Lung-restricted ALK5 inhibition avoids systemic toxicities associated with TGFβ pathway inhibition.

Systemic therapies targeting transforming growth factor beta (TGFβ) or TGFβR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Non-proliferative and Proliferative Lesions of the Non-human Primate ().

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.

BSTP Review of 12 Case Studies Discussing the Challenges, Pathology, Immunogenicity, and Mechanisms of Inhaled Biologics.

The inhalation route is a relatively novel drug delivery route for biotherapeutics and, as a result, there is a paucity of published data and experience within the toxicology/pathology community. In recent years, findings arising in toxicology studies with inhaled biologics have provoked concern and regulatory challenges due, in part, to the lack of understanding of the expected pathology, mechanisms, and adversity induced by this mode of delivery. In this manuscript, the authors describe 12 case studies, comprising 18 toxicology studies, using a range of inhaled biotherapeutics (monoclonal antibodies, fragment antigen-binding antibodies, domain antibodies, therapeutic proteins/peptides, and an oligonucleotide) in rodents, nonhuman primates (NHPs), and the rabbit in subacute (1 week) to chronic (26 weeks) toxicology studies.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Dog.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.

Long-Term Nonclinical Pulmonary Safety Assessment of Afrezza, a Novel Insulin Inhalation Powder.

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control.

Intermittent convection-enhanced delivery of GDNF into rhesus monkey putamen: absence of local or cerebellar toxicity.

Glial cell line-derived neurotrophic factor (GDNF) has demonstrated neurorestorative and neuroprotective effects in rodent and nonhuman primate models of Parkinson's disease. However, continuous intraputamenal infusion of GDNF (100 µg/day) resulted in multifocal cerebellar Purkinje cell loss in a 6-month toxicity study in rhesus monkeys. It was hypothesized that continuous leakage of GDNF into the cerebrospinal fluid compartment during the infusions led to down-regulation of GDNF receptors on Purkinje cells, and that subsequent acute withdrawal of GDNF then mediated the observed cerebellar lesions.

Symposium Summary: "Breathe In, Breathe Out, Its Easy: What You Need to Know About Developing Inhaled Drugs".

Developing inhaled drugs requires knowledge of lung anatomy, cell biology, respiratory physiology, particle physics, and some plumbing. Although dose makes the poison, in the context of an inhaled drug, the "dose" is not easily defined. This lack of clarity around dose poses issues and challenges in the design of inhalation toxicology programs.

Regulatory Forum Opinion Piece*: An Experienced Pathologist Should Be Present at Necropsy for Novel Medical Device Studies.

Necropsies conducted to support medical device development may be conducted in facilities that do not have the infrastructure in place to support Good Laboratory Practice for Nonclinical Laboratory Studies (GLP) studies and for a variety of reasons they may be conducted without a pathologist present at necropsy. However, when a novel medical device or one that is expected to significantly alter tissues is deployed, or when the surgical model confounds interpretation of device effects, it is the opinion of the authors that an experienced pathologist should be present at necropsy.

Regulatory Forum Opinion Piece*: An Experienced Pathologist Need Not Always Be Present at Necropsy for Small Molecule or Biotherapeutic Safety Studies.

It is the author's opinion that an experienced pathologist need not always be present at necropsy for safety studies to support development of large or small molecules. Reasons why an experienced pathologist need not be present in the room for every study as well as the value of necropsy attendance as a training opportunity for less experienced pathologists are presented. However, there are studies for which an experienced pathologist should be in attendance at necropsy and examples of these types of studies are listed.

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.

STP position paper: interpreting the significance of increased alveolar macrophages in rodents following inhalation of pharmaceutical materials.

The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) appointed a working group to address risk assessment for increases in alveolar macrophages following inhalation of pharmaceutical materials. This position paper provides recommendations for inhalation study-specific terminology and interpretation based on literature and information from marketed inhaled drugs. Based on a weight-of-the-evidence approach, and with appropriate consideration of the physical and pharmacological characteristics of the compound, uncomplicated increases in the size or number of alveolar macrophages in nonclinical species are interpreted as nonadverse.

Regulatory forum opinion piece: blind reading of histopathology slides in general toxicology studies.

With the intention of reducing bias, a recent European Food Safety Authority draft guidance document included a recommendation for blinded evaluation of histopathology slides in general toxicology studies (EFSA 2011). Although blinding as to treatment status reduces bias in many types of scientific experiment and is sometimes also appropriate in toxicologic pathology (Holland and Holland 2011), it is most unlikely to help achieve the overall goal of improved human safety when used for routine histopathology evaluation of tissues in general toxicology studies. This is the case because (1) blinding is not applicable to the inductive reasoning process used to identify test article effects in the tissues and would dramatically reduce the chances of these being successfully identified; and (2) in any case, the bias that would be reduced by blinding is actually a bias favoring diagnosis of a toxicological hazard and a conservative safety evaluation, which is appropriate in this context.

C-122, a novel antagonist of serotonin receptor 5-HT2B, prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by sustained elevation of pulmonary arterial pressure that leads to right ventricle failure and death. Pulmonary resistance arterioles in PAH undergo progressive narrowing and/or occlusion. Currently approved therapies for PAH are directed primarily at relief of symptoms by interfering with vasoconstrictive signals, but do not halt the microvascular cytoproliferative process.

Effects of strain and treatment with inhaled aII-trans-retinoic acid on cigarette smoke-induced pulmonary emphysema in mice.

Models of emphysema produced by exposing animals to cigarette smoke (CS) have potential for use in testing treatments of this disease. To better characterize development of emphysema in an animal model, male and female mice of the B6C3F1 and A/J strains were exposed to CS at 250 mg total particulate material (TPM)/m3 for 15 weeks. Emphysema was evident in both strains of mice to differing degrees of severity.

Effects of cigarette smoke exposure and cessation on inflammatory cells and matrix metalloproteinase activity in mice.

B6C3F1 female mice were exposed to cigarette smoke (CS) (250 mg/m3 total particulate material) or filtered air (FA), 6 hours/day, 5 days/week, for 6, 7, or 10 weeks, or to CS for 6 weeks, then FA for 1 or 4 additional weeks. Exposure to CS increased macrophages, neutrophils, lymphocytes, and matrix metalloproteinase (MMP)-2 and MMP-9 content in bronchoalveolar lavage fluid. Partial recovery of most lavage parameters (except lymphocytes) was observed 1 week after cessation of CS exposure with further reductions after 4 weeks, but interstitial inflammation persisted longer.

Multiple modes of responses to air pollution particulate materials in A549 alveolar type II cells.

Exposure to components of air pollution may cause adverse effects on lung cellular and organ functions through several mechanisms. Cell death, altered gene expression including production of cytokines, and modifications of normal cellular processes are possible outcomes that may be independent or coupled. To assess the effects of materials representative of a variety of particulate components of air pollution on lung epithelium, a human cell line of type II origin (A549 cells) was exposed to these materials in vitro.

Animal models of emphysema and their relevance to studies of particle-induced disease.

Emphysema is a pulmonary disease that may be exacerbated by inhaled particles. Over the years, many animal models of emphysema have been developed that may be useful in studying the effects of inhaled particles on humans with emphysema. Models have been described in many species, and many approaches have been described for inducing emphysema.

Animal models of chronic bronchitis and their relevance to studies of particle-induced disease.

Chronic bronchitis is a significant cause of morbidity and mortality. Chronic irritation of the conducting airways by inhaled substances, most importantly cigarette smoke, air pollution, and occupational exposures, is thought to be a key factor in the pathogenesis of chronic bronchitis. Microbial infections have been implicated in acute exacerbations of bronchitis and in its progression.

Effects of concurrent ozone exposure on the pathogenesis of cigarette smoke-induced emphysema in B6C3F1 mice.

Episodic elevation of air pollutants may exacerbate respiratory distress associated with chronic obstructive pulmonary disease (COPD), yet few experiments have been performed to determine how continuously polluted atmospheres may contribute to the etiology of COPD, in general and pulmonary emphysema in particular. This study describes the effects of concurrent exposure to ozone (O(3)) in the pathogenesis of cigarette smoke (CS)-induced emphysema in the mouse. Female B6C3F1 mice were whole-body exposed either to filtered air (FA) or to mainstream CS at a concentration of 250 mg total particulate material/m(3) for 6 h/day, 5 days/wk for 15 or 32 wk.

Mutagenicity and in vivo toxicity of combined particulate and semivolatile organic fractions of gasoline and diesel engine emissions.

Exposure to engine emissions is associated with adverse health effects. However, little is known about the relative effects of emissions produced by different operating conditions, fuels, or technologies. Rapid screening techniques are needed to compare the biological effects of emissions with different characteristics.

Effects of subchronic inhalation exposure of rats to emissions from a diesel engine burning soybean oil-derived biodiesel fuel.

There is increasing interest in diesel fuels derived from plant oils or animal fats ("biodiesel"), but little information on the toxicity of biodiesel emissions other than bacterial mutagenicity. F344 rats were exposed by inhalation 6 h/day, 5 days/wk for 13 wk to 1 of 3 dilutions of emissions from a diesel engine burning 100% soybean oil-derived fuel, or to clean air as controls. Whole emissions were diluted to nominal NO(x) concentrations of 5, 25, or 50 ppm, corresponding to approximately 0.

Structural, functional, and molecular characterization of the SHHF model of heart failure.

Heart failure is a complex multifactorial disease resulting in a myriad of progressive changes at the molecular, cellular, and physiological level. To better understand the mechanisms associated with the development of congestive heart failure, a comprehensive examination of the aging lean male spontaneously hypertensive, heart failure-prone rat (SHHF) was conducted. Myocardial function and structural integrity progressively diminished as evidenced by decreased ejection fraction and increased left ventricular volume measured using echocardiography.

Aberrant CpG island methylation of the p16(INK4a) and estrogen receptor genes in rat lung tumors induced by particulate carcinogens.

Recent studies by our laboratory indicate that the p16(INK4a) gene is frequently methylated in lung tumors induced by genotoxic carcinogens and that the frequency for methylation of the estrogen receptor alpha (ER) gene varies as a function of carcinogenic exposure. The purpose of the current investigation was to define the role of these two genes in lung tumors induced by the particulate carcinogens carbon black (CB), diesel exhaust (DE) or beryllium metal. Methylation of p16 was observed in 59 and 46% of DE and CB tumors, respectively.