Chronic Toxicology Studies Research Articles

A phase 2a study investigating the effects of ritlecitinib on brainstem auditory evoked potentials and intraepidermal nerve fiber histology in adults with alopecia areata.

Reversible axonal swelling and brainstem auditory evoked potential (BAEP) changes were observed in standard chronic (9-month) toxicology studies in dogs treated with ritlecitinib, an oral Janus kinase 3/tyrosine kinase expressed in hepatocellular carcinoma family kinase inhibitor, at exposures higher than the approved 50-mg human dose. To evaluate the clinical relevance of the dog toxicity finding, this phase 2a, double-blind study assessed BAEP changes and intraepidermal nerve fiber (IENF) histology in adults with alopecia areata treated with ritlecitinib. Patients were randomized to receive oral ritlecitinib 50 mg once daily (QD) with a 4-week loading dose of 200 mg QD or placebo for 9 months (placebo-controlled phase); they then entered the active-therapy extension and received ritlecitinib 50 mg QD (with a 4-week loading dose of 200 mg in patients switching from placebo). Among the 71 patients, no notable mean differences in change from baseline (CFB) in Waves I-V interwave latency (primary outcome) or Wave V amplitude on BAEP at a stimulus intensity of 80 dB nHL were observed in the ritlecitinib or placebo group at Month 9, with no notable differences in interwave latency or Wave V amplitude between groups. The CFB in mean or median IENF density and in percentage of IENFs with axonal swellings was minimal and similar between groups at Month 9. Ritlecitinib treatment was also not associated with an imbalanced incidence of neurological and audiological adverse events. These results provide evidence that the BAEP and axonal swelling finding in dogs are not clinically relevant in humans.

Deriving acceptable limits for non-mutagenic impurities in medicinal products – Durational adjustments

ICH Q3A/B guidelines are not intended for application during the clinical research phase of development and durationally adjusted qualification thresholds are not included. A central tenet of ICH Q3A is that lifetime exposure to 1 mg/day of an unqualified non-mutagenic impurity (NMI) is not a safety concern. An analysis of in vivo toxicology data from 4878 unique chemicals with established NO(A)ELs was conducted to determine whether durationally adjusted qualification limits can be supported. Although not recommended in ICH Q3A/B, a conservative approach was taken by using allometric scaling in the analysis. Following allometric scaling of the 5th percentile of the distribution of NO(A)ELs from available chronic toxicology studies, it was reconfirmed that there is a safety basis for the 1 mg/day qualification threshold in ICH Q3A. Additionally, allometric scaling of the 5th percentile of the distribution of NO(A)ELs from sub-acute and sub-chronic toxicology studies could support acceptable limits of 20 and 5 mg/day for an unqualified NMI for dosing durations of less than or greater than one month, respectively. This analysis supports durationally adjusted NMI qualification thresholds for pharmaceuticals that protect patient safety and contribute to 3Rs efforts for qualifying impurities using new approach methods.

Preclinical Testing of Chronic ICA-1S Exposure: A Potent Protein Kinase C-ι Inhibitor as a Potential Carcinoma Therapeutic

Protein kinase C-iota (PKC-ι) is an oncogene overexpressed in many cancer cells including prostate, breast, ovarian, melanoma, and glioma cells. Previous in vitro studies have shown that 5-amino-1-((1R,2S,3R,4R)-2-3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), a PKC-ι-specific inhibitor, has low toxicity in both acute and sub-acute mouse model toxicological testing and is an effective therapeutic against several cancer cell lines showing significant reductions in tumor growth when treating athymic nude mice with xenografted carcinoma cell lines. To further assess ICA-1S as a possible therapeutic agent, chronic mouse model toxicological testing was performed in vivo to provide inferences concerning the long-term effects and possible health hazards from repeated exposure over a substantial part of the animal’s lifespan. Subjects survived well after 30, 60, and 90 days of doses ranging from 50 mg/kg to 100 mg/kg. Heart, liver, kidney, and brain tissues were then analyzed for accumulations of ICA-1S including the measured assessment of aspartate transaminase (AST), alkaline phosphatase (ALK-P), gamma-glutamyl transferase (GGT), troponin, and C-reactive protein (CRP) serum levels to assess organ function. Predictive in vitro/in silico methods were used to predict compound-induced direct hepatocyte toxicity or renal proximal tubular cell (PTC) toxicity in humans based on the high-content imaging (HCI) of compound-treated cells in combination with phenotypic profiling. In conclusion, ICA-1S shows low toxicity in both acute and chronic toxicology studies, and shows promise as a potential therapeutic.

Abstract 1333: Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML

Abstract First generation natural killer (NK) and chimeric antigen receptor (CAR) NK cell products are well-tolerated and have clinical activity (~20-60% CR) in patients with AML. CRs generally increase with the inclusion of CARs or cytokines, increased cell doses, and when Flu/Ara-C lymphodepletion (LD) is used for conditioning. Key clinical limitations have been low PK of the infused NK cells, short durability of the observed responses potentially due to immune evasion of leukemic stem cells (LSCs), and manufacturing challenges precluding higher or multiple doses. SENTI-202 is a next generation allogeneic logic-gated selective CAR NK cell therapy specifically designed to address these limitations and to augment endogenous NK anti-AML activity. SENTI-202 expresses a bivalent activating CAR (aCAR) that targets CD33 and FLT3 on bulk AML blasts and LSCs. As FLT3 is also expressed on healthy hematopoietic stem cells (HSCs), SENTI-202 is further engineered to express an inhibitory CAR that recognizes endomucin expressed on HSCs, thereby conferring protection from aCAR-mediated cytotoxicity even in the presence of FLT3/CD33. SENTI-202 expresses a calibrated-release IL15 that provides both auto- and paracrine cytokine support, mediating expansion, activation, and persistence of SENTI-202 and host immune cells. SENTI-202 demonstrated robust and specific killing of primary AML blasts, LSCs, and AML cell lines in vitro. SENTI-202 protected HSCs from CAR-mediated cytotoxicity while preserving their function. In vivo, SENTI-202 revealed robust efficacy that increased with higher E:T ratio and with 3 weekly vs 1 dose. Preclinically, 3 doses of SENTI-202 that were > 60-fold the planned starting clinical trial dose were well tolerated in acute and chronic toxicology studies with no CAR NK-related body weight, laboratory, or histopathology findings. SENTI-202 non-clinical PK revealed greater than dose proportional exposure, which was ~2-fold greater compared to non-engineered NK cells. Pretreatment of CD33/FLT3 negative AML cell lines with Ara-C resulted in upregulation of CD33 and FLT3 expression, sensitizing cells to robust SENTI-202-mediated killing, providing additional rationale for the use of Ara-C-based LD. In the presence of exogenous IL2, persistence, cytotoxicity, and serial killing activity of SENTI-202 were increased, supporting the use of low dose IL2 to further augment SENTI-202 clinical activity. Taken together, these results support the Phase 1 trial design of SENTI-202-101 in patients with R/R CD33 and/or FLT3 positive malignancies including AML, which uses Flu/Ara-C as LD followed by 3 weekly doses of SENTI-202 and includes the option of enrolling patients into cohorts that additionally receive low dose IL2 following SENTI-202 administration. Citation Format: Deepika Kaveri, Pearley Chinta, Elizabeth Leitner, Lawrence Naitmazi, Gozde Yucel, Mengxi Tian, Niran Almudhfar, Han Deng, Yongshuai Li, Alice Lam, Chen-Ting Lee, Enping Hong, Rochelle Emery, Brian Garrison, Aaron N. Nguyen, Kanya Rajangam. Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1333.

Evaluation of the translation of multiple cardiovascular regulatory mechanisms in the anesthetized dog

The strategic and targeted use of an anesthetized canine cardiovascular model early in drug discovery enables a comprehensive cardiovascular and electrophysiological assessment of potential safety liabilities and guides compound selection prior to initiation of chronic toxicological studies. An ideal model would enable exposure-response relationships to guide safety margin calculations, have a low threshold to initiate, and have quick delivery of decision quality data. We have aimed to profile compounds with diverse mechanism of actions (MoAs) of “non-QT” cardiovascular drug effects and evaluate the ability of nonclinical in vivo cardiovascular models to detect clinically reported effects. The hemodynamic effects of 11 drugs (atropine, itraconazole, atenolol, ivabradine, milrinone, enalaprilat, fasudil, amlodipine, prazosin, amiloride, and hydrochlorothiazide) were profiled in an anesthetized dog cardiovascular model. Derived parameters included: heart rate, an index of left ventricular contractility, mean arterial pressure, systemic vascular resistance, and cardiac output. Species specific plasma protein data was generated (human, dog) and utilized to calculate free drug concentrations. Using the anesthetized dog cardiovascular model, 10 of the 11 drugs displayed the predicted changes in CV parameters based on their primary MoAs and corresponding clinically described effects. Interestingly but not unexpected, 1 of 11 failed to display their predicted CV pattern which is likely due to a delay in pharmacodynamic effect that is beyond the duration of the experimental model (hydrochlorothiazide). The analysis from the current study supports the strategic use of the anesthetized dog model early in the drug discovery process for a comprehensive cardiovascular evaluation with good translation to human.

Characterization of a chloroacetanilide derivative in a chronic oral toxicology study in rats

Introduction. For the treatment of agricultural crops, an original herbicide of selective action was created - propisochlor, a derivative of chloroacetanilide. Its characteristics in an acute toxicological experiment didn’t reveal the features of the pathogenesis of intoxication. The identification of regularities in the manifestation of the toxic properties of propisochlor in various regimens with the oral route of exposure is necessary at the stage of production design. Moreover, it is necessary to substantiate a number of hygienic regulations for safe production and its further application in agriculture, which lies in the plane of solving the leading state tasks aimed at protecting human health and its environment, and meets the requirements of international legislation. A special role in the development of such methods and methods belongs to the sanitary-toxicological study of new xenobiotics and generic molecules, as a result of which threshold and subthreshold doses of pesticide preparations and their active substances are determined. The aim of this study was to characterize propisochlor in a chronic oral toxicology study in rats. To achieve the goal, it was necessary to solve the following tasks: to study the chronic oral effect of the chloroacetanilide derivative on the rat organism; establish a dose that does not have a visible undesirable effect (NOELch); set the lowest dose at which no adverse effects are observed (LOAELch); calculate the allowable daily intake, or allowable daily exposure (ADD, or ADI, or PDE) for a person. Material and methods. The object of testing in this work was a herbicide from the class of chloroacetanilide, propisochlor. The test system in the chronic experiment was white male rats, on which the influence of the test compound was evaluated in dynamics after 1, 3, 6 and 12 months according to the state of integral indicators. Results. The parameters of the oral toxicity of the test compound were determined and the changes in the studied integral indicators were evaluated at various stages of the experiment in a chronic experiment. Conclusion. Based on the comparative characterization of a new herbicide propisochlor in a chronic oral toxicology study in rats, a non-acting dose (NOEL), the lowest effective dose (LOAEL), and an acceptable daily dose for humans (ADI, or PDE) are justified.

Nipocalimab’s Selective Targeting of FcRn and IgG Clearance Preserves Key Immune Functions

Objective To characterize the effect of nipocalimab, a fully human, effectorless IgG1 anti-neonatal Fc receptor (FcRn) monoclonal antibody on immune function. Background Nipocalimab binds to FcRn with high affinity which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections. Design/Methods Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587). Results Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP). Conclusions These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab's selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering.

Multifunctional stimuli-responsive hybrid nanogels for cancer therapy: Current status and challenges.

With cancer research shifting focus to achieving multifunctionality in cancer treatment strategies, hybrid nanogels are making a rapid rise to the spotlight as novel, multifunctional, stimuli-responsive, and biocompatible cancer therapeutic strategies. They can possess cancer cell-specific cytotoxic effects themselves, carry drugs or enzymes that can produce cytotoxic effects, improve imaging modalities, and target tumor cells over normal cells. Hybrid nanogels bring together a wide range of desirable properties for cancer treatment such as stimuli-responsiveness, efficient loading and protection of molecules such as drugs or enzymes, and effective crossing of cellular barriers among other properties. Despite their promising abilities, hybrid nanogels are still far from being used in the clinic, and their available data remains relatively limited. However, many studies can be done to facilitate this clinical transition. This review is critically summarizing and analyzing the recent information and progress on the use of hybrid nanogels particularly inorganic nanoparticle-based and organic nanoparticle-based hybrid nanogels in the field of oncology and future directions to aid in transferring those results to the clinic. This work concludes that the future of hybrid nanogels is greatly impacted by therapeutic and non-therapeutic factors. Therapeutic factors include the lack of hemocompatibility studies, acute and chronic toxicological studies, and information on agglomeration capability and extent, tumor heterogeneity, interaction with proteins in physiological fluids, endocytosis-exocytosis, and toxicity of the nanogels' breakdown products. Non-therapeutic factors include the lack of clear regulatory guidelines and standardized assays, limitations of animal models, and difficulties associated with good manufacture practices (GMP).

Genus Vernonia (Asteraceae): A Promising Source of Antitumor Agents with Pharmacological Potentials

Antitumor research leads to the development of new molecules that act specifically in tumor cells by blocking or inhibiting their molecular targets. New therapeutic approaches for the screening of bioactive compounds present in medicinal plants have received increasing attention due to their chemopreventive and chemotherapeutic properties. In ethnomedicine, plants of the genus Vernonia (Asteraceae) are widely used and some have shown several and interesting biological activities, including anticancer. This present study aimed to document experimental evidence supporting the claimed ethnomedical uses of Vernonia species for the treatment of various types of cancer and also to confirm the anticancer potential of these plants. The compounds isolated from aqueous and alcoholic extracts, as well as fractions from different parts of Vernonia plants have acted as potential anticancer agents that inhibited the proliferation of various types of human cancer cell lines, including cervical cancer cells, melanoma cells, promyelocytic leucemia cells, breast adenocarcinoma cells, ovarian cancer, liver cancer cell, and human lung cancer cells. Studies have correlated the antitumor activity of Vernonia plants by inducing apoptosis and modulating mitochondrial signaling pathways controlled by NF-κB, Bcl-2 and p53, as well as inducing DNA damage and arresting the cell cycle at the S-phase checkpoint by oxidative stress. In conclusion, Vernonia species act as a promising source for drug development. However, further studies are needed to explore the exact mechanism of action, pharmacokinetics, chronic toxicological studies, safe dose consumption, and possible interactions with other herbs.

SELECTIVE TARGETING OF FCRN AND IGG CLEARANCE BY NIPOCALIMAB PRESERVES KEY IMMUNE FUNCTIONS

Autoantibody disorders are caused by immune-system-produced antibodies that can damage the body's tissues or organs. In the case of warm autoimmune hemolytic anemia (wAIHA), pathogenic autoantibodies lead to the destruction of red blood cells. There is an unmet need for innovative targeted therapies that address the underlying pathogenesis of wAIHA in a safe and efficacious way. Nipocalimab is a fully human, aglycosylated, effectorless IgG1 monoclonal antibody that blocks the IgG binding site on the neonatal Fc receptor (FcRn) which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Nipocalimab is in clinical development for the treatment of multiple IgG-mediated autoantibody diseases, including wAIHA (NCT04119050). Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections. Here, we characterize the effect of nipocalimab on immune function. Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587). Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP). These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab's selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering. LEL *, FY *, CJB *, JB *, RX *, AMBP are employed by the Janssen Pharmaceutical Companies of Johnson & Johnson and may hold stock or stock options in Johnson & Johnson; ST * is an employee of Orna Therapeutics; SK * is an employee of Checkmate Pharmaceuticals; JD * is an employee of Faze Medicines; WA * is an employee of Kisbee Therapeutics. *Former employees of Momenta Pharmaceuticals which was acquired by Janssen Pharmaceutical Companies of Johnson & Johnson.

Evaluation of acute toxicity, 28-day repeated dose toxicity, and genotoxicity of Moringa oleifera leaves infusion and powder

Ethnopharmacological relevanceMoringa oleifera Lam. leaves infusion and powder are widely used by population due the nutritional and medicinal potentials, however data regarding safety of use are still inconclusive, leading to prohibition of this plant in some countries. Aim of the studyThe present work investigated the nutritional and phytochemical composition, acute and 28-day repeated dose toxicity, and genotoxicity of M. oleifera leaves infusion and powder. Materials and methodsFor nutritional characterization of leaf powder, it was determined: humidity; mineral residue (ash); total lipid, protein, carbohydrate, and crude fiber contents; and total caloric value. Phytochemical composition was determined by high performance liquid chromatography (HPLC). The acute toxicity assay used Swiss female albino mice and oral administration in a single dose at 2000 and 5000 mg/kg of infusion or powder. The 28-day repeated dose toxicity assay employed female and male mice, with oral administration of infusion or powder at the doses 250, 500 and 1000 mg/kg. The animals were evaluated for body weight, water and feed consumption, biochemical and hematological parameters, and histology of the liver, spleen, and kidneys. In vivo genotoxicity and mutagenicity (2000 mg/kg) were evaluated by the comet assay and the micronucleus test, respectively. ResultsNutritional characterization confirmed that M. oleifera leaves are rich in proteins, carbohydrates, lipids, minerals, and fiber. HPLC indicated the presence of flavonoids and cinnamic derivatives as major polyphenols. Acute toxicity did not reveal alterations in weight gain and water and feed consumptions and no change in biochemical, hematological, and histological parameters. Behavior alterations was observed in the first 2 h after administration at 5000 mg/kg in both treatments. Infusion did not present toxicity when administered for 28 days. Conversely, the powder at 500 and 1000 mg/kg promoted liver and kidney damages observed through biochemical parameters and histopathology. Genotoxicity and mutagenicity were not detected at 2000 mg/kg. ConclusionsThe present study reveals that M. oleifera leaves are an important source of polyphenols and nutrients. Indiscriminate use of both infusion and crude leaf powder above 2000 mg/kg and powder at 500 and 1000 mg/kg are not recommended. Chronic toxicological studies and establishment of preparation protocols are suggested aiming to guarantee the safety in the use of M. oleifera leaves as nutraceutical by population.

Application and toxicity studies of arabinoxylan and \u03b2-d-glucan stearic acid ester composite coatings in extending postharvest storage of peach

Peaches are good source of nutrients and known for their taste and aroma. The highly perishable nature of the peaches tends to decay rapidly during transportation and storage is a serious constraint for efficient transportation and storage. Therefore, the effect of arabinoxylan (AX) and β-D-glucan stearic acid ester (SABG) composite coating material was examined for the postharvest storage quality of peach under storage at 22 ± 2 °C with 85% relative humidity (RH). Both, AX-SABG and shellac (1–2%) coatings significantly reduced the change in the quality attributes like weight loss (1.2–1.4 fold), respiration rate (1.1–1.2 fold), ripening index (1.3–1.5 fold) and firmness (1.3–1.5 fold) during 6 days storage as compared to the uncoated peaches. In addition, AX-SABG (1–2%) coating was more effective in retaining aroma volatiles and reducing disease incidence compared to shellac. Further, acute and chronic toxicological studies have shown no tissue related toxicity and mortality in mice. Our results suggest that AX-SABG as an edible coating has the potential to preserve the fruit quality during 6 days storage at 22 ± 2 °C and extend the postharvest shelf life of peach during storage.

Profiling the Concentration of Reduced and Oxidized Glutathione in Rat Brain Using HPLC/DAD Chromatographic System.

This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman′s reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.

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. Analysis of the data revealed that many of these molecules were associated with a characteristic pattern of toxicity with high levels of immunogenicity. Microscopic changes in the airways consisted of a predominantly lymphoid perivascular/peribronchiolar (PV/PB) mononuclear inflammatory cell (MIC) infiltrate, whereas changes in the terminal airways/alveoli were characterized by simple ("uncomplicated") increases in macrophages or inflammatory cell infiltrates ranging from mixed inflammatory cell infiltration to inflammation. The PV/PB MIC changes were considered most likely secondary to immunogenicity, whereas simple increases in alveolar macrophages were most likely secondary to clearance mechanisms. Alveolar inflammatory cell infiltrates and inflammation were likely induced by immune modulation or stimulation through pharmacologic effects on target biology or type III hypersensitivity (immune complex disease). Finally, a group of experts provide introductory thoughts regarding the adversity of inhaled biotherapeutics and the basis for reasonable differences of opinion that might arise between toxicologists, pathologists, and regulators.

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. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

Congenital Unilateral Renal Aplasia in a Cynomolgus Monkey (Macaca fascicularis) With Investigation Into Potential Pathogenesis.

We describe and characterize unilateral renal aplasia in a cynomolgus monkey (Macaca fascicularis) from a chronic toxicology study adding to the limited histopathology reports of congenital renal anomalies in macaques. In the current case, the affected kidney was macroscopically small and characterized microscopically by a thin cortex with an underdeveloped medulla and an absent papilla. The remnant medulla lacked a corticomedullary junction and contained only a few irregular collecting duct-like structures. The cortex had extensive interstitial mature collagen deposition with fibromuscular collar formation around Bowman's capsules. Due to parenchymal collapse, mature glomeruli were condensed together with occasional atrophic and sclerotic glomeruli. The majority of the cortical tubules were poorly differentiated with only small islands of fully developed cortical tubules present. Histochemical and immunohistochemical stains were utilized to demonstrate key diagnostic features of this congenital defect, to assist with differentiating it from renal dysplasia, and to provide potential mechanistic pathways. Immunostaining (S100, paired box gene 2 [PAX2], aquaporins) of the medulla was compatible with incomplete maturation associated with aplasia, while the immunostaining profile for the cortex (vimentin, calbindin, PAX2-positive cortical tubules, and smooth muscle actin-positive fibromuscular collars) was most compatible with dedifferentiation secondary to degenerative changes.

Neuro-protective Effect of Ayurveda Formulation, Saraswatharishtam, on Scopolamine Induced Memory Impairment in Animal Model

Introduction: In the management of neurodegenerative and neuropsychiatric diseases, memory loss remains very challenging. Saraswatharishtam (SWRT) is an ayurvedic formulation prescribed for several neurological ailments like acute anxiety, partial memory loss, slurred speech, neuro-degenerative diseases like Parkinson’s, Alzheimer’s etc. Methods: Present study explores the possibilities of curative effects of SWRT on Scopolamine induced memory impairment and to compare the effects of commonly used drug Piracetam. Adult Wistar Swiss albino mice were used for this experiment and divided into five different groups for the treatments of the drugs in various combinations. The first group was vehicle treated control while the second group was treated with scopolamine to induce memory impairment. The third groups of animals were treated with Scopolamine where the mice were pretreated with standard neuro-protective drug Piracetam, while the fourth and fifth groups of animals were treated with scopolamine and the animals were pretreated with SWRT at the doses of 200 and 400 mg/kg respectively to compare with the effect of Piracetam. Scopolamine was administered on day eight, only for 45 minutes in all the cases (Group I- Group V). Several neurobehavioral parameters were examined to confirm the neuro-protective effects of SWRT. Results: Saraswatharishtam significantly reversed the memory loss in mice model as revealed from elevated plus maze, radial maze and rectangular maze experiments justifying its neuroprotective potential as observed in usually used synthetic Piracetam. SWRT was found to be nontoxic and very promising antioxidant recorded from acute and chronic toxicological studies. Conclusions: It is suggested that SWRT, could be used as safe and easily available Ayurveda medicine to treat neurological dysfunctions without any side effects. Further translational research is needed to explore its neuro-protective potential.

Evaluating Associations Between Nonclinical Cardiovascular Functional Endpoints and Repeat-dose Cardiovascular Toxicity in the Beagle Dog: A Cross-company Initiative.

Integrating nonclinical in vitro, in silico, and in vivo datasets holistically can improve hazard characterization and risk assessment. In pharmaceutical development, cardiovascular liabilities are a leading cause of compound attrition. Prior to clinical studies, functional cardiovascular data are generated in single-dose safety pharmacology telemetry studies, with structural pathology data obtained from repeat-dose toxicology studies with limited concurrent functional endpoints, eg, electrocardiogram via jacketed telemetry. Relationships between datasets remain largely undetermined. To address this gap, a cross-pharma collaboration collated functional and structural data from 135 compounds. Retrospective functional data were collected from good laboratory practice conscious dog safety pharmacology studies: effects defined as hemodynamic blood pressure or heart rate changes. Morphologic pathology findings (mainly degeneration, vacuolation, inflammation) from related toxicology studies in the dog (3-91 days repeat-dosing) were reviewed, harmonized, and location categorized: cardiac muscle (myocardium, epicardium, endocardium, unspecified), atrioventricular/aortic valves, blood vessels. The prevalence of cardiovascular histopathology changes was 11.1% of compounds, with 53% recording a functional blood pressure or heart rate change. Correlations were assessed using the Mantel-Haenszel Chi-square trend test, identifying statistically significant associations between cardiac muscle pathology and (1) decreased blood pressure, (2) increased heart rate, and between cardiovascular vessel pathology and increased heart rate. Negative predictive values were high, suggesting few compounds cause repeat-dose cardiovascular structural change in the absence of functional effects in single-dose safety pharmacology studies. Therefore, observed functional changes could prompt moving (sub)chronic toxicology studies forward, to identify cardiovascular liabilities earlier in development, and reduce late-stage attrition.

A Pathology Review of the Lower Gastrointestinal Tract in Relation to Ulcerative Colitis in Rats and Cynomolgus Macaques Treated With Ammonium Perfluorooctanoate.

Among many short-term, subchronic, and chronic toxicology studies with ammonium perfluorooctanoate (PFOA), the gastrointestinal tract has not been identified as a target organ for PFOA-related toxicity in laboratory animals where the corresponding serum PFOA concentrations typically approach several orders of magnitude higher than the general human population. These lack of gastrointestinal tract-related findings were in direct contrast to an epidemiological observation where a positive trend was observed for ulcerative colitis, an idiopathic chronic inflammatory condition of the gut, in a Mid-Ohio River community whose drinking water contained higher levels of PFOA. This study was conducted to perform a histological reevaluation of large intestine sections in laboratory animals from 2 long-term toxicological studies: one was with Sprague Dawley rats that received ammonium PFOA in their diet for 2 years and the other one was with cynomolgus macaques that received daily capsules of ammonium PFOA for 6 months. In both studies, there was a lack of histological evidence of treatment-related inflammatory lesions that was suggestive of the occurrence of ulcerative colitis in these laboratory animals even under the most rigorous treatment schedules. These findings do not offer support for the biological plausibility of the epidemiological associations reported.

Neuro-protective Effect of Ayurveda Formulation, Saraswatharishtam, on Scopolamine Induced Memory Impairment in Animal Model

Introduction: In the management of neurodegenerative and neuropsychiatric diseases, memory loss remains very challenging. Saraswatharishtam (SWRT) is an ayurvedic formulation prescribed for several neurological illnesses like acute anxiety, partial memory loss, slurred speech, neuro degenerative diseases like Parkinson’s, Alzheimer’s etc. Methods: Present study explores the possibilities of curative effects of SWRT on Scopolamine induced memory impairment and to compare the effects of commonly used drug piracetam. Adult Wistar Swiss albino mice were used for this experiment and divided into five different groups for the treatments of the drugs in various combinations. The first group was vehicle treated control while the second group was treated with scopolamine to induce memory impairment. The third group of animals were treated with Scopolamine where the mice were pretreated with standard neuroprotective drug Piracetam, while the fourth and fifth group of animals were treated with scopolamine and the animals were pretreated with SWRT at the doses of 200 and 400 mg/kg respectively to compare with the effect of Piracetam. Scopolamine was administered on day eight, only for 45 minutes in all the cases (Group I- Group V). Several neurobehavioral parameters were examined to confirm the neuroprotective effects of SWRT. Results: Saraswatharishtam significantly reversed the memory loss in mice model revealed from elevated plus maze, radial maze and rectangular maze experiments justifying its neuroprotective potential as observed in usually used synthetic Piracetam. SWRT was found to be nontoxic and very promising antioxidant recorded from acute and chronic toxicological studies. Conclusions: It is suggested that SWRT, could be used as safe and easily available Ayurveda medicine to treat neurological dysfunctions without any side effects. Further translational research is needed to explore its neuro-protective potential.