US20100234447A1

US20100234447A1 - Modulation of glucagon receptor expression

Info

Publication number: US20100234447A1
Application number: US12/725,359
Authority: US
Inventors: Susan M. Freier
Original assignee: Isis Pharmaceuticals Inc
Current assignee: Ionis Pharmaceuticals Inc
Priority date: 2003-04-28
Filing date: 2010-03-16
Publication date: 2010-09-16
Also published as: EP1620453A4; US20120295958A1; US20040266714A1; EP2272857A1; EP2327709A3; US8642753B2; US7919476B2; EP2327709A2; JP2012050439A; US7399853B2; WO2004096016A3; EP2327709B1; WO2004096016A2; US20070238690A1; JP2007525945A; US20070238688A1; US20070238689A1; EP1620453A2; US20070238687A1; JP2012050438A

Abstract

Compounds, compositions and methods are provided for modulating the expression of glucagon receptor. The compositions comprise oligonucleotides, targeted to nucleic acid encoding glucagon receptor. Methods of using these compounds for modulation of glucagon receptor expression and for diagnosis and treatment of disease associated with expression of glucagon receptor are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 11/697,280, filed Apr. 5, 2007, which is a divisional of U.S. application Ser. No. 10/832,777, filed Apr. 27, 2004, now U.S. Pat. No. 7,399,853, issued Jul. 15, 2008, which claims benefit of U.S. provisional application 60/466,256, filed Apr. 28, 2003, the entire contents of each is being expressly incorporated herein by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0007USC1SEQ.txt, created on Feb. 9, 2010 which is 208 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulating the expression of glucagon receptor. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding glucagon receptor. Such compounds are shown herein to modulate the expression of glucagon receptor.

BACKGROUND OF THE INVENTION

The maintenance of normal glycemia is a carefully regulated metabolic event. Glucagon, the 29-amino acid peptide responsible for maintaining blood glucose levels in the postabsorbative state, increases glucose release from the liver by activating hepatic glycogenolysis, gluconeogenesis, stimulating lipolysis in adipose tissue, and stimulating insulin secretion. During high blood glucose levels, insulin reverses the glucagon-mediated enhancement of glycogenolysis and gluconeogenesis. In patients with diabetes, insulin is either not available or not fully effective. While treatment for diabetes has traditionally focused on increasing insulin levels, antagonism of glucagon function has been considered as an alternative therapy. As glucagon exerts its physiological effects by signaling through the glucagon receptor, the glucagon receptor has been proposed as a potential therapeutic target for diabetes (Madsen et al., Curr. Pharm. Des., 1999, 5, 683-691).
Glucagon receptor is belongs to the superfamily of G-protein-coupled receptors having seven transmembrane domains. It is also a member of the smaller sub-family of homologous receptors which bind peptides that are structurally similar to glucagon. The gene encoding human glucagon receptor was cloned in 1994 and analysis of the genomic sequence revealed multiple introns and an 82% identity to the rat glucagon receptor gene (Lok et al., Gene, 1994, 140, 203-209.; MacNeil et al., Biochem. Biophys. Res. Commun., 1994, 198, 328-334). Cloning of the rat glucagon receptor gene also led to the description of multiple alternative splice variants (Maget et al., FEBS Lett., 1994, 351, 271-275). Disclosed and claimed in U.S. Pat. No. 5,776,725 is an isolated nucleic acid sequence encoding a human or rat glucagon receptor (Kindsvogel et al., 1998). The human glucagon receptor gene is localized to chromosome 17q25 (Menzel et al., Genomics, 1994, 20, 327-328). A missense mutation of Gly to Ser at codon 40 in the glucagon receptor gene leads to a 3-fold lower affinity for glucagon (Fujisawa et al., Diabetologia, 1995, 38, 983-985) and this mutation has been linked to several disease states, including non-insulin-dependent diabetes mellitus (Fujisawa et al., Diabetologia, 1995, 38, 983-985), hypertension (Chambers and Morris, Nat. Genet., 1996, 12, 122), and central adiposity (Siani et al., Obes. Res., 2001, 9, 722-726).
Inhibiting glucagon function by antagonizing the glucagon receptor has been proposed as a therapeutic target for diabetes. Currently, there are no known therapeutic agents which effectively inhibit the synthesis of glucagon receptor and to date, investigative strategies aimed at modulating glucagon receptor function have involved the use of antibodies, peptidyl antagonists, and small molecules. In addition, targeted disruption of the glucagon receptor gene in mice has shown that, despite a total absence of glucagon receptors and elevated plasma glucagon levels, the mice maintain near-normal glycemia and lipidemia (Parker et al., Biochem. Biophys. Res. Commun., 2002, 290, 839-843). Patent application WO 02/45494 (Allen et al.) discloses transgenic mice comprising mutations in a glucagon receptor gene. Also claimed are agonists or antagonists of glucagon receptor, agents that modulate the function, expression or activity of a glucagon receptor gene, methods of identifying such agents, methods of ameliorating conditions associated with impaired glucose tolerance, methods of identifying agents that affect obesity, weight gain, diabetes, methods of treating obesity or diabetic conditions, and phenotypic data associated with a transgenic mouse comprising a mutation in a glucagon receptor gene.
A glucagon-neutralizing monoclonal antibody has been described that antagonizes glucagon-stimulated signal transduction in part by binding to the glucagon binding site of the glucagon receptor (Buggy et al., Horm. Metab. Res., 1996, 28, 215-219). An antibody which specifically binds to the amino acid sequence of a glucagon receptor has been disclosed and claimed in U.S. Pat. No. 5,770,445 (Kindsvogel et al., 1998).
Several peptidyl antagonists of glucagon receptor have been reported in the art. Six glucagon analogs with N-terminal modifications were designed to have a higher affinity than glucagon for the glucagon receptor (Zechel et al., Int. J. Pept. Protein Res., 1991, 38, 131-138). Two somatostatin analogs have been reported to be inhibitors of glucagon secretion (Rossowski and Coy, Biochem. Biophys. Res. Commun., 1994, 205, 341-346).
Many small molecules have been examined as glucagon receptor antagonists including: [(+)-3,5 diisopropyl-2-(1-hydroxyethyl)-6-propyl-4′-fluoro-1,1′-biphenyl (Bay27-9955) (Petersen and Sullivan, Diabetologia, 2001, 44, 2018-2024), a series of alkylidene hydrazides (Ling et al., Bioorg. Med. Chem. Lett., 2002, 12, 663-666), a series of 4-aryl-pyridines containing both a 3-[(1R)-hydroxyethyl] and a 2′-hydroxy group (Ladouceur et al., Bioorg. Med. Chem. Lett., 2002, 12, 3421-3424), a series of 5-hydroxyalkyl-4-phenylpyridines (Ladouceur et al., Bioorg. Med. Chem. Lett., 2002, 12, 461-464), a series of triarylimidazoles (Chang et al., Bioorg. Med. Chem. Lett., 2001, 11, 2549-2553), a series of 2-pyridyl-3,5-diaryl pyrroles (de Laszlo et al., Bioorg. Med. Chem. Lett., 1999, 9, 641-646), several substituted benzimidazoles (Madsen et al., J. Med. Chem., 1998, 41, 5150-5157), and a series of pyrrolo[1,2-a]quinoxalines (Guillon et al., Eur. J. Med. Chem., 1998, 33, 293-308).
There remains a long felt need for additional agents capable of effectively inhibiting glucagon receptor function. Antisense technology is an effective means for reducing the expression of specific gene products and has proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications. The present invention provides compositions and methods for modulating glucagon receptor expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding glucagon receptor, and which modulate the expression of glucagon receptor. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of glucagon receptor and methods of modulating the expression of glucagon receptor in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of glucagon receptor are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding glucagon receptor. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding glucagon receptor. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding glucagon receptor” have been used for convenience to encompass DNA encoding glucagon receptor, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of glucagon receptor. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.
In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid 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 under conditions in which assays are performed in the case of in vitro assays.
In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
“Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
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. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

B. Compounds of the Invention

According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.
The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620).
Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).
In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes 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 a target nucleic acid and increased stability in the presence of nucleases.
While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.
The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.
In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 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, or 50 nucleobases in length.
In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.
Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.
Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid 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 nucleic acid encodes glucagon receptor.
The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.
Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding glucagon receptor, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).
The terms “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. Similarly, the terms “stop codon region” and “translation termination 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 termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.
The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
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). The 5′ cap site of an mRNA comprises an N7-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 site. It is also preferred to target the 5′ cap region.
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.
Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.
The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.
While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.
Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of glucagon receptor. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding glucagon receptor and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding glucagon receptor with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding glucagon receptor. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding glucagon receptor, the modulator may then be employed in further investigative studies of the function of glucagon receptor, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.
Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).
The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between glucagon receptor and a disease state, phenotype, or condition. These methods include detecting or modulating glucagon receptor comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of glucagon receptor and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics, therapeutics (including prophylaxis) and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding glucagon receptor. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective glucagon receptor inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding glucagon receptor and in the amplification of said nucleic acid molecules for detection or for use in further studies of glucagon receptor. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding glucagon receptor 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 glucagon receptor in a sample may also be prepared.
The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of glucagon receptor is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to an animal a therapeutically effective amount of a glucagon receptor inhibitor. The glucagon receptor inhibitors of the present invention effectively inhibit the activity of the glucagon receptor protein or inhibit the expression of the glucagon receptor protein. In one embodiment, the activity or expression of glucagon receptor in an animal is inhibited by about 10%. Preferably, the activity or expression of glucagon receptor in an animal is inhibited by about 30%. More preferably, the activity or expression of glucagon receptor in an animal is inhibited by 50% or more. Because the compounds herein are inhibitors of glucagon receptor, they are believed to be useful in lowering blood glucose, for example, and in treating conditions associated with glucagon receptor activity, such as high blood glucose and other metabolic conditions such as diabetes (including Type 2 diabetes), obesity, and insulin resistance.
The reduction of the expression of glucagon receptor may be measured, for example, in blood, plasma, serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding glucagon receptor protein and/or the glucagon receptor protein itself.
The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, 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. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, 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.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂component parts.
Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones. Also preferred are oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.
Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_ngroup bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” 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 guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 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 other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further 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 et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Conjugates

Another modification of the 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. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenan-thridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.
Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
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. These 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, increased stability 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. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. 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. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Salts

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. The term “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. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Sodium salts are especially suitable salts of the compounds of the present invention.

G. Formulations

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. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
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. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other pharmaceutical agents which function by a non-antisense mechanism. Examples of such pharmaceutical agents include but are not limited to cancer chemotherapeutic drugs, anti-inflammatory drugs, anti-viral drugs, and compounds for treatment of metabolic diseases such as diabetes, high blood sugar or obesity, or cardiovascular conditions such as elevated blood cholesterol or blood pressure. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially. When used with the compounds of the invention, such pharmaceutical agents may be used individually (e.g., rosiglitazone and oligonucleotide), sequentially (e.g., 5-fluorouracil and oligonucleotide for a period of time followed by methotrexate and oligonucleotide), or in combination with one or more other treatments (e.g., 5-fluorouracil, methotrexate and oligonucleotide, or 5-fluorouracil, radiotherapy and oligonucleotide).
In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES

Example 1

Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O—[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

Oligonucleotide and Oligonucleoside Synthesis

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.
Oligonucleotides: Unsubstituted and substituted phosphodiester (P=0) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.
Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.
3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.
Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.
Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.
3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.
Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.
Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.
Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.
Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.
Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

RNA Synthesis

In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.
Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.
RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.
Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.
The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.
Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).
RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.
[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides
Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides
[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites.
[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]Chimeric Oligonucleotides
[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

Design and Screening of Duplexed Antisense Compounds Targeting Glucagon Receptor

In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target glucagon receptor. The nucleobase sequence of the antisense strand of the duplex comprises at least an 8-nucleobase portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.
For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO.: 824) and having a two-nucleobase overhang of deoxythymidine (dT) would have the following structure:
In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 824) may be prepared with blunt ends (no single strand overhang) as shown:
The RNA duplex can be unimolecular or biomolecular; i.e., the two strands can be part of a single molecule or may be separate molecules. RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.
Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate glucagon receptor expression.
When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides are purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material are similar to those obtained with non-HPLC purified material.

Example 7

Oligonucleotide Synthesis

96 Well Plate Format

Oligonucleotides are synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages are afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages are generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites are purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
Oligonucleotides are cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis

96-Well Plate Format

The concentration of oligonucleotide in each well is assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products is evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the compounds on the plate are at least 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells are routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.
For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells are routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) are obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs are routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells are maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) are obtained from the Clonetics Corporation (Walkersville, Md.). HEKs are routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells are routinely maintained for up to 10 passages as recommended by the supplier.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 is obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal calf serum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

Primary Mouse Hepatocytes

Primary mouse hepatocytes are prepared from CD-1 mice purchased from Charles River Labs. Primary mouse hepatocytes are routinely cultured in Hepatocyte Attachment Media (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone (Sigma), 10 nM bovine insulin (Sigma). Cells are seeded into 96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/well for use in RT-PCR analysis.
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
Treatment with Antisense Compounds:
When cells reached 65-75% confluency, they are treated with oligonucleotide. For cells grown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium is replaced with fresh medium. Cells are harvested 16-24 hours after oligonucleotide treatment.
The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

Analysis of Oligonucleotide Inhibition of Glucagon Receptor Expression

Antisense modulation of glucagon receptor expression can be assayed in a variety of ways known in the art. For example, glucagon receptor mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Protein levels of glucagon receptor can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to glucagon receptor can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

Design of Phenotypic Assays and In Vivo Studies for the Use of Glucagon Receptor Inhibitors Phenotypic Assays

Once glucagon receptor inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of glucagon receptor in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with glucagon receptor inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the glucagon receptor inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or glucagon receptor inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they may not be informed as to whether the medication they are administering is a glucagon receptor inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.
Volunteers may receive either the glucagon receptor inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements may include the levels of nucleic acid molecules encoding glucagon receptor or glucagon receptor protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements may include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.
Information recorded for each patient may include age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.
Volunteers taking part in this study are healthy adults (age 18 to 65 years) and, typically, roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and glucagon receptor inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the glucagon receptor inhibitor show positive trends in their disease state or condition index at the conclusion of the study.
One of ordinary skill will know how to conduct an appropriate clinical trial and will recognize that this is just one of many protocols which may be appropriately used.

Example 12

RNA Isolation

Poly(A)+ mRNA Isolation
Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μl, of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 1404 of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

Real-Time Quantitative PCR Analysis of Glucagon Receptor mRNA Levels

Quantitation of glucagon receptor mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
Probes and primers to human glucagon receptor were designed to hybridize to a human glucagon receptor sequence, using published sequence information (GenBank accession number NM_—000160.1, incorporated herein as SEQ ID NO:4). For human glucagon receptor the PCR primers were:
forward primer: GACACCCCCGCCAATACC (SEQ ID NO: 5)
reverse primer: CCGCATCTCTTGAACACGAA (SEQ ID NO: 6) and the PCR probe was: FAM-TTGGCACCACAAAGT-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:
forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)
reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.
Probes and primers to mouse glucagon receptor were designed to hybridize to a mouse glucagon receptor sequence, using published sequence information (GenBank accession number NM_—008101.1, incorporated herein as SEQ ID NO: 11). For mouse glucagon receptor the PCR primers were:
forward primer: ATTTCCTGCCCCTGGTACCT (SEQ ID NO:12)
reverse primer: CGGGCCCACACCTCTTG (SEQ ID NO: 13) and the PCR probe was: FAM-CCACAAAGTGCAGCACCGCCTAGTGT-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:
forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:15)
reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO:16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

Northern Blot Analysis of Glucagon Receptor mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
To detect human glucagon receptor, a human glucagon receptor specific probe was prepared by PCR using the forward primer GACACCCCCGCCAATACC (SEQ ID NO: 5) and the reverse primer CCGCATCTCTTGAACACGAA (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
To detect mouse glucagon receptor, a mouse glucagon receptor specific probe was prepared by PCR using the forward primer ATTTCCTGCCCCTGGTACCT (SEQ ID NO: 12) and the reverse primer CGGGCCCACACCTCTTG (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

Antisense Inhibition of Human Glucagon Receptor Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human glucagon receptor RNA, using published sequences (GenBank accession number NM_—000160.1, incorporated herein as SEQ ID NO: 4, a concatenation of three contigs from GenBank accession number AC069004.2, incorporated herein as SEQ ID NO: 18, and GenBank accession number AJ245489.1, incorporated herein as SEQ ID NO: 19). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human glucagon receptor mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which HepG2 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.

TABLE 1

Inhibition of human glucagon receptor mRNA levels by chimeric
phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap

		TARGET	TARGET			SEQ
ISIS #	REGION	SEQ ID NO	SITE	SEQUENCE	% INHIB	ID NO

310462	Coding	4	560	ccgcatctcttgaacacgaa	61	20

299881	5′UTR	4	97	ttgagcctcagggcccgcgc	56	21

299882	5′UTR	4	121	gtgtcctcccctgaagctgc	68	22

299883	5′UTR	4	163	gagtggcagagcagcagagc	38	23

299884	5′UTR	4	192	tgtgtgtgtacgctcctccg	76	24

299885	5′UTR	4	198	tcctggtgtgtgtgtacgct	67	25

299886	5′UTR	4	205	aatgcagtcctggtgtgtgt	30	26

299887	5′UTR	4	254	ctgggcagctagctgcctcc	38	27

299888	Start Codon	4	263	ggcatgcctctgggcagcta	73	28

299889	Coding	4	462	ccagcaggaatacttgtcga	43	29

299890	Coding	4	328	ggacctgtggctggcaggcc	72	30

299891	Coding	4	350	aagtccatcacctgagcgga	39	31

299892	Coding	4	361	tctcaaacaggaagtccatc	37	32

299893	Coding	4	366	ccacttctcaaacaggaagt	24	33

299894	Coding	4	386	cactggtcaccgtagagctt	31	34

299895	Coding	4	391	ggtgacactggtcaccgtag	40	35

299896	Coding	4	431	cacaccagctccgtgggagg	35	36

299897	Coding	4	442	aggttctgttgcacaccagc	76	37

299898	Coding	4	453	atacttgtcgaaggttctgt	28	38

299899	Coding	4	539	cggtgttgcactttgtggtg	85	39

299900	5′UTR	19	546	ccctggcagagacagcggca	79	40

299901	Coding	4	552	cttgaacacgaagcggtgtt	83	41

299902	Coding	4	564	gggcccgcatctcttgaaca	87	42

299903	intron: exon	18	15279	cttctgcgagttacagtggc	58	43
	junction

299904	Coding	4	651	ctggacctcaatctcctcgc	43	44

299905	Coding	4	656	tccttctggacctcaatctc	5	45

299906	Coding	4	663	ggccacctccttctggacct	80	46

299907	Coding	4	669	catcttggccacctccttct	39	47

299908	Coding	4	681	gaagctgctgtacatcttgg	71	48

299909	Coding	4	751	cccccaggatggccaaggcg	69	49

299910	Coding	4	830	acggagctggctttcagcac	49	50

299911	Coding	4	866	ctgtagcgggtcctgagcag	48	51

299912	Coding	4	872	ttctggctgtagcgggtcct	54	52

299913	Coding	4	879	gccaattttctggctgtagc	61	53

299914	Coding	4	889	tgaggtcgtcgccaattttc	56	54

299915	Coding	4	898	tgctgacactgaggtcgtcg	63	55

299916	Coding	4	904	gccaggtgctgacactgagg	67	56

299917	Coding	4	966	cacgatgccatattgcatga	59	57

299918	Coding	4	1028	gtggccaggcccagcaggtt	52	58

299919	Coding	4	1122	cagacacttgaccactgccc	40	59

299920	Coding	4	1182	ccgcaggatccaccagaagc	46	60

299921	Coding	4	1210	tgatcaggatggccaggaag	42	61

299922	Coding	4	1228	ggacgaagatgaagaagttg	44	62

299923	Coding	4	1259	cgcagcttggccacgagcag	8	63

299924	Coding	4	1274	tgcatctgccgtgcccgcag	58	64

299925	Coding	4	1291	acttgtagtctgtgtggtgc	34	65

299926	Coding	4	1415	aggtcgaagaagagcttggc	38	66

299927	Coding	4	1528	gcactttgcccaggcgccag	78	67

299928	Coding	4	1539	ctcctcccatagcactttgc	40	68

299929	Coding	4	1608	aaactgcagctccttgctgg	46	69

299930	Coding	4	1636	atgaatcctggctgccacca	70	70

299931	Coding	4	1670	ctagggaggccaccagccaa	49	71

299932	Coding	4	1681	tctcagccaatctagggagg	63	72

299933	Stop Codon	4	1704	tcccagcagggttcagaagg	30	73

299934	5′UTR	19	1747	ttcctgcaggtgacccaatg	50	74

299935	3′UTR	4	1841	tctcgcagacagccacactg	43	75

299936	3′UTR	4	1854	agaggaggcccaatctcgca	79	76

299937	3′UTR	4	1881	tgcaccagggacaaggcagg	0	77

299938	3′UTR	4	1901	tggactcctctgctcacctc	58	78

299939	3′UTR	4	1938	tggcacgcagttcacggcac	54	79

299940	3′UTR	4	1969	acatgggacgtgccgacata	63	80

299941	3′UTR	4	1978	tttccatgcacatgggacgt	63	81

299942	3′UTR	4	1989	gttggaggacatttccatgc	79	82

299943	3′UTR	4	2015	cacggtgaccacttgagctc	18	83

299944	intron	18	11002	agatgtccgtgtttgtcagc	9	84

299945	intron	18	11557	taataactttttaaagaagg	17	85

299946	intron	18	12295	tactacgttgctcgggctgg	23	86

299947	intron	18	14121	agctctgtggctcagttacc	74	87

299948	intron: exon	18	15467	gtgcagcttgctgtggcaca	47	88
	junction

299949	intron	18	16094	cagcaaccgcttggtacagg	100	89

299950	intron: exon	18	17017	agaagttgatctgtgtgaga	29	90
	junction

299951	intron: exon	18	17456	ccagcaggccctggagagac	53	91
	junction

304471	5′UTR	4	100	cctttgagcctcagggcccg	42	92

304472	5′UTR	4	103	gcccctttgagcctcagggc	25	93

304473	5′UTR	4	167	agctgagtggcagagcagca	76	94

304474	5′UTR	4	169	gcagctgagtggcagagcag	75	95

304475	5′UTR	4	190	tgtgtgtacgctcctccgag	73	96

304476	5′UTR	4	194	ggtgtgtgtgtacgctcctc	72	97

304477	5′UTR	4	196	ctggtgtgtgtgtacgctcc	71	98

304478	5′UTR	4	209	gggcaatgcagtcctggtgt	65	99

304479	5′UTR	4	246	ctagctgcctcccacatctg	54	100

304480	5′UTR	4	249	cagctagctgcctcccacat	85	101

304481	5′UTR	4	257	cctctgggcagctagctgcc	44	102

304482	Start Codon	4	262	gcatgcctctgggcagctag	62	103

304483	Coding	4	325	cctgtggctggcaggccagc	68	104

304484	Coding	4	368	ttccacttctcaaacaggaa	24	105

304485	Coding	4	370	gcttccacttctcaaacagg	49	106

304486	Coding	4	375	gtagagcttccacttctcaa	41	107

304487	Coding	4	376	cgtagagcttccacttctca	38	108

304488	Coding	4	395	ttgtggtgacactggtcacc	24	109

304489	Coding	4	407	agcaggctcaggttgtggtg	52	110

304490	Coding	4	534	ttgcactttgtggtgccaag	61	111

304491	Coding	4	535	gttgcactttgtggtgccaa	57	112

304492	Coding	4	536	tgttgcactttgtggtgcca	67	113

304493	Coding	4	537	gtgttgcactttgtggtgcc	75	114

304494	Coding	4	563	ggcccgcatctcttgaacac	87	115

304495	Coding	4	567	gtcgggcccgcatctcttga	81	116

304496	Coding	4	617	tgggaggcatcacgccaagg	60	117

304497	Coding	4	627	catctggcactgggaggcat	48	118

304498	Coding	4	666	cttggccacctccttctgga	74	119

304499	Coding	4	671	tacatcttggccacctcctt	24	120

304500	Coding	4	685	cctggaagctgctgtacatc	71	121

304501	Coding	4	795	attcgcgtggatggcattgc	53	122

304502	Coding	4	848	agcccatcaatgaccagcac	31	123

304503	Coding	4	861	gcgggtcctgagcagcccat	42	124

304504	Coding	4	886	ggtcgtcgccaattttctgg	50	125

304505	Coding	4	893	acactgaggtcgtcgccaat	22	126

304506	Coding	4	900	ggtgctgacactgaggtcgt	60	127

304507	Coding	4	962	atgccatattgcatgaacac	27	128

304508	Coding	4	1032	gagggtggccaggcccagca	56	129

304509	Coding	4	1124	aacagacacttgaccactgc	13	130

304510	Coding	4	1125	gaacagacacttgaccactg	8	131

304511	Coding	4	1158	gttgtcattgctggtccagc	65	132

304512	Coding	4	1168	agaagcccatgttgtcattg	44	133

304513	Coding	4	1187	gggaaccgcaggatccacca	42	134

304514	Coding	4	1230	gcggacgaagatgaagaagt	54	135

304515	Coding	4	1638	agatgaatcctggctgccac	53	136

304516	3′UTR	4	1727	ccagagtccagccctagctg	41	137

304517	3′UTR	4	1732	gggtgccagagtccagccct	48	138

304518	3′UTR	4	1735	tctgggtgccagagtccagc	65	139

304519	3′UTR	4	1736	ctctgggtgccagagtccag	75	140

304520	3′UTR	4	1737	cctctgggtgccagagtcca	74	141

304521	3′UTR	4	1740	acgcctctgggtgccagagt	55	142

304522	3′UTR	4	1760	cagttctgggttgtccagcg	52	143

304523	3′UTR	4	1849	aggcccaatctcgcagacag	74	144

304524	3′UTR	4	1850	gaggcccaatctcgcagaca	80	145

304525	3′UTR	4	1856	ggagaggaggcccaatctcg	66	146

304526	3′UTR	4	1861	tgcagggagaggaggcccaa	63	147

304527	3′UTR	4	1883	tctgcaccagggacaaggca	50	148

304528	3′UTR	4	1891	tgctcacctctgcaccaggg	66	149

304529	3′UTR	4	1893	tctgctcacctctgcaccag	32	150

304530	3′UTR	4	1899	gactcctctgctcacctctg	31	151

304531	3′UTR	4	1905	gccctggactcctctgctca	69	152

304532	3′UTR	4	1932	gcagttcacggcacagcccc	53	153

304533	3′UTR	4	1933	cgcagttcacggcacagccc	30	154

304534	3′UTR	4	1945	gggacactggcacgcagttc	61	155

304535	3′UTR	4	1971	gcacatgggacgtgccgaca	83	156

304536	3′UTR	4	1984	aggacatttccatgcacatg	61	157

304537	3′UTR	4	1986	ggaggacatttccatgcaca	69	158

304538	3′UTR	4	1999	gctctttattgttggaggac	66	159

304539	3′UTR	4	2001	gagctctttattgttggagg	68	160

304540	3′UTR	4	2008	accacttgagctctttattg	40	161

304541	intron	18	3174	ggcagttttggcgtccccag	67	162

304542	intron	18	6670	gagcttcctgcctcttcacg	39	163

304543	intron	18	7544	ggataggatgtgcgtgtcta	42	164

304544	intron	18	7975	ctctctgcctccgatttctt	12	165

304545	intron: exon	18	14888	acaccagctctgcagggtag	75	166
	junction

304546	intron: exon	18	15285	cacctccttctgcgagttac	33	167
	junction

310441	Start Codon	4	258	gcctctgggcagctagctgc	64	168

310442	Coding	4	317	tggcaggccagcagcagcag	87	169

310443	Coding	4	321	tggctggcaggccagcagca	88	170

310444	Coding	4	347	tccatcacctgagcggaggg	55	171

310445	Coding	4	351	gaagtccatcacctgagcgg	36	172

310446	Coding	4	355	acaggaagtccatcacctga	28	173

310447	Coding	4	365	cacttctcaaacaggaagtc	59	174

310448	Coding	4	389	tgacactggtcaccgtagag	18	175

310449	Coding	4	393	gtggtgacactggtcaccgt	12	176

310450	Coding	4	397	ggttgtggtgacactggtca	72	177

310451	Coding	4	403	ggctcaggttgtggtgacac	62	178

310452	Coding	4	452	tacttgtcgaaggttctgtt	44	179

310453	Coding	4	458	caggaatacttgtcgaaggt	40	180

310454	Coding	4	493	tgttggccgtggtattggcg	90	181

310455	Coding	4	497	gagatgttggccgtggtatt	87	182

310456	Coding	4	500	caggagatgttggccgtggt	95	183

310457	Coding	4	532	gcactttgtggtgccaaggc	96	184

310458	Coding	4	540	gcggtgttgcactttgtggt	92	185

310459	Coding	4	544	cgaagcggtgttgcactttg	50	186

310460	Coding	4	548	aacacgaagcggtgttgcac	87	187

310461	Coding	4	556	atctcttgaacacgaagcgg	65	188

310463	Coding	4	588	gggtccacgcacccactgac	50	189

310464	Coding	4	606	acgccaaggctgcccccggg	71	190

310465	Coding	4	660	cacctccttctggacctcaa	31	191

310466	Coding	4	683	tggaagctgctgtacatctt	57	192

310467	Coding	4	687	cacctggaagctgctgtaca	60	193

310468	Coding	4	691	acatcacctggaagctgctg	73	194

310469	Coding	4	695	gtgtacatcacctggaagct	79	195

310470	Coding	4	720	ccccagggacaggctgtagc	86	196

310471	Coding	4	723	ggcccccagggacaggctgt	62	197

310472	Coding	4	860	cgggtcctgagcagcccatc	48	198

310473	Coding	4	864	gtagcgggtcctgagcagcc	58	199

310474	Coding	4	868	ggctgtagcgggtcctgagc	48	200

310475	Coding	4	919	ccgctccatcactgagccag	52	201

310476	Coding	4	923	gccaccgctccatcactgag	41	202

310477	Coding	4	951	catgaacaccgcggccacac	63	203

310478	Coding	4	955	attgcatgaacaccgcggcc	76	204

310479	Coding	4	960	gccatattgcatgaacaccg	66	205

310480	Coding	4	1019	cccagcaggttgtgcaggta	58	206

310481	Coding	4	1025	gccaggcccagcaggttgtg	72	207

310482	Coding	4	1029	ggtggccaggcccagcaggt	83	208

310483	Coding	4	1055	aggctgaagaagctcctctc	71	209

310484	Coding	4	1059	gtagaggctgaagaagctcc	46	210

310485	Coding	4	1063	ccaggtagaggctgaagaag	25	211

310486	Coding	4	1068	gatgcccaggtagaggctga	51	212

310487	Coding	4	1072	agccgatgcccaggtagagg	70	213

310488	Coding	4	1156	tgtcattgctggtccagcac	83	214

310489	Coding	4	1160	atgttgtcattgctggtcca	53	215

310490	Coding	4	1167	gaagcccatgttgtcattgc	45	216

310491	Coding	4	1173	ccaccagaagcccatgttgt	50	217

310492	Coding	4	1176	gatccaccagaagcccatgt	53	218

310493	Coding	4	1185	gaaccgcaggatccaccaga	47	219

310494	Coding	4	1206	caggatggccaggaagacgg	39	220

310495	Coding	4	1209	gatcaggatggccaggaaga	67	221

310496	Coding	4	1219	tgaagaagttgatcaggatg	10	222

310497	Coding	4	1222	agatgaagaagttgatcagg	20	223

310498	Coding	4	1287	gtagtctgtgtggtgcatct	35	224

310499	Coding	4	1290	cttgtagtctgtgtggtgca	63	225

310500	Coding	4	1293	gaacttgtagtctgtgtggt	27	226

310501	Coding	4	1414	ggtcgaagaagagcttggcg	46	227

310502	Coding	4	1417	agaggtcgaagaagagcttg	26	228

310503	Coding	4	1423	tgaggaagaggtcgaagaag	17	229

310504	Coding	4	1669	tagggaggccaccagccaag	53	230

315163	Coding	4	686	acctggaagctgctgtacat	75	231

315164	Coding	4	409	gcagcaggctcaggttgtgg	24	232

315165	Coding	4	1424	ctgaggaagaggtcgaagaa	42	233

315166	Coding	4	398	aggttgtggtgacactggtc	34	234

315167	Coding	4	1212	gttgatcaggatggccagga	47	235

315168	Coding	4	1062	caggtagaggctgaagaagc	40	236

315169	Coding	4	559	cgcatctcttgaacacgaag	48	237

315170	Coding	4	543	gaagcggtgttgcactttgt	61	238

315171	Coding	4	454	aatacttgtcgaaggttctg	16	239

315172	Coding	4	1026	ggccaggcccagcaggttgt	72	240

315173	Coding	4	1070	ccgatgcccaggtagaggct	59	241

315174	Coding	4	496	agatgttggccgtggtattg	79	242

315175	Coding	4	399	caggttgtggtgacactggt	58	243

315176	Coding	4	1420	ggaagaggtcgaagaagagc	26	244

315177	Coding	4	392	tggtgacactggtcaccgta	49	245

315178	Coding	4	402	gctcaggttgtggtgacact	62	246

315179	Coding	4	533	tgcactttgtggtgccaagg	75	247

315180	Coding	4	689	atcacctggaagctgctgta	45	248

315181	Coding	4	956	tattgcatgaacaccgcggc	78	249

315182	Coding	4	1208	atcaggatggccaggaagac	36	250

315183	Coding	4	555	tctcttgaacacgaagcggt	71	251

315184	Coding	4	553	tcttgaacacgaagcggtgt	87	252

315185	Coding	4	1027	tggccaggcccagcaggttg	61	253

315186	Coding	4	871	tctggctgtagcgggtcctg	73	254

315187	Coding	4	498	ggagatgttggccgtggtat	93	255

315188	Start Codon	4	259	tgcctctgggcagctagctg	70	256

315189	Coding	4	1058	tagaggctgaagaagctcct	54	257

315190	Coding	4	348	gtccatcacctgagcggagg	68	258

315191	Coding	4	1292	aacttgtagtctgtgtggtg	39	259

315192	Stop Codon	4	1705	gtcccagcagggttcagaag	31	260

315193	Coding	4	953	tgcatgaacaccgcggccac	73	261

315194	Coding	4	1024	ccaggcccagcaggttgtgc	73	262

315195	Coding	4	1061	aggtagaggctgaagaagct	57	263

315196	Coding	4	1169	cagaagcccatgttgtcatt	47	264

315197	Coding	4	1161	catgttgtcattgctggtcc	0	265

315198	Coding	4	1021	ggcccagcaggttgtgcagg	84	266

315199	Coding	4	400	tcaggttgtggtgacactgg	42	267

315200	Coding	4	1165	agcccatgttgtcattgctg	45	268

315201	Coding	4	363	cttctcaaacaggaagtcca	47	269

315202	Coding	4	550	tgaacacgaagcggtgttgc	83	270

315203	Coding	4	367	tccacttctcaaacaggaag	69	271

315204	Coding	4	353	aggaagtccatcacctgagc	26	272

315205	Coding	4	1071	gccgatgcccaggtagaggc	82	273

315206	Coding	4	1186	ggaaccgcaggatccaccag	36	274

315207	Coding	4	349	agtccatcacctgagcggag	63	275

315208	Coding	4	1221	gatgaagaagttgatcagga	28	276

315209	Coding	4	461	cagcaggaatacttgtcgaa	27	277

315210	Coding	4	463	gccagcaggaatacttgtcg	41	278

315211	Coding	4	320	ggctggcaggccagcagcag	72	279

315212	Coding	4	1183	accgcaggatccaccagaag	59	280

315213	Coding	4	862	agcgggtcctgagcagccca	68	281

315214	Coding	4	565	cgggcccgcatctcttgaac	88	282

315215	Coding	4	1295	cggaacttgtagtctgtgtg	29	283

315216	Coding	4	1177	ggatccaccagaagcccatg	58	284

315217	Stop Codon	4	1706	ggtcccagcagggttcagaa	34	285

315218	Coding	4	1184	aaccgcaggatccaccagaa	55	286

315219	Coding	4	410	ggcagcaggctcaggttgtg	50	287

315220	Coding	4	495	gatgttggccgtggtattgg	86	288

315221	Coding	4	455	gaatacttgtcgaaggttct	37	289

315222	Coding	4	1215	gaagttgatcaggatggcca	39	290

315223	Coding	4	688	tcacctggaagctgctgtac	48	291

315224	Coding	4	959	ccatattgcatgaacaccgc	20	292

315225	Coding	4	863	tagcgggtcctgagcagccc	61	293

315226	5′UTR	4	256	ctctgggcagctagctgcct	28	294

315227	Coding	4	359	tcaaacaggaagtccatcac	17	295

315228	Coding	4	1172	caccagaagcccatgttgtc	15	296

315229	Coding	4	694	tgtacatcacctggaagctg	67	297

315230	Coding	4	494	atgttggccgtggtattggc	52	298

315231	Coding	4	1069	cgatgcccaggtagaggctg	7	299

315232	Coding	4	1178	aggatccaccagaagcccat	83	300

315233	Coding	4	1207	tcaggatggccaggaagacg	52	301

315234	Coding	4	352	ggaagtccatcacctgagcg	60	302

315235	Start Codon	4	261	catgcctctgggcagctagc	65	303

315236	Coding	4	561	cccgcatctcttgaacacga	51	304

315237	Coding	4	323	tgtggctggcaggccagcag	60	305

315238	Coding	4	324	ctgtggctggcaggccagca	43	306

315239	Coding	4	1179	caggatccaccagaagccca	88	307

315240	Coding	4	1223	aagatgaagaagttgatcag	0	308

315241	Coding	4	1289	ttgtagtctgtgtggtgcat	66	309

315242	Coding	4	322	gtggctggcaggccagcagc	47	310

315243	Coding	4	406	gcaggctcaggttgtggtga	44	311

315244	Coding	4	870	ctggctgtagcgggtcctga	61	312

315245	5′UTR	4	255	tctgggcagctagctgcctc	24	313

315246	Coding	4	464	ggccagcaggaatacttgtc	71	314

315247	Coding	4	360	ctcaaacaggaagtccatca	13	315

315248	Coding	4	1060	ggtagaggctgaagaagctc	49	316

315249	Coding	4	1422	gaggaagaggtcgaagaaga	69	317

315250	Coding	4	1416	gaggtcgaagaagagcttgg	32	318

315251	Coding	4	1288	tgtagtctgtgtggtgcatc	30	319

315252	Coding	4	1216	agaagttgatcaggatggcc	17	320

315253	Coding	4	542	aagcggtgttgcactttgtg	55	321

315254	Coding	4	456	ggaatacttgtcgaaggttc	44	322

315255	Coding	4	1419	gaagaggtcgaagaagagct	34	323

315256	Coding	4	460	agcaggaatacttgtcgaag	10	324

315257	Coding	4	404	aggctcaggttgtggtgaca	58	325

315258	Coding	4	538	ggtgttgcactttgtggtgc	58	326

315259	Coding	4	1294	ggaacttgtagtctgtgtgg	30	327

315260	Coding	4	390	gtgacactggtcaccgtaga	19	328

315261	Coding	4	954	ttgcatgaacaccgcggcca	59	329

315262	Coding	4	684	ctggaagctgctgtacatct	61	330

315263	Coding	4	1174	tccaccagaagcccatgttg	1	331

315264	Coding	4	1214	aagttgatcaggatggccag	44	332

315265	Coding	4	1023	caggcccagcaggttgtgca	51	333

315266	Coding	4	920	accgctccatcactgagcca	38	334

315267	Coding	4	1220	atgaagaagttgatcaggat	0	335

315268	Coding	4	554	ctcttgaacacgaagcggtg	78	336

315269	Coding	4	318	ctggcaggccagcagcagca	37	337

315270	Coding	4	499	aggagatgttggccgtggta	97	338

315271	Coding	4	1164	gcccatgttgtcattgctgg	66	339

315272	Coding	4	1217	aagaagttgatcaggatggc	25	340

315273	Coding	4	1064	cccaggtagaggctgaagaa	62	341

315274	Coding	4	1163	cccatgttgtcattgctggt	55	342

315275	Coding	4	547	acacgaagcggtgttgcact	46	343

315276	Coding	4	408	cagcaggctcaggttgtggt	62	344

315277	Coding	4	394	tgtggtgacactggtcaccg	15	345

315278	Coding	4	1020	gcccagcaggttgtgcaggt	83	346

315279	Coding	4	869	tggctgtagcgggtcctgag	33	347

315280	Coding	4	562	gcccgcatctcttgaacacg	77	348

315281	Coding	4	1418	aagaggtcgaagaagagctt	40	349

315282	Coding	4	411	gggcagcaggctcaggttgt	23	350

315283	Coding	4	557	catctcttgaacacgaagcg	40	351

315284	Coding	4	1175	atccaccagaagcccatgtt	38	352

315285	Coding	4	1155	gtcattgctggtccagcact	75	353

315286	Coding	4	566	tcgggcccgcatctcttgaa	74	354

315287	Coding	4	721	cccccagggacaggctgtag	53	355

315288	Coding	4	1162	ccatgttgtcattgctggtc	43	356

315289	Coding	4	1056	gaggctgaagaagctcctct	2	357

315290	Coding	4	549	gaacacgaagcggtgttgca	88	358

315291	Coding	4	362	ttctcaaacaggaagtccat	9	359

315292	Coding	4	1159	tgttgtcattgctggtccag	47	360

315293	Coding	4	457	aggaatacttgtcgaaggtt	55	361

315294	Coding	4	405	caggctcaggttgtggtgac	38	362

315295	Coding	4	1421	aggaagaggtcgaagaagag	19	363

315296	Coding	4	1425	gctgaggaagaggtcgaaga	33	364

315297	Coding	4	546	cacgaagcggtgttgcactt	81	365

315298	Coding	4	1166	aagcccatgttgtcattgct	35	366

315299	Start Codon	4	260	atgcctctgggcagctagct	63	367

315300	Coding	4	690	catcacctggaagctgctgt	63	368

315301	Coding	4	364	acttctcaaacaggaagtcc	31	369

315302	Coding	4	558	gcatctcttgaacacgaagc	44	370

315303	Coding	4	958	catattgcatgaacaccgcg	48	371

315304	Coding	4	1170	ccagaagcccatgttgtcat	33	372

315305	Coding	4	867	gctgtagcgggtcctgagca	50	373

315306	Coding	4	865	tgtagcgggtcctgagcagc	62	374

315307	Coding	4	1022	aggcccagcaggttgtgcag	37	375

315308	Coding	4	692	tacatcacctggaagctgct	41	376

315309	Coding	4	1181	cgcaggatccaccagaagcc	49	377

315310	Coding	4	357	aaacaggaagtccatcacct	21	378

315311	Coding	4	1057	agaggctgaagaagctcctc	49	379

315312	Coding	4	1211	ttgatcaggatggccaggaa	54	380

315313	Coding	4	541	agcggtgttgcactttgtgg	81	381

315314	Coding	4	319	gctggcaggccagcagcagc	75	382

315315	Coding	4	545	acgaagcggtgttgcacttt	68	383

315316	Coding	4	952	gcatgaacaccgcggccaca	80	384

315317	Coding	4	354	caggaagtccatcacctgag	26	385

315318	Coding	4	1180	gcaggatccaccagaagccc	72	386

315319	Coding	4	1213	agttgatcaggatggccagg	33	387

315320	Coding	4	722	gcccccagggacaggctgta	51	388

315321	Coding	4	356	aacaggaagtccatcacctg	0	389

315322	Coding	4	957	atattgcatgaacaccgcgg	56	390

315323	Coding	4	459	gcaggaatacttgtcgaagg	59	391

315324	Coding	4	693	gtacatcacctggaagctgc	79	392

315325	Coding	4	1153	cattgctggtccagcactgg	61	393

315326	Coding	4	358	caaacaggaagtccatcacc	10	394

315327	Coding	4	1031	agggtggccaggcccagcag	27	395

315328	Coding	4	551	ttgaacacgaagcggtgttg	66	396

315329	Coding	4	1171	accagaagcccatgttgtca	47	397

315330	Coding	4	401	ctcaggttgtggtgacactg	14	398

315331	Coding	4	396	gttgtggtgacactggtcac	5	399

As shown in Table 1, SEQ ID NOs 20, 21, 22, 24, 25, 28, 29, 30, 35, 37, 39, 40, 41, 42, 43, 44, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79, 80, 81, 82, 87, 88, 89, 91, 92, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 107, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 124, 125, 127, 129, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 152, 153, 155, 156, 157, 158, 159, 160, 161, 162, 164, 166, 168, 169, 170, 171, 174, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 217, 218, 219, 221, 225, 227, 230, 231, 233, 235, 236, 237, 238, 240, 241, 242, 243, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 261, 262, 263, 264, 266, 267, 268, 269, 270, 271, 273, 275, 278, 279, 280, 281, 282, 284, 286, 287, 288, 291, 293, 297, 298, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 311, 312, 314, 316, 317, 321, 322, 325, 326, 329, 330, 332, 333, 336, 338, 339, 341, 342, 343, 344, 346, 348, 349, 351, 353, 354, 355, 356, 358, 360, 361, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 381, 382, 383, 384, 386, 388, 390, 391, 392, 393, 396 and 397 demonstrated at least 40% inhibition of human glucagon receptor expression in this assay and are therefore preferred. SEQ ID NO: 183, 184, 231, 249, 254, 346, 365 and 392 are presently more preferred. The target regions to which the preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found.

Example 16

Antisense Inhibition of Mouse Glucagon Receptor Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series of antisense compounds were designed to target different regions of the mouse glucagon receptor RNA, using published sequences (GenBank accession number NM_—008101.1, incorporated herein as SEQ ID NO: 11, an mRNA sequence derived from GenBank accession number AF229079.1 with an alternate promoter, incorporated herein as SEQ ID NO: 400, GenBank accession number AF229079.1, incorporated herein as SEQ ID NO: 401, a second mRNA sequence derived from GenBank accession number AF229079.1 with an alternate promoter, incorporated herein as SEQ ID NO: 402, and GenBank accession number AA920726.1, incorporated herein as SEQ ID NO: 403). The compounds are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse glucagon receptor mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which mouse primary hepatocytes were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.

TABLE 2

Inhibition of mouse glucagon receptor mRNA levels by chimeric phosphorothioate
oligonucleotides having 2′-MOE wings and a deoxy gap

		TARGET	TARGET				CONTROL
ISIS #	REGION	SEQ ID NO	SITE	SEQUENCE	% INHIB	SEQ ID NO	SEQ ID NO

148350	5′UTR	11	57	cccacatctggcagaggttg	30	404	1

148355	coding	11	182	ttctcaaacaaaaagtccat	7	405	1

148356	coding	11	193	agagcttccacttctcaaac	63	406	1

148357	coding	11	203	tggtcactatagagcttcca	39	407	1

148359	coding	11	227	agcaggcttaggttgtggtg	39	408	1

148363	coding	11	322	ggcaggaaatgttggcagtg	52	409	1

148366	coding	11	383	ggcccacacctcttgaacac	93	410	1

148368	exon: exon	11	477	ccccttctggacctcgatct	45	411	1
	junction

148371	coding	11	538	ccagggacagactgtagccc	53	412	1

148372	exon: exon	11	589	agtgcagcttcctgaggccc	52	413	1
	junction

148381	coding	11	938	cacttgaccaccacccaggg	47	414	1

148382	coding	11	947	tcaaacagacacttgaccac	0	415	1

148385	coding	11	977	ttgtcattgctggtccagca	57	416	1

148387	coding	11	998	aggatccaccagaatcccat	53	417	1

148390	coding	11	1139	agggtcagcgtggacctggc	45	418	1

148393	coding	11	1226	aagagcttggtggagcgcag	26	419	1

148394	coding	11	1277	tagagaacagccaccagcag	26	420	1

148395	coding	11	1285	ggaaacagtagagaacagcc	26	421	1

148396	exon: exon	11	1299	cacctccttgttgaggaaac	0	422	1
	junction

180446	5′UTR	11	7	ctcctcaggttgcaagggag	15	423	1

180447	5′UTR	11	14	tgcacctctcctcaggttgc	38	424	1

180448	5′UTR	11	25	ctcagagtgtgtgcacctct	54	425	1

180449	5′UTR	11	30	aggtcctcagagtgtgtgca	55	426	1

180450	5′UTR	11	48	ggcagaggttgcacacctag	39	427	1

180451	Start Codon	11	80	ggcatgcctctgggtagcca	40	428	1

180452	Coding	11	141	tggcagacatgacagcacca	5	429	1

180453	Coding	11	192	gagcttccacttctcaaaca	37	430	1

180454	Coding	11	251	cagaccagctcagtaggtgg	45	431	1

180455	Coding	11	291	ggtgtcaggccagcaggagt	58	432	1

180456	Coding	11	359	cggtgctgcactttgtggca	68	433	1

180457	Coding	11	371	ttgaacactaggcggtgctg	69	434	1

180458	Coding	11	410	cgtggccctcgaacccactg	39	435	1

180459	Coding	11	545	aaggcccccagggacagact	56	436	1

180460	Coding	11	572	cccagcaggatgaccagcgc	59	437	1

180461	Coding	11	582	cttcctgaggcccagcagga	40	438	1

180462	Coding	11	650	acagagccagccttgagcac	47	439	1

180463	Coding	11	764	actgtggccactctgcagcc	43	440	1

180464	Coding	11	775	actgcatgatcactgtggcc	60	441	1

180465	Coding	11	785	atgatgccgtactgcatgat	58	442	1

180466	Coding	11	836	agcaggctgtacaggtacac	36	443	1

180467	Coding	11	974	tcattgctggtccagcactg	62	444	1

180468	Coding	11	1011	gacaggaatacgcaggatcc	0	445	1

180469	Coding	11	1079	cgcagcttggccacaagaag	56	446	1

180470	Coding	11	1090	tctgatgggcacgcagcttg	8	447	1

180471	Coding	11	1100	gcatagtgcatctgatgggc	45	448	1

180472	Coding	11	1110	cttgtaatcagcatagtgca	14	449	1

180473	Coding	11	1256	ccctggaaggagctgaggaa	45	450	1

180474	Coding	11	1292	ttgttgaggaaacagtagag	47	451	1

180475	Coding	11	1348	gagctttgccttcttgccat	64	452	1

180476	Coding	11	1360	tttcctcctgaagagctttg	56	453	1

180477	Coding	11	1388	atgtggctgccatggctgct	64	454	1

180478	Coding	11	1435	gctgaagtttctcacaggga	56	455	1

180479	Coding	11	1450	tgcctgcactcataagctga	48	456	1

180480	Coding	11	1470	acagccagtcccactgctgc	41	457	1

180481	Coding	11	1512	ccttgggagactactggcca	56	458	1

180482	Stop Codon	11	1544	caagtggagattcaggtggg	47	459	1

180483	3′UTR	11	1567	ttgaacacaacctgcctagg	9	460	1

180484	3′UTR	11	1575	gccctttcttgaacacaacc	34	461	1

180485	3′UTR	11	1600	atctggctctgggttgtcct	59	462	1

180486	3′UTR	11	1610	ttggccgggcatctggctct	53	463	1

180487	3′UTR	11	1620	ctcttcaaccttggccgggc	66	464	1

180488	3′UTR	11	1646	tacaagctgctgtcttgctg	54	465	1

180489	3′UTR	11	1687	ggcctgtgccaggctaggac	47	466	1

180490	3′UTR	11	1724	gcttctccatcatatccaac	47	467	1

180491	3′UTR	11	1750	aacactcagagttcatagat	51	468	1

180492	3′UTR	11	1756	catgggaacactcagagttc	49	469	1

180493	3′UTR	11	1795	ctgaaggacatatctgggta	51	470	1

180494	intron	401	3953	gtaacaaaggcgagaccaag	36	471	1

180495	intron	401	5396	gaggaagtgtcaccattagg	23	472	1

180496	intron: exon	401	7321	cagaccagctctgtgaaggt	32	473	1
	junction

180497	intron: exon	401	7505	cggtgctgcactgggcatgg	77	474	1
	junction

180498	exon: intron	401	8075	ctgggctcaccccgtcactg	27	475	1
	junction

180499	intron	401	8766	ccaaggatgggcaacctgac	33	476	1

180500	exon: intron	401	9005	ccttaccaaccggaacttgt	2	477	1
	junction

180501	genomic	402	128	cctctcctcaggtgtgctca	3	478	1

180502	genomic	400	10	ccaagcccaaggcctcatga	40	479	1

180503	genomic	400	85	ctcaggctgcagaggaccag	39	480	1

180504	genomic	403	40	taggtctcttccctccactc	4	481	1

As shown in Table 2, SEQ ID NOs 404, 406, 407, 408, 409, 410, 411, 412, 413, 414, 416, 417, 418, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 476, 479 and 480 demonstrated at least 30% inhibition of mouse glucagon receptor expression in this experiment and are therefore preferred. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds shown in Tables 1 and 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found.

TABLE 3

Sequence and position of preferred
target segments identified in glucagon receptor.

	TARGET	TARGET		REV COMP
SITE ID	SEQ ID NO	SITE	SEQUENCE	OF SEQ ID	ACTIVE IN	SEQ ID NO

215734	4	97	gcgcgggccctgaggctcaa	21	H. sapiens	484

215735	4	121	gcagcttcaggggaggacac	22	H. sapiens	485

215737	4	192	cggaggagcgtacacacaca	24	H. sapiens	486

215738	4	198	agcgtacacacacaccagga	25	H. sapiens	487

110316	4	263	tagctgcccagaggcatgcc	28	H. sapiens	488

215800	4	462	tcgacaagtattcctgctgg	29	H. sapiens	489

215741	4	328	ggcctgccagccacaggtcc	30	H. sapiens	490

215746	4	391	ctacggtgaccagtgtcacc	35	H. sapiens	491

215748	4	442	gctggtgtgcaacagaacct	37	H. sapiens	492

110318	4	539	caccacaaagtgcaacaccg	39	H. sapiens	493

215798	19	546	tgccgctgtctctgccaggg	40	H. sapiens	494

215750	4	552	aacaccgcttcgtgttcaag	41	H. sapiens	495

215751	4	564	tgttcaagagatgcgggccc	42	H. sapiens	496

215801	18	15279	gccactgtaactcgcagaag	43	H. sapiens	497

215752	4	651	gcgaggagattgaggtccag	44	H. sapiens	498

215754	4	663	aggtccagaaggaggtggcc	46	H. sapiens	499

215756	4	681	ccaagatgtacagcagcttc	48	H. sapiens	500

215757	4	751	cgccttggccatcctggggg	49	H. sapiens	501

215758	4	830	gtgctgaaagccagctccgt	50	H. sapiens	502

215759	4	866	ctgctcaggacccgctacag	51	H. sapiens	503

215760	4	872	aggacccgctacagccagaa	52	H. sapiens	504

215761	4	879	gctacagccagaaaattggc	53	H. sapiens	505

215762	4	889	gaaaattggcgacgacctca	54	H. sapiens	506

215763	4	898	cgacgacctcagtgtcagca	55	H. sapiens	507

215764	4	904	cctcagtgtcagcacctggc	56	H. sapiens	508

215765	4	966	tcatgcaatatggcatcgtg	57	H. sapiens	509

215766	4	1028	aacctgctgggcctggccac	58	H. sapiens	510

215767	4	1122	gggcagtggtcaagtgtctg	59	H. sapiens	511

215768	4	1182	gcttctggtggatcctgcgg	60	H. sapiens	512

215769	4	1210	cttcctggccatcctgatca	61	H. sapiens	513

215770	4	1228	caacttcttcatcttcgtcc	62	H. sapiens	514

215771	4	1274	ctgcgggcacggcagatgca	64	H. sapiens	515

215774	4	1528	ctggcgcctgggcaaagtgc	67	H. sapiens	516

215775	4	1539	gcaaagtgctatgggaggag	68	H. sapiens	517

215776	4	1608	ccagcaaggagctgcagttt	69	H. sapiens	518

215777	4	1636	tggtggcagccaggattcat	70	H. sapiens	519

215778	4	1670	ttggctggtggcctccctag	71	H. sapiens	520

215779	4	1681	cctccctagattggctgaga	72	H. sapiens	521

215799	19	1747	cattgggtcacctgcaggaa	74	H. sapiens	522

215781	4	1841	cagtgtggctgtctgcgaga	75	H. sapiens	523

215782	4	1854	tgcgagattgggcctcctct	76	H. sapiens	524

215784	4	1901	gaggtgagcagaggagtcca	78	H. sapiens	525

215785	4	1938	gtgccgtgaactgcgtgcca	79	H. sapiens	526

215786	4	1969	tatgtcggcacgtcccatgt	80	H. sapiens	527

215787	4	1978	acgtcccatgtgcatggaaa	81	H. sapiens	528

215788	4	1989	gcatggaaatgtcctccaac	82	H. sapiens	529

215793	18	14121	ggtaactgagccacagagct	87	H. sapiens	530

215794	18	15467	tgtgccacagcaagctgcac	88	H. sapiens	531

215795	18	16094	cctgtaccaagcggttgctg	89	H. sapiens	532

215797	18	17456	gtctctccagggcctgctgg	91	H. sapiens	533

220245	4	100	cgggccctgaggctcaaagg	92	H. sapiens	534

220247	4	167	tgctgctctgccactcagct	94	H. sapiens	535

220248	4	169	ctgctctgccactcagctgc	95	H. sapiens	536

220249	4	190	ctcggaggagcgtacacaca	96	H. sapiens	537

220250	4	194	gaggagcgtacacacacacc	97	H. sapiens	538

220251	4	196	ggagcgtacacacacaccag	98	H. sapiens	539

220252	4	209	acaccaggactgcattgccc	99	H. sapiens	540

220253	4	246	cagatgtgggaggcagctag	100	H. sapiens	541

220254	4	249	atgtgggaggcagctagctg	101	H. sapiens	542

220255	4	257	ggcagctagctgcccagagg	102	H. sapiens	543

220256	4	262	ctagctgcccagaggcatgc	103	H. sapiens	544

220257	4	325	gctggcctgccagccacagg	104	H. sapiens	545

220259	4	370	cctgtttgagaagtggaagc	106	H. sapiens	546

220260	4	375	ttgagaagtggaagctctac	107	H. sapiens	547

110282	4	407	caccacaacctgagcctgct	110	H. sapiens	548

220263	4	534	cttggcaccacaaagtgcaa	111	H. sapiens	549

220264	4	535	ttggcaccacaaagtgcaac	112	H. sapiens	550

220265	4	536	tggcaccacaaagtgcaac	a113	H. sapiens	551

220266	4	537	ggcaccacaaagtgcaacac	114	H. sapiens	552

110289	4	563	gtgttcaagagatgcgggcc	115	H. sapiens	553

220267	4	567	tcaagagatgcgggcccgac	116	H. sapiens	554

220268	4	617	ccttggcgtgatgcctccca	117	H. sapiens	555

220269	4	627	atgcctcccagtgccagatg	118	H. sapiens	556

220270	4	666	tccagaaggaggtggccaag	119	H. sapiens	557

220272	4	685	gatgtacagcagcttccagg	121	H. sapiens	558

220273	4	795	gcaatgccatccacgcgaat	122	H. sapiens	559

220275	4	861	atgggctgctcaggacccgc	124	H. sapiens	560

220276	4	886	ccagaaaattggcgacgacc	125	H. sapiens	561

220277	4	900	acgacctcagtgtcagcacc	127	H. sapiens	562

220279	4	1032	tgctgggcctggccaccctc	129	H. sapiens	563

220282	4	1158	gctggaccagcaatgacaac	132	H. sapiens	564

220283	4	1168	caatgacaacatgggcttct	133	H. sapiens	565

220284	4	1187	tggtggatcctgcggttccc	134	H. sapiens	566

220285	4	1230	acttcttcatcttcgtccgc	135	H. sapiens	567

220286	4	1638	gtggcagccaggattcatct	136	H. sapiens	568

220287	4	1727	cagctagggctggactctgg	137	H. sapiens	569

220288	4	1732	agggctggactctggcaccc	138	H. sapiens	570

220289	4	1735	gctggactctggcacccaga	139	H. sapiens	571

220290	4	1736	ctggactctggcacccagag	140	H. sapiens	572

220291	4	1737	tggactctggcacccagagg	141	H. sapiens	573

220292	4	1740	actctggcacccagaggcgt	142	H. sapiens	574

220293	4	1760	cgctggacaacccagaactg	143	H. sapiens	575

220294	4	1849	ctgtctgcgagattgggcct	144	H. sapiens	576

220295	4	1850	tgtctgcgagattgggcctc	145	H. sapiens	577

220296	4	1856	cgagattgggcctcctctcc	146	H. sapiens	578

220297	4	1861	ttgggcctcctctccctgca	147	H. sapiens	579

220298	4	1883	tgccttgtccctggtgcaga	148	H. sapiens	580

220299	4	1891	ccctggtgcagaggtgagca	149	H. sapiens	581

220302	4	1905	tgagcagaggagtccagggc	152	H. sapiens	582

220303	4	1932	ggggctgtgccgtgaactgc	153	H. sapiens	583

220305	4	1945	gaactgcgtgccagtgtccc	155	H. sapiens	584

220306	4	1971	tgtcggcacgtcccatgtgc	156	H. sapiens	585

220307	4	1984	catgtgcatggaaatgtcct	157	H. sapiens	586

220308	4	1986	tgtgcatggaaatgtcctcc	158	H. sapiens	587

220309	4	1999	gtcctccaacaataaagagc	159	H. sapiens	588

220310	4	2001	cctccaacaataaagagctc	160	H. sapiens	589

220311	4	2008	caataaagagctcaagtggt	161	H. sapiens	590

220312	18	3174	ctggggacgccaaaactgcc	162	H. sapiens	591

220314	18	7544	tagacacgcacatcctatcc	164	H. sapiens	592

220316	18	14888	ctaccctgcagagctggtgt	166	H. sapiens	593

226083	4	258	gcagctagctgcccagaggc	168	H. sapiens	594

226084	4	317	ctgctgctgctggcctgcca	169	H. sapiens	595

226085	4	321	tgctgctggcctgccagcca	170	H. sapiens	596

226086	4	347	ccctccgctcaggtgatgga	171	H. sapiens	597

226089	4	365	gacttcctgtttgagaagtg	174	H. sapiens	598

110281	4	397	tgaccagtgtcaccacaacc	177	H. sapiens	599

226092	4	403	gtgtcaccacaacctgagcc	178	H. sapiens	600

226093	4	452	aacagaaccttcgacaagta	179	H. sapiens	601

226094	4	458	accttcgacaagtattcctg	180	H. sapiens	602

226095	4	493	cgccaataccacggccaaca	181	H. sapiens	603

226096	4	497	aataccacggccaacatctc	182	H. sapiens	604

226097	4	500	accacggccaacatctcctg	183	H. sapiens	605

226098	4	532	gccttggcaccacaaagtgc	184	H. sapiens	606

110288	4	540	accacaaagtgcaacaccgc	185	H. sapiens	607

226099	4	544	caaagtgcaacaccgcttcg	186	H. sapiens	608

226100	4	548	gtgcaacaccgcttcgtgtt	187	H. sapiens	609

226101	4	556	ccgcttcgtgttcaagagat	188	H. sapiens	610

226103	4	588	gtcagtgggtgcgtggaccc	189	H. sapiens	611

226104	4	606	cccgggggcagccttggcgt	190	H. sapiens	612

226106	4	683	aagatgtacagcagcttcca	192	H. sapiens	613

226107	4	687	tgtacagcagcttccaggtg	193	H. sapiens	614

226108	4	691	cagcagcttccaggtgatgt	194	H. sapiens	615

226109	4	695	agcttccaggtgatgtacac	195	H. sapiens	616

226110	4	720	gctacagcctgtccctgggg	196	H. sapiens	617

226111	4	723	acagcctgtccctgggggcc	197	H. sapiens	618

226112	4	860	gatgggctgctcaggacccg	198	H. sapiens	619

226113	4	864	ggctgctcaggacccgctac	199	H. sapiens	620

226114	4	868	gctcaggacccgctacagcc	200	H. sapiens	621

226115	4	919	ctggctcagtgatggagcgg	201	H. sapiens	622

226116	4	923	ctcagtgatggagcggtggc	202	H. sapiens	623

226117	4	951	gtgtggccgcggtgttcatg	203	H. sapiens	624

226118	4	955	ggccgcggtgttcatgcaat	204	H. sapiens	625

226119	4	960	cggtgttcatgcaatatggc	205	H. sapiens	626

226120	4	1019	tacctgcacaacctgctggg	206	H. sapiens	627

226121	4	1025	cacaacctgctgggcctggc	207	H. sapiens	628

226122	4	1029	acctgctgggcctggccacc	208	H. sapiens	629

226123	4	1055	gagaggagcttcttcagcct	209	H. sapiens	630

226124	4	1059	ggagcttcttcagcctctac	210	H. sapiens	631

226126	4	1068	tcagcctctacctgggcatc	212	H. sapiens	632

110302	4	1072	cctctacctgggcatcggct	213	H. sapiens	633

226127	4	1156	gtgctggaccagcaatgaca	214	H. sapiens	634

226128	4	1160	tggaccagcaatgacaacat	215	H. sapiens	635

226129	4	1167	gcaatgacaacatgggcttc	216	H. sapiens	636

226130	4	1173	acaacatgggcttctggtgg	217	H. sapiens	637

226131	4	1176	acatgggcttctggtggatc	218	H. sapiens	638

226132	4	1185	tctggtggatcctgcggttc	219	H. sapiens	639

226134	4	1209	tcttcctggccatcctgatc	221	H. sapiens	640

226138	4	1290	tgcaccacacagactacaag	225	H. sapiens	641

226140	4	1414	cgccaagctcttcttcgacc	227	H. sapiens	642

226143	4	1669	cttggctggtggcctcccta	230	H. sapiens	643

231032	4	686	atgtacagcagcttccaggt	231	H. sapiens	644

231034	4	1424	ttcttcgacctcttcctcag	233	H. sapiens	645

231036	4	1212	tcctggccatcctgatcaac	235	H. sapiens	646

231037	4	1062	gcttcttcagcctctacctg	236	H. sapiens	647

231038	4	559	cttcgtgttcaagagatgcg	237	H. sapiens	648

231039	4	543	acaaagtgcaacaccgcttc	238	H. sapiens	649

231041	4	1026	acaacctgctgggcctggcc	240	H. sapiens	650

231042	4	1070	agcctctacctgggcatcgg	241	H. sapiens	651

231043	4	496	caataccacggccaacatct	242	H. sapiens	652

231044	4	399	accagtgtcaccacaacctg	243	H. sapiens	653

231046	4	392	tacggtgaccagtgtcacca	245	H. sapiens	654

231047	4	402	agtgtcaccacaacctgagc	246	H. sapiens	655

110287	4	533	ccttggcaccacaaagtgca	247	H. sapiens	656

231048	4	689	tacagcagcttccaggtgat	248	H. sapiens	657

231049	4	956	gccgcggtgttcatgcaata	249	H. sapiens	658

231051	4	555	accgcttcgtgttcaagaga	251	H. sapiens	659

231052	4	553	acaccgcttcgtgttcaaga	252	H. sapiens	660

231053	4	1027	caacctgctgggcctggcca	253	H. sapiens	661

231054	4	871	caggacccgctacagccaga	254	H. sapiens	662

231055	4	498	ataccacggccaacatctcc	255	H. sapiens	663

231056	4	259	cagctagctgcccagaggca	256	H. sapiens	664

231057	4	1058	aggagcttcttcagcctcta	257	H. sapiens	665

231058	4	348	cctccgctcaggtgatggac	258	H. sapiens	666

231061	4	953	gtggccgcggtgttcatgca	261	H. sapiens	667

231062	4	1024	gcacaacctgctgggcctgg	262	H. sapiens	668

231063	4	1061	agcttcttcagcctctacct	263	H. sapiens	669

231064	4	1169	aatgacaacatgggcttctg	264	H. sapiens	670

231066	4	1021	cctgcacaacctgctgggcc	266	H. sapiens	671

231067	4	400	ccagtgtcaccacaacctga	267	H. sapiens	672

231068	4	1165	cagcaatgacaacatgggct	268	H. sapiens	673

231069	4	363	tggacttcctgtttgagaag	269	H. sapiens	674

231070	4	550	gcaacaccgcttcgtgttca	270	H. sapiens	675

231071	4	367	cttcctgtttgagaagtgga	271	H. sapiens	676

231073	4	1071	gcctctacctgggcatcggc	273	H. sapiens	677

231075	4	349	ctccgctcaggtgatggact	275	H. sapiens	678

231077	4	463	cgacaagtattcctgctggc	278	H. sapiens	679

231078	4	320	ctgctgctggcctgccagcc	279	H. sapiens	680

231079	4	1183	cttctggtggatcctgcggt	280	H. sapiens	681

231080	4	862	tgggctgctcaggacccgct	281	H. sapiens	682

231081	4	565	gttcaagagatgcgggcccg	282	H. sapiens	683

231083	4	1177	catgggcttctggtggatcc	284	H. sapiens	684

231085	4	1184	ttctggtggatcctgcggtt	286	H. sapiens	685

231086	4	410	cacaacctgagcctgctgcc	287	H. sapiens	686

231087	4	495	ccaataccacggccaacatc	288	H. sapiens	687

231090	4	688	gtacagcagcttccaggtga	291	H. sapiens	688

231092	4	863	gggctgctcaggacccgcta	293	H. sapiens	689

231096	4	694	cagcttccaggtgatgtaca	297	H. sapiens	690

231097	4	494	gccaataccacggccaacat	298	H. sapiens	691

110307	4	1178	atgggcttctggtggatcct	300	H. sapiens	692

231099	4	1207	cgtcttcctggccatcctga	301	H. sapiens	693

231100	4	352	cgctcaggtgatggacttcc	302	H. sapiens	694

231101	4	261	gctagctgcccagaggcatg	303	H. sapiens	695

231102	4	561	tcgtgttcaagagatgcggg	304	H. sapiens	696

231103	4	323	ctgctggcctgccagccaca	305	H. sapiens	697

231104	4	324	tgctggcctgccagccacag	306	H. sapiens	698

231105	4	1179	tgggcttctggtggatcctg	307	H. sapiens	699

231107	4	1289	atgcaccacacagactacaa	309	H. sapiens	700

231108	4	322	gctgctggcctgccagccac	310	H. sapiens	701

231109	4	406	tcaccacaacctgagcctgc	311	H. sapiens	702

231110	4	870	tcaggacccgctacagccag	312	H. sapiens	703

231112	4	464	gacaagtattcctgctggcc	314	H. sapiens	704

231114	4	1060	gagcttcttcagcctctacc	316	H. sapiens	705

231115	4	1422	tcttcttcgacctcttcctc	317	H. sapiens	706

231118	4	542	cacaaagtgcaacaccgctt	321	H. sapiens	707

231119	4	456	gaaccttcgacaagtattcc	322	H. sapiens	708

231122	4	404	tgtcaccacaacctgagcct	325	H. sapiens	709

231123	4	538	gcaccacaaagtgcaacacc	326	H. sapiens	710

231126	4	954	tggccgcggtgttcatgcaa	329	H. sapiens	711

231127	4	684	agatgtacagcagcttccag	330	H. sapiens	712

231129	4	1214	ctggccatcctgatcaactt	332	H. sapiens	713

231130	4	1023	tgcacaacctgctgggcctg	333	H. sapiens	714

231133	4	554	caccgcttcgtgttcaagag	336	H. sapiens	715

231135	4	499	taccacggccaacatctcct	338	H. sapiens	716

231136	4	1164	ccagcaatgacaacatgggc	339	H. sapiens	717

231138	4	1064	ttcttcagcctctacctggg	341	H. sapiens	718

231139	4	1163	accagcaatgacaacatggg	342	H. sapiens	719

231140	4	547	agtgcaacaccgcttcgtgt	343	H. sapiens	720

231141	4	408	accacaacctgagcctgctg	344	H. sapiens	721

231143	4	1020	acctgcacaacctgctgggc	346	H. sapiens	722

231145	4	562	cgtgttcaagagatgcgggc	348	H. sapiens	723

231146	4	1418	aagctcttcttcgacctctt	349	H. sapiens	724

231148	4	557	cgcttcgtgttcaagagatg	351	H. sapiens	725

231150	4	1155	agtgctggaccagcaatgac	353	H. sapiens	726

231151	4	566	ttcaagagatgcgggcccga	354	H. sapiens	727

231152	4	721	ctacagcctgtccctggggg	355	H. sapiens	728

110306	4	1162	gaccagcaatgacaacatgg	356	H. sapiens	729

231154	4	549	tgcaacaccgcttcgtgttc	358	H. sapiens	730

231155	4	1159	ctggaccagcaatgacaaca	360	H. sapiens	731

231156	4	457	aaccttcgacaagtattcct	361	H. sapiens	732

231160	4	546	aagtgcaacaccgcttcgtg	365	H. sapiens	733

231162	4	260	agctagctgcccagaggcat	367	H. sapiens	734

231163	4	690	acagcagcttccaggtgatg	368	H. sapiens	735

231165	4	558	gcttcgtgttcaagagatgc	370	H. sapiens	736

231166	4	958	cgcggtgttcatgcaatatg	371	H. sapiens	737

231168	4	867	tgctcaggacccgctacagc	373	H. sapiens	738

231169	4	865	gctgctcaggacccgctaca	374	H. sapiens	739

231171	4	692	agcagcttccaggtgatgta	376	H. sapiens	740

231172	4	1181	ggcttctggtggatcctgcg	377	H. sapiens	741

231174	4	1057	gaggagcttcttcagcctct	379	H. sapiens	742

231175	4	1211	ttcctggccatcctgatcaa	380	H. sapiens	743

231176	4	541	ccacaaagtgcaacaccgct	381	H. sapiens	744

231177	4	319	gctgctgctggcctgccagc	382	H. sapiens	745

231178	4	545	aaagtgcaacaccgcttcgt	383	H. sapiens	746

231179	4	952	tgtggccgcggtgttcatgc	384	H. sapiens	747

231181	4	1180	gggcttctggtggatcctgc	386	H. sapiens	748

231183	4	722	tacagcctgtccctgggggc	388	H. sapiens	749

231185	4	957	ccgcggtgttcatgcaatat	390	H. sapiens	750

231186	4	459	ccttcgacaagtattcctgc	391	H. sapiens	751

110293	4	693	gcagcttccaggtgatgtac	392	H. sapiens	752

231187	4	1153	ccagtgctggaccagcaatg	393	H. sapiens	753

110319	4	551	caacaccgcttcgtgttcaa	396	H. sapiens	754

231190	4	1171	tgacaacatgggcttctggt	397	H. sapiens	755

63771	11	138	caacctctgccagatgtggg	404	M. musculus	756

63777	11	274	gtttgagaagtggaagctct	406	M. musculus	757

63778	11	284	tggaagctctatagtgacca	407	M. musculus	758

63780	11	308	caccacaacctaagcctgct	408	M. musculus	759

63784	11	403	cactgccaacatttcctgcc	409	M. musculus	760

63787	11	464	gtgttcaagaggtgtgggcc	410	M. musculus	761

63789	11	558	agatcgaggtccagaagggg	411	M. musculus	762

63792	11	619	gggctacagtctgtccctgg	412	M. musculus	763

63793	11	670	gggcctcaggaagctgcact	413	M. musculus	764

63802	11	1019	ccctgggtggtggtcaagtg	414	M. musculus	765

63806	11	1058	tgctggaccagcaatgacaa	416	M. musculus	766

63808	11	1079	atgggattctggtggatcct	417	M. musculus	767

63811	11	1220	gccaggtccacgctgaccct	418	M. musculus	768

95505	400	14	gcaacctgaggagaggtgca	424	M. musculus	769

95506	400	25	agaggtgcacacactctgag	425	M. musculus	770

95507	400	30	tgcacacactctgaggacct	426	M. musculus	771

95508	400	48	ctaggtgtgcaacctctgcc	427	M. musculus	772

95509	400	80	tggctacccagaggcatgcc	428	M. musculus	773

95511	400	192	tgtttgagaagtggaagctc	430	M. musculus	774

95512	400	251	ccacctactgagctggtctg	431	M. musculus	775

95513	400	291	actcctgctggcctgacacc	432	M. musculus	776

95514	400	359	tgccacaaagtgcagcaccg	433	M. musculus	777

95515	400	371	cagcaccgcctagtgttcaa	434	M. musculus	778

95516	400	410	cagtgggttcgagggccacg	435	M. musculus	779

95517	400	545	agtctgtccctgggggcctt	436	M. musculus	780

95518	400	572	gcgctggtcatcctgctggg	437	M. musculus	781

95519	400	582	tcctgctgggcctcaggaag	438	M. musculus	782

95520	400	650	gtgctcaaggctggctctgt	439	M. musculus	783

95521	400	764	ggctgcagagtggccacagt	440	M. musculus	784

95522	400	775	ggccacagtgatcatgcagt	441	M. musculus	785

95523	400	785	atcatgcagtacggcatcat	442	M. musculus	786

95524	400	836	gtgtacctgtacagcctgct	443	M. musculus	787

95525	400	974	cagtgctggaccagcaatga	444	M. musculus	788

95527	400	1079	cttcttgtggccaagctgcg	446	M. musculus	789

95529	400	1100	gcccatcagatgcactatgc	448	M. musculus	790

95531	400	1256	ttcctcagctccttccaggg	450	M. musculus	791

95532	400	1292	ctctactgtttcctcaacaa	451	M. musculus	792

95533	400	1348	atggcaagaaggcaaagctc	452	M. musculus	793

95534	400	1360	caaagctcttcaggaggaaa	453	M. musculus	794

95535	400	1388	agcagccatggcagccacat	454	M. musculus	795

95536	400	1435	tccctgtgagaaacttcagc	455	M. musculus	796

95537	400	1450	tcagcttatgagtgcaggca	456	M. musculus	797

95538	400	1470	gcagcagtgggactggctgt	457	M. musculus	798

95539	400	1512	tggccagtagtctcccaagg	458	M. musculus	799

95540	400	1544	cccacctgaatctccacttg	459	M. musculus	800

95542	400	1575	ggttgtgttcaagaaagggc	461	M. musculus	801

95543	400	1600	aggacaacccagagccagat	462	M. musculus	802

95544	400	1610	agagccagatgcccggccaa	463	M. musculus	803

95545	400	1620	gcccggccaaggttgaagag	464	M. musculus	804

95546	400	1646	cagcaagacagcagcttgta	465	M. musculus	805

95547	400	1687	gtcctagcctggcacaggcc	466	M. musculus	806

95548	400	1724	gttggatatgatggagaagc	467	M. musculus	807

95549	400	1750	atctatgaactctgagtgtt	468	M. musculus	808

95550	400	1756	gaactctgagtgttcccatg	469	M. musculus	809

95551	400	1795	tacccagatatgtccttcag	470	M. musculus	810

95552	401	3953	cttggtctcgcctttgttac	471	M. musculus	811

95554	401	7321	accttcacagagctggtctg	473	M. musculus	812

95555	401	7505	ccatgcccagtgcagcaccg	474	M. musculus	813

95557	401	8766	gtcaggttgcccatccttgg	476	M. musculus	814

95560	11	10	tcatgaggccttgggcttgg	479	M. musculus	815

95561	11	85	ctggtcctctgcagcctgag	480	M. musculus	816

As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of glucagon receptor.
According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 17

Western Blot Analysis of Glucagon Receptor Protein Levels

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to glucagon receptor is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18

Effects of Antisense Inhibition of Glucagon Receptor in Mice on Plasma Glucose Levels and Glucagon Receptor mRNA Reduction: Lean Animals, db/db Mice and ob/ob Mice

In accordance with the present invention, two antisense oligonucleotides targeted to the mouse glucagon receptor, ISIS 148359 (agcaggctta ggttgtggtg, SEQ ID NO: 408) and ISIS 180475 (gagctttgcc ttcttgccat, SEQ ID NO: 452), were evaluated for therapeutic efficacy in art-accepted mouse models of obesity and diabetes. Ob/ob mice have mutations in the leptin gene and are leptin-deficient, while db/db mice have mutations in the leptin receptor gene. The two strains exhibit obesity and diabetes strongly resembling Type 2 diabetes in humans. Tsang, S. H., 1998, P & S Medical Review, Vol. 5, No. 1.
Db/db and ob/ob mice were evaluated over the course of 4 weeks for the effects of ISIS 148359 and ISIS 180475 on serum glucose levels and glucagon receptor mRNA levels, while normoglycemic mice were evaluated for 2 weeks. Control animals received saline treatment (50 mg/kg). The normoglycemic mice were dosed subcutaneously twice a week for 2 weeks with 50 mg/kg of ISIS 148359, ISIS 180475 or saline. The db/db and ob/ob mice were dosed subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 148359, ISIS 180475, saline, the positive control oligonucleotide ISIS 116847 (ctgctagcc tctggatttga, SEQ ID NO: 817) or the negative control oligonucleotide ISIS 141923 (ccttccctga aggttcctcc, SEQ ID NO: 818). The mice were monitored weekly for fed or fasted plasma glucose levels (fasted glucose measured 16 hr after last feeding) and upon termination of the experiment the level of glucagon receptor mRNA in the liver was determined. The data are summarized in Table 4.

TABLE 4

Effects of ISIS 148359 and ISIS 180475 treatment on fed
and fasting glucose levels and glucagon receptor mRNA levels
in normoglycemic mice, db/db mice, and ob/ob mice

Biological			ISIS # Antisense
Marker	mice (time	day of	Oligonucleotides	Controls

Measured	course of study)	treatment	148359	180475	saline	116847	141923

fed	lean mice	−6	221	216	210	N.D.	N.D.
plasma	(2 week)	15	151	130	181	N.D.	N.D.
glucose	db/db mice	−1	294	295	296	295	304
mg/dL	(4 weeks)	5	361	329	460	355	408
		12	375	303	510	303	425
		26	314	222	495	354	493
	ob/ob mice	−1	338	342	343	337	361
	(4 weeks)	12	245	180	426	227	476
		27	168	145	394	205	431
fasted	db/db mice	19	336	232	320	321	298
plasma	(4 weeks)	29	254	150	302	193	262
glucose	ob/ob mice	19	205	132	317	167	245
mg/dL	(4 weeks)	29	178	117	322	189	340
glucagon	lean mice	end	82	93	0	N.D.	N.D.
receptor	(2 week)
% mRNA	db/db mice	end	74	96	0	0	0
reduction	(4 weeks)
	ob/ob mice	end	86	97	0	10	0
	(4 weeks)

These data demonstrate that the antisense oligonucleotides ISIS 148359 and ISIS 180475 targeted to glucagon receptor mRNA are capable of decreasing levels of glucagon receptor mRNA in mouse liver. These data further demonstrate that reduction of glucagon receptor expression is accompanied by a decrease in plasma glucose levels in normoglycemic mice, db/db mice and ob/ob mice. It is important to note that the treated mice become normoglycemic and do not become hypoglycemic. Antisense inhibitors of glucagon receptor are thus believed to be useful therapeutic modalities for treatment of hyperglycemia.

Example 19

Glucagon Receptor Antisense Oligonucleotides Lower Plasma Glucose in ob/ob Diabetic Mice

4 Week Study

C57Bl/eOlaHsd-Lep_ob(ob/ob) male mice were purchased from Harlan (Indianapolis, Ind., USA). Animals were acclimated for one week prior to study initiation. Mice were housed five per cage in polycarbonate cages with filter tops. Animals were maintained on a 12:12 hr light-dark cycle (lights on at 6:00 AM) at 21° C. All animals received de-ionized water ad libitum. ob/ob mice received Purina Diet 5015 ad libitum. Antisense compounds were prepared in normal saline, and the solution was sterilized through a 0.2 μm filter. Animals were dosed with antisense compound solutions or vehicle (saline) twice per week (separated by 3.5 days) via subcutaneous injection. Before the initiation of each study and once weekly during the study, blood was collected by tail clip without anesthesia into EDTA plasma tubes containing trasylol (Serologicals Proteins, Kankakee, Ill., USA) and dipeptidyl peptidase (DPP)-IV inhibitor (Linco Diagnostic Services, St. Charles, Mo., USA). Food intake and body weights were measured weekly. Plasma levels of glucose and triglycerides were determined on the Hitachi 912 clinical chemistry analyzer (Roche, Indianapolis, Ind., USA).
To test the efficacy of antisense inhibitors of glucagon receptor to treat hyperglycemia, 7-8 week-old ob/ob mice were dosed two times per week with antisense inhibitors of glucagon receptor [ISIS 148359 (SEQ ID NO: 408) or ISIS 180475 (SEQ ID NO: 452)], a generic control oligonucleotide (ISIS 141923; SEQ ID NO: 818) whose sequence does not match any known transcripts in the mouse or rat genomes, a mismatch oligonucleotide (ISIS 298682; GCGATTTCCCGTTTTGACCT; SEQ ID NO: 819) whose sequence is identical to ISIS 180475 except for 7 internal bases, or saline twice a week (every 3.5 days) for 4 weeks. All oligonucleotides were administered at 25 mg/kg. Data are the mean values (±SEM where shown) of 8 mice per treatment group. Plasma glucose levels in all mice were approximately 330-370 mg/dl day zero. Whereas hyperglycemia worsened over time in saline- and control oligonucleotide-treated ob/ob mice, animals treated with glucagon receptor antisense compounds showed a dramatic reduction in plasma glucose. At day 12, plasma glucose levels in ob/ob mice treated with control oligonucleotide (ISIS 141923) and saline were approximately 472 and 425 mg/dl, respectively. Plasma glucose levels in mice treated with antisense oligonucleotides ISIS 148359 and ISIS 180475 were 240 and 180 mg/dl, respectively. At day 27, plasma glucose levels in ob/ob mice treated with control oligonucleotide (ISIS 141923) and saline were approximately 435 and 390 mg/dl, respectively. Plasma glucose levels in mice treated with antisense oligonucleotides ISIS 148359 and ISIS 180475 were 165 and 130 mg/dl, respectively. The latter is in the normal range.
A separate study, also using ob/ob mice (as well as db/db mice, lean mice, ZDF rats and lean rats) was also performed in which animals were also dosed subcutaneously every 3.5 days for a total of 9 doses of glucagon receptor antisense compound ISIS 180475 and one or more controls (unrelated control oligonucleotide ISIS 141923, mismatch control oligonucleotide ISIS 298682, and/or saline). The results of this study are shown in Table 5.
At the end of the 4-week treatment period, liver glucagon receptor mRNA was measured (normalized to total RNA in the same samples using Ribogreen) and was found to be reduced by 85-95%. Data are mean values of four mice per treatment group (P<0.05 using Student's t-test).

TABLE 5

Effects of antisense inhibition of glucagon receptor in rodents

	Plasma	Plasma	Plasma	Plasma
	Glucose	Triglycerides	Insulin	Glucagon
Body Weight (g)	(mg/dl)	(mg/dl)	(ng/ml)	(pg/ml)

ob/ob mice
Saline	56.5 ± 1.5	564 ± 118	163 ± 25	35.9 ± 13.8	n.d.
ISIS 180475	54.1 ± 1.6	122 ± 6*	129 ± 7	19.8 ± 9.5	n.d.
db/db mice
ISIS 141923	46.0 ± 0.7	571 ± 29	412 ± 33	n.d.	117 ± 12
ISIS 298682	43.9 ± 1.3	577 ± 65	448 ± 40	n.d.	131 ± 20
ISIS 180475	45.6 ± 0.6	241 ± 37*	121 ± 12*	n.d.	3765 ± 952*
db^+/? lean mice
ISIS 141923	28.0 ± 1.0	196 ± 12	121 ± 7	n.d.	80 ± 1
ISIS 180475	27.6 ± 1.0	164 ± 4*	83 ± 6*	n.d.	362 ± 40*
ZDF rats
ISIS 141923	403 ± 12	417 ± 38	640 ± 105	5.0 ± 1.9	136 ± 7
ISIS 180475	404 ± 8	143 ± 15*	250 ± 25*	4.4 ± 0.5	548 ± 20*
SD lean rats
Saline	344 ± 4	116 ± 3	106 ± 26	2.7 ± 0.3	56 ± 11
ISIS 180475	327 ± 5	104 ± 7	139 ± 58	1.3 ± 0.2*	855 ± 122*

*P < 0.05.
n.d., not determined

Example 20

Glucagon Receptor Antisense Oligonucleotides Lower Plasma Glucose in db/db Diabetic Mice

4 Week Study

C57Bl/KsOlaHsd-Lep_db(db/db) and lean (db^+/+) male mice were purchased from Harlan (Indianapolis, Ind., USA). Animals were acclimated for one week prior to study initiation. Mice were housed five per cage in polycarbonate cages with filter tops. Animals were maintained on a 12:12 hr light-dark cycle (lights on at 6:00 AM) at 21° C. All animals received de-ionized water ad libitum. db/db mice received Purina Diet 5008 ad libitum. Antisense compounds were prepared in normal saline, and the solution was sterilized through a 0.2 μm filter. Animals were dosed with antisense compound solutions or vehicle (saline) twice per week (separated by 3.5 days) via subcutaneous injection. Before the initiation of each study and once weekly during the study, blood was collected by tail clip without anesthesia into EDTA plasma tubes containing trasylol (Serologicals Proteins, Kankakee, Ill., USA) and dipeptidyl peptidase (DPP)-IV inhibitor (Linco Diagnostic Services, St. Charles, Mo., USA). Food intake and body weights were measured weekly. Plasma levels of glucose and triglycerides were determined on the Hitachi 912 clinical chemistry analyzer (Roche, Indianapolis, Ind., USA.
To test the efficacy of antisense inhibitors of glucagon receptor to treat hyperglycemia, 7-8 week-old db/db mice were dosed two times per week with antisense inhibitors of glucagon receptor [ISIS 148359 (SEQ ID NO: 408) or ISIS 180475 (SEQ ID NO: 452)], a generic control oligonucleotide (ISIS 141923) whose sequence does not match any known transcripts in the mouse or rat genomes, a mismatch oligonucleotide (ISIS 298682; SEQ ID NO: 819) whose sequence is identical to ISIS 180475 except for 7 internal bases, or saline for 4 weeks.
Glucose lowering efficacy and target reduction in db/db mice undergoing glucagon receptor antisense treatment were similar to those observed in similarly treated ob/ob mice; furthermore, plasma triglycerides were lowered from 412±33 to 121±12 mg/dl following glucagon receptor antisense treatment (Table 5). These results in db/db mice are similar to those reported in preliminary studies testing glucagon receptor antisense compound ISIS 148359 for 3 weeks [Osborne et al., 2003, Diabetes 52, A129 (abstract)].

Example 21

Glucagon Receptor Antisense Oligonucleotides Lower Plasma Glucose in ZDF Rats

ZDF/GmiCrl-fa/fa (ZDF) male rats were purchased from Charles River Laboratories (Wilmington, Mass., USA). Animals were acclimated for one week prior to study initiation. Rats were housed one per cage in polycarbonate cages with filter tops. Animals were maintained on a 12:12 hr light-dark cycle (lights on at 6:00 AM) at 21° C. All animals received de-ionized water ad libitum. ZDF rats received Purina Diet 5008 ad libitum. Antisense compounds were prepared in normal saline, and the solution was sterilized through a 0.2 μm filter. Seven-week old animals were dosed with antisense compound solutions or vehicle (saline) twice per week (separated by 3.5 days) via subcutaneous injection, for a total of 9 doses (last treatment on day 28), followed by a washout period of equal duration. Oligonucleotide concentration was 25 mg/kg of either glucagon receptor antisense oligonucleotide ISIS 180475 (SEQ ID NO: 452) or negative control oligonucleotide ISIS 141923 (SEQ ID NO: 818). Before the initiation of each study and once weekly during the study, blood was collected by tail clip without anesthesia into EDTA plasma tubes containing trasylol (Serologicals Proteins, Kankakee, Ill., USA) and dipeptidyl peptidase (DPP)-IV inhibitor (Linco Diagnostic Services, St. Charles, Mo., USA). Food intake and body weights were measured weekly. Glucagon receptor mRNA (target) was measured by real-time quantitative RT-PCR from livers of five animals removed from the study at each time point. Rat 36B4 ribosomal phosphoprotein mRNA (“18S RNA”) was measured and used to normalize RNA input. Data are the mean values of five rats per treatment group. In overall comparisons during the treatment period, target reduction by glucagon receptor antisense compound ISIS 180475 was significantly different when compared to control oligonucleotide-treated animals (P<0.05 adjusted using the Tukey method). Liver glucagon receptor mRNA decreased dramatically to 50% of controls within 24 hours after the first dose of ISIS 180475 and to 30% of controls 48 hr following the seventh dose.
For non-fasted plasma glucose levels, rats were treated as described above. Data are the mean values of five rats per treatment group. In overall comparisons during the treatment period, glucose lowering by the glucagon receptor antisense compound ISIS 180475 showed significant difference when compared to control oligonucleotide-treated animals. (P<0.05 adjusted using the Tukey method). The drop in plasma glucose paralleled the drop in glucagon receptor mRNA levels; there was a significant drop in plasma glucose within 48 hours after the initial glucagon receptor antisense dose. After 9 doses, the control oligonucleotide (ISIS 141923) treated rats had plasma glucose levels averaging approximately 417 mg/dl and antisense (ISIS 180475) treated rats had plasma glucose levels averaging approximately 143 mg/dl.
During the washout phase, hyperglycemia and glucagon receptor expression in liver began to rebound within 10 days, but even one month after the final dose, efficacy was still observed as plasma glucose and target mRNA levels in washout animals remained below pre-treatment levels. Glucose lowering achieved by the twice per week dosing schedule and the gradula rebound of glucagon receptor mRNA during the washout period are both consistent with the extended half lives of 2′-methoxyethoxy modified phosphorothioate oligonucleotides (typically ranging from 9 to 19 days according to published reports).
Non-fasted plasma insulin levels were also determined for rats treated as described above. Data are the mean values of five rats per treatment group. No significant changes were observed during the treatment period; however, individual comparisons between glucagon receptor antisense and control oligonucleotide treated animals on day 38 and 56 (washout period) were significant (P<0.05). Plasma insulin levels declined during the treatment phase in both control oligonucleotide and antisense-treated animals. During the washout phase of the control oligonucleotide treated group, insulin levels continued to decline as hyperglycemia progressed. This result is expected since beta-cell failure typically occurs in ZDF rats between 8 and 12 weeks of age. Interestingly, the mild elevation of glucose in glucagon receptor antisense-treated animals during the washout period resulted in a robust rise in plasma insulin to levels nearly as high as at start of study. This is consistent with evidence of preserved beta-cell function.

Example 22

Glucagon Receptor Antisense Oligonucleotides do not Cause Hyperglycemia or Hypoglycemia, in Spite of Hyperglucagonemia

In addition to effects on blood glucose, treatment with the antisense inhibitor of glucagon receptor (ISIS 180475; SEQ ID NO: 452) resulted in marked (and reversible) hyperglucagonemia in both normal and diabetic rodents (Table 5). This level of hyperglucagonemia is similar to that observed in glucagon receptor knockout mice (Parker et al., 2002, Biochem Biophys Res Commun. 290, 839-843; Gelling et al., 2003, Proc. Natl. Acad. Sci. USA., 100, 1438-1443. Because of these high levels of serum glucagon, it was important to determine whether the antisense inhibitors of glucagon receptor might induce hyperglycemia, particularly as hepatic glucagon receptor levels gradually return to normal following treatment withdrawal. It is therefore significant that at no time during the treatment or washout periods did animals with hyperglucagonemia exhibit hyperglycemia. In fact, glucagon receptor antisense-treated animals showed a moderate decrease in fed plasma glucose at all time points tested.
It was also confirmed that antisense treatment also did not cause hypoglycemia. After 4 weeks of antisense treatment, by which time maximum reduction of glucagon receptor expression had been achieved, db^+/?lean mice were subjected to periods of fasting of up to 24 hours. Although the glucagon receptor antisense-treated mice displayed a 10-30% reduction in plasma glucose, at no time did the animals reach adverse levels of hypoglycemia. This is in contrast to Gcgr knockout mice, which become hypoglycemic during periods of fasting. Gelling et al., 2003, Proc Natl. Acad. Sci. U.S.A. 100, 1438-1443.

Example 23

Glucagon Receptor mRNA is Reduced in Islets of Antisense-Treated db/db Mice

Pancreatic islets were isolated from 12-week-old male db/db mice (n=5-6 per treatment group) that had been treated twice per week (every 3.5 days) by subcutaneous injection with saline or glucagon receptor antisense oligonucleotide ISIS 180475 (SEQ ID NO: 452) at 25 mg/kg for a total of 9 doses. Mice were sacrificed by cervical dislocation. The common bile duct was cannulated with a 27-gauge needle and the pancreas was distended with 3 ml of Hank's buffer (Sigma, Taufkirchen, Germany) containing 2% bovine serum albumin (Applichem, Darmstadt, Germany) and 1 mg/ml collagenase (Serva, Heidelberg, Germany). Subsequently, the pancreas was removed and digested in Hank's buffer at 37° C. Islets were purified on a Histopaque-1077™ (Sigma) gradient for 15 min at 750×g. Islets were cultured overnight in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Karlsruhe, Germany). 200 islets from 3 individuals were pooled to give one sample for RNA extraction. Real-time quantitative RT-PCR was used to profile gene expression. Islet glucagon receptor mRNA levels were decreased by approximately 75% in antisense-treated animals compared to saline-treated controls. It should be noted that, in addition to pharmacologic effect of the antisense compound, a compensatory response to hyperglucagonemia or the increased alpha-cell populations in treated animals could contribute to the results observed.

Example 24

Glucagon Receptor Antisense Oligonucleotides Decrease the Number of Functional Glucagon Receptors

To assess whether the reduction in glucagon receptor mRNA correlates with a reduction in functional glucagon receptor number, a homologous competition assay was performed using hepatocyte membranes prepared from mice treated with control or glucagon receptor antisense compounds. ¹²⁵I-glucagon binding was effectively competed by increasing concentrations of unlabeled glucagon in control membrane samples. 15-20 μg of membrane from control oligonucleotide- or glucagon receptor oligonucleotide (ISIS 180475; SEQ ID NO: 452)-treated db/db mice were incubated with 0.1 nM ¹²⁵I-glucagon (2000 Ci/mmol, PerkinElmer, Boston, Mass., USA) and the indicated concentrations of unlabeled glucagon (Eli Lilly and Company, Indianapolis, Ind., USA) in buffer containing 50 mM Hepes, 1 mM MgCl₂, 5 mM EGTA, 0.005% Tween 20, 0.1% BSA, and EDTA-free protease inhibitor cocktail (Roche). Assays were performed under steady state conditions in the presence of excess labeled ligand on 96-well MultiScreen-HV 0.45 μm filter plates (Millipore, Bedford, Mass., USA). Following incubation for 2 hrs at room temperature, plates were rapidly washed by filtration with ice-cold buffer (20 mM Tris, pH 7.4) and dried for 45 min at 50° C. Following the addition of Optiphase Supermix (PerkinElmer), plates were counted on a Wallac Microbeta scintillation counter. Data analyses were performed using GraphPad Prism software and expressed as mean+/−SEM. Data obtained for samples from animals treated with glucagon receptor oligonucleotide neared the limits of detection for the assay and curve-fitting parameters. In order to derive a numerical value for apparent Bmax, the Kd was fixed at the average value (0.69+/−0.2 nM) obtained from the samples from the control antisense-treated animals.
Functional GCGR expression was found to be decreased approximately 85% by glucagon receptor antisense treatment and is in accord with quantitative RT-PCR results.

Example 25

Antisense Inhibitors of Human and Monkey Glucagon Receptor

Dose Response

Based on the screen in Example 15 above, a subset of human glucagon receptor antisense oligonucleotides were chosen for further study. Dose-response studies were conducted for ISIS 315186, 310457, 315324, 315278, 315181, 315297, 315163 and 310456 in both human HepG2 cell cultures and in cynomolgus monkey primary hepatocytes. These six compounds are homologous to both human and cynomolgus monkey glucagon receptor nucleic acid targets. The universal control ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; SEQ ID NO: 820, where N is an equimolar mixture of A, C, G and T, a chimeric 2′ MOE gapmer with a phosphorothioate backbone and with MOEs at positions 1-5 & 16-20) was used as negative control.
The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal calf serum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.
Primary cynomolgus monkey hepatocytes were obtained from CellzDirect (Los Angeles) and plated onto collagen-coated 24-well plates (Costar) at a density of 75,000 cells/well. The culturing medium for these hepatocytes was William's E media (Invitrogen) supplemented with 10% FBS (Invitrogen). Cells were allowed to attach overnight and were then treated with oligonucleotide-Lipofectin mixture for 4 hours. The oligonucleotide-Lipofectin mixture was washed off and then cells were incubated in normal medium.
Cells were treated with oligonucleotide for 20 hours at doses of 1, 5, 10, 25, 50, 100 nM for HepG2 cells and 5, 10, 25, 50, 100 and 200 nM for primary monkey hepatocytes (n=3). RNA was analyzed by RT-PCR to determine percent inhibition of glucagon receptor expression compared to control (ISIS 29848), at each oligonucleotide concentration. The results were plotted to give the IC50, the dose of oligonucleotide which results in 50% reduction of glucagon receptor mRNA levels. Results are shown in Table 6.
TABLE 6

IC50s of glucagon receptor antisense oligonucleotides in human HepG2

cells and in cynomolgus monkey primary hepatocytes (in nM)

SEQ ID IC50 in human IC50 in monkey

ISIS # NO: HepG2 cells (nM) hepatocytes (nM)

315186 254 5 30

310457 184 7 10

315324 392 6 19

315278 346 8 25

315181 249 9 32

315297 365 11 11

315163 231 19 37

310456 183 25 20

Based on these results, three compounds (ISIS 315297, 310457 and 315163) were chosen for study in monkeys.

Example 26

Dose-Ranging Study of Antisense Inhibition of Glucagon Receptor Expression in Cynomolgus Monkeys

A monkey study was performed at SNBL USA, Ltd., Everett, Wash. Forty mature (6-8 years old) male Macaca fascicularis (purpose-bred cynomolgus monkeys) weighing approximately 5-10 kg at the initiation of dosing were used. The animals were individually housed in primary climate-controlled enclosures conforming to the Animal Welfare Act. Animals were offered Purina Mills Laboratory Profiled Fiber Plus® Monkey Diet (Animal Specialties, Hubbard, Oreg.). Occasional fresh fruit and vegetable treats were also offered. Fresh drinking water was available to all animals, ad libitum. Before fasting measurements, food was removed from enclosures between 1630 and 1700 on the afternoon before the scheduled blood draw. After the animals were fasted for at least 16 hours, blood samples for plasma analysis were collected BEFORE dosing or feeding. For fasting analysis, approximately 2.3-2.5 mL of blood was drawn from a peripheral vein. Approximately 1.8 to 2.0 mL was be deposited into a K2-EDTA tube containing DPP-IV inhibitor at 10 μL/mL of blood and trasylol at 250 KIU/mL of blood. Approximately 0.5 mL was deposited into a lithium heparin tube. Once the blood was been deposited into the EDTA plus additives tube, it was inverted to mix and placed on ice within 5 minutes. The blood in the lithium heparin tube was also placed on ice within 5 minutes. Blood samples were centrifuged (2000×g, 15 minutes at 4 to 8° C.) to obtain plasma within 30 minutes of sample collection. The plasma was frozen at or below −70° C. Samples were shipped on dry ice via overnight courier as described below for subsequent analysis. For non-fasted analysis, the animals were given their AM feeding (between 0830 and 0930) on the day of the blood draw. Ninety minutes after feeding, blood was drawn. The number of biscuits remaining were counted at the time of the blood draw. Samples are prepared and shipped as above.
Monkeys were dosed subcutaneously for 10 weeks with ISIS 315297, ISIS 310457 or ISIS 315163 at three concentrations. In week 1, oligonucleotides were given at 2.0, 5.0 and 20 mg/kg/dose (Day 1, 3 and 5); in week 2 through 10, oligonucleotides were given at 1.0, 2.5 and 10 mg/kg/dose (twice weekly starting at Day 8) The larger dose (6, 15 or 60 mg/kg/week, i.e. 3 injections of 2, 5 or 20 mg/kg) was given in week 1 in order to rapidly achieve the desired steady state oligonucleotide concentration. In week 1, compounds were administered 3 times, every other day; for weeks 2-10, compounds were administered twice per week, with at least 2 days between dosings.
Oligonucleotides were given by subcutaneous (SC) injection, using volumes of 0.1-0.3 ml/kg). For each dosing of each animal, the appropriate volume of the relevant ASO solution or of the control/vehicle article was administered subcutaneously using a syringe and needle (27G). The total volume of the relevant dosing solution or the control article was calculated on the basis of the animal's most recent body weight. Multiple injection sites on the upper back (intra-scapular region) of each monkey were employed. During acclimation the skin of the upper back was be shaved and a clock-like grid (points at 12, 3, 6, and 9 o'clock) was tattooed on each animal. Injection points were a minimum of 5 cm apart. The injection sites were rotated so that each site was used for fourth dose, starting with 12 o'clock and rotating clockwise. The needle was inserted away from the dot and angled so that the dose was deposited underneath the dot.
After the 10 week study (approx. 2 days after last dose), animals were euthanized and three 1 to 4 gram samples of liver tissue were removed and individually snap frozen over liquid nitrogen; alternatively, biopsies could be taken from living animals and frozen. Frozen tissues were homogenized in 4M guanidinium isothiocyanate solution and subjected to CsCl centrifugation (150,000×g for 16 hr at 18° C.). The supernatant was removed and the RNA pellet was resuspended in water, following which it was applied to RNEASY mini columns (Qiagen, Valencia Calif.). After purification and quantitation, the tissues were subjected to RT-PCR analysis as described in previous examples using the following primers and probe:
Forward primer

ACTGCACCCGCAACGC (SEQ ID NO: 821)

Reverse primer

CACGGAGCTGGCCTTCAG (SEQ ID NO: 822)

Probe

ATCCACGCGAACCTGTTTGTGTCCTT (SEQ ID NO: 823)

RNA amounts were normalized to 18S RNA levels in the tissue. Results are shown in Table 7, as percent reduction in glucagon receptor mRNA in antisense-treated monkeys compared to saline-treated monkeys.
TABLE 7

Glucagon receptor mRNA reduction in monkey liver after treatment

with antisense inhibitors of glucagon receptor - RT-PCR expt 1

SEQ ID % reduction % reduction % reduction

ISIS # NO: at 2 mg/kg At 5 mg/kg at 20 mg/kg

310457 184 17 31 64

315297 365 2 21 49

315163 231 22 18 47

RNA analysis of the same tissue samples by RT-PCR was repeated independently using the same primer-probe set as above. Results are shown in Table 8 as percent reduction in glucagon receptor mRNA in antisense-treated monkeys compared to saline-treated monkeys.
TABLE 8

Glucagon receptor mRNA reduction in monkey liver after treatment

with antisense inhibitors of glucagon receptor -RT-PCR expt 2

SEQ ID % reduction % reduction % reduction

ISIS # NO: at 2 mg/kg At 5 mg/kg at 20 mg/kg

310457 184 25 23 63

315297 365 18 21 56

315163 231 25 29 44

The results obtained by RT-PCR were confirmed by Northern blot analysis according to standard methods (Example 14). The cDNA probe that was used for northern blots was a 900-base fragment of monkey GCGR generated by RT-PCR from cynomolgus monkey liver. Results are shown in Table 9.

TABLE 9

Glucagon receptor mRNA reduction in monkey liver after treatment
with antisense inhibitors of glucagon receptor - Northern blot

	SEQ ID	% reduction	% reduction	% reduction
ISIS #	NO:	at 2 mg/kg	At 5 mg/kg	at 20 mg/kg

310457	184	8	16	65
315297	365	0	10	38
315163	231	8	30	27

Blood glucose levels were measured in monkeys after treatment with antisense inhibitors of glucagon receptor. Glucose readings were performed using a drop of blood from the blood samples collected as above and read on a One Touch Profile® (Lifescan Inc., a Johnson and Johnson Company). Because normoglycemic (nondiabetic) monkeys were used in this study, no significant changes in blood glucose levels were expected or observed. At no point did animals become hypoglycemic after antisense treatment.
Glucagon levels were measured in plasma of fasted monkeys before (baseline) and after treatment for 5 weeks or 10 weeks with antisense inhibitors of glucagon receptor. Monkeys were anesthetized prior to blood collection to avoid artifacts due to stress. Glucagon levels were determined by radioimmunoassay, ELISA and/or Luminex immunoassay by contract laboratory (Linco, St. Charles Mo.). Results are shown in Table 10.

TABLE 10

Fasted glucagon levels in monkey liver after treatment
with antisense inhibitors of glucagon receptor

				Glucagon	Glucagon
		Antisense	Glucagon	(pg/ml)	(pg/ml)
	SEQ ID	Dose	(pg/ml)	Week 5	Week 10
ISIS #	NO:	(mg/kg)	Baseline	fasted	fasted

Saline			155 ± 31	150 ± 19	250 ± 141
310457	184	2	487 ± 123	278 ± 54	189 ± 36
		5	211 ± 25	179 ± 55	169 ± 26
		20	308 ± 77	580 ± 247	1247 ± 451
315297	365	2	410 ± 94	140 ± 42	133 ± 26
		5	519 ± 58	193 ± 31	204 ± 30
		20	375 ± 87	209 ± 23	276 ± 67
315163	231	2	176 ± 42	152 ± 33	143 ± 26
		5	262 ± 76	203 ± 74	225 ± 95
		20	251 ± 40	257 ± 76	421 ± 197

Glucagon-like peptide 1 (GLP-1) levels were measured in plasma of fasted monkeys before (baseline) and after treatment for 5 weeks or 10 weeks with antisense inhibitors of glucagon receptor. Monkeys were anesthetized prior to blood collection to avoid artifacts due to stress. GLP-1 levels were determined by radioimmunoassay, ELISA and/or Luminex immunoassay by contract laboratory (Linco, St. Charles Mo.). Results are shown in Table 11.

TABLE 11

Fasted GLP-1 levels in monkey liver after treatment
with antisense inhibitors of glucagon receptor

		Antisense	GLP-1	GLP-1 (pM)	GLP-1 (pM)
	SEQ ID	Dose	(pM)	Week 5	Week 10
ISIS #	NO:	(mg/kg)	Baseline	fasted	fasted

Saline			4 ± 2	4 ± 1	5 ± 1
310457	184	2	4 ± 1	3 ± 1	3 ± .41
		5	4 ± 1	4 ± .48	3 ± 1
		20	8 ± 3	17 ± 6	30 ± 15
315297	365	2	3 ± 1	3 ± 1	3 ± .29
		5	4 ± 1	4 ± 1	4 ± 2
		20	11 ± 8	9 ± 4	7 ± 5
315163	231	2	4 ± 1	4 ± 1	4 ± 1
		5	3 ± 1	5 ± 2	3 ± 1
		20	2 ± 0	4 ± .48	5 ± 1

Claims

1. An antisense compound 12 to 50 nucleobases in length comprising at least one of a modified internucleoside linkage, a high affinity modified sugar or a modified nucleobase, wherein the compound targets and specifically hybridizes with at least 90% complementarity within nucleotides 532 to 558 of a nucleic acid molecule encoding a human glucagon receptor having SEQ ID NO:4.

2. The compound of claim 1 which is 15 to 30 nucleobases in length.

3. The compound of claim 1 which is 20 nucleobases in length.

4. The compound of claim 1 having at least 95%. complementarity with a nucleic acid molecule encoding human glucagon receptor having SEQ ID NO: 4.

5. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.

6. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.

7. The compound of claim 1 having at least one 5-methylcytosine.

8. The compound of claim 1 that is a pharmaceutically acceptable salt.

9. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable diluent or carrier.

10. The compound of claim 1 comprising at least one high affinity modified sugar moiety selected from the group consisting of a 2′-O-(2-methoxyethyl), a 2′-O-methyl or a locked nucleic acid.

11. The compound of claim 1 wherein the compound is chimeric, comprising deoxynucleotides in a gap region, at least one high affinity modified sugar in each of a first wing region, and a second wing region, said wing regions flanking the gap region on the 5′ end and the 3′ end, respectively, and at least one phosphorothioate modified internucleoside linkage.

12. The compound of claim 11 wherein the gap segment is ten deoxynucleotides in length, the first and second wing segments are each five nucleotides in length and comprise five 2′-O-(2-methoxyethyl) nucleotides, and each internucleoside linkage in the chimeric oligonucleotide is a phosphorothioate.

13. The compound of claim 1 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

14. The compound of claim 11 further comprising at least one 5-methylcytosine modified nucleobase.

15. The compound of claim 1 comprising a pharmaceutically acceptable salt.

16. The compound of claim 1, wherein the compound targets and specifically hybridizes within nucleotides 532 to 558.

17. A method of inhibiting the expression of human glucagon receptor in cells or tissues comprising contacting said cells or tissues with an antisense compound 12 to 50 nucleobases in length comprising at least one of a modified internucleoside linkage, a high affinity modified sugar or a modified nucleobase, wherein the compound is targeted to a nucleic acid molecule encoding human glucagon receptor and wherein the compound specifically hybridizes with at least 90% complementarity with nucleotides 532 to 558 of a nucleic acid molecule having the sequence SEQ ID NO:4 and inhibits the expression of the human glucagon receptor.

18. A method of treating or delaying the onset of a disease or condition associated with glucagon receptor in a human comprising administering to said human a therapeutically or prophylactically effective amount of an antisense compound 12 to 50 nucleobases in length comprising at least one of a modified internucleoside linkage, a high affinity modified sugar or a modified nucleobase, wherein the compound is targeted to a nucleic acid molecule encoding human glucagon receptor and wherein the compound specifically hybridizes with at least 90% complementarity with nucleotides 532 to 558 of a nucleic molecule having the sequence SEQ ID NO:4, and inhibits the expression of the human glucagon receptor, thereby treating or delaying the onset of the disease or condition.

19. The method of claim 18, wherein the disease or condition is a metabolic disease or condition.

20. The method of claim 19, wherein the metabolic disease or condition is diabetes, obesity, hyperglycemia, primary hyperglucagonemia, insulin deficiency or insulin resistance.

21. The method of claim 20 wherein the diabetes is Type 2 diabetes.

22. The method of claim 19, wherein the metabolic disease or condition is associated with elevated blood glucose levels, elevated blood triglyceride levels or elevated blood cholesterol levels.

23. A method of decreasing blood glucose levels in a human comprising administering to said human a therapeutically or prophylactically effective amount of an antisense compound 12 to 50 nucleobases in length comprising at least one of a modified internucleoside linkage, a high affinity modified sugar or a modified nucleobase, wherein the compound is targeted to a nucleic acid molecule encoding human glucagon receptor and wherein the compound specifically hybridizes with at least 90% complementarity within nucleotides 532 to 558 of a nucleic acid molecule having the sequence SEQ ID NO:4 and inhibits the expression of the human glucagon receptor.

24. The method of claim 23 wherein the blood glucose levels are plasma glucose levels.

25. The method of claim 23 wherein the human suffers from diabetes or obesity.

26. A method of preventing or delaying the onset of an increase in blood glucose levels in a human comprising administering to said human a therapeutically or prophylactically effective amount of an antisense compound 12 to 50 nucleobases in length comprising at least one of a modified internucleoside linkage, a high affinity modified sugar or a modified nucleobase, wherein the compound is targeted to a nucleic acid molecule encoding human glucagon receptor and wherein the compound specifically hybridizes with at least 90% complementarity within nucleotides 532 to 558 of a nucleic acid molecule having the sequence SEQ ID NO:4 and inhibits the expression of the human glucagon receptor.

27. The method of claim 26 wherein the human suffers from diabetes, obesity or insulin resistance.

28. The method of claim 26 wherein the blood glucose levels are plasma glucose levels.

29. The method of claim 17, wherein the compound is at least 95% complementary to SEQ ID NO: 4.

30. The method of claim 17, wherein the compound is 15 to 30 nucleobases in length.

31. The method of claim 17, wherein the compound is 20 nucleobases in length.

32. The method of claim 17, wherein the compound comprises an oligonucleotide.

33. The method of claim 32, wherein the oligonucleotide is chimeric.

34. The method of claim 17, wherein the compound comprises at least one chemical modification.

35. The method of claim 34, wherein the compound comprises at least one modified internucleoside linkage, sugar moiety, or nucleobase.

36. The method of claim 35, wherein the compound comprises at least one 2′-O-(2-methoxyethyl) sugar moiety.

37. The method of claim 35, wherein the compound comprises at least one phosphorothioate internucleoside linkage.

38. The method of claim 35, wherein the compound comprises at least one 5-methylcytosine.

39. A method of claim 33, wherein every internucleoside linkage of the oligonucleotide is a phosphorothioate linkage, nucleobases 1-5 and 16-20 of the oligonucleotide comprise a 2′-O-(2-methoxyethyl) modification and every cytidine residue comprises a 5-methyl modification.

40. The method of claim 17, wherein the compound is a sodium salt.

41. The method of claim 17, wherein the compound is administered as a composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

42. The method of claim 41, wherein the composition farther comprises a colloidal dispersion system.

43. The method of claim 18, wherein administering comprises delivering a plurality of doses.

44. The method of claim 43, wherein each dose of said plurality of doses comprises from about 1 mg about 20 mg of the compound per kg of body weight.

45. The method of claim 17, wherein the compound is 100% complementary to SEQ ID NO:4.

46. The method of claim 17, wherein the compound targets and specifically hybridizes within nucleotides 532 to 558.