CN1871028A - Method for treating pain by administering a nerve growth factor antagonist and an NSAID and composition containing - Google Patents

Abstract

The present invention features methods for treating or preventing pain comprising administering an amount of a nerve growth factor antagonist (such as an anti-NGF antibody) and an amount of an NSAID such that together they provide effective pain relief. The invention also features compositions comprising a nerve growth factor antagonist and an NSAID and kits containing the same.

Description

By using nerve growth factor antagonist and NSAID and the method that contains their combination treatment pain

The cross reference of related application

The sequence number that the application requires on February 19th, 2003 to submit to is that 60/448,823 U.S. Provisional Application and the sequence number submitted on February 19th, 2003 are 60/448,853 U.S. Provisional Application No., and their content is incorporated herein by reference by complete.

Invention field

The application relates to by patient's combined administration nerve growth factor antagonist and NSAID being prevented and treat the method and composition of pain.

Background of invention

Current recommendation comprises that some treatments of using nonsteroid anti-inflammatory drugs (NSAID) are used to ease the pain.The verified NSAID of using shows the feature of pain relief.Yet known have shortcoming with the NSAIDs treatment, comprises deleterious side effect, as GI irritation and kidney and liver toxicity.In addition, even NSAID can not enough alleviate pain in some pain status with the approval therapeutic dose of their maximums.

Nerve growth factor (NGF) is the neurotrophin of at first identifying, it is fully characterized in the growth of periphery and axoneuron and the role in the survival.Proved that NGF is existence crucial during sympathetic and embryo's sensory neuron of periphery and basal forebrain cholinergic neuron are grown and keeps the factor (Smeyne etc., Nature 368:246-249 (1994); Crowley etc., Cell 76:1001-1011 (1994)).NGF raises the expression (Lindsay etc. of neuropeptide in the sensory neuron, Nature 337:362-364 (1989)) and it is active in two kinds of different membrane-bound receptors---TrkA tyrosine kinase receptor and p75 are receptor-mediated, so receptor structurally with other members relevant (Chao etc., Science 232:518-521 (1986)) of Tumor Necrosis Factor Receptors family.

Except the effect in nervous system, show that day by day the outer process of NGF and nervous system is relevant.For example, the NGF that external source is used has shown and has strengthened vascular permeability (Otten etc., Eur.J.Pharmacol.106:199-201 (1984)), strengthen T cell and B cellullar immunologic response (Otten etc., Proc.Natl.Acad.Sci.U.S.A.86:10059-10063 (1989)), induction of lymphocyte differentiation and mastocyte are bred and are caused discharging solvable bio signal (Matsuda etc., Proc.Natl.Acad.Sci.U.S.A.85:6508-6512 (1988) from mastocyte; Pearce etc., J.Physiol.372:379-393 (1986); Bischoff etc., Blood 79:2662-2669 (1992); Horigome etc., J.Biol.Chem.268:14881-14887 (1993)).

NGF is the cell generation by many types, these cells comprise mastocyte (Leon etc., Proc.Natl.Acad.Sci.USA 91:3739-3743 (1994)), bone-marrow-derived lymphocyte (Torcia etc., Cell 85:345-356 (1996)), keratinocyte (Di Marco etc., J.Biol.Chem.268:22838-22846), smooth muscle cell (Ueyama etc., J Hypertens.11:1061-1065 (1993)), fibroblast (Lindholm etc., Eur.J.Neurosci.2:795-801 (1990)), bronchial epithelial cell (Kassel etc., Clin.Exp.Allergy 31:1432-40 (2001)), kidney mesangial cell (Steiner etc., Am.J.Physiol.261:F792-798 (1991)) and skeletal muscle myotube (Schwartz etc., J Photochem.Photobiol.B66:195-200 (2002)).Found the NGF receptor on the various kinds of cell type outside nervous system.For example, on person monocytic cell, T cell and bone-marrow-derived lymphocyte and mastocyte, found TrkA.

Observing the NGF level in people patient and some animal models increases relevant with multiple inflammatory diseases.These diseases comprise systemic lupus erythematosus (Bracci-Laudiero etc., Neuroreport 4:563-565 (1993)), multiple sclerosis (Bracci-Laudiero etc., Neurosci.Lett.147:9-12 (1992)), psoriasis (Raychaudhuri etc., Acta Derm.l ' enereol.78:84-86 (1998)), arthritis (Falcim etc., Ann.Rheum.Dis.55:745-748 (1996)), interstitial cystitis (Okragly etc., J.Urology 161:438-441 (1999)) and asthma (Braun etc., Eur.J Immunol.28:3240-3251 (1998)).

Equally, the rising of NGF level is relevant with hyperpathia and inflammation and observe in many arthritis forms in the peripheral tissues.Suffer from the synovial membrane of patient with rheumatoid arthritis and express high-caliber NGF, and report that the synovial membrane of non-inflammatory patients does not detect NGF (Aloe etc., Arch.Rheum.35:351-355 (1992)).In the inductive rheumatoid arthritis rat of experiment, observe analog result (Aloe etc., Clin.Exp.Rheumatol.10:203-204 (1992)).Be reported in the increase of following mastocyte quantity in the transgenic arthritis mice, NGF level rising (Aloe etc., Int.J TissueReactions-Exp.Clin.Aspects 15:139-143 (1993)).

Have two classes to be used for the treatment of the medicine of pain, each class is by different mechanism effects and have different effects, but all has shortcoming.The first kind comprises nonsteroid anti-inflammatory drugs (NSAID), it is used for the treatment of mild pain, but its therapeutic use is subjected to the restriction of disadvantageous gastrointestinal tract effect, described gastrointestinal tract effect, cause the stomach burn into to form peptic ulcer or duodenitis and colon and nephrotoxicity as life-time service.Second class comprises opioid analgesic (as morphine), be used for the treatment of moderate to serious pain, but their therapeutic use is owing to disadvantageous effect is restricted, described disadvantageous effect such as constipation, nausea and vomiting, respiration inhibition, confusion, renal colic, to the tolerance of life-time service and the danger of addiction.

Obviously need improved pain therapy, it provides the treatment benefit (for example reducing the seriousness and/or the frequency of pain) of raising and/or reduces the influence of the disadvantageous side effect that many current schemes cause.

Complete being incorporated herein by reference of all lists of references (comprising patent application and publication) that this paper is quoted.

Summary of the invention

The present invention is based on NGF antagonist combination NSAID and can effectively treat this discovery of pain.This treatment causes unexpectedly improving pain therapy.In addition, this treatment allows to realize the pain relief of same amount and/or the other forms of enhancing of NSAID pain therapy with littler NSAID dosage usually.

On the one hand, feature of the present invention is the method for treatment (perhaps prevention in other embodiments) pain, and this method comprises uses a certain amount of nerve growth factor antagonist and a certain amount of NSAID to make uniting of they that effective pain relief is provided.The relative quantity of NGF antagonist and NSAID and ratio can change.In some embodiments, enough NGF antagonisies will be used so that allow to reduce to realize the common dosage of the NSAID that the pain relief of same degree is required.In some embodiments, with use the common dosage of enough NGF antagonisies with the required NSAID of the pain relief that allows to realize same degree be reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more than.This minimizing can be reflected in the amount of application (reduction frequency) in the given institute's amount of application used and/or preset time.

On the other hand, the invention provides the method that strengthens the NSAID pain therapy, it comprises the NGF antagonist of co-administered effective dose NSAID and effective dose.Use simultaneously and/or use at different time co-administered as used herein comprising.Use and/or use as composition isolated co-administered also comprising as common preparation (being that NGF antagonist and NSAID exist (combination) in same compositions)." co-administered " used herein comprises any situation, and wherein NSAID and NGF antagonist are applied to individuality with effective dose.Further discuss as this paper, be to be understood that NGF antagonist and NSAID can use with different administration frequencies and/or interval.For example, anti-ngf antibodies can be used weekly, and NSAID can use more continually.Be appreciated that the NGF antagonist can use through identical route of administration or different administration approach with NSAID, and during using, can change different dosage regimens.Can before the pain outbreak, use.

On the other hand, the invention provides the generation that reduces pain in individuality, ease the pain, alleviating pain and/or postpone the generation of pain or the method for progress, described method comprises the NGF antagonist of co-administered effective dose and the NSAID of effective dose.

Method of the present invention is suitable for treatment or prevents any etiologic any pain, comprising the pain of using the NSAID treatment usually.In some embodiments, pain is postoperative pain.In some embodiments, pain is the pain relevant with burn.In other embodiments, pain is the relevant pain of rheumatoid arthritis.In other embodiments, pain is the pain relevant with osteoarthritis.

The NGF antagonist that is suitable for method of the present invention is any reagent that can directly or indirectly cause the NGF biological activity to reduce.In some embodiments, the NGF antagonist is in conjunction with (physiological interaction) NGF (for example antibody), in conjunction with NGF receptor (as trkA receptor and/or p75) and/or reduction (hindering and/or blocking-up) downstream NGF receptor signal transduction (for example inhibitor of the inductive kinase signal of NGF or other downstream signals transduction in the target cell).In other embodiments, the NGF antagonist suppresses the synthetic and/or release of (reduction) NGF.In other embodiments, the NGF antagonist reduces expression or the function of NGF receptor TrkA and/or p75.In another embodiment, the NGF antagonist is not to be the NGF antagonist of TrkA immunoadhesin (being outside the TrkA immunoadhesin).In some embodiments, the NGF antagonist is selected from following any or multiple: anti-ngf antibodies, at the antisense molecule (comprising antisense molecule) of the nucleic acid of coding NGF at the nucleic acid of coding NGF, at antisense molecule, the NGF of NGF receptor (as TrkA and/or p75) suppress chemical compound, NGF analog, in conjunction with TrkA and/or the dominant negative mutant of p75 receptor, anti-TrkA antibody, anti-p75 antibody and the inhibitors of kinases of NGF.In some embodiments, NGF antagonist (as anti-ngf antibodies) is in conjunction with NGF (as hNGF) and not significantly in conjunction with relevant neurotrophin, as NT-3, NT4/5 and/or BDNF.In another embodiment, the NGF antagonist is an anti-ngf antibodies.In other embodiments, anti-ngf antibodies specific bond NGF.In other embodiments, anti-NGF receptor identification people NGF.In other embodiments, anti-ngf antibodies specific bond people NGF.In other embodiments, this antibody and the substantially the same NGF epi-position 6 of one or more antibodies that is selected from MAb911, MAb912 and MAb938 (seeing Hongo etc., Hybridoma 19:215-227 (2000)).In other embodiments, anti-ngf antibodies is through humanization (comprising humanization Mab 911, as described herein antibody E3).In other embodiments, anti-ngf antibodies is antibody E3 (describing as this paper).In other embodiments, anti-ngf antibodies contain antibody E3 one or more CDR (1,2,3,4,5 CDR of E3 or as in some embodiments, be all 6 CDR).In other embodiments, anti-ngf antibodies is behaved.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ ID NO:1) of the variable region of heavy chain shown in the table 1 and the aminoacid sequence (SEQ ID NO:2) of the variable region of light chain shown in the table 2.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ ID NO:1) of the variable region of heavy chain shown in the table 1.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ IDNO:2) of the variable region of light chain shown in the table 2.In other embodiments, described antibody contains the constant region of modification, as the constant region of immunologic inertia, as not causing the cracking of complement-mediated, does not perhaps stimulate the cytotoxicity (ADCC) that relies on antibody.In other embodiments, as Eur.J.Immunol. (1999) 29:2613-2624; The modification constant region of describing among PCT application No.PCT/GB99/01441 and/or the UK patent application No.9809951.8.

In some embodiments, the NGF antagonist is in conjunction with the NGF molecule.In other embodiments, the NGF antagonist is the antibody of specific bond NGF.Yet the NGF antagonist also can be in conjunction with the trkA receptor.The NGF antagonist can be anti-people NGF (anti-hNGF) monoclonal antibody, and it can be in conjunction with hNGF and effectively suppresses combining of hNGF and people TrkA (hTrkA).

The binding affinity of anti-ngf antibodies and NGF (as hNGF) can arrive about 0.80nM for about 0.10nM, and about 0.15 to about 0.75nM and about 0.18 to about 0.72nM.In one embodiment, binding affinity is that about 2pM is to 22pM.In some embodiments, binding affinity arrives for about 10nM.In other embodiments, binding affinity is for being lower than about 10nM.In other embodiments, binding affinity is about 0.1nM or about 0.07nM.In other embodiments, binding affinity is for being lower than about 0.1nM or being lower than about 0.07nM.In other embodiments, binding affinity is about 100nM, about 50nM, about 10nM, one of about 500pM, about 100pM, and perhaps about 50pM is to about 2pM, about 5pM, about 10pM, about 1nM, about 15pM, about 20pM, one of about 40pM, about 50pM.In some embodiments, binding affinity is one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM or about 50pM, or is lower than about 50pM.In some embodiments, binding affinity is for being lower than one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM or about 50pM.In other embodiments, binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM or is higher than about 40pM.As known in the art, binding affinity can be expressed as K _DPerhaps dissociation constant, the binding affinity of increase is corresponding to the K that reduces _DAnti-NGF mouse monoclonal antibody 911 (Hongo etc., Hybridoma 19:215-227 (2000)) is about 10nM with the binding affinity of people NGF, and humanization anti-ngf antibodies E3 (this paper description) is about 0.07nM with the binding affinity of people NGF.

When the NGF antagonist was antibody, antibody can be antibody fragment, comprised the multi-specificity antibody that is selected from Fab, Fab ', F (ab ') 2, Fv fragment, double antibody, single-chain antibody molecule and formed by antibody fragment and the antibody fragment of strand Fv (scFv) molecule.

NSAID can be the on-steroidal anti-inflammatory compound.NSAID is suppressed the Capability Categories of cyclo-oxygenase by them.Cyclo-oxygenase 1 and cyclo-oxygenase 2 are two kinds of main isotypes of cyclo-oxygenase, and most standard NSAID are mixed inhibitors of two kinds of isotypes.Most standard NSAID belong to one of 5 kinds of following structured sorts: (1) propanoic derivatives, and as ibuprofen, naproxen, congex, diclofenac sodium, and ketone ibuprofen; (2) acetogenin is as Tolmetin and slindac; (3) fragrant that acid (fenamicaicid) derivant is as mefenamic acid and that acid of first chlorophenol; (4) biphenylcarboxylic acid derivatives is as diflunisal and flufenisal; (5) oxicam, as piroxim, thiophene oxygen thiazine and isoxicam.

Another kind of other NSAID of selectivity inhibition cyclo-oxygenase 2 has been described.The Cox-2 inhibitor is in for example U.S. Patent No. 5,616,601,5,604,260,5,593,994,5,550,142,5,536,752,5,521,213,5,475,995,5,639,780,5,604,253,5,552,422,5,510,368,5,436,265,5, describe to some extent in 409,944 and 5,130,311, all these documents are incorporated herein and are reference.Some representative cox 2 inhibitor comprises celecoxib (SC-58635), Luo Feikexi, DUP-697, flosulide (CGP-28238), meloxicam, 6-methoxyl group-2-naphthalene acetic acid (6-MNA), MK-966, nabumetone (prodrug of 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614 or their combination.In some embodiments, the invention provides method, wherein aspirin and/or acetominophen and NGF antagonist (as anti-ngf antibodies) are united use.

NGF antagonist and/or NSAID can be applied to individuality by any suitable approach.For example, they can be together or separately and/or simultaneously and/or in turn interior, the Sublingual of interior, intrathoracic, the intraperitoneal of per os, intravenous, Sublingual, subcutaneous, intra-arterial, intramuscular, rectum, spinal column, ventricle, transdermal or use by suction.Using can be (for example intravenous) of general or partial.

On the other hand, feature of the present invention is the compositions that contains nerve growth factor antagonist and NSAID.Nerve growth factor antagonist and NSAID can exist with one or more pharmaceutically suitable carrier or excipient, and perhaps they may reside in the independent compositions.On the other hand, the invention provides the synergistic composition of NGF antagonist and NSAID.

The third aspect, feature of the present invention are the test kits that is used for any method disclosed herein, and described test kit contains NGF antagonist and NSAID.This test kit can contain the description relevant for any method disclosed herein.Described description can comprise NGF antagonist (as anti-NGF-antibody) and NSAID co-administered (promptly use simultaneously and/or use at different time).In some embodiments, NGF antagonist and NSAID are packaging together, but they can be also can be in same container.Thereby in some embodiments, test kit contains the NGF antagonist that is present in the same container and NSAID and about the description of any method disclosed herein.In other embodiments, test kit contains NGF antagonist and the NSAID that is present in the independent container.

In some embodiments, the invention provides the method for the individual pain of treatment, it comprises anti-nerve growth factor (NGF) antibody and the NSAID that individuality is used effective dose.In some embodiments, NSAID is selected from ibuprofen, naproxen, congex, diclofenac sodium, ketone ibuprofen, Tolmetin, slindac, mefenamic acid, meclofenamic acid, diflunisal, flufenisal, piroxim, thiophene oxygen thiazine, isoxicam, celecoxib, Luo Feikexi, DUP-697, flosulide, meloxicam, 6-methoxyl group-2-naphthalene acetic acid, MK-966, nabumetone, nimesulide, NS-398, SC-5766, SC-58215, T-614.In some embodiments, NSAID is an ibuprofen.In some embodiments, anti-ngf antibodies with about 10nm or the affinity that is lower than about 10nm in conjunction with people NGF.In some embodiments, anti-ngf antibodies behaviour antibody.In some embodiments, anti-ngf antibodies is a humanized antibody.In some embodiments, humanized antibody is the antibody that contains variable region of light chain shown in variable region of heavy chain shown in the SEQ ID NO:1 and the SEQ ID NO:2.In some embodiments, pain is postoperative pain.

In some embodiments, the invention provides the pharmaceutical composition of treatment pain, it contains effective dose anti-ngf antibodies and NSAID and pharmaceutically suitable carrier.

In some embodiments, the invention provides the test kit that is used for the treatment of pain, it contains anti-ngf antibodies, NSAID, about co-administered anti-ngf antibodies and the NSAID description with treatment pain.

The accompanying drawing summary

Fig. 1 be illustrated in 10 or 30mg/kg S[+] ibuprofen associating NGF antagonist (anti-NGF antagonist Mab 911; Seeing Hongo etc., Hybridoma 19:215-227 (2000)) accumulation pain score has reduced in the animal of treatment.Animal is divided into two groups (contrast and Antybody therapies).Use NGF antagonist (time=-15 hours) preceding 15 hours of operation with 1mg/kg dosage intraperitoneal.Implement operation as described in the time 0.Perform the operation back 24 hours (among the figure " 0 ") assessment tranquillization pain (resting pain).Use ibuprofen (with 300mg/ml in 45% beta-schardinger dextrin-liquid) to treat all animals then with 10mg/kg or 30mg/kg body weight.The control animal of non-antibody treatment is also with 10mg/kg, 30mg/kg, 100mg/kg and the treatment of 300mg/kg ibuprofen.Ibuprofen is in the nape subcutaneous delivery.Behind the ibuprofen dosage 1 hour, check tranquillization pain.The treatment that adds ibuprofen with the anti-ngf antibodies antagonist is more effective in alleviating tranquillization pain with ibuprofen or anti-ngf antibodies antagonist for treating than only.

Fig. 2 shows with 5mg/kg diclofenac sodium associating NGF antagonist (anti-ngf antibodies Mab 911; See Hongo etc., Hybridoma 19:215-227 (2000)) accumulation pain shot chart in the animal of treatment.Animal is divided into two groups (contrast and Antybody therapies).Use NGF antagonist (time=-15 hours) preceding 15 hours of operation with 1mg/kg dosage intraperitoneal.Implement operation as described in the time 0.Back 24 hours (among the figure " 0 ") assessment tranquillization pain of performing the operation.Treat all animals with diclofenac sodium with 5mg/kg then.The control animal of non-antibody treatment is also treated with 5mg/kg.Diclofenac sodium is in the nape subcutaneous delivery.Behind the ibuprofen dosage 1 hour, check tranquillization pain.

Detailed Description Of The Invention

We have found that NGF antagonist (such as anti-ngf antibodies) and NSAID by co-administered effective dose can prevent or treat pain. Method and composition of the present invention can be used for treatment or pre-pain, comprising any pain of usually using the NSAID treatment. According to the present invention, use nerve growth factor antagonist and NSAID by uniting, can use now the more NSAID treatment pain of low dosage, thereby reduce the possibility of the side effect relevant with the NSAID treatment. In some embodiments, reduce at least about 5% using the common dosage of enough NGF antagonists with the required NSAID of the pain relief that allows to realize same degree, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 90%, or more than.

As described herein, co-administered NSAID and NGF antagonist can also be strengthened NSAID to the treatment of pain.

On the one hand, the invention provides the method for the treatment of or individual (such as mammal, comprising people and the non-human) pain of prevention, comprise the NGF antagonist of co-administered effective dose and the NSAID of effective dose. On the other hand, the invention provides the method for strengthening NSAID treatment in the individuality or pre-pain, the method comprises the NGF antagonist (such as anti-ngf antibodies) of co-administered effective dose and the NSAID of effective dose. On the other hand, the invention provides prevention, ease the pain and/or prevent the method for pain development or progress.

In some embodiments, anti-ngf antibodies can activate its TrkA or p75 acceptor with combination or the establishment NGF of its TrkA or p75 acceptor in vivo in conjunction with NGF and establishment NGF. In some embodiments, the binding affinity of described antibody and NGF is about 0.01 to about 1.00nM, or about 0.05nM is to about 0.25nM. In other embodiments, binding affinity is about 0.1nM or about 0.07nM. In other embodiments, binding affinity is about 1pM, about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 50pM, about 100pM, or more than. In one embodiment, binding affinity is that about 2pM is to 22pM. In some embodiments, this antibody and one or more antibody that are selected from MAb911, MAb912 and MAb938 are substantially the samely in conjunction with NGF epi-position 6. See Hongo etc., Hybridoma 19:215-227 (2000).

Antibody can be antibody fragment, as is selected from multi-specificity antibody that Fab, Fab ', F (ab ') 2, Fv fragment, double antibody, single-chain antibody molecule and antibody fragment form and one or more antibody fragments of scFv (scFv) molecule. Described antibody can also be chimeric, and it can be humanization or people. Described antibody can also be bispecific.

Can be used for representative NSAIDs of the present invention includes, but is not limited to: (1) propanoic derivatives, such as brufen, naproxen, congex, diclofenac and Ketoprofen; (2) acetogenin is such as MCN 2559 and slindac; (3) fenamic acid derivative is such as mefenamic acid and meclofenamic acid; (4) biphenylcarboxylic acid derivatives is such as Diflunisal and flufenisal; (5) oxicam is such as piroxim, thiophene oxygen thiazine and isoxicam. Described another kind of other NSAID, it selectively suppresses COX-2. The Cox-2 inhibitor is in for example U.S. Patent No. 5,616,601,5,604,260,5,593,994,5,550,142,5,536,752,5,521,213,5,475,995,5,639,780,5,604,253,5,552,422,5,510,368,5,436,265,5, describe to some extent in 409,944 and 5,130,311, all these documents are incorporated herein and are reference. Some representative cox 2 inhibitor comprises Sai-Mi-Xi-Bu (SC-58635), Luo Feikexi, DUP-697, Flosulide (CGP-28238), Meloxicam, 6-methoxyl-2-naphthylacetic acid (6-MNA), MK-966, nabumetone (prodrug of 6-MNA), aulin, NS-398, SC-5766, SC-58215, T-614, perhaps their combination. In some embodiments, aspirin and/or acetominophen and NGF antagonist (such as anti-ngf antibodies) are united use.

Method and composition of the present invention can be used for treating any etiologic pain, comprises the pain of acute and chronic ache, any tool inflammatory component, and wherein usually uses any pain of NSAID treatment. The example of pain comprises pain after the operation, postoperative pain (comprising toothache), antimigraine, headache and trigeminal neuralgia and, and wound ache related (comprise traumatic brain injury), neuropathic pain, with flesh-skeletal diseases relevant pain ache related with burn, wound or kidney stone, these diseases such as rheumatoid arthritis, osteoarthritis, splanchnodynia, colitis, pancreatitis, gastritis, ankylosing spondylitis, seronegativity (non-rheumatoid disease) arthropathy, nonarticular rheumatism and joint week illness, and the pain relevant with cancer (comprising " breaking through pain (break-through pain) " and the pain relevant with TCA), peripheral neurophaty and postherpetic neuralgia and the pain of being correlated with sickleshaped cell crisis. The example of the pain of tool inflammatory component (except more described above) comprises rheumatic pain, the pain relevant with catarrh and dysmenorrhoea. In some embodiments, method and composition of the present invention can be used for treatment or prevention postoperative pain and/or cancer pain. In other embodiments, pain is for usually not using the pain indication of NSAID treatment, such as neuropathic pain. In other embodiments, method and composition described herein can be used for treating and/or preventing and the relevant pain of burning. In other embodiments, method and composition described herein can be used for treating and/or preventing the pain relevant with rheumatoid arthritis. In other embodiments, method and composition described herein can be used for treating and/or preventing the pain relevant with osteoarthritis.

On the other hand, the invention provides the kit that is used for the treatment of pain, it contains NGF antagonist (such as anti-ngf antibodies) and the NSAID that is applicable to any method described herein.

General technology

Unless otherwise noted, practice of the present invention will be used molecular biology (comprising recombinant technique), microbiology, cell biology, biochemistry and the immunology routine techniques in the art technology scope. This type of technology is in detail explanation in the literature, such as " Molecular Cloning:A Laboratory Manual ", and second edition, (Sambrook etc., 1989) Cold Spring Harbor Press; " Oligonucleotide Synthesis " (M.J.Gait compiles, 1984); " Methods in Molecular Biology ", Humana Press; " Cell Biology:A Laboratory NOtebook " (J.E. Cellis compiles, 1998) Academic Press; " Animal Cell Culture " (R.I.Freshney compiles, 1987); " Introduction to Cell and Tissue Culture " (J.P.Mather and P.E. Roberts, 1998) Plenum Press; " Cell and Tissue Culture:Laboratory Procedures " (A.Doyle, J.B.Griffiths and D.GNewell compile 1993-1998) J.Wiley and Sons; " Methods in Enzymology " (Academic Press, Inc.); " Handbook of Experimental Immunology " (D.M.Weir and C.C.Blackwell compile); " Gene Transfer Vectors for Mammalian Cells " (J.M.Miller and M.P.Calos compile, 1987); " Current Protocols in Molecular Biology " (E M.Ausubel etc., editor, 1987); " PCR:The Polymerase Chain Reaction " (Mullis etc., editor, 1994); " Current Protocols in Immunology " (J.E.Coligan etc., editor, 1991); " Short Protocols in Molecular Biology " (Wiley and Sons, 1999); " Immunobiology " (C.A.Janeway and P.Travers, 1997); " Antibodies " (P.Finch, 1997); " Antibodies:a practical approach " (D.Catty, editor, IRL Press, 1988-1989); " Monoclonal antibodies:a practical approach " (P.Shepherd and C.Dean, editor, Oxford University Press, 2000); " Using antibodies:a laboratory manual " (E.Harlow and D.Lane, Cold Spring Harbor Laboratory Press, 1999); " The Antibodies " (M.Zanetti and J.D.Capra, editor, Harwood Academic Publishers, 1995).

Definition

" antibody " (can take the plural form Alternate) is as can be by being positioned at least one antigen recognition site specific bond target of immunoglobulin molecules variable region, such as the immunoglobulin molecules of carbohydrate, polynucleotides, lipid, polypeptide etc. As used herein, this term not only comprises complete polyclone or monoclonal antibody, and any other of immunoglobulin molecules that also comprises its fragment (such as Fab, Fab ', F (ab ') 2, Fv), strand (scFv), its mutant, the fusion that comprises antibody moiety, humanized antibody, chimeric antibody, bispecific antibody, single-chain antibody, multi-specificity antibody (for example bispecific antibody) and contain required specific antigen recognition site modified configuration. Antibody comprises the antibody of arbitrary classification, such as IgG, IgA or IgM (or its subclass) and needn't be the antibody of any particular category. The antibody amino acid sequence that depends on the heavy chain immunoglobulin constant domain can be included into immunoglobulin (Ig) different classes of. Immunoglobulin (Ig) mainly contains five classes: IgA, IgD, IgE, IgG and IgM, and several subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA1 and the IgA2 of being further divided in these. Heavy chain constant domain corresponding to dissimilar immunoglobulin (Ig)s is called α, δ, ε, γ and μ. The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling are known.

" monoclonal antibody " refers to predominating of antibody population, and wherein monoclonal antibody comprises the amino acid (natural existence or non-natural exist) that participates in selective binding antigen. The monoclonal antibody high special is for single antigen site. Term " monoclonal antibody " not only comprises complete monoclonal antibody and full length monoclonal antibodies, also comprises its fragment (such as Fab, Fab ', F (ab ') 2, Fv), strand (scFv), its mutant, the fusion that comprises antibody moiety, Humanized monoclonal antibodies, chimeric mAb and contains required specific antigen recognition site and any other modified configuration of immunoglobulin molecules that can conjugated antigen. The mode of the source of antibody or antibody preparation (for example, by hybridoma, bacteriophage select, recombinant expressed, transgenic animals etc.) unrestricted.

The molecule of the immunoglobulin structure that " humanization " antibody refers to have the immunoglobulin (Ig) antigen binding site that basically derives from inhuman species and keeps this molecule based on structure and/or the sequence of human immunoglobulin(HIg). Antigen binding site can contain the complete variable domains that is fused to constant domain or only contain the complementary determining region (CDR) of grafting suitable framework region in the variable domains. Antigen binding site can be modified for wild type or by one or more amino acid substitutions, and is for example modified and more as human immunoglobulin(HIg). Some form of humanized antibody has kept all CDR sequences (for example containing the humanization mouse antibodies from all 6 CDR of mouse antibodies). Other forms of humanized antibody have the one or more CDR (1,2,3,4,5,6) that have change with respect to antibody originally. In some cases, the framework region of human immunoglobulin(HIg) (FR) residue or other residues are replaced by corresponding inhuman residue. In addition, humanized antibody can contain the residue that does not have in receptor antibody or donor antibody.

As used herein, term " nerve growth factor " and " NGF " refer to keep nerve growth factor and its variant (comprise, for example splice variant and protein processing variant) of at least part of activity of NGF. As used herein, NGF comprises all mammalian species native sequences NGF of (comprising people, non-human primate, dog, cat, horse or ox).

" NGF acceptor " refers to the polypeptide of NGF combination or activation. The NGF acceptor comprises TrkA acceptor and the p75 acceptor of any mammalian species (including but not limited to people, dog, cat, horse, primate or ox).

" NGF antagonist " refers to blocking-up, suppresses or reduces the bioactive any molecule of (comprising remarkable reduction) NGF, and the NGF biologically active comprises the downstream pathway by the mediation of NGF signal, such as receptors bind and/or cause cell response to NGF. Term " antagonist " does not contain special Biological mechanism of action, and think and obviously comprise and comprise all possible pharmacology, physiology or biochemical interaction with NGF, it can be with direct or indirect, perhaps with NGF, its acceptor interaction, perhaps by another mechanism, this term also comprises the result of described mechanism that can be by multiple different, chemically different component realization. Representative NGF antagonist includes, but is not limited to anti-ngf antibodies, the antisense molecule (comprising the antisense molecule for coding NGF nucleic acid) for NGF, NGF Inhibitor, NGF analogue, the dominant negative mutant in conjunction with the TrkA acceptor of NGF, TrkA immunoadhesin, anti-TrkA antibody, anti-p75 antibody, for one of TrkA and/or p75 acceptor or both antisense molecules (comprising the antisense molecule for the nucleic acid molecules of coding TrkA or p75) and inhibitors of kinases. For the present invention, should clearly understand term, title, functional status and feature that term " antagonist " comprises all aforementioned definitions, thus NGF self, NGF biologically active (including but not limited to the ability of any aspect of its mediated pain) or bioactive result on any meaningful degree by substantive invalid, reduce or neutralization. In some embodiments, the NGF antagonist in conjunction with (physically interact) NGF (for example antibody), in conjunction with NGF acceptor (such as trkA acceptor or p75 acceptor), reduce (hindering and/or blocking-up) downstream NGF receptor signal transduction and/or suppress (reductions) NGF synthetic, produce and release. In some embodiments, the NGF antagonist is in conjunction with (physically interacting) NGF (for example antibody), in conjunction with NGF acceptor (such as TrkA acceptor and/or p75 acceptor) and/or reduction (hindering and/or blocking-up) downstream NGF receptor signal transduction. In other embodiments, the NGF antagonist is in conjunction with NGF and stop the TrkA receptor dimerization and/or the TrkA autophosphorylation. In other embodiments, the NGF antagonist suppresses or the synthetic and/or generation (release) of reduction NGF. This paper provides the example of all types of NGF antagonists.

As used herein, " anti-ngf antibodies " refers to and can and suppress the NGF biological activity and/or the antibody of the downstream pathway of NGF signal mediation in conjunction with NGF.

" TrkA immunoadhesin " refers to contain the fragment (for example ectodomain of TrkA receptor) of TrkA receptor and the solubility chimeric molecule of immunoglobulin sequences, and it keeps the binding specificity of TrkA receptor.

" biological activity " of NGF is often referred to the ability in conjunction with NGF receptor and/or activation NGF receptor signal approach.Do not add restriction, biological activity comprises following one or more: in conjunction with the ability of NGF receptor (as p75 and/or TrkA); Promote the ability of TrkA receptor dimerizationization and/or autophosphorylation; The ability of activation NGF receptor signal approach; Promote other changes in cell differentiation, propagation, survival, growth and the cytophysiology, comprise that change, synapse generation, synapse function, neurotransmitter and/or the neuropeptide of (for neuron, comprising periphery and axoneuron) neuron morphology discharge the ability of damage regenerated ability in back and mediated pain.

Term " epi-position " is used in reference to (monoclonal or the polyclone) antibody combining site on the antigen (as proteantigen).

As used herein, " treatment " is the method that obtains useful or desirable clinical effectiveness.For the present invention, useful or desirable clinical effectiveness includes, but is not limited to following one or more: the improvement of any aspect of pain or alleviate comprises acute, chronic, inflammatory, nerve or postoperative pain.For the present invention, useful or desirable clinical effectiveness includes, but is not limited to one or more following contents: comprise and reduce severity, alleviate one or more symptoms relevant with pain (any aspect that comprises pain) (as shortening durante dolors and/or reduction pain sensitivity or sensation).

Pain " generation of reduction " refers to seriousness (can comprise that reduction is to the other medicines that are generally used for this situation and/or the needs and/or the amount (for example being exposed to other medicines and/or therapeutic agent) of therapeutic agent), persistent period and/or the frequency (comprise for example postpone or increase the time that arrives pain) of any reduction in individuality.As understood by a person skilled in the art, Different Individual can change to the reaction of treatment, and it is same, for example " reduce the method that pain takes place in individuality " and reflected co-administered NGF antagonist described herein and NSAID described herein, this is based on this use of rational expectation and may causes the pain of this class in this concrete individuality to reduce.

One or more symptoms of " alleviation " pain or pain are represented to compare with NSAID with not co-administered NGF antagonist, the alleviating or improve of one or more symptoms of pain." alleviation " also comprises the persistent period that shortens or reduce symptom.

One or more symptoms that " alleviate " pain or pain are illustrated in to be used according in the individuality of NGF antagonist combination NSAID treatment of the present invention or in the groups of individuals, reduces the degree of one or more undesirable clinical manifestations of pain.

As used herein, the development of " delay " pain represents to postpone, hinder, slow down, stop, stablize and/or postpone the process of pain.Depend on the medical history and/or the individuality for the treatment of, the time span of this delay can change.As it will be apparent to those skilled in the art that enough or significant delay can be played the effect that comprises prevention, so that pain does not take place individuality.With compare without the method, the method for " delay " symptom development be to reduce symptom development probability and/or in given time limit reduction symptom degree methods in the given time limit.These are relatively generally based on the clinical research with remarkable quantity experimenter on the statistics.

" development " of pain or " progress " refer to the initial performance of disease and/or development subsequently.Can detect and assess the progress of pain with the standard clinical techniques of knowing in this area.Yet, development also refer to detect less than progress.About the present invention, development or progress refer to the biological process of symptom." development " comprises generation, recurrence and outbreak." outbreak " or " generation " of pain used herein comprise initial outbreak and/or recurrence.

" effective dose " is for being enough to cause amount favourable or expectation clinical effectiveness (comprise and alleviate or reduce pain perception).For the present invention, the effective dose of NGF antagonist (as anti-ngf antibodies) and NSAID comprises the intensity of the pain that is enough to treat, alleviate, reduce any kind (comprising acute, chronic, inflammatory, nerve or postoperative pain) or the amount of prevent irritation (sensation that comprises nociception and pain).In some embodiments, the effective dose of NSAID and NGF antagonist for the sensitivity threshold value to outside stimulus can be adjusted to the health volunteer in observed NGF antagonist of being on close level and the amount of NSAID.In other embodiments, this level can be not with the health volunteer in observed quite, but with do not accept comparing of therapeutic alliance and reduced.The effective dose of NGF antagonist also comprises to be enough to strengthen the NSAID treatment (therapeutic effect) of pain as described herein or reduce treatment as described herein or the NGF antagonism dosage of the NSAID dosage that prevent irritation is required.As understanding in this area, the effective dose of NGF antagonist combination NSAID can be according to becoming as types of pain (with patient's medical history and other factors, as the type (and/or dosage) of used NGF antagonist and/or NSAID).This paper about the present invention, effective dose can be the required NGF antagonist of realization potentiation and the amount of NSAID antagonist.The present invention up and down the effective dose of this paper antagonist be often referred to and be enough to cause NSAID to strengthen (it can refer to the dosage that reduces again and/or observe certain other beneficial effects) and/or cause amount with the beneficial effect that only uses the NSAID treatment to compare for the treatment of pain effect." effective dose " of NGF antagonist can also cause using the potentiation that NGF antagonist or NSAID compare with only using.

" individuality " is vertebrates, preferred mammal, more preferably people.Mammal includes, but is not limited to letting animals feed, mutation animal (sport animal), house pet, primate, horse, milch cow, Canis familiaris L., cat, mice and rat.

Term " NSAID " refers to the on-steroidal anti-inflammatory compound.Suppressing the ability of cyclo-oxygenase by NSAIDs classifies them.Cyclo-oxygenase 1 and cyclo-oxygenase 2 are two kinds of main isotypes of cyclo-oxygenase, and most standard NSAID are mixed inhibitors of two kinds of isotypes.Most standard NSAID belong to one of 5 kinds of following structured sorts: (1) propanoic derivatives, and as ibuprofen, naproxen, congex, diclofenac sodium, and ketone ibuprofen; (2) acetogenin is as Tolmetin and slindac; (3) fenamic acid derivative is as mefenamic acid and that acid of first chlorophenol; (4) biphenylcarboxylic acid derivatives is as diflunisal and flufenisal; (5) oxicam, as piroxim, thiophene oxygen thiazine and isoxicam.

Described another kind of other NSAID, its selectivity suppresses cyclo-oxygenase 2.The Cox-2 inhibitor is in for example U.S. Patent No. 5,616,601,5,604,260,5,593,994,5,550,142,5,536,752,5,521,213,5,475,995,5,639,780,5,604,253,5,552,422,5,510,368,5,436,265,5, describe to some extent in 409,944 and 5,130,311, quote all these documents as a reference.Some representative cox 2 inhibitor comprises celecoxib (SC-58635), DUP-697, flosulide (CGP-28238), meloxicam, 6-methoxyl group-2-naphthalene acetic acid (6-MNA), Luo Feikexi, MK-966, nabumetone (prodrug of 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; Perhaps their combination.

In some embodiments, aspirin and/or acetominophen and NGF antagonist (as anti-ngf antibodies) are united use.Aspirin is another kind of on-steroidal anti-inflammatory compound.

" associating " used herein used and comprised and use simultaneously and/or use at different time.Use (being that NGF antagonist and NSAID are present in the same compositions) or use as compositions separately co-administered also comprising as common preparation.Be applied to individual any situation with NSAID and NGF antagonist co-administered comprising used herein, and this is used and can simultaneously and/or separately carry out.Further discuss as this paper, be appreciated that the administration frequency that NGF antagonist and NSAID can be different or use at interval.For example, anti-ngf antibodies can be used weekly, and NSAID can use more continually.Be to be understood that and use identical or different route of administration to use NGF antagonist and NSAID.

" postoperative pain " (can be called " cut back " or " pain after the wound " alternately) refer to be meant otch, puncture in wound such as the individuality tissue, cut, tear or pain (comprising no matter being the pain that aggressive or noninvasive all surgical procedures cause) that wound causes or causes.As used herein, postoperative pain does not comprise that the pain of (cause or cause) takes place no outside health wound.In some embodiments, postoperative pain is inner or outside (comprising periphery) pain, and wound, otch, wound, tear or cutting (as traumatic wounds) or deliberately (as surgical incision) generation accidentally.As used herein, " pain " comprises the nociception and the sensation of pain, and pain can be with pain score and the objective and subjective assessment of other method well known in the art.As used herein, postoperative pain comprises allodynia (promptly to normal non-destructive stimulus increased response (promptly harmful impression)) and hyperpathia (promptly normal harmful or unhappy irritant reaction being strengthened), and it can be heat or machinery (sense of touch) in essence again.In some embodiments, pain is characterized as heat sensitivity, mechanical sensitivity and/or tranquillization pain.In some embodiments, postoperative pain comprises through mechanical induction pain or tranquillization pain.In other embodiments, postoperative pain comprises tranquillization pain

When the one side of NSAID treatment improves (not using the NGF antagonist with using NSAID compares), the NSAID treatment " reinforcement " of pain.For example, the effect of NSAID when not having the NGF antagonist, the effect of NSAID treatment pain has strengthened when having the NGF antagonist.As another example, when this use allows better pain relief (for example, when the dosage of the NASIAD that uses can not effectively be treated or be prevented pain), use NGF antagonist and NSAID " to strengthen " with NSAID treatment of pain or prevention by uniting.

The inventive method

About all methods described herein, the reference of NGF antagonist and NSAIDs also comprised contain one or more these combination of agents things.The present invention can be used for treating individual pain, and described individuality comprises all mammals, comprises people and non-human.

On the one hand, the invention provides the method for the individual pain of treatment, it comprises the NGF antagonist (as anti-ngf antibodies) of co-administered effective dose and the NSAID of effective dose.In some embodiments, be reduced by at least about 5% with using the common dosage of enough NGF antagonisies with the required NSAID of the pain relief that allows to realize same degree, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more than 90%.

On the other hand, the invention provides the method that enhancing NSAID treats individual pain, it comprises the NGF antagonist of co-administered effective dose and the NSAID of effective dose.

In some embodiments, pain comprises following any or multiple: pain, postoperative pain (comprising toothache), migraine, headache and the trigeminal neuralgia of acute and/or chronic pain, any tool inflammatory component and ache related with burn, wound or renal calculus, with wound ache related (comprising traumatic craniocerebral injury), neuropathic pain, the pain relevant with sickleshaped cell crisis, with dysmenorrhea or the relevant pain of functional disorder of intestine, with the pain (comprise " breakthrough pain " reach and terminal cancer be correlated with pain) relevant with cancer.In other embodiments, pain is arbitrary pain for the treatment of with NSAID (for example ibuprofen) usually.In other embodiments, pain is the pain relevant with burn.In other embodiments, pain is the pain relevant with rheumatoid arthritis.In other embodiments, pain is the pain relevant with osteoarthritis.

On the other hand, the invention provides prevention, alleviate and/or prevent the method for pain development or progress.Thereby, in some embodiments, use the NGF antagonist before in pain events (as operation), as anti-ngf antibodies and/or NSAID.For example, may cause or be in the activity (as exterior trauma or operation) 30 minutes before, 1 hour, 5 hours, 15 hours, 24 hours that causes pain danger or even longer, as 1 day, a couple of days or even 1 week, 2 weeks, 3 weeks or use the NGF antagonist more than 3 weeks.

With method assessment treatment of pain as known in the art or prevention.Can implement assessment based on objective measurement, as observed behavior, as to the reaction that stimulates, facial expression etc.Can also assess based on subjective measurement, as describe patient's pain with multiple pain yardstick.See Katz etc. for example, Surg ClinNOrth Am. (1999) 79 (2): 231-52; Caraceni etc., J Pain Symptom Manage (2002) 23 (3): 239-55.

The diagnosis or the assessment of rheumatoid arthritis pain well set up in this area.Can implement assessment based on measurement as known in the art, as characterize patient's pain with multiple pain yardstick.See Katz etc. for example, Surg Clin NOrth Am. (1999) 79 (2): 231-52; Caraceni etc., J PainSymptom Manage (2002) 23 (3): 239-55.Also be useful on the yardstick commonly used of measuring morbid state, as American university rheumatoid disease (ACR) (Felson etc., Arthritis and Rheumatism (1993) 36 (6): 729-740), health evaluating questionnaire (HAQ) (Fries etc., (1982) Paulus yardstick (Paulus etc. J.Rheumatol.9:789-793),, Arthritis andRheumatism (1990) 33:477-484), yardstick (AIMS) (Meenam etc., Arthritis and Rheumatology (1982) 25:1048-1053) is measured in influence with arthritis.

The diagnosis or the assessment of osteoarthritis well set up in this area.Can implement assessment based on measurement as known in the art, as use multiple pain yardstick to characterize patient's pain.See Katz etc. for example, Surg Clin NOrth Am. (1999) 79 (2): 231-52; Caraceni etc., J Pain SymptomManage (2002) 23 (3): 239-55.For example, can move (Ambulation) pain yardstick (comprising pain, stiff and body function) and 100mm visual simulation yardstick (VAS) assessment pain and evaluation reaction with WOMAC to treatment.

Be to be understood that when NGF antagonist (as anti-ngf antibodies) and NSAID are co-administered as compositions single or that separate the nerve growth factor antagonist of being given is consistent with desirable effect performance with the ratio of NSAID.In some embodiments, the part by weight of nerve growth factor antagonist and NSAID can be 1: 1.In some embodiments, this ratio can between about 0.001 than about 1 to about 1000 than about 1, about 0.01 than about 1 to about 100 than about 1, perhaps about 0.1 than about 1 to about 10 than about 1.Also imagine other ratios.

Be to be understood that and be used for the treatment of or the amount of nerve growth factor antagonist that prevent irritation is required and NSAID will be not only becomes along with selected particular compound or compositions, also along with the character of route of administration, the disease of being treated, with patient's age and situation and become, and finally determine by the doctor in charge.

The NGF antagonist

Method of the present invention is used the NGF antagonist, it refers to blocking-up, suppresses or reduces the bioactive any molecule of (comprising significantly) NGF, the NGF biological activity comprises the downstream pathway of NGF signal transduction mediation, as in conjunction with and/or cause receptor to the cell response of NGF.Term " antagonist " does not imply the specific mechanisms of any biological agent, and thinks that it obviously comprises with comprising and interact with all possible pharmacology, the physiology and biochemistry of NGF and can be by the described results of interaction of multiple different, chemically different compositions realization.Representative NGF antagonist includes, but is not limited to anti-ngf antibodies, polypeptide (comprise the polypeptide that contains the NGF binding structural domain that derives from anti-ngf antibodies, for example contain the polypeptide that is enough in conjunction with the binding structural domain in the CDR zone of NGF), antisense molecule (comprising antisense molecule) at NGF at the nucleic acid of coding NGF, antisense molecule (comprising antisense molecule) at TrkA and/or p75 receptor at the nucleic acid molecules of coding trkA or p75, NGF suppresses chemical compound, the NGF analog, dominant negative mutation in conjunction with the TrkA receptor of NGF, the TrkA immunoadhesin, anti-TrkA antibody, anti-p75 antibody, and inhibitors of kinases.For the present invention, should clearly understand term, title, functional status and feature that term " antagonist " comprises all aforementioned definitions, thus NGF self, NGF biological activity (including but not limited to the ability of any aspect of its mediated pain) or bioactive result on any meaningful degree by substantive invalid, reduce or neutralization.In some embodiments, the NGF antagonist is in conjunction with (physically interacting) NGF (for example antibody), in conjunction with NGF receptor (as trkA receptor and/or p75 receptor) and/or reduction (hindering and/or blocking-up) downstream NGF receptor signal transduction.Therefore, in some embodiments, the NGF antagonist is in conjunction with (physically interacting) NGF.In other embodiments, the NGF antagonist is in conjunction with NGF receptor (as trkA receptor or p75).In other embodiments, the NGF antagonist reduces (hindering and/or blocking-up) downstream NGF receptor signal transduction (for example inhibitor of signal transduction of kinases).In other embodiments, the NGF antagonist suppresses the synthetic and/or release of (reduction) NGF.In another embodiment, the NGF antagonist is the TrkA immunoadhesin.In another embodiment, the NGF antagonist is different from anti-ngf antibodies.In some embodiments, the NGF antagonist is in conjunction with NGF (as hNGF) and not significantly in conjunction with the related neural nutrient protein, as NT-3, NT4/5 and/or BDNF.In some embodiments, the NGF antagonist is in conjunction with people NGF, and remarkable combination is from the NGF of another invertebrate species (being mammal in some embodiments).In some embodiments, the NGF antagonist is in conjunction with people NGF and one or more NGF from other invertebrate species (being mammal in some embodiments).In some embodiments, the NGF antagonist is in conjunction with NGF and at least a other neurotrophins.In some embodiments, the NGF antagonist is in conjunction with the NGF of mammalian species (as horse or Canis familiaris L.), but remarkable combination is from the NGF of another mammalian species.

Anti-ngf antibodies

In some embodiments of the present invention, the NGF antagonist contains anti-ngf antibodies.Anti-ngf antibodies should demonstrate the feature below any or multiple: (a) in conjunction with NGF and suppress the NGF biological activity and/or the downstream pathway of NGF semiotic function mediation; (b) any aspect that NSAID treated or prevented pain is especially united in any aspect of treatment or prevent irritation; (c) block or weaken NGF receptor activation (comprising trkA receptor dimerizationization and/or autophosphorylation); (d) removing of enhancing NGF; (e) strengthen NSAID to treatment of pain.

Anti-ngf antibodies is as known in the art, sees, for example PCT publication No. W002096458, WO01/78698, WO01/64247, U.S. Patent No. 5,844,092,5,877,016 and 6,153,189; Hongo etc., Hybridoma, 19:215-227 (2000); Cell.Molec.Biol.13:559-568 (1993); GenBank accession number U39608, U39609, L17078 or L17077.

In some embodiments, anti-ngf antibodies specific bond NGF.In other embodiments, anti-ngf antibodies is through humanization (antibody E3 as described herein).In other embodiments, anti-ngf antibodies is antibody E3 (describing as this paper).In other embodiments, anti-ngf antibodies contains one or more CDR (as 1,2,3,4,5 CDR, perhaps in some embodiments, containing all 6 CDR of E3) of antibody E3.In other embodiments, antibody behaviour antibody.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ ID NO:1) of the variable region of heavy chain shown in the table 1 and the aminoacid sequence (SEQID NO:2) of the variable region of light chain shown in the table 2.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ ID NO:1) of the variable region of heavy chain shown in the table 1.In other embodiments, anti-ngf antibodies contains the aminoacid sequence (SEQ ID NO:2) of the variable region of light chain shown in the table 2.In other embodiments, described antibody contains the modification constant region, as immunologic inertia, does not promptly cause the cracking of complement-mediated or does not stimulate the constant region of the cell-mediated toxicity (ADCC) that relies on antibody.In other embodiments, the modification constant region as describing among Eur.J.Immunol. (1999) 29:2613-2624, PCT application No.PCT/GB99/01441 and/or the UK patent application No.9809951.8.In other embodiments, anti-ngf antibodies is U.S.Ser.No.10/745, any antibody of describing in 775.

In some embodiments, anti-ngf antibodies is for being called the humanization mouse anti NGF monoclonal antibody of antibody " E3 ", it contains and comprises A330P331 to heavy chain and the variable region of light chain shown in people's heavy chain IgG2a constant region (aminoacid is numbered with reference to wild type IgG2a sequence, sees Eur.J.Immunol. (1999) 29:2613-2624), people's light chain κ constant region and the table 1 and the table 2 of S330S331 sudden change.

Table 1: variable region of heavy chain

QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVS(SEQ ID NO：1)

Table 2: variable region of light chain

DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRT(SE Q ID NO：2)

The following coding E3 heavy chain or the polynucleotide of E3 variable region of light chain are deposited in American type culture collection (ATCC) on January 8th, 2003.

Material		The ATCC accession number	Preservation day
				Carrier Eb.911.3E	E3 light chain V district	PTA-4893	On January 8th, 2003
Carrier Eb.pur.911. 3E	E3 light chain V district	PTA-4894	On January 8th, 2003
				Carrier Db.911.3E	E3 heavy chain V district	PTA-4895	On January 8th, 2003

Carrier Eb.911.3E is the polynucleotide of the variable region of light chain shown in the coding schedule 2; Carrier Eb.pur.911.3E is the polynucleotide of variable region of light chain shown in the coding schedule 2, and carrier Db.911.3E is the polynucleotide of variable region of heavy chain shown in the coding schedule 1.These polynucleotide constant domain of also encoding.

The technology that has two kinds of definite CDR at least: (1) is based on the method for sequence variations between planting (be Kabat etc. " Sequences of proteins of Immunological Interest) " (the 5th edition, 1991, National Institutes of Health, Bethesda MD)) and (2) based on method (Chothia etc. (1989) the Nature 342:877 of the Crystallographic Study of antigen-antibody complexes; Al-lazikani etc. (1997) J.Molec.Bioi.273:927-948)).As used herein, CDR can refer to the CDR by arbitrary method or two kinds of method combination definition.

In another embodiment, anti-ngf antibodies contains one or more CDR (as 1,2,3,4,5 CDR, perhaps in some embodiments, containing all 6 CDR of E3) of antibody E3.Determining of CDR district is in the art technology scope.CDR can be Kabat, Chothia, perhaps the combination of Kabat and Chothia.

Be used for antibody of the present invention and can comprise that monoclonal antibody, polyclonal antibody, antibody fragment (as Fab, Fab ', F (ab ') 2, Fv, Fc etc.), chimeric antibody, bi-specific antibody, different any other of immunoglobulin molecules of puting together antibody, strand (scFv), its mutant, the fusion rotein that comprises antibody moiety, humanized antibody and containing required specific antigen recognition site modify configuration, comprise the glycosylation variant of antibody, the aminoacid sequence variant of antibody and the antibody of covalent modification.Antibody can be Mus, rat, the people's or any other source (comprising chimeric or humanized antibody).For the present invention, antibody is with the mode and the NGF reaction of the downstream pathway of inhibition NGF and/or the mediation of NGF signal transduction functionality.In one embodiment, antibody is people's antibody of one or more epi-positions on the identification people NGF.In another embodiment, antibody is the mice or the rat antibody of one or more epi-positions on the identification people NGF.In another embodiment, antibody recognition is selected from the one or more epi-positions on the NGF of primates, Rodents, dog, cat, horse and cattle.In another embodiment, described antibody contains modified constant region, as immunologic inertia, does not promptly cause the cracking of complement-mediated or does not stimulate the constant region of the cytotoxicity (ADCC) that relies on antibody.Can assess the ADCC activity with disclosed method in the U.S. Patent No. 5,500,362.In other embodiments, as Eur.J.Immunol. (1999) 29:2613-2624; PCT applies for No.PCT/GB99/01441; And/or the modification constant region of describing among the UK patent application No.9809951.8.

The binding affinity of anti-ngf antibodies and NGF (as hNGF) can arrive about 0.72nM to about 0.75nM, about 0.18 for about 0.01 to about 1Nm, about 0.05 to about 0.25nM, about 0.10 to about 0.80nM, about 0.15.In some embodiments, binding affinity is about 1pM, about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM or is higher than about 40pM.In one embodiment, binding affinity arrives about 22pM for about 2pM.In other embodiments, binding affinity is for being lower than about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM or about 50pM or about 10pM.In some embodiments, binding affinity is about 10nM.In other embodiments, binding affinity is for being lower than about 10nM.In other embodiments, the about 0.1nM of binding affinity or about 0.07nM.In other embodiments, binding affinity is for being lower than about 0.1nM or being lower than about 0.07nM.In other embodiments, binding affinity is that one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM or about 50pM are to one of about 2pM, about 5pM, about 10pM, about 15pM, about 20pM or about 40pM.In some embodiments, binding affinity is one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM or about 50pM, or is lower than about 50pM.In other embodiments, binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, one of about 20pM, about 40pM or is higher than about 40pM.

Mensuration antibody is to measure the segmental affinity of single function Fab of this antibody to a kind of method of the binding affinity of NGF.In order to obtain single function Fab fragment, can cut or recombinant expressed antibody (for example IgG) with papain.By surperficial plasmon resonance (BIAcore3000 ^TMSurface plasmon resonance (SPR) system BIAcore, INC, Piscaway NJ), can determine the segmental affinity of anti-NGF Fab of antibody.Can use N-ethyl-N '-(3-methylamino propyl group)-carbodiimide hydrochloric acid (EDC) and N-hydroxy-succinamide (NHS) activation CM5 chip according to supplier's explanation.People NGF can be diluted in the 10mM sodium acetate (pH4.0) and be expelled to activatory chip with the concentration of 0.005mg/mL.Between each chip channel, use different flowing times, can realize two kinds of antigen density scopes: be used for the 100-200 reacton (RU) of detailed dynamics research, be used for the 500-600RU of Screening test method.Can seal chip with ethanolamine.Regeneration research shown that the mixture of Pierce elution buffer (production number 21004, Pierce Biotechnology, Rockford IL) and 4M NaCl (2: 1) can effectively be removed bonded Fab and in 200 injections the activity of hNGF on the maintenance chip.HBS-EP buffer (0.01M HEPES, pH7.4,0.15 NaCl, 3mM EDTA, 0.005% surfactant P20) is as the running buffer of BIAcore algoscopy.With the serial dilution (K that 1.0-10x estimates that injected the Fab sample of 1 minute purification in 100 μ L/ minutes _D) and allow to reach 2 hours dissociate the time.By ELISA and/or SDS-PAGE electrophoresis, the Fab that uses concentration known (as determining by amino acid analysis) is as the proteinic concentration of standard test Fab.By using the BIAevaluation program with 1: 1 Langmuir (Langmuir) of data fitting combination model (Karlsson, R.Roos, H.Fagerstam, L.Petersson, B. (1994) .Methods Enzyology 6:99-110) obtain kinetics association rate (K simultaneously _On) and the speed (k that dissociates _Off).Can be with K _Off/ k _OnCalculated equilibrium dissociation constant (K _D) value.This scheme is applicable to determines the binding affinity of antibody to the NGF of any species, described NGF comprises the NGF (as mice NGF, rat NGF, primates NGF) of people NGF, another kind of vertebrates (being mammal in some embodiments), and the scheme that is used to use other neurotrophins, as relevant neurotrophin NT3, NT4/5 and/or BDNF.

In some embodiments, described antibodies people NGF, and not significantly in conjunction with the NGF from another invertebrate species (mammal in some embodiments).In some embodiments, the NGF antagonist is in conjunction with people NGF and one or more NGF from other invertebrate species (being mammal in some embodiments).In some embodiments, described antibodies NGF and not with significantly cross reaction of other neurotrophins (as relevant neurotrophin NT3, NT4/5 and/or BDNF).In some embodiments, described antibodies NGF and at least a other neurotrophins.In some embodiments, the NGF of described antibodies mammalian species (as horse or Canis familiaris L.), but not significantly in conjunction with the NGF of another mammalian species.

Epi-position can be continuous or discontinuous.In one embodiment, described antibodies be selected from as Hongo etc., Mab911, MAb912 and the essentially identical hNGF epi-position of MAb 938 antibody described among the Hybridoma, 19:215-227 (2000).In another embodiment, described antibody and MAb 911 are in conjunction with substantially the same hNGF epi-position.Also in one embodiment, described antibody and MAb 909 are in conjunction with substantially the same hNGF epi-position (Hongo etc., preamble).For example, described epi-position can contain the K32 in the hNGF variable region 1 (23-35 amino acids), K34 and E35 residue, F79 and T81 residue in the hNGF variable region 4 (81-88 amino acids), H84 in the variable region 4 and K88 residue, R103 residue between hNGF variable region 5 (94-98 amino acids) and the hNGF C end (111-118 amino acids), E11 residue in the preceding variable region 1 of hNGF (10-23 amino acids), Y52 between hNGF variable region 2 (40-49 amino acids) and hNGF variable region 3 (the 59-66 amino acids), L112 and S113 residue in the hNGF C end, R59 in the hNGF variable region 3 and R69 residue, or the V18 in the preceding variable region 1 of hNGF, one or more in V20 and the G23 residue.In addition, epi-position can comprise variable region 1, variable region 3, variable region 4, variable region 5, N-end regions and/or the C end regions of one or more hNGF.In yet another embodiment, antibody significantly reduces the solvent accessibility of hNGF R103 residue.Though be to be understood that epi-position described above is relevant with people NGF, those of ordinary skill can be compared the structure of people NGF and other species NGF and identify the possible homologue of these epi-positions.

On the one hand, can suppress antibody (for example people's antibody, humanized antibody, mouse antibodies, chimeric antibody) the available expression total length of NGF or the immunogen preparing of part NGF sequence.On the other hand, can use the immunogen that comprised the expression of NGF cell.Spendable immunogenic another example is to comprise the NGF protein of total length NGF or the NGF protein of part.

Available any method known in the art prepares anti-ngf antibodies.As further described herein, the approach of immune host animal and scheme are consistent with the routine techniques of having set up that generally is used for the antibody stimulation and produce.The general technology that is used to produce people and mouse antibodies is known in the art and describes to some extent at this paper.

Expectation can be handled the basis that any mammalian subject of comprising the people or its antibody produced cell are used to produce mammal (comprising the people) hybridoma cell line.Usually, with a certain amount of immunogen (comprising immunogen as described herein) in intraperitoneal, intramuscular, per os, subcutaneous, vola and/or the intradermal vaccination host animal.

Hybridoma can be used Kohler, B. and Milstein, C. the general somatocyte hybriding technology of (1975) Nature 256:495-497 or as pass through Buck, D.W. etc., In Vitro, the improved somatocyte hybriding technology of 18:377-381 (1982) is from lymphocyte and immortalization myeloma cell preparation.In hybridization, can use obtainable myeloma cell line, include, but is not limited to X63-Ag8.653 and from the myeloma cell line at the cell distribution center of California, USA Santiago Salk institute.Substantially, this technology relates to fusion agent (as Polyethylene Glycol) or by well known to a person skilled in the art that the electric hand section merges myeloma cell and lymphoid cell.After the fusion, cell is grown in the growth medium (as trophicardyl-aminopterin-thymidine (HAT) culture medium) to remove the not parental cell of hybridization from merging the culture medium separation and selecting.Interpolation described herein or any culture medium of not adding serum can be used for cultivating the hybridoma of secrete monoclonal antibody.As another alternative approach of cell-fusion techniques, EBV immortalization B cell can be used for producing anti-NGF monoclonal antibody of the present invention.With hybridoma amplification and sub-clone, and pass through the anti-immunogen activity that routine immunization algoscopy step (for example radioimmunoassay, enzymoimmunoassay or fluorescence immunoassay) is measured supernatant as required.

The hybridoma that can be used as the antibody source comprises all parental generation hybridoma derivants, the daughter cell that produces NGF specific monoclonal antibody or its part.

Available known steps is grown in external or body and is produced the hybridoma of this antibody-like.As required, can pass through routine immunization globulin purification process (as ammonium sulfate, gel electrophoresis, dialysis, chromatography, ultrafiltration), in culture medium or body fluid, separate monoclonal antibody.If there is unwanted activity, can be by for example allowing prepared product flow through by adsorbent attached to the immunogen preparing on the solid phase, and with purpose antibody from immunogen eluting or release and remove undesirable activity.Personnel selection NGF or the fragment immunity host animal that comprises target amino acid sequence can produce antibody (for example monoclonal antibody) colony; described target amino acid sequence is conjugated with has immunogenic protein to the species with immunity; as keyhole maple hemocyanin; serum albumin; bovine thyroglobulin or soybean trypsin inhibitor; described puting together used difunctional dose or derivatization reagent, for example maleimide benzoyl sulfosuccinimide ester (puting together by cysteine residues); N-hydroxy-succinamide (passing through lysine residue); glutaraldehyde; succinic anhydride; SOCl ₂Or RIN=C=NR, wherein R and R1 are different alkyl.

As required, can be to purpose anti-ngf antibodies (monoclonal or polyclone) order-checking, the polynucleotide sequence of the anti-ngf antibodies polypeptide of will encoding then is cloned into and is used for the carrier of expressing or breeding.The sequence of coding purpose antibody can remain on the carrier in the host cell, and host cell and freezing so that use later on then can increase.In alternative, polynucleotide sequence can be used for genetic manipulation with " humanization " antibody or improve the affinity or the further feature of antibody.For example, constant region is transformed with similar human constant region, if to avoid antibody to be used for clinical trial and when treatment immunne response the people.May need the genetic manipulation antibody sequence to obtain bigger affinity and bigger inhibition NGF effect to NGF.Obviously, those skilled in the art can carry out one or more polynucleotide changes and still keep its binding ability to NGF anti-ngf antibodies.

Humanized monoclonal antibodies generally has four steps.They are: (1) determines the nucleotide of initial light chain of antibody and weight chain variable domain and the aminoacid sequence of prediction; (2) which antibody framework region design humanized antibody promptly determines to use in the humanization process; (3) Shi Ji humanization methodology/technology; And the transfection and the expression of (4) humanized antibody.See that for example U.S. Patent No. 4,816,567,5,807,715,5,866,692,6,331,415,5,530,101,5,693,761,5,693,762,5,585,089 and 6,180,370.

Described many " humanization " antibody molecules that comprise derived from the antigen binding site of non-human immunoglobulin, comprised the chimeric antibody of relevant complementary determining region (CDR) with rodent or modified rodent V zone and they and the fusion of people's constant domain.See Winter etc. for example, Nature 349:293-299 (1991); Lobuglio etc., Proc.Nat.Acad.Sci.USA 86:4220-4224 (1989); Shaw etc., J Immunol.138:4534-4538 (1987) and Brown etc., Cancer Res.47:3577-3583 (1987).Other list of references has been described before merging with suitable people's antibody constant region, and the rodent CDR of framework region (FR) is supported in grafting to the people.See Riechmann etc. for example, Nature 332:323-327 (1988); Verhoeyen etc., Science 239:1534-1536 (1988) and Jones etc., Nature 321:522-525 (1986).The rodent CDR that provides by recombinant modified rodent framework region has been provided another list of references.See that for example the european patent number publication No. 519,596.These " humanization " molecules are designed to minimize the harmful immunne response at the anti-human antibody molecules of rodent, and described replying limited these parts therapeutic is used in people receptor persistent period and effect.For example, but the engineered antibody constant region, and making it is immunologic inertia (for example not causing the complement cracking).See, for example PCT/GB99/01441; UK Patent Application No.9809951.8.Also the method for applicable other humanized antibody is by Daugherty etc., Nucl.AcidsRes.19:2471-2476 (1991) and U.S. Patent No. 6,180,377,6,054,297,5,997,867,5,866,692,6,210,671 and 6,350,861 and PCT publication No. WO01/27160 open.Additive method is described in U.S. sequence No.10/745,775.

In another alternative, but can obtain fully human antibodies by using through transforming the mice that obtains with the commercial sources of expressing special human normal immunoglobulin.The transgenic animal that design produces more desirable (for example fully human antibodies) or more potent immunne response also can be used for producing humanization or people's antibody.The example of this type of technology is from Abgenix, Inc. (Fremont, Xenomouse CA) ^TMAnd from Medarex, Inc. (Princeton, HuMAb-Mouse NJ) ^And TC Mouse ^

Although obviously top discussion relates to humanized antibody, the General Principle of being discussed can be applicable to for example antibody of Canis familiaris L., cat, primate, horse and cattle that is used for customized.Also obviously can make up one or more aspects of humanized antibody described herein, for example CDR transplanting, framework mutations and CDR sudden change.

In alternative, antibody can and be expressed with any method reorganization preparation known in the art.In another alternative, antibody can be by display technique of bacteriophage reorganization preparation.See that for example U.S. Patent No. 5,565,332,5,580,717,5,733,743,6,265,150, and Winter etc., Annu.Rev.Immunol.12:433-455 (1994).Alternatively, available display technique of bacteriophage (McCafferty etc., Nature 348:552-553 (1990)) is from immunoglobulin variable (V) domain gene repertoire external generation people's antibody or antibody fragment without immune donor.According to this technology, will be cloned in the main or less important coat protein gene of filobactivirus (as M13 or fd) in the antibody V domain gene frame, and as the functional antibodies fragment at the phage particle surface display.Because filamentous particle comprises the single stranded DNA copy of phage genome, based on the selection of antibody function characteristic also cause the encoding selection of the antibody gene that shows these characteristics.Therefore, phage has been imitated some characteristics of B cell.Phage display can carry out in a variety of forms; Summary sees, Johnson for example, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).The V genetic fragment of several sources can be used for phage display.Clackson etc., Nature 352:624-628 (1991) always hang oneself and have separated a collection of different anti- oxazolone antibody in the little combinatorial library at random of V gene of immune mouse spleen.Can make up from the V gene repertoire of not immune people's donor and according to Mark etc., J.Mol.Biol.222:581-597 (1991) or Griffith etc., EMBO J.12:725-734 (1993) technology of describing separate basically at the antibody of synantigen (comprising autoantigen) not.In the natural immunity was replied, antibody gene was with two-forty accumulation sudden change (somatic hypermutation).The variation of some importings will be given higher affinity, and preferably duplicate and break up the B cell of demonstration high-affinity surface immunoglobulin in antigen is attacked subsequently.This natural process can be by using the technical modelling (Marks etc., Bio/Technol.10:779-783 (1992)) that is called " chain reorganization ".In the method, the affinity of " originally " people antibody that obtains by phage display can exist variant (repertoire) repertoire to replace heavy chain successively and light chain V regional gene improves by using available from the V domain gene of immune donor not natural.This technology can produce antibody and the antibody fragment of affinity in the pM-nM scope.Waterhouse etc., Nucl.Acids Res.21:2265-2266 (1993) have described the strategy of the very big phage antibody repertoire of preparation (being also referred to as " mothers in all libraries ").Gene reorganization also can be used for from the rodent animal antibody people's antibody of deriving, and wherein people's antibody has similar affinity and specificity to initial rodent animal antibody.According to the method that is also referred to as " the epi-position marking ", the repertoire of personnel selection V domain gene replaces the heavy chain or the light chain V domain gene of the rodent animal antibody that obtains by display technique of bacteriophage, produces rodent-people's chimera.Antigenic selection causes the people variable region of separation energy restore functionality antigen binding site, i.e. the selection of epi-position domination (marking) gametophyte.When repeating the method, obtain people's antibody (seeing the PCT patent application WO 9306213 that on April 1st, 1993 announced) for alternative residue rodent V domain.The conventional humanization that is different from the rodent animal antibody of transplanting by CDR, this technology provide the framework that do not have the rodent source or the fully human antibodies of CDR residue.Although obviously top discussion relates to humanized antibody, the General Principle of being discussed can be applicable to for example antibody of Canis familiaris L., cat, primate, horse and cattle that is used for customized.

Can be by at first from host animal separation antibody and antibody produced cell, obtain gene order and with gene order recombinant expressed antibody and the preparation antibody of recombinating in host cell (for example Chinese hamster ovary celI).Other available method is to express antibody sequence in plant (for example Nicotiana tabacum L.) or transgenic breast (transgenic milk).The method that is used at the recombinant expressed antibody of plant or Ruzhong is disclosed.See Peeters etc. for example, Vaccine 19:2756 (2001); Lonberg, N. and D.Huszar Int.Rev.Immunol 13:65 (1995); And Pollock etc., J Immunol Methods 231:147 (1999).The method for preparing antibody derivatives (for example humanized antibody, single-chain antibody etc.) is known in the art.

Immunoassay and fluidic cell sorting technology such as fluorescence activated cell sorting (FACS) also can be used for separating the antibody special to NGF.

Antibody can with many different carrier combinations.Carrier can be active and/or inert.The example of known carrier comprises polypropylene, polystyrene, polyethylene, glucosan, nylon, amylase, glass, natural and cellulose, polyacrylamide, agarose and the magnet modified.According to purpose of the present invention, the character of carrier can be solubilized or does not dissolve.Those skilled in the art will know that other suitable carrier of binding antibody or other suitable carrier that the enough normal experiments of energy are determined binding antibody.

With conventional method (for example, can specific bond coding monoclonal antibody heavy chain and the oligonucleotide probe of the light chain gene) DNA of monoclonal antibody of encoding that is easy to separate and check order by using.Hybridoma is as the preferred source of this type of DNA.In case separate, DNA can be placed expression vector, host cell is advanced in transfection then, as Bacillus coli cells, ape COS cell, Chinese hamster ovary (CHO) cell or do not produce the proteinic myeloma cell of immunoglobulin, with synthetic monoclonal antibody in described recombinant host cell.Also can modifying DNA, for example, by the coded sequence replacement homology Mus sequence (Morrison etc., Proc.Nat.Acad.Sci.81:6851 (1984)) of personnel selection heavy chain and light chain constant domain or by immunoglobulin coding sequence is connected with all or part coded sequence of NIg polypeptide.By that way, prepared " chimeric " or " heterozygosis " antibody with the anti-NGF monoclonal antibody of this paper binding specificity.As described herein, the DNA of coding anti-ngf antibodies antagonist (as Humanized anti-human NGF antagonist antibodies) can be used for sending and express the anti-ngf antibodies antagonist by required cell.This paper has further described the DNA delivery technique.

Can use method well known in the art to characterize anti-ngf antibodies.For example, a kind of method is to identify its bonded epi-position, i.e. " epitope mapping ".This area has many methods to be used for epi-position position mapping on the protein and characterizes, comprise for example at Harlow and Lane, " Using Antibodies, aLaboratory Manual ", Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York, the crystal structure of solution antibody-antigenic complex of describing in the Chapter 11 of 1,999 one books, competition assay, gene fragment expression algoscopy and based on the algoscopy of synthetic peptide.In additional examples, epitope mapping can be used for determining the bonded sequence of anti-ngf antibodies.Epitope mapping can obtain from multiple commercial sources, for example Pepscan Systems (Edelhertweg 15,8219 PHLelystad, Holland).Epi-position can be linear epitope, promptly comprises a string aminoacid sequence, or the comformational epitope that forms by the amino acid whose three-dimensional interactions that not necessarily is contained in a string sequence.Separable or synthetic (for example being re-combined into) different length peptide (for example 4-6 amino acid long) at least also is used to use the binding assay of anti-ngf antibodies.In another example, the bonded epi-position of anti-ngf antibodies can be in screening system be determined by using from the overlapping peptide of NGF sequence and the combination of measuring anti-ngf antibodies.According to the gene fragment expression algoscopy, the open reading-frame of coding NGF makes up the also reactivity of mensuration NGF fragment of expressing and the antibody of desiring to detect of fragmentation at random or by special heredity.For example, genetic fragment can produce by PCR, and when having radioactivity aminoacid, external and be translated into protein.Combine with radioactive label NGF is segmental by immuno-precipitation or gel electrophoresis therapy determining antibody then.Also can identify some epi-position by the big library of peptide sequence at random (phage library) that is used in the phage particle surface display.Alternatively, in simple binding assay, test the segmental qualification of overlapping peptide library and tried combining of antibody.In other example, can carry out the mutation of antigen binding structural domain, domain exchange test and the third ammonia scanning mutagenesis to identify that epi-position is in conjunction with required, enough and/or essential residue.For example, available sudden change NGF carries out the domain exchange test, and wherein to be used for self-osculation relevant for NGF, but the sequence of the different protein (as another member of neurotrophin family) of antigenicity replaces the various fragments of (exchange) NGF polypeptide.By combining of assessment antibody and mutant NGF, can assess the bonded importance of specific NGF fragment antagonist.

The other method that can be used for characterizing anti-ngf antibodies is the competition assay with known other antibody in conjunction with same antigen (being that NGF goes up multiple fragment), to determine that whether anti-ngf antibodies is in conjunction with the epi-position identical with other antibody.Competition assay is known to those skilled in the art.As at Hongo etc., to describe among the Hybridoma 19:215-227 (2000), the antibody example that can use in competition assay of the present invention comprises MAb911,912,938.

Other NGF antagonisies

Can use the NGF antagonist outside the anti-ngf antibodies.In embodiment kinds more of the present invention, the NGF antagonist contains at least a antisense molecule, and it can block or reduce function NGF or function trkA and/or p75 receptor expression.The nucleotide sequence of NGF, trkA and p75 is known and obtains from the data base that can openly obtain easily.See Borsani etc. for example, Nuc.Acids Res.1990,18,4020; Accession number NM 002506; Ullrich etc., Nature 303:821-825 (1983).Prepare specific bond NGF, trkA or p75 mRNA routinely and not with the antisense oligonucleotide molecule of other polynucleotide cross reactions.The fixed representative site of target includes, but is not limited to start codon, 5 ' regulatory region, coding region and 3 ' untranslated region.In some embodiments, oligonucleotide length is about 10 to 100 nucleotide, about 15 to 50 nucleotide, is about 18 to 25 nucleotide, and is perhaps more.Oligonucleotide can contain backbone modifications, and the phosphorothioate bond of knowing in this area and 2 '-O is sugar-modified.See for example Agrawal and Zhao (1998), Antisense ﹠amp; Nucleic Acid Drug Development 8,135-139.Representative antisense molecule comprises the NGF antisense molecule of describing in U.S.'s publication No. 20010046959; Also referring to http://www.rna-tec.com/repair.htm.

Alternatively, use clpp gene subtraction, morpholino oligonucleotide, RNAi or ribozyme, method as known in the art can reduce the NGF expression and/or discharge.See Rossi for example, volumes such as J.J., " Intracellular Ribozyme Applications:Principles and Protocols ", Horizon Scientific Press (Duarte, CA, 1999); US 6,506, and 559, WO02/244321, WO 01/192513, WO 01/29058.

In other embodiments, the NGF antagonist comprises at least a NGF and suppresses chemical compound.Used herein, " NGF suppresses chemical compound " refers to be different from the chemical compound of anti-ngf antibodies, and it directly or indirectly reduces, suppresses, neutralizes or eliminates the NGF biological activity.NGF suppresses chemical compound should show any or multiple following feature: (a) in conjunction with NGF and the downstream pathway that suppresses the mediation of NGF biological activity and/or NGF signal transduction functionality; (b) any aspect of using treatment or prevent irritation is especially united with NSAID in any aspect of treatment or prevent irritation; (c) blocking-up or reduction NGF receptor activation (comprising trkA receptor dimerizationization and/or autophosphorylation); (d) removing of increase NGF; (e) suppressing (reduction) NGF suppresses, produces or discharge; (f) pain therapy of enhancing NSAID.Representative NGF suppresses chemical compound and comprises the micromolecule NGF inhibitor of describing in U.S.'s publication No. 20010046959; As inhibition NGF and the bonded chemical compound of describing among the PCT publication No. WO 00/69829 of p75; As inhibition NGF and the bonded chemical compound of describing among the PCT publication No. WO 98/17278 of TrkA/p75.The additional examples that NGF suppresses chemical compound comprises that the NGF that describes in PCT publication No. WO 02/17914, WO 02/20479, the U.S. Patent No. 5,342,942,6,127,401 and 6,359,130 suppresses chemical compound.It is the competitive inhibitor of NGF that other representative NGF suppress chemical compound.See U.S. Patent No. 6,291,247.In addition, those skilled in the art can prepare other micromolecule NGF and suppress chemical compound.

In some embodiments, NGF suppresses chemical compound in conjunction with NGF.Representative target (combination) site includes, but is not limited to the part in conjunction with those NGF of the correct 3D shape of the part of the NGF of TrkA receptor and/or p75 receptor and and part responsible receptor binding moiety adjacent with the receptors bind zone.In another embodiment, NGF suppresses chemical compound in conjunction with NGF receptor (as TrkA and/or p75) and suppress the NGF biological activity.Representative target site comprises those parts in conjunction with TrkA and/or the p75 of NGF.

In comprising micromolecular embodiment, micromolecule can have about 100 to 20,000 dalton, 500 to 15,000 dalton, perhaps one of 1000 to 10,000 dalton's molecular weight.Can obtain the micromolecule library by commercial sources.Can use micromolecule with arbitrary method as known in the art, described method comprises in suction, intraperitoneal, intravenous, intramuscular, subcutaneous, the sheath, in the ventricle, per os, through intestinal, parenteral, intranasal or percutaneous.In some embodiments, when the NGF antagonist is micromolecule, it will be divided into the ratio of 0.1 to 300mg/kg weight in patients 1 to 3 times or multidose is used.For the adult patients of normal type, can use every dose of 1mg to 5g.

In other embodiments, the NGF antagonist contains at least a NGF analog." NGF analog " refers to have similar three dimensional structure with the part of NGF and combine the chemical compound of NGF receptor under external or body physiological condition among the present invention.In one embodiment, the NGF analog is in conjunction with TrkA and/or p75 receptor.Two cyclic peptide of describing in two cyclic peptide that representative NGF analog includes, but is not limited to describe among the PCT publication No. WO 97/15593, the U.S. Patent No. 6,291,247; U.S. Patent No. 6,017, the cyclic compound of describing in 878; With the NGF derived peptide of describing among the PCT publication No. WO89/09225.Can be by the suitable NGF analog of the molecule Modeling and Design of NGF receptors bind, for example method by describing among the PCT publication No. WO98/06048.The NGF analog can be that the combination of arbitrary needs of identical or different structure is with the affinity that is improved and the monomer or the dimer/oligomer of biological effect.

In other embodiments, the invention provides the NGF antagonist of at least a dominant negative mutant that contains TrkA receptor and/or p75 receptor.Thereby thereby those skilled in the art can preparation example such as the dominant negative mutant this receptor of TrkA receptor will be in conjunction with NGF and as " receptor " of catching NGF.Yet dominant negative mutant will not have the normal biological activity of receptor during in conjunction with NGF.Representative dominant negative mutant includes, but is not limited to below with reference to the mutant of describing in the document: Li etc., and Proc.Natl.Acad.Sci.USA 1998,95, and 10884; Eide etc., J.Neurosci.1996,16,3123; Liu etc., J.Neurosci 1997,17, and 8749; Klein etc., Cell 1990,61, and 647; Valenzuela etc., Neuron 1993,10, and 963; Tsoulfas etc., Neuron 1993,10, and 975; With Lamballe etc., EMBO J.1993,12,3083, these lists of references are incorporated herein by reference in full by this paper.Dominant negative mutant can be used with protein form or the expression vector form that dominant negative mutant (for example Tu Bian TrkA receptor) is expressed in vivo.Can use described protein or expression vector with any method as known in the art, as in intraperitoneal, intravenous, intramuscular, subcutaneous, the sheath, in the ventricle, per os, use through intestinal, parenteral, intranasal, percutaneous or by suction.For example, using of expression vector comprises part or systemic administration, comprises that injection, dosage forms for oral administration, particle gun or intubate use and local application.In another embodiment, protein or expression vector directly are applied to sympathetic nerve or sensory trunk or neuroganglion.Those skilled in the art are familiar with using to obtain exogenous proteins expression in vivo of expression vector.See that for example U.S. Patent No. 6,436,908,6,413,942,6,376,471.

The therapeutic combination that can also targeted delivery contains antisense polynucleotides, expression vector or sub-gene group polynucleotide.Receptor-mediated DNA delivery technique is at for example Findeis etc., TrendsBiotechnol. (1993) 11:202; Chiou etc., Gene Therapeutics:Methods AndApplications Of Direct Gene Transfer (J.A.Wolff volume) (1994); Wu etc., J.Biol.Chem. (1988) 263:621; Wu etc., J.Biol.Chem. (1994) 269:542; Zenke etc., Proc.Natl.Acad.Sci. (USA) (1990) 87:3655; Wu etc. describe among J.Biol.Chem. (1991) 266:338 to some extent.Contain the therapeutic combination of 100ng of having an appointment with the local application of gene therapy scheme to the polynucleotide of about 200mg (or more) DNA.In some embodiments, can also use during the gene therapy scheme less than about 500ng, about 500ng is to about 50mg, and about 1 μ g is to about 2mg, and about 5 μ g are to about 500 μ g, and about 20 μ g are to about 100 μ g or higher DNA concentration range.Can use gene delivery vector to send therapeutic polynucleotide of the present invention and polypeptide.Gene delivery vector can be that virus or non-viral source (are generally seen Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; And Kaplitt, Nature Genetics (1994) 6:148).Can use endogenous mammal or allogeneic promoter and/or enhancer to induce the expression of these coded sequences.The expression of coded sequence can be composition or be regulated.

Be used for sending required polynucleotide and be to know in this area at the carrier that the purpose cell is expressed based on virus.Representative carrier based on virus includes, but is not limited to recombinant retrovirus (to be seen, PCT publication No. WO 90/07936 for example, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, U.S. Patent No. 5,219,740,4,777,127, British Patent No. 2,200,651 and European patent No.0345242), based on the carrier of Alphavirus (sindbis virus's carrier for example, Semliki forest virus (ATCCVR-67, ATCCVR-1247), ross river virus (Ross River virus) (ATCC VR-373, ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR1249, ATCC VR-532), and adeno associated virus (AAV) carrier (is seen, for example PCT publication No. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938, WO 95/11984 and WO 95/00655).Also can use as Curiel the DNA that is connected with the deactivation adenovirus that describes among Hum.Gene Ther. (1992) 3:147.

Also can use non-viral delivery vector and method, include, but is not limited to only be connected or unconnected polycation concentration of DNA (seeing Curiel for example, Hum.Gene Ther. (1992) 3:147) with the deactivation adenovirus; The DNA that part connects (seeing Wu for example, J.Biol.Chem. (1989) 264:16985); Eukaryotic cell delivery vector cell (see that for example U.S. Patent No. 5,814,482, PCT publication No. WO 95/07994, WO 96/17072, WO 95/30763 and WO 97/42338) and nuclear charge neutralization or merge with cell membrane.Also available naked DNA.Representative naked DNA introduction method is described in PCT publication No. WO 90/11092 and U.S. Patent No. 5,580,859 to some extent.The liposome that can be used as gene delivery vector is in U.S. Patent No. 5,422,120; Describe to some extent among PCT publication No. WO95/13796, WO 94/23697, WO 91/14445 and the EP patent No.0524968.Other method is at Philip, and Mol.Cell Biol. (1994) 14:2411 reaches at Woffendin, describes to some extent among Proc.Natl.Acad.Sci. (1994) 91:1581.

Obviously, expression vector also can be in order to instruct any expression based on proteinic NGF antagonist (for example anti-ngf antibodies, TrkA immunoadhesin or the like) described herein.For example, the polynucleotide of coding anti-ngf antibodies antagonist also can be used for sending and express the anti-ngf antibodies antagonist at the purpose cell.Obviously, expression vector can be used for instructing the expression of anti-ngf antibodies antagonist.This expression vector can intraperitoneal, in intravenous, intramuscular, subcutaneous, the sheath, in the ventricle, per os, use through intestinal, parenteral, intranasal, percutaneous or by suction.For example, using of expression vector comprises part or systemic administration, comprises injection, dosage forms for oral administration, particle gun or inserts to lead and use and local application.Further discuss as this paper, those skilled in the art are familiar with using to obtain exogenous proteins expression in vivo of expression vector.See that for example U.S. Patent No. 6,436,908,6,413,942,6,376,471.It is as known in the art can blocking (part is to blocking-up fully) NGF and/or bioactive other TrkA receptor fragments of NGF.

In another embodiment, the NGF antagonist contains at least a TrkA immunoadhesin.As used herein, the TrkA immunoadhesin refers to contain the ectodomain (perhaps its part) of TrkA receptor and the solubility chimeric molecule of immunoglobulin sequences, this molecule keep the TrkA receptor binding specificity (in some embodiments, keeping binding specificity basically) and can be in conjunction with NGF.As described herein, the TrkA immunoadhesin can be blocked (reducing and/or inhibition) NGF biological activity.

The TrkA immunoadhesin is as known in the art, and has been found that its blocking-up (reducing or inhibition) NGF combines with the TrkA receptor.See that for example U.S. Patent No. 6,153,189.In one embodiment, contain can be in conjunction with the fusion of TrkA receptor amino acid sequence (aminoacid sequence that perhaps keeps the binding specificity of TrkA receptor basically) and the immunoglobulin sequences (aminoacid that perhaps keeps the binding specificity of TrkA receptor basically) of NGF for the TrkA immunoadhesin.In some embodiments, TrkA receptor behaviour TrkA receptor sequence, and merge with immunoglobulin constant domain sequence.In other embodiments, immunoglobulin constant domain sequence is a heavy chain immunoglobulin constant domain sequence.In other embodiments, the combination of merging (for example, by disulfide bond covalently bound) of two kinds of TrkA receptor-immunoglobulin heavy chains causes homodimer immunoglobulin spline structure.Light chain immunoglobulin can also combine to produce homotrimer or homotype tetramer structure with one of TrkA receptor-immunoglobulin chimera in the dimer of disulfide-bonded or both.The example of suitable TrkA immunoadhesin comprises U.S. Patent No. 6,153, those that describe in 189.

In another embodiment, the NGF antagonist contains at least a anti-TrkA antibody, and its physics that can block, suppress, change and/or reduce NGF and TrkA receptor and/or downstream signal transduction interacts, thereby reduces and/or block the NGF biological activity.Anti-TrkA antibody is as known in the art.Representative anti-TrkA antibody comprises those anti-TrkA antibody of describing in PCT publication No. WO 97/21732, WO00/73344, WO 02/15924 and the U.S. Patent No. 20010046959.In another embodiment, the NGF antagonist comprises at least a anti-p75 antibody, and its physics that can block, suppress and/or reduce NGF and p75 receptor and/or downstream signal transduction interacts, thereby reduces and/or blocking-up NGF biological activity.

In another embodiment, the NGF antagonist comprises at least a inhibitors of kinases, and it can the inhibition downstream signal transduction of kinases relevant with TrkA and/or p75 receptor active.Representative inhibitors of kinases is K252a or k252b, and they are as known in the art and at Knusel etc., J.Neurochem.59:715-722 (1992); Knusel etc., J.Neurochemistry 57:955-962 (1991); Koizumi etc., J.Neuroscience 8:715-721 (1988); Hirata etc., ChemicalAbstracts 111:728 describes among the XP00204135 to some extent, see summary and " 12th CollectiveChemical Substance Index ", p.34237, (5-7 c.3,55-60,66-69), p.34238, c.1 (41-44), c.2 (25-27,32-33), p.3423, c.3 (48-50,52-53); U.S. Patent No. 6,306,849.

If the expection clinician seeks, the NGF antagonist of many other classifications will be identified.

The evaluation of NGF antagonist

Use method as known in the art can identify or characterize anti-ngf antibodies and other NGF antagonisies, thereby detect and/or the bioactive reduction of measuring N GF, improvement or neutralization.For example, U.S. Patent No. 5,766, the kinases receptors of describing in 863 and 5,891,650 activation (KIRA) algoscopy can be used for identifying anti-NGF agent.This ELISA type algoscopy is suitable for coming qualitative or quantitative measurement kinase activity by the autophosphorylation of the kinase domain of measuring receptor protein tyrosine kinases (hereinafter " rPTK ") (for example TrkA receptor), and the potential antagonist of identifying and characterize selected rPTK (for example TrkA).This algoscopy phase I comprises the phosphorylation of the kinase domain of kinases receptors (as the TrkA receptor), and wherein said receptor is present in the eukaryotic cell membrane.Receptor can be the nucleic acid of endogenous recipient or coding receptor, perhaps the receptor construct can be transformed into cell.Usually, first solid phase (for example hole of first assay plate) is by the homologous basically colony bag quilt of these cells (mammal cell line usually), thereby cell adhesion is to solid phase.Usually, thus cell is adhering first solid phase that adheres to naturally.If use " receptor construct ", it contains the fusion of kinases receptors and labeling polypeptide usually.In the ELISA of algoscopy part, be hunted down agent (being generally capture antibody) identification of labeling polypeptide.Then analyte (as candidate's anti-ngf antibodies or other NGF antagonisies) is added in the hole with adherent cell with NGF, thereby tyrosine kinase receptor (for example TrkA receptor) exposes (perhaps contact) in NGF and analyte.This algoscopy can be identified by TrkA part NGF and suppress its active antibody (perhaps other NGF antagonisies).After being exposed to NGF and analyte, with lysis buffer (it contains the solubilising detergent) adherent cell of dissolving and stirring gently, thereby discharge cell lysate, the ELISA part that it can be directly used in algoscopy does not need to concentrate or the clarification cell lysate.

The cell lysate that so prepares is easy to be used for the ELISA stage of algoscopy then.The first step as the ELISA stage, second solid phase (being generally the hole of ELISA titer plate) use with the trapping agent bag of tyrosine kinase receptor specific bond by or for the situation of receptor construct, use trapping agent (usually being capture antibody) bag quilt with the labeling polypeptide specific bond.The bag that carries out second solid phase sticks on second solid phase trapping agent.Trapping agent is generally monoclonal antibody, but as in this paper embodiment, describing also available polyclonal antibody.Then the cell lysate that obtains is exposed to (or contact) Adhesion catching agent, receptor or receptor construct are adhered on (or being captured in) second solid phase.Carry out rinse step then, to remove unconjugated cell lysate, to stay the receptor or the receptor construct of catching.Receptor that will adhere to or catch or receptor construct are exposed to (or contact) anti-phosphotyrosine antibody then, and it identifies phosphorylated tyrosine residue in the tyrosine kinase receptor.In one embodiment, the enzyme (directly or indirectly) of anti-phosphotyrosine antibody and catalysis on-radiation colour reagent change color is puted together.Therefore, can pass through the phosphorylation of the change color measurement receptor of reagent subsequently.Enzyme can directly be puted together with anti-phosphotyrosine antibody, or put together that molecule (biological example element) can be puted together with anti-phosphotyrosine antibody and enzyme can be subsequently by puting together molecule and anti-phosphotyrosine antibody is puted together.At last, measure by the change color of for example colour reagent, anti-phosphotyrosine antibody with catch combining of receptor or receptor construct.

Also can be: (a) combine NGF and suppress the NGF biological activity and/or by the downstream pathway of NGF semiotic function mediation by hatching candidate agent and NGF and monitoring following each or the anti-NGF antagonist of multinomial characterized; (b) blocking-up or reduction NGF receptor activation; (c) removing of increase NGF; (d) suppress NGF receptor activation (comprising TrkA dimerization and/or autophosphorylation); (e) treat, alleviate or any aspect of prevent irritation or osteoarthritis pain, particularly unite use with NSAID; (f) suppressing (reduction) NGF synthesizes, produces or discharge; (g) strengthen NSAID to treatment of pain.In some embodiments, identify anti-NGF antagonist by hatching candidate agent with bioactive reduction of NGF or neutralization that NGF and monitoring combine and/or follow.The cell of the NGF polypeptide of available purification or natural expression or transfection expression NGF polypeptide is implemented binding assay.In one embodiment, binding assay is the competitive binding assay method, wherein assesses candidate's antibody combines NGF with known NGF antagonist competition ability.This algoscopy can be implemented in a variety of forms, comprises the ELISA form.In other embodiments, by hatching candidate agent with NGF and monitor the trkA receptor dimerizationization that combines and follow and/or anti-NGF antagonist is identified in the inhibition of autophosphorylation.

After initial the evaluation, the activity of candidate's anti-ngf antibodies antagonist can further be determined to reach perfect by the directed bioactive bioassary method of known detection.Alternatively, available bioassary method is directly screened material standed for.For example, NGF promotes the discernible variation of many forms in responsive cell.Growth (Urfer etc., Biochem, 36:4775-4781 (1997) that these include, but is not limited to promote the differentiation of PC12 cell and strengthen neurite in these cells; Tsoulfas etc., Neuron10:975-990 (1993)), promote neurite to reply the explant of sensation and sympathetic ganglion certainly to outgrowth (Levi-Montalcini, R. and Angeletti, P.Nerve growth factor.Physiol.Rev.48:534-569,1968), and the neuron that promote to rely on NGF (as the neuronic survival of embryo's dorsal root ganglion, trigeminal ganglion or sympathetic ganglion (Chun ﹠amp for example; Patterson, Dev.Biol.75:705-711 (1977); Buchman ﹠amp; Davies, Development 118:989-1001 (1993)).Therefore, be used to suppress the bioactive algoscopy of NGF and need add analyte cultivation NGF responsive cell, described analyte such as candidate's anti-ngf antibodies and candidate NGF antagonist with NGF.After in due course, measure cell effect (cell differentiation, neurite are to outgrowth or cell survival).

Candidate NGF antagonist blocking-up or in and the bioactive ability of NGF also can suppress the ability of the survival of NGF mediation in the fetal rat dorsal root ganglion survival bioassary method of describing among the Hybridoma 19:215-227 (2000) and assess by monitoring candidate preparation at Hongo etc.Identify method description to some extent in PCT/US2004/01609 of NGF active regulator.

Compositions

Describe as the multiple embodiments of this paper, compositions of the present invention contains the NGF antagonist (as anti-ngf antibodies) and the NSAID of effective dose.In some embodiments, compositions also contains pharmaceutically acceptable excipient.In some embodiments, said composition is used for arbitrary method described herein (as the method for treatment postoperative pain).The example of these compositionss, and their compound method also chapters and sections and following this paper describe to some extent in front.NGF antagonist and NSAID can exist or exist as the compositions of separating with single compositions.Therefore, in some embodiments, NGF antagonist and NSAID are present in the same compositions.In other embodiments, NGF antagonist and NSAID are present in the compositions separately.

On the other hand, the invention provides the synergistic composition of NGF antagonist and NSAID.

In some embodiments, the invention provides the pharmaceutical composition that contains the NGF antagonist that is used for the treatment of pain (as postoperative pain), wherein said application comprises the while and/or uses NSAID successively.In some embodiments, the invention provides the pharmaceutical composition that contains NSAID that is used for the treatment of pain, wherein said application comprises the while and/or uses the NGF antagonist successively.In some embodiments, the invention provides pharmaceutical composition, it contains and is useful on separately, simultaneously and/or the NGF antagonist and the NSAID of sequential therapeutic pain.In some embodiments, the NGF antagonist is anti-ngf antibodies (antibody E3 as described herein).In other embodiments, NSAID is an ibuprofen.In other embodiments, the NGF antagonist is that anti-ngf antibodies and NSAID are ibuprofen.

Be to be understood that described compositions can contain more than one NGF antagonist.For example, compositions can contain more than one member's (for example discerning the mixture of the anti-ngf antibodies of the different epi-positions of NGF) of a class NGF antagonist, and the member of different classes of NGF antagonist (for example anti-ngf antibodies and NGF suppress chemical compound).Other representative compositions contain the identical epi-position of identification more than one anti-ngf antibodies, in conjunction with the anti-ngf antibodies of the different plant species of the different epi-positions of NGF, perhaps different NGF suppresses chemical compound.In other embodiments, described compositions contains one or more NGF antagonisies, and it is selected from the antagonist of transduceing in conjunction with the antagonist (for example antibody) of (physics interactions) NGF, in conjunction with the antagonist and reduction (hindering and/or blocking-up) the downstream NGF receptor signal of NGF receptor (as TrkA receptor or p75 receptor).

Be used for compositions of the present invention can also lyophilized formulations or the form of aqueous solution contain pharmaceutically suitable carrier, excipient or stabilizing agent (Remington: " The Science and Practice ofPharmacy " the 20th edition (2000), Lippincott Williams and Wilkins compile, K.E.Hoover).Acceptable carrier, excipient or stabilizing agent are nontoxic to the receiver under used dosage and concentration, and can contain buffer agent (as phosphoric acid, citric acid, with other organic acid), antioxidant (comprising ascorbic acid and methionine), antiseptic is (as stearyl dimethyl benzyl ammonium chloride, chlorination hexane diamine, benzalkonium chloride, benzene rope chloramines, phenol, butanols or benzylalcohol, alkyl paraben is as methyl parahydroxybenzoate or propyl p-hydroxybenzoate, catechol, resorcinol) Hexalin, the 3-amylalcohol, and metacresol), low-molecular-weight (about 10 residues are following) polypeptide, protein (as serum albumin), gelatin or immunoglobulin, hydrophilic polymer (as polyvinylpyrrolidone), aminoacid is (as glycine, glutamine, agedoite, histidine, arginine or lysine), monosaccharide, disaccharide and other carbohydrate (comprise glucose, mannose or dextrin), chelating agen (as EDTA), sugar is (as sucrose, mannitol, trehalose or Sorbitol), the salifiable equilibrium ion of shape (as sodium), composite metal (for example zinc-protein complex) and/or non-ionic surface active agent are (as TWEEN ^TM, PLURONICS ^TM) or Polyethylene Glycol (PEG).This paper is further describes pharmaceutically acceptable excipient.

Compositions described herein can contain the known extra chemical compound useful to pain therapy.The compositions of NGF antagonist and NSAID and they also can with in order to strengthen and/or other reagent of the effectiveness of additional reagent are united use.

In other embodiments, the invention provides compositions (being described in this paper), it is used for arbitrary method described herein, useful as drug and/or be used to make medicine.

Test kit

The present invention also provides the test kit that is used for the inventive method.Test kit of the present invention comprises one or more containers, and described container contains NGF antagonist (as anti-ngf antibodies), NSAID, and in some embodiments, described test kit also comprises the description of using according to arbitrary method described herein.In some embodiments, test kit contains anti-ngf antibodies (antibody E3 as described herein).In other embodiments, test kit contains anti-ngf antibodies, its comprise antibody E3 one or more CDR (as E3 1,2,3,4,5 or in some embodiments, be all 6 CDR).Test kit can also comprise about suffer from based on individuality whether have pain or whether this individuality be in the explanation of selecting the individuality that is suitable for treating in the danger of pain.In some embodiments, the invention provides the test kit that uses according to arbitrary method described herein, described test kit contains the NGF antagonist.In other embodiments, test kit contains anti-ngf antibodies.In other embodiments, test kit contains humanization anti-ngf antibodies (antibody E3 as described herein).In other embodiments, description comprises that co-administered NGF antagonist and NSAID are with treatment, the explanation that prevents and/or alleviate any pain (as postoperative pain, the pain relevant with burn, rheumatoid arthritis or osteoarthritis).

In some embodiments, test kit contains NGF antagonist (as anti-ngf antibodies), NSAID and about simultaneously and/or use NGF antagonist and the NSAID operation instructions with effective treatment pain successively.In another embodiment, this test kit contains NGF antagonist (as anti-ngf antibodies) and about co-administered NGF antagonist (as anti-ngf antibodies) and the NSAID description with effective treatment pain.In other embodiments, test kit contains NGF antagonist (as anti-ngf antibodies), NSAID (as ibuprofen) and about co-administered NGF antagonist and the NSAID operation instructions with effective treatment pain.Therefore, arbitrary method described herein can be reflected in the operation instructions.

In some embodiments, test kit contains anti-ngf antibodies.In other embodiments, anti-ngf antibodies is the antibody that contains variable region of light chain shown in variable region of heavy chain shown in the table 1 and the table 2.In other embodiments, anti-ngf antibodies is antibody E3 described herein.

NGF antagonist (as anti-ngf antibodies) and NSAID may reside in the disclosed or same container.Be to be understood that test kit can contain a kind of clear and definite compositions or two or more compositionss, wherein a kind of compositions contains the NGF antagonist, and a kind of compositions contains NSAID.

Test kit of the present invention is suitable for packing.Suitable packing includes, but is not limited to bottle, bottle, wide mouthed bottle (jar), resilient packing (for example Mi Feng mylar or plastic bag) etc.Test kit can be chosen the component that provides extra wantonly, as buffer agent and descriptive information.

Generally include the information of dosage, dosage regimen and route of administration about planned treatment about the operation instruction of NGF antagonist.Container can be unit dose, batch package (for example multiple-unit container) or subunit dosage.The operation instructions that provide in the test kit of the present invention are generally the explanation of writing on label or the package insert (for example being included in the paper in the test kit), but the also machine-readable explanation of acceptable (for example description of on magnetic or light storage disks, carrying).

Labelling or package insert point out that said composition is used for the treatment of, alleviates and/or prevent irritation (comprising postoperative pain).Can provide operation instructions to implement any method described herein.

Test kit of the present invention is suitable for packing.Suitable packing includes, but not limited to bottle, bottle, wide mouthed bottle, resilient packing (for example Mi Feng mylar or plastic bag) etc.Also imagine packaged combination and use specific device, as the packing of inhaler, nose application device (for example nebulizer) or device for casting (as Micropump).Test kit can have sterile access port (for example, container can for having the intravenous solution bag or the bottle of the stopper that hypodermic needle can pierce through).Container also can have sterile access port (for example, container can be intravenous solution bag or the bottle with stopper that hypodermic needle can pierce through).At least a activating agent is the NGF antagonist in the compositions, as anti-ngf antibodies.Container can also contain another kind of pharmaceutically active agents.

Test kit can randomly provide extra component, as buffer agent or explanatory information.Usually, the test kit label or the package insert that contain on container and the container or accompany with container.

In some embodiments, the invention provides the product of the inclusions that contains the mentioned reagent box.In some embodiments, test kit contains the information that NGF antagonist (as anti-ngf antibodies) and/or NSAID and indication are used for the treatment of pain (associating mutually).

NGF antagonist and NSAID use and the assessment to treating

Can NGF antagonist and NSAID be applied to individuality by any suitable approach.For example, they can be together or individually in per os, intravenous, Sublingual, subcutaneous, intra-arterial, intramuscular, the spinal column, in the per rectum, intrathoracic, intraperitoneal, ventricle, Sublingual, transdermal or use by suction.They can dosage forms for oral administration, for example uses with the dosage form of tablet, lozenge, capsule, elixir, suspending agent, syrup, wafer, chewing gum, lollipop, suppository or method well known in the art preparation.Those skilled in the art it is evident that embodiment described herein is not intended to restriction but illustrates utilizable technology.

Therefore, in some embodiments, according to known method individuality is used the NGF antagonist, (as anti-ngf antibodies), use as intravenous, for example, as fast injection or by transfusion continuously in a period of time, can pass through in intramuscular, intraperitoneal, brain keel, subcutaneous, intraarticular, the synovial membrane, sheath is interior, per os, suction or local approach.Nebulizer (comprising spray atomization device and ultrasound atomizer) by the available liquid preparation of commercial sources can be used for using.Liquid preparation can directly atomize, and freeze dried powder can atomize after the reconstruct.Alternatively, NGF antagonist (as anti-ngf antibodies) can and be with the atomizing of graduated dose inhaler through the fluorocarbon preparation, perhaps as the lyophilizing and the powder suction of milling.

Site-specific or targeted local delivery technique also can be used to use.The example of site-specific or targeted local delivery technique comprises the multiple implantable depot formulation source (depot source) of NGF antagonist and/or NSAID, perhaps local delivery conduit, as transfusion catheter, inherent conduit, perhaps pain relieving (PCA) technology of needle catheter, synthetic graft, adventitial wrap, part flow arrangement and stent device or other implantable devices, site-specific carrier, direct injection, use patient control or device and/or directly application.See, for example PCT publication No. WO 00/53211 and U.S. Patent No. 5,981,568.

Can use the several formulations of reagent (the NGF antagonist is as anti-ngf antibodies or its fragment).In some embodiments, reagent such as anti-ngf antibodies or its fragment can directly be used.In some embodiments, comprise that the described reagent of anti-ngf antibodies can be several formulations, comprise the preparation that contains pharmaceutically acceptable excipient.Pharmaceutically acceptable excipient is as known in the art, and is the material of being convenient to the relative inertness that pharmaceutically active substance uses.For example, excipient can be given shape or denseness, perhaps as diluent.Suitable excipient (including but not limited to) stabilizing agent, wetting agent and emulsifying agent, the salt that is used to change Morie osmolarity, capsule agent, buffer agent and skin penetration enhancer.Excipient and be used for parenteral and excipient that the outer medicine of parenteral is sent " TheScience and Practice of Pharmacy " such as Remington the 20th edition, propose among the Mack Publishing (2000).

In some embodiments, prepare these reagent (NGF antagonist) to use by injection (for example intraperitoneal, intravenous, subcutaneous, intramuscular or the like).Therefore, these reagent can make up as (saline, Ringer solution, dextrose solution or the like) with pharmaceutically suitable carrier.Concrete dosage regimen, i.e. dosage, selection of time and repeat to depend on concrete individual and individual medical history.Dosage regimen (comprising used NGF antagonist) can change in time.

Can be with any suitable method, comprise by injection (for example intraperitoneal, intravenous, subcutaneous, intramuscular or the like) and use anti-ngf antibodies.Describe as this paper, can also use anti-ngf antibodies by suction.Usually, for using of anti-ngf antibodies, initial candidate's dosage can be about 0.2mg/kg or about 2mg/kg.In some embodiments, depend on factor above-mentioned, daily dose commonly used can be for about 3 μ g/kg to 30 μ g/kg to 300 μ g/kg to 3mg/kg to 30mg/kg to 100mg/kg or above any.For using repeatedly of a couple of days or longer time, according to condition, continued treatment is up to the generation desirable inhibition of disease symptoms or up to enough treatment levels of having realized easing the pain.Representative dosage regimen comprises the initial dosage of using about 2mg/kg, the anti-ngf antibodies of about 1mg/kg maintenance dose weekly then, the perhaps maintenance dose of about 1mg/kg week about.Yet, also can use other dosages, this depends on the pattern of the pharmacokinetics decay that the doctor wishes to realize.For example, imagine administration weekly 1-4 time.Other dosage regimens comprise maximum 1 time of every day, 1 to 4 time weekly, and perhaps lower frequency.In some embodiments, about weekly once, every month about 1 to 4 administered compound.The dosage of anti-ngf antibodies is described to some extent at this paper.Can easily monitor the progress of this treatment by routine techniques and algoscopy.

In some embodiments, when NGF antagonist of the present invention is not antibody, can be divided into 1 to 3 dose and uses, perhaps as disclosed herein using with 0.1 to 300mg/kg weight in patients ratio.In the patient of some normal types, can use about 0.3 to 5.00mg/kg dosage.Concrete dosage, i.e. dosage, selection of time and will depend on concrete individual and individual medical history repeatedly, and the character (as half life and other considerations as known in the art of this reagent) of indivedual reagent.

NSAID can use with the routine dose level up to this class analgesics.In some embodiments, NSAID uses with the level that reduces.The appropriate dosage level will depend on the analgesic effect of selected NSAID, but suitable usually level will be every day about 0.001 to 25mg/kg, every day about 0.005 to 10mg/kg, or every day about 0.05 to 1mg/kg, or lower.This chemical compound can with up to every day 6 times (perhaps more), every day 1 to 4 time scheme use, perhaps can frequency of administration lower.In some embodiments, the NSAID continuous administration is perhaps used (as utilizing PCA) very continually.

When as compositions single or that separate when co-administered, nerve growth factor antagonist is used with the ratio consistent with desirable effect performance with NSAID.In some embodiments, by weight, the ratio of nerve growth factor antagonist and NSAID is about 1: 1.In some embodiments, this ratio can be for about 0.001 than about 1 to about 1000 to about 1, arrives approximately 1 than about 1 to about 100 to about 0.01, or about 0.1 than about 1 to about 10 to about 1.Also imagine other ratios.

Be to be understood that to be used for the treatment of and will be not only become along with selected particular compound or compositions with the amount of required nerve growth factor antagonist of prevent irritation and NSAID, and along with the character of route of administration, the disease of being treated, with the process of patient's age and situation, treatment or stage and become, and will finally determine by the doctor in charge.For example, the optimal dose of NGF antagonist (as anti-ngf antibodies) will depend on used NGF antagonist (perhaps its compositions), pain to be treated type and seriousness, to use that reagent is used to prevent still be therapeutic purposes, former treatment, patient's clinical history and replying and the doctor in charge's decision this reagent.Usually, the clinician will use NGF antagonist (as anti-ngf antibodies), up to reaching the dosage of realizing desirable result.

Experience considers that (as half life) will help the dosage decision usually.For example compatible antibody with human immune system, this antibody of immune system attack that can be used to prolong the half life of antibody and prevent the host as humanized antibody or fully human antibodies.Can determine frequency of administration and adjust with therapeutic process, and frequency of administration usually (but be not must) based on treatment of pain and/or inhibition and/or alleviate and/or postpone.Alternatively, the sustained continuous release formulations of NGF antagonist and/or NSAID can suit.Realize that the several formulations and the device that continue to discharge are well known in the art.

In one embodiment, can in being used the individuality of the active reagent of inhibition NGF, determine NGF antagonist dosage according to experience with treatment pain by one or many.Individuality is given the reagent (for example anti-ngf antibodies) of the inhibition NGF of increment and unites NSAID.In order to assess the effect of treatment, can follow the tracks of the pain indicator.

The method according to this invention, depend on receptor for example physiological condition, use purpose and be treatment still prevention and known other factors of technical staff, can be continuously or intermittence use NGF antagonist and NSAID.Anti-NGF antagonist use can preselected period basically continuously or can be the dosage of a series of interruptions, described preselected period for example before the pain development, during or after; After reaching before; After reaching during this time; During reaching before; Or before the pain development, during and after.For example, can be in wound, cutting, wound, operation, and before may algesiogenic any other incident, between and/or use afterwards.

In some embodiments, can there be more than one NGF antagonisies (as antibody).Antagonist can be identical or different by this.Can there be at least a, different NGF antagonist more than at least two kinds, at least three kinds, at least four kinds, at least five kinds or five kinds.Substantially, these NGF antagonisies have not the complementary activity of adverse effect mutually.The NGF antagonist can be united use with other reagent of rendeing a service in order to enhancing and/or additional reagent.

The NSAID that can have in some embodiments, more than one.NSAID can be identical or different by this.Can there be at least a, different NSAID more than at least two kinds, at least three kinds, at least four kinds, at least five kinds or five kinds.Substantially, these NSAID have not the complementary activity of adverse effect mutually.NSAID can with in order to strengthen and/or other reagent of the effectiveness of additional reagent are united use.

By the antibody and optional pharmaceutically suitable carrier, excipient or stabilizing agent (the Remington:The Science and Practice of Pharmacy the 20th edition (2000) that will have required purity, MackPublishing (2000)) mix, the treatment preparation for preparing NGF antagonist (as antibody) used according to the invention and NSAID with lyophilized formulations or aqueous solution form is to preserve.Acceptable carrier, excipient or stabilizing agent are nontoxic to the receiver under used dosage and concentration, and can contain buffer agent (as phosphoric acid, citric acid, with other organic acid), salt (as sodium chloride), antioxidant (comprising ascorbic acid and methionine), antiseptic is (as stearyl dimethyl benzyl ammonium chloride, chlorination hexane diamine, benzalkonium chloride, benzene rope chloramines, phenol, butanols or benzylalcohol, alkyl paraben is as methyl parahydroxybenzoate or propyl p-hydroxybenzoate, catechol, resorcinol, Hexalin, the 3-amylalcohol, and metacresol), low-molecular-weight (about 10 residues are following) polypeptide, protein (as serum albumin), gelatin or immunoglobulin, hydrophilic polymer (as polyvinylpyrrolidone), aminoacid is (as glycine, glutamine, agedoite, histidine, arginine or lysine), monosaccharide, disaccharide and other carbohydrate (comprise glucose, mannose or dextrin), chelating agen (as EDTA), sugar is (as sucrose, mannitol, trehalose or Sorbitol), the salifiable equilibrium ion of shape (as sodium), composite metal (for example zinc-protein complex) and/or non-ionic surface active agent are (as TWEEN ^TM, PLURONICS ^TM) or Polyethylene Glycol (PEG).

By methods known in the art, as at Epstein etc., Proc.Natl.Acad.Sci.USA82:3688 (1985); Hwang etc., the method for describing in Proc.Natl Acad.Sci.USA 77:4030 (1980) and the U.S. Patent No. 4,485,045 and 4,544,545 preparation comprises the liposome of anti-NGF antagonist (as antibody).Have the liposome that strengthens circulation time and be disclosed in U.S. Patent No. 5,013,556.Can produce useful especially liposome by the lipid composite that anti-phase evaporation comprises the deutero-PHOSPHATIDYL ETHANOLAMINE of phosphatidylcholine, cholesterol and PEG-(PEG-PE).Liposome is extruded to produce the liposome of purpose diameter by the filter membrane of determining the aperture.

Active component also can be captured in the microcapsule of preparation, for example by condensation technique or by interfacial polymerization, for example respectively in colloid drug delivery system (for example liposome, albumin microsphere spheroid, microemulsion, nano-particle and Nano capsule) or the hydroxy methocel in microemulsion or gelatin microcapsule and poly-(methyl methacrylate) microcapsule.This type of technology is disclosed in Remington, and The Science andPractice of Pharmacy the 20th edition is among the Mack Publishing (2000).

Can prepare extended release preparation.The suitable example of extended release preparation comprises the semi-permeable substrate of the solid hydrophobic polymer that comprises antibody, and described substrate is for there being shaped objects (for example film or little former) form.The example that continues release matrix comprises polyester, hydrogel (for example poly-(2-hydroxyethyl meth acrylate) or poly-(vinyl alcohol)), polyactide (U.S. Patent No. 3,773,919), the copolymer of L-glutamic acid and 7-ethyl-L-glutamic acid, nondegradable acetyl ethane-acetic acid ethyenyl ester, degradable lactic acid-ethanol copolymer such as LUPRON DEPOT ^TM(by lactic acid alcohol acid copolymer and the bright Injectable microspheres body of forming than Rayleigh (leuprolide acetate) of acetic acid), sucrose acetate isobutyrate salt and poly--D-(-)-3-hydroxybutyric acid.

The preparation that is used for using in the body must be for aseptic.This is easy to finish by for example aseptic membrane filtration.Place container therapeutic NGF antagonist (as the anti-ngf antibodies) compositions, for example intravenous solution bag or have in the bottle that plug that hypodermic needle can pierce through gives with aseptic access port.

Can be unit dosage forms according to compositions of the present invention,, be used for per os, parenteral or rectal administration, or use by sucking or being blown into as tablet, pill, capsule, powder agent, granule, solution or suspending agent or suppository.

For preparation solid composite (as tablet), mix main active and pharmaceutical carrier, for example conventional tablet composition (as corn starch, lactose, sucrose, Sorbitol, Talcum, stearic acid, magnesium stearate, dicalcium phosphate or natural gum) and other medicines diluent (for example water) comprise solid composite before the prescription of the homogenous mixts of The compounds of this invention or its avirulence officinal salt with formation.Compositionss are when being homogeneity before referring to these prescriptions, the expression active component in compositions homodisperse so that compositions can be easy to be further divided into equivalent effective unit dosage form, as tablet, pill and capsule.Solid composite is further divided into the unit dosage forms that comprises the above-mentioned type of about 0.01mg to about 0.1mg to about 500mg active component of the present invention before this prescription then.Can the tablet or the pill of new compositions be carried out coating or otherwise carry out compound so that the dosage form with prolongation effect advantage to be provided.For example, tablet or pill can comprise internal dose and outside dosage component, and the external dose component is the peplos of interior dosage component.Two kinds of components can be by the intestinal layer separately, this intestinal layer resist under one's belt decompose and allow in layer component complete enter duodenum or postpone discharge.Multiple material can be used for this type of intestinal layer or coating, and these materials comprise the mixtures of material of many polymeric acid and polymeric acid and Lac, spermol and cellulose acetate and so on.

Compositions wherein of the present invention can be mixed with per os or by the liquid form that injection is used and be comprised aqueous solution, suitably seasoned syrup, aqueous or oiliness suspending agent, with Emulsion with edible oil (as Oleum Gossypii semen, Oleum sesami, Oleum Cocois, Oleum Arachidis hypogaeae semen) seasoning, and elixir and similar pharmaceutical carrier.The suitable dispersion or the suspending agent that are used for water slurry comprise synthetic and natural gum (as tragakanta, Radix Acaciae senegalis), alginate, glucosan, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone or gelatin.Active component also can mix the controlled release product of high viscosity, as sucrose acetate isobutyrate or other.These preparations can be used for dosage forms for oral administration, perhaps injection.Injection can cause the part of medicine to be stored, its 1 day by 3 months in local release.

The compositions of using by injection comprises and contains NGF antagonist and NSAID as active component, and follows surfactant (perhaps wetting agent or surfactant) or be those compositionss of Emulsion form (as Water-In-Oil or oil in water emulsion).

Suitable surfactant comprises (particularly) nonionics, as polyoxyethylene sorbitol (Tween for example ^TM20,40,60,80 or 85) and other Sorbitol (Span for example ^TM20,40,60,80 or 85).Compositions with surface activity preparation advantageously comprises 0.05 to 5%, perhaps 0.1 to 2.5% surfactant.Should be appreciated that if necessary, can add other composition, for example mannitol or other pharmaceutically suitable carrier.

With passing through the obtainable lipomul of commercial sources, as Intralipid ^TM, Liposyn ^TM, Infonutrol ^TM, Lipofundin ^TMAnd Lipiphysan ^TMCan prepare suitable Emulsion.Active component be dissolvable in water the premixing emulsion composition or alternatively be dissolved in oil (for example soybean oil, safflower oil, Oleum Gossypii semen, Oleum sesami, Semen Maydis oil or almond oil) and the Emulsion based on mixed phospholipid (for example lecithin, soybean phospholipid or soybean lecithin) and water formation in.Be to be understood that to add other composition that for example glycerol or glucose are to adjust the tension force of Emulsion.Suitable Emulsion generally comprises the oil up to 20% (for example between 5 to 20%).Lipomul can comprise the fat drop of 0.1 to 1.0 μ m (especially 0.1 to 0.5 μ m), and its pH is in 5.5 to 8.0 scope.

In some embodiments, emulsion composition can be by mixed nerve growth factor antagonist and Intralipid ^TMOr the compositions of its component (soybean oil, lecithin, glycerol and water) preparation.

The compositions that is used for sucking or be blown into is included in solution and the suspension and the powder agent of pharmaceutically acceptable aqueous or organic solvent or its mixture.The liquid or solid compositions can comprise suitable as noted above pharmaceutically acceptable excipient.Compositions can be used by per os or nasal respiration approach, is used to produce part or whole body effect.Compositions in preferred aseptic acceptable solvent can atomize by using gases.Atomized soln can directly be breathed or atomising device is connected with face shield, tent or intermittent positive pressure breathing machine from atomising device.Solution, suspension or dust composition can be used (comprising per os or per nasal) from the device of delivery formulation in a suitable manner.

Therapeutic efficiency can be assessed by means commonly known in the art.

The following examples unrestricted the present invention in order to illustrate is provided.

Embodiment

Embodiment 1

Anti-NGF monoclonal antibody associating NSAID treatment postoperative pain

The effect that we unite NSAID (ibuprofen) with the pain model assessment anti-ngf antibodies of artificial hand's postoperative pain.For each experiment, before using be 16 male adult Sprague Dawley rat (Harlan of 200 to 200g with body weight; Indianapolis, IN) indoor food and water unrestrictedly fed at least 1 week under the normal illumination condition.After adapting to 2 hours before the operation in test chamber, rat is divided into two groups: perform the operation for one group and accepted antibody in preceding 15 hours, another group is accepted carrier (5% dextrose/0.45% saline USP) in this time.Use anti-ngf antibodies antagonist 911 (seeing Hongo etc., Hybridoma 19:215-227 (2000)) with the 1mg/kg body weight.Behind all animal surgeries 24 hours with 10,200 and multiple concentration (subcutaneous) administration of ibuprofen of 300mg/kg.

Operation is based on Brennan etc., the method that Pain 64:493-501 (1996) describes.With 2% isoflurane and AIR MIXTURES anesthetized animal, this mixture keeps at intra-operative by nose cone body (nose cone).Prepare the sole of the foot face of right back pawl with polyvinyl pyrrolidone-iodine pad, produce 1-cm central authorities longitudinal cut by skin and fascia, it begins up to toe from distance heel edge 0.5cm.Foot is fixed in the bending position measures with ruler.Sole of the foot flesh is raise with crooked tweezers and vertically cutting.Starting point by muscle is cut muscle to the complete degree of depth between inserting.Perioperative hemorrhage by gauze pad by pressure-controlled.With mattress suture wound closure (5-0 ethicon black monofilament).These are sewed up knotting 5-6 time, and first ties loose knotting.With bacitracin solution wiping wound location.Allow animal to recover and in clean cage, having a rest in preceding 22 hours of on-test of behavior.

For each experiment, animal is divided into two groups (contrast and antibody treatment).Used anti-ngf antibodies in preceding 15 hours in operation.The tranquillization pain (" baseline " in following figure) of assessments in back 22 hours in two groups of performing the operation.Performed the operation back 24 hours, with ibuprofen with 10,30,100 or 300mg/kg (subcutaneous) handle all animals.Ibuprofen is handled and was begun to assess tranquillization pain in back 1 hour.

Operation back different time is assessed tranquillization pain with accumulation pain score.Rat is placed the plastic wire (grid: 8mm of clean plastics cage ²) and allow to adapt to 15 minutes-20 minutes.Numerical range assessment behavior with 0 to 2.If the weight of animal is born at the claw (bleach or be pressed in online assessment by noticing this claw) through cutting, must be divided into 0 so.If claw is the contact skin net only, skin does not bleach or caves in, and must be divided into 1 so.If claw leaves net fully, then must be divided into 2.Observe every animal 1 minute every 5 minutes, continue 30 minutes altogether.The summation of observed 6 scores is used to assess and is cut sufficient pain in 1/2 hour.

These result of experiment show in table 1 and Fig. 1.

Table 1: perform the operation back 1 day with after 1mg/kg anti-ngf antibodies antagonist and 0,10mg/kg, 30mg/kg, 100mg/kg or the processing of 300mg/kg ibuprofen, the accumulation pain score of animal.Data show with meansigma methods (SEM).By the one way analysis of variance analytical data, it is right to utilize Prizm software to be used for the Bonferroni calibration analyte individuality of multiple comparisons then.

	Baseline	10mg/kg	30mg/kg	100mg/kg	300mg/kg
						Mab911	5.4(0.8)	3(0.87)	3.4(0.68)	N/D	N/D
Contrast	8.2(0.72)	7.3(0.73)	6.1(1.08)	5(0.82)	4.4(0.78)
							p＜0.001	p＜0.001	p＜0.05

As shown in table 1, Mab911 (1mg/kg) handle tranquillization pain score significantly be lower than contrast (p＜0.001) without ibuprofen.Similarly, the tranquillization pain score of 1mg/kg Mab911 and 10mg/kg ibuprofen processing significantly is lower than the score of only handling with the 10mg/kg ibuprofen (p＜0.001); The tranquillization pain score that 1mg/kg Mab911 and 30mg/kg ibuprofen are handled significantly is lower than the score of only handling with the 30mg/kg ibuprofen (p＜0.05).Fig. 1 provided with or without the 1mg/kg anti-ngf antibodies handle and with or the tranquillization pain score of the animal of ibuprofen processing that need not multiple dosage.Handle in reducing tranquillization pain than only with ibuprofen or only more effective with anti-ngf antibodies and ibuprofen before the operation with antibody treatment.To understand Mab911 (1mg/kg) and handle associating 10mg/kg ibuprofen at least with only to use the 300mg/kg ibuprofen the same effective.

In order to test effect,, just animal is used carrier or 5mg/kg diclofenac sodium replacement ibuprofen as above-mentioned enforcement experiment with anti-NGF monoclonal antibody 911 associating diclofenac sodium treatment postoperative pains.The result shows in Fig. 2.Compare with only handling, to observe average pain score with 1mg/kg and the processing of 5mg/kg diclofenac sodium and reduce with the 5mg/kg diclofenac sodium.

Although in order understanding clearly, to have described aforementioned invention in more detail, described description and embodiment should not to be interpreted as limitation of the scope of the invention by explanation and embodiment.

Claims (13)

1. treat the method for individual pain, it comprises anti-nerve growth factor (NGF) antibody and the NSAID that this individuality is used effective dose.

2. the process of claim 1 wherein that NSAID is selected from ibuprofen, naproxen, congex, diclofenac sodium, ketone ibuprofen, Tolmetin, slindac, mefenamic acid, meclofenamic acid, diflunisal, flufenisal, piroxim, thiophene oxygen thiazine, isoxicam, celecoxib, Luo Feikexi, DUP-697, flosulide, meloxicam, 6-methoxyl group-2-naphthalene acetic acid, MK-966, nabumetone, nimesulide, NS-398, SC-5766, SC-58215, T-614.

3. the process of claim 1 wherein that NSAID is an ibuprofen.

4. each method among the claim 1-3, wherein anti-ngf antibodies is in conjunction with people NGF.

5. the method for claim 4, wherein anti-ngf antibodies with about 10nM or the binding affinity that is lower than about 10nM in conjunction with people NGF.

6. the process of claim 1 wherein anti-ngf antibodies behaviour antibody.

7. the process of claim 1 wherein that anti-ngf antibodies is a humanized antibody.

8. the method for claim 7, wherein humanized antibody is the antibody that contains the variable region of light chain shown in variable region of heavy chain shown in the SEQ ID NO:1 and the SEQ ID NO:2.

9. the process of claim 1 wherein that pain is postoperative pain.

10. the method for claim 4, wherein pain is postoperative pain.

11. the method for claim 8, wherein pain is postoperative pain.

12. the pharmaceutical composition of treatment pain, it contains anti-ngf antibodies and NSAID and pharmaceutically suitable carrier of effective dose.

13. the test kit of treatment pain, it contains anti-ngf antibodies, NSAID and about co-administered anti-ngf antibodies and the NSAID description with treatment pain.

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