Dieter Weiser

Anti-inflammatory effects of the willow bark extract STW 33-I (Proaktiv®) in LPS-activated human monocytes and differentiated macrophages

INTRODUCTION Willow bark extract is frequently used in the treatment of painful rheumatological diseases, such as arthritis and back pain. Its effect has been attributed to its main component salicin, but pharmacological studies have shown that the clinical efficacy of the willow bark extract cannot be explained by its salicin content alone. Therefore different modes of action have been suggested for the anti-inflammatory effect of willow bark extract. Here, we report in vitro data revelling the effect and mode of action of the aqueous willow bark extract STW 33-I as well as a water-soluble fraction (fraction E [Fr E]) in comparison with well-known non-steroidal anti-inflammatory drugs (NSAIDs) like aspirin (ASA) and diclofenac (Diclo) on pro-inflammatorily activated human monocytes and differentiated macrophages. RESULTS STW 33-I and the water-soluble Fr E showed concentration-dependent and significant anti-inflammatory effects in lipopolysaccharide-activated monocytes. Both inhibited the intracellular protein expression of tumour necrosis factor-alpha (TNFα) as well as the mRNA expression of TNFα and cyclooxygenase 2 (COX-2), and the release of nitric oxide (NO). In addition, apoptosis of pro-inflammatorily activated monocytes was induced. Furthermore, treatment of activated macrophages with STW 33-I inhibited the nuclear translocation of the p65 subunit of the nuclear transcription factor-kappa B (NF-κB p65). CONCLUSIONS The present in vitro investigations suggest a significant anti-inflammatory activity of willow bark water extract STW 33-1 and of its water-soluble fraction by inhibiting pro-inflammatory cytokines (TNFα), COX-2 and nuclear translocation of the transcription factor NF-κB in pro-inflammatorily activated monocytes. Our results provide further evidence for the therapeutic use of STW 33-I in inflammation-related disorders.

Studies on the degradation of human very low density lipoproteins by human milk lipoprotein lipase

Abstract Human milk lipoprotein lipase (LPL) was purified by heparin-Sepharose 4B affinity chromatography. The time required for the purification was approximately 2 h. The acetone-diethyl ether powder of milk cream was extracted by a 0.1% Triton X-100 buffer solution and the extract was applied to the heparin-Sepharose 4B column. The partially purified LPL eluted by heparin had a specific activity of 5120 units/mg which represented a 2500-fold purification of the enzyme. The LPL was found to be stable in the heparin solution for at least 2 days at 4 °C. This enzyme preparation was found to be free of the bile salt-activated lipase activity, esterase activity, and cholesterol esterase activity. The LPL had no demonstrable basal activity with emulsified triolein in the absence of a serum cofactor. The enzyme was activated by serum and by apolipoprotein C-II. The application of milk LPL to studies on the in vitro degradation of human very low density lipoproteins can result in a 90–97% triglyceride hydrolysis. The LPL degraded very low density lipoprotein triglyceride and phospholipid without any effect on cholesterol esters. Of the partial glycerides potentially generated by lipolysis with milk LPL, only monoglycerides were present in measurable amounts after 60 min of lipolysis. These results show that the partially purified human milk LPL with its high specific activity and ease of purification represents a very suitable enzyme preparation for studying the kinetics and reaction mechanisms involved in the lipolytic degradation of human triglyceride-rich lipoproteins.

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: The identification of lipoprotein B as the main lipoprotein degradation product☆

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.

Kinetics of acylglycerol hydrolysis by human milk lipoprotein lipase

The incubation of human plasma very-low-density lipoprotein with human milk lipoprotein lipase results in an almost complete hydrolysis of triacylglycerols. The degradation of these substrates can be described by a consecutive reaction as follows: (Formula: see text), where k1, k2 and k3 are the apparent first-order rate constants of degradation. Using least-squares non-linear curve fitting, k1 and k2 are determined to be directly proportional to enzyme concentration. k1/k2 ratio of 1:12 is similar for both VLDL and trioleoylglycerol substrates of lipoprotein lipase. However, when trioleoylglycerol and rac-1,2-dioleoylglycerol are used as substrates, a direct measurement indicates a k1/k2 ratio of 1:1.5. This result suggests that the intermediary diacylglycerol produced by the lipoprotein reaction is incompletely re-equilibrated with the bulk of the substrate in the assay mixture. The k3 value is not proportional to lipoprotein lipase concentration, and in the enzyme concentration range studied, the value decreases when the enzyme concentration increases.

Herbal Extracts Modulate the Amplitude and Frequency of Slow Waves in Circular Smooth Muscle of Mouse Small Intestine

Background: Herbal preparations like STW 5 (Iberogast®) are widely used drugs in the treatment of dyspepsia and motility-related disorders of the gastrointestinal tract. STW 5 is a phytotherapeutic agent consisting of a fixed mixture of 9 individual plant extracts. The electrophysiological mechanisms of action of STW 5 remain obscure. Aim: The aim of the present study was to investigate whether herbal extracts influence electrophysiological parameters of the small intestine. For this purpose, the resting membrane potential (RMP) and the slow wave rhythmicity of smooth muscle cells of mouse small intestine were observed. Methods: Intracellular recordings of smooth muscle cells of the circular muscle layer of mouse small intestine were performed using standard microelectrode techniques. After dissection of the mucosa, the small intestine was placed in an organ bath and a microelectrode was applied on a circular smooth muscle cell. The RMP and the amplitude of slow waves were measured in millivolts. Results: The RMP of smooth muscle cells was –59 ± 1.3 mV. This RMP was significantly depolarized by STW 5 (9.6 ± 1.6 mV); the depolarizing effects can be mainly attributed to the constituents of matricariae flos, angelicae radix and chelidonii herba. The basal frequency of small intestinal slow waves was 39.5 ± 1.4 min–1 and the amplitude was 23.1 ± 0.9 mV. STW 5 significantly reduced the amplitude and frequency of the slow waves (11.7 ± 0.8 mV; 33.5 ± 3.4 min–1). This effect on slow waves represents the sum of the effects of the 9 phytoextracts. Whereas angelicae radix and matricariae flos completely blocked slow wave activity, Iberis amara increased the frequency and amplitude, chelidonii herba reduced the frequency and amplitude of the slow waves, mentae piperitae folium reduced the frequency and left amplitude unchanged and liquiritae radix, carvi fructus and melissae folium had no effects. Conclusion: Herbal extracts cause changes in smooth muscle RMP and slow wave rhythmicity, up to reversible abolition, by blockade of large conductance Ca2+ channels and other not yet identified mechanisms. In herbal preparations like STW 5 these effects add up to a total effect and this study indicates that herbal preparations which are widely used in dyspepsia and motility-related disorders have characteristic, reproducible, reversible effects on small intestinal electrophysiology.

In vitro anti-proliferative effects of the willow bark extract STW 33-I.

The well-known anti-inflammatory and analgesic effects of the phytopharmacon willow bark extract have been attributed to the content of salicin; however, pharmacological studies have shown that salicin alone, despite being involved in its therapeutic action, cannot fully explain its clinical efficacy. In addition to reducing inflammation and pain, acetylsalicylic acid (ASA, CAS 50-78-2), like other synthetic non-steroidal anti-inflammatory drugs (NSAIDs), has been shown to exert anti-proliferative effects and to induce apoptosis in a variety of cell lines, e.g., colon, stomach, and prostate cancer cells. To investigate the mechanism of action and possible anti-proliferative and proapoptotic effects of willow bark, a water extract (STW 33-I) and a polyphenol rich fraction (fraction E) have been tested by using the colon-carcinoma cell line HT-29. Both, STW 33-I and its fraction E showed significant anti-proliferative and (1) Introduction The most well-known component of willow bark extract is salicin, which is metabolized in vivo to salicylic acid. The standardized aqueous willow bark extract STW 33-I, which is an effective analgesic and anti-inflammatory drug, contains 23-26% total salicin derivatives and additionally flavonoids, condensed tannins and polyphenols. Typical representatives of the flavonoids are glycosides of naringenin, isosalipurpuroside or eriodictyol. In vitro experiments have demonstrated for pro-apoptotic effects on HT-29 cancer cells. Related to the salicin content of the willow bark extract, a higher dosage of ASA was needed. Furthermore, compared to ASA and to diclofenac (Diclo, CAS 15307-79-6), the COX-1 and COX-2 mRNA expressions were influenced differently by STW 33-I and fraction E. ASA and Diclo inhibited both the COX-1 and COX-2 mRNA expressions, whereas STW 33-I and its fraction E increased the COX-1 mRNA expression. In addition to the already well-known anti-inflammatory and analgesic effects, willow bark extract has been found to possess anti-proliferative and pro-apoptotic effects similar to NSAIDs. The different influence of willow bark on the COX-1 and COX-2 mRNA expressions in comparison to NSAIDs might be relevant, e.g., for prevention of undesirable side effects such as gastric erosions.

Evidence for the Effectiveness of STW 5 in an Experimental Model of Ulcerative Colitis

G A A b st ra ct s patients were re-assigned to LL (n=84) or LP (n=86) group. Mean changes from baseline at all treatment weeks were statistically significant in all treatment groups for stool consistency, abdominal discomfort/pain, and SBM frequency (p<0.003). L group had statistically significant improvement in stool consistency compared to P group in almost a half of the 12 weekly visits (p<0.03). Similar significant improvements were observed at Weeks 13, 14, and 15 for the LL group (p<0.05). L group had statistically significant mean reduction from baseline in abdominal discomfort/pain at Weeks 10 and 11 compared to P group (p<0.05). Significant results were further observed at Weeks 13, 15, and 16 for the LL group (p<0.04). These results were not seen in the LP group. The range of increase from baseline in SBM frequency at Period I was 2.1-2.9 and 1.4-2.5 for the L and P groups, respectively. LL group had a range of 2.6-3.2, whereas the range for LP group and P group were 2.0-2.7 and 2.12.5, respectively. CONCLUSION: Lubiprostone provided significant improvement in stool consistency, abdominal discomfort/pain, and SBM frequency for standard treatment of 12 weeks, and consistent results of all outcomes were observed during 16 weeks of treatment. Moreover, longer term treatment provided significant reduction in abdominal discomfort/ pain.

Tu1821 Beyond Acid Suppression: Novel Targets in Gastroesophageal Reflux Disease by Gene Microarray Profiling

elimination groups. Co-administration of PPI was more prevalent in delayed elimination group compared with normal elimination group (90% vs. 62%). No difference was found in NSAIDs or vancomycin co-administration between the groups. Multivariate logistic regression model adjusted for age and gender revealed that co-administration of PPI was an independent factor associated with delayed MTX elimination (OR 12.3, p=0.04). Conclusion: Co-administration of proton pump inhibitors delays elimination of plasma methotrexate in patients with high-dose methotrexate therapy.

Adenosine Receptors: New Targets to Protect Against Tissue Damage in Inflammatory Bowel Symptoms

Irritable bowel syndrome (IBS) is a disease in which, typically, alterations in intestinal motility and visceral hypersensitivity appear to exist, apparently without any organic alteration (Thompson, 1991). Several pathogenic factors responsible for IBS have been suggested. It seems that there are cell factors, which give reason to believe that there is a low-grade intestinal inflammation in this pathology (Ortiz-Lucas et al., 2010). Several cytokines, such as tumour necrosis factor α (TNFα), interleukin 1, and interleukin 6, contribute to the pathogenesis (Ardizzone and Bianchi Porro, 2005; Pizarro et al., 2006). Macrophages are the major producers of TNFα, and, interestingly, they are also highly responsive to TNFα. TNFα has been shown to play a pivotal role in activating the cytokine cascade in many inflammatory diseases and it has been proposed as a therapeutic target for a number of diseases. Consequently, recent strategies for the treatment of intestinal inflammation have primarily targeted the immunopathogenic processes that mediate intestinal inflammation at the cytokine level (Bamias et al., 2003; Sandborm and Targan, 2002). At present, pharmacotherapy represents the mainstay of inflammatory bowel disease management (Stein and Hanauer, 1999). Some anti-inflammatory or immuno-modulating drugs, including salicylates and methotrexate, are able to decrease intracellular adenosine 5 -triphosphate concentrations and raise extracellular adenosine levels. It has been proposed that such properties can significantly contribute to the drugs’ pharmacological actions in inflammatory diseases (Cronstein et al., 1999). Several lines of evidence suggest that adenosine regulates immunity and inflammation (Amann and Peskar, 2002; Montesinos et al., 2007). The wide distribution of adenosine receptors (AR) as well as enzymes for purine metabolism in different gut regions suggests a complex role for this mediator in the regulation of gastrointestinal functions (Antonioli et al., 2008). Adenosine binds to four different types of G protein-coupled cell surface receptors referred to as A1R, A2AR, A2BR, and A3R, each having a unique pharmacological profile, tissue distribution and signalling pathway (Jacobson and Gao, 2006). All known ARs

Mo1091 A Study of the Mechanisms Underlying the Potential Usefulness of STW 5 in Reflux Esophagitis

Proton pump inhibitor (PPI) therapy is the most effective medical treatment for symptom relief in gastro-esophageal reflux disease (GERD). However, up to 40% of patients do not achieve adequate symptom relief, especially those suffering from non-erosive reflux disease (NERD) which does not respond well to PPIs. Therefore, the search continues for other treatment options. STW 5, a multi-component herbal preparation, was shown to relieve heartburn and concomitant reflux symptoms in patients with functional dyspepsia and to prevent inflammation in an acute model of reflux esophagitis (RE), without affecting the pH of the refluxate. The present study assesses the efficacy of STW 5 in a more chronic model of RE, and the underlying mechanisms both In-Vivo and in-vitro. Rats were pretreated for 7 d with STW 5 or omeprazole (as reference drug) before surgical induction of esophagitis. RE was achieved by ligation of the fore-stomach and covering the duodenum near the pylorus with a piece of Nelaton catheter. After recovery, rats were treated for a further 10 d with the drugs and then sacrificed, their esophagi excised, weighed and evaluated macroscopically. Tissue homogenates were used for semi-quantitative determination of cytokines using a cytokine array. Both, STW 5 and omeprazole, improved body weight, tissue damage score and esophageal weight index to similar extents. However, STW 5 had a much more pronounced anti-inflammatory effect. As shown in the cytokine array (fig. 1), STW 5 inhibited the majority of the measured pro-inflammatory cytokines induced by surgical reflux, suggesting a direct anti-inflammatory and/or mucosal protecting action. Since recent evidence indicates that the mucosal damage observed in GERD is due to release of inflammatory mediators from mucosal and sub-mucosal cells in response to bile salts and other substances in the gastric refluxate, the effects of STW 5 on cytokine release from the normal human esophageal cell-line HET-1A in response to stimulation with chenodeoxycholic acid (CDCA) was assessed. Incubation of cells with CDCA caused marked release of CD40ligand, IFN-γ, IL-1ra, IL-6, IL-8 and IL-23, which were all inhibited by co-incubation with STW 5 (0,3 10 μl/ml) as assessed semi-quantitatively (cytokine array, fig.2) and confirmed quantitatively (ELISA), without affecting cell viability. Similar effects were observed when cells were incubated with capsaicin as TRPV1 agonist (shown to be the main receptor involved in acid induced cytokine release from HET-1A cells), which evoked the release of CD40-ligand and IL-23. The present findings suggest that multi-target anti-inflammatory drugs like STW 5 might present an alternative/additional treatment option for GERD patients not responding adequately to PPIs. Further elucidation of its exact mechanism of action might pave the way for a new class of anti-reflux agents.