Effect of N1-dansylspermine and Ro25,6981 on locomotor activity in naive mice and in the reserpinized mouse model of Parkinson's disease.

The effect of N1-dansylspermine, a polyamine analogue and competitive polyamine antagonist, and Ro25,6981, a noncompetitive polyamine antagonist with good affinity and selectivity for the GluN2B subunit, on locomotor activity in naive mice was investigated. Furthermore, the ability of the polyamine antagonists to reverse reserpine-induced hypokinesia was assessed, 24 h after injection of a catecholamine-depleting dose of reserpine (5 mg/kg, subcutaneous), to investigate the therapeutic potential of polyamine antagonists in Parkinson's disease. N1-dansylspermine significantly decreased locomotor activity in naive animals (P<0.001) but caused a mild, but significant increase in locomotor activity in reserpinized mice at the highest dose tested (P<0.05). Ro25,6981 significantly stimulated locomotor activity in naive animals (P<0.001) and had a slight significant stimulatory effect on reserpine-induced hypokinesia (P=0.05). N1-dansylspermine and Ro25,6981 had opposite effects on locomotor activity in naive mice, but both had a mild antiparkinsonian effect in the reserpine model. These findings suggest that antagonism of the polyamine binding site on the GluN2B subunit can reduce hypokinesia, albeit to a limited extent.

The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells.

Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB.

Endothelin-A receptor antagonists prevent amyloid-ß-induced increase in ETA receptor expression, oxidative stress, and cognitive impairment.

Alzheimer's disease is a neurodegenerative disorder associated with abnormal accumulation of amyloid-ß (Aß) which can release endothelin (ET). The present study was conducted to investigate the effect of ET antagonists on Aß-induced changes in ETA and ETB receptor expression, oxidative stress, and cognitive impairment. Male Sprague-Dawley rats were treated with Aß1-40 in the lateral cerebral ventricles and were administered vehicle or ET antagonists for 14 days. Aß treatment produced an increase in ETA receptor expression in the cerebral cortex, hippocampus, and brain stem by 72%, 85%, and 90%, respectively. No change in ETB receptor expression was observed. There was an increase in malondialdehyde (MDA) and decrease in reduced glutathione (GSH) and superoxide dismutase (SOD) levels in Aß-treated rats. In the Morris swim task, Aß treated rats showed a significant impairment in spatial memory. ET receptor antagonists, BQ123, BMS182874, and TAK-044, significantly decreased Aß-induced increase in ETA expression in the cortex, hippocampus, and brain stem. Rats treated with ET antagonists showed significant attenuation of Aß-induced changes in the brain MDA, GSH, and SOD levels. Rats treated with specific ETA receptor antagonists, BQ123 and BMS182874, significantly reduced the cognitive impairment induced by Aß. However, nonspecific ETA/ETB receptor antagonist TAK-044 did not show any improvement in the learning and memory parameter. This study demonstrates that ETA receptor antagonists are effective in preventing cognitive impairment, changes in ETA expression and oxidative stress induced by Aß. It is concluded that ETA receptor antagonists may be useful in improving cognitive impairment due to Alzheimer's disease.

Endothelin-1-induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms.

We have demonstrated recently [Callera, Touyz, Teixeira, Muscara, Carvalho, Fortes, Schiffrin and Tostes (2003) Hypertension 42, 811-817] that increased vascular oxidative stress in DOCA (deoxycorticosterone acetate)-salt rats is associated with activation of the ET (endothelin) system via ETA receptors. The exact source of ET-1-mediated oxidative stress remains unclear. The aim of the present study was to investigate whether ET-1 increases generation of ROS (reactive oxygen species) in DOCA-salt hypertension through NADPH-oxidase-dependent mechanisms. Xanthine oxidase, eNOS (endothelial nitric oxide synthase) and COX-2 (cyclo-oxygenase-2) were also examined as potential ET-1 sources of ROS as well as mitochondrial respiration. DOCA-salt and control UniNX (uninephrectomized) rats were treated with the ETA antagonist BMS182874 (40 mg.day(-1).kg(-1) of body weight) or vehicle. Plasma TBARS (thiobarbituric acid-reacting substances) were increased in DOCA-salt compared with UniNX rats. Activity of NADPH and xanthine oxidases in aorta, mesenteric arteries and heart was increased in DOCA-salt rats. BMS182874 decreased plasma TBARS levels without influencing NADPH and xanthine oxidase activities in DOCA-salt rats. Increased p22(phox) protein expression and increased p47(phox) membrane translocation in arteries from DOCA-salt by rats were not affected by BMS182874 treatment. Increased eNOS and COX-2 expression, also observed in aortas from DOCA-salt rats, was unaltered by BMS182874. Increased mitochondrial generation of ROS in DOCA-salt rats was normalized by BMS182874. ETA antagonism also increased the expression of mitochondrial MnSOD (manganese superoxide dismutase) in DOCA-salt rats. In conclusion, activation of NADPH oxidase does not seem to be the major source of oxidative stress induced by ET-1/ETA in DOCA-salt hypertension, which also appears to be independent of increased activation of xanthine oxidase or eNOS/COX-2 overexpression. Mitochondria may play a role in ET-1-driven oxidative stress, as evidenced by increased mitochondrial-derived ROS in this model of hypertension.

The pre-ischaemic neuroprotective effects of N1-dansyl-spermine in a transient focal cerebral ischaemia model in mice.

The pre-ischaemic neuroprotective potential of a novel polyamine/NMDA antagonist N1-dansyl-spermine (1-5 mg kg(-1)) was studied in a transient focal cerebral ischaemia model in mice in comparison to a reference compound, MK-801 (1 or 3 mg kg(-1)). The intraluminal suture transient middle cerebral artery occlusion (MCAO) model was used. N1-dansyl-spermine and MK-801 were administered (i.p.) 30 min prior to ischaemia. A range of histological and behavioural assessments was employed. N1-dansyl-spermine had a comparable effect to MK-801 at reducing the percentage hemisphere lesion volume (%HLV) at the doses tested. Furthermore, N1-dansyl-spermine reduced the ischaemic brain oedema, which MK-801 did not. N1-dansyl-spermine significantly reversed the decrease of locomotor activity (LMA) caused by the MCAO and showed a significant effect at improving the rotarod performance impaired by MCAO. In contrast, MK-801 had no beneficial effect on sensorimotor function and even worsened the LMA. These results clearly demonstrate the pre-ischaemic neuroprotective effect of N1-dansyl-spermine in a transient focal cerebral ischaemia model.

The pre-ischaemic neuroprotective effect of a novel polyamine antagonist, N1-dansyl-spermine in a permanent focal cerebral ischaemia model in mice.

The polyamine sites on the NMDA receptor complex offer a therapeutic target for focal ischaemia, potentially devoid of most side effects associated with NMDA antagonists. In this study, we investigated the effect of a novel polyamine antagonist, N(1)-dansyl-spermine (0.5-10 mg kg(-1)) in a permanent focal cerebral ischaemia model in mice, and compared its effect to that of MK-801 (0.3-3 mg kg(-1)) following administration 30 min prior to ischaemia. A battery of histological and behavioural tests was employed following permanent middle cerebral artery occlusion to assess any neuroprotective effect. Following middle cerebral artery occlusion, N(1)-dansyl-spermine (1-5 mg kg(-1)) and MK-801 (1 or 3 mg kg(-1)) caused a comparable and significant reduction in the percentage hemisphere lesion volume. Similarly, both drugs significantly reduced oedema and neurological deficit score to a similar extent. Locomotor activity in MCAO mice was not significantly improved by MK-801 or N(1)-dansyl-spermine, although N(1)-dansyl-spermine induced a trend towards significant improvement. Significant improvement in rotarod performance was observed at neuroprotective doses with both drugs. Upon comparison of the profile of effects, N(1)-dansyl-spermine at least matched the effectiveness of MK-801 as a neuroprotective agent in this model. In addition, in sham-operated control mice, N(1)-dansyl-spermine was well tolerated, in contrast to the pronounced adverse effects of MK-801 on locomotor activity and rotarod performance. In conclusion, this study has shown that N(1)-dansyl-spermine is as effective a neuroprotective drug as MK-801 in this model. Moreover, in contrast to MK-801, N(1)-dansyl-spermine could be a promising therapeutic candidate for stroke as it is well tolerated at neuroprotective doses in sham-operated animals.

The neuroprotective effects of N1-dansyl-spermine in the gerbil model of cerebral ischaemia.

The effects of N1-dansyl-spermine, a polyamine antagonist, and ifenprodil, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, were investigated in the gerbil model of global cerebral ischaemia. Transient forebrain ischaemia was induced by 5-min bilateral occlusion of the common carotid arteries. N1-dansyl-spermine (2, 5 and 10 mg/kg) and ifenprodil (30 mg/kg) were administered intraperitoneally 30 min after bilateral carotid artery occlusion. On histological examination, 4 days (96 h) after ischaemia, there was a significant decrease in neuronal density of the hippocampal CA1 subfield. This reduction in neuronal density was attenuated in those animals treated with the 5 or 10 mg/kg dose of N1-dansyl-spermine and those treated with 30 mg/kg ifenprodil. However, unlike ifenprodil, N1-dansyl-spermine failed to attenuate the ischaemia-induced increase in locomotor activity. This demonstrates that polyamines play a significant role in the neuronal damage produced after cerebral ischaemia, while casting doubt on the suggestion that increased locomotor activity correlates with CA1 pyramidal cell damage.

Role of the tissue factor pathway in synovial inflammation.

OBJECTIVE: Clinical and experimental evidence suggests that extravascular fibrin deposition in arthritic joints is prominent and deleterious. The aim of this study was to investigate the contributions of tissue factor (TF) and its inhibitor, TF pathway inhibitor (TFPI), in arthritis. METHODS: Synovial tissue specimens obtained from 10 patients with rheumatoid arthritis (RA) and 12 patients with osteoarthritis (OA) were scored histologically for inflammation and fibrin content. TF and TFPI levels were assayed at antigenic and functional levels. TF messenger RNA (mRNA) levels were determined using RNase protection assays. The effect of TF inhibition in murine antigen-induced arthritis (AIA) was assessed by administering systemically active site-blocked activated factor VIIa (FVIIai). RESULTS: Functional TF activity was significantly increased in synovial membranes from RA patients compared with those from OA patients. In contrast, no difference in TF mRNA and TF antigenic levels was observed between these 2 groups. This discrepancy can be accounted for by TFPI, because we observed a negative correlation between TF activity and TFPI activity. There was a significant difference between the RA and OA groups in terms of synovial inflammation, with more inflammation observed in the RA group. Most importantly, TF activity was associated with fibrin (P = 0.024) and with histologic inflammation (P = 0.03) scores. In AIA, inhibition of TF-induced coagulation by FVIIai led, on day 9 of arthritis, to decreased synovial thickness and decreased articular cartilage damage, although only the latter difference between controls and treated mice reached significance (P < 0.04). Finally, in FVIIai-treated mice, there was a strong negative association between the prothrombin time and intraarticular fibrin deposition. CONCLUSION: Our results show that TF expression in arthritic synovial tissue favors extravascular coagulation and may play a role in inflammation in RA. In this context, TF inhibitors may be of therapeutic value.

Elevated mean arterial pressure in the ovariectomized rat was normalized by ET(A) receptor antagonist therapy: absence of cardiac hypertrophy and fibrosis.

1. The influence of menopause on ventricular function and remodelling remains undefined. The following study examined the effect of ovariectomy on ventricular contractility, cardiac hypertrophy and extracellular matrix protein expression. 2. Elevated circulating levels of the vasoconstrictor endothelin-1 have been reported in post-menopausal women. Moreover, endothelin-1 has been shown to influence blood pressure, ventricular function and cardiac remodelling. In this regard, the potential pathophysiological role of endothelin-1 in the ovariectomized rat was assessed via the administration of the selective endothelin(A) receptor (ET(A)) antagonist BMS-182874. 3. In 3 and 6 week ovariectomized female Sprague - Dawley rats, uterus atrophy was associated with a significant increase in mean arterial pressure, and left ventricular systolic pressure, as compared to sham. By contrast, right ventricular contractile indices were normal in the ovariectomized rat. Despite increased systolic load, left ventricular hypertrophy was not evident, prepro-atrial natriuretic peptide (prepro-ANP) mRNA levels and collagen protein content were similar to sham. 4. The treatment of ovariectomized rats with BMS-182874 (60 mg kg(-1) per day) did not reverse uterus atrophy. However, BMS-182874 normalized mean arterial pressure, and left ventricular systolic pressure in the ovariectomized rat. 5. Thus, despite elevated blood pressure, ovariectomized rats were not associated with either cardiac hypertrophy or fibrosis. Lastly, endothelin-1, acting via the stimulation of the ET(A) receptor represents an integral mechanism implicated in the increase of mean arterial pressure following ovariectomy.

ET(A) receptor antagonist prevents blood pressure elevation and vascular remodeling in aldosterone-infused rats.

Increased endothelin-1 may be associated with elevation of blood pressure (BP) and promotion of vascular hypertrophy, especially in salt-sensitive hypertension. Mineralocorticoid hypertension has been associated with activation of the endothelin system. We evaluated whether in aldosterone-infused rats the selective endothelin type A receptor-antagonist BMS 182874 prevents BP elevation and vascular hypertrophy. Rats were infused with aldosterone (0.75 microg/h) subcutaneously via a mini-osmotic pump and were offered 1% NaCl in the drinking water+/-BMS 182874 (40 mg/kg in food) for 6 weeks. Systolic BP was monitored by the tail-cuff method, and vascular changes of mesenteric arteries were evaluated using a pressurized myograph. Aldosterone-infusion significantly increased BP to 151+/-7 mm Hg compared with controls (108+/-4 mm Hg, P<0.01). BMS 182874 normalized BP (117+/-4 mm Hg). Media cross-sectional area of aorta was significantly increased by aldosterone infusion (P<0.05), and BMS treatment normalized it (P<0.001). Aldosterone infusion increased media width and media-to-lumen ratio of mesenteric resistance arteries (17.6+/-0.4 microm and 7.5+/-0.4%) compared with controls (14.2+/-0.5 microm, P<0.01, and 5.9+/-0.1%, P<0.05). BMS 182874 normalized media and media-to-lumen ratio (15.1+/-0.6 microm and 5.7+/-0.1%, both P<0.01). In conclusion, the endothelin type A receptor antagonist attenuated BP elevation and prevented vascular remodeling or hypertrophy of aorta and mesenteric resistance arteries in aldosterone-infused rats. These results suggest a role for endothelin-1 in BP elevation and structural alterations of large and small vessels in aldosterone and salt-induced hypertension.

Endothelin-A-receptor blockade improves renal function and doubles the lifespan of stroke-prone spontaneously hypertensive rats.

This study assessed whether the orally active endothelin-A- (ETA) receptor antagonist, BMS 182874 (BMS), affects systolic blood pressure (SBP), renal function and survival in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSPs). Eight-week-old male and female SHRSPs were given a high-salt diet (4% NaCl) (n = 6/group). The treatment groups (n = 6/group), also on a high-salt diet, were administered BMS (40 mg/kg/day) which was mixed in the food. In untreated SHRSPs, mean survival was 136 days (range 96-194 days) in males, and 98 days (range 74-112 days) in females. Treatment with BMS increased survival to 223 days in males (range 129-280 days, p <0.01 vs controls) and 156 days (range 124-180, p < 0.05 vs controls) in females. SBP increased to approximately 240 mmHg in all groups. BMS had no effect on SBP in females and a small, but significant (p < 0.05), SBP-lowering effect in males. In control rats, severe renal disease was evident at 16 weeks (proteinuria, 161 +/- 1.3 mg/day). In the treated group, development of proteinuria was significantly delayed (13 +/- 0.5 mg/day at 16 weeks). In conclusion ETA-receptor antagonism prolongs survival and improves renal status, but has little effect on the progression of hypertension. These data suggest that interruption of the endothelin (ET) system by blockade of ETA-receptors may achieve protection from target-organ damage despite lack of a reduction in blood pressure.

Blockade of angiotensin II type 1 receptor and not of endothelin receptor prevents hypertension and cardiovascular disease in transgenic (mREN2)27 rats via adrenocortical steroid-independent mechanisms.

We investigated the role of angiotensin II (Ang II) and endothelin-1 (ET-1) in transgenic (mREN2)27 rats, a model of the monogenic renin-dependent form of severe hypertension and cardiovascular disease. Four-week-old heterozygous male transgenic (mREN2)27 rats (n=24) were matched according to body weight (BW) and blood pressure (BP) and randomly allocated to receive a placebo (group P), the mixed endothelin type A and B receptor antagonist bosentan (100 mg/kg BW PO, group B), the Ang II type 1-specific receptor antagonist irbesartan (50 mg/kg BW PO, group I), or the endothelin type A-selective antagonist BMS-182874 (52 mg/kg BW PO, group BMS). After 4 weeks of treatment, during which BW and BP were measured weekly, animals were euthanized, and the heart, left ventricle, right ventricle, adrenal gland, brain, and kidney were weighed. The plasma levels of adrenocortical steroids were measured by high-performance liquid chromatography. The tension responses of ET-free segments of the thoracic aorta to 5 x 10(-6) mmol/L phenylephrine, 60 mmol/L KCl, and cumulative doses of ET-1 were assessed. The density of ET-1 receptor subtypes in the aorta and vascular structural changes in the mesenteric arterioles (100 to 200 microm ID) were also measured with autoradiography and myography, respectively. Compared with all other groups, group I rats showed significantly (P<0.001) lower systolic BP (group I, 161+/-8 mm Hg; group P, 269+/-23 mm Hg; group B, 275+/-17 mm Hg; and group BMS, 254+/-21 mm Hg), left ventricular weight (2.28+/-0.15 versus 3. 71+/-0.26, 3.38+/-0.27, and 3.96+/-0.51 mg/g BW, respectively), tension responses to vasoconstrictors, and normalized media thickness of the mesenteric arterioles (22.3+/-0.6 versus 25.3+/-0.5, 25.5+/-0.7, and 24.1+/-1.5 microm, respectively). Compared with levels in group P (78+/-25 pmol/mL), plasma aldosterone levels were significantly decreased in group B (51+/-11 pmol/mL) and group I (40+/-16 pmol/mL). Thus, endogenous ET-1 and Ang II contribute to the regulation of aldosterone, but only Ang II is crucial for the development of hypertension and related target organ damage via the Ang II type 1 receptor. Endogenous Ang II does not appear to enhance cardiovascular production of ET-1 in this model of hypertension within the time span of our experiment.

The ETA receptor antagonist, BMS-182874, reduces acute hypoxic pulmonary hypertension in pigs in vivo.

OBJECTIVE: Elevated levels of the potent vasoactive peptide endothelin (ET), have been found in pathophysiological conditions associated with pulmonary hypertension. In this study, we have investigated the effects of the ETA receptor antagonist, BMS-182874, on hypoxic pulmonary hypertension in pigs. METHODS: Pigs were subjected to acute, intermittent 15-min periods of hypoxia (FiO2 0.1). Following a first hypoxia establishing hypoxic baseline values, vehicle or BMS-182874 (10 or 30 mg/kg) was administered i.v. before a second hypoxic period. In separate groups of animals, the effects of the nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NNA) in combination with BMS-182874 (10 mg) during repeated hypoxia were investigated. The ET-1-blocking properties of BMS-182874 were studied in vivo by infusion of ET-1 during normoxia and in vitro using isolated porcine pulmonary arteries. RESULTS: The hypoxia-evoked increase in mean pulmonary artery pressure was reduced by administration of BMS-182874 (10 mg/kg i.v.; from 42 +/- 8 to 34 +/- 4 mmHg, P < 0.05 and 30 mg/kg i.v.; from 38 +/- 4 to 30 +/- 5 mmHg, P < 0.05). In addition, BMS-182874 at 30 mg/kg reduced the pulmonary vascular resistance during hypoxia (from 7.4 +/- 1.5 to 5.3 +/- 1.1 mmHg.min.l-1 P < 0.05). The hemodynamic response to repeated hypoxia was reproducible in control animals and unaffected by the cyclo-oxygenase inhibitor diclophenac (3 mg/kg). Infusion of L-NNA alone resulted in an augmented pulmonary vasoconstriction during hypoxia; pulmonary arterial pressure from 35 +/- 6 to 43 +/- 9 mmHg; P < 0.05 and vascular resistance from 7.2 +/- 1.1 to 9.9 +/- 1.8 mmHg.min.l-1; P < 0.05. L-NNA in combination with BMS-182874 (10 mg/kg) resulted in a hypoxic pulmonary vasoconstriction of similar magnitude as hypoxic baseline. In addition, BMS-182874 reduced the hemodynamic response to ET-1 in normoxic pigs and competitively antagonized the vasoconstrictor effect of ET-1 in isolated porcine pulmonary arteries. CONCLUSIONS: The non-peptide, selective ETA receptor antagonist, BMS-182874, reduces hypoxic pulmonary vasoconstriction in pigs. The reduction in pulmonary vascular response to hypoxia following BMS-182874 is at least partly independent of nitric oxide.

Subcutaneous injection of an analog of neuropeptide FF prevents naloxone-precipitated morphine abstinence syndrome.

There is evidence that neuropeptide FF (NPFF) has antiopiate activity and may play a role in opiate dependence and subsequent abstinence syndrome. A fragment of NPFF was modified at the C-terminal in an effort to convert it to an NPFF antagonist. It was also dansylated at the N-terminal in an effort to render it more lipophilic and increase its penetration of the blood-brain barrier. Third ventricle administration of the resulting compound, dansyl-PQRamide (0.75 microgram and 1 microgram), dose-dependently antagonized the quasi-morphine abstinence activity of NPFF (10 micrograms) in opiate-naive rats. Subcutaneous injection of dansyl-PQRamide (13 mg/kg) in chronically morphine-infused rats attenuated opiate dependence as indicated by prevention of naloxone-precipitated abstinence syndrome. Dansyl-PQRamide displaced radiolabelled ligand from NPFF receptors in a concentration-dependent manner with a Ki of 13 microM, and had a half-life over 300 times longer than NPFF under aminopeptidase digestion.

A search for new anti-herpes virus compounds.

This study reports on the synthesis and the activity of a series of new compounds of nucleoside-type structure. They are characterized for being differently substituted in the aromatic ring of the base (deazoadenosine derivatives) or by bearing a dansyl group in the sugar moiety (dansylthymidine). One molecule belonging to this latter class of compounds (the 3'-0-dansylthymidine) is showing an anti-herpesvirus potential while being active in inhibiting the virus-encoded enzyme thymidine kinase. This finding may represent an important step for the synthesis of new enzyme inhibitors and it is discussed in terms of future developments of more active congeners.