Scientific and Regulatory Policy Committee Points to Consider: Review of the United States Food and Drug Administration (FDA) Guidance on Pathology Peer Review in Nonclinical Toxicology Studies.

In December 2021, the United States Food and Drug Administration (FDA) issued the final guidance for industry titled Pathology Peer Review in Nonclinical Toxicology Studies: Questions and Answers. The stated purpose of the FDA guidance is to provide information to sponsors, applicants, and nonclinical laboratory personnel regarding the management and conduct of histopathology peer review as part of nonclinical toxicology studies conducted in compliance with good laboratory practice (GLP) regulations. On behalf of and in collaboration with global societies of toxicologic pathology and the Society of Quality Assurance, the Scientific and Regulatory Policy Committee (SRPC) of the Society of Toxicologic Pathology (STP) initiated a review of this FDA guidance. The STP has previously published multiple papers related to the scientific conduct of a pathology peer review of nonclinical toxicology studies and appropriate documentation practices. The objectives of this review are to provide an in-depth analysis and summary interpretation of the FDA recommendations and share considerations for the conduct of pathology peer review in nonclinical toxicology studies that claim compliance to GLP regulations. In general, this working group is in agreement with the recommendations from the FDA guidance that has added clear expectations for pathology peer review preparation, conduct, and documentation.

Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels.

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

BDK inhibition acts as a catabolic switch to mimic fasting and improve metabolism in mice.

OBJECTIVE: Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. METHODS: The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr-/-). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. RESULTS: A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr-/- mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice. CONCLUSIONS: These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a "fed signal" to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a "fasting signal" to prolong the catabolic state.

Toxicologic Pathology Forum: Opinion on Obligatory Microscopic Examination of Intermediate-Dose Groups in Toxicity Studies With Biotherapeutics in Cynomolgus Monkeys.

In nonrodent toxicity studies that are usually conducted in cynomolgus monkeys or beagle dogs, the added value of examining all tissues from all dose groups (current practice) versus all tissues in only control and high-dose groups and target tissues in intermediate-dose groups by default, is a subject of debate. A previous retrospective review of 325 nonrodent toxicity studies that included a limited number of biotherapeutics suggested that the evaluation of all tissues from all groups was not justified as a routine practice and recommended the examination of all tissues in control and high-dose groups and only target tissues in intermediate-dose groups. In contrast, the present retrospective review which examined 213 nonrodent studies (212 in cynomolgus monkeys and 1 in dog) from 4 multinational pharmaceutical companies (Bristol-Myers Squibb, Novartis, Pfizer Inc, and Roche) conducted only with biotherapeutics showed that restricting the microscopic examination in intermediate-dose groups to target tissues has the potential to miss findings in 6.6% of studies, possibly impacting the overall study interpretation and conclusion. In conclusion and in the opinion of the authors, all tissues from all dose groups should be examined in toxicity studies with biotherapeutics conducted in nonrodent species.

Human sebum requires de novo lipogenesis, which is increased in acne vulgaris and suppressed by acetyl-CoA carboxylase inhibition.

Sebum plays important physiological roles in human skin. Excess sebum production contributes to the pathogenesis of acne vulgaris, and suppression of sebum production reduces acne incidence and severity. We demonstrate that sebum production in humans depends on local flux through the de novo lipogenesis (DNL) pathway within the sebocyte. About 80 to 85% of sebum palmitate (16:0) and sapienate (16:1n10) were derived from DNL, based on stable isotope labeling, much higher than the contribution of DNL to triglyceride palmitate in circulation (~20%), indicating a minor contribution by nonskin sources to sebum lipids. This dependence on local sebocyte DNL was not recapitulated in two widely used animal models of sebum production, Syrian hamsters and Göttingen minipigs. Confirming the importance of DNL for human sebum production, an acetyl-CoA carboxylase inhibitor, ACCi-1, dose-dependently suppressed DNL and blocked synthesis of fatty acids, triglycerides, and wax esters but not free sterols in human sebocytes in vitro. ACCi-1 dose-dependently suppressed facial sebum excretion by ~50% (placebo adjusted) in human individuals dosed orally for 2 weeks. Sebum triglycerides, wax esters, and free fatty acids were suppressed by ~66%, whereas non-DNL-dependent lipid species, cholesterol, and squalene were not reduced, confirming selective modulation of DNL-dependent lipids. Last, individuals with acne vulgaris exhibited increased sebum production rates relative to individuals with normal skin, with >80% of palmitate and sapienate derived from DNL. These findings highlight the importance of local sebocyte DNL for human skin sebaceous gland biology and illuminate a potentially exploitable therapeutic target for the treatment of acne vulgaris.

The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis.

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

The inhibin B response in male rats treated with two drug candidates.

BACKGROUND: Serum Inhibin B was measured in two studies of known testis-toxic drug candidates. METHODS AND RESULTS: Study 1 was for a compound for Hepatitis C, and utilized a 10-week dosing period, followed by mating and necropsy of half of each group, and then a 12-week recovery period for the remaining animals. At the postmating necropsy, 6 of 15 high-dose males had testis lesions; Inhibin B was significantly reduced in all animals in that group. The mid-dose group had no lesions but significantly reduced serum Inhibin B. At recovery, 9 of 15 high-dose males showed damage in testes; serum Inhibin B levels were not different from controls. Inhibin B appeared to both overreport and underreport testis damage in Study 1. Study 2 was an acute pathogenesis study for an antibacterial compound, using control and two dose levels and multiple time points (days 5, 8, 15, 22, and then untreated until day 71). At each time point blood was sampled from all remaining rats and five/group were killed for histologic evaluation. The low-dose group had minimal to moderate lesions, while serum Inhibin B was never changed. The high-dose animals progressed quickly from minimal lesions to being broadly and moderately affected; serum Inhibin B levels were reduced at days 8 and 15 only. In Study 2, Inhibin B appeared less sensitive than histology, except at the extremes of testis damage, when Inhibin B was routinely low. CONCLUSION: We conclude that in these two studies there was a poor correlation between changes in serum levels of Inhibin B and testis histopathology.

Society of Toxicologic Pathology position paper on pathology image data: compliance with 21 CFR Parts 58 and 11.

The Society of Toxicologic Pathology (STP) has developed the following recommendations for the use of pathology images in compliance with the Code of Federal Regulations (CFR), Volume 21, Part 58 (Good Laboratory Practices [GLP]) and Part 11 (Electronic Records/Signatures). These recommendations include: (1) based on current technologies and practices, pathology images (printed, electronic, or digital) used for data generation (e.g., to make a diagnosis or for morphometric analysis) are raw data that must be authenticated and archived; (2) authentication of an image may be done either by initialing and dating a print of the image or by specifically annotating the electronic image file in compliance with Part 11 regulations; (3) images used for raw data are subject to GLP procedures and controls in order to ensure data integrity including written Standard Operating Procedures, testing/validation of equipment, training of personnel, etc.; (4) validation and/or performance qualification of imaging systems used to support GLP studies must be documented and any exceptions to full validation/qualification must be described in the GLP Compliance Statement for the study; (5) images that are not used for data generation are illustrative images, are not raw data, and generally do not have to be archived; 6) illustrative images should not be used to re-evaluate or supersede the pathologist's diagnosis.

Intralesional lipid-complexed cytokine/superantigen immunogene therapy for spontaneous canine tumors.

These studies sought to determine the gene expression and short-term effects of intralesional lipid-complexed immunogene therapy with constructs encoding Staphylococcus aureus enterotoxin A and canine interleukin-2 (L-SEA/cIL-2) in dogs with tumors of various histotypes, and then to assess the safety and efficacy of repeated L-SEA/cIL-2 injections in dogs with spontaneous soft tissue sarcomas (STS). In the first study, pet dogs with a variety of tumors received a single intralesional injection of L-SEA/cIL-2, and surgical excision was performed 48 h later. In the second study, dogs with histologically confirmed STS were treated weekly for a maximum of 12 weeks with escalating doses of L-SEA/cIL-2. Tumors were then surgically excised and assessed histologically and immunohistochemically. Overall, treatments were well tolerated, with no dose-limiting toxicities encountered. At 48 h, in the single injection study, plasmid DNA was detected in 14 of 16 tumor samples, and plasmid-specific mRNA was detected in 3 of 14. In the multiple injection study, the overall response rate in dogs with STS was 25%, consisting of 3 complete responses (CR) and 1 partial response (PR). Diffuse lymphoplasmacytic inflammation was observed in all tumors from patients experiencing CR or PR, whereas these changes were not evident in tumors from nonresponders. The infiltrate was composed primarily of CD3(+) cells at 48 h from the single-injection study, and was composed of both CD3(+) and CD79a(+) cells at 12 weeks in responding dogs from the multiple-injection study. In conclusion, these studies suggests that intralesional L-SEA/cIL-2 immunotherapy is well tolerated, results in detectable transgene expression in canine tumors, and has antitumor activity in dogs with spontaneous STS.