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BACKGROUND: Bladder cancer is the ninth most common cancer in men. 70% of these tumors are classified as non-muscle invasive bladder cancer and those patients receive 6 intravesical instillations with Mycobacterium bovis BCG after transurethral resection. However, 30% of patients show recurrences after treatment and experience severe side effects that often lead to therapy discontinuation. Recently, another vaccine strain, Salmonella enterica typhi Ty21a, demonstrated promising antitumor activity in vivo. Here we focus on increasing bacterial retention in the bladder in order to reduce the number of instillations required and improve antitumor activity. OBJECTIVE: To increase the binding of Ty21a to the bladder wall by surface labeling of the bacteria with adhesion protein FimH and to study its effect in a bladder cancer mouse model. METHODS: Binding of Ty21a with surface-labeled FimH to the bladder wall was analyzed in vitro and in vivo. The antitumor effect of a single instillation of Ty21a+FimH in treatment was determined in a survival experiment. RESULTS: FimH-labeled Ty21a showed significant (p

Summary Background Topical ionic contraviral therapy (ICVT) with digoxin and furosemide inhibits the potassium influx on which DNA viruses rely for replication. Therefore, ICVT was hypothesized to be a potential novel treatment for cutaneous warts. Objectives To assess the clinical efficacy, safety and tolerability of ICVT in adults with cutaneous warts. The secondary objective was to gain insight into the underlying working mechanism of ICVT. Methods Treatment with ICVT was assessed for efficacy, safety and tolerability in a single‐ centre, randomized, double‐blind, placebo‐controlled phase IIA trial. Eighty adult patients with at least two cutaneous warts (plantar or common) were randomized to one of four treatments: digoxin + furosemide (0·125%), digoxin (0·125%), furosemide (0·125%) or placebo. The gel was administered once daily for 42 consecutive days. Predefined statistical analysis was performed with a mixed‐model ancova. The trial was registered at ClinicalTrials.gov with number NCT02333643. Results Wart size and human papillomavirus (HPV) load reduction was achieved in all active treatment groups. A statistically significant reduction in wart diameter of all treated warts was shown in the digoxin + furosemide treatment group vs. placebo (−3·0 mm, 95% confidence interval −4·9 to −1·1, P = 0·002). There was a statistically significant reduction in the HPV load of all treated warts in the digoxin + furosemide group vs. placebo (−94%, 95% confidence interval −100 to −19, P = 0·03). With wart size reduction, histologically and immunohistochemically defined viral characteristics disappeared from partial and total responding warts. Conclusions This study demonstrates the proof of concept for the efficacy of topical ICVT in adults with cutaneous warts., What's already known about this topic? Cutaneous warts are caused by the human papillomavirus (HPV).Ionic contraviral therapy (ICVT) might be a potential treatment for cutaneous warts.A previous phase I/II open‐label study demonstrated the safety and efficacy of ICVT. What does this study add? Proof of concept for the efficacy of topical ICVT in adults with cutaneous warts.Topical ICVT demonstrates a favourable safety profile, with the effects most pronounced when it is combined in a formulation for common warts.Wart size reduction was related to HPV load reduction measured by quantitative polymerase chain reaction (qPCR) in swabs.qPCR is a valuable disease biomarker for drug development in cutaneous warts. https://doi.org/10.1111/bjd.17803 available online https://www.bjdonline.com/article/

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery., Wenzel et al. developed a flat embedding method for TEM, which increases the fraction of appropriately orientated cells per image, making it easier to quantify cell morphology. They find that tetracycline-treated B. subtilis shows membrane lesions and that tetracycline action is via direct perturbation of bacterial cell membrane in addition to its known ribosome inhibition activity.

This multicentre pilot study investigated the role of peroperative carcinoembryonic antigen (CEA)-specific fluorescence imaging during cytoreductive surgery-hyperthermic intraperitoneal chemotherapy surgery in peritoneal metastasized colorectal cancer. A correct change in peritoneal carcinomatosis index (PCI) owing to fluorescence imaging was seen in four of the 14 included patients. The use of SGM-101 in patients with peritoneally metastasized colorectal carcinoma is feasible, and allows intraoperative detection of tumour deposits and alteration of the PCI. Augmented reality guidance.

Transmission electron microscopy (TEM) is an important imaging technique in bacterial research and requires ultrathin sectioning of resin embedding of cell pellets. This method consumes milli- to deciliters of culture and results in sections of randomly orientated cells. For rod-shaped bacteria, this makes it exceedingly difficult to find longitudinally cut cells, which precludes large-scale quantification of morphological phenotypes. Here, we describe a new fixation method using either thin agarose layers or carbon-coated glass surfaces that enables flat embedding of bacteria. This technique allows for the observation of thousands of longitudinally cut rod-shaped cells per single section and requires only microliter culture volumes. We successfully applied this technique to Gram-positiveBacillus subtilis, Gram-negativeEscherichia coli, the tuberculosis vaccine strainMycobacterium bovisBCG, and the cell wall-lacking mycoplasmaAcholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we examined cellular changes induced by a panel of different antibiotics. Surprisingly, we found that the ribosome inhibitor tetracycline causes significant deformations of the cell membrane. Further investigations showed that the presence of tetracycline in the cell membrane changes membrane organization and affects the peripheral membrane proteins MinD, MinC, and MreB, which are important for regulation of cell division and elongation. Importantly, we could show that this effect is not the result of ribosome inhibition but is a secondary antibacterial activity of tetracycline that has defied discovery for more than 50 years.SignificanceBacterial antibiotic resistance is a serious public health problem and novel antibiotics are urgently needed. Before a new antibiotic can be brought to the clinic, its antibacterial mechanism needs to be elucidated. Transmission electron microscopy is an important tool to investigate these mechanisms. We developed a flat embedding method that enables examination of many more bacterial cells than classical protocols, enabling large-scale quantification of phenotypic changes. Flat embedding can be adapted to most growth conditions and microbial species and can be employed in a wide variety of microbiological research fields. Using this technique, we show that even well-established antibiotics like tetracycline can have unknown additional antibacterial activities, demonstrating how flat embedding can contribute to finding new antibiotic mechanisms.

Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria (N. D. Rawlings and A. Bateman, BMC Genomics 10:437, 2009, https://doi.org/10.1186/1471-2164-10-437). In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis. In addition, pathogenicity of M. marinum is reduced in the ΔpecA mutant, indicating that PecA contributes to virulence., Tuberculosis, one of the world’s most severe infectious diseases, is caused by Mycobacterium tuberculosis. A major weapon of this pathogen is a unique cell wall that protects the pathogen from eradication by the immune system. Mycobacteria have specialized secretion systems, e.g., type VII secretion or ESX systems, to transport substrates across this cell wall. The largest group of proteins that are secreted by these ESX systems are the PE proteins. Previously, it was shown that the N-terminal PE domain of about 100 amino acids is required for secretion. Here, we describe the identification of an aspartic protease, designated PecA, that removes (part of) this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.

Background DNA viruses such as HPV rely on K+ influx for replication. Both digoxin and furosemide inhibit the K+ influx by interacting with cell membrane ion co-transporters (Na+/K+-ATPase and Na+-K+-2Cl− co-transporter-1, respectively). We therefore hypothesized that these two compounds in a topical formulation may be valuable in the treatment of HPV-induced warts. This new approach is called Ionic Contra-Viral Therapy (ICVT). Objective To evaluate systemic exposure, safety and tolerability of ICVT with a combination of furosemide and digoxin after repeated topical application in subjects with common warts. Furthermore, we aimed to evaluate pharmacodynamics effects of ICVT. Methods Twelve healthy subjects with at least four common warts on their hands were included in the study and treated with a fixed dose of 980 mg topical gel containing 0.125% (w/w) digoxin and 0.125% (w/w) furosemide for 7 consecutive days on their lower back to assess safety and systemic exposure. Two warts were treated with 10 mg each and two served as negative controls to obtain preliminary evidence of treatment effect. Results ICVT was well tolerated topically, and there was no evidence of systemic exposure of digoxin or furosemide. There were no clinical relevant safety findings and no serious adverse events (SAEs). A rapid and statistically significant reduction in diameter, height and volume of the warts was already observed at day 14. Conclusion ICVT was found to be safe for administration to humans and 7 days of active treatment showed a statistical significant wart reduction compared to untreated control lesions, clearly indicating pharmacological activity.

To analyze the results of malleostapedotomy performed by applying the self-fixing and articulated titanium piston according to Hausler. Retrospective case review. Tertiary referral center. This study concerns a retrospective analysis of the results of malleostapedotomy with the use of a self-fixing articulated titanium piston in 16 ears of 16 consecutively treated patients between 2005 and 2009. The medical files were used for the acquisition of data on medical and surgical history and to obtain pre- and postoperative audiometry. Diagnosis and outcomes of mainly revision surgeries are presented and compared to the literature. Effect of (revision) malleostapedotomy by evaluating postoperative audiometry and air–bone gap closure. The postoperative air–bone gap closure was ≤ 10 dB in 9/16 (56%) ears and within ≤ 20 dB in 13/16 (81%) ears. The mean postoperative air–bone gap was 14.3 dB HL (0.5–2.0 kHz) and 17.3 dB HL (0.5–4.0 kHz). Postoperatively, there was no increase in bone conduction thresholds larger than 3 dB (0.5–2.0 kHz) and postoperative dizziness was absent or very limited and transient. The malleostapedotomy procedure has become surgically less demanding over time by the technical improvements present in the nowadays available pistons. The design of the self-fixing and articulated titanium piston used in the present group of patients allows a safe and straight-forward malleostapedotomy procedure. Present hearing outcomes match with results presented in the literature.

Background The clinical appearance of cutaneous warts is highly variable and not standardised. Objectives The aim of this study was to develop and validate a reproducible clinical tool for the standardised assessment of cutaneous warts to distinguish these lesions accurately. Methods Nine morphological characteristics were defined and validated regarding intra- and inter-observer agreement. Based on literature and semi-structured interviews, a systematic dichotomous assessment tool, the Cutaneous WARTS diagnostic tool (CWARTS diagnostic tool) was developed. The validation consisted of two independent parts performed with photographs from the recent WARTS-2 trial. In part A, the CWARTS diagnostic tool was tested by 28 experienced physicians who assessed photographs of 10 different warts to investigate inter-observer concordance. In part B, morphological characteristics were validated by blinded and independent scoring of 299 photographs by 6 different observers. Part B also entailed re-assessment of the photographs after at least 1 week. Primary outcome measurement was the intraclass correlation coefficient (ICC). Results Presence of black dots (capillary thrombosis) had the greatest ICC (0.85) for inter-observer agreement in part A, followed by arrangement (0.65), presence of border erythema(0.64) and sharpness of the border (0.60). In part B results were similar for inter-observer agreement with presence of black dots having the highest ICC (0.68), followed by border erythema (0.64), arrangement (0.58) and colour (0.55). For intra-observer agreement, presence of black dots had the highest agreement (0.69), followed by presence of border erythema (0.64) and colour (0.55). Conclusions The wart phenotype can be reliably assessed by the CWARTS diagnostic tool. This article is protected by copyright. All rights reserved.

Purpose Aim of this study was to demonstrate that MDCO-216 (human recombinant Apolipoprotein A-I Milano) does not induce adverse immunostimulation, in contrast to its predecessor, ETC-216, which was thought to contain host cell proteins (HCPs) that elicited an inflammatory reaction. Methods Data were taken from a clinical trial in which 24 healthy volunteers (HV) and 24 patients with proven stable coronary artery disease (sCAD) received a single intravenous dose of MDCO-216, ranging 5–40 mg/kg. Additionally, whole blood from 35 HV, 35 sCAD patients and 35 patients requiring acute coronary intervention (aCAD group) was stimulated ex vivo with MDCO-216 and ETC-216. Results No inflammatory reaction was observed in HV and sCAD patients following MDCO-216 treatment, judging by body temperature, white cell counts, neutrophil counts, C-reactive protein, circulating cytokines (IL-6, TNF-α), and adverse events. In the ex vivo experiment, the geometric means (SD) of the ratio of MDCO-216 stimulated IL-6 over background levels were 0.8 (1.9), 0.7 (1.5), 1.0 (2.0) for respectively HV, sCAD, aCAD. The corresponding ETC-216 stimulated values were 15.8 (2.9), 9.5 (3.6), 3.8 (4.0). TNF-α results were comparable. Because many ETC-216 stimulated samples had cytokine concentrations >ULOQ, ratios were categorised and marginal homogeneity of the contingency table (MDCO-216 versus ETC-216) was assessed with the Stuart-Maxwell test. P-values were ≤0.0005 for all populations. Conclusions MDCO-216 did not induce adverse immunostimulation in HV and sCAD patients, in contrast to ETC-216. Results from the ex vivo stimulation suggests the same holds true for aCAD patients.

BACKGROUND: Cutaneous warts have a cure rate after therapy of no more than approximately 50%. Recently, we developed and validated a standard assessment tool for warts (Cutaneous WARTS diagnostic tool, CWARTS) based on phenotypical characteristics.OBJECTIVES: To assess whether patient and morphological wart characteristics predict the human papillomavirus (HPV) type in a specific wart and whether these characteristics as well as the HPV type predict a favourable treatment response.METHODS: Photographs were used to score nine morphological wart characteristics using the newly developed CWARTS tool. Genotyping of 23 wart-associated HPV types was performed using the hyperkeratotic skin lesion-polymerase chain reaction/multiplex genotyping assay. The results were correlated with a favourable response to treatment with monochloroacetic acid, cryotherapy or a combination of cryotherapy and salicylic acid. Odds ratios were calculated using logistic regression in a generalized estimating equations model.RESULTS: Black dots (capillary thrombosis) strongly predicted the presence of any HPV type in a wart. From all characteristics tested, the HPV type most strongly predicted the treatment response when the warts were treated with monochloroacetic acid or a combination of cryotherapy and salicylic acid with a significantly decreased treatment response if the warts contained HPVs of the alpha genus (HPV2, HPV27 or HPV57). When cryotherapy alone was used for common warts, HPV type did not play a role, but cryotherapy was less effective in the presence of callus and when the wart was located deeper in the skin.CONCLUSIONS: Morphological characteristics of the warts and the HPV genotype influence treatment outcome and thus potentially influence future treatment decisions for common and plantar warts

Bladder cancer is the fourth most common malignancy amongst men in Western industrialized countries with an initial response rate of 70% for the non-muscle invasive type, and improving therapy efficacy is highly needed. For this, an appropriate, reliable animal model is essential to gain insight into mechanisms of tumor growth for use in response monitoring of (new) agents. Several animal models have been described in previous studies, but so far success has been hampered due to the absence of imaging methods to follow tumor growth non-invasively over time. Recent developments of multimodal imaging methods for use in animal research have substantially strengthened these options of in vivo visualization of tumor growth. In the present study, a multimodal imaging approach was addressed to investigate bladder tumor proliferation longitudinally. The complementary abilities of Bioluminescence, High Resolution Ultrasound and Photo-acoustic Imaging permit a better understanding of bladder tumor development. Hybrid imaging modalities allow the integration of individual strengths to enable sensitive and improved quantification and understanding of tumor biology, and ultimately, can aid in the discovery and development of new therapeutics.

Toll-like receptor-4 (TLR4) pathways are major contributors to pathological inflammatory responses induced by tissue damage. NI-0101 is the first monoclonal antibody (mAb) blocking TLR4 signaling. This activity is independent of the ligand type and concentration, therefore, potentially blocking any TLR4 ligands. A phase I single ascending dose study was conducted in 73 healthy volunteers to evaluate NI-0101 tolerability, preliminary safety, pharmacokinetics (PKs), and pharmacodynamics (PDs), in absence and in presence of a systemic challenge with lipopolysaccharide (LPS), a TLR4 ligand. NI-0101 was well tolerated without safety concern. The PK profile was characterized by a half-life of ∼10 days at high concentrations and by a rapid elimination at low concentrations due to expected target-mediated drug disposition. NI-0101 prevented cytokine release following ex vivo and in vivo LPS administration and prevented the C-reactive protein (CRP) increase and the occurrence of flu-like symptoms expected following the in vivo administration of LPS.

Arginine-vasopressin (AVP) is a physiological co-activator of the hypothalamus–pituitary–adrenal (HPA) axis, together with corticotrophin releasing hormone (CRH). A synthetic analogue of AVP, desmopressin (dDAVP), is often used as a pharmacological tool to assess co-activation in health and disease. The relation between dDAVP’s neuroendocrine, cardiovascular, pro-coagulatory, anti-diuretic and non-specific stress effects has not been studied. A randomized, double-blind, placebo-controlled, three-way crossover study was performed in 12 healthy male and female volunteers (6 : 6). dDAVP was administered intravenously as a 10 μg bolus (over 1 min) or a 30 μg incremental infusion (over 60 min). Neuroendocrine, cardiovascular, pro-coagulatory, anti-diuretic effects and adverse events (AEs) were recorded, and autonomic nervous system (ANS) activation evaluated. The incremental infusion reached 1.8-fold higher dDAVP concentrations than the bolus. Neuroendocrine effects were similar for the 10 μg dDAVP bolus and the 30 μg incremental infusion, while cardiovascular and coagulatory effects were greater with the 30 µg dose. Osmolality and ANS activity remained uninfluenced. AEs corresponded to dDAVP’s side-effect profile. In conclusion, the neuroendocrine effects of a 10 μg dDAVP bolus administered over 1 min are similar to those of a 30 μg incremental infusion administered over one hour, despite higher dDAVP concentrations after the infusion. Cardiovascular and coagulatory effects showed clear dose-related responses. A 10 μg dDAVP bolus is considered a safe vasopressinergic function test at which no confounding effects of systemic or autonomic stress were seen.

To develop a limited sampling strategy to determine ciclosporin systemic exposure [area-under-the-curve(AUC)]. This is meant to be the first step in a future study of the relationship between AUC and the biological effects of ciclosporin.The pharmacokinetics of ciclosporin was investigated prospectively following stem cell transplantation (SCT) in 17 children, aged 1.8-16.1 years. Ciclosporin was given twice daily, intravenously over a short infusion of 2 h duration during the early post-SCT period, or orally later on, when oral medication was well tolerated. Parameter estimation was performed using nonlinear mixed effect modelling as implemented in the NONMEM program. Individual empirical Bayes estimates of clearance and distribution volume were correlated with the demographic variables.Pharmacokinetics was described adequately with a two-compartment model with lag time (population estimates: CL = 11.3 l h(-1); V(c) = 16.5 l; V(p) = 59.9 l; t(1/2) absorption = 0.78 h, t(lag) = 0.6 h). The AUCs, determined for the combination of trough level with one time point between 2 and 3 h after dosing, correlated very well with the reference AUC (r(2) = 0.97). No correlation was found between clearance and distribution volume, and the demographic patient variables length, body weight, age and glomerular filtration rate.A two-point limited sampling strategy, in combination with a Bayesian fitting procedure using the pharmacokinetic population model described, can adequately determine the AUC of ciclosporin. Since no correlation between clearance and body weight was found, dosing ciclosporin per kg bodyweight is not supported by the results of this study. We suggest starting with a fixed dose, followed by AUC determination and dose adjustment.

RWJ-800088 is a novel thrombopoietin mimetic peptide for the treatment of thrombocytopenia. The objectives of this study were to evaluate the pharmacokinetics, pharmacodynamics, and safety of ascending doses of RWJ-800088 administered as a single intravenous delivery in a double-blind, placebo-controlled study with five parallel groups of eight healthy human subjects each. Platelet counts and functionality, peripheral stem cells, drug concentrations, and routine laboratory parameters were measured frequently up to day 29, and antibody formation was measured up to days 56-72. At doses > or = 0.75 microg/kg of RWJ-800088, platelet levels showed dose-related elevation as compared to results with placebo. The pharmacokinetic profile was characterized for doses of 2.5 and 3.0 microg/kg, although the dose relationship could not be fully defined. The two highest doses of RWJ-800088 appeared to increase burst-forming units-erythroid and colony-forming unit counts, suggesting some effects on progenitor lineages. RWJ-800088 was well tolerated, with no evidence of antibody formation in this single-dose study. Additional patient studies are warranted to investigate the therapeutic use of this novel peptide.

Introduction The clinical assessment of the myocardial damage caused by anthracyclin (ANT)-therapy is difficult. Therefore a study was performed to evaluate non-invasive markers of anthracyclin-induced cardiac effects, with emphasis on course-to-course variation. Methods Eligible for study participation were patients, without known cardiologic abnormalities who did not use cardiotoxic medication (except for ANT-therapy), who had previously completed at least three cycles of anthracyclin-containing chemotherapy (n = 14) and patients who were ANT-naïve and who were scheduled to receive doxorubicin-containing chemotherapy (n = 12). Seven patients in this last group also completed at least three cycles and were available for follow-up assessments; thus a total population of 21 patients (12F/9M) completed at least three courses ANT-chemotherapy. In these patients blood samples and ECG-recordings were taken within 6 months after completion of ANT-therapy. In 12 patients (10F/2M) assessments were also done before, immediately afterwards and at 24 h after each course of ANT. Results and Conclusions In the patients who completed chemotherapy, NT-proBNP was 277% (n = 21; 95% CI: 86–661%, P

Aims Apolipoprotein A-1 (ApoA-1), based on epidemiology, is inversely associated with cardiovascular (CV) events. Human carriers of the ApoA-1 Milano variant have a reduced incidence of CV disease. Regression of atherosclerotic plaque burden was previously observed on intravascular ultrasound (IVUS) with ETC-216, a predecessor of MDCO-216. MDCO-216, a complex of dimeric ApoA-1 Milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, is being developed to reduce atherosclerotic plaque burden and CV events. We investigated the efficacy and safety of a single infusion of MDCO-216 in healthy volunteers and in patients with coronary artery disease (CAD). Methods and results Twenty-four healthy volunteers and 24 patients with documented CAD received a 2-h infusion of MDCO-216 in a randomized, placebo controlled, single ascending dose study. Five cohorts of healthy volunteers and four cohorts of CAD patients received ApoA-1 Milano doses ranging from 5 to 40 mg/kg. Subjects were followed for 30 days. Dose-dependent increases in ApoA-1, phospholipid, and pre-beta 1 HDL and decreases in ApoE were observed. Prominent and sustained increases in triglyceride, and decreases in HDL-C, endogenous ApoA-1 and ApoA-II occurred at doses >20 mg/kg and profound increases in ABCA1-mediated cholesterol efflux were observed. Other lipid and lipoprotein parameters were generally unchanged. MDCO-216 was well tolerated. Conclusions MDCO-216-modulated lipid parameters profoundly increased ABCA1-mediated cholesterol efflux and was well tolerated. These single-dose data support further development of this agent for reducing atherosclerotic disease and subsequent CV events. [10.1093/ehjcvp/pvv042][1] [1]: /lookup/doi/10.1093/ehjcvp/pvv042

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 μg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs. © 2015 American Society for Clinical Pharmacology and Therapeutics. Chemicals/CAS: alkaline phosphatase, 9001-78-9; carbon, 7440-44-0; Alkaline Phosphatase; alkaline phosphatase, placental; Carbon Radioisotopes; GPI-Linked Proteins; Isoenzymes; Recombinant Proteins

Summary Aim : To investigate a motilin effect on gallbladder volume in healthy volunteers and patients with functional dyspepsia. Methods : Forty-three healthy volunteers and 10 patients with functional dyspepsia received motilin (4 pmol.min/kg) or placebo in four separate double-blind, randomized, placebo-controlled, cross-over studies. The gallbladder volume was measured by ultrasonography. Analysis of variance of the combined data of these studies was performed to investigate a motilin effect on gallbladder volume and potential differences between patients and healthy volunteers. Results : The baseline gallbladder volume was similar for placebo and motilin treatment, as well as for patients and healthy volunteers. Motilin, compared with placebo, significantly decreased the gallbladder volume in healthy volunteers (P = 0.003) and patients (P

Aims The role of motilin in the regulation of upper gastrointestinal (GI) motility is well defined. However, little is known about the effects on the distal GI tract. To investigate the effect of exogenous motilin on rectal function, barostat measurements in the rectum were performed and lower abdominal symptoms were scored. Methods Eight fasted, healthy volunteers were infused intravenously with synthetic motilin or placebo over 90 min in a double-blind, randomized, cross-over design. Rectum volume was measured with a barostat device during constant pressure and during isobaric distensions. Lower abdominal symptoms were scored by visual analogue scales. Plasma motilin concentrations were measured by radioimmunoassay. Results Baseline rectum volumes were similar between treatments: 185 ± 62 mL (motilin) and 136 ± 41 mL (placebo). During the constant pressure procedure, motilin increased rectum volume [area under the effect curve (AUEC)] by 6%[95% confidence interval (CI) −3, 16] of baseline, compared with placebo. During isobaric distensions motilin increased rectum volume (AUEC) by 43 mL (95% CI 0.4, 85; P

Background The optimal dosing strategy for continuous intravenous furosemide infusion is unknown in pediatric patients. Eighteen patients less than 1 year old were studied after cardiac surgery during routine clinical care. The current strategy starts with a continuous infusion of 0.1 mg/kg · h, which may be adapted. Methods A pharmacokinetic-pharmacodynamic model was developed that linked furosemide dose to furosemide serum concentrations, renal function (creatinine clearance), and urine output. Various regimens were simulated that adapt according to urine production. The modified dosing schedule was prospectively tested in a subsequent population of 18 pediatric patients after cardiac surgery. Results Data from the follow-up study suggest that urine production is more controlled for the proposed regimen. Conclusions Both the modeling and simulation results and the follow-up study indicated that a bolus dose of 1 mg/kg followed 6 hours later with a 1- or 2-mg/kg loading dose and a 0.2-mg/kg · h intravenous infusion provides a rational starting point for furosemide therapy after cardiac surgery in pediatric patients less than 1 year old. Adjustment of this regimen every 12 hours in steps of 0.1 mg/kg · h on the basis of clinical assessment should lead to adequate control over urinary output. Clinical Pharmacology & Therapeutics (2002) 72, 383–390; doi: 10.1067/mcp.2002.127608

Aims To investigate the pharmacokinetic and pharmacodynamic interaction of the antithrombotic pentasaccharide fondaparinux (Org31540/SR90107A), given subcutaneously, and oral warfarin in healthy subjects. Methods This study was performed according to a randomised, three-way cross-over, placebo-controlled, double-blind design in 12 healthy male subjects. The treatment consisted of five subcutaneous (s.c.) injections of fondaparinux (4 mg) or placebo at 24 h intervals. Oral dosing of warfarin or placebo was added to the fourth (15 mg) and fifth (10 mg) s.c. injection. Blood samples for pentasaccharide assay, PT and APTT were drawn before the first s.c. dose of the pentasaccharide and over a 6 day period thereafter. Results Fondaparinux administered to healthy male volunteers alone or in combination with oral warfarin was well tolerated and no serious adverse events were observed. No differences were found in the AUC (43 vs 44 mg l−1 h), Cmax (645 vs 678 ng ml−1) or elimination half-life (13.8 vs 14.1 h) of fondaparinux between the pentasaccharide-only and the combination treatment. The effect of warfarin on PT (mean maximal increase: 8.2 s.) was not influenced by the presence of the pentasaccharide (mean maximal increase in PT: 9.1 s.). After all treatments a small rise in APTT was seen. No further differences could be detected in the pharmacodynamic parameters following the three treatments. Conclusions The coadministration of warfarin did not influence the pharmacokinetics of fondaparinux in healthy subjects. PT can still be used to monitor the effect of oral anticoagulants during the switch from antithrombotic treatment with pentasaccharide to full oral anticoagulant therapy.

Aim: To assess non-invasively the dose–response relations for the effects of exogenous motilin on antrum contraction frequency, gall-bladder volume and gastric myoelectrical activity. Methods: In a double-blind, randomized, placebo-controlled, five-way crossover study, 10 fasted healthy volunteers were infused intravenously with synthetic human motilin (0.5, 1, 2 and 4 pmol.min/kg) or placebo for 60 min. Gall-bladder volume and antrum contractions were assessed by ultrasonography and gastric myoelectrical activity by electrogastrography. Motilin concentrations were measured using a radioimmunoassay. Results: Baseline plasma motilin levels (60 pmol/L) were similar for all treatments. Motilin levels increased upon the start of infusion and rapidly returned to baseline after cessation of the infusion. At motilin doses of 2 and 4 pmol.min/kg, the antrum contraction frequency was significantly augmented, with maximum differences of two contractions per 2-min interval compared to placebo, while no changes in gastric myoelectrical activity were observed. Changes in gall-bladder volume were not significantly different for any of the motilin doses compared to placebo. Conclusions: Motilin increased antrum contraction frequency, whereas no effect on gastric myoelectrical activity was observed. Antrum contraction frequency appears to be a useful biomarker for motilin efficacy, and motilin doses of 2 and 4 pmol.min/kg were equally effective.

Patients with germ cell tumors (GCTs) have an excellent prognosis but are at risk for silent fractures. Data on bone mineral density (BMD) after anticancer treatment are scarce.The objective of the study was BMD monitoring in GCT patients treated with or without chemotherapy.We prospectively studied 63 newly diagnosed GCT patients with a median age of 33 years (range 16-70 y) within 3 months of unilateral orchidectomy. Twenty-seven patients (42.9%) had no metastases. Thirty-six patients (57.1%) with metastatic disease received combination chemotherapy.This study was conducted at the outpatient clinic of a single academic institution.We performed dual-energy X-ray absorptiometry scans and collected blood samples on a yearly basis, before and up to 5 years after anticancer treatment.Changes in total hip and lumbar spine BMD, serum concentrations of gonadal hormones, and bone turnover markers were measured.BMD remained normal in stage I patients. In patients with metastatic disease, a significant decrease in lumbar spine BMD (-1.52%; P = .004) and total hip BMD (-2.05%; P.0001) was observed 1 year after chemotherapy and remained stable thereafter for up to 5 years. There was no significant relationship between the observed decrease in BMD and gonadal status, vitamin D status, or cumulative dose of cisplatin or (antiemetic) corticosteroids.Metastatic GCT survivors demonstrate significant bone loss within the first year after curative combination chemotherapy, with no recovery up to 5 years after anticancer treatment. Whether this bone loss is associated with increased fracture risk and whether this could be prevented by bone modifying treatment remains to be established.

Background In Dutch ambulance practice, failure or inability to intubate patients with altered oxygenation and/or ventilation leaves bag-valve mask ventilation as the only alternative, which is undesirable for patient outcome. A novel Laryngeal Mask Airway Supreme (LMA-S) device may be a suitable alternative. Aim To evaluate the effectiveness and suitability of the LMA-S for emergency medical services in daily out-of-hospital emergency practice. Methods After a period of theoretical and practical training of ambulance paramedics in the use of the LMA-S, prospective data were collected on the utilisation of LMA-S in an observational study. Procedures for use were standardised and the evaluation included the number of direct intubation attempts before using LMA-S, attempts required, failure rate and the adequacy of ventilation. Data were analysed taking patient characteristics such as age and indication for ventilatory support into account. Results The LMA-S was used 50 times over a period of 9 months (33 involving cardiorespiratory arrest, 14 primary and three rescue). The LMA-S could be applied successfully in all 50 cases (100%) and was successful in the first attempt in 49 patients (98%). Respiratory parameters showed adequate oxygenation. All paramedics were unanimously positive about the utilisation of LMA-S because of the easiness of the effort of insertion and general use, and emphasised its value as a useful resource for patients in need. Conclusions Ensuring ventilation support by using LMA-S by paramedics in prehospital emergency practice is safe and effective.

During studies on warfarin, heparin and various anticoagulants with novel mechanisms of action, the activated partial thromboplastin time (aPTT) and the (apparent) international normalized ratio (INR) from a bedside monitor (Coagucheck Plus®) were compared with laboratory assay results. Data were compared using the Bland and Altman method of comparison where systematic differences result in significant slopes of the regression line. During heparin treatment, the bedside monitor largely underestimated the aPTT (slope=-0.80). During treatment with the direct thrombin inhibitor napsagatran (slope=0.99), the pentasaccharides Org31540/SR90107A (slope=0.77) and SanOrg34006 (slope=0.35), and warfarin (slope=0.60), the bedside monitor underestimated the aPTT at lower aPTT levels, while at higher aPTT levels it overestimated the laboratory values. The bedside monitor slightly overestimated the INR during treatment with warfarin (slope=0.33). Apparent INR was largely overestimated during treatment with Org31540/SR90107A (slope=1.38), SanOrg34006 (slope=0.97), Napsagatran (slope=1.23), and recombinant tissue factor pathway inhibitor (slope=1.48, P < 0.001 for all regression lines). These results indicate that a substantial disagreement in aPTT or (apparent) INR exists between the bedside monitor and laboratory assay during treatment with the studied 'classic' and novel anticoagulants. The amount of disagreement depended on the anticoagulant given. © 2001 Lippincott Williams & Wilkins. Chemicals/CAS: Anticoagulants; Antithrombins; Fibrinolytic Agents; Heparin, 9005-49-6; Naphthalenes; Oligosaccharides; Org 31540, 104993-28-4; Piperidines; Ro 46-6240; SANORG 34006; Warfarin, 81-81-2

Objective: The effect of oral warfarin on the pharmacokinetics and pharmacodynamics of the synthetic direct thrombin inhibitor napsagatran was investigated. Methods: In an open, randomised, two-way crossover study, 12 healthy male volunteers were infused napsagatran (80 µg/min) for 48 h. Each subject was administered 25 mg warfarin (Coumadin) at the start of the infusion in either the first or second treatment period. Sampling was performed regularly over the treatment period and 24 h thereafter for measurement of plasma levels of napsagatran, activated partial thromboplastin time (APTT) and prothrombin time (PT). Results: The pharmacokinetic parameters of napsagatran were not significantly influenced by co-administration of warfarin. Napsagatran administration was followed by increases in APTT and PT. Co-administration of warfarin increased the AUEC (area under the effect curve) calculated for the period 0–48 h (corrected for baseline) for APTT by 45% (95% CI: 28–65%) and for PT by 438% (95% CI: 272–678%) compared to the treatment with napsagatran alone. Conclusion: Warfarin has no effect on the pharmacokinetics of napsagatran, but has a marked influence on the pharmacodynamic parameters (APTT, PT) of napsagatran. In clinical practice, this interaction between the two compounds should be taken into account. The PT cannot be used to monitor the effect of oral anticoagulants during the switch from this group of direct thrombin inhibitors to full oral anticoagulant therapy.

Objective: The commonly used continuous intravenous (IV) furosemide dosing schedule after cardiac surgery in children is largely empirical and may not be optimal. This may even be more marked in children after cardiac surgery who are haemodynamically unstable, and in whom transient renal insufficiency may occur. A study was performed to obtain an impression regarding which clinically applicable measures may be used to design a rational scheme for continuous IV furosemide therapy in children after cardiac surgery. Subjects and methods: Twelve paediatric patients (5F/7 M, age 0–33 weeks) post-cardiac surgery, who were to receive 3 days of continuous IV furosemide treatment, were included in an open study. Blood and urine samples were taken for furosemide, creatinine, and electrolyte levels, and fractionated urinary output was measured. Furosemide in blood and urine was measured using high performance liquid chromatography (HPLC). Results: The mean starting dose of continuous IV furosemide was 0.093 (±0.016) mg/kg per hour. The mean dose was increased to 0.175 (±0.045) mg/kg per hour per hour on day 2, and changed to 0.150 (±0.052) mg/kg per hour on day 3. Infusion rates were increased from day 1 to day 2 in ten cases, and decreased from day 2 to day 3 in three cases. Serum furosemide levels never exceeded ototoxic levels. The urinary furosemide excretion rate was inversely related to serum creatinine levels. Conclusions: This study extends the observation of the beneficial effects of continuous IV furosemide also to those children who are haemodynamically unstable after cardiac surgery. However, as the effects of furosemide are dependent on renal function, it can be hypothesised that the dosing schedule may be optimised. Contrary to the currently used dosage schedule in which the dose of furosemide is gradually increased over time, it may be more rational to start with a higher dose and adapt this dose (downward) guided by the observed effect (urine output). Because the infusion rate was increased to 0.2 mg/kg per hour in nine out of 12 patients on day 2 and was never increased further, this suggests that a starting rate of 0.2 mg/kg per hour may be optimal.

Aims To study the delivery of 15 mg beclomethasone 17,21-dipropionate (BDP) to the distal part of the small bowel for three oral sustained-release formulations (I-III) and a reference capsule in volunteer ileostomists, and to compare these findings with the in vitro dissolution profiles. Methods Two groups of nine ileostomy volunteers (aged 20–61 years), who were otherwise healthy, were enrolled in the study. The recovery of BDP and its metabolite beclomethasone 17-monopropionate (B17P) in ileostomy effluent was investigated in a cross-over study after administration of formulations I or II or a reference capsule containing micronised BDP, and in a second open study after administration of formulation III. Radio-opaque granules were coadministered for evaluation of gastrointestinal passage. Ileostomy effluents were collected hourly for 10–12 h following drug intake. After marker beads had been counted on X-rays, ileostomy collections were analysed for BDP and its metabolites. Cumulative recovery, lag-time and mean transit time were determined for drug and marker beads. Results Gastrointestinal passage characteristics were similar for all treatments. Total drug recovery was approximately three times higher for the sustained-release formulations than for the reference capsule. Recovery of B17P from stoma fluid samples was significantly lower for formulation III than for formulations I and II. Conclusions The novel oral formulations delivered substantial amounts of steroid drug at the distal small bowel/proximal colon, which may warrant further studies to evaluate clinical applicability.

Aims Application of single methods to assess liver blood flow (LBF) yielded conflicting results on the magnitude and duration of effect on LBF of oral nifedipine and captopril. The aim of this study was to investigate the influence of these drugs on LBF by simultaneous use of ICG infusion and echo-Doppler. Methods The study was performed according to a double-blind, placebo-controlled, randomized, cross-over design in nine healthy male volunteers. After an overnight fast and an equilibration period, subjects received a continuous i.v. indocyanine green (ICG) infusion for 4 h. At presumed ICG steady state (t=45 min), subjects were dosed with oral nifedipine (20 mg), captopril (50 mg) or placebo. During the experiment, blood sampling for ICG assay and measurement of portal venous blood flow (echo-Doppler) took place regularly. Treatments were compared using analysis of variance. Differences are reported with 95% confidence intervals (CI). Results The area under the curves (AUC) for ICG over 1 h and over 3 h after nifedipine were 15% (difference in AUC: +0.6, +7.0 mg l−1 min) and 22% (+7.0, +28.4 mg l−1 min) lower compared with placebo. After captopril, the AUC values were 8–10% lower compared with placebo but the 95% CIs included zero. Portal venous flow was 15% (+5, +86 ml min−1 ) higher compared to placebo after nifedipine but not after captopril (−3%; −49, +33 ml min−1 ). The duration of effect on liver blood flow lasted approximately 2 h but was variable (range: 40–160 min). The time to maximal blood flow increase and the duration of effect after nifedipine were very similar for both measures of LBF. Changes in ICG concentrations could be reasonably well predicted from the changes in portal blood flow. Conclusions Nifedipine increases LBF for a substantial period of time but the effect is variable between subjects. This effect could be detected by both the ICG method and echo-Doppler and the findings of both methods were in agreement. In this respect it is likely that captopril does not influence LBF in healthy volunteers as no effect was detected with either method.

Objective Heart rate variability (HRV) reflects the balance of activities of sympathetic and parasympathetic components of the autonomic nervous system. We compared HRV parameters in response to a prolonged fast in obese versus normal weight humans. In addition, the effect of weight-loss was evaluated in obese individuals. Design Intervention study. Patients The study subjects included 14 nondiabetic obese (12 females/2 males, aged 30 ± 3 years, Body Mass Index (BMI) 35·2 ± 1·2 kg/m2) and 12 lean subjects (10 females/2 males, aged 27 ± 3 years, BMI 23·3 ± 0·5 kg/m2). Measurements HRV was examined 75 min after standardized breakfast and after a 48-h fast in 14 nondiabetic obese and 12 lean subjects. The postprandial measurement was repeated in 12 obese subjects after weight-loss. Results In lean subjects, fasting decreased high-frequency (HF) power by 43% (P

In a recent manuscript Raps et al (1) used the correlation between sex hormone-binding globulin (SHBG) and the normalised APC sensitivity ratio (nAPCsr) as an argument for using SHBG as a useful marker to estimate thrombotic risk of a hormonal contraceptive preparation. They present a log-normal association of SHBG with nAPCsr with an R(2) linear value of 0.476. We had the opportunity to verify this association in the large seven pill study in which nAPCsr was known (2). (c) 2012 International Society on Thrombosis and Haemostasis.

Cyclosporine (CsA) is an immunosuppressive drug requiring dose individualization and regular control due to its highly variable pharmacokinetics. Since gastroparesis may influence the absorption of CsA, a randomized cross-over study was performed to assess the pharmacokinetics and tolerability of a novel microemulsion CsA formulation in comparison with the standard CsA dosage form in six stable pancreas-kidney transplant recipients with scintigraphically proven gastroparesis. The absorption of CsA was investigated during three 2-hr study days during each treatment period, and a full pharmacokinetic profile was done for each formulation. No adverse events or differences in tolerability/safety parameters between the treatments were found. The average AUC (0-->2 hr) was 150% higher after the novel formulation. The coefficient of variation in AUC (0-->2 hr) for both formulations was comparable (37% after the microemulsion and 40% after the standard formulation). The median time at which blood CsA levels exceeded the preceding trough level by 20% was 30 min (range: 30 -> 718 min) after the standard formulation. With approximately the same average dose, the AUCss tau after the microemulsion was 81% higher than the standard formulation, while predose and 12-hr trough levels were similar. The average maximal CsA plasma level after the microemulsion was 396 ng/ml (95% CI: 71-722 ng/ml) higher than after the standard formulation. The median time at which the highest blood levels were observed was 90 min (range: 150 -> 718 min) after the standard formulation. The time profiles of the CsA metabolites followed those of the parent compound. The microemulsion resulted in a higher systemic exposure to CsA than the standard formulation in pancreas-kidney transplant patients with diabetic gastroparesis, but substantial variability in blood concentrations remained.

Summary Introduction There is no clear consensus about tests in vitro that are suitable for evaluating various factor Xa inhibitors. The availability of reversible and irreversible inhibitors further complicates the application of available assays. Methods We evaluated the suitability of the prothrombinase-induced clotting test (PiCT) for fondaparinux and rivaroxaban, as representatives for irreversible and reversible inhibition of factor Xa, with specific attention to preincubation times prior to re-calcification, in the context of automate program limitations. Results We demonstrate that the PiCT assay requires a preincubation step to allow inhibitory activity by fondaparinux. Without this step, inhibition in the test is minimal and lacking sufficient dynamic range. In contrast, to measure the reversible inhibition by rivaroxaban, we found any preincubation introduced an artifact in inhibition as exemplified by a biphasic pattern and only the test without a preincubation phase gave informative results. Conclusion It is concluded that PiCT in its format with two steps is suitable for fondaparinux evaluation, while its format without preincubation (the one-addition format) is suitable for reversible inhibitors such as rivaroxaban. Unfortunately, both types of inhibitors cannot be compared in vitro in a single assay format.

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc(Min) mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis. Cell Death and Differentiation (2012) 19, 1826-1835; doi:10.1038/cdd.2012.68; published online 15 June 2012

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Reactive oxygen species (ROS), like superoxide anion, play an important role in different disease states. • Superoxide dismutase (SOD) acts as a free radical scavenger by catalysing the dismutation of superoxide. • Over the last decade, therapeutic use of SOD has been explored, but the results of these experiments have indicated that this has been of limited value, probably due to unfavourable pharmaceutical characteristics of the compounds. WHAT THIS STUDY ADDS • Single intravenous administrations of PC-SOD (SOD covalently linked to lecithin) in doses up to 80 mg was well tolerated and biologically active for a period of 19 ± 6 h in healthy White volunteers. • This suggests that PC-SOD has pharmaceutically appropriate characteristics and may be a possible protective agent for patients in clinical conditions characterized by acute high radical overload. AIM To study the pharmacokinetics (PK), safety and tolerability of single rising doses up to 80 mg of superoxide dismutase covalently linked to lecithin (PC-SOD) in healthy White volunteers. METHODS This double-blind, placebo-controlled, four-period cross-over study was performed in eight healthy volunteers (four male/four female). Three doses of PC-SOD (20, 40 and 80 mg) and placebo were administered intravenously in randomized order. Serum and urinary PC-SOD concentrations were measured predose and up to 96 h after dosing. In addition to standard safety measurements, the urinary excretion of N-acetyl-β-glucosaminidase, α-glutathione S-transferase (α-GST) and π-GST was measured to evaluate renal function. The PK of PC-SOD was analysed using noncompartmental and compartmental methods. RESULTS All treatments were well tolerated, and no obvious relationship between adverse events and treatment was observed. No effects of PC-SOD on renal function could be detected. Dose normalized Cmax and AUC were not different between the different dosages, indicating linearity of plasma concentrations with dose. Estimated PC-SOD clearance was 2.54 ml min−1[95% confidence interval (CI) 2.07, 2.83]. The terminal half-life was estimated to be 1.54 days (95% CI 0.93, 2.15). SOD activity was elevated above baseline for 19 ± 6 h after the 80-mg dose. CONCLUSIONS Single intravenous administrations of PC-SOD in doses up to 80 mg were well tolerated in healthy White male and female volunteers. With the doses used, SOD activity was linearly related to the dose; after the 80-mg dose it was present for an appreciable period. These findings suggest that it is worthwhile to investigate PC-SOD in clinical conditions characterized by a high radical overload.

It has been suggested that (abdominally) obese individuals are hypersensitive to growth hormone (GH) action. Because GH affects glucose metabolism, this may impact glucose homeostasis in abdominal obesity. Therefore, we studied the effect of GH on glucose metabolism in abdominally obese (OB) and normal-weight (NW) premenopausal women. A 1-h intravenous infusion of GH or placebo was randomly administered to six NW [body mass index (BMI) 21.1 ± 1.9 kg/m2] and six OB (BMI 35.5 ± 1.5 kg/m2) women in a crossover design. Insulin, glucagon, and GH secretion were suppressed by concomitant infusion of somatostatin. Glucose kinetics were measured using a 10-h infusion of [6,6-2H2]glucose. In both groups, similar physiological GH peaks were reached by infusion of GH. GH strongly stimulated endogenous glucose production (EGP) in both groups. The percent increase was significantly greater in OB than in NW women (29.8 ± 11.3 vs. 13.3 ± 7.4%, P = 0.014). Accordingly, GH responsiveness, defined as the maximum response of EGP per unit GH, was increased in OB vs. NW subjects (6.0 ± 2.1 vs. 2.2 ± 1.5 μmol·min−1·mU−1·l−1, P = 0.006). These results suggest that the liver is hyperresponsive to GH action in abdominally obese women. The role of the somatotropic ensemble in the control of glucose homeostasis in abdominal obesity is discussed.

Tissue plasminogen activator (t-PA) binds to the endothelium in vitro [1]. This binding may be crucial in the prevention of thrombus formation. The aim of the present study was to develop a mathematical model to quantify the binding of t-PA to the endothelium in vivo. Nine healthy male volunteers received a continuous low dose infusion with recombinant t-PA (3.75 μg min−1) and, as control, an infusion with indocycanine green (ICG; 0.5 mg min−1), both for 40 min. A circulatory model was developed using non-linear mixed effect modelling. Delays were observed in the ICG concentration profile that were not captured by a one- or two-compartment model. The ICG concentration profile was best described by a three-compartment recirculatory model with a total distribution volume of 3.0 l (s.e. mean 0.3, inter-individual coefficient of variation (CV) 17%) and plasma flow trough the ring of 0.77 l min−1 (s.e. mean 0.20, CV 0%; fixed). t-PA antigen, activity and t-PA/PAI-1 complex profile showed a marked delay in increase at the beginning of the infusion, that was not captured by the circulatory model. Incorporating a reversible and concentration-dependent binding component in the model resulted in an accurate description of the t-PA concentration profile. In the resulting model (Figure 1), binding of t-PA was characterized by an affinity constant of 0.29 ml ng−1 (s.e. mean 0.14, CV 114%) and a concentration of binding sites of 99 ng ml−1 (s.e. mean 12, CV 0%). Figure 1 Recirculatory model for t-PA with saturable endothelial binding. Clearance was estimated at 0.41 l min−1 (s.e. mean 0.02, CV 18%), and endogenous production at 2.06 μg min−1 (s.e. mean 0.07, CV 6%). The in vivo characteristics of the t-PA binding site (Kd ∼ 51 pm) correspond with a high affinity t-PA endothelial binding site described in vitro (Kd 28 pM). The current model provides a means of quantification of t-PA binding and may provide a tool to study the t-PA binding in vivo. Assessing the capacity of the endothelium to bind t-PA and possibly other endogenous substances may serve as a novel tool to assess endothelial function.

We assessed sympathovagal balance in thyrotoxicosis. Fourteen patients with Graves' hyperthyroidism were studied before and after 7 days of treatment with propranolol (40 mg 3 times a day) and in the euthyroid state. Data were compared with those obtained in a group of age-, sex-, and weight-matched controls. Autonomic inputs to the heart were assessed by power spectral analysis of heart rate variability. Systemic exposure to sympathetic neurohormones was estimated on the basis of 24-h urinary catecholamine excretion. The spectral power in the high-frequency domain was considerably reduced in hyperthyroid patients, indicating diminished vagal inputs to the heart. Increased heart rate and mid-frequency/high-frequency power ratio in the presence of reduced total spectral power and increased urinary catecholamine excretion strongly suggest enhanced sympathetic inputs in thyrotoxicosis. All abnormal features of autonomic balance were completely restored to normal in the euthyroid state. β-Adrenoceptor antagonism reduced heart rate in hyperthyroid patients but did not significantly affect heart rate variability or catecholamine excretion. This is in keeping with the concept of a joint disruption of sympathetic and vagal inputs to the heart underlying changes in heart rate variability. Thus thyrotoxicosis is characterized by profound sympathovagal imbalance, brought about by increased sympathetic activity in the presence of diminished vagal tone.

Objective Recently a few cases of long QT syndrome were reported during treatment with cisapride. In most of these cases, risk factors for cardiac arrhythmias or pharmacologic interactions might have been involved, and the role of cisapride remained unclear. Macrolides such as clarithromycin potentially interact with the metabolic elimination of cisapride and have overlapping indication areas. We therefore studied whether combined treatment with clarithromycin and cisapride leads to pharmacokinetic changes and increased QT intervals. Methods The study was an open, randomized, 2-way crossover study with washout periods of 1 week. Twelve healthy volunteers were recruited. Treatments were cisapride (10 mg 4 times a day) for 10 days with concomitant clarithromycin (500 mg twice a day) from days 6 through 10, or clarithromycin (500 mg twice a day) for 10 days combined with cisapride (10 mg 4 times a day) from days 6 through 10. Frequent ECG recordings were performed for 24 hours before drug treatment (baseline). After 5 days of monotherapy and combination therapy, frequent ECG recordings and assessments of plasma drug levels were performed for 24 hours. Results Clarithromycin alone was associated with a minimal increase in QTc intervals. Monotherapy with 10 mg cisapride 4 times a day led to a concentration-dependent QTc elevation, amounting to 6 ms during steady state. Combination of cisapride and clarithromycin caused an average QTc increase of 25 ms above pretreatment values and 3-fold increases in cisapride concentrations. Conclusions QTc elevations after cisapride or clarithromycin alone remained within the normal range of diurnal variation. Coadministration of cisapride and clarithromycin produced a substantial QT prolongation. The data support the recently purported interaction between cisapride and clarithromycin and thus the filed contraindication to combine these drugs. Clinical Pharmacology & Therapeutics (1998) 64, 542–546; doi

Plasma leptin concentrations were measured every 20 min for 24 h in eight normal weight women and in eight upper body and eight lower body obese women matched for body mass index. The circadian rhythm of leptin, which could mathematically be described by a cosine, was characterised by an acrophase just after midnight in all subjects. The amplitude of a cosine fit as well as the average 24-h leptin concentration were increased by 280% and 420%, respectively, in obese compared to normal weight women. All characteristics of leptin concentration profiles were similar in upper body and lower body obese women, except for a significantly higher amplitude in the lower body obese group. Visceral and sc body fat depots were measured using magnetic resonance imaging in all three groups. Average 24-h leptin concentrations were strongly correlated with sc fat (r = 0.84), whereas visceral fat was not an independent predictor of the plasma leptin level. A loss of 50% of the overweight was associated with a 55% decrease in the average 24-h leptin concentrations in obese women (95% confidence interval, 12.3, 26.6), whereas the characteristics of the circadian rhythm of leptin remained unchanged. Finally, it was observed that a fasting plasma leptin concentration is not an acceptable indicator of the average leptin concentration over 24 h.

Echo-Doppler measurements of portal venous blood flow in intrahepatic branches and indocyanine green (ICG) clearance after continuous i.v. infusion as measure for liver blood flow were compared to evaluate the increase in splanchnic blood flow after food intake. It was shown that both methods assessed the changes in flow in a similar manner. Changes in blood flow in intrahepatic portal vein branches measured with echo-Doppler adequately predicted the change in ICG concentrations. Hence, echo-Doppler measurements of hepatic portal blood flow in intrahepatic branches can be used to estimate changes in total liver blood flow.

The pharmacokinetics and cardiovascular effects of the calcium entry blocker nisoldipine (10 mg twice daily) were studied in 6 patients with renal failure (creatinine clearance 23 +/- 9 ml/min) and 6 healthy control subjects after a single dose and 1 week of oral administration. No significant differences in elimination half-life, area under the concentration/time curve, peak plasma drug concentration and time to reach that peak were observed between renal patients and control subjects, and between single-dose and short term administration. The decrease in systolic blood pressure and increase in heart rate were similar in both groups, but the decrease in diastolic blood pressure was more pronounced in the patients. This can be explained by increased haemodynamic sensitivity for nisoldipine. Adverse effects were mainly restricted to the first day of administration.