CN1189210C - Coupling object between CB and biological active peptide or immunoglobhulin or immunological activity original as well as medication usage - Google Patents

Abstract

The present invention provides a coupling object of a choleragen B subunit (CB) as a ligand/carrier and biological active polypeptide or immunoglobulin or immunological activity original which has therapeutic action on cerebral and spinal cord diseases or injuries; the present invention also provides a compound composition containing the coupling object and medicinal auxiliary agents or reinforcing agents, and a preparing and improving process for maintaining the bioactivity of the coupling object. The coupling object of the present invention can pass through the receptor-mediated endocytosis (RME) of the peripheral nerve and can enter the nervus centralis through the transference of the neural axoplasm without passing through the blood-brain barrier. A delivery system of the RME is founded, namely RME-DS nerve transference delivery. The CB coupling object is used for treating and/or preventing the cerebral and spinal cord diseases or injuries, for example, CB-NGF, CB-Anti-betaA and CB-IGF can treat senile dementia, and CB-CNTF, CB-BDNF and CB-MnA can treat motor neuron diseases.

Description

The conjugate of CB and biologically active polypeptide and medical usage thereof

Technical field

The present invention relates to choleragen B subunit as aglucon/carrier and the former conjugate of biologically active polypeptide, immunoglobulin or immunocompetence, and medical usage and preparation method, and the compositions that contains it.

Background technology

Traditional materia medica administration system all is ' menses approach ' (Blood routed deliverysystem) basically: no matter be from initial oral ' following through gastrointestinal blood ' in history, still to modern age and contemporary various injection (i.m, i.v, etc.) ' go into through the intramuscular blood capillary blood follows or directly inject blood vessel '.Tradition research also all is to be absorbed in ' menses approach ' to not seeing through the pharmaceutical preparation of blood brain barrier (BBB), is conceived to BBB itself, manages to change preparation, makes it to be easy to see through the BBB of cerebrovascular endothelial cell.As: the change preparation makes it more have the material that can enter cerebrovascular endothelial cell in fat-soluble, as to change preparation some molecular structures or the coupling to make it to deceive people BBB, and (Science 2002,297:1116-1118).

The approach that medicine is gone into brain is the source problem of central nervous system (CNS) drug design.Medicine and pharmacology history record in modern age: blood brain barrier BBB is that German physician Paul Ehrlich finds (his contribution on bacterial immune is learned once obtained Nobel's medical science in 1908 and physiology's prize) at 1870s; His student Edwin Goldmann finds that thereupon brain essence communicates closely with cerebrospinal fluid CSF, enters the dosing way that the intracerebroventricular of marrowbrain essence and waist such as wear at invasive thereby set up through CSF.For over 100 years more than a hundred years, the medicine and pharmacology of nervus centralis are to follow the source that Ehrlich and Goldmann open up entirely, medicine is followed through blood, the intracerebroventricular that life side manages to make it to pass BBB or make invasive, lumbar puncture etc. [also have the operation of intracerebral transplantation cell in recent years, its surgical operation invasive to patient, can only once implant and transgenic cell often only discharges a kind of trophic factors, can not guarantee that long-term continuing discharges, this all makes it difficulty and gives popularization].This patent invention has been opened up the macromole preparation first and has been gone into brain on the medicine and pharmacology history " neuroreceptor mediates the administration system into born of the same parents " new way of RME-D neural transhipment dispensing, having designed a series of process nerves from the source and entered encephalomyelic new drug, is the invention of great significance of a source innovation.

' focus ' comprehensive dispatch of in August, 2002 U.S. Science (297:1116-1118,2002) attempt to break through the contemporary situation of BBB: the people such as Pardridge of the UCLA that goes before also still also pace up and down, grope on this old road.In recent years, Pardridge utilizes the antibody coupling BDNF (Brain Derived Neurotrophic Factor) of cerebrovascular endothelial cell transferrins (transferrin) receptor or transport protein (transporter), thereby BDNF was stolen into another country BBB, on laboratory animal, treat experimental cerebral ischemia etc., its clinical target is apoplexy [it is the Trojan Horse meter of ' having deceived ' endotheliocyte that the report of Science likens this method to visually, and the character that it is stolen into another country also is described].The shortcoming of this technology: the one,, system's menses administration does not have targeting, and it all gets involved full marrowbrain, and side effect (as: someone reports that BDNF may bring out epilepsy) takes place in multi-functional neurotrophic factor in this case probably; The 2nd,, the normal function of transport protein can be blocked at the antibody of transport protein, thereby brain function may be influenced.This all is that Pardridge will be at the obstacle of clinic trial.The current report of Science in 2002 is indicating that macromole CNS medicine is about to go on the pharmaceutical market of clinical practice, with and coupling to combine be the technological trend of this development.

The existing dosing mode to neurotrophic factor (NT) has three kinds of methods that make it to cross blood brain barrier (BBB), the one, intracerebral ventricle injection, the 2nd, spider hypostegal cavity injection in the canalis spinalis, the 3rd, the capable intracerebral transplantation of cell or tissue of secretion NT (or the spider hypostegal cavity is transplanted) operation.Three kinds all have bigger operation wound, can not do secular or repetitious use.And the present invention uses common intramuscular injection or collunarium, and safety, no wound can be made routine and repeatedly use repeatedly.The required targeting of therapeutic purposes is looked at the intramuscular injection position, and selects corresponding nerve segment position.Preparation (as: the CB-NGF of collunarium, CB-Anti betaA, CB-IGF etc.) can enter the akrencephalon olfactory bulb through nervi olfactory, and then influence brain structures such as basal forebrain district (Basal forebrain area), particularly Meynert basal nuclei (nucleusbasalis of Meynert) cholinergic neuron, Hippocampus and cerebral cortex (treatment Alzheimer ' s disease AD Ah taste extra large Mo Shi alzheimer disease, parkinson or brain illness or damages such as apoplexy, cerebral embolism); Through III, V, VII, XI, the mode administration that XII puts slow releasing capsule to the intramuscular injection or the intramuscular of cranial nerve distributed areas is that targeting ground imports brain stem to the macromolecular drug that treats and/or prevents disease of brain stem; Put slow releasing capsule through the intramuscular injection of the spinal nerves distributed areas of corresponding sections or intramuscular the macromolecular drug that treats and/or prevents diseases of spinal cord or damage is imported spinal cord (CB-CNTF, CB-BDNF, CB-MnA etc. treat motor neuron, CB-BDNF, CB-IGF, CB-Anti Nogo treatment spinal cord injury or paraplegia).

The cure rate of various viral encephalitiss is low, sequela is serious.Though on virusology, can produce many specific antiviral antibodies, the IgG macromole can not or very difficult by BBB, specific treatment antibody can not enter neurocyte, does not bring into play treatment/preventive effect.

Choleragen (Choleragen, i.e. cholera toxin, Cholera Toxin) B subunit or unit (CB, choleragen B subunit) are avirulent receptors bind units, are also referred to as choleragenoid (choleragenoid).At the beginning of the eighties, (Univ.of Pennsylvania is UPenn) with phytohemagglutinin (lectins) such as cholera toxin (CT) and WGA and horseradish peroxidase (HRP) covalent bond, as the neuroanatomy probe greatly U.S. guest for Wan Xuancai.Axoplasmic transport (At) efficient that experimental results show that the receptor-mediated born of the same parents of going into of CT-HRP (RME) is than UPenn laboratory head, and the WGA-HRP that cytobiology professor Dr.N.Ganatas recommends is more sensitive effectively.Ten thousand take the lead in getting rid of the toxicity A subunit of CT after coming back home again, only with CB in conjunction with HRP.CT or CB are as effect high over one hundred times (Wan et al., 1982, the Brain Res.243:215) of axoplasmic transport profitization agent (a potent axonal transport facilitator) than non-RME transhipment.Wan Xuancai and with the neuroanatomy of having found use the neuron dendron of classical Golgi method (Gorky's argentation) in history from not seen, ten thousand called after Golgi-phobic Dendrites, GBD, " disliking Gorky's dendron " (Exp.Neurol 1982,78:167-175; Anat.Rec, 1984,208 (3): 190A; PROC.CAMS﹠amp; PUMC 1986,1:1-10).This is operated in 1984 and obtains the prize of health ministry first class scientific achievement, obtains national science technological progress second prize in 1985.

Sweden immunologist Holmgren has used CB as immunological adjuvant in the early 1990s, with CB-in conjunction with anaphylactogen or antigen (antigen, Ag) conjugate is made vaccine, experimental results show that to bring into play immunoregulation effect, and the sensitization that antigen is produced is suppressed or obtains tolerance (tolerance).To allergy and the autoimmune disease due to sensitinogen or the autoimmune antigen, can obtain curative alleviation (oral or collunarium, the through mucous membrane immune system plays a role) by inoculation CB-Ag.Particularly the immunoadjuvant function of CB (collunarium or oral) was tested in Northern Europe Sweden aspiration crowd, prove CB to the human body safety and nondestructive (Holmgren et al., Am.J.Trop.Med.Hyg.1994,50:5Suppl.42-54).But he never relates to and does not also observe receptor-mediated born of the same parents of going into of CB-GM1 or nervous system disease.

Summary of the invention

The present inventor is through research for many years, find that choleragen B subunit (CB) is as aglucon/carrier and to the medicative biologically active peptide of marrowbrain disease (neurotrophic factor neurotrophic factors and neurotrophins, NT etc.), or antibody immunoglobulin immunoglobulins, IgG connects into conjugate, these conjugates can enter marrowbrain through peripheral nerve, promptly got around blood brain barrier (BBB), is most of scripts to see through BBB, but in experiment, medicative active polypeptide/albumen of marrowbrain disease or IgG etc. have been imported marrowbrain essence, neural transhipment dispensing makes the clinical prevention potentiality of these active substances obtain fully effectively performance, and there have reality to be applied to be clinical, finished the present invention thus.

The invention provides choleragen B subunit as aglucon/carrier and conjugate and the compound thereof former to the medicative biologically active polypeptide of marrowbrain i or I, immunoglobulin or immunocompetence.

In the present invention, described biologically active peptide refers to comprise polypeptide/albumen and neurotrophic factor (neurotrophic factors NT, comprise nerve growth factor nerve growth factor NGF, neurotrophins family and other cytokine families), neuro hormone (neurohormones), neuropeptide (neuropeptides), analgesia peptide and apoptosis inhibitive factor (inhibitors forapoptosis); Immunoglobulin mainly is to the medicative specific antibody IgG of marrowbrain i or I; Immunocompetence is former mainly to be cause the autoimmune of marrowbrain disease former (immunogens) and anaphylactogen (allergens).

Can include, but are not limited to nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), Brain Derived Neurotrophic Factor (BDNF), glial cell derived neurotrophic factor (GDNF), insulin like growth factor (IGF) etc. with neurotrophic factor of the present invention; Neuro hormone has melatonin (melantonin) etc.; The apoptosis inhibitive factor has Bcl 2 etc.

The IgG (anti betaA) (the extra large Mo Shi senile dementia of treatment Ah taste) that anti-beta amyloid 1-42 (betaA) can be arranged the medicative antibody of marrowbrain i or I with the present invention; To the antibody of pain mediator, comprising anti-P material IgG; IgG antibody to the neural virus of parent; The antibody of anti-interleukin-1 receptor; IgG with anti-Nogo albumen (the Nogo albumen that suppresses the nervus centralis regeneration).

Can treat the original motor neuron antigen protein of the autoimmune that causes the marrowbrain disease (MnA) of immunologic pattern motor neuron (as lateral sclerosis myasthenia ALS etc.) with the present invention; Single sialic acid joint glycosides ester 2 (GM2); Acetylcholine n receptor a subunit (at myasthenia gravis) etc.The invention provides two classes with the conjugate of choleragen B subunit (CB) as aglucon/carrier:

1.CB with to the medicative biologically active polypeptide of marrowbrain i or I/proteic conjugate-CB-NT.

In the present invention, to the medicative biologically active polypeptide/albumen of marrowbrain i or I, they are meant with neurotrophic factor (neurotrophic factors, NT) be the nerve growth factor of representative (NGF), neurotrophic factor neurotrophins family (NGF and Brain Derived Neurotrophic Factor BDNF, NT3 etc.), also comprise other several neurotrophic factor family (ciliary neurotrophic factor CNTF, insulin-like growth factor I GF, glial cell line-derived neurotrophic factor GDNF etc.), neuro hormone, neuropeptide and apoptosis inhibitive factor etc.

Be that NT among the present invention comprises nerve growth factor NGF and is other several neurotrophic factor families, ciliary neurotrophic factor CNTF, insulin-like growth factor I GF, glial cell line-derived neurotrophic factor GDNF etc. and neuro hormone, neuropeptide and the apoptosis inhibitive factor etc. of representative with it.The medicative biologically active peptide of marrowbrain i or I/proteic CB coupling preparation is meant with CB-NGF to be the CB-CNTF of representative, CB-BDNF, CB-IGF, CB-GDNF, and CB-Bcl 2, CB-apoptosis inhibitive factor, CB-melatonin, CB-neuro hormone, CB-neuropeptide etc.

2.CB with conjugate-CB-IgG or CB-Ig to medicative antibody of marrowbrain i or I (IgG) or immunocompetence former (immunogen Ig)

In the present invention, the medicative antibody of marrowbrain disease is meant monoclonal or the polyclonal antibody of an immunoglobulin like protein IgG, it includes but not limited to that IgG also comprises IgE, IgM etc.IgG one class includes the anti-P material antibody of anti-pain, anti-beta amyloid 1-42 monoclonal antibody (anti-beta Amyloid 1-42, Anti betaA), the antibody of anti-interleukin-1 receptor (Anti IL-1r), the antibody of antibody, anti-encephalitis and the anti-encephalomyelitis virus of the neural virus of anti-parent and the anti-antibody that suppresses nerve growth albumen Nogo etc.The former Ig of wherein said immunocompetence mainly is meant autoimmune that causes the marrowbrain disease former (immunogens Ig) and anaphylactogen (allergens), comprise: motor neuron antigen protein (MnA) and single sialic acid joint glycosides ester 2 (GM 2), acetylcholine n receptor a subunit etc.

To medicative IgG antibody of marrowbrain i or I or the former CB conjugate of immunocompetence---CB-IgG/Ig is meant with the anti-P material of CB-antibody (CB-Anti P) to be the CB-Anti betaA and the CB-Anti Nogo of representative, CB-Anti IL-1r, CB-MnA etc.

The present invention also provide contain choleragen B subunit as aglucon/carrier with to the former conjugate of the medicative biologically active peptide of marrowbrain disease, immunoglobulin or immunocompetence and the pharmaceutical composition of pharmaceutically acceptable adjuvant and reinforcing agent." reinforcing agent " is meant that strengthening the CB conjugate is absorbed by neuroreceptor or delay some preparations that it is taken away by nose mucus or muscle hemodilution." medicinal adjuvant " be meant pharmaceutical field be commonly referred to be safety, avirulent adjuvant, and they both the abiology activity do not have ill effect yet.These adjuvant for example can comprise lactose, starch, water, alcohol etc.Can contain conjugate of the present invention in the compound of the present invention and replenish adjuvant.

Pharmaceutical composition of the present invention can be made dosage forms such as slow releasing capsule, solution, spray, injection, can adopt form administrations such as parenteral or nasal cavity splash into, spraying.Pharmaceutical composition of the present invention preferably adopts intranasal administration, intramuscular injection or intramuscular to put the mode administration of slow releasing capsule (going into born of the same parents, not menses through the CB-GM of muscle nerve ending 1 mediation).

The present invention relates to above-mentioned coupling preparation at the RME-DS target administration, get around the medical usage that blood brain barrier treats/prevent marrowbrain illness or damage and have solid theory.Accompanying drawing 1 has shown CB-NT, the receptor-mediated cytobiology principle of going into born of the same parents' dispensings (RME-DS) of CB-Ab (IgG).

The invention still further relates to the purposes that above-mentioned conjugate enters the medicine of nervus centralis in preparation through the receptor-mediated born of the same parents of going into of peripheral nervous, axoplasmic transport.Particularly, the ganglioside oligosaccharide of conjugate of the present invention on neurolemma be receptor-mediated goes into born of the same parents, axoplasmic transport, from around enter the central nervous system, and further in marrowbrain, make transcellular transport.

The preparation that the present invention relates to above-mentioned conjugate be used for the treatment of and/or prevention of brain diseases of spinal cord or damage in medical usage.Particularly, described marrowbrain i or I comprises: the degeneration neuropathy, its be selected from the extra large Mo Shi alzheimer disease of Ah taste (Alzheimer ' s dementia, AD), motor neuron (motor neuron disease, MND), parkinson (Parkinson ' s disease, PD), intractable migraine and nervous system autoimmune disease; Cerebral spinal cord injury, it is selected from apoplexy, cerebral thrombosis, cerebral ischemia, cerebral trauma and trauma of spinal cord; Pain; Viral encephalitis and viral encephalomyelitides; The HIV encephalitis.

Pathology-the physiological mechanisms of degeneration maincenter (or periphery) neuropathy or pathogenic process what all have immune disorder or autoimmune to participate in, have clear and definite be autoimmune disease (as: myasthenia gravis, multiple sclerosis and motor neuron) basically.So the targeting neurotrophic treatment that the transhipment of immunomodulating composite nerve is offerd medicine is that alleviation, treatment and prevention now are the best strategy of the degeneration neuropathy of incurable disease.

On the other hand, the choleragen B subunit that the invention still further relates to is as aglucon/carrier and preparation modification method to the former conjugate of the medicative biologically active peptide of marrowbrain disease or antibody, immunocompetence, comprise with choleragen B subunit with to medicative biologically active peptide of marrowbrain i or I or immunoglobulin or immunocompetence is former couples together, and keep the biological activity of conjugate constant.Particularly, couple together with the amino of choleragen B subunit or carboxyl and to the medicative biologically active peptide of marrowbrain i or I or immunoglobulin or immunocompetence former carboxyl or amino; Or import sulfydryl or dithione couples together with disulfide bond.That is to say, the aminoacid of choleragen B subunit with covalently bound with chemistry through bridging agent to the former aminoacid of the medicative biologically active peptide of marrowbrain disease or antibody, immunocompetence, and is kept bioactive improving technology.

The method that connects different albumen/polypeptide in recent years is a lot, and methodology commonly used can be referring to [Chemistry of Protein and Cross-Linking.Wong 1991 CRC Press Inc.; BocaRaton, Fla; And E.Harlow and D.Lan, Eds, Antibodies:A Laboratory Manual.Cold Spring Harbor Laboratory, Cold Spring Harbor.NY, 1988].Can use organic compound such as carboxylic acid esters, acid anhydride class, acyl halide class, acyl azide as bridging agent between albumen/polypeptide, form covalently bound (as combining with lysine amino or with the sulfenyl of albumen/polypeptide etc.) with the reactive group of albumen/polypeptide.Concrete method can be consulted two books.Ideal covalently bound be amino side chain through albumen/polypeptide lysine amino.The choleragen B subunit of pentamer has 9 lysines on each monomer, visible CB is one and suitablely especially does link coupled object.The structure of CB and active situation can be with doing evaluation with combining of single sialic acid joint glycosides GM1 in the coupling conjugate.Preferred improvement is following two kinds of methods in our practice:

Method I (glutaraldehyde GA two step improved method) be to Avrameas and Ternyck and Gonatas etc. improved method (Modification of Avrameas and Temyck 1971, Immunochemistry 8,1175; Gonatas etc., 1979, J.Histochem.Cytochem.27,728; Wan, XST 1986, Proc.CAMS﹠amp; PUMC, 1,1-10; 1984, Anat.Rec.208,3,190A).In recent years for adapting to different active polypeptide again through improvement.The first step: with 1.25% glutaraldehyde activation CB; (60 * 0.9cm) separate activatory CB and the glutaraldehyde that is not connected (or only post and dialysing at 0.15M NaCl, remove not connected glutaraldehyde) through Sephadex G-25 column glue post; Concentrate.Second step: isometric medicative biologically active peptide/albumen of marrowbrain disease or antibody etc. are carried out coupling (noting different isoelectric point, IPs and biological activity site) in spissated in the activatory CB of glutaraldehyde, the adding; Composition through Sephadex G-200 glue post separating and combining attitude and non-binding attitude; Concentrated and stable.

CB-can identify (as PC12) with the cell culture that exsomatizes to the biological activity of the medicative biologically active peptide of marrowbrain disease.

Method II (disulfide bond connection improved method) be to former methods of people such as Friden improvement (Modification from Friden et al., 1993, Science 259,373; Kordower etal., 1994, PNAS USA 91,9077).It is earlier with EDC[1-ethyl-3-(3-dimethylaminoproyl) carbodiimide] act on medicative biologically active peptide/albumen (as NGF or IgG) carboxyl of its lysine easily is connected with trinitride, organic reagent SPDP (the Carlson et al.1978 that the seventies such as reuse Carlson propose, Biochem J 173,723) derivant, pyridine two sulfur propanoic acid hydrazides PDP[pyridyldithiopropionate-hydrazide] enclose a reaction sulfenyl (2-pyridyl-sulfide group) at the carboxyl of biologically active peptide (or IgG and other albumen) lysine.Note different isoelectric point, IPs and biological activity site.Use SATA[N-succinimidyl-S-acetylthioacetate] the lysine amido that acts on the CB makes it connect sulfydryl.Pyridine sulfuration base (2-pyridyl-sulfide group) displacement of sulfydryl on the CB and NGF (or IgG) lysine forms disulfide bond, promptly two protein molecular coupling combinations, discharges 2-thiopyridines (pyridine-2-thione).With protein A-Sepharose column purification coupling conjugate.The biological activity of its trophic factors of coupling conjugate that this method obtains is better.Different CB conjugates all has improvement characteristics and technical know-how separately on the preparation details.

Medicine and pharmacology meaning of the present invention and economic development value

Present inventor's design for the first time on pharmacotherapeutics has proposed: neural transhipment medication, promptly with neuroreceptor mediate into aglucon/carrier of born of the same parents medicine from the peripheral nervous axoplasmic transport enter encephalomyelic " the RME-administration system (and RME-Delivery System, RME-DS).The present inventor has proposed from rationale and methodology first: the neural axoplasmic transport of RME is the transhipment passage of physiological material just, and also preparation medicine etc. enters the main road of marrowbrain essence.The biological development of the twentieth century cellular elements second half has obtained a sequence-specific biologically active peptide/albumen, is referred to as somatomedin, cytokine etc.The NGF of Rita Levi-Montalcini and Stanley Cohen, EGF work obtains Nobel Prize in Physiology or Medicine in 1986, indicates the academic achievement of this wide field.But this sequence macromole polypeptide/albumen but is subjected to the restriction of blood brain barrier BBB to the medical application potentiality of nervus centralis (CNS) marrowbrain illness and damage, can not get performance.Can be still the main flow of current C NS medicine by the small-molecule drug below the 500Da of BBB.The present invention is a sequence-specific biologically active peptide/albumen, and no invasive such as neurotrophic factor, somatomedin, cytokine, apoptosis inhibitive factor are gone into brain and opened up road; For incurable diseases such as senile dementia and motor neuron are brought new hope.

CB-conjugate of the present invention is with characteristics such as its targeting dispensing, non-invasi and advantage and have very big medical significance and economic development value.The data of Science (Vol.297:1116-1118,2002) is estimated: nervus centralis (CNS) medicine is 38,000,000,000 dollars on the world markets in 1998, almost is small-molecule drug entirely; Macromolecular drug does not come into the market as yet, and CNS macromolecular drug of the present invention is as after putting goods on the market through clinic trial, and economic benefit is huge.

Choleragen B subunit of the present invention is as aglucon/carrier and to the medicative biologically active peptide of marrowbrain disease/proteic conjugate, can walk around blood brain barrier (BBB) most of scripts be can not see through BBB the medicative biologically active peptide/albumen of marrowbrain disease such as NT, immunocompetence globulin IgG etc. have been imported marrowbrain essence, make the clinical prevention potentiality of these active substances obtain fully effectively performance.

Be used for clinical or just having a mind to treatment preparation by clinic trial as the marrowbrain disease being had with CB is link coupled biologically active peptide/albumen, antibody or the immunocompetence of treatment/preventive effect is former all to have a large amount of zooperies proofs effective in cure and safe and reliable in known references, having plenty of.And through with the CB coupling after, then become more efficient, safety, nothing infringement ground and enter encephalomyelic present clinical and new drug preparation that market is not had.

Choleragen B subunit (CB) is avirulent receptors bind unit.Itself non-toxic and safe, through our description of test, certain neurotrophic effect is still arranged, this is because the metabolism that CB has activated neurocyte GM1 utilization rate (Liu Z-P and Wan X-C again, 1995, Chinese J.Neuroimmurol.Neurol 2:61).

The explanation of accompanying drawing table

Fig. 1 has shown CB-NT, the receptor-mediated cytobiology principle of going into born of the same parents' dispensings (RME-DS) of CB-Ab (IgG)

Fig. 2 has shown NGF, the active mensuration comparison curves of CB-NGF external biological.Illustrate that coupling does not influence biological activity

Fig. 3 a has shown EXPERIMENTAL EXAMPLE 1, the fleeing incubation period of A-E group water maze laboratory (Escapelatency, El) relatively reach the comparison of Gp behind the Gb and nose dropping treatment before the aged Mus G group CB-NGF nose dropping treatment.

Fig. 3 b has shown EXPERIMENTAL EXAMPLE 1, the swimming distance of A-E group water maze laboratory (Distancetraveled, Dt) relatively reach the comparison of Gp behind the Gb collunarium before the aged Mus G group CB-NGF collunarium.

Fig. 3 c has shown EXPERIMENTAL EXAMPLE 1, A-E group water maze laboratory swimming rate (Dt/El) relatively reach the comparison of Gp behind the Gb collunarium before the aged Mus G group CB-NGF collunarium.

Fig. 4 picture A-F has shown EXPERIMENTAL EXAMPLE 1, A, and B, C, D, E group and F green grass or young crops are organized the morphology of forebrain substrate ChAT (choline second vinegar transferring enzyme) positive neuron less and compare (and seeing Table 1).

Fig. 5 has shown that in EXPERIMENTAL EXAMPLE 2 the anti-P material of CB-rabbit IgG is to the influence of the whipping threshold of pain.Illustrated the anti-P material of rabbit IgG from waist marrow transcellular transport to the sacrum tail marrow that reacts bitterly of control whipping.

Fig. 6 photo 1 has shown in EXPERIMENTAL EXAMPLE 3, with (the cholinesterase group dyeing of the isolating Medulla Sus domestica motor neuron of sucrose gradient ultracentrifugation, 600 times of amplifications) Swine spinalmotoneurons isolated by sucrose gradient ultra-centrifuge method (cholinesterase staining.Magnification, x 600)

Fig. 6 photo 2 has shown in EXPERIMENTAL EXAMPLE 3, the lumbosacral enlargement ventricornu of immunologic injury Lewis rat (cresyl viollet dyeing, 300 times of amplifications) photo right side glial cells hyperplasia, and motor neuron is lost.The?anterior?horn?of?the?lumbosacral?enlargement?of?animmuno-injured?Lewis?rat(Cresyl?violet?staining，x?300)

Fig. 6 photo 3 has shown in EXPERIMENTAL EXAMPLE 3, the lumbosacral enlargement ventricornu of normal control Lewis rat (cresyl viollet dyeing, 250 times of amplifications)

Fig. 6 photo 4 has shown lumbosacral enlargement ventricornu (cresyl viollet dyeing, 250 times of amplifications) photo middle (the anterior angle outside) glial cells hyperplasia of immunologic injury Lewis rat, and motor neuron is lost.

Fig. 6 photo 5 has shown the existing engrain neurocyte (cresyl viollet dyeing, 600 times of amplifications) that withers and become of the lumbosacral enlargement ventricornu of immunologic injury Lewis rat (symptom moderate person)

Fig. 6 photo 6 has shown the lumbosacral enlargement ventricornu of immunologic injury Lewis rat (symptom is heavier, and the person does not die frequently on one's deathbed).Glial cells hyperplasia is arranged, motor neuron degeneration and lose (cresyl viollet dyeing, 600 times of amplifications)

Fig. 6 photo 7 has shown that normal control Lewis rat blood serum (diluting 1: 40) can not dye the anterior angle motor cell of Wistar rat, SABC feminine gender.(ABC method, 600 times of amplifications)

Fig. 6 photo 8 has shown that the serum (diluting 1: 800) of MND animal pattern can be used as first antibody and is used for immunohistochemical reaction, can dye the anterior angle motor cell of Wistar rat, the SABC positive.(ABC method, 600 times of amplifications)

Fig. 6 photo 9 has shown the strong support situation of normal control rat two hind legs.

Fig. 6 photo 10 has shown MND rat model hind leg unablely drags slack situation.

Fig. 6 photo 11 has shown that normal rat tibialis anterior injection 10ul HRP is in the less part of ventricornu labeled cell (400 times of amplifications).

Fig. 6 photo 12 has shown moderate immunologic injury rat tibialis anterior injection 10ul HRP in the more part of ventricornu labeled cell, and is still normal substantially with the axoplasmic transport that shows immunologic injury rat nerve, can transport pharmaceutical preparation (400 times of amplifications).

Fig. 7 has shown in EXPERIMENTAL EXAMPLE 3, the survival comparison diagram of the compound CB-MnA treatment immunologic injury MND rat of CB-CNTF (1-4 group)

Embodiment

Preparation embodiment 1:

The anti-P material of CB-IgG[rabbit] glutaraldehyde GA coupling

CB[pentamer pentamer molecular weight 45-50kDa, List Ltd., US] 3.0mg is dissolved in 1.25%GA[Sigma, the person that is used for the electron microscope specimen] 1ml, 0.1M sodium phosphate (pH6.5) is in room temperature effect spend the night [lower pH can prevent lysine amino self coupling among the CB]; Separate activatory CB and the glutaraldehyde that is not connected (or only post and dialysing, remove not connected glutaraldehyde) through SephadexG-25 column glue post (40 * 0.9cm crosses post with 0.15M NaCl solution) at 0.15M NaCl; The CB component (molecular weight 45-50kDa) that concentrated post liquid with microporous filter membrane is to about 1ml.

The spissated pH to 9.5 that activates CB of carbonate buffer solution (pH9.5) adjusting with 1M adds carbonate buffer solution (pH9.5) 1ml that rabbit anti-P material IgG (molecular weight 150kDa) 9.0mg is dissolved in 1M, acts on 24 hours with activation CB in 4 degree low temperature; Through the Sephadex G-200 glue post (composition of 1.6 * 90cm) separating and combining attitudes (molecular weight 200kDa) and non-binding attitude; Concentrate and stable (add lysine, make into 0.1M).

Preparation embodiment 2:

The GA coupling of CB-NGF

CB[molecular weight 45-50kDa, List Ltd., US] 4mg is dissolved in 1.25%GA[Sigma, the person that is used for the electron microscope specimen] 1ml, 0.1M sodium phosphate (pH6.5) is in room temperature effect spend the night [lower pH can prevent lysine amino self coupling among the CB]; Separate activatory CB and the glutaraldehyde that is not connected (or only post and dialysing, remove not connected glutaraldehyde) through Sephadex G-25 column glue post (40 * 0.9cm crosses post with 0.15M NaCl solution) at 0.15M NaCl; The CB component (molecular weight 45-50kDa) that concentrated post liquid with microporous filter membrane is to about 1ml.

The spissated pH to 9.5 that activates CB of carbonate buffer solution (pH9.5) adjusting with 1M adds NGF (betaNGF, 4mg two chains, molecular weight 26.0kDa, Sigma, N 8133) 2.0mg is dissolved in carbonate buffer solution (pH9.5) 1ml of 1M, with activation CB effect 24 hours in 4 degree low temperature; Through the Sephadex G-200 glue post (composition of 1.6 * 90cm) separating and combining attitudes (molecular weight 80kDa) and non-binding attitude; Concentrate and stable (add lysine, make into 0.1M).

Preparation embodiment 3:

The disulfide bond S-S coupling process of CB-NGF

NGF (beta NGF, two chains, molecular weight 26.0kDa, Sigma, N 8133) 2.0mg is dissolved in 1ml, in the carbonate buffer solution of 1M (pH8.5), slowly adds citraconic anhydride (the Fructus Citri Limoniae acid anhydride of 6.0mg/ml, Sigma US), puts room temperature effect 1 hour with the blocking-up free amine group; Separate Fructus Citri Limoniae acid anhydride and NGF peptide with G10 glue post.Add EDC 20mg at the NGF of 2.0mg/2ml and act on carboxyl, transferring pH earlier is 5, with transferring to 8, puts room temperature 10 minutes; Cross G10 glue post and remove EDC; Concentrating NGF liquid is 2ml; The PDP solution of dropping 20mM (Sigma, US) 0.5ml stirred 30 minutes in NGF liquid at room temperature; Cross SephadexG-25 (0.1M, PBS elute) glue post and remove unnecessary PDP, microporous filter membrane concentrates NGF liquid to 2ml.

(List US) is dissolved in 1ml, and in the carbonate buffer solution of 1M (pH8.5), (Sigma, US) 0.5ml stirred 30 minutes the SATA solution of dropping 20mM at room temperature, made the lysine amido of CB connect sulfydryl 4mg CB; Cross Sephadex G-25 (0.1M, PBS elute) glue post and remove unnecessary SATA, the microporous filter membrane concentrated volume is to 1.5ml.

(sulfhydryl) two solution stirred 30 minutes at room temperature to mix NGF (2pyridyl sulfide) and CB; CB-NGF with protein A-Sepharose post and NGF monoclonal antibody affinity column purification coupling conjugate.Be concentrated into 2ml, packing, quick-freezing is standby in negative 20-40 degree.NGF, the biological activity determination of CB-NGF (S-S coupling) and CB-NGF (GA coupling) is (Fig. 2) relatively

With NGF, CB-NGF (S-S coupling) and CB-NGF (GA coupling) act on the PC-12 cell respectively, look the influence of its pair cell neurite-outgrowth and represent biological activity.

Cultivate the PC-12 cell with 96 hole culture plates (protein I V is adherent in NIUJIAO unit), every hole about 1 * 10k cell, adherent growth adds the NGF of various dose respectively after 6 hours, CB-NGF (S-S coupling) and CB-NGF (GA coupling), influence [200ng/ml, 100ng/ml, the 50ng/ml of test pair cell neurite-outgrowth, 25ng/ml, 12.5ng/ml, 6ng/ml, 3ng/ml, 1ng/ml, 0ng/ml]; After 5 days, each hole of observed and recorded (the 2-3 visual field, every hole) cellular neural projection length surpasses the above cell number of 2 diameters of cell space and the percentage ratio of observation of cell sum, and NGF, the active mensuration comparison curves of CB-NGF external biological are as shown in Figure 2.Presentation of results: CB-NGF (S-S coupling) and CB-NGF (GA coupling) have the biological activity suitable with NGF, and the link coupled CB-NGF of S-S is active in better.

All not influences of the link coupled process of biological activity determination description of test link coupled biologically active peptide of institute and proteic activity.

Fig. 2 has shown NGF, the active mensuration comparison curves of CB-NGF external biological.

Preparation embodiment 4:

The disulfide bond S-S coupling process of CB-CNTF

CNTF (Sigma, C 3835) 400 μ g are dissolved in 1ml, in the carbonate buffer solution of 1M (pH8.5), add that slowly (Fructus Citri Limoniae acid anhydride, Sigma US), put room temperature effect 1 hour with the blocking-up free amine group for the citraconic anhydride of 6.0mg/ml; Separate Fructus Citri Limoniae acid anhydride and CNTF peptide with G10 glue post.Add EDC 20mg at the CNTF of 400mu g/2ml and act on carboxyl, transferring pH earlier is 5.5, with transferring to 8.5, puts room temperature 10 minutes; Cross G10 glue post and remove EDC; Concentrating CNTF liquid is 2ml; The PDP solution of dropping 20mM (Sigma, US) 0.5ml stirred 30 minutes in CNTF liquid at room temperature; Cross Sephadex G-25 (0.1M, PBS elute) glue post and remove unnecessary PDP, microporous filter membrane concentrates CNTF liquid to 2ml.

(List US) is dissolved in 1ml, and in the carbonate buffer solution of 1M (pH8.5), (Sigma, US) 0.5ml stirred 30 minutes the SATA solution of dropping 20mM at room temperature, made the lysine amido of CB connect sulfydryl 4mg CB; Cross Sephadex G-25 (0.1M, PBS elute) glue post and remove unnecessary SATA, the microporous filter membrane concentrated volume is to 1.5ml.

(sulfhydryl) two solution stirred 30 minutes at room temperature for mixed C NTF (2pyridyl sulfide) and CB; CB-CNTF with protein A-Sepharose post and CNTF monoclonal antibody affinity column purification coupling conjugate.Be concentrated into 2ml, packing, quick-freezing is standby in negative 20-40 degree.

Preparation embodiment 5,

The GA coupling preparation of CB-MnA/MnA

Motor neuron antigen protein (MnA): press Engelhardt et al., method (J NeurosciMethods 1985,15:219) do homogenate by cervical part of esophagus and the last chest section spinal cord separation anterior angle motor neuron cell space [seeing Fig. 6 photo 1] of aquatic foods Tu pig, electrophoresis extracts high molecular (150-180kD) albumen 3.0mg as MnA.

With CB[pentamer pentamer molecular weight 45-50kDa, List Ltd., US] 2.0mg is dissolved in 1.25%GA[Sigma, the person that is used for the electron microscope specimen] 1ml, 0.1M sodium phosphate (pH6.5) is in room temperature effect spend the night [lower pH can prevent lysine amino self coupling among the CB]; Separate activatory CB and the glutaraldehyde GA that is not connected (or only post and dialysing, remove not connected glutaraldehyde) through SephadexG-25 column glue post (40 * 0.9cm crosses post with 0.15M NaCl solution) at 0.15M NaCl; The CB component (molecular weight 45-50kDa) that concentrated post liquid with microporous filter membrane is to about 1ml.

The spissated pH to 9.5 that activates CB of carbonate buffer solution (pH9.5) adjusting with 1M adds carbonate buffer solution (pH9.5) 1ml that MnA (molecular weight 150-180kDa) 3.0mg is dissolved in 1M, acts on 24 hours with activation CB in 4 degree low temperature.This CB-MnA and MnA are used for collunarium to bring out mucosa-immune, and CB plays adjuvant effect, make the tolerance of body generation to immunogen MnA, so do not need the composition of separating and combining attitude and non-binding attitude.Only, be concentrated into 2.0ml (this CB-MnA, MnA mixed liquor are equivalent to MnA 3mg/2.0ml) with 0.15M NaCl dialysis.

EXPERIMENTAL EXAMPLE 1:

The CB-NGF collunarium is protected forebrain substrate cholinergic neuron and is improved the experiment of learning and memory

CB (CTB, U.S. List company product), betaNGF (U.S. Sigma company product); The reagent that coupling is used is the product of U.S. Sigma.

15 of Wistar rats (4-5 monthly age) are divided five groups of normal not damaged matched groups of (A-E): A. (n=3); B. false damage matched group (n=3); C. dorsal part Hippocampus damage group (n=3); D. Hippocampus damage+uncombined CB, NGF contrasts collunarium group (n=3), five weeks of treatment phase; E. Hippocampus damage+combined state CB-NGF collunarium group (n=3), five weeks of treatment phase.F. around teenager (1 monthly age) rat and each two CB-NGF collunarium of G old rats (22 monthly age).F makes ChAT immunohistochemical analysis (Fig. 4); The water maze learning and memory behavior that G makes (Gb) and treatment back (Gp) before the nose dropping treatment is (Fig. 3) relatively.

The dosage of NGF collunarium is 25mu g/100ul in combined state and non-binding attitude, day drips once, keeps rat dorsal position 40 minutes so that preparation better enters the olfactory mucosa olfactory cell.

Dorsal part Hippocampus trauma surgery: Chlora Hydrate (400mg/kg, i.p.) anesthesia; The three-dimensional elements of a fix that microsyringe (Hamilton) enters Hippocampus are: 4.5mm after AP bregma, the other 3mm that opens of L center line, DV from the brain surface downwards, promptly-(Quinolinate or ibotenate 10nmol is dissolved in artificial cerebrospinal fluid to the neural poison system of 3mm. sea core acids damage, injection 2.0ul.) micro-injection speed is 0.5/min, and injection finishes the back let the acupuncture needle remain at a certain point 5min.

Sham-operation damage treated animal is injected into Hippocampus artificial cerebrospinal fluid 2.0ul with same anesthesia and location of operation coordinate, and injection finishes the back let the acupuncture needle remain at a certain point 5min.

I. (3c): (D E) all 2 weeks and 2 weeks of treatment beginning back just does water maze laboratory after surgery, does altogether ten times (day) for B, C, and each (day) does the test of 6 backwater labyrinths for surgical injury and treatment group for Morris water maze laboratory Fig. 3 a, 3b in learning and memory experiment.

A, (normal control), B (sham-operation contrast), two groups flee incubation period (Escape latency seeks the time El of platform), is 10sec (SEM=2), several indifferences after testing for 3-4 time; The swimming of finding the platform of fleeing is apart from (Distance traveled Dt) is respectively 210cm (SEM=12), 220cm (SEM=11), no significant difference [p＞0.05]; Swimming rate (Dt/El) is respectively 21cm/sec and 22cm/sec, no significant difference [p＞0.05].

C. change greatly El and Dt of last three parameters of dorsal part Hippocampus damage group all is multiplied, swimming rate (Dt/El) greatly slow down [p＜0.001, ANOVA].Illustrate that learning and memory suffers damage.

D. Hippocampus damage adds non-binding attitude CB, the NGF collunarium, and three parameters and the C damage group of water maze do not have significant difference [p＞0.05, ANOVA].Non-binding attitude CB has been described, NGF is invalid substantially.

E. Hippocampus damage add CB-NGF nose dropping treatment group on three parameters of water maze with A, B, matched group do not have significant difference [p＞0.05, ANOVA].Illustrate that the Hippocampus damage is restored to the infringement of learning and memory.

D, the test of E water maze is compared and also illustrated: a CB-NGF has obvious curative effects.

The juvenile Mus CB-NGF of F collunarium experimental group is not done the water maze behavioral experiment, has only done the neuronic neuroanatomy of forebrain basal area ChAT and has analyzed (see figure 4).

G. organize aged Mus before CB-NGF treatment Gb and treatment afterwards Gp compare, ability of learning and memory also has and improves [p＜0.05, t-test].Prompting CB-NGF can have functions of prevention and health care.(see figure 3)

II. the neuronic neuroanatomy analysis of forebrain substrate choline Acetylase (ChAT) (seeing Fig. 4 and table 1)

Above-mentioned A, B, C, D, E, the animal of F group is all got brain at experiment sacrifice at the 5th weekend and cooks frozen section (20um) [forebrain basal area serial section], does immunocytochemical stain reaction (Vector ABC test kit) with anti-ChAT antibody (CambridgeBiochemicals).

The result is shown in Fig. 4 and digital watch 1:

Fig. 4 picture A shows normal not damaged become Mus matched group forebrain basal area (BFA) ChAT (choline acetyl transferase) immunocytochemical stain;

Fig. 4 photo B has shown that false damage (injection artificial cerebrospinal fluid) becomes Mus matched group BFA, and ChAT immunocytochemical stain and A do not have change more substantially;

Fig. 4 photo C has shown one-tenth Mus dorsal part Hippocampus damage group BFA, and the ChAT immunocytochemical stain greatly weakens, and cellular atrophy and having is lost;

Fig. 4 picture B shows has shown that one-tenth Mus Hippocampus damage+uncombined CB mixings NGF contrast collunarium group BFA ChAT immunocytochemical stain weakens, and cellular atrophy also has and loses [being lighter than C] (five weeks of treatment phases afterwards);

Fig. 4 photo E has shown one-tenth Mus Hippocampus damage+combined state CB-NGF nose dropping treatment group, and the BFAChAT immunocytochemical stain recovers normally (five week of treatment phase back) substantially;

Fig. 4 photo F has shown five week of two CB-NGF collunariums of teenager rat (1 monthly age) back BFA ChAT immunocytochemical stain, strengthens to some extent than A.

Table 1 has shown EXPERIMENTAL EXAMPLE 1, A, B, C, D, the quantification situation of number, size and the one-level dendron of ChAT (choline second vinegar transferring enzyme) positive neuron in the E experimental group forebrain substrate Meynert base nuclear

Table 1: the quantitative situation of number, size and the one-level dendron of ChAT (choline second vinegar transferring enzyme) positive neuron in the forebrain substrate Meynert base nuclear

Group	The number of ChAT positive neuron [is the % of radix with A]	The size of ChAT positive neuron (μ m2) [is the % of radix with A]	One-level dendron number is greater than 3 ChAT positive neuron %
				A. normal control	910±20 [100±2]％	190±14 [100±2]％	90±2％
B. sham-operation	914±20 [100±2]％	188±15 [99±2]％	89±3％
				C. Hippocampus damage	730±35 [82±4]％	142±10 [74±2]％	27±5％
D. non-binding mixing collunarium	780±30 [86.7±4]％	160±10 [84±1]％	34±5％
				The E.CB-NGF nose dropping treatment	918±30 [100±2]％	185±8 [100±2]％	85±4％
F.CB-NGF is to the potentiation of juvenile rat ChAT	948±30 [105±3]％	195±12 [102.6±2]％	97±3％

The experiment of CB-NGF collunarium understands that in particular RME-DS is after olfactory cell-nervi olfactory is gone into brain; NGF is at akrencephalon olfactory bulb transcellular transport; the NGF secondary effect (secondary effect) pointed out in ' diagram of cell biological mechanism ' has taken place: prop up to the side of olfactory bulb projection by forebrain substrate cholinergic neuron, NGF has protected the forebrain substrate cholinergic neuron because of dorsal part Hippocampus damage atrophy degeneration.It is the anatomic model of internationally recognized AD senile dementia that the dorsal part Hippocampus is destroyed damage, and this experiment is the important experiment basis that CB-NGF could treat or improve the AD senile dementia.Senile rat Gb, the experiment of Gp and F teenager rat prompting: CB-NGF have the health-care effect of hypermnesis to the protection of forebrain substrate cholinergic neuron and to the preventive effect of dementia.

EXPERIMENTAL EXAMPLE 2:(sees Fig. 5)

The anti-P material of the CB-rabbit IgG intramuscular injection raising threshold of pain and anti-P material IgG are at the transcellular transport of spinal cord

Experiment

Material: CB (CTB, U.S. List company product), the anti-P material of rabbit IgG (U.S. Sigma company product); The rabbit that coupling is used anti-P material IgG antibody and other reagent are the product of U.S. Sigma; Goat-anti rabbit ABC test kit (U.S. Vector company product).

Each 12 of Wistar rat (4-5 monthly age) and Switzerland mices (2 monthly age), the contrast (n=3) of three groups of each minutes: A group for not dealing with; The B group is played tricks and is handled contrast (n=4): located to inject the anti-P material of non-binding attitude rabbit IgG 20 μ g (n=2) every 1 day in quadriceps femoris (lumbar nerves L 2-3 domination); The non-specific IgG 20 μ g (n=2) of Mus (or mice); C group is experimental group (n=6), every 1 day in the quadriceps femoris place (lumbar nerves L 2-3 domination) inject CB-rabbit anti-P material IgG coupling conjugate (containing 20 μ g rabbit iggs).

The anti-P material of I.CB-rabbit IgG intramuscular injection quadriceps femoris [domination of waist section spinal cord] influences (see figure 5) [whipping react bitterly be spinal cord sacrum rear domination] to the whipping threshold of pain

(C) handled in experiment and the false injection of handling (B) all lasted for three weeks; Around first thoughtful the (week after stopping injection treatment) survey A, B, the whipping threshold of pain of C treated animal, day (inferior) tested 8 times at every turn on every Wendesdays; The stimulation instrument of surveying pain is AusBio G16 type (AusBio Pty Ltd), and stimulus parameter is fixed as N-6.6 (rat), N-4.4 (mice), spacing electric heating causalgia, record whipping pain threshold (sec).

Average 5 (SEM=2) of A group pain threshold, average 5 (SEM=2.5) of B group pain threshold do not have notable difference (p＞0.05).The pain threshold (sec) of A group as basis (being converted to 100%), the pain threshold of C group after processing second week and rise to 180% (SEM=10) and 200% (SEM=12) the 3rd week respectively, with A, B organizes relatively has evident difference (p＜0.05).Stop that there is recovery trend the threshold of pain of all animals (n=3) after the injection treatment, last day the C treated animal average 5 (SEM=2.5) of survey pain pain threshold, show that the threshold of pain recovered normally, with A, B organizes no significant difference (p＞0.05).

The anti-P material of description of test CB-rabbit IgG has anti-pain effect, has obviously improved the threshold of pain.The results are shown in Figure 5.

II. the immunocytochemical assay of the anti-P material of rabbit IgG in spinal cord

Sacrifice A (n=2) the 3rd weekend in experiment, B (n=2), and each 7 of the large and small Mus of C (n=3) group are got waist sacrum tail marrow and cook frozen section (30um), make the immunocytochemistry reaction of spinal cord slice of Vector goat-anti rabbit ABC test kit.Detect the optical density value OD of each treated animal spinal cord slice grey matter (Rexed II-VII layer) immune reaction product with microscope spectrophotometer (Nikon-CA) scanning.A, the OD of B group compares with the C group analysis as 100% (SEM=10).The OD value of result: C group waist marrow is 200% (SEM=10), and the OD value of sacrum tail marrow is respectively 185% (SEM=12), 180% (SEM=12); With A, the B group is compared all has notable difference (p＜0.05).The anti-P material of OD sxemiquantitative description of test rabbit IgG has entered the waist marrow and has had transcellular transport further to arrive the Rexed II-VII layer grey matter of sacrum tail marrow.

The P material is the pain mediator, anti-P material IgG can in and the function of P material and bring into play anti-pain effect.The anti-P material of surrounding injection IgG, its anti-pain effect can only show the part of injection at most momentaryly, and because the dilution that blood follows, effect is also not obvious.In this experiment, the anti-P material of the rabbit IgG that experimental results show that of immunocytochemical assay has really entered the Rexed II-VII layer grey matter of waist sacrum tail marrow in the anti-P material of the CB-rabbit IgG.Injection site (quadriceps femoris) is the domination of waist marrow, and not transcellular axoplasmic transport only arrives the waist marrow; Originally experimental results show that the transcellular transport (reaching sacrum tail marrow from the transhipment of waist marrow) of anti-P material IgG at spinal cord.The antibody that the enters sacrum tail marrow pain mediator P material that neutralized, this is the mechanism place (the whipping motion is the domination of sacrum tail marrow) that the anti-P material of CB-rabbit IgG can improve the animal whipping threshold of pain.Continued the anti-pain effect in a period, can only enter the explanation that is used for of sacrum tail spinal center with anti-P material IgG.

Functional IgG antibody is in the proof once more of nervus centralis transcellular transport, illustrating that input marrowbrain in CB-therapeutic antibodies IgG coupling conjugate targeting ground can have excellent curative, also is that design CB-Anti betaA via intranasal application nervi olfactory enters the experiment basis [beta amyloid 1-42 has a large amount of infrastest documents to the toxicity of brain] of brain treatment senile dementia feasibility.Enter the toxicant betaamyloid 1-42 that brain neutralization causes senile dementia with CB-Anti betaA IgG collunarium, and compound CB-NGF collunarium to enter brain protection's forebrain cholinergic neuron should be that the AD senile dementia is controlled method for root preferably.Current, still there is not the medicine of taking into account AD senile dementia two big pathogeny (cholinergic theory and beta Amyloid toxicity theory), the compound recipe that CB-NGF of the present invention adds CB-Anti betaA provides this medicine for the first time.

EXPERIMENTAL EXAMPLE 3:(sees Fig. 6 photo 1-12 and Fig. 7)

The animal of CB-CNTF and CB-MnA (motor neuron antigen protein) treatment motor neuron

Experiment

The coupling preparation of I.CB-CNTF and CB-MnA (motor neuron antigen protein) (having seen preparation embodiment 4-5): use S-S method and glutaraldehyde GA method respectively.CNTF is available from Sigma; Motor neuron antigen protein (MnA): press Engelhardt et al., method (J NeurosciMethods 1985,15:219) slaughter the cervical part of esophagus of pig by aquatic foods and go up chest section spinal cord and separate anterior angle motor neuron cell space [seeing Fig. 6 photo 1] and do homogenate, electrophoresis extracts high molecular (150-180kD) albumen as MnA.

II. the immune animal model of motor neuron (MND): anterior angle tissue homogenate (SAH) 1ml (containing albumen 6mg) adds the complete freund adjuvant of 1ml and is inoculated in Lewis rat back subcutaneous (10 point) with the Medulla Sus domestica after just butchering (neck and go up the breast section), one week the back do inoculation for the second time with same dose (full freund adjuvant toos many or too much for use).After second week different back myasthenia of limbs of degree, paralysis, weight loss (Fig. 6 photo 9-10) appear promptly, last respiratory failure death.Histopathologic slide checks visible anterior horn motor neurons degeneration and forfeiture; The serum of MND animal pattern (diluting 1: 800) can be used as the anterior angle motor cell that first antibody is used for immunohistochemical reaction, can dyes another rat or mice, the SABC positive (Fig. 6 photo 8) proves the specific antibody that has anti-motor neuron in the serum.Normal control Lewis rat (same adjuvant subcutaneous injection) no any symptom and myeloid tissue's pathological change, its serum (diluting 1: 40) can not dye the anterior angle motor cell of rat or mice, SABC feminine gender (Fig. 6 photo 7) illustrates the specific antibody that does not wherein have anti-motor neuron.

The obvious curative effects [Fig. 7] of III.CB-CNTF and CB-MnA treatment motor neuron:

1 group): the Lewis rat of 5 immunologic injury motor neurons (MND), all dead in 26 days after the SAH immunity inoculation.Immunity inoculation ' slope climbing movement function test ' scoring after one week: [immunity inoculation allows animal climb the long slope of 30 degree 50cm after one week to the 2-0 branch, tests weekly 2 times; The intact animal climbs fluent smooth, and having strong support of hind leg is 5 minutes; The hind leg support force is little, but still can climb 30-40cm, is 4 minutes; 20-30cm is 3 minutes; 10-20cm is 2 minutes; Hind leg supports unable, but can step slightly, is 1 minute; Can not step, hind leg drags Vent, paralysis (as Fig. 6 photo 10) backward, is 0 minute]

2 groups): through the Lewis rat of 5 immunologic injury MND in 2 weeks of CB-MnA/MnA nose dropping treatment, in 4 after the immunity inoculation between 6 weeks dead 2, all the other 3 survivals.[the treatment phase is the 1-3 week after the immunity inoculation; Collunarium dosage is for being equivalent to the CB-MnA/MnA liquid 20ul of MnA 30mu g at every turn, the next day once].Immunity inoculation ' slope climbing movement function test ' scoring after one week: 3-0 branch; The rat of 3 survivals is the 3-4 branch in 6 later scorings of week.Experiment shows that the CB-MnA/MnA collunarium has therapeutic effect.[CB in the CB-MnA/MnA mixed liquor of this experiment has been the adjuvant effect of mucosa-immune, and CB-MnA content is low.Through with 1) serum of group rat is the immunohistochemical reaction that the first antibody of SABC is done the section of this treated animal olfactory bulb, it is faint to dye, and shows that also the afferent retrograde axoplasmic transport of CB-MnA is seldom].

3 groups): through the Lewis rat of 5 immunologic injury MND in 2 weeks of CB-CNTF intramuscular injection for treating in 4 after the immunity inoculation between 6 weeks dead 2, all the other 3 survivals.[the treatment phase is postvaccinal 1-3 week; The dosage of intramuscular injection once, divides 10 quadriceps femoris, biceps brachii m. and neck portion fleshes that are injected in bilateral for be equivalent to the CB-CNTF 100ul of CNTF 4 μ g the next day at every turn].Immunity inoculation ' slope climbing movement function test ' scoring after one week: 3-0 branch; The rat of 3 survivals is the 3-4 branch in 6 later scorings of week.Experiment shows that the CB-CNTF intramuscular injection has therapeutic effect.

4 groups): through the compound again CB-MnA/MnA nose dropping treatment of CB-CNTF intramuscular injection for treating, the Lewis rat that makes 5 immunologic injury MND of 3 courses of treatment in week altogether all survived for 12 weeks none death.The dosage of intramuscular injection for the CB-CNTF 100ul that at every turn is equivalent to CNTF 4 μ g once a day, divide 10 quadriceps femoris, biceps brachii m. and neck portion fleshes that are injected in bilateral].Immunity inoculation ' slope climbing movement function test ' scoring after one week is continuously the 4-5 branch always.Experiment shows that the compound CB-MnA/MnA collunarium of CB-CNTF intramuscular injection has therapeutic effect clearly.

Histopathologic slide checks 1 group), 2 groups) and 3 groups) all visible anterior horn motor neurons degeneration of dead animal and forfeiture.(Fig. 6 photo 2,4,5,6 compares with normal right photo 3)

2 groups), 3 groups) rat of survival and 4 groups) survival rats, sacrifice in 12 weeks and to do the tissue slice inspection, do not find the myelin fiber forfeiture of tangible spinal cord anterior angle motor neuron degeneration and phrenic nerves.With its serum as first antibody (diluting 1: 40) all can not with the spinal motor nerve cell generation immunohistochemical reaction (just like Fig. 6 photo 7) of Wistar rat.

Do the intramuscular injection of equal metering at moderate immunologic injury rat tibialis anterior and normal rat tibialis anterior with the horseradish peroxidase (HRP) of non-binding attitude, do the group reaction that shows the retrograde axoplasmic transport of HRP, both dyeing does not have big difference (Fig. 6 photo 11,12).The cell column of the retrograde labelling of moderate immunologic injury rat does not have big difference at the cell of more section place of cell number (Fig. 6 photo 12) and the less place of normal rat cell number (Fig. 6 photo 11) engrain, even very graver.Even illustrate moderate immunologic injury rat, the function of the axoplasmic transport that drives in the wrong direction still exists, and can transport CB coupling preparation.

In Fig. 7, shown the survival comparison diagram of the compound CB-MnA/MnA treatment of CB-CNTF immunologic injury MND rat.

Square frame: death

Triangle arrow: treatment

Arrow 0: angled tissue (SAH) before the immunity inoculation Medulla Sus domestica

Vertical coordinate: number of animals (the Lewis rat of immunologic injury)

Abscissa: all numbers of surviving

1 group of (red line) MND (motor neuron) immunologic injury model (3-4 week is all dead)

2 groups of (green line) CB-MnA (pig motor neuron antigen protein) treat MND immunologic injury (2 death, 3 survivals)

3 groups of (medium green line) CB-CNTF (ciliary neurotrophic factor) treat MND immunologic injury (2 death, 3 survivals)

4 groups of (thick green line) CB-CNTF and CB-MnA combination therapy MND immunologic injury (all survivals)

Above description of test CB-CNTF, the combination therapy of CB-MnA have made immunologic pattern MND animal model obtain the survival healing, and non-evident effect.

Human MND (lateral sclerosis myasthenia ALS, amyotrophic lateral sclerosis etc.) is very harmful incurable disease, morbidity in 40 to 70 years old, and several whole death in the 3-5, etiology unknown does not still have specific medicament at present.[research work that has is thought: the genovariation of coding Cu/Zn SOD sudismase may be relevant with it].CNTF is found in the experimental work of twentieth century nineties, and BDNF can protect motor neuron, and it is very big because of side effect once to make clinic trial (subcutaneous administration) with CNTF, fails at clinical expansion [Clin Neuropharmacol 1995,18 (6) 515-32; ArchNeurol 1996,53 (2) 141-7].CB-CNTF of the present invention, CB-MnA/MnA compound recipe have very big medical applications and are worth.

EXPERIMENTAL EXAMPLE 3 illustrated the mucosa-immune of the former CB-MnA/MnA of CB adjuvant autoimmune regulate (to the former toleration that produced of autoimmune) compound with the CB-neurotrophic factor in conjunction with controlling agent (CB-CNTF, CB-BDNF etc.) the targeting neurotrophic treatment of neural transhipment dispensing, can be alleviate, treatment and prevention now be the best strategy of the degeneration neuropathy motor neuron ALS of incurable disease etc.

Claims (9)

1. choleragen B subunit is as the conjugate of aglucon/carrier and neurotrophic factor.

2. according to the conjugate of claim 1, wherein said neurotrophic factor is selected from nerve growth factor, ciliary neurotrophic factor, Brain Derived Neurotrophic Factor, glial cell derived neurotrophic factor and insulin like growth factor.

3. pharmaceutical composition, it contains one or more conjugates and the pharmaceutically useful adjuvant and the reinforcing agent of with good grounds claim 1 or 2.

4. according to the preparation method of the conjugate of claim 1 or 2, it is characterized in that choleragen B subunit and neurotrophic factor are coupled together the formation conjugate, and described conjugate keep the biological activity of choleragen B subunit and neurotrophic factor.

5. according to the preparation method of claim 4, it is characterized in that the amino of choleragen B subunit or the carboxyl or the amino process aldehyde radical of carboxyl and neurotrophic factor are coupled together; Or import sulfydryl or dithione couples together with disulfide bond, form conjugate, and described conjugate has kept the biological activity of choleragen B subunit and neurotrophic factor.

6. the conjugate according to claim 1 or 2 is preparing the purposes that enters the medicine of nervus centralis through the receptor-mediated born of the same parents of going into of peripheral nervous, axoplasmic transport.

7. according to the purposes of claim 6, the ganglioside oligosaccharide of wherein said conjugate on neurolemma be receptor-mediated goes into born of the same parents, axoplasmic transport, from around enter the central nervous system, and further in marrowbrain, do the nerve transhipment dispensing of transcellular transport.

8. be used for the treatment of according to the preparation of the conjugate of claim 1 or 2 and/or the purposes of the medicine of prevention of brain diseases of spinal cord or damage.

9. purposes according to Claim 8, wherein said marrowbrain i or I comprises: the degeneration neuropathy, it is selected from the extra large Mo Shi alzheimer disease of Ah taste, parkinson, motor neuron; Cerebral spinal cord injury, it is selected from apoplexy, cerebral thrombosis, cerebral ischemia, cerebral trauma and trauma of spinal cord.

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