Nonclinical Safety Research Articles

Intraepidermal Nerve Fiber Analysis in Human Patients and Animal Models of Peripheral Neuropathy: A Comparative Review.

Analysis of intraepidermal nerve fibers (IENFs) in skin biopsy samples has become a standard clinical tool for diagnosing peripheral neuropathies in human patients. Compared to sural nerve biopsy, skin biopsy is safer, less invasive, and can be performed repeatedly to facilitate longitudinal assessment. Intraepidermal nerve fiber analysis is also more sensitive than conventional nerve histology or electrophysiological tests for detecting damage to small-diameter sensory nerve fibers. The techniques used for IENF analysis in humans have been adapted for large and small animal models and successfully used in studies of diabetic neuropathy, chemotherapy-induced peripheral neuropathy, HIV-associated sensory neuropathy, among others. Although IENF analysis has yet to become a routine end point in nonclinical safety testing, it has the potential to serve as a highly relevant indicator of sensory nerve fiber status in neurotoxicity studies, as well as development of neuroprotective and neuroregenerative therapies. Recently, there is also interest in the evaluation of IENF via skin biopsy as a biomarker of small fiber neuropathy in the regulatory setting. This article provides an overview of the anatomic and pathophysiologic principles behind IENF analysis, its use as a diagnostic tool in humans, and applications in animal models with focus on comparative methodology and considerations for study design.

Nonclinical toxicology studies with sodium taurodeoxycholate: acute and subacute toxicity in dogs

Sodium taurodeoxycholate (TDCA) has been investigated for various inflammatory disorders such as sepsis. We recently evaluated nonclinical safety profile of TDCA using rats infused intravenously. As a series of preclinical safety investigations, we further conducted toxicity studies with TDCA delivered to dogs via intravenous administration under Good Laboratory Practice regulation in this study. In dose range-finding study (dose escalation study), dogs given with TDCA at a dose of 150 mg/kg showed marked changes in clinical signs, hematology, and serum biochemistry. And biochemical markers of liver damage and local skin lesions were observed following intravenous infusion of 100 mg/kg TDCA, suggesting that 100 mg/kg was chosen as the highest dose of TDCA for 4-week repeated-dose toxicity study using dogs. Despite no treatment-related significant changes in body weight, food consumption, ophthalmoscopy, and urinalysis, skin lesions were observed at the injection site of animals administered with higher than 50 mg/kg of TDCA along with biochemical and histopathological changes associated with liver injury. However, most of off-target effects were found to be reversible since these were recovered after stopping TDCA infusion. These findings indicate that the no-observed-adverse-effect-level (NOAEL) for TDCA in dogs was considered to be 5 mg/kg/d. Taken together, our results provide important toxicological profiles regarding the safe dose of TDCA for drug development or clinical application.

Investigations on the Relationship Between Ovarian, Endocrine, and Renal Findings in Nonclinical Safety Studies of the γ-Secretase Inhibitor Avagacestat.

Avagacestat, a gamma (γ)-secretase inhibitor that was in development for treatment of Alzheimer's disease, produced ovarian granulosa-thecal cell tumors in rats and dogs and a glomerulopathy with profound proteinuria in female rats. This report describes the results of follow-up investigative studies, including the use of ovariectomized (OVX) rats, to further characterize these findings and determine their mechanism(s). Ovarian proliferative changes in rats likely resulted from: (1) inhibition of Notch signaling pathways regulating ovarian follicular differentiation/development, characterized microscopically as altered ovarian cyclicity and/or ovarian follicular degeneration; (2) subsequent disruption of the hypothalamic-pituitary-ovarian axis due to ovarian atrophy with decreases in serum estrogen and progesterone (as low as 0.45× and 0.21× controls, respectively); and (3) chronic gonadotropin stimulation and pituitary hypertrophy/hyperplasia in response to the absence of negative feedback. Gonadotropin stimulation in rats was confirmed by increases in serum follicle-stimulating hormone (up to 7.75× controls) and luteinizing hormone (up to 5.84×). A similar nongenotoxic mechanism was likely responsible for the ovarian findings in dogs although changes in serum hormone levels were not detected. The dose- and time-dependent glomerulopathy with progression to chronic progressive nephropathy in female rats appears to be a direct effect of avagacestat and was not ameliorated with coadministration of 17β-estradiol or an antihypertensive (enalapril) and was not present in control OVX rats. In contrast, adrenocortical hypertrophy in female rats was considered secondary to ovarian changes based on the absence of this finding in avagacestat-treated OVX rats and no increase in adrenocorticotropic hormone staining in the pituitary.

PS1301 POTENT ANTI‐TUMOR ACTIVITY OF DUOBODY®‐CD3XCD20 IN PRECLINICAL MODELS IN VITRO AND IN VIVO

Background:DuoBody‐CD3xCD20 (GEN3013) is a bispecific antibody (bsAb) recognizing the T‐cell antigen CD3 and the B‐cell antigen CD20, that triggers potent T‐cell‐mediated lysis of CD20‐expressing cells. DuoBody‐CD3xCD20 is a full‐length bispecific IgG1 generated by controlled Fab‐arm exchange (Labrijn et al. PNAS 2013, Nat Prot 2014), with an effector function‐silenced Fc region.Aims:Preclinical characterization of DuoBody‐CD3xCD20.Methods:T‐cell activation and cytotoxicity induced by DuoBody‐CD3xCD20 in vitro, in comparison with a known CD3xCD20 bsAb, was measured by flow cytometry, 48 hours after incubation of healthy donor T cells with CD20‐expressing cell lines (E:T ratio 2:1). Anti‐tumor activity of DuoBody‐CD3xCD20 in vivo was assessed in xenograft models. In a co‐engraftment model, Raji tumor cells were co‐inoculated with healthy donor PBMCs subcutaneously (SC) in NOD‐SCID mice, followed by intravenous (IV) antibody treatment. In a MiXeno model, JEKO‐1 tumor cells were inoculated SC in NCG mice, followed by intraperitoneal injection of human PBMCs. Treatment was initiated when tumors reached an average size of 100 mm3. Human immune system (HIS mice) were inoculated with Daudi lymphoma cells IV or with Raji lymphoma cells SC, followed by treatment from day 3. Activity of DuoBody‐CD3xCD20 after SC or IV administration to cynomolgus monkeys was assessed as part of nonclinical safety studies.Results:DuoBody‐CD3xCD20 induced potent activation and cytotoxic activity of CD4+ and CD8+ T cells in the presence of CD20‐expressing cells in vitro. T‐cell‐mediated cytotoxicity was observed in a diverse panel of diffuse large B‐cell and mantle cell lymphoma cell lines, with EC50 values ranging from 0.2 to 5.0 pM. Direct comparison in vitro demonstrated that DuoBody‐CD3xCD20 was ∼100‐fold more potent than another CD3xCD20 bsAb that is currently tested in clinical trials. DuoBody‐CD3xCD20 potently inhibited outgrowth of CD20‐expressing tumors (Raji) in vivo, even in the presence of high doses of Fc‐inactive rituximab, demonstrating that the anti‐tumor activity of DuoBody‐CD3xCD20 was not inhibited by an excess of circulating CD20 antibodies that compete for target binding. In addition, DuoBody‐CD3xCD20 induced regression of established mantle cell lymphoma (JEKO‐1) tumors and delayed tumor growth and enhanced survival in the HIS xenograft models. In cynomolgus monkeys, profound and long‐lasting B‐cell depletion (≥ 70 days) from peripheral blood and lymphoid organs was induced by DuoBody‐CD3xCD20 at dose levels > 0.1 mg/kg. B‐cell depletion was reversible, with time to recovery correlating with the treatment dose. A transient decline in peripheral T cells was observed, as well as T cell activation. At the same dose levels, SC and IV administration of DuoBody‐CD3xCD20 induced comparable B cell depletion. Importantly, peak plasma levels were reduced and delayed after SC administration, which was associated with reduced plasma cytokine levels.Summary/Conclusion:DuoBody‐CD3xCD20 induced potent anti‐tumor activity in vitro and in vivo, also in the presence of competing CD20 antibodies. DuoBody‐CD3xCD20 was ∼100‐fold more potent in vitro than another CD3xCD20 bsAb that is in clinical development. SC administration in cynomolgus monkeys induced profound B‐cell depletion, which was associated with reduced plasma cytokine levels compared to IV administration. A First‐in‐Human trial to evaluate the safety and preliminary efficacy of DuoBody‐CD3xCD20 by SC administration in patients with B‐cell malignancies is currently enrolling (NCT03625037).

PS1302 THE CD58-CD2 AXIS CONTROLS KILLING OF B CELLS BY CYTOTOXIC T CELLS

Background: DuoBody-CD3xCD20 (GEN3013) is a bispecific antibody (bsAb) recognizing the T-cell antigen CD3 and the B-cell antigen CD20, that triggers potent T-cell-mediated lysis of CD20-expressing cells. DuoBody-CD3xCD20 is a full-length bispecific IgG1 generated by controlled Fab-arm exchange (Labrijn et al. PNAS 2013, Nat Prot 2014), with an effector function-silenced Fc region. Aims: Preclinical characterization of DuoBody-CD3xCD20. Methods: T-cell activation and cytotoxicity induced by DuoBody-CD3xCD20 in vitro, in comparison with a known CD3xCD20 bsAb, was measured by flow cytometry, 48 hours after incubation of healthy donor T cells with CD20-expressing cell lines (E:T ratio 2:1). Anti-tumor activity of DuoBody-CD3xCD20 in vivo was assessed in xenograft models. In a co-engraftment model, Raji tumor cells were co-inoculated with healthy donor PBMCs subcutaneously (SC) in NOD-SCID mice, followed by intravenous (IV) antibody treatment. In a MiXeno model, JEKO-1 tumor cells were inoculated SC in NCG mice, followed by intraperitoneal injection of human PBMCs. Treatment was initiated when tumors reached an average size of 100 mm3. Human immune system (HIS mice) were inoculated with Daudi lymphoma cells IV or with Raji lymphoma cells SC, followed by treatment from day 3. Activity of DuoBody-CD3xCD20 after SC or IV administration to cynomolgus monkeys was assessed as part of nonclinical safety studies. Results: DuoBody-CD3xCD20 induced potent activation and cytotoxic activity of CD4+ and CD8+ T cells in the presence of CD20-expressing cells in vitro. T-cell-mediated cytotoxicity was observed in a diverse panel of diffuse large B-cell and mantle cell lymphoma cell lines, with EC50 values ranging from 0.2 to 5.0 pM. Direct comparison in vitro demonstrated that DuoBody-CD3xCD20 was ∼100-fold more potent than another CD3xCD20 bsAb that is currently tested in clinical trials. DuoBody-CD3xCD20 potently inhibited outgrowth of CD20-expressing tumors (Raji) in vivo, even in the presence of high doses of Fc-inactive rituximab, demonstrating that the anti-tumor activity of DuoBody-CD3xCD20 was not inhibited by an excess of circulating CD20 antibodies that compete for target binding. In addition, DuoBody-CD3xCD20 induced regression of established mantle cell lymphoma (JEKO-1) tumors and delayed tumor growth and enhanced survival in the HIS xenograft models. In cynomolgus monkeys, profound and long-lasting B-cell depletion (≥ 70 days) from peripheral blood and lymphoid organs was induced by DuoBody-CD3xCD20 at dose levels > 0.1 mg/kg. B-cell depletion was reversible, with time to recovery correlating with the treatment dose. A transient decline in peripheral T cells was observed, as well as T cell activation. At the same dose levels, SC and IV administration of DuoBody-CD3xCD20 induced comparable B cell depletion. Importantly, peak plasma levels were reduced and delayed after SC administration, which was associated with reduced plasma cytokine levels. Summary/Conclusion: DuoBody-CD3xCD20 induced potent anti-tumor activity in vitro and in vivo, also in the presence of competing CD20 antibodies. DuoBody-CD3xCD20 was ∼100-fold more potent in vitro than another CD3xCD20 bsAb that is in clinical development. SC administration in cynomolgus monkeys induced profound B-cell depletion, which was associated with reduced plasma cytokine levels compared to IV administration. A First-in-Human trial to evaluate the safety and preliminary efficacy of DuoBody-CD3xCD20 by SC administration in patients with B-cell malignancies is currently enrolling (NCT03625037).

Good Laboratory Practice: Initial Development, Necessity, and Issues of Data Reliability in Basic Research

This review describes the initial development of good laboratory practice (GLP) and follows the discoveries of quality control problems in labs that conducted tests in U.S. pharmaceutical companies. In addition to introducing the essence of the GLP standards, how the GLP ensures the reconstructability and reproducibility of study results is explained in detail. Issues in nonclinical safety studies in drug development and approaches of the Japanese Pharmaceuticals and Medical Devices Agency to overcome them are also described. It is hoped that this review is helpful not only to those who work on drug development but also to faculties and students who work in academia and are involved in basic research when they attempt to resolve problems related to ensuring the reliability of basic research and research integrity.

Nonclinical Safety Assessment of a Long-Acting Recombinant PEGylated Factor Eight (BAY 94-9027) With a 60 kDa PEG.

BAY 94-9027 (Jivi) is a site-specifically PEGylated human B-domain-deleted (BDD) recombinant factor VIII (FVIII), with a 60 kDa branched PEG molecule attached. The nonclinical safety of BAY 94-9027 was evaluated in a toxicology program that included 2 weeks intravenous (IV) toxicity studies in rats and rabbits, a juvenile toxicity study in rats as well as a 26-week chronic study in rats. Doses of 75, 750, or 2250 IU/kg given every other day for 2 weeks did not elicit any findings related to BAY 94-9027. Specifically, no thrombus formation or histological changes such as cellular vacuolation were seen. In the chronic toxicity study, 40, 400, and 1200 IU/kg of BAY 94-9027 given twice weekly did not induce adverse effects related to BAY 94-9027, and no tissue vacuolation was observed. There was no PEG detected in choroid plexus or other areas of the brain, cerebrospinal fluid or in spleen or kidneys. These results were supported by toxicity studies in rats and rabbits treated with PEG 60 kDa attached to the maleimide linker (PEG-60-Mal-Cys). No findings related to PEG-60-Mal-Cys were seen. These results demonstrate the safety of BAY 94-9027 for long-term use.

Nonclinical safety assessment of AMG 553, an investigational anti-FLT3 CAR-T therapy.

7032 Background: FMS-like tyrosine kinase 3 (FLT3) is a tyrosine-protein kinase involved in hematopoiesis. In acute myeloid leukemia (AML), higher FLT3 transcript levels, independent of the presence of FLT3 mutations, correlate with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells. FLT3 protein is detectable on the cell surface of more than 80% of leukemia isolates from adult AML patients whereas FLT3 mutations are present only in approximately one third of patients. AMG 553 is a novel, investigational, adoptive cellular immunotherapy for the treatment of relapsed refractory AML, consisting of autologous T cells genetically modified ex vivo to express a transmembrane chimeric antigen receptor (CAR) to target FLT3 protein on the surface of AML cells irrespective of FLT3 mutational status. Methods: The nonclinical safety evaluation of AMG 553 includes assessment of FLT3 expression in normal tissues; in vitro assessment of cytotoxicity against human cells with or without FLT3 expression; toxicology studies in cynomolgus monkeys administered autologous T-cells transduced with an anti-FLT3 CAR or administered anti-FLT3 bi-specific T-cell engager (BiTE) antibodies. Results: Assessment of FLT3 expression in normal tissues indicates that FLT3 protein is detected on the cell surface of a subpopulation of human bone marrow hematopoietic stem and progenitor cells; expression in other tissues is cytoplasmic and not anticipated to be accessible to AMG 553. AMG 553-induced cytotoxicity was only observed in a percentage of primary bone marrow CD34+ cells. There was no evidence of CAR-T cell-mediated toxicity, expansion, or persistence in cynomolgus monkeys likely due to restricted cell surface FLT3 protein expression in healthy animals which demonstrates the limited value of such studies for this target. All findings in toxicology studies with anti-FLT3 BiTEs were consistent with the targeted killing of cells expressing FLT3 on the plasma membrane. Conclusions: The nonclinical safety data support AMG 553 as a novel therapeutic to target FLT3 protein on AML cells irrespective of FLT3 mutational status, while only affecting a percentage of normal hematopoietic stem and progenitor cells.

AGEN2373 is a conditionally-active agonist antibody targeting the costimulatory receptor CD137 for the treatment of human malignancies.

e14005 Background: Agonist antibodies targeting CD137 are potent inducers of tumor-reactive T cell proliferation, cytokine production, and cytotoxicity, and have been explored clinically for their ability to enhance immune cell-mediated destruction of tumor cells. Despite early signs of clinical activity, the development of first-generation anti-CD137 antibodies has been hampered by on-target dose-limiting hepatotoxicity or poor clinical activity. Subsequent CD137 therapies have therefore sought to localize CD137 agonism to the tumor microenvironment (TME) to potentially improve tolerability in patients. However, these approaches may also limit the potential benefit of CD137 signaling outside the TME that contributes to anti-tumor immunity, such as T cell priming in tumor draining lymph nodes. Here we describe the pharmacologic and non-clinical safety profile of a next-generation anti-CD137 monospecific antibody, AGEN2373. Methods: Flow cytometry and surface plasmon resonance-based binding, superantigen-driven immune cell activation, and luminescent reporter cell line assays were developed to test the ability of AGEN2373 to induce CD137 signaling. A non-clinical, multi-dose safety study was conducted wherein cynomolgus monkeys were injected intravenously with 100 mg/kg doses of AGEN2373. Results: AGEN2373 engages cell-expressed human and cynomolgus monkey CD137, and provides potent CD137 co-stimulation only in the presence of CD137 ligand and/or Fc gamma receptor-expressing antigen presenting cells. No changes in clinical parameters, importantly no sign of liver toxicity, were observed after administration of AGEN2373 in cynomolgus monkeys. Conclusions: AGEN2373, a novel conditionally-active anti-CD137 agonist antibody that is well-tolerated in non-human primates, is expected to have clinical activity in patients with human malignancies by its ability to selectively enhance tumor immunity in the context of immune co-stimulation.

A first-in-human, multicenter, open label, phase I study in patients with advanced and/or refractory solid malignancies to evaluate the safety of intravenously administered ATOR-1015.

TPS2653 Background: ATOR-1015 is a human bispecific IgG1 antibody targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and the tumor necrosis factor receptor superfamily member 4, OX40 (also known as CD134). Both in vitro and in vivo, ATOR-1015 induces activation of cytotoxic T cells and depletion of regulatory T cells (1). In syngeneic tumor models, using human OX40 transgenic mice cross-reacting with both targets, ATOR-1015 is demonstrated to localize to the tumor. Further, the effects of ATOR-1015 are shown to occur in the tumor area and not in the spleen (1). Treatment with ATOR-1015 also reduces tumor growth and improves survival in several tumor models in mice, including bladder, colon and pancreatic cancer (1). The non-clinical safety profile and the pharmacokinetics were established in cynomolgus monkeys and the data were used for the dosing schedule. Methods: This is a multicenter, open-label, dose escalation study enrolling patients with advanced and/or refractory solid malignancies (NCT03782467). The primary objective of the study is to determine the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D) and to establish the safety profile of ATOR-1015. ATOR-1015 is administered intravenously biweekly as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The study will start with single patient cohorts until grade 2 toxicities are observed, thereafter the study follows a modified 3+3 design. At the MTD/RP2D, or a lower dose, up to 20 patients are planned for additional safety and efficacy evaluation. Study enrollment was initiated in January 2019. A total of up to 53 patients are estimated to be enrolled in the study. (1) Månsson Kvarnhammar et al. Journal for ImmunoTherapy of Cancer 2018; 6(Suppl 1):115. Abstract P683. Clinical trial information: NCT03782467.

Shared Learnings on the New EMA First-in-Human and Early Clinical Trial Guideline: Proceedings From a DIAlogue Session at DIA Europe 2018

The EU is a member of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), and therefore adopts the ICH Guidelines, including the ICH M3 Guideline on Nonclinical Safety Studies. Following the 2016 incident in France with BIA 10-2474, and in light of the substantial evolvement of how early clinical development has been undertaken during the last 10 years, for example, conducting integrated (FIH) studies that include multiple parts (eg, single ascending doses, multiple ascending doses, food effect), EMA decided to update the existing 2007 FIH guideline. The key revisions to the 2007 guideline, now titled "Guideline on Strategies to Identify and Mitigate Risks for First-in-Human and Early Clinical Trials With Investigational Medicinal Products," include additional information. The revision reinforces the importance and impact of pharmacologic data, which supports the intended efficacy of the compound, risk assessment, and protocol design. The updates, effective February 2018, are intended to provide additional guidance and clarity for Sponsors developing FIH and early phase clinical research programs, and ultimately support subject safety. At the 2018 DIA Europe Annual Meeting in Basel, Switzerland, European regulators, industry representatives and academics convened a DIAlogue Session on April 17 to discuss how the revised 2017 guideline is being applied, and to establish recommendations for its application. Using two case studies as examples, the session participants discussed the nonclinical and clinical considerations for applying the newly revised recommendations, and interacted with a panel including regulators and industry representatives. The proceedings from this session reflect practical considerations for the implementation of the revised guideline.

MON-102 Nonclinical Development of AZP-531 (Livoletide): A Peptide Analog of Unacylated Ghrelin for the Treatment of Hyperphagia in Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is a rare and complex endocrine disease characterized by hyperphagia and abnormal food-related behaviors that contribute to severe morbidity and early mortality and to a significant burden on patients and caregivers. Hyperghrelinemia may be involved in the underlying mechanisms of hyperphagia. While ghrelin, the most potent appetite-stimulating hormone, is present at higher concentrations in the plasma of PWS patients compared to age-matched controls, detailed studies of ghrelin and unacylated ghrelin (UAG) reveal a relative deficit of UAG in hyperphagic individuals. UAG is a 28-amino-acid peptide that does not bind the growth hormone secretagogue receptor (GHSR), in contrast to acylated ghrelin. UAG has intrinsic activities that often counteract effects of ghrelin, and exerts its actions through a GHSR-independent mechanism. Livoletide is a cyclic 8-amino-acid analogue of UAG with improved plasma stability and pharmacokinetics. The objective of this nonclinical safety program was to support the clinical development of livoletide including a pivotal Phase 2b/3 clinical trial in patients with PWS. The program was designed to define the safety pharmacology and the chronic toxicologic and toxicokinetic profile, and identify parameters for clinical monitoring of potential adverse effects. Genotoxicity, safety pharmacology, reproductive toxicity, and repeat-dose 13-week toxicology studies were all completed. In the in vivo studies, livoletide was administered subcutaneously consistent with the clinical route of delivery. Livoletide was not cytotoxic or genotoxic. Safety pharmacology studies indicated no treatment-related effects on major physiological systems. Results from preliminary embryo-fetal developmental toxicity studies in rat and rabbit indicated that livoletide at high multiples of the anticipated human exposure is not associated with adverse maternal toxicity, embryo-fetal toxicity or teratogenic potential when administered throughout the period of organogenesis. Repeat-dose toxicity studies of up to 13 weeks’ duration in rats and dogs demonstrated that livoletide is very well-tolerated, with no evidence of systemic toxicity. Cumulative data indicated that livoletide has a wide safety margin relative to planned clinical exposures. The highest chronic doses tested were 75 mg/kg in rat and 30 mg/kg in dog; these were considered to be the NOAELs. These dose levels provided AUC values of ≥50-fold the intended clinical systemic exposure (~1200 ng·h/mL). No anti-livoletide antibodies were detected in any of the toxicology studies. These results confirm the favorable long-term safety profile of livoletide and support the subcutaneous administration of the highest anticipated human clinical dose in the Phase 2b/3 study.

Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison

IntroductionIrwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a “state-of-the-art” assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. MethodsThis assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. ResultsOur survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DiscussionOur survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

Toxicologic Pathology Forum*: Opinion on Sexual Maturity and Fertility Assessment in Long-tailed Macaques ( Macaca fascicularis) in Nonclinical Safety Studies.

If nonhuman primates represent the only relevant species for nonclinical safety evaluation of biotechnology-derived products, male and female fertility effects can be assessed in repeat dose toxicity studies given that sexually mature monkeys are used. This opinion piece provides recommendations for determining sexual maturity and when/how fertility assessments should be conducted in the cynomolgus monkey. Male sexual maturity should be proven by presence of sperm in a semen sample, female sexual maturity by at least two consecutive menstrual bleedings. As per regulatory guidance, default parameters for an indirect assessment of fertility in both sexes are reproductive organ weight and histopathology. Beyond default parameters, daily vaginal swabs are recommended for females, and for males, it is recommended to include blood collections (for potential analysis of reproductive hormones), testis volume sonography, and collection of frozen testis samples at necropsy. Only if there is a cause for concern, blood collection for potential reproductive hormone analysis should be conducted in females and semen analysis in males. In principle, adverse reproductive effects can be detected within 4 weeks of test article administration, depending on study design and reproductive end point chosen. Therefore, there are options for addressing reproductive toxicity aspects with studies of less than 3 months dosing duration. *This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the authors. It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Pilot study on functional performance and acceptability of two new synthetic adhesive male condoms (Wondaleaf): a randomized cross-over trial

ObjectivesThis study compared the condom failure rate, safety and acceptability of two new synthetic adhesive male condoms, Wondaleaf-Cap® (WLC) and Wondaleaf-On-Man® (WLM), with a marketed latex external condom Durex®-Together (DT). Study designWe enrolled healthy married, monogamous, heterosexual condom users in a randomized controlled, cross-over, pilot trial in Malaysia. We randomized participants to six groups with different condom use-orders of the experimental WLC and WLM and control latex condom for four episodes for vaginal sex over 1 month for each condom type. We summarized the clinical and non-clinical failure rate, safety and acceptability of each condom type using descriptive statistics. We tested differences in condom failure and acceptability using generalized estimating equations and repeated measure ANOVA respectively. ResultsWe screened 75 couples and randomized 50 eligible couples. Two couples withdrew before receiving any condom. The remaining used 576 condoms with 192 uses for each condom variant. Clinical failure rates of WLC, WLM and DT were 1.04%, 0% and 0.52%, respectively. Non-clinical failure rates of WLC, WLM and DT were 2.08%, 3.12% and 1.04%, respectively. Removal was found more painful with Wondaleaf products than the DT. Preferences of participants for WLC, WLM and DT were 33.3%, 29.2% and 25%, respectively. Overall, WLC and DT had greater acceptances among male participants than WLM. ConclusionResults of this pilot study support that use of synthetic adhesive male condoms is associated with failure rates similar to those seen with existing latex, and with greater acceptability. A larger study to ascertain non-inferiority is underway. ImplicationsThe availability of synthetic adhesive male condoms may increase the acceptability of condom use. However, removal pain and clinical performance requires further study.

CDER Experience With Juvenile Animal Studies for CNS Drugs.

A survey was undertaken to evaluate juvenile animal studies conducted for drug applications reviewed by the Center for Drug Evaluation and Research between 2009 and 2014. Some conclusions about the nonclinical pediatric safety assessment based on studies performed in support of central nervous system-active compounds are presented here. A total of 44 completed studies from 32 New Drug Applications submitted to the Divisions of Psychiatry and Neurology Products were evaluated. Data on animal species and age range used, endpoints evaluated, and outcomes included in labeling were analyzed. Of the drugs evaluated, all but one had studies conducted in rats. In some cases, a second study in a nonrodent species (dog) was also conducted. Indices of growth and development and standard general toxicity parameters were included in all of the studies. Expanded neurohistopathology evaluations, bone mineral density measurements, and reproductive and neurobehavioral functional assessments were also generally carried out. A variety of neurological and neurobehavioral tests were employed. In the majority of rat studies, the potential for long-term cognitive impairment was evaluated using a complex water maze. Juvenile animal studies provided safety information considered relevant to drug use in children and that was included in labeling for 78% of the applications surveyed. The most commonly reported findings in labeling were for neurobehavioral effects, including changes in locomotor activity, auditory startle habituation, and learning and memory. Of the studies described in labeling with neurobehavioral effects, 54% found these effects to be persistent and to provide evidence of developmental neurotoxicity.

Evolving the Role of Discovery-focused Pathologists and Comparative Scientists in the Pharmaceutical Industry.

GlaxoSmithKline has recently made significant organizational changes to its nonclinical safety, drug metabolism and pharmacokinetic, and laboratory animal science/veterinary functions, with the goal to increase our focus on scientific partnership with the discovery part of the organization. One specific change was bringing together pathologists and comparative medicine veterinarians and scientists into a single functional unit. We describe our early activities (assessing our capabilities and gaps, external benchmarking, listening to our discovery partners, redesigning some of our working practices) aimed at implementing these changes. In addition, early on we held a Discovery Engagement Workshop attended by all pathologists and comparative medicine veterinarians and scientists, as well as selected discovery scientists. The purpose of this workshop was to share learnings from the above activities and devise plans aimed at achieving our overall goal of functional integration: driving pathobiology expertise into drug discovery and increasing the human (translational) relevance of experimental data. This review describes the new organizational structure, the workshop activities, and implementation plans; updates our progress; and considers the opportunity for a pan-industry network of discovery-focused pathologists and comparative medicine veterinarians and scientists.

Toxicologic Pathology Forum: Current Status on the Use of Animal Models of Human Disease in the Pharmaceutical Industry in Japan in Nonclinical Safety Assessment-Opinion Paper.

In nonclinical safety studies for new drug development, healthy animals have been commonly used. However, in some cases, the use of animal models of human disease is considered to be more favorable in evaluating risks in patients. To elucidate the current status of the use of animal models for nonclinical safety assessment, an internal questionnaire from the Japan Pharmaceutical Manufacturers Association and surveys (questionnaire period: August 27 to September 30, 2015) of both common technical documents and review reports of approved drugs (approval period: May 1999 to May 2017) disclosed by the Pharmaceutical and Medical Devices Agency were conducted. Although there were some concerns and limitations raised, the survey results revealed that animal models have been used in nonclinical safety assessment on a case-by-case basis and that nonclinical safety studies using animal models were included in the data packages of several approved drugs in Japan. The survey results also revealed that nonclinical safety studies using animal models have become more frequent in the past few years. In almost all cases, useful information, such as signs of toxicity under disease conditions and mechanisms of toxic change, was obtained from the results of nonclinical studies using animal models. Note: This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the author(s). It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Histopathological Findings in Cynomolgus Macaques ( Macaca fascicularis) Consistent with Secondary Immunological Reaction to Biotherapeutics with an Emphasis on the CNS and Eye.

Biotherapeutics are pharmaceutical products derived from or synthesized by biological systems. Such molecules carry the potential for immunogenicity which may lead to adverse immune responses. The cynomolgus macaque ( Macaca fascicularis) is the species of choice in nonclinical safety assessment of biotherapeutics. The main aim of this study was to confirm whether mononuclear cell infiltrates at specific locations represent a generic effect of biotherapeutics, and therefore the result of their immunogenicity. Following a review of microscopic findings in studies conducted over a 10-year period at one test facility, 15% of biotherapeutics were reported to have such findings. The most commonly affected site was the choroid plexus and less frequently the meninges and ciliary body. The reporting of such findings as test article-related becomes more subjective as the severity and incidence decreases. To assess the accuracy of such associations, a mathematical approach was employed to determine the probability of obtaining the observed results by chance. There was good agreement between this approach and the original findings. In addition to an increased number and size of mononuclear cell infiltrates in the brain, biotherapeutic administration was strongly associated with the presence of plasma cells and eosinophils.