Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists : Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes In the last few years , many efforts have been made to search for potent and selective human A3 adenosine antagonists . In particular , one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family . This class of compounds has been strongly investigated from the point of view of structure-activity relationships . In particular , it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position . In this study , we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes . The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach . Introduction Adenosine is an ubiquitous modulator , which exerts its functions through interaction with four G-protein-coupled receptors classified as A1 , A2A , A2B and A3 -LSB- 1 -RSB- . In recent decades , intensive and successful efforts have been made by medicinal chemists to discover potent and selective ligands -LRB- both agonists and antagonists -RRB- for almost all adenosine receptor subtypes -LSB- 2 , 3 -RSB- . Only the A2B subtype is still without a very potent and selective agonist ligand -LSB- 4 -- 6 -RSB- . At the same time , great progress has been made in the field of human A3 adenosine receptor antagonists . This receptor seems to be associated with cerebroprotection -LSB- 7 , 8 -RSB- and cardioprotection -LSB- 9 -RSB- and effects on the immune and inflammatory systems -LSB- 10 , 11 -RSB- . The A3 adenosine receptor subtype , recently cloned from different species -LRB- e.g. , rat , human , dog , sheep -RRB- -LSB- 12 , 13 -RSB- , is coupled to the modulation of at least two second-messenger systems : inhibition of adenylate cyclase and stimulation of phospholipase C and D -LSB- 12 , 13 -RSB- . In humans , A3 receptors have been found in several organs , such as lung , liver , kidney , heart , and with a lower density , in the brain -LSB- 9 , 12 , 13 -RSB- . This receptor subtype is under examination in relation to its potential therapeutic applications . In particular , antagonists for A3 receptors seem to be useful for the treatment of inflammation or glaucoma -LSB- 14 , 15 -RSB- . In the last 5 years , many efforts have been made in the context of searching for potent and selective human A3 adenosine antagonists . In this field , several different classes of compounds have been proposed , possessing good affinity -LRB- nM range -RRB- and with a broad range of selectivity -LSB- 16 -RSB- . In particular one of the most attractive human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family , reported by our group -LSB- 17 -- 23 -RSB- . In Fig. 1 , the most interesting members of this class of compounds -LRB- 1 -- 3 -RRB- , which display high affinity and selectivity for this receptor subtype , are depicted -LSB- 17 -- 23 -RSB- . Fig. 1Structures and binding profiles of some representative pyrazolo-triazolo-pyrimidines as human A3 adenosine receptor antagonists This class of compounds has been strongly investigated from the point of view of structure-activity relationships . In particular , it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are a small substituent at the N8 position and an unsubstituted phenyl carbamoyl moiety at the N5 position -LSB- 17 , 21 -RSB- . In contrast , when the phenyl carbamoyl moiety -LRB- 2 -RRB- was replaced with a phenylacetyl group -LRB- 3 -RRB- , a significant decrease in affinity at the hA3 adenosine receptors with a simultaneous retention or a slight increase in affinity at the hA2B adenosine receptor subtype was observed -LSB- 24 -RSB- . These data seem to suggest that bond type at the N5 position plays a fundamental role in the receptor recognition . For this reason , we decided to further investigate the effect of the bond nature at this position on affinity and selectivity , synthesizing two new derivatives bearing the benzoyl - -LRB- 4 -RRB- and benzensulfonamido -LRB- 5 -RRB- moieties at the N5 position . Chemistry The designed compounds -LRB- 4 , 5 -RRB- were easily prepared by treating the well-known N8 methyl derivative -LRB- 6 -RRB- -LSB- 18 -RSB- with benzoyl -LRB- 7 -RRB- or benzensulfonyl -LRB- 8 -RRB- chlorides in dioxane in the presence of pyridine at reflux for 16 h -LRB- Scheme 1 -RRB- . Scheme 1Preparation of designed compounds Results and discussion Newly synthesized -LRB- 4 , 5 -RRB- and reference compounds -LRB- 2 , 3 -RRB- were tested at the human A1 , A2A and A3 receptors expressed in CHO cells ; -LSB- 3H -RSB- CCPA -LRB- A1 -RRB- and -LSB- 3H -RSB- NECA -LRB- A2A , A3 -RRB- were used as radioligands in binding assays -LSB- 25 -RSB- . Radioligand binding at A2B adenosine receptors is problematic as no high-affinity ligand is readily available for this subtype . Therefore , inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of affinity of compounds -LRB- Table 1 -RRB- . Table 1Biological profile of synthesized -LRB- 4 , 5 -RRB- and reference -LRB- 2 , 3 -RRB- compoundsCompoundRhA1a -LRB- Ki nM -RRB- hA2Ab -LRB- Ki nM -RRB- hA2Bc -LRB- IC50 nM -RRB- hA3d -LRB- Ki nM -RRB- hA1/hA3hA2A/hA32CONHPh310 -LRB- 295 -- 327 -RRB- 27.7 -LRB- 13.3 -- 57.8 -RRB- 3,440 -LRB- 2,880 -- 4,110 -RRB- 1.80 -LRB- 0.88 -- 3.68 -RRB- 172153COCH2Ph1 ,040 -LRB- 864 -- 1,260 -RRB- 282 -LRB- 201 -- 375 -RRB- 12,620 -LRB- 9,730 -- 16,400 -RRB- 0.92 -LRB- 0.80 -- 1.06 -RRB- 1,1303604 COPh2 ,030 -LRB- 1,710 -- 2,400 -RRB- 879 -LRB- 643 -- 1,200 -RRB- > 30,00015.7 -LRB- 7.85 -- 31.5 -RRB- 129565SO2Ph20 ,700 -LRB- 16,700 -- 25,700 -RRB- 6,060 -LRB- 5,170 -- 7,110 -RRB- > 30,000744 -LRB- 534 -- 1,040 -RRB- 288Data are expressed as geometric means , with 95 % confidence limitsaDisplacement of specific -LSB- 3H -RSB- - CCPA binding at human A1 receptors expressed in CHO cells , -LRB- n = 3 -- 6 -RRB- bDisplacement of specific -LSB- 3H -RSB- - NECA binding at human A2A receptors expressed in CHO cellscIC50 values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing hA2B receptorsdDisplacement of specific -LSB- 3H -RSB- - NECA binding at human A3 receptors expressed in CHO cells A careful examination of the data clearly revealed significant differences in the binding profile of the reference compounds -LRB- 2 , 3 -RRB- compared with the literature data -LRB- see Fig. 1 -RRB- . In fact , in these binding studies , compound 2 was found to be ninefold less potent at the hA3 receptors with a consequent reduction in selectivity versus the other receptor subtypes , in particular versus hA2A -LRB- hA2A/hA3 = 15 -RRB- . In contrast , derivative 3 retained good affinity at the hA3 adenosine receptor and a quite good range of selectivity versus the other receptors in comparison with literature data . Also if these discrepancies in binding profile could be attributed to a different biological method utilized -LRB- e.g. , radioligand -RRB- , the data seem to indicate that the phenylacetyl moiety retains or enhances both potency and selectivity compared with the well-known phenylcarbamoyl chain . A significantly different binding profile was instead observed when the chain at the N5 position was alternatively modified . Introduction of a shorter amide -LRB- 4 -RRB- with respect phenylacetyl chain -LRB- 3 -RRB- led to a loss of potency at all four adenosine receptor subtypes , but a quite good retention of selectivity was observed . In contrast , the presence of a benzensulfonamido moiety at the N5 position induced a substantial loss of both potency and selectivity at the four adenosine receptor subtypes . Molecular modeling studies were performed on the pyrazolo-triazolo-pyrimidine derivatives 2 -- 5 in order to identify the hypothetical binding motif of these N5 analogues and to rationalize their structure-activity relationship . Following our previously reported modeling studies -LSB- 17 -- 23 -RSB- , we built up a refined model of human A3 receptor by using a rhodopsin-based homology modeling -LRB- RBHM -RRB- approach -LSB- 26 -- 29 -RSB- . Moreover , our recently described ligand-based homology modeling methodology -LRB- LBHM -RRB- has been used to simulate the conformational changes induced by ligand binding -LRB- methodological details are summarized in the Experimental Section -RRB- -LSB- 29 , 30 -RSB- . Using this methodology , we found an `` expanded '' conformational model of the human A3 receptor reverse agonist-like state , in which both shape and chemical complementarities have been specifically optimized around each ligand . Considering these new N5 analogues , the molecular volume of transmembrane -LRB- TM -RRB- binding cavity has been changed from 660 -LRB- A3 model obtained by the conventional rhodopsin-based homology modeling -RRB- to 840 Å3 -LRB- expanded A3 model obtained by ligand-based homology modeling -RRB- without altering the conventional rhodopsin-like receptor topology . The binding cavity reorganization induced by ligand binding is due to the conformational change in several amino acid side chains , such as Leu90 -LRB- 3.32 -RRB- , Leu91 -LRB- 3.33 -RRB- , Thr94 -LRB- 3.36 -RRB- , His95 -LRB- 3.37 -RRB- , Ile98 -LRB- 3.40 -RRB- , Gln167 -LRB- EL2 -RRB- , Phe168 -LRB- EL2 -RRB- , Phe182 -LRB- 5.43 -RRB- , Ile186 -LRB- 5.47 -RRB- , Leu190 -LRB- 5.51 -RRB- , Phe239 -LRB- 6.44 -RRB- , Trp243 -LRB- 6.48 -RRB- , Leu244 -LRB- 6.49 -RRB- , Leu264 -LRB- 7.35 -RRB- , and Ile268 -LRB- 7.39 -RRB- . Interestingly , none of the new pyrazoloquinoline antagonists found an energetically stable docking pose in the conventional RBHM-driven A3 model . This is mainly due to the unfavorable topological complementarity among these antagonists and corresponding RBHM-driven TM binding cavity . In particular , highly destabilizing van der Waals interactions -LRB- steric conflicts -RRB- seem to be the reason for absent topological complementarities . These steric conflicts are drastically reduced or completely eliminated after the application of the LBHM approach . Molecular docking studies were carried out for the pyrazolo-triazolo-pyrimidine antagonists 2 -- 4 , using the `` expanded '' conformational state of the receptor . As shown in Fig. 2 , we found a similar binding motif indicating that a common receptor-driven pharmacophore model can be depicted . This finding is in agreement with our previously reported studies -LSB- 17 -- 23 -RSB- . Fig. 2Hypothetical binding motif of the newly synthesized pyrazolo-triazolo-pyrimidine antagonists 2 -- 4 . The most energetically favorable docked conformation of each derivative is viewed from the membrane side facing TM helices 4 and 5 . To clarify the TM cavity , the view of TM4 was omitted . Side chains of some amino acids important for ligand recognition are highlighted . Hydrogen atoms are not displayed Indeed , ligand recognition occurs in the upper region of the TM bundle , and the pyrazolo-triazolo-pyrimidine moiety is surrounded by TMs 3 , 5 , 6 , 7 with the substituent in the N5 position oriented toward the intracellular environment . As shown in Fig. 2 , the furan ring at the 2-position is close to TMs 3 and 7 . Interestingly , an important hydrogen bonding network can be observed in all energetically stable docked conformations of pyrazolo-triazolo-pyrimidine antagonists . In particular His95 -LRB- 3.37 -RRB- and Ser247 -LRB- 6.52 -RRB- are able to interact through hydrogen bonding with the N5-carbonyl oxygen of compounds 2 -- 4 -LRB- 2C =O · His95 ca. 3.0 Å ; 3C =O · Ser247 ca. 2.8 Å ; 4C =O · His95 ca. 2.9 Å . -RRB- These polar amino acids seem to be critical for the recognition of all antagonist structures and for receptor selectivity . In particular , Ser247 -LRB- 6.52 -RRB- of human A3 receptor subtype is not present in the corresponding position of A1 and A2 receptors , where the residue is replaced by a histidine -LRB- His251 in human A1 , His250 in human A2A and His251 in human A2B -RRB- . Histidine side chain is bulkier than serine , and possibly for this reason , large substituents at the N5 position of pyrazolo-triazolo-pyrimidine framework are not well tolerated by A1 and A2 receptor subtypes . In contrast , the hydroxyl group of Ser247 -LRB- 6.52 -RRB- of human A3 receptor is appropriately positioned to form a hydrogen-bonding interaction with the carbonyl oxygen of the N5-amide/ureide group of compounds 2 -- 4 . These observations support the importance of an N5-acyl/carbamoyl group in modulating receptor selectivity . The hydrophobic environment of the five nonpolar amino acids Ile98 -LRB- 3.40 -RRB- , Ile186 -LRB- 5.47 -RRB- , Leu190 -LRB- 5.51 -RRB- , Phe239 -LRB- 6.44 -RRB- , and Leu244 -LRB- 6.49 -RRB- can comfortably accommodate the phenyl ring of all N5-acyl/carbamoyl derivatives . In contrast , the introduction of the N5-sulfonamido moiety , as present in derivative 5 , drastically reduces the affinity at the human A3 receptor . Interestingly , in this specific case molecular docking is not able to find an antagonist pose comparable to those described for the other N5-acyl/carbamoyl derivatives . As shown in Fig. 3 , the rigid tetrahedral configuration associated with the N5-sulfonamido moiety avoids the sampling of energetically favorable antagonist poses in which the phenyl ring is linked to the N5 position in the hydrophobic pocket delimited by Ile98 -LRB- 3.40 -RRB- , Ile186 -LRB- 5.47 -RRB- , Leu190 -LRB- 5.51 -RRB- , Phe239 -LRB- 6.44 -RRB- , and Leu244 -LRB- 6.49 -RRB- . Fig. 3Hypothetical binding motif of the newly synthesized N5-sulfonamido pyrazolo-triazolo-pyrimidine antagonist 5 . The most energetically favorable docked conformation of each derivative is viewed from the membrane side facing TM helices 4 and 5 . To clarify the TM cavity , the view of TM4 was omitted . Hydrogen atoms are not displayed The most stable docking pose of 5 presents the N5-sulfonamido moiety close to TM3 and TM7 , and the phenyl ring linked to N5 position is surrounded by a hydrophobic pocket delimited by Leu90 -LRB- 3.32 -RRB- and Ile268 -LRB- 7.39 -RRB- . This antagonist pose is energetically less stable -LRB- ca. 15 kcal/mol -RRB- with respect to those found for derivatives 2 -- 4 , due to the absence of the stabilizing interactions among the polar residues Thr94 -LRB- 3.36 -RRB- , His95 -LRB- 3.37 -RRB- , and Ser247 -LRB- 6.52 -RRB- and the N5-sulfonamido moiety . Structure superimposition of compounds 4 and 5 is shown in Fig. 4 . Fig. 4Structure superimposition of compounds 4 -LRB- in magenta -RRB- and 5 -LRB- in green -RRB- inside the receptor binding site This severe steric constriction might explain the drastic reduction in affinity of derivative 5 at the human A3 receptor . Conclusions The present study has led to potent and selective human A3 adenosine receptor antagonists belonging to the class of the pyrazolo-triazolo-pyrimidine . In this study , we clarified the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes . In particular , we confirmed the critical function played by the N5-acyl/carbamoyl moiety in receptor recognition . The observed structure-activity relationship was also rationalized by the recently published ligand-based homology modeling approach , indicating that chemical and topological properties of the different ligands might induce important conformational changes in the antagonist-driven binding site of the human A3 adenosine receptor . Experimental section Chemistry General Reactions were routinely monitored by thin-layer chromatography -LRB- TLC -RRB- ion silica gel -LRB- precoated F254 Merck plates -RRB- and products visualized with iodine or potassium permanganate solution . Infrared spectra -LRB- IR -RRB- were measured on a Perkin Elmer 257 instrument . 1H NMR was determined in CDCl3 or DMSO-d6 solutions with a Bruker AC 200 spectrometer ; peak positions are given in parts per million -LRB- 6 -RRB- downfield from tetramethylsilane as internal standard , and J values are given in Hz . Light petroleum ether refers to the fractions boiling at 40 -- 60 °C . Melting points were determined on a Buchi-Tottoli instrument and are uncorrected . Chromatographies were performed using Merck 60 -- 200 mesh silica gel . All products reported showed IR and 1H NMR spectra in agreement with the assigned structures . Organic solutions were dried over anhydrous magnesium sulphate . Elemental analyses were performed by the microanalytical laboratory of Dipartimento di Chimica , University of Trieste . General procedures for the preparation of 5 - -LRB- benzoyl -RRB- amino-8-methyl-2 - -LRB- 2-furyl -RRB- - pyrazolo -LSB- 4,3 -RSB- -1,2,4-triazolo -LSB- 1,5-c -RSB- pyrimidine -LRB- 4 -RRB- and 5-benzensulfonamido-8-methyl-2 - -LRB- 2-furyl -RRB- - pyrazolo -LSB- 4,3-e -RSB- -1,2,4-triazolo -LSB- 1,5-c -RSB- pyrimidine -LRB- 5 -RRB- Amino compound -LRB- 6 -RRB- -LRB- 10 mmol -RRB- was dissolved in freshly distilled dioxane -LRB- 15 mL -RRB- , and benzoylchloride or benzesulfonyl chloride -LRB- 13 mmol -RRB- and pyridine -LRB- 20 mmol -RRB- were added . The mixture was refluxed under argon for 16 h . Then the solvent was removed under reduced pressure and the residue was dissolved in EtOAc -LRB- 30 ml -RRB- and washed twice with water -LRB- 15 mL -RRB- . The organic phase was dried on Na2SO4 and concentrated under reduced pressure . The residue was purified by flash chromatography -LRB- EtOAc-light petroleum 8:2 -RRB- to afford the desired compounds -LRB- 4 , 5 -RRB- as solids . 5 - -LRB- Benzoyl -RRB- amino-8-methyl-2 - -LRB- 2-furyl -RRB- - pyrazolo -LSB- 4,3-e -RSB- -1,2,4-triazolo -LSB- 1,5-c -RSB- pyrimidine -LRB- 4 -RRB- Yield 78 % , pale yellow solid , mp -LRB- EtOAc-light petroleum -RRB- 240 °C ; IR -LRB- KBr -RRB- : 3,425-2,965 , 1,675 , 1,645 , 1,620 , 1,545 , 1,450 cm − 1 ; 1 H NMR -LRB- DMSO d6 -RRB- δ : 4.42 -LRB- s , 3H -RRB- ; 6.65 -LRB- dd , 1H , J = 2 , J = 4 -RRB- ; 7.24 -LRB- d , 1H , J = 4 -RRB- ; 7.41 -- 7.62 -LRB- m , 4H -RRB- ; 7.85 -- 7.98 -LRB- m , 2H -RRB- ; 8.14 -LRB- s , 1H -RRB- ; 9.87 -LRB- bs , 1H -RRB- . Anal . for C18H13N7O2 -LRB- MW 359.34 -RRB- . Calculated : C 50.16 ; H 3.69 ; N 27.29 . Found : C 50.01 ; H 3.61 ; N 27.35 . 5-Benzensulfonamido-8-methyl-2 - -LRB- 2-furyl -RRB- - pyrazolo -LSB- 4,3-e -RSB- -1,2,4-triazolo -LSB- 1,5-c -RSB- pyrimidine -LRB- 5 -RRB- Yield 62 % , white solid , mp -LRB- EtOAc-light petroleum -RRB- > 300 °C ; IR -LRB- KBr -RRB- : 3,375-2,985 , 1,640 , 1,600 , 1,540 , 1,355 , 1,150 cm − 1 ; 1H NMR -LRB- DMSO d6 -RRB- δ : 4.39 -LRB- s , 3H -RRB- ; 6.68 -LRB- dd , 1H , J = 2 , J = 4 -RRB- ; 7.22 -LRB- d , 1H , J = 4 -RRB- ; 7.38 -- 7.59 -LRB- m , 4H -RRB- ; 7.88-8.02 -LRB- m , 2H -RRB- ; 8.11 -LRB- s , 1H -RRB- ; 11.05 -LRB- bs , 1H -RRB- . Anal . for C17H13N7O3S -LRB- MW 395.40 -RRB- . Calculated : C 51.64 ; H 3.31 ; N 24.80 ; S 8.11 . Found : C 51.87 ; H 3.35 ; N 24.97 ; S 8.07 . Biology All pharmacological methods followed the procedures as described earlier -LSB- 25 -RSB- . In brief , membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure . In a first low-speed step -LRB- 1,000 × g -RRB- , cell fragments and nuclei were removed . The crude membrane fraction was sedimented from the supernatant at 100,000 × g . The membrane pellet was resuspended in the buffer used for the respective binding experiments , frozen in liquid nitrogen and stored at − 80 °C . For the measurement of adenylyl cyclase activity only one high-speed centrifugation of the homogenate was used . The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl , pH 7.4 and immediately used for the cyclase assay . For radioligand binding at A1 adenosine receptors , 1 nM -LSB- 3H -RSB- CCPA was used , whereas 30 and 10 nM -LSB- 3H -RSB- NECA were used for A2A and A3 receptors , respectively . Nonspecific binding of -LSB- 3H -RSB- CCPA was determined in the presence of 1 mM theophylline , and in the case of -LSB- 3H -RSB- NECA , 100 pM R-PIA was used . Ki values from competition experiments were calculated with the program SCTFIT -LSB- 31 -RSB- . Radioligand binding at A2B adenosine receptors is problematic as no high-affinity ligand is available for this subtype . Therefore , inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of affinity of compounds . IC50 values from these experiments were converted to Ki values with the Cheng and Prusoff equation -LSB- 32 -RSB- . Computational methodologies All modeling studies were carried out on a 10 CPU -LRB- PIV-3.0 GHZ and AMD64 -RRB- Linux cluster running under openMosix architecture -LSB- 33 -RSB- . Homology modeling , energy calculation , and docking studies were performed using the Molecular Operating Environment -LRB- MOE , version 2006.08 -RRB- suite -LSB- 34 -RSB- . All docked structures were fully optimized without geometry constraints using RHF/AM1 semiempirical calculations . Vibrational frequency analysis was used to characterize the minima stationary points -LRB- zero imaginary frequencies -RRB- . The software package MOPAC -LRB- ver . 7 -RRB- -LSB- 35 -RSB- , implemented in MOE suite , was utilized for all quantum mechanical calculations . Homology model of the human A3 AR Based on the assumption that GPCRs share similar TM boundaries and overall topology , a homology model of the hA3 receptor was constructed . First , the amino acid sequences of TM helices of the A3 receptor were aligned with those of bovine rhodopsin , guided by the highly conserved amino acid residues , including the DRY motif -LRB- D3 .49 , R3 .50 , and Y3 .51 -RRB- and three proline residues -LRB- P4 .60 , P6 .50 , and P7 .50 -RRB- in the TM segments of GPCRs . The same boundaries were applied for the TM helices of the A3 receptor as they were identified from the X-ray crystal structure for the corresponding sequences of bovine rhodopsin -LSB- 36 -RSB- , the CR coordinates of which were used to construct the seven TM helices for the hA3 receptor.The loop domains of the hA3 receptor were constructed by the loop search method implemented in MOE . In particular , loops are modeled first in random order . For each loop , a contact energy function analyzes the list of candidates collected in the segment searching stage , taking into account all atoms already modeled and any atoms specified by the user as belonging to the model environment . These energies are then used to make a Boltzmann-weighted choice from the candidates , the coordinates of which are then copied to the model . Any missing side-chain atoms are modeled using the same procedure . Side chains belonging to residues whose backbone coordinates were copied from a template are modeled first , followed by side chains of modeled loops . Outgaps and their side chains are modeled last.Special caution has to be given to the second extracellular -LRB- EL2 -RRB- loop , which has been described in bovine rhodopsin as folding back over transmembrane helices -LSB- 36 -RSB- and , therefore , limiting the size of the active site . Hence , amino acids of this loop could be involved in direct interactions with the ligands . A driving force for this peculiar fold of the EL2 loop might be the presence of a disulfide bridge between cysteines in TM3 and EL2 . Since this covalent link is conserved in all receptors modeled in the current study , the EL2 loop was modeled using a rhodopsin-like constrained geometry around the EL2-TM3 disulfide bridge . After the heavy atoms were modeled , all hydrogen atoms were added , and the protein coordinates were then minimized with MOE using the AMBER94 force field -LSB- 37 -RSB- . The minimizations were carried out by the 1,000 steps of steepest descent followed by conjugate gradient minimization until the rms gradient of the potential energy was less than 0.1 kcal mol − 1 Å − 1 . Protein stereochemistry evaluation was performed by several tools -LRB- Ramachandran and Chi plots measure phi/psi and chi1/chi2 angles , clash contacts reports -RRB- implemented in MOE suite -LSB- 34 -RSB- . Ligand-based homology modeling We have recently revisited the rhodopsin-based model of the human A3 receptor in its resting state -LRB- antagonist-like state -RRB- , taking into account a novel strategy to simulate the possible receptor reorganization induced by the antagonist-binding -LSB- 28 -RSB- . We called this new strategy ligand-based homology modeling . Briefly , ligand-based homology modeling technique is an evolution of a conventional homology modeling algorithm based on a Boltzmann-weighted randomized modeling procedure adapted from Levitt -LSB- 38 -RSB- with specialized logic for the proper handling of insertions and deletions so that any selected atoms will be included in the energy tests and minimization stages of the modeling procedure . Ligand-based option is very useful when one wishes to build a homology model in the presence of a ligand docked to the primary template , or other proteins known to be complexed with the sequence to be modeled -LSB- 34 -RSB- . In this specific case , both model building and refinement take into account the presence of the ligand in terms of specific steric and chemical features . In order to generate an initial ensemble of ligand poses , a conventional docking procedure -LRB- see next section for details -RRB- with reduced van der Waals radii -LRB- equal to 75 % -RRB- and an increased Coulomb-vdW cutoff -LRB- cutoff on 10 Å ; cutoff on 12 Å -RRB- was performed . For each pose , an homology model is then generated to accommodate the ligand by reorienting nearby side chains . These residues and the ligand are then locally minimized . Finally , each ligand is re-docked into its corresponding low energy protein structures and the resulting complexes are ranked according to MOEScore -LSB- 34 -RSB- . Different quantitative measures of molecular volume of the receptor binding cavities have been carried out by using MOE suite -LSB- 34 -RSB- . Prediction of antagonist-receptor complex stability -LRB- in terms of corresponding pKi value -RRB- and the quantitative analysis for non-bonded intermolecular interactions -LRB- H-bonds , transition metal , water bridges , hydrophobic -RRB- were calculated and visualized using several tools implemented into MOE suite -LSB- 34 -RSB- . Molecular docking of the hA3 AR antagonists All antagonist structures were docked into the hypothetical TM binding site by using the MOE-dock tool , part of the MOE suite . Searching is conducted within a user-specified 3D docking box -LRB- the standard protocol selects all atoms inside 12 Å from the center of mass of the binding cavity -RRB- , using the Tabu Search -LSB- 39 -RSB- protocol -LRB- standard parameters are 1,000 steps/run , 10 attempts/step , and 10 Tabu list length -RRB- , and the MMFF94 force field -LSB- 40 -RSB- . MOE-dock performs a user-specified number of independent docking runs -LRB- 50 in our specific case -RRB- and writes the resulting conformations and their energies in a molecular database file . The resulting docked complexes were subjected to MMFF94 energy minimization until the rms of conjugate gradient was < 0.1 kcal mol − 1 Å − 1 . Charges for the ligands were imported from the MOPAC output files . To better refine all antagonist-receptor complexes , a rotamer exploration of all side chains involved in the antagonist-binding was carried out . Rotamer exploration methodology was implemented in MOE suite -LSB- 34 -RSB- .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

antagonist binding
ligand-based homology modeling


Determinants of potential drug -- drug interaction associated dispensing in community pharmacies in the Netherlands Objective : There are many drug -- drug interactions -LRB- D -- DI -RRB- of which some may cause severe adverse patient outcomes . Dispensing interacting drug combinations should be avoided when the risks are higher than the benefits . The objective of this study was to identify determinants of dispensing undesirable interacting drug combinations by community pharmacies in the Netherlands . Statements on the impact of the article on practice Process and structure characteristics may influence the dispensing of undesirable interacting drug combinations in community pharmacies but probably to a minor degree . Medication surveillance in Dutch pharmacies seems to be effective . Introduction Drug -- drug interactions -LRB- D -- DIs -RRB- are responsible for many adverse patient outcomes . Different studies suggest that D -- DIs may cause up to 3 % of all hospital admissions -LSB- 1 -- 4 -RSB- . A D -- DI is defined as a pharmacokinetic or pharmacodynamic influence of drugs on each other , which may result in desired effects , in reduced efficacy and effectiveness or in increased toxicity -LSB- 5 -RSB- . Although many D -- DIs exist , only a small part of these D -- DIs is clinically relevant -LSB- 6 -- 8 -RSB- . The potential benefits of drug combinations should be weighed against the seriousness of the D -- DI , taking into account the availability of alternatives . Only in cases that the risks associated with the D -- DI are higher than the benefits or if a better alternative is available , the D -- DI should be avoided . In the Netherlands , one of the tasks of the pharmacist is to intervene in case of D -- DIs , which involve a high risk for the patient . Hereto , the pharmacist uses patient characteristics and the medication history . All prescriptions , which are submitted to the pharmacy , are screened on potential interactions with the help of medication surveillance software . These D -- DIs are evaluated by the pharmacist who intervenes if necessary . This task is important but cumbersome , and requires great attention from the pharmacist . The organisational aspects , such as the tuning of the medication surveillance software and instructions of technicians , should be managed by the pharmacist in such a way that in case of D -- DIs with a high risk the pharmacist intervenes . This is important for the prevention of adverse patient outcomes -LSB- 9 -RSB- . The objective of this study was to assess process and structure characteristics associated with the dispensing of interacting drug combinations , which carry a high risk of adverse patient outcomes . Methods Setting The data for this study were retrieved from the Drug Information Project , a division of the Health Care Insurance Board . This is a database containing the reimbursement data from eight health care insurance companies in the Netherlands . The reimbursement data from January 1st , 2001 until October 31st , 2002 were analysed . Eleven potential D -- DIs , that contained a high risk and could be substituted , because a good alternative was available , were selected and counted for each pharmacy in the database . These undesirable potential D -- DIs were mostly interactions between chronically used drugs which can not be interrupted and short-term use of antibiotics or antimycotics , and were selected from the Dutch guidelines for the management of D -- DIs -LRB- Table 1 -RRB- -LSB- 10 , 11 -RSB- . A D -- DI was counted as such , when the chronically used drug was dispensed both in the period 150 days preceding and in the period 150 days after the dispensing of antibiotics or antimycotics for short-term use in the same pharmacy . Pharmacies with less than 5,000 dispensings in the database were excluded . Table 1Number of dispensings in the database of the individual drugs involved , the eleven potential D -- DIs and the calculated ratioDrug -- drug interactionNumber of dispensings drug A × 1,000 -LRB- range -RRB- Number of dispensings drug B × 1,000 -LRB- range -RRB- Number of D -- DIs counted -LRB- range -RRB- Average ratio -LRB- range -RRB- aDrug ADrug B1Erythromycin , clarithromycin , azithromycin , roxithromycinDigoxin440 .8 -LRB- 0 -- 2754 -RRB- 487.0 -LRB- 0 -- 3064 -RRB- 3,993 -LRB- 0 -- 41 -RRB- 1.39 -LRB- 0 -- 18.52 -RRB- 2ItraconazoleDigoxin88 .7 -LRB- 0 -- 349 -RRB- 487.0 -LRB- 0 -- 3064 -RRB- 245 -LRB- 0 -- 7 -RRB- 0.45 -LRB- 0 -- 21.69 -RRB- 3CiprofloxacinTheophylline105 .4 -LRB- 0 -- 769 -RRB- 100.9 -LRB- 0 -- 756 -RRB- 944 -LRB- 0 -- 14 -RRB- 6.39 -LRB- 0 -- 534.38 -RRB- 4Miconazole oral gelAcenocoumarol , fenprocoumon44 .6 -LRB- 0 -- 233 -RRB- 608.2 -LRB- 5 -- 3156 -RRB- 154 -LRB- 0 -- 3 -RRB- 0.38 -LRB- 0 -- 21.30 -RRB- 5ErythromycinCarbamazepine49 .7 -LRB- 0 -- 531 -RRB- 193.6 -LRB- 0 -- 871 -RRB- 35 -LRB- 0 -- 4 -RRB- 0.24 -LRB- 0 -- 40.92 -RRB- 6Erythromycin , clarithromycin , azithromycinDisopyramide426 .6 -LRB- 0 -- 2754 -RRB- 9.4 -LRB- 0 -- 151 -RRB- 61 -LRB- 0 -- 4 -RRB- -- 7Erythromycin , clarithromycinPimozide274 .4 -LRB- 0 -- 2004 -RRB- 57.4 -LRB- 0 -- 394 -RRB- 70 -LRB- 0 -- 15 -RRB- 0.46 -LRB- 0 -- 46.12 -RRB- 8Propranolol , oxprenolol , pindololbeta2-mimetics , inhalation corticosteroids250 .6 -LRB- 1 -- 1075 -RRB- 2,546.9 -LRB- 27 -- 10504 -RRB- 5,127 -LRB- 0 -- 94 -RRB- 0.54 -LRB- 0 -- 12.98 -RRB- 9Erythromycin , clarithromycinCisapride274 .4 -LRB- 0 -- 2004 -RRB- 127.5 -LRB- 0 -- 821 -RRB- 586 -LRB- 0 -- 11 -RRB- 1.16 -LRB- 0 -- 40.45 -RRB- 10Itraconazole , fluconazole , ketoconazoleCisapride199 .9 -LRB- 0 -- 727 -RRB- 127.5 -LRB- 0 -- 821 -RRB- 347 -LRB- 0 -- 12 -RRB- 0.95 -LRB- 0 -- 57.10 -RRB- 11Acenocoumarol , fenprocoumonAzapropazon608 .2 -LRB- 5 -- 3156 -RRB- 8.4 -LRB- 0 -- 164 -RRB- 32 -LRB- 0 -- 19 -RRB- -- aCalculated with Formula 1 Procedure For each pharmacy , we calculated the dispensing-ratios for the eleven potential D -- DIs with Formula 1 . This formula was used because the risk of dispensing a D -- DI between drug A and drug B is dependent on the number of times each drug is dispensed . The more drug A or drug B are dispensed , the higher the risk that these drugs are combined on the basis of chance alone . In case the dispensing of drug A is independent from the dispensing of drug B and the D -- DI is never intervened , the ratio will on average be one . The number of times this ratio was above one was calculated , because a ratio above one might indicate that medication surveillance fails . In this calculation , there were 342 D -- DIs between norfloxacin and theophylline , which were excluded in the analysis because the guidelines concerning the management of this D -- DI were inconsistent . Two groups of pharmacies were selected , and the pharmacists were asked in August 2003 by the Inspectorate for Health Care -LRB- IHC -RRB- to fill in a questionnaire . The first group included pharmacies with a high risk of dispensing these D -- DIs , while the second group consisted of a random sample from the remaining pharmacies . These groups were equal in size . The selection criteria are described in Fig. 1 . The selection criterion for receiving a questionnaire -LRB- ≥ 4 times a ratio > 1 -RRB- was chosen on pragmatic reasons to have enough power for statistical analysis with manageable numbers . A concept questionnaire was composed on basis of a literature search and interviews with experts -LSB- 9 -RSB- . The questions concerned process and structure characteristics of several quality aspects and those questions were selected that could discriminate between high and low quality pharmacies . Mostly questions with objective answers were included , for example about written instructions for technicians , filing of data , tuning of the software -LRB- which signals were shown and which not -RRB- and personnel . The concept questionnaire was tested in three pharmacies and some questions were amended on the basis of their comments . The final questionnaire contained 183 questions , divided into 12 subjects -LRB- Table 2 -RRB- . The questionnaire was accessible via the Internet . Pharmacies who had no access to the Internet received the questionnaire by post . Pharmacies who failed to fill in the questionnaire received reminders at regular intervals . A sample from both groups was visited by the IHC -LRB- Fig. 1 -RRB- . Also here , the selection criterion -LRB- ≥ 5 times a ratio > 1 -RRB- was chosen to have enough power with manageable numbers . Thirty-seven questions from the questionnaire were selected and during the visits these questions were verified . The pharmacies were informed in advance that a selection of the pharmacies would be visited . The selected pharmacies were acquainted after completing the questionnaire . The visiting inspectors were blinded to the number of interacting drug combinations . Fig. 1The selection of the pharmacies receiving a questionnaire and IHC visitTable 2The subjects and number of questions in the questionnaireChapterSubject -LRB- number of questions -RRB- General pharmacy dataOwnership of the pharmacy -LRB- 1 -RRB- , cooperation with other pharmacies -LRB- 1 -RRB- , cooperation with general practitioners -LRB- 1 -RRB- , electronic submission of prescriptions -LRB- 4 -RRB- FacilitiesAlterations -LRB- 2 -RRB- Quality policySetting up and implementing a quality system -LRB- 4 -RRB- , certification -LRB- 2 -RRB- , attitude towards quality management -LRB- 12 -RRB- Quality measurementMeasurement of errors -LRB- 2 -RRB- , complaints -LRB- 1 -RRB- , patient satisfaction -LRB- 2 -RRB- , interventions -LRB- 3 -RRB- , and participation in mystery guest investigations -LRB- 2 -RRB- Receipt procedureNumber of personnel involved in dispensing a receipt -LRB- 2 -RRB- , checks in dispensing a receipt -LRB- 3 -RRB- Medication surveillance -- tuning softwareMedication surveillance system used -LRB- 1 -RRB- , tuning of the system e.g. which signals are showed and which are regarded as irrelevant -LRB- 55a -RRB- , surveillance of pharmacy preparations -LRB- 2 -RRB- Medication surveillance -- organisationThe way technicians are instructed to manage medication surveillance signals -LRB- 5 -RRB- , the way this is supervised -LRB- 2 -RRB- , number of interventions -LRB- 1 -RRB- , use of resources -LRB- 2 -RRB- , participation in courses -LRB- 4 -RRB- , management of the D -- DI between carbamazepine and erythromycin -LRB- 5 -RRB- and between Sulfamethoxazole/trimethoprim and Acenocoumarol -LRB- 7 -RRB- Medication surveillance -- recording managementThe way the management of signals is recorded -LRB- 4 -RRB- Pharmacy preparationsThe way instructions for pharmacy preparations are recorded -LRB- 1 -RRB- , the way pharmacy preparations are supervised -LRB- 3 -RRB- , the number of pharmacy preparations -LRB- 2 -RRB- , the policy regarding analysing pharmacy preparations -LRB- 3 -RRB- Personnel and workloadSubjective workload -LRB- 3 -RRB- , absence through illness -LRB- 1 -RRB- , number of receipts dispensed per technician -LRB- 2 -RRB- , personnel and experience of personnel -LRB- 18 -RRB- Patient careInformation given to patients -LRB- 6 -RRB- , information exchange with hospitals -LRB- 4 -RRB- , participation in health care projects -LRB- 4 -RRB- Farmacotherapeutic consultation groupsParticipation in farmacotherapeutic consultation groups -LRB- 3 -RRB- , agreements made -LRB- 3 -RRB- aAs the questions for the four systems -LRB- Pharmacom , Aposys , Euroned , others -RRB- differed , pharmacists had to fill in only a quarter of these questions Formula 1 : Formula 2 : whereki , ab : number of dispensings of interacting drug combination AB in pharmacy i ; ki , a : number of dispensings of drug A in pharmacy i ; ki , b : number of dispensings of drug B in pharmacy i ; Ni : total number of dispensed drugs known in the database in pharmacy i ; Ntot : total number of dispensed drugs known in the database in all pharmacies Statistical analysis For each pharmacy , dispensing-ratios for the D -- DIs , comparable to the standardised mortality ratio , were calculated using Formula 2 . With this formula , we standardise for the total number of dispensings per pharmacy in the database . In case all pharmacies dispense the D -- DIs in equal numbers , the ratio will be one for all pharmacies , and therefore the ratios have a better comparability . Pharmacies which have only a small number of dispensings in the database , will have extremely high numbers in case they dispense one or a small number of D -- DIs . Therefore , the results were equalized with Bayesian statistics to prevent extreme ups and downs due to low numbers of dispensings -LSB- 12 -RSB- . The pharmacies were divided into two sets . One set was used for the analyses and contained two-third of the pharmacies , the other set was used for the validation of the results obtained in the analyses . In the univariate analysis , correlations were searched between the answers in the questionnaire and these ratios . Correlations are only given if in both sets a correlation was found -LRB- p < 0.01 -RRB- . In the multivariate analysis , models were composed using the analysis set , predicting the dispensing of the interacting drug combinations . The models were validated using the validation set . The number of questions was too large for the multivariate analysis , and only a limited number of questions were selected . From every chapter , those questions were selected that correlated with the other questions and that could discriminate between pharmacies . Results The database contained a total of 100,295,311 dispensings in the selected study period . One thousand one hundred and forty-two pharmacies were recorded in the database with 5,000 or more dispensings . The number of dispensings per pharmacy varied from 5,019 to 264,631 . Because pharmacies receive reimbursements from several health care insurance companies and because not all health care insurance companies were included in the database , these numbers do not correspond with the total number of dispensings per pharmacy . The eleven potential D -- DIs were dispensed 11,594 times . In 5 % , more than one pharmacy was involved . As these D -- DIs could not be assigned to a single pharmacy , they were excluded from further analyses . The number of dispensings and D -- DIs are shown in Table 1 . Disopyramide -LRB- D -- DI number 6 -RRB- and azapropazon -LRB- D -- DI number 11 -RRB- were not dispensed by 44 % and 46 % of the pharmacies , respectively . Therefore , a ratio could not be calculated for these pharmacies and these D -- DIs were excluded from the analyses . The number of times a ratio above one was found was calculated -LRB- Table 1 -RRB- and pharmacies were selected as shown in Fig. 1 . Two hundred and sixty-eight pharmacies were selected to receive a questionnaire and 74 pharmacies were selected for a visit by the IHC . For several reasons , such as recent visitations and duplications in the database , 12 pharmacies were excluded . Eventually , 256 pharmacies received a questionnaire and 62 pharmacies were selected for a visit . Two hundred and forty-six questionnaires were filled in -LRB- response rate 96.1 % -RRB- and 58 -LRB- 93.5 % -RRB- pharmacies were visited after the questionnaire was completed . The judgements during the visits by the IHC were compared with the answers by the pharmacists . In 33 of the 37 verified questions , the IHCs judgement matched in more than 90 % the answer of the pharmacist . Except four questions , the judgement by the IHC was equally more positive and more negative than the answers by the pharmacist . In the univariate analysis , all combinations between the questions and D -- DIs were searched for significant correlations . Two correlations were found with D -- DI number 1 between macrolide antibiotics and digoxin -LRB- Table 3 -RRB- . Pharmacies , which are part of a health care centre dispensed this interacting drug combination more often than other pharmacies . A correlation with the type of medication surveillance system was also found . Pharmacies using the Euroned system dispensed this interacting drug combination more often , while pharmacies using the Pharmacom system dispensed this interacting drug combination less often . Table 3Significant univariate correlations between the questionnaire and the number of dispensings of the D -- DIs between macrolide antibiotics and digoxin -LRB- number 1 -RRB- QuestionCorrelationSignificanceIs the pharmacy part of a health care centre ? -LRB- 1 yes , 2 no -RRB- -LRB- yes n = 18 , no n = 228 -RRB- − 0.1650.009 Which medication surveillance system is used in the pharmacy?Pharmacom -LRB- 1 yes , 0 other -RRB- -LRB- n = 81 -RRB- − 0.2610.000 Aposys -LRB- 1 yes , 0 other -RRB- -LRB- n = 62 -RRB- 0.0880.170 Euroned -LRB- 1 yes , 0 other -RRB- -LRB- n = 89 -RRB- 0.1970.002 For the multivariate analysis , 32 variables were selected , representative of the whole range of questions . These variables were used in the analysis-set to compose models . The adjusted explained variance ranged from 2.6 % to 28.9 % -LRB- Table 4 -RRB- . The model explaining the D -- DI between macrolide antibiotics and digoxin had by far the highest adjusted explained variance . The models were validated in the validation-set , calculating the unexplained variance -LRB- Table 4 -RRB- . The six variables in this model explaining the D -- DI between macrolide antibiotics and digoxin are shown in Table 5 . Table 4Predictability of the models composed in the multivariate analysisD -- DIAdjusted explained variance -LRB- R2 -RRB- -LRB- analysis set -RRB- Unexplained variancea -LRB- validation set -RRB- 128.90.61212.8 − 0.22317.331.547.0 − 0.18514.4 − 0.4176.56.4816.10.68914.0 − 0.43102.60.90 aAn unexplained variance of zero means that the predictability found in the validation set equals the predictability in the analysis-set . The higher the unexplained variance , the worse the predictability in the validation-set compared to the analysis-setTable 5The questions in the multivariate model predicting the dispensing of the D -- DI between macrolide antibiotics and digoxin -LRB- number 1 -RRB- Variable : Answer -LRB- coding -RRB- Direction coefficientConstant3 .3679 Is the pharmacy part of a health care centre ? -LRB- yes n = 18 , no n = 228 -RRB- Yes -LRB- 0 -RRB- versus no -LRB- 1 -RRB- − 2.2749Co-trimoxazole -- acenocoumarol : no appointments were made with the GPs . The drug will be dispensed.Option 1 ` with all GPs ' -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 11 -RRB- ReferenceEight options of choice option 1 ` with all GPs ' and optionOption 2 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 10 -RRB- 1.0308 Eight ` with no GPs ' Option 3 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 4 -RRB- 0.3788 Option 4 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 4 -RRB- − 0.4542 Option 5 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 3 -RRB- 0.9026 Option 6 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 2 -RRB- − 0.5100 Option 7 -LRB- 1 -RRB- versus other option -LRB- 0 -RRB- -LRB- n = 4 -RRB- − 0.1912 Option 8 ` with no GPs ' -LRB- 1 -RRB- versus other option -LRB- 1 -RRB- -LRB- n = 202 -RRB- 0.0886 Are separate signal texts in the medication surveillance program adjusted to the situation in the pharmacy ? -LRB- yes n = 72 , no n = 165 -RRB- Yes -LRB- 0 -RRB- versus no -LRB- 1 -RRB- 0.1793 Is the management of signals traceably recorded on the receipt ? -LRB- yes n = 211 , no n = 35 -RRB- Yes -LRB- 0 -RRB- on the receipt , no not on the receipt -LRB- 1 -RRB- 0.2691 The supervision on management of signals takes place on the basis of signal lists -LRB- yes n = 158 , no n = 86 -RRB- Yes -LRB- 0 -RRB- on the basis of signal lists , no -LRB- 1 -RRB- not on the basis of signal lists0 .0723 How many receipts are dispensed per year divided by the number of fte technicians < 10 -- 4 Discussion In this study , we investigated determinants for the dispensing of 11 undesirable interacting drug combinations . In general , our results are in line with the expectation that the medication surveillance system plays an important role in medication surveillance . Although the 11 potential D -- DIs were counted 11,594 times which suggests that a considerable number of patients is exposed to potential and avoidable adverse patient outcomes , these results should be judged against a background of approximately 100 million dispensings . It is possible that in these cases due to particular circumstances any other option , such as substituting or not dispensing one of the drugs , is a less favourable choice than dispensing the D -- DI . In 5 % of the total number of D -- DIs more than one pharmacy was involved , indicating the importance of communication . For the D -- DI between macrolide antibiotics and digoxin , two determinants were found . Although the type of medication surveillance system was a determinant , this does not mean that the differences are determined by the quality of the system itself because they may also correlate with the attitude of the pharmacists using the systems . The three medications surveillance systems differ in the extent to which communication with other healthcare providers is possible and developments were made in recent years . The Pharmacom system has the most advanced communication possibilities and compared to the other systems , new developments to the Euroned system were modest . Unexpectedly , pharmacies part of a health care centre dispensed this D -- DI more often than other pharmacies . In health care centres , the communication lines between pharmacists and general practitioners are much shorter , suggesting that intervening undesirable D -- DIs will be easier . Possibly , pharmacies which are part of a health care centre oppose the opinions from the general practitioners less often , to avoid harming the cooperation within the health care centre but , of course , there may be several other reasons . For the other eight assessed D -- DIs no determinants were found in the univariate analysis , neither did the models in the multivariate analysis have a good predictability . A possible explanation is that the quality of medication surveillance in community pharmacies in the Netherlands is high . Therefore , the number of pharmacies dispensing high-risk D -- DIs seems to be small . Our study has some potential limitations . First , because we used strict inclusion criteria to prevent false-positive results , it is likely that the number of dispensings of undesirable interacting drug combinations in this study is an underestimation and it is possible that important determinants were not recognized or difficult to assess . In the univariate analyses , only those questions are given which had a significant -LRB- p < 0.01 -RRB- correlation in two independent sets . Although we included 183 questions and nine D -- DIs in the univariate analysis , the possibility of including a significant correlation by chance was small -LRB- on average 0.16 question -RRB- . Second , the reimbursement data from eight health care insurance companies were used . In the Netherlands , these companies work mostly regionally . It is nevertheless not to be expected that the determinants of dispensing interacting drugs differ per region or that pharmacies differ in their management of D -- DIs between patients of different health care insurance companies . Third , from all potential D -- DIs , only 11 -LRB- but highly clinically relevant ones -RRB- were selected for this study . According to the Dutch guidelines , for all 11 combinations the dispensing of an alternative was strongly advised as a good alternative was available . Nevertheless , it is possible that these dispensings were not an error because any other option was not possible . For example , when a patient is hypersensitive to the alternative drug recommended in the guidelines or when the alternative drug is not effective . In these cases , the benefit of both drug therapies should be weighed against the potential risks of the D -- DI . The potential risks can partly be avoided by taking appropriate measures such as monitoring of drug levels . In this study , we could not retrieve why the pharmacist had dispensed the interacting drug combination , and whether the dispensing was erroneous or not . Fourth , the questionnaire was composed on the basis of a literature search and interviews with experts . It is possible that not all characteristics correlating with the dispensing of undesirable interacting drug combinations were disclosed , such as differences in population characteristics between pharmacies . For example , pharmacies with an elderly population using more drugs simultaneously have a higher risk of dispensing interacting drug combinations than pharmacies with a younger population . Also , it is possible that in areas with many general practitioners who use a medication surveillance system for prescribing , the background chance of a D -- DI is much smaller . Fifth , it is possible that the differences between pharmacies were too small compared with the power of this study to distinguish determinants . All associations found in this study were directly related to the management of signals . In our questionnaire , we also included other topics , such as pharmacy preparations and patient care . Future research should focus on the management of a larger variety of signals than the ones in our study and on how D -- DI associated dispensing could be further reduced . Conclusion In conclusion , both medication surveillance systems and being part of a health care centre may play an important role in the management of D -- DIs and the avoidance of adverse patient outcomes . Pharmacies in a healthcare centre dispensed D -- DIs more often . For most D -- DIs , no determinants were found possibly indicating that the quality of medication surveillance in the Netherlands is high .

O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O

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