CN102370985A - Purpose of agonist of natriuretic peptide receptor A in pain management - Google Patents

Purpose of agonist of natriuretic peptide receptor A in pain management Download PDF

Info

Publication number
CN102370985A
CN102370985A CN2010102577814A CN201010257781A CN102370985A CN 102370985 A CN102370985 A CN 102370985A CN 2010102577814 A CN2010102577814 A CN 2010102577814A CN 201010257781 A CN201010257781 A CN 201010257781A CN 102370985 A CN102370985 A CN 102370985A
Authority
CN
China
Prior art keywords
natriuretic peptide
pain
type
receptor
peptide receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN2010102577814A
Other languages
Chinese (zh)
Inventor
张旭
张方雄
刘兴君
鲍岚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institutes for Biological Sciences SIBS of CAS
Original Assignee
Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institutes for Biological Sciences SIBS of CAS filed Critical Shanghai Institutes for Biological Sciences SIBS of CAS
Priority to CN2010102577814A priority Critical patent/CN102370985A/en
Publication of CN102370985A publication Critical patent/CN102370985A/en
Pending legal-status Critical Current

Links

Images

Abstract

The invention provides a purpose of an agonist of natriuretic peptide receptor A (NPR-A) in pain management. Specifically, the invention relates to a purpose of the NPR agonist in the preparations of medicine compositions used for releasing or treating pains. Preferably the agonist is B-type natriuretic peptide (BNP) or atrial natriuretic peptide (ANP). The invention also relates to a medicine composition containing the NPR-A agonist, and a method for screening potential substances that can be used for releasing or treating pains. According to the method, for the first time, a relation between NPR-A mediated NPR-A/PKG/BKCa approach and pains is disclosed; and a medicine, a method and a purpose for treating, releasing or ameliorating pains through the regulation of the activation of the approach are provided.

Description

The purposes of agonist in pain therapy of natriuretic peptide receptor A
Technical field
The invention belongs to biotechnology and field of medicaments.More specifically, the present invention relates to the natriuretic peptide receptor stimulating agent, the agonist (for example Type B natriuretic peptide or atrial natriuretic peptide) of preferred natriuretic peptide receptor A, the purposes in pain therapy.
Background technology
A kind of offending reaction that pain is body to be produced damaged tissue or potential damage is a kind of complex physical mental activity, also is one of modal symptom clinically.It reacts two parts by the pain sensation with pain and forms.It comprises that noxious stimulation acts on the caused pain of body and feels, and body is to the pain reaction (reaction of somatic movement property and/or internal organs vegetative reaction often are attended by intensive emotion color) of noxious stimulation.
The pain sensation can be used as a kind of warning that body comes to harm, and causes the protective reaction of body series of defence property.But then, some violent or secular pain has become a kind of insufferable torment to body.Therefore, analgesia is the vital task that this area researcher is faced.
As everyone knows, neuropeptide and receptor thereof play an important role in transmission of pain with in regulating.Neuropeptide is the endogenous active substance that general reference is present in nervous tissue and participates in the nervous function effect, is one type of special information substance.Their characteristics are low, the active height of content, action range and complicacy, regulate diversified physiological function in vivo, as: the pain sensation, sleep, emotion, Learning and Memory, the differentiation of nervous system itself and growth all receive the adjusting of neuropeptide even.
The dorsal root ganglion small neuron is accepting can to secrete the P material and the calcitonin-gene-related peptide of its expression when pain stimulates, thereby strengthens excitatory synapse transmission (Schaible, 1996, Prog Brain Res 113:423-441).Somatostatin and receptor thereof also are expressed in a part of dorsal root ganglion small neuron; Activate somatostatin receptor and can reduce the nociception that inflammation causes (
Figure BSA00000236191600011
etc.; 1976, Neuroscience 1:131-136; Carlton etc., 2001, Pain 90:233-244; Bar etc., 2004, Neuroscience 127:197-206).
In addition, under normal circumstances, galanin, neurotensin and neuropeptide tyrosine also all are expressed in the dorsal root ganglion neurons, and these neuropeptides also have expression (Todd etc., 1994, JNeurosci 14:774-784 in dorsal horn neurons simultaneously; Simmons etc., 1995, Neurosci Lett 187:119-122; Zhang etc., 1995, JNeurosci 15:2733-2747; Zhang etc., 1997, Proc Natl Acad Sci USA 94:729-734; Zhang etc., 1998, Ann N Y Acad Sci 863:402-413; Polgar etc., 1999, J Neurosci 19:2637-2646).The same with P material, calcitonin-gene-related peptide and somatostatin, these neuropeptides also participate in regulating dorsal root ganglion neurons electrical activity (Zhang etc., 1995, J Neurosci 15:2733-2747; Xu etc., 1997, Proc Natl Acad Sci USA 94:13262-13266).
It should be noted that above these neuropeptides mentioned and receptor thereof around under nerve injury or the inflammation situation, change (Ji etc., 1994, the J Neurosci 14:6423-6434 of significance can take place in their expression in dorsal root ganglion; Ji etc., 1995, Neuroscience 68:563-576; Zhang etc., 1995, J Neurosci 15:2733-2747; Zhang etc., 1998, Ann N Y Acad Sci 863:402-413; Xiao etc., 2002, ProcNatl Acad Sci USA 99:8360-8365), this shows that they are bringing into play potential effect aspect the pathological pain regulating.
Though this area has obtained above-mentioned progress about the research of neuropeptide; But this area still presses for and further work out those unknown neuropeptides and receptor is expressed in dorsal root ganglion; And it expresses the variation that is taken place behind peripheral tissue inflammation, thereby develops treatment or lenitive effective way and method.
Natriuretic peptide is a paracrine factor, comprises four members in the family: atrial natriuretic peptide, Type B natriuretic peptide and C type natriuretic peptide (Potter etc., 2009, Handb Exp Pharmacol:341-366).The Type B natriuretic peptide is also claimed BNF, is expressed and secretion by the ventricle of heart.(atrial natriuretic peptide ANP) is peptide hormone synthetic by atrial muscle cell and that discharge to atrial natriuretic peptide, and it mainly acts on is to make the vascular smooth muscle diastole and promote kidney row sodium, draining.D type natriuretic peptide is 38 amino acid whose polypeptide that from the Naja of a kind of America, extract, and it has the function of regulating cardiovascular system, urinary system, digestive system and reproductive system.
At present; With natriuretic peptide receptor (natriuretic peptide receptor; NPR) be divided into A, B, three hypotypes of C, wherein A receptor (NPR-A) and B receptor (NPR-B) are the guanylate cyclase coupled receptors, after natriuretic peptide and A, the B receptors bind; The guanylate cyclase of activated receptor is active, through rising intracellular cGMP performance physiological effect; C receptor (NPR-C) is non-coupled receptor, mainly mediates the removing of natriuretic peptide.
Natriuretic peptide receptor A belongs to guanylate cyclase receptor, the Type B natriuretic peptide combine with natriuretic peptide receptor A to improve cyclic guanosine 3 in the cell ', 5 '-phosphoric acid (cyclic guanosine 3 '; 5 '-monophosphate; CGMP) level (Misono, 2002, Mol Cell Biochem 230:49-60); Protein kinase (the cGMP-dependent protein kinase that further then activation cGMP relies on; PKG), ion channel and other effector molecule (Hofmann etc., 2006, Physiol Rev 86:1-23).Natriuretic peptide receptor A also is the receptor of atrial natriuretic peptide simultaneously.Natriuretic peptide receptor A mainly comes to light at present and is expressed among cardiovascular, kidney and the adrenal gland, is bringing into play functions (Hofmann etc., 2006, Physiol Rev 86:1-23) such as adjusting vasodilation, natruresis and aldosterone synthesize.
Though the expression of great majority research Type B natriuretic peptide and natriuretic peptide receptor A and effect all are to concentrate on cardiovascular system (Woodard and Rosado, 2007, J Cell Mol Med 11:1263-1271 at present; Potter etc.; 2009; Handb Exp Pharmacol:341-366), there are some researches show also that recently Type B natriuretic peptide and natriuretic peptide receptor A also also have low expression level the central nervous system, and the synapse transmission of adjusting and neuroprotective (Richard and Bourque are arranged; 1996, J Neurosci 16:7526-7532; Kuribayashi etc., 2006, Brain Res1071:34-41; Cao and Yang, 2008, Prog Neurobiol 84:234-248).
Yet this area is never studied natriuretic peptide and (for example Type B natriuretic peptide and natriuretic peptide receptor A) the neural around expression of its receptor and function, does not also know between itself and the pain whether have relation.
In sum; Although this area has had been found that some acting molecules in sensory information transmits at present; Yet also press for research and in the sensory information transmittance process, also have those molecules in action; With the understanding that promotes sensory information is transmitted, the new analgesia strategy analgesic new with exploitation proposed.
Summary of the invention
One of the object of the invention is exactly to disclose peripheral nerve cGMP to raise material (like the natriuretic peptide receptor) is used for alleviating or treating the pharmaceutical composition of pain (especially chronic pain) in preparation purposes.The present invention has also specifically disclosed neural around expression of natriuretic peptide receptor (like natriuretic peptide receptor A) agonist (like the Type B natriuretic peptide) and function.Another object of the present invention provides Type B natriuretic peptide, its receptor and the purposes of regulator in pain therapy thereof.
In first aspect of the present invention, provide peripheral nervous cGMP to raise material is used for alleviating or treating the pharmaceutical composition of pain in preparation purposes.
In an embodiment of the invention, said peripheral nervous cGMP rise material is the natriuretic peptide receptor stimulating agent.
In yet another embodiment of the present invention, said natriuretic peptide receptor stimulating agent is selected from: the agonist of the agonist of natriuretic peptide receptor A, natriuretic peptide receptor B and/or the agonist of natriuretic peptide receptor C.
In yet another embodiment of the present invention, said natriuretic peptide receptor stimulating agent is selected from: natural natriuretic peptide, they have analog or its combination of exciting natriuretic peptide receptor active.
In yet another embodiment of the present invention, said natural natriuretic peptide is selected from: atrial natriuretic peptide, Type B natriuretic peptide, C type natriuretic peptide, D type natriuretic peptide, urodilatin, preceding natriuretic peptide, former-preceding natriuretic peptide or their combination.
In yet another embodiment of the present invention, said analog is selected from: with the link coupled natural natriuretic peptide of PEG; With serum albumin or the link coupled natural natriuretic peptide of its fragment; The variable shearing product of natural natriuretic peptide; The fusion rotein that forms by the annulus of natural natriuretic peptide and aminoterminal and/or c-terminus afterbody, the fusion rotein of preferably partly forming by the non-annularity of a kind of annulus of natural natriuretic peptide and another kind of natural natriuretic peptide.
In yet another embodiment of the present invention, said derivant is selected from; AlbuBNP, CD-NP, ASBNP, ASBNP.1, hBNP1-32, hBNP-054, Nesiritide or their combination.
In yet another embodiment of the present invention, the said natriuretic peptide receptor stimulating agent agonist that is natriuretic peptide receptor A.
In a preference, said natriuretic peptide receptor A is mammiferous natriuretic peptide receptor A.
In another preference, said mammal is selected from: people, orangutan, monkey, dog, rat, mice, cattle, horse, sheep or pig.
In an embodiment of the invention, the agonist of said natriuretic peptide receptor A is selected from: Type B natriuretic peptide or atrial natriuretic peptide.
In a preference, said Type B natriuretic peptide or atrial natriuretic peptide are selected from:
(a) protein of aminoacid sequence shown in protein of aminoacid sequence shown in the SEQ ID NO:1 or the SEQ ID NO:2;
(b) the proteinic conservative variant protein matter or its active fragment that are limited in (a); Or
(c) with aminoacid sequence in (a) through 1-20, preferred 1-10, more preferably the replacement of 1-5 amino acid residue, disappearance or interpolation form, and have activation natriuretic peptide receptor A active, by (a) deutero-protein.
In yet another embodiment of the present invention, the form of said Type B natriuretic peptide or atrial natriuretic peptide is selected from down group: isolating Type B natriuretic peptide or atrial natriuretic peptide, its coded sequence or comprise the carrier of its coded sequence or genetically engineered host cell.
In yet another embodiment of the present invention, said pain is self source property pain.
In yet another embodiment of the present invention, said body source property pain is selected from: neuropathic pain or nociceptive pain.
In a preference; Said neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain, migraine or sciatica are preferably the pain that causes of damage or pathological change by peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN and with the related neurogenic neuralgia of HIV.
In another preference, said nociceptive pain is selected from: body nociceptive pain or internal organs nociceptive pain, preferred cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.
In yet another embodiment of the present invention, said pain is inflammatory pain.
In yet another embodiment of the present invention, said inflammatory pain is selected from: arthritis pain, diabetes complicated peripheral nerve inflammatory pain, peripheral tissues's inflammatory pain or internal organs inflammatory pain.
In yet another embodiment of the present invention, said pain is chronic pain or acute pain, and preferred said chronic pain is the rational pain of chronic susceptibility pain or chronic neuropathic, more preferably chronic susceptibility pain.In yet another embodiment of the present invention, said chronic pain is selected from down group: soft tissue, joint and bone pain: pain behind deformity property pain, skeletal muscle pain, lumbago, myofasical pain syndrome, headache, the burn after various osteoarthritis, the wound; Deep tissue and Encelialgia: cardiovascular pain, ophthalmalgia, actinal surface portion pain, chronic gynecological pain, sexual anhedonia, genitourinary system chronic pain; Nerve and nerve root injury property pain: phantom limb pain after amputation, peripheral nerve property pain, sympathetic reflex dystrophy and sympathetic nerve lasting pain, trigeminal neuralgia and atypical facial pain, nerve root injury and arachnoiditis, PHN; Central pain: the blood vessel injury of brain, spinal cord, like hemorrhage, infraction, vascular malformation; Multiple sclerosis; Traumatic spinal cord injury, brain injury; Syringomyelia and syringobulbia; Tumor; Parkinson disease; Child's pain: growing pain; Old people's pain: cervical spondylosis, prolapse of lumbar intervertebral disc, lumbar spondylolisthesis are pain caused; Or cancerous pain.
In second aspect of the present invention, a kind of pharmaceutical composition is provided, said pharmaceutical composition comprises:
(a) agonist of the natriuretic peptide receptor (for example natriuretic peptide receptor A) of alleviation or treatment pain effective dose;
(b) pharmaceutically acceptable carrier.
In an embodiment of the invention, the agonist of said natriuretic peptide receptor A is selected from: Type B natriuretic peptide or atrial natriuretic peptide.
In a preference, said Type B natriuretic peptide or atrial natriuretic peptide are selected from:
(a) protein of aminoacid sequence shown in protein of aminoacid sequence shown in the SEQ ID NO:1 or the SEQ ID NO:2;
(b) the proteinic conservative variant protein matter or its active fragment that are limited in (a); Or
(c) with aminoacid sequence in (a) through 1-20, preferred 1-10, more preferably the replacement of 1-5 amino acid residue, disappearance or interpolation form, and have activation natriuretic peptide receptor A active, by (a) deutero-protein.
In another preference, the form of said Type B natriuretic peptide or atrial natriuretic peptide is selected from down group: isolating Type B natriuretic peptide or atrial natriuretic peptide, its coded sequence or comprise the carrier of its coded sequence or genetically engineered host cell.
In the third aspect of the invention, the method that provides a kind of screening to can be used for alleviating or treating the potential material of pain, described method comprises:
(1) candidate substances is contacted with the dorsal root ganglion small neuron;
(2) detect candidate substances and whether can activate natriuretic peptide receptor/PKG/BK CaApproach (natriuretic peptide receptor A/PKG/BK for example CaApproach);
If said candidate substances can activate natriuretic peptide receptor/PKG/BK CaApproach shows that then this candidate substances is the potential material of alleviating or treating pain.
In a preference, said natriuretic peptide receptor/PKG/BK CaThe detection of pathway activation is to carry out through testing one or more indexs that are selected from down group: natriuretic peptide receptor expression level, electrical activity, calcium ion level, intracellular cGMP level, known natriuretic peptide receptor A/PKG/BK CaThe inhibitor of approach is to the reverse or the inhibition of this candidate substances effect.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1: Type B natriuretic peptide and natriuretic peptide receptor A expression and the expression behind the periphery tissue inflammation in dorsal root ganglion changes.
Figure 1A: expressed the neuropeptide have significant change and the cluster analysis of neuropeptide receptor after the detected periphery inflammation of gene chip.After the injection of CFA vola, 18 neuropeptides and 7 neuropeptide receptors significantly increase at the expression of waist (L) 4 and L5 dorsal root ganglion, comprise Type B natriuretic peptides and their receptors natriuretic peptide receptor A;
Figure 1B: immunoblotting assay shows with matched group to be compared, the Type B natriuretic peptide and the also corresponding increase of natriuretic peptide receptor A protein level of CFA vola injection back L4 and L5 dorsal root ganglion.Type B natriuretic peptide protein level increases after the periphery inflammation gradually in the dorsal root ganglion, peaks in the 4th day in injection.Natriuretic peptide receptor A the 1st day protein level after injection begins to increase, and arrives the peak in the 2nd day.Be used as last appearance confidential reference items with glycerol triphosphoric acid dehydrogenase (GAPDH); *Compare with normal group p＜0.05.
Fig. 2: Type B natriuretic peptide and natriuretic peptide receptor A expression and the postinflammatory around expression in the dorsal root ganglion small neuron changes.
Fig. 2 A: immunohistochemical staining shows that immunoreation of Type B natriuretic peptide and natriuretic peptide receptor A immunoreation mainly are distributed in L4 and L5 dorsal root ganglion small neuron.The injection of CFA vola is after 2 days, and Type B natriuretic peptide and natriuretic peptide receptor A express at the dorsal root ganglion small neuron to be increased.By the neuronal quantity of Type B natriuretic peptide immunostaining greater than by the neuronal quantity of natriuretic peptide receptor A immunostaining.It is positive that all natriuretic peptide receptor A positive neurons also are the Type B natriuretic peptide.Scale: 50 microns.
Fig. 2 B: rat Type B natriuretic peptide and male L4 of natriuretic peptide receptor A and L5 dorsal root ganglion neurons cell size distribution after 2 days injected in normal and CFA vola.Type B natriuretic peptide and natriuretic peptide receptor A positive neurons elemental size drop on the magnitude range interior (cross-sectional area＜900 square microns) of dorsal root ganglion small neuron.
Fig. 2 C: dorsal root ganglion small neuron Type B natriuretic peptide immunity intensity is increased to 81.64 ± 0.87 (606 neurons) of CFA vola injection after 2 days from 63.56 ± 0.69 (608 neurons) of normal rat.Natriuretic peptide receptor A immune fluorescence intensity also increases to inflammation 51.14 ± 0.67 after 2 days (317 neurons) from normal 58.02 ± 0.83 (324 neurons). * *Compare with normal group p＜0.001.
Fig. 2 D: at L4 and L5 dorsal root ganglion, about respectively 52% and 53% the Type B natriuretic peptide positive neuron natriuretic peptide receptor A that also dyes is injected after 2 days in normal and CFA vola; It is positive that nearly all natriuretic peptide receptor A positive neuron all is the Type B natriuretic peptide.
Fig. 3: the expression of Type B natriuretic peptide in the male dorsal root ganglion small neuron of TrkA.
Fig. 3 A: two dyeing show that the Type B natriuretic peptide is present in the male rat dorsal root ganglion small neuron of TrkA.
Fig. 3 B:B type natriuretic peptide is present in the dorsal root ganglion CGRP positive and the positive small neuron of IB4.Scale: 50 microns.
Fig. 4: the expression of natriuretic peptide receptor A in dorsal root ganglion Toplink small neuron.
Fig. 4 A: two dyeing show that natriuretic peptide receptor A is expressed in the rat dorsal root ganglion small neuron that contains CGRP.
Fig. 4 B: at the cornu dorsale medullae spinalis shallow-layer, natriuretic peptide receptor A positive fiber is also expressed CGRP simultaneously.In addition, natriuretic peptide receptor A immunoreation is also found along vascularity (arrow indication).Scale: 50 microns.
Fig. 5: exist under the glutamic acid condition, the Type B natriuretic peptide is to the excitatoty reduction effect of dorsal root ganglion small neuron.
Fig. 5 A: full cell patch pincers record shows that the rat dorsal root ganglion small neuron of growing up receives the depolarization slope current to stimulate (1 second time; The discharge frequency of the action potential that peak from 100 to 500 skin amperes) brings out is adding the not obviously change of generation after 10 minutes of 100 nanograms/milliliter Type B natriuretic peptides, and adding 10 micromole's glutamic acid has then increased discharge frequency.Yet Type B natriuretic peptide and glutamic acid are handled (100 nanograms/milliliter Type B natriuretic peptides+10 micromole's glutamic acid, 10 minutes) together and have been suppressed neuronal excitability significantly.
Fig. 5 B: the discharge frequency ratio of the dorsal root ganglion small neuron action potential that galvanism causes (after the dosing divided by before the dosing) is adding extracellular fluid (1.13 ± 0.06,22 example) and is adding Type B natriuretic peptide (100 nanograms/milliliter; 1.13 ± 0.09,14 examples) do not have significance difference (p=0.971) between the group.But add slight increase is arranged behind 10 micromole's glutamic acid (1.49 ± 0.14,10 examples, p=0.035).Type B natriuretic peptide and glutamic acid add together the action potential that suppressed to a great extent to bring out (0.08 ± 0.05,21 examples, * *P＜0.001).The Type B natriuretic peptide adds the neuronal excitability that glutamic acid causes and suppresses to be anticipated PKG inhibitor KT5823 (1.81 ± 0.17,11 example) or potassium channel BK CaBlocker iberiotoxin (IBTX) (1.14 ± 0.15,10 example) blocking-up.
Fig. 6: intrathecal injection Type B natriuretic peptide is to the inhibitory action of the inductive acute pain of formalin.
Fig. 6 A: intrathecal injection Type B natriuretic peptide (100 nanograms, 500 nanograms and 2 micrograms; Prior to vola injection of formalin 10 minutes) dose dependent ground reduces formalin inductive biphase pain reaction (P＜0.001, corresponding 100 nanograms, 500 nanograms and 2 microgram Type B natriuretic peptides respectively; Compare ANOVA, 7 routine Mus/groups with the pump pickle group).
The inductive anti-nocuity effect of Fig. 6 B:B type natriuretic peptide can be by injection in advance (prior to intrathecal injection Type B natriuretic peptide 10 minutes) PKG inhibitor KT5823 (500 nanogram) or potassium channel BK in the sheath CaBlocker iberiotoxin (IBTX) (500 nanogram) blocks (first phase: p=0.038 and 0.048; Second phase: P=0.003 and 0.037; For giving Type B natriuretic peptide and KT5823 or IBTX jointly; Give relatively 7 routine Mus/groups separately with the Type B natriuretic peptide), and adopt ATP sensitive potassium channel blocker glibenclamide (Glib) (50 microgram) can not block the effect of Type B natriuretic peptide.The pain behavior that causes of formalin can not be by KT5823, IBTX or Glib individual processing influence (7 routine Mus/group). *P＜0.05 and saline group ratio; #p＜0.05, ##p＜0.01 are given relatively separately with the Type B natriuretic peptide.
Fig. 7: intrathecal injection Type B natriuretic peptide is to the inhibitory action of the inductive chronic inflammatory pain of CFA.
Fig. 7 A: at rat left hind paw vola injection CFA two days later, intrathecal injection Type B natriuretic peptide (2 microgram) increased greatly the radiant heat test the pawl incubation period of contracting ( *P＜0.001, ANOVA compares 7 routine Mus/groups with the pump pickle group).
After Fig. 7 B:CFA vola injection 2 days and 4 days, intrathecal injection Type B natriuretic peptide has increased thermostimulation and has contracted pawl incubation period.Data are picked up from Type B natriuretic peptide 120 fen hour of injection.(P＜0.001 ,=0.039 and 0.211, correspond to the Type B natriuretic peptide and injected the back the 2nd, 4 and 7 day at CFA, compare 7 routine Mus/groups with the pump pickle group).Intrathecal injection KT5823 has then hindered self thermalgesia recovery.Data are picked up from KT5823 120 fen hour of injection.(P=0.056,＜0.001 and＜0.001, correspond to KT5823 and injected the back the 2nd, 4 and 7 day at CFA, compare 7 routine Mus/groups with the pump pickle group). *P＜0.05 draw * *Compare with the saline group p＜0.001.
The specific embodiment
Long-term and deep the discovering of inventor's process: the agonist (for example Type B natriuretic peptide) of natriuretic peptide receptor (for example natriuretic peptide receptor A) can be through activating the receptor-mediated natriuretic peptide receptor/PKG/BK of natriuretic peptide CaApproach reduces pain, thereby can be used for analgesia (especially inflammatory pain or chronic pain).Thus, the inventor provides a kind of New Policy and novel drugs type of alleviating and/or treating pain.The inventor has accomplished the present invention on this basis.
Is example with the inventor about the research of Type B natriuretic peptide and natriuretic peptide receptor A; In this research; The inventor utilize gene chip and immunoassay find Type B natriuretic peptide and natriuretic peptide receptor A at dorsal root ganglion (dorsal root ganglion; DRG) express in, and (complete Freund ' s adjuvant, CFA) vola injects to express in the inductive rat inflammatory pain model increases at complete Freund's adjuvant.Immunohistochemistry result shows that the Type B natriuretic peptide is expressed in trk C (tyrosine kinase receptor A; TrkA) in the positive dorsal root ganglion small neuron; Comprising most of calcitonin-gene-related peptides (calcitoningene-related peptide, CGRP) the positive small neuron of positive small neuron and IB4 (isolectin B4).Natriuretic peptide receptor A then exists only in the positive small neuron of CGRP.
Exogenous Application of B type natriuretic peptide can reduce dorsal root ganglion small neuron irritability, and its effect mainly is through open large conductance calcium activated potassium channel (large-conductance Ca 2+-activated K +Channel, zBK Ca) and realize.In addition, the intrathecal injection Type B natriuresis Toplink pain behavior and the CFA that suppress significantly to cause because of the sole injection of formalin induces the rat thermal hyperalgesia.If give protein kinase (cGMP-dependent protein kinase, PKG) inhibitor KT5823 or potassium channel BK that cGMP relies in the sheath in advance CaBlocker iberiotoxin has then blocked the analgesic activity of Type B natriuretic peptide mediation.Therefore, the Type B natriuretic peptide has oppositely been regulated pain sensation transmission through its presynaptic receptor natriuretic peptide receptor A.This is illustrated in the nocuity sensory nerve path, and activating Type B natriuretic peptide/natriuretic peptide receptor A is treatment or lenitive potential strategy.
Inventor's research has also further shown vivo activation natriuretic peptide receptor/cGMP/PKG/BK CaApproach can be alleviated the pain of object, so uses the suitable receptor-mediated signal path of agonist selective activation nocuity afferent neuron natriuretic peptide further conduct treatment or lenitive potential strategy.
As used herein, have comprised " containing ", " having " or " comprising " " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " belong to the subordinate concept of " containing ", " having " or " comprising " with " by ... formation ".
As used herein, " isolating " is meant that material separates (if crude, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
Pain and classification thereof
As used herein, term " pain " is meant that body to a kind of offending reaction that damaged tissue or potential damage produce, is a kind of complex physical mental activity, also is one of modal symptom clinically.Pain can be divided into body source property (somatogenic) pain and psychogenic (psychogenic) pain.Body source property pain can be divided susceptibility pain (like nociceptive pain, nociceptive pain) and neuropathic pain (Neuropathic Pai) again.Pain can be divided into according to its generation and lasting time and process: acute pain and chronic pain, chronic pain also comprise chronic susceptibility pain or the rational pain of chronic neuropathic.
Neuropathic pain is because the damage or the pathological change of maincenter or peripheral nervous system cause; Include but not limited to the relevant neuralgia of lumbago and backache (lower back pain), neuralgia (neuralgia), fibromyalgia (Fibromyalgia), diabetes (diabetic neuropathic pain, DNP), neuralgia (pain associated with multiple sclerosis), PHN (PHN) and HIV related (neurogenic) neuralgia (HIVNP) etc. that multiple sclerosis is relevant.
Nociceptor property pain is because the intravital nociceptor of people receives machinery, heat, chemical stimulation or damage and causes; Body nociceptor property pain and internal organs nociceptor property pain be can be divided into, cancer pain (cancer-related pain), arthritis pain (arthritic pain), postoperative pain (post-operative pain) and HIV related (nociceptor property) neuralgia (HIVNP) etc. included but not limited to.Wherein, neuralgia (neuralgia) includes but not limited to trigeminal neuralgia (Trigeminal neuralgia), sciatica (sciatic neuralgia) etc.
Pain can be divided into according to its generation and lasting time and process: acute pain and chronic pain.
The acute pain complicacy that to be a group take place rapidly with obvious stimulation factors such as damage or various emergency cases and offending impression, consciousness and affectional experience, with autonomous, psychology and reaction behavior, it comprises pain after operation back, the wound, burn and various Medicine and Surgery emergency case; For example: heart infarction; Acute pancreatitis, biliary colic, renal colic; Acute appendicitis, pathologisch Bruch, acute pain etc. appears in the acute ileus patient.The Therapeutic Method of acute pain comprises: controlled analgesia (PCA) is carried out in the ward, with local anaesthetics and/or opiates medicine in epidural space infusion etc.Normal model such as formaldehyde method, hot plate method or the method for adopting causes the acute pain of animal in mechanism or animal experiment, and gives medicine to be measured immediately, to observe the mitigation of medicine to be measured to acute pain.
Chronic pain is meant that pain continues the long period (as surpassing several weeks～1 month); The progress that surpasses general acute disease; The reasonable time that perhaps surpasses wound healing, or relevant with the chronic pathology process that causes constant pain, and perhaps pain blanking time through several months or several years recurs.The normal complete Freund's adjuvant etc. of adopting is induced the chronic pain that causes animal in mechanism or animal experiment, and after inducing a few days and even several weeks, gives medicine to be measured, to observe the mitigation of medicine to be measured to chronic pain.
The course of disease of chronic pain is long, the cause of disease is complicated; The different structure of maincenter and peripheral nervous system and function unusual; The unify variation of emotion of autonomic nervous system; And social adaptation, life are or/and the reduction of ability to work; The characteristics of spirit, the many-sided mental maladjustment of family have determined the significance of rehabilitation means for the chronic pain treatment.
Chronic pain is not alleviated, can be psychologically, cause harmful effect to the patient on the physiology; The pain that can not alleviate will postpone patient's recovery, prolong the date of being in hospital, and increase burden for patient and family members, strengthen the medical care insurance expense, make it forfeiture work, family, dignity, cause depression, anxiety, suicide, and the maimed colony of psychology is enlarged.Moreover, also increased social factor leading to social instability.Comparatively speaking, it is generally acknowledged that now acute pain is a symptom of disease, and chronic pain is one type of disease.
Chronic pain includes but not limited to: 1. soft tissue, joint and bone pain: pain behind deformity property pain, skeletal muscle pain, lumbago, myofasical pain syndrome, headache, the burn after various osteoarthritis, the wound.2. deep tissue and Encelialgia: cardiovascular pain, ophthalmalgia, actinal surface portion pain, chronic gynecological pain, sexual anhedonia, genitourinary system chronic pain.3. neural and nerve root injury property pain: phantom limb pain after amputation, peripheral nerve property pain, sympathetic reflex dystrophy and sympathetic nerve lasting pain, trigeminal neuralgia and atypical facial bitterly, nerve root injury and arachnoiditis, PHN.4. central pain: the blood vessel injury of brain, spinal cord, like hemorrhage, infraction, vascular malformation; Multiple sclerosis; Traumatic spinal cord injury, brain injury; Syringomyelia and syringobulbia; Tumor; Parkinson disease.5. child's pain: growing pain.6. old people's pain: various cervical spondylosiss, prolapse of lumbar intervertebral disc, lumbar spondylolisthesis etc. are pain caused.7. cancerous pain.
At present, the main method of chronic pain treatment has: the 1. drug therapy of chronic pain, as: use opioid drug, non-opium medicine, adrenocortical hormone, spasmolytic and vitamin medicaments, psychosis, local anesthetic, the neural medicine etc. that destroys; 2. the nerve of chronic pain and local retardance method, as: epidural block therapy, subarachnoid block therapy; The trigeminal nerve block therapy, temporomandibular joint block treatment, suprascapular nerve block treatment; The other nerve root block treatment of vertebra, sciatic nerve blockade therapy, stellate ganglion block therapy etc.; 3. the neurosurgical treatment method of chronic pain; Like neural posterior rhizotomy, rhizotomy in the percutaneous thermocoagulation of low level cranial nerve amputation and cranial nerve, spinal dura mater; The epidural rhizotomy; Root rhizoid amputation behind the selectivity, the percutaneous spinal nerve root is damaged art, percutaneous central fascicle neurotomy; 4. CT guiding hypencephalon stereotactic surgery is damaged art, as: nervi trigeminus is damaged art, and the percutaneous intervertebral disc is cut the suction art, the neural art of damaging of intractable cancer pain in late period, and hypophysis cerebri destroys analgesia, the operation drug rehabilitation; 5. irritation therapy, as: TCNS, spinal cord stimulation trial, brain stimulation method alleviating pain; 6. the psychotherapy of chronic pain like placebo and placebo effect, loosens and biofeedback, a cognitive behavior method, hypnosis pain relieving, behavior therapy; 7. the naturopathy of chronic pain, as: therapy alleviating pains such as ultrasound wave, ultrashort wave, microwave, laser, surperficial heating and cooling, polarized infrared light; Massage and the massage alleviating pain; Training and limitation of the movement alleviating pain; 8. the processing of cancerous pain; 9. additive method, as: intervention property radio frequency heat is coagulated art, A-botulinum toxin injection for curing chronic pain, the radiotherapy of carcinomas pain, chemotherapy, incretotherapy, acupuncture and moxibustion treatment, small needle knife therapy, patient-controlled analgesic therapy (PCA) etc.
Though the whole bag of tricks of known pain therapy and control in this area still presses in this area and develops the active drug that effectively is directed against pain (especially chronic pain).
Peripheral nervous system cGMP raises the activator of material and natriuretic peptide receptor
As used herein, term " Peripheral nervous system cGMP raises material" be meant that the cGMP level that can make after using in the peripheral nervous system rises, thus reach the active substance of alleviating or treating the effect of pain.Among the present invention Peripheral nervous system cGMP raises material and is preferablyThe natriuretic peptide receptor activators.
As used herein, term " natriuretic peptide receptor activators " is meant and can further activates natriuretic peptide receptor/cGMP/PKG/BK thus through activating the natriuretic peptide receptor with the natriuretic peptide receptors bind or through alternate manner CaApproach, thus reach the active substance of alleviating or treating the effect of pain.Comprise in the said term: the activator of the activator of natriuretic peptide receptor A, natriuretic peptide receptor B, the activator of natriuretic peptide receptor C or their combination.The natriuretic peptide receptor stimulating agent that can be used among this paper includes but not limited to: natural natriuretic peptide, they have analog (or derivant) and/or its combination of exciting natriuretic peptide receptor active.
Term " natural natriuretic peptide " is meant naturally occurring natriuretic peptide or their precursor, includes but not limited to: atrial natriuretic peptide, Type B natriuretic peptide, C type natriuretic peptide (for example shown in SEQ ID NO:3), D type natriuretic peptide (for example shown in SEQ ID NO:4), urodilatin, preceding natriuretic peptide or former-preceding natriuretic peptide.Preferred said natural natriuretic peptide is from mammals such as people, dog, Mus, horse, cattle or sheep.
Term " analog with exciting natriuretic peptide receptor active " is meant: derived from the natural agonist of natriuretic peptide receptor, and can with the natriuretic peptide receptors bind, one-step activation natriuretic peptide receptor/cGMP/PKG/BK goes forward side by side CaApproach; Thereby reach the active substance of alleviating or treating the effect of pain; For example, the aminoacid sequence of natural natriuretic peptide is deleted, is blocked, is inserted, is replaced, is replaced, is prolonged and the analog of other albumen fusions etc., and this analog still keeps natural natriuretic peptide BA.Preferably, this analog comprises a natural or artificial natriuretic peptide receptors bind functional domain, and reserve part or all combine the activity of natriuretic peptide.
The analog that is applicable to many natriuretic peptides of the present invention comprises the natriuretic peptide annulus, for example by ANP, BNP, CNP or the DNP circulus that is connected to form of the disulfide bond between two cysteine in the sequence (being connected by disulfide bond with Cys26 and the circulus that forms like people BNP Cys10) separately.Though this ring of some analog also can comprise some replacements that are different from the amino acid residue of natural natriuretic peptide, deletion and/or insert; As long as these amino acid whose replacements, deletion and/or insertion can't weaken this analog BA identical with natural natriuretic peptide, then this analog is equal to natural natriuretic peptide.Such as; Deliver at Schoenfelda etc. article " Mutations in B-type natriuretic peptide mediating receptor-A selectivity; " FEBS Letters 414 (1997) 263-267 have disclosed the variant of some BNP, and these variation physical abilitys combine natriuretic peptide receptor A more specifically.(U.S.Patent?6,525,022?and?U.S.Patent?6,028,055)。In patent WO2006076471A2, disclose this natriuretic peptide ring and contain one or more conservatives replacements, but still kept the natriuretic activity of this ring.Thereby disclosed among the patent WO2006076471A2 and deleted this ring that partial amino-acid has been shortened and still kept associated biomolecule and learn activity.The similar polypeptide that comprises the circulus between people BNP Cys10 and the Cys26 after the 14th the lysine that has disclosed BNP among the WO2006076471A2 is replaced by glycine or arginine has still kept the BA of natural natriuretic peptide.Disclosed the BA that the similar polypeptide of circulus that has changed or deleted the serine of BNP the 19th to 22 has still kept natriuretic peptide among the WO2006076471A2.The analog of the natriuretic peptide of the BA that has natriuretic peptide that discloses among the patent WO2006076471A2 is equally applicable to the present invention.
In addition, be applicable to that natriuretic peptide ring of the present invention and analog thereof can comprise an aminoterminal and/or c-terminus afterbody, like people BNP the 1st to 9 amino acids and the 27th to 32 amino acids, the linear order that the C-terminal of DNP is made up of 15 aminoacid.Afterbody is the aminoacid or the polypeptide of strand that can't change the BA of natriuretic peptide ring.The aminoacid sequence of the afterbody in the analog can and/or insert by other aminoacid replacement, deletion, the relevant BA of short of change native sequences, and this analog is equal to natural natriuretic peptide.These afterbodys can have 1 to a plurality of amino acid whose truncates based on native sequences.Can be selected from the amino acid fragment of following people BNP: 8-9 such as the aminoterminal afterbody, 7-9,6-9,8-9,7-9,6-9,5-9,4-9,3-9,2-9, and 1-9; One or more lysine residues in the amino acid fragment of anyone above-mentioned BNP can be replaced by glycine residue or arginine residues.Similarly, the c-terminus afterbody can be selected from the amino acid fragment of following people BNP: 27-28,27-29,27-30,27-31 and 27-32; One or more lysine residues in the amino acid fragment of anyone above-mentioned BNP can be replaced by glycine residue or arginine residues.
Be applicable to that natriuretic peptide analog of the present invention has also comprised the fusion by annulus and other albumen or the polypeptide of natural natriuretic peptide; Fusion rotein still keeps the BA of exciting natriuretic peptide receptor, the fusion rotein of preferably partly being made up of the non-annularity of a kind of annulus of natural natriuretic peptide and another kind of natural natriuretic peptide.(Journal of the American College of Cardiology Vol.52 such as Ondrej Lisy for example; No.1; 2008 JACC Vol.52; No.1,2008 (1), put down in writing in 2008:60-8) by 22 aminoacid of CNP and 15 fusion rotein that linear C-terminal aminoacid sequence is formed of DNP.
Except that above-mentioned analog, the analog that many technology are formerly disclosed also is applicable to the present invention.For example United States Patent (USP) 5,114,923, and having disclosed the polypeptide that has the natriuresis excretory function is R '-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-R2, and wherein R ' is selected from (H); GIy-; Ser-Gly-; Gly-Ser-Gly-; Gln-Gly-Ser-Gly-; Val-Gln-Gly-Ser-Gly-; Met-Val-Gln-Gly-Ser-Gly-; Lys-Met-Val-Gln-Gly-Ser-Gly; Pro-Lys-Met-Val-Gln-Gly-Ser-Gly; Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-; And R3-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-, wherein R 3Be proteic 102 aminoacid sequences of the people of position 1-99 or c-terminus part, and R 2Be (OH), NH 2, NHR ' or modification wherein partly are independently low alkyl (C 1-4) or R2 be Lys; Lys-Val; Lys-Val-Leu; Lys-Val-Leu-Arg; Lys-Val-Leu-Arg-Arg; Lys-Val-Leu-Arg-Arg-His; Perhaps amino-compound (NH 2, NHR 1Or NR ' modification part ").
U.S. patent 4; 904; The natriuretic peptide analog that discloses in 763 also is applicable to the present invention, for example, and X-Cys PheGIy Arg Lys Met Asp Arg He Ser Ser Ser Ser GIy Leu GIy Cys Lys VaI Leu Arg ArgHis-OH; Wherein X is H, H-Gly-Ser-Gly-or H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly.
U.S. patent 4,904, and 763 have disclosed the natriuretic peptide analog also is applicable to the present invention; For example, X-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gl y-Leu-Gly-Cys--Y, (wherein 2 cysteine are connected by disulfide bond); Wherein X is that H or H-Asp-Ser.-Gly-and Y are Asn-Val-Leu-Arg-Arg-Tyr-OH ,-Asn-Val-Leu-Arg-Arg-OH;-Asn-Val-Leu-Arg-Tyr-OH;-Asn-Val-Leu-Arg-OH ,-Asn-Val-Leu-OH or Asn-Ser-Phe-Arg-Tyr-OH, or the derivant of its salt.Another group is applicable to that analog of the present invention is disclosed in WO8912069.
Another group is applicable to that natriuretic peptide analog of the present invention is disclosed in U.S. patent application .20030109430; Its structural formula is R '-Cys-Phe-Gly-Arg-Arg/Lys-Leu/Met-Asp-Arg-Ile-Lys-Met-Gly/ Ser-Ser-Leu/Ser-Ser-Gly-Leu-Gly-Cys-R2, and wherein R1 is selected from down: (H); GIy-; Ser-Gly-; Asp/Lys/Gly-Ser-Gly-; Arg/His/Gln-Asp/Lys/Gly-Ser-Gly-; Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly-; Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly-; Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly; Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly-; Ser-Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly-; Or one 10 to 109 aminoacid sequence of the upstream portion of pig, dog or people BNP native sequences; R2 is (OH), NH2, and or NR ' R " wherein R ' and R " (is to be Asn/Lys perhaps 1-4C) for independently hanging down alkyl here; Asn/Lys-Val; Asn/Lys-Val-Leu; Asn/Lys-Val-Leu-Arg; Asn/Lys-Val-Leu-Arg-Arg/Lys; Asn/Lys-Val-Leu-Arg-Arg/Lys-Tyr/His; Perhaps amino-compound (NH2 or NR ' R "); if wherein structural formula is that R '-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R2 and R1 are Asp-Ser-Gly-, R2 can not be Asn-Val-Leu-Arg-Arg-Tyr.
Also have one group of analog to be disclosed in U.S. patent application 20020086843; Its structural formula be X-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gl y-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-OH [wherein 2 cysteine residues bridgings] wherein X be H; H-Gly-Ser-Gly-, or H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-.
The derivant that can be used among the present invention also can include, but is not limited to: the active fragment of natural natriuretic peptide, like their annulus; With the link coupled natural natriuretic peptide of PEG, its derivant or their active fragment; With the link coupled natural natriuretic peptide of carrier protein (for example serum albumin or its fragment); The variable shearing product of natural natriuretic peptide, or the like.Preferred said derivant is selected from; AlbuBNP, CD-NP, ASBNP, ASBNP.1, hBNP1-32, hBNP-054, Nesiritide or their combination.
Term " AlbuBNP " is meant people BNP (1-32) sequence of 1～5 copy and the fusion rotein that ripe sero-abluminous N-terminal forms; (Pharmaceutical Research such as Wei Wang for example; Vol.21; No.11, the BNP-HAS and BNP (the 2x)/HAS that are put down in writing in 2004:2105-2111).
Term " CD-NP " is meant C type natriuretic peptide or its active fragment or the fusion rotein that forms with D type natriuretic peptide or its active fragment; Preferred said active fragment is the annulus of said natriuretic peptide; (Journal of the American College of Cardiology Vol.52 such as Ondrej Lisy for example; No.1,2008 JACC Vol.52, No.1; 2008 (1), put down in writing in 2008:60-8) by 22 aminoacid of CNP and 15 fusion rotein that linear C-terminal aminoacid sequence is formed of DNP.
Term " variable shearing product " is meant through altered rna shears functional variable spliced body of natriuretic peptide that (alternative RNA splicing) back produces, and for example Shuchong Pana etc. (Proc Natl Acad Sci U S A.2009,7; 106 (27): 11282-7.Epub on June 18th, 2009) ASBNP, the ASBNP1 that are put down in writing in.
Term " with the link coupled natural natriuretic peptide of PEG, its derivant or their active fragment " is meant the bioactive molecule that is formed by natural natriuretic peptide or derivatives thereof and the coupling of Polyethylene Glycol (PEG) molecule; This coupling can occur in N end, C end or the chain of natural natriuretic peptide or derivatives thereof (like the 3rd K of BNP1-32), for example (Trends Cardiovasc Med.2007 January such as Cataliotti A; 17 (1): the CONJ-hBNP-021 that is put down in writing 10-4), (Circulation, 2008 year October 21 such as Cataliotti A; 118 (17): 1729-36.Epub on October 6th, 2008) hBNP-054 that is put down in writing in, and the natriuretic peptide of the PEGization of being put down in writing among the WO/2006/076471.
Natriuretic peptide derivant of the present invention for example can be:
Figure BSA00000236191600151
Figure BSA00000236191600152
Figure BSA00000236191600153
wherein modify and occur on Lys3, Lys14 or the Lys27 of BNP, preferably occur in Lys3.
Below be available aminoacid sequence among example explanation the present invention with the agonist of natriuretic peptide receptor A, should understand other agonist that also can confirm and obtain other natriuretic peptide receptor similarly.
For example, term " activator of natriuretic peptide receptor A " is meant and can further activates natriuretic peptide receptor A/cGMP/PKG/BK thus through expression, activity and/or the secretion that combines or pass through alternate manner to improve natriuretic peptide receptor A with natriuretic peptide receptor A CaApproach, thus reach the active substance of alleviating or treating the effect of pain.Said activator includes but not limited to: Type B natriuretic peptide or atrial natriuretic peptide.
(B-type natriuretic peptide BNP) has known it is by the myocardial cell secretion to the Type B natriuretic peptide, and (natriuretic peptide receptor-A NPR-A), reduces myocardial fibrosis through activating natriuretic peptide receptor A.Yet, in the state of the art Type B natriuretic peptide and natriuretic peptide receptor A it be not immediately clear in the expression and the effect of somatosensory system.Disclosed the new function of Type B natriuretic peptide in this research, promptly pain has been regulated.
(atrial natriuretic peptide ANP) is peptide hormone synthetic by atrial muscle cell and that discharge to atrial natriuretic peptide, and it mainly acts on is to make the vascular smooth muscle diastole and promote kidney row sodium, draining.
As natriuretic peptide receptor A agonist, Type B natriuretic peptide and atrial natriuretic peptide all cause intracellular cGMP to increase through receptor, and further activation PKG/BKCa plays analgesic activity thereby reduce dorsal root ganglion nervelet irritability.
In the present invention; Used Type B natriuretic peptide or atrial natriuretic peptide can be naturally occurring (such as its can be separated or purification from mammal), also can be (such as producing reorganization Type B natriuretic peptide or atrial natriuretic peptide) of artificial preparation according to the genetic engineering recombinant technique of routine.Preferably, the present invention can adopt the Type B natriuretic peptide or the atrial natriuretic peptide of reorganization.
Any suitable Type B natriuretic peptide or atrial natriuretic peptide all can be used for the present invention.Described Type B natriuretic peptide or atrial natriuretic peptide comprise Type B natriuretic peptide, atrial natriuretic peptide or its bioactive fragment (or be called " active fragment " or " polypeptide of the present invention ") of total length.For example; The aminoacid sequence of said Type B natriuretic peptide can with SEQID NO:1 (SPKMVQGSGC FGRKMDRISS SSGLGCKVLR RH; People's Type B natriuretic peptide sequence) sequence shown in is substantially the same, also can be proteinic conservative variant protein matter or its active fragment of SEQ ID NO:1.For example; The aminoacid sequence of said atrial natriuretic peptide can with SEQ ID NO:2 (SLRRSSCFGG RMDRIGAQSG LGCNSFRY; People's atrial natriuretic peptide sequence) sequence shown in is substantially the same, also can be proteinic conservative variant protein matter or its active fragment of SEQ ID NO:2.
The Type B natriuretic peptide that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form or the aminoacid sequence of atrial natriuretic peptide are also included among the present invention; For example that aminoacid sequence among SEQ ID NO:1 or the SEQ ID NO:2 is individual through 1-20; Preferred 1-10; More preferably the replacement of 1-5 amino acid residue, disappearance or interpolation form, and have activate natriuretic peptide receptor A active, by (a) deutero-protein.Type B natriuretic peptide, atrial natriuretic peptide or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and said sequence through the aminoacid replacement does not influence its activity or kept the activity of its part.Suitably replacement aminoacid is technology well known in the art, and said technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, can not change biological activity basically at the inessential area change single amino acids of a peptide species.See Watson etc., " Molecular Biology of The Gene " (" gene molecule biology ", the 4th edition, 1987, Benjamin/Ku Ming publishing company, The Benjamin/Cummings Pub.Co.P224).
The bioactive fragment of any Type B natriuretic peptide or atrial natriuretic peptide can be applied among the present invention.Here, the implication of bioactive fragment is meant that as a peptide species it still can keep all or part of function of natriuretic peptide of total length.Generally, described bioactive fragment keeps 50% the total length Type B natriuretic peptide or the activity of atrial natriuretic peptide at least.Under preferred condition, said active fragment can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length Type B natriuretic peptide or atrial natriuretic peptide.
Should understand through conservative substituted sequence mentioned above and also can be used for the present invention; Preferred reactive derivative refers to compare with the original acid sequence, has 5 at the most, preferably at the most 3; More preferably at the most 2,1 aminoacid is replaced by similar performance or close aminoacid and is formed polypeptide best.These conservative variation polypeptide preferably carry out the aminoacid replacement according to Table I and produce.
Table I
Initial residue Representational replacement The preferred replacement
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The present invention also can adopt through Type B natriuretic peptide or the atrial natriuretic peptide of modifying or improveing, such as, can adopt Type B natriuretic peptide or the atrial natriuretic peptide modifying or improve in order to promote its half-life, effectiveness, metabolism and/or proteinic effectiveness.Said can be the conjugate of a kind of Type B natriuretic peptide or atrial natriuretic peptide through Type B natriuretic peptide or the atrial natriuretic peptide modified or improve, or it can comprise substituted or artificial aminoacid.Said can be to have less common ground with naturally occurring Type B natriuretic peptide or atrial natriuretic peptide through Type B natriuretic peptide or the atrial natriuretic peptide modified or improve, but also can suppress the pain sensation, and can not bring other harmful effect or toxicity.That is to say that any bioactive version that does not influence Type B natriuretic peptide or atrial natriuretic peptide all can be used among the present invention.
Both can adopt isolating Type B natriuretic peptide or atrial natriuretic peptide among the present invention, and also can adopt its coded sequence, comprise the carrier of its coded sequence or genetically engineered host cell.Those of ordinary skills can prepare and use the Type B natriuretic peptide or the atrial natriuretic peptide of above-mentioned form according to this area general knowledge and routine techniques based on concrete needs.
For example, method well known to those skilled in the art can be used to make up the sequence that contains Type B natriuretic peptide encoding gene and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described DNA sequence can effectively be connected on the suitable promoter in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome binding site and the transcription terminator that translation initiation is used.
Comprise the carrier of above-mentioned suitable gene order and suitable promoter or control sequence, can be used to transform appropriate host cell, so that it can marking protein.Persons skilled in the art all know how to select appropriate carriers, promoter, enhancer and host cell.
Recombiant protein in the above methods can or be secreted into the extracellular at cell inner expression.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) is technological with other various LCs and the combination of these methods.
Pharmaceutical composition
The invention provides a kind of pharmaceutical composition, it contains effective dose (like 0.000001-50wt%; Preferable 0.00001-20wt%; Better, said natriuretic peptide receptor activators 0.0001-10wt%) (for example Type B natriuretic peptide), and pharmaceutically acceptable carrier.Pharmaceutical composition of the present invention can be used for through activating the natriuretic peptide receptor/cGMP/PKG/BK of natriuretic peptide receptor (like natriuretic peptide receptor A) mediation CaApproach, treatment, alleviation or the improvement pain relevant with this approach.In addition, also can unite use with other therapeutic agent or adjuvant simultaneously.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and does not have excessive bad side reaction (like toxicity, stimulation and allergy), promptly has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the carrier of therapeutic agent administration, comprises various excipient and diluent.
Pharmaceutical composition of the present invention contains the natriuretic peptide receptor A activator and the pharmaceutically acceptable carrier of safe and effective amount.This type carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with normal saline or contain glucose and the aqueous solution of other adjuvant prepares through conventional method.Described pharmaceutical composition should be made under aseptic condition.The dosage of active component is the treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.
The effective dose of activator can change with the order of severity of the pattern of administration and disease to be treated etc. in the pharmaceutical composition of the present invention.The selection of preferred effective dose can be confirmed (for example through clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of activator, metabolism, half-life etc.; The patient the order of severity, patient's body weight, patient's immune state, the approach of administration etc. of the disease that will treat.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
PH in the present composition or carrier etc. can be as indicated above, and can be selected with general knowledge as required by those of ordinary skills.
The administering mode of pharmaceutical composition of the present invention has no particular limits, can be whole body or partial.Preferably; Pharmaceutical composition of the present invention can give object through the mode of spinal cord intrathecal injection; Usually, when the dosage of activator of the present invention with about 0.0004-0.008mg/kg the weight of animals (preferable 0.001-0.002mg/kg the weight of animals) gives, can obtain gratifying effect.Also can adopt the means of gene therapy to carry out administration, such as can be directly with activator through delivering medicine to the experimenter such as methods such as injections; Perhaps, can be delivered on the target spot through the ceneme (such as expression vector or virus etc.) that certain approach will carry protein or polypeptide Class Activation agent (for example Type B natriuretic peptide), and make it the expression activity Activator protein.
With the Type B natriuretic peptide is example; Can directly give mammal (such as the people) with described Type B natriuretic peptide albumen; Perhaps; Can the gene of coding Type B natriuretic peptide be cloned in the appropriate carriers (like conventional protokaryon or carrier for expression of eukaryon or viral vector such as herpesvirus vector or adenovirus vector) through the method for routine, described carrier is imported in the cell that can express said albumen or polypeptide, make described cellular expression Type B natriuretic peptide.Can realize the expression of Type B natriuretic peptide through an amount of said cell being incorporated into the suitable position of body of mammals.
The method of the potential material of pain is alleviated or is treated in screening
The potential material that the present invention also provides a kind of screening to can be used for alleviating or treating pain (is specially through activating natriuretic peptide receptor (for example natriuretic peptide receptor A)/PKG/BK CaThe potential material of pain is alleviated or treated to approach) method, described method comprises: (1) contacts candidate substances with the system of expressing natriuretic peptide receptor (for example natriuretic peptide receptor A); (2) detect candidate substances and whether can activate natriuretic peptide receptor (for example natriuretic peptide receptor A)/PKG/BK CaApproach; If said candidate substances can activate natriuretic peptide receptor (for example natriuretic peptide receptor A)/PKG/BK CaApproach shows that then this candidate substances is the potential material of alleviating or treating pain.In an embodiment of the invention, the system of said expression natriuretic peptide receptor A is the dorsal root ganglion small neuron, preferred L4 and/or L5 dorsal root ganglion neurons.
Natriuretic peptide receptor/PKG/BK CaThe detection of pathway activation degree can be carried out through one or more indexs in the test system, and these indexs include but not limited to: natriuretic peptide receptor expression level, electrical activity (for example action potential level and frequency), the inside and outside calcium ion level of cell, intracellular cGMP level, known natriuretic peptide receptor/PKG/BK CaThe inhibitor of approach (natriuretic peptide receptor/PKG/BK for example CaThe inhibitor KT5823 of approach, iberiotoxin) to the reverse or the inhibitory action of this candidate substances.
In optimal way of the present invention, when screening, whether can activate natriuretic peptide receptor/PKG/BK in order to be easier to observe candidate substances CaApproach also can be provided with matched group, and described matched group can be the dorsal root ganglion small neuron that does not add said candidate substances.As optimal way of the present invention, described method also comprises: the potential material to obtaining carries out further cell experiment and/or animal experiment, with further selection and definite for alleviating and the real useful material of treatment pain.
Major advantage of the present invention
Advantage of the present invention mainly comprises:
(1) the present invention has disclosed the natriuretic peptide receptor/PKG/BK of natriuretic peptide receptor (natriuretic peptide receptor A) mediation first CaRelation between approach and the pain, thus a kind of new method of treating, alleviate or improve pain (especially chronic pain) through the activation of regulating this approach is provided;
(2) the invention provides a kind of brand-new sensory information and transmit regulatory mechanism, having proved the Type B natriuretic peptide first has regulating action to the sensory information transmittance process of pain, thereby the new way of an inhibition of pain is provided; And
(3) screening for the treatment pain medication provides good approach.
Based on description of the invention, other advantage of the present invention also is conspicuous for those of ordinary skills.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York, publishing house of cold spring harbor laboratory, New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Material and method
Animal and medicine: all experiments all obtain Chinese Academy of Sciences's Neuroscience Research institute's laboratory animal and public utility is used committee's approval.Male SD rat is provided by Shanghai SLAC Experimental Animal Center, and raises in little time of 12:12/dark cycle room, and room temperature is controlled at 22-26 ℃, animal ad lib and drinking-water.
The Type B natriuretic peptide available from Phoenix Pharmaceuticals (Burlingame, CA).KT5823, iberiotoxin (IBTX), glibenclamide (glibenclamide, Glib), glutamic acid and complete Freund's adjuvant (CFA) available from Sigma Chemical Company (StLouis, MO).Type B natriuretic peptide, iberiotoxin and glutamic acid are dissolved in (concentration is respectively 100 every milliliter of microgram, 100 micromoles and 1 mM) in the distilled water.KT5823 and glibenclamide are dissolved in and are made into 1000 times of mother solutions (concentration is respectively 1 mM and 10 mMs) among the DMSO.
Gene chip is analyzed: 200 microlitre CFA are injected in palm vola behind bull rat (100) bilateral.After injection 0.5,2,4,7 and 14 day (20 every group) draw materials respectively.Take out waist (L) 4 and L5 dorsal root ganglion.(Agilent Technologies Inc., Santa Clara CA) separate dorsal root ganglion from these and extract total RNA, as the synthetic cDNA of template to utilize the total RNA extraction reagent box of Agilent.In vitro transcription has adopted the linear amplification kit of the low ribonucleic acid input of Agilent fluorescence; Synthesizing fluorescently labeled ribonucleic acid is used to gene chip hybridization.Hybridization solution has used Agilent in situ hybridization test kit.The Agilent22k rat oligo microarray system that uses tests.System carries out data analysis with the Agilent microarray scanning.Handle through feature extraction software, data are calculated as log2 ratio, and promptly log2 ratio is considered to raise greater than the gene of 1 (greater than 2 times increase), is considered to downward modulation less than-1 (greater than 2 times minimizing).Utilize Cluster3.0 and TreeView software that the data that filter out are carried out cluster analysis and visual mapping then.
Immunoblotting assay: 200 microlitre CFA are injected in palm vola behind the bull rat bilateral.These mouse were raised 1,2,4 and 7 day respectively, took out L4 and L5 dorsal root ganglion then.RIPA buffer (phosphate buffer, 1%NP-40,0.5% NaTDC, 0.1% sodium lauryl sulphate, Phenylmethanesulfonyl fluoride, aprotinin, vanadic acid sodium) has been used in proteinic extraction.The BCA test kit of Pierce company has been used in concentration determination.The anti-Type B natriuretic peptide antibody of Santa Cruz company, the anti-natriuretic peptide receptor A antibody of Abcam company and the anti-GAPDH antibody of Abcam company are used to immunoblotting assay.Data-measuring is independently tested and the different animal tissue sample from least 3 times.Type B natriuretic peptide or natriuretic peptide receptor A and GAPDH signal intensity rate draw through the ScionImage computed in software.
Immunohistochemistry: bull rat integral body adds 0.02% picric acid with 4% paraformaldehyde and fixes.L4 and L5 dorsal root ganglion and corresponding sections spinal cord are used to frozen section, carry out the anti-natriuretic peptide receptor of the rabbit A (1: 500) of Abcam company and the two marks of goat-anti calcitonin-gene-related peptide (1: 500) of Biogenesis company then and handle.The fluorescent labeling IB4 of every milliliter of 10 microgram of the anti-calcitonin-gene-related peptide of rabbit (1: 500) or Vector Labs company of the anti-TrkA of rabbit (1: 500) or DiaSorin company that does not have the anti-natriuretic peptide receptor of rabbit A (1: 500) or the Santa Cruz company of goat-anti Type B natriuretic peptide (1: 500) that fixed L4 and L5 dorsal root ganglion frozen section be used to two mark Santa Cruz company and Abcam company.
The immunologic opsonin of Type B natriuretic peptide is confirmed that by corresponding immunogen peptide pre-absorption test tissue staining was carried out in the Type B natriuretic peptide antibody and the neutralization of 200 nanogram Type B natriuretic peptide room temperatures that are about to 200 nanograms in two hours again.With original antibody relatively, by in the immunogen peptide with after antibody tissue staining subsequently show as weak reaction.
Electrophysiology: acute isolation 5 age in week rat L4 be used to test with the L5 dorsal root ganglion neurons.Cell is laid down on the slide, and in 2～12 hours, carries out the patch-clamp record.All records are all from dorsal root ganglion small neuron (diameter＜30 micron).The extracellular fluid composition is (mM) as follows: sodium chloride 150, potassium chloride 5, calcium chloride 2.5, magnesium chloride 1, HEPES 10 and glucose 10, pH value 7.4.Liquid is formed (mM) as follows in the electrode: potassium chloride 140, magnesium chloride 1, calcium chloride 2.5, EGTA 5, HEPES 10, ATP 2 and GTP 0.3, pH value 7.3.Full cell record need meet the following conditions: inhale broken preceding greater than 1 G sealing-in resistance; Inhale broken back greater than resting membrane electric potential in 100M input resistance and the cell less than-55mV.In case cell is inhaled brokenly, the voltage clamp pattern changes the current clamp pattern into.Data are to be amplified and the Pulse software collection by the EPC-9 patch clamp amplifier of German HEKA company.
Performance testing: it all is to carry out with the mode of double blinding that medicine injection and animal behavior are observed.In formalin experiment, it is subcutaneous with No. 30 syringe needles the formalin of 50 microlitres 5% to be injected into the bull rat left hind back side.Pain behavior after the observation injection of formalin.Be injected into the every lower limb number of times of lifting at a distance from 5 minutes of claw and be used to quantize pain behavior degree, observation lasts till back 60 minutes of injection.Formalin-induced is lifted the lower limb behavior and can be divided into biphase.Initial acute stage (0～10 minute, the 1st phase) is long chronic reaction (10～60 minutes, the 2nd phase) then.In the inductive chronic inflammatory of CFA was tested bitterly, it is subcutaneous that 50 microlitre CFA are flow into the left back palm.Animal is placed on the glass plate of 2 millimeters thick, uses model to stimulate appearance (Inst. of Biomedicine Engineering Chinese Academy of Medicine) to measure the burning pain incubation period of rat as the radiant heat of BME-410C.Burning pain is identified as from the beginning thermostimulation to the required time of rats withdraw foot reflex generation incubation period.
Statistical analysis: data appear with mean standard error mode.Immunoblotting assay, immunofluorescence and electric physiological data have been carried out Student ' s t check.The performance testing result carries out two-way variance analysis (two-way ANOVA) earlier, carries out Student ' s t then and checks the difference between the comparison different dosing.When p＜0.05, difference has been considered to statistical significance.
Embodiment 1.B type natriuretic peptide and natriuretic peptide receptor A express in dorsal root ganglion and after inflammation, express to be increased
Normal rat and rat CFA injection 0.5,2,4,7 and 14 day the L4 in back and total RNA of L5 dorsal root ganglion are used to the analysis of Agilent 22k rat oligo gene chip.Gene expression data has carried out filtration and statistical analysis.
To be detected 20; In 500 gene probes; Amount to 1257 gene probes (accounting for total probe 6.1%) and be confirmed as apparent the expression and change, promptly in five time points to be detected, have at least once the gene expression of this probe in detecting that rise or the downward modulation more than 2 times taken place with respect to normal structure.Further unigene analyzes 1123 genes having confirmed these probe representatives and (expresses increase for 509; 614 expression decreased); Comprising 18 neuropeptides, (G-protein-coupled receptors is GPCRs) with 1 natriuretic peptide receptor (Burbach for 6 g protein coupled receptors; 2010, Eur J Pharmacol 626:27-48) (Figure 1A).
These neuropeptides and receptor are carried out cluster analysis (Eisen etc., 1998, Proc Natl Acad Sci USA95:14863-14868) (Figure 1A).It should be noted that; Around after the inflammation of tissue; Have 4 pairs of neuropeptides and the expression of receptor in dorsal root ganglion thereof to come to light co-variationization taken place, these 4 neuropeptides be tachykinin (TAC3), neurotensin (NTS), somatostatin (SST) and Type B natriuretic peptide (BNP) (Figure 1A).All these neuropeptides and receptor expression thereof all raise.What is interesting is that the mRNA level of tachykinin and neurotensin is instantaneous rise 2 days, it then is from 0.5 to 14 day long-term raise (Figure 1A) that somatostatin and Type B natriuretic peptide raise.
Though tachykinin, neurotensin and somatostatin and receptor thereof the expression of dorsal root ganglion and the existing before this report of function (Zhang etc., 1995; Malcangio and Bowery, 1999; Thermos etc., 2006), but never expression and the function of B property natriuretic peptides and their receptors in dorsal root ganglion studied in this area.For this reason, inventor's primary study the expression and the function in the pain sensation is regulated of Type B natriuretic peptides and their receptors.
The protein level that the inventor has further investigated Type B natriuretic peptide and natriuretic peptide receptor A around after the inflammation expression in dorsal root ganglion whether change.The result is consistent with chip detection, and immunoblotting shows that the Type B natriuretic peptide brings out in the dorsal root ganglion of periphery inflammation by day increasing at CFA, and (2.61 ± 0.35 times to normal structure, p=0.002) (Figure 1B) to reach peak value at the 4th day.The receptor A albumen of natriuretic peptide increases inflammation the 1st day also that (1.44 ± 0.19 times to normal structure, and p=0.104), and (1.58 ± 0.20 times to normal structure, p=0.045) to reach peak value at the 2nd day.The expression of receptor level of natriuretic peptide is returned datum-plane (Figure 1B) in inflammation after the 7th day.
The above results shows that Type B natriuretic peptide and natriuretic peptide receptor A are expressed in rat dorsal root ganglion, and under the situation that peripheral organization is inflamed, expresses increase.
Embodiment 2.B type natriuretic peptide and natriuretic peptide receptor A are expressed in the dorsal root ganglion small neuron and after inflammation, express to be increased
The inventor utilizes immunohistochemistry further to study Type B natriuretic peptide and the natriuretic peptide receptor A cell distribution at rat dorsal root ganglion.
The result shows: Type B natriuretic peptide and natriuretic peptide receptor A's mainly is to be distributed in dorsal root ganglion small neuron (cross-sectional area＜900 square microns) (Fig. 2 A and 2B).It is positive that 50% dorsal root ganglion neurons is the immunity of Type B natriuretic peptide; 26% dorsal root ganglion neurons is natriuretic peptide receptor A positive (Fig. 2 A).Injection CFA is the 2nd day in the vola; Dorsal root ganglion small neuron Type B natriuretic peptide and natriuretic peptide receptor A immune fluorescence intensity all increase (Fig. 2 A-C) to some extent; And the immuning positive neuron number is not seen change (Fig. 2 A; In the time of 2 days, 50% and 27% dorsal root ganglion neurons is the Type B natriuretic peptide respectively and natriuretic peptide receptor A is positive in inflammation).Therefore, the Type B natriuretic peptide and the natriuretic peptide receptor A up-regulated that cause of inflammation mainly occurs in the dorsal root ganglion small neuron.
Through adopting two fluorescence staining labellings, the inventor has further studied the male dorsal root ganglion neurons cell divide of Type B natriuretic peptides and their receptors.The dorsal root ganglion small neuron is divided into peptidergic neuron (CGRP is positive) and IB4 positive neuron usually.Nerve growth factor receptor TrkA then is expressed in (Silos-Santiago etc., 1995, J Neurosci15:5929-5942 in most of Toplink small neurons and some IB4 positive neurons; Fang etc., 2006, J Neurosci 26:7281-7292).
Ironically, the inventor finds that nearly all natriuretic peptide receptor A positive neuron also expresses the Type B natriuretic peptide simultaneously; And have only half the Type B natriuretic peptide immuning positive neuron also to be natriuretic peptide receptor A positive (Fig. 2 A and 2D) simultaneously.Find that further the immunoreation of Type B natriuretic peptide is present in the positive dorsal root ganglion small neuron of TrkA (Fig. 3 A).Simultaneously the inventor finds that also the immunoreation of Type B natriuretic peptide is present in 95% peptidergic neuron and 89% IB4 positive neuron (Fig. 3 B).And the immunoreation of natriuretic peptide receptor A mainly is at Toplink small neuron (Fig. 4 A) and first and second layers of centripetal fiber of cornu dorsale medullae spinalis (Fig. 4 B).In addition, be distributed widely in cardiovascular organization consistent (Nakayama, 2005, Endocr J52:11-21) with the natriuretic peptide receptor A of previous report, natriuretic peptide receptor A immunoreation also comes to light along vascularity (Fig. 4 B) at spinal cord.
Therefore, the Type B natriuretic peptide is expressed in all dorsal root ganglion Toplink small neurons and the positive small neuron of IB4, and natriuretic peptide receptor A almost is expressed in all Toplink small neurons.
There is Type B natriuretic peptide reduction neuronal excitability under the glutamic acid condition in embodiment 3.
The inventor has further investigated the electrical activity whether the Type B natriuretic peptide can regulate the dorsal root ganglion small neuron.
Research in the past shows, activatory natriuretic peptide receptor A induces the synthetic of cGMP, and the PKG that activates downstream makes potassium channel BK CaBy phosphorylation, increase the open ability of these passages, thereby cause VSMC and mesangial cell hyperpolarization (Williams etc., 1988, Proc Natl Acad Sci USA85:9360-9364; Sansom and Stockand, 1996, Clin Exp Pharmacol Physiol 23:76-82; Tanaka etc., 1998, Naunyn Schmiedebergs Arch Pharmacol 357:705-708).Importantly, use potassium channel BK CaChannel opener NS1619 can be reduced dorsal root ganglion small neuron discharge frequency (Zhang etc., 2003, Neuroscience 122:1003-1011 by report; Chen etc., 2009, J Neurochem110:352-362).
Therefore, infer that the Type B natriuretic peptide may produce inhibitory action to the irritability of dorsal root ganglion small neuron through the potassium channel of regulating downstream.
Yet unexpectedly; The inventor finds to stimulate (1 second persistent period by slope current; High peak currents 100-500 skin ampere) ratio (after the administration/administration before) that brings out the action potential frequency of dorsal root ganglion small neuron is giving 100 nanograms/milliliter Type B natriuretic peptides (1.13 ± 0.09; 14 examples) after ten minutes with add extracellular fluid group (1.13 ± 0.06,22 example) (Fig. 5 A and 5B) more do not take place obviously to change.
The inventor notices potassium channel BK CaThe open endocellular liberation calcium ion that depends on raises and level of membrane potential (Fukao etc., 1999; Schubert and Nelson, 2001; Zhou etc., 2001).Therefore, might not be opened by potassium channel owing to make the endocellular liberation calcium ion concentration increase in the superincumbent experiment.When stimulating when coming to harm, nociceptor discharges excitatory transmitter such as glutamic acid (D ' Mello and Dickenson, 2008); Thereby glutamic acid active ions receptor increases intracellular calcium level (Crawford etc., 2000).Therefore we infer that further there is scalable potassium channel BK under the glutamic acid situation of extracellular in the Type B natriuretic peptide CaOpen.
In order to verify this supposition, we mix at the dorsal root ganglion small neuron and add Type B natriuretic peptide (100 nanograms/milliliter) and glutamic acid (10 micromole).We find after dosing 10 minutes subsequently, and the frequency of the action potential that brings out has reduced by 92.7 ± 7.2% (21 examples respond in the 26 routine neurons), and the cell resting potential does not receive obvious influence (Fig. 5 A and 5B).On the contrary, singly add glutamic acid and just increased action potential frequency (1.49 ± 0.14,10 example) (Fig. 5 A and 5B) slightly.
The inventor adopts PKG inhibitor KT5823 and potassium channel BK CaBlocker iberiotoxin further assesses the regulating action of the neuron activity of natriuretic peptide receptor A mediation.Exist under the glutamic acid condition, can be in the inhibitory action of the inductive dorsal root ganglion small neuron of Type B natriuretic peptide by 1 micromole KT5823 pretreatment (1.81 ± 0.17,11 example) or 100 nanomole iberiotoxin (1.14 ± 0.15,10 example) blocking-up (Fig. 5 B).
Can know that by The above results the Type B natriuretic peptide can pass through PKG/ potassium channel BK under the condition that extracellular glutamic acid exists CaReduce dorsal root ganglion small neuron irritability.
Use the Type B natriuretic peptide in embodiment 4. sheaths and alleviate inflammatory pain
In the present embodiment, the inventor has investigated the Type B natriuretic peptide and could suppress the inflammatory pain behavior.The subcutaneous injection formalin in adult rat vola, a left side can produce to lift the pain behavior that lower limb is a characteristic (Fig. 6 A).The behavior shows as biphase (1～10 minute is first phase, and 10 minutes～60 minutes is the 2nd phase) (Araiza-Saldana etc., 2005, Eur J Pharmacol 527:60-70).With matched group relatively; Intrathecal injection Type B natriuretic peptide (giving in 10 minutes prior to the vola injection of formalin) is to two inhibitory action (p＜0.001 that phase all produces dose dependent of formalin experiment; Corresponding to 100 nanograms, 500 nanograms and 2 microgram Type B natriuretic peptides, the ANOVA check; First phase: p=0.099,0.026 and 0.034, the 2 phase: p=0.007,＜0.001 and=0.004; Corresponding to 100 nanograms, 500 nanograms and 2 microgram Type B natriuretic peptides, student t check, 7 routine Mus/groups) (Fig. 6 A).
The inductive anti-harmful effect of intrathecal injection Type B natriuretic peptide (2 microgram) can be by intrathecal injection PKG inhibitor KT5823 (500 nanogram) or potassium channel BK CaBlocker iberiotoxin (500 nanogram) reverses (giving in 10 minutes prior to the injection of Type B natriuretic peptide), but not by ATP sensitive potassium channel (K ATP) blocker glibenclamide (50 microgram) reverse (the 1st phase: p=0.038,0.048 and 0.800, the 2 phase: p=0.003,0.037 and 0.907; Combined Treatment 2 microgram Type B natriuretic peptides and KT5823 or iberiotoxin, 7 routine Mus/groups; Compare with 2 microgram Type B natriuretic peptide individual processing) (Fig. 6 B).Yet KT5823, iberiotoxin or glibenclamide individual processing can not change the behavior of formalin-induced nocuity (the 1st phase: p=0.549,0.568 and 0.873, the 2 phase: p=0.743,0.906 and 0.764; Corresponding to KT5823 or iberiotoxin or glibenclamide, 7 routine Mus/groups).
Therefore, the Type B natriuretic peptide is through inducing activation PKG/ potassium channel BK CaApproach suppresses formalin and causes the reaction of rat nocuity.
Can inductive chronic inflammatory disease pain model at vola injection CFA, it shows as in the heat radiation test thermal hyperalgesia phenomenon (Ren and Dubner, 1999, ILAR J40:111-118) that shortens to characteristic with the pawl that contracts incubation period.Thermal hyperalgesia performance in the 1st day is the strongest behind vola injection CFA, after 7 to 14 days, recovers gradually then (Fig. 7 A).Periphery inflammation the 2nd day, intrathecal injection Type B natriuretic peptide (2 microgram) alleviated hyperpathia significantly, and promptly showing as the radiant heat test pawl that contracts increases (P＜0.001, ANOVA incubation period; P=0.011,0.023 and 0.032 is corresponding to Type B natriuretic peptide injection 90,120 and 180 minutes, 7 routine rat/groups) (Fig. 7 A).
Periphery inflammation the 2nd day, Application of B type natriuretic peptide also promoted the recovery (P=0.039, corresponding to the injection of Type B natriuretic peptide after 120 minutes, 7 routine Mus/groups) (Fig. 7 B) of thermal hyperalgesia.On the other hand; Intrathecal injection PKG inhibitor KT5823 (500 nanogram) has then delayed self thermal hyperalgesia recovering process, and (KT5823 gives 120 minutes; P=0.056,＜0.001 and＜0.001, corresponded respectively to inflammation the 2nd, 4,7 days, 7 routine Mus/groups) (Fig. 7 B).Yet intrathecal injection Type B natriuretic peptide (2 microgram) does not influence normal rat burning pain incubation period (P=0.310, ANOVA, 7 routine Mus/groups).This result has supported the Type B natriuretic peptide will bring into play inhibitory action needs inflammation-induced glutamic acid to discharge.
Therefore, in acute and chronic inflammation property pain model, in dorsal root ganglion neurons, activating Type B natriuretic peptide signal path can ease the pain.
Discuss
This research has disclosed that Type B natriuretic peptides and their receptors natriuretic peptide receptor A is expressed in the dorsal root ganglion small neuron and its expression raises behind peripheral tissue inflammation.In addition, exist under the glutamic acid condition of extracellular, the Type B natriuretic peptide is through activating PKG/ potassium channel BK CaApproach reduces the irritability of dorsal root ganglion small neuron; But the pain reaction that intrathecal injection Type B natriuretic peptide dose dependent ground reduction formalin and CFA bring out.Therefore, pass through to activate presynaptic natriuretic peptide receptor A/PKG/BK by the excretory Type B natriuretic peptide of nocuity centripetal fiber CaApproach has oppositely been regulated the excitatory synapse transmission.
These discoveries have shown that the Type B natriuretic peptide/natriuretic peptide receptor a-signal transduction path that activates the nocuity centripetal fiber can be a potential treatment pain strategy.
The inhibiting presynaptic mechanism of Type B natriuretic peptide
Type B natriuretic peptides and their receptors natriuretic peptide receptor A is expressed in dorsal root ganglion Toplink small neuron, shows that Type B natriuretic peptide that the activity of dorsal root ganglion Toplink small neuron may discharge through self is from main regulation.Though the autonomous regulatory mechanism of local nerve circuit feedback formula of autoreceptor mediation is well-known; Classical like dopamine and serotonin (Feuerstein; 2008; Handb Exp Pharmacol:289-338); But the polypeptide system this from the main regulation evidence be still limited (
Figure BSA00000236191600281
etc., 2000, Neuropharmacology39:1337-1356).
The inventor finds that Type B natriuretic peptide and natriuretic peptide receptor A are expressed in dorsal root ganglion Toplink small neuron and influence this neuron discharge frequency, shows that natriuretic peptide receptor A can be used as the autoreceptor of some nocuity nervus centripetalis.According to existing result of study, the Type B natriuretic peptide is present in cornu dorsale medullae spinalis centripetal fiber rather than spinal interneuron (Kawata etc., 1989, Neuroscience 33:401-410; Saper etc., 1989, Neurosci Lett 96:29-34).Therefore, the Type B natriuretic peptide possibly discharged by the elementary sensation afferent nerve fiber of cornu dorsale medullae spinalis.Importantly, natriuretic peptide receptor A also is detected and is distributed in the cornu dorsale medullae spinalis centripetal fiber that contains CGRP.Therefore, import a kind of autonomous regulatory mechanism that synapse is transmitted at dorsal root ganglion Toplink small neuron co expression Type B natriuretic peptide and natriuretic peptide receptor A.Yet the Type B natriuretic peptide is also expressed at some IB4 positive small neurons, has reason therefore to think that the Type B natriuretic peptide that is discharged by the positive small neuron of IB4 can affact on the natriuretic peptide receptor A of peptidergic neuron expression with the paracrine mode.
The release that presynaptic is suppressed excitatory neurotransmitter glutamic acid has been considered to a kind of important mechanisms that the pain sensation is regulated.Some neuropeptide such as opioid peptide, somatostatin and neuropeptide tyrosine etc. have been found that suppressing glutamic acid through its teleneuron presynaptic receptor discharges (Tallent, 2008, Results Probl Cell Differ44:177-200).In addition, the result who activates presynaptic ion-type glutamate receptor also plays inhibitory action (Kerchner etc., 2001, J Neurosci 21:59-66) to the release of glutamic acid.
In this research, electrophysiological recording shows that Application of B type natriuretic peptide adds glutamic acid through open potassium channel BK CaCan show the irritability that reduces the dorsal root ganglion small neuron.Therefore corresponding with it the possibility of result has reduced the neurotransmitter release that calcium ion relies on.The inhibitory action of Type B natriuretic peptide needs glutamic acid to participate in this fact and shows that Type B natriuretic peptide function Characteristics is movable dependent.
With the corresponding to result of this viewpoint be that the Type B natriuretic peptide influences the behavior of rat inflammatory pain and to the not influence of normal threshold of pain value; This can be interpreted as in the inflammation-induced body (D ' Mello and Dickenson due to the release glutamate; 2008, Br J Anaesth 101:8-16).
What is interesting is that the inductive presynaptic of most of neuropeptides suppresses by its g protein coupled receptor mediation (Tallent, 2008), and Type B natriuretic peptide receptor is a guanylate cyclase, and does not combine G albumen.
In sum, the Type B natriuretic peptide can suppress presynaptic neurotransmitter release through the signal path that activates natriuretic peptide receptor A mediation.This possibly represent a kind of new mechanism of regulating the nocuity transmission.
Activate Type B natriuretic peptide/natriuretic peptide receptor a-signal path and suppress inflammatory pain
As everyone knows, activating natriuretic peptide A receives cognition to make intracellular cGMP/PKG signal strengthen (Silberbach and Roberts, 2001, Cell Signal 13:221-231).Electric physiology and performance testing result in this research show: the inhibitory action of inductive neuronal excitability of Type B natriuretic peptide and the behavior of nocuity pain can be blocked through PKG inhibitor KT5823, explains that the cGMP/PKG signal is the downstream passages of Type B natriuretic peptide.
Corresponding is that the immunoreation of cGMP also is detected (Vles etc., 2000, Brain Res 857:219-234 at the centripetal fiber of cornu dorsale medullae spinalis with it; De Vente etc., 2006, J Chem Neuroanat 31:263-274).Intracellular cGMP concentration is synthesized with degradation rate is common and regulates.The synthetic of the cGMP of natriuretic peptide receptor A mediation can strengthen (deVente etc., 2006) because of existing phosphodiesterase inhibitor to suppress degraded.Therefore, intrathecal injection phosphodiesterase inhibitor sldenafil (sildenafil) reduces the cGMP degraded and has suppressed the inductive inflammation pain of formalin (Torres-Lopez etc., 2002, Proc West Pharmacol Soc 45:141-143; Araiza-Saldana etc., 2005, Eur JPharmacol 527:60-70).Therefore, the nocuity transmission possibly be affected because of the level change of intracellular cGMP.
In addition, cGMP Signal Regulation neurotransmission can have different effects because of neural kind is different.(nitric oxide is NO) through the transmission of the synthetic promotion pain of the responsive inductive cGMP of guanylate cyclase of nitric oxide signal for a large amount of nitric oxide that fact proved; Can reduce inflammation and neuropathic pain (Schmidtko etc., 2009, Trends Neurosci 32:339-346) significantly and suppress the synthetic of cGMP.Yet the responsive guanylate cyclase of nitric oxide has expressed at neurokinin 1 receptor positive spinal cord projection neuron and two layers of inhibitory interneuron of cornu dorsale medullae spinalis; But do not express (Schmidtko etc. in dorsal root ganglion neurons; 2008a, JNeurosci 28:8568-8576).Therefore; Type B natriuretic peptide and nitric oxide play reverse effect to pain transmission; This possibly be because they activated due to synthesizing of the neuronic cGMP of variety classes; Be that Type B natriuretic peptide signal promotes the synthetic of elementary nervus centripetalis cGMP, and the nitric oxide signal promote dorsal horn neurons cGMP synthetic.
With natriuretic peptide receptor expression mode class seemingly, (PKG type I cGKI) also is present in dorsal root ganglion small neuron and cornu dorsale medullae spinalis centripetal fiber (Qian etc., 1996, J Neurosci 16:3130-3138) to I type PKG.Yet cGKI sudden change Mus is but brought out hyperpathia to formalin-induced nocuity pain and zymosan and reacts and alleviate rather than increase the weight of (Tegeder etc., 2004, Proc Natl Acad Sci USA 101:3253-3257) to some extent.
The spinal cord that the fact that the inventor notices is the cGKI knock-out mice is obviously little, and the neuron number also significantly reduces (Tegeder etc., 2004).Therefore can not get rid of disappearance (Schmidt etc., 2002), and cause having alleviated nocuity reaction (Tegeder etc., 2004) because of cGKI sudden change Mus owing to axon guidance in spinal structure ateliosis and the embryo development procedure.
On the other hand, CRP2 (cysteine-rich protein 2) is as the effector molecule in cGKI downstream, and also coming to light to be expressed in contains in cGKI and the CGRP cornu dorsale medullae spinalis centripetal fiber.CRP2 sudden change Mus then shows as and in the inflammatory pain model, increases the pain reaction, that is to say that CRP2 helps inhibition of pain transmission (Schmidtko etc., 2008b, J Neurosci 28:1320-1330).This viewpoint that meets us is PKG signal path negative regulation pain sensation transmission in the nocuity centripetal fiber.Therefore, activate Type B natriuretic peptide/natriuretic peptide receptor a-signal path and can become a kind of important mechanisms that suppresses inflammatory pain.
Type B natriuretic peptide/the effect of natriuretic peptide receptor A aspect pain therapy
Known that the Type B natriuretic peptide/natriuretic peptide receptor A that activates cardiac ventricles can reduce cardiovascular pathological changes and ventricle fibrosis (Woodard and Rosado, 2007, J Cell Mol Med 11:1263-1271; Potter etc., 2009, Handb Exp Pharmacol:341-366).That measures serum Type B natriuretic peptide concentration increases degree at the clinical diagnosis index that has been used as heart failure, and synthetic Type B natriuretic peptide analog has been used to treat decompensated heart failure (Woodard and Rosado, 2007 in some countries; Potter etc., 2009; Rodseth, 2009, Anaesthesia 64:165-178).
Although this research shows the inflammation of peripheral organization caused dorsal root ganglion small neuron up-regulated expression Type B natriuretic peptide and natriuretic peptide receptor A; Additional application Type B natriuretic peptide still can reduce inflammatory pain, this means that additional application Type B natriuretic peptide can further activate the natriuretic peptide receptor A/PKG/BK of nocuity sensory neuron CaApproach.Therefore, except being applied in heart failure and other disease, the Type B natriuretic peptide still can be used as analgesia molecule or chemical compound with other natriuretic peptide receptor A agonist in pain therapy.
Atrial natriuretic peptide/the latent effect of natriuretic peptide receptor A aspect pain therapy
(atrial natriuretic peptide ANP) is peptide hormone synthetic by atrial muscle cell and that discharge to atrial natriuretic peptide, and it mainly acts on is to make the vascular smooth muscle diastole and promote kidney row sodium, draining.
Natriuretic peptide receptor A agonist also has atrial natriuretic peptide simultaneously except the Type B natriuretic peptide.Similar with the Type B natriuretic peptide, atrial natriuretic peptide also can make intracellular cGMP increase through natriuretic peptide receptor A approach, and further activation PKG/BKCa plays analgesic activity thereby reduce dorsal root ganglion nervelet irritability.
Therefore, atrial natriuretic peptide also has the effect of pain therapy aspect, can be as analgesia molecule or chemical compound in pain therapy.
Conclusion
This research shows that Type B natriuretic peptides and their receptors natriuretic peptide receptor A is expressed in the dorsal root ganglion small neuron and behind peripheral tissue inflammation, expresses to be increased.In addition, exist under the condition of glutamic acid, the Type B natriuretic peptide is through open potassium channel BK CaThe irritability that suppresses the dorsal root ganglion small neuron; Vivo activation natriuretic peptide receptor A/cGMP/PKG/BK CaThe pain that approach can reduce inflammation and cause.
Further research shows that also atrial natriuretic peptide also can make intracellular cGMP increase through natriuretic peptide receptor A approach, further activation PKG/BK CaThereby, reduce dorsal root ganglion nervelet irritability and play analgesic activity.
These results show that the signal path that uses suitable agonist selective activation nocuity afferent neuron natriuretic peptide receptor A to mediate can be used as the potential strategy of pain therapy.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Figure ISA00000236191800011

Claims (22)

1. peripheral nervous cGMP raises material is used for alleviating or treating the pharmaceutical composition of pain in preparation purposes.
2. purposes as claimed in claim 1 is characterized in that, it is the natriuretic peptide receptor stimulating agent that said peripheral nervous cGMP raises material.
3. purposes as claimed in claim 1 is characterized in that, said natriuretic peptide receptor stimulating agent is selected from: the agonist of the agonist of natriuretic peptide receptor A, natriuretic peptide receptor B and/or the agonist of natriuretic peptide receptor C.
4. purposes as claimed in claim 1 is characterized in that, said natriuretic peptide receptor stimulating agent is selected from: natural natriuretic peptide, they have analog or its combination of exciting natriuretic peptide receptor active.
5. purposes as claimed in claim 4 is characterized in that, said natural natriuretic peptide is selected from: atrial natriuretic peptide, Type B natriuretic peptide, C type natriuretic peptide, D type natriuretic peptide, urodilatin, preceding natriuretic peptide, former-preceding natriuretic peptide or their combination.
6. purposes as claimed in claim 4 is characterized in that, said analog is selected from: with the link coupled natural natriuretic peptide of PEG; With serum albumin or the link coupled natural natriuretic peptide of its fragment; The variable shearing product of natural natriuretic peptide; The fusion rotein that forms by the annulus of natural natriuretic peptide and aminoterminal and/or c-terminus afterbody, the fusion rotein of preferably partly forming by the non-annularity of a kind of annulus of natural natriuretic peptide and another kind of natural natriuretic peptide.
7. purposes as claimed in claim 4 is characterized in that said derivant is selected from; AlbuBNP, CD-NP, ASBNP, ASBNP.1, hBNP1-32, hBNP-054, Nesiritide or their combination.
8. purposes as claimed in claim 2 is characterized in that, said natriuretic peptide receptor stimulating agent is the agonist of natriuretic peptide receptor A.
9. purposes as claimed in claim 8 is characterized in that, the agonist of said natriuretic peptide receptor A is selected from: Type B natriuretic peptide or atrial natriuretic peptide.
10. purposes as claimed in claim 9; It is characterized in that the form of said Type B natriuretic peptide or atrial natriuretic peptide is selected from down group: isolating Type B natriuretic peptide or atrial natriuretic peptide, its coded sequence or comprise the carrier of its coded sequence or genetically engineered host cell.
11. purposes as claimed in claim 9 is characterized in that, said Type B natriuretic peptide or atrial natriuretic peptide are selected from:
(a) protein of aminoacid sequence shown in protein of aminoacid sequence shown in the SEQ ID NO:1 or the SEQ ID NO:2;
(b) the proteinic conservative variant protein matter or its active fragment that are limited in (a); Or
(c) with aminoacid sequence in (a) through 1-20, preferred 1-10, more preferably the replacement of 1-5 amino acid residue, disappearance or interpolation form, and have activation natriuretic peptide receptor A active, by (a) deutero-protein.
12. purposes as claimed in claim 1 is characterized in that, said pain is self source property pain or inflammatory pain.
13. purposes as claimed in claim 12 is characterized in that, said self source property pain is selected from: neuropathic pain or nociceptive pain.
14. purposes as claimed in claim 12 is characterized in that, said inflammatory pain is selected from: arthritis pain, diabetes complicated peripheral nerve inflammatory pain, peripheral tissues's inflammatory pain or internal organs inflammatory pain.
15. purposes as claimed in claim 1 is characterized in that, said pain is chronic pain or acute pain.
16. purposes as claimed in claim 15 is characterized in that, said chronic pain is the rational pain of chronic susceptibility pain or chronic neuropathic.
17. purposes as claimed in claim 15; It is characterized in that said chronic pain is selected from down group: soft tissue, joint and bone pain: pain behind deformity property pain, skeletal muscle pain, lumbago, myofasical pain syndrome, headache, the burn after various osteoarthritis, the wound; Deep tissue and Encelialgia: cardiovascular pain, ophthalmalgia, actinal surface portion pain, chronic gynecological pain, sexual anhedonia, genitourinary system chronic pain; Nerve and nerve root injury property pain: phantom limb pain after amputation, peripheral nerve property pain, sympathetic reflex dystrophy and sympathetic nerve lasting pain, trigeminal neuralgia and atypical facial pain, nerve root injury and arachnoiditis, PHN; Central pain: the blood vessel injury of brain, spinal cord, like hemorrhage, infraction, vascular malformation; Multiple sclerosis; Traumatic spinal cord injury, brain injury; Syringomyelia and syringobulbia; Tumor; Parkinson disease; Child's pain: growing pain; Old people's pain: cervical spondylosis, prolapse of lumbar intervertebral disc, lumbar spondylolisthesis are pain caused; Or cancerous pain.
18. a pharmaceutical composition, said pharmaceutical composition comprises:
(a) the natriuretic peptide receptor stimulating agent of alleviation or treatment pain effective dose;
(b) pharmaceutically acceptable carrier.
19. purposes as claimed in claim 18 is characterized in that, said natriuretic peptide receptor stimulating agent is selected from: the agonist of the agonist of natriuretic peptide receptor A, natriuretic peptide receptor B and/or the agonist of natriuretic peptide receptor C
20. pharmaceutical composition as claimed in claim 19 is characterized in that, the agonist of said natriuretic peptide receptor A is selected from: Type B natriuretic peptide or atrial natriuretic peptide.
21. a screening can be used for alleviating or treating the method for the potential material of pain, it is characterized in that, described method comprises:
(1) candidate substances is contacted with the dorsal root ganglion small neuron;
(2) detect candidate substances and whether can activate natriuretic peptide receptor/PKG/BK CaApproach;
If said candidate substances can activate natriuretic peptide receptor/PKG/BK CaApproach shows that then this candidate substances is the potential material of alleviating or treating pain.
22. method as claimed in claim 21 is characterized in that, said natriuretic peptide receptor/PKG/BK CaApproach is natriuretic peptide receptor A/PKG/BK CaApproach.
CN2010102577814A 2010-08-11 2010-08-11 Purpose of agonist of natriuretic peptide receptor A in pain management Pending CN102370985A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2010102577814A CN102370985A (en) 2010-08-11 2010-08-11 Purpose of agonist of natriuretic peptide receptor A in pain management

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2010102577814A CN102370985A (en) 2010-08-11 2010-08-11 Purpose of agonist of natriuretic peptide receptor A in pain management

Publications (1)

Publication Number Publication Date
CN102370985A true CN102370985A (en) 2012-03-14

Family

ID=45790566

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2010102577814A Pending CN102370985A (en) 2010-08-11 2010-08-11 Purpose of agonist of natriuretic peptide receptor A in pain management

Country Status (1)

Country Link
CN (1) CN102370985A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105999229A (en) * 2016-05-07 2016-10-12 上海大学 New application of brain natriuretic peptide BNP
CN111978390A (en) * 2020-08-31 2020-11-24 上海景泽生物技术有限公司 Polyethylene glycol modified rhBNP and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033807A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Therapeutic uses of b-type natriuretic peptide and human growth hormone 1-43
WO2009033824A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a combination of beta-casokinin and cnp-22 as a therapeutic agent
WO2009033733A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of apeptide as a therapeutic agent

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033807A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Therapeutic uses of b-type natriuretic peptide and human growth hormone 1-43
WO2009033824A1 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a combination of beta-casokinin and cnp-22 as a therapeutic agent
WO2009033733A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of apeptide as a therapeutic agent

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘 哲: "《B型钠尿肽最新研究进展》", 《心血管病学进展》 *
张旭: "《病理性疼痛的分子机制》", 《生命科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105999229A (en) * 2016-05-07 2016-10-12 上海大学 New application of brain natriuretic peptide BNP
CN111978390A (en) * 2020-08-31 2020-11-24 上海景泽生物技术有限公司 Polyethylene glycol modified rhBNP and application thereof
CN111978390B (en) * 2020-08-31 2022-10-14 上海景泽生物技术有限公司 Polyethylene glycol modified rhBNP and application thereof

Similar Documents

Publication Publication Date Title
AU2015276203B2 (en) Exendin-4 derivatives as selective glucagon receptor agonists
AU2003237347B2 (en) Treatment of cell proliferative disorders with chlorotoxin
EP0898967B1 (en) Use of transduced myogenic cells for relieving pain and for treating behavioral and perceptive abnormalities
Saperas et al. Central action of recombinant interleukin-1 to inhibit acid secretion in rats
CN103732618A (en) Novel oxyntomodulin derivatives and pharmaceutical composition for treating obesity comprising same
JP2007181465A (en) Polynucleotide encoding macrophage inflammatory protein variants
US20040138204A1 (en) Compositions and methods for pain reduction
US20210008177A1 (en) Elapidae neurotoxin enhances opioid analgesic effect and inhibits opioid induced hyperalgesia and tolerance
RU2595857C2 (en) Agent for prophylactic and/or therapeutic treatment of peripheral neuropathic pain caused by anticancer agent
Fraley et al. Alarin 6-25Cys antagonizes alarin-specific effects on food intake and luteinizing hormone secretion
CN103097404B (en) There is analgesic activity and suppress the new peptides of ASIC passage
CN102370985A (en) Purpose of agonist of natriuretic peptide receptor A in pain management
JP4581115B2 (en) Treatment or prevention agent for pruritic skin diseases
US20070073040A1 (en) Anti-obesity polypeptides
KR102127218B1 (en) Use of compounds in the manufacture of drugs for the treatment of brain glioma
CN113501862B (en) Polypeptide and application thereof in preparation of immunoregulation medicament
WO2008120263A2 (en) Prokineticins receptors antagonists, derivatives and uses thereof
CN114075296A (en) Multifunctional variant protein and fusion protein thereof
KR20130021315A (en) Composition for preventing or treating erectile dysfunction comprising dkk2 protein or gene therefor and use thereof
JPH02207099A (en) Peptide relating to pthrp, preparation and use thereof
TW202019457A (en) New myokines and uses thereof
CN111574590B (en) Polypeptide with anti-tumor function and application thereof
US20040082516A1 (en) Compositions for promoting nerve regeneration
US20210171588A1 (en) Methods For Treatment of Neuropathic Pain
KR20180027924A (en) Pharmaceutical composition for treating muscle atrophy comprising glucagon like-peptide-1, GLP-1 derived peptide, or GLP-1 degradation inhibitor

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20120314