Comparative pharmacokinetics of RAD001 (everolimus) in normal and tumor-bearing rodents.

PURPOSE: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice. METHODS: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity. RESULTS: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation. CONCLUSIONS: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.

First in vivo MRI assessment of a self-assembled metallostar compound endowed with a remarkable high field relaxivity.

{Fe[Gd(2)bpy(DTTA)(2)(H(2)O)(4)](3)}(4-) is a self-assembled, metallostar-structured potential MRI contrast agent, with six efficiently relaxing Gd(3+) centres confined into a small molecular space. Its proton relaxivity is particularly remarkable at very high magnetic fields (r(1) = 15.8 mM(-1) s(-1) at 200 MHz, 37 degrees C, in H(2)O). Here we report the first in vivo MRI feasibility study, complemented with dynamic gamma scintigraphic imaging and biodistribution experiments using the (153)Sm-enriched compound. Comparative MRI studies have been performed at 4.7 T in mice with the metallostar and the small molecular weight contrast agent gadolinium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate ([Gd(DOTA)(H(2)O)](-) = GdDOTA). The metallostar was well tolerated by the animals at the concentrations of 0.0500 (high dose) and 0.0125 (low dose) mmol Gd kg(-1) body weight; (BW). The signal enhancement in the inversion recovery fast low angle shot (IR FLASH) images after the high-dose metallostar injection was considerably higher than after GdDOTA injection (0.1 mmol Gd kg(-1) BW), despite the higher dose of the latter. The high-dose metallostar injection resulted in a greater drop in the spin-lattice relaxation time (T(1)), as calculated from the inversion recovery true fast imaging with steady-state precession (IR TrueFISP) data for various tissues, than the GdDOTA or the low dose metallostar injection. In summary, these studies have confirmed that the approximately four times higher relaxivity measured in vitro for the metallostar is retained under in vivo conditions. The pharmacokinetics of the metallostar was found to be similar to that of GdDOTA, involving fast renal clearance, a leakage to the extracellular space in the muscle tissue and no leakage to the brain. As expected on the basis of its moderate molecular weight, the metallostar does not function as a blood pool agent. The dynamic gamma scintigraphic studies performed in Wistar rats with the metallostar compound having (153)Sm enrichment also proved the renal elimination pathway. The biodistribution experiments are in full accordance with the MR and scintigraphic imaging. At 15 min post-injection the activity is primarily localized in the urine, while at 24 h post-injection almost all radioactivity is cleared from tissues and organs.

Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin.

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

From anatomy to the target: contributions of magnetic resonance imaging to preclinical pharmaceutical research.

In recent years, in vivo magnetic resonance (MR) methods have become established tools in the drug discovery and development process. In this article, the role of MR imaging (MRI) in the preclinical evaluation of drugs in animal models of diseases is illustrated on the basis of selected examples. The individual sections are devoted to applications of anatomic, physiologic, and "molecular" imaging providing, respectively, structural-morphological, functional, and target-specific information. The impact of these developments upon clinical drug evaluation is also briefly addressed. The main advantages of MRI are versatility, allowing a comprehensive characterization of a disease state and of the corresponding drug intervention; high spatial resolution; and noninvasiveness, enabling repeated measurements. Successful applications in drug discovery exploit one or several of these aspects. Additionally, MRI is contributing to strengthen the link between preclinical and clinical drug research.

Combined blockade of endothelin-1 and thromboxane A(2) receptors against postischaemic contractile dysfunction in rat hearts.

1. Endothelin-1 (ET-1) may play a role in myocardial ischaemia/reperfusion injury because both the release and vasoconstrictor effect of ET-1 are increased after ischaemia. Since the increased vasoconstrictor effect of ET-1 can be mediated by ET-1-induced release of thromboxane A(2) (TXA(2)), the aim of this study was to test whether combined blockade of ET and TXA(2) receptors protects the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion. 2. Bosentan (antagonist for ET(A) and ET(B) receptors, 1 microM based on concentration-response curves of ET-1), SQ 30,741 (antagonist of TXA(2) receptors, 0.1 microM), or the combination thereof was administered to isolated perfused rat hearts undergoing 15 min of global ischaemia and 60 min of reperfusion. 3. Neither bosentan or SQ 30,741 alone, nor the combination thereof, improved the incomplete postischaemic recovery of coronary flow, left ventricular developed pressure, phosphocreatine, or ATP. However, they attenuated ischaemia-induced acidosis but this did not translate into a measurable effect on haemodynamic or metabolic variables. 4. Thus, combined blockade of ET and TXA(2) receptors does not protect the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion in isolated perfused rat hearts. This finding suggests that neither ET-1 nor ET-1-induced release of TXA(2) play a major role in the postischaemic recovery of the cardiac contractile function and energy metabolism.

Plasminogen activation in experimental permanent focal cerebral ischemia.

BACKGROUND: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia. MATERIAL AND METHODS: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2). RESULTS: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005). DISCUSSION: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.

Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo.

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.

Transgenic activation of Ras in neurons promotes hypertrophy and protects from lesion-induced degeneration.

Ras is a universal eukaryotic intracellular protein integrating extracellular signals from multiple receptor types. To investigate its role in the adult central nervous system, constitutively activated V12-Ha-Ras was expressed selectively in neurons of transgenic mice via a synapsin promoter. Ras-transgene protein expression increased postnatally, reaching a four- to fivefold elevation at day 40 and persisting at this level, thereafter. Neuronal Ras was constitutively active and a corresponding activating phosphorylation of mitogen-activated kinase was observed, but there were no changes in the activity of phosphoinositide 3-kinase, the phosphorylation of its target kinase Akt/PKB, or expression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). Neuronal Ras activation did not alter the total number of neurons, but induced cell soma hypertrophy, which resulted in a 14.5% increase of total brain volume. Choline acetyltransferase and tyrosine hydroxylase activities were increased, as well as neuropeptide Y expression. Degeneration of motorneurons was completely prevented after facial nerve lesion in Ras-transgenic mice. Furthermore, neurotoxin-induced degeneration of dopaminergic substantia nigra neurons and their striatal projections was greatly attenuated. Thus, the Ras signaling pathway mimics neurotrophic effects and triggers neuroprotective mechanisms in adult mice. Neuronal Ras activation might become a tool to stabilize donor neurons for neural transplantation and to protect neuronal populations in neurodegenerative diseases.

Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice.

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.

Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

Bradykinin-dependent cardioprotective effects of losartan against ischemia and reperfusion in rat hearts.

It is unclear whether losartan, an angiotensin II type 1 (AT1) receptor antagonist, protects the heart against acute ischemia-reperfusion injury. Therefore we evaluated cardiac protection conferred by pre- and postischemic treatment as well as by exclusive postischemic treatment with losartan. Furthermore, we sought to determine both the extent of this protection and its dependence on bradykinin in comparison with quinaprilat, a cardioprotective angiotensin-converting enzyme inhibitor. Cardiac protection was assessed as recovery of coronary flow, left ventricular developed pressure, phosphocreatine, and adenosine triphosphate (ATP) in isolated perfused rat hearts after 15 min of global ischemia and 30 min of postischemic reperfusion. We found that, in hearts pre- and postischemically treated with losartan (1 microM) or quinaprilat (0.1 microM), these variables all recovered significantly better than those in untreated control hearts. In hearts that were only postischemically treated with losartan, these variables also recovered significantly better than those in control hearts. In contrast, in hearts treated with the combination of the bradykinin B2 receptor antagonist Hoe 140 with quinaprilat or losartan, the recovery of the variables no longer differed from that in control hearts. In conclusion, losartan protects the heart against acute ischemia-reperfusion injury. This protection can be achieved by pre- and postischemic treatment as well as by exclusive postischemic treatment with losartan. Furthermore, the extent of this protection is equivalent to that conferred by quinaprilat and, unexpectedly, dependent on bradykinin.

Time course of microglia activation and apoptosis in various brain regions after permanent focal cerebral ischemia in mice.

We investigated the temporal course of microglia activation in different brain regions after permanent middle cerebral artery (MCA) occlusion in mice and compared this microglia response with the appearance of apoptotic cells, Microglia activation and morphological changes of microglial cells were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse CR3 complement receptor. Cells showing morphological and biochemical features of apoptosis were identified using the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and light microscopy. As early as 30 min after onset of MCA occlusion activated microglia with hypertrophic cell bodies and stout processes were detected in the periphery of the ischemic lesion as identified by diffusion-weighted magnetic resonance imaging. A wider distribution and a progressive increase in the number of activated microglia was found with increasing time. Only few TUNEL-positive cells with apoptotic features were observed within the lesion area at 6 h after onset of cerebral ischemia. From 12 h after MCA occlusion onward a tremendous increase in the number of TUNEL-positive cells was found. Within the thalamus from 24 h onward microglia cells with few processes, irregular morphology and fragmented appearance were detected. Microglia activation in the thalamus progressed up to 4 weeks after MCA occlusion, but had declined after 90 days. Neuronal degeneration in the thalamus as determined by anti-neuronal nuclei immunohistochemistry progressed from 6 days after MCA occlusion onward. Only a few TUNEL-positive cells were found in the thalamus. In summary, microglia activation both in the primary cortical lesion area and in the secondarily affected thalamus preceded the manifestation of tissue injury. These observations encourage further studies on the role of microglia in focal cerebral ischemia.

Reperfusion after thrombolytic therapy of embolic stroke in the rat: magnetic resonance and biochemical imaging.

The effect of thrombolytic therapy was studied in rats submitted to thromboembolic stroke by intracarotid injection of autologous blood clots. Thrombolysis was initiated after 15 minutes with an intracarotid infusion of recombinant tissue-type activator (10 mg/kg body weight). Reperfusion was monitored for 3 hours using serial perfusion- and diffusion magnetic resonance imaging, and the outcome of treatment was quantified by pictorial measurements of ATP, tissue pH, and blood flow. In untreated animals, clot embolism resulted in an immediate decrease in blood flow and a sharp decrease in the apparent diffusion coefficient (ADC) that persisted throughout the observation period. Thrombolysis successfully recanalized the embolized middle cerebral artery origin and led to gradual improvement of blood flow and a slowly progressing reversal of ADC changes in the periphery of the ischemic territory, but only to transient and partial improvement in the center. Three hours after initiation of thrombolysis, the tissue volume with ADC values less than 80% of control was 39 +/- 22% as compared to 61 +/- 20% of ipsilateral hemisphere in untreated animals (means +/- SD, P = .03) and the volume of ATP-depleted brain tissue was 25 +/- 31% as compared to 46 +/- 29% in untreated animals. Recovery of ischemic brain injury after thromboembolism is incomplete even when therapy is started as early as 15 minutes after clot embolism. Possible explanations for our findings include downstream displacement of clot material, microembolism of the vascular periphery, and events associated with reperfusion injury.

Exploration of P-type Ca2+ channels as drug targets for the treatment of epilepsy or ischemic stroke.

We investigated the neuroprotective efficacy of the P-type Ca2+ channel antagonist daurisoline against electroshock-induced convulsions in rats and mice, hypoxic/hypoglycemic-induced damage in rat hippocampal slices and brain damage induced by occlusion of the middle cerebral artery (MCA) in rats. Daurisoline applied intravenously (i.v.) (bolus of 1-60 mg/kg) reduced the spontaneous activity of rat cerebellar Purkinje cells in a dose-dependent manner, a result demonstrating activity in the brain with systemic administration of the compound. While this effect reversed rapidly in about 10-20 min following bolus-application of the drug at doses of up to 30 mg/kg, a dose of 60 mg/kg consistently induced a depression of respiration followed by death of the animals. Daurisoline administered at 10-30 mg/kg did not prevent electroshock-induced convulsions in mice or rats, nor did it reduce the neuronal damage in hippocampal slices induced by a hypoxic/hypoglycemic insult in vitro by MCA occlusion in vivo. These observations do not support the hypothesis that P-type Ca2+ channels are promising drug targets for the acute treatment of epileptic convulsions and/or ischemic stroke.

Blood-brain barrier disturbances after rt-PA treatment of thromboembolic stroke in the rat.

We studied the effects of rt-PA (recombinant tissue type-plasminogen activator) treatment on the blood-brain barrier (BBB) after thromboembolic stroke in rat. New MRI methods of diffusion and perfusion imaging to observe the hemodynamic and biophysical effects of thrombolysis were combined with methods for assessment of BBB disturbances. In untreated animals clot embolism produced a rapid drop in MRI perfusion values and the ADC (apparent diffusion coefficient), with subsequent infarction. BBB disturbances, visualised as extravasation of serum proteins on cryostat sections, were manifest in nearly all animals in the borderzone of infarcts. In animals treated with rt-PA 15 min after clot embolism thrombolysis resulted in reperfusion of affected brain regions with subsequent improvement of ADC values. Final lesion size on ADC maps was reduced by 36% relative to untreated animals. However, BBB disturbances were not improved after treatment. To the contrary, rt-PA treated animals showed further regions with serum protein extravasation in the infarcted territories and in distant non-ischemic brain regions. MR imaging with the BBB tracer GdDTPA showed more pronounced and widespread contrast enhancement in the rt-PA treated than in the untreated group. Increased blood-brain barrier disturbances have to be taken into account even when thrombolytic therapy is started very early after the onset of stroke.

Reduction of excitotoxicity-induced brain damage by the competitive NMDA antagonist CGP 40116: a longitudinal study using diffusion-weighted imaging.

The cerebroprotective properties of the competitive N-methyl-D-aspartate (NMDA) antagonist CGP 40116 were evaluated in a rat model of excitotoxicity-induced brain damage using direct intrastriatal injection of quinolinic acid and subsequent (5 or 45 min later) i.p. administration of the drug. Diffusion-weighted magnetic resonance imaging (DWI) was used to follow the temporal lesion growth during the acute phase (4 h) and T2-weighted MRI (T2WI) to quantify vasogenic edema extent 2 days later. For control animals, we found a rapid increase in lesion volume during the first hour followed by a moderate growth over the following hours. The DWI-visible hyperintensity was partially reversible after treatment with CGP 40116. The onset of action of CGP 40116 was immediate. The final outcome (63% reduction of lesion volume within 2-4 h post-surgery) was independent of the time of drug administration. DWI data after 4 h correlated well with those obtained by T2WI 2 days later. DWI is a valuable method for early prediction of the outcome of therapeutic interventions of excitotoxic insults.

Endothelin-1-induced release of thromboxane A2 increases the vasoconstrictor effect of endothelin-1 in postischemic reperfused rat hearts.

BACKGROUND: The release and vasoconstrictor effect of endothelin-1 (ET-1) are increased after myocardial ischemia, suggesting a role for ET-1 in ischemia/reperfusion injury. However, the mechanisms of the increased vasoconstriction by ET-1 are unknown. The aim of this study was to test whether ET-1-induced release of thromboxane A2 (TXA2) contributes to the vasoconstrictor effect of ET-1 in nonischemic hearts and whether such release can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts. METHODS AND RESULTS: ET-1-induced release of TXA2 was assessed by measurement of the concentrations of its stable metabolite thromboxane B2 (TXB2) in the coronary effluent of nonischemic and reperfused isolated rat hearts before and after administration of 0.01 nmol ET-1 using an enzyme immunoassay. The contribution of ET-1-induced release of TXA2 to the vasoconstrictor effect of ET-1 was assessed by measurement of the effects of ET-1 with and without the cyclooxygenase inhibitor indomethacin or the TXA2/endoperoxide receptor antagonist SQ 30,741 using 31P magnetic resonance spectroscopy. In nonischemic hearts, ET-1 led to a small increase in TXB2 in the coronary effluent (3.9 +/- 1.5 pg/mL; n = 3), but neither indomethacin nor SQ 30,741 significantly diminished the vasoconstrictor effects of ET-1 (reduction of coronary flow, 4.0 +/- 0.4 and 4.5 +/- 0.3 mL/min, respectively, versus 4.9 +/- 0.5 mL/min for ET-1 alone; n = 8, 6, and 9, respectively). In postischemic reperfused hearts, however, ET-1 led to a greater increase in TXB2 (13.7 +/- 1.5 pg/mL; P < .05 versus nonischemic hearts; n = 3), and both indomethacin and SQ 30,741 diminished the vasoconstrictor effects of ET-1 (reduction of coronary flow, 2.6 +/- 0.3 and 2.2 +/- 0.3 mL/min, respectively, versus 4.0 +/- 0.1 mL/min for ET-1 alone; n = 8, 8, and 6, respectively; P < .05). Furthermore, indomethacin and SQ 30,741 prevented the detrimental effects of ET-1 on left ventricular developed pressure, intracellular pH, and phosphocreatine during reperfusion. CONCLUSIONS: ET-1-induced release of TXA2 does not significantly contribute to the vasoconstrictor effect of ET-1 in nonischemic hearts but can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts.

CGP 53153: a new potent inhibitor of 5alpha-reductase.

CGP 53153 (N-2-(cyano-2-propyl)-3-oxo-4-aza-5alpha-androst-1-ene-17beta-carb oxamide) is a steroidal inhibitor of 5alpha-reductase, the enzyme which effects the conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT). In vitro, CGP 53153 competitively inhibited rat microsomal 5alpha-reductase from prostate by 50% (IC50) at a concentration of 36nM compared to the reference compound finasteride which inhibited 5alpha-reductase with an IC50 of 11 nM in the same system. In vivo, inhibition of 5alpha-reductase activity was characterized in three different test systems. Inhibition of 5alpha-reductase activity was first assessed in a standard test designed to compare directly the potency of different 5alpha-reductase inhibitors. This test assesses potency through the inhibition of prostate growth in juvenile castrate male rats treated with a standard dose of T-propionate (1 mg/kg, s.c.) and a 5alpha-reductase inhibitor administered orally at various doses for 4 days. CGP 53153 and finasteride significantly reduced T-propionate-mediated prostate growth by about 25% (ED25) compared to T-propionate-treated controls at oral doses of 0.01 and 0.1 mg/kg, respectively. Second, the effects on prostate weight were studied in normal adult male rats treated orally once daily for 14 days with 1, 3 and 10 mg/kg CGP 53153 and with 10 mg/kg finasteride. CGP 53153 significantly reduced prostate weight at 3 and 10 mg/kg by 31% and 37%, respectively, compared to vehicle-treated controls, whereas the dose of 10 mg/kg finasteride did not significantly reduce prostate weight. Third, the effects on prostate volume were studied in normal 6-9-year-old male dogs treated orally once daily with 5 mg/kg CGP 53153 and with 5 mg/kg finasteride for 12 weeks. Prostate volume was monitored with magnetic resonance imaging every 2 weeks beginning 6 weeks before start of the treatment with 5alpha-reductase inhibitors and ending after a recovery period of 8 weeks after termination of treatment. Treatment for 12 weeks with both CGP 53153 and finasteride was equally effective in reducing prostate volume by more than 70% in individual dogs. Anti-androgenic potency of CGP 53153 and finasteride was assessed in juvenile castrate male rats treated with DHT-propionate (1 mg/kg, s.c.) and a 5alpha-reductase inhibitor (p.o.) for 4 days. Neither CGP 53153 nor finasteride given at a dose of 10 mg/kg had any significant effect on DHT-propionate-mediated prostate growth, whereas the reference anti-androgen flutamide given at a dose of 10 mg/kg reduced prostate weight to levels comparable to those seen in untreated castrate animals. For CGP 53153, the dose of 10 mg/kg is 1000-fold higher than the ED25 for 5alpha-reductase inhibition in vivo. In conclusion, both CGP 53153 and finasteride are potent inhibitors of the rat 5alpha-reductase enzyme system in vitro without showing any anti-androgenic effects in vivo. Both CGP 53153 and finasteride were equally potent in reducing prostate volume in aged male dogs, whereas in rats, CGP 53153 is up to 10 times more potent than finasteride in reducing prostate weight as shown in two different rat models.