Cicletanine is an antihypertensive compound with vasorelaxant (1-4) and natriuretic (5-7) properties. The mechanism of the natriuretic action was recently elucidated in rats (5,6). The salidiuretic activity of orally (p.o.) administered racemic cicletanine is due to the dextrorotatory (+)-enantiomer (5), which acts through its sulfoconjugated metabolite: (+)-cicletanine sulfate (6). This urinary metabolite appears to inhibit the apical Na+-dependent Cl-/HCO3- anion exchanger in the cortical diluting segment (6).
In contrast to the natriuretic mechanism just described, the vascular mechanism or mechanisms of cicletanine have not been clearly elucidated. Binding, biochemical, and contractility studies in animals have disclosed two pharmacological receptors for cicletanine in vascular smooth muscle (VSM): (a) the H1-receptor, at which cicletanine acts with a Kb (dissociation constant) of 36 nM(2); and (b) the low Km cyclic GMP phosphodiesterases (PDEs), which are inhibited with micromolar (10-600 μM) concentrations of cicletanine, an action showing partial stereoselectivity in favor of the (-)-enantiomer (3,4). However, the antihypertensive action of cicletanine probably does not result from specific H1 antagonism. Conversely, the inhibition of cyclic GMP PDEs potentiates the vasorelaxant actions of the guanylate cyclase activators sodium nitroprusside (SNP) and atriopeptin II (3,4).
To characterize further these vasorelaxant mechanisms, we tested racemic (±) cicletanine in pithed rats for its ability to modify the vascular responses to different vasocontractant agents. The finding of a reduction of the pressor responses to angiotensin II (AII) caused us to characterize this interaction further by using the resolved (+ and -) enantiomers in both pithed rats and isolated rat aorta.
Screening of vasocontractants in pithed rats treated orally with racemic cicletanine
Male Sprague-Dawley rats weighing 250-270 g were kept for at least 1 week in our animal quarter before the study. The animals were orally dosed three times (24, 16, and 2 h before the determination of dose-response curves to vasocontractants) with cicletanine (50 mg/kg) or its solvent (corn oil, 5 ml/kg).
The rats were anesthetized briefly (5 min) with diethyl ether while the trachea was cannulated and connected to a respirator. They were then pithed by insertion of a stainless-steel rod into the spinal cord through the right eye orbit. The left carotid artery was cannulated to measure blood pressure (BP). Thirty minutes after the rats were pithed, carotid BP achieved a steady state and the following studies were performed.
- Series 1. Cumulative dose-response curves to the α1-adrenoceptor agonist cirazoline were constructed in rats pretreated orally (p.o.) with cicletanine or its solvent or pretreated intravenously (i.v.) with prazosin (30 μg/kg, 5 min before determination of the effect of cirazoline), a standard blocker of α1-adrenoceptors. The dose-response curve was cumulative because each successive dose of cirazoline was administered as soon as the maximal response to the previous dose occurred. In general, the time interval between two contiguous doses was <30 s (8).
- Series 2. Cumulative dose-response curves to the α1-adrenoceptor agonist B-HT 920 were studied as described in series 1. The standard α2-adrenoceptor antagonist used was idazoxan (100 μg/kg i.v.).
- Series 3. Cumulative dose-response curves to angiotensin II (AII) were determined as described in series 1. The standard AII antagonist used was losartan (40 μg/kg i.v.).
- Series 4. Cumulative dose-response curves to vasopressin were determined as described in series 1. The standard antagonist used to block vascular vasopressin1-receptors was (β-mercaptor-β, β-cyclo-pentamethylenepropionyl1, O-Me-Tyr2, Arg8)-vasopressin (SKF 100273, 1 μg/kg i.v.).
- Series 5. Cumulative dose-response curves to serotonin were determined as described in series 1. The standard antagonist of vascular serotonin (S2) receptors mediating pressor effects on stimulation was methysergide (3 μg/kg i.v.).
- Series 6. Frequency-response curves to electrical stimulation of the spinal cord were determined as described in series 1. The parameters of electrical stimulation were 60 V and 0.5 ms at various frequencies (0.125, 0.25, 0.5, 1, 2, and 4 Hz). Before the stimulation was started, the animals received d-tubocurarine (3 mg/kg i.v. delivered in 30 s). The time interval between two consecutive trains of stimuli was 5 min. The standard antagonist used in this study to inhibit the pressor responses to electrical stimulation of the spinal cord was prazosin (100 μg/kg i.v.).
Cicletanine administration. For oral administration, cicletanine was suspended in corn oil (10 mg/ml). The placebo group received corn oil (5 ml/kg). In pilot experiments, we noted that optimal inhibition of pressor responses was observed after three oral administrations.
Reactivity to AII and vasopressin in pithed rats treated orally and intravenously with the resolved (+ and -) cicletanine enantiomers
Male Sprague-Dawley rats weighing 250-270 g were kept at least 1 week in our animal quarters before the study. The animals were dosed three times (24, 16, and 2 h before the determination of dose-response curves to vasocontractants) with either the vehicle (5 ml/kg distilled water with addition of 6% glucose), the (+)-enantiomer or the (-)-enantiomer of cicletanine. These treatments were administered orally to conscious rats. Sixty minutes after the last dose, the animals were anesthetized and pithed as described in the previous section. The left carotid artery was cannulated to measure BP. The pulsatile pressure signals were fed into a tachometer to obtain heart rate, which is not reported since it was not affected by the agonist studied.
After the surgical procedure was finished, a stabilization period of 30-45 min was allowed, during which the cardiovascular parameters measured attained a steady state. The responses to various vasoconstrictor agents were then determined ≈2 h after the oral dosing of (+)-cicletanine, (-)-cicletanine, or their vehicle. In addition, some studies were performed in naive pithed rats treated i.v. with (+)-cicletanine, (-)-cicletanine, or their vehicle 10 min before the responses to vasoconstrictor stimuli were determined.
- Series 1. Cumulative dose-response (maximal increases in mean carotid artery BP) curves to AII were determined in pithed rats that received (-)-cicletanine (20 mg/kg p.o.), (+)-cicletanine (20 mg/kg p.o.) or their vehicle (5 ml/kg p.o.) or (-)-cicletanine (10 mg/kg i.v.), (+)-cicletanine (10 mg/kg i.v.) or their vehicle (1,25 ml/kg i.v.).
- Series 2. Cumulative dose-response (maximal mean carotid BP) curves to vasopressin were determined in rats dosed intravenously or orally with (-)-cicletanine, (+)-cicletanine, or their vehicle.
Isolated rat aortic rings
Preparation of isolated rat aortic rings. Male Wistar rats weighing 300-350 g were kept for 15 days in a humidity- and temperature-controlled room and were fed a standard diet. The animals were fasted the night before the experiment. Rats were killed by cervical dislocation; the thoracic aortas were immediately removed, carefully cleaned, and cut into six rings (3 mm long) that were placed individually between platinum hooks in 20 ml Krebs solution maintained at 37°C and gassed with 95% O2/5% CO2. The Krebs medium contained (in mM) NaCl 118, NaHCO3 25, CaCl2 1.25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2 and glucose 11.5. An initial load of 1 g was applied to the preparations and maintained throughout a 60- to 90-min equilibration period (the incubation media was renewed every 20 min). Tension was recorded on a MacLab (Analog Digital Instruments, Castle Hill, Australia) by Dynamometers UF1 (Piolen Control). We removed the endothelium by gently rubbing the intimal surface of the aorta with a small wood stick. An absence of functional endothelium was indicated by the failure of the preparation precontracted with norepinephrine (NE) to relax to acethylcholine (ACH 2 μM).
Aortic contractility. Aortic rings equilibrated for 60-90 min in the Krebs media were contracted with increasing cumulative concentrations of AII to generate concentration-response curves. Each successive concentration was added when the response to the previous one attained a persistent apparent maximum. Cicletanine and other compounds were preincubated for 20 min before AII was added. To study the effect on AII-dependent aorta contraction, we added the compounds from freshly prepared, concentrated stock solutions in water or dimethyl sulfoxide (DMSO) provided that the final DMSO concentrations themselves had no effect on aorta contractility.
All results are mean ± SEM. In pithed rats, the dose of each agonist producing a pressor effect of 50 mm Hg (ED50 mm Hg) was calculated by fitting simultaneously the individual dose-response curves with a logistic curve (9). This response was located on the straight portion of the dose-response curve. To quantify the rightward displacement of the control dose-response curve after a treatment, dose ratios were used (ED50 mm Hg after a treatment divided by the ED50 mm Hg after vehicle) (10). The vehicle and treatment dose-response curves did not depart notably from parallelism for any calculated dose ratios (described in the Results section).
Statistical differences between two mean values were determined by a non-paired Student's t test. A significant shift (p < 0.05) refers to a significant difference between the ED50 value determined in the drug-treated and the matched vehicle-treated group.
Racemic cicletanine, the resolved (-)- and (+)-enantiomers and the sulfo- and glucuro-conjugated metabolites were obtained from IPSEN (Paris, France). Losartan was a gift from Dupont Merck Pharmaceutical (Wilmington, DE, U.S.A.). Cirazoline was obtained from Synthelabo Recherche (Bagneux, France). SKF 100273 was purchased from ICN Biomedicals France (Orsay, France). B-HT 920 was obtained from Boehringer Ingelheim (Reims, France). Methysergide was purchased from Research Biomedicals International (Natick, MA, U.S.A.). Other chemicals were either from Merck or Sigma (distributed through Coger, Paris, France).
Effects of orally administered racemic cicletanine on the vascular reactivity to various vasoconstrictor agents in pithed rats
Rats were orally treated with racemic (±) cicletanine (50 mg/kg) or vehicle (corn oil) three times: 24, 16, and 2 h before the experimental procedure was performed. Baseline mean BP measured immediately before a dose-response curve to various pressor agonists was determined almost identical in pithed rats treated with cicletanine (58 ± 1 mm Hg, n = 27) or vehicle (59 ± 1 mm Hg, n = 30).
Table 1 shows ED50 values to different vasoconstrictors in rats treated with racemic cicletanine. Some vasoconstrictors were significantly antagonized by cicletanine, particularly AII. Therefore, the dose of AII that increased the mean carotid artery BP by 50 mm Hg was twice as high in cicletanine- than in vehicle-treated animals (ED50 = 0.48 ± 0.012 vs. 0.25 ± 0.007 μg/kg, p < 0.05) (Table 1). Figure 1 further illustrates this antagonism; i.e., in rats treated with cicletanine, the dose-pressor response curve to AII was located to the right of that determined in vehicle treated animals. An intravenous dose (40 μg/kg) of the standard antagonist of AII receptor losartan increased the ED50 mm Hg threefold (ED50 = 0.64 ± 0.01 vs. 0.21 ± 0.01 μg/kg, p < 0.001) (Fig. 1). Therefore, the displacement by cicletanine represented 47.2% of that obtained with losartan (Table 1).
The ED50 value for vasopressin was significantly increased by 58% in animals treated with cicletanine (Table 1). This change in ED50 value was five to six times lower than that obtained with SKF 100273 (1 μg/kg i.v.), a standard antagonist of vasopressin receptors (Table 1). Second, cicletanine treatment slightly displaced to the right the pressor responses evoked by electrical stimulation of the spinal cord. Therefore, the ED50 value for electrical stimulation of the spinal cord was only marginally (+27%) but significantly increased in animals treated with cicletanine (Table 1). This change in ED50 value was five to six times lower than that obtained with prazosin (100 μg/kg i.v.), a blocker of α1-adrenoceptors (Table 1). Third, the dose-pressor response curves to the α1-adrenoceptor agonist cirazoline obtained in rats treated with cicletanine were slightly displaced to the right of those determined in animals that received the vehicle of cicletanine. Therefore, the ED50 value for cirazoline was only marginally (+13%) but significantly increased in animals treated with cicletanine (Table 1). This change in ED50 value was 60 times lower than that obtained with prazosin (30 μg/kg i.v.), a selective and specific α1-adrenoceptor antagonist (Table 1). Finally, cicletanine did not significantly modify the dose-pressor response curve to serotonin or the α2-adrenoceptor agonist B-HT 920 (Table 1).
Effects of the resolved (±) cicletanine enantiomers on the vascular reactivity to AII and vasopressin in pithed rats
The baseline values of mean carotid artery blood BP were very similar among the groups of animals that received (+)-cicletanine, (-)-cicletanine, or their vehicle either orally or intravenously (data not shown).
In rats dosed orally with (+)-cicletanine (20 mg/kg), the dose-response curve to AII was almost coincident with the curve determined in matched vehicle-treated rats (Fig. 2B). By contrast, the oral administration of 20 mg/kg (-)-cicletanine produced a significant threefold rightward shift of the dose-pressor response curve to AII as compared with the matched curve determined in vehicle-treated rats (Fig. 2A). Therefore, the ED50 value for AII was significantly (p < 0.05) increased from 0.19 ± 0.02 to 0.56 ± 0.06 μg/kg in animals treated with (-)-cicletanine.
Similar results were obtained after intravenous administration of cicletanine enantiomers (considering that cicletanine enantiomers are much more soluble in saline than is the racemic compound). Therefore, in rats receiving (+)-cicletanine (10 mg/kg i.v.), the ED50 value and the maxima of the dose-response curves to AII were not significantly different from those determined in the matched vehicle-treated group (Fig. 2B, inset). By contrast, (-)-cicletanine 10 mg/kg i.v. produced a significant rightward twofold shift of the dose-response curve to AII without affecting the maximum (Fig. 2A, inset). Therefore, the ED50 value for AII was significantly (p < 0.05) increased from 0.20 ± 0.02 to 0.39 ± 0.06 μg/kg in animals treated with (-)-cicletanine.
The dose-response curve to vasopressin after 10 mg/kg i.v. and 20 mg/kg p.o. (-)-cicletanine were displaced slightly and similarly (1.5- and 1.6-fold shift, respectively). This shift was significant for the two routes of administration (p < 0.05). By contrast, the shift after oral administration of 20 mg/kg (+)-cicletanine was smaller and not significant (data not shown).
Effects of the resolved (±) cicletanine enantiomers and conjugated metabolites on the vascular reactivity to AII in isolated rat aorta
Figure 3A shows the tension responses to AII in isolated rat aorta preincubated for 20 min with (-)-cicletanine (100 μM) or its vehicle. (-)-Cicletanine induced a very important and significant (p < 0.01) reduction in the effect of AII. Figure 3B shows the percentage of remaining tension at each AII concentration for three different constant doses of (-)-cicletanine. The percentage of inhibition was independent of the AII concentration. Finally, Figure 3C shows that the reciprocal of the percentage of inhibition by (-)-cicletanine was a linear function of the (-)-cicletanine concentration, as predicted for noncompetitive inhibition (described in the Discussion section). KI for (-)-cicletanine, obtained for the intercept with the x axis, was ≈55 μM(Fig. 3C).
(+)-Cicletanine exhibited behavior qualitatively similar to that of (-)-cicletanine, but with lower KI (≈140 μM) (Fig. 3C). Neither (-)-cicletanine sulfate nor (-)-cicletanine glucuronate was able to modify tension responses to AII in isolated rat aorta (data not shown).
Our results demonstrate that racemic cicletanine administered orally three times in a 24-h period can significantly reduce the vascular reactivity to AII and also, although to a lesser extent, to vasopressin. This inhibitory effect appears to be of a specific nature since it was not observed against other pressor agents such as the α1-adrenoceptor agonist cirazoline, the α2-adrenoceptor agonist B-HT 920, or serotonin. Furthermore, cicletanine slightly reduced the pressor effects elicited by electrical stimulation of the spinal cord. Therefore, cicletanine does not appear to exert major effects on peripheral sympathetic nerve function, except for a possible decrease in the liberation of NE from sympathetic nerve endings innervating resistance vessels.
Cicletanine comprises two enantiomers which may independently contribute to the antihypertensive activity of the drug. Our results provide experimental evidence that only one of the two enantiomers effects vascular reactivity to AII and vasopressin. (-)-Cicletanine but not (+)-cicletanine significantly inhibited the pressor response, particularly to AII, and to a lesser extent, to vasopressin.
A single intravenous administration of (-)-cicletanine induced inhibition (of AII and vasopressin pressor effects) similar to that induced by repeated oral administration of racemic or (-)-cicletanine. This may imply a problem of gastrointestinal absorption or metabolism that may influence the antagonism to AII-induced increase in vascular resistance. It is unfortunate that few studies have investigated pharmacokinetics and metabolism of cicletanine in rats.
Pharmacokinetic studies in healthy volunteers have shown that cicletanine is rapidly absorbed and is strongly bound to plasma proteins (90%), with a volume of distribution of 371 and an elimination half-life of 6-8 h; elimination is mixed (renal and hepatic) (11). In the rats bioavailability of racemic (±) cicletanine is reported to be ≅75% and gastrointestinal absorption normally involves passive diffusion that does not appear to be affected by drug chirality (11). Moreover, Johnson and colleagues reported that both radiolabeled enantiomers are well absorbed after oral administration in rats (12,13). Therefore, the presence of an enterohepatic cycle and not poor absorption may explain the lesser efficacy (against AII and vasopressin pressor effects) of oral (-)-cicletanine as compared with that of intravenous (-)-cicletanine.
The literature contains no data on the elimination half-life of plasma cicletanine in rats, which is difficult to determine because the compound undergoes an enterohepatic cycle. On the other hand, whether a correlation exists between blood concentration and effect is not known, since there is evidence of accumulation in vessels. Radiolabeled cicletanine was shown to be taken up by the aorta after oral dosing (1). Moreover, Silver and Cumiskey reported (16) that incubation of denuded aortic smooth muscle rings from spontaneously hypertensive rats with vasorelaxant concentrations (10-100 μM) of cicletanine resulted in a 7- to 12-fold accumulation of cicletanine in the tissue relative to the tissue bath (16; uptake was not stereoselective). Therefore, repeated oral dosing of (-)-cicletanine may be required to obtain accumulation in vessels. However, this hypothesis will require further investigation for validation.
Vasorelaxation in humans can be clearly demonstrated after 3-week treatment, and maximal antihypertensive activity is attained after several weeks of dosing with cicletanine (14,15). As Silver and Cumiskey reported (16), these observations suggest that the slow onset of cicletanine effects could be related to its slow accumulation in vascular muscle.
Our results suggest that (-)-cicletanine should be considered the enantiomer that contributes to the antihypertensive activity of racemic (±) cicletanine, by reducing the vascular reactivity to endogenous pressor substances such as AII and vasopressin. By contrast, (+)-cicletanine is the enantiomer for the renal component of the antihypertensive action of racemic cicletanine (5,6). Thus, the two enantiomeric components of racemic cicletanine concurrently contributes to its therapeutic effects in that (-)-cicletanine reduces the reactivity of the vascular system to AII and vasopressin whereas (+)-cicletanine favors natriuresis.
Because (-)-cicletanine inhibits the pressor effects of AII, we attempted to characterize further this effect by performing studies in isolated conductance vessels, the aorta, which itself does not contribute to the pressor effects of AII. Experiments in isolated rat aorta show that (-)-cicletanine itself (and not its sulfo- or glucuroconjugated metabolites) inhibits the contractile effects of AII.
Previous binding experiments showed that cicletanine apparently does not act through a membrane receptor (1). This finding is compatible with our experimental results in isolated aorta, which clearly indicated noncompetitive inhibition (Fig. 3), i.e., the effect (Ec) of (-)-cicletanine can be described by the following mathematical function: eqn. (1)
where [C] and [AII] are the concentrations of (-)-cicletanine and AII and KI and KA are the apparent dissociation constants for cicletanine and AII, respectively. Equation 1 predicts that each fixed concentration of (-)-cicletanine the Ec tension ratio with the control curve is independent of the AII concentration, i.e.: eqn. (2)
Figure 3B shows that this theoretical prediction was confirmed by the experiments.
Equation 2 can be rearranged as: eqn. (3)
For each (-)-cicletanine concentration of Fig. 3B, a mean Ec tension ratio was obtained. Figure 3C represents the reciprocal of Ec tension ratio as a function of (-)-cicletanine concentration. KI is obtained from the intercept with the x-axis and is 55 and 140 μM for (-)- and (+)-cicletanine, respectively.
These results obtained in isolated rat aorta clearly indicate that (-)-cicletanine is a noncompetitive antagonist of AII receptor stimulation, i.e., that it may act at any of the numerous steps that couple the occupation of the receptor to the final response. The findings of Silver and co-workers (3,4) suggest that the target of (-)-cicletanine can be the cyclic GMP PDEs, particularly the calcium/calmodulin PDE: (-)- and (+)-cicletanine inhibited this enzyme with KI values of 180 and 325 μM, respectively (4). On the other hand, both cicletanine enantiomers accumulate in VSM to concentrations that are 7- to 12-fold higher than those in the external media (16). Therefore, (-)-cicletanine might act on the AII signal transduction pathway by inhibiting calcium/calmodulin PDE. The low-Km cyclic GMP-specific PDE inhibitor zaprinast also inhibited the pressor effects of AII (17).
In in vivo conditions, cicletanine displaced the dose-response curve to AII in an apparent competitive manner, possibly due to the dose of cicletanine studied, which may have been too low to disclose a clear noncompetitive antagonism. Alternatively, the effects of cicletanine on resistance vessel AII-mediated responses may differ from those produced at the level of conductance vessels. The solution of this issue requires further investigation.
In conclusion our results provide evidence that cicletanine can reduce the vascular reactivity to AII and vasopressin, two endogenous vasocontractants that play a key role in several forms of hypertension.
Acknowledgment: We thank R. Vistelle (UFR de Pharmacie, Reims, France) for interest and discussion.
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Keywords:© Lippincott-Raven Publishers
Cicletanine; Vascular smooth muscle; Angiotensin II; Vasopressin