Regulatory authority approaches to deploying quality improvement standards to community pharmacies: insights from the SafetyNET-RX program.

BACKGROUND: Continuous quality improvement (CQI) programs provide an effective means to improve the safety and quality of community pharmacy practice. The role of formal support processes in ensuring the success of these CQI programs is explored in this research using the SafetyNET-Rx project. OBJECTIVE: The primary objectives of this research were to determine how knowledge of, and confidence in, mandated CQI standards differs among pharmacies with access to formal support mechanisms and those without and the challenges faced by both. METHODS: A survey questionnaire was mailed to 179 community pharmacies in Nova Scotia, Canada, in spring 2011. Quantitative results were analyzed using the Mann-Whitney U test for nonparametric data. Qualitative open-ended responses were analyzed using content analysis. RESULTS: Performing the Mann-Whitney U test indicated that a number of differences exist between the 2 groups with respect to: (1) staff knowledge of reporting quality-related events (QREs) to an anonymous database; (2) conducting annual pharmacy safety self-assessments; (3) confidence in meeting these 2 elements; and (4) documenting changes to address QREs. A number of challenges were identified by respondents through the open-ended questions. CONCLUSIONS: This research highlights the value of the active provision of formal support when developing standards related to quality improvement.

Could fresh human tissues play a key role in drug development?

Biopta was founded in 2002, to provide human tissue-based drug development and testing services to the pharmaceutical industry. Although animal tissues are readily available and are relatively inexpensive, they frequently fail to faithfully predict the results seen in the clinic. Human tissues can provide integrated responses to test drugs in a manner more representative than individual cell types or cell lines alone, and more-directly relevant to the species of interest -- Homo sapiens. In order to expand the use of human tissues, however, an improved infrastructure for the collection and distribution of fresh, functional tissues is highly desirable. Moreover, where there is the potential to obtain tissue from various locations, it becomes possible to test tissue that is specific to the site of drug activity. This is important, as differences may occur between the same tissue types in different locations in the body. The detection of adverse effects is greatly helped by knowledge of how existing drugs behave in the human body. These drugs can act as reference compounds, so that new compounds can then be compared, by using standard concentration-response type studies, in a huge variety of tissues, and their effects extrapolated from what is known of the reference compounds.

ER-Golgi network--a future target for anti-cancer therapy.

Tumor cell demise is an important event in the elimination of abnormal malignant cells and provides an important mechanism of natural tumor suppression. Abnormalities incapacitating these finely tuned processes provide a strong advantage for cancer clones to succeed in evading both the physiological control systems and therapeutic intervention. Expanding our knowledge of the molecular "crosstalks" that regulate tumor cell demise is crucial in guiding the successful design of future anti-cancer therapeutics. Although currently available data indicate that elimination of malignant cells often depends on classical apoptotic pathways (mitochondrial and/or death-receptor pathways), the evidence is mounting that alternative apoptotic and non-apoptotic pathways may effectively contribute to tumor cell death. The assumption that every organelle is capable of sensing, amplificating and executing cell death is also a relatively novel and unexplored concept. As recently shown, the secretory pathway can be actively involved in sensing stress stimuli and possibly even initiating and propagating cell death signaling. Experimental evidence indicates that ER and Golgi apparatus can activate both pro-survival (recovery) mechanisms as well as cell suicide programs if the stress-signaling threshold is exceeded. It is thus conceivable that the fragile balance of protein trafficking between various subcellular compartments provides an exceptional therapeutic opportunity. Interestingly, a growing number of reports recognize novel therapeutic targets, including proteins in control of endoplasmic reticulum (ER) and Golgi homeostasis. Further studies are, however, needed to elucidate precise signaling pathways emanating from ER-Golgi compartment. Development of more potent and selective small-molecule drugs that activate ER-Golgi mediated cell demise is also needed. As the interest in the role of ER-Golgi network during cancer cell death has been gaining momentum, we attempt here to critically appraise current status of development of investigational anti-cancer agents that target ER and/or Golgi.

Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs.

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

Multiparameter detection of apoptosis using red-excitable SYTO probes.

Functional assays allowing phenotypic characterization of different cell death parameters at a single-cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent SYTO probes, we provide herein further insights into applicability of novel red-excitable SYTO stains (SYTO 17, 59-64) for multiparameter analysis of cell fate. In particular, SYTO 62 appears to be a spectrally favorable candidate. Using a correlative comparison between SYTO 16, Annexin V, YO-PRO 1, and fluorescently labeled inhibitors of caspases (FLICA), we demonstrate the specificity of SYTO 62 in detection of apoptotic cell death. Used in conjunction with FLICA or Annexin V, SYTO 62 stain proved amenable for multivariate kinetic analysis of apoptotic events. Considering simplicity of staining protocols, low cost, and avoidance of spectral compensation problems, we expect that red-excitable SYTO dyes will find a wide range of cytometric applications.

Evaluation of equine laminar vein function: harvesting, dissection and the use of functional methods to distinguish between veins and arteries.

INTRODUCTION: Pharmacological evaluation of the unique equine laminar microvasculature is crucial to understanding its role in health and in diseases such as laminitis. However, separating the distinctive characteristics of arterial versus venous components of this complex vascular network has previously proved to be extremely difficult. Encased in a hard hoof capsule, isolation of individual blood vessels presents a considerable challenge. Exacerbating this difficulty, the laminar venous network is adapted to sustain high intravascular pressures and consequently has thickened walls, making the normally straightforward visual distinction between arteries and veins problematic. Here we describe a novel harvesting and dissection method coupled with a functional analysis procedure that facilitates distinction of arteries and veins. METHODS: Laminar tissue was recovered from the hoof of euthanized, clinically normal horses by dissection at the coronary band and stored in cold Krebs-Henseleit physiological salt solution prior to further dissection in the laboratory to remove 2 mm segments of vessels 100-500 microm in diameter. Active length tension measurements were made to evaluate optimal conditions for experimentation, and based on the differences in contractility and appearance, an experimental protocol was set up to allow a) initial distinction between arteries and veins and b) in vitro pharmacological evaluation. RESULTS: Active length tension studies clearly revealed the presence of two populations of vessels distinguished by either a large or a lower maximal contraction that subsequent histological evaluation confirmed to be arteries and veins respectively. Functional distinction using relative contractility to 60 mM potassium salt solution then demonstrated equine laminar veins to have increased sensitivity to the agonist endothelin 1 (ET-1) compared to arteries. DISCUSSION: In vitro evaluation of laminar vessels is possible despite anatomical obstacles. Furthermore, a clear distinction can be made between laminar veins and arteries using functional characteristics providing vessels of a similar size range are selected. Utilising these novel procedures, investigators can unambiguously analyse the pharmacological characteristics of equine laminar veins and arteries to decipher the physiological mechanisms responsible for the control of laminar blood flow.

Neurohumoral effects of the new orally active renin inhibitor, aliskiren, in chronic heart failure.

BACKGROUND: Suppression of the renin-angiotensin-aldosterone system (RAAS) is therapeutically valuable in chronic heart failure (CHF). RAAS inhibition can be achieved in a number of ways though an orally active renin inhibitor (RI) has never been studied before. We describe the neurohumoral effects of an RI. METHODS AND RESULTS: 27 patients with NYHA class II or III CHF and an ejection fraction

Functional human tissue assays.

Functional human tissue assays can be used to measure a vast range of physiological effects at the level of the organ, cell or even gene. In relation to drug discovery, such assays have been used in three main areas: discovery biology, in vitro efficacy pharmacology, and safety pharmacology. The most common area to which assays have been applied has been discovery biology, to investigate the mechanisms underlying a novel drug target or to validate that a target identified in a particular tissue is capable of eliciting a physiological response. Furthermore, as the available assays develop, they are considered an important adjunct to routine safety and efficacy pharmacology tests. Such approaches are often superior to extrapolation from animal data.

Evidence for involvement of alpha1D-adrenoceptors in contraction of femoral resistance arteries using knockout mice.

The role of alpha(1D)-adrenoceptors in vasoconstrictor responses to noradrenaline in mouse femoral resistance arteries was investigated using wire myography in alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) and wild-type (WT) mice of the same genetic background.alpha(1D)-KO mice were 2.5-fold less sensitive than WTs to exogenous noradrenaline and BMY 7378 was significantly less potent against noradrenaline in alpha(1D)-KO mice than in WTs, showing a minor contribution of alpha(1D)-adrenoceptors in response to noradrenaline. Prazosin and 5-methyl-urapidil were equally effective against noradrenaline in alpha(1D)-KO and WT mice. Chloroethylclonidine produced a significantly greater attenuation of the response to noradrenaline in alpha(1D)-KO mice than in WTs. Responses to electrical field stimulation (EFS), at 2-20 Hz for 10 s and 0.09 ms pulse width were significantly smaller overall in alpha(1D)-KOs than in WTs although no significant differences were seen at the different frequencies.BMY 7378 produced significantly greater inhibition of responses at 2 and 5 Hz than at higher frequencies in WTs. In alpha(1D)-KOs, this greater sensitivity to BMY 7378 at lower frequencies was not apparent, confirming that the effect of BMY 7378 was due to blockade of alpha(1D)-adrenoceptors. Prazosin and 5-methyl-urapidil had similar inhibitory effects on responses to EFS in alpha(1D)-KO and WT mice. Chloroethylclonidine inhibited responses to EFS to a significantly greater extent in alpha(1D)-KO mice. The present study with alpha(1D)-KO mice shows that alpha(1D)-adrenoceptors contribute to vasoconstrictor responses to exogenous and neurally released noradrenaline in femoral resistance arteries.

Pharmacological characterization of alpha1-adrenoceptors in mouse isolated femoral small arteries.

Arteries were isolated from male DBA/2 mice and mounted on a small vessel wire myograph for isometric recording. Responses to exogenous noradrenaline were inhibited with high affinity by prazosin (pKB, 9.3) and 5-methyl-urapidil (pKB, 9.2) and with low affinity by 8-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378) (pA(2), 6.7). Chloroethylclonidine (10 microM) produced only a small reduction in the maximum response to noradrenaline. Responses to electrical field stimulation were also inhibited with high affinity by prazosin (pIC50, 9.3-9.5) and 5-methyl-urapidil (pIC50, 8.0-8.3). Responses were sensitive to BMY 7378 at low frequencies of stimulation (pIC50 at 2 Hz, 8.2) but not at high frequencies (pIC50 at 20 Hz, 6.5). In conclusion, contractions to exogenous and endogenous noradrenaline in mouse femoral small arteries are mediated mainly by alpha1A-adrenoceptors. alpha1D-adrenoceptors are not involved in responses to exogenous noradrenaline but appear to be activated by neurally released noradrenaline at a low frequency of stimulation.

The clinical relevance of adrenomedullin: a promising profile?

Adrenomedullin (AM) is a peptide that possesses potentially beneficial properties. Since the initial discovery of the peptide by Kitamura et al. in 1993, the literature has been awash with reports describing its novel mechanisms of action and huge potential as a therapeutic target. Strong evidence now exists that AM is able to act as an autocrine, paracrine, or endocrine mediator in a number of biologically significant functions, including the endothelial regulation of blood pressure, protection against organ damage in sepsis or hypoxia, and the control of blood volume through the regulation of thirst. Its early promise as a potential mediator/modulator of disease was not, however, entirely as a result of the discovery of physiological functions but due more to the observation of increasing levels measured in plasma in direct correlation with disease progression. In health, AM circulates at low picomolar concentrations in plasma in 2 forms, a mature 52-amino acid peptide and an immature 53-amino acid peptide. Plasma levels of AM have now been shown to be increased in a number of pathological states, including congestive heart failure, sepsis, essential hypertension, acute myocardial infarction, and renal impairment. These earliest associations have been further supplemented with evidence of a role for AM in other pathologies including, most intriguingly, cancer. In this review, we offer a timely review of our current knowledge on AM and give a detailed account of the putative role of AM in those clinical areas in which the best therapeutic opportunities might exist.

Non-esterified fatty acids impair endothelium-dependent vasodilation in rat mesenteric resistance vessels.

Elevated circulating levels of NEFAs (non-esterified fatty acids) are associated with states of insulin resistance and increased risk of vascular disease. Previous animal and human studies have demonstrated NEFA-induced endothelial dysfunction of large conduit arteries, reversible by the antioxidant ascorbic acid. We therefore investigated the effect of NEFAs on carbachol-induced endothelium-dependent vasodilation of rat resistance arteries in vitro using the technique of wire myography. In addition, we investigated the effect of co-incubation of NEFAs and ascorbic acid. Cumulative concentration-response curves to carbachol (endothelium-dependent vasodilation) and SNAP (S-nitroso-N-acetyl-DL-penicillamine; endothelium-independent vasodilation) were constructed. Those to carbachol were repeated following a 30 min incubation with either oleic acid (10(-4) M) or palmitic acid (10(-4) M), demonstrating significant impairment of endothelium-dependent vasodilation with both [P<0.05, comparison of pD2 values (the negative log concentration of agonist required to effect a 50% response)]. A cumulative concentration-response curve to carbachol was repeated following co-incubation with palmitic acid (10(-4) M) and the antioxidant ascorbic acid (10(-5) M), demonstrating an abolition of the previously observed endothelial dysfunction induced by palmitic acid. There was no impairment of vasodilation to SNAP following NEFA incubation. We conclude that NEFAs directly impair endothelial function in rat resistance arteries via an increase in oxidative stress at the vascular endothelium.

Alpha1-adrenoceptor subtypes involved in vasoconstrictor responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries.

1. The alpha(1)-adrenoceptor subtypes involved in responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries were characterised using a small vessel myograph, with antagonists prazosin (nonsubtype selective), 5-methyl-urapidil (alpha(1A)-selective), BMY 7378 (alpha(1D)-selective) and the alkylating agent chloroethylclonidine (preferential for alpha(1B)-). 2. Prazosin and 5-methyl-urapidil produced rightward shifts of the exogenous noradrenaline concentration - response curve (CRC) with pA(2) values of 9.2 and 9.1 respectively, in agreement with the presence of alpha(1A)-adrenoceptors. BMY 7378 (1 microm) shifted the noradrenaline CRC with an apparent pK(B) of 6.7, in agreement with the presence of alpha(1A)-, but not alpha(1D)-, adrenoceptors. Chloroethylclonidine at 1 microm had no effect and at 10 microm produced only a small reduction (c. 20%) in the maximum response to noradrenaline, indicating little, if any, contribution from alpha(1B)-adrenoceptors. 3. Responses of the rat femoral resistance arteries to electrical field stimulation (EFS) at 5-30 Hz for 10 s and 0.05 ms pulse width were principally due to alpha(1)-adrenoceptor stimulation. Prazosin and 5-methyl-urapidil inhibited EFS-mediated responses with pIC(50)s of 9.3 and 8.2, respectively, consistent with the alpha(1A)-adrenoceptor being the predominant subtype. Responses to EFS at 10-30 Hz were relatively insensitive to BMY 7378 (pIC(50), 6.5-6.7), while responses to 5 Hz were inhibited with a significantly higher pIC(50) of 8.02, suggesting the contribution of alpha(1D)-adrenoceptors. Chloroethylclonidine had no effect on responses to EFS, ruling out the contribution of an alpha(1B)-subtype. In the presence of cocaine, the predominant subtype involved in responses to EFS was the alpha(1A)-adrenoceptor, with a contribution from alpha(1D)-adrenoceptors at low frequency, as seen in the absence of cocaine. However, there was also a significant increase in the sensitivity to BMY 7378 at higher frequencies, suggesting that a further small alpha(1D)-adrenoceptor component may be uncovered in the presence of cocaine. 5. The present study has shown a predominant role of the alpha(1A)-adrenoceptor in contractions due to exogenous noradrenaline and to neurally released noradrenaline in rat femoral resistance arteries. alpha(1D)-Adrenoceptors are not involved in responses to exogenous noradrenaline but appear to be activated by neurally released noradrenaline at a low frequency of stimulation and at higher frequencies in the presence of neuronal-uptake blockade.

Effects of low-dose continuous combined HRT on vascular function in women with type 2 diabetes.

BACKGROUND: Improvement in vascular endothelial function is widley cited as a beneficial effect of hormone replacement therapy (HRT). Women with type 2 diabetes (T2DM) are at increased cardiovascular risk and have impaired endothelial function. Any benefits of HRT on endothelial function in this group are of particular interest. OBJECTIVES: We assessed effects on vascular function of oral 17beta oestradiol (1 mg) and norethisterone (0.5 mg) in postmenopausal women with T2DM. DESIGN: Double-blind, randomised, placebo-controlled trial. ASSESSMENTS: Twenty-eight women had pulse wave velocity (PWV) and adhesion molecules VCAM-1 and ICAM-1 assessed before and after three months' treatment. Twenty-four women also had gluteal fat biopsy for assessment of resistance vessel function (using wire myography). RESULTS: HRT did not affect PWV, VCAM-1, ICAM-1 or carbachol response. Effects of L-NAME and indomethacin on carbachol sensitivity were similar in both groups. CONCLUSIONS: This HRT preparation had no detectable effect on these measures of endothelial function in women with T2DM.

The alpha 1-adrenoceptor profile in human skeletal muscle resistance arteries in critical limb ischaemia.

OBJECTIVE: We recently reported the hyper-responsiveness of human skeletal muscle resistance arteries (SkMRAs) to noradrenaline in critical limb ischaemia (CLI). In this study we investigated the characteristics of alpha(1)-adrenoceptor subtypes and evaluated the agonist affinity and adrenoceptor reserve in the ischaemic arteries. METHODS: Human SkMRAs were isolated from non-ischaemic and ischaemic areas of limbs amputated for CLI. Subcutaneous resistance arteries were isolated from inguinal biopsies from healthy subjects. Arterial segments were mounted on a small vessel wire myograph. RESULTS: Contractile responses to agonists, adrenaline and A-61603 (alpha(1A)-selective) were significantly increased in ischaemic arteries compared to those in non-ischaemic arteries. Receptor inactivation studies indicated an increase in the alpha-adrenoceptor reserve in the ischaemic arteries but the affinity of noradrenaline was unaffected. Healthy subcutaneous arteries had a similar noradrenaline affinity but a higher receptor reserve than skeletal muscle arteries. In the ischaemic arteries, the antagonists prazosin (alpha(1)-selective), 5-methyl-urapidil (alpha(1A)-selective) and BMY 7378 (alpha(1D)-selective) produced rightward shifts in the concentration response curves (CRCs) of noradrenaline giving pK(B)s of 9.6+/-0.3, 8.4+/-0.2 and 7.1+/-0.4, respectively. Pretreatment with 10 microM chloroethylclonidine decreased the contractile responses to noradrenaline and A-61603 to 57+/-7 and 72+/-4% of their respective controls. CONCLUSIONS: These results demonstrate that the ischaemic SkMRAs have an increased alpha-adrenoceptor reserve with no change in the predominant alpha(1A)-adrenoceptor profile.

Endothelial function is preserved in pregnant women with well-controlled type 1 diabetes.

OBJECTIVE: Pregnant women with diabetes mellitus have a higher incidence of adverse pregnancy outcomes. Vascular, and in particular, endothelial function may be significantly modified in diabetes resulting in impaired endothelium-dependent relaxation. This study aims to investigate endothelium-dependent relaxation in pregnant women with pre-existing type I diabetes mellitus. METHODS: Small arteries (mean luminal diameter approximately 295 microm) were isolated from biopsies of subcutaneous fat from pregnant women with pre-existing type I diabetes mellitus, non-diabetic pregnant women, and non-diabetic non-pregnant women. Endothelial and smooth muscle function were determined using wire myography, and the contributions of nitric oxide, vasodilator prostanoid and endothelial hyperpolarisation were studied using specific inhibitors. RESULTS: Arteries obtained from the diabetic pregnant women did not demonstrate any difference in either endothelial or smooth muscle function when compared with non-diabetic pregnant women. The contribution of nitric oxide to endothelium-dependent relaxation was approximately 20% in the pregnant women regardless of whether they were diabetic, and approximately 11% in the non-pregnant women. Endothelial hyperpolarisation appeared to contribute largely to vasorelaxation in human subcutaneous arteries, and was at least twice that of nitric oxide in pregnant women and fivefold greater in non-pregnant women. CONCLUSIONS: This study provides evidence that pregnant women with well-controlled pre-existing type 1 diabetes mellitus have both normal endothelial and smooth muscle function. Endothelium-dependent hyperpolarisation appears to play a large role in vascular relaxation in human subcutaneous resistance arteries. This study suggests that the problems associated with diabetic pregnancies are unlikely to be due to vascular dysfunction.