HK1033420B - Method for lowering blood glucose levels in mammals - Google Patents

Description

The invention relates to a simple method for lowering blood sugar concentration by means of activity-reducing agents (substrates, pseudosubstrates, inhibitors, binding proteins, antibodies, etc.) as claimed 1.

In addition to proteases involved in nonspecific proteolysis, which ultimately leads to the breakdown of proteins into amino acids, regulatory proteases are known to be involved in the functionalization (activation, deactivation, modulation) of endogenous peptide agents [KIRSCHKE, H., LANGNER, J., RIEMANN, S., WIEDERANDERS, B., ANSORGE, S. and BOHLEY, P., Lysosomal cysteine proteases. Excer AN Medica (Ciba Foundation Symposium 75), 15 (1980); KRÄSLUSICH, H.-G. and WIMMER, E., Viral Proteases. GLOREN Ann. Rev. Biochem. 57, 701 (1987); in particular, such proteases have been identified in connection with the immunological research of SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCHK, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH, SCH

Due to the frequency of the amino acid proline in a variety of peptide hormones and the associated structural properties of these peptides, a function analogous to that of signal peptides is being discussed for proline-specific peptides (YARON, A., The Role of Proline in the Proteolytic Regulation of Biologically Active Peptides. Biopolymers 26, 215 (1987); WALTER, R., SIMMONS, W.H. and YOSHIMOTO, T., Proline Specific Endo- and Exopeptid. Moltid. Cell. Biochem. 30, 111 (1980); VANHOOF, G., GOOSS, F., DE MEESTER, I., HENDRIKS, D. and SCHARPE, S., FEB. 73 and their motifs, 9, 95), whereas the particular structure and stability of this peptide is determined by this particular configuration and processing.In contrast, enzymes which act on proline-containing sequences by highly specific structural modification (HIV protease, cyclophylin, etc.) are attractive targets of current drug research. In particular, for the proline-splitting peptides prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPI), relationships between modulation of biological activity of natural peptide substrates and their selective cleavage by these enzymes have been established. Thus, it is likely that PEP plays a role in the immune signalling response and is involved in the development of a drug called DPIS during the treatment of HIV.The use of the active substance in the active substance is not recommended for the treatment of patients with severe allergic reactions.

Similar to the exceptional proline specificity of these enzymes, their high selectivity for the amino acid alanine within typical recognition regions in substrates of these enzymes is discussed, suggesting that alanine-containing peptides can assume similar conformations to structural analogues of proline-containing peptides.

IV and DP IV analogues (e.g. the cytosol DP II has a substrate specificity almost identical to DP IV) occur in the bloodstream where it highly specifically clears dipeptides from the N-terminus of biologically active peptides when proline or alanine are the adjacent N-terminal amino acid residues in their sequence.

The glucose-dependent insulinotropic polypeptides are Gastric Inhibitory Polypeptide 1-42 (GIP1-42) and Glucagon-Like Peptide Amide-1-7-36 (GLP-17-36). Hormones that stimulate glucose-induced insulin secretion by the pancreas (including incretins) are substrates of DP IV because they can break down the dipeptides tyrosinyl alanine and histidyl alanine from the N-terminal sequences of these peptides in vitro and in situ [MENTLEIN, R., GALLWITZ, B., and SCHMIDT, W. , Dipeptidyl peptidase IV hydrolyzes gastric inhibitory polypeptide, glucagon-dehydrogen-17-36) and their N-terminal sequences are responsible for the degradation of Eurydion peptide and methionine in humans.

The reduction of such DP IV and DP IV analogues in vivo to break down such substrates may be effective in suppressing undesirable enzyme activity in laboratory conditions as well as in pathological conditions of mammalian organisms [DEMUTH, H.-U., Recent developments in the irreversible inhibition of serine and cysteine proteases.J. Enyzeme Inhibition 3, 2478 (1990); DEMUTH, H.-U. and HEINS, J., On the catalytic mechanism of dipeptidyl peptidase IV in dipeptidyl peptidase IV (CD 26) in metabolic metabolism and the response (B. BESTON, S. E. Ed., S. E. F. BESTON, Georgetown, Germany, 1995). B. S. KARDON, S. KARDON, S. KARDON, S. T. S., S. KARDON, S. KARDON, S. T. S., S. T. W., R. W., W. W., W. K. W., R. K. W., R. K. W., R. K. W., R. K. W., R. W., R. K. W., R. W., R. K. W., R. W., R. W., R. W., R. K. W., R. W., R. W., R. W., R. W., R. K., R. W., R. W., R. W., R. W., R. W., R. W., R. W., R. W., R. W., R. W., R., R. W., R. W., R., R. W., R., R., R. W., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R

Hyperglycaemia and its causes and consequences (including diabetes mellitus) are currently treated by administering insulin (e.g. from material isolated from bovine pancreas or genetically engineered) to diseased organisms in various dosage forms.All the methods known to date, as well as the more modern ones, are characterised by high material costs, high costs and often significant impairment of the quality of life of the patients.The classic method (daily intravenous insulin injection, especially since the 1930s) treats the acute symptoms of the disease, but after prolonged use leads to, amongst others, severe changes in the blood vessel (arteriosclerosis) and nerve damage [CLAY, Care of Islet cells, P. 76 (1993) ] and islet cells of diabetes.

Recently, the installation of subcutaneous depot implants (insulin is dosed and daily injections are eliminated) and the implantation (transplantation) of intact long-horn cells into the dysfunctional pancreas or other organs and tissues have been proposed.

The use of high-grade, low-molecular-weight oral enzyme inhibitors, on the other hand, is a less expensive alternative to invasive surgical techniques for the treatment of pathological conditions. Such enzyme inhibitors are now being used therapeutically as immunosuppressants, antithrombotics and AIDS virostatics. Chemical design of stability, transport and clearance properties can modify their mode of action and tailor them to individual properties [SANDLER, M. and SMITH, H.J., Hrsg., Design of Enzyme Inhibitors as CAM. Oxford University Press, Oxford (1989); BAIN, J.E., SHEERD, T.A., J.NACHR, L.N., S.J., S.J., S.J., S.J., S.J., S.J., M.A., M.A., M.A., M.A., M.A., M.A., M.A., M.A., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M., M.,

The purpose of the invention is a simple and novel method of lowering blood glucose levels which, according to the invention, can be achieved by causally degrading the endogenous (or additionally exogenously administered) insulinotropic peptides GIP1-42 and GLP-17-36 by DP IV by administration of activity-lowering effectors to a mammalian organism, thereby reducing or delaying the decrease in the concentration of these peptide hormones in the treatment of diabetes mellitus.

The invention is based on the surprising finding that a reduction in DP IV activity in the bloodstream causes a causal effect on blood sugar levels. 1. the reduction in DP IV activity leads to a relative increase in stability of glucose-stimulated or externally administered incretins, i.e. the degradation of incretin in the blood can be controlled by the application of DP IV activity-lowering effectors.2. increased biological instability of incretins leads to a change in endogenous insulin activity.3. the increase in incretin stability achieved by reduction in DP IV activity in the blood induces a subsequent change in glucose and results in a controllable modulation of blood glucose levels by DP IV activity-lowering effectors.

The invention thus concerns activity-lowering effectors of the enzyme dipeptidyl peptidase IV (DP IV) activity for use in lowering blood sugar levels below the glucose concentration in the serum of a mammalian organism characteristic of hyperglycaemia for the relief of diabetes mellitus.

The DP IV inhibitors as applied in the invention can be used in pharmaceutically applicable formulation complexes as inhibitors, substrates, pseudosubstrate, inhibitors of DP IV expression, binding proteins or antibodies to these enzyme proteins or combinations of these different substances which reduce DP IV protein concentration in the mammalian organism. For example, DP IV inhibitors such as the dipeptide derivatives or dipeptide mimetics alanyl-pyrrolidide, isoleucode thiazolidide and the pseudosubstrate N-valyl-ol, Oxy-Benzyl-hydrolamine are described in the literature. Recent developments in the literature on the development of the enzyme are described in [DEMH, H. and H.], and the enzyme is irreversible inhibition of the 24 amino acids.

The method presented represents a novel approach to reducing elevated serum glucose levels in mammals, and is simple, commercially viable and suitable for use in human medicine in the treatment, in particular of diseases based on above-average blood glucose levels.

The activity-lowering agents are administered in the form of pharmaceutical preparations containing the active substance in combination with commonly known carrier materials, e.g. parenterally (e.g. intravenously in saline) or enteral (e.g. orally, formulated with commonly used carrier materials such as glucose).

Depending on their endogenous stability and bioavailability, single or multiple doses of the activity-lowering effectors should be used to achieve the desired normalization of blood glucose levels. e.g. in the case of aminoacyl thiazolidides, such a dose range may be between 1.0 mg and 10.0 mg of effector per kilogram.

The following is a list of the

Figure 1: MALDI-TOF analysis of DP IV-catalyzed hydrolysis of GIP1-42 (b) and GLP7-36 and their inhibition by isoleucyl thiazolidide (a).Figure 2: HPLC analysis of serum presence of GLP-1 metabolites in the presence and absence of DP IV inhibitors isoleucyl thiazolidide in vivo.Figure 3: Effect of DP IV inhibitor isoleucyl thiazolidide on various blood parameters of the i.d. glucose-stimulated rat.

Examples of implementation Example 1: Inhibition of DP IV-catalyzed hydrolysis of incretins GIP1-42 and GLP-17-36 in situ

Both in vitro with purified Enyzm and in situ, e. g. in pooled human serum, the hydrolysis of incretins caused by DP IV activity can be detected or inhibited by inhibitors (Figure 1).

According to the invention, in situ incubation of 30 μM GIP1-42 and 30 μM GLP-17-36 and 20 μM Isoleucyl thiazolidide (1a), a reversible DP IV inhibitor, in 20 per cent serum at pH 7.6 and 30 °C results in complete suppression of enzyme-catalyzed hydrolysis of both peptide hormones within 24 hours (1b and 1c, upper spectra respectively). Synthetic GIP1-42 (5 μM) and synthetic GLP-17-36 (15 μM) were incubated with humanememidum (20 per cent) in 0.1 mM TRIC (1a) for 24 hours. The samples were incubated at pH 7.6 and 30 °C using the accretion approaches (GIP1-42mol for PTP and 2.5 pmol for PTP-17.5f) and analysed as samples in 2 separate samples co-administered with MALDI-D6's. The signal in the range of m/z 4980.1 ± 5.3 corresponds to GIP1-42 (M 4975.6) and m/z 4745.2 ± 5.5 to the DP IV hydrolysis product GIP3-42 (M 4740.4).

In the non-inhibitor approaches, incretins were almost completely degraded during this time (Figures 1b and 1c, lower spectra).

Example 2: Inhibition of degradation of GLP-17-36 by the DP IV inhibitor isoleucyl thiazolidide in vivo.

When comparing the metabolism of native incretins (here GLP-17-36) in the serum of the rat in dependence on the presence of the DP IV inhibitor isoleucyl thiazolidide (i.v. injection of a 1.5 μM inhibitor solution in 0.9% saline solution) with a control, no decrease in the insulin-tropic peptide hormone GLP-17-36 is observed in the inhibitor-treated experimental rats (n = 5) at a concentration of the inhibitor isoleucyl thiazolidide of approximately 0.1 mg/kg in the laboratory during the experimental period (Figure 2).

To detect metabolites in the presence and absence of the DP IV inhibitor (20 minutes after previous intravenous inhibitor or saline administration), the test and control animals were given 50-100 pM 125I-GLP-17-36 (specific activity of approximately 1 μMCi/ pM) intravenously. Blood samples were taken after 2-5 min and plasma extracted with 20% acetone acetyl. The peptide extract was then separated by RP-LCHP and the radioactivity of the fractions analysed on a γ-counter.

Example 3: Modulation of insulin action and reduction of blood glucose levels following intravenous administration of the DP IV inhibitor isoleucyl thiazolidide in vivo.

In rats stimulated by glucose by intraduodenal (i. d.) injection, a time-delayed reduction in glucose levels due to the inhibitory effect may be observed by the i. v. administration of different DP IV effector, e. g. 0.1 mg isoleucyl thiazolidide per kg rat. This effect is dose-dependent and reversible after discontinuation of the 0.05 mg/ min infusion of the DP IV inhibitor isoleucyl thiazolidide per kg rat.

Figure 3 illustrates these relationships with the inhibitor-dependent changes in plasma parameters: A - DP IV activity, B - plasma insulin levels, C - blood glucose levels.

The animals (n = 5, male Wistar rats, 200-225 g) received an initial dose of 1.5 μM isoleucyl thiazolidide in 0.9% saline (A) or equal volumes of 0.9% saline without inhibitor (■) (control group n = 5). The control group continued to receive an infusion of the inhibitor of 0.75 SM/ min over a 30 min test period (*). The control group received an infusion of an inhibitor-free 0.9% saline over the same period. At t=0, the animals received an i.i.d. glucose dose of 1 g/ kg 40 % dextrose solution (w/ v).

Blood samples were taken from all animals at 10-minute intervals.

Glucose measurements were taken in whole blood (Lifescan One Touch II analyzer) while DP IV activity and plasma insulin concentrations were measured.

The insulin assay used herein is sensitive between 10 and 160 mU/ml [PEDERSON, R.A., BUCHAN, A.M.J., ZAHEDI-ASH, S., CHEN, C.B. and BROWN, J.C. Reg. Peptides 3, 53-63 (1982) ]. DP IV activity was determined by spectrophotometry [DEMUTH, H.-U. and HEINS, J., On the catalytic mechanism of dipeptidyl peptidase IV in dipeptidyl peptidase IV (CD 26) in Landes Metabolism and the Immune Response (B. Standischer, Ed.) R.G., Biomedical Publishers, Georgetown, 1-35 (1995) ]. All measurements are given as mean with variation.

Claims (4)

1. Activity-lowering effector of dipeptidylpeptidase IV (DP IV)-enzymatic activity for use in lowering the blood glucose level below the glucose concentration in the serum of a mammalian organism characteristic of hyperglycemia for alleviation of diabetes mellitus, wherein said effector results in the reduced degradation of the endogenous insulinotropic peptides GIP_1-42 and GLP-1_7-36 by DP IV.
2. Activity-lowering effector for use according to claim 1, wherein said effector is a DP IV-inhibitor.
3. Activity-lowering effector for use according to claim 1 or 2, wherein said effector is applied parenterally or enterally.
4. Activity-lowering effector for use according to claim 3, wherein said effector is applied orally together with usual carrier materials.