Derivation of Naïve Human Embryonic Stem Cells Using a CHK1 Inhibitor.

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.

Metabolic Control over mTOR-Dependent Diapause-like State.

Regulation of embryonic diapause, dormancy that interrupts the tight connection between developmental stage and time, is still poorly understood. Here, we characterize the transcriptional and metabolite profiles of mouse diapause embryos and identify unique gene expression and metabolic signatures with activated lipolysis, glycolysis, and metabolic pathways regulated by AMPK. Lipolysis is increased due to mTORC2 repression, increasing fatty acids to support cell survival. We further show that starvation in pre-implantation ICM-derived mouse ESCs induces a reversible dormant state, transcriptionally mimicking the in vivo diapause stage. During starvation, Lkb1, an upstream kinase of AMPK, represses mTOR, which induces a reversible glycolytic and epigenetically H4K16Ac-negative, diapause-like state. Diapause furthermore activates expression of glutamine transporters SLC38A1/2. We show by genetic and small molecule inhibitors that glutamine transporters are essential for the H4K16Ac-negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels, is causal for diapause metabolism and epigenetic state.

Susceptibility of aging mice to listeriosis: Role of anti-inflammatory responses with enhanced Treg-cell expression of CD39/CD73 and Th-17 cells.

Foodborne Listeria monocytogenes (Lm) causes serious illness and death in immunosuppressed hosts, including the elderly population. We investigated Lm susceptibility and inflammatory cytokines in geriatric mice. Young-adult and old mice were gavaged with a Lm strain Lmo-InlAm. Tissues were assayed for Lm burden and splenocytes were analyzed for Th1/Th2/Th17/Treg responses and expression of CD39 and CD73. Old Lm-infected mice lost body-weight dose-dependently, had higher Lm colonization, and showed higher inflammatory responses than Lm-infected young-adult mice. After infection, IL-17 levels increased significantly in old mice whereas IFN-γ levels were unchanged. Levels of IL-10 and Treg cells were increased in infected old mice as compared to infected young-adult mice. Age-dependent enhanced expression of CD39/CD73 was observed in purified Treg prior to infection, suggesting increased baseline adenosine production in old mice. Lm lysate-treated splenocytes from older mice produced significantly higher levels of IL-10, IL17, and IL-1ß, produced less IFN-γ and IL-2, and proliferated less than splenocytes from young-adult mice. Data suggests that older mice maybe more susceptible to Lm infection due to an imbalance of Th cell responses with disproportionate and persistent anti-inflammatory responses. Lm infection enhanced differentiation of proinflammatory Th17 cells, which may also exacerbate pathological responses during listeriosis.

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal.

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.

Extracellular adenosine produced by ecto-5'-nucleotidase (CD73) regulates macrophage pro-inflammatory responses, nitric oxide production, and favors Salmonella persistence.

Surface enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) mediate the synthesis of extracellular adenosine that can regulate immune responses. Adenosine produced by CD39/CD73 acts via adenosine receptors (ARs). CD73 is expressed by a variety of cell types and mediates anti-inflammatory responses. Because efficient innate immune responses are required for clearance of Salmonella infection, we investigated the role of CD73 in macrophage function, including phagocytosis, intracellular killing of Salmonella, and anti-bacterial pro-inflammatory responses to Salmonella-whole cell lysate (ST-WCL) or Salmonella infection. Additionally, RAW 264.7 macrophage mRNA expression of CD39, CD73, and all ARs were measured by qPCR after ST-WCL treatment. Pro-inflammatory cytokine mRNA and nitric oxide (NO) production were quantitated in the ST-WCL treated macrophage with and without CD73-inhibitor (APCP) treatment. Phagocytosis and intracellular killing by peritoneal macrophages from CD73-deficent mice were also evaluated using E. coli BioParticles® and GFP-Salmonella infection, respectively. CD73, CD39, and A2BAR mRNA were predominantly expressed in RAW cells. ST-WCL treatment significantly reduced CD73 expression, suggesting endogenous down-regulation of CD73, and an enhanced pro-inflammatory response. ST-WCL treated and CD73-inhibited macrophages produced more NO and a higher level of pro-inflammatory cytokines than CD73-competent macrophages (e.g. IL-1ß, TNF-α). Phagocytosis of E. coli BioParticles® was significantly higher in the macrophages treated with APCP and in the peritoneal macrophages from CD73-deficent mice as compared to APCP-untreated, and CD73-competent macrophages. Internalized bacteria were more efficiently cleared from macrophages in the absence of CD73, as observed by fluorescence-microscopy and Salmonella-DNA measurement by qPCR from the infected cells. CD73 down-regulation or CD73-inhibition of macrophages during Salmonella infection can enhance the production of pro-inflammatory cytokines and NO production, improving intracellular killing and host survivability. Extracellular adenosine synthesized by CD73 suppresses antibacterial responses of macrophages, which may weaken macrophage function and impair innate immune responses to Salmonella infection.

Genotypic and phenotypic characterization of multidrug resistant Salmonella Typhimurium and Salmonella Kentucky strains recovered from chicken carcasses.

Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Oral exposure to Listeria monocytogenes in aged IL-17RKO mice: A possible murine model to study listeriosis in susceptible populations.

Foodborne Listeria monocytogenes (LM) is a cause of serious illness and death in the US. The case-fatality rate of invasive LM infection in the elderly population is >50%. The goal of this study is to establish a murine model of oral LM infection that can be used as a surrogate for human foodborne listeriosis in the geriatric population. Adult C57BL/6 (wild-type, WT) and adult or old IL17R-KO (knock-out) mice were gavaged with a murinized LM strain (Lmo-InlAm) and monitored for body-weight loss and survivability. Tissues were collected and assayed for bacterial burden, histology, and cytokine responses. When compared to WT mice, adult IL17R-KO mice are more susceptible to LM infection and showed increased LM burden and tissue pathology and a higher mortality rate. Older LM-infected KO-mice lost significantly (p < 0.02, ANOVA) more body-weight and had a higher bacterial burden in the liver (p = 0.03) and spleen as compared to adult mice. Uninfected, aged KO-mice showed a higher baseline pro-inflammatory response when compared to uninfected adult-KO mice. After infection, the pro-inflammatory cytokine, IFN-γ, mRNA in the liver was higher in the adult mice as compared to the old mice. The anti-inflammatory cytokine, IL-10, mRNA and regulatory T-cells (CD4+CD25+h or CD4+Foxp3+) cells in the aged mice increased significantly after infection as compared to adult mice. Expression of the T-cell activation marker, CD25 (IL-2Rα) in the aged mice did not increase significantly over baseline. These data suggest that aged IL17R-KO mice can be used as an in vivo model to study oral listeriosis and that aged mice are more susceptible to LM infection due to dysregulation of pro- and anti-inflammatory responses compared to adult mice, resulting in a protracted clearance of the infection.

Docetaxel As Monotherapy or Combined With Ramucirumab or Icrucumab in Second-Line Treatment for Locally Advanced or Metastatic Urothelial Carcinoma: An Open-Label, Three-Arm, Randomized Controlled Phase II Trial.

PURPOSE: This trial assessed the efficacy and safety of docetaxel monotherapy or docetaxel in combination with ramucirumab (vascular endothelial growth factor receptor 2 antibody) or icrucumab (vascular endothelial growth factor receptor 1 antibody) after progression during or within 12 months of platinum-based regimens for patients with locally advanced or metastatic urothelial carcinoma. PATIENTS AND METHODS: Patients were randomly assigned (1:1:1) to receive docetaxel 75 mg/m(2) intravenously (IV) on day 1 of a 3-week cycle (arm A), docetaxel 75 mg/m(2) IV plus ramucirumab 10 mg/kg IV on day 1 of a 3-week cycle (arm B), or docetaxel 75 mg/m(2) IV on day 1 plus icrucumab 12 mg/kg IV on days 1 and 8 of a 3-week cycle (arm C). Treatment continued until disease progression or unacceptable toxicity. The primary end point was investigator-assessed progression-free survival (PFS). RESULTS: A total of 140 patients were randomly assigned and treated in arms A (n = 45), B (n = 46), or C (n = 49). PFS was significantly longer in arm B compared with arm A (median, 5.4 months; 95% CI, 3.1 to 6.9 months v 2.8 months; 95% CI, 1.9 to 3.6 months; stratified hazard ratio, 0.389; 95% CI, 0.235 to 0.643; P = .0002). Arm C did not experience improved PFS compared with arm A (1.6 months; 95% CI, 1.4 to 2.9; stratified hazard ratio, 0.863; 95% CI, 0.550 to 1.357; P = .5053). The most common grade 3 or worse adverse events (arms A, B, and C) were neutropenia (36%, 33%, and 39%), fatigue (13%, 30%, and 20%), febrile neutropenia (13%, 17%, and 6.1%), and anemia (6.7%, 13%, and 14%, respectively). CONCLUSION: The addition of ramucirumab to docetaxel met the prespecified efficacy end point for prolonging PFS in patients with locally advanced or metastatic urothelial carcinoma receiving second-line treatment and warrants further investigation in the phase III setting.

Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc.

Extracellular adenosine generation in the regulation of pro-inflammatory responses and pathogen colonization.

Adenosine, an immunomodulatory biomolecule, is produced by the ecto-enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) by dephosphorylation of extracellular ATP. CD73 is expressed by many cell types during injury, infection and during steady-state conditions. Besides host cells, many bacteria also have CD39-CD73-like machinery, which helps the pathogen subvert the host inflammatory response. The major function for adenosine is anti-inflammatory, and most recent research has focused on adenosine's control of inflammatory mechanisms underlying various autoimmune diseases (e.g., colitis, arthritis). Although adenosine generated through CD73 provides a feedback to control tissue damage mediated by a host immune response, it can also contribute to immunosuppression. Thus, inflammation can be a double-edged sword: it may harm the host but eventually helps by killing the invading pathogen. The role of adenosine in dampening inflammation has been an area of active research, but the relevance of the CD39/CD73-axis and adenosine receptor signaling in host defense against infection has received less attention. Here, we review our recent knowledge regarding CD73 expression during murine Salmonellosis and Helicobacter-induced gastric infection and its role in disease pathogenesis and bacterial persistence. We also explored a possible role for the CD73/adenosine pathway in regulating innate host defense function during infection.

The GIPC1-Akt1 Pathway Is Required for the Specification of the Eye Field in Mouse Embryonic Stem Cells.

During early patterning of the neural plate, a single region of the embryonic forebrain, the eye field, becomes competent for eye development. The hallmark of eye field specification is the expression of the eye field transcription factors (EFTFs). Experiments in fish, amphibians, birds, and mammals have demonstrated largely conserved roles for the EFTFs. Although some of the key signaling events that direct the synchronized expression of these factors to the eye field have been elucidated in fish and frogs, it has been more difficult to study these mechanisms in mammalian embryos. In this study, we have used two different methods for directed differentiation of mouse embryonic stem cells (mESCs) to generate eye field cells and retina in vitro to test for a role of the PDZ domain-containing protein GIPC1 in the specification of the mammalian eye primordia. We find that the overexpression of a dominant-negative form of GIPC1 (dnGIPC1), as well as the downregulation of endogenous GIPC1, is sufficient to inhibit the development of eye field cells from mESCs. GIPC1 interacts directly with IGFR and participates in Akt1 activation, and pharmacological inhibition of Akt1 phosphorylation mimics the dnGIPC1 phenotype. Our data, together with previous studies in Xenopus, support the hypothesis that the GIPC1-PI3K-Akt1 pathway plays a key role in eye field specification in vertebrates.

Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes.

High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type-specific 3D organization of lincRNA genes.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition.

The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxia-inducible factor 1α (HIF1α) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.

Reprogramming of prostate cancer-associated stromal cells to embryonic stem-like.

BACKGROUND: CD90(+) prostate cancer-associated (CP) stromal cells represent a diseased cell type found only in tumor tissue. They differ from their normal counterpart in gene expression and inductive signaling. Genetic reprogramming by induced pluripotent stem (iPS) cell technology can effectively change adult cells into stem-like cells through wholesale alteration of the gene expression program. This technology might be used to 'erase' the abnormal gene expression of diseased cells. The resultant iPS cells would no longer express the disease phenotype, and behave like stem cells. METHODS: CP stromal cells, isolated from tumor tissue of a surgically resected prostate by anti-CD90-mediated sorting and cultured in vitro, were transfected with in vitro packaged lentiviral expression vectors containing stem cell transcription factor genes POU5F1, LIN28, NANOG, and SOX2. RESULTS: Alkaline phosphatase-positive iPS cells were obtained in about 3 weeks post-transfection at a frequency of 10(-4) . Their colony morphology was indistinguishable from that of human embryonic stem (ES) cells. Transcriptome analysis showed a virtually complete match in gene expression between the iPS and ES cells. CONCLUSIONS: Genes of CP stromal cells could be fully inactivated by genetic reprogramming. As a consequence, the disease phenotype was 'cured'.

Activity, pharmacokinetics and tissue distribution of TLC ELL-12 (liposomal antitumor ether lipid) in rats with transplantable, s.c. methylnitrosourea-induced tumors.

TLC ELL-12 is a liposomal formulation of the novel antineoplastic compound 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH(3)). The purpose of these studies was to evaluate the activity and tissue distribution of L-ET-18-OCH(3) when administered i.v. as TLC ELL-12 to rats bearing solid tumors. Growth-inhibitory activity of L-ET-18-OCH(3) and TLC ELL-12 against methylnitrosourea (MNU)-induced tumors grown in vitro was evaluated. Female Buffalo rats were injected s.c. with transplantable MNU-induced tumor cells. Four days later, animals were treated i.v. with L-ET-18-OCH(3) administered as TLC ELL-12 once daily for 5 consecutive days. Another group of MNU-tumor bearing rats was given a single 12.5 mg/kg dose of TLC ELL-12 containing [14C]L-ET-18-OCH(3) by i.v. injection into a tail vein. The 50% growth inhibitory concentration for TLC ELL-12 against MNU tumor cells in vitro was 63 microM (about 30 microg/ml). Tumor growth was significantly inhibited in ELL-12-treated rats versus controls. After a single dose, whole blood L-ET-18-OCH(3) concentrations declined in a multiphasic fashion with C(max) and terminal half-life values of approximately 91.1 microg L-ET-18-OCH(3)/ml and 13.1 h, respectively. Tumor L-ET-18-OCH(3) levels increased through the first 16-24 h post-dosing to about 23 microg/g and remained elevated at the terminal time point with little evidence of metabolism. Concentration-time profiles for selected tissues indicate rapid distribution of L-ET-18-OCH(3) from the circulation into tissues with highest concentrations in spleen, liver, lungs, kidneys and gastrointestinal tract. L-ET-18-OCH(3) as TLC ELL-12 shows both in vitro and in vivo activity against the MNU tumor line. When i.v. administered, L-ET-18-OCH(3) from ELL-12 is well distributed and slowly eliminated by metabolism in tissues.

Toxicity and disposition of TLC ELL-12 (liposomal antitumor ether lipid) in Sprague-Dawley rats.

TLC ELL-12 is a liposomal formulation of the antineoplastic L-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine [L-ET-18-OCH3 (EL)]. The purpose of these studies was to characterize the toxicity and disposition of [N-methyl-14C] L-ET-18-OCH3 administered as TLC ELL-12. Rats received TLC ELL-12 by i.v. infusion into a tail vein as a single 12.5 or 62.5 mg/kg dose or as five daily doses at 12.5 mg/kg (cumulative dose of 62.5 mg/kg). Whole blood and tissue samples were collected over 24 h, and assayed for total and EL-specific radioactivity. The amounts of radioactivity in urine, bile, injection site and carcass were determined for up to 48 h. TLC ELL-12 was well tolerated in male and female rats in single doses up to 37.5 mg/kg. The minimum lethal dose was 112.5 mg/kg. Doses of 12.5 mg/kg (no observed adverse effects) and 62.5 mg/kg (approximate maximum tolerated dose) were chosen for further study. The pharmacokinetics of EL given as TLC ELL-12 were non-linear with a disproportionate increase in AUC at the higher dose. Daily dosing at 12.5 mg/kg did not result in accumulation in the blood. The highest concentrations of EL at 24 h after dosing were in the spleen and liver. Virtually no radioactivity was recovered in the urine or bile of rats, most remaining in the carcass and injection site (tail). After a 12.5 mg/kg dose of TLC ELL-12, the levels of EL in the blood and most tissues examined were well above the levels that inhibit tumor growth and may therefore be therapeutically active.