Multiple spectroscopic studies on the interaction between olaquindox,a feed additive,and bovine serum albumin

The interaction between olaquindox (OLA) and bovine serum albumin (BSA) was investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy. The results showed that the fluorescence quenching of BSA by OLA was a static quenching process induced by the formation of OLA–BSA complex. The binding constant of OLA–BSA complex was calculated to be 1.299 × 10⁴ L mol⁻¹ (293 K). The negative values of ΔH⁰ and ΔS⁰ indicated that hydrogen bond and van der Waals interactions played major roles in stabilizing the complex. Site probe competition experiments and number of binding sites (n) revealed that OLA could bind to site I in subdomain IIA of BSA, and the binding distance (r) was evaluated to be 3.643 nm according to Förster’s non-radiative energy transfer theory. The results of CD and three-dimensional fluorescence spectra suggested some conformational changes of BSA after OLA binding.     

Study of curcumin and genistein interactions with human serum albumin

Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied polyphenolic compound which has a variety of biological activity as anti-inflammatory and antioxidative agent. Genistein one of the flavonoids found in soybean and chickpeas inhibits DNA strand breaks acting as a direct scavenger of reactive oxygen species. Human serum albumin (HSA) with high affinity binding sites is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interactions of curcumin and genistein with human serum albumin at physiological conditions, using constant protein concentration and various pigment contents. FTIR, UV–Visible, CD and fluorescence spectroscopic methods were used to analyse drug binding mode, the binding constant and the effects of pigment complexation on HSA stability and conformation. Structural analysis showed that curcumin and genistein bind HSA via polypeptide polar groups with overall binding constants of K_curcumin = 5.5 (±0.8) × 10⁴ M⁻¹ and K_genistein = 2.4 (±0.40) × 10⁴ M⁻¹. The number of bound pigment (n) is 1.33 for curcumin and 1.49 for genistein. The HSA conformation was altered by pigment complexation with reduction of α-helix and increase of random coil and turn structures suggesting a partial protein unfolding.     

Preferential binding of insecticide phorate with sub-domain IIA of human serum albumin induces protein damage and its toxicological significance

Phorate, an organophosphorus insecticide is known for its adverse effects on acetylcholinesterase, and other neuronal and pulmonary activities. Most likely, the toxicity of drugs/agrochemicals is modulated through cellular distribution bound to plasma proteins. Therefore, the in vitro interaction of phorate with human serum albumin (HSA) has been investigated, using sensitive techniques like fluorescence spectroscopy and circular dichroism, to ascertain its binding mechanism and toxicological implications. Fluorescence studies revealed the quenching constant (Ksv) as 2.5 × 10⁴ M⁻¹ and binding affinity (Ka) as 2.96 × 10⁴ M⁻¹ (r² = 0.99), with a primary binding site of phorate at sub-domain IIA of HSA. Circular dichroism (CD) data demonstrated a noticeable reduction in secondary structure (α-helical content) of phorate treated HSA. Albumin treated with 200-1000 μM phorate released significant amounts of acid soluble amino and carbonyl groups, whereas higher concentrations resulted in protein fragmentation. It is postulated that the 1′-O and 3-O alkyl groups of phorate have a role in binding with electrophilic centers of Trp 214, and Arg 218/Lys 195, respectively. Moreover, the significant ultrastructural changes, reactive oxygen species (ROS) generation, mitochondrial damage and cell death in phorate treated cultured human amnion epithelial (WISH) cells, elucidated phorate induced cellular toxicity.     

Characterization of interaction and the effect of carbamazepine on the structure of human serum albumin

The binding of carbamazepine (CBZ) to human serum albumin (HSA) was investigated under simulative physiological conditions. In this study, intrinsic fluorescence of tryptophan-214 in HSA was monitored upon the addition of CBZ. Binding constant of CBZ–HSA was calculated by the remarkable static quenching effect of CBZ and found to be (2.081 ± 0.023) × 10⁴ M⁻¹. The fluorimetric results revealed that the hydrophobic interaction was a predominant intermolecular force for stabilizing the complex, which is also in agreement with the results obtained from voltammetric approach. Three site probes, warfarin, ibuprofen and digitoxin, were employed in fluorescence displacement experiments to locate the exact binding site for CBZ in HSA. The alteration in secondary structure of protein in the presence of CBZ was confirmed by the evidences from circular dichroism and FT-IR spectroscopy. Further, the distance r between donor (Trp-214) and acceptor (CBZ) was obtained according to fluorescence resonance energy transfer (FRET).     

Interaction of kaempferol with human serum albumin: A fluorescence and circular dichroism study

The interaction of kaempferol (kaemp), a natural flavonoid to which antioxidative, anti-inflammatory and cardio-protective biological activities have been attributed, with human serum albumin (HSA), the main in vivo transporter of exogenous substances, was investigated by steady-state, synchronous fluorescence and circular dichroism spectroscopies. The binding constant, K, and number of binding sites, n, were computed using literature models that showed satisfactory agreement and revealed a strong interaction (K ∼ 3.5 × 10⁵ M⁻¹, n ∼ 1). The binding process was investigated at temperatures in the range 298–313 K, allowing for the evaluation of the thermodynamic parameters, which indicate the occurrence of hydrogen bonding interactions. The distance between kaemp and the tryptophan residue of HSA was estimated at 2.7 nm using Förster's theory of nonradiative resonance energy transfer. Using circular dichroism we evidenced some degree of HSA defolding upon binding.     

On-line molecular imprinted solid-phase extraction flow-injection fluorescence sensor for determination of florfenicol in animal tissues

A simple and convenient on-line molecular imprinted solid-phase extraction flow-injection fluorescence sensor was developed in this study. Florfenicol (FF)-imprinted polymer was prepared by self-assembly with acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The binding characteristics of the imprinted polymer to florfenicol were evaluated by equilibrium binding experiments, and the morphology of the imprinted polymer was studied by scanning electron microscope (SEM). Fluorescence intensity of 3-p-nitrylphenyl-5-(2′-sulfonophenylazo) rhodanine (4NRASP) and bovine serum albumin (BSA) was inhibited by FF. Under optimum conditions the intensity of fluorescence shows a linear relationship with the concentration of FF over the range of 1.2 × 10⁻⁶ to 2.6 × 10⁻⁵ g mL⁻¹ with a regression equation of ΔI = 23.54 + 17.86c (c 10⁻⁶ g mL⁻¹) (r = 0.9965, n = 6). The low detection limit of FF was found to be 3.4 × 10⁻⁷ g mL⁻¹ according to 11 parallel determinations for the blank solution. The relative standard deviation for the determination of 2.0 × 10⁻⁶ g mL⁻¹ FF solution was 3.5% (n = 11). This proposed sensor could be satisfactorily applied to the determination of FF in liver and meat samples.     

Probing the binding of morin to human serum albumin by optical spectroscopy

Morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one], a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. Its binding to human serum albumin was investigated by fluorescence quenching, fluorescence anisotropy, and UV–vis absorbance under the simulative physiological condition. Fluorescence quenching data show that the interaction of morin with HSA forms a non-fluorescent complex with the binding constants of 1.394 × 10⁵, 1.489 × 10⁵, 1.609 × 10⁵ and 1.717 × 10⁵ M⁻¹ at 292, 298, 303 and 310 K, respectively. The thermodynamics parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 8.97 kJ mol⁻¹ and 129.15 J mol⁻¹ K⁻¹ via van’t Hoff equation. From the spectroscopic results and thermodynamics parameters, it is observed that van der Waals and hydrogen bonds are predominant intermolecular forces when forming the complex. The distance r = 4.25 nm between donor (Trp214) and accepter (morin) was estimated based on the Förster theory of non-radiative energy transfer. The red shift of UV–vis absorbance shows that morin is bound to several amino acids on the hydrophobic pocket of HSA. Moreover, the competitive probes, such as warfarin and ibuprofen (site I and II probes, respectively), reveal that the binding location of morin to HSA in the site I of the hydrophobic pocket, which corresponds to the results of UV–vis absorbance, while morin also binds other lower affinity binding sites on HSA from the fluorescence anisotropy spectroscopy.     

Molecular spectroscopic studies on the interaction between Ractopamine and bovine serum albumin

To investigate the interaction between Ractopamine (RAC), an animal growth promoter, and bovine serum albumin (BSA), three spectroscopic approaches (fluorescence, UV–vis and FT-IR) and three different experiments (two mole-ratio and a Job's methods) were used to monitor the biological kinetic interaction procedure. The Stern–Volmer quenching constants K_SV, the binding constants K_a, and the number of binding sites n at 298, 301 and 304 K were evaluated by molecular spectroscopic approaches. The values of enthalpy (−13.47 kJ mol⁻¹) and entropy (78.39 J mol⁻¹ K⁻¹) in the reaction indicated that RAC bound to BSA mainly by electrostatic and hydrophobic interaction. The site markers competitive experiments indicated that the binding of RAC to BSA primarily took place in site I. The spectra data matrix was further investigated with multivariate curve resolution-alternating least squares (MCR-ALS), and the concentration profiles and the pure spectra for three species (BSA, RAC and RAC-BSA) existed in the kinetic interaction procedure, as well as the apparent equilibrium constants, were obtained.     

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.     

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-d-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC₅₀ around 1 μM at −70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca²⁺ responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells.Memantine was confirmed to be a moderate affinity (IC₅₀ at −70 mV of 0.79 ± 0.02 μM, Hill = 0.92 ± 0.02), strongly voltage-dependent (δ = 0.90 ± 0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 μM - onset τ_fast = 273 ± 25 ms (weight 69%), onset τ_slow = 2756 ± 296 ms, offset τ_fast = 415 ± 82 ms (weight 38%) offset τ_slow = 5107 ± 1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC₅₀ at −70 mV of 0.71 ± 0.03 μM, Hill = 0.84 ± 0.02) but somewhat less pronounced voltage-dependency (δ = 0.79 ± 0.04), slower, single exponential kinetics (ketamine: k_on = 0.15 ± 0.05 × 10⁶ M⁻¹ s⁻¹, k_off = 0.22 ± 0.05 s⁻¹ c.f. memantine following normalization k_on = 0.32 ± 0.11 × 10⁶  M⁻¹ s⁻¹, k_off = 0.53 ± 0.10 s⁻¹) and was fully trapped.The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.     

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.     

Standardized UV–vis spectra as the foundation for a threshold-based,integrated photosafety evaluation

Phototoxicity is a relatively common phenomenon and is an adverse effect of some systemic drugs. The fundamental initial step of photochemical reactivity is absorption of a photon; however, little guidance has been provided thus far regarding how ultraviolet–visible (UV–vis) light absorption spectra may be used to inform testing strategies for investigational drugs. Here we report the results of an inter-laboratory study comparing the data from harmonized UV–vis light absorption spectra obtained in methanol with data from the in vitro 3T3 Neutral Red Uptake Phototoxicity Test. Six pharmaceutical companies submitted data according to predefined quality criteria for 76 compounds covering a wide range of chemical classes showing a diverse but “positive”-enhanced distribution of photo irritation factors (22%: PIF < 2, 12%: PIF 2–5, 66%: PIF > 5). For compounds being formally positive (PIF value above 5) the lowest reported molar extinction coefficient (MEC) was 1700 L mol⁻¹ cm⁻¹ in methanol. However, the majority of these formally positive compounds showed MEC values being significantly higher (up to almost 40,000 L mol⁻¹ cm⁻¹).     

Disposition of smoked cannabis with high Δ-tetrahydrocannabinol content: A kinetic model

Introduction

No model exists to describe the disposition and kinetics of inhaled cannabis containing a high THC dose. We aimed to develop a kinetic model providing estimates of the THC serum concentrations after smoking cannabis cigarettes containing high THC doses (up to 69 mg THC).

Methods

Twenty-four male non-daily cannabis users smoked cannabis cigarettes containing 29.3 mg, 49.1 mg, and 69.4 mg THC. Blood samples were collected over a period of 0-8 h and serum THC concentrations were measured. A two-compartment open model was fitted on the individual observed data.

Results

Large inter-individual variability was observed in the pharmacokinetic parameters. The median pharmacokinetic parameters generated by the model were C_max = 175 ng/mL, T_max = 14 min, and AUC_0-8h = 8150 ng × min/mL for the 69.4 mg THC dose. Median model results show an almost linear dose response relation for C_max/Dose = 2.8 × 10^− 6/mL and AUC_0-8h/Dose = 136 × 10^− 6 min/mL. However, for increasing dose level, there was a clear decreasing trend: C_max/Dose = 3.4, 2.6 and 2.5 × 10^− 6/mL and AUC_0-8h/Dose = 157, 133 and 117 × 10^− 6 min/mL for the 29.3, 49.1 and 69.4 mg dose, respectively. Within the restriction of 8 h of observation, the apparent terminal half life of THC was 150 min.

Conclusion

The model offers insight into the pharmacokinetics of THC in recreational cannabis users smoking cannabis containing high doses of THC mixed with tobacco. The model is an objective method for providing serum THC concentrations up to 8 h after smoking cannabis with a high THC content (up to 23%).     

Thermodynamics of A2B adenosine receptor binding discriminates agonistic from antagonistic behaviour

Thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of 12 ligands (six agonists and six antagonists) to the A_2B adenosine receptor subtype have been determined by affinity measurements carried out on HEK 293 cells stably transfected with human A_2B adenosine receptors at six different temperatures (4, 10, 15, 20, 25, 30 °C) and van’t Hoff plot analysis have been performed. Affinity constants were obtained from saturation experiments of [³H]MRE 2029-F20 or by its displacement in inhibition assays for the other compounds. van’t Hoff plots were essentially linear in the temperature range investigated, showing that the ΔCp° of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 7 ≤ ΔH° ≤ 23 kJ mol⁻¹and 123 ≤ ΔS° ≤ 219 J K⁻¹ mol⁻¹ for agonists and −40 ≤ ΔH° ≤ −20 kJ mol⁻¹ and 10 ≤ ΔS° ≤ 91 J K⁻¹ mol⁻¹ for antagonists indicating that agonistic binding is always totally entropy-driven while antagonistic binding is enthalpy and entropy-driven. In the −TΔS° versus ΔH° plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated.     

Electrochemical DNA nano-biosensor for the study of spermidine–DNA interaction

An electrochemical DNA nano-biosensor is prepared by immobilization of double stranded DNA (dsDNA) onto a mixed self-assembled monolayer (SAM) composed of azide- and hydroxyl-terminated thiols. The SAMs- and dsDNA-modified gold electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of spermidine with DNA was studied by differential pulse voltammetry at the DNA-modified electrode. The decrease in the guanine oxidation peak current was used to study the interaction. The binding constant (K), obtained by differential pulse voltammetry, was 1.85 × 10⁵ M⁻¹. A linear dependence of the guanine peak currents was observed in the range of 1.6–70.4 μM spermidine, with a detection limit of 0.72 μM and r = 0.994 by using differential pulse voltammetry.     

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0 = control, 5 × 10⁻⁸, 10⁻⁷, 2 × 10⁻⁷ or 5 × 10⁻⁷ M) or diisopropylphosphorofluoridate (DFP, 0 = control, 5 × 10⁻⁶, 10⁻⁵, 2 × 10⁻⁵, and 5 × 10⁻⁵ M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 μM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 μM concentrations. No fullerene derivative at 1 μM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.     

Controlled non-invasive transdermal iontophoretic delivery of zolmitriptan hydrochloride in vitro and in vivo

The objective was to investigate the transdermal delivery kinetics of zolmitriptan from an iontophoretic patch system in Yorkshire swine in vivo. Preliminary in vitro experiments showed that cumulative drug transport during a 6-h current application (0.25 mA cm⁻²) was independent of patch load (263.7 ± 92.7, 357.2 ± 85.9, 374.9 ± 74.3 and 335.9 ± 27.7 μg cm⁻² for 7.5, 15, 45 and 90 mg patch loads, respectively; ANOVA, p < 0.05); the steady-state flux was ∼92 μg cm⁻² h⁻¹. The in vivo studies used multistep current profiles to demonstrate (i) rapid drug uptake and (ii) the effect of superposing a bolus input on basal drug levels. In both studies, zolmitriptan was detected in the blood after 2.5 min; drug levels were 7.1  1.7 and 10.4 ± 3.5 ng ml⁻¹ at t = 30min in Studies 1 and 2, respectively. In Study 2, increasing current intensity from 0.2 to 1.4 mA (0.05-0.35 mA cm⁻²) at t = 180 min caused zolmitriptan levels to rise from 9.38 ± 0.93 ng ml⁻¹ at t = 180 min to 13.57 ± 1.85 ng ml⁻¹ at t = 190 min; a ∼50% increase in 10 min. Extrapolation of these results to humans suggests the feasibility of delivering therapeutic amounts of zolmitriptan at faster rates than those from existing dosage forms.     

Improved photostability and reduced skin permeation of tretinoin: Development of a semisolid nanomedicine

The aims of this work were to increase the photostability and to reduce the skin permeation of tretinoin through nanoencapsulation. Tretinoin is widely used in the topical treatment of various dermatological diseases such as acne, psoriasis, skin cancer, and photoaging. Tretinoin-loaded lipid-core polymeric nanocapsules were prepared by interfacial deposition of a preformed polymer. Carbopol hydrogels containing nanoencapsulated tretinoin presented a pH value of 6.08 ± 0.14, a drug content of 0.52 ± 0.01 mg g⁻¹, pseudoplastic rheological behavior, and higher spreadability than a marketed formulation. Hydrogels containing nanoencapsulated tretinoin demonstrated a lower photodegradation (24.17 ± 3.49%) than the formulation containing the non-encapsulated drug (68.64 ± 2.92%) after 8 h of ultraviolet A irradiation. The half-life of the former was seven times higher than the latter. There was a decrease in the skin permeability coefficient of the drug by nanoencapsulation, independently of the dosage form. The liquid suspension and the semisolid form provided K_p = 0.31 ± 0.15 and K_p = 0.33 ± 0.01 cm s⁻¹, respectively (p ? 0.05), while the samples containing non-encapsulated tretinoin showed K_p = 1.80 ± 0.27 and K_p = 0.73 ± 0.12 cm s⁻¹ for tretinoin solution and hydrogel, respectively. Lag time was increased two times by nanoencapsulation, meaning that the drug is retained for a longer time on the skin surface.     

A fluorescence-based in vitro assay for drug interactions with breast cancer resistance protein (BCRP, ABCG2)

Purpose: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein BCRP (ABCG2). Methods: BCRP expression was verified by immunostaining and Western blots in intact porcine brain capillaries, isolated endothelial cells (PBCECs) and in MDCKII-cells over-expressing human wild-type BCRP (MDCKII-hBCRP). Transport of fluorescent mitoxantrone across cells was determined to assess a preferred transport direction. Sensitivity of cultured cells versus mitoxantrone in the absence and in the presence of transport modulators was examined at increasing concentrations of the cytostatic using the AlamarBlue™ assay. In addition, cells were incubated with mitoxantrone in the absence and presence of increasing concentrations of different compounds with the potential to interact with BCRP. Intracellular fluorescence accumulation was measured using a flow cytometer. Results: Isolated capillaries as well as 7-day old PBCECs showed expression of BCRP. Cell sensitivity to mitoxantrone significantly increased in the presence of the BCRP inhibitors KO143 and GF120918. Transport of mitoxantrone across PBCEC monolayers was directed with P_app (apical to basolateral) 5.6 × 10⁻⁶ cm s⁻¹ and with P_app (basolateral to apical) 2.8 × 10⁻⁵ cm s⁻¹. FACS analysis revealed a different extent of fluorescence accumulation dependent on the kind and concentration of BCRP modulating compounds. Conclusions: The mitoxantrone-based assay can be used as a rapid FACS screening system to assess drug interactions with BCRP at the blood-brain barrier and therefore represents a useful tool in drug profiling.     

Atropine maintenance dosage in patients with severe organophosphate pesticide poisoning

Although the importance of atropine in therapy of organophosphate (OP) poisoning is generally recognized, its dosing is a matter of debate. A retrospective analysis of atropine dosing was undertaken in 34 patients who had been enrolled in a clinical study assessing obidoxime effectiveness in OP-poisoning. All patients were severely intoxicated (suicidal attempts) and required artificial ventilation. Atropine was administered routinely by intensive care physicians for life-threatening muscarinic symptoms, with the recommendation to favor low dosage. The pharmacological active enantiomere S-hyoscyamine was determined by a radioreceptor assay.When RBC-AChE activity ranged between 10% and 30%, S-hyoscyamine plasma concentrations of approx. 5 nmol L⁻¹ were sufficient. This concentration could be maintained with about 0.005 mg h⁻¹ kg⁻¹ atropine. Only when RBC-AChE was completely inhibited, therapy of cholinergic crisis required atropine doses up to 0.06 mg h⁻¹ kg⁻¹. Elimination half-life of S-hyoscyamine was 1.5 h, showing occasionally a second slow elimination phase with t_1/2 = 12 h. Malignant arrhythmias were observed in some 10% of our cases, which occurred late and often in the absence of relevant glandular cholinergic signs, when the S-hyoscyamine concentration was below 2.5 nmol L⁻¹. Arrhythmias mostly resolved on reinstitution of atropine.