Life ins't flat: Taking cancer biology to the next dimension

Classically, most cell culture experiments have been performed under adherent 2D conditions. Cells in the human body grow within an organized 3D matrix, surrounded by other cells. The behavior of individual cells is controlled through their interactions with their immediate neighbors and the extracellular matrix. The complex summation of these multiple signals determines whether a given cell undergoes differentiation, apoptosis, proliferation, or invasion. In 2D culture many of these complex interactions are lost. As a result, there are a growing number of studies which report differences in phenotype, cellular signaling, cell migration, and drug responses when the same cells are grown under 2D or 3D culture conditions. One potential application of these techniques is to anticancer drug discovery, which has long been hampered by the lack of good preclinical models. Compounds with good antitumor activity in 2D cell culture models often fail to translate into the clinic. Here we suggest that the response of cancer cells to drugs is determined in part by the 3D tumor microenvironment and discuss models to re-create the 3D tumor microenvironment in vitro. It is likely that the adoption of these and other 3D models will allow us to more closely re-create the behavior of the tumor in vivo which may lead to identifying better anticancer drug candidates at an earlier stage of development.     

Modeling opportunities in comparative oncology for drug development

Successful development of novel cancer drugs depends on well-reasoned scientific drug discovery, rigorous preclinical development, and carefully conceived clinical trials. Failure in any of these steps contributes to poor rates of approval for new drugs to treat cancer. As technological and scientific advances have opened the door to a variety of novel approaches to cancer drug discovery and development, preclinical models that can answer questions about the activity and safety of novel therapies are increasingly necessary. The advance of a drug to clinical trials based on information from preclinical models presupposes that the models convey informative data for future use in human patients with cancer. The study of novel cancer drugs using in vitro models is highly controllable, reproducible, relatively inexpensive, and linked to high throughput. However, these models fail to reproduce many of the complex features of human cancer. Mouse models address some of these limitations but have important biological differences from human cancer. The integration of studies using pet dogs with spontaneously occurring tumors as models in the development path can answer questions not adequately addressed in conventional models and is therefore gaining attention and interest in drug development communities. The study of novel cancer drugs in dogs with naturally occurring tumors allows drug assessment in a cancer that shares many fundamental features with the human cancer condition, and thus provides an opportunity to answer questions that inform the cancer drug development path in ways not possible in more conventional models.     

Three-dimensional bioprinting of stem cell-derived central nervous system cells enables astrocyte growth,vasculogenesis, and enhances neural differentiation/function

Current research tools for preclinical drug development such as rodent models and two-dimensional immortalized monocultures have failed to serve as effective translational models for human central nervous system (CNS) disorders. Recent advancements in the development of induced pluripotent stem cells (iPSCs) and three-dimensional (3D) culturing can improve the in vivo-relevance of preclinical models, while generating 3D cultures though novel bioprinting technologies can offer increased scalability and replicability. As such, there is a need to develop platforms that combine iPSC-derived cells with 3D bioprinting to produce scalable, tunable, and biomimetic cultures for preclinical drug discovery applications. We report a biocompatible poly(ethylene glycol)-based matrix which incorporates Arg-Gly-Asp and Tyr-Ile-Gly-Ser-Arg peptide motifs and full-length collagen IV at a stiffness similar to the human brain (1.5 kPa). Using a high-throughput commercial bioprinter we report the viable culture and morphological development of monocultured iPSC-derived astrocytes, brain microvascular endothelial-like cells, neural progenitors, and neurons in our novel matrix. We also show that this system supports endothelial-like vasculogenesis and enhances neural differentiation and spontaneous activity. This platform forms a foundation for more complex, multicellular models to facilitate high-throughput translational drug discovery for CNS disorders.     

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure–activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.     

Percutaneous permeation of enantiomers and racemates of chiral drugs and prediction of their flux ratios using thermal data: a pharmaceutical perspective

Albeit pharmacological, pharmacokinetic, and toxicological differences between enantiomeric pairs or between the pure enantiomers and racemate of chiral drugs are known to exist for decades, we are just beginning to realize that there are apparent differences between these species with respect to their percutaneous permeation as well. Such differences in permeation are likely to be enhanced when chiral drugs are formulated with chiral excipients, necessitating a careful assessment of the effect of formulation excipients on the permeation as well as the overall therapeutic outcomes. The in vitro transport data from the preclinical investigations, using laboratory animal models and/or in vitro cell culture systems, must be carefully validated in vivo as there are differences between these models and the human skin. Mathematical models such as MTMT that utilize the interdependence of certain physicochemical characteristics and percutaneous permeability have a predictive value in assessing the flux behavior of enantiomers and racemates.     

Strategy for achieving standardized bone models

Reliably producing functional in vitro organ models, such as organ-on-chip systems, has the potential to considerably advance biology research, drug development time, and resource efficiency. However, despite the ongoing major progress in the field, three-dimensional bone tissue models remain elusive. In this review, we specifically investigate the control of perfusion flow effects as the missing link between isolated culture systems and scientifically exploitable bone models and propose a roadmap toward this goal.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.     

Drug discovery in academia

Drug discovery and development is generally done in the commercial rather than the academic realm. Drug discovery involves target discovery and validation, lead identification by high-throughput screening, and lead optimization by medicinal chemistry. Follow-up preclinical evaluation includes analysis in animal models of compound efficacy and pharmacology (ADME: administration, distribution, metabolism, elimination) and studies of toxicology, specificity, and drug interactions. Notwithstanding the high-cost, labor-intensive, and non-hypothesis-driven aspects of drug discovery, the academic setting has a unique and expanding niche in this important area of investigation. For example, academic drug discovery can focus on targets of limited commercial value, such as third-world and rare diseases, and on the development of research reagents such as high-affinity inhibitors for pharmacological "gene knockout" in animal models ("chemical genetics"). This review describes the practical aspects of the preclinical drug discovery process for academic investigators. The discovery of small molecule inhibitors and activators of the cystic fibrosis transmembrane conductance regulator is presented as an example of an academic drug discovery program that has yielded new compounds for physiology research and clinical development. high-throughput screening; drug development; pharmacology; fluorescence; cystic fibrosis transmembrane conductance regulator     

Application of predictive biosimulation within pharmaceutical clinical development: examples of significance for translational medicine and clinical trial design

The challenge of accurately predicting human clinical outcome based on preclinical data has led to a high failure rate of compounds in human clinical trials. A series of methods are described by which biosimulation can address these challenges and guide the design and evaluation of experimental and clinical protocols. Early compound development often proceeds on the basis of preclinical data from animal models. The systematic evaluation possible in a simulation can assist in the critical step of translating the preclinical outcomes to human physiology. Later in the process, clinical trials definitively establish a therapy's beneficial effects, as well as any adverse side effects. Biosimulation allows for the optimal design of clinical trials to ensure that key issues are addressed effectively and efficiently, and in doing so, improves the success rate of the trials.     

Patient-derived model systems and the development of next-generation anticancer therapeutics

Anticancer drug discovery and development using conventional cell line and animal models has traditionally had a low overall success rate. Despite yielding game-changing new therapeutics, 10–20 new molecules have to be brought to the clinic to obtain one new approval, making this approach costly and inefficient. The use of in vitro experimental models based on primary human tumour tissues has the potential to provide a representation of human cancer biology that is closer to an actual patient and to ‘bridge the translational gap’ between preclinical and clinical research. Here, we review recent advances in the use of human tumour samples for preclinical research through organoid development or as primary patient materials. While challenges still remain regarding analysis, validation and scalability, evidence is mounting for the applicability of both models as preclinical research tools.     

Morphological and functional characterization of human induced pluripotent stem cell‐derived neurons (iCell Neurons) in defined culture systems

Pre‐clinical testing of drug candidates in animal models is expensive, time‐consuming, and often fails to predict drug effects in humans. Industry and academia alike are working to build human‐based in vitro test beds and advanced high throughput screening systems to improve the translation of preclinical results to human drug trials. Human neurons derived from induced pluripotent stems cells (hiPSCs) are readily available for use within these test‐beds and high throughput screens, but there remains a need to robustly evaluate cellular behavior prior to their incorporation in such systems. This study reports on the characterization of one source of commercially available hiPSC‐derived neurons, iCell^® Neurons, for their long‐term viability and functional performance to assess their suitability for integration within advanced in vitro platforms. The purity, morphology, survival, identity, and functional maturation of the cells utilizing different culture substrates and medium combinations were evaluated over 28 days in vitro (DIV). Patch‐clamp electrophysiological data demonstrated increased capacity for repetitive firing of action potentials across all culture conditions. Significant differences in cellular maturity, morphology, and functional performance were observed in the different conditions, highlighting the importance of evaluating different surface types and growth medium compositions for application in specific in vitro protocols. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1613–1622, 2015     

Induction of drug metabolizing enzymes: a survey of in vitro methodologies and interpretations used in the pharmaceutical industry--do they comply with FDA recommendations?

The FDA has published guidelines by which to carry out and interpret in vitro induction studies using hepatocytes but do researchers in pharmaceutical companies actually follow these to the letter? In a survey of 30 participants in the pharmaceutical industry, 19 questions were posed regarding the species investigated, methodologies and interpretations of the data. Also addressed was the in-house decision making processes as a result of in vitro induction data. The survey showed that, although the basic methods were similar, no two researchers carried out and interpreted induction assays in exactly the same way. No single method was superior but all included enzyme activities as the major end point. Hepatocytes from animal species were used to confirm animal in vivo data but only human hepatocytes were used to predict human induction responses. If a compound was found to be positive in an in vitro induction assay, few would halt the development of the compound. The majority would consider other properties of the compound (bioavailability, clearance and therapeutic concentrations) and follow the FDA recommendation to conduct clinical drug-drug interaction studies. Overall, the results from this survey indicate that there is no standard pharmaceutical industry method or evaluation criterion by which in vitro assays are carried out. Rather than adhering to the FDA guidelines, some adapt methods and interpretation according to their own experience and need (whether screening or lead optimisation). There was general consensus that studies using human hepatocyte cultures currently provide the best indication of the in vivo induction potential of NCEs. In addition, the assessment of in vitro induction data from the literature suggest that the two-fold induction threshold and the percent of positive control criteria may not be the best methods to accurately assess the in vivo induction potential of a drug. Although the two-fold induction criterion is now obsolete, more predictive models for determining the clinical induction potential are needed. Alternative models are proposed and discussed herein.     

Bringing 3D tumor models to the clinic – predictive value for personalized medicine

Current decision‐guiding algorithms in cancer drug treatment are based on decades of research and numerous clinical trials. For the majority of patients, this data is successfully applied for a systemic disease management. For a number of patients however, treatment stratification according to clinically based risk criteria will not be sufficient. The most effective treatment options are ideally identified prior to the start of clinical drug therapy. This review will discuss the implementation of three‐dimensional (3D) cell culture models as a preclinical testing paradigm for the efficacy of clinical cancer treatment. Patient tumor‐derived cells in 3D cultures duplicate the individual tumor microenvironment with a minimum of confounding factors. Clinical implementation of such personalized tumor models requires a high quality of methodological and clinical validation comparable to other biomarkers. A non‐systematic literature search demonstrated the small number of prospective studies that have been conducted in this area of research. This may explain the current reluctance of many physicians and insurance providers in implementing this type of assay into the clinical diagnostic routine despite potential benefit for patients. Achieving valid and reproducible results with a high level of evidence is central in improving the acceptance of preclinical 3D tumor models.     

The basics of preclinical drug development for neurodegenerative disease indications

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug's mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer's disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.     

Synthesis and pharmacological evaluation of 3-aryl-3-azolylpropan-1-amines as selective triple serotonin/norepinephrine/dopamine reuptake inhibitors

A series of 3-aryl-3-azolylpropan-1-amines was prepared and screened for its capability of inhibiting monoamine reuptake. Analogs with nanomolar potency, good human in vitro microsomal stability, and low drug–drug interaction potential were described. In vivo models were used to evaluate the compound 19r for antidepressive, anxiolytic, and analgesic activity.     

Drug screening model meets cancer organoid technology

Tumor organoids inherit the genomic and molecular characteristics of the donor tumor, which not only bridge the gap between genome and phenotype but also circumvent the disadvantages such as genetic information change by using 2D cell lines and the mouse-specific tumor evolution in patient-derived xenograft (PDX). So, cancer organoid has been widely applied to preclinical drug evaluation, biomarker identification, biological research, and individualized therapy. Besides, cancer organoid can be preserved, resuscitated, passed infinitely, and mechanically cultured on a chip for drug screening; it has become one of the partial models for low/high-throughput drug screening in the preclinical trial in vitro. Therefore, this review presents the recent developments of tumor organoids for drug screening, which will introduce from four aspects, including the stability/credibility, types, application, deficiency and prospect of the tumor organoids model for drug screening.     

1-Oxo-3,4-dihydroisoquinoline-4-carboxamides as novel druglike inhibitors of poly(ADP-ribose) polymerase (PARP) with favourable ADME characteristics

A novel 3,4-dihydroisoquinol-1-one-4-carboxamide scaffold was designed as the basis for the development of novel inhibitors of poly(ADP-ribose) polymerase (PARP). Synthesis of 3,4-dihydroisoquinol-1-one-4-carboxylic acids was achieved using the previously developed protocol based on the modified Castagnoli-Cushman reaction of homophthalic anhydrides and 1,3,5-triazinanes as formaldimine synthetic equivalents. Employment of 2,4-dimethoxy groups on the nitrogen atom of the latter allowed preparation of 2,3-unsubatituted 3,4-dihydroquinolone core building blocks. Iterative synthesis and in vitro biological testing of the amides resulting from the amidation of these carboxylic acids allowed not only drawing important structure-activity generalisations (corroborated by in silico docking simulation) but also the identification of the lead compound, 4-([1,4''-bipiperidine]-1''-carbonyl)-7-fluoro-3,4-dihydroisoquinolin-1(2H)-one, as the candidate for further preclinical development. The lead compound as well as its des-fluoro analog were compared to the approved PARP1 inhibitor, anticancer drug Olaparib, in terms of their molecular characteristics defining druglikeness as well as experimentally determined ADME parameters. The newly developed series demonstrated clear advantages over Olaparib in terms of molecular weight, hydrophilicity, human liver microsomal and plasma stability as well as plasma protein binding. Further preclinical investigation of the lead compound is highly warranted.     

Recent and advanced therapy for oral cancer

Oral cancer is a common and deadly kind of tissue invasion, has a high death rate, and may induce metastasis that mostly affects adults over the age of 40. Most in vitro traditional methods for studying cancer have included the use of monolayer cell cultures and several animal models. There is a worldwide effort underway to reduce the excessive use of laboratory animals since, although being physiologically adequate, animal models rarely succeed in exactly mimicking human models. 3D culture models have gained great attention in the area of biomedicine because of their capacity to replicate parent tissue. There are many benefits to using a drug delivery approach based on nanoparticles in cancer treatment. Because of this, in vitro test methodologies are crucial for evaluating the efficacy of prospective novel nanoparticle drug delivery systems. This review discusses current advances in the utility of 3D cell culture models including multicellular spheroids, patient-derived explant cultures, organoids, xenografts, 3D bioprinting, and organoid-on-a-chip models. Aspects of nanoparticle-based drug discovery that have utilized 2D and 3D cultures for a better understanding of genes implicated in oral cancers are also included in this review.