Increasing the Reuse of Protein Non-Naïve Nonhuman Primates in Pharmaceutical Drug Discovery and Development: An Overview and Industry Position on the Challenges and Benefits.

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Role of signal transducer and activator of transcription 1 in murine allergen-induced airway remodeling and exacerbation by carbon nanotubes.

Asthma is characterized by a T helper type 2 phenotype and by chronic allergen-induced airway inflammation (AAI). Environmental exposure to air pollution ultrafine particles (i.e., nanoparticles) exacerbates AAI, and a concern is possible exacerbation posed by engineered nanoparticles generated by emerging nanotechnologies. Signal transducer and activator of transcription (STAT) 1 is a transcription factor that maintains T helper type 1 cell development. However, the role of STAT1 in regulating AAI or exacerbation by nanoparticles has not been explored. In this study, mice with whole-body knockout of the Stat1 gene (Stat1(-/-)) or wild-type (WT) mice were sensitized to ovalbumin (OVA) allergen and then exposed to multiwalled carbon nanotubes (MWCNTs) by oropharygneal aspiration. In Stat1(-/-) and WT mice, OVA increased eosinophils in bronchoalveolar lavage fluid, whereas MWCNTs increased neutrophils. Interestingly, OVA sensitization prevented MWCNT-induced neutrophilia and caused only eosinophilic inflammation. Stat1(-/-) mice displayed increased IL-13 in bronchoalveolar lavage fluid at 1 day compared with WT mice after treatment with OVA or OVA and MWCNTs. At 21 days, the lungs of OVA-sensitized Stat1(-/-) mice displayed increased eosinophilia, goblet cell hyperplasia, airway fibrosis, and subepithelial apoptosis. MWCNTs further increased OVA-induced goblet cell hyperplasia, airway fibrosis, and apoptosis in Stat1(-/-) mice at 21 days. These changes corresponded to increased levels of profibrogenic mediators (transforming growth factor-ß1, TNF-α, osteopontin) but decreased IL-10 in Stat1(-/-) mice. Finally, fibroblasts isolated from the lungs of Stat1(-/-) mice produced significantly more collagen mRNA and protein in response to transforming growth factor-ß1 compared with WT lung fibroblasts. Our results support a protective role for STAT1 in chronic AAI and exacerbation of remodeling caused by MWCNTs.

Nickel nanoparticles cause exaggerated lung and airway remodeling in mice lacking the T-box transcription factor, TBX21 (T-bet).

BACKGROUND: Nickel nanoparticles (NiNPs) are increasingly used in a variety of industrial applications, including the manufacturing of multi-walled carbon nanotubes (MWCNTs). While occupational nickel exposure is a known cause of pulmonary alveolitis, fibrosis, and cancer, the health risks of NiNPs are not well understood, especially in susceptible individuals such as asthmatics. The T-box transcription factor Tbx21 (T-bet) maintains Th1 cell development and loss of T-bet is associated with a shift towards Th2 type allergic airway inflammation that characterizes asthma. The purpose of this study was to determine the role of T-bet in susceptibility to lung remodeling by NiNPs or MWCNTs. METHODS: Wild-type (WT) and T-bet-/- mice were exposed to NiNPs or MWCNTs (4 mg/kg) by oropharyngeal aspiration (OPA). Necropsy was performed at 1 and 21 days. Bronchoalveolar lavage fluid (BALF) was collected for differential counting of inflammatory cells and for measurement of cytokines by ELISA. The left lung was collected for histopathology. The right lung was analyzed for cytokine or mucin (MUC5AC and MUC5B) mRNAs. RESULTS: Morphometry of alcian-blue/periodic acid Schiff (AB/PAS)-stained lung tissue showed that NiNPs significantly increased mucous cell metaplasia in T-bet-/- mice at 21 days (p < 0.001) compared to WT mice, and increased MUC5AC and MUC5B mRNAs (p < 0.05). MWCNTs also increased mucous cell metaplasia in T-bet-/- mice, but to a lesser extent than NiNPs. Chronic alveolitis was also increased by NiNPs, but not MWCNTs, in T-bet-/- mice compared to WT mice at 21 days (P < 0.001). NiNPs also increased IL-13 and eosinophils (p < 0.001) in BALF from T-bet-/- mice after 1 day. Interestingly, the chemokine CCL2 in the BALF of T-bet-/- mice was increased at 1 and 21 days (p < 0.001 and p < 0.05, respectively) by NiNPs, and to a lesser extent by MWCNTs at 1 day. Treatment of T-bet-/- mice with a monoclonal anti-CCL2 antibody enhanced NiNP-induced mucous cell metaplasia and MUC5AC mRNA levels (p < 0.05), yet marginally reduced NiNP-induced alveolitis. CONCLUSION: These findings identify T-bet as a potentially important susceptibility factor for NiNP exposure and to a lesser extent for MWCNT exposure, and suggests that individuals with asthma are at greater risk.

Role of cyclooxygenase-2 in exacerbation of allergen-induced airway remodeling by multiwalled carbon nanotubes.

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

Nickel nanoparticles enhance platelet-derived growth factor-induced chemokine expression by mesothelial cells via prolonged mitogen-activated protein kinase activation.

Pleural diseases (fibrosis and mesothelioma) are a major concern for individuals exposed by inhalation to certain types of particles, metals, and fibers. Increasing attention has focused on the possibility that certain types of engineered nanoparticles (NPs), especially those containing nickel, might also pose a risk for pleural diseases. Platelet-derived growth factor (PDGF) is an important mediator of fibrosis and cancer that has been implicated in the pathogenesis of pleural diseases. In this study, we discovered that PDGF synergistically enhanced nickel NP (NiNP)-induced increases in mRNA and protein levels of the profibrogenic chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2), and the antifibrogenic IFN-inducible CXC chemokine (CXCL10) in normal rat pleural mesothelial 2 (NRM2) cells in vitro. Carbon black NPs (CBNPs), used as a negative control NP, did not cause a significant increase in CCL2 or CXCL10 in the absence or presence of PDGF. NiNPs prolonged PDGF-induced phosphorylation of the mitogen-activated protein kinase family termed extracellular signal-regulated kinases (ERK)-1 and -2 for up to 24 hours, and NiNPs also synergistically increased PDGF-induced hypoxia-inducible factor (HIF)-1α protein levels in NRM2 cells. Inhibition of ERK-1,2 phosphorylation with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, blocked the synergistic increase in CCL2, CXCL10, and HIF-1α levels induced by PDGF and NiNPs. Moreover, the antioxidant, N-acetyl-L-cysteine (NAC), significantly reduced HIF-1α, ERK-1,2 phosphorylation, and CCL2 protein levels that were synergistically increased by the combination of PDGF and NiNPs. These data indicate that NiNPs enhance the activity of PDGF in regulating chemokine production in NRM2 cells through a mechanism involving reactive oxygen species generation and prolonged activation of ERK-1,2.

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.

BACKGROUND: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). METHODS: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. RESULTS: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. CONCLUSION: This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

14-Day Toxicity Studies of Tetravalent and Pentavalent Vanadium Compounds in Harlan Sprague Dawley Rats and B6C3F1/N Mice via Drinking Water Exposure.

BACKGROUND: The National Toxicology Program (NTP) performed short-term toxicity studies of tetra- and pentavalent vanadium compounds, vanadyl sulfate and sodium metavanadate, respectively. Due to widespread human exposure and a lack of chronic toxicity data, there is concern for human health following oral exposure to soluble vanadium compounds. OBJECTIVES: To compare the potency and toxicological profile of vanadyl sulfate and sodium metavanadate using a short-term in vivo toxicity assay. METHODS: Adult male and female Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice, 5 per group, were exposed to vanadyl sulfate or sodium metavanadate, via drinking water, at concentrations of 0, 125, 250, 500, 1000 or 2000 mg/L for 14 days. Water consumption, body weights and clinical observations were recorded throughout the study; organ weights were collected at study termination. RESULTS: Lower water consumption, up to -80% at 2000 mg/L, was observed at most exposure concentrations for animals exposed to either vanadyl sulfate or sodium metavanadate and was accompanied by decreased body weights at the highest concentrations for both compounds. Animals in the 1000 and 2000 mg/L sodium metavanadate groups were removed early due to overt toxicity. Thinness was observed in high-dose animals exposed to either compound, while lethargy and abnormal gait were only observed in vanadate-exposed animals. CONCLUSIONS: Based on clinical observations and overt toxicity, sodium metavanadate appears to be more toxic than vanadyl sulfate. Differential toxicity cannot be explained by differences in total vanadium intake, based on water consumption, and may be due to differences in disposition or mechanism of toxicity.

Lung deposition and clearance of microparticle and nanoparticle C60 fullerene aggregates in B6C3F1 mice and Wistar Han rats following nose-only inhalation for 13 weeks.

C60 fullerenes (C60) are spherical structures consisting of 60 carbon atoms that are generated via combustion from both natural and anthropogenic sources. C60 are also synthesized intentionally for industrial applications. Individual C60 structures have an approximate diameter of 1nm; however, C60 readily forms aggregates and typically exist as larger particles that range from nanometers to micrometers in diameter. In this report, lung and extrapulmonary tissue deposition and lung clearance of C60 nanoparticles (nano-C60, 50nm) and microparticles (micro-C60, 1µm) were examined in Wistar Han rats and B6C3F1/N mice after nose-only inhalation for 90 days. Exposure concentrations were 0.5 and 2mg/m(3) (nano-C60) and 2, 15, and 30mg/m(3) (micro-C60). For both C60 particle sizes, the C60 lung burden increased proportionally to exposure concentration. The C60 lung burden was greater in both species at all time points following exposure to nano-C60 particle exposure compared to micro-C60 exposure at the common exposure concentration 2mg/m(3). The calculated C60 particle lung retention half-times were similar for both nano-C60 and micro-C60 exposure at 2mg/m(3) in male mice (15-16 days). In contrast, in male rats, the half-time of C60 particles following nano-C60 exposure (61 days) was roughly twice as long as the half-time following micro-C60 exposure (27 days) at the same exposure concentration (2mg/m(3)) and was similar to the clearance following micro-C60 exposure at higher exposure concentrations (15 and 30mg/m(3)). C60 was detected in bronchial lymph nodes but the burden was not quantified due to the high variability in the data. C60 concentrations were below the experimental limit of quantitation (ELOQ) in liver, spleen, blood, brain and kidney tissues. These tissue burden data provide information for comparison between nanometer and micrometer sized C60 particle exposure and will aid in the interpretation of toxicity data.

Respiratory toxicity and immunotoxicity evaluations of microparticle and nanoparticle C60 fullerene aggregates in mice and rats following nose-only inhalation for 13 weeks.

C60 fullerene (C60), or buckminsterfullerene, is a spherical arrangement of 60 carbon atoms, having a diameter of approximately 1 nm, and is produced naturally as a by-product of combustion. Due to its small size, C60 has attracted much attention for use in a variety of applications; however, insufficient information is available regarding its toxicological effects. The effects on respiratory toxicity and immunotoxicity of C60 aggregates (50 nm [nano-C60] and 1 µm [micro-C60] diameter) were examined in B6C3F1/N mice and Wistar Han rats after nose-only inhalation for 13 weeks. Exposure concentrations were selected to allow for data evaluations using both mass-based and particle surface area-based exposure metrics. Nano-C60 exposure levels selected were 0.5 and 2 mg/m3 (0.033 and 0.112 m2/m3), while micro-C60 exposures were 2, 15 and 30 mg/m3 (0.011, 0.084 and 0.167 m2/m3). There were no systemic effects on innate, cell-mediated, or humoral immune function. Pulmonary inflammatory responses (histiocytic infiltration, macrophage pigmentation, chronic inflammation) were concentration-dependent and corresponded to increases in monocyte chemoattractant protein (MCP)-1 (rats) and macrophage inflammatory protein (MIP)-1α (mice) in bronchoalveolar lavage (BAL) fluid. Lung overload may have contributed to the pulmonary inflammatory responses observed following nano-C60 exposure at 2 mg/m3 and micro-C60 exposure at 30 mg/m3. Phenotype shifts in cells recovered from the BAL were also observed in all C60-exposed rats, regardless of the level of exposure. Overall, more severe pulmonary effects were observed for nano-C60 than for micro-C60 for mass-based exposure comparisons. However, for surface-area-based exposures, more severe pulmonary effects were observed for micro-C60 than for nano-C60, highlighting the importance of dosimetry when evaluating toxicity between nano- and microparticles.

An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1ß via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1ß release in vitro and in vivo during allergic inflammation. METHODS: THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses. RESULTS: Priming of THP-1 macrophages with LPS increased pro-IL-1ß and subsequent exposure to MWCNTs induced IL-1ß secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1ß secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1ß. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1ß in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1ß in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1ß mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs. CONCLUSIONS: These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1ß by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.

Innate Immune Responses to Nanoparticle Exposure in the Lung.

The nanotechnology revolution offers enormous societal and economic benefits for innovation in the fields of engineering, electronics, and medicine. Nevertheless, evidence from rodent studies show that biopersistent engineered nanomaterials (ENMs) stimulate immune, inflammatory, and fibroproliferative responses in the lung, suggesting possible risks for lung diseases or systemic immune disorders as a consequence of occupational, environmental, or consumer exposure. Due to their nanoscale dimensions and increased surface area per unit mass, ENMs have a much greater potential to reach the distal regions of the lung and generate ROS. High aspect ratio ENMs (e.g., nanotubes, nanofibers) activate inflammasomes in macrophages, triggering IL-1ß release and neutrophilic infiltration into the lungs. Moreover, some ENMs alter allergen-induced eosinophilic inflammation by immunostimulation, immunosuppression, or modulating the balance between Th1, Th2, and Th17 cells, thereby influencing the nature of the inflammatory response. ENMs also migrate from the lungs across epithelial, endothelial, or mesothelial barriers to stimulate or suppress systemic immune responses.