Exendin-4 derivatives as selective glucagon receptor agonists.

DIVISIONAL SUB-DIRECTOR OF PATENT FUND EXAMINATION BIOTECHNOLOGICAL, PHARMACEUTICAL AND CHEMICAL AREAS

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EMELIA HERNÁNDEZ PRIEGO

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DERIVATIVES OF EXENDINA-4 AS SELECTIVE AGONISTS OF

GLUCAGON RECEIVER

Field of the Invention

The present invention relates to exendin4 peptide analogs that activate the glucagon receptor and its medical use, for example in the treatment of severe hypoglycemia.

Background of the invention

Exendin-4 is a 39 amino acid peptide that 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 a glucagon-like peptide-1 receptor (GLP-1) activator, while not significantly activating the glucagon receptor.

The amino acid sequence of exendin-4 is shown in SEQ ID NO: 1

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂

Glucagon is a 29 amino acid peptide that is released into the bloodstream when circulating glucose is low. The amino acid sequence of glucagon is shown in SEQ ID NO 2.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

During hypoglycemia, when blood glucose levels fall below normal, glucagon sends signals to the liver to break down glycogen and release glucose, causing an increase in blood glucose levels to the normal level. Hypoglycemia is a common side effect in diabetics who are treated with insulin due to elevated blood glucose levels. Thus, the most predominant role of glucagon in glucose regulation is to counteract the action of insulin and maintain blood glucose levels.

Glucagon has an isoelectric point of approximately 7 and therefore is only sparingly soluble (&lt;0.2 mg / ml) in the pH range of 4-8. It is very soluble (&gt; 10 mg / ml) at pH values ​​below 3 or above 9 (Bromer, WW, Handbook of Experimental Pharmacology, Vol. 66/1, 1983). Therefore, the glucagon solutions currently available on the market (GlucaGen® HypoKit, Glucagon emergency rescue kit) are acidic and need to be freshly prepared before use due to the chemical and biophysical instability of glucagon in solution a Low pH (Joshi, AB et al, Int. J. Ph. Sci., 203, 115-125, 2000).

The preparation of glucagon formulations with greater stability compared to commercial kit solutions is described in patent applications WO 9947160, WO 12059762, US 2011/0097386, US 2011/0237510, US 2011/049713, WO 12012460, WO 12122535, US 2012/0071817 and WO 13101749, the contents of which are incorporated herein by reference.

The preparation of stabilized glucagon analogs is described in patent applications WO 14016300, WO 11049713, WO 07056362, WO 08086086 and WO 09155257, the contents of which are incorporated herein by reference.

The use of 4-thiazolylalanine at position 1 of a synthetic peptide has been described in WO 07140284 for GLP-1 receptor agonists. In contrast, the 4-thiazolylalanine of the present invention surprisingly provides very active glucagon receptor agonists with reduced activity in the GLP1 receptor when compared to peptides carrying natural histidine at position 1 (native glucagon).

Brief Description of the Invention

Exendin4 analogs are provided herein that potently and selectively activate the glucagon receptor and show greater solubility at an almost neutral pH and greater chemical stability in solution when compared to natural glucagon. All compounds carry the artificial amino acid 4 thiazolylalanine at position 1. This surprisingly results in greater selectivity towards the glucagon receptor versus the GLP1 receptor when identical compounds that differ from each other only at position 1 (Tza at position 1 instead of His) are compared. The present invention therefore provides highly selective glucagon receptor agonists.

The invention provides a peptide compound having the formula (I):

Tza-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X10-Ser-Lys-Gln-X14-GluSer-Arg-Arg-Ala-Gln-X21-Phe-lle-Glu-Trp-Leu- Leu-Ala-X29-GlyPro-Glu-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R¹ (I)

Χ10 represents an amino acid residue selected from Tyr, Leu, Val, lie, Phe, phenylglycine, 1-naphthylalanine, 2-fluorophenylalanine, cyclohexylglycine and ter-leucine

X14 represents an amino acid residue selected from Leu and Nle

X21 represents an amino acid residue selected from Asp and Glu,

X29 represents an amino acid residue selected from Gly and Thr,

R¹ represents OH or NH₂ or a salt or solvate thereof.

The compounds of the invention are glucagon receptor agonists as determined by observing that they are capable of stimulating the formation of intracellular cAMP after binding to the glucagon receptor. The compounds show at least a relative activity of 0.1%, preferably 0.5%, more preferably 1.0% and even more preferably 10.0% compared to that of natural glucagon in the glucagon receptor.

The compounds of the invention also activate the GLP1 receptor as determined by observing that they are capable of stimulating the formation of intracellular cAMP after binding to the GLP1 receptor. The activity of a given compound of this invention (expressed by its relative activity with respect to the activity of GLP1 in the GLP1 receptor) is below 10%, more preferably below 5% and even more preferably below 2 % compared to the activity of the same compound in the glucagon receptor (expressed by its relative activity with respect to the glucagon activity in the glucagon receptor).

Surprisingly, it was found that the peptide compounds of formula I with 4-thiazolylalanine at position 1 showed greater activation of the glucagon receptor and greater selectivity towards activity on the GLP-1 receptor compared to derivatives having a histidine in this position Histidine is the naturally occurring amino acid in glucagon at position 1 and has been shown to be important for the glucagon receptor activation mechanism (Unson, CG et al, Arch. Biochem. Biophys., 300, 747-750 , 1993).

Additionally, the compounds of the invention preferably have a higher solubility at acidic and / or physiological pH values, for example, at pH 4.5 and / or at pH 7.4 at 25 ° C, preferably at least 0.5 mg / ml, more preferably at least 1.0 mg / ml and even more preferably at least 10.0 mg / ml.

In addition, the compounds of the invention preferably have high stability when stored for 14 days at 50 ° C in solution at pH 7.3 (determined by chromatographic analysis as described in the examples). Preferably, the newly formed degradation products are below 40%, more preferably below 30%, even more preferably below 20%.

In one mode, the R group¹ C-terminal is NH₂.

In an additional mode, the R group¹ C-terminal is OH

An additional modality refers to a group of compounds, where

X10 represents Leu,

X14 represents an amino acid residue selected from Leu and Nle,

X21 represents an amino acid residue selected from Asp and

Glu,

X29 represents an amino acid residue selected from Gly and Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents Tyr,

X14 represents an amino acid residue selected from Leu and

Nle,

X21 represents Glu,

X29 represents an amino acid residue selected from Gly and Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents Val,

Χ14 represents Leu,

X21 represents Glu,

X29 represents an amino acid residue selected from Gly and

Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents lie,

X14 represents an amino acid residue selected from Leu and

Nle,

X21 represents Glu,

X29 represents Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents 1-Naphthylalanine,

X14 represents an amino acid residue selected from Leu and Nle,

X21 represents an amino acid residue selected from Asp and Glu,

X29 represents Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents 2-fluorophenylalanine,

X14 represents an amino acid residue selected from Leu and

Nle,

X21 represents Asp,

X29 represents Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents cyclohexylglycine,

X14 represents an amino acid residue selected from Leu and

Nle,

X21 represents an amino acid residue selected from Asp and

Glu,

X29 represents Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Tyr, Leu, Val, lie, phenylglycine, 1-naphthylalanine, 2-fluorophenylalanine and cyclohexylglycine,

X14 represents Leu,

Χ21 represents an amino acid residue selected from Asp and

Glu,

X29 represents an amino acid residue selected from Gly and

Thr,

R¹ represents OH or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Tyr,

Leu, lie, Phe, 1-naphthylalanine, cyclohexylglycine and ter-leucine,

X14 represents Nle,

X21 represents an amino acid residue selected from Asp and

Glu,

X29 represents Thr,

R¹ represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Leu, Phe, 1-naphthylalanine, 2-fluorophenylalanine and cyclohexylglycine,

X14 represents an amino acid residue selected from Leu and

Nle

X21 represents Asp,

X29 represents Thr,

R1 represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Tyr, Leu, Val, lie, phenylglycine, 1-naphthylalanine, cyclohexylglycine and ter-leucine,

X14 represents an amino acid residue selected from Leu and Nle

X21 represents Glu,

X29 represents an amino acid residue selected from Gly and Thr,

R1 represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Tyr, Leu, Val, He, Phe, phenylglycine, 1-naphthylalanine, 2-fluorophenylalanine, cyclohexylglycine and ter-leucine,

X14 represents an amino acid residue selected from Leu and Nle

X21 represents an amino acid residue selected from Asp and Glu,

X29 represents Thr,

R1 represents OH, or a salt or solvate thereof.

An additional modality refers to a group of compounds, where

X10 represents an amino acid residue selected from Tyr, Leu and Val,

X14 represents Leu,

X21 represents Glu,

X29 represents Gly,

R1 represents OH, or a salt or solvate thereof.

Specific examples of the peptide compounds of formula (I) are the compounds with SEQ ID NO: 3-25 as well as the salts or solvates thereof.

Specific examples of the peptide compounds of formula (I) are the compounds with SEQ ID NO: 3, 5, 6, 9, 15, 20, 23, 24 and 25 as well as the salts or solvates thereof.

In certain embodiments, that is, when the compound of formula (I) comprises genetically encoded amino acid residues, the invention additionally provides a nucleic acid (which may be DNA or RNA) encoding said compound, an expression vector comprising such an acid. nucleic acid and a host cell that contains such a nucleic acid or expression vector.

In a further aspect, the present invention provides a composition comprising a compound of the invention mixed 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, for example a pharmaceutically acceptable salt or solvate, for example a hydrate. In a further aspect, the present invention provides a composition for use in a method of medical treatment, specifically in human medicine.

In certain embodiments, the nucleic acid or expression vector can be used as therapeutic agents, for example 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 this invention and their formulation can essentially be used to treat hypoglycemia, increase blood glucose levels, as an adjuvant therapy with insulin, but also to reduce and maintain body weight, as an antidote for beta poisoning. -blockers and calcium channel blockers and to induce temporary relaxation of the gastro-intestinal system for radiological uses.

Detailed description of the invention

Definitions

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 the generally accepted three-letter codes for other amino acids, such as Nle (Norleucine).

In addition, the following codes were used for the amino acids shown in Table 1:

<td>Structure</td><td>Name</td><td>Code</td>

<td>former N .OH FLN TK OR</td><td>Anine L-4-Thiazolyl</td><td>Tza</td>

<td>Q</td><td>L-Cyclohexylglycine</td><td>chg</td>

<td></td><td>L-Phenylglycine</td><td>Phg</td>

<td>To OH γ or</td><td>L-ter-Leucine</td><td>Tle</td>

<td>.0 \ .HX .OH H_Nf OR</td><td>L-2-Fluorophenylalanine</td><td>2F-Phe</td>

<td>OR Ah X^{OH 4N} lT OR</td><td>L-1-Naphthyl Alanine</td><td>1-Nal</td>

The term native exendina-4 refers to native exendina-4 that has the sequence

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (SEQ ID NO: 1).

The invention provides peptide compounds as defined above.

The peptide compounds of the present invention comprise a linear main chain of aminocarboxylic acids connected by means of peptide bonds, that is carboxamide. Preferably, the aminocarboxylic acids are α-aminocarboxylic acids and more preferably Lo-aminocarboxylic acids, unless otherwise indicated. The peptide compounds comprise a sequence of the main chain of 39 aminocarboxylic acids.

For the avoidance of doubt, in the definitions provided in the present description, it is generally intended that the sequence of the peptide radical differ from that of native exendin-4 in at least one of those positions that are established to allow variation. The amino acids of the peptide radical can be considered consecutively numbered from 1 to 39 in the conventional N-terminal to Cterminal direction. The reference to a position within the peptide radical should be considered accordingly, as the reference to positions within the native exendin-4 molecules and others, for example, in the exendin-4, the His at position 1, the Gly in position 2 ..... the Met in position 14, ... and the Being in position 39.

In a further aspect, the present invention provides a composition comprising a compound of the invention as described herein, or a salt or solvate thereof, mixed with a carrier.

The invention also provides the use of a compound of the present invention for use as a medicament, specifically for the treatment of a condition as described in the present description.

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 person skilled in the art is aware of a variety of different methods for preparing peptides 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 the subsequent isolation and purification. A different way of preparing the peptides is gene expression in a host cell in which the DNA sequence encoding the peptide has been introduced. Alternatively, gene expression can be achieved without using a cellular system. The methods described above can also be combined in any way.

A preferred way of preparing 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, III., 1984; E. Atherton and RC Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989). Solid phase synthesis is initiated by anchoring an N-terminally protected amino acid with its carboxy terminus to an inert solid support that carries a cleavage linker. This solid support can be any polymer that allows the initial amino acid to be coupled, for example a trifyl resin, a chlorotrityl resin, a Wang resin or a Rink resin in which the connection of the carboxy group (or carboxamide for the Rink resin) The resin is acid sensitive (when using the Fmoc strategy). The polymeric support must be stable under the conditions used to deprotect the α-amino group during peptide synthesis.

Once 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 presented by the peptide sequence using appropriate amide coupling reagents, for example BOP, HBTU, HATU or DIC (N, N'-diisopropylcarbodide) / HOBt (1-hydroxybenzotriazole), where BOP, HBTU and HATU are used with tertiary amine bases. Alternatively, the N-terminus released can be functionalized with groups other than amino acids, for example carboxylic acids, etc.

Finally, the peptide is cleaved from the resin and deprotected.

This can be achieved using King's cocktail (DS King, CG Fields, GB Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The raw material can then be purified by chromatography, for example preparative RP-HPLC, if necessary.

Power

As used herein, the term "potency" or "in vitro potency" is a measure of the ability of a compound to activate the receptors for GLP-1 or glucagon in a cell-based analysis. Numerically, it is expressed as the EC50 value, which is the effective concentration of compound that induces a semi-maximal increase in response (eg intracellular cAMP formation) in a dose-response experiment.

Therapeutic uses

The compounds of the invention are glucagon receptor agonists. Such agonists may initially provide a therapeutic benefit to address a clinical need to solve hypoglycemia.

Hypoglycemia induced by anti-hyperglycemic medication, for example insulin treatment, is an important risk in the therapy of T1DM and T2DM to maintain glycemic control. Attempting to achieve close glucose control may increase the risk of hypoglycemia in the outpatient and in the critical care setting. In a healthy state, fasting plasma glucose concentrations are normally above 70 mg / dL. If blood sugar levels fall below this threshold, mild hypoglycemia occurs initially with symptoms that can still be self-treated. These symptoms may include weakness, drowsiness, paleness, blurred vision or a feeling of sadness and unhappiness. Hypoglycemic symptoms also depend on the age of the patient and are predominantly neurological in older people while a change in behavior is frequently observed in children. Hypoglycemic events during the night can result in headache in the morning, poor quality of sleep, lived dreams, nightmares, profuse sweating in bed and restless behavior. Sleepwalking has also been reported during nocturnal hypoglycemia. If blood sugar levels fall further, the consequence may be a serious hypoglycemia event. Severe hypoglycemia is associated with a serum glucose value below 40-50 mg / dL and this event may result in neuroglycopenic symptoms such as seizures or coma that require the assistance of a second person. Hypoglycemia can affect the brain resulting in confusion (abnormal behavior or both, such as inability to complete routine tasks), visual disturbances, seizures and sometimes loss of consciousness. The frequent occurrence of hypoglycemia can result in a reduction in perception thereby significantly increasing the risk of severe hypoglycemia. Severe deep and prolonged severe hypoglycemia can result in death, while the potential mechanisms responsible for hypoglycemia-induced death include brain death and cardiac arrhythmias. On average, patients with T1DM experience 2 episodes of symptomatic hypoglycemia per week and 1 episode of severe hypoglycemia per year. The incidence of hypoglycemia in patients with T2DM treated with insulin is approximately one third of that observed in T1DM. This number may increase in patients with a longer duration of insulin treatment, with comorbidities and with the patients' age.

The treatment of hypoglycemia depends on the duration and intensity of the hypoglycemic event. Mild and moderate hypoglycemia can be easily treated by drinking or eating drinks or foods that contain sugar. Severe hypoglycemia on the other hand, requires the help of another person. While intravenous application of a carbohydrate is restricted to health care professionals, the administration of glucagon as rescue medication can be carried out by any person trained through subcutaneous or intramuscular injection. Glucagon is a peptide hormone that is produced by pancreatic alpha cells and released into the bloodstream when circulating glucose is low. As an islet hormone with effects contrary to those of insulin, glucagon is raising blood glucose levels by stimulating gluconeogenesis and glycogenolysis (while simultaneously inhibiting glycolysis and glycogen synthesis) to avoid a hypoglycemic state.

Two commercial glucagon emergency kits are approved as rescue medication for severe hypoglycemia. The Glucagon Emergency Kit (Eli Lilly and Co, Indianapolis, IN) and the GlucaGen® Hypokit® (Novo Nordisk A / S, Bagsvaerd, Denmark). The kits contain a vial of glucagon powder and a syringe loaded with solvent. The glucagon kit has to be reconstituted before use. The solvent is transferred from the syringe to the vial and the vial is shaken until all the solid has dissolved. The solution is suctioned back into the syringe and after removal of air bubbles from the syringe the kit is ready for administration in the leg or abdomen. The recommended dose is 1 mg of glucagon in 1 mL of sterile water for adults and children weighing more than 25 kg and for children 6 to 8 years of age or older. For children under 25 kg or younger from 6 to 8 years of age, half the dose (0.5 mL) is recommended.

The FDA-approved instructions for both commercially available glucagon products allow only immediate use once the lyophilized powder has been reconstituted in the aqueous solution. Due to the complex procedure comprising different stages to dissolve the lyophilized powder carefully and complete an injection, these products need to be administered to the patients by caregivers or relatives of the patients in case of an emergency situation. Based on these requirements, glucagon remains an underutilized therapeutic approach despite its documented advantages to immediately improve hypoglycemia.

An agonistic product of the glucagon receptor with better stability in solution, as described in this invention, could allow a ready-to-use pen-type device suitable for self-injection by the patient. Beyond its advantages as rescue medication, such a product could offer the opportunity to become a therapy component as an insulin counterpart for glucose optimization.

A different application may be the use in an automatic closed loop artificial pancreas control system with an insulin release and dual pump glucagon receptor agonist as described in this invention. Such an implantable system measures blood glucose subcutaneously and insulin is administered to the patient to bring glucose levels back to a normal level. In contrast, a stabilized glucagon receptor agonist is administered by the artificial pancreas system to prevent glucose levels from falling too low.

Accordingly, the compounds of the invention can be used for the treatment of mild to moderate hypoglycemia or in an event of severe hypoglycemia. In addition, the following forms of hypoglycemia could be treated with the compounds of the invention: that induced by anti-diabetic treatments, for example insulin therapy, reactive or post-prandial hypoglycemia, fasting hypoglycemia, alcohol-induced hypoglycemia, post-hypoglycemia gastric bypass, non-diabetic hypoglycemia and hypoglycemia associated with pregnancy.

As outlined above, glucagon is a hormone with acute effects against insulin, which raises blood glucose levels by stimulating gluconeogenesis and glycogenolysis to avoid a hypoglycemic state. However, recent data in rodents and humans reveal that glucagon could also have beneficial effects on energy balance, body fat mass and nutrient intake. Therefore, the compounds of this invention can be used for a variety of conditions or disorders beyond the treatment of hypoglycemia. The compounds of this invention can be used in combination with other active therapeutic drugs. The relevant therapeutic use includes the treatment or prevention of hypoglycemia, both acute and chronic, diabetes mellitus type 2, the delay in the progression of prediabetes to type 2 diabetes, for example in impaired glucose tolerance states and / or impaired fasting glucose, gestational diabetes, type 1 diabetes mellitus, obesity, diseases associated with being overweight or obese, metabolic syndrome / diabetes, cardiovascular diseases, regulation of appetite and satiety in the treatment of appetite disorders, for example bulimia and maintenance of a reduced body weight after a satisfactory weight loss.

For cases of beta-blocker poisoning, in which symptomatic bradycardia and hypotension are present, a high dose of glucagon is considered the first-line antidote. Therefore, an injection of compounds of the present invention can be used as a defense in an overdose of beta blockers and calcium channel blockers.

An extrahepatic effect of glucagon is the relaxation of smooth muscle cells in the gastrointestinal tract, which includes the stomach, duodenum, small intestine and colon. The compounds of the invention and the pharmaceutical formulation thereof can be used as a relaxant of smooth muscle cells combined with diagnostic imaging techniques for the gastro-intestinal tract, for example radiographs, CT scanning, sonography, formation. MRI imaging and nuclear medicine imaging.

Accordingly, the compounds of this invention and their formulation can be used to treat hypoglycemia, increase blood glucose levels, as an adjuvant therapy with insulin, to reduce and maintain body weight, as an antidote for blocker poisoning. beta and calcium channel blockers and to induce temporary relaxation of the gastro-intestinal system for radiological uses.

Pharmaceutical compositions

The term "pharmaceutical composition" indicates a mixture that contains ingredients that are compatible when mixed and that can be administered. A pharmaceutical composition may include one or more medicinal drugs. Additionally, the pharmaceutical composition may include carriers, solvents, adjuvants, emollients, expanders, stabilizers and other components, whether these ingredients are considered active or inactive. Guidelines for experts in the preparation of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, (20^toed.) ed. AR Gennaro AR, 2000, Lippencott Williams &amp; Wilkins.

The exendin-4 peptide derivatives of the present invention, or salts thereof, are administered together with a pharmaceutically acceptable carrier, diluent, or excipient as part of a pharmaceutical composition. A pharmaceutically acceptable carrier is a carrier that is physiologically acceptable while retaining the therapeutic properties of the substance with which it is administered. Conventional acceptable pharmaceutical carriers and their formulations are known to those skilled in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, (20^to ed.) ed. AR Gennaro AR, 2000, Lippencott Williams &amp; Wilkins. An exemplary pharmaceutically acceptable carrier is physiological saline.

Acceptable pharmaceutical carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral administration (including subcutaneous, intramuscular, intravenous, intradermal and transdermal). The compounds of the present invention will typically be administered parenterally.

The term "salt" or "pharmaceutically acceptable salt" represents salts of the compounds of the invention that are safe and effective for use in mammals. Pharmaceutically acceptable salts may include, but are not limited to, acid addition salts and alkaline salts. Examples of acid addition salts include chloride, sulfate, hydrogen sulfate, (hydrogen) phosphate, acetate, citrate, tosylate or mesylate salts. Examples of alkaline salts include salts with inorganic cations, for example alkali metal or alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts and salts with organic cations such as amine salts. Additional examples of pharmaceutically acceptable salts are described in Remington: The Science and Practice of Pharmacy, (20^toed.) ed. AR Gennaro AR, 2000, Lippencott Williams &amp; Wilkins or in Handbook of Pharmaceutical Salts, Properties, Selection and Use, ed. P.

H. Stahl, CG Wermuth, 2002, published jointly by Verlag Helvética Chimica Acta, Zurich, Switzerland and Wiley-VCH, Weinheim, Germany.

The term "solvate" represents complexes of compounds of the invention or salts thereof with solvent molecules, for example organic solvent molecules and / or water.

The term "therapeutically effective amount of a compound" refers to a non-toxic, but sufficient amount of the compound to provide the desired effect. The amount of a compound of formula (I) necessary to achieve the desired biological effect depends on numerous factors, for example, the specific compound selected, the intended use, the mode of administration and the clinical condition of the patient. The appropriate effective amount in an individual case can be determined by a person skilled in the art using routine experimentation.

The pharmaceutical compositions of the invention are those suitable for parenteral administration (for example subcutaneous, intramuscular, intradermal or intravenous), oral, rectal, topical and peroral (for example sublingual), although the most suitable mode of administration depends on each individual case. of the nature and severity of the condition to be treated and 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; in the form of powders or granules; in the form of a solution or suspension in an aqueous or non-aqueous liquid; or in the form of an oil-in-water or water-in-oil emulsion. They can be provided in a single dose injectable form, for example in pen form. The compositions may be prepared, as already mentioned, by any suitable pharmaceutical method that includes a step in which the active ingredient and the carrier (which may consist of one or more additional ingredients) are contacted.

Combination therapy

In addition to its use as a medication for hypoglycemic events, the compounds of the present invention, glucagon receptor agonists, can be widely combined with other pharmacologically active compounds, such as all drugs mentioned in Rote Liste 2014, for example with all antidiabetics mentioned in Rote Liste 2014, chapter 12, all weight-reducing agents or appetite suppressants mentioned in Rote Liste 2014, chapter 1, all lipid lowering agents mentioned in Rote Liste 2014, chapter 58, all antihypertensives and nephroprotectors, mentioned in Rote Liste 2014, or all diuretics mentioned in Rote Liste 2014, chapter 36.

Combinations of active ingredients can be used especially for a synergistic improvement of the action. They can be applied by separate administration of the active ingredients to the patient or in the form of combined products in which a plurality of active ingredients is present in a pharmaceutical preparation. When the active ingredients are administered by separate administration of the active ingredients, this can be performed simultaneously or successively.

Most of the active ingredients mentioned below are described in USP Dictionary of USAN and International Drug Ñames, US Pharmacopeia, Rockville 2011.

Other active substances that are suitable for such combinations include in particular those that add, for example, a therapeutic effect to one or more active substances with respect to one of the aforementioned indications and / or that allow reducing the dosage of one or more active substances. .

Therapeutic agents that are suitable for combinations include, for example, antidiabetic agents such as:

Insulin and insulin derivatives, for example: Glargina / Lantus® (see www.lantus.com). Glulisine / Apidra®, Detemir / Levemir®, Lispro / Humalog® / Liprolog®, Degludec / DegludecPlus, Aspart, basal insulin and the like (eg LY2963016), PEGylated insulin Lispro (LY2605541), Humulin®, Linjeta, SuliXen®, N105 , Insulin plus Symlina, fast-acting and slow-acting insulins (e.g. Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel insulins, oral, inhalable, transdermal and sublingual (e.g. Exúbera®, Nasulin®, Afrezza , Tregopil, TPM 02, Capsulin, Oral-lyn®, Cobalamin® oral insulin, ORMD-0801, NN1953, VIAtab). Additionally, those Insulin derivatives that are bound to albumin or other protein by means of a bifunctional linker such as HM12460A (insulin LAPS) are also included.

GLP-1, GLP-1 analogs and GLP-1 receptor agonists, for example: Lixisenatida / AVE0010 / ZP10 / Lyxumia, Exenatida / Exendina4 / Byetta / Bydureon / ITCA 650, Liraglutida / Victoza, Semaglutida, Taspoglutida, Albiglutida, Dulaglutida, rExendina-4, CJC-1134-PC-PB105423, T1060C, 11210, 112 , CM-3, GLP-1 Choose, ORMD-0901, NN9924, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, MAR-701, ZP2929, ZP-3022, CAM-2036, DA-15864, ARI-2651, ARI-2255, ExenatidaXTEN and Glucagon-Xten, MAR709, HM1525A, dual GLP1 R / GlucagonR agonists, dual GLP1R / GIPR agonists, triple agonists of GLP1 R / GlucagonR / GIPR, combinations of GLP1R agonists with insulin derivatives such as IDegLira, Lixilan etc.

DPP-4 inhibitors, for example: Alogliptin / Nesina,

Linagliptin / BI-1356 / Ondero / Trajenta / Tradjenta / Trayenta / Tradzenta, Saxagliptin / Onglyza, Sitagliptina / Januvia / Xelevia / Tesave /

Janumet / Velmetia, Vildagliptina, Anagliptina, Gemigliptina, Tenegliptina, Melogliptina, Trelagliptina, DA-1229, MK-3102, KM-223.

SGLT2 inhibitors, for example: Canaglifozina, Dapaglifloxina, Remoglifoxina, Sergliflozina, Empagliflozina, I praglifloxina,

Tofoglifloxin, luseoglifloxin, LX-4211, PF-04971729, RO-4998452, EGT-0001442, DSP-3235.

Biguanides (for example Metformin, Buformin, Fenformin), Thiazolidinodiones (for example Pioglitazone, Rivoglitazone, Rosiglitazone, Troglitazone), dual agonists of PPAR (for example Aleglitazar, Muraglitazar, Tesaglitazar), Sulfonyluremide, Glglibyl amide, for example Tolulbidelamide, Glipizide), Meglitinides (for example Nateglinide, Repaglinide, Mitiglinide), Alphaglucosidase inhibitors (for example Acarbose, Miglitol, Voglibosa), Amylin and amylin analogs (eg Pramlintide, Symlina).

GPR119 agonists (for example GSK-263A, PSN-821, MBX2982, APD-597), GPR40 agonists (for example TAK-875, TUG-424, P-1736, JTT-851, GW9508).

Other suitable combination partners are: Cycloset, 11-beta-HSD inhibitors (for example LY2523199, BMS770767, RG4929, BMS816336, AZD-8329, HSD-016, BI-135585), glucokinase activators (for example TTP-399, AMG-151, TAK-329 ), DGAT inhibitors (for example LCQ-908), protein tyrosine phosphatase 1 inhibitors (for example Trodusquemine), glucose-6-phosphatase inhibitors, fructose-1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, inhibitors of phosphoenol pyruvate carboxykinase, glycogen synthase kinase inhibitors, pyruvate dehydrogenase inhibitors, alpha2 antagonists, CCR-2 antagonists.

Also suitable as combination partners are one or more lipid reducing agents, such as for example: HMG-CoA reductase inhibitors (for example Simvastatin, Atorvastatin), fibrates (for example Bezafibrate, Fenofibrate), nicotinic acid and derivatives of same (for example Niacina),

PPAR- agonists or modulators (alpha, gamma or alpha / gamma) (for example Aleglitazar),

PPAR-delta agonists, ACAT inhibitors (for example Avasimibe), cholesterol absorption inhibitors (for example Ezetimibe), bile acid binding substances (for example cholestyramine), inhibitors of ileal bile acid transport, MTP inhibitors , or modulators of PCSK9.

Compounds that raise HDL such as: CETP inhibitors (for example 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 tr of obesity, such as: Sibutramine, Tesofensin, Orlistat, cannabinoid-1 receptor antagonists, MCH-1 receptor antagonists, MC4 receptor agonists, NPY5 or NPY2 antagonists (eg Velneperit), beta-3 agonists, leptin or leptin mimetics, agonists of the 5HT2c receptor (for example Lorcaserina), or the combinations of bupropiona / naltrexone, bupropiona / zonisamide, bupropiona / phentermine or pramlintide / metreleptin.

Other suitable combination partners are:

Other gastrointestinal peptides such as Peptide YY 3-36 (PYY3-36) or analogs thereof, pancreatic polypeptide (PP) or analogues thereof, GIP receptor agonists or antagonists, ghrelin antagonists or inverse agonists, Xenin and analogs thereof .

On the other hand, combinations with drugs that influence high blood pressure, chronic heart failure or atherosclerosis, such as for example: Angiotensin II receptor antagonists (for example 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, neutral endopeptidase inhibitors, thrombocyte aggregation inhibitors and others or combinations thereof.

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, to prepare a medicament that is suitable for the treatment or prevention of diseases or conditions that may be affected by glucagon receptor binding. This is preferably a disease in the context of the metabolic syndrome, specifically one of the diseases or conditions listed above, more specifically diabetes or obesity or complications thereof.

The use of the compounds according to the invention, or a physiologically acceptable salt thereof, combined with one or more active substances can take place simultaneously, separately or successively.

The use of the compound according to the invention, or a physiologically acceptable salt thereof, combined with another active substance can take place simultaneously or at staggered moments, but particularly in a short time. If administered simultaneously, the two active substances are provided to the patient together; if used at staggered times, the two active substances are provided to the patient in a period of less than, or equal to 12 hours, but specifically less than or equal to 6 hours.

Accordingly, in another aspect, this invention relates to a medicament comprising 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 a physiologically acceptable salt or solvate thereof and the additional active substance with which they are to be combined can both be present together in a formulation, for example a tablet or a capsule, a ready formulation for use in an appropriate syringe or device, a lyophilized product that can be reconstituted before injection or separately in two identical or different formulations, for example in the form of a so-called parts kit.

Brief description of the figures

Figure 1

Blood glucose excursions after subcutaneous administration of GCG or SEQ. ID 5 in terminal anesthetized rats. Values ​​are the mean ± SEM, n = 6-8 rats.

Figure 2

Blood glucose excursions after subcutaneous administration of GCG or SEQ. ID 6 in terminal anesthetized rats. Values ​​are the mean ± SEM, n = 6-8 rats.

Figure 3

SEQ effect. ID 5 subcutaneous and human glucagon on blood glucose in dogs.

Figure 4

SEQ effect. ID 5 subcutaneous and intramuscular on blood glucose in dogs.

Figure 5

SEQ effect. Subcutaneous ID 5 vs. I KNOW THAT. ID 6 on blood glucose in dogs.

Methods

The abbreviations used are the following:

<td>2F-Phe</td><td>2-fluorophenylalanine</td>

<td>AA</td><td>amino acid</td>

<td>cAMP</td><td>adenosine cyclic monophosphate</td>

<td>Boc</td><td>tert-butyloxycarbonyl</td>

<td>BOP</td><td>(b.enzotriazol-1 hexafluorophosphate)</td>

yloxy) tris (dimethylamino) phosphonium

<td>BSA</td><td>bovine serum albumin</td>

<td>tBu</td><td>tert-butyl</td>

<td>Chg</td><td>cyclohexylglycine</td>

<td>CTC</td><td>2-chlorotrityl chloride</td>

<td>DEC</td><td>N, N'-diisopropylcarbodiimide</td>

<td>DIPEA</td><td>Ν, Ν-diisopropilet lamina</td>

<td>DMEM</td><td>Dulbecco's modified Eagle medium</td>

<td>DMF</td><td>dimethylformamide</td>

<td>EDT</td><td>ethanedithiol</td>

<td>FBS</td><td>fetal bovine serum</td>

<td>Fmoc</td><td>fluorenylmethyloxycarbonyl</td>

<td>GCG</td><td>glucagon</td>

<td>GLP-1</td><td>glucagon-related peptide 1</td>

<td>HEY YOU</td><td>2- (1 H-7-azabenzotriazol-1-l) hexafluorophosphate</td>

1,1,3,3-tetramethyluronium

<td>HBSS</td><td>Hank's balanced salt solution</td>

<td>HBTU</td><td>2- (1 H-Benzotriazol-1-yl) -1,1,3,3 hexafluorophosphate</td>

tetramethyl uronium

<td>HEPES</td><td>2- [4- (2-Hydroxyethyl) piperazin-1-yl] ethanesulfonic acid</td>

<td>HOBt</td><td>1-hydroxybenzotriazole</td>

<td>HOSu</td><td>N-hydroxysuccinimide</td>

<td>HPLC</td><td>high performance liquid chromatography</td>

<td>Htrf</td><td>homogeneous fluorescence resolved over time</td>

<td>IBMX</td><td>3-¡sobutil-1-methylxant¡na</td>

<td>Nal</td><td>1-naphthylalanine</td>

<td>PBS</td><td>phosphate buffered saline</td>

<td>PEG</td><td>polietilengl cabbage</td>

<td>Phg</td><td>phenylglycine</td>

<td>RP-HPLC</td><td>high performance liquid chromatography in phase</td>

inverse

<td>sc</td><td>subcutaneous</td>

<td>TFA</td><td>trifluoroacetic acid</td>

<td>Tle</td><td>ter-Leucine</td>

<td>TRIS</td><td>tris (hydroxymethyl) -aminomethane</td>

<td>Trt</td><td>trifilo</td>

<td>Tza</td><td>4-thiazolylalanine</td>

<td>UV</td><td>ultraviolet</td>

General synthesis of peptide compounds

Materials:

For the synthesis of solid phase peptides a resin was used

Fmoc-Ser (tBu) -Wang previously loaded. Fmoc-Ser (tBu) Wang resin was purchased from Novabiochem with a load of 0.3 mmol / g.

Natural amino acids protected with Fmoc were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, Iris Biotech or Bachem. The following conventional amino acids were used in all syntheses: Fmoc-L-Ala-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-L-Asn (Trt) -OH, Fmoc-L-Asp (OtBu) - OH, Fmoc-L-Gln (Trt) -OH, Fmoc-L-Glu (OtBu) -OH, Fmoc-Gly-OH, Fmoc-LHis (Trt) -OH, Fmoc-L-lle-OH, Fmoc-L -Leu-OH, Fmoc-L-Lys (Boc) -OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser (tBu) -OH, Fmoc-LThr (tBu) - OH, Fmoc-L-Trp (Boc) -OH, Fmoc-L-Tyr (tBu) -OH, Fmoc-L-ValOH.

In addition, the following special amino acids were purchased from the same suppliers as before: Fmoc-L-Tza-OH, Fmoc-L-Phg-OH, Fmoc-L-Nal-OH, Fmoc-L-2F-Phe-OH, Fmoc -L-Chg-OH, Fmoc-L-Tle-OH

Solid phase peptide syntheses were performed in a Prelude Peptide Synthesizer (Protein Technologies Inc) using conventional Fmoc chemistry and HBTU / DIPEA activation. DMF was used as solvent. Deprotection: 20% piperidine / DMF for 2 x

2.5 min. Washings: 7 x DMF. Coupling 2: 5: 10 AA 200 mM / HBTU 500 mM / DIPEA 2M in DMF. 2 x for 20 min. Washes: 5 x DMF.

All peptides that had been synthesized were cleaved from the resin with the 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 verified by ESI mass spectrometry.

The crude peptides were purified by a conventional preparative RP-HPLC purification procedure.

Purification procedure by general preparative HPLC:

The crude peptides were purified on an Akta Purifier System or on a Jasco Semiprep HPLC System. Preparative HPLC RP-C18 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 used as eluents. Fractions containing the product were collected and lyophilized to obtain the purified product, typically in the form of TFA salt.

Test of solubility and stability of exendin-4 derivatives:

Before the solubility and stability test of a batch of peptide, its content was determined. Therefore, two parameters were investigated, their purity (HPLC-UV) and the amount of salt loading of the batch (ion chromatography).

For the solubility test, the target concentration was 10 mg / mL of pure compound. Therefore, solutions were prepared from solid samples in different buffer systems with a concentration of 10 mg / mL of compound, based on the previously determined content. HPLC-UV was performed after 2 h of gentle agitation of the supernatant, which was obtained by means of 20 min centrifugation at 4000 rpm.

The solubility was then determined in comparison to the maximum UV areas obtained with a starting solution of the peptide at a concentration of 2 mg / mL in pure water or a variable amount of acetonitrile (optical control that the entire compound had dissolved) .

For the solubility test, analytical chromatography was carried out with a Waters UPLC system on a Waters ACQUITY UPLC® CSH ™ C18 1.7 pm (150 x 2.1 mm) at 50 ° C with gradient elution at a flow rate of 0.5 mL / min and was monitored at 210-225 nm. The gradients were adjusted from 20% B (0-3 min) to 75% B (3-23 min) followed by a 98% B washing stage (23.5-30.5) and a balancing period (31- 37 min at 20% B). Buffer A = 0.5% trifluoroacetic acid in water and B = 0.35% trifluoroacetic acid in acetonitrile. Optionally, the LC was coupled to a Waters LCT Premier ESI-TOF mass spectrometer using the positive ion mode.

For the stability test, the target concentration was 1.0 mg / mL of pure compound in TRIS buffer (50 mM) at pH 7.3 containing m-cresol (30 mM), sodium chloride (85 mM) and Polysorbate 20 (8 p.m). The solution was stored for 14 days at 50 ° C. After that time, the solution was analyzed by UPLC.

For the stability test, an UPLC was carried out in a Waters Acquity UPLC H-Class system with a Waters Acquity UPLC BEH130 C18 1.7 pm (2.1 x 100 mm) column at 40 ° C with a gradient elution at a velocity of flow rate of 0.5 mL / min and was monitored at 215 and 280 nm. The gradients were adjusted from 10% B to 90% B over 19.2 min and then to 90% B for 0.8 min. Buffer A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile.

For the determination of the amount of remaining peptide, the maximum areas of the target compound at 0 and t 14 were compared, resulting in "% remaining peptide", following the equation% remaining peptide = [(maximum area peptide at 14 ) x 100] / peptide of the maximum area at 0.

The “% of normalized purity” is defined by means of the% of relative purity on day 14 with respect to the% of relative Purity at 0 following the equation% of normalized purity = [(% of relative purity at 14) x 100)] / % relative purity at 0

The% relative purity at 0 was calculated by dividing the maximum peptide area at 0 by the sum of all the maximum areas at 0 following the equation% relative purity at 0 = [(maximum area at 0) x 100] / sum of all maximum areas at 0

Similarly, the% relative purity at 14 was calculated by dividing the maximum peptide area at 14 by the sum of all the maximum areas at 14 following the equation% relative purity t 14 = [(maximum area at 14) x 100] / sum of all maximum areas at 14

The potential difference between "% normalized purity" and "% remaining peptide" reflects the amount of peptide that did not remain soluble after stress conditions.

This precipitated product includes non-soluble degradation products, polymers and / or fibrils, which have been removed before centrifugation analysis.

Anionic chromatography:

Apparatus: Dionex ICS-2000, pre / column: Ion Pac AG-18 2 x 50 mm (Dionex) / AS18 2 x 250 mm (Dionex), eluent: aqueous sodium hydroxide, flow: 0.38 mL / min, gradient: 0 -6 min: 22 mM KOH, 6-12 min: 22-28 mM KOH, 12-15 min: 28-50 mM KOH, 15-20 min KOH: 22 mM KOH, suppressor: 2 mm ASRS 300, detection: conductivity .

In vitro cell analysis to determine the effectiveness of the glucagon receptor:

The agonism of the compounds for the respective receptor was determined by functional analyzes that measured the cAMP response of the HEK-293 cell lines stably expressing the human GLP-1 or glucagon receptor.

The cAMP content of the cells was determined using a Cisbio Corp. kit (Cat. No. 62AM4PEC) based on HTRF (time-resolved homogeneous fluorescence). For preparation, the cells were divided into T175 culture flasks and grown overnight almost to confluence in the medium (DMEM / 10% FBS). The medium was then removed and the cells washed with PBS lacking calcium and magnesium, followed by treatment with Accutase proteinase (Sigma-Aldrich No. cat. A6964). The detached cells were washed and resuspended in assay buffer (1 x HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cell density was determined. They were then diluted to 400,000 cells / ml and 25 pl aliquots were dispensed in the 96-well plate wells. For measurement, 25 μΙ of test compound in analysis buffer was added to the wells, followed by incubation for 30 minutes at room temperature. After the 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. The in vitro potency of the agonists was quantified by determining the concentrations that caused a 50% activation of the maximum response (EC50).

Blood glucose profile in anesthetized rats:

Method intended to study a test compound in the hepatic glycogenolysis process. The rats had free access to food until the start of the experiment. It can be established that the increase in blood glucose after administration of glucagon (GCG) or GCG mimetic and that lasted approximately 60 to 90 minutes, was the result of hepatic glycogen breakage induced by GCG or GCG mimetic. The effect of GCG mimetic on hepatic glycogenolysis and subsequent hyperglycemic peak in blood was compared with the effect obtained with a subcutaneous bolus injection of GCG at a dose of 30 pg / kg.

Blood glucose levels were analyzed in anesthetized male Wistar rats as previously described (Herling et al. Am J Physiol. 1998; 274: G1087-93). The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg / kg) and ketamine (10 mg / kg) and a tracheotomy was performed. Anesthesia was maintained for up to 5 hours by subcutaneous infusion of sodium pentobarbital (adjusted to the anesthetic depth of the individual animal; approximately 24 mg / kg / h). Body temperature was controlled with a rectal probe thermometer and the temperature was maintained at 37 ° C by means of a heated surgical table. Blood samples for glucose analysis (10 pl) were obtained from the tip of the tail every 15 minutes. The rats were allowed to stabilize their blood glucose levels after surgery for up to 2 hours. Next, GCG was administered subcutaneously as the reference compound, or the test compound. For GCG a dose of 30 pg / kg was used to induce hepatic glycogenolysis. The SEQ test compound. ID 5 was administered at doses of 10, 20 and 30 pg / kg and the SEQ test compound. ID 6 was administered at doses of 10 and 30 pg / kg.

Blood glucose profile in normoglycemic Beagle dogs:

The normoglycemic male Beagle dogs were fasting overnight before and after the entire experiment. The animals were randomized into groups of n = 6 per group. At the precise time min 0 the animals were treated with individual doses of the test compound or native human glucagon as reference compound. Injectable solutions were prepared immediately before the experiment. The test compound was administered as a single injection through three different routes (sc, im and iv) at doses of 1-100 pg / kg. Blood sampling was performed consecutively by means of puncture in the jugular vein (jugularis vein) before drug administration (= 0 min) and after that up to 240 min. Blood glucose was determined enzymatically (hexokinase method) from whole blood, K-EDTA plasma insulin was analyzed with a dog-specific ELISA.

Examples

The invention is further illustrated by means of the following examples.

Example 1

Synthesis of SEQ ID NO: 25

The solid phase synthesis was carried out on a previously charged FmocSer (tBu) -Wang resin. The Fmoc synthesis strategy with HBTU / DIPEA activation was applied. They were used at position 1 Fmoc-Tza-OH and at position 10 Fmoc-Tle-OH in the solid phase synthesis protocol. The peptide was cleaved from the resin with King cocktail (DS King, CG Fields, GB Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by means of preparative HPLC on a Waters column (Sunfire, Prep C18) using a gradient of acetonitrile / water (both buffers with 0.1% TFA).

Finally, the molecular mass of the purified peptide was confirmed by LC-MS.

Example 2

Synthesis of SEQ ID NO: 24

The solid phase synthesis was carried out on a previously charged FmocSer (tBu) -Wang resin. The Fmoc synthesis strategy with HBTU / DIPEA activation was applied. They were used at position 1 Fmoc-Tza-OH and at position 10 Fmoc-Chg-OH in the solid phase synthesis protocol. The resin cleaved peptide with King cocktail (DS King, CG Fields, GB Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by means of preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile / water gradient (both buffers with 0.1% TFA).

Finally, the molecular mass of the purified peptide was confirmed by LC-MS.

Example 3

Synthesis of SEQ ID NO: 5

The solid phase synthesis was carried out on a previously charged FmocSer (tBu) -Wang resin. The Fmoc synthesis strategy with HBTU / DIPEA activation was applied. FmocTza-OH was used in position 1 in the solid phase synthesis protocol. The resin cleaved peptide with King cocktail (DS King, CG Fields, GB Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by means of preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile / water gradient (both buffers with 0.1% TFA).

Finally, the molecular mass of the purified peptide was confirmed by means of LC-MS.

Similarly, the peptides of SEQ ID NO were synthesized:

3-36, see table 2.

Table 2: list of synthesized peptides and comparison of calculated molecular weight vs. found.

<td></td><td>Mass</td><td>Mass</td>

<td>SEQ ID</td><td>calculated</td><td>found</td>

<td> 3</td><td> 4259.68</td><td> 4259.3</td>

<td> 4</td><td> 4229.66</td><td> 4229.8</td>

<td> 5</td><td> 4279.67</td><td> 4279.7</td>

<td> 6</td><td> 4323.73</td><td> 4323.6</td>

<td> 7</td><td> 4259.68</td><td> 4259.8</td>

<td> 8</td><td> 4273.71</td><td> 4273.7</td>

<td> 9</td><td> 4215.63</td><td> 4216.0</td>

<td> 10</td><td> 4293.70</td><td> 4295.1</td>

<td> 11</td><td> 4357.80</td><td> 4358.2</td>

<td> 12</td><td> 4273.71</td><td> 4272.8</td>

<td> 13</td><td> 4293.70</td><td> 4293.7</td>

<td> 14</td><td> 4273.71</td><td> 4274.3</td>

<td> 15</td><td> 4259.68</td><td> 4259.7</td>

<td> 16</td><td> 4273.71</td><td> 4273.4</td>

<td> 17</td><td> 4323.73</td><td> 4323.4</td>

<td></td><td>Mass</td><td>Mass</td>

<td>SEQ ID</td><td>calculated</td><td>found</td>

<td> 18</td><td> 4311.69</td><td> 4311.3</td>

<td> 19</td><td> 4343.70</td><td> 4343.3</td>

<td> 20</td><td> 4293.70</td><td> 4293.5</td>

<td> 21</td><td> 4311.69</td><td> 4311.4</td>

<td> 22</td><td> 4343.76</td><td> 4343.4</td>

<td> 23</td><td> 4285.72</td><td> 4285.2</td>

<td> 24</td><td> 4299.69</td><td> 4299.0</td>

<td> 25</td><td> 4273.65</td><td> 4273.0</td>

<td> 26</td><td> 4242.64</td><td> 4242.5</td>

<td> 27</td><td> 4212.61</td><td> 4212.5</td>

<td> 28</td><td> 4262.56</td><td> 4262.2</td>

<td> 29</td><td> 4306.68</td><td> 4306.6</td>

<td> 30</td><td> 4242.64</td><td> 4240.1</td>

<td> 31</td><td> 4256.66</td><td> 4256.5</td>

<td> 32</td><td> 4276.65</td><td> 4276.2</td>

<td> 33</td><td> 4340.75</td><td> 4340.2</td>

<td> 34</td><td> 4256.66</td><td> 4256.6</td>

<td> 35</td><td> 4242.64</td><td> 4242.5</td>

<td> 36</td><td> 4256.66</td><td> 4254.1</td>

Example 4: Chemical stability and solubility

The chemical solubility and stability of the peptide compounds were evaluated as described in the methods section.

The results are provided in table 3.

Table 3: Chemical stability and solubility

<td>SEQ ID</td><td>Solubility (pH 7.4) [mg / ml]</td><td>Stability (pH 7.3, 50 ° C. 2 w) [% Purity normalized]</td><td>Stability (pH 7.3, 50 ° C. 2w) [% Peptide remaining]</td>

<td> 2</td><td> &lt;0.2</td><td> 70</td><td>n / a</td>

<td> 1</td><td> &gt; 10.0</td><td> 37</td><td> 33</td>

<td> 3</td><td> &gt; 10.0</td><td> 94</td><td> 92</td>

<td> 6</td><td> &gt; 10.0</td><td> 92</td><td> 94</td>

<td> 5</td><td> &gt; 10.0</td><td> 96</td><td> 88</td>

<td> 9</td><td> &gt; 10.0</td><td> 83</td><td> 70</td>

<td> 15</td><td> &gt; 10.0</td><td> 86</td><td> 75</td>

<td> 20</td><td> &gt; 10.0</td><td> 90</td><td> 90</td>

<td> 23</td><td> &gt; 10.0</td><td> 94</td><td> 92</td>

<td> 24</td><td> &gt; 10.0</td><td> 95</td><td> 90</td>

<td> 25</td><td> &gt; 10.0</td><td> 89</td><td> 82</td>

Example 5: In vitro data on the GLP-1 and glucagon receptor

The potency of the peptide compounds in the GLP-1 and glucagon receptors were determined by exposing the cells expressing the human glucagon receptor (hGLUC R) and the receptor of

Human GLP-1 (hGLP-1 R) to the compounds listed at increasing concentrations and measuring the cAMP formed as described in Methods.

The results for Exendina-4 derivatives with activity in the human GLP-1 receptor (hGLP-1 R) and the human glucagon receptor (hGLUC R) are shown in Table 4.

Table 4: EC50 values ​​of exendin-4 peptide analogs at GLP-1 and glucagon receptors (indicated in pM)

<td>SEQ ID NO</td><td>EC50 hGLP-1 R [pM]</td><td>EC50 hGLUC R [p.m]</td>

<td> 1</td><td> 0.4</td><td> &gt;10000000</td>

<td> 2</td><td> 56.6</td><td> 1.0</td>

<td> 3</td><td> 44333.3</td><td> 1.8</td>

<td> 4</td><td> 3300.0</td><td> 0.7</td>

<td> 5</td><td> 2190.0</td><td> 0.6</td>

<td> 6</td><td> 9300.0</td><td> 0.5</td>

<td> 7</td><td> 4190.0</td><td> 2.4</td>

<td> 8</td><td> 5800.0</td><td> 2.2</td>

<td> 9</td><td> 12200.0</td><td> 2.2</td>

<td> 10</td><td> 45000.0</td><td> 6.0</td>

<td> 11</td><td> 11700.0</td><td> 0.9</td>

<td> 12</td><td> 20000.0</td><td> 1.0</td>

<td> 13</td><td> 32100.0</td><td> 3.1</td>

<td>SEQ ID NO</td><td>EC50 hGLP-1 R [pM]</td><td>EC50 hGLUC R [p.m]</td>

<td> 14</td><td> 52900.0</td><td> 1.2</td>

<td> 15</td><td> 34500.0</td><td> 2.2</td>

<td> 16</td><td> 19700.0</td><td> 0.9</td>

<td> 17</td><td> 6940.0</td><td> 1.0</td>

<td> 18</td><td> 25800.0</td><td> 3.1</td>

<td> 19</td><td> 6640.0</td><td> 0.7</td>

<td> 20</td><td> 38900.0</td><td> 3.8</td>

<td> 21</td><td> 49700.0</td><td> 1.4</td>

<td> 22</td><td> 8570.0</td><td> 1.0</td>

<td> 23</td><td> 50700.0</td><td> 3.5</td>

<td> 24</td><td> 8310.0</td><td> 0.7</td>

<td> 25</td><td> 23100.0</td><td> 0.8</td>

Example 6: Comparison Test

A selection of exendin4 derivatives comprising the artificial amino acid 4-thiazolylalanine at position 1 has been tested in comparison with the corresponding compounds having histidine at position 1. Histidine at position 1 is essential for receptor activation. in glucagon but also in many related peptides including GLP-1 and exendin-4. Therefore, it is surprising that the artificial amino acid 4-thiazolylalanine leads to even greater activation of the receptor compared to identical compounds that have natural histidine at position 1. In addition, the activation of the GLP-1 receptor that counteracts the effect of Glucagon is surprisingly reduced by the introduction of the artificial amino acid 4-thiazolylalanine. This leads to even more selective glucagon receptor agonists with a higher GCG / GLP-1 activity ratio. The compounds of the reference pairs and the corresponding EC50 values ​​in the GLP-1 and Glucagon receptors (indicated in pM) are given in Table 5.

Table 5: Comparison of exendin-4 derivatives comprising the artificial amino acid 4-thiazolylalanine at position 1 vs. Exendin-4 derivatives that have the natural amino acid histidine at position 1. EC50 values ​​at the GLP-1 and Glucagon receptors are indicated in pM.

<td>SEQ ID DO NOT</td><td>Amino acid from position 1</td><td>EC50 hGLP-1R</td><td>EC50 hGlucagon-R</td><td>Reason</td>

<td> 2</td><td>His</td><td> 56.6</td><td> 1.0</td><td> 57: 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 3</td><td>Tza</td><td> 44333.3</td><td> 1.8</td><td> 24630 : 1</td>

<td> 26</td><td>His</td><td> 1240.0</td><td> 9.4</td><td> 132 : 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 4</td><td>Tza</td><td> 3300.0</td><td> 0.7</td><td> 4714 : 1</td>

<td> 27</td><td>His</td><td> 80.8</td><td> 1.3</td><td> 62 : 1</td>

<td>SEQ ID DO NOT</td><td>Amino acid from position 1</td><td>EC50 hGLP-1R</td><td>EC50 hGlucagon-R</td><td>Reason</td>

<td> 5</td><td>Tza</td><td> 2190.0</td><td> 0.6</td><td> 3650 : 1</td>

<td> 28</td><td>His</td><td> 52.4</td><td> 1.0</td><td> 52 : 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 6</td><td>Tza</td><td> 9300.0</td><td> 0.5</td><td> 18600: 1</td>

<td> 29</td><td>His</td><td> 145.0 '</td><td> 0.9</td><td> 161 : 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 7</td><td>Tza</td><td> 4190.0</td><td> 2.4</td><td> 1746 : 1</td>

<td> 30</td><td>His</td><td> 1180.0</td><td> 6.5</td><td> 182 :1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 8</td><td>Tza</td><td> 5800.0</td><td> 2.2</td><td> 2636: 1</td>

<td> 31</td><td>His</td><td> 941.0</td><td> 4.1</td><td> 230 : 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 10</td><td>Tza</td><td> 45000.0</td><td> 6.0</td><td> 7500 : 1</td>

<td> 32</td><td>His</td><td> 18700.0</td><td> 12.0</td><td> 1558: 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 11</td><td>Tza</td><td> 11700.0</td><td> 0.9</td><td> 13000: 1</td>

<td> 33</td><td>His</td><td> 159.0</td><td> 1.1</td><td> 145:1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 12</td><td>Tza</td><td> 20000.0</td><td> 1.0</td><td> 20000 : 1</td>

<td> 34</td><td>His</td><td> 363.0</td><td> 1.4</td><td> 259: 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID DO NOT</td><td>Amino acid from position 1</td><td>EC50 hGLP-1R</td><td>EC50 hGlucagon-R</td><td>Reason</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 15</td><td>Tza</td><td> 34500.0</td><td> 2.2</td><td> 15682: 1</td>

<td> 35</td><td>His</td><td> 934.0</td><td> 7.1</td><td> 132 : 1</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 15</td><td>Tza</td><td> 19700.0</td><td> 0.9</td><td> 21888:1</td>

<td> 36</td><td>His</td><td> 358.5</td><td> 1.4</td><td> 256: 1</td>

Example 7: Effect of SEQ.ID 5 and SEQ.ID 6 on glucose release in anesthetized rats after sc injection

During the pretreatment period of 2 hr blood glucose stabilized at a level of approximately 6 mmol / l, representing normal feeding values ​​in rats. The GCG at the dose of 30 pg / kg caused a rapid increase in blood glucose, which peaked after 30 minutes at blood glucose levels of approximately 10 to 11 mmol / l. The SEQ test compound. ID 5 at doses of 10, 20 and 30 pg / kg subcutaneously caused a dose-dependent increase in blood glucose, which reached a maximum of 30, 45 and 90 min after injection, respectively. The dose of 20 pg / kg of SEQ. ID 5 demonstrated an almost comparable form of blood glucose excursion compared to 30 pg / kg of GCG (Figure 1).

The SEQ test compound. ID 6 at doses of 10 and 30 pg / kg caused a dose-dependent increase in blood glucose, which reached a maximum of 30 and 60 min after injection, respectively. The dose of 10 pg / kg of SEQ. ID 6 demonstrated a more potent blood glucose excursion compared to 30 pg / kg of GCG (Figure 2).

Example 8: Effect of SEQ. ID. 5 and SEQ. ID 6 on glucose release in normogenic Beagle dogs after sc injection

In animals and humans, glucagon injection leads to rapid recruitment of hepatic glycogen that breaks down immediately to glucose. This results in an acute but short-term increase in blood glucose. In normoglycemic Beagle dogs, subcutaneous (sc) injection of 1 pg / kg of human glucagon leads to a rapid increase in blood glucose by 2-3 mmol / L at 15 min. of the sc injection of SEQ. ID 5 and SEQ. ID 6 measures the effect of human glucagon on blood glucose. In dogs, the total net glucose response (change in blood glucose AUC (0-240 min) from the initial moment) after the injection of 1 pg / kg sc of SEQ. ID 5 was similar to that of 1 pg / kg sc of human glucagon. The blood glucose response to SEQ. ID 5 increased depending on the dose until a maximum increase of ~ 3.5-4 mmol / L was reached with 10 pg / kg sc (Figure 3). Beyond this higher dose of SEQ. ID 5 sc did not produce any higher glucose excursion. In dogs, the beginning of the glucose response to SEQ. ID 5 sc was similar to that of human glucagon, although the duration of the glucose response was slightly longer. I KNOW THAT. ID 5 was active in all parenteral routes since subcutaneous, intramuscular and intravenous injections resulted in a rapid and transient increase in blood glucose increase. There was no difference in activity and action profile in blood glucose time between subcutaneous and intramuscular injections of SEQ. ID 5 in dogs (figure 4).

Regarding the induction of a blood glucose response, SEQ. ID 5 and SEQ. ID 6 were similarly active in normoglycemic dogs (figure 5).

Table 10: Sequences

<td>I KNOW THAT. ID</td><td>Sequence</td>

<td> 1</td><td>HGEGTFTSDLSKQMEEEAVRL-FlEWLKNG- GPSSGAPPPS-NH2</td>

<td> 2</td><td>HSQGTFTSDYSKYLDSRRAQD-FVQWLMN- TO H</td>

<td> 3</td><td>Tza-SQGTFTSDLSKQ-Nle-ESRRAQDFIEWLL- ATGPESGAPPPS-OH</td>

<td> 4</td><td>Tza-SQGTFTSDLSKQLESRRAQEF-IEWLLA- GGPESGAPPPS-OH</td>

<td> 5</td><td>Tza-SQGTFTSDYSKQLESRRAQEF-IEWLLA- GGPESGAPPPS-OH</td>

<td> 6</td><td>Tza-SQGTFTSDYSKQLESRRAQEF-IEWLL-AT-GPESGAPPPS-OH</td>

<td>I KNOW THAT. ID</td><td>Sequence</td>

<td> 7</td><td>Tza-SQGTFTSDVSKQLESRRAQEF-IEWLL-AT-GPESGAPPPS-OH</td>

<td> 8</td><td>Tza-SQGTFTSDISKQLESRRAQEF-IEWLL-AT-GPESGAPPPS-OH</td>

<td> 9</td><td>Tza-SQGTFTSDVSKQLESRRAQEF-IEWLLA- GGPESGAPPPS-OH</td>

<td> 10</td><td>Tza-SQGTFTSD-Phg-SKQLESRRAQEFIEWLL- ATGPESGAPPPS-OH</td>

<td> 11</td><td>Tza-SQGTFTSD-1Nal-SKQLESRRAQEFlEWL- LATGPESGAPPPS-OH</td>

<td> 12</td><td>Tza-SQGTFTSDLSKQLESRRAQEF-IEWLLA- TGPESGAPPPS-OH</td>

<td> 13</td><td>Tza-SQGTFTSDFSKQ-Nle-ESRRAQDFIEWLL- ATGPESGAPPPS-OH</td>

<td> 14</td><td>Tza-SQGTFTSDISKQ-Nle-ESRRAQEFIEWLL- ATGPESGAPPPS-OH</td>

<td> 15</td><td>Tza-SQGTFTSDLSKQLESRRAQDF-IEWLL-AT-GPESGAPPPS-OH</td>

<td> 16</td><td>Tza-SQGTFTSDLSKQ-Nle-ESRRAQEFIEWLL- ATGPESGAPPPS-OH</td>

<td> 17</td><td>Tza-SQGTFTSDYSKQ-Nle-ESRRAQEFIEWLL- ATGPESGAPPPS-OH</td>

<td>I KNOW THAT. ID</td><td>Sequence</td>

<td> 18</td><td>Tza-SQGTFTSD-2FPhe-SKQ-Nle-ESRRAQDFl-EW-LLATGPESGAPPPS-OH</td>

<td> 19</td><td>Tza-SQGTFTSD-1Nal-SKQ-Nle-ESRRAQDFlEW- LLATGPESGAPPPS-OH</td>

<td> 20</td><td>Tza-SQGTFTSD-Chg-SKQ-Nle-ESRRAQDFIEW- LLATGPESGAPPPS-OH</td>

<td> 21</td><td>Tza-SQGTFTSD-2FPhe-SKQLESRRAQDFlEW- LLATGPESGAPPPS-OH</td>

<td> 22</td><td>Tza-SQGTFTSD-1Nal-SKQLESRRAQDFlEW-LL-ATGPESGAPPPS-OH</td>

<td> 23</td><td>Tza-SQGTFTSD-Chg-SKQLESRRAQDFIEWL- LATGPESGAPPPS-OH</td>

<td> 24</td><td>Tza-SQGTFTSD-Chg-SKQ-Nle-ESRRAQEFIEW- LLATGPESGAPPPS-OH</td>

<td> 25</td><td>Tza-SQGTFTSD-Tle-SKQ-Nle-ESRRAQEFIEWL- LATGPESGAPPPS-OH</td>

<td> 26</td><td>HSQGTFTSDLSKQ-Nle-ESRRAQDFIEWLL-AT-GPESGAPPPS-OH</td>

<td> 27</td><td>HSQGTFTSDLSKQLESRRAQE-FlEWLLAG- GPESGAPPPS-OH</td>

<td> 28</td><td>HSQGTFTSDYSKQLESRRAQE-FlEWLLA- GGPESGAPPPS-OH</td>

<td>I KNOW THAT. ID</td><td>Sequence</td>

<td> 29</td><td>HSQGTFTSDYSKQLESRRAQE-FlEWLLA-TG-PESGAPPPS-OH</td>

<td> 30</td><td>HSQGTFTSDVSKQLESRRAQE-FlEWLLAT- GPESGAPPPS-OH</td>

<td> 31</td><td>HSQGTFTSDlSKQLESRRAQE-FlEWLLAT- GPESGAPPPS-OH</td>

<td> 32</td><td>HSQGTFTSD-Phg-SKQLESRRAQEFIEWLL- ATGPESGAPPPS-OH</td>

<td> 33</td><td>HSQGTFTSD-1Nal-SKQLESRRAQEFlEWLL- ATGPESGAPPPS-OH</td>

<td> 34</td><td>HSQGTFTSDLSKQLESRRAQE-FlEWLLAT- GPESGAPPPS-OH</td>

<td> 35</td><td>HSQGTFTSDLSKQLESRRAQD-FlEWLLAT- GPESGAPPPS-OH</td>

<td> 36</td><td>HSQGTFTSDLSKQ-NIe-ESRRAQEFlEWLLA- TGPESGAPPPS-OH</td>

CLAIMS