Advertisement

Effects of tanezumab on satellite glial cells in the cervicothoracic ganglion of cynomolgus monkeys: A 26-week toxicity study followed by an 8-week recovery period

Open AccessPublished:February 23, 2019DOI:https://doi.org/10.1016/j.autneu.2019.02.004

      Abstract

      Tanezumab, a humanized monoclonal anti-NGF antibody, has demonstrated efficacy and safety profiles in Phase III clinical trials of chronic pain. In a 24-week study in non-human primates, morphological observations of sympathetic ganglia showed decreased ganglia volume, decreased neuronal size, and increased glial cell density compared with controls after 3 tanezumab treatments. Using stereological techniques to quantify glial cells, the present 26-week study found no significant difference after weekly treatments in total cervicothoracic ganglia satellite glial cell number between placebo- or tanezumab-treated cynomolgus monkeys. These findings suggest that tanezumab treatment does not result in a true gliosis in sympathetic ganglia.

      1. Introduction

      During nervous system development, nerve growth factor (NGF) mediates the survival and proper differentiation of specific populations of sensory and sympathetic neurons through the binding of its receptors tropomyosin-related kinase A (TrkA; high affinity) and p75 neurotrophin receptor (low affinity). However, in the adult, NGF becomes important for pain signaling pathways. NGF levels increase during inflammation and other painful conditions, and signaling pathways downstream of NGF receptor activation result in the activation of transcription factors that control nociceptive gene expression (
      • Mantyh P.W.
      • Koltzenburg M.
      • et al.
      Antagonism of nerve growth factor-TrkA signaling and the relief of pain.
      ).
      Tanezumab is a highly selective and specific humanized IgG2Δa monoclonal anti-NGF antibody that prevents NGF binding to its receptors, and is in development for the treatment of chronic pain (
      • Mantyh P.W.
      • Koltzenburg M.
      • et al.
      Antagonism of nerve growth factor-TrkA signaling and the relief of pain.
      ). Tanezumab (or its murine precursor, muMab911) reduced pain-related behaviors in preclinical animal models of pain (
      • Shelton D.L.
      • Zeller J.
      • et al.
      Nerve growth factor mediates hyperalgesia and cachexia in auto-immune arthritis.
      ;
      • Zahn P.K.
      • Subieta A.
      • et al.
      Effect of blockade of nerve growth factor and tumor necrosis factor on pain behaviors after plantar incision.
      ). In Phase II and Phase III human clinical trials, tanezumab demonstrated efficacy over placebo in chronic pain conditions such as osteoarthritis and chronic lower back pain (
      • Brown M.
      • Murphy F.
      • et al.
      Tanezumab reduces osteoarthritic knee pain: results of a randomized, double-blind, placebo-controlled phase III trial.
      ;
      • Katz N.
      • Borenstein D.G.
      • et al.
      Efficacy and safety of tanezumab in the treatment of chronic low back pain.
      ).
      In studies performed to investigate the effects of tanezumab-induced NGF inhibition on sympathetic ganglia, stereologic and morphologic analyses of cynomolgus monkey sympathetic ganglia after three subcutaneous (SC) 1.2 mg/kg tanezumab injections 8 weeks apart revealed diffusely small neurons compared with controls with no indication of neuronal cell death. Glial cell density appeared increased, likely a result of the decreased average neuronal cell size (
      • Belanger P.
      • Butler P.
      • et al.
      From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
      ). In the present study, stereologic analyses of cervicothoracic ganglia (CTG) from tanezumab-treated adult cynomolgus monkeys were conducted to determine if there was an absolute increase in satellite glial cell (SGC) number (i.e. a true gliosis).

      2. Materials and methods

      2.1 Animal care and use

      Male and female cynomolgus monkeys were kept in a temperature- and humidity-controlled environment. Animals were housed individually in cages that complied with the Animal Welfare Act requirements and suggested guidelines (
      • National Research Council
      The Guide for the Care and Use of Laboratory Animals Washington.
      ). Animals were offered PMI's LabDiet Laboratory Fiber-Plus biscuits twice daily unless fasted for study procedures; water was provided ad libitum. A total of 35 male animals (including 3 spares) weighing 4.0 to 10.8 kg and aged 4.1 to 9.0 years and a total of 38 female animals (including 6 spares) weighing 2.7 to 3.6 kg and aged 5.0 to 6.0 years were selected at the pre-study physical examination. Animals were randomized to groups in a weight-stratified manner to achieve similar group body weight distributions.

      2.2 Tanezumab administration and study design

      Tanezumab, a humanized IgG2Δa monoclonal antibody against NGF, was manufactured and supplied by Pfizer, Inc. Animals were dosed weekly for 26 weeks with tanezumab at: 0 mg/kg (vehicle), 4 mg/kg intravenous (IV), 30 mg/kg IV, or 30 mg/kg SC. Six females and five or six males in each group were terminated and necropsied on Day 177 (the day after the final [26th] dose; post-dosing necropsy). Four females and four males from the control and 30 mg/kg SC groups were followed for 8 weeks following completion of scheduled dosing and were terminated and necropsied on Day 232 (recovery necropsy; Table 1).
      Table 1Study design.
      GroupTanezumab dose (mg/kg/week)
      Animals were administered vehicle (group 1) or tanezumab (groups 2–4) once a week for 26 consecutive weeks.
      RouteNumber of animals
      MalesFemales
      10SC6
      Post-dosing necropsy, Day 177.
       + 4
      Recovery necropsy, Day 232.
      6
      Post-dosing necropsy, Day 177.
       + 4
      Recovery necropsy, Day 232.
      24IV5
      Post-dosing necropsy, Day 177.
      6
      Post-dosing necropsy, Day 177.
      330IV5
      Post-dosing necropsy, Day 177.
      6
      Post-dosing necropsy, Day 177.
      430SC5
      Post-dosing necropsy, Day 177.
       + 4
      Recovery necropsy, Day 232.
      6
      Post-dosing necropsy, Day 177.
       + 4
      Recovery necropsy, Day 232.
      SC, subcutaneous injection; IV, intravenous injection.
      a Animals were administered vehicle (group 1) or tanezumab (groups 2–4) once a week for 26 consecutive weeks.
      b Post-dosing necropsy, Day 177.
      c Recovery necropsy, Day 232.

      2.3 Toxicokinetic analyses

      Toxicokinetic (TK) analyses were conducted in animals administered tanezumab IV or SC (n = 6/sex/dose group for IV administration, n = 10/sex/dose group for SC administration). Systemic exposure (as assessed by the highest drug concentration observed in plasma [Cmax] and the area under the plasma drug concentration-time curve from 0 to 168 h post dose [AUC168]) was similar in males and females across dose groups, therefore, group mean TK parameters are discussed and presented using combined data from both male and female cynomolgus monkeys.

      2.4 Section preparation

      Tissues for stereology investigations were prepared as previously described (
      • Butt M.
      • Evans M.
      • et al.
      Morphologic, stereologic, and morphometric evaluation of the nervous system in young cynomolgus monkeys (Macaca fascicularis) following maternal administration of tanezumab, a monoclonal antibody to nerve growth factor.
      ). Briefly, at necropsy, the monkeys were whole-body perfused with 4% methanol-free formaldehyde. The CTG (stellate ganglia; typically formed by fusion of the inferior cervical and the first thoracic sympathetic ganglia) were initially harvested along with the spinal column and stored in 4% methanol-free formaldehyde. For stereology, CTG were removed from the sympathetic chain, embedded in paraffin and sectioned completely through using an 18-μm block advance. Every 20th section was mounted to a glass slide and stained with hematoxylin and eosin (H&E). Stereologic counts of the satellite glial cells were performed on the post-dosing necropsy control, 30 mg/kg IV, and 30 mg/kg SC males and females; counts were not performed on the animals that received 4 mg/kg IV. Prior to the satellite glial cell analysis, a stereologic assessment of neurons was performed (unpublished data). The satellite glial cell assessment was performed on all animals for which sections were deemed suitable (as part of the blinded evaluation performed by the reviewing pathologist) for the prior neuronal analysis. Table 2 reflects the number of animals in each of the target groups analyzed.
      Table 2Summary data for the stereologic evaluation of cervicothoracic ganglia satellite glial cells from cynomolgus monkeys.
      Dose and routeDosing period
      0 mg/kg

      SC
      4 mg/kg

      IV
      30 mg/kg

      IV
      30 mg/kg

      SC
      Males, n4444
       Mean number of SGC6,490,086NP6,523,6455,780,055
       p value0.98630.7099
      Females, n5435
       Mean number of SGC5,031,581NP4,813,8574,677,406
       p value0.80280.6136
      SC, subcutaneous injection; IV, intravenous injection; SGC, satellite glial cells; NP, stereologic evaluation not performed.

      2.5 Stereologic evaluations

      Stereologic investigations were performed blinded (with the operator/technician and the reviewing pathologist completely unaware of the treatment status of the various animals) using Stereologer® software (Stereology Resource Center, Chester, MD). Sections were analyzed using a systematic random approach: every 40th section through the entirety of the ganglion was examined (section sampling fraction of 1/40) beginning with a randomly selected starting section. Ganglion volume was based on the Cavalieri method (
      • Gundersen H.J.
      • Jensen E.B.
      • et al.
      The efficiency of systematic sampling in stereology—reconsidered.
      ). The total satellite glial cell count was estimated using the optical fractionator method (
      • Mouton P.R.
      Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists.
      ) counting satellite glial cells in a subset (area sampling fraction of approximately 0.529) of each section evaluated. SGCs were only counted if they came into focus within the frame height and only if the center of the cell nucleus was within the dissector frame or touching an inclusion line. SGCs were identified based on their morphologic characteristics on H&E-stained sections.

      2.6 Statistical analysis

      To determine if the differences between groups were statistically significant, a two-tailed, independent t-test was performed. Tanezumab-treated animals were compared to concurrent controls of the same sex.

      3. Results

      3.1 Stereologic evaluations

      3.1.1 Ganglion volume

      Differences in ganglion volume were not statistically significant across treatment groups (males and females; data not shown). Although not statistically significant, average male ganglion volume was lower in the IV 30 mg/kg and SC 30 mg/kg tanezumab groups compared with controls. These results are congruent with those of a previous study, which demonstrated a significant decrease in ganglion volume in male cynomolgus monkeys treated with SC 1.2 mg/kg tanezumab (
      • Belanger P.
      • Butler P.
      • et al.
      From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
      ).

      3.1.2 Satellite glial cell counts

      The average total number of SGCs in the ganglia of the control, 30 mg/kg IV tanezumab, and 30 mg/kg SC tanezumab-treated animals (both male and female) was similar for animals evaluated after the dosing phase (Table 2). There were no statistically significant differences between the control groups and the tanezumab-treated groups.

      3.2 Toxicokinetic analyses

      There were no quantifiable concentrations of tanezumab in plasma samples collected and analyzed from control dose group animals. Mean overall Cmax on Day 169 was 233, 1480 and 1080 μg/mL for the 4 (IV), 30 (IV) and 30 (SC) mg/kg/week dose groups, respectively (Supplemental Table 1). Mean overall AUC168 on Day 169 was 26,000, 160,000 and 162,000 μg·hour/mL for the 4 (IV), 30 (IV) and 30 (SC) mg/kg/week dose groups, respectively (Supplemental Table 1). For the IV dose groups, mean systemic exposure increased with increasing dose in an approximately dose-proportional manner on Days 1, 22, 85 and 169. For the IV and SC dose groups, mean systemic exposure was similar on Days 22, 85 and 169; exposure on these days was higher compared to Day 1. The mean time at which Cmax was first observed (Tmax) was 3.0 h post-dose on all Days for the 4 (IV) and 30 (IV) mg/kg/week dose groups. For the 30 (SC) mg/kg/week dose group, mean Tmax was observed at 67, 37, 33 and 45 h post dose on Days 1, 22, 85 and 169, respectively (Supplemental Table 1).

      4. Discussion

      Prior morphologic studies in sympathetic ganglia have reported an increased density of satellite glial cells in the CTG of tanezumab-treated cynomolgus monkeys compared with controls (
      • Belanger P.
      • Butler P.
      • et al.
      From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
      ). At the end of the current study's dosing period, there was no significant difference in total calculated SGCs between sex-matched controls and male or female monkeys treated with 30 mg/kg IV or 30 mg/kg SC tanezumab. The observed (at the morphologic evaluation) increased density of SGCs was therefore not due to an increase in absolute numbers of SGCs; a true gliosis was not present. The current investigation represents, to the knowledge of the authors, the only quantitative investigation concerning SGCs in animals that have received NGF antibody therapy.
      Changes in sympathetic ganglia, including decreased ganglion volume and decreased neuron size/area, have been reported to occur in association with NGF antibody therapy in animal models (
      • Andrews T.J.
      • Cowen T.
      Nerve growth factor enhances the dendritic arborization of sympathetic ganglion cells undergoing atrophy in aged rats.
      ;
      • Hendry I.A.
      • Campbell J.
      Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment.
      ;
      • Rich K.M.
      • Luszczynski J.R.
      • et al.
      Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury.
      ;
      • Ruit K.G.
      • Osborne P.A.
      • et al.
      Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse.
      ). The current investigation indicates that these changes are not caused by and do not elicit a SGC response. The observed increased SGC density that may be associated with some NGF antibody therapies does not appear to be indicative of a true morphologic change, but rather an apparent change possibly due both to decreased ganglion volume and to decreased average neuron size (
      • Belanger P.
      • Butler P.
      • et al.
      From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
      ), bringing existing cells closer together than normal. Indeed, in the current study, both a trend towards lower ganglion volume and a significant decrease in neuron size/area were observed in tanezumab-treated males compared with placebo (data not shown).
      The results of this study are limited by the criteria used to count SGCs. Satellite glial cells were identified as cells with round to oval, distinct/dark nuclei. Cells with oval nuclei with a long axis more than twice the length of the short axis were not counted since most of these cells were likely either endothelial cells or fibroblasts. The counts may have included some Schwann cells (another glial cell population in sympathetic ganglia), as these cells are not always reliably distinguished from SGCs on paraffin/H&E sections, especially when they occur in close proximity to neurons. Nonetheless, the present study findings were reproducible both within and across treatment groups. Therefore, this possible limitation does not change the overall study conclusion.

      5. Conclusions

      Tanezumab treatment in non-human primates was not associated with an increase in the absolute number of SGCs in sympathetic (cervicothoracic) ganglia as determined using stereologic methods. The apparent observational increase in SGC density in sympathetic ganglia was not considered to represent a true gliosis, as the number of SGCs was similar (not significantly different) between placebo- and tanezumab-treated animals. These results add to the growing body of evidence regarding the preclinical safety profile of tanezumab (
      • Belanger P.
      • Butler P.
      • et al.
      From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
      ;
      • Bowman C.J.
      • Evans M.
      • et al.
      Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in cynomolgus monkeys (Macaca fascicularis).
      ;
      • Butt M.
      • Evans M.
      • et al.
      Morphologic, stereologic, and morphometric evaluation of the nervous system in young cynomolgus monkeys (Macaca fascicularis) following maternal administration of tanezumab, a monoclonal antibody to nerve growth factor.
      ;
      • Gropp K.E.
      • Carlson C.S.
      • et al.
      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.
      ;
      • Zorbas M.
      • Hurst S.
      • et al.
      A multiple-dose toxicity study of tanezumab in cynomolgus monkeys.
      ).
      The following is the supplementary data related to this article.
      • Supplemental Table 1

        Mean overall (combined male + female) plasma toxicokinetic parameters for tanezumab in cynomolgus monkeys after intravenous or subcutaneous administration of tanezumab on Days 1, 22, 85 and 169.

      Acknowledgments

      Editorial support was provided by Joseph Oleynek and Shannon Buckley, PhD, of Engage Scientific Solutions and funded by Eli Lilly & Co and Pfizer. The study was sponsored by Pfizer, Inc.

      Disclosures

      PB, JG, and MZ are employees of Pfizer and own stock and/or stock options in Pfizer. ME and TC are former employees of Pfizer, Inc. ME owns Pfizer shares. MB is an employee of Tox Path Specialists who were paid consultants to Pfizer in connection with the development of this manuscript.

      References

        • Andrews T.J.
        • Cowen T.
        Nerve growth factor enhances the dendritic arborization of sympathetic ganglion cells undergoing atrophy in aged rats.
        J. Neurocytol. 1994; 23: 234-241
        • Belanger P.
        • Butler P.
        • et al.
        From the cover: evaluation of the effects of tanezumab, a monoclonal antibody against nerve growth factor, on the sympathetic nervous system in adult Cynomolgus monkeys (Macaca fascicularis): a stereologic, histomorphologic, and cardiofunctional assessment.
        Toxicol. Sci. 2017; 158: 319-333
        • Bowman C.J.
        • Evans M.
        • et al.
        Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in cynomolgus monkeys (Macaca fascicularis).
        Reprod. Toxicol. 2015; 53: 105-118
        • Brown M.
        • Murphy F.
        • et al.
        Tanezumab reduces osteoarthritic knee pain: results of a randomized, double-blind, placebo-controlled phase III trial.
        J. Pain. 2012; 13: 790-798
        • Butt M.
        • Evans M.
        • et al.
        Morphologic, stereologic, and morphometric evaluation of the nervous system in young cynomolgus monkeys (Macaca fascicularis) following maternal administration of tanezumab, a monoclonal antibody to nerve growth factor.
        Toxicol. Sci. 2014; 142: 463-476
        • Gropp K.E.
        • Carlson C.S.
        • et al.
        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.
        Toxicol. Pathol. 2018; 00192623318772501
        • Gundersen H.J.
        • Jensen E.B.
        • et al.
        The efficiency of systematic sampling in stereology—reconsidered.
        J. Microsc. 1999; 193: 199-211
        • Hendry I.A.
        • Campbell J.
        Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment.
        J. Neurocytol. 1976; 5: 351-360
        • Katz N.
        • Borenstein D.G.
        • et al.
        Efficacy and safety of tanezumab in the treatment of chronic low back pain.
        Pain. 2011; 152: 2248-2258
        • Mantyh P.W.
        • Koltzenburg M.
        • et al.
        Antagonism of nerve growth factor-TrkA signaling and the relief of pain.
        Anesthesiology. 2011; 115: 189-204
        • Mouton P.R.
        Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists.
        Johns Hopkins University Press, Baltimore2002
        • National Research Council
        The Guide for the Care and Use of Laboratory Animals Washington.
        National Academy Press, DC1996
        • Rich K.M.
        • Luszczynski J.R.
        • et al.
        Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury.
        J. Neurocytol. 1987; 16: 261-268
        • Ruit K.G.
        • Osborne P.A.
        • et al.
        Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse.
        J. Neurosci. 1990; 10: 2412-2419
        • Shelton D.L.
        • Zeller J.
        • et al.
        Nerve growth factor mediates hyperalgesia and cachexia in auto-immune arthritis.
        Pain. 2005; 116: 8-16
        • Zahn P.K.
        • Subieta A.
        • et al.
        Effect of blockade of nerve growth factor and tumor necrosis factor on pain behaviors after plantar incision.
        J. Pain. 2004; 5: 157-163
        • Zorbas M.
        • Hurst S.
        • et al.
        A multiple-dose toxicity study of tanezumab in cynomolgus monkeys.
        Regul. Toxicol. Pharmacol. 2011; 59: 334-342