Preclinical Safety Testing Research Articles

Preclinical Analysis of JAA-F11, a Specific Anti–Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper “Humanization of JAA-F11, a Highly Specific Anti–Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis” (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

Ion Channels in Drug Discovery and Safety Pharmacology.

Ion channels are membrane proteins involved in almost all physiological processes, including neurotransmission, muscle contraction, pace-making activity, secretion, electrolyte and water balance, immune response, and cell proliferation. Due to their broad distribution in human body and physiological roles, ion channels are attractive targets for drug discovery and safety pharmacology. Over the years ion channels have been associated to many genetic diseases ("channelopathies"). For most of these diseases the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a number of patients. The search for the development of new and more specific therapeutic approaches is therefore strongly pursued. At the same time acquired channelopathies or dangerous side effects (such as proarrhythmic risk) can develop as a consequence of drugs unexpectedly targeting ion channels. Several noncardiovascular drugs are known to block cardiac ion channels, leading to potentially fatal delayed ventricular repolarization. Thus, the search of reliable preclinical cardiac safety testing in early stage of drug discovery is mandatory. To fulfill these needs, both ion channels drug discovery and toxicology strategies are evolving toward comprehensive research approaches integrating ad hoc designed in silico predictions and experimental studies for a more reliable and quick translation of results to the clinic side.Here we discuss two examples of how the combination of in silico methods and patch clamp experiments can help addressing drug discovery and safety issues regarding ion channels.

Rabbit a reliable human like model in preclinical cardiac electrophysiological safety testing

Rabbit a reliable human like model in preclinical cardiac electrophysiological safety testing

Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury.

Cervical spinal cord injury (SCI) remains an important research focus for regenerative medicine given the potential for severe functional deficits and the current lack of treatment options to augment neurological recovery. We recently reported the preclinical safety data of a human embryonic cell‐derived oligodendrocyte progenitor cell (OPC) therapy that supported initiation of a phase I clinical trial for patients with sensorimotor complete thoracic SCI. To support the clinical use of this OPC therapy for cervical injuries, we conducted preclinical efficacy and safety testing of the OPCs in a nude rat model of cervical SCI. Using the automated TreadScan system to track motor behavioral recovery, we found that OPCs significantly improved locomotor performance when administered directly into the cervical spinal cord 1 week after injury, and that this functional improvement was associated with reduced parenchymal cavitation and increased sparing of myelinated axons within the injury site. Based on large scale biodistribution and toxicology studies, we show that OPC migration is limited to the spinal cord and brainstem and did not cause any adverse clinical observations, toxicities, allodynia, or tumors. In combination with previously published efficacy and safety data, the results presented here supported initiation of a phase I/IIa clinical trial in the U.S. for patients with sensorimotor complete cervical SCI. Stem Cells Translational Medicine 2017;6:1917–1929

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo

Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear-responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC-Pad formulations in human and porcine sera, along with the nanopharmaceutical-mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad-PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA.

A role for in vitro disease models in the landscape of preclinical cardiotoxicity and safety testing

A role for in vitro disease models in the landscape of preclinical cardiotoxicity and safety testing

In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks

Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography–high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC50 values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC50 value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed.

Cardiac Cell Therapies for the Treatment of Acute Myocardial Infarction: A Meta-Analysis from Mouse Studies

Aims: Stem cell-based regenerative therapies for the treatment of ischemic myocardium are currently a subject of intensive investigation. A variety of cell populations have been demonstrated to be safe and to exert some positive effects in human Phase I and II clinical trials, however conclusive evidence of efficacy is still lacking. While the relevance of animal models for appropriate pre-clinical safety and efficacy testing with regard to application in Phase III studies continues to increase, concerns have been expressed regarding the validity of the mouse model to predict clinical results. Against the background that hundreds of preclinical studies have assessed the efficacy of numerous kinds of cell preparations - including pluripotent stem cells - for cardiac repair, we undertook a systematic re-evaluation of data from the mouse model, which initially paved the way for the first clinical trials in this field. Methods and Results: A systematic literature screen was performed to identify publications reporting results of cardiac stem cell therapies for the treatment of myocardial ischemia in the mouse model. Only peer-reviewed and placebo-controlled studies using magnet resonance imaging (MRI) for left ventricular ejection fraction (LVEF) assessment were included. Experimental data from 21 studies involving 583 animals demonstrate a significant improvement in LVEF of 8.59%+/- 2.36; p=.012 (95% CI, 3.7–13.8) compared with control animals. Conclusion: The mouse is a valid model to evaluate the efficacy of cell-based advanced therapies for the treatment of ischemic myocardial damage. Further studies are required to understand the mechanisms underlying stem cell based improvement of cardiac function after ischemia.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.

Preclinical safety testing for morcellation and extraction for an endobag with sealable ports: in vitro pilot study

Electromechanical power morcellation is an important tool of modern laparoscopy. Recent reports on the spread of previously undetected malignancy by power morcellation indicate the need for additional protective devices to reduce this risk. We conducted a study to obtain the first data concerning the safety of an endobag with three closable ports during morcellation and subsequent bag extraction under in vitro conditions, mimicking the settings in our operating theater. The second purpose of the study was to establish a minimal width of the skin incision necessary to safely extract the sealed bag after morcellation. The morcellation test was carried out on 11 stained porcine muscle tissue samples with one additional sample as a control. The insufflation pressure was set at 12mmHg. After filling the endobag with blue dye solution, an additional extraction test was conducted by pulling the closed bag through a template with apertures of various diameters. For each opening, a series of ten bag extractions was carried out. No loss of solid material or fluid was recorded during the morcellation test. The extraction test showed a loss of fluid for template openings smaller than 18mm. The force necessary to extract the bag was inversely related to the width of the aperture. The data suggest that under the evaluated conditions, the use of a closable morcellation bag can considerably improve the patient's safety during morcellation. Further studies are necessary to evaluate the influence of the bag on operating time, intervention costs and complications.

Gene Therapy for Hemoglobinopathies: Tremendous Successes and Remaining Caveats

β -globinopathies (sickle-cell anemia and β-thalassemia) are the most common monogenic disorders worldwide that are potentially amenable to gene therapy. Proof-of-concept studies done 15 years ago, which showed correction of murine models of hemoglobinopathies, have gone through the rigors of preclinical safety and efficacy testing, and are now showing clinical promise both in β-thalassemia and sickle-cell anemia. Lentivirus-mediated gene replacement therapy with erythroid-specific expression of the globin gene appears safe thus far.

The Use of Minipigs for Preclinical Safety Assessment by the Pharmaceutical Industry: Results of an IQ DruSafe Minipig Survey.

The use of minipigs in preclinical safety testing of pharmaceuticals is considered an alternative to the more traditional dog and nonhuman primate (NHP) nonrodent species. Substantial evidence exists to suggest that the anatomy, physiology, and biochemistry of minipigs are similar enough to humans to consider them as valid nonrodent models for pharmaceutical safety testing. Since the utilization of minipigs was last assessed over 5 years ago, the Preclinical Safety Leadership Group (DruSafe) of the International Consortium for Innovation and Quality in Pharmaceutical Development conducted this survey to provide an updated assessment of the utility, perceived value, and impediments to the use of minipigs in preclinical safety testing. Of the 32 participating members of DruSafe, 15 responded to the survey representing both large and small companies. Respondents indicated that the minipig has been utilized mostly for short-term safety assessment studies with dermal, oral, and parenteral routes of administration. Minipigs are widely accepted as appropriate models for cardiovascular assessments and have been used to a limited extent for reproductive toxicology testing. Overall responses indicated that safety testing for large molecules using this species is relatively low due to a lack of background data, reagents or biomarkers, concerns regarding immune system characterization and poor suitability for developmental toxicity assessments. Most companies utilized contract research organizations for definitive safety toxicity assessment studies. Conclusions of this survey indicate that minipig is an acceptable nonrodent species largely limited to studies using small molecules, primarily dermal products, and results are comparable to those reported 5 years ago.

Development of Preclinical Biomarkers Predictive of Safety of Vaginal Microbicides for the Prevention of HIV

Human immunodeficiency virus (HIV) is transmitted predominantly through sexual intercourse. Research suggeststhat the vaginal epithelium acts as a barrier to HIV transmission, but this epithelium can be disrupted, leading toHIV infection and other sexually transmitted infections. Topical microbicides are considered an effective strategy forHIV prevention, and several candidate drugs are currently in clinical trials. However, significant concerns about drugadherence, efficacy, and safety must be addressed in order to develop a safe and effective microbicide. This paper willprovide an overview of the field and strategies to overcome some of the obstacles facing microbicide development,and briefly discuss a research project that focused on one aspect of preclinical microbicide safety testing.

The Use of Minipig in Drug Discovery and Development: Pros and Cons of Minipig Selection and Strategies to Use as a Preferred Nonrodent Species.

The pig was introduced more than 20 years ago in drug development following attempts of finding a species that shares better homology with human than the dog, based on biophysiological parameters. However, miniaturization, standardized breeding, and health status control were required before the pig could find a broader than niche application in pharmaceutical industry. During the years of experience with minipigs in pharmaceutical research and the science evolving rapidly, the selection of a nonrodent animal species for preclinical safety testing became primarily driven by pharmacological (target expression homologous function), pharmacokinetic, and biophysiological considerations. This offered a broad field of application for the minipig, besides the well-established use in dermal projects in all areas of drug development but also in novel approaches including genetically modified animals. In this article, we look at recent approaches and requirements in the optimal selection of a nonrodent model in pharmaceutical development and critically ask how good a choice the minipig offers for the scientist, how did the testing environment evolve, and what are the key requirements for a broader use of the minipig compared to the other well-established nonrodent species like dog or monkey.

Gene therapy for cancer: regulatory considerations for approval.

The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA.

Highly bioavailable curcumin (Theracurmin): Its development and clinical application

Curcumin is a polyphenol with antioxidant and anti-inflammatory properties. It is highly lipophilic and sparingly soluble in water and very little is absorbed when it is ingested; therefore, improving its absorbability is a major priority. We developed a highly bioavailable curcumin called Theracurmin using submicron particle formation and surface controlled technology. In human study, the area under the blood concentration–time curve (AUC) after oral administration of Theracurmin was 27-fold higher than that of commercially available curcumin. Preclinical safety tests were conducted and no adverse effects were confirmed. The effects of Theracurmin on cancer (lung, pancreatic, and prostate), cardiovascular disease (heart disease), vascular function (arterial stiffness and central blood pressure), and bone and cartilage (knee osteoarthritis) were evaluated by collaborating with universities and medical institutions. In this paper, we present the development of Theracurmin and its effects in an animal model as well as in human clinical studies.

Preclinical safety testing of an affinity-optimised MAGE-A10 T cell receptor for adoptive T cell therapy

Meeting abstracts Preclinical safety testing for adoptive immunotherapies utilising T cell receptor-(TCR) engineered T cells relies on stringent target validation and a robust pre-clinical specificity testing strategy. Specific safety concerns include on-target off-tumour toxicity and off-target

Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies.

Despite enormous advances in translational biomedical research, there remains a growing demand for improved animal models of human disease. This is particularly true for diseases where rodent models do not reflect the human disease phenotype. Compared to rodents, pig anatomy and physiology are more similar to humans in cardiovascular, immune, respiratory, skeletal muscle, and metabolic systems. Importantly, efficient and precise techniques for genetic engineering of pigs are now available, facilitating the creation of tailored large animal models that mimic human disease mechanisms at the molecular level. In this article, the benefits of genetically engineered pigs for basic and translational research are exemplified by a novel pig model of Duchenne muscular dystrophy and by porcine models of cystic fibrosis. Particular emphasis is given to potential advantages of using these models for efficacy and safety testing of targeted therapies, such as exon skipping and gene editing, for example, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system. In general, genetically tailored pig models have the potential to bridge the gap between proof-of-concept studies in rodents and clinical trials in patients, thus supporting translational medicine.

Mechanistic Insights in TNF Signaling and Drug-Induced Liver Injury: Towards a Predictive Preclinical Toolbox

There is an increasing body of evidence that various drugs can sensitize hepatocytes to TNF-mediated killing. This interaction is likely due to perturbations of the normal hepatocyte physiology that triggers a pro-apoptotic TNFR pathway when TNF is present in the system. We have focussed on the effects of DILI compounds on endoplasmic reticulum (ER) stress as well as the Nrf2-mediated oxidative stress adaptive toxicity pathways and how these two pathways converge with adverse TNFR signaling. Moreover, we have deepened our understanding on effects of DILI compounds on the TNFR-mediated NF-kappa B regulation. We have integrated primary human hepatocyte transcriptomics data with live cell imaging adaptive stress response GFP-reporter data to unravel in detail the interaction between different adaptive stress pathways and TNF/drug cytotoxic synergy. Moreover we have applied RNA interference screening to identify key modulators of these signaling pathways that define this synergistic toxic interaction. Our data provide suggestions on the toolbox components that can be used in a preclinical drug safety testing phase to assess safety liabilities for a drug/cytokine interplay in the development of DILI. This work is part of the MIP-DILI project supported by the Innovative Medicines Initiative (grant agreement n° 115336), and the FP7 SEURAT-1 DETECTIVE project (grant agreement 266838).

Extended Preclinical Safety, Efficacy and Stability Testing of a Live-attenuated Chikungunya Vaccine Candidate.

We recently described a new, live-attenuated vaccine candidate for chikungunya (CHIK) fever, CHIKV/IRES. This vaccine was shown to be well attenuated, immunogenic and efficacious in protecting against CHIK virus (CHIKV) challenge of mice and nonhuman primates. To further evaluate its preclinical safety, we compared CHIKV/IRES distribution and viral loads in interferon-α/β receptor-incompetent A129 mice to another CHIK vaccine candidate, 181/clone25, which proved highly immunogenic but mildly reactive in human Phase I/II clinical trials. Compared to wild-type CHIK virus, (wt-CHIKV), both vaccines generated lower viral loads in a wide variety of tissues and organs, including the brain and leg muscle, but CHIKV/IRES exhibited marked restrictions in dissemination and viral loads compared to 181/clone25, and was never found outside the blood, spleen and muscle. Unlike wt-CHIKV, which caused disrupted splenic architecture and hepatic lesions, histopathological lesions were not observed in animals infected with either vaccine strain. To examine the stability of attenuation, both vaccines were passaged 5 times intracranially in infant A129 mice, then assessed for changes in virulence by comparing parental and passaged viruses for footpad swelling, weight stability and survival after subcutaneous infection. Whereas strain 181/clone25 p5 underwent a significant increase in virulence as measured by weight loss (from <10% to >30%) and mortality (from 0 to 100%), CHIKV/IRES underwent no detectible change in any measure of virulence (no significant weight loss and no mortality). These data indicate greater nonclinical safety of the CHIKV/IRES vaccine candidate compared to 181/clone25, further supporting its eligibility for human testing.