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.

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.

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.

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.

Tanezumab, a humanized monoclonal antibody against nerve growth factor is in development for treatment of chronic pain. Three nonclinical studies assessed effects of clinically relevant and supratherapeutic doses of tanezumab on the sympathetic nervous system (SNS) of adult nonhuman primates. Study 1 evaluated potential effects of subcutaneous (SC) tanezumab (1.2 mg/kg every 8 weeks [Q8W]) on SNS in cynomolgus monkeys for 3 or 6 months and reversibility or persistence of any effects through a nondosing/recovery period. Study 2 evaluated whether neuronal cell death occurs shortly after a single SC tanezumab injection (1.2 mg/kg). Assessments for these two studies included evaluations of superior cervical and cervicothoracic ganglia for neuronal cell death and morphology. Study 3 evaluated effects of SC tanezumab (1.2 mg/kg Q8W and 30 mg/kg/week) over 6 months on sympathetic control of cardiovascular function. Tanezumab exposure was associated with stereologic changes in sympathetic ganglia, including smaller ganglion volume, and smaller average neuron size/area beginning at 2 weeks and reaching maximal levels by 1 month with no further progression through 6 months. These changes were not associated with clinical signs, completely reversed upon tanezumab withdrawal, and were not considered adverse. Tanezumab had no adverse effects on sympathetic control of cardiovascular function. These data support the conclusion that tanezumab administration for up to 6 months has no adverse effects on SNS morphology or function and does not cause neuronal cell death in adult nonhuman primates.

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.

Anti-NGF monoclonal antibody muMab 911 does not deplete neurons in the superior cervical ganglia of young or old adult rats.

Nerve growth factor (NGF) blocking therapies are an emerging and effective approach to pain management. However, concerns about the potential for adverse effects on the structure and function of the peripheral nervous system have slowed their development. Early studies using NGF antisera in adult rats reported effects on the size and number of neurons in the sympathetic chain ganglia. In the work described here, both young adult (6-8 week) and fully mature (7-8 month) rats were treated with muMab 911, a selective, murine, anti-NGF monoclonal antibody, to determine if systemic exposures to pharmacologically active levels of antibody for 1 month cause loss of neurons in the sympathetic superior cervical ganglia (SCG). State-of-the-art, unbiased stereology performed by two independent laboratories was used to determine the effects of muMab 911 on SCG neuronal number and size, as well as ganglion size. Following muMab 911 treatment, non-statistically significant trends toward smaller ganglia, and smaller and fewer neurons, were seen when routine, nonspecific stains were used in stereologic assessments. However, when noradrenergic neurons were identified using tyrosine hydroxylase (TH) immunoreactivity, trends toward fewer neurons observed with routine stains were not apparent. The only statistically significant effects detected were lower SCG weights in muMab 911-treated rats, and a smaller volume of TH immunoreactivity in neurons from younger rats treated with muMab 911. These results indicate that therapeutically relevant exposures to the anti-NGF monoclonal antibody muMab 911 for 1 month have no effect on neuron numbers within the SCG from young or old adult rats.

Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in cynomolgus monkeys (Macaca fascicularis).

Two intravenous studies with tanezumab, an anti-nerve growth factor monoclonal antibody, were conducted in pregnant cynomolgus monkeys to assess potential effects on pregnancy and pre- and postnatal development. Study 1 evaluated infants up to 12 months of age following weekly maternal dosing (0, 0.5, 4 or 30 mg/kg; 18 per group) from gestation day (GD) 20 through parturition. Study 2 evaluated infants 2 months postnatally following weekly maternal dosing (0, 0.5 or 30 mg/kg; 20-21 per group) from GD 20 through 48. In the absence of maternal toxicity, tanezumab increased stillbirth and post-birth infant mortality/morbidity, decreased infant growth and resulted in microscopic changes in the peripheral sympathetic and sensory nervous system of the infants at all doses. Decreased primary antibody responses and increased incidences in skin changes in infants were also observed. The no-observed-adverse-effect-level for maternal toxicity was 30 mg/kg and <0.5 mg/kg for developmental toxicity.

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.

Tanezumab, an antibody to nerve growth factor, was administered to pregnant cynomolgus monkeys at 0, 0.5, 4, and 30 mg/kg weekly, beginning gestation day (GD) 20 through parturition (∼GD165). Maternal tanezumab administration appeared to increase stillbirths and infant mortality, but no consistent pattern of gross and/or microscopic change was detected to explain the mortality. Offspring exposed in utero were evaluated at 12 months of age using light microscopy (all tissues), stereology (basal forebrain cholinergic and dorsal root ganglia neurons), and morphometry (sural nerve). Light microscopy revealed decreased number of neurons in sympathetic ganglia (superior mesenteric, cervicothoracic, and ganglia in the thoracic sympathetic trunk). Stereologic assessment indicated an overall decrease in dorsal root ganglion (thoracic) volume and number of neurons in animals exposed to tanezumab 4 mg/kg (n = 9) and 30 mg/kg (n = 1). At all tanezumab doses, the sural nerve was small due to decreases in myelinated and unmyelinated axons. Existing axons/myelin sheaths appeared normal when viewed with light and transmission electron microscopy. There was no indication of tanezumab-related, active neuron/nerve fiber degeneration/necrosis in any tissue, indicating decreased sensory/sympathetic neurons and axonal changes were due to hypoplasia or atrophy. These changes in the sensory and sympathetic portions of the peripheral nervous system suggest some degree of developmental neurotoxicity, although what effect, if any, the changes had on normal function and survival was not apparent. Overall, these changes were consistent with published data from rodent studies.

A multiple-dose toxicity study of tanezumab in cynomolgus monkeys.

Nerve growth factor (NGF) is an important mediator of pain and hyperalgesia and has become a target of novel analgesic therapeutics. Tanezumab is a humanized IgG(2) antibody that binds NGF with high affinity and specificity. In a study to assess the toxicity and pharmacokinetic properties of tanezumab in adult, male and female, cynomolgus monkeys following weekly intravenous administration of 1, 10, or 30 mg/kg for up to 26 weeks (followed by an 8-week recovery period), tanezumab was well tolerated with no macroscopic or microscopic effects on those brain, spinal cord, nerve, or ganglia sections evaluated. One fifth of tanezumab-treated monkeys developed an antibody response to tanezumab that prevented maintenance of tanezumab exposure between dosing. In the antibody-negative animals, accumulation of tanezumab was observed; steady state was achieved approximately 8 weeks after the first dose of study drug, and exposure to tanezumab was approximately dose proportional with no observed difference between male and female animals. One monkey died during the study; this monkey had findings suggestive of hypersensitivity reaction. The favorable toxicity and pharmacokinetic profile of tanezumab seen in this study supports its further evaluation for the treatment of pain in clinical practice.

Immunotoxicity evaluation of nelfinavir in rats.

The objective of these investigations was to determine whether exposure to the HIV-1 protease inhibitor nelfinavir compromises immune function in Sprague Dawley rats. Animals (20/sex per group) were exposed orally for 1 or 6 months to nelfinavir at doses of 0 (1% carboxymethylcellulose vehicle), 100, 300 or 1000 mg/kg per day. Animals were observed daily for morbidity/mortality and for clinical signs of toxicity. Body weights were recorded weekly (weeks 1-14) and then monthly thereafter and at study termination. At termination (1 month or 6 months; 10/sex per group), serum was collected and retained for toxicokinetic analysis. The spleen, thymus and liver were removed from each animal and weighed; thymuses and liver were discarded after weighing. Spleens were prepared and immunophenotyping, natural killer (NK) cell activity, and proliferative responses to mitogenic stimuli (e.g., concanavalin A, Salmonella typhimurium) were evaluated. There were no treatment-related effects on immune cell populations (absolute or percent values) or in proliferative responses. At the 1-month interval, a decrease in NK cell activity (0.45-fold control) was noted in male rats at 100 and 1000 mg/kg per day but not at the middle dose of 300 mg/kg per day. Female rats at 1 month were noted for an increase in NK cell activity (1.4-fold control) at 100 mg/kg per day, but there was no difference in the NK response between vehicle-treated animals and those exposed to higher doses of nelfinavir. No effects on NK activity were noted in female animals after 6 months of nelfinavir treatment. Assay difficulties prevented evaluation of male rats at the 6-month interval. Taken together, the absence of a dose-response effect for NK activity in male rats treated for 1 month, the lack of suppressive effects in females treated for either 1 or 6 months, and the unchanged splenic NK cell numbers in nelfinavir-treated animals at both 1 and 6 months suggest that the decreased NK activity noted in male rats at 1 month is not biologically relevant. It was therefore concluded that, under the experimental conditions used, oral treatment with nelfinavir for 1 or 6 months at doses up to 1000 mg/ kg per day is not immunosuppressive in rats. C8hr values following nelfinavir treatment at 1000 mg/kg per day for 6 months were between 1- and 2.7-fold the reported Cmax values in humans.

Lack of respiratory and contact sensitizing potential of the intranasal antiviral drug candidate rupintrivir (AG7088): a weight-of-the-evidence evaluation.

Rupintrivir, also known as AG7088, is a small molecule 3C protease inhibitor designed to target human rhinovirus as a potential intranasal treatment for the common cold. The ability of rupintrivir to induce both respiratory and contact hypersensitivity responses was evaluated using a weight of the evidence approach. A local lymph node assay (LLNA) in mice evaluating concentrations of rupintrivir up to 50% in dimethylformamide showed no evidence of sensitizing capability. An irritation study conducted in rabbits was performed to assess potential dermal irritancy and provide information for worker safety guidelines. The study showed no evidence of skin irritation when the material was placed in direct contact with the skin in a semi-occluded fashion for four days. Quantitative whole body autoradiography (QWBA) following intranasal instillation of the compound into rabbits showed that the compound was retained in the nasal cavity or was swallowed. No radioactivity was observed in the pulmonary regions of these animals. Histopathologic evaluation of the nasopharyngeal tract and the lungs of both rats and dogs exposed by intranasal instillation acutely or following repeated intranasal exposures for 14 (rat) or 28 days (rat and dog) did not reveal any evidence of irritation or inflammation in these regions of the respiratory tract. These data demonstrate that rupintrivir does not cause irritation or inflammatory responses that may precede the development of sensitization of the skin or respiratory tract. It was concluded that the weight of the available toxicologic evidence indicated that rupintrivir was not likely to cause sensitization of either the skin or the respiratory tract in humans following intranasal delivery.

Absence of embryo-fetal toxicity in rats or rabbits following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000mg/kg/day on days 6-17 of gestation and to rabbits at doses of 200, 400, or 1000mg/kg/day on days 7-20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations (C(max)) in rats were comparable to C(max) values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13-0.17x the human C(max) and 0.25-0.5x the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000mg/kg/day. Two rabbits in the 400mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000mg/kg/day on gestation days 7-10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

Absence of reproductive and developmental toxicity in rats following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to produce reproductive toxicity was evaluated in rats administered oral doses of 200, 500, or 1000mg/kg/day. In the fertility and early embryonic development to implantation study, male rats were treated beginning 28 days prior to mating until necropsy and females for 2 weeks prior to mating and through gestation day (GD) 7. In the pre- and postnatal development study, pregnant rats were treated from GD 6 through lactation day (LD) 20. Selected F(1) pups from this study were evaluated in sensory and behavioral tests and were subsequently mated. Pregnant F(1) females were euthanized on GD 20 and their F(2) fetuses were examined. F(1) animals were not directly dosed with the drug. No treatment-related effects were observed on any male reproductive parameters. Administration of nelfinavir did not produce adverse effects on fertility, pregnancy, embryo-fetal development, parturition, or lactation in the F(0) generation. Similarly, no adverse effects of nelfinavir treatment were observed on pre- and postnatal growth, development, reproductive performance and embryo-fetal development in the F(1) offspring. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for developmental and reproductive toxicity in rats was considered to be 1000mg/kg/day, the highest dose tested.