ASPECTS OF THE AMLODIPINE PLEIOTROPY IN BIOCHEMISTRY, PHARMACOLOGY AND CLINICS

ASPECTS OF THE AMLODIPINE PLEIOTROPY IN BIOCHEMISTRY, PHARMACOLOGY AND CLINICS

Rasma Vitolina, Aivars Krauze, Gunars Duburs and Astrida Velena*

Latvian Institute of Organic Synthesis, Laboratory of membrane active compunds and β-diketones, Aizkraukles str. 21, Riga, Latvia, LV-1006

ABSTRACT

Amlodipine is the third generation calcium antagonist, 1,4-dihydropyridine derivative with the prolonged duration of the antihypertensive action, especially blocking L-type Ca²⁺ ion channels. It promotes beneficial therapeutic effect by coronary and other blood vessel diseases and thus delays development of the atherosclerosis. It has several known trade names, the most mentioned is Norvasc. Amlodipine is well tolerated in the clinics, it could be used in combinations with other drugs – diuretics, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, statins. Amlodipine at nanomolar concentrations binds to the voltage-dependent L-type calcium channels. It possesses optimal lipophylicity. Amlodipine also influences the NO-dependent metabolic processes, stimulates NO synthesis and prolongs NO action duration. Results of the studies of the amlodipine pharmacological and clinical properties are summarized in several reviews. The present review contains opinion from the scientific works of the last decades about the multisided or pleiotropic amlodipine mechanisms of action, it contains information about sometimes controversial clinicalstudies of the amlodipine vaso- and cardioprotective activity.

Anti-atherogenic action

INTRODUCTION:Amlodipine is the third generation calcium antagonist, 1, 4-dihydropyridine (1, 4-DHP) compound with prolonged action. It has expressed properties of Ca²⁺ channel (especially L-type and T-type) blockers (CCB). Besides, it exerts multisided (pleiotropic, namely, when a drug has actions other than those for which the compound was specifically indicated or developed) effects on other metabolic processes. It is the basis for the beneficial therapeutic action at heart and vasculature diseases and delays the development of atherosclerosis ^{1 - 11}. This drug is well tolerated by patients. It has no significant side effects. It could be used in the monotherapy, as well in combination with other drugs-diuretics, angiotensine-converting enzyme (ACE) inhibitors, antagonists of the angiotensine II receptors, statins ^{12, 13, 14, 15}.

Amlodipine at nanomolar concentrations binds to the voltage-dependent L-type calcium channels. It has optimal lipophylicity, and it has a free aminogroup, which garantees the good bioavailability ⁹. Amlodipine also influences the NO-dependent metabolic processes too, it stimulates NO synthesis and prolongs NO action duration ¹⁶.

Results of the studies of amlodipine pharmacological and clinical properties are summarized in several reviews ^{9, 12, 17 - 20}.

This review summarizes outlook about experimental materials from the last decades concerning amlodipine pleiotropy in the action mechanism, as well sometimes controversial clinical observations about its cardio-protective and vazoprotective action.

Amlodipine overall characteristics:Amlodipine(Amlodipinum, Drugbank ID: DB00381, http://www. drugbank. ca/drugs/DB00381 ²¹, C₂₃H₂₇N; http://en.wikipedia.org/wiki/Amlodipine ²²), is the third generation dihydropyridine type chiral compound, calcium antagonist with expressed lipophilicity (lgP ~1.90 (exp.), 2.22 (theor.), according other data: 3.00 (exp.), 3.30 (theor.)) currently available in the market and used in the medicinal praxis as a racemate [1:1 mixture of (R)-(+)- and (S)-(–)-amlodipine], its chemical (IUPAC) name is: (RS)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chloro-phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarbo-xylate).

Ca antagonist activity differs for both its enantiomers: S(-)-enantiomer is ~1000 times more active as R(+)-enantiomer. As R(+)-enantiomer is responsible for NO release, the summary amlodipine therapeutic efficacy partially is formed also by R(+)-enantiomer. S(-)-enantiomer completely lacks NO releasing activity.

Amlodipine and Norvasc (amlodipine besylate) structural formulae:

AMLODIPINE

NORVASC (AMLODIPINE BESYLATE)

Amlodipine is a generic (Pfizer patent expired at 2007) drug, and is known with several authorized trade names (about 446 brands, including 24 of that for S-Amlodipine): Agen (Zentiva - Czech Republic), Aken (Kendrick Farmaceutica - Mexico), Amcard (Apex Pharma Ltd. - Bangladesh), Amdepin (Cadila Pharmaceuticals - India), Amdipin (Laboratorios Lafrancol - Colombia), Amlodine (Westfield Pharmaceuticals, a division of InnoGen - Philippines), Amlodac, Amlodis, Amlogard, Amlopine (Berlin (Thailand) Pharmaceutical Industry Co Ltd. - Thailand), Amlopres, Amtas, Amvaz, Coroval, Dailyvasc (XenoPharmaceuticals), Istin(United Kingdom, Ireland), Klodip, Lipinox, Lopin (Edruc Ltd. - Bangladesh), Lotrel, Norvasc (Pfizer - North America and in some European countries), Norvasc and Perivasc (Australia) [http://en. wikipedia.org/wiki/Amlodipine].

In the combined therapy amlodipine is combined either with ACE inhibitors -lisinopril (Hipril, containing 5mg from each of the both drugs, Micro Labs - India), benazepril, captopril, perindopril, or beta-blockers - nebivolol hydrochloride a.o., alfa-1 adrenoreceptor antagonist Terazosin, statins-simvastatin, atorvastatin, valsartan, telmisartan, a.o. (see http://drugs-about.com/ing/amlodipine.html).

Drugstores share many drugs, containing amlodipine (or its besylate, or mesylate, or camsilate, or maleate, or succinate salt forms) (in 3 strengths - 2.5 mg, or 5.0 mg, or 10.0 mg) as the only one single (solely) active substance - Norvasc (Pfizer - North America and in some European countries), Agen (Zentiva), Amlocard (Hexal), Lofral (Mepha), Tenox 5mg (KRKA) (it should not to be mixed with another drug of the same name Tenox (containing as the active substance Temazepam)), Amlodipine Medochemie (Medochemie), Amlodigamma 5 mg tablets (Worwag Pharma); Amlopress (Cipla), Amdepin (Cadila) (India); Amlodipin Olainfarm 5mg tablets and Amlodipin Olainfarm 10mg (Amlodipinum) tablets (Latvia).

In accordance with the international guidelines and therapeutic schemes ‘ESC/ESH 2007 Guidelines for the management of arterial hypertension’, amlodipine is one of the drugs of choise for the prevention and cure of elevated arterial blood tension, stenocardia and other cardiovascular diseases as the monotherapeutics or in the combination with other antihypertensives or antianginal drugs [See http://eurheartj.oxfordjournals. org/content/28/12/1462.full.pdf ²³.

Amlodipine is a 1, 4-dihydropyridine type calcium antagonist (calcium ion antagonist or slow-channel blocker) that inhibits the transmembrane influx of calcium ions into the cells of vascular smooth muscle and cardiac muscle through outer membranes of their cells ^{1, 7, 8}, with a greater effect on vascular smooth muscle cells than on cardiac muscle cells.The contractile processes of cardiac muscle and vascular smooth muscle are dependent on the concentration of calcium ions in cytoplasm. Amlodipine has no influence on serum concentration of Ca²⁺ ions.

Within the physiologic pH range, amlodipine is an ionized compound (pK_a=8.6), and its kinetic interaction with the calcium channel receptor is characterized by a gradual rate of association and dissociation with the receptor binding site, resulting in a gradual onset of effect.

Experimental data suggest that amlodipine binds to both dihydropyridine and non-dihydropyridine binding sites.

Amlodipine is a peripheral arterial vasodilator that acts directly on vascular smooth muscle to cause a reduction in peripheral vascular resistance and reduction in blood pressure.

The precise mechanisms by which amlodipine relieves angina have not been fully delineated, but are thought to include the following:

Exertional angina: In patients with exertional angina, amlodipine reduces the total peripheral resistance (after-load) against which the heart works. The unloading of the heart is thought to decrease ischemia and relieve effort angina by reducing myocardial energy oxygen consumption and oxygen requirements, at any given level of exercise.

Vasospastic angina: Amlodipine has been demonstrated to block the constriction in response to the elevated levels of each calcium, or potassium, or epinephrine, or serotonin, and or thromboxane A₂ analog, and restore blood flow in coronary arteries and arterioles in experimental animal models and in human coronary vessels (in vitro) ^{[9, 22, 24]}. This inhibition of coronary spasm is responsible for the effectiveness of amlodipine in vasospastic (Prinzmetal's or variant) angina. Amlodipine and amlodipine besylate are both calcium blockers, however the former works better with both angina and hypertension while the latter works on hypertension alone.

Preclinical pharmacological investigations: Amlodipine causes stepvise dose-dependent hypotensive effect in the animal experiments, without occurrence of the reflector tachycardy. The maximal decrease of blood presuure is observed in the 8-th hour after the peroral amlodipine application, the hypotensive action duration is about 24 hours ⁷. Prolonged (30 weeks) introduction of amlodipine in SHR rats retards the increase of arterial blood pressure and development of the myocardial hypertrophy ⁸.

Antianginal and antiischemic amlodipine action (by decrease of calcium overload and endothelial dysfunction) is shown in the experiments with dogs ²⁵: the decrease of the infarct zone is observed ²⁶, left ventricule function was not influenced ²⁷. In the experiments on the myocard infarct model (in rats treated with isoproterenol) amlodipine almost normalized tissue protection system (revealing antioxidant properties) ¹⁴.

1. The role of amlodipine and kinins in the NO production and releaseand its implications for the treatment of CVD: In vivo,amlodipine induced coronary vasodilatation, influencing both adenosine- and NO-dependent mechanisms ²⁸. The vasodilatation was reduced through inhibitionof NO synthesis (with use of a substituted arginine molecule),and amlodipine has been shown to increase peripheral and coronaryblood flow ^[1]. Experimental studies of amlodipine in vitro (pigs coronar vessels, pigs endothelial cell culture) revealed increase of the bioavailability of the endothelium to NO, firstly – by increasing NO production, secondly – due to the antioxidant properties of amlodipine, thus prolonging NO action ¹⁶.

Amlodipine (10 micromoles / kg) decreased the resistance of the femoral artery (in vivo perfusion of rabbit femoral artery) and increased NO concentration in the femoral vein (verapamil and nifedipine did not possess such effect, despite these drugs too decreased the resistance of the femoral artery). These amlodipine effects weakened bradykinin B2 receptor antagonist HOE 140, 30 mg / kg ²⁹.

Authors concluded, that vasorelaxating activity of amlodipine partly is NO-dependent, because it was weakened by blockage of NO-synthase (with N(G)-nitro-L-arginine methylester, L-NAME, 1 micromole /kg), however in the presence of verapamil and nifedipine the vasorelaxation was not changed- thus not connected with NO in these experiments (with rabbit femoral artery in vivo). Amlodipine action additionally could be connected with B2 receptor activation and local rise of bradykinin, because, as mentioned, bradykinin B2 receptor antagonist HOE 140 diminished NO increase in the venae caused by amlodipine.

In vitro, amlodipine, unlike nifedipine or diltiazem,caused a dose-dependent release of nitrite, the hydration productof NO. The release of nitriteor inhibition of tissue oxygen consumption was similar in magnitudeto those caused by the 3 ACE inhibitors captopril, enalapril,and ramiprilat. Amlodipine effectively blocks serum ACE activity in vitro in the concentrations near to enalapril ²⁹. Amlodipine not only reduces the resistance of the smooth muscles of the vessels, but relaxes spontaneous contractions of the uterus (in vitro) ³⁰.

The calcium channel blocker amlodipine promotes the unclamping of eNOS from caveolin in endothelial cells ³¹. There are no L-type calcium channels on endothelialcells. As amlodipine activates eNOS, there must be a mechanismindependent of its CCB properties whereby amlodipine releasesNO ¹. Amlodipine enhanced endothelial NO production through a bradykinin-dependent pathway. This ability of amlodipine to stimulate NO synthesis was dose dependent and could not be reproduced by other CCBs (diltiazem, nifedipine), but could be replicated by the ACE inhibitor enalapril ¹.

Amlodipine specifically increased the intracellular concentrationsof endothelial nitric oxide synthase protein, leading to an enhanced rate of NO production. The molecular mechanism for progression of congestive heart failure (CHF) may involve cytokine overexpression.

An adverse event-CHF or death-occurred more commonly in patients with higher IL-6 levels. Amlodipine lowers plasma IL-6 levels in patients with CHF. The beneficial effect of amlodipine in CHF may be due to a reduction of cytokines such as IL-6 ³². Reduction in NO production or the biologic activity of NOcontributes to the development of heart failure, diabetes,atherosclerosis, and hypertension. In states where eNOS isdown-regulated, the activity of the enzyme can be enhanced, anddrugs such as amlodipine, other CCBs, and ACE inhibitors mightconfer benefit by restoring NO production.

NO-dependent regulation of the oxygen consumption in explanted humanhearts were greater when amlodipine was combined with an ACEinhibitor. The combination was found to be more than additive,indicating a true synergism ¹. This can be explained throughan understanding of the role of kinins in the mechanism of actionof both drugs. ACE inhibitors block the breakdown of kinins,and amlodipine stimulates kinin formation by activating or releasingkallikrein ^{1, 12}.

1. Inhibition of cytokine- and free-radical induced endothelial apoptosis: Tumour necrosis factor- (TNF-) is a cytokine that is elevated in atherosclerosis and mediates damage to the vessel wall during the inflammatory process. Cytokines such as TNF- can activate resident macrophages that interfere with the structural integrity of the fibrous cap. Disruption of the heterogeneous fibrous plaque reveals the underlying, highly thrombotic lipid core, leading to acute coronary syndromes in patients with coronary artery disease (CAD). In addition to its effect on plaque destabilisation, TNF- also interferes with endothelial function, leading to the expression of proteins that mediate programmed cell death or apoptosis. Amlodipine has been shown to inhibit TNF--induced endothelial apoptosis in a dose-dependent manner, starting at low nanomolar levels (10.0 nM) ¹.
2. Amlodipine anti-atherogenic action and its mechanisms: In the atherosclerosis animal (rats, rabbits, monkeys, fed with cholesterol-rich food) models amlodipine dose-dependently diminishes the atherosclerotic lesions of vessels ^{9, 11, 17} and cause atheroprotective effect, diminishing the accumulation of the cholesterol into the aorta wall and decreasing lipid peroxidation of the aorta, increasing superoxide dismutase (SOD) activity in the blood and aorta tissue (in the experiments with rabbits, fed with cholesterol, if additionally amlodipine 5 mg/kg/day per os were used), retarding the consumption of the endogenous vitamin E. This effect may be connected with the amlodipine antioxidant properties ³³.

Recent clinical trials also revealed amlodipine antiatherogenic action, mechanism however is not fully understood. In the experiments with the hypertensive SHR stroke-prone rats the observations were made that amlodipine remarkably influenced endothelial NO synthase and proteinkinase cascade, which could be the basis for the vascular protection ³⁴. The ability of amlodipine to retard the development of the atherosclerotic lessions was observed, as well as the regress of atherosclerosis. This is probably connected with the decrease of the oxidative stress and inflammation (in the experiments with mice deficient to apolipoprotein-E – ApoEKO, the amlodipine dose – 3 mg/kg/per day, 10 weeks) ³⁵.

Combining amlodipine (dose - 0.003% (w/w) with food, which is equivalent to 3.5 mg amlodipine / kg living mass/ per day) together with atorvastatin (dose - 0.0035% (w/w) atorvastatine with food, which is equivalent to 4 mg/ kg living mass/ per day) the remarkable increase of the antiatherosclerotic effect was observed ³⁶, as well as decreased mineralization of the atherosclerotic plaques (experiments with APOE*3 Leiden transgenic mice) ³⁷. Atheroprotective amlodipine action partly was observed as the result of the inhibition of glycation end products (AGE) accumulation ³⁸(see also subpart l).

Medication of the experimental animals (kept on atherogenic diet) with amlodipine during 8 weeks decreased total cholesterol, low density lipoprotein cholesterol (LDL), triglyceride (TG) and endothelin level in the plasma and aortic tissue ³⁹.

Amlodipine in 1-10 mkM concentrations (especially in 35 mkM concentration, remarkably exceeding the toxicity level –10 mkM) and other CCB influenced the synthesis of proteoglycans (glucosamineglycans, GAG) in blood vessel walls ⁴⁰: decreased it molmass and charge density. These events influenced the connection of proteoglycans with LDL. In this aspect both stereoisomers of amlodipine are active. In the therapeutic concentrations this effect is less pronounced. Modulation of GAG synthesis is mentioned as one of the initiating factors in the atherosclerosis development.

Amlodipine action is more prolonged as compared with nifedipine a.o. classical CCBs. Amlodipine is in the charged state at physiologic pHlevels (protonated amino group). This positive charge leads to strong electrostatic interactionsof amlodipine with membrane phospholipid head groups. Electrostatic interactions result in the drug’s concentrationsin the membrane at levels >10 000-fold higher than in thesurrounding aqueous environment. There is a sustained reservoir of amlodipine available for bindingover time to calcium channel receptors within the plasma membraneof vascular smooth muscle cells (VSMCs) ^{1, 12}.

It explains the extended duration of the activity of amlodipine comparedwith shorter-acting, less lipophilic and less charged agents ¹. Additionally,the positive charge and strong lipid affinity of amlodipineenable it to inhibit aggregation of modified LDLs, a key stepin foam cell formation mediated by the electronegative propertiesof oxidized lipid. This atheroprotective effect of amlodipinecould not be reproduced by other antihypertensive agents (otherCCBs, ACE inhibitors) that lack such physical chemical characteristics.

In the experiments using the model of mouse obstructive coronary disease (M encephalomyo-carditis virus) amlodipine remarkably decreased histopathological parameters of the myocardial damage and increased life-span, in comparison with the control group. This therapeutic effect could be connected with inhibition of the overproduction of NO ⁴¹. L-type CCBs indicatedthat these drugs were potent and selective vasodilators becauseof their ability to reduce intracellular SMC Ca²⁺ concentration.Increasing basic and clinicaldata suggest that there are non–calcium-related pleiotropicactions of CCBs.

Amlodipine can regulate membrane fluidity andcholesterol deposition, stimulate NO production to recruit itsbiologic actions, act as an antioxidant, and regulate matrixdeposition.

The PREVENT study showed significant clinical benefits of amlodipine therapy, compared with placebo, including a marked reduction in cardiovascular morbidity and significant slowing in the progression of carotid atherosclerosis. These findings support a potentially new therapeutic role for certain third-generation dihydropyridine-type CCBs, such as amlodipine, in the treatment of atherosclerosis. However other authors have reported lack of amlodipine antiatherosclerotic properties ⁴², namely in diabetic animals ⁴³.

1. Amlodipine renoprotective action in the case of the experimental diabetic nephropathy: Amlodipine decreased albuminuria, pathological lessions and accumulation of proteins of the extracellular matrix in the kidneys in experimental diabetic rats with diabetic nephropathy ^{44, 45}.
2. Amlodipine as an platelet aggregation inhibitor: It is shown that amlodipine inhibits platelet aggregation ⁴, which probably is caused by stimulation of NO, because NO is natural inhibitor of the thrombocyte aggregation ⁴⁶. Also other experiments confirm the ability of amlodipine to modulate NO production in the endothelium ^{47, 48, 49}. (See also subpart a)
3. Antioxidant properties of amlodipine: The charged 1, 4-dihydropyridine calcium antagonist amlodipine had the highest affinity (amlodipine > verapamil >> diltiazem) for the membrane bilayer (Kp=10⁴) and produced the largest changes in membrane thermodynamic properties, including a reduction in the thermal phase transition temperature (-11%), enthalpy (-14%) and cooperative unit size (-59%), relative to control phosphatidylcholine liposomes ¹³.

Both in vitro and in vivo studies have shown that amlodipine inhibits oxidative damage of lipids associated with cellularmembranes and lipoprotein particles (LDL) ¹². Under controlled experimentalin vitroconditions (Total Oxyradical Scavenging Capacity (TOSC) assay), amlodipine revealed antioxidant and antiradical activity: inhibited lipid peroxide formation and trapped reactive oxygen species hydroxyl- and peroxyl-radicals (ROS scavenging capacity), atconcentrations as low as 10.0 nM (which is remarkably less as for used as reference (classical) antioxidants – GSH, uric acid and Trolox), independent of the calciumchannel modulation ⁵⁰.

TOSC assay is based on the reaction of artificially generated oxyradicals with α-keto-γ-methiolbutyric acid (KMBA), which is completely oxidized to ethylene. In the presence of an antioxidant molecule, competition with KMBA ensues, resulting in a reduction of free radical generation, hence, lower ethylene production.

Amlodipine showed efficiency as scavenger of peroxyl radicals (TOSC: 5945 ± 544 units/mg) significantly higher (>50%, P <0.001) than that of GSH (2733 ± 636 units/mg), and 70% lower (P < 0.0001) than the value obtained with uric acid (18144 ± 696 units/mg) and trolox (17522 ± 734 units/mg). Of interest, the scavenging capacity of amlodipine towards hydroxyl radicals (1455 ± 154 units/mg) was 320% higher (P <0.00001) than that of GSH (358 ± 112 units/mg), 20% higher than that of uric acid (1198 ± 121 units/mg), and 100% higher than that of trolox (759 ± 143 units/mg).

Amlodipine increased enzyme activity of paraoxonase (PON) and glutathione peroxidase (GSH-Px), however decreased glutathione reductase (GSSG-R) activity and diminished the concentration of the endogenous antioxidant α-tocopherol (vitamin E). Moreover, amlodipine in a concentration of 2 ng/ml decreased the content of malonic dialdehyde (by-product of oxidative stress) and activity of superoxide dismutase in the blood ⁵¹.

Amlodipine antioxidant activity could be related to its as dihydropyridine compound reductant nature or hydrogen donor properties, respectively – the ability of donating protons and electrons to the lipid peroxide molecules, thereby blocking the peroxidation process. Verapamil and amlodipine produced a potent antiischemic effect and reduced the area of myocardial infarction in rats.

The observed changes were accompanied by inhibition of lipid peroxidation. In contrast to verapamil, amlodipine in a dose of 50 ng/ml in vitro decreased hemoglobin affinity for oxygen. Amlodipine however shows no activity on the inhibition of macrophage superoxide release and cell migration, which occurs by decreased TNFα‐induced O₂‐ release.

1. Amlodipine effect on some enzymes and enzyme systems in normal and tumor cells: Amlodipine retarded THP-1 cell (monocyte/macrophage derived human leukemia cell line) adhesion to vascular endothelium via inhibition of protein kinase C signal transduction thus confirmed role of amlodipine in the mutual interaction of monocytes and endothelium, modulation proteinkinase-C and RhoA-dependent mechanisms (RhoA-small proteins of GTPase) ⁵².

Amlodipine and nifedipine stimulated the dead space wound healing in rats. The drugs enhanced normal healing as evidenced by increase in tensile strength of 10 days old granulation tissue. There was neither a significant change in the hydroxyproline level (or collagen) nor a change in the glycosaminoglycan content in granulation tissue. However, lysyloxidase level was increased significantly. The increase in tensile strength could thus be attributed to better cross-linking and maturation of collagen rather than collagen synthesis per se. The drugs were also able to overcome steroid depressed wound healing. It is likely that the prohealing effects may be related to the improved antioxidant status too, since superoxide dismutase (SOD) levels were observed to be higher in the CCB- treated animals ⁵³.

Amlodipine dose-dependently (1-100 nM) inhibited p42/p44 MAPKs activation by bFGF – basal fibroblast growth factor (experiments on the human arteria smooth muscle cells), and this may be a reason for amlodipine antiproliferative activity ^{54, 55, 56}. In the experiments with promyelocyte leukemia HL-60 and MDR HL-60 cells amlodipine augmented stealthy liposomal topotecan (SLT) anticancer activity. The enhanced antitumor activity in the MDR HL-60 cells by the SLT plus amlodipine could be owing to multiple reasons:

a)      synergistic apoptosis inducing effect,

b)      reversal of MDR by amlodipine and

c)      increasing the availability of active lactone of topotecan by the stealthy liposomes.

The apoptosis induced by amlodipine proceeds through initiator caspase 8 and then effectors caspases 3/7 signalling pathway ⁵⁷. The amlodipine antitumor activity in vitro and in vivo was shown in the cell culture of human epidermoid carcinoma A431 cell line, and in mice, the cell proliferation was inhibited ⁵⁸. Amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells ⁵⁹.

Flow cytometric analysis revealed that treatment with amlodipine (20–30 μM, for 24 h) induced G1 phase cell accumulation. The amlodipine-induced G1 arrest was associated with a decrease in phosphorylation of pRB(retinoblastoma protein), a regulator of G1 to S phase transition, reduction of protein levels of cyclin D1 and cyclin dependent kinase 4 (CDK4), G1 specific cell cycle proteins, and increased expression of p21^Waf1/Cip1, an inhibitory protein of CDK/cyclin complexes. In vitro kinase assay revealed that amlodipine significantly decreased CDK2-, CDK4-, and their partners cyclin E- and cyclin D1-associated kinase activities. The amlodipine-induced reductions in cyclin D1 protein expression and in CDK2 kinase activity were reproduced by a dihydropyridine derivative, nicardipine, having an inhibitory effect on A431 cell growth, but not by nifedipine, lacking the antiproliferative activity.

The results demonstrate that amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells through induction of p21(Waf1/Cip1) expression, inhibition of CDK(cycline dependent kinase4)/cyclin-associated kinase activities, and reduced phosphorylation of pRB. Amlodipine prevented the occurence of chronic morphological vasospasm in the femoral arteria (determined by morphometric analysis in rats) ²⁴. Ca²⁺ channel blockers, such as amlodipine, inhibit vascular smooth muscle cell (VSMC) growth through interactions with targets other than L-type Ca²⁺ channels.

The effects of amlodipine on Ca²⁺ movements in thrombin- and thapsigargin-stimulated VSMCs were therefore investigated by determining the variations of intracellular free Ca²⁺ concentration in fura 2-loaded cultured VSMCs. Results indicated that 10–1,000 nM amlodipine inhibited thrombin-induced Ca²⁺ mobilization from a thapsigargin-sensitive pool and thapsigargin-induced Ca²⁺ responses, including Ca²⁺ mobilization from internal stores and store-operated Ca²⁺ entry.

These effects of amlodipine do not involve L-type Ca²⁺ channels and could not be reproduced with 100 nM isradipine, diltiazem, or verapamil. The inhibition by amlodipine of Ca²⁺ mobilization appears therefore to be a specific property of the drug, in addition to its Ca²⁺channel-blocking property. It is suggested that amlodipine acts in this capacity by interacting with Ca²⁺-ATPases of the sarcoplasmic reticulum, thus modulating the enzyme activity. This mechanism might participate in the inhibitory effect of amlodipine on VSMC growth ⁶⁰. In thrombin- and thapsigargin-stimulated SMCs, amlodipine and not isradipine dose-dependently reduced Ca²⁺ mobilization (i.e. Ca²⁺ release from internal stores); these dihydropyridines did not affect either Ca²⁺ influx or ERK 1/2 activation.

In bFGF-stimulated SMCs, amlodipine and isradipine reduced both Ca²⁺ influx and ERK 1/2 activation without affecting Ca²⁺ mobilization. ERK 1/2 activation could also be directly stimulated by the L-type channel agonist Bay K 8644, demonstrating the involvement of voltage-gated Ca²⁺ influx in this process. Most of the observed effects described were obtained with approximately 10 nmol/l amlodipine/isradipine (i.e. concentrations close to the peak plasma level in treated patients) ⁶¹.

Amlodipine is reported ⁵⁸ to inhibit proliferation of human epidermoid carcinoma A431 cells. It specifically attenuated Ca²⁺responses evoked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺-ATPases. The possible mechanism of the antiproliferative action of amlodipine and its antitumor effect on A431 xenografts in nude mice was examined. Amlodipine reduced BrdU incorporation into nucleic acids in serum-starved A431 cells, and the reduction was diminished by uridine 5'-triphosphate (UTP), a phospholipase C (PLC)-linked agonist.

Fluorometric measurement of intracellular free Ca²⁺ concentration revealed that amlodipine blunted the UTP-induced Ca²⁺release from the internal Ca²⁺ stores and consequently Ca²⁺ influx through Ca²⁺-permeable channels on the plasma membrane. Investigations were made whether amlodipine interacts directly with intracellular Ca²⁺ stores in fluo-3-loaded cells. Application of amlodipine in Ca²⁺-free HBSS evoked a transient rise in [Ca²⁺]_i. Thapsigargin applied thereafter caused no rise in [Ca²⁺]_i, and vice versa, suggesting that amlodipine induced Ca²⁺ release from the thapsigargin-sensitive Ca²⁺ stores.

When amlodipine was applied after UTP, an additive rise in [Ca²⁺]_i was observed, while UTP after amlodipine did not cause any increase in [Ca²⁺]_i. This indicates that amlodipine caused Ca²⁺ release from both IP₃-sensitive and IP₃-insensitive Ca²⁺ stores. It were shown that thapsigargin subsequently evoked a rise in [Ca²⁺]_i by UTP, while UTP after thapsigargin did not, indicating that thapsigargin induced Ca²⁺ release from both IP₃-sensitive and IP₃-insensitive Ca²⁺ stores. In contrast, no [Ca²⁺]_i increases were induced by nicardipine or nimodipine, both of which have an antiproliferative effect on A431 cells and also blunted the thapsigargin-induced Ca²⁺ release from internal Ca²⁺ stores and the ensuing Ca²⁺ influx.

In addition, the Ca²⁺-releasing effect of amlodipine was evoked with as low as 3 μM. Although amlodipine alone caused Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores, such an effect was not reproduced by other dihydropyridine Ca²⁺ channel blockers, including nicardipine and nimodipine, despite their antiproliferative effects in the cells. Daily intraperitoneal administration of amlodipine (10 mg/kg) for 20 days into mice bearing A431 xenografts retarded tumor growth and prolonged the survival of mice. These results suggested a potential antitumor action for amlodipine in vitro and in vivo, which may be in part mediated by inhibiting Ca²⁺ influx evoked by the passive depletion of internal Ca²⁺ stores and by PLC-linked agonist stimulation ⁵⁸. Possibly amlodipine mechanism included the carboanhydrase I [E.C. 4.2.1.1] inhibiting activity (as the other dihydropyridines too). This enzyme catalyses reversible hydration of carbon dioxide, with the following increase of pH, which could influence entry of the calcium ions into the cell through calcium channels ^{62, 63, 64}.

A remarkable antiulcer effect of amlodipine was observed, as well as interaction with H2 blocker and proton pump inhibitor in pylorus ligated rats. The similar therapeutic effect was observed by using combinations of small amlodipine and famotidine or omeprazol doses ⁶⁵. Amlodipine caused the antinociceptive action by blocking N-type Ca²⁺ channels at the primary afferent neurons and blocking synaptic transmission in the nociceptive neurons (experiments on mice) ⁶⁶.

Prolonged use of the drug facilitated learning and memory in mice ⁶⁷. Amlodipine retarded the development of pulmonar hypertension (in experiments with rats), diminishing the blood pressure, blocking the decrease of the endothelial NO synthase activity, as well as the result of its own antiinflammatory, antiproliferative and antithrombotic action ⁶⁸.

Amlodipine enchanced the activity of antiepileptic drugs (against pentylenetetrazole-induced seizures, in the experiments in vivo) ⁶⁹.

1. Inhibition of smooth muscle cell proliferation and migration: Vascular smooth muscle cell proliferation and migration are early characteristics of atheroma development and are closely related to vascular diseases. In VSMCs obtained from atherosclerotic plaques, Ca²⁺ transportmechanisms and basal intracellular Ca²⁺ levels are disruptedas a result of increased membrane cholesterol content. Thesechanges have important consequences for atherosclerosis, becausecalcium participates directly in signal transduction pathwaysthat promote SMC proliferation and migration, among other changes.Membrane bilayer swelling was attributed to the accumulationof unesterified cholesterol in the diseased artery, an atherogenic stimulus that leads to increased SMC proliferationowing to the production of growth factors ¹. Amlodipine (in this case only its R(+) stereoisomer), has been shown to effectively inhibit smooth muscle cell proliferation following cholesterol enrichment ¹. There is growing evidence that calcium antagonists, and also amlodipine, have antiproliferative action, inhibiting VSMC growth/proliferation ^{34, 54}.

Their molecular mechanisms were clarified. The antiproliferative action of amlodipine is achieved by induction (increasing the expression) of the p21^(Waf1/Cip1) gene, which may explain beneficial covert effects of this widely used cardiovascular therapeutic drug beyond a more limited role as a vascular relaxant ⁵⁵. Amlodipine inhibits cell proliferation via PKD1(polycystic kidney disease 1)- related pathway ⁵⁶. Polycystic kidney disease 1 (PKD1) is also involved in cell cycle inhibition via p21^(Waf1/Cip1) up-regulation. Authors clarified the involvement of PKD1-related signaling on human coronary artery smooth muscle cells (hCASMCs). Cultured hCASMCs, which constitutively express PKD1, were stimulated with 5% serum.

Amlodipine increased p21^(Waf1/Cip1) expression in a dose- and time-dependent manner, resulting in reduced hCASMC proliferation. The inhibitory effect of amlodipine was mimicked by overexpression of PKD1 and was reversed by a dominant-negative version of PKD1 (R4227X). Immunoblot analysis showed that phosphorylated JAK2 was increased by amlodipine treatment or PKD1 overexpression. A luciferase assay revealed that the overexpression of PKD1 induced STAT1 enhancer activity. These data suggest that PKD1 contributes to the antiproliferative effect of amlodipine on hCASMCs via JAK/STAT signaling and p21^(Waf1/Cip1) up-regulation.

The study was designed ⁵⁴to determine whether mitogen-activated protein kinases (MAPKs) are involved in VSMC proliferation induced by basic fibroblast growth factor (bFGF), and to examine the inhibitory effect of amlodipine. p42/p44 MAPK is mitogen activated proteinkinase, which is related to serine/threonine protein kinases group. It phosphorylates both nuclear and cytoplasmic proteins. MAPK calls proto-oncogene c-fos and c-myc expression in the cell nucleus, and stimulates cell growth and differentiation. Amlodipine dose-dependently (1-100 nM) inhibited p42/p44 MAPKs activation by bFGF – basal fibroblast growth factor, which have expressed mitogen properties, and which is a multifunctional growth factor.

Amlodipine (10 nM) inhibited bFGF-induced human VSMC proliferation, inhibited both short-term and sustained p42/p44 MAPKs activation by bFGF, suggesting that bFGF-induced VSMC proliferation may be related to p42/p44 MAPKs activation, and that the antiproliferative effect of amlodipine may be related to its inhibition of p42/p44 MAPKs activation. Keeping in the mind that amlodipine had antiproliferative ⁵⁴ and antimicrobial ⁷⁰ action, it was found that amlodipine possessed the ability to retard the development of Leishmania donovani (visceral leishmaniosis) infection ⁷¹, both in vitro (in 50% effective concentration for amlodipine and lacidipine is 2 and 2.5 μg/ml, resp.) and in vivo (in mice, by use of amlodipine and lacidipine commercial oral tablets from ‘Sun Pharmaceuticals Ltd.’, in the doses 10 mg/kg weight, which are 4.5-times and 325- times lower doses than LD_50% for amlodipine and lacidipine, respectively).

Drugs were administered once weekly during four weeks. This property is independent from influence on Ca²⁺ transport, and is determined by phenyl-1, 4-dihydropyridine cycle structure.

1. Modulation of vascular cell gene expression and extracellular matrix formation: Amlodipine increases the expression of the cytokine interleukin-6 in human vascular SMC by directly activating its specific gene promoter. Up-regulation of interleukin-6 by amlodipine at low pharmacological concentrations leads to potent antiproliferative effect, independent of calcium channel modulation ³². Amlodipine was also shown to have favourable effects on the synthesis of extracellular matrix (ECM) proteins involved in atherogenesis ^{1, 72}. Disruptions in ECM metabolism may contribute to vascular remodelling during the development and progression of human atherosclerotic lesions.

Matrix metalloproteinases (MMPs) represent a family of enzymes that degrade ECM components in human atherosclerotic plaques, leading to promotion and destabilisation of the lesion. In both fibroblasts and vascular SMC, CCBs have been shown to inhibit the expression of procollagens I, III and IV at nanomolar concentrations ⁷². Matrix metalloproteinases actively participate in VSMCproliferation and vessel-wall remodeling. Recently,various CCBs, including amlodipine, have been reported to modulateMMP activity.

Although the mechanisms of action involvedare still unclear, CCBs might act not only on MMP activity/expressionbut also on transcription of the tissue inhibitor of metalloproteinases(TIMPs) ⁷³. It was hypothesised that calcium antagonists may potentiate the activity of MMPs in arterial aneurysmal disease (AAA) and thus accelerate AAA expansion. In the therapeutic ranges amlodipine significantly enhanced elastin degradation and potentiated MMP-9 activity within the aortic organ cultures ⁷⁴.

1. Influence of amlodipine on collagenolytic activity: In the cell culture (cells of human endothelium) amlodipine, in contrary to nifedipine, significantly decreased the level and slightly inhibited expression of matrix metalloproteinase-1 (MMP-1) and its tissue inhibitor (TIMP-1) in human vascular endothelial cells (ECs), stimulated with interleukin-1 (beta) (IL-1beta), thereby significantly inhibiting collagenolytic activity. This process was not significantly influenced by nifedipine. It seems, that this action too does not depend from the blockage of the Ca²⁺ channels, because the findings revealed that amlodipine, but not nifedipine, inhibited IL-1beta-induced matrix metalloproteinase-1 expression in human ECs ⁷³.
2. Mitochondria as amlodipine drug target:Ca²⁺ accumulation and Ca²⁺ overloading in mitochondria are responsible for the cell abnormality associated with ischemia and reperfusion injury. Study ⁷⁵ was aimed at evaluating the efficacy of the Ca²⁺ channel blocker amlodipine on the mitochondrial Ca²⁺ accumulation, mitochondrial antioxidant status (antioxidants (glutathione) and antioxidant enzymes (superoxide dismutase, catalase)) and mitochondrial respiratory enzymes (malate dehydrogenase, succinate dehydrogenase and NADH dehydrogenase) in ischemia and reperfusion (I/R) induced liver injury. Pretreatment with amlodipine effectively counteracted the alteration in mitochondrial enzymes induced by ischemia-reperfusion liver damage. Transmission electron microscopic (TEM) study confirmed the restoration of cellular normality and the cytoprotective role of amlodipine against I/R induced hepatic injury. On the basis of these findings it may be concluded that amlodipine not only possesses Ca²⁺ channel antagonist properties but it may also reduce the extent of mitochondrial damage by its antioxidant activity.
3. Amlodipine effect on accumulation of advanced glycation end products AGE and RAGE: Recent studies suggested that advanced glycation end products (AGEs) can promote the development of atherosclerotic lesions in a manner similar to oxidatively modified low density lipoproteins. Atheroprotective amlodipine effect partly was explained as the result of the inhibition of AGE accumulation ³⁸. Amlodipine and fluvastatin inhibited the deposition of AGE in aortic wall of rabbits fed with cholesterol (1% in the diet) and fructose (10% in water).

Although amlodipine, a calcium channel blocker, and fluvastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, show antioxidant and atheroprotective effects, the relation of AGEs to their effects is unknown. The exact mechanism of AGE suppression is ambiguous. Inhibitory effects of chronic treatment with amlodipine (5 mg/kg per day) or fluvastatin at a dose insufficient to reduce plasma cholesterol levels (2 mg/kg per day) on the accumulation of AGEs in atherosclerotic aortas of rabbits were studied immunohistochemically.

After eight weeks of treatment, AGEs, namely argpyrimidine, carboxymethyllysine and pyrraline, markedly accumulated with intimal thickening in cholesterol and fructose-fed control rabbits, while the drugs inhibited those changes other than the pyrraline deposition without plasma lipid-lowering effects. Enhanced lipid peroxidation was observed in plasma from cholesterol and fructose-fed rabbits only, and lipid peroxidation was not suppressed by the drugs.

Clinical studies: Amlodipine already has a comparatively long and succesfull history of its clinical use for the cure of hypertonia (hypertension) and coronary ischemic disease.

Analysis of several randomised studies revealed however that amlodipine changed the life span of the patients with coronary insufficience not-significantly.

For patients with vasospastic stable angina pectoris, amlodipine remarkably decreased the count of incidents and decreased the consumption rate of nitroglycerine, and, in comparison with nifedipine, caused less expressed negative inotropic effect ¹⁸.

For patients with arterial hypertension amlodipine improved the endothelial function and remarkably inhibited the development of a.carotis atherosclerosis ¹⁷.

In the wide placebo-controlled ‘crossover GENRES’ study was found the correlation between some fundamental laboratory analysis results and proposed amlodipine (in the dose 5 mg per day, during 4-weeks long period) hypotensive effect ⁷⁶. It was observed however remarkably individual deviations from the mean (medial) parameters. Interestingly, negative correlation between calcium level in the serum and the amlodipine effect was found ⁷⁶. About the reason of this phenomenon some speculative hypotheses were proposed. For example: calcium ions under amlodipine influence concentrate into intracellular structures – organellae.

Medial level of the electrolyte (sodium, chloride and potassium ions) excretion with urine was with the negative correlation according to the lowering of the blood pressure under the amlodipine effect. The same negative correlation was observed between main cholesterol level and the lowering of the blood pressure under the amlodipine effect. Negative correlation was observed also between marker metabolites as uric acid and insuline level and the lowering of the blood pressure under the amlodipine effect. It could be mentioned that decreased sodium intake with the food did not lower amlodipine antihypertensive effect.

Authors concluded that diuretics and CCB are more suitable for the antihypertensive therapy for the patients with hypertension characterised by low renin level.

Amlodipine was used for the long (6 months) period with success for cure of children suffering hypertonia ^{8, 10}.

In case of patients with pulmonary hypertension amlodipine remarkably decreased the resistance of the pulmonary blood vessels, decreased pulmonary blood pressure and improved the function of the right chamber (ventricule), essentially (fundamentally) decreasing the pressure in the right auricle ⁷⁷. Organoprotective and pleiotropiceffects of amlodipine (as maleate) in hypertensive patients with chronic obstructive pulmonary disease were observed ⁴⁵.

After the chronical use of amlodipine the tendency could be observed to increase norepinephrine level in the plasma and to increase sympathic nerve activity, however the changes were not statistically significant, and therefore it was concluded that amlodipine practically did not change the sympathic nerve activity ⁷⁸. However in other clinical studies it was observed the increased sympathic nerve activity after medication with amlodipine (6 weak course), which accompanied the increase of the heart systolic and diastolic volume ⁷⁹.

In the patients with cardiomyopathies of the non-ischaemic nature (medicated with ACE inhibitors, digoxine, diuretics) amlodipine did not influence the existing level of circulating neurohormones and marker substances of the oxidative stress ⁸⁰.

In the patients with NIDDM (2nd type diabetes) amlodipine (5-10 mg, 12 weaks) not only decreased blood pressure, but improved the insuline resistance and decreased the level of TNF-alpha ⁸¹. In the hypertensive patients with NIDDM amlodipine decreased microalbuminuria (urinary albumin excretion – UAE). In the combination with fosinopril the effect was increased as compared for the separate (solely) both drug use ⁸². In the wide clinical investigations (twenty-six prospective studies were identified and 22 manuscripts were included, 802 individuals investigated) nimodipine, amlodipine and other CCBs ability to increase the fibrinolysis (by decrease of the ‘euglobulin clot lysis time’– ECLT) and to increase the activity of tissue plasminogen (tPA) was shown. The elevated fibrinolytic activity is apparently caused by an increase of the tPA antigen (tPA ag) level and a decrease of the plasminogen activator inhibitor-1 antigen (PAI-1 ag). A sensitivity analysis showed that dihydropyridines, but not phenylalkylamines, exerted a fibrinolytic effect. This fibrinolytic effect is not only seen in patients with subarachnoid hemorrhage but also in hypertensive patients.

Thus, calcium antagonists increased fibrinolytic activity ¹⁹. This may add to the beneficial pharmacological effect of calcium antagonists to prevent ischemic events in patients with hypertension and subarachnoid hemorrhage. In the nanomolar concentrations amlodipine remarkably decreased the constitutive and platelet-derived growth factor BB (PDGF-BB)-dependent incorporation of the collagen into the extracellular matrix (ECM), which is formed by vessel smooth muscles and fibroblasts. Expression of the fibrillar (I and III type) collagen and basal membrane (IV type) collagen was blocked and the proteolytic activity of the collagenase (72-kDa type IV) was increased ⁷².

In the multicenter, prospective, randomized, open, blinded end point CAMELOT (The Comparison of Amlodipine vs Enalapril to Limit Occurrences of Thrombosis) study ⁸³, administration of amlodipine for 24 months to normotensive patients with coronary artery disease resulted in a reduction in adverse cardiovascular events, whereas no significant treatment effects were observed with enalapril.

Furthermore, the assessment of atherosclerotic plaques by intravascular ultrasonography showed no progression in plaque volume in patients receiving amlodipine ⁸⁴. These results indicated that not only angiotensin receptor blockers (ARBs), but also amlodipine may have antiatherosclerotic effects. In the randomized, placebo-controlled clinical investigation (coronary angiography for 825 patients) the use of amlodipine however sufficiently did not change the development of the of atherosclerosis (angiographic data), as well did not change life span or frequency of the infarct incidents, as compared with the placebo group ⁸⁵.

However, clinical studies of other authors have suggested the ability (property) of amlodipine to decrease the frequency the incidents of heart and brain attack connected with ischaemia ⁴², and this could not be explained only by the effective antihypertensive activity ⁸⁶.

Prolonged use (till one year) of amlodipine gives good results for the patients with transplanted heart, keeping the blood pressure and kidney functions in the normal level ⁸⁷. Additive action of amlodipine and atorvastatine was shown in the clinical studies with patients with hypertensia & hyperlipidemia. The synergy of both drugs beneficially influenced the cure of patients ^{15, 88}.

The effectiveness and safety of amlodipine has been summarized in many randomized placebo-controlled clinical studies. Amlodipine is comparativelly well tolerated, if used in the doses not more than 10 mg/per day. The observed side effects are small or medium ^{89, 90}, and the medication should be broken only for about 1.5% of patients (in the placebo group 1%).

The main and more frequent side effects are headache and peripheral edema.

The incidence or frequency (in %%) of the amlodipine side effects depended from the dose ⁹⁰:

TABLE 1. THE INCIDENCE OR FREQUENCY (IN %%) OF THE AMLODIPINE SIDE EFFECTS

Other adverse experiences that were not clearly dose related but were reported with an incidence greater than 1.0% in placebo-controlled clinical trials include the following ⁹⁰:

TABLE 2. OTHER ADVERSE EXPERIENCES OF AMLODIPINE THAT WERE NOT CLEARLY DOSE RELATED

More frequently the side effects are expressed for women ^{89, 90}.

The swelling of upper extremitates for children is reported ⁹¹.

Prolonged use of amlodipine could lead to the possibility of the development of parkinsonism ⁹².

Sometimes (<1%) observed other side effects could be connected with various systems of the whole organism. However amlodipine could be used safely for patients with chronic diseases (in the cases of COPD, compensated not-hereditary cardiopathy, pathologies of the peripheral blood vessels, Diabetes Mellitus (DM), changed lipid composition) ⁸⁹.

Some exclusive events with amlodipine overdosage have been reported (extremally low blood pressure, tachycardia, resp. shock and metabolic acidosis), which could be medicated with intravenous injection or infusion of calcium salts ^{93, 94}, however it could be effective not in all cases. At intoxication by amlodipine or other CCBs it is recommended 4-aminopyridine (fampridine) use ⁹⁵.

Amlodipine could be used in the combination therapy together with the other cardio- and vasoprotective acting drugs (positive data have been reported about the combinations with atorvastatin, benazepril, fosinopril, valsartan, telmisartan (TWYNSTA^®), famotidine or omeprazole a.o.).

By combined amlodipine and valsartan (angiotensine-II receptor antagonist) therapy prolonged severe hypotensia could occur, which were medicated with large dosis of insuline, glucagone and naloxone ⁹⁶.

Amlodipine possesses analgesic properties, and it could be used in the combination with other analgetics ⁹⁷.

The internet pages http://www.drugs.com/drug-interactions/amlodipine.html(http://www.drugs.com/drug-interactions/amlodipine.html#ixzz13YzP4sdu) and http://www.drugs.com/drug-interactions/amlodipine. html#ixzz13Z2yqsKA) indicate about 462 drugs (with 2648 brand and generic names), which interfere with amlodipine action. The major drug interaction occurs with 11 drugs, containing as active 2 substances -itraconazole, tizanidine, and assiduity and caution should be kept by use of amlodipine in combination with these drugs. This list is however not fully completed, and rise from month to month.

In this site also 4 diseases were mentioned, when amlodipine could be contraindicated - cardiogenic shock/hypotension, obstructive coronary artery disease,significantly impaired hepatic function and hepatic disease, Chf/Ami- congestive heart failure/myocard infarction.

As it was shown in the text above, amlodipine as effective therapeutics nevertheless has a number of various side effects, therefore new its analogues were and will be designed.

May be, the new original biologically active amlodipine analogues (as mentioned in ^{98, 99} and other) would possess some of the intrinsic for amlodipine signes of pleiotropism.

High cytotoxic activity of some of these compounds against cancerous cells (human lung fibrosarcoma HT-1080 and mouse hepatoma MG-22A), which is connected with intensive generation of the intracellular NO radicals, was revealed already.

CONCLUSION & SUMMARY: Amlodipine is the third generation calcium antagonist, however it is not only specific calcium channel blocker, but also broad spectrum or pleiotropic active drug, possessing the possibility to touch and trap some metabolic functions and systems of the various cells, tissues and organs of the whole organism. Its ability to cure the heart and vascular diseases (hypertension and atherosclerosis), whose pathogenetic factors include excessive constriction of the vessels, thrombosis, inflammation, proliferation, oxidation, confirms the drug multifunctional, or pleiotropic action.

Amlodipine influence on the membrane permeability and cholesterol deposition in the membrane, its antioxidative action, stimulation of the NO release, as well ECM regulation are not directly connected to the decrease of the resistance of the blood vessels – antihypertensive action. The cellular and molecular mechanisms of the observed amendment of the endothelium function, delay of the cardiovascular action disturbances as the result of amlodipine use are videly investigated, however up to now are not fully clear and unequivocal.

Amlodipine could be used in the combined therapy in the combination with other cardiovascular and other organs and tissues targeting drugs: there exist positive data about atorvastatin, benazepril, fosinopril, valsartan, telmisartan, famotidin or omeprazol a.o., the mutual influence is studied for about 462 drugs, and partly it is already in use.

ACKNOWLEDGEMENT: This study was supported by The Latvian National Research Programme 2010. – 2013. ‘Development of new prevention, treatment, diagnostics means and practices and biomedicine technologies for improvement of public health’ (VPP-4).

The authors are grateful to the administration of Latvian IOS for the technical support.

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