Patent Grant
9758561
This application is a 35 U.S.C. §111(a) filing claiming priority under 35 U.S.C. §119(a)-(d) to European Patent Application No. EP2014/14305502.8, filed Apr. 7, 2014, the content of which is incorporated herein by reference in its entirety.
The present invention relates to dual GLP-1/glucagon receptor agonists and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as for reduction of excess food intake. These dual GLP-1/glucagon receptor agonists show reduced activity on the GIP receptor to reduce the risk of hypoglycemia and are structurally derived from exendin-4, a pure GLP-1 receptor agonist.
Pocai et al (Obesity 2012; 20:1566-1571; Diabetes 2009, 58, 2258) and Day et al. (Nat Chem Biol 2009; 5:749) describe dual agonists of the glucagon-like peptide-1 (GLP-1) and glucagon receptors, e.g. by combining the actions of GLP-1 and glucagon in one molecule, which lead to a therapeutic principle with anti-diabetic action and a pronounced weight lowering effect superior to pure GLP-1 agonists, among others due to glucagon-receptor mediated increased satiety and energy expenditure.
Holst (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) describe that GLP-1 receptor agonists, such as GLP-1, liraglutide and exendin-4, have 3 major pharmacological activities to improve glycemic control in patients with T2DM by reducing fasting and postprandial glucose (FPG and PPG): (i) increased glucose-dependent insulin secretion (improved first- and second-phase), (ii) glucagon suppressing activity under hyperglycemic conditions, (iii) delay of gastric emptying rate resulting in retarded absorption of meal-derived glucose.
The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 2.
Liraglutide is a marketed chemically modified GLP-1 analog in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker D J et al, Nature Drug Disc. Rev. 9, 267-268, 2010; Buse, J. B. et al., Lancet, 374:39-47, 2009).
The amino acid sequence of Liraglutide is shown as SEQ ID NO: 4.
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO: 3.
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Recent publications suggest that glucagon has in addition beneficial effects on reduction of body fat mass, reduction of food intake, and increase of energy expenditure (K M Heppner, Physiology & Behavior 2010, 100, 545-548).
GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K-cells following food intake. GIP and GLP-1 are the two gut enteroendocrine cell-derived hormones accounting for the incretin effect, which accounts for over 70% of the insulin response to an oral glucose challenge (Baggio L L, Drucker D J. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132: 2131-2157).
GIP's amino acid sequence is shown as SEQ ID NO: 5.
Peptides which are based on the structures of GLP-1 or glucagon, and bind and activate both the glucagon and the GLP-1 receptor (Hjort et al. Journal of Biological Chemistry, 269, 30121-30124, 1994; Day J W et al, Nature Chem Biol, 5: 749-757, 2009) and suppress body weight gain and reduce food intake are described in patent applications WO 2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO 2011/075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO 2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/087671, WO 2011/087672, WO2011/117415, WO2011/117416, WO 2012/177443 WO 2012/177444, WO 2012/150503, WO 2013/004983, WO 2013/092703, WO 2014/041195 and WO 2014/041375, the contents of which are herein incorporated by reference. The body weight reduction was shown to be superior to pure GLP-1 agonists.
In addition, triple co-agonist peptides which not only activate the GLP-1 and the glucagon receptor, but also the GIP receptor are described in WO 2012/088116 and by V A Gault et al (Biochem Pharmacol, 85, 16655-16662, 2013; Diabetologia, 56, 1417-1424, 2013).
Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng, J. et al., J. Biol. Chem., 267:7402-05, 1992). Exendin-4 is an activator of the GLP-1 receptor, whereas it shows low activation of the GIP receptor and does not activate the glucagon receptor (see Table 1).
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1.
Exendin-4 shares many of the glucoregulatory actions observed with GLP-1. Clinical and nonclinical studies have shown that exendin-4 has several beneficial antidiabetic properties including a glucose dependent enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing down gastric emptying, reduction of food intake and body weight, and an increase in beta-cell mass and markers of beta cell function (Gentilella R et al., Diabetes Obes Metab., 11:544-56, 2009; Norris S L et al, Diabet Med., 26:837-46, 2009; Bunck M C et al, Diabetes Care., 34:2041-7, 2011).
These effects are beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of getting diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.
Relative to GLP-1, exendin-4 is resistant to cleavage by dipeptidyl peptidase-4 (DPP4) resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 45 (Suppl 2):152A (abstract 554), 1996).
Exendin-4 was also shown to be much more stable towards degradation by neutral endopeptidase (NEP), when compared to GLP-1, glucagon or oxyntomodulin (Druce M R et al., Endocrinology, 150(4), 1712-1721, 2009). Nevertheless, exendin-4 is chemically labile due to methionine oxdiation in position 14 (Hargrove D M et al., Regul. Pept., 141: 113-9, 2007) as well as deamidation and isomerization of asparagine in position 28 (WO 2004/035623).
Compounds of this invention are exendin-4 derivatives, which in addition to the agonistic activity at the GLP-1 receptor of native exendin-4 show agonistic activity at the glucagon receptor and which have—among others—the following modification: at position 14 an amino acid carrying an —NH2 group in the side-chain, which is further substituted with a lipophilic residue (e.g. a fatty acid combined with a linker) and at position 27 an Aib.
Bloom et al. (WO 2006/134340) disclose that peptides which bind and activate both the glucagon and the GLP-1 receptor can be constructed as hybrid molecules from glucagon and exendin-4, where the N-terminal part (e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminal part (e.g. residues 15-39 or 25-39) originates from exendin-4. Such peptides comprise glucagon's amino acid motif YSKY in position 10-13. Krstenansky et al (Biochemistry, 25, 3833-3839, 1986) show the importance of these residues 10-13 of glucagon for its receptor interactions and activation of adenylate cyclase.
In the exendin-4 derivatives described in this invention, several of the underlying residues are different from glucagon and the peptides described in WO 2006/134340. In particular residues Tyr10 and Tyr13, which are known to contribute to the fibrillation of glucagon (D E Otzen, Biochemistry, 45, 14503-14512, 2006) are replaced by Leu in position 10 and Gln, a non-aromatic polar amino acid, in position 13. This replacement, especially in combination with isoleucine in position 23 and glutamate in position 24, leads to exendin-4 derivatives with potentially improved biophysical properties as solubility or aggregation behaviour in solution. The non-conservative replacement of an aromatic amino acid with a polar amino acid in position 13 of an exendin-4 analogue surprisingly leads to peptides with high activity on the glucagon receptor, keeping their activity on the GLP-1 receptor (see also WO2013/186240.
Furthermore, we surprisingly found that compounds carrying an Aib amino acid in position 27 show reduced activity on the GIP receptor compared to the corresponding derivatives with Lys at position 27 as in native exendin-4, as shown in Example 5, Table 8. A reduced activation of the GIP receptor is potentially beneficial as there are reports in the literature that high levels of GIP in diabetics might in some cases lead to more frequent episodes of hypoglycemia (T McLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983).
Furthermore, compounds of this invention are exendin-4 derivatives with fatty acid acylated residues in position 14. This fatty acid functionalization in position 14 resulted in exendin-4 derivatives with high activity not only at the GLP-1 receptor, but also at the glucagon receptor, when compared to the corresponding non-acylated exendin-4 derivatives, for example those shown in Example 5, Table 7. In addition, this modification results in an improved pharmacokinetic profile.
It is described in the literature (Murage E N et al., Bioorg. Med. Chem. 16 (2008), 10106-10112), that a GLP-1 analogue with an acetylated lysine at position 14 showed significantly reduced potency on the GLP-1 receptor compared to natural GLP-1.
Compounds of this invention are more resistant to cleavage by neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP4), resulting in a longer half-life and duration of action in vivo, when compared with native GLP-1 and glucagon.
Compounds of this invention preferably are soluble not only at neutral pH, but also at pH 4.5. This property potentially allows co-formulation for a combination therapy with an insulin or insulin derivative and preferably with a basal insulin like insulin glargine/Lantus®.
Native exendin-4 is a pure GLP-1 receptor agonist without activity on the glucagon receptor and low activity on the GIP receptor. Provided herein are exendin-4 derivatives based on the structure of native exendin-4 but differing at ten or more positions as compared to SEQ ID NO: 1 wherein the differences contribute to the enhancement of the agonistic activity at the glucagon receptor. Among other substitutions—methionine at position 14 is replaced by an amino acid carrying an —NH2 group in the side-chain, which is further substituted by a lipophilic residue (e.g. a fatty acid combined with a linker). Furthermore, we surprisingly found that a replacement of the lysine at position 27 by Aib leads to reduced GIP receptor activity compared to the GLP-1 receptor activity. A reduced activation of the GIP receptor is potentially beneficial as there are reports in the literature that high levels of GIP in diabetics might in some cases lead to more frequent episodes of hypoglycemia (T McLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983).
The invention provides a peptidic compound having the formula (I):
The compounds of the invention are GLP-1 and glucagon receptor agonists as determined by the observation that they are capable of stimulating intracellular cAMP formation in the assay system described in Methods.
According to another embodiment the compounds of the invention, particularly with a lysine at position 14 which is further substituted with a lipophilic residue, exhibit at least a relative activity of 0.1% (i.e. EC50<700 pM), more preferably of 1% (i.e. EC50<70 pM), more preferably of 5% (i.e. EC50<14 pM) and even more preferably of 10% (i.e. EC50<7 pM) compared to that of GLP-1(7-36)amide at the GLP-1 receptor. Furthermore, the compounds exhibit at least a relative activity of 0.1% (i.e. EC50<1000 pM), more preferably of 0.5% (i.e. EC50<200 pM) and even more preferably of 1% (i.e. EC50<100 pM) compared to that of natural glucagon at the glucagon receptor.
The term “activity” as used herein preferably refers to the capability of a compound to activate the human GLP-1 receptor and the human glucagon receptor. More preferably the term “activity” as used herein refers to the capability of a compound to stimulate intracellular cAMP formation. The term “relative activity” as used herein is understood to refer to the capability of a compound to activate a receptor in a certain ratio as compared to another receptor agonist or as compared to another receptor. The activation of the receptors by the agonists (e.g. by measuring the cAMP level) is determined as described herein, e.g. as described in the Example 4.
The compounds of the invention preferably have an EC50 for hGLP-1 receptor of 450 pmol or less, preferably of 200 pmol or less, more preferably of 150 pmol or less, more preferably of 100 pmol or less, more preferably of 75 pmol or less, more preferably of 50 pmol or less, more preferably of 25 pmol or less, more preferably of 15 pmol or less, more preferably of 10 pmol and more preferably of 5 pmol or less and/or an EC50 for hGlucagon receptor of 500 pmol or less, preferably of 200 pmol or less, more preferably of 150 pmol or less, more preferably of 100 pmol or less, more preferably of 75 pmol or less and/or an EC50 for hGIP receptor of 250 pmol or more, preferably of 500 pmol or more; more preferably of 1000 pmol or more. It is particularly preferred that the EC50 for both hGLP-1 and hGlucagon receptors is 250 pm or less, more preferably of 200 pmol or less, more preferably of 150 pmol or less, more preferably of 100 pmol or less, more preferably of 60 pmol or less. The EC50 for the hGLP-1 receptor, the hGlucagon receptor and the hGIP receptor may be determined as described in the Methods herein and as used to generate the results described in Example 4.
The compounds of the invention have the ability to reduce the intestinal passage, increase the gastric content and/or to reduce the food intake of a patient. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and also described herein in the Methods.
The compounds of the invention have the ability to reduce blood glucose level, and/or to reduce HbA1c levels of a patient. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and also described herein in the Methods.
The compounds of the invention have the ability to reduce body weight of a patient. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and also described herein in the Methods and in Examples 7 and 8.
It was found that peptidic compounds of the formula (I) particularly those with a lysine at position 14 which is further substituted with a lipophilic residue, showed increased glucagon receptor activation compared to derivatives having the original methionine (from exendin-4) or leucine at position 14 (see Table 7). Furthermore, oxidation (in vitro or in vivo) of methionine is not possible anymore.
It was also found that compounds carrying an Aib amino acid in position 27 show reduced activity on the GIP receptor compared to the corresponding derivatives with Lys at position 27 as in native exendin-4, as shown in Example 5, Table 8. A reduced activation of the GIP receptor is potentially beneficial as there are reports in the literature that high levels of GIP in diabetics might in some cases lead to more frequent episodes of hypoglycemia (T McLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983).
In one embodiment the compounds of the invention have a high solubility at acidic and/or physiological pH values, e.g., at pH 4.5 and/or at pH 7.4 at 25° C., in another embodiment at least 0.5 mg/ml and in a particular embodiment at least 1 mg/ml.
Furthermore, the compounds of the invention preferably have a high stability when stored in solution. Preferred assay conditions for determining the stability is storage for 7 days at 40° C. in solution at pH 4.5 or pH 7.4. The remaining amount of peptide is determined by chromatographic analyses as described in the Examples. Preferably, after 7 days at 40° C. in solution at pH 4.5 or pH 7.4 the remaining peptide is at least 70%, more preferably at least 75%, even more preferably at least 80%.
Preferably, the compounds of the present invention comprise a peptide moiety which is a linear sequence of 39 amino carboxylic acids, particularly α-amino carboxylic acids linked by peptide, i.e. carboxamide bonds.
In a further embodiment, R1 is NH2 and in a further embodiment R1 is OH.
Specific preferred examples for —Z—C(O)—R5 groups are listed in the following Table 2, which are selected from (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-(17-carboxy-heptadecanoyl)amino-butyrylamino]-ethoxy]-ethoxy)-acetylamino}-ethoxy}-ethoxy)-acetyl.
Further preferred are stereoisomers, particularly enantiomers of these groups, either S- or R-enantiomers. The term “R” in Table 2 is intended to mean the attachment site of —Z—C(O)—R5 at the peptide back bone, i.e. particularly the ε-amino group of Lys.
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A further embodiment relates to a group of compounds, wherein
A still further embodiment relates to a group of compounds, wherein
Specific examples of peptidic compounds of formula (I) are the compounds of SEQ ID NO: 6-27, as well as salts or solvates thereof.
Specific examples of peptidic compounds of formula (I) are the compounds of SEQ ID NO: 6-22 and 24-27, as well as salts or solvates thereof.
Specific examples of peptidic compounds of formula (I) are the compounds of SEQ ID NO: 9, 12 and 15 as well as salts or solvates thereof.
In certain embodiments, i.e. when the compound of formula (I) comprises genetically encoded amino acid residues, the invention further provides a nucleic acid (which may be DNA or RNA) encoding said compound, an expression vector comprising such a nucleic acid, and a host cell containing such a nucleic acid or expression vector.
In a further aspect, the present invention provides a composition comprising a compound of the invention in admixture with a carrier. In preferred embodiments, the composition is a pharmaceutically acceptable composition and the carrier is a pharmaceutically acceptable carrier. The compound of the invention may be in the form of a salt, e.g. a pharmaceutically acceptable salt or a solvate, e.g. a hydrate. In still a further aspect, the present invention provides a composition for use in a method of medical treatment, particularly in human medicine.
In certain embodiments, the nucleic acid or the expression vector may be used as therapeutic agents, e.g. in gene therapy.
The compounds of formula (I) are suitable for therapeutic application without an additional therapeutically effective agent. In other embodiments, however, the compounds are used together with at least one additional therapeutically active agent, as described in “combination therapy”.
The compounds of formula (I) are particularly suitable for the treatment or prevention of diseases or disorders caused by, associated with and/or accompanied by disturbances in carbohydrate and/or lipid metabolism, e.g. for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity and metabolic syndrome. Further, the compounds of the invention are particularly suitable for the treatment or prevention of degenerative diseases, particularly neurodegenerative diseases.
The compounds described find use, inter alia, in preventing weight gain or promoting weight loss. By “preventing” is meant inhibiting or reducing when compared to the absence of treatment, and is not necessarily meant to imply complete cessation of a disorder.
The compounds of the invention may cause a decrease in food intake and/or increase in energy expenditure, resulting in the observed effect on body weight.
Independently of their effect on body weight, the compounds of the invention may have a beneficial effect on circulating cholesterol levels, being capable of improving lipid levels, particularly LDL, as well as HDL levels (e.g. increasing HDL/LDL ratio).
Thus, the compounds of the invention can be used for direct or indirect therapy of any condition caused or characterised by excess body weight, such as the treatment and/or prevention of obesity, morbid obesity, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea. They may also be used for treatment and prevention of the metabolic syndrome, diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, or stroke. Their effects in these conditions may be as a result of or associated with their effect on body weight, or may be independent thereof.
Preferred medical uses include delaying or preventing disease progression in type 2 diabetes, treating metabolic syndrome, treating obesity or preventing overweight, for decreasing food intake, increase energy expenditure, reducing body weight, delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes; delaying the progression from type 2 diabetes to insulin-requiring diabetes; regulating appetite; inducing satiety; preventing weight regain after successful weight loss; treating a disease or state related to overweight or obesity; treating bulimia; treating binge eating; treating atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, hepatic steatosis, treatment of beta-blocker poisoning, use for inhibition of the motility of the gastrointestinal tract, useful in connection with investigations of the gastrointestinal tract using techniques such as X-ray, CT- and NMR-scanning.
Further preferred medical uses include treatment or prevention of degenerative disorders, particularly neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ataxia, e.g spinocerebellar ataxia, Kennedy disease, myotonic dystrophy, Lewy body dementia, multi-systemic atrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, spinal muscular atrophy, prion-associated diseases, e.g. Creutzfeldt-Jacob disease, multiple sclerosis, telangiectasia, Batten disease, corticobasal degeneration, subacute combined degeneration of spinal cord, Tabes dorsalis, Tay-Sachs disease, toxic encephalopathy, infantile Refsum disease, Refsum disease, neuroacanthocytosis, Niemann-Pick disease, Lyme disease, Machado-Joseph disease, Sandhoff disease, Shy-Drager syndrome, wobbly hedgehog syndrome, proteopathy, cerebral β-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma, synucleinopathies, tauopathies, frontotemporal lobar degeneration (FTLD), dementia, cadasil syndrome, hereditary cerebral hemorrhage with amyloidosis, Alexander disease, seipinopathies, familial amyloidotic neuropathy, senile systemic amyloidosis, serpinopathies, AL (light chain) amyloidosis (primary systemic amyloidosis), AH (heavy chain) amyloidosis, AA (secondary) amyloidosis, aortic medial amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finnish type (FAF), Lysozyme amyloidosis, Fibrinogen amyloidosis, Dialysis amyloidosis, Inclusion body myositis/myopathy, Cataracts, Retinitis pigmentosa with rhodopsin mutations, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, Hereditary lattice corneal dystrophy, Cutaneous lichen amyloidosis, Mallory bodies, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic (Pindborg) tumor amyloid, cystic fibrosis, sickle cell disease or critical illness myopathy (CIM).
The amino acid sequences of the present invention contain the conventional one letter and three letter codes for naturally occurring amino acids, as well as generally accepted three letter codes for other amino acids, such as Aib (α-aminoisobutyric acid).
The term, native exendin-4″ refers to native exendin-4 having the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 1).
The invention provides peptidic compounds as defined above.
The peptidic compounds of the present invention comprise a linear backbone of amino carboxylic acids linked by peptide, i.e. carboxamide bonds. Preferably, the amino carboxylic acids are α-amino carboxylic acids and more preferably L-α-amino carboxylic acids, unless indicated otherwise. The peptidic compounds preferably comprise a backbone sequence of 39 amino carboxylic acids.
The peptidic compounds of the present invention may have unmodified side-chains, but carry at least one modification at one of the side chains.
For the avoidance of doubt, in the definitions provided herein, it is generally intended that the sequence of the peptidic moiety (I) differs from native exendin-4 at least at one of those positions which are stated to allow variation. Amino acids within the peptide moiety (I) can be considered to be numbered consecutively from 1 to 39 in the conventional N-terminal to C-terminal direction. Reference to a “position” within peptidic moiety (I) should be constructed accordingly, as should reference to positions within native exendin-4 and other molecules, e.g., in exendin-4, His is at position 1, Gly at position 2, . . . , Met at position 14, . . . and Ser at position 39.
An amino acid residue with an —NH2 side chain group, e.g. Lys, Orn, Dab or Dap, is functionalized in that at least one H atom of the —NH2 side chain group is replaced by —Z—C(O)—R5, wherein R5 comprises a lipophilic moiety, e.g. an acyclic linear or branched (C8-C30) saturated or unsaturated hydrocarbon group, which is unsubstituted or substituted e.g. by halogen, —OH and/or CO2H and Z comprises a linker in all stereoisomeric forms, e.g. a linker comprising one or more, e.g. 1 to 5, preferably 1, 2 or 3 amino acid linker groups selected from the group of γ-Glutamate (γE) and AEEAc. Preferred groups R5 comprise a lipophilic moiety, e.g. an acyclic linear or branched (C12-C20) saturated or unsaturated hydrocarbon group, e.g. pentadecanyl, hexadecanyl or heptadecanyl, which is unsubstituted or substituted by CO2H, more preferably pentadecanyl, heptadecanyl or 16-carboxy-hexadecanyl. In one embodiment amino acid linker groups are selected from γE, γE-γE, AEEAc-AEEAc-γE and AEEAc-AEEAc-AEEAc. In another embodiment the amino acid linker group is γE. In another embodiment the amino acid linker group is γE-γE. In another embodiment the amino acid linker group is AEEAc-AEEAc-γE. In another embodiment the amino acid linker group is AEEAc-AEEAc-AEEAc.
In a further aspect, the present invention provides a composition comprising a compound of the invention as described herein, or a salt and/or solvate thereof, in admixture with a carrier.
The invention also provides the use of a compound of the present invention for use as a medicament, particularly for the treatment of a condition as described below.
The invention also provides a composition wherein the composition is a pharmaceutically acceptable composition, and the carrier is a pharmaceutically acceptable carrier.
Peptide Synthesis
The skilled person is aware of a variety of different methods to prepare peptides that are described in this invention. These methods include but are not limited to synthetic approaches and recombinant gene expression. Thus, one way of preparing these peptides is the synthesis in solution or on a solid support and subsequent isolation and purification. A different way of preparing the peptides is gene expression in a host cell in which a DNA sequence encoding the peptide has been introduced. Alternatively, the gene expression can be achieved without utilizing a cell system. The methods described above may also be combined in any way.
A preferred way to prepare the peptides of the present invention is solid phase synthesis on a suitable resin. Solid phase peptide synthesis is a well-established methodology (see for example: Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984; E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989). Solid phase synthesis is initiated by attaching an N-terminally protected amino acid with its carboxy terminus to an inert solid support carrying a cleavable linker. This solid support can be any polymer that allows coupling of the initial amino acid, e.g. a trityl resin, a chlorotrityl resin, a Wang resin or a Rink resin in which the linkage of the carboxy group (or carboxamide for Rink resin) to the resin is sensitive to acid (when Fmoc strategy is used). The polymer support must be stable under the conditions used to deprotect the α-amino group during the peptide synthesis.
After the first amino acid has been coupled to the solid support, the α-amino protecting group of this amino acid is removed. The remaining protected amino acids are then coupled one after the other in the order represented by the peptide sequence using appropriate amide coupling reagents, for example BOP, HBTU, HATU or DIC (N,N′-diisopropylcarbodiimide)/HOBt (1-hydroxybenzotriazole), wherein BOP, HBTU and HATU are used with tertiary amine bases. Alternatively, the liberated N-terminus can be functionalized with groups other than amino acids, for example carboxylic acids, etc.
Usually, reactive side-chain groups of the amino acids are protected with suitable blocking groups. These protecting groups are removed after the desired peptides have been assembled. They are removed concomitantly with the cleavage of the desired product from the resin under the same conditions. Protecting groups and the procedures to introduce protecting groups can be found in Protective Groups in Organic Synthesis, 3d ed., Greene, T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1999).
In some cases it might be desirable to have side-chain protecting groups that can selectively be removed while other side-chain protecting groups remain intact. In this case the liberated functionality can be selectively functionalized. For example, a lysine may be protected with an ivDde ([1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl) protecting group (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603) which is labile to a very nucleophilic base, for example 4% hydrazine in DMF (dimethyl formamide). Thus, if the N-terminal amino group and all side-chain functionalities are protected with acid labile protecting groups, the ivDde group can be selectively removed using 4% hydrazine in DMF and the corresponding free amino group can then be further modified, e.g. by acylation. The lysine can alternatively be coupled to a protected amino acid and the amino group of this amino acid can then be deprotected resulting in another free amino group which can be acylated or attached to further amino acids.
Finally the peptide is cleaved from the resin. This can be achieved by using King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The raw material can then be purified by chromatography, e.g. preparative RP-HPLC, if necessary.
Potency
As used herein, the term “potency” or “in vitro potency” is a measure for the ability of a compound to activate the receptors for GLP-1, glucagon or GIP in a cell-based assay. Numerically, it is expressed as the “EC50 value”, which is the effective concentration of a compound that induces a half maximal increase of response (e.g. formation of intracellular cAMP) in a dose-response experiment.
Therapeutic Uses
Metabolic syndrome is a combination of medical disorders that, when occurring together, increase the risk of developing type 2 diabetes, as well as atherosclerotic vascular disease, e.g. heart disease and stroke. Defining medical parameters for the metabolic syndrome include diabetes mellitus, impaired glucose tolerance, raised fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol and reduced HDL cholesterol.
Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health and life expectancy and due to its increasing prevalence in adults and children it has become one of the leading preventable causes of death in modern world. It increases the likelihood of various other diseases, including heart disease, type 2 diabetes, obstructive sleep apnoe, certain types of cancer, as well as osteoarthritis, and it is most commonly caused by a combination of excess food intake, reduced energy expenditure, as well as genetic susceptibility.
Diabetes mellitus, often simply called diabetes, is a group of metabolic diseases in which a person has high blood sugar levels, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. The most common types of diabetes are: (1) type 1 diabetes, where the body fails to produce insulin; (2) type 2 diabetes, where the body fails to use insulin properly, combined with an increase in insulin deficiency over time, and (3) gestational diabetes, where women develop diabetes due to their pregnancy. All forms of diabetes increase the risk of long-term complications, which typically develop after many years. Most of these long-term complications are based on damage to blood vessels and can be divided into the two categories “macrovascular” disease, arising from atherosclerosis of larger blood vessels and “microvascular” disease, arising from damage of small blood vessels. Examples for macrovascular disease conditions are ischemic heart disease, myocardial infarction, stroke and peripheral vascular disease. Examples for microvascular diseases are diabetic retinopathy, diabetic nephropathy, as well as diabetic neuropathy.
The receptors for GLP-1 and GIP as well as glucagon are members of the family of 7-transmembrane-spanning, heterotrimeric G-protein coupled receptors. They are structurally related to each other and share not only a significant level of sequence identity, but have also similar mechanisms of ligand recognition and intracellular signaling pathways.
Similarly, the peptides GLP-1, GIP and glucagon share regions of high sequence identity/similarity. GLP-1 and glucagon are produced from a common precursor, preproglucagon, which is differentially processed in a tissue-specific manner to yield e.g. GLP-1 in intestinal endocrine cells and glucagon in alpha cells of pancreatic islets. GIP is derived from a larger proGIP prohormone precursor and is synthesized and released from K-cells located in the small intestine.
The peptidic incretin hormones GLP-1 and GIP are secreted by intestinal endocrine cells in response to food and account for up to 70% of meal-stimulated insulin secretion. Evidence suggests that GLP-1 secretion is reduced in subjects with impaired glucose tolerance or type 2 diabetes, whereas responsiveness to GLP-1 is still preserved in these patients. Thus, targeting of the GLP-1 receptor with suitable agonists offers an attractive approach for treatment of metabolic disorders, including diabetes. The receptor for GLP-1 is distributed widely, being found mainly in pancreatic islets, brain, heart, kidney and the gastrointestinal tract. In the pancreas, GLP-1 acts in a strictly glucose-dependent manner by increasing secretion of insulin from beta cells. This glucose-dependency shows that activation of GLP-1 receptors is unlikely to cause hypoglycemia. Also the receptor for GIP is broadly expressed in peripheral tissues including pancreatic islets, adipose tissue, stomach, small intestine, heart, bone, lung, kidney, testis, adrenal cortex, pituitary, endothelial cells, trachea, spleen, thymus, thyroid and brain. Consistent with its biological function as incretin hormone, the pancreatic B-cell express the highest levels of the receptor for GIP in humans. There is some clinical evidence that the GIP-receptor mediated signaling could be impaired in patients with T2DM but GIP-action is shown to be reversible and can be restored with improvement of the diabetic status. While there are many reports that also GIP action on insulin secretion is glucose-dependent, there are also reports in the literature that high plasma levels of GIP might lead to more frequent episodes of hypoglycemia (T McLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983). In addition, plasma GIP levels in obese subjects were reported to be higher than normal, suggesting that GIP might induce obesity and insulin resistance (W Creutzfeldt et al. Diabetologia. 1978, 14, 15-24). This is supported by reports that the ablation of the GIP receptor might prevent those conditions: GIP receptor knock-out mice fed on high-fat diet actually showed a suppression of body weight compared to wild-type mice (K Miyawaki et al. Nat Med. 2002, 8, 738-42), and long-term administration of the GIP receptor antagonist (Pro3)GIP also prevented obesity and insulin resistance in mice (V A Gault et al. Diabetologia. 2007, 50, 1752-62). Therefore, goal of this invention was to provide dual GLP-1/glucagon receptor agonists with reduced activity on the GIP receptor.
Glucagon is a 29 amino acid peptide hormone that is produced by pancreatic alpha cells and released into the bloodstream when circulating glucose is low. An important physiological role of glucagon is to stimulate glucose output in the liver, which is a process providing the major counterregulatory mechanism for insulin in maintaining glucose homeostasis in vivo.
Glucagon receptors are however also expressed in extra-hepatic tissues such as kidney, heart, adipocytes, lymphoblasts, brain, retina, adrenal gland and gastrointestinal tract, suggesting a broader physiological role beyond glucose homeostasis. Accordingly, recent studies have reported that glucagon has therapeutically positive effects on energy management, including stimulation of energy expenditure and thermogenesis, accompanied by reduction of food intake and body weight loss. Altogether, stimulation of glucagon receptors might be useful in the treatment of obesity and the metabolic syndrome.
Oxyntomodulin is a peptide hormone consisting of glucagon with an eight amino acids encompassing C-terminal extension. Like GLP-1 and glucagon, it is pre-formed in preproglucagon and cleaved and secreted in a tissue-specific manner by endocrinal cells of the small bowel. Oxyntomodulin is known to stimulate both, the receptors for GLP-1 and glucagon and is therefore the prototype of a dual agonist.
As GLP-1 is known for its anti-diabetic effects, GLP-1 and glucagon are both known for their food intake-suppressing effects and glucagon is also a mediator of additional energy expenditure, it is conceivable that a combination of the activities of the two hormones in one molecule can yield a powerful medication for treatment of the metabolic syndrome and in particular its components diabetes and obesity.
Accordingly, the compounds of the invention may be used for treatment of glucose intolerance, insulin resistance, pre-diabetes, increased fasting glucose, hyperglycemia, type 2 diabetes, hypertension, dyslipidemia, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or any combination of these individual disease components.
In addition, they may be used for control of appetite, feeding and calory intake, increase of energy expenditure, prevention of weight gain, promotion of weight loss, reduction of excess body weight and altogether treatment of obesity, including morbid obesity.
The compounds of the invention are agonists for the receptors for GLP-1 and for glucagon (e.g. “dual agonists”) with reduced activity on the GIP receptor and may provide therapeutic benefit to address a clinical need for targeting the metabolic syndrome by allowing simultaneous treatment of diabetes and obesity.
Further disease states and health conditions which could be treated with the compounds of the invention are obesity-linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea.
Although all these conditions could be associated directly or indirectly with obesity, the effects of the compounds of the invention may be mediated in whole or in part via an effect on body weight, or independent thereof.
Further, diseases to be treated are neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, or other degenerative diseases as described above.
In one embodiment the compounds are useful in the treatment or prevention of hyperglycemia, type 2 diabetes, obesity.
Compared to GLP-1, glucagon and oxyntomodulin, exendin-4 has beneficial physicochemical properties, such as solubility and stability in solution and under physiological conditions (including enzymatic stability towards degradation by enzymes, such as DPP4 or NEP), which results in a longer duration of action in vivo. Therefore, the pure GLP-1 receptor agonist exendin-4 might serve as good starting scaffold to obtain exendin-4 analogs with dual GLP-1/glucagon receptor agonism.
Nevertheless, also exendin-4 has been shown to be chemically labile due to methionine oxdiation in position 14 as well as deamidation and isomerization of asparagine in position 28. Therefore, stability might be further improved by substitution of methionine at position 14 and the avoidance of sequences that are known to be prone to degradation via aspartimide formation, especially Asp-Gly or Asn-Gly at positions 28 and 29.
Pharmaceutical Compositions
The term “pharmaceutical composition” indicates a mixture containing ingredients that are compatible when mixed and which may be administered. A pharmaceutical composition may include one or more medicinal drugs. Additionally, the pharmaceutical composition may include carriers, buffers, acidifying agents, alkalizing agents, solvents, adjuvants, tonicity adjusters, emollients, expanders, preservatives, physical and chemical stabilizers e.g. surfactants, antioxidants and other components, whether these are considered active or inactive ingredients. Guidance for the skilled in preparing pharmaceutical compositions may be found, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins and in R. C. Rowe et al (Ed), Handbook of Pharmaceutical Excipients, PhP, May 2013 update.
The exendin-4 peptide derivatives of the present invention, or salts thereof, are administered in conjunction with an acceptable pharmaceutical carrier, diluent, or excipient as part of a pharmaceutical composition. A “pharmaceutically acceptable carrier” is a carrier which is physiologically acceptable (e.g. physiologically acceptable pH) while retaining the therapeutic properties of the substance with which it is administered. Standard acceptable pharmaceutical carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins and in R. C. Rowe et al (Ed), Handbook of Pharmaceutical excipients, PhP, May 2013 update. One exemplary pharmaceutically acceptable carrier is physiological saline solution.
In one embodiment carriers are selected from the group of buffers (e.g. citrate/citric acid), acidifying agents (e.g. hydrochloric acid), alkalizing agents (e.g. sodium hydroxide), preservatives (e.g. phenol), co-solvents (e.g. polyethylene glycol 400), tonicity adjusters (e.g. mannitol), stabilizers (e.g. surfactant, antioxidants, amino acids).
Concentrations used are in a range that is physiologically acceptable.
Acceptable pharmaceutical carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The compounds of the present invention will typically be administered parenterally.
The term “pharmaceutically acceptable salt” means salts of the compounds of the invention which are safe and effective for use in mammals. Pharmaceutically acceptable salts may include, but are not limited to, acid addition salts and basic salts. Examples of acid addition salts include chloride, sulfate, hydrogen sulfate, (hydrogen)phosphate, acetate, citrate, tosylate or mesylate salts. Examples of basic salts include salts with inorganic cations, e.g. alkaline or alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts and salts with organic cations such as amine salts. Further examples of pharmaceutically acceptable salts are described in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins or in Handbook of Pharmaceutical Salts, Properties, Selection and Use, e.d. P. H. Stahl, C. G. Wermuth, 2002, jointly published by Verlag Helvetica Chimica Acta, Zurich, Switzerland, and Wiley-VCH, Weinheim, Germany.
The term “solvate” means complexes of the compounds of the invention or salts thereof with solvent molecules, e.g. organic solvent molecules and/or water.
In the pharmaceutical composition, the exendin-4 derivative can be in monomeric or oligomeric form.
The term “therapeutically effective amount” of a compound refers to a nontoxic but sufficient amount of the compound to provide the desired effect. The amount of a compound of the formula (I) necessary to achieve the desired biological effect depends on a number of factors, for example the specific compound chosen, the intended use, the mode of administration and the clinical condition of the patient. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. For example the “therapeutically effective amount” of a compound of the formula (I) is about 0.01 to 50 mg/dose, preferably 0.1 to 10 mg/dose.
Pharmaceutical compositions of the invention are those suitable for parenteral (for example subcutaneous, intramuscular, intradermal or intravenous), oral, rectal, topical and peroral (for example sublingual) administration, although the most suitable mode of administration depends in each individual case on the nature and severity of the condition to be treated and on the nature of the compound of formula (I) used in each case.
Suitable pharmaceutical compositions may be in the form of separate units, for example capsules, tablets and powders in vials or ampoules, each of which contains a defined amount of the compound; as powders or granules; as solution or suspension in an aqueous or nonaqueous liquid; or as an oil-in-water or water-in-oil emulsion. It may be provided in single or multiple dose injectable form, for example in the form of a pen. The compositions may, as already mentioned, be prepared by any suitable pharmaceutical method which includes a step in which the active ingredient and the carrier (which may consist of one or more additional ingredients) are brought into contact.
In certain embodiments the pharmaceutical composition may be provided together with a device for application, for example together with a syringe, an injection pen or an autoinjector. Such devices may be provided separate from a pharmaceutical composition or prefilled with the pharmaceutical composition.
Combination Therapy
The compounds of the present invention, dual agonists for the GLP-1 and glucagon receptors, can be widely combined with other pharmacologically active compounds, such as all drugs mentioned in the Rote Liste 2014, e.g. with all weight-reducing agents or appetite suppressants mentioned in the Rote Liste 2014, chapter 1, all lipid-lowering agents mentioned in the Rote Liste 2014, chapter 58, all antihypertensives and nephroprotectives, mentioned in the Rote Liste 2014, or all diuretics mentioned in the Rote Liste 2014, chapter 36.
The active ingredient combinations can be used especially for a synergistic improvement in action. They can be applied either by separate administration of the active ingredients to the patient or in the form of combination products in which a plurality of active ingredients are present in one pharmaceutical preparation. When the active ingredients are administered by separate administration of the active ingredients, this can be done simultaneously or successively.
Most of the active ingredients mentioned hereinafter are disclosed in the USP Dictionary of USAN and International Drug Names, US Pharmacopeia, Rockville 2011.
Other active substances which are suitable for such combinations include in particular those which for example potentiate the therapeutic effect of one or more active substances with respect to one of the indications mentioned and/or which allow the dosage of one or more active substances to be reduced.
Therapeutic agents which are suitable for combinations include, for example, antidiabetic agents such as:
Insulin and Insulin derivatives, for example: Glargine/Lantus®, 270-330 U/mL of insulin glargine (EP 2387989 A), 300 U/mL of insulin glargine (EP 2387989 A), Glulisin/Apidra®, Detemir/Levemir®, Lispro/Humalog®/Liprolog®, Degludec/DegludecPlus, Aspart, basal insulin and analogues (e.g. LY-2605541, LY2963016, NN1436), PEGylated insulin Lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin plus Symlin, PE0139, fast-acting and short-acting insulins (e.g. Linjeta, PH20, NN1218, HinsBet), (APC-002)hydrogel, oral, inhalable, transdermal and sublingual insulins (e.g. Exubera®, Nasulin®, Afrezza, Tregopil, TPM 02, Capsulin, Oral-lyn®, Cobalamin® oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, Oshadi oral insulin). Additionally included are also those insulin derivatives which are bonded to albumin or another protein by a bifunctional linker.
GLP-1, GLP-1 analogues and GLP-1 receptor agonists, for example: Lixisenatide/AVE0010/ZP10/Lyxumia, Exenatide/Exendin-4/Byetta/Bydureon/ITCA 650/AC-2993, Liraglutide/Victoza, Semaglutide, Taspoglutide, Syncria/Albiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide/HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034. MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, Exenatide-XTEN and Glucagon-Xten.
DPP4 inhibitors, for example: Alogliptin/Nesina, Trajenta/Linagliptin/BI-1356/Ondero/Trajenta/Tradjenta/Trayenta/Tradzenta, Saxagliptin/Onglyza, Sitagliptin/Januvia/Xelevia/Tesave/Janumet/Velmetia, Galvus/Vildagliptin, Anagliptin, Gemigliptin, Teneligliptin, Melogliptin, Trelagliptin, DA-1229, Omarigliptin/MK-3102, KM-223, Evogliptin, ARI-2243, PBL-1427, Pinoxacin.
SGLT2 inhibitors, for example: Invokana/Canaglifozin, Forxiga/Dapagliflozin, Remoglifozin, Sergliflozin, Empagliflozin, Ipragliflozin, Tofogliflozin, Luseogliflozin, LX-4211, Ertuglifozin/PF-04971729, RO-4998452, EGT-0001442, KGA-3235/DSP-3235, LIK066, SBM-TFC-039,
Biguanides (e.g. Metformin, Buformin, Phenformin), Thiazolidinediones (e.g. Pioglitazone, Rivoglitazone, Rosiglitazone, Troglitazone), dual PPAR agonists (e.g. Aleglitazar, Muraglitazar, Tesaglitazar), Sulfonylureas (e.g. Tolbutamide, Glibenclamide, Glimepiride/Amaryl, Glipizide), Meglitinides (e.g. Nateglinide, Repaglinide, Mitiglinide), Alpha-glucosidase inhibitors (e.g. Acarbose, Miglitol, Voglibose), Amylin and Amylin analogues (e.g. Pramlintide, Symlin).
GPR119 agonists (e.g. GSK-263A, PSN-821, MBX-2982, APD-597, ZYG-19, DS-8500), GPR40 agonists (e.g. Fasiglifam/TAK-875, TUG-424, P-1736, JTT-851, GW9508).
Other suitable combination partners are: Cycloset, inhibitors of 11-beta-HSD (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585), activators of glucokinase (e.g. TTP-399, AMG-151, TAK-329, GKM-001), inhibitors of DGAT (e.g. LCQ-908), inhibitors of protein tyrosinephosphatase 1 (e.g. Trodusquemine), inhibitors of glucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrokinase, alpha2-antagonists, CCR-2 antagonists, SGLT-1 inhibitors (e.g. LX-2761), dual SGLT2/SGLT1 inhibitors.
One or more lipid lowering agents are also suitable as combination partners, such as for example: HMG-CoA-reductase inhibitors (e.g. Simvastatin, Atorvastatin), fibrates (e.g. Bezafibrate, Fenofibrate), nicotinic acid and the derivatives thereof (e.g. Niacin), PPAR-(alpha, gamma or alpha/gamma) agonists or modulators (e.g. Aleglitazar), PPAR-delta agonists, ACAT inhibitors (e.g. Avasimibe), cholesterol absorption inhibitors (e.g. Ezetimibe), Bile acid-binding substances (e.g. Cholestyramine), ileal bile acid transport inhibitors, MTP inhibitors, or modulators of PCSK9.
HDL-raising compounds such as: CETP inhibitors (e.g. Torcetrapib, Anacetrapid, Dalcetrapid, Evacetrapid, JTT-302, DRL-17822, TA-8995) or ABC1 regulators.
Other suitable combination partners are one or more active substances for the treatment of obesity, such as for example: Sibutramine, Tesofensine, Orlistat, antagonists of the cannabinoid-1 receptor, MCH-1 receptor antagonists, MC4 receptor agonists, NPY5 or NPY2 antagonists (e.g. Velneperit), beta-3-agonists, leptin or leptin mimetics, agonists of the 5HT2c receptor (e.g. Lorcaserin), or the combinations of bupropione/naltrexone, bupropione/zonisamide, bupropione/phentermine or pramlintide/metreleptin.
Other suitable combination partners are:
Further gastrointestinal peptides such as Peptide YY 3-36 (PYY3-36) or analogues thereof, pancreatic polypeptide (PP) or analogues thereof.
Glucagon receptor agonists or antagonists, GIP receptor agonists or antagonists, ghrelin antagonists or inverse agonists, Xenin and analogues thereof.
Moreover, combinations with drugs for influencing high blood pressure, chronic heart failure or atherosclerosis, such as e.g.: Angiotensin II receptor antagonists (e.g. telmisartan, candesartan, valsartan, losartan, eprosartan, irbesartan, olmesartan, tasosartan, azilsartan), ACE inhibitors, ECE inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, thrombocyte aggregation inhibitors and others or combinations thereof are suitable.
In another aspect, this invention relates to the use of a compound according to the invention or a physiologically acceptable salt thereof combined with at least one of the active substances described above as a combination partner, for preparing a medicament which is suitable for the treatment or prevention of diseases or conditions which can be affected by binding to the receptors for GLP-1 and glucagon and by modulating their activity. This is preferably a disease in the context of the metabolic syndrome, particularly one of the diseases or conditions listed above, most particularly diabetes or obesity or complications thereof.
The use of the compounds according to the invention, or a physiologically acceptable salt thereof, in combination with one or more active substances may take place simultaneously, separately or sequentially.
The use of the compound according to the invention, or a physiologically acceptable salt thereof, in combination with another active substance may take place simultaneously or at staggered times, but particularly within a short space of time. If they are administered simultaneously, the two active substances are given to the patient together; if they are used at staggered times, the two active substances are given to the patient within a period of less than or equal to 12 hours, but particularly less than or equal to 6 hours.
Consequently, in another aspect, this invention relates to a medicament which comprises a compound according to the invention or a physiologically acceptable salt of such a compound and at least one of the active substances described above as combination partners, optionally together with one or more inert carriers and/or diluents.
The compound according to the invention, or physiologically acceptable salt or solvate thereof, and the additional active substance to be combined therewith may both be present together in one formulation, for example a in a vial or a cartridge, or separately in two identical or different formulations, for example as so-called kit-of-parts.
General Synthesis of Peptidic Compounds
Materials
Different Rink-Amide resins (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin, Merck Biosciences; 4-[(2,4-Dimethoxyphenyl)(Fmoc-amino)methyl]phenoxy acetamido methyl resin, Agilent Technologies) were used for the synthesis of peptide amides with loadings in the range of 0.2-0.7 mmol/g.
Fmoc protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, his Biotech, Bachem, Chem-Impex International or MATRIX Innovation. The following standard amino acids were used throughout the syntheses: Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.
In addition, the following special amino acids were purchased from the same suppliers as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mmt)-OH, Fmoc-Aib-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Ala-OH, Boc-L-His(Boc)-OH (available as toluene solvate) and Boc-L-His(Trt)-OH.
The solid phase peptide syntheses were performed for example on a Prelude Peptide Synthesizer (Protein Technologies Inc) or similar automated synthesizer using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as the solvent. Deprotection: 20% piperidine/DMF for 2×2.5 mM Washes: 7×DME Coupling 2:5:10 200 mM AA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DME
In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH or Fmoc-L-Lys(Mmt)-OH was used in the corresponding position. After completion of the synthesis, the ivDde group was removed according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DME The Mmt group was removed by repeated treatment with 1% TFA in dichloromethane. The following acylations were carried out by treating the resin with the N-hydroxy succinimide esters of the desired acid or using coupling reagents like HBTU/DIPEA or HOBt/DIC.
All the peptides that have been synthesized were cleaved from the resin with King's cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% EDT. The crude peptides were then precipitated in diethyl or diisopropyl ether, centrifuged, and lyophilized Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by a conventional preparative RP-HPLC purification procedure.
Alternatively, peptides were synthesized by a manual synthesis procedure: 0.3 g Desiccated Rink amide MBHA Resin (0.66 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter. Resin was swelled in DCM (15 ml) for 1 h and DMF (15 ml) for 1 h. The Fmoc group on the resin was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test (quantitative method) was used for the conformation of removal of Fmoc from solid support. The C-terminal Fmoc-amino acid (5 equiv. excess corresponding to resin loading) in dry DMF was added to the de-protected resin and coupling was initiated with 5 equivalent excess of DIC and HOBT in DME The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). Kaiser test on peptide resin aliquot upon completion of coupling was negative (no colour on the resin). After the first amino acid attachment, the unreacted amino group, if any, in the resin was capped used acetic anhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion of the sequence. After capping, resin was washed with DCM/DMF/DCM/DMF (6/6/6/6 time each). The Fmoc group on the C-terminal amino acid attached peptidyl resin was deprotected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin was washed with DMF/DCM/DMF (6:6:6 time each). The Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.
The remaining amino acids in target sequence on Rink amide MBHA Resin were sequentially coupled using Fmoc AA/DIC/HOBt method using 5 equivalent excess corresponding to resin loading in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). After each coupling step and Fmoc deprotection step, a Kaiser test was carried out to confirm the completeness of the reaction.
After the completion of the linear sequence, the ε-amino group of lysine used as branching point or modification point was deprotected by using 2.5% hydrazine hydrate in DMF for 15 min×2 and washed with DMF/DCM/DMF (6:6:6 time each). The γ-carboxyl end of glutamic acid was attached to the ε-amino group of Lys using Fmoc-Glu(OH)—OtBu with DIC/HOBt method (5 equivalent excess with respect to resin loading) in DMF. The mixture was rotated on a rotor at room temperature for 2 h. The resin was filtered and washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on the glutamic acid was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min (25 ml each). The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.
If the side-chain branching also contains one more γ-glutamic acid, a second Fmoc-Glu(OH)—OtBu used for the attachment to the free amino group of γ-glutamic acid with DIC/HOBt method (5 equivalent excess with respect to resin loading) in DMF. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on the γ-glutamic acid was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min (25 mL). The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.
Palmitic Acid & Stearic Acid attachment to side chains of Glutamic acid: To the free amino group of γ-glutamic acid, palmitic acid or stearic acid (5 equiv.) dissolved in DMF was added and coupling was initiated by the addition of DIC (5 equiv.) and HOBt (5 equiv.) in DMF. The resin was washed with DMF/DCM/DMF (6:6:6 time each).
Final Cleavage of Peptide from the Resin:
The peptidyl resin synthesized by manual synthesis was washed with DCM (6×10 ml), MeOH (6×10 ml) and ether (6×10 ml) and dried in vacuum desiccators overnight. The cleavage of the peptide from the solid support was achieved by treating the peptide-resin with reagent cocktail (80.0% TFA/5% thioanisole/5% phenol/2.5% EDT, 2.5% DMS and 5% DCM) at room temperature for 3 h. Cleavage mixture was collected by filtration and the resin was washed with TFA (2 ml) and DCM (2×5 ml). The excess TFA and DCM was concentrated to small volume under nitrogen and a small amount of DCM (5-10 ml) was added to the residue and evaporated under nitrogen. The process was repeated 3-4 times to remove most of the volatile impurities. The residue was cooled to 0° C. and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the supernatant ether was removed and fresh ether was added to the peptide and re-centrifuged. The crude sample was preparative HPLC purified and lyophilized. The identity of peptide was confirmed by LCMS.
Analytical HPLC/UPLC
Method A: detection at 210-225 nm
Method B: detection at 210-225 nm
General Preparative HPLC Purification Procedure
The crude peptides were purified either on an Akta Purifier System, a Jasco semiprep HPLC System or a Agilent 1100 HPLC system. Preparative RP-C18-HPLC columns of different sizes and with different flow rates were used depending on the amount of crude peptide to be purified. Acetonitrile+0.1% TFA (B) and water+0.1% TFA (A) were employed as eluents. Product-containing fractions were collected and lyophilized to obtain the purified product, typically as TFA salt.
Solubility and Stability-Testing of Exendin-4 Derivatives
Prior to the testing of solubility and stability of a peptide batch, its purity (HPLC-UV) was determined.
For solubility testing, the target concentration was 10 mg pure compound/ml. Therefore, solutions from solid samples were prepared in different buffer systems with a concentration of 10 mg/mL compound based on the previously determined % purity. HPLC-UV was performed after 2 h of gentle agitation from the supernatant, which was obtained by 20 min of centrifugation at 4500 rpm.
The solubility was then determined by comparison of a 0.2 μL-injection with the UV peak areas obtained with a stock solution of the peptide at a concentration of 1.2 mg/mL in DMSO (based on % purity), injecting various volumes ranging from 0.2-2 μl. This analysis also served as starting point (t0) for the stability testing.
For stability testing, an aliquot of the supernatant obtained for solubility was stored for 7 days at 40° C. After that time course, the sample was centrifuged for 20 min at 4500 rpm and 0.2 μL of the supernatant were analysed with HPLC-UV.
For determination of the amount of the remaining peptide, the peak areas of the target compound at t0 and t7 were compared, resulting in “% remaining peptide”, following the equation
% remaining peptide=[(peak area peptide t7)×100]/peak area peptide t0.
The stability is expressed as “% remaining peptide”.
As HPLC/UPLC method Method A has been used, detecting at 214 nm.
In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Efficacy
Agonism of compounds for the receptors was determined by functional assays measuring cAMP response of HEK-293 cell lines stably expressing human GLP-1, GIP or glucagon receptor.
cAMP content of cells was determined using a kit from Cisbio Corp. (cat. no. 62AM4PEC) based on HTRF (Homogenous Time Resolved Fluorescence). For preparation, cells were split into T175 culture flasks and grown overnight to near confluency in medium (DMEM/10% FBS). Medium was then removed and cells washed with PBS lacking calcium and magnesium, followed by proteinase treatment with accutase (Sigma-Aldrich cat. no. A6964). Detached cells were washed and resuspended in assay buffer (1×HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cellular density determined. They were then diluted to 400000 cells/ml and 25 μl-aliquots dispensed into the wells of 96-well plates. For measurement, 25 μl of test compound in assay buffer was added to the wells, followed by incubation for 30 minutes at room temperature. After addition of HTRF reagents diluted in lysis buffer (kit components), the plates were incubated for 1 hr, followed by measurement of the fluorescence ratio at 665/620 nm. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC50).
Bioanalytical Screening Method for Quantification of Exendin-4 Derivatives in Mice and Pigs
Mice were dosed 1 mg/kg subcutaneously (s.c.). The mice were sacrificed and blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours post application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-life were calculated using WinonLin Version 5.2.1 (non-compartment model).
Female Göttinger minipigs were dosed 0.1 mg/kg subcutaneously (s.c.). Blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 24, 32, 48, 56 and 72 hours post application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-life were calculated using WinonLin Version 5.2.1 (non-compartment model).
Gastric Emptying and Intestinal Passage in Mice
Female NMRI-mice of a body weight between 20 and 30 g are used. Mice are adapted to housing conditions for at least one week.
Mice are overnight fasted, while water remains available all the time. On the study day, mice are weighed, single-caged and allowed access to 500 mg of feed for 30 min, while water is removed. At the end of the 30 min feeding period, remaining feed is removed and weighed. 60 min later, a coloured, non-caloric bolus is instilled via gavage into the stomach. The test compound/reference compound or its vehicle in the control group is administered subcutaneously, to reach Cmax when coloured bolus is administered. After another 30 min, the animals are sacrificed and the stomach and the small intestine prepared. The filled stomach is weighed, emptied, carefully cleaned and dried and reweighed. The calculated stomach content indicates the degree of gastric emptying. The small intestine is straightened without force and measured in length. Then the distance from the gastric beginning of the gut to the tip of the farthest traveled intestinal content bolus is measured. The intestinal passage is given as relation in percent of the latter distance and the total length of the small intestine. Comparable data can be obtained for both female and male mice.
Statistical analyses are performed with Everstat 6.0 by 1-way-ANOVA, followed by Dunnetts or Newman-Keuls as post-hoc test, respectively. Differences are considered statistically significant at the p<0.05 level. As post hoc test Dunnet's Test is applied to compare versus vehicle control, only. Newman-Keul's Test is applied for all pairwise comparisons (i.e. versus vehicle and reference groups).
Automated Assessment of Feed Intake in Mice
Female NMRI-mice of a body weight between 20 and 30 g are used. Mice are adapted to housing conditions for at least one week and for at least one day single-caged in the assessment equipment, when basal data are recorded simultaneously. On the study day, test product is administered subcutaneously close to the lights-off phase (12 h lights off) and assessment of feed consumption is directly started afterwards. Assessment included continued monitoring (every 30 min) over 22 hours. Repetition of this procedure over several days is possible. Restriction of assessment to 22 hours is for practical reasons to allow for reweighing of animals, refilling of feed and water and drug administration between procedures. Results can be assessed as cumulated data over 22 hours or differentiated to 30 min intervals. Comparable data can be obtained for both female and male mice.
Statistical analyses are performed with Everstat 6.0 by two-way ANOVA on repeated measures and Dunnett's post-hoc analyses. Differences are considered statistically significant at the p<0.05 level.
Acute and Chronic Effects after Subcutaneous Treatment on Blood Glucose and Body Weight in Female Diet-Induced Obese (DIO) C57BL/6 Mice
C57BL/6 Harlan mice are housed in groups in a specific pathogen-free barrier facility on a 12 h light/dark cycle with free access to water and standard or high-fat diet. After prefeeding on high-fat diet, mice are stratified to treatment groups (n=8), so that each group has similar mean body weight. An age-matched group with ad-libitum access to standard chow is included as standard control group. Before the experiment, mice are subcutaneously (s.c.) injected with vehicle solution and weighed for 3 days to acclimate them to the procedures.
1) Acute effect on blood glucose in fed female DIO mice: initial blood samples are taken just before first administration (s.c.) of vehicle (phosphate buffer solution) or the exendin-4 derivatives (dissolved in phosphate buffer), respectively. The volume of administration is 5 mL/kg. The animals have access to water and their corresponding diet during the experiment. Blood glucose levels are measured at t=0 h, t=1 h, t=2 h, t=3 h, t=4 h, t=6 h and t=24 h (method: Accu-Check glucometer). Blood sampling is performed by tail incision without anaesthesia.
2) Chronic effect on body weight in female DIO mice: mice are treated twice daily s.c. in the morning and in the evening, respectively, at the beginning and the end of the light phase with either vehicle or exendin-4 derivatives for 4 weeks. Body weight is recorded daily. Two days before start of treatment and on day 26, total fat mass is measured by nuclear magnetic resonance (NMR).
Statistical analyses are performed with Everstat 6.0 by repeated measures two-way ANOVA and Dunnetts post-hoc analyses (glucose profile) and 1-way-ANOVA, followed by Dunnetts post-hoc test (body weight, body fat). Differences versus vehicle-treated DIO control mice are considered statistically significant at the p<0.05 level.
Effects of 4 Weeks of Treatment on Glucose, HbA1c and Oral Glucose Tolerance in Female Diabetic Dbdb-Mice
8 week old, female diabetic dbdb-mice of mean non-fasted glucose value of 14.5 mmol/l and a body weight of 37-40 g are used. Mice are individually marked and are adapted to housing conditions for at least one week.
7 days prior to study start, baseline values for non-fasted glucose and HbA1c are determined, 5 days prior to study start, mice are assigned to groups and cages (5 mice per cage, 10 per group) according to their HbA1c values to ensure even distribution of lower and higher values between groups (stratification).
Mice are treated for 4 weeks, by twice daily subcutaneous administration in the morning and the afternoon. Blood samples from the tail tip are obtained for HbA1c on study day 21 and oral glucose tolerance is assessed in the 4th week.
An oral glucose tolerance test is done in the morning without prior extra compound administration to majorly assess the effect of chronic treatment and less of acute compound administration. Mice are fasted for 4 hours prior to oral glucose administration (2 g/kg, t=0 min) Blood samples are drawn prior to glucose administration and at 15, 30, 60, 90, 120, and 180 min Feed is returned after the last blood sampling. Results are represented as change from baseline, glucose in mmol/l and HbA1c in %.
Statistical analyses are performed with Everstat Version 6.0 based on SAS by 1-way-ANOVA, followed by Dunnett's post-hoc test against vehicle-control. Differences are considered statistically significant at the p<0.05 level.
Glucose Lowering in Non-Fasted Female Diabetic dbdb-Mice
Female diabetic dbdb-mice of mean non-fasted glucose value of 20-22 mmol/l and a body weight of 42 g+/−0.6 g (SEM) are used. Mice are individually marked and are adapted to housing conditions for at least one week.
3-5 days prior to study start mice are assigned to groups and cages (4 mice per cage, 8 per group) according to their non-fasted glucose values to ensure even distribution of lower and higher values between groups (stratification). On the study day, mice are weighed and dosed (t=0) Immediately prior to compound administration feed is removed while water remains available, and a first blood sample at a tail incision is drawn (baseline). Further blood samples are drawn at the tail incision at 30, 60, 90, 120, 240, 360, and 480 min.
Statistical analyses are performed with Everstat Version 6.0 based on SAS by 2-way-ANOVA on repeated measures, followed by Dunnett's post-hoc test against vehicle-control. Differences are considered statistically significant at the p<0.05 level.
The invention is further illustrated by the following examples.
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.23 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to literature (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Hereafter Palm-Glu-Glu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method A).
Deconvolution of the mass signals found under the peak with retention time 12.61 min revealed the peptide mass 4581.5 which is in line with the expected value of 4581.1.
The manual synthesis procedure as described in Methods was carried out on a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesis strategy was applied with DIC/HOBt-activation. In position 14 Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Boc)-OH were used. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 10.46 min revealed the peptide mass 4450.5 which is in line with the expected value of 4451.9.
In an analogous way, the peptides listed in Table 3 were synthesized and characterized.
In an analogous way, the following peptides of Table 4 can be synthesized:
Solubility and stability of peptidic compounds were assessed as described in Methods. The results are given in Table 5.
Potencies of peptidic compounds at the GLP-1, glucagon and GIP receptors were determined by exposing cells expressing human glucagon receptor (hGlucagon R), human GIP receptor (hGIP-R) or human GLP-1 receptor (hGLP-1R) to the listed compounds at increasing concentrations and measuring the formed cAMP as described in Methods.
The results are shown in Table 6:
A selection of inventive exendin-4 derivatives comprising a functionalized amino acid in position 14 has been tested versus corresponding compounds having in this position 14 a ‘non-functionalized’ amino acid with otherwise identical amino acid sequence. The reference pair compounds and the corresponding EC50 values at GLP-1, Glucagon and GIP receptors (indicated in pM) are given in Table 7. As shown, the inventive exendin-4 derivatives show a superior activity in comparison to the compounds with a ‘non-functionalized’ amino acid in position 14.
Furthermore, a selection of inventive exendin-4 derivatives comprising an Aib in position 27 has been tested versus corresponding compounds having in this position a lysine residue as in native exendin-4 and otherwise identical amino acid sequence. The reference pair compounds and the corresponding EC50 values at GLP-1, Glucagon and GIP receptors (indicated in pM) are given in Table 8. As shown, the inventive exendin-4 derivatives show a reduced activity on the GIP receptor compared to the corresponding derivatives with Lys at position 27 as in native exendin-4.
Pharmacokinetic profiles were determined as described in Methods. Calculated T1/2 and Cmax values are shown in Table 9.
1) Glucose Profile
After blood sampling to determine the blood glucose baseline level, fed diet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQ ID NO: 6, 50 μg/kg of SEQ ID NO: 7 or phosphate buffered solution (vehicle control on standard or high-fat diet) subcutaneously. At predefined time points, more blood samples were taken to measure blood glucose and generate the blood glucose profile over 24 h (see
2) Body Weight
Female obese C57BL/6 mice were treated for 4 weeks twice daily subcutaneously with 50 μg/kg SEQ ID NO: 6, 50 μg/kg SEQ ID NO: 7 or vehicle. Body weight was recorded daily, and body fat content was determined before the start and after 4 weeks of treatment. Treatment with 50 μg/kg SEQ ID NO: 6 or 50 μg/kg SEQ ID NO: 7 showed a decrease in daily body weight when compared to vehicle DIO control mice (Table 10,
1) Glucose Profile
After blood sampling to determine the blood glucose baseline level, fed diet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQ ID NO: 9 or phosphate buffered solution (vehicle control on standard or high-fat diet) subcutaneously. At predefined time points, more blood samples were taken to measure blood glucose and generate the blood glucose profile over 24 h.
SEQ ID NO: 9 demonstrated a decrease in blood glucose compared to DIO control (
2) Body Weight
Female obese C57BL/6 mice were treated for 4 weeks twice daily subcutaneously with 50 μg/kg SEQ ID NO: 9 or vehicle. Body weight was recorded daily, and body fat content was determined before the start and after 4 weeks of treatment.
Treatment with 50 μg/kg SEQ ID NO: 9 showed a decrease in daily body weight when compared to vehicle DIO control mice (Table 11,
| Number | Date | Country | Kind |
|---|---|---|---|
| 14305502 | Apr 2014 | EP | regional |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5424286 | Eng | Jun 1995 | A |
| 5641757 | Bornstein et al. | Jun 1997 | A |
| 6284727 | Kim et al. | Sep 2001 | B1 |
| 6329336 | Bridon et al. | Dec 2001 | B1 |
| 6344180 | Holst et al. | Feb 2002 | B1 |
| 6410511 | L'Italien et al. | Jun 2002 | B2 |
| 6429197 | Coolidge et al. | Aug 2002 | B1 |
| 6451974 | Hansen | Sep 2002 | B1 |
| 6458924 | Knudsen et al. | Oct 2002 | B2 |
| 6482799 | Tusé et al. | Nov 2002 | B1 |
| 6506724 | Hiles et al. | Jan 2003 | B1 |
| 6514500 | Bridon et al. | Feb 2003 | B1 |
| 6528486 | Larsen et al. | Mar 2003 | B1 |
| 6579851 | Goeke et al. | Jun 2003 | B2 |
| 6593295 | Bridon et al. | Jul 2003 | B2 |
| 6703359 | Young et al. | Mar 2004 | B1 |
| 6706689 | Coolidge et al. | Mar 2004 | B2 |
| 6723530 | Drucker | Apr 2004 | B1 |
| 6821949 | Bridon et al. | Nov 2004 | B2 |
| 6828303 | Kim et al. | Dec 2004 | B2 |
| 6849714 | Bridon et al. | Feb 2005 | B1 |
| 6858576 | Young et al. | Feb 2005 | B1 |
| 6861236 | Moll et al. | Mar 2005 | B2 |
| 6872700 | Young et al. | Mar 2005 | B1 |
| 6884579 | Holst et al. | Apr 2005 | B2 |
| 6887470 | Bridon et al. | May 2005 | B1 |
| 6887849 | Bridon et al. | May 2005 | B2 |
| 6894024 | Coolidge et al. | May 2005 | B2 |
| 6902744 | Kolterman et al. | Jun 2005 | B1 |
| 6924264 | Prickett et al. | Aug 2005 | B1 |
| 6956026 | Beeley et al. | Oct 2005 | B2 |
| 6969702 | Bertilsson et al. | Nov 2005 | B2 |
| 6972319 | Pan et al. | Dec 2005 | B1 |
| 6982248 | Coolidge et al. | Jan 2006 | B2 |
| 6989366 | Beeley et al. | Jan 2006 | B2 |
| 6998387 | Goke et al. | Feb 2006 | B1 |
| 7056734 | Egan et al. | Jun 2006 | B1 |
| 7056887 | Coolidge et al. | Jun 2006 | B2 |
| 7105489 | Hathaway | Sep 2006 | B2 |
| 7105490 | Beeley et al. | Sep 2006 | B2 |
| 7115569 | Beeley et al. | Oct 2006 | B2 |
| 7138375 | Beeley et al. | Nov 2006 | B2 |
| 7138546 | Tang | Nov 2006 | B2 |
| 7141240 | Perfetti et al. | Nov 2006 | B2 |
| 7141547 | Rosen et al. | Nov 2006 | B2 |
| 7144863 | DeFelippis et al. | Dec 2006 | B2 |
| 7153825 | Young et al. | Dec 2006 | B2 |
| 7157555 | Beeley et al. | Jan 2007 | B1 |
| 7179788 | DeFelippis et al. | Feb 2007 | B2 |
| 7189690 | Rosen et al. | Mar 2007 | B2 |
| 7220721 | Beeley et al. | May 2007 | B1 |
| 7223725 | Beeley et al. | May 2007 | B1 |
| 7256253 | Bridon et al. | Aug 2007 | B2 |
| 7259136 | Hathaway et al. | Aug 2007 | B2 |
| 7259233 | Dodd et al. | Aug 2007 | B2 |
| 7259234 | Bachovchin et al. | Aug 2007 | B2 |
| 7265087 | Göke et al. | Sep 2007 | B1 |
| 7271149 | Glaesner et al. | Sep 2007 | B2 |
| 7297761 | Beeley et al. | Nov 2007 | B2 |
| 7312196 | L'Italien et al. | Dec 2007 | B2 |
| 7329646 | Sun et al. | Feb 2008 | B2 |
| 7399489 | Kolterman et al. | Jul 2008 | B2 |
| 7399744 | Mack et al. | Jul 2008 | B2 |
| 7407932 | Young et al. | Aug 2008 | B2 |
| 7407955 | Himmelsbach et al. | Aug 2008 | B2 |
| 7414107 | Larsen | Aug 2008 | B2 |
| 7419952 | Beeley et al. | Sep 2008 | B2 |
| 7442680 | Young et al. | Oct 2008 | B2 |
| 7442682 | Kitaura et al. | Oct 2008 | B2 |
| 7452858 | Hiles et al. | Nov 2008 | B2 |
| 7456254 | Wright et al. | Nov 2008 | B2 |
| 7476652 | Brunner-Schwarz et al. | Jan 2009 | B2 |
| 7507714 | Pan et al. | Mar 2009 | B2 |
| 7521423 | Young et al. | Apr 2009 | B2 |
| 7544657 | Ebbehøj et al. | Jun 2009 | B2 |
| 7563871 | Wright et al. | Jul 2009 | B2 |
| 7576050 | Greig et al. | Aug 2009 | B2 |
| 7585837 | Shechter et al. | Sep 2009 | B2 |
| 7592010 | Rosen et al. | Sep 2009 | B2 |
| 7595293 | Engelund et al. | Sep 2009 | B2 |
| 7595294 | Nestor | Sep 2009 | B2 |
| 7608692 | Prickett et al. | Oct 2009 | B2 |
| 7612176 | Wright et al. | Nov 2009 | B2 |
| 7632806 | Juul-Mortensen et al. | Dec 2009 | B2 |
| 7638299 | Cho et al. | Dec 2009 | B2 |
| 7682356 | Alessi et al. | Mar 2010 | B2 |
| 7683030 | Prickett et al. | Mar 2010 | B2 |
| 7691963 | Prickett et al. | Apr 2010 | B2 |
| 7696161 | Beeley et al. | Apr 2010 | B2 |
| 7700549 | Beeley et al. | Apr 2010 | B2 |
| 7704953 | Herman et al. | Apr 2010 | B2 |
| 7713930 | Brunner-Schwarz et al. | May 2010 | B2 |
| 7723471 | Levy et al. | May 2010 | B2 |
| 7741269 | Young et al. | Jun 2010 | B2 |
| 7749955 | Hansen et al. | Jul 2010 | B2 |
| 7772189 | Herman et al. | Aug 2010 | B2 |
| 7790681 | Hathaway et al. | Sep 2010 | B2 |
| 7799344 | Oberg | Sep 2010 | B2 |
| 7799759 | Rosen et al. | Sep 2010 | B2 |
| 7803404 | Hokenson et al. | Sep 2010 | B2 |
| 7829664 | Tatake et al. | Nov 2010 | B2 |
| 7847079 | Rosen et al. | Dec 2010 | B2 |
| 7858740 | Beeley et al. | Dec 2010 | B2 |
| 7867972 | Ballance et al. | Jan 2011 | B2 |
| 7879028 | Alessi et al. | Feb 2011 | B2 |
| 7888314 | Hathaway et al. | Feb 2011 | B2 |
| 7897560 | Dorwald et al. | Mar 2011 | B2 |
| 7906146 | Kolterman et al. | Mar 2011 | B2 |
| 7928065 | Young et al. | Apr 2011 | B2 |
| 7928186 | Chang | Apr 2011 | B2 |
| 7935786 | Larsen | May 2011 | B2 |
| 7939494 | Khan et al. | May 2011 | B2 |
| 7960341 | Hathaway et al. | Jun 2011 | B2 |
| 7977306 | Rosen et al. | Jul 2011 | B2 |
| 7981861 | Coolidge et al. | Jul 2011 | B2 |
| 7989585 | Dodd et al. | Aug 2011 | B2 |
| 7994121 | Bachovchin et al. | Aug 2011 | B2 |
| 7994122 | Riber et al. | Aug 2011 | B2 |
| 8008255 | Ong et al. | Aug 2011 | B2 |
| 8012464 | Rosen et al. | Sep 2011 | B2 |
| 8026210 | Young et al. | Sep 2011 | B2 |
| 8030273 | Lau et al. | Oct 2011 | B2 |
| 8039432 | Bridon et al. | Oct 2011 | B2 |
| 8057822 | Prickett et al. | Nov 2011 | B2 |
| 8071539 | Rosen et al. | Dec 2011 | B2 |
| 8076288 | Levy et al. | Dec 2011 | B2 |
| 8080516 | Bridon et al. | Dec 2011 | B2 |
| 8084414 | Bridon et al. | Dec 2011 | B2 |
| 8093206 | Bridon et al. | Jan 2012 | B2 |
| 8097239 | Johnsson et al. | Jan 2012 | B2 |
| 8097586 | Lv et al. | Jan 2012 | B2 |
| 8114632 | Melarkode et al. | Feb 2012 | B2 |
| 8114833 | Pedersen et al. | Feb 2012 | B2 |
| 8114958 | Soares et al. | Feb 2012 | B2 |
| 8114959 | Juul-Mortensen | Feb 2012 | B2 |
| 8119648 | Himmelsbach et al. | Feb 2012 | B2 |
| 8143217 | Balkan et al. | Mar 2012 | B2 |
| 8158579 | Ballance et al. | Apr 2012 | B2 |
| 8158583 | Knudsen et al. | Apr 2012 | B2 |
| 8178495 | Chilkoti | May 2012 | B2 |
| 8178541 | Himmelsbach et al. | May 2012 | B2 |
| 8197450 | Glejbol et al. | Jun 2012 | B2 |
| 8211439 | Rosen et al. | Jul 2012 | B2 |
| 8232281 | Dugi et al. | Jul 2012 | B2 |
| 8236760 | Pimentel et al. | Aug 2012 | B2 |
| 8252739 | Rosen et al. | Aug 2012 | B2 |
| 8263545 | Levy et al. | Sep 2012 | B2 |
| 8263550 | Beeley et al. | Sep 2012 | B2 |
| 8263554 | Tatarkiewicz et al. | Sep 2012 | B2 |
| 8268781 | Gotthardt et al. | Sep 2012 | B2 |
| 8278272 | Greig et al. | Oct 2012 | B2 |
| 8278420 | Wang et al. | Oct 2012 | B2 |
| 8288338 | Young et al. | Oct 2012 | B2 |
| 8293726 | Habib | Oct 2012 | B2 |
| 8293869 | Bossard et al. | Oct 2012 | B2 |
| 8293871 | Wright et al. | Oct 2012 | B2 |
| 8299024 | Rabinovitch et al. | Oct 2012 | B2 |
| 8299025 | Alessi et al. | Oct 2012 | B2 |
| 8329419 | Nicolaou et al. | Dec 2012 | B2 |
| 8329648 | Fineman et al. | Dec 2012 | B2 |
| 8338368 | Dimarchi et al. | Dec 2012 | B2 |
| 8343910 | Shechter et al. | Jan 2013 | B2 |
| 8372804 | Richardson et al. | Feb 2013 | B2 |
| 8377869 | Richardson et al. | Feb 2013 | B2 |
| 8389473 | Hathaway et al. | Mar 2013 | B2 |
| 8404637 | Levy et al. | Mar 2013 | B2 |
| 8410047 | Bock et al. | Apr 2013 | B2 |
| 8420604 | Hokenson et al. | Apr 2013 | B2 |
| 8424518 | Smutney et al. | Apr 2013 | B2 |
| 8426361 | Levy et al. | Apr 2013 | B2 |
| 8431685 | Wright et al. | Apr 2013 | B2 |
| 8445647 | Prickett et al. | May 2013 | B2 |
| 8450270 | Dimarchi et al. | May 2013 | B2 |
| 8454971 | Day et al. | Jun 2013 | B2 |
| 8461105 | Wright et al. | Jun 2013 | B2 |
| 8481490 | Tatarkiewicz et al. | Jul 2013 | B2 |
| 8485180 | Smutney et al. | Jul 2013 | B2 |
| 8497240 | Levy et al. | Jul 2013 | B2 |
| 8499757 | Smutney et al. | Aug 2013 | B2 |
| 8546327 | Dimarchi et al. | Oct 2013 | B2 |
| 8551946 | Dimarchi et al. | Oct 2013 | B2 |
| 8551947 | Coolidge et al. | Oct 2013 | B2 |
| 8557769 | Coskun et al. | Oct 2013 | B2 |
| 8557771 | Fan et al. | Oct 2013 | B2 |
| 8569481 | Köster et al. | Oct 2013 | B2 |
| 8575097 | Xu et al. | Nov 2013 | B2 |
| 8580919 | Bossard et al. | Nov 2013 | B2 |
| 8598120 | Soares et al. | Dec 2013 | B2 |
| 8603761 | Nicolaou et al. | Dec 2013 | B2 |
| 8603969 | Levy et al. | Dec 2013 | B2 |
| 8614181 | Juul-Mortensen et al. | Dec 2013 | B2 |
| 8617613 | Wright et al. | Dec 2013 | B2 |
| 8636001 | Smutney et al. | Jan 2014 | B2 |
| 8641683 | Glejbol et al. | Feb 2014 | B2 |
| 8642544 | Alfaro-Lopez et al. | Feb 2014 | B2 |
| 8664232 | Himmelsbach et al. | Mar 2014 | B2 |
| 8669228 | Dimarchi et al. | Mar 2014 | B2 |
| 8673927 | Dugi et al. | Mar 2014 | B2 |
| 8697647 | Levy et al. | Apr 2014 | B2 |
| 8697838 | Dimarchi et al. | Apr 2014 | B2 |
| 8710002 | Rothkopf | Apr 2014 | B2 |
| 8710181 | Christiansen et al. | Apr 2014 | B2 |
| 8716221 | Lv et al. | May 2014 | B2 |
| 8729018 | Chilkoti | May 2014 | B2 |
| 8729019 | Oberg et al. | May 2014 | B2 |
| 8735350 | Shechter et al. | May 2014 | B2 |
| 8748376 | Ludvigsen et al. | Jun 2014 | B2 |
| 8759290 | James | Jun 2014 | B2 |
| 8759295 | Ghosh et al. | Jun 2014 | B2 |
| 8772232 | Lau et al. | Jul 2014 | B2 |
| 8778872 | Dimarchi et al. | Jul 2014 | B2 |
| 8785396 | Leone-Bay et al. | Jul 2014 | B2 |
| 8801700 | Alessi et al. | Aug 2014 | B2 |
| 8809499 | Fan et al. | Aug 2014 | B2 |
| 8816047 | Levetan et al. | Aug 2014 | B2 |
| 8841255 | Chilkoti | Sep 2014 | B2 |
| 8853157 | Knudsen et al. | Oct 2014 | B2 |
| 8853160 | Greig et al. | Oct 2014 | B2 |
| 8877252 | Wright et al. | Nov 2014 | B2 |
| 8877709 | Shechter et al. | Nov 2014 | B2 |
| 8883449 | Kjeldsen et al. | Nov 2014 | B2 |
| 8889619 | Bai et al. | Nov 2014 | B2 |
| 8900593 | Day et al. | Dec 2014 | B2 |
| 8969288 | Dimarchi et al. | Mar 2015 | B2 |
| 8969294 | Bianchi et al. | Mar 2015 | B2 |
| 8980830 | Dimarchi et al. | Mar 2015 | B2 |
| 8981047 | Dimarchi et al. | Mar 2015 | B2 |
| 9018164 | Dimarchi et al. | Apr 2015 | B2 |
| 9181305 | Haack et al. | Nov 2015 | B2 |
| 20010011071 | Knudsen et al. | Aug 2001 | A1 |
| 20010027180 | Isaacs | Oct 2001 | A1 |
| 20010043934 | L'Italien et al. | Nov 2001 | A1 |
| 20020061838 | Holmquist et al. | May 2002 | A1 |
| 20020137666 | Beeley et al. | Sep 2002 | A1 |
| 20020146405 | Coolidge et al. | Oct 2002 | A1 |
| 20030036504 | Kolterman et al. | Feb 2003 | A1 |
| 20030050237 | Kim et al. | Mar 2003 | A1 |
| 20030069182 | Rinella et al. | Apr 2003 | A1 |
| 20030087820 | Young et al. | May 2003 | A1 |
| 20030087821 | Beeley et al. | May 2003 | A1 |
| 20030092606 | L'Italien et al. | May 2003 | A1 |
| 20030119021 | Koster et al. | Jun 2003 | A1 |
| 20030119734 | Flink et al. | Jun 2003 | A1 |
| 20030180287 | Gombotz et al. | Sep 2003 | A1 |
| 20030216287 | Tang | Nov 2003 | A1 |
| 20030220255 | Knudsen et al. | Nov 2003 | A1 |
| 20040023871 | Hiles et al. | Feb 2004 | A1 |
| 20040029784 | Hathaway | Feb 2004 | A1 |
| 20040037826 | Michelsen et al. | Feb 2004 | A1 |
| 20040038865 | Gelber et al. | Feb 2004 | A1 |
| 20040048783 | Brunner-Schwarz et al. | Mar 2004 | A1 |
| 20040097510 | Himmelsbach et al. | May 2004 | A1 |
| 20040209255 | Koster et al. | Oct 2004 | A1 |
| 20040209803 | Baron et al. | Oct 2004 | A1 |
| 20040242853 | Greig et al. | Dec 2004 | A1 |
| 20040266670 | Hiles et al. | Dec 2004 | A9 |
| 20040266678 | Beeley et al. | Dec 2004 | A1 |
| 20040266683 | Hathaway et al. | Dec 2004 | A1 |
| 20040266692 | Young et al. | Dec 2004 | A1 |
| 20050009742 | Bertilsson et al. | Jan 2005 | A1 |
| 20050009847 | Bertilsson et al. | Jan 2005 | A1 |
| 20050009988 | Harris et al. | Jan 2005 | A1 |
| 20050043238 | Young et al. | Feb 2005 | A1 |
| 20050059601 | Beeley et al. | Mar 2005 | A1 |
| 20050096276 | Coolidge et al. | May 2005 | A1 |
| 20050101537 | Beeley et al. | May 2005 | A1 |
| 20050106214 | Chen | May 2005 | A1 |
| 20050143303 | Quay et al. | Jun 2005 | A1 |
| 20050171019 | Young et al. | Aug 2005 | A1 |
| 20050186174 | Bossard | Aug 2005 | A1 |
| 20050197287 | Mack et al. | Sep 2005 | A1 |
| 20050209142 | Bertilsson et al. | Sep 2005 | A1 |
| 20050215469 | Beeley et al. | Sep 2005 | A1 |
| 20050215475 | Ong et al. | Sep 2005 | A1 |
| 20050267034 | Prickett et al. | Dec 2005 | A1 |
| 20050271702 | Wright et al. | Dec 2005 | A1 |
| 20050281879 | Chen et al. | Dec 2005 | A1 |
| 20060003918 | Kim et al. | Jan 2006 | A1 |
| 20060057137 | Steiness | Mar 2006 | A1 |
| 20060069029 | Kolterman et al. | Mar 2006 | A1 |
| 20060073182 | Wong et al. | Apr 2006 | A1 |
| 20060074012 | Hiles et al. | Apr 2006 | A1 |
| 20060079448 | Bertilsson et al. | Apr 2006 | A1 |
| 20060084605 | Engelund et al. | Apr 2006 | A1 |
| 20060094652 | Levy et al. | May 2006 | A1 |
| 20060094653 | Levy et al. | May 2006 | A1 |
| 20060110423 | Wright et al. | May 2006 | A1 |
| 20060135586 | Kozlowski et al. | Jun 2006 | A1 |
| 20060135747 | Levy et al. | Jun 2006 | A1 |
| 20060148713 | Beeley et al. | Jul 2006 | A1 |
| 20060165733 | Betz et al. | Jul 2006 | A1 |
| 20060171920 | Shechter et al. | Aug 2006 | A1 |
| 20060172001 | Ong et al. | Aug 2006 | A1 |
| 20060178304 | Juul-Mortensen et al. | Aug 2006 | A1 |
| 20060183677 | Young et al. | Aug 2006 | A1 |
| 20060183682 | Juul-Mortensen | Aug 2006 | A1 |
| 20060210614 | Quay et al. | Sep 2006 | A1 |
| 20060247167 | Schlein et al. | Nov 2006 | A1 |
| 20060275252 | Harris et al. | Dec 2006 | A1 |
| 20060287221 | Knudsen et al. | Dec 2006 | A1 |
| 20060293232 | Levy et al. | Dec 2006 | A1 |
| 20060293499 | Bentley et al. | Dec 2006 | A1 |
| 20070010424 | Pedersen et al. | Jan 2007 | A1 |
| 20070010656 | Beeley et al. | Jan 2007 | A1 |
| 20070014818 | Betz et al. | Jan 2007 | A1 |
| 20070021336 | Anderson et al. | Jan 2007 | A1 |
| 20070037750 | Young et al. | Feb 2007 | A1 |
| 20070049531 | Knudsen et al. | Mar 2007 | A1 |
| 20070059373 | Oberg | Mar 2007 | A1 |
| 20070059374 | Hokenson et al. | Mar 2007 | A1 |
| 20070065469 | Betz et al. | Mar 2007 | A1 |
| 20070066528 | Beeley et al. | Mar 2007 | A1 |
| 20070092482 | Bossard et al. | Apr 2007 | A1 |
| 20070129284 | Kjeldsen et al. | Jun 2007 | A1 |
| 20070166352 | Wright et al. | Jul 2007 | A1 |
| 20070196416 | Li et al. | Aug 2007 | A1 |
| 20070281940 | Dugi et al. | Dec 2007 | A1 |
| 20080071063 | Allan et al. | Mar 2008 | A1 |
| 20080091176 | Alessi et al. | Apr 2008 | A1 |
| 20080119393 | Beeley et al. | May 2008 | A1 |
| 20080119569 | Wright et al. | May 2008 | A1 |
| 20080125348 | Wright et al. | May 2008 | A1 |
| 20080125349 | Wright et al. | May 2008 | A1 |
| 20080125351 | Wright et al. | May 2008 | A1 |
| 20080125353 | Hiles et al. | May 2008 | A1 |
| 20080125361 | Ludvigsen et al. | May 2008 | A1 |
| 20080171848 | Christiansen et al. | Jul 2008 | A1 |
| 20080176802 | Prickett et al. | Jul 2008 | A1 |
| 20080176804 | Mack et al. | Jul 2008 | A1 |
| 20080200390 | Prickett et al. | Aug 2008 | A1 |
| 20080213288 | Michelsen et al. | Sep 2008 | A1 |
| 20080214467 | Prickett et al. | Sep 2008 | A1 |
| 20080233053 | Gross et al. | Sep 2008 | A1 |
| 20080249007 | Lau et al. | Oct 2008 | A1 |
| 20080249018 | Kolterman et al. | Oct 2008 | A1 |
| 20080249089 | Himmelsbach et al. | Oct 2008 | A1 |
| 20080255159 | Himmelsbach et al. | Oct 2008 | A1 |
| 20080260838 | Hokenson et al. | Oct 2008 | A1 |
| 20080260847 | Wright et al. | Oct 2008 | A1 |
| 20080274952 | Soares et al. | Nov 2008 | A1 |
| 20080280814 | Ludvigsen et al. | Nov 2008 | A1 |
| 20080300171 | Balkan et al. | Dec 2008 | A1 |
| 20080312157 | Levy et al. | Dec 2008 | A1 |
| 20080318865 | Juul-Mortensen | Dec 2008 | A1 |
| 20090011976 | Ludvigsen et al. | Jan 2009 | A1 |
| 20090018053 | L'Italien et al. | Jan 2009 | A1 |
| 20090029913 | Beeley et al. | Jan 2009 | A1 |
| 20090035253 | Wright et al. | Feb 2009 | A1 |
| 20090036364 | Levy et al. | Feb 2009 | A1 |
| 20090043264 | Glejbol et al. | Feb 2009 | A1 |
| 20090054315 | Bock et al. | Feb 2009 | A1 |
| 20090069226 | Ong et al. | Mar 2009 | A1 |
| 20090082255 | Brunner-Schwarz et al. | Mar 2009 | A1 |
| 20090088369 | Steiness | Apr 2009 | A1 |
| 20090098130 | Bradshaw et al. | Apr 2009 | A1 |
| 20090110647 | Richardson et al. | Apr 2009 | A1 |
| 20090111749 | Richardson et al. | Apr 2009 | A1 |
| 20090137456 | Dimarchi et al. | May 2009 | A1 |
| 20090137466 | Anderson et al. | May 2009 | A1 |
| 20090163423 | Young et al. | Jun 2009 | A1 |
| 20090170750 | Kjeldsen et al. | Jul 2009 | A1 |
| 20090176704 | Beeley et al. | Jul 2009 | A1 |
| 20090180953 | Gotthardt et al. | Jul 2009 | A1 |
| 20090186817 | Ghosh et al. | Jul 2009 | A1 |
| 20090186819 | Carrier et al. | Jul 2009 | A1 |
| 20090203597 | Rabinovitch et al. | Aug 2009 | A1 |
| 20090203603 | Baron et al. | Aug 2009 | A1 |
| 20090215688 | Knudsen et al. | Aug 2009 | A1 |
| 20090215694 | Kolterman et al. | Aug 2009 | A1 |
| 20090221485 | James | Sep 2009 | A1 |
| 20090226431 | Habib | Sep 2009 | A1 |
| 20090232775 | Bertilsson et al. | Sep 2009 | A1 |
| 20090232807 | Glaesner et al. | Sep 2009 | A1 |
| 20090232891 | Gelber et al. | Sep 2009 | A1 |
| 20090239796 | Fineman et al. | Sep 2009 | A1 |
| 20090247463 | Wright et al. | Oct 2009 | A1 |
| 20090253625 | Greig et al. | Oct 2009 | A1 |
| 20090258818 | Surolia et al. | Oct 2009 | A1 |
| 20090264352 | Anderson et al. | Oct 2009 | A1 |
| 20090280169 | Leonard | Nov 2009 | A1 |
| 20090280170 | Lee et al. | Nov 2009 | A1 |
| 20090286716 | Knudsen et al. | Nov 2009 | A1 |
| 20090286723 | Levy et al. | Nov 2009 | A1 |
| 20090291886 | Ong et al. | Nov 2009 | A1 |
| 20090298757 | Bloom et al. | Dec 2009 | A1 |
| 20090308390 | Smutney et al. | Dec 2009 | A1 |
| 20090308391 | Smutney et al. | Dec 2009 | A1 |
| 20090308392 | Smutney et al. | Dec 2009 | A1 |
| 20090325860 | Costantino et al. | Dec 2009 | A1 |
| 20100009904 | Lv et al. | Jan 2010 | A1 |
| 20100016806 | Glejbol et al. | Jan 2010 | A1 |
| 20100022455 | Chilkoti | Jan 2010 | A1 |
| 20100029554 | Ghosh et al. | Feb 2010 | A1 |
| 20100041867 | Shechter et al. | Feb 2010 | A1 |
| 20100056451 | Juul-Mortensen et al. | Mar 2010 | A1 |
| 20100087365 | Cherif-Cheikh et al. | Apr 2010 | A1 |
| 20100099619 | Levy et al. | Apr 2010 | A1 |
| 20100137558 | Lee et al. | Jun 2010 | A1 |
| 20100152097 | Wright et al. | Jun 2010 | A1 |
| 20100152111 | Wright et al. | Jun 2010 | A1 |
| 20100168011 | Jennings, Jr. et al. | Jul 2010 | A1 |
| 20100173844 | Ludvigsen et al. | Jul 2010 | A1 |
| 20100185184 | Alessi et al. | Jul 2010 | A1 |
| 20100190699 | Dimarchi et al. | Jul 2010 | A1 |
| 20100190701 | Day et al. | Jul 2010 | A1 |
| 20100190715 | Schlein et al. | Jul 2010 | A1 |
| 20100196405 | Ng et al. | Aug 2010 | A1 |
| 20100197565 | Smutney et al. | Aug 2010 | A1 |
| 20100210505 | Bossard et al. | Aug 2010 | A1 |
| 20100216692 | Brunner-Schwarz et al. | Aug 2010 | A1 |
| 20100240586 | Bao et al. | Sep 2010 | A1 |
| 20100247661 | Hokenson et al. | Sep 2010 | A1 |
| 20100261637 | Spetzler et al. | Oct 2010 | A1 |
| 20100278924 | Oberg et al. | Nov 2010 | A1 |
| 20100292172 | Ghosh et al. | Nov 2010 | A1 |
| 20100317056 | Tiwari et al. | Dec 2010 | A1 |
| 20100317576 | Rothkopf | Dec 2010 | A1 |
| 20100331246 | Dimarchi et al. | Dec 2010 | A1 |
| 20110003004 | Hokenson et al. | Jan 2011 | A1 |
| 20110034373 | Coskun et al. | Feb 2011 | A1 |
| 20110034377 | Young et al. | Feb 2011 | A1 |
| 20110059181 | Hu et al. | Mar 2011 | A1 |
| 20110065633 | Dimarchi et al. | Mar 2011 | A1 |
| 20110065731 | Dugi et al. | Mar 2011 | A1 |
| 20110071076 | Beeley et al. | Mar 2011 | A1 |
| 20110091420 | Liu et al. | Apr 2011 | A1 |
| 20110097386 | Steiner et al. | Apr 2011 | A1 |
| 20110097751 | Nicolaou et al. | Apr 2011 | A1 |
| 20110098217 | Dimarchi et al. | Apr 2011 | A1 |
| 20110112277 | Kozlowski et al. | May 2011 | A1 |
| 20110118136 | Köster et al. | May 2011 | A1 |
| 20110123487 | Chilkoti | May 2011 | A1 |
| 20110129522 | Mevorat-Kaplan et al. | Jun 2011 | A1 |
| 20110136737 | Levy et al. | Jun 2011 | A1 |
| 20110152181 | Alsina-Fernandez et al. | Jun 2011 | A1 |
| 20110152182 | Alsina-Fernandez et al. | Jun 2011 | A1 |
| 20110152185 | Plum et al. | Jun 2011 | A1 |
| 20110166062 | Dimarchi et al. | Jul 2011 | A1 |
| 20110166554 | Alessi et al. | Jul 2011 | A1 |
| 20110171178 | Levetan et al. | Jul 2011 | A1 |
| 20110178014 | Hathaway et al. | Jul 2011 | A1 |
| 20110178242 | Harris et al. | Jul 2011 | A1 |
| 20110190200 | Dimarchi et al. | Aug 2011 | A1 |
| 20110195897 | Kajihara et al. | Aug 2011 | A1 |
| 20110230409 | Knudsen et al. | Sep 2011 | A1 |
| 20110237503 | Alsina-Fernandez et al. | Sep 2011 | A1 |
| 20110237510 | Steiner et al. | Sep 2011 | A1 |
| 20110245162 | Fineman et al. | Oct 2011 | A1 |
| 20110257092 | Dimarchi et al. | Oct 2011 | A1 |
| 20110263496 | Fineman et al. | Oct 2011 | A1 |
| 20110281798 | Kolterman et al. | Nov 2011 | A1 |
| 20110288003 | Dimarchi et al. | Nov 2011 | A1 |
| 20110301080 | Bush et al. | Dec 2011 | A1 |
| 20110301081 | Becker et al. | Dec 2011 | A1 |
| 20110301084 | Lau et al. | Dec 2011 | A1 |
| 20110306549 | Tatarkiewicz et al. | Dec 2011 | A1 |
| 20120004168 | Young et al. | Jan 2012 | A1 |
| 20120021978 | Werner et al. | Jan 2012 | A1 |
| 20120040899 | Costello et al. | Feb 2012 | A1 |
| 20120046222 | Alfaro-Lopez et al. | Feb 2012 | A1 |
| 20120071510 | Leone-Bay et al. | Mar 2012 | A1 |
| 20120071817 | Ward et al. | Mar 2012 | A1 |
| 20120094356 | Chung et al. | Apr 2012 | A1 |
| 20120100070 | Ahn et al. | Apr 2012 | A1 |
| 20120122783 | Dimarchi et al. | May 2012 | A1 |
| 20120135922 | Prickett et al. | May 2012 | A1 |
| 20120136318 | Lanin et al. | May 2012 | A1 |
| 20120148586 | Chou et al. | Jun 2012 | A1 |
| 20120149639 | Balkan et al. | Jun 2012 | A1 |
| 20120157932 | Glejbol et al. | Jun 2012 | A1 |
| 20120172295 | Dimarchi et al. | Jul 2012 | A1 |
| 20120177697 | Chen | Jul 2012 | A1 |
| 20120196795 | Xu et al. | Aug 2012 | A1 |
| 20120196796 | Soares et al. | Aug 2012 | A1 |
| 20120196802 | Lv et al. | Aug 2012 | A1 |
| 20120196804 | Dimarchi et al. | Aug 2012 | A1 |
| 20120208755 | Leung et al. | Aug 2012 | A1 |
| 20120208831 | Himmelsbach et al. | Aug 2012 | A1 |
| 20120209213 | Theucher | Aug 2012 | A1 |
| 20120225810 | Pedersen et al. | Sep 2012 | A1 |
| 20120231022 | Bass et al. | Sep 2012 | A1 |
| 20120238493 | Dimarchi et al. | Sep 2012 | A1 |
| 20120238496 | Fan et al. | Sep 2012 | A1 |
| 20120253023 | Levy et al. | Oct 2012 | A1 |
| 20120258912 | Bentley et al. | Oct 2012 | A1 |
| 20120258985 | Kozlowski et al. | Oct 2012 | A1 |
| 20120264683 | Coskun et al. | Oct 2012 | A1 |
| 20120264684 | Kajihara et al. | Oct 2012 | A1 |
| 20120276098 | Hamilton et al. | Nov 2012 | A1 |
| 20120277154 | Fan et al. | Nov 2012 | A1 |
| 20120283179 | Brunner-Schwarz et al. | Nov 2012 | A1 |
| 20120294855 | Van Cauter et al. | Nov 2012 | A1 |
| 20120295836 | Knudsen et al. | Nov 2012 | A1 |
| 20120295846 | Hagendorf et al. | Nov 2012 | A1 |
| 20120295850 | Tatarkiewicz et al. | Nov 2012 | A1 |
| 20120302501 | Coolidge et al. | Nov 2012 | A1 |
| 20120309975 | Colca et al. | Dec 2012 | A1 |
| 20120316108 | Chen et al. | Dec 2012 | A1 |
| 20120316138 | Colca et al. | Dec 2012 | A1 |
| 20120322725 | Dimarchi et al. | Dec 2012 | A1 |
| 20120322728 | Colca et al. | Dec 2012 | A1 |
| 20120329715 | Greig et al. | Dec 2012 | A1 |
| 20130005664 | Chilkoti | Jan 2013 | A1 |
| 20130023470 | Young et al. | Jan 2013 | A1 |
| 20130023471 | Rabinovitch et al. | Jan 2013 | A1 |
| 20130046245 | Raab et al. | Feb 2013 | A1 |
| 20130053350 | Colca et al. | Feb 2013 | A1 |
| 20130065826 | Soula et al. | Mar 2013 | A1 |
| 20130079277 | Chilkoti | Mar 2013 | A1 |
| 20130079278 | Lau et al. | Mar 2013 | A1 |
| 20130084277 | Arnold et al. | Apr 2013 | A1 |
| 20130085099 | Chilkoti | Apr 2013 | A1 |
| 20130085104 | Chilkoti | Apr 2013 | A1 |
| 20130089878 | Nicolaou et al. | Apr 2013 | A1 |
| 20130090286 | Dimarchi et al. | Apr 2013 | A1 |
| 20130095037 | Gotthardt et al. | Apr 2013 | A1 |
| 20130096258 | Bossard et al. | Apr 2013 | A1 |
| 20130104887 | Smutney et al. | May 2013 | A1 |
| 20130116172 | Dimarchi et al. | May 2013 | A1 |
| 20130116175 | Shechter et al. | May 2013 | A1 |
| 20130118491 | Richardson et al. | May 2013 | A1 |
| 20130123178 | Dimarchi et al. | May 2013 | A1 |
| 20130123462 | Dimarchi et al. | May 2013 | A1 |
| 20130125886 | Richardson et al. | May 2013 | A1 |
| 20130130977 | Wright et al. | May 2013 | A1 |
| 20130137631 | Levy et al. | May 2013 | A1 |
| 20130137645 | Rosendahl | May 2013 | A1 |
| 20130142795 | Bai et al. | Jun 2013 | A1 |
| 20130156849 | De Fougerolles et al. | Jun 2013 | A1 |
| 20130157934 | Dimarchi et al. | Jun 2013 | A1 |
| 20130157953 | Petersen et al. | Jun 2013 | A1 |
| 20130164310 | Annathur et al. | Jun 2013 | A1 |
| 20130165370 | Bock et al. | Jun 2013 | A1 |
| 20130165379 | Kolterman et al. | Jun 2013 | A1 |
| 20130172274 | Chilkoti | Jul 2013 | A1 |
| 20130178411 | Chilkoti | Jul 2013 | A1 |
| 20130178415 | Soula et al. | Jul 2013 | A1 |
| 20130184203 | Alfaro-Lopez et al. | Jul 2013 | A1 |
| 20130184443 | Bentley et al. | Jul 2013 | A1 |
| 20130189365 | Hokenson et al. | Jul 2013 | A1 |
| 20130199527 | Smutney et al. | Aug 2013 | A1 |
| 20130203660 | Day et al. | Aug 2013 | A1 |
| 20130209586 | Hathaway et al. | Aug 2013 | A1 |
| 20130217622 | Lee et al. | Aug 2013 | A1 |
| 20130236974 | De Fougerolles | Sep 2013 | A1 |
| 20130237592 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130237593 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130237594 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130244278 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130244279 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130245104 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130245105 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130245106 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130245107 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130252281 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130253043 | De Fougerolles et al. | Sep 2013 | A1 |
| 20130259923 | Bancel et al. | Oct 2013 | A1 |
| 20130259924 | Bancel et al. | Oct 2013 | A1 |
| 20130266640 | De Fougerolles et al. | Oct 2013 | A1 |
| 20130280206 | Kozlowski et al. | Oct 2013 | A1 |
| 20130281368 | Bilsky et al. | Oct 2013 | A1 |
| 20130281374 | Levy et al. | Oct 2013 | A1 |
| 20130284912 | Vogel et al. | Oct 2013 | A1 |
| 20130288958 | Lau et al. | Oct 2013 | A1 |
| 20130289241 | Bai et al. | Oct 2013 | A1 |
| 20130291866 | Smutney et al. | Nov 2013 | A1 |
| 20130291867 | Smutney et al. | Nov 2013 | A1 |
| 20130296236 | Silvestre et al. | Nov 2013 | A1 |
| 20130303442 | Levy et al. | Nov 2013 | A1 |
| 20130310310 | Liu et al. | Nov 2013 | A1 |
| 20130310538 | Chilkoti | Nov 2013 | A1 |
| 20130331322 | Young et al. | Dec 2013 | A1 |
| 20130336893 | Haack | Dec 2013 | A1 |
| 20130338065 | Smutney et al. | Dec 2013 | A1 |
| 20130338071 | Knudsen et al. | Dec 2013 | A1 |
| 20130345134 | Sauerberg et al. | Dec 2013 | A1 |
| 20140007873 | Smutney et al. | Jan 2014 | A1 |
| 20140011732 | Spetzler et al. | Jan 2014 | A1 |
| 20140014106 | Smutney et al. | Jan 2014 | A1 |
| 20140017208 | Osei | Jan 2014 | A1 |
| 20140031281 | Wright et al. | Jan 2014 | A1 |
| 20140038891 | Prickett et al. | Feb 2014 | A1 |
| 20140056924 | Van Cauter | Feb 2014 | A1 |
| 20140066368 | Mack et al. | Mar 2014 | A1 |
| 20140083421 | Smutney et al. | Mar 2014 | A1 |
| 20140088003 | Wright et al. | Mar 2014 | A1 |
| 20140100156 | Haack et al. | Apr 2014 | A1 |
| 20140107019 | Erickson et al. | Apr 2014 | A1 |
| 20140107021 | Dimarchi et al. | Apr 2014 | A1 |
| 20140120120 | Woo et al. | May 2014 | A1 |
| 20140121352 | Shechter et al. | May 2014 | A1 |
| 20140128318 | Jung et al. | May 2014 | A1 |
| 20140128604 | Himmelsbach et al. | May 2014 | A1 |
| 20140135348 | Dugi et al. | May 2014 | A1 |
| 20140141467 | Tiwari et al. | May 2014 | A1 |
| 20140142037 | Yue | May 2014 | A1 |
| 20140162943 | Alfaro-Lopez et al. | Jun 2014 | A1 |
| 20140187483 | Steiness | Jul 2014 | A1 |
| 20140200183 | Hathaway et al. | Jul 2014 | A1 |
| 20140206608 | Haack et al. | Jul 2014 | A1 |
| 20140206609 | Haack et al. | Jul 2014 | A1 |
| 20140206613 | Rabinovitch et al. | Jul 2014 | A1 |
| 20140206615 | Knudsen et al. | Jul 2014 | A1 |
| 20140212419 | Dimarchi et al. | Jul 2014 | A1 |
| 20140212440 | Jung et al. | Jul 2014 | A1 |
| 20140213513 | Haack et al. | Jul 2014 | A1 |
| 20140213516 | Chilkoti | Jul 2014 | A1 |
| 20140220029 | Michelsen et al. | Aug 2014 | A1 |
| 20140220134 | Zierhut et al. | Aug 2014 | A1 |
| 20140221280 | Bloom | Aug 2014 | A1 |
| 20140221281 | Haack et al. | Aug 2014 | A1 |
| 20140221282 | Sun et al. | Aug 2014 | A1 |
| 20140227264 | Hamilton et al. | Aug 2014 | A1 |
| 20140235535 | Erickson et al. | Aug 2014 | A1 |
| 20140243263 | Rothkopf | Aug 2014 | A1 |
| 20140249299 | Levy et al. | Sep 2014 | A1 |
| 20140308358 | Oberg et al. | Oct 2014 | A1 |
| 20140309168 | Rosendahl | Oct 2014 | A1 |
| 20140315953 | Leone-Bay et al. | Oct 2014 | A1 |
| 20150011467 | Bloom et al. | Jan 2015 | A1 |
| 20150126440 | Day et al. | May 2015 | A1 |
| 20150164995 | Kadereit et al. | Jun 2015 | A1 |
| 20150164996 | Kadereit et al. | Jun 2015 | A1 |
| 20150164997 | Haack et al. | Jun 2015 | A1 |
| 20150166625 | Haack et al. | Jun 2015 | A1 |
| 20150166627 | Kadereit et al. | Jun 2015 | A1 |
| 20150216941 | Bley et al. | Aug 2015 | A1 |
| 20150232527 | Gong et al. | Aug 2015 | A1 |
| 20150315260 | Bossart et al. | Nov 2015 | A1 |
| 20150322128 | Bossart et al. | Nov 2015 | A1 |
| 20150322129 | Bossart et al. | Nov 2015 | A1 |
| 20150368311 | Haack et al. | Dec 2015 | A1 |
| 20160168225 | Haack et al. | Jun 2016 | A1 |
| 20160235855 | Xiong et al. | Aug 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 101538323 | Sep 2009 | CN |
| 101559041 | Oct 2009 | CN |
| 101663317 | Mar 2010 | CN |
| 101798588 | Aug 2010 | CN |
| 101870728 | Oct 2010 | CN |
| 101601646 | Mar 2011 | CN |
| 102100906 | Jun 2011 | CN |
| 102363633 | Feb 2012 | CN |
| 102421796 | Apr 2012 | CN |
| 101444618 | Jun 2012 | CN |
| 102532301 | Jul 2012 | CN |
| 102649947 | Aug 2012 | CN |
| 102816244 | Dec 2012 | CN |
| 102827270 | Dec 2012 | CN |
| 101670096 | Jan 2013 | CN |
| 103304660 | Sep 2013 | CN |
| 103421094 | Dec 2013 | CN |
| 103665148 | Mar 2014 | CN |
| 103833841 | Jun 2014 | CN |
| 103908657 | Jul 2014 | CN |
| 102766204 | Oct 2014 | CN |
| 104926934 | Sep 2015 | CN |
| 1 140 145 | Jul 2005 | EP |
| 0 619 322 | Dec 2005 | EP |
| 1 609 478 | Dec 2005 | EP |
| 1 143 989 | Dec 2006 | EP |
| 1 658 856 | Mar 2010 | EP |
| 1 684 793 | Sep 2011 | EP |
| 1 633 391 | Oct 2011 | EP |
| 2 387 989 | Nov 2011 | EP |
| 1 633 390 | Jan 2012 | EP |
| 2 494 983 | Sep 2012 | EP |
| 2 626 368 | Aug 2013 | EP |
| 2 664 374 | Nov 2013 | EP |
| 1 817 048 | Feb 2014 | EP |
| 2 769 990 | Aug 2014 | EP |
| 2014-227368 | Dec 2014 | JP |
| 10-2012-0137271 | Dec 2012 | KR |
| 10-2012-0139579 | Dec 2012 | KR |
| 10-2014-0018462 | Feb 2014 | KR |
| 10-2014-0058104 | May 2014 | KR |
| 10-2014-0058387 | May 2014 | KR |
| 10-2014-0130659 | Nov 2014 | KR |
| 10-2014-0133493 | Nov 2014 | KR |
| 2009121626 | Feb 2011 | RU |
| 9619229 | Jun 1996 | WO |
| 9805351 | Feb 1998 | WO |
| 9808871 | Mar 1998 | WO |
| 9830231 | Jul 1998 | WO |
| 9907404 | Feb 1999 | WO |
| 9925727 | May 1999 | WO |
| 9925728 | May 1999 | WO |
| 9934822 | Jul 1999 | WO |
| 9943708 | Sep 1999 | WO |
| 9947160 | Sep 1999 | WO |
| 9964061 | Dec 1999 | WO |
| 0015224 | Mar 2000 | WO |
| 0037098 | Jun 2000 | WO |
| 0041546 | Jul 2000 | WO |
| 00041548 | Jul 2000 | WO |
| 0055119 | Sep 2000 | WO |
| 0066629 | Nov 2000 | WO |
| 0071175 | Nov 2000 | WO |
| 0073331 | Dec 2000 | WO |
| 0151078 | Jul 2001 | WO |
| 0216309 | Feb 2002 | WO |
| 0234285 | May 2002 | WO |
| 0267989 | Sep 2002 | WO |
| 03011892 | Feb 2003 | WO |
| 03020201 | Mar 2003 | WO |
| 03061362 | Jul 2003 | WO |
| 03077851 | Sep 2003 | WO |
| 03084563 | Oct 2003 | WO |
| 03092581 | Nov 2003 | WO |
| 03099314 | Dec 2003 | WO |
| 03101395 | Dec 2003 | WO |
| 03105888 | Dec 2003 | WO |
| 03105897 | Dec 2003 | WO |
| 2004004779 | Jan 2004 | WO |
| 2004004780 | Jan 2004 | WO |
| 2004004781 | Jan 2004 | WO |
| 2004005342 | Jan 2004 | WO |
| 2004012672 | Feb 2004 | WO |
| 2004018468 | Mar 2004 | WO |
| 2004035623 | Apr 2004 | WO |
| 2004045592 | Jun 2004 | WO |
| 2004056313 | Jul 2004 | WO |
| 2004056317 | Jul 2004 | WO |
| 2004089280 | Oct 2004 | WO |
| 2004089985 | Oct 2004 | WO |
| 2004105781 | Dec 2004 | WO |
| 2004105790 | Dec 2004 | WO |
| 2005000222 | Jan 2005 | WO |
| 2005000360 | Jan 2005 | WO |
| 2005012347 | Feb 2005 | WO |
| 2005021022 | Mar 2005 | WO |
| 2005046716 | May 2005 | WO |
| 2005048989 | Jun 2005 | WO |
| 2005049061 | Jun 2005 | WO |
| 2005049069 | Jun 2005 | WO |
| 2005054291 | Jun 2005 | WO |
| 2005077072 | Aug 2005 | WO |
| 2005077094 | Aug 2005 | WO |
| 2005081619 | Sep 2005 | WO |
| 2005102293 | Nov 2005 | WO |
| 2005110425 | Nov 2005 | WO |
| 2005115437 | Dec 2005 | WO |
| 2005117584 | Dec 2005 | WO |
| 2005120492 | Dec 2005 | WO |
| 2006017688 | Feb 2006 | WO |
| 2006024275 | Mar 2006 | WO |
| 2006024631 | Mar 2006 | WO |
| 2006029634 | Mar 2006 | WO |
| 2006037811 | Apr 2006 | WO |
| 2006044531 | Apr 2006 | WO |
| 2006051103 | May 2006 | WO |
| 2006051110 | May 2006 | WO |
| 2006066024 | Jun 2006 | WO |
| 2006069388 | Jun 2006 | WO |
| 2006073890 | Jul 2006 | WO |
| 2006074600 | Jul 2006 | WO |
| 2006083254 | Aug 2006 | WO |
| 2006086769 | Aug 2006 | WO |
| 2006097535 | Sep 2006 | WO |
| 2006110887 | Oct 2006 | WO |
| 2006114396 | Nov 2006 | WO |
| 2006125763 | Nov 2006 | WO |
| 2006134340 | Dec 2006 | WO |
| 2006138572 | Dec 2006 | WO |
| 2007019331 | Feb 2007 | WO |
| 2007022123 | Mar 2007 | WO |
| 2007024700 | Mar 2007 | WO |
| 2007033316 | Mar 2007 | WO |
| 2007033372 | Mar 2007 | WO |
| 2007035665 | Mar 2007 | WO |
| 2007047834 | Apr 2007 | WO |
| 2007047922 | Apr 2007 | WO |
| 2007056362 | May 2007 | WO |
| 2007064691 | Jun 2007 | WO |
| 2007065156 | Jun 2007 | WO |
| 2007067964 | Jun 2007 | WO |
| 2007075534 | Jul 2007 | WO |
| 2007109354 | Sep 2007 | WO |
| 2007120899 | Oct 2007 | WO |
| 2007121411 | Oct 2007 | WO |
| 2007128761 | Nov 2007 | WO |
| 2007133778 | Nov 2007 | WO |
| 2007139941 | Dec 2007 | WO |
| 2007140284 | Dec 2007 | WO |
| 2008021133 | Feb 2008 | WO |
| 2008021560 | Feb 2008 | WO |
| 2008023050 | Feb 2008 | WO |
| 2008038147 | Apr 2008 | WO |
| 2008058461 | May 2008 | WO |
| 2008071972 | Jun 2008 | WO |
| 2008073448 | Jun 2008 | WO |
| 2008081418 | Jul 2008 | WO |
| 2008086086 | Jul 2008 | WO |
| 2008098212 | Aug 2008 | WO |
| 2008101017 | Aug 2008 | WO |
| 2008148839 | Dec 2008 | WO |
| 2008152403 | Dec 2008 | WO |
| 2009020802 | Feb 2009 | WO |
| 2009024015 | Feb 2009 | WO |
| 2009029847 | Mar 2009 | WO |
| 2009030771 | Mar 2009 | WO |
| 2009035540 | Mar 2009 | WO |
| 2009055740 | Apr 2009 | WO |
| 2009055742 | Apr 2009 | WO |
| 2009058662 | May 2009 | WO |
| 2009058734 | May 2009 | WO |
| 2009063072 | May 2009 | WO |
| 2009067268 | May 2009 | WO |
| 2009095479 | Aug 2009 | WO |
| 2009099763 | Aug 2009 | WO |
| 2009113099 | Sep 2009 | WO |
| 2009137078 | Nov 2009 | WO |
| 2009137080 | Nov 2009 | WO |
| 2009143014 | Nov 2009 | WO |
| 2009143285 | Nov 2009 | WO |
| 2009152477 | Dec 2009 | WO |
| 2009153960 | Dec 2009 | WO |
| 2009155257 | Dec 2009 | WO |
| 2009155258 | Dec 2009 | WO |
| 2009158704 | Dec 2009 | WO |
| 2010011439 | Jan 2010 | WO |
| 2010013012 | Feb 2010 | WO |
| 2010043566 | Apr 2010 | WO |
| 2010070251 | Jun 2010 | WO |
| 2010070252 | Jun 2010 | WO |
| 2010070253 | Jun 2010 | WO |
| 2010070255 | Jun 2010 | WO |
| 2010071807 | Jun 2010 | WO |
| 2010096052 | Aug 2010 | WO |
| 2010096142 | Aug 2010 | WO |
| 2010102148 | Sep 2010 | WO |
| 2010120476 | Oct 2010 | WO |
| 2010121559 | Oct 2010 | WO |
| 2010123290 | Oct 2010 | WO |
| 2010133675 | Nov 2010 | WO |
| 2010133676 | Nov 2010 | WO |
| 2010138671 | Dec 2010 | WO |
| 2010142665 | Dec 2010 | WO |
| 2010148089 | Dec 2010 | WO |
| 2011000095 | Jan 2011 | WO |
| 2011006497 | Jan 2011 | WO |
| 2011011675 | Jan 2011 | WO |
| 2011012718 | Feb 2011 | WO |
| 2011020319 | Feb 2011 | WO |
| 2011020320 | Feb 2011 | WO |
| 2011024110 | Mar 2011 | WO |
| 2011039096 | Apr 2011 | WO |
| 2011049713 | Apr 2011 | WO |
| 2011052523 | May 2011 | WO |
| 2011056713 | May 2011 | WO |
| 2011058082 | May 2011 | WO |
| 2011058083 | May 2011 | WO |
| 2011075393 | Jun 2011 | WO |
| 2011075514 | Jun 2011 | WO |
| 2011075623 | Jun 2011 | WO |
| 2011080103 | Jul 2011 | WO |
| 2011084453 | Jul 2011 | WO |
| 2011084456 | Jul 2011 | WO |
| 2011084459 | Jul 2011 | WO |
| 2011087671 | Jul 2011 | WO |
| 2011087672 | Jul 2011 | WO |
| 2011088837 | Jul 2011 | WO |
| 2011094337 | Aug 2011 | WO |
| 2011109784 | Sep 2011 | WO |
| 2011117415 | Sep 2011 | WO |
| 2011117416 | Sep 2011 | WO |
| 2011119657 | Sep 2011 | WO |
| 2011143208 | Nov 2011 | WO |
| 2011143209 | Nov 2011 | WO |
| 2011144751 | Nov 2011 | WO |
| 2011153965 | Dec 2011 | WO |
| 2011156407 | Dec 2011 | WO |
| 2011160630 | Dec 2011 | WO |
| 2011162830 | Dec 2011 | WO |
| 2011163012 | Dec 2011 | WO |
| 2011163272 | Dec 2011 | WO |
| 2011163473 | Dec 2011 | WO |
| 2012012352 | Jan 2012 | WO |
| 2012012460 | Jan 2012 | WO |
| 2012015975 | Feb 2012 | WO |
| 2012031518 | Mar 2012 | WO |
| 2012035139 | Mar 2012 | WO |
| 2012050923 | Apr 2012 | WO |
| 2012059762 | May 2012 | WO |
| 2012064892 | May 2012 | WO |
| 2012080471 | Jun 2012 | WO |
| 2012088116 | Jun 2012 | WO |
| 2012088157 | Jun 2012 | WO |
| 2012122535 | Sep 2012 | WO |
| 2012130015 | Oct 2012 | WO |
| 2012138941 | Oct 2012 | WO |
| 2012140647 | Oct 2012 | WO |
| 2012150503 | Nov 2012 | WO |
| 2012158965 | Nov 2012 | WO |
| 2012162547 | Nov 2012 | WO |
| 2012167744 | Dec 2012 | WO |
| 2012169798 | Dec 2012 | WO |
| 2012173422 | Dec 2012 | WO |
| 2012177443 | Dec 2012 | WO |
| 2012177444 | Dec 2012 | WO |
| 2012177929 | Dec 2012 | WO |
| 2013002580 | Jan 2013 | WO |
| 2013004983 | Jan 2013 | WO |
| 2013009545 | Jan 2013 | WO |
| 2013029279 | Mar 2013 | WO |
| 2013041678 | Mar 2013 | WO |
| 2012174478 | May 2013 | WO |
| 2013060850 | May 2013 | WO |
| 2013074910 | May 2013 | WO |
| 2013078500 | Jun 2013 | WO |
| 2013090648 | Jun 2013 | WO |
| 2013092703 | Jun 2013 | WO |
| 2013093720 | Jun 2013 | WO |
| 2013101749 | Jul 2013 | WO |
| 2013104861 | Jul 2013 | WO |
| 2013148871 | Oct 2013 | WO |
| 2013148966 | Oct 2013 | WO |
| 2013151663 | Oct 2013 | WO |
| 2013151664 | Oct 2013 | WO |
| 2013151665 | Oct 2013 | WO |
| 2013151666 | Oct 2013 | WO |
| 2013151667 | Oct 2013 | WO |
| 2013151668 | Oct 2013 | WO |
| 2013151669 | Oct 2013 | WO |
| 2013151670 | Oct 2013 | WO |
| 2013151671 | Oct 2013 | WO |
| 2013151672 | Oct 2013 | WO |
| 2013151736 | Oct 2013 | WO |
| 2013160397 | Oct 2013 | WO |
| 2013163162 | Oct 2013 | WO |
| 2013164484 | Nov 2013 | WO |
| 2013171135 | Nov 2013 | WO |
| 2013177565 | Nov 2013 | WO |
| 2013186240 | Dec 2013 | WO |
| 2013192130 | Dec 2013 | WO |
| 2014012069 | Jan 2014 | WO |
| 2014016300 | Jan 2014 | WO |
| 2014017843 | Jan 2014 | WO |
| 2014017845 | Jan 2014 | WO |
| 2014017849 | Jan 2014 | WO |
| 2014027253 | Feb 2014 | WO |
| 2014027254 | Feb 2014 | WO |
| 2014041195 | Mar 2014 | WO |
| 2014041375 | Mar 2014 | WO |
| 2014056872 | Apr 2014 | WO |
| 2014073842 | May 2014 | WO |
| 2014073845 | May 2014 | WO |
| 2014081872 | May 2014 | WO |
| 2014091316 | Jun 2014 | WO |
| 2014096145 | Jun 2014 | WO |
| 2014140222 | Sep 2014 | WO |
| 2014152460 | Sep 2014 | WO |
| 2014158900 | Oct 2014 | WO |
| 2014170496 | Oct 2014 | WO |
| 2015055801 | Apr 2015 | WO |
| 2015055802 | Apr 2015 | WO |
| 2015067716 | May 2015 | WO |
| 2015086728 | Jun 2015 | WO |
| 2015086729 | Jun 2015 | WO |
| 2015086730 | Jun 2015 | WO |
| 2015086731 | Jun 2015 | WO |
| 2015086732 | Jun 2015 | WO |
| 2015086733 | Jun 2015 | WO |
| 2015100876 | Jul 2015 | WO |
| 2015104314 | Jul 2015 | WO |
| Entry |
|---|
| US 8,729,011, 05/2014, Dimarchi et al. (withdrawn) |
| WHO Cardiovascular guidelines “Prevention of Cardiovascular Disease: Guidelines for assessment and management of cardiovascular risk” accessed at Mar. 16, 2015 at URL who.intJcardiovascular—diseases/guidelines/Full%20text.pdf. |
| Martin, et al. “Neurodegenation in excitotoxcity, global cerebral ischemia, and target deprivation: A perspective on the contributions of aptopsis and necrosis,” Brain Res. Bull, 46:281-309 (1998). |
| United Healthcare, diabetes, http://www.uhc.com/source4women/health—topics/diabetes/relatedinformation/d0f0417b073bf110VgnVCM1000002f10b10a—.htm—referenced Aug. 22, 2013. |
| eMedicine Health, diabetes causes, http://www.onhealth.com/diabetes—health/page3.htm#diabetes—causes (referenced Aug. 22, 2013). |
| St. John Providence Health Center; Preventing Obesity; http://www.stjohnprovidence.org/HealthInfoLib/swarticle.aspx?type=85&id=P07863 (referenced Aug. 22, 2013). |
| Medline Plus, obesity, available at http://www.nlm.nih.gov/medlineplus/obesity.html—(referenced Aug. 22, 2013). |
| Amylin Pharmaceuticals, Inc. (2007) “Byetta: Exenatide Injection,” Product Information. Accessible on the Internet at URL: http://www.accessdata.fda.gov/drugsaffda—docs/label/2008/021773s012lbl.pdf. [Last Accessed Jun. 2, 2014]. |
| Baggio et al. (2007) “Biology of incretins: GLP-1 and GIP,” Gastroenterology. 132:2131-2157. |
| Bjat et al. (Jun. 1, 2013) “A novel GIP-oxyntomodulin hybrid peptide acting through GIP, glucagon and GLP-1 receptors exhibits weight reducing and anti-diabetic properties,” Biochem. Pharmacol. 85:1655-1662. |
| Bhat et al. (Mar. 17, 2013) “A DPP-IV-resistant triple-acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice,” Diabetologia. 56:1417-1424. |
| Biron et al. (2006) “Optimized selective N-methylation of peptides on solid support,” J. Peptide Sci. 12:213-219. |
| Bis et al. (Jun. 27, 2014) “Antimicrobial preservatives induce aggregation of interferon alpha-2a: the order in which preservatives induce protein aggregation is independent of the protein,” Int. J. Pharm. 472:356-361. |
| Braga et al. (2005) “Making Crystals from Crystals: a green route to crystal engineering and polymorphism,” Chem. Commun. 2005:3635-3645. |
| Bunck et al. (Sep. 2011) “Effects of Exenatide on Measures of B-Cell Function After 3 Years in Metformin-Treated Patients with Type 2 Diabetes,” Diabetes Care. 34:2041-2047. |
| Buse et al. (2009) “Liraglutide once a day versus exenatide twice a day for type 2 diabetes: a 26-week randomised, parallel group, multinational, open-label trial (LEAD-6),” The Lacenet. 374:39-47. |
| Chae et al. (2010) “The fatty acid conjugated exendin-4 analogs for type 2 antidiabetic therapeutics,” Journal of controlled Release. 144:10-16. |
| Chhabra et al. (1998) “An Appraisal of New Variants of Dde Amine Protecting Group for Solid Phase Peptide Synthesis,” Tetrahedron Letters. 39:1603-1606. |
| Creutzfeld et al. (1978) “Gastric inhibitory polypeptide (GIP) and insulin in obesity: increased response to stimulation and defective feedback control of serum levels,” Diabetologia. 14:15-24. |
| Day et al. (2009) “A New Glucagon and GLP-1 co-agonist Eliminates Obesity in Rodents,” Nature Chemical Biology. 5(10):749-757. |
| Deacon (2004) “Circulation and degradation of GIP and GLP-1,” Horm. Metab. Res. 36:761-765. |
| Druce et al. (2009) “Investigation of structure-activity relationships of Oxyntomodulin (Oxm) using Oxm analogs,” Endocrinology. 150(4):1712-1722. |
| Drucker et al. (2010) “Liraglutide,” New Reviews—Drug Discovery. 9(4):267-268. |
| Eng et al. (1992) “Isolation and Characterization of Exendin-4, an Exendin-3 Analogue, from Heloderma Suspectum Venom,” The Journal of Biological Chemistry. 267(11):7402-7405. |
| Eng et al. (1996) “Prolonged Effect of Exendin-4 on Hyperglycemia of db/db Mice,” Diabetes. 45:152A. Abstract 554. |
| Ficht et al. (2008) “Solid-phase Synthesis of Peptide and Glycopeptide Thioesters through Side-Chain-Anchoring Strategies,” Chem. Eur. J. 14:3620-3629. |
| Finan et al. (Dec. 8, 2014) “A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents,” Nat. Med. 21(1):27-36.—with supplementary information. |
| Finan et al. (Oct. 30, 2013) “Unimolecular Dual Incretins Maximize Metabolic Benefits in Rodents, Monkeys, and Humans,” Sci. Trans. Med. 5:209RA151. |
| Furman (Mar. 15, 2012) “The development of Byetta (exenatide) from the venom of the Gilo monster as an anti-diabetic agent,” Toxicon. 59:464-471. |
| Gault et al. (2007) “Chemical gastric inhibitory polypeptide receptor antagonism protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat and cafeteria diets,” Diabetologia. 50:1752-1762. |
| Gault et al. (Aug. 1, 2011) “Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with Type 2 diabetes and obesity,” Clin Sci (Lond). 121:107-117. |
| Gentilella et al. (2009) “Exenatide: A Review from Pharmacology to Clinical Practice,” Diabetes, Obesity, and Metabolism. 11:544-556. |
| Göke et al. (1993) “Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells,” J. Biol. Chem. 268:19650-19655. |
| Hadji-Georgopoulos et al. (1983) “Increased gastric inhibitory polypeptide levels in patients with symptomatic postprandial hypoglycemia,” J. Endocrinol. Metabol. 56(4):648-652. |
| Hargrove et al. (2007) “Biological Activity of AC3174, A Peptide Analog of Exendin-4,” Regulatory Peptides. 141:113-119. |
| Heppner et al. (2010) “Glucagon regulation of energy metabolism,” Physiol. Behav. 100:545-548. |
| Hjorth et al. (1994) “Glucagon and Glucagon-like Peptide 1: Selective Receptor Recognition via Distinct Peptide Epitopes,” The Journal of Biological Chemistry. 269(48):30121-30124. |
| Holst (2007) “The physiology of glucagon-like peptide 1,” Physiol. Rev. 87(4):1409-1439. |
| Kaiser et al. (1970) “Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.” Anal. Biochem. 34:595-598. |
| Kamerzell et al. (2011) “Protein—excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development,” Adv. Drug Deily. Rev. 63:1118-1159. |
| Kazakos et al. (2011) “Incretin effect: GLP-1, GIP, DPP4,” Diabetes Res Clin Pract. 93(Suppl 1):S32-S36. et al. (2011) “Incretin effect: GLP-1, GIP, DPP4,” Diabetes Res Clin Pract. 93(Suppl 1):S32-S36. |
| King et al. (1990) “A Cleavage Method which Minimizes Side Reactions Following Fmoc Solid Phase Peptide Synthesis,” International Journal of Peptide Protein Research. 36:255-266. |
| Knudsen et al. (2000) “Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration” J. Med. Chem. 43(9):1664-1669. |
| Kong et al. (2010) “Long acting hyaluronate—exendin 4 conjugate for the treatment of type 2 diabetes,” Biomaterials. 31:4121-4128. |
| Kosinski et al. (Mar. 16, 2012) “The glucagon receptor is involved in mediating the body weight-lowering effects of Oxyntomodulin,” Obesity (Silver Spring). 20:1566-1571. |
| Krstenansky et al. (1986) “Importance of the 10-13 Region of Glucagon for Its Receptor Interaction and Activation of Adenylate Cyclase,” Biochemistry. 25(13):3833-3839. |
| Lee et al. (May 10, 2013) “Hormonal Response to a Mixed-Meal Challenge After Reversal of Gastric Bypass for Hypoglycemia,” J. Clin. Endocrinol. Metab. 98(7):E1208-E1212. |
| Lorenz et al. (2013) “Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity” Bioorg. Med. Chem. Lett. 23(14):4011-4018. |
| McLaughlin et al. (2010) “Reversible Hyperinsulinemic Hypoglycemia after Gastric Bypass: A Consequence of filtered Nutrient Delivery,” J. Clin. Endocrinol. Metabol. 95(4):1851-1855. |
| Meier (Sep. 4, 2012) “GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus,” Nat. Rev. Endocrinol. 8:728-742. |
| Meier et al. (May 21, 2015) “Incretin-based therapies: where will we be 50 years from now?” Diabetologia. 58:1745-1750. |
| Miyawaki et al. (2002) “Inhibition of gastric inhibitory polypeptide signaling prevents obesity,” Nat. Med. 8(7):738-742. |
| Murage et al. (2008) “Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1,” Bioorg. Med. Chem. 16:10106-10112. |
| Nauck et al. (1993) “Additive insulinotropic effects of exogenous synthetic human gastric inhibitory polypeptide and glucagon-like peptide-1-(7-36) amide infused at near-physiological insulinotropic hormone and glucose concentrations,” J. Clin. Endocrinol. Metab. 76:912-917. |
| Norris et al. (2009) “Exenatide Efficacy and Safety: A Systematic Review,” Diabetic Medicine. 26:837-846. |
| Norwegian Institute of Public Health (Dec. 19, 2013) ATC/DDD Index for Cardiovascular System. |
| Oh et al. (2010) “Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives,” Journal of Controlled Release. 141:2-12. |
| Pan et al. (2006) “Design of a long acting peptide functioning as both a glucagon-like peptide-1 receptor agonist and a glucagon receptor antagonist.” Journal of Biological Chemistry. 281(18):12506-12515. |
| Pedersen et al. (2006) “N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon,” Biochemistry. 45:14503-14512. |
| Pocai (2009) “Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice,” Diabetes. 58 (10):2258-2266. |
| Pocai (Dec. 14, 2013) “Action and therapeutic potential of oxyntomodulin,” Molecular Metabolism 3:2412-51. |
| Rentier et al. (Mar. 26, 2015) “Synthesis of diastereomerically pure Lys(Nε-lipoyl) building blocks and their use in Fmoc/tBu solid phase synthesis of lipoyl-containing peptides for diagnosis of primary biliary cirrhosis,” Journal of Peptide Science. 21(5):408-414. |
| Seddon (2004) “Pseudopolymorph: A polemic,” Crystal Growth and Design. 4(6):1087. |
| Tasyurek et al. (Jul. 2014) “Incretins: Their physiology and application in the treatment of diabetes mellitus,” Diabetes Metab. Res. Rev. 30(5):354-371. |
| Vippagunta et al. (2001) “Crystalline Solids,” Advanced Drug Delivery Reviews. 48:3-26. |
| Vojkovsky (1995) “Detection of secondary amines on solid phase,” Peptide Research 8:236-237. |
| Ward et al. (Nov. 2013) “Peptide lipidation stabilizes structure to enhance biological function,” Mol. Metabol. 2 (41):468-479. |
| Yun et al. (Feb. 2012) “Solution Structure of LXXLL-related Cofactor Peptide of Orphan Nuclear Receptor FTZ-F1.” Bulletin of the Korean Chemical Society, 33(2):583-588. |
| European Search Report corresponding to European Patent Application No. 12172010, dated Apr. 19, 2013. |
| European Search Report corresponding to European Patent Application No. 12306232, dated Apr. 19, 2013. |
| European Search Report corresponding to European Patent Application No. 12306647, dated May 22, 2013. |
| European Search Report corresponding to European Patent Application No. 13505222, dated Jul. 15, 2013. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/062090, issued Nov. 24, 2014. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/070882, issued Dec. 1, 2014. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/077307, issued Feb. 12, 2015. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/077310, issued Feb. 2, 2015. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/077312, issued Feb. 13, 2015. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2013/077313, issued Feb. 12, 2015. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077336, dated Feb. 26, 2016. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077337, dated Jun. 14, 2016. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077338, dated Jun. 14, 2016. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077339, dated Jun. 14, 2016. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077340, dated Jun. 14, 2016. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/EP2014/077341, dated Jun. 14, 2016. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/062090, mailed Feb. 7, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/070882, mailed Dec. 5, 2013. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/077307, mailed Feb. 18, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/077310, mailed Feb. 18, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/077312, mailed Feb. 18, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2013/077313, mailed Feb. 18, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077336, mailed Mar. 18, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077337, mailed Apr. 1, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077338, mailed Mar. 26, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077339, mailed May 11, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077340, mailed Mar. 18, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2014/077341, mailed Mar. 18, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2015/057416, mailed Jun. 22, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2015/057417, mailed Jun. 17, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2015/057418, mailed Jun. 19, 2015. |
| Bromer (1983) “Chemical Characteristics of Glucagon,” Handbook of Experimental Pharmacology. 66:1-22. |
| Chen et al. (Jan. 2014) “Hyaluronic acid-based drug conjugates: state-of-the-art and perspectives,” J. Biomed. Nanotechnol. 10(1):4-16. |
| Donnelly (May 2012) “The structure and function of the glucagon-like peptide-1 receptor and its ligands,” Br. J. Pharmacol. 166(1):27-41. |
| Eng et al. (1990) “Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom,” J. Biol. Chem. 265:20259-20262. |
| Ferry, Jr. “Diabetes Health (cont.),” MedicineNet. Accessible on the Internet at URL: http://www.onhelath.com/diabetes—health/page3.htm. [Last Accessed Aug. 22, 2013]. |
| Herling et al. (1998) “Pharmacodynamic profile of a novel inhibitor of the hepatic glucose-6-phosphatase system,” Am. J. Physiol. 274(6 Pt 1):G1087-G1093. |
| Joshi et al. (2000) “The degradation pathways of glucagon in acidic solutions,” Int. J. Pharm. 203(1-2):115-125. |
| Korczyn et al. (2002) “Emerging Therapies in the Pharmacological Treatment of Parkinson's Disease,” Drugs. 62:775-786. |
| Li et al. (Jul. 25, 2012) “Cloning, expressing of Exendin-4 analogue and bioactivity analysis in vivo,” Chinese Journal of Biotechnology. 28(7):877-886. |
| Liu et al. (2011) “Solid phase peptide synthesis and analysis for exendin-4,” China Biotechnology. 31(2):69-73.—English abstract and drawings. |
| Lozano et al. (2013) “Polyarginine nanocapsules: a new platform for intracellular drug delivery,” Journal of Nanoparticle Research. 15:1515. pp. 1-14. |
| Margolis (2004) “Diagnosis of Huntington Disease,” Clin. Chem. 49:1726-1732. |
| Martin et al. (1998) “Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis,” Brain Res. Bull. 46:281-309. |
| Medline Plus “Obesity,” National Insitute of Health. Accessible on the Internet at URL: http://www.nlm.nih.gov/medlineplus/obesity.html. [Last Accessed Aug. 22, 2013]. |
| Robberecht et al. (1986) “Comparative efficacy of seven synthetic glucagon analogs, modified in position 1, 2 and/or 12, on liver and heart adenylate cyclase from rat,” Peptides. 7(1):109-112. |
| Rovo et al. (May 2014) “Rational design of a-helix-stabilized exendin-4 analogues,” Biochemistry. 53(22):3540-3552. |
| Shiau et al. (1998) “The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen,” Cell. 95(7):927-937. |
| St. John Providence Health System “Preventing Obesity in Children,” St. John Providence Health System. Accessible on the Internet at URL: http://www.stjohnprovidence.org/HealthInfoLib/swarticle.aspx?type=85&id=P07863. [Last Accessed Aug. 22, 2013]. |
| Ueda et al. (2010) “Identification of glycosylated exendin-4 analogue with prolonged blood glucose-lowering activity through glycosylation scanning substitution,” Bioorg. Med. Chem. Lett. 20(15):4631-4634. |
| United Healthcare “Diabetes,” United Healthcare. Accessible on the Internet at URL: http://www.uhc.com/source4women/health—topics/diabetes/relatedinformation/d0f0417b073bf110VgnVCM1000002f10b10a.htm. [Last Accessed Aug. 22, 2013]. |
| Unson et al. (1993) “The role of histidine-1 in glucagon action,” Arch. Biochem. Biophys. 300(2):747-750. |
| World Health Organization (2007) “Prevention of Cardiovascular Disease,” WorldHealth Organization. pp. 1-86. |
| European Search Report corresponding to European Patent Application No. 13306712, dated May 27, 2014. |
| European Search Report corresponding to European Patent Application No. 13306713, dated Jun. 12, 2014. |
| European Search Report corresponding to European Patent Application No. 13306714, dated May 28, 2014. |
| European Search Report corresponding to European Patent Application No. 13306715, dated Jun. 12, 2014. |
| European Search Report corresponding to European Patent Application No. 13306716, dated May 27, 2014. |
| European Search Report corresponding to European Patent Application No. 13306717, dated Jun. 3, 2014. |
| European Search Report corresponding to European Patent Application No. 14305503, dated Sep. 23, 2014. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2015/063607, mailed Septmeber 23, 2015. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2016/062496, mailed Aug. 3, 2016. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2016/063332, mailed Aug. 10, 2016. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2016/063339, mailed Aug. 8, 2016. |
| Stoessl et al. (2008) “Potential therapeutic targets for Parkinson's disease,” Expert Opinion on Therapeutic Targets. 12(4):425-436. |
| European Search Report corresponding to European Patent Application No. 13305222, dated Jul. 15, 2013. |
| International Search Report with Written Opinion corresponding to International Patent Application No. PCT/EP2016/063305, dated Oct. 4, 2016. |
| Bayram et al. (Sep. 2014) “Effects of glucagon-like peptide-1 in diabetic rat small resistance arteries,” Journal of Cardiovascular Pharmacology. 64(3):277-84. |
| Brom et al. (Feb. 1, 2014) “Non-invasive quantification of the beta cell mass by SPECT with 111In-labelled exendin,” Diabetologia. 57(5):950-959. |
| Cai et al. (Dec. 2014) “Rb and p107 are required for alpha cell survival, beta cell cycle control and glucagon-like peptide-1 action,” Diabetologia. 57(12):2555-2565. |
| Charokopou et al. (Nov. 2014) “Cost-effectiveness of saxagliptin compared to GLP-1 analogues as an add-on to insulin in the treatment of type 2 diabetes mellitus from a UK health care perspective,” Value in Health. 17(7):A347. Abstract No. PDB89. |
| Chen et al. (Dec. 14, 2013) “Exendin-4 is effective against metabolic disorders induced by intrauterine and postnatal overnutrition in rodents,” Diabetologia. 57(3):614-622. |
| Choi et al. (Jun. 2014) “A long-acting exendin-4 analog conjugate to the human fcfragment reveals low immunogenic potential,” Diabetes. 63(Suppl 1):A259-A260. Abstract No. 1009-P. |
| Clemmensen et al. (Dec. 30, 2013) “GLP-1/glucagon coagonism restores leptin responsiveness in obese mice chronically maintained on an obesogenic diet,” Diabetes. 63(4):1422-1427. |
| De Marinis et al. (Jun. 2014) “Differential action of GLP-1 and GIP on human pancreatic islet function and viability,” Diabetes. 63(Suppl 1):A52. Abstract No. 196-OR. |
| De Marinis et al. (Sep. 2014) “Differential action of GLP-1 and GIP on human pancreatic islet function and viability,” Diabetologia. 57(Suppl 1):S171. Abstract No. 401. |
| Eriksson et al. (Feb. 10, 2014) “Detection of metastatic insulinoma by positron emission tomography with [(68)ga] exendin-4-a case report,” J. Clin. Endocrinol. Metab. 99(5):1519-1524. |
| Eriksson et al. (May 2014) “Effects of the glucagon-like peptide-1 analog exendin-4 on reendothelialization and intimal hyperplasia formation in an animal model of vascular injury,” Arteriosclerosis, Thrombosis, and Vascular Biology. 34(Suppl 1): Abstract No. 515. |
| Gong et al. (Apr. 18, 2014) “Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors,” Neuropharmacology. 84:31-45. |
| Gupta et al. (Sep. 25, 2014) “Mitigation of autophagy ameliorates hepatocellular damage following ischemia reperfusion injury in murine steatotic liver,” Am. J. Physiol. Gastrointest. Liver Physiol. 307(11):G1088-G1099. |
| Jerlhag et al. (Jun. 2014) “A glucagon like peptide-1 analogue reduces alcohol intake and prevents relapse drinking,” Alcoholism: Clinical and Experimental Research. 38(Suppl 1):85A. Abstract No. 0339. |
| Jin et al. (Jun. 24, 2014) “Dipeptidyl peptidase IV inhibitor MK-0626 attenuates pancreatic islet injury in tacrolimus-induced diabetic rats,” PloS one. 9(6):e100798. pp. 1-10. |
| Johnson et al. (Sep. 5, 2014) “A Potent a/β-Peptide Analogue of GLP-1 with Prolonged Action in Vivo,” Journal of the American Chemical Society. 136(37)12848-12851. |
| Kwon et al. (Sep. 2014) “Pharmacological evaluation of once-weekly potentials by combination of long-acting insulin with long-acting exendin4 in an animal model,” Diabetologia. 57(Suppl 1):S398-S399. Abstract No. 972. |
| Li et al. (Apr. 2014) “Vascular protective effect of exendin-4 in experimental models of oxidative stress,” Cytotherapy. 16(4 Suppl):S37-S38. Abstract No. 115. |
| Li et al. (Nov. 5, 2014) “Exendin-4 promotes endothelial barrier enhancement via PKA-and Epac1-dependent Rac1 activation,” American Journal of Physiology. 308(2):C164-C175. |
| Lim et al. (Nov. 18, 2014) “Evaluation of PEGylated Exendin-4 Released from Poly (Lactic-co-Glycolic Acid) Microspheres for Antidiabetic Therapy,” Journal of Pharmaceutical Sciences. 104(1):72-80. |
| Lovshin et al. (Oct. 2014) “Blood pressure-lowering effects of incretin-based diabetes therapies,” Canadian Journal of Diabetes. 38(5):364-71. |
| Lynch et al. (Jun. 24, 2014) “A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation,” Diabetologia. 57(9):1927-1936. |
| Maas et al. (Oct. 2014) “Impact of the mTOR inhibitor Everolimus on peptide receptor radionuclide therapy in a transgenic neuroendocrine tumor mouse model,” European Journal of Nuclear Medicine and Molecular Imaging. 41(Suppl 2):S529. Abstract No. P593. |
| Masjkur et al. (Nov. 4, 2014) “Hes3 is Expressed in the Adult Pancreatic Islet and Regulates Gene Expression, Cell Growth, and Insulin Release,” The Journal of Biological Chemistry. 289(51):35503-35516. |
| Mondragon et al. (Aug. 13, 2014) “Divergent effects of liraglutide, exendin-4, and sitagliptin on beta-cell mass and indicators of pancreatitis in a mouse model of hyperglycaemia,” PloS one. 9(8):e104873. pp. 1-9. |
| Nagai et al. (Sep. 2014) “Effects of sitagliptin on body fat and intrahepatic lipid content in Japanese overweight patients with type 2 diabetes,” Diabetologia. 57(Suppl 1):S356. Abstract No. 876. |
| Patel et al. (Sep. 29, 2014) “Cannabinoid receptor 1 antagonist treatment induces glucagon release and shows an additive therapeutic effect with GLP-1 agonist in diet-induced obese mice,” Canadian Journal of Physiology and Pharmacology. 92(12):975-983. |
| Pathak et al. (Nov. 6, 2014) “Antagonism of gastric inhibitory polypeptide (GIP) by palmitoylation of GIP analogues with N- and C-terminal modifications improves obesity and metabolic control in high fat fed mice”; Molecular and Cellular Endocrinology. 401:120-129. |
| Pi et al. (2014) “[Clinical research progresses on glucagon-like peptide-1 analogs in treatment of diabetes mellitus],” [Jianyan Yixue Yu Linchuang]. 11(6):830-832.—with English machine translation. |
| Qian et al. (Jun. 19, 2014) “Analysis of the interferences in quantitation of a site-specifically PEGylated exendin-4 analog by the Bradford method,” Analytical Biochemistry. 465C:50-52. |
| Roed et al. (Nov. 22, 2013) “Real-time trafficking and signaling of the glucagon-like peptide-1 receptor,” Mol. Cell Endocrinol. 382(2):938-949. |
| Russell et al. (Jun. 2014) “The novel GLP-1-GLP-2 dual agonist ZP-GG-72 increases intestinal growth and improves insulin sensitivity in DIO mice,” Diabetes. 63(Suppl 1):A98. Abstract No. 374-OR. |
| Schattauer GMBH (Jun. 12, 2014) Meeting Abstracts of the Swiss Society of Radiology and the Swiss Society of Nuclear Medicine 2014. Nuklearmedizin. 53(2):A111-A126. |
| Tashiro et al. (Jan. 10, 2014) “A glucagon-like peptide-1 analog liraglutide suppresses macrophage foam cell formation and atherosclerosis,” Peptides. 54:19-26. |
| Tweedie et al. (May 2014) “Exendin-4, a candidate treatment for the clinical management of traumatic brain injury,” Brain Injury. 28(5-6):549-550. Abstract No. 0101. |
| Vioix et al. (Nov. 2014) “Cost-minimisation analysis of dapagliflozin compared to lixisenatide as an add-on to insulin in the treatment of type 2 diabetes mellitus from a UK health care perspective,” Value in Health. 17(7):A348. Abstract No. PDB95. |
| Wang et al. (Jun. 2014) “Microfluidic multiplexer perifusion device for studying islet immunotoxicity,” Diabetes. 63 (Suppl 1):A555. Abstract No. 2181-P. |
| Wu et al. (May 24, 2014) “(64)Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression,” Theranostics. 4(8):770-777. |
| Xu et al. (Feb. 11, 2014) “Exendin-4 alleviates high glucose-induced rat mesangial cell dysfunction through the AMPK pathway,” Cell. Physiol. Biochem. 33(2):423-432. |
| Xu et al. (Sep. 2014) “Insulinoma imaging with glucagon-like peptide-1 receptor targeting probe (18)F-FBEM-Cys (39)-exendin-4,” Journal of Cancer Research and Clinical Oncology. 140(9): 479-1488. |
| Yang et al. (2014) “Design, synthesis and biological evaluation of novel peptide MC62 analogues as potential antihyperglycemic agents,” European Journal of Medicinal Chemistry. 73:105-111. |
| Yang et al. (Jun. 2014) “Exendin-4, an analogue of glucagon-like peptide-1, attenuates hyperalgesia through serotonergic pathways in rats with neonatal colonic sensitivity,” J. Physiol. Pharmacol. 65(3):349-357. |
| Yosida et al. (May 13, 2014) “Involvement of cAMP/EPACTTRPM2 activation in glucose- and incretin-induced insulin secretion,” Diabetes. 63(10):3394-3403. |
| Zhang et al. (Aug. 2014) “GLP-1 ameliorates the proliferation activity of INS-1 cells inhibited by intermittent high glucose concentrations through the regulation of cyclins,” Molecular Medicine Reports. 10(2):683-688. |
| Aramadhaka et al. (Apr. 18, 2013) “Connectivity maps for biosimilar drug discovery in venoms: The case of Gila Monster Venom and the anti-diabetes drug Byetta®,” Toxicon. 69:160-167. |
| Bhavsar et al. (Mar. 2013) “Evolution of exenatide as a diabetes therapeutic,” Curr. Diabetes Rev. 9(2):161-193. |
| Gao et al. (Jun. 4, 2012) “A site-specific PEGylated analog of exendin-4 with improved pharmacokinetics and pharmacodynamics in vivo,” J. Pharm. Pharmacol. 64(11):1646-1653. |
| Gupta (May 2013) “Glucagon-like peptide-1 analogues: An overview,” Indian J. Endocrinol. Metab. 17(3):413-421. |
| Hou et al. (Jan. 23, 2013) “Long-term treatment with EXf, a peptide analog of Exendin-4, improves β-cell function and survival in diabetic KKAy mice,” Peptides. 40:123-132. |
| Kim et al. (Nov. 9, 2012) “Site-specific PEGylated Exendin-4 modified with a high molecular weight trimeric Peg reduces steric hindrance and increases type 2 antidiabetic therapeutic effects,” Bioconjug. Chem. 23(11):2214-2220. |
| Lee et al. (Oct. 17, 2013) “Decanoic acid-modified glycol chitosan hydrogels containing tightly adsorbed palmityl-acylated exendin-4 as a long-acting sustained-release anti-diabetic system,” Acta Biomater. 10(2):812-820. |
| Parkes et al. (Dec. 12, 2012) “Discovery and development of exenatide: the first antidiabetic agent to leverage the multiple benefits of the incretin hormone, GLP-1,” Expert Opin. Drug Discov. 8(2):219-244. |
| Qian et al. (Jul. 1, 2013) “Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site,” Int. J. Pharm. 454(1):553-558. |
| Simonsen et al. (Jan. 11, 2013) “The C-terminal extension of exendin-4 provides additional metabolic stability when added to GLP-1, while there is minimal effect of truncating exendin-4 in anaesthetized pigs,” Regul. Pept. 181:17-21. |
| Sun et al. (Nov. 6, 2013) “Bifunctional PEGylated exenatide-amylinomimetic hybrids to treat metabolic disorders: an example of long-acting dual hormonal therapeutics,” J. Med. Chem. 56(22):9328-9341. |
| Yim et al. (Aug. 8, 2013) “Synthesis and preclinical characterization of [64Cu]NODAGA-MAL-exendin-4 with a Nε-maleoyl-L-lysyl-glycine linkage,” Nucl. Med. Biol. 40(8)1006-1012. |
| Yue et al. (Jan. 28, 2013) “Development of a new thiol site-specific prosthetic group and its conjugation with [Cys(40)]-exendin-4 for in vivo targeting of insulinomas,” Bioconjug. Chem. 24(7):1191-1200. |
| Number | Date | Country | |
|---|---|---|---|
| 20150322128 A1 | Nov 2015 | US |
