Potential toxicity of Schisandra chinensis to water environment: acute toxicity tests with water crustaceans.

Fruits of Schisandra chinensis, an East Asian liana plant, are currently more and more used to produce nutrient supplements that positively affect human health due to the content of various secondary metabolites. On the other hand, these substances because of their bioactivity can cause possible allelopathic or toxic effects concerning other organisms (algae, plants, animals). But the ecotoxicological properties of S. chinensis outside its area of origin have yet to be sufficiently verified. Two crustaceans, Daphnia magna and Thamnocephalus platyurus, were selected as model aquatic organisms to test the potential impact of S. chinensis active compounds on the aquatic environment. Crude water extract from S. chinensis fruits, simulating the natural leakage of active substances in water, was tested in treatments from 0.0045 to 45 mg/L (according to the content of schisandrin as the dominating lignan). Effective concentration (EC50) causing 50% lethal effect for D. magna was established to 0.0448 mg/L after 24 h and 0.0152 mg/L after 48 h. EC50 for T. platyurus reached 0.4572 mg/L after 24 h, i.e. more than ten times higher than for D. magna. This study showed that the potential environmentally relevant concentrations of S. chinensis bioactive compounds could represent a severe risk to aquatic ecosystems.

Bioactive compounds from Schisandra chinensis - Risk for aquatic plants?

Schisandra chinensis is a potential plant for production of nutrient supplements due to adaptogens content. The dominant bioactive substance, lignan schisandrin, has positive effects on human health, but it can cause possible allelopathic effects in relation to other plants. S. chinensis is not native to European ecosystems, and its ecotoxicological properties have not been verified yet. Lemna minor was selected as a model aquatic plant to test its potential impact on the aquatic environment. Crude water extract from S. chinensis fruits, simulating the natural soaking of active substances in a surface water body, was used in treatments from 0.045 to 45 mg/L (according to the content of schisandrin as the dominating lignan). During seven days of cultivation, the growth (number of plants, leaf area, fresh weight) and photosynthetic activity of L. minor fronds were assessed. In low treatments (0.045 and 0.09 mg/L), the extract of S. chinensis did not cause any changes in duckweed growth parameters or photosynthetic performance. Higher treatments (0.45 and 0.9 mg/L) caused significant limitations in plants' number, total leaf area, and fresh weight. The photosynthetic parameters (basal chlorophyll fluorescence, quantum yields) were affected only by 0.9 mg/L. The highest treatment, 45 mg/L, exhibited extreme toxicity to duckweed plants causing their death during the first five days of cultivation. Schisandrin and other bioactive substances extractable from S. chinensis fruits can negatively impact water biota in the case of massive contamination of surface water.

Indol-2-Carboxylic Acid Esters Containing N-Phenylpiperazine Moiety - Preparation and Cholinesterase-inhibiting Activity.

BACKGROUND: The indole derivatives and the N-phenylpiperazine fragment represent interesting molecular moieties suitable for the research of new potentially biologically active compounds. This study was undertaken to identify if indol-2-carboxylic acid esters containing N-phenylpiperazine moiety possess acetylcholinesterase and butyrylcholinesterase inhibitory activity. MATERIALS AND METHODS: The study dealt with the synthesis of a novel series of analogs of 1H-indole-2- carboxylic acid and 3-methyl-1H-indole-2-carboxylic acid. The structure of the derivatives was represented by the indolylcarbonyloxyaminopropanol skeleton with the attached N-phenylpiperazine or diethylamine moiety, which formed a basic part of the molecule. The final products were synthesized as dihydrochloride salts, fumaric acid salts, and quaternary ammonium salts. The first step of the synthetic pathway led to the preparation of esters of 1H-indole-2-carboxylic acid from the commercially available 1H-indole-2-carboxylic acid. The Fischer indole synthesis was used to synthesize derivatives of 3-methyl-1H-indole-2-carboxylic acid. RESULTS AND DISCUSSION: Final 18 indolylcarbonyloxyaminopropanols in the form of dihydrochlorides, fumarates, and quaternary ammonium salts were prepared using various optimization ways. The very efficient way for the formation of 3-methyl-1H-indole-2-carboxylate (Fischer indole cyclization product) was the one-pot synthesis of phenylhydrazine with methyl 2-oxobutanoate with acetic acid and sulphuric acid as catalysts. CONCLUSION: Most of the derivatives comprised of an attached N-phenylpiperazine group, which formed a basic part of the molecule and in which the phenyl ring was substituted in position C-2 or C-4. The synthesized compounds were subjected to cholinesterase-inhibiting activity evaluation, by modified Ellman method. Quaternary ammonium salt of 1H-indole-2-carboxylic acid which contain N-phenylpiperazine fragment with nitro group in position C-4 (7c) demonstrated the most potent activity against acetylcholinesterase.

Antioxidative Activity of 1,3,5-Triazine Analogues Incorporating Aminobenzene Sulfonamide, Aminoalcohol/Phenol, Piperazine, Chalcone, or Stilbene Motifs.

A series of 1,3,5-triazine analogues, incorporating aminobenzene sulfonamide, aminoalcohol/phenol, piperazine, chalcone, or stilbene structural motifs, were evaluated as potential antioxidants. The compounds were prepared by using step-by-step nucleophilic substitution of chlorine atoms in starting 2,4,6-trichloro-1,3,5-triazine. Reactions were catalyzed by Cu(I)-supported on a weakly acidic resin. The radical scavenging activity was determined in terms of %inhibition activity and EC50, using the ABTS method. Trolox and ascorbic acid (ASA) were used as standards. In the lowest concentration 1 × 10-4 M, the %inhibition activity values at 0 min were comparable with both standards at least for 10 compounds. After 60 min, compounds 5, 6, 13, and 25 showed nearly twice %inhibition (73.44-87.09%) in comparison with the standards (Trolox = 41.49%; ASA = 31.07%). Values of EC50 at 60 min (17.16-27.78 µM) were 5 times lower for compounds 5, 6, 13, and 25 than EC50 of both standards (trolox = 178.33 µM; ASA = 147.47 µM). Values of EC50 correlated with %inhibition activity. Based on these results, the presented 1,3,5-triazine analogues have a high potential in the treatment of illnesses caused or related to oxidative stress.

Diazepam filled hard capsules intended for detoxification of patients addicted to benzodiazepines and Z-drugs.

OBJECTIVES: The abuse of benzodiazepines and Z-drugs reduces the quality of life of millions of addicted people worldwide. They cannot be discontinued abruptly due to harmful withdrawal symptoms. Detoxification is usually based on replacement of short/middle acting benzodiazepines or Z-drugs by diazepam and tapering the dose over time. In order to enhance patient adherence to an individual withdrawal plan, suitable diazepam dosage forms have to be available. Hard capsules containing an exact and uniform dose could be used for the relief of symptoms caused by altering the plasma level and overcoming psychogenic stress from the dose reduction. METHODS: This work demonstrates that capsules with a content of diazepam ranging from 2.125mg to 0.492 mg (dose decreasing always by 15%) cannot be easily prepared by standard mortar technology in a pharmacy. To meet mass and content uniformity European Pharmacopoeia criteria, capsules were prepared by improved technology based on the preparation of binary blends of calcium phosphate anhydrous and diazepam in descending concentrations in a high-speed mixer (time 30 s) and densification of about 10% during filling of the capsules. RESULTS: All batches (n=20) prepared by improved technology met the requirement for content uniformity compared with only nine batches prepared by standard mortar blender technology. Based on the process capability index, none of the samples prepared by standard technology fitted pharmacopeia limits at the statistically acceptable level. On the other hand, all batches prepared by improved technology exhibited acceptable process capability index. CONCLUSIONS: We have shown that at least 99.73% of batches prepared by our improved technology would meet the pharmacopoeia limits for content uniformity and are suitable for treatment of this type of addiction.

Nano-selenium and its nanomedicine applications: a critical review.

Traditional supplements of selenium generally have a low degree of absorption and increased toxicity. Therefore, it is imperative to develop innovative systems as transporters of selenium compounds, which would raise the bioavailability of this element and allow its controlled release in the organism. Nanoscale selenium has attracted a great interest as a food additive especially in individuals with selenium deficiency, but also as a therapeutic agent without significant side effects in medicine. This review is focused on the incorporation of nanotechnological applications, in particular exploring the possibilities of a more effective way of administration, especially in selenium-deficient organisms. In addition, this review summarizes the survey of knowledge on selenium nanoparticles, their biological effects in the organism, advantages, absorption mechanisms, and nanotechnological applications for peroral administration.

Chemotherapeutic agents for the treatment of metastatic breast cancer: An update.

Breast cancer is the second greatest cause of death among women worldwide; it comprises a group of heterogeneous diseases that evolves due to uncontrolled cellular growth and differentiation and the loss of normal programmed cell death. There are different molecular sub-types of breast cancer; therefore, various options are selected for treatment of different forms of metastatic breast cancer. However, the use of chemotherapeutic drugs is usually accompanied by deleterious side effects and the development of drug resistance when applied for a longer period. This review offers a classification of these chemotherapeutic agents according to their modes of action and therefore improves the understanding of molecular targets that are affected during treatment. Overall, it will allow the clinician to identify more specific targets to increase the effectiveness of a drug and to reduce general toxicity, resistance and other side effects.

Melatonin and breast cancer: Evidences from preclinical and human studies.

The breast cancer affects women with high mortality and morbidity worldwide. The risk is highest in the most developed world but also is markedly rising in the developing countries. It is well documented that melatonin has a significant anti-tumor activities demonstrated on various cancer types in a plethora of preclinical studies. In breast cancer, melatonin is capable to disrupt estrogen-dependent cell signaling, resulting in a reduction of estrogen-stimulated cells, moreover, it's obvious neuro-immunomodulatory effect in organism was described. Several prospective studies have demonstrated the inverse correlation between melatonin metabolites and the risk of breast cancer. This correlation was confirmed by observational studies that found lower melatonin levels in breast cancer patients. Moreover, clinical studies have showed that circadian disruption of melatonin synthesis, specifically night shift work, is linked to increased breast cancer risk. In this regard, proper light/dark exposure with more selective use of light at night along with oral supplementation of melatonin may have benefits for high-risk women. The results of current preclinical studies, the mechanism of action, and clinical efficacy of melatonin in breast cancer are reviewed in this paper. Melatonin alone or in combined administration seems to be appropriate drug for the treatment of early stages of breast cancer with documented low toxicity over a wide range of doses. These and other issues are also discussed.

The hypoxia-responsive long non-coding RNAs may impact on the tumor biology and subsequent management of breast cancer.

Long non-coding RNAs (lncRNAs) are DNA transcripts longer than 200 nucleotides without protein-coding potential. As they are key regulators of gene expression at chromatic, transcriptional and posttranscriptional level, they play important role in various biological and pathological processes. Dysregulation of lncRNAs has been observed in several diseases including cancer. Breast cancer is heterogeneous disease with many molecular subtypes specific in different prognosis and treatment responses. Hypoxia, a common micro-environmental feature of rapidly growing tumour is associated with metastases, recurrences and resistance to therapy. Aberrant expression of hypoxia related lncRNAs significantly correlates with poor outcomes in cancer patients, as the lncRNAs play an important regulatory role in the breast cancer-cell survival. Thus, a better understanding of lncRNAs role in the hypoxic conditions of breast cancer is crucial for precise understanding of the tumorigenesis, disease features and poor clinical outcome, especially in highly aggressive breast cancer subtypes (HER2-positive and triple-negative types). Moreover, lncRNAs may represent tumour marker predicting prognosis and therapeutic targets improving precise and personalized therapy for better patient´s survival. In this review, we summarize the recent information on lncRNAs in breast cancer with special focus on the hypoxia-responsive lncRNAs and their potential impact on the prognosis, therapy algorithms and individual outcomes. Presented data helps in better understanding of the specific mechanisms predicting new therapeutic agents and strategies for the pharmacological intervention.

Adipokines in neurovascular diseases.

Adipose tissue is now described as an endocrine organ secreting a number of adipokines contributing to the development of inflammation and metabolic imbalance, but also endothelial dysfunction, vascular remodeling, atherosclerosis, and ischemic stroke. Leptin, adiponectin, and resistin are the most studied adipokines which play important roles in the regulation of cardiovascular homeostasis. Leptin and adiponectin mediate both proatherogenic and antiatherogenic responses. Leptin and adiponectin have been linked to the development of coronary heart disease and may be involved in the underlying biological mechanism of ischemic stroke. Resistin, a pro-inflammatory cytokine, is predictive of atherosclerosis and poor clinical outcomes in patients with coronary artery disease and ischemic stroke. The changes in serum levels of novel adipokines apelin, visfatin are also associated with acute ischemic stroke. These adipokines have been proposed as potential prognostic biomarkers of cardiovascular mortality/morbidity and therapeutic targets in patients with cardiometabolic diseases. In this article, we summarize the biologic role of the adipokines and discuss the link between dysfunctional adipose tissue and metabolic/inflammation imbalance, consequently endothelial damage, progression of atherosclerotic disease, and the occurrence of ischemic stroke.

Nitric oxide in the pathophysiology of retinopathy: evidences from preclinical and clinical researches.

Retinopathy is the leading cause of blindness and visual disability in working-aged people. The pathogenesis of retinopathy is an actual and still open query. Alterations contributing to oxidative and nitrosative stress, including elevated nitric oxide and superoxide production, changes in the expression of different isoforms of nitric oxide synthase or endogenous antioxidant system, have been implicated in the mechanisms how this ocular disease develops. In addition, it was documented that renin-angiotensin system has been implicated in the progression of retinopathy. Based on comprehensive preclinical and clinical researches in this area, the role of above-mentioned factors in the pathogenesis of diabetic retinopathy, hypertensive retinopathy and ischaemic proliferative retinopathy is reviewed in this study. Moreover, the genetic susceptibility factors involved in the development of the retinopathy and possible strategies that utilize antioxidants as additive therapy are also highlighted here.

Therapeutical strategies for anxiety and anxiety-like disorders using plant-derived natural compounds and plant extracts.

Anxiety and anxiety-like disorders describe many mental disorders, yet fear is a common overwhelming symptom often leading to depression. Currently two basic strategies are discussed to treat anxiety: pharmacotherapy or psychotherapy. In the pharmacotherapeutical clinical approach, several conventional synthetic anxiolytic drugs are being used with several adverse effects. Therefore, studies to find suitable safe medicines from natural sources are being sought by researchers. The results of a plethora experimental studies demonstrated that dietary phytochemicals like alkaloids, terpenes, flavonoids, phenolic acids, lignans, cinnamates, and saponins or various plant extracts with the mixture of different phytochemicals possess anxiolytic effects in a wide range of animal models of anxiety. The involved mechanisms of anxiolytics action include interaction with γ-aminobutyric acid A receptors at benzodiazepine (BZD) and non-BZD sites with various affinity to different subunits, serotonergic 5-hydrodytryptamine receptors, noradrenergic and dopaminergic systems, glutamate receptors, and cannabinoid receptors. This review focuses on the use of both plant-derived natural compounds and plant extracts with anxiolytic effects, describing their biological effects and clinical application.

Cerium dioxide nanoparticles possess anti-inflammatory properties in the conditions of the obesity-associated NAFLD in rats.

BACKGROUND: Obesity is a risk factor for non-alcoholic fatty liver disease (NAFLD). The disease is associated with impairment of pro/antioxidant equilibrium and the inflammation in liver tissue. The aim of the work was to investigate the anti-inflammatory properties of the nanocrystalline cerium dioxide (nCeO2) on the rat model of NAFLD associated with monosodium glutamate (MSG)-induced obesity. METHODS: The study was carried out on three groups of rats: control, MSG- and MSG+nCeO2. They were injected with saline (control) or MSG. A month after born MSG-rats had been treated with water in a volume of 2.9ml/kg, MSG+CeO2 groups - with CeO2 intragastrically (i.g.). The anthropometric and carbohydrate metabolism parameters, content of proinflammatory cytokines (interleukin (IL)-1ß, IL-12Bp40, interferon-γ (INF-γ)) and anti-inflammatory cytokines (IL-4, IL-10, tumor growth factor-ß (TGF-ß)) were measured by ELISA. RESULTS: We have demonstrated the anti-obesity effect of nanocrystalline cerium dioxide and for the first time its anti-inflammatory properties. Nanoparticles reduced the content of pro-inflammatory cytokines (IL-1ß, IL-12Bp40) in rat serum and restored the level of anti-inflammatory cytokines (IL-4, IL-10, TGF-ß) to the control values. CONCLUSION: The precise mechanisms of this phenomenon remain to be unclear but we suppose they are at least partially associated with the strong anti-oxidant action of studied substance. Nanocrystalline cerium dioxide attenuates the inflammatory processes in rat blood that can prevent obesity complications and liver injury.

Angiomodulators in cancer therapy: New perspectives.

The formation of new blood vessels plays a crucial for the development and progression of pathophysiological changes associated with a variety of disorders, including carcinogenesis. Angiogenesis inhibitors (anti-angiogenics) are an important part of treatment for some types of cancer. Some natural products isolated from marine invertebrates have revealed antiangiogenic activities, which are diverse in structure and mechanisms of action. Many preclinical studies have generated new models for further modification and optimization of anti-angiogenic substances, and new information for mechanistic studies and new anti-cancer drug candidates for clinical practice. Moreover, in the last decade it has become apparent that galectins are important regulators of tumor angiogenesis, as well as microRNA. MicroRNAs have been validated to modulate endothelial cell migration or endothelial tube organization. In the present review we summarize the current knowledge regarding the role of marine-derived natural products, galectins and microRNAs in tumor angiogenesis.

Depot risperidone-induced adverse metabolic alterations in female rats.

Atypical antipsychotics are associated with adverse metabolic effects including weight gain, increased adiposity, dyslipidaemia, alterations in glucose metabolism and insulin resistance. Increasing evidence suggests that metabolic dysregulation precedes weight gain development. The aim of this study was to evaluate alterations in adipokines, hormones and basic serum biochemical parameters induced by chronic treatment with depot risperidone at two doses (20 and 40 mg/kg) in female Sprague-Dawley rats. Dose-dependent metabolic alterations induced by risperidone after 6 weeks of treatment were revealed. Concomitant to weight gain and increased liver weight, an adverse lipid profile with an elevated triglyceride level was observed in the high exposure group, administered a 40 mg/kg dose repeatedly, while the low dose exposure group, administered a 20 mg/kg dose, developed weight gain without alterations in the lipid profile and adipokine levels. An initial peak in leptin serum level after the higher dose was observed in the absence of weight gain. This finding may indicate that the metabolic alterations observed in this study are not consequent to body weight gain. Taken together, these data may support the primary effects of atypical antipsychotics on peripheral tissues.

Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy.

AIM: To study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy. METHODS: Consecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n=20); non-proliferative diabetic retinopathy (NPDR; n=18); proliferative diabetic retinopathy (PDR; n=16)] and healthy controls (n=20) between the ages of 40 and 65years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis. RESULTS: A significant difference was found between the serum levels of anti-MPO antibody in various study groups (p<0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR)=1.079, CI 1.010-1.124, p=0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR=1.008, CI 1.006-5.688, p=0.04). CONCLUSIONS: An increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy.

H2S causes contraction and relaxation of major arteries of the rabbit.

OBJECTIVE: Cardiovascular disease (CVD) caused by atherosclerosis remains a worldwide burden. Hydrogen sulfide is a promising new therapeutic avenue for the treatment of CVD, however reports show exogenous H2S has both vasodilator and vasoconstrictor effects depending on organ examined, and in vitro studies in animal models which are not resistant to developing atherosclerosis are limited. We sought to determine if rabbit arteries constricted or dilated to hydrogen sulfide. MATERIAL AND METHODS: The aorta, carotid, renal and iliac arteries were harvested from New Zealand White rabbits (n=4) and subjected to a concentration response curve to the fast H2S releaser NaHS. In addition, a bolus dose of NaHS was used to determine if further dilation was achievable after maximum dilation to acetylcholine similar to nitric oxide donors. Further, NaHS was used to determine if H2S could impair homocysteine induced endothelial dysfunction. RESULTS: Blood vessels relaxed poorly to NaHS and contracted at higher doses. A bolus dose of NaHS relaxed then contracted the aorta, however a bolus dose of NaHS after maximal relaxation to acetylcholine caused marked contraction. NaHS did not prevent homocysteine induced vascular dysfunction. CONCLUSION: NaHS at low doses caused minor relaxation of rabbit blood vessels, indicating a possible therapeutic benefit for low dose H2S in the cellular milieu.

Glucose Metabolism in Cancer and Ischemia: Possible Therapeutic Consequences of the Warburg Effect.

The Warburg effect states that the main source of energy for cancer cells is not aerobic respiration, but glycolysis-even in normoxia. The shift from one to the other is governed by mutually counteracting enzymes: pyruvate dehydrogenase and pyruvate dehydrogenase kinase (PDK). Anaerobic metabolism of cancer cells promotes cell proliferation, local tissue immunosuppression, resistance to hypoxic conditions, and metastatic processes. By switching glucose back to oxidative metabolism, these effects might be reversed. This can be achieved using PDK inhibitors, such as dichloroacetate. Patients suffering from ischemic conditions might benefit from this effect. On the other hand, the ß-blockers (adrenergic ß-antagonists) often used in these patients appear to improve cancer-specific survival, and nonselective ß-blockers have been shown to promote glucose oxidation. Might there be a link?

Effects of polyphenol compounds melanin on NAFLD/NASH prevention.

BACKGROUND: One of the pathogenic mechanisms of the progression non-alcoholic liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) is the accumulation of reactive oxygen species (ROS). So, antioxidant therapy is necessary for successful treatment of the liver injury. We have paid attention to melanin produced by yeast Nadsoniella nigra strain X-1 as novel antioxidant and anti-inflammatory agents with low toxicity. In current study we aimed to investigate the preventive effect of melanin on the monosodium glutamate (MSG) induced NAFLD model in rats. METHODS: The study was carried out on 45 Wistar rats that were divided into 3 groups: intact, MSG- and MSG+melanin groups (n=15 in each group). Newborn rats of MSG- and MSG+melanin groups were administered with MSG (4mg/g, 8µl/g, subcutaneously) at 2nd-10th days of life. Since the age of 1 month, rats of MSG-group were treated with water (0.25ml/100g), rats of MSG+melanin groups-with melanin (1mg/kg) dissolved in water (0.25ml/100g). INTRODUCTION: had been performed intermittently (two-week courses alternated with two-week breaks) for 3 months. In 4-month rats anthropometrical parameters and visceral adipose tissue (VAT) mass were estimated. To assess morphological changes in liver we used NAS (NAFLD activity score). The content of pro-inflammatory cytokines (interleukin (IL)-1ß, IL-12Bp40, interferon (INF)-γ) and anti-inflammatory cytokines (IL-4, IL-10, tumor growth factor (TGF)-ß) were measured by ELISA. RESULTS: We found significantly lower total score (1.0±0.19 vs 3.33±0.36, p<0.001), degree of steatosis (0.73±0.18 vs 1.80±0.17, p<0.001) and manifestation of lobular inflammation (0.27±0.11 vs 1.20±0.17, p<0.001) due to NAFLD activity score in MSG+melanin group compared to MSG-obesity. NASH we confirmed only in 33.3% of rats with MSG-obesity that was significantly higher than after melanin (6.7%) administration (p=0.033). Melanin administration reduce amount of visceral fat on 44.5% (p<0.001) as compared to MSG-obesity group. Melanin reduced the content of IL-1ß in rat serum and restored the level of anti-inflammatory cytokines (IL-10, TGF-ß) to the control values. CONCLUSION: Thus, the administration of melanin can prevent development of NAFLD/NASH in rats with MSG-induced obesity and can be considered as possible novel therapeutic agents but further studies to confirm its action needed.

IRAP inhibition using HFI419 prevents moderate to severe acetylcholine mediated vasoconstriction in a rabbit model.

Coronary artery vasospasm (constriction) caused by reduced nitric oxide bioavailability leads to myocardial infarction. Reduced endothelial release of nitric oxide by the neurotransmitter acetylcholine, leads to paradoxical vasoconstriction as it binds to smooth muscle cell M3 receptors. Thus, inhibition of coronary artery vasospasm will improve clinical outcomes. Inhibition of insulin regulated aminopeptidase has been shown to improve vessel function, thus we tested the hypothesis that HFI419, an inhibitor of insulin regulated aminopeptidase, could reduce blood vessel constriction to acetylcholine. The abdominal aorta was excised from New Zealand white rabbits (n=15) and incubated with 3mM Hcy to induce vascular dysfunction in vitro for 1h. HFI419 was added 5min prior to assessment of vascular function by cumulative doses of acetylcholine. In some rings, vasoconstriction to acetylcholine was observed in aortic rings after pre-incubation with 3mM homocysteine. Incubation with HFI419 inhibited the vasoconstrictive response to acetylcholine, thus improving, but not normalizing, vascular function (11.5±8.9% relaxation vs 79.2±37% constriction, p<0.05). Similarly, in another group with mild vasoconstriction, HFI419 inhibited this effect (34.9±4.6% relaxation vs 11.1±5.2%, constriction, p<0.05). HFI419 had no effect on control aorta or aorta with mild aortic dysfunction. The present study shows that HFI419 prevents acetylcholine mediated vasoconstriction in dysfunctional blood vessels. HFI419 had no effect on normal vasodilation. Our results indicate a therapeutic potential of HFI419 in reducing coronary artery vasospasm.