Pharmacogenomics of poor drug metabolism in greyhounds: Canine P450 oxidoreductase genetic variation, breed heterogeneity, and functional characterization.

Greyhounds metabolize cytochrome P450 (CYP) 2B11 substrates more slowly than other dog breeds. However, CYP2B11 gene variants associated with decreased CYP2B11 expression do not fully explain reduced CYP2B11 activity in this breed. P450 oxidoreductase (POR) is an essential redox partner for all CYPs. POR protein variants can enhance or repress CYP enzyme function in a CYP isoform and substrate dependent manner. The study objectives were to identify POR protein variants in greyhounds and determine their effect on coexpressed CYP2B11 and CYP2D15 enzyme function. Gene sequencing identified two missense variants (Glu315Gln and Asp570Glu) forming four alleles, POR-H1 (reference), POR-H2 (570Glu), POR-H3 (315Gln, 570Glu) and POR-H4 (315Gln). Out of 68 dog breeds surveyed, POR-H2 was widely distributed across multiple breeds, while POR-H3 was largely restricted to greyhounds and Scottish deerhounds (35% allele frequencies), and POR-H4 was rare. Three-dimensional protein structure modelling indicated significant effects of Glu315Gln (but not Asp570Glu) on protein flexibility through loss of a salt bridge between Glu315 and Arg519. Recombinant POR-H1 (reference) and each POR variant (H2-H4) were expressed alone or with CYP2B11 or CYP2D15 in insect cells. No substantial effects on POR protein expression or enzyme activity (cytochrome c reduction) were observed for any POR variant (versus POR-H1) when expressed alone or with CYP2B11 or CYP2D15. Furthermore, there were no effects on CYP2B11 or CYP2D15 protein expression, or on CYP2D15 enzyme kinetics by any POR variant (versus POR-H1). However, Vmax values for 7-benzyloxyresorufin, propofol and bupropion oxidation by CYP2B11 were significantly reduced by coexpression with POR-H3 (by 34-37%) and POR-H4 (by 65-72%) compared with POR-H1. Km values were unaffected. Our results indicate that the Glu315Gln mutation (common to POR-H3 and POR-H4) reduces CYP2B11 enzyme function without affecting at least one other major canine hepatic P450 (CYP2D15). Additional in vivo studies are warranted to confirm these findings.

Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability.

Dogs are commonly used in human and veterinary pharmaceutical development. Physiologically based pharmacokinetic modeling using recombinant cytochrome P450 (CYP) enzymes requires accurate estimates of CYP abundance, particularly in liver. However, such estimates are currently available for only seven CYPs, which were determined in a limited number of livers from one dog breed (beagle). In this study, we used a label-free shotgun proteomics method to quantitate 11 CYPs (including four CYPs not previously measured), cytochrome P450 oxidoreductase, and cytochrome b5 in liver microsomes from 59 dogs representing four different breeds and mixed-breed dogs. Validation included showing correlation with CYP marker activities, immunoquantified protein, as well as CYP1A2 and CYP2C41 null allele genotypes. Abundance values largely agreed with those previously published. Average CYP abundance was highest (>120 pmol/mg protein) for CYP2D15 and CYP3A12; intermediate (40-89 pmol/mg) for CYP1A2, CYP2B11, CYP2E1, and CYP2C21; and lowest (<12 pmol/mg) for CYP2A13, CYP2A25, CYP2C41, CYP3A26, and CYP1A1. The CYP2C41 gene was detected in 12 of 58 (21%) livers. CYP2C41 protein abundance averaged 8.2 pmol/mg in those livers, and was highest (19 pmol/mg) in the only liver with two CYP2C41 gene copies. CYP1A2 protein was not detected in the only liver homozygous for the CYP1A2 stop codon mutation. Large breed-associated differences were observed for CYP2B11 (P < 0.0001; ANOVA) but not for other CYPs. Research hounds and Beagles had the highest CYP2B11 abundance; mixed-breed dogs and Chihuahua were intermediate; whereas greyhounds had the lowest abundance. These results provide the most comprehensive estimates to date of CYP abundance and variability in canine liver. SIGNIFICANCE STATEMENT: This work provides the most comprehensive quantitative analysis to date of the drug-metabolizing cytochrome P450 proteome in dogs that will serve as a valuable reference for physiologically based scaling and modeling used in drug development and research. This study also revealed high interindividual variation and dog breed-associated differences in drug-metabolizing cytochrome P450 expression that may be important for predicting drug disposition variability among a genetically diverse canine population.

Oral Coadministration of Fluconazole with Tramadol Markedly Increases Plasma and Urine Concentrations of Tramadol and the O-Desmethyltramadol Metabolite in Healthy Dogs.

Tramadol is used frequently in the management of mild to moderate pain conditions in dogs. This use is controversial because multiple reports in treated dogs demonstrate very low plasma concentrations of O-desmethyltramadol (M1), the active metabolite. The objective of this study was to identify a drug that could be coadministered with tramadol to increase plasma M1 concentrations, thereby enhancing analgesic efficacy. In vitro studies were initially conducted to identify a compound that inhibited tramadol metabolism to N-desmethyltramadol (M2) and M1 metabolism to N,O-didesmethyltramadol (M5) without reducing tramadol metabolism to M1. A randomized crossover drug-drug interaction study was then conducted by administering this inhibitor or placebo with tramadol to 12 dogs. Blood and urine samples were collected to measure tramadol, tramadol metabolites, and inhibitor concentrations. After screening 86 compounds, fluconazole was the only drug found to inhibit M2 and M5 formation potently without reducing M1 formation. Four hours after tramadol administration to fluconazole-treated dogs, there were marked statistically significant (P < 0.001; Wilcoxon signed-rank test) increases in plasma tramadol (31-fold higher) and M1 (39-fold higher) concentrations when compared with placebo-treated dogs. Conversely, plasma M2 and M5 concentrations were significantly lower (11-fold and 3-fold, respectively; P < 0.01) in fluconazole-treated dogs. Metabolite concentrations in urine followed a similar pattern. This is the first study to demonstrate a potentially beneficial drug-drug interaction in dogs through enhancing plasma tramadol and M1 concentrations. Future studies are needed to determine whether adding fluconazole can enhance the analgesic efficacy of tramadol in healthy dogs and clinical patients experiencing pain.

Identification of canine cytochrome P-450s (CYPs) metabolizing the tramadol (+)-M1 and (+)-M2 metabolites to the tramadol (+)-M5 metabolite in dog liver microsomes.

We previously showed that (+)-tramadol is metabolized in dog liver to (+)-M1 exclusively by CYP2D15 and to (+)-M2 by multiple CYPs, but primarily CYP2B11. However, (+)-M1 and (+)-M2 are further metabolized in dogs to (+)-M5, which is the major metabolite found in dog plasma and urine. In this study, we identified canine CYPs involved in metabolizing (+)-M1 and (+)-M2 using recombinant enzymes, untreated dog liver microsomes (DLMs), inhibitor-treated DLMs, and DLMs from CYP inducer-treated dogs. A canine P-glycoprotein expressing cell line was also used to evaluate whether (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 are substrates of canine P-glycoprotein, thereby limiting their distribution into the central nervous system. (+)-M5 was largely formed from (+)-M1 by recombinant CYP2C21 with minor contributions from CYP2C41 and CYP2B11. (+)-M5 formation in DLMs from (+)-M1 was potently inhibited by sulfaphenazole (CYP2C inhibitor) and chloramphenicol (CYP2B11 inhibitor) and was greatly increased in DLMs from phenobarbital-treated dogs. (+)-M5 was formed from (+)-M2 predominantly by CYP2D15. (+)-M5 formation from (+)-M1 in DLMs was potently inhibited by quinidine (CYP2D inhibitor) but had only a minor impact from all CYP inducers tested. Intrinsic clearance estimates showed over 50 times higher values for (+)-M5 formation from (+)-M2 compared with (+)-M1 in DLMs. This was largely attributed to the higher enzyme affinity (lower Km) for (+)-M2 compared with (+)-M1 as substrate. (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 were not p-glycoprotein substrates. This study provides a clearer picture of the role of individual CYPs in the complex metabolism of tramadol in dogs.

Tramadol metabolism to O-desmethyl tramadol (M1) and N-desmethyl tramadol (M2) by dog liver microsomes: Species comparison and identification of responsible canine cytochrome P-450s (CYPs).

Tramadol is widely used to manage mild to moderately painful conditions in dogs. However, this use is controversial since clinical efficacy studies in dogs showed conflicting results, while pharmacokinetic studies demonstrated relatively low circulating concentrations of O-desmethyltramadol (M1). Analgesia has been attributed to the opioid effects of M1, while tramadol and the other major metabolite (N-desmethyltramadol, M2) are considered inactive at opioid receptors. The aims of this study were to determine whether cytochrome P450 (CYP) dependent M1 formation by dog liver microsomes is slower compared with cat and human liver microsomes; and identify the CYPs responsible for M1 and M2 formation in canine liver. Since tramadol is used as a racemic mixture of (+)- and (-)-stereoisomers, both (+)-tramadol and (-)- tramadol were evaluated as substrates. M1 formation from tramadol by liver microsomes from dogs was slower than from cats (3.9-fold), but faster than humans (7-fold). However, M2 formation by liver microsomes from dogs was faster than from cats (4.8-fold) and humans (19-fold). Recombinant canine CYP activities indicated that M1 was formed by CYP2D15, while M2 was largely formed by CYP2B11 and CYP3A12. This was confirmed by dog liver microsomes studies that showed selective inhibition of M1 formation by quinidine and M2 formation by chloramphenicol and CYP2B11 antiserum, and induction of M2 formation by phenobarbital. Findings were similar for both (+)-tramadol and (-)-tramadol. In conclusion, low circulating M1 concentrations in dogs is explained in part by low M1 formation and high M2 formation, which are mediated by CYP2D15 and CYP2B11/CYP3A12, respectively.

Caracterización clínica y manejo terapéutico de los ancianos dispensarizados por Asma Bronquial / Clinical characterization and therapeutic management of elderly people registered with bronchial asthma

Introducción: Las enfermedades respiratorias son causa de morbilidad, discapacidad y mortalidad en toda la población, entre estas el Asma Bronquial ha aumentado su frecuencia de presentación en ancianos. Objetivo: Caracterizar clínicamente los pacientes de 60 años y más, dispensarizados por Asma Bronquial en el Policlínico Área Sur de Sancti Spíritus. Material y Método: Se realizó una investigación descriptiva transversal, del 1 de enero al 31 de mayo del 2007, se aplicó un cuestionario sobre síntomas respiratorios, validado por autores cubanos, entrevista individual a profundidad sobre la enfermedad actual y una observación durante el tratamiento por vía inhalada. Resultados: El 31,6 por ciento estuvo entre 65 y 69 años; la edad promedio fue 68,2 + - 3,02 y de estos el 52,5 por ciento fueron mujeres. La tos fue el síntoma menos encontrado, la disnea apareció en reposo en el 75,7 por ciento, fue de intensidad variable en el 68,7 por ciento y apareció de noche en el 56,5 por ciento, utilizaron Salbutamol indiscriminadamente 71 casos y presentaron dificultades en la técnica para el uso del spray el 59,1 por ciento. Conclusiones: Se presentaron síntomas propios de otras enfermedades cardiorrespiratorias que se diferencian del paciente asmático; el inadecuado manejo terapéutico evidenció desconocimiento y falta de adiestramiento del paciente para el autocontrol[AU]

Introduction: Breathing diseases are a cause of morbidity, disability and mortality in the whole population, among them bronchial asthma has increased its incidence rate in elderly people. Objective: To characterize clinically patients of 60 years and over, registered with bronchial asthma in the South Area of Sancti Spíritus. Material and method: A descriptive, cross-sectional investigation was made, from January 1 to May 31 of 2007. A questionnaire about breathing symptoms was applied, validated by Cuban authors; a deep personal interview on the current disease was made, as well as an observation during the inhalation treatment. Results: 120 elderly people were included. 31.6 Percent were in the range from 65 to 69 years, with an average age of 68.2 + - 3,02. Of these, 63 were women. Cough was the least commonly found symptom, Dyspnea developed at rest in 75.7 Percent; it was of variable intensity in 68.7 Percent and developed at night in 56,5 Percent, Salbutamol was used indiscriminately in 71 cases and there were difficulties in the technique for using the spray in 59.1 Percent. Conclusions: There were symptoms typical of other cardiorrespiratory diseases which differ from those of the asthmatic patient. The inadequate therapeutic management showed ignorance and lack of training of the patient for self-control

Algunos factores de riesgos conocidos en enfermedades crónicas no transmisibles / Some known risk factors in non- transmitted chronic diseases

Las enfermedades crónicas no trasmisibles, mayormente están asociadas a factores de riesgos prevenibles comunes relacionados con el estilo de vida. Con el objetivo de identificar la frecuencia de algunos factores de riesgos conocidos, que influyen en la aparición de estas enfermedades, se realizó un proyecto de investigación descriptivo, de tipo transversal, en la población mayor de 15 años, pertenecientes al Policlínico Sur del municipio de Sancti Spíritus, en el período comprendido entre noviembre del 2005 y abril del 2006, incluyéndose hábito de fumar, alcoholismo, sedentarismo y antecedentes personales de Hipertensión Arterial. La población estudiada tiene una distribución similar por sexo y predominan los grupos de edades comprendidos entre 25-54 años de edad, con vínculo laboral estable; el hábito de fumar se presenta en un tercio de los individuos, mayormente entre 45 y 54 años, siendo el sexo masculino el que fuma con más severidad y si han abandonado el hábito lo hacen desde hace menos tiempo, conocen las enfermedades causadas por el cigarrillo, mayormente el cáncer y enfermedades del corazón. El consumo de alcohol estuvo presente en más de la mitad de la población, afectando mayormente al sexo masculino, con las categorías más frecuentes; bebedor social seguido del bebedor riesgo. Por el contrario el sedentarismo se presentó en la tercera parte de los estudiados, siendo más frecuente en el sexo femenino y los antecedentes de Hipertensión Arterial tienen una frecuencia similar no presentando diferencia en cuanto al sexo(AU)

Chronic non- transmited diseases, are mostly associated to common preventable risk factors related with lifestyle. With the objective of identifying the frequency of some well-known risk factors that influence the development of these diseases , a descriptive research project of a cross-sectional type was made in the population older than 15 years, belonging to the South Polyclínic of the municipality of Sancti Spíritus in the period from November of 2005 to April of 2006, with smoking, alcoholism, physical inactivity and personal antecedents of arterial hypertension being included. The population studied has a similar distribution by sex, and the age groups from 25 to 54 years with steady jobs prevail. Smoking is shown in a third of the individuals, mostly between 45 and 54 years, with the male sex being the one that smokes more heavily and if they have quit the habit they have done it more recently and know the diseases caused by cigarette smoking, mostly cancer and heart diseases. Alcohol intake was present in more than half of the population, affecting mostly the male sex, with the most frequent categories being; social drinker followed by the drinker at risk. On the contrary, physical inactivity was observed in the third part of those studied, and more frequently in the female sex. Antecedents of arterial hypertension have a similar frequency and no difference regarding sex(AU)

Determinación y analisis de hematocrito, hemoglobina y ph sanguíneo, pre y pos ejercicio en equinos atletas de salto en Bogotá, Colombia / Determination and analysis of the hematocrit, hemoglobin and blood ph pre and post exercises in jumping athletic horses in Bogota, Colombia

Con el fin de determinar los parámetros fisiológicos del hematocrito y hemoglobina y ácido-base en una población de equinos atletas de salto alto en Bogotá, se realizaron pruebas en la Escuela de equitación del Ejercito Nacional, CESPO y Club los Arrayanes, localizadas al norte de la ciudad de Bogotá a una altura sobre el nivel del mar de 2.600 metros, con una temperatura promedio de 13 grados centígrados. Se conto con 40 equinos hembras y machos, los cuales se encontraron entre los 6 y los 12 años de edad. La selección de los atletas se realizo de acuerdo con la categoría en la que participaran y a la normalidad encontrada al examen físico. En los tiempos de muestreo se determinaron las variables sanguíneas en reposo y después del ejercicio, teniendo en cuenta los siguientes parámetros: Hemoglobina, hematocrito, pH de la sangre venosa, presión de gas carbónico venoso, bicarbonato en sangre venosa. Estos ejemplares seleccionados se hicieron saltar 15 obstáculos a la mano. El calentamiento previo a la ejecución de los saltos, consistió en el galopeo libre por 3 minutos y las realización de los 15 obstáculos ya mencionados. Durante esta investigación se pudo observar que el ejercicio de salto no es suficiente extenuante para producir cambios drásticos en la sangre y pH comparados con los que se presentan en los caballos de carreras o de enduro, sin embargo se observa una diferencia dentro de los 3 grupos de rendimiento: alto, medio y bajo...