Severity of the acute phase response in experimental ulcerative colitis under conditions of the use of vitamin D3 in a novel rectal suppository

Цель - изучение влияния витамина D3 в составе оригинальных ректальных суппозиториев на выраженность острофазового ответа при экспериментальном язвенном колите Методика. Эксперимент выполнен на 49 половозрелых крысах-самцах Wistar. Язвенный колит (ЯК) моделировали двухэтапным (накожным, а затем ректальным) применением 3% спиртового раствора оксазолона. Оригинальные суппозитории на основе смеси полиэтиленгликолей массой 300 мг, содержащие 1500 ME витамина D3, вводили per rectum каждые 12 ч в течение 6 сут. Исследования проводили на 2-е, 4-е и 6-е сут заболевания. Клиническую картину оценивали по адаптированной для крыс шкале Disease activity index (DAI) с учетом массы тела, консистенции и наличия крови в каловых массах. Количество лейкоцитов и нейтрофилов определяли на гематологическом анализаторе и в мазках крови. Функциональную активность нейтрофилов исследовали по поглощению частиц монодисперсного латекса в спонтанном и индуцированном НСТ-тесте. Концентрацию в сыворотке крови C-реактивного белка (С-РБ), IL-6 и IL-8 определяли с помощью иммуноферментного анализа, экспрессию TNF-α в стенке толстой кишки - иммуногистохимическим методом. Результаты. При экспериментальном ЯК на 2-е, 4-е и 6-е сут наряду с прогрессивным повышением DAI зафиксированы изменения показателей острофазового ответа (ООФ): увеличивается экспрессия в толстой кишке TNF-α с максимальным уровнем на 4-е и 6-е сут, повышается концентрация в сыворотке крови IL-6 с максимальным уровнем на 4-е, IL-8 - на 6-е сут; в сыворотке возрастает концентрация С-РБ максимально на 6-е сут, в крови увеличивается общее количество лейкоцитов, число палочкоядерных и сегментоядерных нейтрофилов с максимальным уровнем на 2-е и 4-е сут. Возрастает поглотительная и НСТ-редуцирующая способность нейтрофилов крови с максимальными значениями на 4-е и 6-е сут эксперимента. Применение при экспериментальном ЯК ректальных суппозиториев с витамином D3 приводит на 4-е и 6-е сут наблюдения к снижению DAI, экспрессии TNF-α в толстой кишке и количества нейтрофилов, на 2-е, 4-е и 6-е сут - к уменьшению концентрации IL-6 и С-РБ в сыворотке, поглотительной и НСТ-редуцирующей способности нейтрофилов крови, на 6-е сут - к снижению концентрации IL-8 в сыворотке и общего количества лейкоцитов в крови. Заключение. Применение при экспериментальном ЯК оригинальных ректальных суппозиториев с витамином D3 приводит к снижению индекса активности болезни. Выраженность клинических проявлений ослабевает по мере снижения экспрессии в кишке TNF-α, снижения концентрации в сыворотке IL-6, количества в крови лейкоцитов и нейтрофилов, уменьшения НСТ-редуцирующей способности нейтрофилов. Aim. To study the effect of vitamin D3 in rectal suppositories on severity of the acute phase response (APR) in experimental ulcerative colitis (UC). Methods. Experiments were performed on 49 mature male Wistar rats. UC was induced by cutaneous followed by rectal applications of a 3% oxazolone alcohol solution. Novel polyethylene glycol suppositories (300 mg) containing 1500 IU of vitamin D3 were injected per rectum every 12 hrs for 6 days. Data were collected on days 2, 4, and 6 of UC. The clinical picture was assessed according to the Disease Activity Index (DAI) scale adapted for rats, taking into account body weight, consistency of, and the presence of blood in the feces. The number of blood leukocytes and neutrophils was determined on a hematological analyzer and in blood smears. The functional activity of neutrophils isolated from the blood was evaluated by the absorption of monodisperse latex particles and with the spontaneous and induced NBT test. Serum concentrations of C-reactive protein (CRP), IL-6 and IL-8 were measured by enzyme immunoassay. TNF-α expression in the colon wall was measured by an immunohistochemical method. Results. The following changes in APR variables along with a progressive increase in DAI were recorded on days 2, 4, and 6. Expression of colon TNF-α increased, with maximal values on days 4 and 6. Serum IL-6 increased and reached a maximal value on day 4; and serum IL-8 increased, with a maximum on day 6. Serum CRP increased, with a maximum on day 6. The total number of blood leukocytes and the number of stab and segmented neutrophils increased, with maximal values on days 2 and 4. The absorption and NBT-reducing ability of blood neutrophils increased, with maximal values on days 4 and 6. The use of rectal suppositories with vitamin D3 in experimental UC resulted in decreases in DAI, colon TNF-α expression, and the number of blood neutrophils on days 4 and 6; decreases in serum concentrations of IL-6 and CRP, and the absorption and NBT-reducing ability of blood neutrophils on days 2, 4, and 6; and decreases in the serum concentration of IL-8 and in the total number of blood leukocytes on day 6. Severity of clinical manifestations was alleviated with decreases in the expression of colon TNF-α, the serum concentration of IL-6, the number of blood leukocytes and neutrophils, and the NBT-reducing ability of neutrophils decreases. Conclusion. Application of the original rectal suppositories with vitamin D3 every 12 hours in experimental UC leads to a decrease in the disease activity index. The severity of clinical manifestations weakens as TNF-α expression, serum IL-6 concentration, number of leukocytes and neutrophils in blood, and decrease of neutrophil NST-reducing capacity decrease.

Clinical Pilot Study of Rectal Suppository Containing Combined Extract of Cissus quadrangularis Linn. and Acmella paniculata (Wall ex. DC.) R. K. Jansen in Acute Hemorrhoids

Background. Cissus quadrangularis Linn. (CQ) is a medicinal plant with good evidence for the treatment of hemorrhoids, listed in the Thai National List of Herbal Products in the oral dosage form. Acmella paniculata (Wall ex. DC.) R. K. Jansen. (AP) is a medicinal plant with a local anesthetic effect. Objective. To investigate the potential of rectal suppositories containing CQ and AP extracts to alleviate symptoms of hemorrhoids compared with the commercialized rectal suppository containing hydrocortisone and cinchocaine. Materials and Methods. Hemorrhoid outpatients (n = 105) with different severity grades (I, II, or III) from eight hospitals in northern Thailand were included in this study. Hemorrhoid severity was graded by proctoscopy associated with either anal pain or bleeding related to hemorrhoids or both. The patients were randomly allocated to two groups: CQ-AP group (n = 52) or the commercialized rectal suppository group (n = 53). One suppository was rectally administered twice daily in the morning and at bedtime for seven days. Evaluations were performed by physicians on days 1, 4, and 8 of the study. The primary endpoints were bleeding and prolapse size, while the secondary endpoint was anal pain. Results. Baseline demographics, lifestyle, constipation, number of prolapses, grade of hemorrhoid severity, and duration of experiencing hemorrhoids were comparable in both groups of patients. The effects of CQ-AP and the commercialized rectal suppository on bleeding, prolapse size, and anal pain were comparable. The patients in both groups were satisfied with both products at comparable levels and stated a preference for further use in the case of hemorrhoids recurrence. In terms of safety, the patients in the commercialized rectal suppository group experienced a higher incidence of adverse events, including anal pain and bleeding. Conclusion. Rectal suppositories containing a combined extract of CQ and AP show potential in alleviating hemorrhoidal symptoms with a good safety profile.

Comparative study of specific activity of rectal suppositories with clopidogrel

One of the most effective platelet antiaggregants is clopidogrel, inhibiting platelet activation by selectively binding adenosine diphosphate (ADP) with specific receptors. However, in a number of clinical situations it is necessary to have a pronounced antithrombotic effect in the shortest possible time which gives rise to interest in the transmucose use of the drug, in particular, rectal route. The theoretical preconditions for the development of a rectal administration of clopidogrel are based on the data that the anti-aggregation effect of a substance is carried out by its main metabolite formed after “first hepatic passage”, while clopidogrel itself in this aspect is inactive. On the base of complex physico-chemical, pharmaceutical, biopharmaceutical, rheological and microbiological investigations, the rational composition of clopidogrel rectal dosage form – suppository on a hydrophilic base to prevent atherothrombotic events in patients with myocardial infarction, acute coronary syndrome, ischemic stroke, peripheral arterial occlusion, is proposed by the employees of the Department of Technology of Medications of the Zaporizhzhia State Medical University. The aim of the work is to investigate the specific activity of rectal suppositories with clopidogrel. Materials and methods. Experimental suppositories with clopidogrel 0,075 g for rectal administration were used as the objects in pre-line studies. The ability of clopidogrel to reduce the inhibitory effect of ADP on adenylate cyclase activity and decrease the number of binding sites for 2-methylthio-ADP (analogue of ADP) without altering the receptor topography is used as a base of the method of comparative study of its specific activity in the rectal dosage form (suppository) and in comparison with the reference medication Plavix (SANOFI WINTHROP INDUSTRIE, France) in the tablet form. Studies were performed on white non-linear rats of different sexes with different mass 150–210 g, aged 3.5–5.0 months. Results. Statistically significant differences in the inhibition of induced platelet aggregation were registered after 6 hours of rectal administration of clopidogrel and gastric administration of the reference medication, indicating the effectiveness of the rectal applicative transmucosal route for this active pharmaceutical ingredient (API). On the 5th day of administration, inhibition of induced platelet aggregation significantly increased, and that is supported by literature on the cumulative effect of clopidogrel in daily life. When comparing the digital material of the table, it is obvious that the rectal administration of clopidogrel is more effective in comparison with oral route which is probably due to the rapid delivery of API to the liver and the formation of an active metabolite of clopidogrel inhibiting the induction and spontaneous aggregation of platelets in human’s and animal’s blood. Obtained data provide with sound arguments for development of rectal suppositories with clopidogrel because in clinical care faster achievement of an anti-aggregation effect in patients with acute coronary syndrome is the primary task of clinical pharmacology and pharmacy. Conclusions. Using the biochemical model of pathology, it was established that the applicative semisolid medication of clopidogrel in the form of rectal suppository exhibited inductive anti-aggregation activity and its administration didn’t reveal any side effects and undesirable events. Rectal suppositories with clopidogrel have been shown to exhibit faster reliable anti-aggregation effect in comparison with intragastric administration.

Development, Characterization and In Vivo Pharmacokinetic Assessment of Rectal Suppositories Containing Combination Antiretroviral Drugs for HIV Prevention

Receptive anal intercourse (RAI) contributes significantly to HIV acquisition underscoring the need to develop HIV prevention options for populations engaging in RAI practices. We explored the feasibility of formulating rectal suppositories with potent antiviral drugs for on-demand use. A fixed-dose combination of tenofovir (TFV) and elvitegravir (EVG) (40 mg each) was co-formulated in six different suppository bases (three fat- and three water-soluble). Fat-soluble witepsol H15 and water-soluble polyethylene glycol (PEG) based suppositories demonstrated favorable in vitro release and were advanced to assess in vivo pharmacokinetics following rectal administration in macaques. In vivo drug release profiles were similar for both suppository bases. Median concentrations of TFV and EVG detected in rectal fluids at 2 h were 1- and 2-logs higher than the in vitro IC50, respectively; TFV-diphosphate levels in rectal tissues met or exceeded those associated with high efficacy against rectal simian HIV (SHIV) exposure in macaques. Leveraging on these findings, a PEG-based suppository with a lower dose combination of tenofovir alafenamide (TAF) and EVG (8 mg each) was developed and found to achieve similar rectal drug exposures in macaques. This study establishes the utility of rectal suppositories as a promising on-demand strategy for HIV PrEP and supports their clinical development.

Clinical and morphological characteristics of experimental ulcerative colitis in the conditions of using original rectal suppositories with vitamin D3

Introduction. Vitamin D3 possesses antioxidant, anti-inflammatory, immunomodulatory, and other properties, has been shown to be effective in some autoimmune diseases, which is a prerequisite for studying its effect, when applied locally, on the clinical status and morphology of the site of injury in ulcerative colitis (UC).The aims was to study the effect of vitamin D3 in the composition of original rectal suppositories on the clinical status and morphology of the lesion of colon in experimental UC.Materials and methods. UC was modeled by two-stage administration of oxazolone. Rectal suppositories were prepared on the basis of a 10% aqueous solution of vitamin D3. The clinical status was assessed using the Disease activity index (DAI) scale. In the area of colon damage, the number of neutrophils, lymphocytes, eosinophils, histiocytes, plasmocytes and fibroblasts was examined per mm² d the tissue damage index (TDI) was calculated.Results and Discussion. In experimental UC, DAI increase an ulcerative defect is recorded in the colon, the number of neutrophils, lymphocytes, eosinophils, plasma cells, histiocytes, fibroblasts, TDI increases. The use of original rectal suppositories with vitamin D3 in experimental UC leads to a decrease in DAI, the size of the ulcer and TDI, a decrease in the infiltration of the intestinal wall by neutrophils, lymphocytes, eosinophils and plasma cells, an increase in the infiltration of histiocytes, fibroblasts.Conclusions. In experimental oxazolone-induced colitis, the clinical picture and morphology of the injury site characteristic of UC are recorded. The use of original rectal suppositories with vitamin D3 a total dose of 18,000 IU leads to a decrease in the severity of clinical signs and a decrease in the representation in the colon wall of cells involved in tissue destruction, an increase in the representation of cells mediating repair, which was reflected in a decrease in the area of the ulcer and tissue damage index , the severity of clinical signs according to DAI weakens as lymphocytes and plasma cells decrease in the area of damage to the colon, decrease in the size of the ulcer and decrease in TDI, increase in histiocytes and fibroblasts.

Morphological aspects of the protective effect of newly formulated rectal suppositories with turmeric extract in experimental Crohn’s disease

Введение. Востребованным для лечения болезни Крона (БК) является разработка новых, обоснованных с патогенетических позиций и безопасных лекарственных средств преимущественно локального действия эндогенного или растительного происхождения. В этом отношении привлекает внимание экстракт корневищ Куркумы длинной (Curcuma longa), содержащий куркуминоидный комплекс, обладающий плейотропными эффектами. Ранее нами показано, что экстракт куркумы в составе суппозиториев при экспериментальной БК обладaет иммуномодулирующим и местным антиоксидантным действием, что предполагает влияние экстракта куркумы на морфологию очага повреждения в кишечнике при БК. Цель работы - изучение влияния экстракта куркумы в составе оригинальных ректальных суппозиториев на динамику морфологических изменений, экспрессию миелопероксидазы (МПО) и TNF-a в очаге повреждения толстого кишечника при экспериментальной БК. Методика. Эксперимент выполнен на 49 половозрелых крысах-самцах Wistar. БК моделировали введением per rectum спиртового раствора тринитробензосульфоновой кислоты (ТНБС). Оригинальные ректальные суппозитории массой 300 мг на основе полиэтиленгликолей различной молекулярной массы, содержащие 0,075 мг куркумина из экстракта корневищ Curcuma longa L., вводили per rectum каждые 12 ч в течение 7 сут. Клинический статус оценивали по модифицированной шкале Disease activity index (DAI). Морфометрически в стенке кишечника оценивали размер язвенного дефект, выраженность клеточной инфильтрации, рассчитывали индекс тканевого повреждения (tissue damage index, TDI), оценивали экспрессию МПО и TNF-α. Результаты. При экспериментальной БК на 3-и, 5-е и 7-е сут наблюдения в толстом кишечнике обнаруживались язвенные дефекты, выраженный отек тканей, плотная нейтрофильно-лимфоцитарная инифильтрации с примесью эозинофилов, плазмоцитов, гистиоцитов, фибробластов, наблюдалось формирование грануляционной ткани. Индекс тканевого повреждения возрастал, повышалась экспрессия МПО и TNF-α. Размер язвенных дефектов, выраженность инфильтрации, индекс тканевого повреждения соответствовали клинической картине и индексу DAI. Применение оригинальных ректальных суппозиториев с экстрактом куркумы (0,075 мг) приводит к снижению выраженности клинических и морфологических признаков заболевания, максимальный эффект отмечен на 5-е и 7-е сут наблюдения. Заключение. Установленные протекторные свойства куркумина в составе оригинальных ректальных суппозиториев при БК на доклиническом этапе расширяют современные представления о плейотропных эффектах экстракта куркумы и являются предпосылкой для проведения дальнейших исследований и внедрения новой лекарственной формы в клиническую практику. The development of safe, new, pathogenetically justified medicines, mainly with local effects and of endogenous or plant origin, is of great interest for treatment of Crohn’s disease (CD). In this regard, an extract of rhizomes of Curcuma longa, containing a curcuminoid complex with pleiotropic effects, has attracted attention. We showed previously that having turmeric extract in suppositories for treatment of experimental CD would produce immunomodulatory and antioxidant effects. This suggests that turmeric extract affects the morphology of the CD intestinal lesion. The aim of this work was to study the effect of turmeric extract in the composition of novel rectal suppositories on the morphology and expression of myeloperoxidase (MPO) and TNF- α in colon lesions of experimental CD. Methods. Experiments were performed on 49 sexually mature male Wistar rats. CD was modeled by administration per rectum of an alcohol solution of trinitrobenzenesulfonic acid. The newly formulated rectal suppositories weighing 300 mg and based on polyethylene glycols of various molecular weights and containing 0.075 mg of curcumin obtained from Curcuma longa L. rhizome extract were administered per rectum every 12 hours for 7 days. The clinical status was assessed with the modified disease activity index (DAI) scale. The ulcer defect diameter, cell infiltration, tissue damage index (TDI), MPO expression, and TNF-α expression were evaluated. Results. In experimental CD on the 3rd, 5th, and 7th days of observation, a morphometric assessment of the lesion in the large intestine revealed the presence of ulcerative defects, edema, thick neutrophilic lymphocytic infiltration with an admixture of eosinophils, plasmocytes, histiocytes, fibroblasts, the formation of granulation tissue, increased TDI, and increased expression of MPO and TNF-α. The size of ulcerative defects, the degree of wall infiltration by neutrophils, lymphocytes, plasmocytes, histiocytes, and fibroblasts were recorded. The TDI, the expression of MPO, and TNF-α were associated with the DAI. The use of the novel rectal suppositories produced a maximal effect on the 5th and 7th days of observation. The severity of clinical and morphological signs of the disease were reduced. These signs included ulcer size, TDI, intestinal wall infiltration with neutrophils, lymphocytes, eosinophils, plasmocytes, histiocytes, and fibroblasts, and expression of MPO and TNF-α. Conclusion. The results supplement available data on the pathogenesis, the role of the expression of MPO and TNF-α, and the morphology of the lesion in the clinical status in TNBS-induced CD in rats. The demonstrated, protective properties of curcumin in the composition of the novel rectal suppositories at the preclinical stage of CD expand modern understanding of the pleiotropic effects of turmeric extract and are a prerequisite for further research and the introduction of a new dosage form in clinical practice.

Immunotropic effects of vitamin D3 in original rectal suppositories in experimental ulcerative colitis

Increased incidence of ulcerative colitis (UC) is a prerequisite for searching new therapeutic approaches, primarily with an opportunity of site-directed impact on the colon lesion. UC pathogenesis is associated with dysregulated immune response, and limited effectiveness of basic therapy for the disorder. Vitamin D3 exhibits antioxidant, anti-inflammatory, immunomodulatory and other properties, it has been shown to be effective in some autoimmune diseases, thus prompting us to study its effect on immune status in UC. We aimed for studying the effect of vitamin D3, as a component of original rectal suppositories, upon clinical course and indexes of immune status in experimental UC. UC in rats was modeled with 3% oxazolone solution. The vitamin D3-containing suppositories (1500 IU) weighing 300 mg were administered per rectum every 12 hours for 6 days. On days 2, 4 and 6 of UC, the clinical features were assessed as well as blood leukocyte counts, numbers of CD3+, CD45RA+; absorbing and NBT-reducing abilities of blood neutrophils were determined; IgM, IgG, IL-6 and IL-8 concentrations in serum were also studied.The DAI index increased in non-treated UC, along with raised neutrophil numbers in blood, their absorption and NBT-reducing activity was also increased, the total number of lymphocytes, including CD3+, CD45RA+ became higher, serum concentrations of IgM, IgG, IL-6, IL-8 increased. Local use of vitamin D3 in UC reduces DAI parameters, causes decrease in blood neutrophil counts, reducing and partially restoring absorptive and NBT-reducing abilities of neutrophils, decline of total lymphocyte counts in blood, partially restoring the CD3+ and CD45RA+ numbers, causing decline and partial restoration of serum IgM, IgG, IL-6, IL-8 concentrations. An association between clinical signs and indexes of immune status in UC was established under the conditions of vitamin D3 use. Conclusions: The protective effect of vitamin D3 in UC can be mediated by its antioxidant effect, changes in production of immunoregulatory cytokines, modulation of Th1-, Th2-, Th17-dependent reactions and Treg activity, being a pre-requisite for further studies to clarify the mechanism of vitamin D3 immunotropic action in UC,with an opportunity of using it in clinical practice.