DE4314260A1 - Peptides for the treatment of hypoglycaemia, process for the preparation thereof and the use thereof - Google Patents

Abstract

Peptides of the formula I, H2N-A-B-C-D-Leu-E-F-Tyr, containing 2 to 8 amino acid residues, are suitable for the treatment of hypoglycaemia. These peptides are used in particular for the treatment of physiological states characterized by a blood glucose content which is too low.

Description

Hypoglycemia is a life-threatening condition that increases in the US alone leads to approximately 20,000 emergency admissions to hospitals each year. About 10% Type I diabetics (approximately 100,000 in the United States) suffer at least once per Year of hypoglycemia so that hospital treatment is required. 25% of all diabetics with insulin suffer at least once a year at this condition. It is known that certain short peptides can have an insulin-like effect (EP 0 491 361).

It has now been found that certain peptides are able to To reduce glucose transport and lipogenesis from glucose.

The invention relates to a peptide of the formula I

H₂N-A-B-C-D-Leu-E-F-Tyr (I)

containing 2 to 8 amino acid residues and physiologically acceptable salts of the peptide of formula I; is there
A is a covalent bond or an amino acid residue of Ser, Thr, Asn or Asp, optionally substituted one or more times by aminoalkyl mercaptan,
B is a covalent bond or an amino acid residue of Leu, Arg or Ile, optionally mono- or polysubstituted by aminoalkyl mercaptan,
C is a covalent bond or an amino acid residue of Tyr, His, Asn or Phe, optionally substituted one or more times by aminoalkyl mercaptan,
D is a covalent bond or an amino acid residue of Gln or Asp, optionally substituted one or more times by aminoalkyl mercaptan,
Leu is a covalent bond or an amino acid residue of the amino acid leucine, optionally substituted one or more times by aminoalkyl mercaptan,
E is a covalent bond or an amino acid residue of Glu, Met or Gln, optionally substituted one or more times by aminoalkyl mercaptan,
F is an amino acid residue of Asn or Ser, optionally substituted one or more times by aminoalkyl mercaptan, and
Tyr the amino acid residue of the amino acid tyrosine, optionally substituted one or more times by aminoalkyl mercaptan,
wherein at least one amino acid residue is substituted by an aminoalkyl mercaptan.

Peptides of the formula I are preferred; containing 2 to 4 amino acid residues
A is a covalent bond or an amino acid residue of Ser or Thr, optionally substituted once by amino- (C₁-C₆) alkyl mercaptan,
B is a covalent bond or an amino acid residue of Leu or Arg, optionally substituted once by amino- (C₁-C₆) alkyl mercaptan,
C is a covalent bond or an amino acid residue of Tyr, His or Phe, optionally substituted by amino- (C₁-C₆) alkyl mercaptan,
D is a covalent bond or an amino acid residue of Gln or Asp, optionally substituted by amino- (C₁-C₆) alkyl mercaptan,
Leu is a covalent bond or an amino acid residue of the amino acid leucine, optionally substituted by aminoethyl mercaptan,
E is a covalent bond or an amino acid residue of Glu or Gln, optionally substituted by amino- (C₁-C₆) alkyl mercaptan,
F is an amino acid residue of Asn or Ser, optionally substituted by amino- (C₁-C₆) alkyl mercaptan, and
Tyr the amino acid residue of the amino acid tyrosine, optionally substituted one or more times by amino- (C₁-C₆) alkyl mercaptan,
wherein at least one amino acid residue is substituted by an amino (C₁-C₆) alkyl mercaptan.

Peptides of the formula II are particularly preferred

NH₂-Leu-E-F-Tyr (II);

is there
E is an amino acid residue of Glu, Gln or Met, optionally substituted by an amino (C₁-C₆) alkyl mercaptan,
F is an amino acid residue of Asn or Ser, optionally substituted by an amino (C₁-C₆) alkyl mercaptan, and
Tyr the amino acid residue of the amino acid tyrosine, once substituted by an amino (C₁-C₆) alkyl mercaptan.

A peptide of the formula III is particularly preferred

NH₂-Leu-Glu-Asn-Tyr-R¹ (III)

wherein R¹ is an amino (C₁-C₆) alkyl mercaptan.

Under the term amino (C₁-C₆) alkyl mercaptan are straight-chain or branched hydrocarbons understood to have 1 to 6 carbon atoms contain and which one or more times with an amino group and an SH- Group are substituted, e.g. B. 2-mercaptoethylamine or 2,2- Diaminodiethyl disulfide.

With the term amino acid are the stereoisomeric forms, i. H. D- and L- Forms of the following connections are meant:

Asparagine, arginine, aspartic acid, glutamine, glutamic acid, histidine, Isoleucine, leucine, methionine, phenylalanine, serine, threonine and tyrosine. L-amino acids are preferred. Amino acid residues are derived from the corresponding amino acids and are part of an amino acid chain. The amino acids were briefly spelled in the usual way Spelling. According to their chemical nature, peptides are acid amides and break down into amino acids during hydrolysis.  

The bond between the amino acid residues is a peptide bond. The Bond between the amino acid residues and the amino (C₁-C₆) alkyl mercaptan takes place via

• a) the amino residue,
• b) the alkyl radical or
• c) the mercapto residue

of the amino (C₁-C₆) alkyl mercaptan.

Among physiologically acceptable salts of the peptides of formula I are to understand in particular pharmaceutically usable. Such salts will be e.g. B. of compounds of formula I, which acidic groups, for. B. carboxyl, included, formed with alkali or alkaline earth metals, such as. B. Na, K, Mg and Ca, as well as with physiologically compatible organic amines, such as. B. Triethylamine and tris- (2-hydroxy-ethyl) amine. Compounds of formula I which basic groups, e.g. B. contain an amino group or a guanidino group, form with inorganic acids, such as. B. hydrochloric acid, sulfuric acid or Phosphoric acid, and with organic carboxylic or sulfonic acids, such as. B. Acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid, salts. Compounds in which basic and acidic Groups of the same number form internal salts.

The invention further relates to a process for the preparation of peptides Formula I or its salts, which is characterized in that

• a) a segment with a C-terminal free carboxyl group or its activated derivative with a corresponding segment with N-terminal free amino group or
• b) the peptide builds up gradually, and

optionally one or more in the compound obtained according to (a) or (b) to protect other functions temporarily splits off a protective group and the compounds of the formula I, II or III thus obtained, if appropriate in them transferred physiologically acceptable salt.  

The peptides according to the invention are according to the general methods of Peptide chemistry gradually from the C-terminal end or by coupling Segments produced (Houben-Weyl, Methods of Organic Chemistry, Volume 15 / 1.2). The peptide couplings can e.g. B. according to the mixed method Anhydrides, via active esters, azides or by the carbodiimide method, especially with the addition of reaction accelerating and racemization-preventing substances, such as. B. 1-hydroxybenzotriazoI, N- Hydroxysuccinimide, 3-hydroxy-4-oxo-3.4-dihydro-1.2.3-benzotriazine, N- Hydroxy-5-norbornene-2,3-dicarboximide, further using active Derivatives of 1-hydroxybenzotriazole or anhydrides of phosphorus, phosphonic and phosphinic acids at a reaction temperature of -10 ° C to Boiling point of the solvent, preferably from -5 ° C to 40 ° C. become.

Suitable solvents for this are dimethylformamide, dimethylacetamide, N- Methyl pyrrolidone or dimethyl sulfoxide. If the solubility of the components it also allows solvents such as methylene chloride, chloroform or Tetrahydrofuran can be used.

If for the prevention of side reactions or for the synthesis more specific Required peptides are the functional groups in the side chain of Amino acids are additionally protected by suitable protective groups, with in primarily Arg (Tos), Arg (Mts), Arg (Mtr), Arg (PMV), Asp (OBzl), Asp (OBut), Cys (4-MeBzl), Cys (Asm), Cys (SBut), Glu (Obzl), Glu (Obut), His (Tos), His (Fmoc), His (Dnp), His (Trt), Lys (Cl-Z), Lys (Boc), Met (O), Ser (Bzl), Ser (But), Thr (But), Trp (Mts), Trp (CHO), Tyr (Br-Z), Tyr (Bzl) or Tyr (But) are used become.

Preferred amino protecting groups are those by catalytic hydrogenation cleavable benzyloxycarbonyl (Z) radical which can be cleaved by weak acids 2- (3,5-dimethyloxyphenyl) propyl (2) oxycarbonyl (Ddz) or trityl (Trt) radical and the 9-fluorenylmethyloxycarbonyl- (Fmoc) - which can be split off by secondary amines  Rest used.

The compounds of formula I and their physiologically tolerable salts primarily serve as active ingredients for pharmaceutical preparations Treatment of hypoglycaemia, i.e. physiological conditions caused by blood sugar levels are too low. Hypoglycaemia can be caused, for example, by increased concentrations of Insulin, insulin-like growth factor, sulfonylureas or glibenclamides.

The invention therefore also relates to a pharmaceutical preparation a content of at least one compound of the formula I and / or at least one of their physiologically tolerated salts in dissolved, amorphous and / or crystalline form.

The preparations according to the invention are produced by at least one peptide of formula I with further auxiliaries and / or carriers in a suitable dosage form is brought.

The auxiliaries and carriers come from the group of carriers, Preservatives and other common auxiliary substances.

The preparation has a pH dissolved or suspended in water. Value from about 2.5 to 9.0, in particular from about 4.0 to 8.5, contains a suitable isotonic agent, a suitable preservative and, if appropriate, a suitable buffer and other auxiliary substances.

The entirety of the preparation components apart from the active ingredient form the Preparation carrier.

Suitable isotonic agents are e.g. B. glycerin, glucose, mannitol, NaCl, calcium or magnesium compounds such as CaCl₂ or MgCl₂.  

The auxiliary substances include starches such as. B. potato, corn or Wheat starch, cellulose or their derivatives, in particular microcrystalline Cellulose, silicon dioxide, various sugars such as milk sugar and mannose, Magnesium carbonate and / or calcium phosphate. It is also advantageous to Preparations add additives that affect the taste and smell of the Improve preparations, e.g. B. such as citric acid, vanilin, honey or Bitter almond oil.

Suitable preservatives are e.g. B. phenol, m-cresol, benzyl alcohol, Benzalkonium chloride and / or p-hydroxybenzoic acid ester.

As buffer substances, in particular for setting a pH of about 4.0 to 8.5, e.g. As sodium acetate, sodium citrate or sodium phosphate be used. Otherwise, to adjust the pH value too physiologically harmless diluted acids (typically hydrochloric acid) or Alkali (typically caustic soda) are suitable.

For the purpose of varying the active profile of the preparation according to the invention modified (EP 132 769, EP 132 770) and also unmodified insulin, preferably cattle, pork or human insulin, especially human Insulin.

Preferred drug concentrations are those corresponding to about 1 to 1500, more preferably about 5 to 1000 and in particular about 40 to 400 international units insulin / ml.

The preparations according to the invention can be administered parenterally, not parenterally, e.g. B. applied through the nose, lungs, stomach, intestine or skin, and as an implant become.

The preferred forms of use in humans are intranasal Application or the use of implants.  

Using a dosing atomizer, about 0.02-0.2 ml a buffer solution in which the required amount of the active substance is dissolved in sprayed the nose. In the case of parenteral administration, the dosages can be reduced by about a power of ten compared to the intranasal dose.

The dosage of the peptides according to the invention to be used depends on various factors such as dosage form of the drug and Application type.

The single dose in implants is approximately 1 to 15 mg for a period of 4-8 weeks each. Applied parenterally, 0.5 to 5 mg / day are sufficient.

The invention is explained in more detail below with the aid of examples. The Percentages refer to volume percentages, unless otherwise specified.

example 1 Ddz-Leu-Glu (OtBu) -Asn-Tyr (tBu) -NH-CH₂-CH₂-S-Trt 5 mmol approach

A solution of 4.4 g of Ddz-Leu-Glu (OtBu) -Asn-Tyr (tBu) -OH, 1.65 g of S-trityl cysteamine and 0.82 g of HOObt in 40 ml of dimethylformamide is added at 0 ° C 1.1 g of DCCl are added, the mixture is stirred at 0 ° C. for 60 minutes and left at room temperature overnight. The precipitate is filtered off and the filtrate is concentrated. The residue is dissolved in 100 ml of ethyl acetate and extracted successively with 70 ml of KHSO₄ / K₂SO₄ buffer, saturated NaHCO₃ solution and water. After drying over Na₂SO₄ the ethyl acetate solution is concentrated. The oily residue is dissolved in 30 ml of methanol and slowly added dropwise in 450 ml of water. After standing overnight, the precipitate is filtered off and dried over P₂O₅ under reduced pressure. Yield: 3.69 g. For further purification, the substance is chromatographed on silica gel. A methylene chloride / methanol mixture (965:35) was used as the eluent.
Yield: 1.76 g
Melting point 148-150 ° C
[α] 22 = -18.5 ° (c = 1, in methanol).

Example 2 H-Leu-Glu-Asn-Tyr-NH-CH₂-CH₂-SH

1.57 g of Ddz-Leu-Glu (OtBu) -Asn-Tyr (tBu) -NH-CH₂-CH₂-Trt is dissolved in a mixture of 20 ml of trifluoroacetic acid and 20 ml of ethyl mercaptan. After 4 hours at room temperature, the contents are poured onto 200 ml of water. This mixture is extracted three times with diethyl ether and freeze-dried. The raw substance is chromatographed on Sephadex LH20 (100 × 2.5 cm). A mixture of 4.3 liters of water, 430 ml of n-butanol and 350 ml of glacial acetic acid serves as the eluent.
Yield 510 mg
[α] 22 = -21.9 ° (c = 1, in water).

Example 3

The biological activity of the peptides of formula I according to the invention is determined from preparative isolated fat cells from the rat.

Rat fat cells were prepared as follows:
Epididymal adipose tissue (Wistar rat, 160-180 g, no food restriction) is digested with collagenase and the resulting isolated fat cells are washed several times by flotation.

A) Lipogenesis

This test determines the insulin-stimulable conversion of glucose into Toluene-soluble products (triglycerides, phospholipids, fatty acids), which the Glucose Transport and the Triglyceride (Glycerin-3-P Synthesis, Esterification) / Phospholipid / fatty acid synthesis including insulin Signal transmission cascade required.

Rat fat cells in KRH buffer are incubated with D- [3-3 H] glucose (0.2 mM or 1 mM final concentration) in the presence of 2 nM insulin and the peptide according to the invention for 90 min at 37 ° C. By adding a toluene-soluble scintillation cocktail, cells are disrupted and the lipids are separated from water-soluble products and the incubation medium. After phase separation, the radioactivity incorporated in lipids is determined by scintillation measurement directly without removing the aqueous phase ([3 H] lipid [dpm _* 10 -3]).

Table 1 below shows the results.  

Table 1

B) Glucose transport activity

Isolated plasma membrane vesicles are made from rat fat cells that are present of 2 nM insulin and the peptides according to the invention for 30 min at 25 ° C. had been pre-incubated, in which the fat cells were injected twice Homogenization buffer (20 mM trihydroxyaminomethane (Tris) / HCl, pH 7.4, 1 mM, 4 ° C, ethylenediaminetetraacetate (EDTA), 0.25 M sucrose) and then homogenized in 20 ml of the same buffer (glass Homogenizer with Teflon pestle).

The homogenate is centrifuged (16 000 × g, 15 min), the sediment is in the suspended the same buffer and centrifuged again. The sediment is in 5 ml  Homogenization buffer suspended and on a sucrose cushion (1.12 M Sucrose, 20 mM Tris / HCl, pH 7.4, 1 mM EDTA) layered after Centrifugation (100,000 × g, 70 min) is the interphase with the Plasma membrane vesicles removed with a syringe, with 45 ml buffer diluted and centrifuged again (48,000 x g, 45 min). The sediment is in 10 ml of buffer suspended, centrifuged again and resuspended in 3 ml of buffer.

Isolated plasma membrane vesicles are incubated with 50 μM D- [3-3 H] glucose and L- [1- 1 C] glucose of the same specific radioactivity for 10 seconds at 25 ° C. The mixtures are quickly sucked off through nitrocellulose filters. The filters are washed and dried extensively. Your radioactivity is determined by liquid scintillation measurement. The specific transport (D- [3 H] glucose / L- [13 C] glucose (dpm _* 10 -3) is calculated as the difference between the [3 H] and [13 C] radioactivity.

Table 2 shows the results.  

Table 2

Claims (11)

1. Peptide of the formula I H₂N-ABCD-Leu-EF-Tyr (I) containing 2 to 8 amino acid residues and physiologically tolerable salts of the peptide formula I, here
A is a covalent bond or an amino acid residue of Ser, Thr, Asn or Asp, optionally substituted by one or more aminoalkyl mercaptans,
B is a covalent bond or an amino acid residue of Leu, Arg, or Ile, optionally substituted by one or more aminoalkyl mercaptans,
C is a covalent bond or an amino acid residue of Tyr, His, Asn or Phe, optionally substituted by one or more aminoalkyl mercaptans,
D is a covalent bond or an amino acid residue of Gln or Asp, optionally substituted by one or more aminoalkyl mercaptans,
Leu a covalent bond or an amino acid residue of the amino acid leucine, which is optionally substituted by one or more aminoalkyl mercaptans,
E a covalent bond an amino acid residue of Glu, Met or Gln, optionally substituted by one or more aminoalkyl mercaptans,
F is an amino acid residue of Asn or Ser, optionally substituted by one or more aminoalkyl mercaptans,
Tyr the amino acid residue of the amino acid tyrosine, which is optionally substituted by one or more aminoalkyl mercaptans,
wherein at least one amino acid residue is substituted by an aminoalkyl mercaptan. 2. Peptide according to claim 1, containing 2 to 4 amino acid residues, characterized in that
A is a covalent bond or an amino acid residue of Ser or Thr, optionally substituted once by amino- (C₁- C₆) alkyl mercaptan,
B is a covalent bond or an amino acid residue of Leu or Arg, optionally substituted once by amino- (C₁-C₆) alkyl mercaptan,
C is a covalent bond or an amino acid residue of Tyr, His or Phe, optionally substituted by amino- (C₁- C₆) alkyl mercaptan,
D is a covalent bond or an amino acid residue of Gln or Asp, optionally substituted by amino- (C₁-C₆) alkyl mercaptan,
Leu is a covalent bond or an amino acid residue of the amino acid leucine, optionally substituted by aminoethyl mercaptan,
E is a covalent bond or an amino acid residue of Glu or Gln, optionally substituted by amino- (C₁-C₆) alkyl mercaptan,
F is an amino acid residue of Asn or Ser, optionally substituted by amino- (C₁-C₆) alkyl mercaptan, and
Tyr is the amino acid residue of the amino acid tyrosine, optionally substituted one or more times by amino- (C₁-C₁) alkyl mercaptan,
wherein at least one amino acid residue is substituted by an amino (C₁-C₆) alkyl mercaptan. 3. peptide of the formula II NH₂-Leu-EF-Tyr (II)
E is an amino acid residue of Glu, Gln or Met, optionally substituted by an amino (C₁-C₆) alkyl mercaptan,
F is an amino acid residue of Asn or Ser, optionally substituted by an amino (C₁-C₆) alkyl mercaptan, and
Tyr the amino acid residue of the amino acid tyrosine, once substituted by an amino (C₁-C₆) alkyl mercaptan. 4. peptide of the formula III, NH₂-Leu-Glu-Asn-Tyr-R¹ (III) wherein R¹ is an amino (C₁-C₆) alkyl mercaptan. 5. A process for the preparation of a peptide of formula I, II or III or the salts thereof according to one or more of claims 1 to 4, characterized in that

• a) reacting a segment with a C-terminal free carboxyl group or its activated derivative with a corresponding segment with an N-terminal free amino group or
• b) the peptide builds up gradually, and

optionally one or. in the compound obtained according to (a) or (b) splits off several protecting groups and the compounds thus obtained Formula I, II or III optionally in their physiologically acceptable salt transferred. 6. Pharmaceutical preparation, characterized by an effective Content of at least one peptide of formula I, II or III according to or more of claims 1 to 4 in dissolved, amorphous or crystalline form. 7. Pharmaceutical preparation according to claim 6, characterized by a content of at least one modified or unmodified Insulin or insulin derivative. 8. A process for the preparation of a pharmaceutical preparation Claim 6 or 7, characterized in that at least one Peptide of formula I, II or III with a physiologically acceptable Carrier and, if appropriate, suitable additives and / or auxiliaries in  brings a suitable dosage form. 9. The method according to claim 8, characterized in that one at least one modified or unmodified insulin or Insulin derivative adds. 10. Use of at least one peptide of the formula I, II or III according to one or more of claims 1 to 4 or obtained according to A method according to claim 5 for the manufacture of a pharmaceutical Preparation for the treatment of hypoglycaemia.