Linezolid decreases susceptibility to secondary bacterial pneumonia postinfluenza infection in mice through its effects on IFN-γ.

Influenza infection predisposes patients to secondary bacterial pneumonia that contributes significantly to morbidity and mortality. Although this association is well documented, the mechanisms that govern this synergism are poorly understood. A window of hyporesponsiveness following influenza infection has been associated with a substantial increase in local and systemic IFN-γ concentrations. Recent data suggest that the oxazolidinone antibiotic linezolid decreases IFN-γ and TNF-α production in vitro from stimulated PBMCs. We therefore sought to determine whether linezolid would reverse immune hyporesponsiveness after influenza infection in mice through its effects on IFN-γ. In vivo dose-response studies demonstrated that oral linezolid administration sufficiently decreased bronchoalveolar lavage fluid levels of IFN-γ at day 7 postinfluenza infection in a dose-dependent manner. The drug also decreased morbidity as measured by weight loss compared with vehicle-treated controls. When mice were challenged intranasally with Streptococcus pneumoniae 7 d postinfection with influenza, linezolid pretreatment led to decreased IFN-γ and TNF-α production, decreased weight loss, and lower bacterial burdens at 24 h postbacterial infection in comparison with vehicle-treated controls. To determine whether these effects were due to suppression of IFN-γ, linezolid-treated animals were given intranasal instillations of rIFN-γ before challenge with S. pneumoniae. This partially reversed the protective effects observed in the linezolid-treated mice, suggesting that the modulatory effects of linezolid are mediated partially by its ability to blunt IFN-γ production. These results suggest that IFN-γ, and potentially TNF-α, may be useful drug targets for prophylaxis against secondary bacterial pneumonia following influenza infection.

Clinical outcomes from the CDC's Colorectal Cancer Screening Demonstration Program.

BACKGROUND: Colorectal cancer remains the second leading cause of cancer-related deaths among US men and women. Screening rates have been slow to increase, and disparities in screening remain. METHODS: To address the disparity in screening for this high burden but largely preventable disease, the Centers for Disease Control and Prevention (CDC) designed and established a 4-year Colorectal Cancer Screening Demonstration Program (CRCSDP) in 2005 for low-income, under-insured or uninsured men and women aged 50 to 64 years in 5 participating US program sites. In this report, the authors describe the design of the CRCSDP and the overall clinical findings and screening test performance characteristics, including the positive fecal occult blood testing (FOBT) rate; the rates of polyp, adenoma, and cancer detection with FOBTs and colonoscopies; and the positive predicative value for polyps, adenomas, and cancers. RESULTS: In total, 5233 individuals at average risk and increased risk were screened for colorectal cancer across all 5 sites, including 44% who underwent screening FOBT and 56% who underwent screening colonoscopy. Overall, 77% of all individuals screened were women. The FOBT positivity rate was 10%. Results from all screening or diagnostic colonoscopies indicated that 75% had negative results and required a repeat screening colonoscopy in 10 years, 16% had low-risk adenomas and required surveillance colonoscopy in 5 to 10 years, 8% had high-risk adenomas and required surveillance colonoscopy in 3 years, and 0.6% had invasive cancers. CONCLUSIONS: This report documents the successes and challenges in implementing the CDC's CRCSDP and describes the clinical outcomes of this 4-year initiative, the patterns in program uptake and test choice, and the comparative test performance characteristics of FOBT versus colonoscopy. Patterns in final outcomes from the follow-up of positive screening tests were consistent with national registry data.

Azithromycin increases in vitro fibronectin production through interactions between macrophages and fibroblasts stimulated with Pseudomonas aeruginosa.

OBJECTIVES: Chronic azithromycin therapy has been associated with improved clinical outcomes in patients with cystic fibrosis (CF) who are chronically infected with Pseudomonas aeruginosa. We have previously demonstrated that azithromycin polarizes macrophages towards an alternatively activated phenotype, thereby blunting inflammation associated with infection. Because this phenotype is pro-fibrotic, it is important to evaluate azithromycin's consequential effects upon fibroblast function and extracellular matrix (ECM) protein production. METHODS: We co-cultured macrophages and fibroblasts together and stimulated them by adding P. aeruginosa or lipopolysaccharide to assess the ability of azithromycin to alter the macrophage phenotype, along with the impact exerted upon the production of fibronectin and other effectors that govern tissue remodelling, including transforming growth factor ß (TGFß), matrix metalloproteinase-9 (MMP-9) and arginase. We supported these studies by evaluating the impact of azithromycin treatment on these proteins in a mouse model of P. aeruginosa infection. RESULTS: Azithromycin increased arginase expression in vitro, as well as the activation of latent TGFß, consistent with polarization to the alternative macrophage phenotype. While the drug increased fibronectin concentrations after stimulation in vitro, secretion of the ECM-degrading enzyme MMP-9 was also increased. Neutralization of active TGFß resulted in the ablation of azithromycin's ability to increase fibronectin concentrations, but did not alter its ability to increase MMP-9 expression. In P. aeruginosa-infected mice, azithromycin significantly decreased MMP-9 and fibronectin concentrations in the alveolar space compared with non-treated, infected controls. CONCLUSIONS: Our results suggest that azithromycin's effect on MMP-9 is regulated independently of TGFß activity. Additionally, the beneficial effects of azithromycin may be partially due to effects on homeostasis in which ECM-degrading mediators like MMP-9 are up-regulated early after infection. This may impact the damaging effects of inflammation that lead to fibrosis in this patient population.

Anticancer activity of novel unnatural synthetic isoprenoids.

BACKGROUND: The KRAS oncogene has a high prevalence in solid malignancies. Targeting KRAS and inappropriate activation of the MAPK pathway with novel drugs is of interest. This study developed and screened a library of compounds designed to inhibit KRAS signaling by altering prenyl function. MATERIALS AND METHODS: To screen a library of novel farnesyl analogs for their anticancer activity in human lung cancer and breast cancer cell lines. To evaluate if the designed and actual pharmacology are congruent. RESULTS: Sixty-seven novel compounds were tested and 70% of them screened positive for activity in at least one cell line. Two active compounds inhibited phosphorylation of MAP kinase consistent with KRAS inhibition. CONCLUSION: Although 47 of the 67 novel agents screened positive for activity, none of them were highly potent. However, targeting RAS with compounds that compete with farnesyl and geranylgeranyl modification of the protein remains viable and further work is already underway to create second generation molecules.

Doxorubicin and paclitaxel-loaded lipid-based nanoparticles overcome multidrug resistance by inhibiting P-glycoprotein and depleting ATP.

To test the ability of nanoparticle formulations to overcome P-glycoprotein (P-gp)-mediated multidrug resistance, several different doxorubicin and paclitaxel-loaded lipid nanoparticles were prepared. Doxorubicin nanoparticles showed 6- to 8-fold lower IC(50) values in P-gp-overexpressing human cancer cells than those of free doxorubicin. The IC(50) value of paclitaxel nanoparticles was over 9-fold lower than that of Taxol in P-gp-overexpressing cells. A series of in vitro cell assays were used including quantitative studies on uptake and efflux, inhibition of calcein acetoxymethylester efflux, alteration of ATP levels, membrane integrity, mitochondrial membrane potential, apoptosis, and cytotoxicity. Enhanced uptake and prolonged retention of doxorubicin were observed with nanoparticle-based formulations in P-gp-overexpressing cells. Calcein acetoxymethylester and ATP assays confirmed that blank nanoparticles inhibited P-gp and transiently depleted ATP. I.v. injection of pegylated paclitaxel nanoparticles showed marked anticancer efficacy in nude mice bearing resistant NCI/ADR-RES tumors versus all control groups. Nanoparticles may be used to target both drug and biological mechanisms to overcome multidrug resistance via P-gp inhibition and ATP depletion.

Development of new lipid-based paclitaxel nanoparticles using sequential simplex optimization.

The objective of these studies was to develop Cremophor-free lipid-based paclitaxel (PX) nanoparticle formulations prepared from warm microemulsion precursors. To identify and optimize new nanoparticles, experimental design was performed combining Taguchi array and sequential simplex optimization. The combination of Taguchi array and sequential simplex optimization efficiently directed the design of paclitaxel nanoparticles. Two optimized paclitaxel nanoparticles (NPs) were obtained: G78 NPs composed of glyceryl tridodecanoate (GT) and polyoxyethylene 20-stearyl ether (Brij 78), and BTM NPs composed of Miglyol 812, Brij 78, and d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Both nanoparticles successfully entrapped paclitaxel at a final concentration of 150 microg/ml (over 6% drug loading) with particle sizes less than 200 nm and over 85% of entrapment efficiency. These novel paclitaxel nanoparticles were stable at 4 degrees C over five months and in PBS at 37 degrees C over 102 h as measured by physical stability. Release of paclitaxel was slow and sustained without initial burst release. Cytotoxicity studies in MDA-MB-231 cancer cells showed that both nanoparticles have similar anticancer activities compared to Taxol. Interestingly, PX BTM nanocapsules could be lyophilized without cryoprotectants. The lyophilized powder comprised only of PX BTM NPs in water could be rapidly rehydrated with a complete retention of original physicochemical properties, in vitro release properties, and cytotoxicity profile. Sequential Simplex Optimization has been utilized to identify promising new lipid-based paclitaxel nanoparticles having useful attributes.

Inactivation of the ketoreductase gilU gene of the gilvocarcin biosynthetic gene cluster yields new analogues with partly improved biological activity.

Four new analogues of the gilvocarcin-type aryl-C-glycoside antitumor compounds, namely 4'-hydroxy gilvocarcin V (4'-OH-GV), 4'-hydroxy gilvocarcin M, 4'-hydroxy gilvocarcin E and 12-demethyl-defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4'-OH-analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4'-OH group (gilvocarcins V and E). The structures of the sugar-containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C-glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post-polyketide synthase modifying enzymes, particularly the C-glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.

Generation of new landomycins with altered saccharide patterns through over-expression of the glycosyltransferase gene lanGT3 in the biosynthetic gene cluster of landomycin A in Streptomyces cyanogenus S-136.

Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was over-expressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist of the same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results of the production of landomycin E (which contains three sugars) by the LanGT3- mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene over-expression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity of the new landomycins--compared to known ones--was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length of the deoxysugar side chain was deduced from the results.

Cytotoxic activities of new jadomycin derivatives.

Cytotoxic activities of jadomycin B and five new jadomycin derivatives against four cancer cell lines (HepG2, IM-9, IM-9/Bcl-2 and H460) were evaluated. Jadomycin S was most potent against HepG2, IM-9 and IM-9/Bcl-2 while jadomycin F was most potent against H460. Their potencies correlated with the degrees of apoptosis induced. Structure-activity-relationship analyses clearly demonstrate that the side chains of the oxazolone ring derived from the incorporated amino acids make a significant impact on biological activity. Therefore, jadomycin offers an ideal scaffold to manipulate structure and could be exploited to make many novel bioactive compounds with altered activities.

Generation of novel landomycins M and O through targeted gene disruption.

Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.

The effect of DB-67, a lipophilic camptothecin derivative, on topoisomerase I levels in non-small-cell lung cancer cells.

PURPOSE: To determine the in vitro drug sensitivity of two non-small-cell lung cancer cell lines after treatment with the novel lipophilic camptothecin derivative, 7- tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67), to determine if topoisomerase I protein levels decrease after treatment with DB-67, and to assess the duration and extent of topoisomerase I modulation after DB-67 exposure, in order to provide information about drug resistance that may be useful in determining an appropriate dosing schedule for DB-67. METHODS: The growth inhibition of the non-small-cell lung cancer cell lines A549 and H460 after exposure to DB-67 was evaluated with the MTS assay. A549 and H460 cells were treated for various times with DB-67 and topoisomerase I levels were determined by western blot analysis. In addition, A549 and H460 cells were treated with DB-67 for 24 h and topoisomerase I levels were determined by western blot analysis daily for 1 week after drug removal. RESULTS: DB-67 inhibited the growth of both A549 and H460 cells grown in culture; the A549 cells were more resistant to the cytotoxic effects of DB-67 than H460 cells. Notably, A549 cells had approximately one-half the baseline topoisomerase I than H460 cells. Topoisomerase I protein levels significantly decreased after 8-18 h of exposure to DB-67. Both A549 and H460 cells treated with DB-67 for 24 h had only negligible amounts of topoisomerase I at the end of treatment. However, within 24 h of drug removal topoisomerase I levels returned to near baseline levels in both cell lines. CONCLUSIONS: The decrease in topoisomerase I levels caused by DB-67 may represent a mechanism of resistance to this novel camptothecin derivative. Dosing DB-67 once every 48-72 h may maximize the interaction of the drug with topoisomerase I and should be considered as a potential dosing schedule in the preclinical and clinical development of this compound.