Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics.

Acute and chronic respiratory diseases account for major causes of illness and deaths worldwide. Recent developments of biotherapeutics opened a new era in the treatment and management of patients with respiratory diseases. When considering the delivery of therapeutics, the inhaled route offers great promises with a direct, non-invasive access to the diseased organ and has already proven efficient for several molecules. To assist in the future development of inhaled biotherapeutics, experimental models are crucial to assess lung deposition, pharmacokinetics, pharmacodynamics and safety. This review describes the animal models used in pulmonary research for aerosol drug delivery, highlighting their advantages and limitations for inhaled biologics. Overall, non-clinical species must be selected with relevant scientific arguments while taking into account their complexities and interspecies differences, to help in the development of inhaled medicines and ensure their successful transposition in the clinics.

Pulmonary delivery of dry powders to rats: tolerability limits of an intra-tracheal administration model.

The inhaled route is increasingly developed to deliver locally acting or systemic therapies, and rodent models are used to assess tolerance before clinical studies. Endotracheal intubation of rats with a probe which generates powder aerosols enables controlled administration of drug directly into the respiratory tract. However, preliminary observations of intratracheal powder administration procedures have raised concerns with regard to pulmonary safety. The aim of the present work was to evaluate the safety of intra-tracheal administration of dry powder in a rat model. Sixty animals were administered various volumes of air alone, lactose or magnesium stearate through a Microsprayer(®) (Pencentury, USA). The mass of powder actually delivered to each animal was calculated. Rats were sacrificed immediately after administration, and the lungs, trachea and larynx were removed and examined for gross pathology. The mass of powder delivered varied, the full dose being rarely delivered. About one third of the administration procedures resulted in respiratory failure, and macroscopic pulmonary lesions were observed in about 55% of animals. Lung damages were observed with air alone, lactose and magnesium stearate. In conclusion, artifacts observed with this technique may limit the relevance of the model. These observations are particularly important in the context of regulatory toxicity studies.

Association of Fcγ receptor IIIA variant with a subset of anti-topoisomerase I-positive patients in systemic sclerosis: a descriptive pilot study.

Hypothesizing a pathophysiological role of anti-topoisomerase I antibodies (anti-topo I) through autoantibody-dependent cell-mediated cytotoxicity (ADCC) and cytotoxic effectors expressing receptors for the Fc portion of IgG in systemic sclerosis (SSc), 267 SSc patients (56 with anti-topo I and 102 with anti-centromere antibodies (ACA)) were genotyped for the functional FCGR3A-V158F polymorphism. A descriptive analysis of patients according to their clinical and immunological status and FCGR3A-158 V/F genotypes was performed using multiple correspondence analysis. This descriptive analysis revealed an association between the FCGR3A-158 VV genotype and the presence of anti-topo I. By contrast, no relationship was found between FCGR3A polymorphism and the presence of ACA. SSc patients with anti-topo I appear to be more frequently homozygous for the high-affinity FcγRIIIA-coding allele, suggesting that some autoantibodies may be pathogenic through ADCC.

The utility of a nebulised intra-tracheal rat model of invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis (IPA) is of particular concern to immunodeficient patients, whose mortality rates may exceed 80%. The development of an animal model that faithfully reproduces the pathophysiology of IPA would improve the studies on diagnostic and therapeutic modes, and the use of rats as a possible model for IPA seems to have been largely overlooked. Such a model could be established with the MicroSprayer IA-1B. Male Sprague-Dawley rats (6-8 weeks old) were rendered immunodeficient by cyclophosphamide injections and a protein-deficient diet. On day D0, they were anaesthetised by inhalation of 5% isoflurane and infected by the intra-tracheal aerosolization of 100 microl of an Aspergillus fumigatus spore suspension through a MicroSprayer IA-1B. This inoculation process was simple and rapid, with no deaths observed during or immediately after the procedure. The rats regained consciousness within 1 min. Follow-up data including those for clinical factors (weight changes, mortality rate), biological factors (Aspergillus antigens) and histological factors were consistent with previous studies. The advantages of this model include the ease of animal manipulation, the reproducibility of infection and the potential for repeated blood sampling.

[Nebulized corticosteroids and pediatricians: results of the NUAGES survey]. / Prescription des corticoïdes nébulisés par les pédiatres : résultats de l'enquête NUAGES.

INTRODUCTION: The aim of the study was to analyze the data of the NUAGES survey (a survey on the practice of nebulization in France), concerning the prescriptions of nebulized steroids from 514 pediatricians. MATERIAL AND METHODS: The reason why nebulization was chosen as a delivery route, the diseases motivating the prescription, the age of the patients, the kind of drug used, and the prescription and device modalities were studied. RESULTS: Efficacy in treating various respiratory diseases was the main reason cited for using nebulization, in particular severe persistent asthma (76%). Pediatricians prescribed nebulization mainly to infants (60%). The most frequently used drug was budesonide suspension (89%), but the intravenous route for steroids (18%) and drug admixtures (62%) were also proposed by nebulization. A specific prescription for the nebulizer was given in 75% of the cases, with the type of interface to use specified in 66%. DISCUSSION: Pediatricians consider that nebulization is well adapted to young children. Although the proper steroid is usually chosen, unfortunately, it is often prescribed with other drugs, with 1 prescription out of 4 not following the recommendations. Prescription of the device is not optimal and may compromise the efficacy of the treatment. CONCLUSION: Nebulization is a potential mode of delivery for steroids that is difficult to prescribe and warrants improved pediatrician training.

[A survey of nebulization practices in France conducted in 2004 by the GAT]. / NUAGES: une enquête sur la pratique de la nébulisation en France.

METHODS: A questionnaire was sent to 50 000 general practicioners (GP) and specialists. RESULTS: 4,898 physicians (9.4%) responded, including 59.1% GP, 16.9% pneumologists, 13% pediatricians and about 10% other specialists, ENT, allergologists, and intensivists. The main reason for pneumologists to prescribe nebulization was the efficiency on long term of approved drugs. GP prescribe nebulization for its local effects, using unapproved drugs, on short periods of time, especially in COPD, asthma, bronchitis and tracheitis/laryningitis. Although pneumologists have been trained during their fellowship and do not ask for further education, MG have learned by their own experience and are asking for further education. CONCLUSION: This study should help to develop teaching programs on nebulization with the aim to optimize its practice.

N.U.A.G.E.S: a survey of nebulisation practice in France with regard to ERS guidelines.

UNLABELLED: A survey of nebulisation practice in France was conducted under the aegis of the French respiratory society in 2004. METHODS: Analysis of a questionnaire was obtained from 3674 physicians. RESULTS: A total of 2439 physicians were general practitioners (GPs), 698 were chest physicians, and 537 paediatricians. The main reasons to use nebulisation are (1) for chest physicians efficacy in treating various pathologies with long-term administration (1 wk to 1 month) of approved drugs, and (2) for GP's local action properties. While chest physicians learned about nebulisation during their university training and do not ask for additional information, GPs learned by practical experience or from colleagues and ask for further information. CONCLUSION: This study will help to develop targeted educational programmes on nebulisation practice.

[Investigation on health status of silica exposed workers in "cotto Fiorentino" companies]. / Indagine sullo stato di salute degli esposti a silice nelle aziende del cotto Fiorentino.

The manufactured in "cotto" is typical of Chianti Fiorentino with about 400 employees. In 2005. the UF PISLL encountered an exposure to silica more than TLV, particularly for some tasks; were prescribed interventions of prevention and was undertaken an investigation of the occupational health status with occupational health physician. We observed 227 workers, 208 males and 19 females, with average age of 43 years and average age working of 15 years. The habit of smoking tobacco was higher than for the general population. The assessment of exposure to silica has been detected for 59 workers (mean 0.05 mg / mc); at pulmonary function testing resulted: 10 with airway obstruction and 4 airflow limitation; by 140 chest X - ray acquired 1 was interstitial pulmonary disease and 1 was bronchopneumonia. Among the diseases not related to exposure to silica, emerging 42 cases of low back pain, 28 hearing loss, 7 with hypertension. Non uniformity in health surveillance and diagnostic criteria highlights the need to cooperate between occupational doctor in public prevention and control service and qualified occupational doctor to ensure a standard of quality in the prevention of disease in exposed to silica.

Comparative analysis of different scintigraphic approaches to assess pulmonary ventilation.

A study was carried out to investigate the predictive value of 81-metastable-krypton (81mKr) distribution, high-size 99-metastable-technetium (99mTc) aerosol deposition and low-size 99mTc aerosol (Technegas) deposition on the pulmonary ventilation evaluated by 133-xenon (133Xe) lung scintigraphy, and to assess the correlation between the 81mKr distribution, the 99mTc aerosols deposition, and the respiratory parameters of patients with chronic obstructive pulmonary disease (COPD). Twenty COPD patients were included. The 81mKr, 133Xe, and 99mTc aerosol lung scintigraphies were successively carried out. The 81mKr distribution and 99mTc deposition were compared to the 133Xe distribution at equilibrium and to the 133Xe clearance. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 81mKr and Technegas lung scintigraphies to detect alterations in ventilation revealed by 133Xe were defined. The 81mKr distribution and 99mTc deposition according to respiratory parameters were described using a principal component analysis. Compared to 133Xe distribution, a significantly higher distribution of 81mKr in the upper parts of the lungs in the more severe patients (p = 0.05), a significantly higher deposition of Technegas in the lower parts of the lungs (p = 0.0008), and a significantly higher deposition in the central parts of the high-size 99mTc aerosol were observed (p = 0.0001). The PPV and the NPV were, respectively, 0.54 and 0.58 for 81mKr and 0.54 and 0.55 for Technegas. There was a significant negative correlation between 81mKr distribution and 133Xe clearance (p = 0.0001) between Technegas deposition and 133Xe clearance (p = 0.0007), and between 99mTc diethylene-triamino-penta-acetate (DTPA) deposition and 133Xe clearance (p = 0.001). Both the 81mKr peripheral distribution and Technegas peripheral deposition correlated negatively with increased obstruction, as measured by forced expiratory volume in 1 sec (FEV1). Peripheral deposition of the high-size 99mTc aerosol deposition correlated with the inspiration/expiration time ratio. In conclusion, 81mKr and 99mTc aerosols' lung scintigraphies do not reflect exactly the pulmonary ventilation as measured by 133Xe scintigraphy.

[Dyspnea and morphine aerosols in the palliative care of lung cancer]. / Dyspnée et aérosols de morphine dans les soins palliatifs du cancer broncho-pulmonaire.

OBJECTIVE: To evaluate the effectiveness of morphine aerosols in the treatment of dyspnoea in the palliative care of patients with lung cancer. MATERIALS AND METHODS: During a randomised, double blind, cross-over study 12 patients receiving palliative care for lung cancer and suffering from dyspnoea despite conventional treatments were given, for two periods of 48 hours separated by a 24 hour wash-out period, 4 mls of morphine sulphate and 4 mls of normal saline 4 hourly by a jet nebuliser. Before and after each nebulisation respiratory rate and capillary oxygen saturation were measured and dyspnoea was quantified with the aid of a visual analogue scale by the patient and various other observers (doctors, students, nurses, care assistants and physiotherapists). RESULTS: The aerosols of normal saline and morphine produced the same improvements in the dyspnoea scores independently of the mass nebulised. Furthermore the nebulisations did not produce any significant change in respiratory rate or oxygen saturation. CONCLUSION: The fact that both aerosols lead to a similar improvement in dyspnoea scores suggests that humidification of the airways rather than a pharmacological action may be beneficial in the treatment of dyspnoea in terminally ill patients.

A multivariate statistical analysis to follow the course of disease after infection of cats with different strains of the feline immunodeficiency virus (FIV).

A descriptive multivariate assay is described which is suitable to analyze results of a biological experiment with small sample size but high qualitative and quantitative complexity of variables. This type of assay allows evaluation of multiple variables observed in the course of an experimental virus infection (e.g. viremia, nucleic acid detection, antibody titers, clinical parameters, anti-microbial treatments or vaccination) in a single graph. In our study, a multiple correspondence analysis (MCA) was used to correlate a total of 145 measurements from each of a dozen of variables measured in five groups of three cats infected by five isolates of feline immunodeficiency virus (FIV). Three groups of virus isolates with distinct virulence were defined and correlation between dynamics of lymphocyte subset counts and viral virulence was established. Comparison between the primary stages of illness and follow-up examinations were of prognostic value and are thus helpful for development and monitoring of therapeutic strategies.

Potential antiviral agents. Part II. Synthesis and antiviral evaluation of pyrazinones substituted with acyclic chains.

The synthesis of a series of 4'-substituted hydroxybutyl pyrazine analogues of the anti-herpes compound, acyclovir, is described. The compounds were characterized with 1H and 13C nmr, mass and IR spectroscopy. Antiviral (HSV-1, CMV, Cox B4, HIV-1) properties of these compounds were examined. None of these compounds were active against these viruses.

Synthesis and antiviral evaluation of SATE-foscarnet prodrugs and new foscarnet-AZT conjugates.

The synthesis of a range of di- and triester derivatives of phosphonoformate (PFA; foscarnet) as potential lipophilic, membrane-soluble prodrugs is described. In addition to normal alkyl esters in the carboxylate and phosphonate residues of PFA, the bioreversible S-(pivaloyl)thioethyl (t-butyl-SATE) group was introduced in an attempt to deliver PFA after bioactivation inside the cells. Furthermore, PFA-AZT conjugates were prepared in order to develop combinational drugs. The key synthetic step was in all cases the formation of the P-C bond to build up the different PFA esters. In contrast to the diester derivatives, the triesters of PFA showed high hydrolytic instability during chromatographic purification. The compounds were evaluated in vitro for their ability to inhibit viruses in several tissue culture systems. All PFA alkyl di- and triesters proved poorly active or inactive against human immunodeficiency virus (HIV) and inactive against hepatitis B virus. In contrast, the PFA-AZT conjugates exhibited significant anti-HIV activity. However, this activity was nearly completely lost in thymidine kinase-deficient cells, suggesting a fast unselective chemical hydrolysis of the conjugates to yield the nucleoside analogue AZT in the cell culture medium. Furthermore, no synergistic effect of PFA and AZT was observed.

Discrimination learning and multiple reversals in young adult and older monkeys (Macaca arctoides).

Two groups of five young adult an older stumptailed macaques (Macaca arctoides) were tested on a visual discrimination task followed by a reversal upon attainment of criterion; task and reversal were repeated until 20 reversals with the same pair of objects had been completed. Both groups required more trials to learn the first reversal than the original discrimination, with no significant difference between the groups. Older monkeys tended to show more perseverative errors on early reversals, but a striking improvement in their scores across successive blocks of reversals culminated in performances virtually indistinguishable from those of the young group by the end of testing.

Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus.

Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton-mediated process occurred. The productive infection of SEC may contribute to the progression of the infection.

Evidence of feline immunodeficiency virus replication in cultured Kupffer cells.

OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection.

Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels.

Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.

Differences in performance of young and old monkeys on a visuospatial memory task.

Two groups of stumptailed macaques (Macaca arctoides) were compared in terms of performance on a visuospatial working memory task involving repeated presentations of an array of 15 doors, each of which concealed one food reward at the start of a session. Rewards obtained by the subject were not replaced. Errors were defined as responses to doors previously pushed open during the session. Both groups generally obtained all rewards and showed improved performance across sessions, but old monkeys consistently made more errors than young ones, especially in the latter half of a given session. Further, old monkeys were less diverse in the errors they made, indicating different response strategies by the two groups.

Responses to inter- and intraspecific scent marks in pine martens (Martes martes).

In order to analyze intra and interspecific olfactory discrimination, behavioral responses (sniffing and marking) towards various odors were observed in pine martens (Manes martes). Two adult males and two adult females were tested for intraspecific discrimination of abdominal gland odors and urine. Both sexes sniffed and marked objects carrying their own scent less than unscented objects. There were no differences in sniffing or marking objects impregnated with odors from known and unknown conspecifics of the opposite sex or objects carrying the odor of an unknown male or female. A second experiment with two adult females gave no evidence for interspecific discrimination: no differences emerged when comparing reactions towards marks of pine martens, stone martens (a closely related species), or genets. The most obvious result of this study is the reduced response of pine martens towards their own mark. It is suggested that scent marking in martens may reflect "autocommunication," the primary effect being to familiarize an animal with its environment.