/mL) is found in accepting group 3 animals of 90mg/mL (10mg/mL).The time of the Cmax of all groups is (group 2 is 0.9 hour, and group 3 and 4 is 1.8 hours) when infusion is finished.In accepting any group of 1 animal sample of excipient, do not observe tester.The concentration curve of single administration is in Fig. 3 A (non-logarithmic scale) and Fig. 3 B shown in (logarithmic scale).

Roughly dissect organizing 1 and 3 animal.For carrying out Histological research, collect injection site and processing.Histological research shows that these of the injection site ratio treatment animal of contrast are subject to less impact, when comparing with contrast, and the thicker and variable color of injection site for the treatment of animal.These hints be that tester has larger zest than excipient.In zootomy, do not observe other visual findings.

B. the M in beagle ₄N (CPE) is with and repeated the venoclysis toxicity test in 28 days convalescent 14 days

By being with venoclysis in convalescent 14 days in 28 days to male and female beagle administration CPE (12.5mg/mL is at 20%HP-β-CD, among the 50%PEG 300), to measure the xicity related reversibility of any treatment.At first, article 32, Canis familiaris L. (16/ sex) is divided into 4 groups of (5/ sexes in the group 1 and 4 at random, 3/ sex in the group 2 and 3), and with %22,45 or the 50%PEG300 of 90mg/kg dosage in 20%HP-β-CD (placebo) or tester (CPE) carry out administration.All animals are accepted altogether 14 dosage once a day by venoclysis.After dosed administration, 3 Canis familiaris L./sex/groups were carried out euthanasia at SD16 days, and 2 Canis familiaris L.s of remainder/sex keeps experiencing 28 days convalescent period in the group 1 and 4.

The parameter of assessment comprises mortality rate, clinical observation result, body weight and body weight change, food consumption, ophthalmology, cardiology, clinical pathology, organ weight in this research, and fractographic microcosmic pathology.

CPE treats mortality rate without impact; All animals all survive when stopping according to plan.On body weight, body weight change or food consumption without impact.The visual impact that not to be noted treatment is correlated with.Observe the of short duration evidence of twice reversible CNS active (ataxia) with it the Canis familiaris L. of 90mg/kg.Every other clinic observation result is considered to isolated and occurs, and is irrelevant with treatment.

There is not the relevant cardiology impact of tester.When predose and SD13 days, have ventricular premature contraction with of placebo treatment is male, and at SD1 days, a female body of processing with 45mg/kg CPE had the atrial pulsation that does not conduct to ventricle; These discoveries are not thought to treat relevant.

The relevant impact of obvious tester does not appear in Clinicopathological Parameters.SD15 days, in the buck of processing with 22.5mg/kg CPE, find significantly lower absolute Reticuloyte count; Yet this variation is MIN, and is similar with the predose value, do not think to have biology or toxicology significance.

At SD16 days, have significance,statistical with 22.5 with the male absolute splenocyte weight of 45mg/kg CPE processing and change relevant with the middle individual animals of matched group (placebo).In not significantly difference of relevant organ weight.

Be considered to less important in the damage of the infusion site point discovery of all animals than the venoclysis process at SD16 days when dissecteds, and irrelevant with treatment.Find that in a female body of 45mg/kg CPE group the infusion site reddens, be multifocal, slight, inner membrance, Aldosterone with microscopic examination relatively, many focuses of smooth muscle cell, slight blood vessel hyperplasia, reach many focuses, slight, subacute veins beneath the skin Zhou Yan, shown the inflammation that is produced by the dosed administration technology.Male and the jenny of selecting in placebo and 90mg/kg CPE group shows because the diffusion that the mode of euthanasia causes, slightly congested to the spleen of moderate.The every other macroscopic and microscopic discovery of having noticed in SD16,42 and 43 days dissection is considered to accidental and non-treatment dependency.

In a word, in beagle, with after the CPE venoclysises in 14 days that reach 90mg/kg dosage, do not find disadvantageous treatment relative influence.

C. IV dosage range research in beagle

The wall scroll dog is by the M among IV infusion (10mL/kg/hr) administration 20%HP-β-CD/50%PEG 300 ₄N (12.5mg/mL).The increase of infusion line internal pressure and test recipe have occured to be leaked from syringe.Dosed administration causes salivation, platycoria, red urine, serious ataxia, trembles and spasm.Because observed result is considered to very serious, animal is considered to impending death, and euthanasia is carried out in plan.As if before euthanasia, Canis familiaris L. is recovering; All previous clinic observation results that find significantly descend on seriousness.

Carry out the trial of same dose the wall scroll bitch with the infusion rate of 5mL/kg/hr, cause after the dosed administration inharmonic/unbalanced motion and urinate frequently.Canis familiaris L. recovered in 1 hour 15 minutes, and showed without continuous or other bad clinical indication.

Hero in addition and female body are with 150mg/kg and carry out dosed administration with 125mg/kg subsequently.During dosed administration, serious ataxia, nystagmus, red urine or excrement occur, urinate frequently, sounding, dyspnea, vomiting occur at these dosage levels, and/or salivate.Two animals recover in the 1hr of dosed administration, and do not find subsequently other important clinical sign.

200,150 and 125mg/kg subsidiary clinical, the cage side of observing, dosed administration after, or irregular observed result comprise nose and/or ear slight erythema, drain and change (soft and/or with the feces of mucus) and a case each clear nasal mucus and red vaginal discharge.These find not frequent occurrence, and are considered to common experiment discovery, and are irrelevant with treatment.At SD43 days, customary moving walking haltingly occured in the female body of accepting 125mg/kg during dosed administration, and it is attributable to the use of dosed administration suspender belt, also be considered to treat uncorrelated.

Two other Canis familiaris L. (nature) is added in the testing program, and carries out dosed administration with 75mg/kg/ days, infusion rates 5mL/kg/hr, carries out total 12 dosage in 13 days interval.Observed result is the single observed result SD7 days female rufous urine behind unique dosed administration of noticing; The meaning of this phenomenon is unknown.Clinical observed result is included in the soft excrement in the male body and the white vomitus that bubbles in female body; These are found to be rare or isolated event, do not think relevant with treatment.SD2 days, in male body, found observed result behind the dosed administration of weak limbs, this is attributable to have used the suspender belt constraint.Two Canis familiaris L.s recover from any clinical symptoms of finding in dosed administration 1hr.With M ₄N treats body weight without impact, and does not find the damage that naked eyes can be observed at when dissected.Do not find the observed result that other treatment is relevant.

D. 8 days IV infusion toxicity research that in the Sprague-Dawley rat, carry out

To be dissolved in the M of the 10%HP-β-CD in PEG 300 ₄N (6.25mg/mL) 0,100,150, or the dosage that raises gradually of 200mg/kg once carried out administration with 8mL/kg/hr to male and female Sprague-Dawley rat (3/ each sex) by the IV infusion in per four days.After four times initial infusions, test extends to and comprises and continue 4hr, reach 14 days infusion every day.Do not occur and M ₄The obvious mortality rate that N is relevant.During the excipient administration, find that a male Mus dies from first day dosage, behind the test material of infusion 100mg/kg, female Mus is found to die from the 5th day of test.It is relevant with trier that these death are not thought, because Mus administration tester (only excipient) not, and for the another Mus, under the facts of other rats with the many survivals all over the world of dosed administration in 200mg/kg/ days, this is separate event.

The observed result clinical and that dissect that occurs is less important with respect to port and infusion line.These observed results comprise the swelling of cervical region or axillary fossa, and the constrained motion of hind leg.During continuous every day of this research dosed administration, occurred trembling a male and female rat.In addition, at a hero and a female rat ptosis and hyperkinetic syndrome shape have appearred.These are only clinic observation results relevant with 200mg/kg/ days tester dosage.

Male and female rats all increases in whole duration of test body weight.The highest increased weight appears at after the excipient administration.After excipient or tester administration, the consumption of food is similar.

Limited quantity based on the rat of estimating in this test reaches continuous 8 days with excipient or tester administration up to 200mg/kg/ days dosage, shows what rat can tolerate, only causes some disadvantageous clinic observation results every day behind the dosed administration.The most of disadvantageous clinic observation result who occurs and all dissections find to be considered to for relevant to port and the pipeline of rat dosed administration.When putting to death, all infusion line or from jugular vein, shift out and/or do not find in port/pipeline location patent thing and material (being assumed to tester).

Embodiment 6.M ₄The dissolubility of N in modified cellulose

(w/v) solution is following is prepared as the 10ml 50%HP-β-CD (w/v) of solubilizing agent and/or excipient and 0.5% hydroxypropyl emthylcellulose (" HPMC "): 5.9mL is suitable for placing the glass beaker that contains stirring rod to animal water for injection (" WFI ").This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.5g HP-β-CD is added among the WFI of continuous stirring lentamente, uses spatula with the central authorities of HP-β-CD guiding beaker.This HP-β-CD solution is stirred about 24 hours, perhaps until under the perusal HP-β-CD dissolve fully.Gained solution is measured as about 9.4mL.The WFI of about 0.6mL is added to makes it to reach 10mL in the gained solution, to generate 50%HP-β-CD (w/v) solution.50mgHPMC is added in 50%HP-β-CD solution, stirs 1hr or until HPMC dissolving under the perusal.With the about 1hr of final solution stirring, keep in Dark Place in room temperature subsequently.The method for preparing modified cyclodextrin and modified cellulose can zoom in or out to obtain the HP-β of required volume or concentration-CD/HPMC solution in proportion.Other modified cyclodextrins/modified fibre cellulose solution can similarly make, and for example in said method, HP-β-CD is replaced with the cyclodextrin of other modifications, or HPMC is replaced with the cellulose of other modifications.

With the 10mL M of about 10mg/mL concentration in 50%HP-β-CD/0.5%HPMC ₄N solution is prepared as follows: 50%HP-β that will about 10mL-CD/0.5%HPMC solution is added to and contains in the stirring rod glass beaker.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Will about 100mg M ₄N is added among 50%HP-β-CD/0.5%HPMC lentamente, is added to the central authorities of beaker under the help of spatula.This M ₄N/50%HP-β-CD/0.5%HPMC mixture is stirred about 24 hours, perhaps until all M ₄N evenly suspends and does not have any gathering.This M ₄N/50%HP-β-CD/0.5%HPMC mixture can be at 90 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄N/50%HP-β-CD/0.5%HPMC mixture is at 90 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By with beaker facing to white background, observe granule facing to black background afterwards existence whether, the described M of observable ₄Whether N/50%HP-β-CD/0.5%HPMC mixture exists any undissolved M ₄N.Final M ₄N/50%HP-β-CD/0.5%HPMC solution is in room temperature storage, and keeps in Dark Place.When 90 ℃ of heating, M ₄N can 1mg/mL and 10mg/mL be dissolved in this 50%HP-β-CD/0.5%HPMC prescription, and after cooling, still remain in the solution, the stability under the room temperature was greater than 7 days.Even under 90 ℃, M ₄N is not dissolved in this same recipe with the concentration of 50mg/mL.

Aforesaid method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N.In other cyclodextrin/cellulose solutions, contain M ₄The prescription of N can similarly make, and for example, by in said method HP-β-CD being replaced by other cyclodextrin, perhaps HPMC is replaced by other modified celluloses.

Alcoholic solution is following is prepared as 10ml 5% (w/v) ethyl cellulose (" EC ") of solubilizing agent and/or excipient: 10mL 100% ethanol (" EtOH ") is placed the glass beaker that contains stirring rod, cover circular Teflon

Lid.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.500 (500) mg EC are added in the ethanol of continuous stirring lentamente, use spatula with the lead central authorities of beaker of EC, to avoid the EC powder adherence on walls of beaker.This EC solution is stirred about 2 hours, perhaps until under the perusal EC dissolve fully.Final solution is kept in Dark Place in room temperature.

The method for preparing the modified fibre cellulose solution can zoom in or out to obtain required volume or concentration in proportion.Other modified fibre cellulose solutions can similarly make, and for example in said method, EC are replaced with the cellulose of other modifications.

With the 10mL M of about 20mg/mL concentration in 5%EC (w/v) (" EC " prescription) ₄N solution is prepared as follows: the 5%EC prescription of about 10mL that will as above prepare is added to and contains in the stirring rod glass beaker.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed, and cover circular Teflon Lid.Will about 200mg M ₄N is added in the 5%EC prescription lentamente, is added to the central authorities of beaker under the help of spatula, sticks on the walls of beaker to avoid M4N.This M ₄The N/EC mixture is stirred about 2 hours, perhaps until all M ₄N dissolving or evenly suspend and do not have any gathering.This M ₄The N/EC mixture can be at 60 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄The N/EC mixture is at 60 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By beaker being faced toward white background, seeking granule, the described M of observable facing to black background afterwards ₄Whether N/EC mixture or solution exist any undissolved M ₄N.Final M ₄N/EC solution keeps in Dark Place in room temperature.

Aforesaid method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N, and can increase or reduce heating-up temperature so that M ₄The N dissolving.In other modified celluloses, contain M ₄The prescription of N can similarly make, for example, and HPMC, MC (methylcellulose), and CMC (carboxymethyl cellulose).M ₄The solubility results of N in modified cellulose listed in table 18.

The result shows, M ₄N can be dissolved in the EC prescription and need not heat with 1mg/mL, this solution at room temperature stability greater than 3 days.M ₄N can 10mg/mL concentration dissolve under 40 ℃, and still remains on after cooling in the solution, and this solution stability at room temperature was greater than 3 days.M ₄N can 20mg/mL concentration be dissolved under 60 ℃ in the EC prescription, and still remains on after cooling in the solution, and this solution stability at room temperature was greater than 3 days.M ₄N can 30mg/mL concentration be dissolved under 60 ℃ in the EC prescription, but can not remain in the solution after the cooling.The M of higher concentration ₄N such as 50mg/mL or 100mg/mL level, may be dissolved in the EC prescription at 90 ℃, but can not remain in the solution after cooling.

Table 18 M ₄The dissolubility of N in containing the prescription of modified fibre

Embodiment 7 M ₄The dissolubility of N in water solublity lipid or water-miscible organic solvent

With the 10mL M of about 50mg/mL concentration in Oleum sesami ₄N solution is prepared as follows: about 10mL Oleum sesami is added in the glass beaker that contains stirring rod.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Will about 500mg M ₄N is added in the Oleum sesami lentamente, is added to the central authorities of beaker under the help of spatula, to avoid M ₄N sticks on the walls of beaker.This M ₄N/ Oleum sesami mixture is stirred about 24 hours, perhaps until all M ₄N dissolving or evenly suspend and do not have any caking.This M ₄N/ Oleum sesami mixture can be at 60 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄N/ Oleum sesami mixture is at 60 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By beaker being faced toward white background, seeking granule, the described M of observable facing to black background afterwards ₄Whether N/ Oleum sesami mixture or solution exist any undissolved M ₄N.If the formation crystallization, this M ₄N/ Oleum sesami solution can place to add follows stirring again at 60 ℃ of about 1hr of heating, until all M on the pyromagnetic force platform ₄In molten time solution of N.Final M ₄N/ Oleum sesami solution keeps in Dark Place in room temperature.

Aforesaid method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N, and can raise or reduce heating-up temperature so that M ₄The N dissolving.In other water solublity lipids, contain M ₄The prescription of N can similarly make, for example, and by in said method, Oleum sesami being replaced by Semen Maydis oil, olive oil, soybean oil, Oleum menthae, or other solubilizing agents, and combination.The result is shown in the table 19.

Table 19 demonstration, for example, at 60 ℃, M ₄N can 1mg/mL to the concentration range that reaches 100mg/mL, remove the 100mg/mL level, be dissolved in the Semen Maydis oil, still remain in the solution after the cooling, 1 and 10mg/mL concentration under this stability of solution greater than 3 days, 20,40 and the 50mg/mL level under this stability of solution less than 3 days, this stability of solution was less than 1 day under the 60mg/mL level.In addition, at 60 ℃, M ₄N may be dissolved in the olive oil with the 30mg/mL level, but can not remain in the solution after the cooling.

In Oleum sesami, M ₄N, 60 ℃ of solubilized, still remains in the solution after the cooling with the concentration of 20mg/mL, 30mg/mL and 50mg/mL in room temperature with the 10mg/mL level.Less than 3 days, and 50mg/mL solution stability at room temperature was less than 1 day greater than 3 days, 30mg/mL solution stability at room temperature for 10mg/mL and 20mg/mL stability at room temperature.

With about 60mg/mL concentration at 85% Oleum sesami and 15%Tween

10mL M in 20 ₄N solution is prepared as follows: about 8.5mL Oleum sesami is entered to containing in the stirring rod glass beaker.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Tween with about 1.5mL

20 slowly are added to the central authorities of beaker.Will about 600mg M ₄N is added to Oleum sesami/Tween lentamente

In 20 the mixture, under the help of spatula, be added to the central authorities of beaker, to avoid M ₄N sticks on the walls of beaker.This M ₄N/ Oleum sesami/Tween

20 mixture are stirred about 2 hours, perhaps until all M ₄N dissolving or evenly suspend and do not have any gathering.This M ₄N/ Oleum sesami/Tween

20 mixture can be at 60 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄N/ Oleum sesami/Tween 20 mixture are at 60 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By with beaker facing to white background, afterwards facing to black background, the described M of observable ₄N/ Oleum sesami/Tween

Whether 20 mixture exist any undissolved M ₄N.Final M ₄N/ Oleum sesami/Tween

20 solution keep in Dark Place in room temperature.If the formation crystallization, this M ₄N/ Oleum sesami/Tween

20 solution can place to add to follow on the pyromagnetic force platform and be stirred in 60 ℃ and reheat, until all M ₄In molten time solution of N.

The method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N, and can raise or reduce heating-up temperature so that M ₄The N dissolving.In other water-insoluble lipid binding non-ionic surface active agents, ionic surfactant or water-miscible organic solvent, contain M ₄The prescription of N can similarly make, for example, by in said method with Oleum sesami or Tween

20 are replaced by Semen Maydis oil, olive oil, soybean oil, Oleum menthae, Tween

80, polyethylene glycol 1000 vitamin E succinic acid ester (" TPGS "), lecithin, PEG 300, PEG 400, PEG 400 monolaurates, glycerol, polyvinylpyrrolidone (PVP), propylene glycol (" PG ") or other solubilizing agents, and combination.M ₄The dissolubility result of N in the water-insoluble lipid is shown in the table 19.

Table 19 demonstration, for example, the M of 60mg/mL ₄N is dissolvable in water at 55 ℃ and contains 85% Oleum sesami and 15%Tween

In 20 the prescription, and the M of 40mg/mL ₄N is dissolvable in water in the same recipe at 45 ℃, but cools off afterwards M in these prescriptions ₄N can not remain in the solution.On the contrary, the M of 29mg/mL ₄N is dissolvable in water at 60 ℃ and contains 89% Oleum sesami and 11%Tween

In 20 the slightly different prescription, and still can remain in the solution after the cooling, this solution is at room temperature stable greater than 7 days.

With the 1mL M of about 2.9mg/mL concentration in saline ₄N emulsion is prepared as follows: the polypropylene tube that will about 0.9mL0.9% saline solution places the 1.5mL size.About 0.1mL is present in 89% Oleum sesami, 11%Tween with 29mg/mL concentration M in 20 ₄N solution is added in this polypropylene tube.This M ₄N/ Oleum sesami/Tween

The saline solution of 20 solution or mixture is by thermal agitation 1min.Emulsion is by with this M ₄N/ Oleum sesami/Tween

The preparation in ultrasonic 5 minutes of 20 solution, wherein the amplitude of ultrasonic little end probe is adjusted to 60% maximum probe amplitude.By beaker being faced toward white background, seeking whether have granule, the described M of observable facing to black background afterwards ₄N/ Oleum sesami/Tween

Whether 20 mixture exist any undissolved M ₄N.Final M ₄N/ Oleum sesami/Tween

20 solution keep in Dark Place in room temperature.

The method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N.At other surfactant of other lipid bindings, or contain M in the water-miscible organic solvent ₄The prescription of N can similarly make, for example, by in said method with Oleum sesami or Tween

20 are replaced by Semen Maydis oil, olive oil, soybean oil, Oleum menthae, Tween

80, TPGS, lecithin, PG, PEG 300, PEG400, PEG 400 monolaurates, glycerol, polyvinylpyrrolidone (" PVP ") or other solubilizing agents, and combination.

Table 19 M ₄The dissolubility of N in the water-insoluble lipid

Table 20 shows M ₄The dissolubility result of N in water-miscible organic solvent EtOH, PG, PEG 300, PEG 400, PEG 400 monolaurates, glycerol, PVP and some combination thereof.

Table 20 M ₄The dissolubility of N in water-miscible organic solvent

Embodiment 8 M ₄The dissolubility of N in non-ionic surface active agent

With about 60mg/mL concentration at Tween

10mL M in 20 ₄N solution is prepared as follows: will about 10mL Tween

20 are added in the glass beaker that contains stirring rod.This beaker is placed the magnetic force platform, stirring rod is set stirs with middling speed.Will about 600mg M ₄N is added to Tween lentamente

In 20, under the help of spatula, be added to the central authorities of beaker, to avoid M ₄N sticks on the walls of beaker.This M ₄N/Tween

20 mixture are stirred about 2 hours, perhaps until all M ₄N dissolving or evenly suspend and do not have any gathering.This M ₄N/Tween

20 mixture can be at 60 ℃ of heating 30min.If (perhaps need larger liquor capacity, for more time, for example, this M of 500mL ₄N/Tween 20 mixture are at 60 ℃ of heating 1hr), maybe need to guarantee M ₄The longer time that N fully dissolves.By beaker being faced toward white background, seeking whether have granule, the described M of observable facing to black background afterwards ₄N/Tween Whether 20 mixture exist any undissolved M ₄N.Final M ₄N/Tween

20 solution keep in Dark Place in room temperature.If form crystallization, this M at lay up period ₄N/Tween

20 solution can place to add to follow on the pyromagnetic force platform and be stirred in 60 ℃ and reheat about 1hr, perhaps until all M ₄In molten time solution of N.

The method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N, and can raise or reduce heating-up temperature so that M ₄The N dissolving.In other non-ionic surface active agents, ionic surface active agent or amphiphatic molecule, contain M ₄The prescription of N can similarly make, for example, by in said method with Tween

20 are replaced by Tween

80, other solubilizing agents, and combination.M ₄N is at Tween

20, Tween

80, and Tween 20 and the combination of PEG 400 in the dissolubility result shown in the table 21.

Table 21 demonstration, at room temperature, the M of 1mg/mL concentration ₄N is dissolvable in water Tween

20 or Tween In 80.Tween

M in 20 solution ₄N (" M ₄N/Tween

20 ") at room temperature stability is greater than 7 days, and Tween

M in 80 solution ₄N (" M ₄N/Tween

80 ") at room temperature stability observing was greater than 3 days.At 50 ℃, the M of higher concentration ₄N is dissolvable in water Tween 20 or Tween In 80.In addition, Tween

Reach 60mg/mL concentration or Tween in 20

When reaching 50mg/mL concentration in 80, M after the cooling ₄N still remains in the solution, yet, Tween

When 80mg/mL and 100mg/mL level, be insoluble after the cooling in 20.Observe the M of 1 0mg/mL and 20mg/mL ₄N/Tween

20 solution are at room temperature stable greater than 7 days.Observe the M of 40mg/mL and 80mg/mL ₄N/Tween

20 solution are at room temperature stable less than 3 days.For M ₄N/Tween

80 solution, 10mg/mL solution are observed at room temperature and can be stablized greater than 3 days, and 50mg/mL solution is at room temperature stable less than 1 day.

The result also shows, when 65 ℃ of heating, and M ₄N is dissolvable in water 50%Tween

20 and the combination of 50%PEG 400, M ₄N concentration reaches 60mg/mL.After the cooling, M ₄N still remains in the solution of these prescriptions, and this solution is at room temperature stable greater than 3 days.

Table 22 shows M ₄N is in the concentration of μ g/mL and the respective concentration of measuring with μ M thereof.

Table 21 M ₄The dissolubility of N in non-ionic surface active agent

Table 22 M ₄The concentration conversion table of N

Embodiment 9 contains M ₄The lyophilizing prescription of HP-β-CD of N

M among HP-β-CD ₄N concentration is following being prepared of 120mg lyophilized powder of about 185mg/g (w/w).Use HP-β-CD and the M of equimolar amounts ₄N increases HP-β-CD and M ₄Complexation ratio between the N.With HP-β-CD of about 98mg and the M of about 22.2mg ₄N mixes in the polypropylene tube of 1.5mL size.Will about 0.2mLWFI be added to and contain HP-β-CD/M ₄In the mixture in the polypropylene tube of N mixture of powders, and vibrations 1min is to produce HP-β-CD/M ₄The water slurry of N.This HP-β-CD/M ₄The N suspension is at-20 ℃ of freezing 24hr.Subsequently, with HP-β-CD/M ₄The N suspension with under 1400rpm, the vacuum, 60 ℃ of centrifugal about 2hr, so that all water is removed from suspension.HP-β-CD/M ₄The heavily about 120mg of the dry powder of N complex.Subsequently, with HP-β-CD/M ₄The N powdered compound dissolves or Eddy diffusion in water or other solubilizing agents.Final M ₄N/HP-β-CD powdered compound keeps in Dark Place in room temperature.The method can zoom in or out to obtain the M of volume required or concentration in proportion ₄N.Contain M ₄The prescription of N and other cyclodextrin can similarly prepare, and for example, in said method, HP-β-CD is replaced with other cyclodextrin.Be presented at the presentation of results in the table 1, lyophilization HP-β-CD/M ₄HP-β-the CD/M that obtains behind the N suspension ₄The N composite powder contains about 81.5%HP-β-CD and 18.5%M ₄N.

M among HP-β-CD/HPMC ₄N concentration is following being prepared of 400mg lyophilized powder of about 150mg/g (w/w).About 1mL50%HP-β of as above making-CD/0.5%HPMC solution is added in the polypropylene tube of 1.5mL size.Will about 60mgM ₄N is added in the mixture in the polypropylene tube that contains HP-β-CD/HPMC suspension, and concussion 1min is to produce HP-β-CD/HPMC/M ₄The N suspension.This HP-β-CD/HPMC/M ₄The N suspension is at-20 ℃ of freezing 24hr.Subsequently, with HP-β-CD/HPMC/M ₄The N suspension with under 1400rpm, the vacuum, 60 ℃ of centrifugal about 5hr, so that all water is removed from suspension.HP-β-CD/HPMC/M ₄The heavily about 400mg of the dry powder of N complex.Subsequently, can be with HP-β-CD/HPMC/M ₄The N powdered compound dissolves or Eddy diffusion in water or other solubilizing agents.Final M ₄N/HP-β-CD/HPMC powdered compound keeps in Dark Place in room temperature.This invention can zoom in or out to obtain the M of volume required or concentration in proportion ₄N.Contain M ₄The prescription of N and other cyclodextrin/cellulose solutions can similarly prepare, and for example, in said method, HP-β-CD is replaced with other cyclodextrin, perhaps HPMC is replaced by other modified celluloses.Be presented at the presentation of results in the table 18, lyophilization HP-β-CD/HPMC/M ₄HP-β-the CD/HPMC/M that obtains behind the N suspension ₄The N composite powder contains about 84%HP-β-CD, 1%HPMC and 15%M ₄N.

Embodiment 10 is for delivery of M ₄The test of the IV pipe of N

In this test, various types of tube for transfusions are carried out its compatibility of test and optimize M ₄The vein of N is carried.The test of relative consistency is analyzed based on visual observations and HPLC (high pressure liquid chromatography).

Visual observations is following carries out: the prescription of about 5mL that will test (" formula materials ") is by the long tube for transfusion of about 20cm.At room temperature (25 ℃) remain on Guan Zhongyue 24hr with this formula materials.Subsequently, this formula materials is circulated repeatedly (for example, about 5 times) by this tube for transfusion, and be collected in beaker or the collecting pipe.Check carefully this formula materials sees precipitation or microgranule whether occur, comprises this beaker or collecting pipe are faced toward white background, subsequently facing to black background.

The compatibility test of using HPLC to analyze changes in method along with the purpose of tested tube for transfusion, time span and tube for transfusion.This thermometrically is by M in the solution before and after the different tube for transfusion circulations ₄The amount of N.

The marginal value that determines the compatibility is 90% analytical pure, refers to after finishing with tube for transfusion interaction and test, if about 90% M ₄N remains in the solution, and this material can be considered to the compatibility.Any amount that is lower than 90% marginal value is considered to incompatible tube for transfusion.The result is summarised in the table 23.

This tests demonstration, PTFE (politef) and fluorubber (Barnant company, Barrington, U.S. Yi Linuosi state), polypropylene (Cole-Parmer, Vernon Hills, Illinois, USA), FEP (perfluoroethylene-propylene) (Saint-Gobain, Mickleton, N.J.), PTFE (Cole-Parmer), polyethylene (Intramedic, Becton Dickinson, Nicholas Sparks, Maryland, USA) and platinum process silicone rubber (small size) (Cole-Parmer) for the M that carries in the prescription described herein ₄N has the compatibility.

Table 23 is for delivery of M ₄The compatibility tubing of N

^*Not preferred, but have some compatibility

(intravesicular) administration in the interior after birth of embodiment 11 pharmaceutical compositions

One or more aforementioned pharmaceutical compositions can be with administration in the interior after birth, such as carried out for 6 weeks weekly original position treatment bladder cancer with 800mg dosage.Said composition contains API, such as NDGA chemical compound, for example M ₄N.Described API, for example M ₄N is present among the compositions with every 5mL bottle 200mg, and at 2 ℃-8 ℃ (36

-46

) store in the refrigerator down.With described compositions before experimenter's administration, medicine bottle is taken out from refrigerator, and allows not heat and get back to room temperature.Can be by adding 800mg (20mL) to 0.9% normal saline solution of 55mL, USP dilutes compositions.Carry out Intravesical instillation by interior after birth administration API (when as above diluting, being total to the 75mL volume), so that API keeps 2hr, emptying subsequently.Use aforesaid compatibility tube for transfusion.

If bladder is damaged, inflammation or perforation, suggestion doctor or patient do not contain the compositions of API, because the integrity of forfeiture mucosa can the generation systems absorption.For the patient with severe allergy bladder symptom, care should be used to uses this chemical compound, because pouring into or keeping interim API can cause the irritable bladder symptom.The patient is apprised of, and urine can be light red or rose pink in about 24hr.The 12 human body stages 0 of embodiment, in 8 healthy male subjects bodies ¹⁴The M of C labelling ₄The little dose drug dynamics research of N three intersections

When with inferior therapeutic dose, or with after the meal with the empty stomach condition under single port take dosage or single vein dosage (in table 24, being respectively mode A-C), during to the human body administration, this test is designed to estimate M ₄The absorption of N.This test is Three-way crossover scheme in the healthy male subjects target group, and is made of lasting three about 35 hours experimental stages, each stage by at least 7 days minimum between the dosed administration during the interval.In the process of each experimental stage, behind dosed administration, gather the pharmacokinetics blood sample at particular point in time, and with predetermined time interval collection urine.After the test particular procedure of 24hr was finished behind dosed administration, the experimenter can leave clinical unit.

In this research, M ₄N with the amount of 100 μ g to people's administration.M ₄N by with ¹⁴The a small amount of labelling of C (the per 100 μ g of 3.3kBq), and to volunteer's administration of health.The M of each time oral administration ₄N is included in 0.1mg's in the gel capsule of size 0 ¹⁴The M of C labelling ₄N and 376.8mg glycerin mono-fatty acid ester.The M of single venoclysis ₄N comprises 0.1mg/mL ¹⁴The M of C labelling ₄N, 30% (w/v) HP-β-CD and 25% (v/v) PEG are diluted with water to 1mL to be used for bolus infusion.Collect blood sample and urine sample from each experimenter subsequently, use accelerator mass spectrometry (AMS) analytic sample ¹⁴C content is to measure than M ₄The IV dosage of N, M ₄The oral dose of N at T _MaxThe time the maximum M that occurs ₄N concentration (C _Max), corresponding to testing (AUC _0-t) and total (AUC _0-∞) M of time ₄The gross area under the curve of total absorption of N (AUC), the final half-life (T of each sample _1/2) and each relative and total bioavailability (F _RelAnd F). ¹⁴The pharmacokinetic parameter of C average ± the SD value lists in table 24.M ₄The baseline values of N is to remaining in any remaining M among the experimenter after during between the dosed administration ₄N proofreaies and correct, thereby does not affect the M of any subsequently dosed administration ₄The N level.

Table 24 ¹⁴The meansigma methods of the pharmacokinetic parameter of C ± SD value (baseline calibration)

^aIntermediate value

Mode A: at high fat after the meal early, with 100 μ g ¹⁴The M of C labelling ₄N (3.3kBq) takes dosed administration as single port.

Mode B: after empty stomach overnight, with 100 μ g ¹⁴The M of C labelling ₄N (3.3kBq) takes dosed administration as single port.

Mode C: after empty stomach overnight, with 100 μ g ¹⁴The M of C labelling ₄N (3.3kBq) injects the intravenous solution administration as 1mL.

For the oral dose administration, the high fat morning of 100 μ g after the meal ¹⁴The M of C labelling ₄The C of total 14C behind the N oral administration _MaxValue (C _MaxThe C of total 14C behind=5.94 ± 1.33pmol/L) oral administrations that are lower than after the empty stomach overnight _MaxValue (C _Max=9.29 ± 1.40pmol/L).T _MaxWhat trend towards occurring in the subject on an empty stomach in subject after the meal is slower.In experimenter after the meal, T _Max, i.e. C _MaxTime of occurrence, alterable height and behind dosed administration in 1.5 to the 24hr scopes, in the empty stomach experimenter, T _MaxUsually occur in 1hr (0.50 to 2.00hr scope) behind the dosed administration.Experimenter's AUC after the meal _0-tValue is usually less than on an empty stomach experimenter's value.

Behind the vein dosed administration, as desired, in 8 in 10 experimenters, Cmax (C _Max=10.31 ± 1.77pmol/L) appeared at for the first sampling time (0.08hr behind the dosed administration).In 2 experimenters, T _MaxBe 0.17hr behind the dosed administration.Single port takes total behind the dosed administration among the experimenter after the meal ¹⁴The AUC of C plasma concentration _0-tValue slightly is lower than the value of vein dosed administration.On the contrary, on an empty stomach among the experimenter single port take corresponding AUC behind the dosed administration _0-tThe value that is worth a little higher than vein dosed administration.

The research prescription all has good toleration in oral and intravenously administrable.Without serious or violent ill symptoms, and the experimenter does not have the abort because of the relevant ill symptoms of test of cure.In range estimation or ECG (electrocardiogram), do not find obvious clinical change.

As follows about this research summary, after the meal with empty stomach state oral administration after M ₄The N apparent absorption is very high.When having food, than the empty stomach state, speed and the degree of absorption are lower, C _MaxTime of occurrence on an empty stomach state prolongs.These conclusions based on hypothesis be in the oral administration dosage ¹⁴The M of C labelling ₄N is not decomposed before absorption.

Embodiment 13 M ₄The Supplementary Study of N dissolubility in water-miscible organic solvent

The M that in table 25, lists ₄The evaluated 48hr that reaches of the dissolubility of N in the combination of water-miscible organic solvent.Incubated at room 2,24 and 48hr after, sample is analyzed by rp-hplc (" RP-HPLC "), with quantitative M ₄The dissolubility of N.Be preparation M ₄The N sample will be dissolved in the M of 200 μ L 100mg/mL in the acetone ₄N places the polypropylene microtubule of 1.5mL.Solvent is evaporated 48hr at room temperature, until the sample bone dry.

The mixable organic solvent prescription of preparation water in the 15mL polypropylene centrifuge tube.Prepare each 10mL prescription.Each solvent is added into based on the weight that adopts under 25 ℃ density separately.After of short duration mixing, each prescription filters by cellulose acetate (" the SFCA ") filter that 0.45 μ m does not contain surfactant, and filtrate collection is in clean 15mL pipe.Prescription in room temperature preservation until use.

Combination (table 25, wherein Benz=benzyl alcohol with each prescription of 400 μ L; Crem=Cremophor

EL; The DMA=dimethyl acetylamide; T80=Tween

80) be added in the microtubule, so that M ₄The maxima solubility of N is 50mg/mL.M ₄The dissolubility of N by incubated at room 2,24 and RP-HPLC during 48hr calculate.On each time point, with sample with the centrifugal 2min of 13000rpm to precipitate any solid M ₄N.As shown in Table 25, the prescription condition of test over half can be dissolved M ₄The concentration of N is greater than 10mg/mL.2 with during 48hr, M ₄The dissolubility of N in glycerol can not detect.

Table 25 can with the miscible organic solvent of water in reach the M of 48hr ₄The dissolubility of N

Embodiment 14 M ₄The dissolubility of N in aqueous solution

M ₄N is containing hydroxypropyl HP-β-CD or sulfobutyl ether the beta-schardinger dextrin-((Captisol of SE-β-CD)

, CyDex company, Lenexa, Kan.) aqueous solution in the room temperature in above-described embodiment 13, described of dissolubility under assessment reach 48 hours.HP-β-CD and SE-β-CD solution according to ten part 50% of weight/volume preparation.As described in the embodiment 13 for the preparation of the M of sample ₄N.Analyze each chemical compound that in volumetric flask, is weighed on the Plus balance between 1.0g and the 5.0g at OHAUS.Each sample is settled to 10mL with water for injection (WFI).After hatching 1 hour under 40 ℃, preparation liquid is filtered into clean 15mL pipe by 0.45 μ m SFCA filter.To prepare liquid, to be kept at room temperature for subsequent use.

Shown in table 26, M ₄N is at WFI, 0.9% saline, and the dissolubility in 5% glucose (D5W) is positioned at below the quantitation limit of RP-HPLC method in the whole process of this research.Note the M of increase ₄The N dissolubility is HP-β-CD and Captisol

The function of concentration and time.

Table 26.M ₄N reaches 48 hours dissolubility in aqueous solution

The prescription condition that makes the miscible organic solvent of water greater than 20 kinds can be dissolved M ₄N is to the concentration greater than 10 mg/mL.M ₄N is at WFI, 0.9% saline, and the dissolubility among the 5%D5W is lower than the detectable limit of RP-HPLC method.M ₄The increase of N dissolubility is HP-β-CD and Captisol

The function of concentration and time.

Embodiment 15 M ₄The dissolubility of N in hydroxypropylβ-cyclodextrin

M ₄The water solubility of N in variable concentrations HP-β-CD calculates by the method that Higuchi and Connors (1965) report.In brief, accurate weighing M ₄N and the M that adds with the amount that surpasses its water solubility ₄N and concentration increase progressively the HP-β of (0-350mmol/L)-CD aqueous solution, at lightly together rotation of room temperature (～12rpm) 48hr.Subsequently, with M ₄N/HP-β-CD solution filters by the SFCA filter of 0.45 μ m, and analyzes with RP-HPLC.

Although most of M ₄N/HP-β-CD complex is considered to inclusion complex, and cyclodextrin also known can formation passes through the micellelike structure and non-inclusion complex and the complex aggregation of energy dissolved substance.This phase-solubility curve is not the formation of checking inclusion complex, and only illustrates how the cyclodextrin concentration that constantly raises affects the dissolubility of medicine.M ₄N/HP-β-CD complex formation is nonlinear, but does not study accurate stoichiometry (and stability constant) in the test of this embodiment, but it can be measured by his additive method such as NMR or potentiometry.

Embodiment 16 is M in HP-β-CD/PEG 300 buffer solution ₄The stability of N

60 ℃ hatch after, calculate 10mg/mL M ₄N is (with 75: 25 40%HP-β-CD:40mg/mLM ₄The ratio preparation of N PEG 300) stability in 15mM buffer solution.

The 40%HP-β of buffering-CD solution is prepared based on weight by volume.The M that in sample, uses ₄Description among N such as the embodiment 13 is prepared.Weighing 2.0g HP-β-CD in the 5mL volumetric flask on OHAUS analysis Plus balance.The buffer solution of 1mL 100mM is added in each bottle.With WFI each sample is settled to 5mL.After 40 ℃ hatch 1hr, the SFCA filter of preparation liquid by 0.45 μ m is filtered in the clean 15mL pipe.Prepare liquid in room temperature preservation until use.

The 40%HP-β of 750 μ L-CD buffer solution is placed in the 1.5mL polypropylene microtubule.40mg/mLM with 250 μ L ₄N PEG 300 is added in each microtubule, so that dissolubility is 10mg/mL M ₄N.After coupon is overturn gently, use Orion, 420A type pH meter is measured the pH of each solution.Take out initial equal portions and carry out the RP-HPLC analysis.Subsequently, specimen is placed 60 ℃ of incubators of Precision.Measure M by RP-HPLC ₄The stability of N.At each time point, with 13,000rpm with the centrifugal 2min of sample, to precipitate any solid M ₄N.

Shown in table 27,60 ℃ hatch 14 days after, observe each M ₄The concentration of N solution has slight decline.The RP-HPLC data are the rising of show sample impurity not, and it can illustrate M ₄The degree of N lowering of concentration.Yet, at M ₄Observe the variation that relies on pH in the impurity of N, yet these impurity account for the total peak area less than 0.1%.Between incubation period, almost do not observe the variation (table 27) of apparent sample pH value.

In HP-β-CD/PEG solution, reach 14 days M under 60 ℃ in the table 27 ₄N stability

Observe after hatching 14 days at 60 ℃, which kind of apparent sample pH value or buffer no matter, stability is all unified to descend, such as M ₄Shown in the loss that N reclaims.

Embodiment 17 is M in 11-14mg/mL PEG 300/HP-β-CD solution ₄The stability of N

Be the specification requirement of supporting to make, this test has detected different M ₄The 24hr room temperature stability of the combination of PEG300/HP-β-CD under the N aimed concn.M ₄The sample for subsequent use of N is in 100%PEG300, with the 33-56mg/mL drug level, is prepared as follows under 60 ℃.

The method that employing is described in embodiment 13 and 16 is with M ₄The N bulk drug is dissolved to concentration in PEG 300 be 33,44,48,52,55 and 56mg/mL (w/w), hatches subsequently at least 2hr in 60 ℃.At the above M of 44mg/mL ₄The abundant dissolving of N needs thermal agitation and mixing.With M ₄The N stock solution is filtered by the SFCA filter of 0.45 μ m, and uses among the 30min after preparation.In addition, preparation 40%HP-β-CD (w/v) solution and filtration in aseptic WFI.40%HP-β-CD reserve liquid and M ₄N/PEG 300 reserve liquids mix in the polypropylene microtubule of 1.5mL.When incubated at room 2 and 24hr, measure M with RP-HPLC ₄The stability of N.

M with aequum ₄N adds among 40%HP-β-CD, to produce medicine and the excipient of the listed ultimate density of table 28.Sample at room temperature jiggle (～12rpm).Hatching 2 and during 24hr, sample is with the centrifugal 2min of 13,000rpm, takes out the increment such as 50 μ L and carries out RP-HPLC and analyze.Which kind of target M no matter ₄N or prescription, hatch 24hr after, dissolubility does not almost observe any variation (table 28).

Table 28 is M in 11-14mg/mL PEG 300/HP-β-CD solution ₄The stability of N

Contain 11-14mg/mL M ₄The specimen of N is still stable behind incubated at room 24hr, wherein M ₄N is made into the ultimate density of 25%PEG 300 and 30%HP-β-CD.

Embodiment 18 40mg/mL M ₄The stability of N/PEG 300

Measurement is dissolved in the 40mg/mLM among the 100%PEG 300 ₄N is hatched the stability that reaches 24hr under 30 ℃, 45 ℃ and 60 ℃.The M of 40mg/mL ₄N PEG 300 reserve liquids are prepared according to the method for describing among the embodiment 17 at 60 ℃.Subsequently, take out sample aliquot, and hatch in suitable temperature.Hatch in the process whole, sample rotates with 450rpm.Omnidistance range estimation observed result and the RP-HPLC data of collecting of test.Shown in table 29, after 30 ℃ hatch 6hr, at 40mg/mL M ₄Observe small crystallization in N/PEG 300 prescriptions, occur more behind the 24hr.The formation of crystallization and the M of dissolving ₄The loss of N consistent (table 30).By range estimation observed result and RP-HPLC analysis verification (table 29 and 30), the 40mg/mLM of under 45 ℃ and 60 ℃, hatching ₄It is stable that N/PEG 300 samples keep after the hatching of 24hr.Under any incubation temperature, do not observe the variation of amount or the type of impurity peaks.

Table 29.40mg/mLM ₄The visual appearance of N/PEG 300 stability sample

Table 30.40mg/mL M ₄N/PEG 300 stability sample: RP-HPLC analyzes

30 ℃ hatch 6hr after, at 40mg/mL M ₄Observe small crystallization in N/PEG 300 prescriptions.After 24 hours, observe more crystallizations, and observe the dissolubility M that RP-HPLC analyzes mensuration ₄The loss of N＞5%.

As estimate observed result and RP-HPLC analysis verification, at 45 ℃ and 60 ℃ 40mg/mL M of hatching ₄It is stable that N/PEG 300 samples keep after hatching 24hr.

Those of ordinary skills should be understood that, can make variation and not deviate from theory of the present invention above-mentioned specific embodiment.Therefore, should be understood that the present invention is not limited to disclosed specific embodiment, and be intended to cover the modification within the spirit and scope of the invention that is defined by the following claims.

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Claims (36)

1. compositions that is used for being injected into animal, described compositions comprises acceptable carrier in active pharmaceutical ingredient and the pharmacy, wherein said active pharmaceutical ingredient is EM-1421, described carrier comprise cyclodextrin and be selected from the solubilizing agent of the group that is formed by following substances and excipient at least a: (a) water-miscible organic solvent except dimethyl sulfoxine; If when described water-miscible organic solvent is propylene glycol, described propylene glycol do not contain white vaseline, do not contain xanthan gum and do not contain at least a in glycerol or the glycine, when described water-miscible organic solvent is Polyethylene Glycol, described Polyethylene Glycol exists not containing under ascorbic acid or the butylated hydroxytoluene, and when described Polyethylene Glycol was PEG400, described PEG400 existed not containing under the PEG 8000; (b) ion, nonionic or amphoteric surfactant, if when described surfactant is non-ionic surface active agent, described non-ionic surface active agent exists not containing under the xanthan gum; (c) modified cellulose; (d) the water-insoluble lipid except Oleum Ricini; And the combination in any of described carrier (a)-(d).

2. compositions according to claim 1 is characterized in that, but described compositions intravenous injection enters in the animal.

3. compositions according to claim 1 is characterized in that, described compositions comprises the active pharmaceutical ingredient of 0.1mg to 200mg.

4. compositions according to claim 3 is characterized in that, described compositions comprises the active pharmaceutical ingredient of 10mg, 20mg, 25mg, 30mg, 40mg, 50mg, 60mg, 75mg, 100mg or 200mg.

5. compositions according to claim 1 is characterized in that, the concentration that described active pharmaceutical ingredient exists is 1mg/mL to 200mg/mL.

6. compositions according to claim 5, it is characterized in that the concentration that described catecholic butanes exists is 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 175mg/mL.

7. compositions according to claim 1 is characterized in that, described water-miscible organic solvent is selected from the group that is comprised of polypropylene glycol, Polyethylene Glycol, polyvinylpyrrolidone, ethanol, benzyl alcohol and dimethyl acetylamide.

8. compositions according to claim 7 is characterized in that, described water-miscible organic solvent is Polyethylene Glycol.

9. compositions according to claim 8 is characterized in that, described Polyethylene Glycol is PEG300, PEG 400 or PEG monolaurate.

10. compositions according to claim 8 is characterized in that, the concentration that described Polyethylene Glycol exists is 5%(v/v) to 100%(v/v).

11. compositions according to claim 8 is characterized in that, described Polyethylene Glycol is PEG 300.

12. compositions according to claim 11 is characterized in that, the concentration that described PEG 300 exists is 10%(v/v), 20%(v/v), 30%(v/v), 40%(v/v) or 50%(v/v).

13. compositions according to claim 8 is characterized in that, described Polyethylene Glycol is PEG 400.

14. compositions according to claim 13 is characterized in that, the concentration that described PEG 400 exists is 10%(v/v), 20%(v/v), 30%(v/v), 40%(v/v) or 50%(v/v).

15. compositions according to claim 8 is characterized in that, described Polyethylene Glycol is PEG 400 monolaurates.

16. compositions according to claim 15 is characterized in that, the concentration that described PEG 400 monolaurates exist is 20%(v/v) to 50%(v/v).

17. compositions according to claim 1 is characterized in that, described cyclodextrin comprises the cyclodextrin of unmodified cyclodextrin or modification.

18. compositions according to claim 17 is characterized in that, the cyclodextrin of described modification is selected from the group that is comprised of HP-β-CD and sulfobutyl ether-beta-schardinger dextrin-.

19. compositions according to claim 17 is characterized in that, the concentration that the cyclodextrin of modification exists is 5%(w/v) to 80%(w/v).

20. compositions according to claim 19 is characterized in that, the concentration that the cyclodextrin of modification exists is 15%(w/v), 20%(w/v), 25%(w/v), 30%(w/v), 35%(w/v), 40(w/v) or 50%(w/v).

21. compositions according to claim 1 is characterized in that, described water-miscible organic solvent is propylene glycol.

22. compositions according to claim 1 is characterized in that, described water-miscible organic solvent is glycerol.

23. compositions according to claim 1 is characterized in that, described surfactant is selected from by polysorbate, polyethylene glycol 1000 vitamin E succinic acid ester, and the group that forms with the product of the mol ratio of 35:1 of ethylene oxide,1,2-epoxyethane and Oleum Ricini.

24. compositions according to claim 23 is characterized in that, described surfactant is selected from the group that is comprised of polysorbate20 and polysorbate80.

25. compositions according to claim 23 is characterized in that, the concentration that described surfactant exists is 5%(v/v) to 100%(v/v).

26. compositions according to claim 1 is characterized in that, described modified cellulose is selected from the group that is comprised of ethyl cellulose, hydroxypropyl emthylcellulose, methylcellulose and carboxymethyl cellulose.

27. compositions according to claim 26 is characterized in that, the concentration that described modified cellulose exists is 0.1%(w/v) to 10%(w/v).

28. compositions according to claim 1 is characterized in that, described water-insoluble lipid is fat emulsions.

29. compositions according to claim 28 is characterized in that, the concentration that described fat emulsions exists is 10%(w/v) to 30%(w/v).

30. compositions according to claim 29 is characterized in that, the concentration that described fat emulsions exists is 20%(w/v).

31. compositions according to claim 1 is characterized in that, described water-insoluble lipid is oil.

32. compositions according to claim 31 is characterized in that, described oil is at least a oil that is selected from the group of following material composition: Semen Maydis oil, olive oil, Oleum menthae, soybean oil, Oleum sesami, mineral oil and glycerol.

33. compositions according to claim 1 is characterized in that, described water-insoluble lipid is the fatty acid of esterification.

34. compositions according to claim 1 is characterized in that, described compositions room temperature or 4 ℃ stability greater than 1 day.

35. compositions according to claim 1 is characterized in that, described compositions room temperature or 4 ℃ stability greater than 3 days.

36. compositions according to claim 1 is characterized in that, described compositions room temperature or 4 ℃ stability greater than 7 days.

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