Angiogenic properties of human placenta-derived adherent cells and efficacy in hindlimb ischemia.

OBJECTIVE: Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. METHODS: The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored in vivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. RESULTS: PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. CONCLUSIONS: PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Prospective dose selection and acceleration of paliperidone palmitate 3-month formulation development using a pharmacometric bridging strategy.

AIMS: To prospectively select the dose of the paliperidone palmitate 3-month (PP3M) formulation, using a pharmacometric bridging strategy based on the paliperidone palmitate 1-month (PP1M) formulation previously approved for schizophrenia treatment. METHODS: Pharmacokinetic (PK) data from a 6-month interim analysis of a single dose PP3M Phase I clinical trial was integrated with a previously developed PP1M population-PK model. The model was updated to incorporate formulation as a covariate on absorption parameters and to explore the most critical design element of the Phase III study: the PP1M-to-PP3M dose multiplier for patients switching formulations. Plasma paliperidone concentrations were measured at predetermined intervals during Phase III, enabling comparison of the multiple-dose PK between PP1M and PP3M. Exposure matching was assessed graphically to determine whether paliperidone plasma concentrations from the two formulations overlapped. RESULTS: Prospective steady-state PK simulations revealed that a 3.5 multiple of the PP1M dose would yield a corresponding PP3M dose with comparable exposure. The prospective pharmacometric simulation and observed Phase III PK data agreed closely. Phase III results confirmed the hypothesis that efficacy of PP3M was noninferior to that of PP1M. The similarity in exposures between the two formulations was likely a key determinant of the equivalent efficacy between the two products observed in the Phase III study. CONCLUSIONS: Successful prospective PP3M Phase III clinical trial dose selection was achieved through the use of pharmacometric bridging, without conducting a Phase II study and using only limited Phase I data for PP3M. We estimate that this strategy reduced development time by 3-5 years and may be applicable to other drug development projects.

Antistaphylococcal activities of the new fluoroquinolone JNJ-Q2.

The new broad-spectrum fluoroquinolone JNJ-Q2 displays in vitro activity against Gram-negative and Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant MRSA isolates. Tested with isogenic methicillin-susceptible S. aureus (MSSA) and MRSA strains bearing quinolone-resistant target mutations, JNJ-Q2 displayed MICs ≤ 0.12 µg/ml, values 16- to 32-fold lower than those determined for moxifloxacin. Overexpression of the NorA efflux pump did not impact JNJ-Q2 MICs. Inhibition of S. aureus DNA gyrase and DNA topoisomerase IV enzymes demonstrated that JNJ-Q2 was more potent than comparators against wild-type enzymes and enzymes carrying quinolone-resistant amino acid substitutions, and JNJ-Q2 displayed equipotent activity against both enzymes. In serial-passage studies comparing resistance selection in parallel MRSA cultures by ciprofloxacin and JNJ-Q2, ciprofloxacin readily selected for mutants displaying MIC values of 128 to 512 µg/ml, which were observed within 18 to 24 days of passage. In contrast, cultures passaged in the presence of JNJ-Q2 displayed MICs ≤ 1 µg/ml for a minimum of 27 days of serial passage. A mutant displaying a JNJ-Q2 MIC of 4 µg/ml was not observed until after 33 days of passage. Mutant characterization revealed that ciprofloxacin-passaged cultures with MICs of 256 to 512 µg/ml carried only 2 or 3 quinolone resistance-determining region (QRDR) mutations. Cultures passaged with JNJ-Q2 selection for up to 51 days displayed MICs of 1 to 64 µg/ml and carried between 4 and 9 target mutations. Established in vitro biofilms of wild-type or ciprofloxacin-resistant MRSA exposed to JNJ-Q2 displayed greater decreases in bacterial counts (7 days of exposure produced 4.5 to >7 log(10) CFU decreases) than biofilms exposed to ciprofloxacin, moxifloxacin, rifampin, or vancomycin.

MurF inhibitors with antibacterial activity: effect on muropeptide levels.

MurF catalyzes the last cytoplasmic step of bacterial cell wall synthesis and is essential for bacterial survival. Our previous studies used a pharmacophore model of a MurF inhibitor to identify additional inhibitors with improved properties. We now present the characterization of two such inhibitors, the diarylquinolines DQ1 and DQ2. DQ1 inhibited Escherichia coli MurF (50% inhibitory concentration, 24 microM) and had modest activity (MICs, 8 to 16 microg/ml) against lipopolysaccharide (LPS)-defective E. coli and wild-type E. coli rendered permeable with polymyxin B nonapeptide. DQ2 additionally displayed activity against gram-positive bacteria (MICs, 8 to 16 microg/ml), including methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus isolates and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium isolates. Treatment of LPS-defective E. coli cells with >or=2x MIC of DQ1 resulted in a 75-fold-greater accumulation of the MurF substrate compared to the control, a 70% decline in the amount of the MurF product, and eventual cell lysis, consistent with the inhibition of MurF within bacteria. DQ2 treatment of S. aureus resulted in similar effects on the MurF substrate and product quantities. At lower levels of DQ1 (

Effect of MexXY overexpression on ceftobiprole susceptibility in Pseudomonas aeruginosa.

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, < or =4 microg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to beta-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 microg/ml at baseline but 16 microg/ml posttreatment, unrelated to beta-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical beta-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.

Ghostwriting in biomedicine: a review of the published literature.

Introduction: The systematic review of biomedical ghostwriting has proven challenging due to problems in consistency and in study design. Moreover, authorship guidelines established by the International Committee of Medical Journal Editors (ICMJE) may have inadvertently created opportunities to potentiate ghostwriting. Given continued interest in ghostwriting by the International Society of Medical Publication Professionals (ISMPP) and other organizations, we undertook an analysis of ghostwriting in the biomedical literature.Methods: We searched PubMed (search terms: ghost writ*, ghostwrit*, ghost writer, ghostwriter, ghostwriting and ghost writing). Results, including abstracts, were reviewed for relevance (relationship to ghostwriting in biomedical journals) to aid in removal of inapplicable work and duplicate publications. After review, we consolidated expert opinions for publication professionals.Results: Overlap was poor across search terms; of 181 unique papers identified, most (112/181) were opinion pieces. An increasing number of papers are using the term "ghostwriting" to describe genetics as well as diverse phenomena of misattributed authorship, including "ghost authorship". Eight primary studies and 1 systematic review of ghostwriting incidence were identified, reporting prevalence ranging from <1% to 91%, in varied settings using differing methods and definitions of ghostwriting. Suggestions for avoiding ghostwriting include early consensus building and better definitions of authorship among manuscript teams.Discussion: The prevalence and definition of ghostwriting remain unclear. Increased transparency and auditable authorship practices that align with specific guidelines may aid in the avoidance of ghostwriting. In addition, MeSH or clearer indexing terms may be helpful to separate usages of ghostwriting in scientific settings (e.g. genetic research) versus biomedical publishing.

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

A point mutation in influenza B neuraminidase confers resistance to peramivir and loss of slow binding.

The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.

A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli.

MurF is an essential enzyme of bacterial cell wall biosynthesis. Few MurF inhibitors have been reported, and none have displayed measurable antibacterial activity. Through the use of a MurF binding assay, a series of 8-hydroxyquinolines that bound to the Escherichia coli enzyme and inhibited its activity was identified. To derive additional chemotypes lacking 8-hydroxyquinoline, a known chelating moiety, a pharmacophore model was constructed from the series and used to select compounds for testing in the MurF binding and enzymatic inhibition assays. Whereas the original diverse library yielded 0.01% positive compounds in the binding assay, of which 6% inhibited MurF enzymatic activity, the pharmacophore-selected set yielded 14% positive compounds, of which 37% inhibited the enzyme, suggesting that the model enriched for compounds with affinity to MurF. A 4-phenylpiperidine (4-PP) derivative identified by this process displayed antibacterial activity (MIC of 8 microg/ml against permeable E. coli) including cell lysis and a 5-log(10)-unit decrease in CFU. Importantly, treatment of E. coli with 4-PP resulted in a 15-fold increase in the amount of the MurF UDP-MurNAc-tripeptide substrate, and a 50% reduction in the amount of the MurF UDP-MurNAc-pentapeptide product, consistent with inhibition of the MurF enzyme within bacterial cells. Thus, 4-PP is the first reported inhibitor of the MurF enzyme that may contribute to antibacterial activity by interfering with cell wall biosynthesis.

Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF.

MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-L-Ala-gamma-D-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-L-Ala-gamma-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-gamma-D-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing L-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 microM was identified.

Identification of a dithiazoline inhibitor of Escherichia coli L,D-carboxypeptidase A.

The enzyme L,D-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in Escherichia coli during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 microM. The inhibitor appeared to cause lysis of E. coli during stationary phase, behavior that is similar to a previously described deletion mutant of L,D-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje, EMBO J. 18:4108-4117, 1999). As much as a one-log drop in CFU in stationary phase was observed upon treatment of E. coli with the inhibitor, and the amount of intracellular tetrapeptide substrate increased by approximately 33%, consistent with inhibition of the enzyme within bacterial cells. Stationary-phase targets such as L,D-carboxypeptidase A are largely underrepresented as targets of the antibiotic armamentarium but provide potential opportunities to interfere with bacterial growth and persistence.

Synthesis and antibacterial activity of C-6 carbamate ketolides, a novel series of orally active ketolide antibiotics.

A new series of antibacterial ketolides is reported, which features the use of a C-6 carbamate for tethering the arylalkyl sidechain to the macrolide core. The best members of this series display in vitro and in vivo activity comparable to telithromycin. Partial epimerization at C-2, unobserved in previously reported ketolides, was noted for this series.

Pyrimido[1,2-b]-1,2,4,5-tetrazin-6-ones as HCMV protease inhibitors: a new class of heterocycles with flavin-like redox properties.

The synthesis and biological activity of pyrimidotetrazin-6-ones against HCMV protease is described. The mechanism of action for these inhibitors is the oxidation of several cysteine residues to generate cross-linked enzyme.