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Meindert Danhof - One of the best experts on this subject based on the ideXlab platform.
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Population pharmacokinetic-pharmacodynamic modelling of the anti-hyperalgesic effect of 5'deoxy-N6-cylopentyladenosine in the mononeuropathic rat.
European journal of pharmacology, 2004Co-Authors: Marloes P Schaddelee, Adriaan P Ijzerman, Joost Dejongh, Sue D Collins, Albertus G De Boer, Meindert DanhofAbstract:The objective of this investigation was to characterise the pharmacokinetic-pharmacodynamic correlation of 5'-deoxy-N6-cyclopentyl-adenosine (5'dCPA) in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 5'dCPA (0.30 or 0.75 mg kg(-1)), the time course of the drug concentration in plasma was determined in conjunction with the effect on (1) the mechanical paw pressure and (2) the Von Frey Hair monofilament withdrawal threshold. Population pharmacokinetic-pharmacodynamic analysis was applied to derive individual concentration-effect relationships. For mechanical paw pressure a composite model consisting of an Emax model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the data. The EC50 for the anti-hyperalgesic effect was 178+/-51 ng ml(-1) and the slope of the anti-nociceptive effect 0.055+/-0.008 g ml ng(-1). For the Von Frey Hair monofilament withdrawal threshold responders and non-responders were observed. Typically, in responders, full pain relief was observed at concentrations exceeding 100 ng ml(-1). The high plasma concentrations required for the anti-hyperalgesic effect relative to the receptor affinity are consistent with restricted transport of 5'dCPA to the site of action in the spinal cord and/or the brain.
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Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2'-deoxy-N6-Cyclopentyladenosine in sarin-poisoned rats.
Toxicology and applied pharmacology, 2003Co-Authors: Tjerk J.h. Bueters, Ad P. Ijzerman, Herman P.m. Van Helden, Meindert DanhofAbstract:Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2′-deoxy-N6-Cyclopentyladenosine in sarin-poisoned rats. Bueters, T.J.H., IJzerman, A.P., Van Helden, H.P.M., and Danhof, M. (2003). The objective of the present study was to determine (1) the influence of sarin poisoning (144 μg/kg sc) on the pharmacokinetics and brain distribution of the adenosine A1 receptor partial agonist 2′-deoxy-N6-Cyclopentyladenosine (2′dCPA), and (2) the effect of 2′dCPA (20 mg/kg iv) on the central acetylcholine (ACh) release and protection against sarin toxicity. A five-compartment model successfully described the pharmacokinetic profile of 2′dCPA in blood and brain microdialysate. A covariate analysis revealed that the volume of distribution of 2′dCPA in blood was different in sarin-poisoned rats, 177 ± 7 versus 148 ± 8 ml in control rats. However, the transport of 2′dCPA from blood to the brain was unaffected as reflected by the values of the intercompartmental transport clearances, 0.21 ± 0.02 and 0.21 ± 0.04 μl/min in control and sarin-poisoned rats, respectively. Also the area-under-curve (AUC) ratios of brain microdialysate and blood were identical with values of 0.02 ± 0.001 and 0.02 ± 0.002, respectively, demonstrating the restricted transport of 2′dCPA into the brain in both treatment groups. Treatment of sarin-poisoned rats by 2′dCPA did not adequately prevent the accumulation of ACh in the central nervous system. 2′dCPA delayed the emergence of concomitant symptoms compared to untreated rats, but eventually only 29% of the animals survived 24 h. In conclusion, the pharmacokinetic profile of 2′dCPA in blood was slightly changed by sarin, but not the distribution of 2′dCPA into the brain. The therapeutic efficacy of 2′dCPA against sarin was limited, presumably due to insufficient quantities of 2′dCPA reaching the brain.
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Adenosine A1 receptor agonist N6-Cyclopentyladenosine affects the inactivation of acetylcholinesterase in blood and brain by sarin
The Journal of pharmacology and experimental therapeutics, 2002Co-Authors: Tjerk J.h. Bueters, Ad P. Ijzerman, Herman P.m. Van Helden, Marloes J.a. Joosen, Meindert DanhofAbstract:The objective of the present study was to develop a kinetics of pharmacodynamics model to properly describe and investigate the in vivo interaction between the selective adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), acetylcholinesterase (AChE) in blood and brain, and the AChE-inhibitor sarin (isopropylmethylphosphonofluoridate). The direct interaction of CPA (2 microM) on the inhibition of AChE by sarin was studied in vitro in heparinized rat blood and in 10% (w/v) brain homogenate. CPA did not directly influence the sarin-mediated inactivation of AChE in either system. In sarin-poisoned (144 microg/kg s.c.) rats not treated with CPA, AChE was completely inactivated in blood and brain within 7 min. CPA (2 mg/kg i.m.) treatment, 1 min after sarin administration, caused a small delay in the inhibition of AChE in blood. Treatment with CPA, 2 min before sarin, protected the neuronal AChE partially from being inhibited, but not the enzyme localized in blood. With a dose-response-time model the proportion of the dose of sarin reaching the site of action was estimated to be 48 +/- 12 or 13 +/- 3% after CPA post- or pretreatment, respectively. A correlation between the residual AChE activity in the brain and the incidence of cholinergic symptoms could be established with logistic regression analysis: lower inhibition of AChE in the brain precluded the onset of critical symptoms. In conclusion, CPA affects the concentration of sarin reaching the site of action, which contributes to the protection previously observed in sarin-poisoned rats.
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Therapeutic efficacy of the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA) against organophosphate intoxication.
Archives of toxicology, 2002Co-Authors: Tjerk J.h. Bueters, Bas Groen, Meindert Danhof, Ad P. Ijzerman, Herman P.m. Van HeldenAbstract:The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun (O-ethyl-N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX (O-ethyl-S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion (O,O-diethyl-O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1-2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.
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Selectivity of action of 8-alkylamino analogues of N6-Cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats.
British journal of pharmacology, 1998Co-Authors: E. A. Van Schaick, Adriaan P Ijzerman, H. C. P. F. Roelen, H. E. Tukker, Meindert DanhofAbstract:A1 adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N6-Cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg−1 8-methylaminoCPA (8MCPA), 12.0 mg kg−1 8-ethylaminoCPA (8ECPA), 20.0 mg kg−1 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min−1 kg−1, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg−1 and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8). Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC50) and instrinsic activity (Emax) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with Emax values of −173±14, −131±11 and −71±6 beats min−1 for 8MCPA, 8ECPA and 8BCPA, respectively. These Emax values were significantly lower than the maximal effect of CPA (−208±8 beats min−1). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated Emax values were 63±5, 63±4 and 68±2%, respectively. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC50 values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml−1 and 164±22, 341±76 and 975±190 ng ml−1 for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8). This study demonstrates that partial agonists for the A1 adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.
Adriaan P Ijzerman - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of nucleoside transport proteins by C8-alkylamine-substituted purines.
Journal of Medicinal Chemistry, 2005Co-Authors: Reynier A. Tromp, Ronald F. Spanjersberg, And Jacobien K. Von Frijtag Drabbe Künzel, Adriaan P IjzermanAbstract:4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. Here we report on the synthesis and the biological evaluation of compounds that are less polar than NBTI. Compound screening in our laboratory indicated that introduction of an alkylamine substituent at the C8-position of N6-Cyclopentyladenosine (CPA, 2) led to an increment in affinity for the transport protein. It was investigated whether this would also apply for NBTI derivatives. Two series of C8-alkylamine-substituted compounds were prepared, one in which the nitro group was absent (46−58) and another in which the ribose moiety was replaced by a benzyl group (72−75). Comparison of the biological data of these compounds with 6-benzylthioinosine (4, Ki = 53 nM) and 9-benzyl-6-(4-nitrobenzylsulfanyl)purine (59, Ki = 135 nM) confirmed the hypothesis. The Ki values improved upon elongation of the alkylamine chain from methylamine to n-hexylamine with an optimum for n-pentylamine (50, Ki = 2.3 nM). Substi...
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Population pharmacokinetic-pharmacodynamic modelling of the anti-hyperalgesic effect of 5'deoxy-N6-cylopentyladenosine in the mononeuropathic rat.
European journal of pharmacology, 2004Co-Authors: Marloes P Schaddelee, Adriaan P Ijzerman, Joost Dejongh, Sue D Collins, Albertus G De Boer, Meindert DanhofAbstract:The objective of this investigation was to characterise the pharmacokinetic-pharmacodynamic correlation of 5'-deoxy-N6-cyclopentyl-adenosine (5'dCPA) in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 5'dCPA (0.30 or 0.75 mg kg(-1)), the time course of the drug concentration in plasma was determined in conjunction with the effect on (1) the mechanical paw pressure and (2) the Von Frey Hair monofilament withdrawal threshold. Population pharmacokinetic-pharmacodynamic analysis was applied to derive individual concentration-effect relationships. For mechanical paw pressure a composite model consisting of an Emax model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the data. The EC50 for the anti-hyperalgesic effect was 178+/-51 ng ml(-1) and the slope of the anti-nociceptive effect 0.055+/-0.008 g ml ng(-1). For the Von Frey Hair monofilament withdrawal threshold responders and non-responders were observed. Typically, in responders, full pain relief was observed at concentrations exceeding 100 ng ml(-1). The high plasma concentrations required for the anti-hyperalgesic effect relative to the receptor affinity are consistent with restricted transport of 5'dCPA to the site of action in the spinal cord and/or the brain.
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temperature dependence of the affinity enhancement of selective adenosine a1 receptor agonism a thermodynamic analysis
European Journal of Pharmacology, 2002Co-Authors: Alessandro Dalpiaz, Palmarisa Franchetti, Barbara Pavan, Francoise Ngo Ngos, Adriaan P IjzermanAbstract:Abstract The 2-amino-benzoylthiophene derivatives LUF 5468 [(2-amino-4-ethyl-5-methyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone] and LUF 5484 [(2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone] have been shown to allosterically enhance the adenosine A1 receptor agonist binding. We report a thermodynamic analysis of the agonist affinity obtained at human adenosine A1 receptors, in the presence and absence of LUF 5468 and LUF 5484. Moreover, an analysis of the temperature dependence for association and dissociation rates of N6-cyclohexyladenosine (CHA) binding was performed in the absence and presence of LUF 5484. Thermodynamic data were obtained by affinity measurements performed at different temperatures followed by van't Hoff analysis. The results indicate that the agonist binding is always totally entropy-driven, and that the modulators contribute to decrease the ΔG° , ΔH° and ΔS° values. It is concluded that the enhancers are able to increase the non-bonded interactions of the binding site with agonists as CHA, N6-cyclopentlyladenosine (CPA), 2′-methyl-N6-Cyclopentyladenosine (MeCPA) and 2-chloro-2′methyl-N6-Cyclopentyladenosine (MeCCPA).
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allosteric modulation of the adenosine a1 receptor synthesis and biological evaluation of novel 2 amino 3 benzoylthiophenes as allosteric enhancers of agonist binding
Journal of Medicinal Chemistry, 1999Co-Authors: P A M Van Der Klein, Angeliki Kourounakis, Adriaan P IjzermanAbstract:Novel allosteric enhancers of agonist binding to the rat adenosine A1 receptor are described. The lead compound for the new series was PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone), a compound previously reported by Bruns and co-workers (Mol. Pharmacol. 1990, 38, 950−958). The 4,5-dimethyl group and the benzoyl moiety were targets for further modifications, leading to series of 4,5-dialkyl (12a−g), of tetrahydrobenzo (12h−u), and of tetrahydropyridine (13a−g) derivatives. A number of compounds, in particular 12b, 12e, 12j, 12n, and 12u, proved superior to PD 81,723. Their EC50 values for enhancing the binding of the adenosine A1 receptor agonist N6-Cyclopentyladenosine to the receptor were lower, and/or their antagonistic activity on the adenosine A1 receptor was shown to be diminished.
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Thermodynamics of full agonist, partial agonist, and antagonist binding to wild-type and mutant adenosine A1 receptors.
Biochemical pharmacology, 1998Co-Authors: Alessandro Dalpiaz, Andrea Townsend-nicholson, Margot W. Beukers, Peterr Schofield, Adriaan P IjzermanAbstract:A thermodynamic analysis of the binding of a full agonist (N6-Cyclopentyladenosine), a partial agonist (8-butylamino-N6-Cyclopentyladenosine) and an antagonist (8-cyclopentyltheophylline) to human wild-type and mutant (mutation of a threonine (Thr) to an alanine (Ala) residue at position 277) adenosine A1 receptors expressed on Chinese hamster ovary (CHO) cells, and to rat brain adenosine A1 receptors was undertaken. The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium to rat brain receptors were determined by means of affinity measurements carried out at four different temperatures (0, 10, 20 and 25 degrees) and van't Hoff plots. Two temperatures (0 and 25 degrees) were considered for human receptors. Affinity constants were obtained from inhibition assays on membrane preparations of rat brain and CHO cells by use of the antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as selective adenosine A1 receptor radioligand. As for rat brain receptors, full agonist binding was totally entropy driven, whereas antagonist binding was essentially enthalpy driven. Partial agonist binding appeared both enthalpy and entropy driven. As for human receptors, full agonist affinity was highly dependent on the presence of Thr277. Moreover, affinity to both wild-type and mutant receptors was enhanced by temperature increase, suggesting a totally entropy-driven binding. Antagonist binding did not depend on the presence of Thr277. Antagonist affinity decreased with an increase in temperature, suggesting a mainly enthalpy-driven binding. Partial agonist binding was significantly dependent on the presence of Thr277 at 25 degrees, whereas such a dependence was not evident at 0 degrees. It is concluded that Thr277 contributes only to the binding of adenosine derivatives and that its role changes drastically with the receptor conformation and with the type of agonist (full or partial) interacting with the adenosine A1 receptors.
Alessandro Dalpiaz - One of the best experts on this subject based on the ideXlab platform.
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Fabrication via a nonaqueous nanoprecipitation method, characterization and in vitro biological behavior of N6-Cyclopentyladenosine-loaded nanoparticles
Journal of pharmaceutical sciences, 2009Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Eleonora Vighi, Eliana LeoAbstract:A novel nonaqueous nanoprecipitation method was proposed to achieve the encapsulation of a small weight hydrophilic drug (N6-Cyclopentyladenosine, CPA) in PLGA nanoparticles using a mixture of cottonseed oil and Tween-80 as nonsolvent phase. The nanoparticles were characterized in vitro as concerns size, morphology, drug loading, drug release, and drug stability in human blood. Human retinal pigment epithelium (HRPE) cells were employed to study intracellular accumulation of encapsulated or free CPA with and without unloaded particles, in the presence or absence of an equilibrative nucleoside transporter inhibitor. The particles displayed a mean size lower than 300 nm and a drug loading considerably higher than that found by conventional encapsulation methods. The suitable in vitro release properties permitted to obtain good drug stabilization in the blood. Studies on HRPE cells suggested that CPA can permeate their membrane by both diffusive- and transport-mediated mechanisms. The loaded and unloaded nanoparticles appeared able to increase the permeation rate of the diffusive mechanism, without interfering with the transporter. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4272–4284, 2009
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Solid lipid microparticles for the stability enhancement of the polar drug N6-Cyclopentyladenosine
International Journal of Pharmaceutics, 2007Co-Authors: Alessandro Dalpiaz, Angelo Scatturin, Matteo Mezzena, Santo ScaliaAbstract:Abstract The objective of this study was to prepare solid lipid microparticles (SLMs) loaded with the polar adenosine A 1 receptor agonist N 6 -cyclopentyladenosine (CPA). The microparticles were produced by the conventional hot emulsion technique, using different lipidic carriers (tristearin, glyceryl behenate and stearic acid) and hydrogenated phosphatidylcholine as the surfactant. The controlled release of CPA was achieved only with stearic acid microparticles. This phenomenon has been attributed to direct acid–base interactions between the basic nitrogen atoms of CPA and stearic acid. These SLMs were characterized by release studies, scanning electron microscopy and powder X-ray diffraction analyses. The obtained particles showed proper features in terms of morphology and size distribution (3.2–10.3 μm), with a drug loading of 0.15 ± 0.04%. The influence of the SLMs carrier system on CPA stability was investigated in vitro using human whole blood. The degradation kinetic of microparticle-entrapped CPA was significantly lower from that measured for the free CPA. The overall results indicate that it was possible to achieve the encapsulation and controlled release of a polar drug, such as CPA, within a lipid matrix without resorting to the complex methods generally used for the preparation of these systems.
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temperature dependence of the affinity enhancement of selective adenosine a1 receptor agonism a thermodynamic analysis
European Journal of Pharmacology, 2002Co-Authors: Alessandro Dalpiaz, Palmarisa Franchetti, Barbara Pavan, Francoise Ngo Ngos, Adriaan P IjzermanAbstract:Abstract The 2-amino-benzoylthiophene derivatives LUF 5468 [(2-amino-4-ethyl-5-methyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone] and LUF 5484 [(2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone] have been shown to allosterically enhance the adenosine A1 receptor agonist binding. We report a thermodynamic analysis of the agonist affinity obtained at human adenosine A1 receptors, in the presence and absence of LUF 5468 and LUF 5484. Moreover, an analysis of the temperature dependence for association and dissociation rates of N6-cyclohexyladenosine (CHA) binding was performed in the absence and presence of LUF 5484. Thermodynamic data were obtained by affinity measurements performed at different temperatures followed by van't Hoff analysis. The results indicate that the agonist binding is always totally entropy-driven, and that the modulators contribute to decrease the ΔG° , ΔH° and ΔS° values. It is concluded that the enhancers are able to increase the non-bonded interactions of the binding site with agonists as CHA, N6-cyclopentlyladenosine (CPA), 2′-methyl-N6-Cyclopentyladenosine (MeCPA) and 2-chloro-2′methyl-N6-Cyclopentyladenosine (MeCCPA).
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Synthesis and Study of 5′-Ester Prodrugs of N6-Cyclopentyladenosine, a Selective A1 Receptor Agonist
Pharmaceutical Research, 2001Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Angelo Scatturin, Enea Menegatti, Fabrizio Bortolotti, Carla Biondi, Elisa Durini, Stefano ManfrediniAbstract:Purpose . A series of 5′-esters of N^6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A_1 agonists. Log P values, stability, affinity, and activity toward human adenosine A_1 receptors were evaluated. Methods . An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5′-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A_1 receptor expressed by N^6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3′-5′-cyclic adenosine monophosphate, performing competitive binding assays. Results . All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5′-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A_1 receptor of the intact prodrugs. Conclusions . We propose 5′-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible canditates for CPA prodrugs.
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Synthesis and study of 5'-ester prodrugs of N6-Cyclopentyladenosine, a selective A1 receptor agonist
Pharmaceutical research, 2001Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Angelo Scatturin, Enea Menegatti, Fabrizio Bortolotti, Carla Biondi, Elisa Durini, Stefano ManfrediniAbstract:Purpose. A series of 5′-esters of N6-Cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated.
Herman P.m. Van Helden - One of the best experts on this subject based on the ideXlab platform.
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Correlations between acetylcholinesterase inhibition, acetylcholine levels and EEG changes during perfusion with neostigmine and N6-Cyclopentyladenosine in rat brain.
European Journal of Pharmacology, 2006Co-Authors: Marloes J.a. Joosen, Herman P.m. Van HeldenAbstract:Organophosphate poisoning can result in seizures and subsequent neuropathology. In order to improve treatment strategies in organophosphate intoxication, the relationship between acetylcholinesterase inhibition, extracellular levels of acetylcholine, and electroencephalogram (EEG) changes was investigated during local perfusion of the reversible acetylcholinesterase inhibitor neostigmine in the hippocampus and striatum of freely moving rats. Acetylcholinesterase activity and acetylcholine levels were measured by microdialysis, and EEG signals were recorded from an electrode placed near the microdialysis probe. A non-linear relationship between the acetylcholinesterase activity and the extracellular amount of acetylcholine was found, the latter being approximately three times higher in the striatum than in the hippocampus upon infusion with 10(-4) M neostigmine. Highly accumulated extracellular acetylcholine significantly correlated with significant relative power increases of the EEG-gamma2-band and a significant relative power decrease in the beta2-band. Co-infusion of the adenosine A1 agonist N6-Cyclopentyladenosine partly prevented acetylcholine accumulation, rendered both powers towards control values, and abolished the acetylcholine-EEG correlation. In view of the latter relationship, it is concluded that prevention of acetylcholine accumulation as a concept for neuroprotection in case of organophosphate poisoning, is worth to be further investigated.
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Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2'-deoxy-N6-Cyclopentyladenosine in sarin-poisoned rats.
Toxicology and applied pharmacology, 2003Co-Authors: Tjerk J.h. Bueters, Ad P. Ijzerman, Herman P.m. Van Helden, Meindert DanhofAbstract:Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2′-deoxy-N6-Cyclopentyladenosine in sarin-poisoned rats. Bueters, T.J.H., IJzerman, A.P., Van Helden, H.P.M., and Danhof, M. (2003). The objective of the present study was to determine (1) the influence of sarin poisoning (144 μg/kg sc) on the pharmacokinetics and brain distribution of the adenosine A1 receptor partial agonist 2′-deoxy-N6-Cyclopentyladenosine (2′dCPA), and (2) the effect of 2′dCPA (20 mg/kg iv) on the central acetylcholine (ACh) release and protection against sarin toxicity. A five-compartment model successfully described the pharmacokinetic profile of 2′dCPA in blood and brain microdialysate. A covariate analysis revealed that the volume of distribution of 2′dCPA in blood was different in sarin-poisoned rats, 177 ± 7 versus 148 ± 8 ml in control rats. However, the transport of 2′dCPA from blood to the brain was unaffected as reflected by the values of the intercompartmental transport clearances, 0.21 ± 0.02 and 0.21 ± 0.04 μl/min in control and sarin-poisoned rats, respectively. Also the area-under-curve (AUC) ratios of brain microdialysate and blood were identical with values of 0.02 ± 0.001 and 0.02 ± 0.002, respectively, demonstrating the restricted transport of 2′dCPA into the brain in both treatment groups. Treatment of sarin-poisoned rats by 2′dCPA did not adequately prevent the accumulation of ACh in the central nervous system. 2′dCPA delayed the emergence of concomitant symptoms compared to untreated rats, but eventually only 29% of the animals survived 24 h. In conclusion, the pharmacokinetic profile of 2′dCPA in blood was slightly changed by sarin, but not the distribution of 2′dCPA into the brain. The therapeutic efficacy of 2′dCPA against sarin was limited, presumably due to insufficient quantities of 2′dCPA reaching the brain.
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Cardiovascular effects of the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA) decisive for its therapeutic efficacy in sarin poisoning.
Archives of toxicology, 2003Co-Authors: Marloes J.a. Joosen, Tjerk J.h. Bueters, Herman P.m. Van HeldenAbstract:Mortality and occurrence of cholinergic symptoms upon sarin intoxication (144 µg/kg s.c., ~2×LD50) in rats is completely prevented by treatment with the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA, 2 mg/kg i.m.). Previously, we have shown that CPA treatment altered the distribution of sarin into the brain, presumably through its cardiovascular side effects. Therefore, the objective of the present study was to evaluate the contribution of the cardiodepressant effects of CPA to its therapeutic efficacy against sarin intoxication. Intramuscular treatment of rats with 0.5 and 2.0 mg/kg CPA 1 min after sarin poisoning attenuated most cholinergic symptoms and prevented mortality, which seemed to be directly associated with an immediate strong and long-lasting bradycardia and hypotension caused by CPA. Treatment with lower doses of CPA (0.1 and 0.05 mg/kg i.m.) caused similar levels of bradycardia and hypotension, albeit a few minutes later than at the higher doses of CPA. Upon sarin intoxication, this was correlated with increased incidence of cholinergic symptoms and decreased survival rates. Pretreatment with the peripheral adenosine A1 receptor antagonist 8-p-sulphophenyltheophylline (8-PST, 20 mg/kg i.p.) counteracted the cardiodepressant effects of 0.05 mg/kg CPA almost completely, thereby nearly abolishing its therapeutic efficacy against sarin poisoning. In conclusion, the present results strongly indicate that bradycardia and hypotension induced by the peripheral adenosine A1 receptor play a prominent role in the therapeutic efficacy of CPA in cases of sarin poisoning.
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Adenosine A1 receptor agonist N6-Cyclopentyladenosine affects the inactivation of acetylcholinesterase in blood and brain by sarin
The Journal of pharmacology and experimental therapeutics, 2002Co-Authors: Tjerk J.h. Bueters, Ad P. Ijzerman, Herman P.m. Van Helden, Marloes J.a. Joosen, Meindert DanhofAbstract:The objective of the present study was to develop a kinetics of pharmacodynamics model to properly describe and investigate the in vivo interaction between the selective adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), acetylcholinesterase (AChE) in blood and brain, and the AChE-inhibitor sarin (isopropylmethylphosphonofluoridate). The direct interaction of CPA (2 microM) on the inhibition of AChE by sarin was studied in vitro in heparinized rat blood and in 10% (w/v) brain homogenate. CPA did not directly influence the sarin-mediated inactivation of AChE in either system. In sarin-poisoned (144 microg/kg s.c.) rats not treated with CPA, AChE was completely inactivated in blood and brain within 7 min. CPA (2 mg/kg i.m.) treatment, 1 min after sarin administration, caused a small delay in the inhibition of AChE in blood. Treatment with CPA, 2 min before sarin, protected the neuronal AChE partially from being inhibited, but not the enzyme localized in blood. With a dose-response-time model the proportion of the dose of sarin reaching the site of action was estimated to be 48 +/- 12 or 13 +/- 3% after CPA post- or pretreatment, respectively. A correlation between the residual AChE activity in the brain and the incidence of cholinergic symptoms could be established with logistic regression analysis: lower inhibition of AChE in the brain precluded the onset of critical symptoms. In conclusion, CPA affects the concentration of sarin reaching the site of action, which contributes to the protection previously observed in sarin-poisoned rats.
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Therapeutic efficacy of the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA) against organophosphate intoxication.
Archives of toxicology, 2002Co-Authors: Tjerk J.h. Bueters, Bas Groen, Meindert Danhof, Ad P. Ijzerman, Herman P.m. Van HeldenAbstract:The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun (O-ethyl-N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX (O-ethyl-S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion (O,O-diethyl-O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A1 receptor agonist N6-Cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1-2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.
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Fabrication via a nonaqueous nanoprecipitation method, characterization and in vitro biological behavior of N6-Cyclopentyladenosine-loaded nanoparticles
Journal of pharmaceutical sciences, 2009Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Eleonora Vighi, Eliana LeoAbstract:A novel nonaqueous nanoprecipitation method was proposed to achieve the encapsulation of a small weight hydrophilic drug (N6-Cyclopentyladenosine, CPA) in PLGA nanoparticles using a mixture of cottonseed oil and Tween-80 as nonsolvent phase. The nanoparticles were characterized in vitro as concerns size, morphology, drug loading, drug release, and drug stability in human blood. Human retinal pigment epithelium (HRPE) cells were employed to study intracellular accumulation of encapsulated or free CPA with and without unloaded particles, in the presence or absence of an equilibrative nucleoside transporter inhibitor. The particles displayed a mean size lower than 300 nm and a drug loading considerably higher than that found by conventional encapsulation methods. The suitable in vitro release properties permitted to obtain good drug stabilization in the blood. Studies on HRPE cells suggested that CPA can permeate their membrane by both diffusive- and transport-mediated mechanisms. The loaded and unloaded nanoparticles appeared able to increase the permeation rate of the diffusive mechanism, without interfering with the transporter. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4272–4284, 2009
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temperature dependence of the affinity enhancement of selective adenosine a1 receptor agonism a thermodynamic analysis
European Journal of Pharmacology, 2002Co-Authors: Alessandro Dalpiaz, Palmarisa Franchetti, Barbara Pavan, Francoise Ngo Ngos, Adriaan P IjzermanAbstract:Abstract The 2-amino-benzoylthiophene derivatives LUF 5468 [(2-amino-4-ethyl-5-methyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone] and LUF 5484 [(2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone] have been shown to allosterically enhance the adenosine A1 receptor agonist binding. We report a thermodynamic analysis of the agonist affinity obtained at human adenosine A1 receptors, in the presence and absence of LUF 5468 and LUF 5484. Moreover, an analysis of the temperature dependence for association and dissociation rates of N6-cyclohexyladenosine (CHA) binding was performed in the absence and presence of LUF 5484. Thermodynamic data were obtained by affinity measurements performed at different temperatures followed by van't Hoff analysis. The results indicate that the agonist binding is always totally entropy-driven, and that the modulators contribute to decrease the ΔG° , ΔH° and ΔS° values. It is concluded that the enhancers are able to increase the non-bonded interactions of the binding site with agonists as CHA, N6-cyclopentlyladenosine (CPA), 2′-methyl-N6-Cyclopentyladenosine (MeCPA) and 2-chloro-2′methyl-N6-Cyclopentyladenosine (MeCCPA).
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Synthesis and Study of 5′-Ester Prodrugs of N6-Cyclopentyladenosine, a Selective A1 Receptor Agonist
Pharmaceutical Research, 2001Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Angelo Scatturin, Enea Menegatti, Fabrizio Bortolotti, Carla Biondi, Elisa Durini, Stefano ManfrediniAbstract:Purpose . A series of 5′-esters of N^6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A_1 agonists. Log P values, stability, affinity, and activity toward human adenosine A_1 receptors were evaluated. Methods . An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5′-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A_1 receptor expressed by N^6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3′-5′-cyclic adenosine monophosphate, performing competitive binding assays. Results . All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5′-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A_1 receptor of the intact prodrugs. Conclusions . We propose 5′-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible canditates for CPA prodrugs.
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Synthesis and study of 5'-ester prodrugs of N6-Cyclopentyladenosine, a selective A1 receptor agonist
Pharmaceutical research, 2001Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Angelo Scatturin, Enea Menegatti, Fabrizio Bortolotti, Carla Biondi, Elisa Durini, Stefano ManfrediniAbstract:Purpose. A series of 5′-esters of N6-Cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated.
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Binding thermodynamics and intrinsic activity of adenosine A1 receptor ligands.
Life sciences, 2000Co-Authors: Alessandro Dalpiaz, Barbara Pavan, Angelo Scatturin, Katia Varani, Rita Pecoraro, P.a. BoreaAbstract:A thermodynamic analysis of the binding to rat cortex adenosine A1 receptors of 5'-deoxyribose-N6-Cyclopentyladenosine (full agonist) and several 8-alkylamino homologues of N6-Cyclopentyladenosine (partial agonists) was performed. The intrinsic activity of the compounds was also evaluated by measuring the inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. Standard free energy (deltaG), enthalpy (deltaH ) and entropy (deltaS ) of the binding equilibrium were determined by affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees C). Affinity constants of drug-receptor interactions were obtained by displacement experiments in the presence of 1nM [3H]N6-cyclohexyladenosine. Levels of c-AMP were evaluated by performing competitive binding assays. As the affinity of the ligands was found to increase with temperature enhancement, the binding of full and partial agonists is therefore totally entropy-driven. Standard entropy values of a wide series of adenosine derivatives, including the compounds under examination, are strictly correlated to those of intrinsic activity. Similarly, deltaS values appear correlated to the in vivo ability of the adenosine derivatives to inhibit rat heart rate. Thermodymanic data of adenosine A1 receptor ligands are proposed as an indicator of their pharmacodynamics.
