Effect of particle size on the biodistribution of lipid nanocapsules: Comparison between nuclear and fluorescence imaging and counting

In vivo biodistribution of nanoparticles depends on several physicochemical parameters such as size. After intravenous injection of 25, 50 and 100 nm lipid nanocapsules (LNC) in nude mice bearing HEK293(β₃) tumour xenografts, biodistribution was evaluated by γ-scintigraphy and by γ-counting. The small LNC 25 nm disappeared faster than the larger LNC 50 and 100 nm from the blood circulation due to faster elimination and wider tissue distribution. At 24 h, biodistribution profiles of all these LNC were similar. Low LNC quantities were found in this weak EPR (enhanced permeability and retention) tumour regardless the particle size. Co-injected 50 nm fluorescent DiD-LNC and ^99mTc-LNC allowed direct comparison of biodistribution as evaluated by the two methods. Optical imaging underestimated LNC quantity especially in dark-colored organs that were observed to capture extensive quantities of the particles by γ-counting (i.e. liver, spleen, and kidney).     

Synthesis and characterization of self-assembled CdHgTe/gelatin nanospheres as stable near infrared fluorescent probes in vivo

This work presented a kind of novel near infrared emitting CdHgTe/gelatin nanospheres which were synthesized with Cd(NO₃)₂, Hg(NO₃)₂, NaHTe and a thiol stabilizer in gelatin solution. The self-assembled nanospheres were megranate-like and nearly 40 nm in diameter, with CdHgTe QDs uniformly embedded in gelatin matrix. They exhibited strong fluorescence ranging from 580 to 800 nm that could be tuned by molar ratios of Hg²⁺ and gelatin. The full widths at half-maximum of the emission spectra were in the range of 60–80 nm. Compared with bare CdHgTe QDs, the photostability of this compact complex nanostructure remarkably improved. Moreover, the fluorescence of CdHgTe/gelatin nanospheres was much more resistant to the interference from certain kinds of endogenous biomolecules such as HSA, transferrin and hemoglobin. Further applications of living cells and mouse imaging were demonstrated with an in vivo near infrared fluorescence imaging system. The inherent advantages of high stability as well as high fluorescence intensity make the nanospheres particular interested NIR bioprobe candidates for in vivo imaging studies.     

A novel lactoferrin-modified β-cyclodextrin nanocarrier for brain-targeting drug delivery

The blood–brain barrier (BBB) restricts the transfer and delivery of most drug substances to brain. In this study, a novel nano-drug delivery system for brain-targeting was developed and investigated in vitro and in vivo. Lactoferrin (Lf) was selected as a brain-targeting ligand and conjugated to β-cyclodextrin (β-CD) via the heterobifunctional polyethyleneglycol (PEG) linker NHS-PEG-MAL, yielding Lf conjugated β-cyclodextrin (Lf-CD). UV–vis, FTIR, NMR and transmission electron microscopy (TEM) techniques clearly demonstrated the successful synthesis of Lf-CD nanoparticles with the average diameter of 92.9 ± 16.5 nm. Using near-infrared fluorescent dye IR-775 chloride (IR) as a model compound of poorly water-soluble drugs, IR-loaded Lf-CD nanoparticles (Lf-CD/IR) were successfully prepared with a high entrapment efficiency of 98.1 ± 4.8%. Biodistribution and pharmacokinetics of Lf-CD/IR were evaluated in KM mice after intravenous administration. The results of tissue distribution studies revealed that Lf-CD/IR treatment showed greatly improved BBB transport efficiency. In addition, AUC_0–2 h of IR in brain after Lf-CD/IR treatment was seven fold higher compared with that of IR treatment without Lf-CD nano-carriers, demonstrating that the introduction of Lf-CD drug-delivery system positively resulted in a higher AUC located in brain tissue. These results provide evidence that Lf-CD nanoparticles could be exploited as a potential brain-targeting drug delivery system for hydrophobic drugs and diagnostic reagents which normally fail to pass through the BBB.     

Development of a liquid chromatographic system with fluorescent detection for β-secretase immobilized enzyme reactor on-line enzymatic studies

A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant β-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1–IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the β-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1–IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt = 1.6 min), which was separated from the substrate and selectively detected at λ_exc = 320 and λ_em = 420 nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1–IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA.     

Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte

The inhibitory effects of Zataria multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H₂O₂) production were examined in human monocytes cultured in the presence of 20 mM glucose. Z. multiflora essential oil was extracted by water-distillation and then analyzed by GC–MS. Carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and γ-terpinene (8%) were the main components detected in the essential oil. Cells cultured in the presence of 20 mM glucose showed an increase in NO and H₂O₂ production as well as NO synthase (NOS) and NADH oxidase (NOX) activities compared to cells cultured in the presence of 5 mM glucose. Pretreatment with Z. multiflora essential oil, carvacrol and thymol reduced NO and H₂O₂ production as well as NOS and NOX activities in those cells cultured in the presence of 20 mM glucose. However, p-cymene, linalool and γ-terpinene did not show any such activities. Accordingly, it was concluded that Z. multiflora can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying hyperglycemia and some inflammatory conditions.     

Anti-oxidative effect of Rhodiola imbricata root extract in rats during cold,hypoxia and restraint (C–H–R) exposure and post-stress recovery

Anti-oxidative potential of Rhodiola imbricata root aqueous extract was examined in rats, administered orally at a dose of 100 mg/kg both in single and multiple doses, 30 min prior to cold (5 °C)–hypoxia (428 mmHg)–restraint (C–H–R) exposure. Lipid per-oxidation, anti-oxidant parameters and lactate dehydrogenase (LDH), were studied in blood, liver and muscle of rats on attaining T_rec23 °C during C–H–R exposure and after recovery (T_rec37 °C) from C–H–R induced hypothermia. The results of untreated control rats on attaining T_rec23 °C showed a significant increase in blood, liver and muscle malondialdehyde (MDA) and LDH levels. Hepatic catalase (CAT) and muscle glutathione S-transferase (GST) also increased significantly. Administration of single dose of Rhodiola imbricata root aqueous extract significantly restricted rise in blood MDA, increased blood reduced glutathione (GSH) and superoxide dismutase (SOD) activity with restricted rise in blood, liver and muscle LDH; improved liver and muscle SOD on attaining T_rec23 °C and T_rec37 °C; liver CAT on attaining T_rec23 °C and liver GST during recovery. Multiple doses treatment of the extract further increased blood, liver and muscle GSH and GST levels; restricted increase in LDH on attaining T_rec23 °C and recovery; increased CAT during recovery. Results suggested the anti-oxidant potential of Rhodiola root extract during C–H–R exposure and post-stress recovery and it also maintained cell membrane permeability.     

Preparation, characterization and in vivo assessment of the bioavailability of glycyrrhizic acid microparticles by supercritical anti-solvent process

In this study, glycyrrhizic acid (GA) microparticles were successfully prepared using a supercritical anti-solvent (SAS) process. Carbon dioxide and ethanol were used as the anti-solvent and solvent, respectively. The influences of several process parameters on the mean particle size (MPS), particle size distribution (PSD) and total yield were investigated. Processed particle sizes gradually decreased as temperature and solution flow rate increased. In addition, processed particle sizes increased from 119 to 205 nm as GA concentration increased. However, CO₂ flow rate did not significantly affect particle size. The optimized process conditions were applied, those included temperature (65 °C), pressure (250 bar), CO₂ and drug solution flow rate (15 and 8 mL min⁻¹), drug concentration in ethanol (20 mg mL⁻¹). Microparticles with a span of PSD ranging from 95 and 174 nm, MPS of 128 nm were obtained, and total yield was 63.5%. The X-ray diffraction patterns of glycyrrhizic acid microparticles show apparent amorphous nature. Fourier transform infrared (FT-IR) spectroscopy results show that no chemical structural changes occurred. The in vitro dissolution tests showed that the GA microparticles exhibited great enhancement of dissolution performance when compared to GA original drug. Furthermore, the in vivo studies revealed that the microparticles provided improved pharmacokinetic parameter after oral administration to rats as compared with original drug.     

Hemoglobin adducts as biomarkers of estrogen homeostasis: Elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese Women

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts, including 17β-estradiol-2,3-quinone (E₂-2,3-Q) and 17β-estradiol-3,4-quinone (E₂-3,4-Q), in human hemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derived from female breast cancer patients (n = 143) as well as controls (n = 147) in Taiwan. Our result confirmed that both E₂-2,3-Q- and E₂-3,4-Q-derived adducts, including E₂-2,3-Q-4-S-Hb and E₂-3,4-Q-2-S-Hb, were detected in all breast cancer patients with median levels at 434 (215–1472) and 913 (559–2384) (pmol/g), respectively. Levels of E₂-2,3-Q-4-S-Hb correlated significantly with those of E₂-3,4-Q-2-S-Hb (r = 0.622–0.628, p < 0.001). By contrast, median levels of these same estrogen quinone-derived adducts in healthy controls were 71.8 (35.7–292) and 139 (69.1–453) (pmol/g). This translated to ∼6-fold increase in mean values of E₂-2,3-Q-4-S-Hb and E₂-3,4-Q-2-S-Hb in breast cancer patients compared to those in the controls (p < 0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination of genetic events and environmental factors may modulate estrogen homeostasis and enhance the production of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breast cancer patients.     

Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography–tandem mass spectrometry

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu_n, n = 2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu_n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 × 10⁶ cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H]⁺ ions of MTX and MTXGlu_n to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3 → 294.2. Calibration curves for all analytes were linear in the range 2–250 nM (r² > 0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60 ± 4 to 108 ± 13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Leptin and IL-8: Two novel cytokines screened out in childhood lead exposure

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain. One possible attribution is the immature blood–brain barrier that causes lead exposure in children. Few studies have investigated the cytokine changes caused by this exposure. Novel cytokines were detected by RayBio^® Human Cytokine Antibody Array and validated by enzyme-linked immunosorbent assay. Several children were admitted to West China Second University Hospital, after a serious lead pollution event in longchang, Sichuan, China. A total of 4 children with elevated blood lead levels (BLLs) and 4 children with low BLLs were randomly chosen in the discovery set, and 40 children with elevated BLLs and 40 children with low BLLs were included in the validation set. Leptin and interleukin-8 (IL-8) were identified to be significantly different between children with elevated and low BLLs via RayBio^® Human Cytokine Antibody Array. In the validation set, IL-8 was higher in children with elevated BLLs [median(P₂₅–P₇₅), 117.69(52.31–233.63) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 17.70(10.75–26.52) pg/mL] (p = 0.000). Leptin was lower in children with elevated BLLs [median(P₂₅–P₇₅): 1658.23(1421.86–2606.55) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 4168.68(3246.32–4744.94) pg/mL] (p = 0.000). In children with low BLLs, leptin was higher in children with BLLs < 3 μg/dL (N = 7) [median(P25–P75): 7220.86(4265.72–7555.15) pg/mL] than in children with BLL ≥ 3 μg/dL (N = 33) [median(P₂₅–P₇₅): 4103.86(3163.40–4678.34) pg/mL] (p = 0.026); IL-8 was significantly different in children with BLL < 4 μg/dL (N = 13) [median(P₂₅–P₇₅): 12.49(8.25–14.86) pg/mL] than in children with BLL ≥ 4 μg/dL (N = 27) [median(P₂₅–P₇₅): 21.98(13.64–33.50) pg/mL] (p = 0.013). The results defined specific changes in cytokine expressions to lead exposure, which can be used to explore the mechanism of lead toxicity and monitor lead exposure.     

Determination of nutlin-3a in murine plasma by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)

A sensitive and precise LC–ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10–20,000 ng/mL) or by liquid–liquid extraction (LLE) for the low concentration range (0.25–300 ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4 μm, 75 mm × 2 mm) with an isocratic mobile phase (acetonitrile/5 mM HCOONH₄ = 70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45 min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10–20,000 ng/mL (R² ≥ 0.993) and the LLE method was linear in a range of 0.25–300 ng/mL (R² ≥ 0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were ≤4.5% for PP and were ≤4.9% for LLE. Within-day and between-day accuracies (% error) ranged from 4.8 to −7.9 for PP, and from −0.2 to −8.4 for LLE. The two extraction methods produced equivalent results, allowing use of both within the same study. This method has been applied to the measurement of nutlin-3a concentrations in murine plasma samples obtained from a preclinical pharmacokinetic study.     

Investigation of the photostability properties of memoquin,a quinone derivative for the treatment of Alzheimer's disease

The photostability properties of memoquin, a multifunctional compound in preclinical development for the treatment of Alzheimer's disease (AD) were investigated in solutions exposed to radiations, using a xenon arc lamp to simulate the natural sunlight. Reversed phase liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC–UV/DAD–ESI-MS/MS) was applied to follow the photodegradation and disappearance of memoquin after irradiation. Under optimized chromatographic conditions, memoquin was separated with high resolution from the photoproducts formed in the photoexposed solutions. The results showed that memoquin is more stable at physiological and acid pHs, while it has a slow degradation pattern at more drastic conditions such as basic pH (t_1/2 = 389 min) and in methanolic solutions (t_1/2 = 465 min). In the irradiated solutions the appearance of photoproducts with lower retention times and molecular weight than memoquin was observed, thus indicating that some fragments were lost from its structure. The photodegradation products were characterized by LC–ESI-MS/MS and LC–UV/DAD analysis. The photoreactive centers were found on the amino groups of the side chains while the 1,4-benzoquinone functionality was maintained. Conversely, memoquin was found to be stable in the dark. These results suggest that, with appropriate handling and storage, memoquin's activity is not impaired.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

Quantitative determination of hippuric and benzoic acids in urine by LC–MS/MS using surrogate standards

An LC–MS/MS method for simultaneous determination of hippuric acid (HA) and benzoic acid (BA) in monkey urine after direct injection was developed. Since HA and BA are endogenous compounds in urine, surrogate standards (¹³C₆-hippuric and ¹³C₆-benzoic acid) were employed to generate calibration curves. l-Phenylalanine-ring-D5 served as an internal standard. Multiple reaction monitoring in the negative ionization mode with an APCI source was used for detection of all components in the assay. The developed method is intended for determination of HA and BA in the range of 0.25–250 and 0.1–100 μg/ml, respectively. Weighted (1/x) quadratic regression (r² > 0.99) was used to generate calibration curves. Precision and accuracy of the method were assessed by analyzing 3 quality control samples (concentrations at low, medium, and high range of calibration curve) prepared in monkey urine. Stability for 48 h at room temperature and after 3 freeze–thaw cycles was also evaluated.     

Spectrofluorimetric assessment of Ramipril using optical sensor Samarium ion–doxycycline complex doped in sol–gel matrix

A new, simple, sensitive and selective spectrofluorimetric method for the determination of Ramipril is developed. The Ramipril can remarkably quench the luminescence intensity of the Sm³⁺ ion in Sm³⁺–doxycycline complex at λ_ex = 375 nm in sol–gel matrix. In the same time the intensity of the emission band of the Ramipril in DMSO at 454 nm is increased due to the energy transfer from the Sm³⁺–doxycycline complex to Ramipril in the excited stated. The quenching of luminescence intensity of Sm³⁺–doxycycline complex doped in the sol–gel matrix and the enhancement of the emission band of Ramipril at 454 nm are directly proportion to the concentration of Ramipril with a dynamic ranges of 3.4 × 10⁻⁹–1.0 × 10⁻⁷ mol l⁻¹ and 2.4 × 10⁻⁹–1.0 × 10⁻⁷ mol l⁻¹ and detection limits of 6.0 × 10⁻¹⁰ and 5.2 × 10⁻¹⁰ mol l⁻¹, respectively.     

Identification of dual DNA-PK MDR1 inhibitors for the potentiation of cytotoxic drug activity

Inhibition of DNA repair is an attractive therapeutic approach to enhance the activity of DNA-damaging anticancer chemotherapeutic agents. Similarly, blockade of the multidrug-resistance protein 1 (MDR1) can overcome efflux-mediated resistance. DNA-dependent protein kinase (DNA-PK) is essential for the non-homologous end-joining DNA repair pathway. NU7441 is a potent DNA-PK inhibitor (IC₅₀ = 14 nM) that is used widely to study the effects of DNA-PK inhibition in vitro. In growth inhibition studies, 1 μM NU7441 sensitised vincristine-resistant CCRF-CEM VCR/R leukaemia cells (1200-fold resistant) to a range of MDR1 substrates, including doxorubicin (8-fold, p = 0.03), vincristine (14-fold, p = 0.01) and etoposide (63-fold, p = 0.02), compared with 1.4-fold (p = 0.02), 2.2-fold (p = 0.04) and 3.6-fold (p = 0.01) sensitisation, respectively, in parental CCRF-CEM cells. This difference in NU7441 sensitivity was confirmed in another two parental and MDR1-overexpressing cell line pairs. A doxorubicin fluorescence assay showed that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1 μM NU7441 increased doxorubicin nuclear fluorescence 16-fold. NU7441 and 3 structurally related compounds (NU7742 (an NU7441 analogue that does not inhibit DNA-PK – IC₅₀ > 10 μM), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative – IC₅₀ = 2 nM) and DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative - IC₅₀ = 7 μM)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC–MS. This paper demonstrates that NU7441 is a dual DNA-PK and MDR1 inhibitor, and this extends the therapeutic potential of the compound when used in combination with MDR substrates.     

Effects of γ-glutamyl linker on DPP-IV resistance, duration of action and biological efficacy of acylated glucagon-like peptide-1

Liraglutide, a GLP-1 mimetic has recently been approved for clinical use in obesity-diabetes. The purpose of this study was to assess if acylation of Liraglutide via its γ-glutamyl linker contributes to DPP-IV inhibition and efficacy of the molecule, given that such an approach could be useful in prolonging bioactivity of related peptides. Liraglutide lacking the γ-glutamyl linker (Lira-γGlu) and Liraglutide exhibited enhanced DPP-IV resistance with extension of t_1/2 plus effective cAMP production (EC₅₀: 0.15 ± 0.11 and 0.16 ± 0.11 nM, respectively) compared to GLP-1 (EC₅₀ 3.81 ± 0.80 nM). GLP-1, Lira-γGlu and Liraglutide increased insulin secretion compared to glucose (1.5-3.0-fold; p < 0.05 to p < 0.001). In vivo, Lira-γGlu and Liraglutide significantly lowered plasma glucose when administered 4 and 8 h prior to a glucose load (1.3-1.9-fold; p < 0.05 to p < 0.001). Twice-daily administration of Lira-γGlu and Liraglutide for 14 days significantly decreased food intake (1.2-fold; p < 0.05) and plasma glucose (1.1-1.6-fold; p < 0.05 to p < 0.01) whilst increasing plasma insulin (1.4-1.6-fold; p < 0.05). At 14 days, Lira-γGlu and Liraglutide markedly improved glucose tolerance (1.4-3.4-fold; p < 0.05 to p < 0.001), insulin response to glucose (1.4-1.5-fold; p < 0.05), insulin sensitivity (1.3-1.4-fold; p < 0.05 to p < 0.01), as well as increasing pancreatic insulin content (1.4-fold; p < 0.05). Functional characteristics of Lira-γGlu and Liraglutide are almost indistinguishable, questioning necessity of γ-glutamyl linker in acylation for generation of long-acting incretin mimetics.     

Solid surface spectroscopic methodology for ultra-trace urinary nickel monitoring in smokers and non-smokers’ subjects

Nickel chemical enrichment on nylon membranes previously treated with eosin (eo) is proposed for subsequent quantification by spectrofluorimetry (λ_em = 547 nm, λ_exc = 515 nm). Operational variables which have influence on quantitative metal retention have been studied. At optimal experimental conditions, quantitative recovery was reached (superior to 99%), with a detection limit of 0.13 ng L⁻¹ and quantification limit of 0.44 ng L⁻¹. The calibration sensitivity was of 6 × 10¹³ ng L⁻¹ for the new methodology with a linear range of 0.44–410 ng L⁻¹ Ni(II). The tolerance levels, respect to cations and anions as potential interferents, were studied, with good results. The methodology was validated by standard addition method and satisfactorily applied to urinary nickel determination of 50 subjects including smokers, second hand smokers and non-smokers’ samples without previous treatment. Stability of biological samples was daily studied for a period of 1 month. Within-day precision was better than 0.02 CV. The reproducibility (between-day precision) was also evaluated over 3 days by performing six determinations each day with a CV of 0.052. The different groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test with satisfactory results.     

Pharmacokinetics of prenylflavonoids and correlations with the dynamics of estrogen action in sera following ingestion of a standardized Epimedium extract

To explore pharmacokinetic properties of prenylflavonoids from the Traditional Chinese Medicinal plant Epimedium, three doses of a standardized extract (100, 300 and 600 mg/kg body weight), were administered to ovariectomized rats and serial blood samples were obtained. Serum concentrations of the Epimedium prenylflavonoids icariin, icariside I, icariside II, icaritin and desmethylicaritin were determined by LC–MS/MS. Aliquots of sera were also applied to human cell lines that permanently express ERα and ERβ proteins for the ex vivo measurement of estrogenic activity. All five prenylflavonoids exhibit non-linear dose-dependent increases in the area under concentration versus time curves. Two distinct pharmacokinetic patterns were evident, an early phase wherein icariin and icariside II reached t_max 0.5–1 h, and a late phase wherein icariside I, icaritin and desmethylicaritin peaked at t_max 8 h. Total concentrations of icaritin and desmethylicaritin reached C_max ∼2 μM and ∼0.25 μM respectively. Estrogenic activity in Epimedium-treated rat sera lagged by several hours compared to animals treated with control drug estradiol benzoate, corresponding to the appearance of bioactive metabolites desmethylicaritin, icaritin and icariside I. Following glucuronidase/sulphatase treatment, prolonged estrogenic activity at higher Epimedium doses (300 and 600 mg/kg of body weight) was evident, and correlated with the persistence of micromolar levels of icaritin at the 48–72 h sampling period. The depot effect resulted in time–concentration bioactivity profiles at the three Epimedium doses (area under curve 374, 543, and 771 pM E2 h⁻¹) that exceeded that observed for estradiol benzoate (148 pM E2 h⁻¹). Our study correlated the pharmacokinetics of prenylflavonoids with the dynamics of their estrogenic effects and reveals the potential estrogenicity of this Epimedium extract. This study may aid the development of prenylflavonoids as drugs for menopause and other conditions requiring estrogenic action.