comparative activity of proline-containing dipeptide noopept and inhibitor of dipeptidyl peptidase-4...
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0007 -4888/14/1563�0342 © 2014 Springer Science+Business Media New York
Comparative Activity of Proline-Containing Dipeptide Noopept and Inhibitor of Dipeptidyl Peptidase-4 Sitagliptin in a Rat Model of Developing DiabetesR. U. Ostrovskaya, I. V. Ozerova, T. A. Gudascheva, I. G. Kapitsa, E. A. Ivanova, T. A. Voronina, and S. B. Seredenin
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 156, No. 9, pp. 317-322, September, 2013Original article submitted June10, 2012
Developing diabetes was modeled on adult male Wistar rats by repeated intraperitoneal injec-tions of streptozotocin in a subdiabetogenic dose of 30 mg/kg for 3 days. Proline-containing dipeptide drug Noopept or a standard diabetic drug dipeptidyl peptidase-4 inhibitor sitagliptin was administered per os in a dose of 5 mg/kg before each injection of the toxin and then for 16 days after streptozotocin course. In active control group, spontaneously increase glucose level and reduced tolerance to glucose load (1000 mg/kg intraperitoneally) were observed on the next day after the third administration of toxin. Basal glucose level decreased by day 16, but glucose tolerance remained impaired. Noopept normalized the basal blood glucose level and tolerance to glucose load on the next day after administration of streptozotocin. The effect of Noopept persisted to the end of the experiment. At early terms of the experiment, sitagliptin was somewhat superior to Noopept by the effect on baseline glucose level, but was inferior by the infl uence on glucose tolerance.. By the end of the experiment, Noopept signifi cantly (by 2 times) surpassed sitagliptin by its effect on glucose tolerance.
Key Words: diabetes; streptozotocin; dipeptide preparation Noopept; sitagliptin
V.V. Zakusov Research Institute of Pharmacology, Russian Acade-
my of Medical Sciences, Moscow, Russia. Address for correspon-dence: [email protected]. R. U. Ostrovskaya
The search for effective antidiabetic agents is dictated by high prevalence of diabetes mellitus and lack of effective drugs with minimal side effects. Type 2 dia-betes mellitus is a multifactorial metabolic disorder featured by resistance of insulin-dependent tissue to insulin in combination with pancreatic β-cell failure to compensate it [3]. The so-called incretins, gastro-intestinal hormones, namely glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic poly-peptide (GIP), are proved to participate in enhancing insulin secretion by β cells. Both these peptides have low biological stability, which precludes their thera-peutic application, therefore, their peptidomimetics or inhibitors of dipeptidyl peptidase-4 (DPP-4), an
enzyme hydrolyzing these peptides, were proposed for the treatment of diabetes mellitus [9]. Januvia (si-tagliptin) was the fi rst orally-active inhibitor of DPP-4 registered in USA in 2007. It is now widely used in the Russian Federation.
The concept of dipeptides mimicking molecular structure of nonpeptide drug and active site of the enzyme with appropriate activity is developed at V. V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences [2]. Highly effective nootropic and neuroprotective dipeptide drug Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) was de-veloped based on the structure of piracetam and vaso-pressin [10]. Wide clinical use of the drug (registration number LS-001577) showed its pronounced nootropic activity combined with good tolerability.
Potential anti-diabetic properties of Noopept were studied based on neuroprotective effects exerted by
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Noopept [11] and the idea that some of the neuro-chemical characteristics of β-cells and neuronal tis-sue are similar [13]. Intraperitoneal administration of Noopept reduced the degree of hyperglycemia, weight loss, and increased pain sensitivity caused by single administration of diabetogenic toxin streptozotocin (STZ) in a dose of 45-50 mg/kg [6].
Clinical manifestation of diabetes mellitus is pre-ceded by metabolic disorders in the pancreatic insu-lar tissue; therefore, the development of preparations for pathogenetic therapy aimed at preventing diabetes progression is an urgent problem [3]. Models of re-introduction of small doses of diabetogenic toxin STZ simulating the dynamics of type 2 diabetes mellitus are of interest in this context [4]. Orally active anti-diabetic drugs, including sitagliptin, most effectively prevent aggravation of the disease and thus may be used continuously. Noopept is a substituted dipep-tide. Unlike peptides with more complex chemical structure, dipeptides remain active when administered orally because of specifi c ATP-dependent system me-diating the transport of dipeptides into enterocytes [12]. Oral bioavailability of Noopept was confi rmed by pharmacokinetic analysis [2].
Here we studied the effects of oral administration of Noopept and sitagliptin, the gold standard for dia-betes treatment, on the model of progressive diabetes.
MATERIALS AND METHODS
Experiments were performed on adult male Wistar rats weighing 210-240 g obtained from Stolbovaya Breed-ing Center (Russian Academy of Medical Sciences). The animals were maintained under vivarium condi-tions (10 rats per cage) with free access to standard food and water except for 12 h preceding measure-ment of the basal (fasting) glucose level and glucose tolerance.
Group 1 rats (passive control) were intraperito-neally injected with saline daily for 3 days and then through a gastric tube for the next 16 days. Group 2 rats (active control) received STZ (Sigma), a toxin that causes selective oxidative stress, DNA alkylation, and apoptosis of pancreatic β cells [15]. STZ was injected intraperitoneally in a dose of 30 mg/kg for 3 days [4]. Saline was administered 5 min before each STZ injection and then for 16 days. Group 3 rats (experi-mental animals) received intraperitoneal injections of STZ in a dose of 30 mg/kg for 3 days. Noopept in a dose of 5 mg/kg was administered 5 min before each STZ injection and then for 16 days. This dose was chosen because the intraperitoneal Noopept exhibited antidiabetic activity in a dose of 0.5 mg/kg [6], while our previous experience suggests that transition from parenteral to enteral administration required 10-fold
dose elevation for attaining the same effect. Group 4 rats (experimental animals) received STZ intraperito-neally in a dose of 30 mg/kg for 3 days. Sitagliptin was administered 5 min before STZ in a dose of 5 mg/kg and then for 16 days. The dose of sitagliptin was chosen based on numerous reports [9]. Each group consisted of 12 animals.
Blood glucose levels (One Touch Ultra gluco-meter) were measured in the blood sampled from the caudal vein on day 4 after the fi rst STZ injection, and then on days 12 and 19. Along with measurement of basal (fasting) blood glucose, all the rats were tested for glucose tolerance (1000 mg/kg intraperitoneally). Blood glucose was measurement 1 and 2 h after glu-cose load. The animals were weighed every 3 days.
The index of antihyperglycemic activity (AA) was calculated to characterize the time course of the effects of Noopept and sitagliptin by the formula: glSTZ-(glSTZ+drug)
AA= ×100%, glSTZ-glSwhere glSTZ and glS are blood glucose levels in ac-tive and passive control groups, respectively, and glSTZ+drug is blood glucose in groups 3 and 4.
The significance of differences between the groups was evaluated using Mann–Whitney test after the preliminary Kruskal–Wallis test. The results were considered signifi cant at p≤0.05.
RESULTS
In the passive control group, baseline glucose was 4.9 mmol/liter and 1 h after load it did not differ sig-nifi cantly from the initial level (5.28 mmol/liter). In animals treated with STZ (active control), glucose level spontaneously increased on the next day after the 3rd STZ injection to 10.84 mmol/liter and tolerance to glucose decreased. One hour after load, glucose level was 17.15 mmol/liter and in 2 h it was 17.93±2.05 mmol/liter. On experimental day 12, basal (fasting) glucose level began to decline, but tolerance to glu-cose remained impaired. On experimental day 19, the basal glucose level in this group returned to normal, but glucose tolerance was still lowered (Table 1). Body weight of rats remained unchanged at all stages of the experiment in all the groups.
In group 3 (Noopept), we found marked decrease in basal glucose level (to 5.54 mmol/liter) correspond-ing to the calculated index AA 90.4% on the next day after STZ administration. Importantly Noopept im-proved tolerance to the glucose load at the very begin-ning of the experiment: the indicator of glucose toler-ance did not signifi cantly differ from that in the group of passive control. Indicators of baseline glycemia and glucose 1 h after load are presented in Figure 1.
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In group 3 (Noopept), parameters did not differ from these in the passive control group in 9 days after STZ treatment (experimental day 12), that is, Noopept normalized the basal level of glycemia and glucose tolerance. This was consistent with the increase in AA indicators for both baseline and post-load glyce-mia. At last, the testing of the active control group 16 days after the end of STZ administration showed that spontaneous glycemia level restored along with some improvement in tolerance to glucose load, and the indices in the experimental group (Noopept) did not differ from those in the group of passive control.
In group 4 (sitagliptin), the basal glucose level re-turned to normal on experimental day 4, which corre-sponded to the high AA index (97.9%; Table 1); it was
slightly higher than in group 3. However, the effect of sitagliptin on glucose tolerance was less pronounced than that of Noopept, especially during the fi rst hour (Fig. 2). On experimental day 12 (9 days after the end of STZ administration), the basal glucose level began to decline and tolerance to glucose load was lowered in the group of active control (15 and 6 mmol/liter, 9 and 8 mmol/liter 1 and 2 h after load, respectively). In group 4 (sitagliptin), basal glucose level was de-creased, and tolerance to glucose was better than in the active control group. On experimental day 19 (16 days after the end of STZ administration), the basal glucose level in active control returned to normal, but tolerance to glucose load remained impaired after 1 h manifesting in high glucose level (12 mmol/liter) 1 h after load. In group 4 (sitagliptin), blood glucose dur-ing the load decreased to 8.8 mmol/liter and AA was 43.6% vs. 84.2% in Noopept group, i.e. almost 2-fold lower than in group 3.
Analyzing the used model of diabetes, we should note that it differs fundamentally from the standard model of single toxin administration at higher dosages. In our previous experiments [6] with single injection of STZ in doses of 45-50 mg/kg, basal hyperglycemia was ≥16 mmol/liter even on day 28 after toxin ad-ministration, which corresponded to published reports [15]. In the model of fractional administration, this parameter did not exceed 10.8 mmol/liter in the period of maximum intensity (experimental day 4) and was close to 6 mmol/liter as soon as on day 9 after STZ treatment. The absence of body weight loss charac-teristic of single STZ administration in a higher dose according to our [6] and literature data [15] demon-strated lower symptom severity in case of fractional STZ administration in low doses. Importantly, toler-ance to glucose load remained impaired in delayed periods after the end of fractional STZ administration (day 19), while glucose levels were close to normal. It is known that clinical manifestations of diabetes and basal hyperglycemia arise only when the injury covers the majority of β cells. The stage of glucose tolerance precedes manifestation of developed clinical symptoms of diabetes [3]. The fact that in the group of active control tolerance was impaired against prac-tically normal basal glucose levels suggests that this scheme of STZ administration (30 mg/kg for 3 days) simulates certain similarities to developing diabetes.
Fundamentally important fact obtained in our ex-periments is the antidiabetic activity of Noopept at oral administration. It was also found that Noopept, at least in this model of progressive diabetes, is not inferior to gold standard, i. e. is oral antidiabetic drug sitagliptin. During the fi rst days after the end of toxin injection, the index of antihyperglycemic activity for baseline glucose level was similar for both drugs and glucose
Fig. 1. Effects of Noopept on spontaneous hyperglycemia and
glucose tolerance in the Wistar rat model of progressive diabetes.
Basal glucose levels at fasting and 1 h after glucose administra-
tion on days 4, 12, and 19 after the end of STZ treatment. Light
bars: active control; dark bars: Noopept (5 mg/kg per os). p<0.05
in comparison with: *passive control, +active control.
Fig. 2. Comparison of Noopept (light bars) and sitagliptin (dark
bars) by relative AA (%) upon oral drug administration in the model
of progressive diabetes.
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tolerance was not much better in group 3 (Noopept) than in group 4 (sitagliptin) (Fig. 3). The effi ciency of Noopept by its infl uence on glucose tolerance index was higher than that of sitagliptin by the end of the experiment (Table 1).
As for mechanisms of antihyperglycemic actions of sitagliptin, it is well known that this drug inhibits the enzyme DPP-4 and thereby slows down deactiva-tion of incretins GLP-1 and GIP [9]. However, the effect of both peptides is not confi ned to elevation of circulating insulin level. They produce a pronounced neurotrophic effect by increasing survival and pro-liferation of β-cells [14]. Noopept also increases the expression of neurotrophic factors [5]. Though this ef-fect was demonstrated by us only for the hippocampus and hypothalamus, given the similarity of brain and pancreatic metabolism, in particular reduced expres-sion of NGF in both the pancreas and hypothalamus in STZ-diabetes [13], it is possible that Noopept also enhances NGF expression in the insular tissue. The antioxidant system of the pancreas is considerably weaker than in other organs. Increased lipid peroxi-dation along with defi cient antioxidant systems and accumulation of infl ammatory cytokines contribute to the progression of diabetes [8]. Antioxidant [11] and anti-infl ammatory [1] effects of Noopept have been demonstrated. DPP-4 inhibitors, especially sitagliptin, showed similar properties [7]. Thus, the analysis iden-tifi ed multi-target mechanism of anti-diabetic effects exhibited by Noopept and sitagliptin and showed cer-tain similarity of its components.
The results obtained in this work prove the effi -cacy of Noopept in the model of progressive diabetes and update previously received data [6] on its effec-
TA
BL
E 1
. E
ffects
of N
oop
ep
t and
Sitag
liptin o
n the B
asal B
loo
d G
luco
se L
evels
(m
mo
l/lit
er)
and
Para
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rs o
f H
yp
erg
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ia 1
and
2 h
aft
er
Glu
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se A
dm
inis
tratio
n
(10
00
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/kg
) in
a R
at
Mo
de
l o
f P
rog
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e D
iab
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s (
M±
SE
M)
Gro
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Da
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fte
r S
TZ
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n
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da
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ay 1
9*
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ba
sa
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2 h
ba
sa
l le
ve
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h
Pa
ssiv
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on
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l4
.98
±0
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5.2
8±
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3–
3.8
2±
0.1
75
.50
±0
.18
4.4
6±
0.1
03
.98
±0
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4.7
±0
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Act
ive
co
ntr
ol
(ST
Z+
salin
e)
10
.84
±1
.31
+1
7.1
5±
1.7
4+
17
.93
±2
.05
+6
.74
±1
.40
+1
5.6
7±
1.7
5+
9.8
6±
1.9
0+
5.2
7±
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4+
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.0±
1.9
+
Gro
up
3 (
ST
Z+
No
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t)5
.54
±0
.14
o6
.07
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.39
o6
.15
±0
.25
o3
.91
±0
.12
o5
.18
±0
.26
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.34
±0
.08
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.19
o5
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o
AA
of
No
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t, %
90
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3.3
59
1.7
96
.91
03
10
2.2
73
84
.2
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up
(S
TZ
+si
tag
lipti
n)
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1±
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8o
10
.04
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.30
o7
.25
±1
.25
o4
.80
±0
.13
o6
.04
±0
.80
o5
.90
±0
.43
o4
.78
±0
.29
8.8
1±
0.8
0o
AA
of
sita
glip
tin
, %
97
.95
9.9
83
.56
6.4
94
.67
3.3
37
.94
3.6
No
te.
*Glu
co
se
le
ve
ls w
ere
no
t m
ea
su
red
on
exp
eri
me
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ays 4
an
d 1
9 w
ith
in 2
h a
fte
r g
luco
se
lo
ad
. p0
.05
co
mp
are
d w
ith
+p
assiv
e c
on
tro
l, o
active
co
ntr
ol
(Ma
nn
–W
hitn
ey t
est)
.
Fig. 3. Comparative efficiency of sitagliptin and Noopept (each
5 mg/kg per os) in the Wistar rat model of progressive diabetes.
Parameters were measured on day 4 (day 1 after the end of STZ
treatment). p<0.05 in comparison with: *passive control, +active
control.
R. U. Ostrovskaya, I. V. Ozerova, et al.
346
tiveness in the treatment of this disease. In general, the results indicate that oral dipeptide should be investi-gated as a tool of multipurpose therapy to prevent the progression of diabetes. With that, widespread use of Noopept as nootropic drug revealed the lack of side effects as its most important feature.
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