# Patent

## Title
Uses of NOGO-A inhibitors and related methods

## Abstract

The present invention is directed to Nogo-A antagonists useful for the control of blood glucose or blood insulin levels in a subject and related use and formulation thereof. In particular, the invention is directed to Nogo-A antagonists useful for the prevention, repression or treatment insulin secretion deficiency and related methods and pharmaceutical formulations. In particular, the invention relates to Nogo-A antagonists useful in the treatment of diabetes mellitus.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage application of International Patent Application No. PCT/IB2011/053056, filed Jul. 8, 2011, which claims the benefit of U.S. Provisional Patent Application No. 61/362,876, filed Jul. 9, 2010.


The Sequence Listing for this application is labeled “Seq-List.txt” which was created on Dec. 31, 2012 and is 105 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to substances and compositions thereof useful in the stimulation of insulin secretion, in particular in the treatment of insulin secretion deficiency. In particular, the invention relates to substances and compositions useful in the treatment of diabetes mellitus, in particular type II diabetes.
BACKGROUND OF THE INVENTION
Insulin secretion from islet β-cells is initiated by the cholinergic parasympathetic stimulation of β-cells (the so-called “cephalic phase”) and subsequently potentiated during the enteric “absorptive phase” (D'Alessio et al., 2001, J. Clin. Endocrinol. Metab., 86:1253-1259). In response to mechanical and chemical sensor stimulation along the digestive tract, intestinal hormones like the incretins GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory peptide) potentiate insulin secretion directly and indirectly, through neuronal stimulation (the “incretin effect”) (Karlsson et al., 1992, Eur. J. Pharmacol., 213:145-146; Balkan et al., 2000, Am. J. Physiol. Regul. Integr. Comp. Physiol., 279:R1449-1454). Progressively, nutrient absorption and blood glucose rise stimulate insulin secretion directly (“post absorptive phase”) (Straub et al., 2002, Diabetes Metab. Res. Rev., 18:451-463). Altogether, different secretagogues act synergistically and trigger the adequate biphasic release of insulin from β-cells. These secretagogues reach islet endocrine cells through the vascular and neural networks. Pancreas innervation consists of parasympathetic and sympathetic efferent fibers, which are branches of the parasympathetic vagus nerve and the sympathetic splanchnic nerves. The vagal input stimulates insulin secretion via cholinergic (i.e. mediated by acetylcholine, Ach) or non-cholinergic mechanisms. Sympathetic postganglionic terminal nerves release noradrenaline or other peptides on endocrine cells repressing insulin and somatostatin secretion, and promoting glucagon release. The afferent sensory fibers innervate the periphery of islets and release peptides, like the calcitonin gene-related peptide (CGRP) repressing insulin secretion (Pettersson and Ahren, 1990, Diabetes Res., 15:9-14).
Insulin secretion insufficiency is responsible for diabetes mellitus (DM). There are two major forms of diabetes mellitus: insulin-dependent (Type I) diabetes mellitus (IDDM) which accounts for about 5 to 10% of all cases, and non-insulin-dependent (Type II) diabetes mellitus (NIDDM or T2DM) which accounts for roughly 90% of all cases. In type I diabetes, β-cell loss is due to an autoimmune reaction. In type II diabetes, increased peripheral insulin resistance challenges the functional β-cell mass: after an initial attempt at overriding the increased insulin demand, β-cell function and number decline progressively, resulting in a large spectrum of conditions that require different prescriptions. Diabetes mellitus affects more than 150 million adults and is one of the leading causes of mortality in the world. Generally, when T2DM is diagnosed, global β-cell function is already reduced by about 50%.
Enhancement of insulin secretion in type II diabetic patients is promoted with drugs such as sulfonylureas, thiazolidinediones (TZD) or GLP-1 receptor agonists, but these treatments do not prevent β-cell exhaustion. Oral anti-diabetics (insulin sensitizers and secretagogues) are found useful during the first stages of the disease when insulin resistance predominates and an insulin pancreatic reserve is still available. However, as pancreatic impairment progresses, basal insulin level starts to be an essential parameter to control for achieving metabolic control in patients. At a later stage of T2DM progression, only a basal-bolus regimen of insulin is able to maintain homeostasis in most patients. Currently, no treatment can stably restore a physiological profile of insulin secretion, leading to diabetes progression and development of serious complications.
Therefore, there is huge heath and economical needs for the development of new treatments for managing insulin secretion insufficiency and in particular new treatment for diabetes mellitus, notably T2DM.
Nogo-A, also known as reticulon-4 or neurite outgrowth inhibitor, is a high molecular weight membrane synaptic protein mostly expressed in the central nervous system (CNS), notably in oligodendrocytes and in subsets of neurons (Chen et al., 2000, Nature, 403:434-439). Nogo-A expression is not restricted to the CNS, but is also found in human skeletal muscle cells (Jokic et al., 2005, Ann. Neurol., 57:553-556). In the intact CNS, Nogo-A appears to have a stabilizing, growth controlling role (Montani et al., 2009, J. Biol. Chem., 284:10793-10807). Nogo-A regulates neurite growth and cell migration (Chen et al., 2000, above). In particular, Nogo-A was shown to restrict neuronal regeneration in the injured adult spinal cord and brain, and to limit plastic rearrangements and functional recovery after large CNS lesions (Schwab, 2004, Curr. Opin. Neurobiol., 14:118-124; Cafferty et al., 2006, J. Neurosci., 26:12242-12250). The growth inhibitory action of Nogo-A is mediated by cytoskeletal regulators, such as Rho GTPases or cofilin (Montani et al., 2009, above). Nogo-A and its receptor (NgR) are also found in synapses, where they may influence synapse stability and function (Aloy et al., 2006, Brain Cell Biol., 35, 137-56).
Nogo-A antagonists have been developed to promote CNS axonal regeneration and functional recovery after spinal cord injury (EP1711530; WO2004/052932; Walmsley et al., 2007, Current Pharmaceutical Design, 13(24), pp. 2470-2484(15); Yang et al., 2009, Annals of Neurology, 999, 999A).
SUMMARY OF THE INVENTION
The present invention relates to the unexpected finding that Nogo-A knock-out mice (mice lacking 2 active Nogo-A alleles by the targeted deletion of Rtn4 exon 3) (Simonen et al., 2003, Neuron, 38, 201-211) challenged with different insulin secretagogues present an increased insulin secretion, resulting in a higher glucose clearance, compared with wild type controls. This effect was unexpectedly found to be associated with a stronger pancreatic parasympathetic input and with higher sensitivity of β-cells to cholinergic and GLP-1 stimulation.
A first aspect of the invention provides a Nogo-A antagonist for controlling blood glucose or blood insulin levels in a subject.
A second aspect of the invention relates a use of a Nogo-A antagonist for controlling blood glucose or blood insulin levels in a subject.
A third aspect of the invention relates a use of a Nogo-A antagonist for the preparation of a medicament for the treatment of insulin secretion deficiency, in particular insulin secretion insufficiency.
A fourth aspect according to the invention relates to a method of controlling blood glucose or blood insulin levels in a subject, said method comprising administering in a subject in need thereof an insulin secretory effective amount of a Nogo-A antagonist, or a pharmaceutical formulation thereof.
A fifth aspect of the invention relates to a method of preventing, repressing or treating insulin secretion deficiency, in particular insulin secretion insufficiency in a subject, said method comprising administering in a subject in need thereof a therapeutically effective amount of a Nogo-A antagonist, or a pharmaceutical formulation thereof.
A sixth aspect of the invention relates to a pharmaceutical formulation comprising a Nogo-A antagonist, combined with at least one co-agent useful in the stimulation of insulin secretion or the treatment of diabetes mellitus, and at least one pharmaceutically acceptable carrier.
A seventh aspect of the invention relates to a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment for the preparation of a pharmaceutical composition for controlling blood glucose levels and/or insulin blood levels in a subject, wherein said polypeptide or polypeptide fragment induces an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in said subject.
An eighth aspect of the invention relates to a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment for the preparation of a pharmaceutical composition for the repression or treatment of insulin secretion deficiency in a subject, wherein said polypeptide or polypeptide fragment induces an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in said subject.
A ninth aspect of the invention relates to a Nogo-A polypeptide or a Nogo-A polypeptide fragment or a pharmaceutical formulation thereof for the prevention, the repression or treatment of insulin secretion deficiency in a subject, wherein said polypeptide or polypeptide fragment induces an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in said subject.
A tenth aspect of the invention provides a method for controlling blood glucose or blood insulin levels in a subject, said method comprising administering in a subject in need thereof an amount of a Nogo-A polypeptide or a Nogo-A polypeptide fragment, or a pharmaceutical formulation thereof sufficient to induce an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in said subject.
An eleventh aspect of the invention provides a composition comprising a Nogo-A polypeptide or a Nogo-A polypeptide fragment, or a pharmaceutical formulation thereof that produces, when administered to a subject, an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in said subject.



DESCRIPTION OF THE FIGURES
FIG. 1 shows the expression of Nogo-A in brain hemisphere (B), sciatic nerves (SN), pancreas (P) and islets (I) in 2-month-old wild-type controls (WT) and Nogo-A KO mice (KO), as assessed by RT-PCR compared to β-actin as internal control as described in Example 1. β-actin was used as internal control.
FIGS. 2A-2E show metabolic profile of Nogo-A KO mice (KO) compared to wild-type (WT) as described in Example 2. A: Body weight, n=10, P=NS; B: pancreatic weight, n=5, P=NS; C: Blood glucose in fasted and random fed conditions, n=5-6, P<0.001; D: Plasma insulin in fasted and random fed conditions, n=5-6, P<0.05; E: Plasma glucagon in fasted and random fed conditions, n=5-6, P=NS.
FIGS. 3A-3G show insulin secretion response to glucose alone or supplemented with the cholinergic analogue carbachol in Nogo-A KO mice compared to wild-type (WT) as described in Example 2. A: Blood glucose after i.p. injection of glucose (2g/kg); B: Plasma insulin during i.p. glucose tolerance test; C: Blood glucose after i.p. injection of insulin (0.5U/kg); D: Blood glucose and plasma insulin (E) after i.v. injection of glucose (1g/kg); F: Blood glucose and plasma insulin (G) after i.v. injection of glucose (1g/kg) supplemented with the cholinergic analogue carbachol (0.53μM/kg). n=5-6, * P<0.05, ** P<0.005.
FIGS. 4A-4E show higher parasympathetic input in Nogo-A KO mice after 2-deoxy-D-glucose (2DG) (984 mg/kg)-induced neuroglycopenia compared to wild-type (WT) as described in Example 2 and sensitivity of β-cells from isolated WT and Nogo-A KO islets, as described in Example 3. A: plasma PP levels in fasted state, n=5, P<0.006; B: plasma GIP in random fed state, n=5, * P<0.05, ** P<0.008; C-E Sensitivity of β-cells from isolated WT and Nogo-A KO islets, in response to glucose only (C), n=16 batches of 10 islets, 4 mice, P=NS, in response to glucose supplemented with CC (10 μM) (D), n=15 batches of 10 islets, 4 mice, * P<0.05, ** P<0.005, ***P<0.001, in response to glucose 8.4 mM supplemented with GLP-1(100 nM), n=15 batches of 10 islets, 3 mice, P=0.009 (E).
FIGS. 5A-5C show increased insulin content of Nogo-A KO pancreas exibiting normal β-cell mass as compared to wild-type (WT) as described in Example 2. A: β-cell mass, n=3, P=NS; B: Insulin content, n=4, P<0.05; C: Glucagon content, n=10, P=NS.
FIGS. 6A-6I show sequences disclosed in the application with their respective SEQ ID numbers.
FIGS. 7A-7E show improved insulin secretion after 2 weeks treatment with neutralizing 11C7 anti Nogo-A antibody in diabetic db/db mice as compared to db/db mice treated with anti-BrdU control antibody, as described in Example 4. A-C: Plasma insulin levels (μg/1) in different glucose tolerance tests in vivo. A: after i.p. injection of glucose (1g/kg). n=2-4, * P<0.005; B: after i.v. injection of glucose (1 g/kg) alone, n=3, * P<0.005; C: after injection of glucose (1g/kg) supplemented with the cholinergic analogue carbachol (CC) (0.53μM/kg), n=3, * P<0.05; D: Plasma gastric inhibitory polypeptide (GIP) (pg/ml) in random-fed 11C7 treated db/db mice compared to db/db mice treated with anti-BrdU control antibody. (n=5, P=NS); E:Sensitivity of (β-cells from isolated islets of 11C7 and anti-BrdU control-treated db/db mice in response to glucose supplemented with the cholinergic analogue carbachol (CC) (100μM) or glucagon-like peptide-1 (GLP-1) (100nM) ** P<0.005.



DETAILED DESCRIPTION
The term “insulin secretion deficiency” includes a disease or condition where pancreatic β-cells are unable to secrete sufficient insulin (insulin secretion insufficiency) and insulin secretion dysfunction such as in impaired glucose tolerance and diabetes mellitus, in particular type II diabetes. This term includes relative and absolute deficiency in response to lower insulin sensitivity.
The term “Nogo-A” or “Nogo-A polypeptide” refers to mammalian Nogo-A protein and iso forms and fragments thereof. Nogo-A is also known as reticulon-4 or neurite outgrowth inhibitor. It includes in particular human Nogo-A which can be described by an amino acid sequence as set forth in SEQ ID NO: 1. It further encompasses a protein encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2. As used herein, the term Nogo-A also encompasses rat Nogo-A (SEQ ID NO: 8), bovine Nogo-A (SEQ ID NO: 9) and mouse Nogo-A (SEQ ID NO 10). As used herein, the terms Nogo-A or Nogo-A polypeptide encompass polypeptides having an amino acid sequence such as those described in Oertle et al., 2003, The Journal of Neuroscience, 23(13), 5393-5406, the contents of which is herein incorporated by reference in its entirety, in particular Nogo-A or Nogo-A polypeptide fragments comprising an amino acid sequence selected from the group consisting of amino acid residues 1-131 (SEQ ID NO: 3), 132-939 (SEQ ID NO: 4), 206-501 (SEQ ID NO: 5), 501-680 (SEQ ID NO: 6), 132-206 (SEQ ID NO: 7), 680-939 (SEQ ID NO: 35) and 940-1127 (SEQ ID NO: 36) of SEQ ID NO: 1. In addition, the terms Nogo-A or Nogo-A polypeptide encompass polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence of SEQ ID NO: 1 and which polypeptides are biologically active. In particular, the terms Nogo-A or Nogo-A polypeptide encompass polypeptides substantially homologous to a protein of SEQ ID NO: 1 or an isoform or fragment thereof, e.g. which have an amino acid sequence different from that of native human Nogo-A or Nogo-A fragment because of one or more deletions, insertions or substitutions. Substantially homologous means a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the native amino acid sequences, as disclosed above. The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and/or mathematical calculation, or more easily by comparing sequence information using known computer program used for sequence comparison such as Clustal package version 1.83. It further encompasses polypeptides which may comprise a sequence having at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
The term “Nogo-A antagonist” as used herein refers to any substances that are able to totally or partially inhibit, block, attenuate, or interfere with any pathway elicited, either directly or indirectly, by Nogo-A. Thus, the term “antagonists” is intended to include, but is not limited to, molecules which neutralise the effect of Nogo-A. For example, Nogo-A antagonists include substances which interact with Nogo Receptor (NgR) and any other receptor expressed in β-cells and compete with its ligand Nogo-A. For example, Nogo-A antagonists include small molecules, peptidomimetics, chimaeric proteins, natural or unnatural proteins, nucleic acid derived polymers (such as DNA and RNA aptamers, siRNAs, PNAs, or LNAs), fusion proteins with Nogo-A antagonizing activities, antibody antagonists such as neutralising anti-Nogo-A antibodies, or gene therapy vectors driving the expression of such Nogo-A antagonists.
The term “Nogo-A antibody” as used herein refers to any antibody or variant form thereof, including but not limited to, antibody fragment, domain antibody or single chain antibody capable of selectively binding to Nogo-A protein or fragment thereof. In particular, Nogo-A antibodies include Nogo-A antibody able to bind to the epitopes of mammalian, notably human Nogo-A, in particular, epitopes localized within regions consisting of amino acid residues 1-979 (SEQ ID NO: 37), 342-357 (SEQ ID NO: 38), 544-725 (SEQ ID NO: 39), 567-748 (SEQ ID NO:40), and 610-621 (SEQ ID NO: 41) of SEQ ID NO: 1 or amino acids 101-118 of SEQ ID NO: 9 (SEQ ID NO: 42), or amino acids 623-640 of SEQ ID NO: 8 (SEQ ID NO: 43). A Nogo-A antibody includes murine, chimeric, humanised, or fully human antibodies, genetically engineered or bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies against Nogo-A protein or fragment thereof and the like. Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity. The term “selectively” indicates that the antibodies preferentially recognize and/or bind the target polypeptide or epitope, i.e., with a higher affinity than to any other antigen or epitope, i.e. the binding to the target polypeptide can be discriminated from non-specific binding to other antigens. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard et al., 1949, Ann. N.Y. Acad., 51, 660-672).
The term “antibody antagonists” as used herein refers to any antibody or variant form thereof, including but not limited to, antibody fragment, domain antibody or single chain antibody capable of reducing the activity of a given pathway, enzyme, receptor or ligand, such as a Nogo-A pathway. Antibody antagonists include antibodies in a conventional immunoglobulin format (IgA, IgD, IgE, IgG, IgM), and also fragments thereof or any other “antibody-like” format that binds to human Nogo-A (for example, a single chain Fv fragment, a fragment Fe, a Fd fragment, a Fab fragment, a Fab′ fragment, a F(ab)2 fragment, chimeric antibodies, diabodies, domain antibodies (dAbs) such as described in Holliger et al., 2005, Nature Biotechnology,23(9), 1126-1136 and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen-binding to the polypeptide (e.g., inmmunoadhesins). The terms Fv, Fe, Fd, Fab, or F(ab)2 are used with their standard meanings (Harlow et al., 1988, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press; Dec. 1, 1988 ISBN 978-0879693145).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in some minor amounts. Monoclonal antibodies are highly specific, as being directed against a single antigenic site. The modifier term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
The term “humanised antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin being derived from one or more human or non-immunogenic to humans immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity of the donor immunoglobulin (Queen et al., 1989, Proc. Natl. Acad. Sci. USA, 86:1002910032).
The term “donor antibody” refers to a non-human antibody, which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogues thereof to the humanised antibody, and thereby provide the humanised antibody with the antigenic specificity and neutralising activity characteristic of the donor antibody. A suitable donor antibody may be selected by methods as described in Handbook of Therapeutic Antibodies. Stefan Dübel (Ed:) January 2007. ISBN: 978-3-527-31453-9.
The term “acceptor antibody” refers to an antibody heterologous to the donor antibody, which provides the amino acid sequences of its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the humanised antibody. The acceptor antibody may be derived from any mammal provided that it is non-immunogenic in humans. Preferably, the acceptor antibody is a human antibody. A suitable human acceptor antibody may be selected for its homology to the nucleotide and amino acid sequences of the donor antibody. For example, suitable human acceptor antibody may be found by methods as described in Handbook of Therapeutic Antibodies. Stefan Dübel (Ed:) January 2007. ISBN: 978-3-527-31453-9. Antibodies according to the invention can be generated by immunization of a suitable host (e.g., vertebrates, including humans, mice, rats, sheep, goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggs of birds, reptiles and fish). Determination of immunoreactivity with an immunogenic Nogo-A polypeptide may be made by any of several methods well known in the art, including, e.g., immunoblot assay and ELISA. Modification of such antibodies into therapeutically useful derivatives may be made by methods as described in Handbook of Therapeutic Antibodies. Stefan Dübel (Ed:) January 2007. ISBN: 978-3-527-31453-9.
The term “CDRs” refers to the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. It refers to all three heavy chain CDRs, and/or all three light chain CDRs.
The term “anti-Nogo-A antibody” may be a neutralising anti-Nogo-A antibody or a fragment thereof, such as murine antibodies 2A10 and 2C4(described in WO 2005/016544, now also identified as U.S. Pat. No. 7,780,964, the content of which is incorporated herein by reference in its entirety). The anti-Nogo-A antibody may be a humanised antibody such as a humanised variant of 2A10, for example H20L16, H28L16, H28L13 and H27L16 (as described in WO 2007/068750, now also identified as U.S. Pat. No. 8,362,208 and WO 2010/004031, now also identified as USPGPUB 2011/0268729, the contents of which are incorporated herein by reference in their entirety), a human antibody, or a fragment thereof. Amino acid sequences of the humanised constructs of the heavy chain and light chain variable region of 2A10 are presented as SEQ ID NOs: 26 to 30 herein (FIG. 6G). Full length heavy and light chain humanised variants of 2A10 are presented as SEQ ID NOs: 20 to 25 (FIG. 6F).
The anti-Nogo-A antibody may also be any of the antibodies described in WO 2004/052932, now also identified as U.S. Pat. No. 8,535,666, the content of which is incorporated herein by reference in its entirety. Examples of antibodies disclosed in WO 2004/052932 are 11C7, including humanized variants thereof. The sequence of the variable regions of 11C7 is shown in SEQ ID NOs: 31 and 32 (FIG. 6H). Human anti-Nogo-A antibodies are also described in WO 2005/028508, now also identified as US PGPUB 2009/056509, and in WO 2009/056509, now also identified as U.S. Pat. No. 8,163,285, the contents of which are incorporated herein by reference in their entirety. Specific antibodies disclosed in WO 2009/056509, also identified as U.S. Pat. No. 8,163,285, include the human antibody 6A3, having variable regions as shown in SEQ ID NOs: 33 and FIG. 6H.
In a particular embodiment, an anti-Nogo-A antibody according to the invention is a humanised anti-Nogo-A antibody such as a humanised variant of antibodies described in WO 2004/052932, now also identified as U.S. Pat. No. 8,535,666, WO 2005/061544, now also identified as U.S. Pat. No. 7,780,964, WO 2005/028508, now also identified as U.S. Pat. No. 8,535,666, WO 2007/068750, now also identified as U.S. Pat. No. 8,362,208, WO 2009/056509, now also identified as U.S. Pat. No. 8,163,285, or WO 2010/004031, now also identified as USPGPUB 2011/0268729 or a human anti-Nogo-A antibody, or a fragment thereof. Anti-Nogo-A antibodies particularly useful in the frame or a use or a method according to the present invention include humanised variants of 11C7, 2A10 or 2C4, 6A3 as described in WO 2009/056509, now also identified as U.S. Pat. No. 8,163,285, and H20L16, H27L16, H28L13 and H28L16 as described in WO 2010/004031(USPGPUB 2011/0268729). Preparation of polyclonal and monoclonal “anti-Nogo-A antibody” may be prepared by known techniques in the art, in particular as described in US 2005/0260616 or Oertle et al., 2003, above.
The term “siRNA” refers to small interfering RNA which are double stranded RNA (about 19-23 nucleotides) able to knock down or silence a targeted mRNA from a target gene. Artificial siRNAs can be either chemically synthesized as oligonucleotides or cloned into a plasmid or a virus vector (adenovirus, retrovirus or lentivirus) as short hairpin RNAs to generate a transient or stable transfection in any type of cells (Martin et al., 2007, Ann. Rev. Genomics Hum. Genet., 8:81-108; Kolfschoten et al., 2007, Nat. Clin. Pract. Endocrinol. Metab., 3(12):827-34; Huang et al., 2008, Expert. Opin. Ther. Targets, 12(5), 637-645).
The term “peptidomimetic” is defined as a peptide analog containing non-peptidic structural elements, which peptide is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic lacks classical peptide characteristics such as enzymatically scissile peptide bonds.
A Nogo-A protein, as an isolated, purified or homogeneous protein according to the invention, may be produced by recombinant expression systems as described in Chen et al., 2000, Nature, 403:434-439, or purified from naturally occurring CNS or skeletal muscle cells.
Suitable systems of expression of Nogo-A or Nogo-A variants or fragments, Nogo-A antagonists include transfected Chinese hamster ovary (CHO) cell line.
The term “pharmaceutically acceptable” refers to a carrier comprised of a material that is not biologically or otherwise undesirable.
The term “carrier” refers to any components present in a pharmaceutical formulation other than the active agent and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives and the like.
As used herein, “treatment” and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it for example based on familial history, overweight status or age; (b) inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage.
In particular, treatment of insulin secretion deficiency comprises to normalize or improve an impairment of glucose clearance through the control of blood insulin levels or insulin production.
The term “subject” as used herein refers to mammals. For example, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents and the like.
The term “efficacy” of a treatment or method according to the invention can be measured based on changes in the course of disease or condition in response to a use or a method according to the invention. For example, the efficacy of a treatment or method according to the invention can be measured by an increase of blood levels of insulin by ELISA or a decrease in fasting glycaemia or post-prandial glycaemia by oral glucose tolerance test (OGTT) or fasting glucose test (FPG). For another example, the efficacy of a treatment or method of the invention encompasses a higher neutrally-stimulated insulin secretion in a subject assessed by measuring the plasma insulin levels by ELISA, or by measuring the plasma C-peptide levels by ELISA, and/or a higher parasympathetic input, assessed by measuring the plasma pancreatic polypeptide (PP) levels with the Luminex® xMAP® technology (Rossi et al., 2005, Diabetes 54:1324-1330).
The term “insulin secretory effective amount” as used herein refers to an amount of at least one Nogo-A antagonist or a pharmaceutical formulation thereof according to the invention that elicits a detectable insulin secretion response in a subject that that is being administered the said Nogo-A antagonist.
The term “an antibody response sufficient to neutralize or antagonize endogenous Nogo-A” refers to a protective immune response against Nogo-A activity, e.g., an induction in the production of circulating antibodies that neutralize endogenous Nogo-A and/or an antibody response that prevents, represses or treats insulin secretion deficiency in an individual. For example, individuals in which a protective immune response has been induced can exhibit reduced insulin secretion deficiency and/or reduced risk to develop Type II diabetes as compared to non-immunized control individuals.
Nogo-A Antagonists
Nogo-A antagonists include substances described in the detailed description.
In a particular embodiment, a Nogo-A antagonist according to the invention is a neutralising anti-Nogo-A antibody.
In a further embodiment, a Nogo-A antagonist is a neutralising anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; or mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.
In a further embodiment, a Nogo-A antagonist is a humanised variant of mAb 11C7 neutralising anti-Nogo-A antibody.
In a particular embodiment, a Nogo-A antagonist according to the invention is a siRNA with Nogo-A antagonizing activities.
Alternatively, Nogo-A antagonists can be generated in-vivo in the form of autoantibodies, through the administration of a Nogo-A polypeptide or fragment thereof or a pharmaceutical composition thereof capable of inducing an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in a subject. In this case, either intact Nogo-A, fragments thereof, or preferably synthetic peptides corresponding to epitopes of Nogo-A, could be used as immunogens to elicit an immune response and the production of neutralising autoantibodies against endogeneous Nogo-A in a subject suffering from insulin secretion deficiency. Therapeutic levels of circulating neutralising anti-NogoA autoantibodies could be maintained and controlled by appropriate immunization / booster protocols. A useful precedent is the use of immunization against certain endogenous fertility hormones such as LHRH (luteinizing hormone releasing hormone) and hCG (human chorionic gonadotropin) as methods of fertility regulation, contraception (Talwar, 1997,Human Reproduction Update, 3(4), 301-310; Talwar et al., 1994, Proc. Natl. Acad. Sci. USA., 91, 8532-8536; Amato et al., 2002, J. Clin. Endocr. & Metab., 87(3), 993-997) and treatment of hormone-dependant cancers (Conry et al., 2000, Clinical Cancer Research, 6, 34-41). These methods described in the above references hereby incorporated by reference in their entirety have been proven both effective and reversible in clinical trials. According to another aspect, is provide a composition comprising a Nogo-A polypeptide or fragment thereof capable to induce an antibody response sufficient to neutralize or antagonize endogenous Nogo-A in a subject.
Compositions
The invention provides Nogo-A antagonists, Nogo-A polypeptide or fragment thereof, pharmaceutical compositions thereof, and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient who is suffering from a medical disorder selected from insulin secretion deficiency, in particular diabetes mellitus such as type II diabetes.
According to another aspect, the invention provides Nogo-A antagonists, pharmaceutical compositions thereof and methods for controlling the glucose and/or insulin blood levels in a subject.
In a particular embodiment, the invention provides a pharmaceutical formulation according to the invention for use as a medicament.
Pharmaceutical compositions of the invention can contain at least one Nogo-A antagonist according to the invention in any form described herein. Compositions of this invention may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
The compositions according to the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use by injection or continuous infusion. Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. According to a particular embodiment, compositions according to the invention are injectable.
Compositions of this invention may be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include but are not limited to poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®.
Further materials as well as formulation processing techniques and the like are set out in Part 5 of Remington's Pharmaceutical Sciences, 21st Edition, 2005, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, the content of which is incorporated herein by reference.
Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
Solid compositions of this invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
Compositions of this invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant.
The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials in Remington's Pharmaceutical Sciences. In a particular embodiment, when the Nogo-A antagonist according to the invention is a siRNA, it may be advantageously delivered in encapsulated form into nanoparticles or liposomes such as described in Fenske et al., 2008, Expert Opin, Drug Deliv., 5(1), 25-44; de Fougerolles, 2008, Hum. Gene Ther., 19(2), 125-32; Huang et al., 2008, above, the content of which is herein incorporated by reference in their entirety.
In a particular aspect, the composition to be administered to a subject in order to induce an antibody response sufficient to neutralize or antagonize endogeneous Nogo-A may, optionally, contain an adjuvant and may be delivered in any manner known in the art for the delivery of immunogen to a subject.
Mode of Administration
Compositions of this invention may be administered in any manner including intravenous injection, intraperitoneal injection, subcutaneous injection, oral route, cutaneous application or combinations thereof.
Combination
According to the invention, the Nogo-A antagonist, the Nogo-A polypeptide or fragment thereof used to generate Nogo-A auto-antibodies, and pharmaceutical formulations thereof can be administered alone or in combination with a co-agent useful in the stimulation of insulin secretion or in the treatment of insulin deficiency and/or diabetes mellitus, such as substances useful for treating, stabilizing, preventing, and/or delaying insulin deficiency e.g. for example a co-agent selected from sulfonylureas (for example those described in Campbell, 2009, Diabetic Hypoglycemia, 2(1), 3-10), thiazolidinediones (TZD) (for example those described in Koyama et al., 2003, Bioorg. Med. Chem. Lett., 13, 1801-1804) or GLP-1 receptor agonists (for example exenatide, liraglutide or lixisenatide or those described in Knudsen et al., 2007, PNAS, 104(3), 937-942).
The invention encompasses the administration of a Nogo-A antagonist and pharmaceutical formulations thereof, or of a Nogo-A polypeptide or fragment thereof used to generate Nogo-A auto-antibodies, wherein the Nogo-A antagonist or pharmaceutical formulation thereof, or the Nogo-A polypeptide or fragment thereof used to generate Nogo-A auto-antibodies, is administered to an individual prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful in the stimulation of insulin secretion or in the treatment of insulin deficiency and/or diabetes mellitus (e.g. multiple drug regimens), in a therapeutically effective amount. A Nogo-A antagonist or the pharmaceutical formulation thereof or the Nogo-A polypeptide or fragment thereof used to generate Nogo-A auto-antibodies, that is administered simultaneously with said co-agents can be administered in the same or different composition(s) and by the same or different route(s) of administration.
According to one embodiment, is provided a pharmaceutical formulation comprising a Nogo-A antagonist, combined with at least one co-agent useful in the stimulation of insulin secretion or in the treatment of a disease or a disorder characterized by insulin deficiency, and at least one pharmaceutically acceptable carrier.
According to another embodiment, is provided a pharmaceutical formulation according to the invention wherein the Nogo-A antagonist is a neutralising anti-Nogo-A antibody.
In a further embodiment, is provided a pharmaceutical formulation according to the invention, wherein the Nogo-A antagonist is a neutralising anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; or mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.
In a further embodiment, is provided a pharmaceutical formulation according to the invention, wherein the Nogo-A antagonist is a humanised variant of mAb 11C7 neutralising anti-Nogo-A antibody.
The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
Patients
In an embodiment, patients according to the invention are patients suffering from a disorder selected from insulin secretion deficiency and insulin resistance.
In a further embodiment, patients according to the invention are patients suffering from insulin secretion deficiency.
In another further embodiment, patients according to the invention are suffering from diabetes mellitus.
In another further embodiment, patients according to the invention are suffering from type II diabetes.
Use According to the Invention
In one embodiment of the invention is provided a use of a Nogo-A antagonist for the preparation of a pharmaceutical composition for controlling blood glucose levels and/or insulin blood levels in a subject.
In another embodiment of the invention is provided a use of a Nogo-A antagonist for the preparation of a pharmaceutical composition for the repression or treatment of insulin secretion deficiency.
In a further embodiment, is provided a use of a Nogo-A antagonist for the preparation of a pharmaceutical composition for the prevention, the repression or treatment of diabetes mellitus.
In a further embodiment, is provided a use of a Nogo-A antagonist for the preparation of a pharmaceutical composition for the prevention, the repression or treatment of type II diabetes.
In another embodiment of the invention is provided a method for controlling blood glucose or blood insulin levels in a subject, said method comprising administering in a subject in need thereof an insulin secretory effective amount of a Nogo-A antagonist, or a pharmaceutical formulation thereof.
In another embodiment of the invention is provided a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment, typically a synthetic polypeptide corresponding to epitopes of Nogo-A, for the preparation of a pharmaceutical composition for controlling blood glucose levels and/or insulin blood levels in a subject, wherein said polypeptide or polypeptide fragment induces an antibody response sufficient to neutralize or antagonize endogeneous Nogo-A in said subject.
In another embodiment of the invention is provided a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment, typically a synthetic polypeptide corresponding to epitopes of Nogo-A, for the preparation of a pharmaceutical composition for the repression or treatment of insulin secretion deficiency in a subject, wherein said polypeptide or polypeptide fragment induces an antibody response sufficient to neutralize or antagonize endogeneous Nogo-A in said subject.
In a further embodiment, is provided a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment for the preparation of a pharmaceutical composition for the prevention, the repression or treatment of diabetes mellitus.
In a further embodiment, is provided a use of a Nogo-A polypeptide or a Nogo-A polypeptide fragment for the preparation of a pharmaceutical composition for the prevention, the repression or treatment of type II diabetes.
In another embodiment of the invention is provided a method for controlling blood glucose or blood insulin levels in a subject, said method comprising administering in a subject in need thereof an amount of a Nogo-A polypeptide or a Nogo-A polypeptide fragment, or a pharmaceutical formulation thereof sufficient to induce an antibody response sufficient to neutralize or antagonize endogeneous Nogo-A in said subject.
In a further embodiment of the invention is provided a use or a method according to the invention, wherein the subject is displaying signs or symptoms of a condition involving insulin secretion deficiency.
In another further embodiment of the invention is provided a use or a method according to the invention, wherein the subject is suffering from diabetes mellitus.
In another further embodiment of the invention is provided a use or a method according to the invention, wherein the subject is suffering from type II diabetes.
In a further embodiment of the invention is provided a use or a method according to the invention, wherein the subject is predispose to develop insulin secretion deficiency for example based on familial history, overweight status or age.
In another embodiment, is provided a use or a method according to the invention, wherein the Nogo-A antagonist is a neutralising anti-Nogo-A antibody.
In a further embodiment, is provided a use or a method according to the invention, wherein the Nogo-A antagonist is a neutralising anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; or mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.
In a further embodiment, is provided a use or a method according to the invention according to the invention, wherein the Nogo-A antagonist is a humanised variant of mAb 11C7 neutralising anti-Nogo-A antibody.
Compounds and compositions according to the invention may be useful in the control of blood glucose or blood insulin levels in a subject. In a particular embodiment, compounds and compositions according to the invention may be useful in the repression or treatment of insulin secretion deficiency. In another particular embodiment, compounds and compositions according to the invention may be useful in the repression or prevention or treatment of diabetes mellitus such as type II diabetes.
Examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments represented in the Figures.
EXAMPLES
The following abbreviations refer respectively to the definitions below:
bp (base paired), h (hour), i.v. (intravenous), l (liter), kb (kilobase), μg (microgram), mmol (millimol), mg (milligram), μM (micromolar), ng (nanogram), pg (picogram), BrdU (5-bromo-2′-deoxyuridine), BSA (Bovine serum albumin), CC (carbachol), cDNA (complementary DNA), CGRP (Calcitonin Gene-Related Peptide), ELISA (Enzyme-linked immunosorbent assay), GFAP (Glial Fibrillary Acidic Protein), GIP (gastric inhibitory peptide), GLP-1 (Glucagon-like peptide 1), KO (knock-out), KRBH (Krebs-Ringer bicarbonate HEPES), PFA (Paraformaldehyde), PBS (Phosphate saline buffer), PCR (Polymerase Chain Reaction), PP (pancreatic polypeptide), PVA (Polyvinyl alcohol), RIA (Radioimmunoassay), RT (reverse transcriptase), VMAT2 (Vesicular Monoamine Transporter 2), WT (wild-type).
Example 1
Nogo-A Expression in the Endocrine and the Neural Components of the Pancreas
The expression pattern of Nogo-A was investigated in brain, sciatic nerves, pancreas and isolated islets, in Nogo-A −/− (KO mice) and control C57BL/6J mice, as follows.
In the adult mouse pancreas, the expression of Rtn4 (Nogo-A transcript) was detected by RT-PCR as described below (FIG. 1). Expression levels of Nogo-A in extracts from isolated islets of Langerhans were as high as in brain extracts (FIG. 1). Nogo-A expression in human and rat islets was also observed where Nogo-A protein was exclusively expressed in β-cells and in a subset of PP-cells, while it was absent from α-and β-cells. Nogo-A was also detected in neurons and fibers of intra-pancreatic ganglia, more specifically, in the parasympathetic cell bodies but not in parasympathetic vesicular acetylcholine transporter (VAChT) fibers. Nogo-A was also found in afferent calcitonin gene-related peptide (CGRP) sensory fibers but not in sympathetic neurons positive for vesicular monoamine transporter (VMAT2), or in supporting glial fibrillary acidic protein (GFAP)-expressing Schwann cells.
These results support the unexpected finding that Nogo-A is expressed in β-cells and in neuronal cells influencing insulin secretion, namely, parasympathetic ganglia and sensory CGRP fibers.
Nogo-A −/− Knock-Out Mice
Nogo-A −/− knock out (KO) mice have been generated as described in Simonen et al., 2003, Neuron, 38:201-211. Backcrossed in pure C57BL/6J background, male Nogo-A KO mice were compared with sex matched and age matched C57BL/6J animals. Genotyping was performed by PCR from genomic DNA isolated from tail biopsies using M58 of SEQ ID NO: 11: TGCTTTGAATTATTCCAAGTAGTCC and M101 of SEQ ID NO 12: AGTGAGTACCCAGCTGCAC primers for WT Nogo-A allele (1.4 kb band), M58 and M63 of SEQ ID NO 13: CCTACCCGGTAGAATATCGATAAGC primers for Nogo-A deleted allele (1.2 kb band).
RT-PCR
Total RNA from brain hemisphere (B), sciatic nerve (SN), pancreas (P) and isolated islets (I) of C57BL/6J control mice were extracted with the RNeasy mini kit (Qiagen) and RNeasy micro kit (Qiagen), respectively. cDNA were prepared using Superscript II RT kit (Invitrogen), and thereafter PCR was performed with the Red Taq kit (Sigma) and primers: Nogo-A CCTCTCTGGCAATTCTCTCTAGAAG (SEQ ID NO: 14) and AGGGGCTCGGGCTCAGTGG (SEQ ID NO: 15), Nogo-B CTGAACCAATTCCTCTGATATGGC (SEQ ID NO: 16) and AGGGGCTCGGGCTCAGTGG (SEQ ID NO: 17), Nogo-C TGCTGGAGGGCAGATCGTGGC (SEQ ID NO: 18) and CTGAACCAATTCCTCTGATATGGC (SEQ ID NO: 19).
Example 2
Increased Insulin Secretion and Decreased Glycemia in Nogo-A Knock-Out Mice
Insulin secretion profile was studied in adult Nogo-A KO males and sex- and age-matched C57BL/6J controls (WT) (described in Example 1) as described below.
Blood Glucose & Insulin Release
Two-month-old KO animals were slightly leaner than controls (body weight: WT 28.47±0.86 g, KO 23.82±1.89 g; n=10, P=0.07), despite a normal pancreas-to-body weight ratio (WT 0.99±0.05%, KO 0.86±0.09%; n=5, P=NS) (FIGS. 2A-B). Plasma glucose, insulin and glucagon levels in fasted and random fed conditions were measured as described below. After 16-hour fasting, glucose, insulin and glucagon values were normal in KO mice but in random fed condition (a period during which insulin secretion is stimulated by glucose, gluco-incretins and neurotransmitters), KO animals had significantly lower blood glucose levels (WT 10.66±0.35 mmol/l, KO 8.48±0.31 mmol/l; n=5, P<0.005), higher plasma insulin levels (WT 0.50±0.02 μg/l, KO 1.23±0.28 μg/1; n=6, P<0.05), and normal plasma glucagon levels (WT 60.79±4.62 pg/l, KO 66.54±13.29 pg/l; n=6, P<0.05) (FIG. 2C-E). These results show that Nogo-A KO mice exhibit a lower glycemia associated with a higher insulin secretion after feeding.
In Vivo Stimulation of β-Cell Secretion
Intraperitoneal glucose tolerance tests as described below (i.p.-GTT) showed that Nogo-A KO animals have an improved glucose clearance after glucose administration (2 g/kg) (WT 709.875±72.73 mmol/l/min, KO 378.95±31.17 mmol/1/min; n=4, P=0.006) (FIG. 3A). The stimulated insulin secretion was significantly higher 1 hour after glucose injection in Nogo-A KO animals (FIG. 3B). Insulin sensitivity assessed by measuring the blood glucose after i.p. injection of insulin (0.5 U/kg) being comparable in KO and WT mice (FIG. 3C), the faster glucose clearance in the former was due to a higher insulin secretion. Nogo-A KO mice were exposed to a bolus of glucose (intravenously, 1 g/kg) (FIG. 3D,E) after overnight fasting, while a vagal stimulation was mimicked with carbachol, a cholinergic analogue (FIG. 3F,G). While controls and KO mice corrected similarly the induced hyperglycemia by secreting comparable amounts of insulin (FIG. 3D-E), Nogo-A KO mice supplemented with carbachol (0.53 μM) corrected better the induced glycemia, in association with an improved insulin secretion (FIG. 3F,G).
All together, these results support that in a context of a down-regulation of Nogo-A activity, the cholinergic stimulation of β-cells has a more potent effect on insulin secretion.
Insulin Content Per β-Cell
The morphology of the pancreatic endocrine compartment was studied as described below. The gross histology of Nogo-A KO pancreas, as well as the islet architecture was comparable to that of controls. The islet cell mass was not modified in Nogo-A KO animals. The β-cell mass was also comparable to that of controls (FIG. 5A). Nevertheless, even in absence of β-cell hypertrophy, the pancreatic insulin content assessed by ELISA as described below was 1.2 fold increased (WT 554.65±54.54 ng, KO 763.51±37.67 ng, n=5, P<0.05) (FIG. 5B), suggesting an augmented insulin content per β-cell. The total pancreatic glucagon content was unchanged (WT 338.07±8.92 pg, KO 344.02±4.96 pg; n=9-11, P=NS) (FIG. 5C).
Parasympathetic Input & β-Cell Sensitivity
Along the food ingestion and digestion process, β-cells secrete insulin in response to different secretagogues. These initiate and then potentiate insulin release. Additionally, during the enteric phase, insulin secretion is promoted by the gluco-incretins GIP and GLP-1 released from enteroendocrine cells at stimulatory glucose concentrations.
In order to challenge simultaneously the parasympathetic input as well as the β-cell sensitivity in vivo, the vagally-stimulated insulin secretion was investigated after intravenous injection of 2-deoxy-D-glucose (2DG) (984 mg/kg), a non-metabolizable glucose analogue that blocks intracellular glucose utilization. Competing with D-glucose, 2DG activates the autonomic nervous system through central neuroglycopenia.
In order to assess indirectly the parasympathetic input on islets of Langerhans, the plasma pancreatic polypeptide (PP) levels that are known to be strictly controlled by parasympathetic stimulus, were measured after overnight (ON) fasting, prior and after 2DG i.v. injection as described below. Whereas the plasma PP levels were not different between the two groups before 2DG injection, 30-minutes later, the plasma PP levels of Nogo-A KO animals were higher than those of WT controls (30′: WT 101.08±11.45, KO 187.09±15.33, n=4, P=0.006), indicating that the parasympathetic input was increased (FIG. 4A).
In order to assess the gluco-incretin input, the plasma levels of GIP and GLP-1, were measured in fed conditions, prior and after 2DG injection. GLP-1 levels were undetectable in normal random fed conditions as previously reported (Althage et al., 2008, J. Biol. Chem., 283:18365-18376). Therefore, GIP plasma levels were considered representative of the gluco-incretin input. In fed condition, Nogo-A KO plasma GIP values were lower in Nogo-A KO mice, irrespective of 2DG treatment (0′: WT 58.05±4.90 pg/ml, KO 34.86±3.03 pg/ml, P=0.007; 15′:WT 51.90±4.65 pg/ml, KO 36.55±4.19 pg/ml, P=0.04; n=5) (FIG. 4B), which correlates with lower glycemia observed in random fed Nogo-A KO animals (FIG. 2C).
Sidestepping the incretin effect, the i.v. injection of 2DG did not affect GIP plasma levels of both groups (FIG. 4B).
Together, these results show that i) the incretin effect is unaltered in Nogo-A KO mice, and ii) the pancreatic parasympathetic input is higher, which suggests that the vagal stimulation of insulin secretion is promoted in absence of Nogo-A.
In Vivo Insulin Secretion Assays
For intraperitoneal (i.p.) glucose tolerance tests, overnight fasted animals (n=10) received an i.p. glucose injection (2 g/kg) (Sigma) and blood was collected from the tail vein at 0, 15, 30, 60, 90 and 120 min into centrifuge tubes treated with lithium heparin. For intravenous glucose tolerance test, overnight fasted animals (n=7) were injected in the retro-orbital, intra-bulbar capillary plexus a glucose injection (1 g/kg), optionally supplemented with carbachol (0.53 μM) (Sigma). After isoflurane-induced brief anesthesia, mice blood samples were collected from the retroorbital plexus at 0, 1, 5, 20 and 50 min into centrifuge tubes treated with lithium heparin. Blood glucose was assessed with Glucometer Dex2(Bayer Corporation). Glycemic areas under the curves (AUCs) were measured from time 0 to 120 min, after subtraction of basal glycaemia. After immediate centrifugation, plasma was separated and insulin levels assessed with Ultrasensitive Rat Insulin ELISA kit (Mercodia). After distribution of 25 μl of assay buffer in all wells, 5 μl of standards or samples were added, followed by the further addition of 50 μl of peroxydase-conjugated monoclonal anti-insulin antibody. After a 2-hour incubation at room temperature, wells were washed 6 times with 350 μl of Wash buffer. Then, 200 μl of TMB substrate buffer were added, followed by a 30 min incubation time, stopped by 50 μl of Stop solution and absorbance was measured at 450 nm.
Insulin Tolerance Tests
Animals fasted for 6 hours were intraperitoneally injected with recombinant human insulin (0.5 unit/kg) (Actrapid, Novo Nordisk) and blood glucose was measured from the tail vein at 15, 30 45, 60 and 120 min.
Vagally Stimulated Islet Hormone Secretion
For vagal stimulation of islet hormone secretion, 2-deoxy-D-glucose (2DG) (Sigma) was injected intravenously (984 mg/kg) in 2-month-old adult WT and Nogo-A KO mice (n=5). For PP, mice were fasted overnight and blood samples were collected at 0, 15 and 30 min (Rossi et al., 2005, Diabetes, 54, 1324-1330). For GLP-1 and GIP, mice were kept fed and blood samples were collected at 0 and 10 min (Rossi et al., 2005, above). Blood samples were collected from the retro-bulbar intra-bulbar capillary plexus, into chilled tubes treated with EDTA. For plasma GLP-1 and GIP measurements, according to the manufacturer's protocol, DPP IV inhibitor (Millipore) was added in tubes and blood samples were immediately processed. Plasma was assayed for total GIP, GLP-1 and PP using a MILLIPLEX mouse gut hormone kit (#MGT-78K) (Millipore) and the Bioplex (Bio-Rad) at the Mouse Metabolic Evaluation Facility (MEF, Center for Integrative Genomics, University of Lausanne, Switzerland.
Immunofluorescence and Immunohistochemistry
Collected pancreata were weighted and then rinsed in cold PBS and fixed O/N at 4° C. in PAF 4%. Tissues were dehydrated, embedded in paraffin and sectioned at 5 μm using a microtome. The primary antibodies used for immunostainings were: mouse anti-glucagon (Sigma Cat. No. G2654; 1/1000), guinea pig anti-insulin (Dako Cat. No. IR002; 1/400), rabbit anti-PP (Bachem Cat. No. T-4088; 1/200), rabbit anti-somatostatin (DakoCat. No. A0566; 1/200), rabbit anti-GFAP (Dako Cat. No. IR524; 1/1000), rabbit anti-CGRP (Sigma Cat. No. C8198; 1/1000), rabbit anti-VMAT2 (Phoenix Pharmaceuticals Cat. No. H-V004; 1/100) and rabbit anti-VAChT (Phoenix Pharmaceuticals Cat. No. H-V006; 1/100).
For immunohistochemistry, dewaxed and rehydrated sections were permeabilized in 0.1% TritonX-100, washed and blocked in 3% BSA, 0.1% Tween in PBS. The primary antibodies were incubated overnight. After washing in PBS, sections were incubated with specific secondary antibodies coupled to either Alexa 488 (Molecular Probes) or Cy3 (Jackson Immunoresearch). Both islet cell mass and β-cell mass were assessed by measuring the endocrine synaptophysin-positive area, or the insulin-positive area, on 4 different sections, separated by 200 μm each (n=3), multiplied by the pancreas weight. Specimens were mounted in PVA and examined with a Leica confocal microscope (DM 5500). Tissue surface and cell numbers were measured with NIH Image J 1.60 software.
Protein Extraction and Hormone Content Measurements
Pancreas collected for hormone measurement were homogenized in 5 ml (n=5-11) of acid-ethanol solution (74% ethanol, 1.4% HCl). Samples were sonicated and centrifuged. The supernatants were submitted to immunoassay experiments using either Glucagon RIA kit (Linco) or Ultrasensitive Rat Insulin ELISA kit (Mercodia) for glucagon and insulin content measurements, respectively.
Example 3
In Vitro Sensitivity of β-Cells to Insulin Secretagogues
The responsiveness of β-cells to various secretagogues (namely carbachol and GLP-1) was assayed in vitro on isolated islets as described below.
Insulin secretion was increased in isolated Nogo-A KO islets in response to glucose supplemented with carbachol (CC; 100 nM), as compared with control islets (FIG. 4D). Islets were incubated with glucose (8.4 mM) supplemented with GLP-1. Again, insulin secretion was higher when Nogo-A was absent (WT 1.23±0.17, KO 2.13±0.27; n=11-14, P<0.01; FIG. 4E).
In conclusion, the constitutive inactivation of Nogo-A conferred to metabolically healthy Nogo-A KO animals the ability to correct faster and more efficiently an induced hyperglycemia thanks to an improved insulin secretion. The higher insulin secretion of Nogo-A KO β-cells resulted from a higher parasympathetic input on β-cells and a higher sensitivity of β-cells to the cholinergic agonist carbachol and the glucoincretin GLP-1.
Together, these findings suggest that selective inhibition of Nogo-A could be a novel therapeutic approach to promote stimulated insulin secretion, notably in the treatment of conditions or disorders that can be alleviated by an enhanced pancreatic β-cell insulin secretion such for example the treatment of diabetes mellitus such as in type II diabetic patients.
In Vitro Insulin Secretion Assays
After mice sacrifice, the abdomen was opened and the pancreas exposed. After clamping at the porta hepatis, the main pancreatic duct was cannulated with a butterfly-27G needle and retrogradually injected with 2 ml of collagenase XI (2 mg/ml in HBSS) (Sigma). After dissection, the pancreas was digested in 3 ml of collagenase XI solution for 15 min at 37° C., and finally disrupted upon vigorous tube shaking Islets were then purified on a Histopaque 1119 (Sigma) gradient, washed thrice with Hanks 1×BSA 0.1%, and finally fished twice. Batches of 10 islets were pre-incubated for 30 min in 1 ml KRBH supplemented with BSA 0.1% and 1.4 mM glucose at 37° C. Then, the supernatant was replaced by 0.5 ml KRBH supplemented with BSA 0.1% and glucose 1.4 mM, 2.8 mM, 4.2 mM, 8.4 mM and 16.8 mM was added for another 30 min at 37° C. (n=3-4). Additionally, islets incubated for 30 min in 0.5 ml KRBH containing glucose, BSA 0.1% and another secretagogue, carbachol 10 μM (Sigma) or GLP-1 100 nM (Bachem) (n=3-4). Secreted insulin (μg/1) in the supernatant was measured with Ultrasensitive Rat Insulin ELISA kit (Mercodia). Similar insulin contents between islet batches were assessed after removal of the supernatant and acid-ethanol extraction.
Example 4
Nogo-A Neutralization by a Nogo-A Antagonist
The beneficial effects of an acute neutralization of Nogo-A by intraperitoneal or intravenous administration of a Nogo-A antagonist to promote insulin secretion is assayed in the following in early type II diabetic animals, homozygous mutants for the leptin receptor gene (db/db).
Young 5-week-old diabetic db/db animals in a C57BL/Ks background are treated with Nogo-A antagonist (anti-Nogo-A antibody 11C7 as described above) by intraperitoneal injection (4′900 ng antibody) or intravenously in the retro-orbital capillary plexus, twice over a two-week period and a control anti-BrdU antibody. This anti-Nogo-A antibody 11C7 was earlier described for its ability, when administered intrathecally to CNS, of inhibiting Nogo-A and allowing an enhanced sprouting and regrowth of lesioned axons after spinal cord injury and stroke in adult rats (Caroni et al., 1988, Neuron., 1:85-96). One week after the second injection, the insulinemia, the glucose levels and body weight of treated random fed db/db mice are compared to that of the control group: the insulinemia of db/db fed mice treated with 11C7 tended to be higher compared to the db/db mice treated with control antibody (BrdU 13.94±5.02, 11C7 32.28±8.14, n=3 mice per group, P=NS), but no effect was detected on glucose levels or body weight, most probably due to high insulin resistance of peripheral tissues (data not shown).
After the 2 weeks treatment, db/db animals are submitted to different challenges in vivo as previously described (namely i.p.-GTT, glucose-induced insulin secretion by intravenously a bolus of glucose (1 g/kg), optionally supplemented with carbachol CC)). Plasma insulin levels and total pancreatic insulin content in treated db/db mice are compared to that of the control group. Further, isolated islets from treated db/db are compared to those of the control group as described above. During the i.p. GTT, db/db mice treated with 11C7 anti-Nogo-A antibody presented higher plasma insulin levels at 15 (BrdU 1.25±0.08, 11C7 2.17±0.15, n=3-4, P<0.005) and 60 minutes (BrdU 1.57±0.04, 11C7 2.15±0.07, n=3-4, P<0.005), and 90 min, as compared with db/db mice treated with BrdU (FIG. 7A). Then, the glucose-induced insulin secretion was assessed in anesthesized db/db mice, i.v. injected with glucose (1 g/kg). Upon glucose injection, 11C7-treated db/db mice presented similar plasma insulin levels, compared with BrdU-treated db/db mice (FIG. 7B), similarly to the phenotype of Nogo-A KO, where the glucose-induced insulin secretion was not affected by the inactivation of Nogo-A (FIG. 4C). Also in accordance with the phenotype of Nogo-A KO mice, when the antibody-treated db/db mice were intravenously injected with glucose (1 g/kg) together with the cholinergic analogue carbachol (CC) (0.53 μM), 11C7-treated db/db mice showed an improved insulin secretion (4-fold on average) at 5 and 20 minutes (BrdU 10.65±2.14, 11C7 46.43±8.00, n=3, P<0.05) compared to BrdU-treated db/db mice (FIG. 7C).
In order to investigate the insulin secretion capacity of antibody-treated db/db mice, the pancreatic insulin content by immunoassay was assessed as described in Example 2. The inventors reasoned that the higher insulin secretion observed in db/db mice treated with Nogo-A neutralizing antibody could result from a higher parasympathetic input and/or from higher β-cell cholinergic sensitivity, such as in Nogo-A KO mice. Parasympathetic tone was therefore indirectly assessed by measuring the plasma level of pancreatic polypeptide (PP) after 2-Deoxy-D-glucose (2DG)-induced neuroglycopenia. After overnight fasting, before and after 2DG injection, PP levels were similar in both anti-Nogo-A treated and anti-BrdU treated control db/db mice (data not shown). Similarly, GIP levels were also measured in random-fed animals showing, as in Nogo_a KO mice, that before and after 2DG injection, GIP levels were similar in both anti-Nogo-A treated and anti-BrdU treated control db/db mice (FIG. 7D) (n=5, P=NS). Altogether, these results indicate that the antibody-mediated neutralization of Nogo-A does not affect the vagal and incretin input on β-cells. β-cell responsiveness to secretagogues was studied using isolated db/db islets cultured in the presence of glucose supplemented with CC (100 μM) or GLP-1 (100 nM). As observed with Nogo-A KO islets, islets from anti-Nogo-A treated db/db mice displayed higher insulin secretion in the presence of glucose supplemented with CC (n=7-15, P<0.005), yet their sensitivity to glucose supplemented with GLP-1 was not affected (FIG. 7E).
Altogether, these observations show that the intravenous administration of neutralizing Nogo-A antibody promotes the in vivo insulin secretion in 5-week-old diabetic db/db mice. Contrary to Nogo-A animals, the higher insulin secretion observed in 11C7-treated db/db mice probably resulted from a higher parasympathetic input only, as the parasympathetic input on islets was not altered. Therefore, those results support that reducing Nogo-A activity is of potential benefit for an anti-diabetic treatment, acting on the stimulatory input and the β-cells, by promoting insulin secretion in response to cholinergic stimuli, like after food intake, without hypoglycemic events.
db/db Homozygous Mice
Five weeks old male db/db homozygous mice, BKS.Cg-Dock7 m+/+Leprdb/J mice, are purchased from Charles River Laboratories, L′Arbresle, France for the treatment with a Nogo-A antagonist. All mice are maintained in a temperature-controlled room, on a 12-h light-dark cycle, and fed standard rodent chow ad libitum.
Statistical Analysis.
All results are reported as mean±standard error of the mean (SEM). Groups are compared with independent t tests (unpaired and two-tailed), reported as P values. All tests are performed using the GraphPad Prism software.

## Claims

1. A method of controlling blood glucose or blood insulin levels in a subject, said method comprising administering to a subject in need thereof an insulin secretory effective amount of a neutralising monoclonal anti-Nogo-A antibody, or a pharmaceutical formulation thereof.

2. The method according to claim 1, wherein the subject is suffering from insulin resistance.

3. The method according to claim 1, wherein the subject is suffering from diabetes mellitus.

4. The method according to claim 3, wherein diabetes mellitus is type II diabetes.

5. The method according claim 1, wherein the neutralising monoclonal anti-Nogo-A antibody is a neutralising monoclonal anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; and mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.

6. The method according to claim 1, wherein the neutralising monoclonal anti-Nogo-A antibody is a humanised variant of mAb 11C7.

7. A method of repressing or treating insulin secretion deficiency in a subject, said method comprising administering in a subject in need thereof a therapeutically effective amount of a neutralising monoclonal anti-Nogo-A antibody, or a pharmaceutical formulation thereof.

8. The method according to claim 7, wherein the neutralising monoclonal anti-Nogo-A antibody is a neutralising anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; and mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.

9. The method according to claim 7, wherein the neutralising monoclonal anti-Nogo-A antibody is a humanised variant of mAb 11C7 neutralising anti-Nogo-A antibody.

10. A pharmaceutical formulation comprising a neutralizing monoclonal anti-Nogo-A antibody, combined with at least one co-agent useful in the stimulation of insulin secretion or in the treatment of diabetes mellitus, and at least one pharmaceutically acceptable carrier.

11. The pharmaceutical formulation according to claim 10, wherein the neutralising monoclonal anti-Nogo-A antibody is a neutralising monoclonal anti-Nogo-A antibody selected from humanised variants of mAbs 11C7, 2A10 or 2C4; and mAbs 6A3, H20L16, H27L16, H28L13 and H28L16.

12. The pharmaceutical formulation according to claim 10, wherein the neutralising monoclonal anti-Nogo-A antibody is a humanised variant of mAb 11C7 neutralising monoclonal anti-Nogo-A antibody.