Distribution of care expenditures for men and women with type 2 diabetes treated in primary care in the Netherlands: a case-control study (ZODIAC-59).

OBJECTIVE: This study aims to provide insight into the distribution of care expenditures for patients with type 2 diabetes mellitus (T2DM)- across multiple healthcare service categories and medical specialties-who receive diabetes care in the primary care setting. DESIGN: Observational, matched case-control study. SETTING: In the Netherlands, T2DM-specific care is mainly provided in the primary care setting. However, many patients with T2DM also use secondary care for complications and comorbidities, either related or unrelated to their diabetes. PARTICIPANTS: Patients with T2DM receiving diabetes care in primary care and participating in the Dutch Zwolle Outpatient Diabetes project Integrating Available Care cohort in the year 2011 were matched to persons without T2DM. Matching (1:2 ratio) was performed based on age, gender and socioeconomic status. Clinical data were combined with an all-payer claims database from 2011. RESULTS: In total, 43 775 patients with T2DM were identified of whom 37 240 could be matched with 74 480 controls. Total secondary care expenditures were €94 705 814, with a total annual median expenditure per patient of €2133 (1161 to 3340) for men and €2,535 (1374 to 5105) for women. The largest share of expenditures was on medication (26%), followed by secondary care (23%) and primary care services related (23%) to T2DM. The five most expensive specialties were: cardiology, surgery, internal medicine, orthopaedics and ophthalmology. Care expenditures for T2DM patients were twofold higher than those for persons without T2DM. Healthcare expenditures showed a skewed distribution, indicating that a small part of the studied population is responsible for a considerable part of the costs. CONCLUSIONS: Expenditures among primary care treated T2DM patients are higher than non-diabetic matched controls. Medication is the largest share of T2DM care expenditures. The present study provides insights into healthcare expenditures for T2DM; this may enable more efficient healthcare planning and reimbursement.

In silico and in vitro analysis of genetic variants of the equine CYP3A94, CYP3A95 and CYP3A97 isoenzymes.

Cytochrome P450 enzymes (CYPs) of the equine CYP3A subfamily are predominantly involved in drug metabolism. In this study, genetic variants of the equine CYP3A94, CYP3A95, and CYP3A97 were identified and characterized using in silico modeling and in vitro enzyme kinetics. The genomes of 81 horses were sequenced to obtain the genetic variants. Structural CYP modifications of the most frequent variants were analyzed in silico using the 3D-structures predicted by homology modeling. Enzyme kinetic analyses were performed using testosterone as substrate. Twenty genetic variants were found including five missense variants (CYP3A94:p.Asp217Asn, CYP3A95:p.Asp214His, CYP3A95:p.Ser392Thr, CYP3A97:p.Ile119Thr, CYP3A97:p.Met500Val) with a higher percentage of minor allele frequency (MAF) (range 0.2-0.4). A splice-site variant (c.798 + 1G > A) in CYP3A94, likely to generate a truncated protein, was found in 50% of the horses. CYP3A94:p.Asp217Asn and CYP3A95:p.Asp214His were localized on the CYP F-α-helix, an important region for the substrate interactions in the human CYP3A4. Testosterone 2ß-hydroxylation was diminished in CYP3A94217Asn and CYP3A95392Thr. Ketoconazole inhibited 2ß-hydroxylation differently in the five variants with the most pronounced inhibition obtained for CYP3A95392Thr. In vitro and in silico analyses of genetic variants allow unraveling structural features in equine CYPs that correlate with changes in the CYP activity.

Antimicrobial use for selected diseases in cats in Switzerland.

BACKGROUND: Antibiotic use in human and veterinary medicine is considered a main driver of antimicrobial resistance. Although guidelines to promote appropriate use of antimicrobials in veterinary patients have been developed, antibiotic overprescription is assumed to be a common problem. The goal of this study was to investigate antimicrobial use in cats in Switzerland with acute upper respiratory tract disease (aURTD), feline lower urinary tract disease (FLUTD) and abscesses, and to assess compliance of prescription with consensus guidelines. A total of 776 cases (aURTD, n = 227; FLUTD, n = 333; abscesses, n = 216) presented to two university hospitals and 14 private veterinary practices in Switzerland during 2016 were retrospectively evaluated. Clinical history, diagnostic work-up and antimicrobial prescription (class, dosage, duration) were assessed. RESULTS: A total of 77% (aURTD), 60% (FLUTD) and 96% (abscesses) of the cases received antibiotic therapy; 13-24% received combination or serial therapy. The cats were treated for a median of 7 (abscesses) and 10 days (aURTD, FLUTD). Treatments with potentiated aminopenicillins (40-64%), third generation cephalosporins (25-28%), aminopenicillins (12-24%) and fluoroquinolones (3-13%) were most common. Prescriptions were judged in complete accordance with consensus guidelines in 22% (aURTD), 24% (FLUTD) and 17% (abscesses) of the cases. Antibiotics were prescribed although not indicated in 34% (aURTD), 14% (FLUTD) and 29% (abscesses) of the cases. The presence of lethargy, anorexia or fever in cats with aURTD, and the detection of bacteriuria in cats with FLUTD were significantly associated with antibiotic therapy. Although diagnostic work-up was significantly more common (aURTD: university hospitals, 58%; private practices, 1%; FLUTD: university hospitals, 92%; private practices, 27%) and the use of critically important antibiotics significantly less common at the university hospitals (aURTD, 10%; FLUTD, 14%) compared to private practices (aURTD, 38%; FLUTD, 54%), the frequency of antibiotic treatment was not different between the university hospitals and private practices. CONCLUSIONS: Our results indicate that overprescription of antibiotics in cats in Switzerland is common and accordance with guidelines is poor. The study highlights the need to promote antimicrobial stewardship in small animal medicine.

[Online information system for -phytotherapy in animals]. / Online-Informationssystem für die Phytotherapie bei Tieren.

INTRODUCTION: Phytotherapy becomes increasingly popular in veterinary medicine. To fully exploit the therapeutic potential of medicinal plants and ensure their safe use, knowledge about the effective plant parts and preparations is required. Improper use and overdosage of medicinal plants can be toxic. With www.phytoarznei.ch, we provide an online decision support aid that allows for the retrieval of currently available information on medicinal plants and their use in domestic animals. This decision support system is based on the available literature in the field, which after critical evaluation has been incorporated into a structured database. For each medicinal plant or drug, we have listed therapeutic indications, different application types, organoleptic properties, plant ingredients, pharmacological effects, dosages, duration of treatment, toxicity, legal frame for use in livestock and relevance for doping. A user-friendly access to all data is achieved by means of two search programs, either by entering the plant name or name of the drug in a search field or by selecting the desired animal species and therapeutic application from respective drop-down lists. This database on medicinal plant applications in animals is linked to the poisonous plant database of the University of Zurich and, for marketed preparations, to the Swiss compendium of veterinary medicinal products as well as to an index of related veterinary products.

INTRODUCTION: En médecine vétérinaire aussi, la phytothérapie devient de plus en plus populaire. Exploiter le potentiel thérapeutique des plantes médicinales et assurer leur utilisation en toute sécurité nécessite toutefois une connaissance particulière des parties de plantes ou des préparations efficaces. Une utilisation inappropriée et un surdosage de plantes médicinales peuvent être toxiques. C'est pourquoi nous avons créé un outil de prise de décision en ligne, www.phytoarznei.ch, qui permet un accès rapide aux connaissances actuelles sur les plantes médicinales et leur utilisation sur les animaux. Ce système d'information est basé sur la littérature spécialisée disponible qui a été incorporée, après une évaluation critique, dans une base de données structurée. Les indications thérapeutiques, les modes d'applications, les propriétés organoleptiques, les composants, les effets pharmacologiques, doses, la durée du traitement, la toxicité, les réglementations juridiques chez les animaux de rente ainsi que la pertinence en matière de dopage sont répertoriés pour chaque plante médicinale ou médicament à base de plantes. Deux programmes de recherche fournissent un accès convivial, soit par la saisie du nom de la plante, du nom du médicament ou des ingrédients de la plante dans une zone de recherche, soit en sélectionnant les espèces d'animaux souhaitées ainsi que l'utilisation thérapeutique dans des menus déroulants correspondants. Cette base de données des plantes médicinales est liée avec base de plantes toxiques de l'Université de Zurich et, si des produits finis correspondants sont disponibles, au Compendium des médicaments vétérinaires suisse ainsi qu'à un répertoire de produits vétérinaires.

[AntibioticScout.ch: Decision support for the prudent use of antimicrobials: Application in cattle]. / AntibioticScout.ch: Entscheidungshilfe für den umsichtigen Einsatz von antimikrobiellen Wirkstoffen: Anwendung beim Rind.

INTRODUCTION: The administration of antibiotics in livestock has been criticized for many years, in particular because of an inappropriate use and the appearance of antibiotic residues in the environment, which can promote the emergence and spread of resistant bacteria. However, antibiotics are essential for the successful and sustainable control of bacterial pathogens. With the aim of optimizing the use of antibiotics in food animals and minimizing the prevalence of resistant bacteria, AntibioticScout. ch provides a decision aid for the prudent use of antimicrobial drugs. This approach emphasizes the importance of supportive therapy and the hallmarks of preventive concepts. Procedures to improve animal health and animal welfare in accordance with the principles of good veterinary practice are primary and effective tools to reduce the use of antimicrobial drugs. The necessary reduction in the use of antibiotics must, therefore, be accompanied by appropriate management strategies in animal husbandry. In particular, hygiene, animal welfare and biosecurity measures are crucial to ensure an optimal health status in farm animals.

INTRODUCTION: On discute depuis des années de l'usage des antibiotiques dans l'élevage des animaux de rente, en particulier en ce qui concerne leur utilisation incorrecte et la charge environnementale liée à des résidus d'antibiotiques susceptibles de favoriser l'apparition et la propagation de résistances. Toutefois les antibiotiques sont essentiels pour assurer une lutte efficace et durable contre les maladies d'origine bactérienne. Dans le but d'optimiser l'usage des antibiotiques dans l'élevage des animaux de rente et, par conséquence, de réduire le développement de résistances, AntibioticScout.ch propose une aide à la décision pour un usage prudent de ces substances ("prudent use"). Parallèlement, on attire l'attention sur les traitements adjuvants et sur les mesures de prévention. Des mesures visant à améliorer la santé et le bien-être des animaux en tenant compte des fondements d'une bonne pratique vétérinaire sont des instruments efficaces pour réduire l'usage des antibiotiques. Cette réduction indispensable doit donc être combinée avec des mesures de gestion adéquates dans les élevages. Ce sont en particulier l'hygiène et les conditions d'élevage correctes ainsi que la mise en place de mesures de biosécurité qui sont décisives pour l'optimisation de la santé des troupeaux.

[Efficacy of the combination of glucocorticoids, mycophenolate-mofetil and human immunoglobulin for the therapy of immune mediated haemolytic anaemia in dogs]. / Wirksamkeitsstudie zur Kombinationstherapie von Glukokortikoid, Mycophenolat- Mofetil und humanem Immunglobulin zur Therapie von primärer immun vermittelter hämolytischer Anämie beim Hund.

INTRODUCTION: Idiopathic immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated diseases in dogs with a high mortality rate. In this retrospective study, we examined the effect of mycophenolate- mofetil (MMF) and human intravenous immunoglobulin (hIVIG) in combination with glucocorticoids on canine IMHA patients. Six dogs were treated with prednisolone and MMF (hIVIG-) and in 15 patients hIVIG was added (hIVIG+). There was no significant difference between the groups regarding age, weight, number of blood transfusions or hematocrit on the day of diagnosis. The hIVIG+ group showed a significantly faster recovery of the hematocrit, but this difference was only short-lived. The survival in the first year was similar in both groups and was with 71.5% somewhat higher than in other published studies. The addition of MMF to prednisolone for the treatment of dogs with acute IMHA was well tolerated and seemed to positively affect the course of the disease. Randomized studies are necessary to confirm this observation. Human immunoglobulin had only minimal clinical advantages and no effect on mortality.

INTRODUCTION: Une anémie hémolytique auto-immune idiopathique (IMHA) compte chez le chien au nombre des affections auto-immunes les plus fréquentes avec un taux de mortalité élevé. Dans le cadre de la présente étude rétrospective, nous avons examiné l'effet du mycophénolate mofétil (MMF) et des globulines humaines par voie intraveineuse (hIVIG) en combinaison avec des glucocorticoïdes sur des chiens souffrant d'IMHA. Six chiens ont été traités au moyen d'une combinaison prédnisolone/ MMF (hIVIG­) et chez 15 patients on a en plus utilisé des hlVIG (hIVIG+). Les deux groupes ne se différenciaient pas de façon significative en ce qui concerne l'âge, le poids, le nombre de transfusions sanguines ou l'hématocrite lors du diagnostic. Les patients du groupe hIVIG+ ont montré une normalisation significativement plus rapide mais de courte durée de l'hématocrite. Le taux de survie à une année était similaire dans les deux groupes avec 71,5%, ce qui est un peu plus élevé que dans d'autres études. L'ajout de MMF à la prédnisolone dans le traitement de chiens atteints d'IMHA est bien supporté et semble influencer le cours de la maladie de façon positive. Des études randomisées sont nécessaires pour confirmer ces observations. Les immunoglobulines humaines n'ont eu qu'un effet limité et pas d'influence sur la mortalité.

[AntibioticScout.ch: A decision supporting tool for antimicrobial stewardship: application to companion animal medicine]. / AntibioticScout.ch: Eine Entscheidungshilfe für den umsichtigen Einsatz von antimikrobiellen Wirkstoffen: Anwendung in der Kleintiermedizin.

INTRODUCTION: Bacterial resistances to antimicrobial drugs pose serious public health challenges. The observed increase of resistances is attributed to the uncontrolled, massive and often unnecessary administration of antibiotics both in human and veterinary medicine. To support the responsible use of antimicrobials in animals and help veterinarians selecting the most suitable antimicrobial drugs, we developed the AntibioticScout.ch as a comprehensive decision supporting tool providing online access to the current knowledge of rational antibiotic prescription practices. User-friendly search functions allow for the fast and efficient retrieval of information that is structured in this database by animal species, organ systems and therapeutic indications. In addition, an online form allows to report treatment failures in order to identify problematic cases as well as ensuing risks and take appropriate mitigation measures. The present report describes the workflow of this decision support system applied to the prudent use of antimicrobials in companion animal medicine.

INTRODUCTION: Les résistances bactériennes face aux substances antimicrobiennes placent le système de santé face à de grands défis. L'augmentation des résistances est attribuée à une utilisation mal dirigée, massive et souvent inutile d'antibiotiques tant en médecine humaine qu'en médecine vétérinaire. Afin d'encourager une utilisation responsable des substances antimicrobiennes en médecine vétérinaire et pour soutenir les vétérinaires lors du choix de l'antibiotique adapté, on a créé, avec AntibioticScout. ch, une aide à la décision de grande envergure qui permet de recourir à chaque instant aux principes de prescription reconnus de la médecine fondée sur les faits (Evidence Based Medicine). La banque de données est structurée par espèces animales, systèmes d'organes et indications et un moteur de recherche intuitif garantit de trouver rapidement et efficacement les informations. D'autre part un formulaire en ligne pour l'annonce d'une inefficacité est proposé afin de reconnaitre les cas à problèmes et les risques en vue d'améliorer l'aide au diagnostic. La présente contribution pour but de démontrer l'utilisation d'AntibioticScout pour un usage prudent des antibiotiques en médecine des petits animaux.

[Diagnosis, therapy and prophylactic measures of parturient paresis in dairy cattle: results of an online survey for Swiss veterinarians]. / Diagnose, Therapie und prophylaktische Massnahmen der Gebärparese beim Rind: Ergebnisse der Online-Umfrage bei Schweizer Tierärzten.

INTRODUCTION: The aim of this study was to obtain the diagnostic, therapeutic and prophylactic approach among Swiss veterinary practitioners in cows with parturient hypocalcemia. All members of the Association for Ruminant Health were contacted per e-mail. The survey was completed by 108 (28%) of 393 that were contacted. According to the questionnaire responses, the typical presentation of a parturient paresis cow is a pluriparous middle-yielding dairy cow one day post-partum in sternal recumbency with normal consciousness. The diagnosis is usually based upon the medical history. Therapy of parturient paresis consists of mixed infusions (with calcium, phosphorus, magnesium or glucose) as well as oral preparations with calcium. The veterinarians estimate that 25-50% of the cows treated for parturient paresis need more than one treatment and that one case of parturient paresis costs CHF 200-300. Prophylactic treatments are usually used for cows, which have suffered from parturient paresis in the previous lactation, elder cows (≥ 3 lactations) as well as cows with a high body condition score (> 3.25). Prophylactic measures used by the veterinarians are vitamin D3 injections and oral preparations with calcium. They recommended a special diet, for example a low calcium diet ante-partum.

INTRODUCTION: Le but de la présente enquête en ligne était de relever les méthodes de diagnostic, de traitement et de prophylaxie utilisées en pratique en matière de parésie puerpérale hypocalcémique. Tous les membres de l'Association suisse pour la santé des ruminants ont été contactés par courriel. Sur les 393 questionnaires envoyés, 108 (28%) ont été remplis complètement et exploités. L'anamnèse typique est un animal pluripare avec une production de parésie puerpérale, il est mentionné des animaux pluripares avec une production laitière moyenne, incapables de se lever un jour après le vêlage et présentant un état de conscience normal. Le diagnostic est fréquemment posé sur la base de l'anamnèse. Les vaches laitières concernées sont traitées avec des perfusions mixtes (produits à base de calcium et de phosphore, parfois avec du magnésium et du glucose) et des préparations de calcium orales. Les vétérinaires estiment que 25 à 50% des vaches nécessitent plusieurs traitements et que les coûts totaux par animal de l'ordre de CHF 200 à 300. Du point de vue prophylactique, ce sont principalement les animaux ayant déjà souffert d'une parésie lors de la lactation précédente ainsi que les vaches plus âgées (3ème lactation et plus) et celles présentant un indice de condition élevé (> 3.25) qui sont traitées. Les vétérinaires utilisent pour cette prophylaxie des injections de vitamine D3 ainsi que des préparations orales de calcium et/ou conseillent aux propriétaires une alimentation pauvre en calcium ante partum.

Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4.

Cytochrome P450 enzymes (CYPs) are responsible for the phase I metabolism of drugs, xenobiotics and endogenous substances. Knowledge of single CYPs and their substrates is important for drug metabolism, helps to predict adverse effects and may prevent reduced drug efficacy in polypharmacy. In this study, three equine isoenzymes of the 3A subfamily, the equine flavoprotein NADPH-P450 oxidoreductase (POR), and the cytochrome b5 (CYB5) were cloned, sequenced and heterologously expressed in a baculovirus expression system. Testosterone, the standard compound for characterization of the human CYP3A4, was used to characterize the newly expressed equine CYPs. The metabolite pattern was similar in equine and the human CYPs, but the amounts of metabolites were isoform-dependent. All equine CYPs produced 2-hydroxytestosterone (2-OH-TES), a metabolite never described in equines. The main metabolite of CYP3A4 6ß-hydroxytestosterone (6ß-OH-TES) was measured in CYPs 3A95 and 3A97 with levels close to the detection limit. Ketoconazole inhibited 2-OH-TES in the human CYP3A4 and the equine CYP3A94 and CYP3A97 completely, whereas a 70% inhibition was found in CYP3A95. Testosterone 6ß- and 2-hydroxylation was significantly different in the equine CYPs compared to CYP3A4. The expression of single equine CYPs allows characterizing drug metabolism and may allow prevention of drug-drug interactions.

[AntibioticScout: Online tool for antimicrobial stewardship in veterinary medicine]. / AntibioticScout: Online-Entscheidungshilfe für den umsichtigen Einsatz von antimikrobiellen Wirkstoffen.

INTRODUCTION: Resistances to antimicrobials pose serious public health challenges. This issue concerns both human and veterinary medicine and can only be solved by a multidisciplinary approach. A comprehensive concept is, therefore, being worked out within the StAR (strategy antibiotic resistance) program in order to preserve the effectiveness of antibiotics for humans as well as animals. In this context, the AntibioticScout (www.AntibioticScout. ch) offers a new online tool for the prudent use of antibiotics in veterinary medicine. By involving all stakeholders, the guidelines included in the AntibioticScout will result in a nationwide accepted standard for the treatment of bacterial infections in animals. An additional system for the rapid reporting of cases of suspected lack of efficacy of antimicrobials is integrated to allow early detection of emerging resistance and the immediate launch of risk mitigation measures. A first version of the AntibioticScout for the treatment of dogs, cats and horses is available by the end of 2016. All stakeholders are now invited to contribute to the development of the AntibioticScout decision support.

INTRODUCTION: Les résistances face aux substances antimicrobiennes placent le système de santé face à de grands défis. Ce problème touche aussi bien la médecine humaine que vétérinaire et ne peut être réglé que de façon transversale. C'est pour cette raison qu'a été développé, avec le programme StAR (Strategie Antibiotikaresistenzen), un concept global pour assurer à long terme l'efficacité des antibiotiques aussi bien chez les hommes que chez les animaux. Dans ce contexte, nous mettons à disposition, avec le développement d'AntibioticScout (www.AntibioticScout. ch), un nouvel outil en ligne pour soutenir un usage responsable des antibiotiques en médecine vétérinaire. Avec le concours de représentants de tous les milieux intéressés, il s'agit de développer les lignes directrices d'un standard accepté au plan national pour le traitement des infections bactériennes chez les animaux et de le mettre à disposition dans AntibioticScout. En outre un système d'annonce immédiate en cas d'inefficacité sera mis en place, afin de repérer précocement les risques et de prendre les mesures correspondantes. Une première version d'AntibioticScout pour le traitement des chiens, chats et chevaux est disponible dès fin 2016. Tous les cercles intéressés sont dès maintenant invités à contribuer au développement d'Antibiotic Scout.

High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture.

Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.

[New causes of animal poisoning in Switzerland]. / Neue Ursachen von Tiervergiftungen in der Schweiz.

This retrospective study evaluated the frequency, etiology, therapy and prognosis of animal poisoning registered from 2003 to 2012. The relevant cases reported to the Swiss Toxicological Information Center (STIC) were compared with those from previously examined periods. Human medicines not approved for animals and pesticides represented the most common causes of poisoning in dogs. Novel cases occurred as a consequence of the exposure of dogs to ricinus fertilizers, grape residues from wineries, pepper lachrymatory spray and dry bouillon. Cats are still freequently poisoned by pyrethroid drugs that should be administered only to dogs. Agrochmical products are the main source of toxicities in farm animals. Most poisonings in horses and exotic animals took place due to toxic plants. In addition, two tigers died of a secondary poisoning after ingestion of meat from euthanized calves.

In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95.

Testosterone (TES) 6-ß-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-ß-hydroxytestosterone showed the largest peak. Formation of 6-ß-hydroxytestosterone and 11-ß-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-ß-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture.

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 µM and 6.06 µM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine.

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66µM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26µM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster.

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Assessing the efficiency of a pharmacokinetic-based algorithm for target-controlled infusion of ketamine in ponies.

The objective of this study was to assess a pharmacokinetic algorithm to predict ketamine plasma concentration and drive a target-controlled infusion (TCI) in ponies. Firstly, the algorithm was used to simulate the course of ketamine enantiomers plasma concentrations after the administration of an intravenous bolus in six ponies based on individual pharmacokinetic parameters obtained from a previous experiment. Using the same pharmacokinetic parameters, a TCI of S-ketamine was then performed over 120 min to maintain a concentration of 1 microg/mL in plasma. The actual plasma concentrations of S-ketamine were measured from arterial samples using capillary electrophoresis. The performance of the simulation for the administration of a single bolus was very good. During the TCI, the S-ketamine plasma concentrations were maintained within the limit of acceptance (wobble and divergence <20%) at a median of 79% (IQR, 71-90) of the peak concentration reached after the initial bolus. However, in three ponies the steady concentrations were significantly higher than targeted. It is hypothesized that an inaccurate estimation of the volume of the central compartment is partly responsible for that difference. The algorithm allowed good predictions for the single bolus administration and an appropriate maintenance of constant plasma concentrations.

Stereoselective biotransformation of ketamine in equine liver and lung microsomes.

Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and, in a second step, into R/S-downstream metabolites. In liver and lung microsomes, levels of R-ketamine exceeded those of S-ketamine at all time points and S-norketamine exceeded R-norketamine at time points below the maximum concentration. In liver and lung microsomes, significant differences in the enzyme velocity (V(max)) were observed between S- and R-norketamine formation and between V(max) of S-norketamine formation when S-ketamine was compared to S-ketamine of the racemate. Our investigations in microsomal reactions in vitro suggest that stereoselective ketamine biotransformation in horses occurs in the liver and the lung with a slower elimination of S-ketamine in the presence of R-ketamine. Scaling of the in vitro parameters to liver and lung organ clearances provided an excellent fit with previously published in vivo data and confirmed a lung first-pass effect.

Effects of a non-selective COX inhibitor and selective COX-2 inhibitors on contractility of human and porcine ureters in vitro and in vivo.

BACKGROUND AND PURPOSE: Anti-inflammatory drugs are used in the treatment of acute renal colic. The aim of this study was to investigate the effects of selective COX-2 inhibitors and the non-selective COX inhibitor diclofenac on contractility of human and porcine ureters in vitro and in vivo, respectively. COX-1 and COX-2 receptors were identified in human ureter and kidney. EXPERIMENTAL APPROACH: Human ureter samples were used alongside an in vivo pig model with or without partial ureteral obstruction. COX-1 and COX-2 receptors were located in human ureters by immunohistochemistry. KEY RESULTS: Diclofenac and valdecoxib significantly decreased the amplitude of electrically-stimulated contractions in human ureters in vitro, the maximal effect (Vmax) being 120 and 14%, respectively. Valdecoxib was more potent in proximal specimens of human ureter (EC50=7.3 x 10(-11) M) than in distal specimens (EC50=7.4 x 10(-10) M), and the Vmax was more marked in distal specimens (22.5%) than in proximal specimens (8.0%) in vitro. In the in vivo pig model, parecoxib, when compared to the effect of its solvent, significantly decreased the maximal amplitude of contractions (Amax) in non-obstructed ureters but not in obstructed ureters. Diclofenac had no effect on spontaneous contractions of porcine ureter in vivo. COX-1 and COX-2 receptors were found to be expressed in proximal and distal human ureter and in tubulus epithelia of the kidney. CONCLUSIONS AND IMPLICATIONS: Selective COX-2 inhibitors decrease the contractility of non-obstructed, but not obstructed, ureters of the pig in vivo, but have a minimal effect on electrically-induced contractions of human ureters in vitro.

In vitro effects of bethanechol on smooth muscle preparations from abomasal fundus, corpus, and antrum of dairy cows.

Abomasal displacement has been associated with gastric hypomotility. The supply of prokinetic drugs available to address this problem is insufficient. The goal of the study was to investigate the effect of the muscarinic agonist bethanechol (BeCh) on contractility parameters of smooth muscle preparations from several regions of the bovine abomasum (fundus, corpus, and antrum). Cumulative concentration-response curves were constructed using BeCh in vitro with and without pre-incubation with antagonists targeted at M(2) and M(3) muscarinic acetylcholine receptor (mAChR) subtypes. In all preparations investigated, BeCh induced a significant and concentration-dependent increase in all contractility parameters investigated. The maximal attainable effect (V(max)) was more pronounced in circular specimens, and V(max) of antral specimens in circular orientation were significantly lower when compared to the other preparations. Both antagonists caused a rightward shift of the concentration-response curve, suggesting that the effect of BeCh is mediated at least partly by M(2) and M(3) AChRs.