Bernhard Burian

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

Background Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor. Objective To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC. Methods PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580. Results ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1. Conclusion and Clinical Relevance ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

Abnormal pulmonary arterial pressure limits exercise capacity in patients with COPD

ZusammenfassungZIELE: Das Vorliegen einer pulmonalen Hypertension ist häufig bei Patienten mit chronisch obstruktiver Lungenerkrankung (COPD) anzutreffen. Der mittlere pulmonalarterielle Druck (mPAP) ist in Ruhe oft nur gering erhöht, zeigt aber einen pathologischen Anstieg unter Belastung. Das Ziel dieser Studie ist es, die Leistungsfähigkeit und den pulmonalen Gasaustausch bei COPD Patienten mit und ohne pulmonalarterieller Hypertension zu untersuchen. PATIENTEN UND METHODEN: Bei 42 Patienten mit COPD Grad II-IV (28 Männer, 14 Frauen) wurden eine Bodyplethysmographie, eine symptomlimitierte Fahrradergospirometrie sowie eine Rechtsherzkatheteruntersuchung durchgeführt. RESULTATE: 32 von 42 Patienten (76%) zeigten einen erhöhten mPAP in Ruhe (PH mPAP = 26,8 ± 5,9 mmHg), bei 10 Patienten war der mPAP in Ruhe im Normbereich (NPH, mPAP = 16,8 ± 2 mmHg). Es gab keinen signifikanten Unterschied hinsichtlich der lungenfunktionellen Parameter in beiden Gruppen. Die maximale Sauerstoffaufnahme (VO2max) war signifikant niedriger in der PH Gruppe (785 ± 244 ml/min) im Vergleich zur NPH Gruppe (1052 ± 207 ml/min, p = 0,004). Es zeigte sich in der PH Gruppe eine erhöhte Totraumventilation mit signifikant erhöhtem Atemäquivalent für CO2 (VECO2 47,3 ± 10 vs 38,6 ± 3,5, p = 0,025) und signifikant höherem arterio-endtidalen CO2 Partialdruck [p(a-ET)CO2]. Der pulmonalarterielle Widerstand (PVR) in Ruhe zeigte eine negative Korrelation hinsichtlich der VO2max, VE/VCO2 und dem arterio-endtidalen CO2 Partialdruck [p(a-ET)CO2]. ZUSAMMENFASSUNG: Patienten mit COPD und erhöhter pulmonalarterieller Druckwerte in Ruhe zeigen eine Verschlechterung des pulmonalen Gasaustausches unter Belastung, eine Beeinträchtigung der maximalen Sauerstoffaufnahme und somit eine limitierte Leistungsfähigkeit.SummaryOBJECTIVE: Pulmonary hypertension (PH) is common in patients with chronic obstructive pulmonary disease (COPD). Mean pulmonary artery pressure (mPAP) is often only slightly elevated at rest but is increased by exercise. The purpose of this study was to determine whether abnormal pulmonary artery pressure impairs exercise capacity in patients with COPD. PATIENTS AND METHODS: 42 patients with moderate-to-very-severe COPD (28 men, 14 women) underwent symptom-limited incremental cardiopulmonary exercise testing and also right-heart catheterization at rest. Abnormal pulmonary artery pressure was defined as mPAP > 20 mmHg at rest. RESULTS: Resting mPAP was elevated in 32 patients (PH, mPAP = 26.8 ± 5.9 mmHg) and normal in 10 non-hypertensive (NPH) patients (NPH, mPAP = 16.8 ± 2 mmHg). There were no significant differences in lung function between the PH and NPH groups. Maximum oxygen uptake during exercise (VO2max) was significantly lower in PH (785 ± 244 ml/min) than in NPH (1052 ± 207 ml/min, P = 0.004). Dead-space ventilation (Vd/Vt) was greater in PH (P = 0.05) with higher VE/VCO2 (ratio of minute ventilation to carbon dioxide output = 47.3 ± 10 vs 38.6 ± 3.5, P = 0.025) and significantly higher arterial-end-tidal pCO2 difference [p(a-ET)CO2]. Pulmonary vascular resistance measured at rest correlated significantly with VO2max, VE/VCO2 and p(a-ET)CO2. CONCLUSIONS: In patients with COPD, abnormal pulmonary artery pressure impairs gas exchange, decreases maximum oxygen uptake during exercise and impairs exercise capacity.

Simultaneous assessment of the pharmacokinetics of a pleuromutilin, lefamulin, in plasma, soft tissues and pulmonary epithelial lining fluid.

BACKGROUND Lefamulin is a pleuromutilin antibiotic under evaluation for the treatment of bacterial infections, including respiratory tract infections. Currently, there are no high-quality pharmacokinetic data on drug tissue concentrations of lefamulin available. METHODS A single dose of intravenous lefamulin (150 mg) was given to 12 healthy men. The registered EudraCT number for this study was 2010-021938-54. Lefamulin concentrations were simultaneously measured in plasma, skeletal muscle tissue, subcutaneous adipose tissue and epithelial lining fluid (ELF) over 24 h, and corresponding pharmacokinetic parameters were calculated. Microdialysis was used to measure unbound lefamulin concentrations in skeletal muscle tissue and subcutaneous adipose tissue, which were similar to unbound lefamulin concentrations in plasma. Bronchoalveolar lavage was performed 1, 2, 4 and 8 h post-dose to determine lefamulin concentrations in ELF. RESULTS Unbound lefamulin levels showed a 5.7-fold higher exposure in ELF compared with that in plasma, demonstrating good penetration to the target site. CONCLUSIONS Lefamulin may be an addition to the therapeutic armamentarium for the treatment of infections. Simultaneous measurements of unbound drug concentration can guide target attainment for future therapeutic trials.

Vasoactive intestinal peptide (VIP) receptor expression in monocyte-derived macrophages from COPD patients.

Vasoactive intestinal peptide (VIP) is one of the most abundant molecules found in the respiratory tract. Due to its anti-inflammatory and bronchodilatatory properties, it has been proposed as a novel treatment for chronic obstructive pulmonary disease (COPD). The actions of VIP are mediated via three different G-protein-coupled receptors (VPAC1, VPAC2 and PAC1) which are expressed in the respiratory tract and on immunocompetent cells including macrophages. Alveolar macrophages (AM) are key players in the pathogenesis of COPD and contribute to the severity and progression of the disease. While VPAC1 has been reported to be elevated in subepithelial cells in smokers with chronic bronchitis, little is known about VPAC expression of AM in COPD patients. AM from COPD patients show a strong VPAC1 expression which exceeds VPAC2. A similar receptor expression pattern was also observed in lipopolysaccharide (LPS)-activated monocyte-derived macrophages (MDM) from healthy volunteers and COPD patients. VIP has been shown to down-regulate interleukin 8 (IL-8) secretion significantly in MDM after LPS stimulation. The response to VIP was similar in MDM from COPD patients and healthy volunteers. Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism in response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is dominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling. The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lung of COPD patients.

Penetration of Doripenem into Skeletal Muscle and Subcutaneous Adipose Tissue in Healthy Volunteers

ABSTRACT Sufficient antibiotic concentrations at the infection site are a prerequisite for good bacterial killing. This study was performed to determine pharmacokinetics of doripenem in soft tissues and saliva. Six healthy male volunteers received a single intravenous infusion of 500 mg doripenem over 1 h. The concentrations of doripenem were measured over 8 h in saliva, plasma, and extracellular space fluid of skeletal muscle and subcutaneous adipose tissue employing in vivo microdialysis. Unbound drug concentrations were determined using ultra-high-performance liquid chromatography-tandem mass spectrometry. Maximum concentrations of doripenem were 15.3 ± 6.0 mg/liter in plasma, 9.9 ± 2.3 mg/liter in subcutaneous adipose tissue, 6.6 ± 2.9 mg/liter in skeletal muscle, and 0.5 ± 0.2 mg/liter in saliva. Areas under the concentration-time curve (AUC) from 0 to infinity were 26.3 ± 10.1, 20.4 ± 3.8, 12.8 ± 3.0, and 1.0 ± 0.5 mg · h/liter in plasma, adipose tissue, skeletal muscle, and saliva, respectively. Ratios of AUC in adipose tissue, skeletal muscle, and saliva to those in plasma were 0.84 ± 0.28, 0.53 ± 0.19, and 0.04 ± 0.03, respectively. In all six volunteers, a threshold of ≥40% for “time above MIC,” an index indicative of good antimicrobial activity, was exceeded in adipose tissue for MICs of ≤2 mg/liter and in skeletal muscle for MICs ≤1.5 mg/liter. Doripenem penetrates well into interstitial space fluid of skeletal muscle and adipose tissue, suggesting good antimicrobial activity in infected soft tissues, whereas it is detectable in relatively low concentrations in saliva.

VPAC1 receptor expression in peripheral blood mononuclear cells in a human endotoxemia model

BackgroundVasoactive intestinal peptide (VIP) exerts immune-modulatory actions mainly via VPAC1 receptor stimulation. VPAC1 may be a treatment target of inflammatory diseases, but little is known about the receptor expression profile in immune-competent cells in vivo.Material and methods20 male healthy subjects received a single intravenous bolus of 2ng/kg body weight Escherichia coli endotoxin (LPS). Receptor status was evaluated in peripherial blood cells before and 3, 6 and 24 h after LPS by FACS analysis and q-PCR. VIP plasma concentrations were measured by ELISA.ResultsGranulocytes accounted for 51% of leukocytes at baseline and 58 ± 37% were positive for VPAC1. The granulocyte population increased 2.6 fold after LPS, and a transient down-regulation of VPAC1 to 28 ± 23% was noted at 3 h (p < 0.001), which returned to baseline at 24 hours. Baseline VPAC1 expression was low in lymphocytes (6.3 ± 3.2%) and monocytes (11 ± 9.6%). In these cells, LPS up-regulated VPAC1 at 6 h (13.2 ± 4.9%, p < 0.001) and 24 h (31.6 ± 20.5%, p = 0.001), respectively. Consistent changes were noted for the VIP-receptors VPAC2 and PAC1. VPAC1, VPAC2 and PAC1 mRNA levels were unchanged in peripheral blood mononuclear cells (PBMC). VIP plasma concentration increased from 0.5 ± 0.3 ng/ml to 0.7 ± 0.4 ng/ml at 6 h after LPS (p < 0.05) and returned to baseline within 24 h.ConclusionThe time profile of VPAC receptor expression differs in granulocytes, monocytes and lymphocytes after LPS challenge in humans. Changes in circulating VIP concentrations may reflect innate immune responses.

Clinical Potential of VIP by Modified Pharmaco-kinetics and Delivery Mechanisms

Vasoactive intestinal peptide (VIP) conveys various physiological effects in the digestive tract, nervous and cardiovascular system, airways, reproductive system, endocrine system, and more. A family of specific membrane bound receptors, termed VPAC1, VPAC2, and PAC1, bind VIP and trigger the effects. Many of them are of clinical interest. To date more than two thousand publications suggest the use of VIP in diseases like asthma, erectile dysfunction, blood pressure regulation, inflammation, endocrinology, tumours, etc. Despite this considerable potential, the peptide is not regularly used in clinical settings. A key problem is the short half life of inhaled or systemically administered VIP due to rapid enzymatic degradation. This shortcomings could be overcome with stable derivates or improved pharmacokinetics. A promising strategy is to use biocompatible and degradable depots, to protect the peptide from early degradation and allow for controlled release. This review focuses on aspects of clinical applications of VIP and the idea to use formulations based on biodegradable particles, to constitute a dispersible VIP-depot. Smart particle systems protect the peptide from early degradation, and assist the sustainable cell targeting with VIP for therapeutic or imaging purposes.

An exploratory microdialysis study investigating the effect of repeated application of a diclofenac epolamine medicated plaster on prostaglandin concentrations in skeletal muscle after standardized physical exercise

AIM Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac. METHODS Microdialysis was used to determine the local interstitial concentration of PGE2 and 8-iso-PGF2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days. RESULTS At baseline PGE2 concentrations were 1169 ± 780 pg ml(-1) at rest and 1287 ± 459 pg ml(-1) during dynamic exercise and increased to 2005 ± 1126 pg ml(-1) during recovery. After treatment average PGE2 concentrations were 997 ± 588 pg ml(-1) at rest and 1339 ± 892 pg ml(-1) during exercise. In contrast with the baseline phase no increase in PGE2 concentrations was recorded during the recovery period after treatment (PGE2 1134 ± 874 pg ml(-1)). 8-iso-PGF2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies. CONCLUSIONS We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE2 induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases.