The Nonclinical Assessment of Trans-1,3,3,3-tetrafluoropropene (HFO-1234ze (E)), a Near Zero Global Warming Potential Propellant for Use in Metered Dose Inhalation Products.

HFO-1234ze (E) is proposed as a near zero global warming propellant for use in metered dose inhaled (MDI) products. This paper describes the non-clinical safety assessment in mice, rats, and dogs and supplements previously reported data (genetic toxicology, short-term toxicology, and reproductive toxicology). In all species, HFO-1234ze (E) was only detectable in blood for a short period after dosing with no evidence of accumulation. HFO-1234ze (E) was without any toxicological effects at very high doses in subchronic (13-week mouse) and chronic (39-week dog) studies. Chronic (26-week) administration to rats at very high doses was associated with an exacerbation of rodent progressive cardiomyopathy, a well-documented background finding in rodents. In a 2-generation study, extremely high doses were associated with the early euthanasia of some lactating female rats. This finding was considered to be significantly influenced by a state of negative energy balance, reflecting the specific vulnerability of rats during lactation. These findings are considered to not pose a risk to humans with typical MDI use given they occurred at doses which far exceed those expected in patients. Overall, the nonclinical safety data for HFO-1234ze (E) support its further development as an MDI propellant.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages.

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation. METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1ß response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover. RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1ß release from macrophages, through lysosomal destabilization. IL-1ß secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1ß release was significantly reduced in primary macrophages from ctss(-/-) mice. CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1ß release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses.

Alveolar macrophage (AM) responses are commonly induced in inhalation toxicology studies, typically being observed as an increase in number or a vacuolated 'foamy' morphology. Discriminating between adaptive AM responses and adverse events during nonclinical and clinical development is a major scientific challenge. When measuring and interpreting induced AM responses, an understanding of macrophage biology is essential; this includes 'sub-types' of AMs with different roles in health and disease and mechanisms of induction/resolution of AM responses to inhalation of pharmaceutical aerosols. In this context, emerging assay techniques, the utility of toxicokinetics and the requirement for new biomarkers are considered. Risk assessment for nonclinical toxicology findings and their translation to effects in humans is discussed from a scientific and regulatory perspective. At present, when apparently adaptive macrophage-only responses to inhaled investigational products are observed in nonclinical studies, this poses a challenge for risk assessment and an improved understanding of induced AM responses to inhaled pharmaceuticals is required.

Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals.

There are many ways in which the dose can be expressed in inhalation toxicology studies. This can lead to confusion when comparing results from studies performed in different laboratories. A working party of the Association of Inhalation Toxicologists has reviewed this subject in detail and has collected data from 10 inhalation laboratories and used these data to determine a new algorithm for the calculation of Respiratory Minute Volume (RMV), one of the most important factors in the calculation of delivered dose. The recommendations of the working party for regulatory inhalation toxicology studies with pharmaceuticals are as follows: 1. The dose should be reported as the delivered dose calculated according to the formula: DD = C x RMV x D(xIF)/BW, where DD = delivered dose (mg/Kg); C = concentration of substance in air (mg/L); RMV =respiratory minute volume or the volume of air inhaled in one minute (L/min); D = duration of exposure (min); IF = proportion by weight of particles that are inhalable by the test species, the inhalable fraction (inclusion of this parameter is not essential provided that the aerosol has reasonable respirability for the intended species. If it is included, the way in which it is determined should be clearly stated); BW = bodyweight (Kg). 2. The RMV for mice, rats, dogs and cynomolgus monkeys should be calculated according to the formula:RMV(L/min) = 0.608 x BW(Kg)(0.852). 3. If deposited dose or the amount of material actually retained inthe respiratory tract is presented as supplementary information,the way in which it is calculated should be clearly stated.4. Dose should always be presented in mg/Kg but may also bepresented in other ways, such as mg/unit body surface area, as supplementary information.

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-[(14)C]-butadiene.

Male Sprague-Dawley rats and B6C3F1 mice were exposed to either a single 6h or a multiple (5) daily (6h) nose-only dose of 1,3-[2,3-(14)C]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose-response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1-20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5 x 6 h 1, 5 and 20 ppm 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 0.79 +/- 0.30, 2.90 +/- 1.19, 16.35 +/- 4.8 adducts/10(8) nucleotides and for mice: 2.23 +/- 0.71, 12.24 +/- 2.15, 48.63 +/- 12.61 adducts/10(8) nucleotides. For lung DNA the corresponding values were for rats: 1.02 +/- 0.44, 3.12 +/- 1.06, 17.02 +/- 4.07 adducts/10(8) nucleotides, and for mice: 3.28 +/- 0.32, 14.04 +/- 1.55, 42.47 +/- 13.12 adducts/10(8) nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.

Dose responses for the formation of hemoglobin adducts and urinary metabolites in rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-(14)C]-butadiene.

Blood and urine were obtained from male Sprague-Dawley rats and B6C3F1 mice exposed to either a single 6 h or multiple daily (5 x 6 h) nose-only doses of 1,3-[2,3- (14)C]-butadiene at atmospheric concentrations of 1, 5 or 20 ppM. Globin was isolated from erythrocytes of exposed animals and analyzed for total radioactivity and also for N-(1,2,3-trihydroxybut-4-yl)-valine adducts. The modified Edman degradation procedure coupled with GC-MS was used for the adduct analysis. Linear relationships were observed between the exposures to 1,3-[2,3-(14)C]-butadiene and the total radioactivity measured in globin and the level of trihydroxybutyl valine adducts in globin. A greater level of radioactivity (ca. 1.3-fold) was found in rat globin compared with mouse globin. When analyzed for specific amino acid adducts, higher levels of trihydroxybutyl valine adducts were found in mouse globin compared with rat globin. Average levels of trihydroxybutyl valine adduct measured in globin from rats and mice exposed for 5 x 6 h at 1, 5 and 20 ppM 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 80, 179, 512 pM/g globin and for mice: 143, 351, 1100 pM/g globin. The profiles of urinary metabolites for rats and mice exposed at the different concentrations of butadiene were obtained by reverse phase HPLC analysis on urine collected 24 h after the start of exposure and were compared with results of a previous similar study carried out for 6 h at 200 ppM butadiene. Whilst there were qualitative and quantitative differences between the profiles for rats and mice, the major metabolites detected in both cases were those representing products of epoxide hydrolase mediated hydrolysis and glutathione (GSH) conjugation of the metabolically formed 1,2-epoxy-3-butene. These were 4-(N-acetyl-l-cysteine-S-yl)-1,2-dihydroxy butane and (R)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(S)-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(R)-hydroxybut-3-ene, (S)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, respectively. The former pathway showed a greater predominance in the rat. The profiles of metabolites were similar at exposure concentration in the range 1-20 ppM. There were however some subtle differences compared with results of exposure to the higher 200 ppM concentrations. Overall the results provide the basis for cross species comparison of low exposures in the range of occupational exposures, with the wealth of data available from high exposure studies.

Responses in the respiratory tract of rats following exposure to sulphuric acid aerosols for 5 or 28 days.

Sulphuric acid mists have been classified by the International Agency for Research on Cancer as being carcinogenic to humans based on epidemiological findings of respiratory tract tumours. To determine if early changes in the respiratory tract following exposure to sulphuric acid (H(2)SO(4)) aerosols are consistent with the possible development of tumours after extended periods of exposure, groups of female rats were exposed to respirable aerosols of H(2)SO(4) at target concentrations of 0, 0.2, 1.0 or 5.0 mg m(-3) for 6 h per day for either 5 days or for 5 days a week over a 28-day period. Additional groups exposed to 0 or 5.0 mg m(-3) over the 28-day period were retained after exposure for 4 or 8 weeks to assess recovery. Histopathological examinations and quantitative cell proliferation measurements were conducted on the nasal passages, larynx and lung. Achieved concentrations were 0.3, 1.38 and 5.52 mg m(-3) H(2)SO(4). Histological and cell proliferative changes were confined to the larynx and no effects were seen in the nasal passages or lungs. At the two highest concentrations, squamous metaplasia accompanied by significant cell proliferation was apparent after 5 and 28 days of exposure and there was a reduction in the severity of the pathological changes following the recovery periods. No effects were seen at 0.3 mg m(-3) after 5 days of exposure and only minimal metaplastic change was seen after 28 days in a few animals and was not accompanied by cell proliferation. The toxicological relevance of these findings is discussed.

Pulmonary responses and recovery following single and repeated inhalation exposure of rats to polymeric methylene diphenyl diisocyanate aerosols.

Acute and repeated inhalation exposures (for 28 days) to polymeric methylene diphenyl diisocyanate (PMDI) were performed in rats. Investigations were made at the end of exposures and after 3, 10 and 30 days of recovery following single acute exposures and after 30 days of recovery following 28 days of exposure. Acute exposures to 10, 30 or 100 mg m(-3) PMDI produced clinical signs in all animals that were consistent with exposure to irritant aerosols. An exposure concentration-related body weight loss and increase in lung weight were seen post-exposure, with complete recovery by day 8. The time course of changes in the lung over the initial days following exposure consisted of a pattern of initial toxicity, rapid and heavy influx of inflammatory cells and soluble markers of inflammation and cell damage, increased lung surfactant, a subsequent recovery and epithelial proliferative phase and, finally, a return to the normal status quo of the lung. During these stages there was evidence for perturbation of lung surfactant homeostasis, demonstrated by increased amounts of crystalline surfactant and increased number and size of lamellar bodies within type II alveolar cells. Repeated exposure over 28 days to the less toxic concentrations of 1, 4 or 10 mg m(-3) PMDI produced no clinical signs or body weight changes, but an increase in lung weight was seen in animals exposed to 10 mg m(-3), which resolved following the 30-day recovery period. Other effects seen were again consistent with exposure to irritant aerosols, but were less severe than those seen in the acute study. Analysis of bronchoalveolar lavage fluid revealed similar changes to those seen in the acute study. At both 10 and 4 mg m(-3) PMDI increased numbers of 'foamy' macrophages in lung lavage cell pellet correlated with the increased phospholipid content of the pellet. Changes in lung lavage parameters and electron microscopic evidence again suggested perturbations in surfactant homeostasis. Histologically, bronchiolitis and thickening of the central acinar regions was seen at 10 and 4 mg m(-3), reflecting changes in cell proliferation in the terminal bronchioles and centro-acinar regions. Almost all effects seen had recovered by day 30 post-exposure. Both acute and subacute studies demonstrate rapid recovery of effects in the lung following exposure to PMDI, with no progression of these effects even at concentrations higher than those shown to produce tumours in a chronic study. These findings add weight to the hypothesis that pulmonary tumours seen following chronic exposure to PMDI are most likely due to a combination of the chronic irritant effects of repeated exposure, coupled with the presence of insoluble polyureas formed by polymerization of PMDI (found in studies reported here and previous chronic studies), and therefore acute or short-term exposures to PMDI are likely to be of little concern for long-term pulmonary health.