RU2675693C1 - Method of inhibiting kappa nuclear factor with the use of potassium 5-hydroxynicotinate in a cell culture - Google Patents
Method of inhibiting kappa nuclear factor with the use of potassium 5-hydroxynicotinate in a cell culture Download PDFInfo
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- hydroxynicotinate
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- 238000000034 method Methods 0.000 title claims abstract description 13
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
Description
Изобретение относится к медицине, в частности к экспериментальной фармакологии.The invention relates to medicine, in particular to experimental pharmacology.
По известным литературным источникам – нуклеарный фактор каппа В (NF-κB) на сегодняшний день является одной из перспективных мишеней для разработки инновационных лекарственных средств в рамках развития современной фармакологии и формирования концепции таргетной терапии [Test system for evaluation of the influence of the biological activity of substances on the signal system of NF-κB: focus on the derivatives of 3 hydroxypyridine / Skachilova S.Y., Ragulina V.A., Kostina D.A., Burda Y.E. // Research result: pharmacology and clinical pharmacology. – 2017. – Vol. 3, №2. – Р. 50-56. doi: 10.18413/2313-8971-2017-3-2-50-56]. According to well-known literature, the nuclear factor kappa B (NF-κB) is today one of the promising targets for the development of innovative drugs in the development of modern pharmacology and the formation of the concept of targeted therapy [Test system for evaluation of the influence of the biological activity of substances on the signal system of NF-κB: focus on the derivatives of 3 hydroxypyridine / Skachilova SY, Ragulina VA, Kostina DA, Burda YE // Research result: pharmacology and clinical pharmacology. - 2017 .-- Vol. 3, No. 2. - R. 50-56. doi: 10.18413 / 2313-8971-2017-3-2-50-56].
Семейство транскрипционного фактора NF-κB играет важную роль в жизненном цикле клетки, регулируя процессы пролиферации, апоптоза, воспаления, миграции клеток, ангиогенез, иммунного ответа и прочие [NF-kB transcription factor: a key player in the generation of immune response / P. Tripathi, A. Aggarwal // Current Science. – 2006. – 90 (4). – P. 519-531; NF-κB in aging and disease / J.S. Tilstra, C.L. Clauson, L.J. Niedernhofer [et al.] // Aging and Disease. – 2011. – 2(6). – P. 449-465]. С другой стороны, сигнальная система NF-κB при неадекватной стимуляции приводит к формированию порочных кругов и ключевых патогенетических звеньев развития сердечно-сосудистых заболеваний, в том числе эндотелиальной дисфункции [Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice / R. Kobayasi, E.H. Akamine, A.P. Davel [et al.] // Journal of Hypertension. – 2010 – Vol. 28(10). – P. 2111–2119], заболеваний печени [Muriel, P. NF-κB in liver diseases: a target for drug therapy. / P. Muriel // J. Appl. Toxicol. – 2009. – № 29. – P. 91-100], поджелудочной железы [NF-κB in acute pancreatitis: Mechanisms and therapeutic potential / A. Jakkampudi [et al.] // Pancreatology. – 2016. – Vol. 16, № 4. – P.477-488], онкологических заболеваний [NF-κB, an active player in human cancers / Y. Xia, S. Shen and I.M. Verma // Cancer Immunol Res. – 2014. – 2 (9). – P. 823-830] и коморбидности.The NF-κB transcription factor family plays an important role in the cell life cycle by regulating proliferation, apoptosis, inflammation, cell migration, angiogenesis, the immune response and others [NF-kB transcription factor: a key player in the generation of immune response / P. Tripathi, A. Aggarwal // Current Science. - 2006 .-- 90 (4). - P. 519-531; NF-κB in aging and disease / J.S. Tilstra, C.L. Clauson, L.J. Niedernhofer [et al.] // Aging and Disease. - 2011 .-- 2 (6). - P. 449-465]. On the other hand, the NF-κB signaling system, when inadequately stimulated, leads to the formation of vicious circles and key pathogenetic links in the development of cardiovascular diseases, including endothelial dysfunction [Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice / R. Kobayasi, EH Akamine, A.P. Davel [et al.] // Journal of Hypertension. - 2010 - Vol. 28 (10). - P. 2111–2119], liver diseases [Muriel, P. NF-κB in liver diseases: a target for drug therapy. / P. Muriel // J. Appl. Toxicol. - 2009. - No. 29. - P. 91-100], pancreas [NF-κB in acute pancreatitis: Mechanisms and therapeutic potential / A. Jakkampudi [et al.] // Pancreatology. - 2016. - Vol. 16, No. 4. - P.477-488], cancer [NF-κB, an active player in human cancers / Y. Xia, S. Shen and I.M. Verma // Cancer Immunol Res. - 2014 .-- 2 (9). - P. 823-830] and comorbidity.
Поэтому ингибирование патологической активации NF-κB и предупреждение формирования порочных кругов – ключевой вопрос современной фармакотерапии. В связи с этим, следует отметить актуальность изучения 5-гидроксиникотината калия в качестве ингибитора NF-κB in vitro с оценкой активности p65 субъединицы в мононуклеарных клетках (МНК) крови крыс линии Wistar, стимулированных бактериальным липополисахаридом.Therefore, inhibition of the pathological activation of NF-κB and the prevention of the formation of vicious circles is a key issue in modern pharmacotherapy. In this regard, it should be noted the relevance of studying potassium 5-hydroxynicotinate as an NF-κB inhibitor in vitro with an assessment of the activity of the p65 subunit in mononuclear cells (MNCs) of blood of Wistar rats stimulated with bacterial lipopolysaccharide.
Известен способ ингибирования NF-κB композициями тритерпенов (патент на изобретение RU 2288706, публ. 10.12.2006). Сущность его заключается в ингибировании воспаления путем обеспечения клетки монотерпеновыми композициями, которые ингибируют фактор NF-κB. Композиция может включать дополнительные химические функциональные агенты. Технический результат - повышение эффективности лечения воспалительных состояний, в частности предраковых. A known method of inhibiting NF-κB compositions of triterpenes (patent for invention RU 2288706, publ. 10.12.2006). Its essence is to inhibit inflammation by providing cells with monoterpene compositions that inhibit NF-κB factor. The composition may include additional chemical functional agents. The technical result is an increase in the effectiveness of the treatment of inflammatory conditions, in particular precancerous.
Основным недостатком способа является то, что применяемые терапевтические дозы монотерпеновых/тритерпеновых соединений могут привести к значительным побочным эффектам или к другим неблагоприятным реакциям. У субъектов с далеко зашедшим заболеванием может наблюдаться определенная степень побочных эффектов при применении терапевтических доз монотерпеновых/тритерпеновых соединений.The main disadvantage of this method is that the therapeutic doses of monoterpene / triterpene compounds used can lead to significant side effects or other adverse reactions. In subjects with advanced disease, a certain degree of side effects may be observed with therapeutic doses of monoterpene / triterpene compounds.
Известно зависящее от концентрации влияние (R)- и (S)-флурбипрофена на активацию фактора транскрипции NF-κВ в RAW-клетках (патент на изобретение RU 2250103, публ. 20.04.2005). Анализ гельудерживания показывает, что липополисахарид (ЛПС) (1 мкг/мл) приводит к активации NF-κВ (комплекс р50/р65 NF-κВ). Микромолярные концентрации (R)-флурбипрофена были в состоянии ингибировать эту индуцированную ЛПС активацию NF-κВ.The concentration-dependent effect of (R) - and (S) -flurbiprofen on the activation of the transcription factor NF-κB in RAW cells is known (Patent RU 2250103, publ. 04/20/2005). Gel retention analysis shows that lipopolysaccharide (LPS) (1 μg / ml) leads to activation of NF-κB (p50 / p65 complex NF-κB). Micromolar concentrations of (R) -flurbiprofen were able to inhibit this LPS-induced activation of NF-κB.
Изобретение относится к области фармации, а именно к применению (R)-энантиомеров арилпропионовых кислот. Сущность изобретения - применение названных кислот для получения лекарственных средств, которые ингибируют каскад активации NF-κB. На основе их создана композиция. Технический результат - использование для лечения заболеваний, на которые может терапевтически положительно воздействовать ингибирование образования NF-κB. The invention relates to the field of pharmacy, and in particular to the use of (R) -enantiomers of arylpropionic acids. The essence of the invention is the use of these acids to obtain drugs that inhibit the activation cascade of NF-κB. Based on them, a composition was created. The technical result is the use for the treatment of diseases that can be therapeutically positively affected by the inhibition of the formation of NF-κB.
Недостатком данного изобретения является то, что арилпропионовые кислоты и их производные обладают рядом побочных эффектов при их применении, а именно тошнота, анорексия, метеоризм, запор, изжога, диарея, НПВС-гастропатия, вплоть до эрозивно-язвенных поражений ЖКТ.The disadvantage of this invention is that arylpropionic acids and their derivatives have a number of side effects when used, namely nausea, anorexia, flatulence, constipation, heartburn, diarrhea, NSAID gastropathy, up to erosive and ulcerative lesions of the gastrointestinal tract.
Наиболее близким к заявленному решению является способ ингибирования NF-κB с использованием производных 3-оксипиридина (Test system for evaluation of the influence of the biological activity of substances on the signal system of NF-κB: focus on the derivatives of 3hydroxypyridine / Skachilova S.Y., Ragulina V.A., Kostina D.A., Burda Y.E. // Research result: pharmacology and clinical pharmacology. – 2017. – Vol. 3, №2. – Р. 50-56), включающий добавление бактериального липополисахарида в концентрации 1 мкг/мл к свежевыделенным по стандартной методике на градиенте плотности фиколла МНК крови крыс Wistar, затем добавление к данной смеси исследуемого фармакологического агента, растворенного в фосфатно-солевом буфере, в соответствующей конечной концентрации, отличающийся тем, что в качестве фармакологического агента используют мексидол, этоксидол, 3-гидрокси-2-этил-6-метилпиридиния глутаминат, 3-гидрокси-2-этил-6-метилпиридиния аспарагинат, 3-гидрокси-2-этил-6-метилпиридиния 4-аминобензоат, 3-гидрокси-2-этил-6-метилпиридиния никотинат, 3-гидрокси-2-этил-6-метилпиридиния N-ацетиламиногексаноат, 3-гидрокси-2-этил-6-метилпиридиния N-ацетиламиноацетат, 3-гидрокси-2-этил-6-метилпиридиния N-ацетиламиноглутаминат, 3-гидрокси-2-этил-6-метилпиридиния ацетилсалицилат, бета-гидроксиникотиноилгидразон 2-метил-3-гидрокси-4-формил-5-оксиметилпиридина дигидрохлорид. Наибольшей ингибирующей способностью на ЛПС-индуцированную экспрессию p65 NF-κB в МНК обладали мексидол, этоксидол и бета-гидроксиникотиноилгидразон 2-метил-3-гидрокси-4-формил-5-оксиметилпиридина дигидрохлорид.Closest to the claimed solution is a method of inhibiting NF-κB using 3-hydroxypyridine derivatives (Test system for evaluation of the influence of the biological activity of substances on the signal system of NF-κB: focus on the derivatives of 3hydroxypyridine / Skachilova SY, Ragulina VA, Kostina DA, Burda YE // Research result: pharmacology and clinical pharmacology. - 2017. - Vol. 3, No. 2. - P. 50-56), including the addition of bacterial lipopolysaccharide at a concentration of 1 μg / ml to freshly isolated the standard method on a density gradient of ficoll MNCs of blood of Wistar rats, then adding the studied pharmacological agent to this mixture, p dissolved in phosphate-buffered saline, in the corresponding final concentration, characterized in that mexidol, ethoxidol, 3-hydroxy-2-ethyl-6-methylpyridinium glutamate, 3-hydroxy-2-ethyl-6-methylpyridinium asparaginate are used as a pharmacological agent 3-hydroxy-2-ethyl-6-methylpyridinium 4-aminobenzoate, 3-hydroxy-2-ethyl-6-methylpyridinium nicotinate, 3-hydroxy-2-ethyl-6-methylpyridinium N-acetylaminohexanoate, 3-hydroxy-2- ethyl 6-methylpyridinium N-acetylaminoacetate, 3-hydroxy-2-ethyl-6-methylpyridinium N-acetylaminoglutaminate, 3-hydroxy-2-ethyl-6 -methylpyridinium acetylsalicylate, beta-hydroxynicotinoylhydrazone 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine dihydrochloride. Mexidol, ethoxidol and beta-hydroxynicotinoylhydrazone 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine dihydrochloride had the highest inhibitory ability on LPS-induced expression of p65 NF-κB in MNCs.
Недостатком данного решения является то, что наблюдаемая ингибирующая активность in vitro в отношении сигнального пути NF-κB мексидола, этоксидола и бета-гидроксиникотиноилгидразон 2-метил-3-гидрокси-4-формил-5-оксиметилпиридина дигидрохлорида не достигает целевых значений и более чем в 3,5 раза отличается от значений интактных крыс.The disadvantage of this solution is that the observed in vitro inhibitory activity with respect to the NF-κB signal pathway of mexidol, ethoxidol and beta-hydroxynicotinoylhydrazone 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine dihydrochloride does not reach the target values and more than in 3.5 times different from intact rats.
Задачей предлагаемого изобретения является создание эффективного способа ингибирования нуклеарного фактора каппа В с использованием 5-гидроксиникотината калия в культуре мононуклеарных клеток крови крыс Wistar.The objective of the invention is to provide an effective method of inhibiting the nuclear factor Kappa B using potassium 5-hydroxynicotinate in a culture of mononuclear blood cells of rat Wistar rats.
Техническим результатом предлагаемого изобретения является эффективный способ ингибирования NF-κB с использованием 5-гидроксиникотината калия, подтверждаемый результатами оценки активности p65 субъединицы в МНК крови крыс Wistar, стимулированных бактериальным липополисахаридом, обладающий наиболее благоприятным профилем безопасности по сравнению с рассмотренными аналогами и наиболее высокой ингибирующей активностью NF-κB по сравнению с прототипом.The technical result of the present invention is an effective method of inhibiting NF-κB using potassium 5-hydroxynicotinate, confirmed by the results of evaluating the activity of the p65 subunit in WNar rat blood MNCs stimulated with bacterial lipopolysaccharide, which has the most favorable safety profile compared to the considered analogues and the highest NF inhibitory activity -κB compared to the prototype.
Поставленная задача достигается тем, что предложен способ ингибирования нуклеарного фактора каппа В с использованием 5-гидроксиникотината калия в культуре клеток, включающий добавление бактериального липополисахарида в концентрации 1 мкг/мл к свежевыделенным по стандартной методике на градиенте плотности фиколла МНК крови крыс Wistar, затем добавление к данной смеси исследуемого фармакологического агента, растворенного в фосфатно-солевом буфере, в соответствующей конечной концентрации, а в качестве фармакологического агента используют 5-гидроксиникотинат калия. This object is achieved by the fact that the proposed method of inhibiting the nuclear factor of kappa B using potassium 5-hydroxynicotinate in a cell culture, including adding bacterial lipopolysaccharide at a concentration of 1 μg / ml to freshly isolated Wistar rat MNC density gradient ficoll, then adding to this mixture of the studied pharmacological agent, dissolved in phosphate-buffered saline, in the corresponding final concentration, and as a pharmacological agent I use t of potassium 5-hydroxynicotinate.
Основным преимуществом предлагаемого способа является то, что введение 5-гидроксиникотината калия, в исследуемой in vitro концентрации, 35 мкг/мл, приводит к выраженному ингибированию NF-κB в культуре клеток, что подтверждается результатами оценки активности p65 субъединицы в МНК крови крыс Wistar, стимулированных бактериальным липополисахаридом. Помимо этого, предлагаемый способ обладает наиболее благоприятным профилем безопасности по сравнению с рассмотренными аналогами и наиболее высокой ингибирующей активностью NF-κB по сравнению с прототипом.The main advantage of the proposed method is that the introduction of potassium 5-hydroxynicotinate, in an in vitro concentration studied, 35 μg / ml, leads to a pronounced inhibition of NF-κB in cell culture, which is confirmed by the results of evaluating the activity of the p65 subunit in WNCAR rat blood MNCs bacterial lipopolysaccharide. In addition, the proposed method has the most favorable safety profile compared with the considered analogues and the highest inhibitory activity of NF-κB compared to the prototype.
Способ осуществляется следующим образом.The method is as follows.
При изучении ингибирующей активности в отношении NF-κB были исследованы 5-гидроксиникотинат калия и Мексидол®, ФАРМАСОФТ НПК, Россия (препарат сравнения), которые добавляли к свежевыделенным по стандартной методике на градиенте плотности фиколла (ρ = 1,077, «Панэко», Россия) МНК крови крыс линии Wistar. Исследуемая in vitro концентрация каждого препарата составляет 35 мкг/мл.When studying the inhibitory activity against NF-κB, potassium 5-hydroxynicotinate and Mexidol ® , FARMASOFT NPK, Russia (reference drug) were studied, which were added to the freshly isolated by the standard method on a density gradient of ficoll (ρ = 1,077, Paneko, Russia) MNC blood of rats of the Wistar line. The in vitro concentration studied for each preparation is 35 μg / ml.
В каждой экспериментальной группе 6 крыс, 3 самца и 3 самки. Для исследования взяты крысы без внешних признаков заболевания, прошедшие карантинный режим, массой 225-275 г.In each experimental group, 6 rats, 3 males and 3 females. For the study, rats were taken without external signs of the disease, which passed the quarantine regime, weighing 225-275 g.
Исследование активности NF-κB проводили в 2 мл пробирках типа Eppendorf (Gen Follower, Китай), куда вносили 1мл суспензии МНК крови крыс, 1×106 клеток в 1 мл среды RPMI-1640 («Панэко», Россия) с 5% сыворотки эмбрионов коров (HiClone, США), затем добавляли бактериальный липополисахарид («Пирогенал», ГУ НИИЭМ им. Н.Ф. Гамалеи, Россия) в концентрации 1 мкг/мл, затем добавляли исследуемый препарат, растворенный в фосфатно-солевом буфере, в соответствующей конечной концентрации. Клетки инкубировали в течение 30 мин при 37°С на шейкере, после чего центрифугировали 5 мин при 200g, надосадочную жидкость удаляли, осадок МНК исследовали на активность p65 субъединицы NF-κB в строгом соответствии с инструкцией к набору NFkB p65 TotalMultispecies InstantOne™ ELISA Kit (Invitrogen / Thermo Fisher Scientific, cat. 85-86081-11).A study of the activity of NF-κB was carried out in 2 ml Eppendorf tubes (Gen Follower, China), where 1 ml of a suspension of rat blood MNCs, 1 × 106 cells in 1 ml of RPMI-1640 medium (Paneko, Russia) with 5% embryo serum was added cows (HiClone, USA), then added bacterial lipopolysaccharide (Pyrogenal, NI Gamalei State Research Institute of Nuclear Medicine, Russia) at a concentration of 1 μg / ml, then the test drug dissolved in phosphate-buffered saline was added in the corresponding final concentration. Cells were incubated for 30 min at 37 ° C on a shaker, then centrifuged for 5 min at 200 g, the supernatant was removed, the MNC sediment was examined for p65 subunit activity of NF-κB in strict accordance with the instructions for the NFkB p65 TotalMultispecies InstantOne ™ ELISA Kit ( Invitrogen / Thermo Fisher Scientific, cat. 85-86081-11).
МНК крови крыс в количестве 1×106 лизировали в 1 мл лизирующего буфера, входящего в состав набора, супернатант вносили в лунки 96-луночного планшета из указанного ИФА набора. После нескольких этапов продукты сэндвич-реакции определяли на микропланшетном ридере Multiskan FC (Thermo Fisher, Германия) при 450 нм. Rat blood MNCs in the amount of 1 × 106 were lysed in 1 ml of the lysis buffer included in the kit; the supernatant was added to the wells of a 96-well plate from the indicated ELISA kit. After several steps, the products of the sandwich reaction were determined on a Multiskan FC microplate reader (Thermo Fisher, Germany) at 450 nm.
Для упрощения расчетов относительного влияния исследуемых веществ были выбраны единицы оптической плотности, непосредственно получаемые с микропланшетного ридера, округленные до 2-го знака после запятой. Статистическую обработку результатов проводили в программе MS Excel 2013 с модулем Attestatv.13 с применением метода непараметрической статистики – критерия Крускала-Уоллиса, т.к. распределение активности p65 субъединицы NF-κB в клетках не являлось нормальным.To simplify the calculations of the relative influence of the studied substances, we selected units of optical density directly obtained from the microplate reader, rounded to the 2nd decimal place. Statistical processing of the results was carried out in MS Excel 2013 with the Attestatv.13 module using the nonparametric statistics method - the Kruskal-Wallis test, because the distribution of p65 activity of the NF-κB subunit in cells was not normal.
ПРИМЕР КОНКРЕТНОГО ВЫПОЛНЕНИЯEXAMPLE OF SPECIFIC PERFORMANCE
Результаты оценки ингибирующей NF-κB активности исследуемых препаратов представлены в табл. 1.The results of the evaluation of the inhibitory NF-κB activity of the studied drugs are presented in table. one.
Как видно из табл. 1, наибольшей ингибирующей способностью на ЛПС-индуцированную экспрессию p65 NF-κB в МНК крови крыс Wistar в исследуемых концентрациях обладает 5-гидроксиникотинат калия. Ингибирующая активность NF-κB 5-гидроксиникотината калия в исследуемой конечной концентрации 35 мкг/мл выше на 41%, чем у Мексидола® при индуцированной ЛПС активации NF-κВ.As can be seen from the table. 1, potassium 5-hydroxynicotinate possesses the highest inhibitory ability on LPS-induced expression of p65 NF-κB in the blood MNCs of Wistar rats in the studied concentrations. The inhibitory activity of potassium NF-κB 5-hydroxynicotinate in the studied final concentration of 35 μg / ml is 41% higher than that of Mexidol ® with LPS-induced activation of NF-κB.
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| RU2250103C2 (en) * | 1999-02-24 | 2005-04-20 | Пац Арцнаймиттель-Энтвиклунгсгезельшафт мбХ | EMPLOYMENT OF (R)-ARYLPROPIONIC ACIDS TO PREPARE DRUGS FOR TREATMENT OF HUMANS AND ANIMALS LIABLE TO BE THERAPEUTICALLY AFFECTED VIA NF-κB ACTIVATION INHIBITION |
| RU2288706C2 (en) * | 2000-11-17 | 2006-12-10 | Рисерч Дивелопмент Фаундейшн | INHIBITION OF NF-κB BY TRITERPENE COMPOSITIONS |
| WO2011137587A1 (en) * | 2010-05-06 | 2011-11-10 | Hutchison Medipharma Limited | Cytokine inhibitors |
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| RU2250103C2 (en) * | 1999-02-24 | 2005-04-20 | Пац Арцнаймиттель-Энтвиклунгсгезельшафт мбХ | EMPLOYMENT OF (R)-ARYLPROPIONIC ACIDS TO PREPARE DRUGS FOR TREATMENT OF HUMANS AND ANIMALS LIABLE TO BE THERAPEUTICALLY AFFECTED VIA NF-κB ACTIVATION INHIBITION |
| RU2288706C2 (en) * | 2000-11-17 | 2006-12-10 | Рисерч Дивелопмент Фаундейшн | INHIBITION OF NF-κB BY TRITERPENE COMPOSITIONS |
| WO2011137587A1 (en) * | 2010-05-06 | 2011-11-10 | Hutchison Medipharma Limited | Cytokine inhibitors |
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