Evaluation of clinical safety and tolerance of a Lactobacillus reuteri NCIMB 30242 supplement capsule: A randomized control trial

A significant number of human clinical trials have reported no adverse effects associated with consumption of Lactobacillus reuteri (L. reuteri). In the present study, the clinical safety and toxicology of oral ingestion of supplement capsules containing L. reuteri NCIMB 30242 was investigated. A randomized group of 131 subjects received a dose of 2.9 × 10⁹ CFU L. reuteri NCIMB 30242 capsules (n = 67) or placebo capsules (n = 64) twice daily for 9 weeks. Clinical chemistry and hematological parameters of safety were analyzed. The frequency, duration and intensity of adverse events (AE)s and clinical significance of safety parameters were recorded for both groups. No clinically significant differences between the probiotic capsule and placebo capsule treated groups were detected in either the blood clinical chemistry or hematology results. The frequency and intensity of AEs was similar in the two groups. These results demonstrate that administration of a twice daily dose of 2.9 × 10⁹ CFU was safe and well tolerated in the population evaluated over 9 weeks.     

Evaluation of safety and human tolerance of the oral probiotic Streptococcus salivarius K12: A randomized, placebo-controlled, double-blind study

Streptococcus salivarius is naturally a predominant member of the human oropharynx and the commercial probiotic strain K12 has been consumed for more than a decade. The present study examines the health responses of human volunteers to oral ingestion of high doses of S. salivarius K12.A randomized group of 53 subjects received a dose of 1 × 10¹⁰ cfu S. salivarius K12 (N = 25) or placebo (N = 28) for 28 days, followed by a 28-day wash out period. Blood, urine and saliva samples were collected at baseline and following treatment and analyzed, while the oral and gastrointestinal tolerance of the subjects to the dosing regimen was determined by use of questionnaires. Adverse events (AE)s were recorded for both groups.No statistically significant differences between the probiotic and placebo treated groups were detected in either the blood clinical chemistry or hematology results (P > 0.05). The questionnaire responses of the subjects indicated that both treatments were well tolerated. The frequency and intensity of AEs was similar in the two groups.This data demonstrates that the daily ingestion of S. salivarius K12 over a 28-day period does not adversely affect the human host and supports the safety of its oral delivery in a food-based carrier.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

One-year chronic oral toxicity with combined reproduction toxicity study of a novel probiotic, Bacillus coagulans, as a food ingredient

Some strains of Bacillus coagulans can survive extremes of heat, stomach acid and bile acids, to which commonly consumed probiotics are susceptible. A toxicological safety assessment was published in 2009 on a proprietary preparation of B. coagulans - GanedenBC³⁰™ - a novel probiotic. It was concluded that GanedenBC³⁰™ is safe for chronic human consumption based upon scientific procedures, supported by a safe history of use (Endres et al., 2009).A one-year chronic oral toxicity study combined with a one-generation reproduction study was conducted to further investigate safety of long-term consumption. The one-year study of GanedenBC³⁰™ administered to male and female HsdBrlHan: Wistar rats in their diet showed no signs of toxicity at the highest dose tested. The conclusion from the reproduction toxicity study is that administration of GanedenBC³⁰™ in the diet caused no signs of toxicity in the parental generation (male or female) nor the F1 offspring. Using the lowest NOEL of 1948 mg/kg concluded at the end of the 1-year feeding study, a 100-fold safety factor, a test article concentration of 6.88 × 10¹⁰ CFU (colony forming units) per gram, and an average 70 kg human, it is determined that GanedenBC³⁰™ is safe for chronic consumption at up to 9.38 × 10¹⁰ CFUs per day.     

Effects of adult-onset choline deprivation on the activities of acetylcholinesterase, (Na,K)- and Mg-ATPase in crucial rat brain regions

Choline (Ch) plays an important role in brain neurotransmission, while Ch-deprivation (CD) has been linked to various pathophysiological states. Prolonged ingestion of Ch-deficient diet (CDD) is known to produce CD causing a reduction of rat brain acetylcholine (ACh) levels, as well as memory and growth disorders. The aim of this study was to investigate the effect of a 2-month adult-onset CD on the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)- and Mg²⁺-ATPase in crucial brain regions of male rats. Adult rats were divided into two groups (control and CD). The CD group was fed with CDD for 2-months. At the end of the second month, rats were sacrificed by decapitation and the brain regions were rapidly removed. Enzyme activities were measured spectrophotometrically in the homogenated frontal cortex, hippocampus, hypothalamus, cerebellum, and pons. In CD rats, AChE activity was found statistically significantly increased in the hippocampus and the cerebellum (+28%, P < 0.001 and +46%, P < 0.001, respectively, as compared to control), while it was found unaltered in the other three regions (frontal cortex, hypothalamus and pons). (Na⁺,K⁺)-ATPase activity was found increased by CD in the frontal cortex (+30%, P < 0.001), decreased in both hippocampus and hypothalamus (−68%, P < 0.001 and −51%, P < 0.001, respectively), and unaltered in both cerebellum and pons. No statistically significant changes were observed in the activities of Mg²⁺-ATPase in the frontal cortex and the hypothalamus, while statistically significant increases were recorded in the hippocampus (+21%, P < 0.01), the cerebellum (+85%, P < 0.001) and the pons (+19%, P < 0.05), as compared to control levels. Our data suggest that adult-onset CD can have significant effects on the examined brain parameters in the examined crucial brain regions, as well as that CD is a metabolic disorder towards which different and brain region specific neurophysiological responses seem to occur. Following a 2-month adult-onset CD, the activity of AChE was found to be increased in the hippocampus and the cerebellum and unaltered in the other three regions (frontal cortex, hypothalamus and pons), while Na⁺,K⁺-ATPase activity was found to be increased in the frontal cortex, decreased in both hippocampus and hypothalamus, and unaltered in both cerebellum and pons. Moreover, Mg²⁺-ATPase activity was found to be unaltered in the frontal cortex and the hypothalamus, and increased in the hippocampus, the cerebellum and the pons. The observed differentially affected activities of AChE, (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase (induced by CD) could result in modulations of cholinergic neurotransmission, neural excitability, metabolic energy production, Mg²⁺ homeostasis and protein synthesis (that might have a variety of neurophysiological consequences depending on the brain region in which they seem to occur).     

Interaction of sanguinarine and its dihydroderivative with the Na/K-ATPase. Complex view on the old problem

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na⁺/K⁺-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K_D = 7.2 ± 2.0 μM or 11.7 ± 0.9 μM, while the enzyme in E2 conformation binds only the charged form of SG with a K_D = 4.7 ± 1.1 μM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na⁺/K⁺-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na⁺/K⁺-ATPase can be considered only prior to SG → DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na⁺/K⁺-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.     

The aqueous stability of bupropion

In this study the aqueous stability of bupropion was determined and the pH-degradation profile was obtained. The effects of hydrogen ion, solvent and hydroxide ion concentration are discussed with particular emphasis on the kinetics of degradation of bupropion. Kinetics and degradation of bupropion were determined by HPLC-UV and LC–MS analysis both utilising high pH chromatographic methods. Degradation of bupropion in aqueous solutions follows first-order reaction kinetics. The pH-degradation profile was determined using non-linear regression analysis. The micro- and macro-reaction constants for degradation are presented. Bupropion was most stable in aqueous solutions below pH 5. Degradation was catalyzed by water but mainly by hydroxide ion on the unionised form of bupropion. The energy of activation for decomposition in aqueous solution pH 10.7 I = 0.055 was determined to be 53 kJ mol⁻¹ with a frequency factor of 6.43 × 10¹⁰ s⁻¹. The degradants of bupropion were positively identified and a mechanism of degradation is proposed. The inherent instability of bupropion above pH 5 has implications for its therapeutic use, formulation, pharmacokinetics and use during analysis and storage.     

Evidence for aconitine-induced inhibition of delayed rectifier K(+) current in Jurkat T-lymphocytes

Aconitine (ACO) is a highly toxic diterpenoid alkaloid and known to exert the immunomodulatory action. However, whether it has any effects on ion currents in immune cells remains unknown. The effects of ACO and other related compounds on ion currents in Jurkat T-lymphocytes were investigated in this study. ACO suppressed the amplitude of delayed-rectifier K⁺ current (I_K(DR)) in a time- and concentration-dependent manner. Margatoxin (100 nM), a specific blocker of K_V1.3-encoded current, decreased the I_K(DR) amplitude in these cells and the ACO-induced inhibition of I_K(DR) was not reversed by 1-ethyl-2-benzimidazolinone (30 μM) or nicotine (10 μM). The IC₅₀ value for ACO-mediated inhibition of I_K(DR) was 5.6 μM. ACO accelerated the inactivation of I_K(DR) with no change in the activation rate of this current. Increasing the ACO concentration not only reduced the I_K(DR) amplitude, but also accelerated the inactivation time course of the current. With the aid of minimal binding scheme, the inhibitory action of ACO on I_K(DR) was estimated with a dissociation constant of 6.8 μM. ACO also shifted the inactivation curve of I_K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I_K(DR) was enhanced in the presence of ACO. In Jurkat cells incubated with amiloride (30 μM), the ACO-induced inhibition of I_K(DR) remained unaltered. In RAW 264.7 murine macrophages, ACO did not modify the kinetics of I_K(DR), although it suppressed I_K(DR) amplitude. Taken together, these effects can significantly contribute to its action on functional activity of immune cells if similar results are found in vivo.     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.     

Evaluation of biogenic amines content in chilean reserve varietal wines

Biogenic amines play important roles in many physiological functions, but when they are ingested in high concentrations may produce severe adverse effects. The aim of this research was to evaluate the biogenic amine content in Chilean reserve varietal wines. A high performance liquid chromatography method was optimized and validated to quantify histamine, tyramine, spermine, spermidine, putrescine, cadaverine and phenylethylamine in Chilean wines. Derivatization and chromatographic conditions were optimized using a central composite design. Sixty reserve wines of the most important Chilean grape varieties were analyzed, i.e. Cabernet Sauvignon (n = 11), Merlot (n = 11), Carménère (n = 11), Syrah (n = 10) and Sauvignon Blanc (n = 10), as well as organic wines (n = 7). Biogenic amines content ranged from 2.19 to 65.09 mg L⁻¹, no significant difference (P > 0.05) was observed between Cabernet Sauvignon, Merlot and Carménère but all showed statistically higher (P < 0.05) concentrations than Sauvignon Blanc. Syrah wines showed no difference (P > 0.05) with Cabernet Sauvignon, higher concentrations (P < 0.05) than Sauvignon Blanc and lower than Merlot and Carménère. Regarding biogenic amines profile, putrescine showed the highest concentration in all grape varieties.     

A unified algorithm for predicting partition coefficients for PBPK modeling of drugs and environmental chemicals

The algorithms in the literature focusing to predict tissue:blood PC (P_tb) for environmental chemicals and tissue:plasma PC based on total (K_p) or unbound concentration (K_pu) for drugs differ in their consideration of binding to hemoglobin, plasma proteins and charged phospholipids. The objective of the present study was to develop a unified algorithm such that P_tb, K_p and K_pu for both drugs and environmental chemicals could be predicted. The development of the unified algorithm was accomplished by integrating all mechanistic algorithms previously published to compute the PCs. Furthermore, the algorithm was structured in such a way as to facilitate predictions of the distribution of organic compounds at the macro (i.e. whole tissue) and micro (i.e. cells and fluids) levels. The resulting unified algorithm was applied to compute the rat P_tb, K_p or K_pu of muscle (n = 174), liver (n = 139) and adipose tissue (n = 141) for acidic, neutral, zwitterionic and basic drugs as well as ketones, acetate esters, alcohols, aliphatic hydrocarbons, aromatic hydrocarbons and ethers. The unified algorithm reproduced adequately the values predicted previously by the published algorithms for a total of 142 drugs and chemicals. The sensitivity analysis demonstrated the relative importance of the various compound properties reflective of specific mechanistic determinants relevant to prediction of PC values of drugs and environmental chemicals. Overall, the present unified algorithm uniquely facilitates the computation of macro and micro level PCs for developing organ and cellular-level PBPK models for both chemicals and drugs.     

Individual fumonisin exposure and sphingoid base levels in rural populations consuming maize in South Africa

Low and high oesophageal cancer incidence areas of the former Transkei region of South Africa have been associated with corresponding low and high levels of fumonisin contaminated home-grown maize. This is the first study in South Africa assessing fumonisin B (FB) mycotoxin exposure by quantifying individual maize consumption with weighed food records and FB levels from maize in each participant’s household and concurrently evaluating sphinganine (Sa), sphingosine (So) and Sa/So ratios in plasma and urine of these participants as possible biomarkers of FB exposure. The high consumption of maize in Bizana (n = 36) and Centane (n = 30) of 0.41 ± 0.21 and 0.39 ± 0.19 kg/day, respectively, confirms the reliance on maize as the dietary staple. Mean total FB (FB₁ + FB₂ + FB₃) levels in home-grown maize were 0.495 + 0.880 and 0.665 + 0.660 mg/kg in Bizana and Centane, respectively. Mean fumonisin exposure based on individual consumption was 3.9 ± 7.3 and 4.1 ± 7.6 μg/kg body weight/day, respectively, for Bizana and Centane. The mean combined sphinganine/sphingosine ratios in Bizana and Centane were similar and ranged from 0.10–0.55 in plasma (n = 41) and urine (n = 62). There was no association between sphingoid base levels and/or Sa/So ratios in the plasma and urine and individual fumonisin exposure, negating the sphingoid bases as potential biomarkers of fumonisin exposure in humans.     

Respiratory effects of buprenorphine/naloxone alone and in combination with diazepam in naive and tolerant rats

Respiratory depression has been attributed to buprenorphine (BUP) misuse or combination with benzodiazepines. BUP/naloxone (NLX) has been marketed as maintenance treatment, aiming at preventing opiate addicts from self-injecting crushed pills. However, to date, BUP/NLX benefits in comparison to BUP alone remain debated. We investigated the plethysmography effects of BUP/NLX in comparison to BUP/solvent administered by intravenous route in naive and BUP-tolerant Sprague-Dawley rats, and in combination with diazepam (DZP) or its solvent. In naive rats, BUP/NLX in comparison to BUP significantly increased respiratory frequency (f, P < 0.05) without altering minute volume (V_E). In combination to DZP, BUP/NLX significantly increased expiratory time (P < 0.01) and decreased f (P < 0.01), tidal volume (V_T, P < 0.001), and V_E (P < 0.001) while BUP only decreased V_T (P < 0.5). In BUP-tolerant rats, no significant differences in respiratory effects were observed between BUP/NLX and BUP. In contrast, in combination to DZP, BUP/NLX did not significantly alter the plethysmography parameters, while BUP increased inspiratory time (P < 0.001) and decreased f (P < 0.01) and V_E (P < 0.001). In conclusion, differences in respiratory effects between BUP/NLX and BUP are only significant in combination with DZP, with increased depression in naive rats but reduced depression in BUP-tolerant rats. However, BUP/NLX benefits in humans remain to be determined.     

Behavior and brain enzymatic changes after long-term intoxication with cadmium salt or contaminated potatoes

This study investigated the cadmium (Cd) intoxication on cognitive, motor and anxiety performance of rats subjected to long-term exposure to diet with Cd salt or with Cd from contaminated potato tubers. Potato plantlets were micropropagated in MS medium and transplanted to plastic trays containing sand. Tubers were collected, planted in sand boxes and cultivated with 0 or 10 μM Cd and, after were oven-dried, powder processed and used for diet. Rats were divided into six groups and fed different diets for 5 months: control, potato, potato + Cd, 1, 5 or 25 mg/kg CdCl₂. Cd exposure increased Cd concentration in brain regions. There was a significant decrease in the step-down latency in Cd-intoxicated rats and, elevated plus maze task revealed an anxiolytic effect in rats fed potato diet per se, and an anxiogenic effect in rats fed 25 mg/kg Cd. The brain structures of rats exposed to Cd salt or Cd from tubers showed an increased AChE activity, but Na⁺,K⁺-ATPase decreased in cortex, hypothalamus, and cerebellum. Therefore, we suggest an association between the long-term diet of potato tuber and a clear anxiolytic effect. Moreover, we observed an impaired cognition and enhanced anxiety-like behavior displayed by Cd-intoxicated rats coupled with a marked increase of brain Cd concentration, and increase and decrease of AChE and Na⁺,K⁺-ATPase activities, respectively.     

The insulinotropic effect of fluoroquinolones

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K_ATP channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5 mM). Only when the glucose concentration was stimulatory by itself (10 mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K_ATP channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100 μM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5 mM, whereas other K_ATP channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500 μM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10 mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca²⁺]_i). In the presence of 5 mM glucose gatifloxacin and moxifloxacin at 500 μM but not at 100 μM elevated [Ca²⁺]_i. It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K_ATP channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.     

Predicting safety toleration of pharmaceutical chemical leads: Cytotoxicity correlations to exploratory toxicity studies

The selection and application of appropriate safety screening paradigms could revolutionize the drug discovery process by reducing safety-related attrition. While mechanism specific genotoxicity and safety pharmacology assays are routinely used in screening, the overall value of employing nonspecific cytotoxicity assays remains controversial. A retrospective analysis of safety findings from rat exploratory toxicity studies (4–14 days) utilizing compounds that spanned broad therapeutic targets (protease, transport, G-protein-coupled receptors, and kinase inhibitors, cGMP modulators) demonstrated that safety toleration in vivo could be approximated using cytotoxicity values. A composite safety score was calculated for each compound dose based on findings in each of the following categories: systemic toleration (mortality, food consumption, and adverse clinical signs), clinical chemistry/hematology parameters (deviations from normal ranges), and multiorgan pathology (necrosis or incidence/severity of histologic change). Binning compounds into potent (LC₅₀ < 10 μM) and non-potent (LC₅₀ > 100 μM) cytotoxicants in vitro showed that compared to non-potent cytotoxicants the exposure to potent cytotoxicants in vivo resulted in higher overall severity scores at lower exposures. Correlating overall toleration for individual compounds was further refined when in vivo exposure was considered. When average plasma exposure (Cp_ave) for a compound exceeded its mean lethal concentration (LC₅₀) in vitro (Cp_ave/LC₅₀ > 1), higher overall severity scores were achieved compared to lower exposure margins (Cp_ave/LC₅₀ <0.01). Based on this analysis, the ability to select lead series and individual compounds with better safety characteristics is presented. In summary, cytotoxicity screening can be used to approximate, not define, the safety characteristics of lead pharmaceutical series early in the drug discovery process.     

Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20-30 nm (CNT20) and 40-70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 μg/ml (p < 0.05), which persisted for 24 h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p < 0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p < 0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis.     

Validation of an HPLC-UV method for sorafenib determination in human plasma and application to cancer patients in routine clinical practice

Sorafenib, a new oral multikinase inhibitor with antiangiogenic properties, has demonstrated preclinical and clinical activity against several tumor types. The aims of this study were to validate a method for the measurement of sorafenib in plasma from cancer patients, then to test this method in clinical practice. Following liquid–liquid extraction, the compounds were separated with gradient elution (on a C18 ultrasphere ODS column using a mobile phase of acetonitrile/20 mM ammonium acetate), then detected at 255 nm. The calibration was linear in the range 0.5–20 mg/L. Intra- and inter-assay precision was lower than 7 and 10%, respectively, at 0.5, 3 and 20 mg/L. Plasma sorafenib concentrations were measured in 22 cancer patients (99 samples). The mean trough sorafenib concentration (C_min) and concentration at peak were 4.3 ± 2.5 mg/L (n = 68, CV = 57.5%) and 6.2 ± 3.0 mg/L (n = 31, CV = 47.5%), respectively. Mean sorafenib C_min in eight patients who experienced grade 3 drug-related adverse events was approximately 1.5-fold greater than that observed in the remaining patients (7.7 ± 3.6 mg/L vs. 4.4 ± 2.4 mg/L, P = 0.0083). In conclusion, the method was successfully used in routine practice to monitor plasma concentrations of sorafenib in cancer patients. Finally, large interindividual variability and higher exposure in patients experiencing severe toxicity support the need for therapeutic drug monitoring to ensure an optimal exposure to sorafenib.     

Simultaneous quantification of glycine- and taurine-conjugated bile acids,total bile acids,and choline-containing phospholipids in human bile using H NMR spectroscopy

Bile acids, phospholipids, and cholesterol are the major lipid components in human bile. The composition of bile is altered in various cholestatic diseases, and determining such alterations will be of great clinical importance in understanding the pathophysiology of these diseases. A robust method for the simultaneous quantification of major biliary lipids – glycine-conjugated bile acids (GCBAs), taurine-conjugated bile acids (TCBAs), total bile acids (TBAs) and choline-containing phospholipids (choline-PLs) has been devised using ¹H NMR spectroscopy. Bile samples were obtained from patients with various hepatopancreatobiliary diseases (n = 10) during an endoscopic retrograde cholangiopancreatography (ERCP) examination. Peak areas of metabolite-signals of interest were obtained simultaneously by deconvoluting the experimental spectrum, making the present method robust. GCBAs and TCBAs have been quantified using the peak areas of their characteristic methylene (CH₂) signals resonating at 3.73 and 3.07 ppm, whereas TBA and choline-PLs were quantified using their methyl (CH₃) and trimethylammonium (–N⁺(CH₃)₃) signals resonating at 0.65 and 3.22 ppm respectively. The present method was compared with an NMR-based literature method (which involves dissolving bile in DMSO), and a good correlation was observed between the two methods with regression coefficients – 0.97, 0.99, 0.98 and 0.93 for GCBAs, TCBAs, TBAs, and choline-PLs respectively. This method has the potential to be extended to in vivo applications for the simultaneous quantification of various biliary lipids non-invasively.