RESUMEN
We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.
Asunto(s)
Antígenos CD34/metabolismo , Células de la Médula Ósea/metabolismo , Perros , Vectores Genéticos/genética , Células Madre/virología , Transducción Genética/veterinaria , Animales , Línea Celular , Fibroblastos , Genes/genética , Ratones , Retroviridae/genética , Timo/citología , Transducción Genética/métodosRESUMEN
BACKGROUND AND OBJECTIVES: Benzodiazepine agonists and azaperone derivatives are used clinically as anxiolytics but have different neuroreceptor mechanisms of action. This study evaluated clinical pharmacodynamic approaches to distinguishing these two classes of compounds. METHODS: Healthy volunteers received single oral doses of placebo, the benzodiazepine agonist triazolam (0.25 mg) or the azaperone anxiolytic buspirone (20 mg), in a double-blind, three-way crossover study. Ratings of mood and sedation, performance on the digit symbol substitution test (DSST), and quantitative measures of electroencephalographic (EEG) beta activity (13 to 31.75 cycles/sec) determined by fast-Fourier transform were obtained at multiple times after dosage. RESULTS: Triazolam significantly increased self- and observer-rated sedation, impaired DSST performance, impaired recall, and increased EEG beta activity. Pharmacodynamic changes were significantly intercorrelated; all effects were maximal 1 to 2 hours after dosage but were indistinguishable from placebo by 8 hours. Buspirone did not alter the EEG or DSST performance but did increase self-ratings of sedation and feeling "spacey" and impaired memory function; these effects generally were quantitatively less than with triazolam. Peak plasma triazolam concentrations preceded maximum pharmacodynamic effects; the mean plasma effect site equilibration half-life was 9.4 minutes. Kinetic-dynamic modeling procedures yielded significant relationships between hypothetical effect site triazolam concentrations and pharmacodynamic changes. CONCLUSIONS: Quantitative analysis of the EEG clearly distinguishes a typical benzodiazepine agonist from a nonagonist anxiolytic, in clinically relevant dosage, whose pharmacodynamic actions do not involve benzodiazepine receptor occupancy. EEG effects associated with triazolam are intercorrelated with other pharmacodynamic measures.
Asunto(s)
Encéfalo/efectos de los fármacos , Buspirona/farmacología , Electroencefalografía/efectos de los fármacos , Triazolam/farmacología , Administración Oral , Adulto , Afecto/efectos de los fármacos , Análisis de Varianza , Buspirona/farmacocinética , Método Doble Ciego , Femenino , Humanos , Modelos Lineales , Masculino , Memoria/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Valores de Referencia , Factores de Tiempo , Triazolam/farmacocinéticaRESUMEN
BACKGROUND: Elderly persons frequently appear to be sensitive to the effects of many drugs that depress the central nervous system. We studied the effect of age on the pharmacokinetics and pharmacodynamics of the benzodiazepine hypnotic agent triazolam, now the most frequently prescribed hypnotic drug in the United States. METHODS: Twenty-six healthy young subjects (mean age, 30 years) and 21 healthy elderly subjects (mean age, 69 years) participated in a four-way crossover study. After a single-blind adaptation trial with placebo, each subject received, in random order and in double-blind fashion, single doses of placebo, 0.125 mg of triazolam, and 0.25 mg of triazolam. For 24 hours after the administration of each of the three study medications, plasma triazolam levels were determined and psychomotor performance, memory, and degree of sedation were assessed. RESULTS: Plasma triazolam concentrations increased in proportion to the dose, but the elderly subjects had higher plasma concentrations due to reduced clearance of the drug. The degree of sedation as rated by an observer and the reduction in the subjects' performance on the digit-symbol substitution test were both greater in the elderly than in the young subjects after they were given the same doses. The relation of the plasma triazolam concentration to the degree of impairment was similar for the two groups. As part of the study, information was presented 1 1/2 hours after the administration of the drugs; the subjects' ability to recall the information 24 hours later was impaired by both doses of triazolam, and the percent decrease was similar in the young and elderly groups. CONCLUSIONS: Triazolam caused a greater degree of sedation and greater impairment of psychomotor performance in healthy elderly persons than in young persons who received the same dose. These effects resulted from reduced clearance and higher plasma concentrations of triazolam rather than from an increased intrinsic sensitivity to the drug. On the basis of these results, the dosage of triazolam for elderly persons should be reduced on average by 50 percent.