Cicletanine New insights into its pharmacological actions

General Pharmacology 33 (1999) 7–16

Review article

Cicletanine New insights into its pharmacological actions Leszek Kalinowski a,*, Mirosława Szczepan´ska-Konkel b, Maciej Jankowski a, Stefan Angielski a a

Department of Clinical Biochemistry, Medical University of Gdansk, Debinki 7, 80-211, Gdansk, Poland b Department of Clinical Chemistry, Medical University of Gdansk, Debinki 7, 80-211, Gdansk, Poland Manuscript received February 23, 1998; accepted manuscript September 23, 1998

Abstract Cicletanine ((6)3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c] pyridine) 3-(4-chlorophenyl)-1,3-dihydro7-hydroxy-6-methylfuro-[3,4-c] pyridine) is a novel antihypertensive agent that has been shown to possess vasorelaxant, natriuretic, and diuretic properties in preclinical and clinical studies. The mechanism(s) by which cicletanine induces these biological effects has not been definitely established, although it appears to differ from that of other classes of antihypertensive drugs. The salidiuretic activity appears to be the result of an action of the sulfoconjugated metabolite of cicletanine, which inhibits the apical Na1-dependent Cl2/HCO32 anion exchanger in the distal convoluted tubule. The mechanism of the vasodilating effect of cicletanine seems to be complex; it may include stimulation of vascular prostaglandin synthesis, inhibition of the low Km cyclic GMP phosphodiesterases, and blockade of Ca21 channels either directly or indirectly through a K1-channel opening effect. The drug has also been shown to interact with a-adrenergic, vascular histamine, and muscarinic receptors. We have also reviewed the other vascular effects of the drug, such as stimulation of nitric oxide synthesis and inhibition of both myosin light chain kinase and protein kinase C. Cicletanine protects cardiovascular and renal systems against the injuries induced by hypertension, in addition to its lowering of arterial pressure. Similarly to the vasorelaxant action of cicletanine, the various properties of the drug likely contribute to its protective effect against injury in hypertension.  1999 Elsevier Science Inc. All rights reserved. Keywords: Cicletanine; Antihypertensive effect; a-Adrenergic receptors; Cyclic GMP-phosphodiesterase; K1 channels; Prostacyclin synthesis; Kidney; Natriuresis; Cytoprotective effect

Cicletanine ((6)3-(4-chlorophenyl)-1,3-dihydro-7hydroxy-6-methylfuro-[3,4-c] pyridine) 3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c] pyridine) is a new antihypertensive molecule synthetized by Esanu et al. (1986) in the research laboratories of Institute Henri Beaufour, Le Plessis-Robinson. Its chemical structure is uncommon for an antihypertensive molecule; it is characterized by the presence of a furopyridine group. As shown in Fig. 1, cicletanine possesses an asymmetric carbon and therefore has two enantiomers. Cicletanine demonstrates vasorelaxant, natriuretic, and diuretic properties in preclinical and clinical studies. Studies in animals and humans indicated that the antihypertensive effect of cicletanine is clearly dissociated from a natriuretic effect that appears at high doses. * Corresponding author. Tel.: 148 58 3412915; Fax: 148 58 3449653; E-mail: [email protected].

Cicletanine has also attracted much interest because this drug protects target organs against hypertensive injury, particularly in salt-dependent hypertension. This protective effect is greater than expected from the extent of blood pressure reduction. The mechanism(s) by which cicletanine induces the biological effects has not been definitely established and does not seem to be closely related to that of other classes of antihypertensive drugs. In the past decade, many papers dedicated to this aspect were published. Some of them have revealed partial stereoselectivity in induction of the pharmacological effects by cicletanine enantiomers. The fact that cicletanine is clinically well tolerated may be due to its multifactorial mechanism of action through which it may alter a number of physiological mediators, although exercising only a slight potency on each of them. At variance with other antihypertensive agents that are generally rather toxic to patients, cicletanine is a well-tolerated drug.

0306-3623/99/$–see front matter  1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00257-2

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L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

Fig. 1. Structures and metabolism of (2) and (1) enantiomers of cicletanine. Cicletanine is rapidly and almost fully metabolized into sulfo- and glucuro-conjugated derivatives, which are subsequently excreted by the kidney. After oral administration of (1)cicletanine, the sulfate conjugate is the major urinary component; the ratio of sulfate to glucuronide conjugate is 3. After oral administration of (2)cicletanine, the glucuronide and sulfate conjugates are present in similar amounts (Vistelle et al., 1995). (1)Cicletanine sulfate appears to be the active salidiuretic metabolite of cicletanine (Garay et al., 1995).

Because of the hydrophobic structure of the compound, cicletanine is almost insoluble in water. It penetrates the cells with a particular affinity for blood vessels. Radiolabeled cicletanine was shown to be taken up by the aorta after oral dosing (Chabrier et al., 1988). Moreover, Silver and Cumiskey (1991) reported that incubation of denuded aortic smooth muscle rings from spontaneously hypertensive rats with vasorelaxant concentrations (10–100 mM) of cicletanine resulted in a 7- to 12-fold accumulation of cicletanine in the tissue, compared with the tissue bath; the uptake was not stereoselective. This paper does not consider the specific pharmacological properties of cicletanine in human subjects but gives a brief compilation of its general pharmacology in preclinical studies. 1. Vascular effects of cicletanine Before a consideration of the vascular effects of cicletanine two points are noteworthy:

1. Variability of the drug action may be dependent on the vascular bed and the species studied (Calder et al., 1992a; Chabrier et al., 1988). 2. Several mechanisms, mentioned later, that may explain cicletanine’s relaxing effects on vascular smooth muscle can finally implicate a decrease in cytosolic calcium (Bukoski et al., 1989; Koltai et al., 1990). 1.2. Interactions with a-adrenergic receptors The a-adrenolitic action of cicletanine was shown by using isolated vessels in genetically hypertensive strokeprone rats (Malhebre et al., 1988), deoxycorticosterone (DOCA)-salt-treated rats (Castro et al., 1995), normotensive rats, guinea pigs, and rabbits (Deitmer et al., 1992). Even though this in vitro effect appears to be slight and less potent than phentolamine (a-adrenergic receptor antagonist) (Castro et al., 1995; Mikkelsen et al., 1993), treatment with cicletanine was shown ex vivo to attenuate vascular contractility caused by noradrena-

L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

line in stress and DOCA-salt treated hypertensive rats (Castro et al., 1988; 1990). In noradrenaline-precontracted isolated blood vessels (2)cicletanine had a stronger relaxant effect than did (1)cicletanine (Mikkelsen et al., 1993). It seems that the effect of cicletanine action with a1 receptors is competitive (Chabrier et al., 1988). Cicletanine appears to act more readily when catecholamine levels are elevated. The activity of the sympathetic nervous system has been shown to be enhanced in stress and DOCA-salt treated hypertensive rats. The fact that a drug is more effective in these hypertension models than in another one (two-kidney one-clip Goldblatt model) has to be taken as a preliminary indication that the antihypertensive effect works through a mechanism of action dependent on elevated catecholamine levels (Castro et al., 1988, 1990; Roesler et al., 1986). Cicletanine peripheral adrenergic blocking effect has been well documented by using isolated vessels in different animal experimental models. The compound enhanced the hypotensive effect of the a-adrenergic blocking agent phentolamine in such a way that ineffective doses of phentolamine made otherwise subeffective doses of cicletanine diminish blood pressure. A peripheral sympathicolytic action of cicletanine has been substantiated by the fact that the drug reduced blood pressure elevated by an a1-adrenergic agonist in pithed rats (Castro et al., 1995). In addition, the drug prevented the DOCA-salt-induced progressive depletion of heart noradrenaline that is patent during the development of this model of hypertension in rats. Although a peripheral mechanism of action has been ascertained for cicletanine, a central action cannot be completely ruled out. First, cicletanine is a lipophil compound and might cross the blood-brain barrier to affect a-adrenoceptors in the central nervous system. Second, in contrast with adult spontaneously hypertensive rats (SHRs), cicletanine produced hypotension in young animals made hypertensive either by stress or by DOCA-salt treatment (Castro et al., 1988, 1990). Compounds that do not readily cross the blood-brain barrier are known to enter young animals’ brains more easily when the barrier is still immature. Third, in the DOCAsalt-treatment model of hypertension, the corticosteroid-induced increase in blood-brain barrier permeability could increase the amount of cicletanine entering the brain. 1.2. Inhibition of the low Km cyclic GMP phosphodiesterases Hormones and neurotransmitters can exert actions on vascular smooth muscle cells by altering the intracellular concentration of cyclic nucleotides. The only known route of degrading these second messengers is through the action of the ubiquitous cyclic nucleotide phosphodiesterases. The possibility of producing selec-

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tive inhibitors for specific types of cyclic nucleotide phosphodiesterases has been explored by various investigators to produce novel cardiovascular therapeutic agents. Cicletanine was found to be a mixed (competitive, noncompetitive) inhibitor of both calmodulin-regulated and cyclic GMP-specific phosphodiesterases (PDEs) isolated from guinea pig ventricle and monkey aortic smooth muscle (Silver et al., 1990, 1991). In contrast with cyclic GMP-specific PDE, the stereoselectivity exists with respect to Ca21/CaM PDE (Table 1). In rat aortic smooth muscle contracted with phenylephrine, cicletanine produced concentration-related vasorelaxation (10–600 mM) that occurred at the concentration range corresponding to the inhibition of low Km cyclic GMP PDEs. The racemate and both enantiomers of cicletanine significantly potentiated vasorelaxation by activators of particulate (atrial natriuretic factor; ANF) and soluble (sodium nitroprusside; SNP) guanylate cyclase. Although it does appear that the (1)enantiomer is less effective relative to the racemate and the (2)enantiomer in potentiating ANF (and to the some extent, SNP)-mediated relaxation, the difference is small (Silver et al., 1991). The increase in potency of ANF and SNP for relaxation of vasopressor-contracted aorta in the presence of cicletanine was consistent with the inhibition of both forms of cyclic GMP-PDE isozymes. On the other hand, more recent data suggest that (2)cicletanine should be considered the enantiomer that contributes to the antihypertensive activity of racemic (6)cicletanine, by reducing the vascular reactivity to endogenous pressor substances such as angiotensin II and vasopressin (Alvarez-Guerra et al., 1996; Vargas et al., 1998). The noncompetitive antagonism of (2)cicletanine against endogenous pressor substances may be related to any of numerous steps that couple the occupation of their receptors to the final response. The findings of Silver et al. (1991) indicate that (2)cicletanine is almost twofold more potent than (1)cicletanine against the Ca21/CaM PDE. This may be the more significant that cicletanine has a particular affinity for blood vessels: the drug enantiomers accumulate in vascular smooth muscle to concentrations that are from 7- to 12-fold higher than those in the external media (Silver and Cumiskey, 1991). Therefore, (2)cicletanine might act on the angiotensin II and vasopressin signal transduction pathway by inhibiting Ca21/CaM PDE. The activity of cicletanine as a cyclic GMP-PDE inhibitor in intact muscle and in in vivo models was for the most part similar to that observed with the reference-selective cyclic GMP-PDE inhibitor zaprinast. An apparent discrepancy, however, existed in the relation between potentiation of SNP-mediated cGMP elevation and vasorelaxation with both cicletanine and zaprinast (Silver et al., 1990, 1991). At equipotent (IC50) PDE inhibitory concentrations, both agents produce comparable increases in the aortic tissue content of cy-

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L. Kalinowski et al./General Pharmacology 33 (1999) 7–16 Table 1 Inhibition of calcium/calmodulin and cyclic cGMP-specific PDEs, by cicletanine IC50 value for cyclic GMP PDE activity (mM) Inhibitor

Ca21/CaM PDEa

Cyclic GMP-specific PDEb

Cicletanine (racemate) Cicletanine (1 enantiomer) Cicletanine (2enantiomer) Zaprinast

270 325 180 100

375 375 375 0.30

Source: According to Silver et al. 1991. a In the presence of 10 mM Ca21 plus 1 nM calmodulin (CaM) and 10 mM cyclic GMP. b In the presence of 0.2 mM cyclic GMP.

clic GMP, but zaprinast does not potentiate SNP- or ANF-induced vasorelaxation. Concentrations of cicletanine (100 mM) and zaprinast (30 mM), which were equieffectvie with respect to potentiation of SNPinduced vasorelaxation, both increased cyclic GMP content of the tissue; however, the magnitude of the increase was markedly greater with zaprinast than with cicletanine. The data indicating that inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant actions of guanylate cyclase activators are not straightforward. In studies with 300 mM cicletanine alone on isolated aortic rings, no significant increases in the vessel tissue content of cyclic GMP were noted, even though this concentration of cicletanine produces greater than 50% relaxation. A similar discrepancy in cyclic GMP formation and a potential cyclic GMP-dependent effect has been confirmed in studies on renal glomeruli under conditions of sodium deprivation (Kalinowski et al., 1997). An ANF-induced increase in glomerular filtration rate (GFR) was demonstrated to be blunted during a low sodium diet (Kalinowski et al., 1995; Shenker, 1988). On the other hand, it was possible to restore the increased GFR in response to ANF in the low sodium diet rats by using zaprinast. The lack of a GFR-increasing response to ANF in the low sodium diet rats was shown to be primarly due to increased cyclic GMP hydrolysis in glomeruli (Kalinowski et al., 1995). Cicletanine alone induced nephrogenous cyclic GMP excretion to the same extent in rats on both normal and low sodium diets, whereas the increase in glomerular filtration rate induced by cicletanine was observed only in the normal sodium diet rats. In addition, cicletanine was unable to relax angiotensin IIcontracted glomeruli isolated from low sodium diet rats despite comparable formation of cyclic GMP in glomeruli of rats on both diets. However, infusion of cicletanine increased the ANF-stimulated nephrogenous cyclic GMP excretion in the low sodium diet rats to the level observed in the normal sodium diet rats, which coincided with restoration of the ANF-induced increase in GFR. Furthermore, the data from the in vivo experi-

ments corresponded to the effects of cicletanine and ANF on isolated glomeruli contracted by angiotensin II. Under conditions of sodium deprivation, angiotensin II blunts the renal response to ANF probably by stimulation of Ca21/CaM PDE. Thus, cicletanine might act on the angiotensin II transduction pathway by inhibiting cyclic GMP PDEs in glomeruli, particularly Ca21/CaM PDE. The low-Km cyclic GMP PDE inhibitor zaprinast also inhibited the pressor effects of angiotensin II (Trapani et al., 1991). All the aforedescribed results provide evidence that cicletanine [mainly the (2)enantiomer form of the drug] by inhibition of cyclic GMP PDEs can reduce vascular reactivity to endogenous vasoconstrictans such as angiotensin II and vasopressin, which plays a key role in several forms of hypertension. 1.3. Opening of K1 channels Several features of the activity profile of cicletanine suggest that this drug is a novel potassium channel opener [for a review, see Koltai et al. (1990)]. The concept for the prototype of potassium channel opener cromakalim is that it induces opening of potassium channels, and the hyperpolarization produced in this way inhibits voltage-controlled calcium channels that finally leads to an inhibition of contractile activity. Siegel et al. (1991) reported that cicletanine causes membrane hyperpolarization, which would make the membrane potential more negative and would reduce K1 efflux. Calder et al. (1994) demonstrated that cicletanine increased basal 86Rb (radiolabeled Rb was used as a marker for K1 because of its longer half-life) efflux from isolated guinea pig mesenteric vessels. Deitmer et al. (1992) reported that a potassium channel opening effect does not contribute significantly to the inhibitory effect of cicletanine in some types of vascular smooth muscle preparation (portal vein of rat, guinea pig, and rabbit; aorta of rat and guinea pig; iliac artery and ear artery of rabbit). On the other hand, Calder (1992) demonstrated the inability of cicletanine to relax guinea pig mesenteric vessels constricted with a high potassium concentration (118 mM KCl), but he has

L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

not been able to demonstrate sustained contraction in these vessels with a contraction of potassium ,40 mM. These experiments were done according to Edwards and Weston’s standard screen (Edwards and Weston, 1990) for detecting K1 channel-opening properties, in that a pharmacologic agent affects tension induced by a low external potassium concentration (20 mM) but has no effect on tension changes induced by a high concentration of potassium (e.g., 80 mM KCl), which produces complete depolarization. Therefore, it confirms that cicletanine possesses properties indicative of potassium channel-opening agents, but several classes of K1 channels exist and it is unclear with which channels, if any, cicletanine interacts. Miller and Weston (1991) and Ebeigbe and Cabanie (1991) demonstrated glibenclamide-sensitive cicletanine-induced relaxation in large vessels (human epigastric arteries of internal diameter 1.5–2.5 mm, rat aorta), indicating a possible role of ATP-dependent K1 channels (KATP) in mediating the relaxation. Involvement of KATP channels in the mechanism of cicletanineinduced relaxation of angiotensin II-contracted isolated renal glomeruli from low sodium diet rats has been also postulated (Szczepan´ska-Konkel et al., 1991). These findings could not confirmed by Calder et al. (1992a, 1994), who studied small human subcutaneous and guinea pig mesenteric arteries, both categorized as resistance arteries. Cicletanine-induced vasorelaxations were not affected by pretreatment with glibenclamide, apamin, or phencyclidine, suggesting that neither KATP nor small conductance calcium-dependent K1 channels (KCa) nor voltage-dependent K1 channels (KV) take part in cicletanine-induced vasorelaxation (Calder et al., 1994). These authors indicated that cicletanine acts on large conductance KCa in guinea pig mesenteric arteries. It should be pointed out that most experiments on the interaction of cicletanine with K1 channels was done on vascular smooth muscle cells, therefore directing attention to potential blood-pressure-lowering actions. It is difficult to set up the role of the K1 channels in explaining the depressor action of the compound because in vivo other mechanisms for the antihypertensive effect of cicletanine also are likely to play a role. Moreover, no such data allow the assessment of the action of cicletanine on K1 channels in vascular epithelial cells. Such action is possible, and it could contribute to the depressor effect of the compound. Inconsistent reports concerning the interaction of cicletanine with vascular smooth muscle K1 channels probably result from the presence of various classes of the channels in particular vascular beds. Thus, contribution of this mechanism in cicletanine-induced depression of blood pressure may vary in different regions of the vascular system. 1.4. Interactions with prostacyclin synthesis There is previously reported convincing evidence [for a review, see Koltai et al. (1990)] that cicletanine is

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capable of inducing prostacyclin (PGI2) generation under both in vitro and in vivo conditions. Some reports suggest a stimulation of arachidonic acid release (Deby et al., 1988a), enzymatic activity of PGI2 synthetase release (Deby et al., 1988b), and cholesteryl ester hydrolase activities contributing to cholesterol mobilization (Hajjar and Pomerantz, 1989). The eicosanoid system apparently has a role in the acute vasodilator action of cicletanine, but the relative importance of it varies in different species or at least in different vascular beds. Such reports arise from studies on the influence of prostanoid synthesis inhibitors on cicletanine-induced relaxation in a variety of vascular smooth muscles: indomethacin was ineffective against cicletanine-induced relaxation in isolated human epigastric (Ebeigbe and Cabanie´, 1991) and rat mesenteric arteries and aortae (Bukoski et al., 1989) but caused attenuated response in aortae from DOCA hypertensive rats (Castro et al., 1989) and human subcutaneous resistance arteries (Calder et al., 1992a, 1992b). In more recent studies (Ebeigbe et al., 1994) performed on isolated noradrenaline-precontracted human epigastric arteries, cicletanine caused concentration-dependent relaxations, uninfluenced by the cyclooxygenase inhibitors meclofenamate and indomethacin but significantly attenuated by the specific prostacyclin synthesis inhibitor tranylcypromine. The long-term effects of cicletanine on prostacyclin synthesis remain to be explored. The way by which cicletanine decreases blood pressure by increasing prostacyclin synthesis is unknown. Some authors (Calder et al., 1992a; Silver et al., 1991) suggested that the effects of cicletanine are endothelium independent and that cicletanine may affect prostacyclin synthesis by acting directly on smooth muscle cells in arterioles, even though most of prostacyclin is synthesized in the vascular endothelium. In addition, long-term cicletanine treatment depresses the growth rate of vascular smooth muscle cells in the same concentration range as that for exerting the vasodilating effect (see the section titled “The Cardioprotective and Vasoprotective Effects of Cicletanine”). In conclusion, many data suggest that the eicosanoid system has a role in the acute vasodilation action of cicletanine. In particular, prostacyclin may be implicated in mediating this relaxation. This may also be relevant to the chronic antihypertensive and vasoprotective properties of the drug. 1.5. Other vascular effects of cicletanine At the normally effective concentration range, cicletanine blocks voltage-dependent calcium channels of vascular smooth muscle cells from guinea pig portal vein (Noack and Deitmer, 1993). In these studies, calcium inward current was carried by the voltage-controlled calcium channels that are blocked by nifedipine.

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L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

Table 2 Inhibition of calcium/calmodulin regulated MLCK and PKC by cicletanine IC50 value for kinase inhibition (mM) Inhibitor

MLCK

PKC

Cicletanine (racemate) Cicletanine (1 enantiomer) Cicletanine (2 enantiomer)

1000 960 620

900 850 880

MLCK 5 myosin light chain kinase, PKC 5 protein kinase C. Source: Silver et al. (1991).

Because it was done under voltage clamp conditions, the inhibitory effect of cicletanine is clearly not produced by a change in the membrane potential and therefore cannot be due to potassium channel opening. Cicletanine was reported to be an H1-type histaminergic receptor antagonist in vascular smooth muscle cells (Auguet et al., 1988; Schoeffter et al., 1987), which possesses a relatively low receptor affinity (KD of 36 nM) (Ebeigbe et al., 1989). The effect seems to be stereospecific: only the (2)enantiomer of cicletanine blocks H1 receptors, whereas the (1)enantiomer has a weak effect (Schoeffter et al., 1987). However, it is difficult to define whether the antihypertensive action of cicletanine partly results from specific H1 antagonism. A dose-dependent inhibition by cicletanine of Ca21 mobilization and phosphoinositide turnover, stimulated by histamine, was observed in smooth muscle cells (Lonchampt et al., 1988). Cicletanine was inhibited both by myosin light chain kinase (MLCK) and by protein kinase C (PKC), which are two Ca21-regulated kinases directly involved in regulation of smooth muscle contractile activity (Table 2) (Silver el al., 1991). The threshold concentration of cicletanine for inhibition of MLCK or PKC is from 100 to 300 mM, which does coincide with high, vasorelaxant concentrations. Thus, it is possible that MLCK or PKC inhibition or both by cicletanine can also contribute to in vitro vasorelaxation by cicletanine, although the revelance of this observation to clinical activity is not apparent. The vasorelaxant effect of cicletanine was demonstrated to be partly endothelium dependent; the effect is mediated by nitric oxide and requires a functional K1 channel (Bukoski et al., 1993). In addition, cicletanineinduced relaxation was attenuated in SHRs. Hirawa et al. (1996) confirmed the fact that chronic cicletanine treatment enhances the formation of nitric oxide in rat vessel walls. The authors indicated that the restoration of vasodepressor substance is indicative of the regression of vascular injury and contributes to the antihypertensive effect of cicletanine during chronic treatment. Among possible mechanisms of antihypertensive action of cicletanine, involvement of the renal kallikreinkinin system was postulated by Emond et al. (1992). In

has been shown in these studies that cicletanine treatment prevented the decrease in renal kallikrein biosynthesis in stroke-prone spontaneously hypertensive rats. Moreover, the direct effect of cicletanine on the reninagiotensin system cannot be completely ruled out. Plasma renin activity tended to rise after the administration of cicletanine, as previously reported (Buchholz et al., 1992; Damase-Michel et al., 1991), but the rise was small and not statistically significant except 24 h. On the other hand, the increased synthesis of prostacyclin, as caused by cicletanine, might stimulate renin release from the kidneys (Gerber et al., 1981).

2. Effect of cicletanine on renal function There is a dissociation between a natriuretic effect of cicletanine and the acute systemic effect. Experimental studies conducted in dogs showed that acute administration of cicletanine was followed by natriuretic response in a dose-related manner beginning at the dosage of 1–3 mg/kg; Lee et al. (1991) showed that the threshold dose of cicletanine to induce natriuresis (1 mg/kg) was lower than that needed to reduce mean arterial blood pressure (3 mg/kg). This natriuretic effect of cicletanine was evident at lower doses than those used in earlier studies (Singer et al., 1990; Tarrade and Guinot, 1988). The diuretic effect is characterized by a very moderate kaliuresis compared with natriuresis and diuresis [for a review, see Chabrier et al. (1988)]. Several observations suggest that (1)cicletanine sulfate was the active salidiuretic metabolite of orally given racemic cicletanine in rats (Fig. 1) (Garay et al., 1992, 1995; Vistelle et al., 1995). First, the urinary excretion of (1)cicletanine sulfate was considerably higher than that of (2)cicletanine sulfate. Moreover, the urinary excretion of (2)cicletanine glucuronate was significantly higher than that of (1)cicletanine glucuronate. Further evidence for (1)cicletanine sulfate as the active salidiuretic metabolite of cicletanine was provided by experiments testing the salidiuretic activity of compounds after direct administration into the rats renal artery. In these experiments, (1)cicletanine sulfate was from three to four times as potent in inducing salidiuresis as the (2)enantiomer (Garay et al., 1995). Final evidence for (1)cicletanine sulfate as the active salidiuretic metabolite of cicletanine was provided by measurement of membrane ion fluxes. In rat erythrocytes, (1)cicletanine sulfate was from two to three times as potent in inhibiting the Na1-dependent Cl2/HCO32 anion exchanger as (2)cicletanine sulfate (IC50 5 61 6 3 mM versus 142 6 31 mM) (Garay et al., 1995). A similar Na1-dependent Cl2/ HCO32 anion exchanger seems to be luminally located in the distal convoluted tubule, further suggesting that cicletanine acts at this segment level (Greven, 1995; Hadj-Aissa et al., 1987). The presence of other mechanism(s) of the natri-

L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

uretic action of cicletanine cannot be excluded. Studies in anesthetized rats with the use of free-flow micropuncture techniques revealed cicletanine’s effect in the superficial distal tubule; thus, the drug shares its site of tubular action with that of the thiazide diuretics (Greven, 1995). However, it seems rather unlikely that the Na1-dependent Cl2/HCO32 anion exchanger has a role in cicletanine action in the superficial distal tubule of the kidney, because its presence in this location has never been described. In clearance experiments, the prostaglandin inhibitors were shown to nearly completely abolishe cicletanine’s effect on urinary fluid and electrolyte excretion in spite of the fact that renal hemodynamics improved (Greven et al., 1995). These authors suggest that cicletanine’s diuretic and natriuretic action may be related to a stimulation of renal prostaglandin synthesis.

3. Cardioprotective and vasoprotective effects of cicletanine Jouve et al. (1986, 1988) first reported evidence that cicletanine prevents the usual outcome of life-threatening arrhythmias developing after occlusion of the left circumplex coronary artery in an occlusion/reperfusion dog model; they showed that cicletanine is able to significantly increase the level of 6-keto-PGF1a in venous blood draining from the ischemic area in cicletaninefree dogs while no relevant change occurs. The antiarrhythmic effect of short- and long-term cicletanine treatment was confirmed in isolated Langendorff preparation (Koltai et al., 1992) and in the working rat heart (Ferdinandy et al., 1992; Koltai et al., 1991), but it has been found to be related to a reduction in ischemic/ reperfusion-induced ion shift (Koltai et al., 1992). Thus cicletanine reduced Na1 and Ca21 gain and K1 loss during global ischemia in rat hearts. Tosaki et al. (1991) also demonstrated that the antiarrhythmic effect of cicletanine seen in rats was not associated with an alteration in release of 6-keto-PGF1a or thromboxane during reperfusion but correlated better with alterations in myocardial ion content. Because these observations (Koltai et al., 1992; Tosaki et al., 1991) were made in both acutely and chronically treated isolated perfused hearts, they were indicative of a direct action of the drug on the heart. Further investigations by Szilva´ssy et al. (1993) demonstrated that, in correlation with alterations of cardiac nucleotide contents, cicletanine protects the heart against ventricular overdrive pacing-induced myocardial ischemia in chronically instrumented rabbits; cicletanine increased cyclic GMP and markedly reduced cyclic AMP cardiac content. In recent studies, Ferdinandy et al. (1995) showed that the antiischemic but not the antiarrhythmic effect of cicletanine may involve opening of KATP. There are many observations demonstrating that

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long-term cicletanine treatment attenuates vascular and renal injury induced by hypertension, particularly in the salt-dependent hypertensive model, beyond what could be expected from blood-pressure reduction per se. The various properties of cicletanine likely contribute to its protective effect against injury in hypertension. Cicletanine exhibits antioxidant properties and it directly facilitates the conversion of arachidonate into vasodilatory prostanoids (Uehara et al., 1993). The drug treatment reduces the amounts of low-density lipoprotein cholesterol, probably through enhancement of cholesteryl ester hydrolase activity (Hajjar and Pomerantz, 1989), and this effect contributes to improving renal glomerulosclerosis in Dahl S rats with salt-induced hypertension (Uehara et al., 1997). It has been also demonstrated that long-term cicletanine treatment preserves endothelial cell function, which is manifested by an increase in the biosynthesis of vasodilator prostacyclin and nitric oxide (Hirawa et al., 1996). In addition, cicletanine, in contrast with many other antihypertensive agents, reduces capillary permeability and therefore has a supplemental protective effect on microvasculature (Lehoux and Plante, 1995). Bukoski et al. (1993) showed in vitro that cicletanine retards [3H]thymidine incorporation into deoxyribonucleic acid fragments and decreases the growth of cultured vascular smooth muscle cells. There appears to be a link between the inhibition by cicletanine of the proliferation of vascular smooth muscle cells and cicletanine-induced vasodepressor prostacyclin generation in these cells. Because prostacyclin tempers the proliferation of vascular smooth muscle cells through modulation of the G1 resting period of the proliferating cell cycle, this retardation of cell growth may result in the decrease in medial hypertrophy seen in cicletaninetreated Dahl S rats (Uehara et al., 1991a, 1991b). Moreover, cicletanine per se possesses inhibitory effects on cytoskeleton protein synthesis in vascular smooth muscle cells (Uehara et al., 1991b). This inhibitory effect of cicletanine on the formation of cytoskeleton proteins could partly account for the regression of medial hyperthrophy in Dahl S rats with salt-induced hypertension.

4. Concluding remarks Cicletanine is a new antihypertensive molecule characterized by the presence of a furopyridine group. This structure may be used as a basis for the development of a new family of compounds possessing antihypertensive properties. Cicletanine comprises two enantiomers, [(2)cicletanine and (1)cicletanine], independently contributing to the vasorelaxant and salidiuretic mechanisms of the drug. Despite the fact that cicletanine was intensively studied within the past several years, further investigations are needed to determine the significance of the particular mechanisms of action of this drug with

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respect to its vasodilating and salidiuretic potency. It seems to be reasonable to assess the action of cicletanine on the K1 channels in both vascular and nephron epithelial cells. Potential action of cicletanine in these cell types in vivo could contribute to the final biological effects of the compound. Cicletanine [mainly the (2)enantiomer] reduces the vascular response to angiotensin II and potentiates the vascular actions of guanylate cyclase activators. Thus, it may be useful to apply cicletanine in combination with a low sodium diet to treat hypertension or in the setting of diuretic resistance and sodium and water retension. It appears that other, more detailed information related to the therapeutic benefits of treatment with the racemate and single enatiomers in various forms of hypertension is still needed. Combined antihypertensive and tissue-protective actions of cicletanine seem to be useful in preventing structural changes in the vascular wall or in reducing the severity of intimal proliferation secondary to vascular wall injury associated with established hypertension. However, there is a possibility that the tissue-protective effects of antihypertensive drugs could be dissociated from the effects on blood-pressure reduction. Exploring antihypertensive drugs with such properties would potentially offer new prospects in the field of antihypertensive therapy. Therefore, it seems intriguing to know whether cicletanine, which has relatively weak antihypertensive effects and potent tissue-protective effects, could be a candidate for such a unique antihypertensive drug. To address these interesting points, it will be necessary to expand on further preclinical studies with cicletanine or other furopyridine analogues.

References Alvarez-Guerra, M., Alda, O., Morin, E., Allard, M., Garay, R.P., 1996. Reduction by (2)-cicletanine of the vascular reactivity to angiotensin II in rats. J Cardiovasc Pharmac 28, 564–570. Auguet, M., Delaflotte, S., Hellegouarch, A., Guillon, J.M., Barane´s, J., Pirotzky, E., Clostre, F., Braquet, P., 1988. In vitro cardiovascular antihistamine properties of cicletanine in comparison with diphenhydramine. Drugs Exp Clin Res 14, 149–153. Buchholz, R.A., Brousseau, A., Silver, P.J., 1992. Natriuretic and diuretic effects of cicletanine in conscious, hydrated, normotensive rats. Drugs Dev Res 25, 125–137. Bukoski, R.D., Wagman, D.W., De Wan, P., Xue, H., 1989. Vascular actions of cicletanine. Arch Mal Coeur 82, 45–50. Bukoski, R.D., Bo, J., Xue, H., Bian, K., 1993. Antiproliferative and endothelium-dependent vasodilator properties of 1,3-dihydro-3-pchlorophenyl-7-hydroxy-6-methyl-furo-(3,4c) pyridine hydrochloride (cicletanine). J Pharmac Exp Ther 265, 30–35. Calder, J.A., 1992. Mechanism of antihypertensive action of thiazide diuretics and related drugs: direct vascular effects. J Drug Dev 4, 189–198. Calder, J.A., Schachter, M., Sever, P.S., 1992a. Mechanisms of action of cicletanine in human and guinea pig resistance arteries. J Cardiovasc Pharmac 19, 387–393. Calder, J.A., Schachter, M., Sever, P.S., 1992b. Interaction between

cicletanine and the eicosanoid system in human subcutaneous resistance arteries. J Pharm Pharmac 44, 574–578. Calder, J.A., Schachter, M., Sever, P.S., 1994. Potassium channel opening properties of thiazide diuretics in isolated guinea pig resistance arteries. J Cardiovasc Pharmac 24, 158–164. Castro, A., Florentino, A., Fuentes, J.A., 1988. Antihypertensive effect of non-diuretic doses of cicletanine on a stress-induced model in the rat. Drugs Exp Clin Res 14, 97–101. Castro, A., Parra, L., Fuentes, J.A., 1989. Studies on the mechanism of action of the antihypertensive agent cicletanine. Arch Mal Coeur 82, 51–54. Castro, A., Parra, L., Alsasua, A., Fuentes, J.A., 1990. The anithypertensive agent cicletanine reverses vascular hyperractivity to noradrenaline and cardiac hyperthrophy in DOCA-salt rats. Arch Int Pharmacodyn Ther 307, 109–18. Castro, A., Hernando, F., Parra, L., Puerro, M., Aleixandre, A., Fuentes, J.A., 1995. Further evidence for a peripheral sympathicolytic action of the antihypertensive cicletanine rats. Drugs Exp Clin Res 21, 29–36. Chabrier, P.E., Guinot, P., Tarrade, T., Auguet, M., Cabanie, M., Clostre, F., Etienne, A., Esanu, A., Graquet, P., 1988. Cicletanine. Cardiovasc Drug Rev 6, 166–179. Damase-Michel, C., Girolami, J.P., Bascands, J.L., Tran, M.A., Moatti, J.P., Pecher, C., Berthet, P., Tarrade, T., Montastruc, J.L., 1991. Effects of cicletanine on vasoactive systems in consious sinoartic-denervated dogs. Fund Clin Pharmac 5, 719–732. Deby, C., Bourgain, R.H., Andries, R., Braquet, P., 1988a. Modulation of prostacyclin synthetase by cicletanine and drugs which affect ion transport. Pharmac Res Commun 20, 101–108. Deby, C., Bourgain, R.H., Deby Dupont, G., Pincemail, J., Garay, R., Braquet, P., 1988b. Modulation of in vivo generation of prostanoids by drugs affecting membrane ion transport. Drugs Exp Clin Res 14, 123–134. Deitmer, P., Golenhofen, K., Noack, T., 1992. Comparison of the relaxing effects of cicletanine and cromacalim on vascular smooth muscle. J Cardiovasc Pharmac 20, 35–42. Ebeigbe, A.B., Cabanie´, M., 1991. In vitro effects of cicletanine in pregnancy induced hypertension. Br J Pharmac 103, 1992–1996. Ebeigbe, A.B., Cabanie´, M., Godfraind, T., 1989. Effects of cicletanine on histamine-induced contractions of isolated rabbit mesenteric arteries. Fund Clin Pharmac 3, 223–235. Ebeigbe, A.B., Oguogho, A., Ighoroje, A.D.A., Aloamaka, C.P., 1994. Differential effects of prostanoid synthesis inhibitors on cicletanine-induced relaxation of isolated human epigastric arteries. Clin Auton Res 4, 137–139. Edwards, G., Weston, A.H., 1990. Potassium channel openers. In: Barber, M.S. (Ed.), Pharmaceutical manufacturing international. Sterling Publications, London, pp. 29–32. Emond, C., Bompart, G., Rakotoarivony, J., Bascands, J.L., Pecher, C., Adam, A., Tarrade, T., Berthet, P., Girolami, J.P., 1992. Protective effect of cicletanine on hypertension-induced decreases in the renal kallikrein-kinin and prostaglandin systems in stroke-prone spontaneously hypertensive rats. J Cardiovasc Pharmac 20, 601–608. Esanu, A., 1986. Dossier d’autorisation de mise sur le marche´ (A.M.M.). Cicletanine. Dossier no. 328747.9 (27.7.1986). Socie´te´ de Conceil de Recherches et d’Applications Scientifiques BE 891.797; DE 3.204.596; FR 2.449.406 and 2.499.574; GB 2.092.586; JP 82.150.688; US 4.483.998. Ferdinandy, P., Koltai, M., Tosaki, A., Berthet, P., Tarrade, T., Esanu, A. Braquet, P., 1992. Cicletanine improves myocardial function deteriorated by ischemia/reperfusion in isolated working rat hearts. J Cardiovasc Pharmac 19, 181–189. Ferdinandy, P., Szilva´ssy, Z., Droy-Lefaix, M.T., Tarrade, T., Koltai, M., 1995. KATP channel modulation in working rat hearts with coronary occlusion: effects of cromakalim, cicletanine, and glibenclamide. Cardiovasc Res 30, 781–787. Garay, R.P., Rosati, C., Nazaret, A., Esanu, T., Tarrade, T., Braquet,

L. Kalinowski et al./General Pharmacology 33 (1999) 7–16 P., 1992. Cicletanine sulfate: inhibition of anion transport systems and natriuretic activity. Naunyn-Schmiedenberg’s Arch Pharmac 346, 114–119. Garay, R.P., Rosati, C., Fanous, K., Allard, M., Morin, E., Lamiable, D., Vistelle, R., 1995. Evidence for (1)-cicletanine sulfate as an active natriuretic metabolite of cicletanine in the rat. Eur J Pharmac 274, 175–180. Gerber, J.G., Olson, R.D., Nies, A.S., 1981. Interrelationship between prostaglandins and renin release. Kidney Int 19, 816–821. Greven, J., 1995. Effects of cicletanine on kidney function: 1. Clearance and micropuncture studies in anesthetized rats. J Pharmac Exp Ther 273, 1190–1196. Greven, J., Rahn, A., Bra¨ndle, E., 1995. Effects of cicletanine on kidney function: 2. Role of renal prostaglandins and kinins, and of muscarinic receptors. J Pharmac Exp Ther 273, 1197–1202. Hadj-Aissa, A., Pozet, H., Labetin, M., Zech, P., 1987. Site of renal action of cicletanine in man. In: Puschett, S.B., Greenberg, A. (Eds.), Diuretics II: chemistry, pharmacology and clinical applications. Elsevier, New York, pp. 35–37. Hajjar, D.P., Pomerantz, K.B., 1989. Cicletanine stimulates cholesteryl ester hydrolase activities and prostacyclin production in arterial smooth muscle cells. Arch Mal Coeur 82, 79–84. Hirawa, N., Uehara, Y., Kawabata, Y., Akie, Y., Ichikawa, A., Funahashi, N., Goto, A., Omata, M., 1996. Restoration of endothelial cell function by chronic cicletanine treatment in Dahl salt-sensitive rats with salt-induced hypertension. Hypertens Res 19, 263–270. Jouve, R., Langlet, F., Puddu, P.E., Rolland, P.H., Guillen, J.C., Cano, J.P., Serradimigni, A., 1986. Cicletanine improves outcome after left circumflex coronary artery occlusion-reperfusion in the dog. J Cardiovasc Pharmac 8, 208–215. Jouve, R., Puddu, P.E., Langlet, F., Lanti, M., Guillen, J.C., Rolland, P.H., Serradimigni, A., 1988. Effects of cicletanine in the left circumflex coronary artery occlusion-reperfusion canine model of sudden death: analysis of 107 experiments using Cox’s proportional hazard models. Drugs Exp Clin Res 14, 167–180. Kalinowski, L., Szczepan´ska-Konkel, M., Pawełczyk, T., Bizon, D., Angielski, S., 1995. Inhibition of cGMP-phosphodiesterase restores the glomerular effects of atrial natriuretic factor in low sodium diet rats. Renal Physiol Biochem 18, 254–266. Kalinowski, L., Szczepan´ska-Konkel, M., Jankowski, M., Angielski, S., 1997. Modulation by low sodium intake of glomerular response to cicletanine and atrial natriuretic factor. Br J Pharmac 121, 635–642. Koltai, M., Esanu, A., Braquet, P., 1990. Relaxation of vascular smooth muscle by cicletanine: importance of potassium channels and prostacyclin. In: Fro¨lich, J.C., Fo¨rstermann, U. (Eds.), Klinische pharmakologie [Clinical Pharmacology, Vol. 7: Importance of prostacyclin and potassium channels for the regulation of vascular tone]. W. Zuckschwerdt Verlag, Munich, pp. 106–136. Koltai, M., Tosaki, A., Tarrade, T., Berthet, P., Esanu, A., Braquet, P.G., 1991. Cicletanine: a novel approach to cardioprotection in spontaneously hypertensive rats. Coronary Artery Dis 2, 941–994. Koltai, M., Tosaki, A., Berthet, P., Tarrade, T., Braquet, P.G., 1992. Effect of cicletanine on reperfusion-induced arrhythmias and myocardial ion contents: a comparison with furosemide. Eur Heart J 13, 395–403. Lee, K.C., Cannif, P.C., Hamel, D.W., Pagani, E.D., Gorcyzca, W.P., Ezrin, A.M., Silver, P.T., 1991. Comparative hemodynamic and renal effects of the low Km cGMP phosphodiesterase inhibitors cicletanine and zaprinast in anesthethized dogs. Drug Dev Res 23, 127–144. Lehoux, S., Plante, G.E., 1995. Antihypertensive drugs and endothelial cell function. Prostaglandins Leukot Essent Fatty Acids 54, 65–70. Lonchampt, M.O., Marche, P., Demerle, C., Girard, A., Cabanie´, M., Esanu, A., Chabrier, P.E., Braquet, P., 1988. Histamine H1-receptors mediate phosphoinositide and calcium response in cultured smooth muscle cells: interaction with cicletanine. Agents Actions 24, 255–260.

15

Malhebre, E., Le Hegara, M., Clostre, F., Braquet, P., 1988. Comparison of cicletanine with other antihypertensive drugs in SHR-SP models. Drugs Exp Clin Res 14, 83–88. Mikkelsen, E.O., Sehested, J., Poulsen, S.H., 1993. Comparison of in vitro effects of cicletanine, its enantiomers and major metabolite on rat thoracic aorta. Naunyn-Schmiedeberg’s Arch Pharmac 347, 548–552. Miller, M., Weston, A.H., 1991. Studies on the mode of action of cicletanine in rat blood vessels. Fund Clin Pharmac 5, 408. Noack, T., Deitmer, P., 1993. Effects of cicletanine on whole-cell currents of single smooth muscle cells from the guinea-pig portal vein. Br J Pharmac 109, 164–170. Roesler, J.M., McCafferty, J.P., DeMarinis, R.M., Matthewa, W.D., Hieble, J.P., 1986. Characterization of the antihypertensive activity of SK&F 86466, a selective alpha-2 antagonist, in the rat. J Pharmac Exp Ther 236, 1–7. Schoeffter, P., Ghysel-Burton, J., Cabanie´, M., Godfraind, T., 1987. Competitive and stereoselective histamine H1 antagonistic effect of cicletanine in guinea pig isolated ileum. Eur J Pharmac 136, 235–238. Shenker, Y., 1988. Atrial natriuretic hormone effect on renal function and aldosterone secretion in sodium depletion. Am J Physiol 255, R867–R873. Siegel, G., Schnalke, F., Schultz, G., Stock, G., 1991. K1 channel opening and vascular tone. In: Mulvany, M.J., Aalkjaer, C., Heagerty, A.M., Nyborg, N.C.B., Strangaard, S. (Eds.), Resistance arteries: structure and function. Elsevier, Amsterdam, pp. 130–138. Silver, P.J., Cumiskey, W.R., 1991. Uptake of cicletanine by vascular smooth muscle. Drug Dev Res 23, 275–280. Silver, P.J., Buchholz, R.A., Dundore, R.L., Harris, A.L., Pagani, E.D., 1990. Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine. J Cardiovasc Pharmac 16, 501–505. Silver, P.J., O’Connor, B., Cumiskey, W.R., Van Aller, G., Hamel, L.T., Bentley, R.G., Pagani, E.D., 1991. Inhibition of low Km cGMP phosphodiesterase and Ca11-regulated protein kinases and relationship to vasorelaxation by cicletanine. J Pharmac Exp Ther 257, 382–391. Singer, D.R.J., Markandu, N.D., Sugden, A.L., MacGregor, G.A., 1990. A comparison of the acute effects of cicletanine and bendrofluazide on urinary electrolytes and plasma potassium in essential hypertension. Eur J Clin Pharmac 39, 227–232. Szczepan´ska-Konkel, M., Redlak, M., Angielski, S., 1991. Glibenclamid-sensitive K1 channels are responsible for angiotensin II hypersensitive contraction and atrial natriuretic factor refractoriness of glomeruli in low-sodium rats. Biochem Biophys Res Commun 181, 871–876. Szilva´ssy, Z., Jakab, I., Bor, P., Koltai, M., Tarrade, T., Esanu, A., Braquet, P., Lonovics, J., 1993. Cicletanine attenuates overdrive pacing-induced global myocardial ischemia in rabbits: possible role of cardiac cyclic nucleotides. Coronary Artery Dis 4, 443–452. Tarrade, T., Guinot, P., 1988. Efficacy and tolerance of cicletanine, a new antihypertensive agent: overview of 1226 treated patients. Drugs Exp Clin Res 14, 205–214. Tosaki, A., Hellegouarch, A., Braquet, P., 1991. Cicletanine and reperfusion injury: is there any correlation between arrhythmias, 6-keto-PGF1a, thromboxane B2, and myocardial ion shifts (Na1, K1, Ca11, and Mg11) induced by ischemia/reperfusion in isolated rat heart? J Cardiovasc Pharmac 17, 551–559. Trapani, A.J., Smits, G.J., McGraw, D.E., McMahon, E.G., Blaine, E.H., 1991. Hemodynamic basis for the depressor activity of zaprinast, a selective cyclic GMP phosphodiesterase inhibitor. J Pharmac Exp Ther 258, 269–274. Uehara, Y., Numabe, A., Hirawa, N., Kawabata, Y., Iwai, J., Ono, H., Matsuoka, H., Takabatake, Y., Yagi, S., Sugimoto, T., 1991a. Antihypertensive effects of cicletanine and renal protection in Dahl salt-sensitive rats. J Hypertens 9, 719–728.

16

L. Kalinowski et al./General Pharmacology 33 (1999) 7–16

Uehara, Y., Numabe, A., Kawabata, Y., Nagata, T., Iwai, J., Matsuoka, H., Yagi, S., Takabatake, Y., Sugimoto, T., 1991b. Evidence for medial-mass regression in the vascular wall of Dahl hypertensive rats by cicletanine treatment. J Cardiovasc Pharmac 18, 158–166. Uehara, Y., Kawabata, Y., Hirawa, N., Takada, S., Nagata, T., Numabe, A., Iwai, J., Sugimoto, T., 1993. Possible radical scavenging properties of cicletanine and renal protection in Dahl salt sensitive rats. Am J Hypertens 6, 463–472. Uehara, Y., Hirawa, Y., Kawabata, Y., Akie, Y., Ichikawa, A., Funahashi, N., Goto, A., Omata, M., 1997. Lipid metabolism and renal

protection by chronic cicletanine treatment in Dahl salt-sensitive rats with salt-induced hypertension. Blood Press 6, 180–187. Vargas, F., Alvarez-Guerra, M., Droy-Lefaix, M.T., Garay, R.P., 1998. Inhibition by (2)-cicletanine of the vascular reactivity to angiotensin II and vasopressin in isolated rat vessels. Am J Hypertens 11, 579–584. Vistelle, R., Lamiable, D., Morin, E., Trenque, T., Kaltenbach, M., 1995. Urinary excretion of cicletanine in the rat: stereochemical aspects. Drug Metab Dispos 23, 988–992.