Respiration-based measurement of lung deposition of a fluorescent dextrane aerosol in a marmoset monkey

A technique for measurement of respiration-based lung deposition of an aerosol was investigated and subsequently applied in a pilot study with a marmoset monkey. The technique consisted of an aerosol exposure system for a marmoset using a face mask and a previously constructed monkey chair, a method for recovery of fluorescent dextrane from lung material, and respiration measurement of the marmoset by whole-body plethysmography. In the pilot study, a ketamine-anesthetized marmoset was exposed for 20 min to an FITC-dextrane aerosol atmosphere (200 microg/L air, particle size 1.5 microm mass median aerodynamic diameter [MMAD]). It was found that 3.4% of the inhaled aerosol was deposited in the lungs; the aerosol was distributed over the lung lobes with an higher concentration at the distal side.     

Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates,astemizole and terfenadine

1.?Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes.

2.?Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice.

3.?Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver.

4.?Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes.

5.?These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.     

A comparison of planar scintigraphy and SPECT measurement of total lung deposition of inhaled aerosol.

Planar gamma camera imaging of inhaled aerosol deposition is extensively used to assess the total deposition in the lung. However, validation of the measurements is not straightforward, as gold standard measurements of lung activity against which to compare are not readily available. Quantitative SPECT imaging provides an alternative method for comparison. Four different methods for planar image quantification are compared. Two attenuation correction techniques, thickness measurement and transmission measurement, have been combined with two scatter correction techniques, reduced attenuation coefficient and line-source scatter function convolution subtraction. Each technique has been applied to 10 studies of aerosol deposition of a fine aerosol (mass median aerodynamic diameter 1.8 microm) and 10 studies using a coarse aerosol (mass median aerodynamic diameter 6.5 microm). The total activity in the right lung for each measurement has been compared to the value determined from SPECT imaging on the same subjects. When the thickness measurement and transmission techniques were applied with scatter compensation using a reduced attenuation coefficient, activity was systematically overestimated by 5% in both cases. The corresponding random errors (coefficient of variation) were 8.6% and 6.6%. Separate scatter correction reduced these systemic errors significantly to -1.5% and 2.7%, respectively. The random errors were not affected. All techniques provided assessment of total lung activity with an accuracy and precision that differed by less than 10% compared to the SPECT values. Planar gamma camera imaging provides a good method of assessing total lung deposition of inhaled aerosol.     

Lung Deposition of Droplet Aerosols in Monkeys

Nonhuman primates are often the animal models of choice to study the infectivity and therapy of inhaled infectious agents. Most animal models for inhaled infectious diseases use aerosol/droplets generated by an atomization technique such as a Collison nebulizer that produces particles in the size range of 1 to 3 μm in diameter. There are few data in the literature on deposition patterns in monkeys. Our study was designed to measure the deposition pattern in monkeys using droplets having diameters of 2 and 5 μm using an exposure system designed to expose monkeys to aerosols of infectious agents. Six cynomolgus monkeys were exposed to droplets. The aerosol solution was generated from a Vero cell supernate containing DMEM + 10% fetal bovine serum tagged with Tc-99m radiolabel. Collison and Retec nebulizers were used to generate small and large droplets, respectively. The particle size (as determined from a cascade impactor) showed an activity median aerodynamic diameter (AMAD) of 2.3 and 5.1 μm for the Collison and Retec nebulizer, respectively. The animals were anesthetized, placed in a plethysmography box, and exposed to the aerosol. The deposition pattern was determined using a gamma camera. Deposition in the head airways was 39% and 58% for 2.3- and 5.1-μm particle aerosols, respectively, whereas the deposition in the deep lung was 12% and 8%, respectively. This information will be useful in developing animal models for inhaled infectious agents.     

A comparison of the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases of man with other species

Plasma and red cells from a variety of animal species were used to demonstrate that there is a relationship between the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases in vitro and the effectiveness of carbamate pretreatment against nerve agent poisoning reported in the literature. Decarbamoylation rates were faster in the non-human primates than in the guinea-pig, and carbamate pretreatment is more effective in these species than in the guinea-pig. The data for the decarbamoylation rates of physostigmine-inhibited enzymes suggests that the non-human primates are the best animal model for extrapolation of protection studies from animal species to man. Control values for red cell acetylcholinesterase (AChE) activity (mumol/min/mL blood) using acetylthiocholine (1 mM) were higher in the human (4.98) and the rhesus monkey (4.14) than in the marmoset (0.84) and the guinea-pig (0.83). Plasma cholinesterase (ChE) activity (mumol/min/mL plasma) using butyrylthiocholine (10 mM) was highest in the rhesus monkey (9.29), intermediate in human (5.10) and guinea-pig (6.06), and lowest in the marmoset (4.07). There was a species difference in the relative activity of AChE: ChE in blood, human (65:35), rhesus monkey (45:55), marmoset (30:70) and guinea-pig (20:80). The rate of recovery of red cell AChE and plasma ChE activities, following incubation of whole blood with physostigmine (1 x 10(-7) M), was in the order human greater than rhesus monkey greater than marmoset greater than guinea-pig. During the incubation of red cells with physostigmine there was little recovery of AChE activity for 3-4 hr in any species. During the incubation of plasma with physostigmine there was complete recovery of ChE activity by 2-3 hr in the human and rhesus monkey and a significant recovery by 3 hr in the marmoset and guinea-pig. This suggests that a component of plasma, possibly ChE, was responsible for the degradation of physostigmine, presumably by hydrolysis. There was a marked species difference in the decarbamoylation rates of physostigmine-inhibited enzyme. In the red cell the t1/2 values (min) were 14.8 (human), 21.2 (rhesus monkey), 17.9 (marmoset) and 31.9 (guinea-pig). In the plasma the t1/2 values (min) were 11.2 (human), 32.9 (rhesus monkey), 44.1 (marmoset) and 52.4 (guinea-pig).     

Chronic toxicity of 3,4,3',4'-tetrachlorobiphenyl in the marmoset monkey (Callithrix jacchus)

Cotton top marmoset monkeys (Callithrix jacchus) were orally dosed with 3, 1, 0.1 or 0 mg 3,4,3',4'-tetrachlorobiphenyl (TCB)/kg body weight twice per week for 18-23 weeks. Severe toxicity occurred in the highest dose group. Clinical signs of toxicity were a rapid decrease in body weight, alopecia, abnormal nail growth, nodular enlargement of the nipple area and scaly skin. Haematological analysis of peripheral blood revealed mild leukocytosis and anemia. Biochemical alterations observed were elevated triglyceride levels and cholesterol levels. Histopathology revealed dose dependent changes in a variety of tissues. Squamous metaplasia was found in skin and adnexa as well as in salivary glands. In the stomach, parietal cells were decreased and mucus producing cells were increased. The duodenal mucosa was hyperplastic. Ovaries showed an absence of corpora lutea. In the thyroid follicular cell hyperplasia and hypertrophy were noted. Toxicity was less severe in marmoset monkeys dosed with 1 mg TCB/kg, while minor toxic effects were observed in the animals dosed with 0.1 mg TCB/kg. The marmoset monkey appears to be less sensitive to the toxic action of TCB than the rhesus monkey. The pattern of histological and biochemical changes induced by TCB in marmoset monkeys is comparable to that described in humans and in other primate species exposed to PCBs. The marmoset monkey model may be valuable for investigations on human-related toxicity of PCBs.     

Lung volume is a determinant of aerosol bolus dispersion.

The technique of inhaling a small volume element labeled with particles ("aerosol bolus") can be used to assess convective gas mixing in the lung. While a bolus undergoes mixing in the lung, particles are dispersed in an increasing volume of the respired air. However, determining factors of bolus dispersion are not yet completely understood. The present study tested the hypothesis that bolus dispersion is related, among others, to the total volume in which the bolus is allowed to mix--i.e., to the individual lung size. Bolus dispersion was measured in 32 anesthetized, mechanically ventilated dogs with total lung capacities (TLCs) of 1.1-2.5 L. Six-milliliter aerosol boluses were introduced at various preselected time-points during inspiration to probe different volumetric lung depths. Dispersion (SD) was determined by moment analysis of particle concentrations in the expired air. We found linear correlations between SD at a given lung depth and the individual end-inspiratory lung volume (V(L)). The relationship was tightest for boluses inhaled deepest into the lungs: SD(40) = 0.068 V(L) - 1.77, r(2) = 0.59. Normalizing SD to V(L) abolished this dependency and resulted in a considerable reduction of inter-individual variability as compared to the uncorrected measurements. These data indicate that lung size influences measurements of bolus dispersion. It therefore appears reasonable to apply a normalization procedure before interpreting the data. Apart from a reduction in measurement variability, this should help to separate the effects on bolus dispersion of altered lung volumes and altered mixing processes in diseased lungs.     

A Novel Method to Aerosolize Powder for Short Inhalation Exposures at High Concentrations: Isolated Rat Lungs Exposed to Respirable Diesel Soot

More efficient methods are needed to aerosolize dry powders for short-duration inhalation exposures at high concentrations. There is an increasing need to reach the peripheral lung with dry powder medications as well as with collected ambient aerosol particulates in environmental research projects. In a novel aerosol generator, a fixed volume of compressed air was used to create a short burst of a highly concentrated aerosol in a 300-ml holding chamber. Collected diesel soot was deagglomerated to a fine aerosol with a mass median aerodynamic diameter (MMAD) of 0.55 μm, not much larger than the 0.25 μm MMAD of diesel exhaust particles measured in air. A fine powder such as 3-μm silica particles was completely deagglomerated to an aerosol with a MMAD of 3.5 μm. Immediately after generation, the aerosol was available for exposure at a chosen flow rate by the use of an automated valve system. Tritium-labeled diesel soot was thus used to expose the isolated perfused rat lung at an air concentration of ～3 mg/L and a flow rate of 370 ml/min in a 1-min-long exposure. The lungs were ventilated at 75 breaths/min and a tidal volume of 1.13 ± 0.11 ml (SD, n = 3). Results showed that 19.8 ± 1.1 μg (SD, n = 3) soot was deposited in the lungs. This amount constitutes 9.5% of the amount inhaled and is close to literature data on deposition of similar sized particles in the rat lung. More than 97% of the deposited soot was located distal to the extrapulmonary bronchi, indicating that the system delivers a highly respirable aerosol. The aerosol system is particularly useful for peripheral lung delivery of collected ambient aerosols or dry powder pharmaceuticals following a minimal effort in formulation of the powder.     

The optimal particle size for parasympathicolytic aerosols in mild asthmatics

Background: the optimal particle size of a parasympathicolytic aerosol is unknown. Methods: eight stable asthmatics with a mean FEV₁ of 72% of the predicted value inhaled three types of monodisperse ipratropium bromide aerosols, with particle sizes of 1.5, 2.8 and 5 μm, respectively, and a placebo aerosol. The volunteers inhaled 8 μg ipratropium bromide, after which lung function improvement was determined. The changes in lung function were analysed with repeated measurements ANOVA. Results: according to the changes in FEV₁ and MEF_50/25 the 1.5/2.8 μm aerosol induced significantly better bronchodilatation than the 5 μm aerosol. No particle size effect was noticeable with regard to changes in R_cot, VC, FVC and PEF. Conclusions: in mild asthmatics the particle size of choice for a parasympathicolytic aerosol should be ≤ 2.8 μm.     

Miniaturized nebulization catheters: a new approach for delivery of defined aerosol doses to the rat lung.

A nebulization catheter technique (AeroProbe) was adapted and evaluated as a new approach for pulmonary delivery of defined aerosol doses to the rat lung. The lung distribution profile was evaluated by dosing Evans blue and Nile blue dye, respectively, to isolated and perfused rat lungs (IPL) and to the lungs of anesthetized and tracheal-intubated rats. The intratracheal aerosol dosing was synchronized with the inspiration of the lungs. Immediately after dosing, the lungs were dissected into upper- and lower trachea, bronchi, and parenchyma. The dye was then extracted from the tissue samples to determine the regional distribution and the recovery of the aerosol dose in the lungs. The droplet-size distribution and the weight of the delivered aerosol dose were analyzed with laser diffraction and gravimetric analysis respectively. The recovery of the delivered dose was high, 99 +/- 12 and 105 +/- 1%, respectively, in the in vivo administrations and IPL-experiments. The lung distribution profile after aerosol dosing to anesthetized rats was mainly tracheobronchial. Only 12 +/- 4% of the dose was recovered in the lung parenchyma. However, after aerosol dosing to the IPL, 38 +/- 11% of the dose was distributed to the lung parenchyma. At the settings used, the nebulization catheter aerosolized 1-2 microL of liquid per puff using 1-1.5 mL of air. The droplet-size distribution of the generated aerosols was broad (2-8% <3 microm; 10% <4-7 microm; 50% <10-15 microm; and 90% <20-40 microm). The nebulization catheter technique provides a complement to existing methodology for pulmonary drug delivery in small animals. With this new technique, defined aerosol doses can be delivered into the lungs of rats with no need for aerosol dosimetry.     

Metabolism of the calcium antagonist,mibefradil (POSICORTM,Ro 40-5967). Part III. Comparative pharmacokinetics of mibefradil and its major metabolites in rat,marmoset, cynomolgus monkey and man

1. The metabolism of mibefradil has been examined in rat, marmoset, cynomolgus monkey and man after single and multiple oral administration. 2. Metabolites typically represent between 50 and 80% of the circulating drug-related material after single oral doses of mibefradil to man, rat and marmoset. They arise by a combination of enzymatic processes: cytochrome P450-mediated oxidation at saturated and unsaturated carbon atoms, cytochrome P450-catalysed dealkylation and hydrolysis of the ester side-chain. 3. Plasma levels of mibefradil in the cynomolgus monkey are extremely low as a result of very efficient first-pass hydrolysis of its side-chain to give the corresponding alcohol. Steady-state concentrations of this metabolite are comparable with those of the parent drug in man and marmoset, but are relatively low in rat plasma. 4. Hydroxylation at the benzylic carbon of the tetrahydronaphthyl ring leads to further important metabolites in primates, whereas the products of O - and N -demethylation are found in small amounts in all four species. 5. Estimates of the exposure of the various species to the principal metabolites indicate that the choice of the rat, marmoset and cynomolgus monkey for the toxicological assessment of mibefradil was appropriate.     

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy- 3-(p-nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30–42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1. Received: 18 October 1999 / Accepted: 8 December 1999     

In vitro characterization of nebulizer delivery of liposomal amphotericin B aerosols

Pharmaceutical aerosols have the potential to prevent pulmonary infectious diseases. Liposomal amphotericin B (LAMB, Ambisome, Astellas Pharma US, Deerfield, IL, USA) is approved as an intravenous infusion for empiric treatment of presumed fungal infections in neutropenic, febrile patients, as well as patients infected with Aspergillus, Cryptococcus, and other fungal pathogens. In this study, four different nebulizers were tested for their ability to deliver LAMB in aerodynamic droplet-size ranges relevant to lung deposition by an inertial sampling technique Mass median aerodynamic diameter (MMAD) and fine particle fraction percent <3.3 μm (FPF(3.3)) and <5.8 μm (FPF(5.8)) were determined by cascade impaction during a 2 min sampling period for each of three trials of all nebulizers. The MMADs for all nebulizers ranged from 1.72 ± 0.11 μm to 2.89 ± 0.12 μm; FPF(3.3) and FPF(5.8) were approximately 80% and 90%, respectively. Although all nebulizers appear acceptable for delivery of LAMB, the Pari LC Star and the Aeroeclipse II were considered the best in terms of delivery of aerosol efficiently and the proportion suitable for lung deposition. Additional research on pulmonary delivery and clinical tolerability is warranted.     

Comparison of Calidria Chrysotile Asbestos to Pure Tremolite: Final Results of the Inhalation Biopersistence and Histopathology Examination Following Short-Term Exposure

Calidria chrysotile asbestos, which is a serpentine mineral, has been shown to be considerably less biopersistent than the durable amphibole mineral tremolite asbestos, which persists once deposited in the lung. The initial results of this inhalation biopersistence study in rats that demonstrates this difference were reported in . This article presents the full results through 1 yr after cessation of the 5-day exposure. This study was based upon the recommendations of the European Commission (EC) Interim Protocol for the Inhalation Biopersistence of synthetic mineral fibers (). In addition, the histopathological response in the lung was evaluated following exposure. In order to quantify the dynamics and rate by which these fibers are removed from the lung, the biopersistence of a sample of commercial-grade chrysotile from the Coalinga mine in New Idria, CA, of the type Calidria RG144 and that of a long-fiber tremolite were studied. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 μm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and the histopathological response. As the long fibers have been shown to have the greatest potential for pathogenicity, the aerosol generation technique was designed to maximize the number of long respirable fibers. The chrysotile samples were specifically chosen to have 200 fibers/cm³ longer than 20 μm in length present in the exposure aerosol. These longer fibers were found to be largely composed of multiple shorter fibrils. The tremolite samples were chosen to have 100 fibers/cm³ longer than 20 μm in length present in the exposure aerosol. Calidria chrysotile has been found to be one of the most rapidly cleared mineral fibers from the lung. The fibers longer than 20 μm in length are cleared with a half-time of 7 h. By 2 days postexposure all long fibers have dissolved/disintegrated into shorter pieces. The fibers between 5 and 20μm in length were cleared with a half-time of 7 days. This length range represents a transition zone between those fibers that can be fully phagocytosed and cleared as particles and the longer fibers that cannot be fully engulfed by the macrophage. The fibers/objects shorter than 5 μm in length were cleared with a half-time of 64 days, which is faster than that reported for insoluble nuisance dusts such as TiO₂. By 12 months postexposure, 99.92% of all the remaining chrysotile was less than 5 μm in length. Following the 5 days of repeated exposure to more than 48,000 chrysotile fibers/cm³ (190 fibers L > 20 μm), histopathological examination revealed no evidence of any inflammatory reaction either after the cessation of the last exposure or at any time during the subsequent 12-mo period. This is in marked contrast to the amphibole tremolite, which was also investigated using the same inhalation biopersistence protocol. The long tremolite fibers, once deposited in the lung, remain over the rat's lifetime with essentially an infinite half-time. Even the shorter fibers, following early clearance, also remain with no dissolution or further removal. At 365 days postexposure, there was a mean lung burden was of 0.5 million fibers L > 20 μm and 7 million fibers 5–20 μm in length with a total mean lung burden of 19.6 million fibers. The tremolite exposed rats, even with exposure to 16 times fewer total fibers than chrysotile, showed a pronounced inflammatory response with the rapid development of granulomas as seen at day 1 postexposure, followed by the development of fibrosis characterized by collagen deposition within these granulomas and by 90 days even mild interstitial fibrosis. With the short exposure, this study was not designed specifically to evaluate pathological response; however, it is quite interesting that even so there was such a marked response with tremolite. These findings provide an important basis for substantiating both kinetically and pathologically the differences between chrysotile and the amphibole tremolite. As Calidria chrysotile has been certified to have no tremolite fiber, the results of the current study together with the results from toxicological and epidemiological studies indicate that this fiber is not associated with lung disease.     

Molecular cloning and tissue distribution of marmoset thiopurine S-methyltransferase

The common marmoset (Callithrix jacchus) is a New World monkey that is increasingly used in pharmacological and toxicological studies. Thiopurine S-methyltransferase (TPMT) plays roles in the metabolism of widely used anticancer and anti-inflammatory drugs. Here, we report the isolation and molecular characterization of marmoset TPMT cDNA, which was found to contain an open-reading frame of 245 amino acids that was approximately 92% identical to its human ortholog. Marmoset TPMT was phylogenetically closer to other primate orthologs than to its pig, dog, rabbit, or rodent orthologs. Among the five marmoset tissue types analyzed, marmoset TPMT mRNA was most abundant in kidney and liver, just as human TPMT is. These results suggest that marmoset and human TPMT are similar at the molecular level.     

The Sophia Anatomical Infant Nose-Throat (Saint) model: a valuable tool to study aerosol deposition in infants.

Relatively little is known about the variables that influence lung deposition of inhaled aerosols in children. A model of the upper airways of an infant could be a useful tool to study these variables in vitro. The objective of this study was to construct an anatomically correct model of the upper airways of a young child. A routine three-dimensional (3D) CT scan of the skull and neck of a child was selected that included the airway from the nasal cavity down to the subglottic region. The CT scan was edited to obtain an anatomically correct distinction between air and mucosa. Next, a model was constructed with a stereolithographic technique using a UV-sensitive resin. To validate the model, a 3D CT scan of the model was made and compared to the anatomy of the original image. To study aerosol deposition, the model was connected to a breathing simulator. Medical aerosols were delivered to the model by MDI/spacer during stimulated breathing. An upper airway model was made of a 9-month-old child that needed reconstructive surgery for a skull deformity and with normal anatomy of the upper airways. The nasal airway of the model was open for air passage and the oral airway was closed. The CT scan of the model matched the original in vivo CT scan closely. Aerosol deposition measurements showed that dose passing the model, or lung dose, was comparable with in vivo lung deposition data. We have constructed an anatomically correct model of the upper airways of a child, using a stereolithographic method for in vitro studies of aerosol deposition in young children. This model will be used to obtain insight in aerosol treatment that cannot be obtained in vivo.