U.S. patent application number 11/443389 was filed with the patent office on 2008-07-31 for novel effectors of dipeptidyl peptidase iv.
Invention is credited to Hans-Ulrich Demuth, Konrad Glund, Susanne Kruber, Dagmar Schlenzig.
Application Number | 20080182798 11/443389 |
Document ID | / |
Family ID | 7869159 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080182798 |
Kind Code |
A1 |
Demuth; Hans-Ulrich ; et
al. |
July 31, 2008 |
Novel effectors of dipeptidyl peptidase IV
Abstract
Dipeptide compounds and compounds analogous to dipeptide
compounds that are formed from an amino acid and a thiazolidine or
pyrrolidine group, and salts thereof used in the treatment of
impaired glucose tolerance, glycosuria, hyperlipidaemia, metabolic
acidoses, diabetes mellitus, diabetic neuropathy and nephropathy
and also of sequelae of diabetes mellitus in mammals.
Inventors: |
Demuth; Hans-Ulrich; (Halle,
DE) ; Glund; Konrad; (Halle, DE) ; Schlenzig;
Dagmar; (Halle, DE) ; Kruber; Susanne; (Halle,
DE) |
Correspondence
Address: |
OSI PHARMACEUTICALS, INC.
41 PINELAWN ROAD
MELVILLE
NY
11747
US
|
Family ID: |
7869159 |
Appl. No.: |
11/443389 |
Filed: |
May 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10727209 |
Dec 2, 2003 |
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11443389 |
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10361956 |
Feb 10, 2003 |
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10727209 |
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09723638 |
Nov 28, 2000 |
6548481 |
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10361956 |
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Current U.S.
Class: |
435/212 ;
514/15.4; 514/21.91; 514/6.9; 562/553 |
Current CPC
Class: |
A61K 31/427 20130101;
A61P 3/10 20180101; A61P 43/00 20180101; A61P 3/06 20180101; A61K
31/426 20130101; A61K 31/40 20130101; A61K 45/06 20130101; A61K
31/425 20130101; C07D 295/185 20130101; C07D 277/04 20130101; A61P
13/12 20180101; A61P 1/00 20180101; A61K 31/40 20130101; A61K
2300/00 20130101; A61K 31/425 20130101; A61K 2300/00 20130101; A61K
31/426 20130101; A61K 2300/00 20130101; A61K 31/427 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/19 ;
562/553 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C07C 205/00 20060101 C07C205/00; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 1998 |
DE |
198/23831.2 |
May 28, 1999 |
EP |
PCT/EP99/03712 |
Claims
1. Dipeptide compound formed from an amino acid and a thiazolidine
or pyrrolidine group, and salts thereof.
2. Dipeptide compound according to claim 1, characterised in that
the amino acid is selected from a natural amino acid.
3. Dipeptide compound according to claim 1, characterised in that
at a concentration of 10 .mu.m it brings about a reduction in the
activity of dipeptidyl peptidase IV or DP IV-analogous enzyme
activities of at least 10%.
4. Dipeptide compound according to claim 3, characterised in that
it brings about a reduction in activity of at least 40%.
5. Dipeptide compound according to claim 1, characterised in that
the amino acid is selected from leucine, valine, glutamine,
proline, isoleucine, asparagine and aspartic acid.
6. Dipeptide compound according to claim 1, namely
L-threo-isoleucyl pyrrolidide, L-allo-isoleucyl thiazolidide,
1-allo-isoleucyl pyrrolidide and salts thereof.
7. Dipeptide compound according to claim 1, characterised in that
the salts are organic salts such as acetates, succinates, tartrates
or fumarates, or inorganic acid radicals such as phosphates or
sulphates.
8. Salts of dipeptide compounds according to claim 1, characterised
in that they are present in a molar ratio of dipeptide compound to
salt of 1:1 or 2:1.
9. Fumaric salts of dipeptide compounds according to claim 1.
10. The fumaric salt according to claim 9, consisting of fumaric
salts of L-threo-isoleucyl thiazolidide or fumaric salts of
L-allo-isoleucyl thiazolidide.
11. A pharmaceutical composition comprising least one compound or a
salt thereof according to claim 1 optionally in combination with
one or more pharmaceutically acceptable carriers and/or
solvents.
12. Pharmaceutical composition according to claim 11, wherein the
carrier is a carrier for parenteral or enteral formulations.
13. Pharmaceutical composition according to claim 11, suitable for
oral administration.
14. Pharmaceutical composition according to claim 11, additionally
comprising an active ingredient having hypoglycaemic action.
15. A method of reducing the activity of dipeptidyl peptidase IV or
of dipeptidyl peptidase IV-analogous enzyme activities comprising a
step of administering a pharmaceutically effective amount of a
compound according to claim 1.
16. A method for lowering the blood sugar level in the serum of a
mammal below the glucose concentration that is characteristic of
hyperglycaemia comprising a step of administering a
pharmaceutically effective amount of a compound according to claim
1.
17. A method for the oral treatment of metabolic disorders
associated with diabetes mellitus comprising a step of
administering a pharmaceutically effective amount of a compound
according to claim 1.
18. A method of treatment of impaired glucose tolerance,
glycosuria, hyperlipidaemia, metabolic acidosis, diabetes mellitus,
diabetic neuropathy and nephropathy and also of sequelae of
diabetes mellitus in mammals comprising a step of administering a
pharmaceutically effective amount of a compound according to claim
1.
Description
[0001] The present invention relates to dipeptide compounds and
compounds analogous to dipeptide compounds that are formed from an
amino acid and a thiazolidine or pyrrolidine group, and salts
thereof, referred to hereinafter as dipeptide compounds, and to the
use of the compounds in the treatment of impaired glucose
tolerance, glycosuria, hyperlipidaemia, metabolic acidoses,
diabetes mellitus, diabetic neuropathy and nephropathy and also of
sequelae of diabetes mellitus in mammals.
[0002] The invention therefore relates also to a simple method of
lowering the blood sugar concentration in mammals with the aid of
dipeptide compounds as activity-reducing effectors (substrates,
pseudosubstrates, inhibitors, binding proteins, antibodies etc.)
for enzymes having activity comparable to or identical to the
enzymatic activity of the enzyme dipeptidyl peptidase IV.
[0003] DP IV or DP IV-analogous activity (for example the cytosolic
DP II has a substrate specificity almost identical to DP IV) occurs
in the blood circulation where it splits off dipeptides highly
specifically from the N-terminus of biologically active peptides
when proline or alanine are the adjacent residues of the N-terminal
amino acid in their sequence.
[0004] The glucose-dependent insulinotropic polypeptides:
[0005] gastric inhibitory polypeptide 1-2 (GIP.sub.1-42) and
glucagon-like peptide amide-1 7-36 (GLP-1.sub.7-36), that is to say
hormones that stimulate glucose-induced secretion of insulin by the
pancreas (also called incretins), are substrates of DP IV, since
the latter is able to split off the dipeptides tyrosinyl-alanine
and histidylalanine, respectively, from the N-terminal sequences of
those peptides in vitro and in vivo.
[0006] The reduction of such DP IV and DP IV-analogous enzyme
activity of the cleavage of those substrates in vivo can be used to
bring about effective suppression of undesired enzyme activity
under laboratory conditions and also in the case of pathological
conditions in mammalian organisms. For example, diabetes mellitus
Type II (including adult-onset diabetes) is based on a reduced
secretion of insulin or disorders in the receptor function
resulting inter alia from anomalous incretin concentrations arising
from proteolysis.
[0007] According to the current state of the art, hyperglycaemia
and associated causes and sequelae (including diabetes mellitus)
are treated by the administration of insulin (e.g. material
isolated from bovine pancreas or obtained by genetic engineering
techniques) to the diseased organisms in various forms of
administration. All the methods known hitherto, including more
modern procedures, are distinguished by the requirement of a large
amount of material, by high costs and often by a distinct
impairment of the quality of life of the patients. The conventional
method (daily i.v. insulin injection, customary since the 1930s)
treats the acute symptoms of the disease, but after prolonged use
leads inter alia to serious vascular changes (arteriosclerosis) and
nerve damage.
[0008] More recently the installation of subcutaneous depot
implants (the insulin is released in metered amounts, and daily
injections are unnecessary) and implantation (transplantation) of
intact Langerhan's cells into the functionally impaired pancreatic
gland or into other organs and tissues have been proposed. Such
transplants require a high level of technical resources.
Furthermore, they involve a surgical intervention into the
recipient organism, which is associated with risks, and even in the
case of cell transplants require methods of suppressing or
circumventing the immune system.
[0009] The use of alanyl pyrrolidide and isoleucyl thiazolidide as
inhibitors of DP IV or of DP IV-analogous enzyme activity is
already known from PCT/DE 97/00820 and the use of isoleucyl
pyrrolidide and isoleucyl thiazolidide hydrochloride is already
known from DD 296 075. Isoleucyl thiazolidide, which is used in the
latter prior art, is a natural, that is to say L-threo-isoleucyl
thiazolidide: on the priority date and also on the application date
of the two specifications, only that form, the natural form, of
isoleucyl thiazolidide was available.
[0010] It has been established that those compounds, especially
L-threo-isoleucyl thiazolidide, are good effectors for DP IV and DP
IV-analogous enzyme activities, but the use of that compound may
give rise to certain problems in the case of some patients or some
forms of the disease:
[0011] Depending upon the symptoms and the severity e.g. of
diabetes mellitus it would be desirable, for example, to have
available effectors that have an action different from that of the
known compounds: for example, it is known that diabetes mellitus
patients must be "stabilised" individually in order that their
illness can be treated in an optimum manner. In some cases, for
example, a reduction in the activity by DP IV effectors ought to be
sufficient. It is also possible that too high a level of inhibitor
activity and the permanent administration of the same medicament,
especially in view of the life-long duration of treatment, may
result in undesirable side-effects. Furthermore, it could also be
desirable to improve certain transport properties in order to
increase the rate of absorption of the effectors in vivo.
[0012] The aim of the invention is therefore to provide new
(especially activity-reducing) effectors for the treatment of e.g.
impaired glucose tolerance, glycosuria, hyperlipidaemia, metabolic
acidoses, diabetes mellitus, diabetic neuropathy and nephropathy
and also of sequelae of diabetes mellitus in mammals, and a simple
method of treating such diseases.
[0013] This aim is achieved according to the invention by the
provision of dipeptide compounds or analogues of dipeptides that
are formed from an amino acid and a thiazolidine or pyrrolidine
group, and salts thereof.
[0014] On administration, preferably oral administration, of these
effectors to a mammalian organism, the endogenous (or additionally
exogenously administered) insulinotropic peptides GIP.sub.1-42 and
GLP-1.sub.7-36 (or alternatively GLP-1.sub.7-37 or analogues
thereof) are broken down to a reduced extent by DP IV or DP IV-like
enzymes and therefore the decrease in the concentration of those
peptide hormones or their analogues is reduced or delayed. The
invention is therefore based on the finding that a reduction, in
the DP IV or DP IV-like enzymatic activity acting in the blood
circulation has an effect on the blood sugar level. It has been
found that
[0015] 1. the reduction in DP IV or DP IV-analogous activity leads
to an increase in the relative stability of the glucose-stimulated
or externally introduced incretins (or analogues thereof), that is
to say by administration of effectors of DP IV or DP IV-analogous
proteins it is possible to control the breakdown of incretin in the
blood;
[0016] 2. the increase in the biological breakdown stability of the
incretins (or their analogues) results in a change in the action of
endogenous insulin;
[0017] 3. the increase in the stability of the incretins brought
about by the reduction in DP IV or DP IV-analogous enzymatic
activity in the blood results in a subsequent change in the
glucose-induced insulin action and therefore in a modulation of the
blood glucose level that is controllable by means of DP
IV-effectors.
[0018] Especially suitable for that purpose according to the
invention are dipeptide compounds in which the amino acid is
selected from a natural amino acid, such as, for example, leucine,
valine, glutamine, proline, isoleucine, asparagine and aspartic
acid.
[0019] The administration, where possible oral administration, of
the high-affinity, low molecular weight enzyme inhibitors according
to the invention is a more economical alternative e.g. to invasive
surgical techniques in the treatment of pathological symptoms.
Through a chemical design of stability, transport and clearance
properties, their mode of action can be modified and matched to
individual characteristics.
[0020] As mentioned above, it may be necessary, for example in the
case of the long-term treatment of diabetes mellitus, to provide
effectors having a defined activity with which it is possible to
meet the individual needs of patients and to treat their symptoms.
The dipeptide compounds according to the invention therefore
exhibit at a concentration (of dipeptide compounds) of 10 .mu.M,
especially under the conditions indicated in Table 1, a reduction
in the activity of dipeptidyl peptidase IV or DP IV-analogous
enzyme activities of at least 10%, especially of at least 40%.
Frequently a reduction in activity of at least 60% or at least 70%
is also required. Preferred effectors may also exhibit a reduction
in activity of a maximum of 20% or 30%. Furthermore, the transport
properties of the present compounds, especially by the peptide
transporter Pep T1, are significantly improved.
[0021] Especially preferred dipeptide compounds are
L-allo-isoleucyl thiazolidide and salts thereof. Those compounds
surprisingly exhibit an approximately five-fold improvement in
transport by the peptide transporter Pep T1 in comparison with
L-threo-isoleucyl thiazolidide, while having approximately the same
degree of action with respect to glucose modulation.
[0022] Further preferred compounds are given in Table 1.
[0023] The salts of the dipeptide compounds according to the
invention may be, for example, organic salts such as acetates,
succinates, tartrates or fumarates, or inorganic acid radicals such
as phosphates or sulphates. Special preference is given to the
fumarates, which have an excellent action combined with a
surprisingly high degree of stability towards hydrolysis and are
considerably less soluble than the hydrochlorides. Those properties
are also advantageous from the galenical standpoint.
[0024] Also preferred are L-threo-isoleucyl pyrrolidide and salts
thereof, especially the fumaric salts, and L-allo-isoleucyl
pyrrolidide and salts thereof, especially the fumaric salts.
[0025] The salts of the dipeptide compounds can be present in a
molar ratio of dipeptide(-analogous) component to salt component of
1:1 or 2:1. Such a salt is, for example, (Ile-Thia).sub.2 fumaric
acid.
[0026] Especially preferred salts are the fumaric salts of
L-threo-isoleucyl thiazolidide and L-allo-isoleucyl
thiazolidide.
[0027] The invention accordingly relates to effectors of dipeptidyl
peptidase IV (DP IV) or DP IV analogous enzyme activity and their
use in lowering the blood sugar level in the serum of a mammalian
organism below the glucose concentration that is characteristic of
hyperglycaemia. The invention relates especially to the use of the
effectors of DP IV or DP IV-analogous enzyme activity according to
the invention in preventing or alleviating pathological metabolic
anomalies in mammalian organisms, such as, for example, impaired
glucose tolerance, glycosuria, hyperlipidaemia, metabolic acidoses,
diabetes mellitus, diabetic neuropathy and nephropathy and also
sequelae of diabetes mellitus in mammals. In a further preferred
embodiment, the invention relates to a method of lowering the blood
sugar level in the serum of a mammalian organism below the glucose
concentration that is characteristic of hyperglycaemia,
characterised in that a therapeutically effective amount of at
least one effector of DP IV or DP IV-analogous enzyme activity
according to the invention is administered to a mammalian
organism.
[0028] In a further preferred embodiment, the invention relates to
pharmaceutical compositions, that is to say medicaments, that
comprise at least one compound according to the invention or a salt
thereof, optionally in combination with one or more
pharmaceutically acceptable carriers and/or solvents.
[0029] The pharmaceutical compositions may be, for example, in the
form of parenteral or enteral formulations and may contain
appropriate carriers or they may be in the form of oral
formulations that may contain appropriate carriers suitable for
oral administration. They are preferably in the form of oral
formulations.
[0030] In addition, the pharmaceutical compositions may contain one
or more active ingredients having a hypoglycaemic action, which may
be active ingredients known per se.
[0031] The effectors of DP IV or DP IV-analogous enzyme activity
according to the invention can be used for lowering the blood sugar
level in the serum of a mammalian organism below the glucose
concentration that is characteristic of hyperglycaemia or for the
production of a corresponding medicament.
[0032] The effectors of DP IV or DP IV-analogous enzymes
administered according to the invention can be used in
pharmaceutically acceptable formulations or formulation complexes
as inhibitors, substrates, pseudosubstrates, inhibitors of DP IV
expression, binding proteins or antibodies of those enzyme proteins
or combinations of those different substances that reduce the DP IV
or DP IV-analogous protein concentration in the mammalian organism.
Effectors according to the invention are, for example, DP
IV-inhibitors such as the dipeptide derivatives or dipeptide
mimetics L-allo-isoleucyl thiazolidide and the effectors indicated
in Table 1 and fumaric salts thereof. The effectors according to
the invention enable the treatment of patients and diseases to be
adjusted individually, it being possible especially to avoid
intolerances, allergies and side effects occurring in individual
cases.
[0033] The compounds also exhibit different effectiveness
behaviours over time. As a result, the physician carrying out the
treatment has the opportunity to respond in various ways according
to the individual situation of a patient: he is able, on the one
hand, to set accurately the speed of onset of the action and, on
the other hand, the duration of action and especially the strength
of action.
[0034] The method according to the invention represents a new kind
of procedure for lowering raised blood glucose concentrations in
the serum of mammals. It is simple, capable of commercial
exploitation and suitable for use in therapy, especially of
diseases that are based on above-average blood glucose values, in
mammals and more especially in human medicine.
[0035] The effectors are administered, for example, in the form of
pharmaceutical preparations that comprise the active ingredient in
combination with customary carrier materials known in the prior
art. For example, they will be administered parenterally (e.g.
i.v., in physiological saline) or enterally (e.g. orally,
formulated with customary carrier materials, such as, for example,
glucose).
[0036] Depending upon their endogenous stability and their
bioavailability, the effectors will need to be administered one or
more times per day in order to achieve the desired normalisation of
the blood glucose values. For example, such a dosage range in human
beings may lie in the range of from 0.01 mg to 30.0 mg per day,
preferably in the range of from 0.01 to 10 mg of effector substance
per kilogram of body weight.
[0037] It has been found that as a direct result of the
administration of effectors of dipeptidyl peptidase IV or DP
IV-analogous enzyme activities in the blood of a mammal, by virtue
of the associated temporary reduction in the activity thereof, the
endogenous (or additionally exogenously administered)
insulinotropic peptides gastric inhibitory polypeptide 1-42
(GIP.sub.1-42) and glucagon-like peptide amide-1 7-36
(GLP-1.sub.7-36) (or alternatively GLP-1.sub.7-37 or analogues
thereof) are broken down to a reduced extent by DP IV and DP
IV-like enzymes and thus the decrease in the concentration of those
peptide hormones or their analogues is reduced or delayed. The
increase in the stability of the (endogenously present or
exogenously introduced) incretins or their analogues brought about
by the action of DP IV-effectors with the result that the former
are available in increased amounts for insulinotropic stimulation
of the incretin receptors of the Langerhan's cells in the pancreas,
alters inter alia the effectiveness of the body's own insulin,
which results in a stimulation of the carbohydrate metabolism of
the treated organism.
[0038] As a result, the blood sugar level in the serum of the
organism being treated falls below the glucose concentration that
is characteristic of hyperglycaemia, thus making it possible to
prevent or alleviate metabolic anomalies such as impaired glucose
tolerance, glycosuria, hyperlipidaemia and possible severe
metabolic acidoses and diabetes mellitus, which are clinical
syndromes resulting from raised glucose concentrations in the blood
over a prolonged period.
[0039] Among the number of orally effective anti-diabetics known
from the prior art, such an effective low molecular weight
substance class has been unknown hitherto (with the exception of
the biguanide metformin: molecular weight 130). The molecular
weights of the aminoacyl thiazolidides vary between 146 (glycyl
thiazolidide), 203 (isoleucyl thiazolidide) and 275 (tryptophanyl
thiazolidide). In comparison, the molecular weights of the
sulphonylureas (glibenclamide: 494), the saccharides (acarbose:
630) and the thiazolidinediones (pioglitazon: 586) vary in the
range around 500 to 700 Da. In the body, aminoacyl thiazolidides
are hydrolysed by aminopeptidases and by acidic hydrolysis to form
endogenous substances, such as amino acids and cysteamine, so that
the use of the compounds according to the invention as orally
available anti-diabetics constitutes an enrichment of pharmacy.
[0040] In rats and mice, experimentally induced hyperglycaemia can
be treated to a better than average extent by oral administration
of the compounds used according to the invention (Tables 2 and 3).
The administration of 500 to 1000 times the effective dose did not
result in any demonstrable pathological change during three-week
toxicological experiments on rats and mice
[0041] The advantageous action of compounds according to the
invention on DP IV is shown by way of example in Table 1:
TABLE-US-00001 TABLE 1 Action of various effectors on the
dipeptidyl-peptidase-IV-catalysed hydrolysis of 0.4 mM of the
substrate H-Gly-Pro-pNA at 30.degree. C., pH 7.6 and an ionic
strength of 0.125 Effector affinity % Residual activity of to DP
IV: DP IV in the presence of Effector K.sub.i [nM] 10 .mu.M
effector metformin >>1,000,000 100 glibenclamide
>>1,000,000 100 acarbose >>1,000,000 100
H-Asn-pyrrolidide 12,000 83.1 H-Asn-thiazolidide 3,500 47.2
H-Asp-pyrrolidide 14,000 81.6 H-Asp-thiazolidide 2,900 45.6
H-Asp(NHOH)-pyrrolidide 13,000 88.2 H-Asp(NHOH)-thiazolidide 8,800
54.5 H-Glu-pyrrolidide 2,200 38.5 H-Glu-thiazolidide 610 25.0
H-Glu(NHOH)-pyrrolidide 2,800 44.9 H-Glu(NHOH)-thiazolidide 1,700
36.5 H-His-pyrrolidide 3,500 49.7 H-His-thiazolidide 1,800 35.2
H-Pro-pyrrolidide 4,100 50.2 H-Pro-thiazolidide 1,200 27.2
H-Ile-azididide 3,100 43.8 H-Ile-pyrrolidide 210 12.3
H-L-allo-Ile-thiazolidide 190 10.0 H-Val-pyrrolidide 480 23.3
H-Val-thiazolidide 270 13.6
[0042] It is known that aminoacyl pyrrolidides and aminoacyl
thiazolidides can be broken down by the enzymes proline
aminopeptidase and prolidase present in the mucosa cells of the
small intestine, in serum and in liver cells and that the
thiazolidine ring has a tendency to open in the presence of acids
(for example in the stomach) with the formation of the
corresponding cysteamine derivative [see U.S. Pat. No. 458 407]. It
was therefore surprising to find that the active ingredients have a
dose-dependent effectiveness after peroral administration. The
dose-dependency of the action of L-allo-Ile-thiazolidide on the
serum-DP IV activity after oral administration of L-allo-isoleucyl
thiazolidide to healthy Wistar rats is documented in the following
Table:
TABLE-US-00002 TABLE 2 Residual activity of DP IV in serum towards
0.4 mM of the substrate H-Gly-Pro-pNA at 30.degree. C., pH 7.6 and
an ionic strength of 0.125, after oral administration and in
dependence upon the dose of L-allo-isoleucyl thiazolidide,
determined 30 min after administration of the inhibitor Dose per
experimental animal Residual activity of DP IV in % 0 mg 100 2.5 mg
52 5.0 mg 40 10 mg 28 20 mg 29
[0043] Extremely surprising and desirable is the glucose-reducing
action of the active ingredient L-allo-isoleucyl thiazolidide
according to the invention achieved in the diabetic animal model
after oral administration with synchronous oral glucose stimulation
(Table 3).
[0044] In order to intensify the blood-sugar-reducing action of
various anti-diabetics, use is frequently made of combinations of
different orally effective anti-diabetics. Since the
anti-hyperglycaemic action of the effectors according to the
invention is exhibited independently of other known orally
administrable anti-diabetics, the active ingredients according to
the invention are analogously suitable for use in combination
therapies, in a suitable galenical form, for achieving the desired
normoglycaemic effect.
[0045] Accordingly, the compounds used according to the invention
can be made in a manner known per se into the customary
formulations, such as, for example, tablets, capsules, dragees,
pills, suppositories, granules, aerosols, syrups, liquid, solid and
cream-type emulsions and suspensions and solutions using inert,
non-toxic, pharmaceutically acceptable carriers and additives or
solvents. In such-formulations the therapeutically effective
compounds are in each case preferably present in a concentration of
approximately from 0.1 to 80% by weight, preferably from 1 to 50%
by weight, of the total mixture, that is to say in amounts
sufficient to achieve a dosage within the indicated range.
TABLE-US-00003 TABLE 3 Reduction in the circulating blood glucose
within a period of 60 min after oral administration of 20 .mu.M of
L-allo-Ile thiazolidide to rats of various animal models with a
synchronous glucose tolerance test (data in % based on
normoglycaemic values) Glucose Glucose concentration concentration
in % in % L-allo- Animal model control Ile-thiazolidide-treated
Wistar rat, normal 100 82 Wistar rat (diabetes 2b- 100 73 model,
obese)
[0046] The good absorption of the compounds used according to the
invention by mucosae of the gastrointestinal tract enables a large
number of galenical preparations to be used:
[0047] The substances can be administered as medicaments in the
form of dragees, capsules, bitable capsules, tablets, drops and
syrup, as well as in the form of pessaries and nasal sprays.
[0048] The formulations are produced, for example, by extending the
active ingredient with solvents and/or carriers, optionally using
emulsifiers and/or dispersing agents, and optionally, for example
where water is used as diluent, organic solvents may be used as
auxiliary solvents.
[0049] The following auxiliaries may be mentioned by way of
example: water, non-toxic organic solvents, such as paraffins (e.g.
mineral oil fractions), vegetable oils (e.g. rapeseed oil,
groundnut oil, sesame oil), alcohols (e.g. ethyl alcohol,
glycerol), glycols (e.g. propylene glycol, polyethylene glycol);
solid carriers, such as, for example, ground natural minerals (e.g.
highly dispersed silicic acid, silicates), sugars (e.g. unrefined
sugar, lactose and dextrose); emulsifiers, such as non-ionic and
anionic emulsifiers (e.g. polyoxyethylene fatty acid esters,
polyoxyethylene fatty alcohol ethers, alkylsulphonates and
arylsulphonates), dispersing agents (e.g. lignin, spent sulphite
liquors, methylcellulose, starch and polyvinylpyrrolidone) and
glidants (e.g. magnesium stearate, talcum, stearic acid and sodium
lauryl sulphate) and optionally flavourings.
[0050] Administration is effected in customary manner, preferably
enterally or parenterally, especially orally. In the case of
enteral administration, in addition to containing the mentioned
carriers, tablets may also comprise other additives, such as sodium
citrate, calcium carbonate and calcium phosphate, together with
various supplementary ingredients, such as starch, especially
potato starch, gelatin and the like. It is also possible to use
glidants, such as magnesium stearate, sodium lauryl sulphate and
talcum, for tableting purposes. In the case of aqueous suspensions
and/or elixirs intended for oral uses it is also possible for
various taste correctors or colourings to be added to the active
ingredients in addition to the auxiliaries mentioned above.
[0051] For parenteral administration it is possible to use
solutions of the active ingredients using suitable liquid carrier
materials. In the case of intravenous administration it has
generally proved advantageous to administer amounts of
approximately from 0.01 to 2.0 mg/kg, preferably approximately from
0.01 to 1.0 mg/kg, body weight per day in order to achieve
effective results, and in the case of enteral administration the
dosage is approximately from 0.01 to 2 mg/kg, preferably
approximately from 0.01 to 1 mg/kg, body weight per day.
[0052] Nevertheless in some cases it may be necessary to depart
from the amounts indicated, depending upon the body weight of the
experimental animal or patient or the nature of the administration
route, and also on the basis of the species of animal and its
individual response to the medicament or the intervals at which the
administration is made. In some cases, for example, it may be
sufficient to use less than the above-mentioned minimum amount,
whereas in other cases it will be necessary to exceed the
above-mentioned upper limit. Where relatively large amounts are
administered it may be advisable to divide the amount into several
individual doses over the day. For use in human medicine the same
range of dosage is provided, the comments made above also applying
accordingly.
EXAMPLES OF PHARMACEUTICAL FORMULATIONS
[0053] 1. Capsules having 100 mg of L-allo-isoleucyl thiazolidide
per capsule:
[0054] For about 10,000 capsules, a solution of the following
composition is prepared:
TABLE-US-00004 L-allo-isoleucyl thiazolidide hydrochloride 1.0 kg
glycerol 0.5 kg polyethylene glycol 3.0 kg water 0.5 kg 5.0 kg
[0055] The solution is introduced into soft gelatin capsules in a
manner known per se. The capsules are suitable for chewing or
swallowing.
[0056] 2. Tablets/coated tablets or dragees having 100 mg of
L-allo-isoleucyl thiazolidide:
[0057] The following amounts relate to the production of 100,000
tablets:
TABLE-US-00005 L-allo-isoleucyl thiazolidide hydrochloride, 10.0 kg
finely ground glucose 4.35 kg lactose 4.35 kg starch 4.50 kg
cellulose, finely ground 4.50 kg
[0058] The above constituents are mixed together and then combined
with a solution, prepared from
TABLE-US-00006 polyvinylpyrrolidone 2.0 kg polysorbate 0.1 kg and
water about 5.0 kg
and granulated in a manner known per se by grating the moist mass
and, after the addition of 0.2 kg of magnesium stearate, drying.
The finished tablet mixture of 30.0 kg is processed to form domed
tablets each weighing 300 mg. The tables can be coated or
sugar-coated in a manner known per se.
[0059] The technical data of preferred compounds are given
below.
TABLE-US-00007 Tests on Ile-Thia*fumarate (isomer) and other salts
Substance K.sub.i Mp (.degree. C.) CE (min) MS [.alpha.]H.sub.2O
.fwdarw. L-threo-IT*F 8 * 10.sup.-8 150.sup.DSC 160 203 -10.7 |
(405 nm) | .fwdarw. D-threo-IT*F no inhibition 147 158 203 not | |
determined | .fwdarw. L-allo-IT*F 2 * 10.sup.-7 145-6 154 203 -4.58
| (380 nm) .fwdarw. D-allo-IT*F no inhibition 144-6 150 203 4.5
(380 nm) IT*F = isoleucyl thiazolidide fumarate The NMR and HPLC
data confirm the identity of the substances in question.
[0060] Measurement Conditions for the K.sub.i Determination of the
Substances
TABLE-US-00008 Enzyme: DP IV.sub.porcine kidney, 0.75 mg/ml, 18
U/ml (GPpNA) in 25 mM Tris pH 7.6, 30% ammonium sulphate, 0.5 mM
EDTA, 0.5 mM DTE Stock solution: 1:250 diluted in measuring buffer
Buffer: 40 mM HEPES pH 7.6, I = 0.125 (KCl) Substrate: GPpNA*HCl
Stock solution: 2.1 mM Measuring Perkin-Elmer Bio Assay Reader, HTS
7000 Plus, apparatus: T = 30.degree. C. .lamda. = 405 nm
Measurement 100 .mu.l buffer batch: 100 .mu.l substrate (3
different concentrations 0.8 mM-0.2 mM) 50 .mu.l water/inhibitor (7
different concentrations 2.1 .mu.M-32.8 nM) 10 .mu.l enzyme
[0061] Buffer, water/inhibitor and enzyme were preheated to
30.degree. C. and the reaction was started by the addition of
substrate which was likewise preheated.
[0062] Determinations were carried out four times.
[0063] The measuring time was 10 minutes.
[0064] Melting Point Determination
[0065] Melting points were determined on a Kofler heating platform
microscope from Leica Aktiengesellschaft, the values are not
corrected, or on a DSC apparatus (Heumann-Pharma).
[0066] Optical Rotation
[0067] The rotation values were recorded at different wavelengths
on a "Polarimeter 341" or higher, from the Perkin-Elmer
company.
[0068] Measurement Conditions for the Mass Spectroscopy
[0069] The mass spectra were recorded by means of electrospray
ionisation (ESI) on an "API 165" or "API 365" from the PE Sciex
company.
[0070] The operation is carried out using an approximate
concentration of c=10 .mu.g/ml, substance is taken up in
MeOH/H.sub.2O 50:50, 0.1% HCO.sub.2H, the infusion is effected
using a spray pump (20 .mu.l/min). The measurements were made in
positive mode [M+H].sup.+, the ESI voltage is U=5600V.
[0071] The salts have the following data:
TABLE-US-00009 IT*salt K.sub.i M (gmol.sup.-1) Mp (.degree. C.)
succinate 5.1e-8 522.73 116 tartrate 8.3e-8 352.41 122 fumarate
8.3e-8 520.71 156 hydrochloride 7.2e-8 238.77 169 phosphate 1.3e-7
300.32 105
[0072] Testing the Solubility of Salts of Ile-Thia
[0073] Ile-Thia*fum
[0074] Amount weighed in 10.55 mg corresponds to 0.02 mmol (520.72
g/mol)
[0075] Addition of 100 .mu.l H.sub.2O.sub.dist.
[0076] 100 .mu.l no solution, visually: no surface-wetting from 200
.mu.l successive beginning of solubility at 400 .mu.l complete
dissolution is observed 2.63%
[0077] It is therefore established that this salt is scarcely
wettable and does not decompose.
[0078] Ile-Thia*succ
[0079] Amount weighed in 16.6 mg corresponds to 0.031 mmol (522.73
g/mol)
[0080] Addition of 16 .mu.l H.sub.2O.sub.dist.
[0081] 16 .mu.l no solution, visually: "sucking-up" of the moisture
from 66 .mu.l-1.5 ml no complete dissolution of the substance is
observed
[0082] Ile-Thia*tartrate
[0083] Amount weighed in 17.3 mg corresponds to 0.049 mmol (352.41
g/mol)
[0084] Addition of 100 .mu.l H.sub.2O.sub.dist.
[0085] 100 .mu.l complete dissolution 17.3%
[0086] Ile-Thia*phos
[0087] Amount weighed in 15.5 mg corresponds to 0.051 mmol (300.32
g/mol)
[0088] Addition of 100 .mu.l H.sub.2O.sub.dist.
[0089] 100 .mu.l slight dissolution is observed successive addition
of 100 .mu.l H.sub.2O at 400 .mu.l complete dissolution 3.87%
[0090] Ile-Thia*HCl
[0091] Amount weighed in 16.1 mg corresponds to 0.067 mmol (238.77
g/mol)
[0092] Addition of 100 .mu.l H.sub.2O.sub.dist. at 100 .mu.l
complete dissolution 16.1%
[0093] General Synthesis of Ile-Thia*salt
[0094] The Boc-protected amino acid Boc-Ile-OH is placed in. ethyl
acetate and the batch is cooled to about -5.degree. C.
N-Methylmorpholine is added dropwise, pivalic acid chloride (on a
laboratory scale) or neohexanoyl chloride (on a pilot-plant scale)
is added dropwise at constant temperature. The reaction is stirred
for a few minutes for activation. N-Methylmorpholine (laboratory
scale) and thiazolidine hydrochloride (laboratory scale) are added
dropwise in succession, thiazolidine (pilot-plant scale) is added.
Working-up in the laboratory is effected in conventional manner
using salt solutions, on a pilot-plant scale the batch is purified
with NaOH and CH.sub.3COOH solutions.
[0095] The removal of the Boc protecting group is carried out using
HCl/dioxane (laboratory scale) or H.sub.2SO.sub.4 (pilot-plant
scale).
[0096] In the laboratory the hydrochloride is crystallised from
EtOH/ether.
[0097] On a pilot-plant scale the free amine is prepared by the
addition of NaOH/NH.sub.3. Fumaric acid is dissolved in hot
ethanol, the free amine is added dropwise, and (Ile-Thia).sub.2
fumarate (M=520.71 gmol.sup.-1) precipitates.
[0098] The analysis of isomers and enantiomers is carried out by
electrophoresis.
* * * * *