Fostering Animal Welfare and Advancing 3Rs Principles through the Establishment of a 3Rs Advisory Group.

Based on the current state of science, the use of animals remains essential in bringing safe and effective medicines to patients. Respect for laboratory animal welfare and the application of 3Rs principles (the replacement, reduction, and refinement of animal use in research) are a priority throughout the pharmaceutical industry. Given the rapid pace of development, technological progress, and the emergence of new-approach methodologies (NAMs) in the field of biomedical research, maintaining a leading position in scientific advancements with a focus on the principles of replace, reduce, and refine (3Rs) can be quite challenging. To effectively address these challenges and sustain a prominent position in the scientific community, organizations can derive significant advantages from establishing an internal 3Rs advisory group (3Rs AG). The primary objective of a 3Rs AG is to stay at the forefront of the knowledge of best practices related to the 3Rs principles in the industry. This group plays a crucial role in fostering innovation and facilitating the seamless integration and implementation of 3Rs principles into a company's policies and procedures. The thoughtful reduction in and replacement of animal studies and the refinement of study designs and practices, enabled by a 3Rs AG, can minimize animal use as well as guide resources and positively impact study and data quality. This article provides guidance on how to establish a successful and impactful 3Rs AG.

An Evaluation of the Occupational Health Hazards of Peptide Couplers.

Peptide couplers (also known as amide bond-forming reagents or coupling reagents) are broadly used in organic chemical syntheses, especially in the pharmaceutical industry. Yet, occupational health hazards associated with this chemical class are largely unexplored, which is disconcerting given the intrinsic reactivity of these compounds. Several case studies involving occupational exposures reported adverse respiratory and dermal health effects, providing initial evidence of chemical sensitization. To address the paucity of toxicological data, a pharmaceutical cross-industry task force was formed to evaluate and assess the potential of these compounds to cause eye and dermal irritation as well as corrosivity and dermal sensitization. The goal of our work was to inform health and safety professionals as well as pharmaceutical and organic chemists of the occupational health hazards associated with this chemical class. To that end, 25 of the most commonly used peptide couplers and five hydrolysis products were selected for in vivo, in vitro, and in silico testing. Our findings confirmed that dermal sensitization is a concern for this chemical class with 21/25 peptide couplers testing positive for dermal sensitization and 15 of these being strong/extreme sensitizers. We also found that dermal corrosion and irritation (8/25) as well as eye irritation (9/25) were health hazards associated with peptide couplers and their hydrolysis products (4/5 were dermal irritants or corrosive and 4/5 were eye irritants). Resulting outcomes were synthesized to inform decision making in peptide coupler selection and enable data-driven hazard communication to workers. The latter includes harmonized hazard classifications, appropriate handling recommendations, and accurate safety data sheets, which support the industrial hygiene hierarchy of control strategies and risk assessment. Our study demonstrates the merits of an integrated, in vivo -in silico analysis, applied here to the skin sensitization endpoint using the Computer-Aided Discovery and REdesign (CADRE) and Derek Nexus programs. We show that experimental data can improve predictive models by filling existing data gaps while, concurrently, providing computational insights into key initiating events and elucidating the chemical structural features contributing to adverse health effects. This interactive, interdisciplinary approach is consistent with Green Chemistry principles that seek to improve the selection and design of less hazardous reagents in industrial processes and applications.

Calculating qualified non-mutagenic impurity levels: Harmonization of approaches.

The presence of impurities in drugs is unavoidable. As impurities offer no direct benefit to the patient, it is critical that impurities do not compromise patient safety. Current guidelines on the derivation of acceptable impurity levels leave aspects of calculations open for interpretation, resulting in inconsistencies across industry and regulators. To understand current impurity qualification practices from a safety standpoint, regulatory expectations and the safety risk that impurities pose, the IQ DruSafe Impurities Working Group (WG) conducted a pharmaceutical industry-wide survey. Survey results highlighted areas that could benefit from harmonization, including nonclinical species/sex selection and the application of adjustment factors (i.e., body surface area). Recommendations for alignment on these topics is included in this publication. Additionally, the WG collated repeat-dose toxicity information for 181 starting materials and intermediates, reflective of pharmaceutical impurities, to understand the toxicological risks they generally pose in relation to the drug substance (DS) and the assumptions surrounding the calculation of qualified impurity levels. An evaluation of this dataset and the survey were used to harmonize how to calculate a safe limit for an impurity based on toxicology testing of the impurity when present within the DS.

The performance, reliability and potential application of in silico models for predicting the acute oral toxicity of pharmaceutical compounds.

Acute oral toxicity (AOT) information is utilized to categorize compounds according to the severity of their hazard and used to inform risk assessments for human health and the environment. Given the wealth of historical AOT information and technological advances, in silico models are being created and evaluated as potential tools to predict the AOT of compounds and reduce reliance on animal testing. Utilizing a historical database of AOT data on 371 Bristol Myers Squibb pharmaceutical compounds (PCs) (195 pharmaceutical intermediates and 176 active pharmaceutical ingredients), we evaluated two pioneering in silico AOT programs: the Leadscope Acute Oral Toxicity Model Suite and the Collaborative Acute Toxicity Modeling Suite. These models demonstrated a high degree of agreement with the in vivo results as well as a high level of sensitivity. We found that these models can be effectively utilized to identify PCs which are of low acute oral toxicity (LD50 > 2000 mg/kg), PCs which should not be classified as Dangerous Goods (LD50 > 300 mg/kg), and can assist in identifying a starting dose for in vivo AOT studies. This manuscript provides an evaluation of the performance of these in silico models and proposes use cases where these in silico models can be most confidently and effectively employed.

Considerations for setting occupational exposure limits for novel pharmaceutical modalities.

In order to develop new and effective medicines, pharmaceutical companies must be modality agnostic. As science reveals an enhanced understanding of biological processes, new therapeutic modalities are becoming important in developing breakthrough therapies to treat both rare and common diseases. As these new modalities progress, concern and uncertainty arise regarding their safe handling by the researchers developing them, employees manufacturing them and nurses administering them. This manuscript reviews the available literature for emerging modalities (including oligonucleotides, monoclonal antibodies, fusion proteins and bispecific antibodies, antibody-drug conjugates, peptides, vaccines, genetically modified organisms, and several others) and provides considerations for occupational health and safety-oriented hazard identification and risk assessments to enable timely, consistent and well-informed hazard identification, hazard communication and risk-management decisions. This manuscript also points out instances where historical exposure control banding systems may not be applicable (e.g. oncolytic viruses, biologics) and where other occupational exposure limit systems are more applicable (e.g. Biosafety Levels, Biologic Control Categories).

BCG Vaccinations Upregulate Myc, a Central Switch for Improved Glucose Metabolism in Diabetes.

Myc has emerged as a pivotal transcription factor for four metabolic pathways: aerobic glycolysis, glutaminolysis, polyamine synthesis, and HIF-1α/mTOR. Each of these pathways accelerates the utilization of sugar. The BCG vaccine, a derivative of Mycobacteria-bovis, has been shown to trigger a long-term correction of blood sugar levels to near normal in type 1 diabetics (T1D). Here we reveal the underlying mechanisms behind this beneficial microbe-host interaction. We show that baseline glucose transport is deficient in T1D monocytes but is improved by BCG in vitro and in vivo. We then show, using RNAseq in monocytes and CD4 T cells, that BCG treatment over 56 weeks in humans is associated with upregulation of Myc and activation of nearly two dozen Myc-target genes underlying the four metabolic pathways. This is the first documentation of BCG induction of Myc and its association with systemic blood sugar control in a chronic disease like diabetes.

Evaluation of a tiered in vitro testing strategy for assessing the ocular and dermal irritation/corrosion potential of pharmaceutical compounds for worker safety.

INTRODUCTION: Irritation reactions are a frequently reported occupational illness. The potential adverse effects of pharmaceutical compounds (PCs) on eye and skin can now be assessed using validated in vitro methods. OBJECTIVES: Our overall aim is to reduce animal testing by replacing the historically utilized in vivo test methods with validated in vitro test methods which accurately determine the ocular and dermal irritation/corrosion potential of PCs to inform worker safety within the pharmaceutical space. Bristol-Myers Squibb (BMS) and the Institute for In Vitro Sciences (IIVS) have therefore conceptualized and internally qualified a tiered in vitro testing strategy to inform occupational hazards regarding eye and skin irritation and corrosivity of PCs. For the small scale pre-qualification phase, we paired historical in vivo and newly generated in vitro data for 15 PCs to determine the predictive capacity of in vitro assays already validated for the eye and skin irritation/corrosion endpoints and accepted for certain regulatory submissions. During the post-qualification phase, a group of 24 PCs were subjected exclusively to the developed tiered testing strategy, which is based on three Organisation for Economic Co-operation and Development (OECD) in vitro methods. MATERIALS AND METHODS: The qualified in vitro testing strategy utilizes the Corrositex® assay for the corrosivity (OECD TG 435), the Bovine Corneal Opacity and Permeability (BCOP) assay for ocular irritation (OECD TG 437), and the EpiDerm™ tissue model-based Skin Irritation Test (SIT) for dermal irritation (OECD TG 439). In the first step, the pH of each PC was determined. For compounds with pH extremes ≥11 or ≤2, the Corrositex® assay was generally conducted first. For compound(s) that were incompatible with or were negative in the Corrositex® assay or had pH values between 2 and 11, the BCOP assay and SIT were performed first. RESULTS: The results of the tiered testing strategy's qualification phase demonstrated that the BCOP assay is sensitive enough to identify a wide range of eye irritation/corrosion potentials and its over-prediction rate was considered acceptable to inform occupational hazards and ensure the proper handling practices of PCs. The SIT correctly predicted the skin irritation potential of 14 out of the 15 PCs included in the qualification phase, only over-predicting one PC. In the post-qualification phase, four PCs out of four tested were predicted corrosive by the Corrositex® assay and thus no further testing was needed or conducted. The rest of the PCs were evaluated in the BCOP assay (both neat and as a 20% dilution), with the higher response being used for hazard classification. Four PCs were determined to be severe eye irritants, 1 a moderate irritant, 8 were mild irritants, and 8 were non-irritants. The same set of PCs was evaluated using the SIT and were classified as non-irritants to skin. These results are consistent with the BMS historical in vivo results showing a very low number of PCs as skin irritants. CONCLUSIONS: This tiered in vitro testing strategy, which replaces the use of animal studies, was found to be reasonably accurate in its predictive capacity when compared to historical in vivo results and represents a conservative and reliable platform that can be utilized for the prediction of ocular and dermal irritation/corrosion potential of PCs and for subsequent GHS classification and worker safety hazard communications.

Mapping of Mcs30, a new mammary carcinoma susceptibility quantitative trait locus (QTL30) on rat chromosome 12: identification of fry as a candidate Mcs gene.

Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.

Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells.

BACKGROUND: Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the approach used to select high affinity aptamers for specific macromolecular targets from among the >10(13) oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX)). Using this approach, we identified high affinity aptamers (nanomolar range K(d)) to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa). We also performed preliminary investigation of the aptamer epitopes and their binding characteristics. CONCLUSIONS/SIGNIFICANCE: Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of cancer biopsies and aid in the effective management of pathological conditions (diagnosis, predicted outcome, and treatment options).