Failure Of Clinical Trials Research Articles

Abstract 49: Comparison of subcutaneous and orthotopic tumor growth in syngeneic mouse models of ovarian and renal cancer

Abstract Oncology drug development has markedly lower success rate (3.4%) than other indications (20.9%) as reported by Wong, Siah and Lo in 2019. Animal models are often pointed at as the culprits for failure in clinical trials. While better animal models for cancer are needed and constantly developed, the incentive of drug developers to use those might not be clear, because the regulatory authorities do not require it. Subcutaneous tumors have earned their place in drug development but adding more sophisticated models to the testing panel before entering the clinical phase should improve the success rate. We present here the properties of subcutaneous and orthotopic tumor growth in two syngeneic models. HM-1 murine ovarian cancer cells were inoculated subcutaneously (s.c.) and intraperitoneally (representing the stage of intraperitoneal carcinomatosis) and murine renal adenocarcinoma Renca cells s.c. and orthotopically within the renal capsule. Body weight and tumor growth were followed, and mice were sacrificed according to humane endpoint criteria. To compare tumor growth in s.c. sites and within the abdomen, the tumor weight at sacrifice was divided by the number of days since the inoculation. In the HM-1 model, the mice with intraperitoneal tumor were sacrificed due to build-up of ascites on average six days earlier that the mice with s.c. tumor. In s.c. HM-1 tumor-bearing mice, the reason for sacrifice was tumor ulceration. In the Renca model, the s.c. and intrabursal models were approximately the same length. There was a striking difference in the tumor growth rate (mg/day) in s.c. versus abdominal sites in both models. The growth was 4-5 times faster in the abdominal sites than subcutaneously, highlighting the effect of tumor specific microenvironment. It is easy to understand that having promising results might be easier in a slower growing model. However, since both ovarian and renal cancer show very different growth kinetics within the abdominal cavity, testing drug candidates aimed for those indications in orthotopic models before accessing the clinical trials could give a more relevant evaluation of the potency of the drug candidates. Citation Format: Mari I. Suominen, Justyna Zdrojewska, Yvonne Konkol, Katja M. Fagerlund, Mervi Ristola, Jukka P. Rissanen. Comparison of subcutaneous and orthotopic tumor growth in syngeneic mouse models of ovarian and renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 49.

Abstract 418: Elacridar potentiates sunitinib efficacy in colorectal cancer models

Abstract For unresectable metastatic colorectal cancer (mCRC), the main treatment strategy is combination chemotherapy, targeted therapy, or immunotherapy. Long-term treatment benefit of these therapies is limited to a small subgroup of patients. To improve treatment outcome, many tyrosine kinase inhibitors (TKIs) have been clinically tested in patients with mCRC. Most failed already in early-phase clinical trials due to intrinsic resistance. ATP Binding Cassette Transporter (ABC)B1- and ABCG2-transporters are known contributors to treatment resistance in mCRC. Therefore, we evaluated whether inhibition of these transporters could potentiate the efficacy of TKIs in colorectal cancer (CRC) models. CRC cell lines, HT29, COLO320 and CACO2 and newly established patient-derived tumor organoids (PDTOs) from mCRC biopsies were first analyzed for the presence of ABCB1 and ABCG2 expression. Models that expressed ABCB1 or ABCG2 were pre-incubated with a non-toxic dose of elacridar, a potent transporter inhibitor, and exposed to a concertation range of three TKIs; sunitinib, cediranib and osimertinib. Next, we measured intracellular drug concentrations using LC/MS-MS one hour after TKI exposure (20 µM) with or without pre-incubation of elacridar. Using the fluorescent properties of sunitinib, we performed live-cell imaging to compare intracellular sunitinib distribution in cells that were or were not pre-incubated with elacridar. ABCB1 and ABCG2 expression was observed in all CRC models except for HT29 cells, which did not express ABCB1. Pre-incubation with elacridar significantly improved sunitinib efficacy in CRC cells. The IC50 decreased from 2.15 µM to 1.17 µM (p = 0.04), from 1.32 µM to 0.44 µM (p = 0.03), and from 4.94 µM to 3.43 µM (p = 0.03) for HT29, COLO320 and CACO2 cells respectively. This effect was not observed for cediranib and osimertinib. Although not significant, for both PDTO models tested, the IC50 for sunitinib decreased from 8.04 µM to 3.29 µM and from 6.93 µM to 4.16 µM. Intracellular sunitinib concentrations in the tested CRC cell models after sunitinib exposure ranged from 2.46 mM to 4.13 mM without elacridar and from 0.38 mM µM to 1.31 mM when pre-incubated with elacridar (p < 0.01). Live-cell imaging of CRC cells revealed a different pattern of intracellular sunitinib accumulation with and without pre-incubation with elacridar. Inhibition of multi-drug resistance proteins ABCB1 and ABCG2 with the clinically tested selective transporter inhibitor elacridar potentiates sunitinib efficacy in CRC models. Interestingly, this was accompanied with reduced intracellular sunitinib accumulation. Further research in mCRC PDTOs is warranted to confirm whether this mechanism is involved in intrinsic TKI resistance and therefore contribute to the early-phase failure of clinical trials testing TKIs for mCRC. Citation Format: Dennis Poel, Kirti K. Iyer, Bob van Gasteren, Beau Dagniaux, Erik van den Hombergh, Nielka P. van Erp, Daniele V. Tauriello, Henk M. Verheul. Elacridar potentiates sunitinib efficacy in colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 418.

Abstract 2857: Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC

Abstract Purpose: Patient-derived cells (PDCs) and organoids (PDOs) are constructed from patient tissue to mimic the biological features of patients. In cancer research, PDC/PDOs have been crucial as they can recapitulate tumor mutations. As heterogeneity between traditional cell lines and the human body leads to clinical trial failure, PDC/PDOs are widely used for predicting the preclinical drug efficacy. The aim of this study is to show that the PDC/PDOs can be used as effective tools for screening novel therapies on non-small cell lung cancer (NSCLC). Experimental design: PDC/PDO models from malignant effusions were established as following. We succeeded the establishment with samples which are positive for malignancy in cytology tests and tumor colony formation. Genotypes are analyzed by Sanger sequencing, Whole exome sequencing, or RNA-sequencing. Cell viability assay was performed using currently approved drugs or drugs in clinical development or their combinations. Results: A total of 46 PDCs and 150 PDOs was established from NSCLC patients, including models harboring sensitizing EGFR mutations, ALK fusions, ROS1 fusions, EGFR exon20 insertion, BRAF V600E, and those harboring various resistance mechanisms to EGFR-TKIs (T790M, C797S/C797G, MET amplification), to ALK-TKIs (G1202R), and to ROS1 TKI (G2032R). Osimertinib-resistant YU-1097 harboring EGFR resistance mutation (E19del/T790M/C797S) revealed sensitivity to BLU-945 (IC50, 108nM), a novel fourth-generation EGFR-TKI. A similar inhibition of cell viability was observed with repotrectinib (IC50, 21nM), a next-generation ROS1-TKI and lorlatinib (IC50, 9nM) in YU-1078 harboring CD74-ROS1, whereas more robust tumor regression was seen with repotrectinib in YU1078-derived xenograft model. Amivantamab, a EGFR-MET bispecific antibody, showed a robust activity in YU-1163 and YUO-036 in vitro and in vivo. YU-1077 harboring ALK G1202R solvent-front mutation showed sensitivity to NVL-655, a next-generation ALK-TKI, with a potency > 10-fold than that of lorlatinib. YUO-010 with MET amplification following osimertinib was sensitive to RGEN 5093-M114, a METxMET bispecific antibody-drug conjugate. Conclusions: PDC/PDO models can be utilized for evaluating activity of novel agents and will accelerate novel drug development in NSCLC. Citation Format: Yunjoo Joo, Sewon Park, Ju-hyeon Lee, Mi Ran Yun, Mi Ra Yu, Chun-Bong Synn, Seung Yeon Oh, Eun Ji Lee, Dong Kwon Kim, Seul Lee, Kyumin Lim, Min Hee Hong, Sun Min Lim, Chang Gon Kim, Ji Yun Lee, Jii Bum Lee, Byoung Chul Cho. Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2857.

Immune evaluation of granulocyte-macrophage colony stimulating factor loaded hierarchically 3D nanofiber scaffolds in a humanized mice model.

Background: Immune evaluation of biomaterials for tissue regeneration is a critical preclinical evaluation. The current evaluation criterion (ISO 10993-1 or GB/T 16886) uses rodents to perform the immune evaluation. However, the immune system of rodents is different from humans, the obtained results may not be reliable, which could lead directly to the failure of clinical trials. Granulocyte-macrophage colony-stimulating factor (GM-CSF) shows a great potential application in tissue regeneration by regulating local immune responses. The presented work combines the advantages of GM-CSF (immunoregulation) and hierarchically 3D nanofiber scaffolds (tissue regeneration). Methods: Firstly, we fabricated GM-CSF loaded 3D radially aligned nanofiber scaffolds, and then subcutaneous implantation was performed in humanized mice. The whole scaffold and surrounding tissue were harvested at each indicated time point. Finally, the cell infiltration and local immune responses were detected by histological observations, including H&E and Masson staining and immunochemistry. Results: We found significant cell migration and extracellular matrix deposition within the 3D radially aligned nanofiber scaffold after subcutaneous implantation. The locally released GM-CSF could accelerate the expression of human dendritic cells (CD11c) only 3days after subcutaneous implantation. Moreover, higher expression of human cytotoxic T cells (CD3+/CD8+), M1 macrophages (CD68/CCR7) was detected within GM-CSF loaded radially aligned nanofiber scaffolds and their surrounding tissues. Conclusions: The 3D radially aligned scaffold can accelerate cell migration from surrounding tissues to regenerate the wound area. And the locally released GM-CSF enhances dendritic cell recruitment and activation of cytotoxic T cells and M1 macrophages. Taken together, the GM-CSF loaded 3D radially aligned nanofiber scaffolds have a promising potential for achieving tissue regeneration.

Development on Animal Models for Drug/Chemical Induced Liver Injury

In the human body, the largest gland is the liver and does a lot of essential work of the body. Liver damage is the cause of mortality and increasing day by day. Liver disease is caused by multiple factors, such as an autoimmune condition, toxic chemical exposure, viral infection, and dietary factors. Drug-induced liver injury (DILI) is a critical issue in drug development because DILI causes failures in clinical trials and the withdrawal of approved drugs from the market and leading to pathological changes result, including increase in SGOT, SGPT and bilirubin as well as the free radical generation. In this review, contains the animal model of hepatotoxicity with a different cause, their action mech., and procedure with dose. These models include the toxicity caused by chemical, drug, radiation, metal, diet, and high-fat this will lead to pathological changes resulting in hepatotoxicity.

Differentiated approach to the pharmacotherapy of autism spectrum disorders: biochemical aspects

Autism Spectrum Disorders (ASD) are highly heterogeneous neurodevelopmental disorders caused by a complex interaction of numerous genetic and environmental factors and leading to deviations in the nervous system formation at very early developmental stages. Currently, there are no accepted pharmacological treatments for so-called core symptoms of ASD, such as social communication disorders and restricted and repetitive behavior patterns. The lack of knowledge about biological basis of ASD, the absence of clinically significant biochemical parameters reflecting abnormalities in signaling molecular cascades controlling the nervous system development and functioning, and the lack of methods for selection of clinically and biologically homogeneous subgroups are considered among the causes for the failure of clinical trials of ASD pharmacotherapy. This review considers the possibilities of applying a differentiated clinical and biological approach to the targeted search for ASD pharmacotherapy with an emphasis on biochemical markers associated with ASD and attempts to stratify patients by biochemical parameters. The use of approach such as “target-oriented therapy and assessment of the status of the target before and during the treatment to identify patients with a positive response to treatment” is discussed using the published results of clinical trials as examples. It is concluded that the identification of biochemical parameters for the identification of distinct subgroups among ASD patients requires research on large samples reflecting the clinical and biological diversity of patients with ASD, and the use of unified approaches for such studies. An integrated approach, including clinical observation, clinical-psychological assessment of patient behavior, study of anamnesis and description of individual molecular profiles should become a new strategy for stratifying and subgrouping patients with ASD for clinical pharmacotherapeutic trials, as well as for evaluating its efficiency.

Immune-mediated myogenesis and acetylcholine receptor clustering promote a slow disease progression in ALS mouse models

BackgroundAmyotrophic lateral sclerosis (ALS) is a heterogeneous disease in terms of onset and progression rate. This may account for therapeutic clinical trial failure. Transgenic SOD1G93A mice on C57 or 129Sv background have a slow and fast disease progression rate, mimicking the variability observed in patients.Based on evidence inferring the active influence of skeletal muscle on ALS pathogenesis, we explored whether dysregulation in hindlimb skeletal muscle reflects the phenotypic difference between the two mouse models.MethodsEx vivo immunohistochemical, biochemical, and biomolecular methodologies, together with in vivo electrophysiology and in vitro approaches on primary cells, were used to afford a comparative and longitudinal analysis of gastrocnemius medialis between fast- and slow-progressing ALS mice.ResultsWe reported that slow-progressing mice counteracted muscle denervation atrophy by increasing acetylcholine receptor clustering, enhancing evoked currents, and preserving compound muscle action potential. This matched with prompt and sustained myogenesis, likely triggered by an early inflammatory response switching the infiltrated macrophages towards a M2 pro-regenerative phenotype. Conversely, upon denervation, fast-progressing mice failed to promptly activate a compensatory muscle response, exhibiting a rapidly progressive deterioration of muscle force.ConclusionsOur findings further pinpoint the pivotal role of skeletal muscle in ALS, providing new insights into underestimated disease mechanisms occurring at the periphery and providing useful (diagnostic, prognostic, and mechanistic) information to facilitate the translation of cost-effective therapeutic strategies from the laboratory to the clinic.

GENDER-RELATED VARIATIONS IN PATHOPHYSIOLOGICAL RESPONSES TO METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PNEUMONIA AND SEPSIS.

In preclinical studies, the protective effects of female sex hormones and the immunosuppressive effects of male sex hormones were demonstrated. However, gender-related differences in multiorgan failure and mortality in clinical trials have not been consistently explained. This study aims to investigate gender-related differences in the development and progression of sepsis using a clinically relevant ovine model of sepsis. Adult Merino male (n=7) and female (n=7) sheep were surgically prepared with multiple catheters before the study. To induce sepsis, bronchoscopy instilled methicillin-resistant Staphylococcus aureus into sheep's lungs. The time from the bacterial inoculation until the modified Quick Sequential Organ Failure Assessment (q-SOFA) score became positive was measured and analyzed primarily. We also compared the SOFA score between these male and female sheep over time. Survival, hemodynamic changes, the severity of pulmonary dysfunction, and microvascular hyperpermeability were also compared. The time from the onset of bacterial inoculation to the positive q-SOFA in male sheep was significantly shorter than in female sheep. Mortality was not different between these sheep (14% vs. 14%). There were no significant differences in hemodynamic changes and pulmonary function between the two groups at any time point. Similar changes in hematocrit, urine output, and fluid balance were observed between females and males. The present data indicate that the onset of multiple organ failure and progression of sepsis is faster in male sheep than in female sheep, even though the severity of cardiopulmonary function is comparable over time. Further studies are warranted to validate the above results.

A Novel Rat Model of Embolic Cerebral Ischemia Using a Cell-Implantable Radiopaque Hydrogel Microfiber.

The failure of neuroprotective treatment-related clinical trials, including stem cell therapies, may be partially due to a lack of suitable animal models. We have developed a stem cell-implantable radiopaque hydrogel microfiber that can survive for a long time in vivo. The microfiber is made of barium alginate hydrogel containing zirconium dioxide, fabricated in a dual coaxial laminar flow microfluidic device. We aimed to develop a novel focal stroke model using this microfiber. Using male Sprague-Dawley rats (n=14), a catheter (inner diameter, 0.42 mm; outer diameter, 0.55 mm) was navigated from the caudal ventral artery to the left internal carotid artery using digital subtraction angiography. A radiopaque hydrogel microfiber (diameter, 0.4 mm; length, 1 mm) was advanced through the catheter by slow injection of heparinized physiological saline to establish local occlusion. Both 9.4-T magnetic resonance imaging at 3 and 6 h and 2% 2,3,5-triphenyl tetrazolium chloride staining at 24 h after stroke model creation were performed. Neurological deficit score and body temperature were measured. The anterior cerebral artery-middle cerebral artery bifurcation was selectively embolized in all rats. Median operating time was 4 min (interquartile range [IQR], 3-8 min). Mean infarct volume was 388 mm3 (IQR, 354-420 mm3) at 24 h after occlusion. No infarction of the thalamus or hypothalamus was seen. Body temperature did not change significantly over time (P = 0.204). However, neurological deficit scores before and at 3, 6, and 24 h after model creation differed significantly (P < 0.001). We present a novel rat model of focal infarct restricted to the middle cerebral artery territory using a radiopaque hydrogel microfiber positioned under fluoroscopic guidance. By comparing the use of stem cell-containing versus non-containing fibers in this stroke model, it would be possible to determine the efficacy of "pure" cell transplantation in treating stroke.

Translating precision medicine for autism spectrum disorder: A pressing need.

Autism spectrum disorder (ASD) is a heterogenous group of neurodevelopmental disorders (NDDs) with a high unmet medical need. Currently, ASD is diagnosed according to behavior-based criteria that overlook clinical and genomic heterogeneity, thus repeatedly resulting in failed clinical trials. Here, we summarize the scientific evidence pointing to the pressing need to create a precision medicine framework for ASD and other NDDs. We discuss the role of omics and systems biology to characterize more homogeneous disease subtypes with different underlying pathophysiological mechanisms and to determine corresponding tailored treatments. Finally, we provide recent initiatives towards tackling the complexity in NDDs for precision medicine and cost-effective drug discovery.

Neutrophil Heterogeneity and its Roles in the Inflammatory Network after Ischemic Stroke.

As the first peripheral immune cells to enter the brain after ischemic stroke, neutrophils are important participants in stroke-related neuroinflammation. Neutrophils are quickly mobilized from the periphery in response to a stroke episode and cross the blood-brain barrier to reach the ischemic brain parenchyma. This process involves the mobilization and activation of neutrophils from peripheral immune organs (including the bone marrow and spleen), their chemotaxis in the peripheral blood, and their infiltration into the brain parenchyma (including disruption of the blood-brain barrier, inflammatory effects on brain tissue, and interactions with other immune cell types). In the past, it was believed that neutrophils aggravated brain injuries through the massive release of proteases, reactive oxygen species, pro-inflammatory factors, and extracellular structures known as neutrophil extracellular traps (NETs). With the failure of early clinical trials targeting neutrophils and uncovering their underlying heterogeneity, our view of their role in ischemic stroke has become more complex and multifaceted. As neutrophils can be divided into N1 and N2 phenotypes in tumors, neutrophils have also been found to have similar phenotypes after ischemic stroke, and play different roles in the development and prognosis of ischemic stroke. N1 neutrophils are dominant during the acute phase of stroke (within three days) and are responsible for the damage to neural structures via the aforementioned mechanisms. However, the proportion of N2 neutrophils gradually increases in later phases, and this has a beneficial effect through the release of anti-inflammatory factors and other neuroprotective mediators. Moreover, the N1 and N2 phenotypes are highly plastic and can be transformed into each other under certain conditions. The pronounced differences in their function and their high degree of plasticity make these neutrophil subpopulations promising targets for the treatment of ischemic stroke.

Differentiated Approach to Pharmacotherapy of Autism Spectrum Disorders: Biochemical Aspects

Autism Spectrum Disorders (ASD) are highly heterogeneous neurodevelopmental disorders caused by a complex interaction of numerous genetic and environmental factors and leading to deviations in the nervous system formation at the very early developmental stages. Currently, there are no accepted pharmacological treatments for the so-called core symptoms of ASD, such as social communication disorders and restricted and repetitive behavior patterns. Lack of knowledge about biological basis of ASD, absence of the clinically significant biochemical parameters reflecting abnormalities in the signaling cascades controlling the nervous system development and functioning, and lack of methods for selection of clinically and biologically homogeneous subgroups are considered as causes for the failure of clinical trials of ASD pharmacotherapy. This review considers the possibilities of applying differentiated clinical and biological approaches to the targeted search for ASD pharmacotherapy with emphasis on biochemical markers associated with ASD and attempts to stratify patients by biochemical parameters. The use of such approach as "the target-oriented therapy and assessment of the target status before and during the treatment to identify patients with a positive response to treatment" is discussed using the published results of clinical trials as examples. It is concluded that identification of biochemical parameters for selection of the distinct subgroups among the ASD patients requires research on large samples reflecting clinical and biological diversity of the patients with ASD, and use of unified approaches for such studies. An integrated approach, including clinical observation, clinical-psychological assessment of the patient behavior, study of medical history and description of individual molecular profiles should become a new strategy for stratifying patients with ASD for clinical pharmacotherapeutic trials, as well as for evaluating their efficiency.

Ultrasound-Assisted Synthesis of Piperidinyl-Quinoline Acylhydrazones as New Anti-Alzheimer's Agents: Assessment of Cholinesterase Inhibitory Profile, Molecular Docking Analysis, and Drug-like Properties.

Alzheimer's disease (AD) is one of the progressive neurological disorders and the main cause of dementia all over the world. The multifactorial nature of Alzheimer's disease is a reason for the lack of effective drugs as well as a basis for the development of new structural leads. In addition, the appalling side effects such as nausea, vomiting, loss of appetite, muscle cramps, and headaches associated with the marketed treatment modalities and many failed clinical trials significantly limit the use of drugs and alarm for a detailed understanding of disease heterogeneity and the development of preventive and multifaceted remedial approach desperately. With this motivation, we herein report a diverse series of piperidinyl-quinoline acylhydrazone therapeutics as selective as well as potent inhibitors of cholinesterase enzymes. Ultrasound-assisted conjugation of 6/8-methyl-2-(piperidin-1-yl)quinoline-3-carbaldehydes (4a,b) and (un)substituted aromatic acid hydrazides (7a-m) provided facile access to target compounds (8a-m and 9a-j) in 4-6 min in excellent yields. The structures were fully established using spectroscopic techniques such as FTIR, 1H- and 13C NMR, and purity was estimated using elemental analysis. The synthesized compounds were investigated for their cholinesterase inhibitory potential. In vitro enzymatic studies revealed potent and selective inhibitors of AChE and BuChE. Compound 8c showed remarkable results and emerged as a lead candidate for the inhibition of AChE with an IC50 value of 5.3 ± 0.51 µM. The inhibitory strength of the optimal compound was 3-fold higher compared to neostigmine (IC50 = 16.3 ± 1.12 µM). Compound 8g exhibited the highest potency and inhibited the BuChE selectively with an IC50 value of 1.31 ± 0.05 µM. Several compounds, such as 8a-c, also displayed dual inhibitory strength, and acquired data were superior to the standard drugs. In vitro results were further supported by molecular docking analysis, where potent compounds revealed various important interactions with the key amino acid residues in the active site of both enzymes. Molecular dynamics simulation data, as well as physicochemical properties of the lead compounds, supported the identified class of hybrid compounds as a promising avenue for the discovery and development of new molecules for multifactorial diseases, such as Alzheimer's disease (AD).

Strategy toward Kinase-Selective Drug Discovery.

Kinase drug selectivity is the ground challenge in cancer research. Due to the structurally similar kinase drug pockets, off-target inhibitor toxicity has been a major cause for clinical trial failures. The pockets are similar but not identical. Here, we describe a transformation invariant protocol to identify distinct geometric features in the drug pocket that can distinguish one kinase from all others. We integrate available experimental structures with the artificial intelligence-based structural kinome, performing a kinome-wide structural bioinformatic analysis to establish the structural principles of kinase drug selectivity. We generate the structural landscape from the experimental kinase-ligand complexes and propose a binary network that encapsulates the information. The results show that all kinases contain binary units that are shared by less than seven other kinases in the kinome. 331 kinases contain unique binary units that may distinguish them from all others. The structural features encoded by these binary units in the network represent the inhibitor-accessible geometric space that may capture the kinome-wide selectivity. Our proposed binary network with the unsupervised clustering can serve as a general structural bioinformatic protocol for extracting the distinguishing structural features for any protein from their families. We apply the binary network to epidermal growth factor receptor tyrosine kinase inhibitor selectivity by targeting the gate area and the AKT1 serine/threonine kinase selectivity by binding to the αC-helix region and the allosteric pocket. Finally, we develop the cross-platform software, KDS (Kinase Drug Selectivity), for customized visualization and analysis of the binary networks in the human kinome (https://github.com/CBIIT/KDS).

Biomedical Advances in ABCA1 Transporter: From Bench to Bedside.

ATP-binding cassette transporter A1 (ABCA1) has been identified as the molecular defect in Tangier disease. It is biochemically characterized by absence of high-density lipoprotein cholesterol (HDL-C) in the circulation, resulting in the accumulation of cholesterol in lymphoid tissues. Accumulation of cholesterol in arteries is an underlying cause of atherosclerosis, and HDL-C levels are inversely associated with the presence of atherosclerotic cardiovascular disease (ASCVD). ABCA1 increases HDL-C levels by driving the generation of new HDL particles in cells, and cellular cholesterol is removed in the process of HDL generation. Therefore, pharmacological strategies that promote the HDL biogenic process by increasing ABCA1 expression and activity have been intensively studied to reduce ASCVD. Many ABCA1-upregulating agents have been developed, and some have shown promising effects in pre-clinical studies, but no clinical trials have met success yet. ABCA1 has long been an attractive drug target, but the failed clinical trials have indicated the difficulty of therapeutic upregulation of ABCA1, as well as driving us to: improve our understanding of the ABCA1 regulatory system; to develop more specific and sophisticated strategies to upregulate ABCA1 expression; and to search for novel druggable targets in the ABCA1-dependent HDL biogenic process. In this review, we discuss the beginning, recent advances, challenges and future directions in ABCA1 research aimed at developing ABCA1-directed therapies for ASCVD.

White matter injury, cholesterol dysmetabolism, and APP/Abeta dysmetabolism interact to produce Alzheimer's disease (AD) neuropathology: A hypothesis and review.

We postulate that myelin injury contributes to cholesterol release from myelin and cholesterol dysmetabolism which contributes to Abeta dysmetabolism, and combined with genetic and AD risk factors, leads to increased Abeta and amyloid plaques. Increased Abeta damages myelin to form a vicious injury cycle. Thus, white matter injury, cholesterol dysmetabolism and Abeta dysmetabolism interact to produce or worsen AD neuropathology. The amyloid cascade is the leading hypothesis for the cause of Alzheimer's disease (AD). The failure of clinical trials based on this hypothesis has raised other possibilities. Even with a possible new success (Lecanemab), it is not clear whether this is a cause or a result of the disease. With the discovery in 1993 that the apolipoprotein E type 4 allele (APOE4) was the major risk factor for sporadic, late-onset AD (LOAD), there has been increasing interest in cholesterol in AD since APOE is a major cholesterol transporter. Recent studies show that cholesterol metabolism is intricately involved with Abeta (Aβ)/amyloid transport and metabolism, with cholesterol down-regulating the Aβ LRP1 transporter and upregulating the Aβ RAGE receptor, both of which would increase brain Aβ. Moreover, manipulating cholesterol transport and metabolism in rodent AD models can ameliorate pathology and cognitive deficits, or worsen them depending upon the manipulation. Though white matter (WM) injury has been noted in AD brain since Alzheimer's initial observations, recent studies have shown abnormal white matter in every AD brain. Moreover, there is age-related WM injury in normal individuals that occurs earlier and is worse with the APOE4 genotype. Moreover, WM injury precedes formation of plaques and tangles in human Familial Alzheimer's disease (FAD) and precedes plaque formation in rodent AD models. Restoring WM in rodent AD models improves cognition without affecting AD pathology. Thus, we postulate that the amyloid cascade, cholesterol dysmetabolism and white matter injury interact to produce and/or worsen AD pathology. We further postulate that the primary initiating event could be related to any of the three, with age a major factor for WM injury, diet and APOE4 and other genes a factor for cholesterol dysmetabolism, and FAD and other genes for Abeta dysmetabolism.

The end of the beginning: progress and next steps in KRAS-mutant non-small-cell lung cancer

One of the most significant breakthroughs in oncology in recent years is the ability to target KRAS. KRAS is the most commonly mutated oncogene in human cancer, with greatest frequency in non-small-cell lung cancer (NSCLC), colorectal cancer, and pancreatic cancer. Since the gene's discovery several decades ago it had been considered an undruggable target with many failed clinical trials. The KRAS enzyme is critical for cell growth and survival, remaining inactive when bound to GDP and active when bound to GTP.

A human liver organoid screening platform for DILI risk prediction

Drug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved invitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. HLOs were derived from three separate iPSC lines and benchmarked on two platforms for their ability to model invitro liver function and identify hepatotoxic compounds using biochemical assays for albumin, ALT, AST, microscopy-based morphological profiling, and single-cell transcriptomics: i) HLOs dispersed in 384-well-formatted plates and exposed to a library of compounds; ii) HLOs adapted to a liver-on-chip system. Dispersed HLOs derived from the three iPSC lines had similar DILI predictive capacity as intact HLOs in a high-throughput screening format, allowing for measurable IC50 values of compound cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of toxicity. On-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known hepatoxic drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and displayed steatosis and mitochondrial perturbation, via phenotypic and transcriptomic analysis, on exposure to fialuridine and acetaminophen, respectively. The high-throughput and liver-on-chip systems exhibit enhanced invivo-like functions and demonstrate the potential utility of these platforms for DILI risk assessment. Tenofovir-inarigivr-associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients. Idiosyncratic (spontaneous, patient-specific) drug-induced liver injury (DILI) is difficult to study due to the lack of liver models that function as human liver tissue and are adaptable for large-scale drug screening. Human liver organoids grown from patient stem cells respond to known DILI-causing drugs in both a high-throughput and on a physiological "chip" culture system. These platforms show promise for researchers in their use as predictive models for novel drugs before entering clinical trials and as a potential invitro diagnostic tool. Our findings support further development of patient-derived liver organoid lines and their use in the context of DILI research.

P664 Analysis of Clinical Trial Screen Failures in IBD: Real World Results from the IOIBD

Abstract Background Recruitment rates for phase 2b/3 randomized controlled trials (RCTs) in IBD have substantially dropped over time. Several steps are required prior to successful patient randomization. Initially the physician must propose a trial to a potentially eligible patient during a pre-screening process (step 1). This is followed by patient’s acceptance or refusal (step 2). Finally, after informed consent the patient undergoes trial screening to ensure they meet all eligibility criteria (step 3). Evaluating each step separately, this study aims to assess reasons why IBD patients are not included in RCT and patients’ outcome after screen failure (SF). Methods All IOIBD member physicians (n=58) were invited to participate. To assess steps 1 and 2, consecutive IBD patients in relapse for whom a treatment change was required were prospectively included over a 4-week period. Reasons that prevented the IBD physician offering a sponsored multicenter phase 2b/3 RCT (step 1) and reasons why the patient accepted or refused to participate (step 2) were assessed through a physician and a patient survey, respectively. Reasons for SF (step 3) from the last 6 months, including the 4 weeks of steps 1-2, were collected retrospectively. Results A total of 104 (59 male, 62 CD, mean age of 37.2 years) and 102 patients (58 male, 63 CD, mean age of 40.6 years) from 12 centers were included in steps 1-2 and 3, respectively (Tables 1 and 2). Among 104 patients in relapse for whom a treatment change was required, 41 (39.4%) were offered a RCT. Of the 28 who consented to RCT, 5 failed their screening (SF rate of 17.9%) and 23 were included. Main reason that prevent IBD physicians from offering an RCT (step 1) are shown in Figure 1. After receiving information about RCT, major reasons why patients accepted or refused to participate included the trust they had in their IBD specialist and the risk of being assigned to a placebo, respectively (step 2). Regarding 102 patients included in step 3, main reasons of SF were insufficient disease activity (n=37), concurrent infection (n=15) and dropout (n=12) (Figure 2). Half of SFs could have been avoided by thorough prescreening. After SF, 51 patients were treated with commercially available therapy, 14 were rescreened for the same RCT (after resolution of the issue leading to SF), no treatment was required for 14, 10 were referred to surgery and 6 were screened for another RCT (the outcome was unknown for 7). Conclusion This first multicentric study reported a SF rate of 17.9%. Insufficient disease activity and the risk of assigning the patient to a placebo seem to be barriers to inclusion. Half of SFs could have been avoided by better pre-screening. After SF, most of patients were treated with commercially available therapy.

CSM-Toxin: A Web-Server for Predicting Protein Toxicity

Biologics are one of the most rapidly expanding classes of therapeutics, but can be associated with a range of toxic properties. In small-molecule drug development, early identification of potential toxicity led to a significant reduction in clinical trial failures, however we currently lack robust qualitative rules or predictive tools for peptide- and protein-based biologics. To address this, we have manually curated the largest set of high-quality experimental data on peptide and protein toxicities, and developed CSM-Toxin, a novel in-silico protein toxicity classifier, which relies solely on the protein primary sequence. Our approach encodes the protein sequence information using a deep learning natural languages model to understand "biological" language, where residues are treated as words and protein sequences as sentences. The CSM-Toxin was able to accurately identify peptides and proteins with potential toxicity, achieving an MCC of up to 0.66 across both cross-validation and multiple non-redundant blind tests, outperforming other methods and highlighting the robust and generalisable performance of our model. We strongly believe the CSM-Toxin will serve as a valuable platform to minimise potential toxicity in the biologic development pipeline. Our method is freely available as an easy-to-use webserver.