Examination of betahistine bioavailability in combination with the monoamine oxidase B inhibitor, selegiline, in humans—a non-randomized, single-sequence, two-period titration, open label single-center phase 1 study (PK-BeST)

Background: Betahistine was registered in Europe in the 1970s and approved in more than 80 countries as a first-line treatment for Menière's disease. It has been administered to more than 150 million patients. However, according to a Cochrane systematic review of betahistine and recent meta-analyses, there is insufficient evidence to say whether betahistine has any effect in the currently approved dosages of up to 48 mg/d. A combination with the monoamine oxidase B (MAO-B) inhibitor, selegiline, may increase the bioavailability of betahistine to levels similar to the well-established combination of L-DOPA with carbidopa or benserazide in the treatment of Parkinson's disease. We investigated the effect of selegiline on betahistine pharmacokinetics and the safety of the combination in humans. Methods: In an investigator-initiated prospective, non-randomized, single-sequence, two-period titration, open label single-center phase 1 study, 15 healthy volunteers received three single oral dosages of betahistine (24, 48, and 96 mg in this sequence with at least 2 days' washout period) without and with selegiline (5 mg/d with a loading period of 7 days). Betahistine serum concentrations were measured over a period of 240 min at eight time points (area under the curve, AUC0-240 min). This trial is registered with EudraCT (2019-002610-39) and ClinicalTrials.gov. Findings: In all three single betahistine dosages, selegiline increased the betahistine bioavailability about 80- to 100-fold. For instance, the mean (±SD) of the area under curve for betahistine 48 mg alone was 0.64 (+/-0.47) h*ng/mL and for betahistine plus selegiline 53.28 (+/-37.49) h*ng/mL. The half-life time of around 30 min was largely unaffected, except for the 24 mg betahistine dosage. In total, 14 mild adverse events were documented. Interpretation: This phase 1 trial shows that the MAO-B inhibitor selegiline increases betahistine bioavailability by a factor of about 80 to 100. No safety concerns were detected. Whether the increased bioavailability has an impact on the preventive treatment of Menière's disease, acute vestibular syndrome, or post-BPPV residual dizziness has to be evaluated in placebo-controlled trials

Effects of acetyl-DL-leucine on cerebellar ataxia (ALCAT trial): study protocol for a multicenter, multinational, randomized, double-blind, placebo-controlled, crossover phase III trial

BACKGROUND: Cerebellar ataxia (CA) is a frequent and often disabling condition that impairs motor functioning and impacts on quality of life (QoL). No medication has yet been proven effective for the symptomatic or even causative treatment of hereditary or non-hereditary, non-acquired CA. So far, the only treatment recommendation is physiotherapy. Therefore, new therapeutic options are needed. Based on three observational studies, the primary objective of the acetyl-DL-leucine on ataxia (ALCAT) trial is to examine the efficacy and tolerability of a symptomatic therapy with acetyl-DL-leucine compared to placebo on motor function measured by the Scale for the Assessment and Rating of Ataxia (SARA) in patients with CA. METHODS/DESIGN: An investigator-initiated, multicenter, European, randomized, double-blind, placebo-controlled, 2-treatment 2-period crossover phase III trial will be carried out. In total, 108 adult patients who meet the clinical criteria of CA of different etiologies (hereditary or non-hereditary, non-acquired) presenting with a SARA total score of at least 3 points will be randomly assigned in a 1:1 ratio to one of two different treatment sequences, either acetyl-DL-leucine (up to 5 g per day) followed by placebo or vice versa. Each sequence consists of two 6-week treatment periods, separated by a 4-week wash-out period. A follow-up examination is scheduled 4 weeks after the end of treatment. The primary efficacy outcome is the absolute change in the SARA total score. Secondary objectives are to demonstrate that acetyl-DL-leucine is effective in improving (1) motor function measured by the Spinocerebellar Ataxia Functional Index (SCAFI) and SARA subscore items and (2) QoL (EuroQoL 5 dimensions and 5 level version, EQ-5D-5 L), depression (Beck Depression Inventory, BDI-II) and fatigue (Fatigue Severity Score, FSS). Furthermore, the incidence of adverse events will be investigated. DISCUSSION: The results of this trial will inform whether symptomatic treatment with the modified amino-acid acetyl-DL-leucine is a worthy candidate for a new drug therapy to relieve ataxia symptoms and to improve patient care. If superiority of the experimental drug to placebo can be established it will also be re-purposing of an agent that has been previously used for the symptomatic treatment of dizziness. TRIAL REGISTRATION: The trial was prospectively registered at www.clinicaltrialsregister.eu (EudraCT no. 2015-000460-34) and at https://www.germanctr.de (DRKS-ID: DRKS00009733 )

Sulfoquinovosyldiacylglycerides - antiviral active substances

Die Substanzklasse der Sulfoquinovosyldiacylglyceride (SQDG) stellt eine Gruppe potentiell antiviraler Wirkstoffe aus phototrophen Mikroorganismen dar, an dessen Beispiel in der vorliegenden Arbeit eine erfolgreiche Strategie der Entwicklung von entsprechenden Wirkstoffkandidaten aufgezeigt wird. Ausgehend von Literaturdaten sind potentielle SQDG-bildende Stämme aus den Algenabteilungen Rhodophyta (Porphyridium purpureum), Heterokontophyta (Heterosigma carterae, verschiedene Spezies Phaeodactylum tricorntum), Chlorophyta (Chlorella kessleri, Chlorella salina, Chlorella vulgaris) und Cyanophyta (verschiedene Spezies Arthrospira platensis, Scytonema hofmanni, Nostoc punctiforme) selektiert und zur Biomassegewinnung in 1 l Photobioreaktor-Screening-Modulen kultiviert worden. P. purpureum kann in den Untersuchungen bereits frühzeitig als SQDG-Produzent identifiziert werden. Die Biomasse der Alge bzw. der daraus gewonnene Extrakt wird daher zur Etablierung der Trennverfahren für die Gewinnung von SQDG und deren Strukturcharakterisierung mit matrix assisted laser desorption ionization (MALDI) trap time of flight (ToF) Hybrid MSn Massenspektrometrie eingesetzt. Als Aufreinigungsmethoden der SQDG eignen sich die Dünnschichtchromatographie (DC) als auch ein Adsorptions/ Desorptions-Trennverfahren mit aminopropylmodifiziertem Kieselgel. Im Rahmen von MALDI-MSn-Experimenten können die vorkommenden SQDG von P. purpureum in ihrer Struktur identifiziert werden. Die SQDG-Molekülionen werden im MS1 und die am Glycerol veresterten Acylreste C16:0, C18:2, C20:4 und C20:5 im MS2 bestimmt. Ein zusätzlicher enzymatischer Verdau der SQDG mit der regioselektiven Lipase aus Rhizopus arrhizus resultiert in der Hydrolyse der sn-1-veresterten ungesättigten Fettsäuren. Ableitend daraus kann die massenspektrometrische Eliminierung der ungesättigten Fettsäuren an dieser Position (MS2) geklärt werden. Die Sulfoquinovose als charakteristisches Strukturelement wird im MS3-Experiment bestätigt. Die MALDI-Untersuchungen identifizieren zudem SQDG in den Mikroalgen H. carterae und den Spezies von P. tricorntum sowie den Cyanobakterien S. hofmanii, N. punctiforme und einem Spezies von A. platensis. Die SQDG aus den Mikroalgen variieren in der sn-1-veresterten ungesättigten Fettsäure. An sn-2-Position der SQDG ist in den Mikroalgen ein Palmitoylrest lokalisiert, der einen prokaryotischen Biosyntheseweg der SQDG in eukaryotischen Mikroorganismen kennzeichnet. Die SQDG-produzierenden Cyanobakterien A. platensis und N. punctiforme können SQDG sowohl prokaryotisch als auch eukaryotisch (C18-Fettsäure an sn-2-Position) synthetisieren. Zusätzlich zu den SQDG kann in den selektierten P. tricornutum-Stämmen die Bildung von acylierten Sulfoquinovosyldiacylglyceriden (Ac-SQDG) mit der MALDI ToF Analytik nachgewiesen werden. Die Ac-SQDG besitzen im Gegensatz zu den SQDG an der 2´-Position der Sulfoquinovose eine zusätzlich veresterte Fettsäure. Zur Untersuchung der antiviralen Aktivität der SQDG ist aufgrund der sehr hohen klinischen Relevanz das humane Cytomegalievirus (HCMV) als Targetvirus ausgewählt und eine Immunoperoxidasefärbung (POX) im zellkulturbasierten Assay mit MRC-5-Fibroblasten etabliert worden. Dabei wird die Viruslast über die Detektion der viralen Immediate Early (IE) Proteine ermittelt. IE-Proteine werden direkt nach dem Eindringen des Virus in die Wirtszelle gebildet. Eine Reduktion der detektierten viralen IE-Proteine in den MRC-5-Zellen lässt damit auf eine Inhibierung der Virusreplikation zu einem frühen Zeitpunkt (Adsorption, Fusion) schließen. Für die Reduktion der HCMV-Replikation um 50 % (IC50) kann nach Inkubationen von isolierten SQDG aus verschiedenen phototrophen Mikroorganismen mittels IE-POX-Assay ein Konzentrationsbereich von 33 bis 93 µg ml-1 bestimmt werden. Die SQDG-Fraktion von S. hofmanii (SQDG C18:2/C16:0) zeigt mit einer ermittelten IC50 von 19 µg ml-1eine höhere antivirale Aktivität. Dieser Wert unterscheidet sich damit im Vergleich zur Referenzsubstanz Dextransulfat (IC50 1,6 µg ml-1) um eine Größenordnung. Die Inhibierung der HCMV-Viruslast kann zudem für die isolierten Ac-SQDG-Fraktionen nachgewiesen werden. Untersuchungen zur Optimierung der SQDG-Produktion sind am Beispiel von A. platensis unter Variation der Prozessparameter Bestrahlungsstärke (30, 60, 80, 110, 140 µE m-2 s-1) und Phosphatquelle im Medium durchgeführt worden. Die maximale Raum-Zeit-Ausbeute (RZA) von 0,16 mgSQDG l-1 d-1 resultiert bei einer Kultivierung mit 110 µE m-2 s-1. Durch Sublimation von K2HPO4 durch K2P2O7 im Medium bei entsprechenden Kultivierungsbedingungen kann die RZA um 20 % gesteigert werden.Sulfoquinovosyldiacylglycerides (SQDG) represent a class of potential antiviral agents from phototrophic microorganisms which are used in order to demonstrate a successful strategy for the development of drug candidates. Based on literature studies, potential SQDG-producing strains have been selected from different alga phyla like Rhodophyta (Porphyridium purpureum), Heterokontophyta (Heterosigma carterae, different species of Phaeodactylum tricorntum), Chlorophyta (Chlorella kessleri, Chlorella salina, Chlorella vulgaris) and Cyanophyta (several species of Arthrospira platensis, Scytonema hofmanni, Nostoc punctiforme). The algae have been cultivated for biomass production in 1 l Photobioreactor-Screening-Modules. P. purpureum has been identified as a SQDG-producer during an early stage of the analyses. Therefore the biomass of the alga or rather the obtained extract were used to establish a separation processes in order to purify SQDG and to perform structural characterization using matrix assisted laser desorption ionization (MALDI) trap time of flight (ToF) hybrid MSn mass spectrometry. Appropriate purification methods for SQDG are thin-layer chromatography (TLC) as well as an Adsorption/Desorption separation method with aminopropyl modified silica gel. SQDG-constitutions from P. purpureum has been characterized by MALDI MSn-experiments, whereas MS1 experiments point out the molecular weight. Esterified acyl groups C16:0, C18:2, C20:4 und C20:5 are identified by MS2. Additional enzymatic digestion of SQDG with regio-selective Lipase from Rhizopus arrhizus results in hydrolysis of sn-1-esterified unsaturated fatty acids. Based on this fact, mass spectrometric elimination of unsaturated fatty acids at this position (MS2) can be elucidated. Sulfoquinovose as a characteristic structure element of SQDG is confirmed by MS3-experiments. Using MALDI MSn analyses SQDG could also be identify in extracts of the microalgae H. carterae and in the species of P. tricorntum as well as in the cyanobacteria S. hofmanii, N. punctiforme and one species of A. platensis. SQDG from microalgae vary in their sn-1-esterified unsaturated fatty acid. At sn-2-position of SQDG a palmitoyl residue is localized which marks a prokaryotic biosynthesis path of SQDG in eukaryotic microorganisms. SQDG-producing cyanobacteria A. platensis and N. punctiforme synthesize SQDG via prokaryotic as well as eukaryotic pathway (C18 fatty acid at sn-2-position). In addition to SQDG, the production of acylated sulfoquinovosyldiacylglycerides (Ac-SQDG) can be identified in selected strains of P. tricornutum by MALDI ToF analysis. Contrary to SQDG, Ac-SQDG have an additional esterified fatty acid at 2´-position of sulfoquinovose. The human herpes virus Cytomegalovirus (HCMV) was focussed as target virus concerning antiviral investigations. In order to analyze anti-HCMV activities of SQDGs, a virus-specific in vitro assay based on the inhibition of early steps of infection was established. Thereby, the viral load is determined by detection of viral Immediate Early (IE) proteins. IE proteins are produced in the MRC-5 host cells directly after HCM-virus penetration. The reduction of viral IE-proteins in host cells records an inhibition of an early step of viral replication (viral adsorption/fusion). By using the IE-POX-Assay the incubations of isolated SQDG from various phototrophic microorganisms results in a reduction of HCMV replication of 50 % (IC50) within the used SQDG-concentration range of 33 bis 93 µg ml-1. SQDG fraction of S. hofmanii (SQDG C18:2/C16:0) shows a higher antiviral activity, as a IC50 of 19 µg ml-1 is determined. This value varies just in one order of magnitude compared to reference agent dextransulfate (IC50 1,6 µg ml-1). Beside antiviral activity of SQDG, the inhibition of virus load can also be determined for isolated Ac-SQDG fractions. Optimization studies of SQDG-production have been performed by variation of irradiance (30, 60, 80, 110, 140 µE m-2s-1) and phosphate source in medium, taking A. platensis as an model organism. The maximum space-time yield (STY) of 0.16 mgSQDG l-1 d-1 can be achieved by a cultivation with an irradiance of 110 µE m-2s-1. Furthermore STY can be increased by 20 % at appropriate cultivation conditions by sublimation of K2HPO4 in the medium by K2P2O7

Bortezomib antagonizes microtubule-interfering drug-induced apoptosis by inhibiting G2/M transition and MCL-1 degradation

Inhibition of the proteasome is considered as a promising strategy to sensitize cancer cells to apoptosis. Recently, we demonstrated that the proteasome inhibitor Bortezomib primes neuroblastoma cells to TRAIL-induced apoptosis. In the present study, we investigated whether Bortezomib increases chemosensitivity of neuroblastoma cells. Unexpectedly, we discover an antagonistic interaction of Bortezomib and microtubule-interfering drugs. Bortezomib significantly attenuates the loss of cell viability and induction of apoptosis on treatment with Taxol and different vinca alkaloids but not with other chemotherapeutics, that is, Doxorubicin and Cisplatinum. Importantly, Bortezomib inhibits G2/M transition by inhibiting proteasomal degradation of cell cycle regulatory proteins such as p21, thereby preventing cells to enter mitosis, the cell cycle phase in which they are most vulnerable to antitubulin chemotherapeutics. Consequently, Bortezomib counteracts Taxol-induced mitotic arrest and polyploidy, as shown by reduced expression of PLK1 and phosphorylated histone H3. In addition, Bortezomib antagonizes Taxol-mediated degradation of MCL-1 during mitotic arrest by preventing cells to enter mitosis and by inhibiting the proteasome. Downregulation of MCL-1 is critically required for Taxol-induced apoptosis, as overexpression of a phosphomutant MCL-1 variant, which is resistant to degradation, significantly diminishes Taxol-triggered apoptosis. Vice versa, attenuation of Bortezomib-mediated accumulation of MCL-1 by knockdown of MCL-1 significantly enhances Taxol/Bortezomib-induced apoptosis. Thus, Bortezomib rescues Taxol-induced apoptosis by inhibiting G2/M transition and mitigating MCL-1 degradation. The identification of this antagonistic interaction of Bortezomib and microtubule-targeted drugs has important implications for the design of Bortezomib-based combination therapies

Safety and Efficacy of Acetyl-DL-Leucine in Certain Types of Cerebellar Ataxia: The ALCAT Randomized Clinical Crossover Trial

Importance Cerebellar ataxia is a neurodegenerative disease impairing motor function characterized by ataxia of stance, gait, speech, and fine motor disturbances. Objective To investigate the efficacy, safety, and tolerability of the modified essential amino acid acetyl-DL-leucine in treating patients who have cerebellar ataxia. Design, Setting, and Participants The Acetyl-DL-leucine on Cerebellar Ataxia (ALCAT) trial was an investigator-initiated, multicenter, double-blind, randomized, placebo-controlled, clinical crossover trial. The study was conducted at 7 university hospitals in Germany and Austria between January 25, 2016, and February 17, 2017. Patients were aged at least 18 years and diagnosed with cerebellar ataxia of hereditary (suspected or genetically confirmed) or nonhereditary or unknown type presenting with a total score of at least 3 points on the Scale for the Assessment and Rating of Ataxia (SARA). Statistical analysis was performed from April 2018 to June 2018 and January 2020 to March 2020. Interventions Patients were randomly assigned (1:1) to receive acetyl-DL-leucine orally (5 g per day after 2 weeks up-titration) followed by a matched placebo, each for 6 weeks, separated by a 4-week washout, or vice versa. The randomization was done via a web-based, permuted block-wise randomization list (block size, 2) that was stratified by disease subtype (hereditary vs nonhereditary or unknown) and site. Main Outcomes and Measures Primary efficacy outcome was the absolute change of SARA total score from (period-dependent) baseline to week 6. Results Among 108 patients who were randomly assigned to sequence groups (54 patients each), 55 (50.9%) were female; the mean (SD) age was 54.8 (14.4) years; and the mean (SD) SARA total score was 13.33 (5.57) points. The full analysis set included 105 patients (80 patients with hereditary, 25 with nonhereditary or unknown cerebellar ataxia). There was no evidence of a difference in the mean absolute change from baseline to week 6 in SARA total scores between both treatments (mean treatment difference: 0.23 points 95{\%} CI, -0.40 to 0.85 points). Conclusions and Relevance In this large multicenter, double-blind, randomized, placebo-controlled clinical crossover trial, acetyl-DL-leucine in the investigated dosage and treatment duration was not superior to placebo for the symptomatic treatment of certain types of ataxia. The drug was well tolerated; and ALCAT yielded valuable information about the duration of treatment periods and the role of placebo response in cerebellar ataxia. These findings suggest that further symptom-oriented trials are needed for evaluating the long-term effects of acetyl-DL-leucine for well-defined subgroups of cerebellar ataxia. Trial Registration EudraCT 2015-000460-34

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines

The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells.

Cytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells.CIK cells were activated using IL-2 (CIK(IL-2)) or IL-15 (CIK(IL-15)) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay.CIK(IL-2) cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIK(IL-15) cells remained low. Further analysis of CIK(IL-15) cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIK(IL-15) cell-mediated cytotoxicity. In this context, CD3 (+) CD8 (+) CD25 (+) CD56(-) CIK(IL-15) cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 (+) CIK(IL-15) cells, which showed the highest cytotoxic potential against ALL cells.This study provides the first evidence that CIK(IL-15) cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

<div><p>We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of <em>MDR1</em> and <em>MRP1</em>, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.</p> </div

PI103 sensitizes RD cells to DOX-induced apoptosis.

<p><b>A)</b> Levels of phospho-Akt, Akt, phospho-S6 ribosomal protein and S6 ribosomal protein were measured by Western blot analyses in RD, TP5014 and HT1080 cells after treatment with 1 µM or 3 µM PI103 as indicated. <b>B)</b> Upper panel: Proliferation was estimated by BrdU incorporation after treatment with 0.5 µM DOX and/or 3 µM PI103. Middle panel: Apoptosis was analyzed by FACS after treatment with 1 µM DOX and/or 3 µM PI103 or solvent. Data represent mean+SEM of at least three independent experiments performed in triplicates. Comparisons were made with ANOVA/Tukey’s testing. *<i>P</i><0.05 compared to cells treated with solvent; #<i>P</i><0.05 compared to cells treated with either drug alone. Lower panel: Protein levels of phospho-Akt, Akt and caspase 3 in cells treated with 1 µM DOX and/or 3 µM PI103 or respective solvents.</p