Uncoupling of Cytochrome P450 2B6 and stimulation of reactive oxygen species production in pharmacogenomic alleles affected by interethnic variability.

Cytochrome P450 mediated substrate metabolism is generally characterized by the formation of reactive intermediates. In vitro and in vivo reaction uncoupling, results in the accumulation and dissociation of reactive intermediates, leading to increased ROS formation. The susceptibility towards uncoupling and altered metabolic activity is partly modulated by pharmacogenomic alleles resulting in amino acid substitutions. A large variability in the prevalence of these alleles has been demonstrated in CYP2B6, with some being predominantly unique to African populations. The aim of this study is to characterize the uncoupling potential of recombinant CYP2B6*1, CYP2B6*6 and CYP2B6*34 metabolism of specific substrates. Therefore, functional effects of these alterations on enzyme activity were determined by quantification of bupropion, efavirenz and ketamine biotransformation using HPLC-MS/MS. Determination of H2O2 levels was performed by the AmplexRed/horseradish peroxidase assay. Our studies of the amino acid substitutions Q172H, K262R and R487S revealed an exclusive use of the peroxide shunt for the metabolism of bupropion and ketamine by CYP2B6*K262R. Ketamine was also identified as a trigger for the peroxide shunt in CYP2B6*1 and all variants. Concurrently, ketamine acted as an uncoupler for all enzymes. We further showed that the expressed CYP2B6*34 allele results in the highest H2O2 formation. We therefore conclude that the reaction uncoupling and peroxide shunt are directly linked and can be substrate specifically induced with K262R carriers being most likely to use the peroxide shunt and R487S carrier being most prone to reaction uncoupling. This elucidates the functional diversity of pharmacogenomics in drug metabolism and safety.

Nutrimetric Validation of Solanidine as Dietary-Derived CYP2D6 Activity Marker In Vivo.

CYP2D6 is involved in the metabolism of many drugs. Its activity is affected by pharmacogenetic variability leading to highly polymorphic phenotypes between individuals, affecting safety and efficacy of drugs. Recently, solanidine, a steroidal alkaloid from potatoes, and its metabolites, has been identified as a dietary-derived activity marker for CYP2D6. The intraday variability in plasma within individuals has not been studied yet in healthy subjects. As part of a CYP phenotyping cocktail study with 20 healthy participants, plasma concentrations of solanidine, 4-OH-solanidine and 3,4-secosolanidine-3,4-dioic acid (SSDA) were determined using a sensitive liquid chromatography-mass spectrometry method in urine and in plasma at timepoints 0, 2.5, 5, 8, and 24 hours after intake of test substances. The participants were phenotyped for CYP2D6 with oral metoprolol (12.5 mg) with 15 plasma sampling points over 24 hours (DRKS00028922). Metabolic ratios (MRs) of metabolite to parent plasma concentrations were formed from single timepoints and the area under the curve (AUC). All participants were genotyped for CYP2D6. The intra-individual variability of the CYP2D6 metabolite SSDA was highly stable with a median SD of 11.62% over 24 hours. MR SSDA/solanidine was more variable (median SD 31.90%) but correlated significantly at all measured timepoints with AUC MR α-OH-metoprolol/metoprolol. The AUC MR SSDA/solanidine showed a significant linear relationship with the genetically predicted CYP2D6 activity score. This study substantiates the MR SSDA/solanidine as CYP2D6 activity marker. The high correlation with metoprolol MR indicates a valid prediction of the CYP2D6 phenotype at any timepoint during the study day.

Two subtypes of compulsive sexual behavior disorder.

Models explaining addictive behaviors such as the Interaction of Person-Affect-Cognition-Execution (I-PACE) model emphasize the importance of reinforcement mechanisms for developing and maintaining these behaviors, including compulsive sexual behavior disorder (CSBD) as well as personal characteristics as vulnerability factors. This study aimed to determine whether there are CSBD subtypes distinguished by reinforcement sensitivity. We hypothesize that one subtype is sensitive to positive reinforcement (C+subtype) and one is sensitive to negative reinforcement (È»-subtype). We calculated a cluster analysis with data from 62 patients with CSBD and tested differences between the identified clusters by t-test. The sample consisted only of men. Cluster variables were: the sensitivity to the Behavioral Inhibition and Approach System (BIS/BAS), the severity of depressive symptoms (BDI-II), the severity of Trait Anxiety (STAI-T), Sexual Sensation Seeking (SSSS), Thrill- and Adventure-Seeking (SSS-V subscale), Disinhibition (SSS-V subscale), Experience Seeking (SSS-V subscale), and Boredom Susceptibility (SSS-V subscale). Between-cluster differences were analyzed for Trait Sexual Motivation (TSMQ) and Sexual Compulsivity (SCS). The results showed a two-cluster solution with cluster 1 representing patients sensitive to negative reinforcement (È»-subtype) and cluster 2 representing patients sensitive to positive reinforcement (C+subtype). No significant difference in symptom severity of Sexual Compulsivity between clusters was found. Cluster 2 showed higher Importance of Sex and a higher motivation to seek sexual encounters than cluster 2. We found a two-cluster solution regarding reinforcement sensitivity in patients with CSBD. This may have clinical implications regarding individual therapy by focusing on the underlying maintenance mechanisms.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Pharmacogenetic and drug interaction aspects on ketamine safety in its use as antidepressant - implications for precision dosing in a global perspective.

Ketamine and its enantiomer S-ketamine (esketamine) are known to produce rapid-onset antidepressant effects in major depression. Intranasal esketamine has recently come onto the market as an antidepressant. Besides experience from short-term use in anaesthesia and analgesia, the experience with ketamine as long-term medication is rather low. The use of ketamine and esketamine is limited due to potential neurotoxicity, psychotomimetic side effects, potential abuse and interindividual variability in treatment response including cessation of therapy. Therefore, taking a look at individual patient risks and potential underlying variability in pharmacokinetics may improve safety and dosing of these new antidepressant drugs in clinical practice. Differential drug metabolism due to polymorphic cytochrome P450 (CYP) enzymes and gene-drug interactions are known to influence the efficacy and safety of many drugs. Ketamine and esketamine are metabolized by polymorphic CYP enzymes including CYP2B6, CYP3A4, CYP2C9 and CYP2A6. In antidepressant drug therapy, usually multiple drugs are administered which are substrates of CYP enzymes, increasing the risk for drug-drug interactions. We reviewed the potential impact of polymorphic CYP variants and common drug-drug interactions in antidepressant drug therapy affecting ketamine pharmacokinetics, and the role for dose optimization. The use of ketamine or intranasal esketamine as antidepressants demands a better understanding of the factors that may impact its metabolism and efficacy in long-term use. In addition to other clinical and environmental confounders, prior information on the pharmacodynamic and pharmacokinetic determinants of response variability to ketamine and esketamine may inform on dose optimization and identification of individuals at risk of adverse drug reactions.

Does Intracellular Metabolism Render Gemcitabine Uptake Undetectable in Mass Spectrometry?

The ergothioneine transporter ETT (formerly OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC-MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC-MS/MS measurements when we expressed the Na+/nucleoside cotransporter CNT3 (SLC28A3). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2'-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.

[Impact of the COVID-19 pandemic on urology residency training programs in Germany]. / Auswirkungen der COVID-19 Pandemie auf die urologische Weiterbildung in Deutschland.

BACKGROUND: Several international medical societies reported a negative impact on urology residency training programs due to the COVID-19 pandemic. OBJECTIVES: The aim of this study was to investigate the impact of the pandemic on urological residency in Germany. MATERIALS AND METHODS: From the 20th of May 2020 until the 20th of June 2020, a Germany-wide online survey on the continuing residency training was distributed via the members of the working group, social media (Facebook, Twitter, Instagram) and the German Society of Residents in Urology (GeSRU e.V.) newsletter. The survey covered 3 topics: 1) basic characteristics of the participants, 2) general and 3) subjective influence of the COVID-19 pandemic on clinics and further residency training. RESULTS: A total of 50 residents took part in the survey; 54% were women. The median age was 31 years. Most of the participants were in their 2nd (22%) and 5th (26%) year of training and worked in a university hospital (44%) or in a clinic of maximum care (30%). 38% of the respondents stated that they only served urological emergencies during the COVID-19 pandemic. For 28% this meant a very large delay (80-100%) in the specialisation, while 28% stated only a minor impact. 66% documented training impairments caused by fewer operations, low patient numbers in the outpatient department (50%), congress (50%) and workshop (44%) cancellations. 46% of residents reported direct contact with COVID-19 patients while 10% were deployed on interdisciplinary IMC units. Numerous physical distancing and hygiene measures have been implemented by the clinics. CONCLUSION: On average, around 50% of the urology residents indicated significant restrictions in training due to the COVID-19 pandemic in Germany. The delay in training cannot currently be measured in units of time, but it can be assumed that training for residents during the pandemic is likely to be of a lower quality compared to previous generations.

[Osteoprotection in the management of metastatic prostate cancer: real-world data from Germany and decision guidance]. / Osteoprotektion als Baustein der Therapie des metastasierten Prostatakarzinoms: Behandlungsrealität in Deutschland und Entscheidungspfade zur Therapieoptimierung.

OBJECTIVE: Osteoprotective medications are a key element not only in the management of bone metastases of castration-resistant prostate cancer (mCRPC) but also of hormone-sensitive prostate cancer (mHSPC). Additionally, osteoprotective drugs can prevent androgen deprivation-induced bone loss. The aim of this study was to illustrate the practice pattern of osteoprotection for prostate cancer patients in Germany. MATERIAL AND METHODS: We designed an online survey consisting of 16 questions. The survey was sent to the nation-wide working groups "Oncology" and "Uro-Oncology" as well as to colleagues from the departments of urology of University Hospital Schleswig-Holstein (Campus Lübeck), Academic Hospital Brunswick and Technical University of Munich. Furthermore, we developed flow charts for decision guidance for osteoprotection within the different stages of prostate cancer. RESULTS: Our analysis demonstrates a routine use of osteoprotection in the management of bone metastases of mCRPC. In contrast, osteoprotective medications are less often used for the treatment of bone metastases of mHSPC and for the prevention of androgen deprivation-induced bone loss. Our flow charts depict the different dosages and intervals for the administration of osteoprotective drugs in the different stages of prostate cancer. CONCLUSIONS: Osteoprotection is not only confined to mCRPC with bone metastases. It plays a crucial role in the management of all stages of metastatic prostate cancer.

RLC score (R status, lymphovascular invasion, C-reactive protein) predicts survival following radical cystectomy for muscle-invasive bladder cancer.

BACKGROUND: CRP-based scoring systems were found to correlate with survival in patients with urooncologic diseases. Our retrospective single-centre study aimed to confirm CRP as a prognostic parameter in patients with bladder cancer (BCa) undergoing radical cystectomy (RC) and, based on the findings, to develop our own outcome score for muscle-invasive bladder cancer (MIBC) patients undergoing RC in order to identify patients with a high risk of mortality. MATERIAL AND METHODS: A total of 254 patients who underwent RC at Hanover Medical School between 1996 and 2007 were reviewed with a follow-up until autumn 2013. The clinicopathologic parameters assessed included age, co-morbidities, pre-/postoperative serum levels of CRP, leukocytes, haemoglobin, creatinine, urinary diversion, tumour grading, staging, lymph node status, lymph node density (LND), lymphovascular invasion (LVI), metastases, and resection margin status. The Chi-square test was used for univariate analyses. Kaplan-Meier estimates and the log-rank test were used for survival analyses. Regarding outcome, overall survival (OS) was assessed. RESULTS: The multivariate analysis excluding lymph node (LN)-positive and metastatic patients at time of RC showed a significant association of R status (R; p < 0.001), LVI (L; p = 0.021) and preoperative CRP level > 5 mg/l (C; p = 0.008) with OS. Based on these parameters, the RLC score was developed. The median OS in the intermediate, high-risk and very high-risk groups according to the RLC score was 62, 22, and 6.5 months, respectively. The score had a high predictive accuracy of 0.752. CONCLUSION: The RLC score identifies BCa patients at a higher risk of overall mortality after RC. Overall, our study supports the role of CRP in prognostic score models for BCa.

Hyperosmolarity stimulates transporter-mediated insertion of estrone sulfate into the plasma membrane, but inhibits the uptake by SLC10A1 (NTCP).

Many drugs are largely hydrophobic molecules; a transporter might conceivably insert these into the plasma membrane. At least 18 transporters from diverse families have been reported to transport the model compound estrone sulfate alias estrone-3-sulfate (E3S). Out of these, we recently examined SLC22A11 (OAT4). We concluded from a comparison of E3S and uric acid transport that SLC22A11 does not translocate E3S into the cytosol, but into the plasma membrane. Here we present a hyperosmolarity alias hypertonicity assay to differentiate transport mechanisms. Human transporters were expressed heterologously in 293 cells. Solute uptake into intact cells was measured by LC-MS. Addition of mannitol or sucrose led to rapid cell shrinkage, but cell viability after 60 min in hyperosmolar buffer was not impaired. A decrease in substrate accumulation with increasing osmolarity as observed here for several substrates and the transporters SLC22A11, ETT (SLC22A4), OCT2 (SLC22A2), OAT3 (SLC22A8), and MATE1 (SLC47A1) suggests regular substrate translocation into the cytosol. An increase as observed for E3S transport by SLC22A11, OAT3, MATE1, SLC22A9, and SLC10A6 implies insertion into the membrane. In marked contrast to the other E3S transporters, the bile acid transporter SLC10A1 (NTCP, Na+ taurocholate co-transporting polypeptide) showed a decrease in the accumulation of E3S in hyperosmolar buffer; the same was observed with taurocholic acid. Indeed, our data from several functional assays strongly suggest that the transport mechanism is identical for both substrates. Apparently, a unique transport mechanism has been established for SLC10A1 by evolution that ensures the transport of amphipathic, detergent-like molecules into the cytosol.

[Opportunities and risks of a contemporary online presence for urologists]. / Chancen und Grenzen eines zeitgemäßen Onlineauftrittes für Urologen.

BACKGROUND: An adequate online presence is essential for any medical practice. Studies have shown that patients increasingly use the internet for medical information, to search for physicians and to use online services. Expert associations and journals use social media to maximise their online reach. OBJECTIVES: This study presents chances and risks of an online presence for urologists. RESULTS: A professional and visually appealing website is key to modern doctor-patient communication. When developing a professional digital identity, one must consider technical aspects as well as legal requirements. Recommendations and guidelines have been put in place to give guidance, e. g. on social media strategies or the development of a websites content design. Medical professionals need in-depth consultation, especially regarding the complex legal requirements. CONCLUSION: Content published online must be handled thoughtfully - no matter what digital medium is used. It is advisable to strictly separate private and professional online presences. Furthermore, the goals regarding an online presence should be regularly reevaluated and, if necessary, adjusted.

[The importance of digitalisation in urology]. / Stellenwert der Digitalisierung im urologischen Alltag.

The Internet has shaped and changed society like no other technology. Culturally, the emergence of the Internet is being described as having the same impact on society as the invention of printing. In 2018, more than 4 billion people had access to the Internet. Among all Internet users, approximately 80 % search the Internet for health-related information, with cancer being the most frequently searched condition. Patients rate the Internet as the second most helpful source of information, outranked only by consultation with a medical doctor. There are more than 2.6 billion active social media users. Among urological residents, 97 % use social media on a regular basis. Digitalisation has the potential to strengthen patients' health literacy and optimise patient care, especially in the oncologic field. In summary, digitalisation bears an enormous potential for the field of urology.

Eosinophilic oesophagitis--a guide for primary care.

BACKGROUND: Eosinophilic oesophagitis (EoE) is an increasingly recognised inflammatory disorder of the oesophagus. The incidence of this chronic, food-triggered disease is rising in Australia and diagnosis should be considered in patients who complain of dysphagia. OBJECTIVE: This article provides an overview of the pathogenesis, evaluation and management of EoE. It is intended as a useful tool for general practitioners (GPs) in the primary care setting and to raise awareness of EoE. DISCUSSION: EoE is an increasingly prevalent allergic disorder of the oesophagus. It is characterised clinically by symptoms of oesophageal dysfunction and microscopically by eosinophilic inflammation of the oesophagus. Treatment involves avoiding trigger foods and corticosteroid therapy, which may be necessary intermittently or long term, given the relapsing nature of the condition. EoE can significantly impair quality of life and GPs are key to recognising the disease and directing patients' long-term care.

Patient perceptions of nonmelanoma skin cancer before diagnosis.

BACKGROUND: There is a paucity of data regarding patient perceptions of nonmelanoma skin cancer (NMSC). OBJECTIVE: To describe patients' perceptions of skin lesions before a diagnosis of NMSC. METHODS: This was a descriptive study in a private practice setting. Patients with a previous biopsy of NMSC who presented for treatment were eligible. A self-administered questionnaire assessed what patient perceptions of lesions diagnosed as NMSCs had been before they were aware of the diagnosis. Medical records were reviewed for tumor type, size, and location. RESULTS: One hundred sixty-three consecutive patients undergoing treatment for NMSC completed the questionnaire. The most common initial impressions of the lesion were skin cancer (20%), acne (19%), sore (10%), unknown (9%), dry skin (7%), age spot (6%), and injury (6%). Seventy-two percent of patients were the first to notice the lesion. Patients with a history of skin cancer were more likely to think the lesion was a skin cancer on initial impression (28% vs. 8%) (p < .001). CONCLUSIONS: Understanding how patients perceive their skin cancers may aid in targeting educational strategies and increase awareness of skin cancer risk. Our data suggest that there are important subtleties in self-identification that may need to be taken into consideration in any educational campaign targeting NMSC.

A randomized controlled trial of a controlled breathing protocol on heart rate variability following myocardial infarction or coronary artery bypass graft surgery.

OBJECTIVES: To determine whether a controlled breathing programme increases heart rate variability following an acute myocardial infarction and/or coronary artery bypass graft surgery. RATIONALE: Heart rate variability is reduced following a myocardial infarction, and low heart rate variability is associated with a high mortality risk. By changing tidal volume and rate of breathing, individuals can alter beat-to-beat heart rate variability. It is hypothesized that heart rate increases with inspiration and decreases with exhalation, and that deep slow breathing enhances respiratory sinus arrhythmia, increasing heart rate variability. DESIGN: Randomized controlled trial. SETTING: Cardiac rehabilitation programme at a large academic medical centre in North Texas. SUBJECTS: From 2001 to 2005, 44 patients, age 46-65 years, who had a myocardial infarction and/or undergone coronary artery bypass graft surgery 1-8 weeks previously and were referred to the Cardiac Rehabilitation Program. INTERVENTION: Patients were randomized to either usual cardiac rehabilitation or cardiac rehabilitation with controlled breathing (6 breaths/min for 10 minutes twice daily during the eight-week treatment period). MAIN MEASURES: Weekly measurements of total power and standard deviation of the mean normal to normal RR interval (SDNN), and fortnightly measurements of respiratory sinus arrhythmia were taken using Biocom Technologies Heart Rhythm Scanner and Tracker software. RESULTS: No significant difference in change were seen between groups in SDNN (P = 0.3984), baseline respiratory sinus arrhythmia (P = 0.6556) or total power (P = 0.6184). CONCLUSION: Results suggest participation in the controlled breathing programme offered no additional benefit in increasing heart rate variability following myocardial infarction or coronary artery bypass graft surgery. However, 77% of study patients were on heart rate-lowering medications, which may have masked changes in heart rate variability.