Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants.

Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives.

Good Practices in Model-Informed Drug Discovery and Development: Practice, Application, and Documentation.

This document was developed to enable greater consistency in the practice, application, and documentation of Model-Informed Drug Discovery and Development (MID3) across the pharmaceutical industry. A collection of "good practice" recommendations are assembled here in order to minimize the heterogeneity in both the quality and content of MID3 implementation and documentation. The three major objectives of this white paper are to: i) inform company decision makers how the strategic integration of MID3 can benefit R&D efficiency; ii) provide MID3 analysts with sufficient material to enhance the planning, rigor, and consistency of the application of MID3; and iii) provide regulatory authorities with substrate to develop MID3 related and/or MID3 enabled guidelines.

A randomized double blind, placebo controlled phase 2 trial of BIIL 284 BS (an LTB4 receptor antagonist) for the treatment of lung disease in children and adults with cystic fibrosis.

BACKGROUND: Airway inflammation, mediated in part by LTB4, contributes to lung destruction in patients with cystic fibrosis (CF). LTB(4)-receptor inhibition may reduce airway inflammation. We report the results of a randomized, double-blind, placebo-controlled study of the efficacy and safety of the leukotriene B(4) (LTB(4))-receptor antagonist BIIL 284 BS in CF patients. METHODS: CF patients aged ≥6 years with mild to moderate lung disease were randomized to oral BIIL 284 BS or placebo once daily for 24 weeks. Co-primary endpoints were change in FEV(1) and incidence of pulmonary exacerbation. RESULTS: After 420 (155 children, 265 adults) of the planned 600 patients were randomized, the trial was terminated after a planned interim analysis revealed a significant increase in pulmonary related serious adverse events (SAEs) in adults receiving BIIL 284 BS. Final analysis revealed SAEs in 36.1% of adults receiving BIIL 284 BS vs. 21.2% receiving placebo (p = 0.007), and in 29.6% of children receiving BIIL 284 BS vs. 22.9% receiving placebo (p = 0.348). In adults, the incidence of protocol-defined pulmonary exacerbation was greater in those receiving BIIL 284 BS than in those receiving placebo (33.1% vs. 18.2% respectively; p = 0.005). In children, the incidence of protocol-defined pulmonary exacerbation was 19.8% in the BIIL 284 BS arm, and 25.7% in the placebo arm (p = 0.38). CONCLUSIONS: While the cause of increased SAEs and exacerbations due to BIIL 284 BS is unknown, the outcome of this trial provides a cautionary tale for the administration of potent anti-inflammatory compounds to individuals with chronic infections, as the potential to significantly suppress the inflammatory response may increase the risk of infection-related adverse events.

Modeling and simulation in clinical pharmacology and dose finding.

The breakout session 2 of the European Medicines Agency/European Federation of Pharmaceutical Industries and Associations Modeling and Simulation (M&S) workshop focused on two topics: when and how M&S should be used and would be accepted by the authorities for the dose-regimen selection; and when and how M&S can be applied to register a dosing regimen without the need for a specific study. Each topic was introduced by an industry and regulatory perspective, followed by case examples for illustration (Table 1).CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e29; doi:10.1038/psp.2013.5; advance online publication 27 February 2013.

A combined pharmacometric analysis of dabigatran etexilate in healthy volunteers and patients with atrial fibrillation or undergoing orthopaedic surgery.

Dabigatran etexilate is the orally bioavailable pro-drug of dabigatran, a direct thrombin inhibitor. Using data from eight clinical studies in healthy volunteers and patients with non-valvular atrial fibrillation (AF) or undergoing orthopaedic surgery (OS), population pharmacokinetic (PK) and pharmacodynamic (PD) models were developed to investigate whether the PK and PD of dabigatran differ across different populations. In both healthy volunteers (n=80) and patients (n=1,965), the PK of dabigatran was best described by a two-compartment disposition model with first-order absorption and elimination. Renal function was the only covariate shown to have a clinically relevant impact on dabigatran exposure. The patient PK model was successfully applied in predicting exposure observed in the RE-LY trial evaluating dabigatran treatment in patients with non-valvular AF. The relationship between dabigatran plasma concentrations and activated partial thromboplastin time in healthy volunteers and patients (n=762) was best described with a combination of a linear model and a maximum effect (Emax) model, consistent with previous reports. PK/PD relationships were robust across the various populations tested and were not affected by any of the covariates examined. In summary, the PK of dabigatran is sufficiently consistent to allow extrapolation of data generated in healthy volunteers to patients with AF or undergoing OS.

Population pharmacokinetic analysis of the oral thrombin inhibitor dabigatran etexilate in patients with non-valvular atrial fibrillation from the RE-LY trial.

BACKGROUND: Dabigatran etexilate (DE) is an orally absorbed prodrug of dabigatran, a thrombin inhibitor that exerts potent anticoagulant and antithrombotic activity. OBJECTIVES: To characterize the pharmacokinetics of dabigatran in patients with non-valvular atrial fibrillation (AF) from the Randomized Evaluation of Long-term Anticoagulant Therapy (RE-LY) trial and to quantify the effect of selected factors on pharmacokinetic (PK) model parameters. PATIENTS AND METHODS: A total of 27 706 dabigatran plasma concentrations from 9522 patients who received DE 110 or 150 mg twice daily were analyzed with non-linear mixed-effects modeling. RESULTS: The pharmacokinetics of dabigatran were best described by a two-compartment disposition model with first-order absorption. The covariates creatinine clearance (CRCL), age, sex, heart failure and the ethnic subgroup 'South Asian' exhibited statistically significant effects on apparent clearance of dabigatran. Body weight and hemoglobin significantly influenced the apparent volume of distribution of the central compartment. Concomitant medication with proton-pump inhibitors, amiodarone and verapamil significantly affected the bioavailability. However, all of the statistically significant factors that were identified, except for renal function status, showed only small to moderate effects (< 26% change in exposure at steady state). On the basis of simulations from the final population PK model, a dose of 75 mg twice daily would result in similar exposure for severely renally impaired patients with CRCL of 15-30 mL min(-1) and patients with normal renal function receiving 150 mg twice daily. CONCLUSIONS: The analysis provides a thorough PK characterization of dabigatran in the AF patient population from RE-LY. None of the covariates investigated, with the exception of renal function, warrants dose adjustment.

Prediction of neutropenia-related effects of a new combination therapy with the anticancer drugs BI 2536 (a Plk1 inhibitor) and pemetrexed.

This study investigated the feasibility of predicting the neutropenia-related effects of a therapy that combines the investigational drug BI 2536 (inhibitor of Polo-like kinase 1) and pemetrexed, an approved anticancer drug. Predictions were arrived at using the pharmacokinetic/pharmacodynamic (PK/PD) parameters of each of the drugs obtained from monotherapy studies and assuming that the neutropenic effect is additive when the drugs are administered as a combination therapy. Subsequently, a PK/PD model was developed to determine whether this assumption of additive effect was reasonable in relation to these two drugs. All analyses and simulations were performed using the population approach in NONMEM, version VI.

Population pharmacokinetic modelling and simulation of single and multiple dose administration of meloxicam in cats.

The objectives of these investigations were: first, to describe the pharmacokinetic properties of meloxicam in cats following single and multiple oral administration and secondly, to simulate different oral dosage regimes for meloxicam in cats after multiple dose administration to illustrate and evaluate those dosage regimes for the alleviation of inflammation and pain in cats. Six healthy domestic short hair cats were treated orally with various dosage regimes (0.05-0.2 mg/kg/day). Plasma samples were collected at predefined times and quantitatively analysed using liquid/liquid extraction followed by reverse phase HPLC with UV-detection. Meloxicam plasma concentration data were analysed using the population pharmacokinetic approach (software: NONMEM). The final model was used to simulate different dosage regimes. The plasma concentration-time profiles of meloxicam in cats after oral single and multiple dose administration were best described by an open one-compartment model with first-order absorption and first-order elimination. Pharmacokinetic parameters were estimated to be 0.00656 L/h/kg for the total apparent body clearance (CL/F), 0.245 L/kg for the apparent volume of distribution (V/F), 1.26 1/h for the absorption constant (K(A)) and 25.7 h for the mean plasma terminal half-life. Simulations showed that the median trough steady-state concentrations of 228 ng/mL were reached after five, one or 6 days following a single initial dose of 0.05, 0.1 and 0.2 mg/kg each followed by 0.05 mg/kg/day.

Detection and specific targeting of hypoxic regions within solid tumors: current preclinical and clinical strategies.

Poor oxygenation of solid tumors is a major indicator of adverse prognosis after standard treatment, e.g. radiotherapy. This observation founded on intratumoral pO(2) electrode measurements has been supported more recently by studies of injected hypoxia markers (pimonidazole, EF5) or hypoxia-related proteins (hypoxia-inducible factor-1alpha, carbonic anhydrase IX) detected immunohistochemically. Alternative approaches include imaging of tumor hypoxia by nuclear medicine studies and the measurement of hypoxia-related proteins (osteopontin) in patient plasma. Low oxygen levels as found in tumors are rarely observed in normal tissues. The presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but as an opportunity for tumor-specific treatment. Classic approaches to normalize tumor oxygenation involve the breathing of modified gas mixtures and pharmacologic modification of blood flow as in the "accelerated radiotherapy, carbogen, nicotinamide" (ARCON) scheme. Specific killing of hypoxic tumor cells can potentially be achieved by hypoxia-selective cytotoxins (model substance tirapazamine), which has shown promise in head and neck cancer. Direct targeting of hypoxia-related molecules such as hypoxia-inducible factor-1alpha, the central regulator of the hypoxic response in tumor cells, is an attractive approach currently tested in preclinical models. For clinical applications, the appropriate combination of hypoxia detection for patient selection with a hypoxia-specific treatment is essential. A therapeutic benefit has been suggested for the selection of patients by plasma osteopontin level and treatment with the hypoxic radiosensitizer nimorazole in addition to radiotherapy, for selection by F-misonidazole positron-emission tomography (PET) and treatment with tirapazamine in addition to chemoradiation and for selection by pimonidazole immunohistochemistry and ARCON treatment, all in head and neck cancer.

Contribution of the active metabolite M1 to the pharmacological activity of tesofensine in vivo: a pharmacokinetic-pharmacodynamic modelling approach.

BACKGROUND AND PURPOSE: Tesofensine is a centrally acting drug under clinical development for Alzheimer's disease, Parkinson's disease and obesity. In vitro, the major metabolite of tesofensine (M1) displayed a slightly higher activity, which however has not been determined in vivo. The aims of this investigation were (i) to simultaneously accomplish a thorough characterization of the pharmacokinetic (PK) properties of tesofensine and M1 in mice and (ii) to evaluate the potency (pharmacodynamics, PD) and concentration-time course of the active metabolite M1 relative to tesofensine and their impact in vivo using the PK/PD modelling approach. EXPERIMENTAL APPROACH: Parent compound, metabolite and vehicle were separately administered intravenously and orally over a wide dose range (0.3-20 mg kg(-1)) to 228 mice. Concentrations of tesofensine and M1 were measured; inhibition of the dopamine transporter was determined by co-administration of [(3)H]WIN35,428 as the pharmacodynamic measure. KEY RESULTS: Pharmacokinetics of tesofensine and M1 were best described by one-compartment models for both compounds. Nonlinear elimination and metabolism kinetics were observed with increasing dose. The PK/PD relationship was described by an extended E(max) model. Effect compartments were used to resolve observed hysteresis. EC(50) values of M1, as an inhibitor of the dopamine transporter, were 4-5-fold higher than those for tesofensine in mice. CONCLUSIONS AND IMPLICATIONS: The lower potency of M1 together with approximately 8-fold higher through steady-state concentrations suggest that M1 did contribute to the overall activity of tesofensine in mice.

Use of achiral/chiral SFC/MS for the profiling of isomeric cinnamonitrile/hydrocinnamonitrile products in chiral drug synthesis.

A directly coupled achiral/chiral SFC/MS method has been developed for the profiling of a three-step stereoselective synthesis of cinnamonitrile and hydrocinnamonitrile intermediates. Semipurified reaction mixtures were screened in one step to determine the diastereomeric/enantiomeric composition of the final product as well as to identify any remaining E/Z isomers present from the starting material. The coupled achiral/chiral column combination was found to significantly enhance the separation of both enantiomers and diastereomers, without adding significantly to the overall analysis time. This analytical technique should prove to be generally useful for the profiling of isomeric reaction products in chiral drug synthesis.

Effects on the immune system.

Marijuana and other exogenous cannabinoids alter immune function and decrease host resistance to microbial infections in experimental animal models and in vitro. Two modes of action by which delta9-tetrahydrocannabinol (THC) and other cannabinoids affect immune responses have been proposed. First, cannabinoids may signal through the cannabinoid receptors CB1 and CB2. Second, at sites of direct exposure to high concentrations of cannabinoids, such as the lung, membrane perturbation may be involved. In addition, endogenous cannabinoids or endocannabinoids have been identified and have been proposed as native modulators of immune functions through cannabinoid receptors. Exogenously introduced cannabinoids may disturb this homoeostatic immune balance. A mode by which cannabinoids may affect immune responses and host resistance maybe by perturbing the balance of T helper (Th)1 pro-inflammatory versus Th2 anti-inflammatory cytokines. While marijuana and various cannabinoids have been documented to alter immune functions in vitro and in experimental animals, no controlled longitudinal epidemiological studies have yet definitively correlated immunosuppressive effects with increased incidence of infections or immune disorders in humans. However, cannabinoids by virtue of their immunomodulatory properties have the potential to serve as therapeutic agents for ablation of untoward immune responses.

Response of Chinese hamster v79 multicellular spheroids exposed to high-energy carbon ions.

Chinese hamster V79-379A spheroids 200 +/- 30 microm (+/- SD) in diameter were irradiated in agitated medium in different oxygen atmospheres with (1) 227 MeV/nucleon (12)C(+6) ions (plateau region) to model tissue in the entrance channel during therapy, (2) carbon ions in the extended Bragg peak modeling tissue in the target volume, or (3) X rays as a reference modality. Cell survival curves were similar for modes (1) and (3), indicating the absence of a contact effect and the presence of a pronounced oxygen effect with oxygen enhancement ratios (OERs) of 2.8 and 2.9, respectively. In contrast, the oxygen effect was substantially smaller in mode (2) with an OER of 1.4. Under normal or restricted oxygen supply conditions (external pO(2) = 145 or 0 mmHg), the relative biological effectiveness (RBE) was 2.1 or 4.3, respectively, for Bragg-peak irradiation. This modality induced apoptosis and a dose-dependent accumulation of cells in G(2)/M phase even at pO(2) = 0 mmHg. The volume ratios of treated to untreated spheroids exhibited cyclic variations after heavy-particle treatment that were not directly attributable to cell cycle synchronization. In summary, the results suggest that carbon ions in the extended Bragg peak are more effective than conventional X rays, particularly under hypoxic conditions.

Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation.

An in vitro model of multi-step activation, in which cells of macrophage lineage are driven sequentially through inflammatory, primed, and fully activated states, was employed to assess for cannabinoid receptor expression. Murine and rat peritoneal macrophages, murine RAW264.7 and P388D, macrophage-like cells, and neonatal rat brain cortex microglia expressed the cannabinoid receptor type 2 (CB2) differentially in relation to cell activation. The CB2 was undetectable in resident peritoneal macrophages, present at high levels in thioglycolate-elicited inflammatory and interferon gamma (IFNgamma)-primed peritoneal macrophages, and detected at significantly diminished levels in bacterial lipopolysaccharide (LPS)-activated peritoneal macrophages. A comparable pattern of differential expression of the CB2 was noted for murine macrophage-like cells and neonatal rat brain cortex microglia. The cannabinoid receptor type 1 (CB1) was not detected in peritoneal macrophages or murine macrophage-like cells regardless of cell activation state but was present in neonatal rat microglia at low levels. These results indicate that levels of the CB2 in cells of macrophage lineage undergo major modulatory changes in relation to cell activation. Furthermore, since inflammatory and primed macrophages express the highest levels of CB2, the functional activities of macrophages when in these respective states of activation may be the most sensitive to the action of cannabinoids.

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma.

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane-2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile-0.12 M NH4H2PO4-tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5-150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze-thaw cycles and after 7 months frozen storage below -20 degrees C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.

Enhancement of tumor radioresponse by docetaxel: Involvement of immune system.

Docetaxel, a potent chemotherapeutic drug and a strong enhancer of tumor radioresponse, possesses immunomodulating properties. We previously reported that 40% of murine tumors responding to docetaxel by growth delay showed heavy infiltration with macrophages or lymphocytes. The present study explored the effect of whole body irradiation on antitumor action of docetaxel alone or docetaxel plus tumor irradiation. Mice bearing 8-mm MCa-K mammary carcinoma in the leg received 33 mg/kg docetaxel i.v., 5 to 65 Gy tumor irradiation, or both (radiation given 24 h after docetaxel). Docetaxel delayed tumor growth and enhanced the efficacy of radiation: it dramatically reduced TCD50 (radiation dose yielding 50% tumor cure) from the control value of 38.6 Gy to 11.8 Gy, for an enhancement factor of 3.27. In addition to enhancing tumor radioresponse, docetaxel decreased the lung metastatic rate in mice with local tumor control from 26% in mice receiving radiation alone to 11% in mice receiving docetaxel plus radiation. Docetaxel induced heavy infiltration of tumors with lymphocytes, determined 2-4 days after treatment: the percentage of lymphocytes increased from the control value of <2% to 27% in mice that received docetaxel 3 days earlier. This increase was due to the influx of helper/inducer T-lymphocytes and natural killer cells. Immunosuppression of tumor-bearing mice with 6 Gy whole-body irradiation prior to tumor isotransplantation reduced docetaxel-induced lymphocyte infiltration of tumors, antitumor and anti-metastatic action of docetaxel, and docetaxel-induced enhancement of tumor radioresponse. Thus, our results showed that docetaxel stimulates tumor infiltration with immune cells, which then participate in antitumor action of docetaxel alone or when combined with radiotherapy.

Controlling the spread of varicella zoster in the hospitalized patient.

In the health care setting, knowledge of communicable diseases is critical in the prevention of transmission. Communicable diseases transmitted by the respiratory route pose several difficult problems for control. One such disease, frequently overlooked in adults, because it is considered a childhood disease, is varicella zoster (chickenpox). Recognition of the disease and knowledge about its epidemiology, control, treatment, and follow-up care are necessary for the health care provider to prevent the spread of disease.

Rocky Mountain spotted fever: a warm weather problem.

Rocky Mountain spotted fever occurs primarily in the warm weather months of April through September in the southeastern and south central United States. It is transmitted by the bite of ticks infected with the etiologic Rickettsia. This article reviews the epidemiology, clinical manifestations, diagnosis and therapy of Rocky Mountain spotted fever. Emphasis is placed on the dilemmas of early diagnosis and the necessity of early and often empirical antibiotic treatment of suspected cases.