CA2435038A1 - Inhibition of erk to reduce or prevent tolerance to and dependence on opioid analgesics - Google Patents

Abstract

Inhibition of mitogen-activated protein kinases (MAPKs), or more particularly extracellular signal regulated kinases (ERKs) inhibits the development of tolerance to opioid analgesics. Therefore, methods for reducing tolerance, reducing the risk of physical dependence, reducing hyperalgesia, reducing the symptoms of opioid withdrawal or inhibiting pain sensitization are described.
MAPK inhibition at the spinal level represents a powerful treatment modality for chronic pain, blocking both neural sensitization induced by pain and factors limiting the effectiveness of opioids, the strongest analgesics currently in use to treat chronic pain.

Description

DESCRIPTION
INHIBITION OF ERK REDUCES OR PREVENTS BOTH TOLERANCE TO
AND DEPENDENCE ON OPIOID ANALGESICS AND SENSITIZATION
AFTER PAINFUL STIMULATION
BACKGROUND OF THE INVENTION
This application claims priority to U.S. Provisional Patent Application Serial Number 60/264,336, which is specifically incorporated by reference. The government may own rights in the present invention pursuant to grant number DA11500 from The National Institutes of Health.
I. Field of the Invention The present invention relates generally to the fields of anesthesiology and more specifically pain management. More particularly, it provides a variety of compositions and methods involving ERK inhibition for implementation with respect to treatment of chronic pain, opioid tolerance and dependence, withdrawal symptoms and pain sensitization.
II. Description of Related Art Tolerance and dependence remain major impediments to the use of opioids in chronic pain states. Tolerance is a group of homeostatic changes in specific neural circuitry that decreases analgesic efficacy and leads to physical dependence upon opioids. Morphological, electrophysiological, and neurochemical changes are all seen in these circuits (Nestler et al., 1997). Several CNS regions have been identified as important mediators of tolerance and physical dependence, such as the nucleus accumbens (NAcc), ventral tegmental area (VTA), arcuate nucleus (Arc), amygdala (Amy), periaqueductal grey (PAG), locus coeruleus (LC), rostral ventromedial medulla (RVM), and nucleus tractus solitarius (NTS) (Koob et al., 1992), as well as the spinal cord (Gutstein et al., 1993). At the cellular level, opioid receptor function is altered.
Receptor desensitization and downregulation have been observed after opioid administration and proposed as mechanisms underlying tolerance development (Cox, 1991). Desensitization is an acute decrease in signaling responses. This phenomenon occurs within a matter of minutes, which does not parallel the time course of ChrOIllc tolerance development. However, desensitization could potentially be the first in a complex series of layered adaptations leading to chronic tolerance. Receptor downregulation or internalization has also been suggested to play a role in tolerance development. However, for many years behavioral studies demonstrated morphine tolerance with no changes in receptor density, and recent in vitro studies have shown that while all agonists can induce desensitization and tolerance, receptor internalization and downregulation only occurs with certain opioids (Whistler et al., 1998). Thus, it appears that complex intracellular signaling events leading to long-term phenotypic changes may be primarily responsible for the development of tolerance and dependence.
Many of the known adaptations associated with tolerance and dependence represent alterations in signals mediating acute opioid effects. In spite of many recent advances, the understanding of this process is incomplete. It is known that opioid receptors generally couple to G; and Go classes of G proteins and acutely inhibit adenylylate cyclase (AC), inhibit calcium conductance and activate a potassimn conductance, leading to hyperpolarization of the cell (Childers, 1991).
Opioids may also activate protein kinase C (PI~C). PI~C activates the NMDA receptor, a known mediator of tolerance and dependence. GRy subunits, NO/cGMP and calcium/calmodulin kinase pathways have also been implicated in opioid signaling. With chronic administration, some pathways undergo adaptive changes that appear to oppose the acute effects of opioids. For instance, in some brain areas acute opioid administration decreases, while chronic application increases AC activity (Nestler et al., 1997). The resultant changes in protein kinase A (PKA) activation leads to changes in the activity of other proteins regulating gene expression and opioid signaling. These changes are held in check by continued opioid administration, but unmasked when opioids are withdrawn, leading to hyperexcitability and increased signaling in affected systems. These changes may underlie the clinical withdrawal syndrome.
While some biochemical and anatomical data relevant to tolerance and dependence have been generated, results are conflicting and no behavioral studies necessary to determine clinical relevance have been performed. Also, the role ERK may play in the genesis of chronic pain is completely unexplored. The role of RGS
proteins in tolerance and dependence are totally unknown. It is therefor an object of this invention to demonstrate a method for reducing tolerance and dependence on opioids used for the reduction of chronic pain SUMMARY OF THE INVENTION
Thus, the present invention contemplates the spinal inhibition of ERK. This is used in the treatment of postoperative or chronic pain, either as a sole agent or in combination with spinally administered substance such as morphine.
The present invention is directed at methods and compositions for patients involving an ERK inhibitor and relating to preventing tolerance to an analgesic; reducing tolerance to an analgesic; reversing tolerance to an analgesic; preventing or reducing physical dependence (addiction) on an analgesic as well as reducing the risk of physical dependence on an analgesic; reducing or inhibiting pain sensitization;
reducing or inhibiting hyperalgesia; preventing or reducing the symptoms of withdrawal from an analgesic; and other symptoms, conditions, or diseases involving the mitogen-activated protein (MAP) pathways, particularly pathways involving an extracellular signal-regulated kinase (ERK), which is a MAP kinase (MAPK). It is also contemplated that embodiments discussed with respect to one of the methods or compositions discussed herein may be implemented with respect to any of the other methods and compositions discussed herein. ERK may be implemented with respect to any of the methods and compositions of the invention that specify a MAP kinase or MAPK.
Addiction is characterized by a continuous craving for an opioid and the need to use it for effects other than pain relief. Physical dependence is used herein interchangeably with addiction. Tolerance refers to a patient's decreased response to the continued use of a drug or the patient's need for increasing doses to maintain a constant response. A patient who is physically dependent on an opioid may suffer from withdrawal when the opioid intake is reduced. Opioid withdrawal symptoms include:

craving, dilated pupils, diarrhea, elevated blood pressure, fever, insomnia, irritability, lacrimation, nausea, piloerection, restlessness, rhinorrhea, tachycardia, vomiting, and yawning.
In some embodiments an analgesic is involved. As used herein, an analgesic refers to a medication that reduces or eliminates pain. Examples of analgesics include opioid analgesics, nonopioid analgesics (NSAIDs and acetaminophen), and adjuvant analgesics. In preferred embodiments, an analgesic may be an opiate or opioid, which are used interchangeably to refer to a compound containing opium or one of more of its derivatives. Opiates are sedative narcotics, both natural and synthetic, that include a number of substances, including substances referred to as' morphine, heroin, codeine, hydromorphone, oxycodone, meperidine (Demerol), diphenoxylate, hydrocone, fentanyl (Sublimaze), and propoxyphene (Darvon). It is contemplated that methods and compositions of the invention may directed at these particular opiates.
The present invention concerns also ERK inhibitors. Two ERKs have been identified: ERKl and ERK2. Herein, "ERK" encompassed both forms. The cDNA
sequence and amino acid sequence of human ERKl corresponds with SEQ ID NO:1 and SEQ ID N0:2, respectively. The cDNA sequence and amino acid sequence of human ERKZ corresponds with SEQ ID N0:3 and SEQ ID N0:4, respectively. It is contemplated that embodiments of the present invention may involve either ERKl or ERK2, or both. .An "ERK inhibitor" refers to a substance that reduces or eliminates ERK
activity, that is, the ability to phosphorylate an ERIC substrate. Embodiments of the invention include an ERK inhibitor that inhibits or reduces ERK protein expression, amount of ERK protein or level of ERK translation, amount of ERK transcript or level of ERK transcription, stability of ERK protein or ERK transcript, half life of ERff~ protein or ERK transcript, prevents the proper localization of an ERK protein or transcript;
reduces or inhibits the availability of ERK polypeptide, reduces or inhibits ERK activity;
reduces or inhibits ERIC, binds ERK protein, or inhibits or reduces the post-translational modification of ERIC, including its phosphorylation.
In some embodiments of the present invention, the ERK inhibitor is a chemical compound. The chemical compound may be SL-327, UOI26, PD098059, or PD184352.

In some embodiments the inhibitor is U0126. Alternatively, the inhibitor may be a polypeptide such as a mutated ERK polypeptide or a mutated MEK polypeptide. In some embodiments, the mutated polypeptide is a dominant negative, such as a ERK or MEK polypeptide lacking kinase activity, that is, it is unable to phosphorylate a 5 substrate. Specifically contemplated as part of the invention is a mutated MEK
polypeptide that is unable to phosphorylate an ERK polypeptide, even under conditions that would allow for phosphorylation. In some embodiments of the invention, the ERK
polypeptide comprises at least 10, 25, 50, 75, or 100 contiguous amino acids of SEQ ID
NO:2 or SEQ ID N0:4. Alternatively, in other embodiments, a MEK polypeptide comprises at least 10, 25, 50, 75, or 100 contiguous amino acids of SEQ ID
N0:6 or SEQ ID N0:8.
In other embodiments of the invention, the inhibitor comprises a nucleic acid molecule. It is contemplated that the inhibitor may be an ERK antisense nucleic acid, such that it is complementary to an ERK-encoding nucleic acid and/or to an ERK
control region and/or to an ERK intron. In other embodiments, the inhibitor is a nucleic acid encoding at least part of an ERK polypeptide. Further embodiments include a nucleic acid that encodes a polypeptide inhibitor, such as a mutated ERK or MEK
polypeptide.
In some embodiments, a nucleic acid comprises a sequence identical or complementary to at least 30, 50, 70 or 100 contiguous nucleotides of SEQ ID N0:1, SEQ ID
N0:3, SEQ ID NO:S, or SEQ ID N0:7. It is specifically contemplated that a nucleic acid of the present invention may be complementary to all or part of SEQ ID NO:l, SEQ ID
N0:3, SEQ ID NO:S, or SEQ ID N0:7. A nucleic acid is "strictly complementary" if the nucleic acid is completely complementary.
The invention includes vectors, including plasmids, and expression constructs that include nucleic acid molecules of the present invention. Specifically contemplated are viral vectors, including an adenovirus vector, an AAV vector, a retrovirus vector, a herpesvirus vector, a lentivirus vector, or vaccinia virus vector. Vectors of the invention may be employed to deliver a nucleic acid, such as one that encodes an ERK
inhibitor or an ERK antisense nucleic acid, to a cell. Delivery of a vector to a cell may involve a Iipid composition. Any type of cell is contemplated as a target of the present invention, however, neurons and other neuronal cells are specifically contemplated. Any cells in the spine are also specifically contemplated as targets for the present invention.
Patients who may benefit from methods and compositions of the present invention include patients who are in chronic pain, have or will undergo surgery, have taken or will take an opiate or other analgesic, experience or will experience pain, have or are susceptible to having pain sensitivity, have or are susceptible to having hyperalgesia, as well as,patients who are addicted or are physically dependent on opiates, patients who are at risk for opiate addiction, patients who have tolerance to an analgesic, and patients who may suffer or are suffering from opiate withdrawal symptoms.
A
patient is administered an "effective amount" of an ERK. inhibitor, which means the patient is given an amount of an ERIC inhibitor effective to effect a particular result. The result includes: preventing tolerance to an analgesic; reducing tolerance to an analgesic;
reversing tolerance to an analgesic; preventing or reducing physical dependence (addiction) on an analgesic as well as reducing the risk of physical dependence on an analgesic; reducing or inhibiting pain sensitization; reducing or inhibiting hyperalgesia;
preventing or reducing the symptoms of withdrawal from an.analgesic; and inhibition or reduction in ERK or MEI~ activity andlor expression levels.
In some embodiments, as part of the methods of the claimed invention, there is a step of identifying a patient in need of treatment. Such a patient may be in need of prevention, reduction, or reversal of tolerance to an analgesic; in need of reducing or preventing physical dependence on an opioid; in need of reducing, eliminating, or preventing hyperalgesia or pain sensitization; or in need of preventing or reducing opioid withdrawal symptoms experienced by the patient. Another step in embodiments of some methods is evaluating the extent to which the ERK inhibitor was effective after it has been administered to the patient. After an ERK inhibitor is administered, a patient may be evaluated for a reduction in tolerance to an analgesic, for reversal in tolerance to an analgesic, for prevention of tolerance to an analgesic, for reduction of physical dependence on an analgesic such as an opioid, for elimination of physical dependence on an analgesic, for reduction or elimination of hyperalgesia, for reduction or elimination of pain sensitization, for reduction or elimination of symptoms of opioid withdrawal, which includes reduction in the severity of those symptoms.

In some cases, reduction of tolerance is to such an extent as to effect a reversal of tolerance to an analgesic. Likewise, reduction of physical dependence or addition on an opioid may be to such an extent that elimination of physical dependence is effected. A
reduction or elimination of physical dependence may also effect a reduction or elimination of withdrawal symptoms.
In further embodiments, the patient is administered another compound in addition to an effective amount of an ERK inhibitor. A patient may be given an analgesic or opiate; an opiate such as methadone, buprenorphine, or levoacetyl methadol; a-adrenergic receptor agonist-such as clonidine; a tranquilizer; a benzodiazepine, such as Librium or Valium; or a combination thereof. In some cases, the patient is being given an opiate to reduce pain, and the ERK inhibitor is administered to reduce or prevent tolerance. In other cases, the patient is addicted to an opiate and the opioid is given to the patient to reduce withdrawal symptoms. In some cases, an addicted patient may also be given non-opioid compounds to reduce withdrawal symptoms or to reduce pain.
An ERK inhibitor and additional compounds may be administered to the patient at the same time, and in some embodiments, in the same formulation.
Alternatively, the ERK inhibitor may be administered or given before the other compound, or the other compound may be given to the patient before the ERK inhibitor. In some embodiments, there may be not more than minutes, l, 2, 3, 4, 5, 6, 12, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, 4, or 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8,9 10, 11, or 12 months between administration of an ERK inhibitor and the other compounds. Moreover, there may be multiple administrations of each compound singly or the compounds in combination.
In some instances, a patient is operated on prior to or at the same time as administration of an ERK inhibitor.
The ERK inhibitor may be administered to the patient in a number of ways. It may be administered to the patient intravenously. In other embodiments it is administered directly to the spine of the patient. In some cases, administration involves a catheter, such as a spinal catheter or an intrathecal catheter.

The ERK inhibitor and any other compound administered to the patient in conjunction with the inhibitor may be formulated in a pharmaceutically or pharmacologically acceptable compound. In some embodiments, the inhibitor is formulated in a composition comprising (3-cyclodextrin.
In still further embodiments of the invention, methods of screening for ERIC
inhibitors are included. Methods of the invention may be implemented by first identifying a compound as an ERK inhibitor and then administering it. Methods of identifying an ERK inhibitor are well known to those of skill in the art and include, but are not limited to, those methods disclosed herein and in the references cited herein.
As used herein, "any integer derivable therein" means a integer between the numbers described in the specification, and "any range derivable therein"
means any range selected from such numbers or integers.
As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 - The basal ERK activation is determined by immunoblotting for neuro2A
cells for the time after the precipitated withdrawal of Naloxone. Chronic fentanyl suppressed ERK activation by 20%
FIG. 2 - The in vivo inhibition of tolerance by spinal infusion of the ERK
cascade inhibitor U0126 was determined -by determining tail flick latency. Animals received spinal catheters infusing either saline (S), 70% DMSO vehicle (V) or U0126 in vehicle (V/LT0126).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention The present invention overcomes deficiencies in the art by establishing the role of MAPK and MAPK inhibition in areas relevant to opioid tolerance and dependence, withdrawal, hyperalgesia, and pain sensitization. Methods are given for the reduction of tolerance, dependence, withdrawal symptoms, and inhibition of pain sensitization. The inventor has shown the correlation in behavioral response in intact animals with supporting biochemical and neuroanatomical findings.
II. Opioid Receptors Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and cam also induce physical dependence (Knob et al., 1992). The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal and immune functions (Olson et al., 1989). Opioids exert their actions by binding to specific membrane-associated receptors located throughout the central and peripheral nervous system (Pert et al., 1973). The endogenous ligands of these opioid receptors have been identified as a family of more than 20 opioid peptides that derive from the three precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin (Hughes et al., 1975;
Akil, et al., 1984). Although the opioid peptides belong to a class of molecules distinct from the opioid alkaloids, they share common structural features including a positive charge juxtaposed with an aromatic ring that is required for interaction with the receptor (Bradbury et al., 1976).
Pharmacological studies have suggested that there are numerous classes of opioid receptors, including those designated 8, K, and p. (Simon, 1991; Lutz et al., 1992). The classes differ in their affinity for various opioid ligands and in their cellular distribution.

The different classes of opioid receptors are believed to serve different physiological functions (Olson et al., 1989; Simon, 1991; Lutz and Pfister, 1992). However, there is substantial overlap of function as well as of distribution. Biochemical characterization of opioid receptors from many groups reports a molecular mass of about 60,000 Da for all 5 three subtypes, suggesting that they could be related molecules (Loh et al., 1990).
Moreover, the similarity between the three receptor subtypes is supported by the isolation of (i) anti-idiotypic monoclonal antibodies competing with both ~, and 8 ligands but not competing with x ligands (Gramsch et al., 1988; Coscia et al., 1991) and (ii) a monoclonal antibody raised against the purified ~. receptor that interacts with both ~. and 10 x receptors (Bero et al., 1988).
Morphine interacts principally with ~ receptors and peripheral administration of this opioid induces release of enkephalins (Bertolucci et al., 1992). The F
receptors bind with the greatest affinity to enkephalins and have a more discrete distribution in the brain than either ~ ox x receptors, with high concentrations in the basal ganglia and limbic regions. Thus, enkephalins may mediate part of the physiological response to morphine, presumably by interacting with 8 receptors. Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase I~+ conductance, and inactivate Ca2~ channels through a pertussis toxin-sensitive mechanism (Puttfarcken et al., 1988; Attali et al., 1989; Hsia et al., 1984).
These results and others suggest that opioid receptors belong to the large family of cell surface receptors that signal through G proteins (Di Chiara et al., 1992; Loh et al., 1990).
Many cell surface receptor/transmembrane systems consist of at least three mem-brane-bound polypeptide components: (a) a cell-surface receptor; (b) an effector, such as an ion channel or the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding regulatory polypeptide or G protein, that is coupled to both the receptor and its effector.
G protein-coupled receptors mediate the actions of extracellular signals as diverse as light, odorai~ts, peptide hormones and neurotransmitters. Such receptors have been identified in organisms as evolutionarily divergent as yeast and man. Nearly all G
protein-coupled receptors bear sequence similarities with one another, and it is thought that all share a similar topological motif consisting of seven hydrophobic (and potentially a-helical) segments that span the lipid bilayer (Dohlman et al., 1987; Dohlman et al., 1991).
G proteins consist of three tightly associated subunits, a, (3 and 'y (1:1:1) in order of decreasing mass. Following agonists binding to the receptor, a conformational change is transmitted to the G protein, which causes the Ga-subunit to exchange a bound GDP
for GTP and to dissociate from the (3y-subunits. The GTP-bound form of the a-subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G protein molecules, and from the stimulation by Ga GTP of many catalytic cycles of the effector.
The family of regulatory G proteins comprises a multiplicity of different a-subunits (greater than twenty in man), which associate with a smaller pool of (3- and ~y-subunits (greater than four each) (Strathmann and Simon, 1991). Thus, it is anticipated that differences in the a-subunits probably distinguish the various G protein oligomers, although the targeting or function of the various a-subunits might also depend on the (3~y subunits with which they associate (Strathmann and Simon, 1991).
Improvements in cell culture and in pharmacological methods, and more recently, use of molecular cloning and gene expression techniques have led to the identification and characterization of many seven-transmembrane segment receptors, including new sub-types and sub-sub-types of previously identified receptors. The al and a2-adrenergic receptors once thought to each consist of single receptor species, are now known to each be encoded by at least three distinct genes (Kobilka et al., 1987; Regan et al., 1988;
Cotecchia et al., 1988; Lomasney, 1990). In addition to rhodopsin in rod cells, which mediates vision in dim light, three highly similar cone pigments mediating color vision have been cloned (Nathans et al., 1986A; and Nathans et al., 1986B). All of the family of G protein-coupled receptors appear to be similar to other members of the family of G
protein-coupled receptors (e.g., dopaminergic, muscarinic, serotonergic, tachykinin, etc.), and each appears to share the characteristic seven-transmembrane segment topography.

Opioid receptors are known to be sensitive to reducing agents, and the occurrence of a disulfide bridge has been postulated as essential for ligand binding (Gioannini et al., 1989). For rhodopsin, muscarinic, and l3-adrenergic receptors, two conserved cysteine residues in each of the two first extracellular loops have been shown critical for stabilizing the functional protein structure and are presumed to do so by forming a disulfide bridge. Structure/function studies of opioid ligands have shown the importance of a protonated amine group for binding to the receptor with high affinity.
The binding site of the receptor might, therefore, possess a critical negatively charged counterpart.
Catecholamine receptors display in their sequence a conserved aspartate residue that has been shown necessary for binding the positively charged amine group of their ligands.
III. Opioids It is now well known that opioids such as heroin and morphine exert their effects by mimicking naturally occurnng substances, termed the endogenous opioid peptides or endorphins. We have learned a great deal about the basic biology of the endogenous opioid system, and have come to appreciate the molecular and biochemical complexity of this system, its widespread anatomy, and its diversity. These diverse functions subsume a 'housekeeping role' in the body. They include the best-known sensory role, prominent in inhibiting responses to painful stimuli, a modulatory role in gastrointestinal, endocrine and autonomic functions; an emotional role, evident in the powerful rewarding and addicting properties of opioids; and a cognitive role of opioids in the modulation of learning and memory. Scientific study has revealed the opioid system to be complex and subtle, with a great diversity in endogenous ligands (over a dozen), yet with only four major receptor types.
The term opioid refers to all compounds in a generic sense related to opium.
The word opium is derived from opos, the Greek word for juice, since the medicine was derived from the juice of the opium poppy, papaver somniferum. Opiates are drugs derived from opium, and include the natural products morphine, codeine, thebaine, and many semi-synthetic congeners derived from them. Endogenous opioid peptides (EOPs) are the naturally synthesized ligands for opioid receptors. The term endor~phira is used synonymously with EOP, but also refers to a specific endogenous opioid, 13-endorphin.

The term narcotic was derived from the Greek word for stupor. At one time, it referred to any drug that induced sleep, but then became associated with opioids.
The three major opioid receptor types, ~,, S, and x have been extensively studied.
The more recently discovered nociceptin/orphanin FQ receptor (N/OFQ receptor;
also initially described as the opioid receptor-like 1 (ORL-1) or "orphan" opioid receptor has added a new dimension to the study of opioids. Recently, a new nomenclature system has been proposed to reflect the consideration of this receptor as part of the opioid receptor family. It has been suggested by the ILTPHAR Nomenclature Committee that these receptors be referred to as the OP (opioid peptide) receptor family and individual receptors be called the ~ or MOP, 8 or DOP, x or KOP, and NJOFQ or NOP
receptors.
These names will be used interchangeably throughout the specification.
Most of the clinically used opioids are relatively selective for MOP
receptors, reflecting their similarity to morphine. However, it is important to note that drugs that are relatively selective at standard doses will interact with additional receptor subtypes when given at sufficiently high doses, leading to possible changes in their pharmacological profile. This is especially true as doses are escalated to overcome tolerance. Some drugs, particularly mixed agonist-antagonist agents, interact with more than one receptor class at usual clinical doses. The actions of these drugs are particularly interesting, since they may act as an agonist at one receptor and an antagonist at another.
Transient administration of opiates leads to a phenomenon termed acute tolerance, whereas sustained administration leads to the development of "classical" or chronic tolerance. Tolerance simply means the decrease in effectiveness of a drug with repeated exposure. Recent studies have focused on cellular mechanisms of acute tolerance. Several investigators have shown that short-term desensitization probably involves phosphorylation of the MOP and DOP receptors via protein kinase C
(Mestek et al. 1995; Narita et al. 1995; Ueda et al. 1995). A number of other kinases also have been implicated, including protein kinase A and beta adrenergic receptor kinase MARK
(Pei et al. 1995; Wang et al. 1994) Traditionally, long-term tolerance is thought to be associated with increases in adenylyl cyclase activity - a counter-regulation opposite to the decrease in cyclic AMP
levels seen after acute opioid administration (Sharma et al. 1977). An interesting study showed that chronic ~, opioid treatment caused superactivation of adenylyl cyclase (Avidor-Reiss et al. 1996). This effect was prevented by pretreatment with pertussis toxin, demonstrating involvement of G;~o proteins, and also by co-transfection with scavengers of G protein- ~3y dimers, indicating a role for this complex in superactivation.
Alterations in levels of cyclic AMP clearly bring about numerous secondary changes, as reviewed by Nestler (Nestler and Aghaj anian 1997).
a. Effects of Clinically Used Opioids Morphine and most other clinically used opioid agonists exert their effects through MOP receptors. These drugs affect a wide range of physiological systems, including, analgesia, mood, rewarding behavior, respiratory, cardiovascular, gastrointestinal, and neuroendocrine function. Delta opioid compounds are also potent analgesics in animals, and in isolated cases have proved useful in humans (Coombs et al.
1985). The main barrier to the clinical use of b agonists is that most of these compounds are peptidergic and unable to cross the blood-brain barrier, thus requiring intraspinal administration. However, much effort is currently being devoted to the development of clinically useful 8 agonist compounds. Kappa selective agonists produce analgesia that has been shown in animals to be mediated primarily at spinal sites.
Respiratory depression and miosis may be less severe than with ~ agonists. Instead of euphoria, Dagonists produce dysphoric and psychotomimetic effects (Pfeiffer et al.
1986). In neural circuitry mediating both reward and analgesia, ~, and K agonists have been shown to have antagonistic effects (see below). Mixed agonist -antagonist compounds were developed for clinical use with the hope that they would have less addictive potential and less respiratory depression. In practice, it has turned out that for the same degree of analgesia, the same intensity of side effects will be observed. A "ceiling effect", limiting the amount of analgesia attainable, is often seen with these compounds. Some drugs of this class, such as pentazocine and nalorphine, can produce severe psychotomimetic effects that are not naloxone reversible (which suggest that they are not mediated through classical opioid receptors). Also, these drugs can precipitate withdrawal in opioid tolerant patients. For these reasons, the clinical use of these compounds is relatively limited.
In human beings, morphine-like drugs produce analgesia, drowsiness, changes in 5 mood, and mental clouding. A significant feature of the analgesia is that it occurs without loss of consciousness. When therapeutic doses of morphine are given to patients with pain, they report that the pain is less intense, less discomforting, or entirely gone;
drowsiness commonly occurs. In addition to relief of distress, some patients experience euphoria.
When morphine in the same dose is given to a normal, pain-free individual, the experience may be unpleasant. Nausea is common, and vomiting also may occur.
There may be feelings of drowsiness, difficulty in mentation, apathy, and lessened physical activity. As the dose is increased, the subj ective, analgesic, and toxic effects, including ~ respiratory depression, become more pronounced. Morphine does not have anticonvulsant activity and usually does not cause slurred speech, emotional lability , or significant motor incoordination.
Afzalgesia. The relief of pain by morphine-like opioids is relatively selective, in that other sensory modalities are not affected. Patients frequently report that the pain is still present, but that they feel more comfortable. Continuous dull pain is relieved more effectively than sharp intermittent pain, but with sufficient amounts of opioid it is possible to relieve even the severe pain associated with renal or biliary colic.
Simultaneous administration of morphine at both spinal and supraspinal sites results in synergy in analgesic response, with a reduction in the total dose of morphine necessary to produce equivalent analgesia at either site alone. The mechanisms responsible for spinal/supraspinal synergy are readily distinguished from those involved with supraspinal analgesia (Pick et al. 1992a). In addition to the spinal/supraspinal synergy, synergistic ~,/~.- and ~,/b-receptor interactions also have been observed within the brainstem between the periaqueductal gray, locus coeruleus, and nucleus raphe magnus (Rossi et al. 1993).

While opioids are primarily used clinically for their pain modulatory properties, they produce a host of other effects. This is not surprising in view of the wide distribution of opioids and their receptors, both in the brain and in the periphery.
Opioids can produce muscular rigidity in human beings; alter the equilibrium point of the hypothalamic heat-regulatory mechanisms; inhibit the release of gonadotropin-releasing hormone (GnRH) and corticotropin-releasing factor (CRF) in the hypothalamus; cause constriction of the pupil by an excitatory action on the parasympathetic nerve innervating the pupil; produce convulsions in animals;
depress respiration, at least in part by virtue of a direct effect on the brainstem respiratory centers; depress the cough reflex, at least in part by a direct effect on a cough center in the medulla; cause nausea and vomiting by direct stimulation of the chemoreceptor trigger zone for emesis, in the area postrema of the medulla; cause orthostatic hypotension and fainting upon rising from a supine position; decrease the secretion of hydrochloric acid in the gastrointestinal tract; . diminishes biliary, pancreatic, and intestinal secretions in the small intestine; diminishes or abolishes propulsive peristaltic waves in the colon; inhibit gastrointestinal propulsive activity in the bowels; increase the pressure in the common bile duct; increase the tone and amplitude of contractions of the ureter; and cause dilatation of cutaneous blood vessels in the skin. Opioids have been shown to modulate immune function both via direct, receptor-mediated effects on immune cells and indirectly via centrally mediated neuronal mechanisms (Gomez-Flores and Weber 2000; Sharp and Yaksh 1997). The overall effects of opioids on immune function appear to be suppressive, with increased susceptibility to infection and tumor spread observed in experimental studies.
b. Tolerafzce a~zd Physical Depezzdeztce The development of tolerance and physical dependence with repeated use is a characteristic feature of all the opioid rugs. Tolerance to the effect of opioids or other drugs simply means that over time, the drug loses its effectiveness and an increased dose is required to produce the same physiological response. Dependence refers to a complex and poorly understood set of changes in the homeostasis of an organism that cause a disturbance of the homeostatic set point of the organism if the drug is stopped. This disturbance is often called witlad~awal. Addiction is a behavioral pattern characterized by compulsive use of a drug and overwhelming involvement with its procurement and use. T Tolerance and dependence are physiological responses seen in all patients and are not predictors of addiction (see Chapter 24). These processes appear to be quite distinct.
For example, cancer pain often requires prolonged treatment with high doses of opioids, leading to tolerance axed dependence. Yet, abuse in this setting is very unusual (Foley 1993). Neither the presence of tolerance and dependence nor the fear that they may develop should ever interfere with the appropriate use of opiaids. Opioids can be discontinued in dependent patients once the need for analgesics is gone without subjecting them to withdrawal. Clinically, the dose can be decreased by 10-20 % every other day and eventually stopped without signs and symptoms of withdrawal.
In vivo studies in animal models demonstrate the importance of other neurotransmitters and their interactions with opioid pathways in the development of tolerance to morphine. Blockade of glutamate actions NMDA (N-methyl-D-aspartate) antagonists blocks morphine tolerance. Since NMDA antagonists have no effect on the potency of morphine in naive animals, their effect cannot be attributed to potentiation of opioid actions. Interestingly, the clinically used antitussive dextromethorphan has been shown to function as an NMDA antagonist. In animals, it can attenuate opioid tolerance development and reverse established tolerance (Elliott et al. 1994). Nitric oxide production, possibly induced by NMDA receptor activation, also has been implicated in tolerance, as inhibition of nitric oxide synthase (NOS) also blocks morphine tolerance , development (Kolesnikov et al. 1993). Administering NOS inhibitors to morphine-tolerant animals may also in certain circumstances reverse tolerance. Although the NMDA antagonists and nitric oxide synthase inhibitors are effective against tolerance to morphine and 8 agonists such as DPDPE, they have little effect against tolerance to the K
agonists. Dependence seems to be closely related to tolerance, since the same treatments that block tolerance to morphine also block dependence. Other related signaling systems are also being actively investigated as mediators of opioid tolerance and dependence.
The selective actions of drugs on tolerance and dependence demonstrate that specific mechanisms can be targeted to minimize these two unwanted actions.
c. Morphine a,ad Related Opioids Any opioid can be used conjunction with the MAPK inhibitor of the current invention in providing treatment for chronic pain and/or reducing tolerance, the risk of physical dependence, hyperalgesia, or the symptoms of opioid withdrawal or inhibiting pain sensitization. The opioid can be used in a patient who needs pain relief, or as a part of a maintenance programs for the treatment of addicts.
Because the laboratory synthesis of morphine is difficult, the drug is still obtained from opium or extracted from poppy straw. Opium is obtained from the unripe seed capsules of the poppy plant, Papavef~ somf~iferum. The milky juice is dried and powdered to make powdered opium, which contains a number of alkaloids. Only a few-morphine, codeine, and papaverine-have clinical usefulness. These alkaloids can be divided into two distinct chemical classes, pher2anth~enes and behzylisoquinolines. The principal phenanthrenes are morphine (10% of opium), codeine (0.5%), and thebaine (0.2%). The principal benzylisoquinolines are papaverine (1.0%), which is a smooth muscle relaxant, and noscapine (6.0%).
Many semisynthetic derivatives are made by relatively simple modifications of morphine or thebaine. Codeine is methylmorphine, the methyl substitution being on the phenolic hydroxyl group. Thebaine differs from morphine only in that both hydroxyl groups are methylated and that the ring has two double bonds. Thebaine has little analgesic action, but is a precursor of several important 14-OH compounds, such as oxycodone and naloxone. Certain derivatives of thebaine are more than 1000 times as potent as morphine (e.g. etorphine). Diacetylmorphine, or heroin, is made from morphine by acetylation at the 3 and 6 positions. Apomorphine, which also can be prepared from morphine, is a potent emetic and dopaminergic agonist.
Hydromorphone, oxymorphone. hydrocodone, and oxycodone also are made by modifying the morphine molecule.
In addition to morphine, codeine, and the semisynthetic derivatives of the natural opium alkaloids, a number of other structurally distinct chemical classes of drugs have pharmacological actions similar to those of morphine. Clinically useful compounds include the morphinans, benzomorphans. methadones, phenylpiperidines, and propionanilides. Although the two-dimensional representations of these chemically diverse compounds appear to be quite different, molecular models show certain common characteristics; these are indicated by the heavy lines in the structure of morphine shown above. Among the important properties of the opioids that can be altered by structural modification are their affinity for various species of opioid receptors, their activity as agonists versus antagonists, their lipid solubility, and their resistance to metabolic breakdown. For example, blockade of the phenolic hydroxyl at position 3, as in codeine and heroin, drastically reduces binding to ,u receptors; these compounds are converted to the potent analgesics morphine and 6-acetyl morphine, respectively, ira vivo.
Codeine. In contrast to morphine, codeine is approximately 60% as effective orally as parenterally, both as an analgesic and as a respiratory depressant.
Codeine has an exceptionally low affinity for opioid receptors, and the analgesic effect of codeine is due to its conversion to morphine.
Tramadol: Tramadol is a synthetic codeine analogue that is a weak p, opioid agonist. Part of its analgesic effects are produced by inhibition of norepinephrine and serotonin reuptake. Tramadol appears fo be as effective as other weak opioids.
In the treatment of mild to moderate pain, tramadol is as effective as morphine or meperidine.
However, for the treatment of severe or chronic pain tramadol is less effective.
Tramadol is as effective as meperidine in the treatment of labor pain and may cause less neonatal respiratory depression.
Heroin. Heroin (diacetylmorphine) is rapidly hydrolyzed to 6-monoacetylmorphine (6-MAM), which, in turn is hydrolyzed to morphine. Both heroin and 6-MAM are more lipid soluble than morphine and enter the brain more readily.
Current evidence suggests that morphine and 6-MAM are responsible far the pharmacological actions of heroin. Heroin is mainly excreted in the urine, largely as free and conjugated morphine.
Levorphanol. Levorphanol (LEVO-DROM012AN) is the only commercially available opioid agonist of the morphinan series. The d-isomer (dextrorphan) is relatively devoid of analgesic action, but may have inhibitory effects at NMDA
receptors. The pharmacological effects of levorphanol closely parallel those of morphine. However, clinical reports suggest that it may produce less nausea and vomiting. The nonanalgesic isomer dextrorphan possesses considerable antitussive activity.
Meperidine and congeners. Meperidine and some of its congeners are 5 phenylpiperidine derivatives. Meperidine is predominantly a ~, agonist, and it exerts its chief pharmacological action in the CNS and the neural elements in the bowel.
The use of meperidine has diminished in recent years due to concerns over metabolite toxicity.
For this reason, meperidine is no longer. recommended for the treatment of chronic pain, and should not be used in doses greater than 600 mg/48 hrs. Useful congeners of 10 meperidine include diphenoxylate and loperaminde.
Fentanyl, Sufentanil Citrate and congeners. Fentanyl and sufentanil citrate are synthetic opioids related to the phe-nylpiperidines. Fentanyl is a ~, agonist and is about-100 times as potent as morphine as an analgesic. The actions of fentanyl and its 15 congeners are similar to those of other p agonists. Fentanyl is a popular drug in anesthetic practice due to the shorter time to peak analgesic effect, rapid termination of effect after small bolus doses, and relative cardiovascular stability.
Sufentanil is a closely related compound, with similar analgesic properties. Two compounds that were designed to be shorter acting derivatives include alfentanil and remifentanil.
Both 20 alfentanil and remifentanil have a more rapid onset of analgesic action, with onset of analgesic effects paralleling the T%Zkeo of 1-1.5 minutes. However, because it is metabolized similarly to fentanyl and sufentanil, with a T%keo of 1-2 hours, the duration of action of alfentanil is dependent on both the dose and length of administration. he pharmacological properties of alfentanil and remifentanil are similar to the other fentanyl congeners.
Methadone and Congeners. Methadone is a Iong lasting ~ agonist with pharmacological properties qualitatively similar to those of morphine. The outstanding properties of methadone are its effective analgesic activity, its efficacy by the oral route, . its extended duration of action in suppressing withdrawal symptoms in physically dependent individuals, and its tendency to show persistent effects with repeated administration Miotic and respiratory-depressant effects can be detected for more than 24 hours after a single dose and, upon repeated administration, marked sedation is seen in some patients.
Levomethadyl acetate (LRAM) Levomethadyl acetate is a congener of methadone that recently has been approved for use in maintenance programs for the treatment of heroin addicts. The drug is thought to act, in part, by its conversion to active metabolites, which explains its slow onset and protracted duration of action.
Propoxyphene. Propoxyphene is structurally related to methadoneT Its analgesic effect resides in the dextro isomer, d-propoxyphene (dextropropoxyphene).
However, levopropoxyphene seems to have some antitussive activity. Although slightly less selective than morphine, propoxyphene binds primarily to ~-opioid receptors and produces analgesia and other CNS effects that are similar to those seen with morphine-like opioids. It is likely that at equianalgesic doses the incidence of side effects such as nausea, anorexia, constipation, abdominal pain, and drowsiness would be similar to those of codeine. As an analgesic, propoxyphene is about one-half to two thirds as potent as codeine given orally.
d. Opioid Agohists The drugs described in this section differ from morphine clinically-used ~, agonists in that they are not full agonists at all opioid receptor populations. However, they can be used in conjunction with the MAPI~ inhibitor, or can be the drugs to which tolerance, dependence, or addiction is to be reduced with the MAPK inhibitor of the current invention. Drugs such as nalorphine, cyclazocine, and nalbuphine and butorphanol are competitive ~, antagonists, but exert their analgesic actions by primarily working as agonists at K agonism.receptors. Pentazocine qualitatively resembles these drugs, but it may be a weaker ~, antagonist or partial ~, agonist while retaining its K-agonist activity.
Pentazocine. Pentazocine was synthesized as part of a deliberate effort to develop an effective analgesic with little or no abuse potential. It has both agonistic actions and weak opioid antagonistic activity. The pharmacology of pentazocine has been reviewed by (Brogden et al. 1973). The pattern of CNS effects produced by pentazocine is generally similar to that of the morphine-like opioids, including analgesia, sedation, and respiratory depression. The analgesic effects of pentazocine are due to agonistic actions at ~. opioid receptors.
Nalbuphine. Nalbuphine is related structurally to both naloxone and oxymorphone. It is an agonist/antagonist opioid with a spectrum of effects that qualitatively resembles those of pentazocine; however, nalbuphine is a more potent antagonist at ~, receptors and is less likely to produce dysphoric side effects than is pentazocine.
Butorphanol. Butorphanol is a morphinan congener with a profile of actions similar to those of pentazocine. In postoperative patients, a parenteral dose of 2 to 3 mg of butorphanol produces analgesia and respiratory depression approximately equal to that produced by 10 mg of morphine or 80 mg of meperidine; the onset, peak, and duration of action are similar to those that follow the administration of morphine. The plasma half life of butorphanol is about 3 hours.
Buprenorphine. Buprenorphine is a semisynthetic, highly lipophilic opioid derived from thebaine. It is 25 to 50 times more potent than morphine.
Buprenorphine produces analgesia and other CNS effects that are qualitatively similar to those of morphine, and is used in the management of heroine addicts.
Meptazinol, dezocine, nalorphine, levallorphan and nalmefene are also considered. Similarly, any administration of an opioid may be in combination with a NSAIDs.
e. Treatmeftt or Opioid Overdosage Opioid antagonists and other drugs used to tread opioid overdose can be used in conjunction with the MAPI~ inhibitors of this invention. It is conceived that these drugs can be given simultaneously or sequentially with a MAPK inhibitor to treat addiction.

Naloxone hydrochloride (NARCAN) is used to treat opioid overdose. As discussed earlier, it acts rapidly to reverse the respiratory depression associated with high doses of opioids. However, it should be used cautiously since it also can precipitate withdrawal in dependent subjects, and undesirable cardiovascular side effects.
By carefully titrating the dose of naloxone, it usually is possible to antagonize the respiratory depressant actions without eliciting a full withdrawal syndrome.
The duration of action of naloxone is relatively short, and it often must be given repeatedly or by continuous infusion. Opioid antagonists also have been effectively employed to decrease neonatal respiratory depression secondary to the administration of intravenous or intramuscular opioids to the mother.
IV. Mitogen-activated protein kinases Mitogen-activated protein kinases (MAPS) are regulated by tyrosine and threonine phosphorylation via phosphorylation by MEI~s. There are two well characterized and highly related classical MAPKs in mammalian cells identified as Extracellular signal Regulated Kinases (ERKs). The sequences encoding them are defined herein as SEQ m NO: 1 (ERKl) and SEQ ID NO: 3 (ERK2). Other protein kinases have been identified including SAPK or JNK, p38 MAPK, ERK3, ERKS and ERK6. These stress-induced protein kinases are also known to be regulated by tyrosine and threonine phosphorylation. The inhibition of MAPKs can be used to decrease dependence, tolerance, withdrawal symptoms and addiction of opioids as well as inhibit pain sensitization.
The sequence around the activating phosphorylation sites in MAPK, SAPK, and p38 are distinct. MAPK contains Thr-Glu-Tyr, SAPK contains Thr-Pro-Tyr and p38, ERK6/ and SAPK3 each contain Thr-Gly-Tyr. The kinases described above are all targeted by upstream enzymes including MEKl/2, SEKl and MKK3/6.
(http://kinase.oci.utoronto.calMap/MAPK.html) a. ERK
A recently defined signaling system, the extracellular signal regulated kinase (ERK) are important regulators of neuronal function. ERKs regulate a diverse array of functions including cell growth and proliferation, differentiation, synaptic plasticity and apoptosis. Multiple pathways to ERF regulation have been realized, and include the use of classic second messengers such as cyclic adenosine monophosphate (cAMP), protein kinase A (PFA) diacylglycerol (DAG) and calcium (Grewal et al., 1999).
The ERF signaling cascade, is an attractive candidate as a element capable of integrating some of the diverse signals and coordinating resultant changes in gene expression and protein function. This downstream cascade couples extracellular signals to Long-term changes in function by phosphorylation of cytoplasmic and nuclear targets, such as nuclear transcription factors, by ERF. At the core of this system is a module of three kinases that phosphorylate each other sequentially - Raf, MEF, and ERK, the effector molecule of the cascade. ERK has been shown to subsume other trophic and adaptive functions in the nervous system (Segal et a1.1996), and all of the putative opioid signaling mediators have been shown to interact with the ERF cascade. Gutstein et al.
(1997) and others have shown in vitro that ERK is acutely activated by opioids. ERF
activation can occur independently of receptor internalization (Framer et a1.2000), and may desensitize the opioid receptor (Polakiewicz et al. 1998). Relatively little work has been done characterizing the effects of chronic opioids on ERF activation in vitro or in vivo, and no prior studies have examined the involvement of ERK in tolerance and dependence at the behavioral level. One group reported that acute morphine administration did not change ERF activity in the VTA, while chronic morphine induced modest increases in activity (Nestler et a1.1996). In contrast, another group reported that chronic morphine suppressed ERF activation in many relevant brain regions, and precipitated withdrawal markedly stimulated ERK activation (Schulz et a1.1998).
ERF activation can lead to the activation of numerous transcription factors and cellular proteins relevant to opioid pharmacology, including CREB and AP-1 binding proteins (c-fos, c-jun). Recently, an intriguing class of proteins has been described, the regulator of G protein signaling (RGS) family. These proteins increase the rate of GTP
hydrolysis by Gl proteins, diminishing receptor signaling responses (De Vries et al.
2000). In yeast, some of these proteins are directly induced by ERK
activation, however this mechanism has not yet been investigated in mammalian systems. Many of these proteins have other intriguing properties. Some neurally-expressed RGS
proteins have a GI -like (GGL) domain that tightly binds the neural-specific Go o subunit, suggesting an additional. mechanism of signal modulation. Another family, initially cloned by Snow et al. (1997), contains a PDZ membrane anchoring domain, a PTB domain capable of interacting with tyrosine phosphorylated proteins, a Rap-binding domain (see below), and a cytoskeletal protein interacting domain. Thus, this class of RGS
proteins could 5 serve a "scaffolding" function, associating relevant signaling molecules.
Regulation of RGS mRNA levels in brain in response to . seizures, amphetamines, and other physiological processes has been described, and behavioral relevance is suggested by the increased .anxiety phenotype in RGS2 null mice (Oliveira-do-Santos et al.).
10 Previous work has suggested commonalties in signaling mechanisms responsible for opioid tolerance and hyperalgesia. Both effects have been shown to involve activation of PKC, NO/cGMP, and NMDA receptor signaling (Mayer et al . 1999).
Gutstein et al. (1995) and others have shown that the presence of chronic pain decreases the analgesic effect of opioids. Recently, ERK was shown to play a role in the 15 development of hyperalgesia. Ji et al. (1999) have shown that ERK is rapidly activated in spinal cord after acute noxious stimuli, and that this activation is partially NMDA
'receptor dependent. ERK inhibition diminished the second, but not the first, phase of the formalin response, and appeared to code stimulus intensity. The apparent paradox of ERK being rapidly activated by both pain and opioids could be explained in one of 20 several ways. First, rapid ERK activation by opioids has yet to be described in vivo, thus could potentially be only an in vitro phenomenon.r Also, it is possible that acute opioid administration could inhibit ERK in some cells, depending on the signaling molecules present. For example, acute cAMP irahibitioh can stimulate ERK activity via Ras and Raf, while in some neural cells, cAMP activation is required to activate ERK
via a Rap-25 1/Raf dependent mechanism (Grewal et al. 1999). If this were the case, tolerance development would lead to increased ERK activation, and possibly a reduction in analgesia. A third possibility is that activation of descending supraspinal opioid systems could inhibit ERK activation at the spinal level. None of these possibilities have been previously investigated.
The ERK signaling cascade can be instigated by signals to ERKs via the small G
proteins Ras and Rapl. The Ras-dependent pathway functions in both neuronal and non-neuronal cells. In this pathway cAMP is inhibitory, possibly via the PKA-dependent phosphorylation of Raf 1. The Rapl pathway is excitatory; In contrast to Ras, the actions of Rap 1 are dictated by the expression of B-Raf. Without B-Raf, Rap 1 antagonizes Ras-dependent signaling, but with B-Raf, Rapl can positively couple to ERKs. Rapl is activated by CAMP by either direct stimulation of GEFs or with PKA. In the ERK signaling cascade, the kinase MEK then phosphorylates the ERK.
b. MEK
MEK is a dual-specificity kinase that phosphorylates the tyrosine and threonine residues on ERK required for activation. Two related genes encode MEKl (SEQ ID
NO:S) and MEK2 (SEQ ID NO:7) (Zheng et al., 1993). The two MEKs differ in their binding to ERKs and, possibly, in their activation profiles. MEKs are specific to the ERK signaling cascade and do not phosphorylate either SAPK or p38 MAPK. In the ERK signaling cascade, Rapl is activated by cAMP by either direct stimulation of GEFs or with PKA. MEK is phosphorylated at two serine residues. After MEK
phosphorylation, ERK is phosphorylated (http://kinase.oci.utoronto.ca/lVlap/MEK.html).
Thus, any inhibition of MEK will inhibit the phosphorylation of ERK.
Kinases downstream in the cascade pathway and block the targets of ERK are also an aspect of the current invention. Inhibition of these kinases could have the same effect of opioid tolerance and dependence as inhibition of ERK. These targets include:
H89, HA1077, Rottlerin , KN62, LY 294002 (Lilly) and Quercetin V. Genes and DNA Segments Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding MAPKs, or more particularly ERKs and the creation and use of recombinant host cells through the application of DNA technology, that express a wild-type, polymorphic or mutant MAPKs. Other aspects of the present invention concern isolated DNA segments and recombinant vectors encoding MAPK
inhibitors, MEK, and inhibitors or negative dominants of MEK. Sequences of SEQ ID NO:l, 3, and 7, and biologically functional equivalents thereof are used in the current invention.
The DNA segments and recombinant vectors encoding MAPKs can be used in the invention, for example for creating dominant negatives, for screening of MAPK
inhibitors and determining the effects of gene disruption on tolerance and dependence.

Genes that express RGS can be used in the current invention for determination of the regulation of candidate RGS proteins during tolerance development and withdrawal.
The present invention concerns DNA segments, isolatable from mammalian cells, such as mouse or human cells, that are free from total genomic DNA and that are capable of expressing a protein, polypeptide or peptide that has MAPK activity or is capable of inhibiting MAPK, or more particularly ERK. As used herein, the term "DNA
segment"
refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding MAPK refers to a DNA
segment that contains wild-type, polymorphic or mutant MAPK coding sequences yet is isolated away from, or purified free from, total mammalian genomic DNA.
Included within the teen "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, ,phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified MAPK gene refers to a DNA segment including MAPK protein, polypeptide or peptide coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene"
'is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and engineered segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutants of MAPK encoded sequences.
"Isolated substantially away from other coding sequences" means that the gene of interest, in this case the MAPK, or more particularly ERK genes, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a MAPK protein, polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID N0:2, corresponding to the MAPK designated "human MAPK".
The term "a sequence essentially as set forth in SEQ ID N0:2" means , for example, that the sequence substantially corresponds to a portion of SEQ ID
N0:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID N0:2. This applies with respect to SEQ ID
NOs: 4, 6 and 8.
The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have about 70%, about 1S 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, about 70% to about 80%, and more preferably about 81 % and about 90%; or even more preferably, between about 91 and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID N0:2, 4, 6 and 8 will be sequences that are "essentially as set forth in SEQ ID N0:2, 4, 6 and 8", provided the biological activity of the protein is maintained.
In particular embodiments, the biological activity of a MAPK protein, polypeptide or peptide, or a biologically functional equivalent, comprises binding to one or more proteases, particularly serine proteases. In specific embodiments, the biological activity of a MAPK protein, polypeptide or peptide, or a biologically functional equivalent, comprises inhibition of the activity of one or more proteases, particularly serine proteases, through binding. A preferred protease activity that may be inhibited by a MAPK protein, polypeptide or peptide, or a biologically functional equivalent, is inhibition of the ability or rate of protealytic cleavage catalyzed by the protease.

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:l, 3, 5 AND 7. The term "essentially as set forth in SEQ ID N0:1, 3, 5 or 7" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID
NO:1, 3, 5 or 7, respectively and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID N0:1, 3, 5 or 7, respectively. Again, DNA
segments that encode proteins, polypeptide or peptides exhibiting MAPK
activity will be most preferred.
The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine and serine, and also refers to codons that encode biologically equivalent amino acids. For optimization of expression o.f MAPK in human cells, the codons are shown in Table 1 in preference of use from left to. right. Thus, the most preferred codon for alanine is thus "GCC", and the least is "GCG" (see Table 1 below). Codon usage for various organisms and organelles can be found at the website http://www.kazusa.or.jp/codon/, incorporated herein by reference, allowing one of skill in the art to optimize codon usage for expression in various organisms using the disclosures herein. Thus, it is contemplated that codon usage may be optimized for other animals, as well as other organisms such as a prokaryote (e.g., an eubacteria, an archaea), an eukaryote (e.g., a protist, a plant, a fungi, an animal), a virus and the like, as well as organelles that contain nucleic acids, such as mitochonch~ia or chloroplasts, based on the preferred codon usage as would be known to those of ordinary skill in the art.

Table 1-Preferred Human DNA
Colons Amino Acids Colons Alanine Ala A GCC GCT GCA GCG

Cysteine Cys C TGC TGT

Aspartic acidAsp D GAC GAT

Glutamic acidGlu E GAG GAA

PhenylalaninePhe F TTC TTT

Glycine Gly G GGC GGG GGA GGT

Histidine His H CAC CAT

Isoleucine Ile I ATC ATT ATA

Lysine Lys K AAG AAA

Leucine Leu L CTG CTC TTG CTT CTA TTA

Methionine Met M ATG

Asparagine Asn N AAC AAT

Proline Pro P CCC CCT CCA CCG

Glutamine Gln Q CAG CAA

Arginine Arg R CGC AGG CGG AGA CGA CGT

Serine Ser S AGC TCC TCT AGT TCA TCG

Threonine Thr T ACC ACA ACT ACG

Valine Val V GTG GTC GTT GTA

Tryptophan Trp W TGG

Tyrosine Tyr Y TAC TAT

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, 5 and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein, polypeptide or peptide activity where an amino acid sequence expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i. e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences that have about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, about 70% to about 80%, and more preferably about 81 % and about 90%; or even more preferably, between about 91 % and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO:1, 3, 5 and 7 will be sequences that are "essentially as set forth in SEQ ID NO:1, 3, 5 and 7".
a. Nucleic Acid Hybridization The nucleic acid sequences disclosed herein also have a variety of uses, such as for example, utility as probes or primers in nucleic acid hybridization embodiments.
Contiguous sequences from MAPK sequences can be used, for example, to form dominant negatives of the MAPK, used to screen for inhibitor function.
Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ
ID
NO:l, 3, 5 and 7. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarily rules.
As used herein, the term "complementary sequences" means nucleic acid sequences that are complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ
ID NO: 1 under stringent conditions such as those described herein.
As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terms "low stringency" or "low stringency condition(s)."
As used herein "stringent condition(s)" or "high stringency" are those conditions that allow hybridization between or within one or more nucleic acid strands) containing complementary sequence(s), but precludes hybridization of random sequences.
Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvents) in a hybridization mixture.
It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature andlor high ionic strength. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCI at temperatures of about 37°C to about 55°C. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. In another example, a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about SS°C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application. For example, in other embodiments, hybridization may be achieved under conditions of, 50 mM Tris-HCl (pH 8.3), 75 mM KCI, 3 mM MgCl2, 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C. Other hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, at temperatures ranging from approximately 40°C to about 72°C.
Accordingly, the nucleotide sequences of the disclosure may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. Depending on the application envisioned, it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
For example, nucleic acid fragments may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID NO:l, 3, 5 and 7, such as, for example, about 8, about 10 to about 14, or about 15 to about 20 nucleotides, and that are chromosome sized pieces, up to about 1,000,000, about 750,000, about 500,000, about 250,000, about 100,000, about 50,000, about 20,000, or about 10,000, or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases, as well as DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths of these lengths listed above, i. e., any range derivable therein and any integer derivable therein such a range) are also contemplated to be useful.

For example, it will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 10, 11, 12, 13, 14, 1S, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2S, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3S, 36, 37, 38, 39, 40, S 41, 42, 43, 44, 4S, 46, 47, 48, 49, S0, S1, S2, S3, S4, SS, S6, S7, S8, S9, 60, 6S, 70, 7S, 80, 8S, 90, 9S, 100, 10S, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000;
5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002, 15,000, 20,000 and the like.
Various nucleic acid segments may be designed based on a particular nucleic acid sequence, and may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all nucleic acid segments can be created:
1S nton+y where n is an integer from 1 to the last number of the sequence and y is the length of the nucleic acid segment minus one, where n + y does not exceed the last number of the sequence. Thus, for a 10-mer, the nucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and/or so on. For a 1S-mer, the nucleic acid segments correspond to bases 1 to 1 S, 2 to 16, 3 to 17 .., and/or so on. For a 20-mer, the nucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and/or so on. In certain embodiments, the nucleic acid segment may be a probe or primer. As used herein, a "probe" generally refers to a nucleic acid used in a detection method or composition. As used herein, a "primer"
generally refers to a nucleic acid used in an extension or amplification method or composition.

The use of a hybridization probe of between 17 and 100 nucleotides in length, or in some aspect of the invention even up to 1-2 Kb or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
In general, it is envisioned that the hybridization probes described herein will be 5 useful both as reagents in solution hybridization, as in PCRT"", for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fxed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The selected conditions will 10 depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc. ). Following washing of the hybridized surface to remove non-specifically bound probe molecules, hybridization is detected, or even quantified, by means of the label.
b. Nucleic Acid Amplification Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989).
The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA
is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification.
Pairs of primers that selectively hybridize to nucleic acids corresponding to ~5 MAPI~ genes are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term "primer", as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCRT"") which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each incorporated herein by reference in entirety.
Briefly, in PCRT"", two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA
polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA
into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in WO 90/07641, filed December 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.
Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPA No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCRTM, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Application No. PCT/LJS87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA
polymerase. The polymerase will copy the replicative sequence that can then be detected.
An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio~-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i. e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA
that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
The original template is annealed to another cycling probe and the reaction is repeated.
Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/LTS89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA
molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase . (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "I~lenow" fragment of E.
coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
Miller et al., PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA
("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i. e., new templates are not produced from the resultant RNA
transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, 1990, incorporated herein by reference).
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention.

c. Nucleic Acid Detection In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention such as all or part of SEQ ID NO: 1, 3, 5 or 7, or an ERK nucleic acid inhibitor in combination with an appropriate means, such as a label, for determining 5 hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of . being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as unease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme 10 tags, colorimetric indicator substrates are known that can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In embodiments wherein nucleic acids are amplified, it may be desirable to 15 separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989).
20 Alternatively, chromatographic techniques may be employed to effect separation.
There are many kinds of chromatography which may be used in the present invention:
adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
25 Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under LTV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the 30 appropriate stimulating spectra, following separation. .

In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of IO skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al., 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding.
Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
One example of the foregoing is described in U.S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
Other methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation.
Historically, a number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis ("DGGE"), restriction enzyme polymorphism analysis, chemical and enzymatic cleavage methods, and others.
The more common procedures currently in use include direct sequencing of target regions amplified by PCR~M (see above) and single-strand conformation polymorphism analysis ("SSCP") Another method of screeiung for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.
U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, axe compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
Currently available RNase mismatch cleavage assays, including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA
probes.
Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A. Other investigators have described the use of an E.
coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufFciently high.
The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. The assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription. Originally, the templates for irz vitro transcription were recombinant plasmids containing bacteriophage promoters. The probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single-stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA
that was present) is recovered and analyzed on a polyacrylamide gel.
The RNase Protection assay was adapted for detection of single base mutations.
In this type of RNase A mismatch cleavage assay, radiolabeled RNA probes transcribed in vitro from wild-type sequences, are hybridized to complementary target regions derived from test samples. The test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCRTM), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch") can be recognized and cleaved in some cases by single-strand specific ribonuclease.
To date, RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage.
There are recent descriptions of using the MutS protein and other DNA-repair enzymes for detection of single-base mismatches.
d. Clohi~ag MAPK Gehes The present invention contemplates cloning MAPK, or more particularly ERK
genes or cDNAs from animal (e.g., mammalian) organisms. A technique often employed by those skilled in the art of protein production today is to obtain a so-called "recombinant" version of the protein, to express it in a recombinant cell and to obtain the protein, polypeptide or peptide from such cells. These techniques are based upon the "cloning" of a DNA molecule encoding the protein from a DNA library, i.e., on obtaining a specific DNA molecule distinct from other portions of DNA. This can be achieved by, for example, cloning a cDNA molecule, or cloning a genomic-like DNA
molecule.
The first step in such cloning procedures is the screening of an appropriate DNA
library, such as, for example, from a mouse, rat, monkey or human. The screening protocol may utilize nucleotide segments or probes that axe designed to hybridize to cDNA or genomic sequences of MAPKs from protists. Additionally, antibodies designed to bind to the expressed MAPK proteins, polypeptides, or peptides may be used as probes to screen an appropriate mammalian DNA expression library.
Alternatively, activity assays may be employed. The operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, in Sambrook et al. (1989), incorporated herein by reference.
Moreover, as the present invention encompasses the cloning of genomic segments as well as cDNA
molecules, it is contemplated that suitable genomic cloning methods, as known to those in the art, may also be used.
As used herein "designed' to hybridize" means a sequence selected for its likely ability to hybridize to a mammalian MAPK gene, for example due to the expected high degree of homology between the human MAPK gene and the MAPK genes from other mammals. Also included are segments or probes altered to enhance their ability to hybridize to or bind to a mammalian MAPK gene. Additionally, these regions of homology also include amino acid sequences of 4 or more consecutive amino acids selected and/or altered to increase conservation of the amino acid sequences in comparison to the same or similar region of residues in the same or related genes in one or more species. Such amino acid sequences may derived from amino acid sequences encoded by the MAPK gene and particularly from the isolated sequences of SEQ
ID
N0:2, 4, 6 and 8.
General methods for screening a mammalian DNA library are exemplified by, but not limited to, the methods detailed in Example 1 herein below. Nucleotide probes may derived from nucleotide sequences from the human MAPK sequence, and more particularly from the isolated sequences of SEQ ID NO:l, 3, 5 and 7. Such sequences may be used as probes for hybridization or oligonucleotide primers for PCRTM.

Designing such sequences may involve selection of regions of highly conserved nucleotide sequences between various species for a particular gene or related genes, relative to the general conservation of nucleotides of the gene or related genes in one or more species. Comparison of the amino acid sequences conserved between one or more 5 species for a particular gene may also be used to determine a group of 4 or more consecutive amino acids that are conserved relative to the protein encoded by the gene or related genes. The nucleotide probe or primers may there be designed from the region of the gene that encodes the conserved sequence of amino acids.
10 One may also prepare fusion proteins, polypeptides and peptides, e.g., where the MAPK proteinaceous material coding regions axe aligned within the same expression unit with other proteins, polypeptides or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteinaceous compostions that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Encompassed by the invention are DNA segments encoding relatively small peptides, such as, for example, peptides of from about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 2I, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; as set forth in SEQ ID N0:2, 4, 6 and 8 and also laxger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID N0:2, 4, 6 and 8, and any range derivable therein and any integer derivable therein such a range. In addition to the "standaxd" DNA and RNA
nucleotide bases, modified bases are also contemplated for use in particular applications of the present invention. A table of exemplary, but not limiting, modified bases is provided herein below.

Table 2 Modified Bases . ~
~.. "".

Abbr. Modified Abbr. Modified base description base description ac4c 4-acetylcytidine Mam5s2u 5-methoxyaminomethyl-2-thi ouridine chm5u 5-(carboxyhydroxylmethyl)uMan q Beta,D-mannosylqueosine ridine Cm 2'-O-methylcytidine Mcm5s2u 5-methoxycarbonylmethyl-2-t hiouridine CmnmSs 5-carboxymethylaminomethyMcmSu 5-methoxycarbonylmethylurid 2u 1-2-thioridine ine CmnmSu 5-carboxymethylaminomethyMoSu 5-methoxyuridine luridine D Dihydrouridine Ms2i6a 2-methylthio-N6-isopentenyla denosine Fm 2'-O-methylpseudouridineMs2t6a N-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)carbam oyl)threonine gal Beta,D-galactosylqueosineMt6a N-((9-beta-D-ribofuranosylpu q rine-6-yl)N-methyl-carbamoyl )threonine Gm 2'-O-methylguanosine My Uridine-5-oxyacetic acid methylester I Inosine o5u Uridine-5-oxyacetic acid (v) I6a N6-isopentenyladenosineOsyw Wybutoxosine mla 1-methyladenosine P Pseudouridine mlf 1-methylpseudouridine Q Queosine m 1 1-methylguanosine s2c 2-thiocytidine g mlI 1-methylinosine s2t 5-methyl-2-thiouridine m22g 2,2-dimethylguanosine s2ix 2-thiouridine m2a 2-methyladenosine s4u 4-thiouridine m2g 2-methylguanosine T 5-methyluridine m3c 3-methylcytidine t6a N-((9-beta-D-ribofuranosylpu rive-6-yl)carbamoyl)threonine m5c 5-methylcytidine Tm 2'-O-methyl-5-methyluridine m6a N6-methyladenosine Um 2'-O-methyluridine TABLE 2 - (Continued) m7g 7-methylguanosine ~ Yw Wybutosine MamSu 5-methylaminomethyluridine X 3-(3-amino-3-carboxypropyl) uridine, (acp3)u VII. Recombinant Vectors, Host Cells and Expression Recombinant vectors form an important further aspect of the present invention.
The term "expression vector or construct" means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a proteinaceous molecule, but it need not be, such as in the case of ERK
antisense inhibitors. Thus, in certain embodiments, expression includes both transcription of a MAPK gene and translation of a RNA into the MAPK gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs. The antisense construct can be, for example an ERK
antisense nucleic acid. A recombinant vector can also be used for delivery of the MAPK
inhibitor of the current invention.
Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller polypeptide or peptide, is positioned under the transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned", "under control" or "under txanscriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
The promoter may be in the form of the promoter that is naturally associated with an MAPK, or more particularly ERK gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein (PCRTM technology is disclosed in U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference).
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an MAPK
gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, protist, or mammalian cell, and/or promoters made by the hand of man that are not "naturally occurring", i.e., containing difference elements from different promoters, or mutations that increase, decrease, or alter expression.
Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins, polypeptides or peptides.
At least one module in a promoter generally functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation.
Typically, these are located in the region 30-110 by upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase promoter, the spacing between promoter elements can be increased to 50 basepairs apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
- The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.
In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the instant nucleic acids. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression are contemplated as well, provided that the levels of expression are sufficient for a given purpose. Tables 3 and 4 below list several elementslpromoters which may be employed, in the context of the present invention, to regulate the expression of an MAPK gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof.
Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA.
This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA
with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA
synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities.
Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Additionally any promoterlenhancer combination (as per the Eukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch~ could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain 10 bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
Table 3 - Promoter and Enhancer Elements Promoter/Enhancer References Immunoglobulin Heavy Banerji et al., 1983; Gilles et al., Chain 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler et al., 1987; Weinberger et al., 1984;
I~iledjian et al., 1988; Porton et al.;

Immunoglobulin Light Queen and Baltimore, 1983; Picard and Chain Schaffner, T-Cell Receptor Luria et al., 1987; Winoto and Baltimore, 1989;
Redondo et al.; 1990 HLA DQ a and DQ (3 Sullivan and Peterlin, 1987 (3-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn and Mani.atis, 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 ReceptorGreene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman et al., 1989 TABLE 3 (Continued) (3-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine KinaseJaynes et al., 1988; Horlick and Benfield,1989;
Johnson et al., 1989 Prealbumin (Transthyretin)Costa et al., 1988 Elastase I Ornitz et al., 1987 Metallothionein Karin et al., 1987; Culotta and Hamer, Collagenase Pinkert et al., 1987; Angel et al., Albumin Gene Pinkert et al., 1987; Tronche et al., 1989,1990 a-Fetoprotein Godbout et al., 1988; Campere and Tilghman, 'y-Globin Bodine and Ley, 1987; Perez-Stable and Constantini, (3-Globin Trudel and Constantini, 1987 c-fos c-HA-ras Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Hirsh et al., 1990 Molecule (NCAM) al-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse or Type I CollagenRipe et al., 1989 Glucose-Regulated ProteinsChang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Laxsen et al., 1986 Human Serum Amyloid Edbrooke et al., 1989 A
(SAA) Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived GrowthPech et al., 1989 Factor TABLE 3 (Continued) Duchenne Muscular Klamut et al., 1990 Dystrophy I

SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986;
Kadesch and Berg, 1986; Wang and Calame, 1986;

Ondek et al., 1987; Kuhl et al., 1987;

Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975;

Vasseur et al., 1980; Katinka et al., 1980, 1981;

Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986;

Satake et al., 1988; Campbell and Villarreal, Retroviruses Kriegler and Botchan, 1982, 1983;

Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986;

Celander and Haseltine, 1987; Thiesen et al., 1988;

Celander et al., 1988; Choi et al., 1988; Reisman and Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;
Spandidos and Wilkie, 1983; Spalholz et al., 1985;
Lusky and Botchan, 1986; Cripe et al., 1987; Gloss et al., 1987;

Hirochika et al., 1987; Stephens and Hentschel, 1987 Hepatitis B virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986;

Shaul and Ben-Levy, 1987; Spandau and Lee, 1988;

Vannice and Levinson, 1988 TABLE 3 (Continued) Human ImmunodeficiencyMuesing et al., 1987; Hauber and Cullan, 1988;

Virus Jakobovits et al., 1988; Feng and Holland, 1988;

Takebe et al., 1988; Rosen et al., 1988;

Berkhout et al., 1989; Laspia et al., 1989; Sharp and Marciniak, 1989; Braddock et al., 1989 Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986 Gibbon Ape Leukemia Holbrook et al., 1987; Quinn et al., Virus 1989 Table 4 - Inducible Elements Element Tnducer References MT II Phorbol Ester (TFA) Palmiter et al., 1982;

Heavy metals Haslinger and I~arin, 1985; Searle et al., 1985;

Stuart et al., 1985;

Imagawa et al., 1987, Karin et al., 1987;

Angel et al., 1987b;

McNeall et al., 1989 MMTV (mouse mammary Glucocorticoids Huang et al., 1981;

tumor virus) Lee et al., 1981;
Majors and Varmus, 1983;

Chandler et al., 1983;

Lee et al., 1984;

Ponta et al., 1985;

Sakai et al., 1988 ~i-Interferon Poly(rI)x Tavernier et al., Poly(rc) TABLE 4 (Continued) Adenovirus 5 E2 Ela Imperiale and Nevins, Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 Resendez et al., cc-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., MHC Class I Gene H-2~cbInterferon Blanar et al., 1989 HSP70 Ela, SV40 Large T Taylor et al., 1989;
Antigen Taylor and Kingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, Tumor Necrosis FactorFMA Hensel et al., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., Hormone a Gene 1989 Turning to the expression of the MAPK proteinaceous molecules of the present invention, once a suitable clone or clones have been obtained, whether they be cDNA
based or genomic, one may proceed to prepare an expression system. The engineering of DNA segments) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may , be employed in the expression of the proteinaceous molecules of the present invention.
Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into proteinaceous molecules. Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude or more larger than the cDNA gene. However, it is contemplated that a genomic version of a particular gene may be employed where desired.

In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and 10 the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
15 a. ~lntisense and Ribozymes In some embodiments of the invention, an antisense nucleic acid can be used as an ERIC inhibitor. The term "antisense nucleic acid" is intended to xefer to the oligonucleotides complementary to the base sequences of DNA and RNA. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target 20 nucleic acid and interfere with transcription, RNA processing, transport and/or translation. Targeting double-stranded (ds) DNA with oligonucleotide leads to triple-helix formation; targeting RNA will lead to double-helix formation. An antisense nucleic acid may be complementary to SEQ ID NQ: l, 3, 5 or 7, complementary to an ERK encoding sequence or to ERK non-coding sequences.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNAs, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries (splice junctions) of a gene. It is contemplated that the most effective antisense constructs may include regions complementary to intron/exon splice junctions. Thus, antisense constructs with complementarily to regions within 50-200 bases of an intron-exon splice junction may be used. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used.
One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated.
For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed.
These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence.
Although shorter oligomers are easier to make and increase ih vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more base pairs will be used. One can readily determine whether a given antisense nucleic acid is effective at targeting of the corresponding host cell gene simply by testing the constructs irc vitro to determine whether the endogenous gene's function is affected or whether the expression of related genes having complementary sequences is affected.
In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines.
Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).
As an alternative to targeted antisense delivery, targeted ribozymes may be used.
The term "ribozyme" refers to an RNA-based enzyme capable of targeting and cleaving particular base sequences in oncogene DNA and RNA. Ribozymes either can be targeted directly to cells, in the form of RNA oligo-nucleotides incorporating ribozyme sequences, or introduced into the cell as an expression construct encoding the desired ribozymal RNA. Ribozymes may be used and applied in much the same way as described for antisense nucleic acids.
Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (I~im and Cech, 1987; Gerlack et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;
Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., 1992). Recently, it was reported that ribozymes elicited genetic changes in some cell lines to which they were applied; the altered genes included the oncogenes H-ras, c fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme. In light of the information included herein and the knowledge of one of ordinary skill in the art, the preparation and use of additional ribozymes that are specifically targeted to a given gene will now be straightforward.
Several different ribozyme motifs have been described with RNA cleavage activity (reviewed in Symons, 1992). Examples that would be expected to function equivalently for the down regulation of MAPKs include sequences from the Group I self splicing introns including tobacco ringspot virus (Prody et al., 1986), avocado sunblotch viroid (Palukaitis et al., 1979; Symons, 1981), and Lucerne transient streak virus (Forster and Symons, 1987). Sequences from these and related viruses are referred to as hammerhead ribozymes based on a predicted folded secondary structure.
Other suitable ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpin ribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993) and hepatitis ~ virus based ribozymes (Perrotta and Been, 1992). The general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988;
Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavage site on a given target RNA. Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology axe required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they axe separated by a specific sequence which is the cleavage site.
For hammerhead ribozymes, the cleavage site is a dinucleotide sequence on the target RNA, uracil (U) followed by either an adenine, cytosine or uracil (A,C or U;
Perriman, et al., 1992; Thompson, et al., 1995). The frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16. Therefore, for a given target messenger RNA
of 1000 bases, 187 dinucleotide cleavage sites axe statistically possible. The message for IGFBP-2 targeted here are greater than 1400 bases long, with greater than 260 possible cleavage sites.
Designing and testing ribozymes for efficient cleavage of a taxget RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al. (1994) and Lieber and Strauss (1995), each incorporated by reference. The identification of operative and preferred sequences for use in MAPI~-targeted ribozymes is simply a matter of preparing and testing a given sequence, and is a routinely practiced "screening" method known to those of skill in the art.
A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

It is proposed that MAPK, or more particularly ERK may be co-expressed with other selected proteinaceous molecules, wherein the proteinaceous molecules may be co-expressed in the same cell or MAPK gene may be provided to a cell that already has another selected proteinaceous molecule. Co-expression may be achieved by 5 co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of either of the respective DNA. Alternatively, a single recombinant vector may be constructed to include the coding regions for both of the proteinaceous molecules, which could then be expressed in cells transfected with the single vector. In either event, the term "co-expression" herein refers to the expression of both the MAPK gene and the 10 other selected proteinaceous molecules in the same recombinant cell.
As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding MAPK, has been introduced. Therefore, engineered cells are 15 distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adj acent to a promoter not naturally associated with the particular introduced gene.
To express a recombinant MAPK protein kinase, whether mutant or wild-type, in accordance with the present invention one would prepare an expression vector that comprises a wild-type, or mutant MAPK proteinaceous molecule-encoding nucleic acid under the control of one or more promoters. To bring a coding sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (i. e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein, polypeptide or peptide. This is the meaning of "recombinant expression" in this context.
Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein, polypeptide or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.
Certain examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Ser~~atia marcescens, and various Pseudomohas species.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E.
coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.
In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEMTM-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E. coli LE392.
Further useful vectors include pIN vectors (Inouye et al., 1985); and pGEX
vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with (3-galactosidase, ubiquitin, and the like.
Promoters that are most commonly used in recombinant DNA construction include the (3-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.

While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.
The following details concerning recombinant protein production in bacterial cells, such as E. coli, are provided by way of exemplary information on recombinant protein production in general, the adaptation of which to a particular recombinant expression system will be known to those of skill in the art.
Bacterial cells, for example, E. coli, containing the expression vector are grown in any of a number of suitable media, for example, LB. The expression of the recombinant proteinaceous molecule may be induced, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media.
The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.
If the recombinant proteinaceous molecule is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as [3-mercaptoethanol or DTT
(dithiothreitol).
Under some circumstances, it may be advantageous to incubate the proteinaceous molecule for several hours under conditions suitable for the proteinaceous molecule to undergo a refolding process into a conformation which more closely resembles that of the native proteinaceous molecule. Such conditions generally include low proteinaceous molecule concentrations, less than 500 mg/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the proteinaceous molecule.
The refolding process can be monitored, for example, by SDS-PAGE, or with antibodies specific for the native molecule (which can be obtained from animals vaccinated with the native molecule or smaller quantities of recombinant proteinaceous molecule). Following refolding, the proteinaceous molecule can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
For expression in Saccharorhyces, the plasmid YRp7, for example, is commonly used. This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No.
44076 or PEP4-1. The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate-MAPK, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol MAPK
2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate-MAPK, and enzymes responsible for maltose and galactose utilization.
In addition to micro-organisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more MAPK coding sequences.
Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN
and MDCK cell lines. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of proteinaceous products may be important for the function of the proteinaceous molecule.
Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteinaceous molecules.
Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign proteinaceous molecule expressed.
Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.SK promoter). Further, it is also possible, and may be S desirable, to utilize promoter or control sequences normally associated with the MAPK
gene, provided such control sequences are compatible with the host cell systems.
A number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 2S0 by sequence extending from the HihdIII site toward the BglI site located in the viral origin of replication.

In cases where an adenovirus is used as an expression vector, the coding sequences may be ligated to an adenovirus transcription/ translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
Insertion in a non-essential region of the viral genome (e.g., region E1, E3, or E4) will result in a recombinant virus that is viable and capable of expressing MAPK in infected hosts.
Specific initiation signals may also be required for efficient translation of MAPK
protein, polypeptide or peptide coding sequences. These signals include the ATG
~S initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements and transcription terminators.

In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly A
addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the proteinaceous molecule at a position prior to transcription termination.
For long-term, high-yield production of a recombinant MAPK protein, polypeptide or peptide, stable expression is preferred. For example, cell lines that stably express constructs encoding an MAPK protein, polypeptide or peptide may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then axe switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
A number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) genes, in tk-, hgprt' or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neomycin (neo), that confers resistance to the aminoglycoside G-41 ~; and hygromycin (hygro), that confers resistance to hygromycin.
Animal cells can be propagated ih vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i. e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower proteinaceous molecule production than adherent cells.
- Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteinaceous molecules. Two suspension culture reactor designs are in wide use - the stirred reactor and the airlift reactor.
The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
It is contemplated that the MAPI~ proteins, polypeptides or peptides of the invention may be "overexpressed", i. e., expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or proteinaceous molecule purification.
However, simple and direct methods are preferred, for example, those involving SDS/PAGE
and proteinaceous composition staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A
specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific proteinaceous molecule in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
VIII. Methods of Gene Transfer In order to mediate the effect of transgene expression in a cell, it will be necessary to transfer the expression constructs (e.g., a therapeutic construct) of the present invention into a cell. Such transfer may employ viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions of gene or nucleic acid transfer, including transfer of antisense sequences.
The mammalian MAPK genes are incorporated into an adenoviral infectious particle to mediate gene transfer to a cell. Additional expression constructs encoding other therapeutic agents as described herein may also be transferred via viral transduction using infectious viral particles, for example, by transformation with an adenovirus vector of the present invention as described herein below.
Alternatively, retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a genes) of interest. Thus, in one example, viral infection of cells is used in order to deliver therapeutically significant genes to a cell.
Typically, the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus. Though adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below.
a. Adenoviral Veetors A particular method for delivery of the expression constructs for MAPK
inhibition involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
"Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences su~cient to (a) support packaging of the construct and/or (b) to ultimately express a tissue and/or cell-specific construct that has been cloned therein.

The expression vector comprises a genetically engineered form of adenovirus.
Knowledge of the genetic organization and/or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and/or Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because .-adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
Also, adenoviruses are structurally stable, and/or no genome rearrangement has been detected after extensive amplification.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and/or high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and/or packaging.
The early (E) andlor late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (ElA
and/or E1B) encodes proteins responsible for the regulation of transcription of the viral genome and/or a few cellular genes. The expression of the E2 region (E2A and/or E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and/or host cell shut-off (Renan, 1990).
The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and/or all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and/or provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and/or examine its genomic structure.

Generation and/or propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and/or constitutively expresses E1 proteins (ElA and/or E1B; Graham et al., 1977).
Since the 5 E3 region is dispensable from the adenovirus genome (Jones and/or Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 and/or both regions (Graham and/or Prevec, 1991). Recently, adenoviral vectors comprising deletions in the E4 region have been described (U.S. Patent 5,670,488, incorporated herein by reference).
In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA.
Combined with the approximately 5.5 kb of DNA that is replaceable in the El and/or E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, and/or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.
Helper cell lines may be derived from human cells such as embryonic kidney cells, muscle cells, hematopoietic cells and/or other embryonic mesenchymal and/or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells and/or other monkey embryonic mesenchymal and/or epithelial cells.
Recently, Racher et al. (1995) disclosed improved methods for culturing 293 cells and/or propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (SO ml) in a 250 ml Erlenmeyer flask and/or left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and/or shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25%
of the final volume) and/or adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and/or shaking commenced for another 72 h.
S Other than the requirement that the adenovirus vector be replication defective, and/or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes and/or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a adenovirus about which a great deal of biochemical and/or genetic information is known, and/or it has historically been used for most constructions employing adenovirus as a vector.
As. stated above, the typical vector according to the present invention is replication defective and/or will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the transforming construct at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
The polynucleotide encoding the gene of interest may also be inserted in Iieu of the deleted E3 region in E3 replacement vectors as described by I~arlsson et al. (1986) and/or in the E4 region where a helper cell line and/or helper virus complements the E4 defect.
Adenovirus growth and/or manipulation is known to those of skill in the art, and/or exhibits broad host range in vitro and/or ih vivo. This group of viruses can be obtained in high titers, e.g., 109 to 1011 plaque-forming units per ml, and/or they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have Iow genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and/or therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and/or vaccine development (Grunhaus and/or Horwitz, 1992; Graham and/or Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and/or Perricaudet, 1991a; Stratford-Perricaudet et al., 1991b; Rich et al., 1993).
Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz andlor Gerard, 1993) and/or stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Recombinant adenovirus andlor adeno-associated virus (see below) can both infect and/or transduce non-dividing hyman primary cells.
b. AA h Vectors Adeno-associated virus (AAV) is an attractive vector system for use in the cell transduction of the present invention as it has a high frequency of integration and/or it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) and/or i~ vivo. AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986;
Lebkowski et al., 1988; McLaughlin et al., 1988). Details concerning the generation and/or use of rAAV
vectors are described in U.S. Patent No. 5,139,941 and/or U.S. Patent No.
4,797,368, each incorporated herein by reference.
Studies demonstrating the use of AAV in gene delivery include LaFace et al.
(1988); Zhou et al. (1993); Flotte et al. (1993); and/or Walsh et al. (1994).
Recombinant AAV vectors have been used successfully for in vitro and/or ih vivo transduction of marker genes (Kaplitt et al., 1994; Lebkowski et al., 1988; Samulski et al., 1989; Yoder et al., 1994; Zhou et al., 1994; Hermonat andlor Muzyczka, 1984; Tratschin et al., 1985;
McLaughlin et al., 1988) and/or genes involved in human diseases (Flotte et al., 1992;
Luo et al., 1994; Ohi et al., 1990; Walsh et al., 1994; Wei et al., 1994).
AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus and/or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991).
rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and/or Smith, 1994). When a cell carrying an AAV
provirus is superinfected with a helper virus, the AAV genome is "rescued"
from the chromosome and/or from a recombinant plasmid, and/or a normal productive infection is established (Samulski et al., 1989; McLaughlin et al., 1988; I~otin et al., 1990;
Muzyczka, 1992).
Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) and/or an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference).
The cells are also infected and/or transfected with adenovirus and/or plasmids carrying the adenovirus genes required for AAV helper function. rAAV virus stocks made in such fashion are contaminated with adenovirus which must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation).
Alternatively, adenovirus vectors containing the AAV coding regions and/or cell lines containing the AAV coding regions and/or some and/or all of the adenovirus helper genes could be used (Yang et al., 1994; Clark et al., 1995). Cell lines carrying the rAAV
DNA as an integrated provirus can also be used (Flotte et al., 1995).
c. Retroviral Vectors Retroviruses have promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and/or cell types and/or of being packaged in special cell-lines (Miller, 1992).
The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and/or directs synthesis of viral proteins.
The integration results in the retention of the viral gene sequences in the recipient cell and/or its descendants. The retroviral genorne contains three genes, gag, pol, and/or env that code for capsid proteins, polymerase enzyme, and/or envelope components, respectively.
A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and/or 3' ends of the viral genome. ~ These contain strong promoter and/or enhancer sequences and/or are also required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and/or env genes but without the LTR and/or packaging components is constructed (Maim et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and/or packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged info viral particles, which are then secreted into the culture media (Nicolas and/or Rubenstein, 1988;
Temin, 1986;
Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and/or used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and/or stable expression require the division of host cells (Paskind et al., 1975).
Concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Gene delivery using second generation retroviral vectors has been reported.
I~asahara et al. (1994) prepared an engineered variant of the Moloney marine leukemia virus, that normally infects only mouse cells, and/or modified an envelope protein so that the virus specifically bound to, and/or infected cells bearing the erythropoietin (EPO) 5 receptor. This was achieved by inserting a portion of the EPO sequence into an envelope protein to create a chimeric protein with a new binding specificity.
d. Herpesvirus Because herpes simplex virus (HSV) is neurotropic, it has generated considerable 10 interest in treating nervous system disorders. Moreover, the ability of HSV
to establish latent infections in non-dividing neuronal cells without integrating in to the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency makes HSV an attractive vector. And though much attention has focused on the neurotropic applications of HSV, this vector also can 15 be exploited for other tissues given its wide host range.
Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, 20 strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations.
HSV also is relatively easy to manipulate and can be grown to high titers.
Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI
25 and in a lessened need for repeat dosings. For a review of HSV as a gene~therapy vector, see (Glorioso et al., 1995).
HSV, designated with subtypes 1 and 2, are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. The large, complex, double-stranded DNA genome encodes for 30 dozens of different gene products, some of which derive from spliced transcripts. In addition to virion and envelope structural components, the virus encodes numerous other proteins including a protease, a ribonucleotide reductase, a DNA polymerase, a ssDNA
binding protein, a helicase/primase, a DNA dependent ATPase, dUTPase and others.
HSV genes from several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion (Honess and Roizman, 1974; Honess and Roizman, 1975; Roizman and Sears, 1995). The expression of a genes, the first set of genes to be expressed after infection, is enhanced by the virion protein number 16, or a-transducing factor (Post et al., 1981; Batterson and Roizman, 1983; Campbell et al., 1983). The expression of (3 genes requires functional a gene products, most notably ICP4, which is encoded by the a4 gene (DeLuca et al., 1985). y genes, a heterogeneous group of genes encoding largely virion structural proteins, require the onset of viral DNA
synthesis for optimal expression (Holland et al., 1980).
In line with the complexity of the genome, the life cycle of HSV is quite involved. In addition to the lytic cycle, which results in synthesis of virus particles and, eventually, cell death, the virus has the capability to enter a latent state in which the genome is maintained in neural ganglia until some as of yet undefined signal triggers a recurrence of the lytic cycle. Avirulent variants of HSV have been developed and are readily available for use in gene therapy contexts (U.S. Patent 5,672,344).
e. Lentiviral T~eetors Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and ehv, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus:
SIV. Lentiviral vectors have been generated by multiply attenuating the HIV
virulence genes, for example, the genes env, vif, vpr, vpu and ref are deleted making the vector biologically safe.
Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both i~ vivo and ex vivo gene transfer and expression of nucleic acid sequences. The lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the e~cv gene encodes viral envelope glycoproteins. The 5' and 3' LTR's serve to promote transcription and polyadenylation of the virion RNA's. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, f~ev, vpu, nef and vpx.
Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
Lentiviral vectors are known in the art, see Naldini et al., (1996); Zufferey et al., (1997), U.S. Patents 6,013,516 and 5,994,136. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art.
Thus, the relevant genes are cloned into the selected vector and then used to transform the target I
cell of interest.
Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well ~as rev and tat is described in U.S. Patent 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can 30~ provide a nucleic acid encoding a viral ehv to produce a packaging cell.
Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The ehv preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By -- inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
The vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney marine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney marine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.
The heterologous or foreign nucleic acid sequence is linked operably to a regulatory nucleic acid sequence. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. The heterologous nucleic acid sequence may also be under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene. Marker genes may be utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts.
Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate, etc.
and cell surface markers.
The vectors are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral particles that contain the vector genome.
Methods for transfection or infection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct.
f. Vaceinia Virus Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA
genome of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal repeats of about 10.5 kb flank the genome. The maj ority of essential genes appear to map within the central region, which is most highly conserved among poxviruses. Estimated open reading frames in vaccinia virus number from 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common.
At least 25 kb can be inserted into the vaccinia virus genome (Smith and Moss, 1983). Prototypical vaccinia vectors contain transgenes inserted into the viral thymidine kinase gene via homologous recombination. Vectors are selected on the basis of a tk-phenotype. Inclusion of the untranslated leader sequence of encephalomyocarditis virus, the level of expression is higher than that of conventional vectors, with the transgenes accumulating at 10% or more of the infected cell's protein in 24 h (Elroy-Stein et al., 1989).
g. Polyoma vzruses The empty capsids of papovaviruses, such as the mouse polyoma virus, have received attention as possible vectors for gene transfer (Barn et al., 1979), first described the use of polyoma empty when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particle was protected from the action of pancreatic DNase. Slilaty and Aposhian (1983) described the use of those reconstituted particles for transferring a transforming polyoma DNA fragment to rat FIII

cells. The empty capsids and reconstituted particles consist of all three of the polyoma capsid antigens VPl, VP2 and VP3 and there is no suggestion that pseudocapsids consisting of only the major capsid antigen VP1, could be used in genetic transfer.
(Montross et al., 1991), described only the major capsid antigen, the cloning of 5 the polyoma virus VP1 gene and its expression in insect cells. Self assembly of empty pseudocapsids consisting of VP1 is disclosed, and pseudocapsids are said not to contain DNA. It is also reported that DNA inhibits the in vitro assembly of VP 1 into empty pseudocapsids, which suggests that said pseudocapsids could not be used to package exogenous DNA for transfer to host cells. The results of (Sandig et al., 1993), showed 10 that empty capsids incorporating exogenous DNA could transfer DNA in a biologically functional manner to host cells only if the particles consisted of all three polyoma capsid antigens VPl, VP2 and VP3. Pseudocapsids consisting of VP1 were said to be unable to transfer to exogenous DNA so that it could be expressed in the host cells, probably due the absence of Caa+ ions in the medium in which the pseudocapsids were prepared.
15 Haynes et al. (1993) discuss the effect of calcium ions on empty VPl pseudocapsid assembly.
U.S. Patent 6,046,173, issued on April 4, 2000, and entitled "Polyoma virus pseudocapsids and method to deliver material into cell," reports on the use of a pseudocapsid formed from papovavirus major capsid antigen and excluding minor capsid 20 antigens, which pseudocapsid incorporates exogenous material for gene transfer.
h. Other hiral Vectors Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as sindbis virus and/or cytomegalovirus.
25 They offer several attractive features for various mammalian cells (Friedmann, 1989;
Ridgeway, 1988; Baichwal and/or Sugden, 1986; Cougar et al., 1988; Horwich et al., 1990).
With the recent recognition of defective hepatitis B viruses, new insight was 30 gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and/or reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions df the genome could be replaced with foreign genetic material. Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and/or pre-surface coding sequences. It was cotransf~cted with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT
gene expression was detected for at least 24 days after transfection (Chang et al., 1991).
i. Modified Viruses In still further embodiments of the present invention, the nucleic acids to be delivered are housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and/or deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and/or against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and/or class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus ih vitro (Roux et al., 1989).
j. Nou-viral Trahsfer DNA constructs of the present invention are generally delivered to a cell, in certain situations, the nucleic acid to be transferred is non-infectious, and can be transferred using non-viral methods.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells are contemplated by the present invention. Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an -organism, as described herein or as would be known to one of ordinary skill in the art.
Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); by electroporation (IJ.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;
Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 197;
Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95106128; U.S. Patent Nos. 5,610,042;
5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA
uptake (Potrykus et al., 1985). Through the application of techniques such as these, organelle(s), cell(s), tissues) or organisms) may be stably or transiently transformed.
Once the construct has been delivered into the cell the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
In a particular embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al., 1997). These DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Using the (3-lactamase gene, Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving "lipofection" technology.
In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ]. This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989).
In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet fiuther embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and i~ vivo, then they are applicable for the present invention.
Other vector delivery systems which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
Receptor-mediated gene targeting vehicles generally consist of two components:
a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al., 1990).
Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, the human prostate-specific antigen (Watt et al., 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue.
In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied fox i~ vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of CaP04 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of 5 CaP04 precipitated plasmids results in expression of the transfected genes.
It is envisioned that DNA encoding a CAM may also be transferred in a similar manner in vivo and express CAM.
Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the 10 ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., I987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of 15 biologically inert substances such as tungsten or gold beads.
IX. Proteinaceous Compositions In certain embodiments, the present invention concerns novel compositions or methods comprising at least one proteinaceous molecule. The proteinaceous molecule 20 may be a MAPK such as ERK1 (SEQ ID NO: 2) or ERK2 (SEQ ID N0:4), MEK1 (SEQ
ID NO: 6) or MEK2 (SEQ ID NO: 8). Also, the proteinaceous molecule may be a MAPK inhibitor or more preferably an ERK or ERK inhibitor, or a delivery agent. The proteinaceous molecule may also be a mutated MAPK. The proteinaceous molecule may also be used, for example, as an MAPK or ERK inhibitor, in a pharmaceutical 25 composition for the delivery of a therapeutic agent or as paxt of a screening assay in the determination of MAPK inhibition. As used herein, a "proteinaceous molecule,"
"proteinaceous composition," "proteinaceous compound," "proteinaceous chain"
or "proteinaceous material" generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a 30 polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the "proteinaceous" terms described above may be used interchangeably herein.

In certain embodiments the size of the at least one proteinaceous molecule may comprise, but is not limited to, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 l, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino molecule residues, and any range derivable therein.
As used herein, an "amino molecule" refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
Accordingly, the term "proteinaceous composition" encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not Limited to those shown on Table S below.
TABLE

Modified and Unusual Amino Acids Abbr.Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3- Aminoadipic acid Hyl Hydroxylysine Bala /3-alanine, (3-Amino-propionicAHyI alto-Hydroxylysine acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4- Aminobutyric acid, piperidinic4Hyp 4-Hydroxyproline acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIIe aLlo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobuiyric acid MeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2'-Diaminopimelic acid NLe Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine 5 In certain embodiments the proteinaceous composition comprises at least one protein, polypeptide or peptide, such as a MAPK. In fzuther embodiments the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
As used herein, the term "biocompatible" refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Organisms include, but are not limited to, Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions. In preferred embodiments, biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or ~8 peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials. The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http:/lwww.ncbi.nlm.nih.gov~. The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art.
Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
In certain embodiments a proteinaceous compound may be purified. Generally, "purified" will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.
In certain embodiments, the proteinaceous composition may comprise at least one antibody. An ERK inhibitor may comprise all or part of an antibody that specifically recognizes .ERKl or ERK2. It, is contemplated that antibodies to specific tissues may bind the tissues) and foster tighter adhesion of the glue to the tissues after welding. As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

The term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in S the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference).
It is contemplated that virtually any protein, polypeptide or peptide containing component may be used in the compositions and methods disclosed herein.
However, it is preferred that the proteinaceous material is biocompatible. In certain embodiments, it is envisioned that the formation of a more viscous composition will be advantageous in that will allow the composition to be more precisely or easily applied to the tissue and to be maintained in contact with the tissue throughout the procedure. In such cases, the use 1 S of a peptide composition, or more preferably, a polypeptide or protein composition, is contemplated. Ranges of viscosity include, but are not Limited to, about 40 to about I00 poise. In certain aspects, a viscosity of about 80 to about 100 poise is preferred.
Proteins and peptides suitable for use in this invention may be autologous proteins or peptides, although the invention is clearly not limited to the use of such autologous proteins. As used herein, the term "autologous protein, polypeptide or peptide" refers to a protein, polypeptide or peptide which is derived or obtained from an organism. Organisms that may be used include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungal, a plant, 2S or a prokaryotic organism, with a selected animal or human subject being preferred. The "autologous protein, polypeptide or peptide" may then be used as a component of a composition intended for application to the selected animal or human subject.
In certain aspects, the autologous proteins or peptides are prepared, for example from whole plasma of the selected donor. The plasma is placed in cubes and placed in a freezer at about -80°C for at least about 12 hours and then centrifuged at about 12,000 times g for about 1 S minutes to obtain the precipitate. The precipitate, such as fibrinogen may be stored for up to about one year (Oz, 1990).

X. Protein Purification To prepare a composition comprising the MAPK inhibitor, or more preferentially, the ERK inhibitor it may be desirable to purify the components or variants thereof. According to one embodiment of the present invention, purification of a peptide 5 comprising the MAPK inhibitor can be utilized ultimately to operatively link this domain with a selective agent. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be fiu-ther purified using 10 chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A
particularly efficient method of purifying peptides is fast protein liquid chromatography or even 15 HPLC.
Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide, such as a MAPK inhibitor. The term "purified protein or peptide" as used herein, is intended to 20 refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A
purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
25 Generally, "purified" will refer to a protein or peptide composition, such as the MAPK. inhibitor, that has been subjected to fractionation to remove. various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the 30 composition, such as constituting about 50%, about 60%, about 70%, about ~0%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis;
and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
P

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution.
Elution occurs by changing the conditions to those in which binding will not occur (e.g., alter pH, ionic strength, and temperature. ).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agaxose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
The ligand should be coupled in such a way as to not affect its binding properties. The ligand also should provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
a. Synthetic Peptides The present invention also describes a MAPI~ inhibitor, including an fusion protein, for use in various embodiments of the present invention. The peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewaxt and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Peptides with at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or up to about 100 amino acid residues are contemplated by the present invention.
The compositions of the invention may include a peptide comprising a MAPK, a MAPK inhibitor, or a MEK that has been modified to enhance its activity or to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S.
patent 5,028,592, incorporated herein by reference, protected peptides often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise peptides that include all L-amino acids, all D-amino acids, or a mixture thereof. The use of D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
XI. Screens for-Mammalian MAPK
In still further embodiments, the present invention provides methods for identifying new compounds that modulate MAPK, or more preferably ERK activity, which may be termed as "candidate substances." "Modulating compounds" or "compounds that modulate MAPK activity" is meant to refer to substances that enhance, inhibit, or alter the activity of MAPK. Such altered activity includes, but is not limited to, changes in binding preferences for target substrates, particularly for serine proteases, and changes in proteinaceous molecule-proteinaceous molecule interactions of MA.PK
that may occur. It is contemplated that such screening techniques will prove useful in the general identification of any compound that will serve the purpose of modulating MAPK, or more particularly ERK activity.
MAPK modulators identified will have utility in methods involved in serine 5 proteases, and are also contemplated for therapeutic uses. Modulators that affect MAPK
affinity for the serine proteases or MAPK proteinaceous molecule-proteinaceous molecule interaction with other proteins or proteases, are also contemplated.
For example, the ability to specifically modulate MAPK activity is envisioned to be useful in cancer. Further, the impact of any possible adverse effects by a modulator of MAPK
10 activity can be limited or otherwise controlled by the more specific administration of the modulator to a tumor site, such as by direct application to a tumor or cancerous tissues.
It is further contemplated that useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds. In fact, it may prove to be the case that 15 the most useful pharmacological compounds for identification through application of the screening assays will be non-peptidyl in nature and, e.g., which will serve to modulate MAPK activity through a tight binding or other chemical interaction. Candidate substances may be obtained from libraries of synthetic chemicals, or from natural samples, such as rain forest and marine samples.
1. Modulation of MAPK
To identify a MAPK modulator using a MAPK protease inhibitor assay, one would simply conduct parallel or otherwise comparatively controlled protease inhibitor assays and identify a compound that modulates MAPK protease inhibitor activity. The candidate screening assay is quite simple to set up and perform. After obtaining a relatively purified preparation of MAPK protein, polypeptide or peptide, either from native or recombinant sources, one will simply admix a candidate substance with the MAPK preparation, under conditions that would allow MAPK to perform its function but for inclusion of a modulating substance.
For example, one will typically desire to include within the admixture an amount of a serine protease, although other substrates may be used, such as other proteases. In any event, one would measure the ability of the candidate substance to alter protease inhibition by the MAPK protein, polypeptide, or peptide in the presence of the candidate substance. In general, one will desire to measure or otherwise determine the activity of the relatively purified MAPK in the absence of the added candidate substance relative to the activity in the presence of the candidate substance in order to assess the relative modulating capability of the candidate substance.
XII. Pharmaceutical Preparations Pharmaceutical compositions of the present invention comprise an effective amount of one or more MAPK, or more particularly one or more ERK inhibitor or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
The preparation of an pharmaceutical composition that contains at least one MAPK
inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed.
Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifiuzgal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, . .lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional caxrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The MAPK inhibitor may comprise different types of carriers depending on whether it is to be administered in solid or liquid form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intradermally, subcutaneously, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredients) in a composition and appropriate doses) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1 % of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
In other non-limiting .examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kglbody weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
In non-limiting examples of a derivable range from the numbers listed hexein, a range of about 5 mg/kg/body weight to about 100 mg/kglbody weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
The MAPK inhibitor may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such 1 S organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, fox example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose;
or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of D1VIS0 as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
Administration of opioids in the epidural or intrathecal space provides more direct access to the first pain-processing synapse in the dorsal horn of the spinal cord.
This permits the use of doses substantially lower than those required for oral or parenteral administration (see Table 23-7). Systemic side effects are thus decreased.
However, epidural opioids have their own dose-dependent side effects, such as itching, nausea, vomiting, respiratory depression, and urinary retention. The use of hydrophilic opioids such as preservative-free morphine (DURAMORPH) permits more rostral spread of the compound, allowing it to directly affect supraspinal sites. As a consequence, after intraspinal morphine, delayed respiratory depression can be observed for as long as 24 hours after a bolus dose. While the risk of delayed respiratory depression is reduced with more lipophilic opioids it is not reduced. Extreme vigilance and appropriate monitoring is required for all patients receiving intraspinal narcotics.
Nausea and vomiting are also more prominent symptoms with intraspinal morphine. However, supraspinal analgesic centers can also be stimulated, possibly leading to synergistic analgesic effects.
Analogous to the relationship between systemic opioids and NSAIDS, intraspinal narcotics are often combined with local anesthetics. This permits the use of lower concentrations of both agents, minimizing local anesthetic complications of motor blockade and the opioid induced complications listed above. Epidural administration of opioids have become popular in the management of postoperative pain, and for providing analgesia for labor and delivery. Lower systemic opioid levels are achieved with epidural opioids, leading to less placental transfer and less potential for respiratory depression of the newborn (Shnider and Levinson 1987). Intrathecal ("spinal"
anesthesia) administration of opioids as a single bolus is also popular for acute pain management.
XIII. Combinational Therapy It is an aspect of this invention that the MAPK inhibitor can be used in combination with another agent, preferably an opioid. The MAPK inhibitor may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i. e., within less than about a minute) as the MAPK nucleic acid construct or proteinaceous molecule. In other aspects, one or more agents may be administered within of from about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about I9 hours, about 20 hours, about 2I hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, to about 48 hours or more prior to and/or after administering the MAPK inhibitor. In certain other embodiments, an agent may be administered within of from about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about days, about 16 days, about 17 days, about 18 days, about 19 days, about 20, to about 21 days prior to and/or after administering the MAPK inhibitor. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about I S 8 weeks or more) lapse between the respective administrations.
Various combinations may be employed, the MAPK inhibitor is "A" and the secondary agent, such as an morphine, is "B":
AB/A B/AB BB/A A/A/B A/BB B/A/A ABBB BIABB
B/BB/A BB/AB AlABB ABlAB ABB/A BB/A/A
B/AB/A B/A/AB A/A/A/B B/A/A/A AB/A/A A/A/B/A
Administration of the therapeutic expression constructs of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy.

Opioid analgesics that can be used in conjugation with the MAPK inhibitor include, but are not limited to morphine, morphine sulphate, tramadol, codeine, levorphanol, meperidine and congeners such as diphenoxylate and loperaminde, sufentanil citrate and congeners such as alfentanil and remifentanil, methadone and congeners, levomethadyl acetate (LAAM), propoxyphene, butorphanol, eptazocine, fentanyl, fentanyl citrate, flupirtine, hydromorphone and oxycodone,. Other opioid compounds that may be used include, but are not limited to pentazocine, nalbuphine, butorphanol, buprenorphine, meptazinol, ~ dezocine, nalorphine, levallorphan and nalmefene, morphine-6-glucuronide, morphine (DepoMorphine, AERx Pain 20 Management System, Multipor technology), morphine sulphate, pulmonary-delivered morphine sulphate, and other morphine-like compounds including conorfone, propiram fumarate, various strength opioid analgesics using OROS technology, various strength analgesics using Geomatrix technology, fentanyl, AERx Pain Management System, buprenorphine, asimadoline, TRK-820, LEF (BCH-3963), loperamide, oxycodone and oxycodone combinations (i.e. oxycodone + ibuprofen or oxycodone +
paracetamol), DPI-3290, ADL-10-0101, Xorphanol, TSN-09, and a combination of NMDA antagonist and an opioid compound, (i.e. dextromethorphan + hydrocodone, dextromethorphan +
morphine and dextromethorphan + oxycodone + paracetamol) ("Advances in Pain Management" February 2000 Scrip Reports).
IX. MAPK Inhibitors The present invention also provides inhibitors of MAPK, or more preferably, inhibitors of ERKl or ERK2. The inhibitors may be purified by methods well known in the art including a variety of chromatograpluc methods. Preferred ERK
inhibitors contemplated as useful in this invention include, but are not limited to:
(a) U0126, (1,4-diamino-2,3-dicyano-1,4- bis[2-aminophenylthioJ butadiene) available from DuPont and places such as Cell Signal, or derivatives of U0126, such as those comprising replacing the amino substitution of the phenyl ring with, for example: S, CN, CH3, =O, I, F, OCH3, OCH2CH3, CHZOH, CHaOMe, CH20Et, CHZSEt, CH2SPr, CHZSPr, CH2SCH2CH=CH2, CH20SnBu, and CHaSCH2CH2NMea.

S ~ ~ S

(b) SL-327, a structural analog of U0126, and (c) PD 184352 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide which is from Parke-Davis.
Other MAPK inhibitors contemplated as useful in this invention include:
(d) PD 098059, [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], which can be written as [2'-amino-3'-methoxyflavone] and is from Parke Davis (e) K252a (R=H), an indolocarbazole alkaloid isolated from Nocardiopsis (Kase et al., 1986) H
O
H3C iiu~..
HO
Cbzlne or derivatives wherein R is CH~OH, CH20Me, CH20Et, CH2SEt, CH2S"Pr, CH2S'Pr, CH2SCHaCH=CH2, CH20S"Bu, and CH2SCH2CHaNMe2 (Kaneko et al., 1997), (f) CEP-1347, a JNK kinase inhibitor from Cephalon which is also known as KT7515, a derivative of the indolocarbazole K252a, (g) SB 203580, or 4- (4-fluorophenyl)-2- (4methylsulfinylphenyl)-5- (4-pyridyl)-1H-imidazole which is a p38 MAPK inhibitor from SmithKline Beecham, and (h) SB 202190, 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole which is a p38 MAPK inhibitor from SmithKline Beecham.
As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
As used herein the specification, the terms "Figure" and "FIG." are used interchangably XV. Examples The following example is included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1-The Effects of Claronic Opioids on ERIPActivation In Vitro N2A neuroblastoma cell lines stably transfected with the ~, opioid receptor were grown in 10%FCS/DMEM. Cells were treated with 1 M fentanyl on day one, with the dose doubled for 7 days. Control cells were treated with an equivalent volume of water daily. On day 7, the cells were washed twice with control media, and then placed in control media containing 1M naloxone. Unwashed day 7 water treated cells served as baseline controls, while unwashed fentanyl treated cells documented the effect of chronic opioid treatment alone. ERK activity was assessed by immunoblotting with phospho-lOS
ERK antibody followed by enhanced chemifluorescent detection. Data were analyzed by one-way ANOVA followed by the Dunnett post-hoc test, with p<O.OS considered significant.
Chronic treatment with fentanyl reduced baseline ERF activity to 86% of water S treated controls (FIG. 1). After withdrawal of Fentanyl, opioid treated cells exhibited increases in ERF activity (FIG. l; *-p<O.OS). This activation peaked at 60 minutes and remained elevated 120 minutes after fentanyl withdrawal.
These results indicate that the ERK signaling system may play a role in the development of opioid dependence and possibly the associated withdrawal syndrome.
The time course of ERF activation after opioid withdrawal is consistent with a role for ERK in the behavioral effects seen in the withdrawal syndrome. These results are consistent with prior immunocytochemical (ICC) findings (Schulz et al. 1998).
Example 2- Inhibition of Tolerance b Spinal Infusion Ih Vivo The inhibition of tolerance by spinal infusion of the ERF cascade inhibitor 1 S U0126 was determined in an in vivo experiment (FIG. 2). Like most previously available ERIC inhibitors, U0126 does not cross the blood-brain barrier. 21 male Sprague- Dawley rats had intrathecal catheters implanted under halothane anesthesia and were allowed to recover for a week. Animals received spinal catheters infusing either saline (S), 70% DMSO vehicle (V) or U0126 in vehicle (V1U0126) Initially, one 7S mg morphine (M) or a placebo (P) pellet was implanted, and on day 3, animals received 3 additional pellets of the same type. U0126 did not cause analgesia or interfere with the analgesic actions of opioids. Tolerance development was assessed using tail flick latency. On day 7, withdrawal was precipitated using naloxone (2mg/kg, i.p.) and withdrawal symptoms observed. Results were then analyzed by repeated measures 2S ANOVA and the Tukey-Framer post-hoc test with p<O.OS considered significant.
U0126 alone did not possess analgesic properties and did not augment morphine analgesia (FIG. 2). After 48 hours, there was a trend toward inhibition of tolerance by U0126; after the second pelleting, this trend reached statistical significance. However, the DMSO vehicle also mildly inhibited tolerance development, but did not posses analgesic properties. These results demonstrate the ERF inhibition reduces the development of opioid tolerance in rats. It is not surprising that withdrawal symptoms were not reduced in the present study, as many of these symptoms are thought to be mediated supraspinally (Miyamoto et al., 1993a and b). The confounding effect of DMSO on tolerance inhibition will be avoided by the use of novel solubilizing agents.
For further studies, systemically available ERIC. cascade inhibitor, SL-327, which can be solublized in a non-toxic carrier (13-cyclodextrin) can be used.
Example 3- ICC for Activated ERK
An ICC has been developed for activated ERIC (Anti-Active MAPK, Promega, 1:500). FIG. 3 demonstrates robust ERK activation in the hypothalamic paraventricular nucleus 1 hour after osmotic stress.
Example 4 - Tlae Effect of Chronic Morphine Administration oh RGS4 mRNA Levels The effect of chronic morphine administration on RGS4 mRNA levels. RGS4 was chosen because of its expression almost exclusively in brain and association with G;
subunits. A paradigm was employed that also produces behavioral sensitization to the locomotor effects of morphine. 10 mg/kg morphine was given to rats daily for two weeks, followed by a challenge dose of 10 mg/kg two weeks later. mRNA levels were determined one hour after the challenge dose. Decreased RGS4 mRNA was observed the red nucleus, an area associated with motor activation (data not shown), which could provide an explanation for opioid stimulatory effects. In contrast, increased mRNA was observed in the dorsal central gray (CGD; FIG. 4), an area associated with analgesic responses. It has long been known that when given chronically, tolerance can develop to some behavioral effects of opioids (i.e., analgesia), while sensitization can develop to other effects (i.e., motor activity). This intriguing fording of differential RGS
4 regulation in .distinct brain regions suggests a mechanistic explanation for these .
divergent behavioral consequences of chronic opioid administration.
Exaynple 5 - ERKlhlzibitiou Reduces Opioid Tolerance iu Rats Extracellular signal-related kinase(ERK) is a member of the mitogen-activated protein kinase(MAPK) superfamily, which are involved in cell regulation. We and others have shown that ERK is activated by p opioids and is the only signaling system activated by acute opioid administration. ERK activation by opioids exhibit both acute and chronic desensitization, suggesting a potential role for ERIC in opioid tolerance and dependence.
The present study determined whether inhibition of ERK by U0126 would affect opioid tolerance development. Methodology: 21 male Sprague-Dawley rats had intrathecal S catheters implanted under halothane anesthesia and recovered for 1 week.
Subsequently, either a 75 mg morphine sulfate or placebo pellet was implanted and a primed osmotic minipump infused 0.1M U0126, DMSO vehicle or saline was connected to the catheter.
Tolerance development was assessed using tail flick latency. Three days after the initial pelleting, 3 additional pellets of the same type were implanted. On day 7, withdrawal was precipitated using naloxone and withdrawal symptoms observed. Results were then analyzed by repeated measures ANOVA and Tukey-T~ramer posthoc test with p<p.05 considered significant. Results: UO126 alone did not possess analgesic properties and did not augment morphine analgesia. After 48 hours, there was a trend toward inhibition of tolerance by UOI26; after the second pelleting, this trend reached statistical significance. However, the DMSO vehicle also mildly inhibited tolerance development, but did not posses analgesic properties. These results demonstrate the ERK
inhibition reduces the development of rat opioid tolerance.
Example 6 - Acute Admihistratioh of Opioids and Psyclaostimulafzts Alter RGS 4 mRNA Levels Groups of six male Sprague-Dawley rats were given i.p. injections of either morphine (10 mg/kg), cocaine (25 mglkg), amphetamine (5 mgfkg), or saline.
Sixty minutes later, the brains were quickly removed, frozen, sectioned at 10 ~,m, slide mounted, and subsequently fixed in paraformaldehyde. In situ hybridization histochemistry was then performed using a cRNA probe for RGS 4. Slides were exposed on x-ray film for 1-11 days. Subsequently, slides were counterstained with cresyl violet to aid in anatomical localization. Autoradiograms were analyzed using a Northern Light imaging station and MCID/MS+ image analysis software.
Ih Situ Hybridization: a 618 by fragment of rat RGS 4 (courtesy of R.
Mackenzie) subcloned into the TA cloning site of pCR 2.0 (Invitrogen) was used to generate the cRNA probe. After linearization with hindIII, this DNA was transcribed from the T7 promoter using T7 polymerase in a standard labeling reaction containing 125 ~.Ci35S-UTP to produce a 388 by probe. Labeled probe was separated from free nucleotide over a G50-50 Sephadex column.
Brain sections were removed from storage and fixed in 4% formaldehyde for one hour. Slides were washed in 2X SSC for 10 min and treated with proteinase I~
(1 ~,g/ml in 100 mM tris-HCl, pH+8.0) for 10 min at 37°C. Slides were then washed in O.1M
triethanolamine with 0.25% acetic anhydride for 10 min and 2X SSC for 5 min before being dehydrated through graded alcohols. Sections were then hybridized with 1.5 x 10 6 dpm of 35S-labeled riboprobe in a standard 50% hybridization buffer using glass coverslips. Hybridization was allowed to continue for 16 hr at 55°C.
Sections were then rinsed in 2X SSC, treated with RnaseA (200 ~glml in 10 mM Tris-HC1, 0.5 M
NaCl, pH-8.0) for 60 min at 37°C and subsequently washed in 2X, 1X, 0.5X, and 0.25X SSC
for 5 min each, and then O.1X SSC for 60 min at 60°C.
Autoradiograms were captured using a Northern Light (Imaging Research) illuminator and a Sony XC-77 CCD camera with a Nikor SSmm lens. Background grayscale density values were determined for each region, and the average background value plus 3.5 times the standard deviation of the background pixel values was used to set a threshold value for signal detection. Integrated optical density (IOD) values were determined for regions of interest by multiplying the number of pixels above threshold by their grayscale levels. Statistical significance was determined by ANOVA
followed by the Student-Newman-I~euls post-hoc test. P<0.05 was required for statistical significance.
Acute treatment with morphine, cocaine, and amphetamine increased RGS 4 mRNA levels in the rostral nucleus accumbens by 27, 22 and 25% respectively (Table 1). All three treatments also increased mRNA levels in the dorsal central gray (morphine 59%, cocaine 41% and amphetamine 58%). In contrast, in the caudal reticulotegmental pontine nucleus and locus coeruleus, RGS 4 mRNA levels were decreased in response to morphine and cocaine treatment, but not amphetamine (see Table 6). Due to a problem in the data analysis system, some changes initially reported in the abstract (decreases in RGS 4 in hippocampus in response to morphine, cocaine, and amphetamine, increase in medial mammilary nucleus in response to morphine, increases in dorsal raphe in response to morphine and cocaine) were not substantiated upon subsequent re-analysis.
Previously reported percent changes in nucleus accumbens, dorsal central gray, reticulotegmental nucleus and locus coeruleus were recalculated. The data below represent these corrected values.

Treatment Nucleus Dorsal ReticulotegmentalLocus Accumbens Central GreyNucleus Coeruleus ~~

Morphine " 27% ~ 59% ~ 31 % 22 Cocaine ~ 22% ' 41% ~ 50% ~ 26 Amphetamine ~ 25% ' S8% NS NS

Y<U.US vs. saline control; N~-Not W gniticant These findings demonstrate that acute administration of opioids and psychostimulants alter RGS 4 mRNA levels in relevant brain regions. The nucleus accumbens mediates rewarding and reinforcing properties of drugs of abuse, while the dorsal. central gray plays a role in defensive responses and analgesia. The increases in RGS 4 mRNA seen in these areas in response to all drugs could be a mechanism for acute desensitization of these responses. The locus coeruleus is involved in analgesia, level of arousal and is a mediator of opioid dependence, while the reticulotegmental nucleus is involved in motor activation. Decreases in RGS 4 levels in these areas could underlie locomotor stimulatory effects of opioids and cocaine. The reasons why amphetamine does not decrease mRNA levels in these areas are not known, but suggest that drugs of abuse may utilize different signaling mechanisms to mediate their effects.
The differential regulation of RGS 4 mRNA levels in different brain regions is intriguing, and could provide an explanation for the observation that while desensitization develops to some effects of drugs of abuse, sensitization to other effects may occur in parallel. In sum, these findings suggest that RGS 4 could be involve din regulating clinically relevant signaling responses to both opioids and psychostimulants.

Prophetic Example 7- Irz Vitro Effects of Chronic Opioid Treatment and Withdrawal on ERKActivation Initially, in vitro studies will further delineate the time course of ERK
activation after opioid withdrawal. N2A cells will be treated with increasing fentanyl doses for 7 days, starting with 20 nM and doubling daily (final dose, 1 ~,M). Cells will then be rinsed, and 1 ~M naloxone applied. Cells will be lysed and processed for phospho-ERK
immunoblotting as previously described (Gutstein et a1.1997) prior to withdrawal and at 5,15,30, 60, 120, 240, minutes and 24 hours after naloxone. To determine role of NMDA receptors in tolerance and withdrawal-induced ERK activation, a parallel study will be performed with MK-SOl added to the media daily.
Prophetic Example 8 - In Vitro Effects of Claronic Opioid Treatment and Withdrawal on EXKActivation For the in vivo studies, tolerance will be induced in rats by injecting 10 rizg/kg morphine (saline control) i.p. for 10 days as described (Trujillo et al.
1991). 30 min.
prior to morphine or saline, animals will be pretreated with 100 mg/kg i.p of the systemically bioavailable and highly selective ERK cascade inhibitor SL-327, vehicle, or saline control. On day 10, withdrawal will be precipitated 1 hr. after morphine injection with 2 mg/kg naloxone i.p. ERK activation will be determined by ICC in CNS
areas associated with tolerance and dependence (VTA, NAcc, Arc, Amy, PAG, NTS, LC, RVM, and lumbar spinal cord dorsal horn) prior to withdrawal and after naloxone at the time of maximal ERK modulation defined in pilot studies. As above, a parallel study to determine the effects of NMDA receptor inhibition on ERK activation during tolerance and withdrawal will be performed, using 0.1 mg/kg MK-801 and saline as pretreatments.
Prior studies strongly imply that NMDA antagonism should decrease ERK
activation. If it does not, that would suggest opioids and noxious stimuli activate ERK by different mechanisms. It is also possible that ICC may not be sensitive enough to detect low, yet physiologically relevant, levels of ERK activation. If increased sensitivity is needed, the region of interest will be microdissected and immunoblotted for phospho-ERK.

Proplzetic Example 9 - Effects of ERK Inhibition and Gene Disruption on Tolerance and Dependence In hivo.
The same experimental procedures as in Examples 9 and 10 can be used to study the in vivo effects of ERK inhibition and gene disruption on tolerance and dependence, with tolerance evaluated daily by tail flick latency (TFL). On day I0, withdrawal will be precipitated as above, and withdrawal signs (wet dog shakes, escape jumps, burrowing, lacrimation, ptosis, weight loss) recorded. Appropriate controls will be utilized to determine whether SL-327 unmasks "hidden" analgesia in tolerant animals, and therefore whether ERIC is required for the expression as well as the development of opioid tolerance (Trujillo et al. 1991). It will also be important to determine whether established opioid tolerance and dependence can be reversed by ERK.
inhibition. In this study, tolerance will be induced as above, then morphine treatments will be preceded by SL-327 or vehicle control pretreatment for an additional 7 days. If reversal of tolerance is obsezved, withdrawal will be precipitated and signs recorded.
Prophetic Example 10 - Tolerance and Dependence in ERK-1 knockout mice Evaluating the development of tolerance and dependence in ERK-I knockout mice and controls will further support the current invention. It is possible that ERK 2 could compensate for the loss of ERK l, leaving tolerance and dependence unaffected.
However, creation of an ERIC 1 and 2 null mouse is underway. If this proves lethal, behavior will be evaluated in ERKl null/ERK 2 heterozygotes. Consideration is also being given to creating a neural specific ERK l and 2 knockout using the cre/lox system and the synapsin promoter.
Prophetic Example 11 - Changes in RGS Expression with Opioid Tolerance and Withdrawal This example can be used to determine the regulation of candidate RGS proteins during tolerance development and withdrawal, and whether ERIC activation mediates these changes. The SL327 tolerance paradigm used previously will also be employed here. mRNA levels in relevant CNS areas will be determined by in situ hybridization (ISH) as previously described after 10 days of treatment, and at the time of maximal alteration in mRNA levels (as determined by pilot studies) observed after opioid withdrawal.. RGS proteins from 4 relevant families (Ross et a1.2000) have been chosen for initial analysis. RGS 2 and RGS 4 will be evaluated from the "classical"
subfamily.
RGS 2 has been shown to interact with opioids in vitro (Potenza 1999) , and the availability of a RGS 2 knockout will facilitate behavioral evaluation. The same caveats mentioned above may apply, as the receptor specificity of RGS proteins is not clearly defined, and others may be able to compensate for the loss of RGS 2. However, the intriguing behavioral phenotype of the RGS 2 knockout suggests that meaningful results may be obtained. From the GGL subfamily RGS 9 and 11, will be examined due to their limited but relevant anatomical distribution (striatum/Nacc and LC, respectively). RGS
12 has been chosen from the "scaffolding" subfamily, which appears selective for Go.
Recent evidence suggests that the GaZ subunit could mediate opioid tolerance (I~elleher et a1.1999, so RGS Z will also be evaluated. Interesting findings will direct the evaluation of additional RGS proteins. ISH will be used initially, as robust mRNA
regulation has been observed in several studies, and currently available RGS
antibodies do not work well in ICC. Extremely little is known about the physiological regulation of protein levels or modifications of RGS proteins. However, as reagents are developed, changes in protein levels and post-translational modifications will be evaluated. It is possible that ERK may not regulate some RGS mRNAs, or may only regulate responses in discrete areas. There is evidence suggesting other cascades homologous to ERIC could be involved, and relevant signaling modulators are available if needed.
Prophetic Example 12 - The effects of ERK inlzibitiorz and gene disruption oh lzyperalgesia This experiment will determine whether spinal ERK activation mediates chronic inflammatory hyperalgesia and determine whether opioids can block this response. The first study will determine whether ERIC. inhibition can block the development of hyperalgesia. Animals will receive intrathecal (i.t.) catheters and allowed to recover.
The animals will then undergo implantation of primed osmotic minipumps infusing either SL-327lvehicle, vehicle alone, or saline, and have catheters flushed with the same solution. Four hours later, 0.2 ml complete Freund's adjuvant (CFA) will be injected in the right hindpaw. Hyperalgesia will be assessed by paw withdrawal pressure (PWP).
The second study will determine the time course of ERK activation after CFA
injection.

Under brief halothane anesthesia, animals will be injected with either CFA or saline in the right hindpaw and lumbar spinal cords processed for ICC 2, S, 15, 30, 60, 120, and 240 minutes and 1 and 7 days after injection. The third study will determine whether ERK inhibition can reverse previously established hyperalgesia, and whether there is a "critical window" for this effect. Animals will be implanted with i.t.
catheters and allowed to recover. Animals will subsequently be injected with CFA, and primed minipumps infusing either SL-327 or vehicle will be implanted 2,4, 24, and 48 hours, and 7 days after CFA injection. Hyperalgesia will be monitored daily using PWP. It is possible that earlier or later time points may need to be used. The final study will determine whether pain-induced ERK activation can be inhibited by opioids.
Opioids can cause analgesia by activating descending supraspinal circuitry and by direct effects in the dorsal horn. This suggests that systemic and spinal opioids could modulate spinal ERK differently, so both routes need to be evaluated. For systemic administration, animals will undergo implantation of one morphine (or placebo) sustained release pellet.
For spinal administration, 5 days after catheter implantation, .a primed osmotic minipump infusing morphine (or placebo) will be implanted. Four hours after morphine or placebo is begun, CFA will be injected in the right hindpaw (or sham injection), and lumbar spinal cords processed for ICC at time points determined by the results of the second study. If ERK activation after CFA is sustained, it will be intriguing to see whether the effect of morphine an ERK activation changes as tolerance develops. While inhibition of the second phase of formalin responses suggests that ERK may be involved in the genesis of chronic pain, it is possible that ERIC may only be involved in acute nociceptive signaling. The current design will allow the inventors to block these acute changes, and if they trigger a series of adaptations leading to chronic pain, a long-lasting decrease in hyperalgesia will be observed. If the role of ERK is strictly limited to acute pain signaling, all of the above studies can be performed acutely, using formalin injection as previously described (Ji et al. 1999) and acute morphine administration.
**********
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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SEQUENCE LISTING
<110> GUTSTEIN, HOWARD B.
<120> INHIBITION OF ERIC REDUCES OR PREVENTS BOTH TOLERANCE TO
AND DEPENDENCE ON OPIOID ANALGESICS AND SENSITIZATION
AFTER PAINFUL STIMULATION
<130> UTFC:696W0 <140> UNKNOWN
<141> 2002-01-24 <150> 60/264,336 <151> 2001-O1-25 <l60> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 3732 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (73)..(1212) <400> 1 cgttcctcgg cgccgccggg gccccagagg gcagcggcag caacagcagc agcagcagca 60 gcgggagtgg ag atg gcg gcg gcg gcg get cag ggg ggc ggg ggc ggg gag 111 Met Ala Ala Ala Ala Ala Gln Gly Gly Gly Gly Gly Glu ccc cgt aga acc gag ggg gtc ggc ccg ggg gtc ccg ggg gag gtg gag 159 Pro Arg Arg Thr Glu Gly Val Gly Pro Gly Val Pro Gly Glu Val Glu atg gtg aag ggg cag ccg ttc gac gtg ggc ccg cgc tac acg cag ttg 207 Met Val Lys G1y Gln Pro Phe Asp Val Gly Pro Arg Tyr Thr Gln Leu cag tac atc ggc gag ggc gcg tac ggc atg gtc agc tcg gcc tat gac 255 Gln Tyr Ile Gly Glu Gly Ala Tyr Gly Met Val Ser Ser Ala Tyr Asp cac gtg cgc aag act cgc gtg gcc atc aag aag atc agc ccc ttc gaa 303 His Val Arg Lys Thr Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu cat cag acc tac tgc cag cgc acg ctc cgg gag atc cag atc ctg ctg 351 His Gln Thr Tyr Cys Gln Arg Thr Leu Arg Glu Ile Gln Ile Leu Leu cgc ttc cgc cat gag aat gtc atc ggc atc cga gac att ctg cgg gcg 399 Arg Phe Arg His Glu Asn Val Ile Gly Ile Arg Asp Ile Leu Arg Ala tcc acc ctg gaa gcc atg aga gat gtc tac att gtg cag gac ctg atg 447 Ser Thr Leu Glu Ala Met Arg Asp Val Tyr Ile Val Gln Asp Leu Met gag act gac ctg tac aag ttg ctg aaa agc cag cag ctg agc aat gac 495 Glu Thr Asp Leu Tyr Lys Leu Leu Lys Ser Gln Gln Leu Ser Asn Asp catatc tgctac ttcctctaccag atcctg cggggcctcaag tacatc 543 HisIle CysTyr PheLeuTyrGln IleLeu ArgGlyLeuLys TyrIle cactcc gccaac gtgctccaccga gatcta aagccctccaac ctgctc 591 HisSer AlaAsn ValLeuHisArg AspLeu LysProSerAsn LeuLeu agcaac accacc tgcgaccttaag atttgt gatttcggcctg gcccgg 639 SerAsn ThrThr CysAspLeuLys IleCys AspPheGlyLeu AlaArg attgcc gatcct gagcatgaccac accggc ttcctgacggag tatgtg 687 IleAla AspPro GluHisAspHis ThrGly PheLeuThrGlu TyrVal getacg cgctgg taccgggcccca gagatc atgctgaactcc aagggc 735 AlaThr ArgTrp TyrArgAlaPro GluIle MetLeuAsnSer LysGly tatacc aagtcc atcgacatctgg tctgtg ggctgcattctg getgag 783 TyrThr LysSer TleAspIleTrp SerVal GlyCysIleLeu AlaGlu atgctc tctaac cggcccatcttc cctggc aagcactacctg gatcag 837.

MetLeu SerAsn ArgProIlePhe ProGly LysHisTyrLeu AspGln ctcaac cacatt ctgggcatcctg ggctcc ccatcccaggag gacctg 879 LeuAsn HisIle LeuGlyIleLeu GlySer ProSerGlnGlu AspLeu aattgt atcatc aacatgaaggcc cgaaac tacctacagtct ctgccc 927 AsnCys IleIle AsnMetLysAla ArgAsn TyrLeuGlnSer LeuPro tccaag accaag gtggettgggcc aagctt ttccccaagtca gactcc 975 SerLys ThrLys ValAlaTrpAla LysLeu PheProLysSer AspSer aaagcc cttgac ctgctggaccgg atgtta acctttaacccc aataaa 1023 LysAla LeuAsp LeuLeuAspArg MetLeu ThrPheAsnPro AsnLys cggatc acagtg gaggaagcgctg getcac ccctacctggag cagtac 1071 ArgIle ThrVal GluGluAlaLeu AlaHis ProTyrLeuGlu GlnTyr tatgac ccgacg gatgagccagtg gccgag gagcccttcacc ttcgcc 1119 TyrAsp ProThr AspGluProVal AlaGlu GluProPheThr PheAla 335 340 , 345 atg gag ctg gat gac cta cct aag gag cgg ctg aag gag ctc atc ttc 1167 Met Glu Leu Asp Asp Leu Pro Lys Glu Arg Leu Lys Glu Leu Ile Phe cag gag aca gca cgc ttc cag ccc gga gtg ctg gag gcc ccc tag 1212 Gln Glu Thr Ala Arg Phe Gln Pro Gly Val Leu G1u Ala Pro cccagacaga catctctgca ccctggggcc tggacctgcc tcctgcctgc ccctctcccg 1272 ccagactgtt agaaaatgga cactgtgccc agcccggacc ttggcagccc aggccggggt 1332 ggagcatggg cctggccacc tctctccttt gctgaggcct ccagcttcag gcaggccaag 1392 gccttctcct ccccacccgc cctccccacg gggcctcggg agctcaggtg gccccagttc 1452 aatctcccgc tgctgctgct gctgcgccct taccttcccc agcgtcccag tctctggcag 1512 ttctggaatg gaagggttct ggctgcccca acctgctgaa gggcagaggt ggagggtggg 1572 gggcgctgag tagggactca gggccatgcc tgcccccctc atctcattca aaccccaccc 1632 tagtttccct gaaggaacat tccttagtct caagggctag catccctgag gagccaggcc 1692 gggccgaatc ccctccctgt caaagctgtc acttcgcgtg ccctcgctgc ttctgtgtgt 1752 ggtgagcaga agtggagctg gggggcgtgg agagcccggc gcccctgcca cctccctgac 1812 ccgtctaata tataaatata gagatgtgtc tatggctgaa aaaaaaaaaa aaaacgttcc 1872 tcggcgccgc cggggcccca gagggcagcg gcagcaacag cagcagcagc agcagcggga 1932 gtggagatgg cggcggcggc ggctcagggg ggcgggggcg gggagccccg tagaaccgag 1992 ggggtcggcc cgggggtccc gggggaggtg gagatggtga aggggcagcc gttcgacgtg 2052 ggcccgcgct acacgcagtt gcagtacatc ggcgagggcg cgtacggcat ggtcagctcg 2112 gcctatgacc acgtgcgcaa gactcgcgtg gccatcaaga agatcagccc cttcgaacat 2172 cagacctact gccagcgcac gctccgggag atccagatcc tgctgcgctt ccgccatgag 2232 aatgtcatcg gcatccgaga cattctgcgg gcgtccaccc tggaagccat gagagatgtc 2292 tacattgtgc aggacctgat,ggagactgac ctgtacaagt tgctgaaaag ccagcagctg 2352 agcaatgacc atatctgcta cttcctctac cagatcctgc ggggcctcaa gtacatccac 2412 tccgccaacg tgctccaccg agatctaaag ccctccaacc tgctcagcaa caccacctgc 2472 gaccttaaga tttgtgattt cggcctggcc cggattgccg atcctgagca tgaccacacc 2532 ggcttcctga cggagtatgt ggctacgcgc tggtaccggg ccccagagat catgctgaac 2592 tccaagggct ataccaagtc catcgacatc tggtctgtgg gctgcattct ggctgagatg 2652 ctctctaacc ggcccatctt ccctggcaag cactacctgg atcagctcaa ccacattctg 2712 ggcatcctgg gctccccatc ccaggaggac ctgaattgta tcatcaacat gaaggcccga 2772 aactacctac agtctctgcc ctccaagacc aaggtggctt gggccaagct tttccccaag 2832 tcagactcca aagcccttga cctgctggac cggatgttaa cctttaaccc caataaacgg 2892 atcacagtgg aggaagcgct ggctcacccc tacctggagc agtactatga cccgacggat 2952 gagccagtgg ccgaggagcc cttcaccttc gccatggagc tggatgacct acctaaggag 3012 cggctgaagg agctcatctt ccaggagaca gcacgcttcc agcccggagt gctggaggcc 3072 ccctagccca gacagacatc tctgcaccct ggggcctgga cctgcctcct gcctgcccct 3132 ctcccgccag actgttagaa aatggacact gtgcccagcc cggaccttgg cagcccaggc 3192 cggggtggag.catgggcctg gccacctctc tcctttgctg aggcctccag cttcaggcag 3252 gccaaggcct tctcctcccc acccgccctc cccacggggc ctcgggagct caggtggccc 3312 cagttcaatc tcccgctgct gctgctgctg cgcccttacc ttccccagcg tcccagtctc 3372 tggcagttct ggaatggaag ggttctggct gccccaacct gctgaagggc agaggtggag 3432 ggtggggggc gctgagtagg gactcagggc catgcctgcc cccctcatct cattcaaacc 3492 ccaccctagt ttccctgaag gaacattcct tagtctcaag ggctagcatc cctgaggagc 3552 caggccgggc cgaatcccct ccctgtcaaa gctgtcactt cgcgtgccct cgctgcttct 3612 gtgtgtggtg agcagaagtg gagctggggg gcgtggagag cccggcgccc ctgccacctc 3672 cctgacccgt ctaatatata aatatagaga tgtgtctatg gctgaaaaaa aaaaaaaaaa 3732 <210> 2 <211> 379 <2l2> PRT
<213> Homo sapiens <400> 2 Met Ala Ala Ala Ala Ala Gln Gly Gly Gly Gly Gly Glu Pro Arg Arg Thr Glu Gly Val Gly Pro Gly Val Pro Gly Glu Val Glu Met Val Lys Gly Gln Pro Phe Asp Val Gly Pro Arg Tyr Thr Gln Leu Gln Tyr Ile G1y Glu Gly Ala Tyr Gly Met Val Ser Ser Ala Tyr Asp His Val Arg Lys Thr Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr Tyr Cys Gln Arg Thr Leu Arg Glu Ile Gln Ile Leu Leu Arg Phe Arg His Glu Asn Val Ile Gly Ile Arg Asp Ile Leu Arg Ala Ser Thr Leu Glu Ala Met Arg Asp Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp Leu Tyr Lys Leu Leu Lys Ser Gln Gln Leu Ser Asn Asp His Ile Cys Tyr Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Ser Asn Thr Thr Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Ile Ala Asp Pro Glu His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys Ser Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser Asn Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His Ile Leu Gly Ile Leu Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile Ile Asn Met Lys Ala Arg Asn Tyr Leu Gln Ser Leu Pro Ser Lys Thr Lys Val Ala Trp Ala Lys Leu Phe Pro Lys Ser Asp Ser Lys Ala Leu Asp Leu Leu Asp Arg Met Leu Thr Phe Asn Pro Asn Lys Arg Ile Thr Val Glu Glu Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro Thr Asp Glu Pro Val Ala Glu Glu Pro Phe Thr Phe Ala Met Glu Leu Asp Asp Leu Pro Lys Glu Arg Leu Lys Glu Leu Ile Phe Gln Glu Thr Ala Arg Phe Gln Pro Gly Val Leu Glu Ala Pro <210> 3 <211> 1611 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (328)..(1410) <400> 3 acataatttc tggagccctg taccaacgtg tggccacata ttctgtcagg aaccctgtgt 60 gatcatggtc tggatctgca acacgggcca.ggccaaagtc acagatcttg agatcacagg 120 tggtgttgag cagcaggcag gcaggcaatc ggtccgagtg gctgtcggct cttcagctct 180 ccgctcggcg tcttccttcc tctcccggtc agcgtcggcg gctgcaccgg cggcgggcag 240 tcctgcggga ggggcgacaa gagctgaggc gcggccgccg agcgtcgagc tcagcgcggc 300 ggaggcggcg gcggcccggc agccaac atg gcg gcg gcg gcg gcg gcg ggc gcg 354 Met Ala Ala Ala Ala Ala Ala Gly Ala ggc ccg gag atg gtc cgc ggg cag gtg ttc gac gtg ggg ccg cgc tac 402 Gly Pro G1u Met Val Arg Gly Gln Val Phe Asp Val Gly Pro Arg Tyr acc aac ctc tcg tac atc ggc gag ggc gcc tac ggc atg gtg tgc tct 450 Thr Asn Leu Ser Tyr Ile Gly Glu Gly Ala Tyr Gly Met Val Cys Ser get tat gat aat gtc aac aaa gtt cga gta get atc aag aaa atc agc 498 Ala Tyr Asp Asn Val Asn Lys Val Arg Val Ala Ile Lys Lys Ile Ser ccc ttt gag cac cag acc tac tgc cag aga acc ctg agg gag ata aaa 546 Pro Phe G1u His Gln Thr Tyr Cys Gln Arg Thr Leu Arg Glu Ile Lys atcttactgcgcttc agacat gagaacatc attggaatc aatgacatt 594 IleLeuLeuArgPhe ArgHis GluAsnIle IleGlyIle AsnAspIle attcgagcaccaacc atcgag caaatgaaa gatgtatat atagtacag 642 IleArgAlaProThr IleGlu GlnMetLys AspValTyr IleValGln gacctcatggaaaca gatctt tacaagctc ttgaagaca caacacctc 690 AspLeuMetGluThr AspLeu TyrLysLeu LeuLysThr GlnHisLeu agcaatgaccatatc tgctat tttctctac cagatcctc agagggtta 738 SerAsnAspHisIle CysTyr PheLeuTyr GlnIleLeu ArgGlyLeu l25 130 l35 aaatat atccattca getaacgtt ctgcac cgtgacctc aagccttcc 786 LysTyr IleHisSer AlaAsnVal LeuHis ArgAspLeu LysProSer aacctg ctgctcaac accacctgt gatctc aagatctgt gactttggc 834 AsnLeu LeuLeuAsn ThrThrCys AspLeu LysIleCys AspPheGly ctggcc cgtgttgca gatccagac catgat cacacaggg ttcctgaca 882 LeuAla ArgValAla AspProAsp HisAsp HisThrGly PheLeuThr 170 175 l80 185 gaatat gtggccaca cgttggtac aggget ccagaaatt atgttgaat 930 GluTyr ValAlaThr ArgTrpTyr ArgAla ProGluIle MetLeuAsn tccaag ggctacacc aagtccatt gatatt tggtctgta ggctgcatt 978 SerLys GlyTyrThr LysSerIle AspIle TrpSerVal GlyCysIle ctggca gaaatgctt tccaacagg cccatc tttccaggg aagcattat 1026 LeuAla GluMetLeu SerAsnArg ProIle PheProGly LysHisTyr cttgac cagctgaat cacattttg ggtatt cttggatcc ccatcacaa 1074 LeuAsp GlnLeuAsn HisIleLeu GlyIle LeuGly~Ser ProSerGln gaa gac ctg aat tgt ata ata aat tta aaa get agg aac tat ttg ctt 1122 G1u Asp Leu Asn Cys Ile Ile Asn Leu Lys Ala Arg Asn Tyr Leu Leu tct ctt cca cac aaa aat aag gtg cca tgg aac agg ctg ttc cca aat 1170 Ser Leu Pro His Lys Asn Lys Val Pro Trp Asn Arg Leu Phe Pro Asn get gac tcc aaa get ctg gac tta ttg gac aaa atg ttg aca ttc aac 1218 Ala Asp Ser Lys Ala Leu Asp Leu Leu Asp Lys Met Leu Thr Phe Asn cca cac aag agg att gaa gta gaa cag get ctg gcc cac cca tat ctg 1266 Pro His Lys Arg Ile Glu Val Glu Gln Ala Leu Ala His Pro Tyr Leu gag cag tat tac gac ccg agt gac gag ccc atc gcc gaa gca cca ttc 1314 Glu Gln Tyr Tyr Asp Pro Ser Asp Glu Pro Ile Ala Glu Ala Pro Phe aag ttc gac atg gaa ttg gat gac ttg cct aag gaa aag cta aaa gaa 1362 Lys Phe Asp Met Glu Leu Asp Asp Leu Pro Lys Glu Lys Leu Lys Glu cta att ttt gaa gag act get aga ttc cag cca gga tac aga tct taa 1410 Leu Ile Phe Glu Glu Thr Ala Arg Phe Gln Pro Gly Tyr Arg Ser atttgtcagg acaagggctc agaggactgg acgtgctcag acatcggtgt tcttcttccc 1470 agttcttgac ccctggtcct gtctccagcc cgtcttggct tatccacttt gactcctttg 1530 agccgtttgg aggggcggtt tctggtagtt gtggctttta tgctttcaaa gaatttcttc 1590 agtccagaga attcactggc c 1611 <210> 4 <211> 360 <212> PRT
<213> Homo sapiens <400> 4 Met Ala Ala Ala Ala Ala Ala Gly Ala Gly Pro Glu Met Val Arg Gly Gln Val Phe Asp Val Gly Pro Arg Tyr Thr Asn Leu Ser Tyr Ile Gly Glu Gly Ala Tyr Gly Met Val Cys Ser Ala Tyr Asp Asn Val Asn Lys Va1 Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr Tyr Cys Gln Arg Thr Leu Arg Glu Ile Lys Ile Leu Leu Arg Phe Arg His Glu Asn Ile Ile Gly Ile Asn Asp Ile Ile Arg Ala Pro Thr Ile Glu Gln Met Lys Asp Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp Leu Tyr Lys Leu Leu Lys Thr Gln His Leu Ser Asn Asp His Ile Cys Tyr Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn 130 135 l40 Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Thr Thr 145 150 l55 160 Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ala Asp Pro Asp His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys Ser I1e Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser Asn Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His Ile Leu Gly Ile Leu Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile Ile Asn Leu Lys Ala Arg Asn Tyr Leu Leu Ser Leu Pro His Lys Asn Lys Val Pro Trp Asn Arg Leu Phe Pro Asn Ala Asp Ser Lys Ala Leu Asp Leu Leu Asp Lys Met Leu Thr Phe Asn Pro His Lys Arg Ile Glu Val 290 295 ~ 300 Glu Gln Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro Ser Asp Glu Pro Ile Ala Glu Ala Pro Phe Lys Phe Asp Met Glu Leu Asp Asp Leu Pro Lys Glu Lys Leu Lys Glu Leu Ile Phe Glu Glu Thr Ala Arg Phe Gln Pro G1y Tyr Arg Ser <210> 5 <211> 8307 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (706) . . (7710) <400> 5 gaattccctc cattatcatc tgctttcttg ggactgcgct gatttccggg attaacggca 60 gcacatcatt gacacagctg atgggatcag ggatcatatg ggcagttotc gccgcactgt 120 tttatgcgtt caccacttta ttgggaaaag gcattcacaa tctcagtcct tacacgacca 180 ccttcttgca gaoaggttta ggggttat~ca tcctgattcc gtttattcac tttggcgctt 240 ttgcggacct atctcaggga aactggatta tggtggtatc taccggcatc atccatacgg 300 gtattgtata tttgctgttt tttgacagtt tacgattttt atcaacgaaa tttatttcaa 360 tcattgtatt totcgacccc gctgtcgcga tcgtgctcga taccgtcttc acaggcttcc 420 gccctgacct ctatcaaacg cttggcatcg taatgatctt tgcgggcatg gccttgacgc 480 ttgtcaggag gcaggggaag gcgaatgtga cagctgaggg tacggatatt gaacaaatac 540 aataaaaaat gtaaaaagcc tatgcggcct ttttttgttt taggtcaatt gactctcgct 600 aatccttaaa ataagataaa ttttctagaa aaatattgta atgatatttc agtctagtta 660 agattattga gtaaatatta cttttattac aaaaggagag aggaa atg aaa aaa aga 717 Met Lys Lys Arg aag agg cga aac ttt aaa agg ttc att gca gca ttt tta gtg ttg get 765 Lys Arg Arg Asn Phe Lys Arg Phe Ile Ala Ala Phe Leu Val Leu Ala tta atg att tca tta gtg cca gcc gat gta cta gca aaa tct aca gaa 8l3 Leu Met Ile Ser Leu Val Pro Ala Asp Val Leu A1a Lys Ser Thr Glu gaa gag aat gga aac cgt att get gcc gat gat cca gaa gaa act ctt 86l Glu Glu Asn Gly Asn Arg Ile Ala Ala Asp Asp Pro Glu Glu Thr Leu cagaaagaacaa actgaggaa getgtacct tttgatcca aaagatata 909 GlnLysGluGln ThrGluGlu AlaValPro PheAspPro LysAspIle aacaaagagggt gagatcacc tctgaaaga acagaaaat acaaaactc 957 AsnLysGluGly GluIleThr SerGluArg ThrGluAsn ThrLysLeu tattatgaaggt gacggtgtc tataaacag gaagtatac cttgatcct 1005 TyrTyrGluGly AspGlyVal TyrLysGln GluValTyr LeuAspPro attcatacaaag gaaacaccg gatgccgac tgggaagac atttctcct 1053 I1eHisThrLys GluThrPro AspAlaAsp TrpGluAsp IleSerPro gagttaaaagag tcgacaagc aaacaagtc gagacagag aacgetatt 1101 GluLeuLysGlu SerThrSer LysGlnVal GluThrGlu AsnAlaIle ttgaattcagat tttcaaaag caaatgaag aacgggctt tatgcgact 1149 LeuAsnSerAsp PheGlnLys GlnMetLys AsnGlyLeu TyrAlaThr tttgagcataat gaccataag gttacatat tcactagca gaagcgaaa 1197 PheGluHisAsn AspHisLys ValThrTyr SerLeuAla GluAlaLys ggcccgaataaa acttcactt actccgaaa gatacctca gcagattat 1245 GlyProAsnLys ThrSerLeu ThrProLys AspThrSer AlaAspTyr aagactgacagt aatgagatt gtgtatccg gatgtgttt ccgaatatt 1293 LysThrAspSer AsnGluIle ValTyrPro AspValPhe ProAsnIle l85 190 195 gat ctt cag act ttc aca ttc aat gaa aac ata aaa gaa gat tta gtc 1341 Asp Leu Gln Thr Phe Thr Phe Asn Glu Asn Ile Lys Glu Asp Leu Val ctt cat cag tat aat gga tac aac aca ttt aca ttt cag tta aaa acc 1389 Leu His Gln Tyr Asn Gly Tyr Asn Thr Phe Thr Phe Gln Leu Lys Thr gat ttg cag get aaa gaa caa gag gat gga tcc att gat ttt tct gat 1437 Asp Leu Gln Ala Lys G1u Gln Glu Asp Gly Ser Ile Asp Phe Ser Asp gaa aaa gga aaa gtt gtt ttt tct gtg cct aag cct ttt atg acg gat 1485 G1u Lys Gly Lys Val Val Phe Ser Val Pro Lys Pro Phe Met Thr Asp tca aaa cta gat gag ctt tcc ggc gag gtt gaa cgg tca gac aaa gtc 1533 Ser Lys Leu Asp Glu Leu Ser Gly Glu Val Glu Arg Ser Asp Lys Val agc tat aag ctg gaa aag aat gaa gaa ggg tat ttg ctt cat ctt acg 1581 Ser Tyr Lys Leu Glu Lys Asn Glu Glu Gly Tyr Leu Leu His Leu Thr gca gac gag aac tgg ctg aaa gac cca gaa cgc gta tat cca gta tct 1629 Ala Asp Glu Asn Trp Leu Lys Asp Pro Glu Arg Val Tyr Pro Val Ser atc gac ccg tca act tct tta tct gtt tca tcg gat acg ttt gtc atg 1677 Ile Asp Pro Ser Thr Ser Leu Ser Val Ser Ser Asp Thr Phe Val Met agt gcc tac ccg acg acg aac tat tcg gcc tcc tca caa aaa tgg gat 1725 Ser Ala Tyr Pro Thr Thr Asn Tyr Ser Ala Ser Ser Gln Lys Trp Asp get aat tta aag gcc tat gta ctg aaa acg ggc tat tat gac aaa aca 1773 Ala Asn Leu Lys Ala Tyr Val Leu Lys Thr Gly Tyr Tyr Asp Lys Thr aca ggc aca aac tat gcg ttc atg aaa ttt aac aac cta aag cca att 1821 Thr Gly Thr Asn Tyr Ala Phe Met Lys Phe Asn Asn Leu Lys Pro Ile caa aat atg act gtc act aaa get aca tta aaa aca tac gta gcc cat 1869 Gln Asn Met Thr Val Thr Lys Ala Thr Leu Lys Thr Tyr Val Ala His tcc tat tat gga aca aag gca acc ggc ctt tgg ctg gat acg gtc aac 1917 Ser Tyr Tyr Gly Thr Lys Ala Thr Gly Leu Trp Leu Asp Thr Val Asn agc aat tat gat aat gca aaa gtg aca tgg aac acc aaa ccg gca tct 1965 Ser Asn Tyr Asp Asn Ala Lys Val Thr Trp Asn Thr Lys Pro Ala Ser aaa aat atc gga aaa gcc gac gtc cat aaa gga cag tgg gcg agt tat 2013 Lys Asn Ile Gly Lys A1a Asp Val His Lys Gly Gln Trp Ala Ser Tyr gat gtg acc get get gta aag tca tgg aac tcc ggc ggg gcc aac tat 2061 Asp Val Thr Ala Ala Val Lys Ser Trp Asn Ser Gly Gly Ala Asn Tyr gga ttt aag ctt cat aca aac gga aac ggc aag gaa tat tgg aaa aaa 2109 Gly Phe Lys Leu His Thr Asn Gly Asn Gly Lys Glu Tyr Trp Lys Lys cta att tct tca gcg aac tcc gcc aat aaa cca tat att gaa gtg acg 2157 Leu Ile Ser Ser Ala Asn Ser Ala Asn Lys Pro Tyr Ile Glu Val Thr tataccattcca aagggaaac acacctaca atcaaa gcctatcac aac 2205 TyrThrIlePro LysGlyAsn ThrProThr IleLys AlaTyrHis Asn ggcgattccact ggttatttc gatatcagc tggaaa aaggttgaa ggc 2353 GlyAspSerThr GlyTyrPhe AspIleSer TrpLys LysValGlu Gly gcaaaaggatat aaggtatgg atttacaat ggtaag gaatatcaa gcg 2301 AlaLysGlyTyr LysValTrp TleTyrAsn GlyLys GluTyrGln A1a atttctgcaggc aacgtcacg tcgtggagc acaaaa ggtaaaaaa atc 2349 IleSerAlaGly AsnValThr SerTrpSer ThrLys GlyLysLys Ile tggcctacgagc gcagaaatt gcctccaag agatat aaactgcat ctg 2397 TrpProThrSer AlaGluTle AlaSerLys ArgTyr LysLeuHis Leu gacggcaaggat ggagetgaa ttggcatta gatcca tccccggtc tat 2445 AspGlyLysAsp GlyAlaGlu LeuAlaLeu AspPro SerProVal Tyr aagaattccggc ggaagctat getacaagc aaaaac tattggatt ggc 2493 LysAsnSerGly GlySerTyr AlaThrSer LysAsn TyrTrpI1e Gly gtcagtgcgata tttgaccaa ggtgaagga gcgatg tccgcaccg gca 2541 ValSerAlaIle PheAspGln GlyGluGly AlaMet SerAlaPro Ala aagcccgttatc cctaatgta ggaaaagca caagca ccgtcagca aag 2589 LysProValIle ProAsnVal GlyLysAla GlnAla ProSerAla Lys 615 . 620~ 625 ggctacaataat gggaatgcc acaggatac ttcgac ctttcttgg aaa 2637 GlyTyrAsnAsn GlyAsnAla ThrGlyTyr PheAsp LeuSerTrp Lys getgtatccggt gcaaccggc tataaggtt caggtg ttcaatggg aaa 2685 AlaValSerGly AlaThrGly TyrLysVal GlnVal PheAsnGly Lys ggctttgagaca cttgatctc ggaaatcag acgtct tggaccaca aaa 2733 GlyPheGluThr LeuAspLeu GlyAsnGln ThrSer TrpThrThr Lys gggaaa aagatc tggccgacaagt gcagaaatc aaggcggga aaatat 2781 GlyLys LysIle TrpProThrSer AlaGluIle LysAlaGly LysTyr getctt caccta aaggacggaagc ggagcagag ctgccgatc aatccc 2829 AlaLeu HisLeu LysAspGlySer GlyAlaGlu LeuProIle AsnPro ggacca acctat aaaaacgccggg ggagatgga gccaaaaga aattac 2877 GlyPro ThrTyr LysAsnAlaGly GlyAspGly AlaLysArg AsnTyr tcgttt aaaatt atcgcatacaac aaagacggc gaagccatt gcatcc 2925 SerPhe LysIle IleAlaTyrAsn LysAspGly GluAlaIle AlaSer ccgget getacc ccggcacttcct gatatcget aggccaaag aatgta 2973 ProAla AlaThr ProAlaLeuPro AspIleAla ArgProLys AsnVal actggc tatttg tatacaaataca aaatcgagt caaacgggt tatgtg 3021 ThrGly TyrLeu TyrThrAsnThr LysSerSer GlnThrGly TyrVal aatttg atatgg gaaaaggttcaa aatgcgaag ggctataaa gttaac 3069 AsnLeu IleTrp GluLysVa1Gln AsnAlaLys GlyTyrLys ValAsn atttac aacggt aaagaatatcag tcctttgat gttggtgat getgat 3117 IleTyr AsnGly LysGluTyrGln SerPheAsp ValGlyAsp AlaAsp cattgg acaacc caaaataaaaac atctggccg acttctgag gaaatc 3165 HisTrp ThrThr GlnAsnLysAsn IleTrpPro ThrSerGlu GluIle aaagcc ggaagc tataagcttcat acagatgga aaaggcggg gaatta 3213 LysAla GlySer TyrLysLeuHis ThrAspGly LysGlyGly GluLeu gccctc gatcca tcacctgtctat aacaatgca aatgggaat tacaaa 3261 AlaLeu AspPro SerProValTyr AsnAsnAla AsnGlyAsn TyrLys gggaag aagaat tattctttcacg cttgtcgcc tatgatgcg aatggt 3309 GlyLys LysAsn TyrSerPheThr LeuValAla TyrAspAla AsnGly gaaacg attcca acggcgcccttt aacccaacg ttccatgaa ggcgca 3357 GluThr IlePro ThrAlaProPhe AsnProThr PheHisGlu GlyAla gaattc ctaggt acagaagagtac tggtctatc attgatatc ccaagc 3405 GluPhe LeuGly ThrGluGluTyr TrpSerIle IleAspIle ProSer ggccag ttaaat ggtgcgacaggc aatgtcatt gtgaatgaa gaagat 3453 GlyGln LeuAsn GlyAlaThrGly AsnValIle ValAsnGlu GluAsp ttg tca att gat gga cgc ggt ccg gga ctt ggc tta tcg aga acc tac 3501 Leu Ser Ile Asp Gly Arg Gly Pro Gly Leu Gly Leu Ser Arg Thr Tyr aac agt ctt gac agc tct gat cac tta ttt gga caa ggc tgg tat get 3549 Asn Ser Leu Asp Ser Ser Asp His Leu Phe Gly Gln Gly Trp Tyr Ala gat gca gaa aca tct gtc atc tca acg gat ggc ggg gca atg tat atc 3597 Asp Ala Glu Thr Ser Val Ile Ser Thr Asp Gly Gly Ala Met Tyr Ile gat gaa gac get acg aca cat cga ttc acg aaa aaa gcc gat ggc acg 3645 Asp Glu Asp A1a Thr Thr His Arg Phe Thr Lys Lys Ala Asp Gly Thr tat cag ccg ccg acc ggt gta tat ctt gaa tta aca gaa aca get gat 3693 Tyr Gln Pro Pro Thr Gly Val Tyr Leu Glu Leu Thr Glu Thr Ala Asp cag ttc ata tta aaa aca aag gat caa acc aac get tat ttc aat aaa 3741 Gln Phe Ile Leu Lys Thr Lys Asp Gln Thr Asn Ala Tyr Phe Asn Lys 1000 1G''05 1010 aaa ggc ggc aaa ctc caa aag gta gtg gat ggc cat aac aat gca act 3789 Lys Gly Gly Lys Leu Gln Lys Val Val Asp Gly His Asn Asn Ala Thr gtc tat act tac aat gac aaa aat caa ctg acc gcc att aca gat gca 3837 Val Tyr Thr Tyr Asn Asp Lys Asn Gln Leu Thr Ala Ile Thr Asp Ala tca ggc cgc aag ctt aca ttt acc tat gat gaa aac ggc cat gtg acg 3885 Ser Gly Arg Lys Leu Thr Phe Thr Tyr Asp Glu Asn Gly His Val Thr tcc atc aca gga cct aag aat aag aaa gtc acc tac tca tat gag aat 3933 Ser Ile Thr Gly Pro Lys Asn Lys Lys Val Thr Tyr Ser Tyr Glu Asn gat ttg ctg aag aag gtt aca gat aca gac ggg act gtc aca agc tat 3981 Asp Leu Leu Lys Lys Val Thr Asp Thr Asp Gly Thr Val Thr Ser Tyr gac tac gat agt gaa ggg cgc ctt gtc aaa caa tat tca get aac agt .4029 Asp Tyr Asp Ser Glu Gly Arg Leu Val Lys Gln Tyr Ser Ala Asn Ser acc gaa gca aag cct gtc ttt acc gaa tat cag tat agc ggt cac cgt 4077 Thr Glu Ala Lys Pro Val Phe Thr Glu Tyr Gln Tyr Ser Gly His Arg ctc gaa aaa gcg atc aac get aaa aaa gaa aca tat gtc tac agc tat 4125 Leu Glu Lys Ala Ile Asn Ala Lys Lys Glu Thr Tyr Val Tyr Ser Tyr gat get gat aaa aag acg ctt ctc atg aca cag cca aat ggc cgg aaa 4173 Asp Ala Asp Lys Lys Thr Leu Leu Met Thr Gln Pro Asn Gly Arg Lys gta cag tat ggc tat aat gaa gcg gga aat ccg att cag gtc att gat 4221 Val Gln Tyr G1y Tyr Asn Glu Ala Gly Asn Pro Ile Gln Val Ile Asp gat gcg gaa ggg tta aaa att aca acg aat aca aag tat gaa ggc aac 4269 Asp Ala Glu Gly Leu Lys Ile Thr Thr Asn Thr Lys Tyr Glu Gly Asn aat gtt gta gaa gat gta gat cca aat gac gtc ggc acc gga aaa gca 4317 Asn Val Val Glu Asp Val Asp Pro Asn Asp Val Gly Thr Gly Lys Ala acggaaagctatcaa tatgac aaagatggc aacgtt acgtcggttaaa 4365 ThrGluSerTyrGln TyrAsp LysAspGly AsnVal ThrSerValLys gatgcatatggaact gagaca tacgaatac aacaaa aacaacgatgtc 4413 AspAlaTyrGlyThr GluThr TyrGluTyr AsnLys AsnAsnAspVal acaaagatgaaggat accgag ggcaatgtc acggat attgettatgac 4461 ThrLysMetLysAsp ThrGlu GlyAsnVal ThrAsp IleAlaTyrAsp ggactggatgetgta tccgaa accgatcag agcgga aaatcatcgtca 4509 GlyLeuAspAlaVal SerGlu ThrAspG1n SerGly LysSerSerSer gcagetgtttatgat aaatat ggaaaccag attcaa tcgagcaaagat 4557 AlaAlaValTyrAsp LysTyr GlyAsnGln IleGln SerSerLysAsp tta tca get tca act aat att ttg aaa gat gga agc ttt gaa get caa 4605 Leu Ser Ala Ser Thr Asn Ile Leu Lys Asp Gly Ser Phe Glu Ala Gln aaa tcc ggc tgg aat ctg act gcc tct aaa gac agg cgc aaa att tca 4653 Lys Ser Gly Trp Asn Leu Thr Ala Ser Lys Asp Arg Arg Lys Ile Ser gtc atc gcc gat aaa agc gga gtt ctt tcc gga tca aag gca ttg gag 4701 Val Ile Ala Asp Lys Ser Gly Val Leu Ser Gly Ser Lys Ala Leu Glu gtc ttg agc caa tca acg tct gcc gga aca gat cat ggg tat tcg tca 4749 Val Leu Ser Gln Ser Thr Ser Ala Gly Thr Asp His Gly Tyr Ser Ser gca aca caa aca gtt gag ctg gag ccg aat aca acc tat aca tta agc 4797 Ala Thr Gln Thr Val Glu Leu Glu Pro Asn Thr Thr Tyr Thr Leu Ser ggg aaa atc aaa aca gat ctt gcg aaa tca aga gcc tat ttt aac att 4845 Gly Lys Ile Lys Thr Asp Leu A1a Lys Ser Arg Ala Tyr Phe Asn Ile gat ttg cga gac aaa gat caa aaa cga att caa tgg att cac aat gaa 4893 Asp Leu Arg Asp Lys Asp Gln Lys Arg Tle Gln Trp Ile His Asn Glu tac agt gca tta gcc gga aaa aat gac tgg aca aaa cgg caa atc act 4941 Tyr Ser Ala Leu Ala Gly Lys Asn Asp Trp Thr Lys Arg Gln Ile Thr ttt acg act cca gcg aat gca ggc aaa get gtt gtg tat atg gaa gtc 4989 Phe Thr Thr Pro Ala Asn Ala Gly Lys Ala Val Val Tyr Met Glu Val gatcataaa gataaa gatggc aaaggaaaagca tggtttgat gaagtc 5037 AspHisLys AspLys AspGly LysGlyLysAla TrpPheAsp GluVal cagctggaa aaaggc gaagta tcatcaagctat aacccagtt caaaac 5085 GlnLeuGlu LysGly GluVal SerSerSerTyr AsnProVal GlnAsn agcagcttc acatca gcaacg gaaaactggaat gtaagcgga gcatcc 5133 SerSerPhe ThrSer AlaThr GluAsnTrpAsn ValSerGly AlaSer gttgattct gaagaa ggcttt aatgatgatgta tcattaaaa getgcc 5181 Va1AspSer GluGlu GlyPhe AsnAspAspVal SerLeuLys AlaAla cggaccagt gcgtcc caagcc ggatcagtaaca aaacaaacc gttgtc 5229 ArgThrSer AlaSer GlnAla GlySerValThr LysGlnThr ValVal ttagggcaa agcgca aatgac aaaccggtttat ctcacactg acagga 5277 LeuGlyGln SerAla AsnAsp LysProVa1Tyr LeuThrLeu ThrGly atgtcgaaa gcaagc agcgtc aaatttacagat gaaaaagac tattct 5325 MetSerLys AlaSer SerVal LysPheThrAsp GluLysAsp TyrSer ctgcaggcg aatgtc acttac getgacggcagc acaggcatc tacaat 5373 LeuGlnAla AsnVal ThrTyr AlaAspGlySer ThrGlyI12 TyrAsn gcgaaattc ccttct ggaaca caggaatggaac cgagcaget gtcgtc 5421 AlaLysPhe ProSer GlyThr GlnGluTrpAsn ArgA1aAla ValVal 1560 ~ 1565 1570 atccctaaa acaaag ccgatc aataaagtcgat atcagtatt ctgttc 5469 I1eProLys ThrLys ProIle AsnLysValAsp Ile~SerIle LeuPhe caaaagagt gcgaca ggcaca gtctggtttgat gatatccgt ttgatt 5517 GlnLysSer AlaThr GlyThr ValTrpPheAsp AspIleArg LeuIle gaaggaagc cttctg actaaa agcacgtatgac agtaatggc aactat 5565 GluGlySer LeuLeu ThrLys SerThrTyrAsp SerAsnGly AsnTyr gtg acc aag gaa gag gac gaa cta ggt tac gcc act tca act gat tac 5613 Val Thr Lys Glu Glu Asp Glu Leu Gly Tyr Ala Thr Ser Thr Asp Tyr gat gaa act ggt aag aaa acg tct gaa aca gat gca aaa ggt gaa aaa 5661 Asp Glu Thr Gly Lys Lys Thr Ser Glu Thr Asp Ala Lys Gly Glu Lys aca acc tat acg tat gat cag gca gac caa ttg aca aac atg acg ctc 5709 Thr Thr Tyr Thr Tyr Asp Gln Ala Asp Gln Leu Thr Asn Met Thr Leu tct aac ggc acg tct atc ctt cac agc tat gat aag gaa ggc aat gag 5757 Ser Asn Gly Thr Ser Ile Leu His Ser Tyr Asp Lys Glu Gly Asn Glu gtaagcaaaaccatc cgggcagga gcagatcaa acctat aaattcgaa 5805 ValSerLysThrIle ArgAlaGly AlaAspGln ThrTyr LysPheGlu tatgatgtcatgggc aagcttgtc aaaacaact gaccct ctcggcaat 5853 TyrAspValMetGly LysLeuVal LysThrThr AspPro LeuG1yAsn gtgttagcaagcgaa tatgatgcc aacagcaac ctgacc aaaaccatt 5901 Va1LeuAlaSerGlu TyrAspAla AsnSerAsn LeuThr LysThrIle tcaccaaatggcaac gaagtgtct ctttcttat gatgga acagaccgg 5949 SerProAsnGlyAsn GluVa1Ser LeuSerTyr AspGly ThrAspArg gtgaaatcaaagtcg tataacggt acagaaaag tacatt ttcacgtat 5997 ValLysSerLysSer TyrAsnGly ThrGluLys TyrTle PheThrTyr gacaaaaatggaaat gaaacgtcg gtcgtcaat aaagag cagaacacc 6045 AspLysAsnGlyAsn GluThrSer ValValAsn LysGlu GlnAsnThr aca aag aaa cgg aca ttt gac aac aaa aac cgt tta acc gaa ctg aca 6093 Thr Lys Lys Arg Thr Phe Asp Asn Lys Asn Arg Leu Thr Glu Leu Thr gac~cgc ggc ggc agc caa act tgg aca tat cct agt gac tcc gat aag 6141 Asp Arg Gly Gly Ser G1n Thr Trp Thr Tyr Pro Ser Asp Ser Asp Lys ctg aaa acc ttc tcg tgg att cat ggg gac caa aaa gga acg aat caa 6189 Leu Lys Thr Phe Ser Trp Ile His Gly Asp G1n Lys Gly Thr Asn Gln ttt acg tat aac aag ctg gat caa atg att gag atg aaa gac agt aca 6237 Phe Thr Tyr Asn Lys Leu Asp Gln Met Ile Glu Met Lys Asp Ser Thr tcc tca tat tca ttt gat tat gat gaa aat gga aac gtc caa acc ttt 6285 Ser Ser Tyr Ser Phe Asp Tyr Asp Glu Asn Gly Asn Val Gln Thr Phe att acg ggc aac ggc ggc gga aca agt ttt tca tat gac gaa aga aac 6333 Ile Thr Gly Asn Gly Gly Gly Thr Ser Phe Ser Tyr Asp Glu Arg Asn ctt gta agc tcg ctg cat atc ggt gac aaa aac ggc gga gac att ctg 6381 Leu Val Ser Ser Leu His Ile Gly Asp Lys Asn Gly Gly Asp Ile Leu acg gaa agc tat gaa tat gat gcc aac ggc aac cgc aca acg ata aac 6429 Thr Glu Ser Tyr Glu Tyr Asp Ala Asn Gly Asn Arg Thr Thr Ile Asn agc tca gca agc ggc aag gtg caa tat gag tat ggg aag ctg aat cag 6477 Ser Ser Ala Ser Gly Lys Val Gln Tyr Glu Tyr Gly Lys Leu Asn Gln ctt gtc aaa gaa acc cat gaa gac ggt act gtt att gaa tac aca tat 6525 Leu Val Lys G1u Thr His Glu Asp Gly Thr Val Ile Glu Tyr Thr Tyr gat gga ttc gga aac cgg aaa acc gtt aca act ata aaa gac ggc tca 6573 Asp Gly Phe Gly Asn Arg Lys Thr Val Thr Thr Ile Lys Asp Gly Ser agc aaa acg gtg aac get tcc ttc aac atc atg aac cag ctg aca aaa 6621 Ser Lys Thr Val Asn Ala Ser Phe Asn Ile Met Asn Gln Leu Thr Lys gtc aat gat gaa agc atc tct tac gat aaa aac ggc aac cgc aca tcc 6669 Val Asn Asp G1u Ser Ile Ser Tyr Asp Lys Asn Gly Asn Arg Thr Ser gac ggc aaa ttc aca tat acg tgg gat gca gaa gat aac ctg acc gcc &717 Asp Gly Lys Phe Thr Tyr Thr Trp Asp Ala Glu Asp Asn Leu Thr Ala gtt acc aaa aaa ggc gaa gac aag cca ttc gca aca tat aag tat gac 6765 Val Thr Lys Lys Gly Glu Asp Lys Pro Phe Ala Thr Tyr Lys Tyr Asp gaa aaa gga aac aga atc caa aaa acc gtc aac gga aaa gtc acg aac 6813 Glu Lys Gly Asn Arg Ile Gln Lys Thr Val Asn Gly Lys Val Thr Asn tac ttc tac gac gga gac age ttg.aac gtc ttg tat gaa aca gac gcg 6861 Tyr Phe Tyr Asp Gly Asp Ser Leu Asn Val Leu Tyr Glu Thr Asp Ala gac aac aac gtc aca aaa tca tac acg tac gga gac agc ggc cag ctg 6909 Asp Asn Asn Val Thr Lys Ser Tyr Thr Tyr Gly Asp Ser Gly Gln Leu tta tcc tac aca gaa aat ggt aag aaa tat ttc tat cac tac aac get 6957 Leu Ser Tyr Thr Glu Asn Gly Lys Lys Tyr Phe Tyr His Tyr Asn Ala 2070 ' 2075 2080 cac ggc gac atc atc gcg atc tca gac agc act gga aaa acc gtt gcc 7005 His Gly Asp Ile Ile Ala Ile Ser Asp Ser Thr Gly Lys Thr Val Ala aag tat caa tac gac gca tgg gga aat cca aca aaa acc gaa gca agc 7053 Lys Tyr Gln Tyr Asp Ala Trp Gly Asn Pro Thr Lys Thr Glu Ala 5er gac gaa gta aaa gac aat cgc tac cgc tac gca ggc tac caa tac gac 7101 Asp Glu Val Lys Asp Asn Arg Tyr Arg Tyr Ala Gly Tyr Gln Tyr Asp gaa gaa acc ggc ctg tac tat ctc atg gcc cgc tac tac gag cca aga 7149 Glu Glu Thr Gly Leu Tyr Tyr Leu Met Ala Arg Tyr Tyr Glu Pro Arg aac ggc gtg ttc cta tcg ctc gac cca gac ccg ggc agc gac gga gat 7197 Asn Gly Val Phe Leu Ser Leu Asp Pro Asp Pro Gly Ser Asp Gly Asp tca ttg gat cag aat ggg tat get tat gga aat aat aac cca gtg atg 7245 Ser Leu Asp Gln Asn Gly Tyr Ala Tyr Gly Asn Asn Asn Pro Val Met aat gtt gat ccg gac ggg cat tgg gta tgg ctt gtg gta aat gcc gga 7293 Asn Val Asp Pro Asp Gly His Trp Val Trp Leu Val Val Asn Ala Gly ttt gca gcc tat gat gga tat aag gca tat aaa tca ggt aaa gga tgg 7341 Phe Ala Ala Tyr Asp Gly Tyr Lys Ala Tyr Lys Ser Gly Lys Gly Trp aaa ggt get get tgg gca get get tct aat ttt ggt cct ggg aag ata 7389 Lys Gly Ala Ala Trp Ala Ala Ala Ser Asn Phe Gly Pro Gly Lys Ile ttt aag ggt get agt cgc gcc tat aag ttt acg aag aag gca gtt aaa 7437 Phe Lys Gly Ala Ser Arg Ala Tyr Lys Phe Thr Lys Lys Ala Val Lys atc aca ggt cat aca aga cat gga ctt aat caa tct ata gga aga aat 7485 Ile Thr Gly His Thr Arg His Gly Leu Asn Gln Ser Ile Gly Arg Asn gga ggc cgt gga gtc aat tta agg gcc aaa ctg aat get gtg cga agc 7533 Gly Gly Arg Gly Val Asn Leu Arg Ala Lys Leu Asn Ala Val Arg Ser cct aaa aaa gta ata aaa caa cct aat ggg gca acg aaa tat gtt gga 7581 Pro Lys Lys Val Ile Lys Gln Pro Asn Gly Ala Thr Lys Tyr Val Gly aaa aaa get aca gtt gtt tta aat aaa agg ggt aaa gta atc aca get 7629 Lys Lys Ala Thr Val Val Leu Asn Lys Arg Gly Lys Val Ile Thr Ala tat gga agt tca aga get aag ggt tct aaa cac gta ttc cat aca cac 7677 Tyr Gly Ser Ser Arg Ala Lys Gly Ser Lys His Val Phe His Thr His gga aaa gga aat aaa tct aag aga agg cgt taa taaggttaag cgaggggggg 7730 Gly Lys Gly Asn Lys Ser Lys Arg Arg Arg atgacaacta ccctcactta aactttaaat gaggaggaaa tatggccaaa ataaaagatg 7790 attgtataga acttgaatta acaccgagaa ggtatcaaga gttggacgat gatccattta 7850 ttttatctgt atttgaattg ttggaaaata agaaagccgt cgttcgcgat ttttctgctg 7910 ttttattaga aagtgaatat aaggtgttga tatctgggat cgaaactatg ataaaaggaa 7970 atcaagacag tatcagttta gagactattg aaccattctt atttcttagt attgatcaag 8030 aaaacggaaa ttacagaatt aagattaaaa ttatatttga tgattataaa gaaagtaagt 8090 cgaactccaa cttgttcgaa attaattgta atgaagaaaa gctagagtct tttgtgaccg 8150 ctttaaaatt aaatcttgaa aatacaaaaa atccttctaa acttccttaa ttataaataa 8210 tttctgcatt ggataaaaat ggggcattta ttgatggaac attaaataag agatgttacc 8270 atcctctctt atttgtatga caaaagaatc taaaaat 8307 <210> 6 <211> 2334 <212> PRT
<213> Homo sapiens <400> 6 Met Lys Lys Arg Lys Arg Arg Asn Phe Lys Arg Phe Ile Ala Ala Phe Leu Val Leu Ala Leu Met Ile Ser Leu Val Pro Ala Asp Val Leu Ala Lys Ser Thr Glu Glu Glu Asn Gly Asn Arg Ile Ala Ala Asp Asp Pro Glu Glu Thr Leu Gln Lys Glu Gln Thr Glu Glu Ala Val Pro Phe Asp Pro Lys Asp Ile Asn Lys Glu Gly Glu Ile Thr Ser Glu Arg Thr Glu Asn Thr Lys Leu Tyr Tyr Glu Gly Asp G1y Val Tyr Lys Gln Glu Val Tyr Leu Asp Pro Ile His Thr Lys Glu Thr Pro Asp Ala Asp Trp Glu Asp Ile Ser Pro Glu Leu Lys Glu Ser Thr Ser Lys Gln Val Glu Thr Glu Asn Ala Ile Leu Asn Ser Asp Phe Gln Lys Gln Met Lys Asn Gly Leu Tyr Ala Thr Phe Glu His Asn Asp His Lys Val Thr Tyr Ser Leu Ala Glu Ala Lys Gly Pro Asn Lys Thr Ser Leu Thr Pro Lys Asp Thr Ser Ala Asp Tyr Lys Thr Asp Ser Asn Glu Ile Val Tyr Pro Asp Val Phe Pro Asn Ile Asp Leu Gln Thr Phe Thr Phe Asn Glu Asn Ile Lys Glu Asp Leu Val Leu His Gln Tyr Asn Gly Tyr Asn Thr Phe Thr Phe Gln Leu Lys Thr Asp Leu Gln Ala Lys Glu Gln Glu Asp Gly Ser Ile Asp Phe Ser Asp Glu Lys Gly Lys Val Val Phe Ser Val Pro Lys Pro Phe Met Thr Asp Ser Lys Leu Asp Glu Leu Ser Gly Glu Val Glu Arg Ser Asp Lys Val Ser Tyr Lys Leu Glu Lys Asn Glu Glu Gly Tyr Leu Leu His Leu Thr Ala Asp Glu Asn Trp Leu Lys Asp Pro Glu Arg Va1 Tyr Pro Val Ser Ile Asp Pro Ser Thr Ser Leu Ser Val Ser Ser Asp Thr Phe Val Met Ser Ala Tyr Pro Thr Thr Asn Tyr Ser Ala Ser Ser Gln Lys Trp Asp Ala Asn Leu Lys Ala Tyr Val Leu Lys Thr Gly Tyr Tyr Asp Lys Thr Thr Gly Thr Asn Tyr Ala Phe Met Lys Phe Asn Asn Leu Lys Pro Ile Gln Asn Met Thr Val Thr Lys Ala Thr Leu Lys Thr Tyr Val Ala His Ser Tyr Tyr Gly Thr Lys Ala Thr Gly Leu Trp Leu Asp Thr Val Asn Ser Asn Tyr Asp Asn Ala Lys Val Thr Trp Asn Thr Lys Pro Ala Ser Lys Asn Ile Gly Lys Ala Asp Val His Lys Gly G1n Trp Ala Ser Tyr Asp Val Thr Ala Ala Val Lys Ser Trp Asn Ser G1y Gly Ala Asn Tyr Gly Phe Lys Leu His Thr Asn Gly Asn Gly Lys Glu Tyr Trp Lys Lys Leu Ile Ser Ser Ala Asn Ser Ala Asn Lys Pro Tyr Ile Glu Val Thr Tyr Thr Ile Pro Lys Gly Asn Thr Pro Thr I1e Lys Ala Tyr His Asn Gly Asp Ser Thr Gly Tyr Phe Asp Ile Ser Trp Lys Lys Val Glu Gly Ala Lys Gly Tyr Lys Val Trp Ile Tyr Asn Gly Lys Glu Tyr Gln Ala Ile Ser Ala Gly Asn Val Thr Ser Trp Ser Thr Lys Gly Lys Lys Ile Trp Pro Thr Ser Ala Glu Ile Ala Ser Lys Arg Tyr 545 ' 550 555 560 Lys Leu His Leu Asp Gly Lys Asp Gly Ala Glu Leu Ala Leu Asp Pro Ser Pro Val Tyr Lys Asn Ser Gly Gly Ser Tyr Ala Thr Ser Lys Asn Tyr Trp Ile Gly Val Ser Ala Ile Phe Asp Gln Gly Glu Gly Ala Met Ser Ala Pro Ala Lys Pro Val Ile Pro Asn Val Gly Lys Ala Gln Ala Pro Ser Ala Lys Gly Tyr Asn Asn Gly Asn Ala Thr Gly Tyr Phe Asp Leu Ser Trp Lys Ala Val Ser Gly Ala Thr Gly Tyr Lys Va1 Gln Val Phe Asn Gly Lys Gly Phe Glu Thr Leu Asp Leu Gly Asn Gln Thr Ser Trp Thr Thr Lys Gly Lys Lys Ile Trp Pro Thr Ser Ala Glu Tle Lys Ala Gly Lys Tyr Ala Leu His Leu Lys Asp Gly Ser Gly Ala Glu Leu Pro Ile Asn Pro Gly Pro Thr Tyr Lys Asn Ala Gly Gly Asp Gly Ala Lys Arg Asn Tyr Ser Phe Lys Ile Ile Ala Tyr Asn Lys Asp Gly Glu Ala Ile Ala Ser Pro Ala Ala Thr Pro Ala Leu Pro Asp Ile Ala Arg Pro Lys Asn Va1 Thr Gly Tyr Leu Tyr Thr Asn Thr Lys Ser Ser Gln Thr Gly Tyr Val Asn Leu ale Trp Glu Lys Val Gln Asn Ala Lys Gly Tyr Lys Val Asn Ile Tyr Asn Gly Lys Glu Tyr Gln Ser Phe Asp Val Gly Asp Ala Asp His Trp Thr Thr Gln Asn Lys Asn Ile Trp Pro Thr Ser Glu Glu Ile Lys Ala Gly Ser Tyr Lys Leu His Thr Asp Gly Lys Gly Gly Glu Leu Ala Leu Asp Pro Ser Pro Val Tyr Asn Asn Ala Asn Gly Asn Tyr Lys Gly Lys Lys Asn Tyr Ser Phe Thr Leu Val Ala Tyr Asp A1a Asn Gly Glu Thr Ile Pro Thr Ala Pro Phe Asn Pro Thr Phe 865 870 875 gg0 His Glu Gly Ala Glu Phe Leu Gly Thr Glu Glu Tyr Trp Ser Ile Ile Asp Ile Pro Ser Gly Gln Leu Asn Gly Ala Thr Gly Asn Val Ile Val Asn Glu Glu Asp Leu Ser Ile Asp Gly Arg Gly Pro Gly Leu Gly Leu Ser Arg Thr Tyr Asn Ser Leu Asp Ser Ser Asp His Leu Phe Gly Gln Gly Trp Tyr Ala Asp A1a Glu Thr Ser Val Ile Ser Thr Asp Gly Gly Ala Met Tyr Ile Asp Glu Asp Ala Thr Thr His Arg Phe Thr Lys Lys Ala Asp Gly Thr Tyr Gln Pro Pro Thr Gly Val Tyr Leu Glu Leu Thr Glu Thr Ala Asp Gln Phe Ile Leu Lys Thr Lys Asp G1n Thr Asn Ala Tyr Phe Asn Lys Lys Gly Gly Lys Leu Gln Lys Val Va1 Asp Gly His Asn Asn Ala Thr Val Tyr Thr Tyr Asn Asp Lys Asn Gln Leu Thr Ala Ile Thr Asp Ala Ser Gly Arg Lys Leu Thr Phe Thr Tyr Asp Glu Asn Gly His Val Thr Ser Ile Thr Gly Pro Lys Asn Lys Lys Val Thr Tyr 1060. 1065 ~ 1070 Ser Tyr Glu Asn Asp Leu Leu Lys Lys Val Thr Asp Thr Asp Gly Thr Val Thr Ser Tyr Asp Tyr Asp Ser Glu Gly Arg Leu Val Lys Gln Tyr Ser Ala Asn Ser Thr Glu Ala Lys Pro Val Phe Thr Glu Tyr Gln Tyr Ser Gly His Arg Leu Glu Lys Ala Tle Asn Ala Lys Lys Glu Thr Tyr 1125 ' 1130 1135 Val Tyr Ser Tyr Asp Ala Asp Lys Lys Thr Leu Leu Met Thr Gln Pro Asn Gly Arg Lys Val Gln Tyr Gly Tyr Asn Glu Ala Gly Asn Pro Ile Gln Val Ile Asp Asp Ala Glu Gly Leu Lys Ile Thr Thr Asn Thr Lys Tyr Glu Gly Asn Asn Val Val Glu Asp Val Asp Pro Asn Asp Val Gly Thr Gly Lys Ala Thr Glu Ser Tyr Gln Tyr Asp Lys Asp Gly Asn Val Thr Ser Val Lys Asp Ala Tyr Gly Thr Glu Thr Tyr Glu Tyr Asn Lys Asn Asn Asp Val Thr Lys Met Lys Asp Thr Glu Gly Asn Val Thr Asp Ile Ala Tyr Asp Gly Leu Asp Ala Val Ser Glu Thr Asp Gln Ser Gly Lys Ser Ser Ser Ala Ala Val Tyr Asp Lys Tyr Gly Asn Gln Ile Gln Ser Ser Lys Asp Leu Ser Ala Ser Thr Asn Ile Leu Lys Asp Gly Ser Phe Glu Ala Gln Lys Ser Gly Trp Asn Leu Thr Ala Ser Lys Asp Arg Arg Lys Tle Ser Val Ile Ala Asp Lys Ser Gly Val Leu Ser Gly Ser Lys Ala Leu Glu Val Leu Ser Gln Ser Thr Ser Ala Gly Thr Asp His Gly Tyr Ser Ser Ala Thr Gln Thr Val Glu Leu Glu Pro Asn Thr Thr Tyr Thr Leu Ser Gly Lys Ile Lys Thr Asp Leu Ala Lys Ser Arg Ala Tyr Phe Asn Tle Asp Leu Arg Asp Lys Asp Gln Lys Arg Ile Gln Trp Ile His Asn Glu Tyr Ser Ala Leu Ala Gly Lys Asn Asp Trp Thr Lys Arg Gln Ile Thr Phe Thr Thr Pro Ala Asn Ala Gly Lys Ala Val Val Tyr Met Glu Val Asp His Lys Asp Lys Asp Gly Lys Gly Lys Ala Trp Phe Asp Glu Val Gln Leu Glu Lys Gly Glu Val Ser Ser Ser Tyr Asn Pro Val Gln Asn Ser Ser Phe Thr Ser Ala Thr Glu Asn Trp Asn Val Ser Gly Ala Ser Val Asp Ser Glu Glu Gly Phe Asn Asp Asp Val Ser Leu Lys Ala Ala Arg Thr Ser Ala Ser Gln Ala Gly Ser Val Thr Lys Gln Thr Val Val Leu Gly Gln Ser Ala Asn Asp Lys Pro Val Tyr Leu Thr Leu Thr Gly Met Ser Lys Ala Ser Ser Val Lys Phe Thr Asp Glu Lys Asp Tyr Ser Leu Gln Ala Asn Val Thr Tyr Ala Asp Gly Ser Thr Gly Ile Tyr Asn Ala Lys Phe Pro Ser Gly Thr Gln G1u Trp Asn Arg Ala Ala Val Val Ile Pro Lys Thr Lys Pro Ile Asn Lys Val Asp Ile Ser Ile Leu Phe Gln Lys Ser Ala Thr Gly Thr Val Trp Phe Asp Asp Ile Arg Leu Ile Glu Gly Ser Leu Leu Thr Lys Ser Thr Tyr Asp Ser Asn Gly Asn Tyr Val Thr Lys Glu Glu Asp Glu Leu Gly Tyr Ala Thr Ser Thr Asp Tyr Asp Glu Thr Gly Lys Lys Thr Ser Glu Thr Asp Ala Lys Gly Glu Lys Thr Thr Tyr Thr Tyr Asp Gln Ala Asp Gln Leu Thr Asn Met Thr Leu Ser Asn Gly Thr Ser Ile Leu His Ser Tyr Asp Lys Glu Gly Asn Glu Val Ser Lys Thr Ile Arg Ala Gly Ala Asp Gln Thr Tyr Lys Phe Glu Tyr Asp Val Met Gly Lys Leu Val Lys Thr Thr Asp Pro Leu Gly Asn Val Leu Ala Ser Glu Tyr Asp Ala Asn Ser Asn Leu Thr Lys Thr Ile Ser Pro Asn Gly Asn Glu Val Ser Leu Ser Tyr Asp Gly Thr Asp Arg Val Lys Ser Lys Ser Tyr Asn Gly Thr Glu Lys Tyr Ile Phe Thr Tyr Asp Lys Asn Gly Asn Glu Thr Ser Val Val Asn Lys Glu Gln Asn Thr Thr Lys Lys Arg Thr Phe Asp Asn Lys Asn Arg Leu Thr Glu Leu Thr Asp Arg Gly Gly Ser Gln Thr Trp Thr Tyr Pro Ser Asp Ser Asp Lys Leu Lys Thr Phe Ser Trp Ile His Gly Asp Gln Lys Gly Thr Asn Gln Phe Thr Tyr Asn Lys Leu Asp Gln Met Ile Glu Met Lys Asp Ser Thr Ser Ser Tyr Ser Phe Asp Tyr Asp Glu Asn Gly Asn Val Gln Thr Phe Ile Thr Gly Asn Gly Gly Gly Thr Ser Phe Ser Tyr Asp Glu Arg Asn Leu Val Ser Ser Leu His Ile Gly Asp Lys Asn Gly Gly Asp Ile Leu Thr Glu Ser Tyr Glu Tyr Asp Ala Asn Gly Asn Arg Thr Thr Ile Asn Ser Ser Ala Ser Gly Lys Val Gln Tyr Glu Tyr Gly Lys Leu Asn Gln Leu Val Lys Glu Thr His Glu Asp Gly Thr Val Ile Glu Tyr Thr Tyr Asp Gly Phe Gly Asn Arg Lys Thr Val Thr Thr Tle Lys Asp Gly Ser Ser Lys Thr Val Asn Ala Ser Phe Asn Ile Met Asn Gln Leu Thr Lys Val Asn Asp Glu Ser Ile Ser Tyr Asp Lys Asn Gly Asn Arg Thr Ser Asp Gly Lys Phe Thr Tyr Thr Trp Asp Ala Glu Asp Asn Leu Thr Ala Val Thr Lys Lys Gly Glu Asp Lys Pro Phe Ala Thr Tyr Lys Tyr Asp Glu Lys Gly Asn Arg Ile Gln Lys Thr Val Asn Gly Lys Val Thr Asn Tyr Phe Tyr Asp Gly Asp Ser Leu Asn Val Leu Tyr Glu Thr Asp Ala Asp Asn Asn Val Thr Lys Ser Tyr Thr Tyr Gly Asp Ser Gly Gln Leu Leu Ser Tyr Thr Glu Asn Gly Lys Lys Tyr Phe Tyr His Tyr Asn Ala His Gly Asp Ile Ile Ala Ile Ser Asp Ser Thr Gly Lys Thr Val Ala Lys Tyr Gln Tyr Asp Ala Trp Gly Asn Pro Thr Lys Thr Glu Ala Ser Asp Glu Val Lys Asp Asn Arg Tyr Arg Tyr Ala Gly Tyr Gln Tyr Asp Glu Glu Thr Gly Leu Tyr Tyr Leu Met Ala Arg Tyr Tyr Glu Pro Arg Asn Gly Val Phe Leu Ser Leu Asp Pro Asp Pro Gly Ser Asp Gly Asp Ser Leu Asp Gln Asn Gly Tyr Ala Tyr Gly Asn Asn Asn Pro Val Met Asn Val Asp Pro Asp Gly His Trp Val Trp Leu Val Val Asn Ala Gly Phe Ala Ala Tyr Asp Gly Tyr Lys Ala Tyr Lys Ser Gly Lys Gly Trp Lys Gly Ala Ala Trp Ala Ala Ala Ser Asn Phe Gly Pro G1y Lys Ile Phe Lys G1y Ala Ser Arg Ala Tyr Lys Phe Thr Lys Lys Ala Val Lys Ile Thr Gly His Thr Arg His Gly Leu Asn Gln Ser Ile Gly Arg Asn G1y Gly Arg Gly Val Asn Leu Arg Ala Lys Leu Asn Ala Val Arg Ser Pro Lys Lys Val Ile Lys Gln Pro Asn Gly Ala Thr Lys Tyr Val Gly Lys Lys Ala Thr Val Val Leu Asn Lys Arg Gly Lys Val Ile Thr Ala Tyr Gly Ser Ser Arg Ala Lys Gly Ser Lys His Val Phe His Thr His Gly Lys Gly Asn Lys Ser Lys Arg Arg Arg <210> 7 <211> 1576 <212> DNA
<213> Homo sapiens <400> 7 gaattcgagc cgaccgaccg ctcccggccc gccccctatg ggccccggct agaggcgccg 60 ccgccgccgg cccgcggagc cccgatgctg gcccggagga agccggtgct gccggcgctc 120 accatcaacc ctaccatcgc cgagggccca tcccctacca gcgagggcgc ctccgaggca 180 aacctggtgg acctgcagaa gaagctggag gagctggaac ttgacgagca gcagaagaag 240 cggctggaag cctttctcac ccagaaagcc aaggttggcg aactcaaaga cgatgacttc 300 gaaaggatct cagagctggg cgcgggcaac ggcggggtgg tcaccaaagt ccagcacaga 360 ccctcgggcc tcatcatggc caggaagctg atccaccttg agatcaagcc ggccatccgg 420 aaccagatca tccgcgagct gcaggtcctg cacgaatgca actcgccgta catcgtgggc 480 ttctacgggg ccttctacag tgacggggag atcagcattt gcatggaaca catggacggc 540 ggctccctgg. accaggtgct gaaagaggcc aagaggattc ccgaggagat cctggggaaa 600 gtcagcatcg cggttctccg gggcttggcg tacctccgag agaagcacca gatcatgcac 660 cgagatgtga agccctccaa catcctcgtg aactctagag gggagatcaa gctgtgtgac 720 ttcggggtga gcggccagct catagactcc atggccaact ccttcgtggg cacgcgctcc 780 tacatggctc cggagcggtt gcagggcaca cattactcgg tgcagtcgga catctggagc 840 atgggcctgt ccctggtgga gctggccgtc ggaaggtacc ccatcccccc gcccgacgcc 900 aaagagctgg aggccatctt tggccggccc gtggtcgacg gggaagaagg agagcctcac 960 agcatctcgc ctcggccgag gccccccggg cgccccgtca gcggtcacgg gatggatagc 1020 cggcctgcca tggccatctt tgaactcctg gactatattg tgaacgagcc acctcctaag 1080 ctgcccaacg gtgtgttcac ccccgacttc caggagtttg tcaataaatg cctcatcaag 1140 aacccagcgg agcgggcgga cctgaagatg ctcacaaacc acaccttcat caagcggtcc 1200 gaggtggaag aagtggattt tgccggctgg ttgtgtaaaa ccctgcggct gaaccagccc 1260 ggcacaccca cgcgcaccgc cgtgtgacag tggccgggct ccctgcgtcc cgctggtgac 1320 ctgcccaccg tccctgtcca tgccccgccc ttccagctga ggacacgtgg cgcctccacc 1380 caccctcctg cctcaccctg cggagagcac cgtggcgggg cgacagcgca tgcaggaacg 1440 ggggtctcct ctcctgccag tcctggccgg ggtgcctctg gggacgggcg acgctgctgt 1500 gtgtggtctc agaggctctg cttccttagg ttacaaaaca aaacagggag agaaaagcaa 1560 aaaaaaaaaa aaaaaa 1576 <210> 8 <211> 400 <212> PRT
<2l3> Rattus norvegicus <400> 8 Met Leu Ala Arg Arg Lys Pro Val Leu Pro Ala Leu Thr Ile Asn Pro Thr Ile A1a Glu Gly Pro Ser Pro Thr Ser Glu Gly Ala Ser G1u Ala Asn Leu Val Asp Leu Gln Lys Lys Leu Glu Glu Leu Asp Leu Asp Glu Gln Gln Arg Lys Arg Leu Glu Ala Phe Leu Thr Gln Lys Ala Lys Val Gly Glu Leu Lys Asp Asp Asp Phe Glu Arg Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly Val Val Thr Lys Ala Arg His Arg Pro Ser Gly Leu Ile Met Ala Arg Lys Leu I1e His Leu Glu Ile Lys Pro Ala Val Arg Asn Gln Ile Ile Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu His Met Asp Gly Gly Sex Leu Asp Gln Val Leu Lys Glu Ala Lys Arg Ile Pro Glu Asp Tle Leu Gly Lys Val Ser Ile Ala Val Leu Arg Gly Leu Ala ~Tyr Leu Arg Glu Lys His Gln Ile Met His Arg Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly Thr His Tyr Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Leu Ala Ile Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys Glu Leu Glu Ala Ser Phe Gly Arg Pro Val Val Asp Gly Ala Asp Gly Glu Pro His 5er Val Ser Pro Arg Pro Arg Pro Fro Gly Arg Pro Ile Ser Gly His Gly Met Asp Ser Arg Pro Ala Met Ala Ile Phe Glu 305 310 3l5 320 Leu Leu Asp Tyr Ile Val Asn Glu Pro Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Ser Asp Phe Gln Glu Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu Lys Leu Leu Thr Asn His Ala Phe Ile Lys Arg Ser Glu Gly Glu Glu Val Asp Phe Ala Gly Trp Leu Cys Arg Thr Leu Arg Leu Lys Gln Pro Ser Thr Pro Thr Arg Thr A1a Val

Claims (66)

CLAIMS:

1. A method of reducing tolerance to an analgesic in a patient comprising administering to the patient an effective amount of an ERK inhibitor to reduce tolerance to the analgesic, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

2. The method of claim 1, wherein the analgesic is an opioid.

3. The method of claim 2, wherein the opioid is morphine or fentanyl.

4. The method of claim 1, wherein the patient has chronic pain.

5. The method of claim 1, further comprising administering to the patient an analgesic.

6. The method of claim 1, wherein the patient has been administered an analgesic.

7. The method of claim 1, further comprising identifying a patient needing a reduction in tolerance to the analgesic.

8. The method of claim 1, further comprising evaluating the patient for a reduction in tolerance to the analgesic after the inhibitor is administered.

9. The method of claim 5, wherein the analgesic and the ERK inhibitor are administered to the patient at the same time.

10. The method of claim 9, wherein the analgesic and the ERK inhibitor are comprised in formulation together.

11. The method of claim 1, wherein the inhibitor is SL-327, U0126, PD098059, or PD 184352.

12. The method of claim 11, wherein the inhibitor is U0126.

13. The method of claim 1, wherein the inhibitor is a nucleic acid.

14. The method of claim 13, wherein the inhibitor is an ERK antisense nucleic acid.

15. The method of claim 13, wherein the nucleic acid comprises a sequence identical or complementary to at least 30 contiguous nucleotides of SEQ ID NO:1 or SEQ
ID
NO:3.

16. The method of claim 15, wherein the nucleic acid is complementary to at least 30 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3.

17. The method of claim 16, wherein the nucleic acid is complementary to at least 50 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3.

18. The method of claim 15, wherein the nucleic acid encodes at least part of an ERK
polypeptide.

19. The method of claim 18, wherein the ERK polypeptide comprises at least 10 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4.

20. The method of claim 18, wherein the ERK polypeptide is mutated.

21. The method of claim 20, wherein the kinase domain of the ERK polypeptide is mutated and the ERK polypeptide is unable to phosphorylate a substrate.

22. The method of claim 1, wherein the inhibitor comprises an MEK polypeptide with a mutation in the kinase domain, wherein the MEK polypeptide is unable to phosphorylate an ERK polypeptide.

23. The method of claim 15, wherein the sequence is comprised in a vector.

24. The method of claim 23, wherein the vector is comprised in a composition comprising a lipid.

25. The method of claim 23, wherein the vector is a viral vector.

26. The method of claim 25, wherein the viral vector is an adenovirus vector, an AAV vector, a retrovirus vector, a herpesvirus vector, a lentivirus vector, or vaccinia virus vector.

27. The method of claim 1, wherein the inhibitor is administered to the patient intravenously.

28. The method of claim 1, wherein the inhibitor is administered to the spine of the patient.

29. The method of claim 1, wherein the inhibitor is administered to the patient via a catheter.

30. The method of claim 1, wherein the inhibitor is formulated in a composition comprising .beta.-cyclodextrin.

31. A method of reversing tolerance to an analgesic in a patient comprising administering to the patient an effective amount of an ERK inhibitor to reduce tolerance to the analgesic, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

32. A method of reducing tolerance to an analgesic in a patient comprising administering an effective amount of an ERK inhibitor to reduce tolerance to the analgesic, wherein the ERK inhibitor comprises:
a) SL-327, U0126, PD098059, or PD184352; or b) a nucleic acid molecule comprising a nucleic acid sequence complementary or identical to at least 30 contiguous nucleic acids of SEQ
ID NO:1 or SEQ ID NO:3.

33. The method of claim 32, further comprising identifying a patient in need of reduction of tolerance to an analgesic.

34. The method of claim 32, further comprising evaluating the patient for a reduction in tolerance to the analgesic after the inhibitor is administered.

35. A method of reducing physical dependence on an opioid in a patient comprising:
a) administering the opioid to the patient;
b) administering to the patient an effective amount of an ERK inhibitor to inhibit ERK, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

36. The method of claim 35, wherein the opioid is morphine or fentanyl.

37. The method of claim 35, further comprising administering a composition comprising an opioid to the patient.

38. The method of claim 37, wherein the opioid is methadone, buprenorphine, or levoacetyl methadol.

39. The method of claim 35, further comprising administering to the patient a .alpha.-2 adrenergic agonist, a tranquilizer, or a benzodiazepine.

40. The method of claim 35, wherein the patient has used an opioid prior to administration of the inhibitor.

41. The method of claim 35, further comprising identifying a patient with physical dependence on an opioid.

42. The method of claim 35, further comprising evaluating the patient for symptoms of physical dependence after the inhibitor is administered.

43. The method of claim 35, wherein the opioid and the ERK inhibitor are administered to the patient at the same time.

44. The method of claim 43, wherein the opioid and the ERK inhibitor are comprised in formulation together.

45. The method of claim 35, wherein the inhibitor is SL-327, U0126, PD098059 or PD 184352.

46. The method of claim 45, wherein the inhibitor is U0126.

47. The method of claim 35, wherein the inhibitor is administered to the patient intravenously.

48. The method of claim 35, wherein the inhibitor is administered to the spine of the patient.

49. The method of claim 35, wherein the inhibitor is administered to the patient via a catheter.

50. The method of claim 35, further comprising performing surgery on the patient.

51. The method of claim 50, wherein the inhibitor is administered postoperatively to the patient.

52. The method of claim 50, wherein the inhibitor is administered pre-operatively to the patient.

53. The method of claim 35, wherein the patient has chronic pain.

54. The method of claim 35, wherein the inhibitor is formulated in a composition comprising .beta.-cyclodextrin.

55. A method of inhibiting pain sensitization in a patient comprising administering to the patient an effective amount of an ERIC inhibitor to reduce pain in the patient, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

56. A method of reducing hyperalgesia in a patient comprising administering to the patient an effective amount,of an ERK inhibitor to reduce pain in the patient, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

57. The method of claim 56, further comprising identifying a patient with hyperalgesia.

58. The method of claim 56, further comprising evaluating the patient for a reduction in hyperalgesia after the inhibitor is administered.

59. A method of reducing the symptoms of opioid withdrawal in a patient exposed to an opioid comprising administering to the patient an effective amount of an ERK
inhibitor to reduce the withdrawal symptoms, wherein the inhibitor is comprised in pharmaceutically acceptable formulation.

60. The method of claim 59, wherein the opioid is morphine or heroin.

61. The method of claim 59, further comprising administering a composition comprising an opioid to the patient.

62. The method of claim 61, wherein the opioid is methadone, buprenorphine, or levoacetyl methadol.

63. The method of claim 59, further comprising administering to the patient an .beta.-2 adrenergic agonist, a tranquilizer, or a benzodiazepine.

64. The method of claim 59, wherein the patient has taken an opioid prior to administration of the inhibitor.

65. The method of claim 59, further comprising identifying a patient with physical dependence on an opioid.

66. The method of claim 59, further comprising evaluating the patient for symptoms of opioid withdrawal.