Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Photodynamic therapy enhances liposomal doxorubicin distribution in tumors during isolated perfusion of rodent lungs.

BACKGROUND: Photodynamic therapy (PDT) at low drug-light conditions can enhance the transport of intravenously injected macromolecular therapeutics through the tumor vasculature. Here we determined the impact of PDT on the distribution of liposomal doxorubicin (Liporubicin™) administered by isolated lung perfusion (ILP) in sarcomas grown on rodent lungs. METHODS: A syngeneic methylcholanthrene-induced sarcoma cell line was implanted subpleurally in the left lung of Fischer rats. Treatment schemes consisted in ILP alone (400 μg of Liporubicin), low-dose (0.0625 mg/kg Visudyne®, 10 J/cm(2) and 35 mW/cm(2)) and high-dose left lung PDT (0.125 mg/kg Visudyne, 10 J/cm(2) and 35 mW/cm(2)) followed by ILP (400 μg of Liporubicin). The uptake and distribution of Liporubicin in tumor and lung tissues were determined by high-performance liquid chromatography and fluorescence microscopy in each group. RESULTS: Low-dose PDT significantly improved the distribution of Liporubicin in tumors compared to high-dose PDT (p < 0.05) and ILP alone (p < 0.05). However, both PDT pretreatments did not result in a higher overall drug uptake in tumors or a higher tumor-to-lung drug ratio compared to ILP alone. CONCLUSIONS: Intraoperative low-dose Visudyne-mediated PDT enhances liposomal doxorubicin distribution administered by ILP in sarcomas grown on rodent lungs which is predicted to improve tumor control by ILP.

Short and long-term effects of azadirachtin A on development and egg production of Rhodnius prolixus

Azadirachtin A was given through a blood meal to 4th-instar larvae and to adult females of Rhodnius prolixus. Development (ecdysis) and egg production were inhibited in a dose-dependent manner. Long-term experiments with subsequent four feedings on azadirachtin-free blood were performed with 4th-instar larvae and with adult females. Only in the low-dose azadirachtin larval groups (0.01 and 0.1 microng/ml of blood), development was partially restored; after a single 1.0 microng/ml treatment about 50% of the treated larvae were still alive 120 days later without any adult emergence. Similarly fed females had a dose-dependent lower survival and egg deposition rate. The results are discussed in relation to the mode of azadirachtin A action.

Patients with non-insulin depending diabetes mellitus and metabolic syndrome are suboptimal treated in swiss primary care.

The prevalence of complicated hypertension is increasing in America and Europe. This survey was undertaken to assess the status quo of primary care management of hypertension in patients with the high-risk comorbid diseases metabolic syndrome (MetS) and/or type 2 diabetes mellitus (non-insulin depending diabetes mellitus (NIDDM)). Data of anti-hypertensive treatment of 4594 Swiss patients were collected over 1 week. We identified patients with exclusively NIDDM (N = 95), MetS (N = 168), and both (N = 768). Target blood pressure (TBP) attainment, frequency of prescribed substance-classes, and correlations to comorbidities/end-organ damages were assessed. In addition, we analyzed the prescription of unfavorable beta-blockers (BB) and high-dose diuretics (Ds). In NIDDM, Ds (61%), angiotensin receptor blockers (ARBs) (40%), and angiotensin converting enzyme inhibitors (ACEIs) (31%) were mostly prescribed, while in MetS, drugs prevalence was Ds (68%), ARBs (48%), and BB (41%). Polypharmacy in patients with MetS correlated with body mass index; older patients (>65 years) were more likely to receive dual-free combinations. TBP was attained in 25.2% of NIDDM and in 28.7% of MetS patients. In general, low-dose Ds use was more prevalent in NIDDM and MetS, however, overall, Ds were used excessively (NIDDM: 61%, MetS: 68%), especially in single-pill combination. Patients with MetS were more likely to receive ARBs, ACEIs, CCBs, and low-dose Ds than BBs and/or high-dose Ds. Physicians recognize DM and MetS as high-risk patients, but select inappropriate drugs. Because the majority of patients may have both, MetS and NIDDM, there is an unmet need to define TBP for this specific population considering the increased risk in comparison to patients with MetS or NIDDM alone.

The management of iron deficiency in inflammatory bowel disease--an online tool developed by the RAND/UCLA appropriateness method.

BACKGROUND: Iron deficiency is a common and undertreated problem in inflammatory bowel disease (IBD). AIM: To develop an online tool to support treatment choice at the patient-specific level. METHODS: Using the RAND/UCLA Appropriateness Method (RUAM), a European expert panel assessed the appropriateness of treatment regimens for a variety of clinical scenarios in patients with non-anaemic iron deficiency (NAID) and iron deficiency anaemia (IDA). Treatment options included adjustment of IBD medication only, oral iron supplementation, high-/low-dose intravenous (IV) regimens, IV iron plus erythropoietin-stimulating agent (ESA), and blood transfusion. The panel process consisted of two individual rating rounds (1148 treatment indications; 9-point scale) and three plenary discussion meetings. RESULTS: The panel reached agreement on 71% of treatment indications. 'No treatment' was never considered appropriate, and repeat treatment after previous failure was generally discouraged. For 98% of scenarios, at least one treatment was appropriate. Adjustment of IBD medication was deemed appropriate in all patients with active disease. Use of oral iron was mainly considered an option in NAID and mildly anaemic patients without disease activity. IV regimens were often judged appropriate, with high-dose IV iron being the preferred option in 77% of IDA scenarios. Blood transfusion and IV+ESA were indicated in exceptional cases only. CONCLUSIONS: The RUAM revealed high agreement amongst experts on the management of iron deficiency in patients with IBD. High-dose IV iron was more often considered appropriate than other options. To facilitate dissemination of the recommendations, panel outcomes were embedded in an online tool, accessible via http://ferroscope.com/.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

Renal perfusion evaluation with contrast-enhanced ultrasonography.

BACKGROUND: Contrast-enhanced ultrasonography (CEUS) is a novel imaging technique that is safe and applicable on the bedside. Recent developments seem to enable CEUS to quantify organ perfusion. We performed an exploratory study to determine the ability of CEUS to detect changes in renal perfusion and to correlate them with effective renal plasma flow. METHODS: CEUS with destruction-refilling sequences was studied in 10 healthy subjects, at baseline and during infusion of angiotensin II (AngII) at low (1 ng/kg/min) and high dose (3 ng/kg/min) and 1 h after oral captopril (50 mg). Perfusion index (PI) was obtained and compared with the effective renal plasma flow (ERPF) obtained by parallel para-aminohippurate (PAH) clearance. RESULTS: Median PI decreased from 188.6 (baseline) to 100.4 with low-dose AngII (-47%; P < 0.02) and to 66.1 with high-dose AngII (-65%; P < 0.01) but increased to 254.7 with captopril (+35%; P > 0.2). These changes parallelled those observed with ERPF, which changed from a median of 672.1 mL/min (baseline) to 572.3 (low-dose AngII, -15%, P < 0.05) and to 427.2 (high-dose AngII, -36%, P < 0.001) and finally 697.1 (captopril, +4%, P < 0.02). CONCLUSIONS: This study demonstrates that CEUS is able to detect changes in human renal cortical microcirculation as induced by AngII infusion and/or captopril administration. The changes in perfusion indices parallel those in ERPF as obtained by PAH clearance.

Hypertension: which aspects of hypertension should we impact on and how?

Cardiovascular complications may, to a large extent, be prevented by lowering blood pressure in hypertensive patients. International recommendations currently stress the importance of reaching values of below 140/90 mmHg in each patient or even lower in the case of concomitant diabetes or renal impairment. It is currently considered crucial to control the systolic pressure as well as the diastolic pressure, in particular because the relationship between cardiovascular risk and blood pressure is closer for the systolic than the diastolic value. An increase in systolic pressure is in itself a sign of the stiffening of the arterial tree. In most patients, the target pressure may only be reached by combining several different antihypertensive agents. In the STRATHE Study, a greater antihypertensive efficacy, in particular on systolic pressure, was obtained by instituting treatment with a fixed low-dose combination of an angiotensin-converting enzyme inhibitor (perindopril) and a diuretic (indapamide), in comparison with other therapeutic strategies based on single-agent therapy. Fixed-dose antihypertensive combinations have now become a validated option for initiating antihypertensive treatment.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Quetiapine dosage in bipolar disorder episodes and mixed states

OBJECTIVE: Although the maximal quetiapine doses in the published studies were restricted to 800 mg/day, higher quetiapine doses are not unusual in clinical practice. The aim of the present study was to evaluate the effectiveness, tolerability and clinical reasons associated to the use of high dosage of quetiapine (>800 mg), when used under routine clinical conditions, in a sample of bipolar disorder and schizoaffective bipolar inpatients. METHODS: Charts of all bipolar and schizoaffective adult inpatients, who had received quetiapine for a mood episode between 1999 and 2005 were retrospectively reviewed. These charts also included the assessment of manic and depressive symptoms on admission and at discharge using the Beck-Rafaelsen Mania Scale (MAS) and the Montgomery Asberg depression rating scale (MADRS), respectively. RESULTS: Data of 50 patients were analyzed. The overall F in repeated measures ANOVA revealed a significant MAS scores reduction between admission and discharge. MAS scores reduction did not differ between the high and low quetiapine groups. Similarly, a significant MADRS reduction was found. Again, no differences between the high and the low dose group were found. Logistic regression analysis of the 50 patients revealed only mixed episodes predicted high quetiapine dosage. CONCLUSIONS: The present study confirms quetiapine efficiency and tolerability in the treatment of bipolar episodes, even in doses > to 800 mg and found a link between quetiapine doses and mixed episodes

Infusion of atrial natriuretic peptide to patients with congestive heart failure.

Atrial natriuretic peptides (ANP) exert vasodilating and natriuretic actions. The present study was undertaken to test the effect of low dose infusions of synthetic ANP on hemodynamic and humoral variables of patients with severe heart failure. Eight patients, aged 26 to 71 years, with severe congestive heart failure due to ischemic heart disease or idiopathic dilated cardiomyopathy were included in the study. Synthetic human (3-28) ANP was infused at doses ranging from 0.5 to 2 micrograms/min for up to 3 h. Pulmonary capillary wedge pressure fell from 24 +/- 1 to 16 +/- 2 mm Hg (mean +/- SEM) (p less than 0.01) and cardiac index tended to rise from 2 +/- 0.2 to 2.3 +/- 0.2 L/min/m2 (NS), while blood pressure and heart rate did not change. One patient experienced a marked drop in pulmonary capillary wedge and arterial blood pressure that necessitated the administration of saline. ANP infusion did not alter plasma renin activity or plasma aldosterone, norepinephrine, or vasopressin levels. It decreased plasma epinephrine levels from 0.472 +/- 0.077 to 0.267 +/- 0.024 nmol/L (p less than 0.05). Plasma ANP levels were markedly elevated in all patients before initiating the infusion. They had no predictive value for the hemodynamic response to exogenous ANP. No correlation was observed between the hemodynamic effects of ANP and those induced by the subsequently administered converting enzyme inhibitor captopril, which seemed to improve cardiac function more consistently.(ABSTRACT TRUNCATED AT 250 WORDS)

Gadolinium Enhanced MR Coronary Vessel Wall Imaging at 3.0 Tesla.

Purpose. We evaluated the influence of the time between low-dose gadolinium (Gd) contrast administration and coronary vessel wall enhancement (LGE) detected by 3T magnetic resonance imaging (MRI) in healthy subjects and patients with coronary artery disease (CAD). Materials and Methods. Four healthy subjects (4 men, mean age 29 ± 3 years and eleven CAD patients (6 women, mean age 61 ± 10 years) were studied on a commercial 3.0 Tesla (T) whole-body MR imaging system (Achieva 3.0 T; Philips, Best, The Netherlands). T1-weighted inversion-recovery coronary magnetic resonance imaging (MRI) was repeated up to 75 minutes after administration of low-dose Gadolinium (Gd) (0.1 mmol/kg Gd-DTPA). Results. LGE was seen in none of the healthy subjects, however in all of the CAD patients. In CAD patients, fifty-six of 62 (90.3%) segments showed LGE of the coronary artery vessel wall at time-interval 1 after contrast. At time-interval 2, 34 of 42 (81.0%) and at time-interval 3, 29 of 39 evaluable segments (74.4%) were enhanced. Conclusion. In this work, we demonstrate LGE of the coronary artery vessel wall using 3.0 T MRI after a single, low-dose Gd contrast injection in CAD patients but not in healthy subjects. In the majority of the evaluated coronary segments in CAD patients, LGE of the coronary vessel wall was already detectable 30-45 minutes after administration of the contrast agent.

Aquaporin expression after thrombin preconditioning in ischemic mouse brain

Background: Aquaporin-4 (AQP4), a water channel, is induced early after stroke.The role of AQP4 in the development and resolution of oedema after stroke remainsdebated. The absence of AQP4 in KO-mice reduces the cytotoxic oedema formationbut in contrast aggravates the vasogenic edema. Thrombin at high dose is known toinduce an oedema and at a low dose (thrombin preconditioning, TPC), to inducetolerance to ischemia. We studied the expression of AQPs in ischemic mouse brainsafter TPC and correlation with oedema formation.Methods: For thrombin preconditioning (TPC), mice were injected intracerebroventricularlywith a low dose of thrombin (0.1U in 2?l), followed 24 hours laterby a 30 min transient middle cerebral occlusion (MCAo). AQP4 expression wasevaluated by immunohistochemistry 1h and 48h after ischemia and correlated withoedema formation in vehicle injected and TPC mice.Results: After TPC, oedema formation, assessed by hemispheric enlargement, wassignificantly attenuated at 1h (4.5 ± 2% vs 11.0 ± 5% in CTL, p<0.05, n=8),which was confirmed by wet weight/dry weight ratio (79.6 ± 0.3% vs 80.1 ± 0.1in ctl, p<0.05, n=0.05). At the same time-point, AQP4 expression was significantlyincreased in TPC mice, (148.9% of the control, P<0.05, n=6) in the ischemicstriatum. The oedema was still reduced at 48h after stroke onset in TPC mice. At48h, the level of expression for AQP4 was still higher for TPC animal although notreaching significance (NS). The lesion size was significantly reduced at 48h afterstroke in TPC mice (5.1 ± 1.6 vs 10.6 ± 1.8 mm2 in CTL, n=5).Discussion: The correlation between the early induction of AQP4 and the decreaseof oedema formation in TPC mice suggests that the induction of AQP4 preventsthe development of oedema.Funding: FNS #3100A0-108001, #3200 68306.02 & #3100A0-112484 and Swiss-Heart foundation.

Ganciclovir Exposure under a 450 mg Daily Dosage of Valganciclovir for the Prevention of Cytomegalovirus Disease in Kidney Transplant Recipients.

Background: It is suggested that a low dose of valganciclovir can be equally effective than a standard dose for cytomegalovirus (CMV) prophylaxis after kidney transplantation. The aim of our study was to determine the ganciclovir exposure observed under a routine daily dosage of 450 mg valganciclovir in kidney transplant recipients with a wide range of renal function. Methods: In this prospective study, kidney transplant recipients with a GFR MDRD above 25 mL/min at risk for CMV (donor or recipient seropositive for CMV) received a dose of valganciclovir (450 mg daily) prophylaxis for 3 months. Ganciclovir levels at trough (Ctrough) and at peak (C3h) were measured monthly. Ganciclovir exposure (AUC0-24) was estimated using Bayesian non-linear mixed-effect modelling (NONMEM) and compared between 3 groups of patients according to their kidney function: GFRMDRD 26-39 mL/min (Group 1), GFRMDRD 40-59 mL/min (Group 2) and GFRMDRD 60-90 mL/min (Group 3). CMV DNAemia was assessed during and after prophylaxis using PCR. Results: Thirty-six patients received 450 mg daily of valganciclovir for 3 months. Median ganciclovir C3h was 3.9 mg/L (range: 1.3-7.1) and Ctrough was 0.4 mg/L (range 0.1-2.7). Median (range) AUC0-24 of ganciclovir was 59.3 mg.h/L (39.0-85.3) in Group 1 patients, 35.8 mg.h/L (24.9-55.8) in Group 2 patients and 29.6 mg.h/L (22.0- 43.2) in Group 3 patients (p<0.001). Anemia was more common in Group 1 patients compared to patients on the other groups (p=0.01). No differences in other adverse events according to ganciclovir exposure were observed. CMV DNAemia was not detected during prophylaxis. After discontinuing prophylaxis, CMV DNAemia was seen in 8/34 patients (23.5%) and 4/36 patients (11%) developed CMV disease. Conclusion: A routine dosage of valganciclovir achieved plasma levels of ganciclovir in patients with GFR>60 mL/min similar to those previously reported using oral ganciclovir. A daily dose of 450 mg valganciclovir appears to be acceptable for CMV prophylaxis in most kidney transplant recipients.

Population pharmacokinetic modelling and evaluation of different dosage regimens for darunavir and ritonavir in HIV-infected individuals.

OBJECTIVES: Darunavir is a protease inhibitor that is administered with low-dose ritonavir to enhance its bioavailability. It is prescribed at standard dosage regimens of 600/100 mg twice daily in treatment-experienced patients and 800/100 mg once daily in naive patients. A population pharmacokinetic approach was used to characterize the pharmacokinetics of both drugs and their interaction in a cohort of unselected patients and to compare darunavir exposure expected under alternative dosage regimens. METHODS: The study population included 105 HIV-infected individuals who provided darunavir and ritonavir plasma concentrations. Firstly, a population pharmacokinetic analysis for darunavir and ritonavir was conducted, with inclusion of patients' demographic, clinical and genetic characteristics as potential covariates (NONMEM(®)). Then, the interaction between darunavir and ritonavir was studied while incorporating levels of both drugs into different inhibitory models. Finally, model-based simulations were performed to compare trough concentrations (Cmin) between the recommended dosage regimen and alternative combinations of darunavir and ritonavir. RESULTS: A one-compartment model with first-order absorption adequately characterized darunavir and ritonavir pharmacokinetics. The between-subject variability in both compounds was important [coefficient of variation (CV%) 34% and 47% for darunavir and ritonavir clearance, respectively]. Lopinavir and ritonavir exposure (AUC) affected darunavir clearance, while body weight and darunavir AUC influenced ritonavir elimination. None of the tested genetic variants showed any influence on darunavir or ritonavir pharmacokinetics. The simulations predicted darunavir Cmin much higher than the IC50 thresholds for wild-type and protease inhibitor-resistant HIV-1 strains (55 and 550 ng/mL, respectively) under standard dosing in >98% of experienced and naive patients. Alternative regimens of darunavir/ritonavir 1200/100 or 1200/200 mg once daily also had predicted adequate Cmin (>550 ng/mL) in 84% and 93% of patients, respectively. Reduction of darunavir/ritonavir dosage to 600/50 mg twice daily led to a 23% reduction in average Cmin, still with only 3.8% of patients having concentrations below the IC50 for resistant strains. CONCLUSIONS: The important variability in darunavir and ritonavir pharmacokinetics is poorly explained by clinical covariates and genetic influences. In experienced patients, treatment simplification strategies guided by drug level measurements and adherence monitoring could be proposed.