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Matrix metalloproteinases have been in the scope of pharmaceutical drug discovery for decades as promising targets for drug development. Until present, no modulator of the enzyme class survived clinical trials, all failing for various reasons. Nevertheless, the target family did not lose its attractiveness and there is ever more evidence that MMP modulators are likely to overcome the hurdles and result in successful clinical therapies.This review provides an overview of past efforts that were taken in the development of MMP inhibitors and insight into promising strategies that might enable drug discovery in the field in the future. Small molecule inhibitors as well as biomolecules are reviewed.Despite the lack of successful clinical trials in the past, there is ongoing research in the field of MMP modulation, proving the target class has not lost its appeal to pharmaceutical research. With ever-growing insights from different scientific fields that shed light on previously unknown correlations, it is now time to use synergies deriving from biological knowledge, chemical structure generation, and clinical application to reach the ultimate goal of bringing MMP derived drugs on a broad front for the benefit of patients into therapeutic use.

Given the vast number of venomous and poisonous animals, it is surprising that only relatively few animal-derived toxins have been explored and made their way into marketed drugs or are being investigated in ongoing clinical trials. In this review, we highlight marketed drugs deriving from animal toxins as well as ongoing clinical trials and preclinical investigations in the field. We emphasize that more attention should be paid to the rich supply of candidates that nature provides as valuable starting points for addressing serious unmet medical needs.

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure–activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in‐depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure–activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long‐time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12′000 mutants showed no signs of resistance development to the synthetic derivatives., A structure–activity relationship of the natural product Leucinostatin A was performed to result in two highly potent compounds against the protozoa Trypanosoma brucei with decreased toxic effects in vitro and in vivo. Those compounds did not form any resistance by long‐time sublethal exposure and their mode of action lies in the destabilization of the inner mitochondrial membrane.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parain-fluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application., IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae.

Owing to their structural diversity, peptides are a unique source of innovative active ingredients. However, their development has been challenging because of their disadvantageous pharmacokinetic (PK) properties. Over the past decade, many attempts have been made to improve the oral bioavailability of peptide drugs. In this review, we highlight the most recent and promising techniques aimed at the improvement of the oral bioavailability of peptides. The most recent findings will influence future approaches of pharmaceutical companies in the development of new, more efficient, and safer orally delivered peptides.

Introduction: Symphytum officinale L. (comfrey, Boraginaceae) is a cultivated or spontaneously growing medicinal plant that is traditionally used for the treatment of bone fractures, hematomas, muscle pains and joint pains. A wide range of topical preparations and dried roots for ex tempore applications are marketed in European drug stores or pharmacies. Objective: The aim of this study was to perform the qualitative and quantitative analysis of pyrrolizidine alkaloids (PAs) and phenolic compounds in the hydroethanolic extracts of 16 commercial comfrey root batches purchased from 12 different European countries. Methods: Liquid chromatography hyphenated with high-resolution tandem mass spectrometry (LC-HRMS/MS) was used for the profiling of PAs and phenolic compounds, whereas LC-MS/MS and liquid chromatography with diode array detection (LC-DAD) were used for their quantification. Results: 20 PAs (i.e. intermedine, lycopsamine, acetylintermedine, acetyllycopsamine, symphytine, symphytine-N-oxide), 17 phenolic compounds (i.e. caffeic and rosmarinic acids, rabdosiin, globoidnan A, globoidnan B) and 9 nonphenolic compounds (sugars, organic and fatty acids) were fully or partly annotated in the analysed samples. In addition, the quantitative analyses revealed that globoidnan B, rabdosiin and globoidnan A are new phenolic markers that can be used together with rosmarinic acid and PAs for the quality control of commercial comfrey root batches. Conclusions: This study brings new insights into the phytochemical complexity of S. officinale, revealing not only numerous toxic PAs, but also a significant number of valuable phenolic compounds that could contribute to the bioactivities of comfreybased preparations

Sentrin-specific proteases (SENPs) are responsible for the maturation of small ubiquitin-like modifiers (SUMOs) and the deconjugation of SUMOs from their substrate proteins. Studies on prostate cancer revealed an overexpression of SENP1, which promotes prostate cancer progression as well as metastasis. Therefore, SENP1 has been identified as a novel drug target against prostate cancer. Herein, we report the discovery and biological evaluation of potent and selective SENP1 inhibitors. A structure-activity relationship (SAR) of the newly identified pyridone scaffold revealed allosteric inhibitors with very attractive in vitro ADMET properties regarding plasma binding and plasma stability for this challenging target. This study also emphasizes the importance of biochemical mode of inhibition studies for de novo designed inhibitors.

Ethnopharmacological relevance The Brazilian Cerrado is a biome with a remarkable diversity of plant species, many of which are used mainly by local communities as a source of treatment to several pathologic processes, especially for the treatment of wounds. However, no systematic review exists focusing on the plants used in this respect and on the appropriate pharmacological investigations that substantiate the actions that are reported. This study revisits the traditional use of medicinal plants from the Brazilian Cerrado in the treatment of wounds and the pharmacological characteristics of the reported plant species. Method ology: For the present article, previous studies on plants of the Brazilian Cerrado used for wound healing carried out between 1996 and 2018 were researched on various academic databases (PubMed, Elsevier, Springer, Lilacs, Google Escolar, and Scielo). Results A total of 33 studies were carried out on 29 plant species distributed into 18 families, mainly Fabaceae or Leguminosae (9), Bignoniaceae (2), Asteraceae (2), Euphorbiaceae (2). Considering the great diversity of Cerrado plants, only a small number of wound healing studies were carried out between 1996 and 2018. It was observed that there is a large gap between experimentation assay and traditional use. There are only few connections between the form of use by the population and the experiments conducted in the laboratory. We found that only about 12% of these studies considered to use the methodologies, or at least in parts, to obtain extracts such as those used in folk medicine. Approximately 37% of the experiments were performed using the bark as well as the same ratio for leaves, 6% using the fruits, and 9% using the seeds, roots or flowers. In several studies, there are reports of chemical constituents such as flavonoids and tannins, followed by steroid terpenes, saponins, and fatty acids, and alkaloids. However, approximately 35% of the studies did not supply information about compounds present in the preparation or the effect which could be attributed to these agents in respect to wound healing. Regarding treatment, most of the studies employed a topical treatment, though intraperitoneal and oral treatment were also described using either topical, oil-based formulations, but also gel- or saline-based formulations. Conclusions Although, there has been an increase in knowledge about the biological actions of plants from Cerrado biome, the scientific basis for the traditional use of these local medicinal plants in wound healing does not provide sufficient information on the efficacy of the treatment, the molecular mechanisms, or, in particular, the effective doses used and the drug interactions. Thus, focused research investigating these hypotheses from traditional knowledge is necessary to prove the evidence of the potential pharmacological action.

Introduction: The development of drug candidates with a defined selectivity profile and a unique molecular structure is of fundamental interest for drug discovery. In contrast to the costly screening of large substance libraries, the targeted de novo design of a drug by using structural information of either the biological target and/or structure-activity relationship data of active modulators offers an efficient and intellectually appealing alternative. Areas covered: This review provides an overview on the different techniques of de novo drug design (ligand-based drug design, structure-based drug design, and fragment-based drug design) and highlights successful examples of this targeted approach toward selective modulators of therapeutically relevant targets. Expert opinion: De novo drug design has established itself as a very efficient method for the development of potent and selective modulators for a variety of different biological target classes. The ever-growing wealth of structural data on therapeutic targets will certainly further enhance the importance of de novo design for the drug discovery process in the future. However, a consistent use of the terminology of de novo drug design in the scientific literature should be sought.

Ethnopharmacological relevance Symphytum officinale L. (comfrey, Boraginaceae) has been traditionally used for millennia in joint distortions, myalgia, bone fractures and hematomas. However, key activity-determining constituents and molecular mechanisms underlying its use have not been completely elucidated. Aim of the study The objective of this study was to isolate and identify the major compounds from a hydroethanolic root extract of S. officinale and evaluate their antioxidant potential, alongside their effect on the cytokine production of ex vivo stimulated neutrophils, thus providing scientific support for the traditional use of comfrey root. Material and methods Four caffeic acid oligomers were isolated from comfrey roots by liquid-liquid chromatography, their structures being established by MS and NMR analyses. In vitro antioxidant evaluation was performed by DPPH and ABTS assays. The cytotoxicity of isolated compounds was established by flow cytometry. The effect on cytokine release, such as interleukin (IL)-1β, IL-8 and tumor necrosis factor alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (ELISA). Results The main constituents found in comfrey root were represented by four caffeic acid oligomers, namely globoidnan B (1), rabdosiin (2), rosmarinic acid (3) and globoidnan A (4). Rabdosiin, globoidnans A and B were isolated for the first time from S. officinale. In the in vitro antioxidant tests, compound 2 was the most active, with EC50 values in DPPH and ABTS assays of 29.14 ± 0.43 and 11.13 ± 0.39, respectively. Neutrophils’ viability over the tested concentration domain of 12.5–50 μM was not altered. At 50 μM, all compounds significantly inhibited IL-1β release, with compound 3 (45.60% release vs. LPS stimulated neutrophils) being the most active, followed by compounds 1 (53.85%), 2 (69.89%) and 4 (60.68%). Conclusions The four caffeic acid oligomers reported in S. officinale root may contribute to the overall anti-inflammatory activity for which comfrey preparations are used in traditional medicine.

De novo drug discovery is still a challenge in the search for potent and selective modulators of therapeutically relevant target proteins. Here, we disclose the unexpected discovery of a peptidic ligand 1 by X-ray crystallography, which was auto-tailored by the therapeutic target MMP-13 through partial self-degradation and subsequent structure-based optimization to a highly potent and selective β-sheet peptidomimetic inhibitor derived from the endogenous tissue inhibitors of metalloproteinases (TIMPs). The incorporation of non-proteinogenic amino acids in combination with a cyclization strategy proved to be key for the de novo design of TIMP peptidomimetics. The optimized cyclic peptide 4 (ZHAWOC7726) is membrane permeable with an IC50 of 21 nm for MMP-13 and an attractive selectivity profile with respect to a polypharmacology approach including the anticancer targets MMP-2 (IC50 : 170 nm) and MMP-9 (IC50 : 140 nm).

The natural phenomenon of drug resistance is a widespread issue that hampers the performance of drugs in many major clinical indications. Antibacterial and antifungal drugs are affected, as well as compounds for the treatment of cancer, viral infections, or parasitic diseases. Despite the very diverse set of biological targets and organisms involved in the development of drug resistance, the underlying molecular mechanisms have been identified to understand the emergence of resistance and to overcome this detrimental process. Detailed structural information on the root causes for drug resistance is nowadays frequently available, so next-generation drugs can be designed that are anticipated to suffer less from resistance. This knowledge-based approach is essential for fighting the inevitable occurrence of drug resistance.

The de novo design of molecules from scratch with tailored biological activity is still the major intellectual challenge in chemical biology and drug discovery. Herein we validate natural-product-derived fragments (NPDFs) as excellent molecular seeds for the targeted de novo discovery of lead structures for the modulation of therapeutically relevant proteins. The application of this de novo approach delivered, in synergy with the combination of allosteric and active site binding motifs, highly selective and ligand-efficient non-zinc-binding (3: 4-{[5-(2-{[(3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzoic acid) as well as zinc-binding (4: 4-({5-[2-({[3-(3-carboxypropoxy)phenyl]methyl}carbamoyl)eth-1-yn-1-yl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl}methyl)benzoic acid) uracil-based MMP-13 inhibitors presenting IC50 values of 11 nM (3: LE=0.35) and 6 nM (4: LE=0.31).

Matrix metalloproteinase 7 (MMP-7) is a member of the MMP superfamily and is able to degrade extracellular matrix proteins such as casein, gelatin, fibronectin and proteoglycan. MMP-7 is a validated target for the development of small molecule drugs against cancer. MMP-13 is within the enzyme class the most efficient contributor to type II collagen degeneration and is a validated target in arthritis and cancer. We have developed the dual MMP-7/-13 inhibitor ZHAWOC6941 with IC50-values of 2.2 μM (MMP-7) and 1.2 μM (MMP-13) that is selective over a broad range of MMP isoforms. It spares MMP-1, -2, -3, -8, -9, -12 and -14, making it a valuable modulator for targeted polypharmacology approaches.

Zugehöriger Artikel: https://digitalcollection.zhaw.ch/handle/11475/2857

In the present study, robust and validated HPLC and UHPLC methods for the quantitative determination of frangulins A and B (3 and 4) and glucofrangulins A and B (1 and 2) in the bark of Frangula alnus have been developed. The HPLC method allowed the separation of the analytes in 25 min and the UHPLC method in just 13 min. The HPLC method used an MN Nucleodur C 18 125 × 4 mm column with 3 μm particles, while the UHPLC method used a Waters Acquity UPLC BEH C18, 100 × 2.1 mm column with 1.7 μm particles. Mobile phase A consisted of water and 1.25 mL/L phosphoric acid (85%), while mobile phase B consisted of CH3CN/MeOH (20:80). The flow rates were set to 1mL/min for the HPLC method and 0.4 mL/min for the UHPLC method, with the column temperature held at 50°C and the detection wavelength being 435 nm for either method. A fractional factorial design was used to check the robustness of the methods. The resolution of the analytes was never less than 1.5 when the factors were varied in the tested range. The conditions for ultrasonic extraction were optimized by response surface methodology and found to be 68% CH 3 CN in the extraction solvent, 35°C extraction temperature, and a duration of 25 min. The extraction procedure was determined to be very robust against small deviations of these factors.

Objective: We aimed to determine the association between the stepwise increase in the sustained viral response (SVR) and Swiss and United States (US) market prices of drug regimens for treatment-naive, genotype 1 chronic hepatitis C virus (HCV) infection in the last 25 years. We identified the following five steps in the development of HCV treatment regimens: 1) interferon (IFN)-α monotherapy in the early '90s, 2) IFN-α in combination with ribavirin (RBV), 3) pegylated (peg) IFN-α in combination with RBV, 4) the first direct acting antivirals (DAAs) (telaprevir and boceprevir) in combination with pegIFN- α and RBV, and 5) newer DAA-based regimens, such as sofosbuvir (which is or is not combined with ledipasvir) and fixed-dose combination of ritonavir-boosted paritaprevir and ombitasvir in combination with dasabuvir. Design: We performed a linear regression and mean cost analysis to test for an association between SVRs and HCV regimen prices. We conducted a sensitivity analysis using US prices at the time of US drug licensing. We selected randomized clinical trials of drugs approved for use in Switzerland from 1997 to July 2015 including treatment-naïve patients with HCV genotype 1 infection. Results: We identified a statistically significant positive relationship between the proportion of patients achieving SVRs and the costs of HCV regimens in Switzerland (with a bivariate ordinary least square regression yielding an R2 measure of 0.96) and the US (R2 = 0.95). The incremental cost per additional percentage of SVR was 597.14 USD in Switzerland and 1,063.81 USD in the US. Conclusion: The pricing of drugs for HCV regimens follows a value-based model, which has a stable ratio of costs per achieved SVR over 25 years. Health care systems are struggling with the high resource use of these new agents despite their obvious long-term advantages for the overall health of the population. Therefore, the pharmaceutical industry, health care payers and other stakeholders are challenged with finding new drug pricing schemes to treat the entire population infected with HCV.

The European Pharmacopeia (Ph. Eur.) contains herbal monographs for Sennae folium and fructus. The current spectrophotometric assay is time consuming and unspecific. An HPLC method for the official Sennae monographs is under discussion. The aim of this study is to optimize an HPLC method for anthraquinone aglyca to be robust and valid with optimal separation and analysis time. The currently proposed Ph. Eur. draft method is compared to a method developed by [2]: Senna pods and leaves from different suppliers were extracted by ACN/0.1% NaHCO3. Solid-Phase-Extraction is applied for method [2], only. Nucleodur 100-3 C18 column and H2O/ACN/MeOH in a gradient was used as stationary and mobile phase with detection at 435nm. It was found that the Ph Eur draft method delivers in the range of 5-20% w/w higher results for anthraquinone content in the herbal drug material calculated as Sennoside B. Overestimation due to coeluting peaks or analyte losses due to SPE could explain the differences in content. Therefore, a rational approach for optimization was attempted using the software Drylab®. Based on 12 initial runs with varying conditions in the entire variable parameter space (column temperature, mobile phase and gradient), the in-silico tool suggests further experiments. The tracking of the changes in retention time of the target analyte peaks is a crucial prerequisite and requires both DAD and MS data of each peak. The result of the in-silico supported optimization is the identification of a suitable operation region of the method parameters shown in Fig. 1a. The transfer of the method from UHPLC to HPLC resulted in an acceptable separation at 43°C and a gradient of A. water and B. MeOH/ACN (98/2 v/v) both containing 0.1% formic acid of 0/30, 8.5/30, 28/48, 30/99 [min/%B] (Fig. 1b). From our study we conclude that Drylab® is a useful LC method optimization tool. However, the peak assignment of all peaks in the chromatograms is a challenge for plant extracts.

The genera Verbascum and Ajuga with multiple traditional therapeutical uses are rich in phenolic compounds with potential anti-inflammatory, antioxidant and immunomodulatory properties [1]. The aim of this study was to screen the phytochemical and biological potential of the species V. nigrum L. and A. chia Schreb. for prevention and treatment of nonmelanoma skin cancer as part of a joint research project on multitarget activity of plant extract [2]. Methanol (MeOH) extraction of the aerial parts of A. chia and V. nigrum was followed by fractionation by RP-C18 flash chromatography. The phytochemical characterization was assessed by UPLC-DAD-ESI-MS and HPTLC. The latter was linked to (bio)autographic acetylcholinesterase inhibition and DPPH assays. Cytotoxic evaluation of HaCaT and A431 cells treated with extracts and fractions was done by MTT assay, followed by immunofluorescence microscopy. V. nigrum extract contains potential AchE inhibitors. Both plants show presence of antioxidant compounds. Based on UV and MS data we identified for A. chia with high probability luteolin-7-O-glucoside, apigenin and verbascoside and for V. nigrum, verbascoside, harpagoside and possible aucubin derivatives. V. nigrum extract, 100% MeOH and 50% MeOH fractions induced A431 cell apoptosis and decreased the cell invasion. The A. chia extract did not exhibit any cytotoxicity on A431 cell line. None of extracts exhibited cytotoxicity on HaCaT cells. Acknowledgements: Sciex-HMSch CRUS Switzerland No. 13.218 & N. Ciocirlan and V. Ghendov for plant material identification (Botanical Garden of Academy of Sciences of Moldova). References: [1] Nichols JA et al. Arch Dermatol Res 2010; 302: 71 – 8 [2] Millsop J et al. Dermatol Res Pract 2013; 2013: 837152

For the European Pharmacopoeia (Ph. Eur.) herbal monograph draft of Cassia angustifolia Vahl. and Cassia senna L. leaves and pods, a safety limitation of aloe-emodin and rhein was proposed, due to toxicological concerns. A quantitative, analytical method of the anthraquinone aglycones in all Ph. Eur. monographed herbal laxatives is of interest. A rational method development for the aglycones aloe-emodin, rhein, emodin, chrysophanol, and physcion in five herbal drugs was realized by using 3D chromatographic modelling (temperature, solvent, and gradient time) and design of experiment (DOE) software (DryLab® 4). A methodical approach suitable for the challenging peak tracking in the chromatograms of the herbal drugs in dependence on the changes in the chromatographic conditions is described by using a combination of mass spectroscopy (MS) data (UHPLC-QDa), UV/Vis-spectra, and peak areas. The model results indicate a low robust range and showed that with the selected chromatographic system, small interferences could not be averted. The separation achieved shows a pure UV/Vis spectrum for all aglycones except for chrysophanol in Aloe barbadensis and emodin in Cassia angustifolia fruit. A gradient with the best resolution of the aglycones in all five drugs is proposed, and its suitability demonstrated for the quantification of aglycones in these herbal drugs.

The incorporation of fluorine atoms into functional molecules is of wide interest in synthetic organic chemistry as well as cognate disciplines. In particular, in medicinal chemistry, there is a strong desire to positively influence the physicochemical molecular properties of drug compounds by introducing fluorine into biologically active molecules. Here, we present targeted fluoro positioning as the key design principle of converting a weak matrix metalloproteinase‐13 (MMP‐13) inhibitor into a very potent (IC 50=6 nm) and highly selective (selectivity factors of >1000 over MMP‐1, 2, 3, 7, 8, 9, 10, 12, 14) inhibitor with excellent plasma and microsomal stability, and no binding to the hERG channel (hERG: human ether‐a‐go‐go related gene).

Zugehöriger Artikel: https://digitalcollection.zhaw.ch/handle/11475/2857, The Front Cover picture shows the design of the highly potent and selective inhibitor ZHAWOC5684 bound to its target protein matrix metalloproteinase-13 (MMP-13). The key design principle involves the structure-based positioning of a single fluorine atom to probe water-mediated interactions within the allosteric binding site. In combination with a zinc-binding carboxylic acid fragment blocking the active site this approach delivers a drug-like small molecule inhibitor of MMP-13 with excellent microsomal and plasma stability.

In vitro cultivation is considered as a prospective supplementary agriculture and season independent approach for supply of plant raw material [1]. We report here on intermediate results of research activities that aim at development of an in vitro collection of medicinal and aromatic plants from the Balkan region. Optimizations of medium composition as well as development of callus suspension and root cultures are being conducted in order to stimulate production of secondary metabolites with phytopharmaceutical value. Bioprocess control strategies are needed for rapid and cost effective screening for selection of highly productive in vitro lines for later scale up in bioreactor conditions. HPTLC fingerprinting coupled with DPPH ß carotene/linoleic acid bleaching [2] as well as bioautographic Xanthine Oxidase Inhibition [34] assays were applied in order to assess secondary metabolite and active compound content simultaneously. Different in vitro lines of Balkan endemic Sideritis scardica Griseb. Pulsatilla montana ssp. balcana essential oil bearing Artemisia alba Turra as well as several representatives of the Hypericum genus differing in their biosynthetic capacity for hypericines have been studied. Hypericine non producing Hypericum calycinum L. showed superior antioxidant activity as compared with the other species. In vitro cultured S. scardica originating from wild accessions showed higher activity than from field cultivation. From all tested species A. alba showed several inhibitory zones in the HPTLC coupled Xanthine Oxidase Inhibition screening. Further analysis of these zones is feasible by coupled HPTLC MS. The applied bioautographic bioprocess control methods provide for rapid and efficient targeting of prospective highly producing in vitro lines for future up scaling especially when a large number of culture variants have to be handled. Acknowledgements: SNF No. IZEBZ0_142989 and SD MEYS No. DO2 – 1153

Artemisia alba Turra (Artemisia Asteraceae) is widespread in the southern and south eastern parts of Europe – Spain Italy and Balkan peninsula [1]. The aerial parts of Artemisia alba Turra have been traditionally utilized as a stomach digestive and tonic in the form of a decoction [2]. The literature survey revealed few reports concerning the non volatile components of this species: santonin nerolidol derivatives and artalbic acid were isolated from the aerial parts whereas the roots were shown to contain a sesquiterpene coumarin ether [3]. The aerial parts of field cultivated Artemisia alba Turra were initially defatted with hexane and then extracted successively with chloroform and methanol. Further CC and PTLC purification of the chloroform extract resulted in the isolation of 10 new compounds with germacrane oplopane eudesmane guaiane and bisabolane carbon skeletons. Their structures were elucidated by NMR spectroscopy (1H NMR 1H 1H COSY HSQC HMBC NOESY) and MS. It was found that the isolated compounds are highly oxygenated and contained 2 – 5 oxygen bearing functions (OH OOH and OAc). The main components were germacrane derivatives with C 10 exomethylene group and C 3 and C 9 substituted positions. Additional oxygenated positions were C 1 C 2 C 4 or C 12 carbons. The dominant component among the isolated compounds was 1 hydroperoxy 3 hydroxy 9 acetoxy germacra 5 10(14) diene.

Erschienen im Book of Abstracts von Planta Medica, Band 79 (Artikel 13/PK44).

Professor Dr. Rainer Riedl discusses his international and interdisciplinary research into the design of synthetic molecules to improve the development of drugs for a range of conditions.

The Center for Organic and Medicinal Chemistry at the Zurich University of Applied Sciences in Switzerland is collaborating with partners across industry and academia to create novel treatment options for patients.

Since the 1970s, the limitations of two dimensional (2D) cell culture and the relevance of appropriate three dimensional (3D) cell systems have become increasingly evident. Extensive effort has thus been made to move cells from a flat world to a 3D environment. While 3D cell culture technologies are meanwhile widely used in academia, 2D culture technologies are still entrenched in the (pharmaceutical) industry for most kind of cell-based efficacy and toxicology tests. However, 3D cell culture technologies will certainly become more applicable if biological relevance, reproducibility and high throughput can be assured at acceptable costs. Most recent innovations and developments clearly indicate that the transition from 2D to 3D cell culture for industrial purposes, for example, drug development is simply a question of time.

In previous research ofArtemisia alba Turra we reported that stimulation of root developmentin vitro was related to domination of monoterpenoids while callus formation and rooting inhibition led to the prevalence of sesquiterpenoids in the essential oils of the aerial parts [1]. Interestingly these two essential oil types correlated to the results of other authors concerning the oils of contrasting wild accessions of this species [2]. Here we study the biochemical parameters ofA. alba response to exogenous indole 3 butyric acid (IBA) and benzyl adenine (BA) treatmentin vitro. The aerials of plants with a well developed root system (monoterpenoid domination in the oils in PGR lacking control as well as in IBA rooting stimulated media) were characterized by elevated CAT (EC 1.11.1.6) APX (EC 1.11.1.11) and GPOX (EC 1.11.1.7) levels as compared with aerials of the plants from the “sesquiterpenoid group” (where combinations of IBA and BA led to suppressed rooting and callusogenesis). Interestingly the plants from the “intermediate oil type” (bearing the terpenoid features of both root and callus forming plants) were characterized by a considerable drop of PAL (EC 4.3.1.24) CAT and GR (EC 1.8.1.7) and a drop of polyphenolics leading to marked elevation of lipid peroxidation and oxidative stressin vitro. In addition electrophoretic profiles indicated differing enzymatic activities for the aerial root and callus tissue samples. Modification of the essential oil profile through affecting morphological development in vitro will further be utilized for the targeted delivery of plant biomass with desired properties.

Inula britannica is used as an anti inflammatory anti bacterial anti hepatitic and anti tumor agent [1]. Species of the Sideritis genus possess anti inflammatory antiulcerative antimicrobial vulnerary antioxidant antispasmodic anticonvulsant analgesic and carminative properties [2]. Sideritis scardica is a Balkan endemic species traditionally employed as expectorant for the treatment of pulmonary emphysema and angina pectoris [3]. In vitro culture was initiated by axillary buds formation of surface sterilized stem explants of I. britannica collected from its wild habitat in Bulgaria and of S. scardica – from sterile germinated seeds of the commercial cultivar Sofia 2 distributed on the Bulgarian market. Vitamin and plant growth regulators (PGR) were modified in order to optimize biomass production and polyphenolics accumulation in vitro. In I. britannica long term growth of stock cultures was achieved in PGR free medium. Biomass and polyphenolics formation were successfully stimulated by supplementation of Gamborg vitamins and a combination of N 6 Benzyladenine (BA) and Naphthylacetic acid. For Sofia 2 cultivar PGR free medium resulted in a low multiplication index has been unfavourable for long term maintenance (over 4 weeks) and required more frequent subculture in order to avoid necrosis of explants. Addition of PGR allowed for optimization of biomass production with only slight reduction of the proportion of high molecular flavonoids as a part of total polyphenolic content.

In vitro secondary metabolite production requires a fine balance between biomass formation and expression of the biosynthetic capacity of the species. Comparison of the metabolite spectra of the in vitro to ex situ material is a crucial benchmark in this process. HPTLC methods known from similar plant species from the Artemisia and Hypericum genera [1 2] have been adapted to extracts with varying polarity of different plant parts from ex situ and in vitro samples of Artemisia alba and Hypericum richeri. The main differences and similarities of the secondary metabolite and bioactive constituent profiles between in situ and in vitro produced plant material were assessed. The results of this study revealed that A. alba can be maintained successfully long term in medium lacking plant growth regulators (PGR) whereas H. richeri requires cytokinin supplementation in order to stimulate axillary bud multiplication and sustain growth in vitro. It was also established that N6 Benzyladenine (BA) strongly stimulated multiplication index and its individual application led to inhibition of rooting for both species. While the combination of BA and indole 3 butiric acid was found to be favorable for both biomass and polyphenolics stimulation in A. alba enhanced growth led to the drop of polyphenols and flavonoids in H. richeri. At the same time hypericins were observed in significant levels in vitro. Further research is in progress to clarify the distinctive features of the biochemical and physiological response to PGR treatment as a model system for affecting antioxidant metabolites production in vitro.

Cytotoxicity assays in vitro (MTT test) showed that the different breast cancer cell lines Kpl-1, MCF-7 and Mfm-223 respond differently to the mistletoe (Viscum album L.) preparations Iscador®. Quercus (Qu), Abies (A), Malus (M) and Pinus (P). In order to determine the differences in the responsiveness of the cells more exactly, the gene expression profiles were determined by cells, which were treated with Mistletoe extracts, compared with untreated control cells. Such differences can be analysed in more detail by looking at the gene expression using Human Whole Genome microarray chips (41,000 genes). The results of the transcriptome analyses suggested that Iscador preparations influenced the overregulation of genes regarding immune defense, stress response, apoptosis and cell-cell adhesion pathways. Within the Mfm-223-Zellen was the Genexpression in all 9 metabolic pathways evident. The Mfm-223 cells included all effects found in MCF-7 and Kpl-1. The MCF-7 cells were affected on the genes which are involved in cell-cell contacts whereas Kpl-1 responded to the mistletoe extracts by changing the mRNA levels of the immune and stress response pathways. Concerning the effects of the mistletoe extract, we conclude that Iscador Qu and M have a greater influence on the immune defense and stress response genes whereas Iscador A tends to affect the cell-cell adhesion and cytoskeleton pathways. In summary, cDNA microarray analyses give us information on whether a cancer cell is sensitive to mistletoe extracts in relation to how many genes are significantly overrepresented after mistletoe treatment, and whether a particular mistletoe extract is more effective on a specific cancer cell than the other preparation.

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure-activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in-depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure-activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long-time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12'000 mutants showed no signs of resistance development to the synthetic derivatives.