CN103108657A - A method of increasing the effect of an activated-potentiated form of an antibody - Google Patents

A method of increasing the effect of an activated-potentiated form of an antibody Download PDF

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CN103108657A
CN103108657A CN2011800443324A CN201180044332A CN103108657A CN 103108657 A CN103108657 A CN 103108657A CN 2011800443324 A CN2011800443324 A CN 2011800443324A CN 201180044332 A CN201180044332 A CN 201180044332A CN 103108657 A CN103108657 A CN 103108657A
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antibody
leu
pro
uld
ala
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奥列格·伊里奇·爱泼斯坦
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Priority claimed from RU2010129294/15A external-priority patent/RU2542414C2/en
Priority claimed from RU2010129290/15A external-priority patent/RU2525155C2/en
Priority claimed from RU2010129298/15A external-priority patent/RU2526153C2/en
Priority claimed from RU2010129295/15A external-priority patent/RU2531049C2/en
Priority claimed from RU2010129291/15A external-priority patent/RU2525156C2/en
Priority claimed from RU2010129292/15A external-priority patent/RU2523557C2/en
Priority claimed from RU2010130356/15A external-priority patent/RU2542453C2/en
Priority claimed from RU2010130358/15A external-priority patent/RU2519695C2/en
Priority claimed from RU2010130348/15A external-priority patent/RU2531048C2/en
Priority claimed from RU2010130353/15A external-priority patent/RU2542445C2/en
Application filed by Individual filed Critical Individual
Priority claimed from PCT/IB2011/002350 external-priority patent/WO2012007845A2/en
Publication of CN103108657A publication Critical patent/CN103108657A/en
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Abstract

The preset invention provides a method of increasing the effect of an activated-potentiated form of an antibody to an endogenous biological molecule by combining said endogenous biological molecule with an activated-potentiated form of an antibody to endothelial NO-synthase. The present invention also provides a pharmaceutical composition comprising a) an activated-potentiated form of an antibody to an endogenous biological molecule, and b) an activated-potentiated form of an antibody to NO synthase.

Description

Improve the method for effect of the antibody of active augmented form
Technical field
The present invention relates to improve the method for effect of the antibody of active augmented form (activated-potentiated form); The invention still further relates to the pharmaceutical preparation of the anti-endothelial NO synthase antibody of the anti-endogenous biomolecule antibody that comprises active augmented form and active augmented form.
Background technology
Nitric oxide (NO) is to have demonstrated the gaseous molecular that plays a role in the signal transduction of different biological process.The NO of endothelium derivation is the key molecule of regulating antiotasis (vascular tone), and itself and contacting already of angiopathy are approved.NO plays inhibitory action to many known processes relevant to atheromatous plaque (atherosclerotic plaque) formation, and described process comprises mononuclear cell adhesion, platelet aggregation and vascular smooth muscle cell proliferation.Another important function of endothelial NO is that the oxidative stress (oxidative stress) that the protection blood vessel wall is not subjected to be induced by the oxidation product of himself metabolite and lipid and lipoprotein damages.Endothelial function disturbance occured in the atherosclerotic stage very early.Therefore, local NO availability deficiency may be the final road (final common pathway) altogether that promotes mankind's atherogenesis.Except the effect in blood vessel endothelium, the NO availability has also showed the regulating action to lipoprotein metabolism.The negative correlation of the plasma concentration of NO metabolite and plasma total cholesterol levels and low density lipoprotein, LDL (LDL) cholesterol level level has been reported, and high density lipoprotein (HDL) improves vascular function in hypercholesterolemiapatients patients.The NO disappearance has material impact in disease progression.Diabetes rise relevant with the M ﹠ M of mainly being accelerated to cause by the atheromatosis development.And report shows that the pulmonary function of diabetics weakens.Someone proposes insulin resistant can cause airway inflammation.Habib etc., Nitric Oxide Measurement From Blood To Lungs, Is There A Link? Pak J Physiol2007; 3 (1).
Nitric oxide is synthetic by L-arginine by nitricoxide synthase (NO synthase) by endothelium.The NO synthase occurs with different subtype, comprises composing type (cNOS) and induction type (iNOS).Composing type NO synthase is present in normal endothelial cell, neuron and some other tissue.
Dr.Oleg I.Epshtein has found the therapeutic effect of extreme dilute form (or the extremely low form) antibody (active augmented form) through homeopathic therapeutic method's technique.U.S. the patent No. 7,700, and 096 discloses the anti-endothelial NO synthase antibody of homeopathic therapeutic method's augmented form.The anti-endothelial NO synthase antibody of described homeopathic therapeutic method's augmented form in the Russian Federation and other country with trade name Sell.
Exist for the method for the effect of the antibody that improves active augmented form and continue demand.
Summary of the invention
According to an aspect, the invention provides a kind of method of effect of the anti-endogenous biomolecule antibody that improves active augmented form, described method comprises the anti-endothelial NO synthase of the anti-endogenous biomolecule antibody of described active augmented form and active augmented form antibody combined.Preferably, method of the present invention aspect comprises the patient who gives to utilize the antibody of described active augmented form to treat this complex.
In a modification, the anti-endogenous biomolecule antibody of described active augmented form is anti-S-100 protein antibodies.In another modification, the anti-endogenous biomolecule antibody of described active augmented form is anti-prostate specific antigen antibody.In another modification, the anti-endogenous biomolecule antibody of described active augmented form is anti-insulin receptor antibody.In another modification, the anti-endogenous biomolecule antibody of described active augmented form is antiangiotensin receptor II antibody.
According to another aspect, the invention provides pharmaceutical composition, described pharmaceutical composition comprises a) the anti-endogenous biomolecule antibody of active augmented form; And b) the anti-NO synthase antibody of active augmented form.Preferably, described pharmaceutical composition comprises pharmaceutically acceptable solid-state carrier.Preferably, the anti-endothelial NO synthase antibody of described active augmented form comprises the mixture of C12, C30 and C200 homeopathic therapeutic method diluent (homeopathic dilutions), and described impregnation mixture is to described solid-state carrier.The anti-endogenous biomolecule antibody of described active augmented form can be monoclonal antibody, polyclonal antibody or natural antibody.Preferably, described anti-human insulin receptor antibody is polyclonal antibody.
What be taken into account is, described pharmaceutical composition comprises by continuous Radix Achyranthis Bidentatae dilution (successive centesimal dilutions) and accompanies by the anti-endogenous biomolecule antibody of the active augmented form that vibration prepares at every turn when diluting.What be taken into account equally is that anti-endothelial NO synthase antibody is monoclonal antibody, polyclonal antibody or natural antibody.Particularly preferably being described anti-endothelial NO synthase antibody is polyclonal antibody.What be taken into account is that the anti-endothelial NO synthase antibody of described active augmented form accompanies by vibration when diluting and diluting at every turn by continuous Radix Achyranthis Bidentatae and prepares.
Description of drawings
Fig. 1: the impact of expression test formulation on the rat plasma glucose level, described rat suffers from the diabetes that streptozotocin (streptozotocin) is induced.
Fig. 2: be illustrated in the glucose tolerance test of rat, test formulation is in injection impact on area under the concentration-time curve (AUC) index (indicator) in the time of the 14th day, and described rat suffers from the diabetes that streptozotocin is induced.
Fig. 3: the impact of expression test formulation on the rat plasma glucose level, described rat suffers from spontaneous non-insulin-dependent diabetes mellitus.
Fig. 4: be illustrated in the glucose tolerance test of rat, test formulation is in injection impact on area under the concentration-time curve (AUC) index in the time of the 28th day, and described rat suffers from spontaneous non-insulin-dependent diabetes mellitus.
The specific embodiment
With reference to appending claims, the present invention is limited.Consider claims, following glossary provides relevant definition.
Term as used herein " antibody " means the particular space that is bonded to specifically another molecule and polar structure, and therefore is defined as and the particular space of another molecule and the immunoglobulin of polar structure complementation.In claims, cited antibody can comprise complete immunoglobulin or its fragment, can be natural antibody, polyclonal antibody or monoclonal antibody, and can comprise a plurality of classes and isotype, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM etc.The fragment of immunoglobulin can comprise Fab, Fv and F (ab ') 2And Fab ' etc.Odd number " antibody (antibody) " comprises plural number " antibody (antibodies) ".
With respect to antibody cited herein, term " active augmented form " or " augmented form " are respectively used to represent that the homeopathic therapeutic method of antibody initial soln strengthens product arbitrarily." homeopathic therapeutic method's reinforcement " expression utilizes hahnemannian method to give homeopathic therapeutic method's effect (potency) to the initial soln of related substance.Although be not limited to this, " homeopathic therapeutic method's reinforcement " can comprise for example in conjunction with external treatment, the especially vertically serial dilution of the repetition of (machinery) vibration.In other words, according to homeopathic therapeutic method's technology, the initial soln of antagonist carries out that continuous repeating diluted and the solution of each acquisition is repeatedly vertically vibrated.The preferred concentration range for that antibody is in the initial soln in solvent (preferred water or water-ethanol admixture) be approximately 0.5mg/ml to about 5.0mg/ml.The preferred process for preparing each component (being antibody-solutions) is: use antibody primary matrix solution (primary matrix solution) (mother tincture, mother tincture) to be diluted respectively 100 12, 100 30With 100 2003 kinds of water diluents doubly or the mixture of water-pure diluent are equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent (C12, C30 and C200); Perhaps use antibody primary matrix solution to be diluted respectively 100 12, 100 30With 100 503 kinds of water diluents doubly or the mixture of water-pure diluent are equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent (C12, C30 and C50).At U.S. Patent number 7,572, the example that the homeopathic therapeutic method strengthens has been described in 441 and 7,582,294, by reference its content whole is incorporated into this paper and be used for described purpose.Simultaneously, term " active augmented form " is used in claims, and term " very low dose " is used in an embodiment.Term " very low dose " becomes industry slang in the field that the material by research and use homeopathic therapeutic method dilution and augmented form produces.Term " very low dose " mean term " the active reinforcement " form supporting to use in claims fully and with claims in term " the active reinforcement " form used synonym basically.
In other words, when having three factors, antibody is in " the active reinforcement " or " reinforcement " form.At first, the product of the preparation method accepted extensively for the homeopathic therapeutic method field of the antibody of " active strengthen " form.Secondly, the antibody of " the active reinforcement " form must possess the definite biological activity of method of accepting extensively by modern pharmacology.The 3rd, the biological activity that " the active reinforcement " antibody of form shows can not be explained by the existence of the molecular forms antibody in homeopathic therapeutic method's method end-product.
For example, the continuous several times dilution that can stand by the antibody that makes separation initial, that be in molecular forms associating external action (as mechanical oscillation) of the antibody of active augmented form prepares.External treatment in concentration reduction process also can be ultrasonic by for example being exposed to, electromagnetism or other physical factor are completed.V.Schwabe, " Homeopathic medicines ", M., 1967, U.S. Patent number 7,229, homeopathic therapeutic method's intensifying method that the homeopathic therapeutic method field is accepted extensively was described 648 and 4,311,897 (by reference its content whole is incorporated into this paper and be used for described purpose).This process makes the molecular concentration of initial molecular forms antibody evenly reduce.Repeat this process until obtain homeopathic therapeutic method's effect of expectation.For independent antibody, can determine required homeopathic therapeutic method's effect by intermediate dilute liquid is carried out biological test in the pharmacology model of expectation.Although be not limited to this, " homeopathic therapeutic method's reinforcement " can comprise the serial dilution of the repetition of for example being combined with external treatment, especially vertical (machinery) oscillating phase.In other words, according to homeopathic therapeutic method's technology, the initial soln of antagonist (initial solution) carries out that continuous repeating diluted and the solution of each acquisition is repeatedly vertically vibrated.The preferred concentration range for that antibody is in the initial soln in solvent (preferred water or water-ethanol admixture) be approximately 0.5mg/ml to about 5.0mg/ml.The preferred process for preparing each component (being antibody-solutions) is: use antibody primary matrix solution (mother tincture) to be diluted respectively 100 12, 100 30With 100 2003 kinds of water diluents doubly or the mixture of water-pure diluent are equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; Perhaps use antibody primary matrix solution (mother tincture) to be diluted respectively 100 12, 100 30With 100 503 kinds of water diluents doubly or the mixture of water-pure diluent are equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C50.For example at U.S. Patent number 7,229, in 648 and 4,311,897, also provide the example that how to obtain to expect effect, incorporate it into this paper with way of reference and be used for described purpose.The process that is applicable to " the active reinforcement " as herein described form antibody will be described hereinafter in further detail.
About with the homeopathic therapeutic method, the human experimenter being treated existing many disputes.Although the present invention relies on accepted homeopathic therapeutic method's method to obtain the antibody of " active strengthen " form, it not merely depends on and carries out the homeopathic therapeutic method prove its activity in the human experimenter.The present inventor finds and sufficient proof in accepted pharmacology model beyond expectationly, carry out the continuous several times dilution and the solution that finally obtains has clear and definite activity by the antibody of starting molecule form, and irrelevant with the existence of trace molecular forms antibody in the target diluent.The antibody of " active strengthen " form that this paper is provided test its biological activity in the pharmacological activity model of accepting extensively, carry out in the in vivo test of described test in suitable experiment in vitro or suitable animal model.The experiment that hereinafter further provides provides the bioactive evidence in this class model.Mankind's clinical research provides the activity that observes in animal model to be converted into fully human treatment's evidence equally.Form that the human research provides equally " the active reinforcement " described herein is for the specific human diseases for the treatment of or the effective evidence of obstacle (disorder), and described disease or obstacle are widely accepted in medical science and are pathological conditions (condition).
Equally; the antibody of " the active reinforcement " required for protection form is only contained solution or solid preparation, and the biological activity of described solution or solid preparation can not be made an explanation by the existence of molecular forms antibody remaining in initial, starting soln (starting solution).In other words, although can comprising the initial molecular forms antibody of trace, the antibody of " the active reinforcement " form also is taken into account, but because the concentration of molecular forms antibody remaining after serial dilution is extremely low, so the biological activity that those skilled in the art can not will observe in accepted pharmacology model with the reasonability of any degree is owing to remaining molecular forms antibody.Although the present invention is not bound to any specific theory, the biological activity of " the active reinforcement " of the present invention form antibody is not owing to initial molecular forms antibody.Preferably described " the active reinforcement " form antibody is in liquid form or solid form, and the concentration of wherein said molecular forms antibody is lower than the detectability of received analytical technology (as capillary electrophoresis and high performance liquid chromatography).Particularly preferably described " the active reinforcement " form antibody is in liquid form or solid form, and the concentration of wherein said molecular forms antibody is lower than Avogadro's number.In the materia medica of molecular forms therapeutant, usually make dose-response curve, in this curve, map to given experimenter or in the concentration of the external active medicine of testing with pharmacology's level of response.The medicine floor level that produces any detectable response is called as threshold dose (threshold dose).Be taken into account especially and preferably, the antibody of " the active reinforcement " form with lower than the concentration of threshold dose of the molecular forms antibody in given biological model comprise molecular antibody (if any).
This combination drug compositions on the one hand can be in liquid form or solid form according to the present invention.The method preparation that in pharmaceutical composition, contained each active augmented form antibody is accepted by the homeopathic therapeutic method field by initial molecular forms antibody.Initial antibody can be monoclonal antibody or the polyclonal antibody according to the known method preparation, and described known method is Immunotechniques for example, G.Frimel, M., " Meditsyna ", 1987, the 9-33 pages; " Hum.Antibodies.Monoclonal and recombinant antibodies, 30years after ", Laffly E., Sodoyer R. work, 2005, Vol.14., N1-2. described in the 33-55 page, incorporates its content into this paper by reference.
Monoclonal antibody can be by obtaining as hybridoma technology.The initial step of described method comprises based on the principle of having developed in the polyclonal antiserum preparation process carries out immunity.The further step of work comprises preparing to produce to have the hybrid cell of mutually homospecific antibody cloning.Its separation use separately prepares method identical in situation with polyclonal antiserum and carries out.
Polyclonal antibody can obtain by the active immunity of animal.For this purpose, for example make suitable animal (as rabbit) accept a series of injections of suitable antigen (for example NO synthase).The immune system of animal produces corresponding antibody, collects from animal with known method.This process makes it possible to prepare the serum that is rich in monospecific antibody.
If necessary, the serum that comprises antibody for example can carry out purification by using affinity chromatography, the separation of salt precipitation fractionation or ion exchange chromatography.Can be with the parent material of resulting purified, serum that be rich in antibody as the active augmented form antibody of preparation.The resulting preferred concentration range for that is in the initial soln of the antibody in solvent (preferred water or water-ethanol admixture) be approximately 0.5mg/ml to about 5.0mg/ml.
The preferred process for preparing each component of combination drug of the present invention is: use antibody primary matrix solution to be diluted respectively 100 12, 100 30With 100 50The mixture of 3 kinds of water-pure diluents doubly is equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C50; Perhaps use antibody primary matrix solution to be diluted respectively 100 12, 100 30With 100 200The mixture of 3 kinds of water-pure diluents doubly is equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.For the preparation solid dosage forms, the solid-state carrier utilization is processed by the expectation diluent that homeopathic therapeutic method's method obtains.For obtaining the solid unit dosage form of complex of the present invention, with each diluent, carrier mass is flooded.Be the compound dosage form of preparation expectation, two kinds of impregnation sequences all are fit to.
In preferred embodiment, be that animal for corresponding antigens (being NO synthase and endogenous biomolecule) produces polyclonal antibody for the preparation of the parent material of the active augmented form that comprises complex of the present invention.For obtaining the anti-NO synthase polyclonal antibody of active augmented form, the antigen of expectation can be injected in laboratory animal, preferred rabbit as immunogen.Anti-NO synthase polyclonal antibody can use the whole molecule of the cattle NO synthase of following sequence to obtain:
SEQ?ID?NO：1
Met?Gly?Asn?Leu?Lys?Ser?Val?Gly?Gln?Glu?Pro?Gly?Pro?Pro?Cys
1 5 10 15
Gly?Leu?Gly?Leu?Gly?Leu?Gly?Leu?Gly?Leu?Cys?Gly?Lys?Gln?Gly
16 20 25 30
Pro?Ala?Ser?Pro?Ala?Pro?Glu?Pro?Ser?Arg?Ala?Pro?Ala?Pro?Ala
31 35 40 45
Thr?Pro?His?Ala?Pro?Asp?His?Ser?Pro?Ala?Pro?Asn?Ser?Pro?Thr
46 50 55 60
Leu?Thr?Arg?Pro?Pro?Glu?Gly?Pro?Lys?Phe?Pro?Arg?Val?Lys?Asn
61 65 70 75
Trp?Glu?Leu?GLys?er?Ile?Thr?Tyr?Asp?Thr?Leu?Cys?Ala?Gln?Ser
76 80 85 90
Gln?Gln?Asp?Gly?Pro?Cys?Thr?Pro?Arg?Cys?Cys?Leu?GLys?er?Leu
91 95 100 105
Val?Leu?Pro?Arg?Lys?Leu?Gln?Thr?Arg?Pro?Ser?Pro?Gly?Pro?Pro
106 110 115 120
Pro?Ala?Glu?Gln?Leu?Leu?Ser?Gln?Ala?Arg?Asp?Phe?Ile?Asn?Gln
121 125 130 135
Tyr?Tyr?Ser?Ser?Ile?Lys?Arg?Ser?GLys?er?Gln?Ala?His?Glu?Glu
136 140 145 150
Arg?Leu?Gln?Glu?Val?Glu?Ala?Glu?Val?Ala?Ser?Thr?Gly?Thr?Tyr
151 155 160 165
His?Leu?Arg?Glu?Ser?Glu?Leu?Val?Phe?Gly?Ala?Lys?Gln?Ala?Trp
166 170 175 180
Arg?Asn?Ala?Pro?Arg?Cys?Val?Gly?Arg?Ile?Gln?Trp?Gly?Lys?Leu
181 185 190 195
Gln?Val?Phe?Asp?Ala?Arg?Asp?Cys?Ser?Ser?Ala?Gln?Glu?Met?Phe
196 200 205 210
Thr?Tyr?Ile?Cys?Asn?His?Ile?Lys?Tyr?Ala?Thr?Asn?Arg?Gly?Asn
211 215 220 225
Leu?Arg?Ser?Ala?Ile?Thr?Val?Phe?Pro?Gln?Arg?Ala?Pro?Gly?Arg
226 230 235 240
Gly?Asp?Phe?Arg?Ile?Trp?Asn?Ser?Gln?Leu?Val?Arg?Tyr?Ala?Gly
241 245 250 255
Tyr?Arg?Gln?Gln?Asp?GLys?er?Val?Arg?Gly?Asp?Pro?Ala?Asn?Val
256 260 265 270
Glu?Ile?Thr?Glu?Leu?Cys?Ile?Gln?His?Gly?Trp?Thr?Pro?Gly?Asn
271 275 280 285
Gly?Arg?Phe?Asp?Val?Leu?Pro?Leu?Leu?Leu?Gln?Ala?Pro?Asp?Glu
286 290 295 300
Ala?Pro?Glu?Leu?Phe?Val?Leu?Pro?Pro?Glu?Leu?Val?Leu?Glu?Val
301 305 310 315
Pro?Leu?Glu?His?Pro?Thr?Leu?Glu?Trp?Phe?Ala?Ala?Leu?Gly?Leu
316 320 325 330
Arg?Trp?Tyr?Ala?Leu?Pro?Ala?Val?Ser?Asn?Met?Leu?Leu?Glu?Ile
331 335 340 345
Gly?Gly?Leu?Glu?Phe?Ser?Ala?Ala?Pro?Phe?Ser?Gly?Trp?Tyr?Met
346 350 355 360
Ser?Thr?Glu?Ile?Gly?Thr?Arg?Asn?Leu?Cys?Asp?Pro?His?Arg?Tyr
361 365 370 375
Asn?Ile?Leu?Glu?Asp?Val?Ala?Val?Cys?Met?Asp?Leu?Asp?Thr?Arg
376 380 385 390
Thr?Thr?Ser?Ser?Leu?Trp?Lys?Asp?Lys?Ala?Ala?Val?Glu?Ile?Asn
391 395 400 405
Leu?Ala?Val?Leu?His?Ser?Phe?Gln?Leu?Ala?Lys?Val?Thr?Ile?Val
406 410 415 420
Asp?His?His?Ala?Ala?Thr?Val?Ser?Phe?Met?Lys?His?Leu?Asp?Asn
421 425 430 435
Glu?Gln?Lys?Ala?Arg?Gly?Gly?Cys?Pro?Ala?Asp?Trp?Ala?Trp?Ile
436 440 445 450
Val?Pro?Pro?Ile?Ser?GLys?er?Leu?Thr?Pro?Val?Phe?His?Gln?Glu
451 455 460 465
Met?Val?Asn?Tyr?Ile?Leu?Ser?Pro?Ala?Phe?Arg?Tyr?Gin?Pro?Asp
466 470 475 480
Pro?Trp?Lys?GLy?Ser?Ala?Thr?Lys?Gly?Ala?Gly?Ile?Thr?Arg?Lys
481 485 490 495
Lys?Thr?Phe?Lys?Glu?Val?Ala?Asn?Ala?Val?Lys?Ile?Ser?Ala?Ser
496 500 505 510
Leu?Met?Gly?Thr?Leu?Met?Ala?Lys?Arg?Val?Lys?Ala?Thr?Ile?Leu
511 515 510 525
Tyr?Ala?Ser?Glu?Thr?Gly?Arg?Ala?Gln?Ser?Tyr?Ala?Gln?Gln?Leu
526 530 535 540
Gly?Arg?Leu?Phe?Arg?Lys?Ala?Phe?Asp?Pro?Arg?Val?Leu?Cys?Met
541 545 550 555
Asp?Glu?Tyr?Asp?Val?Val?Ser?Leu?Glu?His?Glu?Ala?Leu?Val?Leu
556 560 565 570
Val?Val?Thr?Ser?Thr?Phe?Gly?Asn?Gly?Asp?Pro?Pro?Glu?Asn?Gly
571 575 580 585
Glu?Ser?Phe?Ala?Ala?Ala?Leu?Met?Glu?Met?Ser?Gly?Pro?Tyr?Asn
586 590 595 600
Ser?Ser?Pro?Arg?Pro?Glu?Gln?His?Lys?Ser?Tyr?Lys?Ile?Arg?Phe
601 605 610 615
Asn?Ser?Val?Ser?Cys?Ser?Asp?Pro?Leu?Val?Ser?Ser?Trp?Arg?Arg
616 620 625 630
Lys?Arg?Lys?Glu?Ser?Ser?Asn?Thr?Asp?Ser?Ala?Gly?Ala?Leu?Gly
631 635 640 645
Thr?Leu?Arg?Phe?Cys?Val?Phe?Gly?Leu?GLy?Ser?Arg?Ala?Tyr?Pro
646 650 655 660
His?Phe?Cys?Ala?Phe?Ala?Arg?Ala?Val?Asp?Thr?Arg?Leu?Glu?Glu
661 665 670 675
Leu?Gly?Gly?Glu?Arg?Leu?Leu?Gln?Leu?Gly?Gln?Gly?Asp?Glu?Leu
676 680 685 690
Cys?Gly?Gln?Glu?Glu?Ala?Phe?Arg?Gly?Trp?Ala?Lys?Ala?Ala?Phe
691 695 700 705
Gln?Ala?Ser?Cys?Glu?Thr?Phe?Cys?Val?Gly?Glu?Glu?Ala?Lys?Ala
706 710 715 720
Ala?Ala?Gln?Asp?Ile?Phe?Ser?Pro?Lys?Arg?Ser?Trp?Lys?Arg?Gln
721 725 730 735
Arg?Tyr?Arg?Leu?Ser?Thr?Gln?Ala?Glu?Gly?Leu?Gln?Leu?Leu?Pro
736 740 745 750
Gly?Leu?Ile?His?Val?His?Arg?Arg?Lys?Met?Phe?Gln?Ala?Thr?Val
751 755 760 765
Leu?Ser?Val?Glu?Asn?Leu?Gln?Ser?Ser?Lys?Ser?Thr?Arg?Ala?Thr
766 770 775 780
Ile?Leu?Val?Arg?Leu?Asp?Thr?Ala?Gly?Gln?Glu?Gly?Leu?Gln?Tyr
781 785 790 795
Gln?Pro?Gly?Asp?His?Ile?Gly?Ile?Cys?Pro?Pro?Asn?Arg?Pro?Gly
796 800 805 810
Leu?Val?Glu?Ala?Leu?Leu?Ser?Arg?Val?Glu?Asp?Pro?Pro?Pro?Pro
811 815 820 825
Thr?Glu?Ser?Val?Ala?Val?Glu?Gln?Leu?Glu?Lys?GLys?er?Pro?Gly
826 830 835 840
Gly?Pro?Pro?Pro?Ser?Trp?Val?Arg?Asp?Pro?Arg?Leu?Pro?Pro?Cys
841 845 850 855
Thr?Leu?Arg?Gln?Ala?Leu?Thr?Phe?Phe?Leu?Asp?Ile?Thr?Ser?Pro
856 860 865 870
Pro?Ser?Pro?Arg?Leu?Leu?Arg?Leu?Leu?Ser?Thr?Leu?Ala?Glu?Glu
871 875 880 885
Pro?Ser?Glu?Gln?Gln?Glu?Leu?Glu?Thr?Leu?Ser?Gln?Asp?Pro?Arg
886 890 895 900
Arg?Tyr?Glu?Glu?Trp?Lys?Trp?Phe?Arg?Cys?Pro?Thr?Leu?Leu?Glu
901 905 910 915
Val?Leu?Glu?Gln?Phe?Pro?Ser?Val?Ala?Leu?Pro?Ala?Pro?Leu?Leu
916 920 925 930
Leu?Thr?Gln?Leu?Pro?Leu?Leu?Gln?Pro?Arg?Tyr?Tyr?Ser?Val?Ser
931 935 940 945
Ser?Ala?Pro?Asn?Ala?His?Pro?Gly?Glu?Val?His?Leu?Thr?Val?Ala
946 950 955 960
Val?Leu?Ala?Tyr?Arg?Thr?Gln?Asp?Gly?Leu?Gly?Pro?Leu?His?Tyr
961 965 970 975
Gly?Val?Cys?Ser?Thr?Trp?Leu?Ser?Gln?Leu?Lys?Thr?Gly?Asp?Pro
976 980 985 990
Val?Pro?Cys?Phe?Ile?Arg?Gly?Ala?Pro?Ser?Phe?Arg?Leu?Pro?Pro
991 995 1000 1005
Asp?Pro?Tyr?Val?Pro?Cys?Ile?Leu?Val?Gly?Pro?Gly?Thr?Gly?Ile
1006 1010 1015 1020
Ala?Pro?Phe?Arg?Gly?Phe?Trp?Gln?Glu?Arg?Leu?His?Asp?Ile?Glu
1021 1025 1030 1035
Ser?Lys?Gly?Leu?Gln?Pro?Ala?Pro?Met?Thr?Leu?Val?Phe?Gly?Cys
1036 1040 1045 1050
Arg?Cys?Ser?Gln?Leu?Asp?His?Leu?Tyr?Arg?Asp?Glu?Val?Gln?Asp
1051 1055 1060 1065
Ala?Gln?Glu?Arg?Gly?Val?Phe?Gly?Arg?Val?Leu?Thr?Ala?Phe?Ser
1066 1070 1075 1080
Arg?Glu?Pro?Asp?Ser?Pro?Lys?Thr?Tyr?Val?Gln?Asp?Ile?Leu?Arg
1081 1085 1090 1095
Thr?Glu?Leu?Ala?Ala?Glu?Val?His?Arg?Val?Leu?Cys?Leu?Glu?Arg
1096 1100 1105 1110
Gly?His?Met?Phe?Val?Cys?Gly?Asp?Val?Thr?Met?Ala?Thr?Ser?Val
1111 1115 1120 1125
Leu?Gln?Thr?Val?Gln?Arg?Ile?Leu?Ala?Thr?Glu?Gly?Asp?Met?Glu
1126 1130 1135 1140
Leu?Asp?Glu?Ala?Gly?Asp?Val?Ile?Gly?Val?Leu?Arg?Asp?Gln?Gln
1141 1145 1150 1155
Arg?Tyr?His?Glu?Asp?Ile?Phe?Gly?Leu?Thr?Leu?Arg?Thr?Gln?Glu
1156 1160 1165 1170
Val?Thr?Ser?Arg?Ile?Arg?Thr?Gln?Ser?Phe?Ser?Leu?Gln?Glu?Arg
1171 1175 1180 1185
His?Leu?Arg?Gly?Ala?Val?Pro?Trp?Ala?Phe?Asp?Pro?Pro?Gly?Pro
1186 1190 1195 1200
Asp?Thr?Pro?Gly?Pro
1201 1205
Anti-NO synthase polyclonal antibody can use the whole molecule of the people NO synthase of following sequence to obtain:
SEQ?ID?NO：2
Met?Gly?Asn?Leu?Lys?Ser?Val?Ala?Gln?Glu?Pro?Gly?Pro?Pro?Cys
1 5 10 15
Gly?Leu?Gly?Leu?Gly?Leu?Gly?Leu?Gly?Leu?Cys?Gly?Lys?Gln?Gly
16 20 25 30
Pro?Ala?Thr?Pro?Ala?Pro?Glu?Pro?Ser?Arg?Ala?Pro?Ala?Ser?Leu
31 35 40 45
Leu?Pro?Pro?Ala?Pro?Glu?His?Ser?Pro?Pro?Ser?Ser?Pro?Leu?Thr
46 50 55 60
Gln?Pro?Pro?Glu?Gly?Pro?Lys?Phe?Pro?Arg?Val?Lys?Asn?Trp?Glu
61 65 70 75
Val?GLys?er?Ile?Thr?Tyr?Asp?Thr?Leu?Ser?Ala?Gln?Ala?Gln?Gln
76 80 85 90
Asp?Gly?Pro?Cys?Thr?Pro?Arg?Arg?Cys?Leu?GLys?er?Leu?Val?Phe
91 95 100 105
Pro?Arg?Lys?Leu?Gln?Gly?Arg?Pro?Ser?Pro?Gly?Pro?Pro?Ala?Pro
106 110 115 120
Glu?Gln?Leu?Leu?Ser?Gln?Ala?Arg?Asp?Phe?Ile?Asn?Gln?Tyr?Tyr
121 125 130 135
Ser?Ser?Ile?Lys?Arg?Ser?GLys?er?Gln?Ala?His?Glu?Gln?Arg?Leu
136 140 145 150
Gln?Glu?Val?Glu?Ala?Glu?Val?Ala?Ala?Thr?Gly?Thr?Tyr?Gln?Leu
151 155 160 165
Arg?Glu?Ser?Glu?Leu?Val?Phe?Gly?Ala?Lys?Gln?Ala?Trp?Arg?Asn
166 170 175 180
Ala?Pro?Arg?Cys?Val?Gly?Arg?Ile?Gln?Trp?Gly?Lys?Leu?Gln?Val
181 185 190 195
Phe?Asp?Ala?Arg?Asp?Cys?Arg?Ser?Ala?Gln?Glu?Met?Phe?Thr?Tyr
196 200 205 210
Ile?Cys?Asn?His?Ile?Lys?Tyr?Ala?Thr?Asn?Arg?Gly?Asn?Leu?Arg
211 215 220 225
Ser?Ala?Ile?Thr?Val?Phe?Pro?Gln?Arg?Cys?Pro?Gly?Arg?Gly?Asp
226 230 235 240
Phe?Arg?Ile?Trp?Asn?Ser?Gln?Leu?Val?Arg?Tyr?Ala?Gly?Tyr?Arg
241 245 250 255
Gln?Gln?Asp?GLy?Ser?Val?Arg?Gly?Asp?Pro?Ala?Asn?Val?Glu?Ile
256 260 265 270
Thr?Glu?Leu?Cys?Ile?Gln?His?Gly?Trp?Thr?Pro?Gly?Asn?Gly?Arg
271 275 280 285
Phe?Asp?Val?Leu?Pro?Leu?Leu?Leu?Gln?Ala?Pro?Asp?Glu?Pro?Pro
286 290 295 300
Glu?Leu?Phe?Leu?Leu?Pro?Pro?Glu?Leu?Val?Leu?Glu?Val?Pro?Leu
301 305 310 315
Glu?His?Pro?Thr?Leu?Glu?Trp?Phe?Ala?Ala?Leu?Gly?Leu?Arg?Trp
316 320 325 330
Tyr?Ala?Leu?Pro?Ala?Val?Ser?Asn?Met?Leu?Leu?Glu?Ile?Gly?Gly
331 335 340 345
Leu?Glu?Phe?Pro?Ala?Ala?Pro?Phe?Ser?Gly?Trp?Tyr?Met?Ser?Thr
346 350 355 360
Glu?Ile?Gly?Thr?Arg?Asn?Leu?Cys?Asp?Pro?His?Arg?Tyr?Asn?Ile
361 365 370 375
Leu?Glu?Asp?Val?Ala?Val?Cys?Met?Asp?Leu?Asp?Thr?Arg?Thr?Thr
376 380 385 390
Ser?Ser?Leu?Trp?Lys?Asp?Lys?Ala?Ala?Val?Glu?Ile?Asn?Val?Ala
391 395 400 405
Val?Leu?His?Ser?Tyr?Gln?Leu?Ala?Lys?Val?Thr?Ile?Val?Asp?His
406 410 415 420
His?Ala?Ala?Thr?Ala?Ser?Phe?Met?Lys?His?Leu?Glu?Asn?Glu?Gln
421 425 430 435
Lys?Ala?Arg?Gly?Gly?Cys?Pro?Ala?Asp?Trp?Ala?Trp?Ile?Val?Pro
436 440 445 450
Pro?Ile?Ser?GLys?er?Leu?Thr?Pro?Val?Phe?His?Gln?Glu?Met?Val
451 455 460 465
Asn?Tyr?Phe?Leu?Ser?Pro?Ala?Phe?Arg?Tyr?Gln?Pro?Asp?Pro?Trp
466 470 475 480
Lys?Gly?Ser?Ala?Ala?Lys?Gly?Thr?Gly?Ile?Thr?Arg?Lys?Lys?Thr
481 485 490 495
Phe?Lys?Glu?Val?Ala?Asn?Ala?Val?Lys?Ile?Ser?Ala?Ser?Leu?Met
496 500 505 510
Gly?Thr?Val?Met?Ala?Lys?Arg?Val?Lys?Ala?Thr?Ile?Leu?Tyr?Gly
511 515 510 525
Ser?Glu?Thr?Gly?Arg?Ala?Gln?Ser?Tyr?Ala?Gln?Gln?Leu?Gly?Arg
526 530 535 540
Leu?Phe?Arg?Lys?Ala?Phe?Asp?Pro?Arg?Val?Leu?Cys?Met?Asp?Glu
541 545 550 555
Tyr?Asp?Val?Val?Ser?Leu?Glu?His?Glu?Thr?Leu?Val?Leu?Val?Val
556 560 565 570
Thr?Ser?Thr?Phe?Gly?Asn?Gly?Asp?Pro?Pro?Glu?Asn?Gly?Glu?Ser
571 575 580 585
Phe?Ala?Ala?Ala?Leu?Met?Glu?Met?Ser?Gly?Pro?Tyr?Asn?Ser?Ser
586 590 595 600
Pro?Arg?Pro?Glu?Gln?His?Lys?Ser?Tyr?Lys?Ile?Arg?Phe?Asn?Ser
601 605 610 615
Ile?Ser?Cys?Ser?Asp?Pro?Leu?Val?Ser?Ser?Trp?Arg?Arg?Lys?Arg
616 620 625 630
Lys?Glu?Ser?Ser?Asn?Thr?Asp?Ser?Ala?Gly?Ala?Leu?Gly?Thr?Leu
631 635 640 645
Arg?Phe?Cys?Val?Phe?Gly?Leu?GLys?er?Arg?Ala?Tyr?Pro?His?Phe
646 650 655 660
Cys?Ala?Phe?Ala?Arg?Ala?Val?Asp?Thr?Arg?Leu?Glu?Glu?Leu?Gly
661 665 670 675
Gly?Glu?Arg?Leu?Leu?Gln?Leu?Gly?Gln?Gly?Asp?Glu?Leu?Cys?Gly
676 680 685 690
Gln?Glu?Glu?Ala?Phe?Arg?Gly?Trp?Ala?Gln?Ala?Ala?Phe?Gln?Ala
691 695 700 705
Ala?Cys?Glu?Thr?Phe?Cys?Val?Gly?Glu?Asp?Ala?Lys?Ala?Ala?Ala
706 710 715 720
Arg?Asp?Ile?Phe?Ser?Pro?Lys?Arg?Ser?Trp?Lys?Arg?Gln?Arg?Tyr
721 725 730 735
Arg?Leu?Ser?Ala?Gln?Ala?Glu?Gly?Leu?Gln?Leu?Leu?Pro?Gly?Leu
736 740 745 750
Ile?His?Val?His?Arg?Arg?Lys?Met?Phe?Gln?Ala?Thr?Ile?Arg?Ser
751 755 760 765
Val?Glu?Asn?Leu?Gln?Ser?Ser?Lys?Ser?Thr?Arg?Ala?Thr?Ile?Leu
766 770 775 780
Val?Arg?Leu?Asp?Thr?Gly?Gly?Gln?Glu?Gly?Leu?Gln?Tyr?Gln?Pro
781 785 790 795
Gly?Asp?His?Ile?Gly?Val?Cys?Pro?Pro?Asn?Arg?Pro?Gly?Leu?Val
796 800 805 810
Glu?Ala?Leu?Leu?Ser?Arg?Val?Glu?Asp?Pro?Pro?Ala?Pro?Thr?Glu
811 815 820 825
Pro?Val?Ala?Val?Glu?Gln?Leu?Glu?Lys?Gly?Ser?Pro?Gly?Gly?Pro
826 830 835 840
Pro?Pro?Gly?Trp?Val?Arg?Asp?Pro?Arg?Leu?Pro?Pro?Cys?Thr?Leu
841 845 850 855
Arg?Gln?Ala?Leu?Thr?Phe?Phe?Leu?Asp?Ile?Thr?Ser?Pro?Pro?Ser
856 860 865 870
Pro?Gln?Leu?Leu?Arg?Leu?Leu?Ser?Thr?Leu?Ala?Glu?Glu?Pro?Arg
871 875 880 885
Glu?Gln?Gln?Glu?Leu?Glu?Ala?Leu?Ser?Gln?Asp?Pro?Arg?Arg?Tyr
886 890 895 900
Glu?Glu?Trp?Lys?Trp?Phe?Arg?Cys?Pro?Thr?Leu?Leu?Glu?Val?Leu
901 905 910 915
Glu?Gln?Phe?Pro?Ser?Val?Ala?Leu?Pro?Ala?Pro?Leu?Leu?Leu?Thr
916 920 925 930
Gln?Leu?Pro?Leu?Leu?Gln?Pro?Arg?Tyr?Tyr?Ser?Val?Ser?Ser?Ala
931 935 940 945
Pro?Ser?Thr?His?Pro?Gly?Glu?Ile?His?Leu?Thr?Val?Ala?Val?Leu
946 950 955 960
Ala?Tyr?Arg?Thr?Gln?Asp?Gly?Leu?Gly?Pro?Leu?His?Tyr?Gly?Val
961 965 970 975
Cys?Ser?Thr?Trp?Leu?Ser?Gln?Leu?Lys?Pro?Gly?Asp?Pro?Val?Pro
976 980 985 990
Cys?Phe?Ile?Arg?Gly?Ala?Pro?Ser?Phe?Arg?Leu?Pro?Pro?Asp?Pro
991 995 1000 1005
Ser?Leu?Pro?Cys?Ile?Leu?Val?Gly?Pro?Gly?Thr?Gly?Ile?Ala?Pro
1006 1010 1015 1020
Phe?Arg?Gly?Phe?Trp?Gln?Glu?Arg?Leu?His?Asp?Ile?Glu?Ser?Lys
1021 1025 1030 1035
Gly?Leu?Gln?Pro?Thr?Pro?Met?Thr?Leu?Val?Phe?Gly?Cys?Arg?Cys
1036 1040 1045 1050
Ser?Gln?Leu?Asp?His?Leu?Tyr?Arg?Asp?Glu?Val?Gln?Asn?Ala?Gln
1051 1055 1060 1065
Gln?Arg?Gly?Val?Phe?Gly?Arg?Val?Leu?Thr?Ala?Phe?Ser?Arg?Glu
1066 1070 1075 1080
Pro?Asp?Asn?Pro?Lys?Thr?Tyr?Val?Gln?Asp?Ile?Leu?Arg?Thr?Glu
1081 1085 1090 1095
Leu?Ala?Ala?Glu?Val?His?Arg?Val?Leu?Cys?Leu?Glu?Arg?Gly?His
1096 1100 1105 1110
Met?Phe?Val?Cys?Gly?Asp?Val?Thr?Met?Ala?Thr?Asn?Val?Leu?Gln
1111 1115 1120 1125
Thr?Val?Gln?Arg?Ile?Leu?Ala?Thr?Glu?Gly?Asp?Met?Glu?Leu?Asp
1126 1130 1135 1140
Glu?Ala?Gly?Asp?Val?Ile?Gly?Val?Leu?Arg?Asp?Gln?Gln?Arg?Tyr
1141 1145 1150 1155
His?Glu?Asp?Ile?Phe?Gly?Leu?Thr?Leu?Arg?Thr?Gln?Glu?Val?Thr
1156 1160 1165 1170
Ser?Arg?Ile?Arg?Thr?Gln?Ser?Phe?Ser?Leu?Gln?Glu?Arg?Gln?Leu
1171 1175 1180 1185
Arg?Gly?Ala?Val?Pro?Trp?Ala?Phe?Asp?Pro?Pro?Gly?Ser?Asp?Thr
1186 1190 1195 1200
Asn?Ser?Pro
1201 1203
In order to obtain anti-NO synthase polyclonal antibody, can use equally the fragment of NO synthase, described fragment for example is selected from following sequence:
SEQ?ID?NO：3
Pro?Trp?Ala?Phe
1192 1195
SEQ?ID?NO：4
Gly?Ala?Val?Pro
1189 1192
SEQ?ID?NO：5
Arg
1185
His?Leu?Arg?Gly?Ala?Val?Pro?Trp?Ala?Phe?Asp?Pro?Pro?Gly?Pro
1186 1190 1195 1200
Asp?Thr?Pro?Gly?Pro
1201 1205
SEQ?ID?NO：6
Ala?Phe?Asp?Pro?Pro?Gly?Pro
11941195 1200
Asp?Thr?Pro?Gly?Pro
1201 1205
SEQ?ID?NO：7
His?Leu?Arg?Gly?Ala?Val?Pro?Trp?Ala?Phe?Asp
1186 1190 11951196
SEQ?ID?NO：8
His?Leu?Arg?Gly?Ala?Val?Pro?Trp?Ala?Phe?Asp?Pro?Pro?Gly?Pro
1186 1190 1195 1200
Asp?Thr?Pro?Gly?Pro
1201 1205
The example process for preparing initial anti-NO synthase polyclonal antibody can be described below.Before blood sampling 7-9 days, will expect antigen through 1-3 intravenous injection to rabbit, to increase the polyclonal antibody level in Sanguis Leporis seu oryctolagi stream.In case after immunity, blood sample collection is with the test antibody level.Usually, the soluble antigen immunoreation reaches top level in 40-60 days after antigen is injected for the first time.After first immune end cycle, rabbit has the convalescence of 30 days, carries out immunity again through 1-3 time other intravenous injection afterwards.
For obtaining to comprise the antiserum of expecting antibody, the rabbit blood from rabbit after the collection immunity also is placed in the 50ml centrifuge tube.With wooden spoon (spatula), formed product grumeleuse on tube wall is removed and rod is placed in the grumeleuse of tube hub.Then blood is positioned in cooler and spends the night at the temperature of 40 ℃.Second day removes the clot on spoon, with remaining liq centrifugal 10min under 13000 rev/mins.Supernatant liquid is the target antiserum.It is yellow that the antiserum that obtains is generally.The NaN that adds 20wt% to antiserum 3Be 0.02% and store to using in freezing state at the temperature of-20 ℃ to ultimate density, perhaps do not add NaN 3And store at the temperature of-70 ℃ to using.For the anti-IFN-γ antibody of separate targets from antiserum, following solid phase adsorption sequentially is well suited for:
Use the NaCl of 0.15M to dilute 2 times the 10ml rabbit anti-serum, add afterwards 6.26g Na 2SO 4, mixed being incorporated in hatched under 4 ℃ 12-16 hour.Precipitate is removed through centrifugal, dilute in the 10ml phosphate buffer and use identical buffer dialysed overnight at ambient temperature.After removing precipitate, with the solution application of sample to the DEAE-cellulose column after the phosphate buffer balance.By at the 280nm place, the antibody fraction being determined in the optical density measurement of eluent.
Use affinity chromatography, be attached to by the anti-NO synthase antibody that will obtain on the insoluble matrix of chromatographic media, and use subsequently dense aqueous saline solution eluting, isolated thick antibody is carried out purification.
The buffer solution of gained is used as the starting soln of homeopathic therapeutic method's dilution process, to be used for preparing the antibody of active augmented form.The preferred concentration of the initial matrix solution of the anti-NO synthase of the rabbit of antigen purification polyclonal antibody is 0.5-5.0mg/ml, preferred 2.0-3.0mg/ml.
Anti-endogenous biomolecule polyclonal antibody can be equally by using the adjuvant preparation to above-mentioned for the similar method of the method for anti-endothelial NO synthase antibody.
With the starting soln of gained buffer solution as homeopathic therapeutic method's dilution process, to be used for preparing the antibody of active augmented form.
each component of the complex of active augmented form can be strengthened to prepare through the homeopathic therapeutic method by initial soln, preferably use by serial dilution and come the following method of proportional reduction concentration and in conjunction with external action: with each formerly solution (preceding solution) (being begun by initial soln) serial dilution of 1 part in 9 parts (ten times of dilutions), or serial dilution is in 99 parts (Radix Achyranthis Bidentatae dilutions), or serial dilution is in the neutral flux of 999 parts (thousand times of dilutions), be that approximately 0.5mg/ml comes initial to the initial soln that is in the antibody in solvent (preferred water or water-ethanol admixture) of about 5.0mg/ml with concentration range.Repeatedly vertically vibration when described external action preferably includes each dilution (dynamization, dynamization).Preferably independent container is used for follow-up each time dilution, until required effort levels or the coefficient of dilution.This method is widely accepted in the homeopathic therapeutic method field.Referring to for example V.Schwabe, " Homeopathic medicines ", M., 1967, the 14-29 pages are incorporated it into by reference this paper and are used for described purpose.
For example, in order to prepare the 1,200 times of diluent (being expressed as C12), the initial matrix solution that is the anti-NO synthase antibody of 3.0mg/ml with 1 part of concentration is diluted in 99 parts of neutral water solvents or water-alcohol solvent (preferred 15% ethanol), and carries out repeatedly subsequently (more than 10 times) vertical vibration to make the 100 times of diluent (being expressed as C1).The 200 times of diluent (C2) prepared by the 100 times of diluent C1.This process is repeated 11 times, thereby make the 1,200 times of diluent C12.Therefore, the 1,200 times of diluent C12 represents the solution that obtains for 12 times by serial dilution in 99 parts of neutral fluxes of initial matrix solution in being in different vessels of the antibody that is 3.0mg/ml with 1 part of concentration to be equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12.Carry out similar procedure with the corresponding coefficient of dilution, obtain required diluent.Intermediate dilute liquid can be tested with detection of active in the living model of expectation.Comprise the preferred active augmented form antibody of complex of the present invention and be C12, C30 and the C200 diluent of each active augmented form.When with the mixture of multiple homeopathic therapeutic method's diluent (being mainly the Radix Achyranthis Bidentatae diluent) of active substance during as biological activity liquid component, each component of compositions (as, C12, C30, C50, C200) prepare according to said process respectively, until (for example obtain second from the bottom part of diluent, respectively until C11, C29 and C199), then add in a container according to compositions of mixtures each component with 1 part, and with the solvent of aequum (as, with 97 parts to carry out the Radix Achyranthis Bidentatae dilution) mix.
Can be with active substance as multiple homeopathic therapeutic method's diluent, use such as the mixture of ten times and/or Radix Achyranthis Bidentatae diluent (D20, C30, C100 or C12, C30, C50 or C12, C30, C200 etc.), its effect is by at suitable living model, for example test diluent in model herein described in the embodiment, thereby determines in the mode of experiment.
Strengthen and process that concentration reduces in, any similar external action process that can be replaced by with vertically vibrating that outer exposed is extremely ultrasonic, accept in electromagnetic field or homeopathic therapeutic method field.
Can by with the aqueous solution of the active component of active augmented form or the mixture of water-alcohol solution (mix and use with the form of liquid dosage form with the ratio of 1: 1), pharmaceutically acceptable solid-state carrier being flooded, prepare the solid unit dosage form of pharmaceutical composition of the present invention.Perhaps, available each required diluent carries out continuous impregnating to carrier.
The pharmaceutical composition that preferably is in solid unit dosage form is by the granule preparation of pharmaceutically acceptable carrier, described granule use in advance the water diluent of active augmented form antibody or water-pure diluent saturated.Solid dosage forms can be any dosage form known in pharmaceutical field, comprises tablet, capsule, lozenge and other.As nonactive ingredient, can use other monosaccharide, oligosaccharide and the polysaccharide that use in glucose, sucrose, maltose, starch, dextrinose, hydroxyl isomaltulose and pharmacy, also can use the process mixture of above-mentioned nonactive ingredient and other pharmaceutically acceptable excipient, described excipient such as hydroxyl isomaltulose, polyvinylpolypyrrolidone, cyclamate (sodium cyclamate), saccharin sodium, anhydrous citric acid etc. comprise lubricant, disintegrating agent, binding agent and coloring agent.Preferred carrier is lactose and hydroxyl isomaltulose.Pharmaceutical dosage form can further comprise the drug excipient of standard, for example microcrystalline Cellulose, magnesium stearate and citric acid.
The example of preparation solid unit dosage form is in following elaboration.Be the preparation solid oral dosage form, with the 100-300 μ m granule of lactose with aqueous solution or the water-alcohol solution of the anti-endogenous biomolecule antibody of the anti-NO synthase antibody of the antihistamine antibody of active augmented form, active augmented form and active augmented form, with the 1kg antibody-solutions, the ratio of 5kg to 10kg lactose (1: 5 to 1: 10) is flooded.In order to realize dipping effect, make lactose granule fluidized-bed reactor (as, " the H ü ttlin Pilotlab " of H ü ttlin GmbH) accept saturated lavation (saturation irrigation) in the fluidized bubbling bed in, the air flow via heating carries out drying at lower than the temperature of 40 ℃ subsequently.To be placed in blender with the dried particles (10-34 weight portion) of the saturated estimator of active augmented form antibody, and mix together with " unsaturation " pure lactose of 25-45 weight portion (be used in the situation that do not reduce therapeutic efficiency, reduce costs, simplify and accelerate the purpose of process) and the magnesium stearate of 0.1-1 weight portion and the microcrystalline Cellulose of 3-10 weight portion.The flaky substance that obtains is evenly mixed, and by directly dry-pressing formed (as, in the Korsch-XL400 tablet machine) carry out tabletting, thereby form the spherical pellet of 150-500mg, preferred 300mg.After tabletting, obtain the pill of 300mg, described pill is with the water-alcohol solution of the complex of active augmented form antibody saturated (3.0-6.0mg/ ball).Be in the form of the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent for each component of the complex that carrier is flooded, be preferably the form of the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
Although the present invention is not subjected to any concrete one theory, to think, active augmented form antibody as herein described does not comprise the molecular forms antibody that enough has owing to the bioactive amount of this type of molecular forms.The biological activity of combination drug of the present invention (complex, pharmaceutical composition) is proved absolutely in appended embodiment.
Preferably, for therapeutic purposes, preferred administration every day of complex of the present invention 1-4 time, preferred administration every day 2 times, each administration comprises the compound dosage form of 1-2 unit.
With reference to appended non-limiting example, the present invention is further detailed.
Embodiment
Embodiment 1
Use the radioligand method, studied compound formulation and component thereof external to the standard part [ 3H] the recombinate effect of combination of σ 1 receptor of pentazocine (pentazocine) and people, described compound formulation comprises the anti-brain specific proteins of rabbit S-100 polyclonal antibody (anti-S100) and the anti-endothelial NO synthase polyclonal antibody (anti-eNOS) of affinity purification of the active augmented form of very low dose (ULD), and above-mentioned active augmented form antibody is by to initial matrix solution (concentration: 2.5mg/ml) carry out super dilution (super-dilution) (100 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture (ratio is 1: 1) (the anti-eNOS of the anti-S100+ of ULD) of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; The component of described compound formulation refers to the anti-brain specific proteins of the rabbit through the antigen affinity purification S-100 polyclonal antibody (anti-S100) of the active augmented form of very low dose (ULD), and it is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; The component of described compound formulation also refers to the anti-endothelial NO synthase polyclonal antibody of rabbit (the anti-eNOS of ULD) of the active augmented form of very low dose, and it is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.Use the distilled water (mixture of homeopathic therapeutic method's diluent C12+C30+C200) of reinforcement as the contrast of test formulation.
Sigma-1 (σ 1) receptor is intracellular receptor, is arranged in central nervous system cell, most of peripheral tissues cell and immunologically competent cell.Receptor demonstrates by many psychotropic drugs and causes the unique ability that shifts.The kinetics of σ 1 receptor is with to act on the various impacts that produce to the preparation of σ 1 receptor directly related.These impacts comprise reinvents (formation of Lipid Rafts (raft)) and lipid transhipment/metabolic adjusting to active channel, exocytosis (ecocytosis), signal conduction, plasma membrane.These all can promote neuronic plasticity in brain.Exist evidence to show that σ 1 receptor all has regulating action to whole main neurotransmitter systems: norepinephrine system, serotonin system, dopamine system, cholinergic system and the adjustable glutamic acid effect of NMDA.σ 1 receptor plays an important role in the pathophysiology of neurodegenerative diseases (for example Alzheimer and parkinson disease), spirit and affective disorder (psychiatric and affective disorders) and apoplexy; σ 1 receptor is participation in learning and Memory Process also.Thus; the capability list of drug influence part and σ 1 acceptor interaction effect understands the existence of neuroprotective, ischemia resisting, anxiety, antidepressant and asthenia component in its pharmacological activity scope, and described pharmacological activity allows to include these medicines in consideration as the effective preparation that is particularly useful for treating cerebrovascular disease.
During test (to measure total binding), the compound formulation (the anti-eNOS of the anti-S100+ of ULD) of 20 μ l or the anti-S100 antibody of ULD of 10 μ l or the anti-NOS antibody of ULD of 10 μ l are transferred in culture medium.Therefore, during the test compound preparation, the amount that is transferred to the anti-eNOS of the anti-S100+ of ULD in test panel (test basin) is identical with the amount of the anti-S100 antibody of ULD of testing as unitary agent and the anti-NOS antibody of ULD, and this also allows each independent component of effect and its of preparation is compared.The fortified water of 20 μ l and 10 μ l is transferred in culture medium.
In addition, also shifted the cell membrane homogenate (human leukemia T lymphocyte series) of the Jurkat cell line of 160 μ l (about 200 μ g protein); And finally shifted the 20 tritium-labeled radioligands of μ l [ 3H] pentazocine (15nm).
In order to measure non-specific binding, the 20 unlabelled part haloperidol of μ l (10 μ M) are transferred in culture medium, replace preparation or fortified water.
Hatched 120 minutes and used glass fiber filter (GF/B under 22 ℃ in the Tris-HCl of 50mM buffer (pH=7.4), Packard) after the filtration, use scintillation counter (Topcount, Packard) and flicker mixture (Microscint0, Packard) measure radioactivity.Specific binding (in test or contrast) is calculated by the difference of total binding (in test or contrast) with non-specific binding.
Result represents (table 1) with the percentage ratio that specific binding in contrast (using distilled water in contrast) suppresses.
Table 1.
Figure BDA00002919547700241
Preparation and fortified water to the standard part [ 3H] the recombinate impact of combination of σ 1 receptor of pentazocine and people
Attention: The % of specific binding in contrast=(specific binding in the specific binding/matched group in the test group) * 100%;
The % that in contrast, specific binding suppresses=100%-(specific binding in the specific binding/matched group in the test group) * 100%.
The result of test compounds reflects that the inhibition more than 50% represents the significance impact; The inhibition of 25%-50% represents slightly-moderate influence; It is non-significance impact that test compounds is considered to lower than 25% inhibition, and belongs to the category of background level.
Therefore, the situation of this test model show the anti-eNOS compound formulation of the anti-S100+ of ULD compare its each independent component (the anti-S100 of ULD and the anti-eNOS of ULD) suppress the standard radioligand [ 3H] pentazocine and people the more effective in conjunction with the aspect of σ 1 receptor that recombinate; The anti-S100 of ULD that is transferred to 10 μ l of test panel suppressed the standard radioligand [ 3H] pentazocine and the people combination of σ 1 receptor of recombinating, but affect intensity inferior to the anti-eNOS compound formulation of the anti-S100+ of ULD; Be transferred to test panel 10 μ l the anti-eNOS of ULD to the standard radioligand [ 3H] recombinate the not impact of combination of σ 1 receptor of pentazocine and people; The fortified water that is transferred to 10 μ l of test panel or 20 μ l to the standard radioligand [ 3H] recombinate the not impact of combination of σ 1 receptor of pentazocine and people.
Embodiment 2
Alzheimer (AD) is to obscure and the neurodegenerative diseases of emotion changes as feature take cognitive function decline, memory decay, consciousness.Although the main cause of this pathology is thought the accumulation of amyloid beta (beta amyloid) now, cause forming amyloid beta speckle and neurofibrillary tangles in cerebral tissue; AD is equally with the shortage of cholinergic system.This is to set up AD model this basis of the most common mode under the help of cholinergic system antagonist hyoscyamine (scopolamine) in animal.Can block learning capacity to the middle injection of laboratory animal (being generally rat or mice) hyoscyamine, and cause memory decay.
Use the whole bag of tricks to estimate the cognitive function of rat and mice, comprised the Morris water maze.Being in the nature of this test is released at difference the have muddy water container of (cloudy water) with animal, and described animal is forced to seek hiding fixed platform.The advantage of this method is the process (about the quasi formation in platform space school, no matter animal is put in water wherein) that it allows the training of researcher monitor animal, so that evaluation memory intensity (testing when removing platform) for this reason.
Studied the impact of combination drug compositions of the present invention in the rat that suffers from hyoscyamine amnesia (Scopolamine amnesia), described compositions comprises the anti-brain specific proteins of the rabbit through antigen affinity purification S-100 polyclonal antibody (anti-S100) and the anti-endothelial NO synthase polyclonal antibody (anti-eNOS) of the active augmented form of very low dose (ULD), and it is by to storing the super dilution 100 of storing solution (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain, be equivalent to Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200 (the anti-eNOS of the anti-S100+ of ULD).
In the research of the anti-eNOS medicine of the anti-S100+ of ULD to the effect of the rat (model of Alzheimer) that suffers from the hyoscyamine amnesia, having used 48 Rj:Wistar (Han) is male rat (heavy 180-280g).In 4 days, be the hyoscyamine (n=36) (amnesia that hyoscyamine is induced) of 0.5mg/kg to described rat skin lower injection normal saline (n=12, normal (intact)) or dosage.The rat that suffers from the amnesia that hyoscyamine induces is divided into three groups, and gastric gives distilled water (7.5ml/kg, n=12, matched group 1) or the anti-S100 (7.5ml/kg of ULD, n=12, group 2) or the anti-eNOS of the anti-S100+ of ULD (7.5ml/kg, n=12, group 3) 9 days (front 4 days of injection hyoscyamine, rear 1 day of lower 4 days of hyoscyamine background and last hyoscyamine injection).
Training in the Morris water maze is in 4 days of injection hyoscyamine, and after testing drug is injected 60 minutes, and (4 sequential tests (sequential tests) were separated by 60 seconds) carried out in the hyoscyamine administration after 30 minutes.The Morris labyrinth is bulge (diameter 150cm, high 45cm), is filled to 30cm place (26-28 ℃) with water.At distance container edge 18cm place, hiding platform (diameter 15cm) is arranged, be embedded in the following 1.5cm of water level place.Make platform invisible by the muddy water that adds non-toxicity dyestuff (for example, milk powder) to make.For each test, animal is put into the labyrinth in one of initial point, described initial point equates with hiding platform distance, and allows animal to find platform.If animal can't be found platform in 120 seconds, animal is placed on platform, placed 60 seconds, and restart test.At the test period of four random orders, animal begins by twice, labyrinth from each starting point.Test record is used for seeking the distance of platform and the incubation period of seeking platform on video-tape and in each experiment of subsequent analysis.Carried out following test at the 5th day: remove platform from the labyrinth, and give rat the free floating of 60 seconds.Record spends in platform once in time of part.
Give the learning capacity that hyoscyamine has significantly worsened animal.In matched group, the distance that animal spends in time of seeking on platform and animal seeks platform swimming significantly increases (table 2 and table 3).Described test shows that the memory of animal in matched group worsens: compare with the intact animal, the local spended time that in this group, animal once was positioned at platform is (table 4) still less.Give improve (table 2, table 3, the table 4) that the anti-S100 of ULD does not cause studying parameter.Give the certain improvement of the anti-eNOS of the anti-S100+ of ULD in causing learning, thereby cause incubation period (table 2) and the coverage distance (table 3) of the training inner platform hunting time of 4 days to shorten, and cause being reflected as the memory improvement (table 4) of the local spended time increase that once was positioned at platform.
The incubation period that table 2. platform is sought, second
* *-have significance, p＜0.05 with the difference of normal group
Table 3. is sought the distance that platform needs, cm
Figure BDA00002919547700271
* *-have significance, p＜0.05 with the difference of normal group
Table 4. spends in platform once in the time of locating, second
Figure BDA00002919547700272
* *Has significance with the difference of normal group, p＜0.05
Therefore, in the model of Alzheimer, give the anti-eNOS of the anti-S100+ of compound ULD and give the anti-S100 of ULD and compare more effective with vehicle (vehicle).
Embodiment 3
The anti-brain specific proteins of rabbit through the antigen affinity purification S-100 polyclonal antibody (anti-S100) of the active augmented form of very low dose (ULD) and anti-endothelial NO synthase polyclonal antibody (anti-eNOS) are treated the cerebral infarction that is caused by prefrontal lobe brain cortex light bolt (prefrontal cerebrocortical photothrombosis) carried out preclinical study in rat, described active augmented form antibody is by to initial matrix solution (concentration: 2.5mg/ml) carry out super dilution (100 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture (ratio is 1: 1) (the anti-eNOS of the anti-S100+ of ULD) of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
Acute cerebrovascular disease (apoplexy) is number three in the fatal cause of disease of developed country, and is one of the main reasons (Gusev E.I., 2003 of mankind's deformity; Janardhan V., Qureshi A.I., 2004).
Photoinduced thrombotic model meets nearly all requirement of Focal Cerebral Ischemia (focal cerebral ischemia) experimental model.The method of being set up by Watson (Watson B. etc., 1985) is that the light of 560nm is to the effect of the phytochrome rose bengal (Bengalrose) in the introducing blood flow based on wavelength.Produce the active oxygen form, make endotheliocyte and platelet viscosity increase, and form grumeleuse occluding vascular chamber.Inducing the method for Ischemia Brain Damage by photoinduced thrombosis is simple technically, and near the clinical form of ischemia apoplexy.The very large advantage of this model is that it is non-invasive, does not namely need craniotomy (craniotomy), and therefore copies more accurately the clinical manifestation of cerebral thrombosis.
37 male Wistar rats are (heavy: 150-180g; Age: 2-3 month) be used for the anti-eNOS of the anti-S100+ of following research: ULD in the effect of the rat that suffers from the cerebral infarction that is caused by prefrontal lobe brain cortex light bolt.Induce the bilateral focal ischemia damage of rat prefrontal lobe brain cortex by photochemistry thrombosis method, described method is set up and is improved by I.V.Viktorov (Romanova G.A. etc., 1998) by Waston (Watson B.D. etc., 1985).Rose bengal (3% solution) is injected into the jugular vein (n=37) (anesthesia: chloral hydrate 300mg/kg, intraperitoneal) of anesthetized rat.Use fibre bundle (diameter 3cm) that light beam is conducted to the skull surface of left and right brain hemisphere prefrontal cortex (frontal cortex) top to induce the light bolt from Halogen light (24V, 250W).The rat (n=6) of sham-operation (sham-operated) does not just give rose bengal with same procedure operation, is not exposed to halogen light yet.Normal group comprises 6 rats.
Inducing before apoplexy 5 days and after 9 days, the following preparation of rat of suffering from the light bolt: distilled water (contrast-light bolt group, every day 5ml/kg, n=12), the anti-S100 of ULD (every day 5ml/kg, n=7) or the anti-eNOS of the anti-S100+ of ULD (every day 5ml/kg, n=6).Rear the 8th day of operation (or sham-operation), carry out condition passive avoidance reflection (CPAR) test, to estimate learning capacity and the memory of rat.Rat is placed in the device that is comprised of lighting point and the camera bellows that is connected, in described camera bellows, animal is applied in the foot shock (electric foot-shock) of 0.45mA, and therefore the preferential camera bellows of selecting becomes dangerous usually.Development in the reflection of second day test condition passive avoidance.And, rat is placed in lighting case.Record enters the incubation period of camera bellows first.If rat avoids entering camera bellows for a long time, draw the following conclusions: this rat is remembered described danger (electric shock).The incubation period that enters camera bellows is longer, and memory is just better.
At the 9th day, the apoplexy lesion volume (volume of the stroke lesion) of evaluation portion experimental group rat from the morphology.
In control rats, the light bolt causes a large amount of apoplexy of formation regional, and therefore causes memory to weaken: the CPAR reproduction is compared with normal rat and has been worsened 9.6%, compares with the sham-operation rat and has worsened 22.9% (table 5).Compare with contrast-light bolt group, give the anti-S100 of ULD and reduced by 42.2% apoplexy volume, and improved 14.0% memory.Compare with contrast-light bolt group, give the anti-eNOS of the anti-S100+ of ULD more effective: the apoplexy volume has reduced 44.0%, and trained reflex reproduces and improved 33.4%.
Therefore, compare with the anti-S100 of one-component preparation ULD, give the anti-eNOS compound formulation of the anti-S100+ of ULD more effective.
Table 5
Figure BDA00002919547700291
Embodiment 4
Compositions is studied in the SHR of hypertension model rat, and described compositions is the compositions of the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of " activity " augmented form and active augmented form.
The compositions of solution form is studied in the SHR of hypertension model rat, and described compositions is the compositions of the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of " activity " augmented form and active augmented form.Suffering from hypertensive SHR to 40 is that male rat (weighing 350 ± 50g, age 4.5-5 month) is studied, and described rat is divided into 4 groups, every group of each 10 animals.
In 28 days, animal is done following the processing.The anti-human Angiotensin II AT1 receptor C-terminal fragment antibody (mixture of water diluent C12, C30 and C200) of the active augmented form of group 1:2.5ml/kg accompanies by the distilled water of 2.5ml/kg; The anti-endothelial NO synthase antibody (mixture of water diluent C12, C30 and C200) of the active augmented form of group 2:2.5ml/kg accompanies by the distilled water of 2.5ml/kg; The combination drug compositions (water diluent C12, the C30 of each component and the mixture of C200) of group 3:5ml/kg; And the distilled water of group 4:5ml/kg.
Give once in a week and for the last time medicine after 9 hours, measure the systolic pressure (SBP) of the rat that wakes by means of arteria caudalis indirect method (using faciola (cuff)).
All test composition all shows to have antihypertensive effect (p＜0.05): at the 28th day, compare with initial level, the systolic pressure (SBP) in group 1 has reduced by 20.6%; Reduced by 14.4% in group 2; Reduced by 27.6% in group 3.In matched group 4, SBP compares with initial value and is changed to 1.6%.These results demonstrate the combination drug compositions and have obvious Synergistic Hypotensive Effects.
Embodiment 5
Be studied in NISAG rat with compositions in hypertension model, described compositions is the compositions of the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and active augmented form.
Be studied in the NISAG rat of compositions in hypertension model with the solution form, described compositions is the compositions of the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and active augmented form.Be male rat (heavy 300g to 50 NISAG that suffer from heritability stress sensitive Arterial Hypertention (hereditarystipulated stress-sensitive arterial hypertension), 4 months ages) study, described rat is divided into 5 groups, every group of each 10 animals.
In 28 days, by oral, once a day, give following medicine to animal: the anti-human Angiotensin II AT1 receptor C-terminal fragment antibody (mixture of diluent C12, C30 and C200) of the active augmented form of group 1:2.5ml/kg accompanies by the distilled water of 2.5ml/kg; The anti-endothelial NO synthase antibody (mixture of diluent C12, C30 and C200) of the active augmented form of group 2:2.5ml/kg accompanies by the distilled water of 2.5ml/kg; The combination drug compositions (homeopathic therapeutic method's water diluent C12, the C30 of each component and the mixture of C200) of group 3:5ml/kg; The drugs compared (losartan, losartan) of group 4:5ml/kg (10ml/kg dosage); And the distilled water of group 5:5ml/kg.
Twice weekly, after giving ULD antibody and losartan 2-6 hour, by arteria caudalis indirect method (use faciola), systolic pressure (SBP) is measured.Table 6 shows that the NISAG that records by indirect method is the variation dynamic (dynamics) of systolic pressure in rat.
Table 6
Embodiment 6
Experimentation is studied the effect of the anti-endothelial NO synthase antibody complex (the anti-eNOS of the anti-IR+ULD of ULD) of the glucagon receptor beta subunit C-terminal fragment antibody of the anti-endothelial NO synthase antibody through the antigen affinity purification through the glucagon receptor beta subunit C-of antigen affinity purification terminal fragment antibody (the anti-IR of ULD), very low dose (the anti-eNOS of ULD) of very low dose and very low dose and very low dose, and the anti-IR of described ULD is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain, the anti-eNOS of described ULD is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain.
Under study for action, 150 male Wistar rats (heavy 250-300g during the research beginning, age 3.5-4 month) have been used.10 rats are normal.All the other rats are carried out streptozotocin intravenous injection (diabetes experimental model) with the dosage of 50mg/kg.The injection streptozotocin is after 72 hours, select plasma glucose levels to be not less than the rat of 12mmol/l, be divided into 7 groups (every group of each 20 rats), gave respectively each preparation according to following mode in 21 days: distilled water (5ml/kg/ days, once a day, intragastric administration), insulin
Figure BDA00002919547700321
(8 units/kg/ days, subcutaneous administration), rosiglitazone (Rosiglitazone)
Figure BDA00002919547700322
(8mg/kg/ days, every day twice, intragastric administration), the anti-IR of ULD are (in the amount of 5ml/kg/ days, 2.5ml/kg/ my god, once a day, intragastric administration), the anti-eNOS of the anti-IR+ULD of ULD (5ml/kg/ days, intragastric administration) and rosiglitazone once a day, And insulin
Figure BDA00002919547700324
The anti-eNOS of the anti-IR+ULD of co-administered or ULD and insulin
Figure BDA00002919547700325
Normal rat is accepted the distilled water of same volume.When rat ejection preparation the 7th day, the 14th day and the 21st day, utilize " glucose FKD " test kit (Russia), with enzyme process (glucose oxidase method), the fasting plasma glucose level is measured.
In research the 14th day (give preparation the 14th day), carry out oral glucose tolerance test (OGTT) according to standard method (Du Vigneaud and Karr, 1925).Rat was cut off the water supply 18 hours.Before test, 60min finally gives its test substances.Normal rat is accepted the distilled water of same volume.Mode by oral 50%w/w D/W (1g/kg rat weight) gives glucose.Use " glucose FKD " test kit (OOO " Pharamaceutical and clinical diagnostics ", Russia, www.fkd.ru) serum glucose in the tail vein sample is measured, described measurement is carried out 0,30,60,90, during 120min.Calculated blood sugar concentration to area (AUC) under the averaged curve of time.
The injection streptozotocin causes the plasma glucose concentration of rat to compare phenomenal growth (18mmol/l to 3.5mmol/l, p＜0.05) with the intact animal.In the anti-IR group of ULD, at the 7th, 14,21 day of ejection preparation, glucose level was compared the average 22%-28% that reduced with matched group; Yet difference does not reach the statistical significance level.The anti-IR of ULD is more effective with the anti-eNOS complex of ULD: glucose level the experiment the 14th day with the 21st day 47% and 42% (the comparing p＜0.05 with matched group) that descended separately.The reference preparation rosiglitazone has reduced glucose level equally at the 14th day and the 21st day that tests; Yet effect only reached statistical significance (36%, compare p＜0.05 with matched group) on the 14th day in experiment.
1/2 insulin of injection effective dose (in preliminary study selected) has reduced glucose level (being down to the Normal group level) (Fig. 1) most effectively at whole viewing durations.What should include consideration in is, has used under study for action the short-acting insulin preparation, and plasma glucose is measured in rear 1 hour in its injection, and this has also affected the effect of 1/2 insulin dose to blood sugar level.In view of this background, can not determine fully to unite use insulin and rosiglitazone or insulin and the anti-eNOS complex of the anti-IR+ of ULD effect how.
Glucose-tolerant imbalance (Glucose tolerance disturbance) (body reduces the utilization of glucose) is one of diagnosis and the most important index for the treatment of diabetes.In the intact animal, in the oral glucose tolerance test (ejection preparation the 14th day), when individually dosed, the anti-eNOS of the anti-IR+ULD of compound formulation ULD and insulin have strengthened glucose tolerance most effectively.Rosiglitazone has also reduced concentration to area under time graph (having strengthened glucose tolerance); Yet its effect is compared with matched group and be there is no statistical significance (Fig. 2).
Embodiment 7
Experimentation is studied the effect of the anti-endothelial NO synthase antibody complex (the anti-eNOS of the anti-IR+ULD of ULD) of the glucagon receptor beta subunit C-terminal fragment antibody of the anti-endothelial NO synthase antibody through the antigen affinity purification through the glucagon receptor beta subunit C-of antigen affinity purification terminal fragment antibody (the anti-IR of ULD), very low dose (the anti-eNOS of ULD) of very low dose and very low dose and very low dose, and the anti-IR of described ULD is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain, the anti-eNOS of described ULD is by carrying out super dilution (100 to initial matrix solution 12, 100 30With 100 200Doubly) obtain.
Under study for action, 36 male Goto-Kakizaki rats (heavy 250-280g during the research beginning, age 10-12 week) have been used.The rat of this strain is characterised in that spontaneous non-insulin-dependent diabetes mellitus.Animal was divided into 3 groups (each 12 rats) and accepted following preparation in 28 day: distilled water (5ml/kg, once a day, intragastric administration) or the anti-IR (2.5ml/kg of ULD, once a day, intragastric administration) or the anti-eNOS (5ml/kg of the anti-IR+ULD of ULD, once a day, intragastric administration).By means of glucose analyser (Beckman, Fullerton, California, the U.S.), before the start injection preparation and during the 4th, 8,12,16,20,24,28 day of ejection preparation, plasma glucose levels is measured.At the 28th day, carry out glucose tolerance test (glucose p.o., 1g/kg).
The anti-IR of injection ULD causes significantly (p＜0.05) decline of rat plasma glucose level; Yet, use the anti-eNOS complex of the anti-IR+ULD of ULD more effective (comparing p＜0.001 with matched group) (Fig. 3).
Described result is confirmed (Fig. 4) by the glucose tolerance test data that ejection preparation carried out in the time of 28 days.The anti-IR of injection ULD causes glucose tolerance to improve (compare with matched group, AUC has reduced by 44%, the non-statistical significance).Simultaneously, this parameter (AUC) that is caused by the injection anti-IR+ULD of ULD anti-eNOS complex be reduced to 62%, and itself and matched group are in a ratio of (p＜0.05) of statistical significance.
Embodiment 8
Used following preparation: with the 300mg tablet (3mg/ sheet) of water-alcohol solution dipping, described water-alcohol solution is the water-alcohol solution of the anti-brain specific proteins of the rabbit through the antigen purification S-100 polyclonal antibody of active augmented form, described antibody is in very low dose (the anti-S100 of ULD), by initial soln (concentration is 2.5mg/ml) is carried out super dilution (100 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; 300mg tablet (6mg/ sheet) with the water-alcohol solution dipping that contains pharmaceutical composition, described water-alcohol solution is the anti-brain specific proteins of the rabbit S-100 polyclonal antibody (anti-S100) of affinity purification of active augmented form and the water-alcohol solution of anti-eNOS polyclonal antibody (anti-eNOS), described antibody is in very low dose (ULD), by initial soln (concentration is 2.5mg/ml) is carried out super dilution (100 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture (the anti-eNOS of the anti-S100+ULD of ULD) of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; 300mg tablet (3mg/ sheet) with the water-alcohol solution dipping, described water-alcohol solution is the anti-eNOS polyclonal antibody of the rabbit through antigen purification of active augmented form, described antibody is in very low dose (the anti-eNOS of ULD), by initial soln (concentration is 2.5mg/ml) is carried out super dilution (100 12, 100 30With 100 200Doubly) obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; And as the 300mg tablet of placebo, this tablet contains excipient: 267mg lactose (lactose monohydrate), 30mg microcrystalline Cellulose, 3mg magnesium stearate.
In motion sickness (kinetosis) model or movement disorders/motion sickness (motion diseases/motion sickness) (being caused by multiple vestibule vegetalitas obstacle), the effect of drugs treatment dizzy (dizzy) and other motion sickness symptom is estimated.Dizziness is the typical sign of vestibular analysor (vestibular analyzer) damage, described damage has multiple origin, comprises the malfunction of vestibular nerve and cochlea system, the circulatory disturbance of vertebral basilar system, pathology of central nervous system (CNS) etc.Dizzy performance as motion sickness, with other vestibule vegetalitas obstacle, comprise the reaction of three types: vestibule-motor reaction (nystagmus (nystagmus) and deflection reaction (reaction of deviation)), vestibule-sensory reaction (except dizziness, nystagmus (or rotation afterreaction), the defensive action) and vegetative reaction (feel sick, vomit, sweat, cardiopalmus, hotness and pulse and fluctuation of blood pressure).
In order to test the anti-kinetosis performance of various compositionss, carry out the comparative study of double-blind placebo-controlled contrast (Double blind placebo controlled) in parallel group, described group is comprised of 15 healthy experimenters: have slight (n=5; 33%) or moderate (n=10; 67%) motion sickness resistance, the age is the masculinity and femininity in 15-60 year (33.3 ± 0.75 years old mean age).Give to group 1 that the anti-eNOS of the anti-S100+ of ULD, group 2 give the anti-S100 of ULD, group 3 gives anti-eNOS.
For the situation of skimulated motion disease and the effect of evaluation study medicine, used the most suitable motion sickness model of also being approved most-test with the Coriolis acceleration (continuous cumulative effect of accelerations by Coriolis, CCEAC) of continuous accumulative effect.All research experimenters are no more than 5 minutes to the initial toleration of CCEAC test.Use multiple diagnostic method to carry out record to the vestibule vegetalitas obstacle that (CCEAC) is caused by kinetic effect (kinetic effect), described diagnostic method comprises that quantitative assessment (Halle scale), the heart rate variability (HRV) of experimenter's inspection, vestibule vegetalitas obstacle sensitivity are analyzed and the self evaluation of functional condition (WBAM-happiness (well-being), activity (activity) and emotion (mood)).Effect standard as the treatment of carrying out, to the toleration of kinetic effect and dynamically estimating of return period degree, and estimated the variation of sensation-motor reaction (nystagmus) index sign, HRV index (utilizing Biocom Wellness scanning system, by the international standard exploitation of AWS, LLC expert association sick according to heart of Europe and North America electrophysiology association) and WBAM data.Feature, sign and time limit (term) that safety criterion occurs for the interior possible side effect (AE) relevant with taking in medicine during treating; The impact of drugs on the response time (TSMR) of the characteristics index (reaction of moving target (RMO)) of central nervous system (CNS) function, simple motion; Dynamically (heart rate (HR), systolic pressure and diastolic pressure (SBP, DBP), the Stange test of health and functional factor; Exercise tolerance (exercise tolerance) (index of Harvard step test (step-test))).After utilizing the anti-eNOS complex of the anti-S100 of ULD and ULD to carry out single dose administration and carry out time-histories administration in 7 days (course administration) post-evaluation safety.
All experimenters do not take in any medicine in before participating in research one month.After the experimenter is screened, it is randomized into (the anti-eNOS of the group anti-S100+ of 1:ULD in 4 groups; The anti-S100 of group 2:ULD; The anti-eNOS of group 3:ULD; And organize 4: placebo).
When research the 1st day (access 1), experimenter's Elementary Function and psycho-physical state carried out record, give subsequently the experimenter 5 tablets of corresponding ULD antibody tablets, and give subsequently the CCEAC test.Test time is carried out record; By means of multiple work up, AE and the vestibule vegetalitas obstacle that relates to motion sickness detected.In ensuing 2-6 days, give the medicinal tablet three times of 1 regulation of experimenter every day.When the 7th day (access 2), give the experimenter dosage identical with the 1st day (access 1).Before CCEAC test and carry out afterwards multiple diagnosis research.Described research is carried out in such a way: seminar only carries out work to an experimenter.Described research is carried out in morning abreast, is generally people's participation every days 4, and each one gives medicine or placebo.Three weeks for the cleaning phase (washout period), when the cleaning phase finishes, gave new medicine or placebo according to regulation to the experimenter in each group afterwards; (time-histories of access 1, medicine is absorbed to repeat research cycle; Access 2).Therefore, each experimenter participates in 4 research cycles under study for action.That is each experimenter who participates in each group has the cleaning phase in three weeks between each cycle.This has just allowed researcher to even up the individual character of (level) test person to the impact of therapeutic effect.According to research approach, carried out the pharmaceutical efficacy analysis to having completed the data of absorbing all test subject (n=15) of drugs all processes.
Under the background of single day picked-up drugs, the sign factor of the motion sickness symptom after kinetic effect (CCEAC) (evidence factors) (dizzy, nauseating, without activity, palor, perspiration etc.) shows, roughly the same motion sickness state has appearred in all research experimenters, and do not had a significant difference (table 7, access 1) in all groups according to the symptom sign of the vegetative function obstacle of Halle Measuring scale assessing by doctor-researcher.Yet the kinetic effect that produces the similar movement disease symptoms is not identical in four groups, and depends on the medicine (table 8, access 1) that the research experimenter absorbs.Absorption in 1 day of the anti-eNOS preparation of the anti-S100+ of ULD causes the most obvious anti-kinetosis effect, and it shows as: not only tolerance time is significantly longer (104.10 ± 13.14 seconds in the CCEAC test; Comparatively speaking, the anti-S100 group of ULD is 68.50 ± 6.57 seconds; The anti-eNOS group of ULD is 75.00 ± 6.79 seconds; Placebo group is 61.30 ± 3.15 seconds); And nystagmus time the shortest (being respectively 9.90 ± 1.20 seconds, 13.50 ± 1.51 seconds, 16.10 ± 1.68 seconds and 13.30 ± 1.12 seconds); And Quick-return maximum (being respectively 96.90 ± 13.54 seconds, 194.20 ± 18.45 seconds, 202.50 ± 21.72 seconds and 241.70 ± 38.41 seconds).
Recorded roughly similar index in access 2 after accepting the medicine of one section time-histories.In order to reach the symptom (table 7, access 2) similar to motion sickness, the longest kinetic effect time is put on accept the anti-eNOS compositions of the anti-S100+ of ULD (table 8, the access 2) experimenter of 7 days.The anti-eNOS compositions of the anti-S100+ of ULD the most significantly anti-kinetosis effect shows as nystagmus time (9.50 ± 1.38 seconds, p＜0.01) and (117.90 ± 15.65 seconds persistent period return period; P＜0.01) remarkable shortening.Compare with placebo group, it is better that the anti-kinetosis effect of the anti-S100 of one-component preparation ULD shows as following index: CCEAC test toleration, nystagmus time and turnaround time (table 8, access 1 and access 2), however the effect of the anti-S100 of ULD is inferior to the anti-eNOS compositions of the anti-S100+ of ULD.Do not have significant difference because CCEAC tests to compare with placebo group with the result of follow-up return period, the anti-eNOS of one-component preparation ULD does not demonstrate anti-kinetosis effect (table 8, access 1 and access 2).The comparative analysis that the CCEAC test index of the anti-eNOS group of the anti-S100+ of the ingestion of drugs ULD of 1 day and the anti-S100 group of ULD is carried out shows, make the adding of the anti-eNOS of ULD that toleration to kinetic effect has improved 52%, nystagmus time reduced by 27% and make the return period after kinetic effect finishes shorten 50%, shortened 49% comprising the dizzy persistent period.Yet, the contribution of the anti-eNOS component of ULD maximum is the effect of compound formulation (the anti-eNOS compositions of the anti-S100+ of ULD) has been introduced in the time-histories absorption of medicine, it shows as: compare with the result of the anti-S100 of ULD group, the toleration of kinetic effect and nystagmus aspect the persistent period (in each parameter) exceed 30%.In addition, compare with the anti-S100 of one-component preparation ULD, when taking in the anti-eNOS compositions of the anti-S100+ of ULD, compare increase degree larger (described increase represents by the variation that CCEAC tests toleration and these two indexs of nystagmus persistent period) in the access effect of 2 o'clock with the data of access 1, be respectively 30% and 4% increase (the anti-S100 group of ULD is respectively 21% and 0%).When estimating the effect of medicine anti-kinetosis performance, pay particular attention to the stability that medicine may affect autonomic nervous system (ANS), especially change the balance of sympathetic nerve and parasympathetic nervous subsystem.Due to this purpose, when each is accessed, under static state and carry out functional test (breathe and upright test) time and all the HRV parameter is analyzed.
Table 7. carries out the Halle scale index of the rear preparation according to using of CCEAC test
Figure BDA00002919547700381
Table 8. is dynamic according to the index of the CCEAC test of the preparation of using
Figure BDA00002919547700391
Attention:
1Used Kruskal-Wallis to check to determine significant difference between group.If the significant difference that relatively demonstrates p＜0.05 between group is used the Mann-Whitney check.
* relatively has significant difference with placebo, p＜0.05;
* and placebo relatively have significant difference, p＜0.01;
* * and placebo relatively have significant difference, p＜0.001.
*Relatively has significant difference, p＜0.05 with the anti-eNOS of the anti-S100+ of ULD;
* *Relatively has significant difference, p＜0.01 with the anti-eNOS of the anti-S100+ of ULD;
* * *Relatively has significant difference, p＜0.001 with the anti-eNOS of the anti-S100+ of ULD.
Before CCEAC test with afterwards, under resting state, the HRV of (sitting posture) analyzes (table 9) and detects the tendency that has the SDNN enhancing in accepting the experimenter of drugs, show due to the impact of parasympathetic nervous on heart rhythm, cause heart rate variability to increase.In all treatment groups, as the response to kinetic effect, sign increases from the RMS-SD value of the activity of the parasympathetic nervous assembly of main regulation.In the group of accepting the anti-eNOS compositions of the anti-S100+ of ULD and the anti-S100 of ULD, show that HF increases, and shows that equally autonomic balance is to the movement of parasympathetic chain.Therefore, carry out the CCEAC test in all groups after, parasympathetic nervous all promotes to some extent on the impact of heart rate.
HRV parameter when table 9. experiment participant is static before and after dynamic behavior
Figure BDA00002919547700401
Attention: * and placebo relatively have significant difference, p≤0.05;
# and baseline parameter relatively have significant difference, p≤0.05.
The analysis that the HRV that is in transition state (transition states) is carried out shows, compares with placebo with the anti-S100 of ULD, and absorption in 1 day of the anti-eNOS compositions of the anti-S100+ of ULD has increased the response time (13.9 ± 1.14; P≤0.05) and stabilization time (24.2 ± 1.28; P≤0.05).Same factor has exceeded the value of placebo group and value after kinetic effect, and this has proved that drug combination has a positive role (toleration to the body change in location strengthens) aspect ANS reactive.In breath test, between maximum heart rate and minimal heart rate, minimum difference has confirmed that two subsystems of ANS obtain better balance (25.1 ± 2.66 heart beatings/min, p≤0.05) after the acceptance anti-eNOS compositions of the anti-S100+ of ULD of a day.When the time-histories treatment in a week finishes, in the group of accepting the anti-eNOS compositions of the anti-S100+ of ULD, notice equally the stabilizing effect (table 10 and table 11) of the rear ANS balance of CCEAC test (uprightly test and breath test).
Research participant's HRV parameter in the upright test of carrying out before and after table 10. dynamic behavior
Attention: * and placebo relatively have significant difference, p≤0.05;
* relatively have significant difference, p≤0.05 with the anti-S100 of ULD.
Research participant's HRV parameter in the breath test of carrying out before and after table 11. dynamic behavior
Figure BDA00002919547700431
Attention: * and placebo relatively have significant difference, p≤0.05
Self evaluation result by research participant experimenter's functional status (happiness, activity and emotion) of carrying out shows that the experimenter in all groups has provided " on average " score (table 12) for each parameter, described self evaluation by the research participant in the treatment beginning with when finishing, carry out after (CCEAC test) simulated in motion sickness.Therefore, under the background that medicine is taken in, the CCEAC toleration is satisfied.Compare with the data of placebo group, when taking in end in the 7th day, observe the highest rate of increase (higher than 10%) in the anti-eNOS compositions of the anti-S100+ of ULD group.
The self evaluation dynamic state of parameters of table 12. research participant's functional condition (happiness-activity-emotion)
Safety analysis comprises the experimenter's who is derived from all participation researchs data.Notice that at viewing duration the research preparation is had good toleration.Do not identify the side effect relevant to administration.All experimenters in each seminar have completed the treatment in the research approach fixed time limit; Nobody withdraws from advance.
According to the result of physical examination (index that comprises heart rate, systolic pressure and diastolic pressure), and according to Harvard step test data, described experimenter is not recorded to any abnormality (table 13) during studying.All variations that detect all are no more than normal range.In this case, all experimenters are in the satisfied happiness of subjective report.
Before and after table 13. dynamic behavior, research participant's body parameter and exercise tolerance is dynamic
Figure BDA00002919547700451
Figure BDA00002919547700461
Except the hematodinamics parameter, safety and the negative influence possible to the central nervous system for the evaluation studies medicine, in the experimenter, following physiological parameter is checked: RMO (moving target reaction), SMRT (simple reaction time), RA (attention field (range of attention)), span of attention (attention span, AS) and the attention coefficient of stability (ASF).In addition, carry out Stange and tested to estimate hypoxic tolerance.
According to the result (table 14) that obtains, absorption in 1 day or time-histories are taken in for the parameter of assessing does not have the significance impact.During twice access, before and after the CCEAC test, sensorimotor coordination (SMRT, RMO) is compared all as broad as long with the result of placebo group.Be presented at before and after the CCEAC test for the data of the amount of attention and this class sophisticated functions of stability, drugs does not all change intensity, and the change of attention is compared as broad as long with placebo group.
The analysis of the standard movement of holding one's breath test shows that experimenter's hypoxic tolerance has the tendency (table 14) of enhancing.When holding the breath, the persistent period of taking in Stange test after all drugs has all extended.Yet, only have the absorption of the anti-eNOS complex composition of the anti-S100+ of ULD just to demonstrate significantly long holding one's breath the time (after baseline place 68.1 ± 18.8 seconds and CCEAC test 91.7 ± 27.4 seconds after kinetic effect; P＜0.05).When using Gench test (Stange test) (holding one's breath during expiration, p＞0.05), noticed equally the enhancing of hypoxic tolerance.
Before and after table 14. dynamic behavior, research participant psycho-physical state parameter is dynamic
Figure BDA00002919547700471
Therefore, use the effect that studies have shown that the anti-eNOS complex composition of the anti-S100+ of ULD and the anti-S100 of one-component preparation ULD of experimental motion sickness.Drugs strengthened the experimenter after the clinical and physiologic effect of skimulated motion disease to the stability of kinetic effect, make experimenter's motion sickness show as more slight clinical progress and the experimenter recovered after treatment stops sooner.In addition, show that the anti-kinetosis effect of complex composition (the anti-eNOS compositions of the anti-S100+ of ULD) has improved the effect of one-component.In experimental motion sickness, the anti-eNOS complex composition of the anti-S100+ of ULD strengthens in time-histories is taken in to some extent at the vestibule autonomy of controlling body and the effect in sensory reaction.The anti-eNOS of ULD that it should be noted that the unitary agent form does not have the protection effect to motion sickness, yet can significantly improve the effect of latter's anti-kinetosis when with the anti-S100 of ULD associating, this take in 1 day of medicine and in short-term journey confirmed in taking in.In the anti-eNOS compositions of the anti-S100+ of ULD, observe at the reactivity of regulating parasympathetic nervous affect ANS and sympathetic division and ANS best ability aspect the instantaneous process (transient processes) of the adaptive capacity under the motion sickness state (strengthen to body position the toleration of variation) suddenly, this is also the pith of medicine anti-kinetosis performance.When being used as the preparation of anti-kinetosis, when comprising executable operations function (operator function), the anti-eNOS compositions of the anti-S100+ of ULD and the anti-S100 one-component of ULD preparation are safe, and health and psycho-physical parameter are not had adverse effect.
The anti-eNOS complex composition of the anti-S100+ of ULD and the anti-S100 of ULD can recommend for the motion sickness (comprising seasick, airsick and carsick) in the people's prevention with slight and moderate stability and alleviation movement disorders.Described complex composition is safe, and the quality of vocational activity is free from side effects.
Embodiment 9
The performance that is used for the treatment of spiritual organic syndrome (psychoorganic syndrome) for the combination drug compositions to the application is studied, and has used the tablet of heavy 300mg.Described tablet is to comprise the water-alcohol solution dipping (6mg/ sheet) of pharmaceutical composition, described water-alcohol solution is the anti-brain specific proteins of the rabbit S-100 polyclonal antibody (anti-S100) of affinity purification of active augmented form of very low dose (ULD) and the water-alcohol solution of anti-endothelial NO synthase polyclonal antibody (anti-eNOS), and it is by with the super dilution 100 of initial soln (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain, be equivalent to the mixture (" the anti-eNOS of the anti-S100+ of ULD ") of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
The matched group patient accepts the tablet (6mg/ sheet) that 300mg floods with the water-alcohol solution that comprises pharmaceutical composition, described water-alcohol solution is the water-alcohol solution of the anti-brain specific proteins of rabbit S-100 polyclonal antibody (anti-S100) of affinity purification of the active augmented form of very low dose (ULD), and it is by with the super dilution 100 of initial soln (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain.
This research comprises the patient of the spiritual organic syndrome that is diagnosed with the wound initiation.The organic syndrome of spirit is characterised in that following three levy (triad): weakness of memory, intelligence decline (loop of intelligence) and emotion (Walther Buel three levies) out of control.
Research is parallel group of clinical trial of open label stochastic comparison, treatment effect and the safety (first group patient take in ULD anti-S100 preparation, second group patient take in ULD anti-S100+ anti-eNOS preparation) of test to the patient of the spiritual organic syndrome suffering from wound and cause.
Research comprise 6 diagnosis suffer from spiritual organic syndrome, the age is patient's (70.83 ± 21.95 years old mean age) of 35 years old-90 years old.
Following following including in exclusion standard verifies patient compliance:
Inclusive criteria:
1. the diagnosis of being confirmed by medical history, neurologic examination and medical records suffers from the traumatic encephaiopathy with spiritual organic syndrome or suffers from the patient of the encephalopathy (vascular and traumatic) of the complicated cause of disease with spiritual organic syndrome.
2. follow the unconverted patient for the treatment of (concomitant therapy) at least one month before access 1.
3. follow treatment to need not to change within the whole observation period.
4. do not need the immunoregulation medicament prescription in ensuing 6 months.
5. has the patient who is enough to the educational level that fully exchanges with researcher and research coordination person.
6. be evaluated as patient reliable and all clinical interviews, test and program that ready execution is stipulated by researcher in scheme.
7. the patient who has effective home address.
Exclusion standard:
1. any cerebral surgery operation is arranged in medical history.
2. acute myocardial infarction.
3. hemorrhagic apoplexy.
4. diagnosis suffers from psychosis, two-phase obstacle or schizoaffective disorder in medical history.
5. the severe depression obstacle of the standard in the depression module of spiritual interview neural according to the simple and clear world (MINI).
6. medical factor/the situation of the test result of the patient in researcher may affect research or further feature.
7. the answer of " I " joint is " 2A ", " 2B ", " 2C " or " 3 " (possess the active suicidal idea that certain enforcement purpose does not possess practical plans, perhaps possess the active suicidal idea of practical plans and purpose) in the depressed questionnaire of Beck.
8. in medical history, autoimmune disease is arranged.
9. liver acute injury or severe cirrhosis (being the C class according to Child-Pugh).
10. uncorrected dysthyroidism.
11. lose the compensatory Arterial Hypertention in medical history.
12. serious or lose cardiovascular disease, hepatopathy, nephropathy, disease of metabolism, respiratory disorder or the hematologic disease of compensatory, Symptomatic peripheral blood vessel or other and may affect the patient at researcher and participate in studying medical science or the psychiatry situation of being in hospital and extending or again be admitted to hospital during maybe may causing studying.
13. disease and the situation that may stop the patient to participate in studying in research worker.
14. taken in the medicine that comprises the anti-eNOS of ULD or comprised the medicine of the anti-S100 of ULD before participating in research.
15. take in the antidepressants (comprising vegetalitas and homeopathic therapeutic method's preparation) of any type.
16. take in the antianxiety drugs (comprising vegetalitas and homeopathic therapeutic method's preparation) of any type.
17. take in immunomodulator (comprising vegetalitas and homeopathic therapeutic method's preparation).
Accepted the whole body steroid therapy 18. access 0 in front 1 month.
19. in participating in comprising the medicine of the anti-eNOS of ULD or comprising the research of medicine of the anti-S100 of ULD, if the patient takes at least one dosage particles.
20. participated in other clinical research in 1 month before adding this research.
21. during studying and last take in gestation, suckling in 1 month after drugs, can not fully practise contraception.
22. there is allergy/intolerance in any drug component, comprises lactose intolerance.
23. the patient takes in anesthetics and tranquilizer, suffers from ethanol dependence or mental sickness.
24. the patient is directly involved in the office worker at center of the research of carrying out and/or patient to be the research center office worker's directly related with the research of carrying out kinsfolk.Described " kinsfolk " is husband (wife), father and mother, children and brother (sister).
25. participating in test at researcher maybe may collect reparation or participate in Judicial procedure.
To meeting after the above-mentioned patient who includes in exclusion standard determines, described patient is randomized into two seminar: patient's group (3 patients that accept the anti-S100 of ULD, the women is 33.33%, and the male is 66.66%, and the mean age is 71.33 ± 16.25 years old); And patient's group (3 patients, women are 66.66%, and the male is 33.33%, and the mean age is 70.33 ± 30.66 years old) of accepting the anti-eNOS of the anti-S100+ of ULD.
Carried out 5 access in this research process, treatment stage continues to access 4, average 84 ± 5 days consuming time from accessing 1.Access 4 (the 84th ± 5 days) are the First terminal point of research, also have afterwards tracing study.Track phase from access 4 continue to the access 5 (average 168 ± 5 days).
In safety analysis, comprised that all participate in the patient's (n=6) of research data.Recorded the good toleration of medicine in research process.Be not recorded to side effect.The patient of all seminar has completed the treatment according to scheme; Nobody drops by the wayside.
Estimated the effect of the anti-eNOS preparation of the anti-S100+ of ULD to following aspect: the main clinical indication of spiritual organic syndrome and symptom (the neural spiritual application form of NPI, intensity part (intensity section)), intensity (the neural spiritual application form of NPI of the misery followed of the personnel of care of patients, painful partly (distress section)) and cognitive function of patients (simple and easy Mental status schedule, MMSE).Access 4 o'clock, found improvement in the key symptoms of the organic syndrome of spirit, for example the intensity of the neural spiritual application form of NPI partly exists statistical significance to reduce and (is down to 69.0 ± 6.24 by 91.0 ± 15.13, p＜0.05), the painful partial fraction of the neural spiritual application form of NPI reduces (being down to 36.33 ± 3.21, p＜0.05 by 44.33 ± 17.78) (table 15).
Be not recorded to improvement clinically in only accepting patient's group of the anti-S100 of ULD.
And when treatment finished, the difference between patient's group on the gross score of the neural spiritual application form intensity part of NPI was statistical significance, p＜0.05.
Table 15.
Figure BDA00002919547700531
*-compare p＜0.05 with baseline; #-with compare p＜0.05
Therefore, observe the anti-eNOS combination drug of the anti-S100+ of ULD compositions in the clinical research of carrying out main clinical indication and the symptom of the organic syndrome of spirit had good effect, and tend to affect the cognitive function of spiritual organic syndrome.Also confirmed in addition to have good Drug tolerance.Be not recorded to the side effect relevant to medicine.
Embodiment 10
The performance that is used for the treatment of Alzheimer for the combination drug compositions to the application is studied, and has used the tablet of heavy 300mg.Described tablet is to comprise the water-alcohol solution dipping (6mg/ sheet) of pharmaceutical composition, described water-alcohol solution is the anti-brain specific proteins of the rabbit S-100 polyclonal antibody (anti-S100) of affinity purification of active augmented form of very low dose (ULD) and the water-alcohol solution of anti-endothelial NO synthase polyclonal antibody (anti-eNOS), and it is by with the super dilution 100 of initial soln (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain, be equivalent to the mixture (ratio is 1: 1) (" the anti-eNOS of the anti-S100+ of ULD ") of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
The matched group patient accepts the tablet (3mg/ sheet) that 300mg floods with the water-alcohol solution that comprises pharmaceutical composition, described water-alcohol solution is the water-alcohol solution of the anti-brain specific proteins of rabbit S-100 polyclonal antibody (anti-S100) of affinity purification of the active augmented form of very low dose (ULD), and it is by with the super dilution 100 of initial soln (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
This research comprises the patient who is diagnosed with Alzheimer.Alzheimer is characterised in that dementia (the stable infringement of acquired dementia, cognitive activity accompanies by to a certain degree the loss of the knowledge of previous acquisition and practical skill, cannot or hardly obtain new knowledge).
Research is for the clinical trial of open label stochastic comparison, and test is to suffering from slight patient's to moderate Alzheimer's disease treatment effect and safety in 2 parallel group (the anti-S100 preparation of ULD and the anti-eNOS preparation of the anti-S100+ of ULD).
Research comprise 6 diagnosis suffer from slightly to moderate Alzheimer's disease, the age is patient's (59.0 ± 3.58 years old mean age) in 55-64 year.
Following following including in exclusion standard verifies patient compliance:
Inclusive criteria is as follows:
1. suffers from slightly patient to moderate Alzheimer's disease by what medical history, neurologic examination and medical records were confirmed.
2. follow the unconverted patient for the treatment of at least one month before access 1.
3. follow treatment to need not to change within the whole observation period.
4. do not need the immunoregulation medicament prescription in ensuing 6 months.
5. has the patient who is enough to the educational level that fully exchanges with researcher and research coordination person.
6. be evaluated as patient reliable and all clinical interviews, test and program that ready execution is stipulated by researcher in scheme.
7. the patient who has effective home address.
Exclusion standard is as follows:
1. any cerebral surgery operation is arranged in medical history.
2. acute myocardial infarction.
3. hemorrhagic apoplexy.
4. diagnosis suffers from psychosis, two-phase obstacle or schizoaffective disorder in medical history.
5. the severe depression obstacle of the standard in the depression module of spiritual interview neural according to the simple and clear world (MINI).
6. medical factor/the situation of the test result of the patient in researcher may affect research or further feature.
7. the answer of " I " joint is " 2A ", " 2B ", " 2C " or " 3 " (possess the active suicidal idea that certain enforcement purpose does not possess practical plans, perhaps possess the active suicidal idea of practical plans and purpose) in the depressed questionnaire of Beck.
8. in medical history, autoimmune disease is arranged.
9. liver acute injury or severe cirrhosis (being the C class according to Child-Pugh).
10. uncorrected dysthyroidism.
11. lose the compensatory Arterial Hypertention in medical history.
12. serious or lose cardiovascular disease, hepatopathy, nephropathy, disease of metabolism, respiratory disorder or the hematologic disease of compensatory, Symptomatic peripheral blood vessel or other and may affect the patient at researcher and participate in studying medical science or the psychiatry situation of being in hospital and extending or again be admitted to hospital during maybe may causing studying.
13. disease or the situation that may stop the patient to participate in studying in research worker.
14. taken in the medicine that comprises the anti-eNOS of ULD or comprised the medicine of the anti-S100 of ULD before participating in research.
15. take in the antidepressants (comprising vegetalitas and homeopathic therapeutic method's preparation) of any type.
16. take in the antianxiety drugs (comprising vegetalitas and homeopathic therapeutic method's preparation) of any type.
17. take in immunomodulator (comprising vegetalitas and homeopathic therapeutic method's preparation).
Accepted the whole body steroid therapy 18. access 0 in front 1 month.
19. in participating in comprising the medicine of the anti-eNOS of ULD or comprising the research of medicine of the anti-S100 of ULD, if the patient takes at least one dosage particles.
20. participated in other clinical research in 1 month before adding this research.
21. during studying and last take in gestation, suckling in 1 month after drugs, can not fully practise contraception.
22. there is allergy/intolerance in any drug component, comprises lactose intolerance.
23. the patient takes in anesthetics and tranquilizer, suffers from ethanol dependence or mental sickness.
24. the patient is directly involved in the office worker at center of the research of carrying out and/or patient to be the research center office worker's directly related with the research of carrying out kinsfolk.Described " kinsfolk " is husband (wife), father and mother, children and brother (sister).
25. participating in test at researcher maybe may collect reparation or participate in Judicial procedure.
To meeting after the above-mentioned patient who includes in exclusion standard determines, described patient is randomized into two seminar: patient's group (3 patients that accept the anti-S100 of ULD, the women is 100%, the male is 0%, mean age is 59.0 ± 3.6 years old) and patient group (3 patients of accepting the anti-eNOS of the anti-S100+ of ULD, the women is 66.66%, and the male is 33.33%, and the mean age is 59.0 ± 4.36 years old).
Carried out 5 access in this research process, treatment stage continues to access 4, average 84 ± 5 days consuming time from accessing 1.Access 4 (the 84th ± 5 days) are the First terminal point of research, also have afterwards tracing study.Track phase from access 4 continue to the access 5 (average 168 ± 5 days).
In safety analysis, comprised that all participate in the patient's (n=6) of research data.Recorded the good toleration of medicine in research process.Be not recorded to side effect.The patient of all seminar has completed the treatment according to scheme; Nobody drops by the wayside.
Estimated the effect of the anti-eNOS preparation of the anti-S100+ of ULD to following aspect: the main clinical indication of Alzheimer and symptom (the neural spiritual application form of NPI, the intensity part), the intensity of the misery followed of the personnel of care of patients (the neural spiritual application form of NPI, painful part) and cognitive function of patients (simple and easy Mental status schedule, MMSE).Access 4 o'clock, found improvement in the key symptoms of Alzheimer, for example the intensity of the neural spiritual application form of NPI partly exists statistical significance to reduce (being down to 12.0 ± 3.46, p＜0.05 by 24.33 ± 4.73) (table 16).
Also found the painful trend that reduces of personnel of care of patients, and the trend that the activity of patient's daily life reduces when treatment finishes (yet there is no any statistical significant difference, may be because the patient's number that participates in research is less).
In addition, found the trend that cognitive function improves, this can be increased to confirmation in 26.66 ± 1.53 minutes by 23.66 ± 3.21 from the MMSE mark, yet described difference fails to reach the value of statistical significance equally when treatment finishes, may be also less relevant with sample size.
In accepting patient's group of the anti-S100 of ULD, do not demonstrate in same endpoint and improve tendency, the improvement of the MMSE mark of the non-significance of statistics is only arranged: be increased to 23.33 ± 0.58 minutes by 22.66 ± 0.58.
And when treatment finished, the difference between patient's group on the MMSE gross score was statistical significance, p＜0.05.
Table 16.
Figure BDA00002919547700571
*-compare p＜0.05 with baseline; #-with compare p＜0.05
Therefore, observe the anti-eNOS combination drug of the anti-S100+ of ULD compositions in the clinical research of carrying out main clinical indication and the symptom of Alzheimer had good effect, and tend to affect the cognitive function of Alzheimer.Also confirmed in addition to have good Drug tolerance.Be not recorded to the side effect relevant to medicine.
Embodiment 11
Group 1-active medicine group: give the tablet (6mg/ sheet) of 300mg with the water-alcohol solution dipping, described water-alcohol solution is the water-alcohol solution of following material: the anti-brain specific proteins of the rabbit through the antigen purification S-100 polyclonal antibody (anti-S100) of the active augmented form of very low dose (ULD) and anti-endothelial NO synthase polyclonal antibody (anti-eNOS) (" the anti-eNOS of the anti-S100+ULD of ULD "), and by to the super dilution 100 of initial soln (concentration is 2.5mg/ml) 12, 100 30With 100 200Doubly obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
Group 2-comparable group: give the tablet (3mg/ sheet) of 300mg with the water-alcohol solution dipping, described water-alcohol solution is the water-alcohol solution of following material: the anti-brain specific proteins of the rabbit through the antigen purification S-100 polyclonal antibody (the anti-S100 of ULD) of the active augmented form of very low dose (ULD), and by to the super dilution 100 of initial soln 12, 100 30With 100 50Doubly obtain, be equivalent to the mixture of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C50.
Group 3-matched group (placebo): give the tablet that 300mg contains excipient (267mg lactose monohydrate, 30mg microcrystalline Cellulose and 3mg magnesium stearate).
The anti-eNOS of the anti-S100+ of active medicine ULD is compared double-blind placebo-controlled-comparative study in the effect aspect not enough Attention Deficit Hyperactivity Disorder (ADHD) patient for the treatment of attention, described research is carried out in child's (9.3 ± 0.24 years old mean age) in 146 6-12 year, and described child is randomized into three groups of carrying out different treatments.Within 12 weeks, the patient (n=46) of group 1 accepts the anti-eNOS compositions of the anti-S100+ of ULD, every day twice, each 2; The member of comparable group 2 (n=50) accepts the anti-S100 of ULD, every day twice, each 2; The member of matched group 3 (n=50) accepts twice every day, each 2.All patients that research comprises have significant ADHD performance clinically, and this is confirmed on the balloon score of ADHD symptom evaluation scale (ADHDRS-IV-home edition): be 33.80 ± 0.92 in group 1; Be 32.5 ± 1.14 in group 2; And to organize in 3 be 33.6 ± 0.91.Most children are characterised in that the ADHD according to the medium severity of CGI-ADHD-severity questionnaire.The gross score of this scale is: group is to be 4.0 ± 0.03 minutes and to organize in 3 be 4.0 ± 0.00 minutes in 4.0 ± 0.02 minutes, group 2 in 1.Therefore, at initial period, the patient of three groups has comparable ADHD severity index.According to the result of neurological, clinical-laboratory and instrumental examination when research is registered, any patient does not detect extremely.In the treatment in 12 weeks, the patient is visited 6 times by the doctor.In this process, doctor-researcher has recorded dynamic changes of strength (gross score of ADHDRS-IV-home edition scale) and the disease severity (based on the CGI-ADHD-severity) of ADHD clinical manifestation, and the safety for the treatment of is write out a prescription, treated and estimate in supervision.
The analysis that the effect of 12 weeks treatment in three groups carried out shows, in the child of the anti-eNOS combination treatment of anti-S100+ with ULD, the gross score of ADHDRS-IV-home edition scale is compared the child who descends over 25% with initial score be 75% (n=36); This numerical value is 66% (n=33) in the patient of the anti-S100 treatment with ULD, accepting in the child of placebo is 56% (n=28).Three horizontal gradients that the situation of considering is improved (ADHDRS-IV scale gross score compare with baseline the reduction value for＜25%, 25-49.9% or 〉=50%), the effect difference that demonstrates between each groups of more detailed assessment is shown in Table 17.Notice in the group 1 of taking in the anti-eNOS of the anti-S100+ of ULD, 52% child compares gross score and has reduced more than 50% with baseline, produced remarkable improvement; Notice child's (Comparatively speaking, in the group 3 of taking in placebo, 8% patient only being arranged) of 34% in the group 2 of taking in the anti-S100 of ULD.
The significance of comparing ADHD clinical meaning (clinical implications) with original state reduces (p＜0.001) and occurs in all three observation groups at treatment 2 Zhou Houyi.In the patient of group 1 and group 2, owing to not only accessing about screening (screening), and when comparing with the index of the group 3 of taking in placebo, the significant difference between ADHDRS-IV-home edition gross score assert, therefore wherein actively dynamically more remarkable.In the treatment of coming weeks, begin to increase with the effect of the anti-eNOS compositions of the anti-S100+ of ULD and the anti-S100 of one-component preparation ULD treatment, in the active medicine group significantly (p＜0.05).ADHDRS-IV-home edition scale gross score reduction value in the child of the group 1 of taking in the anti-eNOS of the anti-S100+ of ULD is 16.5 minutes, is 12.4 minutes (being 6.3 minutes in the group 3 of absorption placebo by contrast) in the patient of the group 2 of taking in the anti-S100 of ULD.Result as the treatment of 12 weeks, compare with baseline, intensity on the ADHD clinical meaning has reduced almost half (48.8%) in the child of the anti-eNOS combination treatment of anti-S100+ with ULD, and has reduced in the patient with the anti-S100 of ULD more than 1/3rd (38.2%).
The picked-up anti-eNOS compositions of the anti-S100+ of ULD or the anti-S100 of ULD affect the two class symptoms of ADHD, the dynamic confirmation that this has been estimated by ADHDRS-IV-home edition scale two parts.In addition, compare with the effect of the anti-S100 treatment of unitary agent ULD, the influence degree of the treatment of the anti-eNOS compositions of the anti-S100+ of ULD aspect meaning intensity (intensity of implications) and attention deficiency and excessively moving/impulsion is significantly higher.
The active treatment effect of the anti-eNOS of the anti-S100+ of active medicine ULD and the anti-S100 of comparative drug ULD shows (table 17) in the patient treatment evaluation of result of ADHD severity evaluation scale (CGI-ADHR-severity).After the treatment of 3 months, in the anti-eNOS of the anti-S100+ of ULD group almost 1/4th patient's disease severity be down to slightly even Min. by moderate, this has been descended by the meansigma methods of CGI-ADHR-severity scale and 15% has been confirmed (to be down to 3.4 ± 0.06 by 4.0 ± 0.02; P＜0.001).Lower slightly with the anti-S100 treatment of the unitary agent ULD effect of 3 months, be shown as-10% (being 5% in placebo group by contrast) in CGI-ADHR-severity scale.Safety analysis has comprised the patient's of all participation researchs data.In the whole monitoring stage, the anti-eNOS of the anti-S100+ of active medicine ULD has with the anti-S100 of comparative formulations ULD the toleration that can compare with placebo.In a patient in a patient in the group of taking in the anti-S100 of ULD in (headache weakens in the research 4th week) and placebo group, (sleep-walking is during the observation second month) reported side effect.These side effect and treatment there is no and contact.In addition, observed a routine acute respiratory disease in therapeutic process, also irrelevant with treatment.All patients in seminar have completed treatment according to the plan that research approach is formulated; Nobody withdraws from advance.There is no pathological change in the replicate analysis process of patient body inspection and laboratory parameters, this has also confirmed the safety of the treatment studied.
Physical examination result (heart rate, SBP, DBP, body temperature) according to the patient is not recorded to any pathological change during treatment.According in the analytical scale of access and difference in comparable group do not reach statistical significance, do not exceed the limit of physiological allowable deviation (allowable deviation) yet.High treatment compliance (adherence to therapy) has also further shown effectiveness and the safety of research preparation.When treatment finished in three month, described compliance respectively does for oneself 99.8 ± 1.15% and 99.8 ± 2.25% (taking in by contrast, in the group 3 of placebo is 74.6 ± 2.54%) in the group 1 of taking in the anti-eNOS of the anti-S100+ of ULD and the group 2 of taking in the anti-S100 of ULD.
Therefore, studies have shown that the anti-eNOS compositions of the anti-S100+ of ULD and the anti-S100 of one-component preparation ULD suffer from effect and the safety aspect the child of ADHD in treatment.In the process in 12 weeks, observe the most significant therapeutic effect in combination drug (the anti-eNOS of the anti-S100+ of ULD), this is actively dynamically confirmed by clinical symptoms in great majority (75%) child.The anti-eNOS compositions of the anti-S100+ of ULD all has correcting to the two class symptoms of ADHD, and notices that therefore attention disorders significantly reduces with crossing to move all in ADHD patient.
When table 17. finishes in the treatment in 12 weeks, the gross score of ADHDRS-IV-home edition scale is dynamic
Relatively have significant difference with placebo group:
##p＜0.01。
The sign of the ADHD clinical meaning of table 18.ADHDRS-IV-home edition scale is dynamic
Figure BDA00002919547700612
Figure BDA00002919547700621
Figure BDA00002919547700631
Attention: relatively have significant difference with baseline parameter:
*p＜0.05，**p＜0.01，***p＜0.001。
Relatively have significant difference with placebo group:
#p＜0.05， ##p＜0.01， ###p＜0.001。
Relatively have significant difference with the anti-S100 group of ULD:
&p＜0.05。
The ADHD severity horizontal dynamic that table 19. is represented by CGI-ADHD-severity scale
Relatively has significant difference with baseline parameter: * * p＜0.01, * * * p＜0.001.
Embodiment 12
In the chronic heart failure human patients, following preparation is carried out double blinding, placebo-contrast clinical research, so that the key parameter of CHF pathology is estimated: the complex of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form.
In the research of 6 months by a definite date, 80 patients (functional classification (FC) is the CHF of II-IV level, and left ventricular ejection fraction (LVEF) is less than 40%) 4 are divided into treatment group and matched group.Do not stop background treatment (bisoprolol beta blocker, ACE inhibitor enalapril, aspirin (unless taboo is arranged); Still allow giving of diuretic, nitrate/salt and digoxin).The antiangiotensin II AT1 receptor C-terminal fragment antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the group 1 active augmented form of acceptance (3 slices/day, n=20).The anti-endothelial NO synthase antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the group 2 active augmented form of acceptance (3 slices/day, n=20).Group 3 accept to comprise simultaneously the anti-endothelial NO synthase antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and active augmented form the combination drug compositions (3 slices/day, n=20).The group 4 accept placebo (3 slices/day, n=20).Described group has comparability aspect following preliminary research parameter: age and sex, and the severity of disease (CHF and LVEF classification) and persistent period.
Before treatment and after treatment, the impact (to CHF process and progress very important) of the medicine that evaluation gives in the patient on reconstructing blood vessel and endothelial function disturbance.The pulse wave velocity (pulse wave velocity) of neck-femoral artery (CF) (elastic artery) section by tremulous pulse and neck-radial artery (CR) (muscular artery) section (PWV) (" Colson " system) on described medicine the impact in the reconstructing blood vessel process estimate.
Table 20 shows pulse wave velocity dynamic of neck-femoral artery (CF) (elastic artery) section of tremulous pulse and neck-radial artery (CR) (muscular artery) section.
Table 20
Figure BDA00002919547700651
(^) expression initial value;
(﹠amp; ) represent that administration began rear 6 months;
(*) expression and initial value relatively have in p value＜0.05 difference that o'clock can check;
(#) represent relatively have in p value＜0.05 with the group of accepting ULD antiangiotensin II AT1 receptor C-terminal Segment A b the difference that o'clock can check;
($) represent relatively have in p value＜0.05 with the group of accepting the anti-endothelial NO synthase Ab of ULD the difference that o'clock can check;
(1) ULD represents very low dose;
(2) Ab represents antibody.
After the treatment of 6 months, aspect the rigidity (stiffness) of muscular artery, only organize 3 demonstrate being proved of pharmaceutical composition required for protection impact.The group 3 accepting the group 1 of ULD antiangiotensin II AT1 receptor C-terminal fragment antibody and accept combination drug compositions of the present invention is demonstrating the increase that has been proved aspect the rigidity of elastic artery.
Embodiment 13
In the chronic heart failure human patients, following preparation is carried out double blinding, placebo-contrast clinical research, estimate with the key metrics to quality of life: the complex of the antiangiotensin II AT1 receptor C-terminal fragment antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and the anti-endothelial NO synthase antibody (being in the mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form.
In the research of 6 months by a definite date, 80 patients (functional classification (FC) is the CHF of II-IV level, and left ventricular ejection fraction (LVEF) is less than 40%) 4 are divided into treatment group and matched group.Do not stop background treatment (bisoprolol beta blocker, ACE inhibitor enalapril, aspirin (unless taboo is arranged); Still allow giving of diuretic, nitrate/salt and digoxin).The antiangiotensin II AT1 receptor C-terminal fragment antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the group 1 active augmented form of acceptance (3 slices/day, n=20).The anti-endothelial NO synthase antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the group 2 active augmented form of acceptance (3 slices/day, n=20).Group 3 accept to comprise simultaneously the anti-endothelial NO synthase antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of the antiangiotensin II AT1 receptor C-terminal fragment antibody (mixture of homeopathic therapeutic method's diluent C12, C30 and C200) of active augmented form and active augmented form the combination drug compositions (3 slices/day, n=20).The group 4 accept placebo (3 slices/day, n=20).Described group has comparability aspect following preliminary research parameter: age and sex, and the severity of disease (CHF and LVEF classification) and persistent period.Before treatment and after treatment, Morphologic Parameters and the physical sport toleration of Quality of Life (Minnesota questionnaire and Kansas questionnaire), heart are estimated.
Table 21 shows result of study with the dynamic form for the treatment of effect underlying parameter.
After the treatment of 6 months, group 1 patient (with the treatment of ULD antiangiotensin II AT1 receptor C-terminal fragment antibody) demonstrates the remarkable improvement of quality of life, the improvement of Assessment of Left Ventricular Systolic Function and the raising of physical sport toleration.The reduction that group 2 has demonstrated being proved of anxiety aspect quality of life and level of depression (using the Kansas questionnaire to estimate).This studies confirm that with combination drug compositions combined standard CHF of the present invention treatment (under study for action, in the group 3 all parameters all be shown as the positive dynamic patient who has been proved give) and has obtained optimum therapeutic effect.
In pharmaceutical composition of the present invention (combination drug), the antiangiotensin II AT1 receptor C-terminal fragment antibody of active (reinforcement) form and the complex of anti-endothelial nitric oxide synthase (NO synthase) antibody provide unforeseeable synergistic therapeutic effect, this means the impact that has strengthened reconstructing blood vessel and endothelial function disturbance, this is very important for CHF process and progress; Also strengthened the impact that patients ' life quality is improved; And having strengthened impact on Morphologic Parameters and the physical sport toleration of heart, this is confirmed by clinical trial.
The results are shown in Table 21.
Table 21
Figure BDA00002919547700681
*, * *, * * *-p value difference＜0.05,0.01 and 0.001;
#-represents relatively have in p value＜0.05 with the group of accepting ULD antiangiotensin II AT1 receptor C-terminal Segment A b the difference that o'clock can check;
$, it is 0.05 and the difference that can check in 0.01 o'clock that $ $-represent and the group of accepting the anti-endothelial NO synthase Ab of ULD have more respectively in the p value;
(1)-ULD represents very low dose;
(2) the Ab meaning is antibody;
(3) " Minnesota " expression Minnesota questionnaire;
(4) " Kansas " expression Kansas questionnaire;
(5) HADS represents the HADS gross score;
(6) FC CHF represents the functional classification of Patients with Chronic Heart Failure;
(7) FF LV represents the left ventricular function mark.
Embodiment 14
For the pharmaceutical composition of studying proposal in the character aspect the treatment Benign Prostatic Hypertrophy, used the water-alcohol solution saturated pill (6mg/ ball) of 300mg to comprise pharmaceutical composition, described pharmaceutical composition is the anti-prostate specific antigen polyclonal antibody of rabbit (anti-PSA) and the anti-endothelial NO synthase polyclonal antibody (anti-eNOS) of affinity purification of the active augmented form of very low dose (ULD), by to the super dilution 100 of initial matrix solution 12, 100 30With 100 200Doubly make, be equivalent to the mixture (the anti-eNOS of the anti-PSA+ of ULD) of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200; And the water-alcohol solution saturated pill (3mg/ ball) of 300mg to comprise pharmaceutical composition, described pharmaceutical composition is the anti-prostate specific antigen polyclonal antibody of rabbit (anti-PSA) of affinity purification of the active augmented form of very low dose (ULD), by to the super dilution 100 of initial matrix solution 12, 100 30With 100 200Doubly make, be equivalent to the mixture (the anti-PSA of ULD) of Radix Achyranthis Bidentatae homeopathic therapeutic method diluent C12, C30 and C200.
Benign prostatic hyperplasia (BPH) is one of obstacle of the most often occurring in the male (Bruskewitz R.C., 2003; Rosen R., 2003): on the one hand, the frequency of pointing out BPH in the epidemiological study that Russia carries out is from 11.3% being increased to gradually 80 years old 81.4% (Gorilovskiy L.M., 1999) in the people among 40-49 year people; On the other hand, the demography of being undertaken by WHO studies confirm that the phenomenal growth in the crowd more than 60 years old, has surpassed the growth of any other age group.
The cardinal symptom of benign prostatic hyperplasia is lower urinary tract symptom, can cause significant discomfort and quality of life to reduce (Bruskewitz R.C., 2003; Lepor H., O ' Leary M.P., 2005).In severe case, this disease can be with complication, for example acute urinary retention, urinary tract infection, erythuria (erythruria) and renal failure (Stepanov V.N., 1999; Jacobsen S.J., 1997; Lepor H., 2004).BPH is (Bruskewitz R.C., 2003 relevant to patient's erectile dysfunction also; Daly MP, 2005).
The pharmaceutical composition that contains the anti-eNOS of the anti-PSA+ULD of ULD and the pharmaceutical composition that contains the anti-PSA of ULD are carried out more parallel group of research of open label in the treatment effect of improving aspect the urinary disturbance that benign prostatic hyperplasia (BPH) causes with safety, and described research comprises that 40 bases include in/the selected patient of exclusion standard.The patient is divided into 2 groups at random, and one group of patient (n=21) accepts the anti-eNOS of the anti-PSA+ of ULD of three 1 balls every day during 12 weeks, and another group (n=19) is accepted the anti-PSA of ULD of three 1 balls every day during 12 weeks.Comparable on age, BPH severity of symptoms, urinary system parameter and prostate volume between two groups.
The patient age that comprises of research more than 45 years old, have the BPH medical history, wherein, the corresponding symptom of lower urinary tract is no less than 6 months, IPSS 〉=13, prostate volume are according to endorectal ultrasonography inspection 〉=30cm 3, peak urinary flow rate degree 〉=4ml/s also≤the remaining volume of urine of 15ml/s and Minimum Residual equals 125ml, PSA level≤4ng/ml.Necessary inclusive criteria is not taken in following medicine in medical records: be finasteride (finasteride), dutasteride (dutasteride) or other experimental drug in front 6 months including research in; Be α 1-adrenoceptor blocker (adrenoreceptor blocker) and medical herbs (herbal medication) including in front 4 weeks of research; Be any PDE5 type inhibitor and the treatment of other erectile dysfunction including in front 4 weeks of research.
Research does not comprise following patient's: once went through invasive method treatment BPH, and comprised transurethral prostatic resection, thermotherapy (thermotherapy), per urethra acupuncture ablation (transurethral needle ablation), stent forming art and other; Suffer from that malignancy disease, acute urinary retention, vesical calculus, urethral stricture, MarionShi are sick, the urogenital infections of active inflammatory phase and other.
The clinical efficacy of pharmaceutical composition is assessed by the improvement of lower urinary tract clinical symptoms, and described assessment is used IPSS questionnaire (international prostate gland symptoms scoring), urinary system parameter (maximum and average urine Flow Velocity, the volume of urinating, remaining volume of urine) and estimated based on the prostate volume that per urethra ultrasonic (TU) data draw; And erectile function is estimated based on the data that IIEF questionnaire (international erectile function index) obtains.Result of study shows in table 22 and table 23.
Table 22.
Figure BDA00002919547700711
1-molecule is the patient's number (n) that demonstrates improvement, and denominator is the total patient's number (N) in research.
The problem 7 of table 23.IPSS questionnaire and obstructive dynamic with the irritative symptoms subscale
*-compare p＜0.05 with baseline; *-compare with baseline p＜0.01; * *-compare with baseline p＜0.001
##-compares p＜0.01 with the anti-PSA of ULD
2-shown the decline % that compares with baseline, cell mean
The anti-PSA of data acknowledgement ULD that provides and the anti-eNOS of the anti-PSA+ULD of ULD can effectively treat lower urinary tract symptom, improve average and the peak urinary flow rate degree, improve Quality of Life (table 22).The treatment time-histories is not grown (12 week), does not therefore all observe reducing of prostate volume in any seminar.The anti-PSA of ULD does not affect the volume of urinating, and it only increases in 52.6% patient, compares with baseline value, and this group on average demonstrates the non-statistical significance ground, given volume degree ground of urinating and reduces 11.8ml (5.4%).Simultaneously, have 71.4% to demonstrate the increase of the volume of urinating in the patient with the anti-eNOS of the anti-PSA+ULD of ULD treatment, and compare the average external volume value added with baseline be 48.3ml (23.7%).
According to obstructive and irritative symptoms and the dynamic analysis demonstration of nocturia sign (problem 7 of IPSS) that the IPSS subscale carries out, two kinds of pharmaceutical compositions all help to reduce obstructive and irritative symptoms and nocturia symptom.Simultaneously, compare with the anti-PSA of ULD, the anti-eNOS of the anti-PSA+ of ULD is more effective in minimizing lower urinary tract irritative symptoms (28.2% pair 40.3%, p＜0.05) and urgent micturition at night (2.0% pair 37.7%) aspect.
Should be noted that with the anti-PSA of ULD and compare, the anti-eNOS of the anti-PSA+ULD of ULD improve aspect patient's erectile function equally more effective.In the anti-eNOS group of the anti-PSA+ULD of ULD, total IIEF of patient (international erectile dysfunction index) mark has improved 19% (having improved 10.5% in the anti-PSA group of ULD), the meansigma methods that the IIEF mark improves in the anti-eNOS group of the anti-PSA+ULD of ULD is 8%, Comparatively speaking, be 4.5% in the anti-PSA group of ULD.
Described pharmaceutical composition demonstrates excellent security performance, does not observe the side effect relevant to the medicine that gives in research process.
Therefore, compare with the anti-PSA of ULD, demonstrate better effect aspect the urological problems that the anti-eNOS of the anti-PSA+ULD of ULD is caused by benign prostatic hyperplasia in treatment.In addition, compare with the anti-PSA of ULD, the anti-eNOS of the anti-PSA+ULD of ULD is demonstrating larger good effect aspect patient's erection function.
Figure IDA00002919548100021
Figure IDA00002919548100031
Figure IDA00002919548100041
Figure IDA00002919548100051
Figure IDA00002919548100061
Figure IDA00002919548100071

Claims (15)

1. the method for the effect of an anti-endogenous biomolecule antibody that improves active augmented form, described method comprises the anti-endothelial NO synthase of the anti-endogenous biomolecule antibody of described active augmented form and active augmented form antibody combined.
2. the method for claim 1, described method further comprise with through described associating and the complex patient that gives to utilize the antibody of described active augmented form to treat.
3. method as claimed in claim 2, wherein, the anti-endogenous biomolecule antibody of described active augmented form is anti-S-100 protein antibodies.
4. method as claimed in claim 2, wherein, the anti-endogenous biomolecule antibody of described active augmented form is anti-prostate specific antigen antibody.
5. method as claimed in claim 2, wherein, the anti-endogenous biomolecule antibody of described active augmented form is anti-insulin receptor antibody.
6. method as claimed in claim 2, wherein, the anti-endogenous biomolecule antibody of described active augmented form is antiangiotensin receptor II antibody.
7. pharmaceutical composition, described pharmaceutical composition comprises: a) the anti-endogenous biomolecule antibody of active augmented form; And b) the anti-NO synthase antibody of active augmented form.
8. pharmaceutical composition as claimed in claim 7, described pharmaceutical composition further comprises pharmaceutically acceptable solid-state carrier.
9. pharmaceutical composition as claimed in claim 8, wherein, the anti-endothelial NO synthase antibody of described active augmented form is in the form of mixtures of C12, C30 and C200 homeopathic therapeutic method diluent, and described impregnation mixture is to described solid-state carrier.
10. pharmaceutical composition as claimed in claim 7, wherein, the anti-endogenous biomolecule antibody of described active augmented form is monoclonal antibody, polyclonal antibody or natural antibody.
11. pharmaceutical composition as claimed in claim 10, wherein, described anti-endogenous biomolecule antibody is polyclonal antibody.
12. pharmaceutical composition as claimed in claim 7, wherein, the anti-endogenous biomolecule antibody of described active augmented form all accompanies by vibration when diluting and diluting at every turn by continuous Radix Achyranthis Bidentatae and prepares.
13. pharmaceutical composition as claimed in claim 7, wherein, described anti-endothelial NO synthase antibody is monoclonal antibody, polyclonal antibody or natural antibody.
14. pharmaceutical composition as claimed in claim 8, wherein, described anti-endothelial NO synthase antibody is polyclonal antibody.
15. pharmaceutical composition as claimed in claim 9, wherein, the anti-endothelial NO synthase antibody of described active augmented form all accompanies by vibration when diluting and diluting at every turn by continuous Radix Achyranthis Bidentatae and prepares.
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Application publication date: 20130515

RJ01 Rejection of invention patent application after publication