Opinion on Maintaining In-House GLP Status for Toxicologic Pathology in Pharmaceutical and (Agro)Chemical Development.

Many pharmaceutical companies have recently elected to stop maintaining good laboratory practices (GLP) status of their R&D sites. Similar discussions have also been engaged in the (agro)chemical industry. This opinion paper examines the pros and cons of maintaining facility GLP status for the purposes of performing the pathology interpretation or peer reviews of GLP studies internally. The toxicologic pathologist provides gross and histomorphologic evaluation and interpretation of nonclinical exploratory and regulatory studies during drug and (agro)chemical development. This assessment significantly contributes to human risk assessment by characterizing the toxicological profile and discussing the human relevance of the findings. The toxicologic pathologist is a key contributor to compound development decisions (advancement or termination) and in the development of de-risking strategies for backup compounds, thus playing a critical role in helping to reduce the late attrition of drugs and chemicals. Maintaining GLP compliance is often perceived as a costly and cumbersome process; a common and short-term strategy to reduce the costs is to outsource regulatory toxicity studies. However, there are significant advantages in maintaining the GLP status for toxicologic pathology activities in-house including the sustainable retention of internal pathology expertise that has maintained the necessary training needed to manage GLP studies. [Box: see text].

Toxicologic Pathology Forum*: Opinion on Integrating Innovative Digital Pathology Tools in the Regulatory Framework.

Digital pathology is defined as the ability to examine digitized microscopic slides and to generate qualitative and quantitative data. The field of digital pathology is rapidly evolving and has the potential to revolutionize toxicologic pathology. Techniques such as automated 2-D image analysis, whole slide imaging, and telepathology are already considered "mature" technologies and have been used for decades in exploratory studies; however, many organizations are reluctant to use digital pathology in regulatory toxicology studies. Innovative technologies using digitized slides including high-content imaging modalities and artificial intelligence are still under development but are increasingly used in toxicologic pathology. While software validation requirements are already described, clear guidance for application of these rules to the digital pathology field are few and the acceptance of these technologies by regulatory authorities remains necessary for successful adoption of digital pathology into the mainstream of toxicologic pathology. This topic was discussed during a roundtable at the 2018 Annual Congress of the French Society of Toxicologic Pathology. This opinion article summarizes the discussion regarding the current questions and challenges on the integration of innovative digital pathology tools within a good laboratory practice framework and is meant to stimulate further discussion among the toxicologic pathology community. *This is an opinion article submitted to the Toxicologic Pathology Forum and does not constitute an official position of the Society of Toxicologic Pathology or the journal Toxicologic Pathology. The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of their respective agencies and organizations. The Toxicologic Forum is designed to stimulate broad discussion of topics relevant to regulatory issues in Toxicologic pathology. Readers of Toxicologic Pathology are encouraged to send their thoughts on these articles or ideas for new topics to toxicologicpathologyforum@toxpath.org .

Specificities of the Skin Morphology in Juvenile Minipigs.

The Göttingen minipig is recognized by the scientific community and by health authorities as the animal model of choice to evaluate dermally applied drugs under development. Young adults of approximately 4 months of age are most generally chosen to participate in dermal pharmacology and toxicology studies, and recently, minipigs have been proved to be also suitable for juvenile studies. A complete anatomical cartography of the skin morphology of juvenile male and female minipigs from postnatal day 1 (PND1) to twelve weeks of age was performed measuring the thickness of skin layers for each anatomical location and time point. Overall, the neonatal skin of minipigs (PND1 and PND8) shows prominent cellularity, similar to that seen in human neonates, and the morphology of the skin of older animals is considered similar to that of young adult minipigs. Epidermal thickness varies only minimally over the period; whereas, the dermal and more markedly, the subcutaneous thicknesses increase over time.

Using a New Photo Scale to Compare Product Integration of Different Hyaluronan-Based Fillers After Injection in Human Ex Vivo Skin.

BACKGROUND: The rheological properties of HA products have been investigated thoroughly, and these properties have been used to predict the clinical performance of HA fillers. It has been suggested that firm gels have a better ability to withstand deformation, and softer gels have been claimed to integrate and spread more into the tissue since they are perceived to deform more easily. However, the scientific published data regarding product integration of filler products with different physicochemical properties is limited. Thus, there is a need to improve the understanding regarding links between rheological properties of the gel material and the clinical performance. OBJECTIVE: The objectives of this study were: to develop and validate a photo scale for assessment of product distribution after intradermal injection, and to evaluate if product differences, such as overall rheological properties, gel particle size, swelling factor, and cohesivity effect the product distribution into the tissue after intradermal injections. MATERIAL AND METHODS: Intradermal injections of HA fillers were performed in ex vivo human abdominal skin samples. The skin samples were processed for histological evaluation. In order to evaluate the product integration after intradermal injection and compare the results between different products a 5-grade product integration scale (from 0 to 4) was developed based on representative microphotographs. The scale was validated and used for the evaluation of integration of the different products used in the study. The results were correlated with the rheological properties of the different products. RESULTS: G', the elastic modulus, is one important rheological parameter. Strong and firm gels have higher G' than weak and soft gels. When plotting the G' to mean product integration score in human skin obtained in the study, there was a statistically significant correlation with products with lowest G' having the highest integration score and products with high G' having the lowest integration scores. No statistical correlation could be seen when analyzing the score versus particle size, swelling factor, and cohesivity. CONCLUSION: The degree of product integration can be assessed and scored according to a 5-grade visual scale based on representative microphotographs. Products with different rheological properties distribute differently when injected into the skin. Firmer gel texture resulted in more targeted product integration whiles softer gel texture resulted in distributed product integration. J Drugs Dermatol. 2018;17(9):982-986.

Spontaneous testicular tubular hypoplasia/atrophy in the Göttingen minipig: a retrospective study.

The aim of this retrospective study was to assess the incidence and severity of tubular atrophy/hypoplasia in the testes of 104 control Göttingen minipigs aged 4.5 to 15 months. The finding was termed "tubular hypoplasia/atrophy" according to published descriptions for the dog. It included different microscopic changes that were considered part of a continuum, namely seminiferous epithelium vacuolation, presence of multinucleated germ cells in the tubular lumen, and decreased numbers (hypospermatogenesis) or absence of germ cells. This retrospective study demonstrates that tubular hypoplasia/atrophy is present in more than 70% of Göttingen minipigs and can occur at a marked severity in control animals. It correlated with a higher level of cell debris and a decrease in sperm content in the epididymides and with lower absolute and relative testes weights. There was no correlation with the weight of other sexual organs, total bodyweight, or age, which demonstrates that this change was not related to sexual immaturity. The distinction between this background finding and a compound-related effect could be challenging for the pathologist.

Transcriptional modulations by RXR agonists are only partially subordinated to PPARalpha signaling and attest additional, organ-specific, molecular cross-talks.

Nuclear hormone receptors (NR) are important transcriptional regulators of numerous genes involved in diverse pathophysiological and therapeutic functions. Following ligand activation, class II NR share the ability to heterodimerize with the retinoid X receptor (RXR). It is established that RXR activators, rexinoids, transactivate several peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in a PPARalpha-dependent manner. We hypothesized that, once activated, RXR might signal through quiescent NR other than PPARalpha, in an organ-specific manner. To study this putative phenomenon in vivo, we developed an array of 120 genes relevant to the class II NR field. The genes were selected using both published data and high-density screenings performed on RXR or PPARalpha agonist-treated mice. Wild-type C57BL/6J and PPARalpha-deficient mice were treated with fenofibrate (PPARalpha activator) or LGD1069 (RXR activator). Using our customized array, we studied the hepatic, cardiac, and renal expression of this panel of 120 genes and compared them in both murine genotypes. The results obtained from this study confirmed the ability of an RXR agonist to modulate PPARalpha-restricted target genes in the liver and the kidney. Furthermore, we show that various organ-specific regulations occurring in both genotypes (PPARalpha +/+ or -/-) are highly indicative of the ability of RXR to recruit other class II NR pathways. Further development of this molecular tool may lead to a better understanding of the permissiveness of class II nuclear receptor dimers in vivo.

RXR activators molecular signalling: involvement of a PPAR alpha-dependent pathway in the liver and kidney, evidence for an alternative pathway in the heart.

(1) In this study we compared the molecular signalling elicited by rexinoids, selective retinoid X receptor (RXR)-activators, in several organs (i.e. liver, kidney, heart) and in hepatocytes of various species. (2) RXR plays the pivotal role of a hetero-dimerization partner for the members of the class II subset of nuclear receptors which regulate the transcription of numerous target genes, following chemical activation. Several of these selective activators are currently used to treat hyperlipidaemia (fibrates), type II diabetes (glitazones), or skin disorders (retinoic acid). Although these therapeutic pathways are not fully elucidated, receptor activation is considered a pre-requisite for efficacy. Therefore RXR, which accepts numerous dimeric partners, is considered a worthwhile pharmacological target. (3) We analysed a number of biochemical and molecular responses to rexinoids which were given orally to mice. Our results showed a prominent involvement of the peroxisome proliferator-activated receptor (PPARalpha) as a majority of the observed hepatic and renal regulations were abolished in PPARalpha-knockout animals. Therefore we documented the species-specificity of these rexinoid actions which were reproduced in rat primary hepatocyte cultures but not in cultures of rabbit or human origin. Conversely, we established that the regulation of the pyruvate dehydrogenase kinase (PDK4) gene in the heart, by rexinoids, is independent of PPARalpha expression. (4) Our results support the obligatory expression of the active, although quiescent, PPARalpha to sustain a subset of relevant regulations attributable to rexinoids in the liver and kidney. Their cardiac molecular signalling unveiled an alternate transduction pathway and therefore opens new prospects in the therapeutic potential of rexinoids.

UVB-induced skin inflammation and cutaneous tissue injury is dependent on the MHC class I-like protein, CD1d.

CD1d is a major histocompatibility complex class 1-like molecule that regulates the function and development of natural killer T (NKT) cells. Previously, we identified a critical role for the CD1d-NKT cell arm of innate immunity in promoting the development of UVB-induced p53 mutations, immune suppression, and skin tumors. Sunburn, an acute inflammatory response to UVB-induced cutaneous tissue injury, represents a clinical marker for non-melanoma skin cancer (NMSC) risk. However, the innate immune mechanisms controlling sunburn development are not considered relevant in NMSC etiology, and remain poorly investigated. Here we found that CD1d knockout (CD1d(-/-)) mice resist UVB-induced cutaneous tissue injury and inflammation compared with wild-type (WT) mice. This resistance was coupled with a faster epithelial tissue healing response. In contrast, the skins of UVB-irradiated invariant NKT cell-knockout (Jα18(-/-)) and NKT cell-deficient (TCRα(-/-)) mice, which express CD1d but are deficient in CD1d-dependent NKT cells, exhibited as much cutaneous tissue injury and inflammation as WT mice. In the absence of NKT cells, CD1d-deficient keratinocytes, dendritic cells, and macrophages exhibited diminished basal and stress-induced levels of pro-inflammatory mediators. Thus, our findings identify an essential role for CD1d in promoting UVB-induced cutaneous tissue injury and inflammation. They also suggest sunburn and NMSC etiologies are immunologically linked.