Kathryn E. Gropp

A Long-Acting FGF21 Molecule, PF-05231023, Decreases Body Weight and Improves Lipid Profile in Non-human Primates and Type 2 Diabetic Subjects

FGF21 plays a central role in energy, lipid, and glucose homeostasis. To characterize the pharmacologic effects of FGF21, we administered a long-acting FGF21 analog, PF-05231023, to obese cynomolgus monkeys. PF-05231023 caused a marked decrease in food intake that led to reduced body weight. To assess the effects of PF-05231023 in humans, we conducted a placebo-controlled, multiple ascending-dose study in overweight/obese subjects with type 2 diabetes. PF-05231023 treatment resulted in a significant decrease in body weight, improved plasma lipoprotein profile, and increased adiponectin levels. Importantly, there were no significant effects of PF-05231023 on glycemic control. PF-05231023 treatment led to dose-dependent changes in multiple markers of bone formation and resorption and elevated insulin-like growth factor 1. The favorable effects of PF-05231023 on body weight support further evaluation of this molecule for the treatment of obesity. Longer studies are needed to assess potential direct effects of FGF21 on bone in humans.

Bone and Joints

Abstract The skeleton fulfills several needs in an animal. It serves as a scaffold on which muscles can act to produce locomotion, provides protection for vital structures, supplies an internal source for minerals, and harbors an environment that supports hematopoiesis. Due to slow and regimented turnover, the current state and the history of a bone coexist. The gross and microscopic structure of each bone depends on its location in the skeleton and the resulting profile of biomechanical forces, as well as on the species, sex, and age. Knowledge of how bone physiology varies due to each of these factors is key in recognizing and understanding bone pathology. In this chapter, basic bone biology, including anatomy, physiology, and recent advances in our knowledge of cell–cell signaling, is covered in the earlier sections. Next, modalities used to fully analyze the health of the skeleton are reviewed, including biomarker evaluation of serum or urine, imaging techniques ranging from radiology to computed tomography, biomechanical testing, routine decalcified microscopic evaluation, and specialized undecalcified microscopy with bone histomorphometry. Animal models, both those found naturally and those generated by genetic or surgical manipulation, can offer great insight into a pathologic process as long as the strengths and limitations of the model are fully appreciated. A review of how bone responds to injury is used to demonstrate the potential range of responses to injurious and restorative events, and the interrelationships between bone cells, is followed by a summary of mechanisms of bone toxicity by various prototypical substances. Many effects on bone in toxicity studies reflect the desired pharmacology of a therapeutic agent, even if the skeleton is not the intended site of action.

Once-weekly administration of a long-acting FGF21 analogue modulates lipids, bone turnover markers, blood pressure, and body weight differently in obese hypertriglyceridemic subjects and in non-human primates

To assess the safety, tolerability, pharmacokinetics and pharmacodynamics of PF‐05231023, a long‐acting fibroblast growth factor 21 (FGF21) analogue, in obese people with hypertriglyceridaemia on atorvastatin, with or without type 2 diabetes.

Serum Natriuretic Peptides as Differential Biomarkers Allowing for the Distinction between Physiologic and Pathologic Left Ventricular Hypertrophy

Given the proven utility of natriuretic peptides as serum biomarkers of cardiovascular maladaptation and dysfunction in humans and the high cross-species sequence conservation of atrial natriuretic peptides, natriuretic peptides have the potential to serve as translational biomarkers for the identification of cardiotoxic compounds during multiple phases of drug development. This work evaluated and compared the response of N-terminal proatrial natriuretic peptide (NT-proANP) and N-terminal probrain natriuretic peptide (NT-proBNP) in rats during exercise-induced and drug-induced increases in cardiac mass after chronic swimming or daily oral dosing with a peroxisome proliferator-activated receptor γ agonist. Male Sprague-Dawley rats aged 8 to 10 weeks were assigned to control, active control, swimming, or drug-induced cardiac hypertrophy groups. While the relative heart weights from both the swimming and drug-induced cardiac hypertrophy groups were increased 15% after 28 days of dosing, the serum NT-proANP and NT-proBNP values were only increased in association with cardiac hypertrophy caused by compound administration. Serum natriuretic peptide concentrations did not change in response to adaptive physiologic cardiac hypertrophy induced by a 28-day swimming protocol. These data support the use of natriuretic peptides as fluid biomarkers for the distinction between physiological and drug-induced cardiac hypertrophy.

Nerve growth factor inhibition with tanezumab influences weight-bearing and subsequent cartilage damage in the rat medial meniscal tear model

Objective To investigate whether the effects of nerve growth factor (NGF) inhibition with tanezumab on rats with medial meniscal tear (MMT) effectively model rapidly progressive osteoarthritis (RPOA) observed in clinical trials. Methods Male Lewis rats underwent MMT surgery and were treated weekly with tanezumab (0.1, 1 or 10 mg/kg), isotype control or vehicle for 7, 14 or 28 days. Gait deficiency was measured to assess weight-bearing on the operated limb. Joint damage was assessed via histopathology. A second arm, delayed onset of treatment (starting 3–8 weeks after MMT surgery) was used to control for analgesia early in the disease process. A third arm, mid-tibial amputation, evaluated the dependency of the model on weight-bearing. Results Gait deficiency in untreated rats was present 3–7 days after MMT surgery, with a return to normal weight-bearing by days 14–28. Prophylactic treatment with tanezumab prevented gait deficiency and resulted in more severe cartilage damage. When onset of treatment with tanezumab was delayed to 3–8 weeks after MMT surgery, there was no increase in cartilage damage. Mid-tibial amputation completely prevented cartilage damage in untreated MMT rats. Conclusions These data suggest that analgesia due to NGF inhibition during the acute injury phase is responsible for increased voluntary weight-bearing and subsequent cartilage damage in the rat MMT model. This model failed to replicate the hypotrophic bone response observed in tanezumab-treated patients with RPOA.

Skeletal Muscle Toolbox

Evaluation of skeletal muscle frequently combines morphologic and morphometric techniques. As is the case with many organ systems, skeletal muscle has limited responses to insult or injury. Over the past several years, crucial interactions between skeletal muscle, bone, and the nervous system have been described. The aim of this lecture was to give attendees the necessary background information in basic skeletal muscle morphology, important species differences, introduction to skeletal muscle biomarkers, approaches to morphologic and morphometric evaluation, and examples of background findings and typical responses of skeletal muscle to insult or injury.

Effects on Cancellous Bone in the Metaphysis

Cancellous bone has high metabolic activity compared to many other bone compartments and can be affected not only by changes in physeal activity but also by perturbations in homeostasis caused by changes in physiology or on-target pharmacology. Examples of several types of resulting morphologic findings were presented; if known, the pathways causing morphologic changes were discussed.

No Bones About It: The Challenges and Rewards of Osteopathology

When examining bones and joints, toxicologic pathologists and those working on experimental models have the advantage of being able to examine sections from an equal number of agematched control animals that are sectioned in the same plane as treated animals. Diagnostic pathologists have the advantage of input from clinicians who may have radiographs and, in some cases, other types of imaging data. Despite these advantages, bones and joints are often not favorite tissues, largely because of the tremendous heterogeneity in the appearance of normal features that can make lesions hard to distinguish. This heterogeneity is influenced by many variables, including but not limited to plane of section, differences in appearance of the various bones of the body and their associated structures (ligaments, tendons, menisci), age of the individual, and the fact that abnormal structures (eg, periarticular osteophytes) can assume a normal appearance through remodeling over time, thus requiring the pathologist to have expertise in the normal morphology (particularly the shape) of the affected site to appreciate changes due to chronic disease. Depending on the age of the animal, there may be the presence or absence of growth plates, remodeling in the cutback zone and endosteum, irregularities around ligament insertions and nutrient vessels, and a variable amount of new bone in the periosteum of the metaphysis. Whenever possible, plane of section should be standardized for comparison between affected and control tissues, particularly when histomorphometry measurements are taken. This is nicely exemplified in the article on animal models of osteoarthritis by McCoy 10 in this issue, in which an ideal, mid-coronal section of a murine stifle joint is included for orientation. Age of the individual can dramatically affect the healing potential of joint tissues, as evidenced by studies of osteochondrosis, a disease that develops only in immature individuals. Due to the remarkable healing properties of epiphyseal cartilage (discussed by Olstad et al 14,15 in the studies on osteochondrosis), most lesions occurring in animals with this disease are subclinical and known to heal spontaneously. Largely because it is not possible to biopsy these sites and confirm their features in the human disease, subclinical lesions of osteochondrosis in humans often are misinterpreted by MD radiologists as ‘‘ossification variants’’ rather than evidence of disease. Conversely, lesions of osteoarthritis occurring in older individuals, in whom the articular cartilage undergoes chronic degenerative changes, remain static at best and usually progress (Figs. 1, 2). Preparing bones for histopathology can be problematic if careful handling is not followed throughout the process. This includes allotting sufficient time for fixation, collecting a sample of an appropriate thickness, rinsing to remove ‘‘bone dust,’’ and using an appropriate decalcifying agent in which the end point can be monitored to prevent over-decalcification. The processing schedule should allow good penetration of the solutions and impregnation of paraffin. At microtomy, using positively charged or coated slides ensures good adherence of the tissue. Finally, it is important to optimize the staining procedure to achieve crisp nuclear and cytoplasmic details. Most sections are routinely stained with hematoxylin and eosin, but broad horizons of color (with diagnostic significance) are available in bone and joint sections stained with safranin O (see Olstad et al, 14 Fig. 18) or toluidine blue, cationic dyes that stain proteoglycans and glycosaminoglycans in cartilage (Figs. 3–5). For those who keep their polarizing lens handy, examining the birefringence of collagen in bone is another scintillating reward and allows one to easily distinguish mature, lamellar bone from newly formed, woven bone (Figs. 6, 7).

Effects of Monoclonal Antibodies against Nerve Growth Factor on Healthy Bone and Joint Tissues in Mice, Rats, and Monkeys: Histopathologic, Biomarker, and Microcomputed Tomographic Assessments:

Tanezumab, an anti-nerve growth factor (NGF) antibody, is in development for management of chronic pain. During clinical trials of anti-NGF antibodies, some patients reported unexpected adverse events requiring total joint replacements, resulting in a partial clinical hold on all NGF inhibitors. Three nonclinical toxicology studies were conducted to evaluate the effects of tanezumab or the murine precursor muMab911 on selected bone and joint endpoints and biomarkers in cynomolgus monkeys, Sprague-Dawley rats, and C57BL/6 mice. Joint and bone endpoints included histology, immunohistochemistry, microcomputed tomography (mCT) imaging, and serum biomarkers of bone physiology. Responses of bone endpoints to tanezumab were evaluated in monkeys at 4 to 30 mg/kg/week for 26 weeks and in rats at 0.2 to 10 mg/kg twice weekly for 28 days. The effects of muMab911 at 10 mg/kg/week for 12 weeks on selected bone endpoints were determined in mice. Tanezumab and muMab911 had no adverse effects on any bone or joint parameter. There were no test article–related effects on bone or joint histology, immunohistochemistry, or structure. Reversible, higher osteocalcin concentrations occurred only in the rat study. No deleterious effects were observed in joints or bones in monkeys, rats, or mice administered high doses of tanezumab or muMab911.

Chapter 23 – Bone and Joints

The skeletal system (bones and joints) fulfills several needs in an animal. The skeleton serves as a scaffold on which muscles can act to produce locomotion, provides protection for vital structures, supplies an internal source for minerals, and harbors an environment that supports hematopoiesis. Due to slow and regimented turnover, the current state and the history of a bone coexist. The gross and microscopic structure of each bone depends on its location in the skeleton and the resulting profile of biomechanical forces, as well as on the species, sex, and age. Knowledge of how bone physiology varies due to each of these factors is key in recognizing and understanding bone pathology. In this chapter, basic bone and joint biology, including anatomy, physiology, recent advances in our knowledge of cell–cell signaling, and bone biomarkers are covered in the earlier sections. Animal models, both those found naturally and those generated by genetic or surgical manipulation, can offer great insight into a pathologic process as long as the strengths and limitations of the model are fully appreciated. A review of how bones and joints respond to injury is used to demonstrate the potential range of responses to injurious and restorative events, and the interrelationships between bone cells, is followed by a summary of mechanisms of bone toxicity by various prototypical substances. Many effects on bone in toxicity studies reflect the desired pharmacology of a therapeutic agent, even if the skeleton is not the intended site of action.