Receiv ed: May 4, 2012 Accept ed after revision: May 29, 2012 Published online: September 13, 2012
Characterization of New Organic Nitrate Hybrid Drugs Covalently Bound to Valsartan and Cilostazol
Maike Knorra,b Michael Hausdinga,b Eberhard Schulza Matthias Oelzea
Robert Rümmlera Alexandra Schuffa Steffen Dauba Jörg Schreinera
Swenja Kröller-Schöna Philip Wenzela,b Tommaso Goria Karl Burginc
Dirk Sartord Armin Scherhagd Thomas Münzela Andreas Daibera
aKardiologie, II. Medizinische Klinik, Universitätsmedizin der Johannes-Gutenberg-Universität Mainz, and bCenter of Thrombosis and Hemostasis, Mainz, Germany; cMaxia Strategies LLC and dCardiolynx AG, Basel, Switzerland
Bac kground and Purpose: Organic nitrates represent a group of nitrovasodilators that are clinically used for the treatment of ischemic heart disease. With the present stud-ies we synthesized and characterized new organic nitrate hybrid molecules. Compounds CLC-1265 (valsartan mono-nitrate) and CLC-1280 (valsartan dinitrate) are derivatives of the angiotensin receptor blocker valsartan, with CLC-1265 containing a single organic nitrate linker and CLC-1280 also containing a second, different linker. Compounds CLC-2000 (cilostazol mononitrate) and CLC-2100 (cilostazol dinitrate) are nitrate derivatives of the phosphodiesterase III inhibitor cilostazol. All compounds are designed as hybrid molecules, potentially combining the NO-donating properties of or-ganic nitrates with the AT1-blocking activity of valsartan or the phosphodiesterase-III–inhibiting effect of cilostazol. Experimental Approach: The properties of new drugs were assessed by isometric tension recording, inhibition of plate-let aggregation and formation of mitochondrial reactive
stable angina pectoris [1]. The impact of these drugs on cardiac preload and the subsequent reduction in car-diac oxygen consumption are thought to play a central role in the improvement in exercise tolerance associated with the treatment with these drugs. Moreover, organic nitrates are also employed as adjunct (or alternative) of angiotensin-converting enzyme inhibitors for the treat-ment of chronic heart failure, e.g. isosorbide dinitrate (ISDN)/hydralazine [2]. As well, the coronary vasodila-tory properties of nitrates, and a mild antiaggregant ef-fect, explain their benefit in the setting of acute coronary syndromes such as unstable angina and myocardial in-farction. Their principle of action is based on an enzy-matic bioactivation process with subsequent formation of nitric oxide (NO) or a related species (NO from nitro-sothiol or NO-metal complex [3]), which results in the activation of soluble guanylyl cyclase and an increase in the second-messenger cGMP, in turn leading to vasodi-lation [4, 5].
While glyceryl trinitrate (GTN) and other nitrates are widely employed in the therapy and prevention of stable angina, the large clinical trials in the setting of myocar-dial infarction conducted 15 years ago revealed only mild effects on mortality and prognosis for patients with coro-nary artery disease. In line with these findings, guidelines suggest that organic nitrates should be used only for the relief of angina in patients with coronary artery disease who are refractory to other therapies (e.g. β-blocker or calcium channel antagonists) [6]. In addition the intro-duction of newer antianginal medications such as ranola-zine or ivabradine may lead to a further reduction of the use of organic nitrates in the next years. Another reason for a reduction in the prescription rate of nitrates is based on serious side effects (e.g. oxidative stress, endothelial dysfunction and nitrate tolerance) of some but not all members of this class of drugs [7, 8].
A promising strategy to overcome the disadvantages of organic nitrates is the covalent link between the ni-trate function and another vasoactive compound poten-tially combining the advantages of both drugs, so-called hybrid molecules [9]. There are several reports in the literature on nitrate hybrid molecules: nicorandil, a po-tassium channel opener with nitrate function [10, 11]; 2NTX-99, a thromboxane synthase inhibitor and throm-boxane receptor antagonist and NO-releasing group [12]; GT-094, a nonsteroidal anti-inflammatory drug with NO function [13]; the antifungal drug ketoconazole with a diazen-1-ium-1,2-diolate or an organic nitrate moiety [14]; NO-donor-tacrine hybrids as hepatopro-tective anti-Alzheimer drug candidates [15]; the NicOx
compound nitroaspirin, reviewed in Gresele and Momi [16]; NO-releasing celecoxib analogs, inhibitors of in-ducible cyclooxygenase [17]; a vitamin E analog with NO donor function [18]. Probably we could not list all ex-amples that have been published during the last few years but our list provides an overview of the diversity of NO hybrids. The idea behind this research is to develop new beneficial functions of established drugs or to improve known principles of action but also to suppress side ef-fects of these drugs.
With the present study we synthesized and charac-terized new nitrate hybrid molecules based on covalent binding of the nitrate function to valsartan and cilo-stazol. The basic vascular action of these drugs was char-acterized by isometric tension recording, assessment of tachyphylaxis by repeated challenges, oxidative stress induction in isolated cardiac mitochondria and antiag-gregatory effects in platelet-rich plasma.
ammonium nitrate] and CLC-1275 [chloromethyl 2-(nitrooxy)-ethylcarbamate] were prepared by Siegfried Ltd. (Zofingen, Switzerland). Linker CLC-1273 (1,4-butanediol mononi-trate) was prepared by Labor für Prozessforschung (Zürich, Switzerland). CLC-1265 (valsartan mononitrate) and CLC-1280 (valsartan dinitrate) were prepared by Siegfried Ltd. Cilostazol was obtained from Betapharma (Shanghai, China), and CLC-2000 (cilostazol mononitrate) and CLC-2100 (cilostazol di-nitrate) were prepared by Solvias (Basel, Switzerland). For detailed protocols of the synthesis, see the online supplemen-tary data (for all online suppl. material, see www.karger.com/doi/10.1159/000339861). Valsartan was obtained from Sigma-Aldrich or Fluka. All compounds were dissolved in dimethyl-sulfoxide using a sonifier (Bandelin Sonoplus) where necessary. Sodium nitroprusside (SNP), ISDN (50% with 50% lactose) and isosorbide-5-mononitrate (ISMN) were of analytical grade and obtained from Sigma-Aldrich or Fluka. All other chemicals were purchased from Sigma-Aldrich or Fluka at the highest pu-rity available.
Source and Care of AnimalsFor all experiments, male Wistar rats (6 weeks old, 250 g)
were obtained from Charles River Laboratories (Sulzfeld, Germany) and cared for in the University of Mainz animal care facility. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. All animal experimental procedures were approved by the local ethics committee (Landesuntersuchungsamt RLP, Koblenz, Germany).
Pharmacology 2012;90:193–204 195Valsartan and Cilostazol Nitrate Derivatives
Isometric Tension Studies in Rat AortaIsometric tension studies were performed in vitro to as-
sess the vasodilative properties of the test compounds, and isolated rat aortic rings were prepared as described in Munzel et al. [19] and Daiber et al. [20]. Briefly, aortic ring segments were preconstricted with phenylephrine (PE; 0.5–1.0 μmol/l) to approximately 50% of the maximal tone in response to KCl. After reaching a stable plateau, linkers, hybrid drugs as well as classical nitrovasodilators were added at half-logarithmic con-centrations until the resting tension (prior PE) was reached. After several washout steps, the aortic ring segments were challenged a second time with the drugs for assessment of in vitro tachyphylaxis. For a detailed protocol, see the online sup-plementary methods.
Detection of Oxidative Stress in Rat Heart MitochondriaThe potential of the test compounds to induce oxidative
stress was examined in a series of experiments following in vitro exposure of isolated cardiac mitochondria [20]. Briefly, the formation of reactive oxygen and nitrogen species (RONS) was measured by chemiluminescence enhanced by L-012 [8-amino-5-chloro-7-phenylpyrido(3,4-D)pyridazine-1,4-(2H,3H)dione sodium salt; 100 μmol/l]. The reaction was initi-ated by adding the complex II substrate succinate (5 mmol/l). Chemiluminescence was measured over 5 min using a Lumat LB9507 from Berthold Technologies, and expressed as counts per 30 s at 5 min. For a detailed protocol, see the online supple-mentary methods.
Measurement of Platelet AggregationAll preparation and treatments of human samples were in ac-
cordance with the Declaration of Helsinki and were granted by the local institutional Ethics Committee (Landesärztekammer Rheinland-Pfalz, Germany). Platelet-rich plasma was prepared from human blood from healthy volunteers by centrifuga-tion for 20 min at 250 g. For measurement of aggregation, the platelet-rich plasma stock (4–6 × 108 platelets/ml) was diluted to a final concentration of 2 × 108 platelets/ml in Tyrode’s buf-fer (ready to use from Sigma plus bicarbonate), transferred to a glass tube that was placed in an aggregometer Chrono-Log (Probe & Go, Endingen, Germany) and kept at 37°C. Aggregation was triggered under continuous stirring by ADP (2 μmol/l). The test compounds or the solvent dimethylsulf-oxide (1 v/v%) were preincubated for 5 min together with the platelets in the glass tubes in order to allow bioactivation of the nitrate drugs prior to addition of the ADP.
Statistical AnalysisResults are expressed as means ± SEM. Two-way ANOVA
(with Bonferroni’s or Dunn’s correction for comparison of multiple means) was used for comparisons of vasodilator po-tency and efficacy. One-way ANOVA (with the same post-hoc tests) was used for comparisons of RONS detection by chemi-luminescence, PE-induced vasoconstriction and platelet ag-gregation. p values <0.05 were considered significant.
Results
New Organic Nitrate Hybrid Molecules of Valsartan and CilostazolCovalent binding of the linker CLC-1270 to valsartan
resulted in the formation of the valsartan mononitrate CLC-1265 (fig. 1). Additional binding of the linker CLC-1275 to CLC-1265 yielded the valsartan dinitrate CLC-1280. Reaction of the linker CLC-1275 and cilostazol led to the formation of the cilostazol mononitrate CLC-2000 and the cilostazol dinitrate CLC-2100 (fig. 1). In addi-tion, a cilostazol trinitrate was formed and two other valsartan nitrate derivatives, CLC-2200 and CLC-2050, were synthesized (for structures, see online suppl. fig. 1). Linker CLC-1275 was designed for relatively rapid onset of NO-mediated activity. It contains an internal carba-mate structure that can readily undergo hydrolysis, re-leasing the nitrate-containing moiety as the new organic nitrate, aminoethyl nitrate (AEN), discussed below. This linker was used to form two nitrate derivatives of the phosphodiesterase III inhibitor cilostazol.
Vasodilatory Properties of Organic Nitrate LinkersAs a first step, the vasodilatory activities of the indi-
vidual linkers were compared to two well-established organic nitrates, ISDN and ISMN. As shown in figure 2, ISMN showed the lowest vasodilatory efficacy with a potency (pD2) of 3.9, while the dinitrate, ISDN, had a potency of 5.2 (table 1). The pD2 value is expressed as the negative log of the EC50. These results are consistent with previously published reports and reflect the known tendency for increased vasodilatory efficacy with an increasing number of nitrate groups (ISMN < ISDN). All three nitrate linkers induced vasodilation with pD2 values consistent with their acting as NO-donating or-ganic mononitrates [21]. Linkers CLC-1270 and CLC-1273 had similar pD2 values of 4.6 and 4.9, respectively (fig. 2; table 1). While both these linkers have a nitrate attached to carbon 4 of a butyl chain, the substitution groups on carbon 1 are distinct. CLC-1273 contains a simple hydroxyl group, while CLC-1270 contains a me-thylamine (see chemical structures, fig. 1). Linker CLC-1273 has not been used for the preparation of any of the valsartan and cilostazol compounds in the present study but was used by others to synthesize naproxcinod [22]. Linker CLC-1273 is included in these experiments only as a comparator. The most potent of the linkers is CLC-1275, which had a pD2 of 6.2 (fig. 2; table 1). This linker includes a carbamate structure, which is relatively more reactive than the other linkers and may be important for
Pharmacology 2012;90:193–204196 Knorr et al.
facilitating the biotransformation of nitrate into NO. In addition, this linker is based on an AEN, which was pre-viously demonstrated to display a superior vasodilatory potency over all known mononitrates [21, 23] and rep-resents the organic nitrate moiety of the nitrate hybrid potassium channel opener nicorandil.
Vasodilatory Properties of Nitrate Derivatives of ValsartanThe concentration-relaxation curves for the valsartan-
based compounds are shown in figure 3. The curves from figure 2 for linkers CLC-1270 and CLC-1275, as well as for ISDN, are reproduced in this figure for comparison
Valsartan CLC-1265 CLC-1280
N
O
HOOC
NN
HN
N
N
O
NNHN
N
N
CH3
O2NO
N
O
NNN
N
N
CH3
O2NO
O
NH
O
O2NO
Linker CLC-1270
ONO2NH
H3C
Linker CLC-1273
ONO2HO
Linker CLC-1275
NH
ONO2O
O
CI
ISDN
O
O ONO2
O2NO
H
H
ISMN
O
O ONO2
HO
H
HSodium nitroprusside
Fe
NO
CNCN
NC
NC
CNNa2
Cilostazol
N
O
N N
NN
O
CLC-2000
N
O
NN
N
N
O
O
O NH
ONO2
CLC-2100
N
O
NN
N
N
O
O
O N O NH
ONO2ONO2
O
Fig. 1. Structures of test compounds.
Pharmacology 2012;90:193–204 197Valsartan and Cilostazol Nitrate Derivatives
(see broken lines). As anticipated, valsartan showed little effect on vasodilation, consistent with its known mecha-nism of action. At the maximum concentration tested (this was the limit of solubility), only about 20% relax-ation was achieved. When linker CLC-1270 is attached to the valsartan structure to produce CLC-1265, the vasodi-latory efficacy is increased to approximately halfway be-tween native valsartan and the free CLC-1270 linker (also this compound was used at the limit of solubility). This suggests that the addition of the linker is indeed confer-ring some NO-mediated effects to CLC-1265, on top of the valsartan effects. While the precise chemical transfor-mation of CLC-1265 has not yet been confirmed under these conditions, it is likely that the tertiary amine struc-ture attaching the linker is cleaved only slowly from the full CLC-1265 compound in vitro (see chemical structure, fig. 1). This is consistent with the rather low vasodilatory potency relative to the free CLC-1270 linker, and, indeed, this was the intention behind selecting linker CLC-1270 for this compound. Alternatively, it is possible that NO is
bioactivated directly, albeit slowly, from the intact CLC-1265 molecule, without the linker being cleaved from the full compound. Addition of a second nitrate, in the form of linker CLC-1275, further enhances the vasodilatory ef-ficacy. Linker CLC-1275 is added to the tetrazole ring of compound CLC-1265 to produce the dinitrate CLC-1280 (see chemical structure, fig. 1). The pD2 for CLC-1280 was quite similar to that of ISDN, at 5.4, but it was more potent at low concentrations with noticeable vasodila-tion already occurring at concentrations as low as log –7. Unlike linker CLC-1270, linker CLC-1275 was intended to be rapidly cleaved from the rest of the molecule. It contains a carbamate group that should be readily hydro-lyzed to yield AEN. AEN has been shown to be a potent vasodilator, without inducing oxidative stress or nitrate tolerance [23]. As with CLC-1265, the precise chemical transformation of CLC-1280 in vitro has not yet been confirmed; however, the markedly enhanced vasodilatory potency relative to CLC-1265 suggests that the cleavage of linker CLC-1275 is indeed relatively rapid.
Vasodilatory Properties of Nitrate Derivatives of CilostazolIn addition to cilostazol, sodium nitroprusside (SNP)
and two organic nitrates, ISMN and ISDN, were included
Fig. 2. Vasodilator potency of nitrate linkers CLC-1270, CLC-1273, CLC-1275 and classical organic nitrates. Vascular function was de-termined by isometric tension recordings in PE-preconstricted rat aortic ring segments. a First concentration-relaxation curves for the nitrovasodilators. b Second concentration-relaxation curves for the nitrovasodilators (repeated challenge upon washout). The data are means ± SEM of 8–20 aortic ring segments. a p < 0.05 ver-sus CLC-1270 and CLC-1273; b p < 0.05 versus ISDN.
Table 1. Vasodilatory potency pD2 of test compounds
Potency is –log EC50 (where EC50 is the concentration that causes half-maximal relaxation). Constriction was induced by PE, then the 1st concentration-relaxation curve (CRC) with the vaso-dilator was performed, followed by 3 washout steps, then the 2nd constriction with PE was induced followed by a 2nd concentra-tion-relaxation curve.
a Some compounds did not induce 100% relaxation and could not be used at higher concentrations due to limited solubility.
Linker CLC-1270, n = 8Linker CLC-1273, n = 8Linker CLC-1275, n = 12ISDN, n = 8ISMN, n = 20
a–8 –6 –4 –2
0
50
100
aa
log(vasodilator) (mol/l)
Rela
xatio
n (%
)
a,b
Linker CLC-1270, n = 8Linker CLC-1273, n = 8Linker CLC-1275, n = 12ISDN, n = 8ISMN, n = 12
b–8 –6 –4 –2
log(vasodilator) (mol/l)
0
50
100
a,ba
a
Rela
xatio
n (%
)
Pharmacology 2012;90:193–204198 Knorr et al.
in the in vitro experiments as comparators. Figure 4a shows the concentration-relaxation curve for each com-pound. Clearly, the most potent compound was SNP, with a pD2 of 8.2, which is consistent with its known ef-fects on vasodilation. Both organic nitrates, ISDN and ISMN, also demonstrated efficacy in inducing vasodi-lation, but with markedly less potency (pD2 of 5.2 and 3.9, respectively). The pD2 value is the negative log of the concentration that induces 50% vasodilation. Part of cilostazol’s clinical efficacy is due to its vasodilatory ac-tivity, which in these experiments was similar to ISDN (pD2 of 5). The addition of a single NO-donating moi-ety to cilostazol to produce CLC-2000 shifted the con-centration-relaxation curve to the left by about one half
log unit, with a corresponding pD2 of about 5.6 (table 1). This supports the hypothesis that vasodilatory potency is increased by the addition of linker CLC-1275 to the base cilostazol moiety.
Interestingly, the addition of a second NO-donating moiety did not confer greater potency, but rather shift-ed the concentration-relaxation curve back to the right. This compound, CLC-2100, with two linkers attached in series had a similar pD2 as the base cilostazol compound, about 4.9. The second linker is attached to the carbamate amine of the first linker, resulting in two closely spaced carbamate structures. The precise chemical reactions leading to the release of the two linkers has not been determined conclusively, and several possible scenarios exist. The overall reactivity of the double linker may be less than that of the single linker, slowing the release of the linkers from cilostazol. Alternatively, unlike the single linker, which should release the potent vasodilator AEN, the double linker could release a different organic nitrate, with reduced vasodilatory potential. Lehmann and co-workers have shown that length of the carbon chain and the properties of attached functional groups have strong influences on the pD2 of organic mononitrates [21, 24]. For a description of the vasodilatory properties of ci-lostazol trinitrate and the modified cilostazol nitrates CLC-2050 and CLC-2200, see the online supplementary extended results and figures 1 and 2.
Effect of Repeated Challenges of the New Organic Nitrate Derivatives on Their Vasodilatory Potency (Induction of in vitro Tachyphylaxis)One of the limitations of organic nitrates in the
clinical setting is that they induce tolerance not only to themselves, but also to other nitrate compounds (cross-tolerance). In the case of nitroglycerine (i.e. GTN), the onset of tolerance is very rapid and is evident even in the in vitro setting. GTN-mediated vasodilation is accompa-nied by high levels of oxidative stress, which subsequent-ly inactivates mitochondrial aldehyde dehydrogenase, the enzyme responsible for the high-potency bioactiva-tion of NO from GTN. As a preliminary screen of nitrate tolerance in vitro, the concentration-relaxation curve for each compound was repeated after washing the test com-pound out of the chamber and reconstricting with PE. The results for the three free linkers, ISDN and ISMN are shown in figure 2b. For the three linkers and ISDN, the concentration-relaxation curves upon rechallenge were not significantly different from the initial curves (com-pare with fig. 2a, statistical analysis not shown). However, for ISMN the second concentration-relaxation curve was
Fig. 3. Comparison of vasodilator potency of nitrate linkers CLC-1270, CLC-1275 and ISDN versus valsartan and valsartan-based nitrate compounds. Vascular function was determined by isometric tension recordings in PE-preconstricted rat aortic ring segments. a First concentration-relaxation curves for the ni-trovasodilators (broken lines, curves for linkers and ISDN were reproduced from fig. 2a). b Comparison of first and second con-centration-relaxation curves for the valsartan-based compounds (broken lines, curves for valsartan-based compounds were re-produced from a). The data are means ± SEM of 8–14 aortic ring segments. a p < 0.05 versus CLC-1270 and CLC-1273; b p < 0.05 versus ISDN and CLC-1280; c p < 0.05 versus CLC-1265; d p < 0.05 versus CLC-1280; e p < 0.05 versus first concentration-relaxation curve.
–8 –6 –4
0
50
100
Linker CLC-1270, n = 8Linker CLC-1275, n = 12ISDN, n = 8Valsartan, n = 12CLC-1265, n = 12CLC-1280, n = 12
a,b,c
a,b,c
a,b
d
log(vasodilator) (mol/l)
–8 –6 –4
log(vasodilator) (mol/l)
Rela
xatio
n (%
)
0
50
100
CLC-1265 1st, n = 12CLC-1280 1st, n = 12
Valsartan 1st, n = 12
Valsartan 2nd, n = 14CLC-1265 2nd, n = 12CLC-1280 2nd, n = 12
e
Rela
xatio
n (%
)
a
b
Pharmacology 2012;90:193–204 199Valsartan and Cilostazol Nitrate Derivatives
significantly shifted as compared with the first (p < 0.05 for two data points, –4 and –3.5).
The results for the valsartan-based compounds are shown in figure 3b, with the results of the first assess-ment reproduced from figure 3a for comparison (see broken lines). As with the linkers, the rechallenge curves for valsartan and CLC-1265 were almost superimposable over the initial curves. Only CLC-1280 showed signs of reduced vasodilatory efficacy in the second concentra-tion-relaxation curve; the curve was shifted to the right by about 1 log unit. While the magnitude of shift is simi-lar to that seen with GTN following in vitro exposure, several of the hallmark features of GTN-induced toler-ance [20] have not been observed with CLC-1280. This suggests the underlying cause of the shift is not nitrate tolerance (see results below, discussion and table 2 for further explanation).
The concentration-relaxation curves upon rechallenge of SNP and the two organic nitrates are shown in figure 4b. The concentration-relaxation curves from the initial exposure are reproduced in this figure for comparison
(see broken lines). SNP, which is known to induce toler-ance rapidly when administered as continuous infusion in patients, was clearly shifted to the right in the second con-centration-relaxation curve, with the pD2 falling from 8.2 to 7.3 (fig. 4b, table 1). On the other hand, the less potent organic nitrates ISDN and ISMN showed little change in their concentration-relaxation curve upon rechallenge (as reported previously by Koenig et al. [25]), suggesting that the induction of clinical tolerance for these compounds may require more time to manifest, for example through some translational and/or transcriptional processes. As with SNP, all three of the cilostazol-based compounds showed a clear right shift of the concentration-relaxation curve upon rechallenge (fig. 4c). This was not expected for cilostazol, as it is not known to induce tolerance in the clinical setting. Interestingly, the concentration-relax-ation curve for CLC-2000 upon rechallenge displayed a paradoxical vasoconstriction at concentrations less than 10–6. The basis of this effect is unclear, and may reflect an experimental artifact. Additional studies may be needed to clarify this observation.
Table 2. Half-maximal relaxation (EC50), ‘tachyphylaxis score’ and effects of test compounds on PE-induced vasoconstriction
EC50 is the concentration that causes half-maximal relaxation. Constriction was induced by PE, then the 1st concentration-relaxation curve (CRC) with the vasodilator was performed, followed by 3 washout steps, then the 2nd constriction with PE was induced followed by a 2nd concentration-relaxation curve. The ratio of EC50 2nd CRC and EC50 1st CRC provides the n-fold loss in vasodilatory potency upon repeated challenge with the vasodilator (classically interpreted as ‘tachyphylaxis’). The 1st PE-induced constriction (PIC) was prior to the 1st vasodilator concentration-relaxation curve, the 2nd PE-induced constriction was made after the 1st vasodilator concentration-relaxation curve (prior to the 2nd).
a Some compounds did not induce 100% relaxation and could not be used at higher concentrations due to limited solubility.
Pharmacology 2012;90:193–204200 Knorr et al.
Effect of the New Organic Nitrate Derivatives on PE-Induced VasoconstrictionFigure 5 shows the average tension induced by PE
before determining the first and second concentra-tion-relaxation curves for each compound. Except for CLC-1280, the PE-induced tension prior to the second concentration-relaxation curve was consistently about the same or slightly less than the tension induced prior to the first curve (fig. 5a; table 2). In the case of CLC-1280, the PE-induced tension was significantly reduced prior to the second concentration-relaxation curve determina-tion. This is in marked contrast to the increased sensitiv-ity to PE seen with GTN-induced nitrate tolerance [26]. For the two classical organic nitrates ISDN and ISMN, as well as the three linkers CLC-1270, CLC-1273 and CLC-1275 as well as the valsartan mononitrate CLC-1265, the PE-induced tension prior to the second concentration-relaxation curve was only slightly less than the tension
induced prior to the first curve. In contrast, for all three cilostazol-based compounds, as well as for SNP, the PE-induced tension was significantly reduced prior to the second concentration-relaxation curve determination (fig. 5b; table 2). This is again in marked contrast to the increased sensitivity to PE seen with GTN-induced ni-trate tolerance.
Induction of Mitochondrial Oxidative Stress in Response to the Linkers and Organic Nitrate DerivativesVascular oxidative stress is a well-known side effect
of nitrate tolerance, both contributing to and resulting from induction of tolerance. Therefore, induction of RONS (e.g. superoxide anion radicals, peroxynitrite an-ions or nitrogen dioxide radicals) in response to in vitro challenges with the test compounds was assessed in iso-lated mitochondria by L-012-enhanced chemilumines-cence. The data are based on experiments with cardiac
Fig. 4. Comparison of vasodilator potency of classical nitrovasodilators SNP, ISMN and ISDN versus cilostazol and cilostazol-based nitrate compounds. Vascular function was determined by isometric tension recordings in PE-preconstricted rat aortic ring segments. a First concentration-relaxation curves for the nitrovasodilators (broken line, curve for ISMN was reproduced from fig. 2a). b Comparison of first and second concentration-relaxation curves for SNP, ISDN and ISMN (broken lines, curves for nitrovasodilators were reproduced from a). c Comparison of first and second concentration-relaxation curves for the cilostazol-based compounds (broken lines, curves for cilostazol-based compounds were reproduced from a). The data are means ± SEM of 12–28 (a), 8–24 (b) and 12–28 (c) aortic ring segments. a p < 0.05 versus ISDN; b p < 0.05 versus cilostazol; c p < 0.05 versus CLC-2100; d p < 0.05 versus first concentration-relaxation curve.
a–10 –8 –6 –4 –2
0
50
100
SNP, n = 16ISMN, n = 20CLC-2000, n = 28ISDN, n = 24Cilostazol, n = 12CLC-2100, n = 14
a,b,c
a
log(vasodilator) (mol/l)–10 –8 –6 –4 –2
log(vasodilator) (mol/l)
Rela
xatio
n (%
)
c
0
50
100
Rela
xatio
n (%
)
b
0
50
100
Rela
xatio
n (%
)
SNP 1st, n = 16ISMN 1st, n = 20
ISMN 2nd, n = 12
ISDN 1st, n = 24SNP 2nd, n = 8
ISDN 2nd, n = 24
d
d
–8 –7 –6 –5 –4
CLC-2000 1st, n = 28Cilostazol 1st, n = 12
CLC-2100 1st, n = 14Cilostazol 2nd, n = 12CLC-2000 2nd, n = 28CLC-2100 2nd, n = 14
d
d
d
log(vasodilator) (mol/l)
Pharmacology 2012;90:193–204 201Valsartan and Cilostazol Nitrate Derivatives
mitochondria from at least 6 rats on 3 different days (fig. 6). There was a slight trend toward a concentration-de-pendent increase in the induction of RONS for valsar-tan and the linkers CLC-1273 and CLC-1275, whereas a substantial increase in RONS formation was observed for linker CLC-1270, which induced greater than 15-fold higher RONS at 1,000 μmol/l as compared to basal levels (fig. 6a). The two compounds CLC-1265 and CLC-1280 induced a small increase in RONS production, but this was not concentration dependent, and the highest levels
of induction (approx. twice the control level) were seen at the lowest concentration tested, 10 μmol/l. It is inter-esting that while both of the compounds contain linker CLC-1270, neither of them induced RONS production similar to that of the free linker. The relatively slow re-lease of linker CLC-1270 from the valsartan moiety may provide some protection from RONS formation induced by high levels of free linker.
It is also noteworthy that while linker CLC-1270 in-duced high levels of RONS formation, it did not induce
Fig. 5. Effects of first nitrovasodilator challenge on subsequent second PE-induced constriction. a Effect of first concentration-relaxation curve of ISMN, ISDN, linkers, valsartan and valsartan-based compounds on PE-mediated constriction upon washout. b Effect of first concentration-relaxation curve of SNP, ISMN, ISDN, cilo-stazol and cilostazol-based compounds on PE-mediated constriction upon washout. The data are means ± SEM of 8–28 aortic ring segments. * p < 0.05 versus first PE-induced constriction.
Fig. 6. Effects of nitrovasodilators on oxidative stress in isolated cardiac mitochondria. Mitochondrial RONS formation was assessed by L-012-enhanced chemiluminescence (ECL). a Comparison of linkers, valsartan and valsartan-based compounds. b Comparison of classical nitrates, cilostazol and cilostazol-based compounds. The data are means ± SEM of 6–12 independent measurements per group. * p < 0.05 versus solvent control (basal).
Mito
chon
dria
l RO
NS
form
atio
nby
L-0
12 E
CL (c
ount
s/30
s)
Basa
l
10 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
0
0
20,000
40,000
60,000
80,000
CLC-1270
CLC-1273
CLC-1275
Val-sartan
CLC-1265
CLC-1280
(μm
ol)
*
*
Mito
chon
dria
l RO
NS
form
atio
nby
L-0
12 E
CL (c
ount
s/30
s)
0
5,000
10,000
15,000
20,000
*
*
*
* * **
Basa
l
10 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
010 100
1,00
0
ISDN ISMN GTN Cilostazol CLC-2000
CLC-2100
(μm
ol)
a b
SNPISMN
ISDN
Cilosta
zol
CLC-2000
CLC-2100
0
1
2
3
4
**
**
PE-in
duce
d te
nsio
n (g
)
CLC-1270
CLC-1273
CLC-1275
Valsarta
n
CLC-1265
CLC-1280
0
1
2
3
4 1st–before drug2nd–after drug
*PE
-indu
ced
tens
ion
(g)
a b
Pharmacology 2012;90:193–204202 Knorr et al.
tolerance to itself (fig. 2). Conversely, the reduced dila-tory potency seen with compound CLC-1280 during rechallenge (fig. 3) was not accompanied by appreciable levels of RONS formation. Together, these observations suggest that the mechanisms underlying the induction of RONS formation and the reduced vasodilatory potential of CLC-1280 following rechallenge are not related to each other and are not indicative of typical nitrate tolerance.
In separate experiments, ISDN induced a slight, but dose-dependent increase in RONS formation, with 1,000 μmol/l inducing about a 100% increase relative to basal levels and GTN even led to a 3-fold increase in RONS at the highest concentration of the drug (fig. 6b). In contrast, ISMN caused no increase in RONS formation. No such induction was seen with cilostazol, CLC-2000 or CLC-2100, and if anything there was a trend toward decreasing levels of RONS induction by these compounds (fig. 6b).
Inhibition of Platelet Aggregation by Linkers and Organic Nitrate DerivativesADP-induced aggregation in platelet-rich plasma
was inhibited by the direct NO donor diethylamine NONOate in a concentration-dependent fashion reach-ing maximal inhibition between 1 and 10 μmol/l (fig. 7a). Valsartan and the linker CLC-1270 showed no di-minished aggregation, even not at the highest employed concentration of 100 μmol/l. The linker CLC-1275 and CLC-1280 displayed significant antiaggregatory effects at 100 μmol/l. The classical mononitrate ISMN displayed only moderate (but not significant) inhibitory effects on
platelet aggregation, while GTN significantly diminished ADP-induced aggregation at 100 μmol/l with only minor effects at 10 μmol/l (fig. 7b). In contrast, all cilostazol compounds were efficient antiaggregatory drugs at 100 μmol/l. Cilostazol itself and CLC-2100 even significantly inhibited platelet aggregation at 10 μmol/l, while CLC-2000 displayed only moderate (but not significant) ef-fects at this concentration.
Discussion
Compounds CLC-1265 and CLC-1280 are nitrate derivatives of the angiotensin receptor blocker valsar-tan, potentially combining the vasodilatory properties of organic nitrates with the antihypertensive properties of angiotensin receptor blockers. Compounds CLC-2000 and CLC-2100 are nitrate derivatives of cilostazol, a phosphodiesterase III inhibitor indicated for symptom-atic relief of intermittent claudication. Cilostazol’s precise mechanism of action is not fully understood. While ci-lostazol is already a vasodilator, compounds CLC-2000 and CLC-2100 were designed to provide additional va-sodilation through their NO donor linkers with the goal to increase clinical efficacy. Except CLC-1265, these compounds contain the linker CLC-1275, that is rapidly cleaved releasing AEN, which was shown to exert potent vasodilatory effects [21], without relying on the activity of mitochondrial aldehyde dehydrogenase for conver-sion to NO, and without inducing in vitro tachyphylaxis
Fig. 7. Effects of nitrovasodilators on ADP-induced aggregation in platelet-rich plasma. Platelet aggregation was assessed by light transmission spectroscopy. a Comparison of the direct NO donor diethylamine NONOate (DEA/NO), linkers, valsartan and the valsartan dinitrate CLC-1280. b Comparison of classical nitrates, cilo-stazol and cilostazol-based compounds. (A typical set of aggregation curves is shown in online suppl. fig. 3.) The data are means ± SEM of 4–6 independent measurements per group. * p < 0.05 versus solvent control (dimethylsulfoxide).
0 0.1 1 10 100
0
50
100Valsartan
CLC-1270
CLC-1275
CLC-1280
DEA/NO
Cilostazol
CLC-2000
CLC-2100
ISMN
GTN*
****
**
Concentration (μmol/l)
AD
P-in
duce
d ag
greg
atio
n (%
)
0 0.1 1 10 100
0
50
100
*
***
**
Concentration (μmol/l)
*
a b
AD
P-in
duce
d ag
greg
atio
n (%
)
Pharmacology 2012;90:193–204 203Valsartan and Cilostazol Nitrate Derivatives
against itself and cross-tolerance to other organic nitrates [23].
The in vitro vasodilation experiments show a clear, but somewhat modest, increase in the vasodilatory po-tency of CLC-1265 over native valsartan, supporting the hypothesis that NO is bioactivated from the attached linker CLC-1270, either directly or after the linker has been cleaved from the parent compound. Compound CLC-1280, on the other hand, shows a clear and signifi-cant increase in vasodilatory potency in vitro, support-ing the proposed chemical conversion to AEN from the CLC-1275 linker. Additional studies should expand our understanding of the biochemical transformation of CLC-1265 and CLC-1280. While both compounds ex-hibit a clear NO-mediated vasodilation, neither induces clear evidence of acute oxidative stress or typical nitrate tolerance in vitro, although with both compounds a clear shift to lower potencies was observed upon repeated chal-lenges, a common way to test for in vitro tachyphylaxis. The most striking evidence that this phenomenon was different from authentic tachyphylaxis came from the observation that the right shift of the second concentra-tion-relaxation curve was not shared by the nitrate linker molecules, which were used for the synthesis of the hy-brid molecules. Therefore, the most reliable explanation for this ‘pseudotachyphylaxis’ is that the high lipophilic-ity of some hybrid molecules prevents complete removal during the washout phase and confers a long-lasting an-tivasoconstriction that was not overcome by the second PE bolus (table 2).
The RONS measurements in isolated cardiac mito-chondria revealed that none of the new nitrate hybrid drugs induced mitochondrial oxidative stress in response to acute challenges, as observed and previously reported for GTN [20]. ISDN, in contrast to ISMN, induced mod-erate mitochondrial RONS as previously reported [20]. Among the linkers, only CLC-1270 alone, but not when bound to valsartan, led to a substantial increase in mi-tochondrial oxidative stress, for which we have to date no biochemical explanation. There may be some direct interaction between this linker and the mitochondrial respiratory chain leading to transfer of electrons to mo-lecular oxygen.
As a second readout for the activation of the NO/cGMP pathway, we used platelet aggregation. The ag-gregation data obtained in ADP-stimulated platelet-rich plasma was in accordance with those obtained by isometric tension recordings. The direct NO donor di-ethylamine NONOate was the most potent antiaggrega-tory drug in this setting, followed by the cilostazol-based
compounds, which may be best explained that platelet aggregation is highly sensitive to cGMP levels [27]. As expected from the isometric tension recording data, the linker CLC-1275 and its hybrid drug CLC-1280, both containing the potent vasodilator AEN [23], showed appreciable antiaggregatory effects at higher concentra-tions. In contrast, the linker CLC-1270 showed no in-hibitory effect on platelet aggregation in accordance with the rather weak vasodilatory potency in isolated aortic ring segments. The rather disappointing antiaggrega-tory effect of GTN may be based on the lack of adequate bioactivating enzymes in platelet-rich plasma (there is only marginal mitochondrial aldehyde dehydrogenase activity in platelets [Daiber, unpubl. observation]), and plasma xanthine oxidoreductase activity may be not high enough for fast release of NO or a related species. Finally, valsartan and ISMN showed no inhibition of platelet ag-gregation as expected from the rather weak vasodilating properties observed in isometric tension recordings.
Conclusions and Clinical Implications
Overall, the new hybrid compounds presented here may develop as future therapeutic alternatives broaden-ing the indications for valsartan or cilostazol alone. In the case of hypertensive patients being treated receiv-ing valsartan, benefits are to be expected with respect to improved blood pressure control, and in those with symptomatic coronary heart disease a reduction of an-gina pectoris without additional prescription of organic nitrate and without induction of nitrate tolerance, which may blunt these effects. In patients with symptomatic pe-ripheral artery disease not adequately controlled or re-sponding to cilostazol, treatment with CLC-2000 might result in improved clinical symptoms and exercise capac-ity. However, proof of these expected clinical benefits will require respective clinical studies in humans, which are planned to be conducted after completion of the further pharmacology and toxicology development program for these compounds.
Acknowledgements
We thank Anja Conrad, Angelica Karpi and Nicole Glas for expert technical assistance. The present work was supported by continuous funding by Cardiolynx AG, Basel, Switzerland (to A.D. and T.M.), in part by a grant by the Federal Ministry of Education and Research (BMBF 01EO1003; to A.D. and P.W.) and in part by a stipend from the Stiftung Mainzer Herz (to M.K.).
Pharmacology 2012;90:193–204204 Knorr et al.
Disclosure Statement
D.S. and A.S. are employees of Cardiolynx AG, Basel, Switzerland.
A.D. and T.M. received research grants from Cardiolynx AG, Basel, Switzerland.
K.B. was contracted by Cardiolynx AG to write the medical reports for the organic nitrate hybrid drugs.
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