RESUMO
Targeting adenoviral vectors for cystic fibrosis gene therapy to the human airways with minimal exposure to alveoli would avoid adverse reactions and maximize response. At present, to deliver gene therapy vectors, large volumes of fluid are instilled or nebulized as aerosols. Either approach would likely cause alveolar exposure and increases the potential for side effects. We describe a coarse spray delivery device that precisely and reproducibly delivers the viral vector to the human airways to treat a small region of the airways for clinical trials. An endoscopic washing pipe (Olympus) that can be inserted into the channel of a bronchoscope was used. To minimize the escape of the therapeutic material downstream from the site of administration, we restricted the volume delivered to <150 microl (to prevent bulk flow), and used large droplets. Their high velocity further enhanced the probability of impaction in the vicinity of the nozzle. A pneumatic dosing system (Kahnetics) was used to reproducibly deliver the spray. The droplet size distribution was determined by laser diffraction and confirmed by cascade impaction: 190-microm volume median diameter with 1% mass <10 microm. The localization of the spray was studied in hollow cast models of human airways. 99mTc-sulfur colloid was used as a radiolabeled marker for these studies. Localization of the deposited spray was determined by scintigraphy and by measuring the radioactivity exiting the terminal airways. In the lung casts the spray was localized to one or two generations over an approximately 2-cm2 area. We conclude that delivery of large droplet sprays limits exposure to a few generations and may be useful in topical gene delivery clinical trials.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Pulmão , Administração por Inalação , Aerossóis , Linhagem Celular , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Relação Dose-Resposta a Droga , Humanos , Pulmão/anatomia & histologia , Modelos Anatômicos , Tamanho da Partícula , Coloide de Enxofre Marcado com Tecnécio Tc 99m/farmacologiaRESUMO
We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.
Assuntos
Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Terapia Genética/métodos , Traqueia/metabolismo , Adenoviridae/genética , Adolescente , Adulto , Estudos de Coortes , Regulador de Condutância Transmembrana em Fibrose Cística/administração & dosagem , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , DNA Complementar/genética , Relação Dose-Resposta a Droga , Epitélio/metabolismo , Feminino , Vetores Genéticos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Recombinação Genética , Fatores de TempoRESUMO
Long-term retention of particles in airways is controversial. However, precise anatomic localization of the particles is not possible in people. In this study the anatomic location of retained particles after shallow bolus inhalation was determined in anesthetized, ventilated beagle dogs. Fifty 30-cm(3) boluses containing monodisperse 2.5-micron polystyrene particles (PSL) were delivered to a shallow lung depth of 81-129 cm(3). At 96 h before euthanasia, red fluorescent PSL were used; at 24 h, green fluorescent PSL and (99m)Tc-labeled PSL were used. Clearance of (99m)Tc-PSL was measured during the next 24 h. Sites of particle retention were determined in systematic, volume-weighted random samples of microwave-fixed lung tissue. Precise particle localization and distribution was analyzed by using gamma counting, conventional fluorescence microscopy, and confocal microscopy. Within 24 h after shallow bolus inhalation, 50-95% of the deposited (99m)Tc-PSL were cleared, but the remaining fraction was cleared slowly in all dogs, similar to previous human results. The three-dimensional deposition patterns showed particles across the entire cross-sectional plane of the lungs at the level of the carina. In these locations, 33 +/- 9.9% of the retained particles were found in small, nonrespiratory airways (0.3- to 1-mm diameter) and 49 +/- 10% of the particles in alveoli; the remaining fraction was found in larger airways. After 96 h, a similar pattern was found. These findings suggest that long-term retention in airways is at the bronchiolar level.
Assuntos
Pulmão/anatomia & histologia , Pulmão/fisiologia , Mecânica Respiratória/fisiologia , Aerossóis , Animais , Cães , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Tamanho da Partícula , Alvéolos Pulmonares/anatomia & histologia , Alvéolos Pulmonares/diagnóstico por imagem , Alvéolos Pulmonares/fisiologia , Tecnécio , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
PURPOSE: To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. METHODS: Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. RESULTS: Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300: 1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. CONCLUSIONS: Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.
Assuntos
Aerossóis/química , Anticorpos Monoclonais/química , Excipientes/química , Carboidratos/química , Química Farmacêutica , Estabilidade de Medicamentos , Humanos , Imunoglobulina E/química , Imunoglobulina E/imunologia , Cinética , Lactose/química , Tamanho da Partícula , Pós/químicaRESUMO
We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.
Assuntos
Anticorpos Anti-Idiotípicos/química , Excipientes/química , Manitol/química , Proteínas/química , Estabilidade de Medicamentos , HumanosRESUMO
PURPOSE: To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. METHODS: Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). RESULTS: Spray-dried powders with a moisture level (approximately 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. CONCLUSIONS: Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.
Assuntos
Aerossóis/química , Anticorpos Monoclonais/química , Desoxirribonucleases/química , Pós/química , Administração por Inalação , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Umidade , Tamanho da Partícula , VácuoRESUMO
Knowledge of the local and regional doses of inhaled particulates is crucial for inhalation therapy and for understanding the progression of pulmonary disease. We studied the deposition pattern of radioactively tagged particles in rats with chronic bronchitis. Rats were exposed to sulfur dioxide (SO2; 236 +/- 14 ppm) for 5 h/d, 5 d/wk for 7 wk to produce chronic bronchitis (CB). Control rats were exposed to room air. The control animals gained 85% more weight over the 7-wk period than did the CB rats. Five control and five CB rats were then exposed for 30 min to an insoluble 99mTc-labeled aerosol. The animals were killed within 5 min after the exposure period. The lungs were excised, dried at total lung capacity (TLC), and sliced into 1 mm sections. The distribution of the radiolabeled particles retained in the lungs was determined in two ways. First, autoradiographs were made of the distribution of the radioactivity throughout a lung slice. Autoradiographs were quantified by image analysis to determine the amount of radioactivity (relative density of the film) associated with airway versus parenchyma (ratio of airway to parenchyma density). The lung slices were then dissected into pieces, the weight and radioactivity content of each piece was measured, and its evenness index (EI) was calculated. This type of analysis enables the homogeneity of particle deposition throughout the lungs to be assessed. If deposition were totally uniform, the average EI would be 1.0 with an SD = 0. The total amount of radioactivity retained in the lungs was similar in control and CB rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aerossóis , Bronquite/patologia , Pulmão/patologia , Aerossóis/farmacocinética , Animais , Câmaras de Exposição Atmosférica , Autorradiografia , Bronquite/metabolismo , Doença Crônica , Pulmão/metabolismo , Masculino , Consumo de Oxigênio , Ratos , Ratos Sprague-DawleyRESUMO
Tracheal instillation of pentamidine in a surfactant vehicle may be an effective direct method of antibiotic delivery to the lungs. In 10 healthy hamsters, we compared the pulmonary distribution of 99mTc sulfur colloid (TcSC) mixed with pentamidine, using as a vehicle either surfactant (n = 5) or saline (n = 5). Each animal was instilled with 0.25 ml/kg of suspension containing 0.0018 mCi TcSC and pentamidine mixed with either surfactant or saline. After 4 h of spontaneous respiration, the lungs were excised, inflated to TLC, dried, and sliced into 3-mm cross sections from apex to base. Autoradiographs were examined to evaluate 99mTc distribution. The surfactant group had detectable radioactivity in 93% of all slices compared with 72% in the saline group (p = 0.02). Six slices per animal (43% of total) and their corresponding autoradiographs were analyzed for distribution of radioactivity. Lung slice area was determined by planimetry, and autoradiograph area was determined by video densitometry. We calculated the fraction of each lung slice with detectable radioactivity. The surfactant group had 41% of the lung slice areas exposed compared with 21% in the saline group (p = 0.02). The coefficient of variation of radioactive intensities within each slice was used as an index of spatial uniformity. There was a trend towards more uniform distribution in the surfactant group, with a narrower range of variation of intensities (1.51 to 2.56) than the saline group (1.95 to 6.47). We conclude that a surfactant vehicle significantly increases airspace deposition of TcSC and pentamidine instilled intratracheally in normal hamster lungs, and may improve uniformity of spread.
Assuntos
Pulmão/diagnóstico por imagem , Pentamidina/administração & dosagem , Surfactantes Pulmonares/administração & dosagem , Coloide de Enxofre Marcado com Tecnécio Tc 99m/administração & dosagem , Animais , Autorradiografia , Cricetinae , Portadores de Fármacos , Pulmão/metabolismo , Mesocricetus , Pentamidina/farmacocinética , Surfactantes Pulmonares/farmacocinética , Cintilografia , Cloreto de Sódio , Coloide de Enxofre Marcado com Tecnécio Tc 99m/farmacocinética , TraqueiaRESUMO
We assessed the retention and clearance of inhaled particles in six anatomic compartments of the respiratory tract. Hamsters were exposed for 45 min to 0.9-micron fluorescent latex particles either at rest (n = 9) or while running on a laddermill (n = 9). Oxygen consumption, which was used to estimate minute ventilation, was continuously monitored. Three animals from each group, rest and exercise, were killed at 10 min, 24 h, or 7 days after the exposure. Morphometric techniques were used to determine the number of particles retained in nose and oropharynx (NOSE), trachea and extrapulmonary airways, intrapulmonary conducting airways, respiratory bronchioles, alveolar ducts (AD), and alveoli (ALV). At 10 min, total particle retention increased linearly as a function of O2 consumption (slope = 1.4 +/- 0.3 x 10(6) particles.ml-1.g-1.h-1, P less than 0.015). Exercised hamsters retained 4.4 times more total particles in their NOSE than rested hamsters, but parenchymal retention (AD + ALV) was unaffected. After 7 days, 95% of the particles were cleared from the NOSE, 80% from the trachea and extrapulmonary airways, 44% from intrapulmonary conducting airways and respiratory bronchioles, and 16% from AD and ALV. There was evidence of particle redistribution from AD to ALV during the 1st day. We conclude that exercise enhances the deposition of 0.9-micron particles in the upper respiratory tract but not in the parenchyma. Subsequently, the deposited particles are cleared at varying rates depending on the lung compartment.
Assuntos
Poluentes Atmosféricos/toxicidade , Respiração/fisiologia , Animais , Cricetinae , Masculino , Mesocricetus , Tamanho da Partícula , Esforço Físico/fisiologia , Fenômenos Fisiológicos Respiratórios , Sistema Respiratório/anatomia & histologia , Distribuição TecidualRESUMO
Does the restraint required for head or nose-only exposure of rodents to inhaled aerosols or gases alter their breathing pattern? And does prior exercise training, which may increase muscle strength, affect this response to restraint? To answer those questions, we measured breathing pattern in 11 adult male hamsters while they were either 1) free to move in small cages or 2) closely restrained in head-out cones. The measurements were repeated after hamsters spent 6 wk either sedentary in standard cages or in cages with exercise wheels. Hamsters were placed in a plethysmograph to measure respiratory frequency (f) and tidal volume (VT). Their product is minute volume (V). When restrained, f and V were 1.9 and 1.7 times, respectively, greater than when hamsters were free, but VT did not change. After 6 wk, the sedentary group responded differently to restraint; f increased 3-fold, VT decreased by one-half, and V increased 1.6-fold. Exercised hamsters increased f 2.3-fold and decreased VT by one-third; V increased by 1.5-fold. In inhalation studies, changes in breathing pattern would significantly influence the amount of material inhaled, the fraction retained, and thus the amount and distribution of material deposited in the lungs.
Assuntos
Mecânica Respiratória/fisiologia , Animais , Cricetinae , Masculino , Mesocricetus , Condicionamento Físico Animal , Restrição Física , Volume de Ventilação Pulmonar/fisiologiaRESUMO
Evaluating the response of animals to a toxic or therapeutic agent requires the knowledge of the dose of the agent in the respiratory tract. Dose is the amount retained in the lungs. It is the difference between the amount deposited and the amount cleared. Many factors influence the amount of and the site of deposition in the respiratory tract. For particles, the characteristics of the aerosol, most importantly size, the physiology of the animal, most importantly breathing pattern, and the geometry of the respiratory tract all play a role in determining local dose. These factors and their relative importance in determining regional deposition are discussed. Finally, ways in which dose can be measured in the respiratory tract are explored. These range from simple estimates of total dose using whole lung digests to the precise localization of dose in well-defined lung compartments using morphometry or in cellular or subcellular compartments using electron energy loss spectroscopy. The techniques utilized by an investigator depend on the amount of resolution desired. Their use and implementation in inhalation studies are the key to fully understanding the response of an animal to an inhaled toxic or therapeutic agent.
Assuntos
Aerossóis , Pulmão/metabolismo , Animais , Doença Crônica , Humanos , Pneumopatias/metabolismo , Taxa de Depuração Metabólica , Modelos Biológicos , RespiraçãoRESUMO
Does exercise alter the redistribution and clearance of particles from the lungs? Sedentary hamsters and hamsters that were exercise trained by voluntary wheel running for the previous 5 wk were exposed to a 198Au-labeled aerosol for 25 min. Six trained and 6 sedentary animals were killed within 5 min after the exposure (day 0); the same number were killed 5 days later. The trained hamsters ran ad libitum during those 5 days. The lungs of all animals were excised, dried at total lung capacity, sliced into 1-mm-thick sections, and dissected into pieces that were counted for radioactivity and weighed. On day 0, trained hamsters had 80% more particles per milligram of lung than sedentary hamsters, although both were exposed under identical conditions of restraint. After five days, exercising hamsters cleared 38% of the particles present at day 0, whereas sedentary animals removed only 15%. Significant clearance was observed from the middle lung regions of sedentary hamsters and from all lung regions in exercising hamsters. We conclude that exercise can enhance the redistribution and clearance of particles from the lungs; the mechanisms responsible are as yet unclear.
Assuntos
Pulmão/fisiologia , Atividade Motora/fisiologia , Aerossóis , Animais , Cricetinae , Radioisótopos de Ouro/administração & dosagem , Radioisótopos de Ouro/farmacocinética , Masculino , Mesocricetus , Taxa de Depuração Metabólica , Distribuição TecidualRESUMO
How does pulmonary emphysema affect aerosol deposition? Groups of awake hamsters with emphysema (intratracheal elastase, 0.2 mg/100 g body wt) and age-matched controls (intratracheal saline) were exposed for 30 minutes to an insoluble radioactive aerosol (0.45 mu aerodynamic diameter) at 30, 60, or 90 days after instillation. Immediately after exposure, the animals were sacrificed. The lungs were excised, dried at total lung capacity, and sliced into 1-mm thick sections. Each slice was cut into pieces, which were counted for radioactivity and weighed. Then a measure of the uniformity of deposition, the evenness index (EI), was calculated. With perfect uniformity, all EIs would be one. We found fewer particles in the emphysematous, as compared with the control, lungs at 60 or 90 days after elastase instillation. The deposited particles were distributed less uniformly throughout the emphysematous lungs than in the control lungs. In controls, the standard deviation (SD) of the EI distribution (mean 1.0) averaged 0.33 for the three times studied. In elastase animals, the SD increased to 0.48 at 30 days, and at 60 days and 90 days the distributions were no longer normally distributed. This increased heterogeneity of deposition was also manifested as a loss of the normal apex-base gradient observed in control animals, an increase in the amount of nonventilated parenchyma, enhanced airway deposition, and an altered lobar deposition pattern. Morphometric analysis showed an increase in the mean linear intercept (MLI) of emphysematous lungs as compared with control lungs. However, the author found no correlation between MLI, a measure of emphysema, and EI, a measure of deposition, quantified in the same lung pieces. It is concluded that the emphysematous lesions produced by elastase markedly alter the deposition of an inhaled submicrometric aerosol. Factors that may contribute to these changes include airway obstruction and differences in breathing pattern in emphysematous as compared with control animals.
Assuntos
Pulmão/patologia , Enfisema Pulmonar/patologia , Aerossóis , Animais , Cricetinae , Histocitoquímica , Masculino , Mesocricetus , Elastase Pancreática/análise , Traqueia/enzimologiaRESUMO
Regional clearance of submicrometric aerosol during 5 days was studied in control hamsters and in those with diffuse interstitial pulmonary fibrosis. Fibrosis was induced by treatment with bleomycin (1.6 U/kg) and oxygen (70% for 72 h) 80 days earlier. Diseased and control animals were exposed for 30 min to an insoluble colloidal 198Au aerosol (AMAD, 0.62 micron; sigma g, 1.35). Five diseased and 6 control animals were killed immediately (Day 0); 6 diseased and 6 control animals were killed 5 days later (Day 5). All lungs were excised, dried at TLC, sliced into 1-mm sections, and dissected into pieces. When compared with deposition in Day 0 control lungs, 26.3% of the particles had been cleared 5 days after exposure from the control lungs. Diseased animals cleared 44.6% of the particles (p less than 0.05, compared with Day 0 diseased animals). Examination of regional clearance on the basis of lobar differences, apex to base levels, and presence of airways also reflected increased clearance in the fibrotic animals. The parenchymal distribution of particle retention at Day 0 was less uniform in the fibrotic animals than in the control animals and was not significantly altered after 5 days of clearance in either group. Particle clearance may be greater in diseased lungs because of accelerated alveolar-bronchiolar transport of particles or particle-containing macrophages.
Assuntos
Aerossóis/metabolismo , Fibrose Pulmonar/metabolismo , Respiração , Animais , Cricetinae , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mesocricetus , Tamanho da Partícula , Fibrose Pulmonar/patologia , Fatores de TempoRESUMO
Aerosol retention was studied in hamsters 30, 60, and 90 days after the initiation of interstitial pulmonary fibrosis by a combination of bleomycin (bleo), 0.16 U/100 g body weight given intratracheally, and O2, for 72 h. Groups of bleo-O2-treated and control animals were exposed (awake) for 25 min to a 99mTc-labeled insoluble aerosol (activity median aerodynamic diameter, 0.45 micron; geometric standard deviation, 1.3). Within 5 min after exposure, the hamsters were killed and their lungs were excised and dried at total lung capacity, and sliced into 1-mm sections. Slices were dissected into pieces, and an evenness index (EI) was calculated for each piece (formula: see text). With uniformity of retention, all Els would be 1. The distribution of Els in control animals had a mean of 1.0 and a SD of 0.27; 0% of the Els were less than or equal to 0.20. Total retention diminished and was less uniform in bleo-O2-treated animals. At 30 days, the SD increased to 0.62, and 6% of the Els were less than or equal to 0.20. At 60 and 90 days, nonuniformity decreased but was still greater than that in the control animals (SD60 = 0.42, SD90 = 0.36). When examined histologically, individual pieces with low Els had more disease than those with high Els. Local decreases in compliance caused by fibrosis may have altered regional ventilation and retention. Our data also correlate with the progression of fibrosis from a focal lesion at 30 days to a more diffuse lesion at 90 days.
Assuntos
Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Aerossóis , Animais , Autorradiografia , Bleomicina/farmacologia , Cricetinae , Pulmão/patologia , Masculino , Mesocricetus , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Enxofre , Tecnécio , Coloide de Enxofre Marcado com Tecnécio Tc 99m , Fatores de TempoRESUMO
General anesthesia was used to produce nonventilated areas of the lung, and aerosol inhalation was used to locate these areas, assuming that no aerosol deposits in a nonventilated region. Male Syrian golden hamsters were anesthetized with pentobarbital sodium (90 mg/kg), which reduced respiratory frequency, tidal volume, minute volume, and O2 consumption to 61, 41, 24, and 36%, respectively, of the corresponding awake levels. Awake and anesthetized hamsters were exposed to the aerosol for 30 min; then the lungs were excised, dried at total lung capacity, sliced into sections, and dissected into pieces. Autoradiographs were made of slices, and the activity and weight of pieces were determined. The evenness index (EI), a measure of the uniformity of retention, was calculated for each piece. With complete uniformity of retention, all EI's would be 1.0. In awake animals, only 0.2% (by wt) of the lungs had little or no retention (EI's less than 0.20). More particles deposited in the apex than in the base of the lungs. General anesthesia for extended periods of time with no deep breaths alters ventilation and therefore the distribution of aerosol retention. Many regions of the lungs in the anesthetized animals received few or no particles (11.6% of lungs had EI less than 0.20); however, no consistent pattern was observed in the location of these areas from animal to animal. The apex-to-base gradient for retention in these animals was also reversed. Radioactive aerosols can be used as probes to indicate the extent and distribution of nonventilated areas in the lungs.
Assuntos
Aerossóis , Anestesia Geral , Cricetinae/fisiologia , Animais , Autorradiografia , Estado de Consciência , Pulmão/análise , Masculino , Mesocricetus , Respiração , Distribuição TecidualRESUMO
In order to investigate some of the characteristics of superoxide anion release from alveolar macrophages, the effects of substances known to influence superoxide release from polymorphonuclear leukocytes (PMN) were studied in rat alveolar macrophages. There is a relatively small, but constant, amount of superoxide released from alveolar macrophages at rest. The amount released increases 5- to 6-fold and becomes maximal in about 20-30 min following exposure to unopsonized zymosan particles. The rate of superoxide release is maximal only 2 min after exposure of the cells to particles, i.e., long before particle uptake is complete. In addition to particles, release of superoxide anion can be stimulated by phorbol-12-myristate-13-acetate (PMA). Lectins and chemotactic factors, which stimulate release in PMN, have little or no effect in alveolar macrophages. Superoxide release during exposure to zymosan appears to be dependent upon extracellular Ca++. Also, the release mechanism can be affected by the addition of cyclic AMP or various protein modifiers to the medium. Since many of these findings differ from those reported by others for PMN, the control of superoxide anion release from alveolar macrophages and PMN is probably different.
Assuntos
Macrófagos/metabolismo , Oxigênio/metabolismo , Alvéolos Pulmonares/citologia , Superóxidos/metabolismo , Animais , Ânions , Células Cultivadas , Concanavalina A/farmacologia , AMP Cíclico/farmacologia , Citocalasina B/farmacologia , Lectinas/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Teofilina/farmacologia , Zimosan/farmacologiaRESUMO
Previous studies have demonstrated an important role for the pituitary gland in the regulation of hepatic drug and steroid metabolism. The present studies were carried out to determine the relationship between the pituitary gland and the actions of gonadal hormones on hepatic drug metabolism in rats. Testosterone administration to castrated male rats increased hepatic microsomal cytochrome P-450 concentrations and enhanced the rates of ethylmorphine demethylation and benzo(a)pyrene hydroxylation. Estradiol treatment, on the other hand, lowered cytochrome P-450 levels and decreased the rates of aniline, ethylmorphine and benzo(a)pyrene metabolism in orchiectomized rats. However, when given to hypophysectomized male rats, neither testosterone nor estradiol affected cytochrome P-450 levels or enzyme activities. Similarly, 5alpha-dihydrotestosterone administration increased hepatic drug metabolism only in the presence of the pituitary gland. The effects of both testosterone and estradiol were fully demonstrable in the absence of the thyroid or adrenal glands, excluding the need for either organ. The results indicate an absolute dependence on the pituitary gland for gonadal hormone (testosterone and estradiol) actions on hepatic mixed-function oxidases.