The continuing evolution of torsades de pointes liability testing methods: Is there an end in sight?

Drug-induced torsades de pointes (TdP) is a syndrome that includes a potentially lethal cardiac arrhythmia. It has been identified as a possible adverse drug reaction (ADR) for drugs which affect the repolarization processes of the heart. In order to predict the potential for TdP liability, regulatory guidelines have been developed which require that new drugs be safety screened. Unfortunately, however, despite this requirement there are no validated preclinical models with TdP incidence as a hard endpoint. Therefore, surrogate biomarkers are used. The most common and eliciting the most discussion/controversy among cardiovascular scientists is the duration of the QT interval of the ECG. Since no single model is available to wholly assess drug-induced TdP liability, safety pharmacologists employ a battery of complementary preclinical models in order to develop an integrated risk assessment (IRA). Ideally, the IRA should be comprised of the results from the effects of the new chemical entity (NCE) on the human ether-a-go-go related (hERG) gene assay (actually a screen for block of the hERG gene product, the inward rectifying K current, IKr) and ECG effects in the conscious canine. However, since neither model is ideal the findings are generally supplemented by conduct of several additional experimental in vitro assays, namely the rabbit left ventricular wedge preparation, Langendorff isolated rabbit heart or isolated canine Purkinje fibre; nevertheless, as with many preclinical models, there is only limited validation and a resultant lack of general acceptance. Institution of regulatory guidance documents such as ICH S7A and S7B in conjunction with heightened awareness of the electrophysiological mechanisms that may be responsible for the development of TdP has led to a sharp fall in proarrhythmic compounds securing licensing, but at what costs? Supplementary experimental assays have furthered our understanding of drug-induced torsadogenesis, and it is now recognized that TdP is a multicausal event. This means that a perceived “positive” torsadogenic risk using one of the aforementioned models does not necessarily guarantee proarrhythmia. There has been an overall fall in the total number of NCEs pursued through development due to strict regulatory guidelines. Here we suggest that regulatory barriers can be alleviated by improving the integrated risk approach. But this requires better validation and deployment of existing preclinical models together with the invention of more precise and accurate models.     

Comparison of post-junctional α-adrenoceptors in iris dilator muscle of humans, and albino and pigmented rabbits

The relative potency of -adrenoceptor agonists and the dissociation constants of competitive antagonists were studied to characterize the post junctional -adrenoceptor of the human iris dilator muscle. The data obtained from human iris dilator tissue was compared to that from rabbit. The iris dilator muscle was mounted in an organ bath and tension changes were recorded. (–)-Norepinephrine, (–)-phenylephrine (PE), oxymetazoline and p-aminoclonidine caused contractile responses in albino rabbit, pigmented rabbit and human iris dilator muscle in a concentration-dependent manner. The imidazoline molecules were partial agonists. In rabbit iris dilator, desensitization occurred to repeated oxymetazoline application at an interval of 1 h but recovery to the agonist activity was complete in about 3 h. Exposure to cocaine (10 mol/l), hydrocortisone (100 mol/l) and U-0521, a catechol-O-methyltransferase inhibitor (100 pmol/l), significantly potentiated the response to norepinephrine by 92-, 32- and 7 fold in iris dilator tissue of albino rabbit, pigmented rabbit and human, respectively. After block of uptake₁ and uptake₂, the EC₅₀ values of norepinephrine in the albino rabbit, pigmented rabbit and human iris dilator did not differ and ranged from 99 to 195 nmol/l. Small but significant potentiation by uptake blockers was also observed in the responses to PE in the albino rabbit or pigmented rabbit iris dilator. The average maximum tension induced by 100 mol/l PE was 96 ± 11 mg (n = 10), 197 ± 11 mg (n = 11), 45 ± 5 mg (n = 27) in albino rabbit, pigmented rabbit and human iris dilator, respectively.In human iris dilator, the responses to PE were competitively antagonized by prazosin, 5-methylurapidil and phentolamine with apparent pKB values of 7.3, 6.6 and 7.5, respectively. The pK _B values of the prazosin-PE interaction in iris dilator of albino and pigmented rabbit were 8.6 and 6.4, respectively.These results suggest that the post-junctional -adrenoceptors in iris dilator may be similar to that in pigmented rabbit iris. The -adrenoceptor of the human or pigmented rabbit iris dilator may be characterized as _1L-adrenoceptor subtype. The -adrenoceptor of albino rabbit iris dilator appears to be a high affinity subtype.Furthermore, albino rabbit may not be the best strain for the drug research which is relevant to human ocular therapeutics.     

Expression of distinct α1-adrenoceptor phenotypes in the iris of pigmented and albino rabbits

Background and purpose:

The expression of multiple pharmacological phenotypes including α_1L-adrenoceptor has recently been reported for α₁-adrenoceptors. The purpose of the present study was to identify α₁-adrenoceptor phenotypes in the irises of pigmented and albino rabbits.

Experimental approach:

Radioligand binding and functional bioassay experiments were performed in segments or strips of iris of pigmented and albino rabbits, and their pharmacological profiles were compared.

Key results:

[³H]-silodosin at subnanomolar concentrations bound to intact segments of iris of pigmented and albino rabbits at similar densities (approximately 240 fmol·mg⁻¹ protein). The binding sites in the iris of a pigmented rabbit were composed of a single component showing extremely low affinities for prazosin, hydrochloride [N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053)] and 5-methylurapidil, while two components with high and low affinities for prazosin, RS-17053 and 5-methylurapidil were identified in irises from albino rabbits. In contrast, specific binding sites for [³H]-prazosin were not clearly detected because a high proportion of non-specific binding and/or low affinity for prazosin occurred. Contractile responses of iris dilator muscle to noradrenaline were antagonized by the above ligands, and their antagonist affinities were consistent with the binding estimates at low-affinity sites identified in both strains of rabbits.

Conclusions and implications:

A typical α_1L phenotype with extremely low affinity for prazosin is exclusively expressed in the iris of pigmented rabbits, while two distinct phenotypes (α_1A and α_1L) with high and moderate affinities for prazosin are co-expressed in the iris of albino rabbits. This suggests that a significant difference in the expression of phenotypes of the α₁-adrenoceptor occurs in the irises between the two strains of rabbits.     

The selectivity of ��-adrenoceptor agonists at human ��1-, ��2- and ��3-adrenoceptors

Background and purpose:

There are two important properties of receptor–ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor–ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 β-adrenoceptor agonists at the three human β-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.

Experimental approach:

Stable clonal CHO-K1 cell lines, transfected with either the human β₁, β₂ or β₃-adrenoceptor, were used, and whole-cell [³H]-CGP 12177 radioligand binding and [³H]-cAMP accumulation were measured.

Key results:

Several agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the β₂-adrenoceptor over the β₁ or β₃), while others (e.g. isoprenaline) had little affinity–selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for β₁; clenbuterol, AZ 40140d, salbutamol for β₂) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the β₁- and β₃-adrenoceptors.

Conclusions and implications:

There are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.This article is commented on by Kenakin, pp. 1045–1047 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00764.x     

The Role of IL-1�� in Nicotine-Induced Immunosuppression and Neuroimmune Communication

Although a number of inflammatory cytokines are increased during sepsis, the clinical trials aimed at down-regulating these mediators have not improved the outcome. These paradoxical results are attributed to loss of the “tolerance” phase that normally follows the proinflammatory response. Chronic nicotine (NT) suppresses both adaptive and innate immune responses, and the effects are partly mediated by the nicotinic acetylcholine receptors in the brain; however, the mechanism of neuroimmune communication is not clear. Here, we present evidence that, in rats and mice, NT initially increases IL-1β in the brain, but the expression is downregulated within 1–2 week of chronic exposure, and the animals become resistant to proinflammatory/pyrogenic stimuli. To examine the relationship between NT, IL-1β, and immunosuppression, we hypothesized that NT induces IL-1β in the brain, and its constant presence produces immunological “tolerance”. Indeed, unlike wild-type C57BL/6 mice, chronic NT failed to induce immunosuppression or downregulation of IL-1β expression in IL-1β-receptor knockout mice. Moreover, while acute intracerebroventricular administration of IL-1β in Lewis (LEW) rats activated Fyn and protein tyrosine kinase activities in the spleen, chronic administration of low levels of IL-1β progressively diminished the pyrogenic and T cell proliferative responses of treated animals. Thus, IL-1β may play a critical role in the perception of inflammation by the CNS and the induction of an immunologic “tolerant” state. Moreover, the immunosuppressive effects of NT might be at least partly mediated through its effects on the brain IL-1β. This represents a novel mechanism for neuroimmune communication.     

Low Doses of 17��-Estradiol and 17��-Estradiol Facilitate, Whereas Higher Doses of Estrone and 17��- and 17��-Estradiol Impair, Contextual Fear Conditioning in Adult Female Rats

Estrogens are known to exert significant structural and functional effects in the hippocampus of adult rodents. In particular, 17β-estradiol can improve, impair, or have no effect on hippocampus-dependent learning and memory depending on dose and time of administration. The effects of other forms of estrogen, such as estrone and 17α-estradiol, on hippocampus-dependent learning have not been as thoroughly investigated. Therefore, the purpose of this study was to investigate the effects of 17β-estradiol, estrone, and 17α-estradiol at three different doses on two different tasks: hippocampus-dependent contextual fear conditioning and hippocampus-independent cued fear conditioning. Adult ovariectomized female rats were injected with one of the estrogens at one of the three doses 30 mins before conditioning to assess the rapid effects of these estrogens on acquisition. Twenty-four hours later memory for the context was examined and 1 h later memory for the cue (tone) was assessed. Levels of synaptophysin were examined in the dorsal hippocampus of rats to identify a potential synaptic correlate of hormonal effects on contextual fear conditioning. Low 17β-estradiol and 17α-estradiol enhanced, whereas high 17β-estradiol and 17α-estradiol impaired, contextual fear conditioning. Only the middle dose of estrone severely impaired contextual fear conditioning. Estrogens did not alter performance in the hippocampus-independent cued task. Synaptophysin expression was increased by estrone (at a middle and high dose) and 17β-estradiol (at a middle dose) in the CA3 region of the hippocampus and was not correlated with cognition. The results of this study indicate that estradiol can positively or negatively influence hippocampus-dependent learning and memory, whereas estrone impairs hippocampus-dependent learning and memory in a dose-dependent manner. These results have important therapeutic implications, as estrone, a main component of a widely used hormone replacement therapy, was shown to have either a negative effect or no effect on learning and memory. It may be possible to use 17α-estradiol and lower doses of estrogens as potential alternatives in hormone replacement therapies.     

Functional change of hypothalamus-pituitary- adrenocortical axis in cerebral hemorrhage in rabbits and patients

To investigate the correlation and its clinical significance between the functional change of hypothalamus-pituitary-adrenocortical axis (HPA) and cerebral hemorrhage by radioimmunoassay (RIA) to detect the serious content of cortisol (Cor) and adrenocorticotropic hormorne (ACTH), we observed the functional change of the HPA dynamically in rabbit and patient of cerebeal hemorrhage.The result showed that the serous     

Methadone maintenance,QTc and torsade de pointes: Who needs an electrocardiogram and what is the prevalence of QTc prolongation?

Introduction and Aims. High‐dose methadone has been associated with rate‐corrected QT (QTc) prolongation and ‘torsade de pointes’. The Medicines and Healthcare products Regulatory Agency (MHRA) advise electrocardiograms (ECGs) for patients on methadone with heart/liver disease, electrolyte abnormalities, concomitant QT prolonging medications/CYP3A4 inhibitors or prescribed methadone >100 mg daily. The percentage of patients fulfilling MHRA criteria for ECG monitoring and prevalence of QT prolongation in patients who had an ECG was assessed. Design and Methods. A cross‐sectional study of opioid‐dependent patients prescribed opioid maintenance that completed a screening questionnaire prior to referral for an ECG. MHRA criteria were assessed in the referred group. The automated QTc score was analysed with methadone dose, substance use and QT risk factors. Results. Of 155 patients screened; 57.4% (n = 89) fulfilled MHRA criteria for ECG monitoring (75.5% (n = 117) if cocaine included as QT prolonging drug). Eighty‐three (53.5%) had ECGs; 19.3% (n = 16) prescribed QT prolonging medication, 22.9% (n = 19) prescribed >100 mg methadone and 47% (n = 39) used cocaine. Mean QTc interval was 429.0 ms (SD 26.4, 351–489). Eighteen per cent exceeded QTc gender‐specific thresholds (≥450 ms men and≥470 ms women). Linear regression found total daily methadone dose (β = 0.318, P = 0.003) and stimulant use (β = ?0.213, P = 0.043) predictive of QTc length. Discussion. Over half to three‐quarters of methadone maintenance patients fulfilled MHRA criteria for ECG monitoring, which has costly implications. QTc prolongation prevalence was 18.1% with no ‘clinically significant’ QTc prolongation >500 ms or torsade de pointes known to be present. Methadone dose and stimulant use were associated with longer QTc intervals. Further research on the clinical management of QTc prolongation with methadone is required.[Mayet S, Gossop M, Lintzeris N, Markides V, Strang J. Methadone maintenance, QTc and torsade de pointes: Who needs an electrocardiogram and what is the prevalence of QTc prolongation? Drug Alcohol Rev 2011;30:388–396]     

Effects of ρ-Da1a a peptidic α1A-adrenoceptor antagonist in human isolated prostatic adenoma and anaesthetized rats

Background and Purpose

ρ-Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α_1A-adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ-Da1a on prostatic function, both in vivo and in vitro.

Experimental Approach

ρ-Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α_1A-adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ-Da1a and tamsulosin on phenylephrine (PHE)-induced increases in intra-urethral (IUP) and arterial pressures (AP) in anaesthetized rats, following i.v. or p.o. administration.

Key Results

On COS cells expressing human α_1A-adrenoceptors and on human prostatic strips, ρ-Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ-Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pK_B values for tamsulosin and ρ-Da1a for this effect were similar. With regards to AP, ρ-Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 μg·kg⁻¹), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 μg·kg⁻¹.

Conclusions and Implications

ρ-Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ-Da1a is more uroselective than tamsulosin. ρ-Da1a is the most selective peptidic antagonist for α_1A-adenoceptors identified to date and could be a new treatment for various urological diseases.     

Activation of PPAR�� Attenuates IFN�� and IL-1��-induced Cell Proliferation in Astrocytes: Involvement of IL-6 Independent Pathway

The present study demonstrates the effect of fibrates, agonists of PPARα on cytokines-induced proliferation in primary cultured astrocytes. Alone or combination treatment with cytokines, such as IL-1β (10 ng/ml), IFNγ (10 ng/ml), and TNF-α (10 ng/ml) cause a significant increase of cell proliferation in a time-dependent manner. Treatment of astrocytes with bezafibrate and fenofibrate (0, 5, and 10 µM) reduced the IFNγ and IL-1β-induced cell proliferation in a dose-dependent manner. To address the involvement of IL-6 on the IFNγ and IL-1β-induced cell proliferation, released IL-6 level was measured. IFNγ and IL-1β cause an increase of released IL-6 protein level in a time-dependent manner. Furthermore, pretreatment with IL-6 antibody (0, 0.1, 1, 2.5, and 5 ng/ml) dose-dependently inhibited the IFNγ and IL-1β-induced cell proliferation. However, bezafibrate and fenofibrate did not affect increased mRNA and protein levels of IL-6 in IFNγ and IL-1β-stimulated astrocytes. Taken together, these results clearly suggest that activation of PPARα attenuates the IFNγ and IL-1β-induced cell proliferation through IL-6 independent pathway.     

Chemical Biology, Molecular Mechanism and Clinical Perspective of ��-Secretase Modulators in Alzheimer��s Disease

Comprehensive evidence supports that oligomerization and accumulation of amyloidogenic Aβ42 peptides in brain is crucial in the pathogenesis of both familial and sporadic forms of Alzheimer''s disease. Imaging studies indicate that the buildup of Aβ begins many years before the onset of clinical symptoms, and that subsequent neurodegeneration and cognitive decline may proceed independently of Aβ. This implies the necessity for early intervention in cognitively normal individuals with therapeutic strategies that prioritize safety. The aspartyl protease γ-secretase catalyses the last step in the cellular generation of Aβ42 peptides, and is a principal target for anti-amyloidogenic intervention strategies. Due to the essential role of γ-secretase in the NOTCH signaling pathway, overt mechanism-based toxicity has been observed with the first generation of γ-secretase inhibitors, and safety of this approach has been questioned. However, two new classes of small molecules, γ-secretase modulators (GSMs) and NOTCH-sparing γ-secretase inhibitors, have revitalized γ-secretase as a drug target in AD. GSMs are small molecules that cause a product shift from Aβ42 towards shorter and less toxic Ab peptides. Importantly, GSMs spare other physiologically important substrates of the γ-secretase complex like NOTCH. Recently, GSMs with nanomolar potency and favorable in vivo properties have been described. In this review, we summarize the knowledge about the unusual proteolytic activity of γ-secretase, and the chemical biology, molecular mechanisms and clinical perspective of compounds that target the γ-secretase complex, with a particular focus on GSMs.     

��2-adrenoceptor agonist clenbuterol reduces infarct size and myocardial apoptosis after myocardial ischaemia/reperfusion in anaesthetized rats

Background and purpose:

Considerable evidence indicates that the β₂-adrenoceptor agonist clenbuterol decreases apoptosis in a rodent model of ischaemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischaemia/reperfusion (I/R) in anaesthetized rats.

Experimental approach:

Rats were randomly assigned to the following groups: (i) sham (ii) I/R (iii) clenbuterol + I/R (iv) ICI 118551 + clenbuterol + I/R (v) metoprolol + clenbuterol + I/R (vi) metoprolol + I/R (vii) pertussis toxin + clenbuterol + I/R. Under anaesthesia, left anterior descending coronary artery was occluded for 30 min followed by reperfusion for 2 h.

Key results:

Compared with the control I/R group,the clenbuterol (0.5 mg·kg⁻¹, i.p.) group had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity, increased superoxide dismutase activity, and decreased malondialdehyde (MDA) level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as indicated by the reduction of terminal deoxynucleotidyltransferase end labelling-positive staining, Bax/Bcl-2 mRNA and caspase-3 protein expression. The G_i-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551(a selective β₂-adrenoceptor antagonist) inhibited the effects of clenbuterol mentioned above. The β₁-adrenoceptor agonist metoprolol had similar effects to clenbuterol but failed to reduce MDA and improve SERCA activity. When administered together, metoprolol and clenbuterol did not induce synergistic effects.

Conclusions and implications:

Clenbuterol pretreatment provides significant cardioprotection against ischaemia/reperfusion injury and this is mediated by the β₂-adrenoceptor–G_i-protein signalling. A combination of the β₂-adrenoceptor agonist clenbuterol and the β₁-antagonist metoprolol did not lead to a synergistic anti-apoptotic effect.     

Minimal α1- and α2-adrenoceptor-mediated coronary vasoconstriction in the anaesthetized swine

Summary -Adrenoceptor-mediated coronary vasoconstriction contributes to the initiation and aggravation of experimental and clinical myocardial ischaemia. However, the extent of ₁- and ₂-adrenoceptor-mediated constriction has not been characterized in the porcine coronary circulation despite the frequent use of this experimental model.Fifteen swine were anaesthetized with either -chloralose, enflurane or isoflurane to determine the amount of -adrenoceptor-mediated coronary constriction elicited by either the selective ₁-adrenoceptor agonist methoxamine or the selective ₂-adrenoceptor agonist azepexole. The left anterior descending coronary artery was cannulated and perfused by an external pump delivering constant blood flow from the carotid artery. Following bilateral cervical vagotomy and ß-adrenoceptor blockade with propranolol (2 mg kg^–1), graded dosages of either one of the -adrenoceptor agonists (9–45 g kg^–1 min^–1) were infused into the coronary perfusion line while coronary arterial pressure (CAP) was measured through a distal side arm of the cannula to detect changes in coronary vascular resistance. Infusion of the -adrenoceptor agonists was terminated when systemic arterial pressure increased. Sonomicrometers were used to measure anterior left ventricular wall thickening for the assessment of regional contractile function. During methoxamine infusion, no increase in vascular resistance was observed during -chloralose, enflurane or isoflurane anaesthesia, whereas the infusion of azepexole increased CAP from 103 ± 31 mmHg to 120 ± 35 mmHg (-chloralose), from 101 ± 16 mmHg to 122 ± 11 mmHg (enflurane) and from 84 ± 20 mmHg to 94 ± 19 mmHg (isoflurane), respectively. In four additional swine anaesthetized with enflurane, the intracoronary infusion of the full catecholamine agonist noradrenaline in the presence of propranolol (6 mg kg^–1) increased CAP from 98 ± 10 to 105 ± 10 mmHg prior to an increase in regional left ventricular function or systemic arterial pressure.These results indicate that there are no ₁- and relatively little ₂-adrenoceptor-mediated coronary constrictive effects in swine. Furthermore, neither -adrenoceptor agonist produced any detectable change in regional myocardial contractile function, regardless of the anaesthesia used.Supported by the German Research Foundation (He 1320/3-2). Dr. Guth is the recipient of a scholarship from the Alexander von Humboldt-Foundation. Send offprint requests to G. Heusch at the above address     

Does diabetes affect the intensity of staining of interstitial cells and neuronal tissue in the bladder,prostate and urethra of rabbits?

We compared the intensity of staining of interstitial cells (ICs) and neural tissue in the lower urinary tract of rabbits with diabetes with the intensity in normal subjects. Diabetes was induced by injecting alloxane (65mg/kg) in adult male rabbits. After 3 days, rabbits with a blood glucose level >300 mg/dL were considered to have diabetes. After 8 weeks, the rabbits were killed, and tissue specimens from the bladder, prostate and urethra were obtained. ICs were stained with anti-human CD117 (c-kit) rabbit polyclonal antibody, and neural tissue was stained with synaptophysin. The streptavidin-biotin method was used for immunohistochemical staining. The intensity of c-kit and synaptophysin staining were scored as negative (0), weak (+), moderate (++), and strong (+++). Staining intensity of ICs and neural tissue was assessed and compared in tissues obtained from rabbits with diabetes (n=8) and from control subjects (n=7). Although staining intensity of both ICs and neural tissue was found to be significantly decreased in the bladder tissue of rabbits with diabetes compared to that in the control group (p=0.0001 [ICs] and p=0.021 [neural tissue]), no significant differences in staining intensity of ICs and neural tissue in the urethra and in the prostate was found when rabbits with diabetes were compared to the control group. Diabetes may cause dysfunction of the lower urinary tract, particularly in the urinary bladder, as shown by the staining intensity of ICs and neural tissue.     

Haemodynamic effects of endothelin receptor antagonist,tezosentan, in tumour necrosis factor-α treated anaesthetized rats

Administration of tumour necrosis factor-alpha (TNF-alpha) produces progressive reduction in cardiac output (CO) by affecting preload, afterload and cardiac contractility. We have examined the effect of an endothelin receptor antagonist, tezosentan (1, 3 or 10 mg/kg), on CO, heart rate (HR), blood pressure (BP), mean circulatory filling pressure (P(mcf)), resistance to venous return (RVR), arterial resistance (AR), dP/dt, stroke volume (SV), plasma levels of NO(2)(-)/NO(3)(-), and inducible nitric oxide synthase (iNOS) activity in lungs, ex vivo, following treatment with TNF-alpha (30 microg/kg) in anaesthetized rats. Treatment with TNF-alpha alone resulted in significant reduction in CO (40+/-4%), dP/dt (24+/-2%), P(mcf) (24+/-2%), BP (21+/-3%) and SV (38+/-5%) ( n=6; mean +/- SEM), and significant increases in RVR (38+/-9%) and AR (45+/-6%). There were no significant changes in HR or in plasma levels of NO(2)(-)/NO(3)(-) in animals treated with TNF-alpha but there was a modest but significant increase in iNOS activity. Tezosentan alone did not have any effect on haemodynamics, plasma levels of NO(2)(-)/NO(3)(-) or iNOS activity. Tezosentan at the highest dose abolished the effects of TNF-alpha on dP/dt, AR, and RVR. In animals treated with a combination of TNF-alpha and highest dose of tezosentan CO, P(mcf), BP, and SV were reduced by 28+/-5%, 16+/-3%, 21+/-4%, and 27+/-5%, respectively. Tezosentan was able to inhibit the negative impact of TNF-alpha on AR and dP/dt but not on P(mcf). It is likely that the negative impact of TNF-alpha on CO in tezosentan-treated animals could be entirely attributed to reduction in preload.     

α1-Adrenoceptor subtype mediating contraction of the smooth muscle in the lower urinary tract and prostate of rabbits

Summary The -adrenoceptor subtype mediating contraction of the smooth muscle in the urinary bladder base (trigone), proximal urethra and prostate isolated from male rabbits was investigated by comparing the responsiveness to -adrenoceptor agonists and antagonists under condition where -adrenoceptors and neuronal and extraneuronal uptakes were inhibited. Noradrenaline (non-selective), phenylephrine (₁-selective) and clonidine (₂-selective) caused a dose-dependent contraction in the trigone, urethra and prostate. Phenylephrine acted as a full agonist whereas clonidine was a partial agonist. YM-12617 and prazosin (₁-selective), phentolamine (non-selective) and yohimbine (₂-selective) produced dose-dependent shifts to the right of the dose-response curves for noradrenaline, phenylephrine and clonidine in the all three tissues. YM-12617 (pA₂=9.77, 9.67 and 9.73 for trigone, urethra and prostate, respectively), prazosin (pA₂=8.26, 8.20 and 8.08), phentolamine (pA₂=7.67, 7.62 and 7.45) and yohimbine (pA₂=6.30, 6.30 and 5.94) showed constant pA₂ values irrespective of the agonists and tissues used, suggesting that only a single subclass of -adrenoceptors is present. The actual pA₂ values for these antagonists are comparable to those reported previously in tissues said to contain mainly ₁-adrenoceptors. Thus, we concluded that the postsynaptic -adrenoceptors of the rabbit trigone, urethra and prostate mediating contraction belong to the ₁-subtype.     

Biomarkers,metabonomics, and drug development: Can inborn errors of metabolism help in understanding drug toxicity?

Application of "omics" technology during drug discovery and development is rapidly evolving. This review evaluates the current status and future role of "metabonomics" as a tool in the drug development process to reduce the safety-related attrition rates and bridge the gaps between preclinical and clinical, and clinical and market. Particularly, the review looks at the knowledge gap between the pharmaceutical industry and pediatric hospitals, where metabonomics has been successfully applied to screen and treat newborn babies with inborn errors of metabolism. An attempt has been made to relate the clinical pathology associated with inborn errors of metabolism with those of drug-induced pathology. It is proposed that extending the metabonomic biomarkers used in pediatric hospitals, as "advanced clinical chemistry" for preclinical and clinical drug development, is immediately warranted for better safety assessment of drug candidates. The latest advances in mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy should help replace the traditional approaches of laboratory clinical chemistry and move the safety evaluation of drug candidates into the new millennium.