Effect of the endothelin receptor antagonist, SB 209670 on cyclosporine-induced nephrotoxicity

Ketoconazole was used as an adjuvant to conventional 3-component immunosuppression in 56 patients with transplanted kidney. While receiving ketoconazole postoperative patients can be given reduced doses of cyclosporin (1.5-2 times). Long after the operation cyclosporin doses can be reduced 3-4-fold. Acute rejection occurred less frequently. This means that reduced cumulative steroid dose is needed for management of the rejection. In the dose above 100 mg/day, ketoconazole is able to enhance nephrotoxic effect of cyclosporin. A complication--acute renal dysfunction--is attributed to iatrogenic hypoaldosteronism induced by combination of large-dose cyclosporin + ketoconazole.     

Blockade of radiocontrast-induced nephrotoxicity by the endothelin receptor antagonist, SB 209670

The effect of the novel nonpeptide endothelin (ET) receptor antagonist, (+/-)-SB 209670, on the renal effects of the high osmolar tri-iodinated ionic contrast media, Hypaque, was evaluated in anesthetized dogs in the presence or absence of the cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.v.). Hypaque alone (5 ml/kg, i.v.) resulted in a marked diuresis and natriuresis but little change in either renal blood flow or renal vascular resistance. When the ET receptor antagonist, (+/-)-SB 209670, was infused into the renal artery at a dose that inhibited ET-induced renal vasoconstriction, Hypaque resulted in a significant increase in renal blood flow and decrease in renal vascular resistance. In the presence of indomethacin, Hypaque caused a significant increase in renal vascular resistance which was abolished by (+/-)-SB 209670. The data indicate that the radiocontrast media, Hypaque, can cause renal vasoconstriction which may be mediated by ET.     

SB209670, a potent endothelin receptor antagonist, prevents or delays axonal degeneration after spinal cord injury

We developed a rat spinal cord transection injury model and investigated whether endogenous endothelin takes part in axonal degeneration after injury, by using a potent nonselective endothelin receptor antagonist, SB209670. Light microscopic analysis showed that axonal degeneration of the spinal cord was clearly observed one week after injury, supported by immunohistochemical study with anti-neurofilament antibody. Electron microscopic observation showed enlargement and shrinking of spinal axons in the injured sites one week after injury. Application of SB209670 to the lesion sites markedly inhibited axonal damage after injury. These results suggest that endogenous endothelin plays a role in axonal degeneration after spinal cord injury and that SB209670 prevents or delays the axonal degeneration after CNS damage.     

Nonpeptide endothelin receptor antagonists. XI. Pharmacological characterization of SB 234551, a high-affinity and selective nonpeptide ETA receptor antagonist

The present study describes the pharmacological profile of ((E)-alpha-[[1-butyl-5-[2-[(2-carboxyphenyl)methoxy]-4-methoxy-phenyl ]-1H-pyrazol-4-yl]methlene]-6-methoxy-1,3-benzodioxole-5-propanoic acid) (SB 234551), a high-affinity, nonpeptide endothelin type A (ETA)-selective receptor antagonist. In human cloned ETA and endothelin type B (ETB) receptors, SB 234551 produced a concentration-dependent displacement of [125I]-endothelin-1 with Ki values of 0.13 and 500 nM, respectively. SB 234551 elicited concentration-dependent, rightward competitive shifts in the endothelin-1 concentration-response curves in isolated rat aorta and isolated human pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 1.9 and 1.0 nM, respectively. SB 234551 antagonized ETB receptor-mediated vasoconstriction in the isolated rabbit pulmonary artery, as demonstrated by concentration-dependent, rightward shifts in the sarafotoxin S6c concentration-response curves (Kb = 555 nM). SB 234551 produced weak functional inhibition of sarafotoxin S6c-mediated endothelium-dependent relaxation (IC50 = 7 microM). SB 234551 (10 microM) had no significant effect against contraction produced by several other vasoactive agents and did not significantly influence radioligand binding to a number of diverse receptors. SB 234551 (0. 1-1.0 mg/kg i.v.) dose-dependently inhibited the pressor response to exogenous endothelin-1 in conscious rats. In vivo pharmacokinetic analysis in the rat demonstrated that SB 234551 was rapidly absorbed from the GI tract with a bioavailability of 30%. SB 234551 had a plasma half-life of 125 min and a systemic clearance of 25.0 ml/min/kg. The present study demonstrates that SB 234551 is an antagonist with high affinity for the ETA receptor, while sparing the ETB receptor. SB 234551 is a new pharmacological tool that should assist in the elucidation of the role of endothelin in pathophysiology.     

Affinity and selectivity of PD156707, a novel nonpeptide endothelin antagonist, for human ET(A) and ET(B) receptors

6-Thioguanine (6TG) a cytostatic antimetabolite is currently used to treat patients with cancer, in particular leukemias. However, one drawback of such use is the development of 6TG resistance. Hypoxanthine-guanine phosphoribosyl transferase (Hprt) plays a crucial role in the bioactivation of 6TG. Loss of Hprt has been associated with the resistance of leukemias to 6TG chemotherapy, however, nothing has been known about the effect of Hprt status on tissue specific toxicity of 6TG in vivo. We determined the effect of Hprt status on the tissue-specific toxicity of 6TG in vivo in transgenic Hprt-deficient mice. The approximate lethal dose for Hprt-deficient mice was 23-fold higher than for the wild-type. Serum biochemical analyses of 6TG-treated wild-type mice showed elevated serum enzyme levels characteristic of liver damage whereas the levels in Hprt-deficient 6TG-treated mice were within normal physiological limits. Histopathological examination of tissues from wild-type and from Hprt-deficient mice showed contrasting spectrums of microscopic lesions. Wild-type mice had loss of hematopoietic cells from bone marrow starting at the lowest dose of 25 mg/kg 6TG whereas Hprt-deficient mice had normal bone marrow and spleen even at doses of 720 mg/kg 6TG. Wild-type mice also experienced severe loss of epithelial cells from the gastrointestinal tract starting at 50 mg/kg; however, the gastrointestinal tract of Hprt -/- mice remained unaffected. Wild-type livers revealed atrophy and necrosis at doses of 25 mg/kg 6TG although Hprt -/- livers displayed no effect until 507 mg/kg. In this study we show that Hprt-deficient mice had 6TG-resistant bone marrow and there are several other factors contributing to 6TG resistance in patients. Because variations among people exist in terms of their 6TG sensitivity, determining 6TG sensitivity of lymphocytes prior to 6TG chemotherapy and restricting treatment to 6TG-sensitive patients may improve the efficacy.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).     

Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons

We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.     

Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons

Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABAA receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABAA receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABAA receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABAA subtype of receptors, is able to affect the development of the hippocampus.     

Effect of an endothelin antagonist on hemodynamic responses to angiotensin II

We determined changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of angiotensin II (Ang II) in conscious, unrestrained normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after pretreatment with bosentan, a nonselective endothelin antagonist. Blood pressure was recorded by radiotelemetry and cardiac output by ultrasonic transit-time flow probes. Bosentan per se failed to affect basal blood pressure and evoked only small changes in cardiac output and total peripheral conductance in both strains. The pressor effects of Ang II were exaggerated in SHR compared with WKY. Strikingly, bosentan pretreatment blunted the increases in blood pressure, the fall in cardiac output, and the decreases in conductance evoked by lower doses of Ang II but not higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. These data suggest that endothelin contributes to the hemodynamic effects of Ang II in both SHR and WKY and that endothelin may contribute to the exaggerated pressor responsiveness of SHR to Ang II.     

Biochemical effects of losartan, a nonpeptide angiotensin II receptor antagonist, on the renin-angiotensin-aldosterone system in hypertensive patients

We investigated the effects of angiotensin II (Ang II) type 1 receptor blockade with losartan on the renin-angiotensin-aldosterone system in hypertensive patients (supine diastolic blood pressure, 95 to 110 mm Hg). Qualifying patients (n = 51) were allocated to placebo, 25 or 100 mg losartan, or 20 mg enalapril. Blood pressure, plasma drug concentrations, and renin-angiotensin-aldosterone system mediators were measured on 4 inpatient days: end of placebo run-in, after first dose, and 2 and 6 weeks of treatment. Plasma drug concentrations were similar after the first and last doses of losartan. At 6 weeks, 100 mg losartan and 20 mg enalapril showed comparable antihypertensive activity. Four hours after dosing, compared with the run-in day, 100 mg losartan increased plasma renin activity 1.7-fold and Ang II 2.5-fold, whereas enalapril increased plasma renin activity 2.8-fold and decreased Ang II 77%. Both drugs decreased plasma aldosterone concentration. For losartan, plasma renin activity and Ang II increases were greater at 2 than at 6 weeks. Effects of losartan were dose related. After the last dose of losartan, plasma renin activity and Ang II changes were similar to placebo changes by 36 hours. These results indicate that long-term blockade of the feedback Ang II receptor in hypertensive patients produces modest increases of plasma renin activity and Ang II that do not appear to affect the antihypertensive response to the antagonist.     

PD 142893, SB 209670, and BQ 788 selectively antagonize vascular endothelial versus vascular smooth muscle ET(B)-receptor activity in the rat

The purpose of this study was to determine whether vascular endothelial and vascular smooth-muscle endothelin ET(B) receptors could be quantitatively differentiated by PD 142893 (PD), SB 209670 (SB), and BQ 788 (BQ) in the same species by using closely matched experimental conditions. The isolated perfused rat kidney (vascular smooth muscle) and isolated perfused rat mesentery (vascular endothelium) were challenged with increasing bolus doses of sarafotoxin S6c in the absence and presence of antagonist. PD, SB, and BQ produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the kidney. PD and SB also produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the mesentery. In contrast, BQ produced an insurmountable antagonism. Schild-derived pA2 values for PD and SB were significantly greater for inhibiting endothelial versus smooth-muscle ET(B) receptors. Furthermore, PD and SB differed in their relative potency between the two assays. Because BQ produced an insurmountable antagonism in the mesentery, it was not possible quantitatively to compare the antagonist activity in the two assays. These results indicate that PD, SB, and BQ selectively antagonize endothelial ET(B)-receptor activity over smooth-muscle ET(B)-receptor activity.     

Effect of the endothelin receptor antagonist bosentan on postischemic liver microcirculation

One mechanism evoked by ischemia is endothelin mediated vasoconstriction of the hepatic vascular bed. Postischemic sinusoidal constriction leads to microcirculatory disturbances and reduced blood flow, thereby causing local hypoxia and liver damage. The aim of the study was to avoid the constrictive response of sinusoids by blocking endothelin receptors. MATERIAL AND METHODS: In an in vivo ischemia-reperfusion-model (21 Wistar rats, 250-300 g) with portal decompression by a splenocaval shunt, hepatic ischemia was induced for 30 min by cross clamping of the hepatoduodenal ligament. The effect of the endothelin receptor antagonist bosentan (10 mg/kg bw i.v.) injected before ischemia was assessed by in vivo microscopy. Microhemodynamic studies, including the sinusoidal perfusion rate, diameters of hepatic sinusoids and postsinusoidal venules, leukocyte endothelium interactions and leukocyte velocity were performed. RESULTS: After ischemia sinusoidal diameters and diameters of postsinusoidal venules were significantly reduced to 76 +/- 7% and 86 +/- 10%, respectively, in the non-treatment group, but dilated to 109 +/- 6% and 118 +/- 8% in the group treated with endothelin receptor antagonist (p < 0.001). Increased percentage of postischemic permanent adherent leukocytes could be diminished in sinusoids and more markedly in venules by therapy (p < 0.001). Leukocyte velocity was decreased to 69 +/- 9% in the treatment group ( p < 0.001). Perfusion rate could be improved to 90 +/- 2% compared to 83 +/- 5% in the untreated group (p < 0.01). Systemic arterial blood pressure was not affected by administration of the receptor antagonist. CONCLUSION: These data indicate that the endothelin receptor antagonist treatment results in prevention of postischemic sinusoidal constriction. Perfusion rate could be improved due to dilation of sinusoids and diminished leukocyte adhesion, but leukocyte velocity was reduced by 31%.     

Expression of endothelin(A) receptors in human gliomas and meningiomas, with high affinity for the selective antagonist PD156707

OBJECTIVE: Endothelin (ET) immunoreactivity, ET production, and specific ET receptors have been identified in the brain. Changes in ET concentration or receptor expression have been implicated in the pathophysiological changes in vasospasm after subarachnoid hemorrhage and in cerebral neoplasia. In this study, we have characterized the ET(A) and ET(B) receptor subtypes present in human normal cerebral cortex (NCC) and two common central nervous system tumors, i.e., meningioma (MNG) and glioblastoma multiforme (GBM). A knowledge of the ET receptor subtypes present may provide a novel therapeutic target for newly developed ET antagonists. METHODS: Saturation, competition, and autoradiographic studies were performed with the subtype-specific radioligands 125I-PD151242 and 125I-BQ3020, to characterize the ET(A) and ET(B) receptors present in NCC, MNG, and GBM. RESULTS: NCC expresses high-affinity ET(A) receptors on pial and intraparenchymal vessels and high-affinity ET(B) receptors on glia and neurons. MNGs express mainly (85%) high-affinity ET(A) receptors in a diffuse pattern, whereas GBMs express high-affinity ET(A) receptors on the neovasculature and ET(B) receptors on the nonvascular elements. The ET antagonist PD156707 (Kd = 0.059 nmol/L) showed a higher affinity for the ET(A) receptor subtype than did bosentan (Kd = 1.1 nmol/L). CONCLUSION: ET(A) receptors are expressed in high concentrations in MNGs and in the vasculature of NCC and GBMs. The ET(A)-selective antagonist PD156707 may be of potential therapeutic value in vascular and neoplastic diseases of the central nervous system.     

Binding of gamma-aminobutyric acidA receptors to tubulin

Tubulin cofractionated with gamma-aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7-1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate-gel electrophoresis of affinity-purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.     

Tachykinin NK2 receptors: characterization in the rat small intestine by the use of a peptide agonist and a nonpeptide antagonist as radioligands

OBJECTIVE: A rare family pedigree is described with a multigenerational history of an early onset, non-Alzheimer's dementia consistent with autosomal dominant inheritance. Information on five generations, with 26 suspected or proven cases of dementia, are presented. METHOD: Previous work on the family was collated and verified. The pedigree was updated. Thirty-three family members agreed to be interviewed for the present study. Standardised clinical information was obtained using the Cambridge Mental Disorders of the Elderly Examination (CAMDEX) National Adult Reading Test (NART), vocabulary and digit substitution subscales of the Wechster adult intelligence scale-revised edition (WARS-R). Abbot samples were taken for biochemical and genetic analysis. RESULTS: Fifteen males and 11 females have been affected. The age of onset of dementia in those for whom data were available (n = 12) ranged from 39 to 64 years with a mean of 53 years. The duration of illness ranged from 4 to 14 years and the age at death ranged from 49 to 69 years (mean 62 years). Autopsy data exist for nine cases. In one case the neuropathology was consistent with Alzheimer's disease. In two cases the diagnosis of Pick's disease was made on the basis of frontal or frontal/temporal lobe atrophy without Pick bodies or cells. One case diagnosed as Pick's disease had frontal/temporal lobe atrophy with cells resembling Pick's bodies. In the five remaining cases there were no distinctive neuropathological features to differentiate the type of dementia. CONCLUSIONS: The importance of recognising familial dementia, collating information on multiple generations and prospectively collecting standardised data is discussed.     

Stereoselective synthesis of a novel and bifunctional endothelin antagonist, IRL 3630

IRL 3630 (3), a single enantiomer of IRL 3461 with more potency was identified. Coupling reaction of the racemic fragment (1) with the chiral (L)-valinesulfonamide (2) under a biphasic solvent system (CH2Cl2-H2O) successfully led to the predominant formation of the desired isomer (3) with concomitant isomerization of 1. IRL 3630, N-butanesulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-+ ++isoxazolyl) -phenyl]-(D)-alanyl]-(L)-valineamide, is a highly potent and bifunctional (ETA + ETB) antagonist [Ki(ETA) = 1.5 nM, Ki(ETB) = 1.2 nM].     

Evaluation of influence of 24-hour cold preservation on endothelin production and on endothelin receptors in the bone vasculature

Endothelin-1 is a vasoactive peptide produced by the vascular endothelium. It is one of the most potent endogenous vascular smooth muscle constrictors. Two subtypes of the endothelin receptor have been cloned and sequenced and denoted endothelin-A and endothelin-B. The aim of this study was to define the influence of cold ischemia on the production of endothelin-1 and on the endothelin receptors. Two different preservation techniques (cold storage only and cold storage with microperfusion with University of Wisconsin solution) also were compared. The study was performed in an in vitro bone perfusion model to isolate the vascular endothelium from blood components. The production of endothelin-1 by the bone vasculature was not altered after 24 hours of cold ischemia. No contractions were observed with S6c, a selective endothelin-B agonist, and this effect was not influenced by cold ischemia. The response mediated by the endothelin-A receptor was increased significantly, an effect that was not influenced by preservation with University of Wisconsin solution. This latter finding was the only significant alteration in the vascular function detected in the in vitro model after 24 hours of cold ischemia. With regard to the pharmacologic properties of endothelin-1, this mediated response could be implicated in the pathogenesis of vasospasm.     

Chronic effects of a nonpeptide corticotropin-releasing hormone type I receptor antagonist on pituitary-adrenal function, body weight, and metabolic regulation

CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis and modulator of autonomic nervous system activity, also participates in the regulation of appetite and energy expenditure. Antalarmin, a pyrrolopyrimidine compound, antagonizes CRH type 1 receptor-mediated effects of CRH, including pituitary ACTH release, stress behaviors, and acute inflammation. We administered antalarmin chronically to evaluate its effects on hypothalamic-pituitary-adrenal axis function and metabolic status. Adult male rats were treated twice daily with 20 mg/kg of i.p. antalarmin or placebo over 11 days. The animals were weighed; plasma ACTH, corticosterone, leptin, and blood glucose levels were determined; and morphometric analyses were performed to determine adrenal size and structure, including sizing, histochemistry, immunohistochemistry, and electron microscopy. Leptin messenger RNA expression in peripheral fat was analyzed by Northern blot. Antalarmin decreased plasma ACTH (mean +/- SD, 2.62 +/- 0.063 pg/ml) and corticosterone concentrations (10.21 +/- 1.80 microg/dl) compared with those in vehicle-treated rats [respectively, 5.3 +/- 2.0 (P < 0.05) and 57.02 +/- 8.86 (P < 0.01)]. Antalarmin had no significant effect on body weight, plasma leptin, or blood glucose concentrations or fat cell leptin messenger RNA levels. The width of the adrenal cortex of animals treated with antalarmin was reduced by 31% compared with that in controls without atrophy of the gland. On the ultrastructural level, adrenocortical cells were in a hypofunctional state characterized by reduced vascularization, increased content of lipid droplets, and tubulovesicular mitochondria with fewer inner membranes. The apoptotic rate was increased in the outer zona fasciculata of animals treated with the antagonist (26.6 +/- 3.58%) compared with that in placebo-treated controls (6.8 +/- 0.91%). We conclude that chronic administration of antalarmin does not affect body weight, carbohydrate metabolism, or leptin expression, whereas it reduces adrenocortical function mildly, without anatomical, clinical, or biochemical evidence of causing adrenal atrophy. These results are promising for future uses of such an antagonist in the clinic.     

The discovery and structure-activity relationships of nonpeptide, low molecular weight antagonists selective for the endothelin ET(B) receptor

The systematic modification of the ETA selective N-(5-isoxazolyl)benzene-sulfonamide endothelin antagonists to give ETB selective antagonists is reported. The reversal in selectivity was brought about by substitution of the 4-position with aryl and substituted aryl groups. Of all the aromatic substituents studied, the para-tolyl group gave rise to the most active and selective ETB antagonist. Larger substituents caused a decrease in both ETB activity and selectivity. A similar trend was observed by substitution at the 5-position of the N-(5-isoxazolyl)-2-thiophenesulfonamide ETA receptor antagonists. The para-tolyl group was again found to be optimal for the ETB activity and selectivity. The structural features that were found to be favorable for binding to the ETB receptor, that is, the presence of a linear, conjugated pi-system of definite shape and size, have been successfully incorporated into the design of ETB selective polycyclic aromatic sulfonamides antagonists.