The Influence of Nebulized Drugs on Nasal Ciliary Activity.

INTRODUCTION: Nebulized drugs are used in the treatment of cystic fibrosis (CF) lung disease, asthma, and COPD, and increasingly also in other chronic lung diseases. Their use in CF is reasonably evidence based, but this is not so for use in other orphan diseases. Potential side effects often have not been studied. Therefore, we evaluated the influence of nebulized drugs on ciliary activity in an in vitro model. METHODS: We constructed an in vitro nebulization model to examine the effect of drugs on ciliary activity. The model was validated by testing solutions with known neutral, positive, or negative effect on ciliary beat frequency (CBF). Next, the influence on CBF of other inhaled drugs was tested. RESULTS: Nebulization of NaCl 0.9% had no influence on CBF, and was used as paired neutral control in further experiments. Salbutamol (Ventolin(®)) had a ciliostimulatory effect (CBF +18%, CBF at t0-t10-t60 7.1-8.5-8.6 Hz, p = 0.002), while hypertonic saline (CBF - 11%, CBF at t0-t10-t60 6.5-5.1-5.9 Hz, p = 0.018) and dry air (CBF -10%, CBF at t0-t10-t60 6.8-5.8-6.1 Hz, p = 0.008) had a cilioinhibitory effect. Nebulization of tobramycin inhaled solution (TOBI(®)) (p = 0.662), colistimethate (Colistineb(®)) (p = 0.369), rhDNAse (Pulmozyme(®)) (p = 0.069), ceftazidim (Glazidim(®)) (p = 0.875), and aztreonam (Cayston(®)) (p = 0.435) did not affect CBF. Obracin(®), a tobramycin containing solution manufactured for intravenous use, had a negative effect on CBF (CBF - 21%, CBF at t0-t10-t60 6.9-5.2-4.5 Hz, p = 0.004). CONCLUSION: Inhaled drugs that are used off-label might have an influence on ciliary activity. This must be taken into account when prescribing these drugs for non-CF indications.

A Proposed In Vitro Method to Assess Effects of Inhaled Particles on Lung Surfactant Function.

The lung surfactant (LS) lining is a thin liquid film covering the air-liquid interface of the respiratory tract. LS reduces surface tension, enabling lung surface expansion and contraction with minimal work during respiration. Disruption of surface tension is believed to play a key role in severe lung conditions. Inhalation of aerosols that interfere with the LS may induce a toxic response and, as a part of the safety assessment of chemicals and inhaled medicines, it may be relevant to study their impact on LS function. Here, we present a novel in vitro method, based on the constrained drop surfactometer, to study LS functionality after aerosol exposure. The applicability of the method was investigated using three inhaled asthma medicines, micronized lactose, a pharmaceutical excipient used in inhaled medication, and micronized albumin, a known inhibitor of surfactant function. The surfactometer was modified to allow particles mixed in air to flow through the chamber holding the surfactant drop. The deposited dose was measured with a custom-built quartz crystal microbalance. The alterations allowed the study of continuously increasing quantified doses of particles, allowing determination of the dose of particles that affects the LS function. The tested pharmaceuticals did not inhibit the function of a model LS even at extreme doses--neither did lactose. Micronized albumin, however, impaired surfactant function. The method can discriminate between safe inhaled aerosols--as exemplified by the approved inhaled medicines and the pharmaceutical excipient lactose--and albumin known to impair lung functionality by inhibiting LS function.

Human sinonasal explant system for testing cytotoxicity of intranasal agents.

BACKGROUND: Intranasal agents play a critical role in the management of sinonasal disorders. There are ongoing efforts to develop new intranasal medications to combat sinonasal disease. Some intranasal agents, however, can have cytotoxic effects on human sinonasal tissue. In order to facilitate safe drug discovery, we developed a simple and reliable in vitro screening assay using human sinonasal explants to measure the cytotoxic profiles of intranasal agents. METHODS: We obtained sinonasal tissues from several regions of the nasal cavity from 12 patients undergoing endoscopic sinonasal surgery. These tissues were cultured on polytetrafluoroethylene membrane in serum-free growth medium. We determined the biochemical properties of these explants by measuring extracellular lactate dehydrogenase (LDH) levels and performing histological analyses over a period of 1 to 2 weeks. We then examined the cytotoxic profiles of 13 intranasal agents by measuring extracellular LDH levels using the human sinonasal explant system. RESULTS: Sinonasal explants exhibited a rapid reduction in extracellular LDH levels indicating stabilization in the culture environment within 2 days. Histological analysis showed maintenance of good cellular architecture for up to 2 weeks. The explants displayed intact epithelium and expressed ßIII-tubulin and Ki-67. Of the 13 tested intranasal agents, 1% zinc sulfate (ZnSO(4) ), 5% ZnSO(4) , and Zicam application were cytotoxic. CONCLUSION: Based on the unique biochemical properties of the human nasal explant culture system, we developed a simple and reliable in vitro screening assay to determine the cytotoxic profiles of various intranasal agents by examining extracellular LDH levels and histopathology.

Mikania laevigata syrup does not induce side effects on reproductive system of male Wistar rats.

Mikania laevigata (Asteraceae) is a native plant from South America and popularly used as antispasmodic and to treat respiratory diseases. Coumarin is the major chemical substance found in this plant, which have been shown to have antifertility activity in female rats. This study evaluates the toxicity of the exposure to the Mikania laevigata syrup using coumarin as chemical marker on reproductive endpoints in male Wistar rats. Endpoints including reproductive organs weight, sperm and spermatids numbers and sperm morphology were evaluated. Animals were treated daily with Mikania laevigata syrup (3.5; 7.0 and 14.0mg/kg of coumarin) during 90 days by oral gavage. No alterations were observed in body and organ weights, sperm and spermatids numbers as well as sperm morphology of the male rats after the exposure to the Mikania laevigata syrup. Results therefore suggest absence of male reproductive toxicity of the Mikania laevigata syrup at tested doses.

Effect of instillation into lung of autologous blood on pulmonary function and tracheobronchial wash cytology.

This study aimed at measuring the functional consequences and the pulmonary cytology changes following a simulation of pulmonary haemorrhage. Pulmonary function tests including lobeline-induced hyperventilation, cytology of tracheo-bronchial wash (TBW) and thoracic radiographs were performed before, as well as 1, 7, 14 and 28 days after, the instillation of 300 ml of blood into the lungs of 4 horses deemed free of exercise-induced pulmonary haemorrhage (Group 1). Control data (Group 2) were obtained by instilling the same volume of saline into the lungs of the same horses in a crossover design (control). The instillation of blood or saline resulted in an increase in the number of neutrophils in the TBW. Thoracic radiographs showed increased opacity in the caudodorsal region of the lungs in 4/4 (Day 1) and 2/4 horses (Day 7), in Group 1, and in 2/4 (Day 1) and 0/4 horses (Day 7) in the control group. These changes were attributed to the instillation procedure rather than the nature of the instilled material. Breathing mechanics and arterial blood gases at rest were not affected in either Groups 1 or 2. However, the maximal expiratory peak flow recorded during lobeline-induced hyperventilation was significantly lower (P<0.05) and the total pulmonary resistance significantly higher (P<0.05) on Day 1 in Group 1, but not Group 2. These observations suggest that expiratory flows might be partly limited in bleeders when breathing at high airflow.

Potentiation of acetaminophen hepatotoxicity and mortality by doxapram in mice.

Whether a single dose of doxapram (DOP) can modulate the acute toxicity and the hepatotoxicity induced by acetaminophen (AA) was examined. Pretreatment with DOP (40 mg/kg, i.p.) 30 min prior to the administration of AA dose-dependently potentiated the lethality of AA in both native mice and mice fasted for 18 h, and the potentiating activity was greater in fasted mice than in native mice. The hepatotoxicity of AA was assessed by plasma transaminases activity (glutamyl oxaloacetic transaminase, GOT; glutamyl pyruvic transaminase, GPT) and the amount of plasma lipid peroxides at 6, 12, 18, 24, 36 and 48h after the administration of AA and histopathological examination of liver sections at 24 h after the administration of AA. DOP (40 mg/kg, i.p.) did not increase the plasma transaminase activity or the lipid peroxides level significantly, whereas AA administration to DOP-treated animals produced earlier maximal elevation of transaminase and lipid peroxide values compared to AA alone. These findings indicate that mortality and hepatotoxicity of AA is potentiated by DOP in mice.

Time-dependent autoimmune response of dichloroacetyl chloride in female MRL +/+ mice.

Welders are exposed to dichloroacetyl chloride (DCAC) when trichloroethene (TCE) is used as a degreasing agent. Human exposure to TCE and tetrachloroethane can also lead to formation of DCAC in situ through metabolism. Due to its strong acylating property, it can bind with cellular macromolecules and act as hapten and consequently may elicit autoimmune (Al) response. Earlier, we reported that both TCE and DCAC induce/accelerate Al response in MRL +/+ mice, and DCAC even at 50 fold lower concentration induced greater Al responses. These studies, however, were conducted at a single time point (six weeks of treatment) and therefore necessitate a time-dependent characterization of this DCAC-induced Al response. Female MRL +/+ were given ip treatments of 0.2 mmol/kg DCAC in 100 microliters of corn oil every 4th day, while controls received an equal volume of corn oil only. DCAC treatment resulted in spleen weight increases at all time points whereas serum IgG showed significant increases at 4, 6 and 8 weeks of treatment. Serum autoantibodies, i.e., antinuclear antibodies, anti-single stranded DNA antibodies and anticardiolipin antibodies showed positive responses only after 4 weeks of treatment. However, the optimal responses were observed at 6 weeks and subsequently the responses diminished (at 8 weeks). The DCAC-specific antibodies showed a pattern similar to autoantibodies, i.e., an optimal response at 6 weeks of treatment. Our results thus suggest that DCAC under the current experimental conditions induces an optimal Al response at 6 weeks of treatment and further emphasize the usefulness of MRL +/+ mice in studying chemical-induced autoimmunity.

Considerations for toxicology studies of respiratory drug products.

The standard approaches for the preclinical development of chronically administered drugs also apply to most respiratory drugs. Modifications from the standard preclinical development plan, however, may be necessary if the drug is administered intranasally or by inhalation. Administration by these routes may result in airway toxicity and the intended patient population is often particularly susceptible. Current and former representatives of the Division of Pulmonary Drug Products (CDER, U.S. FDA) present this article to describe general principles of preclinical development for respiratory drug indications. The article addresses drugs intended for administration by the intranasal or inhalation routes. The article describes the types of studies recommended, considers the initial human dose, and discusses dose-escalation strategies in clinical trials. Other areas of special concern with intranasal or inhalation administration include immunotoxicity, reproductive toxicity, types of dosing apparatus, excipients and extractables, and formulation changes. The approaches described in this article are intended as general information and should be adapted to the scientific considerations and circumstances of a particular drug under development.

Lipidosis of the dorsal root ganglia in rats treated with an almitrine metabolite.

Toxic effects of a detriazinyl metabolite of almitrine (DTMA) were evaluated in rats and on cultured rat macrophages. In rats daily treated with DTMA for 16 weeks, spastic gaits with heel-lifting appeared, and lamellated and/or crystalloid bodies formed in sensory neurons, satellite cells, Schwann cells, and vascular endothelial cells of the dorsal root ganglia. The lysosomal lamellated bodies, which were not induced by almitrine, were produced also in cultured rat macrophages exposed to over 1 x 10(-5) M DTMA.