Anti-oxidative effect of the tyrosine kinase inhibitor nintedanib: a potential therapy for chronic lung allograft dysfunction?

Background: The long-term survival after lung transplantation (LTx) is often limited by the development of chronic lung allograft dysfunction (CLAD). Increased oxidative stress has been found to occur in chronic lung allograft dysfunction because of several risk factors, e.g. immunological factors or drug related factors. The aim of this study was to investigate the anti-oxidative effect of the receptor tyrosine kinase (RTK) inhibitor nintedanib on immunologically induced oxidative stress and on drug induced oxidative stress.Methods: In-vivo studies were used for investigation of immunologically induced oxidative stress: Immunohistochemistry of transglutaminase-2 (TGM-2) was used to figure out a potential anti-oxidative effect of receptor tyrosine kinase inhibitor nintedanib in a rat model of allogeneic left LTx. In-vitro studies were used for investigation of drug induced oxidative stress: Cell viability assay, 2'7'-dichlorodihydrofluorescein diacetate (DCFDA) and immunofluorescence of transglutaminase-2 were disposed to examine the potential impact of nintedanib on cyclosporin A (CsA) treated lung fibroblasts of the rat.Results: In-vivo studies: Allogeneic transplanted animals without drug interaction showed severe chronic rejection and an excessive expression of TGM-2, whereas the application of nintedanib significantly decreased the number of TGM-2 positive cells. In-vitro studies: Concentrations of CsA ranging from 250 ng/ml to 500 ng/ml demonstrated oxidative stress caused by an increased production of reactive oxygen species (ROS) and an overexpression of TGM-2 without inducing apoptosis in cells. Concentrations of more than 1000 ng/ml led to a considerable decrease of cellularity. 30 min-pre-incubation with nintedanib at a concentration between 25 and 100 nM reduced generation of intracellular ROS and expression of TGM-2.Conclusion: These results demonstrate a downregulation of ROS and TGM-2 by pretreatment with the receptor tyrosine kinase inhibitor nintedanib and present its potential anti-oxidative and immunomodulatory effect in the treatment of chronic lung allograft dysfunction.

Synergism of imatinib, vatalanib and everolimus in the prevention of chronic lung allograft rejection after lung transplantation (LTx) in rats.

Chronic lung allograft dysfunction (CLAD) still remains a major drawback in the outcome following lung transplantation (LTx). New therapeutic strategies are warranted. Growth factors and their receptors like platelet-derived growth factor-receptor (PDGFR) and vascular endothelial growth factor-receptor (VEGFR), may play a crucial role in the development of CLAD, especially bronchiolitis obliterans (BO) and vasculopathy. In this study, we used an orthotopic left lung transplantation model from Fischer (F344) to Wystar Kyoto (WKY) rats to investigate the effect of the receptor tyrosine kinase inhibitor (RTKI) vatalanib alone, the dual combination of vatalanib and imatinib and a triple therapy consisting of vatalanib, imatinib and the mammalian target of rapamycin inhibitor (mTORI) everolimus on the development of CLAD after LTx in rats. With this trial we demonstrated that monotherapy with vatalanib attenuated mild and severe chronic vascular rejection, whereas dual therapy (vatalanib and imatinib) after LTx also showed a significant reduction of chronic bronchiolar rejection and interstitial fibrosis. By adding everolimus, the effect of vatalanib and imatinib could additionally be increased. In conclusion, the combination of mTORI and RTKIs might be a possible strategy in the prevention of CLAD and BO.

The activity of nintedanib in an animal model of allogenic left lung transplantation resembling aspects of allograft rejection.

AIM OF THE STUDY: The prevention and treatment of chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx) remain unsatisfactory. Growth factors may play an important role in the development of CLAD. This study evaluated the effects of nintedanib, a receptor tyrosine kinase inhibitor, in the treatment of CLAD after experimental LTx. MATERIALS AND METHODS: A rat model of left lung allo-transplantation (Fisher 344 to Wistar Kyoto) was used to evaluate the effect of nintedanib (50 mg/kg per day) on the development of CLAD. Therapy with nintedanib began 2 days before LTx and ended on postoperative day (POD) 20 (n = 6) or 60 (n = 6). Nontreated animals who underwent LTx (n = 12) were used as controls, whereas naïve lungs (n = 24) served as reference for physiological healthy organs without transplantation damage or medical effects. Acute and chronic rejection were evaluated on POD 20 and 60, respectively. RESULTS: Immunohistologic analysis showed a decrease in growth factors/receptors on POD 60 (nintedanib-treated vs. nontreated controls: platelet-derived growth factor (PDGF) A: [P ≤ 0.001]; PDGF receptor-α: [P ≤ 0.001]; vascular endothelial growth factor (VEGF) A: [P ≤ 0.001]; VEGF receptor-2: [P ≤ 0.001]). However, no reductions in fibrotic changes were observed in nintedanib-treated allografts compared with nontreated allografts. Although nintedanib treatment started before LTx none of the animals showed impaired wound healing. No dehiscence of the sutures of the bronchus, vessels or skin, or stenosis of the bronchus was found. CONCLUSION: In conclusion, while nintedanib reduced the expression of growth factors/receptors in a rat LTx model, a reduction in fibrotic alterations was not observed at POD 60.

Everolimus's influence on persistent acute rejection after experimental lung transplantation.

BACKGROUND: In lung transplantation, acute rejection episodes increase the risk of chronic rejection. Therefore treatment of acute rejection needs to be optimized for better long-term outcome of lung-transplantation and patient survival. OBJECTIVES: The aim was to verify whether an inhibitor of the mammalian target of rapamycin (Everolimus) contained the extent of persistent acute rejection after left lung allo-transplantation in rats. MATERIAL AND METHODS: Rats (F344-to-WKY) with a high grade of acute rejection were treated with methylprednisolone (10mg/kg, postoperative days 14-16) alone or in combination with everolimus (2.5 mg/kg, postoperative days 14-30). The rats were killed on postoperative day 20 and 30. Infiltration of inflammatory cells (ED1, CD11a, CD18) and activation of endothelial cells (ICAM-1) were measured by immunohistochemistry RESULTS: Everolimus treatment significantly reduced the number of ICAM-1 positive small vessels (66%; p<0.05) and suppressed the infiltration of leucocytes (CD11a (64%), CD18 (42%); p<0.05) and macrophages (ED1; 22%) in the allografts on POD 30. Despite this clear anti-inflammatory effects, lung allografts still showed severe acute vascular rejection in combination with high grade small airway inflammation. CONCLUSIONS: The shown anti-inflammatory effects of Everolimus could not delay the progression of acute rejection in rat lung allografts.

Pharmacokinetics of a self-microemulsifying drug delivery system of tacrolimus.

This study evaluated the pharmacokinetics of tacrolimus (Tac) in a novel self-microemulsifying drug delivery system (SMEDDS) for improved oral administration. SMEDDS Tac consisted of ethyl oleate as the oily phase, Solutol HS 15 as the surfactant and glycofurol as the co-surfactant and contained 0.5mg/mL tacrolimus. Blood and tissue concentrations of tacrolimus from two study groups (oral application of SMEDDS Tac and Prograf®) were determined using ELISA technique following tacrolimus administration in rats. There was no difference between area under the whole blood concentration-time curve in the SEDDM Tac group and the Prograf® group. Maximum concentrations of the drug were three times higher (P<0.05) in the SEDDM Tac group accompanied by a 3-fold earlier peak time. Elimination half-life was significantly lower in the SEDDM Tac group. Application of SMEDDS Tac increased tissue accumulation. Already after 15 min, Tac levels of small intestine, liver, kidney, spleen, heart and bone marrow were significantly higher in the SMEDDS Tac group than in the Prograf® group (P<0.05). However, the Tac concentration in the kidney was significantly lower in the SMEDDS Tac group. Formulation of SMEDDS did not affect blood-brain barrier function. The SMEDDS is a potentially useful method for a local delivery of Tac to target organs. The selection of the optimum SMEDDS Tac composition might have advantage as an alternative oral dosage form for Tac.

Impact of minimized extracorporeal circulation on outcome in patients with preoperative anemia undergoing coronary artery bypass surgery.

Preoperative anemia and low hematocrit during cardiopulmonary bypass have been associated with worse outcome in patients undergoing cardiac surgery. The minimized extracorporeal circulation (MECC) allows a reduction of the negative effects associated with conventional extracorporeal circulation (CECC). In this study, the impact of the MECC on outcome of anemic patients after coronary artery bypass grafting (CABG) was assessed. Between January 2004 and December 2011, 1,945 consecutive patients with preoperative anemia underwent isolated CABG using CECC (44.8%) or MECC (55.2%). The cutoff point for anemia was 13 g/dl for men and 12 g/dl for women. The postoperative creatine kinase and lactate levels were significantly lower in the MECC group (p < 0.001). There was no difference in postoperative blood loss between the groups. However, the intraoperative and postoperative transfusion requirements were significantly lower in the MECC group (p < 0.05). Furthermore, MECC patients had lower incidences of postoperative acute renal failure, and low cardiac output syndrome, shorter intensive care unit lengths of stay and reduced 30-day mortality (p < 0.05). In conclusion, a reduced postoperative mortality, lower transfusion requirements, and less renal and myocardial damage encourage the use of MECC for CABG, especially in the specific high-risk subgroup of patients with anemia.

Suppression of bronchiolitis obliterans in allogeneic rat lung transplantation--effectiveness of everolimus.

BACKGROUND: Chronic rejection (CR) after lung transplantation (LTX) manifests pathologically by fibrotic airway remodelling and bronchiolitis obliterans (BO). The role of the mammalian target of rapamycin inhibitor everolimus in preventing this process is poorly understood. METHODS: A rat model of left lung allo-transplantation (Fisher 344 to Wistar Kyoto) was used to analyze the effect of everolimus (2.5 mg/kg/day) on the development of CR. Drug therapy began on postoperative day (POD) 0, 7 and 14 characterizing different grade of acute rejection (AR) of the allograft before drug treatment. RESULTS: Non-treated recipients developed severe acute rejection (AR) and first signs of CR on POD 20 and a pronounced CR on POD 60. On POD 20, only application of everolimus from POD 0 to 60 significantly reduced acute inflammatory infiltration (p<0.001). Independent of treatment scheme, everolimus suppressed the development of early signs of chronic alterations (POD 20). However, neither early (POD 7-60) nor late (POD 14-60) application of everolimus affected the progression of CR (POD 60). Only its initial treatment (POD 0-60) inhibited the development of BO and vasculopathy (p<0.001). An additional finding was a decrease in body weight after drug application. CONCLUSION: The effectiveness of everolimus after rat LTX depended on the grade of inflammation of the allograft before initiation of drug treatment. Only allografts with no or low grade AR benefit from long-term treatment with everolilmus in the prevention of BO after LTX. It could be speculated that conversion to an everolimus-based immunosuppression after LTX might only be successful in patients free of BO.

Impact of the severity of acute rejection in the early phase after rat lung transplantation on the effectiveness of mycophenolate mofetil to treat chronic allograft rejection.

OBJECTIVES: Prevention and treatment of bronchiolitis obliterans (BO) after lung transplantation (LTX) remains disappointing. The high prevalence and the critical detection of early mild acute lung rejection aggravated optimum immunosuppressive therapy. In this study, two hypotheses were investigated: (i) mycophenolate mofetil (MMF) prevented the development of BO after rat LTX. (ii) The effectiveness of MMF depended on the degree of acute rejection (AR) at the time of drug initiation. METHODS: A rat model of left lung allo-transplantation (Fisher 344 to Wistar Kyoto) was used to evaluate the basic effect of MMF (30 mg/kg body weight per day) on the development BO. MMF therapy began on Days 0, 7 and 14 after transplantation characterizing different degree of AR of the allograft at the time of drug initiation and ended on Day 60 after transplantation. Non-treated animals were used as controls. Histopathological alterations were analysed according to the International Society for Heart and Lung Transplantation (ISHLT). RESULTS: MMF treatment of allografts with high-grade AR (ISHLT-A3-4/B2R; from Day 14 to 60) failed. All animals developed vascular fibrosis and BO. Promising long-term outcome was shown after treatment of mild to moderate acute rejecting of allografts (MMF from Day 7 to 60). The proportion of animals with high grade acute airway inflammation was significantly reduced (33%; P<0.05). All animals were free of BO and chronic vascular alterations (90%). MMF (Day 0-60) significantly reduced the proportion of animals with severe acute vascular rejection and high grade airway inflammation (17 and 0%, respectively; P<0.05, each). None of the animals treated with MMF from Day 0 to 60 developed chronic rejection (CR). CONCLUSIONS: MMF significantly reduced AR and CR after rat LTX. However, only allografts with no or mild AR at the time of drug initiation benefited from MMF treatment. In addition, the appearance of early signs of fibroproliferative alterations in the airway structures might prevent a successful long-term outcome.