Antifungal efficiency of miconazole and econazole and the interaction with transport protein: A comparative study

Abstract

Context: Miconazole (MIZ) and econazole (ECZ) are clinically used as antifungal drugs.

Objective: The drug effect and binding property with transport protein human serum albumin of MIZ and ECZ were studied.

Materials and methods: The antifungal efficiency was investigated by microdiluting drug solutions from 0 to 48?μmol?L^?1 through microcalorimetry and voltammetry studies. Transmission electron microscopy was used for morphological observations of C. albicans. The interaction with HSA was studied by electrochemical methods, fluorescence spectrometry, electron microscopy, and molecular simulation.

Results: IC₅₀ of MIZ and ECZ for C. albicans were obtained as 19.72 and 29.90?μmol?L^?1. Binding constants of MIZ and ECZ with HSA of 2.36?×?10⁴ L?mol^?1 and 3.73?×?10⁴ L?mol^?1 were obtained. After adding MIZ solution of 12 and 40?μmol?L^?1, the peak currents increased to 4.887 and 6.024?μA. The peak currents of C. albicans in the presence of 20 and 48?μmol?L^?1 ECZ were 4.701 and 5.544?μA. The docking scores for MIZ and ECZ of the best binding conformation in site I and site II were 5.60, 4.79, 5.63, and 5.85.

Discussion and conclusion: Strong inhibition to the metabolism of C. albicans and destructive effect was proved for both drugs. The lower IC₅₀, growth rate constant of C. albicans, and higher peak current, reveal stronger antifungal activity of MIZ. Both drugs show an efficient quenching effect to intrinsic fluorescence residues of protein. MIZ mainly binds on site I while ECZ on site II. Molecular modeling experiments give further insight of the binding mechanism.     

Inhibitory activities of baicalin against renin and angiotensin-converting enzyme

Context: Baicalin has been characterized as the active compound and quality control marker in Scutellaria baicalensis Georgi, traditionally used as a hypotensive herb.

Objectives: To investigate the inhibitory activities of baicalin against renin and angiotensin-I converting enzyme (ACE) and their molecule mechanism of interactions.

Methods: The fluorescence method using renin substrate 1(R-2932) and the spectroscopy method by Cushman were used to determine renin and ACE activities, respectively. The fluorescence quench techniques were used to characterize their interactions.

Results: The results showed that baicalin inhibited renin activity with an IC₅₀ value of 120.36 µM and inhibited ACE activity with an IC₅₀ value of 2.24?mM in vitro. The fluorescence emission of both renin and ACE were efficiently quenched by baicalin and a complete quenching was achieved at a high concentration of baicalin. Furthermore, baicalin was more effective in quenching the fluorescence of renin (K_SV?=?60?×?10³ M^?1) than ACE (K_SV?=?17.1?×?10³ M^?1). The quenching of fluorescence of renin and ACE involved static interactions, which was characterized by the formation of quencher–enzyme complex. The baicalin–renin complex formed through three-sites binding including the active site with a binding constant of 796.15?×?10¹³ M^?1, but there was only one binding site for the baicalin–ACE complex with a much smaller binding constant of 6.8?×?10⁵ M^?1.

Conclusion: The inhibition activity of baicalin against renin was a result of the formation of stable complex through multisites binding including the active site, which could explain the higher inhibitory efficiency.     

Interaction of 3′ -O-caffeoyl d-quinic acid with human serum albumin

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the viewpoint of thermodynamics and mechanism of binding at pH 6.0. The association constants (K_a) for the HSA-CGA interaction at 10, 25 and 40° C were 6.0 × 10⁴, 9.0 × 10³ and 2 × 10⁴ M^?1, resulting in AG of -6.21, -5.80, -6.32 kcal/mol, respectively. These high K_a -values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 mm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the α-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4°C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 ± 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 × 10⁴ M^?1and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA?CGA?>QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.     

The binding of quinidine to protein fractions of normal human sera.

In contrast to previous assumptions, albumin is not the only protein in normal human serum responsible for binding quinidine. Human serum proteins were fractionated by gel filtration and floatation. Quinidine binding was determined by equilibrium dialysis. The binding to low (LDL) and high (HDL) density lipoproteins exhibited two binding sites on each protein, and the dissociation constants K and number of binding sites n were calculated. LDL: K₁ = 2 × 10^?5, n₁ = 1 and K₂ = 5·2 × 10^?4, n₂ = 97. HDL: K₁ = 1·9 × 10^?5, n₁ = 0·1 and K₂ = 1·1 × 10^?3, n₂ = 14·7.     

In-vitro Binding of Propiverine Hydrochloride and Some of its Metabolites to Serum Albumin in Man

The distribution and pharmacological action of propiverine, a bladder spasmolytic agent, are affected by the extent of plasma-protein binding. Because attempts to assess the albumin-binding of propiverine have produced conflicting results, the binding parameters of the drug and some of its metabolites to serum albumin in man have been re-evaluated. In man propiverine is bound to serum albumin at a single site with high affinity (K_A1 = 1.45 × 10⁴ L mol^?1) and at least two sites with low affinity (K_A2 = 2.5 × 10² L mol^?1). The metabolites of propiverine, namely M2 (dealkylated propiverine), M5 (the N-oxide of propiverine) and M6 (the N-oxide of M2), are less firmly bound to serum albumin; this is considered to be non-specific binding. Binding experiments with human serum revealed that there are additional binding proteins. At therapeutic plasma levels the extent of binding was calculated to be 90, 15, 60, and 20% for propiverine and the metabolites M2, M5, and M6, respectively. The strong binding of propiverine to serum proteins controls its availability to the liver. Because the metabolites are not tightly bound to serum proteins, after metabolism of propiverine its metabolites are easily eliminated.     

Plasma Protein Binding of the Experimental Antitumour Agent Acridine-4-carboxamide in Man,Dog, Rat and Rabbit

Abstract— The plasma binding of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC) was investigated in-vitro by equilibrium dialysis for 3 h at 37°C against isotonic phosphate buffer (pH 7·35) using [³H]AC. There were significant species differences with the smallest % free fraction (mean ± s.d.) occurring in human plasma (3·4 ± 0·2), followed by dog (8·1 ±0·4), mouse (14·8 ± 0·8), rat (16·3 ± 0·9) and rabbit (20·2 ± 0·7). In plasma from healthy individuals (n = 5), the % free fraction ranged from 2·7 to 3·8. In physiological solutions of human proteins, the greatest binding was observed for α-acid glycoprotein (AAG) (0·75 g L^?1) with a mean free fraction of 24·1 ± 2·2%, followed by albumin (40 g L^?1) with 31·6 ± 0·7 and 39·8 ± 2·5% for fatty-acid-free and globulin-free, respectively. There was also some binding to globulins (5 g L^?1) with a mean % free fraction of 70·3 ± 1·6 and 84·8 ± 2·2 for Conn's fraction I and IV, respectively. Binding data from the displacement of [³H]AC by increasing concentrations of AC in human AAG (0·75 g L^?1) or albumin solution (40 g L^?1) indicated that AAG had 10-fold greater binding affinity for AC (K_a, 7·8 × 10⁴ m^?1) compared with albumin (K_a, 6·8 × 10³ m^?1). In human plasma enriched with AAG there was a significant negative linear correlation (r = 0·932; P < 0·001) between % AC free fraction and increasing AAG concentration over the range 0·6–4·5 g L^?1. Small but significant (P < 0·05) increases in AC free fraction occurred in the presence of various metabolites (50 and 100 μm) but, of those tested, only N-monomethyl-acridine carboxamide increased the free fraction to the same extent as parent AC.     

阿司匹林与人血清白蛋白的相互作用研究

目的以光谱技术研究阿司匹林分子与人血清白蛋白(HSA)间结合作用机制。方法通过荧光光谱法确定阿司匹林对HSA的荧光猝灭机制。由Lineweaver-Burk双倒数作图法确定反应的解离常数。根据热力学方程讨论两者间主要的作用力类型。结合同步荧光技术考察阿司匹林对人血清白蛋白构象的影响。结果阿司匹林对HSA的荧光猝灭机制为静态猝灭。在37℃和25℃时阿司匹林与HSA的解离常数分别为KD37=1.44×10-3mol.L-1,KD25=1.96×10-3mol.L-1。结合反应热力学参数为ΔH=-19.73kJ.mol-1,△G=-16.21kJ.mol-1,ΔS=-11.77kJ.mol-1。结论两者结合的主要作用力类型是范德华力。阿司匹林与白蛋白结合后使蛋白质构象发生变化。     

Solid-state stability and compatibility studies of clavulanate potassium

Abstract

The kinetic and thermodynamic parameters of degradation of clavulanate potassium in the solid state were studied by using a reversed phase high performance liquid chromatography (RP-HPLC) method. The degradation of clavulanate potassium was a first-order reaction depending on the substrate concentration at an increased relative air humidity (RH) and in dry air. The dependence ln k?=?f(1/T) became the ln k?=?(0.026?±?166.35)–(2702.82?±?1779.43)(1/T) in dry air and ln k?=?(1.65?±?100.40)?×?10³?(5748.81?±?3659.67)(1/T) at 76.4% RH. The thermodynamic parameters E_a, ΔH^≠a, ΔS^≠a of the degradation of clavulanate potassium in the solid state were calculated. The dependence ln k?=?f (RH%) assumed the form ln k?=?(8.78?±?5.75) 10?^?2 (RH%) + (2.64?×?10^?8?±?40.41). The compatibility of clavulanate potassium with commonly used excipients was studied at an increased temperature and in dry air. The geometric structure of molecule, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals were also determined in order to predict the structural changes and reactive sites in clavulanate potassium during degradation and compatibility studies in the solid state. The ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR) and Raman spectra of degraded samples of the compound were analyzed.     

Codissolution of calcium hydrogenphosphate and sodium hydrogencitrate in water. Spontaneous supersaturation of calcium citrate increasing calcium bioavailability

The sparingly soluble calcium hydrogenphosphate dihydrate, co-dissolving in water during dissolution of freely soluble sodium hydrogencitrate sesquihydrate as caused by proton transfer from hydrogencitrate to hydrogenphosphate, was found to form homogenous solutions supersaturated by a factor up to 8 in calcium citrate tetrahydrate. A critical hydrogencitrate concentration for formation of homogeneous solutions was found to depend linearly on dissolved calcium hydrogenphosphate: [HCitr^2?] = 14[CaHPO₄] - 0.05 at 25 °C. The lag phase for precipitation of calcium citrate tetrahydrate, as identified from FT-IR spectra, from these spontaneously formed supersaturated solutions was several hours, and the time to reach solubility equilibrium was several days. Initial calcium ion activity was found to be almost independent of the degree of supersaturation as determined electrochemically. The supersaturated solutions had a pH around 4.7, and calcium binding to hydrogencitrate as the dominant citrate species during precipitation was found to be exothermic with a determined association constant of 357 L mol^?1 at 25 °C for unit ionic strength, and ΔH° = ?22 ± 2 kJ mol^?1, ΔS° = ?26 ± 8 J K^?1 mol^?1. Calcium binding to hydrogencitrate and, more importantly, to citrate is suggested to decrease the rate of precipitation by lowering the driving force of precipitation, and becoming important for the robust spontaneous supersaturation with perspectives for design of functional foods with increased calcium bioavailability.     

Interaction of myo-inositol hexaphosphate (MIHP) with β-globulin from Sesamum indicum L.

β-Globulin, the low molecular weight protein fraction from Sesamum indicum L., interacts with myo-inositol hexaphosphate (MIHP) maximally at pH 3.0, with concomitant precipitation up to 85 ± 2% at an MIHP concentration of 8 × 10^?4m . The kinetics of interaction as followed by stopped-flow spectrophotometry suggested the reaction to be of pseudo first-order, having an initial fast step followed by a relatively slow step of rate constants 1.9 × 10^?2 s^?1 and 1.2 × 10^?2 s^?1 respectively at 1 × 10^?4m MIHP concentration. The analysis of the complex indicated the presence of polymer as seen in sedimentation velocity experiment. This was accompanied by conformational change of a three-fold decrease in β-structure and also an increase in fluorescence emission intensity accompanied with a red shift from 330 to 334 nm. Stoichiometric analysis of MIHP binding suggested four independent binding sites for MIHP, with a free energy change, ΔG^o= -5.1 kcal mol^?1 resulting from a binding constant of 3.6 × 10³m -1.     

Studies on interaction between Vitamin B12 and human serum albumin

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV–vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (K_sv) are 3.99 × 10⁴, 4.33 × 10⁴, 4.76 × 10⁴ and 5.16 × 10⁴ M⁻¹ at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters ΔH^Θ = −13.38 kJ mol⁻¹, ΔS^Θ = 66.73 J mol⁻¹ K⁻¹ were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r = 5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the α-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.     

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.

2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (K_m/K_i ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of K_m/K_i ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (K_m/K_i ratio of 8.0 versus 4.0).

3. The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.

4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (K_i?=?0.37?μM; K_m/K_i ratio about 73). Inhibition was not enhanced at parathion concentrations near the K_i by a preincubation step with NADPH.

5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (K_s?=?7.7?μM, ΔA_max?=?2.2?×?10^?2?a.u.?nmol CYP^?1; ΔA_max/K_s 2.86?×?10³?a.u. nmol?CYP^?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.

6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

     

The interaction of plant-growth regulators with serum albumin: Molecular modeling and spectroscopic methods

The affinity between two plant-growth regulators (PGRs) and human serum albumin (HSA) was investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that paclobutrazol (PAC) could bind on both site I and site II in HSA where the interaction was easier, while uniconazole (UNI) could not bind with HSA. Furthermore, the results of fluorescence spectroscopy, three-dimensional (3D) fluorescence spectroscopy and circular dichroism (CD) spectroscopy suggested that PAC had a strong ability to quench the intrinsic fluorescence of HSA. The binding affinity (K_b) and the amounts of binding sites (n) between PAC and HSA at 291 K were estimated as 2.37 × 10⁵ mol L⁻¹ and 1, respectively, which confirm that PAC mainly binds on site II of HSA. An apparent distance between the Trp214 and PAC was 4.41 nm. Additionally, the binding of PAC induced the conformational changes of disulfide bridges of HSA with the decrease of α-helix content. These studies provide more information on the potential toxicological effects and environmental risk assessment of PGRs.     

Binding of Sulfinpyrazone and its Metabolites in Human Serum and in Solutions of Human Serum Albumin

Abstract: The binding of sulfinpyrazone, its sulfone metabolite and its sulfide metabolite to serum protein was studied by equilibrium dialysis. At 20 μg/ml 99.1% of the parent compound was bound in serum, whereas 99.8% of the sulfide and 98.3% of the sulfone were bound at this concentration. The binding of the three compounds were studied in diluted serum and in solutions of human serum albumin (HSA). There was no evidence of binding to proteins other than albumin. The association constants to primary and secondary binding sites and the number of binding sites were calculated. For the sulfide a lower K₁-value in serum (0.76·10⁶ M^?1) than in the HSA solution (1.8·10⁶ M^?1) indicated the possible presence of a competitively bound substance in serum. In undiluted serum no displacing effect of the sulfide on sulfinpyrazone binding was found when both compounds were present in a concentration of 20 μg/ml, but in a HSA solution a pronounced sulfide induced displacement of the sulfinpyrazone from its primary binding site was shown. Acetylation of HSA depressed the binding of sulfinpyrazone but in undiluted serum there was no other effect on sulfinpyrazone binding by the addition of acetylsalicylic acid than could be explained by the displacing effect of salicylic acid. At concentrations at 20 μg/ml of sulfinpyrazone and above 50 μg/ml of the displacing agent significant displacement was demonstrated with phenylbutazone, tolbutamide and salicylic acid.     

Serum albumin and lipoproteins as the quinidine binding molecules in normal human sera.

To substantiate the binding of quinidine in human sera and predict variations of binding dissociation constants and number of binding sites were determined for separate serum proteins. Human sera were fractionated by gel filtration and ultracentrifugation, and binding was evaluated by equilibrium dialysis at pH 7·30 at 20° and 37° in a Krebs-Ringer phosphate buffer. Quinidine was bound to all serum lipoproteins and to serum albumin. The binding was influenced by the buffer composition. In sodium phosphate buffer there were two separate binding sites for quinidine on LDL and HDL, while there was only one detectable binding site on VLDL and HDL in a Krebs-Ringer phosphate buffer. On LDL also there appeared to be one binding site but it exhibited a positive cooperative binding effect at lower concentrations of quinidine. This effect was assumed to be caused by inorganic ions of the Krebs-Ringer phosphate buffer. At a therapeutic level of quinidine in normal human serum the concentration of quinidine bound to serum proteins was 1·062 × 10^?5 M. Calculated from the evaluated binding parameters VLDL contributed with 0·101 × 10^?5 M of this binding, LDL with 0·143 × 10^?5 M, HDL with 0·083 × 10^?5 M and albumin with 0·699 × 10^?5 M.     

Cis-resveratrol glucuronidation kinetics in human and recombinant UGT1A sources

1. It was hypothesized that cis-resveratrol glucuronidation contributes to a greater extent to in-vitro disposition of total resveratrol than previously assumed. To this end, the kinetic data for cis-resveratrol glucuronidation are reported.

2. Glucuronidation assays were conducted in human liver and intestinal microsomes and in uridine diphosphate-glucuronosyltransferases (UGTs) UGT1A1, UGT1A6, UGT1A9, and UGT1A10. Kinetic parameters were estimated for the major cis-resveratrol-3-O-glucuronide (cis-R3G). Substrate inhibition was observed with apparent V_max, K_m and K_i of 6.1?±?0.3/27.2?±?1.2 nmol min^?1 mg^?1, 415?±?48.1/989.9?±?92.8 and 789.6?±?76.3/1012?±?55.9?μM in human intestinal microsomes (HIMs) and UGT1A6, respectively (estimate?±?standard error (SE)). Biphasic kinetics were observed in human liver microsomes (HLMs), while sigmoidal kinetics were seen in UGT1A9 (V_max?=?11.92?±?0.2 nmol min^?1 mg^?1; K_m?=?360?μM; n?=?1.27?±?0.07). The 4′-O-glucuronide (cis-R4′G) exhibited atypical kinetics in HLM, HIM, UGT1A1, and UGT1A10. UGT1A9 catalysed cis-R4′G formation at high substrate concentrations (V_max?=?0.33?±?0.015 nmol min^?1 mg^?1; K_m?=?537.8?±?67.8?μM).

3. In conclusion, although the rates of formation of cis-R3G in HLM and UGT1A9 were higher than those for trans-R3G, the contribution to total resveratrol disposition could not be determined fully due to atypical kinetics observed.

     

Dynamics of microparticles inside lipid vesicles: movement in confined spaces

Microparticles and nanoparticles used in drug delivery frequently depend on their movement in confined spaces such as cells. Liposomes containing small numbers of 1-µm diameter polystyrene particles were used to study the dynamics of their movement within the confined space of the liposome interior. The analysis of the trajectories of single and multiple entrapped particles revealed that the particles were largely localized toward the periphery of the liposome with a rare presence in the centre. Interparticle interactions were studied by calculating interparticle distances, ranging from close to zero to around 8 µm with a mean of ～4 µm. The diffusion coefficient of a single entrapped particle was D?=?0.27?×?10^?9 cm² s^?1 when compared with 5.1?×?10^?9 cm² s^?1 free in water. When more than one particle was entrapped, the calculated diffusion coefficients were D?=?0.61?×?10^?9 cm² s^?1 for two particles, D?=?1.26?×?10^?9 cm² s^?1 for three particles, and D?=?1.3?×?10^?9 cm² s^?1 for multiple particles). Particle movement was found to be distinctly faster at the periphery (average velocity 21.4 μm s^?1) than at the centre of the vesicle (average velocity 14.2 μm s^?1). These results demonstrate the significance of particle–particle interactions as well as particle–surface interactions, which is evident here in some systems by particle aggregation close to the liposome membrane.     

In vivo and in vitro Inhibition of the Metabolism of N-Alkyl-substituted Amphetamines in Rat by Ferrocenylisopropylamine

Abstract

1. Ferrocenylisopropylamine (FIPA) inhibits the elimination of amphetamines in rat. The half-life of isopropylamphetamine was increased from approx. 30 to 85–100 min after administration of FIPA.

2. With isolated, perfused, rat liver, the half-lives of isopropylamphetamine, biamphetamine and benzylamphetamine were increased from 5–20 min to about 200 min by equimolar amounts of FIPA, indicating that the prolonging effect of FIPA is due to interference at the metabolic level.

3. Experiments with hepatic microsomal suspensions demonstrated that FIPA competitively inhibits the oxidative N-dealkylation of isopropylamphetamine; the K_i of FIPA is 4·1 × 10^?6 M.

4. Binding of isopropylamphetamine and FIPA to cytochrome P-450 was studied using hepatic microsomes of phenobarbital-treated rats. Isopropylamphetamine caused a type I, and FIPA a type II difference spectrum; FIPA showed a much higher binding affinity (K_s = 1·24 × 10^?2 M) than isopropylarnphetamine (K_s = 0·96 × 10^?3 M). FIPA acts as a modifier of the spectral changes induced by isopropylamphetamine.

5. Results suggest that the competitive inhibition of the N-dealkylation of N-alkylamphetamines, and thus the prolonging of their action, by FIPA is related to competition for binding to cytochrome P-450.     

Comparison of lenograstim and filgrastim on haematological effects after autologous peripheral blood stem cell transplantation with high-dose chemotherapy

SUMMARY

Objective: To compare the efficacy of lenograstim and filgrastim on haematological recovery following an autologous peripheral blood stem cell transplantation (PBSCT) with high-dose chemotherapy.

Methods: A retrospective case-controlled study.

Results: Absolute neutrophil count (ANC) recovery above 0.5?×?10⁹?l^?1 and white blood cell (WBC) recovery above 4?×?10⁹?l^?1 for 3 consecutive days was achieved earlier with filgrastim than with lenograstim ((13.2?±?8.0 vs 19.0?±?10.0 days, p?=?0.004), (16.9?±?9.7 vs 29.9?±?16.6 days, p?=?0.001), respectively). The platelet recovery above 20 x 10⁹/l was also achieved earlier with filgrastim than with lenograstim (19.5?±?11.6 vs

27.2?±?13.8 days, p?=?0.006). Furthermore, filgrastim-treated patients received fewer days of granulocyte colony simulating factor (G-CSF) administration (12.5?±?7.0 vs 18.6?±?8.5 days, p?=?0.001) and spent less time in hospital (23.7?±?10.9 vs 32.0?±?17.6 days, p?=?0.009). Duration of antibiotic administration was also significantly shorter in the filgrastim group (13.6?±?7.6 vs 29.1?±?19.8 days, p?=?0.001). Conclusion: In patients undergoing PBSCT following high-dose chemotherapy, filgrastim significantly reduced the duration of neutropenia, thrombocytopenia and days of G-CSF administration, and led to earlier hospital discharge compared with lenograstim.     

The effect of DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) on monoamine oxidase activities in tissues of the rat

The in vitro inhibition of monoamine oxidase (MAO) in rat liver, and of the clorgyline-resistant amine oxidase (CRAO) in rat heart and aorta, by DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) has been studied. Inhibition of each enzyme activity was independent of prolonged preincubation, was reversed by dialysis, and also Ackermann-Potter plots were consistent with reversible inhibition. Simple linear competitive inhibition of MAO-A and MAO-B was observed, with K_i values of 6 × 10^?6 and 8 × 10^?5 m, respectively. CRAO was inhibited in a mixed, non-competitive manner (K_i of 3·2 × 10^?5 and 7·8 × 10^?6 m in heart and aorta, respectively) which conformed to a kinetic model in which the binding of DSP-4 to CRAO increased the affinity of substrate binding, but prevented product formation. The possible significance of these results for the in vivo actions of the drug is discussed.