The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation
Aim. To investigate changes in pro-inflammatory status of laboratory rats after introduction of antiviral substances with interferon-inducing action: 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline and tilorone. Methods. MCP-1 content, blood leukocyte counts and functional activity of phagocy...
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| Cite this: | The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation / G.V. Antonovych, N.M. Zholobak, M.O. Shibinska, M.Ya. Spivak // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 264-271. — Бібліогр.: 17 назв. — англ. |
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Antonovych, G.V. Zholobak, N.M. Shibinska, M.O. Spivak, M.Ya. 2019-06-12T12:25:24Z 2019-06-12T12:25:24Z 2015 The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation / G.V. Antonovych, N.M. Zholobak, M.O. Shibinska, M.Ya. Spivak // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 264-271. — Бібліогр.: 17 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008EA https://nasplib.isofts.kiev.ua/handle/123456789/152566 547,752:547,83,615.281.8+616.002 Aim. To investigate changes in pro-inflammatory status of laboratory rats after introduction of antiviral substances with interferon-inducing action: 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline and tilorone. Methods. MCP-1 content, blood leukocyte counts and functional activity of phagocytes were measures using flow cytometry techniques. Complement content was determined in microtest based upon hemolysis of sensitized erythrocytes. Results. Both oral and intraperitoneal applications of the substances were characterized by an elevation in monocytic counts, potentiated metabolic reserve of phagocytic cells, increased MCP-1 and complement content in serum. In contrast to tilorone, after introduction of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline substantial raise in circulating neutrophil counts and their phagocytic activity was not determined, while MCP-1 and complement responses were significantly lower compared to reference substance. Conclusion. Although both substances are IFN-inducers with pluripotent immunostimulatory action, the tested derivative was characterized by a less pronounced elevation of complement activity, MCP-1 content and neutrophil counts. This implies that application of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline can strengthen innate antiviral resistance with minimized risks of potential autoimmunological adverse effects. Мета. Дослідити вплив 6-(2-морфолін-4-іл-етил)-6H-індоло [2,3-b]хіноксаліна (антивірусної сполуки з інтерферон-індукуючою дією) на про-запальний статус лабораторних щурів. Методи. Концентрація MCP-1, кількість лейкоцитів в крові та функціональна активність фагоцитів визначали цитофлюориметричними методами. Активність системи комплементу оцінювалась в мікротесті на основі гемолізу сенситизованих еритроцитів. Результати. Застосування перорального та парентерального способу введення сполук супроводжувалось збільшенням кількості моноцитів, потенціонуванням метаболічного резерву фагоцитуючих клітин, підвищенням вмісту MCP-1 і компонентів комплементу в сироватці крові. Після введення 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]хіноксаліну достовірного збільшення кількості циркулюючих нейтрофілів або їх фагоцитарної активності не спостерігалось, в той час як рівні MCP-1 і компонентів комплементу були значно нижчими, ніж у препарату порівняння – тилорону. Висновки. ІХоча обидві сполуки є індукторами ІФН з плюрипотентною імуностимулюючою дією, тестоване похідне призводило до менш вираженого підвищення активності комплементу, нижчого вмісту MCP-1 та кількісті нейтрофілів. Даний факт свідчить на користь того, що застосування 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]хіноксаліну для посилення неспецифічного антивірусного імунітету супроводжуватиметься нижчим ризиком розвитку побічних аутоімунологічних реакцій. Цель. Изучить влияние 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина (антивирусного соединения с интерферон-индуцирующим действием) на про-воспалительный статус лабораторных крыс. Методы. Концентрация MCP-1, количество лейкоцитов в крови и функциональная активность фагоцитов определялись цитофлюориметрическими методами. Активность системы комплемента оценивали в микротесте, основанном на гемолизе сенситизированных эритроцитов. Результаты. Как пероральный, так и парентеральный способы введения соединений приводили к увеличению количества моноцитов, потенционированию метаболического резерва фагоцитирующих клеток, повышению содержания MCP-1 и компонентов комплемента в сыворотке крови. После введения 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина достоверного увеличения численности циркулирующих нейтрофилов либо их фагоцитарной активности не наблюдалось, в то время как уровни MCP-1 и компонентов комплемента были значительно ниже, чем у препарата сравнения – тилорона. Выводы. Хотя оба соединения представляют собой индукторы ИФН с плюрипотентным иммуностимулирующим действием, тестируемое производное приводило к менее выраженному повышению активности комплемента, меньшему содержанию MCP-1 и количеству нейтрофилов. Данный факт свидетельствует о том, что применение 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина для усиления неспецифического противовирусного иммунитета будет нести более низкие риски развития побочных аутоиммунологических реакций. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Biomedicine The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation Вплив 6-(2-морфолін-4-іл-етил)-6H-індоло [2,3-b]хіноксаліна на біомаркери запалення Влияние 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина на биомаркеры воспаления Article published earlier |
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| title |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| spellingShingle |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation Antonovych, G.V. Zholobak, N.M. Shibinska, M.O. Spivak, M.Ya. Biomedicine |
| title_short |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| title_full |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| title_fullStr |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| title_full_unstemmed |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| title_sort |
effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6h-indolo [2,3-b]quinoxaline upon biomarkers of inflammation |
| author |
Antonovych, G.V. Zholobak, N.M. Shibinska, M.O. Spivak, M.Ya. |
| author_facet |
Antonovych, G.V. Zholobak, N.M. Shibinska, M.O. Spivak, M.Ya. |
| topic |
Biomedicine |
| topic_facet |
Biomedicine |
| publishDate |
2015 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Вплив 6-(2-морфолін-4-іл-етил)-6H-індоло [2,3-b]хіноксаліна на біомаркери запалення Влияние 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина на биомаркеры воспаления |
| description |
Aim. To investigate changes in pro-inflammatory status of laboratory rats after introduction of antiviral substances with interferon-inducing action: 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline and tilorone. Methods. MCP-1 content, blood leukocyte counts and functional activity of phagocytes were measures using flow cytometry techniques. Complement content was determined in microtest based upon hemolysis of sensitized erythrocytes. Results. Both oral and intraperitoneal applications of the substances were characterized by an elevation in monocytic counts, potentiated metabolic reserve of phagocytic cells, increased MCP-1 and complement content in serum. In contrast to tilorone, after introduction of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline substantial raise in circulating neutrophil counts and their phagocytic activity was not determined, while MCP-1 and complement responses were significantly lower compared to reference substance. Conclusion. Although both substances are IFN-inducers with pluripotent immunostimulatory action, the tested derivative was characterized by a less pronounced elevation of complement activity, MCP-1 content and neutrophil counts. This implies that application of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline can strengthen innate antiviral resistance with minimized risks of potential autoimmunological adverse effects.
Мета. Дослідити вплив 6-(2-морфолін-4-іл-етил)-6H-індоло [2,3-b]хіноксаліна (антивірусної сполуки з інтерферон-індукуючою дією) на про-запальний статус лабораторних щурів. Методи. Концентрація MCP-1, кількість лейкоцитів в крові та функціональна активність фагоцитів визначали цитофлюориметричними методами. Активність системи комплементу оцінювалась в мікротесті на основі гемолізу сенситизованих еритроцитів. Результати. Застосування перорального та парентерального способу введення сполук супроводжувалось збільшенням кількості моноцитів, потенціонуванням метаболічного резерву фагоцитуючих клітин, підвищенням вмісту MCP-1 і компонентів комплементу в сироватці крові. Після введення 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]хіноксаліну достовірного збільшення кількості циркулюючих нейтрофілів або їх фагоцитарної активності не спостерігалось, в той час як рівні MCP-1 і компонентів комплементу були значно нижчими, ніж у препарату порівняння – тилорону. Висновки. ІХоча обидві сполуки є індукторами ІФН з плюрипотентною імуностимулюючою дією, тестоване похідне призводило до менш вираженого підвищення активності комплементу, нижчого вмісту MCP-1 та кількісті нейтрофілів. Даний факт свідчить на користь того, що застосування 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]хіноксаліну для посилення неспецифічного антивірусного імунітету супроводжуватиметься нижчим ризиком розвитку побічних аутоімунологічних реакцій.
Цель. Изучить влияние 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина (антивирусного соединения с интерферон-индуцирующим действием) на про-воспалительный статус лабораторных крыс. Методы. Концентрация MCP-1, количество лейкоцитов в крови и функциональная активность фагоцитов определялись цитофлюориметрическими методами. Активность системы комплемента оценивали в микротесте, основанном на гемолизе сенситизированных эритроцитов. Результаты. Как пероральный, так и парентеральный способы введения соединений приводили к увеличению количества моноцитов, потенционированию метаболического резерва фагоцитирующих клеток, повышению содержания MCP-1 и компонентов комплемента в сыворотке крови. После введения 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина достоверного увеличения численности циркулирующих нейтрофилов либо их фагоцитарной активности не наблюдалось, в то время как уровни MCP-1 и компонентов комплемента были значительно ниже, чем у препарата сравнения – тилорона. Выводы. Хотя оба соединения представляют собой индукторы ИФН с плюрипотентным иммуностимулирующим действием, тестируемое производное приводило к менее выраженному повышению активности комплемента, меньшему содержанию MCP-1 и количеству нейтрофилов. Данный факт свидетельствует о том, что применение 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]хиноксалина для усиления неспецифического противовирусного иммунитета будет нести более низкие риски развития побочных аутоиммунологических реакций.
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| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152566 |
| citation_txt |
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo [2,3-b]quinoxaline upon biomarkers of inflammation / G.V. Antonovych, N.M. Zholobak, M.O. Shibinska, M.Ya. Spivak // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 264-271. — Бібліогр.: 17 назв. — англ. |
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264
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 4. P. 264–271
doi: http://dx.doi.org/10.7124/bc.0008EA
UDC 547,752:547,83,615.281.8+616.002
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo
[2,3-b]quinoxaline upon biomarkers of infl ammation
G. V. Antonovych1, N. M. Zholobak1, M. O. Shibinska2, M. Ya. Spivak1
1 D. K. Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154, Academika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 O. V. Bogatsky's Physico-chemical Institute, NAS of Ukraine
86, Lustdorfskaya dor., Odessa, Ukraine, 65080
g.antonovych@gmail.com
Aim. To investigate changes in pro-infl ammatory status of laboratory rats after introduction of antiviral
substances with interferon-inducing action: 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline
and tilorone. Methods. MCP-1 content, blood leukocyte counts and functional activity of phagocytes
were measures using fl ow cytometry techniques. Complement content was determined in microtest based
upon hemolysis of sensitized erythrocytes. Results. Both oral and intraperitoneal applications of the
substances were characterized by an elevation in monocytic counts, potentiated metabolic reserve of
phagocytic cells, increased MCP-1 and complement content in serum. In contrast to tilorone, after intro-
duction of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline substantial raise in circulating neu-
trophil counts and their phagocytic activity was not determined, while MCP-1 and complement re-
sponses were signifi cantly lower compared to reference substance. Conclusion. Although both substanc-
es are IFN-inducers with pluripotent immunostimulatory action, the tested derivative was characterized
by a less pronounced elevation of complement activity, MCP-1 content and neutrophil counts. This im-
plies that application of 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline can strengthen innate
antiviral resistance with minimized risks of potential autoimmunological adverse effects.
K e y w o r d s: 6H-indolo[2,3-b]quinoxalines, infl ammation, interferon, MCP-1, complement.
© 2015 G. V. Antonovych et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Introduction
Indolo[2,3-b]quinoxalines belong to a group of low
molecular substances with a tetracyclic heteroaromat-
ic core. A wide range of biological activity has been
reported for different derivatives of this group, incud-
ing the anti-infl ammatory, anti-cancerogenic, antifun-
gal effects, and in case of planar confi guration – the
antiviral action and antitumor activity. In recent years
we have searched for new antiviral substances among
several newly-synthesized and previously known in-
doloquinoxalines. One of them – 6-(2-morpholin-4-
yl-ethyl)-6H-indolo[2,3-b]quinoxaline (hereinafter –
S1) – has demonstrated the low toxicity, prolonged
interferon (IFN) induction in vivo, antiviral activity in
vitro in both prophylactic and therapeutic application
schemes, as well as an ability to potentiate the func-
tional activity of murine peritoneal cells [1,2]. Such
spectrum of biological properties allowed us to con-
sider S1 an immunomodulating substance. To further
characterize the compound it was expedient to exam-
ine in more detail the changes caused by it in the im-
mune system of laboratory animals, with particular
focus on the pro-infl ammatory biomarkers. Tilorone
hydrochloride, a potent IFN-inducer in rodents, was
chosen as a reference drug.
Materials and Methods
Substances and dosage
S1 and tilorone were kindly provided by Dr Sergey
Lyakhov (Department of Medical Chemistry, A.V. Bo-
265
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline upon biomarkers of infl ammation
gatsky Physico-Chemical Institute, NAS of Uk rai-
ne). The substances were administered orally and
in traperitoneally (i.p.) as solutions in sterile water
according to the conventional methods. The optimal
doses were determined in the preliminary studies of
IFN-induction and were as follows: for tilorone –
125 mg/kg orally, 12.5 mg/kg i.p.; for S1 – 60 mg/kg
orally, 10 mg/kg i.p. An additional 30 mg/kg dose
was used for the oral administration of S1, since it
was able to induce IFN in a wider concentration
range than the reference drug.
Animals and sampling
To evaluate the effect of studied substances upon the
immune system, the changes in the following pa ra-
meters were measured: immune cell counts in cir cu-
lating blood, phagocytic and metabolic activity of
neutrophils and monocytes, complement hemolytic
activity, levels of monocyte chemotactic protein-1
(MCP-1). The experiment was performed on 40-day
old male Wistar rats weighing 130–170 g. Group
formation and marking of animals were held for 2
days prior to the substance administration in order to
minimize the stress. The animals were kept in large
cages with free access to water and standard diet.
Handling procedures were conducted according to
European Convention for the Protection of Vertebrate
Animals (Strasbourg, 2006). Each group consisted
of 7–10 rats.
The blood samples were collected from the ca-
rotid arteries on I, III, VII and X day after the sub-
stances administration. Part of the blood, not less
than 3 ml, was gathered into tubes with sodium hep-
arin («Vacuette», Austria); remaining blood was col-
lected in the centrifuge tubes for serum. Purifi ed se-
rum was frozen within 30 minutes after the blood
collection and stored at –20 °C.
Measurement techniques
The numbers of lymphocytes, monocytes and granu-
locytes were measured relatively to Flow-Count
standardized fl uorospheres using a EPICS XL fl ow
cytometer (Beckman Coulter). Preliminary staining
of monocytes with HIS48 antibody (BD PharMin-
gen) was performed to distinguish them from lym-
phocytic pool, whereas granulocytes were differenti-
ated from other cells by their light scattering proper-
ties. The phagocytic activity and the intensity of
oxidative burst inside phagocytes were assessed in
whole blood environment using a pH-rodo test sys-
tem (Life Technologies). Prior to the measurements,
erythrocytes were lysed with Q-Prep solutions (Be-
ckman Coulter).
The determination of MCP-1 levels in serum was
also performed using the FlowCytomix (eBiosci-
ence) test system according to the manufacturer’s
instructions. The level of complement activity was
established in a microtest [3] based on the hemolysis
of sensitized sheep erythrocytes.
Statistics and representation
Statistical analysis was performed in Microsoft Ex-
cel 2010. The same program was used along with
OriginPro 8.1 for graphical representation of the re-
sults. Due to the small sampling size, a non-paramet-
rical Mann-Whitney test was applied to assess the
differences between animal groups. The differences
Fig. 1. Effect of the intraperitoneal and oral substance adminis-
tration upon the level of monocyte chemotactic protein-1
266
G. V. Antonovych, N. M. Zholobak, M. O. Shibinska, M. Ya. Spivak
were considered signifi cant at P < 0.05. The results
are presented as medians, error bars denote the abso-
lute maximum and minimum.
Results and discussion
1. Effect upon MCP-1 levels
The monocyte chemotactic protein-1 is a major che-
mokine that determines the extent to which a mono-
cyte/macrophage link is involved in the overall im-
mune response [4]. It was shown to be more potent
than other macrophage chemoattractants [5]. Apart
from the recruitment of monocytes to the sight of
infl ammation and stimulation of their proliferation,
MCP-1 also attracts T-cells, plays a part in the polar-
ization of Th0 lymphocytes into Th1/Th2 subpopu-
lations, and affects the activity of NK- and memory
T-cells [6]. Most MCP-1 producing cells are also ab-
le to produce IFN and other cytokines. That is why
after introduction of immunomodulatory substances,
the changes in this chemokine level were expected.
Interestingly, however, that in case of S1 a maximum
MCP-1 level was detected only on the third day after
the substance administration (Fig. 1), whereas
tilorone was characterized by a more classical dy-
namics: a peak was observed on the fi rst day of mon-
itoring period, with subsequent gradual decrease.
These trends were characteristic of both oral- and in-
traperitoneal administration routes. On day X of
monitoring, the differences between tested and con-
trol animal groups were not signifi cant, therefore the
data are not provided.
Up-regulation of MCP-1, observed in the animals
treated with S1, presumably has a secondary nature,
taking into account short lifespan of the chemokine
in vivo and the above described dynamics of its con-
tent. In our experiments on mice [2] it was previ-
ously shown that IFN production reaches maximum
on the third day after the administration of S1, which
coincides in time with the peak MCP-1 levels. How-
ever, although interferons, especially IFN-γ, are
known to induce the MCP-1 synthesis, other factors
are probably involved in the MCP triggering.
The results, obtained on the animals that received
i.p. injections, were characterized by a high level of
variation, which did not allow us to state signifi cant
differences between the substances starting from the
third day of monitoring. However, estimating the
median and maximum MCP-1 levels, detected in the
animals stimulated with S1 and tilorone, we can as-
sume a more pronounced chemokine potentiation by
the reference drug. When administered orally, a sig-
nifi cantly lower MCP-1 production was noted for a
30 mg/kg dose of S1, although the IFN-inducing ef-
fect of this smaller dose is comparable to that of 60
mg/kg dosage and tilorone (data not provided).
2. The activity of complement system
The complement system is a collection of soluble
proteins that circulate in blood in a non-activated
form, playing a part in both native and adaptive im-
munity. Our understanding of its role in the antiviral
defense has slightly increased in the recent years. In
Fig. 2. Complement activity after the intraperitoneal and oral ad-
mi nistration of the substances
267
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline upon biomarkers of infl ammation
addition to the opsonization and agglutination of vi-
rions, the lysis of virus-infected cells and the chemot-
actic functions, it was shown that complement com-
ponents C3a, C5a and their inactive precursor C3
also play a role in the activation of cytotoxic T lym-
phocytes [7, 8], NK-, and B-cells [9, 10], as well as
in the prolongation of their lifespan. The method,
used by us for assessing the activity of complement
system, does not allow characterization of the chang-
es in its individual components. However, the ob-
tained results (Fig.2) in general indicate an ability of
the studied compounds to increase the overall com-
plement hemolytic activity.
The activation effect caused by tilorone in both
routes of administration was signifi cantly higher
than that of S1 on the fi rst and third days of monitor-
ing. S1, administered intraperitoneally, did not lead
to a substantial rise of the complement activity rela-
tive to the control animal group. Moreover, the oral
application of this substance in 30 mg/kg dose was
characterized by a lower hemolytic activity com-
pared to tilorone. The dynamics of complement ac-
tivity resembled that of the MCP-1 production: max-
imal levels were detected mostly on the third day of
observation, except for the oral tilorone application,
characterized by an earlier peak. At the end of moni-
toring period the differences between all animal
groups were insignifi cant.
3. Changes in immune
cell counts
The numbers of circulating lymphocytes, mono-
cytes and neutrophils in the blood were established
on day VII of observation. Both administration
methods of the reference drug and indoloquinoxa-
line derivative did not lead to signifi cant changes in
the number of lymphocytes, therefore correspond-
ing data are not provided. Under tilorone infl uence
neutrophils were signifi cantly increased relative to
the control (Fig. 3, 4) and in case of the oral admin-
istration – relatively to S1 as well. The increased
numbers of circulating monocytes were observed
for both drugs, regardless of administration route.
However, the oral administration of S1 in 30 mg/kg
dose did not affect the number of monocytes, or the
effect lasted less than seven days. It should be not-
ed, that in rodents a great percentage of monocytes
is deposited in the spleen and can be recruited into
the blood fl ow more effectively, than in humans
[11]. The recruitment of neutrophils also took place,
but had a limited range.
In general, the results obtained resemble a typical
response to an immune-modulating drug. The chang-
es in absolute lymphocytic counts are rare and occur
only after serious immunological disturbances, op-
erative interventions or oncological conditions. For
these reasons we did not expect to reveal them. On
the other hand, neutrophils and monocytes are much
Fig. 4. Number of circulating phagocytes on the day VII after
the oral substance administration
Fig. 3. Number of circulating phagocytes on the day VII after
the intraperitoneal substance administration
268
G. V. Antonovych, N. M. Zholobak, M. O. Shibinska, M. Ya. Spivak
more labile, mobile cells. An increase in their num-
bers is a common consequence of the cytokine boost.
Thus, the oral treatment of rats with tilorone, which
was proved to be more effi cient than S1 at enhancing
complement activity and MCP-1 levels, logically led
to higher neutrophil counts. The same dependency
can be marked between two oral doses of S1: a
smaller 30 mg/kg dose is characterized by a weaker
effect upon the MCP-1 and complement system,
causing a minimal change in cell number. Interest-
ingly, however, i.p. injection of S1 leads to a signifi -
cant increase of monocyte counts relative to the con-
trol, although corresponding treatment did not alter
the complement activity and only poorly elevated
the MCP-1 content.
4. Infl uence upon functional
activity of phagocytes
An increase in phagocytic activity of neutrophils
relative to control was observed in both methods of
tilorone administration and lasted from the fi rst to
the seventh day of monitoring period (Fig. 5, 6). A
similar effect of the tested indoloquinoxaline after
i.p. injection was less pronounced, and in case of
oral administration required a bigger dose to mani-
fest. These results correlate with indicators of neu-
trophil metabolic activity: oxidative burst was sig-
nifi cantly intensifi ed on day III and VII after oral
tilorone administration, whereas for S1 such poten-
tiation was more brief and observed only in case of a
higher oral dose.
The studied substances mainly did not alter the per-
centage of phagocytizing monocytes. A rise in mono-
cytic absorptive capacity was notable only on the day
VII of observation in the animals, treated orally with
tilorone (P < 0,05). In other animal groups the changes
in phagocytic activity of monocytes were not signifi -
cant, however the substances managed to potentiate
the intensity of their oxidative burst. In particular, un-
der the 60 mg/kg oral dose of S1, as well as after the
intraperitoneal injection of this substance, the mono-
cyte metabolic activity was enhanced on the III and
VII days of observation (Fig. 5, 6). A similar effect of
the reference drug was notable only on the third day
for both routes of administration.
Fig. 5. Functional activity of circulating phagocytes after the
intraperitoneal injection of substances
Note: The left vertical axis corresponds to the cellular absorp-
tion activity, the right Y-axis displays the oxidative activity
Fig. 6. Functional activity of circulating phagocytes after the
oral substance administration
Note: The left vertical axis corresponds to the cellular absorp-
tion activity, the right Y-axis displays the oxidative activity
269
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline upon biomarkers of infl ammation
E. coli bacteria, used as an object for phagocytosis
in this experiment, were not antibody-opsonized.
That is why the above-described rise of the cellular
phagocytic activity cannot be linked with the com-
plement system, up-regulated by the studied com-
pounds. However, according to the classical works
[12, 13] the complement components can modulate
the oxidative burst intensity without antibody medi-
ation. Obviously, this parameter is even more depen-
dent upon the MCP-1 content. In this respect it
should be noted that although S1 induced a lower
MCP-1 production than tilorone, it managed to po-
tentiate the metabolic activity of monocytes as ef-
fectively as the reference drug. The ability of S1 to
stimulate the oxidative intensity was demonstrated
earlier on murine peritoneal macrophages [2]. In this
study S1, administered intraperitoneally, prompted a
faster and more pronounced tetrazolium reduction by
macrophages than a greater tilorone dosage. This may
seem not to correlate with observations of the current
work, however here a system response of the cells is
evaluated instead of a local one, and circulating mono-
cytes were studied instead of mature macrophages.
Conclusions
The application of immuno-stimulating agents with
the purpose to strengthen the antiviral defense mecha-
nisms as a rule leads to the pluripotent effects. 6-(2-
Morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline
as a low molecular substance is likely to react with the
membrane and the cytoplasmic pattern recognition
receptors or even bind to DNA, as was shown for
some structurally similar indoloquinoxalines [14].
Such activity can result in several vectors of intracel-
lular signaling and subsequent effects. The described
changes in the immunological parameters of the ani-
mals, treated with tilorone and S1, are interconnected
and altogether indicate the activation of innate immu-
nity. From one sight, this can be considered as a posi-
tive effect, but from the other point of view all the
studied parameters are referred to as the pro-infl am-
matory markers. A pathological role of complement
and MCP-chemokines is well recognized in athero-
sclerosis, myocarditis, systemic lupus erythematosus,
whereas the elevated activity of phagocytes is most
undesirable during certain pulmonary conditions and
after organ transplantation [15].
These facts do not imply strictly a negative charac-
ter of the up-regulated immunological parameters. In
fact, they all remained within- or close to normal
physiological range. The potentiated hemolytic activ-
ity indicates an elevated content of the non-activated
complement components, which in the normal condi-
tions are unable to cause a direct physiological action.
The intensifi cation of oxidative burst is another re-
serve that the organism acquired but does not normal-
ly use. It is a depiction of the increased metabolic re-
sources required to form the reactive oxygen species
(ROS), which would not be engaged until necessary.
Therefore, all possibly undesirable biological effects
that can be mediated by the complement system and
ROS are not likely to manifest without preliminary
individual auto-immunological predisposition. MCP-1,
in turn, is too potent as a chemokine to be left unno-
ticed. Its elevation in case of S1, however, was rela-
tively brief. An increase in monocyte and neutrophil
counts has a more lasting effect, especially taking into
account their high absorptive potential.
According to the work [16], human, murine and
rat monocytes can be divided into two main po pu-
lations:pro-infl ammatory and classical. They pass
their level of infl ammatory inclination to macrophag-
es, into which they differentiate. In mice all mono-
cytes that are recruited into the blood fl ow from the
bone marrow are Ly6C+ (analog of human Cd14+
CD16+ cells), demonstrating the secretion of ROS,
TNFα, nitric oxide, IL-1β, large amounts of type 1
interferon and high phagocytic capacity. The expres-
sion of Ly6C+ later diminishes, turning cells into
classical Ly6C- monocytes with an anti-infl ammato-
ry cytokine profi le [17]. The stimulatory signals,
caused by S1 and tilorone, can prevent this process,
causing a long-time infl ammatory predisposition in
some percent of phagocytes. This consequence to
certain extent is inevitable for all medications that
induce the endogenous cytokine production. Never-
theless, potential adverse effects in patients with al-
lergic and immunological disorders should be taken
into account during the subsequent pre-clinical and
clinical testing of S1.
270
G. V. Antonovych, N. M. Zholobak, M. O. Shibinska, M. Ya. Spivak
An important observation is that the different dos-
es and application routs of S1 can lead to a stronger
or weaker elevation of the infl ammatory markers. In
particular, a lower oral dose – 30 mg/kg, which in
our previous experiment induced the prolonged IFN
production, was characterized by minimal infl uence
upon the numbers and function of phagocytizing
cells in blood, the complement activity and MCP-1
content. It means that the tested indoloquinoxaline
can potentiate the antiviral immunity with minimal
infl ammatory effects.
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Вплив 6-(2-морфолін-4-іл-етил)-6H-індоло
[2,3-b]хіноксаліна на біомаркери запалення
Г. В. Антонович, Н. М. Жолобак,
М. О. Шибінська, М. Я. Співак
Мета. Дослідити вплив 6-(2-морфолін-4-іл-етил)-6H-індоло
[2,3-b]хіноксаліна (антивірусної сполуки з інтерферон-ін ду-
куючою дією) на про-запальний статус лабораторних щурів.
Методи. Концентрація MCP-1, кількість лейкоцитів в кро ві
та функціональна активність фагоцитів визначали цитофлю-
ориметричними методами. Активність системи комплемен-
ту оцінювалась в мікротесті на основі гемолізу сенситизова-
них еритроцитів. Результати. Застосування перорального
та парентерального способу введення сполук супроводжу-
валось збільшенням кількості моноцитів, потенціонуванням
метаболічного резерву фагоцитуючих клітин, під вищенням
вмісту MCP-1 і компонентів комплементу в сироватці крові.
Після введення 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]
хіноксаліну достовірного збільшення кількості циркулюю-
чих нейтрофілів або їх фагоцитарної активності не спосте-
рігалось, в той час як рівні MCP-1 і компонентів комплемен-
ту були значно нижчими, ніж у препарату порівняння – ти-
лорону. Висновки. ІХоча обидві сполуки є індукторами ІФН
з плюрипотентною імуностимулюючою дією, тестоване по-
хідне призводило до менш вираженого підвищення актив-
ності комплементу, нижчого вмісту MCP-1 та кількісті ней-
трофілів. Даний факт свідчить на користь того, що застосу-
271
The effect of antiviral substance 6-(2-morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline upon biomarkers of infl ammation
вання 6-(2-морфолін-4-іл-етил)-6H-індоло[2,3-b]хіноксаліну
для посилення неспецифічного антивірусного імунітету су-
проводжуватиметься нижчим ризиком розвитку побічних
аутоімунологічних реакцій
Ключов і слова: 6H-індоло[2,3-b]хіноксалін, запалення,
інтерферон, MCP-1, комплемент.
Влияние 6-(2-морфолин-4-ил-этил)-6H-индоло
[2,3-b]хиноксалина на биомаркеры воспаления
Г. В. Антонович, Н. М. Жолобак,
М. О. Шибинская, Н. Я. Спивак
Цель. Изучить влияние 6-(2-морфолин-4-ил-этил)-6H-ин до -
ло[2,3-b]хиноксалина (антивирусного соединения с интер-
ферон-индуцирующим действием) на про-воспалительный
статус лабораторных крыс. Методы. Концентрация MCP-1,
количество лейкоцитов в крови и функциональная актив-
ность фагоцитов определялись цитофлюориметрическими
методами. Активность системы компле мента оценивали в
микротесте, основанном на гемолизе сенситизированных
эритроцитов. Результаты. Как пероральный, так и паренте-
ральный способы введения соединений приводили к увели-
чению количества моноцитов, потенционированию метабо-
лического резерва фагоцитирующих клеток, повышению со-
держания MCP-1 и компонентов комплемента в сыворотке
крови. После введения 6-(2-морфолин-4-ил-этил)-6H-ин-
доло[2,3-b]хиноксалина достоверного увеличения числен-
ности циркулирующих нейтрофилов либо их фагоцитарной
активности не наблюдалось, в то время как уровни MCP-1 и
компонентов комплемента были значительно ниже, чем у
препарата сравнения – тилорона. Выводы. Хотя оба соеди-
нения представляют собой индукторы ИФН с плюрипотен-
тным иммуностимулирующим действием, тестируемое про-
изводное приводило к менее выраженному повышению ак-
тивности комплемента, меньшему содержанию MCP-1 и ко-
личеству нейтрофилов. Данный факт свидетельствует о том,
что применение 6-(2-морфолин-4-ил-этил)-6H-индоло[2,3-b]
хиноксалина для усиления неспецифического противови-
русного иммунитета будет нести более низкие риски разви-
тия побочных аутоиммунологических реакций.
Ключевые слова: 6H-индоло[2,3-b]хиноксалины, вос па-
ление, интерферон, MCP-1, комплемент.
Received 07.06.2015
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