CN101340932A - Carrier for medicaments for obtaining oral bioavailability - Google Patents
Carrier for medicaments for obtaining oral bioavailability Download PDFInfo
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- CN101340932A CN101340932A CNA2005800244861A CN200580024486A CN101340932A CN 101340932 A CN101340932 A CN 101340932A CN A2005800244861 A CNA2005800244861 A CN A2005800244861A CN 200580024486 A CN200580024486 A CN 200580024486A CN 101340932 A CN101340932 A CN 101340932A
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention relates to a protein complex consisting of at least one hemagglutinin from at least one of the Clostridium botulinum types A, B, C, D, E, F or G and a polypeptide Hc conjugate, whereby the polypeptide Hc conjugate is comprised of a selected polypeptide bound to the heavy chain or the N-terminal fragment of botulinum toxin.
Description
The present invention relates to by at least a hemagglutinin and protein complex that polypeptide-Hc-conjugate (conjugate) is formed, wherein hemagglutinin derives from least one type in bacillus botulinus (Clostridium botulinum) A, B, C, D, E, F or the G type, and polypeptide-Hc-conjugate is connected to the heavy chain of botulinum toxin (botulinum toxin) or its amino terminal fragment by the polypeptide chain of selecting and constitutes.
Though multiple pharmaceutical agent all has high activity, because these materials can not be by oral administration, and can only parenteral, i.e. drug administration by injection, their treatment practicality has just greatly been weakened.Especially, the oral administration of protein reagent can't be realized, because these materials can't their action site of by oral route arrival.Can run into two obstacles that are difficult to overcome behind the oral protein: the degeneration environment in gastrointestinal tract causes protein inactivation, a large amount of protease impels the polypeptide degraded, even albumen mass-energy is resisted these situations, polymer substance can not be gone beyond intestinal mucosal barrier and enter blood and arrive action site.Protein is cut by protease in the harmonization of the stomach small intestinal respectively, and cleaved products aminoacid and peptide are absorbed or drain.Thereby if oral administration, pharmaceutical grade protein can not tell on.
People have carried out many effort overcoming these defectives, and protein reagent can be utilized by the oral thing of making a living.Here mention certain methods: have with protease inhibitor cocktail protection reagent and avoid proteoclastic trial.At this on the one hand, adopt protease inhibitor, as pressing down enzyme peptide (aprotinin), bestatin (bestatin), puromycin, soybean trypsin inhibitor, with protein reagent administration simultaneously; Be intended to prevent from degraded and protein reagent can be destroyed to absorb by doing like this.
Simultaneously also the someone has attempted adjusting local pH value in stomach/intestinal by recipe design.Even also relate to protein reagent itself, with by amino acid whose chemical modification being increased amino acid whose stability and improving absorbability.For the latter, people's intention is passed through palmitoylation by for example strengthening lipotropy (lipophily)) realize.For example with insulin and two preferendum oligomer couplings (hexyl of NOBEXCorporation company-insulin list conjugate (monoconjugates)).Another kind of means are to strengthen mucosa permeability, for example, and by granting chelating agen and surfactant respectively, as sodium lauryl sulphate, sodium deoxycholate.This design also comprises grants spissated low molecular vehicle molecule (for example, 4-(4-2-(2-hydroxybenzoyl))-aminophenyl)-butanoic acid simultaneously).Another kind of means attempt using specific transporting mechanism in intestinal wall.In this respect, exist the transporting mechanism that absorbs vitamin B12, people are intended to by protein reagent and vitamin B12 coupling are used for protein reagent with this mechanism.Up to the present, all these different means are not to produce that get the nod, oral biological available pharmaceutical grade protein.
The another kind of oral biological available method of protein that provides has been described in WO 03/101484.Describe according to it, the C-terminal residue of botulinum toxin heavy chain connects with polypeptide.The C-terminal residue it is said and can mediate the transportation of passing epithelial membrane.For oral this hybrid protein, this hybrid protein can be mixed with the auxilin (auxiliary proteins) that centers on botulinum toxin natively.
In addition, the description of WO 02/05844 provides available protein of oral biology and low-molecule drug, they are integrated in the complex, this complex be in the botulinum toxin complex of bacillus botulinus at least a hemagglutinin with may be nontoxic, the complex of (non-hemagglutinating) protein (NTNH) of non-hemagglutination.But verified, the polypeptide of integration molecular weight＜50kDa, its effectiveness still is not enough to satisfy profitable commercial requirement.
Thereby the problem to be solved in the present invention just provides a kind of available method of the oral biology of polypeptide that makes.
This problem is solved by the subject content that is defined in this patent claims.
The following drawings graphic extension the present invention.
Fig. 1 is the result's of rat glucose tolerance test a sketch map.Oral insulin-Hc-the conjugate given with (gavage) 2U of 4 rats is arranged, and have 4 rats (animals) to give the insulin-Hc-conjugate that is incorporated into (insulin complex substance) in the complex with 2U.Glucose with 2g/kg after 60 minutes stimulates (challenge) these rats.Other has 3 groups of lumbar injection 0.1U, 0.6U, 2U insulin, also gives and the 2g/kg glucose simultaneously.Measured glucose level every about 30 minutes.
The sketch map of Fig. 2 shows, insulin-Hc-conjugate, is incorporated into the effect comparative result of the insulin of insulin-Hc-conjugate in the complex (insulin complex substance) and abdominal cavity using.The term harmonization that this experiment condition and Fig. 1 adopt.The result shows is area (AUC) under the glucose concentration curve.
In this application, term " protein complex " is meant such carrier, and the polypeptide of other selection can be transported in the blood system of humans and animals by this carrier.Described protein complex is made up of at least a hemagglutinin and the possibility protein nontoxic, non-hemagglutination (NTNH) of clostridium botulinum toxin complex, and this botulinum toxin complex derives from least a A, B, C, D, E, F or G type bacillus botulinus.Described hemagglutinin and NTNH refer to be present in exist in the clostridium (Clostridia), form the protein of complex with botulinum toxin natively.Yet, for clarity, it is to be noted that described protein complex does not comprise botulinum toxin.
In this application, term " botulinum toxin complex " is meant the native protein aggregation of A, B, C, D, E, F or G type from bacillus botulinus, comprises botulinum toxin, hemagglutinin and protein nontoxic, non-hemagglutination (NTNH).
In this application, term " polypeptide " or " polypeptide of selecting " are meant the peptide of being made up of at least 2 aminoacid.Described polypeptide can be linear, cyclic or branched.In addition, described polypeptide can be made up of the amino acid chain that surpasses, and wherein these chains can be connected to each other, and for example connect by disulfide bond.And described polypeptide can comprise the aminoacid of modification and the modification after the common translation, as glycosylation.Described polypeptide can be the polypeptide that has pharmacology or immunocompetent polypeptide or be used for diagnostic purpose, for example antibody.
In this application, term " carrier " is meant the amino terminal fragment of the complete heavy chain of botulinum toxin (Hc) or its heavy chain, and this botulinum toxin is selected from A, B, C, D, E, F or G BOTULINUM TOXIN TYPE A A complex.
In this application, term " polypeptide-Hc-conjugate ", " Hc-conjugate " or " conjugate " are meant the carrier that is connected to the polypeptide of selection with covalent bond.For example, insulin-Hc-conjugate is meant by insulin and is connected to heavy chain of botulinum toxin or the molecule that its N-terminal fragment is formed.
In this application, term " new complex (neocomplex) " is meant the complex of the protein complex that is integrated with the Hc-conjugate therein.
Bacillus botulinus (Clostridium botulinum) has developed effective mechanism and transport the protein oral route and enter organism, and wherein said protein is absorbed by target cell subsequently.Described protein refers to the most malicious known up to now material: clostridium botulinum toxin (Clostridium botulinumtoxin) claims botulinum toxin (botulinum toxin) hereinafter again.Under field conditions (factors), botulinum toxin and many other being present in the botulinum toxin complex by the bacillus botulinus expressed protein.If the botulinum toxin complex is granted by oral, this macromolecule neurotoxin of botulinum toxin will be absorbed in intestinal so, and the target cell of arriving soon after, the i.e. motor neuron of motor end plate.At its action site, this neurotoxin stops acetylcholine to discharge, thereby causes the paralysis of related muscles.
Bacillus botulinus is divided into 7 sero-group: A, B, C, D, E, F, G type according to their toxin.Described toxin refers to the protein that molecular weight is about 150,000 dalton (Da).Usually botulinum toxin complex and contaminated food are together taken in, and through intestinal absorption, arrive its action site, i.e. motor end plate.
Botulinum toxin is made up of two subunits.Each subunit is fulfiled function separately: heavy chain (molecular weight 100kDa) is attached to neurocyte high special, makes light chain can shift the Cytoplasm that (translocation) enters cell.Heavy chain in natural botulinum toxin (Hc) is received light chain (Lc) by disulfide-bridged, two.Light chain serves as protease, cuts those protein of being responsible for the fusion of excretion vesicles and neuron membrane (snare protein matter).Thereby cause excretion vesicles can not discharge acetylcholine: the activation of muscle is blocked.
Article two, chain all protease cut original synthetic polypeptide and produce.In the clostridium of some types, cutting takes place by the protease (A, C type, part Type B) of clostridium self; And in other types, cutting then only occurs over just in receptor's the intestinal in (trypsin) or the tissue.Exist and the heavy chain of do not mix any light chain or complete toxin is a totally nontoxic with unpack format; Itself can not the block nerves cell in the release of acetylcholine.
Also synthetic many other protein of clostridium, they form complex (botulinum toxin complex) with botulinum toxin, and it is stable in sour environment also to prevent this neurotoxin degeneration and by proteasome degradation, this external enwergy is ingested by intestinal mucosa.
Described other protein is called complex proteins matter hereinafter, refer to some hemagglutinins and have about 120, the protein nontoxic, non-hemagglutination (NTNH) of the molecular weight of 000Da.These other protein form protein complex.In A type clostridium botulinum toxin complex, following hemagglutinin has been described: have about 16, the Ha2 of 900Da molecular weight, have approximately 21, the Ha3a of 000Da molecular weight has about 52, the Ha3b of 000Da molecular weight and have about 35, the Hal of 000Da molecular weight.
B to G BOTULINUM TOXIN TYPE A A complex with similar formal construction.With the botulinum toxin type B complex is example.In this type, except that NTNH, also described about 70, the HA-70 of 000Da, about 17, the Ha-17 of 000Da and about 33,000Da Ha-33 (referring to Bhandari, M.et al. (1997) Current Microbiology 35, pp.207-214).
In addition, East, ((1994) System Appl.Microbiol.17 pp.306-312) has described the Type B Ha-33 sequence of making comparisons with A type and C type sequence to A.K.et al..C type and D type have equally also been described, except that Ha 33 (=Hal), similar with the A type, also have about 33, the Ha3b of 000Da, or about 53, the Ha3b of 000Da, about 22-24, the Ha3a of 000Da and about 17, the Ha2 of 000Da is (referring to Inoue, K.et al. (1999) Microbiology 145, pp.2533-2542).
Surprisingly, the inventor finds that the heavy chain of botulinum toxin is only arranged in rebuilding (reconstitution) experiment, and promptly meaning does not have light chain, by individually or to be incorporated in the protein complex quantitatively with the link coupled form of polypeptide.
Polypeptide can be coupled to the heavy chain of clostridium botulinum toxin with chemical method, and is incorporated in the protein complex of being made up of at least a complex proteins matter.There is the number of chemical method can be used for protein is coupled to described heavy chain.Many bifunctional reagents that two different proteins are connected are arranged.Preferably use the reagent that forms disulphide bridges with the cysteine of coupling object (coupling partner).Subsequently, carry out binding with carrier, heavy chain in the step of back.Be suitable for described bonded reagent such as SPDP (N-succinyl-3-[2-dithio pyridine-] propionate) (N-succinimidy-3-[2-pyridyldithio]-propionate) or DTDP (4,4 '-dithiodipyridine) (4,4 '-dithiodipyridine), it forms disulphide bridges and does not have sept (spacer) between protein.Described coupling has following advantage, promptly can be cut under reducing condition in vivo, for example is cut by thioredoxin system in Cytoplasm.In this case, select such coupling, make heavy chain be integrated into protein complex and disturb, and the biological activity of polypeptide is kept.Perhaps, can realize the coupling of heavy chain and polypeptide by in suitable expression system that two kinds of peptides are synthetic as recombination fusion protein.
The described polypeptide that is connected in carrier preferably has pharmacology or immunocompetent polypeptide, and its utilization is Orally administered according to protein complex of the present invention, and it may have treatment or prophylactic activity.The polypeptide of described selection can be for example hormone, cytokine, enzyme, somatomedin, antigen, antibody, inhibitor, receptor stimulating agent or receptor antagonist or thrombin (coagulation factors).In this, polypeptide be recombinant production or or isolating from their natural origin be not conclusive.Preferred polypeptide is an insulin, erythropoietin, interferon, interleukin, the HIV-protease inhibitor, GM-CSF (granulocyte/macrophage stimulation factor), NGF (nerve growth factor), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), fiber proenzyme activator, TPA (tissue plasminogen activator) for example, renin inhibitor, human growth factor, IGF (insulin like growth factor), vaccine such as tetanus vaccine, hepatitis B vaccine, diphtheria vaccine, antibody is herceptin (anti-Her2 antibody) for example, anti-TNF (tumor necrosis factor) antibody, anti-EGF receptor antibody, VEGF antibody, anti-IgE antibodies, anti-CD11a antibody, calcitonin, urokinase, streptokinase, angiogenesis inhibitor, Factor IX, factor Xa antagonist, inhibitors of metalloproteinase.
As the polypeptide of diagnostic purpose can be for example antibody or part, and wherein said polypeptide can have label.For label, the label that can be detected in human or animal body can be considered arbitrarily.Preferred label is an isotope, for example C
13, or radioactive marker.The antibody of labelling can be used for the detection of tumor; The part of labelling can be used for for example detection of pathologic (pathological) receptor.
The carrier that is connected in polypeptide is integrated in the described protein complex.Protein complex is by at least a hemagglutinin, and needs, and at least a NTNH forms.In this, described hemagglutinin and NTNH are selected from the botulinum toxin complex of naturally occurring A, B, C, D, E, F or G type bacillus botulinus.Yet described protein complex can contain the composition that is different from its natural composition, for example can only be made up of hemagglutinin and does not have NTNH protein.In addition, described protein complex can be made up of the hemagglutinin kind of lacking than naturally occurring botulinum toxin complex, preferably form by three kinds of different hemagglutinin kinds, preferably form by two kinds, especially preferably be made up of a kind of hemagglutinin kind, wherein protein complex can contain or not contain NTNH protein in all cases.And described protein complex can be made up of the hemagglutinin mixture of the one or more kinds of different serotypes and/or the NTNH protein of different serotypes.
Preferred protein complex is consistent with the naturally occurring protein complex that comes from bacillus botulinus A, B, C, D, E, F or G type (not containing botulinum toxin), for example has the protein complex of Ha1, the Ha2, Ha3a, Ha3b and the NTNH that come from the bacillus botulinus Type B.And, described protein complex can be made up of Ha1, Ha2, Ha3a and NTNH, form by Ha1, Ha2, Ha3b and NTNH, also can form by Ha1, Ha3a, Ha3b and NTNH, can form by Ha2, Ha3a, Ha3b and NTNH in addition, form by Ha1, Ha2 and NTNH, form by Ha1, Ha3a and NTNH, form by Ha1, Ha3b and NTNH, form by Ha2, Ha3a and NTNH, form by Ha2, Ha3b and NTNH, form, perhaps further form by the combination in any of listed complex proteins matter by Ha3a, Ha3b and NTNH.In addition, described protein complex can be made up of a kind of and NTNH in the hemagglutinin; Described in addition protein complex can be made up of the combination of listed hemagglutinin and not contain NTNH.According to illustrative Type B protein complex, further preferred A, C, D, E, F or the hemagglutinin of G type and/or the protein complex of NTNH.
The method of producing protein complex of the present invention that provides also is provided another aspect of the present invention, this method comprises the following steps: a) to separate respectively at least a A from bacillus botulinus in 2.0 to about 6.5 pH value scope, B, C, D, E, the botulinum toxin complex of F or G type, b) improve pH value in the scope of about 7.0-about 10.0, c) remove each clostridium botulinum toxin in the complex proteins matter by chromatography, d) the complex proteins matter that obtains in step c) is mixed with the polypeptide-Hc-conjugate of selection, perhaps e) is separated in the complex proteins matter that obtains in the step c) and at least a complex proteins matter mixed with polypeptide-Hc-conjugate, f) with step d) or e) mixture be about 6.5-about 2.0 to pH value, it is about 6.0 to be preferably about 4.0-, the buffer dialysis of preferred especially pH value 6.0.
Complex proteins matter can be separated from natural botulinum toxin complex.Separation method exemplifies as follows: at first, separating the botulinum toxin complex from clostridium under acid ph value, in about 2.0 to about 6.5 pH value scope, in about 4.0 to about 6.5 pH value scope, is 6.0 at pH value especially preferably preferably.PH value brings up to about 7.0 to about 10.0, preferably bring up to about 7.0 to about 8.0 after, remove botulinum toxin by the chromatography of using always in the protein chemistry method.Described method is feasible, because this complex o'clock is stable in pH value＜6.5, and under neutral and alkali condition, disintegrates (disintegrate) respectively, and the release toxin.The complex proteins matter that does not then contain toxin can be mixed with polypeptide-Hc-conjugate, can be by commonly used to buffer in the protein chemistry method, especially preferably the method to phosphate, acetate or citrate buffer dialysis is reduced to about 2.0 in about 6.5 scope with pH value, preferably be reduced to about 4.0 in about 6.0 scopes, especially preferably being reduced to pH value is 6.0.Contain the protein complex of polypeptide-Hc-conjugate thereby form, and provide oral bioavailability for selected polypeptide thus.
Other chromatography, concentration method and sedimentation method that are commonly used in the protein chemistry method also can be used for separating of complex proteins matter.
Because its DNA sequence is known, described complex proteins matter also can be carried out recombinant production by the DNA recombinant technique in the specific host biology.So the complex proteins matter of producing can also show modification, this means that they can be the derivants of complex proteins matter.In this, modification is not only to mean disappearance, interpolation, insertion or replacement, also means amino acid whose chemical modification, for example methylate or acetylation and the translation after modification, as glycosylation or phosphorylation.At different host expresses desired proteins is well-known to those skilled in the art, need not set forth respectively at this.In this, the required complex proteins matter of described protein complex can be distinguished in host living beings or express simultaneously.Preferably, described reorganization complex proteins matter is in antibacterial, for example produce in escherichia coli (E.coli), bacillus subtilis (Bacillus subtilis) or the clostridium difficile (Clostridium difficile), perhaps in eukaryotic cell, for example at Chinese hamster ovary celI, in insect cell, as use baculovirus (Baculovirus) system, perhaps in yeast, produce.According to above-mentioned method, described complex proteins matter can be separated and be mixed with the polypeptide conjugate of selecting.In addition, polypeptide-Hc-conjugate and the described complex proteins matter selected can be expressed simultaneously as fusion rotein in host living beings.Particularly preferably being described each complex proteins matter produces by YAC in yeast at the same time or separately with polypeptide-Hc-conjugate of selecting.
In addition, can form by the complex proteins matter of recombinant production with from the mixture of the isolating complex proteins matter of natural meat poison toxin complex according to protein complex of the present invention.
Be to illustrate by the following examples, should not be construed as restrictive of the present invention.
Embodiment 1: the heavy chain that separates A type clostridium botulinum toxin
Cultivate A type clostridium botulinum toxin (strains A TCC 3502) according to the method for publishing, and carry out following variation (referring to Das Gupta; Sathyamoorthy, 1984, Toxicon 22, pp.415-424).Behind the growth 72h, add 3N sulphuric acid contratoxin and precipitate.Extract precipitate and remove nucleic acid, then use the ammonium sulfate precipitation toxin.After dissolving and dialysis, carry out DEAE-sepharose ion-exchange chromatography (2.6x15cm) at pH6.0.With the toxin of 150mM NaCl elution of bound, it is further carried out ion-exchange chromatography to 50mM Tris/HCl dialysis and use sepharose Q post (2.6x10.0cm).With this neurotoxin of NaCl gradient (0-300mM NaCl) eluting.Merge the fraction that contains this neurotoxin, to 10mM sodium ascorbyl phosphate (NaPhosphate), the pH7.0 dialysis.With sepharose S-post (1.6x11cm) on the dialysis solution, with NaCl gradient (0-300mMNaCl) eluting.Obtain the high-purity neurotoxin through ion-exchange chromatography.
(referring to Kozaki et al.1981, J.Med.Sci.Biol.34 pp.61-68) separates neurotoxin heavy chain, and carries out following variation according to publishing document.In this, to borate/phosphate buffer, pH 8.5 dialysis are attached on the post (1x5cm) of having filled QAE-Sephadex then with highly purified A type neurotoxin.Contain the borate of 10mM DTE/phosphate buffer washing with other, then 3ml borate/phosphate buffer and the chromatographic column that contains 150mM DTE and 2M carbamide with other is incubated overnight.Use borate/phosphate buffer+10mM DTE+2N carbamide eluting light chain subsequently, then with 200mM NaCl eluting and the same blended heavy chain of buffer.In order to remove the remaining incomplete active neurotoxin of removing, the level wheel cylinder that merges is crossed affinity column four times.Coupling in this affinity chromatograph column filling sepharose of antibody of anti-A type clostridium botulinum toxin light chain.After described chromatography, can not mixed the heavy chain that any light chain or its natural toxin are arranged.Even under high concentration, activity test does not show its activity (the mice diaphragm is measured (diaphragm assay)) yet.
Embodiment 2: the complex proteins matter of preparation Type B bacillus botulinus
The complex proteins matter of Type B bacillus botulinus is (referring to Evans et al., 1986 according to the method for publishing; European Journal of Bioch.154, pp.409-416) isolating with Type B bacillus botulinus (Okra strain) fermentation back, some steps have been done change in the said method:
As embodiment 1, after the biomass of extracting Acid precipitation, from extract, be settled out nucleic acid, from supernatant, be settled out toxic botulinum toxin complex with ammonium sulfate.Precipitate is resuspended in 0.05M sodium citrate+1mM EDTA of pH 5.5, and the DEAE-sephadex purification by chromatography is then used in dialysis, and wherein polymer composite is not incorporated into chromatographic column (5x12cm) and flows through chromatographic column quantitatively.The eluate that will contain complex is to 50mM Tris/HCl, and 1mM EDTA is after the pH7.9 dialysis, carry out chromatography with sepharose Q-chromatographic column, wherein complex proteins matter is not incorporated into chromatographic column, and neurotoxin keeps being incorporated into chromatographic column, and only by the salt gradient eluting.Go up at Q Hyper D post (2.6x13cm) described complex proteins matter is carried out further purification, wherein chromatographic column is with identical buffer (50mM Tris/HCl, 1mM EDTA) balance.Do not contain the protein complex of toxin with sodium chloride gradient (0-300mM NaCl) eluting.
Remove last micro-neurotoxin with affinity chromatography.For this purpose, the solution pump that will contain complex is crossed post four times, wherein chromatographic column contain with the link coupled sepharose of anti-Type B bacillus venenosus neurotoxin antibody (IgG fraction) as affinity substrate.Then, in activity test (mice hemidiaphragm mensuration), no longer record its biological activity (toxicity).
Embodiment 3: the protein that the heavy chain of A type clostridium botulinum toxin is integrated into the Type B bacillus botulinus
Complex
100 μ g (34 μ l) embodiment 2 from the highly purified complex proteins matter of Type B and mixing of 200 μ g (690 μ l) embodiment 1 from isolating A type heavy chain.To containing the 1150mM sodium phosphate of 150mMNaCl, the pH6.0 salt buffer was 2-8 ℃ of dialysis 3 days with mixture.Then, get 450 μ l, add 150 μ l 4M ammonium sulfate and precipitate.Under such deposition condition, the heavy chain that is not integrated into protein complex persists in the solution, and protein complex (integrating or be not integrated with heavy chain) is precipitated out quantitatively.After the overnight incubation, centrifugation is resuspended in 120 μ l sodium ascorbyl phosphates, mm NaCl, 2mM EDTA, pH6.0 then again.Carry out the formation that " gel filtration " detects protein complex by BioSep S-3000.The chromatography collection of illustrative plates only shows single peak (is about 500,000 dalton places at molecular weight).Sds polyacrylamide gel electrophoresis shows that the peak fraction contains the heavy chain of Type B complex proteins matter and botulinum toxin type A simultaneously.
Embodiment 4: the heavy chain that insulin is attached to botulinum toxin type A
Synthetic SPDP-insulin
With the insulin of 12.5mg (Roche, reorganization) be dissolved in 6.3ml buffer (the 10mM sodium carbonate, pH=6.9).For this purpose, inject 3 μ l citaconanhydride (Fluka) with the sealing alpha-amido, described mixture is at room temperature hatched, and observe its pH value with pipettor.Keep pH value at 6.8-7.0 by adding 1M NaOH.Then, with reactant mixture to the 50mM sodium phosphate, 100mM sodium chloride, pH=7.3 is 4 ℃ of following dialysed overnight.
With SPDP (N-succinyl-3-[2-dithio pyridine-] propionate of 6.2mg, Pierce) be dissolved in the DMF of 500 μ l.The described solution of 274 μ l is added the insulin derivates (regulating pH=8.3) of 6.3ml, on blender, hatch this mixture 1 under the room temperature
1/
2H.To the 50mM sodium phosphate, 100mMNaCl, still residual SPDP is removed in the pH=7.3 dialysis.In order to remove blocking group, to water dialysis (2h at room temperature), more at room temperature to 10mM HCl dialysis 5
1/
2H.At last, to the 50mM sodium phosphate, 100mM sodium chloride, 4mM EDTA, the pH=7.3 dialysed overnight (final volume of SPDP solution: 7ml).
Produce insulin-Hc-conjugate
With 25mg according to embodiment 1 isolating heavy chain and 8mg insulin-SPDP (final volume: 41.5mg) mix, hatch 4 days (convertible blender (end-over-endmixer)) at 4 ℃ from botulinum toxin type A.In order to remove not link coupled insulin-SPDP, to 50mM Tris/HCl, 250mMNaCl, 1mM EDTA dialyses, and the flexible pipe of wherein dialysing (hose) exclusion limit is 50kD.
With this product of Western engram analysis.Identified～protein of 100kD molecular weight with anti-insulin antibody.Thereby it represents the conjugate (=insulin-Hc-conjugate) of heavy chain and insulin.Arrived the activity of insulin in vitro detection with the 3T3 cell.
Embodiment 5: insulin-Hc-conjugate is incorporated in the protein complex of Type B bacillus botulinus
Insulin-Hc-conjugate (embodiment's 4) of 7.4mg is mixed in 52ml with the protein complex (purified according to embodiment 2) of 21mg, under 4 ℃ to the 50mM sodium phosphate, 250mM NaCl, 1mM EDTA, pH6.0 dialysis 4 days.Use BioSep S-3000 chromatographic column (Phenomenex) to analyze the sample of 500 μ l by gel filtration.Only～600kD molecular weight place shows a macromolecule peak.By Western engram analysis peak fraction sample.The use anti-insulin antibody shows that insulin (as insulin-Hc-conjugate) has been integrated into protein complex (the complex proteins matter by bacillus botulinus is formed).
Embodiment 6: use glucose tolerance test new complex of test insulin in rat
In rat, carry out zoopery, detect the effectiveness of the insulin-Hc-conjugate (being called the new complex of insulin hereinafter) be integrated into protein complex after oral.
The group of 4 rats is given with the new complex of insulin or to being handled with free insulin-Hc-conjugate (not having complex proteins matter).Undressed animal in contrast.The insulin dose of each group is identical, all is the every animal of 2U/.Solution with gavage give with.As other contrast, give and 0.1U the insulin of 0.6U and 2.0U by lumbar injection.1h behind the new complex of administration of insulin and free insulin-Hc-conjugate stimulates with glucose (2g/kg is oral).At this time point, positive control is also injected (0.1U, the insulin of 0.6U and 2.0 units).30,60,120 and 180min after take a blood sample, measure glucose level.Behind the 60min, in control animal, reached the maximum concentration of glucose of about 140mg/ml, and in the animal groups of having handled with the new complex of insulin, concentration has only increased seldom (referring to Fig. 1).(area under curve, AUC), the effect of the new complex of insulin and the insulin of intraperitoneal injection are (Fig. 2) that can compare if the progress of attention concentration of glucose changes.
In the animal of only handling with insulin-Hc-conjugate, concentration of glucose is the same with the matched group performance: concentration is strong to raise up to the time point of 60min.Can infer that thus independent insulin-Hc-conjugate is to not influence of insulin level; Thereby, use the insulin-Hc-conjugate that is not integrated into protein complex and be unsuitable for making the oral insulin biology to utilize.
Embodiment 7: compare the integrate body of insulin-Hc-conjugate and be connected in botulinum toxin heavy chain C end
The insulin of end
C-terminal fragment (the M of recombinant expressed A type clostridium botulinum toxin heavy chain in escherichia coli
r≈ 50kD; Claim the H1 fragment hereinafter again).For this purpose, the DNA sequence of C-terminal fragment aminoacid 871-1296 (NIINT......ERPL) that is connected in the botulinum toxin type A of His label is cloned among the carrier pBN29 4772; With this carrier transformed into escherichia coli N15[pREP4] (Quiagen), use the Ni-NTA-sepharose post to express fragment with the affinity chromatography purification.
With mixing of 100 μ g from the complex proteins matter that does not contain toxin of Type B bacillus botulinus and the recombinant C of 200 μ g (340 μ l)-terminal fragment, 2-8 ℃ to the 50mM sodium ascorbyl phosphate, 150mMNaCl, pH6.0 dialysis 3 days.Then, use H
2O postreaction thing adds the 4M ammonium sulfate precipitation of 150 μ l to 450 μ l.Under such deposition condition, the C-terminal fragment that is not integrated into protein complex persists in the solution, and protein complex (integrating or be not integrated with the C-terminal fragment) quantitatively precipitates.After being incubated overnight, centrifugation is resuspended in the 50mM sodium ascorbyl phosphate of 150 μ l, 150mM NaCl, 2mM EDTA, pH6.0 then again.Get 100 μ l and cross the non-binding C-terminal fragment that Biosep S-3000 solvent resistant column is separated into described complex and may exists as pollutant.Eluting goes out 2 peaks: first peak (12.2min) represents described protein complex.Free C-terminal fragment-fragment is represented at a very little peak.Check first peak with SDS-PAGE; Only there is described complex proteins matter; Thereby when ammonium sulfate precipitation, the C-terminal fragment is not integrated into described complex but remains in the solution with uncombined state.In a word, the C-terminal fragment of botulinum toxin can not be incorporated in the described complex.
Embodiment 8: the biological activity that compares insulin-H1-conjugate and insulin-Hc-conjugate
Produce the C-terminal fragment (H1 fragment) of botulinum toxin type A heavy chain according to embodiment 7 described methods.Behind derivatization, 13mg H1 fragment is mixed with 8mg insulin-SPDP.Method with similar embodiment 4 is produced insulin-SPDP.After 24 hours, to 50mM Tris/HCl, 250mM NaCl, 1mM EDTA dialyse and remove non-link coupled insulin-SPDP 4 ℃ of insulations.
Coexisting, it is the same to integrate heavy chain (embodiment 5) in the complex, at 4 ℃ to the 50mM sodium ascorbyl phosphate, 250mM NaCl, 1mM EDTA, pH6.0 dialysis 3.8mg (H1-insulin-conjugate) and 21mg do not contain the protein complex (according to embodiment 2 purification gained) 5 days of toxin.Utilize zoopery to check these mixture.
The group that 4 rats are formed with insulin-Hc-conjugate (embodiment 5), or with insulin-H1-conjugate+complex proteins matter, or is handled with saline solution.Preceding two groups dosage is every animal 2U insulin.1h after the administration stimulates (0.5g/kg lumbar injection) with glucose.Measurement of glucose levels behind the 30min shows that the animal groups blood sugar level of accepting the salt processing is 170 ± 12mg/dl, and only rises to 115 ± 14mg/dl to the animal groups with the new complex of HC-conjugate.Initial value at the time point of glucose injection is 85 ± 14mg/dl.At last, have the not any influence of demonstration in zoopery of H1-insulin-conjugate of complex proteins matter.
Embodiment 9: use the insulin-conjugate with the terminal heavy chain that shortens of C-
C-is terminal to shorten 30 amino acid whose chains in order to obtain, and prepares chromosomal DNA from the culture of A type bacillus botulinus (ATCC 3502).By pcr amplification, with the coding light chain gene fragment clone (among the pQE-BoNT (A)-L), wherein this fragment contains the thrombin cleavage site in addition at annular section to plasmid pQE60.Generate the terminal genetic fragment that shortens 30 amino acid whose chains of coding C-by pcr amplification from chromosomal DNA.With described gene fragment clone BoNT (A)-L encoding gene in expression plasmid pQE-BoNT (A)-L segmental 3 ' terminal (pQE-BoNT (A) A)-L H30min).Express strain M15[pREP4 with described plasmid transformation escherichia coli] (Qiagen).Induce with 500 μ M IPTG (25 ℃ are spent the night), then cell lysis is used the Ni-NTA-sepharose column chromatography.From the shortening of such acquisition 30 amino acid whose toxin, as separation heavy chain H30min as described in the embodiment 1.With heavy chain H30min similar to Example 4ly with the insulin coupling, and in the complex that does not contain toxin that is incorporated into from the Type B bacillus botulinus similar to Example 5.
With glucose tolerance test test synthetic new complex like this, 4 animals are used in test, with compare (as described in the embodiment 8) that does not have complex.Give when being equivalent to the dosage of 2U insulin, blood sugar level rises to 110 ± 18mg/dl behind the 30min, and (initial value is 90 ± 5mg/dl) and rise to 168 ± 14mg/dl in the control animal.
Claims (12)
1. protein complex, be made up of at least a hemagglutinin and the polypeptide-Hc-conjugate that derives from least one type in bacillus botulinus A, B, C, D, E, F or the G type, wherein polypeptide-Hc-conjugate is connected to the heavy chain of botulinum toxin or its N-terminal fragment by the polypeptide chain of selecting and constitutes.
2. according to the protein complex of claim 1, wherein hemagglutinin is the mixture that derives from the hemagglutinin of at least one type in A, B, C, D, E, F or the G type bacillus botulinus.
3. according to the protein complex of aforementioned each claim, wherein protein complex also contains protein nontoxic, non-hemagglutination.
4. according to the protein complex of aforementioned each claim, wherein heavy chain separates a kind of in A, B, C, D, E, F or G BOTULINUM TOXIN TYPE A A complex.
5. according to the protein complex of aforementioned each claim, wherein heavy chain is recombinant expressed in suitable expression system.
6. according to the protein complex of aforementioned each claim, wherein polypeptide is hormone, cytokine, somatomedin, antigen, antibody, inhibitor, receptor stimulating agent or receptor antagonist or thrombin.
7. according to the protein complex of aforementioned each claim, wherein polypeptide of Xuan Zeing and heavy chain or its N-terminal fragment are to recombinate in the mode of fusion rotein to produce.
8. according to each described protein complex of claim 1-6, wherein the polypeptide of Xuan Zeing is connected by chemical bond with heavy chain or its N-terminal fragment.
9. protein complex according to Claim 8, wherein chemical bond is a disulfide bond.
10. protein complex according to Claim 8, wherein chemical bond is a peptide bond.
11. produce the method for the protein complex of aforementioned each claim, the method comprising the steps of:
A) in pH value is the scope of 2.0-6.5, separate the botulinum toxin complex from bacillus botulinus;
B) set-up procedure a) in the pH value of isolating botulinum toxin complex to the scope of 7.0-10.0;
C) from complex proteins matter, remove botulinum toxin by chromatography;
D) the complex proteins matter that obtains in the step c) is mixed with polypeptide-Hc-conjugate; Perhaps
E) be separated in the complex proteins matter that obtains in the step c), and at least a complex proteins matter is mixed with polypeptide-Hc-conjugate;
F) to pH value be the buffer dialysis step d) or the e of 6.5-2.0 scope) mixture.
12. according to the purposes of each described protein complex of claim 1-10 as transport agent, its be used to transport have pharmacological activity, immunocompetent polypeptide, or as the polypeptide of purpose of diagnosis.
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| DE102004035606.8 | 2004-07-22 | ||
| DE102004035606A DE102004035606A1 (en) | 2004-07-22 | 2004-07-22 | Carrier for drugs for obtaining oral bioavailability |
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| EP (1) | EP1768701A2 (en) |
| JP (1) | JP2008506724A (en) |
| KR (1) | KR20070047786A (en) |
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| AU (1) | AU2005266739A1 (en) |
| BR (1) | BRPI0513715A (en) |
| CA (1) | CA2574124A1 (en) |
| DE (1) | DE102004035606A1 (en) |
| IL (1) | IL180229A0 (en) |
| MX (1) | MX2007000421A (en) |
| NO (1) | NO20070775L (en) |
| RU (1) | RU2006147284A (en) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108137654A (en) * | 2015-05-15 | 2018-06-08 | 内布拉斯加大学董事会 | Suitable for molecule to be deliverrf into the engineered clostridium botulinum toxin of selected cell |
| WO2024109534A1 (en) * | 2022-11-24 | 2024-05-30 | 武汉禾元生物科技股份有限公司 | Human serum albumin-insulin conjugate and preparation method therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008169166A (en) * | 2007-01-14 | 2008-07-24 | Tokyo Univ Of Agriculture & Technology | Sugar-binding polypeptide, composite material and drug delivery system |
| JP2009132686A (en) * | 2007-10-26 | 2009-06-18 | Okayama Univ | Polypeptide derived from botulinum toxin and method for detecting botulinum toxin |
| WO2009131435A1 (en) * | 2008-04-23 | 2009-10-29 | Erasmus University Medical Center Rotterdam | Linker containing bungarotoxin and a binding peptide |
| US20130085267A1 (en) * | 2009-12-18 | 2013-04-04 | Allergan, Inc. | Stabilization of Therapeutic Agents to Facilitate Administration |
| US9119274B2 (en) | 2011-07-15 | 2015-08-25 | Nxp B.V. | Resonant converter control |
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| GB8914984D0 (en) * | 1989-06-29 | 1989-08-23 | Animal Health Inst | Nucleotide sequences |
| EP0866718A1 (en) * | 1995-12-13 | 1998-09-30 | President And Fellows Of Harvard College | Use of toxin peptides and/or affinity handles for the delivering compounds into cells |
| GB9617671D0 (en) * | 1996-08-23 | 1996-10-02 | Microbiological Res Authority | Recombinant toxin fragments |
| GB9824282D0 (en) * | 1998-11-05 | 1998-12-30 | Microbiological Research Agenc | Delivery of superoxide dismutase to neuronal cells |
| DE19856052A1 (en) * | 1998-12-04 | 2000-06-08 | Deutsches Krebsforsch | Conjugate for enrichment in neuronal cells |
| US6776990B2 (en) * | 1999-04-08 | 2004-08-17 | Allergan, Inc. | Methods and compositions for the treatment of pancreatitis |
| US6641820B1 (en) * | 2000-01-19 | 2003-11-04 | Allergan, Inc. | Clostridial toxin derivatives and methods to treat pain |
| US6500436B2 (en) * | 2000-01-19 | 2002-12-31 | Allergan, Inc. | Clostridial toxin derivatives and methods for treating pain |
| US6670322B2 (en) * | 2000-06-01 | 2003-12-30 | Wisconsin Alumni Research Foundation | Method of targeting pharmaceuticals to motor neurons |
| DE10035156A1 (en) * | 2000-07-19 | 2002-02-07 | Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh | New protein complex containing complex protein from botulinum toxin, useful for oral delivery of therapeutic polypeptide or low molecular weight pharmaceutical |
| US20040220100A1 (en) * | 2000-07-21 | 2004-11-04 | Essentia Biosystems, Inc. | Multi-component biological transport systems |
| WO2003061694A1 (en) * | 2001-05-10 | 2003-07-31 | Seattle Genetics, Inc. | Immunosuppression of the humoral immune response by anti-cd20 antibodies |
| GB0112687D0 (en) * | 2001-05-24 | 2001-07-18 | Microbiological Res Authority | Pharmaceutical use of secreted bacterial effector proteins |
| JP2005538954A (en) * | 2002-05-31 | 2005-12-22 | トーマス・ジェファーソン・ユニバーシティ | Compositions and methods for transepithelial transport of molecules |
| JP2007070225A (en) * | 2003-07-25 | 2007-03-22 | Yukako Fujinaga | Pharmaceutical formulation containing component derived from bacterium of clostridium |
| US20050129677A1 (en) * | 2003-12-10 | 2005-06-16 | Shengwen Li | Lipid rafts and clostridial toxins |
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2004
- 2004-07-22 DE DE102004035606A patent/DE102004035606A1/en not_active Withdrawn
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- 2005-07-22 CA CA002574124A patent/CA2574124A1/en not_active Abandoned
- 2005-07-22 JP JP2007521791A patent/JP2008506724A/en active Pending
- 2005-07-22 KR KR1020077004107A patent/KR20070047786A/en not_active Application Discontinuation
- 2005-07-22 CN CNA2005800244861A patent/CN101340932A/en active Pending
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- 2005-07-22 WO PCT/DE2005/001290 patent/WO2006010360A2/en active Application Filing
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108137654A (en) * | 2015-05-15 | 2018-06-08 | 内布拉斯加大学董事会 | Suitable for molecule to be deliverrf into the engineered clostridium botulinum toxin of selected cell |
| WO2024109534A1 (en) * | 2022-11-24 | 2024-05-30 | 武汉禾元生物科技股份有限公司 | Human serum albumin-insulin conjugate and preparation method therefor |
Also Published As
| Publication number | Publication date |
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| MX2007000421A (en) | 2007-03-28 |
| EP1768701A2 (en) | 2007-04-04 |
| RU2006147284A (en) | 2008-08-27 |
| IL180229A0 (en) | 2007-07-04 |
| WO2006010360A3 (en) | 2007-12-27 |
| AU2005266739A1 (en) | 2006-02-02 |
| KR20070047786A (en) | 2007-05-07 |
| CA2574124A1 (en) | 2006-02-02 |
| NO20070775L (en) | 2007-04-18 |
| WO2006010360A2 (en) | 2006-02-02 |
| DE102004035606A1 (en) | 2006-03-30 |
| BRPI0513715A (en) | 2008-05-13 |
| JP2008506724A (en) | 2008-03-06 |
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