Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity ...profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile, was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity.
Environmental enrichment in rodents may improve animal well-being but can affect neurologic development, immune system function, and aging. We tested the hypothesis that wood block enrichment affects ...the interpretation of traditional and transcriptomic endpoints in an exploratory toxicology
testing model using a well-characterized reference compound, cyclophosphamide. ANOVA was performed to distinguish effects of wood block enrichment separate from effects of 40 mg/kg cyclophosphamide treatment. Biologically relevant and statistically significant effects of wood block enrichment
occurred only for body weight gain. ANOVA demonstrated the expected effects of cyclophosphamide on food consumption, spleen weight, and hematology. According to transcriptomic endpoints, cyclophosphamide induced fewer changes in gene expression in liver than in spleen. Splenic transcriptomic
pathways affected by cyclophosphamide included: iron hemostasis; vascular tissue angiotensin system; hepatic stellate cell activation and fibrosis; complement activation; TGFβ-induced hypertrophy and fibrosis; monocytes, macrophages, and atherosclerosis; and platelet activation. Changes
in these pathways due to cyclophosphamide treatment were consistent with bone marrow toxicity regardless of enrichment. In a second study, neither enrichment nor type of cage flooring altered body weight or food consumption over a 28-d period after the first week. In conclusion, wood block
enrichment did not interfere with a typical exploratory toxicology study; the effects of ingested wood on drug level kinetics may require further consideration.
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As the development processes mature for large molecule biologics, additional technologies to assess potential safety issues would be favorable, especially as related to target ...engagement. ABT‐207 is a humanized antibody that inhibits repulsive guidance molecule C (RGMc). The antibody has possible application to treat anemia of chronic disease. In this study, we used an affinity maturated variant of ABT‐207 (PR‐1285929; h5F9‐AM8) to examine the toxicological effects of RGMc inhibition. We applied hepatic whole genome transcriptomic profiling using the Affymetrix platform to investigate the effects of PR‐1285929 and to examine whether there were toxicologically relevant pathway perturbations. Female Sprague Dawley rats were treated for 3 weeks (i.v.) with PR‐1285929 at doses of 0.02, 0.20, 2.0, or 20.0 mg/kg/week. At the end of the dosing period, the rats were sacrificed, and liver was collected for histology and gene expression analysis (rat RAE230 2.0 array). Histological examination showed mild to moderate dose dependent accumulation of hepatic iron as determined by Prussian blue staining. This effect was histopathologically assessed as non‐adverse since no other effects (e.g. inflammation or degeneration) were observed. Global gene expression profiling revealed a relatively minor impact on the hepatic transcriptome. Levels of hepatic hepcidin (Hamp) mRNA, the main regulator of serum iron homeostasis, declined in excess of 20‐fold in a dose dependent manner. This finding was also reflected in liver Hamp mRNA expression as measured using RT‐PCR and decreased serum hepcidin protein levels. Interestingly, RGMc is a main regulator of hepcidin, and thus, its inhibition is likely associated with the reduced Hamp. At the transcript level, a minor oxidative stress response induction, mostly related to glutathione metabolism, was apparent at the highest dose (20 mg/kg), but lower doses revealed few consistent changes in these genes. Only minor modulations were apparent with respect to select BMPs and ferroportin. No other toxicologically relevant pathways were consistently up‐ or down‐regulated. This experimental example shows the potential utility of transcriptomic profiling to investigate plausible mechanisms of cellular changes mediated by biologic therapies and can serve as a method for risk mitigation in the development of protein pharmaceuticals.
Support or Funding Information
Author Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication
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Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B ...cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01
had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.
Loss of donor-mediated immune antitumor activity after allogeneic hematopoietic stem-cell transplantation (HSCT) permits relapse of hematologic cancers. We hypothesized that immune checkpoint ...blockade established by targeting cytotoxic T-lymphocyte-associated protein 4 with ipilimumab could restore antitumor reactivity through a graft-versus-tumor effect.
We conducted a phase 1/1b multicenter, investigator-initiated study to determine the safety and efficacy of ipilimumab in patients with relapsed hematologic cancer after allogeneic HSCT. Patients received induction therapy with ipilimumab at a dose of 3 or 10 mg per kilogram of body weight every 3 weeks for a total of 4 doses, with additional doses every 12 weeks for up to 60 weeks in patients who had a clinical benefit.
A total of 28 patients were enrolled. Immune-related adverse events, including one death, were observed in 6 patients (21%), and graft-versus-host disease (GVHD) that precluded further administration of ipilimumab was observed in 4 patients (14%). No responses that met formal response criteria occurred in patients who received a dose of 3 mg per kilogram. Among 22 patients who received a dose of 10 mg per kilogram, 5 (23%) had a complete response, 2 (9%) had a partial response, and 6 (27%) had decreased tumor burden. Complete responses occurred in 4 patients with extramedullary acute myeloid leukemia and 1 patient with the myelodysplastic syndrome developing into acute myeloid leukemia. Four patients had a durable response for more than 1 year. Responses were associated with in situ infiltration of cytotoxic CD8+ T cells, decreased activation of regulatory T cells, and expansion of subpopulations of effector T cells in the blood.
Our early-phase data showed that administration of ipilimumab was feasible in patients with recurrent hematologic cancers after allogeneic HSCT, although immune-mediated toxic effects and GVHD occurred. Durable responses were observed in association with several histologic subtypes of these cancers, including extramedullary acute myeloid leukemia. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01822509.).
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Whole blood is an easily accessible tissue that allows for non‐invasive, real‐time monitoring of compound exposure and effects. Using blood for transcriptional profiling presents ...technical challenges, particularly because PAXgene tubes require 2.5mL of blood. We have developed a reproducible method of RNA stabilization and isolation to be used for transcriptional profiling from as little as 25μL of whole blood. To demonstrate the validity of this technique, we used an acute inflammation model and treated rats with lipopolysaccharide (LPS, IV) for 2h. Varied amounts of whole blood (25‐500μL) were collected from each rat and immediately added to Qiazol or collected directly into PAXgene tubes. Both techniques yielded abundant, high‐quality RNA. Total RNA (50ng) was processed using the NuGEN Ovation RNA Amplification System, then hybridized to Affymetrix Rat230_2 microarrays. Statistical analysis of microarray intensity values revealed high correlation between blood collected using PAXgene tubes and all volumes of blood added to Qiazol (r = 0.8, p=0.001, FC=1.5). Expression analysis of both methods showed little discordance in IL‐10 signaling, IFN signaling, and LPS‐induced inflammatory response. The evidence from this novel blood collection technique suggests as little as 25μL of whole blood can be used in preclinical toxicology and pharmacology studies for future identification of novel biomarkers.
Dibromoacetic acid (DBAA), a by-product formed during disinfection of drinking water, alters spermatogenesis in rats through defective spermiation. The mechanism underlying this toxicity is not fully ...understood. In this study, gene expression data generated with microarrays from testes were used to generate a mechanistic understanding of DBAA-induced testicular toxicity. Testes were collected from male Sprague-Dawley rats dosed orally for 1 and 4 days with DBAA at 250 mg/kg/day. At both time points, DBAA administration induced delayed spermiation in Stage X tubules and regulated the expression of a small number of genes, including a mild but consistent downregulation of cytochrome P450c17α (CYP17) mRNA, an enzyme expressed by Leydig cells and essential for the production of testicular androgens. Downregulation of CYP17 was confirmed at the protein level and its biological significance was substantiated by demonstrating reduced testicular testosterone levels in DBAA-dosed rats. Furthermore, testosterone production by human chorionic gonadotrophin (hCG)-stimulated rat primary Leydig cells was reduced following treatment with 100 μM DBAA. Collectively, these results indicate that DBAA can directly target rat Leydig cells and downregulate testicular CYP17 expression with a resulting decreased testicular testosterone production. This disruption of testicular steroidogenesis is likely to contribute to the mechanism of failed spermiation observed in rats following exposure to DBAA.PUBLICATION ABSTRACT
Background
Approximately one-fifth of patients with colorectal cancer present with hepatic metastases. There are limited prospective data on the outcomes of synchronous combined liver and bowel ...surgery and liver-first or bowel-first routes where contemporary chemo(radio)therapy is integrated into management.
Methods
Between 1 April 2014 and 31 March 2017, 125 patients with colorectal cancer and synchronous liver metastases were recruited. Data are reported on pathway-specific outcomes, including perioperative complications, treatment completion, and overall and disease-free survival. The study was registered with ClinicalTrials.gov (NCT02456285).
Results
There was no difference in age, body mass index, or Charlson score between surgical groups. Neoadjuvant chemotherapy was used in 50 (40%) patients for a mean duration of 4.6 months (standard deviation SD 5.4), and mean time from completion of chemotherapy to surgery was 2.6 months (SD 1.9). Complications were similar between patients completing the synchronous and staged pathways (
p
= 0.66). Mean total inpatient stay was 16.5 days (SD 8.1) for staged surgery compared with 16.8 days (SD 10.3) for the synchronous group (
t
-test;
p
= 0.91). There was no difference in time to treatment completion between pathways. Thirty six (35%) patients were disease-free at 12 months, with no significant difference between groups (Chi-square,
p
= 0.448). Quality of life was similar in all surgical groups.
Conclusions
Perioperative complications and oncological and healthcare occupancy outcomes are equivalent between patients completing staged and synchronous pathways for the management of patients with colorectal cancer and synchronous liver metastases. Future studies should focus on optimizing the criteria for pathway selection, incorporation of cancer genomics data, and patient (user) preferences.
Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further ...immunization, we demonstrate germinal centre B (B
) cells that last for at least 6 months. A 186-fold increase in B
cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of B
cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding B
cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells
. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous B
cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.
Anthropogenic infrastructure can negatively affect wildlife through direct mortality and/or displacement behaviors. Some tetranoids (grouse spp.) species are particularly vulnerable to tall ...anthropogenic structures because they evolved in ecosystems void of vertical structures. In western North America, electric power transmission and distribution lines (power lines) occur in sagebrush (Artemisia spp.) landscapes within the range of the greater sage-grouse (Centrocercus urophasianus; sage-grouse). The U.S. Fish and Wildlife Service recommended using buffer zones near leks to mitigate the potential impacts of power lines on sage-grouse. However, recommended buffer distances are inconsistent across state and federal agencies because data are lacking. To address this, we evaluated the effects of power lines on sage-grouse breeding ecology within Utah, portions of southeastern Idaho, and southwestern Wyoming from 1998-2013. Overall, power lines negatively affected lek trends up to a distance of 2.7 and 2.8 km, respectively. Power lines died not affect lek persistence. Female sage-grouse avoided transmission lines during the nesting and brooding seasons at distances up to 1.1 and 0.8 km, respectively. Nest and brood success were negatively affected by transmission lines up to distances of 2.6 and 1.1 km, respectively. Distribution lines did not appear to affect sage-grouse habitat selection or reproductive fitness. Our analyses demonstrated the value of sagebrush cover in mitigating potential power line impacts. Managers can minimize the effects of new transmission power lines by placing them in existing anthropogenic corridors and/or incorporating buffers at least 2.8 km from active leks. Given the uncertainty we observed in our analyses regarding sage-grouse response to distribution lines coupled with their role in providing electric power service directly to individual consumers, we recommend that buffers for these power lines be considered on a case-by-case basis. Micrositing to avoid important habitats and habitat reclamation may reduce the potential impacts of new power line construction.
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