Synthesis and antitumor properties of the myelopeptide mp-1

Article abstract of DOI:10.1134/S1068162012040073

The bone marrow-derived peptide Phe-Leu-Gly-Phe-Pro-Thr (MP-1) has been synthesized by the classical methods of peptide chemistry in solution, and its antitumor properties have been studied. It has been shown that MP-1 enhances the efficacy of the cytosta

Full text of DOI:10.1134/S1068162012040073

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 4, pp. 354–359. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © L.A. Fonina, E.M. Treshchalina, R.G. Belevskaya, A.A. Az’muko, M.A. Efremov, L.A. Sedakova, E.A. Kirilina, 2012, published in Bioorganicheskaya
Khimiya, 2012, Vol. 38, No. 4, pp. 406–412.
Synthesis and Antitumor Properties of the Myelopeptide MPꢀ1
L. A. Foninaa, E. M. Treshchalinac, R. G. Belevskayaa, A. A. Az’mukob, M. A. Efremova,
, 1
L. A. Sedakovac, and E. A. Kirilinaa
aShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997 Russia
bResearch Institute of Experimental Cardiology, Russian Cardiology Scientific and Production Association,
Ministry of Health of the Russian Federation, Moscow, Russia
cResearch Center of Experimental Therapy and Diagnosis of Tumors, Blokhin Center of Oncology,
Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478 Russia
Received November 17, 2011; in final form, November 18, 2011
Abstract—The bone marrowꢀderived peptide PheꢀLeuꢀGlyꢀPheꢀProꢀThr (MPꢀ1) has been synthesized by
the classical methods of peptide chemistry in solution, and its antitumor properties have been studied. It has
been shown that MPꢀ1 enhances the efficacy of the cytostatic therapy of lympholeukosis P388, increases the
latent period of the growth of P388 tumors implanted in irradiated mice, and reduces the recurrence of the
breast adenocarcinoma Caꢀ755 in mice after the surgery.
Keywords: myelopeptide MPꢀ1, antitumor activity, immunodepression
DOI: 10.1134/S1068162012040073
1
INTRODUCTION
balance of CD4/CD8 cells (T helpers/T suppressors),
which results in the restoration of the level of antibody
formation [7].
The hexapeptide PheꢀLeuꢀGlyꢀPheꢀProꢀThr
belongs to the group of endogenous regulatory peptides
(myelopeptides) produced by bone marrow cells and is
called myelopeptideꢀ1. The studies of the biological
activity and the mechanisms of action of individual
myelopeptides revealed that each of six peptides isolated
to date produces a specific action on a particular type of
immunocompetent cell, accomplishing the immunoꢀ
correction effects by natural mechanisms [1, 2].
Because it has been shown earlier that MPꢀ1 norꢀ
malizes the disturbed balance of the major regulatory
subpopulations of CD4/CD8 cells, which is characꢀ
teristic, among other things, for the tumor growth, it
was of interest to study the ability of this peptide to
produce the antitumor action: to inhibit the growth of
various tumors under the conditions of immunodefiꢀ
ciency induced by irradiation or postsurgical stress.
It was shown earlier that MPꢀ1 is an immunomodꢀ
ulator that normalizes the antibody formation in aniꢀ
mals with immunodeficiencies of different etiology,
caused in particular by radiation, cytostatics, and antiꢀ
biotics [3–5].
The goal of the present work was to study the
immunocorrection and antitumor properties of MPꢀ1
under the conditions of immunodeficiency induced by
both irradiation or surgical stress and the development
of malignant tumors.
The in vivo modeling of directed deficiencies in
mice by selective elimination of T and B cells demonꢀ
strated that the immunocorrection effect of MPꢀ1 is
accomplished through a population of Tcells [4]. A
study of the binding of fluoresceinꢀlabeled MPꢀ1 to
the surface of lymphoid cells by the method of double
fluorescence staining revealed that the target cell of
this regulatory peptide is CD4+ lymphocyte, i. e., T
helper [6]. The mechanism of the immunocorrection
effect is based on its ability to normalize the disturbed
RESULTS AND DISCUSSION
MPꢀ1 (PheꢀLeuꢀGlyꢀPheꢀProꢀThr) was syntheꢀ
sized by the classical methods of peptide chemistry in
solution by the scheme (3 + 3). Nꢀ and
tripeptides were obtained by successive elongation of
peptide chains at the ꢀterminus by the method of
activated ꢀoxysuccinimide esters. The tripeptides
obtained were condensed by the carbodiimide
method. The ꢀamino groups of amino acids were
Cꢀterminal
N
N
α
Abbreviations: CP, cisplatin; DMF,
N,Nꢀdimethylformamide;
blocked by Bocꢀ and Zꢀsubstituents, carboxylic
groups were protected by salt formation, and the
ꢀterminal carboxylic group of threonine was proꢀ
tected by amidation. The target hexapeptide MPꢀ1
was isolated and purified by reversedꢀphase chromaꢀ
HPLC, highꢀperformance liquid chromatography; LTP, lifetime
prolongation; MLT, mean lifetime; MPꢀ1, myelopeptideꢀ1; s.c.,
subcutaneously; ITG, inhibition of tumor growth.
Corresponding author: phone: (495) 330ꢀ72ꢀ56; eꢀmail: kiriliꢀ
naelen@yandex.ru.
С
1
354

SYNTHESIS AND ANTITUMOR PROPERTIES OF THE MYELOPEPTIDE MPꢀ1
355
Table 1. Efficacy of MPꢀ1 monotherapy and the application of MPꢀ1 in combination with CP in mice with hypodermic
lympholeukosis P388
Preparation
Dose, g/mouse
V
mean (mm3)&
ITG, %&
MLT, day
LTP, %
–
Control
MPꢀ1
CP
–
461; 2948; 6348
–
21.1
21.5
24.7
27.8
32.8
33.7
–8
1
8
×
×
×
×
×
10
10
10
73; 1132; 2111 84*; 62*; 67*
–8
–7
–8
60; 362; 993
39; 175; 750
9; 66; 367
86*; 91*; 86*
91*; 94*; 88*
28*
31*
32*
39*
CP
1.6
8
–8
–8
CP + MPꢀ1
10 + 1
×
×
10
10
98**; 97**; 94**
99**; 97**; 95**
–7
CP + MPꢀ1
1.6
10 + 1
2; 79; 335
&
Note: The three numbers correspond to the results obtained on day 11, 14, and 18 after the tumor inoculation, respectively. In a tenfold
3
course of MPꢀ1, the interval between injections was 24 h; difference from the control: *
mean size of the tumor in mm ; MLT, mean lifetime; LTP, lifetime prolongation.
p
< 0.05, **
p
< 0.01; V
(mm ), the
mean
3
Table 2. Effect of MPꢀ1 on the inoculability of lympholeukosis P388 under stress induced by irradiation (4.5 Gy)
Day after the implantation of P388 (death of animals, %)
Number Dose of MPꢀ1,
of group
g/mouse
14
0
15
60
16
70
17
90
18
19
20
21
22
23
24
100
1
–
(control group)*
–5
2
3
4
5
1
1
1
1
×
×
×
×
10
10
10
10
0
0
0
0
44
40
30
0
56
50
40
0
67
70
50
40
100
80
–6
–7
–8
90
90
60
90
90
60
90
90
60
100
90
60
100
80
40
70
100
* Administration of the physiological solution.
tography; it was characterized by the data of analytical and induced a maximum ITG, by 86–94%, which
HPLC, mass spectrometry, and sequencing.
remained throughout the week at a level of 86–88%; in
this case, the lifetime of mice increased by 28–31%. In
groups with combined therapy, ITG after the injection
In the first series of experiments, the effect of MPꢀ1
on the growth and development of P388 lympholeukosis
and the efficacy of this peptide in the combined cytostatic
antitumor therapy were studied. The antitumor effect of
MPꢀ1 was estimated using the cytostatic drug CP as posꢀ
itive control, which is applied in clinical practice.
The antitumor activity of MPꢀ1 and CP was estiꢀ
mated in vivo on different types of implanted mouse
tumors (lympholeukosis P388 and breast adenocarciꢀ
noma Ca755), obtained from the bank of tumor strains
of the Blokhin Russian Oncological Research Center.
of CP (at doses of
MPꢀ1 (at a single dose of
8 × ×
10–8 and 1.6 10–7 g/mouse) and
1 ×
10–8 g/mouse for 10 days)
increased to 94–95% by the end of the first week after
the treatment. The lifetime of mice in these groups
increased by 32 and 39%, i. e., was almost 1.5 times
greater than in the case of the CP monotherapy.
Thus, it is evident that the combined therapy of
lympholeukosis P388 by CP and MPꢀ1 more strongly
suppresses the tumor and increases the survival of aniꢀ
mals than the monotherapy with each of these prepaꢀ
rations.
Experiments were performed on mice with s.c.
implanted lympholeukosis P388. CP was injected sinꢀ
gly intraperitoneally at doses of 8 and 4 mg/kg, which
cause marked immunodepression. The treatment with
CP was started 24 h after tumor inoculation. MPꢀ1
Then the antitumor and prophylactic effects of
MPꢀ1 on the growth and development of lympholeuꢀ
kosis P388 under the conditions of immunosuppresꢀ
sion induced by radiation was studied. Mice of the
BDF1 line were injected with MPꢀ1 for 5 days (at an
was injected s.c. at a single dose of
1 ×
10–8 g/mouse
daily beginning from day 2 to day 11 after tumor
implantation. The results of the experiments are preꢀ
sented in Table 1.
interval of 24 h) at doses of 1 × ×
10–8 –1 10–5 g/mouse.
Then mice were irradiated at a dose of 4.5 Gy, and
after 24 h 103 lympholeukosis P388 cells were s.c.
implanted; the injections of MPꢀ1 were continued for
another five days after implantation. The results of the
It was shown that the MPꢀ1 monotherapy induced
a 84% reduction of tumor growth by the last day of
treatment and did not influence the survival of mice.
CP in the monotherapy at doses of 8 × ×
10–8 and 1.6
10–7 g/mouse was nearly equal in efficiency to MPꢀ1 experiments are presented in Table 2.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 4
2012

356
FONINA et al.
Table 3. Effect of MPꢀ1 on spontaneous recidivation of breast carcinoma Caꢀ755 in mice under the conditions of immuꢀ
nodepression induced by surgical stress
Administration of MPꢀ1,
10–8 g/mouse
Number of mice
(total number)
Number of mice
with tumor recurrence
Recidivation, %
1
×
– (control group)•
34
28
32
22
13
14
65
days 2–11 after the operation
46
for 5 days before and after the operation
44*
•
Note: Administration of physiological solution daily for 10 days. *
p
< 0.05.
It is evident from the data in Table 2 that, in mice only the therapeutic effect of MPꢀ1 was studied,
of the control group, tumors appeared on day 15 (the whereas according to Scheme 2, the prophylactic
latent period). In experimental groups 2–4, tumors effect of the peptide on the spontaneous recidivation
appeared also on day 15; however, they were found in of the breast carcinoma Caꢀ755 was also examined.
a lesser number of animals. In group 4, tumors began The animals of the control group were injected with
to appear even later (on day 17). The injection of the physiological solution. The size of recurrent
MPꢀ1 for 10 days delayed the appearance of tumors to Caꢀ755 carcinoma was measured on day 10 after the
22–24 days. The maximum delay in the inoculability removal of the tumor. The results are presented in
of leukosis was 6 days (33–35%) at a single dose of Table 3.
MPꢀ1 of 1 ×
10–8 g/mouse, which is rather much, havꢀ
ing regard to a short lifespan of mice with this tumor.
It follows from the data in Table 3 that spontaneous
recurrences in the control group after tumor removal
were observed in 65% of cases. In the group of animals
receiving MPꢀ1 for 10 days after tumor removal the
number of spontaneous recurrences decreased to 46%,
which is 1.4 times less than in the control (Scheme 1).
In the group of mice that received MPꢀ1 during the
maximum possible immunodepression, for 5 days
prior to the operation (neoadjuvant therapy) and for
5 days after the operation (adjuvant therapy), the numꢀ
ber of spontaneous recurrences decreased reliably to
44%, which is nearly 1.5 times less than in the control.
These results allow one to conclude that MPꢀ1 proꢀ
duces a prophylactic effect: its injection distinctly
slows down the appearance of tumors, prolongs the
life of experimental animals, and decreases the level of
radiationꢀinduced immunodepression in mice.
In the second series of experiments, the antitumor
and prophylactic effects of MPꢀ1 on the spontaneous
and artificial recidivation of tumors under the condiꢀ
tions of immunodepression induced by surgical stress,
the removal of breast adenocarcinoma Caꢀ755 in mice.
It is known that the postsurgical stress is accompanied
by the stimulation of metastasizing and reappearance of
tumors. The onset of the exponential phase in tumor
growth, when tumors drastically increase in size within
a short period of time, corresponds to a decrease in the
immunological control of the organism, i. e., proꢀ
nounced immunodepression [8].
In a study of the effect of MPꢀ1 on artificial recidꢀ
ivation,
a suspension of Caꢀ755 tumor cells
(50 mg/mouse) was repeatedly implanted into mice
on days 10–11 after the operation. The mice of the
control and experimental groups received MPꢀ1
according to the same protocol as in the previous
series of experiments. The size of the recurrent Caꢀ755
carcinoma was measured on day 7 after the implantaꢀ
tion of the tumor. The results are presented in Table 4.
The experiments showed that the artificial recidiꢀ
vation in the control group occurred in 100% of cases
after the removal of the tumor. The administration of
MPꢀ1 with a therapeutic purpose (on days 2–11 after
the operation) did not lead to a decrease in the number
of recurrences. At the same time, the peptide adminisꢀ
tered with the preventive purpose during the maxiꢀ
mum possible immunodepression (5 days prior to, and
5 days after the operation) decreased the number of
artificial recurrences of the breast carcinoma Caꢀ755
to 67% (by 33% less than in the control).
Cells of the Caꢀ755 tumor (50 mg/mouse) were s.c.
implanted into female mice of BDF1 hybrids. When
the mean volume of the tumor reached 700–900 mm3,
they were removed under thiopental narcosis. Mice in
which subcutaneous recurrences appeared on day 10
after the surgical removal of the tumor formed a group
of “spontaneous recidivation.” In a study of the effect
of MPꢀ1 on the spontaneous recurrence of breast adeꢀ
nocarcinoma Caꢀ755, mice of the first experimental
group received MPꢀ1 at a dose of
1 ×
10–6 g/mouse
beginning from day 2 to day 11 after operation
(Scheme 1). The mice of the second experimental
group were injected with MPꢀ1 at a preventive dose of
1
×
10–8 g/mouse for five days prior to, and for 5 days
Thus, the study of the effect of MPꢀ1 on the growth
after, the operation (Scheme 2). Thus, the animals of of implanted tumors under the conditions of radiaꢀ
both experimental groups received MPꢀ1 for 10 days, tionꢀinduced immunodepression and after the occurꢀ
and the difference was that, according to Scheme 1, rence of recidivation under the conditions of surgical
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 4
2012

SYNTHESIS AND ANTITUMOR PROPERTIES OF THE MYELOPEPTIDE MPꢀ1
357
Table 4. Effect of MPꢀ1 on artificial recidivation of breast carcinoma Caꢀ755 in mice under the conditions of immunodeꢀ
pression induced by surgical stress
Number of mice
(total number)
Number of mice
with tumor recurrence
–8
Administration of MPꢀ1, 1
×
10 g/mouse
Recidivation, %
– (control group)*
15
17
18
15
17
12
100
100
67
days 2–11 after the operation
for 5 days before and after the operation
* Administration of the physiological solution daily for 10 days.
stress showed that MPꢀ1 exhibits the preventive Japan) on an Ultrasphere C 18 column (4.6 × 250 mm)
immunocorrection effect. The peptide reliably proꢀ in a gradient of acetonitrile concentration in 0.1% triꢀ
longed the lifespan of experimental animals, fluoroacetic acid (0–80%, 32 min). The elution rate
decreased the level of radiationꢀinduced immunodeꢀ was 1.6 mL/min, and the detection was at 214 and
pression, increased the time of tumor inoculability in 280 nm.
mice, and reduced the recidivation of the tumor after
its surgical removal.
Preparative HPLC of terminal and intermediate pepꢀ
tides was carried out on a column of Diasorb C 16ꢀT
(
50
× 250 mm) in a gradient of buffer C in buffer A
The preventive effect of MPꢀ1, which manifested
itself in the postsurgical period, is evidently related to
its immunocorrection effect during immunodepresꢀ
sion, which appears in animals in operation stress. The
postoperative immunodepression promotes the
resumption of tumor growth, and MPꢀ1 provides the
restoration of immunity, thereby reducing the probaꢀ
bility of recurrence. The data obtained indicate that
MPꢀ1 holds promise in complex cancer therapy as an
immunocorrection agent to prevent the recurrence in
oncology patients who underwent surgery, intensive
radiation or chemotherapy. MPꢀ1 is an endogenous
lowꢀmolecularꢀweight immunoregulatory peptide
with the known primary structure and mechanism of
action; the peptide holds promise as the basis for the
design of newꢀgeneration drugs that target impaired
immune functions and produce no side effects.
(10–50%, 120 min; buffer A, 0.01 M CH3COONH4
;
buffer C, 80% acetonitrile in buffer A). The flow rate
was 50 mL/min, and the detection was at 226 nm.
Fractions corresponding to the main peak were comꢀ
bined, acetonitrile was evaporated, and the resulting
solution was diluted with water and lyophilized.
Synthesis of HꢀPheꢀLeuꢀGlyꢀPheꢀProꢀThrꢀNH2
(MPꢀ1). HꢀLeuꢀGlyꢀOH
× TFA. DMF (60 mL) and
BocꢀLeuꢀONSu (6.58 g, 0.02 mol) were added to a
solution of glycine (1.65 g, 0.022 mol) in 1 M NaOH
(22 mL). The reaction mixture was stirred for 12 h at
20
solved in water (100 mL) and extracted with diethyl
ether ( 30 mL). The water layer was acidified with
5% H2SO4, and the target compound was extracted
with ethyl acetate ( 50 mL). The ethyl acetate soluꢀ
°С, DMF was evaporated, and the residue was disꢀ
3
×
2
×
tion was washed with water to pH 5 and evaporated.
The oily residue was dissolved in TFA (70 mL) and
kept for 1 h at room temperature, the acid was distilled
in vacuo, and the residue was triturated with ether and
filtered. The resulting precipitate was dried in a dessiꢀ
cator over KOH. Yield: 5.44 g (90%).
EXPERIMENTAL
Commercially available amino acids and their
derivatives (Reanal, Hungary; Fluka, Switzerland) or
derivatives obtained by standard methods were used.
The purity of compounds obtained at the intermediate
stages of the synthesis was controlled by TLC on silica
gel plates (Merck, Germany) in the following solvent
chloroform–methanol–32%
acid 60 : 45 : 20 (A), chloroform–methanol–32%
acetic acid 5 : 3 : 1 (B), chloroform–methanol–32%
ZꢀPheꢀLeuꢀGlyꢀOH (1). A solution of HꢀLeuꢀ
GlyꢀOH
was added to
×
TFA (5.44 g, 0.018 mol) in DMF (60 mL)
ꢀmethylmorpholine (2 mL, 0.018 mol)
N
systems:
acetic
and ZꢀPheꢀONSu (7.15 g, 0.018 mol). The reaction
mixture was stirred for 12 h at 20°С, DMF was evapoꢀ
rated, and the residue was dissolved in ethyl acetate
(100 mL) and washed with 5% H2SO4 (50 mL) and
acetic acid 15 : 4 : 1 (C), nꢀbutanol–acetic acid–water
3 : 1 : 1 (D), and chloroform–methanol–acetic acid
9 : 1 : 0.5 (E). Compounds were detected using ninhyꢀ
water (3 × 50 mL) to pH 5. The ethyl acetate solution
was evaporated and crystallized from diethyl ether.
Yield: 7.97 g (93%).
drin or
Analytical HPLC was carried out using an
LCꢀ10ADvp chromatographic system (Shimadzu, HꢀThr(Bzl)ꢀNH2 (4.6 g, 0.022 mol) in DMF (60 mL),
oꢀtolidine.
HꢀProꢀThr(Bzl)ꢀNH2
× TFA. To a solution of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 4
2012

358
FONINA et al.
BocꢀProꢀONSu (6.26 g, 0.020 mol) was added, and
Molecular weight of MPꢀ1 was determined by mass
the mixture was stirred for 12 h at 20 . The reaction spectrometry on a Thermo Bioanalysis Vision 2000
°
С
mixture was evaporated, and the residue was dissolved device (England) and was 681.
in ethyl acetate (100 mL) and extracted with a satuꢀ
Antitumor efficacy of MPꢀ1 was estimated on mice
rated NaHCO3 solution (2 × 50 mL) and 5% H2SO4
with s.c. implanted lympholeukosis P388 cells. MPꢀ1
was injected s.c. at a single dose of
1 ×
10–5 g/mouse
(2 50 mL). The ethyl acetate solution was washed
×
with water to pH 5, and ethyl acetate was evaporated.
The oily residue was dissolved in TFA (70 mL), kept
for 1 h at 20°С, evaporated, and triturated with ether.
The precipitate was filtered and dried in a dessicator
over KOH. Yield: 7.96 g (95%).
daily beginning from day 2 to day 11 after tumor
implantation. Experiments on the combined antituꢀ
mor effect of MPꢀ1 were performed using CP as a
cytostatic agent. CP was injected intraperitoneally at
doses of 8 and 4 mg/kg, which induce a marked immuꢀ
nodepression. The treatment with CP was started 24 h
after the tumor implantation. The efficacy of treatꢀ
ment in both cases was estimated from standard
parameters, ITG and LTP (both in percent). These
parameters indicate the effect of preparations on
tumor size and the life of mice.
BocꢀPheꢀProꢀThr(Bzl)ꢀNH2. To a solution of
HꢀProꢀThr(Bzl)ꢀNH2 (7.96 g, 0.019 mol) in DMF
(60 mL),
Nꢀmethylmorpholine (2.1 mL, 0.019 mol)
and BocꢀPheꢀONSu (6.9 g, 0.019 mol) were added,
and the mixture was stirred for 12 h at room temperaꢀ
ture. The reaction mixture was evaporated, and the
residue was dissolved in ethyl acetate (100 mL) and
Vc – Vexp
ITG = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ × 1 0 0 ,
Vc
washed with a saturated NaHCO3 solution (
2 × 50 mL
and 5% H2SO4 (2 50 mL). The ethyl acetate solution
×
where Vc and Vexp are the volume of the tumor in the
control and experiment;
was washed with water to pH 5, evaporated, and crysꢀ
tallized from diethyl ether. Yield: 9.38 g (89%).
LTc – LTexp
LTP = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ × 100,
LTc
HꢀPheꢀProꢀThr(Bzl)ꢀNH2
of BocꢀPheꢀProꢀThr(Bzl)ꢀNH2 (9.38 g, 0.017 mol) in
TFA (70 mL) was kept for 1 h at 20 , evaporated, and
the oily residue was triturated with ether and filtered.
The filtrate was dried in a dessicator over KOH. Yield:
9.62 g (99.9%).
× TFA (2). A solution
°
С
where LTc and LTexp are the lifetime of mice in the
control and experiment.
Irradiation of mice (total dose of 4.5 Gy) was perꢀ
formed on a Stebelꢀ3A device at a dose rate of
5 Gy/min.
ZꢀPheꢀLeuꢀGlyꢀPheꢀProꢀThr(Bzl)ꢀNH2. Pepꢀ
tide (
(1.9 mL), and HONSu (1.04 g, 0.009 mol) were added
to a solution of peptide ( ) (9.62 g, 0.017 mol) in DMF
(100 mL). The solution was cooled to –25 , and
1) (7.97 g, 0.017 mol), Nꢀmethylmorpholine
Prophylactic effect of MPꢀ1 on oncogenesis under
the conditions of immunodeficiency induced by irraꢀ
diation (4.5 Gy) was estimated from the delay of the
100% inoculability of leukosis compared with the conꢀ
trol group (unirradiated animals). Lympholeukosis
P388 cells capable of forming well palpable hypoderꢀ
mic tumors at the early stage of development were
used as an implantable tumor. Studies were carried out
on BDF1 mice into which 103 lympholeukosis P388
cells in a volume of 0.2 mL were implanted 24 h after
irradiation. Each group contained no less than eight
animals. MPꢀ1 was injected s.c. in a physiological
solution, beginning from day 5 before tumor implanꢀ
tation and for the next five days after the implantation
2
°С
dicyclohexylcarbodiimide (3.68 g, 0.178 mol) was
added under stirring. The reaction mixture was kept
for 12 h at 8°С. The resulting dicyclohexylurea was filꢀ
tered. DMF was evaporated in vacuo, and the residue
was dissolved in 100 mL of a mixture of ethyl acetate
and butanol 1 : 1 and washed with a saturated NaHCO3
solution (2 × 50 mL) and 5% H2SO4 (2 × 50 mL). The
ethyl acetate–butanol solution was washed with water
to pH 5, evaporated, and crystallized from ether.
Yield: 13.06 g (85%).
at single doses from
1 × 1 ×
10–8 to 10–5 g/mouse.
HꢀPheꢀLeuꢀGlyꢀPheꢀProꢀThrꢀNH2. The peptide
ZꢀPheꢀLeuꢀGlyꢀPheꢀProꢀThr(Bzl)ꢀNH2 (13.06 g,
Prophylactic and therapeutic effects of MPꢀ1 on
0.0145 mol) was dissolved in methyl alcohol (100 mL) the recurrence of tumors under surgical stress were estiꢀ
and hydrated over 10% Pd/C until the starting comꢀ mated on a model of solid nonmetastatic breast adenoꢀ
pound disappeared, which was monitored by TLC. carcinoma Caꢀ755. The scheme of the experiment
The catalyzer was filtered, and the solution was evapꢀ involved the s.c. inoculation of a suspension of Caꢀ755
orated. The target peptide was purified by HPLC on a cells into BDF1 female mice (50 mg/mouse). Then,
column (50
×
250 mm) of Diasorb Cꢀ16ꢀT in a gradiꢀ mice having tumors of a volume of 700–900 mm3 were
ent of concentration (0–50%, 120 min) of buffer B in selected, and the tumors were removed under thiopenꢀ
buffer A. The flow rate was 50 mL/min, and detection tal narcosis. Two schemes of the tumor recurrence
was at 226 nm. Acetonitrile was evaporated, and the experiment were used: spontaneous recidivation (forꢀ
resulting solution was diluted with water and lyoꢀ mation of tumor nodes at the site of the surgical wound
philized. The yield of the amide MPꢀ1 was 6.16 g on day 10 after the removal of the primary tumor in
(62%).
mice) and artificial recidivation, the formation of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 4
2012

SYNTHESIS AND ANTITUMOR PROPERTIES OF THE MYELOPEPTIDE MPꢀ1
359
tumor nodes after the second inoculation (10–11 days
ACKNOWLEDGMENTS
after the removal of the primary node) of suspended
Caꢀ755 cells (50 mg/mouse). With regard to these
schemes, six groups of mice were formed, 15–34 aniꢀ
mals in each.
This work was supported by the program of the Preꢀ
sidium of the Russian Academy of Sciences “Molecuꢀ
lar and cellular biology.”
Spontaneous recidivation. On day 10 after the
removal of the primary tumor in mice, tumor nodes at
the site of the surgical wound developed. MPꢀ1 was
REFERENCES
1. Petrov, R.V., Mikhailova, A.A., and Fonina, L.A., Ross.
Khim. Zh., 2005, vol. 49, no. 1, pp. 55–63.
2. Mikhailova, A.A., Fonina, L.A., Kirilina, E.A.,
Gur’yanov, S.A., Efremov, M.A., and Petrov, R.V., Regꢀ
ulatory Peptides, 2003, vol. 114, pp. 183–187.
3. Mikhailova, A.A., Fonina, L.A., Kirilina, E.A., Shaꢀ
nurin, S.Yu., Gur’yanov, S.A., Malakhov, A.A., Nesmeyaꢀ
nov, V.A., and Petrov, R.V., Regulatory Peptides, 1994,
vol. 53, pp. 203–209.
4. Shanurin, S.Yu., Gur’yanov, S.A., and Mikhailova, A.A.,
Immunologiya, 1993, no. 2, pp. 17–19.
5. Shanurin, S.Yu., Radiobiologiya, 1994, vol. 34, pp. 4–5.
6. Petrov, R.V., Mikhailova, A.A., and Kirilina, E.A.,
Folia Biologica (Praha), 1994, no. 40, pp. 455–461.
7. Kirilina, E.A., Mikhailova, A.A., Malakhov, A.A.,
injected s.c. at a single dose of 1 ×
10–8 g/mouse daily
beginning from day 2 to day 11 after the operation
(Scheme 1) or for 5 days before and 5 days after the
operation (Scheme 2); after 10 days, the development
of new nodes was controlled by palpation. The mice of
control groups received injections of the physiological
solution from day 2 to day 11 after the removal of the
primary tumor.
Artificial recidivation was caused by the repeated
implantation of Caꢀ755 cells against the background
of the injection of MPꢀ1. MPꢀ1 was injected s.c. at a
single dose of 1 ×
10–8 g/mouse according to Schemes 1
or 2 (see above). Seven days after the repeated implanꢀ
tation of Caꢀ755 cells, the development of tumor
nodes was controlled.
Gur’yanov, S.A., and Efremov, M.A., Immunologiya
1998, no. 4, pp. 26–29.
,
8. Kozlov, A.M., Peretolchina, N.M., Kiselev, S.M.,
Saprykina, N.S., and Mikhailova, L.M., Rukovodstvo
po eksperimental’nomu (doklinicheskomu) izucheniyu
novykh farmakologicheskikh veshchestv (Guidelines for
Experimental (Preclinical) Studies of New Pharmacoꢀ
logical Agents), Moscow: Meditsina, 2005.
Statistical processing of data was carried out using
the STATGRAPH software package and a package of
functions for the statistical processing of the Excel
program.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 4
2012