Widespread genetic heterogeneity of human ribosomal RNA genes.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

Phase 2 study of vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-paclitaxel in patients with untreated metastatic pancreatic adenocarcinoma.

BACKGROUND: The Hedgehog (Hh) signalling pathway is overexpressed in pancreatic ductal adenocarcinoma (PDA). Preclinical studies have shown that Hh inhibitors reduce pancreatic cancer stem cells (pCSC), stroma and Hh signalling. METHODS: Patients with previously untreated metastatic PDA were treated with gemcitabine and nab-paclitaxel. Vismodegib was added starting on the second cycle. The primary endpoint was progression-free survival (PFS) as compared with historical controls. Tumour biopsies to assess pCSC, stroma and Hh signalling were obtained before treatment and after cycle 1 (gemcitabine and nab-paclitaxel) or after cycle 2 (gemcitabine and nab-paclitaxel plus vismodegib). RESULTS: Seventy-one patients were enrolled. Median PFS and overall survival (OS) were 5.42 months (95% confidence interval [CI]: 4.37-6.97) and 9.79 months (95% CI: 7.85-10.97), respectively. Of the 67 patients evaluable for response, 27 (40%) had a response: 26 (38.8%) partial responses and 1 complete response. In the tumour samples, there were no significant changes in ALDH + pCSC following treatment. CONCLUSIONS: Adding vismodegib to chemotherapy did not improve efficacy as compared with historical rates observed with chemotherapy alone in patients with newly diagnosed metastatic pancreatic cancer. This study does not support the further evaluation of Hh inhibitors in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01088815.

Preclinical evaluation of the maximum tolerated dose and toxicokinetics of enteric-coated lantibiotic OG253 capsules.

Clostridium difficile associated disease (CDAD) is the leading infectious cause of antibiotic-associated diarrhea and colitis in the United States. Both the incidence and severity of CDAD have been increased over the past two decades. We evaluated the maximum tolerated dose (MTD) and toxicokinetics of OG253, a novel lantibiotic in development for the treatment of CDAD. OG253 was orally administered to Wistar Han rats as enteric-coated capsules in a one-day dose escalation study, followed by a seven-day repeated dose toxicokinetics study. All three doses of OG253 (6.75, 27 and 108 mg/day) were generally well-tolerated with no treatment-related clinical signs, alterations in body weight or food consumption in both one-day acute tolerability and seven-days repeated dose tolerability and toxicokinetics study. OG253 capsule administration neither significantly alter the weight of organs nor affect the hematology, coagulation, clinical biochemistry parameters and urine pH compared to placebo capsule administered rats. LC-MS/MS analysis did not detect OG253 in the plasma, indicating that OG253 is not absorbed into the blood from the rat gastrointestinal tract. Glandular atrophy of the rectal mucosa was noticed in two out of six rats administered with a high dose of OG253. Surprisingly, we found that OG253 treatment significantly lowered both serum cholesterol and triglyceride levels in both sexes of rats. Overall, there was a 29.8 and 61.38% decrease in the serum cholesterol and triglyceride levels, respectively as compared to placebo-treated rats. The well-tolerated high dose of OG253 (425.7 mg/kg/day) is recommended as the MTD for safety and efficacy studies. Further preclinical study is needed to evaluate the safety profile of OG253 under longer exposure.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) redox function negatively regulates NRF2.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.

Superior therapeutic efficacy of nab-paclitaxel over cremophor-based paclitaxel in locally advanced and metastatic models of human pancreatic cancer.

BACKGROUND: Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC. METHODS: We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC. RESULTS: Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer. CONCLUSIONS: Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

Heterogeneity of pancreatic cancer metastases in a single patient revealed by quantitative proteomics.

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.

A preclinical and clinical study of mycophenolate mofetil in pancreatic cancer.

A high throughput screening for anticancer activity of FDA approved drugs identified mycophenolic acid (MPA), an inhibitor of inositol monophosphate dehydrogenase (IMPDH) as an active agent with an antiangiogenesis mode of action. Exposure of pancreatic cancer cell lines to MPA resulted in growth inhibition and reduced the expression of VEGF that was reversed by supplementing the media with guanosine supporting and IMPDH-dependant mechanism. In preclinical in vivo study, MPA showed a moderate inhibition of tumor growth in a panel of 6 human derived pancreatic cancer xenografts but reduced the expression of VEGF. To investigate the effects of MPA in human pancreatic cancer, a total of 12 patients with resectable pancreatic cancer (PDA) received increasing doses of mycophenolate mofetil (MMF) in cohorts of 6 patients each from 5-15 days prior to surgical resection. Treatment was well tolerated with one episode of grade 1 muscle pain, one episode of grade 2 lymphopenia (2 gr/day dose) and one episode of grade 2 elevantion in LFT (all in the 2 gr./day dose). Patients recovered from surgery uneventfully with no increased post-operative complications. Assessment of CD31, VEGF, and TUNEL in resected specimens compared to a non treated control of 6 patients showed no significant variations in any of the study endpoints. In conclusion, this study shows the feasibility of translating a preclinical observation to the clinical setting and to explore a drug mechanism of action in patients. MPA, however, did not show any hints of antiangiogenesis of anticancer clinical activity questioning if this agent should be further developed in PDA.

Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer.

PURPOSE: S-trans,trans-Farnesylthiosalicylic Acid (FTS, salirasib) inhibits Ras-dependent cell growth by dislodging all isoforms of Ras, including mutant Ras, from the plasma membrane. This study evaluated the activity, safety, and toxicity of salirasib in preclinical models and patients with metastatic pancreatic adenocarcinoma (PDA). PATIENTS AND METHODS: In the preclinical study, salirasib was tested, alone and in combination with gemcitabine, in patient derived xenografts (PDX) of PDA. In the clinical study, treatment-naïve patients with advanced, metastatic PDA were treated with a standard dose schedule of gemcitabine and salirasib 200-800 mg orally (PO) twice daily (bid) for 21 days every 28 days. Tissue from preclinical models and patients' biopsies were collected pre-treatment and on Cycle (C) 1, Day (D) 9 to characterize the effect of gemcitabine and salirasib on activated Ras protein levels. Plasma samples for pharmacokinetics were collected for salirasib administered alone and in combination. RESULTS: Salirasib inhibited the growth of 2/14 PDX models of PDA and modulated Ras signaling in these tumors. Nineteen patients were enrolled. No DLTs occurred. Common adverse events included hematologic and gastrointestinal toxicities and fatigue. The median overall survival was 6.2 months and the 1 year survival 37 %. In 2 patients in whom paired tissue biopsies were available, Ras and KRas protein levels were decreased on C1D9. Salirasib exposure was not altered by gemcitabine and did not correlate with PD outcomes. CONCLUSION: The combination of gemcitabine and salirasib appears well-tolerated, with no alteration of salirasib exposure, and exerted clinical and PD activity in PDA.

Discovery and Evaluation of Novel Angular Fused Pyridoquinazolinonecarboxamides as RNA Polymerase I Inhibitors.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into the 47S ribosomal RNA (rRNA) precursor. Further processing produces the 28S, 5.8S, and 18S rRNAs that are assembled into mature ribosomes. Many cancers exhibit higher Pol I transcriptional activity, reflecting a need for increased ribosome biogenesis and protein synthesis and making the inhibition of this process an attractive therapeutic strategy. Lead molecule BMH-21 (1) has been established as a Pol I inhibitor by affecting the destruction of RPA194, the Pol I large catalytic subunit. A previous structure-activity relationship (SAR) study uncovered key pharmacophores, but activity was constrained within a tight chemical space. This work details further SAR efforts that have yielded new scaffolds and improved off-target activity while retaining the desired RPA194 degradation potency. Pharmacokinetic profiling was obtained and provides a starting point for further optimization. New compounds present additional opportunities for the development of Pol I inhibitory cancer therapies.

Integrated preclinical and clinical development of mTOR inhibitors in pancreatic cancer.

BACKGROUND: The purpose of this work was to determine the efficacy of inhibiting mammalian target of rapamycin (mTOR) in pancreatic cancer preclinical models and translate preclinical observations to the clinic. METHODS: Temsirolimus (20 mg Kg(-1) daily) was administered to freshly generated pancreatic cancer xenografts. Tumour growth inhibition was determined after 28 days. Xenografts were characterised at baseline by gene expression and comparative genomic hybridisation. Patients with advanced, gemcitabine-resistant pancreatic cancer were treated with sirolimus (5 mg daily). The primary end point was 6-month survival rate (6mSR). Correlative studies included immunohistochemistry assessment of pathway expression in baseline tumours, drug pharmacokinetics (PKs), response assessment by FDG-PET and pharmacodynamic effects in peripheral-blood mononuclear cells (PBMCs). RESULTS: In all, 4 of 17 xenografts (23%) responded to treatment. Sensitive tumours were characterised by gene copy number variations and overexpression of genes leading to activation of the PI3K/Akt/mTOR pathway. Activation of p70S6K correlated with drug activity in the preclinical studies. Sirolimus was well tolerated in the clinic, showed predictable PKs, exerted pathway inhibition in post-treatment PBMCs and resulted in a 6mSR of 26%. No correlation, however, was found between activated p70S6K in tumour tissues and anti-tumour effects. CONCLUSION: Sirolimus activity in pancreatic cancer was marginal and not predicted by the selected biomarker.

Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). METHODS: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against ß1 integrin and FAK as well as a small-molecule FAK inhibitor. RESULTS: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of ß1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. CONCLUSION: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

Small-Molecule Inhibition of Axl Targets Tumor Immune Suppression and Enhances Chemotherapy in Pancreatic Cancer.

Activation of the receptor tyrosine kinase Axl is associated with poor outcomes in pancreatic cancer (PDAC), where it coordinately mediates immune evasion and drug resistance. Here, we demonstrate that the selective Axl kinase inhibitor BGB324 targets the tumor-immune interface to blunt the aggressive traits of PDAC cells in vitro and enhance gemcitibine efficacy in vivo Axl signaling stimulates the TBK1-NFκB pathway and innate immune suppression in the tumor microenvironment. In tumor cells, BGB324 treatment drove epithelial differentiation, expression of nucleoside transporters affecting gemcitabine response, and an immune stimulatory microenvironment. Our results establish a preclinical mechanistic rationale for the clinical development of Axl inhibitors to improve the treatment of PDAC patients.Significance: These results establish a preclinical mechanistic rationale for the clinical development of AXL inhibitors to improve the treatment of PDAC patients. Cancer Res; 78(1); 246-55. ©2017 AACR.

Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTENConclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639-47. ©2017 AACR.

A phase 2 trial of personalized cytotoxic therapy based on tumor immunohistochemistry in previously treated metastatic pancreatic cancer patients.

BACKGROUND: The choice of a regimen in metastatic pancreatic cancer patients following progression on 1st line therapy is empiric and outcomes are unsatisfactory. This phase II study was performed to evaluate the efficacy of therapy selected by immunohistochemistry (IHC) in these patients following progression after one or more therapies. METHODS: Eligible patients underwent a percutaneous biopsy of a metastatic lesion and treatment selection was determined by IHC. The study required 35 evaluable patients (power of 86%) for detecting a true 1-year survival rate of >20%. RESULTS: A tumor biopsy was performed in 48 of 49 accrued patients. Study therapy was not given (n=13) either due to insufficient tumor on biopsy (n=8) or due to worsening cancer related symptoms after biopsy (n=5). The demographics of evaluable patients (n=35) are male/female (59%/41%), with age range 34-78 years (median 63 years). Patients had 1-6 prior regimens (median of 2). The most common IHC targets were topoisomerase 1 or 2, thymidylate synthase, excision repair cross-complementation group 1 protein (ERCC1), and osteonectin secreted protein acidic and rich in cysteine (SPARC). Commercially available treatment regimens prescribed included FOLFIRI, FOLFOX, irinotecan, and doxorubicin. The response (RECIST) was 9%, the median survival was 5.6 months (94% CI, 3.8-8.2), and the 1-year survival was 20% (95% CI, 7-33%). CONCLUSIONS: In all patients, IHC assays resulted in identification of at least two targets for therapy and a non-cross resistant regimen could be prescribed for therapy with evidence of some benefit. An IHC based treatment strategy is feasible and needs validation in larger studies.

The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, ß1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

Effect of the ET(B) receptor agonist, IRL-1620, on paclitaxel plasma pharmacokinetics of breast tumor rats.

Endothelin (ET)-B receptors are expressed in human breast carcinoma. We previously demonstrated that intravenous administration of the ET(B) receptor agonist, IRL-1620, to tumor-bearing rats, increased blood perfusion and enhanced delivery of paclitaxel to breast tumor tissue. The present study was conducted to determine whether IRL-1620 alters the pharmacokinetics of paclitaxel. Breast tumor-bearing rats were given 0.3 ml/kg saline or 3 nmol/kg IRL-1620 by intravenous (iv) administration. Fifteen minutes after saline or IRL-1620, 40 microCi/rat 3H-Paclitaxel was administered iv and serial plasma samples were collected until 24 hrs. 3H-Paclitaxel radioactivity in the plasma samples was measured by liquid scintillation counting. Data were fit to a three-compartment model and pharmacokinetic parameters were generated using WinNonlin software. IRL-1620 did not produce any change in the plasma paclitaxel pharmacokinetics of tumor-bearing rats. The AUC(0-infinity) (9.43 +/- 3.18 microg-hr/ml), clearance (0.69 +/- 0.17 l/hr/kg), volume of distribution (10.31 +/- 4.54 l/kg), and half-life (1.0 +/- 0.32 hrs) of paclitaxel were similar between rats treated with saline or IRL-1620. In conclusion, the ET(B) receptor agonist, IRL-1620, does not alter paclitaxel plasma pharmacokinetics and, therefore, could be used to augment the delivery of paclitaxel to the tumor tissue.

Effect of homeopathic treatment on gene expression in Copenhagen rat tumor tissues.

BACKGROUND: Increasing evidence suggests that the inability to undergo apoptosis is an important factor in the development and progression of prostate cancer. Agents that induce apoptosis may inhibit tumor growth and provide therapeutic benefit. In a recent study, the authors found that certain homeopathic treatments produced anticancer effects in an animal model. In this study, the authors examined the immunomodulating and apoptotic effects of these remedies. MATERIALS AND METHODS: The authors investigated the effect of a homeopathic treatment regimen containing Conium maculatum, Sabal serrulata, Thuja occidentalis, and a MAT-LyLu Carcinosin nosode on the expression of cytokines and genes that regulate apoptosis. This was assessed in prostate cancer tissues, extracted from animals responsive to these drugs, using ribonuclease protection assay or reverse transcription polymerase chain reaction. RESULTS: There were no significant changes in mRNA levels of the apoptotic genes bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, FasL, or the cytokines interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, IL-2, and interferon-gamma in prostate tumor and lung metastasis after treatment with homeopathic medicines. CONCLUSIONS: This study indicates that treatment with the highly diluted homeopathic remedies does not alter the gene expression in primary prostate tumors or in lung metastasis. The therapeutic effect of homeopathic treatments observed in the in vivo experiments cannot be explained by mechanisms based on distinct alterations in gene expression related to apoptosis or cytokines. Future research should explore subtle modulations in the expression of multiple genes in different biological pathways.

Homeopathic medicines do not alter growth and gene expression in prostate and breast cancer cells in vitro.

BACKGROUND: Homeopathy is an alternative medical system practiced in all parts of the world. Although several theories are proposed to explain the mechanisms of action, none are scientifically verified. In this study, the authors investigate the effect of selected homeopathic remedies often used to treat prostate and breast cancer. MATERIALS AND METHODS: The authors investigated the effect of the homeopathic medicines Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias, Phytolacca, and Carcinosin on prostate and breast cancer cell (DU-145, LNCaP, MAT-LyLu, MDA-MB-231) growth and on gene expression that regulates apoptosis, using MTT and multiprobe ribonuclease protection assay. RESULTS: None of the homeopathic remedies tested in different potencies produced significant inhibitory or growth-promoting activity in either prostate or breast cancer cells. Also, gene expression studies by ribonuclease protection assay produced no significant changes in mRNA levels of bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, or FasL after treatment with homeopathic medicines. CONCLUSIONS: The results demonstrate that the highly diluted homeopathic remedies used by homeopathic practitioners for cancer show no measurable effects on cell growth or gene expression in vitro using currently available methodologies.

Can homeopathic treatment slow prostate cancer growth?

BACKGROUND: Homeopathy is a complementary medicine widely used around the world. Despite extensive use of homeopathy for cancer and other serious conditions with reported success, clinical and laboratory research has been equivocal, and no rigorous research has been done on cancer. In 1999, the US National Cancer Institute evaluated the effects of homeopathic treatment of cancer from a clinic in India and has released a request for protocols to conduct further research into this treatment. Therefore, the authors conducted a series of carefully controlled laboratory studies evaluating the effects of commonly used homeopathic remedies in cell and animal models of prostate cancer. STUDY DESIGN: One hundred male Copenhagen rats were randomly assigned to either treatment or control groups after inoculation with prostate tumor cells. METHODS: Prostate tumor cells DU-145, LNCaP, and MAT-LyLu were exposed to 5 homeopathic remedies. Male Copenhagen rats were injected with MAT-LyLu cells and exposed to the same homeopathic remedies for 5 weeks. In vitro outcomes included tumor cell viability and apoptosis gene expression. In vivo outcomes included tumor incidence, volume, weight, total mortality, proliferating cell nuclear antigen (PCNA) expression, apoptotic cell death (terminal deoxynucleotidyl transferase mediated d-uridine triphosphate nick end labeling), and gene expression (rAPO-multiprobe). RESULTS: There were no effects on cell viability or gene expression in 3 prostate cell lines with any remedies at any exposure time. There was a 23% reduction in tumor incidence (P < .0001), and for animals with tumors, there was a 38% reduction in tumor volume in homeopathy-treated animals versus controls (P < .02). At time of killing, experimental animals with tumors had a 13% lower average tumor weight (P < .05). Tumors in these treated animals showed a 19% increase in apoptotic cell death (P < .05) and reduced PCNA-positive cells. CONCLUSIONS: The findings indicate that selected homeopathic remedies for the present study have no direct cellular anticancer effects but appear to significantly slow the progression of cancer and reduce cancer incidence and mortality in Copenhagen rats injected with MAT-LyLu prostate cancer cells.

Determination of endothelin antagonist BMS182874 in plasma by high-performance liquid chromatography.

A simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of BMS182874 (BMS) in mouse plasma. The drug was extracted from plasma by a liquid-liquid extraction process. The method consists of reversed-phase chromatography using a Thermo Hypersil-Keystone RP-18 5 microm, 250 x 2.1 mm column and UV spectrophotometer detection at 255 nm. The mobile phase consists of 45% (v/v) acetonitrile: 55% (v/v) trifluoroacetic acid (0.015% v/v; pH 3.0) at a flow rate of 0.6 ml/min. Validity of the method was studied and the method was precise and accurate with a linearity range from 100 ng/ml to 1000 ng/ml. The extraction efficiency was found to be 81, 84 and 87% for 100, 500 and 1000 ng/ml, respectively for spiked drug in plasma. The limit of quantification and limit of detection were found to be 50 and 10 ng/ml, respectively in plasma. Within-day and between-day precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 4%. The assay was also used to analyze samples collected during animal studies. The suitability and robustness of the method for in vivo samples were confirmed by analysis of BMS from mouse plasma and tissues dosed with BMS.