Human protective response induced by meningococcus B vaccine is mediated by the synergy of multiple bactericidal epitopes.

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.

Potentially inappropriate prescribing and the risk of adverse drug reactions in critically ill older adults

Background: Potentially inappropriate medication (PIM) use in the elderly is associated with increased risk of adverse drug reactions (ADRs), but there is limited information regarding PIM use in the intensive care unit (ICU) setting. Objective: The aim of the study is to describe the prevalence and factors associated with the use of PIM and the occurrence of PIM-related adverse reactions in the critically ill elderly. Methods: This study enrolled all critically ill older adults (60 years or more) admitted to medical or cardiovascular ICUs between January and December 2013, in a large tertiary teaching hospital. For all patients, clinical pharmacists listed the medications given during the ICU stay and data on drugs were analyzed using 2012 Beers Criteria, to identify the prevalence of PIM. For each identified PIM the medical records were analyzed to evaluate factors associated with its use. The frequency of ADRs and, the causal relationship between PIM and the ADRs identified were also evaluated through review of medical records. Results: According to 2012 Beers Criteria, 98.2% of elderly patients used at least one PIM (n=599), of which 24.8% were newly started in the ICUs. In 29.6% of PIMs, there was a clinical circumstance that justified their prescription. The number of PIMs was associated with ICU length of stay and total number of medications. There was at least one ADR identified in 17.8% of patients; more than 40% were attributed to PIM, but there was no statistical association. Conclusions: There is a high prevalence of PIM used in acutely ill older people, but they do not seem to be the major cause of adverse drug reactions in this population. Although many PIMs had a clinical circumstance that led to their prescription during the course of ICU hospitalization, many were still present upon hospital discharge. Therefore, prescription of PIMs should be minimized to improve the safety of elderly patients (AU)

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pH-Dependent dissolving nano- and microparticles for improved peroral delivery of a highly lipophilic compound in dogs.

RR01, a new highly lipophilic drug showing extremely low water solubility and poor oral bioavailability, has been incorporated into pH-dependent dissolving particles made of a poly(methacrylic acid-co-ethylacrylate) copolymer. The physicochemical properties of the particles were determined using laser-light-scattering techniques, scanning electron microscopy, high-performance liquid chromatography, and x-ray powder diffraction. Suspension of the free drug in a solution of hydroxypropylcellulose (reference formulation) and aqueous dispersions of pH-sensitive RR01-loaded nanoparticles or microparticles were administered orally to Beagle dogs according to a 2-block Latin square design (n = 6). Plasma samples were obtained over the course of 48 hours and analyzed by gas chromatography/mass spectrometry. The administration of the reference formulation resulted in a particularly high interindividual variability of pharmacokinetic parameters, with low exposure to compound RR01 (AUC0-48h of 6.5 microg x h/mL and coefficient of variation (CV) of 116%) and much higher Tmax, as compared to both pH-sensitive formulations. With respect to exposure and interindividual variability, nanoparticles were superior to microparticles (AUC0-48h of 27.1 microg x h/mL versus 17.7 microg x h/mL with CV of 19% and 40%, respectively), indicating that the particle size may play an important role in the absorption of compound RR01. The performance of pH-sensitive particles is attributed to their ability to release the drug selectively in the upper part of the intestine in a molecular or amorphous form. In conclusion, pH-dependent dissolving particles have a great potential as oral delivery systems for drugs with low water solubility and acceptable permeation properties.

Pharmacokinetics and metabolism of formestane in breast cancer patients.

Formestane (Lentaron(R), 4-hydroxyandrostenedione) is a steroidal aromatase inhibitor used for treatment of advanced breast cancer. Clinically, it is administered as a depot form once fortnightly by intramuscular (i.m.) injection. To investigate the pharmacokinetics, bioavailability and metabolism of the drug, seven patients received single 250 mg i.m. doses of commercial formestane on Days 0, 21, 35, 49 and 63 of this trial. On Day 63, three of the patients received an additional single intravenous (i.v.) pulse dose of 1 mg of 14C-labelled formestane. The plasma kinetics after i.m. dosing confirmed a sustained release of formestane from the site of injection. Within 24-48 h of the first dose, the circulating drug reached a C(max) of 48.0+/-20.9 nmol/l (mean+/-S.D.; N=7). At the end of the dosing interval, after 14 days, the plasma concentration was still at 2.3+/-1.8 nmol/l. The kinetic variables did not significantly change during prolonged treatment. Intramuscular doses appear to be fully bioavailable. Following i.v. injection of 14C-formestane, the unchanged drug disappeared rapidly from plasma, the terminal elimination half-life being 18+/-2 min (N=3). Plasma clearance, CL was 4.2+/-1.3 l/(h kg) and the terminal distribution volume V(z) was 1.8+/-0.5 l/kg. The drug is mainly eliminated by metabolism, renal excretion of metabolites accounting for 95% of dose. The excretory balance of 14C-compounds in urine and faeces totals up to 98.9+/-0.8% of the i.v. dose after 168 h. The 14C-compounds in plasma and urine were separated by HPLC, and three major metabolites were submitted to structural analysis by MS, NMR and UV spectroscopy. One of the metabolites is the direct 4-O-glucuronide of formestane. The other two represent 3-O-sulfates of the exocons 3beta,4beta-dihydroxy-5alpha-androstane-17-one and 3alpha,4beta-dihydroxy-5alpha-androstane-17-one, their ratio being 7:3. These exocons are formed by stereoselective 3-keto reduction, accompanied by reduction of the 4,5-enol function. The exocons do not inhibit human placental aromatase activity in vitro.

Oral bioavailability of a poorly water soluble HIV-1 protease inhibitor incorporated into pH-sensitive particles: effect of the particle size and nutritional state.

The new chemical entity CGP 70726, a very poorly water-soluble HIV-1 protease inhibitor, was incorporated into pH-sensitive nanoparticles and microparticles made of the poly(methacrylic acid-co-ethylacrylate) copolymer Eudragit((R)) L100-55. The particles were characterized in terms of morphology, size distribution, drug loading, production yield and dispersion state of the drug inside the polymeric matrices. Aqueous dispersions of the particles were administered orally to Beagle dogs against a suspension of free drug (control formulation) all at a dose of 100 mg/kg. Oral administration was conducted in the absence and presence of food. Plasma concentrations and pharmacokinetic parameters were determined within 8 h post-dose. While no measurable absorption of the drug resulted after administration of the control formulation, substantial systemic exposure to the compound was obtained with both kinds of pH-sensitive formulations. The selective release of CGP 70726 in a highly dispersed/amorphous state and creation of high concentrations close to its absorption site was thought to account for this positive result. The largest areas under the plasma concentration-time curve (AUC) were obtained in the fasted state, with slightly better performance of the microparticles over the nanoparticles, in both nutritional states (7.8+/-1.5 versus 5.8+/-0. 8 micromol.h/l in the fasted state; 4.4+/-1.4 versus 2.00+/-0.5 micromol.h/l in the fed state). With these results, the potential of pH-sensitive particles for the oral delivery of HIV-1 protease inhibitors with low water solubility was confirmed.

Proteolysis of human calcitonin in excised bovine nasal mucosa: elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI).

PURPOSE: Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. METHODS: The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. RESULTS: Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. CONCLUSIONS: Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.

Pharmacokinetics of a novel HIV-1 protease inhibitor incorporated into biodegradable or enteric nanoparticles following intravenous and oral administration to mice.

CGP 57813 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease. This lipophilic compound was successfully entrapped into poly(D,L-lactic acid) (PLA) and pH sensitive methacrylic acid copolymers nanoparticle. The intravenous administration to mice of PLA nanoparticles loaded with CGP 57813 resulted in a 2-fold increase of the area under the plasma concentration-time curve, compared to a control solution. An increase in the elimination half-life (from 13 to 61 min) and in the apparent volume of distribution (1.7-3.6 L/kg) was observed for the nanoparticle incorporated compound vs control solution. Following oral administration, only nanoparticles made of the methacrylic acid copolymer soluble at low pH provided sufficient plasma levels of CGP 57813. In vitro, these nanoparticles dissolved completely within 5 min at pH 5.8. PLA nanoparticles, which are insoluble in the gastrointestinal tract, did not provide significant plasma concentrations of CGP 57813. From these observations, one can conclude that the passage of intact PLA nanoparticles across the gastrointestinal mucosa appears to be very low.

Prolactin lowering activity of the retinoid Ro 14-9706 affecting lactation and pup survival.

The arotinoid Ro 14-9706, though devoid of any teratogenic potential, was found to reduce dose dependently the survival of pups when their mothers were treated with toxic doses during days 6-15 of gestation. The increased mortality was primarily seen during early lactation. When pups derived from treated mothers were nursed by control foster mothers unexposed to the drug, their survival was significantly improved indicating that the increased mortality was not solely due to fetal drug exposure. When pups derived from untreated mothers were fostered by dams that were exposed to the arotinoid during pregnancy, a significant pup mortality (p < 0.01) was observed, suggesting that the nursing behaviour of lactating dams was seriously affected. This impairment could be linked to a prolactin-suppressive activity of the arotinoid during lactation which was also seen during pregnancy. Other pituitary hormones, however, were not affected by the compound. Although the drug induced pronounced structural alterations in mitochondria of adrenocortical cells, visualized by light microscopy as extended vacuolization in the zona fasciculata and reticularis, this pathological finding did not translate into functional impairment of steroidogenesis. Thus, the arotinoid Ro 14-9706 exhibits in rats a prolactin-suppressive activity which affects lactation and subsequently pup survival. This particular endocrinological interference is a new phenomenon and uncommon for retinoids.

Comparative teratogenicity of three retinoids: the arotinoids Ro 13-7410, Ro 13-6298 and Ro 15-1570.

Three retinoids of the arotinoid series, namely the free carboxylic acid Ro 13-7410, its ethyl ester Ro 13-6298, and the new arotinoid ethyl sulfone Ro 15-1570, were tested for their embryotoxic and teratogenic activity in rats. The retinoids were administered orally on either day 9 or 13 of gestation. Treatment on day 9 of gestation resulted mainly in malformations of the head and the trunk; whereas, on day 13 limb malformations were prominent. Ro 13-7410 and Ro 13-6298 were about 1000 times more embryotoxic and teratogenic than retinoic acid but induced a similar malformation pattern to retinoic acid. In contrast, the sulfur-containing arotinoid Ro 15-1570 was active at similar dose levels to retinoic acid but caused a peculiar malformation pattern on day 13 of gestation. This finding supports the hypothesis that the arotinoid ethyl sulfone Ro 15-1570 has unique biological properties, inducing no bone toxicity in adult rats and distinctly affecting limb development.

Effects of vitamin D derivatives on soft tissue calcification in neonatal and calcium mobilization in adult rats.

The activity of 18 vitamin D analogs on soft tissue calcification and growth impairment in neonatal rats and their effect on bone calcium mobilization, intestinal calcium absorption and binding to intestinal 1,25-dihydroxyvitamin D3 receptors in adult rats were compared. Depending on the chemical modification of the vitamin D parent compounds, they could be separated into active and inactive analogs. Cholecalciferol and ergocalciferol were similarly active, but epimerization of ergocalciferol at carbon 23 caused loss of activity. Hexafluorination at carbon 26 and 27 and the introduction of a double bond at carbon 22 or 23 had no or little effect on the activity. The loss of activity was caused by the introduction of a triple bond at carbon 23 and by hydroxylation at carbon 23, 26 or 28. The differentiation of human promyelocytic leukemia cells (HL-60) induced by these derivatives was used as a parameter for antitumour activity. All six analogs, which markedly affected calcium metabolism, were highly active in HL-60 cells. However, at least three derivatives were highly active in the antitumour test but failed to induce hypercalcemia. Thus, these results indicate that it could be possible to develop medically useful vitamin D derivatives devoid of hypercalcemic side-effects.

Concentración sérica de alfa-1-antitripsina en diversas enfermedades hepáticas / Serum concentration of alpha-1-antitrypsin in different hepatic diseases

Se investigó la concentración sérica de alfa-1-antitripsina (A1AT) en 195 pacientes con diversas hepatopatías y en 43 adultos sanos. En todas las enfermedades hepáticas estudiadas se encontró un incremento significativo de la A1AT sérica respecto al grupo control (p < 0,001; hepatitis fulminante p < 0,05) confirmando que esta glicoproteína es un reactante de fase aguda. En las hepatitis fulminantes la concentración sérica de A1AT fue similar a la encontrada en los pacientes con hepatitis viral aguda sin insuficiencia hepática. La mayor elevación de la A1AT correspondió a los pacientes con obstrucción biliar y tumores hepáticos (primitivos y metastásicos) registrándose diferencias significativas respecto a las hepatopatías agudas y crónicas necrótico-inflamatorias (hepatitis, cirrosis, alcoholismo) (p < 0,001); a su vez en los pacientes con hígado metastásico la A1AT sérica fue mayor que en el resto de la población estudiada (p < 0,004 vs obstrucción biliar; p < 0,05 vs hepatoma). El 96% de las metástasis hepáticas presentaron valores de A1AT mayores de 400mg% registrándose diferencias significativas con el resto del grupo. La determinación de la A1AT sérica puede ser de utilidad para el diagnóstico de los tumores hepáticos malignos; de acuerdo a nuestros hallazgos el dosaje de esta glicoproteína carece de valor en el diagnóstico del hepatocarcinoma y en la insuficiencia hepática aguda