Abivertinib inhibits megakaryocyte differentiation and platelet biogenesis / 医学前沿

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton's tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34+ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34+ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294

ABSTRACT Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4). Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

Palbociclib induces cell cycle arrest and senescence of human renal tubular epithelial cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of palbociclib on cell cycle progression and proliferation of human renal tubular epithelial cells.@*METHODS@#Human renal tubular epithelial cell line HK-2 was treated with 1, 5, 10, and 20 μmol/L of palbociclib, and the changes in cell proliferation and viability were examined by cell counting and CCK8 assay. EDU staining was used to assess the proliferation of HK-2 cells following palbiciclib treatment at different concentrations for 5 days. The effect of palbociclib on cell cycle distribution of HK-2 cells was evaluated using flow cytometry. SA-β-Gal staining and C12FDG senescence staining were used to detect senescence phenotypes of HK-2 cells after palbociclib treatment at different concentrations for 5 days. The relative mRNA expression levels of P16, P21, and P53 and the genes associated with senescence-related secretion phenotypes were detected by RT-PCR, and the protein expressions of P16, P21 and P53 were detected by Western blotting.@*RESULTS@#Palbociclib inhibited HK-2 cell proliferation and induced cell cycle arrest in G1 phase. Compared with the control cells, HK-2 cells treated with high-dose (10 μmol/L) palbociclib exhibited significantly suppressed cell proliferation activity, and the inhibitory effect was the most obvious on day 5 (@*CONCLUSIONS@#Palbociclib induces HK-2 cell senescence by causing cell growth arrest and delaying cell cycle progression.

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Total testosterone levels and the effect of sildenafil on type 2 diabetics with erectile dysfunction

Hypotestosteronemia has been reported in approximately half of type 2 diabetic men in general. This study aims to assess serum total testosterone levels in type 2 diabetics with erectile dysfunction and to correlate the degree of improvement between sildenafil citrate and testosterone levels. A cross sectional and prospective comparative interventional study was conducted at the Diabetic Clinic of Assalam Teaching Hospital in Mosul, during the period from January 1, 2009 through to December 31, 2011. The study enrolled 120 type 2 diabetic males with erectile dysfunction who were analyzed with regard to age, duration of diabetes, duration and severity of erectile dysfunction, serum total testosteron levels and the degree of response to sildenafil citrate in terms of testosterone levels. The data were statistically analyzed using the independent two-sample Student t test, X[2] test and Pearson correlation test. A jp-value of <0.05 was considered statistically significant. Thirty six percent of type 2 diabetic males with erectile dysfunction were found to have low serum testosterone levels. Hie hypotestosteronemic and normotestosteronemic subgroups were not significantly different in terms of mean age, duration of diabetes, reduction of libido, and reduction in erectile function. The rate and the degree of improvement of erection by sildenafil in the normo-and-hypotestosteronemic respondents were not significantly different, but the degree of improvement by sildenafil was significantly correlated to testosterone levels among the hypotestosteronemic group. Hypotestosteronemia was found in 36% of type 2 diabetic males with erectile dysfunction. The degree of improvement of erectile dysfunction by sildenafil was directly proportional to the serum testosterone levels among the hypotestosteronemic group. Therapeutic supplement with testosterone preparation in the hypotestosteronemic diabetics with erectile dysfunction may improve their response to sildenafil

Imatinib activity on Schistosoma mansoni

Imatinib, a drug used for treatment of human chronic myeloid leukaemia, due to its activity against protein kinases, has been also evaluated in vitro against Schistosoma mansoni showing high schistosomicidal activity. In the present experiments imatinib activity in vitro was confirmed at the doses of 25 µM, 50 µM and 100 µM. The first drug activity observed with the lower dose was interruption of egg-laying and with the higher dosages was the death of the worms. In mice infected with S. mansoni no activity was found even with 1,000 mg/kg/day, 500 mg/kg/day, single oral dose or when administered for three consecutive days. This is another example of the difference of results related to in vitro and in vivo trials using S. mansoni worms.

Receptores plaquetários P2Y12: importância na intervenção coronariana percutânea / P2Y12 platelet receptors: importance in percutaneous coronary intervention

As plaquetas estão envolvidas em vários processos biológicos, desde o combate a agentes infecciosos até a coordenação do controle da permeabilidade vascular e angiogênese. Entretanto, o seu principal foco de ação consiste na modulação da cascata de coagulação. A intervenção coronariana percutânea é um procedimento com alto risco trombogênico, que induz a ativação plaquetária e de monócitos, devido à lesão direta do endotélio e pelo contato de estruturas trombogênicas com o sangue, levando ao aumento da atividade inflamatória, tanto no local do dano vascular coronariano como de forma sistêmica. Os receptores plaquetários P2Y12 desempenham papel central na amplificação da agregação induzida por todos os agonistas plaquetários, como a adenosina difosfato, o colágeno, tromboxano A2, adrenalina e serotonina. Por esse motivo, têm sido o principal alvo das drogas antiplaquetárias. Apesar de atuarem no mesmo receptor, características farmacocinéticas e farmacodinâmicas distintas conferem peculiaridades a cada agente.

Apart from their role in hemostasis and thrombosis, platelets are involved in many other biological processes such as wound healing and angiogenesis. Percutaneous coronary intervention is a highly thrombogenic procedure inducing platelets and monocytes activation through endothelial trauma and contact activation by intravascular devices. Platelet P2Y12 receptor activation by adenosine diphosphate facilitates non-ADP agonist-mediated platelet aggregation, dense granule secretion, procoagulant activity, and the phosphorylation of several intraplatelet proteins, making it an ideal drug target. However, not all compounds that target the P2Y12 receptor have similar efficacy and safety profiles. Despite targeting the same receptor, the unique pharmacologic properties of each of these P2Y12 receptor-directed compounds can lead to very different clinical effects.

Effect of acute administration of sildenafil to rats with detrusor overactivity induced by chronic deficiency of nitric oxide

Purpose Recently, the effect of phosphodiesterase inhibitors (PDE5i) in the lower urinary tract symptoms (LUTS) associated to benign prostatic hyperplasia have been studied thoroughly. However, it remains unclear how the PDE5i improve LUTS. Therefore, the aim of the present study was to evaluate the potential of acute administration of the PDE5i sildenafil to improve detrusor overactivity (DO) induced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), an nitric oxide sinthase (NOS) inhibitor, in rats. Materials and Methods Twenty-seven MALE adult Wistar Rats were divided into the following groups: (1) control, (2) L-NAME, (3) sildenafil alone, and (4) L-NAME + sildenafil. The NOS blocker L-NAME (20 mg/rat/day) was given in the drinking water. Sildenafil (100µg/kg) was administrated intravenously (i.v.) acutely, diluted in cremophor, propylene glycol and water. All animals underwent to anesthetized cystometograms. Results The chronic and systemic administration of L-NAME markedly increased the number of non voiding contractions (2.62 (± 0.89)), and frequency of micturition (1.97 (± 0.78)), as well increased volume threshold (2.83 mL (± 1.64)) compared with control group, the number of non voiding contractions (1.17 (± 0.75)), frequency of micturition (1.08 (± 0.65)) and volume threshold (1.16 mL (± 0.38)), p < 0.001, p = 0.01, and p = 0.04, respectively. Sildenafil infusion decreased the number of micturition cycles significantly from the baseline to end point (-0.93 (± 0.34)) in nitric oxide (NO) deficient animals compared with sildenafil infusion alone (control) in animals with normal NO level (0.13 (± 0.25)), p = 0.03. Conclusion Systemic reduction of nitric oxide causes detrusor overactivity and acute infusion of sildenafil reduces the number of micturition cycles in chronic NO-deficient rats. .

Sildenafil citrate protects skeletal muscle of ischemia-reperfusion injury: immunohistochemical study in rat model

PURPOSE: To investigate the effect of sildenafil citrate (SC) on skeletal muscle ischemia-reperfusion (IR) injury in rats. METHODS: Adult male Wistar rats were randomized into three groups: vehicle-treated control (CTG), sildenafil citrate-treated (SCG), and sham group (SG). CTG and SCG had femoral artery occluded for 6 hours. Saline or 1 mg/kg of SC was given 5.5 hours after occlusion. SG had a similar procedure without artery occlusion. Soleus muscle samples were acquired 4 or 24h after the reperfusion. Immunohistochemistry caspase-3 analysis was used to estimate apoptosis using the apoptotic ratio (computed as positive/negative cells). Wilcoxon rank-sum or Kruskal-Wallis tests were used to assess differences among groups. RESULTS: Eighteen animals were included in the 4h reperfusion groups and 21 animals in the 24h reperfusion groups. The mean apoptotic ratio was 0.18±0.1 for the total cohort; 0.14±0.06 for the 4h reperfusion groups and 0.19±0.08 for the 24h groups (p<0.05). The SCG had lower caspase-3 ratio compared to the control groups at the 24h reperfusion time point (p<0.05). CONCLUSION: Sildenafil citrate administration after the onset of the ischemic injury reduces IR-induced cellular damage in skeletal muscle in this rat hindlimb ischemia model.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Evaluation of the effect of sildenafil and/or doxazosin on Benign prostatic hyperplasia-related lower urinary tract symptoms and erectile dysfunction

To verify the association between lower urinary tract symptoms [LUTS] and erectile dysfunction [ED] and evaluate the influence of sildenafil and doxazosin either as single agents or combined on both symptoms. A prospective randomized study including 150 patients presented with LUTS caused by BPH in association with clinically diagnosed ED, with age equal or more than 45 years from April 2010 to April 20011. They were categorized into three comparative groups each one containing 50 patients. These groups were comparable regarding pretreatment international prostate symptoms score [IPSS] and international index of erectile function [IIEF]. The patients of the first group were given sildenafil 50 mg as monotherapy, those of the second group were given doxazosin 2 mg and those of the third group were given combination of both drugs for 4 months for each group. The main post-treatment parameters for assessment and comparison include assessment of patient's symptoms by repeated IPSS and IIEF, uroflowmetry and assessment of PVR. The statistics was done by use of the chi-square test Pre-treatment parameters were assessed and compared between the three groups. After 4 months of treatment, the comparative parameters were applied to all groups and the differences were measured post-treatment regarding IPSS, erectile function score, uroflowmetry, and post-void residual [PVR] urine. Sildenafil alone caused mild improvement in IPSS, more improvement in IIEF score, and little effect on flow rate and PVR urine. Doxazosin alone caused more improvement in IPSS, flow rate and PVR urine and less improvement in IIEF score. A combination of both sildenafil and doxazosin caused more improvement in all of the comparative parameters than when each drug was given alone. There is a strong relationship between LUTS and ED. Doxazosin or sidenafil as a single drug could be used in treating mild or mild to moderate symptoms but more severe symptoms may usually need a combination of both drugs

Antimicrobial effect of para-alkoxyphenylcarbamic acid esters containing substituted N-phenylpiperazine moiety

In current research, nine basic esters of para-alkoxyphenylcarbamic acid with incorporated 4-(4fluoro-/3-trifluoromethylphenyl)piperazin-1-yl fragment, 6i-6m and 8f-8i, were screened for their in vitro antimicrobial activity against Candida albicans, Staphylococcus aureus and Escherichia coli, respectively. Taking into account the minimum inhibitory concentration assay (MIC), as the most active against given yeast was evaluated 8i (MIC = 0.20 mg/mL), the most lipophilic structure containing para-butoxy and trifluoromethyl substituents. Investigating the efficiency of the compounds bearing only a single atom of fluorine and appropriate para-alkoxy side chain against Candida albicans, the cut-off effect was observed. From evaluated homological series, the maximum of the effectiveness was noticed for the stucture 6 k (MIC = 0.39 mg/mL), containing para-propoxy group attached to phenylcarbamoyloxy fragment, beyond which the compounds ceased to be active. On the contrary, all the tested molecules were against Staphylococcus aureus and Escherichia coli (MICs > 1.00 mg/mL) practically inactive.

Imatinib atenua a fibrose miocárdica em associação com a inibição da atividade do PDGFRα / Imatinib attenuates myocardial fibrosis in association with inhibition of the PDGFRα activity

FUNDAMENTO: O Imatinib é um inibidor do receptor tirosina-quinase que foi confirmada como exercendo um efeito inibidor sobre a atividade do receptor do PDGF, fator de crescimento plaquetário (PDGFRα e PDGFRβ). OBJETIVO: Investigar o efeito protetor do Imatinib na fibrose miocárdica em acetato de deoxicorticosterona (DOCA)/ratos com hipertensão induzida por sal. MÉTODOS: Sessenta ratos Sprague-Dawley machos, uninefrectomizados foram distribuídos em três grupos: ratos controles (grupo CON): grupo deoxicorticosterona (grupo DOCA); grupo deoxicorticosterona e Imatinib (grupo DOCA IMA). A Pressão Arterial Sistólica (PAS) foi medida quinzenalmente. Foi estudada a porção apical do ventrículo esquerdo. Foram empregados: coloração vermelho sirius, coloração de hematoxilina-eosina, imuno-histoquímica e ensaio de western blot. RESULTADOS: A PAS nos grupos DOCA e IMA+DOCA foi maior que no grupo CON nos dias 14 e 28. Os animais do grupo DOCA apresentaram fibrose intersticial e perivascular grave no dia 28, e as expressões de PI, PIII, tenascina-C e fibronectina foram significativamente maiores que nos grupos DOCA+IMA e CON. Quando comparados com o grupo CON, os grupos DOCA e DOCA+IMA apresentaram resposta inflamatória de tecido miocárdico e infiltração de monócitos/macrófagos de diferentes graus. As expressões proteicas do PDGF-A, PDGF-C e PDGFRα foram significativamente maiores nos grupos DOCA e DOCA+IMA que no grupo CON, mas a expressão proteica do p-PDGFRα no grupo DOCA+IMA foi menor que no DOCA. CONCLUSÃO: O Imatinib pode exercer efeitos inibitórios sobre a fibrose miocárdica em ratos com hipertensão induzida por DOCA/sal, os quais podem ser atribuídos à inibição da atividade do PDGFR-α.

BACKGROUND: Imatinib is a tyrosine kinase receptor inhibitor that has been confirmed to exert inhibitory effect on the platelet derived growth factor PDGF receptor (PDGFRα and PDGFRβ) activity. OBJECTIVE: To investigate the protective effect of imatinib on the myocardial fibrosis in deoxycorticosterone-acetate (DOCA)/salt induced hypertensive rats. METHODS: Sixty male uninephrectomized Sprague-Dawley rats were assigned to three groups: control rats (CON group); deoxycorticosterone group (DOCA group); deoxycorticosterone and imatinib group (DOCA+IMA group). Systolic blood pressure (SBP) was measured biweekly. The apical portion of the left ventricle was studied. Sirius-Red staining, Hematoxylin-Eosin staining, immunohistochemistry and Western blot assay were employed. RESULTS: SBP in the DOCA group and DOCA+IMA group was higher than that in the CON group on day 14 and 28. Animals in the DOCA group showed severe interstitial and perivascular fibrosis on day 28, and the expressions of PI, PIII, tenascin-C and fibronectin were significantly higher than those in the DOCA+IMA group and CON group. When compared with the CON group, myocardial tissue inflammatory response and monocyte/macrophage infiltration of different degrees were observed in the DOCA group and DOCA+IMA group. Protein expressions of PDGF-A, PDGF-C and PDGFRα were signiflcantly higher in the DOCA and DOCA+IMA groups than those in the CON group, but the p-PDGFRα protein expression in the DOCA+IMA group was lower than that in the DOCA group. CONCLUSION: Imatinib can exert inhibitory effects on myocardial fibrosis in DOCA/salt induced hypertensive rats, which may be attributed to the inhibition of PDGFR-α activity.

Nilotinib as first-line therapy for chronic myeloid leukemia.

Tyrosine Kinase Inhibitors brought a revolution in the management of chronic myeloid leukemia. Long term disease free survival became a reality for the majority of patients. With the identification of imatinib resistance and its implications, roles of newer targeted therapy molecules came into focus. Nilotinib data has matured and shows the fulfillment of earlier promise - even in first line therapy. This review provides insight into the place of this molecule in the first line management of chronic myeloid leukemia.

Influence of sildenafil and buflomedil on survival of randomized flaps in rats: an experimental study / Influência do buflomedil e do sildenafil na sobrevivência de retalhos randomizados em ratos: estudo experimental

BACKGROUND: Microcirculation dysfunction, as a consequence of localized vascular insufficiency, is considered to be one of the dominant causes of surgical flap necrosis. Several vasoactive drugs have been tested for the pharmacological treatment of tissue ischemia, with varying degrees of success. This study aimed to assess the impact of buflomedil and sildenafil on the viability of random skin flaps in rats. METHODS: Caudally pedicled skin flaps (10 x 3 cm) were created on the backs of rats. The animals were randomly assigned, in groups of 10, to three treatment groups: one group served as the vehicle control group, one group received buflomedil (10 mg/kg/d, orally), and a third group received the same dosage of sildenafil. Following seven days of dosing, the animals were sacrificed, and the viable flap area was determined. RESULTS: The average viable flap area for each group was: 16.2 ± 3.56 cm² (control group), 17.69 ± 2.54 cm² (buflomedil group), and 18.28 ± 3.74 cm² (sildenafil group). Data analysis by the Kruskal-Wallis test failed to show a statistically significant difference between the three groups. CONCLUSIONS: Neither buflomedil nor sildenafil showed a reduction in the necrotic area of random skin flaps in rats.

INTRODUÇÃO: A insuficiência no aporte sanguíneo e a consequente disfunção gerada no fluxo da microcirculação são consideradas causas dominantes de sofrimento de um retalho cirúrgico. Várias drogas vasoativas têm sido testadas para o tratamento farmacológico da isquemia tecidual, porém com graus variáveis de sucesso. Este estudo teve como objetivo avaliar a influência do buflomedil e do sildenafil na viabilidade de retalhos cutâneos ao acaso, em ratos. MÉTODO: Foram confeccionados retalhos cutâneos no dorso de ratos, com dimensões de 10 x 3 cm e base caudal. Foram utilizados 30 ratos, divididos em três grupos de 10 ratos cada: um grupo que recebeu apenas o veículo da solução (grupo controle); um grupo que recebeu buflomedil (grupo buflomedil); e um terceiro grupo que recebeu sildenafil (grupo sildenafil). A via de administração foi a oral e a dose foi de 10 mg/kg/dia para cada droga, durante sete dias. Ao final desse período, os animais foram sacrificados, sendo realizada a determinação das áreas viáveis dos retalhos. RESULTADOS: A média das áreas viáveis dos retalhos foi de 16,2 ± 3,56 cm² para o grupo controle, de 17,69 ± 2,54 cm² para o grupo buflomedil, e de 18,28 ± 3,74 cm² para o grupo sildenafil. A análise dos dados pelo teste de Kruskal-Wallis não demonstrou significância estatística entre os três grupos. CONCLUSÕES: A utilização do buflomedil e do sildenafil demonstrou não diminuir a área de necrose de retalhos randomizados em ratos.

Effects of sildenafil on the viability of random skin flaps / Os efeitos do sildenafil na viabilidade de retalhos cutâneos randômicos

PURPOSE: To assess the viability of McFarlane skin flaps in rats with administration of sildenafil. METHODS: Twenty Wistar rats were distributed into two groups: Control (dorsal skin flap, subdermal application of saline solution at 0.9 percent) and Study (dorsal skin flap, subdermal application of sildenafil). Seven days after the surgery, flaps were photographed and graphically rendered. Then, they were analyzed with AutoCAD software. Three biopsies (proximal, medial and distal) of each flap were collected for histological analysis. RESULTS: Macroscopic analysis showed that animals of the study group had greater necrotic areas (p=0.003) in the dorsal skin flaps. Additionally, histological analysis of the distal third of these flaps showed a tendency to less granulated tissue formation in animals treated with sildenafil. CONCLUSION: Sildenafil subdermally was associated with lower viability of the random skin flap in rats.

OBJETIVO: Avaliar a viabilidade de retalhos cutâneos de ratos à McFarlane após a administração de sildenafil. MÉTODOS: Vinte ratos Wistar foram distribuídos em dois grupos: Controle (confecção do retalho cutâneo dorsal, aplicação subdérmica de solução salina a 0,9 por cento) e Estudo (confecção do retalho cutâneo dorsal, aplicação subdérmica de sildenafil). Sete dias após a operação, os retalhos foram fotografados e representados graficamente, para serem analisados com o programa AutoCad. Três biópsias (cranial, média e caudal) foram coletadas de cada retalho, para análise histológica. RESULTADOS: A análise macroscópica evidenciou que os animais do grupo Estudo apresentaram maiores áreas de necrose (p=0,003) nos retalhos cutâneos dorsais. Além disso, a análise histológica dos terços distais dos retalhos mostrou uma tendência à formação de menos tecido de granulação nos animais que receberam o sildenafil. CONCLUSÃO: O sildenafil subdérmico esteve associado com uma pior viabilidade tecidual dos retalhos cutâneos dorsais de ratos.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Estudio in vivo del efecto angiogénico de inhibidor de fosfodiesterasa para aplicación posterior en injerto dermoepidérmico / Angiogenic effect of sildenafil in a chicken egg model

Background: Angiogenesis is a complex process. Phosphodiesterase inhibitors may have a direct angiogenic effect. Aim: To determine if phosphodiesterase inhibitors have angiogenic properties, using a chicken egg model. Material and Methods: We used 44 fertilized chicken eggs. A methylcellulose filter was placed over their allantocorionic membrane. This preparation was instilled with different solutions. Group A (Control) received 30 u.1 of saline solution, Group B, C and D received 30 jul of a solution made of saline solution and sildenafil at different concentrations of 0.33, 1 and 3.3 u-g/ul, respectively. At day 12 the filters were removed, prepared for histologic analysis, and the number of capillaries in an area of 2250 urn² were blindly counted. Statistical analysis was made using variance analysis (ANOVA) with Bonferroni technique (p < 0.001). Results: The number of capillaries counted, per 2250 urn², in Groups A, B, C, and D were 11.1 +/- 0.5, 15.4 +/- 1.2, 16.6 +/- 0.8 and 19.2 +/- 0.9, respectively. The number of capillaries of groups B, C and D were significantly higher than those of group A (control). Moreover, there was a linear relationship between the number of capillaries and sildenafil dose (p < 0.001). Conclusions: In this experiment, sildenafil had a potent angiogenic effect.

Introducción: El proceso de angiogenesis es un proceso complejo. El uso de factores proangiogénicos está bien establecido. En este estudio se trató de averiguar si los inhibidores de fosfodiesterasa, además de su rol vasodilatador, tendrían un efecto angiogénico en los tejidos para evaluar su potencial uso terapéutico futuro en injerto dermoepidérmico. Hipótesis: Se plantea como hipótesis que el inhibidor de fosfodiesterasa tiene un efecto angiogénico directo. Material y Método: Se utilizaron 44 huevos de pollo fecundados obtenidos del Instituto de Salud Pública (ISP), a los cuales se les implantó un disco de metilcelulosa sobre la membrana alantocoriónica, a los que luego se les instiló distintas soluciones: Grupo A control, se instiló 30li1 de solución fisiológica. Grupo B, C y D 30li1 de solución con Citrato de Sildenafil a una concentración de 0,33 Lig/u.1, 1 ug/Lil y 3,3 ug/ul respectivamente. Al día 12 se removieron los discos y se fijaron para análisis histológico y se contaron de manera ciega los capilares en área de 2.250 um². Análisis estadístico con método de análisis de varianza (ANOVA) con técnica de Bonferroni (p < 0,001). Resultados: En Grupo A control, existía un promedio de 11,09 capilares/2.250um² DS 0,52. Grupo B 15,35 capilares/2.250 um² DS 1,19. Grupo C 16,62 capilares/2.250 Lim² DS 0,82. Grupo D 19,2 capilares/2.250 um² DS 0,89. Se encontró que el numero de capilares era significativamente mayor en los Grupos B, C y D en relación a control (p < 0,001). Además se observó diferencias estadísticamente significativas entre todos los grupos que recibieron tratamiento con dosis progresivas del Citrato de Sildenafil (p < 0,001). Conclusión: Se observó un efecto angiogénico del inhibidor de fosfodiesterasa utilizado, lo que podría ser aplicado en modelo para estudiar angiogénesis en injertos.