Generation of cynomolgus monkey airway, liver ductal, and kidney organoids with pharmacokinetic functions

To assess the potential value of the 6-day subrenal capsule (SRC) assay in preclinical evaluation of new drugs using serially xenografted human tumors as source of tumor tissue, we studied the response of 31 human tumor lines (8 malignant melanomas, 12 sarcomas, 9 lung carcinomas and 2 colon carcinomas) to relevant standard drugs and to a new imidazotetrazine, Mitozolomide. Mitozolomide was found to be the most active drug tested in 50% of the lung carcinomas and as active as CCNU in melanomas. The activity of the standard anticancer drugs against subrenal grafts closely resembled the patterns seen with the same tumors in the clinic. In further attempts to validate the procedure, sensitivity profiles of some tumors were concurrently determined in the subcutaneous (s.c.) nude mouse model. In 11 out of the 12 tumors, the two assays selected the same drug as being the most active and in most of these tumors the two procedures gave the same ranking for the different drugs. Also, when the relative sensitivities of a series of melanoma xenografts to each of two drugs (DTIC and CCNU) were tested, the two assays gave the same ranking of the xenografts for each drug. The concordance between the two assays and the fact that the s.c. nude mouse assay reflects the chemosensitivity of the parent tumor in patients, suggest that the application of the 6-day SRC assay to xenografted tumors is a valid and useful procedure permitting rapid preclinical evaluation of new drugs to be carried out at relatively low cost.

BackgroundBuruli ulcer (BU) caused by Mycobacterium ulcerans is the world's third most common mycobacterial infection. There is no vaccine against BU and surgery is needed for patients with large ulcers. Although recent experience indicates combination chemotherapy with streptomycin and rifampin improves cure rates, the utility of this regimen is limited by the 2-month duration of therapy, potential toxicity and required parenteral administration of streptomycin, and drug-drug interactions caused by rifampin. Discovery and development of drugs for BU is greatly hampered by the slow growth rate of M. ulcerans, requiring up to 3 months of incubation on solid media to produce colonies. Surrogate markers for evaluating antimicrobial activity in real-time which can be measured serially and non-invasively in infected footpads of live mice would accelerate pre-clinical evaluation of new drugs to treat BU. Previously, we developed bioluminescent M. ulcerans strains, demonstrating proof of concept for measuring luminescence as a surrogate marker for viable M. ulcerans in vitro and in vivo. However, the requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments.Methodology/Principal FindingFor this study, we engineered M. ulcerans strains that express the entire luxCDABE operon and therefore are autoluminescent due to endogenous substrate production. The selected reporter strain displayed a growth rate and virulence similar to the wild-type parent strain and enabled rapid, real-time monitoring of in vitro and in vivo drug activity, including serial, non-invasive assessments in live mice, producing results which correlated closely with colony-forming unit (CFU) counts for a panel of drugs with various mechanisms of action.Conclusions/SignificanceOur results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity.

Impact of obese levels on the hepatic expression of nuclear receptors and drug-metabolizing enzymes in adult and offspring mice

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.

Changes in the expression of drug-metabolizing enzymes and transporters can alter the pharmacokinetics of drugs, potentially affecting their efficacy and safety. In this study, we investigated the effects of decreased multidrug resistance–associated protein (MRP) 2 expression on the gene expression of other drug-metabolizing enzymes and transporters. Variations in the mRNA expression of drug-metabolizing enzymes and transporters were observed in MRP2-knockdown human hepatocellular carcinoma HepG2 cells and the liver of MRP2-deficient Eisai hyperbilirubinemic rats (EHBR). Both models showed decreased mRNA and protein expression of sulfotransferase (SULT) 1E1, a phase II drug-metabolizing enzyme, suggesting a relationship between the transcriptional regulation of MRP2 and SULT1E1. The plasma levels of bilirubin, bile acids, and cholesterol were higher in EHBR than in control Sprague-Dawley rats. Treatment with chenodeoxycholic acid (CDCA), a primary bile acid, reduced SULT1E1 mRNA expression in HepG2 cells and suppressed human SULT1E1 promoter activity in a luciferase reporter assay using HepG2 cells. CDCA is a known agonist of the farnesoid X receptor (FXR), and transcriptome analysis of the EHBR liver also suggested FXR activation, as inferred from changes in its target gene expression. These findings suggest that decreased MRP2 expression causes coordinated changes in the SULT1E1 gene expression via FXR activation by endogenous substances. These indirect changes in the expression of drug-metabolizing enzymes or transporters should be considered during drug development and in clinical practice.Significance StatementThis study investigated compensatory or coordinated changes in gene expression of drug-metabolizing enzymes and transporters in multidrug resistance–associated protein (MRP) 2–knockdown HepG2 cells and in the liver of MRP2-deficient rats. Decreased expression of MRP2 affects the gene expression of drug-metabolizing enzymes and transporters, including a decrease in SULT1E1, likely through nuclear receptor activation by endogenous molecules.

In this time of escalating costs there is a compelling need to minimize the costs in time, money and valuable resources spent on preclinical evaluation of new drugs. International harmonisation of requirements is a major step in this direction.

Positron emission tomography (PET) and 11C-raclopride were used to measure the occupancy of central dopamine D2 receptors by a new neuroleptic, CP-88,059-1. In a double blind dose escalation study, seven healthy male subjects received a predose of between 2 mg and 60 mg CP-88,059-1, 5 h before PET scanning. One additional subject was assigned to placebo predose. Receptor occupancy was defined as the percentage reduction in binding potential compared with that seen in the subject predosed with placebo and with that seen in seven unmedicated normal volunteers previously studied. Binding of 11C-raclopride decreased in a dose dependent manner, and 85% dopamine D2 receptor occupancy was achieved with the highest dose of CP-88,059-1. The findings confirm that brain dopamine D2 receptors are blocked by CP-88,059-1 and suggest that an effective antipsychotic dose will be between 20 mg and 40 mg. The study high-lights the potential of positron emission tomography in the preclinical evaluation of new drugs.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States and the world; with no Food and Drug Administration–approved pharmacological treatment available, it remains an area of unmet medical need. In nonalcoholic steatohepatitis (NASH), the most important predictor of clinical outcome is the fibrosis stage. Moreover, the Food and Drug Administration recommends that clinical trials for drugs to treat this disease include patients with fibrosis stage 2 or greater. Therefore, when using animal models for investigating the pathophysiology of NAFLD and for the preclinical evaluation of new drugs, it is important that the animals develop substantial fibrosis. The aim of this study was to develop a mouse model of NAFLD that replicated the disease in humans, including obesity and progressive liver fibrosis. Agouti yellow mutant mice, which have hyperphagia, were fed a Western diet and water containing high‐fructose corn syrup for 16 weeks. Mice became obese and developed glucose intolerance. Their gut microbiota showed dysbiosis with changes that replicate some of the changes described in humans with NASH. They developed NASH with activity scores of 5–6 and fibrosis, which was stage 1 after 16 weeks, and stage 3 after 12 months. Changes in liver gene expression assessed by gene‐set enrichment analysis showed 90% similarity with changes in human patients with NASH. Conclusion: Ay mice, when fed a Western diet similar to that consumed by humans, develop obesity and NASH with liver histology, including fibrosis, and gene expression changes that are highly similar to the disease in humans.

BACKGROUND: The 5-year survival rate of patients with recurrent epithelial ovarian cancer is only 30%, in part due to relapse and resistance to first-line platinum-based chemotherapy. Even with marked progress toward understanding ovarian cancer biology, the translation of research findings into new therapies is still an enormous barrier to progress. Recent data suggests a 90% failure rate for new oncology drugs in the clinic. Development and preclinical testing of new cancer therapies has been limited by the availability of clinically relevant models that recapitulate the molecular and phenotypic characteristics of primary ovarian cancers. To overcome this barrier, we have initiated collaborative project between Stephenson Cancer Center (SCC) and Oklahoma Medical Research Foundation (OMRF) with the goal to generate a biobank of patient-derived xenograft (PDX) models derived directly from ovarian cancer patients undergoing therapy. RESULTS: PDX tumor models are generated in the Patient-Derived Xenograft and Preclinical Therapeutics (PDX-PCT) Core at OMRF and used to improve preclinical evaluation of new drugs towards more personalized medicine. We have collected blood samples and tumor tissues from consenting patients having primary debulking surgery at Stephenson Cancer Center since 2015 (>140 unique patients). Fresh tumor tissue is minced into small tumor chunks (~2 mm) and implanted into immunocompromised mice. PDXs are expanded for 3-4 passages and characterized based on tumor type, histology and molecular characteristics. Immunohistochemical and molecular analysis showed that histology of the original patient tumors as well as the expression of commonly used markers for high-grade serous ovarian cancer such as cytokeratin, PAX8 and WT1 are perfectly maintained in the derivative PDX lines. Genotyping of patients' tumors and their corresponding PDXs using Illumina's Exome Array revealed that single nucleotide polymorphism (SNP) profile found in the original tumors is perfectly maintained in PDX lines. One of the PDX limitations is that human tumor stromal and immune cells are being replaced with mouse equivalents upon in vivo propagation of PDXs, which negatively affects quality of genomic or proteomic analyses. To overcome this challenge, we optimized a method to deplete mouse cells from PDX tumors by magnetic cells separation. Our data showed that ovarian cancer PDXs contain around 28-54% of human tumor cells, where the rest are mouse cells. We also optimized protocols to utilize pure population of human tumor cells isolated from PDXs to transduce these with lentiviral vectors expressing GFP and/or luciferase to generate luciferized PDXs. Luciferized ovarian PDXs are excellent models used for non-invasive orthotopic tumor growth and drug response monitoring. CONCLUSIONS: Collection of our PDXs illustrates heterogeneity and diversity of human ovarian tumors. However, each PDX maintains essential molecular features of the original patients' tumor. Our goal is to generate clinically faithful ovarian tumor models that will provide a platform to learn more about cancer biology and to screen these for new better therapies. Citation Format: Magdalena Cybula, Katherine Moxley, Lin Wang, Luyao Wang, Magdalena Bieniasz. UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-021.

Lower vertebrate and invertebrate models of Alzheimer's disease – A review

Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease. Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.

This review aimed to review the biological, pharmacological, and phytochemical aspects of the genus Haloxylon. Plants of the genus Haloxylon have been used for a long time in traditional medicine, and they are distributed in the western Mediterranean region to the Middle East, Iran, Mongolia, Burma, and southwest China. The studied parts of Haloxylon species include aerial parts, leaves, branches, seeds, roots, rhizosphere, soil, and whole plants, used to treat several diseases, including sexual disorders, hepatobiliary disorders, eye disorders, skin diseases and hemorrhoids, diarrhea, and effective in the treatment of various ailments such as snake bite, stomach ache, diabetes, wounds, earache and sciatica pain, windbreak dune fixation, feeding of livestock and firewood. Till now, no review on the genus Haloxylon has been conducted. This review aimed to provide updated information on the genus Haloxylon, including traditional medicinal uses, valorization and exploitation of medicinal plants, phytochemistry, botanical characterization, pharmacological and toxicological research focusing on the medicinal properties of several Haloxylon species, especially their antioxidant, antibacterial, anti-inflammatory, cytotoxic and antifungal activities, as well as the effect of each bioactive molecule isolated from these species and their pharmacological use, including the preclinical evaluation of new drugs. The present work was conducted using various scientific databases, including Science Direct, Scopus, PubMed, Google Scholar, etc. Correct plant names were verified from plantlist.org. The results of this search were interpreted, analyzed, and documented based on the obtained bibliographic information. Among all species of the Chenopodiaceae family, 6 species of the Haloxylon genus have approved antioxidant activity, 5 species have antibacterial activity, 3 species have anti-inflammatory activity, 2 species have cytotoxic activity, and 3 species have antifungal activity. The majority of the chemical constituents of this plant include flavonoids, alkaloids, phenols, saponins, glycosides, and tannins. Among them, the main bioactive constituents would be present in the alkaloid fraction. The study of more than 9 Haloxylon plants has identified more than 46 compounds. Pharmacological research proved that crude extracts and some pure compounds obtained from Haloxylon had activities for the treatment of different diseases. The objective of the present study was focused on antioxidant, antibacterial, anti-inflammatory, cytotoxic and antifungal diseases. From the study of the phytochemistry of the Haloxylon family, it was concluded that all studied plants had active compounds. Among them, 11 isolated molecules have medicinal activities with antioxidant properties, 10 molecules showed antibacterial effects, more than 6 molecules have anti-inflammatory properties, more than 9 isolated molecules have medicinal activities against cytotoxic diseases, and more than 28 molecules have antifungal effects. Therefore, the safety of Haloxylon herbal medicine should be considered a top priority in the early stages of development and clinical trials. Several previously conducted studies have validated multiple traditional uses of Haloxylon species. Further research is needed on Haloxylon plants before they can be fully utilized in the clinic as a potent drug candidate, as researchers are mainly focusing on alkaloids, diterpenoids, and triterpenoids, whereas there are many other types of compounds that may possess novel biological activities.

Gene fusions involving ETS transcription factors (predominantly ERG and ETV1) and PTEN deletions are prevalent in the prostate cancer genome. This report describes a novel mouse model that overexpresses ERG and lacks PTEN with the majority of mice developing prostate tumors by 6 mo. Biological mechanisms suggest increased/altered binding of the male hormone receptor in the genome. This model will be useful in pre-clinical evaluation of new drugs targeting these common prostate cancer genomic alterations.

ABSTRACTGambling is a harmless pastime for many, but for some it can become problematic with serious social and financial consequences. To date, no pharmacological treatments for gambling disorder have been approved. Progress in this regard is undoubtedly hampered by the lack of established preclinical models that allow for the screening of the potential efficacy of new approaches. The Rat Gambling Task (rGT), based on the Iowa Gambling Task in humans, is a model of some of the decision-making processes involved in gambling. The purpose of the present review is to summarize the literature to date on the use of the rGT for preclinical testing of pharmacological agents. First, the rGT is described and compared to the IGT. Next, validity is examined to establish the rGT as a viable model for preclinical evaluation of new drugs. Finally, the available data on the effects of pharmacological challenges on the rGT are reviewed focusing on dopamine, norepinephrine, serotonin and opioid systems. It is concluded that the rGT may provide a viable preclinical model for new drug development for the treatment of gambling.

Plants of the genus Euphorbia have long been used as traditional medicine in China, Europe, America, Turkey, India, Africa, Iran, and Pakistan for their great medicinal value and health benefits. Their stems, leaves, roots, and latex are widely used to treat respiratory infections, body and skin irritations, digestive disorders, inflammatory infections, body pain, snake or scorpion bites, pregnancy, sensory disturbances, microbial and anticancer diseases. This review aims to provide updated information on the genus Euphorbia, including traditional medicinal uses, valuation and exploitation of medicinal plants, phytochemistry, botanical characterization, pharmacological and toxicological research focused on the medicinal properties of several Euphorbias in particular their antibacterial, anti-tumor, and cell manifestations, in addition to the effect of each isolated bioactive molecule from these species and their pharmacological use including preclinical evaluation of new drugs. This work was conducted using scientific databases such as: PubMed, Google Scholar, Scopus, Science Direct, etc. In addition, ChemDraw software has been used for the drawing of chemical molecules. The correct names of the plants were confirmed from plantlist.org. The results of this review research were interpreted, analyzed and documented based on the bibliographical information obtained. Among all the species of the Euphorbiaceae family, 15 species have been demonstrated to exhibit anticancer activity, 21 species have antibacterial activity, and 10 species have cytotoxic activity. The majority of the chemical constituents of this plant include triterpenoid glycosides, diterpenoids, flavonoids, and the 4α-methyl steroids. Among them, the main bioactive constituents are present in the diterpenoid fraction. The study of more than 33 steroid plants identified more than 104 compounds. Pharmacological research proved that the crude extracts and some pure compounds obtained from Euphorbia had activities for the treatment of different diseases. The objective of the present study was focused on cytotoxic, antibacterial and antitumor diseases. The study of the phytochemistry of the Euphorbia families led to the conclusion that all the plants studied had active compounds, of which 27 plants were characterized by their cytotoxic effects, 7 had antibacterial effects and 10 plants had anti-tumor activities. Therefore, the safety of Euphorbia herbal medicine should be considered a top priority in the early stages of development and clinical trials. Many previous studies have validated many traditional uses of Euphorbia species. The latex of some Euphorbia species seems to be toxic however, studies dealing with the safety and quality of these species are still incomplete. Extensive studies are needed on the Euphorbia plants before they can be fully used in clinics as a potent drug candidate, as researchers are focusing mainly on diterpenoids and triterpenoids, while there are many other types of compounds that may possess new biological activities.