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  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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  • C12N2310/3511—Conjugate intercalating or cleaving agent

Definitions

• the invention relates to antisense oligonucleotides for metabotropic glutamate receptor type 1 (mGluRi).
• Metabolic glutamate receptor type 1 (mGluRi), is a member of group I mGluRs and is positively coupled to phosphatidylinositol (PI) hydrolysis. Activation of this type of receptor ultimately leads to activation of protein kinase C (PKC) and increased concentrations of intracellular Ca 2+ , thereby causing initiation of cellular excitation.
• PKC protein kinase C
• Glutamate neurotoxicity has been shown to be involved in several models of ischemia. For example, in in vitro models mimicking conditions of hypoxia/ischemia and glucose deprivation in brain and spinal cord slices, it has been shown that glutamate release is increased, and this effect is attenuated by glutamate receptor antagonists (Nakai et al (1999) Eur J Pharmacol 366(2-3): 143-150; Kimura et al (1998) J Pharmacol Exp Ther 285(1): 178-185; Reyes et al (1998) Brain Res 782(1-2): 212-218). Neuronal damage in these models is attenuated by agents that inhibit glutamate release (Culmsee et al (1998) Eur J Pharmacol 342(2-3): 193- 201).
• PKC has also been implicated in both focal and global ischemic neuronal injury, and inhibition of PKC has been shown to attenuate measures associated with ischemic injury (Nakane et al (1998) Brain Res 782: 290-296; Sieber et al (1998) Stroke 29(7): 1445-1452). It has also been suggested that Ca 2+ is one of the triggers involved in ischemic cell death (Kristian and Siesjo (1998) Stroke 29: 705-718; Stys (1998) J Cerebral Blood Flow &Metabolism 18: 2-25).
• Glutamate has also been implicated in ocular ischemia associated with glaucoma, retinal ischemia due to central artery occlusion, anterior ischemic optic neuropathy, and possibly also optic neuritis, optic nerve trauma and AIDS (Sucher et al ( ⁇ 997)Vision Res 37: 3483-3493; Lagreze et al (1998) Investigative Ophthalmology & Visual Science 39: 1063-1066; Duarte et al (1998) Gen Pharmacol 30: 289-295). Actions at mGluRs may play an important role (Duarte et al (1998) supra). Moreover, inhibition of PKC (associated with activity at group I mGluRs) has been shown to significantly attenuate damage in a rat model of ocular ischemia (Hicks et al (1998) Gen Pharmacol 30: 265-273).
• Antagonists at NMDA receptors have been shown to ameliorate the extent of the infarction with cerebral ischemia (Lees (1 97) Neurology 49(5 suppl 4): S66-S69; Dyker and Lees (1999) Stroke 30: 986-992).
• CNS central nervous system
• brain and spinal cord The mechanisms of neuronal injury in central nervous system (CNS; brain and spinal cord) appear to be very similar to those observed in ischemia.
• glutamate excitotoxicity plays an important role in CNS trauma, as may be sustained by a blow to the head, spinal cord injury, whiplash, noise-induced hearing loss, etc.
• mGluRi antagonists afforded significant neuroprotection either alone, or in combination with NMDA receptor antagonists (Mukhin et al (1997) NeuroReport 8: 2561- 2566), whereas a group I mGluR agonist exacerbated neuronal injury.
• EAAs excitatory amino acids
• Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, mitochrondrial encephalomyopaythies, spinocerebellar degeneration syndromes, motor neuron diseases, and even schizophrenia have been linked to glutamate excitotoxicity (Massieu and Garcia (1998) Neurobiology (Budapest) 6: 99-108; Arias et al (1998) Neurobiology (Budapest) 6: 33-43; Bittigau and Ikonomidou
• mGluRi is positively coupled to PI hydrolysis, and activation of these receptors leads to the activation of protein kinase C (PKC). Knockdown of mGluRi has been shown to inhibit the activation of PKC (Fundytus et al (1999) Society for Neuroscience Abstracts 25: 449). Inhibition of PKC, with antisense oligonucleotides targeting PKC, has been shown to inhibit tumor growth (Dean et al (1996) Cancer Res 56: 3499-3507).
• a major problem associated with the treatment of advanced cancer is the control of pain. This difficulty is exacerbated by the fact that different types of pain such as somatic, visceral, incident and neuropathic are not all equally amenable to current analgesic therapies. Often, neuropathic pain is only partially relieved by the most common analgesics, opioids (Cherny et al (1994) Neurology 44: 857-861; MacDonald (1991) Recent Results in Cancer Res 121: 24- 35; McQuay et al (1992) Anesthesia 47: 757-767). Opioid therapy is problematic for 30% of cancer patients who experience pain, and of these, 2/3 have neuropathic pain. Currently, the most common analgesics are opioid drugs. However, administration of opioids is often associated with undesirable side effects such as sedation, respiratory depression, nausea, vomiting, constipation, pruritis, renal toxicity.
• Oligonucleotides complementary to, and designed to hybridize to, a target mRNA transcript via Watson-Crick base pairing are used. Formation of an oligonucleotide-mRNA duplex leads to mRNA inactivation and inhibition of protein synthesis.
• the present invention provides antisense oligonucleotides directed toward human mGluRi which are useful in the treatment of diseases and disorders involving glutamate and/or mGluRi activity imbalance, including but not limited to stroke, ischemia, pain, etc.
• An object of the present invention is to provide antisense oligonucleotides for metabotropic glutamate receptor type 1 (mGluRi).
• antisense oligonucleotides of human metabotropic glutamate receptor type I there is provided antisense oligonucleotides of human metabotropic glutamate receptor type I.
• AS treatment induced a 43.85% decrease of mGluRi protein compared to vehicle treatment in neuropathic rats, and a 39.22% decrease in mGluRi protein compared to vehicle treatment in sham-operated rats.
• the amount of mGluRi protein in lumbar spinal cord from MS-treated rats was not decreased (and even appeared to be increased) from vehicle-treated rats.
• AS treatment induced a 24.62% decrease in neuropathic rats, and a 17.80% decrease in mGluRi protein in sham- operated rats.
• Figure 3 Nerve-injury induced hyperalgesia and allodynia.
• B Cold water test in post-treatment group Mean increase in number of responses after nerve injury, both prior to i.t. infusion (day 4), and after i.t. infusion (days 8, 12 and 18).
• D von Frey hair test in post-treatment group Mean percent decrease in 50% response threshold in grams. On day 4 after nerve injury, prior to i.t. drug infusion, all neuropathic rats showed a large decrease in 50% response threshold.
• AS-treated neuropathic rats had a significantly lower decrease in response latency from days 8 to 18 after nerve injury.
• Figure 5 [3H]PDBu binding.
• FIG. 10 Inflammation (CFA)-induced hyperalgesia and allodynia. Change in response to heat (A, B) and mechanical (C, D) stimulation of the CFA injected and contralateral hindpaws on days 1 to 8 after CFA injection.
• CFA Inflammation
• C Von-Frey test in pre-treated group Mean percent decrease in 50% response threshold in ACSF-, AS- and MS-treated rats.
• Post-hoc Fisher's LSD t-tests indicated that the decrease in 50% response threshold in the injected hindpaw was attenuated in AS-treated rats, compared to ACSF- and MS-treated rats.
• FIG. 11 Western Blot Analysis. Sample Western blots and histogram summary results from Western blot analysis of lumbar spinal cords taken from naive rats (no treatment), and ACSF-, AS- and MS-treated CFA-injected rats after 7 days of oligonucleotide infusion. A Peak binding density of mGluRi IgG in lumbar spinal cords taken from naive, and ACSF-,
• Post-hoc Fisher's LSD t-tests indicated that vehicle-treated cells showed significantly more mortality at 48 hours compared to all other time points. Fisher's LSD t- tests also showed that vehicle-treated cells displayed higher mortality at 48 hours than either AS- or MS-treated cells.
• Post-hoc Fisher's LSD t-tests indicated that vehicle- treated cells displayed significantly higher mortality at 72 and 168 hours than at either 48 or 96 hours.
• Fisher's LSD t-tests also indicated that vehicle-treated cells displayed significantly higher mortality at 72 and 168 hours than AS-treated cells, and significantly higher mortality at 72 hours than MS-treated cells.
• Fisher's LSD t-tests also indicated that AS-treated cells displayed significantly higher mortality at 48 hours than vehicle-treated cells, but significantly less mortality at all other time points.
• FIG. 19 Western blot analysis and sample Western blot for Sequence ID #3.
• This antisense (AS) treatment induced a decrease in mGluRi protein production in comparison to the missense (MS) control treatment, as indicated by area under the curve for mGluRi IgG binding density.
• FIG. 20 Western blot analysis and sample Western blot for Sequence ID #5.
• This antisense (AS) treatment induced a decrease in mGluRi protein production in comparison to the missense (MS) control treatment, as indicated by area under the curve for mGluRi IgG binding density.
• FIG. 21 Western blot analysis and sample Western blot for Sequence ID #7.
• This antisense (AS) treatment induced a decrease in mGluRi protein production in comparison to the missense (MS) control treatment, as indicated by area under the curve for mGluRi IgG binding density.
• Figure 22 Western blot analysis and sample Western blot for Sequence ID #1.
• Antisense (AS) treatment induced a 56.93% decrease in amount of mGluRi protein as indicated by area under the curve for mGluRi IgG binding density.
• Figure 23 Western blot analysis and sample Western blot for Sequence ID #1.
• Antisense (AS) treatment induced a 72.21% decrease in amount of mGluRi protein as indicated by area under the curve for mGluRi IgG binding density.
• Table 1 Groups for animal infusion
• “Corresponds to” refers to a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
• the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
• the nucleotide sequence "TATAC” corresponds to a reference sequence "TATAC” and is complementary to a reference sequence "GTATA".
• Naturally-occurring refers to the fact that an object can be found in nature.
• a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
• Nucleic acid refers to DNA and RNA and can be either double stranded or single stranded.
• the invention also includes nucleic acid sequences which are complementary to the claimed nucleic acid sequences.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• Oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
• Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
• Polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
• the term includes single and double stranded forms of DNA or RNA.
• Protein refers to a whole protein, or fragment thereof, such as a protein domain or a binding site for a second messenger, co-factor, ion, etc. It can be a peptide or an amino acid sequence that functions as a signal for another protein in the system, such as a proteolytic cleavage site.
• Other biochemistry and chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill, San Francisco).
• antisense oligonucleotides are designed that are complementary to specific regions of the rat mGluRi gene.
• antisense oligonucleotides are designed that are complementary to specific regions of the human mGluRi gene, wherein the mGluRi gene can be mGluR ⁇ ⁇ gene.
• the human and rat genes are purported to share significant homology, the antisense sequences used in rats do not target the human gene.
• antisense oligonucleotide sequences of the present invention are listed below. It should be apparent to one skilled in the art that other antisense oligonucleotide sequences that are complementary to specific regions of the human mGluRi gene are within the scope of the present invention.
• Coding region bases 236-3820 (gene is 4074 bp) (location indicated in parentheses)
• Coding region bases 1-3585 (gene is 6384 bp) (location indicated in parentheses)
• Coding region bases 1-2721 (gene is 3670 bp) (location indicated in parentheses)
• Coding region bases 371-3091 (gene is 3295 bp long) (location indicated in parentheses)
• SEQ ID#36 (395-411) 5'-CAA AAA GAT CGC TGG GAA-3' SEQ ID#37: (3278-3295) 5'-GAA AAG GTC AGG CTC TTG-3' SEQ ID#38: (3272-3289) 5'-GTC AGG CTC TTG CCA GAG-3' SEQ ID#39: (3266-3283) 5'-CTC TTG CCA GAG CCT TGG-3'
• Targeting an antisense compound to the mGluRi mRNA or gene, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding mGluRi.
• the targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result.
• a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene.
• start codon and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding mGluRi, regardless of the sequence(s) of such codons.
• start codon region and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.
• stop codon region and “translation termination codon region” refer to a portion of such mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
• Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5* cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.
• 5'UTR 5' untranslated region
• 3'UTR 3' untranslated region
• the 5' cap of an mRNA comprises an N 7 -methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage.
• the 5' cap region of an mRNA is considered to include the 5* cap structure itself as well as the first 50 nucleotides adjacent to the cap.
• the 5' cap region may also be a preferred target region.
• introns regions, known as "introns,” which are excised from a transcript before it is translated.
• exons regions
• mRNA splice sites i.e., intron-exon junctions
• intron-exon junctions may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease.
• Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.
• oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
• hybridization means hydrogen bonding, which may be
• adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
• “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
• oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
• “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
• An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
• Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with seventeen specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.
• antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics.
• the antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases.
• Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides).
• a nucleoside is a base-sugar combination.
• the base portion of the nucleoside is normally a heterocyclic base.
• the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
• Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
• the phosphate group can be linked to either the 2 3' or 5' hydroxyl moiety of the sugar.
• the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
• the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
• the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
• the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
• the antisense oligonucleotides comprise modified oligonucleotide backbones which may or may not include phosphorus atoms.
• both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
• the base units are maintained for hybridization with an appropriate nucleic acid target compound.
• an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
• nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• Teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
• Modified oligonucleotides may also contain one or more substituted sugar moieties. Other preferred modifications include 2'-methoxy (2'-O ⁇ CH3), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2 -fluoro (2'-F).
• Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
• Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
• nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guamne, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5 -halo particularly 5-bromo, 5 -trifluoromethyl and
• nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch etal, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15,
• nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
• oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
• moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al ( 1989) Proc Natl Acad Sci USA 86: 6553-6556), cholic acid (Manoharan et al (1994) BioorgMed Chem Lett 4: 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al (1992) Ann NY Acad Sci 660: 306-309; Manoharan et al (1993) BioorgMed Chem Lett 3: 2765-2770), a thiocholesterol (Oberhauser et al, Nucl.
• Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra etal, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923-937.
• the present invention also includes antisense compounds which are chimeric compounds.
• "Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
• oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
• An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
• RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
• RNA target Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
• Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
• Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
• the antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
• Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
• the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
• the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
• prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
• prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 to Imbach et al.
• pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
• the antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits.
• an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of mGluRi is treated by administering antisense compounds in accordance with this invention.
• the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier.
• antisense compounds and methods of the invention may also be useful prophylactically, e.g., to reduce pain, to minimize glutamate neurotoxicity and/or excitotoxicity associated with stroke, ischemia, CNS trauma, neurodegenrative disorders, gastrointestinal disorders or to inhibit tumor formation, for example.
• the antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding mGluRi, enabling sandwich hybridization and other assays to easily be constructed to exploit this fact.
• Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding mGluRi can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of mGluRi in a sample may also be prepared.
• the present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention.
• the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
• Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
• Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration.
• Phosphodiester oligonucleotides do appear to reach their cellular target, as they are internalized within cells (Stein and Cheng, 1993), and are stable in CSF (Wahlstedt, 1994).
• Vehicle- or phosphodiester-bonded antisense and missense oligonucleotides, targeting either rat or human mGluRi are infused intrathecally (i.t.; at the L4 to L5 lumbar region) in rats, via a catheter attached to an osmotic mini-pump, for 14 days (the maximum time of infusion allowed by current osmotic mini-pump technology, which is longer than the 3 -day length of treatment employed in human trials), in doses of, for example, 5, 50, 500 and 1000 ⁇ g/day.
• Antisense- and missense-treated animals are compared to vehicle-treated controls, as well as to rats not given infusion. Table 1 outlines the setup of one example of such an experiment using 18 experimental groups which corresponds to a total of
• Rats' motor coordination is assessed using the rotarod and righting reflex tests, which are well known to workers skilled in the art.
• rats are placed on a rotating rod (1 revolution per second) and their ability to stay on this rod is assessed as a function of how long they remain on the rod (up to a cutoff time of 60 sec).
• rats are placed on their backs, and the latency to return to a standing position is measured. Motor function in antisense- and missense-treated rats are compared to motor function in both vehicle-infused and non-infused control animals.
• Rats' sensitivity to heat, cold and mechanical stimulation are assessed with the plantar test, cold water test and von Frey hair test respectively, according to procedures known in the art.
• the plantar test the plantar surface of the rat's hindpaw is stimulated with a point of radiant heat, and the latency for paw withdrawal is measured.
• the cold water test the rat is standing in 1 cm deep, 1°C water for 75 sec, and latency to lift a hindpaw, latency to jump, frequency of hindpaw lifts and cumulative duration of hindpaw lifts is recorded.
• the von Frey hair test the plantar surface of the rat's hindpaw is stimulated with thin filaments, using an up-down method (Chaplan etal, (1994) J. Neurosci.
• Rats are housed individually in plastic metabolic cages for 1 hour prior to collection of blood and CSF samples. Urine samples are collected from these cages at 1, 6 and 12 hours, then every 24 hours after initiation of drug infusion in separate groups of rats. Collection continues until 4 days after termination of drug infusion.
• Blood approximately 1ml, is collected from a venous catheter implanted in the vena cava (Yashpal, Gauthier and Henry, 1985) using a heparinized 1 ml syringe at 1, 6 and 12 hour, then every 24 hours after beginning drug infusion, until 4 days after termination of drug infusion.
• To replenish blood volume an equal volume of physiological saline is injected immediately following collection.
• the blood plasma is collected according to standard methods known in the art and frozen at -70°C until analyzed.
• a second, shorter, intrathecal catheter (extending to the thoracic region) is attached to the catheter used to infuse oligunucleotides, and implanted at the same time (Jhamandas, Yaksh, Harty, Szolcsanyi and Go, 1984). CSF is collected via this second catheter (10-12 ⁇ l) at the same time points as blood. CSF samples are immediately frozen at -70°C until analysis.
• rats are euthanized and spinal cords and brains collected as described below. If pharmacokinetic analysis indicates that oligonucleotides reach systemic circulation (i.e. they are present in blood and/or urine) we will also collect heart, lungs, liver, spleen and kidneys.
• oligonucleotides in CSF, plasma and urine samples The amount of oligonucleotide in biological samples can be determined using high performance liquid chromatography (HPLC) with ultraviolet (UV) detection.
• HPLC high performance liquid chromatography
• UV ultraviolet
• the HPLC system may be a Hewlett Packard Series 1050, consisting of a gradient pump, autoinjector and variable-wavelength UV detector.
• anion-exchange chromatography is performed using a 20 x 1 mm I.D. guard column (Upchurch Scientific, Oak Harbor, WA), handpacked with spherical 13 mm Dionex Nucleopak PA- 100 support (Dionex Chromatography, Sunnyvale, CA).
• the mobile phase composition will initially be set at 60% A, 10% B, 30% C, at a flow rate of 1 ml/min, then be brought to 30% A, 40% B, 30% C over 1.2 min, and held for 0.8 min.
• Oligonucleotides are detected spectrophotometrically at a wavelength of 267 nm.
• CSF, plasma and urine samples can be diluted using 100 ⁇ l of 0.5% NP-40 (Sigma, St. Louis, MO) in 0.9% saline. Samples are then centrifuged at 12 000 * g for 10 min, and 50 to 100 ⁇ l of supernatant will be injected directly onto the HPLC system
• Standard curves for antisense and missense oligonucleotides in CSF, plasma and urine, in concentrations from 0.05 to 8 ⁇ M, are prepared daily. These calibration curves are linear, with less than 16% coefficient of variation. Concentrations as low as 0.01 ⁇ M were detected using this method (Qian etal, (1997) J. Pharmacol. Exp. Ther. 282, 663-670).
• Mean oligonucleotide concentration is calculated for each sample, and then for each group at each time point.
• the area under the curve is obtained using Lagrange polynomial integration from time zero to the last measured sample time, with extrapolation to infinity using the least- squares terminal slope method with the NCOMP computer program (Qian et al, (1 97) J. Pharmacol. Exp. Ther. 282, 663-670). From the areas and terminal slopes, clearance (CL), steady state volume of distribution (V ⁇ ), half-life (t ⁇ /2) and maximum concentration (C ⁇ x ) are determined. Data is analyzed with one, two and three compartments and the best fit will be adopted. These data are indicative of the stability of the oligonucleotides in CSF, blood and urine.
• Staining in spinal cords and brains from vehicle-treated rats is compared to staining in spinal cords and brains from non-infused (normal) controls. Staining in spinal cords and brains from oligonucleotide-treated rats will be compared to that of both vehicle-infused and non-infused controls.
• oligonucleotide is present in blood and/or urine samples
• the heart, lung, liver, spleen, and kidneys are also collected as per their specifications. These tissues are examined for inflammation, edema, hemorrhage, degeneration, necrosis, changes in mitochondria (e.g. swelling), using standard techniques known to those skilled in the art.
• the antisense oligonucleotides of the present invention Before using the antisense oligonucleotides of the present invention in human patients, it is necessary to first verify that they are not toxic to human cells, and that they do not promote tumor growth. It is also useful to verify that the antisense targeting human mGluRi gene will arrest production of receptor protein in human cells, especially cells of the human central nervous system. It should be readily understood by a worker skilled in the art that any human cell line which expresses metabotropic glutamate receptors, for example, human glial and neuroblastoma cells (Lee et al (1995) Proc Natl Acad Sci USA 92: 8083-8087), can be used in validation studies. (i) Toxicity in human cell culture
• Antisense oligonucleotides directed against mGluRi are unlikely to promote tumor growth; by inhibiting mGluRi expression, the activation of PKC will be inhibited (Schoepp and Conn (1993) Trends Pharmacol Sci 14: 13-25), and inhibition of PKC (with oligonucleotides) has been shown to inhibit tumor growth (Dean et al (1996) Cancer Res 56: 3499-3507). Standard techniques known in the art can be used in order to confirm that the antisense oligonucleotides of the present invention do not cause tumorgenicity in human cells. Generally, the antisense oligonulceotides are monitored for their ability to affect the growth of a malignant human cell line and compared to negative control cells. A specific example of a tumorgenicity validation test is provided below.
• antisense and missense oligonucleotides on the ability of a malignant human neuroblastoma cell line, for example SK-N-AS, obtained from ATCC, to grow in soft agar can be monitored in order to test for tumorigenicity in central nervous system cells.
• Cell cultures are grown as described above in growth media containing from 0.2 to 20 mM antisense or missense oligonucleotides, or artificial CSF, and seeded at 5 x 10 5 cells per 60 mm dish
• oligonucleotide treated cells are compared to untreated and artificial CSF treated cells (an increased rate of growth indicates tumorigenic effects). Because there is a small possibility that the oligonucleotides of the present invention can reach systemic circulation, this test can also be performed in breast (MB-157; ATCC) and lung (DMS-114; ATCC) cancer cell lines. Breast and lung cancer cells are a good choice because these two types of cancer also commonly metastasize to the central nervous system. Unacceptable tumorigenicity is indicated by >20% excess growth of oligonucleotide treated cells relative to controls.
• a test that can be used to further test for tumorgenicity is a thymidine incorporation test using these cell lines to assess rate of DNA synthesis.
• the cells are incubated with [ 3 H]thymidine (2 ⁇ Ci well) for 24 hours, beginning at the 24 th hour of culture. Cells are harvested and radioactivity is counted (cpm/well), for example, using a matrix 9600 gas counter (Packard Instrument Company, Meriden, CT). Data from oligonucleotide-treated cells can be compared to control values. Radioactivity counts >20% greater than controls will constitute unacceptable tumorigenicity.
• test antisense oligonucleotide does not cause toxicity or tumorgenicity it is usefiil to confirm its efficacy in vivo prior to its use in clinical trials.
• the techniques used to study efficacy in vivo are standard techniques well known in the art.
• human cells expressing metabotropic glutamate receptors are treated with the test oligonucleotide and the amount of mGluRi produced is compared to that produce in control cells, which are prepferrably cells treated with a missense oligonucleotide.
• antisense oligonucleotides of the present invention would be tested in Clinical Trials in order to obtain regulatory approval for therapeutic use.
• Clinical Trials An example of standard procedures carried out in a clinical trial is presented below.
• the palliative care physician can identify patients for whom opioid therapy is problematic, and who are scheduled to receive intrathecal (i.t.) drug adminstration for pain control. These patients can be approached in order to explain the goals of the study and obtain written consent from those patients who wish to participate in the study. An expert palliative care physician can then assess the patient. For example, only those patients diagnosed as presenting with symptoms of neuropathic pain can be included in the study. These patients may have pure neuropathic pain, or mixed pain (neuropathic pain plus either somatic or visceral). Those presenting with somatic or visceral pain only would not be included at this time, although such patients can be added to other studies. Patients presenting with either meningitis or arachnoiditis can not be included in the study.
• the treatment plan can be as follows:
• the patient's physician can determine if intrathecal opioids are contraindicated or unlikely to relieve the pain. If this is the case, the patient will be randomly assigned to receive either one of the three antisense doses or vehicle.
• Intrathecal opioids are administered until their effectiveness is clear. If the opioids provide sufficient pain relief, the patient can be maintained on this treatment and not included in the study. If the opioids provide some but not adequate pain relief (as determined by the patient), the opioid infusion can be maintained and supplemented by either one of the three antisense doses or vehicle (randomly determined). If the opioids provide no noticeable pain relief, or relief only with intolerable side effects, the intrathecal opioid will be discontinued and the patient will be randomly assigned to receive either one of the three antisense doses or vehicle. It is estimated that approximately 300 patients per year are treated for pain in the palliative care units. Of these, opioid therapy is problematic for 30%, and of these 30%, 2/3 have neuropathic pain.
• One example of a study group would contain eight patients per dose group for a total of 32 patients. Due to ethical considerations, patients must be maintained on their current pain and cancer treatments. A worker skilled in the art would realize that the effectiveness of the antisense may be influenced by concurrently prescribed medications. However, testing these interactions is not the focus of the study. The primary outcome measure is whether addition of antisense oligonucleotide to the treatment improves pain control. This is the first part of a larger study, and future projects can be performed to study drug interactions if the antisense proves to be an effective analgesic.
• MQOL McGill Quality of Life Questionnaire
• the MQOL measures physical symptoms; physical, psychological and existential well-being; support; and overall quality of life.
• SDS Symptom Distress Scale
• Performance status and overall motor function can be assessed using the Edmonton Functional Assessment Tool (EFAT) (Kaasa et al (1997) JPain and Sympt Manage 13: 10-19).
• MMSE Folstein Mini-Mental State Examination
• Sensitivity to hot and cold stimuli can be tested by recording patients' rating of intensity on a visual analog scale of thermal stimuli produced by a 9 cm 2 peltier-type contact thermode (Morin and Bushnell (1998) Pain 74: 67-73).
• Cold temperatures can range from -5°C to 23°C, while heat temperatures will range from 41°C to 48°C (Morin and Bushnell (1998) Pain 74: 67-73).
• Mechanical sensitivity can be assessed by calculating the 50% response threshold (in grams) to von Frey hair stimulation.
• Intrathecal administration of oligonucleotides is a preferred mode of delivery for two reasons.
• Second, intrathecal administration of antisense oligonucleotides has been shown to be effective against neuropathic pain in rats.
• patients can be approached for whom oral or subcutaneous administration of analgesics is ineffective, and who are scheduled to receive intrathecal treatment. Patients should continue with their normally scheduled analgesic therapy while in the study.
• Studies presented herein show that intrathecal morphine injection induces analgesia more effectively in antisense-treated neuropathic rats than in vehicle- or missense-treated neuropathic rats.
• a range of doses of antisense oligonucleotides (0 (vehicle), 25 ⁇ g/day, 50 ⁇ g/day, and 100 ⁇ g/day; i.e. 4.167 ⁇ g/hr at the highest dose) can be tested.
• the pre-clinical trials presented herein indicate that a missense oligonucleotide, which is a control for antisense drug mechanism, has no behavioral or physiological effect. It is possible to exclude missense, because one placebo is already included (vehicle, i.e. artificial CSF), in the initial clinical trials if the lack of physiological effect is borne out by our pre-clinical toxicology and protein analysis studies.
• An exemplary dose range for antisense oligonucleotide treatment includes the dose (50 ⁇ g/day).
• the design of the study can be double blinded. Although as few as 4 subjects can be used to obtain reliable pharmacokinetic data (Glover et al (1997) J Pharmacol Exp Ther 282: 1173-1180), 8 patients can be used per dose (for a total of 32 patients out of an available 60 per year).
• Intrathecal catheterization of human patients are ideally performed by an anaesthetist.
• a polyesther catheter (PortexTM) is inserted subarachnoidally.
• lumbar puncture (18 G Tuohy needle) is performed at an interspace between the second and the fifth lumbar vertebra using a lateral approach.
• the catheter is inserted 3-4 cm, and tunneled subcutaneously around the flank to the portal site over the lower ribs.
• the catheter can be fixed with a transparent self-adhesive dressing (TegadermTM). All procedures should be performed using local anesthetic infiltration and intravenous sedation (e.g.
• fentanyl, diazepam when needed.
• the catheter is connected to a computerized CADD infusion pump (DeltecTM, Pharmacia) designed to give a continuous infusion of drug at a rate of 10 ⁇ l/hr (Gestin et al (1997) Ann Fr Anesth R ⁇ anim 5: 346-350) and the infusion can be started immediately. If clinical signs of arachnoiditis or meningitis are found, as monitored by the primary care nurse, a physician should be contacted immediately and the catheter removed and cultured for bacterial analysis. Patients who complete the study should be infused continuously with antisense oligonucleotide for 3 days (72 hours).
• oligonucleotides should be monitored. Samples of CSF, blood and urine can be collected at regular intervals for chemical analysis. The pharmacokinetic data can be analyzed in collaboration with an expert clinical pharmacologist.
• One CSF sample (1 ml) is collected prior to the commencement of the study. Subsequent 1 ml samples are collected 1 h, 6 h, 24 h, 48 h and 72 h after the initiation of drug infusion, then once per day until the pain returns up to a maximum of 1 week after cessation oligonucleotide treatment..
• the oligonucleotides can be administered centrally and are, therefore, unlikely to enter systemic circulation.
• phosphodiester-bonded oligonucleotides are degraded quickly in blood.
• blood samples can be collected at regular intervals. Blood (10-15 ml) can be collected once prior to, and 2, 10, 15, 30, 60, 90, and 120 min, and 3, 6, 12, 24, 43 and 72 h after we begin drug infusion, and then once per day until the pain returns up to a maximum of 1 week after cessation oligonucleotide treatment.
• urine samples at regular intervals.
• Urine samples will be collected prior to, and again 1 h, 6 h, 24 h, 48 h and 72 h after initiation of drug infusion, and then once per day until the pain returns up to a maximum of 1 week after cessation oligonucleotide treatment.
• Samples can be prepared using standard techniques known in the art and the amount of oligonucleotide present can be determined using HPLC with UV detection. Pharmacokinetic analysis can be performed as described for samples collected from rats.
• Pain can be assessed using 0 - 10 numerical rating scales for average pain, pain at rest, and pain upon movement during the past 24 hours. These can be completed prior to infusion, and daily after the initiation of drug infusion for 10 days (i.e. the 3 days of drug infusion, plus 7 days after cessation of drug infusion). Pain can be assessed biweekly during weeks 2, 3 and 4 post-infiision, weekly thereafter until the pain returns up to a maximum of 13 weeks. Cognitive status (MMSE) and symptom distress (SDS) can be assessed whenever the numerical pain rating scales are completed.
• MMSE cognitive status
• SDS symptom distress
• the complete BPI, EFAT, sensitivity to thermal and mechanical stimuli, and MQOL can be completed prior to and at 72 hour after initiation of infusion, one week after initiation of infusion, and weekly thereafter until pain returns up to a maximum of 13 weeks post-infusion.
• the primary outcome measure can be the pain rating obtained with numerical rating scale of average pain in the past 24 hours.
• the score for "average pain" at 72 hours after the initiation of the infusion can be categorized as mild (0-4), moderate (5-6) or severe (7-10) (Serlin et al (1995) Pain 61: 277-284) to determine the percentage of patients in each category at each dose level.
• Secondary measures can include symptoms and side effects as rated by the SDS, EFAT and MMSE. Patients can be assessed as to whether symptoms and side effects decreased, stayed the same or increased, compared to pre-treatment scores, during the course of oligonucleotide treatment.
• the MQOL can also be administered to evaluate the patients' overall satisfaction with care and quality of life. The optimum outcome is good pain relief, without an increase in symptoms and side effects, or cognitive or functional impairment.
• mGluRi spinal knockdown of mGluRi reduces cold and hot hyperalgesia and mechanical allodynia in neuropathic rats; knockdown of mGluRi restores morphine sensitivity, and reduces NMDA sensitivity in neuropathic rats; and enhanced PKC activity associated with nerve injury is reversed by knockdown of mGluRi.
• Intrathecal (i.t.) catheters were inserted using a lumbar catheterization method (Storkson et al, (1996) J Neurosci Methods 65: 167-172).
• the catheter was attached to an Alzet osmotic mini- pump (ALZA Model 2001TM) containing either artificial cerebrospinal fluid, antisense (AS) oligonucleotide solution, or missense (MS) oligonucleotide solution.
• ALZA Model 2001TM Alzet osmotic mini- pump
• An antisense (AS: 5'-GAG CCG GAC CAT TGT GGC-3') oligonucleotide was designed that is complementary to base pairs 371-388 of the rat mGluRi gene RATGPCR.
• a missense (MS: 5'-GAG CCG AGC ACT GTG TGC-3') oligonucleotide was designed by taking the AS sequence and mismatching four base pair couples. Oligonucleotides were purchased from Medicorp Inc (Montreal, QC).
• Vehicle, AS and MS were continuously infused i.t. for 7 days, via the catheter, in a volume of 1 ⁇ l hr -1.
• the daily dose of AS and MS was 50 ⁇ g day -1. This dose of AS and MS oligonucleotides was not found to produce any motoric or sedative side-effects, as examined using placing, righting and grasping reflexes.
• Morphine (SabexTM, QC) was dissolved in 0.9% saline, and injected intrathecally (i.t.), via lumbar puncture between vertebrae L4 and L5, in doses of 3, 10 or 30 ⁇ g in 20 ⁇ l .
• Rats were quickly decapitated, and their spinal cords were pressure ejected and rapidly frozen. Spinal cords were stored at -70°C until analysis. The lumbar enlargement section of spinal cords were homogenized in Tris buffer containing protease inhibitors. Group I mGluRs are most likely found in the dorsal horn in lumbar spinal cord, with some expression in the intermediate gray matter and ventral horn (Hargett et al, (1998) Soc Neurosci Abstr 24: 1869). Concentration of protein in each sample was determined using the method of Bradford (Bradford, (1976) Anal Biochem 72: 248-254). The concentration of protein in each sample fell on the linear portion of the curve.
• the primary antibody was later tagged with a peroxidase-conjugated anti- rabbit antibody (secondary antibody; Jackson Laboratories).
• the secondary antibody was detected by chemiluminescence (Boehringer Mannheim) and the membranes were apposed to Kodak Biomax MR film for one minute. Band density was measured using Alpha Imager software.
• the mGluRi is a protein of approximately 133 -142kD (Houamed et al, (1991) Science 252: 1318-1321; Martin et al, (1992) Neuron 9: 259-270; Masu et al, (1991) Nature 349: 760-765).
• DHPG 3,5-dihydroxyphenylglycine
• SNBs spontaneous nociceptive behaviors
• SNBs included elevation of tail, licking of tail, elevation of hindpaws and licking of hindpaws. The time spent in each of these behaviors was combined into one "time spent exhibiting SNBs" score. Data were analyzed by ANOVA followed by post-hoc Fisher's LSD t-tests on significant results.
• NMDA intrathecal NMDA
• NMDA (Sigma, Oakville, ON) was injected intrathecally (i.t.), via an i.t. catheter, in doses of 1.67 and 2.5 nmol in 20 ⁇ l. Rats were observed for a period of 8 min and the time spent exhibiting SNBs was recorded (time spent favoring paws, agitation, licking and biting paws).
• neuropathic hyperalgesia and allodynia In rats that were pre-treated with i.t. oligonucleotides, cold, hot and mechanical sensitivity were measured prior to any treatment (baseline) and again 4, 8, 12 and 16 days after nerve injury. In rats that were post-treated with i.t. oligonucleotides, cold, hot and mechanical sensitivity were measured prior to any treatment (baseline) and again 4 days after nerve injury (but before i.t. treatment), as well as 8, 12 and 18 days after nerve injury.
• Cold sensitivity was measured by placing rats in a 1 cm deep 1 °C cold water bath for 75 sec, and counting the number of responses (lifting of hindpaw).
• Cold hyperalgesia was assessed by calculating the increase in number of responses, compared to baseline, from days 4 to 16 or 18 after nerve injury.
• Heat sensitivity was measured by applying focussed radiant heat to the glass under the plantar surface of the hindpaw and measuring the latency for the rat to withdraw its paw (Hargreaves et al, (1988) Pain 32: 77-88). A cut-off latency of 20 sec was used to prevent tissue injury. Heat hyperalgesia was assessed by calculating the percent decrease in latency (fro ⁇ Tbaseline) on days 4 to 16 or 18 after nerve injury.
• data from the pre-treatment schedule were analyzed by repeated measures ANOVA with nerve injury condition and i.t. treatment as independent groups factors, and days post nerve injury as a repeated measures factor.
• Data from the post-treatment schedule were analyzed by repeated measures ANOVA with i.t. treatment as the independent groups factor and days post nerve injury as the repeated measures factor.
• Significant results were further analyzed with post-hoc Fisher's LSD t-tests.
• mice were tested by measuring their latency to withdraw their tail from 55 °C water. A cut-off latency of 10 sec was used to prevent tissue injury. Tail flick latencies were measured prior to any treatment (na ⁇ ve rats before oligonucleotide infusion or nerve injury; BLl), and again 4 days after nerve injury (after 7 days of oligonucleotide infusion; BL2). In ACSF-treated neuropathic rats, BL2 (2.44 ⁇ 0.12) was slightly, but significantly higher than BLl (2.01 ⁇ 0.22) (p ⁇ 0.05), indicating that the rats were not hyperalgesic after sciatic nerve injury.
• BLl and BL2 were not different in oligonucleotide-treated rats versus vehicle-treated rats, this suggests that antisense treatment did not affect motor function and the ability to respond on this test.
• [ 3 H]-phorbol-12,13-dibutyrate ([ 3 H]PDBu) binding assay (Olds et al, (1989) Science 245: 866-869; Woriey et al, (1986) J Neurosci 6: 199-207) was used to demonstrate the amount of membrane-bound (i.e. activated) PKC. It has previously been shown that [ 3 H]PDBu binding is enhanced in spinal cord in rats with a sciatic nerve injury (Mao et al, (1992) Brain Res 588: 144-149). Traditionally, it is assumed that PKC has to be transported to the membrane and bind DAG to be activated. Although this does not provide absolute quantification of PKC activity, it does provide some indication of the activation of PKC.
• Pre-treatment Three days prior to nerve injury, rats were implanted with i.t. catheters attached to mini osmotic pumps containing either vehicle, or 50 ⁇ g day _1 AS or MS. Nerve injury was induced three days later, and rats were tested 4, 8, 12 and 16 days after nerve injury for cold hyperalgesia, hot hyperalgesia and mechanical allodynia. Oligonucleotides were infused for a total of 7 days (from 3 days prior to until 4 days after nerve injury) . This pre- treatment schedule was employed to determine whether mGluRi is involved in the development of hyperalgesia and/or allodynia associated with nerve injury. Western blot analysis, [ 3 H]PDBu binding, morphine analgesia and NMDA sensitivity were assessed in rats with this treatment schedule (pre-treatment).
• Post-treatment A separate group of rats was used to determine whether mGluRi is involved in the maintenance of neuropathic pain, and whether AS oligonucleotide knockdown of mGluRi could reverse hyperalgesia and/or allodynia associated with an established neuropathy. Briefly, nerve injury was induced by placing a polyethylene cuff around one sciatic nerve as described above. Because there was no effect of oligonucleotide treatment in sham-operated rats when it was given as a pre-treatment, and would be expected to have the greatest effect, a sham-operated group was not included in the post-treatment test. Rats were confirmed to be neuropathic (i.e.
• Heat sensitivity in neuropathic rats Heat sensitivity was measured by stimulating the plantar surface the hindpaw with a focused radiant heat source and measuring the latency of the rats to withdraw the paw.
• ACSF- and MS-treated neuropathic rats displayed a large decrease from baseline in withdrawal latency to radiant heat stimulation of the ipsilateral paw, as compared to sham-operated rats ( Figure 3e).
• AS-treated neuropathic rats this decrease was significantly less, compared to ACSF- and MS-treated neuropathic rats ( Figure 3e).
• Figure 5 A is a histogram summary of [ 3 H]PDBu binding density from all the slides in each treatment group
• Figure 5B is a computer-generated image showing [ 3 H]PDBu binding in a single representative slide from each treatment group. Because mGluRi is positively coupled to PI hydrolysis, and thus to activation of PKC, the involvement of PKC in neuropathic pain was examined by measuring the binding of [ 3 H]phorbol, 12, 13-dibutyrate ([ 3 H]PDBu) in lumbar spinal cord slices of rats four days after nerve constriction (7 days of oligonucleotide infusion).
• Intrathecal infusion of our AS oligonucleotide greatly decreased mGluRi protein in lumbar spinal cord, and slightly decreased it in the brain. Further, using 12 days post-infusion, the amount of mGluRi protein in AS-treated neuropathic rats had recovered to levels similar to ACSF-treated rats, indicating that the effect was reversible. As a functional correlate of a decrease in mGluRi protein, it was shown that AS-treated rats displayed significantly fewer DHPG-induced SNBs. Moreover, AS oligonucleotide treatment significantly decreased cold hyperalgesia, mechanical allodynia and heat hyperalgesia of the ipsilateral hindpaw of neuropathic rats.
• spinal PKC ⁇ is translocated to the membrane in neuropathic rats (Mao et al, (1995) Neurosci Lett 198: 75-78), inhibition of PKC in the spinal cord reduces morphine tolerance and neuropathic pain (Mao et al, (1995) Pain 62: 259-274; Mao et al, (1992) Brain Res 588: 144-149), and PKC ⁇ knockout mice do not develop neuropathy (Malmberg et al, (1997) Science 278: 279-283). The activation of PKC may underlie the effects seen with inhibition of either mGluRi or NMDA receptors.
• neuropathic pain is often difficult to treat, being only partially relieved by high doses of opioids (Cherny etal (1994) Neurology 44: 857-861; MacDonald (1991) Recent Results in Cancer Res 111: 24-35; McQuay et al (1992) Anesthesia 47: 757-767).
• ACSF- and MS-treated neuropathic rats were shown to be less sensitive to the analgesic effects of intrathecally administered morphine, while mGluRi AS-treated neuropathic rats displayed a normal analgesic response to morphine.
• knockdown of mGluRi at the spinal level prevented the development of morphine insensitivity in neuropathic rats.
• Activation of group I mGluRs has been shown to enhance activity at NMDA receptors (Bleakman et al (1992) Mol Pharmacol 42: 192-196) via a PKC-mediated mechanism (Chen and Huang (1992) Nature 356: 521-523; Harvey and Collingridge (1993) Br J Pharmacol 109: 1085-1090; Raymond et al (1994) J Physiol Paris 88: 181-192), and NMD A receptors have been shown to contribute to nociception in animal models of persistent nociception (Chaplan et al (1997) J Pharmacol Exp Ther 280: 829-838; Mayer et al (1995) .
• NMDA receptor activity would be enhanced in neuropathic rats, and that neuropathic rats would therefore be more sensitive to the excitatory effects of NMDA injected intrathecally.
• mGluRi AS oligonucleotide treatment would attenuate this enhanced NMDA receptor activity, and thus NMDA sensitivity, in neuropathic rats.
• neuropathic rats with nerve injury displayed increased nociceptive behaviours in response to intrathecal injection of NMDA, and that this effect was reversed by mGluRi AS oligonucleotide treatment.
• mGluRi AS treatment induced a reduction in the number of NMDA receptors, this is highly unlikely.
• unmodified, phosphodiester bonded oligonucleotides were used, which have generally been shown not to have non-sequence-specific effects.
• mGluRi AS treatment reduced the number of NMDA receptors, the response to i.t.
• NMDA would likely be blunted in the AS-treated rats compared to ACSF-treated sham-operated rats.
• the time spent exhibiting nociceptive behaviours was virtually identical for the two groups of rats.
• rats with nerve injury were confirmed to be more responsive to spinal administration of NMDA, and this could be attributed to mGluRi-associated mechanisms.
• mGluRi AS oligonucleotide treatment had no significant effect in sham-operated rats or in the contralateral paw of neuropathic rats, indicating that mGluRi is involved in chronic neuropathic pain, but not in the mediation of acute nociceptive stimuli.
• This is in agreement with a previous study where the blockade of mGluRi with selective antibodies alleviated cold hyperalgesia in neuropathic rats, but had no effect on response latency to focused radiant heat in naive rats, nor on formalin- induced pain scores (Fundytus et al (1998) Soc Neurosci Abstr 23: 1013).
• PS phosphorothioate
• knockdown of mGluRi reverses nerve-injury induced insensitivity to morphine, as well as reducing neuropathic pain
• the knockdown of mGluRi will be useful as therapy for neuropathic pain in the clinic. It may be used to alleviate pain directly, or as an adjunct to opioid analgesic therapy.
• An antisense (AS) oligonucleotide (AS: 5'-GAG CCG GAC CAT TGT GGC-3') was designed to be complementary to base pairs 371-388 of the rat mGluRi gene, RATGPCR.
• a control missense (MS) oligonucleotide was designed that had exactly the same bases as the AS sequence, with four base pairs mismatched (MS: 5 '-GAG CCG AGC AC5 GTG TGC-3').
• Rats were rendered neuropathic by placing a 2 mm length of PE90 polyethylene tubing around one sciatic nerve (Mosconi and Kruger (1996) Pain 64: 37-57).
• Rats were continuously infused for 7 days (from days 5 to 12 after nerve injury), with either artificial CSF (ACSF), or 50 ⁇ g/day AS or MS oligonucleotides.
• Mechanical sensitivity Mechanical sensitivity
• Example II Mechanical sensitivity was measured as described in Example I. Testing was performed prior to any surgery or treatment (baseline), 4 days after nerve injury, and 8, 12 and 18 days after nerve injury. Mechanical allodynia was assessed by calculating the percent decrease in 50% response threshold compared to baseline.
• Heat sensitivity was measured, as described in Example I, prior to any surgery or treatment (baseline), 4 days after nerve injury, and 8, 12 and 18 days after nerve injury. Heat hyperalgesia was assessed by calculating the percent decrease in response latency compared to baseline.
• Cold sensitivity was measured as described in Example I. Testing was performed prior to any surgery or treatment (baseline), 4 days after nerve injury, and 8, 12 and 18 days after nerve injury. Cold hyperalgesia was assessed by calculating the increase in response frequency compared to baseline.
• Figure 7 depicts mechanical sensitivity in neuropathic rats treated with AS, MS or ACSF.
• Figure 8 shows heat sensitivity in neuropathic rats treated with AS, MS or ACSF.
• Figure 9 illustrates the cold sensitivity in neuropathic rats treated with AS, MS or ACSF.
• AS, MS or ACSF Four days after nerve injury, prior to drug infusion, all rats displayed cold hyperalgesia, as indicated by a large increase in the number of responses in the cold water bath. Drug infusion began on day 5. From days 8 to 18 after nerve injury, ACSF and MS treated continued to display cold hyperalgesia. Conversely, cold hyperalgesia was reversed in AS treated rats, as indicated by a reduced frequency of responding.
• mGluRi is a viable new target for drug development in the treatment of neuropathic pain.
• EXAMPLE III ANTISENSE KNOCKDOWN OF MGLURj ALLEVIATES HYPERALGESIA DUE TO CHRONIC INFLAMMA ⁇ ON IN RATS
• Rats were male Wistar rats weighing 325-375 grams at the beginning of the experiment. Rats were housed 3 to 4 per cage with food and water freely available, with a 12: 12 hour ligh dark cycle (lights on at 06:00 hours).
• Intrathecal (i.t.) catheters were inserted using a lumbar catheterization method (Storkson, Kjorsvik, Tjolsen and Hole, 1996). The catheter was attached to an Alzet osmotic mini-pump (ALZA Model 2001) containing either ACSF, antisense (AS) oligonucleotide solution, or missense (MS) oligonucleotide solution. Rats were infused intrathecally for 7 days.
• CFA complete Freund's adjuvant
• Oligonuceotides An antisense (AS: 5'-GAG CCG GAC CAT TGT GGC-3') oligonucleotide was designed that was complementary to base pairs 371-388 of the rat mGluRi gene RATGPCR.
• Vehicle, AS and MS were continuously infused i.t., via the catheter, in a volume of 1 ⁇ l/hr.
• the daily dose of AS and MS was 50 ⁇ g/day. Effective knockdown of receptors has been achieved with doses as low as 1 ⁇ g/day, up to doses as high as 720 ⁇ g/day (Wahlestedt, 1994). This dose of AS and MS oligonucleotides was not found to produce any motoric or sedative side-effects, as examined using placing, righting and grasping reflexes.
• Pre-treatment Three days before injection of CFA rats were implanted with intrathecal catheters attached to osmotic mini-pumps containing either ACSF, AS or MS solution. Heat and mechanical sensitivity was measured prior to any surgery or injection (baseline), and again 1, 2, 4, 6 and 8 days after CFA injection. This treatment schedule was employed to see if mGluRi is involved in the development of inflammatory pain. Western blot analysis was carried out on lumbar spinal cords from rats in this treatment group. Post-treatment: A separate group of rats was injected with CFA, followed by implantation of intrathecal catheters attached to osmotic mini-pumps containing either ACSF, AS or MS solution 2 days later.
• Heat and mechanical sensitivity was measured prior to any surgery or injection (baseline), and again 1, 2, 4, 6 and 8 days after CFA injection. This treatment was employed to determine if mGluRi was involved in the maintenance of inflammatory pain, and whether antisense oligonucleotide knockdown of spinal mGluRi could reverse pain due to an established inflammatory injury.
• Heat sensitivity was measured by applying focussed radiant heat to the plantar surface of the hindpaw and measuring the latency for the rat to withdraw its paw (Hargreaves et al (1988) Pain 32: 77-88). Heat hyperalgesia was assessed by calculating the percent decrease in latency (from baseline) on days 1 to 8 after CFA injection. Data were analyzed by repeated measures ANOVA with i.t. treatment as the independent groups factor and days post CFA injection as the repeated measures factor. Significant results were further analyzed with post-hoc Fisher's LSD t-tests.
• the Western blot analysis was performed as described in the previous example except that the proteins were separated by polyacrylamide gel electrophoresis using a 5% polyacrylamide gel rather than a 7.5% polyacrylamide gel.
• oligonucleotides in cultures of a non-tumorigenic human cell line that contains group I mGluRs, for example SH-SY5Y, obtained from American Type Culture Collection (ATCC, Rockland, MD ) (Lee et al (1995) Proc Natl Acad Sci USA 92: 8083-8087), were monitored.
• Cells were grown to confluency in RPMI- 1640 media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 ⁇ g/ml streptomycin (all from Sigma, St.
• FBS fetal bovine serum
• penicillin G 100 U/ml penicillin G
• streptomycin all from Sigma, St.
• Treatment media was growth media with either antisense or missense oligonucleotide (dissolved in artificial CSF) added in a concentration of 0.2 to 20 mM, preferrably 10 ⁇ M. Because of the seriousness of this application (human treatment) higher concentrations were tested than what would likely be used in either rat or human CSF for oligonucleotide treatment. In rats, the highest dose was 1000 ⁇ g/day.
• the maximum concentration reached at the end of the 14 day infusion would be 14000 ⁇ g/2000 ⁇ l of CSF (a rat has approximately 2 ml of CSF).
• the molecular weight of oligonucleotide obtained from Upstate Biotechnology is 5813.9 g/mol. Therefore, the maximum concentration for CSF in the rat was 1.20 x IO "3 ⁇ M (or 1.20 x IO "6 mM).
• Cell viability was assessed at 24, 48, 72 and 168 hours of treatment using the trypan blue dye exclusion method. Trypan blue dye is excluded from live cells with intact membranes, but stains dead cells.
• the cells were counted in a hemocytometer (see above) with a long-distance phase contrast microscope. Live cells were clear and dead cells were blue. Oligonucleotide treated cell cultures were compared to non-treated cultures, and artificial CSF treated cultures to determine the percentage of cells which died within each time period. Unacceptable toxicity is indicated by >20% more cell death in oligonucleotide treated cells relative to controls.
• oligonucleotides in cultures of a non-tumorigenic human cell line that contains group I mGluRs, for example SH-SY5Y, obtained from American Type Culture Collection (ATCC, Rockland, MD ) (Lee et al (1995) Proc Natl Acad Sci USA 92: 8083-8087), were monitored.
• Cells were grown to confluency in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 ⁇ g/ml streptomycin (all from Sigma, St.
• FBS fetal bovine serum
• penicillin G 100 U/ml penicillin G
• streptomycin all from Sigma, St.

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