Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue, 2,6-dichloro-4-aminophenol iloprost

We synthesized a fluorescent probe, 2,6-dichloro-4-aminophenol iloprost or dichlorohydroxyphenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%. This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (HSA). Energy transfer from the tryptophan residue to the phenol moiety of DCHPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate of DCHPA-iloprost binding to HSA. As DCHPA-iloprost showed similar binding characteristics similar to those of iloprost and prostacyclin and competed with iloprost for HSA binding sites, we used DCHPA-iloprost as a probe to locate the binding domain of prostacyclin (PGI2) in HSA. The distance between the tryptophan indole and the phenol group of DCHPA-iloprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI2 and stabilizes its activity could be derived from this model.     

Effect of 7-fluoro prostacyclin,a stable prostacyclin analogue,on cAMP accumulation and prostaglandin binding in mastocytoma P-815 cells

The effect of 7-fluoro proscyclilin (PGI₂-F), a chemically stable analogue of prostacyclin, on cAMP accumulation in and [³H]PGE binding to mastocytoma P-815 cells was compared with those of the Na salt and methyl ester of prostacyclin (PGI₂Na or PGI₂Me), which are rapidly inactivated in aqueous solution or metabolized in the tissue.PGIF was as effective as PGI₂Me, and slightly less effective than PGI₂Na in stimulating cAMP accumulation in mastocytoma cells and rabbit platelets. PGI₂F was also more stable than PGI₂Me or PGI₂Na, and retained its original cAMP elevating activity even after incubation with or without cells for 4 h at 37°C. Cells which had been exposed to PGI₂F and then washed free of unbound reagent continued to produced cAMP for more than 3 h. PGI₂F was also as effective as PGE₁ or PGE₂ in displacing [³H]PGE₂ bound to the cells. Non-competitive inhibition by PGI₂F or PGI₂Me of [³H]PGE₂ binding to the cells, with apparent K_is of 1.29 μM and 1.13 μM, respectively, indicates the presence of different receptors for PGE₂ and for PGI₂F or PGI₂Me in mastocytoma P-815 cells.     

Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of iloprost at a dose of 10 μg, at a rate of 0.1 μg/kg/h, twice daily for two weeks.The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen . In the one patient tested, thermography revealed an increase in skin temperature by more than 2°C.Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.     

Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy.

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of Iloprost at a dose of 10 micrograms, at a rate of 0.1 micrograms/kg/h, twice daily for two weeks. The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen in vitro. In the one patient tested, thermography revealed an increase in skin temperature by more than 2 degrees C. Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI₂) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI₂ is inactivated under physiological conditions it has not been possible to demonstrate specific PGI₂ binding to the rat stomach. Therefore a stable PGI₂ analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE₂ nor 6 keto PGF_1α displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 × 10⁻¹¹ M and 7.1 × 10⁻⁸ M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI₂ was less potent than unlabelled Iloprost in displacing ³H-Iloprost from its binding site. The addition of PGE₂ to the incubation medium resulted in an increase in ³H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI₂-like agents but not for either PGE₂ or 6 keto PGF_1α.     

The use of a prostacyclin analogue, [3H]iloprost,for studying prostacyclin-binding sites on human platelets and neuronal hybrid cells

The stable prostacyclin analogue [³H]iloprost has been used for labelling prostacyclin-binding sites on human platelets and NCB-20 neuronal hybrid cells. The ligand-binding properties of the sites have been determined and correlate well with stimulation of cAMP synthesis in NCB-20 cells and inhibition of aggregation in human platelets.     

Probing the binding mechanism of capecitabine to human serum albumin using spectrometric methods,molecular modeling,and chemometrics approach

Capecitabine as a prodrug of 5-Fluorouracil plays an important role in the treatment of breast and gastrointestinal cancers. Herein, in view of the importance of this drug in chemotherapy, interaction mechanism between Capecitabine (CAP) and human serum albumin (HSA) as a major transport protein in the blood circulatory system has been investigated by using a combination of spectroscopic and molecular modeling methods. The fluorescence spectroscopic results revealed that capecitabine could effectively quench the intrinsic fluorescence of HSA through a static quenching mechanism. Evaluation of the thermodynamic parameters suggested that the binding process was spontaneous while hydrogen bonds and van der Waals forces played a major role in this interaction. The value of the binding constant (K_b = 1.820 × 10⁴) suggested a moderate binding affinity between CAP and HSA which implies its easy diffusion from the circulatory system to the target tissue. The efficiency of energy transfer and the binding distance between the donor (HSA) and acceptor (CAP) were determined according to forster theory of nonradiation energy transfer as 0.410 and 4.135 nm, respectively. Furthermore, UV–Vis spectroscopic results confirmed that the interaction was occurred between HSA and CAP and caused conformational and micro-environmental changes of HSA during the interaction. Multivariate curve resolution-alternating least square (MCR-ALS) methodology as an efficient chemometric tool was used to separate the overlapped spectra of the species. The MCR-ALS result was exploited to estimate the stoichiometry of interaction and to provide concentration and structural information about HSA-CAP interactions. Molecular docking studies suggested that CAP binds mainly to the subdomain IIA of HSA, which were compatible with those obtained by experimental data. Finally, molecular dynamics simulation (MD) was performed on the best docked complex by considering the permanence and flexibility of HSA-CAP complex in the binding site. MD result showed that CAP could steadily bind to HSA in the site I based on the formation of hydrogen bond and π-π stacking interaction in addition to hydrophobic force.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

Stereoselective kinetics of warfarin binding to human serum albumin: effect of an allosteric interaction

Kinetic and equilibrium binding studies were performed on the interaction of warfarin enantiomers with human serum albumin (HSA) in the absence and presence of lorazepam acetate (LoAc) enantiomers. Binding kinetics were followed by recording changes in the fluorescence of warfarin upon binding to HSA using the stopped-flow technique. The binding of (R)-warfarin displayed an exponentially increasing fluorescence, satisfying the two-step mechanism reported previously for the racemate, i.e., a diffusion controlled pre-equilibrium is followed by a slower rearrangement of the complex. In the case of (S)-warfarin, the signal was biphasic: a fast fluorescence enhancement was followed by a slow decline. The different kinetic features indicate that the equilibrium conformations of the [(S)-warfarin-HSA] and [(R)-warfarin-HSA] complexes are achieved via different mechanisms. The phenomenon was seen in buffers of different pH and compositions. Equilibrium binding measurements indicated significantly lower molar intrinsic fluorescence for the (S)-warfarin complex, suggesting differences in the microenvironments of the bound enantiomers. In the presence of (S)-LoAc, the allosterically enhanced binding of (S)-warfarin manifested itself in accelerated relaxation kinetics. In accordance with the low molar intrinsic fluorescence determined for the stable ternary complex, the amplitude of the decline in fluorescence became larger.     

Stimulation of prostaglandin D2 receptors on human platelets by analogs of prostacyclin.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.     

Effects of TRK-100, a stable prostacyclin analogue, on regulation of cyclic AMP metabolism in platelets

TRK-100 is a chemically stable analogue of prostacyclin and effective in inhibiting platelet aggregation when orally administered in experimental animals. In the present study we compared the potency of TRK-100 with those of PGI2 and PGE1 to cause an activation of adenylate cyclase activity in rat and human platelet membranes. TRK-100 was half as effective as PGI2, and 10 times more effective than PGE1 in both platelet membranes. TRK-100 also induced an activation of phosphodiesterase activity when directly added to intact platelets probably as a feedback mechanism of intracellular cAMP level like PGI2 did. TRK-100 would mimic PGI2 in the regulation of cAMP metabolism.     

Anti-coagulant rodenticide binding properties of human serum albumin: a biochromatographic approach

In this paper, the anti-coagulant rodenticide-human serum albumin (HSA) binding was investigated using a perturbation method to calculate the solute distribution isotherms. It was shown that rodenticide can bound either on the benzodiazepine HSA site with low affinity (site I) or on the warfarin HSA site with high affinity (site II). The thermodynamic parameters of this association were calculated for the two HSA binding sites. For the site II, the rodenticide-HSA association was governed enthalpically whereas for the site I, this one was driven entropically. Moreover, the role of the magnesium (Mg(2+)) and calcium (Ca(2+)) on this association was carried out. It was clearly demonstrated that the rodenticide affinity for the site I was not affected by modifying the bulk solvent surface tension whereas for the site II the association constant increased strongly with the Mg(2+) or the Ca(2+) concentration in the bulk solvent. These results showed that the rodenticide-HSA affinity and thus the rodenticide toxicological effect depends on the Mg(2+) or Ca(2+) concentration.     

Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.     

Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application

For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.     

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2)

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.     

Effects of taprostene, a chemically stable prostacyclin analogue, in patients with ischaemic peripheral vascular disease: a placebo controlled double-blind trial

Thirty patients with ischaemic peripheral vascular disease and intermittent claudication were randomly allocated to receive either placebo or taprostene, a chemically stable prostacyclin analogue, intravenously at a rate of 25 ng/kg/min for 6 hours daily on 5 consecutive days. Taprostene produced a significant (p less than 0.05) increase in absolute walking time compared to placebo on one day after infusion and at 1, 4 and 8 weeks (14% vs 2.8%) later. Taprostene also produced a significant (p less than 0.05) increase in the pain-free walking time compared to placebo in the follow-up period (8 weeks after infusion: 23% vs 3.8%). During the infusion period systolic and diastolic blood pressure decreased (p less than 0.05) and heart rate was accelerated (p less than 0.05) in the taprostene treated group whereas no change was monitored in the placebo group. The ankle/brachial Doppler index was unaffected by taprostene. The platelet half-life was significantly (p less than 0.05) prolonged following taprostene-infusion (72.6 +/- 9.35 vs 77.9 +/- 7.44 hours). However, no change on platelet half-life was found in the placebo group (p less than 0.05). Various measures of platelet function parameters followed in vitro (ADP-induced aggregation, platelet sensitivity to PGI2, PGE1, PGD1 and taprostene, concentrations of platelet factor 4 and beta-thromboglobulin) showed no change with taprostene. Measures of circulating platelet aggregates and endothelial cells count showed no changes during the 2 months follow-up period too. It is assumed that taprostene may be of clinical benefit in patients with ischaemic peripheral vascular disease. However, future investigations have to be carried out to assess the optimal dose regime.