Nonclinical Safety Research Articles

Biodistribution and safety of a live attenuated tetravalent dengue vaccine in the cynomolgus monkey.

The first licensed dengue vaccine is a recombinant, live, attenuated, tetravalent dengue virus vaccine (CYD-TDV; Sanofi Pasteur). This study assessed the biodistribution, shedding, and toxicity of CYD-TDV in a non-human primate model as part of the nonclinical safety assessment program for the vaccine. Cynomolgus monkeys were given one subcutaneous injection of either one human dose (5log10 CCID50/serotype) of CYD-TDV or saline control. Study endpoints included clinical observations, body temperature, body weight, food consumption, clinical pathology, immunogenicity, and post-mortem examinations including histopathology. Viral load, distribution, persistence, and shedding in tissues and body fluids were evaluated by quantitative reverse transcriptase polymerase chain reaction. The subcutaneous administration of CYD-TDV was well tolerated. There were no toxicological findings other than expected minor local reactions at the injection site. A transient low level of CYD-TDV viral RNA was detected in blood and the viral genome was identified primarily at the injection site and in the draining lymph nodes following immunization. These results, together with other data from repeat-dose toxicity and neurovirulence studies, confirm the absence of toxicological concern with CYD-TDV and corroborate clinical study observations.

Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RA-Deficiency.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP. To facilitate the translation of PMT therapy to human hPAP patients, a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human CSF2RA-cDNA driven from an EF1α short promoter (Lv.EFS.CSF2RAcoop), and a series of nonclinical efficacy and safety studies were performed in cultured macrophage cell lines and primary human cells. Studies in cytokine-dependent Ba/F3 cells demonstrated efficient transduction, vector-derived CD116 expression proportional to vector copy number, and GM-CSF-dependent cell survival and proliferation. Using a novel cell line constructed to express a normal GM-CSF receptor β subunit and a dysfunctional α subunit (due to a function-altering CSF2RAG196R mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RAcoop transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RAcoop transduction of healthy primary CD34+ cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RAcoop reconstituted GM-CSF receptor α expression, restoring GM-CSF signaling in hPAP macrophages, and had no adverse effects in the intended target cells, thus supporting testing of PMT therapy of hPAP in humans.

Identification of appropriate QTc formula in beagle dogs for nonclinical safety assessment

A number of drugs belonging to different therapeutic classes cause increase in QT interval duration, and this change has been associated with ventricular arrhythmias. Investigation of changes in QT intervals in toxicity studies in dogs is therefore of potential value. Estimation of a direct effect of drugs on the duration of the QT interval can be confused by drug-induced increases in heart rate. The objective of this evaluation was to identify an appropriate correction formula by comparing different formulae that could appropriately correct changes in QT interval in conscious beagle dogs in toxicology studies. Most commonly used QTc (QT correction) formulae are derived from human observations, like Bazett's formula and thus are not applicable for other species like dogs, where the normal values of heart rate is higher compared to humans. Using our historical data, we have established and compared different correction formulas and found that Van de Water's formula is the most appropriate for dog under conditions stated. However, there is no universally accepted formula for QTc calculation in dogs, and hence each organization should have its own formula, based on the analysis of data obtained from the strain used in its own experimental conditions.

Non-clinical Safety and Efficacy of an AAV2/8 Vector Administered Intravenously for Treatment of Mucopolysaccharidosis Type VI

In vivo gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. We recently demonstrated that AAV8-mediated liver gene transfer is effective in animal models of mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disease that is caused by arylsulfatase B (ARSB) deficiency. In preparing for a first-in-human trial, we performed non-clinical studies to assess the safety of intravenous administrations of AAV2/8.TBG.hARSB produced under good manufacturing practice-like conditions. No toxicity was observed in AAV-treated mice, except for a transient increase in alanine aminotransferase in females and thyroid epithelial hypertrophy. AAV2/8.TBG.hARSB biodistribution and expression confirmed the liver as the main site of both infection and transduction. Shedding and breeding studies suggest that the risk of both horizontal and germline transmission is minimal. An AAV dose-response study in MPS VI mice was performed to define the range of doses to be used in the clinical study. Overall, these data support the non-clinical safety and efficacy of AAV2/8.TBG.hARSB and pave the way for a phase I/II clinical trial based on intravascular infusions of AAV8 in patients with MPS VI.

Nonclinical safety assessment of SPX-101, a novel peptide promoter of epithelial sodium channel internalization for the treatment of cystic fibrosis

Background: ENaC inhibition has long been an attractive therapeutic target for the treatment of cystic fibrosis. However, previous attempts at developing ENaC inhibitors have been unsuccessful due to complications arising from systemic circulation of the compounds. Here, we describe the preclinical toxicology assessment of a new inhaled peptide promoter of ENaC internalization delivered as a nebulized aerosol.Methods: Preclinical assessment of SPX-101 safety was determined using an in vitro hERG assay, bolus injection of SPX-101 in a canine cardiovascular and respiratory safety pharmacology model and 28-day inhalation toxicology studies of nebulized drug in rats and dogs.Results: SPX101 had no effects on the respiratory, cardiac or central nervous systems. The 28-day inhalation toxicology studies of nebulized SPX-101 in rats and dogs revealed no drug-related adverse events. Plasma levels of SPX-101 peaked less than 1 h after the end of treatment in rats and were below the limit of detection in canine models.Conclusions: SPX-101, a novel peptide promoter of ENaC internalization, elicited no adverse effects at doses up to the MFD and in excess of the highest preclinical efficacious and expected clinical doses. In contrast to channel blockers like amiloride and derivative small molecules, SPX-101 does not achieve significant systemic circulation, thus doses are not limited due to toxic side effects like hyperkalemia and weight loss.

Abstract DDT01-04: Discovery and development of H3B-6545: A novel, oral, selective estrogen receptor covalent antagonist (SERCA) for the treatment of breast cancer

Abstract Mutations in the estrogen receptor (ER) are detected in up to 30% of patients that initially respond but subsequently relaps to anti-endocrine therapies. ERα mutations, likely through constitutively activating ERα, can functionally confer resistance to existing classes of endocrine therapies. Current endocrine therapies are only partially effective in the ERα mutant setting and a significant proportion of endocrine-therapy resistant breast cancer metastases continue to remain dependent on ERα signaling for growth/survival indicating a critical need to develop the next generation of ERα antagonists that can overcome ERα wild-type and mutant activity. Here we describe a novel series of compounds with a unique mode of inhibition that potently target both wild-type and mutant ERα. These compounds are Selective Estrogen Receptor Covalent Antagonists (SERCAs) that inactivate the estrogen receptor by targeting a cysteine that is not present in other nuclear hormone receptors, leading to a unique biological and activity profile differentiated from Selective Estrogen Receptor Modulators (SERMs) and Selective Estrogen Receptor Degraders (SERDs). Using structure-based drug design approaches we have identified a first-in-class clinical candidate, H3B-6545. H3B-6545 is a highly selective small molecule that potently antagonizes wild-type and mutant ERα in biochemical and cell based assays. In vitro comparisons with standard of care and other experimental agents confirm increased cell potency of H3B-6545 under continuous as well as washout treatment conditions. In vivo, once daily oral dosing of H3B-6545 shows potent activity and superior efficacy to fulvestrant in the MCF-7 xenograft model with maximal antitumor activity at doses >10x below the maximum tolerated dose in mice. In addition, H3B-6545 shows superior antitumor activity to tamoxifen and fulvestrant in patient derived xenograft models of estrogen receptor positive breast cancer including models carrying ERα mutations In non-clinical safety studies in rat and monkeys, H3B-6545 is well tolerated across a broad dose range and at exposures that significantly exceed those required for efficacy in mouse xenograft models. In summary, H3B-6545 is a first-in-class, orally available and potent selective estrogen receptor covalent antagonist with a compelling preclinical efficacy and safety profile that is being developed for the treatment of breast cancer. Citation Format: Peter G. Smith, Xiaoling Puyang, Craig Furman, Guo Zhu Zheng, Deepti Banka, Michael Thomas, Vanitha Subramanian, Sean Irwin, Nicholas Larsen, Benjamin Caleb, Craig Karr, Jeremy Wu, Morgan O’Shea, Joyce Yang, Allison Davis, Amy Kim, Nathalie Rioux, Victoria Rimkunas, Huilan Yao, Crystal MacKenzie, Pavan Kumar, Sherri Smith, Sean Eckley, Andrew Hart, George Lai, Christopher Rowbottom, Peter Fekkes, Silvia Buonamici, Dominic Reynolds, Lihua Yu, Tarek Sahmoud, Markus Warmuth, Lorna Mitchell, Ping Zhu, Manav Korpal. Discovery and development of H3B-6545: A novel, oral, selective estrogen receptor covalent antagonist (SERCA) for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-04. doi:10.1158/1538-7445.AM2017-DDT01-04

Nonclinical safety of astilbin: A 4-week oral toxicity study in rats with genotoxicity, chromosomal aberration, and mammalian micronucleus tests

Astilbin is an active flavonoid compound isolated from Rhizoma Smilacis Glabrae. It has been widely used as an anti-hepatic, anti-arthritic, and anti-renal injury agent. However, its safety has not yet been established. The objective of this study was to evaluate 4-week repeated oral toxicity and genotoxicity of astilbin. We examined oral toxicity in Sprague-Dawley rats after daily oral administration of astilbin at 50, 150, and 500 mg/kg for 4 weeks. Negative control animals received the same volume of the solvent. Astilbin administration did not lead to death, body weight gain, food consumption, or adverse events. There were no significant differences in toxicity between the astilbin and control group; we observed no toxic effects on hematological or urinalysis parameters, biochemical values, organ weight, or histopathological findings. We assessed the genotoxicity of astilbin with the Ames test (TA97a, TA98, TA100, TA102, and TA1535), chromosomal aberration assay (using Chinese hamster ovary cells), and mammalian micronucleus test (in mice). We found no genotoxicity in any tested strains. The no-observed-adverse-effect level (NOAEL) for astilbin in the 4-week repeated oral toxicity study in rats was greater than 500 mg/kg body weight/day, regardless of gender. Results also suggested that astilbin does not have genotoxicity potential.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Mini-Review: Toxic Tendinopathy.

Toxic tendinopathy is a rare but reproducible complication in humans, given agents of four drug classes: aromatase inhibitors, fluoroquinolone antibiotics, glucocorticoids (long-term regimens), and statins. Toxic tendinopathy in humans has been linked less consistently to treatment with anabolic steroids, antiretroviral agents (mainly protease inhibitors), metalloproteinase inhibitors (MMPI), and isotretinoin. Classic drug-induced tendinopathies appear as "tendinosis" (i.e., progressive tendon degeneration without inflammation), although cases associated with aromatase inhibitors exhibit mainly tenosynovitis. Any tendon may be affected, but fluoroquinolones, glucocorticoids, and statins most frequently affect large load-bearing tendons in the lower limb, especially the calcaneal ("Achilles") tendon-which ruptures in approximately 30 to 40% of cases. The time to symptom onset ranges from days (fluoroquinolones) to weeks, months, or even years. The pathogenesis is incompletely understood, but proposed mechanisms include apoptosis of tenoblasts and tenocytes, deficient tenocyte function (leading to abnormal extracellular matrix maintenance and repair as well as disrupted intercellular signaling), and structural disintegration (via a combination of increased expression of lytic enzymes, lessened cholesterol content in cell membranes, and neoangiogenesis within highly ordered tendon tissue). Nonclinical safety assessment of therapeutic candidates in these drug classes should incorporate tendon routinely as a protocol-specified tissue for pathology evaluation.

Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant.

A multicenter phase I trial of the DNA-dependent protein kinase (DNA-PK) inhibitor M3814 in patients with solid tumors.

2556 Background: Agents that generate breaks in DNA are frequently used as cancer therapeutics. These agents induce different forms of DNA damage including double-strand breaks (DSBs), which are the most lethal if left unrepaired. M3814 targets tumor cell growth and survival by inhibiting DNA-PK, which is part of a critical DSB DNA damage repair mechanism. The purpose of the phase I, first in man trial was to evaluate the dose-limiting toxicity (DLT), establish a recommended phase II dose (RP2D), and assess the pharmacokinetic (PK) profile and single-agent clinical activity of M3814. Methods: Patients (pts) with potential aberrations in the DNA-damage and repair systemwere included. A standard 3+3 design was implemented with a starting dose of M3814 of 100 mg once daily, determined based on non-clinical safety. M3814 was given continuously and DLT was evaluated after 3 weeks. Throughout the trial rich PK sampling was taken. Tumor evaluation was performed every second cycle and treatment continued until progression, unacceptable toxicity, pt wish, or physician decision. Results: A total of 25 pts were enrolled at 6 dose levels (DL). Three pts were enrolled per DL, except at 300 and 400 mg BID where 9 and 4 pts were enrolled, respectively. At 300 mg BID one DLT (several low grade adverse events [AEs] lasting > 1 week) was seen. No DLTs were observed at 400 mg BID, which was declared as the RP2D; further dose escalation was not possible due to an impurity in the drug. An additional 6 pts were included at the RP2D. The most frequent AEs were nausea, vomiting, decreased appetite, constipation, diarrhea, pyrexia, fatigue, and rash, all seen in > 20% of pts. No pts discontinued due to AE and no grade 4 AEs were reported. Six pts (20%) had stable disease for at least 18 weeks; no pt had a partial remission. PK analysis demonstrated high variability of exposure with a tendency for skin rash in pts with the highest exposure. Conclusions: M3814 was found to be safe and tolerable at doses up to 400 mg BID, with limited single-agent activity in the studied population. Clinical evaluation of M3814 is ongoing in combination with radiotherapy as well as chemo-radiotherapy and planned in combination with chemotherapy. Clinical trial information: NCT02316197.

GCT1021-01, a first-in-human, open-label, dose-escalation trial with expansion cohorts to evaluate safety of Axl-specific antibody-drug conjugate (HuMax-Axl-ADC) in patients with solid tumors (NCT02988817).

TPS2605 Background: HuMax-AXL-ADC is an antibody-drug conjugate (ADC) composed of an Axl-specific human monoclonal immunoglobulin G1 (IgG1κ) conjugated via a protease-cleavable valine-citrulline linker to the microtubule disrupting agent monomethyl auristatin E (MMAE). In vivo, HuMax-AXL-ADC demonstrated therapeutic anti-tumor efficacy in patient-derived xenograft models representing a variety of solid cancers, including pancreas, thyroid, lung, esophageal, cervical cancers and malignant melanoma. The non-clinical safety profile and pharmacokinetics (PK) of a once every 3 weeks (1Q3W) dosing schedule were established in cynomolgus monkeys. Methods: The primary objective of this trial is to determine the MTD and to establish the safety profile of HuMax-AXL-ADC in a mixed population of patients with specified solid tumors: ovarian, cervical, endometrial, thyroid cancer, NSCLC, and malignant melanoma. The trial consists of two parts, a phase I dose escalation part and a phase IIa expansion part. The dose escalation part explores two different dosing regimens: the first investigates doses from 0.3 up to 2.8 mg/kg to be administered 1Q3W. The second investigates doses in the range of 0.45 to 1.4 mg/kg to be administered weekly for 3 weeks followed by one treatment-free week (3Q4W dosing schedule). The second arm has a delayed start to inform a safe starting dose: when at least 8 patients have been evaluated for dose limiting toxicities, the 1.5 mg/kg cohort of the 1Q3W arm has been declared safe, and the predicted PK parameters of the starting dose in the 3Q4W arm are below pre-defined limits, the 3Q4W arm will be initiated. The 1Q3W arm follows a modified Bayesian Continuous Reassessment Method including escalation with overdose control in up to 41 patients on up to 7 main and 4 intermediate dose levels while the 3Q4W arm is run as a standard 3+3 trial design on up to 5-6 dose levels. In the phase IIa expansion part, further safety and biological activity data will be generated in selected indications using cohorts of 22 patients (11+11 patients in each cohort applying the Simon’s two-stage design). Clinical trial information: NCT 02988817.

Capillary microsampling in nonclinical safety assessment: practical sampling and bioanalysis from a CRO perspective.

Microsampling has the 3R benefits of refining blood collection techniques while reducing the number of animals required for rodent safety assessment studies. There are significant scientific benefits of correlating study findings with systemic exposure and consequently, there is an industry drive to utilize microsampling in regulatory toxicology studies. This article will focus on capillary microsampling and will discuss the initial considerations before using capillary microsampling, study design and some practicalities of sample collection. From a bioanalytical perspective, the impact of different sample collection and storage workflows on the design of method validation experiments, the suitability of using microsampling for unstable analytes and aspects supporting analysis of peptides, large molecules and for biomarker applications will be discussed.

Toxicogenomic module associations with pathogenesis: a network-based approach to understanding drug toxicity

Despite investment in toxicogenomics, nonclinical safety studies are still used to predict clinical liabilities for new drug candidates. Network-based approaches for genomic analysis help overcome challenges with whole-genome transcriptional profiling using limited numbers of treatments for phenotypes of interest. Herein, we apply co-expression network analysis to safety assessment using rat liver gene expression data to define 415 modules, exhibiting unique transcriptional control, organized in a visual representation of the transcriptome (the ‘TXG-MAP’). Accounting for the overall transcriptional activity resulting from treatment, we explain mechanisms of toxicity and predict distinct toxicity phenotypes using module associations. We demonstrate that early network responses complement traditional histology-based assessment in predicting outcomes for longer studies and identify a novel mechanism of hepatotoxicity involving endoplasmic reticulum stress and Nrf2 activation. Module-based molecular subtypes of cholestatic injury derived using rat translate to human. Moreover, compared to gene-level analysis alone, combining module and gene-level analysis performed in sequence identifies significantly more phenotype-gene associations, including established and novel biomarkers of liver injury.

Translational science approach for assessment of cardiovascular effects and proarrhythmogenic potential of the beta-3 adrenergic agonist mirabegron

IntroductionTranslational assessment of cardiac safety parameters is a challenge in clinical development of beta-3 adrenoceptor agonists. The preclinical tools are presented that were used for assessing human safety for mirabegron. MethodsStudies were performed on electrical conductance at ion channels responsible for cardiac repolarization (IKr, IKs, Ito, INa, and ICa,L), on QT-interval, subendocardial APD90, Tpeak-end interval, and arrhythmia's in ventricular dog wedge tissue in vitro and on cardiovascular function (BP, HR, and QTc) in conscious dogs. ResultsIn conscious dogs, mirabegron (0.01–10mg/kg, p.o.) dose-dependently increased HR, reduced SBP but DBP was unchanged. Propranolol blocked the decrease in SBP and attenuated HR increase at 100mg/kg mirabegron. Mirabegron, at 30, 60, or 100mg/kg, p.o., had no significant effect on the QTc interval. In paced dog ventricular wedge, neither mirabegron nor metabolites M5, M11, M12, M14, and M16 prolonged QT, altered transmural dispersion of repolarization, induced premature ventricular contractions, or induced ventricular tachycardia. Mirabegron nor its metabolites inhibited IKr, IKs, Ito INa, or ICa,L at clinically relevant concentrations. DiscussionUp to exposure levels well exceeding human clinical exposure no discernible effects on ion channel conductance or on arrhythmogenic parameters in ventricular wedge resulted for mirabegron, or its main metabolites, confirming human cardiac safety findings. In vivo, dose-related increases in HR with effects markedly higher than seen clinically, was mediated in part by cross-activation of beta-1 adrenoceptors. This non-clinical cardiac safety test program therefore proved predictive for human cardiac safety for mirabegron.

Cardiac contractility: Correction strategies applied to telemetry data from a HESI-sponsored consortium

IntroductionQT has a long history of heart rate (HR) correction but limited investigations have been undertaken to assess the impact of cardiovascular parameters on left ventricular (LV) contractility in drug safety testing. Cardiac contractility is affected by preload (Cyon-Frank-Starling law), afterload (Anrep effect) and HR (Bowditch effect). We evaluated multi-parameter correction methods to help with dP/dtmax interpretation. MethodologyModeling was undertaken using data from dogs in single or double 4×4 Latin square studies. Correction models (16 fitting formulas×2 modeling approaches (universal and individualized)×2 correction approaches (linear or proportional)) were evaluated. 3D/2D cloud analysis of the beat-to-beat data for the control, pimobendan, and either itraconazole or atenolol groups were used to evaluate correlations between parameters and derive an optimal correction method. ResultsCardiac contractility (i.e., dP/dtmax) was best correlated to HR and systolic LV pressure with a correlation coefficient of 0.8. In decreasing order, dP/dtmin, mean arterial blood pressure (BP), systolic BP, diastolic BP, arterial pulse pressure and LV end diastolic pressure (LVEDP) showed a reduced correlation to dP/dtmax. Subject-specific models improved the correction by up to 14% when compared to universal correction models. The non-linear correction model was superior to the linear model. DiscussionResults suggest that the optimal correction formula for dP/dtmax would be subject-specific, non-linear and would include HR and LV systolic pressure. Correcting contractility for HR and systolic LV pressure may enhance data interpretation in non-clinical drug safety assessments. Similar correction methods could be evaluated for other species used in safety pharmacology.

Distribution of FcRn Across Species and Tissues

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I type molecule that binds to, transports, and recycles immunoglobulin G (IgG) and albumin, thereby protecting them from lysosomal degradation. Therefore, besides the knowledge of FcRn affinity, FcRn protein expression is critical in understanding the pharmacokinetic behavior of Fc-containing biotherapeutics such as monoclonal antibodies. The goal of this investigation was to achieve for the first time a comparative assessment of FcRn distribution across a variety of tissues and species. FcRn was mapped in about 20 tissues including placenta from human and the most frequently used species in non-clinical safety testing of monoclonal antibodies (mouse, rat, cynomolgus monkey). In addition, the FcRn expression pattern was characterized in two humanized transgenic mouse lines (Tg32 and Tg276) expressing human FcRn under different promoters, and in the severe combined immunodeficient (SCID) mouse. Consecutive sections were stained with specific markers, namely, anti-CD68 for macrophages and anti-von Willebrand Factor for endothelial cells. Overall, the FcRn expression pattern was comparable across species and tissues with consistent expression of FcRn in endothelial cells and interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. The human FcRn transgenic mouse Tg276 showed a different and much more widespread staining pattern of FcRn. In addition, immunodeficiency and lack of IgG in SCID mice had no negative effect on FcRn expression compared with wild-type mice.

2-Year animal carcinogenicity results for crisaborole, a novel phosphodiesterase 4 inhibitor for atopic dermatitis

BackgroundCrisaborole is a novel, topical nonsteroidal, anti-inflammatory, phosphodiesterase 4 (PDE4) inhibitor for the treatment of mild to moderate atopic dermatitis. ObjectiveAs part of a nonclinical safety testing program, these 2-year studies tested the carcinogenic potential of crisaborole. MethodsCrisaborole ointment, 2%, 5%, or 7%, was applied once daily topically to mice, and crisaborole was administered orally to rats at doses of 30, 100, or 300mg/kg/day for up to 104 weeks. Systemic exposure to crisaborole and its metabolites, moribundity/death, clinical signs, and tumor formation were assessed in each study. ResultsCrisaborole treatment was not tumorigenic in mice at any of the doses administered and did not increase the incidence of neoplastic or nonneoplastic microscopic lesions compared with controls. Oral administration of crisaborole at the high dose (300mg/kg/day) to female rats increased the incidence of treatment-related benign granular cell tumors in the distal reproductive tract (uterus with cervix and vagina) but did not cause moribundity/death. ConclusionCrisaborole was well tolerated and not tumorigenic in mice. It was not tumorigenic in male rats at 300mg/kg/day at exposures that were 3× the human area under the concentration-time curve (AUC24) and was nontumorigenic in female rats at 100mg/kg/day at exposures that were 1× the human AUC24.

Regulatory Forum Opinion Piece*: Use and Utility of Animal Models of Disease for Nonclinical Safety Assessment: A Pharmaceutical Industry Survey.

An Innovation and Quality (IQ) Consortium focus group conducted a cross-company survey to evaluate current practices and perceptions around the use of animal models of disease (AMDs) in nonclinical safety assessment of molecules in clinical development. The IQ Consortium group is an organization of pharmaceutical and biotechnology companies with the mission of advancing science and technology. The survey queried the utilization of AMDs during drug discovery in which drug candidates are evaluated in efficacy models and limited short-duration non-Good Laboratory Practices (GLP) toxicology testing and during drug development in which drug candidates are evaluated in GLP toxicology studies. The survey determined that the majority of companies used AMDs during drug discovery primarily as a means for proactively assessing potential nonclinical safety issues prior to the conduct of toxicology studies, followed closely by the use of AMDs to better understand toxicities associated with exaggerated pharmacology in traditional toxicology models or to derisk issues when the target is only expressed in the disease state. In contrast, the survey results indicated that the use of AMDs in development is infrequent, being used primarily to investigate nonclinical safety issues associated with targets expressed only in disease states and/or in response to requests from global regulatory authorities.

Inflammation and immunogenicity limit the utility of the rabbit as a nonclinical species for ocular biologic therapeutics

The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species. Such studies are often confounded by the development of anti-drug antibodies and severe ocular inflammation, the latter requiring studies to be terminated prematurely for animal welfare reasons. Notably, these confounding factors preclude the interpretation of safety. Nonclinical toxicology programs should be designed with consideration of ethical animal use and 3Rs principles (Replacement, Reduction and Refinement). The experience of several pharmaceutical sponsors, demonstrating that toxicology studies of ocular (intravitreal and topical ocular) biologic drug candidates in the rabbit are of limited interpretive value, calls into question the utility of such studies in this species and indicates that such studies should not be conducted.