| Doxazosin, a quinazoline derivative, was produced firstly by Pfizer andsold in Denmark at1988. It is a new highly selective α1-adrenoceptorantagonist, with the effect of lowering blood pressure by dilating blood vesselthrough selectively and competitively block postsynaptic α1-adrenoceptor,while have no impact on postsynaptic or presynaptic α2-adrenoceptor. It couldalso relax both resistant and capacity vessels, reactively inhibit heart rate andimprove microcirculation, so decrease preload and afterload of the heart. It’susing in the treatment of lower urinary tract symptom （LUTS） due to benignprostatic hyperplasia （BPH）has been approved by FDA, furthermore, it coulddecrease the mass of prostate by inducing apoptosis. Additionally, doxazosincould decrease risk of coronary heart disease by benefiting lipid metabolismthrough decrease blood levels of total triglyceride （TG） and cholesterol （TC）,simultaneously increase high density lipoprotein （HDL） and decrease lowdensity lipoprotein （LDL）. However, Antihypertensive and Lipid-LoweringTreatment to Prevent Attack Trial （ALLHAT）, a large clinical study carryingout recently, showed that risk of cardiovascular disease in patients randomizedto doxazosin group increased by25%; while risk of heart failure was doubled,if compared to chlorthalidone group. Due to this result, the doxazosin treatmentarm of the study was terminated prematurely. Consequently, in the SeventhReport of the Joint National Committee on Prevention, Detection, Evaluation,and Treatment of High Blood Pressure （JNC7） guidelines, alpha-blockers werenot recommended for the routine treatment of hypertension. However,alpha-blockers were still widely used in treatment of hypertension as add-onmedicine, especially for hypertension patients with BPH.Alfuzosin, same with doxazosin, is a quinazoline compound with highlyselective α1-adrenoceptor block property. A chiral carbon atom exists in molecular structure of either doxazosin or alfuzosin, while both the twomedicines are used in clinical in their racemic enantiomers [（±）doxazosin and（±）alfuzosin]. Presently the exact effect of doxazosin and its enantiomers onisolated heart is not clear, so we studied the relationship between chiralstructure of doxazosin and alfuzosin and their effects on heart rate andcontractile force in the isolated mouse atrium. The result showed that doxazosinsignificantly affected the heart rate and contraction of the isolated mouseatrium, and induced cardiac arrest at high concentration. Chiral structure ofdoxazosin has an obvious effect on its bioactivity. laevo isomer of doxazosinhas positive inotropic action, while dextroisomer has negative effect. Alfuzosin,however, decreased the HR slightly without chiral recognition in the isolatedmouse atrium. It was reported that blood levels of hormone andpro-inflammatory cytokines related to heart function and cardiac contractility.Brain natriuretic peptide （BNP）, nuclear factor-κB （NF-κB） and interleukin-6（IL-6） are all biomarkers of heart failure. BNP is a kind of hormone mainlysecreted by ventricular wall when over stretched, also has diuretic andnatriuretic effect. NF-κB belongs to the category of "rapid-acting" primarytranscription factors, play an important role in expression of inflammatorymediator and immune reaction genes. IL-6is an important proinflammatorycytokine, it`s serum concentration is independently related to myocardialcontractility. In this study we explored the impact of doxazosin enantiomers onplasma levels of NT-proBNP, IL-6and NF-κB.Alternative pathways could mediate the inotropic or chronotropic effectsof doxazosin and its enantiomers at post-receptor levels. In order to reveal themechanism of different effect of doxazosin enantiomers on heart, we alsoinvestigated the influence of different pathway inhibitors, such as atropine,propranolol, indomethacin, phenoxybenzamine, verapamil, methylene blue andH-89, on the intropic effects of different doxazosin enantiomers in the isolatedrat left atrium strips.Binding to plasma proteins plays a major role in drug therapy as the bounddrug is difficult to pass through blood vessel wall and cell membrane, whereas the unbound drug can cross the capillary wall to reach the cellular target as wellas metabolic tissue. A change in drug binding to plasma protein significantlyaffects its pharmacokinetic and pharmacodynamic properties. Because thedifference in the protein binding property between enantiomers often causes thedifference in their pharmacokinetic characters, enantioselective protein bindingstudy is essential to the understanding of their pharmacology, safety andclinical efficacy. Hence, it is necessary to stereoselectively detect and quantifyeach enantiomer of （±）doxazosin in biological media. In the present study, theequilibrium dialysis technique has been utilized to determine the plasma proteinbinding of the enantiomers of （±）doxazosin to the rat, dog and human plasma.Chiral HPLC-FL methods have been validated and employed to measure thedrug concentration on each side of equilibrium.Part I Effects of doxazosin enantiomers on heart rate, contractile force andplasma heart failure biomarkersIn this part, the isolated mouse atrium strip were used to study therelationship between chiral structure of doxazosin, alfuzosin and their effectson heart, namely heart rate and contractile force. In order to understand theinfluence of different optical enantiomers of doxazosin on cardiac function ofrats, we studied the effect of long-term doxazosin or its enantiomersadministration on normal rat plasma NT-proBNP, NF-κB and IL-6levels.1. Cardiac arrest effect of （±）doxazosin and （±）alfuzosin enantiomers onisolated mouse right atriumAmong16samples in （+）doxazosin group,5cases of cardiac arrest wereinduced at concentration of30μmol·L-1, so cardiac arrest rate was31.3%.（±）Doxazosin induced one cardiac arrest at30μmol·L-1, while （-）doxazosininduced one cardiac arrest at concentration of10μmol·L-1and30μmol·L-1respectively. No cardiac arrest was induced in （-）alfuzosin,（+）alfuzosin,（±）alfuzosin group and control group.2. Impact of （±）doxazosin,（±）alfuzosin and their enantiomers on heart rate ofisolated mouse right atriumHeart rates of isolated right atrium before administration of the drug were nearly same among all groups （P>0.05），solvent has no effect on HR of isolatedright atrium （P>0.05）.（+）doxazosin and （±）doxazosin at used concentrationssignificantly decreased the heart rate in a concentration-dependent manner（P<0.01）； HRs were decreased by （83.47±12.23）%and （74.97±15.66）%respectively induced by （+）doxazosin and （±） doxazosin at concentration of30μmol·L-1. The inhibition effect on HR of the isolated right atrium induced by（+）doxazosin was much stronger than （±）doxazosin at the same concentration（P<0.01）.（-）Doxazosin has no effect on HR of isolated right atrium at3μmol·L-1（P>0.05）, while the inhibition effect on HR was significantly inferiorto （+）doxazosin at the same concentration （P<0.01）.（-）Alfuzosin and（+）alfuzosin (10and30μmol·L-1) decreased HR significantly, which were（13.05±7.27）%and （14.27±9.75）%respectively at concentration of30μmol·L-1,while （±）alfuzosin decreased HR only at30μmol·L-1，and the three drugsinhibited HR at about the same degree.3Different effect of （±）doxazosin,（±）alfuzosin and their enantiomers oncontractile force of isolated mouse left atrium.Contractile force of left atrium in different groups were nearly same（P>0.05）.The solvent has no effect on contractile force of isolated mouse leftatrium （P>0.05）.（±）Doxazosin and （-）doxazosin at concentrations of3,10and30μmol·L-1significantly strengthen contractile force of isolated mouse leftatrium （P<0.05） by a concentration-dependent manner. The positive inotropiceffect of （-）doxazosin (10and30μmol·L-1) was stronger than （±）doxazosin ofthe same concentration （P<0.05）. In contrast,（+）doxazosin has mild butsignificant negative inotropic effect （P<0.05）, while （±）alfuzosin and itsenantiomers have no effect on contractile force of isolated mouse left atrium（P>0.05）.4Effect of long-term administration of doxazosin and its enantiomers onplasma NT-proBNP of ratsPlasma NT-proBNP concentrations in control group,（-）doxazosin group,（+）doxazosin group and（±）doxazosin group were475.55±150.21ng·L-1、635.00±343.52ng·L-1、375.55±127.09ng·L-1and424.12±134.19ng·L-1 respectively. NT-proBNP concentration was lower in either （±）doxazosin groupor （+）doxazosin group than in control group, while concentration in（-）doxazosin group was higher than in control group, but the difference has nostatistical significance by single factor analysis of variance between groups（P>0.05）. Plasma NT-proBNP concentration in （-）doxazosin group wassignificantly higher than in （+）doxazosin group （P<0.05）.5Effect of long-term administration of doxazosin and its enantiomers onplasma NF-κB of ratsPlasma NF-κB concentration in control group,（-）doxazosin group,（+）doxazosin group and （±）doxazosin group was888.46±343.36ng·L-1,705.77±193.34ng·L-1,1140.00±365.26ng·L-1and910.38±276.39ng·L-1respectively. NF-κB concentration in either （±）doxazosin group or（+）doxazosin group was higher than in control group, while concentration in（-）doxazosin group was lower than in control group, but there was no statisticalsignificance by single factor analysis of variance （P>0.05）. Plasma NT-proBNPconcentration in （-）doxazosin group was significantly lower than in（+）doxazosin group （P<0.05）.6Effects of long-term administration of doxazosin and its enantiomers onplasms IL-6of ratsPlasma IL-6concentration of control group,（-）doxazosin group,（+）doxazosin group and （±）doxazosin group were47.1±23.9ng·L-1,71.5±23.7ng·L-1,59.4±23.1ng·L-1and70.2±37.4ng·L-1respectively. Difference amongthe four groups has no statistic significance （P>0.05）.These results demonstrate that the inhibit effect of （±）doxazosin and itsenantiomers on heart rate was stronger than that of （±）alfuzosin and itsenantiomers， and there exist chiral difference.（-）Doxazosin has positiveinotropic effect while （+）doxazosin has mild but significant negative inotropiceffect,（±）alfuzosin and its enantiomers with same concentration have no effecton contractile force of isolated left atrium. Chiral structure of doxazosin has anobvious effect on its bioactivity. Alfuzosin, however, decreases the HR slightlywithout chiral recognition in the isolated mouse atrium. Long-term administration of （-）doxazosin,（+）doxazosin and （±）doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP,NF-κB and inflammatory cytokines IL-6. Plasma NF-κB concentration in（+）doxazosin was higher than （-）doxazosin group, which showed that（+）doxazosin maybe affect immune and inflammation system.Part II Mechanism of contractile force change induced by doxazosinenantiomers in isolated rat atriumIn this part, isolated rat left atrium strips were used to study the effect ofdifferent doxazosin enantiomers on myocardial contractile force. In order toprobe the exact mechanism of doxazosine enatiomers inotropic effects, isolatedrat left atrium were pretreated by different blocker such as atropine, propranolol,phenoxybenzamine, indomethacin, verapamil, methylene blue or H-89.1Inotropic effects of （-）doxazosin and （+）doxazosin on isolated rats left atriumSolvent has no effect on contractile force of isolated rats right atrium.（P>0.05）；（-）doxazosin has significant positive inotropic effect on isolated ratsright arium （P<0.05），percentages increase of contractile force induced by（-）doxazosin of3,10and30μmol·L-1were （5.57±20.95）%,（6.96±39.77）%and （67.46±30.09）%respectively （P<0.05）. On the contrary,（+）doxazosin hasnegative inotropic effect, the decrease in percentages of contractile forceinduced by （+）doxazosin of3,10,30μmol·L-1were (-15.61±14.15)%,（-36.99±23.58）%and （-57.48±56.01）%respectively.2Impact of atropine and propranolol on cardiac inotropic effects of（-）doxazosin and （+）doxazosinAfter the isolated rat atrium were pretreated with atropine and propranolol,the solvent has no effect on contractile force. The percentages increase ofcontractile force induced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （13.02±16.66）%,（53.65±41.02）%and （70.87±52.66）%respectively. The decrease in percentage of contractile force induced by（+）doxazosin at concentrations of3,10and30μmol·L-1were （-8.02±21.43）%,（-21.60±21.50）%and （-63.75±24.14）%respectively.3Impact of phenoxybenzamine on cardiac inotropic effects of （-）doxazosin and （+）doxazosinAfter the isolated rat atrium were pretreated with phenoxybenzamine, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （13.71±14.39）%,（35.48±11.90）%and （71.62±37.20）%respectively. The decrease in percentage of contractile force induced by（+）doxazosin were (-14.08±23.48)%,（-33.08±17.85）%and （-60.73±17.21）%respectively. After pretreated with ethanol, the solvent has no effect oncontractile force. The percentages increase of contractile force induced by（-）doxazosin at concentration of3,10and30μmol·L-1were （19.05±7.14）%,（49.27±28.71）%and （71.21±35.57）%respectively. The decrease in percentageof contractile force induced by （+）doxazosin were (-10.40±14.76)%,（-23.73±26.18）%and （-65.63±6.52）%respectively.4Impact of indomethacin on cardiac inotropic effects of （-）doxazosin and（+）doxazosinAfter the isolated rat atrium was pretreated with indomethacin, the solventhas no effect on contractile force. The percentages increase of contractile forceinduced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （19.87±13.06）%,（55.73±18.00）%and （60.25±35.25）%respectively. The decrease inpercentages of contractile force induced by （+）doxazosin were （-7.23±13.39）%,（-23.73±26.18）%and （-65.63±6.52）%respectively. After pretreated withethanol, the percentage increase of contractile force induced by （-）doxazosin atconcentrations of3,10and30μmol·L-1were （19.05±7.14）%,（49.27±28.71）%and （71.21±35.57）%respectively. The decrease in percentage of contractileforce induced by （+）doxazosin were (-10.40±14.76)%,（-23.73±26.18）%and（-65.63±6.52）%respectively.5Impact of verapamil on cardiac inotropic effects of （-）doxazosin and（+）doxazosinAfter the isolated rat atrium was pretreated with verapamil, the solvent hasno effect on contractile force. The percentage increase of contractile forceinduced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （24.81±20.49）%,（75.98±23.00）%and （131.15±17.56）%respectively. Thedecrease in percentage of contractile force induced by （+）doxazosin atconcentration of3,10and30μmol·L-1were （-9.60±17.19）%,（-33.47±9.43）%and （-72.23±14.65）%respectively.6Impact of methylene blue on cardiac inotropic effects of （-）doxazosin and（+）doxazosinAfter the isolated rat atrium was pretreated with methylene blue, thesolvent has no effect on contractile force. The percentages increase ofcontractile force induced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （36.26±9.82）%,（65.99±26.27）%and （16.02±35.33）%respectively. The decrease in percentage of contractile force induced by（+）doxazosin at concentrations of3,10μmol·L-1were （-5.57±17.32）%,(-10.54±25.09)%,（-58.27±15.47）%respectively.7Impact of H-89on cardiac inotropic effects of （-）doxazosin and （+）doxazosinAfter the isolated rat atrium was pretreated with H-89, the solvent has noeffect on contractile force. The percentages increase of contractile forceinduced by （-）doxazosin at concentrations of3,10and30μmol·L-1were （28.96±9.49）%,（81.93±20.75）%and （101.53±51.95）%respectively. The decreasein percentage of contractile force induced by （+）doxazosin was(-17.71±16.37)%,（-30.76±18.38）%and （-67.67±10.74）%respectively.These results demonstrated that the positive inotropic effect of（-）doxazosin on isolated rat left atrium was independent of its α-receptorblocking effect, also did not related to M-receptor, β-receptor, PG enzyme.L-type Ca2+channel, while intracellular cGMP and PKA maybe participatedpartly in the positive inotropic effect of （-）doxazosin. But, cardial myocyteα-receptor, M-receptor, β-receptor, L-type Ca2+channel, PG enzyme, cGMP orPKA maybe not related to the negative inotropic effect of （+）doxazosin.Part III Stereoselective binding of doxazosin enantiomers to plasmaproteins from rat, dog and humanBinding to plasma proteins plays a major role in drug therapy. A change indrug binding to plasma protein significantly affects its pharmacokinetic and pharmacodynamic properties. Because the difference in the protein bindingproperty between enantiomers often causes the difference in theirpharmacokinetic characters, enantioselective protein binding study is essentialto the understanding of their pharmacology, safety and clinical efficacy. Hence,it is necessary to stereoselectively detect and quantify each enantiomer of（±）doxazosin in biological media. Here, we report a first application of chiralHPLC methods using a chiral stationary phase column in conjunction withequilibrium dialysis for the simultaneous determination of the protein bindingof each enantiomer of doxazosin in rat, dog and human plasma after addition ofthe racemate in vitro.1Method validationThe chiral HPLC methods for the separation of doxazosin enantiomers werevery selective.Prazosin,（-）doxazosin and （+）doxazosin were clearly wellresolved from the matrix components under the chromatographic conditionsemployed, and the two enantiomers of doxazosin were baseline resolved fromeach other.The calibration curves （weight1/x2） of the two enantiomers of doxazosinwere linear with correlation coefficients greater than0.9990over theconcentration range of50to1600ng·mL-1for plasma and12.5to200ng·mL-1for PBS. The precision and accuracy of the method were also investigated. Theprecision （relative standard deviation, RSD） based on five repetitive injectionsat drug concentrations of0.2,0.5and1μg·mL-1was less than3.2%（intra-day）and6.3%（inter-day） for the plasma extracts of all three species. The accuracywas found to be in the range of90.5%-102.4%for all plasma and PBS buffermatrices at three drug concentration levels. The detection limit, at asignal-to-noise ratio of3, in plasma of all three species was about0.2ng·mL-1for doxazosin. The enantiomers of doxazosin were stable during the entirecourse of the study including sample preparation, centrifugation and HPLCassay.2Optimization of equilibrium dialysis conditionsThe time taken to reach equilibrium for doxazosin between plasma and isotonic PBS was investigated. Isotonic PBS spiked with （±）doxazosin (800ng·mL-1) was dialyzed against pooled human plasma at0,4,8,15, and20h.（-）Doxazosin and （+）doxazosin crossed the dialysis membrane rapidly andreached a steady-state level in human plasma within15h at37°C. Theequilibrium time for dog or rat plasma was the same to that for human plasma.Therefore, equilibrium dialysis duration was15h in the subsequent equilibriumdialysis experiments at incubation temperature of37°C （body temperature）.3Difference in protein binding between the enantiomersIn the concentration range between200and800ng·mL-1of （±）doxazosin,the percentage of plasma protein binding of enantiomers in the three specieswere89.4%-94.3%for （-）doxazosin and90.9%-95.4%for （+）doxazosin. Theresults showed that either （-）doxazosin or （+）doxazosin was highly bound toplasma proteins, which is consistent with the previous investigationsdetermining the binding of racemic doxazosin. Furthermore, Cb values of（-）doxazosin were significantly smaller than those of （+）doxazosin （P<0.05） ineach plasma from the three species at the used concentrations of （±）doxazosin.Difference in Cb between the two enantiomers became larger with theincreased concentration of （±）doxazosin regardless of the species used.Moreover, the stereoselectivity of doxazosin in dog plasma was moresignificant than that in rat plasma and human plasma. The mean Cb values(ng·mL-1) of （-）doxazosin vs （+）doxazosin for dog plasma in the PBS contained（±）doxazosin200,400, and800ng·mL-1were200.0vs243.0,398.6vs477.3,and845.1vs1006.8, respectively. Overall, enantioselective binding ofdoxazosin enantiomers to plasma proteins from rat, dog and human wasobserved, and （+）doxazosin exhibited a higher protein-binding capacity than（-）doxazosin in all three species.A difference in plasma protein-binding capacity between the twoenantiomers may lead to their pharmacokinetic behavior being different fromeach other. As drug clearance from the blood is directly proportional to the itsfree fraction in plasma, a higher unbound （-）doxazosin concentration in humanplasma observed in the present study might partly explain the phenomenon reported by Liu et al. They found that the human plasma concentration of（-）doxazosin is lower than that of （+）doxazosin after administration of theracemate. Indeed, in our laboratory, the different plasma concentration of thetwo enantiomers in dog and rat was observed after administration of（-）doxazosin and （+）doxazosin. Therefore, the discrepancy for plasmaprotein-binding capacity between （-）doxazosin and （+）doxazosin is a commonreason for their different pharmacokinetic parameters in rat, dog and human.4Difference in protein binding among three speciesIn the case of200ng·mL-1of racemic doxazosin added to the PBS, nosignificant difference in percentage of plasma protein binding among threespecies was found. However, the percentage of plasma protein binding in dogplasma was significantly smaller than that in human plasma at400and800ng·mL-1of racemic doxazosin （P<0.05and P<0.01）, which is consistent withthe previous report that plasma protein binding of （±）doxazosin in human washigher than that in dog. Since total protein concentrations of the pooled plasmafrom rat, dog and human were61.81,51.35and57.75mg·mL-1respectively;the value of percentage of plasma protein binding should be corrected with thevalue of protein assay. The corrected percentage of plasma protein binding ofthe enantiomers at a given concentrations was variable in different species:dog> human> rat （P<0.01）.These results demonstrate that either （-）doxazosin or （+）doxazosin washighly bound to plasma proteins. Moreover, protein binding of doxazosinenantiomers in human, dog and rat plasma revealed that there was a significantdifference in bound fractions, i.e., a higher protein-binding capacity of（+）doxazosin than （-）doxazosin, which might be one of the reasons for anobviously difference in plasma concentration between doxazosin enantiomersin human orally administered （±）doxazosin. Additionally, a pronounced speciesdifference in the plasma protein binding was found in the present study, with ahigher protein-binding capacity in human than dog. Since the total proteinconcentrations of the plasma were significantly different among rat, dog andhuman, the corrected percentage of plasma protein binding was calculated in the study, which indicates an order of dog> human> rat （P<0.01）. Therefore,the findings of a stereoselective plasma protein binding between （-）doxazosinand （+）doxazosin and a pronounced species difference in the plasma proteinbinding of rat, dog and human should be useful for explaining thepharmacokinetic characters of chiral doxazosin.ConclusionDoxazosin has obvious effects on the heart rate and cardiac contractility ofisolated mouse atrium. Doxazosin could induce cardiac arrest reaction at highconcentration, chiral structure of doxazosin has obvious influence on its activity.In contrast, alfuzosin only slightly inhibited the mouse heart rate; chiralstructure of alfuzosin has no obvious effect on its cardiac effects. Long-termadministration of （-）doxazosin,（+）doxazosin and （±）doxazosin did not increaseor decrease plasma level of heart failure biomarker, namely NT-proBNP, NF-κB and IL-6. Plasma NF-κB concentration in （+）doxazosin was higher than（-）doxazosin group, which showed that （+）doxazosin maybe affect immune andinflammation system.The positive inotropic effect of （-）doxazosin on isolated rat left atrium wasindependent of its α-receptor blocking effect, also did not related to M-receptor,β-receptor, PG enzyme. L-type Ca2+channel, while intracellular cGMP andPKA maybe participated partly in the positive inotropic effect of （-）doxazosin.But, cardial myocyte α-receptor, M-receptor, β-receptor, L-type Ca2+channel,PG enzyme, cGMP or PKA maybe not related to the negative inotropic effectof （+）doxazosin.Either （-）doxazosin or （+）doxazosin was highly bound to plasma proteins.Moreover, protein binding of doxazosin enantiomers in human, dog and ratplasma revealed that there was a significant difference in bound fractions, i.e., ahigher protein-binding capacity of （+）doxazosin than （-）doxazosin, whichmight be one of the reasons for an obviously difference in plasma concentrationbetween doxazosin enantiomers in human orally administered （±）doxazosin.Additionally, a pronounced species difference in the plasma protein bindingwas found in the present study, with a higher protein-binding capacity in human than dog. Since the total protein concentrations of the plasma were significantlydifferent among rat, dog and human, the corrected percentage of plasma proteinbinding was calculated in the study, which indicates an order of dog> human>rat （P<0.01）. Therefore, the findings of a stereoselective plasma protein bindingbetween （-）doxazosin and （+）doxazosin and a pronounced species difference inthe plasma protein binding of rat, dog and human should be useful forexplaining the pharmacokinetic characters of chiral doxazosin. |
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