A Metric Fractionator for Estimation of Total Cell Number in Paraffin-Embedded Flat or Hollow Organs.

The physical fractionator is a convenient and practical solution for estimation of total cell number in a regulatory toxicology setting because it is insensitive to shrinkage allowing for paraffin processing/embedding and does not require measurement of the reference or organ volume. The principle involves sampling a known fraction of an organ in one or more steps and counting the total number of cells present in the final sample, physical disector section pairs. The total cell number in the organ is estimated by multiplying the cell count in the final fraction by the inverse of the sampling fraction(s). The key feature of the design is that tissue shrinkage due to paraffin processing occurs before the organ is uniformly sampled. Another requirement is that thermal expansion or contraction is avoided during the preparation of disector sections from the individual embedded subsamples, which ensures that the disector sections represent a known constant fraction. This vertical physical fractionator with subsampling is a simple and fast estimator to obtain precise and robust estimates of total cell number in large flat or hollow organs that do not prolong routine necropsy procedures. It is compatible with paraffin processing, avoids exhaustive sectioning, and allows for the collection of routine histopathology sections.

Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.

Bone Therapeutics: Safety Considerations, Session Summary.

This session was a series of presentations focused on safety considerations for late stage or currently marketed bone therapeutic agents. The first presentation was an overview of a major regulatory requirement in the nonclinical filing package for bone therapeutics, studies designed to assess the impact of an agent on bone quality. Two presentations focused on safety issues associated with drugs whose primary mechanism of action is inhibition of bone resorption. Typical findings associated with this class of agents in general and reproductive toxicology studies were reviewed, highlighting INHAND (International Harmonization of Nomenclature and Diagnostic Criteria) nomenclature. This was followed by an overview of safety issues that have been identified largely through clinical experience. Similar presentations followed emphasizing safety and regulatory issues associated with classes of drugs whose primary mechanism of action is stimulation of bone formation known broadly as bone anabolic agents. The major focus of these discussions was carcinogenicity risk assessment. The final presentation was an introduction to a rapidly evolving area in bone therapeutics, treatment of rare genetic bone diseases, and the developmental challenges associated with these indications and novel therapeutic modalities.

Carcinogenicity risk assessment of romosozumab: A review of scientific weight-of-evidence and findings in a rat lifetime pharmacology study.

Romosozumab is a humanized immunoglobulin G2 monoclonal antibody that binds and blocks the action of sclerostin, a protein secreted by the osteocyte and an extracellular inhibitor of canonical Wnt signaling. Blockade of sclerostin binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) allows Wnt ligands to activate canonical Wnt signaling in bone, increasing bone formation and decreasing bone resorption, making sclerostin an attractive target for osteoporosis therapy. Because romosozumab is a bone-forming agent and an activator of canonical Wnt signaling, questions have arisen regarding a potential carcinogenic risk. Weight-of-evidence factors used in the assessment of human carcinogenic risk of romosozumab included features of canonical Wnt signaling, expression pattern of sclerostin, phenotype of loss-of-function mutations in humans and mice, mode and mechanism of action of romosozumab, and findings from romosozumab chronic toxicity studies in rats and monkeys. Although the weight-of-evidence factors supported that romosozumab would pose a low carcinogenic risk to humans, the carcinogenic potential of romosozumab was assessed in a rat lifetime study. There were no romosozumab-related effects on tumor incidence in rats. The findings of the lifetime study and the weight-of-evidence factors collectively indicate that romosozumab administration would not pose a carcinogenic risk to humans.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Skeletal Tissues (Bones, Joints, and Teeth).

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is an initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the skeletal tissues and teeth of laboratory rats and mice, with color photomicrographs illustrating examples of many common lesions. The standardized nomenclature presented in this document is also available on the internet (http://www.goreni.org/). Sources of material were databases from government, academic and industrial laboratories throughout the world.

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.

Inhibition of both sclerostin and DKK1 results in novel skull findings in the rat and non-human primate that is not observed with inhibition of sclerostin alone.

Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone.

Reconstruction of Remodeling units reveals positive effects after 2 and 12 months of Romosozumab treatment.

Romosozumab treatment results in a transient early increase in bone formation and sustained decrease in bone resorption. Histomorphometric analyses revealed that the primary bone-forming effect of romosozumab is transient early stimulation of modeling-based bone formation on cancellous and endocortical surfaces; preclinical studies have demonstrated that romosozumab may affect changes in the remodeling unit resulting in positive bone balance. To further investigate the effects of romosozumab on bone balance, month 12 (M12) and M2 (to analyze early effects) unpaired bone biopsies from the FRAME clinical trial were analyzed using remodeling site reconstruction to assess whether positive changes in bone balance on cancellous/endocortical surfaces may contribute to the progressive improvement in bone mass/structure and reduced fracture risk in osteoporotic women at high fracture risk. At M12, bone balance was higher with romosozumab vs placebo on cancellous (+6.1 µm vs +1.5 µm; p = 0.012) and endocortical (+5.2 µm vs -1.7 µm; p = 0.02) surfaces; higher bone balance was due to lower final erosion depth (40.7 µm vs 43.7 µm; p = 0.05) on cancellous surfaces and higher completed wall thickness (50.8 µm vs 47.5 µm; p = 0.037) on endocortical surfaces. At M2, final erosion depth was lower on the endocortical surfaces (42.7 µm vs 50.7 µm; p = 0.021) and slightly lower on the cancellous surfaces (38.5 µm vs 44.6 µm; p = 0.11) with romosozumab vs placebo. Sector analysis of early endocortical formative sites revealed higher osteoid thickness (29.9 µm vs 19.2 µm; p = 0.005) and mineralized wall thickness (18.3 µm vs 11.9 µm; p = 0.004) with romosozumab vs placebo. These evolving bone packets may reflect early stimulation of bone formation that contributes to the increase in completed wall thickness at M12. These data suggest that romosozumab induces a positive bone balance due to its effects on bone resorption and formation at the level of the remodeling unit, contributing to the positive effects on bone mass, structure, and fracture risk.

Romosozumab treatment has a dual effect on bone, adding new bone and reducing bone loss. In the FRAME clinical trial, romosozumab increased bone mass and strength, and reduced fracture risk in postmenopausal women with osteoporosis. Addition of new bone occurs early in treatment and rapidly on cancellous and endocortical bone surfaces where bone resorption is not ongoing. However, it remains unclear if romosozumab affects bone loss or gain in areas where bone resorption is ongoing (remodeling units), contributing to a further positive bone balance. Here, we examined whether changes at the remodeling unit occur early (2 months) and/or late (12 months) in treatment, using bone biopsies from patients treated with romosozumab or placebo in FRAME. At month 2, a combination of lower bone resorption and higher bone gain on endocortical surfaces resulted in a positive bone balance with romosozumab versus placebo. At month 12, bone balance was positive with romosozumab versus placebo due to lower bone resorption on cancellous surfaces and greater bone gain on endocortical surfaces. This demonstrates that romosozumab induces a positive bone balance at remodeling units early in treatment leading to overall gains observed later, contributing to the positive effects of romosozumab on bone mass and structure.

Society of toxicologic pathology position paper: review series: assessment of circulating hormones in nonclinical toxicity studies: general concepts and considerations.

This is an introductory paper to a series of papers intended to provide the basis for understanding the contribution of endocrine axis disruption or dysfunction to the pathogenesis of morphological findings and to aid in the interpretation of study outcomes. This is the first in this series of guidance papers prepared by the Working Group and outlines general concepts of study design and assay conduct and validation for hormone studies in general.

Efficacy and Safety of FX201, a Novel Intra-Articular IL-1Ra Gene Therapy for Osteoarthritis Treatment, in a Rat Model.

Osteoarthritis (OA) is a disabling, degenerative disease characterized by progressive cartilage and bone damage. There remains a need for local therapies that, following a single injection, can provide long-term pain relief and functional improvement and potentially delay disease progression. FX201 is a novel, intra-articular (IA), interleukin-1 receptor antagonist (IL-1Ra) gene therapy in development for the treatment of OA. In this study, we assessed the efficacy, biodistribution, and safety of helper-dependent adenovirus (HDAd)-ratIL-1Ra, the rat surrogate of FX201, and the biodistribution of FX201, in the anterior cruciate ligament transection (ACLT) rat OA model. A single IA injection of HDAd-ratIL-1Ra administered 7 days post-ACLT mitigated OA-related changes to cartilage, bone, and the synovial membrane at week 12 following surgery. Furthermore, FX201 and HDAd-ratIL-1Ra persisted for at least 92 days in the injected joint and proximal tissues with minimal evidence of vector spreading peripherally. Finally, HDAd-ratIL-1Ra showed a favorable safety profile without any local or systemic adverse effects. In conclusion, HDAd-ratIL-1Ra demonstrated local therapeutic and disease-modifying effects and was well tolerated, supporting further clinical development of FX201.

Modeling-Based Bone Formation After 2 Months of Romosozumab Treatment: Results From the FRAME Clinical Trial.

The bone-forming agent romosozumab is a monoclonal antibody that inhibits sclerostin, leading to increased bone formation and decreased resorption. The highest levels of bone formation markers in human patients are observed in the first 2 months of treatment. Histomorphometric analysis of bone biopsies from the phase 3 FRAME trial (NCT01575834) showed an early significant increase in bone formation with concomitant decreased resorption. Preclinical studies demonstrated that most new bone formation after romosozumab treatment was modeling-based bone formation (MBBF). Here we analyzed bone biopsies from FRAME to assess the effect of 2 months of romosozumab versus placebo on the surface extent of MBBF and remodeling-based bone formation (RBBF). In FRAME, postmenopausal women aged ≥55 years with osteoporosis were randomized 1:1 to 210 mg romosozumab or placebo sc every month for 12 months, followed by 60 mg denosumab sc every 6 months for 12 months. Participants in the bone biopsy substudy received quadruple tetracycline labeling and underwent transiliac biopsies at month 2. A total of 29 biopsies were suitable for histomorphometry. Using fluorescence microscopy, bone formation at cancellous, endocortical, and periosteal envelopes was classified based on the appearance of underlying cement lines as modeling (smooth) or remodeling (scalloped). Data were compared using the Wilcoxon rank-sum test, without multiplicity adjustment. After 2 months, the median percentage of MBBF referent to the total bone surface was significantly increased with romosozumab versus placebo on cancellous (18.0% versus 3.8%; p = 0.005) and endocortical (36.7% versus 3.0%; p = 0.001), but not on periosteal (5.0% versus 2.0%; p = 0.37) surfaces, with no significant difference in the surface extent of RBBF on all three bone surfaces. These data show that stimulation of bone formation in the first 2 months of romosozumab treatment in postmenopausal women with osteoporosis is predominately due to increased MBBF on endocortical and cancellous surfaces. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Recommendations for the evaluation of pathology data in nonclinical safety biomarker qualification studies.

A set of best practices for the conduct of histopathology evaluation in nonclinical safety studies was endorsed by the Society of Toxicologic Pathology (STP) in 2004. These best practices indicate that the study pathologist should have knowledge of the treatment group and access to all available study-related data for the animal from which the tissue was obtained. A new set of best practices for the conduct of histopathology review for safety biomarker qualification for nonclinical studies has been endorsed by the STP and is summarized in this document. These best practices are generally similar to those for nonclinical safety studies, specifically that the pathologist be "unblinded" or have access to study data. Although histopathology evaluation in biomarker qualification studies must be performed without knowledge of novel biomarker data, the study pathologist(s) should be involved in the attendant meta-analyses of these data. Blinded evaluation is an experimental tool in biomarker qualification studies that is appropriate only when well-defined criteria for specific histopathologic findings are identified prior to blinded review. Additionally, this paper also considers the management of bias, the use of a tiered evaluation approach, the importance of using qualified pathologists and standard reporting, and the management of spontaneous findings.

Modern pathology methods for neural investigations.

This session at the 2010 joint symposium of the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP) explored modern neuropathology methods for assessing the neurotoxicologic potential of xenobiotics. Conventional techniques to optimally prepare and evaluate the central and peripheral neural tissues while minimizing artifact were reviewed, and optimal schemes were set forth for evaluation of the nervous system during both routine (i.e., general toxicity) studies and enhanced (i.e., specialized neurotoxicity) studies. Stereology was introduced as the most appropriate means of examining the possible impact of toxicants on neural cell numbers. A focused discussion on brain sampling took place among a panel of expert neuroscientists (anatomists and pathologists) and the audience regarding the proper balance between sufficient sampling and cost- and time-effectiveness of the analysis. No consensus was reached on section orientation (coronal sections of both sides vs. a parasagittal longitudinal section with several unilateral hemisections from the contralateral side), but most panelists favored sampling at least 8 sections (or approximately double to triple the current complement) in routine toxicity studies.

Sclerostin Downregulation Globally by Naturally Occurring Genetic Variants, or Locally in Atherosclerotic Plaques, Does Not Associate With Cardiovascular Events in Humans.

Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.6 ± 10.4 years). Sclerostin staining was absent in most plaques (67%), and when detected, it was of reduced intensity compared with normal aorta and was located in deeper regions of the plaque/wall but was not observed in areas considered relevant to plaque stability (fibrous cap and endothelium). Additionally, genetic variants associated with lifelong reduced sclerostin expression were explored for associations with phenotypes including those related to bone physiology and CV risk factors/events in a population-based phenomewide association study (PheWAS). Natural genetic modulation of sclerostin by variants with a significant positive effect on bone physiology showed no association with lifetime risk of MI or stroke. These data do not support a causal association between the presence of sclerostin, or its inhibition, in the vasculature and increased risk of serious cardiovascular events. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Choice of morphometric methods and consequences in the regulatory environment.

In certain cases, quantitative tissue structural data derived from tissue sections may be required to make critical decisions in the drug development or risk assessment process. Most frequently, these questions center on test article-related effects on cell number. In this opinion article, the limitations of estimating cell number by standard cell or nuclear profile counts from sections/blocks collected for routine histopathology are discussed from both a scientific and regulatory perspective and contrasted with the robust, sensitive, statistically based methods of design-based stereology. Specific existing industry practices are reviewed. Recent advances in stereological theory, software, hardware, and automated immunohistochemical staining now make it feasible to implement unbiased stereological methods to assess test article-related effects on cell number in a regulatory toxicology setting. These design-based stereological methods for counting cells are recommended when the quantification of small changes in cell number is critical to the risk assessment or decision-making process. These methods provide levels of sensitivity and statistical guarantees of accuracy that no other currently available tissue section-based methodology can provide.

Design-based stereology: introduction to basic concepts and practical approaches for estimation of cell number.

In regulatory toxicology studies, qualitative histopathological evaluation is the reference standard for assessment of test article-related morphological changes. In certain cases, quantitative analysis may be required to detect more subtle morphological changes, such as small changes in cell number. When the detection of subtle test article-related morphological changes is critical to the decision-making process, sensitive quantitative methods are needed. Design-based stereology provides the tools for obtaining accurate, precise quantitative structural data from tissue sections. These tools have the sensitivity necessary to detect small changes by combining statistical sampling principles with geometric analysis of the tissue microstructure. It differs from other morphometric methods based on tissue section analysis by providing estimates that are statistically valid, truly three-dimensional, and referent to the entire organ. Further, because the precision of the stereological analysis procedure can be predicted, studies can be designed and powered to detect subtle, potentially toxicologically significant changes. Although stereological methods have not been widely applied in toxicologic pathology, recent advances have made it feasible to implement these methods in a regulatory toxicology setting, particularly methods for estimation of total cell number.

Protein extraction of formalin-fixed, paraffin-embedded tissue enables robust proteomic profiles by mass spectrometry.

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)-embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT-embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis.

p38 MAPK inhibition reduces aortic ultrasmall superparamagnetic iron oxide uptake in a mouse model of atherosclerosis: MRI assessment.

OBJECTIVE: Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE-/- mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake. METHODS AND RESULTS: ApoE-/- mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (increased 103%, P<0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (increased 6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment. CONCLUSIONS: The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II-infused apoE-/- mice.

Clinically Relevant Doses of Sclerostin Antibody Do Not Induce Osteonecrosis of the Jaw (ONJ) in Rats with Experimental Periodontitis.

Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte-secreted protein, sclerostin. Sclerostin functions to inhibit the Wnt signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature-induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ-like lesions in ovariectomized rats. Beginning 8 weeks post-ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200 µg/kg zoledronic acid (ZA), a potent bisphosphonate at 100-fold the osteoporosis dose, or 5 mg/kg sclerostin antibody (Scl-Ab) at the osteoporotic dose. EP was initiated at week 12 and maintained for the remainder of the study. Scl-Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA-treated rats developed histologic features of ONJ, whereas Veh-treated controls did not. Scl-Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ-like lesions, in ovariectomized rats with EP. © 2018 American Society for Bone and Mineral Research.