Patent Application
20150259416
The International Diabetes Federation (IDF) has estimated that the number of people with diabetes in 2011 was 366 million and this number is expected to grow to 550 million by 2030. The majority of patients are suffering from type-2 diabetes mellitus (T2DM). There is a link between obesity, insulin resistance and the development of T2DM, but the precise underlying mechanisms remain unknown and it becomes paramount to identify potential therapeutic targets that can ultimately address this crucial problem.
Classical target tissues subjective to insulin resistance in T2DM are muscle, liver and fat. These peripheral tissues maintain glucose homeostasis by effectively responding to insulin and failure of insulin to activate its receptors and the downstream signaling cascades result in defect glucose handling. In addition, insulin can stimulate its receptors on the pancreatic β-cell and thereby directly contribute to a positive feedback loop for insulin biosynthesis and secretion.
Apolipoprotein CIII (apoCIII) is a lipoprotein synthesized mainly in the liver, and to a lesser extent in the small intestine. ApoCIII resides on apoB lipoproteins and high density lipoproteins (HDL) and regulates their metabolism by inhibiting lipoprotein lipase (LPL). The expression of apoCIII is increased by insulin deficiency and insulin resistance and it has also been shown that hyperglycemia induces apoCIII transcription. On top of high levels of circulating triglycerides, mice overexpressing apoCIII are more susceptible to high-fat diet induced diabetes. In humans, elevated levels of circulating apoCIII are associated with insulin resistance. Furthermore, patients with type-1 diabetes mellitus (T1DM) were found to have elevated levels of serum apoCIII. This resulted in a higher activity of the voltage-gated Ca2+-channels, increased cytoplasmic free Ca2+ concentration ([Ca2+]i) and apoptosis, an effect that was blocked by lowering apoCIII.
The invention as disclosed herein provides a method for treating or limiting development of diabetes, comprising administering to a subject in need thereof an amount effective of a composition comprising a compound of formula A-B, wherein A is a pancreatic β cell targeting moiety, and B is an inhibitor of expression and/or activity of Apolipoprotein CIII (apoCIII), protein kinase A (PKA), Src kinase, and/or β1 integrin.
In another aspect, a composition of formula A-B is provided, wherein A is a pancreatic β cell targeting moiety, and B is an inhibitor of Apolipoprotein CIII (apoCIII), protein kinase A (PKA), Src kinase, or β1 integrin. In an additional aspect, the composition is of formula A-B-C, wherein C is a compound that permits cell entry of the composition.
In various embodiments, the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides.
In various embodiments, the PKA or Src kinase inhibitor is selected from the group consisting of PP1 analogs, PP2, adenosine 3′,5′-cyclic monophosphorothioate-R, adenosine 3′,5′-cyclic monophosphorothioate H-7, adenosine 3′,5′-cyclic monophosphorothioate H-8, adenosine 3′,5′-cyclic monophosphorothioate H-9, and adenosine 3′,5′-cyclic monophosphorothioate H-89.
In various embodiments, the β1 integrin inhibitor is selected from the group consisting of anti-β1 integrin antibody, anti-β1 integrin aptamer, β1 integrin small interfering RNA, β1 integrin small internally segmented interfering RNA, β1 integrin short hairpin RNA, β1 integrin microRNA, and β1 integrin antisense oligonucleotides.
In various embodiments, the pancreatic β cell specific targeting moiety comprises a moiety that that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), orphan G protein-coupled receptor GPR44(GPR44), SerpinB10 (PI-10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36 kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon-like peptide 1 receptor (GLP1R), and transmembrane protein 27 (TMEM27). In further embodiments, the pancreatic β cell specific targeting moiety comprises a moiety selected from the group consisting of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PP), exendin-4, naphthylalanine and naphthylalanine derivatives.
Specific embodiments of the invention will become evident from the following more detailed description of certain embodiments and the claims.
All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.
All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.
In a first aspect, the present invention provides compositions of formula A-B is provided, wherein A is a pancreatic β cell targeting moiety, and B is an inhibitor of Apolipoprotein CIII (apoCIII), protein kinase A (PKA), Src kinase, or β1 integrin. In an additional aspect, the composition is of formula A-B-C, wherein C is a compound that permits cell entry of the composition.
The inventors have surprisingly discovered that obesity is associated with a local production of apoCIII in pancreatic β-cells. The examples provided herein demonstrate that apoCIII is locally expressed and increased in islets from ob/ob mice that have reduced insulin signaling in their beta cells due to insulin resistance. As these islets contain>90% β-cells, most of the apoCIII expression is derived from the insulin-secreting β-cells. Thus, the compositions of the inventon can be used, for example, in treating or limiting development of diabetes.
As used herein, an “inhibitor” of expression and/or activity of ApoCIII, PKA, Src kinase, and/or β1 integrin includes compounds that reduce the transcription of ApoCIII, PKA, Src kinase, and/or β1 integrin DNA into RNA, compounds that reduce translation of the ApoCIII, PKA, Src kinase, and/or β1 integrin RNA into protein, and compounds that reduce the function of ApoCIII, PKA, Src kinase, and/or β1 integrin protein. Such inhibiting can be complete inhibition or partial inhibition, such that the expression and/or activity of the ApoCIII, PKA, Src kinase, and/or β1 integrin is reduced, resulting in a reduced ability to increase intracellular calcium concentration. Such inhibitors are selected from the group consisting of antibodies that bind to ApoCIII, PKA, Src kinase, and/or β1 integrin; aptamers that can interfere with ApoCIII, PKA, Src kinase, and/or β1 integrin activity; antisense oligonucleotides directed against the ApoCIII, PKA, Src kinase, and/or β1 integrin protein, DNA, or mRNA; small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNA) or small internally segmented interfering RNAs (sisiRNA) directed against the ApoCIII, PKA, Src kinase, and/or β1 integrin protein, DNA, or mRNA, and any other chemical or biological compound that can interfere with ApoCIII, PKA, Src kinase, and/or β1 integrin activity.
In various embodiments, the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides.
In various embodiments, the PKA or Src kinase inhibitor is selected from the group consisting of PP1 analogs, PP2, adenosine 3′,5′-cyclic monophosphorothioate-R, adenosine 3′,5′-cyclic monophosphorothioate H-7, adenosine 3′,5′-cyclic monophosphorothioate H-8, adenosine 3′,5′-cyclic monophosphorothioate H-9, and adenosine 3′,5′-cyclic monophosphorothioate H-89.
In various embodiments, the β1 integrin inhibitor is selected from the group consisting of anti-β integrin antibody, anti-β integrin aptamer, β1 integrin small interfering RNA, β1 integrin small internally segmented interfering RNA, β1 integrin short hairpin RNA, β1 integrin microRNA, and β1 integrin antisense oligonucleotides.
The pancreatic β cell targeting moieties of the compositions permit targeting of the composition to pancreatic β cells upon administration to a subject in need thereof. In some embodiments the pancreatic β targeting moiety can comprise an antibody, receptor, receptor ligand and the like, which preferentially and/or specifically bind to pancreatic β cells. One or more targeting moieties can be attached to the inhibitor to provide a composition that is capable targeting the composition to pancreatic β cells. In various embodiments, targeting moieties include, but are not limited to peptides that preferentially bind pancreatic β cells, antibodies that bind pancreatic β cells, and receptor ligands that bind pancreatic β cells.
In one embodiment, the pancreatic β cell specific targeting moiety comprises one or more peptides or other moieties that preferentially bind pancreatic β cells, selected from the group consisting of glucagon-like peptide-1 (GLP-1 SEQ ID NO: 73), glucagon-like peptide-2 (GLP-2 SEQ ID NO: 74), peptide YY (PYY SEQ ID NO: 3), neuropeptide Y (NPY SEQ ID NO: 4), pancreatic peptide (PPY SEQ ID NO: 5), exendin-4 (SEQ ID NO: 6), naphthylalanine and naphthylalanine derivatives (sequences shown in Table 1).
In other embodiments, the pancreatic β cell specific targeting moiety comprises a moiety that that selectively binds a pancreatic β cell protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2; SEQ ID NO: 7), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1 SEQ ID NO: 8), orphan G protein-coupled receptor GPR44(GPR44 SEQ ID NO: 9), SerpinB10 (PI-10 SEQ ID NO: 10), FXYD domain containing ion transport regulator 2 (FXYD2 SEQ ID NO: 11), Tetraspanin-7 (TSPAN7 SEQ ID NO: 12), gap junction protein, delta 2, 36 kDa (GJD2 SEQ ID NO: 13), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2 SEQ ID NO: 14), prokineticin receptor 1 (PROKR1 SEQ ID NO: 15), glutamate receptor, metabotropic 5 (GRM5 SEQ ID NO: 16), neuropeptide Y receptor Y2 (NPY2R SEQ ID NO: 17), glucagon-like peptide 1 receptor (GLP1R SEQ ID NO: 18), and transmembrane protein 27 (TMEM27 SEQ ID NO: 19).
golgi brefeldin A resistant
Homo sapiens
When the targeting moiety comprises an antibody, such antibodies can be polyclonal or monoclonal. The antibodies can be humanized, fully human, or murine forms of the antibodies. Such antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). In some embodiments, additional amino acid residues may be added to either the N- or C-terminus of the antibody or antibody fragment. When the targeting moiety comprises an aptamer, such aptamers can be oligonucleic acid or peptide molecules that bind to a specific target molecule. Methods of constructing and determining the binding characteristics of aptamers are well known in the art, and the aptamers can be isolated from random libraries or they can be previously identified peptides.
Attaching the pancreatic β cell targeting moiety to the inhibitor may be accomplished by any chemical reaction that will bind the two molecules so long as the pancreatic β cell targeting moiety and the inhibitor retain their respective activities. In one embodiment, both the β cell targeting moiety and the inhibitor comprise polypeptides, and the composition comprises a recombinant fusion protein. In another embodiment, both the β cell targeting moiety and the inhibitor comprise polynucleotides, and the composition comprises a recombinant nucleic acid. In other embodiments, a linkage between the pancreatic β cell targeting moiety and the inhibitor can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies, to other molecules. For example, representative, non-limiting examples of coupling agents can be organic compounds such as thioesters, carbodiimides, succinimide esters, disocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines.
In another embodiment, the compositions are of general formula A-B-C, wherein C is a compound that permits cell entry of the composition. In one embodiment, the compound that permits cell entry of the composition may comprise a cell penetrating peptide. In other embodiments, the compound that permits cell entry of the composition may comprise a non-peptidic cell penetrating compound. Cell penetrating peptides or non-peptidic cell penetrating compounds facilitate uptake of the composition into the pancreatic β cells targeted by the targeting moiety of the composition. The cell penetrating compound can be linked to components A-B of the composition by any suitable technique that retains the activities of each component, as discussed herein. In one embodiment, each of A-B-C are polypeptides and the composition comprises a fusion protein.
Exemplary cell penetrating peptides include, but are not limited to RRASAP (SEQ ID NO: 20) LRRASAP (SEQ ID NO: 21) WLRRASAP (SEQ ID NO: 22) RRATAP (SEQ ID NO: 23) LRRATAP (SEQ ID NO: 24) WLRRATAP (SEQ ID NO: 25) RRAYAP (SEQ ID NO: 26) LRRAYAP (SEQ ID NO: 27) WLRRAYAP (SEQ ID NO: 28) RRADAP (SEQ ID NO: 29) LRRADAP (SEQ ID NO: 30) WLRRADAP (SEQ ID NO: 31) RRAEAP (SEQ ID NO: 32) LRRAEAP (SEQ ID NO: 33) WLRRAEAP (SEQ ID NO: 34) RRASAPRRASAP (SEQ ID NO: 35) LRRASAPLRRASAP (SEQ ID NO: 36) WLRRASAPWLRRASAP (SEQ ID NO: 37) RRATAPRRATAP (SEQ ID NO: 38) LRRATAPLRRATAP (SEQ ID NO: 39) WLRRATAPWLRRATAP (SEQ ID NO: 40) RRAYAPRRAYAP (SEQ ID NO: 41) LRRAYAPLRRAYAP (SEQ ID NO 42) WLRRAYAPWLRRAYAP (SEQ ID NO: 43) RRADAPRRADAP (SEQ ID NO: 44) LRRADAPLRRADAP (SEQ ID NO: 45) WLRRADAPWLRRADAP (SEQ ID NO: 46) RRAEAPRRAEAP (SEQ ID NO: 47) LRRAEAPLRRAEAP (SEQ ID NO: 48) WLRRAEAPWLRRAEAP (SEQ ID NO: 49), GRKKRRQRRRPPQ (SEQ ID NO:50); AYARAAARQARA (SEQ ID NO:51); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:52); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:53); PLSSISRIGDP (SEQ ID NO:54); AAVALLPAVLLALLAP (SEQ ID NO:55); AAVLLPVLLAAP (SEQ ID NO:56); VTVLALGALAGVGVG (SEQ ID NO:57); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO:58); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:59); KLALKLALKALKAALKLA (SEQ ID NO:60); KETWWETWWTEWSQPKKKRKV (SEQ ID NO:61); KAFAKLAARLYRKAGC (SEQ ID NO:62); KAFAKLAARLYRAAGC (SEQ ID NO:63); AAFAKLAARLYRKAGC (SEQ ID NO:64); KAFAALAARLYRKAGC (SEQ ID NO:65); KAFAKLAAQLYRKAGC (SEQ ID NO:66), AGGGGYGRKKRRQRRR (SEQ ID NO:67); YGRKKRRQRRR (SEQ ID NO:68); and YARAAARQARA (SEQ ID 69).
The individual components of the composition can be made by any suitable means known to those of skill in the art. Once the composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
In a second aspect, the present invention provides an isolated nucleic acid encoding a fusion protein or recombinant nucleic acid of any embodiment or combination of embodiments of the compositions of the invention, operatively linked to a promoter. Thus, in some embodiments the fusion protein comprises a fusion protein of any embodiment or combination of embodiments of peptidic components A-B (and optionally C) of the compositions of the invention. In other embodiments, components A-B of the compositions of the invention are each polynucleotides, and thus the isolated nucleic acid encodes a recombinant nucleic acid composition of the invention. The isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. The term “operatvely linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
In a third aspect, the present invention provides recombinant expression vectors comprising an isolated nucleic acid of the invention. The term “vector” as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term “vector” includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms “plasmid” and “vector” may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
In a fourth aspect, the present invention provides recombinant host cells comprising an expression vector of any embodiment or combination of embodiments of the invention. The phrase “recombinant host cell” (or “host cell”) as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein. A wide variety of host cell expression systems can be used to express the antibody-like binding proteins of the invention, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express an antibody-like binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the antibody-like binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the antibody-like binding protein can be recovered.
The term “transformation” as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term “transfection” as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
In a fifth aspect, the invention provides methods for treating or limiting development of diabetes, comprising administering to a subject in need thereof an amount effective of a composition comprising a compound of formula A-B, wherein A is a pancreatic β cell targeting moiety, and B is an inhibitor of expression and/or activity of Apolipoprotein CIII (apoCIII), protein kinase A (PKA), Src kinase, and/or β1 integrin. Thus, any embodiment or combination of embodiments of the compositions disclosed herein may be used in the present invention.
In various embodiments, the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides.
In various embodiments, the PKA or Src kinase inhibitor is selected from the group consisting of PP1 analogs, PP2, adenosine 3′,5′-cyclic monophosphorothioate-R, adenosine 3′,5′-cyclic monophosphorothioate H-7, adenosine 3′,5′-cyclic monophosphorothioate H-8, adenosine 3′,5′-cyclic monophosphorothioate H-9, and adenosine 3′,5′-cyclic monophosphorothioate H-89.
In various embodiments, the β1 integrin inhibitor is selected from the group consisting of anti-β1 integrin antibody, anti-β1 integrin aptamer, β1 integrin small interfering RNA, β1 integrin small internally segmented interfering RNA, β1 integrin short hairpin RNA, β1 integrin microRNA, and β1 integrin antisense oligonucleotides.
In various embodiments, the pancreatic β cell specific targeting moiety comprises a moiety that that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), orphan G protein-coupled receptor GPR44(GPR44), SerpinB10 (PI-10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36 kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon-like peptide 1 receptor (GLP1R), and transmembrane protein 27 (TMEM27). In further embodiments, the pancreatic β cell specific targeting moiety comprises a moiety selected from the group consisting of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PP), exendin-4, naphthylalanine and naphthylalanine derivatives.
As used herein, “pancreatic β cells” are any population of cells that contains pancreatic β islet cells. Such pancreatic β islet cell populations include the pancreas, isolated pancreatic islets of Langerhans (“pancreatic islets”) and isolated pancreatic β islet cells.
In one embodiment, the method is for treating diabetes. In this embodiment, the subject has been diagnosed with type 1 or type 2 diabetes. As used herein, “diabetes” is characterized by insufficient or no production of insulin by the pancreas, leading to high blood sugar levels.
As used herein, “treating diabetes” means accomplishing one or more of the following: (a) reducing the severity of the diabetes or diabetic complications; (b) limiting or preventing development of diabetic complications; (c) inhibiting worsening of diabetic complications or of symptoms characteristic of diabetes; (d) limiting or preventing recurrence diabetic complications or of symptoms characteristic of diabetes; (e) limiting or preventing recurrence of diabetic complications or of symptoms characteristic of diabetes in patients that were previously symptomatic.
Symptoms characteristic of diabetes that can be treated by the methods of the invention include, but are not limited to, elevated blood glucose levels, decreased insulin production, insulin resistance, proteinuria, and impaired glomerular clearance. Diabetic complications that can be treated according to the methods of the invention include, but are not limited to, complications in the nerves (such as diabetic neuropathy) and complications associated with smooth muscle cell dysregulaton (including but not limited to erectile dysfunction, bladder dysfunction, and vascular complications including but not limited to atherosclerosis, stroke, and peripheral vascular disease)
In another embodiment, the method is for limiting development of diabetes. In this aspect, the subject is at risk of type 1 or type 2 diabetes, and a benefit is to limit development of diabetes and/or diabetic complications. Any subject at risk of developing diabetes can be treated, including but not limited to subjects with one or more of, metabolic syndrome, known genetic risk factors for diabetes, a family history of diabetes, and obesity.
In a further embodiment, the methods for treating or limiting development of diabetes and/or diabetic complications further comprises treating those individuals that have been identified as overexpressing apoCIII compared to control. Increases in apoCIII expression precede development of diabetic complications, and thus this embodiment permits early detection of suitable patients for treatment using the methods of the invention.
As used herein, “overexpression” is any amount of apoCIII expression above control. Any suitable control can be used, including apoCIII expression levels from a subject known not to be suffering from diabetes, or previously determined standardized expression levels of apoCIII from a population of similar patient samples. Any amount of increased apoCIII expression relative to control is considered “overexpression”; in various embodiments, the overexpression comprises at least 10%, 20%, 50%, 100%, 200%, or greater increased apoCIII expression compared to control. In a preferred embodiment, apoCIII expression is detected in blood or serum samples. In one embodiment to evaluate the levels of apoCIII in sera, albumin is removed from serum samples using standard techniques, such as via use of Montage Albumin Deplete Kit (Millipore) or AlbuSorb™ (Biotech Support Group). The collected sera samples can then be freeze-dried overnight and run on sep-Pak C18. The eluted proteins can be freeze-dried and thereafter dissolved in 100 μL 0.1% TFA and run on an ACE C18 10- ×0.21 -cm column 20-60%, and the area under the curve, where apoCIII elutes, evaluated. ApoCIII can be identified using any suitable technique, including but not limited to MALDI mass spectrometry.
As used herein, the term “subject” or “patient” is meant any subject for which therapy is desired, including humans, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, insects, horses, chickens, and so on. Most preferably, the subject is human.
The therapeutic may be administered by any suitable route, including but not limited to oral, topical, parenteral, intranasal, pulmonary, or rectal in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier. The therapeutic may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The therapeutic may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
In another embodiment, the composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the composition of the invention has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into or next to the pancreas, and delivery of the desired molecule can be via diffusion, timed-release bolus, nano-containers or continuous administration.
The dosage range depends on the choice of the compound, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art
Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The therapeutic composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).
The therapeutic compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody-like binding protein.
Animals. Age-matched ob/ob (Lepob/Lepob) and ob/lean (Lepob/Lep+) on a C57BL/6J background were obtained from a breeding colony at Karolinska Institutet, Stockholm, Sweden. Mice used in experiments were genotyped. All experiments were performed on mice, between 4 to 12 weeks of age. The animals were housed in a temperature- and humidity-controlled room with 12-hour light:dark cycles, regular chow and water ad libitum. Animal care and experimentations were carried out according to the Animal Experiment Ethics Committee at Karolinska Institutet.
Cell Culture. MIN6-m9 cells, between passage 35 and 42, were cultured in DMEM containing 11.1 mM glucose and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 10% FCS, and 75 μM β-mercaptoethanol at 5% CO2 and 37° C. MIN6 cells were transfected using Lipofectamine (Invitrogen, USA) with the control expression construct containing tdTomato or c-Myc tagged mouse apoCIII construct for 24 hours. All experiments were performed 48 h after transfection.
Metabolic Studies. Non-fasted glucose was measured with a glucose meter (Accu-Chek™ Advantage, Roche Diagnostics, USA) and serum insulin by the ArcDia™ 2-photon fluorescence excitation microparticle fluorometry (TPX) assay for insulin (ArcDia Group, Finland).
Pancreatic islet isolation. Animals were euthanized by cervical dislocation and the pancreas perfused with 3 mL of 1 mg/mL collagenase A (Roche, USA) in Hank's balanced salt solution (HBSS) (Sigma, Sweden) buffer supplemented with 0.2% BSA and 25 mM HEPES buffer. Pancreas was thereafter extracted and digested at 37° C. for 20 min. Islets were handpicked and were either immediately used for mRNA/protein analysis or were cultured overnight in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine as well as 100 U/ml and 100 μg/ml of penicillin and streptomycin, respectively.
Dispersion of pancreatic islets into single cells. Isolated islets were washed with HBSS without Ca2+ and Mg2+ and dispersed into single cells by incubation with Accutase in PBS containing 0.5 mM EDTA (Innovative Cell Technologies, Cytotech, Denmark). Cells were kept free floating in Petri dishes in the same medium as for islets.
RNA isolation and quantitative RT-PCR analysis. Total RNA was isolated from sorted cells or isolated pancreatic islets using the RNAeasy™ Micro Kit (Qiagen, Germany). Briefly, cells were lysed by first using RLT lysis buffer followed by a spin Qiashredder™ lysis. For liver and intestine RNA isolation, tissue/cell disruption was first carried out by a hand-held rotor-stator homogenizer in RLT lysis buffer followed by 1000×g centrifugation for 5 minutes. The supernatant was subsequently transferred to a Qiashredder™ column to complete the lysis prior to the RNAeasy™ Mini Kit application (Qiagen, Germany). All lysates were applied to the RNAeasy™ spin column and the subsequent RNA isolation and on column DNAse treatment were carried out according to manufacturer recommendations. Total RNA was reverse transcribed at 37° C. with Multiscribe™ (Applied Biosystems, USA). The expression of all genes was measured by real-time quantitative PCR (qPCR) with Taq™ SYBR Supermix with ROX (Invitrogen, USA) on an ABI7900HT instrument (Applied Biosystems, USA). β-actin was used as an endogenous control with 1 cycle at 95° C. for 10 min followed by 40 cycles of tandem 95° C. for 30 seconds and 60° C. for 1 minute. In all cases, unless otherwise stated, gene-specific intron spanning primers were used and the PCR melting curve produced one single peak corresponding to a specific single amplified product.
Western Blotting. Islets or cells were washed with PBS, lysed with RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, EDTA-free protease inhibitor cocktail, 1 μg/ml pepstatine and leupeptin). Lysates were passed 5 times through a syringe needle (0.33×13 mm/29 Gx½) followed by 30 minutes incubation on a rotator at 4° C. Homogenates were spun at 10,000×g for 10 minutes and the protein amount was determined in the supernatants by the BCA method (Pierce). Equal amounts of protein (25-50 μg) were separated over a 4-12% Bis-Tris gel with MES buffering system (Invitrogen, USA). Proteins were subsequently electrotransferred to PVDF membrane. In case of the phosphospecific antibodies, the membranes were probed with the respective antibodies and then stripped and reprobed with antibodies recognizing the respective total protein levels. Rabbit anti-FoxO1, rabbit anti-phospho-FoxO1, Akt and phospho-Akt antibody were purchased from Cell Signaling Technology (Danvers, USA). Further antibodies used were rabbit anti-apoCIII antibody (Santa-Cruz, USA), guinea-pig c-peptide (Abcam, UK), mouse anti-cMyc (Santa Cruz, USA) and mouse anti-tubulin (Abcam, UK). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibodies using the ECL system (Amersham, USA).
Immunocytochemistry. To visualize Myc-tagged apoCIII in MING cells, post-transfected cells were cultured onto glass cover slips prior to 3% PFA fixation for 30 minutes. Subsequently, cells were incubated in antibodies specific for c-Myc (mouse monoclonal, Santa-Cruz, USA) and C-peptide (rabbit polyclonal, Cell Signaling, USA) in 10% Goat serum blocking buffer, overnight at 4° C. with gentle shaking Cells were incubated with Alexa 488 anti-mouse (Santa-Cruz, USA) and Alexa 546 anti-rabbit (Cell Signaling, USA) in blocking buffer for 1 hour followed by washing and mounting using VectaShield™ mounting media with DAPI (Vector Labs, USA). The human islets were fixed in 4% paraformaldehyde for 15 minutes, washed in PBS and incubated in 20% sucrose/PBS overnight at 4° C. Thereafter they were embedded in Tissue-Tek® OCT™ (Sakura, The Netherlands) and cryosectioned (10 μm). The sections were permeabilized in 0.3% Triton-X/PBS for 15 minutes, treated with 3% H2O2 block for 10 minutes and washed in PBS and subsequently blocked in 10% goat serum. Immunohistochemical staining was performed using goat anti-human apoCIII (1:100, Academy Biomedical, USA) overnight incubation at 4° C., followed by Alexa™ Flour 488-labeled rabbit anti-goat (1:1000, Invitrogen) for 30 minutes at room temperature. Counterstaining was performed with DAPI (1:1000; Sigma-Aldrich). Following the above-described method, a blockage of goat anti-human apoCIII was also performed as a control by using the goat anti-human apoCIII pretreated over night at 4° C. with apoCIII protein (immunizing peptide).
Measurements of [Ca2+]i. Changes in [Ca2+]i were recorded in islets after a 16 hour incubation period with 0.07 mg/ml apoCIII antisense or an inactive control (Isis Pharmaceuticals, USA) in RPMI 1640 medium. The basal medium used for islet perifusion experiments was a HEPES buffer (pH 7.4), containing: 125 mM NaCl, 5.9 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, and 25 mM HEPES, 0.1% BSA supplemented with either 3 mM glucose, 11 mM glucose or 25 mM KCl. Islets were attached to coverslips using Puramatrix™ Hydrogel (BD Biosystem, USA), loaded with 2 μM fura-2 acetoxymethyl ester (Molecular Probes, USA) and mounted on an inverted epifluorescence microscope (Zeiss, Axiovert™ 135) connected to a Spex Industries Fluorolog system for dual-wavelength excitation fluorimetry. The emissions due to the two excitation wavelengths of 340 and 380 nm were used to calculate the fluorescence ratio 340/380, reflecting changes in [Ca2+]i. To compensate for possible variations in output of light intensity from the two monochromators, each experiment also included measurement of a 360/360 ratio. Every experiment was normalized by dividing all fluorescence ratios by the corresponding 360/360 ratio.
Caspase assay. Activity of capase 3/7 was determined using SensoLyte™ Homogeneous Rh110 Caspase 3/7 assay kit according to manufacturer's instructions, with modifications (AnaChem, USA). Briefly, islets or cells were harvested, lysed using lysis buffer (AnaChem, USA) and the protein quantified using BCA method. 10 μg of protein was loaded in a black 384-well plate and topped up with the appropriate amount of assay buffer (AnaChem, USA) containing Rh110 Caspase 3/7 Substrate. Plate was incubated in the dark for 1 hour at 24° C. Fluorescence intensity at Ex/Em=490/520 nm was measured to determine the relative caspase activity.
RNA analysis. 5′-RACE was performed by using ob/ob islets (1.5 μg RNA) Ambion FirstChoice™ RACE-ready cDNA kit (Invitrogen, USA). PCR was carried-out following manufacturer's instructions. Platinum Taq polymerase (Invitrogen, USA) was used in RACE-PCR. Primer sequences for apoCIII-outer (primary amplification): 5′ GGAGGGGTGAAGACATGAGA-3′ (SEQ ID NO: 70); apoCIII-inner (nested amplification): 5′ TCTGAAGTGATTGTCCATCCAG-3′ (SEQ ID NO: 71). An aliquot of the first PCR (outer PCR) was used for subsequent nested PCR (inner PCR). Amplified cDNA were gel purified and subcloned into the pCRII-TOPO vector (Invitrogen, USA) and sequenced with M13 reverse and M13 forward primers.
Statistical analysis. Studies were repeated at least three times. For individual experiment, the number of animals used (n) is included in each figure legend in parenthesis. All results are expressed as mean±SEM (indicated by error bars). Statistical analyzes were performed with either GraphPad™ Prism 5 or Microsoft Excel 2007. A student's t-test or one-way ANOVA (Tukey's post-hoc) were used as appropriate. P values <0.05 were considered statistically significant.
To determine the changes in insulin signaling in ob/ob mouse islets, which consist of more than 90% β-cells, the phenotypic changes were tracked in these mice at 4, 8 and 12 weeks of age, measuring parameters such as body weight, non-fasting blood glucose and insulin concentrations. Age-matched ob/lean mice that were routinely genotyped from 4 weeks onwards were used as controls. Since the ob/ob mouse is a known transient hyperglycemia model with increasing hyperglycemia up to 14 weeks of age, the study was limited to the first 12 weeks of age and found that both weight and non-fasting blood glucose levels progressively increased from 4- to 12-weeks of age (
With prolonged hyperinsulinemia, insulin signaling at the islet level may be compromised, similar to what is seen in the β-cell-specific insulin receptor knockout mouse ((βIRKO) and the β-cell-specific PI3K subunit p85 β knockout mouse (βPik3r1). The mRNA expression levels of several genes known to be involved downstream of islet insulin signaling was determined. The expression of gck, irs1, irs2, vamp2, snap25 and rab27a were shown to be controlled by insulin receptor (IR) activated phosphatidylinositol 3-kinase (PI3K) activity. All genes were down-regulated in 12 weeks old ob/ob islets (
One of the ways in which insulin induces changes in gene expression is through the phosphorylation cascade of Akt and its downstream target forkhead transcription factor FoxO1. Insulin-mediated activation of Akt leads to the phosphorylation of FoxO1 with its nuclear exclusion and loss of transcriptional activity. This signaling cascade has been shown to be crucial in maintaining β-cell function and proliferation during either insulin resistance or insult/injury. Levels of phospho-Akt and phospho-FoxO1 in ob/ob islets were determined at 4-, 8- and 12-weeks of age. There was a significant reduction in phospho-Akt (Ser473) and phospho-FoxO1 (Ser256) in the 12-weeks old ob/ob islet as compared to the islets at 4-weeks of age (
Insulin receptor-mediated repression of FoxO1 is pivotal in maintaining the balance between carbohydrate and fat metabolism in hepatocytes. Reduced FoxO1 phosphorylation, and subsequent activation, in insulin-resistant hepatocytes promote the expression of apoCIII, a modulator of circulating triglycerides, glucose 6 phosphatase a key gluconeogenic enzyme, and mttp1, a microsomal triglyceride transfer protein that is involved in lipoprotein assembly. To explore if a similar mechanism occurs in ob/ob islets with declining levels of phospho-FoxO1, the mRNA expression levels of Foxo1 target genes apoCIII, g6pc2 (islet specific glucose-6-phosphatase) and mttp 1 were determined. There was a significant increase in ob/ob islet mRNA expression of all three genes across time (
There was no significant difference in liver and intestine apoCIII expression in ob/ob mice at the age-groups studied (
The presence of apoCIII in pancreatic islet has not been observed before and is interesting since this apolipoprotein is known to be involved in both β-cell dysfunction and T1DM. A proteomic characterization of single pancreatic islets showed presence of apoCIII. However, this can be the result of either systemic circulating apoCIII residing within the microvasculature of pancreatic islets or, as shown here, a local production of apoCIII within the islet itself. Comparatively, the expression of apoCIII in ob/lean pancreatic islets is about 3000-fold lower compared to the liver (
The source of apoCIII within the islets was determined. The endocrine portion of the ob/ob islet is made up of approximately 90% β-cells, making this cell type a prime candidate for apoCIII expression. To examine this, a flow cytometry protocol originally optimized to sort out rat β-cells from the total pancreatic islet cell milieu was used. Subsequent characterization of sorted β-cells revealed enriched expression of insulin, low expression of somatostatin and undetectable expression of glucagon and pancreatic polypeptide (
Exogenously added human apoCIII induces apoptosis in the rat insulin secreting cells lines RINm5F and INS-1E and in primary mouse β-cells. It was therefore important to determine if a local production of apoCIII likewise affected mouse β-cell function and apoptosis. Hampered by the ability of the antibody to recognize apoCIII in mouse liver sections and lysates, a c-Myc tagged mouse apoCIII construct was used that allows determining the localization of Myc-apoCIII within β-cells. Myc-apoCIII was detectable in media of the Myc-apoCIII-transfected cells after a 24-hour culture, indicating that the synthesized apoCIII is being released into the extracellular medium (
To explore the effect of increased endogenous apoCIII expression within islets from 12-weeks old ob/ob mice, knockdown experiments using antisense oligonucleotides specific for apoCIII and an inactive control were performed. The expression of apoCIII in the islets exposed to antisense nucleotides for 18-hours was 20% compared to control islets (
It was determined if reducing endogenous apoCIII affects apoptosis in ob/ob islets. Although ob/ob islets are hyperplastic with massive β-cell proliferation, it does not rule out the presence of apoptosis in these islets; therefore the level of apoptosis was determined in freshly isolated islets of ob/ob mice at weeks 4-, 8- and 12 (when phospho-FoxO1 declines) by measuring the activity of caspase 3/7. Apoptosis in ob/ob islets was significantly higher in 12-weeks old ob/ob islets as compared to islets at 4 weeks of age (
These results show that obesity is associated with a local production of apoCIII in pancreatic β-cells and that increased levels of apoCIII leads to insulin resistance, increased [Ca2+]i and β-cell apoptosis; therefore identifying local islet production of apoCIII as a novel factor for mediating β-cell apoptosis in insulin resistant states of T2DM. The effects are reversible as reducing endogenouse β-cell apoCIII normalizes [Ca2+]i and improves β-cell survival. Hence, locally enhanced apoCIII can interfere with β-cell function and survival and have an impact on development of the T2DM phenotype. The results demonstrate that apoCIII is locally expressed and increased in islets from ob/ob mice that have reduced insulin signaling. As these islets contain>90% β-cells, most of the apoCIII expression is derived from the insulin-secreting β-cells. ApoCIII induced an increase in cytoplasmic free Ca2+ concentration and β-cell apoptosis that was abrogated by lowering apoCIII levels or blocking the voltage-gated Ca2+-channels. This makes apoCIII a prime target that links β-cell insulin resistance to reduction in β-cell mass in T2DM. Consequently, inhibition of β-cell apoCIII expression constitutes a novel mechanism to maintain β-cell function and survival.
In summary, insulin signaling in β-cells is essential to keep apoCIII expression low. When insulin signaling is impaired at the islet level, FoxO1 translocation into the nucleus induces changes in gene expression which include, among other alterations, a promotion of apoCIII transcription. The increase in local production of apoCIII subsequently leads to an increase in [Ca2+]i which in turn results in an increase in β-cell apoptosis. Hence, preventing insulin resistance at the islet level is crucial to preserve β-cell mass, especially during the late phase of T2DM where β-cells are progressively lost. Islet apoCIII is a key player in the promotion of β-cell apoptosis and lowering local β-cell production of apoCIII may provide a new therapeutic for T2DM.
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/779508 filed Mar. 13, 2013, incorporated by reference herein in its entirety.
