Hypotensive Effect Of Doxazosin And Related Mechanism In Conscious Rats

Doxazosin currently used as add-on therapy for hypertension is racemicmixture of (-)doxazosin and (+)doxazation. Previously we reported that rankorder of acute hypotensive activity was (-)doxazosin <(±)doxazosin <(+)doxazosin in anesthetized rats. However, contributions of the twoenantiomers to hypotensive effect of long-term administration of (±)doxazosinin conscious animals are unknown. Here we observed hypotensive responsesto doxazosin enantiomers in conscious rats, and analyzed (±)doxazosin,(-)doxazosin and (+)doxazosin concentrations in plasma byHPLC-fluorescence method. α1-Adrenoceptor blocking activity of the3agentswas investigated in isolated rat caudal artery.Part I Effects of long-term administrations of (-)doxazosin,(+)doxazosinand (±)doxazosin on blood pressure in conscious ratsAnimals were allowed to habituate to the animal maintenance facilitiesfor a period of at least7days before the beginning of the experiments.40ratswere randomly divided into four groups of10each. Group I served as solventcontrol, and the rats in groups II, III and IV were administered orally8mg/kgof (-)doxazosin,(+)doxazosin and (±)doxazosin once daily for12weeks,respectively. During the11th week of drug administration, systolic bloodpressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP)and heart rate were measured at0,2,4,8and12h after a long-termadministration of8mg/kg (±)doxazosin in the conscious and nonfasted rats.The time required to complete the measurements in40animals was5days1Effects of long-term administrations of doxazosin and its enantiomerson blood pressure in nonfasted conscious ratsThe systolic blood pressure, diastolic blood pressure, mean bloodpressure did not change significantly at2,4,8and12h after solvent administration (P>0.05). In the rat treated with a long-term administration of(±)doxazosin for12weeks, the SBP decreased significantly at2h(123.03±3.63mmHg),4h (123.93±6.53mmHg),8h (102.15±3.68mmHg)and12h (113.86±5.28mmHg) after the last administration of8mg/kg(±)doxazosin, as compared to pre-drug baseline level (144.57±4.18mmHg;P<0.01). Hypotensive effects on DBP and MBP by long-term administrationof (±)doxazosin were similar to that on SBP. The results of statisticalcomparison between solvent group and (±)doxazosin group also indicatedsignificant hypotensive effects on SBP, DBP and MBP by (±)doxazosin.(+)Doxazosin had similar hypotensive effects as (±)doxazosin, but themaximal decreases (8h after the last administration) in SBP, DBP and MBPinduced by (+)doxazosin were significantly smaller than that by (±)doxazosinat the same dose (P<0.05). A long-term administration of (-)doxazosinproduced significant, but mild, hypotensive effects on the SBP (112.67±6.56mmHg), DBP (72.34±4.24mmHg) and MBP (85.46±4.96mmHg)8h after thelast administration, as compared to pre-drug baseline levels (132.81±4.83mmHg,87.15±3.58mmHg and102.00±3.96mmHg)(P<0.05and0.01).However, the maximal decreases (8h after the last administration) in SBP,DBP and MBP induced by (-)doxazosin were not significantly different fromthat by solvent (P>0.05).2Effects of long-term administrations of doxazosin and its enantiomerson heart rate in nonfasted conscious ratsA long-term administration of (-)doxazosin,(+)doxazosin or(±)doxazosin did not affect HR significantly, as compared to pre-drug baselinelevel (P>0.05), however results of statistical comparison between solventgroup and (±)doxazosin group indicated a significant positive chronotropiceffect at8h (336.61±16.57bpm vs427.43±34.4bpm)and12h (332.24±22.68bpm vs435.15±25.85bpm) after the last administration of (±)doxazosin(P<0.05and0.01). Part II Effects of long-term administrations of doxazosin and itsenantiomers on general situation of the ratAnimals were allowed to habituate to the animal maintenance facilitiesfor a period of at least7days before the beginning of the experiments.40ratswere randomly divided into four groups of10each. Group I served as solventcontrol, and the rats in groups II, III and IV were administered orally8mg/kgof (-)doxazosin,(+)doxazosin and (±)doxazosin once daily for12weeks,respectively. Effect of long-term administrations of doxazosin and itsenantiomers on general situation were observed.1Effects of long-term administrations of doxazosin and its enantiomerson rat body weightBefore long-term oral administrations of solvent,(-)doxazosin,(+)doxazosin and (±)doxazosin, the body weights of4groups were notsignificantly different from each other (P>0.05). Body weights of the solventgroup at the end of the3rd,6th,9th and12th week were449.2±12.38g,489.8±14.55g,516±14.04g and539.7±14.69g, which were significantlygreater than the body weight (359.9±6.98g) before administration of thesolvent (P<0.01). Long-term administrations of (-)doxazosin,(+)doxazosinand (±)doxazosin for12weeks did not significantly affect body weight gain ofthe rat (P>0.05).2Effects of long-term administrations of doxazosin and its enantiomerson food consumption of the ratsBefore long-term oral administrations of solvent,(-)doxazosin,(+)doxazosin and (±)doxazosin, the food consumption of the4groups was notsignificantly different from each other (P>0.05). There was no significantdifference in food consumption among solvent,(-)doxazosin,(+)doxazosinand (±)doxazosin groups during the12week (except at the10th week)(P>0.05). Food consumption of the (-)doxazosin group or (+)doxazosin groupwas significantly greater than solvent group at the end of the10th week(P<0.05). There was no significant difference in food consumption between(±)doxazosin group and solvent group.3Effects of long-term administrations of doxazosin and its enantiomers onkidney function CRTN, UA and UREA levels of solvent group after a long-termadministration were33.2±1.15μmol/L,70.80±9.31μmol/L and5.28±0.27mmol/L. A long-term administration (12weeks) of (-)doxazosin,(+)doxazosinor (±)doxazosin did not affect the level of CRTN, UA, UREA significantly, ascompared to solvent group (P>0.05).4Effects of long-term administrations of doxazosin and its enantiomerson liver functionALT, AST, TP, ALB, GLO and A/G levels of solvent group after along-term administration were36.30±3.56U/L,154.30±19.42U/L,57.31±1.71g/L,28.67±0.77g/L,28.64±1.06g/L and1.00±0.02. A long-termadministration (12weeks) of (-)doxazosin,(+)doxazosin or (±)doxazosin didnot affect the levels of ALT, AST, TP, ALB, GLO and A/G significantly, ascompared to solvent group (P>0.05).5Effects of long-term administrations of doxazosin and its enantiomerson GLU and serum lipid levels (T-CHO、TG、HDL-C、LDL-C、VLDL-C)GLU, T-CHO, TG, HDL-C, LDL-C and VLDL-C levels of solvent groupafter a long-term administration were5.85±0.38mmol/L,1.15±0.04mmol/L,0.37±0.05mmol/L,0.33±0.01mmol/L,0.24±0.02mmol/L and0.17±0.02mmol/L. The level of GLU was not changed by the agents. Serum T-CHOlevel, TG level and VLDL-C level of the rats in solvent group,(-)doxazosingroup,(+)doxazosin group and (±)doxazosin group were not significantlydifferent from each other (P>0.05). A long-term treatment with (+)doxazosinincreased HDL-C level, and a long-term treatment with (-)doxazosindecreased LDL-C level significantly (P<0.01).A long-term administration (12weeks) of (-)doxazosin,(+)doxazosin or(±)doxazosin did not affect the level of CK significantly, as compared tosolvent group (P>0.05).Part III Plasma drug concentrations corresponding to maximalhypotensive responses to doxazosin and its enantiomers orallyadministered in the ratTime-dependent hypotensive effects and maximum hypotensive effect induced by single oral administration of8mg/kg (±)doxazosin were studied.To clarify the possible influence of fasting state on plasma drug concentration,a difference in plasma drug concentration-time curves between fasted andnonfasted rats was studied. We measured the plasma concentrationscorresponding to maximal hypotensive responses to (-)doxazosin,(+)doxazosin and (±)doxazosin for long-term administration by HPLC.1Changes in blood pressure and heart rate in conscious rats after a singleadministration of (±)doxazosinThe systolic blood pressure, diastolic blood pressure, mean bloodpressure and heart rate did not change significantly at2,4,8and12h aftersolvent administration (P>0.05). The SBP in rats treated with8mg/kg(±)doxazosin decreased significantly4h after administration, as compared topre-drug baseline level (P<0.05), but there was no significant differencebetween each individual datum (racemic doxazosin group) and its respectivecontrol value (solvent group). A single administration of (±)doxazosin did notaffect HR significantly (P>0.05), and the values of HR were389.43±23.31,343.02±14.06,359.16±17.18,359.16±17.18and344.99±9.23(bpm) beforeand2h,4h,8h,12h after the administration, respectively2Effect of food on the plasma drug concentrations measured after asingle administration of (±)doxazosinPlasma (±)doxazosin concentration quickly increased to254.61±27.69ng/ml at0.5h, then reached its peak level (306.97±43.47ng/ml) at1h afteroral administration in fasted rats. Its concentration was still maintained at ahigher level (296.10±40.97ng/ml) at2h, and decreased to30.22±16.53ng/mlat12h in fasted rats. Although the plasma concentration of (±)doxazosin innonfasted rats was slightly lower than that in fasted rats0.5h,1h and2h afteroral administration of the same dose, drug concentration-time curve for(±)doxazosin in fasted rats was not significantly different from that innonfasted rats (P>0.05). In addition, stable (±)doxazosin concentrations weremaintained at4h (170.75±27.05ng/ml) and6h (169.87±40.29ng/ml) afteroral administration in nonfasted rats. 3Analysis of plasma drug concentrations corresponding to maximalhypotensive responses to the last administration of doxazosin enantiomers inthe ratPlasma drug concentrations were measured8h after the lastadministration of (-)doxazosin,(+)doxazosin and (±)doxazosin, and theconcentration of (-)doxazosin,(+)doxazosin or (±)doxazosin was18.26±3.55ng/ml,177.11±20.66ng/ml or113.18±13.21ng/ml. The concentrations of(-)doxazosin and (±)doxazosin were significantly lower than (+)doxazosin(P<0.05and0.01), and the concentration ratio of (-)doxazosin/(+)doxazosinwas0.10. A chiral separation of (±)doxazosin-containing plasma samples wasfurther performed, and the (-)doxazosin and (+)doxazosin components were12.01±2.45ng/ml and96.56±9.94ng/ml, respectively. The concentration ratioof (-)doxazosin/(+)doxazosin in plasma from the rats orally administered(±)doxazosin was0.12.Part Ⅳ α1-Adrenoceptor blocking activity of doxazosin and itsenantiomers in the isolated rat caudal arteryIn solvent control group, there were no significant differences in Emaxvalues among the second, third, fourth and fifth set of concentration-responsecurves for NA (1.75±0.05g,1.76±0.07g,1.80±0.07g and1.93±0.06g;P>0.05), and EC50values for NA calculated from the4concentration-responsecurves were not significantly different from each other (data not shown).Before treatment with (-)doxazosin,(+)doxazosin and (±)doxazosin, thevalues of Emax or EC50obtained from the concentration-response curves forNA were not significantly different from each other (P>0.05).(-)Doxazosin(0.003,0.03and0.3μmol/L),(+)doxazosin and (±)doxazosin (0.001,0.01and0.1μmol/L) produced a shift to the right of the concentration-response curvesfor NA (Figure8) without significant changes in the Emax values (P>0.05).Slope of the Schild plot for (-)doxazosin,(+)doxazosin or (±)doxazosin(0.607±0.028,0.647±0.018or0.716±0.028) was significantly different fromunity (P<0.05), indicating that the three agents non-competitively inhibited theconcentration-response curves for NA in the isolated rat caudal artery. The pKBvalue of (-)doxazosin (8.032±0.039) was significantly smaller (P<0.01)than that of (+)doxazosin (8.995±0.032) or (±)doxazosin (8.694±0.032), andthe pKBvalue of (±)doxazosin was significantly smaller than that of(+)doxazosin (P<0.01).ConclusionThe effect of (+)doxazosin against α1-adrenoceptor is significantly higherthan that of (-)doxazosin and (±)doxazosin. However the hypotensive effect of(±)doxazosin of long-term administration higher than (+)doxazosin inconscious rats. The hypotensive effect of (-)doxazosin were not significantlydifferent from that by solvent. These results suggest that (-)doxazosincomponent is absolutely necessary to ensure the potent hypotensive effect ofracemic doxazosin administered for12weeks in conscious rats. And(+)doxazosin increased HDL-C level while (-)doxazosin decreased LDL-Clevel significantly.