Evaluation Of New Drug Candidates Research Articles

PET mapping of receptor occupancy using joint direct parametric reconstruction.

In this work, we evaluate a novel joint direct parametric reconstruction framework to directly estimate distributions of RO and other kinetic parameters in the brain from a pair of baseline and postdrug injection dynamic PET scans. The proposed method combines the use of regularization on RO maps with alternating optimization to enable estimation of occupancy even in low binding regions. Simulation results demonstrate the quantitative improvement of this method over conventional approaches in terms of accuracy and precision of occupancy. The proposed method is also evaluated in preclinical in-vivo experiments using 11C-MK6884 and a muscarinic acetylcholine receptor 4 positive allosteric modulator drug, showing improved estimation of receptor occupancy as compared to traditional estimators. The proposed joint direct estimation framework improves RO estimation compared to conventional methods, especially in intermediate to low-binding regions. This work could potentially facilitate the evaluation of new drug candidates targeting the CNS.

Effect of Systemic Administration of CD4+ T cells and Local Administration of T-cell Stimulants on T-cell Activity in Psoriatic Skin Xenografts on NOG Mice.

Immunodeficient mice engrafted with psoriatic human skin are widely used for the preclinical evaluation of new drug candidates. However, the T-cell activity, including the IL23/IL17 pathway, declines in the graft over time after engraftment, which likely affects the study data. Here, we investigated whether the T-cell activity could be sustained in xenografted psoriatic skin by local stimulation of T cells or systemic injection of autologous CD4 + T cells. We surgically transplanted human psoriatic skin from 5 untreated patients onto female NOG mice. Six days after surgery, mice received an intraperitoneal injection of autologous human CD4+ T cells, a subcutaneous injection under the grafts of a T-cell stimulation cocktail consisting of recombinant human IL2, human IL23, antihuman CD3, and antihuman CD28, or saline. Mice were euthanized 21 d after surgery and spleens and graft biopsies were collected for analysis. Human T cells were present in the grafts, and 60% of the grafts maintained the psoriatic phenotype. However, neither local T-cell stimulation nor systemic injection of autologous CD4+ T cells affected the protein levels of human IL17A, IL22, IFN γ, and TNF α in the grafts. In conclusion, NOG mice seem to accept psoriatic skin grafts, but the 2 approaches studied here did not affect human T-cell activity in the grafts. Therefore, NOG mice do not appear in this regard to be superior to other immunodeficient mice used for psoriasis xenografts.

Advances in the screening of antimicrobial compounds using electrochemical biosensors: is there room for nanomaterials?

The abusive use of antimicrobial compounds and the associated appearance of antimicrobial resistant strains are a major threat to human health. An improved antimicrobial administration involves a faster diagnosis and detection of resistances. Antimicrobial susceptibility testing (AST) are the reference techniques for this purpose, relying mainly in the use of culture techniques. The long time required for analysis and the lack of reproducibility of these techniques have fostered the development of high-throughput AST methods, including electrochemical biosensors. In this review, recent electrochemical methods used in AST have been revised, with particular attention on those used for the evaluation of new drug candidates. The role of nanomaterials in these biosensing platforms has also been questioned, inferring that it is of minor importance compared to other applications.

Experimental Comparison of Primary and hiPS-Based In Vitro Blood–Brain Barrier Models for Pharmacological Research

In vitro model systems of the blood–brain barrier (BBB) play an essential role in pharmacological research, specifically during the development and preclinical evaluation of new drug candidates. Within the past decade, the trend in research and further development has moved away from models based on primary cells of animal origin towards differentiated models derived from human induced pluripotent stem cells (hiPSs). However, this logical progression towards human model systems from renewable cell sources opens up questions about the transferability of results generated in the primary cell models. In this study, we have evaluated both models with identical experimental parameters and achieved a directly comparable characterisation showing no significant differences in protein expression or permeability even though the achieved transendothelial electrical resistance (TEER) values showed significant differences. In the course of this investigation, we also determined a significant deviation of both model systems from the in vivo BBB circumstances, specifically concerning the presence or absence of serum proteins in the culture media. Thus, we have further evaluated both systems when confronted with an in vivo-like distribution of serum and found a notable improvement in the differential permeability of hydrophilic and lipophilic compounds in the hiPS-derived BBB model. We then transferred this model into a microfluidic setup while maintaining the differential serum distribution and evaluated the permeability coefficients, which showed good comparability with values in the literature. Therefore, we have developed a microfluidic hiPS-based BBB model with characteristics comparable to the established primary cell-based model.

Tumours modulate the systemic vascular response to anti-angiogenic therapy.

Toxicologic evaluation of new drug candidates routinely utilizes healthy animals. In oncology, there remains a limited understanding of the effects of novel test candidates in a diseased host. For vascular modulating agents (VMAs), an increased understanding of preclinical tumour–host interaction, and its potential to exacerbate or alleviate ‘off‐target’ effects of anti‐angiogenic administration, could aid in the prediction of adverse clinical outcomes in a defined cancer patient. We have previously reported that the implantation and growth of a range of human‐ and mouse‐derived tumours leads to structural vascular and, potentially, functional signalling changes within host mouse endocrine tissues, indicating possible roles for tumour‐ and host‐derived cytokines/growth factors and the liberation of myeloid‐derived suppressor cells in this phenomenon. Here, we further demonstrate that the growth of the Calu‐6 xenograft is associated with a resistance to VMA‐induced mouse peripheral endocrine vascular rarefaction (toxicity), with potential functional impact, notably with respect to mixed tyrosine kinase inhibition. The pathogenesis of these findings indicates a potential role for both tumour‐ and host‐derived basic fibroblast growth factor (bFGF), with associated upregulation in the intra‐tumoural autotaxin‐lysophosphatic acid signalling axis.

Recapitulation of First Pass Metabolism Using 3D Printed Microfluidic Chip and Organoid.

The low bioavailability of oral drugs due to first pass metabolism is a major obstacle in drug development. With significant developments in the field of in vitro organ modeling and microfluidic chip three-dimensional (3D) printing, the challenge is to apply these for the production and evaluation of new drug candidates. This study aimed to produce a microfluidic chip to recapitulate and assess the feasibility of the first pass metabolism. The infill condition of the polycarbonate transparent filament and layer height was optimized to visualize and maintain the organoid or spheroid on the chip. Next, the chip was fabricated using a 3D printer after a computer-aided design (CAD). The chip consisted of three wells of different heights. The small intestinal (SI) organoid and colorectal adenocarcinoma spheroids were placed on the second and third wells, respectively. No additional equipment was assembled, and the tilted tunnel was connected to each well to transport the material by gradient force. The chip was fabricated using 50% and 0.1 um thickness. Among the three different prototypes of chip (chips 1, 2, and 3), the highest distribution of plasmids in the Matrigel of the second well was observed in Chip 2 at 48 h. The effect of first pass metabolism was analyzed using docetaxel. In the chip without an SI organoid, there was a marked decrease in the viability of colorectal adenocarcinoma spheroids due to drug efficacy. However, in the chip with the SI organoid, no significant change in viability was observed because of first pass metabolism. In conclusion, we presented a simple, fast, and low-cost microfluidic chip to analyze the efficacy change of candidate drug by the first pass metabolism.

Considerations and Pitfalls in Selecting the Drug Vehicles for Evaluation of New Drug Candidates: Focus on in vivo Pharmaco-Toxicological Assays Based on the Rotarod Performance Test.

Purpose - During the discovery and development of new drugs, compounds with low aqueous solubility pose special challenges in their pharmacological evaluation and, therefore, the selection of appropriate vehicles to administer the compounds of interest is determinant for the quality of the results generated during the in vivo non-clinical studies. This work aimed to evaluate the motor deficit (as a surrogate of neurotoxicity) of several administration/delivery vehicles through the rotarod performance test. Methods - Trained male CD-1 mice were intraperitoneally administered with the following vehicles: dimethyl sulfoxide (DMSO), aqueous sodium chloride (NaCl) 0.9%, aqueous carboxymethylcellulose (CMC) 0.5%, polyethylene glycol (PEG)-400, propylene glycol (PG), and solutions of these vehicles containing 5% and 10% DMSO. Results - It was observed that the aqueous vehicles (NaCl 0.9% and CMC 0.5%) did not affect the performance of the animals on the rod. On the other hand, a vehicle consisting solely of DMSO led to significant motor impairment and only a small improvement was recorded over time. Additionally, a strong neuromotor toxicity was observed in the early evaluation points of the experiment using vehicles constituted by PG and PEG-400 or by mixtures of PG/DMSO (5% and 10%) and PEG-400/DMSO (5% and 10%). Conclusion - This study provides useful data about the neurotoxicity inherent to several vehicles frequently used in non-clinical pharmaco-toxicological assays, aiming to draw especial attention to the need of a careful selection of drug vehicles in order to avoid the impact of such confounding variables on the accuracy of the results and in decision-making processes.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform.

With great advances in the field of in vitro brain modelling, the challenge is now to implement these technologies for development and evaluation of new drug candidates. Here we demonstrate a method for culturing three-dimensional networks of spontaneously active neurons and supporting glial cells in a microfluidic platform. The high-throughput nature of the platform in combination with its compatibility with all standard laboratory equipment allows for parallel evaluation of compound effects.

Abstract 1224: A patient derived xenograft (PDX) platform for development of next generation KIT kinase inhibitors in imatinib-resistant gastrointestinal stromal tumors (GIST)

Abstract Despite best efforts in the treatment of gastrointestinal stromal tumors (GIST), patients continue to face a poor prognosis. That said, introduction of imatinib into clinical practice has vastly improved outcomes for KIT positive patients. However, as with many kinases, resistance has developed into a clinical dilemma, rendering the drug ineffective. A number of resistance mutations have previously been identified including the well known D816 activating mutation. To overcome this challenge, we have established a set of GIST patient derived xenograft models to recapitulate the original patient tumor, including genetics of resistance. In this work, we show four primary GIST samples which were selected based on mutational and clinical profile. PDX models were developed in immunocompromised mice and were further characterized by immunohistochemistry, additional sequencing and pharmacological efficacy. We then evaluated the efficacy of chemotherapeutic agents in these models. We describe imatinib resistance mutations, and demonstrate in vivo efficacy of dasatinib over imatinib in the resistant GIST PDX model. Take together, this data validates these GIST PDX models as a novel platform for the evaluation of new drug candidates to better delay and circumvent resistance now found in the clinic. Citation Format: Chelsea Mullins, Jill Ricono, Patrick Carson, Gaston Habets, Rafe Shellooe, Hoa Nguyen, Thomas Broudy, Cyrus Mirsaidi, Praveen Nair. A patient derived xenograft (PDX) platform for development of next generation KIT kinase inhibitors in imatinib-resistant gastrointestinal stromal tumors (GIST). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1224. doi:10.1158/1538-7445.AM2014-1224

Antipsychotic, antidepressant, and cognitive-impairment properties of antipsychotics: rat profile and implications for behavioral and psychological symptoms of dementia

Many dementia patients exhibit behavioral and psychological symptoms (BPSD), including psychosis and depression. Although antipsychotics are frequently prescribed off-label, they can have marked side effects. In addition, comparative preclinical studies of their effects are surprisingly scarce, and strategies for discovery of novel pharmacotherapeutics are lacking. We therefore compared eight antipsychotics in rat behavioral tests of psychosis, antidepressant-like activity, and cognitive impairment as a basis for preclinical evaluation of new drug candidates. The methods used in this study include inhibition of MK-801-induced hyperactivity, forced swim test (FST), passive avoidance (PA), spontaneous locomotor activity, and catalepsy. The drugs exhibited antipsychotic-like activity in the MK-801 test but with diverse profiles in the other models. Risperidone impaired PA performance, but with some dose separation versus its actions in the MK-801 test. In contrast, clozapine, olanzapine, lurasidone, and asenapine showed little or no dose separation in these tests. Aripiprazole did not impair PA performance but was poorly active in the MK-801 test. Diverse effects were also observed in the FST: chlorpromazine was inactive and most other drugs reduced immobility over narrow dose ranges, whereas clozapine reduced immobility over a wider dose range, overlapping with antipsychotic activity. Although the propensity of second-generation antipsychotics to produce catalepsy was lower, they all elicited pronounced sedation. Consistent with clinical data, most currently available second-generation antipsychotics induced cognitive and motor side effects with little separation from therapeutic-like doses. This study provides a uniform in vivo comparative basis on which to evaluate future early-stage drug candidates intended for potential pharmacotherapy of BPSD.

Fabrication of Paclitaxel Nanocrystals by Femtosecond Laser Ablation and Fragmentation

Nanonization, which involves formulating the drug powder as nanometer-sized particles, is a known method to improve drug absorption and allow the intravenous administration of insoluble drugs. This study investigated a novel femtosecond (fs) laser technique for the fabrication of nanocrystals in aqueous solution of the insoluble model drug paclitaxel. Two distinct methods of this technology, ablation and fragmentation, were investigated and the influence of laser power, focusing position and treatment time on the particle size, drug concentration, and degradation was studied. The colloidal suspensions were characterized with respect to size, chemical composition, morphology, and polymorphic state. Optimal laser fragmentation conditions generated uniformly sized paclitaxel nanoparticles (<500 nm) with quantifiable degradation, while ablation followed by fragmentation was associated with a larger polydispersity. Laser treatment at higher powers produced smaller particles with larger amount of degradation. The crystalline morphology of the drug was retained upon nanonization, but the anhydrous crystals were converted to a hydrated form, a phenomenon also observed during bead milling. These findings suggest that drug nanocrystals can be produced with fs laser technology using very little drug quantities, which may be an asset for preclinical evaluation of new drug candidates.

Nanonization of megestrol acetate by laser fragmentation in aqueous milieu

Nanonization is a simple and effective method to improve dissolution rate and oral bioavailability of drugs with poor water solubility. There is growing interest to downscale the nanocrystal production to enable early preclinical evaluation of new drug candidates when compound availability is scarce. The purpose of the present study was to investigate laser fragmentation to form nanosuspensions in aqueous solution of the insoluble model drug megestrol acetate (MA) using very little quantities of the drug. Laser fragmentation was obtained by focusing a femtosecond (fs) or nanosecond (ns) laser radiation on a magnetically stirred MA suspension in water or aqueous solution of a stabilizing agent. The size distribution and physicochemical properties of the drug nanoparticles were characterized, and the in vitro dissolution and in vivo oral pharmacokinetics of a laser fragmented formulation were evaluated. A MA nanosuspension was also prepared by media milling for comparison purpose. For both laser radiations, smaller particles were obtained as the laser power was increased, but at a cost of higher degradation. Significant nanonization was achieved after a 30-min fs laser treatment at 250 mW and a 1-h ns laser treatment at 2500 mW. The degradation induced by the laser process of the drug was primarily oxidative in nature. The crystal phase of the drug was maintained, although partial loss of crystallinity was observed. The in vitro dissolution rate and in vivo bioavailability of the laser fragmented formulation were similar to those obtained with the nanosuspension prepared by media milling, and significantly improved compared to the coarse drug powder. It follows that this laser nanonization method has potential to be used for the preclinical evaluation of new drug candidates.

The immunology of tuberculosis: From bench to bedside

Tuberculosis (TB) is an international public health priority and kills almost two million people annually. TB is out of control in Africa due to increasing poverty and HIV coinfection, and drug-resistant TB threatens to destabilize TB control efforts in several regions of the world. Existing diagnostic tools and therapeutic interventions for TB are suboptimal. Thus, new vaccines, immunotherapeutic interventions and diagnostic tools are urgently required to facilitate TB control efforts. An improved understanding of the immunopathogenesis of TB can facilitate the identification of correlates of immune protection, the design of effective vaccines, the rational selection of immunotherapeutic agents, the evaluation of new drug candidates, and drive the development of new immunodiagnostic tools. Here we review the immunology of TB with a focus on aspects that are clinically and therapeutically relevant. An immunologically orientated approach to tackling TB can only succeed with concurrent efforts to alleviate poverty and reduce the global burden of HIV.

Building a Tiered Approach to In Vitro Predictive Toxicity Screening: A Focus on Assays with In Vivo Relevance

One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

The isolated rabbit heart and Purkinje fibers as models for identifying proarrhythmic liability

Introduction Delayed ventricular repolarization is associated with rare, but often fatal, polymorphic tachyarrhythmias named Torsades de Pointes. ICH S7B guideline recommends an integrated approach for cardiovascular preclinical evaluation of new drug candidates, including action potential assays (as a Purkinje fiber test) but also proarrhythmia models. The aim of this preliminary study was to compare the respective value of two preclinical in vitro rabbit cardiac preparations—the Purkinje fiber and the isolated perfused heart (Langendorff method)—based on effects of dofetilide, a selective IKr inhibitor. Methods Transmembrane action potentials from rabbit Purkinje fibers were recorded using a conventional intracellular glass microelectrode. Electrocardiograms from rabbit isolated hearts were evaluated for QRS, QT and T wave durations (Tpeak–Tend). The pacing protocol was the same for both preparations (basal rate of 80 bpm and pacing of 40, 60 and 140 bpm). Dofetilide was tested in both systems at concentrations of 1, 3 and 10 nmol/L. Results In Purkinje fibers dofetilide induced a concentration- and reverse use-dependent increase in action potential durations measured at 50 and 90% of repolarization. At 10 nmol/L, only 3/10 fibers showed early after depolarizations. In the isolated heart model, dofetilide also induced a similar concentration- and reverse use-dependent increase in QT-interval. From 3 nmol/L, major changes in T wave morphology, R-on-T extrasystoles and TdP were observed, mainly at low rate. Prior to arrhythmias, T wave shape and duration were markedly altered suggesting an increase in the heterogeneity of cardiac ventricular repolarization. Conclusions The effects of dofetilide were comparable in the two models for delayed repolarization but the isolated heart appears to be a better predictor for arrhythmias and a unique in vitro model to assess arrhythmogenic potential of QT prolonging compounds at least when associated with IKr/hERG inhibition.

Development and evaluation of an in vivo assay in Caenorhabditis elegans for screening of compounds for their effect on cytochrome P450 expression

Most drugs and xenobiotics induce the expression of cytochrome P450 (CYP) enzymes, which reduce the bioavailability of the inducer and/or co-administered drugs. Therefore, evaluation of new drug candidates for their effect on CYP expression is an essential step in drug development. The available methods for this purpose are expensive and not amenable to high-throughput screening. We developed a fluorescence-based in vivo assay using transgenic Caenorhabditis elegans worms that express the green fluorescent protein (GFP) under the control of various CYP promoters. Using this assay, we found striking similarities between the worm CYPs and their human orthologs in their response to treatment with various drugs. For example,the antibiotic rifampicin, one of the strongest inducers of the human gene CYP3A4, was the strongest inducer of the worm ortholog CYP13A7. Since worms can be easily grown in liquid medium in microtitre plates, the assay described in this paper is suitable for the screening of a large number of potential lead compounds in the drug discovery process.

In vitro approaches to investigate mechanism-based inactivation of CYP enzymes

Mechanism-based inactivation (MBI) of human drug-metabolising CYP enzymes is an important consideration in the preclinical ADME evaluation of new drug candidates. In this report, the in vitro approaches used to investigate MBI of CYP enzymes are described, with an emphasis on the characterisation required to assess potential drug–drug interactions. Recent disparities in MBI data between in vitro test systems are also reviewed, highlighting the limitations of Escherichia coli-expressed human recombinant CYP in the prediction of drug–drug interactions that arise via MBI.

Expedient synthesis of deuterium‐labelled amides within micro‐reactors

AbstractThe pharmaceutical industry relies heavily on the synthesis of small quantities (10–500 mg) of stable, isotopically labelled compounds in the evaluation of new drug candidates for metabolism studies. As a result of the phenomenal cost of labelled materials even the preparation of small quantities can be extremely expensive.In this paper, for the first time, we report that micro‐reactor technology may be used to prepare stable deuterium‐labelled compounds by conducting all optimization experiments using unlabelled precursors and simply substituting the labelled derivatives once the optimization is complete. Here, we wish to present a simple, general procedure for the synthesis of amides containing isotopic labels demonstrated using [C‐2H3]acetyl chloride 1. The reaction is carried out within a micro‐reactor set‐up which we believe offers superiority over other reported methods viz requiring stoichiometric quantities of reagents, high containment of the system and generality of the technique, obtaining products in high yields. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Drug‐Drug, Drug—Dietary Supplement, and Drug—Citrus Fruit and Other Food Interactions: What Have We Learned?

Serious drug-drug interactions have contributed to recent U.S. market withdrawals and also recent nonapprovals of a few new molecular entities. Many of these interactions involved the inhibition or induction of metabolizing enzymes and efflux transporters, resulting in altered systemic exposure and adverse drug reactions or loss of efficacy. In addition to drug-drug interactions, drug-dietary supplement and drug-citrus fruit interactions, among others, could also cause adverse drug reactions or loss of efficacy and are important issues to consider in the evaluation of new drug candidates. This commentary reviews (1). the current understanding of the mechanistic basis of these interactions, (2). issues to consider in the interpretation of study results, and (3). recent labeling examples to illustrate the translation of study results to information useful for patients and health care providers.

Toxicological and Pharmacological Evaluation of New Drug Candidates by in Vitro Robotic High-Throughput Cell Assays

A high-throughput cell-based fluorescence screening assay was used to determine the cytotoxic activity of various cytostatic reference agents, unknown compounds of natural sources and pharmaceutic standard excipients in different cell lines such as HEK-293 and DLD-1 cells. In this paper we exemplarily show results for a selection of compounds and excipients. The cytostatic agent piposulfan has a much higher cytotoxic activity in DLD-1 cells (EC50=0.001 mg/mL) than in HEK-293 cells (EC50=0.3 mg/mL) in contrast to busulfan and vinblastine where the EC50 values are close together in both cell lines. From 113 tested compounds from natural sources the cytotoxic activity of 75 compounds showed no difference in both cell lines, 34 of them had a higher activity in HEK-293 cells than in DLD-1 cells and 4 compounds showed less cytotoxic activity in HEK-293 cells than in DLD-1 cells. We demonstrate the rather potent cytotoxic action of Cremophor EL; this finding is in accordance with previously published observations. The presented results demonstrate that the used assay system is reliable, enables the ranking of the cytotoxic potential of compounds of various chemical classes, and allows the determination of cell type-specific cytotoxicity. (JALA 2004;9:159-62)