Drug-Biomacromolecule Interaction XII: Comparative binding study of sulfaethidole to bovine serum albumin by equilibrium dialysis,fluorescence probe technique,uv difference spectrophotometry and circular dichroism

Binding of sulfaethidole to bovine serum albumin was studied by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism. Equilibrium dialysis method enabled us to estimate the total number of drug binding sites of albumin molecule. For sulfaethidole, albumin had 6 primary and 40 secondary binding sites. The primary and secondary binding constants were 0.9×10⁵ M⁻¹ and 0.2×10⁶ M⁻¹, respectively. 1-Anilino-8-naphthalenesulfonate (ANS) and 2-(4′-hydroxylbenzeneazo)-benzoic acid (HBAB) were used as the fluorescence probe and the uv spectrophotometric probe, respectively. In fluorescence probe technique, results indicated that the number of higher affinity drug binding site of albumin was 1 and the number of lower affinity drug binding sites of albumin was 3, and the primary and secondary drug binding constants for bovine serum albumin were 2.15×10⁵ M⁻¹ and 1.04×10⁵ M⁻¹, respectively. In uv difference spectrophotometry, binding sites were 3 and binding constant was 1.88×10⁵ M⁻¹. The above results suggest that several different methods should be used in ompensation for insufficient information about drug binding to albumin molecule given by only one method.     

Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin

Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 × 10⁵ M⁻¹ for PFOA and 2.2 × 10⁴ M⁻¹ for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-l-proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow’s binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 × 10⁶ M⁻¹ and 2.2 × 10⁶ M⁻¹. At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 × 10⁶ M⁻¹ and 1.2 × 10⁶ M⁻¹, whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of quinidine on the binding of [3H]-ouabain and [3H]-digoxin by human lymphocytes

Summary To explore the molecular basis of the glycoside-quinidine interaction, the in vitro effect of quinidine on the binding of [³H]-ouabain and [³H]-digoxin to Na⁺ K⁺ ATPase receptors on human mononuclear cells was investigated. The maximum [³H]-ouabain binding capacity was 45.7±9.4×10³ molecules/cell in pure lymphocyte preparations (n=8) and 75.5±7.3×10³ molecules/cell in mixtures of mononuclear cells (n=8). These parameters were not influenced by 10⁻⁵ M quinidine. In eight equilibrium experiments with pure lymphocytes, the dissociation constant of [³H]-ouabain increased from 0.79±0.26×10⁻⁸ M in the absence of 10⁻⁵ M quinidine to 1.56±0.74×10⁻⁸ M in its presence (p<0.01), indicating that the affinity of the drug was decreased. Similar findings were observed using mixed mononuclear cells. In five uptake and release experiments, quinidine decreased the association rate constant of [³H]-ouabain from 3.15±0.36×10⁴ M⁻¹×s⁻¹ to 2.01±0.37×10⁴ M⁻¹ s⁻¹ (p<0.01), whereas the dissociation rate constant was not affected. A therapeutic concentration of quinidine does not affect the number of glycoside receptors on lymphocytes, but it does appear to reduce fractional receptor occupancy by both [³H]-ouabain and [³H]-digoxin at lower tracer concentrations. This finding is compatible with the clinical observation that quinidine reduces the distribution volume of digoxin.     

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na⁺/K⁺-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na⁺/K⁺ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na⁺ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K⁺ and 30 to 33° C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC₅₀ value) amounted to ∼ 5 × 10^–7 M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC₅₀ values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 × 10^–6 M and 1.1 × 10^–5 M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na⁺/K⁺ pump inhibition (K′_D value) were calculated to be ∼ 10^–6 M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K′_D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K′_D value for 22-allyl-digitoxigenin was even larger and amounted to ∼ 4 × 10^–5 M. The K′_D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (∼ 9 × 10² s^–1 M^–1) was smaller than the association rate constant of digitoxigenin (∼ 2 × 10⁴ s^–1 M^–1), whereas the apparent dissociation rate constants of both compounds were similar (∼ 4 × 10^–3 s^–1). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (∼ 3 × 10³ s^–1 M^–1) and a larger dissociation rate constant (∼ 8 × 10^–2 M^–1). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na⁺/K⁺ pump by affinity labeling. Received: 2 June 1997 / Accepted: 17 September 1997     

HYDRODYNAMIC AND QUASI-ELASTIC LIGHT SCATTERING STUDIES ON THE 12S GLOBULIN FROM RAPESEED*

The 12S globulin, one of the major storage proteins of rapeseeds, has the following physico-chemical constants, as determined by ultracentrifugation, quasi-elastic light scattering measurements and gel chromatography: sedimentation coefficient s⁰_{20, w}= 12.7 times 10^-13 s; diffusion coefficient (quasi-elastic light scattering) D⁰_{20, w}= 3.8 × 10^-7 cm² s^-1; Stokes radius (by quasi-elastic light scattering) R_s= 5.7 nm and (by gel chromatography) R_s= 5.5 nm; partial specific volume (calculated from the amino acid composition) v?= 0.729 mlg^-1; molecular weight M_s, D = 300, 000 daltons, M_{s, Rs}= 294,000 daltons (R_s from the gel chromatography); frictional ratio f/fo = 1.28.     

Binding of a new multidrug resistance modulator,S9788, to human plasma proteins and erythrocytes

Summary The interactions of S9788 with human plasma proteins have been investigatedin vitro by an erythrocyte partitioning technique that allows an estimation of the plasma proteins and erythrocytes binding parameters. S9788 was 98% bound to plasma and blood. Lipoproteins bound S9788 with high affinities (binding constants of 0.645, 12.8 and 87.0×10⁶M⁻¹ for, HDL, LDL and VLDL, respectively) and accounted for more than 55% of the total circulating S9788. Albumin and alpha₁-acid glycoprotein also bound S9788 with lower binding constants of 0.022 and 0.245×10⁶ M⁻¹. S9788 was mainly distributed in the plasma blood compartment (75–80%) with blood-to-plasma concentrations ratio of 0.6 to 0.7. These results indicate that,in vivo, the fraction of blood S9788 available for tissue diffusion,i.e., the free drug fraction in blood, should depend on lipoprotein concentration in plasma.     

Brain Uptake of Nonsteroidal Anti-Inflammatory Drugs: Ibuprofen, Flurbiprofen, and Indomethacin

Purpose To determine the roles of blood–brain barrier (BBB) transport and plasma protein binding in brain uptake of nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, flurbiprofen, and indomethacin. Methods Brain uptake was measured using in situ rat brain perfusion technique. Results [¹⁴C]Ibuprofen, [³H]flurbiprofen, and [¹⁴C]indomethacin were rapidly taken up into the brain in the absence of plasma protein with BBB permeability–surface area products (PS_u) to free drug of (2.63 ± 0.11) × 10⁻², (1.60 ± 0.08) × 10⁻², and (0.64 ± 0.05) × 10⁻² mL s⁻¹ g⁻¹ (n = 9–11), respectively. BBB [¹⁴C]ibuprofen uptake was inhibited by unlabeled ibuprofen (K_m = 0.85 ± 0.02 mM, V_max = 13.5 ± 0.4 nmol s⁻¹ g⁻¹) and indomethacin, but not by pyruvate, probenecid, digoxin, or valproate. No evidence was found for saturable BBB uptake of [³H]flurbiprofen or [¹⁴C]indomethacin. Initial brain uptake for all three NSAIDs was reduced by the addition of albumin to the perfusion buffer. The magnitude of the brain uptake reduction correlated with the NSAID free fraction in the perfusate. Conclusions Free ibuprofen, flurbiprofen, and indomethacin rapidly cross the BBB, with ibuprofen exhibiting a saturable component of transport. Plasma protein binding limits brain NSAID uptake by reducing the free fraction of NSAID in the circulation.     

Rational Design of a Dual-Mode Optical and Chemical Prodrug

Purpose The purpose of this study is to demonstrate the rational design and behaviour of the first dual-mode optical and chemical prodrug, exemplified by an acetyl salicylic acid-based system.Methods A cyclic 1,4-benzodioxinone prodrug was synthesised by reaction of 3,5-dimethoxybenzoin and acetyl salicoyl chloride with pyridine. After purification by column chromatography and recrystallization, characterization was achieved using infrared and NMR spectroscopies, mass spectrometry, elemental analysis and single crystal X-ray diffraction. Light-triggered drug liberation was characterised via UV-visible spectroscopy following low-power 365 nm irradiation for controlled times. Chemical drug liberation was characterised via UV-visible spectroscopy in pH 5.5 solution.Results The synthetic method yielded pure prodrug, with full supporting characterisation. Light-triggered drug liberation proceeded at a rate of 8.30 × 10⁻² s⁻¹, while chemical, hydrolytic liberation proceeded independently at 1.89 × 10⁻³ s⁻¹. The photochemical and hydrolytic reactions were both quantitative.Conclusions This study demonstrates the first rational dual-mode optical and chemical prodrug, using acetyl salicylic acid as a model, acting as a paradigm for future dual-mode systems. Photochemical drug liberation proceeds 44 times faster than chemical liberation, suggesting potential use in drug-eluting medical devices where an additional burst of drug is required at the onset of infection.     

Voltammetric Determination of Mexidol

A new voltammetric method for determining mexidol in pharmaceuticals is proposed. Using this technique, mexidol was determined in model solutions (with R _s = of 1 – 6%) and in a ready-to-use preparation. The analytical range of mexidol determination using a glassy carbon composite electrode in 0.1 M H₂SO₄ extends from 4.8 × 10⁻³ to 1.8 × 10⁻² M. The detection limit is 1.9 × 10⁻³ M. The proposed procedure can be used for the quality control in the drug production technology. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 8, pp. 51 – 52, August, 2005.     

Content of antioxidants in plant extracts obtained by supercritical extraction

The content of natural antioxidants in extracts of medicinal plants, fruits, and seeds obtained by supercritical fluid extraction was determined by a microcalorimetric technique using the model reaction of induced cumene oxidation. All tested extracts can be divided into two groups according to their antioxidant activity. The first group includes extracts containing strong antioxidants with high inhibition rate constants; the second group includes preparations containing week antioxidants capable of reducing the oxidation rate. Among the preparations studied, the maximum concentrations of antioxidants were found in the extracts of sage (10.0 × 10⁻² mole/kg), rosemary (5.3 × 10⁻² mole/kg), cranberry (1.4 × 10⁻² mole/kg), and carrot (1.2 × 10⁻² mole/kg). The rate constants of reactions between antioxidants and peroxide radicals were calculated (k ₇ = (0.4 − 9.0) × 10⁴ liter/(mole sec)). With respect to this value, several types of natural antioxidants can be distinguished in each extract. The most active antioxidants are those contained in the rosemary extract, their reaction rate constants being about 2.3 × 10⁵ liter/(mole sec). It is suggested that the most active components are tocopherols, polyphenols, and quinones. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 4, pp. 29–33, April, 2006.     

An in vitro approach for demonstrating the critical role of serum albumin in the detoxication of the carbamate carbaryl at in vivo toxicologically relevant concentrations

The hydrolysis of carbaryl by bovine serum albumin (BSA) was studied at toxicologically relevant concentrations (range 15–300 μM) in order to determine the role of this protein in the detoxication of the carbamate in vivo. The 1-naphthol released during the hydrolysis of carbaryl was monitored using gas chromatography coupled with mass spectrometry. BSA hydrolyzed carbaryl in a time-progressive way. The hydrolysis was also dependent of enzyme (1.0, 2.5, 5.0 and 7.0 mg ml⁻¹) and substrate (range between 15 and 1,000 μM) concentration. The estimated turnover number and Michaelis–Menten constant were 1.6 × 10⁻⁴ s⁻¹ and 430 μM, respectively. Thus, the second order rate constant was 0.37 M⁻¹ s⁻¹. At enzyme concentrations of 7.0 mg ml⁻¹ and substrate concentrations ranging between 50 and 300 μM about 80% of substrate was hydrolyzed in 3 h. At lower substrate concentrations (15 and 30 μM carbaryl) also significant hydrolysis was detected at the highest enzyme concentration, even when these substrate concentrations were 30 and 15 times lower than the Michaelis–Menten constant. Although the efficacy of the enzymatic hydrolysis is low, the extrapolation of our results to the physiological albumin high concentrations (around 40 mg ml⁻¹) suggests that the hydrolysis of carbaryl by serum albumins plays a critical role in the detoxication of this carbamate at in vivo toxicologically relevant concentrations. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Effects of a new class of no-releasing NSAIDs on platelets and isolated arteries

A new class of nitroderivatives of non-steroidal anti-inflammatory drugs has recently been synthesized (Nicox Ltd., London, UK). In order to improve gastric tolerance of the parent compound, a side-chain, able to release nitric oxide, has been added to the core structure of the molecule. We studied in vitro the effects of nitrofenac and two NO-aspirins (NCX 4215 and NCX 4016) on platelets and isolated arteries to identify any possible effect due to the release of nitric oxide or to the inhibition of cyclo-oxygenase activity. Nitrofenac induced a dose-dependent relaxation both with intact (46% with 1×10⁻³ mol/L) and endothelium-denuded (75% with 1×10⁻³ mol/L) rings of rat aorta precontracted with epinephrine, while diclofenac did not affect this contraction (0% relaxation in intact and 22% in rubbed arteries). Pretreatment with diclofenac 1×10⁻³ mol/L significantly increased the vasorelaxant effects of nitrofenac at each drug concentration, both in intact (86% with 1×10⁻³ mol/L) and rubbed preparations (89%). NO-aspirins, unlike acetylsalicylic acid, were able to relax both intact and endothelium-denuded rings of rat aorta (100% relaxation). Methylene blue and oxyhaemoglobin completely reversed the relaxation induced by nitrofenac and NO-aspirins, both in rubbed and intact aortic rings. Both NO-aspirins exhibited antiaggregating properties in archididonic acid-stimulated human platelets, measured using a turbidimetric method (NCX 4215, 1×10⁻³ mol/L: 70% inhibition; NCX 4016 1×10⁻⁴ mol/L: 100%), NCX 4016 proving as effective as acetylsalicylic acid 1×10⁻⁵ mol/L. Thrombin-induced platelet aggregation was inhibited in acetylsalicylic acid-treated platelets (NCX 4215, 1×10⁻³ mol/L: 50%, NCX 4016, 1×10⁻⁴ mol/L: 92%). NCX 4016 was also able to provent thrombin-induced intracellular free calcium increase, effect not observed with acetylsalicylic acid. In vitro thromboxane A₂ production in human platelets, assayed RIA as thromboxane B₂ serum concentration, was reduced by NCX 4215, 1×10⁻³ mol/L (76%) and virtually abolished by NCX 4016 5×10⁻⁵ mol/L (95% inhibition). These results demonstrate in vitro the antiaggregating activity of NO-aspirins, NCX 4016 being more active than NCX 4215, and the vasorelaxant effects of all the tested molecules. The mechanism involved in two-fold: release of nitric oxide and inhibition of cyclo-oxygenase.     

Clonazepam release from core-shell type nanoparticlesin vitro

AB-type amphiphilic copolymers (abbreviated as LE) composed of poly (L-leucine) (PLL) as the A component and poly (ethylene oxide) (PEO) as the B component were synthesized by the ring-opening polymerization of L-leucine N-carboxy-anhydride initiated by methoxy polyoxyethylene amine (Me-PEO-NH₂) and characterized. Core-shell type nanoparticles were prepared by the diafiltration method. Particle size distribution obtained by dynamic light scattering was dependent on PLL composition and the size for LE-1, LE-2 and LE-3 was 369.6±267, 523.4±410 and 561.2±364 nm, respectively. Shapes of the nanoparticles observed by transmission electron microscope (TEM) were almostly spherical. The critical micelle concentration (CMC) of the nanoparticles determined by a fluorescence probe technique was dependent on the composition of hydrophobic PLL, and the CMC for LE-1, LE-2 and LE-3 was 2. 0×10⁻⁶, 1.7×10⁻⁶ and 1.5×10⁻⁶ (mol/l), respectively. Clonazepam release from core-shell type nanoparticles in vitro was dependent on PLL composition and drug loading content.     

Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

Pharmacokinetics and distribution of 6-mercaptopurine administered intravenously in children with lymphoblastic leukaemia

Objective: The pharmacokinetics of 6-mercaptopurine, including cerebrospinal-fluid (CSF) distribution, and the erythrocyte 6-thioguanine nucleotide concentrations were determined in children randomised to receive intravenous mercaptopurine for acute lymphoblastic leukaemia (ALL), according to the EORTC protocol ALL n°58881. Results: After 1 month of oral treatment at a dose of 50 mg · m⁻² · day⁻¹, the pharmacokinetic parameters were determined after the first i.v. administration of 1 g · m⁻² (bolus dose of 0.2 g · m⁻² followed by an 8-h infusion of 0.8 g · m⁻²) in 11 patients: systemic clearance was 23.02 l · h⁻¹, volume of distribution was 0.75 l · kg⁻¹, and elimination half-life was 1.64 h. The erythrocyte thioguanine concentrations were measured in the same 11 patients and increased significantly between the beginning and the end of infusion (10 pmol × 10⁸ packed RBC) or within 24 h of infusion (223 pmol × 10⁸ packed RBC). The CSF concentration was 3.78 μmol · l⁻¹, 1–6 h after the beginning of infusion (n=28) and the CSF to plasma ratio was 0.15 (n=16). In patients receiving the oral dose of 50–165 mg · m⁻² · day⁻¹ of 6-mercaptopurine, CSF concentrations were below 0.18 μmol · l⁻¹, 1–24 h after drug intake (n=67), and the CSF to plasma ratio was not calculated. Conclusion: Following the i.v. administration of 6-mercaptopurine, we observed high CSF concentrations of 6-mercaptopurine and an acute increase of erythrocyte thioguanine nucleotide concentrations. The clinical trial (EORTC protocol ALL n°5881), comparing the oral and i.v. administrations of mercaptopurine, will demonstrate if the i.v. administration reduces the incidence of CNS relapses. Received: 15 August 1996 / Accepted in revised form: 8 April 1997     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

Kinetics of inhibition of soluble peripheral nerve esterases by PMSF: a non-stable compound that potentiates the organophosphorus-induced delayed neurotoxicity

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus carbamates and sulfonyl fluorides) is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work, kinetic data were obtained for phenylmethylsulfonyl fluoride (PMSF) tested at different concentrations incubated for up to 3 h with soluble fraction of chicken peripheral nerve. PMSF is a protease and esterase inhibitor causing protection or potentiation of the organophosphorus-induced delayed neuropathy and is unstable in water solution. The target of the promotion effect was proposed to be a soluble esterase not yet identified. A kinetic model equation was deduced assuming a multienzymatic system with three different molecular phenomena occurring simultaneously: (1) inhibition, (2) spontaneous chemical hydrolysis of the inhibitor and (3) ongoing inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied for analyzing the experimental data. The best-fitting model is compatible with a resistant component (16.5–18%) and two sensitive enzymatic entities (both 41%). The corresponding second-order rate constants of inhibition (ki = 12.04 × 10⁻² and 0.54 × 10⁻² μM⁻¹ min⁻¹, respectively) and the chemical hydrolysis constant of PMSF (kh = 0.0919 min⁻¹) were simultaneously estimated. These parameters were similar to those deduced in fixed-time inhibition experiments. The consistency of results in both experiments was considered an internal validation of the methodology. The results were also consistent with a significant ongoing inhibition. The proportion of enzymatic components showed in this work is similar to those previously observed in inhibition experiments with mipafox, S9B and paraoxon, demonstrating that this kinetic approach gives consistent results in complex enzymatic systems.     

Microbial transformation of aniline to acetaminophen

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were screened. Among them,Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed betweenS. rimosus (N-cetylation function) andS. aureofaciens (p-hydroxylation function) and also betweenS. lividans andS. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixedS. rimosus (pro⁻ his⁻) andS. aureofaciens (ilv⁻) protoplasts with 40% (w/v) polyethylene glycol 3350 (PEG) for 3 min gave 8.3×10⁻⁷ of fusion frequency. Treatment of mixedS. lividans (pant⁻) andS. globisporus (leu⁻) protoplasts with 50% (w/v) PEG for 3 min at 30°C gave 1.2×10⁻⁶ of frequency. Among the fused strains, up to 40–50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts ofS. aureofaciens (2% frequency) lost their p-hydroxylation function.     

Estimation of catecholamines in daily urine by stripping voltammetry

A voltammetric method for the quantitative estimation of adrenaline (A) and noradrenaline (NA) in daily urine was developed using a carbon glass electrode. The lower detection limits for A and NA concentrations were 1 × 10⁻⁹ g/ml. The relative standard deviation for the range of concentrations 1 × 10⁻⁹ to 1 × 10⁻⁵ M was no greater than 0.1. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 2, pp. 51–53, February, 2008.     

Ion-selective sensors for determining ampicillin and oxacillin in biological fluids and pharmaceuticals

Ion-selective sensors based on organic ion exchangers of the ampicillin (oxacillin)-tetradecylammonium (Am-TDA; Ox-TDA) type sensitive to antibiotics of the penicillin group have been developed. The ratio of components (1: 1) and the solubility products of ion associates (8.5 × 10⁻⁸ and 5.2 × 10⁻¹⁰ for Am-TDAand Ox-TDA, respectively) are determined. According to thermoanalytical data, both Am-TDAand Ox-TDAion associates are individual substances not containing water. The organic ion exchangers do not undergo changes on heating up to 70°C. It is established that the electrode functions are linear in the interval from 1 × 10 ⁻⁵ to 1 × 10⁻¹ M for all antibiotics studied. The slopes of the electrode functions for Am and Ox are 58 × 1 and 60 × 1, respectively, being close to the theoretical values for singly charged ions. Methods for the determination of Am and Ox in the blood serum and saliva and in various medicinal forms (tablets, powders in bottles, etc.) are described. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 3, pp. 53–55, March, 2006.