WO2002009736A1 - Methods and agents for treating persistent pain - Google Patents

Abstract

Disclosed are methods and an animal model for modulating persistent pain in a subject, and for identifying novel agents capable of ameliorating persistent pain. The methods of the invention are based on the finding that enhanced activation of NMDA receptor function in the forebrain, in particular by increased expression of the NR2B subunit, is associated with an increase in long-term reaction to a persistent pain stimulus.

Description

METHODS FOR MODULATING PERSISTENT PAIN AND FOR IDENTIFYING AGENTS TO TREAT PERSISTENT PAIN

This application claims priority to U.S. Provisional Application No. 60/221,029, filed July 27, 2001, the entirety of which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of neurobiology. In particular, the invention provides assay methods for identification of substances useful for the treatment of persistent pain, and methods for modulating persistent pain, which relate to down-regulation of NMD A receptor activity in the forebrain.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application to more fully describe the state of the art to which this invention pertains. Full citations of any publications not fully cited within the specification may be found at the end of the specification. The disclosure of each such publication is incorporated by reference herein.

NMDA (N-methyl-D-aspartate) receptors mediate a slow, voltage- dependent synaptic current at glutamatergic synapses throughout the central nervous system (CΝS). Calcium influx through ΝMDA receptors triggers a wide range of brain processes, including the synaptic plasticity associated with memory formation, neuronal death in ischemia, and central sensitization during persistent pain. Functional ΝMDA receptors contain heteromeric combinations of the ΝR1 subunit plus one or more of Ν 2A-D, of which the NR2A and NR2B subunits are the major NR2 subtypes found in forebrain structures. Compared to NRl-NR2B-containing heteromers, NMDA receptors formed by co-expression NR1 and NR2A in heterologous cells mediate currents that decay three to four times faster. Forebrain NMDA receptors are composed almost exclusively of NR1 and NR2B subunits at birth, gradually incorporating more NR2A subunits during postnatal development. This developmental decrease in the NR2B:NR2A ratio, complete by the third or fourth postnatal week in rodents, parallels a decrease in the duration of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs). In transgenic mice with forebrain-targeted NR2B overexpression, these developmental changes in NMDA receptor kinetics were reversed (Tang et al., Nature 401: 63-69, 1999). NR2B subunit expression, driven by the α-calcium/calmodulin- dependent protein kinase U, was observed extensively throughout the cerebral cortex, striatum, amygdala, and hippocampus, but not in the thalamus, brainstem, cerebellum or spinal cord (Tang et al., 1999, supra). Presumably by increasing the NR2B-.NR2A ratio in NMDA heteromeric complexes, this manipulation led to marked alterations in the physiology of hippocampal synapses (Tang et al., 1999, supra). First, in mature, cultured hippocampal neurons, NMDA receptors mediated more slowly decaying currents and four-fold greater charge transfer in transgenic versus wild-type cells. Second, in hippocampal slices prepared from adult mice, hippocampal CAl synapses exhibited increased susceptibility to long-term potentiation in NR2B transgenic animals. As adults, these NR2B transgenic mice exhibited superior performance on a battery of learning- and memory-related behavioral tasks compared to wild-type adults (Tang et al., 1999, supra). As mentioned above, the functionality of the NMDA receptor is also involved in the perception of pain in mammals. Pain can occur following acute or chronic injury to the peripheral or central nervous system, arising from a variety of causes that include traumatic injury or inflammation. Persistent pain can result from either of these causes, and is often more difficult to manage than acute pain, using conventional analgesics.

Attempts have been made to treat persistent pain with antagonists of the NMDA receptor. For instance, keta ine, dextromethorphan and CPP (3-(2- carboxypiperizin-4-yl)-propyl-l-phosphonic acid) have been reported to produce relief in several neuropathies, including postherpetic neuralgia, central pain caused by spinal cord injury, and phantom limb pain (Kristensen et al., Pain 51: 249-253, 1992; Eide et al., Pain 61: 221-228, 1995; Knox et al., Anaesth. Intensive Care 23:620-622, 1995; Max et al., Clin. Neuropharmacol. 18: 360-368 1995). However, these agents also induce unacceptable side effects at analgesic doses, including hallucination, dysphoria and disturbances of cognitive and motor function (Boyce et al., Neuropharmacol. 38: 611-623, 1999). It has been reported that an NR2B-selective NMDA antagonist, CP-

101,606, has potent analgesic activity in rat hyperalgesia and nociceptive tests at doses that do not cause behavioral abnormality (Taniguchi et al., Br. J. Pharmacol. 122: 809- 812, 1997). It was shown through binding of radiolabeled drug that CP-101,606 was most dense in regions of the central nervous system in which NR2B subunits are selectively expressed (i.,e., the hippocampus). However, the drug was found to be distributed throughout the brain and spinal cord, however, so it was not discerned whether the drug's specific effect on NR2B in the spinal cord or the brain contributed more significantly to the observed analgesic effect. It has been shown that NR2B subunits, in addition to their distribution in the brain, are distributed throughout the rat lumbar spinal cord, mainly in fibers in laminae I and JJ of the dorsal horn (Boyce et al., \999, supra).

Thus, NR2B-targeted antagonists appear to be attractive candidates for the treatment and management of pain. However, the specific mechanisms by which NR2B-specific agonists act in vivo to exert an analgesic effect is not clear; hence a rational basis for testing NR2B-targeted compounds for their antinociceptive effect is presently unavailable. It would be an advance in the art to identify in vivo the means by which NR2B-specific antagonists act as antinociceptive agents, and to utilize this information for the management of pain.

SUMMARY OF THE INVENTION

The present invention provides assay methods for identification of substances useful for the treatment of persistent pain, and methods for modulating persistent pain, which relate to down-regulation of NMDA receptor activity in the forebrain. According to one aspect of the invention, a method of reducing or eliminating persistent pain in a patient in need of such treatment is provided. The method comprises modifying NMDA receptors in neural synapses of the patient's forebrain to decrease channel decay time in the NMDA receptors, the modification resulting in reduction or elimination of the persistent pain in the patient.

According to another aspect of the invention, a genetically altered non- human animal is provided, which displays increased sensitivity to persistent pain as compared with an equivalent, but unaltered animal. The animal expresses a gene encoding NR2B to a greater extent in its forebrain than does the equivalent, but unaltered animal.

According to another aspect of the invention, a method is provided for identifying compounds that inhibit long-term reaction to a persistent pain stimulus in a subject by decreasing expression of NR2B genes in the forebrain of the subject. This method comprises providing a chimeric DNA construct comprising an NR2B promoter operably linked to a reporter gene, contacting the chimeric DNA construct with a test compound suspected of down-regulating the NR2B promoter, and measuring expression of the reporter gene, a decrease in the expression being indicative that the test compound inhibits long-term reaction to a persistent pain stimulus in the subject.

According to another aspect of the invention, an in vivo assay for identifying compounds that inhibit long-term reaction to a persistent pain stimulus by down-regulating NMDA receptors in the forebrain of a subject is provided. This method comprises: (a) providing a non-human transgenic animal that expresses an NR2B transgene in its forebrain; (b) treating the transgenic animal with a test compound suspected of down-regulating NMDA receptor function; and (c) directly or indirectly measuring NMDA function in the forebrain of the treated animal as compared with an equivalent untreated animal, a decrease in NMDA in the forebrain of the treated animal being indicative that the test compound inhibits long-term reaction to persistent pain by down-regulating NMDA receptor function in the forebrain of the subject.

Other features and advantages of the present invention will be understood by reference to the drawings, detailed description and examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Pharmacological inhibition of forebrain NMDA receptors. (Fig. 1 A) The tail-flick latency was similar before (4.1 ± 0.4 sec) and 10 min after (4.0 ± 0.3 sec) MK-801 injection i.c.v. (n = 7 mice). (Fig. IB) The hot-plate latency was similar before (10.2 ± 0.8 sec) and 10 min after (9.8 ± 1.0 sec) MK-801 injection (n = 7 mice). (Fig. 1C) Behavioral responses to subcutaneous injection of formalin into one hindpaw were significantly decreased in mice injected with MK-801 i.c.v. 10 min previously (filled circles; n = 7) compared to untreated, control mice (open circles; n = 9). (Fig. ID) Injection of MK-801 60 min after formalin injection (between phases 2 and 3) inhibited phase 3 responses (filled circles; n = 4) compared to the same control mice displayed in Fig. 2C. (Fig IE) Summarized results from Figs. 2C and 2D. * indicates significant difference from control.

Figure 2. Forebrain-targeted NR2B overexpression enhances NMDA receptor-mediated synaptic responses in the ACC and insular cortex but not spinal cord. (Fig. 2 A) Diagram of a frontal cortical slice taken from an adult mouse, showing the placement of recording and stimulating electrodes in the ACC and insular cortex.(Fig. 2B) Traces of NMDA receptor-mediated fEPSPs recorded from the ACC and insular cortex in the presence of 20 μM CNQX (see panel C for scale bars). (Fig. 2C) Bath application of 100 μM AP-5 completely (upper traces) and reversibly (lower trace) blocked NMDA receptor-mediated fEPSPs in the ACC. Similar results were found in insular cortex (not shown). (Fig. 2D) The input (stimulation intensity, 200 msec duration)-output (fEPSP slope) relationship in the ACC (open squares, wild- type, n = 8; filled squares, transgenic, n = 12) and insular cortex (open circles, wild- type, n = 6; filled circles, transgenic, n = 9) reveals enhanced NMDA receptor- mediated responses in transgenic relative to wild-type mice (p < 0.001). Examples of fEPSP traces recorded from the ACC in wild-type and transgenic animals are shown above. We also integrated fEPSPs in the ACC and found an increased area-under-the- curve for transgenic (33.2 ± 4.3 mV'ms) relative to wild-type mice (19.1 ± 3.3 mV»ms, p < 0.05). Similar results were found in the insular cortex (transgenic, 33.1 ± 6.1 rnV-ms vs. wild-type, 17.0 ± 2.7 mV'ms; p < 0.05). (Fig. 2E) Diagram of a spinal slice showing the placement of intracellular recording and stimulating electrodes. (Fig. 2F) Traces of EPSPs recorded in the presence of 20 μM CNQX, after bath application of 100 μM AP-5 (upper traces) and after washout (lower trace). (Fig. 2G) Summarized data of the input (stimulating intensity)-output (EPSP slope) for NMDA receptor-mediated responses in the spinal cord dorsal horn (open squares, wild-type, n = 4; filled squares, transgenic, n = 6). Examples of EPSP traces recorded from dorsal horn neurons in wild-type and transgenic animals are shown above.

Figure 3. Acute nociceptive responses in wild-type and NR2B overexpressing mice. (Fig. 3A) The spinal nociceptive tail-flick reflex latency was similar in wild-type (6.64 ± 0.35 sec; n = 7) and NR2B transgenic mice (6.16 ± 0.31 sec; n = 9). (Fig.3B) The hot-plate response latency was similar in wild-type (50 °C, 31.8 ± 2.3 sec; 52.5 °C, 17.7 + 1.6 sec; 55 °C, 11.3 ± 1.5 sec; n = 6-12) and NR2B transgenic mice (50 °C, 31.3 ± 3.0 sec; 52.5 °C, 19.4 ± 2.1 sec; 55 °C, 12.8 ± 1.8 sec; n = 6-11). (Fig. 3C) The cold-plate response latency was similar in wild-type (20.6 ± 2.9 sec; n = 12) and NR2B transgenic mice (19.3 ± 3.1 sec; n = 11). Figure 4. c-Fos expression in different areas of the central nervous system. Numbers of c-Fos positive cells in the anterior cingulate cortex (ACC), the SI and S2 regions of somatosensory cortex (SI, 2), insular cortex, the CAl subfield of the hippocampus, the periaqueductal gray (PAG), and spinal cord laminae I- VI (SCDH) are illustrated in wild-type and NR2B transgenic mice with hindpaw injection of saline (control; n = 4 wild-type, 6 transgenic) or formalin (n = 4 wild-type, 4 transgenic). Cells were counted contralateral to the injected hindpaw except in the spinal cord, where cells were counted ipsilaterally. * indicates significant difference from wild-type and transgenic controls; f additionally indicates significant difference from wild-type, formalin-treated mice. Figure 5. Enhanced behavioral responses to formalin or CFA injection in NR2B transgenic mice. (Fig. 5 A) The number of seconds during which wild-type (open squares, n = 16) and N 2B transgenic mice (filled squares, n = 9) were engaged in nociceptive behavioral responses to hindpaw formalin injection were plotted in 5 min intervals. (Fig. 5B) Summarized results from (Fig. 5A). * indicates significant difference from wild-type mice. (Fig. 5C) The responses of animals to a mechanical stimulus (a 0.4 mN von Frey fiber applied to the dorsum of a hindpaw) that elicited no responses before dorsal hindpaw CFA injection were recorded 1 and 3 days after injection. The data were plotted as percent positive responses to stimulation of the ipsilateral or contralateral hindpaw (relative to side of injection) for wild-type (open squares, n = 4) and NR2B transgenic (filled squares, n = 4) mice. * indicates significant difference between wild-type and transgenic mice in the indicated conditions.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions: Various terms relating to the present invention are used hereinabove and also throughout the specifications and claims.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed. The term "operably linked" or "operably inserted" means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

The terms "promoter", "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3¹ direction) coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A "vector" is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

The term "nucleic acid construct" or "DNA construct" is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA" or "transgene". Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.

The term "selectable marker gene" refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.

The term "reporter gene" refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.

A "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA (transgene) may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. If germline cells are stably transformed, the transformation may be passed from one generation of animals arising from the germline cells, to the next generation. In this instance, the transgene is referred to as being inheritable.

The term "subject" as used herein refers to a human subject or a non- human animal subject, or it may refer to any other living organism. The term "patient" may be used interchangeably for the term "subject." The term "acute pain" refers to an unpleasant sensation induced by noxious stimuli. It is short-lasting and can occur without any tissue injury.

The term "persistent pain" refers long-lasting, unpleasant sensations that are often related to tissue injury. Persistent pain lasts long after the initial noxious stimulus producing acute pain is gone, and may persist from days to years. Other definitions are found in the description set forth below.

II. Description:

NMDA receptors play an important role in long-term plasticity in the brain. In persistent pain, central sensitization requires activation of spinal NMDA receptors, but a role for forebrain NMDA receptors heretofore had not been established. In accordance with the present invention, it has now been shown that intracerebro ventricular injection of an NMDA receptor antagonist inhibits behavioral responses of animal subjects to subcutaneous formalin injection (a standard model for inflammatory pain), leaving acute nociceptive reflexes intact, hi transgenic animals with forebrain-targeted overexpression of the NMDA receptor subunit NR2B (a manipulation previously shown to augment charge transfer through NMDA channels), formalin-induced c-Fos expression was enhanced in pain-related forebrain structures. In addition, transgenic animals displayed selectively exaggerated late-phase formalin- induced behavioral responses. These results are believed to be the first evidence that genetic modification of forebrain NMDA receptor kinetics can influence nociceptive processing.

Utilizing the above-summarized discoveries, the present invention provides a method to reduce or eliminate persistent pain in a patient in need of such treatment. The method involves down-regulating charge transfer through NMDA channels in the forebrain of the patient. As described hereinabove and in greater detail in the examples, it has been shown through the forebrain-targeted administration of NMDA antagonists and through the behavioral studies of transgenic animals with augmented NMDA channel charge transfer in the forebrain, that such treatment results in the reduction or alleviation of persistent pain.

The activity of NMDA receptors in the forebrain can be down- regulated in a variety of ways. It is believed that any means by which NMDA function in the forebrain can be down-regulated will yield the same antinociceptive effect as observed in subjects treated with NR2B antagonists in their forebrains. For instance, methods to inhibit NMDA receptor function in the forebrain include, but are not limited to: (1) forebrain-targeted administration of compounds that act directly or indirectly to reduce NMDA receptor function, preferably, but not exclusively, by targeting NR2B; (2) modulating expression of NMDA receptor subunits at the transcriptional (e.g., promoter) and/or translational level (which may include down- regulation of NR2B gene expression or up-regulation of upstream transcription factors for other NR2 subunits, including NR2A, C or D, to reduce the ratio of NR2B to other NR2 subunits in the forebrain); and (3) use of agents that act inside cells of the forebrain at the intracellular domains of the NMDA receptor, to interfere with the interaction between the receptor and its downstream targets.

In a preferred embodiment, NR2B subunits are targeted for negative regulation. In one embodiment, this is accomplished by forebrain-targeted administration of NR2B-selective antagonists, examples of which are already available. Alternatively, somatic cells of subjects may be stably or transiently transformed with a vector encoding an antisense molecule or ribozyme, or a transcription suppressing protein, for instance, designed to inhibit expression of NR2B. Such "DNA therapy", for instance would comprise targeted administration of an NR2B expression- inhibiting vector which, upon delivery to the target cells, would inhibit the production of NR2B, thus reducing the pool of NR2B subuit available for incorporation into NMDA receptors. DNA therapy to transiently produce such NR2B expression- inhibiting molecules in targeted brain locations is accomplished according to methods well known in the art. For instance, the forebrain may be selectively targeted by using a promoter that is specific for gene expression in that region, such as a promoter derived from the CaMKII gene, whose activity has been demonstrated to be restricted to the forebrain region (Mayford et al., Cell 81, 891-904, 1995).

The present invention also provides an animal model system useful for the discovery of new substances that can reduce or alleviate persistent pain. This animal model system comprises NR2B transgenic animals. As exemplified by the NR2B transgenic mice described herein, these animals exhibit enhanced late-phase response to a persistent pain stimulus, such as the inflammation that results from injection with formalin or CFA.

The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Examples of animals preferred for use in the present invention include, but are not limited to, rodents, most preferably mice and rats, as well as cats, dogs, dolphins and primates.

A "transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term "transgenic animal" is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule, i.e., a "transgene". The term "transgene", as used herein, refers to any exogenous gene sequence which is introduced into both the somatic and germ cells or only some of the somatic cells of a mammal. This molecule maybe specifically targeted to defined genetic locus, or be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term "germline transgenic animal" refers to a transgenic animal in which the transgene was introduced into a germline cell, thereby conferring the ability to transfer the transgene to offspring. If such offspring in fact possess the transgene then they, too, are transgenic animals.

The transgene of the present invention includes without limitation, the entire coding region of an NR2B gene, or its complementary DNA (cDNA), or chimeric genes containing part or all of a NR2B coding region, whose expression in the forebrain is driven by a tissue specific promoter. It is preferable, but not essential, that the NR2B coding sequence used in the transgene be of the same species origin as the transgenic animal to be created.

Nucleic acid sequences encoding NR2B have been reported for several species. Examples include mouse (GenBank Accession No. U60210), and human (GenBank Accession No. NM 000834).

The promoter is comprised of cis-acting DNA sequences capable of directing the transcription of a gene in the appropriate tissue environment and, in some cases, in response to physiological regulators. The promoter preferred for use in the present invention is derived from the aCaMKII gene, whose activity has been demonstrated to be restricted to the forebrain region (Mayford et al., Cell 81 : 891-904, 1995). Other promoters are also known to direct the expression of exogenous genes to specific cell-types in the brain. Promoters useful for stem cell transformation, wherein tissue specificity is needed, include any promoter whose endogenous genes are expressed in the target cell of interest; e.g., the pkcγ promoter, the telencephalin promoter, the neuronal enolase promoter and the prp promoter. For somatic transformation, tissue specific promoters may or may not be needed. Thus, constitutive promoters, such as the CMV promoter or the β-actin promoter should prove useful for somatic transformation. Methods to obtain transgenic, non-human mammals are known in the art. For general discussions, see, e.g., Joyner, "Gene Targeting," IRL Press, Oxford, 1993; Hogan et al. (Eds.), "Manipulating the Mouse Embryo - A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1994; and Wasserman & DePamphilis, "A Guide to Techniques in Mouse Development," Academic Press, San Diego CA, 1993. One method for introducing exogenous DNA into the germline is by microinjection of the gene construct into the pronucleus of an early stage embryo (e.g., before the four-cell stage) (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 5016, 1981; Brinster et al., Proc. Natl. Acad. Sci. USA 82, 4438, 1985). The detailed procedure to produce NR2B transgenic mice by this method has been described (Tsien et al, Cell 87: 1317-26, 1996). Another method for producing germline transgenic mammals utilizes embryonic stem cells. The DNA construct may be introduced into embryonic stem cells by homologous recombination (Thomas et al., Cell 51: 503, 1987; Capecchi, Science 244: 1288, 1989; Joyner, et al, Nature 338: 153, 1989) in a transcriptionally active region of the genome. A suitable construct may also be introduced into the embryonic stem cells by DNA-mediated transfection, such as electroporation

(Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, 1999). Detailed procedures for culturing embryonic stem cells and methods of making transgenic mammals from embryonic stem cells may be found in Teratocarcinomas and Embryonic Stem Cells, A practical Approach, ed. E. J. Robertson (JJ L Press, 1987).

Other methods for producing germline transgenic animals are being developed currently. For instance, instead of eggs being the recipients of exogenous DNA, sperm are now being genetically manipulated.

In any of the foregoing methods of germline transformation, the construct may be introduced as a linear construct, as a circular plasmid, or as a viral vector which may be incorporated and inherited as a transgene integrated into the host genome. The transgene may also be constructed so as to permit it to be inherited as an extrachromosomal plasmid. The term "plasmid" generally refers to a DNA molecule that can replicate autonomously in a host cell. Transgenic animals also may be obtained by infection of neurons either in vivo, ex vivo, or in vitro with a recombinant viral vector carrying an NR2B gene. Suitable viral vectors include retroviral vectors, adenoviral vectors and Herpes simplex viral vectors, to name a few. The selection and use of such vectors is well known in the art.

The present invention also provides a variety of assays and other methods, which utilize the inventors' discovery of the effect of NMDA receptor function on the enhancement of persistent pain.

One useful assay is an in vitro assay for identifying compounds that inhibit persistent pain by decreasing expression of NR2B genes. This assay involves the following basic steps: (1) provide a chimeric DNA construct comprising an NR2B promoter operably linked to a reporter gene; (2) contact the chimeric DNA construct with a test compound suspected of down-regulating the NR2B promoter, and (3) measure expression of the reporter gene. A decrease in the expression of the reporter gene in the presence of the test compound indicates that the test compound will be useful in the management of persistent pain by decreasing the expression of NR2B genes.

The NR2B transgenic animals of the invention maybe used for several in vivo assays. For instance, an in vivo assay, useful for identifying compounds that negatively affect activation of NMDA receptors in a mammal, comprises the following steps: (1) provide an NR2B transgenic animal; (2) treat the transgenic animal with a test compound suspected of negatively affecting activation NMDA receptors; and (3) directly or indirectly (i.e., biochemically or by behavioral tests) measure a change in activity of the treated animal as compared with the untreated animal, a negative change being indicative that the test compound down-regulates activation of NMDA receptors in the animal. This assay can be extended by measuring behavioral responses to persistent pain stimuli in the transgenic animals, inasmuch as such responses will be more robust in these animals as compared with non-transgenic animals, and differences caused by various test compounds will be more apparent.

It will be appreciated that assays similar to the in vivo assays discussed above can be developed easily in cultured cells. For instance, cultured neuronal or non-neuronal cells may be transformed with a DNA construct for expression of NR2B, optionally together with NR1 subunit or other NR2 subunits (NR2A, NR2C, NR2D), and those cells used for various biochemical and physiological assays to assess the changes resulting from the presence of the transgene. In another embodiment, cells or tissue slices from NR2B transgenic animals may be utilized for a similar purpose.

The following example is set forth to illustrate embodiments of the invention. It is not intended to limit the scope of the invention in any way.

EXAMPLE 1

Genetic Enhancement of Persistent Pain by Forebrain NR2B Overexpression

This example sets forth genetic evidence that forebrain NMDA receptors are important for persistent pain in mice. Forebrain-targeted NR2B overexpression led to selectively exaggerated late-phase nociceptive behavioral responses after peripheral formalin injection, a widely used and clinically relevant animal model of pathologically prolonged pain after tissue injury (Dubuisson and Dennis, Pain 4: 161-174, 1977; Tjolsen et al., Pain 79: 105-111, 1992).

Experimental Procedures:

Animals. Both wild-type and NR2B transgenic mice were prepared as described by Tang et al., 1999. Adult male mice weighing 15-29 g were used. Behavioral experiments were carried out in a quiet environment and performed during the day. Room temperature was always maintained at 20°C. All behavioral experiments were performed blind.

Acute behavioral nociceptive tests.

Tail-flick test. The spinal nociceptive tail-flick reflex was evoked by focused, radiant heat applied to underside of the tail. The latency to reflexive removal of the tail away from the heat was measured by a photocell timer to the nearest 0.1 sec. The mean tail-flick latency was calculated as the average of 3-4 measurements performed at 10 min intervals.

Hot-plate test. Mice were placed on a thermally-controlled metal plate (Columbia Instruments; Columbus, Ohio). The time between placement of a mouse on the plate and licking or lifting of a hindpaw was measured with a digital timer. Mice were removed from the hot plate immediately after the first response. The mean hot-plate latency was calculated as the average of 3-4 measurements performed at 10 min intervals.

Cold-plate test. Mice were placed in a plastic container (21cm diameter x 21cm high) resting on a bed of ice. The temperature of the ice surface was monitored with a digital thermometer and maintained at 0°C by placing additional ice around the container. The time between placement of a mouse on the cold plate and the first jumping or licking of the hindpaw was measured with a digital timer. Mice were removed from the plate after the first response. The mean cold-plate latency was calculated as the average of 3-4 measurements performed at 10 min intervals.

Formalin-induced persistent pain. Formalin (5%, lOμl) was injected subcutaneously into the dorsal side of a hindpaw. Mice respond by licking the inj ected hindpaw, and this behavior is typically concentrated in three distinct phases: a first phase (0-10 min), a second phase (10-60 min) and a third, late phase (60-120 min) (Kim et al, Brain Res. 829: 185-189, 1999). The total time spent licking or biting the injected hindpaw was recorded during each 5 min interval over the course of2 hr. Intracerebro ventricular drug injection. Intracerebro ventricular injection of MK-801 was carried out by standard methods. To avoid possible stress during the injection, mice were always lightly anesthetized with halothane (2 %); recovery from anesthetic was complete in 2-3 min. The injection was monitored by observing the movement of air behind the solution. The injection sites were confirmed by examining tissue sections prepared after experiments.

Immunostaining. At 120 min after hindpaw formalin or saline injection, mice were deeply anesthetized with halothane (3-4% in a gaseous mixture of 70% N₂O and 30% O₂) and perfused through the ascending aorta with 50 ml of saline, followed by 200 ml of cold 0.1 M phosphate buffer containing 4% paraformaldehyde. Cryostat-cut brain sections (30 μm) were immunocytochemically processed with anti-c-Fos rabbit antibody (1:20,000; Oncogene Science, Uniondale, NY) using Vectastain reagents and developed with DAB/nickel/GOD. Controls, performed by replacing primary antibody with 1% NGS in this protocol, exhibited no staining. Anatomical terminology is based on the atlas of Franklin and Paxinos.

The mean number of c-Fos labeled cells in each nucleus of interest was calculated using sections from 4-6 mice. The investigator responsible for plotting and counting the labeled cells was blind to the experimental situation of each animal.

Data analysis. Results were expressed as mean ± standard error of the mean (SEM). Statistical comparisons were performed with the use of one- or two- way analysis of variance (ANOVA), and the post-hoc Scheffe F-test was used to identify significant differences. In all cases, P < 0.05 was considered statistically significant.

Results:

We examined the responses of awake, adult mice to acute and persistent nociceptive stimuli, testing the effects of manipulating NMDA receptor function through both pharmacological and genetic approaches. To study acute nociception, we recorded the behavioral responses of mice in tail-flick, hot-plate, and cold-plate tests. To study inflammation-induced persistent pain, we tested the responses of mice to peripheral, subcutaneous formalin injection into a single hindpaw. The formalin test is a widely used animal model of tissue injury and inflammatory pain. Nociceptive behavioral responses to formalin, which include licking the injected hindpaw (see Experimental Procedures), occur in three distinct phases in mice: Phase 1 (0-10 min after injection) responses are thought to be due to a peripheral inflammatory response at the site of injection. Phase 2 (10-60 min) occurs after central sensitization in the spinal cord and requires ongoing activity from the periphery. Phase 3 (60-120 min) responses, which are normally less severe in comparison to the preceding phases (Kim et al., 1999, supra), occur by an unknown mechanism.

Using these nociceptive tests, we first examined the contribution of forebrain NMDA receptors by injecting the selective NMDA receptor antagonist MK- 801 (dizocilpine) intracerebroventricularly (i.c.v.) before or during experiments. Second, we tested the effects of forebrain-targeted NR2B overexpression both on nociceptive behavior and on the pattern of neuronal activation produced after formalin injection, using immediate early gene (IEG) expression to map neuronal activity at the cellular level. Animals from two different transgenic lines were studied; these lines were indistinguishable in NMDA receptor physiology, synaptic plasticity, and memory-related behavioral tasks (Tang et al., 1999, supra), and exhibited similar results in our experiments. Therefore, results from the two lines were pooled.

Pharmacological inhibition of forebrain NMDA receptors. To determine whether forebrain NMDA receptors contribute to acute or persistent nociception, we applied the potent, non-competitive NMDA receptor antagonist MK- 801 i.c.v. 10 min prior to behavioral experiments. We monitored acute nociception by measuring the latency of a mouse's response to two stimuli: focused heating of the tail (tail-flick test) and placement on a 55°C metal plate (hot-plate test). Consistent with a previous report (Suh et al., Eur. J. Pharmacol. 263: 217-221, 1994), MK-801 (1 μg/5 μl) affected the latency to response in neither test (n = 7 mice in each test) (Figure 1 A, B).

However, MK-801 pretreatment did decrease behavioral responses to formalin injection (5%, 10 μl; n = 7) compared to control mice not treated with MK- 801 (n = 9, Figure 1C, E). Mice receiving saline injection i.c.v. responded no differently to formalin than the untreated controls (data not shown). The decreases in behavioral responses were observed in all three phases. These data suggest that while forebrain NMDA receptors do not contribute to acute nociceptive reflexes, they do contribute to persistent pain after formalin injection.

To test whether phase 3 responses could be manipulated independently of phases 1 and 2, we injected MK-801 i.c.v. 60 min after hindpaw formalin injection; whereas phase 1 and 2 responses were indistinguishable between these mice and mice not treated with MK-801, phase 3 responses were inhibited after delayed MK-801 injection as much as when MK-801 injection was given prior to formalin treatment (n = 4, Figure ID, E). These data suggest that phase 3 responses could be inhibited independently of phases 1 and 2 and that inhibition of phase 3 responses did not require a long period of time for MK-801 to diffuse away from the site of injection, consistent with a forebrain-selective site of action.

While these experiments suggest that forebrain NMDA receptor activation contributes to formalin-induced persistent pain, there are some limitations. First, i.c.v. injection does not permit any direct control of the brain areas affected by MK-801. Second, NMDA antagonists possess significant psychotomimetic and neurotoxic properties, potentially confounding the interpretation of behavioral data. Third, blocking NMDA receptors does not provide any information about what electrophysiological parameters of NMDA receptor function may be important for persistent pain. Transgenic overexpression of NR2B in the forebrain and acute nociception. To test whether prolonging forebrain NMDA receptor-mediated EPSCs and enhancing charge transfer through the channels would affect the nociceptive responses of mice, we made use of mice with forebrain-targeted NR2B overexpression. Although these mice exhibited the alterations in hippocampal synaptic physiology and enhanced performance on a series of behavioral tasks associated with learning and memory described above, mating behavior, body weights, and open-field behavior were indistinguishable between wild-type and transgenic littermates, and transgenic mice exhibited no seizures (Tang et al., 1999, supra). First, we examined how forebrain-targeted NR2B overexpression affected NMDA receptor function in two pain-related forebrain areas, the anterior cingulate cortex (ACC) and insular cortex. Brain slices of these areas were prepared from adult mice, and excitatory postsynaptic field potentials (fEPSPs) were recorded upon local electrical stimulation (Fig. 2A). In each region, after blockade of AMP A and kainate receptors by CNQX (20 μM), a slow fEPSP was observed (Fig. 2B) that could be entirely and reversibly blocked by the NMDA receptor antagonist AP-5 (100 μM; Fig. 2C). Consistent with the high levels of NR2B transgene expression found throughout the cerebral cortex in transgenic mice, these mice, compared to wild-type mice, exhibited enhanced NMDA receptor-mediated fEPSPs in both the ACC and insular cortex (Fig. 2D).

In contrast, the spinal cord dorsal horn exhibited no changes in NMDA receptor-mediated synaptic transmission. Intracellular recordings were performed from dorsal horn neurons in slices from adult mice, and EPSPs were evoked by dorsal root stimulation (Fig. 2E). Slow, AP-5 -sensitive EPSPs, isolated in the presence of CNQX (20 μM) (Fig. 2F), were of similar magnitude in wild-type and transgenic slices (Fig. 2G). These findings provide direct evidence that spinal NMDA receptor function was not altered by forebrain-targeted NR2B overexpression.

To determine whether forebrain NR2B overexpression affected acute nociception, we compared the responses of wild-type and transgenic mice in the tail- flick, hot-, and cold-plate tests. No significant difference in tail-flick response latency was observed (wild-type, n = 7 mice; transgenic, n = 9 mice; Fig. 3 A), indicating that spinal nociceptive transmission was not significantly altered in transgenic mice. Hotplate response latencies were measured at three different plate temperatures (50, 52.5, and 55 °C), and no differences were found between wild-type and NR2B transgenic mice (wild-type, n = 6-12 mice; transgenic, n = 6-11 mice; Fig. 3B). Likewise, no difference in response latency was observed between groups of mice placed on a cold- plate (0°C) (wild-type, n = 12 mice; transgenic, n = 11 mice; Fig. 3C). These results indicate that behavioral responses to noxious heat or cold were indistinguishable between NR2B transgenic and wild-type mice.

Patterns of c-Fos expression in wild-type and NR2B transgenic mice after formalin injection. We next compared the responses of transgenic and wild-type mice to formalin injection, first, by examining the pattern of neuronal activation induced in each case. Neuronal c-Fos expression has been widely used as an indicator of neuronal activity induced by various stimuli. Previous studies in rats have demonstrated that formalin injection induced neuronal c-Fos expression and that this expression is believed to be related to nociception, particularly in the spinal cord, hi adult wild-type mice, we found that subcutaneous injection of formalin (5%, 10 μl) into the dorsum of a hindpaw induced c-Fos expression in various regions of the mouse brain related to nociceptive transmission and modulation (wild-type, saline- injected, n = 4; wild-type, formalin-injected, n = 6). The results listed here describe increases in c-Fos expression in formalin-injected wild-type mice compared to saline-injected wild-type mice. Unless otherwise indicated, similar c-Fos expression patterns were observed in brain regions both ipsilateral and contralateral to the injected hindpaw, with contralateral c-Fos expression being somewhat more pronounced. Among forebrain areas, formalin injection induced prominent c-Fos staining in the anterior cingulate cortex (ACC; Figure 4), lateral septal nucleus, secondary motor cortex, some nuclei in the amygdaloid complex (medial, basolateral, and cortical nuclei), piriform cortex, retrosplenial cortex, several midline thalamic nuclei (lateral habenular, paraventricular, mediodorsal, centromedial, paracentral, and anterodorsal nuclei) and various hypothalamic nuclei (paraventricular, periventricular, supraoptic, and dorsomedial nuclei). Less prominent but significant c-Fos expression was observed in the somatosensory cortex (SI, S2, and hindlimb areas) (Figure 4), the hippocampal CAl (Figure 4) and CA3 subfields, insular cortex (Figure 4), accumbens nucleus, and lateral preoptic area. A small amount of formalin-induced c-Fos expression was observed in other thalamic nuclei, including the lateral posterior nucleus, posterior group, ventral lateral, ventral posterolateral, ventral posteromedial, and ventral medial nuclei. No c-Fos expression was observed in the caudate-putamen nucleus. In the midbrain, formalin-induced tissue injury resulted in augmented c-Fos expression within all subareas of the periaqueductal gray (PAG; Figure 4), dorsal raphe, mterpeduncular nucleus, mesencephalic reticular formation, superficial gray layer of the superior colliculus, and inferior colliculus. hi the brainstem, c-Fos expression was also observed in the locus coeruleus, parabrachial nucleus, and rostroventral medulla. Finally, abundant c-Fos expression was observed in the dorsal horn of lumbar spinal cord, particularly in superficial laminae and the deep neck of the dorsal horn ipsilateral to the injected hindpaw, with much smaller but nonetheless evident expression contralaterally.

To determine whether forebrain-targeted NR2B overexpression may affect formalin-induced c-Fos expression, we examined the pattern of c-Fos immunoreactivity induced in NR2B transgenic mice after formalin injection in experiments done in parallel to those described above. In saline-injected control animals (n = 4), baseline c-Fos expression was indistinguishable between wild-type and transgenic mice (Figure 4). After formalin treatment (n = 4), the most prominent findings occurred in the ACC, insular cortex, and hippocampal CAl subfield, all of which exhibited significantly more c-Fos immunoreactivity in transgenic than wild- type formalin-treated mice (Figure 4). Similar results were noted in the lateral septal nucleus, hippocampal CA3 subfield, and accumbens nucleus. No significant differences in c-Fos staining between transgenic and wild-type mice after formalin injection were found in somatosensory cortex (Figure 4), amygdala, thalamus, or hypothalamus. In good accord with the restriction of NR2B overexpression to the forebrain, no significant differences in formalin-induced c-Fos expression between transgenic and wild-type mice were observed in the midbrain, brainstem, or spinal cord.

Consistent with NR2B overexpression directly affecting c-Fos expression in response to formalin injection, the brain areas in which significantly greater c-Fos expression was observed in transgenic than in wild-type mice were areas in which NR2B overexpression was present (Tang et al., 1999, supra). Moreover, these areas of greater c-Fos expression encompassed some parts of the limbic system and other forebrain areas known to be important for the central processing of nociceptive information, including, in particular, the ACC, lateral septal nucleus, hippocampus, and insular cortex. Importantly, no NR2B-related changes in gene expression were observed in second or third order sensory neurons or in descending brainstem analgesic systems, suggesting that alterations in the forebrain response to formalin injection had no ramifications on neuronal activity in the brainstem or spinal cord.

Enhancement of nociceptive responses in NR2B transgenic mice. Because increased c-Fos expression in the forebrain may not necessarily correspond to an increased perception of pain, we tested whether the behavioral responses to formalin injection were affected in transgenic mice. We found that while both phase 1 and phase 2 responses were similar between wild-type and NR2B transgenic mice (Figure 5), phase 3 responses were selectively enhanced in transgenic mice (wild-type, n = 16; transgenic, n = 9, Figure 5), with a peak comparable to that of phase 2. Similar results were found in the two lines of transgenic mice (line 1, n = 6; line 2, n = 3). These results suggest that forebrain NR2B overexpression selectively enhanced delayed nociceptive behavioral responses to hindpaw formalin injection. hi addition, we tested the effects of hindpaw injection of CFA in wild- type and transgenic mice. Application of a 0.4 mN von Frey fiber to the dorsum of a hindpaw elicited no response in untreated mice, but at one and three days after CFA injection (50%, 10 μl) into the dorsum of a single hindpaw, mice responded to stimulation of either the same (ipsilateral) or, to a lesser extent, the contralateral hindpaw by hindpaw withdrawal. This mechanical allodynia, or display of nociceptive response to a previously non-noxious mechanical stimulus, was significantly enhanced in NR2B transgenic mice (Fig. 5C).

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

Claims

We claim:

1. A method of reducing or eliminating persistent pain in a patient in need of such treatment, which comprises modifying NMDA receptors in neural synapses of the patient's forebrain to decrease channel decay time in the NMDA receptors, the modification resulting in reduction or elimination of the persistent pain in the patient.

2. The method of claim 1, comprising modifying the NMDA receptor function in the patient's forebrain with a chemical compound.

3. The method of claim 2, wherein the chemical compound specifically targets NR2B subunits of NMDA receptors in the patient's forebrain.

4. The method of claim 5, comprising inhibiting production of NR2B receptor subunits in the patient's forebrain, resulting in a decreased amount of NR2B subunit as compared with the amount produced by an equivalent, but untreated patient.

5. A genetically altered non-human animal having increased sensitivity to persistent pain as compared with an equivalent, but unaltered animal, wherein the animal expresses a gene encoding NR2B to a greater extent in its forebrain than does the equivalent, but unaltered animal.

6. The genetically altered animal of claim 5, wherein the animal over- expresses an endogenous NR2B gene.

7. The genetically modified animal of claim 5, wherein the animal expresses a transgene encoding NR2B.

8. The genetically altered animal of claim 5, selected from the group consisting of mouse, rat, cat, dog, dolphin and non-human primate.

9. The genetically altered animal of claim 5, wherein the genetic alteration is inheritable.

10. A method of identifying compounds that inhibit long-term reaction to a persistent pain stimulus in a subject by decreasing expression of NR2B genes in the forebrain of the subject, which comprises providing a chimeric DNA construct comprising an NR2B promoter operably linked to a reporter gene, contacting the chimeric DNA construct with a test compound suspected of down-regulating the NR2B promoter, and measuring expression of the reporter gene, a decrease in the expression being indicative that the test compound inhibits long-term reaction to a persistent pain stimulus in the subject.

11. An in vivo assay for identifying compounds that inhibit long-term reaction to a persistent pain stimulus by down-regulating NMDA receptors in the forebrain of a subject, which comprises: a) providing a non-human transgenic animal that expresses an NR2B transgene in its forebrain; b) treating the transgenic animal with a test compound suspected of down-regulating NMDA receptor function; and c) directly or indirectly measuring NMDA function in the forebrain of the treated animal as compared with an equivalent untreated animal, a decrease in NMDA in the forebrain of the treated animal being indicative that the test compound inhibits long-term reaction to persistent pain by down-regulating NMDA receptor function in the forebrain of the subject.

12. The method of claim 11 , wherein the NMDA receptor function is measured biochemically.

13. The method of claim 12, wherein the NMDA receptor function is measured by measuring peak amplitude or channel decay time of NMDA receptors.

14. The method of claim 11, wherein the NMDA receptor function is measured using behavioral tests of long term response to a persistent pain stimulus.