Effects of endothelin receptor antagonists on the progression of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage renal disease in European countries and is associated with an enhanced renal synthesis of endothelin (ET)-1. ETs are - beside their potent vasoconstrictor properties - very potent profibrotic acting paracrine hormones especially in the kidney. METHODS: We analyzed in rats with streptozotocin-induced diabetes the effects of an ETA-type (ETA) receptor antagonist (LU 135252) in comparison to a combined ETA/ETB receptor antagonist (LU 224332) on the expression of interstitial and glomerular collagen type I, III and IV as well as on fibronectin and laminin by quantitative immunohistochemistry using a computer-aided image analysis system. Global glomerular matrix deposition was analyzed after PAS staining. In addition to the morphometric examination of the kidneys, we also investigated GFR, urinary albumin and total protein excretion. The diabetic rats were treated for 36 weeks. RESULTS: Treatment with either LU 135252 or LU 224332 normalized the amount of PAS-positive material within the glomeruli. The expression of glomerular fibronectin and type IV collagen was increased 36 weeks after induction of diabetes. The overexpression of these two matrix proteins within the glomeruli of diabetic rats was completely abolished by both ET receptor antagonists, whereas protein excretion was only reduced by about 50% as compared to diabetic rats without treatment. CONCLUSION: The present study indicates that ETA receptor antagonists as well as combined ETA/ETB receptor antagonists reduce proteinuria and completely normalize the renal matrix protein expression in hyperglycemic rats with streptozotocin-induced diabetes. The antifibrotic effect seems to be mediated via the ETA receptor. ET receptor antagonists might be a new approach in the treatment of diabetic nephropathy.

Adult human mesangial cells (HMCs) express endothelin-B-receptors which mediate endothelin-1-induced cell growth.

Endothelin (ET) receptor antagonists are nephroprotective in renal damage models of the rat. It is unknown whether ET receptor antagonists are also beneficial in human renal diseases. Major differences exist between the ET systems in rats and humans, therefore this study was designed to characterize the ET receptors expressed on human adult mesangial cells (HMCs). HMCs cultures are a surrogate model for the development of glomerulosclerosis. Binding experiments with [125I]ET-1 in the presence or the absence of the test compounds [endothelin-1, -3 (ET-1, ET-3), sarafotoxin 6c (S6c), or BQ123] revealed an affinity (IC50 values) of 10.5 nm for ET-1 and 87.6 nm for ET-3. The affinities of the ET(B) agonist S6c and the ET(A) antagonist BQ123 were 85.9 nm and > 10 microm, respectively. Thus, the ET receptor on HMCs shows an ET(B)-like pharmacology, but in contrast to the classical ET(B)-receptor the affinities are low. No affinity for BQ123 up to > 10 microm excludes the presence of ET(A)-receptors. Functional studies using microfluorimetry (fura-2 method) showed comparable biphasic calcium signals induced by 10 nm ET-1, ET-3 and S6c. This effect could not be inhibited by BQ123, but by the ET(B) antagonist BQ788. Reverse transcriptase polymerase chain reaction (RT-PCR) studies under different culture conditions showed that both ET(A)- and ET(B)-receptor mRNAs are expressed in HMCs. The amount of ET(A)-receptor mRNA increased 2.7-fold and that of the ET(B)-receptor mRNA 7.1-fold after stimulation with 10% fetal calf serum (FCS). ET-1, ET-3 and S6c stimulated HMCs growth (ET-1 > S6c > ET-3), but the magnitude of the effect of ET-1 is lower than reported in rat mesangial cells (rat MCs). The effect on HMCs growth could be inhibited by BQ788, but not by BQ123. Our data provide evidence for the expression of ET(B)-receptors on HMCs that are functionally active. This finding differs from the ET receptor expression in rat MCs as reported by others.

The selective endothelin-A-receptor antagonist LU 135.252 inhibits the direct arrhythmogenic action of endothelin-1.

Besides being a strong vasoconstrictor, endothelin-1 (ET-1) also causes severe ventricular arrhythmias. The aim of our study was to differentiate between the vasoconstrictor and arrhythmogenic actions of ET-1 by using the selective endothelin-A-(ETA) receptor antagonist LU 135.252 (LU). A bolus injection of 5 mg/kg LU was administered to 10 anesthetized mongrel dogs in group A. The 30 min intracoronary ET-1 infusion was started 20 min after the LU bolus at a rate of 60 pmol/min. In the control group (group B, n = 8) only ET-1 was administered (60 pmol/min). The left anterior descending coronary artery blood flow (CBF), cardiac output, electrocardiograph (ECG) and arterial blood pressure were monitored. Two monophasic action potential duration (MAPD) catheters were placed onto the left ventricular epicardium (LVEP) and into the right ventricular endocardium (RVEND) to follow electrophysiologic changes. No significant changes were observed in blood pressure (0 min vs 30 min: group A, 99.0 +/- 4.5 vs 90.0 +/- 5.2 mmHg, p = NS; group B, 103 +/- 6 vs 104 +/- 3 mmHg, p = NS), cardiac output (0 min vs 30 min: group A, 3.5 +/- 0.7 vs 3.2 +/- 0.8 l/min, p = NS; group B, 3.6 +/- 0.4 vs 3.3 +/- 0.3 l/min, p = NS), and MAPD90 (0 min vs 30 min: group A, LVEP, 241 +/- 11 vs 260 +/- 14 ms; RVEND, 233 +/- 5 vs 239 +/- 8 ms, p = NS), whereas a significant decrease was observed in CBF (deltaCBF 30 min: group A, -28 +/- 2%, p < 0.05; group B, -32 +/- 3%, p < 0.05). In group A ventricular fibrillation (VF) occurred once. Ventricular premature contractions (VPCs) and short, nonsustained ventricular tachycardias (nsVTs) were observed in seven cases. Early after depolarizations and a MAPD90 increase were observed in the control group B (0 min vs 30 min: LVEP, 244 +/- 10 vs 292 +/- 12 ms; RVEND, 255 +/- 9 vs 290 +/- 8 ms) accompanied by VPCs, incessant nsVTs. Sustained VT and VF were evident in seven cases. Our results indicate, that the applied single bolus injection of LU effectively prevents ET-1-induced major ventricular arrhythmias, whereas it has no effect on coronary vasoconstriction. These data support the notion that ET-1 possesses a direct arrhythmogenic action.

Endothelin-A-receptor antagonist LU 135.252 inhibits the formation of ventricular arrhythmias caused by intrapericardial infusion of endothelin-1.

Intrapericardial endothelin-1 (ET-1) infusion causes dose-dependent severe ventricular arrhythmias. We examined the effects of the endothelin-A- (ETA) receptor antagonist LU 135.252 (LU) on ET-1-induced arrhythmias on six open-chest mongrel dogs. Ten minutes after an intravenous bolus of LU (5 mg/kg), ET- 1 (33 pmol/kg/min) was given into the pericardial space for 30 min (LU group). Six dogs received ET-1 infusion without LU treatment (control group). Mean arterial blood pressure (MAP), cardiac output, electrocardiograph (ECG), right ventricular endocardial and epicardial (RVEND, RVEP), and left ventricular endocardial and epicardial (LVEND, LVEP) monophasic action potential durations (MAPDs) were recorded. No significant changes were observed in MAP and cardiac output. MAPD90s did not change significantly in the LU group (basic vs ET 20min: RVEP, 186 +/-7 vs 190 +/- 7; LVEP, 189 +/- 8 vs 201 +/- 11; RVEND, 191 +/- 10 vs 192 +/- 9; LVEND, 199 +/- 11 vs 203 +/- 11 ms), while significant MAPD90 prolongation was found in all investigated regions of the control group (ET start vs ET 20 min: LVEP, 174 +/- 3 vs 208 +/- 10*; RVEND, 206 +/- 9 vs 241 +/- 12* ms, *p < 0.05). No early after depolarization (EAD) was observed in the LU group, while EADs occurred in three controls. In the LU group, we have not found any significant arrhythmias except nonsustained ventricular tachycardias (nsVTs) in one animal. In the control group incessant nsVTs were observed in six, sustained VTs (sVTs) in four and ventricular fibrillation (VF) in two instances. Significant ST-elevation was observed in all animals in the LU and control groups (LU: 6.7 +/- 2.1 mV; control: 10.1 +/- 2.0 mV, p = NS). In conclusion, the arrhythmogenic action and the main electrophysiological effects of pericardial ET-1 infusion, MAPD prolongation and EAD formation, are inhibited by LU. However, LU could not prevent the ischemic changes resulting from ET-1 infusion.

Long-term effects of the endothelin(A) receptor antagonist LU 135252 and the angiotensin-converting enzyme inhibitor trandolapril on diabetic angiopathy and nephropathy in a chronic type I diabetes mellitus rat model.

Diabetic angiopathy is a serious problem in antidiabetic therapy. We wanted to investigate whether treatment with the endothelin(A) receptor antagonist LU 135252 or with the angiotensin-converting enzyme inhibitor trandolapril might prevent angiopathy in long-term type I diabetes mellitus. Six groups of male Wistar rats were investigated: untreated age-matched control rats, healthy controls treated with trandolapril (0.3 mg/kg), healthy controls treated with LU 135252 (100 mg/kg), untreated diabetic rats, and diabetic rats treated with either trandolapril or LU 135252. Rats were rendered diabetic by injection of streptozotozin. Duration of the disease was 6 months. Thereafter, rats were sacrificed, and hearts, kidneys, and a mesenterial loop were removed. Hearts and kidneys were processed histologically; the mesenterial loop was perfused with saline at constant pressure for investigation of microvessels using microvideoangiometry while treated with either 30 mM KCl, 1 microM acetylcholine, or 1 microM sodium nitroprusside. All diabetic rats developed hyperglycemia without differences among these three groups. Diabetic rats exhibited marked anemia, which was significantly antagonized by both treatments. The heart capillaries/muscle fibers ratio was decreased significantly in diabetic animals, which was prevented fully by both treatments. Renal glomerular diameter was increased in diabetic rats. This was significantly antagonized by LU 135252 but not by trandolapril. Deposition of homogeneous eosinophilic material within the glomeruli was nearly completely prevented by LU 135252. The acetylcholine-induced vasodilation in mesenteric microvessels was significantly attenuated in diabetic rats, which was significantly antagonized by both treatments. We conclude that both angiotensin and endothelin seem to contribute to the development of diabetic angiopathy and that, in addition to angiotensin-converting enzyme inhibition, blockade of endothelin(A) receptors may be an interesting new approach to antiangiopathic therapy.

Discovery and synthesis of (S)-3-[2-(3,4-dimethoxyphenyl)ethoxy]-2- (4,6-dimethylpyrimidin-2-yloxy)-3,3-diphenylpropionic acid (LU 302872), a novel orally active mixed ET(A)/ET(B) receptor antagonist.

Structural variation of the endothelin A-selective antagonist (S)-3-methoxy-2-(4,6-dimethoxypyrimidin-2-yloxy)-3, 3-diphenylpropionic acid (LU 135252) led to analogues which retain ET(A) affinity but exhibit substantial ET(B) affinity as well. The most active derivative obtained is (S)-3-[2-(3, 4-dimethoxyphenyl)ethoxy]-2-(4,6-dimethylpyrimidin-2-yloxy)- 3, 3-diphenylpropionic acid (LU 302872), which can be prepared in enantiomerically pure form in eight steps via an acid-catalyzed transetherification. It has a K(i) = 2.15 nM for binding to the ET(A) receptor and a K(i) = 4.75 nM for binding to the ET(B) receptor, is orally available, and antagonizes the big ET-induced blood pressure increase in rats and the big ET-induced bronchospasm in guinea pigs each time at a dose of 10 mg/kg.

The ET(A) receptor blocker LU 135252 prevents chronic transplant nephropathy in the "Fisher to Lewis" model.

The effect of the orally highly bioavailable and specific endothelin A (ET(A)) receptor antagonist LU 135252 was assessed in a model of chronic renal allograft nephropathy. Kidneys of Fisher rats were orthotopically grafted to Lewis rats. Fisher autografts and kidneys after uninephrectomy served as controls. All animals received low-dose cyclosporin A (CsA; 1.5 mg/kg body wt) for 10 d after surgery. Allotransplanted animals were then randomized to receive standard diet or a diet designed to deliver 30 mg of LU 135252/kg body wt per d for 35 wk. BP was monitored telemetrically. Treatment with LU 135252 did not affect systolic or diastolic pressure. Indices of glomerulosclerosis (GSI), and tubulointerstitial and vascular damage were measured. Chronic transplant nephropathy was almost completely prevented by LU 135252 compared with untreated allografts or kidneys of uninephrectomized controls, i.e., GSI 0.7 +/- 0.12 versus 1.6 +/- 0.25 (P < 0.001) versus 0.7 +/- 0.06 (P < 0.001). Allograft weight and serum creatinine were significantly lower in treated versus untreated animals. The results are consistent with the notion that ET(A) receptor-mediated events play a role in the genesis of chronic transplant nephropathy.

The Endothelin-A antagonist LU 135 252 supresses ischemic ventricular extrasystoles and fibrillation in pigs and prevents hypoxic cellular decoupling.

The endothelin-A (ETA) antagonist LU 135 252 (1 mg/kg, n = 10) or saline (control, n = 10) was injected i.v. into anesthetized pigs 15 min before occlusion of the last third of the left anterior descending coronary artery (LAD) for up to 90 min. Then, or when ventricular fibrillation occurred, the ischemic mass was determined and amounted to about 13% of ventricular mass in all groups. Heart rate, QT interval, blood pressure, and the left ventricular contractility parameter LV dp/dtmax were not altered by LU in the 15 min pretreatment period. The lower dose of the ETA antagonist had only marginal antiarrhythmic effects. At the 3 mg/kg dose, LU prolonged the time of regular sinus rhythm within the first 20 min of ischemia by 50% (mean +/- SEM: 12 +/- 2 min in control vs. 18 +/- 1 after LU; p < 0.05) and reduced the number of ventricular extrasystoles by 87% (54 +/- 18 vs. 7 +/- 3; p < 0.05). The total incidence of ventricular fibrillation (VF) (80% vs. 50%; p = 0.17) and also the incidence of late VF (ischemia > 20 min) was reduced by 3 mg/kg LU (78% vs. 38%; p = 0.12). In vitro, LU (10(-6) mol/L) prevented the hypoxia-induced (N2 gassing) impairment of intercellular coupling, measured as the delay between a direct stimulus and a distal action potential in guinea pig papillary muscles.

LU 302 872 and its racemate (LU 224 332) show balanced endothelin-A/B receptor affinity, high oral activity, and inhibit human prostate tissue contractions.

LU 302 872 (racemate LU 224 332) is a new glycerinic acid derivative related to the selective ETA receptor antagonist LU 135 252. LU 302 872 exhibits high and balanced affinity to ETA and ETB receptors (Ki 2.2 and 5.8 nmol/L), whereas LU 135 252 is ETA-selective (Ki 1.4 and 184 nmol/L). Two hours after oral treatment of rats with 10 mg/kg of LU 302 872 or of LU 135 252, the big ET-1-induced (20 micrograms/kg i.v.) blood pressure increase is inhibited by 59 +/- 8% or 52 +/- 2% (n = 6-8; p < 0.05 vs. control), whereas bosentan is without effect (-6 +/- 7%; n = 6). In guinea pigs, 10 mg/kg p.o. of LU 302 872 inhibited the big ET-1 (20 micrograms/kg i.v.)-induced bronchospasm (reduction in respiratory volume) by 78 +/- 7% (n = 6; p < 0.05), whereas the ETA antagonist LU 135 252 was ineffective (0.2 +/- 37%; n = 6). Hence, a high oral effectiveness of the new ETA/B antagonist could be demonstrated in two species for both an ETA- or an ETB-mediated response. In human prostate tissue (excised during cystectomy in bladder cancer patients), ET-1 and in most cases, the ETB agonist sarafotoxin 6c (S6c) caused contractions of similar magnitude but more sustained than that of norepinephrine (10(-6) mol/L). A high concentration (10(-5) mol/L) of the ETA antagonist LU 135 252 only moderately attenuated ET contractions. The ETA/B antagonist LU 302 872 or its racemate, LU 224 332, dose-dependently inhibited ET-1-induced contractions. S6C dose-response curves, too, were shifted to the right or suppressed by the combined ETA/B antagonist (10(-6) mol/L LU 224 332). LU 302 872 may be a suitable candidate for testing in benign prostate hyperplasia (BPH).

Renal endothelin system in diabetes: comparison of angiotensin-converting enzyme inhibition and endothelin-A antagonism.

An activated renal endothelin (ET) system is implicated in the pathogenesis of renal fibrosis, as recently shown in ET-1 transgenic mice. Because progressive renal fibrosis is also a major finding in diabetic nephropathy, we analyzed the activity of the renal ET system in rats with streptozotocin-induced diabetes mellitus and the effect of blocking the ETA receptor, using the orally active ETA antagonist LU 135252. The effects of long-term treatment with LU 135252 were compared with those of an ACE inhibitor. Plasma and urinary ET-1 concentrations were measured. Progression of diabetic nephropathy was analyzed by measuring urinary albumin and protein excretion. Urinary ET-1 excretion was significantly elevated as early as 7 days after induction of diabetes and increased further. The daily urine volume was significantly correlated with urine ET-1 excretion. Treatment with LU 135252 significantly decreased the ET-1 excretion by more than 50%, whereas ACE inhibition resulted only in a mild decrease. Albumin excretion was significantly decreased after ACE inhibition, whereas ETA inhibition resulted in a nonsignificant decrease. Urinary ET and albumin excretion probably reflect independent mechanisms of renal damage in diabetes.

Nephroprotection of an ET(A)-receptor blocker (LU 135252) in salt-loaded uninephrectomized stroke-prone spontaneously hypertensive rats.

The present study was designed to assess whether the orally active and highly specific endothelin A (ET(A)) receptor antagonist LU 135252 affects progressive renal dysfunction in a hypertensive rat model of renal damage, ie, the uninephrectomized (UNX) stroke-prone spontaneously hypertensive rat (SHRsp). The animals were examined on a normal salt (0.25%) diet and, to sensitize the kidney to hypertensive injury, also on a high salt (3%) diet. Stereological methods were used to quantify indices of glomerulosclerosis, vascular damage, and tubulointerstitial damage. Treatment with LU 135252 (100 mg/kg body wt) did not affect systolic blood pressure (BP) in animals on a normal salt diet during the whole period of the experiment (18 weeks) or in salt-loaded animals until week 10; subsequently, BP was slightly but significantly lower in salt-loaded UNX-SHRsp given LU 135252. Between weeks 6 and 12, 40% of the untreated UNX-SHRsp on a high salt diet, but none on a standard salt diet, died; such mortality was completely prevented by LU 135252. Indices of renal damage were more abnormal in salt-loaded UNX-SHRsp compared with UNX-SHRsp on a normal salt diet. Development of glomerulosclerosis and tubulointerstitial and vascular damage in UNX-SHRsp on high salt was completely prevented by LU 135252. The respective indices were no longer significantly different from those of salt-loaded sham-operated SHRsp controls. In the less severely damaged kidneys of UNX-SHRsp on normal salt, treatment with LU 135252 tended to ameliorate the indices, but the difference was not statistically significant. The results document a role of the ET system, specifically of ET(A) receptors, in the development of progressive renal injury in salt-loaded UNX-SHRsp. LU 135252 completely prevented death and renal damage resulting from salt loading.

The paracrine endothelin system: pathophysiology and implications in clinical medicine.

Apart from the initially described vasoconstriction, endothelins have been shown to cause a variety of biological activities in non-vascular tissues. A rapidly growing body of data supports the concept of endothelin as a paracrine acting hormone. In this review, we will discuss the impact of this local endothelin system for various cardiovascular pathophysiological states, especially atherosclerotic vascular disease, restenosis, myocardial infarction, congestive heart failure, and arterial hypertension. In addition, the endothelin system is a modulator of renal function via its binding to abundant receptors in renal tissue and by the ability of renal endothelial and epithelial cells to synthesize and release endothelin. In the kidney, endothelin may function as a paracrine/autocrine factor in the regulation of renal blood flow, glomerular haemodynamics, and sodium and water homeostasis. The renal endothelin system is involved in kidney diseases such as impaired renal function in liver cirrhosis, cyclosporin toxicity, acute renal failure and renal glomerular and interstitial fibrosis. Therapeutic approaches with new orally active endothelin receptor antagonists are also discussed.

Discovery and optimization of a novel class of orally active nonpeptidic endothelin-A receptor antagonists.

A novel class of endothelin-A receptor ligands was discovered by high-throughput screening. Lead structure optimization led to highly potent antagonists which can be synthesized in a short sequence. The compounds are endothelin-A-selective, are orally available, and show a long duration of action.

Structure-activity relationship of verapamil analogs and reversal of multidrug resistance.

We studied the relationship between the chemical structure and multidrug resistance (MDR) reversal activity of racemic verapamil (VER) and 14 VER analogs (VAs). The LoVo-R human colon carcinoma cell line was used as an experimental model. This cell line exhibited a typical MDR phenotype and overexpressed the MDR1 gene products. Key structural features were identified as being related to MDR reversal and cytotoxic activity. In particular, we demonstrated that the methoxy groups in the VER molecule structure [1.7-Bis-(3.4-dimethoxyphenyl)-3-methylaza-7-cyan-8-methyl-n onane] prevented cytotoxicity when the VAs were used alone, whereas the 7-cyan-8-methyl groups were important for MDR reversal activity and interaction with P-glycoprotein (P-gp). Among the VAs tested, the most active compounds were gallopamil, R-isomer of VER (R-VER), and nor-VER, which potentiated doxorubicin (DOX) cytotoxicity by 52.3 +/- 7.2 (n = 3 +/- SD), 38.9 +/- 6.4 (n = 4 +/- SD), and 35.4 +/- 4.3 (n = 3 +/- SD) times, respectively. The reversal activity of these compounds was similar to that of VER, which enhanced DOX cytotoxicity by 41.3 +/- 5.0 (n = 3 +/- SD) times. The potentiation of DOX cytotoxicity was associated with an increase in DOX uptake in LoVo-R cells and with an increased [3H]azidopine P-gp photolabeling inhibition. Some compounds that had a high reversal potency (i.e. R-VER and nor-VER) showed a lower calcium antagonist activity than VER, and seem useful candidates for the treatment of MDR in cancer patients.

Receptor selectivity of endothelin antagonists and prevention of vasoconstriction and endothelin-induced sudden death.

The new endothelin (ET) receptor antagonist LU 127043 shows higher ETA affinity than BQ 123, Ro 46-2005, and BMS 182874, with a Ki of 6 nmol/L vs. 19, 28, and 57 nmol/L. ETA/ETB selectivity of LU 127043 of about 160 is comparable to that of BQ 123 (200) and is much greater than that of Ro 46-2005 (0.93) and SB 209670 (0.74). In rabbit aortic segments, LU 127043 shows ET antagonistic potency similar to that of BQ 123 and BMS 182874 (pA2 7.34 vs. 7.36 and 7.09), whereas SB 209670 is more potent (9.80). In rats, LU 127043 completely prevents the ET-1-induced sudden death due to coronary constriction, as indicated by a pronounced T-wave increase. With i.v. pretreatment, LU 127043 is as effective as SB 209670, whereas it is three times more active using 4 h oral pretreatment. Even 8 h after oral administration, LU 127043, in contrast to SB 209670, provides dose-dependent protection. Hence, LU 127043 is an example of a selective ETA antagonist with high oral availability and long duration of action. Because the in vivo efficacy of other high affinity ET antagonists is relatively low, further optimization for therapeutic use should concentrate on pharmacokinetic properties.

Oedema reduction by levemopamil in focal cerebral ischaemia of spontaneously hypertensive rats studied by magnetic resonance imaging.

The effect of treatment with the Ca2+ channel blocker and 5-HT2 receptor antagonist levemopamil (recommended INN for (S)-emopamil) on the extent of ischaemic brain oedema was studied by magnetic resonance imaging in vivo. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent middle cerebral artery occlusion. The treatment consisted of slow intravenous injections of an aqueous solution of levemopamil given immediately after middle cerebral artery occlusion and again 2 h and 4 h later. One group of animals (n = 17) received 3 x 2 mg/kg of levemopamil (total dose: 6 mg/kg) and another group (n = 13) received 3 x 4 mg/kg (total dose: 12 mg/kg). Saline was administered to the controls (n = 16) at corresponding times. High-resolution T2-weighted spin echo images were obtained 24 h after middle cerebral artery occlusion from two transversal brain planes (4.5 mm and 6.5 mm dorsal to the interaural line). Dose-dependent reductions of brain oedema were achieved in both brain planes. The lower dose of levemopamil reduced the extent of oedema significantly (P < 0.05) by 20 +/- 3.7% in the upper and by 21 +/- 3.8% in the lower brain plane as compared to the controls (means +/- S.E.M.). The higher dose diminished the extent of oedema in the same planes by 30 +/- 3.5% and 31 +/- 4.0%, respectively. Dose-dependent reductions of infarct size, as determined by vital tissue staining using 2,3,5-triphenyltetrazolium chloride (TTC), were observed in the levemopamil-treated groups. Body temperature was not affected by levemopamil, suggesting direct cerebroprotection by this drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of multi-drug resistance in human KB cell lines by structural analogs of verapamil.

Several structural analogs of verapamil were studied for their ability to reverse multi-drug resistance (MDR) in human KB cell lines. D595, D792 and verapamil completely reversed resistance to colchicine and adriamycin. D595 and D792 had a higher reversing potency than verapamil. Devapamil, gallopamil, emopamil and D528 partially reversed MDR. The reversing potency of a drug did not correlate with its calcium antagonistic activity. No differences in reversing potency between (R)-isomers, (L)-isomers and the racemic forms were observed in the case of both verapamil and emopamil. (R)-verapamil, which has less calcium antagonistic activity and less in vivo toxicity than racemic verapamil, and D792, which has higher reversing potency and less in vivo toxicity than racemic verapamil, should be suitable for clinical applications to overcome drug resistance in cancer patients.

Enhancement of the activity of immunotoxins by analogues of verapamil.

Verapamil has been shown to enhance immunotoxin activity but only at concentrations that are too high for in vivo use. Therefore, four structural analogues of verapamil (D792, D595, D528, and Sz45) were evaluated for their ability to enhance the in vitro activity of immunotoxins made with either ricin A chain or Pseudomonas exotoxin. The following immunotoxins were used: HB21-PE and 454A12-rRTA which recognize the human transferrin receptor; and 260F9-rRTA which reacts with human ovarian carcinoma and breast carcinoma cells. The activities of the immunotoxins were determined in ovarian carcinoma cells and in KB cells using inhibition of either protein synthesis or colony formation as a measure for the cytotoxicity of the immunotoxins. Each of the four analogues enhanced the activity of ricin A-immunotoxins in a dose-dependent manner. D792 and D595 also increased the activity of IIB21-PE. Low concentrations of either Sz45 or D528 enhanced the activity of HB21-PE, but high concentrations of these two analogues either had less enhancing potency than low concentrations or even decreased the activity of HB21-PE. Specificity of enhancement by the analogues was shown by competition of the activity of the immunotoxins by the corresponding antibody and by inactivity of an irrelevant immunotoxin. The amount of enhancement ranged from 2-fold to greater than 60-fold and was dependent on the cell line and on the experimental conditions. The enhancing ability of the drugs did not correlate with their calcium-antagonistic activity. When compared with verapamil, D792 and D595 had greater enhancing potency with regard to both ricin A-immunotoxins and Pseudomonas exotoxin-immunotoxins. Greater enhancing potency and less in vivo toxicity makes D792 a candidate for use in the enhancement of immunotoxins in vivo.

(S)-emopamil, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 1st communication: pharmacological profile.

(S)-Emopamil ((2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitril e hydrochloride) is a novel compound of the phenylalkylamine group of calcium antagonists. (S)-Emopamil was tested in comparison to verapamil and gallopamil for calcium and serotonin antagonism and for cerebroprotective activity in acute hypoxia/ischemia. In receptor binding studies with (S)-3H-devapamil, (S)-emopamil exhibited distinct affinity to the verapamil binding site of the calcium channel. In rat cerebrocortical membranes, its affinity (Ki = 38 nmol/l) equalled that of verapamil and gallopamil (Ki = 49 and 27 nmol/l, respectively), whereas it was somewhat weaker in guinea pig skeletal muscle membranes. Comparing (S)-emopamil to its (R)-enantiomer, there was no clear stereoselectivity. Additionally, (S)-emopamil showed very high affinity to the cerebral serotonin S2 receptor; its Ki value (4.4 nmol/l) for 3H-ketanserin displacement being substantially lower than that of verapamil and gallopamil (Ki = 177 and 242 nmol/l, respectively). This feature is clearly stereoselective; (S)-emopamil's affinity was distinctly higher than that of the (R)-enantiomer (Ki = 58 nmol/l). The functional significance of (S)-emopamil's receptor affinity was tested in rat aortic strips. (S)-Emopamil's serotonin antagonistic efficacy (EC50 = 4.5 nmol/l) was an order of magnitude higher than that of verapamil and gallopamil. (S)-Emopamil has a less potent calcium antagonistic effect (EC50 = 270 nmol/l) on the aorta than verapamil and gallopamil (EC50 = 35 and 14 nmol/l, respectively). In isolated electrically driven (1 Hz) left atria of guinea pigs, (S)-emopamil inhibited contractile force at a much higher concentration (EC50 = 29 mumol/l) than verapamil and gallopamil (EC50 = 1.1 and 0.19 mumol/l, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gallopamil, diltiazem and nifedipine on the loss of K+ from ischaemic pig hearts.

K+ release into the extracellular space was investigated during repeated 6-min coronary occlusions before and after the intravenous administration of cardiovascular active doses of gallopamil (0.02; 0.05 mg/kg), diltiazem (1.0; 2.0 mg/kg) or nifedipine (0.01; 0.05 mg/kg) to anaesthetized pigs. [K+]e was measured epicardially using silver valinomycin electrodes calibrated in vivo. During control occlusions [K+]e- rose steeply in all groups, from a pre-ischaemic baseline value of about 3.5 mmol/l reaching a plateau value within the ischaemic period. This response was reproducible in an untreated control group. Gallopamil reduced the ischaemic K+ efflux dose dependently and significantly 10 min after injection; the higher dose also did 60 min after injection. Diltiazem had less effect on K+ efflux 10 min after administration and an effect was no longer detectable after 60 min. Nifedipine did not significantly inhibit the ischaemic K+ loss. Besides these differences in the direct protection of the ischaemic myocardium, the Ca2+ antagonists also had the following effects on the haemodynamic profile. Diltiazem and gallopamil significantly prolonged PQ intervals whereas nifedipine caused a shortening accompanied by a significant increase in heart rate. Blood pressure and LV dP/dtmax were significantly reduced by all compounds, but to a different degree. Diltiazem reduced blood pressure to a greater extent than did nifedipine and gallopamil. LV dP/dtmax was comparably reduced by gallopamil and diltiazem, while nifedipine had less effect. Thus, gallopamil exerted pronounced protective effects on the ischaemic pig heart.