WO2017059205A1

WO2017059205A1 - Combination therapy

Info

Publication number: WO2017059205A1
Application number: PCT/US2016/054685
Authority: WO
Inventors: Sanjay Bhanot
Original assignee: Ionis Pharmaceuticals, Inc.
Priority date: 2015-09-30
Filing date: 2016-09-30
Publication date: 2017-04-06
Also published as: CN108026529A

Abstract

Methods for the treatment of conditions associated with Type 2 diabetes with a combination of an antisense oligonucleotide targeting GCGR and metformin are provided.

Description

COMBINATION THERAPY

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 BIOL0279WOSEQ_ST25.txt created September 29, 2016, which is 32 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. FIELD

The present embodiments provide combinations of an antisense oligonucleotide targeted to glucagon receptor (GCGR) and metformin. Methods of using such combinations to treat, prevent, or ameliorate conditions associated with metabolic disorders; particularly disorders associated with diabetes are also provided. Such methods can be useful for treating, preventing, or ameliorating conditions associated with metabolic disorders; particularly disorders associated with diabetes.

BACKGROUND

Type 2 diabetes is characterized by impaired insulin secretion and/or action, and many subjects also exhibit inappropriate levels of circulating glucagon in the fasting and postprandial state. An increase in the glucagon/insulin ratio is likely an important determinant of the hyperglycemia seen in type 2 diabetes patients (Baron et al., Diabetes. 1987. 36: 274-283). Lack of suppression of postprandial glucagon secretion in subjects with T2DM also plays an important role in the pathogenesis of postprandial hyperglycemia (Henkel et al., Metabolism. 2005. 54: 1168-1173).

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with Type 2 diabetes mellitus (T2DM) and even though there are currently several therapies available, many patients remain sub-optimally controlled (Datamonitor, 2006). Alternative treatments that suppress uncontrolled hepatic glucose production and help restore normal hepatic glucose metabolism are therefore still needed and could offer therapeutic benefit to patients with T2DM.

SUMMARY

Several embodiments provided herein relate to the discovery that inhibiting glucagon receptor (GCGR) can be useful for treating diabetes. In certain embodiments, methods are provided for the combined use of antisense compounds targeting GCGR with metformin for the treatment of diabetes. The combined use provided achieves an additive or greater-than-additive, effect in treating diabetes in an individual compared to treatment with metformin alone or antisense compound alone. The combined use provided achieves an additive or greater-than additive effect on the endpoints in an individual compared to treatment with metformin alone or antisense compound alone. In certain embodiments, the additive, or greater-than-additive, effect is achieved with toxicity that is no greater than the additive effect of the toxicities of each drug. In certain embodiments, antisense compounds targeting GCGR are useful for treating diabetes, such as Type 2 diabetes, at unexpectedly low doses for an antisense compound. Such doses are unexpectedly low for treating diabetes with an antisense compound.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI reference sequence records are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Compounds described by ISIS number (ISIS #) indicate a combination of nucleobase sequence, chemical modification, and motif.

"2'-0-methoxyethyl" (also 2'-MOE and 2'-0(CH₂)₂-OCH₃) refers to an O-methoxy-ethyl modification at the 2' position of a furanosyl ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.

"2'-MOE nucleoside" (also 2'-0-methoxyethyl nucleoside) means a nucleoside comprising a 2'- MOE modified sugar moiety.

"2 '-substituted nucleoside" or "2 -modified nucleoside" means a nucleoside comprising a 2'- substituted or 2'-modified sugar moiety. As used herein, "2 '-substituted" or "2 -modified" in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

"3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3 '-most nucleotide of a particular compound. "5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5 '-most nucleotide of a particular compound.

"5-methylcytosine" means a cytosine with a methyl group attached to the 5 position.

"About" as applied to dosing amounts means within ±10% of a value. For example, if it is stated, "the dose is an amount in the range of about 50-100 mg," it is implied that the dose is an amount in the range of 40-110 mg. In another example, if it is stated that the dose is an amount of "about 50 mg," it is implied that the dose can be from 40 mg to 60 mg.

"Adiposity" or "Obesity" refers to the state of being obese or an excessively high amount of body fat or adipose tissue in relation to lean body mass. The amount of body fat includes concern for both the distribution of fat throughout the body and the size and mass of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese. The term "Obesity" as used herein includes conditions where there is an increase in body fat beyond the physical requirement as a result of excess accumulation of adipose tissue in the body. The term "obesity" includes, but is not limited to, the following conditions: adult-onset obesity; alimentary obesity; endogenous or inflammatory obesity; endocrine obesity; familial obesity; hyperinsulinar obesity; hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroid obesity; lifelong obesity; morbid obesity and exogenous obesity.

"Administration" or "administering" refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

"Administered concomitantly" or "co-administration" means administration of two or more compounds in any manner in which the pharmacological effects of both are manifest in the patient.

Concomitant administration does not require that both compounds be administered in a single pharmaceutical composition, in the same dosage form, by the same route of administration, or at the same time. The effects of both compounds need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive. Concomitant administration or co-administration encompasses administration in parallel or sequentially.

"Amelioration" refers to an improvement or lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression or severity of one or more indicators of a condition or disease. The progression or severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

"Antisense compound" means a compound comprising an antisense oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, ribozymes, siR As, shR As, ssRNAs, and occupancy-based compounds.

"Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

"Antisense mechanisms" are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

"Antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence that is at least partially complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof.

"Bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety. "Bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

"Chemical modification" in a compound describes the substitutions or changes through chemical reaction, of any of the units in the compound. "Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase. "Modified oligonucleotide" means an oligonucleotide comprising at least one modified intemucleoside linkage, a modified sugar, and/or a modified nucleobase.

"Chemically distinct region" refers to a region of a compound that is in some way chemically different than another region of the same compound. For example, a region having 2'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0-methoxyethyl

modifications. "Chimeric antisense compounds" means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

"Cholesterol" is a sterol molecule found in the cell membranes of all animal tissues. Cholesterol must be transported in an animal's blood plasma by lipoproteins including very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). "Plasma cholesterol" refers to the sum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/or non-esterified cholesterol present in the plasma or serum.

"Co-administration" means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

"Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

"Contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to each other in a sequence.

"Designing" or "Designed to" refer to the process of designing an antisense compound that specifically hybridizes with a selected nucleic acid molecule.

"Diabetes mellitus" or "diabetes" is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

"Diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles.

"Diluent" means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition may be a liquid, e.g. saline solution.

"Dose" means a specified quantity of a compound or pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose may require a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In other embodiments, the compound or pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.

"Dosing regimen" is a combination of doses designed to achieve one or more desired effects.

"Dosage unit" means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized ISIS 449884. In certain embodiments, a dosage unit is a vial containing reconstituted ISIS 449884.

"Duration" means the period of time during which an activity or event continues. For example, the duration of a loading phase is the period of time during which loading doses are administered. For example, the duration of the maintenance phase is the period of time during which maintenance doses are administered.

"Dyslipidemia" refers to a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

"Effective amount" means the amount of compound sufficient to effectuate a desired physiological outcome in an individual in need of the compound. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

"Efficacy" means the ability to produce a desired effect.

"Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

"Gapmer" means an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings." "GCGR" means any nucleic acid or protein of GCGR. "GCGR nucleic acid" means any nucleic acid encoding GCGR. For example, in certain embodiments, a GCGR nucleic acid includes a DNA sequence encoding GCGR, an RNA sequence transcribed from DNA encoding GCGR (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding GCGR. "GCGR mRNA" means an mRNA encoding a GCGR protein. The target may be referred to in either upper or lower case.

"GCGR specific inhibitor" refers to any agent capable of specifically inhibiting GCGR RNA and/or GCGR protein expression or activity at the molecular level. For example, GCGR specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of GCGR RNA and/or GCGR protein.

"Glucose" is a monosaccharide used by cells as a source of energy and inflammatory intermediate. "Plasma glucose" refers to glucose present in the plasma.

"Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

"Hyperlipidemia" or "hyperlipemia" is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low density lipoproteins, very low density lipoproteins and triglycerides.

"Hypertriglyceridemia" means a condition characterized by elevated triglyceride levels.

"Identifying" or "selecting an animal with metabolic disease" means identifying or selecting a subject having been diagnosed with a metabolic disease, or a metabolic disorder; or, identifying or selecting a subject having any symptom of a metabolic disease, including, but not limited to, metabolic syndrome,

hyperglycemia, hypertriglyceridemia, hypertension increased insulin resistance, decreased insulin sensitivity, above normal body weight, and/or above normal body fat or any combination thereof. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring blood-pressure, measuring body fat, measuring body weight, and the like.

"Immediately adjacent" means there are no intervening elements between the immediately adjacent elements of the same kind (e.g. no intervening nucleobases between the immediately adjacent nucleobases).

"Inhibiting GCGR" means reducing expression of GCGR mRNA and/or protein levels in the presence of a GCGR antisense compound, including a GCGR antisense oligonucleotide, as compared to expression of GCGR mRNA and/or protein levels in the absence of a GCGR antisense compound, such as an antisense oligonucleotide.

"Individual" means a human or non-human animal selected for treatment or therapy.

"Inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.

"Insulin resistance" is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduces glucose uptake whereas insulin resistance in liver reduces glucose storage, with both effects serving to elevate blood glucose. High plasma levels of insulin and glucose due to insulin resistance often leads to metabolic syndrome and type 2 diabetes.

"Insulin sensitivity" is a measure of how effectively an individual processes glucose. An individual having high insulin sensitivity effectively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.

"Intemucleoside linkage" means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. "Modified intemucleoside linkage" means any intemucleoside linkage other than a naturally occurring, phosphate intemucleoside linkage. Non -phosphate linkages are referred to herein as modified intemucleoside linkages.

"ISIS 449884" means an antisense oligonucleotide having the nucleobase sequence

"GGTTCCCGAGGTGCCCA", incorporated herein as SEQ ID NO: 1, where each intemucleoside linkage is a phosphorothioate intemucleoside linkage, each cytosine is a 5-methylcytosine, and each of nucleosides 1-3 and 14-17 comprise a 2'-MOE moiety. ISIS 449884 is complementary to nucleobases 7270-7286, 7295- 7311, 7319-7335, 7344-7360, 7368-7384, 7392-7408, 7416-7432, and 7440-7456 of the sequence of RefSeqNo. NW_926918.1 truncated from nucleobases 16865000 to 16885000, incorporated herein as SEQ ID NO: 2.

"Linked nucleosides" means adjacent nucleosides linked together by an intemucleoside linkage.

"Lipid-lowering therapy" or "lipid lowering agent" means a therapeutic regimen provided to a subject to reduce one or more lipids in a subject. In certain embodiments, a lipid-lowering therapy is provided to reduce one or more of ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject. Examples of lipid-lowering therapy include statins, fibrates, and MTP inhibitors. "Metabolic disease" or "metabolic disorder" refers to a condition characterized by an alteration or disturbance in metabolic function. "Metabolic" and "metabolism" are terms well known in the art and generally include the whole range of biochemical processes that occur within a living organism. Metabolic diseases or disorders include, but are not limited to, obesity, diabetes, hyperglycemia, prediabetes, non- alcoholic fatty liver disease (NAFLD), metabolic syndrome, insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia or a combination thereof.

"Metabolic syndrome" means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).

"Mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned. For example, nucleobases including but not limited to a universal nucleobase, inosine, and hypoxanthine, are capable of hybridizing with at least one nucleobase but are still mismatched or non-complementary with respect to nucleobase to which it hybridized. As another example, a nucleobase of a first oligonucleotide that is not capable of hybridizing to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatch or non-complementary nucleobase.

"Mixed dyslipidemia" means a condition characterized by elevated cholesterol and elevated triglycerides.

"Modulating" refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating GCGR RNA can mean to increase or decrease the level of GCGR RNA and/or GCGR protein in a cell, tissue, organ or organism. A "modulator" effects the change in the cell, tissue, organ or organism. For example, a GCGR antisense compound can be a modulator that decreases the amount of GCGR RNA and/or GCGR protein in a cell, tissue, organ or organism.

"MOE" means methoxyethyl.

"Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.

"Motif means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.

"Natural" or "naturally occurring" means found in nature. "Non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

"Nucleic acid" refers to molecules composed of monomelic nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double -stranded nucleic acids.

"Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid. As used herein a "naturally occurring nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A "modified nucleobase" is a naturally occurring nucleobase that is chemically modified. A "universal base" or "universal nucleobase" is a nucleobase other than a naturally occurring nucleobase and modified nucleobase, and is capable of pairing with any nucleobase.

"Nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or

oligonucleotide independent of any sugar or intemucleoside linkage.

"Nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.

"Off-target effect" refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

"Oligomeric compound" means a compound comprising a single oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.

"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. Unless otherwise indicated, oligonucleotides consist of 8-80 linked nucleosides. "Modified oligonucleotide" means an oligonucleotide, wherein at least one sugar, nucleobase, or intemucleoside linkage is modified. "Unmodified oligonucleotide" means an oligonucleotide that does not comprise any sugar, nucleobase, or intemucleoside modification.

"Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular

administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. "Pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as PBS or water-for-injection.

"Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

"Pharmaceutical agent" means a compound that provides a therapeutic benefit when administered to an individual.

"Pharmaceutical composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more compounds or salt thereof and a sterile aqueous solution.

"Pharmaceutically acceptable derivative" encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

"Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.

"Phosphorus moiety" means a group of atoms comprising a phosphorus atom. In certain

embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.

"Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.

"Prevent" refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely.

"Prodrug" means a compound in a form outside the body which, when administered to an individual, is metabolized to another form within the body or cells thereof. In certain embodiments, the metabolized form is the active, or more active, form of the compound (e.g., drug). Typically conversion of a prodrug within the body is facilitated by the action of an enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) present in cells or tissues, and/or by physiologic conditions.

"Reduce" means to bring down to a smaller extent, size, amount, or number. "RefSeq No." is a unique combination of letters and numbers assigned to a sequence to indicate the sequence is for a particular target transcript (e.g., target gene). Such sequence and information about the target gene (collectively, the gene record) can be found in a genetic sequence database. Genetic sequence databases include the NCBI Reference Sequence database, GenBank, the European Nucleotide Archive, and the DNA Data Bank of Japan (the latter three forming the International Nucleotide Sequence Database Collaboration or INSDC).

"Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

"RNAi compound" means an antisense compound that acts, at least in part, through RISC or Ago2, but not through RNase H, to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single -stranded RNA (ssRNA), and microRNA, including microRNA mimics.

"Segments" are defined as smaller or sub-portions of regions within a nucleic acid.

"Side effects" means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system

abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

"Single-stranded" in reference to a compound means the compound has only one oligonucleotide. "Self-complementary" means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligonucleotide, wherein the oligonucleotide of the compound is self-complementary, is a single-stranded compound. A single-stranded compound may be capable of binding to a complementary compound to form a duplex.

"Sites," are defined as unique nucleobase positions within a target nucleic acid.

"Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.

"Specifically inhibit" a target nucleic acid means to reduce or block expression of the target nucleic acid while exhibiting fewer, minimal, or no effects on non-target nucleic acids reduction and does not necessarily indicate a total elimination of the target nucleic acid's expression.

"Statin" means an agent that inhibits the activity of HMG-CoA reductase.

"Sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. "Unmodified sugar moiety" or "unmodified sugar" means a 2'-OH(H) furanosyl moiety, as found in RNA (an "unmodified RNA sugar moiety"), or a 2'-H(H) moiety, as found in DNA (an "unmodified DNA sugar moiety"). Unmodified sugar moieties have one hydrogen at each of the Γ, 3 ', and 4' positions, an oxygen at the 3 ' position, and two hydrogens at the 5 ' position. "Modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety or a sugar surrogate. "Modified furanosyl sugar moiety" means a furanosyl sugar comprising a non- hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain

embodiments, a modified furanosyl sugar moiety is a 2 '-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.

"Synergy" or "synergize" refers to an effect of a combination that is greater than additive of the effects of each component alone at the same doses.

"Target gene" refers to a gene encoding a target.

"Targeting" or "targeted" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

"Target nucleic acid," "target R A," "target R A transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by antisense compounds.

"Target region" means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

"Target segment" means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. "5 ' target site" refers to the 5 '-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment.

"Therapeutic lifestyle change" means dietary and lifestyle changes intended to lower fat /adipose tissue mass and/or cholesterol. Such change can reduce the risk of developing heart disease, and may include recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity. "Therapeutically effective amount" means an amount of a compound, pharmaceutical agent, or composition that provides a therapeutic benefit to an individual.

"Treat" refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.

Certain Embodiments

In certain embodiments, there is provided a method of treating diabetes comprising administering concomitantly to an individual in need thereof a therapeutically effective combination of a modified oligonucleotide targeting GCGR and metformin. In certain embodiments, the diabetes is Type 2 diabetes (T2DM). In certain embodiments, the diabetic individual suffers from hyperglycemia that is uncontrolled by metformin.

In certain embodiments, there is provided a method of treating diabetes comprising administering concomitantly to an individual in need thereof a modified oligonucleotide targeting GCGR and metformin in amounts that in combination are effective in treating said diabetes. In certain embodiments, the diabetes is

T2DM. In certain embodiments, the said amounts results in additive or greater-than-additive improvements of the symptoms of diabetes in the individual. In certain embodiments, the said amounts cause synergistic improvements of the symptoms of diabetes in the individual. In certain embodiments, the combination reduces plasma glucose levels. In certain embodiments, the combination increases active GLP-1 levels. In certain embodiments, the combination increases total GLP-1 levels. In certain embodiments, the combination increases glucagon levels. In certain embodiments, the combination reduces HbAlc levels. In certain embodiments, the combination reduces fructosamine levels. In certain embodiments, the combination improves glucose tolerance. In certain embodiments, the combination reduces insulin resistance. In certain embodiments, the combination improves insulin sensitivity.

In certain embodiments, the antisense compounds targeting GCGR are useful for treating diabetes, such as Type 2 diabetes, at unexpectedly low doses for an antisense compound. In certain embodiments, the antisense compound comprises a modified oligonucleotide. In certain embodiments, the dose is 50 mg/week of the modified oligonucleotide. In certain embodiments, the dose is 75 mg/week of the modified oligonucleotide. In certain embodiments, there is provided a method of enhancing the therapeutic effectiveness of metformin in treating diabetes comprising administering to an individual in need thereof with a therapeutically effective combination of a modified oligonucleotide targeting GCGR and metformin. In certain embodiments, the individual is diabetic. In certain embodiments, the diabetes is Type 2 diabetes.

In any of the foregoing embodiments, the antisense compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 7270-7286, 7295-7311, 7319-7335, 7344-7360, 7368-7384, 7392-7408, 7416-7432, and 7440-7456 of SEQ ID NO: 2, wherein the nucleobase sequence is complementary to SEQ ID NO: 2.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 1 or consists of the sequence of SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide is 100% complementary to SEQ ID NO: 2.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the sequence of any of the GCGR antisense oligonucleotides described in US. Pat. No. 7,750, 142 or U.S. Pat. No. 8,865,674, both of which are herein incorporated by reference in their entirety In certain embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide. In certain embodiments, the modified oligonucleotide comprises at least one modified intemucleoside linkage. In several embodiments, each intemucleoside linkage is a phosphorothioate intemucleoside linkage. In certain embodiments, at least one nucleoside comprises a modified sugar, such as a bicyclic sugar including, but not limited to, a 4'- CH₂-0-2' bridge or a 4'-CH(CH₃)-0-2' bridge. In certain embodiments, the modified sugar comprises a 2'-0(CH₂)₂-OCH₃ group. In certain embodiments, at least one nucleoside comprises a modified nucleobase, such as a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises:

a 5 '-wing consisting of 1 to 5 linked nucleosides;

a 3 '-wing consisting of 1 to 5 linked nucleosides; and

a gap between the 5'-wing and the 3'-wing consisting of 8 to 12 linked 2'-deoxynucleosides;

wherein at least one of the 5'-wing and the 3'-wing comprises at least one bicyclic nucleoside or one 2'- substituted nucleoside. In certain embodiments, the 2 '-substituted nucleoside comprises a 2'-0(CH₂)₂-OCH₃ group or a 2'-0-CH₃ group. In certain embodiments, the bicyclic nucleoside comprises a 4'- CH₂-0-2' bridge or a 4'-CH(CH₃)-0-2' bridge.

In certain embodiments, the modified oligonucleotide is single -stranded, consisting of 17 linked nucleosides, having the nucleobase sequence of SEQ ID NO: 1, and comprising:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of 3 linked nucleosides; and

a 3' wing segment consisting of 4 linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment; wherein each nucleoside of each wing segment comprises a 2'MOE nucleoside; wherein each intemucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is a 5'-methylcytosine.

In certain embodiments, the modified oligonucleotide is ISIS 449884 and can be named by accepted oligonucleotide nomenclature, showing each 3'-0 to 5'-0-linked phosphorothioate diester intemucleotide linkage as follows:

2'-0-(2-methoxyethyl)-i^?-thioguanylyl-(3'-0→-5'-0)-2'-0-(2-methoxyethyl)-i^?-thioguanylyl (3'- 0→5'-0)-2'-0-(2-methoxyethyl)-5-methyl-i^?-thiouridylyl-(3'-0→-5'-0)-2'-deoxy-5-methyl-i^?- miothymidylyl-(3'-0→5'-0)-2'-deoxy-5-me^

thiocytidylyl-(3 '-0→5 '-0)-2'-deoxy-5 -methyl - -thiocytidylyl -(3 '-0→5 '-0)-2'-deoxy- -mioguanylyl-(3 '- 0→5'-0)-2'-deoxy- i^?-thioadenylyl-(3'-0→-5'-0)-2'-deoxy-i^?-thioguanylyl-(3'-0→-5'-0)-2'-deoxy-i^?- thioguanylyl-(3 '-0→5 '-0)-2'-deoxy-i^?-thiothymidylyl-(3 '-0→5 '-0)-2'-deoxy- thioguanylyl-(3 ' -0→5' -0)- - 0-(2-methoxyethyl)-5 -methyl - -thiocytidylyl -(3 '-0→5 '-0)-2'-0(2-methoxyethyl)-5 -methyl - -thiocytidylyl <3'^→5'^)-2'-0 2-methoxyethyl)-5-methyl- -thiocytidylyl -(3'-0→5'-0)-2'-O-(2-methoxyethyl)- adenosine, 16 sodium salt.

In certain embodiments, the modified oligonucleotide is ISIS 449884 and has the following structure:

In certain embodiments, the modified oligonucleotide described herein is administered in the form of a dosage unit (e.g., injection, infusion, etc.) in a pharmaceutical composition. In certain embodiments, such pharmaceutical compositions comprise an antisense oligonucleotide, including but not limited to ISIS 449884, in an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg. It will be understood that the aforementioned amounts of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069). In certain embodiments, metformin is administered in the form of a dosage unit (e.g., injection, infusion, etc.) in a pharmaceutical composition. In certain embodiments, such pharmaceutical compositions comprise metformin in an amount of any of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 1550 mg, about 2000 mg, about 2500 mg, about 2550 mg, or about 3000 mg. It will be understood that the aforementioned amounts of metformin can be readily represented as milligrams of metformin per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069).

In certain embodiments, a combination of an antisense oligonucleotide, including but not limited to ISIS 449884 and metformin is administered concomitantly. In certain embodiments, the antisense oligonucleotide, including but not limited to ISIS 449884, is in an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg. In certain embodiments, the antisense oligonucleotide is in the dose range 15-30 mg. In certain embodiments, the antisense

oligonucleotide is in the dose range of 30-60 mg. In certain embodiments, the dose range is 60-80 mg. In certain embodiments, the dose range is 80-100 mg. In certain embodiments, metformin is in an amount of any of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 1550 mg, about 2000 mg, about 2500 mg, about 2550 mg, or about 3000 mg. In certain embodiments, the dose range for metformin is 500-3000 mg. It will be understood that the aforementioned amounts of antisense oligonucleotide or metformin can be readily represented as milligrams of the compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069).

In certain embodiments, the antisense oligonucleotide is concomitantly administered at the same time as the dose of metformin. In certain embodiments, the antisense oligonucleotide is concomitantly

administered before the dose of metformin. In certain embodiments, the antisense oligonucleotide is concomitantly administered after the dose of metformin. In certain embodiments, the treatment with antisense oligonucleotide is started after the treatment with metformin has been started. In certain embodiments, the treatment with antisense oligonucleotide is started at the same time as the treatment with metformin. In certain embodiments, the treatment with antisense oligonucleotide is started before the treatment with metformin has been started. The compositions described herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions described herein. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and/or aromatic substances and the like which do not deleteriously interact with the

oligonucleotide (s) of the formulation.

Antisense oligonucleotides may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense oligonucleotides can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense oligonucleotide having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'- terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003.

Certain treatments

In certain aspects there is provided a method of treating an individual suffering from diabetes comprising concomitantly administering to the individual a combination of an antisense compound complementary to human GCGR and metformin. In certain embodiments the antisense compound complementary to human GCGR is as described herein or as disclosed in US. Pat. No. 7,750, 142 or U.S. Pat. No. 8,865,674. In certain embodiments the antisense compound complementary to human GCGR is ISIS 449884.

In certain embodiments the diabetes is Type 2 diabetes mellitus (T2DM).

In certain aspects there is provided a combination of an antisense compound complementary to human GCGR and metformin for use in treating T2DM. In certain embodiments the antisense compound complementary to human GCGR is as described herein or as disclosed in US. Pat. No. 7,750, 142 or U.S. Pat. No. 8,865,674. In certain embodiments the antisense compound complementary to human GCGR is ISIS 449884. In certain embodiments the diabetes is Type 2 diabetes mellitus (T2DM).

In certain aspects there is an antisense compound complementary to human GCGR for use in a method of treating diabetes in an individual in need thereof, wherein the method comprises concomitantly administering to the individual with a combination of the antisense compound and metformin. In certain embodiments the antisense compound complementary to human GCGR is as described herein or as disclosed in US. Pat. No. 7,750,142 or U.S. Pat. No. 8,865,674. In certain embodiments the antisense compound complementary to human GCGR is ISIS 449884. In certain embodiments the diabetes is Type 2 diabetes mellitus (T2DM).

Certain aspects are directed to use of a combination of an antisense compound complementary to human GCGR and metformin for the manufacture of a medicament for treating diabetes. In certain embodiments the antisense compound complementary to human GCGR is as described herein or as disclosed in US. Pat. No. 7,750,142 or U.S. Pat. No. 8,865,674. In certain embodiments the antisense compound complementary to human GCGR is ISIS 449884. In certain embodiments the diabetes is Type 2 diabetes mellitus (T2DM).

Certain Dosing Regimens

In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen. In certain such embodiments, the dosing regimen is effective to treat diabetes and acceptably tolerable in an individual. In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide targeted to GCGR and metformin. In certain embodiments, a pharmaceutical combination comprises an antisense oligonucleotide targeted to GCGR and metformin. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 1. In certain embodiments, the antisense oligonucleotide is ISIS 449884.

In certain embodiments, the doses of the antisense oligonucleotide, including but not limited to ISIS

449884, administered are about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg. It will be understood that the aforementioned doses of antisense oligonucleotide can be readily represented as milligrams of the antisense compound per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069). In certain embodiments, the dose of metformin is in an amount of any of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 1550 mg, about 2000 mg, about 2500 mg, about 2550 mg, or about 3000 mg. It will be understood that the aforementioned amounts of metformin can be readily represented as milligrams of metformin per kilogram of the subject's body weight per week (mg/kg/wk) by simply dividing the amount by the subject's body weight per week. In certain embodiments, body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069).

In certain embodiments, a combination of an antisense oligonucleotide, including but not limited to ISIS 449884 and metformin is administered concomitantly. In certain embodiments, the antisense oligonucleotide, including but not limited to ISIS 449884, is in an amount of any of about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg. In certain embodiments, metformin is in an amount of any of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 1550 mg, about 2000 mg, about 2500 mg, about 2550 mg, or about 3000 mg.

In certain embodiments, in the dosing regimen, the administration of the antisense oligonucleotide is at the same time as the administration of metformin. In certain embodiments, in dosing regimen, the administration of the antisense oligonucleotide is before the administration of metformin. In certain embodiments, in the dosing regimen, the administration of the antisense oligonucleotide is after the administration of metformin. In certain embodiments, in the dosing regimen, the treatment with antisense oligonucleotide is started after the treatment with metformin has been started. In certain embodiments, in the dosing regimen, the treatment with antisense oligonucleotide is started at the same time as the treatment with metformin. In certain embodiments, in the dosing regimen, the treatment with antisense oligonucleotide is started before the treatment with metformin has been started.

In certain embodiments, dose, dose frequency, and duration of the treatment period may be selected to achieve a desired effect within 1 to 26 weeks. In certain embodiments, the dose is the same and the dose frequency is varied to achieve the desired effect within 1 to 26 weeks. In certain embodiments, the dose increases over time and the dose frequency remains constant. In certain embodiments, it is desirable to achieve a desired effect as quickly as possible. In certain embodiments, a high dose and/or high dose frequency may be desirable. In certain embodiments, the dose may be administered in one administration or injection. In certain embodiments, the dose may be administered in more than one administration or injection. In certain embodiments, doses, dose frequency, and duration of the treatment period may be selected to achieve an acceptable safety profile. For example, in certain embodiments, such variables may be selected to mitigate toxicity of the pharmaceutical composition. In certain embodiments, such variables are selected to mitigate liver toxicity. In certain embodiments, such variables are selected to mitigate renal toxicity. In certain embodiments, such variables are selected to mitigate prolongation of aPTT, complement pathway activation, pro-inflammatory effects, thrombocytopenia or neutropenia.

In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal. In certain embodiments, a safety profile is not acceptable when ALT is 5-10 times the upper limit of normal, and bilirubin is elevated two or more times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal. In certain embodiments, an acceptable safety profile comprises ALT elevations that are above three times the upper limit of normal, but do not exceed five times the upper limit of normal, and bilirubin elevations that do not exceed two times the upper limit of normal. In certain embodiments, when administration of a pharmaceutical composition results in ALT elevations that are above three times the upper limit of normal, the dose and/or dose frequency is adjusted to mitigate the ALT elevation.

In certain embodiments, the treatment period lasts from one day to the lifetime of the individual. In certain embodiments, the treatment period lasts 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days as measured from administration of the first dose to the last dose administered to the individual. In certain embodiments, the treatment period lasts 1 week, 2 weeks, 3, weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks as measured from administration of the first dose to the last dose administered to the individual. In certain embodiments, the treatment period lasts 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, or 12 months as measured from administration of the first dose to the last dose administered to the individual. In certain embodiments, the treatment period lasts 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 1 1 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, or 50 years as measured from administration of the first dose to the last dose administered to the individual. In certain embodiments, the treatment period lasts as long as the dose continues to be needed, effective, and tolerated. In certain embodiments where the treatment period includes more than one dose, the doses administered during the treatment period are all the same as one another. In certain embodiments, the doses administered during the treatment period are not all the same. In certain embodiments, the doses increase over time. In certain embodiments, the doses decrease over time.

In certain embodiments, a dose is administered by parenteral administration. In certain embodiments, the parenteral administration is subcutaneous administration. In certain embodiments, the parenteral administration is intravenous infusion.

In certain embodiments, the doses during the treatment period are about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2.0 mg, about 2.1 mg, about 2.2 mg, about 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3.0 mg, about 3.1 mg, about 3.2 mg, about 3.3 mg, about 3.4 mg, or about 3.5 mg of the antisense compound per kilogram of the subject's body weight. In certain embodiments, the subject's body weight is calculated as the ideal body weight using the Devine formula (Pai, M.P. and Paloucek, F.P. Ann. Pharmacol. 2000. 34: 1066-1069).

In certain embodiments, doses, dose frequency, and duration of the treatment period may be selected to achieve a desired safety profile. For example, in certain embodiments, such variables may be selected to mitigate toxicity of the pharmaceutical composition. In certain embodiments, such variables are selected to mitigate liver toxicity. In certain embodiments, such variables are selected to mitigate renal toxicity. In certain embodiments, such variables are selected to mitigate prolongation of aPTT, complement pathway activation, pro-inflammatory effects, thrombocytopenia or neutropenia.

In certain embodiments, doses, dose frequency, and duration of the treatment period may be adjusted from time to time to achieve a desired effect. In certain embodiments, individuals are monitored for effects (therapeutic and/or toxic effects) and doses, dose frequency, and/or duration of the treatment period may be adjusted based on the results of such monitoring.

In certain embodiments, the pharmaceutical composition comprises an antisense oligonucleotide and metformin. In certain embodiments, the antisense oligonucleotide has the nucleobase sequence of SEQ ID NO: 1. In certain embodiments, the antisense oligonucleotide is ISIS 449884.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mR A. In certain embodiments, the target nucleic acid is a pre-mR A. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

Nucleotide sequences that encode GCGR include, without limitation, the following: RefSeqNo.

NM_000160.3 (incorporated herein as SEQ ID NO: 3) and RefSeqNo. NW_926918.1 truncated from nucleotides 16865000 to 16885000 (incorporated herein as SEQ ID NO: 2).

Compositions and Methods for Formulating Pharmaceutical Compositions

Compounds described herein may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more compounds or a salt thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compounds comprise or consist of a modified oligonucleotide. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS.

Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

A compound described herein targeted to GCGR nucleic acid can be utilized in pharmaceutical compositions by combining the compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising a compound targeted to GCGR nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the compound comprises or consists of a modified oligonucleotide provided herein. Pharmaceutical compositions comprising compounds provided herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of a compound which are cleaved by endogenous nucleases within the body, to form the active compound. In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent. NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

While the present invention has been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. Each of the references, RefSeqNos, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

Example 1: Phase 2 randomized double blind, stratified, placebo-controlled study of ISIS 449884 in combination with metformin

The effect of administration of GCGR antisense oligonucleotide, ISIS 449884, in patients with Type 2 diabetes on a stable treatment of metformin is being studied in an on-going clinical trial.

Subjects

The target study population is 60 patients with Type 2 diabetes being treated with metformin for a minimum of 3 months prior to screening evaluation and with a BMI > 25.0 kg/m².

Dose Regimen

This study is a multicenter study of two doses of ISIS 449884 at 50 mg/week (Cohort A) and 75 mg/week (Cohort B) in combination with metformin versus metformin plus placebo. Patients participating in this study must be on a stable dose of metformin (at least 1,500 mg/day) for a minimum of 3 months prior to screening evaluations and will be required to continue their stable dose unchanged throughout the study. Patients will be stratified based on their screening HbAlc levels (< 9.0% versus > 9.0%).

The study will include a < 3-week Screening Period, a 3-week Pre-Treatment Period, followed by a

26-week Treatment Period of once weekly subcutaneous (SC) dosing and a 24-week Post-Treatment period. Patient eligibility for the study will be determined during the Screening and Pre-Treatment Periods. Patients will be randomized during the Pre-Treatment Period.

Patients will receive their assigned Study Drug (ISIS 449884 or placebo) via SC injection once weekly from week 1 through week 26 (days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106, 113, 120, 127, 134, 141, 148, 155, 162, 169, and 176). The Study Drug will be administered as a SC injection by a trained professional. The Dosing information is further described in the Table below.

Table 2: Study Drug Dosing Information

Safety and clinical laboratory evaluations will be performed on a weekly basis from Week 1 to Week

14 and bi-weekly from Week 15 to Week 26. Blood samples will be taken periodically during the patients' visits for parameters including, but not limited to, fasting C-peptide, fasting insulin, fasting plasma glucose (FPG), fasting glucagon, fasting active Glucagon-like Peptide-1 (GLP-1), fasting total GLP-1, HbAlc, lipids and TSH. Body weight, BMI, Waist/Hip Circumference will also be measured periodically. The plasma pharmacokinetics of ISIS 449884 will be assessed periodically.

Patients will undergo four oral glucose tolerance test (OGTT) procedures during the Pre-Treatment, Treatment, and Post-Treatment Periods. For each OGTT procedures, patients will begin fasting the evening before the test. The procedure will be conducted the next day (after 10 hours of fasting), when blood samples will be collected. After an indwelling catheter is inserted and a 30-minute period is allowed to remove any effects linked to the stress induced by venipuncture, blood sampling will begin at -10 and -5 minutes before the glucose solution is consumed. The glucose solution (75 grams in a 300 mL solution) will be consumed within a 5 minute period. Blood samples for glucagon, active GLP-1, total GLP-1, C-peptide, insulin, and glucose analysis will be drawn during the test every 30 minutes during the 2 hours of the OGTT.

Results

This study was a multicenter study of two doses of ISIS 449884 at 50 mg/week (Cohort A) and 75 mg/week (Cohort B) in combination with metformin versus metformin plus placebo. Patients participating in this study were on a stable dose of metformin (at least 1,500 mg/day) for a minimum of 3 months prior to screening evaluations and continued their stable dose unchanged throughout the study. Patients were stratified based on their screening HbAlc levels (< 9.0% versus > 9.0%).

Patients received their assigned Study Drug (ISIS 449884 or placebo) via SC injection once weekly from week 1 through week 26 (days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106, 113, 120, 127, 134, 141, 148, 155, 162, 169, and 176). A total of 39 patients were enrolled in Cohort A and 40 patients were enrolled in Cohort B (see Table 2 above).

Levels of the primary endpoint, HbAlc, were measured on week 27. The results are presented in the Table below and demonstrate a reduction of >2% in HbAlc in patients given the 75 mg dose and >1% reduction in patients given the 50 mg dose. There was up to 3.4% reduction in some patients at the 75 mg dose.

Table 3: Mean change (% baseline) in HbAlc

ALT levels were also measured regularly and did not show any elevations in level outside the expected range for antisense oligonucleotides. Overall, the study demonstrated that both doses of ISIS 449884 had a favorable safety profile and were well tolerated.

Claims

WHAT IS CLAIMED IS:

1. A method of treating diabetes, comprising administering to an individual in need thereof a dose of a modified antisense oligonucleotide targeting GCGR and a dose of metformin, wherein said doses of said modified GCGR antisense oligonucleotide and said metformin are effective in combination in treating diabetes in the individual.

2. The method of claim 1, wherein the dose of the modified antisense oligonucleotide and/or the dose of the metformin in combination is less than the therapeutically effective amount of modified antisense oligonucleotide alone and/or the therapeutically effective amount of metformin alone in treating diabetes in the individual.

3. The method of claim 1 or 2, wherein the modified oligonucleotide and metformin in combination provide a greater-than-additive effect compared to the modified oligonucleotide alone or the metformin alone in treating diabetes in the individual.

4. The method of any preceding claim, wherein the modified oligonucleotide and metformin synergize in combination to treat diabetes in the individual.

5. The method of any preceding claim, wherein the modified antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1.

6. The method of any preceding claim, wherein the dose of modified antisense oligonucleotide is 50 mg per week.

7. The method of any preceding claim, wherein the dose of modified antisense oligonucleotide is 75 mg per week.

8. The method of any preceding claim, wherein the dose is administered for at least 1-26 weeks.

9. The method of any preceding claim, wherein the dose is administered to the subject one time per week.

10. The method of any preceding claim, wherein the method of treating diabetes reduces plasma glucose levels.

11. The method of any preceding claim, wherein the method of treating diabetes increases active GLP-1 levels.

12. The method of any preceding claim, wherein the method of treating diabetes increases glucagon

levels.

13. The method of any preceding claim, wherein the method of treating diabetes reduces HbAlc

concentration levels.

14. The method of any preceding claim, wherein the method of treating diabetes reduces fructosamine levels.

15. The method of any preceding claim, wherein the method of treating diabetes improves glucose

tolerance.

16. The method of any one of claims 1-15, wherein the modified oligonucleotide is a single -stranded modified oligonucleotide.

17. The method of any one of claims 1-15, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.

18. The method of claim 17, wherein each intemucleoside linkage of the modified oligonucleotide is a phosphorothioate intemucleoside linkage.

19. The method of any one of claims 1-18, wherein at least one nucleoside of the modified

oligonucleotide comprises a modified sugar.

20. The method of claim 19, wherein at least one modified sugar is a bicyclic sugar.

21. The method of claim 20, wherein the bicyclic sugar comprises a 4'- CH₂-0-2' bridge, a 4'-CH(CH₃)- 0-2' bridge or a 2'-0(CH₂)₂-OCH₃ group.

22. The method of any one of claims 1-21, wherein at least one nucleoside of the modified

oligonucleotide comprises a modified nucleobase.

23. The method of claim 22, wherein the modified nucleobase is a 5'-methylcytosine.

24. The method of any one of claims 1-22, wherein the modified oligonucleotide is single-stranded

consisting of 17 linked nucleosides having a nucleobase sequence consisting of SEQ ID NO: 1, comprising:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of 3 linked nucleosides; and

a 3' wing segment consisting of 4 linked nucleosides;

25. A method of treating diabetes, comprising administering to an individual in need thereof 50 mg per week of ISIS 449884 and a 500-3000 mg daily dose range of metformin, wherein said doses are effective in combination in treating diabetes in the individual.

26. The method of claim 25, wherein the dose of metformin is 1500 mg per day.

27. A method of treating diabetes, comprising administering to an individual in need thereof 75 mg per week of ISIS 449884 and a 500-3000 mg daily dose range of metformin, wherein said doses are effective in combination in treating diabetes in the individual.

28. The method of claim 27, wherein the dose of metformin is 1500 mg per day.

29. A combination for treating diabetes by administering to an individual in need thereof, comprising a dose of a modified antisense oligonucleotide targeting GCGR and a dose of metformin, wherein said doses of said modified GCGR antisense oligonucleotide and said metformin are effective in combination in treating diabetes in the individual.

30. The combination of claim 29, wherein the dose of the modified antisense oligonucleotide and/or the dose of the metformin in combination is less than the therapeutically effective amount of modified antisense oligonucleotide alone and/or the therapeutically effective amount of metformin alone in treating diabetes in the individual.

31. The combination of claim 1 or 2, wherein the modified oligonucleotide and metformin in combination provide a greater-than-additive effect compared to the modified oligonucleotide alone or the metformin alone in treating diabetes in the individual.

32. The combination of any preceding claim, wherein the modified oligonucleotide and metformin

synergize in combination to treat diabetes in the individual.

33. The combination of any one of claims 29-32, wherein the modified antisense oligonucleotide

comprises the nucleotide sequence of SEQ ID NO: 1.

34. The combination of any one of claims 29-33, wherein the dose of modified antisense oligonucleotide is 50 mg per week.

35. The combination of any one of claims 29-33, wherein the dose of modified antisense oligonucleotide is 75 mg per week.

36. The combination of any one of claims 29-35, wherein the dose is administered for at least 1-26 weeks.

37. The combination of any one of claims 29-36, wherein the dose is administered to the subject one time per week.

38. The combination of any one of claims 29-37, wherein the combination reduces plasma glucose levels.

39. The combination of any one of claims 29-38, wherein the combination increases active GLP-1 levels.

40. The combination of any one of claims 29-39, wherein the combination increases glucagon levels.

41. The combination of any one of claims 29-40, wherein the combination reduces HbAlc concentration levels.

42. The combination of any one of claims 29-41, wherein the combination reduces fructosamine levels.

43. The combination of any one of claims 29-42, wherein the combination improves glucose tolerance.

44. The combination of any one of claims 29-43, wherein the modified oligonucleotide is a single- stranded modified oligonucleotide.

45. The combination of any one of claims 29-44, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.

46. The combination of claim 45, wherein each intemucleoside linkage of the modified oligonucleotide is a phosphorothioate intemucleoside linkage.

47. The combination of any one of claims 29-46, wherein at least one nucleoside of the modified

oligonucleotide comprises a modified sugar.

48. The combination of claim 47, wherein at least one modified sugar is a bicyclic sugar.

49. The combination of claim 48, wherein the bicyclic sugar comprises a 4'- CH₂-0-2' bridge, a 4'- CH(CH₃)-0-2' bridge or a 2'-0(CH₂)₂-OCH₃ group.

50. The combination of any one of claims 29-49, wherein at least one nucleoside of the modified

oligonucleotide comprises a modified nucleobase.

51. The combination of claim 50, wherein the modified nucleobase is a 5 '-methylcytosine.

52. The combination of any one of claims 29-51, wherein the modified oligonucleotide is single -stranded consisting of 17 linked nucleosides having a nucleobase sequence consisting of SEQ ID NO: 1, comprising:

a gap segment consisting of ten linked deoxynucleosides;

a 5' wing segment consisting of 3 linked nucleosides; and

a 3' wing segment consisting of 4 linked nucleosides;

wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment; wherein each nucleoside of each wing segment comprises a 2'MOE nucleoside; wherein each intemucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is a 5 '-methylcytosine.

53. A combination for treating diabetes, comprising administering to an individual in need thereof 50 mg per week of ISIS 449884 and a 500-3000 mg daily dose range of metformin, wherein said doses are effective in combination in treating diabetes in the individual.

54. The combination of claim 53, wherein the dose of metformin is 1500 mg per day.

55. A combination for treating diabetes, comprising administering to an individual in need thereof 75 mg per week of ISIS 449884 and a 500-3000 mg daily dose range of metformin, wherein said doses are effective in combination in treating diabetes in the individual.

56. The combination of claim 55, wherein the dose of metformin is 1500 mg per day.