The Complex Mechanisms and the Potential Effects of Statins on Vascular Calcification: A Narrative Review.

Vascular calcification (VC) is a complex process of calcium deposition on the arterial wall and atherosclerotic plaques and involves interaction between vascular smooth muscle cells, inflammatory and VC mediators. The latter are independent predictors of cardiovascular morbidity and mortality and potential targets of pharmaceutical therapy. This paper is a narrative review of the complex mechanisms of VC development and in this context the potential anti-atherosclerotic effects of statins. At the initial stages of atherosclerosis VC correlates with atherosclerosis burden and in the long-term with cardiovascular morbidity and mortality. A plethora of animal and clinical studies have proposed statins as the cornerstone of primary and secondary prevention of atherosclerotic cardiovascular disease. Based on coronary computed tomography data, high doses of statins may have negligible or even positive effects on the progression of coronary artery calcification. Growing data support an increase in atherosclerotic plaque calcification in peripheral arteries (e.g., carotids), after long-term, statin-therapy. Despite the paradox of increasing VC, those effects of statins have been associated with higher plaque stability, reducing the risk of consequent adverse events. Statins seem to promote a "favorable" atherosclerotic calcification, suppressing atherosclerotic lesion expansion and their vulnerability. More studies are required to clarify the underlying mechanisms.

Exercise training inhibits atherosclerosis progression and reduces VE-cadherin levels within atherosclerotic plaques in hypercholesterolemic mice.

Vascular endothelial-cadherin (VE-cadherin), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) have emerged as key-factors of atherogenesis. The aim of this study was to evaluate the effects of exercise training (ET) on those key-factors in relation to the progression of atherosclerotic lesions in hypercholesterolemic mice. Thirty male, apoE knockout (apoE-/-) mice were randomly assigned to the following equivalent groups: 1) CO-control: High-fat diet (HFD) administration for 12 weeks. 2) EX-exercise: HFD administration as in CO, and during the last 4 weeks (9th -12th week) ET on treadmill (5sessions/week, 60min/session). At the end of study, blood samples were obtained and all mice were sacrificed. Aortic roots were excised and analysed regarding the percentage of aortic stenosis, and the relative concentrations of collagen, elastin, VE-cadherin, MMP-8,-9 and TIMP-1,-2 within the atherosclerotic lesions. Aortic stenosis was significantly lower in the EX than the CO group (39.63 ± 7.22% vs 62.04 ± 8.55%; p < 0.001), along with considerable increase in fibrous cap thickness and of collagen and elastin contents within plaques (p < 0.05). Compared to controls, exercised-treated mice showed reduced intra-plaque relative concentrations of VE-cadherin (15.09 ± 1.89% vs 23.49 ± 3.01%, p < 0.001), MMP-8 (8.51 ± 2.24% vs 18.51 ± 4.08%, p < 0.001) and MMP-9 (12.1 ± 4.86% vs 18.88 ± 6.23%, p < 0.001). Inversely, the relative concentrations of TIMP-1 and TIMP-2 in the ET group were considerably higher by 62.5% and 31.2% than in the EX group (p < 0.05), respectively. Finally, body weight and lipids concentrations did not differ between groups at the end of the study (p > 0.05). ET treatment induced regression of established atherosclerotic lesions in apoE-/- mice and improved their stability. Those effects seemed to be mediated by favourable modification of VE-cadherin, MMPs and TIMPs.

NEK11 as a candidate high-penetrance melanoma susceptibility gene.

BACKGROUND: A proportion of patients diagnosed with cutaneous melanoma reports a positive family history. Inherited variants in CDKN2A and several other genes have been shown to predispose to melanoma; however, the genetic basis of familial melanoma remains unknown in most cases. The objective of this study was to provide insight into the genetic basis of familial melanoma. METHODS: In order to identify novel melanoma susceptibility genes, whole exome sequencing (WES) analysis was applied in a Dutch family with melanoma. The causality of a candidate variant was characterised by performing cosegregation analysis in five affected family members using patient-derived tissues and digital droplet PCR analysis to accurately quantify mutant allele frequency. Functional in-vitro studies were performed to assess the pathogenicity of the candidate variant. RESULTS: Application of WES identified a rare, nonsense variant in the NEK11 gene (c.1120C>T, p.Arg374Ter), cosegregating in all five affected members of a Dutch family. NEK11 (NIMA-related Kinase 11) is involved in the DNA damage response, enforcing the G2/M cell cycle checkpoint. In a melanoma from a variant carrier, somatic loss of the wildtype allele of this putative tumour suppressor gene was demonstrated. Functional analyses showed that the NEK11 p.Arg374Ter mutation results in strongly reduced expression of the truncated protein caused by proteasomal degradation. CONCLUSION: The NEK11 p.Arg374Ter variant identified in this family leads to loss-of-function through protein instability. Collectively, these findings support NEK11 as a melanoma susceptibility gene.

The cardiovascular-protective properties of saffron and its potential pharmaceutical applications: A critical appraisal of the literature.

Saffron, the dried stigma of Crocus sativus L., is used in traditional medicine for its healing properties and the treatment of various pathological conditions. The present literature review aimed to summarize and evaluate the preclinical and clinical data regarding the protective effects and mechanisms of saffron and its main components (crocin, crocetin, safranal) on cardiovascular risk factors and diseases. Many in vitro and animal studies have been conducted implicating antioxidant, hypolipidemic, anti-diabetic, and antiinflammatory impact of saffron and its constituents. Notably, there is evidence of direct atherosclerosis regression and stabilization in valid atherosclerosis-prone animal models. However, current clinical trials have shown mostly weak effects of saffron and its constituents on cardiovascular risk factors: (a) Modest lowering of fasting blood glucose, without significant reduction of HbA1c in type 2 diabetic patients, (b) moderate/controversial hypolipidemic effects, (c) negligible hypotensive effect, and (d) inconsistent modification of metabolic syndrome parameters. There are important drawbacks in clinical trial design, including the absence of pharmacokinetic/pharmacodynamic tests, the wide variance of doses and cohorts' characteristics, the small number of patients, the short duration. Therefore, large, properly designed, high-quality clinical trials, focusing on specific conditions are required to evaluate the biological/pharmacological activities and firmly establish the clinical efficacy of saffron and its possible therapeutic uses in cardiovascular diseases.

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-ß-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism.

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-ß-cyclodextrin (2-HP-ß-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-ß-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-ß-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Exploring the interactions of irbesartan and irbesartan-2-hydroxypropyl-ß-cyclodextrin complex with model membranes.

The interactions of irbesartan (IRB) and irbesartan-2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) complex with dipalmitoyl phosphatidylcholine (DPPC) bilayers have been explored utilizing an array of biophysical techniques ranging from differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), ESI mass spectrometry (ESI-MS) and solid state nuclear magnetic resonance (ssNMR). Molecular dynamics (MD) calculations have been also conducted to complement the experimental results. Irbesartan was found to be embedded in the lipid membrane core and to affect the phase transition properties of the DPPC bilayers. SAXS studies revealed that irbesartan alone does not display perfect solvation since some coexisting irbesartan crystallites are present. In its complexed form IRB gets fully solvated in the membranes showing that encapsulation of IRB in HP-ß-CD may have beneficial effects in the ADME properties of this drug. MD experiments revealed the topological and orientational integration of irbesartan into the phospholipid bilayer being placed at about 1nm from the membrane centre.

The Complementary Effects of Dabigatran Etexilate and Exercise Training on the Development and Stability of the Atherosclerotic Lesions in Diabetic ApoE Knockout Mice.

Aim: To determine the complementary effects of dabigatran etexilate (DE), exercise training (ET), and combination (DE + ET) on the development and stability of the atherosclerotic lesions in diabetic apoE knockout (apoE-/-) mice. Methods: In 48 male apoE-/- diabetic mice, streptozotocin (STZ) was induced for 5 consecutive days. Mice received a high-fat diet (HFD) for 8 weeks and then were randomized into four groups (1. Control/CG, 2. DEG: HFD with DE, 3. ETG: ET on treadmill, 4. DE + ETG: combination DE and ET treatment). At the end of the eighth week, all mice were euthanatized and morphometry of the aortic lesions at the level of aortic valve was obtained. Collagen, elastin, MCP-1, TNF-a, matrix metalloproteinases (MMP-2,-3,-9), and TIMP-1 concentrations within plaques at the aortic valve were determined. Results: All active groups had significantly smaller aorta stenosis (DEG:7.9 ± 2.2%, ETG:17.3 ± 5.3%, DE + ETG:7.1 ± 2.7%) compared to CG (23.3 ± 5.5% p < 0.05), reduced the relative intra-plaque content of MCP-1, macrophages, MMP-3, and MMP-9, and considerably increased collagen, elastin, and TIMP-1 (p < 0.05). Group 4 showed the most pronounced results (p < 0.05). Both DEG and DE + ETG significantly reduced MMP-2 and TNF-a concentrations compared to ETG and CG (p < 0.010). Conclusion: DE and ET treatment of diabetic apoE-/- mice resulted in complementary amelioration of atherosclerotic lesions development and stability, mediated by the anti-inflammatory modulation of both DE and ET.

Low-pass whole-genome and targeted sequencing of cell-free DNA from cerebrospinal fluid in pediatric patients with central nervous system tumors.

Background: Central nervous system tumors are the most common pediatric solid tumors and the most frequent cause of cancer-related morbidity in childhood. Significant advances in understanding the molecular features of these tumors have facilitated the development of liquid biopsy assays that may aid in diagnosis and monitoring response to therapy. In this report, we describe our comprehensive liquid biopsy platform for detection of genome-wide copy number aberrations, sequence variants, and gene fusions using cerebrospinal fluid (CSF) from pediatric patients with brain, spinal cord, and peripheral nervous system tumors. Methods: Cell-free DNA was isolated from the CSF from 55 patients, including 47 patients with tumors and 8 controls. Results: Abnormalities in cell-free DNA were detected in 24 (51%) patients including 11 with copy number alterations, 9 with sequence variants, and 7 with KIAA1549::BRAF fusions. Positive findings were obtained in patients spanning histologic subtypes, tumor grades, and anatomic locations. Conclusions: This study demonstrates the feasibility of employing this platform in routine clinical care in upfront diagnostic and monitoring settings. Future studies are required to determine the utility of this approach for assessing response to therapy and long-term surveillance.

Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors.

We designed a liquid biopsy (LB) platform employing low-pass whole genome sequencing (LP-WGS) and targeted sequencing of cell-free (cf) DNA from plasma to detect genome-wide copy number alterations (CNAs) and gene fusions in pediatric solid tumors. A total of 143 plasma samples were analyzed from 19 controls and 73 patients, including 44 bone or soft-tissue sarcomas and 12 renal, 10 germ cell, five hepatic, and two thyroid tumors. cfDNA was isolated from plasma collected at diagnosis, during and after therapy, and/or at relapse. Twenty-six of 37 (70%) patients enrolled at diagnosis without prior therapy (radiation, surgery, or chemotherapy) had circulating tumor DNA (ctDNA), based on the detection of CNAs from LP-WGS, including 18 of 27 (67%) patients with localized disease and eight of 10 (80%) patients with metastatic disease. None of the controls had detectable somatic CNAs. There was a high concordance of CNAs identified by LP-WGS to CNAs detected by chromosomal microarray analysis in the matching tumors. Mutations identified in tumor samples with our next-generation sequencing (NGS) panel, OncoKids®, were also detected by LP-WGS of ctDNA in 14 of 26 plasma samples. Finally, we developed a hybridization-based capture panel to target EWSR1 and FOXO1 fusions from patients with Ewing sarcoma or alveolar rhabdomyosarcoma (ARMS), respectively. Fusions were detected in the plasma from 10 of 12 patients with Ewing sarcoma and in two of two patients with ARMS. Combined, these data demonstrate the clinical applicability of our LB platform to evaluate pediatric patients with a variety of solid tumors.

m.3685T > C is a novel mitochondrial DNA variant that causes Leigh syndrome.

Variants in the mitochondrial genome can result in dysfunction of Complex I within the electron transport chain, thus causing disruptions in oxidative phosphorylation. Pathogenic variants in the MT-ND1 (NADH:ubiquinone oxidoreductase core subunit 1) gene that result in Complex I dysfunction are a known cause of Leigh syndrome. The patient is a 4-yr-old female who initially presented with generalized tonic-clonic seizures, with other symptoms of Leigh syndrome becoming apparent after the seizures. A three-generation pedigree revealed no family history of mitochondrial disorders. Laboratory studies were remarkable for elevated blood lactate, alanine, and GDF15. T2-weighted magnetic resonance imaging (MRI) revealed bilateral asymmetric signal hyperintensities in the basal ganglia, specifically in the bilateral putamen and right caudate. Magnetic resonance spectroscopy showed regionally elevated glucose and lactate. Mitochondrial respiratory chain enzyme analysis on skin fibroblasts demonstrated slightly reduced Complex I function. A 16-gene dystonia panel and chromosomal microarray analysis did not identify any disease-causing variants. Combined exome and mitochondrial genome sequencing identified the m.3685T > C (MT-ND1 p.Tyr127His) variant with 62.3% heteroplasmy with no alternative cause for the patient's condition. Mitochondrial genome sequencing of the mother demonstrated that the m.3685T > C variant occurred de novo. The m.3685T > C variant is absent from population databases. The tyrosine 127 residue is highly conserved, and several nearby pathogenic variants in the MT-ND1 gene have been previously associated with Leigh syndrome. We propose that the m.3685T > C variant is a novel mitochondrial DNA variant that causes Leigh syndrome, and we classify this variant as likely pathogenic based on currently available information.

Development and Pharmacokinetics of a Novel Acetylsalicylic Acid Dry Powder for Pulmonary Administration.

Aspirin is an historic blockbuster product, and it has been proposed in a wide range of formulas. Due to exacerbation risks, the pulmonary route has been seldom considered as an alternative to conventional treatments. Only recently, owing to overt advantages, inhalable acetylsalicylic acid dry powders (ASA DPI) began to be considered as an option. In this work, we developed a novel highly performing inhalable ASA DPI using a nano spray-drying technique and leucine as an excipient and evaluated its pharmacokinetics compared with oral administration. The formulation obtained showed remarkable respirability and quality features. Serum and lung ASA DPI profiles showed faster presentation in blood and higher retention compared with oral administration. The dry powder was superior to the DPI suspension. The relative bioavailability in serum and lungs claimed superiority of ASA DPI over oral administration, notwithstanding a fourfold lower pulmonary dose. The obtained ASA DPI formulation shows promising features for the treatment of inflammatory and infectious lung pathologies.

Pharmacokinetic Characteristics of Nebulized Colistimethate Sodium Using Two Different Types of Nebulizers in Critically Ill Patients with Ventilator-Associated Respiratory Infections.

Background: Rising antimicrobial resistance has led to a revived interest in inhaled colistin treatment in the critically ill patient with ventilator-associated respiratory infection (VARI). Nebulization via vibrating mesh nebulizers (VMNs) is considered the current standard-of-care, yet the use of generic jet nebulizers (JNs) is more widespread. Few data exist on the intrapulmonary pharmacokinetics of colistin when administered through VMNs, while there is a complete paucity regarding the use of JNs. Methods: In this study, 18 VARI patients who received 2 million international units of inhaled colistimethate sodium (CMS) through a VMN were pharmacokinetically compared with six VARI patients who received the same drug dose through a JN, in the absence of systemic CMS administration. Results: Surprisingly, VMN and JN led to comparable formed colistin exposures in the epithelial lining fluid (ELF) (median (IQR) AUC0-24: 86.2 (46.0-185.9) mg/L∙h with VMN and 91.5 (78.1-110.3) mg/L∙h with JN). The maximum ELF concentration was 10.4 (4.7-22.6) mg/L and 7.4 (6.2-10.3) mg/L, respectively. Conclusions: Based on our results, JN might be considered a viable alternative to the theoretically superior VMN. Therapeutic drug monitoring in the ELF can be advised due to the observed low exposure, high variability, and appreciable systemic absorption.

Molecular Dynamics Protocols for the Study of Cyclodextrin Drug Delivery Systems.

Hypertension treatment is a current therapeutic priority as there is a constantly increasing part of the population that suffers from this risk factor, which may lead to cardiovascular and encephalic episodes and eventually to death. A number of marketed medicines consist of active ingredients that may be relatively potent; however, there is plenty of room to enhance their pharmacological profile and therapeutic index by improving specific physicochemical properties. In this work, we focus on a class of blood pressure regulators, called sartans, and we present the computational scheme for the pharmacological improvement of irbesartan (IRB) as a representative example. IRB has been shown to exert increased pharmacological action compared with other sartans, but it appears to be highly lipophilic and violates Lipinski rule (MLogP >4.15). To circumvent this drawback, proper hydrophilic molecules, such as cyclodextrins, can be used as drug carriers. This chapter describes the combinatory use of computational methods, namely molecular docking, quantum mechanics, molecular dynamics, and free energy calculations, to study the interactions and the energetic contributions that govern the IRB:cyclodextrin association. We provide a detailed computational protocol, which aims to assist the improvement of the pharmacological properties of sartans. This protocol can also be applied to any other drug molecule with diminished hydrophilic character.

Applications of NMR in Drug:Cyclodextrin Complexes.

NMR spectroscopy is an effective technique, applicable for studying bioactive materials or drug delivery systems in order to obtain comprehensive details related to structural and dynamic characteristics at atomic resolution. The applications of NMR spectroscopy have been increased considerably as a result of the combination of advancement in technological NMR instrumentation and scientific knowledge. This chapter is dedicated to highlight the applications of NMR spectroscopy in drug:cyclodextrin complexes using both liquid- and solid-state NMR spectroscopy.

Application of Neutralization and Freeze-Drying Technique for the Preparation of the Beneficial in Drug Delivery 2-Hydroxypropyl-ß-Cyclodextrin Complexes with Bioactive Molecules.

Bioavailability of active substances is of great importance for the formulation of a drug product, as it actually reflects drug absorption and achievement of the optimum pharmacological effect. A great number of chemical compounds with excellent pharmacological properties possess low solubility and permeability values, ending in low bioavailability in the human body after administration (especially after per os administration). CDs are oligosaccharides that possess biological properties similar to their linear counterparts, but some of their physicochemical properties differ. They are enhancing bioavailability and solving problems of absorption for poorly soluble lipophilic drugs by forming water-soluble inclusion complexes. For this reason, they are widely used in drug delivery systems (Carrier et al. J Control Release 123:78-99, 2007; Kurkov and Loftsson, Int J Pharm 453:167-80, 2013). The main purpose of this chapter is to show a protocol for the preparation of drug:CDcomplex delivery systems.

Nasal powders of quercetin-ß-cyclodextrin derivatives complexes with mannitol/lecithin microparticles for Nose-to-Brain delivery: In vitro and ex vivo evaluation.

Quercetin, a flavonoid with possible neuroprotective action has been recently suggested for the early-stage treatment of Alzheimer's disease. The low solubility and extended first pass effect render quercetin unsuitable for oral administration. Alternatively, brain targeting is more feasible with nasal delivery, by-passing, non-invasively, Blood-Brain Barrier and ensuring rapid onset of action. Aiming to increase quercetin's disposition into brain, nasal powders consisting of quercetin-cyclodextrins (methyl-ß-cyclodextrin and hydroxypropyl-ß-cyclodextrin) lyophilizates blended with spray-dried microparticles of mannitol/lecithin were prepared. Quercetin's solubility at 37 °C and pH 7.4 was increased 19-35 times when complexed with cyclodextrins. Blending lyophilizates in various ratios with mannitol/lecithin microparticles, results in powders with improved morphological characteristics as observed by X-ray Diffraction and Scanning Electron Microscopy analysis. In vitro characterization of these powders using Franz cells, revealed rapid dissolution and permeation 17 (methyl-ß-cyclodextrin) to 48 (hydroxypropyl-ß-cyclodextrin) times higher than that of pure quercetin. Ex vivo powders' transport across rabbit nasal mucosa was found more efficient in comparison with the pure Que. The overall better performance of quercetin-hydroxypropyl-ß-cyclodextrin powders is confirmed by ex vivo experiments revealing amount of quercetin permeated ranging from 0.03 ± 0.01 to 0.22 ± 0.05 µg/cm2 for hydroxypropyl-ß-cyclodextrin and 0.022 ± 0.01 to 0.17 ± 0.04 µg/cm2 for methyl-ß-cyclodextrin powders, while the permeation of pure quercetin was negligible.

Charting the structural and thermodynamic determinants in phenolic acid natural product - cyclodextrin encapsulations.

Cyclodextrins are pliable platforms that have served to optimize the pharmaceutic profile of numerous compounds and to enhance the stability of natural food additives. Caffeic and rosmarinic acid are natural products with proven health benefits, though their full therapeutic potential has not been exploited. To enhance their pharmaceutic profile, we developed cyclodextrin-based formulates and unveiled their thermodynamic and structural principles. The complexes' stoichiometry was determined by ESI-MS. Solid-state and liquid NMR spectroscopy revealed the interactions and the topographical location of the caffeic and rosmarinic acid inside the cyclodextrin cavity. The theoretically analyzed HP-ß-CD's degree of substitution (DS) of caffeic and rosmarinic acids can explain the intensities obtained by 2D NOESY experiments. The thermodynamics and the affinity of the complexes were evaluated through isothermal titration calorimetry. In addition, the rosmarinic and caffeic acids as, also, their complexes showed considerable antimicrobial activity against common food spoilage and pathogenic bacteria. The generated data could provide the basis to understand the structural and thermodynamic determinants implicated in natural products - CD recognition and to develop platforms for the optimization of their pharmaceutical and stability profiles in order to be utilized as safe and stable natural antimicrobial food additives.Communicated by Ramaswamy H. Sarma.

Analysis of CRISPR-Cas9 screens identifies genetic dependencies in melanoma.

Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.

Antihypertensive activity and molecular interactions of irbesartan in complex with 2-hydroxypropyl-ß-cyclodextrin.

Irbesartan (IRB) exerts beneficial effects either alone or in combination with other drugs on numerous diseases, such as cancer, diabetes, and hypertension. However, due to its high lipophilicity, IRB does not possess the optimum pharmacological efficiency. To circumvent this problem, a drug delivery system with 2-hydroxypropyl-ß-cyclodextrin (2-HP-ß-CD) was explored. The 1:1 complex between IRB and 2-HP-ß-CD was identified through ESI QTF HRMS. Dissolution studies showed a higher dissolution rate of the lyophilized IRB-2-HP-ß-CD complex than the tablet containing IRB at pH = 1.2. DSC results revealed the differences of the thermal properties between the complex and various mixtures consisting of the two components, namely IRB and 2-HP-ß-CD. Interestingly, depending on the way the mixture preparation was conducted, different association between the two components was observed. Molecular dynamics (MD) simulations predicted the favorable formation of the above complex and identified the dominant interactions between IRB and 2-HP-ß-CD. In vitro pharmacological results verified that the inclusion complex not only preserves the binding affinity of IRB for AT1R receptor, but also it slightly increases it. As the complex formulation lacks the problems of the tablet, our approach is a promising new way to improve the efficiency of IRB.

Neuropeptide urocortin 1 and its receptors are expressed in the human liver.

The corticotropin-releasing hormone (CRH) system, consisting of CRH, urocortins (Ucns), their receptors CRH(1) and CRH(2), and CRH-binding protein, holds the principal role in mediating the response to stress stimuli. Besides their expression in the brain, CRH neuropeptides and receptors have been found in multiple peripheral sites. We investigated the expression of the CRH system in the human liver, using RT-PCR and immunohistochemistry. Ucn 1 gene transcripts were amplified in all the human liver biopsies examined. Ucn 1 immunoreactivity was localized in hepatocytes. CRH(1) and CRH(2(alpha)) receptor gene expression was also found, and receptor protein had a similar distribution to Ucn 1. Finally, Ucn 1 and CRH receptor expression was demonstrated in hepatic biopsies from a variety of liver pathologies, including primary or metastatic liver carcinoma and cirrhosis. We conclude that the CRH system is expressed by human liver under normal and pathological conditions, Ucn 1 being the major ligand. Further study is required to unfold the biological role of these effectors in liver physiology and pathogenesis, as they may act in an autocrine manner through activation of the CRH receptors expressed locally.