[Gene-specific treatment approaches in amyotrophic lateral sclerosis in the present and future]. / Genspezifische Therapieansätze bei amyotropher Lateralsklerose in Gegenwart und Zukunft.

Amyotrophic lateral sclerosis (ALS) is monogenic in up to 10% of cases. Various mutation types result in a loss of function, a gain of toxicity or a combination of both. Due to the continuous development of gene-specific approaches, the treatment of the various ALS forms is no longer a dream. Depending on the underlying mutation type and pathomechanism, different antisense oligonucleotide (ASO)-based or viral strategies are available. The SOD1 and C9ORF72 genes are the most frequently mutated ALS genes in Germany and their mutations most likely predominantly lead to a gain of toxicity. For both genes, specific ASOs were developed binding to the respective mRNAs and leading to their degradation and are now being tested in clinical trials after excellent efficacy in the related ALS mouse models, with promising interim results. For the sporadic form of ALS there are also gene-specific approaches that compensate pathomechanisms and are a promising therapeutic option. In this article, gene-specific therapeutic developments in ALS as well as possible pitfalls and challenges are discussed in detail.

Quantitative analysis of flavanones and chalcones from willow bark.

Willow bark extracts are used for the treatment of fever, pain and inflammation. Recent clinical and pharmacological research revealed that not only the salicylic alcohol derivatives, but also the polyphenols significantly contribute to these effects. Quantitative analysis of the European Pharmacopoeia still focuses on the determination of the salicylic alcohol derivatives. The objective of the present study was the development of an effective quantification method for the determination of as many flavanone and chalcone glycosides as possible in Salix purpurea and other Salix species as well as commercial preparations thereof. As Salix species contain a diverse spectrum of the glycosidated flavanones naringenin, eriodictyol, and the chalcone chalconaringenin, a subsequent acidic and enzymatic hydrolysis was developed to yield naringenin and eriodictyol as aglycones, which were quantified by HPLC. The 5-O-glucosides were cleaved with 11.5% TFA before subsequent hydrolysis of the 7-O-glucosides with an almond ß-glucosidase at pH 6-7. The method was validated with regard to LOD, LOQ, intraday and interday precision, accuracy, stability, recovery, time of hydrolysis, robustness and applicability to extracts. All 5-O- and 7-O-glucosides of naringenin, eriodictyol and chalconaringenin were completely hydrolysed and converted to naringenin and eriodictyol. The LOD of the HPLC method was 0.77 µM of naringenin and 0.45 µM of eriodictyol. The LOQ was 2.34 µM of naringenin and 1.35 µM for eriodictyol. The method is robust with regard to sample weight, but susceptible concerning enzyme deterioration. The developed method is applicable to the determination of flavanone and chalcone glycosides in willow bark and corresponding preparations.

Novel neurological and immunological targets for salicylate-based phytopharmaceuticals and for the anti-depressant imipramine.

Inflammatory processes are increasingly recognised to contribute to neurological and neuropsychatric disorders such as depression. Thus we investigated whether a standardized willow bark preparation (WB) which contains among other constituents salicin, the forerunner of non-steroidal antiphlogistic drugs, would have an effect in a standard model of depression, the forced swimming test (FST), compared to the antidepressant imipramine. Studies were accompanied by gene expression analyses. In order to allocate potential effects to the different constituents of WB, fractions of the extract with different compositions of salicyl alcohol derivative and polyphenols were also investigated. Male Sprague Dawley rats (n=12/group) were treated for 14 days (p.o.) with the WB preparation STW 33-I (group A) and its fractions (FR) (groups FR-B to E) in concentrations of 30 mg/kg. The FRs were characterized by a high content of flavone and chalcone glycosides (FR-B), flavonoid glycosides and salicyl alcohol derivatives (FR-C), salicin and related salicyl alcohol derivatives (FR-D) and proanthocyanidines (FR-E). The tricyclic antidepressant imipramine (20 mg/kg) (F) was used as positive control. The FST was performed on day 15. The cumulative immobility time was significantly (p<0.05) reduced in group A (36%), group FR-D (44%) and by imipramine (16%) compared to untreated controls. RNA was isolated from peripheral blood. RNA samples (group A, group FR-D, and imipramine) were further analysed by rat whole genome microarray (Agilent) in comparison to untreated controls. Quantitative PCR for selected genes was performed. Genes (>2 fold, p<0.01), affected by WB and/or FR-D and imipramine, included both inflammatory (e.g. IL-3, IL-10) and neurologically relevant targets. Common genes regulated by WB, FR-D and imipramine were GRIA 2 ↓, SRP54 ↓, CYP26B ↓, DNM1L ↑ and KITLG ↓. In addition, the hippocampus of rats treated (27 d) with WB (15-60 mg/kg WB) or imipramine (15 mg/kg bw) showed a slower serotonin turnover (5-hydroxyindol acetic acid/serotonin (p<0.05)) depending on the dosage. Thus WB (30 mg/kg), its ethanolic fraction rich in salicyl alcohol derivatives (FR-D) (30 mg/kg) and imipramine, by being effective in the FST, modulated known and new targets relevant for neuro- and immunofunctions in rats. These findings contribute to our understanding of the link between inflammation and neurological functions and may also support the scope for the development of co-medications from salicylate-containing phytopharmaceuticals as multicomponent mixtures with single component synthetic drugs.

Contribution of flavonoids and catechol to the reduction of ICAM-1 expression in endothelial cells by a standardised Willow bark extract.

INTRODUCTION: A quantified aqueous Willow bark extract (STW 33-I) was tested concerning its inhibitory activity on TNF-α induced ICAM-1 expression in human microvascular endothelial cells (HMEC-1) and further fractionated to isolate the active compounds. RESULTS: At 50 µg/ml the extract, which had been prepared from Salix purpurea L., decreased ICAM-1 expression to 40% compared to control cells without showing cytotoxic effects. Further liquid-liquid partition revealed an ethyl acetate phase with potent reduction of ICAM-1 expression to 40% at 8 µg/ml. This fraction was comprehensively characterised by the isolation of flavanone aglyca and their corresponding glycosides, chalcone glycosides, salicin derivatives, cyclohexane-1,2-diol glycosides, catechol and trans-p-coumaric acid. All compounds were investigated for their activity on TNF-α induced ICAM-1 expression. The flavonoid and chalcone glycosides were not active up to 50 µM, whereas catechol and eriodictyol at the same concentration showed a significant reduction of ICAM-1 expression to 50% of control. Interestingly, other isolated flavanone aglyca like taxifolin, dihydrokaempferol and naringenin showed only weak or moderate inhibitory activity. Eriodictyol was a minor compound in the extract, whereas the catechol content in the extract (without excipients) reached 2.3%, determined by HPLC. One of the isolated cyclohexan-1,2-diol glucosides, 6'-O-4-hydroxybenzoyl-grandidentin, is a new natural compound. CONCLUSION: As catechol is quantitatively important in Willow bark extracts it can be concluded from the in vitro data that not only flavonoids and salicin derivatives, but also catechol can probably contribute to the anti-inflammatory activity of Willow bark extracts.

Prediction of adverse events by in vivo gene expression profiling exemplified for phytopharmaceuticals containing salicylates and the antidepressant imipramine.

BACKGROUND AND OBJECTIVE: Gene expression profiles of Sprague-Dawley (SD) rats treated with a standardized willow bark extract (WB), its salicin rich ethanol fraction (EtOH-FR) or the tricyclic antidepressant imipramine were evaluated for their potential to induce adverse events. Treatments had shown antidepressant-like effects. METHODS: Gene expression profiles (Agilent Whole Genome Array, n=4/group) obtained from the peripheral blood of male SD rats treated with WB (STW 33-I), EtOH-FR (30 mg/kg bw) or imipramine (20 mg/kg bw) were analysed comparatively by the Ingenuity Systems Programme, which allows to conduct model calculations of thresholds for theoretical potential adverse events (AE). RESULTS: The number of genes regulated by the three treatments were 1673 (WB), 117 (EtOH-FR) and 1733 (imipramine). The three treatments related to 47 disease clusters. The WB extract reached the threshold for a potential AE in one disease cluster (cardiac hypertrophy), whereas the EtOH-FR exceeded the threshold in 5 disease clusters (cardiac arteriopathy and stenosis, glomerular injury, pulmonary hypertension, alkaline phosphatase levels ⇑). Imipramine treatment hit 13 disease clusters: tachycardia, palpitation, myocardial infarction, arrhythmias, heart block, precipitation of congestive heart failure; urinary retention, altered liver functions. Those correspond to known potential adverse events. Glomerular injury and altered liver functions are part of the side effect profile of salicylic acid derivatives in agreement with the findings for the salicin rich EtOH-FR. CONCLUSION: There is no linear relationship between the number of constituents of a drug (preparation) and the number of different targets hit in a biological system on the gene expression level. Therefore, the number of genetic targets in a biological system does not necessarily increase with the complexity of the treatment corresponding to the non-linear behaviour of biological systems. Regarding gene expression levels AE of single treatments are not necessarily additive in combination treatments. The applied method appears to be an interesting screening tool for the prediction of potential AE. The phenomena that imipramine crossed the potential threshold for AEs several times whereas the WB extract did reach the threshold level only once, however not backed by clinical data for this AE, deserves to be further investigated. It questions the commonly assumed principle that substances with low number or without AE will have a poor efficacy.