Hatsumi Aki

Drug binding to human erythrocytes in the process of ionic drug-induced hemolysis: Flow microcalorimetric approaches

Erythrocyte hemolysis induced by cationic phenothiazine derivatives and anionic non-steroidal anti-inflammatory drugs was compared, by flow microcalorimetry, with respect to thermodynamic characteristics for drug binding to intact human erythrocytes. Phenothiazines having high hemolytic activities bound strongly to erythrocyte cells, inducing an immediate hemolytic action characterized by an endothermic heat effect prior to saturating available binding sites. The thermodynamic observable delta H and delta S fell within the ranges of -119 to -65.1 kJ/mol and -308 to -128 J/mol/K, respectively, for these cationic species. There was a linear relationship between the hemolytic activity and the degree of exothermicity of delta H which was enhanced significantly by the presence of a halogen atom(s) at the C-2 position of the phenothiazine nucleus in the order of H less than Cl less than CF3. Anti-inflammatory drugs, however, bound to quite different sites in the erythrocytes with lower affinities and higher capacities than cationic drugs. The latter was characterized by small negative delta H (-17.3 to -7.1 kJ/mol) and positive delta S (10 to 41 J/mol/K). In the calorimetric profiles observed during hemolysis by anionic drugs, two stages were seen: the first, an exothermic process, arising from drug binding to the erythrocytes; the second, an endothermic process, corresponding to the heat of dilution of hemoglobin released from erythrocytes. Hemolysis occurred after the binding sites on the erythrocytes were saturated with drugs. Our data suggest that the binding activities of ionic drugs, such as the amounts of the bound drug and their binding energies to erythrocytes, contribute to the hemolysis.

Competitive binding of drugs to the multiple binding sites on human serum albumin : A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.

Application of differential flow microcalorimetry for study of drug interactions in the blood system

A compact differential flow microcalorimeter has been developed to investigate biomolecular reactions, especially drug interactions in the blood system. The calorimeter is an adiabatic type and consists of a twin-cell structure, each mixing part having a volume of 60 microliters. Both the precision and accuracy of the instrument have been evaluated by dilution of sucrose solutions to be 0.1-0.5% at a heat effect of 100-10 microW. The resolution is approximately 0.5 microW (less than 10(-3) Torr). The heat produced in erythrocyte hemolysis induced by chlorpromazine hydrochloride (CPZ) and the binding heat of CPZ to human blood components viz., intact erythrocytes, erythrocyte membranes, serum albumin and plasma were measured. The heat effect of hemolysis was endothermic and related to the quantity of free hemoglobin released from erythrocytes. The overall binding of CPZ to blood components was, however, an exothermic process. The thermodynamic and binding parameters were computed directly from the calorimetric data by use of a nonlinear least squares regression method, assuming a one-class binding model, and the stoichiometry of the binding reaction was determined.

Thermodynamic aspects of the molecular recognition of drugs by human serum albumin

Abstract Binding sites on human serum albumin (HSA) for some typical anionic drugs (Site I- and Site II-bound drugs) have been thermodynamically characterized by flow microcalorimetry. The binding and the thermodynamic parameters were computed directly from the calorimetric titration data at 37°C in a phosphate buffer (pH 7.4) using one- and two-class binding models. From compensation analysis by plotting the molar enthalpy change (ΔHm,i) versus the molar free energy change (ΔGm,i), and the molar entropy change (ΔSm,i) for every class of HSA binding sites, drug binding was classified into groups S1, S2 and S3, which agreed with those of fatty acid binding. Groups S1 and S2 included high-affinity binding sites (the first class of binding sites) and low-affinity binding sites (the second class of binding sites) for Site II-bound drugs, respectively, and group S3 contained the high-affinity binding sites for Site I-bound drugs. In each group, the ΔHm,i − ΔSm,i plot gave an excellent linear relationship, where the value of T β calculated from the intercept intercept interpreted the molecular recognition by HSA as a quantitative measure of the hydrophobic interaction upon complex formation. Groups S1 and S2 were characterized by large negative values of ΔHm,i and ΔSm,i, reflecting van der Waals interaction and hydrogen bonding formation in low dielectric media. The main force stabilizing the binding complex in group S3 was hydrophobic interaction characterized by small negative ΔHm,i values, minor or positive ΔSm,i values, and a large positive value of TΔS0 (32.4 kJ mol−1).

Microcalorimetric Study for the Binding of Ionic Drugs to Human Erythrocytes and the Ghost Membranes

Abstract— The binding of phenothiazine derivatives (as cationic drugs) and non‐steroidal anti‐inflammatory drugs (as anionic drugs) to human erythrocytes and ghost membranes has been compared with respect to their thermodynamic characteristics, by flow microcalorimetry at pH 7.4 and 37°C. From enthalpy‐entropy correlation, it was shown that anionic and cationic drugs are bound to different binding sites on the membranes. Phenothiazines bind to a single common site of the erythrocyte membranes with relatively high binding affinities (K = 104‐105 M−1). The binding is entropy‐driven and characterized by a small negative enthalpy (ΔH) and a positive entropy change (ΔS), reflecting hydrophobic interactions. However, the binding reaction for the intact erythrocytes shows large negative values for both AH and AS. The values of K for the membranes and ΔH for the intact erythrocytes increased with the increase of the hydrophobic character of the substituent group at the C‐2 position of the phenothiazine nucleus (H < Cl < CF3). The results indicate that phenothiazines bind and/or penetrate to the inner membranes of the erythrocytes and react with intracellular components such as haemoglobin, while anti‐inflammatory drugs bind to the surface protein on the membranes with a lower affinity (K = 103 M−1) than phenothiazines, reflecting the small negative ΔH and positive ΔS for the interaction with intact erythrocytes.

Anti‐inflammatory effects of 5‐aminosalicylic acid conjugates with chenodeoxycholic acid and ursodeoxycholic acid on carrageenan‐induced colitis in guinea‐pigs

Two epimeric bile acid conjugates, 5‐aminosalicylic acid‐chenodeoxycholic acid (5‐ASA‐CDCA) and 5‐aminosalicylic acid‐ursodeoxycholic acid (5‐ASA‐UDCA), were synthesized to deliver 5‐ASA to the large intestine by oral administration. The movement of the conjugates down the gastrointestinal tract and the anti‐inflammatory effects on ulcerative colitis were investigated by administering the conjugates to guinea‐pigs with an inflammatory bowel disease induced by 2% degraded carrageenan solution. The conjugates were protected from deconjugation in stomach and small intestine and reached the caecum and the colon, where 5‐ASA was more easily liberated from 5‐ASA‐CDCA than from 5‐ASA‐UDCA. The conjugates at doses equivalent to 50 or 150 mg kg−1 5‐ASA were orally administered once a day for 4 weeks from the 15th day after starting carrageenan treatment. The body weights and the bleeding scores of occult blood in faeces were measured during the experiment. The number of ulcers in the caecum and the colon were counted after killing the guinea‐pigs at the end of the experiment. Rapid onset of efficacy was shown by a significant reduction in bleeding scores within a week after administration of the conjugates. Treatment with the lower dose of 5‐ASA‐CDCA showed a recovery of body weight and a significantly decreased number of ulcers in the caecum, and the ulcers in the colon had completely disappeared by the end of the experiment. There was a good correlation found between the number of ulcers in the caecum and the bleeding scores of occult blood in faeces. The findings indicate that both conjugates were sufficiently delivered to the large intestine without deconjugation and that the lower dose of 5‐ASA‐CDCA is enough for treatment of ulcerative colitis in colonic inflammatory bowel diseases.

Population pharmacokinetics of phenobarbital by mixed effect modelling using routine clinical pharmacokinetic data in Japanese neonates and infants: an update.

What is known and objective:  Optimal use of phenobarbital in the neonatal population requires information regarding the drug’s pharmacokinetics and the influence of various factors, such as different routes of administration, on the drug’s disposition. However, because of sampling restrictions, it is often difficult to perform traditional pharmacokinetic studies in neonates and infants. This study was conducted to establish the role of patient characteristics in estimating doses of phenobarbital for neonates and infants using routine therapeutic drug monitoring data.

Mechanism for the inhibition of the acid degradation of ampicillin by 2-hydroxypropyl-β-cyclodextrin

The formation of inclusion complexes between amoxicillin (AMPC) and 2-hydroxypropyl-β-cyclodextrin (HPCD) was investigated by isothermal microcalorimetry and molecular dynamics simulation to evaluate the inhibitory effects on the degradation of AMPC in aqueous solutions at various pH. The process depended significantly on the ionic species of AMPC in the solution. In a strong acid solution, cationic AMPC and HPCD formed two different types of inclusion complexes with a 1:1 stoichiometry: the first-type had a high association constant K1 of 4.0-8.0·103 M-1 and included the penam ring of AMPC in the HPCD cavity (Mode I), while the second-type with a K2 of 1.0·103 M-1 contained the phenyl group of AMPC (Mode II). Furthermore, a complex with a 1:2 (AMPC:HPCD) stoichiometry was realized in a two-step reaction and was characterized by a smaller K1:2of 4.0·102 M-1 and larger negative enthalpy and entropy changes than the complexes with a 1:1 stoichiometry. Since the β-lactam ring of AMPC could be protected by inclusion with HPCD in the 1:2 complex and Mode I of 1:1 complexes, the degradation of AMPC in the presence of HPCD was approximately four times slower than in its absence at pH 1.2 and 37°C. In weak acid and neutral solutions, zwitterionic AMPC and HPCD formed only one type of inclusion complex with a 1:1 stoichiometry, where the phenyl group was included (Mode II). AMPC was very stable in these solutions (t1/2=226 h at pH=6.0) and there is little significant difference in the degradation rate between complexed AMPC and uncomplexed AMPC. Thus, the results indicated that the inclusion complex of AMPC with HPCD, effectively increasing the stability of AMPC in a strong acidic solution like that the stomach, would be useful for eradicating Helicobacter pylori infection and as a drug delivery system.

Biothermodynamic characterization of monocarboxylic and dicarboxylic aliphatic acids binding to human serum albumin: A flow microcalorimetric study

Thermodynamic parameters have been evaluated for the binding of unbranched monocarboyxlic aliphatic acids (MCAs) of 4 to 16 carbons (MC4 to MC16) and dicarboxylic aliphatic acids (DCAs) of 4 to 16 carbons (DC4 to DC16) to human serum albumin (HSA) on the basis of microcalorimetric measurement at pH 7.4 and 37 degrees C by computer-fitting to single- and two-class binding models. Long-chain MCAs (MC10 to MC16) and DCAs (DC14 and DC16) had the first class of binding sites with high affinity (large binding constant) of 10(5) to 10(6) M-1 and the second class with lower affinity and high capacity (large numbers of binding sites). Short- or medium-chain MCAs and DCAs bound to HSA at some low affinity binding sites. The binding constants of MCAs were ten times larger than those of DCAs. All the relationships between the thermodynamic parameters and alkyl-chain length of the acids showed clear-cut inflections in their plots around eight or nine methylene units. The free energy change of the first class of binding sites (- delta G1) became more negative with an increment of -1.0 kJ mol-1 CH2(-1) as the alkyl-chain length increased, but there were steep rises between MC9 and MC11 with -2.90 kJ mol-1 CH2(-1) and between DC9 and DC12 with -2.02 kJ mol-1 CH2(-1). The enthalpy change (- delta H) increased at the rate of -7.4 kJ mol-1 CH2(-1) to the maximum at MC9 and DC10, then decreased due to hydrophobicity of the alkyl-chains. From compensation analyses (delta H vs. delta S and delta G), HSA binding sites were characterized into three groups.

High-yield automated synthesis of [18F]fluoroazomycin arabinoside ([18F]FAZA) for hypoxia-specific tumor imaging

The aim of this study was to develop an efficient fully automated synthesis method to achieve a high radiochemical yield of [(18)F]FAZA with a small amount of precursor. A small cartridge containing 25mg of the QMA resin was prepared and evaluated to obtain [(18)F]F(-) in a small quantity of base (K(2)CO(3)), which might allow the use of a small amount of precursor. The labeling and hydrolyzing conditions for [(18)F]FAZA synthesis were also investigated manually. No-carrier-added [(18)F]F(-) was trapped on the small QMA cartridge and eluted with a mixture of Krytofix 222 (2.26 mg, 6.0 μmol) and K(2)CO(3) (0.69 mg, 5.0 μmol) in 70% MeCN (0.4 mL). The automated synthesis of [(18)F]FAZA was optimally performed with a modified NIRS original synthesis system for clinical use, by labeling 2.5mg (5.2 μmol) of the precursor in DMSO (0.4 mL) at 120°C for 10 min, and then by hydrolyzing the (18)F-labeled intermediate with 0.1M NaOH (0.5 mL) at room temperature for 3 min. Using the above condition, the [(18)F]FAZA injection was obtained with a high radiochemical yield of 52.4±5.3% (decay-corrected, n=8) within 50.5±1.5 min.