Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures

Mass spectrometric characterization of urinary hydrafinil metabolites for routine doping control purposes

Little information on the human metabolism and urinary elimination of hydrafinil (9-fluorenol) exists. In order to support preventive anti-doping activities concerning compounds such as hydrafinil, a pilot elimination study was conducted with three healthy male volunteers receiving a single oral dose of 50 mg of hydrafinil. Urine samples were collected prior to and up to 72-h post-administration and were subjected to both gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, which allowed for the identification of the intact drug as well as Phase I and Phase II metabolites, primarily hydroxylated and/or glucuronidated or sulfo-conjugated hydrafinil. The identity of these metabolites was corroborated by high-resolution/high-accuracy tandem mass spectrometry, and the applicability of routine doping control workflows for the detection of hydrafinil and its main metabolites was assessed. Therefore, two findings of hydrafinil and its metabolites were recorded, which concerned out-of-competition doping control samples and, hence, were not pursued with confirmatory analyses. Yet, the initial testing procedure results indicate that hydrafinil might require consideration in sports drug testing programs to ensure its detection, if classified as prohibited by the World Anti-Doping Agency (WADA)

Superior Therapeutic Index in Lymphoma Therapy: CD30(+) CD34(+) Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack

Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30(+) HSPCs while eliminating CD30(+) lymphoma cells. Consequently, normal hemato-and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30(+) HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30(+) malignancies leaving healthy activated lymphocytes and HSPCs unaffected

Elimination profiles of microdosed ostarine mimicking contaminated products ingestion

The possibility of nutritional supplement contamination with minute amounts of the selective androgen receptor modulator (SARM) ostarine has become a major concern for athletes and result managing authorities. In case of an adverse analytical finding (AAF), affected athletes need to provide conclusive information, demonstrating that the test result originates from a contamination scenario rather than doping. The aim of this research project was to study the elimination profiles of microdosed ostarine and characterize the time-dependent urinary excretion of the drug and selected metabolites. Single- and multi-dose administration studies with 1, 10, and 50 mu g of ostarine were conducted, and collected urine samples were analyzed by LC-MS/MS following solid-phase extraction or enzymatic hydrolysis combined with liquid-liquid extraction. In the post-administration samples, both the maximum urine concentrations/abundance ratios and detection times of ostarine and its phase-I and phase-II metabolites were found to correlate with the administered drug dose. With regard to the observed maximum levels of ostarine, the time points of peak urinary concentrations/abundance ratios, and detection windows, a high inter-individual variation was observed. However, the study demonstrated that a single oral dose of as little as 1 mu g can be detected for up to 9 (5) days by monitoring ostarine (glucuronide), and hydroxylated metabolites (especially M1a) appear to offer a considerably shorter detection window. The obtained data on ostarine (metabolite) detection times and urinary concentrations following different administration schemes support the interpretation of AAFs, in particular when scenarios of proven supplement contamination are discussed and supplement administration protocols exist

Depletion of Numb and Numblike in Murine Lung Epithelial Cells Ameliorates Bleomycin-Induced Lung Fibrosis by Inhibiting the beta-Catenin Signaling Pathway

Idiopathic pulmonary fibrosis (IPF) represents the most aggressive form of pulmonary fibrosis (PF) and is a highly debilitating disorder with a poorly understood etiology. The lung epithelium seems to play a critical role in the initiation and progression of the disease. A repeated injury of lung epithelial cells prompts type II alveolar cells to secrete pro-fibrotic cytokines, which induces differentiation of resident mesenchymal stem cells into myofibroblasts, thus promoting aberrant deposition of extracellular matrix (ECM) and formation of fibrotic lesions. Reactivation of developmental pathways such as the Wnt-beta-catenin signaling cascade in lung epithelial cells plays a critical role in this process, but the underlying mechanisms are still enigmatic. Here, we demonstrate that the membrane-associated protein NUMB is required for pathological activation of beta-catenin signaling in lung epithelial cells following bleomycin-induced injury. Importantly, depletion of Numb and Numblike reduces accumulation of fibrotic lesions, preserves lung functions, and increases survival rates after bleomycin treatment of mice. Mechanistically, we demonstrate that NUMB interacts with casein kinase 2 (CK2) and relies on CK2 to activate beta-catenin signaling. We propose that pharmacological inhibition of NUMB signaling may represent an effective strategy for the development of novel therapeutic approaches against PF

Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans

Effective virus neutralization in HSV-1 KOS infected eyes by mAb 2c.

<p>Representatively, six mice from each group were killed at day 5 post infection. Primary infected eyes were homogenized and inspected for viral loads using a standard plaque assay. Statistical analysis was undertaken with a nonparametric ANOVA test. Comparisons were considered significant at *<i>P</i> < 0.05. Error bars represent the SEM.</p

Influence of mAb 2c treatment on the cellular and humoral immune response to HSV-1 of corneally infected mice.

<p>The reduced virus titers correlate with reduced anti-HSV immune responses. The total numbers of cells in draining lymph nodes (DLN) <b>(A)</b> and spleens <b>(B)</b> and the relative antibody titers in 10 µl tear fluids <b>(C)</b> and 60 µl sera <b>(D)</b> of mice on day 14 post infection are shown. Each dot represents a single mouse. Differences between the groups were statistically significant by a nonparametric ANOVA one way test (*<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001). Error bars represent the SEM.</p

MAbs 2c and hu2c restrict cell-to-neuron <b>(A-F)</b> and neuron-to-cell spread of HSV-1 <b>(G-L)</b>.

<p>For the investigation of cell-to-neuron spread, epithelial C127I cells (arrows) were infected with the reporter virus HSV-1(17⁺)Lox-Che. At 14 hpi cells were detached, treated with the indicated antibodies and co-cultured with uninfected DRG sensory neurons (arrowheads) <b>(A-E)</b>. For the investigation of neuron-to-cell transmission, DRG neurons were infected with HSV-1(17⁺)Lox-Che. At 24 hpi, GFP-transfected, uninfected C127I were detached, treated with the indicated antibodies and co-cultured with the infected neurons <b>(G-K)</b>. For both settings, cells were fixed as indicated at 7, 22 and 30 hpi with PHEMO and labeled with anti-β-tubulin-III. Images were acquired with a confocal microscope. For the quantification of the cell-to-neuron <b>(F)</b> or neuron-to-cell transmission <b>(L)</b>, the values of mCherry-fluorescence were determined in a defined area within neurons <b>(F)</b> or GFP-positive C127I cells (G). (A-E and G-K) Scale bar is 10 µm. <b>(F and L)</b>. Error bars show standard error of the mean (SEM) of one representative experiment. Differences between the groups were statistically significant by a nonparametric ANOVA one way test (***<i>P</i> < 0.001).</p