CN101389319A - Methods of treating influenza viral infections - Google Patents

Abstract

Methods are described for treating an influenza viral infection or associated diseases, disorders or mechanisms in a subject, comprising administering to the subject a therapeutically effective amount of a catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof: wherein R1 and R2 each independently represents a hydrogen, a lower alkyl, a lower acyl, an alkylene, or -OR1 and -OR2 each independently represents an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof; R3, R4, R5, R6, R10, R11, R12 and R13 each independently represents a hydrogen, or a lower alkyl; and R7, R8 and R9 each independently represents a hydrogen, -OH, a lower alkoxy, a lower acyloxy, an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, or any two adjacent groups together may be an alkyene dioxy; with the proviso in certain circumstances that where one of R7, R8 and R9 represents a hydrogen, then -OR1, -OR2 and the other two of R7, R8 and R9 do not simultaneously represent -OH.

Description

The method of treatment influenza infection

The cross reference of related application

The rights and interests of 60/775, No. 869 U.S. Provisional Application that the application requires to submit on February 23rd, 2006 and 60/776, No. 043 U.S. Provisional Application submitting on February 23rd, 2006, its disclosed content is all incorporated among the application by reference at this.

Background technology

Influenza virus is the main source of infection of multiple disease, and it can cause serious cold symptoms and often cause breathing the pneumonia of lacking of proper care and/or can be fatal.Different according to the serum type of nucleoprotein and memebrane protein, influenza virus is divided into three types, i.e. A type, Type B and C type.In the influenza virus of these types, annual popular type is influenza virus A and influenza virus B.Influenza virus A has two kinds of glycoproteins on the surface of its peplos, i.e. hemagglutinin (HA) and neuraminidase (NA), and therefore it is divided into hypotype according to proteinic antigenicity (antigenecities) based on this, for example H1N1, H2N2 and H3N2.Influenza virus B and influenza virus C only have a kind of hypotype separately.

Influenza virus A has experienced great change aspect antigenicity, annual all than the viral prevalence of other type.The antiviral agent that is used for influenza virus A is known, but because it can not deal with the sudden change of virus usually, so it does not make us satisfied fully.The unable sudden change of dealing with virus of antiviral agent is likely because the serious antigenic variation of virus.

All A type influenza virus comprise the virus that those cause people's seasonal epidemics regularly, are unsettled in heredity and are suitable for evading host's defence well.Influenza virus lacks " correction " and repair the mechanism of the mistake that takes place between replicative phase.Because these mistakes that do not obtain correcting, when it duplicated in human body and animal body, the genomic constitution of virus changed, and existing bacterial strain is replaced by a kind of new antigenic variant.The antigen of these influenza virus As is formed continue, permanent and be generally little change and be called as antigen " drift ".

The trend that the frequent and permanent antigen of influenza virus experience changes needs the influenza situation and the annual composition of adjusting influenza vaccines in the monitoring whole world constantly.

Influenza virus has the advantages that other popular key health is considered.That is, influenza virus A comprises that being derived from different types of hypotype can replace or reset (reassort) genetic stew and fusion.This rearrangement process is called as antigenic drift, produces the new hypotype that is different from two kinds of parent viruses.Because human do not have immunity to new hypotype, and do not have currently available vaccines, existing vaccines to grant asylum, antigenic drift has caused highly fatal pandemic influenza in history.For it is taken place, new hypotype need have be derived from the human body influenza virus be easy to one continue during be passed to another people's gene from a people.

It has been generally acknowledged that the advantage that antigenic drift occurs relates to and infected other domestication kind of mankind that live very closely of the various bacterial strains of influenza virus.For example, the virus that pig both had been easy to infect fowl also is easy to infect mammiferous virus, comprises human bacterial strain.Therefore, it can be used as a kind of " blender " and is used for capturing (scrambling) genetic stew from people's virus and the virus of fowl, causes occurring new hypotype.Yet, the recent another kind of possible mechanism of having confirmed to occur antigenic drift.Shown that the mankind itself can be used as a kind of blender that new hypotype occurs.

Avian influenza virus subtype is planted in current known existence ten five (15).Owing to several reasons, the H5N1 hypotype needs to pay close attention to especially.The H5N1 sudden change is rapid, and has document to show that it has the tendency of obtaining gene from the virus that infects other animal species.Verified its of twice incident (occasions) by Hong Kong 1997 and 2003 causes the ability of human serious disease.Ended in 14th to December in 2005 since then, World Health Organization (WHO) by experiment the chamber make a definite diagnosis 138 routine human infection H5N1 bird flus.In this 138 example, 71 examples are dead.

In addition, experimentation has shown that being derived from this viral separator has very high pathogenic and can cause human serious disease.In addition, the birds that infected the survival of H 5 N 1 avian influenza hypotype drain virus at least ten (10) sky, thereby have promoted the further propagation of virus in live-bird market and the migratory bird.The propagation of infecting in the bird has increased the chance of human direct infection.Along with the past of time, if more people is infected, as while infected person influenza bacterial strain and bird flu bacterial strain, so human probability as the blender that new hypotype occurs increases, and it has enough human genes who is easy to be passed to from a people another people.This will indicate the beginning of flu outbreak.In history, when new virus subtype occurs and is easy to be passed to another man-hour from a people, can expect and take place three to four times in average each century of flu outbreak.Flu outbreak be uncertain.Most of influenza experts are consistent to think that another time flu outbreak is inevitably, and may soon take place.

Though it is considerable producing the experience of influenza vaccines; particularly as the annual variation that changes with the virus that adapts to the distribution that causes owing to antigenic drift of vaccine composition, for produce any significant amount, can resist new virus subtype and protective any new vaccine may need four months at least.

Therefore, be used for the treatment of the compositions of influenza infection symptom usually to those patient's administrations of being infected.Consider the enhanced pathogenic of emerging influenza virus bacterial strain, treat or to alleviate the influenza infection symptom more and more important.

Though there is the method for various treatment influenza infection symptoms, many methods are always ineffective for the hypotype of the emerging bacterial strain that comprises fowl, and manyly cause deleterious side effect.Therefore, there are the needs new and more efficient methods that are used for the treatment of influenza infection in the art.The present invention has satisfied this needs.

Summary of the invention

The present invention relates to by giving the method for catechol butane (catecholic butane) or its pharmaceutically acceptable salt treatment influenza infection.Though wish not to be bound by any particular theory, can think that method of the present invention both can be used for reducing host's the duplicating or grow and also can reduce the various diseases that accompany influenza infection or the appearance and/or the seriousness of disorder of influenza virus.

One embodiment of the invention comprise a kind of method for the treatment of influenza infection in the study subject.Described method comprises catechol butane or its pharmaceutically acceptable salt of using the general formula (I) of effective therapeutic dose to study subject:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl or alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together; Condition is to work as R ₇, R ₈And R ₉One of expression during hydrogen, so-OR ₁,-OR ₂With R ₇, R ₈And R ₉In other both expression-OH simultaneously not.Replace or unsubstituted amino acid residue is preferably at its carboxyl terminal with its pharmaceutically acceptable salt and links to each other with aromatic ring.

Another embodiment of the present invention comprises a kind of method for the treatment of influenza infection in the study subject.Described method comprises to the nordihydroguaiareticacid acid derivatives of the general formula of study subject drug treatment effective dose (II) or its pharmaceutically acceptable salt:

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃, or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; R ₁₈And R ₁₉Difference is expression-H or low alkyl group independently; Condition is R ₁₄, R ₁₅, R ₁₆And R ₁₇Be not simultaneously-OH.Replace or unsubstituted amino acid residue is preferably at its carboxyl terminal with its pharmaceutically acceptable salt and links to each other with aromatic ring.

Another embodiment of the present invention comprises a kind of method for the treatment of influenza infection in the study subject, and described method comprises nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt of using the general formula (III) of effective therapeutic dose to study subject:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, condition is R ₂₀, R ₂₁, R ₂₂And R ₂₃Be not simultaneously-OH.Replace or unsubstituted amino acid residue is preferably at its carboxyl terminal with its pharmaceutically acceptable salt and links to each other with aromatic ring.

Another embodiment of the present invention comprises a kind of method for the treatment of influenza infection in the study subject.Described method comprises the following compositions to study subject drug treatment effective dose, described compositions contains and is selected from by three-O-methyl nordihydroguaiaretic acid (NDGA), four-O-methyl N DGA, four-glycyl NDGA, four-dimethyl glycyl NDGA, or the catechol butane of the group of its pharmaceutically acceptable salt composition, and pharmaceutically acceptable carrier or excipient.

Another embodiment of the present invention comprises the method for H5N1 subtype influenza viral infection in a kind of people's of treatment study subject.Described method comprises the nordihydroguaiareticacid acid derivatives of people's study subject oral administration general formula (III) or its pharmaceutically acceptable salt, the amount of use for about 10mg/kg to the every dosage of about 375mg/kg:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

Another embodiment of the present invention comprises a kind of the inhibition because influenza infection and the method for proinflammatory cytokine in the inducing cell.Described method comprises catechol butane or its pharmaceutically acceptable salt of the general formula I of pair cell effective dosage:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together.

Another embodiment of the present invention comprises a kind of the inhibition because influenza infection and the method for short scorching lipid medium in the inducing cell.Described method comprises catechol butane or its pharmaceutically acceptable salt of the general formula I of pair cell effective dosage:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together.

Another embodiment of the present invention comprises a kind of method that suppresses to induce owing to H5N1 subtype influenza viral infection the struma necrosin ﹠ (TNF-α) in the macrophage.Described method comprises the nordihydroguaiareticacid acid derivatives of the general formula of macrophage effective dosage (III) or its pharmaceutically acceptable salt:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

In addition, another embodiment of the present invention comprises a kind of the inhibition owing to H5N1 subtype influenza viral infection is induced prostaglandin E in the macrophage ₂(PGE ₂) method.Described method comprises to the nordihydroguaiareticacid acid derivatives of the general formula of macrophage effective dosage (III) or its pharmaceutically acceptable salt:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

Another embodiment of the present invention comprises a kind of catechol butane or its pharmaceutically acceptable salt that contains general formula I, and by using catechol butane or its pharmaceutically acceptable salt to treat the test kit of the description of study subject influenza infection.

According to following disclosed content, other aspects of the present invention, feature and advantage will be conspicuous, disclosed content comprise detailed description of the present invention with and preferred embodiment and claims.

Description of drawings

When read in conjunction with the accompanying drawings, can understand foregoing invention content and following detailed description of the present invention better.In order to demonstrate the invention, present embodiment preferred shown in the drawings.Yet, should be appreciated that, the invention is not restricted to shown accurate arrangement and means.

In the accompanying drawings:

Fig. 1 produces TNF-diagram not for using RAW 264.7 mouse macrophages to be induced at different time by lipopolysaccharide (LPS) under various conditions.

Fig. 2 is the inductive apoptotic diagram of TNF-born of the same parents in the C3HA fibroblast under various conditions.

Fig. 3 is for using RAW 264.7 macrophages by lipopolysaccharide-induced generation PGE under various conditions ₂Diagram.

Fig. 4 is for using RAW 264.7 macrophages by lipopolysaccharide-induced generation PGE under various conditions _{2 α}Diagram.

Fig. 5 is for using RAW 264.7 macrophages by lipopolysaccharide-induced generation PGE under various conditions _{1 α}Diagram.

Fig. 6 A and 6B are for using the diagram of RAW 264.7 macrophages by lipopolysaccharide-induced generation cytokine under various conditions according to antibody chip research.

Fig. 7 comprises the diagram of EM-1421 to the effect of duplicating of the influenza virus A/WS/33 in the mdck cell, wherein organizes A and organizes B to have shown same data, but have linear and logarithm y axle (linear and log y-axes) respectively.

Fig. 8 comprises the diagram of EM-1421 to the effect of duplicating of the influenza virus A/WS/33 in RAW 264.7 macrophages, wherein organizes A and organizes B to have shown same data, but have linear and logarithm y axle respectively.

Fig. 9 comprises the diagram of EM-1421 to the effect of duplicating of the influenza virus A/WS/33 in the RAW264.7 macrophage that used the EM-1421 processing before viral infection, wherein organizes A and organizes B to have shown same data, but have linear and logarithm y axle respectively.

Figure 10 is according to low infection multiplicity (MOI) model system during by RAW264.7 mouse macrophage infective virus and/or use EM-1421 to handle the diagram of the TNF-α that produces.

Figure 11 uses the diagram of RAW264.7 mouse macrophage to the dose response experiments of generation TNF-α according to low infection multiplicity (MOI) model system.

Figure 12 is a diagram of using RAW 264.7 mouse macrophages that the time-histories that produces TNF-α is tested according to low infection multiplicity (MOI) model system.

Figure 13 is according to high infection multiplicity (MOI) model system during by RAW 264.7 mouse macrophage infective virus and/or use EM-1421 to handle the diagram of the TNF-α that produces.

Figure 14 uses the diagram of RAW 264.7 mouse macrophages to the dose response experiments of generation TNF-α according to high infection multiplicity (MOI) model system.

Figure 15 is a diagram of using RAW 264.7 mouse macrophages that the time-histories that produces TNF-α is tested according to high infection multiplicity (MOI) model system.

Figure 16 produces PGE for use the RAW264.7 macrophage to be induced by viral infection under various conditions according to low infection multiplicity (MOI) model system ₂Diagram.

Figure 17 produces PGE for use the RAW264.7 macrophage to be induced by viral infection under various conditions according to high infection multiplicity (MOI) model system ₂Diagram.

Figure 18 is induced the diagram that produces cytokine for use RAW 264.7 macrophages under various conditions according to antibody chip research by viral infection.

The specific embodiment

The inventor has been found that in the present invention the catechol butane can be used for treating influenza infection.Described catechol butane has general formula (I) or its pharmaceutically acceptable salt:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl or alkylene respectively independently, or-OR ₁And OR ₂Represent amino acid residue or its salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, unsubstituted or the amino acid residue or its pharmaceutically acceptable salt that replace, or can form any two adjacent groups of alkylene dioxy base together; Condition is to work as R ₇, R ₈And R ₉One of expression during hydrogen, so-OR ₁,-OR ₂With R ₇, R ₈And R ₉In other both expression-OH simultaneously not.Preferably, replacement or unsubstituted amino acid residue or its pharmaceutically acceptable salt link to each other with aromatic ring at its carboxyl terminal.This acacatechin butane can combine the pharmaceutical composition that can be made into multiple delivery pathways to produce with pharmaceutically acceptable carrier or excipient.

In another embodiment of the present invention, the catechol butane has general formula (I), wherein R ₁And R ₂Expression-H, low alkyl group, lower acyl independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₇, R ₈And R ₉Be independently-H ,-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Condition is that described catechol butane is not NDGA.

In another embodiment of the present invention, the catechol butane has general formula (I), wherein R ₁And R ₂Expression-H, low alkyl group, lower acyl independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₇, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₈And R ₉Be independently-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Condition is that described catechol butane is not NDGA.

In an also embodiment of the present invention, the catechol butane has general formula (I), wherein R ₁And R ₂Be independently-CH ₃Or-(C=O) CH ₂N (CH ₃) ₂Or its salt.

In another embodiment of the present invention, the catechol butane has general formula (I), wherein R ₈And R ₉Be independently-OCH ₃Or-O (C=O) CH ₂N (CH ₃) ₂Or its salt.

In another embodiment of the present invention, the catechol butane has general formula (I), wherein R ₁And R ₂Be independently-CH ₃The CH of ,-(C=O) ₂N (CH ₃) ₂Or-(C=O) CH ₂N ⁺H (CH ₃) ₂Cl ^-And R ₈And R ₉Be independently-OCH ₃,-O (C=O) CH ₂N (CH ₃) ₂Or-(C=O) CH ₂N ⁺H (CH ₃) ₂Cl ^-

In an also embodiment of the present invention, the catechol butane has general formula (I), wherein R ₁And R ₂Independently expression-H or-CH ₃, R ₈And R ₉Be independently-OH or-OCH ₃, condition is that described catechol butane is not NDGA.

In of the present invention one different embodiment, the catechol butane has general formula (I), wherein R ₁And R ₂Respectively do for oneself-CH ₃, R ₈And R ₉Respectively do for oneself-OCH ₃

In an optional embodiment, be used for catechol butane according to the method for embodiment of the present invention for having NDGA derivant or its pharmaceutically acceptable salt of following general formula (II):

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, R ₁₈And R ₁₉Expression-H or such as the alkyl of low alkyl group independently respectively, for example-CH ₃Or-CH ₂CH ₃Condition is R ₁₄, R ₁₅, R ₁₆And R ₁₇Be not simultaneously-OH.Preferably, replacement or unsubstituted amino acid residue link to each other with aromatic ring at its carboxyl terminal with its pharmaceutically acceptable salt.

The present inventor has found unexpectedly that the compositions that is essentially pure preparation that contains at least a NDGA derivant can be used for the treatment of influenza infection effectively.Because the NDGA derivant is to be used for other purpose administration at first, and the treatment influenza is a kind of unexpected understanding, and this discovery is serendipitous and is unexpected.

Preferably, the NDGA derivant that is used in embodiment of the present invention has aforesaid general formula (II), wherein R ₁₄, R ₁₅, R ₁₆And R ₁₇Difference is expression-OH, lower alkoxy independently, for example, and-OCH ₃, low-grade acyloxy, for example ,-O (C=O) CH ₃, or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, be-OH but can not distinguish simultaneously; R ₁₈And R ₁₉Expression-H or such as the alkyl of low alkyl group independently, for example ,-CH ₃Or-CH ₂CH ₃In one embodiment, R ₁₈And R ₁₉Can all be-H ,-CH ₃Or-CH ₂CH ₃Preferably, work as R ₁₄, R ₁5, R ₁₆And R ₁₇In one or more expressions unsubstituted or the amino acid residue that replaces or its salt the time, described residue links to each other with aromatic ring at carboxyl terminal.

The catechol butane of the present invention of suitable dosage form, when needing with pharmaceutically acceptable carrier or excipient, can be by being selected from the study subject that one or more approach that comprise following group are administered to this treatment of one or more needs safely: intranasal administration; Oral administration; Inhalation; Subcutaneous administration; Transdermal administration; Intravenously administrable; Oral administration; The intraperitoneal administration; Eye drops; Administration near the eyes; The intramuscular administration; Drug delivery implant; Infusion and central vein administration.

In addition, described catechol butane can be with suitable solution, suspension, semisolid or solid form, or by above-mentioned one or more approach with Liposomal formulation, nanoparticle formulations or micellar preparation safely to patient's administration of this treatment of one or more needs.

In addition, the catechol butane of Liposomal formulation, nanoparticle formulations or micellar preparation can be imbedded in the biodegradable polymer formulations and administration safely, for example by subcutaneous implantation.

In one embodiment of the invention, being used for route of administration of the present invention is not by parenteral, and the parenteral among the present invention refers to intravenously administrable, intramuscular administration, subcutaneous administration, transdermal administration and intraperitoneal administration.

The present invention has also described a kind of pharmaceutical composition of the feature that contains the catechol butane that is used for the treatment of influenza, described compositions is made into to discharge as described above or administration, for example, with tablet, capsule, hydrophilic or hydrophobic liquid, such as the powder that produces by lyophilizing, aerosol form or with moisture water-soluble composition, hydrophobic composition, liposome composition, such as micelle composition based on polysorbate80 or diblock copolymer, Nanoparticulate compositions, polymer composition, the cyclodextrin inclusion compound compositions, emulsion or based on the lipids form of the nano-particle that is called as " lipid core (lipocores) ".

In addition, the present invention also provides a kind of pharmaceutical composition that contains the catechol butane that is used for the treatment of influenza, described compositions is made into to be used for that oral or injection discharges with pharmaceutically acceptable carrier, and described carrier comprises at least a of the solubilizing agent that is selected from the group of being made up of following substances and excipient: (a) water-miscible organic solvent; (b) cyclodextrin (comprising modified cyclodextrin); (c) ion, nonionic or amphoteric surfactant; (d) modified cellulose; (e) water-fast lipid; And the combination in any of carrier (a)-(e).

According to embodiment of the present invention, the catechol butane can be in conjunction with one or more other medicament or medicine medication.It can with other medicament or medicine simultaneously, medication before or after other medicament or medicine medication.In specific embodiment, the catechol butane can be in conjunction with one or more other antiinflammatory medications.Described other antiinflammatory is selected from following group, comprising: (1) serotonin receptor antagonist; (2) serotonin receptor agonist; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitor; (6) tachykinin receptor antagonists comprises neurokinin 1 and neurokinin 2 receptor subtype antagonisies; (7) calcitonin gene related peptide (CGRP) receptor antagonist; (8) interleukin-1 receptor antagonist; (9) act on the enzyme inhibitor that arachidonic acid metabolite synthesizes path, comprise (a) inhibitor of phospholipase enzymes, comprise PLA ₂Obform body inhibitor and PLC and obform body inhibitor (b) cyclooxygenase-2 inhibitors, and (c) lipoxygenase inhibitor; (10) prostaglandin receptor antagonist comprises eicosanoid EP-1 and EP-4 receptor subtype antagonist and thromboxane receptor subtype antagonist; (11) LTRA comprises leukotriene B ₄Receptor subtype antagonist and leukotriene D ₄The receptor subtype antagonist; (12) opioid receptor agonist comprises μ opium, sheet opium and kappa opioid receptor subtype agonist; (13) purinoceptor agonist and antagonist comprise P _2XReceptor antagonist and P _2YReceptor stimulating agent; (14) adenosine triphosphate (ATP)-sensitive potassium channel opener.

In other embodiments, the catechol butane can be in conjunction with one or more other influenza medicament medication, for example second of general formula I kind of catechol butane or its pharmaceutically acceptable salt, amantadine, Ao Simiwei, Peramivir, rimantadine, zanamivir or arbidol.

The present invention has also described a kind of feature for preparing the method for pharmaceutical composition of the present invention, described method comprises preparation or the catechol that is essentially purified form butane is provided, described composition is combined with pharmaceutically acceptable carrier or excipient, preparing compositions with the mode that the administering mode of hope conforms to.

In addition, the invention still further relates to and comprise the aforesaid compositions that is used for the treatment of influenza or the test kit of preparation, described compositions is made into to discharge as mentioned above, include but not limited to intranasal administration, suction, oral administration, topical, intravenously administrable, intraperitoneal administration and other parenteral, alternatively, comprise doser that is used for this type of administration and the operation instruction that is used for this type of administration.

Definition:

Unless the application has definition in addition, all technology and the scientific terminology that uses among the application has the identical implication of usually understanding with the technical field of the invention personnel of implication.The present invention may be better understood according to following specific implication.

The term that uses among the present invention " activating agent ", " chemical compound " and " medicine " refer to one or more catechol butane, comprise the NDGA derivant, with and pharmaceutically acceptable salt.

The term that uses among the present invention " alkylene dioxy base " refers to methylene (or substituted methylene) dioxy base or vinyl (or substituted vinyl) dioxy base.

Be suitable for " buffer agent " of the present invention and comprise the buffer agent of any routine in present technique field, for example Tris, phosphate, imidazoles and heavy carbonate.

" carrier " that uses among the present invention refers to a kind of formulation auxiliary agents of nontoxic solid, semisolid or liquid filler, diluent, medium, excipient, solubilizing agent, capsule material or any general type, and comprises all components outside the active pharmaceutical ingredient in the compositions.Described carrier can contain other medicament, for example wetting agent or emulsifying agent, or pH buffer agent.When needing, can add other material, for example antioxidant, wetting agent, viscosity stabiliser and similar medicament.

" cyclodextrin " that uses among the present invention refers to a kind of cyclodextrin of non-modified or the cyclodextrin of modification, the any modified cyclodextrin that includes but not limited to alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin and contain its modification part, for example HP-(" HP-β-CD ") or sulfobutyl ether beta-schardinger dextrin-(" SBE-β-CD ").Cyclodextrin has 6 (alpha-cyclodextrins), 7 (beta-schardinger dextrin-s) and 8 (gamma-cyclodextrin) sugar usually, each sugar has maximum three substituent groups, therefore may have 0-24 one-level substituent group (the one-level substituent group is defined as the substituent group that directly links to each other with the cyclodextrin ring).The cyclodextrin modification of using among the present invention or non-modified can have one-level substituent group or other modification part of any proper number and position.

The term " cytokine " of using among the present invention " refer to many parahormones, low-molecular-weight any protein by all kinds emiocytosis, described protein in immunomodulating and inflammatory reaction process, regulate immunoreactive intensity with persistent period and mediated cell communicating by letter to cell.The example of cytokine comprises chemotactic factor, interleukin, lymphokine and other signaling molecule, for example tumor necrosis factor and interferon etc.

The term that uses among the present invention " chemotactic factor " refers to relevant molecule on a group little, the most basic, structure, described molecule by stride film with one group of 7-, g protein coupled receptor interacts and regulates the leukocytic cell transportation of all kinds.Chemotactic factor also plays essence in immune development, dynamic equilibrium and function aspects, and it is to central nervous system's cell and relate to angiogenesis or endotheliocyte that angiogenic growth suppresses has influence.

The term that uses among the present invention " interleukin " or " IL " refer to by the synthetic one group of multi-functional cytokine of lymphocyte, mononuclear cell, macrophage and some other cell.

The term that uses among the present invention " lymphokine " refers to the one group of cytokine that is discharged by activated lymphocyte, and it mediates immunoreation.

The term that uses among the present invention " interferon " refers to by the excretory one group of glycoprotein of vertebrate cells by such as the external medicament of virus, antibacterial, parasite and tumor cell multiple attack being made a response.Interferon promotes immunoreation and the resistance of giving the external medicament of antagonism, for example, propagation, the breeding that hinders intracellular parasite by suppressing normal cell and malignant cell, strengthen macrophage and granulocytic phagocytosis, improve natural killer cell activity and have several other immunoloregulation functions.

The term that uses among the present invention " tumor necrosis factor " or " TNF " refer to a kind of mainly by the excretory cytokine of macrophage.TNF can combine with its receptor TNFRSF1 A/TNFR1 and TNFRSF 1B/TNFBR and work by this receptor.This cytokine participates in the adjusting of multiple bioprocess, comprises cell proliferation, differentiation, apoptosis, lipid metabolism and cohesion.This cytokine relates to multiple disease, comprises autoimmune disease, insulin resistance and cancer.The TNF that increases that produces when influenza infection also relates to the performance of disease, imbalance or the syndrome relevant with viral infection (definition vide infra).

The term that uses among the present invention " unsubstituted or replace amino acid residue or its salt " refers to-OR ₁,-OR ₂One of or other suitable R group, the salt that is used for the prescription of catechol butane of the present invention for the amino acid residue of the salt of the amino acid residue of amino acid residue or replacement or amino acid residue or replacement, include 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, the 5-hydroxylysine, 4-Hydroxyproline, desiodothyroxine, 3-Methyl histidine, ε-N-methyllysine, ε-N, N, the N-trimethyl lysine, aminoadipic acid, Gla, phosphoserine, phosphothreonine, phosphotyrosine, the N-methylarginine, N-acetyllysine, and N, the amino acid residue of N-dimethyl-replacement, or its pharmaceutically acceptable salt.

The term that uses among the present invention " low alkyl group " refers to C ₁-C ₆Alkyl, it can be a straight chain or branched, and comprises one or more unsaturated carbon-carbon bonds alternatively.

The term that uses among the present invention " lower acyl " refers to C ₁-C ₆Acyl group, it can be a straight chain or branched, and comprises one or more unsaturated carbon-carbon bonds alternatively.

" NDGA makes and refers to nordihydroguaiaretic acid the term that uses among the present invention.

The term that uses among the present invention " NDGA derivant " refers to one or more chemical compounds that have general formula (II) respectively, or its pharmaceutically acceptable salt:

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-OH, lower alkoxy, low-grade acyloxy independently, or amino acid residue unsubstituted or that replace, or its pharmaceutically acceptable salt, but can not be simultaneously separately-OH; R ₁₈And R ₁₉Be independently-H or such as the alkyl of low alkyl group.For example, described term comprises wherein R ₁₄, R ₁₅, R ₁₆And R ₁₇Respectively do for oneself-OCH ₃, or respectively do for oneself-O (C=O) CH ₃And R ₁₈And R ₁₉Respectively do for oneself-chemical compound of H or the low alkyl group of respectively doing for oneself.In an embodiment of the present invention, R ₁₈And R ₁₉Respectively do for oneself-CH ₃Or-CH ₂CH ₃

" the pharmaceutically acceptable carrier " that uses among the present invention refers to a kind of formulation auxiliary agents of filler, diluent, capsule material or any general type of nontoxic solid, semisolid or liquid." pharmaceutically acceptable carrier " is nontoxic in dosage and the concentration used to the receptor, and can coexist with other composition in the preparation.For example, preferably, the carrier that is used to contain the preparation of catechol butane of the present invention does not contain the known chemical compound that can the contained catechol butane of infringement of oxidant and other.Suitable carriers includes but not limited to water, glucose, glycerol, saline, ethanol, buffer agent, dimethyl sulfoxine, polyoxyethylene castor oil (Cremaphor EL) and compositions thereof.Carrier can contain other medicament, for example solubilizing agent, wetting agent or emulsifying agent, or pH buffer agent.When needing, can add other material, for example antioxidant, wetting agent, viscosity stabiliser and similar medicament.

The pharmaceutically acceptable salt that uses among the present invention comprises acid-addition salts (forming with the free amine group of polypeptide), and it forms with the mineral acid of all example hydrochloric acids or phosphoric acid or such as acetic acid, mandelic acid, oxalic acid and tartaric organic acid.The salt that forms with free carboxyl also can be by obtaining such as the inorganic base of the hydroxide of sodium, potassium, ammonium, calcium or ferrum with such as the organic base of 2-aminopropane., trimethylamine, 2-ethylaminoethanol and histidine.

The term that uses among the present invention " pharmaceutically acceptable excipient " comprises carrier, adjuvant or diluent or other auxiliary substance, for example the material of those public routine in the art that can easily obtain.For example, pharmaceutically acceptable auxiliary substance comprises pH regulator and buffer agent, soaks into and press regulator, stabilizing agent, wetting agent etc.

The term that uses among the present invention " study subject ", " host " and " patient " are used interchangeably, refer to the animal of using combination treatment of the present invention, include but not limited to troglodyte, people, fowl, cat, dog, horse, rodent, cattle, pig, sheep, goat, mammals farm-animals, mammals motion animal and mammals house pet.

The catechol butane that relates to that uses among the present invention " is essentially pure " chemical compound refers to the chemical compound (hereinafter referred " non-NDGA material ") that is substantially free of non-catechol butane of the present invention.Be substantially free of and refer at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% do not contain non-NDGA material.

The term that uses among the present invention " treatment ", " processing " etc. refer to pharmacology and/or the physiologic effect that obtains hope.Described effect can be preventative with regard to completely or partially preventing a kind of situation or disease or its symptom, and/or can be curative with regard to partially or even wholly treating a kind of situation or disease and/or being attributable to regard to the side effect of described situation or disease.Therefore, for example " treatment " comprise mammal, especially any treatment of people's situation or disease, comprise: (a) prevent a kind of situation or the disease or the appearance of its symptom of study subject, have described situation or disease but described study subject has the tendency N of described situation or disease; (b) suppress described situation or disease or its symptom, for example, stop its development; And (c) remove, alleviate or improve described situation or disease or its symptom, for example, make described situation or disease or its resolution of symptoms.

The term that uses among the present invention " treatment effective dose " or " effective dose " refer to the amount of activating agent, chemical compound or the medicine of biology of being sought by researcher, veterinary, doctor or other clinician, the tissue system or the study subject of study subject caused hope or medical response.The reaction of wishing comprises blocking-up, prevents, alleviates or alleviates the viral infection that is present in the study subject of being treated.In certain embodiments, the reaction of hope comprises the alleviating of at least a or multiple symptom, imbalance or disease of the influenza infection of the study subject in the treatment.In some other embodiment, the reaction of hope comprises the minimizing of counting of influenza virus of the study subject in the treatment or the inhibition of duplicating or growing.

Those skilled in the art will recognize that " the treatment effective dose " that be used for activating agent of the present invention can be along with multiple factors vary, for example specific study subject, for example age, body weight, diet, health status etc., look for the seriousness and the complication of the viral infection situation of treatment or prevention, the administering mode of activating agent, the specific activating agent of use etc.Program that can operative norm is given the administering effect of study subject with activating agent with assessment, thereby makes those skilled in the art determine to give the effective amount of the activating agent of study subject.For example, the syndrome that can infect from study subject Test Virus before or after giving activating agent, for example fever or inflammation etc., or viral count.In addition, also can use such as investigation or the technology of animal model is used for the treatment of with evaluation or the curative effect of the activating agent of prophylaxis of viral infections.

When a numerical range is provided, unless context is clearly pointed out in addition, be appreciated that each intermediate value between the upper and lower bound of described scope, to 1/10 (to the tenthof the unit of the lower limit) of lower limit unit, and other any statement or the intermediate value in described scope all be included in the present invention.Except the boundary of clearly getting rid of in described scope, these upper and lower bounds more among a small circle can be included in the described littler scope independently, and are also included within the present invention.When described scope comprises one or two of described boundary, got rid of any of those boundaries that comprised or two scope and be also included among the present invention.

All publications of mentioning among the present invention comprise patent, patent application and journal of writings, and all integrally by reference mode is incorporated among the present invention, comprise its reference paper of quoting also by reference mode incorporate among the present invention.The publication of being discussed among the present invention only provides its disclosed content before the applying date of the present invention.Do not have content to be interpreted among the present invention and admit that the present invention does not have the right prior to this type of publication according to invention formerly.In addition, the date of the publication that is provided may be different from the actual publication date, and it may need to be confirmed independently.

Must be noted that, unless the singulative that uses among the present invention " a ", " an " and the clearly pointed out in addition in context " speech comprise plural number refer to object.Therefore, for example, mention that " a kind of chemical compound " comprises multiple this compounds, mention " described catechol butane " comprise refer to one or more catechol butane with and derivant well known by persons skilled in the art.

Only provide in the embodiment of hereinafter describing of the present invention, in no case should be construed to limitation of the present invention in the mode of example.

The preparation of catechol butane:

Can prepare catechol butane of the present invention by the method for any routine.For example, can this compounds of preparation as described below: United States Patent (USP) 5,008,294 (people such as Jordan, on April 16th, 1991 issued); United States Patent (USP) 6,291,524 (people such as Huang, calendar year 2001 JIUYUE issued in 18th); People such as Hwu (people such as Hwu, " antiviral activity of the first that methylates dihydroguaiaretic acid acid (nordihydroguaiaretic acids).1. synthetic, the structure of the HIV trans-activation of Tai Te adjusting (Tat-regulated) are discerned and are suppressed.Pharmaceutical chemistry magazine (J.Med.Chem) 4/ (16): 2994-3000 ＂ (1998)); Or people such as McDonald, (McDonald, people such as R.W., the synthetic and active anticancer of first dihydroguaiaretic acid acid (NDGA) and analog." cancer therapy drug design (Anti-Cancer Drug Des.), 16:261-270 (2001)).

In one embodiment of the invention, a kind of catechol butane, four-O-methyl N DGA, be also referred to as meso-1,4-two (3, the 4-Dimethoxyphenyl)-2,3-dimethylbutane, Te Laimeipu Cole (terameprocol), EM-1421 or M4N (being shown below) prepare by the following method: the methanol solution of a kind of NDGA of containing of preparation and potassium hydroxide in reaction flask.Add dimethyl sulfate then to reaction flask, reaction is proceeded.Last water makes the product precipitation with the reactant cool quickly.By filtering and the described precipitation of dry separation in vacuum drying oven.Then with compound dissolution in dichloromethane and toluene solution, subsequently by the alumina column purification.Remove by rotary evaporation and to desolvate, in isopropyl alcohol, precipitate broken up and pass through isolated by filtration.Dry cake in vacuum drying oven.Make four-O-methyl N DGA (M by the described filter cake that in isopropyl alcohol, refluxes ₄N) crystallization and by filtering isolation of crystalline again.

In certain embodiments of the invention, some catechol butane, for example G of the present invention ₄N, be also referred to as meso-1,4-two [3,4-(dimethylamino acetoxyl group) phenyl]-(2R, 3S)-dimethylbutane or four-dimethyl glycyl NDGA (being shown below), or its hydrochlorate and similar compound with aminoacid replacement base, also can prepare according to the method for routine, for example, as United States Patent (USP) 6,417,234 is described.

Compositions:

The present invention also provides the compositions that comprises drug component, and described compositions comprises catechol butane and pharmaceutically acceptable carrier or excipient.These compositionss can comprise the buffer agent of selecting according to the purposes of the hope of catechol butane, also can comprise other material that is suitable for desirable purposes.Those skilled in the art can easily select a kind of suitable reducing that is suitable for desirable purposes, and wherein numerous buffers is known in the art.In some cases, described compositions can comprise pharmaceutically acceptable excipient, and wherein multiple excipient is known in the art.Being suitable for pharmaceutically acceptable excipient of the present invention is described in multiple publication, comprise, Gennaro (Gennaro for example, A., " Lei Mingdun: pharmacy science with put into practice (Remington:The Science and Practice of Pharmacy) " " the 19th edition; Li Pupingkete, William; Louis Wilkins publishing house (Lippincott, Williams ， ﹠amp; Wilkins) (1995)); People such as Ansel (Ansel, people such as H.C., " pharmaceutical dosage form and drug delivery system (Pharmaceutical Dosage Forms and Drug Delivery Systems eds.) ", the 7th edition, Li Pupingkete, William; Louis Wilkins publishing house (Lippincott, Williams ， ﹠amp; Wilkins) (1995)); And Kibbe (Kibbe, A.H., handbook of pharmaceutical excipients (Handbook ofPharmaceutical Excipients), the 3rd edition.American pharmaceutical association (Amer.PharmaceuticalAssoc)).

Prepare compositions of the present invention according to possible administering mode.Therefore, if described compositions is intended to by intranasal or inhalation, for example, for this purpose, described compositions can be converted to common in the art powder or aerosol form.Other preparation for example is used for oral or gastrointestinal discharges, and is also using in the art usually.

The compositions that is used for administration of the present invention can form the preparation or the powder of solution, suspension, tablet, pill, capsule, sustainable release.

Be suitable for compositions or preparation oral or that injection discharges and additionally comprise the pharmaceutical composition that contains the catechol butane that is used for the treatment of influenza, wherein said compositions is made with pharmaceutically acceptable carrier, wherein said carrier comprises at least a solvent and the excipient that is selected from following group, and described group comprises: (a) water-miscible organic solvent; (b) cyclodextrin (comprising modified cyclodextrin); (c) ion, nonionic or amphoteric surfactant; (d) modified cellulose; (e) water-fast lipid; And the conjugate of any carrier (a)-(e).

Water-miscible organic solvent can be preferably but not necessarily is dimethyl sulfoxide also.Unrestriced exemplary water-miscible organic solvent comprises Polyethylene Glycol (" PEG "), for example, PEG 300, PEG400 or PEG 400 monolaurates, propylene glycol (" PG "), polyvinylpyrrolidone (" PVP "), ethanol, benzyl alcohol or dimethyl acetylamide.Preferably, for some embodiment, when water-miscible organic solvent was PG, PG existed when not having white vaseline, xanthan gum (being also referred to as HANSHENGJIAO and Hydrargyri Oxydum Rubrum glue), glycerol or glycine at least a.When water-miscible organic solvent is PEG, for some embodiment, PEG is preferably and does not exist ascorbic acid or butylated hydroxytoluene not to exist when (" not existing "), for some embodiment, when PEG is PEG400, preferably, PEG400 exists when not having Polyethylene Glycol 8000.

Cyclodextrin or modified cyclodextrin can be alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, HP-β-CD or SBE-β-CD without limitation.

Ion, nonionic or amphoteric surfactant can comprise, for example be surfactant without limitation such as polyethylene glycol oxide Span-20 (being also referred to as polysorbate), it is a kind of non-ionic surface active agent, for example, polysorbate20 and polysorbate80 commercially availablely are

20 or

80, poly-divinyl 1000 succinic acid (" TPGS ") of D-alpha tocopherol, glycerin mono-fatty acid ester (being also referred to as glycerin mono-fatty acid ester (glyceryl monooleate)), its for a kind of esterification fatty acid or a kind of ethylene oxide,1,2-epoxyethane and Oleum Ricini be 35: 1 product with mol ratio, commercially availablely be

EL.Preferably, for some embodiment, when surfactant was non-ionic surface active agent, non-ionic surface active agent existed when not having xanthan gum.

The non-limiting example of modified cyclodextrin comprises ethyl cellulose (" EC "), hydroxypropyl emthylcellulose (" HPMC "), methylcellulose (" MC ") or carboxymethyl cellulose (" CMC ").In one embodiment of the invention, the catechol butane can dissolve in modified cellulose, and modified cellulose can dilute in ethanol (" EtOH ") before use.

Water-fast lipid comprises, for example, oil or oils, for example Oleum Ricini, Oleum sesami or Oleum menthae, wax or wax class, for example Cera Flava or palm wax (carnuba wax), and blended fat emulsifier compositions, for example

(Pharmacia ﹠amp; Upjohn company now is a Pfizer company), use according to the recommendation of maker.For example, adult's dosage of recommendation is for being no more than 2g fat/kg body weight/day (10%, 20% and 30%

Be respectively 20mL/kg, 10mL/kg and 6.7mL/kg).Think 10%

1,000mL contains: pure Oleum Glycines 100g, pure lecithin 12g, anhydrous glycerol 22g, the water that is used to inject complement to 1,000mL.Use sodium hydroxide to regulate pH value to about 8.

20%1,000mL contains: pure Oleum Glycines 200g, pure lecithin 12g, anhydrous glycerol 22g, the water that is used to inject complement to 1,000mL.Use sodium hydroxide to regulate pH value to about 8.

30%1,000mL contains: pure Oleum Glycines 300g, pure lecithin 12g, anhydrous glycerol 16.7g, the water that is used to inject complement to 1,000mL.Use sodium hydroxide to regulate pH value to about 7.5.These Product is stored in the room temperature of the control that is lower than 25 ℃, should be not freezing.For the embodiment of some ejection preparation, oil is a kind of oil of non-Oleum Ricini, and for the embodiment of some oral formulations, Oleum Ricini exists when not having Cera Flava or palm wax.

In one embodiment of the invention, the catechol butane dissolves in different carriers or dissolves in different carriers and dilutes to form a kind of animal that is used for, and comprises the fluid composition of human oral.For example, aspect of this embodiment, the catechol butane is dissolved in the water-miscible organic solvent, for example PEG 300, PEG 400 or PEG 400 laurates (" PEG chemical compound ") or be dissolved among the PG.In another embodiment, compound dissolution of the present invention in modified cyclodextrin, for example HP-β-CD or SBE-β-CD.In another embodiment, compound dissolution of the present invention and/or be diluted in the combination preparation of a kind of PEG of containing chemical compound and HP-β-CD.In yet another embodiment, compound dissolution of the present invention is in the modified cellulose such as HPMC, CMC or EC.In an embodiment also, compound dissolution of the present invention had both contained in the combination preparation that modified cyclodextrin also contains modified cellulose at another kind, for example, and HP-β-CD and HPMC or HP-β-CD and CMC.

In an also embodiment of the present invention, compound dissolution of the present invention is in ion, nonionic or amphoteric surfactant, for example 20, 80, TPGS or esterified fatty acid.For example, chemical compound of the present invention can only be dissolved in TPGS or

In 20, or be dissolved in such as TPGS and PEG 400 or 20 and the compositions of PEG 400 in.

In another embodiment, compound dissolution of the present invention in water-fast lipid, for example wax, fat emulsifier, for example

Or oil.For example, chemical compound of the present invention can only be dissolved in the Oleum menthae, or be dissolved in Oleum menthae with

In the compositions of PEG 400, or in the compositions of Oleum menthae and PEG 400, or Oleum menthae and

Compositions in, or in the compositions of Oleum menthae and Oleum Ricini.

Certainly, EC can replace or add to replace HPMC or the CMC in the above-mentioned example; PEG300 or PEG 400 monolaurates can replace or add to replace the PEG400 in the above-mentioned example;

80 can replace or add to replace in the above-mentioned example

20, can replace or add to replace Oleum menthae or the Oleum Ricini in the above-mentioned example such as other oil of Semen Maydis oil, olive oil, Oleum Glycines, mineral oil or glycerol.

In addition, can heat, for example, in the process of preparation any of these compositions, be heated to about 30 ℃ and arrive about 90 ℃ temperature so that the dispersive equably suspension of compound dissolution of the present invention or acquisition The compounds of this invention.

In another embodiment, the catechol butane can not followed any carrier or use with solid form oral with carrier.In one embodiment, chemical compound of the present invention at first is dissolved in a kind of as in the liquid-carrier of above-mentioned example, makes a kind of solid composite as the Orally administered composition administration subsequently.For example, compound dissolution of the present invention is in the modified cyclodextrin such as HP-β-CD, and the described compositions of lyophilizing is to produce a kind of oral powder that is fit to.

In yet another embodiment, compound dissolution of the present invention or be suspended in the TPGS solution suitably heats to obtain homodisperse solution or suspension.

When cooling, described compositions becomes like butter, is fit to oral.In another embodiment, compound dissolution of the present invention adds Cera Flava with preparation waxy solid compositions in oil.

Generally speaking, in the process of preparation oral formulations, before adding other excipient, at first dissolve chemical compound of the present invention to produce the more compositions of high stability.Unsettled preparation is undesirable.Unsettled liquid preparation forms crystalline precipitate or two phase liquid usually.Unsettled solid preparation is revealed as granular or block usually, contains flowing liquid sometimes.Best solid preparation is revealed as slick, uniform, and has little fusion temperature scope.Generally speaking, the ratio of the excipient in the preparation can influence stability.For example, sclerosing agent very little, for example Cera Flava is for the oral formulations too soft (runny) of exquisiteness.

Therefore, generally speaking, for liquid preparation of the present invention, the excipient of use should be the good solvent of catechol butane compound of the present invention, for example M ₄N.In other words, excipient should just can dissolve the catechol butane without heating.Excipient also should coexist so that it can form a kind of stable solution, suspension or emulsion each other independently with the catechol butane.In addition, generally speaking, with regard to solid preparation of the present invention, the excipient of use should be the catechol butane good solvent with avoid the caking and uneven preparation.Too soft or inhomogeneous for fear of undesirable solid preparation quality, even excipient should can coexist each other so that it also can form slick homogeneous solid when not having the catechol butane.

Therapeutic Method:

Find that catechol butane and this theme inventive compositions can be used as therapeutic agent when hope provides treatment for the patient who is subjected to influenza infection.

Stride species when infecting when what pay close attention to from bird to generalized mammal people especially,, can treat multiple animal reservoir, comprise the mankind and non-human animal, for example the bird when bird flu according to subject methods of the present invention.When these terms are widely used when comprising Carnivora animal (for example Canis familiaris L. and cat), rodent (for example Cavia porcellus and rabbit) and comprising the mammals organism of other animal of cattle, goat, horse, sheep, rabbit, pig and primate (for example people, chimpanzee and monkey) with description, this type of host is " mammal " or " mammal (mammalian) " usually.In many embodiments, the host behaves.Animal model has influence for experimentation, for example is provided for treating the model of human diseases.In addition, the present invention also is applicable to treatment of animals.

The preparation of administration, dosage and approach:

As mentioned above, the activating agent of effective dose is given host or study subject.Usually, compositions of the present invention can contain and is lower than about 1% to about 99% activating agent, catechol butane promptly of the present invention; Alternatively, the present invention can contain 5% to about 90% the activating agent of having an appointment.In addition, the present invention also provides compositions, in described compositions, such as containing NDGA derivant, for example M ₄The activating agent of the catechol butane of N is with according to animal, and for example people's body weight gives study subject to be less than about 0.1mg/kg to about 400mg/kg or higher oral dose, for example the people.More specifically, only as nonrestrictive example, can use from about 0.01 to the dosage of the scope of about 400mg/kg body weight by any suitable route of administration treatment study subject, for example be lower than about 0.01mg/kg, 0.05mg/kg, 0.1mg/kg, 0.5mg/kg, 1.0mg/kg, 2.5mg/kg, 5.0mg/kg, 10mg/kg, 15mg/kg, 25mg/kg, 50mg/kg, 100mg/kg, 150mg/kg, 200mg/kg, 250mg/kg, 300mg/kg, 350mg/kg or 400mg/kg or higher.

The proper dosage of administration depends on study subject to be treated, for example the state of age, disease or the situation of the overall key health situation of study subject, study subject, the body weight of study subject.Generally speaking, about 0.1mg can give the child to about 500mg, and about 0.1mg extremely about 5g can be grown up.Described activating agent can single dose or more generally with multiple dosed administration.For given medicament, those skilled in the art can easily determine preferred dosage in several ways.Those skilled in the art set up dose-effect curve by the test of routine can easily determine other effective dosage.Certainly, the amount of medicament will change according to the concrete medicament that uses.

As dosage, the care-giver will react the administration frequency of determining activating agent according to age, body weight, disease condition, key health situation and patient.Therefore, as long as the needs of determining as conventional, described medicament can be continuously, off and on, once a day or repeatedly or administration during other is suitable.

Catechol butane of the present invention or activating agent can be incorporated in in the several formulations to be used for the treatment of administration.More specifically, catechol butane of the present invention can be by making pharmaceutical composition in conjunction with suitable, pharmaceutically acceptable carrier or diluent, and can make the preparation of solid, semisolid, liquid or gas form, for example tablet, capsule, powder, aerosol, liposome, nano-particle, microgranule, ointment, solution, suppository, injection, inhalant and aerosol.

Similarly, can be accomplished in several ways the administration of activating agent, administration in for example oral, oral administration, rectally, intranasal administration, intravenously administrable, subcutaneous administration, intramuscular administration, the trachea, topical, a matter administration, transdermal administration etc., or by sucking or implanting.Especially; the systematically administration of nano-particle, capsule and lipid formulations; comprise non-oral and through intranasal; and through between matter, oral, topical, transdermal administration; by sucking or implant, the biological activity and the stability that for example be used for drug targeting, strengthen drug bioavailability and protect medicine.The drug disposition retention time that the medicine that combines nano-particle of the present invention can expection can obtain to prolong.

Aspect pharmaceutical dosage form, activating agent can its pharmaceutically acceptable salt form administration, or it also can be individually or suitably associating, and is used in combination with the other medicines reactive compound.Following method and excipient are exemplary, never have restricted.

For oral formulations, activating agent can be separately or is combined with solution or form of suspension with proper additive and to use as liquid, or use as solid with tablet, powder, microgranule or capsular form, for example, additive with routine uses, for example lactose, mannitol, corn starch or potato starch; Use with binding agent, for example crystalline cellulose, cellulose derivative, arabic gum, corn starch or gelatin; With disintegrating agent, for example corn starch, potato starch or sodium carboxymethyl cellulose; With lubricant, for example Pulvis Talci or magnesium stearate; And use if desired, with diluent, buffer agent, wetting agent, antiseptic and flavoring agent.

Pharmaceutically acceptable excipient, for example medium, adjuvant, carrier or diluent are general in the art.Suitable excipient medium is, for example water, saline, glucose, glycerol, ethanol etc. with and compositions.In addition, if desired, described medium can comprise the auxiliary substance of trace, for example pH regulator and buffer agent, osmotic pressure regulator, stabilizing agent, wetting agent or emulsifying agent.The practical methods for preparing this type of dosage form be to those skilled in the art known maybe will be conspicuous.For example, referring to the Lei Mingdun pharmaceutical science, Mike publishing company, Easton, Pennsylvania (Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania), and the 17th edition, 1985.Under any circumstance, the compositions of administration or preparation all will contain the state of medicament to obtain to wish of q.s in the study subject of receiving treatment.

Described activating agent can by in aqueous solution or nonaqueous solvent for example in vegetable oil or other similar oil, comprise in Semen Maydis oil, Oleum Ricini, synthctic fat acid glyceride, high-grade aliphatic ester or the propylene glycol injection made in its dissolving, suspension or emulsifying; If desired, the additive with routine uses for example solubilizing agent, isotonic agent, suspending agent, emulsifying agent, stabilizing agent and antiseptic.Be used for the suitable treatment preparation that gastrointestinal tract sends according to catechol butane of the present invention and also comprise the disclosed various injectable carrier/excipient preparations of following document: the U.S. Provisional Patent Application that on January 27th, 2005 submitted to, and the International Application PCT/US2006/000287 of submission on January 27th, 2006, its name is called " preparation that is used for the catechol butane injection that contains the NDGA chemical compound is gone into animal ", international publication number is WO2006/081364A2, date of publication is on August 3rd, 2006, and its full content is incorporated among the present invention by reference.

Activating agent can use with aerosol form and pass through inhalation.Chemical compound of the present invention can be gone into the acceptable propellant of pressurization, for example dichlorodifluoromethane, propane, nitrogen etc. by system.

In addition, described activating agent can be mixed and made into suppository with the multiple alkali such as emulsifying alkali or water-soluble alkali.Chemical compound of the present invention can pass through the administration of suppository per rectum.Described suppository can comprise medium, for example cocoa butter, Carbowax (carbowaxes) and Polyethylene Glycol, and described suppository can melt when body temperature, and solidifies when room temperature.

Can be provided for the unit dosage forms of oral or rectally, for example syrup, elixir and suspension, each dosage unit wherein, for example teaspoonful, tablespoonful, tablet or suppository comprise the compositions of the scheduled volume that contains one or more activating agents.Similarly, be used for injecting or the unit dosage forms of intravenously administrable can comprise activating agent, normal saline or other pharmaceutically acceptable carrier as the compositions of aseptic aqueous solution.

The term that uses among the present invention " unit dosage forms " refers to and is suitable as the discrete unit (physically discrete units) of physical property that single dose is used for the study subject of humans and animals, each unit contains the chemical compound of the present invention of scheduled volume, and the amount of being calculated is enough to produce desirable effect with pharmaceutically acceptable diluent, carrier or medium.The specification that is used for new unit dosage forms of the present invention depends on the specific compound of use and the effect that desire obtains, and with the host in the relevant pharmacodynamics of each chemical compound.

The present invention includes the test kit of activating agent with many times or unit dose.In this type of test kit, except the container of the compositions that contains the NDGA derivant that many times or unit dose are housed, it can be a kind of informational package insert that has directions for use, described description described medicine aspect the relevant pathological condition of treatment use and the benefit followed, in the present invention, pathological condition is an influenza, and especially is influenza H5N1 hypotype.

The preparation of nano-particle (" NP "):

The present invention includes the preparation of catechol butane NP preparation.Being suitable for many different NP preparations of the present invention can prepare according to method for releasing.By control molecular weight, copolymer ratios, drug loading, diameter of particle and porosity and manufacturing conditions, the NP preparation can be different according to the drug release curve of hope.The NP preparation also can be different according to using polymer, stabilizing agent and surfactant in production technology.Different excipient also has different effects to ingestion of medicines, medicine distribution and the persistency of medicine in blood plasma in vivo.Those of ordinary skills can determine the characteristic or the feature of wishing, therefore determine the suitable NP preparation that uses.

The polymeric matrix of NP must satisfy biocompatibility, bioavailability, mechanical strength and easy to handle standard.The most famous polymer that is used for this purpose is biodegradable poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer (" PLGAs ").

NP among the present invention can pass through the method preparation of any routine in present technique field.In one embodiment, for example, can be as preparation as described in the following document: people such as Lockman (Lockman, people such as P.R. are used for discharging by blood brain barrier the nanotechnology of medicine " The industry of drug development pharmacopedics(DrugDevelopment Indus.Pharmacy), 28 (1): 1-13, (2002)).The manufacturing process of this type comprises, for example emulsion polymerisation, interfacial polymerization, precipitation thinner evaporation and solvent deposition.

In the emulsion polymerization process of preparation NP of the present invention, described polymerization process comprises from single monomeric unit constructs a polymer chain, for example, and as described in following document: Kreuter (Kreuter, J., nano-particle ", The pharmaceutical technology encyclopedia(Encyclopedia of PharmaceuticalTechnology), Swarbick, J.; Boylan, J.C.Eds.; Marseille Er Deke company (MarcelDekker) (New York, 1994), 165-190 page or leaf, (1994)).Formed the thing triggering by free radical or ion after, for example by using high-energy ray, ultraviolet or hydroxide ion, being aggregated in room temperature can automatically take place.In case polymerization is finished, filter and neutralization solution.Polymer formation comprises from about 100 to 10 ⁷The micelle of individual polymer molecule and drop.In this process, do not need surfactant and stabilizing agent usually.In addition, this process can be in organic facies but not aqueous phase finish.

NP of the present invention also can prepare by interfacial polymerization process, for example, and as described in following document: Khouri (Khouri, people such as A.I., the exploitation that is used to make the new technology of gathering isobutyl cyanide acrylate nano-particle ", International Journal of Pharmaceutical Medicine(Int.J.Pharm.), 28:125 (1986)).In this process, when water and organic facies when homogenizing, emulsifying or Micro Fluid merge under the mechanical agitation of big moment, monomer is used to produce polymer and polymerization takes place.For example, by lipophilic catechol butane is mixed in organic facies with monomer, dissolving mixt and mixture is added water lentamente by a tubule prepare the paracyanogen base acrylate/nano granule that contains the catechol butane when constantly stirring in oil.Can automatically form the capsule of 200-300nm then by anionic polymerisation.The variation of this process comprises benzyl benzoate, acetone and phospholipid added and contains in the organic facies of monomer and medicine, for example, and as described in following document: people such as Fessi (Fessi, people such as H., " behind solvent exchange by interface deposition preparation Nano capsule " International Journal of Pharmaceutical Medicine(Int.J.Pharm.), 55:R1-R4, (1989)).This produces a kind of preparation, does not degrade before protecting it to arrive target tissue by incapsulating at medicine described in the said preparation.

Can be when the preparation NPs use in oil degeneration and the desolvated process such as the macromole of albumin and gelatin.In oily emulsifying degenerative process, can macromole be trapped in the organic facies by homogenizing.In case be trapped, described macromole is induced to aqueous phase lentamente by stirring constantly.Then, can for example make by introducing the nano-particle sclerosis that two kinds of immiscible phases form by crosslinked with acetaldehyde or by thermal denaturation.

Alternatively, macromole can form NPs by " desolvation ".In the desolvation process, macromole is dissolved in a kind of solvent, and described macromole is present in the solvent with structure expansible, that curl.Then, by changing environment, for example pH, electric charge or use are induced closely to curl expansible macromole such as alcoholic acid desolvating.Then, described macromole can be by and sclerosis crosslinked and fixing with acetaldehyde.Before crosslinked, the NDGA chemical compound can be adsorbed or be bound to macromole, thereby derivant is absorbed in the microgranule of new formation.

Can handle solid lipid NP by high pressure homogenize.Solid lipid NP has the advantage that can be sterilized and steam be pressed and it has the solid matrix that sustained release is provided.

The present invention also comprises the NP with different medicine-carrying methods.NP can be the gluey NP of the finely dispersed therein solid of medicine.NP can be solid NP, and medicine links to each other with the outside of NP, for example by absorption.NP can be absorbed in wherein Nano capsule for the messenger drug thing.NP can also be the gluey NP of solid, has finely dispersed medicine therein and is used for target administration to the cell surface part of suitably organizing.

The size of NP can be relevant with the curative effect that is used for given release mode.NP is generally about 10nm to about 1000nm; Alternatively, NP can for about 30nm to about 800nm; Further be generally about 60nm to about 270nm; Even further be that about 80nm is to about 260nm; Or about 90nm is to about 230nm, or about 100nm about 195nm extremely.The size of several factor affecting NP, all factors can be adjusted by those of ordinary skills, for example, the pH of the solution that in polymerization process, uses, the concentration of amount of start trigger (for example heating or radiation etc.) and monomeric unit.Measure by the size that photon correlation spectroscopy can carry out NP by using light scattering.

Alternatively, the NP among the present invention, for example polysaccharide NP or albumin NP can apply one deck lipid coatings.For example, polysaccharide NP can be crosslinked with phosphate (anionic) and quaternary ammonium salt (cationic) part, is with or without lipid bilayer, for example a kind of lipid bilayer that contains dipalmitoyl phosphatidyl choline and cholesterol coating.Other polymer/stabilizing agent comprises but is not limited to: Oleum Glycines; Maltodextrin; Poly-butyl cyanide acrylate; Butyl cyanide acrylate/dextran 70kDa, polysorbate-85; Poly-butyl cyanide acrylate/dextran 70kDa, polysorbate-85; Stearic acid; Polymethyl methacrylate.

For example make the NP preparation that contains the catechol butane be used for the treatment of influenza by intravenously administrable by being adsorbed to NP.Undesirably taken in by reticuloendothelial cell for fear of these NP preparations, NP can be coated with surfactant or use the preparation of magnetic response material.

Therefore, alternatively, surfactant can be used in combination with NP.For example, poly-butyl cyanide acrylate NP can use together with dextran-70,000 stabilizing agent and as the Polyoxyethylene Sorbitan Monooleate of surfactant.Other surfactant comprises but is not limited to: polysorbate-20,40 or 60; Poloxamer (Poloxamer) 188; Lipid coatings-dipalmitoyl phosphatidyl choline; Epikuron 200; Poloxamer (Poloxamer) 338; Polaxamine 908; Polaxamer 407.For example, Polyaxamine 908 can be used as a kind of surfactant and uses the absorption to NP with the RES in minimizing liver, spleen, lung and the bone marrow.

Described magnetic response material can be Magnet (Fe ₃O ₄), it can be incorporated in to the compositions that is used for preparing NP.These magnetic responses NP can externally be guided by magnet.

In another embodiment, can prepare NP (Mu of the present invention as the mixture of Mu and the described use polylactide-co-glycolide of Feng copolymer (" PLGAs ") and d-alpha-tocopherol cetomacrogol 1000 succinate (vitamin E TPGS or TPGS), L and Feng, S.S., " a kind of be used for anti-cancer medicine paclitaxel (

) the new sustained release agent of medicine: the PLGA nano-particle that contains vitamin E TPGS." The sustained release magazine(J.Control.Rel.) 86:33-48 (2003)).The latter can also be used as emulsifying agent except as the host material.

Capsule forms the preparation of carrier:

Present invention resides in the catechol butane that forms in the micelle carrier, wherein said micelle is prepared by conventional method in the art.The example of this type of preparation method is as described in the following document: for example Liggins (Liggins, R.T. and Burt, H.M., g " is used to prepare the polyether-polyester diblock copolymer of paclitaxel polymer micelle administration system." The drug release studies progress(Adv.Drug Del.Rev.54:191-202, (2002)); (people such as Zhang is " as the progress of the amphiphilic diblock copolymer of the micelle carrier of paclitaxel for people such as Zhang." International Journal of Pharmaceutical Medicine(Int.J.Pharm.) 132:195-206, (1996)); And Churchill (Churchill, J.R. and Hutchinson, F.G., " the amphipathic copolymer of biodegradable." United States Patent (USP) 4,745,160, (1988)).In these class methods, the polyether-polyester block copolymer is for having the amphipathic copolymer of hydrophilic (polyethers) part and hydrophobic part (polyester), and it is as forming micellar carrier.

The micelle of another kind of type is, for example form by both having the AB-type block copolymer that hydrophilic segment also has a hydrophobic part, because its amphipathic nature, it is known forming micellar structure in aqueous medium, for example as following document description: Tuzar (Tuzar, Z. and Kratochvil, P., " block in the solution and graft copolymer micelle.", The colloid surface scientific advance(Adv.Colloid InterfaceSci.) 5:201-232. (1976)); And people such as Wilhelm (" poly-(styrene-ethylene oxide,1,2-epoxyethane) block copolymer micelle in the water forms: fluorescent probe research for Wilhelm, people such as M..", Macromolecule24:1033-1040 (1991)).Do not consider that high-load dewatering medicament is incorporated in in the micelle kernel, these polymer micelles can keep gratifying water stability.The micelle of the scope of these about in size＜200nm can reduce non-selective RES effectively and purify and demonstrate enhanced permeability and retentivity.

In addition, for example, can use MePEG 1900 and poly-(D, L-lactide)-6-methoxy poly (ethylene glycol) (MePEG:PDLLA) diblock copolymer of 5000 preparations.Use stannous octoate (0.25%) as catalyst, be reflected at 160 ℃ and can allow to carry out 3 hours.Yet, if reaction allowed to carry out about 6 hours, can use temperature low as 130 ℃, if or reaction only allowed to carry out about 2 hours, can use temperature high as 190 ℃.

In one embodiment, use Kohori, people's such as F. (1998) method (Kohori, people such as F., " preparation and the characteristic that contain the thermal sensitivity block copolymer micelle of poly-(N-N-isopropylacrylamide-b-D, L-lactide).", The sustained release magazine(J.Control.Rel.) 55:87-98, (1998)) pass through radical polymerization process, can use N-N-isopropylacrylamide (" IPAAm ") (emerging people (Kohjin), Tokyo, Japan) and DMAA (" DMAAm ") (with light pure chemistry product company (Wako PureChemicals), Tokyo, Japan) poly-(IPAAm-DMAAm) copolymer of preparation terminal hydroxy group.The copolymer that makes can be dissolved in the cold water, and by two kinds of ultrafiltration membrance filters, separation (cut-off) molecular weight is 10,000 and 20,000 copolymer.Polymer solution at first is 20,000 diffusion barrier filtration by molecular weight.Then, be that 10,000 diffusion barrier filters filtrate by molecular weight again.The result can obtain the fraction of three kinds of molecular weight, low-molecular-weight fraction, middle molecular weight fraction and high molecular fraction.Then by the terminal hydroxy group of the poly-IPAAm-co-DMAAm copolymer of middle molecular weight fraction by D, the ring-opening polymerisation of L-lactide can synthetic segmented copolymer.The poly-IPAAm-co-DMAAm-b-that makes gathers (D, the L-lactide) copolymer can be as Kohori, F. wait the described method (Kohori of people (1999), F. wait the people, " use the thermosensitive polymer micelle control amycin cytotoxic activity of forming by poly-(N-N-isopropylacrylamide-co-N; N-DMAA)-b-poly-(D, L-lactide).", Colloid surface chemistry: bioelectric interface(Colloids Surfaces B:Biointerfaces) 16:195-205, (1999)) preparation.

The catechol butane can be loaded in the micellar kernel, and prepares micelle simultaneously by dialysis.For example, the hydrochlorate of catechol butane can be dissolved in the N,N-dimethylacetamide (" DMAC ") and add triethylamine (" TEA ").Poly-(D, the L-lactide) block copolymer of poly-(IPAAm-co-DMAAm)-b-can be dissolved among the DMAC, and can add distilled water.The solution of catechol butane and block copolymer solution can be in mixed at room temperature, and using the isolated molecule amount then is 12,000-14,000 dialyzer (

2 spectrum health care industry company (spectrumMedical Indus.), California, the U.S.) pass through distill water dialysis at 25 ℃.See above, as Kohori, people such as F. (1999) are described, can be by using the micro-filtration membrane (ANODISC in 20nm aperture ^TMFertile China international (Whatman International)) filter poly-(D, L-lactide) micelle of poly-(IPAAm-co-DMAAm) copolymer-b-that purification combines the catechol butane.

Contain the preparation of the multivesicular liposome of catechol butane:

Can prepare multivesicular liposome (" MVL ") by any conventional method in the art, for example the multiple emulsion process of describing as Mantriprgada (Mantriprgada, S., " system be used for sustained-release administration liposome (

Technology)." liposome progress (Prog Lipid Res.) 41:392-406, (2002)).In brief, in multiple emulsion process, at first be equipped with " Water-In-Oil " emulsion by dissolving amphipathic lipids system, the phospholipid that for example contains at least a neutral fat plastid, the triglyceride of one or more volatilizations in organic solvents for example, with first kind of immiscible aqueous solution composition and hydrophobic catechol butane, for example hydrophobic catechol butane adds this liposome component.Emulsifying mixt mixes with second kind of immiscible aqueous solution composition then to form water in oil emulsion then, is suspended in second kind of solvent spherolite in the aqueous solution composition by mechanical mixture with formation then.Described solvent spherolite will be included in the multiple drop that has wherein dissolved the catechol butane.Remove organic solvent from spherolite then, usually by evaporation, by decompression or above suspension or wherein feed air-flow.After solvent was removed fully, spherolite became MVL, for example the DepoFoam microgranule.When not comprising the neutral fat plastid in this method, will form common multilamellar vesicle or unilamellar vesicle but not MVL.

The catechol butane preparation that is used for oral administration:

Some catechol butane are water solublity, hydrophilic compounds, for example G ₄N.The present invention includes the preparation of the hydrophilic compounds in pharmaceutically acceptable carrier or the excipient and such as the administering mode of oral formulations, the aqueous solution form of chemical compound for example, perhaps chemical compound can as the powder administration, perhaps be made tablet by lyophilizing, perhaps chemical compound is incapsulated.

Chemical compound among the present invention can be the casing sheet.Preparation among the present invention can be by slow release and/or sustained release, and comprising slows down discharges or acceleration release.

Can be included in the amount of the catechol butane in the oral formulations according to the dose titration of the hope of giving the study subject administration.This kind is adjusted in those of ordinary skills' the technical merit.

Some catechol butane are hydrophobic or hydrophilic chemical compound, for example M ₄N.Can promote dissolution velocity or the pharmaceutically acceptable carrier of the scope absorption that can promote enteral lipophilic compound of chemical compound in the intestinal aqueous topical fluids by using.Lipid carrier is being known in the art, for example, and as Stuchlik described (Stuchlik, M. and Zak, S., the k " lipid medium that is used for oral administration.", Biomedical paper(Biomed.Papers) 145 (2): 17-26, (2001)).Preparation of the present invention can be used as the liquid oral administration and maybe can pack in various types of capsules.

In one embodiment, the present invention includes a kind of preparation that contains lipophilic catechol butane, by this type of compound dissolution is made into oral administration at the butane of catechol described in the triacylglycerol, described then preparation is loaded into capsule for oral.Triacylglycerol is to have long-chain and/or the medium-chain fatty acid molecule that links to each other with glycerol molecule.The scope of long-chain fatty acid is from about C ₁₄To C ₂₄, can in common fat, find.The scope of medium-chain fatty acid is from about C ₆To C ₁₂, can in Oleum Cocois or palm-kernel oil, find.Be suitable for triacylglycerol of the present invention and comprise structured lipid, described structured lipid contains the mixture that short-chain fatty acid or medium-chain fatty acid or both contain, the esterification on identical glycerol molecule of described short-chain fatty acid or medium-chain fatty acid.

In another embodiment of the invention, one or more surfactants can be joined in the mixture of catechol butane and lipid carrier, so that medicine exists with the meticulous drop of oil/surfactant mixture.Surfactant can promote that Oily preparation disperses to be diluted in the gastrointestinal fluid.

The present invention also comprise the microemulsion form that contains hydrophilic surfactant and oil the catechol butane be used for oral preparation.Described microemulsion microgranule can be for containing the surfactant micella of dissolved oil and medicine.

Catechol butane preparation in the solid lipid nano-particles preparation also is suitable for oral administration.Can use in the art that the mode of any routine prepares solid lipid nano-particles, for example, see above, as Stuchlik, M. and Zak, S. (2001) is described.

In one embodiment, can prepare solid lipid nano-particles in the homogenizing process in heat by lipid in the fusing of high temperature homogenizing.In this process, solid lipid is melted, and the catechol butane is dissolved in the lipid of fusing.The lipid melt mixed of the disperse medium of preheating and drug loading then, mixture mixes to form rough pre-emulsion with homogeneous agent (homogenisator).Carry out high pressure homogenize to produce oil/water-nano-emulsion in the temperature that is higher than the lipid fusing point then.The cooling nano-emulsion to room temperature to form solid lipid nano-particles.

In another embodiment of the present invention, can in cold homogenizing process, prepare solid lipid nano-particles.In this process, the lipid dissolving, the catechol butane is dissolved in the lipid of fusing.In liquid nitrogen or dry ice, solidify the lipid of drug loading then.At the microgranule of grinding factory abrasive solid medicine lipid with formation 50-100 μ m.In cold aqueous solution disperse medium, disperse lipid particles then, at room temperature or subambient temperature homogenizing to form solid lipid nano-particles.

The present invention also comprises the preparation of the lipotropy catechol butane of the liposome that is used for oral administration or micelle.Can using in the art, the mode of any routine prepares these preparations.Micelle is generally the lipid monolayer vesicle, and hydrophobic medicine combines with hydrophobic region on the monolayer in described vesicle.Liposome is generally the phospholipid bilayer vesicle.Lipophilic catechol butane will be positioned at the central authorities of these vesicles usually.

The preparation according to the suitable catechol butane that is used for oral administration of the present invention in addition is described at following document: the serial number that on January 27th, 2005 submitted to is 60/647,495 U.S. Provisional Patent Application, and submission on January 27th, 2006, name is called the international application of " oral formulations that is used to comprise the catechol butane administration of NDGA chemical compound ", attorney docket is 682714-9WO, and its full content is incorporated the present invention by reference into.

The catechol butane preparation that is used for intranasal administration:

The present invention includes the catechol butane preparation that is used for intranasal administration with and the intranasal administration mode.Advantageously, intranasal administration can be than the activating agent that gathers higher concentration by intravenously administrable in brain.In addition, the liver that avoided at the study subject of accepting medicine of this administering mode and the problem of enteral first pass metabolism.

Amount that can absorbed activating agent partly depends on the dissolubility of the medicine in the mucus, and described mucus is a kind of compositions that contains have an appointment 95% serum albumin, glycoprotein, lipid and electrolytical aqueous solution.Generally speaking, along with the lipotropy enhancing of activating agent of the present invention, the drug level among the CSF also increases.For example, see (Minn, people such as A., " medicine is transferred in the mammal brain: the intranasal approach with and specific dysbolismus.", The drug targeting magazine(J.Drug Target), 10:285-296, (2002)).

Hydrophilic catechol butane can be dissolved in the pharmaceutically acceptable carrier, for example saline, phosphate buffer or phosphate buffer salt.In one embodiment, as described, the 0.05M phosphate buffer of pH7.4 can be used as carrier, for example, (" intranasal administration of the water-soluble prodrug by levodopa (L-Dopa) promotes its general administration and CNS specific administration to people such as Kao for Kao, people such as H.D..", Drug research(Pharmaceut.Res.), 17 (8): 978-984, (2000)).

When using medicament, can make the intranasal administration optimization of medicament of the present invention by the position of adjusting study subject.For example, patient's head can differently orientate as vertical-90, lie on the back-90 °, lie on the back-45 ° or lie on the back-70 ° to obtain optimum efficiency.

The carrier of catechol butane compositions can and can coexist with the activating agent in the compositions for pharmaceutically acceptable any material.When carrier was liquid, it can be hypotonic or isoosmotic than the nose juice, and pH about 4.5 to about 7.5 scope.When carrier was powder type, it was also in acceptable pH scope.

The carrier components that is used for intranasal administration can selectively comprise and can promote activating agent to enter the lipophilic substance that brain absorbs by nasal mucosa and by the olfactory nerves approach.The example of this type of lipophilic substance includes but not limited to ganglioside and Phosphatidylserine.In described compositions, can comprise one or more lipophilic adjuvants, for example with micelle form.

The intranasal that is used for the treatment of influenza can prepare for the activating agent pharmaceutical composition of study subject administration by usual manner in the art, for example, and as United States Patent (USP) 6,180, described in 603.For example, compositions of the present invention can be made powder, microgranule, solution, aerosol, drop, nano-particle or liposome.Except activating agent, described compositions can contain suitable adjuvant, buffer agent, antiseptic, salt.Solution such as the intranasal drop can contain antioxidant, buffer agent etc.

Pass through drug delivery implant:

Catechol butane of the present invention can be used for the treatment of to study subject by the surgical operation drug delivery implant, for example contains the subcutaneous implantation of the biodegradable polymer of catechol butane.Except surgical operation, this treatment can combine with the treatment of other routine.

Therefore, biodegradable polymer of the present invention can for may be dissolved in the tissue fluid, to polymer or the copolymer of host tissue without any toxicity or unfavorable effect.Preferably, polymer or grant human body by the monomer of its synthetic polymer by FDA and take.Have the monomeric copolymer of different solubility characteristics and be preferably with the kinetics of control degradation, for example improve a kind of monomer with respect to other monomeric ratio with the control dissolution velocity.

In one embodiment, polymer is 1, the copolymer of 3-pair-(to the carboxyl phenoxy group) propane and decanedioic acid [p (CPP:SA)], described as following document: Fleming A.B. and Saltzman, the pharmacokinetics of carmustine implant (Pharmacokinetics of the CarmustineImplant), clinical pharmacokinetics (Clin.Pharmacokinet), 41 (6): 403-419 (2002); And Brem, H. and Gabikian, P., " the biodegradable polymer implant that is used for the treatment of brain tumor." sustained release magazine (J.Control.Rel.) 74:63-67, (2001)).In another embodiment, polymer is the copolymer of Polyethylene Glycol (" PEG ") and decanedioic acid, as people such as Fu described (Fu, people such as J., " the new polymer support that is used for control drug release after sucking or injecting." biomaterial (Biomaterials), 23:4425-4433, (2002)).

The polymeric drug delivery system both applicable to hydrophobic catechol butane of the present invention also applicable to hydrophilic catechol butane of the present invention.The catechol butane can combine with biodegradable polymer and implant by surgical operation.Some polymer compositions also can be used for vein of the present invention or anapnotherapy.

Pass through inhalation

Catechol butane of the present invention can be by sucking general ground and/or being administered to lung partly.The inhalation of medicine obtains high drug level and does not cause the toxic method of general basically as a kind of in the tissue of lung, and follows method also as a kind of general that realizes medicine and accepted widely.The technology that is used to prepare this type of preparation is being known in the art.Can see the effect of treatment pneumonopathy by hydrophobic catechol butane of this mode administration or hydrophilic catechol butane.

Suck pulmonary administration for passing through, catechol butane of the present invention can be made into dry powder, aqueous solution, liposome, nano-particle or polymer, and for example as the aerosol administration.The hydrophilic preparation can absorb also by the alveolar surface, and intravasation supplies the general utilization.

In one embodiment, see above as Fu, described preparation of people such as J. (2002) and use contain the polymer of activating agent of the present invention.For example, polymer of the present invention can be the polymer of decanedioic acid and Polyethylene Glycol (" PEG "), or the copolymer of poly-(lactic acid-co-ethylene glycol) acid (" PLGA ") or polymine (" PEI ") and poly-l-lysine (" PLL ").

In another embodiment, described (Choi, people such as W.S. are " by the proteinic inhalation of alcohol suspension as people such as Choi.", NAS's journal(Proc.Natl.Acad.Sci.USA), 98 (20): 11103-11107, (2001)), the catechol butane that is used for inhalation can be dissolved in salt or ethanol before atomizing sucks and takes.

In another embodiment, when the mode with present technique field routine prepare, during as the dry powder administration, medicament of the present invention is also effective, for example, as people such as Patton described (Patton, people such as J.S., " sucking insulin, " The drug release studies progress(Adv.Drug Deliv.Rev.), 35:235-247 (1999) (2001)).

Present invention resides in the assistance that embeds the microprocessor in the drug release device and discharge the catechol butane down, for example, SmartMist ^TMThing and AERx ^TMFor example as Gonda, people such as I. (1998) are described, " the suction delivery system with compliance and disease control ability." Sustained release impurity(J.Control.Rel.) 53:269-274.

Catechol butane of the present invention and compositions are administered for any influenza virus of treatment.In some preferred embodiment, influenza pandemic strain to be treated is the fowl strain.In some preferred embodiments, influenza infection to be treated is based on the strain of H5N1 fowl.In some preferred embodiment of the present invention, catechol butane and compositions are given the people's study subject that has infected the bird flu epidemic strain by administration.In addition, in certain preferred aspects, catechol butane and compositions are given the people's study subject that suffers human influenza and bird flu MOI by administration.After reading content disclosed in this invention, those skilled in the art will recognize that by taking preparation of the present invention and can treat and/or alleviate other disease condition and/or symptom.

Though be not subject to any specific influenza pathogenesis or the theory of symptomatic reaction, can think that the influenza infection in the human body causes the proinflammatory cytokine dysregulation.The Clinical symptoms of serious people H5N1 disease is consistent with the cytokine dysregulation that virus causes.Though think that all influenza infections cause proinflammatory cytokine, but H5N1/97 virus causes much higher the transcribing of proinflammatory cytokine (Cheung CY than human influenza A virus subtype H3N2 or H1N1, Poon LL, people such as Lau AS, " proinflammatory cytokine of the human body macrophage that causes by influenza A (H5N1) virus: a kind of rare serious human disease's mechanism? " Lancet(Lancet), 2002360 (9348): 1831-7).The specific cytokine that produces is the interferon beta (Id.) in the main external deutero-macrophage of mononuclear cell of TNF-α (being also referred to as " TNF " in the present invention and in the accompanying drawing) and people.

Influenza infection presents serious flu-like symptoms usually, and causes breathing imbalance and/or fatal pneumonia usually.The patient who has infected the H5N1 influenza subtype is because the symptom of acute respiratory distress and multiple organ dysfunction syndrome complicates the constitutional virus bronchopneumonia.Lymphopenia and erythrophagoytosis have become the performance of significance in some this class patients.The symptom of erythrophagoytosis and acute respiratory distress and multiple organ dysfunction syndrome is relevant with the cytokine dysregulation usually.The reactive erythrophagoytosis symptom of the rising that accompanies by proinflammatory cytokine IL-6, IFN-γ and TNF-α concentration has been described in the report after death of the death relevant with H5N1 in 1997.There are many diseases relevant or disorder, include but not limited to encephalitis after asthma, pneumonia, the influenza, bacterial myositis, the Electrocardiographic variation of heart, bronchitis, pulmonary tuberculosis, cancer, rheumatic arthritis, osteoarthritis, scleroderma, systemic lupus erythematosis, cystic fibrosis, cachexia, whole myasthenia imbalance, heart failure, parkinson disease, amyotrophic lateral sclerosis or Green-barre syndrome with influenza infection.

People's H5N1 virus in 2003 is similar to people H5N1/97 separated strain, and it causes the excessive generation of proinflammatory cytokine by the deutero-macrophage of person monocytic cell in external demonstration.Produce a large amount of TNF-α (Guan Y, Poon LL, people such as Cheung CY, " H5N1 influenza: the deformation protein threat of being very popular by in the constitutional human macrophage, inducing with the similar genotypic H5N1 virus that is derived from fowl of Human virus." NAS's journal (Proc Natl Acad Sci U S A), 2004101 (21): 8156-61).Therefore, think that the increase of level of the TNF-α that is derived from macrophage and other cytokine is relevant with the seriousness of the disease of patient that has infected influenza A, especially with carry H5N1 disease bird flu " patient's the uncommon clinical manifestation and the severity of disease relevant.Systemic inflammatory responses, multiple organ dysfunction, acute respiratory distress syndrome, reactive erythrophagoytosis and lymphopenia are the prominent features with patient of serious H5N1 disease.

TNF-α causes that apoptotic ability is known.Because apoptosis is critical, therefore cause that apoptotic activity also is the pathogenesis of influenza for effective influenza virus is duplicated.Human influenza virus and bird flu virus effectively duplicate all relevant (the Wurzer WJ of rise with TNF superfamily member TRAIL and FasL, Ehrhardt C, " NF-/F-of tumor necrosin relative death inducing ligand (TRAIL) and Fas/FasL relies on and induces (NF-kappaB-dependent induction) is critical for effective influenza virus breeding to people such as Pleschka S." Journal of biological chemistry(J Biol Chem), 2004279 (30): 30931-7).

In addition, though be not subject to any specific theory, but advised and thought that generation corresponding to the proinflammatory cytokine of influenza virus gene group RNA increases by the Toll sample receptor on the immune cell membrane sending signal (Diebold SS, Kaisho T, people such as Hemmi H " rely on the congenital antiviral response of the TLR7 mediation identification of single stranded RNA." Science(Science), 2004303 (5663): 1529-31).Use a kind of lipopolysaccharide (" LPS ") of bacterial endotoxin to stimulate macrophage also to cause producing proinflammatory cytokine, for example TNF-α, IL-1, IL-6, IL-10 and such as the short scorching lipid medium and the platelet activating factor of prostaglandin, leukotriene.Shown that the cytokine that produces in response to LPS is to work by Toll sample receptor pathway, be similar to influenza virus reaction (Takeda K, Kaisho T and Akira S. " Toll sample receptor." " Immunology year comments Opinion(Annu Rev Immunol) ", 200321:335-76).

Such as general formula (I), (II) and catechol butane (III), for example, M ₄N or G ₄N, and the compositions that contains with good grounds one or more catechol butane of the present invention can suppress TNF-α and other short scorching biotic factor, prostaglandin E when the LPS of the macrophage of response Mus monocyte derived or viral infection ₂And the generation of other short scorching lipid medium.Produce a large amount of TNF-α because the macrophage cell line (RAW 264.7) of Mus monocyte derived is compared with the constitutional human macrophage can induce in response to LPS, so it has represented the suitable pattern of the people's effect of drugs in a kind of TNF of the prediction system.

Referring now to following non-restrictive example the present invention is described in more detail.

Embodiment 1

The catechol butane of general formula (I), i.e. M are used in research ₄N to the effect that produces TNF-α by inductive RAW 264.7 macrophages of LPS to measure M ₄N suppresses the inductive ability of TNF-α.Can use with the similar method of the method for this embodiment and measure any catechol butane of general formula (I) producing the effect of any proinflammatory cytokine in the macrophage that stimulates at any LPS.

As shown in Figure 1 and hereinafter described, M ₄The inductive TNF-α of LPS that N suppresses in RAW 264.7 macrophages crosses expression, is 57% inducing back 10 hours maximum amount of suppression.

More specifically, about being used to measure M ₄N suppresses the inductive method of TNF-α by LPS, perhaps stays 1.5 x 10 ⁵Individual macrophage is not handled (contrast) or is used LPS (1 μ g/ml), M ₄N (25 μ M) or two kinds of chemical compounds are cultivated in different time points together.RAW 264.7 cells are the Mus mononuclear phagocyte.The LPS that uses is derived from salmonella minnesota R595 (Salmonella minnesotaR595), it can be from Li Site Biotechnology Experiment chamber (the List BiologicalLaboratories of joint-stock company, Inc.) (Campbell, California (Campbell, CA)) obtains.Use Mus TNF-α specific immune to detect then by the TNF-alpha levels in the interpolation mensuration culture supernatant that is derived from standard curve.All tests all carry out twice, and under every kind of situation, Error Graph (errorbars) in-less-than symbol size (symbol size).

Buy the RAW264.7 macrophage from ATCC, in the DMEM that has added 10% hyclone (FBS) (Dulbecco ' s modified Eagles medium), remain on 37 ℃ in cultivation and the carbon dioxide 8%.FBS is available from Atlanta biological product company (Atlanta Biologicals) (Atlanta, Georgia (Atlanta, GA)), and all other medium components are available from SIGMA Ai Erke company (Sigma Aldrich) (St.Louis Blues, the Missouri State (St.Louis, MO)).For the preparation of TNF, gather cell by trypsinization, centrifugal, counting is placed 105 cells of 1.5 x and incubated overnight in 24 hole tissue culturing plates.There are not/exist 25 μ MM then ₄N uses lipopolysaccharide (LPS) the time point irritation cell as shown in Figure 1 of every milliliter 1 gram.Before being added into the hole, dissolving LPS and Ultrasonic Pulverization in tissue culture medium (TCM).In DMSO, prepare M ₄The N storage liquid before being added into the hole, is diluted in culture medium then.The supernatant that collect to produce, 8, centrifugal two minutes of 000rpm is to remove cell and fragment ,-20 ℃ of storages.Use is from (the R﹠amp of development system joint-stock company; D Systems Inc.) (the TNF-alpha levels in the culture supernatant is measured in the immune detection agent for Minneapolis, Kang Nitikai (Quantikine) Mus TNF-α/TNFSF1A that the Minnesota State (Minneapolis, MN)) buys.Described detection agent is the ELISA that catches of sandwich type.The hole provides the polyclonal antibody of the specific affinity purification that is used for Mus TNF-α of pre-bag quilt.Add supernatant to the hole, hatch, any TNF-α of existence is caught by sessile antibody.After the cleaning, the enzyme of adding and anti-TNF-α capture antibody carries out second incubation step then.Clean-out opening adds substrate solution then once more.The division of substrate produces blue solution, then described solution flavescence when adding stop buffer.Use BMG POLARstar galaxy microwell plate reader (galaxymicroplate reader) to measure optical density at 450nm.The reorganization Mus TNF-α solution that standard is provided by manufacturer is measured the level of TNF-α in the culture supernatant to produce standard curve by the interpolation that is derived from standard curve.Shown in Fig. 1 have a few and all carry out twice, meansigma methods is used for quantitatively.

Using phorbol myristyl acetas (phorbol myristyl acetate) (PMA) or A23187 (Calcium ionophore), but not after LPS induces, in RAW 264.7 macrophages, do not observing and pass through M ₄N is to producing the inhibition of TNF-α.Think that PMA and A23187 right and wrong play a role specifically, are independent of cell surface receptor and most of signal transduction process.Therefore, though be not subject to any particular theory, M ₄N suppresses LPS and induces and produce ability that TNF-gives birth to and can derive from the stream signal and the influence in the stage of activation to the TNF reaction, rather than to the influence of the downstream process being responsible for the synthetic of TNF-α and discharging.Therefore, can be sure of M ₄The beneficial effect that N infects treatment H5N1 and not causing to producing the potential deleterious non-specific decline of TNF-α.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress to produce too much the inductive TNF-α of response LPS, explanation when influenza infection by improving the level of TNF-α, described chemical compound and relevant catechol butane and NDGA derivant can be used for the treatment of disease or when influenza infection by the alpha mediated disorder of the TNF-that increases.

Embodiment 2

The catechol butane of general formula (I), i.e. M are used in research ₄N to the effect of the inductive apoptosis of TNF-α in the mouse fibroblast cell to measure M ₄N suppresses the apoptosis-induced ability of TNF-α.Can use any catechol butane of measuring general formula (I) with the similar method of the method for this embodiment to TNF-α in the cell of any kind apoptosis-induced effect.

Influenza infection is induced and is produced TNF-α, and TNF-α pro-apoptosis bioactivity is known.Influenza virus needs apoptosis effectively duplicating, and the inductive apoptosis of blocking-up TNF-α can reduce that influenza virus is duplicated and disease.

As shown in Figure 2 and hereinafter described, M4N suppresses the inductive apoptosis of TNF-α to the TNF sensitivity consumingly by cycloheximide.The C3HA mouse fibroblast cell does not exist/exists NDGA (25 μ M) or M ₄N (50 μ M) with the people recombinate TNF-α (20ng/ml), cycloheximide (CHI) (10ng/ml) or both hatch.Add all chemical compounds simultaneously and handled 6 hours.During last half an hour, add rhodamine (Rhodamine) 123, use BMGPOLARstar galaxy fluorescence analyser (galaxy flourimeter) to measure fluorescence.

More specifically, the C3HA cell in the DMEM that has added 10% hyclone (FBS) (Dulbecco ' s modified Eagles medium), cultivate and the carbon dioxide 8% in remain on 37 ℃.C3HA cell line is the mouse fibroblast cell cell line by the deutero-3T3 sample of C3H Mus.FBS is available from Atlanta biological product company (Atlanta, Georgia), and all other medium components are available from SIGMA Ai Erke company.Detect for apoptosis, gather cell by trypsinization, centrifugal, counting adds 1.5 x 10 in each hole of the flat 96 hole ELISA Plate of culture medium ⁴Individual cell.There are not/exist nordihydroguaiaretic acid (25 μ M) or M ₄During N (50 μ M), before adding TNF (20ng/ml), CHI (10g/ml) or both, allow cell adhesion at least six (6) hour.The people recombinates TNF-α available from Pei Puteke (Peprotech) (roach, New Jersey (Rocky Hill, NJ)), and CHI is available from EMD bioscience joint-stock company (EMD Biosciences Inc.) (Santiago, California (San Diego, CA)).The both is dissolved in the culture medium.NDGA also is dissolved in the culture medium, and M ₄The N storage liquid prepares in DMSO, dilutes in culture medium then, before being added into the hole, dilutes in culture medium then.Adding total amount simultaneously is all chemical compounds of 200 microlitres, allows to hatch six (6) hours.Rhodamine to the ultimate density of adding 50 μ L during last 30 (30) minutes of hatching is each hole of 2 μ g/ml.(Eugene, Oregon (Eugene, OR)) also dilutes in culture medium rhodamine available from molecular probe joint-stock company (Molecular Probes Inc.).Rhodamine is hidden by great-hearted mitochondrion, and the key health cell of living presents strong mitochondrion fluorescence.On the contrary, because apoptotic cells can experience the mitochondrial permeability conversion usually, thereby mitochondrion loses its transmembrane potential, so fluorescence can weaken in having experienced apoptotic cells.As a result, rhodamine fluorescence weakens in apoptotic cells sharp.Use BMG POLARstar galaxy microplate when setting excitation wavelength 492 nanometers and emission wavelength 538 nanometers respectively, to measure fluorescence intensity then.All points all carry out three times, calculate cell death percentage ratio according to following equation:

[(control value-experiment value)/control value] * 100

The result of this embodiment shows that the catechol butane of general formula (I) can suppress the inductive apoptosis of TNF-α, illustrate that described chemical compound and relevant catechol butane and NDGA derivant can be used for reducing influenza virus the duplicating of host cell, and need apoptosis and effectively duplicate.

Embodiment 3

The catechol butane of general formula (I), i.e. M are used in research ₄N is to producing prostaglandin E by inductive RAW 264.7 macrophages of LPS ₂(" PGE ₂"), prostaglandin F _{1 α}(" PGF _1G") and prostaglandin F _{2 α}(" PGF _{2 α}") effect to measure M ₄N suppresses the ability of excessive generation prostaglandin when the response influenza infection.Can use with the similar method of the method for this embodiment and measure any catechol butane of general formula (I) producing the effect of any short scorching lipid medium in the macrophage that stimulates at any LPS.

In fact, prostaglandin be in a organized way with organ in autocrine and the paracrine lipid medium found.It is synthetic by essential fatty acid in cell, for example gamma-Linolenic acid, arachidonic acid and eicosapentaenoic acid.It acts on various kinds of cell, for example except endotheliocyte, uterine cell and mastocyte, causes the platelet cell of polymerization or depolymerization, causes the vascular smooth muscle cell of shrinking or expanding, and causes painful logical spinal neuron.Prostaglandin has multiple effect, includes but not limited to muscle contraction and induces inflammation.Other effect comprises the moving and cell adjusting and controlling growth of calcium current.

PGE2 acts on PGH2 (PGH2) by the prostaglandin E synzyme and produces, and PGH2 is obtained by fatty acid by the effect of Cycloxygenase (COX-1 and COX-2).At influenza infection between the emergence period, PGE ₂Induced.Infected person influenza virus sub-strain H3N2 can increase the release (MizumuraK of PGE2 in the bronchial epithelial cell, Hashimoto S, people such as Maruoka S, " the effect of the arachidonic acid-induction PGE2 of the mitogen activated protein kinase in the influenza virus from bronchial epithelial cell." Clinical and experimental allergic(Clin Exp Allergy), 200333 (9): 1244-51).

Can be by comprising PGH ₂, PGE ₂Or PGD ₂Three kinds of different substrates produce PGF by three kinds of approach _2GPGF _2GCause smooth muscle contraction, it is active relevant with childbirth with asthma.

Prostacyclin is also referred to as PGI ₂, its effect of passing through the PGI synthase is by PGH ₂Produce, it is expressed by many cell types widely.Prostacyclin is loose dose of a kind of important effective vasodilation and smooth muscle in the multiple biological respinse such as inflammation and childbirth.Yet the prostacyclin instability obtains reliable measurement result by the stable derivatives that measurement is called as the prostacyclin of PGF1 α (6-keto-PGF1 α) usually.

As shown in Figure 3 and hereinafter described, M ₄N is to the inductive PGE of LPS ₂Generation have strong inhibitory action.In Fig. 3, M ₄N (25 μ M) has presented in RAW 246.7 macrophages LPS has been induced PGE ₂The strong inhibition that produces.Use separately LPS (1 μ g/ml) in the time period that illustrates, or with 25 μ M M ₄N is used in combination the processing macrophage.Use prostaglandin E then ₂Immune detection (R﹠amp; D system company, Minneapolis, the Minnesota State) detect PGE by supernatant ₂The data that illustrate for 2-4 measured value of each time point on average+/-SEM.At 6,10 and 16 hours, the inhibition level was respectively 72,64 and 80%.M ₄The inhibitory action of N all continues in culture fluid in during whole 16 hours.

As shown in Figure 4 and hereinafter described, M ₄N is to the inductive PGF of LPS _{2 α}Generation also have strong inhibitory action.In Fig. 4, after 16 hours, detect the PGF of 15ng/ml from RAW264.7 macrophage culture supernatant with LPS (1 μ g/ml) stimulation _{2 α}Use LPS (1 μ g/ml) and M when simultaneously ₄N (25 μ M) handled RAW 264.7 macrophages in the time of 16 hours, PGF _{2 α}Generation be suppressed.According to twice experiment by M ₄The meansigma methods % that N (25 μ M) suppresses is 82%.Use PGF _{2 α}The ELISA test kit (detection design company, An Juebai, Michigan (Assay Designs, Ann Arbor, MI)) measure PGF in the RAW 264.7 macrophage culture supernatant by ELISA _{2 α}Value.The data that illustrate be twice independent experiment average+/-SEM.

As shown in Figure 5 and hereinafter described, M ₄N is to the inductive PGF of LPS _{1 α}Generation also have some inhibitory action, in Fig. 5, use LPS (1 μ g/ml) to stimulate after 16 hours, from RAW 264.7 macrophage culture supernatant, detect the PGF1 of 5-6ng/ml _αWhen using LPS (1 μ g/ml) and M ₄N (25 μ M) handles RAW 264.7 macrophages in the time of 16 hours simultaneously, PGF _{1 α}Generation be suppressed.Inhibition meansigma methods % according to twice test is 41%.Use PGF _{1 α}ELISA test kit (R﹠amp; D system company, Minneapolis, the Minnesota State) measure PGF in the RAW 264.7 macrophage culture supernatant by ELISA _{1 α}Level.The data that illustrate are the independently mean+/-SEM of RAW 264.7 tests of basis.

For confirm shown in Fig. 5 to producing PGF _{1 α}(a kind of PGI ₂The index of/prostacyclin) moderate relatively suppression ratio is not owing to some test errors, has measured in these RAW 264.7 macrophage culture supernatant yet and has produced PGE ₂Inhibition %.Consistent with the result among Fig. 3, detect PGE in these supernatant ₂Inhibition surpass 90%, this shows observed to producing PGF _{1 α}Moderate suppression ratio can not owing to cell, LPS and M ₄The test error that N is relevant.

Because be also referred to as the M of EM-1421 ₄N has strong inhibitory action to the generation of Prostaglandins and Leukotrienes, and it can be suitable for treating the inflammatory conditions in the lung particularly well, the asthma that causes by influenza infection for example, and its tendency depends on lipid medium.Different with the PGF synthase with the PGE synthase, the PGI synthase can relatively have resistance to EM-1421, and it has illustrated a large amount of generation PGF when having EM-1421 _{1 α}Reason.

More specifically, about being used to measure M ₄N is to PGE ₂The method of the effect that produces is bought the RAW264.7 macrophage, and cultivates and keep according to the program of describing among the embodiment 1.The generation of finishing PGE2 in the cell by trypsinization collection cell is also centrifugal.The pair cell counting is placed 1.5 x 10 in 24 hole tissue culturing plates ⁵Individual cell and incubated overnight.Use 1 μ g/mi LPS time point irritation cell as shown in Figure 5 when not having/existing 25 μ M then.Before being added into the hole, dissolving LPS and supersound process in tissue culture medium (TCM).In DMSO, prepare M ₄The N stop buffer before being added into the hole, dilutes in culture medium then.The supernatant that collect to produce, 8, centrifugal two minutes of 000rpm is to remove cell and fragment ,-20 ℃ of storages.The prostaglandin E that use is bought from development system joint-stock company ₂Prostaglandin E in the immune detecting measuring culture supernatant ₂Level.Described detection is the ELISA of state of conflict.The prostaglandin E that exists in the supernatant ₂Combine Mus monoclonal anti PGE2 antibody with the PGE2 competition of the alkali phosphorus enzyme labelling of fixed amount.The complex that produces is combined with the bonded goat anti-mouse antibody of microtiter well.After the cleaning, add colorific substrate and carry out quantitatively with amount to desmoenzyme.Use BMG POLARstar galaxy microwell plate reader to measure optical density at 405nm.The prostaglandin E of standard is provided by manufacturer ₂Solution is measured prostaglandin E in the culture supernatant to produce standard curve by the interpolation that is derived from standard curve ₂Level.Have a few and all carry out twice, meansigma methods is used for quantitatively.

Use similar method to measure M ₄N is to PGF _{1 α}And PGF _{2 α}The effect that produces.

The result of this embodiment shows that the too much prostaglandin that the catechol butane of general formula (I) can suppress to respond LPS and stimulates produces, and illustrates when described chemical compound and relevant catechol butane and NDGA derivant can be used for the treatment of at influenza infection by high level prostaglandin mediated disease or disorder.

Embodiment 4

The catechol butane of general formula (I), i.e. M are used in research ₄N to the effect of one group of cytokine producing by RAW 264.7 macrophages to measure inducing and M by the LPS stimulated cells factor ₄N suppresses inductive ability.

In this research, used antibody (" Ab ") chip technology.As shown in Figure 6 and hereinafter described, M ₄N is inhibited to the inductive several production of cytokines of LPS.Though the production of cytokines level is low generally, handles (" control " group among Fig. 6 A) without LPS stimulation or EM-1421 and in the supernatant that derives from the RAW264.7 macrophage, detect many cytokines.Though several production of cytokines reduce (KC, BLC, IL-4, IL-9, MIP-1 α, M1P-1 γ and IL-12p40p70) and two kinds of production of cytokines increase (IL-1 α and MIG), but after using EM-1421 to handle with final concentration 25 μ M (" EM-1421 " group among Fig. 6 A), this kind production of cytokines pattern is kept generally.This has strengthened EM-1421 is safe clinical evidence.

What LPS caused many cytokines (among Fig. 6 B " LPS " group) rolls up (〉 20%), comprise RANTES, IL-1 α, IL-2, TIMP-1, TIMP-2, TNF-α, IL-6, MCP-1, sTNFRI, sTNFRII, IL-12p40, MIP-1 α and G-CSF.Under several situations, these increase by EM-1421 (" LPS+EM-1421 " group among Fig. 6 B) partially or even wholly offsets.In the cytokine that high level produces, it is about 20% that EM-421 suppresses the inductive IL-1 α of LPS, and TNF-α is about 24%, and MCP-I is about 33%, sTNFRI about 63% and sTNFRII about 20%.In the cytokine of low-level generation, it is about 100% that EM-421 suppresses the inductive I-TAC of LPS, and IL-2 is about 100%, and TIMP-1 is about 30%, and TIMP-2 is about 100%, BLC about 100% and IL-3 about 100%.Yet, because these cytokines with low-level generation, therefore predict that the meaning of these observed results is difficult.Enjoyably, EM-1421 does not suppress the generation of the inductive RANTES of LPS, IL-6, IL-12p70, MIP1-α and G-CSF, and increases the generation about 43% of the inductive IL-12p40p70 of LPS.

More specifically, about being used to measure M ₄The method of the effect that the N pair cell factor produces has been used " Mus inflammation chip-1 " (battle array photo bio technical concern company, Atlanta, Georgia (RayBiotech, Inc., Atlanta, GA)) in described method.Buy the RAW264.7 macrophage, cultivate, use LPS (1 μ g/ml) to stimulate, use EM-1421 (25 μ M) to handle, according to the collection cell of describing among the embodiment 1.Use Mus inflammation chip-1 according to the description of manufacturer, use or do not use LPS (1 μ g/ml) stimulation and use or do not use EM-1421 (25 μ M) to handle the cytokine in the culture supernatant of measure R AW264.7 macrophage.In brief, use nitrocellulose Ab chip to hatch the about 2h of culture supernatant, clean, contact, use the ECL hydroponics, and contact with the X-ray film with second kind of Ab solution.The autoradiography of scanning chip.Use Photoshop (Adobe) to analyze and measure the mean pixel gray scale of each array position.In Fig. 6 A and Fig. 6 B, marked the meansigma methods of copy-point (duplicate spots).Positive control point is about 100 units on each chip, and SEM is lower than 10% for all copy-points, and SEM is lower than 1% for most of copy-points.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress to respond that LPS stimulates, except TNF-α, also have the generation too much of other several cytokines, illustrate that described chemical compound and relevant catechol butane and NDGA derivant can be used for the treatment of when influenza infection by high level cytokine mediated disease or disorder.

Embodiment 5

The catechol butane of general formula (I), i.e. M are used in research ₄N to the work of the influenza strain A/WS/33 that produces by mdck cell and RAW264.7 macrophage in order to measure M ₄N suppresses the ability of influenza virus growth in these cells.Any catechol butane that can be used for measuring general formula (I) with the similar method of the method for this embodiment at the cell of any kind to the growth of any strain of influenza virus or the effect of duplicating.

A/WS/33 be a kind of can be from American type culture collection (American TypeCulture Collection) (ATCC) (Manassas, continent, Virginia (Manassas, VA)) commercial influenza virus A strain that obtains.It separates from the patient who suffers from influenza.The host of the A/WS/33 that recommends comprises Embryo Gallus domesticus, ferret and Mus.

Mdck cell is the epithelioid cell that is derived from the kidney of the normally adult female Cocker of appearance.It demonstrates supports various types of viruses, comprises the growth of influenza A virus.Use mdck cell to produce the A/WS/33 of high titre deposit (stocks) and be used for the quantity of quantitative assay with the infective virus in the culture supernatant of experiment with measuring.Determine to be used for mdck cell and not cause that the maximum concentration of the EM-1421 of toxic action is 25 μ M.Determined that multiple quantitative analysis method duplicates with monitoring A/WS/33, includes but not limited to cytopathy (TCID ₅₀), speckle, immunity focus on (immunofocus) and immunofluorescence assay.

As shown in Figure 7, group A and group B have shown same data, but have linear and logarithm y axle respectively, and concentration is that the A/WS/33 that the EM-1421 of 25 μ M suppresses in the mdck cell duplicates about 75%.Be generally used for making the visual logarithmic plot of titration of virus greatest differences to show that the inhibitory action of EM-1421 is lower than 1 log unit.

As shown in Figure 8, group A and group B have shown same data, but have linear respectively and logarithm y axle, do not suppress duplicating of A/WS/33 in RAW 264.7 macrophages at the EM-1421 of different test concentrations (3 μ M, 6 μ M, 12 μ M and 25 μ M).On the contrary, EM-1421 has promoted to produce A/WS/33 by RAW 264.7.Yet in addition, this effect relatively moderately promptly is lower than 1 logarithmic growth (Fig. 8 B).Use EM-1421 pretreatment RAW 264.7 cells also to promote the growth of A/WS/33.Shown in group A among Fig. 9 and group B, the concentration of EM-1421 described facilitation when 25 μ M is especially remarkable, and the time point at 36 hours records 1 logarithmic increment.

EM-1421 suppresses the growth of mdck cell influenza strain A/WS/33, but promotes the growth of RAW264.7 macrophage.Under two kinds of situations, these effects are all relatively appropriate, i.e. about 1 the logarithmic variation of virus titer.For fully determining the effect that EM-1421 duplicates influenza virus, need to check other virus and cell type.In addition, whether influence virus is significantly loaded in order to determine these effects, and in vivo test is essential.

More specifically, about being used to measure M ₄N is to the method for the effect of the A/WS/33 that produced by mdck cell or RAW 264.7 macrophages, and mdck cell or RAW 264.7 macrophages are inoculated with 0.001 or 0.002 infection multiplicity (MOI) respectively.Except check sample does not add the medicine, influenza infection is taking place after 30 minutes, EM-1421 is added into cell with the concentration of hope, and keeps this concentration during whole test.Collect culture supernatant at desired time point.

Use then based on the infective virus in immune these supernatant of focus detection quantitative analysis of MDCK.Mdck cell (5 x 10 tile in 24 orifice plates ⁵/ hole), and in the Virus culture base incubated overnight, described culture medium contains: contain 10% hyclone (Atlanta biological product company, the Atlanta, Georgia) DME basal medium (#10-013-CV, (Media technology company, Virginia He Endeng (MediaTech, Hemdon VA)), 25mM HEPES buffer (#25-060-CL, Media technology company), 1:100 antibiotic/antifungal solution (#A5955-Sigma-Aldrich, St. Louis, the Missouri State (St.Louis Mo)), 1.8 μ g/ml hyclone (#A7906 Sigma-Aldrich) and 2mg/ml trypsin #3740, Wo Xindun company (Worthington), New Jersey Lakewood (Lakewood NJ)).In not having the same culture medium of hyclone, clean twice cell then.In 30 minutes, add the serial dilutions of the supernatant that contains virus then, use then and contain 0.6% Tragacanth (#104792, MP biological medicine joint-stock company, Soren, Ohio (MP Biomedicals Inc., Solon OH)) the Corticovirus culture medium.After hatching 24 and 48 hours, extract covering out, use PBS cleaning cell and use the acetone/methanol of 50:50 to fix.Use anti-HA antibody staining cell with focus detection then.

For measuring M ₄N wishes that to being produced the effect of A/WS/33 by pretreated RAW 264.7 macrophages of EM-1421, at first using the EM-1421 of concentration hatched RAW 264.7 macrophages 2 hours.Be 0.002 o'clock use A/WS/33 incubated cell at MOI then.EM-1421 is present in the cell culture fluid and keeps same concentration during whole test.Collect culture supernatant at desired time point.Use above-mentioned then based on the infective virus in immune these supernatant of focus detection quantitative analysis of MDCK.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress duplicating of influenza virus in some host cells, illustrates that described chemical compound and relevant catechol butane and NDGA derivant can be used for suppressing duplicating of host influenza virus or grow.

Embodiment 6

The catechol butane of general formula (I), i.e. M are used in research ₄N produces the effect of TNF-α to measure M to the RAW264.7 macrophage by infection influenza strain A/WS/33 ₄N suppresses because the inductive ability of TNF-α that influenza infection causes.Can use with the similar method of the method for this embodiment and measure any catechol butane of general formula (I) in the macrophage that has infected any influenza virus, producing the effect of any proinflammatory cytokine.

Two kinds of model systems in this research, have been used, low multiple infection model and high multiple infection model.In low multiple infection model, use the very infection of the influenza virus of low dosage (MOI=0.002) startup RAW 264.7 macrophages, described then influenza virus was distributed in the whole culture fluid during ensuing 24-48 hour.This model is attempted near observed situation in the body during natural infection.Yet in this model, infection must be carried out in comprising the tryptic infection culture medium that does not contain serum of 2 μ g/ml.Find when RAW 264.7 cells when its growth medium (DEM that contains 10%FCS) is converted to the infection culture medium that does not contain serum, infection culture medium autostimulation macrophage also improves cytokine and the background value of the generation of lipid medium.Trypsin is included in and infects in the culture medium, because influenza virus is from cell propagation to cell (in the body or external), it must work by extracellular protease.

In high MOI model, use the influenza virus (MOI=5) of high dose to start infection, guarantee that all cells in fact promptly and side by side infect described influenza virus.After starting these infection, observe strong viral hemagglutinin dyeing 8 hours.In remaining the common culture medium of low value, infects the background value of cytokine and lipid medium.Yet, under these conditions, do not produced infective virion.Lacking viral ionic generation and high influenza virus starts dosage and means that this model keeps off in intravital influenza infection.

Therefore, low MOI and high MOI model all are used to obtain influenza virus and the EM-1421 more complete visual angle to the metabolic effect of RAW 264.7.

Figure 10-12 has described the result of low MOI detection model.In Figure 10, when RAW 264.7 cells when its growth medium is converted into the infection culture medium that does not contain serum, cell produces the TNF-α (" culture medium " bar) of about 750pg/ml, and it is higher than the 100pg/ml that records usually when cell remains in the growth medium.EM-1421 (25 μ M) reduce separately this be worth about 67%, offset significantly relevant with culture medium (" EM-1421 " bar) conversion " stress " or " activation " signal.As reporting, find that influenza infection causes the value of TNF-α to increase (" influenza " bar).In this research, the value of the TNF-α that records behind influenza virus infection (strain A/WS/33) increases about 80-85% usually.EM-1421 (25 μ M) strain has fully been blocked the increase (" influenza/EM-1421 " bar) that the inductive TNF-α of this influenza virus produces.

Figure 11 has described the result of dose-response test when the EM-1421 of variable concentrations.The EM-1421 culture medium separately or culture medium and ultimate density is low as influenza infection 0.1 μ M all suppresses the increase of the generation of TNF-α together.The concentration that increases EM-1421 causes that inhibition increases.

Figure 12 shows the time-histories result of experiment.Inoculate or do not inoculating influenza strain A/WS/33 (" influenza "), and using or do not using EM-1421 (" EM-1421 ") incubated cell.The amount of TNF-α in the time point determining culture supernatant that illustrates in the drawings.As if the inhibitory action of finding EM-1421 is right after inducing of TNF-α, and inducing of TNF-α all kept suppressing during whole 24 hours.

Figure 13-15 has described the result of high MOI detection model.RAW 264.7 cells produce the TNF-α of about 100pg/ml when not having viral infection and use EM-1421 (" culture medium " bar) to handle.EM-1421 (25 μ M) reduces this separately and is worth about 35% (" EM-1421 " bar).In addition, find that influenza infection causes that TNF-α value increases, and illustrates about 135% (" influenza " bar).EM-1421 (25 μ M) has also fully blocked the increase (" influenza/EM-1421 " bar) that the inductive TNF-α of influenza virus produces.Figure 14 has described the result of dose response test.The EM-1421 of about 10 μ M of ultimate density and 25 μ M suppresses TNF-α output respectively by influenza infection increases about 34% and 60%.Figure 15 shows the result of time-histories test.As if the inhibitory action of EM-1421 is right after inducing of TNF-α, and inducing at 12 hours and 24 hours of TNF-α suppresses 51% and 55% respectively.

As mentioned above, M ₄N suppresses the expression excessively of the inductive TNF-α of influenza virus in the RAW264.7 macrophage consumingly in low and high MOI model system.Therefore, M ₄N may suppress to have infected influenza virus similarly, has especially infected the TNF-α response in the human macrophage of H5N1 influenza virus sub-strain.TNF-α has one of pivotal role person in the inflammatory reaction fatal usually in the lung of the fatal H5N1 subtype influenza of height in infection.Therefore, by the imbalance of control cytokine, EM-1421 can reduce pneumonia and the death relevant with the mortality influenza infection significantly, and reduces people H5N1 severity of disease.The time-histories test shows that EM-1421 suppresses inducing of TNF-α in early days in infection, shows that EM-1421 may work with the synthetic and/or release of inhibition TNF-α, but not causes the degraded of TNF-α.

More specifically, about the low MOI model that uses, 1.5 x 10 ⁵Individual RAW 264.7 macrophages/hole is tiled in the DME culture medium that contains 10% FCS (#D5648, Sigma Aldrich, St.Louis Blues, the Missouri State) and spends the night.Remove culture medium, in the viral growth culture medium (the DME yl that contains trypsin, 2.5%HEPES buffer agent and the 0.2%BSA of 2 μ g/ml) to use MOI be that the virus inoculation (strain A/WS/33) of 0.002 200 μ l is replaced, and allow virus absorption 30 minutes.The volume that increases culture medium then is to 1ml.When volume increased to 1ml, the ultimate density of EM-1421 increased to 25 μ M.Do not contain virus and the hole of EM-1421 (" culture medium ") or only contain EM-1421 (" EM-1421 ") and be used as " simulated infection " hole, its hole with infection is subjected to identical processing but does not have virus.After hatching about 24 hours, collect culture supernatant, detect TNF-α by ELISA.The data that illustrate be two independent trialss average+/-SEM, twice multiple infection carried out in each test.All ELISA points detect twice.

In the high MOI model that uses, 1.5 x 10 ⁵Individual RAW 264.7 macrophages/hole is tiled in the DME culture medium that contains 10% FCS spends the night.Remove culture medium, using MOI in containing the DME culture medium of 10% FCS is that the virus inoculation (strain A/WS/33) of 5 200 μ l is replaced, and allows virus absorption 30 minutes.The volume that increases culture medium then is to 1ml.When volume increased to 1ml, the ultimate density of EM-1421 increased to 25 μ M.The hole of not containing virus and the hole of EM-1421 (" culture medium ") or only containing EM-1421 (" EM-1421 ") is used as " simulated infection " hole, and its hole with infection is subjected to identical processing but does not have virus.After hatching about 24 hours, collect culture supernatant, detect TNF-α by ELISA.The data that illustrate be two independent trialss average+/-SEM, twice multiple infection carried out in each test.All ELISA points detect twice.

In the dose response test, when the volume of culture medium increased to 1ml, the ultimate density that is added into the EM-1421 of culture medium was 0.1,1,10 or 25 μ M.

In the time-histories test, after hatching about 4,12 or 24 hours behind the cell culture fluid inoculation influenza virus, collect culture supernatant, detect TNF-α by ELISA.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress to produce in response to the too much TNF-α of influenza infection, illustrates that described chemical compound and relevant catechol butane and NDGA derivant can be used for the treatment of when influenza infection by high-level TNF-alpha mediated disease or disorder.

Embodiment 7

The catechol butane of general formula (I), i.e. M are used in research ₄N produces PGE to RAW 264.7 macrophages that infect influenza strain A/WS/33 ₂Effect to measure M ₄N suppresses to induce PGE by influenza infection ₂Ability.Can use with the similar method of the method for this embodiment and measure any catechol butane of general formula (I) in the macrophage that has infected any influenza virus, producing the effect of any short scorching lipid medium.

As Figure 16 and 17 and hereinafter described, M ₄Crossing of the inductive PGE2 of influenza expressed in N inhibition RAW 264.7 macrophages.Therefore, M ₄N may suppress to have infected influenza virus similarly, has especially infected the TNF-α response in the human macrophage of H5N1 influenza virus sub-strain.In addition, by the imbalance of control cytokine, M ₄N can be used to reduce people's H5N1 severity of disease.

Also do not study the PGE during influenza infection widely ₂Generation.In this research, both used low MOI model also to use high MOI model.

Figure 16 has described the result of low MOI detection model.When the RAW264.7 cell when its growth medium is converted to the infection culture medium that does not contain serum, cell produces the PGE of about 1ng/ml ₂(" culture medium " bar).EM-1421 (25 μ M) induced strong individually goes out this value (" EM-1421 " bar).Influenza infection can repeatedly cause PGE ₂Value increase about 30% (" influenza " bar).EM-1421 (25 μ M) has also blocked the inductive PGE of this influenza virus consumingly ₂The increase (" influenza/EM-1421 " bar) that produces.

Figure 17 has described the result of high MOI detection model.RAW 264.7 cells produce low-down PGE when not having viral infection and use EM-1421 (" culture medium " bar) to handle ₂Value (about 75pg/ml).EM-1421 (25 μ M) increases PGE separately ₂The about twice of value (" EM-1421 " bar).Yet, because PGE ₂In " culture medium " and " EM-1421 " hole, produce low-levelly, therefore predict that the meaning of these observed results is difficult.Influenza infection causes PGE ₂Level show to increase severely and to add, and increases about 1,300% shown in the figure to about 1,100pg/ml (" influenza " bar).EM-1421 (25 μ M) reduces the inductive PGE of influenza virus ₂About 32% (" influenza/EM-1421 " bar) of increasing of generation.

Lipid medium role in the inductive pneumonia of influenza virus is not characterized as yet well.The result who obtains in this research has disclosed new information.In the model of low MOI (Figure 16), the infection culture medium that does not contain serum causes the PGE of high background value ₂Generation; Influenza infection causes PGE ₂Value extra little and repeatably increase; EM-1421 had both infected by culture medium and had also fully suppressed PGE by influenza infection ₂The increase of generation.The result of low MOI model and observed by EM-1421 to the inductive PGE of LPS ₂The strong inhibition that produces is consistent.On the contrary, high MOI model (Figure 17) has disclosed influenza infection to PGE ₂Strong inducing (low background), but EM-1421 induces this medium inhibition is only arranged.Carried out other test to illustrate by low MOI model system and the observed different result of high MOI model system.

More specifically, about the low MOI model that uses, 1.5 x 10 ⁵Individual RAW264.7 macrophage/hole is tiled in the DME culture medium that contains 10% FCS spends the night.Remove culture medium, in the viral growth culture medium (the basic DME that contains 2 μ g/ml trypsin, 2.5%HEPES buffer and 0.2%BSA) to use MOI be the virus inoculation (strains A/WS/33) replace, and allow virus absorption 30 minutes of 0.002 200 μ l.The volume that increases culture medium then is to 1ml.When volume increased to 1ml, the ultimate density of EM-1421 increased to 25 μ M.The hole of not containing virus and the hole of EM-1421 (" culture medium ") or only containing EM-1421 (" EM-1421 ") is used as " simulated infection " hole, and its hole with infection is subjected to identical processing but does not have virus.After hatching about 24 hours, collect culture supernatant, detect PGE by ELISA ₂The data that illustrate be two independent trialss average+/-SEM, twice multiple infection carried out in each test.All ELISA points detect twice.

In the high MOI model that uses, 1.5x10 ⁵Individual RAW 264.7 macrophages/hole is tiled in the DME culture medium that contains 10% FCS spends the night.Remove culture medium, using MOI in containing the DME culture medium of 10% FCS is the virus inoculation (strains A/WS/33) replace, and allow virus absorption 30 minutes of 5 200 μ l.The volume that increases culture medium then is to 1ml.When volume increased to 1ml, the ultimate density of EM-1421 increased to 25 μ M.The hole of not containing virus and the hole of EM-1421 (" culture medium ") or only containing EM-1421 (" EM-1421 ") is used as " simulated infection " hole, and its hole with infection is subjected to identical processing but does not have virus.After hatching about 24 hours, collect culture supernatant, detect PGE by ELISA ₂The data that illustrate be two independent trialss average+/-SEM, twice multiple infection carried out in each test.All ELISA points detect twice.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress the PGE in response to influenza infection ₂Too much generation, illustrate by improving PGE ₂The described chemical compound of level and relevant catechol butane and NDGA derivant can be used for the treatment of when influenza infection by high-level PGE ₂The disease or the disorder of mediation.

Embodiment 8

The catechol butane of general formula (I), i.e. M are used in research ₄The work that N produces the cytokine of RAW 264.7 macrophages is in order to measure inductive production of cytokines and the M except TNF-α of influenza virus ₄N is to inducing the inhibition ability of generation.

In this research, used antibody (" Ab ") chip technology.As shown in figure 18, in 40 kinds of cytokines, chemotactic factor, receptor and the protease on chip, 8 kinds in this test, have been detected.In addition, under low MOI condition, the RAW264.7 cell is converted to from growth medium and contains the tryptic infection culture medium that does not contain serum and induce and produce high-caliber TNF-α, high-caliber MIP-1 γ and the relative moderate increase of sTNFRII and chemotactic factor MIP-1 level.Influenza infection is the generation of inducing cell factor G-CSF also, does not detect cytokine G-CSF in the check sample culture medium.EM-1421 (25 μ M) has blocked the many kinds in these effects.As the generation of the inductive TNF-α of influenza virus, EM-1421 has fully blocked the generation of inductive TNF-α of culture medium and MIP-1 γ.It is about 60% that the generation of the inductive M1P-1 γ of influenza virus is blocked, and the generation of G-CSF is fully blocked.On the contrary, EM-1421 does not suppress the generation of inductive sTNFRII of influenza virus or MCP-1.

Carry out ELISA and detect cytokine interferon-(IFN-β) and IL-6.Do not comprise IFN-β on chip, influenza virus infection A can induce this cytokine.Yet, under low (A) and two kinds of conditions of high (B) MOI, from the culture supernatant of infection cell, do not detect the IFN-β (data not shown goes out) of significant quantity after 24 hours at virus inoculation.Influenza virus A induce the ability of interferon-be that the height strain relies on (347:52), obviously strain A/WS/33 is non-derivant for people 2006 such as Hayman, virusology (Virology).EM-1421 does not induce the generation of IFN-β yet.

Induce IL-6 though reported some influenza strain, not detecting IL-6 and induce as described above from chip analysis, this shows the derivant that strain A/WS/33 neither this cytokine.After infecting strain A/WS/33, all do not have to detect high IL-6 value at low (A) or high (B) MOI by the ELISA detection method, this has confirmed the result of chip analysis.Under low MOI condition, after using EM-1421, detect low IL-6 value.Yet because the value very low (10pg/ml) of IL-6, the meaning of predicting this observed result is difficult.

Except TNF-α, the chip analysis that we carry out also discloses the generation that EM-1421 also blocks inductive MIP-1 γ of influenza virus and G-CSF.It is a kind of chemotactic factor that MIP1-γ is also referred to as CCL9, its active relevant with the many cell processes that comprise pneumonia (people such as Rosenblum-Lichtenstein, 2006, U.S.'s respiratory system cell and molecular biology magazine(Am.J.Resp.Cell.Mol.Biol.) 35:415).G-CSF is being crucial aspect the generation of regulating neutrophilic granulocyte, the Mus that lacks this gene show the neutrophil infiltration level that enters in the lung reduction (people such as Gregory, 2006, Blood(Blood), early than the electronic edition of print journal).EM-1421 has further increased the support that can prevent pulmonary's inflammation relevant with influenza to EM-1421 to the inhibition of these two kinds of molecules.Several cytokines and the chemotactic factor of the subsidiary influenza infection of having reported do not induced in the influenza A/WS/33 strain of Shi Yonging in these trials, comprises IFN-β, IL-6 and RANTES.The capacity variation of influenza virus A strain aspect its inducing cell factor and chemotactic factor is extensive.Test is carried out with other influenza strain, and A/PR/8/34 has for example reported that it also induces many guided cell factors and chemotactic factor (people such as Wareing, 2004, leukocyte magazine biology (J.Leukoc.Biol.) 76:886) except inducing TNF-α.

More specifically, about being used to measure M ₄The method of the generation of the N pair cell factor has been used " Mus inflammation chip-1 " in described method, battle array photo bio technical concern company, Atlanta, Georgia).According to the program of describing among the embodiment 1, buy the RAW264.7 macrophage, cultivate, use LPS (1 μ g/ml) to stimulate, use EM-1421 (25 μ M) to handle, gather cell.After the A/WS/33 influenza virus A of culture medium (the basic DME that contains 2mg/ml trypsin, 2.5%HEPES buffer and 0.2%BSA), 0.002 MOI or influenza virus and EM-1421 are hatched 24 hours from RAW264.7 macrophage culture fluid (1.5 x 10 ⁵Cells/well) collects supernatant.According to the cytokine in the description use Mus inflammation chip-1 measurement supernatant of manufacturer.In brief, use nitrocellulose Ab chip to hatch the about 2h of culture supernatant, clean, contact, use the ECL hydroponics, and contact with the X-ray film with second kind of Ab solution.The autoradiography of scanning chip.Use Photoshop (Adobe) to analyze and measure the mean pixel gray scale of each chip position.Two meansigma methodss that repeat a little to 8 kinds of testing products are mapped.Other 32 kinds of products that do not detect on the chip are not shown.Positive control point is about 100-110 unit on each chip, and SEM is lower than 5% between repeating a little.Lymph (Lymph.)=lymphocyte chemotactic factor (LCF) (lymphotactin).

More specifically, about the ELISA method of using that is used for detecting generation IFN-β or IL-6,1.5 x 10 by RAW 264.7 macrophages ⁵Individual RAW 264.7 macrophages/hole is tiled in 24 orifice plates of the DME culture medium that contains 10% FCS spends the night.Under low MOI testing conditions, remove culture medium, in the viral growth culture medium (the basic DME that contains trypsin, 2.5%HEPES buffer and 0.2% BSA of 2 μ g/ml) to use MOI be that the virus inoculation (strain A/WS/33) of 0.002 200 μ l is replaced, and allow virus absorption 30 minutes.Under the high MOI testing conditions that uses, 1.5 x 10 ⁵Individual RAW 264.7 macrophages/hole is tiled in 24 orifice plates of the DME culture medium that contains 10% FCS spends the night.Remove culture medium, using MOI in containing the DME culture medium of 10% FCS is that the virus inoculation (strain A/WS/33) of 5 200 μ l is replaced, and allows virus absorption 30 minutes.The volume that increases culture medium then is to 1ml.When volume increased to 1ml, the ultimate density of EM-1421 increased to 25 μ M.The hole of not containing virus and the hole of EM-1421 (" culture medium ") or only containing EM-1421 (" EM-1421 ") is used as " simulated infection " hole, and its hole with infection is subjected to identical processing but does not have virus.After hatching about 24 hours, collect culture supernatant, detect IFN-β or IL-6 by ELISA.The data that illustrate are the meansigma methods of two independent trialss, and twice multiple infection carried out in each test.Not shown part, SEM is lower than the sign size.The ELISA point detects twice.

The result of this embodiment shows that the catechol butane of general formula (I) can suppress to remove in response to influenza infection the too much generation of other several cytokines of TNF-α, illustrates that described chemical compound and relevant catechol butane and NDGA derivant can be used for the treatment of when influenza infection by high-caliber described cytokine mediated disease or disorder.

The catechol butane of embodiments of the invention explanation general formulas (I) can suppress the proinflammatory cytokine that causes by influenza infection, for example excessive generation of TNF-α, and short scorching lipid medium, PGE ₂Excessive generation, for example, and the catechol butane of formula (I) also can reduce the alpha mediated apoptosis of TNF-, thereby reduces duplicating of influenza virus in the host cell.These presentation of results catechol butane can be used for treating influenza infection and relevant disease and imbalance.

Be appreciated that those skilled in the art can change above-mentioned embodiment and do not depart from wide in range inventive concept of the present invention.Therefore, be appreciated that to the invention is not restricted to disclosed specific embodiment, but be intended to be included in the interior modification of the spirit and scope of the invention that claims limit.

Claims (106)

1. method for the treatment of influenza infection in the study subject, described method comprises catechol butane or its pharmaceutically acceptable salt of using the general formula (I) of effective therapeutic dose to study subject:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together; Condition is to work as R ₇, R ₈And R ₉One of expression during hydrogen, so-OR ₁,-OR ₂With R ₇, R ₈And R ₉In other both expression-OH simultaneously not.

2. method according to claim 1 is characterized in that, described catechol butane or its pharmaceutically acceptable salt comprise one or more route of administration administrations of following group by being selected from: intranasal administration; Oral; Inhalation; Subcutaneous administration; Transdermal administration; Intravenously administrable; Oral administration; The intraperitoneal administration; Eye drops; Administration near the eyes; The intramuscular administration; Drug delivery implant; Infusion and central vein administration.

3. method according to claim 1 is characterized in that, described catechol butane or its pharmaceutically acceptable salt are by oral or intravenously administrable.

4. method according to claim 1, it is characterized in that, described catechol butane or its pharmaceutically acceptable salt are by a kind of compositions administration, and described compositions comprises catechol butane or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier or excipient.

5. method according to claim 4 is characterized in that, described pharmaceutically acceptable carrier or excipient comprise: (a) water-miscible organic solvent; (b) cyclodextrin or modified cyclodextrin; (c) ion, nonionic or amphoteric surfactant; (d) modified cellulose; Or (e) water-fast lipid; Or the combination in any (a)-(e).

6. method according to claim 4, it is characterized in that described pharmaceutically acceptable carrier or excipient comprise at least a or mixture of following substances: dimethyl sulfoxide (DMSO), phosphate-buffered saline, saline, lipid based formulation, Liposomal formulation, nanoparticle formulations, micellar preparation, water soluble preparation, biodegradable polymer, aqueous solution preparation, hydrophobic preparations, lipid base carrier, polymer formulations, cyclodextrin, modified cyclodextrin, slow releasing preparation, surfactant, edible fat or edible oil.

7. method according to claim 6, it is characterized in that described nanoparticle formulations comprises at least a or mixture of following substances: poly-(DL-lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer, polyvinyl alcohol, D-alpha-tocopherol cetomacrogol 1000 succinate and poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer-mono methoxy-poly-(Polyethylene Glycol).

8. method according to claim 6; it is characterized in that; described Liposomal formulation comprises at least a or mixture of following substances: lecithin, cholesterol, PEG-DPPE, distearoylphosphatidylcholine, cholesterol and PEG-DPPE, and a kind of preparation that comprises following substances: 1-2-two oleoyls-sn-glycerol-3-phosphocholine, 1-2-two palmityls-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt, cholesterol, triolein and tricaprylin.

9. method according to claim 6 is characterized in that, described pharmaceutically acceptable carrier or excipient comprise at least a or mixture of following substances: Semen Maydis oil, Oleum Ricini, Oleum Arachidis hypogaeae semen or dimethyl sulfoxide.

10. method according to claim 6, it is characterized in that, described polymer formulations is selected from and comprises a kind of composition of following group: 1, and 3-is two-(to the carboxyl phenoxy group) propane, decanedioic acid, poly-(ethylene-vinyl acetate) copolymer and poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer.

11. method according to claim 4 is characterized in that, described pharmaceutically acceptable carrier or excipient make catechol butane or its pharmaceutically acceptable salt adopt with at least a or mixed form in high local concentrations and the slow release during certain.

12. method according to claim 4, it is characterized in that described compositions is to be selected to comprise a kind of form of following group: powder, aerosol, cream, ointment, gelinite, tablet, capsule, pill, capsule sheet, microgranule, syrup, solution, collutory, elixir, emulsion, suppository, suspension, spray and drop.

13. method according to claim 1 is characterized in that, described catechol butane is dissolved in before administration in saline, dimethyl sulfoxide or the ethanol.

14. method according to claim 1 is characterized in that, described catechol butane or its pharmaceutically acceptable salt comprise second kind of medicament combination medicine-feeding of following group with being selected from: the second influenza agent, antiinflammatory, anti-infective with and compositions.

15. method according to claim 14 is characterized in that, described antiinflammatory is selected from the group of the anti-inflammatory drug that comprises corticosteroid and non-steroidal.

16. method according to claim 14 is characterized in that, described anti-infective is selected from the group that comprises antibiotic medicine, ethanol and polyvidone.

17. method according to claim 14, it is characterized in that, the described second influenza agent be selected from second kind of catechol butane comprising general formula (I) or its pharmaceutically acceptable salt, amantadine,, the group of oseltamivir, Peramivir, rimantadine, zanamivir and arbidol.

18. method according to claim 14 is characterized in that, before administration catechol butane or its pharmaceutically acceptable salt, basically simultaneously or described second medicament of administration afterwards.

19. method according to claim 1 is characterized in that, R ₁And R ₂Expression-H, low alkyl group, lower acyl independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₇, R ₈And R ₉Be independently-H ,-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

20. method according to claim 1 is characterized in that, R ₁And R ₂Expression-H, low alkyl group, lower acyl independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₇, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₈And R ₉Be independently-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at carboxyl terminal.

21. method according to claim 20 is characterized in that, R ₁And R ₂Be independently-CH ₃Or-(C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

22. method according to claim 20 is characterized in that, R ₈And R ₉Be independently-OCH ₃Or-O (C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

23. method according to claim 20 is characterized in that, R ₁And R ₂Be independently-CH ₃The CH of ,-(C=O) ₂N (CH ₃) ₂Or-(C=O) CH ₂N ⁺H (CH ₃) ₂Cl ^-, and R ₈With R9 be independently-OCH ₃,-O (C=O) CH ₂N (CH ₃) ₂Or-O (C=O) CH ₂N ⁺H (CH ₃) ₂2Cl ^-

24. method according to claim 20 is characterized in that, R ₁And R ₂Be independently-H or-CH ₃, and R ₈And R ₉Be independently-OH or-OCH ₃, condition is that described catechol butane is not NDGA.

25. method according to claim 20 is characterized in that, R ₁And R ₂Be independently-CH ₃, and R ₈And R ₉Be independently-OCH ₃

26. method according to claim 1, it is characterized in that described catechol butane or its pharmaceutically acceptable salt are given the study subject administration to be selected from the group that comprises following amount: about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 2.5, about 5.0, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, the each dosage of about 400mg/kg body weight.

27. method according to claim 1 is characterized in that, described influenza infection is to be caused by bird flu virus.

28. method according to claim 27 is characterized in that, described bird flu virus is influenza virus sub-strain H5N1.

29. method according to claim 1 is characterized in that, described study subject is behaved.

30. method according to claim 1 is characterized in that, described treatment influenza infection comprises blocking-up, prevents, alleviates or alleviates disease relevant with influenza infection in the study subject or disorder.

31. method according to claim 30, it is characterized in that described disease relevant or the disorderly group that comprises systemic inflammatory responses, multiple organ dysfunction, acute respiratory distress syndrome, reactive erythrophagoytosis and lymphopenia that is selected from influenza infection.

32. method according to claim 30, it is characterized in that described disease relevant with influenza infection or disorder are encephalitis after asthma, pneumonia, the influenza, bacterial myositis, the Electrocardiographic variation of heart, bronchitis, pulmonary tuberculosis, cancer, rheumatic arthritis, osteoarthritis, scleroderma systemic lupus erythematosis, cystic fibrosis, cachexia, whole myasthenia imbalance, heart failure, parkinson disease, amyotrophic lateral sclerosis or Green-barre syndrome.

33. method according to claim 1 is characterized in that, described treatment influenza infection comprises inhibition, prevention or reduces the growth of influenza virus in the study subject.

34. a method for the treatment of influenza infection in the study subject, described method comprise nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt of using the general formula (II) of effective therapeutic dose to study subject:

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, R ₁₈And R ₁₉Difference is expression-H or low alkyl group independently; Condition is R ₁₄, R ₁₅, R ₁₆And R ₁₇Be not simultaneously-OH.

35. method according to claim 34 is characterized in that, R ₁₄, R ₁₅, R ₁₆And R ₁₇Expression-OCH respectively ₃

36. method according to claim 34 is characterized in that, R ₁₄, R ₁₅, R ₁₆And R ₁₇Represent amino acid residue and its pharmaceutically acceptable salt unsubstituted or that replace respectively, amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

37. method according to claim 36 is characterized in that, described amino acid residue unsubstituted or that replace comprises glycine residue and its pharmaceutically acceptable salt unsubstituted or that replace.

38. method according to claim 34 is characterized in that, R ₁₈And R ₁₉Difference is expression-CH independently ₃Or-CH ₂CH ₃

39. method according to claim 34 is characterized in that, described nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt comprise one or more route of administration administrations of following group by being selected from: intranasal administration; Oral; Inhalation; Subcutaneous administration; Transdermal administration; Intravenously administrable; Oral administration; The intraperitoneal administration; Eye drops; Administration near the eyes; The intramuscular administration; Drug delivery implant; Infusion and central vein administration.

40., it is characterized in that described nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt are by oral or intravenously administrable according to the described method of claim 39.

41. method according to claim 34, it is characterized in that described nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt are given the study subject administration to be selected from the group that comprises following amount: about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 2.5, about 5.0, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, the each dosage of about 400mg/kg body weight.

42. method according to claim 34 is characterized in that, described influenza infection is caused by bird flu virus.

43., it is characterized in that described bird flu virus is influenza virus sub-strain H5N1 according to the described method of claim 42.

44. method according to claim 34 is characterized in that, described study subject is behaved.

45. a method for the treatment of avian influenza in the study subject, described method comprise nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt of using the general formula (III) of effective therapeutic dose to study subject:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, condition is R ₂₀, R ₂₁, R ₂₂And R ₂₃Be not simultaneously-OH.

46., it is characterized in that R according to the described method of claim 45 ₂₀, R ₂₁, R ₂₂And R ₂₃Difference is expression-OCH independently ₃

47., it is characterized in that R according to the described method of claim 45 ₂₀, R ₂₁, R ₂₂And R ₂₃Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively, amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

48., it is characterized in that described amino acid residue unsubstituted or that replace comprises glycine residue or its pharmaceutically acceptable salt unsubstituted or that replace according to the described method of claim 47.

49., it is characterized in that described nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt comprise one or more route of administration administrations of following group by being selected from: intranasal administration according to the described method of claim 45; Oral; Inhalation; Subcutaneous administration; Transdermal administration; Intravenously administrable; Oral administration; The intraperitoneal administration; Eye drops; Administration near the eyes; The intramuscular administration; Drug delivery implant; Infusion and central vein administration.

50., it is characterized in that described nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt are by oral or intravenously administrable according to the described method of claim 49.

51. according to the described method of claim 45, it is characterized in that described nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt are given the study subject administration to be selected from the group that comprises following amount: about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 2.5, about 5.0, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, the each dosage of about 400mg/kg body weight.

52., it is characterized in that described influenza infection is caused by bird flu virus according to the described method of claim 45.

53., it is characterized in that described bird flu virus is influenza virus sub-strain H5N1 according to the described method of claim 52.

54., it is characterized in that described study subject is behaved according to the described method of claim 45.

55. method for the treatment of influenza infection in the study subject, described method comprises the following compositions of using effective therapeutic dose to study subject, described compositions contains the catechol butane that is selected from the group that comprises three-O-methyl nordihydroguaiaretic acid (NDGA), four-O-methyl N DGA, four-glycyl NDGA, four-dimethyl glycyl NDGA, or its pharmaceutically acceptable salt, and pharmaceutically acceptable carrier or excipient.

56., it is characterized in that described catechol butane or its pharmaceutically acceptable salt comprise one or more route of administration administrations of following group by being selected from: intranasal administration according to the described method of claim 55; Oral; Inhalation; Subcutaneous administration; Transdermal administration; Intravenously administrable; Oral administration; The intraperitoneal administration; Eye drops; Administration near the eyes; The intramuscular administration; Drug delivery implant; Infusion and central vein administration.

57., it is characterized in that described catechol butane or its pharmaceutically acceptable salt are by oral or intravenously administrable according to the described method of claim 56.

58. according to the described method of claim 55, it is characterized in that described catechol butane or its pharmaceutically acceptable salt are given the study subject administration to be selected from the group that comprises following amount: about 0.01, about 0.05, about 0.1, about 0.5, about 1.0, about 2.5, about 5.0, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, the each dosage of about 400mg/kg body weight.

59., it is characterized in that described influenza infection is caused by bird flu virus according to the described method of claim 55.

60., it is characterized in that described bird flu virus is influenza virus sub-strain H5N1 according to the described method of claim 55.

61., it is characterized in that described study subject is behaved according to the described method of claim 55.

62., it is characterized in that described pharmaceutically acceptable carrier or excipient comprise oil according to the described method of claim 55.

63., it is characterized in that described pharmaceutically acceptable carrier or excipient comprise polyoxyethylene castor oil, ethanol and salt according to the described method of claim 55.

64., it is characterized in that described compositions comprises three-O-methyl N DGA or the four-O-methyl N DGA of each dosage at least about 7mg according to the described method of claim 55.

65. method for the treatment of H5N1 subtype influenza viral infection in people's study subject, described method comprises oral nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt of using the general formula (III) of effective therapeutic dose for people's study subject, and the amount of use is for about 0.01 to the each dosage of about 400mg/kg body weight:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

66., it is characterized in that the method for described treatment H5N1 subtype influenza viral infection comprises blocking-up, prevents, alleviates or alleviate disease or the disorder of following people's study subject H5N1 subtype influenza viral infection according to the described method of claim 65.

67. according to the described method of claim 66, it is characterized in that relevant with H5N1 subtype influenza viral infection described disease or the disorderly group that comprises systemic inflammatory responses, multiple organ dysfunction, acute respiratory distress syndrome, reactive erythrophagoytosis and lymphopenia that is selected from people's study subject.

68. according to the described method of claim 65, it is characterized in that described disease or disorder relevant with H5N1 subtype influenza viral infection in people's study subject are encephalitis after asthma, pneumonia, the influenza, bacterial myositis, the Electrocardiographic variation of heart, bronchitis, pulmonary tuberculosis, cancer, rheumatic arthritis, osteoarthritis, scleroderma, systemic lupus erythematosis, cystic fibrosis, cachexia, whole myasthenia imbalance, heart failure, parkinson disease, amyotrophic lateral sclerosis or Green-barre syndrome.

69., it is characterized in that the method for described treatment H5N1 subtype influenza viral infection comprises inhibition, prevention or reduces the growth of H5N1 subtype influenza virus in people's study subject according to the described method of claim 65.

70. an inhibition is because influenza infection and the method for proinflammatory cytokine in the inducing cell, described method comprises that pair cell uses catechol butane or its pharmaceutically acceptable salt of the general formula of effective dose (I):

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together.

71., it is characterized in that described proinflammatory factor is selected from the group that comprises chemotactic factor, interleukin (IL), lymphokine, tumor necrosis factor (TNF) and interferon (IFN) according to the described method of claim 70.

72. according to the described method of claim 71, it is characterized in that described proinflammatory factor is selected from and comprises that TNF-α, macrophage infect the group that strengthens albumen 1 γ (MIP-1 γ), granulocyte colony-stimulating factor (G-CSF), IL-1 α, monocyte chemoattractant protein 1 (MCP-1), interferon-induced T cell α chemoattractant (I-TAC), IL-2, metalloproteases-1 tissue inhibiting (TIMP-1), TIMP-2, B cell chemotactic factor (BLC), IL-3 and active T cellular expression that regulate, normal and excretory chemotactic factor (RANTES).

73., it is characterized in that described proinflammatory factor is TNF-α according to the described method of claim 72.

74., it is characterized in that described cell is a macrophage according to the described method of claim 70.

75., it is characterized in that described macrophage is a human macrophage according to the described method of claim 74.

76., it is characterized in that described influenza virus is a bird flu virus according to the described method of claim 70.

77., it is characterized in that described bird flu virus is influenza virus sub-strain H5N1 according to the described method of claim 76.

78., it is characterized in that R according to the described method of claim 70 ₁And R ₂Be independently-H, low alkyl group, lower acyl, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₇, R ₈And R ₉Be independently-H ,-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

79., it is characterized in that R according to the described method of claim 70 ₁And R ₂Be independently-H, low alkyl group, lower acyl, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₇, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₈And R ₉Be independently-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

80., it is characterized in that R according to the described method of claim 79 ₁And R ₂Be independently-CH ₃Or-(C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

81., it is characterized in that R according to the described method of claim 79 ₈And R ₉Be independently-OCH ₃Or-O (C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

82., it is characterized in that R according to the described method of claim 79 ₁And R ₂Be independently-CH ₃The CH of ,-(C=O) ₂N (CH ₃) ₂Or-(C=O) CH ₂N ⁺H (CH ₃) ₂Cl ^-, and R ₈And R ₉Be independently-OCH ₃,-O (C=O) CH ₂N (CH ₃) ₂Or-O (C=O) CH ₂N ⁺H (CH ₃) ₂2Cl ^-

83., it is characterized in that R according to the described method of claim 79 ₁And R ₂Be independently-H or-CH ₃And R ₈And R ₉Be independently-OH or-OCH ₃

84., it is characterized in that R according to the described method of claim 79 ₁And R ₂Be independently-CH ₃And R ₈And R ₉Be independently-OCH ₃

85. an inhibition is because influenza infection and the method for short scorching lipid medium in the inducing cell, described method comprises that pair cell uses catechol butane or its pharmaceutically acceptable salt of the general formula of effective dose (I):

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together.

86. 5 described methods is characterized in that according to Claim 8, described short scorching lipid medium is prostaglandin or leukotriene.

87. 6 described methods is characterized in that according to Claim 8, described prostaglandin is selected from and comprises prostaglandin E ₂(PGE ₂), prostaglandin F _{1 α}(PGF _{1 α}), prostaglandin F _{2 α}(PGF _{2 α}), PGH ₂(PGH ₂) and the group of prostacyclin.

88. 7 described methods is characterized in that according to Claim 8, described prostaglandin is PGE ₂

89. 5 described methods is characterized in that according to Claim 8, described cell is a macrophage.

90. 9 described methods is characterized in that according to Claim 8, described macrophage is a human macrophage.

91. 5 described methods is characterized in that according to Claim 8, described influenza virus is a bird flu virus.

92., it is characterized in that described bird flu virus is influenza virus sub-strain H5N1 according to the described method of claim 91.

93. 5 described methods is characterized in that R according to Claim 8 ₁And R ₂Be independently-H, low alkyl group, lower acyl, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently; R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₇, R ₈And R ₉Be independently-H ,-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

94. 5 described methods is characterized in that R according to Claim 8 ₁And R ₂Be independently-H, low alkyl group, lower acyl, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄Be low alkyl group independently, R ₅, R ₆, R ₇, R ₁₀, R ₁₁, R ₁₂And R ₁₃Be independently-H; R ₈And R ₉Be independently-OH, lower alkoxy, low-grade acyloxy or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace; Amino acid residue wherein unsubstituted or that replace links to each other with aromatic ring at its carboxyl terminal.

95., it is characterized in that R according to the described method of claim 94 ₁And R ₂Be independently-CH ₃Or-(C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

96., it is characterized in that R according to the described method of claim 94 ₈And R ₉Be independently-OCH ₃Or-O (C=O) CH ₂N (CH ₃) ₂Or its pharmaceutically acceptable salt.

97., it is characterized in that R according to the described method of claim 94 ₁And R ₂Be independently-CH ₃The CH of ,-(C=O) ₂N (CH ₃) ₂Or-(C=O) CH ₂N ⁺H (CH ₃) ₂Cl ^-And R ₈And R ₉Be independently-OCH ₃,-O (C=O) CH ₂N (CH ₃) ₂Or-O (C=O) CH ₂N ⁺H (CH ₃) ₂2C ^-

98., it is characterized in that R according to the described method of claim 94 ₁And R ₂Be independently-H or-CH ₃, and R ₈And R ₉Be independently-OH or-OCH ₃

99., it is characterized in that R according to the described method of claim 94 ₁And R ₂Be independently-CH ₃, and R ₈And R ₉Be independently-OCH ₃

100. an inhibition is owing to H5N1 subtype influenza viral infection is induced the method for tumor necrosis factor in the macrophage, described method comprises nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt of using the general formula (III) of effective dose to macrophage:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

A kind of inhibition is owing to H5N1 subtype influenza viral infection is induced prostaglandin E in the macrophage ₂(PGE ₂) method, described method comprises nordihydroguaiareticacid acid derivatives or its pharmaceutically acceptable salt of using the general formula (III) of effective dose to macrophage:

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Expression-OCH respectively ₃

A kind of catechol butane of general formula I or test kit of its pharmaceutically acceptable salt of containing:

R wherein ₁And R ₂Represent hydrogen, low alkyl group, lower acyl, alkylene respectively independently, or-OR ₁With-OR ₂Represent amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace respectively independently; R ₃, R ₄, R ₅, R ₆, R ₁₀, R ₁₁, R ₁₂And R ₁₃Represent hydrogen or low alkyl group respectively independently; And R ₇, R ₈And R ₉Represent independently respectively hydrogen ,-OH, lower alkoxy, low-grade acyloxy, unsubstituted or the amino acid residue or its salt that replace, or can form any two adjacent groups of alkylene dioxy base together; Condition is to work as R ₇, R ₈And R ₉One of expression during hydrogen, so-OR ₁,-OR ₂With R ₇, R ₈And R ₉In other both expression-OH simultaneously not; And by using the description of catechol butane or its pharmaceutically acceptable salt treatment study subject influenza infection.

According to the described test kit of claim 102, it is characterized in that described test kit comprises nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt of general formula (II):

R wherein ₁₄, R ₁₅, R ₁₆And R ₁₇Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, R ₁₈And R ₁₉Difference is expression-H or low alkyl group independently; Condition is R ₁₄, R ₁₅, R ₁₆And R ₁₇Be not simultaneously-OH; And by using the description of NDGA derivant or its pharmaceutically acceptable salt treatment study subject influenza infection.

According to the described test kit of claim 103, it is characterized in that described test kit comprises nordihydroguaiaretic acid (NDGA) derivant or its pharmaceutically acceptable salt of general formula (III):

R wherein ₂₀, R ₂₁, R ₂₂And R ₂₃Respectively independently expression-OH ,-OCH ₃,-O (C=O) CH ₃Or amino acid residue or its pharmaceutically acceptable salt unsubstituted or that replace, condition is R ₂₀, R ₂₁, R ₂₂And R ₂₃Be not simultaneously-OH.

According to the described test kit of claim 104, it is characterized in that R ₂₀, R ₂₁, R ₂₂And R ₂₃Difference is expression-OCH independently ₃And the description that is used for the treatment of H5N1 subtype influenza viral infection in people's study subject.

According to the described test kit of claim 102, it is characterized in that described test kit also comprises and is used for giving the doser of study subject administration with catechol butane or its pharmaceutically acceptable salt.

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