Alternative testing in drug research and development-The validation issue.

As ECVAM (the European Centre for the Validation of Alternative Methods) intensifies its work, it is worth discussing the aspects of alternative testing in drug research and development as well as the implication of validating tests-and the possible role of ECVAM in this. Substituting animal in vivo tests with alternative testing has always been a major target in the pharmaceutical industry, for ethical and practical reasons. In vitro tests have an important role, especially in the first phase of drug discovery (the substance-finding phase) but to a lesser extent in safety testing. In the further development of a new drug, validation becomes more important for safety tests than for the initial screening and substance-finding tests. That also implies that diverse safety and toxicity testing of pharmaceuticals should be based on worldwide accepted and validated protocols. ECVAM has a role here, in informing the scientific and regulatory community about promising (validated) tests in drug discovery or development and pressing for worldwide harmonization, especially of safety testing.

Effects of different antihypertensive drug classes on survival in animal models.

Data on the influence of antihypertensive drug treatment on mortality of hypertensive rats are reviewed. Dihydropyridine calcium antagonists, verapamil, the angiotensin-converting enzyme (ACE) inhibitor captopril, and a triple combination of reserpine, hydralazine, and chlorothiazide normalized or markedly prolonged survival. Captopril was less effective in sodium chloride-induced, low-renin Dahl rat hypertension. Dihydralazine prolonged but did not nearly normalize survival. The K(+)-channel activator minoxidil was relatively ineffective. Data on diuretics or beta-blockers are insufficient or unavailable. Calcium antagonists nitrendipine and nimodipine and the ACE inhibitor captopril improved survival and prevented vascular lesions and calcinosis even at doses that failed to achieve normotension. All drugs that normalized survival also reduced heart weights. Minoxidil invariably increased heart weights and failed to improve survival. (Di)hydralazine assumed an intermediate position.

Pharmacological basis for use of calcium channel antagonists in hypertension.

The major mechanism of antihypertensive action of Ca2+ channel antagonists is the inhibition of Ca2+ entry into the vascular smooth muscle cell. Ca2+ channels are stabilized by these drugs in a mode unavailable for opening. The inhibition of Ca2+ entry leads to vasodilatation and lowering of arterial pressure. The advantages of Ca2+ channel antagonists in hypertension include improvement of blood supply to vital organs, diuretic activity, coronary vasodilatation, reduction in heart size and vascular cytoprotective effect.

Suppression of renin-angiotensin system in the heart of spontaneously hypertensive rats.

Renin-like enzyme and angiotensin converting enzyme (ACE) were identified and their specific activities measured in cardiac tissues of spontaneously hypertensive rats (SHR) and their Wistar-Kyoto (WKY) normotensive controls. In addition, the enzyme activities were determined following administration of hypotensive drugs. The pH optima of cardiac renin-like enzymes were identical with those in vascular walls, the specific activity being higher in the heart. Cardiac ACE revealed similarities with the venous wall enzyme. The highest specific cardiac renin-like activity was found in the septum and that of ACE in atria/auricles. Both enzyme values were lower in the hearts of SHR than in those of normotensive controls. Following nifedipine treatment, specific renin-like activities increased in all cardiac structures studied (P less than 0.01); with nitrendipine and muzolimine less pronounced elevations were obtained. Administration of these three hypotensive drugs resulted in a stimulation of ACE in all the cardiac structures except in atria/auricles, where their activities were lowered.

Effect of hypotensive agents on the renin-angiotensin system in vascular walls of spontaneously hypertensive rats.

Previous investigations have shown that the renin-angiotensin system (RAS) is activated in vascular walls of spontaneously hypertensive rats (SHR). The present study was undertaken to determine whether antihypertensive drugs attenuate this activation. Two calcium channel blockers, nifedipine and nitrendipine, and the diuretic muzolimine were applied to SHR for 2-4 weeks, and angiotensin (ANG) I-forming angiotensinogenase (AIFA) and ANG I converting enzyme (ACE) activities were determined. The values for both enzymes were elevated in arterial tissues of SHR (P less than 0.01), whereas in venous walls AIFA activity was decreased (P less than 0.01). All hypotensive drugs reduced arterial ACE activities in SHR (nifedipine and muzolimine: P less than 0.01; nitrendipine: P less than 0.05). Angiotensin I-forming angiotensinogenase activity was increased following treatment with nifedipine (P less than 0.01) but reduced by nitrendipine (P less than 0.05); with muzolimine, no significant alterations were observed. The results obtained indicate that in SHR, stimulation of vascular wall ACE is abolished following treatment with hypotensive agents, and the effect is independent of their mode of action. It is assumed that the activation of vascular ACE is not caused by, but rather a reaction to, elevated blood pressure.

Comparison of the effects of antihypertensive drugs on pre- and postglomerular vessels of the hydronephrotic kidney.

The in vivo renal microvascular effects of three antihypertensive drugs: nitrendipine, a calcium entry blocker, and two direct-acting vasodilator agents, hydralazine and minoxidil, were compared. The dilator responses of pre- and postglomerular vessels were directly observed after the topical or intravenous administration of each drug using the hydronephrotic kidney preparation and television microscopy. When applied directly to the kidney to avoid changes in systemic blood pressure, all three drugs preferentially dilated the preglomerular vessels. The largest increases in preglomerular vessel diameters were observed with nitrendipine followed by hydralazine and minoxidil, in the order listed. These drugs produced little change in efferent arteriole diameters though acetylcholine evoked significant dilator responses of the same vessels. When infused intravenously to lower blood pressure, the predominant effect of the drugs was on the afferent arteriole. No significant changes in the efferent arteriole diameter were observed with any drug. These data demonstrate that these vasodilators, which act primarily at preglomerular sites, probably increase glomerular capillary pressure, but to differing degrees.

Effects of salt loading and nitrendipine on dihydropyridine receptors in hypertensive rats.

It has already been emphasized that salt-dependent hypertension might be especially sensitive to calcium antagonists, and it has recently been shown that binding at different receptor types might be altered by dietary sodium chloride. Therefore, it was of interest to find the extent to which the specific membrane-associated dihydropyridine (DHP) receptors are influenced by salt loading and/or nitrendipine treatment. Groups of 10-12 salt-sensitive (S/JR) or salt-resistant (R/JR) Dahl rats and spontaneously hypertensive rats (SHRSP) or normotensive Wistar-Kyoto rats (WKY) received diets containing low (0.4%) or high (8%) NaCl for 21 days; treated animals received 300 ppm nitrendipine additionally with the diet. Blood pressure of R/JR and WKY did not differ markedly on the different diets, whereas in hypertensive rat strains, salt loading increased blood pressure after 3 weeks. High salt resulted in an increased number of cardiac DHP receptors in SHRSP; the addition of nitrendipine enhanced DHP receptor capacity in the heart and brain membranes of both strains (SHRSP and S/JR). No major changes in affinity were observed. Augmentation in the number of DHP receptors as analyzed in a follow-up interim observation study on SHR occurred more rapidly in heart than in brain tissue. It can be concluded that an altered number of the DHP receptor sites, and not changes in affinity, might be essential during treatment with calcium antagonists. The DHP receptor sites, and not changes in affinity, might be essential during treatment with calcium antagonists. The DHP receptor density can be modified by high salt intake, as shown in salt-loaded SHRSP.

Nifedipine corrects the blunted renal response to saline loading in hypertension-prone SBH rats.

The elimination of an acute oral saline load is markedly blunted in adult Sabra hypertension-prone (SBH) rats compared with hypertension-resistant Sabra normotensive (SBN) rats. Within 2 h, urinary output and the excretion of sodium and potassium are significantly reduced, while urine osmolality is markedly elevated in SBH rats. The long-term administration of nifedipine, 20-30 mg/kg body weight enhanced the diuretic and natriuretic response to saline loading in members of both strains. The effect was significantly more pronounced in SBH, especially in adult animals where the diuretic and natriuretic response averaged 150 and 130% of control, while in SBN the enhanced response was 50 and 20%, respectively. As a result of the disparate effect of nifedipine in the two strains, the blunted response of SBH was abolished. The mechanism of the preferential response to nifedipine of SBH rats remains to be determined.

Renal effects of 1,4-dihydropyridines in animal models of hypertension and renal failure.

Renal effects of 1,4-dihydropyridine (DHP)-type calcium antagonists (nitrendipine and nisoldipine) were analyzed in diverse conditions, such as long-term antihypertensive treatment, acute saline-loading, and acute renal failure in rats. In spontaneously hypertensive rats (SHR), 60-week treatment with nitrendipine resulted in normotensive blood pressure values without increasing body weight, an indicator of salt-water retention, or increasing plasma renin activity and plasma aldosterone concentration compared with the untreated rats. After acute saline-loading of normotensive or hypertensive rats, administration of calcium antagonists nitrendipine and nisoldipine increased urinary volume and sodium excretion. This was in contrast to the effects observed with the vasodilator minoxidil, with which salt-water retention was shown. In acute renal failure induced by 60-min renal ischemia in uninephrectomized rats, administration of nisoldipine decreased mortality rate and improved kidney function. The increase in renal tissue calcium content and the decrease in ATP content associated with the renal failure was abolished by nisoldipine treatment. In conclusion, renal protective effects are present with DHP-type calcium antagonists; however, mechanisms in situations such as hypertension or acute renal failure might be different and deserve further analysis.

Calcium channel blocker nisoldipine limits ischemic damage in rat kidney.

The effects of the calcium channel blocker nisoldipine on renal function after 60 min. normothermic ischemia and contralateral nephrectomy were studied in male Wistar rats. Nisoldipine (300 ppm) was given in a standard diet as well as one hour prior to ischemia (10 mg./kg. orally). Survival, serum urea, serum creatinine, urine volume and creatinine clearance were used to test the effectiveness of the drug. Nisoldipine treatment resulted in the survival of all animals (compared to 66.6 per cent in the untreated group) and improved immediate and long term (14 days) renal function. The drug given post ischemia only was not effective, suggesting that nisoldipine must be present in the kidney during ischemia. The beneficial effects of the drug in postischemic acute renal failure may be attributed in part to effects on postischemic renal hemodynamics. Additional direct effects on ischemic renal epithelial cells, presumably by inhibiting transmembrane calcium fluxes, cannot be excluded.

Influence of a calcium-enriched diet on salt-induced hypertension in rats.

Addition of dietary calcium exerts antihypertensive effects in spontaneously hypertensive rats (SHR), which can be intensified by a parallel increase of sodium in the diet. It was of interest to what extent calcium addition to a high salt diet might modify salt-dependent hypertension in salt-sensitive Dahl rats (S/JR). Groups of six S/JR and seven salt-resistant Dahl rats (R/JR) received, when 4 weeks old, diets containing differing sodium and calcium concentrations. A further group received a calcium-enriched diet supplemented with the calcium antagonist nitrendipine. A higher calcium content in the diet did not change the effects resulting from an 8% NaCl diet, with regard to heart weight and laboratory parameters. Salt-sensitive Dahl rats on a calcium-enriched NaCl diet had a more rapid development of hypertension than S/JR on a normal calcium/high salt diet or S/JR on a calcium-enriched diet supplemented with nitrendipine. Salt-resistant Dahl rats did not differ significantly with regard to blood pressure development on any diet. In contrast to the effect in SHR, dietary calcium has therefore no antihypertensive effect on salt-induced hypertension. A moderate increase in the calcium content of the diet does not alter blood pressure lowering effects of calcium antagonists.

Inhibition of human red blood cell Na+, K+-cotransport by various "high ceiling" diuretics.

The rank order of diuretic efficacy of furosemide analogs, e.g. bumetanide and piretanide, in humans is reflected better by their ability to inhibit Na+, K+-cotransport in human red blood cells than by their natriuretic activity in rats. High ceiling diuretics which are structurally unrelated to sulfamoyl diuretics, e.g. muzolimine, tizolemide, MK 447, may be similarly effective in rat and man, but by acting via other mechanisms cannot be detected by use of the Na+, K+-cotransport system. On the other hand, a possible conversion of such compounds to metabolites active in the cotransport system cannot be ruled out. In contrast to ethacrynic acid, the weak inhibitory activity of muzolimine on the Na+, K+-cotransport was not potentiated by cysteine. These results suggest that the diuretic activity of muzolimine is not caused by inhibition of Na+, K+-cotransport.

Chronic treatment with nifedipine prevents development of hypertension and abnormal red cell Na+ transport in Dahl-S-rats.

Transmembrane Na+ transport was measured in erythrocytes of salt resistant (DR) and salt sensitive (DS) Dahl rats, fed either a standard (0.4%) or high (0.8%) NaCl diet, or a high NaCl diet containing 300 ppm of the calcium entry blocker nifedipine. Whereas salt-loaded DS became severely hypertensive, those treated with nifedipine remained normotensive. With a low NaCl diet, total Na+ efflux and Na+ pump activity was lower in DS than in DR. Cotransport was more active in DS than in DR. After excess Na+ intake, total Na+ efflux was markedly reduced in DS, but normal in nifedipine-treated DS. Cell Na+ content was increased by 52% in salt-loaded DS, but not so in DS chronically treated with nifedipine. Na+ pump was completely suppressed in salt-loaded DS, but normal in DR and rats treated with nifedipine. These results suggest that Na+ pump inhibition might be involved in the development of Dahl rat hypertension, and that prevention of salt-induced hypertension by chronic nifedipine treatment also prevents suppression of erythrocytic Na+ pump. This suggests a casual link between its biochemical and therapeutic action.

Characteristic changes of plasma proteins in the Dahl hypertensive rat strain (DS) during the development of hypertension.

Several recent studies have reported the existence of additional plasma proteins in essential hypertensive patients and strains of genetically hypertensive rats. The aim of this work was to look for similar changes in a model of salt induced hypertension, the Dahl salt sensitive (DS) rat, using the sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. No additional proteins were found when different staining techniques were employed (Coomassie, silver). However, during the development of hypertension a characteristic change in the plasma protein pattern of DS rats occurred, which could not be detected in the majority of normotensive control animals. Treatment with nifedipine inhibited both the development of hypertension and this specific change in the plasma proteins, in spite of continuous dietary salt loading. It is postulated that the plasma protein changes reflect a regulatory phenomenon of hypertension.

Salt-induced hypertension in the 'Sabra' rat strain: influence of nifedipine treatment.

The effects of chronic dietary salt-loading and nifedipine therapy on hypertension-prone (SBH), -resistant (SBN) and parental (SB) Sabra rats were investigated. Salt diet for 12 weeks resulted in a sustained hypertension and heart hypertrophy only in SBH. Nifedipine therapy (300 p.p.m. = 300 mg/kg of food) introduced after week 7 on a salt diet, achieved small changes in salt-loaded SBN and SB rats, but resulted in a marked decrease in blood pressure in SBH rats within 1 week and in a regression of cardiac hypertrophy. Plasma renin activity rose slightly in nifedipine treated SB and SBN rats, but decreased significantly in treated SBH rats. Histopathological investigations revealed hypertensive vasculopathy in three out of nine untreated SBH rats, whereas there were no morphological changes in the treated rats.

Healing of malignant hypertensive arteriopathy in Dahl rats by nifedipine.

Pathomorphological studies were undertaken to investigate the therapeutic effect of the Ca2+-antagonist nifedipine on malignant hypertensive arteriopathy in Dahl salt-sensitive rats. The individual course of disease was followed by comparing pre-treatment biopsies of the mesenteric arteries with post-treatment findings at necropsy. Within seven weeks, continuous therapy with nifedipine resulted in healing of early vascular lesions and in partial repair of the more advanced ones. Under normalization of blood pressure, vascular fibrinoid exacerbations were prevented and existing intramural fibrin insudates were completely or partially removed. Lamellar fibroelastosis of the intima occurred as a characteristic of repair.

Beneficial effect of muzolimine in postischemic acute renal failure in rats.

Evidence has been presented in the past that muzolimine might act at a localization differently to sulfamoyl-type diuretics and/or with a different or additional mechanism. This is further supported by the fact that at the maximum of the dose-response curves for muzolimine and furosemide in rats, a combination of maximal oral doses still results in significantly higher sodium excretion. To further substantiate that this aspect of muzolimine is of relevance, e. g. for the therapy of acute renal failure, muzolimine treatment by food or implanted osmotic minipumps was employed in an obstructive model of severe renal ischemia in rats. Acute renal ischemia was induced in Wistar rats by clamping the left renal pedicle for 60 minutes with a microsurgery clamp. The right kidney had been removed four days before ischemia. Clearance data were obtained on the first, third and on the ninth to fourteenth days after ischemia in the surviving animals. Renal ischemia resulted in anuria, increased mortality and impaired renal function with histopathologically apparent tubular obstruction in the untreated controls. Treatment with muzolimine by food (in a concentration of 800 ppm for four days) and additional oral gavage one hour prior to ischemia prevented the sequelae of ischemia to a great extent. Similar beneficial effects could be obtained by therapeutic implantation of osmotic minipumps ensuring administration of 0.44 micrograms muzolimine/h per animal. These results in rats further support the suggestion that muzolimine might act differently to sulfamoyl-diuretics. Furthermore, they strongly implicate muzolimine as the diuretic of choice in acute renal failure.