CN101228178A - Compositions and methods for treating or preventing overweight or obesity with zinc-charged protein fragments - Google Patents

Abstract

Compositions and methods for treating or preventing, for example, overweight or obesity are provided. Compositions provided comprise zinc-charged, protease digested serum or milk proteins. Compositions are administered in a therapeutically effective amount to treat or prevent, for example, overweight or obesity.

Description

Use and carry the treatment of zinc protein fragments or prevent overweight or fat composition and method

The application is the part continuation application of No. 11/315,810, the U. S. application submitted on December 22nd, 2005, and the latter is the part continuation application of No. 11/114,792, the U. S. application submitted on April 26th, 2005.The application requires the right of priority of above-mentioned each application, and the full content that the application comprises above-mentioned each application for your guidance.

1. invention field

The present invention relates to be used for the treatment of or prevention of obesity or overweight composition and method.Particularly, the invention discloses preparation carry zinc, through the protein mixture that is derived from serum for example or breast of protease digestion and use the method for this mixture to the patient.

2. background of invention

Obesity is serious healthy prevailing disease.According to CDC (CDC) and national health and nutrition inspection investigation (NHANES) 1999-2000, estimate at 64% U.S. grownup and be that overweight or fat (overweight finger body surface index (BMI) is 25 or bigger, obesity refers to that BMI is 30 or bigger), and should numeral also increase.In 15 years, fat prevalence rate in the grownup increases above 30% in the past.Even it is shocking that more in 20 years, children and teen-age fat prevalence rate have increased by 100% in the past.Overweight and obese individuals is suffered from body illness such as high blood pressure, hypertension, high blood cholesterol levels, diabetes, insulin resistance, glucose does not tolerate, coronary heart disease and even psychological disorders as danger increase (the National Institutes of Health:Clinical Guidelineson the Identification of depressed and eating disorder, Evaluation, and Treatment of Overweight and Obesity inAdults, 1998, Bethesda, Maryland:Department of Health and Human Services, National Institutes of Health, National Heart, Lung, and Blood Institute; Mokdad etc., 2001, JAMA 289:76-79).

When the people became obesity, two kinds of situations can take place their intravital adipocyte (adipocyte): the volume of these cells and number both can increase.The intravital adipocyte number of people only increases at the people Shi Caihui that obtains to weigh sb. greatly.Body weight for humans follow the progressively increase or the reduction of adipocyte mean sizes usually than minor swing.Yet, the overall dimension that exists adipocyte to grow.Along with body weight obviously increases, produce new adipocyte by inoblast.Unfortunately, when the people loses by going on a diet when weighing sb. greatly, adipocyte only can shrink size, and its number but can not reduce.It is identical that the adipocyte number keeps in the human body of losing weight, this facts explain so difficulty of losing weight of why losing weight and keeping being obtained.Can more effectively reach these targets by the number that reduces adipocyte.

Present anti-obesity treatment concentrates on by medicine depress appetite, erstricted diet to be taken in, and perhaps uses the operation of losing weight in the most radical method.The popular appetite suppressant can be divided into two classes: the norepinephrine energy medicine such as the Phenylpropanolamine (Acutrim that influence the brain appetite center, Dexatrim), with the serotonergic medicine that influences the brain satiety center such as Phenfluoramine and dexfenfluramine (Williamson, 1999, JAMA281:278-279).Yet research shows that the neurophysiology of feed behavior is very complicated, and this makes that appetite inhibiting is difficult to realize.Simultaneously, Phenfluoramine and derivative thereof are with very deleterious side effect, and this forces these materials of forbidding.Attempt the patient that erstricted diet takes in and be difficult to finish the program of losing weight usually, and when they return old feed custom, tend to restore to the original state.Too much food is converted into the fat that replenishes the adipocyte that shrinks owing to losing weight rapidly.The size that reduces stomach by operation can the significant limitation food intake.Yet such operation has the side effect of himself, only recommends the individuality of serious obesity.In the liposuction program, can only from some body portion, remove fat.The result is to stay the fat quantity that the adipocyte in other position such as neck and the forearm can raise and lack of proper care, thereby produce ugly appearance.

Therefore be desirable to provide the non-intrusion type that works by the number that the reduces adipocyte therapy of losing weight.If adipocyte that can the target whole body (rather than only in limited zone) then will be especially useful.

3. summary of the invention

The present invention part has been based on having found to can be used for reducing weight increase, even composition and method that the whose body weight that needs is reduced.Be not subjected to the restriction of any invention scope, believe that the compositions and methods of the invention can induce the apoptosis of the adipocyte in the individuality.Be not subjected to the restriction of any invention scope, believe that also the compositions and methods of the invention can regulate the insulin receptor activity in the individuality.Following example has proved that the compositions and methods of the invention can reduce the body weight of individual human effectively.

On the one hand, the invention provides the composition that can be used for methods described herein.In certain embodiments, described composition is the form of pharmaceutical unit dosage form.In certain embodiments, described composition is the form of dietetic product unitary dose.In certain embodiments, described composition is the form of dietary supplements, foodstuff additive or food compositions.In certain embodiments, composition of the present invention comprise carry zinc, through the serum protein or the milk-protein of protease digestion.In certain embodiments, described proteolytic enzyme is papoid.In certain embodiments, described serum protein or milk-protein are selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, commentaries on classics be lost albumen and α-fetoprotein.

On the other hand, the invention provides method with one or more combination treatment individualities of the present invention.In certain embodiments, the invention provides the method for treatment or prevention of obesity.In certain embodiments, the invention provides treatment or prevent overweight method.In certain embodiments, the invention provides the method that reduces weight increase.In certain embodiments, the invention provides the method for losing weight of inducing.Described method comprises step from the present composition of significant quantity to individuality that use.In preferred embodiments, the invention provides treatment or the overweight or fat method of prevention people.

On the other hand, the invention provides the apoptotic method of composition induced lipolysis as herein described of using.Described method comprises the step that adipocyte is contacted with composition of the present invention.Described cell can be any adipocyte, comprises interior, the external or ex vivo adipocytes of body.In certain embodiments, described adipocyte is in the individual body.In certain embodiments, described adipocyte is in the human body.

Also on the one hand, the invention provides the method and composition that is used for the treatment of obesity, they can directly reduce the number of adipocyte by apoptosis.This method is different from the ordinary method that reduces body weight, and described ordinary method comprises depress appetite, limit food product absorption or remove adipocyte from the single zone of health.Composition of the present invention comprise through protease digestion carry the zinc protein fragments, they can treat or prevent the fat or overweight of the individuality that needs effectively.

On the other hand, this paper provides the method for preparing the present composition.

4. accompanying drawing summary

Fig. 1 is the weight increase stabilization of mouse after using composition of the present invention;

Fig. 2 is that individual human every day twice (3mg/kg body weight) is taken composition of the present invention losing weight after ten days;

Fig. 3 is that the present composition is to the active inhibition of insulin receptor;

Fig. 4 A is the adipocyte with 20 Therewith/ml present composition treatment;

Fig. 4 B is the adipocyte with 2 Therewith/ml present composition treatment;

Fig. 5 reduces with respect to the weight increase that contrasts with the obesity mice that the present composition is treated;

Fig. 6 is when treating with the present composition and the weight increase minimizing of treatment back obesity mice after some days;

Fig. 7 is losing weight with the rat of present composition oral administration;

Fig. 8 is with the interior oil droplet minimizing of the rats'liver (fatty liver) of present composition treatment;

Fig. 9 is amounting to the body weight for humans minimizing in the test in 60 days;

Figure 10 is that body weight for humans reduces the function as dosage;

Figure 11 is that the body weight for humans in month reduces;

Figure 12 is losing weight of six individual humans;

Figure 13 A-C is that the blood-glucose of using the individual human of the present composition reduces.

5. detailed Description Of The Invention

Present patent application has been described new compositions and preparation method thereof and treatment or prevention of obesity and overweight Method. Especially, described composition comprise be derived from serum for example or breast carry zinc protein and egg thereof Plain boiled water solution fragment.

5.1 definition

When being used for this paper, except as otherwise noted, term " protein " refers to comprise one or more many The molecule of peptide chain or its fragment. Because can use for example one or more sugar or oligosaccharides modified polypeptide chain, So term " protein " clearly comprises glycoprotein. Except as otherwise noted, term " protein " does not have The size restriction is arranged. Particularly, this term comprises the egg through protease digestion from for example serum or breast The white matter fragment comprises glycoprotein. The preferred protease that is used for digestion comprises papain.

When being used for this paper, except as otherwise noted, term " treatment " refers to alleviate or reduces and treated The seriousness of the symptom that disease or illness are relevant. For example, term " treatment is fat " and " treating overweight " Refer to alleviate and fat or overweight one or more relevant symptoms.

When being used for this paper, except as otherwise noted, term " control " refer to keep with controlled disease or The seriousness of the symptom that illness is relevant reduces or avoids and by the relevant symptom of control disease or illness.

When being used for this paper, except as otherwise noted, term " prevention " refers to reduce acquisition by prevent disease Or illness or its symptom dangerous or postpone its generation. For example, term " prevention of obesity " and " prevention Overweight " refer to reduce with the dangerous of fat or overweight relevant one or more symptoms or postpone its generation.

When being used for this paper, " individuality " is animal, is the people usually.In preferred embodiments, described individuality is the people, for example the patient.

When being used for this paper, except as otherwise noted, " treatment is the amount effectively " of term compound and " effectively amount " refer to that this amount enough provides the treatment benefit in treatment of diseases, prevention and/or control, to postpone one or more symptoms relevant with disease to be treated or illness or it is minimized.Term " treatment is amount effectively " and " effectively amount " can comprise symptom or the reason that can improve overall treatment, reduce or avoid disease or illness or the amount that improves the result of treatment of another kind of curative.

Term " is used " jointly and " co-administered " comprises the while, walks abreast or use two kinds of curatives in order, and does not have concrete time limitation.In one embodiment, two kinds of medicines appear in the individual body simultaneously, perhaps bring into play their biology or result of treatment simultaneously.In one embodiment, described two kinds of curatives are in the identical composition or unit dosage.In another embodiment, described two kinds of curatives are in independently in composition or the unit dosage.

Term " dietary supplements " refers to that Public Law 103-417 was defined as the material of dietary supplements on October 25th, 1994 according to healthy and education bill the 3rd part of dietary supplementation in 1994.Dietary supplements is an orally administrable prod, wherein comprises " dietary ingredient " that be used for replenishing diet." dietary ingredient " comprise of the present invention year zinc, through the composition of protease digestion and one or more optional other dietary ingredients, for example VITAMIN, mineral substance, herbal medicine or other medicinal plant, amino acid and material such as enzyme, organ-tissue, body of gland and metabolite.Dietary supplements can also be extract or enriched material, and can find in many forms such as tablet, capsule, soft capsule, soft gelatin capsule, liquid or powder.

5.2 invention embodiment

The present invention relates to be used for the treatment of or prevention of obesity or overweight composition and method.In certain embodiments, described composition and method can reduce individual intravital adipocyte number by apoptosis.This method is different from the ordinary method that reduces body weight, comprises depress appetite, erstricted diet absorption or remove adipocyte in the single zone of health.

Apoptosis is cell active self-destruction that gene the instructs process of going out, and is also referred to as " apoptosis ".The sign of this process is that cellular contraction, cytolemma change and dna break.Therefore apoptosis is different from the death that is caused by cell injury, and the death that is caused by cell injury is called as necrosis.

Shown in the past that fetal serum had proteolytic enzyme susceptibility apoptosis induction activity to various cancer cell and laboratory animal tumour.Simultaneously, from serum, isolate zinc-binding protein, be called Pp63 glycophosphoproteins or α-2-HS-glycoprotein (AHSG).The Pp63 glycophosphoproteins of purifying can be induced the cancer cell apoptosis.Can greatly strengthen this apoptosis activity by loading Pp63 glycophosphoproteins with zinc.In addition, show that also digestion carries the peptide fragment mixture that the zinc Pp63 glycophosphoproteins produced and also can induce cancer cell apoptosis (Yu, CL and Tsai, M.H. (2001) Cancer Letters 166:173-184; Yu, CL and Tsai, M.H. (2001) Anticancer Research 21:1839-1856).

Except Pp63 glycophosphoproteins, fetus and adult animals (ox, sheep, mouse and rat) and human blood clearly also comprise other zinc-binding protein, comprise α-1-acid glycoprotein (AAG), α-1-antitrypsin (AAT).Now disclose these albumen: when with loading zinc, these protein (Pp63 glycophosphoproteins, AAG and AAT) have the apoptosis induction activity to 3T3-L1 mouse adipose cell lines.In addition, the fragment that obtains by these protein of protease digestion also can be induced the apoptosis of 3T3-L1 mouse adipose cell lines.On the contrary, if do not load zinc, then under the same conditions, no matter protein still be fragment all can not be apoptosis-induced.Although do not wish to be subjected to the theoretical restriction of any concrete operation, believe that carrying zinc partly is the basis of the therapeutic activity of present method composition.Described in lower part, the composition that carries the zinc gained has and the diverse zinc that combines of for example natural sera albumen or milk-protein.

Further open now, except serum, anti-obesity proteins matter fragment can be derived from breast.When carrying zinc with zinc and protease treatment full milk with acquisition, proteolytic enzyme can digest milk-protein.Gained carries apoptosis zinc, also can induce 3T3-L1 clone through the milk-protein of protease digestion.

For anti-obesity character in the test body, will be applied to laboratory animal from the fragment intravenously that protease digestion carries zinc AAG gained.Although feed with specific high fat diet, the body weight of described animal does not increase, and does not suffer harmful side effect yet.For further test anti-obesity character, give individual human oral twice AAG fragment every day.After ten days, individual human lost weight 4.4 pounds.

5.3 composition and preparation thereof

This paper provides and can be used at the individuality treatment that needs are arranged or prevention of obesity or overweight composition.

In certain embodiments, composition of the present invention comprise significant quantity be used for the inventive method carry zinc, through the serum protein or the milk-protein of protease digestion.In certain embodiments, described amount can be treated obesity effectively.In certain embodiments, described amount can be treated overweight effectively.In certain embodiments, described amount can effectively prevent obesity.In certain embodiments, described amount can effectively prevent overweight.In certain embodiments, described amount can reduce whose body weight effectively increases.In certain embodiments, described amount can induce whose body weight to reduce effectively.In certain embodiments, described amount induced lipolysis apoptosis effectively.In certain embodiments, described amount can be regulated the insulin receptor activity effectively.

Any source that described protein can be known perfectly well from those skilled in the art.In certain embodiments, from serum, separate described protein.In certain embodiments, separate described protein from the Ruzhong.Described serum or breast can be from animals, for example ox, sheep, mouse, rat or people.Described serum can be to grow up or fetal serum.Can obtain and/or prepare described breast or serum according to any technology that those skilled in the art know perfectly well.

In certain embodiments, can be according to technology well known by persons skilled in the art, from the recombinant sources preparation with separate described protein.In certain embodiments, can prepare described protein according to synthetic or semi-synthetic technology well known by persons skilled in the art.

Described breast or serum protein can be to effective any breast of methods described herein or serum protein.In certain embodiments, described serum protein or milk-protein are selected from: α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin and Transferrins,iron complexes.In certain embodiments, described serum protein or milk-protein are α-2-HS-glycoprotein.In certain embodiments, described serum protein or milk-protein are α-1-acid glycoprotein.In certain embodiments, described serum protein or milk-protein are α-1-antitrypsins.In certain embodiments, described serum protein or milk-protein are albumin.In certain embodiments, described serum protein or milk-protein are Transferrins,iron complexess.

In certain embodiments, described composition comprises multiple serum protein or the milk-protein that is selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.In certain embodiments, described composition comprises two kinds of serum proteins or the milk-protein that is selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.In certain embodiments, described composition comprises three kinds of serum proteins or the milk-protein that is selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.In certain embodiments, described composition comprises four kinds of serum proteins or the milk-protein that is selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.In certain embodiments, described composition comprises five kinds of serum proteins or the milk-protein that is selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.

In composition of the present invention, described serum protein or milk-protein are (zinc-charged) that carries zinc.As described below, the composition that carries the zinc gained has the diverse zinc that combines of trace zinc that may comprise with natural sera or breast.Any technology that can know perfectly well according to those skilled in the art is that described serum protein or milk-protein load zinc.Example technique for example is described in United States Patent (USP) 5,994, No. 298,6,258, No. 779,6,720, No. 311 and 6,737, No. 402, and the full content of these documents is included into this paper as a reference.By removing coupled ion such as metal ion, protein is contacted with zine ion, can load zinc for protein.Can remove coupled ion by any technology that those skilled in the art know perfectly well, described technology for example comprise dialyse, filtration, sequestrant etc.In certain embodiments, sequestrant by making protein and significant quantity such as EDTA or EGTA contact and remove coupled ion.Example condition comprises that for example about 5mM is used for about 40 Jian protein to about 100mM EDTA.In certain embodiments, after removing coupled ion, from protein, remove sequestrant by standard technique such as filtration, dialysis, chromatography or molecular sieve.

Contact by any type of zinc, can load protein with zinc with zinc loading protein significant quantity.The exemplary forms of zinc comprises zinc salt such as zinc chloride, zinc acetate and zinc sulfate.Among the work embodiment below, load protein with zinc acetate.Example condition comprises for example about 50 to about 500mM zinc salt, is used for 40 Jian protein as zinc acetate, zinc chloride or zinc sulfate.Actual conditions comprises that about 50 are used for 40 Jian protein to about 500mM zinc acetate.In certain embodiments, from protein, remove excessive zinc by standard technique such as filtration, dialysis, chromatography or molecular sieve.

In composition of the present invention, described protein is through protease digestion.Can be with the described protein of protease digestion before loading zinc, in the process of loading zinc or after loading zinc.Among the work embodiment below, after protease digestion, load zinc.Can to be that those skilled in the art are known can produce the effective segmental any proteolytic enzyme of the inventive method described proteolytic enzyme.Example proteolytic enzyme comprises papoid.Under the effective segmental condition, protein is contacted the inventive method suitable the generation with proteolytic enzyme.In certain embodiments, make protein contact about two hours or the longer time at 37 ℃ with papoid.In certain embodiments, from protein, remove papoid by standard technique such as filtration, dialysis, chromatography or molecular sieve.In certain embodiments, can be by other technology well known by persons skilled in the art such as freeze-thaw technological assimilation protein.

In certain embodiments, described herein year zinc, through the serum protein of protease digestion or milk-protein size basically less than 100kDa, 50kDa, 25kDa, 20kDa, 15kDa, 10kDa, 5kDa or 3kDa.Term " size basically less than " refer to be no more than 50%, 75%, 90% or 95% through the serum protein of protease digestion or milk-protein fragment greater than this size restriction.Can according to any technology that those skilled in the art know perfectly well will carry zinc, divide size through the serum protein of protease digestion or milk-protein, described technology for example comprise filter, dialysis, chromatography or molecular sieve.

By standard technique, comprise hereinafter described those composition to be mixed with the described form of lower part, for example dietary supplements, foodstuff additive, food compositions, nutraceutical composition or pharmaceutical composition.

5.4 using method

This paper provides one or more combination treatments of the present invention or prevention of obesity or the overweight method used.

In certain embodiments, this paper provides the method for using one or more combination treatment obesities of the present invention.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to treat fat step.In certain embodiments, this paper provides the method with one or more composition prevention of obesity of the present invention.Described method comprises to the individuality that needs are arranged uses the step of the present composition of significant quantity with prevention of obesity.

In certain embodiments, this paper provides the method for using one or more combination treatments of the present invention overweight.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to treat overweight step.In certain embodiments, this paper provides with the overweight method of one or more composition preventions of the present invention.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to prevent overweight step.

In certain embodiments, this paper provides the method for using one or more compositions of the present invention to reduce weight increase.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to reduce the step of weight increase.In certain embodiments, this paper provides the method with one or more composition prevention weight increase of the present invention.Described method comprises to the individuality that needs are arranged uses the step of the present composition of significant quantity with the prevention weight increase.

In certain embodiments, this paper provides and has used one or more compositions of the present invention to induce the method for losing weight.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to induce the step of losing weight.In certain embodiments, described losing weight is for medical reasons.In certain embodiments, described losing weight is that the cosmetic gonosome heavily reduces.

In certain embodiments, this paper provides treatment or prevention to relate to the overweight or fat disease or the method for illness.Described disease or illness comprise that for example high blood pressure, hypertension, high blood cholesterol levels, diabetes, insulin resistance, glucose do not tolerate, coronary heart disease and psychological pathology such as depression and eating disorder.Described method comprises to the individuality that needs are arranged uses the present composition of significant quantity to treat or to prevent the step of described disease or illness.

In certain embodiments, this paper provides the apoptotic method of one or more composition induced lipolysis of the present invention of using.Described method comprises makes adipocyte contact with apoptosis-induced step with the present composition of significant quantity.Described adipocyte can be any adipocyte well known by persons skilled in the art, comprises interior, the external or ex vivo adipocytes of body.In certain embodiments, described adipocyte may be intraindividual.

In certain embodiments, this paper provides and has used one or more compositions of the present invention to regulate the method for Cuticle of cell growth factor receptors (EGFR) function.Described method comprises contacts to regulate the step of receptor active the cell of expressing EGFR with the present composition of significant quantity.The cell of described expression EGFR can be any cell of expression EGFR well known by persons skilled in the art, comprises the cell of the expression EGFR that body is interior, external or stripped.In certain embodiments, the cell of described expression EGFR may be intraindividual.

In certain embodiments, this paper provides the method for using one or more compositions of the present invention to regulate cell Regular Insulin function of receptors.Described method comprises that the cell that makes the expression of insulin acceptor contacts with the present composition of significant quantity to regulate the step of receptor active.The cell of described expression of insulin acceptor can be any cell of expression of insulin acceptor well known by persons skilled in the art, comprises the cell of the expression of insulin acceptor that body is interior, external or stripped.In certain embodiments, the cell of described expression of insulin acceptor may be intraindividual.

5.4.1 individual crowd

Described individuality may be any individuality that needs the inventive method.In certain embodiments, described individuality is an animal.In certain embodiments, described individuality is the people.In certain embodiments, described individuality is the patient.

In certain embodiments, described individuality is the people who is in the fat danger.In certain embodiments, described individuality is fat people.When being used for this paper, " obesity " refers to be thought fat individuality by the practitioner in the art.In certain embodiments, " obesity " refers to that body surface index (BMI) is 30kg/m ²Or bigger people.

In certain embodiments, described individuality is the people who is in the overweight danger.In certain embodiments, described individuality is overweight people.When being used for this paper, " overweight " refers to be thought overweight individuality by the practitioner in the art.In certain embodiments, " overweight " refers to that body surface index (BMI) is 25kg/m ²Or bigger people.

In certain embodiments, described individuality is that BMI is greater than 30kg/m ², 35kg/m ², 40kg/m ², 45kg/m ²Or 50kg/m ²The people.In certain embodiments, described individuality is that BMI is less than 25kg/m ²The people, for example be that the practitioner in the art thinks that the BMI that need to reduce body weight or weight increase is less than 25kg/m ²The people.

In certain embodiments, described individuality is healthy.In certain embodiments, described individuality is healthy except that overweight or fat.In certain embodiments, described individuality is unsound.In certain embodiments, described individuality is still unsound except that overweight or fat.

In certain embodiments, described individuality is healthy or unsound, can use composition of the present invention to reduce weight increase or to induce and lose weight for any reason.

5.4.2 associating

On the other hand, the invention provides to the individual co-administered composition of the present invention that needs being arranged and can effectively treat or second medicine of prevention of obesity, with treatment or prevent overweight, realization is lost weight or reduce the method for weight increase.

In certain embodiments, described second medicine can be regulated α-melanotropin (α-MSH).In some embodiments, described second medicine is selected from selectivity serotonin reuptake inhibithors (SSRI), serotonin 2C excitomotor and serotonin 1B excitomotor.In other embodiments, described second medicine is selected from for example Bupropion, fluoxetine, fluvoxamine, Sertraline, paroxetine, citalopram, escitalopram, sibutramine, duloxetine and Venlafaxine and pharmacy acceptable salt or prodrug.In other embodiments, described second medicine can suppress the activity of neuropeptide tyrosine.In other embodiments, described second medicine is selected from NPY antagonistic, ghrelin antagonistic and degree albumen (leptin).In some embodiments, described second medicine can exciting NPY Y2 acceptor.In some embodiments, described second medicine is the npy receptor antagonistic.Other embodiment of the present invention comprises such embodiment, and wherein said second medicine is selected from γ-An Jidingsuan (GABA) inhibitor, GABA receptor antagonist and GABA passage antagonistic.Described GABA inhibitor can be the another kind of medicine that 5-HT 1b excitomotor maybe can suppress the GABA neuronal activity.In other embodiments, described second medicine is the Dopamine HCL reuptake inhibithors.Phentermine is the example of Dopamine HCL reuptake inhibithors.In certain embodiments, described second medicine is a noradrenaline reuptake inhibitor.The example of noradrenaline reuptake inhibitor comprises thionisoxetine and Reboxetine.Other embodiment comprises that wherein said second medicine is the embodiment of dopamine agonist.Can on market, obtain some dopamine agonists, comprise Cabergoline, amantadine, lisuride, pergolide, Ropinirole, pramipexole and bromocriptine.In other embodiments, described second medicine is norepinephrine releasing agent such as Diethylpropion, or blended Dopamine HCL/noradrenaline reuptake inhibitor such as Tomoxetine hydrochloride.In some other embodiment, described second medicine is the 5-HT1b excitomotor, for example sumatriptan, almotriptan, naratriptan, frovatriptan, Rizatriptan, zolmitriptan and elitriptan.In other embodiments, described second medicine is an anticonvulsive drug.Described anticonvulsive drug can be selected from zonisamide, topiramate, Sodital, lorazepam, clonazepam, chlordiazepoxide, tiagabine, gabapentin, prophenytoin, Phenytoin Sodium Salt, Carbamzepine, valproate, felbamate, Levetiracetam, oxcarbazepine, lamotrigine, methsuximide and ethosuximide.In some embodiments, described second medicine is the Cannabined receptor antagonistic.

In certain embodiments, described second medicine itself can be the combination of two or more medicines.For example, described second medicine can be that Dopamine HCL absorbs Depressant again and norepinephrine absorbs Depressant again, for example combination of Mazindol.Perhaps, described second medicine can be SSRI and noradrenaline reuptake inhibitor, for example the combination of sibutramine, Venlafaxine and duloxetine.

5.5 dietary supplements and pharmaceutical composition

Composition of the present invention can use with the form of food, foodstuff additive, dietary supplements, dietetic product or medicine.Described composition can comprise carrier, thinner or vehicle.According to desired use, described carrier, thinner or vehicle can be selected as being fit to people or animal doctor's use, foodstuff additive, supplement or drug use.

In certain embodiments, with the mixture of breast in composition of the present invention is provided.In certain embodiments, the form with dry powder provides composition of the present invention.Can be according to the dry composition of the present invention of any technology well known by persons skilled in the art, described technology comprises freeze-drying, lyophilize, spraying drying and evaporation drying.Described powder can comprise composition of the present invention and optional any carrier described herein or well known by persons skilled in the art, thinner, vehicle or other additive.In certain embodiments, the form with liquid provides composition of the present invention.Described liquid can comprise composition of the present invention and optional any carrier described herein or well known by persons skilled in the art, thinner, vehicle or other additive.Described liquid can be that those skilled in the art think useful any volume, for example makes things convenient for the volume of administration and route of administration.

When being used for this paper, " food " or " food compositions " is the material of mainly being made up of protein, carbohydrate and/or fat, and it is used to organic body to keep growth, reparation and vital process and supplying energy.Food can also comprise supplementation material such as mineral substance, VITAMIN and seasonings.Term " food " comprises the beverage that is fit to human or animal's consumption.When being used for this paper, " foodstuff additive " comprise direct and indirect additive according to the definition of FDA in 21 CFR 170.3 (e) (1).

Can the composition that this paper provided be mixed with soft gelatin capsule, tea, tablet etc. with the standard compounding process.Referring to for example Remington ' s Pharmaceutical Sciences, the 18th edition, MackPublishing, Easton Pa. (1990).As described herein, composition of the present invention can be mixed with dietary supplements or pharmaceutical preparation, be used to be applied to individual with treatment or prevention of obesity or overweight.

The suitable form of the present composition comprises foodstuff additive, food compositions (comprising drink composition) and dietary supplements.Composition of the present invention can be added in the various food for consumption simultaneously.As foodstuff additive, can use composition of the present invention according to the mode identical with the general food additive, therefore only composition of the present invention need be mixed with other food component.

Should be realized that, dietary supplements and the nonessential use formulation composition identical with pharmaceutical composition, or have aseptic and other FDA requirement identical with pharmaceutical composition.Described dietary supplements can be a liquid form, and for example solution, syrup or suspension perhaps can be the product forms that is used for before use with water or any other suitable liquid reorganization.Can prepare described liquid preparation by conventional means, for example tea, healthy beverage, dietary beverage, liquid concentrate or dissolve in tablet, capsule, pill or the powder of liquid prepare beverage thereby can be dissolved in the liquid by tablet, capsule, pill or the powder that will dissolve in liquid and consume the gained beverage.Perhaps, described dietary supplements can adopt tablet or capsular form, described form is by the conventional means preparation and randomly comprise other dietary supplements, comprise VITAMIN, mineral substance, other herbal medicine supplement, tackiness agent, weighting agent, lubricant, disintegrating agent or wetting agent, as described above those.Can be by means commonly known in the art with tablet coating.In preferred embodiments, described dietary supplements can adopt capsule or form of powder, is used for oral consumption in the liquid for being dissolved in.

The edible dosage of activeconstituents in disease or treatment of conditions or prevention will change according to the seriousness and the route of administration of disease to be treated or illness.Described dosage and possible administration frequency also will change according to human consumer or patient's age, body weight, reaction and passing medicine history.By considering these factors, those skilled in the art can easily select suitable dosage regimen.

The amount of mixing the present composition in the foodstuff products will depend on the kind of food and required effect.Usually, the amount of the contained present composition of single supply accounts for about 0.1% to about 100% or about 0.1% to about 50% or about 0.5% to about 20% of food compositions.In certain embodiments, the amount of the contained present composition of foodstuff products accounts for about 1% to about 10% of food compositions.

The example of food includes but not limited to candy that candy such as sweeting agent (rock sugar, gelifying agent, jam etc.), natural gum, beans are stuck with paste, cured or candy (biscuit, dessert etc.), steam candy, cocoa or the cocoa product (chocolate and cocoa) of molding, freezing candy (ice-creams, ice etc.), beverage (fruit juice, soft drink, soda pop), healthy beverage, health bar and tea (green tea, black tea etc.).

Also comprise the packing that comprises food, dietary supplements or medicine and indicate the label that has The compounds of this invention in the scope of the present invention.Randomly, described label can be indicated the beneficial property and the working instructions of composition of the present invention, the present composition.

In preferred embodiments, the composition of Gong using is pharmaceutical composition or single unit dosage.Pharmaceutical composition and single unit dosage can comprise prevention or treatment one or more preventions of significant quantity or curative and one or more pharmaceutically acceptable carriers, vehicle or thinner usually.In the linguistic context of specific embodiments and this paper, term " pharmaceutically acceptable " refers to by federation or state government rule mechanism approval or lists in American Pharmacopeia or other generally acknowledged pharmacopeia is used for animal, perhaps more specifically is used for the people's.The thinner that term " carrier " refers to use with curative, adjuvant (for example freund's adjuvant (fully and not exclusively)), vehicle or vehicle.Described pharmaceutical carrier can be a sterile liquid, and for example water and oil comprise oil, animal, plant or synthetic those of originating, for example peanut oil, soybean oil, mineral oil, sesame wet goods.When intravenously was used described pharmaceutical composition, water was preferred carrier.Salt brine solution, glucose solution and glycerine solution also can be used as liquid vehicle, especially for injectable solution.The example of suitable pharmaceutical carrier be described in E.W.Martin's " Remington ' sPharmaceutical Sciences ".Nutraceutical composition can but must not comprise one or more activity or non-activity composition, described composition must not thought pharmaceutically acceptable by the current practitioner of this area.

According to those skilled in the art's judgement, can use medicine of the present invention or nutraceutical composition by any approach, that these approach include but not limited to is oral, in the intravenously, stomach, in the duodenum, intraperitoneal or Intraventricular.

Typical medicine or nutraceutical composition and formulation comprise one or more vehicle.Suitable vehicle is known to the technician of pharmaceutical field, and the non-limitative example of appropriate excipients comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, lime, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, dry skimmed milk, glycerine, propylene glycol, water, ethanol etc.Whether concrete vehicle is fit to mix in medicine or nutraceutical composition or the formulation, and this depends on various factors well known in the art, and described factor includes but not limited to that formulation will be applied to patient's mode and the concrete activeconstituents in the formulation.If desired, described composition or single unit dosage can also comprise a small amount of wetting agent or emulsifying agent or pH buffer reagent.

Lactose free composition of the present invention can comprise vehicle well known in the art, for example.List in the vehicle of American Pharmacopeia (USP) SP (XXI)/NF (XVI).Usually, the lactose free composition comprise activeconstituents and medicine compatible with tackiness agent/weighting agent pharmaceutically acceptable amount and lubricant.The lactose free formulation of example comprises activeconstituents, Microcrystalline Cellulose, pregelatinized Starch and Magnesium Stearate.

The present invention comprises also and uses medicine or nutraceutical composition and the formulation that contains one or more compounds that described compound can reduce the speed that activeconstituents will decompose.Described compound is referred to herein as " stablizer ", includes but not limited to antioxidant such as xitix, pH buffer reagent or salt buffer agent.

Described medicine or nutraceutical composition and single unit dosage can adopt forms such as solution, suspension, emulsion, tablet, pill, capsule, powder, slow release formulation.Oral dosage form can comprise N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate of standard vector such as pharmaceutical grade etc.Described composition and formulation will comprise the prevention or the curative of prevention or treatment significant quantity, be preferably purified form, and the carrier of appropriate amount, thereby be provided for correctly being applied to patient's form.Described formulation should be fit to mode of administration.In preferred embodiments, described pharmaceutical composition or single unit dosage are aseptic, are suitable for administration to individuality, preferred animal individual, more preferably mammalian subject, most preferably individual human.

It is compatible with its expection route of administration that medicine of the present invention or nutraceutical composition are formulated into.The example of route of administration includes but not limited to that parenteral uses, for example in intravenously, intracutaneous, subcutaneous, intramuscular, subcutaneous, oral, oral cavity, the sheath, in the suction, nose, in the transdermal, part, saturating mucous membrane, tumour, in the synovia and rectal administration.In specific embodiments, according to conventional procedure be mixed with described composition in suitable intravenously, subcutaneous, intramuscular, oral, the nose or topical application to human medicine or nutraceutical composition.In one embodiment, according to conventional procedure the pharmaceutical composition preparation is used for subcutaneous administration to human.Usually, being used for the composition that intravenously uses is to be dissolved in the solution that aseptic grade is opened aqueous buffer.When essential, described composition can also comprise solubilizing agent and local anaesthetics such as lignocaine, to reduce the pain of injection position.

The example of formulation includes but not limited to: tablet; The capsule sheet; Capsule such as soft elastic gelatin capsule; Cachet; Tablet; Lozenge; Dispersion; Suppository; Ointment; Paste (paste); Patch; Powder; Dressing; Emulsifiable paste; Plaster; Solution; Paster; Aerosol (for example nose spraying or inhalation); Gel; Be fit to oral or mucosal administration to patient's liquid dosage form, comprise suspension (for example water-based or non-aqueous liquid suspension, O/w emulsion or water-in-oil liquid emulsion), solution and elixir; Suitable parenteral is applied to patient's liquid dosage form; Be fit to the sterile solid (for example crystal or unformed solid) that parenteral be applied to patient's liquid dosage form with recombinating to provide.

The composition of combination dosage form, shape and type will change according to their purposes usually.For example, compare with the formulation that is used for the identical illness of chronic treatment, the formulation that is used for the acute treatment illness can comprise one or more relatively large compositions.And the treatment effective dosage forms of various disease or illness type may be different.Similarly, compare with the oral dosage form that is used for the treatment of same disease or illness, parenteral dosage form can comprise more a spot of one or more activeconstituentss.These and other mode that the present invention includes concrete formulation will differ from one another, and this will be to understand easily for those skilled in the art.Referring to for example Remington ' s Pharmaceutical Sciences, 18th ed.Mack Publishing, Easton PA (1990).

Usually, the composition components that comprises described composition can separately provide, and is perhaps admixed together in unit dosage, for example as the sealed vessel such as the lyophilized powder in ampoule or the anther sac of indication activeconstituents quantity or there is not aqueous concentrate.When will be by inculcating when using composition, can be with comprising sterile pharmaceutical grade water or the brinish infusion bottle distributes it.In the time will using composition, an ampoule sterile water for injection or a salt solution can be provided, thereby use preceding mixing element by injection.

The amount of the present composition that the exemplary dosage form of using in the inventive method is contained is about 0.1mg extremely about 400mg/ days, 1mg to 100mg/ sky or 1mg to 15mg/ sky, gives as single dosage once a day or as the divided dose of whole day.Concrete formulation of the present invention comprises about 0.1,0.2,0.25,0.3,0.5,0.75,1.0,2.0,2.5,3.0,5.0,7.5,10.0,15.0,20.0,25.0,40.0,50.0,60.0 or the 100mg composition.Exemplary dosage form is that protein concn is 3,5,7.5 or the 15ml liquid composition of 0.1mg/ml.

Be fit to the Orally administered pharmaceutical composition that is used for the inventive method and can be used as discrete formulation existence, such as but not limited to tablet (for example chewable tablet), capsule sheet, capsule and liquid (for example seasoning syrup).Described formulation comprises the activeconstituents of predetermined amount, can be by well known to a person skilled in the art the preparation of medicament method.Generally referring to Remington ' s Pharmaceutical Sciences, the 18th edition, MackPublishing, Easton PA (1990).

In certain embodiments, oral dosage form is a solid, and prepares with no water constituent under anhydrous condition, as the detailed description in the upper section.Yet scope of the present invention has exceeded the anhydrous solid oral dosage form.Therefore, this paper has also described other form.

According to the conventional medicine compounding process, by with the activeconstituents in the homogeneous mixture in the typical oral dosage form of the incompatible preparation of at least a vehicle group.Vehicle can adopt form widely, and this depends on the dosage form that hope is used.For example, the vehicle that is adapted at using in liquid oral or the aerosol dosage forms includes but not limited to water, ethylene glycol, oil, alcohol, seasonings, sanitas and tinting material.The vehicle example that is adapted at using in the solid oral dosage form (for example powder, tablet, capsule and capsule sheet) includes but not limited to starch, sugar, Microcrystalline Cellulose, thinner, granulating agent, lubricant, tackiness agent and disintegrating agent.

The vehicle example that can be used for oral dosage form of the present invention includes but not limited to tackiness agent, weighting agent, disintegrating agent and lubricant.The tackiness agent that is suitable for pharmaceutical composition and formulation includes but not limited to W-Gum, yam starch or other starch, gelatin, natural and synthetic rubber such as gum arabic, sodiun alginate, Lalgine, other alginate, the powdery tragacanth, guar gum, Mierocrystalline cellulose and derivative thereof (ethyl cellulose for example, rhodia, calcium carboxymethylcellulose, Xylo-Mucine), polyvinylpyrrolidone, methylcellulose gum, pregelatinized Starch, (for example model 2208 for Vltra tears, 2906,2910), Microcrystalline Cellulose and composition thereof.

The weighting agent example that is used for pharmaceutical composition as herein described and formulation includes but not limited to talcum, lime carbonate (for example particle or powder), Microcrystalline Cellulose, Solka-floc, glucose mixture, kaolin, N.F,USP MANNITOL, silicic acid, sorbyl alcohol, starch, pregelatinized Starch and composition thereof.In the pharmaceutical composition of the present invention the content of tackiness agent or weighting agent account for usually described pharmaceutical composition or formulation about 50 to about 99 weight percents.

The suitable form of Microcrystalline Cellulose includes but not limited to as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA) material of Chu Shouing and composition thereof.Concrete tackiness agent is as the Microcrystalline Cellulose of AVICEL RC-581 sale and the mixture of Xylo-Mucine.Suitable anhydrous or low humidity vehicle or additive comprise AVICEL-PH-103 ^TMWith starch 1500LM.

The tablet of disintegration when composition of the present invention uses disintegrating agent to be exposed to aqueous environments to be provided at.The tablet that comprises too many disintegrating agent may disintegration when storing, and comprise very little the tablet of disintegrating agent may not can with required speed or disintegration under required condition.Therefore, thus should use the not many disintegrating agent that does not also damage the sufficient quantity that activeconstituents discharges very little to form solid oral dosage form of the present invention.Used disintegration dosage can easily be discerned by those of ordinary skills based on the type of formulation and change.Typical pharmaceutical composition comprises about 0.5 disintegrating agent to about 15 weight percents, is specially about 1 disintegrating agent to about 5 weight percents.

The disintegrating agent that can be used for pharmaceutical composition of the present invention and formulation includes but not limited to agar-agar, Lalgine, lime carbonate, Microcrystalline Cellulose, croscarmellose sodium, polyvinylpolypyrrolidone, polacrilin potassium, primojel, potato or tapioca (flour), pregelatinized Starch, other starch, clay, other phycocolloid, other Mierocrystalline cellulose, natural gum and composition thereof.

The lubricant that can be used for pharmaceutical composition of the present invention and formulation includes but not limited to calcium stearate, Magnesium Stearate, mineral oil, light mineral oil, glycerine, sorbyl alcohol, N.F,USP MANNITOL, polyoxyethylene glycol, other glycol, stearic acid, sodium lauryl sulphate, talcum, hydrogenated vegetable oil (for example peanut oil, Oleum Gossypii semen, Trisun Oil R 80, sesame oil, sweet oil, Semen Maydis oil and soybean oil), Zinic stearas, ethyl oleate, Laurate ethyl, agar and composition thereof.Extra lubricant comprises for example syloid silica gel (AEROSIL200, by W.R.Grace Co.of Baltimore, the MD manufacturing), the aerosol that condenses of synthetic silica is (by Degussa Co.of Piano, the TX sale), CAB-O-SIL (by Cabot Co.of Boston, the high thermal silicon dioxide product that MA sells) and composition thereof.If all use, the then pharmaceutical composition that mixes less than them usually of the content of lubricant or about 1 weight percent of formulation.

5.6 Shi Yongjiliang ﹠amp; Frequency

The amount that effectively prevents, treats, controls or alleviate the composition in the inventive method of illness or its one or more symptoms changes according to the character of disease or illness and the route of administration of seriousness and activeconstituents.Described frequency and dosage also will change according to every patient's particular case, depend on the concrete therapy of being used (for example curative or prophylactic agent), the seriousness of illness, disease or illness, route of administration, and patient's age, body weight, reaction and passing medicine history.Can be by being derived from the extrapolated effective dose of dose-response curve external or the animal model test system.

The example dosage of the present composition comprises the gross protein/kilogram individuality or the example weight (for example about 1 microgram/kilogram is to about 10 mg/kg) of milligram or microgram amount.For compositions for use of the present invention, be applied to the patient dosage can for 0.001mg/kg to about 100mg/kg.In certain embodiments, the dosage that is applied to the patient is that 0.001mg/kg is to about 10mg/kg.In certain embodiments, by proteinic weight in the composition, dosage is that about 0.02mg/kg is to about 4.0mg/kg weight in patients.In the embodiment of true protein (for example recombinant protein or commercially available protein) as raw material, dosage can be in the lower end of described scope.In by breast or the proteinic embodiment of serum pref, dosage can be in the upper end of described scope.

Usually, recommendation dosage range every day that is used for the composition of illness described herein in the inventive method is about 0.1mg extremely about 100mg/ days, as single dose or the multidose of every day.The practitioner in the art can determine actual dose according to for example Individual Age, body weight, BMI or other factors.In certain embodiments, total every day dosage range can for about 0.1mg to about 25mg/ days, more specifically be about 1mg extremely about 15mg/ days.

Described composition can be used as single dosage once a day and uses, and perhaps uses as the divided dose in the whole day.In some embodiments, divided dose administered twice every day dosage every day to equate.In certain embodiments, use dosage every day three times every day.In specific embodiments, divided dose every day of equating to use dosage every day three times.In specific embodiments, divided dose every day of equating to use dosage every day four times.

Institute is described herein, weight be preferably based on carry zinc, through the proteinic weight of protease digestion.

In certain embodiments, can repetitive administration every day composition of the present invention.In certain embodiments, using can be at interval at least 1 day, 2 days or 3 days.

The significant quantity of composition described herein will provide the treatment benefit and can not cause toxicity basically.

By the standard drug program in cell cultures or the laboratory animal, for example by determining LD ₅₀(lethal dose of 50% colony) or LD ₁₀₀(lethal dose of 100% colony) can determine the toxicity of composition.Dosage ratio between toxicity and the result of treatment is a therapeutic index.Compound with high therapeutic index is preferred.The data that obtain from these cell cultures mensuration and zooscopy can be used to illustrate the non-toxic scope of human.The dosage of compound described herein preferably is in the scope of circulation composition, and this scope comprises having low toxicity or nontoxic effective dose.Dosage can change in this scope, depends on used formulation and used route of administration.Individual doctor can select definite formulation, route of administration and dosage (referring to for example Fingl etc., 1996, In:The Pharmacological Basisof Therapeutics, the 9th edition, the 2nd chapter, the 29th page, Elliot M.Ross) according to patient's illness.

6. embodiment

6.1 embodiment 1: carry the proteinic preparation of zinc

Method 1: 40 milliliters of serum (from various animals) were cultivated 1 hour with 8ml 0.1M EDTA or EGTA (water-soluble).By with 4 liters of deionized water dialysis 3 hours, remove EDTA or EGTA.To cultivate at least 4 hours through the serum of EDTA or EGTA processing with zinc acetate (final Zn concentration is 50mM).By centrifugal disgorging.With deionized water 40ml serum is dialysed two-wheeled to remove excessive zinc: with 10 liters of deionized waters dialysis 18 hours, dialysed again 4 hours with 4 liters of fresh deionized waters then for the first time.

Method 2: 40 milliliters of breasts (from various animals) were cultivated 1 hour with 8ml 0.1M EDTA or EGTA (water-soluble).By with 4 liters of deionized water dialysis 3 hours, remove EDTA or EGTA.To cultivate at least 4 hours through the breast that EDTA handles with zinc acetate (final Zn concentration is 50mM).By centrifugal disgorging.With deionized water the 40ml breast is dialysed two-wheeled to remove excessive zinc: with 10 liters of deionized waters dialysis 18 hours, dialysed again 4 hours with 4 liters of fresh deionized waters then for the first time.

Method 3: at room temperature, 10ml salt brine solution with 25mM EDTA or EGTA is cultivated protein soln (α-2-HS-glycoprotein (AHSG or Pp63 glycophosphoproteins), α-1-acid glycoprotein (AAG), α-1-antitrypsin (AAT), albumin, Transferrins,iron complexes and each 40 Jian of α-fetoprotein) 1 hour, described salt brine solution comprises 2mM HEPES, and pH 7.0.By being that the molecular sieve of 10KDa repeats to remove by filter EDTA or EGTA with molecular weight cut-off.With the 250mM zinc acetate protein soln (40 Jian) was cultivated 18 hours.Remove excessive zine ion by concentrating four-wheel with 20 times of volume salt brine solutions (pH 7.0), all use molecular sieve at every turn, this causes dilution factor is 6 * 10 ^-6Zine ion.Determine that by the 10KDa membrane filtration liquid of protein soln free zinc ion concentration is about 5-10 Jian, observed under this concentration does not have cytotoxicity before.

Method 4: at room temperature, 10ml salt brine solution with 5mM EDTA or EGTA is cultivated protein soln (α-2-HS-glycoprotein (AHSG or Pp63 glycophosphoproteins), α-1-acid glycoprotein (AAG), α-1-antitrypsin (AAT), albumin, Transferrins,iron complexes and each 40 Jian of α-fetoprotein) 1 hour, described salt brine solution comprises 2mM HEPES, and pH 7.0.By being that the molecular sieve of 10KDa repeats to remove by filter EDTA or EGTA with molecular weight cut-off.With 80 Jian zinc acetates protein soln (40 Jian) was cultivated 18 hours.

Method 5: at room temperature, 10ml salt brine solution with 25mM EDTA or EGTA is cultivated protein soln (α-2-HS-glycoprotein (AHSG or Pp63 glycophosphoproteins), α-1-acid glycoprotein (AAG), α-1-antitrypsin (AAT), albumin, Transferrins,iron complexes and each 40 Jian of α-fetoprotein) 1 hour, described salt brine solution comprises 2mM HEPES, and pH 7.0.By being that the molecular sieve of 10KDa repeats to remove by filter EDTA or EGTA with molecular weight cut-off.With the 50mM zinc acetate protein soln (40 Jian) was cultivated 18 hours.By with 1000 times of volumes of deionized water dialysis 2 hours, remove excessive zine ion.

6.2 embodiment 2: carry zinc protein with protease digestion

Method 1: with papoid (2,500 units are from papaya milk) the zinc serum (40m1) that carries of above-mentioned preparation was cultivated 2 hours at 37 ℃.Is that 3KDa arrives high molecular sieve collection fragment to 10KDa by making the cultivation mixture by molecular weight cut-off.

Method 2: with papoid (2,500 units are from papaya milk) the zinc breast (40ml) that carries of above-mentioned preparation was cultivated 2 hours at 37 ℃.Is that 3KDa arrives high molecular sieve collection fragment to 10KDa by making the cultivation mixture by molecular weight cut-off.

Method 3: at 37 ℃ with papoid (468 units, from papaya milk) the zinc protein (5ml comprises α-2-HS-glycoprotein (AHSG or Pp63 glycophosphoproteins), α-1-acid glycoprotein (AAG), α-1-antitrypsin (AAT), albumin, Transferrins,iron complexes and each 40 Jian of α-fetoprotein) that carries of above-mentioned preparation was cultivated 2 hours.By making the cultivation mixture is the molecular sieve collection fragment of 3KDa by molecular weight cut-off.

6.3 embodiment 3: zooscopy

To keep three A/J mouse (6 weeks are big, the 18-20 gram) of high fat diet and random water to be placed in the cage.After 4 days, with the amount of 2mg/Kg body weight, give the AAG fragment that injection prepares as stated above in the mouse tail vein, continue 6 days.Measure body weight every day.

The AAG fragment suppresses the mouse weight increase

As shown in Figure 1, injection according to the method described above the AAG fragment of preparation can cause that the mouse weight increase stablizes (5-11 days).The mouse body weight finishes to keep in back 6 days constant (12-17 days) in the described fragment of injection.

6.4 embodiment 4: human experimentation

To continue ten days from composition twice Orally administered individual human that keeps normal diet of giving every day of the serum for preparing as stated above.The dosage that gives is the 0.02mg/kg body weight, but useful range can for 0.02mg/kg body weight for example to the 4mg/kg body weight.Measure body weight every day.

Protein fragments from serum can cause that body weight for humans reduces

Fig. 2 shows and takes for twice individual human every day from composition (3mg/kg body weight) the losing weight after ten days of the serum of preparation as stated above.Find to keep the individual human of normal diet losing 4.4 pounds after 10 days with described fragment treatment.

6.5 embodiment 5: composition of the present invention suppresses mouse and rat insulin acceptor

Make cell lysates contact the present composition of pressing embodiment 2 preparations from mouse adipose cell lines 3T3-L1 and rat insulin acceptor (Sigma).As shown in Figure 3, composition of the present invention can suppress the insulin receptor signal activity in the described clone, and this shows (K-LISA PTK screening reagent box, Calbiochem catalog number (Cat.No.) 539701) by the colorimetric ELISA assay method that is used for tyrosine kinase activity.

In Fig. 3 A, the hole 1﹠amp that provides; 2 have the basic tyrosine kinase activity to described 3T3-L1 cell lysates.Add composition of the present invention and can suppress basic tyrosine kinase activity (hole 3﹠amp; 4; Two parts).When Regular Insulin being added in the adipocyte lysate, the tyrosine kinase activity of IR stronger (the hole 5﹠amp that becomes; 6).Add composition of the present invention and can end most of insulin-induced IR tyrosine kinase activity (hole 7﹠amp; 8).

In Fig. 3 B, the hole 1﹠amp that provides; 2 have the basic tyrosine kinase activity to described rats'liver insulin receptor.Add composition of the present invention and can suppress basic tyrosine kinase activity (hole 3﹠amp; 4; Two parts).When adding Regular Insulin, the tyrosine kinase activity of IR stronger (the hole 5﹠amp that becomes; 6).Add composition of the present invention and can end most of insulin-induced IR tyrosine kinase activity (hole 7﹠amp; 8)

6.6 embodiment 6: composition of the present invention reduces the human insulin level

Present embodiment provides the influence of the present composition to the hematochemistry of individual human.Composition of the present invention can significantly reduce individual fasting insulin level.

In the experimental phase, individual human was taken in the present composition according to embodiment 2 preparations in one month.In to the photograph stage, the individual human of the present composition is not taken in monitoring in one month.

3 different time points blood sample collections in this month.Use the ELSA assay method to check the fasting Regular Insulin and the IGF-1 level of each sample (double).

The insulin level of experimental phase is 1.38+/-0.04 Xin/ml.The insulin level in contrast stage is 2.02+/-0.07 Xin/ml.Composition of the present invention has reduced by 32% with the insulin level of individuality.

The IGF-1 level of experimental phase be 96+/-7.8ng/ml.The IGF-1 level in contrast stage be 98+/-2.4ng/ml.Composition of the present invention does not obviously change the IGF-1 level.

In addition, measured individual hematochemistry mutually and in experiment to the photograph process.Obviously, composition of the present invention has obviously reduced individual uric acid (from 9.4 to 7.3), has obviously reduced individual triglyceride level (from 178 to 95) and slightly improved individual HDL (from 37 to 44).

6.7 embodiment 7: the apoptosis of composition induced lipolysis cell of the present invention

Present embodiment provides the influence of the present composition to adipocyte.Than under the low dosage, the oil droplet that composition of the present invention can suppress in the adipocyte forms.Under higher dosage, composition of the present invention can be apoptosis-induced.

Preceding adipocyte is induced to be divided into adipocyte.Under the dosage of 20 Therewith/ml, the composition of the embodiment of the invention 2 caused apoptosis (referring to Fig. 4 A) in 6 to 8 hours.

Preceding adipocyte is induced to be divided into adipocyte.Under the dosage of 2 Therewith/ml, the oil droplet that the composition of the embodiment of the invention 2 can reduce or eliminate in the adipocyte forms (referring to Fig. 4 B).

6.8 embodiment 8: composition of the present invention reduces the weight increase of high fat diet mouse

Present embodiment provides the influence of the mouse of composition of the present invention to feeding with high fat diet.Composition of the present invention can obviously reduce the weight increase in the experiment periods.

Before experiment, feed mouse (n=6) with inducing obesity with high fat diet.With vehicle and high fat diet treatment control mice.By peritoneal injection (3 days), be intravenous injection (7 days) then, with combination treatment experiment mice of the present invention.

The weight increase of control mice (average 16%) is the twice of the weight increase (average 7.69%) of experiment mice.In this experiment, peritoneal injection does not reduce weight increase, but intravenous injection has obviously reduced weight increase (Fig. 5) really.

6.9 embodiment 9: the minimizing of high fat diet mouse weight increase is to the dependency of present composition dosage

Present embodiment provides the influence of the mouse of the present composition to feeding with high fat diet.With the identical mouse of combination treatment of the present invention, stop then handling, carry out and the body weight change of not carrying out described treatment with observation.Composition of the present invention is used in back two days in the application neutralization and has obviously been reduced weight increase.

Before experiment, feed mouse with inducing obesity with high fat diet.Treat experiment mice (8 days) with composition of the present invention by intravenous injection, in remaining fate, treat then with the contrast intravenous injection.

In using the process of the present composition or at least subsequently two days, the mouse body weight does not increase.After with combination treatment of the present invention several days, mouse recovered weight increase (Fig. 6).

6.10 embodiment 10: the present composition is at the intravital Orally active of rat

Present embodiment provides the present composition at the intravital Orally active of experimental rat.

By every day mouth raise, with composition of the present invention (n=3) or the contrast (water; N=3) treatment rat.

As shown in Figure 7, obviously reduced the weight increase of experimental rat with composition oral administration treatment of the present invention.

6.11 embodiment 11: the minimizing of fatty liver in the rat of present composition treatment

In fatty liver, fat is accumulated in liver cell.Simple fatty liver can not damaged liver usually.When fat account for liver at least 10% the time, the patient just has fatty liver.Its most normal obesity, insulin resistant and hyperlipidaemia followed, these all are the members of metabolism syndrome.The possible explanation of fatty liver comprises that fat transferred to the liver from the health other parts, and perhaps the fat extraction thing that offers liver from intestines increases.Another explanation is because liver can not be converted into fat the form that can be eliminated, so fat is just accumulated.The research of carrying out in 2004 hints that nearly 1/3 U.S. grownup who accepts investigation has fatty liver.

Press embodiment 10 described treatment rats.Staining agent oil red O with oil droplet dyes the liver sample.As shown in Figure 8, obviously reduced by the oil droplet deposition in the treatment rats'liver with the Orally administered treatment of the present composition.

6.12 embodiment 12: composition of the present invention reduced people's body weight in 60 days

Present embodiment provides the long term human in 60 days to lose weight.

In 60 days, individual human three oral 5mg compositions of the present invention every day.As shown in Figure 9, individual in 60 days 16 pounds of losss of weight progressively.Individuality does not experience total side effect.

6.13 embodiment 13: composition of the present invention is in the intravital effective dose of individual human

Present embodiment provides and has reduced the influence of the present composition at the intravital dosage of individual human of embodiment 12.

Individual human is taken in 3ml (0.1mg/ml), 10ml (0.1mg/ml) and 15ml (0.1mg/ml) composition of the present invention.Independently using each dosage to identical individual human in 7 days.As shown in figure 10,15ml dosage causes losing weight fully, but 3 and 10ml dosage do not have.

This experiment and should individuality in, 15ml dosage is more effective than 3ml or 10ml dosage.

6.14 embodiment 14: two doses every day of the present composition reduces people's body weight

Present embodiment provides the long term weight when taking for twice individual human every day of embodiment 12 to reduce.

In 32 days, individual human twice oral 7.5ml every day (0.1mg/ml) composition of the present invention.A dosage is taken in the morning, and second dosage is taken between the lights.As shown in figure 11, individual in 32 days loss of weight 8 pounds.Initial weight is 144 pounds, and final weight is 136 pounds.

Under this individuality and this experiment condition, every day twice dosage with every day three times dosage the same effective.

6.15 embodiment 15: composition of the present invention reduces people's body weight

As previously described in the embodiment, present embodiment provide altogether in four months people's long term weight reduce.As previously mentioned, dosage is 3mg/ days, 10mg/ days or 15mg/ days.As previously mentioned, dosage is used three times or is used every day twice every day.

In time of four months altogether, individual human (55 years old age) loss of weight 29 pounds.His final body fat is 16% (22.4 pounds), and his final lean mass is 83.6% (114.6 pounds).Hematochemistry demonstration minimizing uric acid (9.4 to 7.3), total cholesterol (256 to 225) and triglyceride level (178 to 126).

In further research, when using composition of the present invention, lose weight for five in six individual humans.Figure 12 provides the relative body surface index of losing weight to increase.Individuality response with normal or low BMI is less, and the individuality with higher BMI demonstrates bigger response.

6.16 embodiment 15: composition of the present invention reduces people's body weight

Present embodiment provides the blood glucose levels of individual human after using the present composition to reduce.

In an experiment, individual take in 15ml (0.1mg/ml) composition of the present invention or contrast dividing in other day.After 30 minutes, the individual 15g glucose of taking in.Monitoring of blood glucose in 140 minutes.As shown in FIG. 13A, composition of the present invention has obviously reduced the glucose peak after glucose is taken in.

In second experiment, individuality has been finished dining.Divide having meal one hour in other day after, individually take in 15ml (0.1mg/ml) composition of the present invention or contrast.Take in composition or contrasting monitoring of blood glucose in back 140 minutes.Shown in Figure 13 B, after using composition of the present invention, individual blood glucose levels has returned to baseline quickly.

In the 3rd experiment, individual overnight fasting.At monitoring of blood glucose in 240 minutes of at 6 in the morning.In 8: 11 morning, individual 15ml (0.1mg/ml) composition of the present invention of taking in.Shown in Figure 13 C, composition of the present invention has temporarily reduced the individual down baseline blood glucose levels of fasting state.

Therefore, composition of the present invention can reduce with the blood-glucose peak value after the sucrose challenge, can quicken to make the blood-glucose after the dining to return to baseline, and can at least temporarily reduce the baseline blood glucose levels under the fasting state.

All publication, patents and patent applications that this specification sheets is quoted all are included into this paper as a reference, as specifically and individually indicating each independent open or patent application to be included into this paper as a reference.Although for the clear purpose of understanding, by illustrating and described for example with describing in detail the invention of front, but by instruction of the present invention, those skilled in the art will easily understand, under the situation of the spirit or scope that do not deviate from appended claims, can carry out some change and modification to it.

Claims (34)

1. composition, its comprise induced lipolysis apoptosis treatment significant quantity carry zinc, through serum protein or milk-protein and acceptable carrier, vehicle or the thinner of protease digestion.

2. the composition of claim 1, it is a dietary supplements.

3. the composition of claim 1, it is foodstuff additive or food compositions.

4. the composition of claim 1, it is a pharmaceutical composition.

5. the composition of claim 1, it is a nutraceutical composition.

6. the composition of claim 1, wherein said through protease digestion carry the zinc serum protein or milk-protein is selected from: α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.

7. the composition of claim 1, it comprises two kinds of being selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein and carries zinc serum protein or milk-proteins through protease digestion.

8. the composition of claim 1, it comprises three kinds of being selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein and carries zinc serum protein or milk-proteins through protease digestion.

9. the composition of claim 1 wherein saidly carries zinc serum protein or milk-protein less than 10 kilodaltons through protease digestion.

10. the composition of claim 1 wherein saidly carries zinc serum protein or milk-protein less than 3 kilodaltons through protease digestion.

11. the composition of claim 1, it does not comprise the protein fragments greater than 10 kilodaltons basically.

12. the composition of claim 1, it does not comprise the protein fragments greater than 3 kilodaltons basically.

13. to individual treatment or prevention of obesity or the overweight method that needs arranged, this method comprises step from each composition among the claim 1-12 of significant quantity to described individuality that use.

14. the method for claim 13, wherein said significant quantity are enough to induce the apoptosis of the adipocyte of described individuality.

15. the method for claim 13, wherein said individuality is the people, and described significant quantity is at least about 0.02mg/kg.

16. the method for claim 13, wherein said individuality is the people, and described significant quantity mostly is about 4mg/kg whose body weight most.

17. the method for claim 13, wherein said individuality is the people, and described significant quantity is that about 0.02mg/kg is to about 4mg/kg.

18. the method for claim 13, wherein said individuality is the people, and described significant quantity is about 1mg/ days to about 400mg/ days.

19. reduce weight increase or induce the method for losing weight in the individuality that needs is arranged, this method comprises step from each composition among the claim 1-12 of significant quantity to described individuality that use.

20. the apoptotic method of induced lipolysis, this method comprise the step that each composition contacts among the claim 1-12 that makes adipocyte and significant quantity.

21. regulate the method for EGF-R ELISA, this method comprises the step that each composition contacts among the claim 1-12 that makes EGF-R ELISA and significant quantity.

22. regulate the method for insulin receptor, this method comprises the step that each composition contacts among the claim 1-12 that makes insulin receptor and significant quantity.

23. each method for compositions among the preparation claim 1-12, this method comprises the steps:

Described serum protein or milk-protein are contacted with sequestrant;

B. remove described sequestrant;

Described serum protein or milk-protein are contacted with zinc;

Described serum protein or milk-protein are contacted with described proteolytic enzyme; With

Described serum protein or milk-protein are contacted with described carrier, vehicle or thinner.

24. the method for claim 21, it also comprises with described serum protein or milk-protein and greater than the step of the molecular separation of 10 kilodaltons.

25. the method for claim 21, it also comprises with described serum protein or milk-protein and greater than the step of the molecular separation of 3 kilodaltons.

26. the method for claim 21, wherein said serum protein or milk-protein are in salt brine solution, concentration is 40 Jian.

27. the method for claim 21, wherein said sequestrant are about 25 to about 50mM EDTA or EGTA.

28. the method for claim 21 is wherein by filtering or dialysing and remove described sequestrant.

29. the method for claim 21, wherein said zinc is the form of about 250mM zinc acetate.

30. the method for claim 21, wherein said proteolytic enzyme are papoid.

31. the method for claim 21, wherein said serum protein or milk-protein are selected from α-2-HS-glycoprotein, α-1-acid glycoprotein, α-1-antitrypsin, albumin, Transferrins,iron complexes and α-fetoprotein.

32. to there being the people who needs to treat or prevention of obesity or overweight method, this method comprises step from composition to the people that use, said composition comprise about 0.02mg/kg to about 4mg/kg carry zinc, through the serum protein or the milk-protein of protease digestion, and pharmaceutically acceptable carrier, vehicle or thinner.

33. carry zinc, through the serum protein of protease digestion or the milk-protein purposes in the individual fat or overweight medicine of preparation treatment or prevention.

34. among the claim 1-12 each carry zinc, through the serum protein of protease digestion or the milk-protein purposes in the individual fat or overweight medicine of preparation treatment or prevention.

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