A novel Ca<Superscript>2+</Superscript>-dependent phospholipase D from <Emphasis Type="Italic">Streptomyces tendae</Emphasis>, possessing only hydrolytic activity

An extracellular phospholipase D (PLD _St ) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD _St was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60°C, and the enzyme was stable at or below 60°C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca²⁺, and the maximum activity was at 2 mM CaCl₂. The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 μmol min⁻¹ mg⁻¹, respectively. More importantly, PLD _St could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD _St does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD _St may therefore be a novel type of PLD enzyme.     

Effect of nitroxyalkyl derivatives of fullerenylproline on the activity of CA<Superscript>3+</Superscript>-ATPase of sarcoplasmic reticulum

The effect of nitroxyalkyl derivatives of fullerenylproline methyl ester on the enzymatic activity of Ca²⁺-ATPase of sarcoplasmic reticulum (SR) has been studied. It is shown that hybrid derivatives of C₆₀ fullerene are capable of inhibiting the activity of Ca²⁺-ATPase of SR. The mononitrate inhibits the hydrolytic activity of the enzyme with K _i = 1.92 × 10⁻⁶ M; active Ca²⁺ transport, with K _i = 3.79 × 10⁻⁶ M. The dinitrate inhibits ATP hydrolysis with K _i = 2.38 × 10⁻⁸ M; Ca²⁺ transport, with K _i  = 3.08 × 10⁻⁸ M. Fullerenylproline methyl ester does not affect the enzymatic activity of Ca²⁺-ATPase. Based on these data it is possible to predict the possible fields of application for hybrid fullerene derivatives as potential drugs.     

Suitability of <Emphasis Type="Italic">Daphnia similis</Emphasis> as an alternative organism in ecotoxicological tests: implications for metal toxicity

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 × 10⁵ and 1 × 10⁶ cells ml⁻¹ on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL⁺ showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC₅₀ determination. LC₅₀ for D. similis was 5.15 × 10⁻⁷ mol l⁻¹ dissolved Cd without algal cells, whereas with 1 × 10⁵ cells ml⁻¹, it was significantly higher (7.15 × 10⁻⁷ mol l⁻¹ dissolved Cd). For Cr, the 48 h-LC₅₀ value of 9.17 × 10⁻⁷ mol l⁻¹ obtained for the cladoceran in tests with 1 × 10⁶ cells ml⁻¹ of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 × 10⁻⁷ mol l⁻¹ dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.     

The combined effect of copper and low pH on antioxidant defenses and biochemical parameters in neotropical fish pacu, <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis> (Holmberg, 1887)

Copper sulfate is widely used in aquaculture. Exposure to this compound can be harmful to fish, resulting in oxidative metabolism alterations and gill tissue damage. Pacu, Piaractus mesopotamicus, (wt = 43.4 ± 3.35 g) were distributed in experimental tanks (n = 10; 180 l) and exposed for 48 h to control (without copper addition), 0.4Cu (0.4 mg l⁻¹), 0CupH (without copper addition, pH = 5.0) and 0.4CupH (0.4 mg l⁻¹, pH = 5.0). In liver and red muscle, the superoxide dismutase (SOD) was responsive to the increases in the aquatic copper. The plasmatic intermediary metabolites and hematological variables in the fish of group 0.4Cu were similar to those of the control group. Conversely, the exposure to 0.4CupH caused an increase in the plasmatic lactate, number of red blood cells (RBC) and hemoglobin (Hb). Plasmatic copper concentration [Cu_p] increased in group 0.4Cu and 0.4CupH, which is higher in group 0.4CupH, suggests an effect of water pH on the absorbed copper. Exposure to 0.4Cu and 0.4CupH resulted in a reduction in the Na⁺/K⁺-ATPase activity and an increase in metallothionein (MT) in the gills. Exposure to 0CupH caused a decrease in glucose and pyruvate concentrations and an increase in RBC, Hb, and the branchial Na⁺/K⁺-ATPase activity. These responses suggest that the fish triggered mechanisms to revert the blood acidosis, save energy and increase the oxygen uptake. MT was an effective biomarker, responding to copper in different pHs and dissolved oxygen. Combined-factors caused more significant disturbance in the biomarkers than single-factors.     

Effects of inorganic lead in vitro on ion exchanges and respiratory metabolism of rat kidney cortex

The effects of Pb²⁺ added in vitro to tissue slices, isolated tubules and isolated mitochondria of rat kidney cortex have been studied. Slices were depleted of K⁺ and loaded with Na⁺, Cl⁻ and water by pre-incubation at 1° C, and reversal of these changes was then induced by incubation under metabolically favourable conditions. The net reaccumulation of K⁺ was reduced by a maximum of 30% when Pb²⁺ was present in the medium, the maximal effect being caused by 200 μM Pb²⁺. Lead also caused a reduction of Na⁺ extrusion which was approximately equimolar with its effect on K⁺, but it did not affect the extrusion of Cl⁻ and water. The initial rates of the net, active movements of K⁺ and Na⁺ were not altered by Pb²⁺, divergence from control values only being noted after 15–30 min incubation. The O₂ consumption and the ATP content were 25–30% lower in slices incubated with 200 μM Pb⁺ than in control slices; the effect on ATP content was not observed until incubation had continued for 30 min. In tubules isolated from the renal cortex, the rate of respiration (50%) and ATP content (30%) were also partly reduced by 200 μM Pb²⁺. The consumption of O₂ by mitochondria isolated from the cortex was much more sensitive to Pb²⁺ added in vitro than the respiration of intact cells; the rate of respiration in state 3 (presence of phosphate acceptor) and the respiratory control ratio were drastically reduced, with half-maximal inhibition at 30 and 20 μM Pb²⁺ respectively. Comparison of the effects of Pb²⁺ on energy metabolism and ion transport of the slices with the corresponding effects of antimycin A and ouabain suggests that Pb²⁺ inhibited K⁺ and Na⁺ transport mainly as a consequence of a primary inhibition of the provision of ATP.     

Effect of Ions on Agitation- and Temperature-Induced Aggregation Reactions of Antibodies

Purpose  The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies. Methods  Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG₂ mAbs. The C_H2 domain melting temperature (T _m1) and reduced valence (z*) of the mAbs were measured. Results  Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F⁻ < Cl⁻ < Br⁻ < I⁻ < SCN⁻ ~ ClO₄ ⁻) and correlated with a decrease in T _m1 and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li⁺, Na⁺, K⁺, Rb⁺, or Cs⁺) had no effect on T _m1, z* or aggregation upon agitation. Conclusions  The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution. R. Matthew Fesinmeyer and Sabine Hogan have contributed equally to this work.     

Lack of pharmacokinetic interaction between ropinirole and theophylline in patients with Parkinson's disease

Objective: Ropinirole and theophylline have the potential to interact, because they use the same hepatic cytochrome P450 (CYP1A2) as their major metabolic pathway. The present study investigated the effect of steady-state oral theophylline on the pharmacokinetics of ropinirole at steady state and the effect of steady-state ropinirole on the pharmacokinetics of a single intravenous (i.v.) dose of theophylline, both in patients with idiopathic Parkinson's disease (PD). Methods: Pharmacokinetic parameters (AUC and C_max) for i.v. theophylline were compared before and after a 4-week period of oral treatment with ropinirole (2 mg t.i.d.) in 12 patients with PD. Patients were then maintained at this dose of ropinirole, and oral theophylline was co-administered at doses of up to 300 mg b.i.d. The parameters AUC, C_max and t_max for ropinirole were compared before, during and after oral theophylline co-treatment. Results: Co-administration of ropinirole did not significantly change the pharmacokinetics of i.v. theophylline (mean AUC with and without ropinirole: 68.6 μg · h⁻¹ · ml⁻¹ and 70.0 μ· h⁻¹ · ml⁻¹, respectively; mean C_max with and without ropinirole: 11.07 μ g · ml⁻¹ and 11.83 μg · ml⁻¹, respectively). Similarly, there were no significant changes in ropinirole pharmacokinetics when the drug was co-administered with oral theophylline (mean AUC for ropinirole with and without theophylline: 21.91 ng · h⁻¹ · ml⁻¹ and 22.09 ng · h⁻¹ · ml⁻¹, respectively; mean C_max for ropinirole with and without theophylline: 5.65 ng · ml⁻¹ and 5.54 ng · ml⁻¹, respectively; median t_max for ropinirole with and without theophylline: 2.0 h and 1.5 h, respectively). Conclusion: These results suggest a lack of significant pharmacokinetic interaction between the two drugs at current therapeutic doses. Received: 10 August 1998 / Accepted in revised form: 27 January 1999     

Effects of flavoxate hydrochloride on voltage-dependent Ba<Superscript>2+</Superscript> currents in human detrusor myocytes at different experimental temperatures

The inhibitory effects of flavoxate hydrochloride (piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba²⁺ currents (I _Ba) in human detrusor myocytes were investigated at different temperatures using conventional whole-cell patch-clamp techniques. When the bath-solution temperature was increased from 22°C to 30°C, I _Ba peak amplitude was enhanced by approximately twice at several test potentials. Neither the I _Ba threshold nor the membrane potentials for the I _Ba maximum peak amplitude was affected by the temperature change. The concentration-response curves of flavoxate at both 30°C (K _i  = 5.1 μM) and 37°C (K _i  = 4.6 μM) were slightly shifted to the left in comparison with that at 22°C (K _i  = 10.3 μM). Similar results were also obtained in the presence of nifedipine (K _i  = 14 nM at 22°C vs. K _i  = 2.5 nM at 30°C and K _i  = 2.1 nM at 37°C). Altering the bath-solution temperature from 22°C to 30°C shifted the steady-state inactivation curve of I _Ba at −90 mV to the left. At 30°C, the steady-state inactivation curve of I _Ba in the presence of flavoxate was also shifted to the left in comparison with that in the absence of flavoxate. Either 3-isobutyl-1-methylxanthine (IBMX) or theophylline, a phosphodiesterase inhibitor, caused little effects on I _Ba, although cyclic nucleotides (dibutyryl cAMP and 8-Br-cGMP) inhibited I _Ba. These results suggest that the inhibitory actions of flavoxate on I _Ba in human detrusor myocytes were slightly changed at different experimental temperatures and that flavoxate directly blocked voltage-dependent L-type Ca²⁺ channels, not through the inhibition of phosphodiesterase activity pathway.     

Miniaturized Rotating Disk Intrinsic Dissolution Rate Measurement: Effects of Buffer Capacity in Comparisons to Traditional Wood’s Apparatus

Purpose  The objective was to investigate the feasibility of using a miniaturized disk intrinsic dissolution rate (IDR) apparatus to determine the Biopharmaceutics Classification System (BCS) solubility class, and to develop an approach where IDR measurements performed in media of different buffer capacity could be compared. Methods  The disk IDR values of 14 model drugs were determined at 37°C in US Pharmacopeia buffers at pH 1.2, 4.5, and 6.8. As little as 5 mg of drug were compressed in a die, with surface area of 0.071 cm², with the die assembly rotated at 100 rpm in 10 mL media. Drug concentration was measured by an in situ fiber optic ultraviolet method. The solubilities and pK_as were determined, and used to simulate dissolution profiles with a convective-diffusion-with-chemical-reaction model. Results  The disk IDR values spanned six orders of magnitude (0.00014 to 114 mg min⁻¹ cm⁻²). The comparison of the miniaturized disk IDR values to published results using traditional dissolution bath apparatus indicated r ² = 0.99. Conclusions  The results demonstrate that using 100-fold less drug does not sacrifice the quality of the measurement, and lends support to an earlier study Yu et al. (Int. J. Pharm. 270:221–227, 2004) that the disk IDR measurement may possibly serve as a surrogate for the BCS solubility classification. Part 4 in the API-Sparing Dissolution Method series from pION. Berger et al. (28) is Part 3 in the series.     

Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells

Purpose Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB. Materials and Methods Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB. Results MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K _m of 6.5 ± 1.1 mM and V _max of 340 ± 60 nmol mg⁻¹min⁻¹; GHB: K _m of 2.07 ± 0.79 mM, V _max of 27.6 ± 9.3 nmol mg⁻¹min⁻¹, and a diffusional clearance of 0.54 ± 0.15 μl mg⁻¹min⁻¹), but not sodium-dependence. α-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K _i) of 0.28 ± 0.1 mM, and 0.19 ± 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4. Conclusion These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.     

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation

Objective: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. Results: In human liver microsomes (n=7), desmethylnaproxen formation was observed with a mean k_M of 92 (21) μmol · l⁻¹, V_max of 538 pmol · min⁻¹ · mg⁻¹ protein and C_int2 (reflective of a second binding site) of 0.36 μl · min⁻¹ · mg⁻¹ protein. This C_int2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this␣reaction by sulfaphenazole, (apparent Ki= 1.6 μmol · l⁻¹), warfarin (apparent Ki=27 μmol · l⁻¹), piroxicam (apparent Ki=23 μmol · l⁻¹) and tolbutamide (apparent Ki=128 μmol · l⁻¹). No effect was observed when α-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 μmol · l⁻¹, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The k_M and V_max for P450 1A2 were 189.5 μmol · l⁻¹ and 7.3 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Likewise, estimates of k_M and V_max for P450 2C9 were 340.5 μmol · l⁻¹ and 41.4 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Reliable estimates of k_M and V_max could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. Conclusion: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form. Received: 16 September 1996 / Accepted in revised form: 20 December 1996     

Acute and chronic effects of sodium and potassium on the tropical freshwater cladoceran <Emphasis Type="Italic">Pseudosida ramosa</Emphasis>

In this study, the toxicities of sodium and potassium to the tropical freshwater cladoceran Pseudosida ramosa were assessed. Acute toxicity tests on this species showed that the 48-h LC₅₀ of Na⁺ was 556 mg l⁻¹, while that of K⁺ was 17.7 mg l⁻¹. Long-term exposure of female P. ramosa to sodium reduced the total number of survivors from 10 to 6 at a concentration of 249 mg l⁻¹, 21-day fecundity from 20.4 to 14.3 eggs female⁻¹ at concentrations ranging from 72 to 249 mg l⁻¹, 21-day fertility from 20.1 to 6.5 neonates female⁻¹ at concentrations ranging from 25 to 249 mg l⁻¹. Furthermore, fecundity of each brood from the second to the fifth was significantly lower at 249 mg l⁻¹ and fertility of each brood from the first to the fifth at concentrations ranging from 25 to 249 mg l⁻¹. A significant decrease in fertility was associated with an increase in the number of aborted eggs. Long-term exposure to potassium decreased the 21-day fecundity of P. ramosa from 14.2 to 10.8 eggs female⁻¹ at a concentration of 11 mg l⁻¹ and fertility (fourth brood only) at 6.2 and 11 mg l⁻¹. Tropical reservoirs located near areas where the soil is overloaded with fertilizers and ferti-irrigation with vinasse already show concentrations of Na⁺ and K⁺ very close to those producing sub-lethal long-term effects on P. ramosa. A possible consequence is that organisms of the aquatic biota cannot adapt and freshwater taxa may become locally extinct, transferring dominance to salt-tolerant taxa.     

Temperature dependency of the release and bioavailability of nicotine from a nicotine vapour inhaler; in vitro/ in vivo correlation

Objective: To investigate the temperature dependency of the dose released and the plasma levels of nicotine from a vapour inhaler. Methods: In an open, randomised, three-way cross-over pharmacokinetic study 18 healthy subjects inhaled nicotine for 20 min (80 inhalations) every hour for 10 h (11 administrations) at three different environmental temperatures: 20°, 30° and 40 °C. In the in vitroexperiment, 5, 10, 15 and 20 l air were forced through the inhaler. With a 15 l air volume, the average amount of nicotine released was 1.44, 3.49, 4.80 and 6.99 mg at 10 °C, 22 °C, 29 °C and 40 °C, respectively. The maximum dose released at the highest temperature (40 °C) and the largest air volume investigated (20 l) was approximately 7.5 mg. Results: In vivo peak plasma levels obtained at 30° and 40 °C were 29.7 and 34.0 ng · ml⁻¹, compared with 22.5 ng · ml⁻¹ at ambient room temperature (20 °C). At 20 °C, the area under the plasma concentration–time curve (AUC) of the last dosing interval was 20.5 ng · ml⁻¹ · h. At 30 °C and 40 °C, the AUCs were 26.5 and 30.3 ng · ml⁻¹ · h, respectively. The results thus showed a mean increase of the in vivo AUC by 29% at 30 °C and by 48% at 40 °C compared with the AUC at 20 °C. These increases should be compared to the in vitro results, showing a mean increase of 59% and 122%, respectively, at 30° and 40 °C. The in vitro results also showed that a relatively larger fraction of the dose was released into the first 5 l of air at the higher temperatures, at 40 °C, about 50% of the total amount released into 20 l. Conclusion: It was concluded that the in vitro/in vivo discrepancy was most probably due to increased aversive effects at elevated temperatures, causing the subjects to inhale smaller puff volumes. Further, the inhaler would not produce nicotine plasma levels exceeding those achieved following cigarette smoking, even in a hot climate. Received: 20 September 1996 / Accepted in revised form: 5 March 1997     

Purification and characterization of α-amylase in Moroccan locust,Dociostaurus maroccanus Thunberg (Orthoptera: Acrididae) and its inhibition by inhibitors from Phaseolus vulgaris L.

An α-amylase with molecular weight of 73?kDa was purified from midgut of Dociostaurus maroccanus using ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The optimal pH and temperature were 6 and 45?°C, respectively. As calculated using Lineweaver–Burk plots, the Km was about 0.62?mM and the Vmax was 1.113 (μmol/min/mg protein). Mn²⁺, Hg⁺, Zn²⁺ and ethylene diamine tetraacetic acid (EDTA) decreased α-amylase activity of D. maroccanus, whereas the addition of K⁺, Na⁺, Fe²⁺, Ba⁺, Mg²⁺, Ca²⁺ and Co²⁺ increased enzyme activity. Alpha-amylase inhibitors (AI1, AI2) with molecular weights of 43?kDa and 29?kDa, respectively, were purified from the seeds of Phaseolus vulgaris and its inhibitory effect on purified α-amylase of D. maroccanus was investigated. These inhibitors inhibited the D. maroccanus gut α-amylase activity significantly.     

Influence of amitriptyline,chlorpromazine, and imipramine on the activity of phosphohydrolases

We have studied the effects of the antidepressants amitriptyline and imipramine and the neuroleptic chlorpromazine on the enzymatic function of Ca²⁺-activating-Mg²⁺-dependent ATPase of sarcoplasmic reticulum (SR), Na⁺, K⁺-dependent ATPase of endoplasmic reticulum (ER), and phosphodiesterase of cyclic adenosine monophosphate (cAMP). It is shown that interference with the enzymatic function (decrease of [Ca²⁺]/[ATP]) or inhibition of active transport of calcium ions by Ca²⁺, Mg²⁺-ATPase of SR by the psychotropic drugs leads to impairment of Ca²⁺ transport into the SR vesicule, which increases the force of muscle contraction. This effect decreases with increasing concentrations of amitriptyline and imipramine and with decreasing concentration of chlorpromazine, which is evidence for different mechanisms of action of Ca²⁺, Mg²⁺-ATPase of SR. Amitriptyline was a noncompetitive reversible inhibitor of Ca²⁺, Mg²⁺-ATPase of SR with an ATP hydrolysis inhibition constant K_i = 1.8 × 10⁻⁴ M and an active calcium transport inhibition constant K_i = 0.7 × 10⁻⁴ M. The goal of the investigation was to study the molecular mechanisms of action of amitriptyline, imipramine, and chlorpromazine. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 9, pp. 3–10, September, 2008.     

Determination of the relative bioavailability of nedocromil sodium to the lung following inhalation using urinary excretion

Objective: To determine the effects of cimetidine on the steady-state pharmacokinetics and pharmacodynamics of atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Methods: Twelve healthy subjects participated in a randomized two-way crossover study. Each subject received atorvastatin 10 mg every morning for 2 weeks and atorvastatin 10 mg every morning with cimetidine 300 mg four times a day for 2 weeks, separated by a 4-week washout period. Steady-state pharmacokinetic parameters (based on an enzyme inhibition assay) and lipid responses were compared. Results: Pharmacokinetic parameters and lipid responses were similar following administration of atorvastatin alone and atorvastatin with cimetidine. Mean values for C_max (the maximum concentration) were 5.11 ng · eq · ml⁻¹ and 4.54 ng eq · ml⁻¹, for t_max (the time to reach maximum concentration) 2.2 h and 1.3 h, for AUC_0–24 (area under the concentration-time curve from time 0 h to 24 h) 58.6 ng eq · h · ml⁻¹ and 58.5 ng eq · h · ml⁻¹, and for t_1/2 (terminal half-life) 10.1 h and 17.0 h, respectively, following administration of atorvastatin alone and atorvastatin with cimetidine. Following treatment with atorvastatin alone and atorvastatin with cimetidine, mean values for the percentage change from baseline for total cholesterol were −29.5% and −29.9%, for low-density lipoprotein (LDL) cholesterol −41.0% and −42.6%, for high-density lipoprotein (HDL) cholesterol 6.3% and 5.8%, and for triglycerides −33.8% and −25.8%, respectively. Conclusions: The rate and extent of atorvastatin absorption and the effects of atorvastatin on LDL-cholesterol responses are not influenced by coadministration of cimetidine. Received: 17 February 1997 / Accepted in revised form: 3 November 1997     

Population analysis of the non-linear red blood cell partitioning of draflazine following various infusion durations

Objective: The pharmacokinetics and non-linear red blood cell partitioning of the nucleoside transport inhibitor draflazine were investigated in 19 healthy male and female subjects (age range 22–55 years) after a 15-min i.v. infusion of 1 mg, immediately followed by infusions of variable rates (0.25, 0.5 and 1 mg · h⁻¹) and variable duration (2–24 h). Methods: The parameters describing the capacity-limited specific binding of draflazine to the nucleoside transporters located on erythrocytes were determined by NONMEM analysis. The red blood cell nucleoside transporter occupancy of draflazine (RBC occupancy) was evaluated as a pharmacodynamic endpoint. Results: The population typical value for the dissociation constant K _d (%CV) was 0.648 (12) ng · ml⁻¹ plasma, expressing the very high affinity of draflazine for the erythrocytes. The typical value of the specific maximal binding capacity B_max (%CV) was 155 (2) ng · ml⁻¹ RBC. The interindividual variability (%CV) was moderate for K _d (38.9%) and low for B_max (7.8%). As a consequence, the variability in RBC occupancy of draflazine was relatively low, allowing the justification of only one infusion scheme for all subjects. The specific binding of draflazine to the red blood cells was a source of non-linearity in draflazine pharmacokinetics. Steady-state plasma concentrations of draflazine virtually increased dose-proportionally and steady state was reached at about 18 h after the start of the continuous infusion. The t_1/2βaveraged 11.0–30.5 h and the mean CL from the plasma was 327 to 465 ml · min⁻¹. The disposition of draflazine in whole blood was different from that in plasma. The mean t_1/2β was 30.2 to 42.2 h and the blood CL averaged 17.4–35.6 ml · min⁻¹. Conclusion: Although the pharmacokinetics of draflazine were non-linear, the data of the present study demonstrate that draflazine might be administered as a continuous infusion over a longer time period (e.g., 24 h). During a 15-min i.v. infusion of 1 mg, followed by an infusion of 1 mg · h⁻¹, the RBC occupancy of draflazine was 96% or more. As the favored RBC occupancy should be almost complete, this dose regimen could be justified in patients. Received: 6 February 1997 / Accepted in revised form: 12 May 1997     

Safety and pharmacokinetics of a single oral dose of amisulpride in healthy elderly volunteers

Objective: Amisulpride is a substituted benzamide neuroleptic, which binds selectively to dopamine D₂ and D₃ receptors, mainly in the limbic structures. States of delusion and agitation occur frequently in the population aged more than 65 years, especially in demented patients and this sometimes requires the use of neuroleptics. The objectives of this study were to determine the safety and the pharmacokinetic profile of 50 mg of amisulpride administered orally as a single dose to elderly volunteers. Methods: Twenty healthy volunteers (10 men and 10 women) aged 65–79 years were included in this open trial. Frequent measurements of blood pressure and heart rate were made and ECG and blood samples were performed up to 72 h after drug intake. Results: The overall clinical and cardiovascular safety was satisfactory. The mean C_max of the racemate amisulpride in elderly people was 64.1 ± 6.7 ng · ml⁻¹, and was not different from the value of 56 ± 4.1 ng · ml⁻¹ in young subjects. As with the C_max, the mean values of t_1/2 and AUC in elderly people (15.6 ± 1.3 h and 667 ± 51 ng · ml⁻¹· h, respectively) were not different to values observed in young subject (respectively 11.7 ± 0.5 h and 603 ± 25 ng · ml⁻¹· h). Conclusions: A single oral dose of amisulpride was well tolerated and showed a similar pharmacokinetic profile in healthy elderly and young subjects. However, these findings should be confirmed after multiple dosing in a larger population in order to establish the lack of need of dosage adjustment in this elderly population. Received: 13 October 1997 / Accepted in revised form: 11 March 1998     

The inhibition by di(2-ethylhexyl)-phthalate of <Emphasis Type="Italic">erg</Emphasis>-mediated K<Superscript>+</Superscript> current in pituitary tumor (GH<Subscript>3</Subscript>) cells

DEHP (bis(2-ethylhexyl)-phthalate) known to be an endocrine-disrupting chemical is a widely used phthalate. Little information regarding the effects of phthalate esters on ion currents is available. In this study, the effects of DEHP and other phthalate esters (DBEP: di(2-butoxyethyl)-phthalate and DMGP: di(2-methylglycol)-phthalate) on ion currents were investigated in pituitary GH₃ cells. Hyperpolarization-elicited K⁺ currents in GH₃ cells bathed in high-K⁺, Ca²⁺-free solution were examined to evaluate the effects of DEHP, DBEP, and DMGP on the ether-à-go–go-related-gene (erg) K⁺ current (I _K(erg)). Addition of DEHP to GH₃ cells suppressed the amplitude of I _K(erg) in a concentration-dependent manner with an IC₅₀ value of 16.3 μM. With a two-pulse protocol, addition of DEHP shifted the activation curve of I _K(erg) to a depolarized potential by approximately 10 mV with no change in the rate of I _K(erg) deactivation. This compound did not have any effects on delayed rectifier K⁺ current in GH₃ cells, while 4-aminopyridine-3-methanol (100 μM) suppressed this current significantly. DBEP (30 μM) had little or no effect on I _K(erg), while DMGP (30 μM) slightly reduced it. In inside-out configuration, DEHP (30 μM) applied to the bath slightly reduced the activity of large-conductance Ca²⁺-activated K⁺ channels. DEHP (30 μM) increased the frequency of spontaneous action potentials (APs); however, this compound at the same concentration had no effect on AP firing in KCNH2 siRNA-transfected GH₃ cells. The effects described herein can contribute to their actions on functional activity of endocrine or neuroendocrine cells if similar results are found in vivo.