Effects of dendritic core-shell glycoarchitectures on primary mesenchymal stem cells and osteoblasts obtained from different human donors.

The biological impact of novel nano-scaled drug delivery vehicles in highly topical therapies of bone diseases have to be investigated in vitro before starting in vivo trials. Highly desired features for these materials are a good cellular uptake, large transport capacity for drugs and a good bio-compatibility. Essentially the latter has to be addressed as first point on the agenda. We present a study on the biological interaction of maltose-modified poly(ethyleneimine) (PEI-Mal) on primary human mesenchymal stem cell, harvested from reaming debris (rdMSC) and osteoblasts obtained from four different male donors. PEI-Mal-nanoparticles with two different molecular weights of the PEI core (5000 g/mol for PEI-5k-Mal-B and 25,000 g/mol for PEI-25k-Mal-B) have been administered to both cell lines. As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 µg/ml to 1 mg/ml and periods of 24 h up to 28 days. Studies conducted by different methods of microscopy as light microscopy, fluorescence microscopy, transmission-electron-microscopy and quantitative assays (LDH and DC-protein) indicate as well a good cellular uptake of the nanoparticles as a particle- and concentration-dependent impact on the cellular macro- and micro-structure of the rdMSC samples. In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B. At higher concentrations PEI-25k-Mal-B is toxic and induces a directly observable mitochondrial damage. The alkaline phosphatase assay (ALP), has been conducted to check on the possible influence of nanoparticles on the differentiation capabilities of rdMSC to osteoblasts. In addition the production of mineralized matrix has been shown by von-Kossa stained samples. No influence of the nanoparticles on the ALP per cell has been detected. Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples. To summarize, while featuring a good cellular uptake, PEI-5k-Mal-B induces only minimal adverse effects and features clearly superior biocompatibility compared to the larger PEI-25k-Mal-B.

Calu-3 model under AIC and LCC conditions and application for protein permeability studies.

Broad area of respiratory epithelium with mild surface conditions is an attractive possibility when trans-mucosal delivery of protein drugs is considered. A mucus and cellular barrier of respiratory epithelium can be modelled in vitro by Calu-3 cell line. We have monitored morphology and barrier properties of Calu-3 culture on permeable supports while developing into liquid covered or air interfaced and mucus lined cellular barrier. Besides morphological differences, cultures differed in electrical resistance and permeability to proteins as well. The accelerated permeability to proteins in these models, due to permeability modulator MP C16, was examined. The effect on electrical resistance of cellular layer was rapid in both cultures suggesting easy access of MP C16 to cells even though its overall impact on cell permeability was strongly reduced in mucus covered culture. Differences in properties of the two models enable better understanding of protein transmucosal permeability, suggesting route of transport and MP C16 modulator action.

The genotoxic and teratogenic effects of maltitol in rats.

In the present study, the genotoxic and cytotoxic effects of the low-caloric artificial sweetener maltitol, which is a sugar alcohol (polyol), were investigated in the bone marrow cells of rats using the chromosome aberration (CA) test. In addition, the teratogenicity and embryotoxicity of maltitol was also investigated in rats. To reveal the genotoxicity and cytotoxicity of maltitol, rats were intraperitoneally administered 2.5, 5 and 10 g/kg body weight (bw) concentrations of maltitol for 6, 12 and 24 h treatment period. The pregnant females were intraperitoneally treated with 1, 2 and 4 g/kg bw/day concentrations of maltitol during the first 7 days of gestation (first trimester) to investigate the teratogenicity of maltitol. The embryos were collected after killing the dams by cervical dislocation under ether anaesthesia on gestation day 19. Maltitol did not induce the CA and did not decrease the mitotic index in bone marrow cells of rats at all concentrations and treatment periods. In addition, maltitol was not teratogenic; however, it decreased the foetuses weight and at the highest dose (4 g/kg bw) caused growth retardation.

Efficacy and toxicity of intravenous iron in a mouse model of critical care anemia*.

OBJECTIVE: Anemia is common in critically ill patients, due to inflammation and blood loss. Anemia can be associated with iron deficiency and low serum hepcidin levels. However, iron administration in this setting remains controversial because of its potential toxicity, including oxidative stress induction and sepsis facilitation. The objective of this work was to determine the efficacy and toxicity of iron administration using a mouse model mimicking critical care anemia as well as a model of acute septicemia. DESIGN: Prospective, randomized, open label controlled animal study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 and OF1 mice. INTERVENTIONS: Intraperitoneal injection of zymosan inducing generalized inflammation in C57BL/6 mice, followed in our full model by repeated phlebotomies. A dose equivalent to 15 mg/kg of ferric carboxymaltose was injected intravenously on day 5. To assess the toxicity of iron in a septicemia model, OF1 mice were simultaneously injected with iron and different Escherichia coli strains. MEASUREMENTS AND MAIN RESULTS: To investigate the effect of iron on oxidative stress, we measured reactive oxygen species production in the blood using luminol-amplified chemiluminescence and superoxide dismutase 2 messenger RNA levels in the liver. These markers of oxidative stress were increased after iron administration in control mice but not in zymosan-treated mice. Liver catalase messenger RNA levels decreased in iron-treated control mice. Iron administration was not associated with increased mortality in the septicemia model or in the generalized inflammation model. Iron increased hemoglobin levels in mice fed with a low iron diet and subjected to phlebotomies and zymosan 2 wks after treatment administration. CONCLUSIONS: Adverse effects of intravenous iron supplementation by ferric carboxymaltose seem to be minimal in our animal models. Furthermore, iron appears to be effective in correcting anemia, despite inflammation. Studies of efficacy and safety of iron in critically ill patients are warranted.

Cardiovascular, liver, and renal toxicity associated with an intravenous ferric carboxymaltose similar versus the originator compound.

BACKGROUND: Ferric carboxymaltose (FCM) is a stable, non-dextran-based intravenous iron complex used to treat iron deficiency of various etiologies. As FCM is a nonbiological complex drug and cannot be fully characterized by physicochemical analyses, it is important to demonstrate in nonclinical models that FCM similars (FCMS) have similar biodistribution. MATERIALS AND METHODS: A total of 30 nonanemic rats were treated weekly with 40 mg iron/kg body weight intravenous FCM, FCMS, or isotonic saline (controls) for 4 weeks. Blood pressure, liver enzymes, and renal function were evaluated. In liver, heart, and kidney tissue, markers for oxidative stress (malondialdehyde to assess lipid peroxidation and antioxidant enzymes) and inflammation (TNFα and IL6) were measured. Iron deposits were localized. RESULTS: The FCMS-treated group had significantly lower blood pressure, higher liver enzymes, increased proteinuria, and reduced creatinine clearance versus the FCM and control groups by day 29. Serum iron and transferrin saturation were significantly higher with FCMS versus FCM or controls. Iron deposition was altered in FCMS-treated animals, with decreased ferritin deposits and iron deposition outside the physiological storage compartments. Markers for lipid peroxidation and antioxidant-enzyme activity were significantly increased after FCMS administration versus FCM and controls, as were inflammatory markers. CONCLUSION: Results from this blinded nonclinical study demonstrated significant differences between the originator FCM and this FCMS.

Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells.

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

Bacterial reversion assay and micronucleus test carried out on hydrogenated glucose syrups 'Malti-Towa' (powder) and maltitol crystal.

Two preparations of maltitol (4-O-alpha-D-glucopyranosyl-D-sorbitol), hydrogenated glucose syrups and maltitol crystal, were examined for genotoxic potential by a battery of short-term tests. In the bacterial reversion assay, maltitol induced no detectable revertants in any of the tester strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, or Escherichia coli WP2/pKM101 at doses of 0.5-50 mg per plate with and without rat liver S9 mix. In the micronucleus test, no significant increase in the frequency of micronucleated erythrocytes was observed in bone marrow of mice after administration of the two preparations at 3.75-30 g per kg by gastric intubation.

Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorption-enhancing mechanism of n-lauryl-beta-D-maltopyranoside.

We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.

Assessment of the extent of oxidative stress induced by intravenous ferumoxytol, ferric carboxymaltose, iron sucrose and iron dextran in a nonclinical model.

Intravenous (i.v.) iron is associated with a risk of oxidative stress. The effects of ferumoxytol, a recently approved i.v. iron preparation, were compared with those of ferric carboxymaltose, low molecular weight iron dextran and iron sucrose in the liver, kidneys and heart of normal rats. In contrast to iron sucrose and ferric carboxymaltose, low molecular weight iron dextran and ferumoxytol caused renal and hepatic damage as demonstrated by proteinuria and increased liver enzyme levels. Higher levels of oxidative stress in these tissues were also indicated, by significantly higher levels of malondialdehyde, significantly increased antioxidant enzyme activities, and a significant reduction in the reduced to oxidized glutathione ratio. Inflammatory markers were also significantly higher with ferumoxytol and low molecular weight iron dextran rats than iron sucrose and ferric carboxymaltose. Polarographic analysis suggested that ferumoxytol contains a component with a more positive reduction potential, which may facilitate iron-catalyzed formation of reactive oxygen species and thus be responsible for the observed effects. Only low molecular weight iron dextran induced oxidative stress and inflammation in the heart.

The new generation of intravenous iron: chemistry, pharmacology, and toxicology of ferric carboxymaltose.

An ideal preparation for intravenous iron replacement therapy should balance effectiveness and safety. Compounds that release iron rapidly tend to cause toxicity, while large molecules can induce antibody formation and cause anaphylactic reactions. There is therefore a need for an intravenous iron preparation that delivers appropriate amounts of iron in a readily available form but with minimal side effects and thus with an excellent safety profile. In this paper, a review is given on the chemistry, pharmacology, and toxicology of ferric carboxymaltose (FCM, Ferinject), a stable and robust complex formulated as a colloidal solution with a physiological pH. The complex is gradually taken up mainly from the hepatic reticulo-endothelial system (RES), followed by effective delivery of iron to the endogeneous transport system for the haem synthesis in new erythrocytes, as shown in studies on the pharmacodynamics and pharmacokinetics with radio-labelled FCM. Studies with radio-labelled FCM also demonstrated a barrier function of the placenta and a low transfer of iron into the milk of lactating rats. Safety pharmacology studies indicated a favourable profile with regard to cardiovascular, central nervous, respiratory, and renal toxicity. A high maximum non-lethal dose was demonstrated in the single-dose toxicity studies. Furthermore, based on the No-Observed-Adverse-Effect-Levels (NOAELs) found in repeated-dose toxicity studies and on the cumulative doses administered, FCM has good safety margins. Reproductive and developmental toxicity studies did not reveal any direct or indirect harmful effects. No genotoxic potential was found in in vitro or in vivo studies. Moreover, antigenicity studies showed no cross-reactivity of FMC with anti-dextran antibodies and also suggested that FCM does not possess sensitizing potential. Lastly, no evidence of irritation was found in local tolerance studies with FCM. This excellent toxicity profile and the high effectiveness of FCM allow the administration of high doses as a single infusion or bolus injection, which will enhance the cost-effectiveness and convenience of iron replacement therapy. In conclusion, FCM has many of the characteristics of an ideal intravenous iron preparation.

Pharmacodynamics and safety of ferric carboxymaltose: a multiple-dose study in patients with iron-deficiency anaemia secondary to a gastrointestinal disorder.

This multiple-dose Phase I/II study provided pharmacodynamics and pharmacokinetics data on the therapeutic benefit of ferric carboxymaltose (FCM, Ferinject) and evaluated the safety and tolerability of this intravenous (i.v.) iron preparation. Two doses of iron as FCM were given as i.v. infusion over 15 min, 500 mg iron given once weekly for up to 4 weeks (Cohort 1) or 1000 mg iron weekly for 2 weeks (Cohort 2), in patients with a total requirement > or = 1000 mg iron (total cumulative maximum dose < or = 2000 mg iron). Adults with moderate to severe, stable iron-deficiency anaemia (IDA) (haemoglobin [Hb] < or = 11.0 g/dl, serum ferritin < 100 microg/l, transferrin saturation [TSAT] < 16%) due to a gastrointestinal (GI) disorder were included. Pharmacodynamics variables: proportion of patients achieving values within the reference range for Hb (men: 14.0-18.0 g/dl, women: 12.0-16.0 g/dl), serum ferritin (20-500 microg/l), TSAT (16-45%) and proportion of patients with an increase in Hb of at least 2.0 g/dl. Pharmacokinetics variables: total serum iron levels at time of maximum serum iron concentration during the fast elimination phase and at trough time-points. Safety assessments: the incidence of adverse events (AEs) and changes in vital signs, physical examinations, and clinical laboratory parameters. In Cohorts 1 and 2, 14/20 (70%) versus 19/26 (73%) of patients completed the study. Individual calculated iron deficits were 1000-2100 mg. The mean cumulative dose of FCM in Cohorts 1 and 2 was 1800 mg and 1563 mg iron, respectively. At baseline, patients in both cohorts had similar Hb levels (mean 8.7 g/dl in both cohorts). More than 97% of patients demonstrated a clinically meaningful increase in Hb levels (> or = 1.0 g/dl) during the study. By the week 4 follow-up visit, an increase of at least 2.0 g/dl was achieved by 15/20 (75%) and by 19/26 (73.1%) patients in Cohorts 1 and 2, respectively, and the mean increase in Hb was 3.2 g/dl in Cohort 1 and 3.3 g/dl in Cohort 2. By day 28, 3/6 (50%) patients in Cohort 1 had achieved normal Hb levels, and by the 4-week post-treatment followup visit 7/19 patients (37%) in Cohort 1 and 12/25 (48%) in Cohort 2 had reached Hb levels within the reference range. Serum ferritin levels increased rapidly at the start of treatment and remained in the reference range throughout the study; increases were greater in Cohort 2. Mean baseline TSAT values were similar in both cohorts (24.2% in Cohort 1, 20.7% in Cohort 2), and were within the reference range at the week 4 follow-up visit for 41.0 and 39.1% of the patients in Cohorts 1 and 2, respectively. The incidence of AEs occurring after the first administration of FCM (treatment-emergent AEs, TEAE) was generally low and similar in Cohorts 1 (11/20 [55.0%]) and 2 (13/26 [50.0%]). Most TEAEs were mild; only 2/ 20 patients (10.0%) in Cohort 1 and 3/26 (11.5%) in Cohort 2 had TEAEs of moderate intensity. There were no AEs of severe intensity, serious AEs, or deaths. Most AEs were considered by the investigator to be unrelated or unlikely to be related to the study medication. Since accumulation of serum iron was not observed, a dosing interval of 3-4 days (500 mg iron) or 1 week (1000 mg iron) was demonstrated to be adequate. The increase in serum ferritin and TSAT at the 4-week follow-up visit is indicative of a repletion of the iron stores. The results suggest that doses up to 1000 mg i.v. iron administered as FCM over 15 min arewell tolerated and effective in the treatment of patients with IDA due to a GI disorder.

The cytogenetic effects of food sweetener maltitol in human peripheral lymphocytes.

The effects of the low-calorie artifical sweetener maltitol (E965), a sugar alcohol (Polyol), on sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus formation (MN) were investigated in human peripheral lymphocytes. Maltitol did not induce SCE at all concentrations (1.25, 2.5, and 5 mg/mL) and treatment periods (24 and 48 h). Maltitol induced CA, although not statistically significantly. Maltitol induced the frequency of MN at 24 and 48 h in a non-dose-dependent manner. In addition, maltitol did not decrease the replication index (RI) and the mitotic index (MI) at all concentrations and treatment periods. Maltitol did not alter the pH and osmolality of the medium. In conclusion, it can be concluded that maltitol has a weak genotoxic potential and it appears non-cytotoxic to human peripheral lymphocytes in vitro.

Effectiveness and toxicity screening of various absorption enhancers using Caco-2 cell monolayers.

We studied the enhancing and toxic effects of five different absorption enhancers on the transport of FITC-dextran with an average molecular weight of 4000 (FD-4) across Caco-2 cell monolayers, and their enhancing effects were also compared with those in rat intestine. The enhancing and cytotoxic properties of these enhancers were characterized using the following tests: measurement of the permeability coefficients of FD-4 and the transepithelial electrical resistance (TEER) in Caco-2, the release of cytosolic lactate dehydrogenase (LDH) and intracellular mitochondrial dehydrogenase (MDH) activity. All the absorption enhancers increased the permeability of FD-4 across Caco-2 cell monolayers and a good relationship was observed between the enhancement and their toxic effects. However, EDTA and Na-Cap were effective for improving the transport of FD-4 across Caco-2 cells without serious cytotoxicity. At concentrations with low cytotoxicity, various absorption enhancers exihibited reversible effects on the TEER values in Caco-2 cell monolayers, except for 50 mM sodium salicylate (Na-Sal). Moreover, we obtained a good correlation between the enhancement of these enhancers in Caco-2 cell monolayers and in rat large intestine. This finding indicated that the effectiveness of absorption enhancers in the Caco-2 monolayer system was similar to an in vivo rat system. Therefore, the screening system using Caco-2 cell monolayers is useful for examining the effectiveness and toxicity of absorption enhancers.