Pharmakologie und Medizinische Chemie Uracil- und Uracilnucleotid-bindender Membranproteine

Im Rahmen der vorliegenden Arbeit wurden zwei Teilprojekte bearbeitet, die sich mit Uracil- bzw. Uracilnucleotid-bindenden Membranproteinen befassen. Im ersten Teilprojekt wurde ein für die Nucleobase Uracil hochaffines und selektives Bindeprotein in der Zellwand des gram-negativen Bakteriums Achromobacter xylosoxidans identifiziert und charakterisiert. Mittels Radioligand-Bindungsstudien wurde das Bindungsverhalten von [3H]Uracil durch Sättigungs-, Kompetitionsexperimente und kinetische Experimente untersucht. Es wurde eine Methode etabliert, das Uracil-Bindeprotein unter Erhalt seiner Bindungsfähigkeit für Uracil aus der Membran zu solubilisieren. Im Rahmen einer Kooperation mit Dr. Sonja Hess (California Institute of Technology, Pasadena, CA, USA) wurden Proteomik-Studien durchgeführt, in welchen das Solubilisat nach proteolytischem Verdau massenspektrometrisch analysiert wurde und eine Entschlüsselung der Aminosäuresequenzen der erhaltenen Peptide erfolgte. Ein Vergleich mit einer Protein-Datenbank für Proteobakterien führte zu dem Ergebnis, dass es sich bei dem identifizierten Uracil-Bindeprotein möglicherweise um ein hochaffines ABC-Transporter assoziiertes periplasmatisches Bindeprotein handeln könnte. Achromobacter xylosoxidans besiedelt natürlicherweise das Erdreich und Gewässer, wird aber auch in Wasserleitungen, Dialyseflüssigkeiten und Chlorhexidinlösungen gefunden. Vor allem bei immunsupprimierten Patienten kann dieses vielfach antibiotikaresistente Bakterium komplizierte Nosokomialinfektionen verursachen. Im zweiten Teilprojekt wurden Serien von Anthrachinon-, Adenosin-, Uridin- und Tetrazol-Derivaten an den G-Protein-gekoppelten P2Y-Rezeptor-Subtypen 2, 4 und 6 in funktionellen Assays zur Messung der intrazellulären Calcium-Freisetzung pharmakologisch charakterisiert. Diese Rezeptoren spielen eine wichtige pathophysiologische Rolle im menschlichen Organismus, und es besteht ein dringender Bedarf an hochaffinen und selektiven Agonisten und Antagonisten. In dieser Arbeit konnten für jeden der drei Rezeptor-Subtypen affine und moderat selektive Antagonisten mit Aktivitäten im einstelligen mikromolaren Konzentrationsbereich gefunden, für die Wirkung wichtige Strukturmerkmale identifiziert und Struktur-Wirkungsbeziehungen herausgearbeitet werden

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use.

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA\u27s Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete\u27s in-competition urine sample represents a substantial risk of an antidoping rule violation

Pilot study on the effects of intravesical oxybutynin hydrochloride instillations on the validity of doping control urine samples

According to class M2.1 of the World Anti-Doping Agency (WADA) Prohibited List, the manipulation of doping control urine samples to alter their integrity and validity is prohibited both in- and out-of-competition. However, some paraplegic athletes with an overactive bladder need to be regularly treated with anti-cholinergic and anti-spasmodic drugs such as oxybutynin, which are often administered intravesically to reduce the substantial side effects observed after oral application. So far, it remains unclear whether such bladder instillations have a negative impact on analytical procedures and thus represent an anti-doping rule violation. Within this pilot study, urine samples were collected from five paraplegic athletes before and after an intravesical oxybutynin hydrochloride instillation. The samples were routinely tested for the presence of performance-enhancing drugs and afterwards fortified with 25 model compounds representing different classes of doping agents (anabolic agents, cannabinoids, diuretics, glucocorticoids, hormone and metabolic modulators, and stimulants) at low and medium concentrations. Additionally, the pH value and specific gravity were measured and the presence of oxybutynin was qualitatively determined by gas chromatography-mass spectrometry (GC-MS). In initial testing procedures, all samples were tested negative. Oxybutynin was present in most of the samples but found to have no significant effect on the detectability of the 25 model compounds subsequently added to each urine specimen. Therefore, it can be concluded that intravesical instillations with oxybutynin hydrochloride do not alter the integrity and validity of doping control urine samples

Ophthalmic drug therapy in professional athletes-what is allowed, what is doping?

At first sight, ophthalmic drugs are not necessarily suspected of being used as performance-enhancing agents in competitive sports. However, there are some restrictions that must be noted, especially when certain drugs are taken orally. A structured analysis of the doping relevance of ophthalmic drugs was performed on the basis of a literature review, taking into account the current guidelines of the National Anti-Doping Agency of Germany and the World Anti-Doping Agency. Use of the most common ophthalmological drug groups is possible without restrictions, especially in the case of topical application. An exception is the oral use of diuretics, which is prohibited at all times. For glucocorticoids, topical application to the eye is allowed, but oral application is prohibited in competition. A similar restriction applies to the use of epinephrin, for which all forms of systemic application are prohibited in competition. When taking beta-blockers, the sport being played is the determining factor: use is prohibited in billiards, archery, darts, golf, motor sports, shooting sports, skiing/snowboarding, ski jumping, freestyle aerials/half pipe, snowboard half pipe/big air, and underwater sports. For shooting and archery, beta-blockers are also prohibited outside of competitions. Athletes with pre-existing ophthalmological conditions should seek comprehensive advice froman ophthalmologist before using any medication, and together they should choose a permissible active ingredient, consider the appropriate route of application, and, if necessary, apply for a Therapeutic Use Exemption. It is currently not known how many of the national and international athletes have to be treated with eye drops

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete's in-competition urine sample represents a substantial risk of an antidoping rule violation