Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERRα, C-Abelson, and as well as their relevant small-molecule compounds in PD models, which will shed light on a clue on exploiting more potential targeted small-molecule drugs tracking PD treatment in the near future.     

CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics

The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as Cyp, Abcb1, Oatp1b2 gene knockout rats. These new rat models are not only widely used in the study of drug metabolism, chemical toxicity, and carcinogenicity, but also promote the study of DMPK related mechanism, and further strengthen the relationship between drug metabolism and pharmacology/toxicology. This review systematically introduces the advantages and disadvantages of CRISPR-Cas9, summarizes the methods of establishing DMPK rat models, discusses the main challenges in this field, and proposes strategies to overcome these problems.     

Targeting RAS phosphorylation in cancer therapy: Mechanisms and modulators

RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an ‘undruggable’ feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.     

Therapeutic regulation of autophagy in hepatic metabolismOA

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ thatgoverns body energy metabolism. Autophagy’s role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. T...     

The development and validation of methods for evaluating the immune system in preweaning piglets

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.     

Human leukocyte antigen and idiosyncratic adverse drug reactions

A clinical association between a specific human leukocyte antigen (HLA) allele and idiosyncratic adverse drug reactions (IADRs) is a strong indication that IADRs are mediated by the adaptive immune system. For example, it is well-established that HLA-B*15:02 and HLA-B*57:01 are associated with carbamazepine-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and abacavir-induced hypersensitivity/flucloxacillin-induced liver injury, respectively. Drug-specific T-cells whose response is restricted by specific HLA risk alleles have been detected from IADR patients, also suggesting an adaptive immune pathogenesis. T-cells from carbamazepine SJS/TEN patients are activated by direct pharmacological interaction between carbamazepine and HLA-B*15:02 expressed on antigen presenting cells (APCs). Abacavir-specific, HLA-B*57:01-restricted T-cells are activated by APCs presenting peptides which are only displayed by the HLA molecule when abacavir is bound during peptide loading. Finally, HLA-B*57:01-restricted activation of T-cells from patients with flucloxacillin-induced liver injury is dependent on processing of drug protein adducts. Based on these observations, it is now possible to utilize blood from healthy drug-naïve volunteers to study the priming of naïve T-cells to drugs. Future development of these methodologies may lead to the development of assays that predict intrinsic immunogenicity of drugs and chemicals at the preclinical stage of drug development.     

Emerging aspects of nanotoxicology in health and disease: From agriculture and food sector to cancer therapeutics

Nanotechnology is an evolving scientific field that has allowed the manufacturing of materials with novel physicochemical and biological properties, offering a wide spectrum of potential applications. Properties of nanoparticles that contribute to their usefulness include their markedly increased surface area in relation to mass, surface reactivity and insolubility, ability to agglomerate or change size in different media and enhanced endurance over conventional-scale substance. Here, we review nanoparticle classification and their emerging applications in several fields; from active food packaging to drug delivery and cancer research. Nanotechnology has exciting therapeutic applications, including novel drug delivery for the treatment of cancer. Additionally, we discuss that exposure to nanostructures incorporated to polymer composites, may result in potential human health risks. Therefore, the knowledge of processes, including absorption, distribution, metabolism and excretion, as well as careful toxicological assessment is critical in order to determine the effects of nanomaterials in humans and other biological systems. Expanding the knowledge of nanoparticle toxicity will facilitate designing of safer nanocomposites and their application in a beneficial manner.     

Graphical display of histopathology data from toxicology studies for drug discovery and development: An industry perspective

Histopathology data comprise a critical component of pharmaceutical toxicology studies and are typically presented as finding incidence counts and severity scores per organ, and tabulated on multiple pages which can be challenging for review and aggregation of results. However, the SEND (Standard for Exchange of Nonclinical Data) standard provides a means for collecting and managing histopathology data in a uniform fashion which can allow informatics systems to archive, display and analyze data in novel ways. Various software applications have become available to convert histopathology data into graphical displays for analyses. A subgroup of the FDA-PhUSE Nonclinical Working Group conducted intra-industry surveys regarding the use of graphical displays of histopathology data. Visual cues, use-cases, the value of cross-domain and cross-study visualizations, and limitations were topics for discussion in the context of the surveys. The subgroup came to the following conclusions. Graphical displays appear advantageous as a communication tool to both pathologists and non-pathologists, and provide an efficient means for communicating pathology findings to project teams. Graphics can support hypothesis-generation which could include cross-domain interactive visualizations and/-or aggregating large datasets from multiple studies to observe and/or display patterns and trends. Incorporation of the SEND standard will provide a platform by which visualization tools will be able to aggregate, select and display information from complex and disparate datasets.     

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS–STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.     

A novel quinazolinone chalcone derivative induces mitochondrial dependent apoptosis and inhibits PI3K/Akt/mTOR signaling pathway in human colon cancer HCT-116 cells

We have synthesized a novel quinazolinone chalcone derivative (QC) and first time reported its in-vitro and in-vivo anticancer potential. It inhibited the cell proliferation of different cancer cell lines like PC-3, Panc-1, Mia-Paca-2, A549, MCF-7 and HCT-116. It induces apoptosis as measured by several biological endpoints such as apoptotic body formation, evident by Hoechst and scanning electron microscopy, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), reduction of Bcl-2/Bax ratio, activation of caspase-9, caspase-3 and PARP-1 (poly-ADP Ribose polymerase) cleavage in HCT-116 cells. In spite of apoptosis, QC significantly hammers the downstream and upstream signaling cascade of PI3K/Akt/mTOR pathway and cell cycle regulator Skp-2, p21 and p27. Interestingly, QC induces the S and G2/M phase of HCT-116 cells at experimental doses. QC inhibits the tumor growth of Ehrlich ascites carcinoma (EAC), Ehrlich tumor (ET, solid) and sarcoma-180(solid) mice models. Furthermore, it was found to be non-toxic as no animal mortality (0/7) occurred during experimental doses. The present study provides an insight of anticancer potential of QC, which may be useful in managing and treating cancer.     

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer,in Sprague Dawley rats and Gottingen minipigs

PurposeExtensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer ¹²³I-CMICE-013 to support applications for clinical trials.MethodsSprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.ResultsThe acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.ConclusionThe lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.     

Targeting glutamine utilization to block metabolic adaptation of tumor cells under the stress of carboxyamidotriazole-induced nutrients unavailability

Tumor cells have unique metabolic programming that is biologically distinct from that of corresponding normal cells. Resetting tumor metabolic programming is a promising strategy to ameliorate drug resistance and improve the tumor microenvironment. Here, we show that carboxyamidotriazole (CAI), an anticancer drug, can function as a metabolic modulator that decreases glucose and lipid metabolism and increases the dependency of colon cancer cells on glutamine metabolism. CAI suppressed glucose and lipid metabolism utilization, causing inhibition of mitochondrial respiratory chain complex I, thus producing reactive oxygen species (ROS). In parallel, activation of the aryl hydrocarbon receptor (AhR) increased glutamine uptake via the transporter SLC1A5, which could activate the ROS-scavenging enzyme glutathione peroxidase. As a result, combined use of inhibitors of GLS/GDH1, CAI could effectively restrict colorectal cancer (CRC) energy metabolism. These data illuminate a new antitumor mechanism of CAI, suggesting a new strategy for CRC metabolic reprogramming treatment.     

Benzyl alcohol protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes but causes mitochondrial dysfunction and cell death at higher doses

Acetaminophen (APAP) hepatotoxicity is a serious public health problem in western countries. Current treatment options for APAP poisoning are limited and novel therapeutic intervention strategies are needed. A recent publication suggested that benzyl alcohol (BA) protects against APAP hepatotoxicity and could serve as a promising antidote for APAP poisoning. To assess the protective mechanisms of BA, C56Bl/6J mice were treated with 400 mg/kg APAP and/or 270 mg/kg BA. APAP alone caused extensive liver injury at 6 h and 24 h post-APAP. This injury was attenuated by BA co-treatment. Assessment of protein adduct formation demonstrated that BA inhibits APAP metabolic activation. In support of this, in vitro experiments also showed that BA dose-dependently inhibits cytochrome P450 activities. Correlating with the hepatoprotection of BA, APAP-induced oxidant stress and mitochondrial dysfunction were reduced. Similar results were obtained in primary mouse hepatocytes. Interestingly, BA alone caused mitochondrial membrane potential loss and cell toxicity at high doses, and its protective effect could not be reproduced in primary human hepatocytes (PHH). We conclude that BA protects against APAP hepatotoxicity mainly by inhibiting cytochrome P450 enzymes in mice. Considering its toxic effect and the loss of protection in PHH, BA is not a clinically useful treatment option for APAP overdose patient.     

Identification of cytochrome P450 isoforms involved in the metabolism of Syl930, a selective S1PR1 agonist acting as a potential therapeutic agent for autoimmune encephalitis

Syl930 is a novel sphingosine-1-phosphate receptor subtype 1 (S1PR₁) agonist for the treatment of autoimmune encephalitis with promising receptor selectivity and little risk of bradycardia. Syl930 could be reversibly converted to its phosphorylated metabolite, acting as the active form to provide therapeutic effects, but eliminated principally in the form of oxidative metabolites. The aim of the present study was to identify the cytochrome P450 isoforms (CYPs) responsible for the oxidative metabolism of Syl930. Considerable production of hydroxylated metabolite (Syl930-M1) was found in both rat blood and tissue homogenates in vivo and in vitro. Moreover, another hydroxylated metabolite, Syl930-M2, was detected in human, beagle dog and cynomolgus monkey liver microsomes with significant differences in the K_m, V_max and CL_int of the metabolites among species. CYP1A1, CYP2J2, CYP4F2 and CYP3A4 were identified to be the major CYPs mediated in the hydroxylation of Syl930 by using 14 recombinant human CYPs, selective chemical inhibitors and monoclonal antibodies against CYPs. The multiple CYPs mediated oxidation was believed to be one of the reasons for the relatively short elimination half-life of Syl930.     

Targeting a cryptic allosteric site of SIRT6 with small-molecule inhibitors that inhibit the migration of pancreatic cancer cells

SIRT6 belongs to the conserved NAD⁺-dependent deacetylase superfamily and mediates multiple biological and pathological processes. Targeting SIRT6 by allosteric modulators represents a novel direction for therapeutics, which can overcome the selectivity problem caused by the structural similarity of orthosteric sites among deacetylases. Here, developing a reversed allosteric strategy AlloReverse, we identified a cryptic allosteric site, Pocket Z, which was only induced by the bi-directional allosteric signal triggered upon orthosteric binding of NAD⁺. Based on Pocket Z, we discovered an SIRT6 allosteric inhibitor named JYQ-42. JYQ-42 selectively targets SIRT6 among other histone deacetylases and effectively inhibits SIRT6 deacetylation, with an IC₅₀ of 2.33 μmol/L. JYQ-42 significantly suppresses SIRT6-mediated cancer cell migration and pro-inflammatory cytokine production. JYQ-42, to our knowledge, is the most potent and selective allosteric SIRT6 inhibitor. This study provides a novel strategy for allosteric drug design and will help in the challenging development of therapeutic agents that can selectively bind SIRT6.     

In vitro ocular metabolism and bioactivation of ketoconazole in rat,rabbit and human

Oral ketoconazole is clinically administered for treatment of severe cases for fungal keratitis. Pharmacodynamics and efficacy of oral and topical (ocular) ketoconazole have been explored in rabbit. However, metabolism of ketoconazole in the eye in any species is not well explored in any preclinical species or human. An understanding of ocular drug metabolism in the eye is crucial for ocular therapeutics to facilitate the risk assessment and development of potential drug candidates for the clinic. We aimed to investigate the metabolism of ketoconazole in rat, rabbit and human ocular S9 fractions. Metabolism in liver S9 fractions was also studied for a direct comparison. Eleven putative metabolites were identified in the in vitro incubations. Of these metabolites, six were present in rat ocular S9 whereas eight were present in rabbit and human ocular matrices. Metabolic pathways in rabbit and human ocular fractions suggested the formation of reactive intermediates in rabbit and human liver and ocular S9 incubations, which was confirmed with trapping studies. Herein, we report eight human ocular metabolites of ketoconazole for the first time. To the best of our knowledge, this is the first report of ocular metabolic pathways and ocular bioactivation of ketoconazole in preclinical species and human.     

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18–156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.     

Antioxidant power as biochemical endpoint in bread for screening and early managing quality and toxicant-related safety anomalies in food production

Flaxseeds are both a food ingredient and a natural source of antioxidants (e.g. lignans, PUFAs) and pro-oxidant contaminants (e.g. cadmium): the variable mixture of anti- and pro-oxidant substances may impact on the redox homeostasis of flaxseed-enriched foods. The antioxidant power is studied here as biochemical activity of flaxseeds in white wheat bread and as endpoint for possible screening of anomalous variations of bioactive mixtures (antioxidants vs. prooxidants) in food matrices.A bioprobe assay based on the superoxide dismutase (SOD) enzyme (6 channels of the multiprobe bioelectronic platform BEST) was performed on white wheat bread with and without flaxseeds. Nine BEST channels were simultaneously used for validation and monitoring of measuring conditions (temperature, pH, conductivity). Findings were compared with quantitative analysis of antioxidants and pro-oxidant contaminants. Organic and aqueous extracts of both bread types were examined in parallel.The SOD-probe detected the difference in antioxidant power given by 10% flaxseed, thus supporting the use of antioxidant power detected by bioenzymatic screening as sensitive biochemical endpoint. Mixtures of bioactive molecules in foods generate biochemical activities that can be monitored as time-effective indicators of invariability, which is pivotal in the daily control of anomalies in food production and therefore in the protection of consumers′ health.