Assessing the interplay between off-target promiscuity, cytotoxicity, and tolerability in rodents to improve the safety profile of novel anti-malarial plasmepsin X inhibitors.

Within drug development, high off-target promiscuity as well as potent cytotoxicity, are associated with a high attrition rate. We investigated the safety profile of novel plasmepsin X (PMX) inhibitors for the treatment of malaria. In our screening cascade, a total of 249 PMX compounds were profiled in a panel of in vitro secondary pharmacology assays containing 44 targets (SafetyScreen44™ panel) and in a cytotoxicity assay in HepG2 cells using ATP as an endpoint. Six of the lead compounds were subsequently tested in a 7-day rat toxicology study, and/or in a cardiovascular study in guinea pigs. Overall, compounds with high cytotoxicity in HepG2 cells correlated with high promiscuity (off-target hit rate >20%) in the SafetyScreen44™ panel and were associated with poor tolerability in vivo (decedents, morbidity, adverse clinical signs, or severe cardiovascular effects). Some side effects observed in rats or guinea pigs could putatively be linked with hits in the secondary pharmacological profiling, such as the M1 or M2 muscarinic acetylcholine receptor, opioid µ and/or κreceptors or hERG/CaV1.2/Na+ channels, which were common to > 50% the compounds tested in vivo. In summary, compounds showing high cytotoxicity and high promiscuity are likely to be poorly tolerated in vivo. However, such associations do not necessarily imply a causal relationship. Identifying the targets that cause these undesirable effects is key for early safety risk assessment. A tiered approach, based on a set of in vitro assays, helps selecting the compounds with highest likelihood of success to proceed to in vivo toxicology studies.

A Perfused In Vitro Human iPSC-Derived Blood-Brain Barrier Faithfully Mimics Transferrin Receptor-Mediated Transcytosis of Therapeutic Antibodies.

Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood-brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm2), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.

Discovery and Characterization of Potent, Efficacious, Orally Available Antimalarial Plasmepsin X Inhibitors and Preclinical Safety Assessment of UCB7362.

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.

Setup of human liver-chips integrating 3D models, microwells and a standardized microfluidic platform as proof-of-concept study to support drug evaluation.

Human 3D liver microtissues/spheroids are powerful in vitro models to study drug-induced liver injury (DILI) but the small number of cells per spheroid limits the models' usefulness to study drug metabolism. In this work, we scale up the number of spheroids on both a plate and a standardized organ-chip platform by factor 100 using a basic method which requires only limited technical expertise. We successfully generated up to 100 spheroids using polymer-coated microwells in a 96-well plate (= liver-plate) or organ-chip (= liver-chip). Liver-chips display a comparable cellular CYP3A4 activity, viability, and biomarker expression as liver spheroids for at least one week, while liver-plate cultures display an overall reduced hepatic functionality. To prove its applicability to drug discovery and development, the liver-chip was used to test selected reference compounds. The test system could discriminate toxicity of the DILI-positive compound tolcapone from its less hepatotoxic structural analogue entacapone, using biochemical and morphological readouts. Following incubation with diclofenac, the liver-chips had an increased metabolite formation compared to standard spheroid cultures. In summary, we generated a human liver-chip model using a standardized organ-chip platform which combines up to 100 spheroids and can be used for the evaluation of both drug safety and metabolism.

The Promise of AI for DILI Prediction.

Drug-induced liver injury (DILI) is a common reason for the withdrawal of a drug from the market. Early assessment of DILI risk is an essential part of drug development, but it is rendered challenging prior to clinical trials by the complex factors that give rise to liver damage. Artificial intelligence (AI) approaches, particularly those building on machine learning, range from random forests to more recent techniques such as deep learning, and provide tools that can analyze chemical compounds and accurately predict some of their properties based purely on their structure. This article reviews existing AI approaches to predicting DILI and elaborates on the challenges that arise from the as yet limited availability of data. Future directions are discussed focusing on rich data modalities, such as 3D spheroids, and the slow but steady increase in drugs annotated with DILI risk labels.

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

Inhibition of gamma-thrombin-induced human platelet aggregation by histone H1subtypes and H1.3 fragments.

Human platelets are differentially activated by varying concentrations of alpha-thrombin or by beta- and gamma-thrombin via three thrombin receptors, PAR-1, PAR-4 and GPIbalpha.It is likely that the development of a normal or abnormal hemostatic event in humans is dictated, in part, by the selective activation of these receptors. The ability to differentially inhibit these thrombin receptors could, therefore, have clinical significance. We have previously demonstrated that histone H1 selectively inhibits the PAR-4 receptor. In the current study we investigated whether five subtypes of the H1 molecule or fragments of the H1.3 subtype differentially inhibited the PAR-4 receptor. PAR-4 inhibition by all H1 subtypes was saturated at 1 uM with no statistical difference observed with the five H1 subtypes tested. Of the five fragments generated from the H1.3 molecule only one had significant inhibitory activity against PAR-4. The C-terminal fragment, N.1, generated by the proteolysis of the parent molecule by Asp-N endoproteinase (Aeromonas proteolytica) at the single aspartate residue, showed the same level of PAR-4 inhibition as the intact H1.3 at 1 uM concentrations. Removal of two N-terminal amino acids (Asp-Val as determined by MALDI analysis) from the N.1 fragment further enhanced its inhibitory activity. These studies may help to develop specific drugs to differentially inhibit the platelet thrombin receptors.

Cytokine mediated proliferation of cultured sea turtle blood cells: morphologic and functional comparison to human blood cells.

Blood cells from three different sea turtle species were cultured for approximately 3 weeks in nutrient medium supplemented with recombinant human cytokines known to induce terminal maturation of human hematological stem cells. Cultured turtle erythrocytes were translucent, approximately 10x larger than human erythrocytes, contained a single fluorescent inclusion body, contained nuclear epsilon (embryonic) globin proteins, and, absent of organelles while fresh cells contained few, but well defined mitochondria. Cells with basophilic cytoplasm and in all stages of proliferation were observed in cytokine-supplemented cultures and appeared to possess active protein synthesis. Cultured thrombocytes aggregated in response to agonists for at least 8 days, post-collection, contained P-selectin in the nucleus of 6 day cultured cells which appeared to be released after activation with collagen, and after 6 days had no organelles or open canalicular-like system (OCS) while freshly isolated cells demonstrated few, if any organelles but had a well developed OCS. The response of turtle cells to apparently homologous but unnatural human cytokines and the sustained biological properties of thrombocytes identify this suspension culture system as a powerful tool to explore the evolution of cell types and molecular components of hematopoiesis and hemostasis.

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood.

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.

ICOS-B7 homologous protein interactions are necessary for mercury-induced autoimmunity.

After exposure to subtoxic doses of heavy metals such as mercury, H-2(s) mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of one of the members of the CD28-B7 costimulation families, ICOS-B7 homologous protein (B7h), in the regulation of mercury-induced autoimmunity. The expression of ICOS on T cells was more enhanced in susceptible A.SW mice than in non-responsive C57BL/6 and DBA/2 mice after HgCl(2) treatment. Furthermore, in A.SW mice treated with HgCl(2), administration of a blocking anti-ICOS Ab effectively inhibited anti-nucleolar autoantibodies and total serum IgE production. Taken together, these results indicate that the ICOS-B7h costimulation pathway is required for this autoimmune syndrome and suggest that targeting this pathway might have therapeutic benefits for human autoimmune diseases.

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct alpha-, beta- and gamma-thrombin.

The development of drugs to neutralize the action of thrombin has to date focused on the alpha form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active alpha-thrombin which may be further hydrolyzed to beta- and gamma-thrombin. While all three forms of the enzyme retain catalytic activities, only alpha-thrombin is presumed to be physiologically important. The beta- and gamma-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that beta- and gamma-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173-21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. Beta-thrombin, like gamma-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the beta-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For beta-thrombin and gamma-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that alpha-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that alpha-thrombin may be rapidly converted to beta-thrombin and/or gamma-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by alpha-, beta- and gamma-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits alpha-thrombin , is a slightly weaker inhibitor of beta-thrombin and a very weak inhibitor of gamma-thrombin -induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, alpha(1)-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only beta- and gamma-thrombin , primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like beta- and gamma-thrombin , activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to gamma-thrombin than to alpha-thrombin. The analysis of the crystalline structures of alpha-, gamma-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.

Radiation enhancement by gemcitabine-mediated cell cycle modulations.

The purpose of this study was to investigate the exact dose dependency and time dependency of the radiation-enhancing effect of gemcitabine (2',2'difluoro desoxycytidine [dFdC]) in in vitro experiments (HeLa cells: cancer of the uterine cervix, #4197 cells: oropharyngeal squamous cell carcinoma), and to correlate this effect with the underlying changes in cell cycle distribution. Cell viability was determined fluorometrically after exposure to dFdC (0-20.0 micro mol/l), irradiation (0-37.5 Gy), and both modalities. Combining both therapies, cells were exposed to dFdC (0-10.0 micro mol/l) for 24 hours before further treatment and irradiated (0-30 Gy) immediately afterwards with or without removal of dFdC. For cell cycle analysis by flow cytometry, cells were irradiated (0-40 Gy) or treated with dFdC (0.012-1.0 micro mol/l, 24-48 hours). Additionally, cells were exposed to dFdC (2.0 micro mol/l, 0-4 hours). Cell cycle kinetics were evaluated using bromodeoxyuridine (BrdU) (10 micro mol/l) S-phase labeling, given either 30 minutes before or in the last hour of dFdC treatment (2.0 micro mol/l, 0-6 hours). The fluorometric assay revealed that dFdC enhances radiation-induced cytotoxicity at marginally toxic or nontoxic concentrations (<37 nmol/l). Radiation resulted in the anticipated G2/M arrest already at 2 Gy. DFdC induced concentration and exposure time-dependent cell cycle changes that were better resolved using BrdU, demonstrating a pronounced S-phase arrest already at 12 nmol/l. BrdU-pulse labeling revealed that the cell cycle block occurred at the G1/S boundary. Our data reconfirm the already known radiation enhancement, the S-phase specific activities of dFdC, and the relevance of the synchronized progression of cells through the S-phase with regard to the radiosensitizing properties of low-dose dFdC. However, we could demonstrate that before progressing in the S-phase, cells were blocked and partially synchronized at the more radiosensitive G1/S boundary. Furthermore, cells progressing past the block might accumulate proapoptotic signals caused by both radiation and dFdC, which will also results in cell death.

Radioiodinated (I-125) monoclonal antibody 425 in the treatment of high grade glioma patients: ten-year synopsis of a novel treatment.

The present report is the follow-up of patients enrolled in a phase II clinical trial using I-MAb 425 as an adjuvant treatment for high grade gliomas. Patient median survivals support published data from an earlier preliminary report. From January 29, 1987 to January 25, 1997, 180 patients diagnosed with astrocytoma with anaplastic foci (AAF) and glioblastoma multiforme (GBM) were treated as outpatients with an average of three weekly intravenous or intraarterial injections of radiolabeled MAb 425. The mean dose was 140 mCi (5.2 GBq). Only one patient who received a single dose of more than 60 mCi (2.2 GBq) experienced acute toxicity. Patients received prior surgery and radiation therapy, with and without chemotherapy. Overall median survival for patients with GBM and AAF was 13.4 and 50.9 months, respectively, with Karnofsky Performance Status (KPS) ranging from 40 to 100 and age ranging from 11 to 75 years. Prognostic factors (KPS and age) correlated positively with increased survival, with KPS the most important determinant of median survival. Data analysis was performed on patients followed 5 years or longer. We conclude that the administration of I-MAb 425 with intensive medical management demonstrates a significant increase in median survival and should be considered a therapeutic regimen for the management of patients with high grade gliomas.

[Combined radiotherapy and gemcitabine. Evaluation of clinical data based on experimental knowledge]. / Kombination von Radiotherapie und Gemcitabine. Bewertung der klinischen Daten auf der Basis experimenteller Erkenntnisse.

BACKGROUND: In experimental studies the nucleoside analog Gemcitabine (2',2' difluorodesoxycytidine) clearly demonstrates radiation enhancing properties. After describing the pharmacological Gemcitabine-related data and the clinical studies regarding combined radiochemotherapy and taking under consideration the in-vitro data and the results provided by animal models, this overview is aimed to draw clinically relevant conclusions, resulting in the improvement of treatment approaches. MATERIALS AND METHODS: The available literature data regarding the metabolism and the mechanism of action, the evaluation of possible schedules of administration, and combined radiochemotherapy including Gemcitabine has been reviewed. Publications reporting experimental data in vitro and in vivo as well as our own experimental results have been incorporated. RESULTS: In clinical phase I and II studies, the favorable tumor response is accompanied by a high incidence of grade III-IV toxicities whereby the maximum-tolerated dose (MTD) of the various schedules of administration used is always lower compared to the MTD of single-agent treatment. In in-vitro and in-vivo data addressing the description and the evaluation of the radiation enhancing mechanism (especially influence on cell cycle, depletion of the dATP pool, induction of apoptosis, inhibition of DNA synthesis, reduction of DNA repair) this effect is already observed with non and moderately toxic Gemcitabine concentrations and depends on drug concentration and exposure time. Independent of the fractionation effect of radiotherapy, the radiation enhancement is persistent for at most 72 hours after the end of drug exposure. Taking under consideration the single dose per day and the target volume, a prolonged infusion and/or a twice-weekly administration of Gemcitabine at low concentration each and simultaneous radiotherapy are presumably considered to resemble the experimental data. CONCLUSION: It is without doubt that data provided by clinical studies are of highest relevance for the evaluation of an optimized radiochemotherapy with Gemcitabine. However, although it is often difficult to transfer experimental data into the clinical situation, these data offer the possibility to develop an improved schedule of administration in patient treatment based on rational evidence in tumor biology.