UA81299C2 - Method for treating pain by administering a nerve growth factor antagonist and an nsaid and composition containing thereof - Google Patents

Abstract

The present invention features methods for treating or prevailing pain comprising administering an amount of a nerve growth factor antagonist (such as an anti-NCF- 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

Description of the invention

This application claims priority over prior applications |US Patent No. 60/448823, filed Feb. 19, 2003, and 60/448853, filed Feb. 19, 2003|, which are fully incorporated herein by reference.

The invention relates to methods and compositions for the prevention or treatment of pain in a patient by introducing a combination of a nerve growth factor antagonist and NSAIDs3.

Currently, a number of treatment methods are recommended for pain relief, involving the administration of 70 non-steroidal anti-inflammatory drugs (NSAIDs3). Administration of NSAIDs has been shown to exhibit pain-relieving properties. However, there are known drawbacks to NSAID3 treatment, including unwanted side effects such as gastrointestinal and kidney irritation and liver toxicity. Moreover, NSAIDs cannot achieve adequate pain relief even in their maximum therapeutically acceptable doses in some painful conditions. 12 Nerve growth factor (NER) was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. It was shown that MORE is a crucial factor for survival and support during the development of peripheral sympathetic and embryonic sensory neurons and cholinergic neurons of the base of the forebrain.

Stgeomeu, ey a!., Seii! 76:1001-1011 (19943). MORE exerts a stimulatory regulation on the expression of neuropeptides in sensory neurons Ts(ipazau, ei aY., Mayte 337:362-364 (1989)), and its activity is mediated through 2 different receptors bound to the membrane, the TKA tyrosine kinase receptor and the p7 receptor /5, which is structurally related to other members of the tumor necrosis factor receptor family.

In addition to its effects on the nervous system, MOE has become increasingly involved in processes outside the nervous system. For example, it was shown that exogenously introduced MOG increases the permeability of blood vessels (ONep, ей ай, Ге)

Eshig. 9U. Rpaptasoi. 106:199-201 (1984)), enhances T- and B-cell immune responses (OMep, ei ai.,, Rhos. Mai.

Asai. All together. O.5.A. 86:10059-10063 (1989)), causes differentiation of lymphocytes and proliferation of mast cells and causes the release of soluble biological signals from mast cells (Maizida, ei ai., Rgos. Mai). Asad from

BOTH. 85:6508-6512 (1988); Reagse, ei a), .RPuvioi 372:379-393 (1986); Vizspoi, ei ai., Viosy Me. 30 79:2662-2669 (1992); Nogidote, ei ai., 9. Vioi Spet. 268:14881-14887 (19933). The SEA is produced by a number of Ge types! cells, including mast cells Ts(eop, ey aiY.,, Rgos. Mai. Asai. Zsi. O.5BA.A. 91:3739-3743 (1994),

B-lymphocytes (Togsia, ei ai., Sei! 85:345-356 (1996), keratinocytes (Oi Masgo, ei ai.,, U. Vioiyi. Spet. sch 268:22838-22846)), smooth muscle cells (euata, ei aiYi., 9. Nuregiepe. 11:1061-1065 (1993)), fibroblasts "--

Shepapoit, ey aiYi., Eig. 9. Meshghozsi. 2:795-801 (1990)), bronchial epithelial cells (Kazzei, ei ai., 35 ip. Ehr. IPegau 31:1432-40 (2001)), renal mesangial cells (Zieipeg, ei aiI, At. 3. RpNuvzio). so 261:g792-798 (1991))| and tubules of skeletal muscles (Zspmagil, ei a, U. RIioiospet. RpoiobrioI. U 66:195-200 (2002))І. MOR receptors have been found on various cell types outside the nervous system. For example, TKA has been found on monocytes, T- and B-lymphocytes, and human mast cells. "

The relationship between elevated MO levels and various inflammatory conditions has been observed in patients as well as in 70 several experimental animal models. These inflammatory conditions include systemic lupus erythematosus (SLE).

From" Meishgivsi Hek. 147:9-12 (1992)), psoriasis (Kauspatsanpygi, ei aiYi.,, Asia Yuegt. IepegeoYi. 78:84-86 (1998), arthritis Tsraisiti, eiig aiYi., App. KPpect. YuOiv. 55:745- 748 (1996)), interstitial cystitis | OKgadiuu, ей ай., 5.

Ohio 161:438-441 (1999))| and asthma IVgatsp, ei ai., Yeshig. In Ittipoi. 28:3240-3251 (1998). 45 Therefore, the increased level of MOE in peripheral tissues is associated with hyperalgesia and inflammation, and it has been observed in a number of forms of arthritis. The synovial membrane of patients affected by rheumatoid arthritis - expresses high levels of MOP, while it was reported that in the non-inflamed synovial membrane of MOE, C|AIoe, ei a), Agsy is not detected. KPesht. 35:351-355 (1992)). Similar results were observed in rats with experimentally induced rheumatoid arthritis. Ex. Kdeshitaiyo!. 10:203-204 (1992), (Te) 20 Elevated MOE levels were reported in arthritic transgenic mice along with an increase in the number of mast cells. 9. Tizvye Keasiope-Ehr. Siip. Azresiv 15:139-143 (1993)|. c There are 2 general categories of drug therapy for pain management, each working by different mechanisms and producing different effects, and both have disadvantages. The first category includes non-steroidal anti-inflammatory drugs (NSAIDs3), which are used to treat minor pain but whose therapeutic use is limited by adverse effects on the gastrointestinal tract, such as mucosal erosion

HF) stomach lining, formation of peptic ulcer or inflammation of the duodenum and colon and toxic effect on the kidneys with long-term use. The second category includes opioid analgesics, such as morphine, which are used to treat moderate to severe pain but whose therapeutic use is limited due to adverse effects such as constipation, nausea and vomiting, respiratory depression, psychiatric disorders, 60 renal colic, resistance to long-term use and the risk of addiction.

Clearly, there is a need for improved pain management that provides improved therapeutic benefit (eg, reduced intensity and/or frequency of pain) and/or reduces the incidence of unwanted side effects caused by many current treatment regimens.

All references in this description, including patent applications and publications, are incorporated by reference in their entirety.

The present invention is based on the discovery that MOPE antagonists are effective in the treatment of pain in combination with

NSAIDs3. Such therapy results in unexpectedly enhanced pain management. In addition, such therapy generally provides the possibility of using a reduced NSAID dosage to obtain the same amount of pain reduction and/or other forms of NSAID pain treatment enhancement3.

In a first aspect, the present invention features a method of treating (or in other embodiments preventing) pain comprising administering an amount of a nerve growth factor antagonist and an amount of

NSAIDs3 so that in combination they provide effective pain relief. The relative amounts and ratio of MOE antagonist and NSAID may vary. In some embodiments, an amount of 70 MOP antagonist is administered sufficient to allow for a reduction in the usual NSAID dose required to obtain the same degree of pain relief. In some embodiments, an amount of MOB antagonist is administered sufficient to provide a reduction in the usual dose of NSAID3 required to obtain the same degree of pain relief by at least about 596, at least about 1095, at least about 2095, at least , by about 3095, at least about y/5B By 4095, at least by about 5095, at least by about 6095, at least by about 70905, at least by about 8095, or at least by about 9095, or more. This reduction may occur in terms of the amount administered in a given administration and/or the amount administered during a given time period (reduced frequency).

In another aspect, the invention features methods of enhancing NSAID pain treatment involving the administration of an effective amount of an NSAID in combination with an effective amount of an MOR antagonist. As used herein, the term "combined administration" includes simultaneous administration and/or administration at different times. Administration in combination also includes administration in the form of a joint composition (ie, the MOE antagonist and the NSAID are present (combined) in the same composition) and/or administration in the form of separate compositions. As used herein, the term "administration in combination" encompasses any circumstance in which an NSAID and a MOG antagonist are ADMINISTERED in an effective amount to an individual. As discussed further in this description, it is clear that the antagonist

MOBs and NSAIDs can be administered with different frequency and/or administration intervals. For example, anti-MOG antibody i) can be administered 1 time/week, while NSAIDs can be administered more often. It is understood that the MOE antagonist and the NSAID can be administered by the same route of administration or by different routes of administration, and that the different routes of administration may change over the course of the administration(s). The introduction can take place before the onset of pain.

In another aspect, the invention provides methods for reducing the frequency of pain, alleviating pain, reducing pain and/or delaying the development or progression of pain in an individual, and said methods c involve the administration of an effective amount of a MOE antagonist in combination with an effective amount of NSAID3.

The methods of the invention are suitable for the treatment or prevention of any pain of any etiology, including -- pain for which the use of NSAIDs is generally prescribed3. In some embodiments, the pain is postoperative pain. In some embodiments, the pain is pain associated with a burn. In other embodiments, the pain is pain associated with rheumatoid arthritis. In other embodiments, the pain is pain associated with osteoarthritis.

Antagonist MOP, acceptable for use in the methods of the invention, is any agent that can "directly or indirectly lead to reduced biological activity of MOP. In some embodiments, the MOB antagonist binds (physically interacts with) a MOE (e.g., an antibody), binds to a MO receptor (such as the IiKA receptor and/or p75), and/or reduces (inhibits and/or blocks) downstream signaling from MOG receptors (for example, inhibitors of kinase signaling or other downstream signaling caused by MOPE in the target cell). In other embodiments, the MO antagonist inhibits (reduces) the synthesis and/or release of MOP. IN

In other embodiments, the MOG antagonist reduces the expression or function of TKA and/or p75 MORE receptors. IN

Go! in another embodiment, the MORE antagonist is a MORE antagonist that is not a TKA immunoadhesin (ie, different from a TKA immunoadhesin). In some embodiments, the MOE antagonist is selected from any one or more of the following: an anti-MOE antibody, an antisense molecule directed at a nucleic acid encoding MOG (including an antisense molecule directed at a nucleic acid encoding MOB), 5p an antisense molecule directed at MOP receptors (such as TKA mabo p75), a substance that inhibits MOB, and a structural analog of MOP, a dominant-negative mutation of the TKA receptor and/or p75 that binds MOE, an antibody

Ge) against TITKA, antibody against p75 and kinase inhibitor. In some embodiments, an MORG antagonist (such as an anti-MOR antibody) binds MORE (such as AMOB) and does not significantly bind to related neurotrophins such as MT-3, MT-4/5 and/or VOME. In another embodiment, the MORE antagonist is an antibody against two MORE. In still other embodiments, an anti-MORE antibody specifically binds to MORE. In still other embodiments, the anti-MOI antibody recognizes human MOP. In still other embodiments, the antibody is against MOG

F) specifically binds human MOB. In yet another embodiment, the antibody specifically binds the same MOB 6 epitope as an antibody selected from one or more of the following: Mar 911, Mab 912, and Mab 938 See. Nopodo, et al., Nubgidota 19:215-227 (2000) In still other embodiments, the anti-MOP antibody is humanized (including a humanized Mab 911, such as the EZ antibody described herein). In still other embodiments, the anti-MOB antibody is an EZ antibody (as described herein). In other embodiments, the anti-MORE antibody includes one or more SCO antibody EZ (eg, 1, 2, 3, 4, 5, or, in some embodiments, all 6 SKO with EZ). In other embodiments, the anti-MORE antibody is human. In still other embodiments, the anti-MORE antibody includes the amino acid sequence of the variable region of the heavy chain 65 shown in Table 1 (5EO IU MO:1) and the amino acid sequence of the variable region of the light chain shown in Table 2 (ZEO IU MO:2). In still other embodiments, the anti-MORE antibody includes the amino acid sequence of the heavy chain variable region shown in Table 1 (ZEO IO MO:1). In still other embodiments, the antibody against MOB includes the amino acid sequence of the variable region of the light chain shown in Table 2 (ZEO IO MO:2). In still other embodiments, the antibody includes

A modified constant region, such as a constant region that is immunologically inert, for example, does not trigger complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ACC). In other embodiments, the constant region is modified as described in |(Eig. .).

And so on. (1999) 29:2613-2624; PCT applications MoPCT/5899/01441; and/or in a UK patent application

Meo9809951. 81. 70 In some embodiments, the MOPE antagonist binds to the MOB molecule. In still other embodiments, the MORE antagonist is an antibody that specifically binds to MORE. However, the MOE antagonist can alternatively bind to the TKA receptor. The MOR antagonist can be a monoclonal antibody against human MOR (anti-pMOR), which is able to bind AMOE and effectively inhibit the binding of MOB to human TTKA (TKA).

The binding affinity of an anti-MORE antibody to a MORE (such as AIMORG) can be from about 0.1OnM to about 0.75Nm, from about 0.15Nm to about 0.75Nm, and from about 0.18nM to about 0.72nM. In one embodiment, the binding affinity is between about 2 pM and 22 pM. In some embodiments, the binding affinity is approximately 100 nm. In other embodiments, the binding affinity is less than about 1OnNM. In other embodiments, the binding affinity is about lm or about 0.07 nM. In other embodiments, the binding affinity is less than about 0 1nM or less than about 0.07nM. In other embodiments, the binding affinity is from about 100 pM, about 5 pM, about 1 pM, about 1 nM, about 500 pM, about 100 pM, or from about 5O0 pM to any of about 2 pM, about b5 pM, about 1 pM, about 15 pM, about 20 pM, about 40pKM or about 5O0pKM. In some embodiments, the binding affinity is any value from about 100 nM, about 5 nM, about 1 nM, about 1 nM, about 500 pkm, about 100 pM, or about 500 pM or less than about 5 pM. In some embodiments, the affinity of i) binding is less than any value of T00NM, about 5ONM, about 1ONM, about 1nM, about 5O00pM, about 100 pM, or about 5OpM. In still other embodiments, the binding affinity is about 2pM, about bpM, about 1OpM, about 15pM, about 20pM, Ge! of about 40pKM or more than about 40pKM. As is well known in the art, binding affinity can be expressed in terms of K p or dissociation constant, and increased binding affinity corresponds to Me reduced Ku . The binding affinity of the murine monoclonal antibody 911 against MSE |Nopoo eyi a!., Nubgidota p 19:215-227 (2000)) to human MOR is approximately 10M, and the binding affinity of the humanized antibody "- against MOE EZ (described in of this description) with human MORE is approximately 0.07 nM.

In cases where the MOPE antagonist is an antibody, the antibody may be an antibody fragment, including an antibody fragment selected from the group consisting of Bar, Rab, (ab)', RU, diatyl, single-chain antibody molecules, and multispecific antibodies. formed from antibody fragments, and a single-chain Em molecule (csEm). "

An NSAID can be any non-steroidal anti-inflammatory compound. NSAIDs are classified by their ability to inhibit cyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are the 2 main isoforms of cyclooxygenase, and the most standard NSAIDs are mixed inhibitors of both isoforms. The most standard NSAIDs3 belong to one of the following five structural categories: (1) propionic acid derivatives, such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid derivatives such as tolmetin and slindac; (3) fenamic acid derivatives such as mefenamic acid and meclofenamic acid; (4) derivatives of biphenylcarboxylic acid, such as diflunisal and flufenizal; and (5) oxicams such as piroxicam, sudoxicam, and

Me isoxicam. - another class of NSPZ was described, which selectively inhibit cyclooxygenase 2. SOC-2 inhibitors were described, for example, in | (US patents Momo 5616601; 5604260; 5593994; 5550142; ; 5436265; 5409944 and 5130311), all of which are incorporated herein by reference. Certain (se) 20 illustrative COX-2 inhibitors include celecoxib (5C-58635), rofecoxib, OVOR-697, flozulide (CsoR-28238), meloxicam, b-methoxy-2-naphthylacetic acid (6-MMA), MK-966 , nabumeton (prodrugs for 6-MMA), nimesulide, with Mm5-398, 50С-5766, 5С-58215, T-614; or combinations thereof.

In some embodiments, the invention provides methods in which aspirin/or acetaminophen is used in combination with a MOB antagonist (such as an anti-MOB antibody).

The MOE antagonist and/or NSAID can be administered to an individual by any suitable route. For example, their

GF) can be administered together or separately and/or simultaneously, and/or sequentially, orally, intravenously, sublingually, subcutaneously, intraarterially, rectally, into the spinal cord, intrathoracically, intraperitoneally, into the ventricles of the brain, sublingually, transdermally, or by inhalation.

Administration can be systemic, that is, intravenous or localized. 60 In the second aspect, this invention characterizes compositions that include a nerve growth factor antagonist and

NSAIDs3. The nerve growth factor antagonist and NSAIDs may be present together with one or more pharmaceutically acceptable carriers or excipients, or may be present in separate compositions. In another aspect, the present invention provides a synergistic composition of an antagonist of MORE and NSCLC3.

In the third aspect, the present invention characterizes a set for use in any of the ways disclosed in this description, and the specified set provides an antagonist of MOE and NSPZ3. The set may, in addition, include instructions for any of the methods described in this description. The instructions may include administration of an antagonist

A MOR (such as an anti-MOR antibody) in combination with an NSAID (ie, simultaneous administration and/or administration at different times). In some embodiments, the MORE antagonist and the NSAIDs are packaged together, but they may or may not be in the same container. Thus, in some embodiments, the kit includes a MOB antagonist and

NSAIDs3, present in the same container, and instructions for use in any of the ways described in this description. In other embodiments, the kit includes the MOE antagonist and NSPZ3Z in separate containers.

In other embodiments, the invention provides a method of treating pain in an individual, which involves administering to the individual an effective amount of an anti-nerve growth factor (NGF) antibody and an NSAID. In other embodiments, the NSAID is selected from the group consisting of ibuprofen, naproxen, naprosyn, diclofenac, ketoprofen, tolmetin, slindac, mefenamic acid, meclofenamic acid, diflunisal, flufenizal, piroxime, sudoxicam, isoxicam, celecoxib, rofecoxib, YUSHR-697, flozulide, meloxicam, b-methoxy-2-naphthylacetic acid, MK-966, nabumetone, nimesulid, M5-398, 5C-5766, 50-58215, T-614. In some embodiments, the NSAID is ibuprofen. In some embodiments, the anti-MOB antibody binds human MOB. In some embodiments, the anti-MORE antibody binds human MOB with a binding affinity of about 1OnM or less than about 1OnM. In some embodiments, the anti-MOG antibody is a human antibody. In some embodiments, the anti-MORE antibody is a humanized antibody. In some embodiments, the humanized antibody is an antibody comprising the heavy chain variable region shown in ZEO IU MO/1 and the light chain variable region shown in ZEO IU

MO:2. In some embodiments, the pain is postoperative pain.

In some embodiments, the invention provides a pharmaceutical composition for the treatment of pain comprising an effective amount of an anti-MOB antibody and an NSAID and a pharmaceutically acceptable carrier.

In some embodiments, the invention provides a kit for the treatment of pain, including an anti-MOB antibody, an NSAID, and instructions for administering an anti-MOB antibody in combination with an NSAID for the treatment of pain.

Figure 1 shows that the cumulative pain score is reduced in animals treated with i)

ZIbuprofen in a dose of 10 or ZOmg/kg in combination with an MOE antagonist (the antagonist against MOE Ma 911; see

Kopodo, ei ai., Nubrgidota 19:215-227 (2000)). The animals were divided into two groups (control and antibody-treated). The MOR antagonist was administered 15 hours before the operation, intraperitoneally (zu zo (time - -15 hours) at a dose of mg/kg. The operation was performed as described at time 0. Pain at rest was assessed 24 hours after the operation (0" on the graph) All animals were then treated with ibuprofen (30mg/ml in 45965 liquid b" beta-cyclodextrin) at a concentration of 1mg/kg or 3mg/kg of body weight. Animals that did not receive treatment with the antibody were also treated with ibuprofen at a dose of 1mg/kg, 3mg/kg /kg, 100mg/kg and 100mg/kg. Ibuprofen was injected subcutaneously into the back of the neck. Pain at rest was assessed 1 hour after administration of the ibuprofen dose. --

Treatment with an anti-MOE antagonist plus ibuprofen is more effective in reducing pain than treatment with either ibuprofen alone or an anti-MOE antagonist antibody alone.

Fig. 2 is a graph showing the cumulative pain score in animals treated with diclofenac at a dose of mg/kg in combination with a MORE antagonist (Mar 911 antibody against MOB antagonist; see

Kopoo, ei ai., Nubgidota 19:215-227 (200031). The animals were divided into two groups (control and those receiving

Treatment with antibodies). The MOR antagonist was administered 15 hours before the operation, intraperitoneally (time - 15 hours) in a dose of Tmg/kg. The operation was performed as described at time 0. Pain at rest was assessed 24 hours after . operations (0" on the graph). Then, all animals were treated with diclofenac at a dose of 5 mg/kg of body weight. Animals that did not?" were treated with an antibody, and were also treated with diclofenac at a dose of 5 mg/kg.Diclofenac was injected subcutaneously into the back of the neck.

Pain at rest was assessed 1 hour after diclofenac administration.

Go! Applicants have discovered that pain can be prevented or treated by administering an effective amount of a MOG antagonist (such as an anti-MORE antibody) in combination with an NSAID3. The methods and compositions of this invention can be used for the treatment or prevention of pain, including any pain for which the use of NSAIDs is generally prescribed. Using a nerve growth factor antagonist NSAIDs in combination, according to this invention, it is now possible to treat pain with a lower dose of NSAIDs, thereby reducing the likelihood of side effects associated with NSAID treatment3. In some embodiments, the implementation is introduced

Ge) an amount of MOR antagonist sufficient to provide the possibility of reducing the usual dose of NSAID required to obtain the same degree of pain relief effect by at least about 5960, at least about 1095, at least about 2095, at least about 3095, at least about 5095, at least about 6095, at least about 7095, at least about 9095, or more. (F) NSAID pain treatment can also be enhanced, as described herein, by administration of an NSAID in combination with a MOR antagonist.

In one aspect, the invention provides methods of treating or preventing pain in an individual (such as a mammal, both human and non-human) comprising administering an effective amount of a MOB antagonist in combination with an effective amount of an NSAID3. In another aspect, the invention provides methods of enhancing treatment or prevention

M5AO pain in an individual, which involves the administration of an effective amount of a MOB antagonist (such as an anti-MORE antibody) in combination with an effective amount of an NSAID3. In another aspect, the invention provides methods of preventing, alleviating and/or preventing the development or progression of pain. 65 In some embodiments, the anti-MOB antibody is capable of binding MOB and effectively inhibiting binding

MOB with its receptor TtgKsA or p75 ip mimo, or effectively inhibit the activation of MOPE of its receptor TKA or p75. In some embodiments, the binding affinity of the anti-MORE antibody is from about 0.01 to about 1.008M or from about 0.05nM to about 0.25nM. In other embodiments, the binding affinity is about 1pM, about 2pM, about 5pM, about 1OpM, about 15pM, about 20pM, about 5O0pM, about 100pM, or more. In one embodiment, the binding affinity is between about 2 pM and 22 pM. In some embodiments, the antibody binds essentially the same MOB epitope 6 as an antibody selected from one or more of the following: Ma 911, Mab 912, and Mab 938 |see.

Nopoo, ei ai., Nubgidota 19:215-227 (2000).

The antibody may also be an antibody fragment, such as an antibody fragment selected from one or more of the following fragments: ERab, Raf, E(ar)", Em, diatyl, single-chain antibody molecules and multispecific antibodies formed from antibody fragments, and single-chain molecule Em (csEm). The antibody can also be chimeric, and it can be humanized or human. The antibody can also be bispecific.

Illustrative NSAIDs that can be used in the present invention include, but are not limited to, (1) /5 derivatives of propionic acid, such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid derivatives such as tolmetin and slindac; (3) fenamic acid derivatives such as mefenamic acid and meclofenamic acid; (4) derivatives of biphenylcarboxylic acid, such as diflunisal and flufenizal; and (5) oxicams such as piroxicam, sudoxicam, and isoxicam. Another class of NSPS3 was described, which selectively inhibit cyclooxygenase 2. SOC-2 inhibitors were described, for example, in | (US patents Momo 5616601; 5604260; 5593994; 5950142; ; 5409944 and 5130311), all of which are incorporated herein by reference. Certain illustrative COX-2 inhibitors include celecoxib (5C-58635), rofecoxib, OSHR-697, flozulide (COR-28238), meloxicam, b-methoxy-2-naphthylacetic acid (6-MMA), MK-966, nabumetone (prodrugs for 6-MMA), nimesulide, M5-398, 50-5766, 50-58215, T-614; or combinations thereof. In some embodiments, aspirin and/or acetaminophen are used in combination with a MORE antagonist (such as an anti-MORE antibody).

The methods and compositions of this invention can be used to treat pain of any etiology, (8) including acute and chronic pain, any pain with an inflammatory component, and pain for which NSAIDs are commonly prescribed3. Examples of pain include postoperative pain, pain after surgery (including dental pain), migraine, headache and trigeminal neuralgia, pain associated with a burn, wound or kidney stone, pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with musculoskeletal disorders such as rheumatoid arthritis, Me) osteoarthritis, visceral pain, colitis, pancreatitis, gastritis, ankylosing spondylitis, sero-negative c (non-rheumatoid) arthropathies, non-articular rheumatism and peri-articular disorders and cancer-related pain (including "attack-like pain" and pain related to the terminal stages of cancer), peripheral neuropathy, --

With postherpetic neuralgia and pain associated with sickle cell crisis. Examples of pain with an inflammatory component (in addition to some of the types of pain described above) include rheumatic pain, pain associated with mucositis, and dysmenorrhea. In some embodiments, the methods and compositions of this invention are used to treat or prevent postoperative pain and/or cancer pain. In other embodiments, the pain is a pain indication for which NSAIDs are generally not prescribed, such as neuropathic pain. In other embodiments, the methods and compositions described herein are used to treat and/or prevent burn-related pain. In other variants of implementation. methods and compositions described in this description are used for the treatment and/or prevention of pain, and associated with rheumatoid arthritis. In other embodiments, the methods and compositions described herein are used to treat and/or prevent pain associated with osteoarthritis.

In another aspect, the invention provides compositions and kits for the treatment of pain comprising a MOG antagonist

Go! (such as an anti-MORE antibody) and NSCLC3, suitable for use in any of the methods described herein. - In the absence of other instructions, the practice of this invention should use the usual methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are within the capabilities of specialists in this field. Such techniques are fully explained in the literature, such as Imoyekshag siopipd: A Iarogaigu Mapia), zesopa eaiiop (ZatrgooK ei ai., 1989) Soia

Ge) Zrgipd Nagrog Rgevzv; Oiidopisieoyide 5upipeviz (M.9U. Saiy, ey. 1984); Meijodz ip Moiesshiag Vioiodu, Nytapa

Rgezv; Seii Vioiodu: A Iarogaiogu MoierookK (E. Seiiiv, ed.,, 1998) Asadetis Rgevzv; Apita! Se Siyige (K..

Egezppeu, eid., 1987); Ipigodisiop io Seyi apa Tivvtse SyMyge (OR MaShyeg apa R.E. Korbegiv, 1998) Review

Rgezv; Se apa Tivvtse SiMyige: Iarogaigu Rgosedigez (A. Yuouie, 9.V. giv, apa 0.0. Memei, eav., 1993-8) U. MPeu apa ope; Meiyodz ip Epgutoiodu (Asadetis Rgevzv, Ips.); Napdrsok oi Ekhregitepia | IttypoYodu (F) (O.M. Uueig apa S.S. ViasKuueyi, edv.); Sepe Tgapeieg Mesiogz og Mattaiyap SeiYiv ().M. MiMPeg apa M.R. Saiov, ka edz., 1987); Systepi Rgoiosoiiv ip Moiesshag Vioodu (H.M. A! Yezibrei ei aiYi., edv., 1987); RSK: Tpe Roiutegaze

Spaiip Keasiyop, (Miiiyiv ei ai., ev. 1994); Siptepi Rgoiosoiiv ip Ittypoiodu (9.E. Soiidap eii ai., edv., bo 1991); Zpog Rgoisoii ip Moiesshag Vioiyudu (UUpeu apa Zopv, 1999); Ittipobrioyudu (S.A. dapemau apa R.

Tgamege, 1997); Apiirodiev (R. Ripspy, 1997); Apiirodiev: a rgasiisai arrgoaspy (OO. Satsu., eid., IC Rgezv, 1988-1989); Moposiope! apiibodiev: a rgasiisai arrgoaspy (R. ZPperpega apa S Oeap, eadv.,, Ozhkhoga Opimegaiu

Rgezv, 2000); Ovipd apirodiev: and Iabogaiiu taptsua! (E. Nagomzh apa O. Gape (Soii Zrgipd Nagbrog Iarogaifiu

Rgezzv, 1999); Tne Apiirodiez (M. 7apetsi apa 9.0. Sarga, edv., Naglood Asadetis Rybiizpegv, 1995)). 65 Definition "Antibody" (used interchangeably in the plural form) is an immunoglobulin molecule,

able to specifically bind to a target, such as a hydrocarbon, polynucleotide, lipid, polypeptide, etc., with the help of at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used in this description, the term covers not only intact polyclonal or monoclonal antibodies, but also their fragments (such as Rab, Rar, K(ar)), Em, single chain (SEU), their mutants, hybrid proteins that include the antibody part, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (eg, bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the desired specificity.An antibody includes an antibody of any class, such as dO , IDA or DM (or its/o subclass), Its antibody does not have to be from any particular class. Depending on the amino acid sequence of the antibody constant domain of its heavy chains, immunoglobulins can be classified into different classes. There are 5 main classes of immunoglobulins: DA, 90, IDE, Id and IM, and several of them are possibly, in addition, divided into subclasses (isotypes), for example, IDDsi, Ids2, IdsZ, Id c4, IdAi! and IdDA2. The heavy chain constant domains, which correspond to different classes of immunoglobulins, are called alpha, delta, /5 epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. "Monoclonal antibody" refers to a homogeneous population of antibodies in which the monoclonal antibody is composed of amino acids (naturally occurring and artificially occurring) that participate in selective antigen binding. Monoclonal antibodies are highly specific, and they are directed against one antigenic site. The term "monoclonal antibody" covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Rab, Rab", K(ar)", Em,) single-chain (Zsgm), their mutants, hybrid proteins that include an antibody moiety, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the required specificity and ability to bind to an antigen. No limitation is intended as to the source of the antibody or the manner in which it is obtained (e.g. , hybridoma, with phage selection, recombinant expression, transgenic animals, etc.) "Humanized" antibodies refer to a molecule that has an antigen-binding site that is essentially i) devoid of immunoglobulin from non-human species, and a different structure immunoglobulin molecule based on the structure and/or sequence of a human immunoglobulin The antigen binding site may include either complete in variable domains, merged into constant domains, or only complementarity-determining regions (CDOs), Ge! zo are transplanted to the corresponding framework regions in the variable domains. Antigen binding sites can be wild-type or modified with one or more amino acid substitutions, for example, modified to closely resemble human immunoglobulin. Some forms of humanized antibodies retain all the sequences of c

SOC (e.g., a humanized murine antibody that contains all 6 SOCs from mouse antibodies). Other forms of humanized antibodies have one or more SOCs (1, 2, 3, 4, 5, 6) that are modified relative to the original antibody. --

In some cases, framework region (EC) residues or other human immunoglobulin residues are substituted with corresponding non-human residues. In addition, humanized antibodies may include residues that are not detected in the recipient antibody or in the donor antibody.

As used herein, the term "nerve growth factor" and "MORE" refers to nerve growth factor and its variants (including, for example, splicing variants and protein processing variants) that have at least some of the activity of MORE. The term "MOE" as used herein includes all species from the native MOE sequence of mammals, including humans, non-human primates, dogs, cats, horses, or cows. "MOR receptor" refers to a polypeptide that binds to or is activated by MOR. MOG receptors;" include the TKA receptor and the p75 receptor of any mammalian species, including but not limited to humans, dogs, cats, horses, primates, or cows. "MORE antagonist" refers to any molecule that blocks, inhibits, or reduces (including significantly)

Go! biological activity of MOB, including downstream pathways mediated by MOP signaling, such as binding to receptors and/or eliciting a cellular response to MOP. The term "antagonist" does not imply - any specific mechanism of biological action, and it is considered that it clearly includes and covers all possible co-pharmacological, physiological and biochemical interactions with MOR, both direct and indirect, or interacting with 5r MOB, by its receptor, or by some other mechanism, and its effects, which can be achieved by different and chemically disparate compositions.Illustrative MOR antagonists include, but are not limited to, an antibody

Ge) against MORE, an antisense molecule directed to MORE (including an antisense molecule directed to the nucleic acid encoding MORE), a compound that inhibits MORE, a structural analog of MORE, a dominant-negative mutation of the TKA receptor that binds MORE, and TKA immunoadhesin, an antibody against p/5, an antisense molecule directed at either or both TKA and/or p/5 receptors (including an antisense molecule directed at the nucleic acid encoding TIKA or p7b5) and a kinase inhibitor. For the purpose of this invention

F) it must be clearly understood that the term "antagonist" encompasses all previously identified terms and functional states and characteristics by which the MOB itself, the biological activity of the MORE (including but not limited to its ability to mediate any aspect of pain) or sequences of biological activities are essentially canceled, reduced or neutralized to any significant extent. In some embodiments, an MOR antagonist binds (physically interacts) with a MOR (e.g., an antibody), binds to a MOR receptor (such as the TKA receptor or p75 receptor), reduces (inhibits and/or blocks) downstream signaling of the MOR receptor, and/or inhibits (reduces) synthesis, production or release of MOP. In some embodiments, the MOG antagonist binds (physically interacts) with a MOP (e.g., an antibody), binds to a MOB receptor (such as the TITKA 65 receptor or the p7b5 receptor), and/or reduces (inhibits and/or blocks) downstream transmission MOP receptor signals. In another embodiment, the MORE antagonist binds to MORE and prevents TKA receptor dimerization and/or TKA autophosphorylation. In other embodiments, the MOB antagonist inhibits or reduces MOP synthesis. In other embodiments, the MORE antagonist inhibits or reduces synthesis and/or production (release). Examples of MOE antagonist types are provided in this description.

As used herein, the term "anti-MO antibody" refers to an antibody that is capable of binding to MOB and inhibiting the biological activity of MOP and/or the downstream pathway(s) mediated by MOP signaling. "Immunoadhesin TITKA" refers to a soluble chimeric molecule that includes a fragment of the TKA receptor, for example, the extracellular domain of the TKA receptor and an immunoglobulin sequence that retains the binding specificity of the TKA receptor. The "biological activity" of a MORE generally refers to the ability to bind MOR receptors and/or activate MOB receptor signaling pathways. Without limitation, biological activity includes any one or more of the following: the ability to bind MOPE receptors (such as p7Ub and/or TKA); the ability to promote dimerization and/or autophosphorylation of TKA receptors; the ability to activate signaling pathways /5 receptors of MOP; the ability to promote cellular differentiation, proliferation, survival, growth, and other changes in cellular physiology, including (in the case of neurons, including peripheral and central neurons) changes in neuronal morphology, synaptogenesis, synaptic function, release of neurotransmitters and/or neuropeptides, and regeneration after damage and the ability to mediate pain.

The term "epitope" is used to refer to binding sites for (monoclonal or polyclonal) antibodies on antigens, such as protein antigens.

The term "treatment" as used herein is an approach to obtain favorable or desired clinical results. For purposes of the present invention, favorable or desired clinical outcomes include, but are not limited to, one or more of the following: alleviation or relief of any aspect of pain, including acute, chronic, inflammatory, neuropathic, or postoperative pain. For the purposes of this invention, favorable or desired clinical outcomes include, but are not limited to, one or more of the following: including a reduction in severity, relief of one or more pain-related symptoms, including and) any aspect of pain (such as decrease in duration of pain and/or decrease in pain sensitivity or pain sensation). "Reducing the incidence" of pain means any reduction in the severity (which may include a reduction in the need for and/or amount (eg exposure) of other agents and/or treatments commonly used for these conditions, duration and/or frequency (including, for example, delaying or increasing the time to onset of pain in an individual). As will be understood by those skilled in the art, individuals may vary in their response to treatment, and also, for example, the manner in which pain occurs in of an individual" reflects the administration of an MOR antagonist described herein in combination with an NSAID, as --

335 is described herein, based on a reasonable expectation that such administration is likely to cause such a reduction in incidence in that particular individual. "Alleviation" of pain or one or more symptoms of pain means a reduction or amelioration of one or more symptoms of pain, compared to the absence of the administration of a MOB antagonist in combination with an NSAID3. "Relief" also includes shortening or decreasing the duration of the symptom. "Alleviating" pain or one or more symptoms of pain means reducing the degree of one or more undesirable clinical manifestations of pain in an individual or population of individuals treated with a MOB antagonist in combination with an NSAID according to the invention. ;" As used herein, the term "pain delay" means delaying, hindering, slowing, delaying, stabilizing, and/or delaying the progression of pain. This delay can be of varying duration, depending on the medical history and/or the individuals receiving the treatment. As is obvious

Go! to one skilled in the art, a sufficient or substantial delay may actually encompass prevention in that the individual does not develop pain. A method that "delays" the development of a symptom is a method that reduces - the probability of the development of a symptom in a given time frame and/or reduces the degree of symptoms in a given time frame, compared to the lack of application of the method. Such comparisons are usually based on clinical studies using a statistically significant number of individuals. "Development" or "progression" of pain refers to the initial manifestations and/or subsequent progression of the disorder. Development

Ge) pain can be detected and evaluated using standard clinical techniques that are well known in the art. However, development also refers to progression, which may be undetectable. For the purposes of this invention, development or progression refers to the biological course of symptoms. "Development" dv Includes emergence, relapse and onset. As used herein, the term "onset" or "occurrence" of pain includes initial onset and/or recurrence. (F) "Effective amount" is an amount sufficient to produce favorable or desired clinical results, including relief or reduction of pain. For purposes of the present invention, an effective amount of an MOR antagonist (such as an anti-MOR antibody) and an NSAID includes an amount sufficient to treat, relieve, reduce, or prevent pain (including pain perception and pain sensation) of any type, including acute, chronic , inflammatory, neuropathic or postoperative pain. In some embodiments, an effective amount of an NSAID and an MOR antagonist capable of modulating the threshold of sensitivity to external stimuli to a level comparable to that observed in healthy individuals. In other embodiments, this level may not be comparable to the level observed in healthy 65 individuals, but is reduced compared to individuals who did not receive the combination therapy. An effective amount of MOPE antagonist also includes an amount of MOPE antagonist sufficient to enhance pain treatment

NSAID3 (of therapeutic effect) as described herein, or to reduce the dose of NSAID3 required for the treatment or prevention of pain as described herein. According to art knowledge, the effective amount of a MOPE antagonist in combination with an NSAID may vary, among other things, depending on the type of pain (and patient history), as well as other factors such as the type (and/or dosage) or antagonist used MORE and/or NHS3). An effective amount in the context of this invention may also be such an amount of MOB antagonist and NSAID that synergism is achieved. An effective amount of an antagonist in the context of the present invention generally means an amount sufficient to result in an enhancement of the therapeutic effect of the NSAID33 administered for pain relief (which in turn means that a reduced 7/0 dose and/or any observed other beneficial effect) and/or lead to a beneficial effect, compared to treatment with one NSAID. An effective amount of an MOR antagonist may also result in a synergistic effect compared to administration with a single MOE or NSAID3 antagonist. The subject is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, domestic animals, primates, horses, cows, dogs, mice and rats.

The term "NSAID3" refers to a non-steroidal anti-inflammatory compound. NSAIDs are divided into categories depending on their ability to inhibit cyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are the two major cyclooxygenase isoforms, and most standard NSAIDs are mixed inhibitors of these two isoforms. Most standard NSAIDs fall into one of the following five structural categories: (1) 2o propionic acid derivatives such as ibuprofen, naproxen, naprosiv, diclofenac, and ketoprofen; (2) acetic acid derivatives such as tolmetin and slindac; (3) fenamic acid derivatives such as mefenamic acid and meclofenamic acid; (4) derivatives of biphenylcarboxylic acid, such as diflunisal and flufenizal; and (5) oxicams such as piroxicam, sudoxicam, and isoxicam.

Another class of NSPZ was described, which selectively inhibit cyclooxygenase 2. SOC-2 inhibitors were described, for example, in I (US patents Momo 5616601; 5604260; 5593994; 55550142; ; 5436265; 5409944 and 5130311), all of which are incorporated herein by reference. Certain (8) illustrative COX-2 inhibitors include celecoxib (5C-58635), rofecoxib, OVOR-697, flozulide (CcoR-28238), meloxicam, b-methoxy-2-naphthylacetic acid (6-MMA), MK-966, nabumetone (prodrugs for 6-MMA), nimesulide,

Mm5-398, 50S-5766, 5S-58215, T-614; or combinations thereof. Gee! In some embodiments, aspirin and/or acetaminophen are used in combination with a MOG antagonist (such as an anti-MORE antibody). Aspirin is another type of non-steroidal anti-inflammatory compound. Me

As used herein, the term "in combination" includes simultaneous administration and/or administration at different times. Administration in combination also includes administration in the form of a joint composition (ie, the MOPE antagonist and the NSAID are present (combined) in the same composition) and/or administration in the form of separate compositions. --

As used herein, the term "combination administration" is intended to encompass any circumstance in which an NSAID and a MOE antagonist are administered in an effective amount to an individual, which may occur simultaneously and/or separately. As discussed further in this description, it is clear that the MOE antagonist and the NSAID can be administered at different frequencies and/or intervals of administration. For example, an anti-MORE antibody can be administered once/week, while an NSAID can be administered more frequently. It is clear that the MOE antagonist and the NSAID can be administered by the same route of administration or by different routes of administration. in c. "Postoperative pain" (which is interchangeably called "pain after an incision" or "post-traumatic pain"). refers to pain that occurs or originates from an external injury, such as a cut, puncture, cut, tear, etc.? or a wound to an individual's tissue (including pain resulting from surgical procedures, whether invasive or non-invasive). The term "postoperative pain" as used herein does not include pain that

Occurs (appears or occurs) without external physical trauma. In some embodiments, postoperative pain is internal or external (including peripheral) pain, and the wound, cut, injury, laceration, or incision may occur accidentally (as in a traumatic wound) or intentionally (as in an operative incision). As used herein, the term "pain" includes the perception of pain feeling pain, and pain is possible

GI should be assessed objectively using pain scores and other methods well known in the field.

As used herein, the term "postoperative pain" includes allodynia (i.e., increased ic response (i.e., pain perception) in response to a normally non-painful stimulus) and hyperalgesia (i.e., increased

Ge reaction to a usually painful or unpleasant stimulus), which, in turn, can be thermal or mechanical (tactile) in nature. In some embodiments, the pain is characterized by thermal sensitivity, mechanical sensitivity, and/or pain at rest. In some embodiments, the implementation of postoperative pain

Includes mechanically induced pain or pain at rest. In other embodiments, postoperative pain includes pain at rest.

F) NSAID pain treatment is "intensified" when the aspect of NSAID treatment improves (compared to administration of NSAID without administration of MOR antagonist). For example, the effectiveness of NSAID treatment can be increased in the presence of a MOR antagonist relative to the effectiveness of NSAIDs in the absence of a MOR antagonist. As another example, the treatment or prevention of NSAID pain can be "enhanced" by the use of a MORE antagonist in combination with

NSAIDs when this use provides the possibility of better pain relief (for example, when a dose of NSAID3 is used that does not provide the possibility of effective treatment or prevention of pain).

With respect to all methods described herein, references to MOPE antagonists and NSAIDs also include compositions that include one or more of these agents. The present invention can be used to treat 65 pain in individuals, including all mammals, both human and non-human.

In one aspect, the invention provides methods of treating pain in an individual, comprising administering an effective amount of a MOB antagonist (such as an anti-MOP antibody) in combination with an effective amount of an NSAID3. In some embodiments, an amount of MOPE antagonist is administered sufficient to provide a reduction in the usual NSAID dose required to produce the same degree of pain relief by at least about 595, at least about 1095, at least about 2095, at least , at least about 3095, at least about 5095, at least about 6095, at least about 7095, at least about 8095, or at least about 9095, or more.

In another aspect, the invention provides methods of enhancing NSAID pain treatment in an individual comprising administering an effective amount of a MOPE antagonist in combination with an effective amount of an NSAID3.

In some embodiments, the pain includes one or more of the following: acute and/or chronic pain, any pain with an inflammatory component, postoperative pain (including dental pain), migraine, headache and trigeminal neuralgia, pain associated with burn, wound, or kidney stone pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with sickle cell crisis, pain associated with dysmenorrhea or bowel dysfunction, and pain associated with with cancer (including "pain attack" and pain associated with the terminal stages of cancer). In other embodiments, the pain is pain that is commonly treated with NSAIDs (such as ibuprofen). In other embodiments, the pain is associated with a burn. In other embodiments, the pain is associated with rheumatoid arthritis. In other embodiments, the pain is associated with osteoarthritis.

In another aspect, the invention provides methods of preventing, alleviating and/or preventing the development or progression of pain. Thus, in some embodiments, an MOR antagonist, such as an anti-MOB antibody, and/or

NSAIDs are administered before a painful event (such as surgery). For example, the MOE antagonist can be administered in 30 minutes, in 1 hour, in 5 hours, in 10 hours, in 15 hours, in 24 hours, or even more, for example, in 1 day, several days, or even 1 week, 2 weeks, With weeks or more before activities likely to cause pain or risk of causing pain, such as external trauma or surgery.

Treatment or prevention of pain is evaluated using methods well known in the art. Evaluation i) can be performed on the basis of an objective indicator, such as observation of behavior, such as reaction to stimuli, facial expressions, and the like. The assessment can also be based on subjective indicators, such as the patient's description of pain using various pain scales |(|see, for example, Kai? ei ai!., 5!igo Siip Ge!

My At. (1999) 79 (2):231-52; Sagasepi ey a). In Rat Zutiot Mapade (2002) 23(3):239-55).

The diagnosis and assessment of pain in rheumatoid arthritis is well established in the field. The evaluation can be performed on the basis of indicators known in the field, such as the patient's description of pain using various pain scales | see, for example, Kai ei aiYi., Z!igd Siip Moi At. (1999) 79 (2):231-52; Sagasepi ei ai. in

Raip Zuthiot Mapade (2002) 23 (3): 239-55). There are also scales for measuring the pathological state, which are commonly (87 zv) USED, such as (She Ategisap SoPede oi KPeitaiyuiodu (ASK) (Reivop, ei ai., Agiigyiv apa so

КПпеитаиизт (1993) 36 (6)) (729-740) (She Neaky Avzezztepi Optsevzioppaiye (NAS) (Rgiev, ei ai., (1982) 9.

KPPeptaiyoi!. 9:789-793), She Rashivz Zsaie (Rashiv, ei ai., Agiyiv apa KPeiytaiyizt (1990) 33: 477-484), apa

She Agiigyiz Itrasi Meazige Zsaiie (AIM5) (Meepap, ei ai., Agigiyz apa Kpeitaiiodu (1982) 253(1048-1053)|.

The diagnosis and assessment of osteoarthritis pain is well established in the field. The assessment can be performed on the basis of indicators known in the field, such as the patient's description of pain using various pain scales | see, for example, Kai ei aiI., Zygu Sip Moi At. (1999) 79 (2):231-52; Sagasepi ey a). In Raip Zutiot

Mapade (2002) 23(3):239-55). For example, the MOMAS postoperative pain scale (including pain, stiffness, and physical function) and the 100 mm visual analog scale (MA5) can be used to assess pain and assess response to treatment.

It is clear that when a MORE antagonist (such as an anti-MORE antibody) and an NSAID are administered in combination, or in

Go! in the form of one or a separate composition (compositions), the antagonist of the nerve growth factor and NSAIDs are presented in a ratio that is consistent with the manifestation of the desired effect. In some embodiments, the weight ratio between the nerve growth factor antagonist and the NSAID can be about 1 to 1. In some embodiments, the weight ratio can be from about 0.001 to about 1 and from about 1000 to about 1, from about 0.01 to about 1 and from about 100 to about 1, or from about 0.1 to se) about 1 and from about 10 to about 1. Other ratios are contemplated.

Ge) It should be understood that the amount of nerve growth factor antagonist and NSAID33 required for use in the treatment or prevention of pain will vary not only with the particular compounds or compositions selected, but also with the route of administration, the nature of the condition to be treated, and age and condition of the patient, and

In the end, it will be chosen at the discretion of the attending physician.

MOG Antagonists (F) The methods of the invention use a MOP antagonist, which refers to any molecule that blocks, inhibits, or reduces (including significantly) the biological activity of MOP, including downstream pathways mediated by MOP signaling, such as binding to receptors and/or cellular reaction caused by MOP. The term "antagonist" does not imply a specific mechanism of irrelevant biological action, and is considered to clearly include and encompass all possible pharmacological, physiological, and biochemical interactions with MORs and their effects, which may be achieved by a variety of different and chemically distinct, Illustrative MOE antagonists include, but are not limited to, an anti-MOP antibody, a polypeptide (including a polypeptide comprising a MOP-binding domain derived from an anti-MOP antibody, e.g., a binding domain comprising 65 SOC regions, sufficient for binding MORP), an antisense molecule directed to the MORE (including an antisense molecule directed to the nucleic acid encoding the MORP), an antisense molecule,

directed at both TKA and/or p75 receptors (including antisense molecules directed at the nucleic acid molecule encoding TKA or p7b5) a compound that inhibits MOPE, a structural analogue of MOB, a dominant-negative mutation of the TKA and/or p75 receptor, which binds MOR, an antibody against TKA, which binds

MOR, TtTKA immunoadhesin, anti-TKA antibody, anti-p75 antibody, and kinase inhibitor. For the purpose of the present invention, it is to be understood that the term "antagonist" encompasses previously identified terms, names, and functional states and characteristics by which the MOP itself, the biological activity of the MOB (including, but not limited to, the ability to mediate any aspect of pain) or the effects of biological activity are essentially nullified, reduced or neutralized to any significant extent. In some embodiments, an MOR antagonist (eg, an anti-MOR antibody) binds (physically interacts with) MOR, binds to a receptor

MOR (such as the TgTKA receptor and/or p75) and/or reduces and/or blocks) MOR receptor downstream signaling.

Accordingly, in some embodiments, the MORE antagonist binds (physically interacts with) the MORE. In other embodiments, the MOP antagonist binds to a MOB receptor (such as the TKA receptor and/or p75).

In other embodiments, the antagonist reduces (inhibits and/or blocks) downstream signaling of the MORG receptor (eg, kinase inhibitor signaling). In other embodiments, the antagonist inhibits (reduces) the synthesis and/or release of MOP. In another embodiment, the MORE antagonist is a TKA immunoadhesin. In another embodiment, the MOP antagonist is different from the anti-MOP antibody. In some embodiments, the MOR antagonist binds MOR (such as MOB) and does not significantly bind to related neurotrophins such as MT-3, MT-4/5 and/or VOME. In some embodiments, the antagonist binds human

MOB does not significantly bind MOP from other vertebrate species (in some embodiments, mammals). In some embodiments, the MORE antagonist binds human MOR as well as one or more MOBs from other vertebrate species (in some embodiments, mammals). In some embodiments, the MORE antagonist binds MOBE as well as at least one other neurotrophin. In some embodiments, the MORE antagonist binds to the MOG of a mammalian species, such as a horse or dog, but does not significantly bind to the MOB of other mammalian species. high school

Antibodies against MOG

In some embodiments of the invention, the MORE antagonist includes an anti-MORE antibody. An anti-MOE antibody i) must exhibit any one or more of the following characteristics: (a) binding to MOE and inhibiting the biological activity of MOE and/or the downstream pathway(s) mediated by MOE signaling function; (B) treatment or prevention of any aspect of pain, particularly in combination with NSAIDs; (c) blockade or Ge! zo Reduction of activation of MOP receptors (including dimerization and/or autophosphorylation of IgKA receptors); (4) increasing the output of MOB; (c) increasing the pain treatment of NSAIDs3. Me

Antibodies against MOB are known in the field | see, for example, applications PCT MoMo UMO 02096458; UUO 01/78698, UMO s 01/64247, US MoMo patents 5844092, 5877016 and 6153189; Nopoo eyi a!., Nubgidota, 19:215-227 (2000); SeiI. Moyes.

Vioi 13:559-568 (1993); SepVapk access numbers 039608, 039609, I 17078 or I 17077). --

In some embodiments, an anti-MORE antibody specifically binds to MORE. In yet another embodiment, the antibody is humanized (such as the EP antibody described herein). In some embodiments, the anti-MOB antibody is an EZ antibody (as described herein). In other embodiments, the anti-MORE antibody includes one or more SCO antibody EZ (eg, 1, 2, 3, 4, 5, or, in some embodiments, all 6 SKO with EZ). In other embodiments, the anti-MORE antibody is human. In yet another embodiment, the anti-MORE antibody comprises the heavy chain variable region amino acid sequence shown in Table 1 (5EO IU MO:1) and the light chain variable region amino acid sequence shown in Table 2 (ZEO IU MO:2) . In still other embodiments, the anti-MORE antibody includes;" the amino acid sequence of the variable region of the heavy chain shown in Table 1 (ZEO IO MO:1). In still other embodiments, the antibody against MOB includes the amino acid sequence of the variable region of the light chain shown in Table 2 (ZEO IU MO:2). In still other embodiments, the antibody includes a modified constant region, such as a constant region that is immunologically inert, for example, does not trigger complement-mediated lysis or does not stimulate antibody-dependent cell-mediated cytotoxicity (ACC). In other embodiments, the constant region is modified as described in |(Eig. .). to Ittipoi. (1999) 29:2613-2624; PCT applications MoPCT/5899/01441; and/or in a UK patent application

Me9809951.8). In other embodiments, the anti-MORE antibody is any antibody described in application no.

Ge) In some embodiments, the anti-MOB antibody is a humanized murine anti-MORE monoclonal antibody, called an "EZ" antibody, that includes the constant region of the heavy chain of human Idslg containing the following mutations: AZZOR31 to 53305331 (numbering of amino acids with reference to the sequence of dv 9c2a of the wild type (see Eig. U. Ittipoi. (1999) 29:2613-2624)); kappa of the constant region of the human light chain and the variable regions of the heavy and light chains are shown in Tables 1 and 2.

F) name) 60 b5

Table 1

Because the variable region of the heavy chain

OMOGOB5OROY MUKRUETI ZITSTUVO EBLOUO MMK ORROKO EMO USOSTT b RUMBAUKKVKUPOKOTOUKMOVZIKIZ5UTAARTAUUUSAVOSU UHATZUUVOU MU

OSTI UTUV (ZEO TO MO).

Table 2 15 .

The variable region of the light chain

PIOMTOB5RZVI ZAUSRKUTI SVABOVIZMM GU IN OOKROKARKIL TU UTUVENYA

SURBZKEZO5O5OTOETETIV5IOREOTATU USOOBNTI RUTESOSTKI EQVT (SVO

Yu MO-2).

And I'm nervous, tt tzhju s

Table 1 o

І Variable region of the heavy chain (22) from OMOGOB5ORSIMUKROETI ZITSTUZSEZ SUPI MUKORROKO EM ooo b»

RUMBAUKKVKUPOKOTOUKMOVZIKIZ5UTAARTAUUUSAVOSU UHATZUUVOU MU sch

SOT UTU (BE IU MO). m d)

Table 2

The variable region of the light chain "- o, s PIOMTOB5RZVI ZAUSRKUTI SVABOVIZMM GU U OOKROKARKIL TU UTUVENYA khz" OURUKEVO5BOBSTOETETIV5I ORE ATUUSOOENTI RUTESOSTKI EK VT(5VO

Yu MO-2).

Ge | The following polynucleotides encoding the variable region of the EZ heavy chain or the EZ light chain were deposited in the ATCC on January 8, 2003.

F p

Vector EB.911.3E is a polynucleotide encoding the variable region of the light chain shown in Table 2; Vector EB.rig.911.3E is a polynucleotide encoding the variable region of the light chain shown in Table 2, and Vector OB.911.3E is a polynucleotide encoding the variable region of the heavy (F) chain shown in Table 1. These polynucleotides also encode constant domains. d There are at least two methods for determining SOC: (1) an approach based on cross-species variability of sequences (ie Karai et al). bZedcepsez oi Rgoivipv oi Ittipoiodisa! Ipiegevi, (5 ey., in 1991, Maiopai! Ipzishiez oi Neay, Veipezda Ma); and (2) an approach based on crystallographic studies of antigen-antibody complexes. (1989) Maishge 342:877; AIII-Iagikapi eb ai (1997) 9. Moyes. Vio)|. 273:927-948))|. As used herein, the term "SOC" may refer to SOCs determined by either approach or a combination of both approaches.

In another embodiment, the anti-MOE antibody includes one or more EZ antibody SOCs (eg, 1, 5, 2, 3, 4, 5, or in some embodiments, all 6 EZ3 SOCs). The definition of SOK areas is quite well known in this field. SOCs can be Cabai, Spoipia, or a combination of CarBbai and Spoipia.

Antibodies that can be used in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Rab, Rab, (ab), Em, Es, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies , single-chain (SsEm), their mutants, hybrid proteins that

Include an antibody part, humanized antibodies and any modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants and covalently modified antibodies.

Antibodies can be mouse, rat, human, or of any other origin (including chimeric and humanized antibodies). For the purposes of this invention, the antibody interacts with MOPE in a way that inhibits MOG 70 and/or downstream pathways mediated by the MOPE signaling function. In one embodiment, the antibody is a human antibody that recognizes one or more epitopes on the human MOR. In another embodiment, the antibody is a mouse or rat antibody that recognizes one or more epitopes on the human MOB. In another embodiment, the antibody recognizes one or more epitopes on a MORP selected from the group consisting of: rodents, dogs, cats, horses, and cows. In another embodiment, the antibody includes a 7/5 Modified constant region, such as a constant region that is immunologically inert (ie, does not trigger complement-mediated lysis) or does not stimulate antibody-dependent cell-mediated cytotoxicity (ACC). AOSS activity can be assessed using methods disclosed in US Pat

MO55003621. In other embodiments, the constant region is modified as described in (Enshg. 9. Pgapypo). (1999) 29:2613-1624; publication PCT MoPCT/Z899/01441 and/or in the British patent application Mo9809951.81.

The binding affinity of an anti-MORE antibody to a MORE (such as AIMORG) can be from about 0.01 nM to about 1 nM, from about 0.05 nM to about 0.25 nM, from about 0.1 nM to about 0.80 nM, from about 0 .15nm to about 0.75nm and from about 0.18nM to about 0.72nM. In other embodiments, the binding affinity is about TpM, about 2 pM, about bpM, about 1OpM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM. In one embodiment, the binding affinity is between about 2 nM and 22 pM. In other embodiments, the binding affinity is less than about 100 nM, about 5 nM, about 1 nM, about 1 nM, about i) 5O0OpM, about 100 pM, about 5OpM, about TOpM. In some embodiments, the binding affinity is about 1OnNM. In other embodiments, the binding affinity is less than about 1 ONM. In other embodiments, the binding affinity is about 0.1 nM or about Ge! with 0.07nNM. In other embodiments, the binding affinity is less than about 0.1 nM or less than about 0.07 nM. In other embodiments, the binding affinity is any of from about 100 OM, Me) about 5 OM, about 1 OM, about 1 NM, about 500 pM, about 100 pM, about 5OpM to any of about 2 pM, about bpM, about 1OpM , about 15 pM, about 20 pM or about 40 pM. In some embodiments, the binding affinity is any of about 1000nm, -- about 50nm, about 10nm, about 1nm, about 500pM, about 100pM, or about 5OpM, or less than about 5O0pM. In still other embodiments, the binding affinity is about 2pM, about 5pM, about 1OpM, about 15pM, about 20pM, about 40pM, or more than about 4OpkmM.

One way to determine the binding affinity of antibodies to MOB is to measure the affinity of Monofunctional Rar antibody fragments. To obtain monofunctional Rab fragments, an antibody with c (for example, Id) can be cleaved with papain or expressed recombinantly. The affinity of the Bar vs. fragment

The MOR of antibodies can be determined by surface plasmon resonance (a device for determining surface plasmon resonance (SRK) ViAsoge Z00Otm, VIAsoge, IMS, Rivsazhchau M) CM5 chips can be activated with M-ethyl-M'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EOS) and M-hydroxysuccinimide (MH) according to the supplier's instructions. Human MORE can be diluted in 10 mM sodium acetate at a pH of 4.0 and injected on top of the activated chip at a concentration of 0.005 mg/ml. Using variable flow time through separate channels of the chip, two ranges of antigen density can be achieved: 100-200 reaction units (RU) for detailed kinetic studies and 500-600 Ki for screening analyses. The chip can be blocked with ethanolamine. Regeneration studies showed that a mixture of Riegse elution buffer (product Mo21004, Riegse Violyesppood I) and 4M Maa (271) efficiently removed bound Rab while maintaining on-chip NMOE activity for 200 injections. Buffer ee, NBBVZ-ER (0.01M NERE5, pH 7.4, 0.15 Mass 3mM EDTA, 0.00595 surface-active substance Pg20) (Che) is used as a working buffer for ViIAsoge analyses. Serial dilution (0.1-10 x certain Kr) of purified Rab samples are injected during their in a concentration of 1O0Omcl/min. and dissociation time values of up to 2 hours are allowed. Concentrations of RGar proteins are determined by EIG IZA and/or 505-RACE electrophoresis using Rab known

Concentrations (as determined by amino acid analysis) as a standard. Kinetic association rates (K op) and dissociation rates (Koi) are obtained simultaneously by fitting the data to the 1:1 binding model (Kagizvop, K. Koov, N. Radegziat, I. Reyigesop, V. (1994). Meiyodz Epgutoiodu 6:99-110) using the VIA evaluation program. The values of the equilibrium dissociation constant (Ko) can be calculated in the form of K ov/Kol. This protocol is suitable for use in determining the binding affinity of an antibody to MOR of any species, including human MOR, other vertebrate (in some embodiments, mammalian) MOR (such as mouse MOR, rat

MOR, primate MOE) and for use with other neurotrophins such as related neurotrophins

MTZ, MTA/5 and/or VOME.

In some embodiments, the antibody binds human MO and significantly does not bind MO from other vertebrate species (in some embodiments, mammals). In some embodiments, the antibody binds human 65 MOP as well as one or more MOBs from other vertebrate species (in some embodiments, mammals such as rodents). In still other embodiments, the antibody binds MOB and does not significantly cross-react with other neurotrophins (such as related neurotrophins MTZ, MT4/5 and/or VOME). In some embodiments, the antibody binds MOR as well as at least one other neurotrophin. In some embodiments, the antibody binds to MOE from a mammalian species, such as a horse or dog, but does not significantly bind to MOB from other mammalian species.

Epitopes can be continuous or discontinuous. In one embodiment, the antibody binds substantially the same AMOBE epitope as an antibody selected from the group consisting of MAB 911, MAB 912, and MAB 938, as described in (Nopdo et al., Nubgidota 19:215 -227 (2000).In another embodiment, the antibody binds substantially the same epitope of PMOR as MAB 911. In yet another embodiment, the antibody binds substantially the same epitope of PMOR as MAB 909 |see Nopoo, ey a!., above). For example, the epitope may include one or more of: KZ2, KZ4 and EZ5 within the variable region 1 (amino acids 23-35) of the MORE; residues E79 and T81 within variable region 4 (amino acids 81-88) of PMS; H8v4 and KV8 residues within variable region 4; residue K103 between variable region 5 (amino acids 94-98) of AMOBE and C-terminus (amino acids 111-118) of YAMOK; residue E11 within prevariable region 1 (amino acids 10-23) of AMOE; U52 between variable region 2 7/5 (amino acids 40-49) of PMOBE and variable region C (amino acids 59-66) of MON; residues 1112 and 5113 within

C-end of AIMOE; residues of KBU and K69 within the variable region Z AMOE; or residues MIV, M20, and (323) within prevariable region 1 of PMOR. In addition, the epitope may include one or more of variable region 1, variable region C, variable region 4, variable region 5, the M-terminal region, and/or the C- the end

MOR. In yet another embodiment, the antibody significantly reduces the solvent accessibility of the K103 residue of MOG.

It is clear that although the epitopes described above refer to human MOE, one of ordinary skill in the art can match the structures of human MOE with the MOE of other species and identify likely copies of these epitopes.

In one aspect, antibodies (eg, human, humanized, murine, chimeric) that can inhibit MOB can be obtained using immunogens that express the full length or partial sequence of MOB. In another aspect, an immunogen comprising a cell that overexpresses a MOP can be used. Another example of an immunogen that can be used is a MOE protein that contains full-length MOE or a portion of the MOE protein. and)

Antibodies against MORE can be obtained by any method known in the art. The route and scheme of immunization of the host animal are generally consistent with accepted and conventional techniques for stimulation and production of antibodies, as further described in this description. General techniques for the production of human and mouse antibodies are known to the successful Ge! from the field and are described in this description.

It is contemplated that any mammalian individual, including humans, or antibody-producing cells derived from them, can be manipulated to serve as a basis for the production of a hybridoma cell line from mammals, including humans. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally with some amount of immunogen, including that --

C5 As described in this description. co

Hybridomas can be obtained from lymphocytes and immortalized cells of myeloma using the general technique of hybridization of somatic cells IKoiieg, V. Apa Miivivii, S. (1975) Maishge 256:495-497|) or in a modification (Vysk, O.MU., ей аЙ. n Miigo, 18:377-381 (1982)). In hybridization, available myeloma lines can be used, including, but not limited to, X63-A98.653 and lines from Bak Ipziyshe, Se! Oivigirshiop Sepieg, « zap Oiedo, Saiiii., OBA. In general, the technique involves the fusion of myeloma cells and lymphoid cells using a fusogen, such as polyethylene glycol, or electrical means well known to those skilled in the art. . After fusion, the cells are separated from the fusion medium and grown in a selective growth medium. such as hypoxanthine-aminopterin-thymidine (HAT) medium to remove unhybridized parental cells. For the cultivation of hybridomas secreting monoclonal antibodies, you can use any of the media described in this description with or without the addition of serum. As another alternative to the fusion technique

Go! cells, it is possible to use B-cells, immortalized by EBM (Epstein-Barr virus), for the production of monoclonal antibodies against MOPE of this invention. If desired, the hybridomas are propagated and subcloned, and the supernatants are analyzed for anti-immunogenic activity by conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescent 5or immunoassay). se) Hybridomas that can be used as a source of antibodies include all derived, progeny cells

Ge) parental hybridomas that produce monoclonal antibodies specific for MOPE or their parts.

Hybridomas that produce such antibodies can be grown ip migo or ip mimo using known procedures. If desired, monoclonal antibodies can be isolated from culture media or biological fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by obtaining on adsorbents made from an immunogen attached to a solid phase and eluting or releasing the desired antibodies from the immunogen. Immunization of the host animal with a human

MOR or a fragment containing a target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, for example, hemocyanin in keyhole or saucer form, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, a complex maleimidobenzoyl ester of sulfosuccinimide (linked bond through cysteine residues), M-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI" or K'M-C-ME, where K and KE! represent different alkyl groups, can give a population of 65 antibodies (for example, monoclonal antibodies).

If desired, the anti-MOE antibody (monoclonal or polyclonal) of interest can be sequenced, and the polynucleotide sequence encoding the anti-MOP antibody polypeptide can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and the host cells can then be propagated and frozen for future use.

Application. Alternatively, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody.

For example, the constant region can be genetically engineered to more closely resemble human constant regions to avoid an immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the 7/0 sequence of the antibody to obtain greater affinity for MOB and greater efficacy in inhibiting MOB. It will be obvious to a person skilled in the art that one or more polynucleotide changes can be made in an antibody against

MOB and yet retain its ability to communicate with the MOB.

There are 4 general steps for humanizing a monoclonal antibody. They are: (1) determination of the nucleotide and the predicted amino acid sequence of the light and heavy variable domains of the original /5 antibody, (2) the design of the humanized antibody, i.e. the decision of which framework region of the antibody to use during the humanization process; (3) valid methodologies!/methodologies of humanization; and (4) transfection and expression of the humanized antibody |see, e.g., US Patent Nos. 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. A number of "humanized" antibody molecules have been described that include an antigen binding site derived from a superhuman immunoglobulin, including chimeric antibodies having rodent M regions or modified rodent M regions and their complementarity-determining regions (CDOs) fused to with human constant domains | see, for example, UMipigeg ei a. Mayte 349:293-299 (1991), Horidia ei aY. Rgos. May Asad All together. OBA 86:4220-4224 (1989), Zpnam/ ei aiYi. 9d. Ittipoi. 138:4534-4538 (1987) and Vgomp ei a! Sapseg Kez. 47:3577-3583 (1987)). Other references describe a rodent SOC grafted to a human framework region (RC) prior to fusion with the constant domain of the corresponding human antibody, see, for example, Kiesptapp et al. Mayte 332:323-327 (1988), Megpoeup ei a). Science 239:1534-1536 (1988) and d9opev her a). Maishge 321:522-525 (1986)). Another reference describes recombinantly maintained rodent SOCs and) coated rodent framework regions (see, for example, European Patent Publication Mo519596). These "humanized" molecules are designed to minimize the unwanted immunological reaction to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of these He! from parts of human recipients. For example, the constant region of an antibody can be engineered by genetic engineering so that it is immunologically inert (eg, does not trigger lysis by complement (see, for example, PCT/5899/01441; UK patent application Mo 9809951.81). Other methods of humanizing antibodies , which can also be used, disclosed (Oatsopegiu ei ai., Mysi. Acid5. Kev. 19:2471-2476 (1991) and in patents

US MoMo 6180377, 6054297, 5997867, 5866692, 6210671, 6350861, and in PCT publication Me01/27160. Other methods of food" are described in the US patent application under the serial number Mo10/745775).

In yet another alternative, fully human antibodies can be obtained using commercially available mice that have been genetically engineered to express specific human immunoglobulin proteins.

Transgenic animals that are designed to produce a more desirable (eg, fully human antibody) or more appropriate immune response can also be used to generate humanized or "human" antibodies. Examples of such technology are Hepotoizetm from Ardepykh, Ips. (Egetopi, SA) and NIiIMAB-Moiseo and TO Moshetm from the village of Meadageh, Ips. (Rhypseiop, Moscow State University). and It is apparent that although the above discussion relates to humanized antibodies, the general principles discussed are applicable to the preparation of antibodies for use in, for example, dogs, cats, primates, horses, and cows. In addition, it is apparent that one or more of the described herein description of aspects of humanization of antibodies can

For example, SOC transplantation, scaffold mutation and SOC mutation can be combined. Alternatively, antibodies can be produced recombinantly and expressed using any method known in the art. Alternatively, antibodies can be produced recombinantly by phage display technology | see, for example, US Patents MoMo 5565332, 5580717, 5733743, 6265150 and Mipieeg ei ai!., Appy. to Kemu And so on. 12:433-455 (1994)). Alternatively, it is possible to use the technology of phage reproduction |MsSaiPepu ei 5o a. Mayte 348:552-553 (1990) for the production of human antibodies and ip mygo antibody fragments from immunoglobulin variable (M) domain gene repertoires from immunized donors. According to this technique, the (Che) genes of the M domain of the antibody are cloned in-frame or into the gene of either the large or small coat protein of a filamentous bacteriophage, such as M13 or ha, and are displayed as functional antibody fragments on the surface of the phage particle. Due to the fact that the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selections based on the functional properties of the antibody also lead to the selection of a gene encoding an antibody that exhibits these properties. Thus, the phage mimics some of the properties of the B cell. Reproduction of phage can be performed in various formats (review | see, for example, in the publication doppzop, Kemip 5. apa Spizumei, ko OBamii 9., Sshitepi Oriop ip ZigisishgaI! Vioiodu Z, 564-571 (1993)). Several sources of M gene segments can be used for phage reproduction. |Siaskop ei ai., Maishge 352:624-628 (1991)| isolated a diverse array of antibodies against oxazolone from a small, random combinatorial library of M genes obtained from the spleens of immunized mice. A repertoire of M genes can be constructed from immunized human donors, and antibodies to a variety of antigens (including autoantigens) can be isolated essentially following the techniques described in

IMagk ei ai., U. Moi. VioI. 222:581-597 (1991) or SgiyAi ei ai., EMVO 3. 12:725-734 (1993)). During a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the b5 introduced changes will confer higher affinity, and B cells expressing high-affinity surface immunoglobulin preferentially replicate and differentiate upon administration of a subsequent permissive dose of antigen. This natural process can be mimicked using a technique known as "chain shuffling" IMagkv5, ei ai., Vio/HTesppoi. 10:779-783 (1992) In this method, the affinity of "primary" human antibodies obtained by phage reproduction can be improved by sequentially replacing the heavy and light chain M region genes with repertoires of variants (repertoires) of naturally occurring B domain genes obtained from non-immunized donors . This technique enables the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for producing very large repertoires of phage antibodies (also known as "the source of all libraries") has been described (Muafekgpoize ei aiYi., Mysi. Asiaz Kev. 21:2265-2266 (1993)|.

Gene shuffling can also be used to generate human antibodies from rodent antibodies, where the human /o antibody has affinities and specificity similar to the original rodent antibody. According to this method, which is also called "epitope imprinting", the M domain gene of the heavy or light chain antibody of rodents obtained by the phage reproduction technique is replaced by a repertoire of human M domain genes, creating rodent-human chimeras. Selection on the antigen leads to the selection of human, variable regions capable of restoring the site that binds the functional antigen, that is, the epitope controls (imprints) the choice of partner. When the 7/5 process is repeated to replace the remaining rodent M domain, a human antibody is produced |see patent application PCT UUO 9306213, published on April 1, 1993). In contrast to traditional humanization of rodent antibodies, which do not have the framework or residues of rodent-derived SOCs. Obviously, although the above discussion relates to humanized antibodies, the general principles discussed are applicable to obtaining on demand antibodies for use in, for example, dogs, cats, primates, horses and cows.

Antibodies can be produced recombinantly by first isolating antibodies and antibody-producing cells from host animals, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another method that can be used is the expression of the antibody sequence in plants (for example, tobacco) or transgenic milk. Methods of recombinant expression of antibodies in plants or milk have been disclosed | see, for example, Reeig5, ei aI. Massipe Sci 19:2756 (2001); Gopregd, M. apa 0. Nyzglag IpiKem. Ittypo! 13:65 (1995); and RoyosK, et al), 9) Ittypo!i Meipodvs 231:147 (1999)). Methods of obtaining derivative antibodies, for example, humanized, single-chain, etc., are known (8) in this field.

Immunoassays and flow cytometry sorting techniques, such as fluorescence-activated cell sorting (RGAS5), can also be used to isolate antibodies that are specific for MOP. Gee! zo Antibodies can be bound to many different carriers. Carriers can be active and/or inert.

Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, Me) glass, natural and modified cellulose, polyacrylamides, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Those skilled in the art are aware of other suitable media for binding antibodies or ways to establish them using routine experimentation. --

The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to the genes encoding the heavy and light chains of the monoclonal antibodies). Hybridoma cells serve as the preferred source of such DNA.

After isolation, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. soybean cells, CO5 monkey cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. for the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the coding sequence with the constant domains of the human heavy and light chain instead of homologous mouse sequences. aI., Rhos. May Asad All together. 81:6851 (1984)| or by covalent attachment to an immunoglobulin encoding all or part of the coding sequence on a non-immunoglobulin polypeptide. Thus, "chimeric" or "hybrid" antibodies are obtained that have the binding specificity of the monoclonal antibody described in this description

Go! against MOR. DNA encoding an antagonistic antibody against MOB (such as a humanized antagonistic antibody against human MOP) can be used to deliver and express an antagonistic antibody against human MOP by a desired cell as described herein. DNA delivery methods are further described in this description. anti-MOP antibodies can be characterized using methods well known in the art. 5r For example, one method is to identify the epitope to which it binds, called "Ik epitope mapping". There are many methods known in the art to map and characterize epitope localization

Ge on proteins, including resolution of the antibody-antigen complex crystal structure, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11

Nagpomzh apa IGape, Ovipd Apiirodiez, and I arogaigu Mapia!i, Soii Zrgipd Nagrog I arogaiogu Rgevzv, Soii 5rgipd ov Nagrog, Memu Hogk, 1999). In an additional example, epitope mapping can be used to determine the sequence to which anti-MOR antibody binds. Epitope mapping is commercially available from many sources,

F) for example, |Rerzsap Zuvietz (Edeipegimed 15, 8219 RN I eiusiad, Tpe Me Ppepapa3z)). The epitope can be a linear epitope, that is, contained in one fragment of amino acid sequencing, or a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in one fragment of sequencing. Peptides of different lengths (for example, at least 4-6 amino acids long) can be isolated or synthesized (for example, recombinantly) and used for binding assays with an antibody against MOP. In another example, the epitope to which the anti-MORE antibody binds can be determined by systematic screening using overlapping peptides derived from the MOR sequence and determining binding by the anti-MORE antibody. According to the analyzes of expressed gene fragments, the open reading frame encoding the MOB is fragmented either randomly or by specific genetic constructs, and the reactivity of the expressed MOB fragments with the antibody to be tested is determined. Gene fragments can, for example,

to be produced by PCR (polymerase chain reaction), and then to be transcribed and translated into the IP protein in the presence of radioactive amino acids. Then the binding of the antibody to radioactively labeled MOB fragments is determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using large libraries of random peptide sequences found on the surface of phage particles (phage libraries). Alternatively, a certain library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, antigen-binding domain mutagenesis, domain exchange experiments, and scanning alanine mutagenesis can be performed to identify the required residues sufficient and/or required for epitope binding. 7/0 For example, domain exchange experiments can be performed using mutant MORE in which various fragments of the MORE polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct, protein (such as another member of the neurotrophin protein family). By evaluating the binding of an antibody to a mutant MOP, it is possible to estimate the value of a certain MOB fragment for antibody binding.

Another method that can be used to characterize an anti-MORE antibody is to use 7/5 competition assays with other antibodies known to bind to the same antigen, i.e., different fragments on the MORE, to determine whether binding an anti-MOB antibody with the same epitope as other antibodies is formed. Competition assays are well known to those skilled in the art. Examples of antibodies that can be used in competition assays for the present invention include Mar 911, 912, 938 as described in (Nopoo, et al. Publications, Nubgidota 19:215-227 (2000) .

Other antagonists of MOG

MORE antagonists other than anti-MORE antibodies can be used. In some embodiments of the invention, the MORE antagonist includes at least one antisense molecule capable of blocking or reducing the expression of a functional MORE, or a functional YKA and/or p75 receptor. Nucleotide sequences of MO, (KA and p7b5) are known and easily available from public databases | see, for example,

Vogzapi ey ai!, Mys. Asiaz Cavs. 1990, 18, 4020; Assezviop Mytbreg MM 002506; Physiology, Medicine 303:821-825 (1983)). It is well-known to obtain antisense oligonucleotide molecules that specifically bind mRNA and)

MOP, KA or p7/5 without cross-interaction with other polynucleotides. Illustrative sites of endowment include, but are not limited to, the initiation codon, regulatory regions 5, coding sequence, and untranslated region 3. In some embodiments, the oligonucleotides are about 10 to He! of 100 nucleotides, have a length of about 15 to 50 nucleotides, have a length of about 18 to 25 nucleotides, or more. Oligonucleotides may include backbone modifications, such as, for example, Me) phosphorothioate linkages, and modifications of 2-0 sugars well known in the art (see, e.g., Aadgazhma! apa c 7pao (1998), Apiizepze 5 Misieis) Asia Ogyd YuOemeiortepi 8, 135-139). Illustrative antisense molecules include MO antisense molecules described in (US Patent Publication No. Mo20010046959; see also (7 "7

NEru/LMmllmy, gpa-ies.sot/geraig. sweat. co

Alternatively, the expression and/or release of MOPE can be reduced using gene silencing, morpholino oligonucleotides, mRNA, or ribosomes, methods that are well known in the art. See, for example,

Kozvi, 9). ey ai., eav., "IpigaseishShiag Kirogute Arriisayope: Rgipsirievg apa Rgoiosoiiv, "Nogigop Zsiepiyis

Rgezz (Otsagie, SA, 1999); 5 6506559; UMO 02/244321; MO 01/192513; MO 01/290581. "

In other embodiments, the MOPE antagonist includes at least one compound that inhibits MOB. As used herein, the term "MORE-inhibiting compound" refers to a compound, other than an anti-MORE antibody, that directly or indirectly reduces, inhibits, neutralizes, or eliminates the biological activity of MORE. ;" A compound that inhibits MOB must exhibit any one or more of the following characteristics: (a) binds to MOB and inhibits the biological activity and/or downstream pathway(s) mediated by the MOB signaling function; (B) treatment or prevention of any aspect of pain, particularly in combination

Go! with NSAIDs3; (c) blocking or reducing activation of MOB receptors (including dimerization and/or autophosphorylation of IiKA receptors); (4) increasing the output of MOB; (e) inhibition (reduction) of synthesis, - production or release of MOB; (Her) increased pain treatment NSAID3. Illustrative compounds that inhibit MOB, which include small molecule inhibitors of MOPE, described in (US patent publication Mo20010046959); compounds that inhibit the binding of MOB to p7b5, as described in (publication PCT MoUUO 00/69829); compounds that inhibit the binding of MOB to TIKA/p75, as described in (publication PCT MoUUO 98/17278)|. Additional examples of compounds that

Ge) inhibit MORE, include compounds described in (publications PCT MoMo MO 02/17914, UMO 02/20479, in US patents

MoMo 5342942, 6127401 and 6359130). Other illustrative compounds that inhibit MOE are compounds that are competitive inhibitors of MOE |see US patent Mob291247). In addition, a person skilled in the art can obtain two other small molecule compounds that inhibit MOR.

In some embodiments, a compound that inhibits MOP binds MOP. Illustrative site allocation

F) (binding) include, but are not limited to, the portion of the MORE that binds to the TtKA receptor and/or the p7b5 receptor, and those portions of the MORE that are adjacent to the receptor-binding region and that are partially responsible for the correct three-dimensional shape of the receptor-binding part. In another embodiment, the compound inhibits MOB because it binds to MOB receptors (such as TKA and p75) and inhibits the biological activity of MOB. Illustrative sites of endowment include those parts of TKA and/or p75 that bind to MOP.

In an embodiment that includes a small molecule, the small molecule can have a molecular weight of about any value between 100 and 20,000 daltons, between 500 and 15,000 daltons, or between 1,000 and 10,000 daltons. Small molecule libraries are commercially available. Small molecules can be administered using 65 any means known in the art including inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, subcutaneous, intraventricular, oral,

enterally, parenterally, intranasally or dermally. In some embodiments, when the MOG antagonist is a small molecule, it is administered in an amount of 0.1 to 0.1 mg/kg of patient weight, divided into 1-3 or more doses. For an adult patient with a normal body weight, doses in the range from 1 mg to 5 g per 1 dose can be administered.

In other embodiments, the MOB antagonist includes at least one structural analog of MOB. "Structural analogues of MOE" in the present invention refer to compounds that have the same three-dimensional structure in the form of a part of the structure of MOB and which bind to the MOE receptor under physiological conditions ip miigo or ip mimo.

In one embodiment, the structural analog of MOPE binds to the TKA receptor and/or p75. Illustrative /o bstructural analogues of MORG include, but are not limited to, bicyclic peptides described in (publication PCT MoUUO 97/15593); bicyclic peptides described in (US patent Mob291246); cyclic compounds described in (US Pat

Mo60178781, and peptides obtained from MOP are described in (PCT publication MoUuO89/09225). Corresponding structural analogues

MORE can also be designed and synthesized using molecular modeling of MOR receptor binding, for example, by the method described in (publication PCT MoUUO 98/06048). Structural analogues of MOG /5 can be monomers or dimers/oligomers in any desired combination of their or other structures to obtain improved affinities and biological effects.

In other embodiments, the invention provides a MOB antagonist, including at least one dominant-negative mutant of the TIKA receptor and/or the p75 receptor. One skilled in the art can obtain dominant-negative mutants of, for example, the TgKA receptor so that the receptor will bind MOP, and thus act as a "sink" for MOP uptake. However, dominant-negative mutants will not have the normal biological activity of the receptor (such as the TKA receptor) after binding to MOP. Illustrative dominant-negative mutants include, but are not limited to, mutants described in the following references: Mai! Asad

All together. BOTH 1998, 95, 10884; Eide ei aiYi, 9U. Meygossy 1996, 16, 3123; ish ei aiYi, U. Metsygozsi 1997, 17, 8749;

KiIviip ei ai, SeiYi 1990, 61, 647; MaYeplceia ei ai, Metsygop 1993, 10, 963; Tvotsiaz ey ai, Metsygop 1993, 10, sch 975; and I atbaiIe ei ai, EMVO 3. 1993, 12, 3083), each of which is fully incorporated herein by reference.

Dominant-negative mutants can be introduced in protein form or in the form of an expression vector, so that i) a dominant-negative mutant (for example, a mutant TKA receptor) is expressed ip mimo. The protein or expression vector can be administered using any means known in the art, such as intraperitoneal, intravenous, intramuscular, subcutaneous, submucosal, ventricular, or intraventricular administration. from the brain, orally, enterally, parenterally, intranasally, dermally, or by inhalation. For example, administration of an expression vector includes local or systemic administration, including injection, oral b» administration, administration in the form of particles using an injection gun or catheter, and local administration. with

In other embodiments, the protein or expression vector is administered directly into a sympathetic or sensory trunk or ganglion. One skilled in the art is familiar with the introduction of expression vectors to obtain the expression --

From the exogenous protein IP mimo | see, for example, US patents MoMo 6436908; 6413942; 63764711. co

Targeted delivery of therapeutic compositions containing antisense polynucleotide, expression vector or subgenomic polynucleotides can also be used. Receptor-mediated methods of DNA delivery are described, for example, in (publications Ripdeiz ei ai, Tgepaz Vioiespto! (1993) 11:202; Spioi ei a).

Seli Tpegaretsiisv: Meijodz Apa Arriisayope OYI Oigesi Sepe Tgapvezieg (UA. Moi, ey.) (1994); MUy ei ai, 9. "

Vio! Spent 1988) 263:621; MUm ei ai, U. Vioi. Spent (1994) 269:542; 7epKe ey ay, Rgos. May Asai. All together. (OA) U s (1990) 87:3655; MUy ei ai., 9. Vioi. Spent (1991) 266:338). Therapeutic compositions containing polynucleotide. are administered in the range of about 10 Ohms to about 200 Ohms (or more) DAILY for local administration into the i? gene therapy protocols. In some embodiments, concentration ranges of less than about 5O0ng, from about 50Ong to about 5BOng, from about 1µg to about 2mg, from about 5µg to about 5O0µg, and from about 20µg to about

Go! 100mcg or more per DNA. Therapeutic polynucleotides and polypeptides of this invention can be delivered using gene delivery carriers. Carriers of gene delivery can be of viral or non-viral origin - Yediv. in general TsdoPu, Sapseg Sepe Tpegaru (1994) 1:51; Kitiga, Nytap Sepe Tpegaru (1994) 5:845; Soppeiu, ko Nytap Sepe TPpegaru (1995) 1:185; and Karii, Mayte Sepeiyisv (1994) 6:148). The expression of such coding sequences can be induced using endogenous mammalian promoters and/or enhancers or heterologous promoters and/or enhancers. The expression of the coding sequence can be either constitutive,

Ge or adjustable.

Viral-based vectors for delivery of the desired polynucleotide and expression in the desired cell are well known in the art. Illustrative viral-based vectors include, but are not limited to, recombinant retroviruses |see, for example, PCT publications MoMo UUO 90/07936; UMO 94/03622; MO 93/25698; MO 93/25234; MO 93/11230; UMO 93/10218; MO 91/02805; US MoMo patents 5219740, 4777127; British patent Mo2200651 and

F) Spanish patent Mo03452421, vectors based on alpha virus (for example, vectors based on Sindbis virus, Semlika forest virus (ATOS MK-67; ATCC Mr-1247), Rose River virus (ATSC MK-373; ATCC Mr-1246 ) and the virus of Venezuelan equine encephalitis (ATSS MK-923; ATS Mt-1250; ATSS MkK-1249; ATSS Mr-532), and in adeno-associated virus (AAM) | see, for example, PCT MoMo UUO publications 04/12649 , UMO 93/03769, MO 93/19191; MO 94/28938; MO 95/11984 and MUO 95/00655). It is also possible to use the introduction of DNA associated with a killed adenovirus, as described in the publication of Sigiei, Nit. Sepe TNeg. (1992) 3:147).

Non-viral delivery vehicles can also be used, including, but not limited to, polycationic, condensed DNA conjugated to a single killed adenovirus |see, e.g., Sigii Nit. Sepe Tpeg. (1992) 65 3:147), DNA conjugated with a ligand |see, for example, MUy, 9. Vioi Spet. (1989) 264:16985); carrier cells for the delivery of eukaryotic cells (see, for example, US patent Mo5814482; publications PCT MoMo UUO 95/07994; MO

96/17072; MO 95/30763; and UMO 97/42338) and neutralization of the nucleic charge or fusion with cell membranes. Deproteinized DNA can also be used. Illustrative ways of introducing deproteinized

DNA is described in (publication PCT Mo M/O 90/11092 and in US patent Mo 5580859). Liposomes that can act as gene delivery carriers are described in (US patent Mo 5422120; PCT publications MoMo UUO 95/13796; UMO 94/23697; MO 91/14445 and Spanish patent Mo0524968). Additional approaches are described in (publication Rniir, Moi. SeiI. Visi. (1994) 14:24119 and in the publication UuoPepaip, Rgos. May). Asad-zsi. (1994) 91:15811.

It is also apparent that the expression vector can be used to direct the expression of any of the protein-based MOG antagonists described herein (eg, anti-MOP antibody, TIKA immunoadhesin, etc.). 7/0 For example, a polynucleotide encoding an antagonistic antibody against MOB can also be used to deliver and express an antagonistic antibody against MOB in a desired cell. Obviously, the expression vector can be used to direct the expression of an antagonistic antibody against MOP. The expression vector can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, subcutaneously, into the ventricles of the brain, orally, enterally, parenterally, intranasally, dermally, or by inhalation. 7/5 For example, the introduction of expression vectors includes local or systemic introduction and local introduction. As further discussed in this description, one skilled in the art is familiar with the introduction of expression vectors to obtain expression of an exogenous ip mimo protein | see, for example, US patents MoMo 6436908; 6413942 and 6376471). In this area, other fragments of the TKA receptor are known, which are able to block MOPE (from partial to complete blockade) and/or the biological activity of MOPE.

In another embodiment, the MOB antagonist includes at least one TKA immunoadhesin.

As used herein, TKA immunoadhesins refer to soluble chimeric molecules comprising the TKA receptor extracellular domain (or a portion thereof) and an immunoglobulin sequence that retains the binding specificity (in some embodiments, substantially retains the binding specificity) of the TKA receptor and is able to communicate with the MOR. TKA immunoadhesin is capable of blocking (reducing and/or suppressing) the biological activity of MOR, as described in this description.

TgKA immunoadhesins are known in the art, and have been found to block (reduce or inhibit) the binding of MOPE to the TKA receptor (see, for example, US Patent Mob153189). In one embodiment, the TIKA immunoadhesin comprises a fusion of the amino acid sequence of the TKA receptor capable of binding MOPE (or an amino acid sequence that essentially preserves the binding specificity of the TtKA receptor) and the sequence of an immunoglobulin (or an amino acid that essentially preserves the binding specificity of the TtKA receptor) TKA receptor binding). In some embodiments, the TgKA receptor is a sequence of the human TgKA receptor, and the fusion occurs with the sequence of the immunoglobulin constant domain. In other embodiments, the sequence of the constant Gα domain of the immunoglobulin is the sequence of the constant domain of the heavy chain of the immunoglobulin. In other embodiments, the association of two TITKA immunoglobulin heavy chain receptor fusions (for example, via -

Covalent coupling with a disulfide bond (bonds)) leads to a structure similar to the homodimeric He) immunoglobulin. The light chain of the immunoglobulin can, in addition, be associated with one or both chimeras of the immunoglobulin receptor TICkA in a disulfide-linked dimer to obtain a homotrimeric or homotetrameric structure. Examples of suitable TKA immunoadhesins include the immunoadhesins described in (US patent Mob153189). "

In another embodiment, the MOG antagonist includes at least one anti-TKA antibody capable of blocking, inhibiting, altering and/or reducing the physical interaction of MORE with the THKA receptor and/or downstream signaling, whereby the biological activity of MORE is reduced and/or or blocked. Antibodies against "" TKA are known in the art. Illustrative anti-TKA antibodies include antibodies described in (PCT publications

MoMo UMO 97/21732, MO 00/73344, UMO 02/15924 and US patent publication Mo20010046959). In another embodiment, the MOE antagonist includes at least one anti-p75 antibody capable of blocking, inhibiting and/or reducing the physical interaction of MOE with the p75 receptor and/or downstream signaling, whereby the biological activity of MOE is reduced and/or blocked. - In another embodiment, the MOR antagonist includes at least one kinase inhibitor capable of inhibiting the downstream transmission of kinase signals associated with the activity of TKA and/or p75 receptors. Illustrative 5or kinase inhibitors are K252a or K252B, which are known in the art and are described in (publications Kpyvei! ei is, ap, 9. Metshospet. 59:715-722 (1992); Kpive! ei aiYi, U. Mephospetivigh 57: 955-962 (1991);

Ge) Meipgossiepse 8:715-721 (1988); Nigaia ei ai, Spetisa! Abzigasiv 111:728, ХРОО204135, see resume and 121p

SoPesiime SPetis! Zirveiapse Ipadeh, p. 34237, with 3 (5-7), 55-60, 66-69), p. 34238, p. 1 (41-44), p. 2 (25-27, 32-33), p. 3423, p. 3 (48-50, 52-53); US patent Mob6306849).

It is expected that the clinician will be able to identify a number of other categories of MOR antagonists when searching.

Identification of MOG Antagonists iF) Antibodies against MOB and other MOE antagonists can be identified or characterized using methods known in the art to detect and/or measure the reduction, alleviation or neutralization of the biological activity of MOG. For example, the receptor kinase activation assay (RIKA), described in US Patent Nos. 5,766,863 and 5,891,650), can be used to identify anti-MOR agents. This EPISA-type assay is suitable for qualitative or quantitative measurement of kinase activation by measuring autophosphorylation of the kinase domain of a receptor protein tyrosine kinase (hereafter referred to as "TRTK"), e.g., the TITKA receptor, as well as for the identification and characterization of potential antagonists of a selected gRTK, e.g., TKA. The first stage of the analysis involves phosphorylation of the kinase domain of the kinase receptor, for example, b5 of the TKA receptor, and the receptor is present in the cell membrane of a eukaryotic cell. The receptor may be an endogenous receptor or a nucleic acid encoding the receptor, or the receptor construct may be transformed into a cell. Typically, the first solid phase (for example, the well of the first analytical tablet) is covered with a substantially homogeneous population of such cells (usually mammalian cell lines) so that the cells adhere to the solid phase. Often the cells are attached to the substrate and, with this, they naturally attach to the first solid phase. If a "receptor construct" is used, it usually includes a fusion of a kinase receptor and a signal polypeptide. The signal polypeptide is recognized by a capture agent, often a capture antibody, in the EGISA portion of the assay. A test substance, such as a prospective anti-MOB antibody or other MOB antagonist, is then added along with the MOE to a cell-attached well so that a tyrosine kinase receptor (e.g., a TKA receptor) is exposed to (or in contact with) the MOB and the substance. that /o is analyzed. This assay enables the identification of antibodies (or other MOP antagonists) that inhibit the activation of TKA by its MOP ligand. After contact with the MOE and the analyte, the attached cells are solubilized using a lysis buffer (which contains a solubilizing detergent) and gentle mixing, releasing a cell lysate that can be subjected directly to the EHISA portion of the assay without the need to concentrate or illuminate the cell lysate

The cell lysate obtained in this way is then ready to be subjected to the EGISA stage of analysis. As the first step in the EHI5ZA stage, the second solid phase (usually the well of the EHIZA titration plate) is coated with a capture agent (often a capture antibody) that specifically binds to the tyrosine kinase receptor or, in the case of a receptor construct, to a signal polypeptide. The coating of the second solid phase is carried out so that the capture means is attached to the second solid phase. The capture agent is generally a monoclonal antibody, but as described in the examples herein, polyclonal antibodies may also be used. The resulting cell lysate is then exposed to or contacted with an attachment capture agent so that the receptor or receptor construct is attached to (or captured in) the second solid phase. A washing step is then performed to remove unbound cell lysate, leaving the captured receptor or receptor construct. The attached or captured receptor or receptor construct is then exposed to or contacted with an anti-phosphotyrosine antibody that identifies phosphorylated tyrosine residues in the receptor tyrosine kinase.

In one embodiment, the anti-phosphotyrosine antibody is linked (directly or indirectly) to i) an enzyme that catalyzes the change in color of a non-radioactive color reagent. Accordingly, the phosphorylation of the receptor can be measured by the subsequent change in the color of the reagent. The enzyme can be directly linked to an antibody against phosphotyrosine, or a conjugating molecule (eg, biotin) can be conjugated to an antibody He! zo against phosphotyrosine, and the enzyme can be further linked to an antibody against phosphotyrosine by means of a conjugation molecule. Finally, the binding of the anti-phosphotyrosine antibody to the captured Me receptor or receptor construct is measured, for example, by a color change in a color reagent. with

MOB antagonists can also be identified by incubating a promising agent with MORE and monitoring any one or more of the following characteristics: (a) binding to MOB and inhibiting the biological activity of MORE and/or the downstream pathway(s) mediated by the signaling function MOR; (B) so blocking or reducing the activation of MOB receptors; (c) increasing the output of MOB; (a) inhibition of MOPE receptor activation (including dimerization and/or autophosphorylation of TKA); (e) treatment, relief or prevention of any aspect of pain, particularly in combination with NSAIDs; (Her) 50 inhibition (reduction) of synthesis, production or release of MOB; (9) enhancement of NSAID pain treatment3. In some embodiments, the antagonist "

MOR is identified by incubation of a promising agent with MOR and monitoring of binding and accompanying reduction or neutralization of MOR biological activity. Binding analysis can be performed with purified . MOPE polypeptide(s) or cells that naturally express or are transfected to express and? polypeptide (polypeptides) MOB. In one embodiment, the binding assay is a competitive binding assay that assesses the ability of a prospective antibody to compete with a known antagonist

MOR for linking MOR. The analysis can be performed in various formats, including the EGISA format. In others

Go! in variants of implementation, the MOPE antagonist is identified by incubating a promising agent with MOB and monitoring the concomitant inhibition of dimerization and/or autophosphorylation of the TtKkA receptor. - After initial identification, the activity of a promising antagonist against MOE can be further confirmed and refined by biological assays, which are known to test targeted types of biological activity. 5or Alternatively, biological assays can be used to directly screen candidates. For example, MOPE contributes to a number of morphologically recognizable changes in the responding cells. These include, but are not limited to, facilitation

Ge) differentiation of RS 12 cells and enhancement of neurite growth from these cells (Opeg ei ai!., Viospet. 36:4775-4781 (1997); Tvotsaz ei aII., Meshgop 10:975-990 (1993)), promotion of neurite growth from explants of responsive sensory and sympathetic ganglia, (Hemi-MopiaiYisipi, K. Apdeieci, R. Megme dgouy Tasiog. RnuzioI. Chem. 48, 534-569, 1968) and promoting the survival of neurons dependent on MOR, such as neurons of the embryonic dorsal root ganglion, trigeminal ganglion, or sympathetic ganglion |eg, Syp 4: Raitzeggop,

F) Oem. Axes 75:705-711, (1977); (1993) (1993). Thus, the assay for inhibiting the biological activity of MORE involves culturing MORE-responsive cells with MORE plus an analyte, such as a promising MORE antibody and a promising MORE antagonist. After a suitable time, the reaction of the cells (cell differentiation, neurite outgrowth or cell survival) should be analyzed.

Determining the ability of a prospective MOPE antagonist to block or neutralize biological activity

MOR can also be performed by monitoring the ability of a promising agent to inhibit MOG-mediated survival in an embryonic rat dorsal root ganglion survival bioassay as described in 65B (Copdo et al., Nubgidota 19:215-227 (2000)). The method of identification of modulators of MOE activity is described in |PCT/О52004/016091.

Compositions

Compositions of the invention include an effective amount of a MOB antagonist (such as an anti-MOP antibody) and NSAIDs, as described in various embodiments described herein. In some embodiments, the compositions additionally contain a pharmaceutically acceptable excipient. In some embodiments, the composition is intended for use in any of the methods described herein (such as methods of treating postoperative pain). Examples of such compositions, as well as how to compose them, are also described in the previous section and below. The antagonist of MORE and NSAIDs can be present in the same composition or be present in the form of separate compositions. Accordingly, in some embodiments, the MOE antagonist and the NSAID are present in the same tyu! of the same composition. In other embodiments, the MOE antagonist and the NSAID are present in separate compositions.

In another aspect, the invention provides a synergistic composition of a MORE and NSPZ3Z antagonist.

In some embodiments, the invention provides pharmaceutical compositions comprising a MOPE antagonist for use in the treatment of pain (such as postoperative pain), wherein said use includes simultaneous and/or sequential administration of NSAIDs. In some embodiments, the invention provides pharmaceutical 7/5 Compositions comprising NSAIDs for the treatment of pain, wherein said use includes simultaneous and/or sequential administration of a MOR antagonist. In some embodiments, the invention provides pharmaceutical compositions comprising a MOB antagonist and an NSAID for separate, simultaneous, and/or sequential administration for the treatment of pain. In some embodiments, the MOR antagonist is an anti-MOR antibody (such as an EZ antibody as described herein). In other embodiments, the NSAID is ibuprofen. In still other embodiments, the MORE antagonist is an anti-MORE antibody, and the NSAID is ibuprofen.

It is understood that the composition may include more than one MOR antagonist. For example, a composition may include more than one member of a class of MORE antagonists (eg, a mixture of anti-MOB antibodies that recognize different epitopes of MOB), as well as members of different classes of MORE antagonists (eg, anti-MOB antibodies and a compound that inhibits MOR). Other illustrative compositions include more than one antibody against MOB that recognize the same epitope(s), different types of antibodies against MOB that bind to different epitopes of MOB, or different compounds that inhibit MOB. In other embodiments, the composition includes one or more antagonists and)

MOPs selected from the group consisting of an antagonist that binds to (physically interacts with) a MOP (e.g., an antibody), an antagonist that binds to a MOB receptor (such as a TIkKA receptor or a p7B receptor), and an antagonist that reduces (inhibits and/or blocks) downstream signaling of the MOP receptor. Gee! zo The composition used in the present invention may, in addition, include pharmaceutically acceptable carriers, excipients or stabilizers (Ketipdiop: Te Zsiepse ap Rgasiise oi Rpagtasu 201 Ea. (2000) Iirripsoi Me

MUyShiate apa UMiikip5, Ea. K.E. Noomeg)|), in the form of lyophilized compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients in the dosages and concentrations used and may include buffers such as phosphate, citrate and other organic acids; antioxidants, --

35 Including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; co-hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues), proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, p-va) with mannose or dextran; chelating agents such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, 7p-protein complexes); ;" or non-ionic surface-active substances, such as TMUEEM tm, RI KOS tm or polyethylene glycol (PEb).

Pharmaceutically acceptable excipients are further described herein.

The compositions described herein may contain additional compounds known to be useful in the treatment of pain. The antagonist of MORE and NSAIDs and their compositions can also be used in combination with other means that serve to enhance and/or supplement the effectiveness of the means. - In other embodiments, the present invention provides compositions (described in this description) for use in any of the ways described in this description, or in the context of use as a medicinal product, and/or for use in the manufacture of a medicinal product. o Kits (Che) The invention also provides kits for use in these methods. Kits of the invention include one or more containers that include a MOR antagonist (such as an anti-MOR antibody), an NSAID, and in some embodiments, further include instructions for use according to any of the methods described herein. In some embodiments, the kit includes an anti-MOB antibody (such as an EZ antibody described herein). In other embodiments, the kit includes an anti-MOP antibody that includes one or more (F, SOC antibodies, EZ (eg, 1, 2, 3, 4, 5, or, in some embodiments, all 6 SOCs with EZ). The kit may, to further include a description of selecting an individual to be treated based on identifying whether the individual has pain or is at risk of pain In some embodiments, the invention provides kits for use with any of the methods described in this description, and the specified set includes a MOP antagonist.

In still other embodiments, the kit includes an anti-MOR antibody. In still other embodiments, the kit includes a humanized anti-MOB antibody (such as the EZ antibody described herein). In still other embodiments, the instructions include a description of administering a MOB antagonist in combination with an NSAID for the treatment, prevention, and/or relief of any pain (such as postoperative pain, pain associated with a burn, b5 rheumatoid arthritis, or osteoarthritis).

In some embodiments, the kit includes a MOE antagonist (such as an anti-MOP antibody), an NSAID, and instructions for administering the MOE antagonist and an NSAID simultaneously and/or sequentially to effectively treat pain. In another embodiment, the kit includes a MOPE antagonist (such as an anti-MOR antibody) and instructions for administering a MOB antagonist (such as an anti-MOR antibody) and an NSAID in combination with each other to effectively treat pain. In other embodiments, the kit includes a MOPE antagonist (such as an anti-MOB antibody), and

NSAIDs (such as ibuprofen) and instructions for administration of a MORE antagonist and an NSAID in combination with each other for effective pain management. Accordingly, any methods described in this description may be reflected in the instructions.

In some embodiments, the kit includes an anti-MOP antibody. In other embodiments, the 7/0 anti-MOP antibody is an antibody comprising the heavy chain variable region shown in Table 1 and the light chain variable region shown in Table 2. In still other embodiments, the antibody against

MOR is an EZ antibody as described herein.

The MOB antagonist (such as an anti-MORE antibody) and the NSAID can be present in separate containers or in the same container. It is understood that the kit may include one single composition or two or more compositions, 7/5 wherein one composition includes a MORE antagonist and one composition includes an NSAID.

The kits of this invention are in the appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (eg sealed Muiag bags or plastic bags) and the like. Sets may optionally contain additional components such as buffers and interpretation information.

Instructions for use of a MOPE antagonist generally include information on dosage, regimen, and route of administration for the intended treatment. Containers can be standard doses, bulk packages (eg, multidose packages) or substandard doses. The instructions provided in the kits of the invention are usually written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions are also acceptable (e.g., instructions on a magnetic or optical storage disk ).

The label or package insert indicates that the composition is used for the treatment, relief and/or and) prevention of pain (including postoperative pain). Instructions may be provided to perform any of the methods described herein.

The kits of this invention are in the appropriate packaging. Appropriate packaging includes, but not Ge! zo is limited to vials, bottles, cans, flexible packaging (such as Muiag sealed bags or plastic bags) and the like. Also contemplated are packages for use in combination with a special device, such as an inhaler, a device for intranasal administration (eg, a nebulizer), or an infusion device, such as a minipump. The kit may have a sterile access channel (e.g., the container may be a bag or vial of intravenous solution having a stopper that can be pierced by a needle --

For subcutaneous injections). The container may also have a sterile access channel (for example, the container may be a bag or vial of an intravenous solution having a stopper pierced by a hypodermic needle). At least one active agent in the composition is a MORE antagonist, such as an antibody against MORG. The container may additionally include a second pharmaceutically active agent. "

Sets may optionally provide additional components such as buffers and interpretation information. c c Typically, a set includes a container and a label or package insert(s) on or associated with the container.

In some embodiments, the invention provides manufactured articles that include the contents described above;" sets In some embodiments, the kits include a MORE antagonist (such as an anti-MORE antibody) and/or

NSAIDs3 with information indicating use for the treatment of pain (in combination with each other).

Administration of an MOR antagonist and an NSAID and assessment of treatment

Go! The antagonist of MORE and NSAIDs can be administered to an individual by any acceptable route. For example, they can be administered together or separately, orally, intravenously, sublingually, subcutaneously, intraarterially, intramuscularly, intranasally, rectally, intrathoracically, intraperitoneally, into the ventricles of the brain, transdermally, or by inhalation. they can be administered orally, for example, in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, chewing gum, lollipops, suppositories, or the like, obtained by procedures recognized in the art. It will be obvious to a specialist in this field that

Although the examples described in this specification are not intended to be limiting, but merely illustrative of the techniques that E.

Accordingly, in some embodiments, a MORE antagonist, such as an anti-MORE antibody, is administered to an individual according to known methods, such as intravenously, for example, as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, spinally

F) brain, subcutaneous, intra-articular, intra-synovial, sub-membranous, oral, inhalation or local routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid compositions can be sprayed directly, and lyophilized powder can be sprayed after dissolution.

Alternatively, a MORE antagonist (such as an anti-MORE antibody) can be administered as an aerosol using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized and ground powder.

Site-specific or targeted local delivery techniques can also be used for administration. 65 Examples of site-specific or targeted local delivery techniques include various implantable depot sources of MOR and/or NSAID3 antagonist, or local delivery catheters such as infusion catheters, implantable or needle catheters, synthetic grafts, adventitial wraps, shunts or other implantable devices, site-specific carriers, direct injection, the use of a patient-controlled analgesia (PCA) technique or device, and/or direct application |see, for example, PCT Publication Mo 00/53211 and US Patent Mo59815681I.

Various compositions of agents (MOR antagonists), such as antibodies against

MOR, or their fragments. In some embodiments, agents such as anti-MOR antibodies, or fragments thereof, can be administered without additives. In some embodiments, means comprising an antibody against MOPE may be in various compositions, including compositions comprising a pharmaceutically acceptable excipient. 7/0 Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the introduction of a pharmacologically active substance. For example, an excipient can add shape or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, osmolality changing salts, encapsulating agents, buffers and skin permeation enhancers. Excipients, as well as compositions for parenteral and non-parenteral delivery of 7/5 drugs are outlined in the Ketipdiop, ei aII., Te Zsiepse apa Rgasiise oi Rpaptasu 201p Ea.

Mask Rybiizpipo (2000)).

In some embodiments, these agents (MOR antagonists) are formulated into compositions for injection (for example, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, 20 dextrose solution, and the like. The specific dosage schedule, ie dose, time of administration and repetition, will depend on the specific individual and the medical history of that individual. The administration regimen (including the MOPE antagonist(s) used) may vary over time.

Antibodies against MOPE can be administered using any suitable method, including injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Antibodies against MOG can be administered by inhalation as described in this description. In general, for administration of anti-MOE antibodies, the initial possible dosage may be about 0.2 mg/kg or about 2 mg/kg. In some embodiments of implementation (8), the usual daily dosage may be in the range of about 300 µg/kg to 300 µg/kg, to 3000 µg/kg, to

Zmg/kg, up to ZOmg/kg, up to 10Omg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs or until therapeutic levels sufficient to reduce pain are achieved. An illustrative schedule of administration includes administration of an initial dose of approximately 2 mg/kg followed by a weekly maintenance dose of approximately mg/kg of anti-MOB antibody or followed by a maintenance dose of approximately mg/kg every other week. However, other administration schemes can be used, depending on the type of pharmacokinetic decay that the physician wishes to achieve. Other input schemes --

Зз5 Include the scheme up to 1 time a day, from 1 to 4 times a week, or less often. In some embodiments, the compounds are administered about once a week, about 1 to 4 times a month. Dosage of antibodies against MOB is described in this description. The course of treatment is easily monitored by conventional methods and analyses.

In some embodiments, when it is not an antibody, the MOPE antagonist according to the invention can be administered in an amount of 0.1 to ZbOmg/kg of the patient's body weight, divided into 1-3 doses or as disclosed in this description. In some adult patients of normal body weight, doses ranging from approximately 0.3 to 5.00 mg/kg can be administered. The specific dosage scheme, that is, the dose, time of administration and repetition, will depend on . of a specific individual and the medical history of this individual, as well as from the properties of individual means and? (such as the half-life of the agent and other considerations well known in the art).

3 NSAID can be administered at dosage levels up to the usual dosage levels for such analgesics. In some embodiments, the NSAID is administered at a reduced level. Appropriate dosage levels will depend on

Go! the analgesic effect of the selected NSAID, but generally acceptable levels are about 0.001 to 25 mg/kg/day, about 0.005 to 10 mg/kg/day, or about 0.05 to 1 mg/kg/day, or less. The compound can be administered according to the scheme up to 6 times a day (or more), 1-4 times a day, or it can be administered less often. In some embodiments, the NSAID is administered continuously or very frequently (eg, as in RA).

When administered in combination either as one or as a separate composition(s), nerve growth factor antagonists and NSAIDs are present in a ratio that is consistent with the manifestation of the desired effect. In some

Ge) in embodiments, the ratio by weight between the nerve growth factor antagonist and the NSAID will be about 1 to 1. In some embodiments, the ratio may be between about 0.001 to about 1 and about 1000 to about 1, about 0.01 to about 1 and 100 to about 1, or vv about 0.1 to about 1 and about 10 to about 1. Other ratios are contemplated. It should be understood that the amount of nerve growth factor antagonist and NSAID3Z required for use in the treatment of ai

F) pain prevention will vary not only with the specific compounds or compositions selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, the course or stage of treatment, and ultimately, it will to be determined at the discretion of the attending physician. For example, because the appropriate dosage of a MOR antagonist (such as an anti-MOR antibody) will depend on the MOR antagonist(s) (or combinations thereof) used, the type and severity of pain that is intended to be treated, and whether the agent is administered prophylactically or therapeutically, previous therapy, the patient's clinical history and reaction to the drug and at the discretion of the attending physician. Typically, the clinician will administer a MOG antagonist (such as an anti-MOP antibody) until a dosage that achieves the desired effect is reached. 65 Empirical considerations such as half-life will generally influence dosage determination.

For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and selected during the course of treatment, and is generally, but not necessarily, based on treating and/or suppressing, and/or alleviating, and/or delaying the onset of pain. Alternatively, sustained release compositions of a MORE antagonist and/or an NSAID may be appropriate. Various compositions and devices for achieving sustained release are known in the art.

In one embodiment, the dosage for the MOE antagonist can be determined empirically in individuals who have received one or more administrations of an agent that inhibits MOP activity to treat pain. 70 Individuals are administered increasing doses of an agent that inhibits MOPE, such as an anti-MOB antibody, in combination with an NSAID3. To assess the effectiveness of treatment, you can monitor the pain index.

Administration of the MOB antagonist and/or NSAID in accordance with the method of this invention can be continuous or intermittent, depending, for example, on the physiological state of the recipient. From whether the purpose of administration is therapeutic or prophylactic, and from other factors known to experienced practitioners. /5 The administration of the MOR antagonist can be essentially continuous for a preselected period of time, or it can be made as a series of doses administered at intervals, for example, either before, during, or after the development of pain; before and after; during and after; before and during; or before, during, and after the development of pain.

For example, the administration can be carried out before, during and/or after a wound, cut, trauma, surgery and any other phenomenon that is likely to cause the development of pain.

In some embodiments, more than one MOP antagonist, such as an antibody, may be present.

Antagonists can be the same or different from each other. At least 1, at least 2, at least 3, at least 4, at least 5 or more different MOR antagonists may be present. In general, these MOE antagonists have additive activity that does not adversely affect each other.

MOE antagonists can also be used in combination with other means that serve to enhance and/or supplement the effectiveness of the means.

In some embodiments, more than one NSAID33 may be present. NSAIDs can be the same i) or different from each other. At least 1, at least 2, at least 3, at least 4, at least 5 or more different NSAIDs3 may be present. In general, these NSAIDs have additional activity that does not adversely affect each other. NSAIDs can also be used in combination with other Ge! with means that serve to strengthen and/or supplement the effectiveness of the means (means).

Therapeutic compositions of the MORE antagonist (such as an antibody) and NSAIDs used in accordance with the present invention are prepared for storage by mixing an antibody of the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers. ,

Tne Zsiepse apa Rgasiise oi Rpagtasu 201 Ea. Musk Rybiizpipd (2000)) in the form of lyophilized compositions or (-і-7

From AQUEOUS solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride, antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylarabenes such as methyl- or propylparaben; catechol; resorcinol; "cyclohexanol; 3-pentanol and m-cresol); polypeptides of low molecular weight (less than about 10 residues), suture) with proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; ;" monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextran; chelating agents such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, 2p-protein complexes); and/or non-ionic surfactants

Go! substances such as TUMEEM tm, RI OKOMISztm or polyethylene glycol (RES).

Liposomes containing a MORE antagonist (such as an antibody) are prepared by methods known in the art, such as those described in (publications Erzieyip, ei aiYi, Rhos. Mai. Asad. Zsi. OBA 82:3688 (1985); Nuapo, ei AI, ka Rgos. Mai! Asad. Zsi. OA 77:4030 (1980); and US MoMo patents 4485045 and 4544545). Liposomes with an increased circulation time of 5° are disclosed in (US patent No. 5013556). Particularly useful liposomes can be generated by reverse phase evaporation with a lipid composition including phosphatidylcholine, cholesterol and

Ge) derivatized RES phosphatidylethanolamine (RES-RE). Liposomes are extruded through filters with a certain pore size to obtain liposomes with the desired diameter.

Active ingredients can also be enclosed in microcapsules obtained, for example, by coacervation or interfacial polymerization methods, for example, respectively, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, in colloidal drug delivery systems (for example, liposomes, iF), albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are disclosed in (the guide Ketipadiop, ei aiY., Te 5siepse apa Rgasiise oi Rpagtasu 201p Ed. Mask Rybiizpipo (2000)).

Extended-release formulations are available. Suitable examples of sustained-release agents include semipermeable matrices of solid hydrophobic polymers containing an antibody, and the matrices are presented in the form of products having a certain shape, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol)), polylactides (US patent Mo3773919), copolymers of I-glutamic acid and 7-ethyl-I-glutamate, non-degradable ethylene vinyl acetate, biodegradable lactic acid-glycolic acid copolymers, b5, such as GORKOM OEROT tm (injectable microspheres consisting of a lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetisobutyrate and poly-Y-(-)- 3-hydroxybutyric acid.

The compositions to be used for the introduction of ip mimo should be sterile. This is easily done, for example, by filtering through sterilizing filtration membranes. Therapeutic compositions of a MOR antagonist (such as an anti-MOR antibody) are generally contained in a container having a sterile access channel, for example, a bag with a solution for intravenous injections or a vial having a stopper pierced by a hypodermic needle.

The compositions according to this invention can be presented in the form of standard dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for 7/0 oral, parenteral or rectal administration, or administration by inhalation or insufflation.

To obtain solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example, conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or resins, and other pharmaceutical diluents , for example, with water, to form a solid precomposed composition, 7/5 containing a homogeneous mixture of the compound of this invention or its non-toxic pharmaceutically acceptable salt. When these pharmaceutical compositions are called "homogeneous" it is meant that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily divided into equally effective standard dosage forms such as tablets, pills and capsules. This solid pre-formulated composition is then divided into standard dosage forms of the type described above, containing from about 0.01mg to about 00.1mg to about 500mg of the active ingredient of this invention. Tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form having the advantage of prolonged action.

For example, a tablet or pill may include an internal dosage component and an external dosage component, the latter being presented in the form of a shell around the former. These two components can be separated by an enteric layer, which serves to resist destruction in the stomach and allows the content of the internal component to pass intact into the duodenum or delay its release. A variety of materials can be used for such enteric layers or coatings, and such materials i) include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

Liquid forms in which the compositions of this invention can be included for oral administration or Ge! by injection, include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical carriers . Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural resins such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin. Active ingredients (87 of 5 May also be included in controlled-release products with high viscosity, such as sucrose acetate isobutyrate or others. These compositions can be used either for oral administration or for injection. Injection can lead to formation of a local depot agent, which is released locally during a period from 1 day to 3 months.

Compositions for injection include compositions containing a MOE antagonist and an NSAID as active "ingredients in combination with a surface-active agent (or wetting agent or surface-active 7) substance) or in emulsion form (in the form of a water-in-water emulsion) oils or oils in water).

Suitable surface-active agents include, in particular, nonionic agents such as polyoxyethylene sorbitans with (eg, Tmeeptm 20, 40, 60, 80 or 85) and other sorbitans (eg, Zraptm 20, 40, 60, 80 or 85).

Compositions with a surface-active agent can contain from 0.05 to 595 surface-active agent, or from 01 to 2.595. It should be understood that, if necessary, other ingredients can be added, for example, mannitol or other pharmaceutically acceptable carriers.

Suitable emulsions can be obtained using commercially available fat emulsions, such as Ipigairiam, - POrosuptm, Iphopcigoi"m, | Iroipapiptm and Ipirpuzaptm. The active ingredient can either be dissolved in a pre-mixed emulsion composition or, alternatively, it can be dissolved in oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and an emulsion formed after mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids, and Che ) or soybean lecithin) and water. It should be understood that other ingredients, such as glycerin or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions should normally contain up to 2095 oil, e.g. 5 to 2095. A fat emulsion may include droplets of fat from 0.1 to 1.0 µm, in particular from 0.1 to 0.5 µm, and have a pH in the range of 5.5 to 8.0.

In some embodiments, the compositions are compositions obtained by mixing the nerve growth factor antagonist with Ipigaiiridtm or its components (soybean oil, egg phospholipids, glycerin and water). eat) Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or their mixtures and powders. Liquid or solid compositions may contain appropriate pharmaceutically acceptable excipients as described above. The compositions are administered by the oral or intranasal respiratory route for local or systemic effect. Compositions in mostly sterile pharmaceutically acceptable solvents can be sprayed using gases. Sprayed solutions may be inhaled directly from the nebulizer, or the nebulizer may be attached to a face mask, tent, or intermittent positive pressure ventilator. Compositions 65 in the form of a solution, suspension or powder can be administered (including orally or intranasally) from devices that deliver the composition in an appropriate manner.

Treatment effectiveness can be assessed by methods well known in the art.

The following examples are presented to illustrate and not to limit the invention.

Examples

Example 1

Treatment with a monoclonal antibody against MOB in combination with NSAIDs for postoperative pain

Applicants used a pain model that mimics postoperative pain to evaluate the efficacy of an anti-MORE antibody in combination with the NSAID, ibuprofen. For each experiment, 16 male adult Zrgadce rats

OBauleu with a body weight of 200 to 220 g (Napap; Ipaiaparoiiv, IM) were kept under normal light conditions of 7/0 for at least 1 week before use with unrestricted access to food and water. After a 2-hour acclimatization period in the test chambers on the day of surgery, rats were divided into two groups: one group received the antibody 15 hours before surgery, the other at this time received the vehicle (595 dextrose/0.4596 saline

Pharmacopoeia of the United States). Antibody 911 against MOB antagonist |see Nopoo, ei ai., Nubgidota 19:215-227 (2000)| administered in a dose of Tmg/kg of body weight. Ibuprofen was administered in various concentrations ranging from 10, 30, 100 to 10 mg/kg 7/5 (subcutaneously) 24 hours after surgery to all animals.

The operation was based on the procedure described by (Vheppap, et al., Research 64:493-501 (1996)). Animals were anesthetized with 295 isoflurane and air mixture, which was maintained during surgery, through the nasal funnel.

The plantar surface of the right hind paw was prepared with a swab with povidone-iodine, a central longitudinal incision 1 cm long was made through the skin and fascia, starting 0.5 cm from the edge of the heel and continuing to 2 toes. Measurements were made with a ruler with the foot held in a bent position. The plantaris muscle was lifted using tweezers with bent branches, and a longitudinal incision was made in it. The muscle was cut to the full depth, between the place of its departure and the area of attachment. Bleeding was stopped during the entire operation by applying pressure with a gauze tampon. The wound was closed with two mattress sutures (black monofilament thread "E(Pisop" 5-0). These suture threads were tied with knots 5-6 times at the first knot, which was loosely tight. The wound area was wiped with a solution of bacitracin. The animals were given the opportunity to recover and rest in clean cages for 22 hours before behavioral i) testing began.

For each experiment, animals were divided into two groups (control and antibody-treated).

The antibody against MORE was administered 15 hours before the operation. Pain at rest was assessed 22 hours after Ge! from the operation in both groups (initial state' in the following graphs). 20 hours after surgery, all animals were then treated with ibuprofen at a dose of 10, 30, 100 or ZbOmg/kg (subcutaneously). Pain at rest was assessed starting 1 hour after treatment with ibuprofen. with

Pain at rest was assessed at various times after surgery using a cumulative pain score.

Rats were placed on a plastic grid (grid: mm?) in a transparent plastic cage and given the opportunity to acclimatize for 15-20 minutes. The behavior was evaluated on a scale from 0 to 2. A score was given if the animal rested on the cut paw, according to the evaluation data, if it was noted that the paw was pale or rested on the grid. A score of 17 was given if the animal spared the paw when the skin was just touching the grid without paleness or pitting of the skin. A score of 2 was given if the paw was kept completely without touching the grid. Each animal was observed for 1 minute every 5 minutes for 30 minutes. A sum of 6 70 points (total 0-12) obtained within 1/2 hour was used to assess pain in the cut foot. - c The results of these experiments are shown in Table 1 and in Fig.1.

AND"

ZOmg/kg, 10Omg/kg or ZOOmg/kg 1 day after surgery. Data are shown as mean value (standard deviation). Data were analyzed using one-way analysis of variance, and then individual pairs were analyzed using the Wopieggopi (ee) correction for multiple comparisons using Rgilt software with 1 variance | scrap 00 zom | zoomkye 00 zom z

F and 3e) As shown in Table 1, the pain score at rest for rats treated with Ma 911 (at a dose of 1 mg/kg) was significantly lower than in the control without ibuprofen (p<0.001). Similarly, pain score at rest when treated with Mar 911 at a dose of mg/kg and 1Omg/kg of ibuprofen was significantly lower than when treated with ibuprofen alone at a dose of 1Omg/kg (re0.001); and pain score at rest when treated with Mab 911 at a dose of mg/kg and 0.00 mg/kg of ibuprofen was significantly lower than when treated with ibuprofen alone at a dose of 0.00 mg/kg (p«0.05). Fig. 1 shows the point assessment of pain at rest, measured in animals treated with an antibody against MOB in a dose of mg/kg and without it and treated with different doses of ibuprofen and without it. Preoperative treatment with an antibody against

MPA and ibuprofen is more effective in reducing pain at rest than ibuprofen alone or treatment with 60 antibody alone. It is believed that treatment with Mab 911 (mg/kg) in combination with ibuprofen at a dose of 10mg/kg is at least as effective as ibuprofen alone at a dose of 300mg/kg.

To test the effect of anti-MOB monoclonal antibody 911 treatment in combination with diclofenac for the treatment of postoperative pain, experiments were performed as described above, except that the animals were administered vehicle or diclofenac at mg/kg instead of ibuprofen. The results are shown in Fig.2. 65 A reduction in pain score was observed for the mean value with treatment with both 911 mg/kg and diclofenac 5 mg/kg, compared with treatment with diclofenac 5 mg/kg alone.

Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

Claims (15)

2 Formula of the invention 1. A method of treating pain in an individual, comprising administering to the individual an effective amount of an anti-nerve growth factor antibody (MORE) and an NSAID3, wherein the anti-(MORE) antibody and an NSAID in combination provide 70 effective pain relief. 2. The method according to claim 1, in which the NSAID is selected from the group consisting of ibuprofen, naproxen, naprosyn, diclofenac, ketoprofen, tolmetin, sulindac, mefenamic acid, meclofenamic acid, diflunisal, flufenizal, piroxicam, sudoxicam, isoxicam, celecoxib, rofecoxib . C. The method of claim 1, wherein the NSAID is ibuprofen. 4. The method according to claims 1, 2 or 3, in which the anti-MORE antibody binds human MOR. 5. The method of claim 4, wherein the anti-MORE antibody binds human MO with a binding affinity of about 10 nM or less than about 10 nM. 6. The method according to any one of claims 1-5, in which the anti-MORE antibody is a human antibody. 7. The method according to any one of claims 1-5, in which the anti-MORE antibody is a humanized antibody. 8. The method according to claim 7, in which the humanized antibody is an antibody that includes the variable region of the heavy chain shown in ZEO IU MO:1 and the variable region of the light chain shown in ZEO IU MO:2. 9. The method according to any one of claims 1-8, in which the pain is postoperative pain. high school 10. The method according to item 1, where the individual is a person. 11. A pharmaceutical composition for the treatment of pain in an individual, comprising an effective amount of antibody (o) against MOB and NSAIDs and a pharmaceutically acceptable carrier. 12. The pharmaceutical composition according to claim 11, in which the anti-MORE antibody is an antibody that includes the variable region of the heavy chain shown in the ZEO IUO MO:1 and the variable region of the light chain shown in the ZO V FAILURE MO:2. The pharmaceutical composition of claim 11, wherein the individual is a human. b" 13. A kit for treating pain in an individual comprising an anti-MOE antibody and an NSAID and instructions for administering an anti-MOE antibody in combination with an NSAID to treat pain in the individual. 14. The kit according to claim 14, in which the anti-MORE antibody is an antibody that includes the variable region - c5 of the heavy chain shown in ZEO I MO and the variable region of the light chain shown in ZEO II MO:2. co 15. The kit of claim 14, wherein the individual is a human. shi s ;" (ee) - ime) o 50 iChe) Ф) ime) 60 b5