HK1019204B - Use of dipeptidyl peptidase iv inhibitors for lowering the blood glucose level in mammals - Google Patents

Description

Use of dipeptidyl peptidase IV effectors to lower blood glucose levels in mammals

The present invention relates to a simple method for lowering blood glucose concentration by means of effectors (substrates, pseudo-substrates, inhibitors, binding proteins, antibodies and the like) that inhibit enzymes having similar or identical activity to the enzymatic activity of dipeptidyl peptidase IV.

Proteases that cause specific degradation of proteins, in addition to proteases that are not specifically proteolyzed, are well known and are involved in the functional regulation (activation, inactivation, or regulation) of endogenous peptides [ kirchke, h., lannner, j.riemann, s., WIEDERANDERS, b., ANSORGE, s. and BOHLEY, p., lysosomal cysteine proteases. Excerpta, medical (Ciba basic monograph 75), 15 (1980); KR * ussich, h. -g. and WIMMER, e., viral protease. annual review of biochemistry 57, 701(1987) ].

So-called invertases, signalases or enkephalinases have been found in immune system studies and neuropeptide studies [ GOMEZ, s., glucchankof, p., LEPAGE, a., MARRAKCHI, n. and COHEN, p. proceedings of the national academy of sciences usa 85, 5468 (1988); ANSORGE, s. and SCH * N, e., histochemistry 82, 41(1987) ].

Since the amino acid proline is very abundant in many peptide hormones and thus determines certain structural properties of these peptides, proline-specific peptidases are discussed as an enzyme with similar function to signal peptidases [ YARON, a., the role of proline in the proteolytic regulation of biologically active peptides. Biopolymer (1987)26, 215; WALTER, r., SIMMONS, w.h. and YOSHIMOTO, t., proline-specific endopeptidase and exopeptidase, molecular cell biochemistry 30, 111 (1980); VANHOOF, G., GOOSSENS, F., DEMEESTER, I., HENDRIKS, D., and SCHARPS., proline motifs and their bioprocessing. FASEB journal 9, 736(1995)]. Due to its exceptional structure, proline in this peptide determines their conformation and stability to prevent degradation by non-specific proteases. [ KESSLER, H., conformation and biological activity. Application chemistry 94, 509(1982)]. In contrast, enzymes with highly specific capacity for proline containing sequences (including HIV-proteases, cyclophylins, etc.) are attractive targets for medicinal chemistry. In particular, post-proline cleaving peptidases, such as the activity of Prolyl Endopeptidase (PEP) and dipeptidyl peptidase iv (dp iv), are linked to the modulation of the biological activity of natural peptide substrates and their selective cleavage by these enzymes. PEP has been shown to be involved in memory and learning, and DPIV is involved in signal transduction during immune responses [ ISHIURA, s., [ TSUKAHARA, t.tabera, t.shimlzu, t.aragaak and SUGITA, h., (1990)260, 131; HEGEN, m., niedobtek, g., KLEIN, c.e., STEIN, h. and FLETSCHER, b., journal of immunology 144, 2908(1990)]。

In addition to their outstanding specificity similar to that of high proline, there is a high selectivity in the recognition regions typical of the substrates of these enzymes for the ability to selectively recognize the amino acid alanine. Accordingly, it is believed that alanine-containing peptides may structurally adopt a conformation similar to structurally similar proline-containing peptides. Shortly before, such properties of proline-containing peptide chains have been through point mutations of the genes (proline-exchanged alanine) [ DODGE r.w. and SCHERAGA, h.a., folding and unfolding kinetics of proline-to-alanine mutants of bovine pancreatic ribonuclease a, biochemistry 35(5)1548(1996) ].

Where the highly active dipeptide is released from the N-terminus of a biologically active peptide, the activity of DP IV-or DP IV analogs-present in the blood circulation (e.g., cytosolic DP II has nearly the same substrate specificity as DP IV) is highly specific when proline or alanine are adjacent residues of the N-terminal amino acid in the sequence. Thus, this enzyme is involved in the regulation of the activity of polypeptides in vivo [ VANHOOF, G., COOSSENS, F., DE MEESTER, I., HENDRIKS, D.and SCHARPS., proline motifs and their bioprocessing, FASEB journal 9, 736(1995)]。

Sugar-dependent insulin-releasing peptide: stomach inhibitory polypeptide 1-42 (GIP)_1-42) And glucagon-like peptide amide-17-36 (GLP-1)_7-36) They are hormones that stimulate glucose-induced insulin secretion from the pancreas (incretins) -are substrates of DP IV because they release the dipeptides tyrosyl-alanine and histidyl-alanine from the N-terminal sequences of these peptides in vitro and in vivo [ MENTLEIN, r. Dipeptidyl peptidase IV hydrolyzes gastric inhibitory polypeptides, glucagon-like peptide-1 (7-36) amide, peptide histidine methionine and is responsible for their degradation in human serum, European journal of biochemistry 214, 829(1993)]。

In mammals, reducing cleavage of this substrate by DPIV-or DPIV analogue-enzyme activity in vivo under laboratory conditions and pathological conditions may be useful for effective inhibition of undesirable enzymatic activity [ deuth, h. -u., recent advances in irreversible inhibition of serine and cysteine proteases, journal of enzyme inhibition, 3, 249-278 (1990); DEMUTH, h. -u. and HEINS, j., catalytic mechanism of dipeptidyl peptidase IV (CD26) in its metabolism and immune response (b. fleischer, eds.) r.g. landes, biopharmaceutical press, Georgetown, 1-35 (1995). For example, type II true diabetes (as well as geriatric diabetes) is based on impaired insulin secretion or dysfunction of the receptor, which is the cause of abnormal secretion concentrations determined by enzymatic digestion among other factors [ BROWN, j.c., DAHL, m., KWAWK, s., MCINTOSH, c.h.s., OTTE, s.c and PEDERSON, r.a. peptide 2, 241 (1981); SCHMLDT, w.e.siegel, e.g., GALLWITZ, b.kummel, h.e., EBERT, R and CREUTZFELDT, w.e., the identification of insulinotropic activity of fragments derived from gastric inhibitory polypeptides, diabetes physiology 29, 591A (1986); adelphorst, k, HEDEGAARD, B.B., KNUDSEN, L.B. and KIRK, o., studies of the structural activity of glucagon-like peptides, journal of biochemistry 296, 6275(1994) ].

According to the prior art, hyperglycemia and the etiologies and sequelae associated therewith are treated by administering insulin to a patient using different administration forms (e.g., materials isolated from bovine or porcine pancreas or obtained by genetic engineering). The hitherto known and more modern treatment methods represent a high expenditure and a strong impairment of the physical condition of the patient. The classical approach (daily intravenous insulin injections, already used since the thirty years) deals with acute symptoms, but after longer application will lead to vascular deformation (arteriosclerosis) and neurological damage [ LACY, p., state of inter-pulse cell transplantation, diabetes care 16(3)76(1993) ].

More modern approaches, such as the installation of subcutaneous depot implants (successful metering of insulin release, elimination of daily injections) and the implantation of langerhans cells in intact intervenous regions (transplants) in dysfunctional pancreas or other organs and tissues, are being tested. However, such transplants are expensive. In addition, they represent a high risk of surgical rejection and require immunosuppression or evasion of the immune response when using transplantation methods. [ LACY, P., treatment of diabetes with transplanted cells, U.S. science 273(1)40-46(1995) ].

In contrast to the above-mentioned therapies, low molecular weight enzyme inhibitors which are as orally effective as possible are a very effective alternative to, for example, invasive surgical procedures in the management of pathological phenomena. Such enzyme inhibitors find use in therapy as antihypertensive agents, immunosuppressive agents, and anti-aids agents. By chemical engineering of the stability, transcription and fission properties of molecules, their efficacy can be improved and they can be adapted to individual differences between organisms. SANDLER, m. and SMLTH, h.j., eds., design of enzyme inhibitors as drugs, oxford university press, oxford university (1989); murRoe, J.E., SHEPHEAD, T.A., JUNGHEIM, L.N., HORNBACK, W.J., HATCH, S.D., MUESTNG, M.A., WISKERCHEN, M.A., SU, K.S., CAMPANALE, K.M., BAXTER, A.J., and COLACINO, J.M., effective oral HIV-1 protease inhibitors comprising a non-coding D-amino acid, biopharmaceutical chemical communication 5(23)2987 (1995).

The object of the present invention is to provide a simple and novel method for lowering blood glucose levels, wherein an effector of an enzyme is administered to a mammal, wherein said effector induces a decrease in the activity of the DP IV-enzyme or of the DPIV analogue-enzyme, which results in the endogenous (or exogenously administered) insulinotropic peptide gastroinhibitory polypeptide 1-42 (GIP)_1-42) And glucagon-like peptide amide-17-36 (GLP-1)_7-36Or analogs of these peptides). Thus, a decrease in the concentration of these peptide hormones or their analogs will be prevented or delayed.

The present invention is based on the surprising discovery that: a reduced activity of the dipeptidyl peptidase DPIV enzyme or DPIV analogue-enzyme in the blood circulation will lead to an improved glucose tolerance. We observed that:

1. reducing the dipeptidyl peptidase DPIV-or DPIV analog-enzyme activity results in a relatively increased stability of glucose-stimulated endogenously released or exogenously administered incretins (or analogs thereof), and thus the degradation of incretins in the blood can be controlled by administering an activity-inhibiting effector of a DPIV-or DPIV analog-protein.

2. The increased stability of the incretins (or analogues thereof) towards biodegradation results in an improved effect of the insulin action.

3. An increase in the stability of incretins caused by a decrease in the activity of the dipeptidyl peptidase DPIV-or DPIV analogue-enzymes will lead to a subsequent change in the glucose-induced insulin action and thereby to a blood glucose level which can be controllably regulated by means of the DPIV effector.

The present invention therefore relates to the use of an activity inhibiting effector of the enzymatic activity of a dipeptidyl peptidase DPIV-or DPIV analogue-for lowering blood glucose levels below glucose concentrations in the blood of a mammal which are characteristic of high blood glucose levels. The invention relates in particular to the use of DPIV-or DPIV analogue-enzyme activity effectors for preventing or alleviating pathological allergies in the metabolism of mammals such as glucosuria, hyperlipidemia, metabolic acidosis and diabetes. In a more preferred embodiment, the present invention relates to a method of lowering blood glucose concentration in blood of a mammal below a glucose concentration indicative of elevated blood glucose levels. Characterized in that a therapeutically effective amount of an effector of dipeptidyl peptidase DPIV-or DPIV analogue-enzyme activity is administered to a mammal.

In a second preferred embodiment, the invention relates to an activity inhibitory effector of the dipeptidyl peptidase DPIV-or DPIV analogue-enzyme activity for use in a method of lowering blood glucose concentrations in the blood of a mammal below a level indicative of an elevated blood glucose concentration.

According to the invention, the administered DPIV-and DPIV analogue-enzyme activity inhibiting effectors that reduce the concentration of DPIV-and DPIV analogue-proteins in mammals are used as enzyme inhibitors, substrates, pseudo-substrates, inhibitors of DPIV-expression products, binding proteins or antibodies to said enzyme proteins or as combinations of these different compounds in a formulation complex that can be used as a medicament. Effectors according to the invention are, for example, DPIV inhibitors such as dipeptide derivatives or dipeptide mimetics such as alanyl-pyrrolidine amide (pyrolidid), isoleucyl-thiazolidine, and the pseudo-substrate N-desmyl-prolyl, O-benzoylhydroxylamine. These compounds are known from the literature [ DEUTH, H. -U., recent advances in irreversible inhibition of serine and cysteine proteases, J.ENZYME INHIBITION 3,249(1990) ], or can be synthesized according to methods described in the literature.

The method according to the present invention is a novel method of reducing elevated blood glucose concentrations in the blood of a mammal. The method is simple, pharmaceutically useful and suitable for the preparation of a medicament for the treatment of diseases, especially human diseases, which are caused on the basis of exceeding normal blood glucose levels.

The effectors are administered in the form of pharmaceutical preparations which comprise the active ingredient and customary carrier materials known from the prior art. They are administered, for example, parenterally (intravenously, in physiological saline solution) or enterally (orally, formulated with customary carrier materials).

Depending on the endogenous stability and bioavailability of the effector, one or more administrations are performed to achieve the desired normalization of blood glucose concentration. For example, in the case of aminoacyl-thiazolidine, the dosage may vary from 1.0 to 10.0mg of effector substance/kg.

Examples

Example 1 in vivo incretin GIP-_1-42And GLP-1_7-36Inhibition of DPIV-catalyzed hydrolysis

The hydrolysis of incretins by enzymatic activity of DPIV-and DPIV analogue-or its inhibition by means of inhibitors can be demonstrated in vivo using purified enzymes or in situ using pooled human serum (figure 1).

According to the invention, GIP was incubated in vivo at 30. mu.M_1-42Or 30. mu.M GLP-1_7-36And 20. mu.M isoleucyl-thiazolidine (1a), complete inhibition of the enzymatic hydrolysis of the two peptide hormones was achieved within 24 hours (1b and 1c, see respective upper panels), the above incubations being reversible DP IV-inhibitors in 20% mixed serum at pH7.6 and 30 ℃. At pH7.6 and 30 deg.C, the synthesized gastric inhibitory polypeptide GIP_1-42(5. mu.M) and synthetic GLP-1_7-36(15 μ M) inHuman serum (20%) in 0.1mM TRICINE buffer was incubated for 24 hours. After different time intervals, remove incubation assay samples (for GIP)_1-422.5pmol for GLP-1_7-367.5 pmol). The samples were co-crystallized using 2 ', 6' -dihydroxyacetophenone as a matrix and analyzed by MALDI-TOF-mass spectrometry. The spectrum (panel l) shows the accumulation of 250 individual laser shots per sample.

(1b) Signals in the range m/z 4980.1 + -5.3 correspond to GIP_1-42(M4975.6), and a signal in the range M/z 4745.2. + -. 5.5 corresponding to the DPIV-hydrolysate GLP-1_{3- 42}(M4740.4)。

(1c) The signal m/z 3325.0 + -1.2 corresponds to GLP-1_7-36(M3297.7), and the signal M/z 3116.7. + -. 1.3 corresponds to the DPIV-hydrolysate GLP-1_9-36(M3089.6)。

In a reference assay sample which does not contain any inhibitor, the incretins are almost completely degraded during this time (FIGS. 1b and 1c, see the spectra below).

Example 2 inhibition of GLP-1 by the DPIV-inhibitor isoleucyl-thiazolidine in vivo_7-36Degradation of

Native incretins (here GLP-1) in rat serum were followed up in comparison to a control_7-36) Is dependent on the DPIV-inhibitor isoleucyl-thiazolidine (i.v. injection of 0.9% saline solution containing 1.5. mu.M inhibitor). Insulinotropic peptide hormone GLP-1 in inhibitor-treated test animals (N ═ 5) tested at a concentration of 0.1mg inhibitor isoleucyl-thiazolidine per kg of test mice_7-36No degradation was observed during the experiment (figure 2).

In order to detect incretin metabolites in the presence and absence of DPIV-inhibitors, the test and reference animals were further injected intravenously with 50-100pM of inhibitor or saline 20 minutes after the start of intravenous administration¹²⁵I-GLP-1_7-36(approximately 1. mu. Ci/pM specific activity). Incubating for 2-5 min and collecting bloodSamples were taken and protoplasm was extracted using 20% acetonitrile. Subsequently, the peptide extract was separated on RP-HPLC and the samples were analyzed for radioactivity on a gamma-counter. The measured data are expressed as counts per minute (cpm) relative to the maximum. Example 3 modulation of insulin action and reduction of blood glucose levels following intravenous administration of the DPIV-inhibitor isoleucyl-thiazolidine in vivo.

In mice stimulated by an enteral (i.d.) injection of glucose, a decrease in glucose levels due to the effect of the inhibitor, which is delayed in time, can be observed by in vivo administration of different DPIV-effectors, e.g. 0.1mg isoleucyl-thiazolidine per kg weight of the mouse. This effect is dose dependent and is reversible upon discontinuation of the injection of 0.05 mg/min of the DPIV-inhibitor isoleucyl-thiazolidine per kg weight of the mouse. In contrast to the intraduodenal glucose-stimulated test animals, no comparable effect was observed after intravenous administration of the same amounts of glucose to the inhibitor-treated and control animals. Figure 3 shows the changes in these inhibitor-dependent protoplasmic parameters: a-DPIV-activity, B-protoplasm-insulin level, C-blood glucose level.

The test animals (N.sub.5, male Wistar-mice, 200-225g) initially obtained 1.5. mu.M isoleucyl-thiazolidine (. tangle-solidup.) in 0.9% saline solution or the same volume of 0.9% saline solution (■) without inhibitor (control, n.sub.5). The experimental group obtained an additional injection of 0.75 μ M/min of inhibitor over the 30 minute experimental period: (^*). The control group was injected with 0.9% non-inhibitor saline solution during the same time interval. All animals were intraduodenally administered a glucose solution (w/v) at a glucose dose of 1g/kg 40% at a start time T ═ 0.

Blood samples were collected from all test animals at 10 minute intervals. When the DPIV-activity and insulin concentration in the protoplasts were analyzed, the glucose content was analyzed using whole blood (Lifescan One Touch II analyzer).

The insulin assay employed is sensitive in the range of 10 to 160mU/ml [ PEDERSON, r.a., BUCHAN, a.m.j., ZAHEDI-ASH, s., CHEN, c.b. and BROWN, j.c.reg.peptides.3, (1982) ]. Determination of DPIV-activity by spectroscopic analysis [ deuth, h. -u., and heinsij., catalytic mechanism of dipeptidyl peptidase IV (CD26) in metabolic and immune responses, (b. fleischer, eds.) r.g. landes, biomedical press, Georgetown, 1-35(1995) ]. All data are given as mean plus standard deviation.

Claims (4)

1. Use of an activity inhibiting effector of dipeptidyl peptidase IV or dipeptidyl peptidase IV-like-enzyme activity for the preparation of a medicament for lowering blood glucose levels in the serum of a mammal below glucose concentrations characteristic of hyperglycemia.

2. Use according to claim 1, characterized in that the medicament is for preventing or alleviating the metabolic pathological metamorphosis in mammals: glucosuria, hyperlipidemia, metabolic acidosis and diabetes.

3. Use according to claim 1, wherein the effector capable of inhibiting the activity of dipeptidyl peptidase IV or dipeptidyl peptidase IV-like-enzymes can be an inhibitor of said enzymes, a substrate, a pseudo-substrate, an inhibitor of dipeptidyl peptidase-expressors, a binding protein or antibody to these enzyme proteins, or a combination of said effectors.

4. Use according to claim 1, characterized in that the effector is isoleucyl-thiazolidine.