WO2002058687A2 - Inhibition of erk to reduce or prevent tolerance to and dependence on opioid analgesics - Google Patents
Inhibition of erk to reduce or prevent tolerance to and dependence on opioid analgesics Download PDFInfo
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- WO2002058687A2 WO2002058687A2 PCT/US2002/002128 US0202128W WO02058687A2 WO 2002058687 A2 WO2002058687 A2 WO 2002058687A2 US 0202128 W US0202128 W US 0202128W WO 02058687 A2 WO02058687 A2 WO 02058687A2
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- A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/485—Morphinan derivatives, e.g. morphine, codeine
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
Definitions
- 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.
- 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).
- NNS 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).
- NAcc nucleus accumbens
- VTA ventral tegmental area
- Arc arcuate nucleus
- Amygdala Amy
- PAG locus coeruleus
- RVM rostral ventromedial medulla
- NTS nucleus tractus solitarius
- opioid receptor function is altered. Receptor desensitization and dowiiregulation have
- Desensitization is an acute decrease in signaling responses. This phenomenon occurs within a matter of minutes, which does not parallel the time course of chronic tolerance development. However, desensitization could potentially be the first in a complex series of layered adaptations leading to chronic tolerance. Receptor dowiiregulation 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.
- opioid receptors generally couple to Gj and G 0 classes of G proteins and acutely inhibit adenylylate cyclase (AC), inhibit calcium conductance and activate a potassium conductance, leading to hyperpolarization of the cell (Childers, 1991).
- Opioids may also activate protein kinase C (PKC).
- PKC activates the NMDA receptor, a known mediator of tolerance and dependence.
- Gp ⁇ subunits, NO/cGMP and calcium/calmodulin kinase pathways have also been implicated in opioid signaling.
- PKA protein kinase A
- 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).
- MAP mitogen-activated protein
- 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.
- an analgesic refers to a medication that reduces or eliminates pain.
- analgesics include opioid analgesics, nonopioid analgesics (NSAIDs and acetaminophen), and adjuvant analgesics.
- 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: ERK1 and ERK2.
- ERK encompassed both forms.
- the cDNA sequence and amino acid sequence of human ERK1 corresponds with SEQ ID NO:l and SEQ ID NO:2, respectively.
- the cDNA sequence and amino acid sequence of human ERK2 corresponds with SEQ ID NO:3 and SEQ ID NO:4, respectively. It is contemplated that embodiments of the present invention may involve either ERK1 or ERK2, or both.
- An "ERK inhibitor” refers to a substance that reduces or eliminates ERK activity, that is, the ability to phosphorylate an ERK 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 ERK 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 ERK, binds ERK protein, or inhibits or reduces the post-translational modification of ERK, including its phosphorylation.
- the ERK inhibitor is a chemical compound.
- the chemical compound may be SL-327, U0126, PD098059, or PD184352.
- the inhibitor is U0126.
- the inhibitor may be a polypeptide such as a mutated ERK polypeptide or a mutated MEK polypeptide.
- 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 substrate.
- a mutated MEK polypeptide that is unable to phosphorylate an ERK polypeptide, even under conditions that would allow for phosphorylation.
- the ERK polypeptide comprises at least 10, 25, 50, 75, or 100 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4.
- a MEK polypeptide comprises at least 10, 25, 50, 75, or 100 contiguous amino acids of SEQ ID NO:6 or SEQ ID NO: 8.
- 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 nucleib acid that encodes a polypeptide inhibitor, such as a mutated ERK or MEK polypeptide.
- a nucleic acid comprises a sequence identical or complementary to at least 30, 50, 70 or 100 contiguous nucleotides of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO: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 NO:3, SEQ ID NO:5, or SEQ ID NO: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.
- 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 lipid 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 ERK 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 MEK activity and/or expression levels.
- a patient in need of treatment there is a step of identifying a patient in need of treatment.
- 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.
- 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.
- reduction of tolerance is to such an extent as to effect a reversal of tolerance to an analgesic.
- 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.
- 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; ⁇ -2 adrenergic receptor agonist — such as clonidine; a tranquilizer; a benzodiazepine, such as Librium or Naliu ; or a combination thereof.
- the patient is being given an opiate to reduce pain, and the ERK inhibitor is administered to reduce or prevent tolerance.
- the patient is addicted to an opiate and the opioid is given to the patient to reduce withdrawal symptoms.
- 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.
- 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.
- 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 ⁇ -cyclodextrin.
- methods of screening for ERK 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.
- 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.
- 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 (N) or U0126 in vehicle (N/U0126).
- S saline
- N DMSO vehicle
- N/U0126 U0126 in vehicle
- 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.
- Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and can also induce physical dependence (Koob 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,
- 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
- opioid receptors there are numerous classes of opioid receptors, including those designated ⁇ , K, and ⁇ (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 three subtypes, suggesting that they could be related molecules (Loh et al, 1990).
- Morphine interacts principally with ⁇ receptors and peripheral administration of this opioid induces release of enkephalins (Bertolucci et al, 1992).
- the ⁇ receptors bind with the greatest affinity to enkephalins and have a more discrete distribution in the brain than either ⁇ or K receptors, with high concentrations in the basal ganglia and limbic regions.
- enkephalins may mediate part of the physiological response to morphine, presumably by interacting with ⁇ receptors.
- opioid receptors Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase K + conductance, and inactivate Ca 2+ 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).
- 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, odorants, 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 ⁇ -helical) segments that span the lipid bilayer (Dohlman et al, 1987; Dohlman et al, 1991).
- G proteins consist of three tightly associated subunits, ⁇ , ⁇ and ⁇ (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 G ⁇ -subunit to exchange a bound GDP for GTP and to dissociate from the ⁇ -subunits.
- the GTP -bound form of the ⁇ -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 G ⁇ - GTP of many catalytic cycles of the effector.
- the family of regulatory G proteins comprises a multiplicity of different ⁇ - subunits (greater than twenty in man), which associate with a smaller pool of ⁇ - and ⁇ - subunits (greater than four each) (Strathmann and Simon, 1991).
- ⁇ -subunits greater than twenty in man
- ⁇ - and ⁇ - subunits greater than four each
- 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).
- rhodopsin, muscarinic, and ⁇ -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.
- opioids such as heroin and morphine exert their effects by mimicking naturally occurring substances, termed the endogenous opioid peptides or endorphins.
- endogenous opioid peptides or endorphins 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.
- 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.
- EOPs Endogenous opioid peptides
- endorphin is used synonymously with EOP, but also refers to a specific endogenous opioid, ⁇ -endorphin.
- 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.
- N/OFQ receptor nociceptin/orphanin FQ receptor
- ORL-1 opioid receptor-like 1
- orphan opioid receptor-like 1
- OP opioid receptor-like 1
- 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 ⁇ ARK (Pei et al. 1995; Wang et al.
- Respiratory depression and miosis may be less severe than with ⁇ agonists.
- P agonists instead of euphoria, P agonists produce dysphoric and psychotomimetic effects (Pfeiffer et al. 1986).
- ⁇ and agonists In neural circuitry mediating both reward and analgesia, ⁇ and 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 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.
- analgesia In human beings, mo hine-like drugs produce analgesia, drowsiness, changes in 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.
- Morphine does not have anticonvulsant activity and usually does not cause slurred speech, emotional lability , or significant motor incoordination.
- Analgesia The relief of pain by mo ⁇ hine-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.
- 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
- 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.
- T 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 withdrawal.
- 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.
- 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.
- NMDA N-methyl-D-aspartate
- 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.
- NOS inhibitors are effective against tolerance to morphine and ⁇ agonists such as DPDPE, they have little effect against tolerance to the 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.
- 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.
- opium is obtained from the unripe seed capsules of the poppy plant, Papaver somniferum.
- 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.
- alkaloids can be divided into two distinct chemical classes, phenanthrenes and benzylisoquinolines.
- 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%).
- 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.
- morphine 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.
- 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.
- 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 is a synthetic codeine analogue that is a weak ⁇ opioid agonist. Part of its analgesic effects are produced by inhibition of norepinephrine and serotonin reuptake. Tramadol appears to be as effective as other weak opioids. In the treatment of mild to moderate pain, tramadol is as effective as morphine or meperidine.
- Tramadol is as effective as meperidine in the treatment of labor pain and may cause less neonatal respiratory depression.
- Heroin is rapidly hydrolyzed to 6- monoacetylmorphine (6-MAM), which, in turn is hydrolyzed to mo ⁇ hine. Both heroin and 6-MAM are more lipid soluble than mo ⁇ hine and enter the brain more readily.
- Levorphanol Levo ⁇ hanol (LENO-DROMORAN) is the only commercially available opioid agonist of the mo ⁇ hinan series.
- the d-isomer (dextro ⁇ han) is relatively devoid of analgesic action, but may have inhibitory effects at NMDA receptors.
- the pharmacological effects of levo ⁇ hanol closely parallel those of mo ⁇ hine. However, clinical reports suggest that it may produce less nausea and vomiting.
- the nonanalgesic isomer dextro ⁇ han possesses considerable antitussive activity.
- Meperidine and congeners Meperidine and congeners. Meperidine and some of its congeners are 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 meperidine include diphenoxylate and loperaminde.
- Fentanyl, Sufentanil Citrate and congeners are synthetic opioids related to the phe-nylpiperidines.
- Fentanyl is a ⁇ agonist and is about- 100 times as potent as mo ⁇ hine as an analgesic.
- the actions of fentanyl and its congeners are similar to those of other ⁇ agonists.
- Fentanyl is a popular drug in
- 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 alfentanil and remifentanil have a more rapid onset of analgesic action, with onset of analgesic effects paralleling the of 1-1.5 minutes.
- alfentanil is metabolized similarly to fentanyl and sufentanil, with a TJ ⁇ k e o 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 long lasting ⁇ agonist with pharmacological properties qualitatively similar to those of mo ⁇ hine.
- 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.
- LAAM Levoraethadyl acetate
- Propoxyphene is structurally related to methadoner Its analgesic effect resides in the dextro isomer, J-propoxyphene (dextropropoxyphene). However, levopropoxyphene seems to have some antitussive activity. Although slightly less selective than mo ⁇ hine, propoxyphene binds primarily to ⁇ -opioid receptors and produces analgesia and other CNS effects that are similar to those seen with mo ⁇ hine- 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.
- Opioid Agonists differ from mo ⁇ hine clinically-used ⁇ agonists in that they are not full agonists at all opioid receptor populations. However, they can be used in conjunction with the MAPK 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 nalo ⁇ hine, cyclazocine, and nalbuphine and buto ⁇ hanol 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 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 mo ⁇ hine-like opioids, including analgesia, sedation, and respiratory depression. The analgesic effects of pentazocine are due to agonistic actions at ⁇ opioid receptors.
- Nalbuphine is related structurally to both naloxone and oxymo ⁇ hone. 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.
- Buto ⁇ hanol is a mo ⁇ hinan congener with a profile of actions similar to those of pentazocine. In postoperative patients, a parenteral dose of 2 to 3 mg of buto ⁇ hanol produces analgesia and respiratory depression approximately equal to that produced by 10 mg of mo ⁇ hine or 80 mg of meperidine; the onset, peak, and duration of action are similar to those that follow the administration of mo ⁇ hine. The plasma half-life of buto ⁇ hanol is about 3 hours.
- Buprenorphine Bupreno ⁇ hine is a semisynthetic, highly lipophilic opioid derived from thebaine. It is 25 to 50 times more potent than mo ⁇ hine. Bupreno ⁇ hine produces analgesia and other CNS effects that are qualitatively similar to those of mo ⁇ hine, and is used in the management of heroine addicts.
- Meptazinol dezocine
- nalo ⁇ hine nalo ⁇ han
- nalmefene any administration of an opioid may be in combination with a NSATDs.
- Opioid antagonists and other drugs used to tread opioid overdose can be used in conjunction with the MAPK 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.
- MAPKs Mitogen-activated protein kinases
- ERKs Extracellular signal Regulated Kinases
- SEQ ID NO: 1 ERK1
- SEQ ID NO: 3 ERK2
- Other protein kinases have been identified including SAPK or JNK, p38 MAPK, ERK3, ERK5 and ERK6.
- SAPK or JNK ERK1
- ERK2 Extracellular signal Regulated Kinases
- Other protein kinases have been identified including SAPK or JNK, p38 MAPK, ERK3, ERK5 and ERK6.
- SAPK or JNK p38 MAPK
- ERK3, ERK5 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.
- 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 MEK1/2, SEK1 and MKK3/6.
- ERK extracellular signal regulated kinase
- ERKs 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 ERK regulation have been realized, and include the use of classic second messengers such as cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) diacylglycerol (DAG) and calcium (Grewal et al, 1999).
- cAMP cyclic adenosine monophosphate
- PKA protein kinase A
- DAG diacylglycerol
- calcium Gibrewal et al, 1999.
- the ERK 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 ERK.
- cytoplasmic and nuclear targets such as nuclear transcription factors
- ERK nuclear transcription factors
- At the core of this system is a module of three kinases that phosphorylate each other sequentially - Raf, MEK, 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 al.1996), and all of the putative opioid signaling mediators have been shown to interact with the ERK cascade. Gutstein et al.
- ERK activation can occur independently of receptor internalization (Kramer et ⁇ /.2000), and may desensitize the opioid receptor (Polakiewicz et al. 1998). Relatively little work has been done characterizing the effects of chronic opioids on ERK activation in vitro or in vivo, and no prior studies have examined the involvement of ERK in tolerance and dependence at the behavioral level.
- acute mo ⁇ hine administration did not change ERK activity in the VTA, while chronic mo ⁇ hine induced modest increases in activity (Nestler et al.1996).
- chronic mo ⁇ hine suppressed ERK activation in many relevant brain regions, and precipitated withdrawal markedly stimulated ERK activation (Schulz et al.1998).
- ERK 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).
- CREB and AP-1 binding proteins c-fos, c-jun.
- G protein signaling RGS
- RGS proteins 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.
- this class of RGS proteins could 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).
- 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 Rapl are dictated by the expression of B-Raf. Without B-Raf, Rapl 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.
- MEK is a dual-specificity kinase that phosphorylates the tyrosine and threonine residues on ERK required for activation.
- Two related genes encode MEK1 (SEQ ID NO:5) 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.
- 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/Map/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
- 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, polymo ⁇ hic 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, 5 and 7, and biologically functional equivalents thereof are used in the current invention.
- 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.
- 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, polymo ⁇ hic or mutant MAPK coding sequences yet is isolated away from, or purified free from, total mammalian genomic DNA. Included within the tenn "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.
- 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.
- 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.
- the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating 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 NO:2, corresponding to the MAPK designated "human MAPK".
- sequence essentially as set forth in SEQ ID NO:2 means , for example, that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2. This applies with respect to SEQ ID NO:2.
- biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have about 70%, about 11%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%o, 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 NO:2, 4, 6 and 8 will be sequences that are "essentially as set forth in SEQ ID NO:2, 4, 6 and 8", provided the biological activity of the protein is
- 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.
- 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.
- 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 NO:l, 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:l, 3, 5 or 7, respectively and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:l, 3, 5 or 7, respectively.
- DNA segments that encode proteins, polypeptide or peptides exhibiting MAPK activity will be most preferred.
- 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.
- the codons are shown in Table 1 in preference of use from left to. right.
- GCC codon for alanine
- GCG codon usage for various organisms and organelles can be found at the website http://www.kazusa.or.jp/codon/, inco ⁇ orated herein by reference, allowing one of skill in the art to optimize codon usage for expression in various organisms using the disclosures herein.
- 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 mitochondria or chloroplasts, based on the preferred codon usage as would be known to those of ordinary skill in the art.
- a prokaryote e.g., an eubacteria, an archaea
- an eukaryote e.g., a protist, a plant, a fungi, an animal
- organelles that contain nucleic acids, such as mitochondria or chloroplasts
- amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, 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.
- 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.
- nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarily rules.
- 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.
- hybridization 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).”
- stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) 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 NaCl 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 solvent(s) in a hybridization mixture.
- hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions.
- 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 55°C.
- hybridization may be achieved under conditions of, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 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 KCl, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
- 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.
- nucleic acid segments of the present invention 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.
- 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.
- intermediate lengths means any length between the quoted ranges, such as 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 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.
- nucleic acid segments may be designed based on a particular nucleic acid sequence, and may be of any length.
- an algorithm defining all nucleic acid segments can be created: nto n + 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.
- the nucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and/or so on.
- nucleic acid segments correspond to bases 1 to 15, 2 to 16, 3 to 17 ...
- the nucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and/or so on.
- the nucleic acid segment may be a probe or primer.
- a probe generally refers to a nucleic acid used in a detection method or composition.
- a primer generally refers to a nucleic acid used in an extension or amplification method or composition.
- 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.
- 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.
- the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
- the test DNA or RNA
- the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
- This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
- the selected conditions will 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.).
- hybridization is detected, or even quantified, by means of the label.
- 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.
- the RNA is whole cell RNA and is used directly as the template for amplification.
- primers that selectively hybridize to nucleic acids corresponding to MAPK 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.
- 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.
- the amplification product is detected.
- the detection may be performed by visual means.
- the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of inco ⁇ orated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).
- PCRTM polymerase chain reaction
- PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
- An excess of deoxynucleoside friphosphates 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.
- 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, inco ⁇ orated herein by reference. Polymerase chain reaction methodologies are well known in the art.
- LCR ligase chain reaction
- Qbeta Replicase described in PCT Application No. PCT/US87/00880, inco ⁇ orated herein by reference, may also be used as still another amplification method in the present invention.
- 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 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)
- RCR Repair Chain Reaction
- SDA Strand Displacement Amplification
- RCR Repair Chain Reaction
- Target specific sequences can also be detected using a cyclic probe reaction (CPR).
- 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.
- 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.
- 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).
- a capture moiety e.g., biotin
- a detector moiety e.g., enzyme
- an excess of labeled probes are added to a sample.
- 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.
- 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, inco ⁇ orated herein by reference).
- TAS transcription-based amplification systems
- NASBA nucleic acid sequence based amplification
- 3SR Genomerase binding amplification
- 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.
- 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.
- 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.
- an RNA polymerase such as T7 or SP6.
- 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 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).
- RNA reverse transcriptase
- 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).
- RNase H ribonuclease H
- 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 "Klenow" 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 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, inco ⁇ orated herein by reference).
- 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 hybridization.
- appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
- enzyme 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.
- nucleic acids are amplified, it may be desirable to separate the amplification product from the template and the excess primer for the pu ⁇ ose of determining whether specific amplification has occurred.
- amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989).
- chromatographic techniques may be employed to effect separation.
- chromatography There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
- 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 UV light.
- the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
- visualization is achieved indirectly.
- 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.
- 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.
- detection is by Southern blotting and hybridization with a labeled probe.
- the techniques involved in Southern blotting are well known to those of 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.
- DGGE denaturing gradient gel electrophoresis
- restriction enzyme polymo ⁇ hism analysis restriction enzyme polymo ⁇ hism analysis
- chemical and enzymatic cleavage methods and others.
- the more common procedures currently in use include direct sequencing of target regions amplified by PCRTM (see above) and single-strand conformation polymo ⁇ hism analysis (“SSCP").
- SSCP single-strand conformation polymo ⁇ hism analysis
- Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA RNA heteroduplexes.
- 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, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
- 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 sufficiently 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.
- the templates for in 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.
- 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.
- 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.
- Cloning MAPK Genes 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 are 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), inco ⁇ orated herein by reference.
- suitable genomic cloning methods as known to those in the art, may also be used.
- “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 NO:2, 4, 6 and 8.
- 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 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 then be designed from the region of the gene that encodes the conserved sequence of amino acids.
- MAPK proteinaceous material coding regions are aligned within the same expression unit with other proteins, polypeptides or peptides having desired functions, such as for purification or immunodetection pu ⁇ oses (e.g., proteinaceous compostions that may be purified by affinity chromatography and enzyme label coding regions, respectively).
- proteins, polypeptides or peptides having desired functions such as for purification or immunodetection pu ⁇ oses (e.g., proteinaceous compostions that may be purified by affinity chromatography and enzyme label coding regions, respectively).
- 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 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 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 NO:2, 4, 6 and 8 and also larger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO:2, 4, 6 and 8, and any range derivable therein and any integer derivable therein such a range.
- 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.
- 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.
- expression includes both transcription of a MAPK gene and translation of a RNA into the MAPK gene product.
- 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.
- 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 transcriptional 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 inco ⁇ orated herein by reference).
- 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.
- 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), inco ⁇ orated 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.
- promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp 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.
- a human cell 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.
- a promoter might include either a human or viral promoter.
- 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.
- CMV cytomegalovirus
- 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 pu ⁇ ose.
- Tables 3 and 4 below list several elements/promoters 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.
- enhancers 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.
- Eukaryotic Promoter Data Base EPDB http://www.epd.isb-sib.ch/
- any promoter/enhancer combination 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 bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
- 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 segment(s) 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.
- 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.
- polyadenylation signal 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 the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells.
- a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
- an antisense nucleic acid can be used as an ERK inhibitor.
- the term "antisense nucleic acid” is intended to refer 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 nucleic acid and interfere with transcription, RNA processing, transport and/or translation. Targeting double-sfranded (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 NO: 1, 3, 5 or 7, complementary to an ERK encoding sequence or to ERK non-coding sequences.
- 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.
- 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.
- genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
- 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.
- 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 in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization.
- oligonucleotide 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 in vitro to determine whether the endogenous gene's function is affected or whether the expression of related genes having complementary sequences is affected.
- 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).
- targeted ribozymes may be used.
- 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 inco ⁇ orating 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 (Kim 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.
- IGS internal guide sequence
- Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
- 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.
- sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon etal, 1991; Sarver et al, 1990; Sioud etal, 1992).
- RNA cleavage activity examples 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.
- ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al, 1992; Yuan and Altman, 1994), hai ⁇ in ribozyme structures (Berzal-Herranz etal, 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).
- Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are 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 are separated by a specific sequence which is the cleavage site.
- 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 are 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 target 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 inco ⁇ orated by reference. The identification of operative and preferred sequences for use in MAPK-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.
- 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 co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of either of the respective DNA.
- 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.
- the term "co-expression” herein refers to the expression of both the MAPK gene and the other selected proteinaceous molecules in the same recombinant cell.
- 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 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 adjacent to a promoter not naturally associated with the particular introduced gene.
- an expression vector that comprises a wild-type, or mutant MAPK proteinaceous, molecule-encoding nucleic acid under the control of one or more promoters.
- 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.
- 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.
- prokaryotic hosts are E. coli strain RR1, E. coli L ⁇ 392, E. coli , E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, protofrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.
- 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.
- 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.
- 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.
- 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.
- pIN vectors Inouye et al, 1985
- pGEX vectors for use in generating glutafhione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
- GST glutafhione S-transferase
- Other suitable fusion proteins are those with ⁇ -galactosidase, ubiquitin, and the like.
- Promoters that are most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase), lactose and tryptophan (tip) 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.
- ⁇ -lactamase penicillinase
- lactose lactose
- tip tryptophan
- Bacterial cells for example, E. coli, containing the expression vector are grown in any of a number of suitable media, for example, LB.
- 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 inco ⁇ oration of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.
- 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 ⁇ -mercaptoethanol or DTT (dithiothreitol).
- urea e.g. 8M
- chaotropic agents such as guanidine hydrochloride
- reducing agents such as ⁇ -mercaptoethanol or DTT (dithiothreitol).
- 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).
- 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.
- 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 -phosphogly cerate 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.
- 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.
- 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.
- cultures of cells derived from multicellular organisms may also be used as hosts.
- any such cell culture is workable, whether from vertebrate or invertebrate culture.
- 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.
- 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
- metallothionein promoter e.g., metallothionein promoter
- mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
- 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 250 bp sequence extending from the HindTTT site toward the Bgll site located in the viral origin of replication.
- 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 El, 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 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.
- polyadenylation site e.g., 5 -AATAAA-3'
- 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.
- a recombinant MAPK protein, polypeptide or peptide For long-term, high-yield production of a recombinant MAPK protein, polypeptide or peptide, stable expression is preferred.
- cell lines that stably express constructs encoding an MAPK protein, polypeptide or peptide may be engineered.
- host cells can be transformed with vectors controlled by appropriate expression confrol elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are 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 he ⁇ es simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) genes, in tk “ , hgprt " or aprt " cells, respectively.
- tk simplex virus thymidine kinase
- hgprt hypoxanthine-guanine phosphoribosyltransferase
- aprt adenine phosphoribosyltransferase
- antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), that confers resistance to methofrexate; gpt, that confers resistance to mycophenolic acid; neomycin (neo), that confers resistance to the aminoglycoside G-418; and hygromycin (hygro), that confers resistance to hygromycin.
- dhfr dihydrofolate reductase
- gpt that confers resistance to mycophenolic acid
- neomycin that confers resistance to the aminoglycoside G-418
- hygromycin hygro
- Animal cells can be propagated in 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.
- suspension cultured cells have limitations, such as tumorigenic potential and lower proteinaceous molecule production than adherent cells.
- 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.
- the MAPK proteins, polypeptides or peptides of the invention may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in cells.
- 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.
- the mammalian MAPK genes are inco ⁇ orated 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 fransduction using infectious viral particles, for example, by transformation with an adenovirus vector of the present invention as described herein below.
- retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest.
- viral infection of cells is used in order to deliver therapeutically significant genes to a cell.
- the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus.
- adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below.
- a particular method for delivery of the expression constructs for MAPK inhibition involves the use of an adenovirus expression vector.
- 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 sufficient 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.
- 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).
- 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.
- 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.
- ITRs inverted repeats
- the early (E) and/or late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
- the El region (El A and/or EIB) encodes proteins responsible for the regulation of transcription of the viral genome and/or a few cellular genes.
- the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
- MLP major late promoter
- TPL 5'-tripartite leader
- 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 El proteins (El A and/or EIB; Graham etal, 1977).
- 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.
- 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 are monkey embryonic mesenchymal and/or epithelial cells.
- Racher et al. (1995) disclosed improved methods for culturing 293 cells and/or propagating adenovirus.
- 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 ⁇ m, the cell viability is estimated with trypan blue.
- Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
- the medium is then replaced with 50 ml of fresh medium and/or shaking initiated.
- 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.
- 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.
- the typical vector according to the present invention is replication defective and/or will not have an adenovirus El region.
- it will be most convenient to introduce the transforming construct at the position from which the El - coding sequences have been removed.
- 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 lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson 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 in vivo.
- This group of viruses can be obtained in high titers, e.g., 10 9 to 10 ⁇ 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 low 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).
- trachea instillation Rosenfeld et al, 1991; Rosenfeld et al, 1992
- muscle injection Rogot et al, 1993
- peripheral intravenous injections Herz and/or Gerard, 1993
- stereotactic inoculation into the brain Le Gal La Salle et al, 1993
- Recombinant adenovirus and/or adeno-associated virus can both infect and/or transduce non-dividing hyman primary cells.
- Adeno-associated virus is an attractive vector system for use in the cell fransduction 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 in 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 inco ⁇ orated herein by reference.
- AAV vectors have been used successfully for in vitro and/or in vivo fransduction of marker genes (Kaplitt et al, 1994; Lebkowski et al, 1988; Samulski et al, 1989; Yoder et al, 1994; Zhou et al, 1994; Hermonat and/or 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 he ⁇ es virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992).
- another virus either adenovirus and/or a member of the he ⁇ es virus family
- 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 is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and/or Smith, 1994).
- 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 etal, 1988; Kotin etal, 1990; Muzyczka, 1992).
- 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 inco ⁇ orated 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; inco ⁇ orated 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).
- 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, 99 Clark et al, 1995).
- Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al, 1995).
- Refroviruses 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 refroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-sfranded 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 genome 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).
- 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.
- a packaging cell line containing the gag, pol, and/or env genes but without the LTR and/or packaging components is constructed (Mann et al, 1983).
- 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).
- HSV he ⁇ es simplex virus
- HSV Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, inco ⁇ oration 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, strength, etc.) makes it possible to confrol 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 and in a lessened need for repeat dosings.
- HSV as a gene therapy vector, see (Glorioso et al, 1995).
- HSV HSV
- 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-sfranded DNA genome encodes for dozens of different gene products, some of which derive from spliced transcripts.
- 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 ⁇ genes is enhanced by the virion protein number 16, or ⁇ - fransducing factor (Post et al, 1981; Batterson and Roizman, 1983; Campbell et al, 1983).
- the expression of ⁇ genes requires functional a gene products, most notably ICP4, which is encoded by the ⁇ 4 gene (DeLuca et al, 1985).
- ⁇ 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).
- HSV 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).
- Lentiviral Vectors are complex refroviruses, which, in addition to the common retroviral genes gag, pol, and env, 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, vi vpr, vpu and nef ve deleted making the vector biologically safe.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in 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 refroviruses: 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 franscriptase), a protease and an integrase; and the env 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 vi vpr, tat, rev, 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 ⁇ l., (1996); Zufferey et ⁇ l.,
- the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for inco ⁇ orating 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
- 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, inco ⁇ orated 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 provide a nucleic acid encoding a viral env to produce a packaging cell.
- Infroducing 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 env preferably is an amphotropic envelope protein which allows fransduction of cells of human and other species.
- 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 murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine 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.
- 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 fransgene.
- 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, methofrexate, 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.
- 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, Gin 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.
- Vaccinia 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-sfranded 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 majority 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.
- Prototypical vaccinia vectors contain fransgenes 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 fransgenes accumulating at 10% or more of the infected cell's protein in 24 h (Elroy- Stein et fl/., 1989).
- the empty capsids of papovaviruses have received attention as possible vectors for gene transfer (Barr 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 Fill 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 VPl, could be used in genetic transfer.
- viral vectors may be employed as expression constructs in the present invention.
- Vectors derived from viruses such as Sindbis virus and/or cytomegalovirus. They offer several attractive features for various mammalian cells (Friedmann, 1989;
- Chang et al. recently introduced the chloramphenicol acetylfransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and/or pre-surface coding sequences. It was cotransfected 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).
- CAT chloramphenicol acetylfransferase
- 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 refrovirus vectors was recently developed based on the chemical modification of a refrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
- 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 ⁇ e 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 inco ⁇ orated herein by reference), including microinjection (Harlan and Weinfraub, 1985; U.S. Patent No. 5,789,215, inco ⁇ orated herein by reference); by electroporation (U.S. Patent No.
- the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites.
- 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).
- 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.
- 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).
- 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.
- 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.
- 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).
- HVJ hemagglutinating virus
- the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et ⁇ /., 1991).
- HMG-1 nuclear nonhistone chromosomal proteins
- 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 in vivo, then they are applicable for the present invention.
- receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells. 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.
- 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).
- ASOR asialoorosomucoid
- transferring Wang and Wu, 1990
- the delivery vehicle may comprise a ligand and a liposome.
- a ligand and a liposome For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
- 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.
- 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.
- 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 for in vivo use as well.
- Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of CaPO 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 CaPO 4 precipitated plasmids results in expression of the fransfected 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 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, 1987). 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 biologically inert substances such as tungsten or gold beads.
- the present invention concerns novel compositions or methods comprising at least one proteinaceous molecule.
- the proteinaceous molecule may be a MAPK such as ERKl (SEQ ID NO: 2) or ERK2 (SEQ ID NO:4), MEKl (SEQ ID NO: 6) or MEK2 (SEQ ID NO: 8).
- 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 composition for the delivery of a therapeutic agent or as part of a screening assay in the determination of MAPK inhibition.
- 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 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.
- 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 11, 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,
- 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.
- the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
- the sequence may comprise one or more non-amino molecule moieties.
- the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
- 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 5 below.
- the proteinaceous composition comprises at least one protein, polypeptide or peptide, such as a MAPK.
- the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
- 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.
- biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or 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://www.ncbi.nlm.nih.gov/).
- Genbank and GenPept databases http://www.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.
- various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
- a proteinaceous compound may be purified.
- 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.
- 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 tissue(s) and foster tighter adhesion of the glue to the tissues after welding.
- 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.
- 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') , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
- DABs single domain antibodies
- Fv single chain Fv
- scFv single chain Fv
- the techniques for preparing and using various antibody-based constructs and fragments are well known in 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; inco ⁇ orated herein by reference).
- any protein, polypeptide or peptide containing component may be used in the compositions and methods disclosed herein.
- the proteinaceous material is biocompatible.
- 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.
- the use 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 100 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.
- 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, or a prokaryotic organism, with a selected animal or human subject being preferred.
- 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.
- the autologous proteins or peptides are prepared, for example from whole plasma of the selected donor. The plasma is placed in tubes 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 15 minutes to obtain the precipitate. The precipitate, such as f ⁇ brinogen may be stored for up to about one year (Oz, 1990).
- a composition comprising the MAPK inhibitor, or more preferentially, the ERK inhibitor it may be desirable to purify the components or variants thereof.
- purification of a peptide 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 further purified using 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; isoelecfric focusing.
- a particularly efficient method of purifying peptides is fast protein liquid chromatography or even 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.
- purified protein or peptide as used herein, is intended to 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.
- 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.
- substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, 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.
- 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.
- High Performance Liquid Chromatography 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 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.
- the sole factor determining rate of flow is the size.
- Gel chromatography is unsu ⁇ assed 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 adso ⁇ tion, 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 contaimng 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 agarose 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.
- affinity chromatography 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.
- the present invention also describes a MAPK 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, Stewart and Young, (1984); Tam et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each inco ⁇ orated 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.
- 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, inco ⁇ orated 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.
- 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.
- 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 MAPK that may occur. It is contemplated that such screening techniques will prove useful in the general identification of any compound that will serve the pvupose of modulating MAPK, or more particularly ERK activity.
- MAPK modulators identified will have utility in methods involved in serine 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 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.
- useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds.
- 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.
- MAPK modulator using a MAPK protease inhibitor assay
- protease inhibitor assay 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.
- 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.
- 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.
- 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.
- 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.
- pharmaceutically 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, inco ⁇ orated herein by reference.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, abso ⁇ tion 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, 18fh Ed. Mack Printing Company, 1990, pp. 1289-1329, inco ⁇ orated herein by reference).
- preservatives e.g., antibacterial agents, antifungal agents
- isotonic agents e.g., abso ⁇ tion delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents
- 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, inco ⁇ orated 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 ingredient(s) in a composition and appropriate dose(s) for the individual subject.
- compositions may comprise, for example, at least about 0.1% of an active compound.
- 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.
- a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram kg/body 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.
- a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
- the composition may comprise various antioxidants to retard oxidation of one or more component.
- 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, fhimerosal or combinations thereof.
- parabens e.g., methylparabens, propylparabens
- chlorobutanol phenol
- sorbic acid fhimerosal 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 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, frimethylamine, histidine or procaine.
- 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, for 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.
- isotonic agents such as, for example, sugars, sodium chloride or combinations thereof.
- Sterile injectable solutions are prepared by inco ⁇ orating 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.
- dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
- 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 DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
- 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.
- prolonged abso ⁇ tion of an injectable composition can be brought about by the use in the compositions of agents delaying abso ⁇ tion, such as, for example, aluminum monostearate, gelatin or combinations thereof.
- opioids in the epidural or infrathecal 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.
- epidural opioids have their own dose-dependent side effects, such as itching, nausea, vomiting, respiratory depression, and urinary retention.
- hydrophilic opioids such as preservative-free mo ⁇ hine (DURAMORPH) permits more rostral spread of the compound, allowing it to directly affect supraspinal sites.
- the MAPK inhibitor can be used in combination with another agent, preferably an opioid.
- the MAPK inhibitor may precede or follow the other agent freatment by intervals ranging from minutes to weeks.
- 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.
- 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.
- 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 19 hours, about 20 hours, about 21 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.
- 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 15 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.
- the MAPK inhibitor is "A” and the secondary agent, such as an mo ⁇ hine, is "B":
- 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 hype ⁇ roliferative cell therapy.
- Opioid analgesics that can be used in conjugation with the MAPK inhibitor include, but are not limited to mo ⁇ hine, mo ⁇ hine sulphate, tramadol, codeine, levo ⁇ hanol, meperidine and congeners such as diphenoxylate and loperaminde, sufentanil citrate and congeners such as alfentanil and remifentanil, methadone and congeners, levomethadyl acetate (LAAM), propoxyphene, buto ⁇ hanol, eptazocine, fentanyl, fentanyl citrate, flupirtine, hydromo ⁇ hone and oxycodone,.
- LAAM levomethadyl acetate
- opioid compounds that may be used include, but are not limited to pentazocine, nalbuphine, buto ⁇ hanol, bupreno ⁇ hine, meptazinol, dezocine, nalo ⁇ hine, levallo ⁇ han and nalmefene, mo ⁇ hine-6-glucuronide, mo ⁇ hine (DepoMo ⁇ hine, AERx Pain Management System, Multipor technology), mo ⁇ hine sulphate, pulmonary-delivered mo ⁇ hine sulphate, and other mo ⁇ hine-like compounds including conorfone, propiram fumarate, various strength opioid analgesics using OROS technology, various strength analgesics using Geomatrix technology, fentanyl, AERx Pain Management System, bupreno ⁇ hine, asimadoline, TRK-820, LEF (BCH-3963), loperamide, oxycodone and oxycodone combinations (i.e.
- oxycodone + ibuprofen or oxycodone + paracetamol DPI-3290, ADL-10-0101, Xo ⁇ hanol, TSN-09, and a combination of NMDA antagonist and an opioid compound, (i.e. dextrometho ⁇ han + hydrocodone, dextrometho ⁇ han + mo ⁇ hine and dextrometho ⁇ han + oxycodone + paracetamol) ("Advances in Pain Management" February 2000 Scrip Reports).
- 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 chromatographic methods.
- Preferred ERK inhibitors contemplated as useful in this invention include, but are not limited to:
- MAPK inhibitors contemplated as useful in this invention include:
- 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- (4methylsulfmylphenyl)-5- (4- ⁇ yridyl)-lH- imidazole which is a p38 MAPK inhibitor from Smi hKline Beecham, and
- a or “an” may mean one or more.
- the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
- another may mean at least a second or more.
- 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.
- the cells were washed twice with control media, and then placed in control media containing IM 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- ERK antibody followed by enhanced chemifluorescent detection. Data were analyzed by one-way ANOVA followed by the Dunnett post-hoc test, with p ⁇ 0.05 considered significant.
- Sprague- Dawley rats had infrathecal 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 (V/U0126) Initially, one 75 mg mo ⁇ hine (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 ANOVA and the Tukey-Kramer post-hoc test with p ⁇ 0.05 considered significant.
- FIG. 3 demonstrates robust ERK activation in the hypothalamic paravenfricular nucleus 1 hour after osmotic stress.
- RGS4 chronic mo ⁇ hine administration on RGS4 mRNA levels.
- RGS4 was chosen because of its expression almost exclusively in brain and association with Gj subunits.
- a paradigm was employed that also produces behavioral sensitization to the locomotor effects of mo ⁇ hine. 10 mg/kg mo ⁇ hine 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 RGS 4 mRNA was observed in the dorsal cenfral gray (CGD; FIG. A), an area associated with analgesic responses.
- CCD dorsal cenfral gray
- Extracellular signal-related kinase is a member of the mitogen-activated protein kinase(MAPK) superfamily, which are involved in cell regulation.
- MAPK mitogen-activated protein kinase
- ERK activation by opioids exhibit both acute and chronic desensitization, suggesting a potential role for ERK in opioid tolerance and dependence.
- the present study determined whether inhibition of ERK by UO126 would affect opioid tolerance development. Methodology: 21 male Sprague-Dawley rats had infrathecal catheters implanted under halothane anesthesia and recovered for 1 week.
- 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 K (1 ⁇ g/ml in 100 mM tris-HCl, ⁇ H+8.0) for 10 min at 37°C. Slides were then washed in 0.1M 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 ⁇ g/ml in 10 mM Tris-HCl, 0.5 M NaCl, ⁇ H-8.0) for 60 min at 37°C and subsequently washed in 2X, IX, 0.5X, and 0.25X SSC for 5 min each, and then 0.1X SSC for 60 min at 60°C.
- RnaseA 200 ⁇ g/ml in 10 mM Tris-HCl, 0.5 M NaCl, ⁇ H-8.0
- Acute treatment with mo ⁇ hine, 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 (mo ⁇ hine 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 mo ⁇ hine and cocaine treatment, but not amphetamine (see Table 6).
- 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 al.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-801 added to the media daily.
- tolerance will be induced in rats by injecting 10 mg/kg mo ⁇ hine (saline control) i.p. for 10 days as described (Trujillo et al. 1991). 30 min. prior to mo ⁇ hine 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 confrol. On day 10, withdrawal will be precipitated 1 hr. after mo ⁇ hine 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.
- VTA tolerance and dependence
- NAcc NAcc
- Arc Amy
- PAG NTS
- LC LC
- RVM lumbar spinal cord dorsal horn
- 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.
- Example 9 Effects of ERK Inhibition and Gene Disruption on Tolerance and Dependence In Vivo.
- 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.
- ISH in situ hybridization
- RGS proteins from 4 relevant families Ross et ⁇ /.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.
- RGS 9 and 11 will be examined due to their limited but relevant anatomical distribution (sfriatum/Nacc and LC, respectively).
- RGS 12 has been chosen from the "scaffolding" subfamily, which appears selective for G 0 .
- G ⁇ z subunit could mediate opioid tolerance (Kelleher et al.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 ERK could be involved, and relevant signaling modulators are available if needed.
- 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 ERK inhibition can block the development of hyperalgesia. Animals will receive infrathecal (i.t.) catheters and allowed to recover. The animals will then undergo implantation of primed osmotic minipumps infusing either SL-327/vehicle, 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).
- PWP paw withdrawal pressure
- the second study will determine the time course of ERK activation after CFA injection. Under brief halo hane anesthesia, animals will be injected with either CFA or saline in the right hindpaw and lumbar spinal cords processed for ICC 2, 5, 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.
- animals will undergo implantation of one mo ⁇ hine (or placebo) sustained release pellet.
- a primed osmotic minipump infusing mo ⁇ hine (or placebo) will be implanted.
- 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.
- ERK activation after CFA is sustained, it will be intriguing to see whether the effect of mo ⁇ hine on 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 ERK 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 mo ⁇ hine administration.
- Banerji et al Cell, 27:299, 1981. Banerji et al, Cell, 35:729, 1983.
- Nicolas et al In: Vectors: A survey of molecular cloning vectors and their uses, Stoneham: Butterworth, 494-513, 1988. Nicolau et al, Biochim. Biophys. Acta, 721:185-190, 1982.
- Vasseur et al Proc. Natl. Acad. Sci, 77:1068, 1980.
- Wagner et al Proc. Natl. Acad. Sci, 87(9):3410-3414, 1990.
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EP02717370A EP1377279A2 (en) | 2001-01-25 | 2002-01-25 | Inhibition of erk to reduce or prevent tolerance to and dependence on opioid analgesics |
AU2002248381A AU2002248381A1 (en) | 2001-01-25 | 2002-01-25 | Inhibition of erk to reduce or prevent tolerance to and dependence on opioid analgesics |
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FR2837103A1 (en) * | 2002-03-15 | 2003-09-19 | Rytek | Use of compound that controls the opening of intestinal epithelium tight junctions for prevention and cure of hyperalgesic intestinal disorders |
WO2003077893A2 (en) * | 2002-03-15 | 2003-09-25 | Rytek | Compositions for treating digestive functional pathologies |
FR2844201A1 (en) * | 2002-09-06 | 2004-03-12 | Rytek | Use of compound that controls the opening of intestinal epithelium tight junctions for prevention and cure of hyperalgesic intestinal disorders |
US20100210709A1 (en) * | 2007-10-09 | 2010-08-19 | Howard Gutstein B | Methods of Treatment of Opioid Tolerance, Physical Dependence, Pain and Addiction With Inhibitors of Certain Growth Factor Receptors |
US8273708B2 (en) | 2004-12-09 | 2012-09-25 | Lionel Bueno | Methods of treatment of ocular surface pathologies |
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WO2015041533A1 (en) | 2013-09-20 | 2015-03-26 | Stichting Het Nederlands Kanker Instituut | Rock in combination with mapk-pathway |
WO2015156674A2 (en) | 2014-04-10 | 2015-10-15 | Stichting Het Nederlands Kanker Instituut | Method for treating cancer |
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WO2017058007A1 (en) | 2015-10-01 | 2017-04-06 | Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis | Histone deacetylase inhibitors for the use in the treatment of drug resistant melanoma |
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WO2002002097A2 (en) * | 2000-06-30 | 2002-01-10 | Sweatt J David | Methods for treating seizure disorders by inhibiting mapk pathway activation with nitriles |
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CA2435038A1 (en) | 2002-08-01 |
EP1377279A2 (en) | 2004-01-07 |
WO2002058687A3 (en) | 2003-10-09 |
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